03-Seminars in Fetal & Neonatal Medicine-June 2014 PDF

Review
Counseling women and men regarding exposures to reproductive

and developmental toxicants before conception or women during
pregnancy
Robert L. Brent
*
Thomas Jefferson University, Alfred I. duPont Hospital for Children, Room 308, A/R Building, PO Box 269, Wilmington, DE 19899, USA
Keywords:
Birth defects
Counseling
Development
Environmental toxicants
Pregnancy
Teratology principles
s u m m a r y
It should be apparent that determining the reproductive risks of an exposure during pregnancy or the
cause of a childs congenital malformations is not a simple process. It involves a careful analysis of the
medical and scientic literature pertaining to the reproductive toxic effects of exogenous agents in
humans and animals, as well as an evaluation of the exposure and the biological plausibility of the
concern of an increased risk or a causal connection between the exposure and a childs congenital
malformations. It also involves having available a detailed physical examination of the malformed infant
or child and a review of the scientic literature pertaining to genetic and environmental causes of the
malformations in question. Abridged counseling on the basis of supercial and incomplete analyses is a
disservice to the family. Experienced counselors understand that their primary task is to educate the
pregnant women or family members concerning the risk of an environmental exposure. The counselor
should advise them on the options available, but not on which option to select.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
In 1944, during World War II, I received my draft notice for in-
duction into the armed forces, which would occur in mid 1945. At
the age of 15 years, I applied to the University of Rochester, since I
had passed all the high school senior courses which included
advanced physics, biology and chemistry. In June 1944, I was a
freshman at the University and obtained a part-time position at the
Manhattan Project Research facility in Rochester in the genetics and
embryology divisions. Whereas all the employees at the University
of Rochester were investigating the biological effects of ionizing
radiation and the entire research staff had the highest level of se-
curity clearance, none of the staff were aware that researchers in
Los Alamos were attempting to develop an atomic bomb. After the
war ended in 1945, the Manhattan Project became one of the
Atomic Energy Commission (AEC) facilities at the University. In
1949, I entered medical school but continued my embryology ra-
diation research. The head of the division, Dr James Wilson, offered
me the opportunity to be his graduate student in the summer of
1950 and Wilson spent the summer intensively training me in all
the experimental techniques used in that division. At the end of the
summer, Dr Wilson announced that he had accepted a position at
the University of Cincinnati. Rather than close the division, the
Administration appointed me the Head of the Division. I had a
budget, a technician and secretarial support and I taught the Uni-
versity of Rochester medical students embryology during the next
four years while I was in Rochester completing my medical and
graduate degrees. During these four years my national and inter-
national counseling effort began at the University of Rochester.
The only other major AEC facility in the country interested in
radiation effects on reproduction was at Liane Russells excellent
laboratory in Oak Ridge, Tennessee. However, she was extensively
involved in pursuing her research. So the calls and letters all came
to Rochester, and then to the Massachusetts General Hospital
where I was a resident physician and the Walter Reed Army Insti-
tute of Research where I was the head of the Radiation Biology
Section for my two years of Army service from 1955 until 1957. I
arrived at The Jefferson Medical College in 1957 and have been
there for 56 years. In the 1950s there were practically no educa-
tional programs in medical school or graduate school that per-
tained to the evaluation of the risks of reproductive and
developmental toxicants or counseling families with regard to the
presence or absence of risks from these exposures. The concept of
professional counseling was still in its infancy.
* Tel.: 1 302 651 6880; fax: 1 302 651 6888.
E-mail address: rbrent@nemours.org.
Contents lists available at ScienceDirect
Seminars in Fetal & Neonatal Medicine
j ournal homepage: www. el sevi er. com/ l ocat e/ si ny
1744-165X/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.siny.2013.09.008
Seminars in Fetal & Neonatal Medicine 19 (2014) 139e152
2. The stages of medical and graduate school education in the
USA: When did counseling with regard to reproductive and
developmental toxicant exposure become a recognized and
necessary skill?
Medical education in the USA received a major impetus
following the publication of Abraham Flexners (1910) monograph
that was commissioned by the Carnegie Foundation for the
Advancement of Teaching [1]. Prior to being contacted by the Car-
negie Foundation, Flexner graduated fromJohns Hopkins in 1885 at
the age of 19 and crystallized in his mind the components of quality
higher educational programs: small classes, personal attention and
hands-on teaching. He returned to Louisville and founded a med-
ical school using these principles. The graduates proved to be em-
issaries of what quality education can accomplish. It was these early
successes that convinced the Carnegie Foundation that Flexner was
the scholar who could improve medical education. Following the
implementation of the Flexner Report many rural medical schools
closed. Many physicians are unaware that Flexner was not a
physician nor did he have an advanced degree. However, he was a
brilliant teacher and scholar.
Over the next 50 years the Flexner model of medical education
evolved into the bioscience model of medical education and med-
ical practice. High quality basic science education and research
could provide all the answers, so that physicians could diagnose,
ameliorate, treat or cure medical problems with which they
encountered.
Unfortunately, the bioscience model is incomplete and can
result in a signicant portion of the patient population being
dissatised with their care. This was evident to George Engle at the
University of Rochester. He was trained in psychiatry and internal
medicines and published many articles about the biopsychosocial
model of health care delivery that reected his interest in psy-
chosomatic medicine [2]. At the University of Rochester Engle
established the medical psychiatric liaison service staffed by in-
ternists and psychiatrists. Engle indicated that he would prefer
having physicians with behavioral training rounding on the other
clinical services rather than on the psychiatry service. Engle was
adamant that you cannot ignore the impact of the environment on
the patients disease or the behavioral defenses available to the
patient. It was clear that Engle believed that compassion and
empathy were important components of the biopsychosocial
model of medical care.
Carl Rogers [3e5] is probably the most important contributor to
the elements of proper counseling since he emphasized the hu-
manistic approach to psychological counseling. If the patients or
contacts do not sense that the counselor is compassionate or
empathetic, their interaction will be less than satisfactory. Exhib-
iting genuine compassion and empathy results in a client-centered
interaction with much greater success in properly communicating
and educating the contact. The client, patient or contact has to
believe that the counselor believes that the contact deserves
respect, which is demonstrated by exhibiting compassion and re-
gard for the contact (unconditional, positive regard) [4]. The
fundamental precepts of Rogerian counseling include congruence
(being genuine in ones concern), empathy, and unconditional
positive regard [4]. A widely accepted component of genuine
concern in medical counseling is the responsibility of the counselor
to provide core knowledge of the evidence addressing the issue in
question. To be genuinely concerned is to seek and provide reliable
information. A further adaptation of these principles involves
providing an unbiased discussion of the facts surrounding the
problem being addressed. Empathy requires some knowledge of,
and sensitivity to, the social and cultural position of the persons
being counseled.
During the rst 50 years of the twentieth century, the rules of
professional counseling were rarely articulated or taught. It was
only after the writings of Engle and Rogers that the essential fea-
tures of professional counseling were legitimized, whether it per-
tained to psychotherapy, medical care and especially for counseling
contacts concerning reproductive and developmental risks from
environmental exposures.
3. The history of providing counseling to pregnant women
exposed to reproductive and developmental toxicants
(teratogenesis, congenital malformations), and men and
women with preconception exposures (mutagenesis in the
gametes and in the offspring in the next generation)
Individual counseling is part of the practice of clinical medi-
cine. However, at the beginning of the twentieth century there
were very few individuals prepared to counsel patients with re-
gard to the risk of reproductive, developmental and mutagenic
toxicants.
Our laboratory has provided consultations dealing with the risk
of various environmental toxicant exposures during or before
pregnancy since 1950. In 1960 a group 60 scientists interested in
birth defects met at the Slone Kettering Institute in New York City
to discuss their common interests in the causes of birth defects
and decided to create a Birth Defects Society. In 1961 many of
those scientists met in Cincinnati where the charter of the Tera-
tology Society was drafted. Within the Teratology Society a pro-
portion of the membership was interested in providing counseling
to the patient population. There were diverse opinions as to
whether individuals who provided counseling should receive
training and pass a certication examination. That issue was never
resolved. However, the problem was partially solved in the 1990s
when the Organization of Teratology Services was formed, which
frequently meets with the Teratology Society at the latters annual
meeting. In 2005, the name of OTIS was changed to the Organi-
zation of Teratology Information Specialists. In Europe the Euro-
pean Teratology Society (founded in the 1970s) and European
Network of Teratology Information Services (ENTIS) dene the
counseling organizations in Europe having active scientic and
counseling programs, as also happens in Japan and Australia. But
none of these organizations professionally certies individuals to
provide counseling, although some members may be certied
genetic counselors.
In North America there are many OTIS members and branches.
The Mother Risk program (Motherrisk.org) headed by Dr Gideon
Koren is located at the Hospital for Sick Children and the University
of Toronto. In San Diego, Dr Kenneth Lyon Jones heads the largest
program in California; and in Seattle, the TERIS program was
initiated with a federal grant obtained by Dr Jan Friedman. With the
advent of the Internet, consulting has become more rapid and
efcient. In 2012 the Ask the Expert (ATE) website of the HPS
(Health Physics Society) received about 2 400 000 hits. More than
700 000 prepared answers to questions were downloaded. More
than 1800 contacts were still quite anxious after reading the
website answers and requested a personal consultation. During
2012 our laboratory received its 25 000th consultation. We also
receive consultations by letter, telephone and e-mail unrelated to
the HPS website. From this extensive experience we have learned
that many physicians and other health care counselors are not
prepared to counsel patients concerning reproductive and devel-
opmental risks. Approximately 6e10% of the contacts concerned
about various environmental toxicants had been provided inaccu-
rate information that stimulated unwarranted anxiety that could
have resulted in an unnecessary interruption of a wanted
pregnancy.
R.L. Brent / Seminars in Fetal & Neonatal Medicine 19 (2014) 139e152 140
4. Role of the counselor in advising families
The counselor must be cognizant of many patients belief that
congenital malformations are caused by a drug or medication taken
during pregnancy. Counseling patients about reproductive risks
requires a signicant degree of both knowledge and skill. Physi-
cians must also realize that erroneous counseling by inexperienced
health professionals may be a stimulus to non-meritorious litiga-
tion [6e8].
Unfortunately, some individuals have assumed that if a drug or
chemical causes birth defects in an animal model or in an in-vitro
system at a high dose, then it has the potential for producing
birth defects at any dose. This may be reinforced by the fact that
many teratology studies reported in the literature using several
doses have not determined the no-effect dose.
Ignoring the basic tenets of teratology (Box 1) appears to occur
most frequently in the evaluation of environmental toxic exposures
where the exposure was very low or unknown and the agent has
been reported to be teratogenic at a very high dose or a maternally
toxic dose in animal modes. In most but not all instances, the actual
population exposure is revealed to be orders of magnitude below
the threshold dose observed in animal studies. This has occurred
with 2,4,5,-trichlorophenoxyacetic acid (2,4,5-T, also known as
Agent Orange), polychlorinated biphenyls (PCBs), lead, cadmium,
pesticides, herbicides, veterinary hormones and some industrial
exposures.
Unfortunately, environmental disasters have been responsible
for birth defects or pregnancy loss in exposed populations (methyl
mercury in Japan, PCBs in the Orient, organic mercury in the Middle
East, lead poisoning in the nineteenth and early twentieth cen-
turies) and there are examples of teratogenic drugs and chemicals
having been introduced (Table 1) [10e17]. Therefore, we can never
generalize as to whether a chemical or drug is safe or hazardous
unless we know the magnitude and stage of the exposure.
Before their baby is born, parents may be concerned about the
risks of various environmental exposures. If the child is born with
congenital malformations they may question whether there was a
causal relationship with an environmental exposure.
4.1. Scholarly evaluation
When a counselor responds to a parents inquiry (What caused
my childs birth defect?), the physician should respond in the same
scholarly manner that would be utilized inperforming a differential
diagnosis for any clinical problem. Physicians have a protocol for
evaluating complex clinical problems; i.e. fever of unknown origin,
failure to thrive, congestive heart failure, or respiratory distress
(Box 2).
If a mother of a malformed infant had some type of exposure
during pregnancy, such as a diagnostic radiological examination or
medication utilized during pregnancy, the consulting physician
should not support or suggest the possibility of a causal relation-
ship before performing a complete evaluation (Box 3). Likewise, if a
pregnant woman who had not yet delivered had some type of
exposure during pregnancy, the consulting physician should not
support or suggest the possibility that the fetus is at increased risk
before performing a complete evaluation.
As mentioned previously, only a small percentage of birth de-
fects is due to prescribed drugs, chemicals and physical agents
(Tables 2 and 3). Even when the drug is listed as a teratogen, it has
to have been administered during the sensitive period of devel-
opment for that drug and above the threshold dose for producing
teratogenesis. Furthermore, the malformations in the child should
be the malformations that are included in the teratogenic syn-
drome produced by that drug. It should be emphasized that a
recent analysis pointed out that there are no drugs with measurable
teratogenic potential in the list of the 200 most prescribed drugs in
the USA [17].
After a complete examination of the child and a review of the
genetic and teratology medical literature, the clinician must decide
on whether the childs malformations are due to a genetic cause or
an environmental toxin or agent (Boxes 1 and 2; Table 1). The
clinician may not be able to conclude, denitively or presumptively,
the etiology of the childs birth defects. This information must then
be conveyed to the patient in an objective and compassionate
manner. A similar situation exists if a pregnant woman has been
exposed to a drug, chemical or physical agent, since the mother will
want to know the risk of that exposure to her unborn child. If one
wishes to answer the generic question, Is a particular environ-
mental drug, chemical or physical agent a reproductive toxicant?
then a formal approach is recommended that includes a ve-part
evaluation as described in Box 4.
5. Deciencies in counseling education and methodology
Some physicians and other health professionals misinform their
patients regarding the magnitude of the risk of environmental
toxicant exposure during pregnancy [23,29]. Ratnapalan et al. [29]
surveyed a large number of general physicians and obstetricians
regarding the risk of abdominal computed tomography (CT) scan to
pregnant women during the 6th week of gestation (Table 4).
Experienced counselors understand that their primary task is to
educate the pregnant women or family members concerning the
risk of an environmental exposure. The counselor should advise
Box 1
Principles of teratology.
When evaluating studies dealing with the reproductive and
developmental effects of any environmental agent, impor-
tant principles should guide the analysis of reproductive
human and animal studies. Paramount to this evaluation is
the application of the basic science principles of teratology
and developmental biology:
a
1. Exposure to teratogens follows a toxicological doseere-
sponse curve. There is a threshold below which no effect
will be observed, and, as the dose of the teratogen is
increased, both the severity and frequency of reproduc-
tive effects will increase (deterministic effect; tissue
reaction).
2. The period of exposure is critical in determining what
effects will be produced and whether any effects can be
produced by a known teratogen. Some teratogenic ef-
fects have a broad period of sensitivity, others a very
narrow one.
3. Even the most potent teratogenic agent cannot produce
every malformation.
4. Most teratogens have a conned group of congenital
malformations that occur after exposure during a critical
period of embryonic development. This conned group
of malformations is referred to as the syndrome that
describes the agents teratogenic effect.
5. Whereas a group of malformations may suggest the
possibility of certain teratogens, they cannot denitively
conrm the causal agent. On the other hand, the pres-
ence of certain malformations can eliminate the possi-
bility that a particular teratogenic agent was responsible.
a
Source: Brent [52].
Table 1
Developmental toxicants: risks of congenital malformations and abortion in the human.
Developmental
toxicant
Reported effects or associations and estimated risks Comments
a
Alcohol Fetal alcohol syndrome: intrauterine growth retardation, maxillary hypoplasia, reduction in width
of palpebral ssures, characteristic but not diagnostic facial features, microcephaly, mental
retardation. An increase in spontaneous abortion has been reported but since mothers who abuse
alcohol during pregnancy have multiple other risk factors, it is difcult to determine whether this is
a direct effect on the embryo. Consumption of 6 oz of alcohol or more per day constitutes a high risk
but it is likely that detrimental effects can occur at lower exposures.
Quality of available information: good
to excellent.
Direct cytotoxic effects of ethanol and
indirect effects of alcoholism. Whereas
a threshold teratogenic dose is likely it
will vary in individuals because of a
multiplicity of factors.
Aminopterin,
methotrexate
Microcephaly, hydrocephaly, cleft palate, meningomyelocele, intrauterine growth retardation,
abnormal cranial ossication, reduction in derivatives of rst branchial arch, mental retardation,
postnatal growth retardation. Aminopterin can induce abortion within its therapeutic range; it is
used for this purpose to eliminate ectopic embryos. Risk from therapeutic doses is unknown but
appears to be moderate to high.
Quality of available information: good.
Anticancer, antimetabolic agents; folic
acid antagonists that inhibit
dihydrofolate reductase, resulting in
cell death.
Androgens Masculinization of female embryo: clitoromegaly with or without fusion of labia minora. Non-
genital malformations are not a reported risk. Androgen exposures which result in masculinization
have little potential for inducing abortion. Based on animal studies, behavioral masculinization of
the female human will be rare.
Effects are dose and stage dependent;
stimulates growth and differentiation of
sex steroid receptor-containing tissue.
Angiotensin-converting
enzyme (ACE)
inhibitors
The therapeutic use of ACE inhibitors has neither a teratogenic effect nor an abortigenic effect in the
rst trimester. Since this group of drugs does not interfere with organogenesis, they can be used in a
woman of reproductive age; if the woman becomes pregnant, therapy can be changed during the
rst trimester without an increase in the risk of teratogenesis. Later in gestation these drugs can
result in fetal and neonatal death, oligohydramnios, pulmonary hypoplasia, neonatal anuria,
intrauterine growth retardation, and skull hypoplasia. Risk is dependent on dose and length of
exposure.
Antihypertensive agents; adverse fetal
effects are related to severe fetal
hypotension over a long period of time
during the second or third trimester.
Caffeine Caffeine is teratogenic in rodent species with doses of 150 mg/kg. There are no convincing data that
moderate or usual exposures (300 mg per day or less) present a measurable risk in the human for
any malformation or group of malformations. On the other hand, excessive caffeine consumption
(exceeding 300 mg per day) during pregnancy is associated with growth retardation and embryonic
loss.
Quality of available information: fair to
good.
Behavioral effects have been reported
and appear to be transient or
temporary; more information is needed
concerning the population with higher
exposures.
Carbamazepine Minor craniofacial defects (upslanting palpebral ssures, epicanthal folds, short nose with long
philtrum), ngernail hypoplasia, and developmental delay. Teratogenic risk is not known but likely
to be signicant for minor defects. There are too few data to determine whether carbamazepine
presents an increased risk for abortion. Since embryos with multiple malformations are more likely
to abort, it would appear that carbamazepine presents little risk because an increase in these types
of malformations has not been reported.
good.
Anticonvulsant; little is known
concerning mechanism. Epilepsy may
itself contribute to an increased risk for
fetal anomalies.
Cocaine Preterm delivery; fetal loss; placental abruption; intrauterine growth retardation; microcephaly;
neurobehavioral abnormalities; vascular disruptive phenomena resulting in limb amputation,
cerebral infarctions and certain types of visceral and urinary tract malformations. There are fewdata
to indicate that cocaine increases the risk of rst trimester abortion. The low but increased risk of
vascular disruptive phenomena due to vascular compromise of the pregnant uterus would more
likely result in mid-gestation abortion or stillbirth. It is possible that higher doses could result in
early abortion. Risk for deleterious effects on fetal outcome is signicant; risk for major disruptive
effects is low, but can occur in the latter portion of the rst trimester as well as the second and third
trimesters.
good.
Cocaine causes a complex pattern of
cardiovascular effects due to its local
anesthetic and sympathomimetic
activities in the mother. Fetopathology
is likely to be due to decreased uterine
blood ow and fetal vascular effects.
Because of the mechanism of cocaine
teratogenicity, a well-dened cocaine
syndrome is not likely. Poor nutrition
accompanies drug abuse and multiple
drug abuse is common.
Chorionic villous
sampling (CVS)
Low, but increased risk of orofacial malformations and limb reduction defects of the congenital
amputation type as seen in vascular disruption malformations in some series. The risk of abortion
following CVS is quite low.
Quality of available information: fair.
Excessive bleeding from the chorion is
probably related in part to the
experience of the operator. Further
research is necessary to determine
whether CVS is safer for the fetus at
certain stages of gestation.
Coumarin derivatives Nasal hypoplasia; stippling of secondary epiphysis; intrauterine growth retardation; anomalies of
eyes, hands, neck; variable central nervous system anatomical defects (absent corpus callosum,
hydrocephalus, asymmetrical brain hypoplasia). Risk from exposure 10e25% during 8th to 14th
week of gestation. There is also an increased risk of pregnancy loss. There is a risk to the mother and
fetus from bleeding at the time of labor and delivery.
Anticoagulant; bleeding is an unlikely
explanation for effects produced in the
rst trimester. Central nervous system
defects may occur any time during
second and third trimesters and may be
related to bleeding.
Cyclophosphamide Growth retardation, ectrodactyly, syndactyly, cardiovascular anomalies, and other minor anomalies.
Teratogenic risk appears to be increased but the magnitude of the risk is uncertain. Almost all
chemotherapeutic agents have the potential for inducing abortion. This risk is dose-related; at the
lowest therapeutic doses the risk is small.
Anticancer, alkylating agent; requires
cytochrome P450 mono-oxidase
activation; interacts with DNA,
resulting in cell death.
Diethylstilbestrol (DES) Clear cell adenocarcinoma of the vagina occurs in about 1:1000 to 10 000 females who were exposed
in utero. Vaginal adenosis occurs in about 75% of females exposed in utero before the 9th week of
pregnancy. Anomalies of the uterus and cervix may play a role in decreased fertility and an increased
incidence of prematurity although the majority of women exposed to DES in utero can conceive and
deliver normal babies. In-utero exposure to DES increased the incidence of genitourinary lesions and
good.
Synthetic estrogen; stimulates estrogen
receptor-containing tissue, may cause
Table 1 (continued)
Developmental
toxicant
a
infertility in males. DES can interfere with zygote survival, but it does not interfere with embryonic
survival when given in its usual dosage after implantation. Offspring who were exposed to DES in
utero have an increased risk for delivering prematurely, but do not appear to be at increased risk for
rst trimester abortion.
misplaced genital tissue which has a
greater propensity to develop cancer.
Diphenylhydantoin Hydantoin syndrome: microcephaly, mental retardation, cleft lip/palate, hypoplastic nails and distal
phalanges; characteristic but not diagnostic facial features. Associations documented only with
chronic exposure. Wide variation in reported risk of malformations but appears to be 10%. The few
epidemiological data indicate a small risk of abortion for therapeutic exposures for the treatment of
epilepsy. For short-term treatment, i.e. prophylactic therapy for a head injury, there is no
appreciable risk.
Quality of available information:
fair to good.
Anticonvulsant; direct effect on cell
membranes, folate, and vitamin K
metabolism. Metabolic intermediate
(epoxide) has been suggested as the
teratogenic agent.
Electromagnetic
elds (EMFs)
The data pertaining to video display terminals indicate that the EMF exposures from these units do
not present an increased risk for abortion or congenital malformations. The data on power line and
appliance exposures are too varied to drawany conclusions, although the risks appear to be small or
non-existent. Human exposures to video display terminals and power lines are quite low and are
unlikely to have reproductive effects.
Pregnant animals exposed to EMF do
not exhibit consistent or reproducible
reproductive effects. There are still
questions about biologic effects for
frequencies and wave forms of
magnetic elds that have not been
adequately studied.
Fluconazole Fluconazole, a triazole antifungal agent used to treat mycotic infections, is a widely administered
medication for vaginal candidiasis. In this latter treatment, it is prescribed as a single dose of 150 mg
and has not been associated with increased abnormalities or congenital defects in pregnancy series
[18e20] Fluconazole is also used to treat more serious mycotic infections at doses of 400e800 mg/
day on a continuous basis. During the 1990s, four case reports of infants exposed to this high-dose
regimen of uconazole had a distinct and consistent pattern of malformations [21] The constellation
of defects consisted of craniosynostosis, orbital hypoplasia, and the rather unique skeletal
manifestations of humeral radial synostosis and femoral bowing.
Infectious agents The cytotoxic effects and inammatory responses resulting from fetal infections interfere with
organogenesis and/or histogenesis. Fetopathic syndromes are related to the specic tissue
localization, pathologic characteristics of the infectious agent and the duration of the infection in the
embryo and fetus. In some instances the infection may be debilitating to the mother and indirectly
result in pregnancy loss.
Cytomegalovirus (CMV) Fetal cytomegalovirus infection presents an increased risk for abortion but it does not appear that
maternal genital infection increases the risk of abortion. Fetal infection occurs in about 20% of
maternal infections. Intrauterine growth retardation: risk of brain damage is moderate after fetal
infection early in pregnancy; characteristic parenchymal calcication.
to excellent.
CMV damages organs principally by
cellular necrosis.
Herpes simplex virus Generalized organ infections, microcephaly, hepatitis, eye defects, vesicular rash. Maternal infection
can be transmitted in utero or perinatally. Herpes simplex 2 is one of the few infections where it is
agreed that the risk of abortion is increased.
Fetal anomalies associated with herpes
simplex virus infection appear to be due
to disruption rather than malformation.
Human
immunodeciency
virus (HIV)
The overall risk of vertical transmission of HIV is 25e40%. Fetal HIV infection and asymptomatic
maternal HIV infection are not associated with adverse effect on fetal growth or development.
Symptomatic maternal HIV infection, other sexually transmitted diseases and opportunistic
infections may increase the risk of low birth weight or perinatal morbidity.
good.
Prophylactic treatment with zidovudine
does not appear to cause permanent
adverse effects in the fetus.
Parvovirus B19 Infection can result in erythema infectiosum in children but in the fetus can result in hydrops fetalis
and fetal death; congenital anomalies are likely to be very rare. The risk for stillbirth with hydrops
has been clearly substantiated. An increased risk for abortion has been suggested, but is more
difcult to substantiate.
good.
Fetal infection is not frequent. Infection
of red blood cell precursors causes
severe anemia.
Rubella virus Greater than 80% incidence of embryonic infection with exposure in rst 12 weeks, 54% at 13e14
weeks, 25% at end of second trimester, and 100% at term. Defects include mental retardation,
deafness, cardiovascular malformations, cataracts, glaucoma, microphthalmia. Diabetes mellitus or
rubella panencephalitis may develop later in life. The abortigenic risk of maternal rubella is
uncertain.
Quality of available information:
excellent.
Rubella has an afnity for specic
tissues. Damage is caused by mitotic
inhibition, cell death and interference
with histogenesis by repair processes,
resulting in calcication and scarring.
Syphilis Defects in 50% of offspring after early exposure to primary or secondary syphilis and 10% after late
exposures. Defects include maculopapular rash, hepatosplenomegaly, deformed nails,
osteochondritis at joints of extremities, congenital neurosyphilis, abnormal epiphyses,
chorioretinitis. Syphilis can increase the incidence of abortion.
Fetal pathology is associated with
maturation of the fetal immune system
at about the 20th week.
Toxoplasmosis Hydrocephaly, microphthalmia, chorioretinitis. Risk is predominantly associated with pregnancies
in which the mother acquires toxoplasmosis. Epidemiological studies do not indicate that
toxoplasmosis increases the incidence of early abortion, but congenital toxoplasmosis may be
responsible for the stillbirth of severely affected fetuses.
to excellent.
Toxoplasmosis is unlikely to contribute
to the risk of repeated abortion.
Varicella-zoster Skin and muscle defects; intrauterine growth retardation; limb reduction defects. No measurable
increased risk of early teratogenic effects. Incidence of maternal varicella during pregnancy is low
but risk of severe neonatal infection is high if maternal infection occurs in last week of pregnancy.
There does not appear to be an increased risk of rst trimester abortion.
good.
Virus infection of fetal tissues can cause
cellular necrosis.
Venezuelan equine
encephalitis
Hydroencephaly; microphthalmia; central nervous system destructive lesions; luxation of hip.
There are not enough data to determine whether infection presents an increased risk of abortion.
Quality of available information: poor
to fair.
Infection can cause cellular necrosis in
fetal tissues but fetal infection is rare.
(continued on next page)
Table 1 (continued)
Developmental
toxicant
a
Lead There is no indication that serum lead levels <50 mg% result in congenital malformations in exposed
embryos and fetuses but the developing central nervous system in the fetus and child may be
susceptible to lead toxicity, resulting in decreased IQ and behavioral effects. Lead levels >50 mg%
result in anemia, and encephalopathy can have serious effects on central nervous system
development. Lead levels <50 mg% do not increase the risk of abortion.
Whereas there are human studies
indicating small deciencies in IQ in
patients with lead levels >10 mg%, there
could be other explanations for these IQ
differences. Furthermore, pathological
ndings have not been described in the
brain at these levels.
Lithium carbonate Although animal studies have demonstrated a clear teratogenic risk, the effect in humans is
uncertain. Early reports indicated an increased incidence of Ebsteins anomaly, other heart and great
vessel defects, but as more studies are reported the strength of this association has diminished.
Lithium levels within the therapeutic range (<1.2 mg%) do not increase the risk of abortion.
good.
Antidepressant; mechanism has not
been dened.
Maternal conditions
Diabetes Caudal hypoplasia or caudal regression syndrome; congenital heart disease; anencephaly. Vascular
lesions in long-standing diabetics may produce placental dysfunction and result in fetal growth
retardation. Documented signicant increased risk of abortion. The risk is greatest in untreated
diabetics or in patients who are poorly controlled; insulin therapy protects the fetus.
Results of in-vitro studies suggest that
diabetic embryopathy has a
multifactorial etiology. Adverse fetal
effects have not been demonstrated
with gestational diabetes.
Endocrinopathy If condition is compatible with pregnancy, effects are similar to those following administration of
high or low doses of the hormone. Hypo- and hyperthyroidism may increase the risk of abortion.
Cushings disease, pituitary tumors, hypothalamic tumors and androgen-producing tumors do not
appear to increase the risk of abortion, but may contribute to infertility.
Receptor-mediated exposures to high
levels of hormone.
Nutritional deprivation Central nervous system anomalies; intrauterine growth restriction; increased morbidity. In some
instances of teratogenesis, abnormal nutrition may be the nal common mechanism. Severe
malnutrition can contribute to pregnancy loss at any stage of pregnancy.
good.
The high mitotic rate of the fetus in
general and the central nervous system
in particular is very sensitive to severe
alterations in nutrient supply.
Phenylketonuria Mental retardation; microcephaly; intrauterine growth retardation. Documented signicant
increased risk of abortion. The risk is greatest in pregnant women who were not treated for their
phenylketonuria.
Very high levels of phenylalanine
interfere with embryonic cell
metabolism.
Mechanical problems Birth defects such as club feet, limb reduction defects, aplasia cutis, cranial asymmetry, external ear
malformations, midline closure defects, cleft palate and muscle aplasia. Submucosal and intramural
myomata, amniotic bands, multiple implantations, bid uterus or infantile uterus which contribute
to mechanical problems do not result in early abortions.
Physical constraint can result in
distortion and a reduction in blood
supply and is more frequent in
pregnancies with multiple conceptuses,
abnormal uterus, amniotic
abnormalities, certain placental
abnormalities and oligohydramnios.
Methyl mercury Minamata disease: cerebral palsy, microcephaly, mental retardation, blindness, cerebellar
hypoplasia. Does not appear to decrease fertility unless the mother becomes clinically ill from
methyl mercury poisoning. At low exposures the teratogenic effect predominates and there are few
human data to indicate the risk of abortion.
Organic mercurials accumulate in lipid
tissue causing cell death due to
inhibition of cellular enzymes,
especially sulfhydryl enzymes. Since
most cases result from accidental
environmental exposure, risk
estimation is usually retrospective.
Methylene blue Hemolytic anemia and jaundice in neonatal period after exposure late in pregnancy. There may be a
small risk for intestinal atresia but this is not yet clear. No indication of increased risk of abortion.
to fair.
Used to mark amniotic cavity during
amniocentesis.
Misoprostol Misoprostol is a synthetic prostaglandin analog that has been used by millions of women for illegal
abortion. A low incidence of vascular disruptive phenomenon, such as limb reduction defects and
Moebius syndrome, has been reported.
Classical animal teratology studies
would not be helpful in discovering
these effects, because vascular
disruptive effects occur after the period
of early organogenesis.
Mycophenolate mofetil This drug is a highly efcient immunosuppressant used to prevent organ rejection after organ
transplantation. The agent, which blocks purine synthesis in lymphocytes, raised denite concern
when congenital defects were reported to the National Transplantation Pregnancy Registry: three
infants all exposed through the rst trimster exhibited congenital malformations, two with cleft lip
with cleft palate, and all three with microtia; one of the infants had a diaphragmatic defect, a
malformation seen in rats and rabbits treated with mycophenolate mofetil.
Quality of the data: fair.
Investigators raise concerns regarding
teratogenicity.
Oxazolidine-2,4-diones
(trimethadione,
paramethadione)
Fetal trimethadione syndrome: V-shaped eye brows, low-set ears with anteriorly folded helix, high-
arched palate, irregular teeth, CNS anomalies, severe developmental delay. Wide variation in
reported risk. Characteristic facial features are documented only with chronic exposure. The
abortifacient potential has not been adequately studied, but appears to be minimal.
to excellent.
Anticonvulsants; affects cell membrane
permeability. Actual mechanism of
action has not been determined.
D-Penicillamine Cutis laxa, hyperexibility of joints. Condition appears to be reversible and the risk is low. There are
no human data on the risk of abortion.
good.
Copper chelating agent; produces
Table 1 (continued)
Developmental
toxicant
a
copper deciency inhibiting collagen
synthesis and maturation.
Polychlorinated
biphenyls
Cola-colored babies: pigmentation of gums, nails and groin; hypoplastic deformed nails;
intrauterine growth retardation; abnormal skull calcication. Although abortion can be induced at
high exposures, most human exposures from environmental contamination are unlikely to increase
the risk of abortion.
Environmental contaminants;
polychlorinated biphenyls and
frequently occurring contaminants are
cytotoxic. Body residues in exposed
women can affect pigmentation in
offspring for up to 4 years after
exposure.
Progestins Masculinization of female embryo exposed to high doses of some testosterone-derived progestins
and may interact with progesterone receptors in the liver and brain later in gestation. The dose of
progestins present in modern oral contraceptives presents no masculinization or feminization risks.
All progestins present no risk for non-genital malformations. Many synthetic progestins and natural
progesterone have been used to treat luteal phase deciency, embryos implanted via in-vitro
fertilization, threatened abortion or bleeding in pregnancy with variable results. Conversely,
synthetic progestins that interfere with progesterone function may cause early pregnancy loss; RU-
486 (mifepristone) is presently used specically for this purpose.
Stimulates or interferes with sex steroid
receptor-containing tissue.
Radioactive isotopes Tissue- and organ-specic damage is dependent on the radioisotope element and distribution, i.e.
131
I administered to a pregnant woman can cause fetal thyroid hypoplasia after the 8th week of
development. Radioisotopes used for diagnosis present no risk for inducing abortion because the
dose to the embryo and implantation site is too low. There may be unusual circumstances wherein
isotopes are introduced into the abdominal cavity in a pregnant woman for the treatment of
malignancy. If the resulting dose to the embryo or fetus is substantial, the risk for abortion is
increased.
to excellent.
Higher doses of radioisotopes can
produce cell death and mitotic delay.
Effect is dependent on dose,
distribution, metabolism, and
specicity of localization.
Radiation
(external irradiation)
Microcephaly; mental retardation; eye anomalies; intrauterine growth retardation; visceral
malformations. Teratogenic risk depends on dose and stage of exposure. Exposures from diagnostic
procedures present no increased risk of abortion, growth retardation or malformation. No
measurable risk with exposures for 5 rad (5 mGy) of X-rays at any stage of pregnancy. In contrast,
exposure of the pregnant uterus to therapeutic doses of ionizing radiation signicantly increases the
risk of aborting the embryo; the fetus is more resistant.
to excellent.
Diagnostic and therapeutic agents;
produce cell death and mitotic delay.
Retinoids, systemic
(isotretinoin,
etrentinate)
Increased risk of central nervous system, cardio-aortic, ear and clefting defects. Microtia, anotia,
thymic aplasia and other branchial arch, aortic arch abnormalities and certain congenital heart
malformations. Exposed embryos are at greater risk for abortion. This is plausible since many of the
malformations, such as neural tube defects, are associated with an increased risk of abortion.
Used in treatment of chronic
dermatoses. Retinoids can cause direct
cytotoxicity and alter programmed cell
death; affect many cell types but neural
crest cells are particularly sensitive.
Retinoids, topical
(tretinoin)
Epidemiological studies, animal studies and absorption studies in humans do not suggest a
teratogenic risk. Regardless of the risks associated with systemically administered retinoids, topical
retinoids present little or no risk for intrauterine growth retardation, teratogenesis or abortion
because they are minimally absorbed and only a small percentage of skin is exposed.
Quality of available information: poor.
Topical administration of tretinoin in
animals in therapeutic doses is not
teratogenic, although massive
exposures can produce maternal
toxicity and reproductive effects. More
importantly, topical administration in
humans results in non-measurable
blood levels.
Smoking and nicotine Placental lesions; intrauterine growth retardation; increased postnatal morbidity and mortality.
While there have been some studies reporting increases in anatomical malformations, most studies
do not report an association. There is no syndrome associated with maternal smoking. Maternal or
placental complications can result in fetal death. Exposures to nicotine and tobacco smoke are a
signicant risk for pregnancy loss in the rst and second trimester.
to excellent.
While tobacco smoke contains many
components, nicotine can result in
vascular spasm vasculitis which has
resulted in a higher incidence of
placental pathology.
Sonography
(ultrasound)
No conrmed detrimental effects resulting from medical sonography. The levels and types of
medical sonography that have been used in the past have no measurable risks. The present clinical
use of diagnostic ultrasound presents no increased risk of abortion.
to excellent.
It appears that if the embryonic
temperature never exceeds 39

C, there
is no measurable risk.
Streptomycin Streptomycin and a group of ototoxic drugs can affect the eighth nerve and interfere with hearing; it
is a relatively low risk phenomenon. There are not enough data to estimate the abortigenic potential
of streptomycin. Because the deleterious effect of streptomycin is limited to the eighth nerve, it is
unlikely to affect the incidence of abortion.
good.
Long-duration maternal therapy during
pregnancy is associated with hearing
deciency in offspring.
Tetracycline Bone staining and tooth staining can occur with therapeutic doses. Persistent high doses can cause
hypoplastic tooth enamel. No other congenital malformations are at increased risk. The usual
therapeutic doses present no increased risk of abortion to the embryo or fetus.
Antibiotic; effects seen only if exposure
is late in the rst or during second or
third trimester, since tetracyclines have
to interact with calcied tissue.
Thalidomide Limb reduction defects (preaxial preferential effects, phocomelia), facial hemangioma, esophageal or
duodenal atresia, anomalies of external ears, eyes, kidneys, and heart, increased incidence of
neonatal and infant mortality. The thalidomide syndrome, although characteristic and recognizable,
can be mimicked by some genetic diseases. Although there are fewer data pertaining to its
abortigenic potential, there appears to be an increased risk of abortion.
to excellent.
Sedativeehypnotic agent. The etiology
of thalidomide teratogenesis has not
been denitively determined.
themon the options available, but not on which option to select. On
the contrary, in one survey, up to 6% of all physicians would
recommend medical termination of pregnancy for women who had
undergone a single CT at 6 weeks of gestation and 27% of all phy-
sicians surveyed were uncertain if they would recommend a
medical termination of pregnancy (Box 2) [29]. In case of a toxi-
cological exposure, the counselor should attempt to establish a
good understanding of the exposure in question and its timing. A
list of information required to provide high quality counseling to
those exposed is listed in Boxes 1 and 3. The pervasive problem is
that many physicians and counselors tell the patient or family what
to do. In many instances the counselor (i) has no expertise con-
cerning the risks of an exposure, or (ii) the counselor knows the
risks, but he/she does not take the time to educate the patient about
the risks.
6. Counseling: What are the reproductive or developmental
concerns of pregnant women and/or family members
concerning the risk of alleged or suspected toxicological
exposures during pregnancy or before pregnancy? Role of the
counselor
These concerns include (i) birth defects (congenital malforma-
tions), (ii) pregnancy loss (miscarriage or spontaneous abortion),
(iii) growth retardation, (iv) prematurity, (v) neurobehavioral ef-
fects (decreased IQ, small head size, convulsive disorders, autism,
attention decit hyperactivity disorder) and cancer. All of these
effects except for cancer and mutations in the F
1
generation are
deterministic effects (threshold effects, tissue reaction effects)
(Table 5). The term tissue reaction effects is a new term adopted
because it conveys the concept that these effects involve exposure
to tissues indicating that many cells have to be affected, which
explains why there is a threshold belowwhich there is no increased
risk (Table 5).
Mutagens have the potential to induce cancer or genetic effects
by altering one cell, and therefore theoretically do not have a
threshold [20e44]. Recent discussions of the universal application
of the linear-no-threshold hypothesis (LNTH) indicate that there is
disagreement as to whether the LNTH is applicable at very low
exposures to environmental toxicants and with protracted toxicant
exposures (Table 5) [35e37].
7. The role of the counselor
The classical approach for the evaluation of risks from in-utero
environmental exposures is to review the available epidemiolog-
ical studies and appropriate animal models, and to evaluate these
data fromthe standpoint of biologic plausibility or biologic common
sense (Box 4) [26]. In order to performan analysis outlined in Box 4,
the drug, chemical or physical agent has to have been distributed or
sold for a period of years so that epidemiological data, animal toxi-
cology data and in-vitro studies are available for analysis.
7.1. Evaluating the allegation of teratogenicity
When confronted with the question of the teratogenicity of
certain environmental agents, the question can be posed from
various vantage points:
1. Evaluating the risk of reproductive toxicity of an environmental
agent.
2. Evaluating the suggestion that an environmental agent was
responsible for an individual childs birth defect or other
reproductive effects.
3. Evaluating the cause of congenital malformations in a particular
child or a group of children.
4. Determining whether to publish a case report of a patient or a
cluster of patients with a particular congenital malformation or
a constellation of congenital malformations that may be asso-
ciated with an environmental agent [38].
If one wishes to answer the generic question, Is a particular
environmental drug, chemical or physical agent a reproductive
toxicant?, then a formal approach is recommended that includes a
Table 1 (continued)
Developmental
toxicant
a
Thyroid: iodides,
radioiodine,
antithyroid drugs
(propylthiouracil),
iodine deciency
Fetal hypothyroidism or goiter with variable neurologic and aural damage. Maternal
hypothyroidism is associated with an increase in infertility and abortion. Maternal intake of 12 mg
of iodide per day increases the risk of fetal goiter.
Fetopathic effect of endemic iodine
deciency occurs early in development.
Fetopathic effect of iodides, antithyroid
drugs and radio iodine involves
metabolic block, decreased thyroid
hormone synthesis and gland
development.
Toluene Intrauterine growth retardation; craniofacial anomalies; microcephaly. It is likely that high
exposures from abuse or intoxication increase the risk of teratogenesis and abortion. Occupational
exposures should present no increase in the teratogenic or abortigenic risk. The magnitude of the
increased risk for teratogenesis and abortion in abusers is not known because the exposure in
abusers is too variable.
to fair.
Neurotoxicity is produced in adults who
abuse toluene; a similar effect may
occur in the fetus.
Valproic acid Malformations are primarily neural tube defects and facial dysmorphology. The facial characteristics
associated with this drug are not diagnostic. Small head size and developmental delay have been
reported with high doses. The risk for spina bida is about 1% but the risk for facial dysmorphology
may be greater. Because therapeutic exposures increase the incidence of neural tube defects, one
would expect a slight increase in the incidence of abortion.
Anticonvulsant; little is known about
the teratogenic action of valproic acid.
Vitamin A The same malformations that have been reported with the retinoids have been reported with very
high doses of vitamin A (retinol). Exposures <10 000 IU present no risk to the fetus. Vitamin A in its
recommended dose presents no increased risk for abortion.
High concentrations of retinoic acid are
cytotoxic; it may interact with DNA to
delay differentiation and/or inhibit
protein synthesis.
Vitamin D Large doses given in vitamin D prophylaxis are possibly involved in the etiology of supravalvular
aortic stenosis, eln faces, and mental retardation. There are no data on the abortigenic effect of
vitamin D.
Quality of available information: poor.
Mechanism is likely to involve a
disruption of cell calcium regulation
with excessive doses.
a
Quality of available information is modied from TERIS [22].
ve-part evaluation as described in Box 4 [26]. This formal
approach is utilized only when a drug or chemical has been utilized
and sold for a period of many years and, during that time, studies on
epidemiology, animals and pharmacokinetics have been performed
[30e37].
If, on the other hand, one is concerned about the reproductive
effects of an environmental agent in an individual patient, the
question may sometimes be answered without the benet of
epidemiological studies, dosimetry or animal studies, utilizing the
basic principles of teratology, reproductive toxicology and genetics.
Key factors in such an evaluation are having an experienced clini-
cian who is knowledgeable about the genocopies that can mimic
environmental teratogens, who is aware of the importance of dose
or exposure and who is aware of the basic principles of develop-
mental biology and teratology. A clinician trained and experienced
in these elds may have a decisive role in utilizing the principles of
teratology (Box 1) and biological plausibility in evaluating the
allegation that a particular environmental agent was responsible
for a childs congenital malformations [10,28,38e40,42,44,45,52].
7.2. Counselors must have knowledge of known or possible
reproductive toxins
Reproductive and developmental counselors should have been
educated and prepared to discuss background risks of birth defects
and the broad subject of the etiology of developmental birth defects
Box 2
Responsibilities of the counselor.
1. Questions submitted by patients or contacts regarding
environmental toxicant exposures should never be
described as silly, dumb or unnecessary. Every response
should attempt to dignify the question as appropriate.
However, the counselor should provide scientic expla-
nations as to why the contacts concerns are or are not
substantiated by the available facts. The counselor is an
educator.
2. It is difcult for many counselors to comprehend the
anguish, heartache, fear and concern in the hearts and
minds of the contacts when they are concerned about the
health of their fetus from exposures to environmental
toxicants. The degree of fear is related to the mental state
of the contact as well as the type and magnitude of the
exposure to the environmental toxicant.
3. Many counselors do not understand that their profes-
sional goal should be to educate the contact about the
reproductive or developmental risks that may result from
a particular environmental exposure, not to advise them
what decision they should select.
4. Many novice counselors do not realize that it is their re-
sponsibility to provide the contacts with the background
risk that they face, even when there are no increased risks
from the exposure. The contacts are requested to keep
the counselor informed. Do they have any more ques-
tions? Please keep in touch, send a picture. The consul-
tation is signed with Warm regards. It should be made
clear to the contact that the counselor has functioned as
an educator. The counselor does not advise contacts on
what decision to select, only the options that are avail-
able. Yet, many contacts thank the counselor for telling
them what to do, even though they have not been
advised on which available option to select.
5. It is important to permanently save a written record of the
statements of the contact and the counselor.
6. Each consultation that denitively determined that their
reproductive or developmental risks are not increased
ends with this statement:
Your risk for birth defects or miscarriage is not
increased above the background risks that all healthy
pregnant women face. The background risks for preg-
nant women with no personal or family history of
reproductive or developmental problems is 3% for
birth defects and 15% for miscarriage. All pregnant
women face these risks, many of which we cannot yet
prevent.
We wish them good luck with their pregnancy and to
keep in touch. If the contact asks about the risks of
mental retardation, cancer or other effects, these
background risks are discussed also. The answers are
directed specically to their questions.
If the contact is concerned about cancer in her
offspring from either preconception or postconception
exposures, it is most important to educate the contact
regarding the high background incidence of cancer in
the population. Potentially lethal cancers occur in 23%
of the population, which dwarfs the incidence of envi-
ronmentally induced cancer. It is important to point out
that the spontaneous occurrence of cancer, which in-
creases with age due to mutation that occurs in the
dividing somatic cells, is the most important etiology
of human cancer.
Box 3
Minimum information required to provide adequate counseling
to women or couples regarding concerns about the risks of
environmental toxicant exposures before or during the
pregnancy.
a
1. Is the contact pregnant, possibly pregnant or planning to
become pregnant?
2. If the contact is pregnant, does she know the date she
became pregnant? Does she know the date of the rst
day of her last menstrual period?
3. Does she know the date of conception from other sour-
ces: an ultrasound that timed the pregnancy or a date
when intercourse took place that is consistent with other
information about timing?
4. Are there historical pregnancy risks (birth defects,
miscarriage, etc.) for the mother or the family e for
example, a history of miscarriages, birth defects, infer-
tility or serious illnesses in the contact, spouse, or par-
ents or siblings?
5. Does the contact know the name of the environmental
toxicant to which she or her spouse was exposed? Is it a
drug, chemical or physical agent (such as radiation or
heat)? What was the amount and length of the exposure
before or during pregnancy? Provide the dates and
amount (dose) of the exposure. The Environmental Pro-
tection Agency (EPA) has prepared toxicological evalu-
ations of chemicals using animal studies and now high-
throughput in-vitro studies. The EPA may even have
estimated a reference dose exposure, which is an expo-
sure that has no deterministic reproductive effects.
6. Has the woman or couple sought advice from another
counselor about the developmental risks of this expo-
sure? Please provide details of the response.
7. The needs of the person seeking counseling are the only
operative issues to be considered.
a
Adapted from Brent [23].
and reproductive problems (Tables 2 and 3). Counselors must have
knowledge of known or possible reproductive toxins and be
familiar with howthe medial literature dealing with teratogens can
be accessed [22,46e55].
Table 1 provides a list of known or alleged reproductive toxin or
teratogenic agents. The TERIS andOTIS websites are more up-to-date,
as is the genetic website of the National Library of Medicine (OMIM).
Each counselor may have many consultations in their les;
however, it is not the purpose of this publication to provide
numerous examples. On the Health Physics website, Ask the Expert
(ATE), there are more than 60 prototype questions. NCRP 174,
published in 2013 [52], has an appendix with scores of consultation
questions and answers that pertain to the preconception and
postconception risks of ionizing radiation, ultrasound, non-ionizing
electromagnetic elds, CT scans and magnetic resonance imaging.
A few examples of responses to contacts exposed to environ-
mental toxicants are presented below. A preconception and post-
conception toxicant exposure is discussed, as well as a contact who
is concerned about the etiology of the childs birth defects. Another
frequently asked question pertains as to whether a cluster of similar
birth defects in a small geographic area is due to an environmental
toxicant.
(1) Preconception exposure: The father or the mother in the
family may have been exposed to a toxicological exposure before
the pregnancy has occurred. One of the future parents could have
been treated with radiation therapy or chemotherapy for cancer
that they survived; or one of the parents had multiple diagnostic X-
rays as a child or adolescent. The family is concerned about the
genetic effects of prior alleged toxicological mutagenic exposures at
home or at work. Of the thousands of consultations we have
received, about 20% are from future parents concerned about pre-
conception toxicological exposures. Some of the exposures have
occurred many years before they contemplated having a family.
Genetic studies of the offspring of patients exposed to muta-
genic agents are largely negative. However, we know that muta-
tions have been produced, but the risk of viable offspring with an
Table 3
Reproductive risks per million recognized pregnancies.
a
Reproductive risks Frequency
Immunologically and clinically diagnosed spontaneous
abortions per million conceptions (<20% has lethal
malformations or chromosome abnormalities that
cause abortion before the rst month of gestation).
350 000
Clinically recognized spontaneous abortions per million
clinically recognized pregnancies. Spontaneous
abortion after the rst missed menstrual period.
150 000
Genetic diseases per million births 110 000
Multifactoral or polygenic genetic environmental
interactions)
90 000
Dominantly inherited disease 10 000
Autosomal and sex-linked genetic disease 1200
Cytogenetic (chromosomal abnormalities) 5000
New mutations in the developing ova or sperm prior
to conception
3000
Major malformations (genetic, unknown, environmental) 30 000
Prematurity (Ireland: 55 000; USA: 124 000) 69 000
Fetal growth restriction 30 000
Stillbirths (>20 weeks; wide range in prevalence because
of variables contributed by cultures, socio-economic
factors, race, prenatal medical care)
4000e20 900
Infertility 7% of couples
a
Source: Brent [9].
Table 2
Etiology of human congenital malformations.
a
Suspected cause Percent of total
Unknown 65
Polygenic
Multifactorial (geneeenvironment interactions)
Spontaneous errors of development
Synergistic interactions of teratogens
Genetic 15e25
Autosomal and sex-linked inherited genetic disease
Cytogenetic (chromosomal abnormalities)
New mutations
Environmental (total) 10
Maternal conditions: alcoholism; diabetes;
endocrinopathies; phenylketonuria; smoking and
nicotine; starvation; nutritional decits
4
Infectious agents: rubella, toxoplasmosis, syphilis,
herpes simplex, cytomegalovirus, varicella-zoster,
Venezuelan equine encephalitis, parvovirus B19
3
Mechanical problems (deformations): amniotic band
constrictions; umbilical cord constraint; disparity in
uterine size and uterine contents
1e2
Chemicals, prescription drugs, high-dose ionizing
radiation, hyperthermia
2
a
Source: Brent [9].
Box 4
Evaluation of potential for developmental toxicity in the human:
evidence of a possible causal relationship.
a
Epidemiological studies
Controlled epidemiological studies consistently demon-
strate an increased incidence of a particular spectrum of
embryonic and/or fetal effects in exposed human
populations.
Secular trend data
Secular trends demonstrate a positive relationship between
the changing exposures to a common environmental agent
in human populations and the incidence of a particular
embryonic and/or fetal effect.
Animal developmental toxicity studies
An animal model can be developed which mimics the hu-
man developmental effect at clinically comparable expo-
sures. Since mimicry may not occur in all animal species,
animal models are more likely to be developed once there is
good evidence for the embryotoxic effects reported in the
human. Developmental toxicity studies in animals are
indicative of a potential hazard in general rather than the
potential for a specic adverse effect on the fetus when
there are no human data on which to base the animal
experiments.
Doseeresponse relationship
Developmental toxicity in humans increases with dose
(exposure) and the developmental toxicity in animals oc-
curs at a dose that is pharmacokinetically (quantitatively)
equivalent to the human exposure.
Biological plausibility
The mechanisms of developmental toxicity are understood
and the effects are biologically plausible.
a
Modied from Brent et al. [25], Brent [26,27], and Shepard
[28].
increased incidence of new mutations or cancer is so low that you
would need very large populations of exposed individuals to
demonstrate the increased risk.
Thereis noconvincingdirect evidence of germline mutationseen
as heritable disease in the offspring of humans and attributable to
ionizing radiation and mutagenic chemicals and drugs, yet these
mutagens clearly induce mutations in microbes and somatic cells of
rodents and humans, and in offspring of exposed mice. It would be
unwise to ignore the possibility of human germ-cell mutations,
especially since progress in human genetics may be able to address
these issues in the future. Preconception exposure of the ovaries or
testes to toxicological agents or lowexposure to mutagenic drugs or
chemicals is a very low risk phenomenon, especially for exposures
fromdiagnostic radiological procedures. There is no risk for sterility.
Since the vulnerable irradiated ova will have been ovulated in two
menstrual cycles (2 months) and the irradiated sperm replaced in
twospermatogenesis cycles (4months), it is best that the familywait
that period of time before attempting conception. If pregnancy oc-
curs during these windows of time, the environmental toxicant
mutagenic risks are miniscule (Box 3) compared with spontaneous
risk of germ cell mutations. There are numerous published studies
on the offspring of parents who have been exposed to high dose
radiation, chemotherapy and other toxicological agents that are
proven mutagens; if the exposure is high enough, infertility may
result. However, if fertility is intact, the offspring do not manifest an
increase in cancer or hereditary diseases [31,34,56e77]. One addi-
tional consideration is that many deleterious mutations (sponta-
neous or as a result of preconceptiontoxicityexposure) wouldnot be
expressed as effects in the offspring because they are lethal to the
developing ova or sperm or to the very young developing embryo
because of defective ova or sperm, a result that has beendescribedas
biological ltration [9,42].
Down syndrome has been extensively studied as a possible
consequence of diagnostic X-ray procedures but an association has
not been established [83]. Some medical studies have been plagued
by differential recall bias between mothers of affected and normal
children, as well as publication bias in not reporting negative
ndings [84].
Studies of cancer survivors are particularly important because
they are numerous and, most importantly, because the timing and
dose of their exposure to radiation (and potentially mutagenic
chemicals) is accurately documented. The rates of genetic defects in
offspring of survivors and of sibling controls were not statistically
signicant [34,56,59e69,72e82]. In a population of Danish children
born after their parents treatment with radiation, the risk of
congenital malformation was increased but was not statistically
signicant [risk ratio: 1.2 (95% condence interval: 0.9e1.8;
n 36)] [85] and there was no evidence of a correlation between
birth defects and gonadal dose, reconstructed on individual patient
treatment records (mean doses were 1.3 Gy to ovaries and 0.5 Gy to
testes) [34,79]. These data suggest that the agents and doses to
which these individuals have been exposed do not induce trans-
missible mutations in human spermatogonial stem cells and
resting oocytes at a frequency high enough to be detected over the
background of spontaneous mutations (Table 3).
(2) The spouse is pregnant or considering pregnancy and is
concerned about medications that she has been prescribed or is
taking, chemicals in the workplace, whether she should have an X-
ray suggested by her physician, concern about the power lines near
her home, the microwave oven in her kitchen, or numerous
frightening messages on the Internet. The most common concern is
whether their child may be bornwith birth defects. Miscarriage and
mental retardation are next on their list, or whether their future
child is at increased risk for developing cancer.
If the family is concerned about medications prescribed for the
pregnant spouse or other environmental exposures that have been
extensively studied, the counselor may be able to determine
whether there extensive toxicological data have been published
and are available to the counselor. Table 1 lists about 60 environ-
mental toxicants that have been studied to determine their ability
to affect the developing embryo/fetus or that are alleged to have
affected the developing embryo/fetus. If the concern is about a
chemical exposure, the family may not know the name of the
chemical or the exposure, which makes it difcult or impossible to
provide an adequate risk assessment. Several websites may be of
assistance (CDC, OMIM, TERIS; see Box 5).
After obtaining the toxicological information, the following
process should be initiated as suggested in Boxes 2 and 3.
(3) Since the risk of birth defects is 3% and miscarriage occurs in
15% of pregnancies in women after the rst missed menstrual
period, the family may contact the counselor to explain the etiology
of the developmental or reproductive problem in their child (Box 3,
Table 2).
Determining the etiology of birth defects or miscarriage in an
individual infant or pregnancy can be a difcult task, especially if
the description of the malformation is provided to the counselor by
the parent or the family physician. Second, this task could be very
Table 4
Survey of obstetricians and family physicians from Ontario, Canada, on medical
termination of pregnancy in women who underwent radiography or computed to-
mography (CT) during early pregnancy.
a
Medical termination of pregnancy
recommended
Percentage of respondents
Family physicians
(n 208)
Obstetricians
(n 65)
Radiography CT Radiography CT
Yes 1 6 0 5
Not sure 25 39 6 25
No 74 55 94 70
a
Source: Ratnapalan et al. [29].
Table 5
Stochastic and threshold doseeresponse relationships of diseases produced by environmental agents.
Phenomenon Pathology Site Diseases Risk Denition
Stochastic (LNTH
a
) Damage to a single cell
may result in disease
DNA
(genes and chromosomes)
Cancer, germ cell mutation Some risk exists at all
dosages; at low doses,
risk may be less
than spontaneous risk
The incidence of the
disease increases but
the severity and nature
of the disease remain
the same
Threshold
(deterministic effect)
(tissue reaction effect)
Multicellular injury Multiple, variable etiology,
affecting many cell and
organ processes
Malformation, growth
retardation, death, toxicity,
etc.
No increased risk below
the threshold dose
Both the severity and
incidence of the disease
increases with dose
LNTH, linear no-threshold hypothesis.
a
Modied from Brent [9].
time consuming for an individual counselor. It is probably more
appropriate to refer the family to a good genetic or dysmorphology
clinic.
(4) Determining whether to publish a case report of a patient or
a cluster of patients with a particular congenital malformation or a
constellation of congenital malformations that may be associated
with an exposure to an environmental agent [38].
Clinical evaluation of a child with congenital malformations by
an experienced and well-trained physician who is familiar with the
elds of developmental biology, teratology, epidemiology, and ge-
netics may be a simple or complex task. Too often, the entire
emphasis is placed on epidemiological data that may be meager or
insufcient for a rational conclusion when clinical ndings that are
readily available can provide denitive answers with regard to the
etiology of a childs malformations or the merits of an environ-
mental etiology. Robert W. Miller [10] coined the term the alert
physician because he observed that discoveries of toxic environ-
mental situations or agents were made by observant clinicians. A
review article by Brent [36] indicated that most new teratogens
were discovered by alert counselors, physicians or scientists, not by
epidemiological investigations. Epidemiological investigations
could be initiated in order to conrm the alert scientists pre-
sumptive ndings. Shepard emphasized that, if the teratogenic
syndrome observed in a group of toxicant-exposed patients was
rare and unique, the nding was worth pursuing because the
toxicant may be a new teratogen [28].
The editor of the American Journal of Medical Genetics requested
a teratologist to determine whether a submitted article should be
published since several pregnant women had been exposed to
misoprostol and the offspring had malformations that could be
produced by a vascular disruptive agent. The reviewer recom-
mended that the article should be published in order to stimulate
further research concerning this drugs potential teratogenicity and
to alert health care workers about the fact that misoprostol might
be a new teratogen [38]. A clinician trained and experienced in
these elds can have a decisive role in utilizing the principles of
teratology (Box 4) and biological plausibility in evaluating the
allegation that a particular environmental agent was responsible
for a childs congenital malformations [9,28,38e40,42,44,45]. Two
excellent publications that discuss the utilization of teratology
principles and biologic plausibility have been published by Graham
et al. [45] and Carey et al. [44].
8. Conclusion
During the past century and especially since the end of World
War II, there have been many advances and discoveries in the elds
of teratology, developmental biology, genetics, radiology, obstet-
rics, reproductive toxicology and many other elds, improving the
care and diagnosis of children with birth defects. In the early years
of the twentieth century, medical education improved in the USA
through the efforts of the Carnegie Foundation and Abraham
Flexner. However, there were no educational programs for training
professionals on how to deal with environmental toxicants and
their risks or on dealing with patients or families who were con-
cerned about these risks. It was George Engle who introduced the
biopsychosocial model of health care, which taught that one should
not ignore the patients environmental and social defenses because
these determine how the patient will respond to the care program.
If the patient or contact does not sense that the counselor is
compassionate or empathetic, the care program will falter. Engle
and Rogers legitimized counseling. Rogers put the nal stamp on
this philosophy by indicating that the counselors task was to
convince the contact that the contacts best interests were the
counselors highest priority (unconditional positive regard) [4].
The Teratology Society was chartered in 1961 and was the rst
group of clinicians and scientists who were interested in birth de-
fects. An offspring of the Teratology Society, OTIS, was more inter-
ested in counseling but never developed a program that certied
counselors. Genetics became a board-certied specialty that did
certify counselors. Similar birth defect organizations were formed
in Europe, Japan and Australia.
The review includes a summary of the drugs, chemicals and
physical agents that have been documented to result in congenital
malformations and reproductive effects when pregnant women are
exposed during pregnancy. The principles of teratology were also
summarized and emphasize that: (i) no teratogenic agent can be
described qualitatively as a teratogen, since a teratogenic exposure
must include not only the agent, but also the dose and the time in
pregnancy when the exposure occurs; (ii) even agents that have
been demonstrated to result in malformations cannot produce
every type of malformation; (iii) known teratogens can be pre-
sumptively identied by the spectrum of malformations they pro-
duce; (iv) it is easier to exclude an agent as a cause of birth defects
than to denitively conclude that it was responsible for birth de-
fects; (v) when evaluating the risk of exposures, the dose is a crucial
component in determining the risk; (vi) teratogenic agents followa
toxicological doseeresponse curve e this means that each
teratogen has a threshold dose, below which there is no risk of
teratogenesis, no matter when in pregnancy the exposure
occurred; (vii) the evaluation of a child with congenital malfor-
mations cannot be adequately performed unless it is approached
with the same scholarship and detail as is any other complicated
medical problem; (viii) each physician must recognize the conse-
quences of providing erroneous reproductive risks to pregnant
women exposed to drugs and chemicals during pregnancy or
alleging that a childs malformations are due to an environmental
agent without performing a complete and scholarly evaluation.
When a counselor responds to a parents inquiry (What caused
my childs birth defect?), the physician should respond in the same
scholarly manner that would be utilized inperforming a differential
diagnosis for any clinical problem. Physicians have a protocol for
evaluating complex clinical problems. If a mother of a malformed
infant had some type of exposure during pregnancy, such as a
Box 5
Websites.
CDC e Centers for Disease Control
www.cdc.gov/pregnancy.2009
OMIM e Online Mendelian Inheritance of Man
http://www3./ncbi.nlm.nih.gov/omim
Human Genome Project
www.geomics.energy.gov
TERIS e Teratogen Information System
http://apps.medical.washington.edu/teris/teris1a.aspx
(you need an ID and password, which can be obtained from
your university library or hospital if they have a subscrip-
tion). Shepard TH. Catalogue of teratogenic agents. 8th ed.
Baltimore: Johns Hopkins University Press; 1995 (now on
the TERIS website).
Health Physics Society
http://hps.org/documents/risk_ps010-2.pdf
OTIS e Organization of Teratology Information Specialists
www.mothertobaby.org
diagnostic radiological examination or medication during preg-
nancy, the consulting physician should not support or suggest the
possibility of a causal relationship before performing a complete
evaluation. Likewise, if a pregnant woman who had not yet deliv-
ered had some type of exposure during pregnancy, the consulting
physician should not support or suggest the possibility that the
fetus is at increased risk before performing a complete evaluation.
Only a small percentage of birth defects are due to prescribed drugs,
chemicals and physical agents.
Conict of interest statement
None declared.
Funding sources
None.
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Further reading
[1] Brent RL. Non-genital malformations following exposure to progestational
drugs: the nal chapter of an erroneous allegation. Birth Defects Res A Clin Mol
Teratol 2005;73:906e18.
[2] Shepard TH. Letter to the editor. Teratology 1995;51:228.
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Review
Urgent global opportunities to prevent birth defects
Vijaya Kancherla
a
, Godfrey P. Oakley Jr.
a,
*
, Robert L. Brent
b
a
Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA, USA
b
Alfred I. DuPont Hospital for Children, Jefferson Medical College, Wilmington, DE, USA
Keywords:
Birth defects
Congenital abnormalities
Population surveillance
Prevalence
Prevention
s u m m a r y
Birth defects are an urgent global health priority. They affect millions of births worldwide. But their
prevalence and impact are largely under-ascertained, particularly in middle- and low-income countries.
Fortunately, a large proportion of birth defects can be prevented. This review examines the global
prevalence and primary prevention methods for major preventable birth defects: congenital rubella
syndrome, folic acid-preventable spina bida and anencephaly, fetal alcohol syndrome, Down syndrome,
rhesus hemolytic disease of the fetus and the newborn; and those associated with maternal diabetes, and
maternal exposure to valproic acid or iodine deciency during pregnancy. Challenges to prevention ef-
forts are reviewed. The aim of this review is to bring to the forefront the urgency of birth defects pre-
vention, surveillance, and prenatal screening and counseling; and to help public health practitioners
develop population-based birth defects surveillance and prevention programs, and policy-makers to
develop and implement science-based public health policies.
2013 Published by Elsevier Ltd.
1. Introduction
Birth defects are one of the leading causes of infant mortality in
the world, contributing to more than 3 million deaths among
children aged <5 years [1]. Their impact is immeasurable, having
life-long health and economic implications for the affected indi-
vidual, the family, and society. Middle- and low-income countries
have twice the prevalence and mortality associated with birth de-
fects compared with developed countries [1]. When addressing
mortality among people aged <50 years, birth defects are among
top contributors.
A recent report on the global burden of disease ranked birth
defects as the 17th most common cause of disability-adjusted life-
years (DALYs), amounting to 39 million DALYs. This came as an
improvement since 1990, when birth defects ranked 13th among
the leading causes of DALYs and contributed to a total of 54 million
DALYs [2]. These statistics indicate that birth defects prevention
efforts in the last two decades were benecial, and there is a need
to further intensify their surveillance and prevention. Only then can
we achieve maximum reduction in birth defects-associated DALYs
in a global context. Also, there is an increasing consensus on
shifting focus to non-communicable diseases worldwide, which are
now leading the burden of disease and rising in prevalence.
Lack of population-based surveillance and prenatal care pro-
grams contributes to gaps in our knowledge of birth defects in the
developing world. Even though they appear to be rare, birth defects
are a major public health priority at a population level. As with
polio and smallpox, many birth defects are preventable and can be
completely eliminated by timely primary prevention.
We have reviewed current literature on a group of major birth
defects that are highly preventable. These birth defects include
congenital rubella syndrome, folic acid-preventable spina bida and
anencephaly, fetal alcohol syndrome, Down syndrome, and rhesus
hemolytic disease of the fetus and newborn. We have also reviewed
birth defects associated with maternal diabetes, those with in-utero
exposures tovalproic acid, andmaternal iodine deciency. Our aimis
to identify their prevalence in the populations and challenges to
primary preventionstrategies. We hope that this reviewwill serve as
a resource to address modiable risk factors for preventable birth
defects and provide an update on current research in the eld. Public
health practitioners and epidemiologists can use this review as an
update on population-based birth defects surveillance and preven-
tion programs. Our review may be helpful to policy-makers to
implement science-based policies such as mandatory food fortica-
tion, and universal prenatal screenings and vaccinations.
2. Congenital rubella syndrome
Congenital rubella syndrome is a group of birth defects that result
from maternal infection to rubella virus during pregnancy. Affected
infants suffer with cataracts, hearing loss, congenital heart defects,
* Corresponding author. Address: Department of Epidemiology, Rollins School of
Public Health, Emory University, 1518 Clifton Rd NE, Room 4007, Atlanta, GA 30322,
USA. Tel.: 1 678 613 6918; fax: 1 404 325 6918.
E-mail address: gpoakley@mindspring.com (G.P. Oakley).
1744-165X/$ e see front matter 2013 Published by Elsevier Ltd.
and both physical and mental retardation. Severity of these defects
depends on the time at which the fetus is exposed to the infection in
utero, with the highest risk during the rst trimester. Congenital
rubella syndrome also poses a high risk of transmission from the
affectedinfant duringtherst year after their birth, contributingtothe
spread of infection to those in their household or close contact. First
identied in 1941, congenital rubella syndrome has been associated
with a high infant mortality worldwide [3]. Introduction of rubella
vaccination during the early 1970s and effective implementation of
mass vaccination programs in several World Health Organization
(WHO) member countries in recent years has led to about 80%
reduction in rubella cases, and consequently congenital rubella syn-
drome [4]. However, its prevention has not been complete due to
challenges associated with birth and transmission rates in different
countries [5]. Congenital rubella syndrome has been completely
eliminated in the USA as of December 2011 [6], and an ongoing sur-
veillance monitors the success of this program [7], whereas middle-
and low-income countries in Africa, the Western Pacic, and South
East Asia, where the vaccination rates are below 70%, have signi-
cantly high rates of rubella infections among women of childbearing
age [8]. Because of these gaps in primary prevention, about 120 000
rubella cases still occur eachyear in the world [4]. China, Bangladesh,
Poland, Bosnia and Herzegovina, and Ukraine document the highest
proportion of congenital rubella cases, with an estimated total of
100 000 births affected each year [9]. Some of the challenges to total
prevention of congenital rubella syndrome are variability in vaccina-
tioncoveragewithinandbetweencountries. Countries withhighbirth
rate require more than 80% vaccine coverage among women of
childbearing age, thus requiring additional vaccination campaigns.
Also, population isolation in low-income countries hinders vaccina-
tion programs [5]. The WHO has strongly urged countries that are
lacking rubella immunization programs to accelerate their efforts to
prevent rubella and congenital rubella syndrome [10]. Complement-
ingcurrent immunizationprograms withselectiverubellavaccination
among girls and young women is recommended [3].
3. Folic acid-preventable spina bida and anencephaly
Folic acid-preventable spina bida and anencephaly (FAPSBA)
are common birth defects affecting the central nervous system. In
spite of unequivocal evidence from controlled trials in 1991 con-
rming the effectiveness of folic acid in prevention of spina bida
and anencephaly [11], each year about 246 000 cases of FAPSBA
occur globally [1]. Countries that have implementedmandatoryfolic
acid fortication of food have seen a signicant reduction in the
prevalence of FAPSBA [12e14]. But not all countries implement
mandatory fortication policies that promote adequate folic acid by
womenof reproductive age[15]. Bell andOakley[16] have estimated
the proportion of FAPSBA cases that can be prevented worldwide,
suggesting that 75% of all neural tube defects can be prevented
through folic acid fortication, and when there is a good coverage of
populationwithaccess tothe country-specic forticationprogram.
Accordingly, less than one-quarter of total preventable cases of
FAPSBAwere prevented worldwide. The number of cases of FAPSBA
prevented have increased from 9% in 2006 [16] to 15% in 2012 [17];
however, more needs to be done to prevent remaining cases, which
amount to 200 000 cases eachyear globally. There is an urgent need
for those countries with the majority of these cases, such as India
[18] and China [19,20], to implement mandatory folic acid forti-
cation policies and to promote consumption among women of
reproductive age. Some countries opt to offer folic acid supplements
to adolescent girls and women, instead of fortifying centrally pro-
cessed food. Such a prevention strategy based on folic acid supple-
ment intake is shown to be effective in only 50% of women who
adhere to the program[21]. It has also been shown that supplement
programs for high-risk women with a history of neural tube defect-
affected pregnancy, even if successful, prevent only a small fraction
(2e5%) of FAPSBA [21]. Thus, optimal prevention of FAPSBA is
achievable only through mandatory fortication of centrally pro-
cessed food. There are many challenges for mandatory fortication
programs in low- and middle-income countries: fortication pro-
grams are known to be dynamic and inuenced by industry and
consumptionpatterns, lackof political will toimplement mandatory
fortication, resistance from the milling industry to purchase and
disseminate folic acid premix in the our, isolation and individual
dietary practices of communities that limit intake of centrally pro-
cessed our, and concerns about safety, cost-effectiveness, and
impact on consumer choice. In countries that implement fortica-
tion, compliance and uptake have to be monitored periodically.
Overall, assessment of serum folate concentrations among women
of reproductive age can serve as an easy and cost-effective way to
identifyriskfor FAPSBAandimplement primarypreventionFigure 1.
4. Fetal alcohol syndrome
Fetal alcohol syndrome is a structural and neurodevelopmental
group of disorders in individuals with prenatal exposure to alcohol.
Those affected suffer lifelong disability, with no cure. There is a wide
variation in the prevalence of fetal alcohol syndrome worldwide, and
numbers of cases have steadily increasedinthe last decade [22]. Inthe
USA, the estimated prevalence is 2e7 cases per 1000 live births [23].
Studies fromEurope [24], Africa [25e27], Israel [28], Australia [29,30],
and Russia [31] show a much higher prevalence. The highest preva-
lence todate has beenreportedfromhigh-alcohol-consumingregions
of South Africa (90 per 1000 births) [32]. The WHO has recently
convenedtostudytheburdenof fetal alcohol syndrome indeveloping
countries using in-school screeners. Worldwide, 5e10% of all preg-
nancies are at risk for alcohol-related birth defects [33,34]. Recent
studies inthe USAshowthat almost 50%of reproductive-agedwomen
used alcohol, which included about 8% of pregnant women; while
binge drinking is prevalent in15%of non-pregnant womenand1.4%of
pregnant women [35]. Drinking during pregnancy is also a major
concern in low- and middle-income countries [36e38]. As maternal
alcohol use during pregnancy is frequently under-ascertained during
the prenatal period, this estimate may be much higher in reality [39].
Behavioral modication among reproductive-aged women should be
the central theme in the prevention of fetal alcohol syndrome [40].
Some challenges to prevention of fetal alcohol syndrome include
cultural and societal attitudes concerning alcohol use, women with
high-risk behaviors entering prenatal care late in pregnancy, and lack
of, or inaccessible, preventive and counseling services in several
countries. Addressing barriers such as guilt and embarrassment
among women regarding their alcohol use will also be an important
challenge in assessing the burden [41]. It has been shown that a ma-
jority of health care providers fail to address the effects of alcohol
abuseor todiagnosefetal alcohol syndromedue tolackof trainingand
knowledge [42,43]. Assessment of maternal alcohol use during
pregnancy should become a standard and routine measure to aid in
risk assessment for the fetus and for counseling and treatment of
alcohol use. Finally, an integrated approach should be developed to
provide health, social, and referral services in a culturally adopted
setting to those affected with alcohol-related birth defects.
5. Maternal age and Down syndrome
Down syndrome is the most common chromosomal abnormality
in newborns. Affected individuals have high rates of intellectual
disability and several birth defects. Down syndrome was rst char-
acterized in the mid-1800s, and advanced maternal age was deter-
minedas the most signicant risk factor inthe early 1930s [44]. Non-
V. Kancherla et al. / Seminars in Fetal & Neonatal Medicine 19 (2014) 153e160 154
Figure 1. The status of global prevention of folic acid-preventable spina bida and anencephaly, 2012 [17].
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disjunctionerrors inchromosomes and/or biological agingof oocytes
in mothers of advanced age are suggested as probable causes for
Down syndrome [45]. Despite improved knowledge and screening,
about 5400 births in the USA are affected with Down syndrome each
year [45]. The prevalence of Downsyndrome has increasedinthe last
two decades, with an increasing trend in delaying childbirth globally
[46]. Middle- and low-income countries record a large proportion of
births to women of advanced age, while reporting twice as many
cases of Down syndrome as developed countries [1,47]. On the con-
trary, countries where family planning is well-promoted show early
and better-spaced pregnancies, and have lower births to women of
advanced maternal age. European countries that have national
screening policies for Down syndrome (within the rst trimester)
identify a large number of prenatally diagnosed Down syndrome
cases, as compared to countries without such policies [48]. A large
majority of prenatally diagnosed Down syndrome cases (w90%) are
medically terminated. Socio-economic and technological disparities
hinder the success of prenatal screening programs inbothdeveloped
and developing countries. Both individual and societal changes that
encourage lowering the age of conception among women will serve
as the single most cost-efcient andeffective intervention to prevent
Down syndrome in future births. Implementation of universal
screening and counseling (both pre and post conception) is urgently
required in middle- and low-income countries. Improving organi-
zational andcultural factors to reduce variationinprenatal screening
will better contribute towards Downsyndrome preventioninseveral
countries [48].
6. Rhesus hemolytic disease of the fetus and newborn
Several red-cell antigens have been identied in association with
the hemolytic disease of the fetus and the newborn (HDFN). Among
these, antigen RhD is one of the most common. Incompatibility be-
tween RhD-negative mother and RhD-positive fetus during preg-
nancy will result inproduction of antibodies to the fetal RhDantigens
(also termed as alloimmunization) [49]. Pregnancies complicated by
alloimmunizationhave a highriskof fetal andneonatal morbidityand
mortality in future pregnancies, and in the absence of timely pro-
phylaxis can lead to fetal jaundice, hydrops (requiring intrauterine
transfusions), fetal anemia, and stillbirths in about 2.4 per 1000 live
births [50]. The mortality rate associated with HDFN in a developed
country such as the UK is about 6 per 100 000 live births or 50 deaths
per year [51], and is expected to be much higher among developing
countries. The alloimmunization is completely treatable by early
identication and administering anti-D immune globulin to the
expectant mother. Anti-D immune globulin has been in use since the
1960s and has resulted in marked reduction in the alloimmunization
to RhD antigen in pregnant women [52]. Lacking prophylaxis, it is
estimated that at least one in every six RhD-negative women deliv-
ering an RhD-positive baby would be at risk [53]. To understand the
racial distribution of this burden, w15% of Caucasians, 3e5% of
African-Americans, 8% Asian-Indians, and <1% of Asians have RhD-
negative blood groups [50,54,55]. The prevalence of RhD-negative
blood group in Europeans is relatively high, and varies between 11%
and21%. For example, inwesterncountries, as inEnglandandWales, it
isestimatedthat w17%of thepregnant womenareRhD-negative, and,
among these, 59% are at risk for alloimmunization [56]. Non-invasive
methods have gained prominence and have aided signicantly in the
reduction of the affected cases. However, several limitations exist.
Improper screening for RhD-negative status, limited prenatal care
facilities, and failure of identication and treatment with immune
globulin among high-risk pregnancies has contributed to persistence
of the problem around the world. Developed countries have imple-
mented successful screening programs to identify RhD-negative
women early in pregnancy and administer the immune globulins to
prevent HDFN; however, there is a wide variation in both screening
methods and clinical management policies among different countries
[57]. On the other hand, most developing countries lack screening.
According to the March of Dimes, 100% of cases with HDFN can be
prevented by immune prophylaxis [1]. In sub-Saharan Africa the fre-
quency of RhD-negative women is: Nigeria 4.4%, Guinea 4.1%, Kenya
3.9%, western Uganda 3.6%, and Cameroon 2.4%; yet there is limited
prophylaxis available due to several challenges including poor
research, limited access, affordability of anti-Dimmune globulin, high
prevalence of illegal abortions, and poor documentation of medical
histories on previous pregnancies, all of which are associated with
highrates of HDFN-relatedmorbidityandmortalityintheregion[58e
63]. Inorder to achieve reductioninthe deaths associated withHDFN,
there is an urgent need for prenatal screening for RhD antigens, uni-
versal access of immune prophylaxis to all women who test RhD
negative, better documentation of reproductive histories, and pro-
motion of post-partum prophylaxis to mothers at risk.
7. Maternal diabetes associated birth defects
Maternal insulin-dependent diabetes and adverse glycemic
control in pregnancy has been consistently associated with a high
risk of birth defects in the offspring both in developed and devel-
oping countries [1]. A three-fold increased risk has been noted for
major cardiovascular, renal, and gastrointestinal defects in infants
born to diabetic mothers compared with their counterparts [64e
66]. Overweight and obesity are signicant predisposing factors
that lead to diabetes, and both children and adults are at risk
worldwide [67]. Despite advances in medical management, birth
defects associated with maternal diabetes are a common occur-
rence. In the USA w100 000 babies are born to diabetic mothers.
The prevalence estimates for diabetes-associated birth defects
range between 2 and 6 per 1000 births in high-income countries,
and are expected to be several times greater in low- and middle-
income countries [1]. Pregnant mothers with higher levels of
HbA1c at conception have a greater risk of delivering a baby with
birth defects as compared to pregnant mothers with normal levels.
Better glycemic control before pregnancy is shown to reduce the
risk signicantly [68]. Unfortunately, more than one-third of
women of reproductive age with diabetes are unaware of their
condition [69]; this proportion may be incrementally more in
developing countries [70]. However, prevention of diabetes-
associated birth defects is possible [71]. Selectively monitoring
women with juvenile diabetes can be part of the solution. Also,
screening women for high Gamma-hydroxybutyrate (GHb) con-
centration will help identify high-risk pregnancies, and prevention
efforts can be focused accordingly [72]. Both public- and patient-
oriented approaches in lifestyle modication are needed. Aware-
ness of diabetes and its pregnancy-related risk must be improved
among women of reproductive age worldwide. Population-based
screening for women with undiagnosed diabetes is also an urgent
need. Challenges exist in terms of feasibility and funding for uni-
versal screening. Africa and South East Asia, which face the highest
burden of diabetes, have a dearth of primary health centers that can
provide oral hypoglycemic agents to pregnant diabetic women [73].
Other challenges include failure to adhere to, or respond to, treat-
ment among those who are on diabetes medications [74]. A multi-
pronged approach with increased awareness among health care
professionals, periodic screening, and management, has been
shown to have a positive impact on prevention [75,76]. As a pri-
mordial prevention, governments should devise health policies
that promote both education and healthy lifestyles. Political will,
advocacy by health care professionals, and involvement from the
community are important next steps to boost prevention of
diabetes-associated birth defects [74].
8. Valproic acid
A positive association between valproic acid (an anti-epileptic
drug) taken in early pregnancy and having offspring with spina
bida was rst shown in the year 1982. This association was highly
signicant, with a 20-fold increase in risk of spina bida to babies
born to mothers who were exposed to valproic acid in the rst
trimester compared with those born to mothers who were unex-
posed [77]. Several subsequent studies further conrmed this as-
sociation, along with newndings of valproic acid teratogenicity in
association with other major birth defects, including cardiac, uri-
nary, and oro-facial defects in the fetus [77e84]. Following these
studies, many countries have revised their prescription guidelines
for women with epilepsy and have recommended judicious use of
valproic acid during pregnancy for epilepsy disorders, advising less
teratogenic forms of anti-epileptic drugs to women of childbearing
age [85e88]. However, other uses of valproic acid, such as for pain,
migraine, andbipolar disorder make themhighlylikely tostay inthe
market, and be prescribed to women of childbearing age without an
epilepsy indication [89]. There is a high risk of off-label use of this
drug in developing countries. The US Food and Drug Administration
has made valproic acid a class X drug for migraines, where the risks
involved in using valproic acid inpregnant women clearly outweigh
potential benets. Within the USA, it was estimated that w40 cases
of spina bida have occurred due to valproic acid use [90], and about
4 per 1000 prescriptions are still written for valproic acid for non-
epilepsy conditions [89]. The March of Dimes Global report on birth
defects (2006) indicated that the risk of such exposure is evenhigher
in developing countries where valproic acid use may be uncon-
trolled and more common compared with more expensive and less
teratogenic anticonvulsive drugs [1,91]. Thus, it becomes an urgent
necessity to educate not only neurologists, but psychiatrists and
other practitioners to eliminate valproic acid prescriptions among
women of childbearing age, and that its use is seldom justied, for
bothepilepsyandnon-epilepsy indications. Additionally, awareness
needs tobe brought bothamongthe medical communityandamong
the population about the teratogenic effects of this drug, so women
in developing countries are not exposed to valproic acid.
9. Iodine deciency
Iodine deciency affects w2 billion people globally and is a large
concern for women of childbearing age. Each year, about 50 million
children are born to iodine-decient mothers, and about 40% of these
children suffer signicant intellectual disability [92]. In 2007, the
WHO reported a high prevalence of iodine deciency in both devel-
oped and developing regions of the world, and not just conned to
remoteandmountainous regions, aspreviouslythought [93]. National
nutritional surveys from the USA estimate that w35% of pregnant
women have at least mild iodine deciency [94]. To address this
problem, the American Thyroid Association recommends 150 mg per
day of iodine intake for pregnant and lactating women, starting early
at preconception [95,96]. Pregnancy confers an increased need for
iodine, [97] and its insufciency has been associated with cretinism,
intellectual delays, birth defects, and other suboptimal outcomes in
theinfant [95,98]. Iodinedeciencyisalsothemost important causeof
preventable intellectual disability in infants worldwide [96]. Dietary
intake is the only source of iodine; however, prenatal vitamins and
supplements also contain iodine and should be recommended to
womenof childbearing age. Use of iodizedsalt has beenimplemented
in several countries to reduce iodine deciency. But only 70% of
households worldwide have access to iodized salt, and there is a need
to promote it extensively to achieve optimal intake [99]. Known
challenges tosuccessful iodizationincludeuse of cheaper non-iodized
salt, limitation in quality control during salt production, compliance
failures, personal preferences, lack of awareness by households about
benets of iodized salt, market uctuations, and poor enforcement of
regulations mandating iodization [92]. Political commitment and salt
iodization legislation are also important to move ahead. There is a
need to identify optimal indicators for iodine deciency, improved
screening of women of childbearing age, and monitoring iodine con-
tent insalt. As most people are limitingtheir salt intake toreduce their
risk of hypertension, other vehicles have to be explored to deliver
needed iodine to populations worldwide [92]. Taking these timely
steps, we may be able to prevent a majority of babies from the ill
outcomes associated with iodine deciency.
10. Other opportunities for prevention
A recent review summarized a list of preventable birth defects
(Box 1) [100]. This prevention can be achieved by screening for
chromosome abnormalities and genetic disease, vaccinations,
screening for teratogenic infectious diseases such as human im-
munodeciency virus and gonorrhea, and limiting or eliminating
the use of all known teratogenic drugs (e.g. anticoagulants, reti-
noids, and thalidomide). We should also be vigilant of new te-
ratogens. Parents should be informed of preconception diagnostic
choices including amniocentesis, chorionic villous sampling,
maternal serum monitoring, and ultrasound monitoring to di-
agnose identiable genetic diseases and serious birth defects.
Box 1
Prevention of congenital malformation and reproductive effect
[100].
1. Rubella vaccination.
2. Folic acid and vitamin B
12
supplementation: 400 mg and
6 mg per day.
3. Supplementation of iodide to decient patients and
populations.
4. Meticulous diabetic control.
5. Competent diagnosis and management of maternal
hypothyroidism.
6. Screening for chromosome abnormalities and genetic
disease.
7. Hepatitis B vaccination for at-risk women.
8. Human immunodeciency virus screening and
treatment.
9. Screening for Neisseria gonorrhoea, Chlamydia tra-
chomatis, group B streptococcus.
10. Vaccination of patients with group B streptococcus.
11. Maternal phenylalanine management for maternal
phenylketonurics.
12. Management or discontinuation of oral use of antico-
agulants, anticonvulsants, retinoids, thalidomide and
all known teratogens.
13. Recognize that new teratogens can be represented in the
next new drug or chemical exposure: angiotensin-
converting enzyme inhibitors, angiotensin II receptor
blockers, mycophenolate, tumor necrosis factor blockers.
14. Amniocentesis, chorionic villus sampling, maternal
serum monitoring, ultrasound monitoring to diagnose
identiable genetic diseases and serious birth defects in
order for the parents to be informed about their options.
15. Maternal smoking and alcohol cessation.
16. Obesity control in itself reduces the risk of birth defects
and decreases the risk of developing diabetes.
17. Immunization against known teratogenic infections.
Botulina toxin vaccine for the pregnant mother to pre-
vent infant botulism.
18. Malaria vaccine to prevent malaria in pregnancy which
increases the risk of miscarriage.
Timely counseling on high-risk behavioral and lifestyle choices
such as maternal smoking, alcohol, caffeine, recreational drugs, and
obesity and overweight is needed in relation to their adverse effects
on pregnancy. While there are many prevention strategies to
reduce the incidence of birth defects worldwide, there is not
enough space allotted in this article for such extensive review.
11. Conclusion
Opportunities to prevent birth defects are abundant, but there
are also many challenges in the process. Pediatricians, researchers,
and public health practitioners in the eld of birth defects should
intensify prevention efforts, and undertake comprehensive and in-
tegrated efforts to accelerate the pace of prevention at a global scale
for preventable birth defects. Help and commitment from govern-
mental and non-governmental organizations, in a complementary
manner, is crucial to developing affordable and feasible prevention
programs. Only then can we achieve our goal of bringing current
rates of major preventable birth defects close to nil.
We have witnessed the global eradication of polio, which is
almost of the same magnitude as the current number of cases of
folic acid-preventable spina bida. Similarly, an integrated
approach from the government and non-governmental agencies
has resulted in global eradication of smallpox. Lessons from suc-
cessful programs can be shared. Birth defects prevention is
achievable by combating socio-economic inequities and disparities
in health care for women of reproductive age. Need-driven and
impact-oriented approaches with improved education, prophy-
laxis, and prenatal care are needed. There exists an urgent need to
translate research ndings to policy.
Nowthat developed countries are on their path towards success,
priority should now be placed on middle- and low-income coun-
tries. Funding for birth defects surveillance is urgently required to
plan and develop a functional primary prevention infrastructure for
future births. Over time, the health and economic returns, both at
micro and macro levels, would be signicant. Political will and
commitment by communities are an absolute necessity to initiate
and sustain birth defects prevention on a global scale.
None declared.
Funding sources
None.
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Practice points
Global programs to prevent birth defects are an urgent
global health priority.
Congenital rubella syndrome and folic acid-preventable
spina bida and anencephaly could be eliminated from all
countries by implementing prevention programs already
shown to eliminate these birth defects in a few countries.
Effective intervention programs must be developed and
implemented to prevent the three-fold increase in major
malformations among women with insulin-dependent
diabetes mellitus, as well as birth defects and develop-
mental disorders from in-utero exposure to alcohol.
Valproic acid should be limited to persons with epilepsy
who have been shown not to have control with at least one
other anticonvulsant, and should never be the rst-line
treatment for epilepsy for women of reproductive age.
Active, effective intervention programs promoting
optimal maternal birth age are needed to reduce the
current Down syndrome epidemic in developed countries
and to prevent the epidemic in countries that have yet to
experience it.
There is a need to further intensify birth defects surveil-
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Political will and commitment by communities are inte-
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Review
Vaccines and pregnancy: Past, present, and future
Sonja A. Rasmussen
a,
*
, Amelia K. Watson
b
, Erin D. Kennedy
c
, Karen R. Broder
d
,
Denise J. Jamieson
e
a
Inuenza Coordination Unit, Ofce of Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
b
University of Georgia, Athens, GA, USA
c
Immunization Services Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
d
Immunization Safety Ofce, Division of Healthcare Quality Promotion, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease
Control and Prevention, Atlanta, GA, USA
e
Division of Reproductive Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention,
Atlanta, GA, USA
Keywords:
Newborns
Pregnant women
Safety
Vaccine
s u m m a r y
Vaccination during pregnancy with certain vaccines can prevent morbidity and mortality in pregnant
women and their infants. However, previous recommendations often focused on the potential risks of
vaccines to the fetus when used during pregnancy. In recent years, additional data have become available
on the absence of increased risks for adverse events associated with vaccines when administered during
pregnancy and on their benets to mothers and infants. Currently two vaccines e (i) inactivated inu-
enza, and (ii) tetanus toxoid, reduced diphtheria toxoid and acellular pertussis (Tdap) e are recom-
mended for use by all pregnant women by the United States Advisory Committee on Immunization
Practices. Here we review the history of vaccination during pregnancy, the current status of recom-
mendations for vaccination during pregnancy in the USA, and the potential for future advances in this
area, including key barriers that must be overcome to accommodate these advances.
Published by Elsevier Ltd.
1. Introduction
Vaccinations have had a substantial impact on decreasing
morbidity and mortality in the USA and the world, and because of
this impact, vaccinations are considered one of the top ten public
health achievements in the twentieth century [1]. Vaccination
during pregnancy has the potential to protect not only the mother,
who may be at increased risk of morbidity and mortality from in-
fectious disease, but also her infant through maternal antibodies
that are transferred across the placenta during the second half of
pregnancy. In addition, mothers vaccinated during pregnancy are
less likely to transmit an infection to their infant after birth.
Because of their immature immune systems, young infants are
often particularly vulnerable to infectious diseases, and their ability
to mount an immune response to vaccinations is often inadequate
[2]. Past discussions regarding the use of vaccines during pregnancy
often focused on potential risks to the infant, rather than on their
benets. In recent years, data have accumulated on the benets of
certain vaccines to infants when given to their mothers during
pregnancy and on the absence of increased risks for adverse events
associated with administration of vaccines during pregnancy. Here
we review the history of the use of vaccines during pregnancy,
what is known about the risks and benets of vaccines during
pregnancy on the mother and her infant, and the increasing
emphasis in recent years on the benets of certain vaccines when
given during pregnancy. We also discuss future advances in the
area of vaccines and pregnancy with the potential to improve
maternal and infant health.
2. Past
The concept that vaccination during pregnancy could protect a
newborn infant is not a newone. As early as 1879, it was recognized
that newborn infants born to mothers who received Jennerian
vaccination (smallpox vaccination) during pregnancy were pro-
tected from vaccinia virus early in life [3]. The protective effect of
maternally transferred antibodies on offspring was shown in 1892
using a mouse model [4]. In humans, it was recognized early in the
twentieth century that many infants were resistant to some com-
mon infectious diseases during the rst few months of life, espe-
cially if the mother was known to have had the disease [5], and this
* Corresponding author. Address: 1600 Clifton Road, MS A-28, CDC, Atlanta, GA
30333, USA. Tel.: 1 404 639 2297.
E-mail address: skr9@cdc.gov (S.A. Rasmussen).
1744-165X/$ e see front matter Published by Elsevier Ltd.
protection was correlated with the presence of maternal antibodies
that were passed transplacentally from mother to the fetus [6].
Vaccination with tetanus toxoid during pregnancy has been
used for many years to protect infants born to women in developing
countries fromneonatal tetanus. Observational studies in the 1960s
provided evidence that neonatal tetanus could be prevented with
tetanus toxoid vaccination during pregnancy, and a double-blind
controlled trial subsequently provided conrmation: the neonatal
tetanus death rate was 7.8 deaths per 100 births among infants
born to mothers in the control arm, compared to no deaths among
infants born to mothers who received two or three injections with
tetanus toxoid more than 6 weeks apart [7]. These results subse-
quently led to the World Health Organizations Maternal and
Neonatal Tetanus Elimination Initiative, a program focused on
tetanus toxoid vaccination of pregnant and childbearing age
women and promotion of hygienic deliveries. This initiative has
resulted in a 93% reduction in neonatal deaths due to tetanus [8].
Much of the previous guidance on vaccinations during preg-
nancy emphasized concerns that vaccine exposure could place the
fetus or infant at risk or could be perceived as doing so; however, as
additional data on benets of vaccines during pregnancy and on the
absence of increased risks for adverse events after maternal
vaccination have accumulated, that emphasis has changed. The
history of recommendations by the Advisory Committee on Im-
munization Practices (ACIP), a group of experts that develops rec-
ommendations on use of vaccines in the USA [9], related to
inuenza vaccination during pregnancy is illustrative. Inactivated
inuenza vaccination of pregnant women and other groups at
increased risk for inuenza-associated complications was rst
recommended by the Surgeon General in 1960 [10], but many
subsequent recommendations were less supportive. In 1990, the
ACIP noted that an increased risk of inuenza-associated compli-
cations in pregnant women had not been documented, except
during pandemics. Thus, only pregnant women with underlying
conditions were recommended to receive inuenza vaccination,
and waiting to vaccinate until after the rst trimester was a
reasonable precaution to minimize any concern over the theoretical
risk of teratogenicity [11]. In 1995, based on case reports and other
studies, the ACIP recommendation was extended to women
without underlying conditions, but only during the third trimester
of pregnancy [12]. In 1997, after a study had shown an increased
risk of inuenza-associated hospitalizations among pregnant
women during interpandemic periods, especially during the sec-
ond and third trimesters [13], ACIP recommended second and third
trimester vaccination [14]. The 1997 recommendations noted that
many experts considered inuenza vaccination to be safe in all
trimesters of pregnancy, but that delaying vaccination until after
the rst trimester would avoid coincidental association of the
vaccine with rst trimester spontaneous abortions [14]. In 2004,
ACIP extended inuenza vaccination to all pregnant women
regardless of pregnancy trimester [15]. This recommendation fol-
lowed the severe 2003e2004 inuenza season during which many
cases of severe inuenza among pregnant women were reported
and missed opportunities for vaccination were identied [16,17].
3. Present
Current ACIP recommendations regarding vaccination during
pregnancy (Table 1) are based on considerations of the benets of
vaccination during pregnancy to mother and infant and the po-
tential risks. Because of concerns about possible harm to the fetus,
pregnant women have been typically excluded from premarketing
clinical trials of medications and vaccines; thus, most data become
available through postmarketing observational studies [43]. In
developing recommendations for pregnant women, ACIP considers
whether the vaccine is inactivated or live: inactivated viral or
bacterial vaccines or toxoids have been considered to be safe during
pregnancy, whereas live vaccines are considered potentially to be of
concern because of the theoretical risk of transmission of the live
vaccine component to the fetus [18].
3.1. Vaccines recommended for all pregnant women by ACIP
ACIP currently recommends two inactivated vaccines for all
pregnant women, inactivated inuenza vaccine and the tetanus
toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap)
vaccine. For both vaccines, evidence suggests that vaccination
during pregnancy provides protection to the infant in early life
because of maternally transferred antibodies [44,45].
Pregnant women are at increased risk for complications asso-
ciated with seasonal and pandemic inuenza because of changes in
their immune, respiratory, cardiovascular and other systems during
pregnancy [2,46]. Infants aged <6 months are also at increased risk
of inuenza-associated complications [2] and are unable to receive
inuenza vaccine because they fail to mount an adequate immune
response; thus, maternal vaccination also protects these vulnerable
infants from inuenza [47]. Finally, infants born to women who
become severely ill with inuenza during pregnancy are at
increased risk of adverse birth outcomes, such as low birth weight,
small for gestational age, and preterm birth [47]. Consistent with
these ndings, recent studies have suggested that infants born to
women who received inactivated inuenza vaccine during preg-
nancy have a lower risk of these adverse birth outcomes, compared
with infants born to unvaccinated women or to women who
received another vaccine [47].
Although inuenza vaccination has been recommended for
pregnant women regardless of trimester since 2004, an increased
focus has recently been placed on the importance of inuenza
vaccination during pregnancy. The nding that pregnant women
were at higher risk for inuenza-associated complications during
the 2009 H1N1 pandemic likely played a role in this change in focus
[48,49]. Shortly before the pandemic, results from a randomized
controlled trial in Bangladesh were published that demonstrated a
63% reduction in inuenza illness in infants in the rst 6 months of
life following maternal inuenza vaccination during pregnancy,
compared with infants born to women who had received pneu-
mococcal polysaccharide vaccine [44].
Observational studies conducted to assess potential risks to the
mother or infant associated with inactivated inuenza vaccination
(including with 2009 H1N1 vaccine) during pregnancy have been
reassuring: no safety concerns have been identied among mothers
or their infants [50e53]. However, data from the rst trimester are
limited; thus, future research should concentrate on these early
pregnancy exposures [51,52].
After the introduction of vaccination against pertussis, the
number of pertussis cases in the USA reported to the US Centers for
Disease Control and Prevention (CDC) dropped substantially to
fewer than 10 000 by 1965. However, gradual increases in the
numbers of cases occurred in the 1980s, and in 2012, 48 277 cases
were reported, the highest number since 1955 [54]. The highest
incidence is reported in infants aged <1 year; among the 20 re-
ported deaths in 2012, 16 were in infants aged <1 year and 15 were
in infants aged <3 months [54].
Following licensure of Tdap vaccine by the US Food and Drug
Administration (FDA) in 2005, ACIP recommended that women
receive Tdap before pregnancy; for women not vaccinated before
pregnancy, a dose of Tdap in the immediate postpartumperiod was
recommended. ACIP also recommended vaccination of persons
who have or who anticipate having close contact with an infant
aged <12 months (a strategy called cocooning) as an approach to
S.A. Rasmussen et al. / Seminars in Fetal & Neonatal Medicine 19 (2014) 161e169 162
Table 1
Summary of current US Advisory Committee on Immunization Practices (ACIP) recommendations for use of vaccines during pregnancy (adapted from the Centers for Disease
Prevention and Control [18]).
Vaccine Indication (routine,
travel, other) [19e21]
Type [22] ACIP recommendation
a
Recommended for all pregnant women
Inactivated inuenza vaccine Routine Inactivated viral Women who are or will be pregnant during inuenza season should
receive inactivated inuenza vaccine [23].
Tetanus toxoid, reduced diphtheria toxoid,
and acellular pertussis (Tdap)
Routine Inactivated bacterial Health care personnel should administer a dose of Tdap during each
pregnancy irrespective of the patients prior history of receiving
Tdap. Optimal timing for Tdap administration is between 27 and 36
weeks of gestation although Tdap may be given at any time during
pregnancy [24].
Allowed during pregnancy by ACIP in some
circumstances
Anthrax e post-event Other Inactivated bacterial In a post-event setting that poses a high risk for exposure to
aerosolized Bacillus anthracis spores, pregnancy is neither a
precaution nor a contraindication to post-exposure prophylaxis.
Pregnant women at risk for inhalation anthrax should receive
anthrax vaccine [25].
Hepatitis A Routine Inactivated viral The safety of hepatitis A vaccination during pregnancy has not been
determined; however, because hepatitis A vaccine is produced from
inactivated hepatitis A virus (HAV), the theoretical risk to the
developing fetus is expected to be low. The risk associated with
vaccination should be weighed against the risk for hepatitis A in
pregnant women who might be at high risk for exposure to HAV
[26].
Hepatitis B Routine Inactivated viral
(recombinant)
Pregnancy is not a contraindication to vaccination. Limited data
suggest that developing fetuses are not at risk for adverse events
when hepatitis B vaccine is administered to pregnant women.
Available vaccines contain non-infectious hepatitis B virus surface
antigen and should cause no risk of infection to the fetus [27].
Japanese encephalitis (JE) Travel Inactivated viral Practitioners should use caution when considering the use of JE
vaccine in pregnant women. Vaccination with JE vaccines usually
should be deferred because of a theoretical risk to the developing
fetus. However, pregnant women who must travel to an area in
which risk for JE is high should be vaccinated if the benets
outweigh the risks of vaccination to the mother and developing
fetus [28].
Meningococcal conjugate (MCV4/MenACWY) Routine Inactivated bacterial Pregnancy should not preclude vaccination with MenACWY if
indicated [29].
Meningococcal polysaccharide (MPSV4) Travel Inactivated bacterial Pregnancy should not preclude vaccination with MPSV4, if indicated
[29].
Inactivated polio vaccine (IPV) Routine Inactivated viral Although no adverse effects of IPV have been documented among
pregnant women or their fetuses, vaccination of pregnant women
should be avoided on theoretical grounds. However, if a pregnant
woman is at increased risk for infection and requires immediate
protection against polio, IPV may be administered in accordance
with the recommended schedules for adults [30,31].
Pneumococcal polysaccharide (PPSV23) Routine Inactivated bacterial The safety of pneumococcal polysaccharide vaccine during the rst
trimester of pregnancy has not been evaluated, although no adverse
consequences have been reported among newborns whose mothers
were inadvertently vaccinated during pregnancy [18,32].
Rabies Travel/other Inactivated viral Because of the potential consequences of inadequately managed
rabies exposure, pregnancy is not considered a contraindication to
postexposure prophylaxis. Certain studies have indicated no
increased incidence of abortion, premature births, or fetal
abnormalities associated with rabies vaccination. If the risk for
exposure to rabies is substantial, pre-exposure prophylaxis also
might be indicated during pregnancy [33].
Smallpox (vaccinia) e post-event setting Other Live viral Pregnant women who have had a denite exposure to smallpox
virus and are, therefore, at high risk for contracting the disease,
should be vaccinated [34].
Tetanus and diphtheria (Td) Routine Inactivated bacterial
toxoids
Wound management: if a Td booster is indicated for a pregnant
woman, health care providers should administer Tdap. Unknown or
incomplete tetanus vaccination: to ensure protection against
maternal and neonatal tetanus, pregnant women who never have
been vaccinated against tetanus should receive three vaccinations
containing tetanus and reduced diphtheria toxoids. The
recommended schedule is 0, 4 weeks, and 6e12 months. Tdap
should replace one dose of Td, preferably between 27 and 36 weeks
of gestation to maximize the maternal antibody response and
passive antibody transfer to the infant [24].
Yellow fever (YF) Travel Live viral Pregnancy is a precaution for YF vaccine administration, compared
with most other live vaccines, which are contraindicated in
pregnancy. If travel is unavoidable, and the risks for YF vaccine
protect infants from pertussis [55]. However, cocooning has been
challenging to implement, and a decision analysis showed that
Tdap vaccination during pregnancy would avert more infant
pertussis cases, hospitalizations, and deaths than postpartum
vaccination or cocooning strategies [56]. Thus, in 2011, after review
of data on the risks and benets, ACIP recommended Tdap vacci-
nation during pregnancy, preferably during the third or late second
trimester (after 20 weeks gestation), for women who had not
previously been vaccinated [45].
Based on further review of data, including information that
pertussis antibody levels wane after a year, ACIP altered their
recommendation in 2012 to recommend Tdap vaccine during each
pregnancy, regardless of previous vaccination [24]. Although ACIP
noted that Tdap vaccine may be administered any time during
pregnancy, third trimester vaccination (at least 2 weeks before
birth to allow time for an adequate maternal immune response)
was recommended to allow the highest levels of maternal
antibodies to be transferred across the placenta to provide infant
protection [24]. Whereas review of limited safety data on Tdap
vaccination during pregnancy has not identied concerns related to
adverse maternal or infant outcomes [57], no data are available on
repeat vaccination, although reassuring data were available for use
of tetanus toxoid-containing vaccines during pregnancy [24].
However, ACIP noted that the benets of Tdap vaccination during
every pregnancy outweighed the potential risks for adverse events
[24]. Enhanced adverse event monitoring for administration of
Tdap vaccination during each pregnancy is ongoing [24].
3.2. Vaccines contraindicated or not recommended for use during
pregnancy by ACIP
In contrast to the two inactivated vaccines that are recom-
mended for all pregnant women, most live vaccines are specically
contraindicated or not recommended for use during pregnancy by
Table 1 (continued)
Vaccine Indication (routine,
travel, other) [19e21]
Type [22] ACIP recommendation
a
exposure are felt to outweigh the vaccination risks, a pregnant
woman should be vaccinated. If the risks for vaccination are felt to
outweigh the risks for YF virus exposure, pregnant women should
be issued a medical waiver to fulll health regulations. Although no
specic data are available, a woman should wait 4 weeks after
receiving YF vaccine before conceiving [35].
No specic recommendation
Haemophilus inuenzae type b (Hib) Routine Inactivated bacterial ACIP has not published pregnancy recommendations for Hib at this
time [20].
Pneumococcal conjugate (PCV13) Routine Inactivated bacterial,
conjugate
ACIP has not published pregnancy recommendations for PCV13 at
this time. Use of PCV13 is limited among women of childbearing age
[18].
Typhoid Travel Two vaccines available: live
bacterial and inactivated
bacterial forms
No data have been reported on the use of any of the three typhoid
vaccines among pregnant women [36].
Not recommended or contraindicated during pregnancy
Anthrax e pre-event Other Inactivated bacterial In a pre-event setting, in which the risk for exposure to aerosolized
Bacillus anthracis spores is presumably low, vaccination of pregnant
women is not recommended and should be deferred until after
pregnancy [25].
Bacillus CalmetteeGurin (BCG) Other Live attenuated Although no harmful effects to the fetus have been associated with
BCG vaccine, its use is not recommended during pregnancy [37].
Human papillomavirus (HPV) Routine Inactivated viral HPV vaccines are not recommended for use in pregnant women. If a
woman is found to be pregnant after initiating the vaccination
series, the remainder of the three-dose series should be delayed
until completion of pregnancy. If a vaccine dose has been
administered during pregnancy, no intervention is needed [38].
Live-attenuated inuenza (LAIV) Routine Live attenuated viral LAIV is not recommended for use during pregnancy [23].
Measlesemumpserubella (MMR) Routine Live attenuated viral MMR vaccine and its component vaccines should not be
administered to women known to be pregnant or attempting to
become pregnant. Because of the theoretical risk to the fetus when
the mother receives a live virus vaccine, women should be
counseled to avoid becoming pregnant for 28 days after receipt of
MMR vaccine [39].
Smallpox (vaccinia) e pre-event setting Other Live viral Because of the limited risk but severe consequences of fetal
infection, smallpox vaccine should not be administered in a pre-
event setting to pregnant women or to women who are trying to
become pregnant. Before vaccination, women of childbearing age
should be asked if they are pregnant or intend to become pregnant
in the next 4 weeks; women who respond positively should not be
vaccinated [40].
Varicella Routine Live viral Because the effects of the varicella virus vaccine on the fetus are
unknown, pregnant women should not be vaccinated. Non-
pregnant women who are vaccinated should avoid becoming
pregnant for 1 month after each injection [41].
Zoster Routine Live viral Zoster vaccine is not recommended for use in pregnant women,
although these women are unlikely to be in the vaccine target age
group. The effects of the live, attenuated varicella zoster virus-based
vaccine on the fetus are unknown. Women should avoid becoming
pregnant for 4 weeks following zoster vaccination [42].
a
Recommendations are verbatim from ACIP references.
ACIP (Table 1). These include Bacillus CalmetteeGurin (BCG)
vaccine (sometimes recommended for children in countries with a
high prevalence of tuberculosis), live attenuated inuenza vaccine
(LAIV), measlesemumpserubella (MMR) vaccine, varicella vaccine,
zoster vaccine, and smallpox vaccine in a pre-event setting. In
addition, because of the theoretical risk of infection with the live
attenuated virus in live vaccines, ACIP recommends that pregnancy
should be avoided for the month following vaccination for several
of these vaccines (i.e. MMR, smallpox, varicella and zoster vaccines)
[39e42]. Prenatal screening for evidence of immunity to rubella
and varicella is recommended, given the risks of adverse fetal ef-
fects associated with rubella and varicella, and increased risk of
varicella-associated complications during pregnancy. Women
without evidence of rubella or varicella immunity should be
vaccinated postpartum before discharge from the health care fa-
cility, with a second dose of varicella vaccine provided 4e8 weeks
later. Although live vaccines are believed to pose a theoretical risk,
the actual observed risks appear to be low, based on limited
observational data of exposures to LAIV, MMR, and varicella vac-
cines [39,50,58,59]. Data from an ongoing pregnancy registry of
women exposed to varicella zoster virus-containing vaccines are
reassuring; no cases of congenital varicella syndrome and no
increased risk for other birth defects have been observed. However,
the small sample size of exposed women in the registry does not
allow exclusion of risk [60,61].
Of the live vaccines, only smallpox vaccine has been clearly
associated with fetal infection when the vaccine was administered
during pregnancy. However, even though millions of pregnant
women received smallpox vaccine during prior large-scale vacci-
nation programs, only w50 cases of fetal vaccinia following
smallpox vaccination during pregnancy were reported [62]. Addi-
tionally, data on smallpox vaccine exposures during pregnancy,
collected in a pregnancy registry after the USA initiated smallpox
vaccination programs among military and certain civilian workers
in 2003, were also reassuring. Among 376 women who inadver-
tently received smallpox vaccine during pregnancy or who became
pregnant within 28 days of vaccination (most were vaccinated near
the time of conception), no cases of fetal vaccinia and no increased
risks for other adverse pregnancy outcomes were observed [59].
Two inactivated vaccines, human papillomavirus (HPV) vaccine
and anthrax vaccine (in a pre-event setting), are not recommended
for use during pregnancy by ACIP, in both cases because of the
absence of immediate benet to a pregnant woman and her infant.
HPV is a sexually transmitted virus that can cause genital warts and
cervical and other cancers. FDA has licensed two types of HPV
vaccines (bivalent and quadrivalent). ACIP recommends routine
vaccination with three doses of the HPV vaccine over a 6-month
period for females aged 11 or 12 years and for females aged 13e
26 years of who did not receive any or all of the series when they
were younger [38]. Even though both HPV vaccines are inactivated
vaccines, they are not recommended during pregnancy because of
the lack of immediate benet to the woman or her infant when
given during pregnancy. However, data regarding inadvertent
pregnancy exposures to HPV vaccine from pre-licensure clinical
trials and from post-licensure pregnancy registries are reassuring
[63]. If pregnancy is recognized after initiating the vaccination se-
ries, the remainder of the series should be delayed until after
pregnancy, but no intervention is needed if HPV vaccine was given
during pregnancy [38]. A pregnancy registry has been established
to collect data on pregnancy exposures to the quadrivalent HPV
vaccine [60].
Although anthrax can rarely occur naturally after exposure to
animals or their tissues infected with Bacillus anthracis, the anthrax
cases that occurred in the USA in 2001 following exposures
distributed through the mail demonstrate anthraxs potential as a
bioterrorism agent. Anthrax vaccine adsorbed (AVA) vaccine is
currently approved for use in the USA for pre-exposure prophylaxis
and contains no live or dead bacteria. AVA is approved as a ve-
dose regimen for pre-exposure prophylaxis, and can also be used
as a component of a post-exposure prophylaxis regimen under an
Emergency Use Authorization [25].
Because of the low risk for anthrax in a pre-event setting, ACIP
does not recommend vaccination during pregnancy. A study of
3465 infants born to women in the military inadvertently exposed
to AVA vaccine during the rst trimester of pregnancy showed that
rst trimester exposure was associated with slightly higher odds of
birth defects, compared with women who were vaccinated later
during pregnancy. However, the nding was statistically signicant
only when alternative referent groups (women who were never
vaccinated or vaccinated after pregnancy) were used. When spe-
cic birth defects were evaluated, only one birth defect, atrial septal
defect (ASD), was associated with AVA exposure; however, the code
used to identify ASD also included patent foramen ovale, an
obligatory opening in the atrial septumin the fetus that may persist
after birth, especially in preterm infants. This nding was no longer
statistically signicant when preterm infants with ASD as the only
reported defect were excluded from the analysis [64]. ACIP review
of these data concluded that AVA is not associated with an
increased risk for birth defects and can be administered during
pregnancy, but that vaccination should be deferred until after
pregnancy unless a woman has an exposure that places her at
immediate risk of disease [25].
3.3. Vaccines allowed during pregnancy by ACIP in some
circumstances
According to ACIP, many vaccines (Table 1) may be used in
certain situations in which the benets to the pregnant woman are
believed to outweigh the potential risks. In these situations, the
risks of maternal infection and of a poor outcome if the woman
becomes infected are believed to be high, and no evidence of harm
from the vaccine is available. Vaccines in this category include
several routinely recommended vaccines [hepatitis A and B,
meningococcal conjugate and meningococcal polysaccharide,
inactivated polio, rabies, pneumococcal polysaccharide (PPSV23),
and tetanus and diphtheria (Td) vaccines] and two vaccines rec-
ommended for travelers (Japanese encephalitis and yellow fever
vaccines). As discussed previously, two vaccines, anthrax and
smallpox, are recommended for use during pregnancy only in the
post-event setting, when risks for maternal illness are signicant
[25,34].
Nearly all vaccines that are allowed by ACIP in some circum-
stances are inactivated; although information regarding safety of
these vaccines is limited, the available data are reassuring [65,66].
Collection of data through pregnancy exposure registries is ongoing
for hepatitis B and meningococcal vaccines [60].
However, one vaccine allowed by ACIP in some circumstances,
yellow fever (YF) vaccine, is a live vaccine. The YF virus is trans-
mitted to humans from the bite of an infected mosquito; YF is
endemic in sub-Saharan Africa and tropical South America. Illness
with YF ranges from mild febrile illness to severe disease with
hepato-renal dysfunction; case fatality among persons with severe
disease is 20e50%. Because of the risk for severe disease, YF vaccine
is recommended for persons aged 9 months who are traveling to
or living in areas with a risk of YF transmission, and is required for
entry into certain countries [35]. The most up-to-date information
regarding vaccines for travelers is located at the CDC website [19].
The limited data available on use of YF vaccine during pregnancy
suggest that YF virus may be transmitted across the placenta, albeit
rarely, based on the presence of IgM antibodies to the YF virus in
the cord blood of one of 81 infants whose mothers had received YF
vaccine during pregnancy [35]. A recent systematic review identi-
ed eight studies (four using active and four using passive sur-
veillance methods) of YF vaccine during pregnancy [67]. Among
1381 women included in the studies using active surveillance, no
increased risk for adverse outcomes was observed. Three of the four
studies using passive surveillance methods were reassuring; the
fourth showed a higher rate of spontaneous abortions in a small
study (n 39) of women vaccinated with YF vaccine during preg-
nancy, but this nding was not statistically signicant [67].
Given that YF vaccine is a live vaccine and that limited data are
available, ACIP considers pregnancy to be a precaution for YF vac-
cine administration; vaccination is recommended during preg-
nancy if travel is unavoidable and the risks for contracting YF
(based on issues such as location of travel, season, and anticipated
activities while traveling) are felt to outweigh the potential risks of
vaccination. Similar to most other live vaccines, non-pregnant
women receiving YF vaccine are recommended to wait 4 weeks
before conceiving [35].
YF vaccine effectiveness among pregnant women may be less
than that among nonpregnant women and appears to vary by
pregnancy trimester, with later pregnancy appearing to be associ-
ated with lower rates of seroconversion. Thus, serologic testing
should be considered for women vaccinated during pregnancy [35].
3.4. No specic recommendations for use during pregnancy by ACIP
For a fewvaccines, ACIP has not made specic recommendations
during pregnancy because of inadequate data or limited use during
pregnancy. These include Haemophilus inuenzae type b (Hib) con-
jugate, pneumococcal conjugate (PCV13), and typhoid vaccines. Hib
vaccine is not routinely recommended for persons aged >5 years.
However, it is recommended under certain circumstances, that is,
among persons aged >5 years of age who are unvaccinated or
partially vaccinated with Hib vaccine and have leukemia, malignant
neoplasms, anatomic or functional asplenia (including sickle cell
disease), human immunodeciency virus infection, or other
immunocompromising conditions [20]. PCV13 is recommended in a
four-dose series for children aged <2 years, and its use among
women of childbearing age is limited; recommendations for
adolescent andadult age groups are limitedto persons whohave not
previously received PCV13 and who have certainmedical conditions
(i.e. sickle cell disease, HIVinfectionor other immunocompromising
condition, cochlear implant, or cerebrospinal uid leak) [20]. Two
types of typhoid vaccines are available in the USA, an oral live-
attenuated vaccine and an injectable inactivated vaccine [68].
These vaccines provide some protection against typhoid fever, a
sometimes severe illness caused by the bacterium Salmonella
enterica serotype Typhi acquired by consumption of contaminated
food or water. The vaccine is recommended for travelers to devel-
oping countries, including parts of Asia, Africa, the Caribbean, and
Central and South America [19]. ACIP has not published typhoid
vaccine recommendations specically for pregnant women; how-
ever, avoiding live attenuated vaccine seems prudent, given theo-
retical concerns associated with vaccination with live vaccines.
4. Future
Future areas of importance in the area of vaccines and preg-
nancy include: (i) improving uptake for vaccines currently recom-
mended for all women during pregnancy; (ii) improving the
availability of information about the safety of vaccines adminis-
tered during pregnancy (including both pre-licensure and post-
licensure data); and (iii) advancing the development and use of
other vaccines that could protect mothers and their infants.
To improve the uptake of vaccines that are currently recom-
mended for all women during pregnancy, future work needs to
identify and address barriers to pregnant women receiving rec-
ommended vaccinations. Despite ACIP recommendations, inacti-
vated inuenza and Tdap vaccination coverage among pregnant
women is considerably lower than desired. In recent years, signif-
icant improvements in inuenza vaccination coverage among
pregnant women have been observed, from 11% during the 2001e
2002 inuenza season to 38e50% during the 2010e2011 inuenza
season, depending on the survey tool used [69]. However, sub-
stantial efforts will be needed to reach the Healthy People 2020
goal of 80% of pregnant women vaccinated with inuenza vaccine
each year [70]. Given that ACIP recommendations regarding Tdap
vaccine during pregnancy are recent, estimates of coverage
following these recommendations are scant, but appear to be quite
low: in an Internet panel survey of 1231 women conducted
following the 2011 ACIP recommendation, only 2.6% of women
reported receiving Tdap vaccine during their recent pregnancy [71].
Offer and recommendation of vaccines by a health care provider
have been shown to be critical to vaccine uptake; for example,
pregnant women whose health care providers recommended and
offered inuenza vaccination are more likely to report being
vaccinated (73.6%) compared with pregnant women who received
only a recommendation but no offer of vaccination (47.9%) and
women who received neither a recommendation nor an offer
(11.1%) [72]. Several strategies have been recommended to improve
vaccine coverage for recommended vaccines during pregnancy.
These include: reminder/recall systems; standing orders; entry of
vaccine administration information into immunization information
systems; review of immunization status at patient visits; ongoing
education of health care providers who administer vaccines;
ensuring that health care providers receive reimbursement for
vaccine administration; assessment of immunization coverage
rates in provider practices; and increasing vaccine demand by
improving public and provider awareness [73,74]. A recent Amer-
ican College of Obstetricians and Gynecologists Committee Opinion
discusses the importance of integrating immunizations into clinical
practice [75]. This document and the organizations website [76]
provide guidance to obstetrician-gynecologists about becoming
routine vaccinators, and have great potential for improving uptake
of recommended vaccines in the future.
Monitoring for adverse events associated with vaccines among
all populations is important to detect rare adverse events that
might not be observed in pre-licensure clinical trials [77]; however,
post-licensure monitoring is especially important for pregnant
women who are often excluded from clinical trials [43]. This
monitoring addresses vaccine safety concerns that are often raised
by pregnant women and their health care providers. Several stra-
tegies have been established for vaccine safety monitoring among
pregnant women in the USA. The Vaccine Adverse Event Reporting
System (VAERS) is a spontaneous reporting surveillance system
administered by the CDC and FDA since 1990. Through VAERS, re-
ports are collected from vaccine manufacturers, health care pro-
viders, and vaccine recipients to identify possible safety concerns;
however, most of the time, VAERS cannot determine whether a
vaccine is the cause of an adverse event. VAERS has been used to
monitor adverse events associated with several vaccines when
used during pregnancy [57,78e80]. Another method used to assess
vaccine safety is the Vaccine Safety Datalink, a collaborative effort
between the CDC and nine health care systems across the USA. This
system can be used to identify a large retrospective cohort of
women vaccinated and not vaccinated with a particular vaccine
during pregnancy; this systemhas been used to assess the effects of
inactivated inuenza vaccine during pregnancy [53,81,82]. Another
system recently established to better understand the effects of
vaccines during pregnancy is the Vaccines and Medications in
Pregnancy Surveillance System (VAMPSS) [83]. VAMPSS uses two
complementary study designs, a prospective cohort and a casee
control study. The prospective cohort arm compares pregnancy
outcomes of women who received a vaccine or medication to
women who were not exposed for a variety of pregnancy outcomes
(e.g. spontaneous abortion, fetal death, preterm birth, small for
gestational age, pre-eclampsia, and presence of a malformation).
The caseecontrol study design focuses on infants with birth defects
identied through hospitals and birth defects surveillance systems:
mothers of babies with and without birth defects are interviewed
regarding their pregnancy exposures and potential confounding
factors. VAMPSS has recently been used to analyse the prenatal
effects of 2009 H1N1 monovalent and trivalent inactivated inu-
enza vaccines [84,85]. The Clinical Immunization Safety Assess-
ment (CISA) project is a collaboration between CDC and seven
medical centers that conducts post-licensure vaccine safety clinical
research and provides consultation to healthcare providers with
vaccine safety questions about patients residing in the USA,
including pregnant women [86,87].
Another important area of future progress in vaccines and
pregnancy is to include pregnant women in pre-licensure clinical
trials for vaccines that are likely to be recommended during preg-
nancy. In the past, pregnant women have been excluded from
clinical trials of medications and vaccines because of the potential
risk to the fetus [43]. However, this leaves women and their health
care providers without data to guide use of these agents during
pregnancy, and collecting these data following licensure often takes
many years [88]. For these reasons, most vaccines (and most
medications) are listed as FDA pregnancy category C (i.e. no
adequate and well-controlled studies in pregnant women are
available), including several types of inactivated inuenza vaccine
[18]. In 2010, a meeting was convened by the National Institutes of
Health (NIH) to address ethical, regulatory, and scientic issues
raised by inclusion of pregnant women in clinical research studies
[89]. In addition, NIH also recently held a series of meetings to
guide the development of future clinical trial protocols of vaccines
in pregnant women [90,91]. From these meetings, it is expected
that future studies of vaccines and medications that are intended
for use among pregnant women will include pregnant women and
will be conducted in an ethical and scientically valid manner.
Identication of other vaccines that might protect infants early
in life from infectious disease morbidity and mortality if adminis-
tered during pregnancy is another key area for the future. For
example, young infants are particularly vulnerable to the effects of
group B streptococcus (GBS). GBS is the leading cause of sepsis and
meningitis among newborns in the rst week of life (early-onset
GBS) in the USA. Infants with GBS aged 7e89 days (late-onset
GBS) present more often with meningitis, with lower mortality
than that seen with early-onset disease, but with a signicant risk
of long-term sequelae. Although maternal screening and intra-
partum antibiotic prophylaxis treatment for GBS has decreased the
number of cases of early-onset GBS disease in the USA by 80% [92],
GBS continues to present a signicant burden on infant health, and
development of antibiotic-resistant GBS strains is of concern [93].
Development of a GBS vaccine for use during pregnancy could have
substantial public health impact: modeling data suggest that
maternal GBS vaccination could potentially prevent 4% of preterm
births, more than 60% of early-onset GBS and 70% of late-onset GBS
disease [94]. Ongoing research on the development of a safe,
effective GBS vaccine for use has shown promising results [93].
Several other vaccines are under investigation for use during
pregnancy, including meningococcal polysaccharide, pneumo-
coccal conjugate and polysaccharide, Hib conjugate and poly-
saccharide, and respiratory syncytial virus vaccines [93,95].
Healy [93] has identied a number of factors to be considered
before implementation of a maternal immunization programwith a
new vaccine, including safety, immunogenicity, timing of immu-
nization, biological considerations (including whether maternal
immunization will decrease the infants immune response to vac-
cines), and implementation (education of health care providers and
the public, acceptability of vaccines during pregnancy, and logistics
and nancing). Regulatory issues and concerns about litigation
pose signicant barriers to the use of currently recommended
vaccines and to the development of new vaccines for use during
pregnancy [96]. Unfortunately, the question of whether the Na-
tional Vaccine Injury Compensation Program e a program estab-
lished by the National Childhood Vaccine Injury Act of 1986 to
provide no-fault monetary awards for adverse events caused by
covered vaccines e includes in-utero injuries that occur following
vaccination of pregnant women has not yet been resolved [97].
In summary, benets of maternal vaccination for preventing
maternal and infant morbidity and mortality have been demon-
strated, in particular, with maternal vaccination with inuenza and
tetanus toxoid vaccines. This strategy has been expanded to
pertussis, and could be benecial to prevent other diseases, espe-
cially those with a substantial burden in the rst fewmonths of life.
Several barriers to developing vaccines for administration during
pregnancy and to optimally implementing maternal vaccination
programs remain. However, recent progress e in terms of addi-
tional data on safety and effectiveness of vaccines during preg-
nancy, improved integration of maternal vaccination into clinician
practice, and advances toward including pregnant women in clin-
ical trials for relevant vaccines e hold promise for the future of
vaccination during pregnancy.
Practice points
Health care providers who care for pregnant women
should review their vaccination history as part of routine
prenatal care, and should recommend and offer appro-
priate vaccines.
All women who are or will be pregnant during the inu-
enza season should receive inactivated inuenza vaccine
regardless of trimester.
All pregnant women should receive a dose of Tdap vac-
cine during each pregnancy, regardless of prior history of
vaccination. Ideally the vaccine is administered between
27 and 36 weeks of gestation although it may be given at
any time during pregnancy.
Research directions
Although safety data frominactivated inuenza vaccine in
pregnancy have been reassuring, data fromvaccination in
the rst trimester of pregnancy are limited, and therefore
additional safety data from use early in pregnancy are
needed.
Patient and health care provider barriers to receiving
recommended vaccines in pregnancy need to be explored
in order to develop more effective interventions to in-
crease adherence to vaccination recommendations.
In addition to safety data, more information is needed
about the potential for new and existing vaccines to pro-
tect infants early in life from infectious disease morbidity
and mortality.
The ndings and conclusions in this report are those of the
authors and do not necessarily represent the ofcial position of the
Centers for Disease Control and Prevention.
Funding sources
None.
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Review
The National Birth Defects Prevention Study: How to communicate
data
Anthony R. Scialli
*
Tetra Tech Sciences, 2200 Wilson Blvd Ste 400, Arlington, VA 22201, USA
Keywords:
Caseecontrol studies
Causality
Cleft palate
Teratology
Epidemiology
s u m m a r y
The National Birth Defects Prevention Study is a population-based caseecontrol study. The study has
actively sought to identify children with any of 34 specied types of malformation. The mothers of
affected and unaffected children have been interviewed with regard to demographic information, life-
style factors, and exposures. A large number of published studies have appeared and continue to appear
on diverse exposures and outcomes. An example of such a study identied an increased odds ratio for
ondansetron use among the mothers of children with cleft palate. Possible explanations for associations
between exposures and outcomes are chance, error, and causation. The ondansetronecleft palate as-
sociation may have arisen by chance given the large number of comparisons made in the study. Error
appears unlikely as an explanation of the association. The assessment of causation in teratology uses a
systematic evaluation based on the Hill criteria or similar criteria of Shepard or Brent.
1. Introduction
Because we counsel families about the risk for developmental
abnormalities from exposures, we nd ourselves in the business of
interpreting data. Our pregnant patients who have been exposed to
a medication or an environmental chemical want to know, Is it
safe?, and our patients who have had a child with a developmental
problemwant to know, What caused this problem? The published
literature does not give us a direct answer to these questions.
Instead, the literature gives us data, and we have to bring our
expertise and analytical skills to bear on turning the data into an-
swers that are useful to our patients. In this paper, I describe the
National Birth Defects Prevention Study (NBDPS), which has
become an important source of data, and I suggest how these data
can be incorporated into our practices using as an example a study
of medications for nausea and vomiting of pregnancy.
2. What is the National Birth Defect Prevention Study?
The NBDPS is an ongoing population-based caseecontrol study
sponsored by the US Centers for Disease Control and Prevention
(CDC, Atlanta, GA, USA). The study saw its origins in an act of
Congress in 1996 that directed CDC to establish Centers for Birth
Defects Research and Prevention. These centers include the birth
defects surveillance and research programs of Arkansas, California,
Georgia, Iowa, Massachusetts, New Jersey, New York, North Car-
olina, Texas, and Utah.
The centers identied infants with any of 34 congenital
malformations or types of malformation (Box 1). The selection
and classication of abnormalities followed detailed guidelines
that were used by all centers in the program [1]. These guidelines
provided criteria for case denition and policies for the exclusion
of genetic syndromes and for the categorization of infants with
multiple malformations. There were differences between centers
in whether stillborn infants and elective terminations of prena-
tally diagnosed malformations were included [2]. Control chil-
dren were randomly selected from birth certicates or birth
hospitals. Mothers were interviewed by female personnel using a
standard computer-assisted telephone interview in English or
Spanish. Prior to the interview, each mother was mailed an in-
formation packet, a calendar that included the dates of her
pregnancy, and a money order for $20. Interviewing was stopped
on 31 March 2013.
Questions were asked about exposures that occurred fromthree
months before conception through the end of the pregnancy. Most
questions were structured with coded response lists, but some
questions were open-ended, for example questions about occupa-
tion and occupational exposures. Interviews were performed no
sooner than 6 weeks after the estimated date of delivery and no
later than 24 months after the estimated date of delivery, with a
target date of 6 months after the estimated date of delivery [2]. The
information collected by the maternal interview is summarized in
* Tel.: 1 571 527 1709.
E-mail address: Tony.Scialli@TetraTech.com.
Box 2. There were also questions about paternal demographic and
lifestyle factors. After the interview, women were sent a kit for
collecting buccal cells from the child, if living, and the parents, and
parents were sent another $20 money order. These cells are being
used for genetic studies.
The data collected from study participants were pooled and
are available to all participating centers. Investigators at the
centers have published and continue to publish study data, often
in conjunction with additional authors. Since the rst results
appeared in 2001, there have been more than 170 publications
covering diverse exposures including medications, diet, obesity,
and lifestyle factors among others (see Appendix).
Box 1
Malformations included in the National Birth Defects Prevention
Study
a
Cardiovascular
Anomalous pulmonary venous return
Conotruncal heart defects
Heterotaxy
Hypoplastic left heart syndrome
Obstructive heart defects
Septal heart defects
Single ventricle
Central nervous system
Anencephaly
DandyeWalker malformation
Encephalocele
Holoprosencephaly
Spina bida
Eye
Anophthalmia/microphthalmia
Congenital cataract
Glaucoma
Ear
Anotia/microtia
Orofacial
Choanal atresia
Cleft lip
Cleft palate
Gastrointestinal
Biliary atresia
Esophageal atresia and tracheo-esophageal stula
Intestinal atresia
Genitourinary
Hypospadias (2nd or 3rd degree)
Renal agenesis (bilateral)
Musculoskeletal
Bladder exstrophy
Cloacal exstrophy
Craniosynostosis
Diaphragmatic hernia
Gastroschisis
Limb deciency
Omphalocele
Sacral agenesis/caudal regression
Non-system-specic
Amniotic band sequence
a
Terminology as presented on the NBDPS web site at http://
www.nbdps.org/aboutus/bd.html.
Box 2
Categories of information collected by maternal interview
a
Maternal health
Illnesses
Diabetes
Fever
Hypertension
Respiratory illnesses
Seizure disorder
Urinary tract infection
Other illnesses
Injuries
Medication exposure (prescription and non-prescription)
X-ray exposure
Pregnancy
Assisted reproductive techniques
Contraception
Nausea and vomiting
Prenatal care
Pregnancy history
Substance use and diet
Alcohol
Caffeine
Food supplements
Illicit drugs
Tobacco
Vitamins
Assessment of diet
Home and work
Hot tub/sauna
Military service
Occupation
Residence
Water
Bathing
Drinking water sources
Swimming pool use
Other water uses
a
Adapted from Yoon et al. [2]
A.R. Scialli / Seminars in Fetal & Neonatal Medicine 19 (2014) 170e176 171
3. Epidemiology study designs
Epidemiology studies can be descriptive, that is, reporting sta-
tistics on the incidence or prevalence of a condition, or they can
evaluate associations between exposures and outcomes. Two
frequently used designs that evaluate associations between expo-
sures and outcomes are cohort studies and caseecontrol studies.
Cohort studies dene two or more groups based on their exposures
or other potential risk factors and measure the occurrence of out-
comes of interest in the group members. Caseecontrol studies
dene groups by outcome and measure the previous occurrence of
exposures or other risk factors of interest. As indicated above, the
National Birth Defects Prevention Study is a caseecontrol study.
There are strengths and weaknesses of each type of design.
Because cohort studies dene group membership based on expo-
sure, ascertainment of exposure is, in theory, straightforward. For
unusual outcomes, however, cohort study designs require large
samples in order to have the power to detect a meaningful differ-
ence between exposed and unexposed groups in the prevalence of
the outcome. Caseecontrol studies dene group by outcomes, so a
study population is enriched by including only individuals with the
outcome of interest. In caseecontrol studies, it can be a challenge to
reliably ascertain exposure status, which can be based on maternal
recall, medical/pharmacy record evaluation, or insurance claims.
Errors in ascertaining exposure status may be systematic, meaning
that they are more likely in one group (e.g. cases) than the other,
which can affect the ndings of the study.
In caseecontrol studies, the end-points that can be evaluated
are limited to those selected by the investigators prior to the
collection of data. Exposures that cause an increase in birth defect
risk usually do so with respect to a syndrome or pattern of birth
defects. A criticism of caseecontrol studies is that they address
individual birth defects rather than syndromes or patterns of birth
defects. For example, exposure to isotretinoin during pregnancy
can be associated with micrognathia, ear abnormalities, and con-
otruncal heart defects. A study of isotretinoin using the National
Birth Defects Prevention Study would be expected to identify an
association with conotruncal heart defects and possibly with
microtia/anotia, but would not identify an association with micro-
gnathia, which is not a malformation included in the study. Iden-
tication of the isotretinoin syndrome, then, might not arise from
this study. Elements of a proposed syndrome could be investigated,
but some prior knowledge of these elements would be needed in
order to direct the investigators attention to the right combination
of abnormalities.
4. Risk estimates
Epidemiology studies assess associations between exposures
and outcomes. An estimate of the strength of an association is
called a risk estimate. In a cohort study, the risk estimate may be
expressed as a relative risk. The term risk is used for the propor-
tion of affected individuals in a population. Relative risk is a ratio
between the risk in one population (often an exposed group) and
the risk in another population (often an unexposed group). In a
caseecontrol study, relative risk can be approximated by the odds
ratio if the incidence of the outcome in the population is not too
high. In the context of a caseecontrol study, an odds is the like-
lihood of having been exposed (the number of exposed individuals
divided by the number of unexposed individuals), and an odds ratio
is the odds of having been exposed in cases (affected individuals)
divided by the likelihood of having been exposed in controls.
A risk estimate of 1 indicates no association between the
exposure and the outcome. For a caseecontrol study, a risk estimate
of 1 means that affected individuals are no more or less likely to
have been exposed to the risk factor of interest than unaffected
individuals. Risk estimates of >1 may indicate an increase in risk,
and risk estimates of <1 may indicate a decrease in risk. However,
the risk estimate is not used by itself because of the recognition that
there will be variability in the risk estimate as the putative asso-
ciation is examined in different studies of similar populations. The
variability or precision of the risk estimate can be expressed as a
condence interval (CI), often a 95% CI. The 95% CI is the range of
risk estimates that would be expected in 95% of repetitions of the
study if the study were repeated an innite number of times in
similar populations. Any value within the 95% CI could be the true
risk of the exposureeoutcome relationship under consideration.
A related concept is the estimation of the likelihood that an
apparent association arose by chance. This estimate is expressed as
a P-value. P 0.05 indicates a 5% likelihood that an association
arose by chance. No P-value guarantees that an association must
have arisen by chance, and no P-value guarantees that an associa-
tion could not have arisen by chance. A 95% CI that includes 1.0 is
consistent with the conclusion that the putative association is not
statistically signicant at P < 0.05. If a 95% CI excludes 1.0, it is
statistically signicant. It cannot be concluded that the association
could not have arisen by chance, only that it is less likely to have
done so than if the 95% CI included 1.0.
5. Multiple comparisons
As an example of data from the National Birth Defects Preven-
tion Study, we now consider a paper from 2012 on medications
used to treat nausea and vomiting of pregnancy [3]. The study
evaluated a subset of the available malformation end-points within
the NBDPS, including cleft lip with or without cleft palate, cleft
palate, neural tube defects, and second- or third-degree hypospa-
dias. According to the study authors, 75 different medications and a
number of herbal products were used for nausea and vomiting of
pregnancy. Medications were grouped into classes by a research
pharmacist for the purposes of analysis. The classes included
antihistamine antiemetics, other antihistamines, antihistamine
antiemetics plus vitamin B
6
, phenothiazines, prokinetics, 5-
hydroxytryptamine-3 (5-HT
3
)-receptor antagonists, coke syrup,
bismuth subsalicylate, antacids, histamine H
2
-receptor blockers,
proton pump inhibitors, pyridoxine (vitamin B
6
), steroids, and
herbal products.
The results for cleft palate showed an elevated odds ratio for
ondansetron (a 5-HT
3
-receptor antagonist) adjusted for maternal
age, race or ethnicity, education, parity, plurality, previous miscar-
riage, smoking, body mass index, infant sex, folic acid use, use of
unknown antiemetic, study site, and expected year of delivery. The
adjusted odds ratio was 2.37 with a 95% condence interval of 1.18e
4.76. This number tells us that after adjustment, children with cleft
palate in the study were 2.37 times as likely to have mothers
reporting the use of ondansetron as children without birth defects.
The 95% CI excluded unity. This study also reported a statistically
signicant association between steroid use and hypospadias, but
we now concentrate on the ondansetronecleft palate ndings.
Does this study mean that use of ondansetron during pregnancy
causes cleft palate in the offspring? When an association is iden-
tied in a study, causation is one possible explanation, but there are
two other possible explanations: chance and error.
The nding of a 95% CI that excludes unity suggests that the
likelihood of chance explaining the ndings is <5%, but, when there
are multiple comparisons in the study, the interpretation changes.
Consider a comparison of two groups of people, say a group of
epidemiologists and a group of obstetricians. A large number of
measurements are taken from these groups e for example, height,
weight, intercanthal distance, IQ, etc. e until there are 20
independent measurements that are intended for comparison. If
statistical testing of the comparisons uses a P-value of 0.05, it is
implied that we will accept a comparison as being statistically
signicant if there is less than a 1 in 20 (5%) chance that a difference
arose by chance. If it became apparent that the only difference
between the group of epidemiologists and the group of obstetri-
cians was a higher IQ in the obstetricians, we would not be so
condent that the difference was a real nding as opposed to a
chance nding. Epidemiologists might, indeed, insist that such a
difference must have arisen by chance.
The relationship between the number of independent compar-
isons and the likelihood of a chance association with an apparent P-
value of 0.05 is given by the equation
P 1 0:95
n
;
where P is the likelihood of a chance nding, and n is the number of
independent comparisons. This relationship is shown graphically in
Fig. 1. As the number of comparisons increases, the likelihood of a
chance nding being identied as statistically signicant at P <0.05
increases asymptotically towards 1. In the study of nausea and
vomiting of pregnancy, the NBDPS evaluated 75 different medica-
tions and four different birth defect groups, potentially giving 300
comparisons. The likelihood of nding a chance association to be
statistically signicant for 300 independent comparisons is
0.99999979247.
In fact, there were fewer than 300 independent comparisons,
because not all medications were counted and not all exposures
were independent of one another. The study authors estimated that
they made 70 comparisons, which would give a likelihood of 1 e
(0.95)
70
97% likelihood of identifying a chance association as
statistically signicant. All the same, it is not possible to say that the
association between cleft palate and ondansetron must be due to
chance e only that it could be. The study authors recognized this
possibility and wrote, Perhaps of most importance is the possibility
of chance as an explanation for the statistically signicant associ-
ations that we observed.
6. Error
The term error is used to include features of a study or of
biology that predispose the study to giving the wrong answer. Error
is sometimes divided into bias and confounding.
6.1. Bias
Bias is a feature of the study design that increases the likelihood
of the study giving the wrong result. A commonly mentioned
source of error is recall bias, which in birth defect studies refers to
the likelihood that the parents of affected children will search their
memories more diligently than parents of unaffected children and
will be more likely to report an exposure, whether or not the
exposure actually occurred. Recall bias does not refer to non-
differential memory errors, i.e. both case parents and control par-
ents make mistakes in their reporting. If memory errors are non-
differential, the effect on the study will be to make it harder to
show a difference between the groups. Differential errors such as
recall bias, on the other hand, predispose a study to show a dif-
ference that may not be real.
It is unlikely that the ondansetron nding was due to recall bias,
because mothers had the opportunity to over-recall many other
drug exposures and to over-recall exposures for other defects, not
just cleft palate. The nding that the association was present just
for ondansetron and just for cleft palate makes recall bias a less
convincing explanation.
Response bias is another potential source of error. If a small
proportion of invited subjects agreed to participate, the question
arises whether the participants had a special story to tell. In other
words, perhaps the basis for their participation was some connec-
tion in their minds between the exposure of interest and the
outcome of interest. In such instances, the participants might not
faithfully represent the larger universe of affected or exposed
individuals.
The NBDPS has evaluated the extent to which participants in the
control group represent the general population from which they
were drawn [4]. The control participation rate was 65.8%. The
participating control pregnancies did not differ frompregnancies in
the background population with respect to maternal age, maternal
education, previous live births, trimester during which prenatal
care began, or diagnosis of diabetes. Infants born to participants
were less likely to weigh <2500 g at birth, less likely to be born at
<37 weeks of gestation, and less likely to be multiples (twins,
triplets, or more). There were small differences between the
participating controls and the background population in maternal
race/ethnicity, paternal age, and maternal smoking. A comparison
of the participating control women and the invited non-
participating control women showed participants to be more
likely to be white non-Hispanic, aged 30 years, to have completed
more than a high school education, and to have started prenatal
care in the rst trimester. Participants were less likely than invited
non-participants to have had more than three previous live births
and to be smokers. The other differences between participating
control women and the general population were also seen in the
non-participating invited control women and did not differ by the
decision to participate.
In the antiemetic study, there was no information about the
response rate of invited cases and controls, so possible response
bias cannot be assessed based on participation rates. However,
response bias does not appear to be a likely explanation for the
ondansetron ndings. It is implausible that participation was based
on a perceived relationship between ondansetron and cleft palate
without also affecting perceived relationships between other de-
fects and ondansetron or other drugs.
Fig. 1. The relationship between the number of independent comparisons being made
in a study and the likelihood of nding a statistically signicant difference at P 0.05
by chance alone.
Another type of bias is diagnosis bias, sometimes called ascer-
tainment bias. If women with a particular exposure are more likely
to have their children examined for a particular birth defect, that
birth defect might be more likely to be diagnosed in the exposed
group. As an example, if anxious women are more likely to compel
their childrens pediatricians to order an echocardiogram than
women who are not anxious, heart defects that might otherwise go
undetected might be ascertained with greater frequency in the
offspring of women exposed to medications used to treat anxiety.
In the antinauseant paper we are discussing, it is unlikely that
women who were treated with ondansetron would be more likely
to prompt a thorough palate examination in their children. Of
course, after a study such as this one is published, it is possible that
practices will change and that a history of ondansetron exposure
will, in fact, prompt a more thorough palate examination in
newborns.
6.2. Confounding
Whereas bias is a feature of study design, confounding is a
feature of the biology of an association that may escape consider-
ation by investigators. In birth defect studies, it is frequent practice
for investigators to consider whether there is confounding by
exposure to tobacco, alcohol, and other medications and to adjust
for potential confounders. If, for example, pregnant women who
drink alcohol are more likely to be nauseated than women who
abstain, women who are treated for nausea may have babies with
alcohol-related malformations. The malformations might be
blamed on antinauseant medication rather than on alcohol unless
the results are adjusted for exposure to alcohol.
A related source of confounding is the illness or indication for
which a medication is being used. If we accept that maternal diabetes
mellitus causes birth defects, a study of antidiabetic medications
might showan association between these medications and birth de-
fects that is mediated in reality by the underlying maternal disease.
Is it likely that confounding by indication explains the ondan-
setron results? Could it be that something about maternal nausea
and vomiting causes cleft palate in the offspring? Perhaps, but this
study did not show a statistically signicant increase in cleft palate
associated with other antinauseants, making confounding by
indication a less likely explanation. Perhaps ondansetron is pref-
erentially used in women with particularly severe nausea and
vomiting, which might explain the apparent association with cleft
palate. There are no data with which to evaluate this possibility.
7. Causation
We come now to the question a patient might ask us in light of
the NBDPS: does ondansetron use during pregnancy cause cleft
palate? We should be clear from the outset that single studies do
not establish causation. Rather, causation is considered using evi-
dence from multiple sources, and causation analysis considers
several elements.
Criteria for causation were specied in the mid-1960s by Sir
Austin Bradford Hill [5] and an advisory committee to the US Sur-
geon General with regard to cigarette-related disease [6]. These
viewpoints, as Sir Austin called them, are listed in Box 3. The Hill
criteria have given rise to variations and interpretations in the half-
century since they were published. Sir Austin used examples
largely from occupational exposures and cancer. In the eld of
teratology, notable contributions came from Drs Thomas Shepard
[7] and Robert Brent [8]. These authors included components of the
Hill list modied to apply to teratology. The Public Affairs Com-
mittee of the Teratology Society published principles to be applied
to causation analyses in a litigation context [9].
I review below some of the Hill criteria in terms of how they
might be used to assess the relationship of ondansetron therapy
during pregnancy and cleft palate. There is no threshold number of
criteria required for a conclusion of causation, but the more criteria
that are met, the more condence we have that a conclusion of
causation is correct.
7.1. Strength of the association
Strength of the association refers to the size of the risk estimate.
The higher the risk estimate, the less likely it is that chance or error
can explain the result. How high a risk estimate is needed? Dr
Shepard suggested that a risk estimate of 6 would show a strong
association [7]. In the antinauseant study, the risk estimate for
ondansetron and cleft palate was 2.37 with a 95% CI that started at
1.18, which is not very much above 1.0. As discussed above, chance
might explain this association based on the multiple comparisons
that were made.
7.2. Consistency of the association
The demonstration of a consistent association in different pop-
ulations is an important element of causation analysis, and if an
association appears only once, we cannot be condent that the
association is causal. In considering ondansetron, there are three
studies other than the NBDPS that can be used to evaluate a
possible association with cleft palate.
A paper from teratology information services in Canada and
Australia compared pregnancy outcome among 176 women who
called with concerns about ondansetron and the same number of
women who called with concerns about exposures that were not
believed to increase the risk of birth defects [10]. There were 169
live births in the ondansetron group among which six children had
major malformations compared with three malformed infants
among 160 live births in the comparison group (P 0.52). The
malformations in the ondansetron group included three instances
of hypospadias and one child each with duplication of the renal
collecting system, pulmonic stenosis, and duodenal atresia.
Although these results might appear reassuring with respect to
cleft palate, the risk estimate in the NBDPS predicts an increase in
the incidence of cleft palate fromabout 6/10 000 to 15/10 000. Such
Box 3
The Hill criteria
a
1. Strength of the association (the likelihood that the as-
sociation is not due to chance, bias, or confounding).
2. Consistency of the association (the association is repro-
duced in different populations).
3. Specicity (uniqueness of the association both with
respect to the exposure and with respect to outcome).
4. Temporal relationship (the putative cause comes before
the effect).
5. Coherence (the association is compatible with related
knowledge).
6. Biologic gradient (there is a doseeresponse effect).
7. Biologic plausibility (the association does not violate
known principles).
8. Experiment (reducing the putative cause reduces the
effect).
9. Analogy (evidence is similar to that for similar cause
eeffect relationships).
a
Adapted from Hill [5]
an increase could not have been detected with the limited sample
in the teratology information service study.
A study using the Swedish Medical Birth Registry reported an
odds ratio for use of antiemetic medications in infants with oro-
facial clefts of 0.75 (95% CI: 0.55e1.03) [11]. Although this odds ratio
is not increased, the result applies to antinauseant medications as a
group and lacks the requisite specicity for conclusions about
ondansetron. Indeed, there were few instances of ondansetron use
among these drugs.
A study from Denmark using a prescription registry to identify
exposures and a patient registry to identify outcomes reported that
among 1233 women believed to have been exposed to ondansetron
in the rst trimester, there was no increase in major malformations
compared with an unexposed group [12]. Although individual
malformations were not analysed, there were no instances of cleft
palate among the 1233 infants with presumed maternal ondanse-
tron exposure. This sample is much larger than the sample in the
teratology information service study, but 1233 pregnancies is still
not sufcient to identify an increase in cleft palate from6/10,000 to
15/10,000. The issue of consistency remains open.
7.3. Specicity
The report of the NBDPS satises the requirement for specicity.
The malformation under consideration was cleft palate e not all
malformations or even all facial clefts e and the exposure was
ondansetron, not all antiemetics.
7.4. Temporality
The requirement that the putative cause comes before the effect
seems easily to be satised by the NBDPS; however, it is possible to
be more critical in requiring the temporal relationship between
exposure and outcome to make embryological sense. In the study
on antinauseants, women were considered exposed if they took a
medication any time in the rst trimester, but not all birth defects
can be caused by an exposure any time in the rst trimester, and
some birth defects are caused by exposures after the rst trimester.
Some authors recommend considering exposures during specic
critical periods as relevant for specic malformations [13]. Using
this approach, ondansetron exposures during the third month after
the last menstrual period might be considered informative for cleft
palate and exposures at other times might be considered uninfor-
mative. This approach has not been widely adopted.
7.5. Biological gradient
It is expected that as the dose level of an exposure increases, the
response will increase, either in terms of frequency or severity of
adverse effects. The NBDPS paper on antinauseants has no infor-
mation on dose level, and this criterion cannot be evaluated.
7.6. Biological plausibility
The requirement for biological plausibility is considered by
some people to be a low hurdle; as long as the proposed causal
association does not involve unicorns or Martians, it might be
considered biologically plausible. However, in teratology, it is
customary to require either an experimental animal model for the
putative causal relationship or a well-established mechanism by
which the causal association could be mediated. With respect to
ondansetron, a 5-HT
3
receptor antagonist, nonclinical embryofetal
toxicology studies did not show an increase in cleft palate or in any
other malformation at up to 4 mg/kg body weight in rats and up to
10 mg/kg body weight in rabbits. The human intravenous dose is
0.45 mg/kg body weight/day [14,15].
There are no published data on possible developmental toxicity
of other 5-HT
3
inhibitors. Mice with knockout of the 5-HT
3
receptor
are healthy [16]. Because cleft palate precludes suckling in mice, the
viability of knockout mice is evidence that antagonism of this re-
ceptor does not produce cleft palate.
8. Counseling
Counseling patients who are contemplating exposure to
ondansetron during pregnancy can take the approach that there are
alternatives in the treatment of nausea and vomiting. One small
study showed no difference in the effectiveness of treatment with
promethazine versus ondansetron for patients hospitalized with
excessive nausea and vomiting of pregnancy [17]. Ondansetron is
expensive compared to promethazine, but it is less sedating.
Favorable experience has also been published for the combination
of droperidol and diphenhydramine in the management of exces-
sive nausea and vomiting of pregnancy [18].
Patients who have been exposed to ondansetron can be advised
that the increase in risk identied by the NBDPS has not been
conrmed. Moreover, the absolute risk is small, with an increase of
<0.09% in the incidence of cleft palate.
9. Conclusions
The NBDPS is a large, population-based caseecontrol study that
has collected information in a systematic manner on the exposures
and lifestyle characteristics of the mothers and fathers of children
with and without congenital malformations. The study has pro-
duced a large number of publications on a variety of exposures. As
is true of all caseecontrol studies, the outcomes under study are
selected before the data are collected, limiting the ability of the
study to identify new birth defect syndromes. Nonetheless, casee
control studies provide important information to be used in the
consideration of birth defect causation questions. These questions
are not decided by a single study but are addressed using infor-
mation from multiple sources.
Dr Scialli has provided testimony for the defense in Paxil
liti-
gation. Paxil
is marketed by GlaxoSmithKline, which also markets

the Zofran
brand of ondansetron. Dr Scialli has not consulted or

testied on Zofran
or generic ondansetron.
Appendix A. Supplementary data
Additional further reading sources related to this article can be
found online at http://dx.doi.org/10.1016/j.siny.2013.09.007.
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Review
Birth defects and assisted reproductive technologies
Joe Leigh Simpson
*
March of Dimes, 1275 Mamaroneck Avenue, White Plains, NY 10605, USA
Keywords:
Assisted reproductive technologies (ART)
Birth defects
Hypospadias
Imprinting
Intracytoplasmic sperm injection (ICSI)
In-vitro fertilization (IVF)
s u m m a r y
Assisted reproductive technologies (ART) using in-vitro fertilization (IVF) account for w1% of births in
the USA and as much as 3e4% in Europe or Australia. Initially studies involved infants prospectively
examined in an early cohort of US births, with salutary results. Later studies began to show the frequency
of birth defects to be increased. In meta-analysis, odds ratio was >1.0, with the 95% condence limit not
extending to <1.0. Although ART are associated with a 30% increase in birth defects; subfertile couples
achieving pregnancy without ART show a 20% increase. It thus appears that the increase in birth defects
is due less, if at all, to ART protocols per se than to the biological perturbations that generated the
infertility that necessitated ART to achieve pregnancy. There is consensus that traditional IVF and
intracytoplasmic sperm injection (ICSI)/IVF show the same overall risk notwithstanding increased sex
chromosome abnormalities in both procedures and increased hypospadias in ICSI. No other organ system
seems disproportionately affected. There is no additive risk in ART twins compared with non-ART twins,
nor in embryos having been cryopreserved. The increased risk observed had not appeared to dissuade
couples from attempting to have their own children.
2014 Published by Elsevier Ltd.
1. Introduction
Assisted reproductive technologies (ART) using in-vitro fertil-
ization (IVF) account for about 1% of births in the USA and as much
as 3e4% in Europe or Australia. More than ve million babies are
estimated worldwide to have been born through ART. Scientic and
medical advances are increasing pregnancy rates, and the preva-
lence of ART births can condentially be expected to continue to
increase. Even prior to the pioneering efforts of Edwards and
Steptoe that resulted in the rst success in 1978, concern had been
raised over whether infants born by ART would universally be
abnormal. This fear has largely been mitigated, and attention is
now focused on more nuanced questions.
The initial studies involved infants carefullyexamined inanearly
cohort of US births, withresults salutaryalthoughsample sizes were
small [1]. Population-based studies from Australia by Lancaster [2]
showed a 2.9% frequency, reassuringly that expected for the gen-
eral population. The rst largepopulationstudy was byWestergaard
et al. [3], who compared 2245 ART births in 1994e1995 with 2245
controls. The odds ratio (OR) was 1.04 with the 95% condence in-
terval (CI) 0.78e1.39 (non-signicant). Other studies of this era
generally failed to show deleterious effects (Table 1) [3e5]. Studies
reported in the 2000s beganto showfrequency of birth defects to be
increased (Table 2). The OR in these studies was often >1.0, with the
95% CI not extending across the 1.0 isobar [6e15]. Thus, there
evolved the present consensus that increased birth defects are
indeed positively associated with ART. The major question at pre-
sent is whether this increase is due toART protocols per se or merely
reective of the biological perturbations that generated the infer-
tility that necessitated ART to achieve pregnancy.
In this communication, we shall rst consider the frequency of
congenital anomalies in offspring of ART pregnancies, stratied by
specic subgroups depending on technique. We shall also comment
on the pitfalls that make difcult denitive conclusions concerning
the explanation for anomalies detected.
2. Frequency of anomalies in ART pregnancies
Almost 50 cohort studies have addressed the question of
anomalies in ART pregnancies, as referenced elsewhere [16e19].
Initial studies naturally focused on IVF alone, because not until the
mid-1990s could male infertility be managed by intracytoplasmic
sperm injection (ICSI) followed by traditional IVF. In the 1990s and
early 2000s, one could condently conclude the overall risk was
not, say, a two- or three-fold increase above the accepted popula-
tion baseline of 2e3%. However, more precise statements could not
be made.
Notwithstanding general reassurance, this author and others
pointed out pitfalls that could sway opinions on safety in either
* Tel.: 1 914 997 4610.
E-mail address: jsimpson@marchofdimes.com.
1744-165X/$ e see front matter 2014 Published by Elsevier Ltd.
direction [20,21]. It was in particular appreciated that power to
detect a signicantly increased frequency of anomalies was not
possible due to small sample size.
Table 2 shows later population-based studies on which this
altered conclusion began to be based [6e15]. All were based on
registries that recorded cohorts of births, from which those with
anomalies could be stratied. A caveat is that the denition of birth
defects among registries varies, usually involving International
Classication of Diseases (ICD)-based diagnosis codes not well
suited to distinguish major from minor anomalies. ICD codes at
birth are not robust in detecting subtle anomalies not externally
visible, nor in excluding inherited syndromes or chromosomal
anomalies. Neither is plausibly due to ART. Not always followed is
the pragmatic, accepted, denition of a major birth defect as one
that causes death, functional impairment or (if structural) requires
surgery.
Reviewing several studies will sufce as illustrative. In 2005
Klemetti et al. [7] published Finnish registry of IVF and ICSI cases
delivered in 1996e1999, nding the adjusted OR for all birth de-
fects to be 1.31 (95% CI: 1.10e1.57), when comparing 4459 cases and
27 078 controls. In 2007, Pinborg et al. [9] used Danish registry data
to derive an OR of 1.24 (95% CI: 1.09e1.43). In 2005 Kallen et al.
[6,12] published an analysis of Swedish registries (1982e1999) data
and in 2010 analysed cases for the years 2001e2007. The latter
study showed OR of 1.25 (95% CI: 1.5e1.37) based on 15 570 cases
[12].
Outside of Scandinavia, the region generating greatest attention
is Australia, betting the countrys sentinel role in developing ART.
In 2002 Hansen et al. reported Western Australia cases, reprising
this in 2012 [14,22]. Their 2012 report of Western Australia registry
data involved ART cases delivered 1994e2002, an interval of sig-
nicance given many changes having since occurred in laboratory
methodology. This study was laudatory, however, in trying to take
into account anomalies in pregnancy terminations. Both the 2911
ART cases and the 210 997 non-ART cases were followed for 6
years. A major birth defect was found in 8.7% of ART cases and 5.4
of non-ART cases (OR: 1.53; 95% CI: 1.30e1.79). Rates were not
different in unlike sex twins (obligatory dizygosity) (OR: 1.08; 95%
CI: 0.77e1.51). The 5.4% birth defects in the control group is at odds
with general acceptance, absent comprehensive laboratory as-
sessments such as array comparative genomic hybridization. These
authors concluded also that there has been a decrease in the
prevalence of birth defects compared with their earlier cohort
(1994e1998) [14,23]. One explanation may simply be greater
appreciation between major and minor anomalies. Plausible sci-
entic reasons include changes in culture media, better regulation
of heat and CO
2
in incubators, and differential ovulation stimula-
tion regimes.
A second Australian group extending interval of ascertainment
to 5 years was that of Davies et al. [13], who used registry data of
308 974 births in South Australia (Adelaide). Minor defects were
excluded unless they required treatment or were disguring.
Among 6163 ART offspring were 513 with anomalies (8.4%),
compared with 5.8% in non-ART births. The adjusted OR was 1.28
(95% CI: 1.16e1.41). OR was 1.26 for IVF alone, and 1.77 for ICSI/IVF.
It would be of interest to knowthe temporal sequence in which the
cumulative absolute frequency of birth defects occurred, i.e.
ascertainment at birth versus later.
Of special interest to US readers is the report of Kelley-Quon
et al. [15], who used the California Patient Discharge Linked Birth
Cohort Database that lists anomalies by ICD-9 codes. This study
involved 4795 infants born in 2006e2007 after ART, compared with
46 025 naturally conceived in the same interval. The overall rate of
major congenital abnormalities was 9.0% vs 6.6% (OR: 1.25; 95% CI:
1.12e1.39; P < 0.001). Incidence of birth defects this high bespeaks
inclusion of many minor anomalies. In this authors opinion, this
alone casts doubt on conclusions that ORs were signicantly
increased for certain organ-specic anomalies: eye, head and neck,
heart and genitourinary track.
These limitations notwithstanding, meta-analyses reached
similar arithmetic conclusions: Rimm et al. [16]: 1.29 (95% CI:
1.01e1.67); Hansen et al. [17,19], risk ratio: 1.32 (95% CI: 1.24e
Table 1
Earlier population-based studies of anomalies in assisted reproductive technology pregnancies.
Study Years of sample accrual Adjusted OR (95% CI) Statistical signicance Country
Dhont et al. [4] 1986e2002 1.25 (0.96e1.64) No Belgium
Westergaard et al. [3] 1994e1995 1.04 (0.78e1.39) No Sweden
Anthony et al. [5] 1995e1996 1.03 (0.86e1.23) No Netherlands
OR, odds ratio; CI, condence interval.
The initial population-based studies depend on data in registries. Ascertainment varied between studies with respect to duration of time in which anomalies were sought and
denition of major defects. The interval of accumulated ART registry cases does not in any report correspond to the extant laboratory and ovulation stimulation protocols used
in 2014.
Table 2
Later population-based studies on anomalies in assisted reproductive technology (ART) pregnancies (2005e2013).
Study Years of sample accrual Adjusted OR (95% CI) Statistical signicance Country
Kallen et al. [6] 1982e2001 1.44 (1.32e1.57) Yes Sweden
Davies et al. [13] 1986e2002 1.24 (1.09e1.41) Yes Australia (Adelaide)
Halliday et al. [11] 1991e2004 1.36 (1.19e1.55) Yes Australia (Parkville)
Hansen et al. [14] 1994e2002 1.53 (1.30e1.79) Yes Australia (Perth)
Pinborg et al. [9] 1995e2000 1.24 (0.97e1.58) No Denmark
Klemetti et al. [7] 1996e1999 1.31 (1.10e1.57) Yes Finland
Ombolet et al. [8] 1997e2003 1.11 (0.08e1.58) No Belgium
Kallen et al. [12] 2001e2007 1.25 (1.15e1.37) Yes Sweden
Kelley-Quon et al. [15] 2006e2007 1.25 (1.21e1.39) Yes USA (California)
Fuji et al. [10] 2006 1.17 (0.81e1.69) No Japan
These population-based studies depend on data in registries. Ascertainment varied with respect to duration of time in which anomalies were sought and denition of major
defects. The interval of accumulated ART registry cases does not in any report correspond to the extant laboratory and ovulation stimulation protocols used in 2013.
J.L. Simpson / Seminars in Fetal & Neonatal Medicine 19 (2014) 177e182 178
1.42); Wen et al. [18]: 1.37 (95% CI: 1.26e1.48) (Table 3). Similar
results are not surprising because the same studies were generally
analyzed.
3. Anomalies by organ system
Because of sample size limitations, individual anomalies have
generally been assessed only by pooling anomalies by organ sys-
tem. Yet within a given system there is a plethora of different
anomalies of difcult etiologies egenetic and otherwise. Collapsing
all such anomalies to assess a single organ system is convenient for
generating statistical power, but this does not obviate the meth-
odological ow.
Hypospadias is the only organ system in which OR has been
consistently shown to be increased. An odds ratio of 3.0 (95% CI:
1.09e6.50) was reported by Wennerholmet al. [23] and 1.5 (95% CI:
1.0e2.1) by Ericson and Kallen [24]. Klemetti et al. [7] also showed
increased hypospadias.
A potential explanation for increased hypospadias in ART is
heritable low testosterone, also adversely affecting spermatogen-
esis. Recurrence risk for hypospadias is 2e5% in rst degree rela-
tives (sons). This could lead to transmission of a polygenic trait
(hypospadias). There was no signicant difference in the meta-
analysis by Wen et al. [18] One confounder is that indications for
ICSI are now much less stringent than when ICSI was initially
introduced, including many cases analyzed by Davies (1986e2002)
[13]. ICSI now constitutes half of all ART cases in the USA and even
more in some venues.
The only other organ system seriously implicated is cardiac.
Sala et al. [25] found a signicant increase, but in general other
authors have found no increase. Special pitfalls exist in assessing
cardiac anomalies. A patent ductus arteriosus is normal at birth,
but soon closes in full-term infants; in premature infants persis-
tence is normal, and surgical ligation is routine. Ventricular septal
defects are not usually evident at birth but manifest about one
week after birth when cardiac circulation is adapted for postnatal
life. Taking these temporal changes into account is not facile, and
beyond the charge of most if not all registries recording birth
defects.
4. Methods of data analysis
Determining whether anomalies are increased in ART could be
based on data from a single center or from a registry. It is fash-
ionable for many to use pooled data (systematic reviews or meta-
analysis), and consider this approach superior.
Meta-analysis is based mostly on obvious strength of sample
size compared with single center studies. Center-specic studies
are thus marginalized. However, the latter can utilize checklist
comparisons by a limited pool of examiners, an approach not
possible in population registries. Indeed, it is studies with less
systematic assessments that more often showed no increase in
birth defects. Meta-analysis has other limitations, namely lack of
consensus on inclusion criteria. In meta-analysis the axiom is that
all studies are comparable, and thus suitable for pooling.
Consider, however, the laudatory reports of Hansen et al. [17,19]
The latter exhaustive report excluded non-English reports, re-
ports in which no birth defects were found in either cases or
controls, and reports in which birth defects ascertainment
extended to a later age. Numbers of studies deemed suitable were
45, similar to Wen et al. (n 46) [18]. Yet Rimm et al. [16]
accepted only 19 studies.
Although the validity of meta-analysis in many circumstances
is strong, this is more arguable in ART and birth defects. In ART
virtually no units utilize similar protocols. Yet many different
laboratories contribute to a single registry, to say nothing of
differences between registries. Different authors must reach
their own opinion as to whether preset criteria are met. De-
cisions on inclusion criteria may be discrepant at different points
in time, even by the same author. For example, upon reassess-
ment 8 years later Hansen et al. [17,19] concluded that many
studies accepted as valid in their 2006 study were no longer
acceptable.
One widely used decision tree is to include or exclude series in
which follow-up extended beyond the newborn period. (Separate
analysis would be more appropriate.) Thus excluded is the report
of Davies et al. [13], despite being an excellent population-based
study that extended to 5 years of age and contained ascertain-
ment from multiple sources. Another study excluded for the same
reason has been Bonduelle et al. [27] who showed increased sex
chromosomal abnormalities in ICSI. These cannot be detected in
registries because physical abnormalities are usually not evident at
birth.
In some meta-analyses it is sometimes decided that certain
studies are given higher quality scores and, hence, greater
weight. For example, Hansen et al. [19] assigned a quality 1 score
(best) to 11 of 13 registry-based studies, and only seven
population-based studies were not scored quality 1. Among 32
clinic samples, only one was assigned quality 1. Many of the latter
were indeed of insufcient sample size, but numbers alone should
not be the sole criterion for high quality because quality of clinical
examination is less than ideal in registries. Consistent clinical
acumen is not possible among all examiners, some of whom lack
familiarity in distinguishing major from minor anomalies. It is
difcult but not impossible to form a collaborative network of
clinical centers to assess anomalies consistently among centers,
but not impossible (see the reports from the South American
Registry ECLAMC [27]). Another problemwith registry data is long
interval during which cases were accumulated. Anomalies were
initially probably more vigorously sought in ART cases than con-
trols [20,21]. This bias is probably less so now, but the earlier bias
is still relevant because registry data being used for currently
published meta-analyses are often based on deliveries even
extending as far back as the 1980s. Paradoxically, the larger the
study (greater power), the more ancient and now atypical cases
included.
The above criticisms are not meant to denigrate work pursued to
unravel a difcult problem, but to remind the reader that crisp data
suitable for analysis do not exist.
5. Pitfalls in assessing presence of birth defects
This author has expressed concern on pitfalls precluding data
accumulated and used for comparison of ARToutcomes [20,21]. Box
1 lists common pitfalls so far discussed, a few aspects of which are
highlighted in the following subsections.
Table 3
Reported meta-analyses on birth defects and assisted reproductive technologies.
Study Year Studies accepted OR (95% CI)
Rimm et al. [16] 2004 19 1.29 (1.01e1.67)
Hansen et al. [17] 2005 25
a
1.29 (1.21e1.37)
Wen et al. [18] 2012 46 1.37 (1.26e1.48)
Hansen et al. [19] 2013 45
a
1.32 (1.24e1.42)
a
Many studies considered acceptable in the 2005 review by Hansen et al. [17]
were no longer considered acceptable in their 2013 report [19].
5.1. Denition of anomalies
The major source of confusion is probably inconsistent deni-
tion of an anomaly (birth defect). How precisely does one distin-
guish major from minor anomalies? Should one rely on studies in
which minor anomalies are collapsed into a single category with
major anomalies? In fact, criteria for distinguishing major from
minor anomalies are well known to geneticists and well codied,
but generally not familiar to clinicians outside medical genetics
[28]. Rigor of physical examination is an issue because geneticists
record more minor anomalies than non-genetic pediatricians.
Relying upon pooled data when the primary outcome (birth de-
fects) is not assessed identically is inherently hazardous. Examiners
not surprisingly have variable skills, training and interest; thus,
criteria cannot be assumed to be comparable across centers,
studies, or countries. This becomes immediately evident by noting
variable rates of anomalies in controls, namely signaled by rates
considerably higher than the accepted 2e4% major birth defects at
birth and shown in checklist-based studies [29].
5.2. ART protocols
There is a lack of conformity in ovulation stimulation regimens,
a plethora of different culture media, and multiple embryo incu-
bation techniques. Standardization does not exist, and commer-
cially utilized media list only components and not concentrations.
5.3. Interval of pregnancy ascertainment
Especially egregious is, as noted, assuming that results derived
from methods used long ago are still generalizable to the
contemporary patient, a concern raised earlier. The most contem-
porary study involves 2006e2007 cycles, well behind currently
used techniques like vitrication [15]. The long interval between
pregnancies analyzed and publication data makes generalizability
to women undergoing ART in 2014 arguable.
5.4. Interval for anomaly surveillance after birth
Another problemis the varying length of surveillance after birth.
Birth defects registries traditionally are restricted to anomalies
ascertained in the neonatal period (before hospital discharge).
However, several excellent single-center studies extend to 5 years,
providing information not possible with registries (e.g. sex chro-
mosomal abnormalities) [13,26]. Certain organ-specic ART-related
anomalies could be manifested only in the newborn period and
other only later. Data analyzed in meta-analysis should ideally be
stratied by length of surveillance.
5.5. Lack of true control group
What is the control group? In theory, the proper control should
consist of women who require ART but who become pregnant
without ART, ideally within the time dened as normal to become
pregnant (12 months). Obviously, this group does not exist. Thus,
no gold standard randomized clinical trials or caseecontrol study
can be conducted. Instead, published reports compare ART out-
comes to naturally conceived offspring. The closest to a proper
control group seems to be a subfertile couple (inability to conceive
by 12 months) who conceive naturally while awaiting ART. Other
options include same-sex couples using a surrogate, but sample
sizes are inadequate.
6. Anomalies in singleton versus twin gestation
Consensus exists that overall birth defects are increased in sin-
gletons, but the same cannot be said for multiple gestations of
which twins constitute by far the largest group. When adjustment
was made for zygosity by Hansen et al. [19], OR was 1.26 (95% CI:
0.9e1.60). Anomaly rates were not different between ART and non-
ART twins. In unlike sex twins (obligatory dizygosity), OR was 1.08
(95% CI: 0.77e1.51). Recall that twins naturally conceived have long
been known to show increased birth defects, particularly if
monozygotic. Prior to the era of ART, half of all like-sexed twins
were monozygotic. Now the percentage should be lower. Interest-
ingly, pretermbirth in ARToffspring is considered to be increased in
singleton births but not in twin gestations [30,31]. Perhaps infer-
tility (and, hence, likelihood of embryo implantation) is greater in
women whose embryos are more likely to be abnormal and, hence,
less likely to sustain two embryos. Or, perhaps, singletons had a co-
twin who underwent unrecognized demise (vanishing twin) that
nonetheless exerted a persistent deleterious effect, perhaps
involving the decidua [32].
In conclusion, anomalies in both non-ART twins and in ART
twins are increased compared with singleton controls, but an ad-
ditive risk in ART is surprisingly unproven at present.
7. Anomalies in ICSI/IVF versus IVF alone
Initially only IVF per se was assessed for association with birth
defects. As stated earlier, no statistically signicant association was
reported during the 1990s. With utilization in the 1990s of ICSI (i.e.
ART using ICSI followed by traditional IVF), increased birth defects
began to be reported. With ICSI the frequency of sex chromosomal
abnormalities appears increased in prenatal samples (amniocen-
teses) as well as hypospadias [7,23,24]. Otherwise, anomaly rates
have appeared similar in ICSI and non-ICSI IVF, rendering overall
anomaly rates not signicantly different. The exception was Davies
Box 1
Pitfalls in experimental design and interpretation in assessing
birth defects in assisted reproductive technology (ART) offspring.
A. Ascertainment
Including or not including anomalies in stillborns.
Including or not including anomalies in terminated
fetuses.
Including or not including anomalies detectable on
ultrasound but not externally visible at birth.
Varying duration of surveillance.
Varying aggressiveness of surveillance.
B. Denition of anomalies
Minor anomalies versus major anomalies.
Failure to utilize pre-set denitions and check lists (i.e.
minimizing individual observer variability).
Determining when a minor anomaly becomes major
(e.g. requiring surgery).
C. Methodology
Failure to stratify analyses into isolated anomalies
versus multiple anomalies (syndromic) disorders.
Failure to recognize (exclude) heritable disorders
whose etiology is not plausibly ART-related.
Generalization of conclusions to a contemporary
population when data were derived in a prior era that
used now-outdated protocols.
Application of meta-analysis when studies are of dis-
similar design (unavoidable given lack of standardized
protocols).
et al. [13], showing increased risk only in ICSI offspring (OR: 1.47;
95% CI: 1.15e1.89).
ICSI couples have the confounder of having increased fre-
quencies of balanced translocations, not only in males (2.05%) but
female (1.38%) sex chromosomal abnormalities, and inversions
were also found not infrequently. Thus, two subfertile individuals
presumably constitute a single infertile couple. The frequency of
balanced translocations in men undergoing ICSI is higher in oligo-
spermic (4%) than in azoospermic men (1%). By analogy the same
principle may hold for single mutant genes, certain men under-
going ICSI having a pleiotropic mutation one component of which is
infertility and another predisposition to a structural anomaly.
Chromosomal abnormalities as assessed by preimplantation ge-
netic diagnosis are increased in ICSI-assisted embryos [32].
In conclusion, sex chromosomal abnormalities and hypospadias
are considered increased in ICSI compared with IVF alone.
8. Cryopreservation
Data are limited concerning whether cryopreservation affects
birth defects in liveborns, but at present data are reassuring. Loss of
cell(s) from cleavage stage biopsy is clearly deleterious to embryo
survival, as shown by decreased pregnancy rates for embryos that
had loss of one cell upon thawing [33]. Similarly, removal of more
than two cells fromcleavage stage embryos reduced the pregnancy
rate [34]. These statements are not applicable for trophectoderm
biopsy where a dozen or more cells are removed. Fortunately, loss
of cells at either cleavage stage or blastocyst stage does not result in
liveborn anomalies because the all or none effect exists. Embryos
surviving to live birth are not affected long term because when loss
of a single cell occurs at this stage of embryogenesis the function of
that cell can be subsumed by another because all cells are totipo-
tential. In their own cohort Hansen et al. [22] claimed to observe a
trend favoring fresh rather than frozen singleton IVF; however, this
was not found in twins or ICSI pregnancies. Davies et al. [13] found
no differences at all.
This author counsels that neither benet nor harm exists for
cryopreservation with respect to birth defects.
9. Is imprinting an explanation for increased birth defects?
The biological basis for increased frequency of birth defects in
ART is unclear. Heritable single gene or polygenic factors conferring
infertility could have a pleiotropic effect on offspring, but this
cannot explain the magnitude of the increase.
There also seems little reason to postulate de-novo chromo-
somal arrangements in vitro or accumulation of the requisite
multiple mutations at multiple loci that would constitute a poly-
genic/multifactorial explanation for isolated structural anomalies.
A plethora of variables in ART technology (vicissitudes of culture
media; ovulation stimulation) could, however, perturb embryonic
differentiation through altered gene expression.
Given lack of obvious explanations, the biological mechanism
most often invoked is perturbations of imprinting. A myriad of
growth factors are included, and these alone give any biologists
pause. A disturbance in methylation precluding scheduled tran-
scription and translation would yield differences in gene products.
Some genes would be expressed that were not originally scheduled
to do so, and the converse.
In 2002, de Braun et al. [35] reported that seven cases with the
autosomal dominant BeckwitheWiedeman syndrome (BWS) were
associated with either IVF alone (n 2) or ART (n 5). In BWS the
usual molecular basis lies inoverexpressionof thepaternal allele; the
maternal allele is ordinarily not expressed in BWS or normal chil-
dren. This usuallyoccurs as result of uniparental (paternal) disomy. In
ART-related cases studied by de Braun et al. [35], however, pertur-
bation was usually due not to increased paternal expression due to
uniparental disomy (UPD), but rather to unscheduled expression of
the maternal allele normally quiescent (hypomethylation of LIT1). A
second study from the UK generated not dissimilar ndings [36].
Other data have been reviewed by Vermeiden et al. [37].
Hansen et al. [14] gathered data in a cohort of pooled known
imprinting disorders: BWS, RusseleSilver syndrome, PradereWilli
syndrome, pseudohypoparathroidism, UPD for chromosome 14,
maternal hypomethylation syndrome. There were four occurrences
in ART cases (0.2%) versus 30 in non-ART (0.01%); OR was 8.21 (95%
CI: 2.81e24.00). On the other hand, a study of Danish registries, for
selecting a list of purported imprinting disorders, found no increase
among ART cases [38]. This argues against imprinting as an
omnibus explanation for increased OR in ART offspring.
In conclusion, invoking imprinting due to ART protocols is a
popular and plausible explanation for the imprinting disorders
listed alone. However, imprinting seems an implausible explana-
tion for isolated structural anomalies (e.g. facial clefts, cardiac).
Moreover, if ART perturbs imprinting only in selected disorders, the
absolute increase will be low. Given an OR of 8, there would be little
arithmetic (absolute) risk in ART offspring, compared with the
general population. Thus, if the sole explanation for the increased
rate of birth defects in ART offspring is imprinting, this is either
disturbed in disorders not yet appreciated or the phenomenon acts
in ways not appreciated (e.g. enhancers in non-coding regions).
10. Subfertility, infertility and birth defects
The likely cause of increased frequency of birth defects in ART is
underlying subfertility [39]. The sentinel observation is that the
frequency of birth defects in infertile couples who belatedly (>12
months unprotected intercourse) achieve pregnancy without ART
is almost the same as that in couples requiring ART [40,41]. In the
study by Zhu et al. [40], OR was 1.17 (95% CI: 1.0e1.36) for infertile
couples who conceived naturally while awaiting ART. Criteria
consisted of requiring more than 12 months of unprotected sex to
succeed. Davies et al. [13] likewise reported increased birth defects
in offspring of women who were subfertile but who conceived
without ART (OR: 1.29; 95% CI: 0.99e1.68). Of special note, women
who had prior offspring with ART but who conceived spontane-
ously in a later cycle had offspring with increased birth defects (OR:
1.25; 95% CI: 1.01e1.56).
In conclusion subfertility is a major factor in, if not the sole
explanation for, ART-related birth defects. Rimm et al. [41] believe
that almost all the increased risk observed in ART offspring reects
selection bias of subfertile couples, who, without medical assis-
tance, would never achieve pregnancy. Hansen et al. [19] disagree,
and indeed one must continue to ferret out potential technology-
related causes. However, like Pinborg et al. [9] and Rimm et al.
[41], this writer believes that selection bias seems more likely.
11. Conclusion
Assisted reproductive technology is associated with a small (OR:
1.3) increase in birth defects. This should be communicated to pa-
tients prior to undergoing ART. The counselor may also wish to
communicate concern over pitfalls in data used to derive these
opinions, but must realize that perfection cannot be achieved in
experimental design because the ideal control group cannot be
constructed. It is often instructive to remind all couples contem-
plating pregnancy that the baseline anomaly rate is 2e3%,
compared with 3e4% in ART. The consensus to be communicated is
that both traditional IVF as well as ICSI/IVF show the same
increased risk, except for increased sex chromosome abnormalities
and hypospadias in ICSI. Otherwise, no particular organ system
seems disproportionately affected. No additive risk seems to exist
in ART twins compared with non-ART twins, nor in embryos pre-
viously cryopreserved. Overall, the increased risk observed e irre-
spective of etiology e seems unlikely to dissuade couples from
attempting to have their own children.
None declared.
Funding sources
None.
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Review
A historical and practical review of rst trimester aneuploidy
screening
Melissa L. Russo
*
, Karin J. Blakemore
Maternal Fetal Medicine, McKusickeNathans Institute of Genetic Medicine, Johns Hopkins University, School of Medicine, Baltimore, MD, USA
Keywords:
Aneuploidy
First trimester screening
Free b-human chorionic gonadotropin
Maternal serum screening tests
Nuchal translucency measurement
Pregnancy-associated plasma protein A
s u m m a r y
There have been tremendous advancements over the past three decades in prenatal screening for
aneuploidy and we have changed our practice from screening by maternal age alone to combined rst
trimester screening and circulating cell-free fetal DNA. We currently use the nuchal translucency and
biochemical markers of free b-hCG and PAPP-A to determine the risk of fetal aneuploidy. The primary
goal is to identify higher risk women for fetal aneuploidy early in pregnancy and give them the option to
pursue invasive testing in a timely manner if desired.
1. Introduction
The past 30 years have produced numerous discoveries and
advances in prenatal screening for cytogenetic disorders in the
fetus: ultrasound imaging, maternal serum biochemical markers,
and isolation of cell free fetal DNA in maternal serum. These ad-
vancements are responsible for the options currently available for
prenatal screening for aneuploidy in the rst trimester. The pur-
pose of this review article is historical and practical. Its historical
aspect chronicles the origins of and innovations in rst trimester
screening, demonstrating how current screening for fetal aneu-
ploidy has come into practice. On a practical side, it will highlight
the available options for rst trimester screening, indications for
screening, rst trimester screening in twin pregnancies and discuss
the additional knowledge that can be obtained from a rst
trimester combined screen.
2. Origins of prenatal screening for fetal aneuploidy
Prior to the 1980s, the primary method to identify women at
risk for aneuploidy was based on the concept of increased risk with
advanced maternal age. Dr Lionel Penrose was the rst to recognize
this concept in the 1930s when he observed that there was a sig-
nicant association between increasing maternal age and birth of a
Down syndrome child [1]. With the advent of cytogenetic analysis
on cultured amniocytes in the 1970s, all women aged >36 years
were offered amniocentesis to diagnose potential fetal aneuploidy.
Maternal age was a poor screening test in isolation because it only
identied 25e30% of fetal aneuploidy.
The rst recommendation for prenatal aneuploidy screening in
the general population stemmed fromthe observation by Merkatz
et al. [2] in 1984 that maternal serum a-fetoprotein (MSAFP) in
the second trimester was signicantly lower in a woman with a
trisomy 18 fetus. Simultaneously, MSAFP was suggested by Cuckle
[3] as a screening test for Down syndrome in the general popu-
lation. This study modeled a mathematical algorithm that com-
bined maternal age and MSAFP in the second trimester to detect
40% of cases of Down syndrome with a false-positive rate of 6.8%.
This led to the rst protocols for fetal aneuploidy screening in the
general population across all maternal ages. After observations
with MSAFP, altered maternal serum levels in affected pregnan-
cies were observed in other analytes: free b-human chorionic
gonadotropin (b-hCG), inhibin A and unconjugated estriol (uE
3
)
[4e6]. Wald et al. [7] combined the maternal serum markers of b-
hCG, a-fetoprotein (AFP) and uE
3
with maternal age in the triple
screen in 1988. Overall, the mean levels of these analytes were
expressed as a multiple of the expected value for gestational age
based on a logelinear regression in the controls, multiples of the
median (MoM). The mean for the analytes b-hCG, AFP and uE
3
in a
second trimester pregnancy affected with Down syndrome were
2.1, 0.7 and 0.7 MoM respectively. Looking across all maternal
ages, the triple screen detection rate for Down syndrome in the
second trimester was 60% with a false-positive rate of 5%, essen-
tially doubling the detection rate fromage alone. Haddowet al. [8]
examined dimeric inhibin A as yet another marker to be added to
the second trimester screening prole and this was later dubbed
* Corresponding author. Address: Maternal Fetal Medicine, McKusickeNathans
Institute of Genetic Medicine, Johns Hopkins University, School of Medicine, 600
North Wolfe Street, Phipps Building, Suite 228, Baltimore, MD 21287, USA. Tel.: 1
410 502 9893; fax: 1 410 614 8305.
E-mail address: mrusso5@jhmi.edu (M.L. Russo).
the quadruple screen. Using age and the four maternal serum
analytes, the detection rate for Down syndrome was 75% at a
false-positive rate (FPR) of 5%.
3. First trimester maternal serum markers of free b-hCG and
pregnancy-associated plasma protein A (PAPP-A)
The goals for improving prenatal screening for aneuploidy
following the advent of the triple and quadruple screen were to
achieve a higher detection rate, a lower FPR, reassurance to patients
with a low risk and to offer diagnostic testing as early as possible.
Thus, with the development of a maternal serum screening test for
the general population, a logical next question was the feasibility of
implementing prenatal screening earlier in pregnancy. Chorionic
villus sampling (CVS) had been introduced in the 1980s, an invasive
procedure that allowed for prenatal diagnosis in the rst trimester,
adding impetus to move screening to an earlier gestational age [9].
The clear advantages of a screening test in the rst trimester
included earlier reassurance to low risk patients, more time to
consider options for diagnosis, and the possibility of earlier, safer
termination in affected pregnancies.
Spencer et al. [10] published their initial ndings of free b-
hCG as a marker for trisomies 21 and 18 in the rst trimester
and showed that the MoM value of free b-hCG in trisomy 21 was
signicantly greater and the median value in trisomy 18 was
signicantly lower than that in the unaffected controls. The idea
of using free b-hCG was rst proposed by Bogart et al. [11] who
compared the ability of free versus total hCG to detect chro-
mosomally abnormal fetuses in the second trimester. Based on
Bogarts work, Spencer et al. had also examined free b-hCG in
the second trimester. Compared with total hCG, free b-hCG was
a better marker for detection of trisomy 21. Macri and Spencer
[12] then expanded on their earlier paper using free b-hCG as an
analyte from 9 to 13 weeks of gestation and showed that free b-
hCG was signicantly elevated (2.20 MoM) in trisomy 21 preg-
nancies. They concluded that this maternal serum analyte would
serve as a good marker for Down syndrome in the rst trimester.
Around this time, it was discovered that PAPP-A was lower in
pregnancies with fetal aneuploidy. Brambati et al. [13] were the
rst to publish this nding in 13 cases of trisomy 21 at 8e12 weeks
of gestation. The PAPP-A levels were 5th percentile. Wald et al.
[14] expanded on these ndings, concluding that PAPP-A was a
useful marker for trisomy 21 in the rst trimester (0.23 MoM).
Muller et al. [15] further established PAPP-A as a potential useful
marker for trisomy 21 in the rst trimester with their study
examining PAPP-A levels in blood samples originally collected for
toxoplasmosis testing in France (median PAPP-A value for trisomy
21 was 0.42 MoM).
4. Fetal nuchal translucency: a novel approach to screen for
aneuploidy
The British physician, John Langdon Down, was the rst to
describe Down syndrome in 1866 [16]. He noted: The skin has a
slight dirty yellowish texture and is decient in elasticity, giving the
impression of being too large for the body.
In the 1990s, Nicolaides et al. [17] pondered the observation of
the fullness of the neck in Down syndrome neonates and the as-
sociation of nuchal edema/cystic hygroma on second trimester ul-
trasound in fetuses with chromosomal abnormalities. Nicolaides
hypothesized that increased nuchal thickness on ultrasound in the
rst trimester could be a marker for fetal aneuploidy. In a pro-
spective study, 827 pregnant women who chose to have diagnostic
testing underwent transabdominal ultrasound at 10e14 weeks of
gestation to evaluate the uid behind the neck in their fetuses.
Visualizing a sagittal section of the crownerump length, the
maximum thickness of subcutaneous translucency between the
skin and soft tissue overlying the cervical spine was measured. In
the 51 fetuses with a nuchal translucency thickness of 3e8 mm, the
incidence of chromosomal abnormalities was 35%. In the fetuses
with smaller measurements (n 776), by contrast, 1% had chro-
mosomal abnormalities. He also noted that the abnormal uid
collection had, for the most part, resolved by the second trimester.
In this study, Nicolaides reported that an increased nuchal trans-
lucency was associated with an increased risk for chromosomal
defects. He introduced the term nuchal translucency (NT) into the
vocabulary of genetics counselors, obstetricians, ultrasound tech-
nicians, radiologists, maternal fetal medicine specialists and aca-
demic medicine. This article transformed the landscape for prenatal
screening.
Based on Nicolaides study, another prospective study by
Brambati et al. [18] was performed to evaluate the technical and
practical aspects of a screening program in the general population.
In women undergoing CVS between 8 and 13 weeks of gestation,
the NT was measured for maximum thickness. These investigators
were among the rst to introduce a standardized protocol: (i) two
different observers scrutinize the ultrasonographic images of the
posterior fetal contour in sagittal plane with the requirement to
distinguish between amnion and fetal skin; and (ii) an abnormal
cut-off value of 3 mm. In 70 fetuses with NT above the cut-off,
18.6% had chromosomal disorders versus 1.7% in the normal NT
group. Brambati et al. afrmed that the NT increased with
increasing gestational age and that an increased NT was associated
with chromosomally abnormal fetuses; he also addressed the need
for quality control and standardization with measurement of NT
screening.
Pandyas and Nicolaides group examined 1015 fetuses with NTs
3 mm and found that the incidence of chromosomal abnormal-
ities, namely, trisomy 21, 18 and 13, was signicantly associated
with increased fetal NT and additively with maternal age at 10e14
weeks of gestation [19]. Another study by Pandya et al. [20]
demonstrated that fetal NT increases with increasing crowne
rump length, and that the likelihood of trisomy 21 varies with the
degree by which a given NT deviates from the normal median at a
given crownerump length. Instead of having a specic numerical
cut-off for the NT, they used the cut-off of >95th percentile above
the normal median and detected 77% of fetuses with trisomy 21 and
78% of other chromosomal abnormalities using only maternal age
plus NT. For quality control, a subgroup analysis showed good
reproducibility of NT measurements when ultrasound examina-
tions were performed by different sonographers, all trained in the
same fashion.
Snijders et al. [21] published one of the most comprehensive
studies on fetal aneuploidy screening with maternal age and nuchal
translucency from the Fetal Medicine Foundation First Trimester
Screening Group. This trial, conducted at 22 centers, examined
96 127 cases of rst trimester NT screening with known genetic
outcomes. The median maternal age for the study group was 31
years; there was a preponderance of older-age women in this study.
The NT was above the 95th percentile for a given crownerump
length in 71.8% of 326 trisomy 21 pregnancies, and in 70.5% of 325
other chromosomally abnormal pregnancies versus 4.4% of normal
pregnancies. With the addition of maternal age, a risk cut-off of 1 in
300 or higher was found in 82.2% of trisomy 21 pregnancies, 77.8%
of other chromosomally abnormal pregnancies and 8.3% of normal
pregnancies.
Another important contribution of this study was the estab-
lishment of criteria by the Fetal Medicine Foundation to achieve a
uniform 10e14-week scan among many different operators and
institutions [21]. These criteria include: a proper sagittal view;
M.L. Russo, K.J. Blakemore / Seminars in Fetal & Neonatal Medicine 19 (2014) 183e187 184
magnication (fetus occupies at least 75% of the image); distinction
between fetal skin and amnion; and reporting the maximum
measurement obtained. The Nuchal Translucency Education and
Quality Review program (NTQR) was developed in 2004 with
criteria from the Society for Maternal Fetal Medicine similar to that
of the Fetal Medicine Foundation for nuchal translucency
screening, and these criteria have since been implemented in North
America and Canada.
Another study on quality control by Whitlow et al. [22] exam-
ined howthe head in an extended or exed position distorts the NT
measurement. From this publication came the recommendation to
measure the fetal neck in a neutral position, as this is the most
reproducible.
5. Combination of biochemistry and nuchal translucency in
the rst trimester
Brambati et al. [23] rst reported the combination of free b-hCG
and PAPP-A, attaining a detection rate of 78.9% for trisomy 21 in a
small series of 13 cases of trisomy 21 and 89 unaffected controls
using just these maternal serum analytes, plus maternal age. Noble
et al. [24] were among the rst to combine biochemistry and the NT
plus age, nding that free b-hCG and NT were two independent
parameters; thus their likelihood ratios could be combined. This
combined screening trial had a detection rate for trisomy 21 of 85%
at a false-positive rate (FPR) of 5%.
Two years later, Orlandi et al. [25] published one of the rst
studies that prospectively assessed the combination of maternal
age, biochemical screening tests of free b-hCG and PAPP-A, and NT
to determine risk for fetal aneuploidy in the rst trimester. The
free b-hCG and PAPP-A values were divided by respective gesta-
tional day-specic median levels to determine the MoM for each
analyte. Trisomy 21 pregnancies were associated with elevated
free b-hCG and low PAPP-A, and trisomy 18 pregnancies were
associated with low free b-hCG and PAPP-A. The likelihood ratios
for each analyte, the NT, and the patients prior age-related risk
were multiplied together to determine a nal risk ratio, and high
risk was determined to be 1 in 380. With this combination, the
detection rate for trisomy 21 was 87% with a FPR of 5% whereas
the detection rate for trisomy 18 was 76% with FPR of 1%. Orlandi
et al.s study also demonstrated the utility of dried blood tech-
nology for specimen collection. The use of the dried blood spot
simplied and allowed for greater exibility in collection, shipping
and processing.
Spencer et al. [26] examined the combined rst trimester
screening for trisomy 21 in a retrospective analysis, and expedited
the process to a one-stop clinic employing new technology that
allowed for results of biochemical analysis within 30 min of
obtaining a blood sample. This technology resulted in precise
measurements with good reproducibility and allowed for com-
plete early fetal assessment in one visit. This study also demon-
strated that each marker was independent; when combined they
obtained a detection rate for trisomy 21 of 89% with FPR of 5%.
Spencer et al. re-conrmed their conclusions from earlier studies
that risk algorithms needed to be created that were adjusted for
gestational age because detection rates varied at different gesta-
tional ages.
Krantz et al. [27] reported on the rst trimester combined
screenings ability to detect trisomy 18 in addition to trisomy 21.
They used the dried blood spot technology previously described,
and a standardized protocol outlined by the Fetal Medicine Foun-
dation for nuchal translucency measurements. Krantz et al. showed
a detection rate for trisomy 21 of 87.5% with FPR of 4.5% in women
aged <35 years and detection rate of 92% with FPR of 14.3% in
women aged 35 years. For trisomy 18, there was a detection rate
of 100% with FPR of 0.4e1.4% for women aged <35 and 35 years
respectively.
Wapner et al. [28] published the rst multicenter trial in the USA
for the combined rst trimester screening for trisomies 21 and 18.
This study, the BUN trial, demonstrated that across multiple cen-
ters this screening method was reproducible and appropriate for
use in clinical practice. The detection rate for trisomy 21 was 78.7%
with FPR of 5%. The detection rate for trisomy 18 was 90.9% with
FPR of 2%.
6. Comparison trials of different screening methods
Once it was demonstrated that combined rst trimester
screening was feasible and applicable to the general population,
the next question was the comparison of combined rst
trimester screening to second trimester screening to determine
whether one test was superior. Numerous studies showed that
the detection rates for trisomy 21 in the rst trimester, with a
xed FPR of 5%, ranged from 76% to 91% and this was thought to
be better than the detection rates in the second trimester [25e
29]. In any comparison of rst and second trimester screening,
however, it is important to recognize that the overestimate in
detection rates in the rst trimester is because the incidence of
affected pregnancies is higher and a certain percentage of
pregnancies detected by early prenatal screening will result in
spontaneous fetal loss as gestation advances. A study by Dunstan
and Nix [30], for example, provides a methodology to compare
detection rates between the rst and second trimester screening
tests for trisomy 21 by taking into account fetal loss rates in the
rst trimester and incorporating this information into the com-
parison. Dunstan and Nix summarized that rst trimester
screening could only be determined to be superior to second
trimester screening if the aneuploidy detection rate was at least
8.3% higher.
During this time, the integrated screen was introduced by Wald
et al. [31] and used markers from the rst and second trimester to
provide a single estimate of aneuploidy risk revealed during the
second trimester. The detection rate for trisomy 21 was 85% with a
much lower FPR of 0.9%.
Two large, multicenter, prospective trials in the UK followed:
the Serum, Urine and Ultrasound Screening Study (SURUSS), and
in the USA, the First-Trimester and Second-Trimester Screening or
Both for Down Syndrome (FASTER) trial. These trials sought to
compare all of the different available screening methods and
determine the most effective and safest method for screening for
trisomy 21. In the SURUSS trial, the most effective screening test
was the integrated test with a detection rate of 85% and a FPR of
0.9% [32]. For the same detection rate of 85%, the serum inte-
grated, combined rst trimester screen and quadruple second
trimester screen had FPRs of 3.9%, 4.3% and 6.2% respectively. The
FASTER trial had similar results. At a xed detection rate of 85%,
the FPRs of the rst trimester combined, serum integrated, fully
integrated, and second trimester quadruple screen were, respec-
tively, 4.8%, 4.4%, 0.8% and 7.3% [33]. From these trials, it was
concluded that the choice of screening test is individualized and
depends on the womans gestational age, whether she desires a
denitive diagnosis in the rst trimester versus the best overall
screening test.
7. History of cell-free fetal DNA from maternal blood in
detection of aneuploidy
Non-invasive aneuploidy screening with cell-free fetal DNA
from maternal plasma is a relatively novel clinical methodology;
however, the concept of isolating fetal cells and fetal cell-free DNA
from maternal blood was rst discovered more than 50 years ago
[34]. Preliminary endeavors towards non-invasive prenatal
screening were focused on detection and isolation of intact fetal
cells from the maternal blood using uorescence-activated cell
sorting [35,36]. One study used isolated, nucleated fetal red blood
cells via uorescent in-situ hybridization (FISH) analysis to detect
trisomy 18 [37]. A large, multicenter trial by the National Institute of
Child Health and Human Development (NIFTY) used intact fetal
nucleated red blood cells with cell sorting, FISH and polymerase
chain reaction to detect 74% of cases of fetal aneuploidy with FPR of
0.6e4% [38]. This method of aneuploidy screening was comparable
with prenatal serumscreening, but had higher specicity. Before its
introduction into the clinical arena, further advancement was
deemed necessary.
Cell-free fetal DNA in maternal plasma was reported by Lo
et al. [39] in 1997. This discovery paved the way for subsequent
developments of non-invasive screening for aneuploidy. Circu-
lating cell-free DNA comprises 3e6% of total cell-free maternal
DNA. It is derived primarily from the placenta and is cleared
from maternal blood within hours after delivery. Cell-free fetal
DNA was rst used for gender determination with X-linked
disorders and for fetal Rh(D) genotyping with Rh-negative
pregnant women.
In 2008, massive parallel sequencing revolutionized the detec-
tion of cell-free fetal DNA [40e42]. With this shotgun sequencing-
based approach, the number of cell-free DNA fragments was
quantied and fetal aneuploidy was detectable. This technological
advance led to several large trials to explore the validity and
feasibility of aneuploidy screening with cell-free fetal DNA [43e46].
Each of these clinical trials had slightly different methods for
analysis of cell-free fetal DNA fractions. After these large clinical
trials, cell-free fetal DNA screening was implemented into clinical
practice for women at risk for fetal aneuploidy.
8. Practical application and other aspects of rst trimester
screening for aneuploidy
There are many options currently available for aneuploidy
screening: the combined rst trimester screen, quadruple screen,
integrated screen or sequential screen. Additionally, for pregnan-
cies at high risk for aneuploidy, cell-free fetal DNA testing is now
being offered. It is important to recognize that these tests are not
diagnostic and each test has associated false negatives and false
positives. If a patient wants to have a denitive diagnosis of
aneuploidy, chorionic villus sampling or amniocentesis are
currently the only available options.
A secondary marker in the rst trimester screen for aneu-
ploidy is the presence of the nasal bone. The incorporation of the
nasal bone into the rst trimester screen improves the detection
rate for trisomy 21 with xed FPR, or retains a 90% detection rate
with a simultaneous 10-fold decrease in the FPR from 5% to 0.5%
[47].
First trimester screening in twin pregnancies is a possible and
practical option. There are several factors to take into consider-
ation with twins. The NTs can be used to determine an individual
risk to each fetus; however, serum markers can only be used to
obtain a pregnancy-specic risk. If there is a positive screen
result, then invasive testing is generally offered for each fetus.
In addition to its use as a screening tool for aneuploidy, the
rst trimester combined screen has several other applications.
The rst trimester screen can accurately establish estimated date
of connement and chorionicity in multiple gestations, which is
very important for obstetrical management. The rst trimester
evaluation of the nuchal translucency is, moreover, a valued
examination to screen for congenital cardiac defects,
diaphragmatic hernia, some skeletal dysplasias and other ge-
netic conditions. In the future, rst trimester screening may
evolve to incorporate the newer technology of cell-free fetal DNA
along with maternal serum analytes and NT measurement by
ultrasound.
9. Conclusions
First trimester screening for aneuploidy is a valuable tool for the
obstetrician and indications and methods of screening have
evolved over the last quarter century. The primary goal of rst
trimester screen is to identify higher risk women for fetal aneu-
ploidy and give them the option to pursue diagnostic testing in a
timely manner if desired.
None declared.
Funding sources
None.
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Review
Selective serotonin reuptake inhibitors in human pregnancy: On the
way to resolving the controversy
Asher Ornoy
a, b,
*
, Gideon Koren
b, c
a
Hebrew University Hadassah Medical School, Jerusalem, Israel
b
Israeli Teratology Information Service, Israel Ministry of Health, Jerusalem, Israel
c
Motherisk Program, Division of Clinical Pharmacology/Toxicology, Department of Pediatrics, Hospital for Sick Children, Toronto, Canada
Keywords:
Major anomalies
Neonatal effects
Persistent pulmonary hypertension
Neurodevelopmental effects
Selective serotonin reuptake inhibitors
(SSRIs)
s u m m a r y
There has been an increase in the use of selective serotonin reuptake inhibitors (SSRIs) during pregnancy.
However, in the last 10 years, in spite of a vast literature regarding use in pregnancy there seems to be
some confusion as to the possible risk of these drugs, especially related to cardiovascular anomalies. In
addition, there are data on developmental follow-up studies that raise the question of possible slight
developmental and neurobehavioral problems. The purpose of the present review is therefore to criti-
cally summarize the current evidence for the risk/benet analysis of SSRI use in human pregnancy.
Although most studies have not shown an increase in the overall risk of major malformations, several
have suggested that the use of SSRIs may be associated with a small increased risk for cardiovascular
malformations. However, newcompelling evidence shows that this apparent increased risk occurs also in
women with untreated depression, highlighting the probable ascertainment bias involved in many of
these studies. Persistent pulmonary hypertension of the newborn (PPHN) has also been described with
an absolute risk of <1%; however, here too, higher rates were described among offspring of women with
untreated depression. Poor neonatal adaptation has been described in up to 30% of neonates exposed to
SSRIs late in pregnancy. Of the few postnatal developmental follow-up studies, there are no signicant
developmental problems. The literature on SSRIs in pregnancy is somewhat confusing but when ana-
lysing all prospective cohort data there seems to be no demonstrable increase in the rate of major
anomalies or developmental disorders. When evaluating the risk/benet ratio of SSRI treatment in
pregnancy, the risk associated with treatment discontinuation e e.g. higher frequency of relapse,
increased risk of preterm delivery and postpartum depression e appear to outweigh the potential, un-
proven risks of treatment. Moreover, maternal depression may negatively affect the childs development,
emphasizing the importance of prevention by appropriate treatment during pregnancy with the least
minimal effective dose.
1. Introduction
Selective serotonin reuptake inhibitors (SSRIs) are widely pre-
scribed for the treatment of depression, anxiety and other disor-
ders. The prevalence rates of depression during pregnancy have
been estimated as 7.4%, 12.8% and 12.0%, for the rst, second and
third trimesters, respectively, and up to 20% of women may have a
depressive episode in the rst postpartum year [1,2]. Indeed, the
frequency of the reported use of antidepressants in pregnancy
increased from 2.5% in 1998 to >8% in 2006, as reported by the
American National Birth Defects Study [2,3]. One important group
of drugs comprises the SSRIs, rst introduced for clinical use in the
1980s. They have better tolerability and safety compared with rst
generation antidepressants, e.g. tricyclic antidepressants, and are
safer in overdose exposures. They exert their effects by inhibiting
the presynaptic plasma membrane serotonin transporter which
mediates the reuptake of serotonin into the presynaptic terminal.
Thus, treatment with an SSRI initially blocks reuptake and results in
enhanced and prolonged serotonergic neurotransmission due to an
increase in synaptic dopamine concentrations [4,5]. All SSRIs,
including those with an additional effect on norepinephrine reup-
take (SNRIs), share a similar mechanism of action despite having
different chemical structures. SSRI use in pregnancy has increased
over the years and in the USA is w6% [4,5]. SSRIs readily cross the
* Corresponding author. Address: Israeli Teratology Information Service, Israel
Ministry of Health and Laboratory of Teratology, Department of Anatomy and Cell
Biology, Hebrew University Hadassah Medical School, PO Box 12272, Jerusalem
91120, Israel. Tel./fax: 972 2675 8430.
E-mail address: asher.ornoy@mail.huji.ac.il (A. Ornoy).
human placenta [6,7] and are also secreted, although in relatively
small amounts, in human milk. SSRIs are the most studied anti-
depressants in pregnancy with >33 000 pregnancies reported in
different studies [4]. Nevertheless there are still conicting views
on the risks of these drugs during pregnancy. When judging the use
of these drugs in pregnancy one should consider the possible
overall effects on the fetus in relation to risks of untreated
depression or anxiety for the mother and offspring.
The purpose of the present review is to summarize and evaluate
the risk/benet analysis of SSRI use during human pregnancy,
including neonatal effects and neurodevelopmental outcome with
special emphasis on prospective cohort studies.
2. Human studies in pregnancy: Major congenital anomalies
and cardiac defects
There are numerous prospective, retrospective and caseecon-
trol studies on the possible effects of SSRI exposure in pregnancy
and in the neonatal period. There seems to be no specic syndrome
or any specic type of malformation related to any of the SSRIs.
However, a possible association between the use of several SSRIs in
pregnancy and cardiac anomalies [8e15] as well as a fewrare major
malformations (neural tube defects, craniosynostosis, omphalocele,
cystic kidney or hypospadias) [15e18] have been described in some
studies. In the positive studies, there was some dominance of
ventricular septal defects and right ventricular outow tract
obstruction defects. Paroxetine was the drug involved with most of
these associations. Many of the prospective studies were under-
powered for associations between exposure and specic malfor-
mations, and retrospective studies had serious potential biases. The
relative risk in positive studies was generally <2 and the lower
range of the 95% condence interval was often close to one. Many of
the studies with positive ndings on SSRIs in pregnancy have been
prescription registries, and women may not have actually taken the
drugs, as shown clearly in the Swedish registry [19]. Exact timing of
exposure during sensitive periods is often problematic, although
exposure preceded the outcome. The ndings in regard to the type
of cardiac malformations are inconsistent although ventricular
septal defect and related anomalies prevail. Results even differ in
studies from the same database published at different time-points.
On the other hand, many other studies have not shown an in-
crease in the overall risk of major malformations [20e28] or an
association with cardiac anomalies. Although the number of
negative studies seem to be higher with larger numbers of exposed
women, it may be difcult to judge, at least with regards to the
possible increased rate of cardiac anomalies. One should also
remember that in most cases the possible contribution to the un-
derlying maternal disease was not controlled for [4,29]. Hence,
rather than reporting the data of individual studies it may be more
accurate to describe the results of the meta-analyses and of
different reviews published on this topic.
2.1. Meta-analyses
Several meta-analyses on paroxetine exposure in pregnancy and
cardiovascular malformations have demonstrated an increased rate
of cardiac anomalies but their signicance is not simple [30e32]. In
the meta-analysis published by Bar-Oz et al. [30] summarizing
studies from 1985 to 2006, rst trimester paroxetine exposure was
associated with a signicant increase in the risk of cardiac anom-
alies. The authors compared the outcome of 2575 pregnancies us-
ing paroxetine with 14 026 non-exposed pregnancies and found an
overall odds ratio of 1.72. However, they also found that women
using antidepressants in pregnancy had signicantly more ultra-
sound examinations in pregnancy and postnatal echocardiograms
compared with women who did not. Hence, detection bias was
suggested as a contributing factor to the observed risk of cardio-
vascular malformations with paroxetine. It is therefore difcult to
conclude from this publication that SSRIs increased the rate of
congenital anomalies. Wurst et al. [31] in their meta-analysis found
an increased risk for combined cardiac defects and aggregated
congenital defects with rst trimester paroxetine use, analysing 20
studies that met the inclusion criteria. They based some of their
results on the data previously published by Bar Oz et al. in their
meta-analysis. The overall prevalence odds ratio was 1.46 for car-
diac defects and 1.24 for aggregated congenital anomalies. Hence,
the risk seems to be low. Moreover, opposing commentaries on this
meta-analysis were recently published stating that the meta-
analysis did not include several important, negative studies [32,33].
In contrast to these publications, there are several meta-
analyses where no increase in cardiac anomalies was observed
following exposure to different SSRIs including paroxetine [34e37].
In the meta-analysis of three caseecontrol studies and six cohort
studies including a large number of paroxetine exposures pub-
lished by OBrien et al. [34], no increased risk of congenital mal-
formations was associated with paroxetine. Cardiac malformation
rates were similar and within population norms. A meta-analysis
discussing the effects of uoxetine in pregnancy did not show any
increase in major congenital anomalies [35]; in another meta-
analysis of seven studies on the effects of older and newer SSRIs
the relative risk for major malformation was 1.01 [36]. A similar
meta-analysis of different SSRIs examining the studies published
from 1990 to 2005 also did not nd any increased risk of major
congenital anomalies [37].
The denition of cardiovascular malformations varied among
studies, some including small septal defects, whereas others
excluded them. The inconsistency across these studies may be
explained by differences in study design, by confounding factors,
e.g. maternal underlying psychiatric disorder, co-administered
medications, lifestyle factors (smoking, drinking), or maternal
obesity and diabetes.
3. New evidence towards resolution of the controversy
A recent, very large national population-based study from
Denmark seems to have largely resolved this controversy [38]. The
authors analysed the association between SSRI use and major
congenital malformations, with focus on cardiac defects. Impor-
tantly, unlike scores of other studies previously mentioned, the
authors also identied a group of women with depression who
avoided taking their SSRIs in pregnancy. Using the Danish Medical
Birth Registry, the authors identied 848 786 pregnancies, 4183
having been exposed to an SSRI throughout the rst trimester, and
806 pregnancies of depressed women who had avoided use of
these drugs during pregnancy. Risks of cardiac malformations were
similar for pregnancies exposed to an SSRI throughout the rst
trimester [adjusted odds ratio (OR): 2.01; 95% condence interval
(CI): 1.60e2.53], and for pregnancies with paused SSRI treatment
during pregnancy (1.85; 1.07e3.20) (P 0.94). The authors found
similar increased risks of specic cardiac malformations for the
individual SSRIs. Just as important, the authors found no dosee
response association [38].
This study has documented that the apparent association be-
tween SSRI use and cardiac malformations may be confounded by
indications. Specically, the authors quoted research published in
2007 showing that women with depression and anxiety have a 2e
3-fold higher likelihood of having ultrasound and echocardiogram
in their babies than healthy women, and, hence, much higher
likelihood of detecting cardiac anomalies [29]. Moreover, women
with depression and anxiety are signicantly more likely than
A. Ornoy, G. Koren / Seminars in Fetal & Neonatal Medicine 19 (2014) 188e194 189
healthy women to attend emergency departments with their in-
fants, where there may be more opportunity to detect a heart
murmur and follow it up [29]. This source of ascertainment bias
has not been sought or addressed in any previous study. Moreover,
this bias is further augmented by the widely publicized news of
SSRIs causing malformations, facilitating more babies of women
on SSRI to be examined but with no similar urge to test children of
healthy women.
These new data have major implications for pregnant women
with depression, as large numbers of them discontinue much-
needed antidepressants, with risk of exacerbation of symptoms,
hospitalization, suicide attempts and postpartum depression.
4. Animal studies
Reproductive and teratogenicity studies with SSRIs were carried
out on mice, rats, rabbits, or dogs administered uoxetine paroxe-
tine or sertraline during organogenesis [39e41]. These studies did
not show an increased risk of teratogenesis even with high doses
that caused maternal toxicity, although there was some decrease in
neonatal survival and growth [41]. This is in contrast with effects
shown in rat and mouse whole embryo cultures [42e44] or cardiac
myocyte cultures [45]. For example, sertraline, uoxetine and
amitriptyline produced in cultured mouse embryos craniofacial
malformations [42]. Paroxetine (1 mM) was shown to decrease
serotonin-mediated proliferation of dissociated rat embryonic
cardiac myocytes [44]. Fluoxetine was found to adversely affect cell
viability and differentiation to cardiomyocytes at higher concen-
trations than achieved clinically in a dose-dependent manner using
mouse embryonic stem cell system [46]. In contrast with these in-
vitro studies, in-vivo animal studies have not supported an asso-
ciation between in-utero exposure to SSRIs and major anomalies
[39e41,46] or any interference with fetal hormonal status or
circadian rhythms [47]. Moreover, the lack of animal teratogenicity
of SSRIs in spite of the fact that serotonin plays an important role in
the prenatal development of the brain additionally supports the
notion that SSRIs are not human teratogens.
In summary, despite having more than 33 000 reported preg-
nancy outcomes after prenatal exposure to various SSRIs the
overall scientic evidence has not fullled the criteria for proof of
human teratogenicity of SSRIs, especially the seven criteria set by
Shepard [48]. Whereas the differences in the design of these
studies and the conicting results are confusing, new data strongly
suggest that the over-representation of cardiac anomalies may
reect an uncontrolled ascertainment bias. Additional studies have
merit only if they can include a comparison group of untreated
depressed women. In our view, the current data do not support
teratogenicity of SSRIs.
4.1. Clinical practice
There seems to be no increase in the rate of major congenital
anomalies in offspring of mothers using SSRIs during pregnancy.
Hence, there does not seem to be a clear indication to perform fetal
echocardiography in search for congenital heart defects.
5. Miscarriage, intrauterine growth restriction (IUGR),
preterm delivery, and stillbirth
Although most studies reported on the rate of major anomalies
following SSRI exposure, other possible pregnancy complications
were also presented. There was a slight increase in the miscarriage
risk in two meta-analyses [49e51]. However, in the prospective
cohort studies that were reviewed, corrections were not performed
for the effect of earlier gestational age at contact e which is an
important confounder e and the possible effects of the underlying
maternal disease were not ascertained [4,50]. SSRIs alone,
serotonin-norepinephrine reuptake inhibitors alone, and combined
use of antidepressants were all associated with an increase in
miscarriage, and the SSRIs paroxetine and venlafaxine were mainly
associated with increased miscarriage risk.
6. Prematurity and reduced birth weight
In a Finnish study, there was no increase in the rate of preterm
delivery, small for gestational age (SGA) or low birth weight [25].
The risk of both low birth weight and preterm delivery was
increased in infants who were born to mothers who had received
SSRI therapy [27]. Infants exposed to SSRIs had shorter gestations
and lower birth weights than non-exposed infants [51]. The
increased risk of low birth weight remained signicant, even when
maternal illness severity was accounted for. The adjusted OR for
preterm delivery was doubled in SSRI-exposed women compared
with two groups of women who had not used SSRIs during preg-
nancy, one with psychiatric history, and another without [52]. In
another study, the risk of preterm delivery was not signicantly
increased among SSRI users, but the risk of SGA offspring was
increased among women who continued SSRI use beyond the rst
trimester [53]. In a study from the Qubec Pregnancy Registry, no
association was found between SSRIs and the risk of SGA regardless
of trimester of exposure [54]. In other studies, there was an
increased risk for preterm delivery only among women exposed to
SSRIs in the second or third trimesters [15,54,55], or to benzodi-
azepines [56] with no increased risk for lowbirth weight or SGA. As
stated previously, the underlying psychiatric disorder is a potential
confounder in most of these studies.
In summary, associations were found in some studies between
the use of SSRIs during pregnancy and risk of miscarriage, IUGR or
preterm delivery. Most of these studies are potentially confounded
by the gestational age at initial contact and the underlying psy-
chiatric disorder.
Due to the possibility that SSRIs may increase the rate of
spontaneous abortions, prematurity or IUGR, fetal growth and
well-being should be carefully monitored in the second half of
pregnancy.
7. Neonatal effects
Poor neonatal adaptation syndrome has been described initially
following prenatal exposure to various SSRIs, in the third trimester
of pregnancy in up to 30% of pregnancies [57e63]. Neonatal with-
drawal syndromes associated with SSRIs are characterized by irri-
tability, abnormal crying, tremor, respiratory distress, tachypnoea,
jitteriness, lethargy, poor tone or colour and rarely convulsions.
Most symptoms are mild and transient. These symptoms are
similar to those observed following late exposure to many psy-
chotropic drugs, opioids and alcohol.
8. Persistent pulmonary hypertension of the newborn
Several studies have suggested an association between maternal
use of SSRIs late in pregnancy and an increased risk of persistent
pulmonary hypertension of the newborn (PPHN) [15,64e67]. The
absolute risk of PPHN was <1%. None of the studies to date
described neonatal death from PPHN associated with SSRI. This is
different fromthe 10e15% mortality rate of PPHNfromother causes
(diaphragmatic hernia, cardiac malformations, aspiration). Other
studies, possibly underpowered, did not nd such an association
[66] and in a recent study it was found that PPHN was associated
with the mode of delivery, specically caesarean delivery prior to
the onset of labour, but not with SSRI use in the second half of
pregnancy [67]. A recent Norwegian study found that untreated
depression increased the risk of PPHN.
In summary, considering the conicting results it is obvious that
if the use of SSRIs in pregnancy is associated with PPHN, the ab-
solute risk is <1% for PPHN, and should not be a reason to dis-
continue SSRI in late pregnancy.
The newborn infant of mothers using SSRIs during the second
half of pregnancy should be followed up in the rst several days
post delivery to diagnose withdrawal symptoms or pulmonary
hypertension. Mothers should be told to look for clinical symptoms
that may appear even several days after their release from hospital.
9. Neurodevelopmental effects
Since SSRIs cross the human placenta [6,7], they are expected
to be able to reach the fetal brain. Indeed, as with other psycho-
tropic drugs, a relatively high percentage of the newborns of SSRI-
treated mothers demonstrate some degree of withdrawal symp-
toms. Hence, it may be expected that intrauterine exposure to
SSRIs and other psychotropic drugs might have long-term neu-
robehavioral and neurodevelopmental consequences. However,
since the postnatal environment where the child is raised has a
major effect on its development [68], one has to be careful not to
automatically ascribe such effects to in-utero exposure. In that
context it is important to note that even when the pregnant
mother is heroin dependent, the cognitive ability of the offspring
depends on the postnatal environment; in adopted, heroin-
exposed children that are raised in a favorable environment the
cognitive ability up to the age of 6 years is normal [69]. In contrast,
unexposed children raised in an unfavorable environment have
neurodevelopmental delay [69]. However, heroin-exposed chil-
dren developed a high rate of attention decit hyperactivity dis-
order at school age with specic behavioural consequences, in
spite of being raised in a good environment [70]. Hence, in order
to fully assess the effects on long-term development, children
must be followed up to adolescence and perhaps even to adult-
hood. Such long-term studies are missing for most psychotropic
drugs. Studies carried out on the neurodevelopmental effects of
SSRIs should also take into account the possible effects of maternal
mental illness on the development of the child.
The existing data regarding neurodevelopmental outcome are
conicting, as there seem to be positive and negative ndings.
A recent prospective study comparing the fetal motor behav-
iour of 96 women who took SSRIs, 37 women who discontinued
their SSRIs at the beginning of pregnancy and 130 controls
demonstrated that SSRIs during pregnancy affect the neuro-
behavioral development of the human fetus [71]. Fetuses exposed
to SSRIs exhibited dose-related increased motor activity in the
second and third trimester of pregnancy and disrupted sleep
especially in the third trimester. Whether these changes might
affect postnatal behaviour is not clear.
In contrast, several negative studies have been published. A
cohort of children of mothers exposed in pregnancy to uoxetine
(55 children), to tricyclic antidepressants (80 children) or unex-
posed were compared with an unexposed control group of 84
children and examined at 16 and 86 months of age. No differences
were found among the groups in their cognitive ability, language
development or behavior [72e74]. When the results were adjusted
to maternal IQ and socio-economic status, a negative correlation
was found between the childs IQ and language ability with the
duration of maternal depression and the number of depression
episodes [73]. No signicant differences in neurobehavioral scores
were found between children whose mothers were taking uoxe-
tine during pregnancy and non-exposed children [73]. In a later
study the same group also examined children of depressed mothers
who received no treatment or who were treated with venlafaxine
(an SNRI); they again found that the degree of maternal disease was
an important factor in the future development of the child [74].
Normal development was also observed in a small group of 11
children exposed in pregnancy to citalopram and followed up to 1
year [75]. In another prospective study developmental assessment
was performed at 2 and 8 months of age on 46 infants exposed
prenatally to SSRIs compared with 23 controls. No signicant dif-
ferences between SSRI-exposed and unexposed infants were found,
and there was no difference in the exposed infants between those
with poor neonatal adaptation (30%) and with normal neonatal
adaptation. Abnormal internalizing or externalizing behaviours
were observed in children of mothers with depression or anxiety
whether exposed or unexposed prenatally to SSRIs [76,77].
Klinger et al. [78] compared the neurodevelopmental outcome of
30 children born to mothers who used SSRIs in pregnancy and who
exhibited neonatal abstinence syndrome (NAS) with 52 children
who did not exhibit abstinence symptoms; no difference was found
between the two groups except a higher rate of social problems in
those with NAS. Although the intelligence of those without NAS was
higher than in those with NAS, the difference was not statistically
signicant. The authors concluded that there was no evidence of
cognitive impairment following prenatal exposure to SSRI.
Although the majority of studies showed no neuro-
developmental sequelae in children prenatally exposed to SSRIs,
there are some studies that showed slight developmental delay,
mainly in their motor function.
Mental developmental indices were similar in children whose
mothers were diagnosed with major depressive disorder treated or
untreated in pregnancy. However, children exposed to SSRIs scored
lower on the psychomotor developmental indices and the motor
quality factor of the behavioral rating scale compared with unex-
posed children [79]. In a follow-up study using a psychomotor
developmental test (Boel), abnormal test was more frequent in
children prenatally exposed to antidepressants compared with
unexposed [80]. In another neurobehavioral assessment study,
newborns prenatally exposed to SSRIs had abnormal outcomes
including increased motor activity, fewer changes in behavioral
state, and abnormal sleep patterns [81].
Childrens developmental milestones were assessed using a
questionnaire at 6 and 19 months of age. Second or third trimester
exposure to antidepressants was associated with delayed gross
motor developmental milestones, though still within normal range,
compared with unexposed children [82]. These children also closed
the developmental gaps with increasing age.
In summary, in most studies that focused on the possible neu-
rodevelopmental effects of prenatal SSRI exposure, there was no
conclusive evidence for an increased risk of adverse long-term ef-
fects. This can best be demonstrated by the study of Gentile [83]
who summarized data on negative and positive studies related to
the neurodevelopmental outcome of exposed children. Up to 2005
there were 11 studies of 306 children that found no effect of the
drugs and two studies with 81 children describing mainly slightly
abnormal motor development. We should, however, remember
that all neurodevelopmental studies related to SSRIs in pregnancy
report ndings in young children. Hence effects on learning abili-
ties, attention span and future mental illness may still be found in
spite of relatively normal early development.
10. Possible association with autism spectrum disorders
(ASD)
Serotonin has been related to ASD for many years. Elevated
levels of serotonin in the platelets in about one-third of children
with ASD have been described by many investigators. Since sero-
tonin is an important neurotransmitter involved in fetal brain
development, the question whether prenatal exposure to SSRIs is
associated with ASDis pertinent [84]. However, there are only a few
studies showing such a possible association. In a recent population-
based caseecontrol study, a two-fold increased risk of ASD was
found with prenatal exposure to SSRIs [85]. The authors compared
the rate of prenatal exposure to antidepressants in 298 children
with ASD and in 1507 control children, nding that 6.7% of mothers
used antidepressants in the ASD group compared with only 3.3% in
the controls. No increased risk was found when mothers with
depression were not treated. In addition, a very recent study
showed that a history of maternal depression was associated with
an increased rate of ASD [86]. A similar increase was found
following the use of SSRIs or other antidepressants. The increase
was observed only in cases of high-functioning ASD. It seems from
these few studies that further studies are needed to verify the
suggested association which may well be because of the underlying
maternal disease.
It is advisable that paediatricians of children exposed in utero to
SSRIs will keep in mind the possibility for neurodevelopmental
problems in these children.
11. Is there a reason to discontinue SSRIs during pregnancy?
11.1. Risk of treatment discontinuation
When evaluating the risk/benet ratio of SSRI treatment in
pregnancy, the risks associated with treatment discontinuation
should also be considered. Abrupt discontinuation of psychotropic
drugs in pregnancy may be associated with physical and psycho-
logical adverse effects [87]. SSRI treatment discontinuation during
pregnancy was also associated with a higher frequency of relapse
[88]. Depression is associated with an increased risk for preterm
delivery, and the risk of preterm delivery increases with increasing
severity of depression [88e91]. Treated women have lower
depressive symptom scores and better functioning. These risks
should be a factor in the decision-making with regard to treatment
continuation during pregnancy. Moreover, many of the develop-
mental studies have demonstrated the negative impact of maternal
depression on infant development, irrespective of the mode of
treatment during pregnancy.
12. Conclusions
Clinicians are faced with the difcult risk/benet consideration
of either making a recommendation to treat or not to treat maternal
depression or anxiety with SSRIs in pregnancy. In the eld of
teratology, decisions on new medications during pregnancy often
need to be made with insufcient experience on their safety in
human pregnancy. In the case of SSRIs in pregnancy, despite
extensive available studies on their use, quality is more important
than quantity, and data are still not conclusive.
It is clear that fetal well-being should be an important consid-
erationwhen decision for treatment is made. However, the decision
must also depend on the benet to the pregnant mother of the
treatment. It is also important to take into account the signicance
of maternal disease for the neurobehavioral development of the
child, because there is no question as to the importance of good
maternal physical and mental health to the childs development.
Hence, treatment of mothers with mental illness treated with SSRIs
should continue throughout pregnancy with frequent maintenance
evaluation of the mothers mental status, with proper adjustment
to the dose that should be the minimally effective dose.
In summary, most studies on the use of SSRIs during pregnancy
support the viewthat they are not major human teratogens. A small
increased risk for cardiovascular anomalies, especially with par-
oxetine, cannot be excluded. There appears to be a small increased
risk for miscarriages, which may be associated with the underlying
maternal condition. Neonates of mothers treated with SSRIs should
be closely followed up after delivery as there is an increased risk of
transitory neonatal effects. There is no conclusive evidence for
adverse long-term neurodevelopmental effects of prenatal SSRI
exposure. Discontinuation of treatment may pose risks, e.g. higher
frequency of relapse and increased risk of preterm delivery. Hence,
the general benet of treatment seems to outweigh the potential
small risk of untoward effects on the embryo, fetus or mother.
None declared.
Funding sources
None.
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Review
Abbreviated assessment of bisphenol A toxicology literature
Rochelle W. Tyl
*
RTI International, Hermann 124, 3040 Cornwallis Road, Research Triangle Park, NC 27709-2194, USA
Keywords:
Basic research studies
Bisphenol A
Good laboratory practices (GLPs)
Guideline studies
Non-monotonic doseeresponse (NMDR)
curves
Premature/neonatal sick infants and BPA
Risk assessment
Route-dependent BPA exposure/metabolism
s u m m a r y
Bisphenol A (BPA), synthesized in 1891, is produced in quantities of >2 million metric tons annually for
polycarbonate plastics, epoxy resins and food contact applications. BPA can be a weak estrogen mimic,
and is ubiquitous in humans (in 93% US population; in 96% US pregnant women). European/US food/drug
agencies conclude that current BPA levels present no risk to the general population (some include in-
fants/children); basic endocrine disruption (ED) researchers state that entire populations are at risk from
these levels. The US Food and Drug Administration banned BPA in baby bottles in 2012 not based on
safety concerns; the US Environmental Protection Agency and its UK counterpart concurred. Basic ED
researchers report reproductive/developmental effects from perinatal BPA exposure in mice at very low
doses, e.g. 2 ng/g body weight (0.002 mg/kg body weight), with non-monotonic doseeresponse (NMDR)
curves, using few animals per group and few groups; contract research organizations, in good laboratory
practice- and guideline-compliant large studies in rats and mice, report no low-dose effects or NMDR
curves. The argument rages!
1. Introduction
Bisphenol A (BPA) was synthesized in 1891, recognized as an
articial estrogen in the 1930s, and was used briey to enhance
cattle/poultry growth and for estrogen replacement in women.
World BPA production was >2.2 million metric tons in 2009. US
consumption in 2003 was 856 000 tons: 72% polycarbonate plas-
tics, 21% epoxy resins, and 5% food-contact applications. BPAs
chemical name is 4,4
0
-dihydroxy-2,2-diphenyl propane, its formula
is (CH
3
)
2
C(C
6
H
4
OH)
2
(or C
15
H
16
O
2
), its CAS No. is 80-05-7, and its
molecular structure is shown in Fig. 1.
BPA, with hydroxyphenol groups, can act as a weak estrogen
mimic, and is considered by some to be an endocrine disruptor
(ED). ScienceDaily [1] reported detectable BPA in 96% of US preg-
nant women; a US Centers for Disease Control and Prevention
survey, 2003e2004, indicated that 92.6% of the US population aged
6 years had detectable urinary BPA [2].
2. Health effects and regulatory activities
European/US food/drug agencies conclude that current BPA
levels present no risk to the general population. Basic ED re-
searchers state that entire populations may suffer adverse health
effects from current BPA levels. The Endocrine Society [3] cited
adverse effects of endocrine-disrupting chemicals (EDCs), espe-
cially BPA. In 2012, the FDA banned BPA in baby bottles, stating that
this action was not based on safety concerns and that the agency
continues to support the safety of BPA for use in products that hold
food. In 2011, the Chief Scientist of the UKs Food Standards Agency
cited a 2011 EPA study with 20 human volunteers consuming three
meals of canned food (BPA lining), and tested hourly for urinary BPA
for 24 h, concluding: This [study] corroborates other independent
studies and adds to the evidence that BPA is rapidly absorbed,
detoxied (to non-estrogenic metabolites), and eliminated from
humans e therefore is not a health concern. EPA concurred that
BPA is not a health concern.
In 1998, vom Saal et al. [4] orally administered 2 or 20 ng BPA/g
body weight to pregnant mice on gestational days 11e17; 2 ng BPA/
g increased male offspring preputial gland size, but reduced
epididymal size (these organs developed fromdifferent embryonic
tissues, proffered as explanation), with no effects at 20 ng BPA/g
(non-monotonic doseeresponse: NMDR). Twenty nanograms BPA/
g body weight decreased efciency of daily spermproduction (DSP/
g testis) 20% in offspring males (but not DSP). Howdeshell et al. [5]
(vom Saals laboratory) reported that BPA accelerated mouse pu-
berty; this has yet to be conrmed.
Ryan et al. [6] (Grays EPA laboratory) reported that after peri-
natal exposures ethinyl estradiol (but not BPA) altered sexually
dimorphic behavior, puberty, fertility and anatomy of offspring
female LongeEvans (LE) rats. vom Saal et al. [7] responded that LE
* Tel.: 1 919 541 5972; fax: 1 919 541 6499.
E-mail address: rwt@rti.org.
rat was insensitive, and awed experimental design reveals the
need for guidelines requiring appropriate positive controls in
endocrine disruption research. Gray et al. [8] in a categorical
rebuttal stated that these rat strains are excellent models to study
potential xenoestrogens, and regulatory agencies consider their
studies useful for risk assessment, whereas many of the positive
low-dose studies were inadequate or . results have not been
replicated ..
Myers et al. [9] criticized good laboratory practices (GLPs) and
guideline-compliant studies, with BPA as case study, and argued
that GLPs cannot be used as criteria for selecting data for risk
assessment. Tyl [10] responded, using her three-generation BPA
study to compare strengths/weaknesses of basic exploratory
research vs guideline-compliant studies for hazard evaluation/risk
assessment. Tyl et al. [11] used Charles River (CR) SpragueeDawley
rats, with seven groups, 30/sex/group, dietary BPA at 0, 0.015e
7500 ppm(0, 0.001e500 mg/kg/day) to F0eF3 adults, exceeding US
EPA OPPTS 837.3800 test guidelines. Adult F0/F1/F2 systemic no
observed adverse effect level (NOAEL) was 75 ppm (5 mg/kg/day);
reproductive/postnatal NOAELs were 750 ppm (50 mg/kg/day), no
treatment-related effects in low-dose region (0.001e5 mg/kg/day),
and no NMDRs for any parameter, across generations, in either sex.
Since BPA exposure caused reproductive effects only in presence of
profound systemic toxicity at 7500 ppm (500 mg/kg/day), BPA was
not considered a selective rat reproductive toxicant.
Regulators appeared pleased with this study and used it in de-
liberations. A German EPA contingent visited Tyls laboratory in
2001 examining study data/documentation, reporting no ndings
on study protocol, performance, results or conclusions. Basic re-
searchers responded that CD rat was the wrong animal model,
insensitive to EDCs, and no concurrent positive control was used.
We chose CR/CD-1 mice (used by basic researchers) for our
follow-on BPA reproductive toxicity evaluation, performing one-
[12] and two-generation [13] dietary E
2
studies (six doses) under
OECD guidelines 415/416, to determine systemic, reproductive/
developmental parameters/estrogen responsivity in CD-1 mice. We
then performed a dietary BPA GLP/guideline-compliant CD-1
mouse two-generation study, with seven groups at 0, 0.018
(0.003 mg/kg/day, with reported low-dose effects) to 3500 ppm
BPA (600 mg/kg/day, with anticipated adult systemic toxicity), and
dietary E
2
(0.5 ppm; 0.08 mg/kg/day) as concurrent positive con-
trol, all with 28/sex/group.
E
2
conrmed CD-1 mouse estrogen sensitivity, increasing F0/F1
adult female uterus cervix vagina weights, accelerating pre-
weaning vaginal patency acquisition, decreasing normal/increasing
abnormal F1 female estrous cycles, and increasing absolute and/or
relative pituitary, thyroid (F1 only), liver, and kidney weights in F1/
F1 E
2
males. At 0.018e300 ppm BPA there were no effects on F0/F1/
F2 reproductive/developmental parameters; no NMDRs at any
dose. Systemic NOAEL was 30 ppm BPA (w5 mg/kg/day); repro-
ductive/developmental NOAELs were 300 ppm (w50 mg/kg/day);
effects only at 600 mg/kg/day (with profound systemic toxicity),
like rats. Nor was BPA considered a selective mouse reproductive/
developmental toxicant [14].
This time, basic researchers did not criticize our animal model,
but stated that something in study design (guideline-compliant
EPA/OECD design) and/or study execution affected study animals
(in Tyls opinion, a patently ridiculous accusation). Our rat/mouse
studies were audited in 2009 by the FDA, again with no ndings on
design, conduct, results or conclusions of either study.
In 2010, Stump et al. [15] reported a guideline-compliant
developmental neurotoxicity study of dietary BPA on gestational
day 0epostnatal day (PND) 21 in rats, at 0, 0.15e2250 ppm. F1
offspring exhibited no treatment-related neurobehavioral effects,
neuropathology, or altered brain morphometry, at any dose. Based
on maternal/offspring body weight decrements at 750 and
2250 ppm, systemic toxicity NOAEL was 75 ppm. There were no
treatment-related effects <75 ppm and no NMDRs at any dose.
Developmental neurotoxicity NOAEL was 2250 ppm; with no evi-
dence that BPA is a developmental neurotoxicant in rats.
Newborn mouse route-dependent BPA exposure/metabolism
was conrmed by Draganov et al. [16], dosing PND 3 female mice
once with [H
3
]BPA, 400 mg/kg, by gavage or subcutaneous injection,
50 mice/route. Blood was collected fromve pups/route/time-point
at 5, 10, 20, 30 min, 1 through 4, 6 or 24 h post dosing, and plasma
analysed for free BPA, BPA glucuronide (BPA-G) and BPA sulfate
(BPA-S). Neonatal female mice metabolized BPA to BPA-G, BPA-S
and other inactive metabolites, after both routes, but plasma free-
BPA was three-fold lower following oral dosing, with faster BPA
transport into/out of plasma; BPA-G was major component at every
time-point through 24 h; after subcutaneous injection, free BPA
was the major component at the earliest time-point; free BPA was
three-fold higher and total BPA C
max
four-fold higher vs oral dosing.
Doerge et al. [17] at the National Center for Toxicological
Research measured BPA serum pharmacokinetics in adult female
CD-1 mice following intravenous injection. Deuterated BPA
(100 mg/kg body weight) distinguished administered from envi-
ronmental BPA. BPA pharmacokinetics indicated rapid distribution
of unconjugated BPA from circulation (t
1/2
0.2 h) and terminal
elimination (t
1/2
0.8 h). They concluded that BPA serum/plasma/
tissue kinetics were consistent with rapid equilibrium and un-
derscore . non-persistent nature of BPA [17], and urinary BPA is
not appropriate for internal estimates, since BPA is rapidly conju-
gated, excreted, and rarely found in serum/plasma, although it is a
reasonable surrogate for ingested BPA [18,19].
vom Saal et al. published small studies in mice with low-dose
BPA (<50 mg/kg/body weight), reporting male reproductive organ
changes, including increased prostate weights [4,20]. The ACC
Bisphenol A Task Group and European Chemistry Industry Council
responded by funding robust studies to replicate vomSaals work in
rats/mice. Neither study found low-dose/NMDR BPA effects in
systemic/reproductive organs, including no increased prostate
weights, in rats or mice [11,14]. Basic researchers expressed
concern that funding might have affected study outcomes!
In 2006, the National Institute of Environmental Health Sciences
(NIEHS) sponsored a meeting of thirty-eight of the worlds leading
scientic experts on bisphenol A
0
(all basic researchers), who
assessedcondence levels for BPAndings inmore than700studies;
assessments were integrated into a consensus statement [21].
Also in 2006, the National Toxicology Program (NTP, NIEHS)
began a BPA review within CERHR (Center for the Evaluation of
Risks to Human Reproduction). CERHR convened scientists, inten-
tionally not BPA experts, who applied selection criteria for scientic
literature to include, which excluded some academic studies; aca-
demic scientists accused them of bias in . selection procedures.
CERHR [22] issued draft conclusions in August 2007, reporting
reasons for concern about BPAs impact on neurodevelopment in
fetuses/newborns at estimated current human exposure range, but
little concern about reproductive effects. Academic BPA experts
responded [23]. The nal NTP-CERHR Expert Panel Report on BPA
was published in 2008 by Chapin et al. [24] e an excellent, thor-
ough review.
Fig. 1. Molecular structure of bisphenol A.
R.W. Tyl / Seminars in Fetal & Neonatal Medicine 19 (2014) 195e202 196
In 2003, Hunt et al. [25] reported increased meiotic distur-
bances, including aneuploidy, in mouse oocytes, after exposure to
damaged caging material from inadvertent use of harsh detergent.
They repeated results by intentionally damaging cages and water
bottles, and later administering BPA daily, to conrm that low BPA
levels (as released from damaged cages, etc.) disrupted female
meiosis. They reported that effects were dose dependent, induced
by environmentally relevant BPA doses, and suggested that BPA is a
potent meiotic aneugen.
In 2008, Pacchierotti et al. [26] re-evaluated BPA aneugenic ef-
fects on mouse germ/bone-marrow cells after acute (one BPA dose,
six or seven daily administrations), subchronic (7-week BPA
drinking water), or chronic oral/dietary exposure. They reported no
signicant induction of hyperploidy/polyploidy . in oocytes and
zygotes at any treatment condition; only increased metaphase II
oocytes with prematurely separated chromatids after chronic
exposure, with no irreversible consequence upon . delity of
chromosome segregation during . second meiotic division. Male
mice exhibited no delay of meiotic divisions and no indication of
hyperploidy and polyploidy after 6 exposure days; daily oral BPA
did not increase micronucleus frequencies in mouse bone-marrow
polychromatic erythrocytes. They concluded that our results do
not add evidence to .suspected aneugenic activity of BPA .other
factors or co-factors should be considered to explain the unex-
pected burst of meiotic abnormalities previously attributed to
accidental BPA exposure.
In 2009, Hunt et al. [27] published a mini-review: Bisphenol A
experience, including aneuploidy, disrupted mouse oogenesis,
male/female reproductive tract decits, abnormal fetal oocytes,
pregnancy complications, mammary/prostate gland morphologic
changes, increased adult malignancies, and altered brain sexual
development at very low BPA doses, <50 mg/kg/day, the EFSA TDI,
FDA ADI, and EPA reference dose; none was subsequently
conrmed.
Many multi-generation studies have reported no low-dose BPA
adverse reproductive effects. Hunt et al. [27] states: These multi-
generation tests have the advantage of being conducted under
strict quality control guidelines for Good Laboratory Practice (GLP),
enabling regulators to document data quality in courts of law.
However, adherence to GLP guidelines .provides no assurance of
the soundness of the data analysis and interpretation. Importantly,
for EDC testing, standard multi-generation test protocols have well-
recognized limitations, using methodologies developed long ago.
Effects detectable by contemporary techniques, e.g. immunouo-
rescence, reverse transcriptaseepolymerase chain reaction and
methylation assays, would likely go unnoticed.
The present author was an invited member of the EPA Endocrine
Disruptor Screening and Testing Advisory Committee (EDSTAC),
1996e1998, which led to the EDS Program; (http://epa.gov/endo/
index/htm), and revised/new test guidelines and protocols. Revised
multi-generation protocols include more endocrine-sensitive out-
comes (e.g. estrous cyclicity, pubertal landmarks, sperm measures,
more detailed histopathologic reproductive assessments, hormone
analyses, etc.) [US Environmental Protection Agency, Health Effects
Test Guidelines, OPPTS 870.3800, Reproduction and Fertility Effects,
August, 1998 (http://www.epa.gov/opptsfrs/publications/OPPTS_
Harmonized/870_Health_Effects_TestGuidelines/Series/870-3800.
pdf), and OECD 416].
In 2008, Calafat et al. [2] examined urine samples from 41 pre-
mature sick infants in two hospital neonatal intensive care units
(NICUs) for free/conjugated BPA. BPA was detected in all urine
samples (LOD 0.4 mg/L), >95% conjugated in >75% premature in-
fants, with no differences in free/conjugated BPA concentrations
from breastfed (or fed breast milk) (n 4) vs formula-fed (n 14)
infants.
Teeguarden and Hanson-Drury [28] at the DOE Pacic North-
west National Laboratory examined 150 human exposure studies
(>30 000 individuals in 19 countries, including women/infants),
and 130 toxicity studies which reported use of BPA low doses
(most 10e1000-fold higher than human exposures), concluding
that biologically active BPA is at such low blood concentrations
(parts per trillion) that it is beneath toxicologists current ability
to detect it (which raises concerns about possible sample
contamination in studies reporting high BPA blood levels), and that
BPA exposure [is] too low to cause problems.
Concerns that BPA contributes to estrogenic compound mixture
effects were also considered unfounded, since human BPA expo-
sures are very low and BPA is completely/almost completely/
rapidly conjugated in gut/liver before access to general circulation
and other organs [29], even in sick NICU newborns [30].
Some proponents of inverted U-shaped NMDR curves believe
that BPA effects occur at very low/very high doses (absent/dimin-
ished at intermediate doses), which contribute to non-repeat-
ability of some studies; and that small original research studies
were correct and many, more robust, repeat studies were wrong,
for a variety of design reasons [9,31].
Sharpes [32] comments are germane: Fundamental, repetitive
work on bisphenol A has sucked in tens, probably hundreds, of
millions of dollars from government bodies and industry which, at
a time when research money is thin ., looks increasingly like an
investment with a nil return .I hope that [this opinion piece] will
remind us all of the central importance . attached to . repeat-
ability of experiments and how we should react when a study
proves to be unrepeatable. As scientists, we all like our ideas and
hypotheses to be . correct; yet, there is equal merit in being .
wrong. This is the .trusted way via which scientic understanding
moves onward . our own convictions and presumptions cannot
stand in its way.
Newbold et al. [33] reported that subcutaneous injections to CD-
1 mice on PND 1e5 at 0e1000 mg BPA/kg/day resulted in cystic
ovaries in all groups; increased incidence only at 100 mg BPA/kg/
day. In 2009, Newbold et al. [34] used the same study design and
mouse strain, but administered BPA on gestational days 9e16 at 0e
1000 mg/kg/day, reporting offspring cystic ovaries in all groups,
increased incidence only at 1 mg/kg/day (P < 0.05).
In 2010, Beronius et al. [35] examined 10 BPA risk assessments,
concluding that differences in conclusions regarding human health
risks of BPA between risk assessments are largely due to differing
opinions concerning the reliability and relevance of studies
reporting effects of low-doses. Governmental agencies stated that
low-dose data were not reliable or relevant; Chapel Hill experts
concluded that (their) low-dose data were reliable and relevant.
European Chemicals Bureau (2003), European Commission Health
& Consumer Protection Directorate-General (2002), Opinion of
Scientic Committee on Food on BPA (2002) and NTP CERHR BPA
Monograph (2008) concluded that, although these data were not
sufciently reliable or relevant, they cannot be dismissed or
deemed irrelevant for human health assessment (!). Beronius et al.
[35] concluded only that differences in risk assessment policy and
process expert judgment resulted in drastically different conclu-
sions e from risk to the entire population to no risk to any part of
the population e neither likely nor reasonable.
In a critical evaluation of key evidence on . human health
hazards of exposure to bisphenol A, the Advisory Committee of the
German Society of Toxicology [36] stated that criticism of Tyl rat
and mouse multi-generation studies was scientically not justied
and recently published additional data further support the reli-
ability of the two- and three-generation studies demonstrating lack
of estrogen-dependent effects at/belowdoses on which current TDI
is based. They stated positive results from some explorative
studies have not been conrmed in subsequent studies with higher
numbers of animals or a priori dened hypotheses, and rodent
data can well be used as a basis for human risk evaluation. They
concluded: available evidence indicates that BPA exposure repre-
sents no noteworthy risk to the health of the human population,
including newborns and babies [36].
Viguie et al. [37] evaluated fetal BPA exposure and consequences
on mother/newborn thyroid functions in sheep (gestation/thyroid
regulation similar to humans). Pregnant ewes were injected sub-
cutaneously daily with 5 mg/kg/day BPA, gestational day 28 till
term. Unconjugated BPA was low/similar in pregnant ewes, new-
borns and mothers. BPA glucuronide was w1300-fold higher than
BPA in amniotic uid/cord blood. Total thyroxine (T
4
) was
decreased 30% in BPA-exposed ewes and newborn cord/jugular
blood, with free plasma T
4
reduced in newborn jugular vein blood;
therefore, BPA prenatal exposure is associated with newborn hy-
pothyroidism. Viguie et al. suggest that this effect, if conrmed
would constitute a major issue for BPA risk assessment. This
author could nd no subsequent publications at other doses, no
control/conrmatory data, no information on offspring conse-
quences, etc.
3. Epigenetics
Epigenetics examines gene expression changes/cellular pheno-
type, by mechanisms other than DNA sequence changes (muta-
tions), controlling gene activation/inactivation. Epigenetic changes
occur normally during cellular differentiation as initially totipotent
cells become pluripotent and then specialized/differentiated, some
stable and transmitted to subsequent generations.
Since thyroid hormone (TH) is closely associated with brain
development/function and BPAwas reported to affect TH, Yaoi et al.
[38] examined low-dose BPA effects on mouse forebrain develop-
ment, performing genome-wide analysis study (GWAS) of epi-
genomic alterations on embryonic day 12.5/14.5 brains, after
maternal daily 20 mg BPA/kg/day subcutaneous injections starting
day E0, using telencephalons from 15 (genomic DNA) and six fe-
tuses (RNA preparation) from three dams/group (treated/not
treated), and restriction landmark genomic scanning to detect DNA
methylation changes of cytosine residues in CPG sites of epigenetic
modications. They scanned CpG methylation at 2500 Notl loci
[cleavage sites in CpG islands (CGI)], representing 48 (de)methyl-
ated unique loci: reporting methylation status at most loci was
primarily developmental-stage specic; mRNA expression of two
functionally related genes changed with development, and with
prenatal BPA exposure. In both genes, changes in transcription
correlated well with changes in Notl methylation status. The au-
thors concluded that epigenetic alterations in promoter-associated
CGI after BPA exposure may underlie some effects on brain
development.
Epigenetic modications (acetylation/methylation histone
marks, DNA cytosine nucleotide methylation) correlate with (con-
trol?) transcriptional status of many (all?) genes [39]. Skinner et al.
[40] stated that most environmental toxicants do not alter DNA
sequences, but do alter the epigenome. If environmental toxicants
(e.g. EDCs) alter the somatic cell epigenome, this may cause/pro-
mote disease in exposed individual(s), but not transmitted to next
generation(s). But if toxicants modify germ-line epigenome, then
altered epigenome, and promoted disease, can/may be transmitted
to subsequent offspring generations.
Many diseases cannot be explained by Mendelian genetics.
Identical twins (with essentially identical genotypes) can have
different disease frequencies. The percentage of diseases from ge-
netic abnormalities is small; e.g. 5% of breast cancers are from
known genetic mutations. Most cancers have no known genetic
cause, and almost half of mutated tumor-suppressor genes causing
familial cancers are inactivated by promoter-region epigenetic
hypermethylation. Recent GWAS reported <1% disease incidence
from specic genetic polymorphisms. Environmental exposures
must be considered in conjunction with epigenetic mechanisms.
Other non-Mendelian diseases appearing familial, but not involving
classic genetic transmission, include breast/prostate cancer, dia-
betes, autism, neurological disorders, and metabolic disease.
Nutrition and environmental toxicants, especially endocrine dis-
ruptors can affect/promote disease [40]. Disruption of normal DNA
methylation patterns and/or post-translational covalent histone
modications are implicated in various cancers, from arsenic
exposure, and, putatively, BPA-induced epigenetic modications
resulting in mouse developmental alterations (see below).
Dolinoy et al. [41] suggested that fetal origins of adult disease
include epigenetic modications from developmental exposures,
which may/can inuence adult disease susceptibility; since peri-
natal BPA exposure is associated with adult obesity, breast/pros-
tate cancer and altered reproductive function, perhaps epigenetic
modications are responsible e especially since epigenome is
particularly susceptible to dysregulation during embryogenesis
when normal developmental DNA methylation/chromatin
patterning occur. Therefore, they examined maternal BPA effects on
fetal epigenome by feeding female a/a mice a phytoestrogen-free
diet 50 mg BPA/kg for 2 weeks premating, mating with A
vy
/a
males, through gestation and lactation; 16 control and 17 BPA-
exposed litters. Agouti gene in viable A
vy
mice is a metastable epi-
allele, epigenetically affecting black eumelanin (a) or yellow
phaeomelanin (A) production; epigenetic cytosine methylation at
CpG sites varies among genetically identical A
vy
/a mice. Maternal
dietary BPA did not affect litter size, pup survival, weaning weight,
genotype or sex ratio, but it shifted coat color distribution of
genetically identical PND 22 A
vy
/a offspring toward yellow coat
color phenotype (P 0.007); 21% BPA-exposed offspring were
classied as yellow vs 10% controls; 9.6% BPA-exposed offspring
were classied as pseudo-agouti vs 18.3% controls. Authors
measured DNA methylation at CpG sites in promoter region of A
vy
IAP offspring; BPA decreased (P 0.004) mean percentage
methylation at these sites. They concluded that methylation at A
vy
IAP . mediates BPA effect(s) on A
vy
/a coat color; prenatal BPA
promotes offspring DNA hypomethylation at multiple loci with
metastable epialleles. To evaluate effects of maternal nutritional
supplements on offspring hypomethylated-epigenome from BPA,
additional pregnant a/a mice, exposed to dietary 50 mg BPA/kg,
were fed diets supplemented with methyl donors folic acid, B
12
,
betazine, and choline (14 litters), or genistein (13 litters) during
prematingeperinatal BPA exposures. Neither supplemented diet
affected litter size, pup survival, weanweight, genotypic ratio or sex
ratio, but both supplemented diets restored coat color distribution
of BPA-exposed offspring to that in control litters. Maternal nutri-
tional supplementation negated BPA-induced hypomethylation;
CpG methylation at A
vy
IAP of BPA-exposed/methyl donor-supple-
mented offspring was equivalent to control offspring methylation
pattern (P 0.25). Although genistein is not a methyl donor at
mouse levels comparable with humans on high soy diets, it
counteracted BPA-induced hypomethylation (P 0.46), presum-
ably by hypermethylating offspring genome.
Salian et al. [42] reasoned that e since steroid hormones/re-
ceptors are critical to spermatogenesis, steroid receptor co-
regulators play major roles in steroid receptor functioning, and
BPA is reported to impair spermatogenesis e co-regulator expres-
sion changes should be associated with impaired spermatogenesis.
They investigated whether perinatal BPA exposure changed
testicular steroid receptor/coregulator expression proles. Preg-
nant F0 female rats (eight per group) were gavaged daily at 0, 1.2 or
2.4 mg BPA/kg/day, on gestational age 12ePND 21; adult F1eF3
offspring testes were evaluated by immunohistochemical locali-
zation of testicular receptors co-activator-1 (SRC-1), G-receptor
integrating protein-1 (GRIP-1), p300/CBP/co-integrator-associated
protein (p/CIP) and nuclear co-repressor (NCoR). Authors re-
ported reduced SRC-1 and NCoR and increased p/CIP and GRIP-1
expression in F1 testes (exposed prenatally) and a similar
pattern in F2/F3 offspring testes (not exposed). Any consequences
were not reported. They concluded that perinatal exposure of male
rats to BPA leads to transgenerational perturbations in . expres-
sion prole of testicular steroid receptor coregulators.
Wolstenholme et al. [43] characterized F1 offspring social be-
haviors fromF0 mice exposed to dietary 5 mg BPA/kg/day from7 to
10 days pre-mating till gestational day 18.5, when six F0 dams were
terminated (three control/three BPA-exposed) and their F1 fetuses
extracted, frozen, F1 brains and later F4 fetal (four control and four
BPA litters) brains collected. Plasma BPA levels in exposed F0 dams
were similar to human exposure ranges. Remaining pregnant F0
BPA dams delivered; F1 offspring were fostered to just-delivered
control dams, and maintained on control diet through 4th
offspring generation. The authors evaluated BPA-exposed F1 and
unexposed F4 offspring for social interactions, elevated plus maze
and social preference tests. F1 prenatally BPA-exposed pups
decreased social interactions (social, investigative and play solicit-
ing), increased elevated plus maze activities, and social preference
tests, but unexposed F4 offspring (from BPA-exposed F0 dams)
increased social behavior, decreased non-social behavior and
exhibited no effects on play-soliciting/investigative behaviors. F1
BPA-exposed embryonic brains on gestational day 18.5 had reduced
transcript levels for oxytocin and vasopressin; decreased vaso-
pressin transcript levels persisted to both F4 sexes; oxytocin tran-
script was reduced in F4 males only, with no signicantly altered
transcripts for interaction between diet and sex. The authors
concluded that doses of BPA, only during gestation, had immediate
and long-lasting transgenerational effects on mRNA in brain and
social behaviors. In this authors opinion, small sample sizes, dia-
metrically opposite behavioral responses (social/non-social) in
exposed F1 versus unexposed F4 offspring, and no signicant sex/
diet transcript changes, do not provide convincing evidence of
BPAs supposed transgenerational effects (although persistent
transcript changes are interesting).
The NTP CERHR BPA monograph [24] and FDA Update [44] indi-
cated some concern about the potential effects of BPA on the brain,
behavior and prostate gland in fetuses, infants and young children.
Wolstenholme et al. [45] discussed role of Bisphenol A in shaping
the brain, epigenome and behavior, reiterating work that prenatal
BPA exposures advanced puberty, increased prostatic growth,
altered pubertal mammary gland development, and permanently
changed morphology/functionality of mouse female reproductive
tract and ovaries, although none has been conrmed. BPA was sug-
gested to compromise brain sexual development [46,47].
Kundakovic and Champagne [48] suggested that molecular
mechanisms underlying long-lasting effects from perinatal BPA
exposure, likely involve disruption of epigenetic programming .
of gene expression during development, resulting in altered brain
development, sexual differentiation (sexual dimorphism), neuro-
biological behaviors (anxiety, response to novelty, learning and
memory), and immune function; effects could extend to subse-
quent generations (see above). They suggested that since proposed
molecular mechanisms may be altered by maternal gestational
BPA exposure, resulting in altered expression of specic offspring
genes, it is important to know whether BPA epigenetic effects (not
yet conrmed) are related to its estrogenic activity, and to deter-
mine which downstream effector proteins could mediate DNA
methylation changes by BPA (not yet conrmed), highlighting basic
research studies indicating effects of prenatal BPA exposure on
offspring brain, behavior and immune system, role of epigenetic
pathways in these effects, and transgenerational implications of
EDC exposures (none yet conrmed). This author concurs only that
more work should be done on the role(s) of epigenetic program-
ming and disruption consequence(s).
Basic researchers state that parental BPA exposure can impact
the next generation e a transgenerational effect. However, if F0
dams are exposed during their pregnancies, F1 offspring are
exposed in utero, so F1 effects are not transgenerational; to conrm
transgenerational effects the effect(s) would have to occur in F2 or
later generations with no direct/indirect offspring BPA exposures.
The NIH scientic panel concluded that, due to lack of repro-
ducibility of low-dose experiments, they had minimal concern for
BPA reproductive effects, but did have higher concern for neural
and behavioral BPA effects [24].
In-vitro/small basic research studies report adverse outcomes,
many exhibiting NMDRs in studies with more than two doses, non-
relevant subcutaneous/intravenous injection, and doses 100e10
000-fold those of human exposures. Studies reporting higher than
expected BPA levels in animal blood, urine, tissues, etc., may have
contamination from damaged plasticware: BPA, released into food/
water, taken up by animals, and rapidly glucuronidated (and/or
sulfated) in gastrointestinal tract/liver, before systemic exposure.
Even in sick premature NICU newborns, over 95% of the BPA
detected in urine was glucuronidated in most of the babies [2].
Perinatal BPA reportedly altered DNA methylation status and
subsequent malignancy development in offspring breast and
prostate; Ho et al. [49] considered that DNA methylation may be
one mechanism for reprogramming early prostate; hypo-
methylation of phosphodiesterase type IV variant 4 (PDE4D4)
promotor, in rat prostate after neonatal BPA at an environmentally
relevant dose, was associated with increased susceptibility to
prostate carcinogenesis. Chen and Tang [50] concluded that of
particular importance will be the exploration of molecular and
behavioral changes that occur in response to environmentally
relevant low doses of BPA in animals and humans.
Yeo et al. [51] evaluated early development of mammalian
cortical neurons, when high intracellular chloride levels are pre-
sent. As neurons mature, chloride levels drop w75% because po-
tassium chloride co-transporter protein, Kcc2, removes chloride
ions from cells. If chloride remains high, this can damage neural
circuits and compromise ability of developing nerve cells to migrate
to proper brain positions. When cortical neurons were exposed
in vitro to 100 nM BPA, chloride was reduced by only w50%, with
greater effects in female neurons. The authors proposed that BPA
shuts down Kcc2 gene, reducing Kcc2 protein, delaying chloride
removal. In addition, MECP2, a gene which makes MECP2 protein
important for normal brain function, is also BPA responsive. When
exposed to BPA in vitro, neural MECP2 was higher and bound to
Kcc2 gene at a higher rate, which might . shut Kcc2 down. BPA
also increased chloride in migrating neurons (female neurons more
affected). Authors concluded that BPA disrupts this gene expres-
sion by epigenetic mechanisms ., which could . play a role in
pathogenesis of human neurodevelopmental disorders. Their
ndings raised the question (at least to ScienceDaily [1]) whether
BPA contributes to neural developmental disorders such as Rett
syndrome, a severe autism spectrum disorder in girls, with MECP2
gene mutations [52]. Experts noted (Round-Up [53]), this study
was in vitro with BPA administered directly to neurons, at one dose,
hundreds to thousands of times higher than humans would be
exposed to, and therefore would not provide any linking/sup-
porting evidence because of lack of credibility with the dose.
The European Food Safety Authority [54] stated that human BPA
risks during perinatal period are minor because rapid metabolic
clearance by rst-pass glucuronide metabolism minimizes human
internal exposure to free BPA vs rodents in which low-dose effects
have been observed. However, Ginsberg and Rice [55] argued that
BPA glucuronide deconjugation to active BPA occurs prenatally by
placental (and other tissue) b-glucuronidase, and BPA sulfate
deconjugation by arylsulfatase C to active BPA starts early post
partum in liver/gut; biomonitoring studies and laboratory experi-
ments document free BPA in rat and human maternal, placental and
fetal tissues indicating human BPA exposure is not negligible; risk
assessors should dene human BPA doseeresponse with pharma-
cokinetic models including deconjugation.
There is growing concern that prenatal BPA exposure may affect
brain development, behavior and emotions. Studies have suggested
that rodent fetal and/or lactational BPA exposure alters adult
offspring behavior [56,57], possibly caused by neurotransmitter
changes [58,59].
4. BPA exposures during pregnancy in children
Braun et al. [60] investigated the impact of gestational/child-
hood BPA on behavior/executive function of 3-year-olds, using a
prospective Cincinnati cohort of 244 mothers and their children.
They characterized gestational/childhood BPA using maternal BPA
urinary concentrations during gestation, at delivery, and in 1-, 2-
and 3-year-olds. (Urinary BPA is a reasonable surrogate for ingested
BPA, but not for internal exposure [18].) BPA was detected in >97%
of maternal gestational/childhood urine samples. Each 10-fold in-
crease in maternal gestational urinary BPA concentration was
associated with more anxious and depressed behavior in daugh-
ters; null/negative changes in sons. Interestingly, the authors
concluded that associations between childhood BPA and neuro-
behavior were largely null and not modied by child gender.
Selgrade et al. [61] identied one study relating maternal uri-
nary BPA during pregnancy and delivery, and childhood wheeze
frombirth to 3 years [62]. Maternal gestational urinary BPAwas not
associated with child wheeze at birth to 3 years. Whenprenatal BPA
was separated into above and below the median, BPA above the
median was positively associated with offspring wheeze at 6
months, but not at 3 years. Maternal prenatal urinary BPA at 16
weeks had borderline association with offspring wheeze, but
offspring wheeze was not associated with maternal urinary BPA at
26 weeks of gestation or at delivery. Selgrade et al. considered these
effects to be borderline and transient at best.
CHAMACOS (Center for Health Assessment of Mothers and
Children of Salinas) longitudinal birth cohort study examined
environmental exposures/health among women/children of Sali-
nas, California, beginning in 1999e2000. In 2013, Chevrier et al.
[63] reported on maternal pregnancy urinary BPA and maternal/
neonatal thyroid function, measuring maternal urinary BPA during
rst/second half of pregnancy, and free thyroxine (T
4
), total T
4
, and
thyroid stimulating hormone (TSH) in pregnant women and
offspring. They reported no statistically signicant association
between the mean of two pregnancy BPA measurements and
maternal thyroid levels, but BPA measurement closer to thyroxine
measurement was signicantly associated with reduced maternal
T
4
. Maternal BPA was associated with reduced TSH in sons (not
daughters) especially when BPA was measured in third trimester.
The authors concluded that exposure to BPA during pregnancy .
reduced total T
4
in pregnant women and decreased TSH in male
neonates, a nding which may have implications for fetal and
neonatal development. (T
4
was reduced only in mothers, and is
usually associated with increased TSH which stimulates T
4
syn-
thesis e not with decreased TSH).
A second CHAMACOS cohort paper [64] evaluated effects of
prenatal BPA on childrens body mass. They reported that offspring
girls, age 9 years, exposed to highest prenatal BPA concentrations,
had lower body weights for their heights and were less likely to be
obese, vs girls with lowest prenatal BPA exposures; boys were
unaffected. Nine-year-olds showed a positive association between
current BPA urinary concentrations and current obesity. The au-
thors suggested that these obese children may have higher urinary
BPA levels because they eat more packaged foods (packaging con-
taining BPA) and/or they ingest more calories, so higher BPA levels
could be a result of dietary habits resulting in obesity, rather than
being a cause of obesity. The authors will follow these children
through puberty.
Columbia Center for Childrens Environmental Health recruited
568 pregnant women; mothers during third trimester pregnancy
and their children at ages 3, 5, and 7 years provided spot urine
samples. Wheeze in previous year was assessed by questionnaires
at 5e7 years; asthma determined by a physician once at 5e12 years.
FENO (fractional exhaled nitric oxide, measuring airway inam-
mation) was measured at 7e11 years. Maternal pregnancy urinary
BPA concentrations were associated inversely with offspring
wheeze at 5 years; offspring urinary BPA concentrations at 3 years
were associated positively with wheeze at 5 and 6 years; urinary
BPA concentrations were associated with wheeze, both at 7 years,
and increased FENO values; and urinary BPA concentrations at 3, 5
and 7 years were associated with asthma. The authors stated that
this is the rst report of an association between postnatal urinary
BPA concentrations and asthma in children [65]; an association
perhaps, but no apparent causal relationship. (Did the authors look
for other adverse agent[s] in urine spots? Were there any blood/
serum analyses? Were there BPA-unexposed controls?) Of the 11
authors, seven disclosed potential conict of interest.
Environmental Health Perspectives Journal Online recently re-
ported that FDA has decided to ban bisphenol A (BPA)-based epoxy
resins as coatings in infant formula packaging, saying that . use
has largely been abandoned by manufacturers anyway (EHP Noon
News: Med page today, 18th July 2013).
In welcome newsteps, basic researchers are using more animals
per group and more test groups [66], and regulatory researchers
are adding more cutting-edge endpoints to their large, robust
studies.
5. Conclusions
ED presence/effects are major societal concerns. Sound repro-
ducible science by academic, governmental, regulatory, industrial
and other researchers must address concerns including environ-
mental EDC levels, hazard/risk, better testing protocols, mecha-
nisms of EDC action, and epigenetic role(s) in normal/abnormal
development.
Sharpe [32] argues that scientic facts usually evolve mean-
ing/importance over time, which is important for scientic prog-
ress, and that BPA research has been trying to go through this
process but has become literally bogged down in the mire of con-
troversy, much of which stems from.earliest ndings and seems
to have little to do with . current state of the science. Sharpe
contrasts large, robust BPA studies in rats/mice using oral exposure
with no low-dose effects, some with positive controls, vs small
basic research studies with low-dose BPA effects, noting the
problem of low-dose effects and inverted U-shaped NMDR curves
. in these studies.
Unfounded/wild accusations/criticisms by academic researchers
toward regulatory/industrial studies/authors must stop. We must
work collegially to resolve scientic concerns, potential, real (and
unreal) ED risks in general, and for specic EDCs (e.g. BPA),
especially to humans, other mammals, other vertebrates, in-
vertebrates (and plants?) in our earthly biome.
There is no conict of interest.
Funding sources
The BPA and E
2
studies performed at RTI were guideline- and
GLP-compliant works for hire (RTI is a Contract Research Organi-
zation) funded by the American Plastics Council of the American
Chemistry Council, which played no role in this producing manu-
script. The time to write this manuscript was provided by the RTI
Fellows Program (Dr Tyl is a Distinguished Fellow in DART).
Acknowledgments
The author wishes to thank RTI Internationals Fellows Program
for the time to write this review, the senior and technical staff in the
Pharmacology and Toxicology and Animal Research Facility groups
for their extraordinary dedication and expertise on the BPA and E
2
studies discussed, and Ms Kathy Ancheta and Ms Rebecca Hipp for
their valued assistance in the production of this review, including
their patient typing and retyping of this manuscript.
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Review
Carcinogenic risks of prenatal ionizing radiation
Robert L. Brent
*
Thomas Jefferson University, Alfred I. duPont Hospital for Children, Room 308, A/R Building, PO Box 269, Wilmington, DE 19899, USA
Keywords:
Cancer
Carcinogenic risks
Fetus
Ionizing radiation
s u m m a r y
The risk of cancer in offspring who have been exposed to diagnostic X-ray procedures while in utero has
been debated for 55 years. High doses at high dose rates to the embryo or fetus (e.g. >0.5 Gy) increase
the risk of cancer. This has been demonstrated in human epidemiology studies as well as in mammalian
animal studies. Most pregnant women exposed to diagnostic X-ray procedures or the diagnostic use of
radionuclides receive doses to the embryo or fetus <0.1 Gy. The risk of cancer in offspring exposed in
utero at a low dose such as <0.1 Gy is controversial and has not been determined.
1. Introduction
In 1950e51, I was working in the radiation embryology section
of the University of Rochester Medical Centers Atomic Energy
Project. We had submitted an abstract to the Anatomy Society
meetings in Detroit [1]. The completed manuscript that was sub-
mitted to Cancer Research was titled, Cancer induced in rat em-
bryos by roentgen irradiation. The editors rejected the manuscript
and stated that if the embryos had developed cancer, there would
have been a much higher mortality. So we changed the title to
Neoplasia induced in rat embryos by roentgen irradiation and the
manuscript was accepted [2]. We examined the tumors as they rst
appeared and continued to grow (Fig. 1). Many of the tumors
became anaplastic and contained many undifferentiated cells with
a high mitotic index. At birth, most of the tumors were gone.
However, there were a few pyknotic cell remnants that were still
present. We followed 300 radiated survivors and controls for 4
years and these irradiated animals did not have a higher incidence
of cancer than the controls.
We put this project aside with the tentative conclusion that the
embryo was less vulnerable to the carcinogenic effects of low ex-
posures of ionizing radiation than the postnatal animal.
Liane Russell [3,4] and our laboratory [5,6] had already
described the all or none phenomenon, which indicated that the
pre-somite mammalian embryo was less vulnerable to the terato-
genic effects of ionizing radiation. The embryo was very vulnerable
to the lethal effects of radiation; however, the surviving embryos
did not have an increased risk of birth defects.
When Alice Stewart published her research results, a 60-year
controversial discussion was initiated. Stewart et al. [7e10] sug-
gested that the human embryo was more vulnerable to the leuke-
mogenic effects of radiation and in later publications concluded
that other childhood cancers also occur more frequently in persons
exposed in utero to diagnostic radiologic procedures (primarily
pelvimetry) (Fig. 2). These authors initially estimated that a 1e2 rad
in-utero radiation exposure increases the risk of leukemia devel-
oping in the offspring by a factor of 1.5 to 2.0 over the natural
incidence. This incidence is considerably greater than the increase
resulting from 2 rad delivered to an adult population. In fact, an
increase in the incidence of leukemia after an adult population
exposure of 2 rad would be difcult to document, even for very
large population groups [11,12]. Dr Stewart became a spokesperson
for anti-radiation groups. She appeared as a plaintiff expert in ra-
diation litigation and was even a plaintiff expert against her own
country in a case before the World Court inwhich Ireland was suing
the UK, claiming that a British nuclear facility (Sellaelds Fuel
Handling Plant) was contaminating the Irish sea and causing
increased cases of birth defects and cancer in the inhabitants on the
east coast of Ireland. After more than a decade of litigation the
World Court decided in favor of the UK [13]. Dr Stewart claimed
that the embryo was many times more vulnerable to the carcino-
genic effects of radiation than children and she was critical of sci-
entists who disagreed with her [8,14].
As a medical and graduate student and part-time instructor, I
did not have time to further pursue the question of the resistance of
the embryo to the carcinogenic effects of radiation. However, there
were many publications exposing animals to carcinogenic agents.
In particular, urethan (urethane; ethyl carbamate) was used by
Klein [15] and Vesselinovitch et al. [16] to produce neoplasia in
rodents. Only a few of the investigators utilizing urethan exposed
pregnant animals to this carcinogenic agent. Klein [15] reported
* Tel.: 1 302 651 6880; fax: 1 302 651 6888.
E-mail address: rbrent@nemours.org.
that cesarean-delivered mice exposed in utero had signicantly
fewer lung tumors than animals treated postnatally. Signicantly
more tumors per lung were observed in mice injected with urethan
at 47 days of age than at birth, suggesting an increased suscepti-
bility with age. Vesselinovitch et al. [16] exposed pregnant mice on
multiple days in mid pregnancy (days 12e18). The incidence of liver
and lung tumors was signicantly higher in mice exposed to this
carcinogen at the end of gestation. Neonatally treated animals
developed all of the tumor types more readily than those exposed
to the carcinogen in utero and also developed leukemia which did
not occur in the in-utero-exposed population. The urethan animal
studies reinforced the animal studies from our laboratory, which
indicated that the fetus had lower carcinogenic risks from muta-
genic or carcinogenic agents when compared to the postnatal an-
imals vulnerability.
2. Human studies concerning the vulnerability of the embryo
to the carcinogenic risks of ionizing radiation
Lilienfeld [17] reviewed the epidemiologic considerations with
respect to leukemogenesis. His results, conrmed by others [18e
21] support the thesis that diagnostic radiation absorbed in utero
was associated with an increased risk of leukemia. Six of nine
studies reported in Lilienfelds paper indicate a 1.3e1.8-fold in-
crease in the risk of leukemia after diagnostic radiation exposure in
utero. Lilienfeld states: When one considers the variety of control
groups used and the sampling variability, the results are remark-
ably consistent in showing an excess frequency of leukemia among
children of radiation-exposed pregnant mothers [17]. Diamond
et al. [22] conrmed and extended the observation of a three-fold
increased incidence of leukemia in children exposed to diagnostic
radiation in utero. Interestingly, this effect did not occur in the
African-American population. When MacMahon [23] extended his
studies, the 1.5-fold excess leukemia incidence remained, but the
excess in other childhood cancers was no longer present (Table 1).
Fig. 1. (A, B) Fifteen-day-old rat fetuses exposed to 1.5 Gy at day 9 post conception. The arrows in 1b are pointing to tumors under the scalp that can be seen in histological sections
in 3a, 3b, and 3c. The tumor growths are derived from outgrowths of the radiated neural tube as seen in 2a, 2b and 2c. Some of the growths dedifferentiated into more aggressive-
appearing tissues (4). At the time of the birth of the fetuses almost all the growths had regressed except for a few remnants of pyknotic cells located between the brain and the scalp
(5a, 5b, and 5c). All photographs are reproduced with permission from Wilson et al. [2].
100
10
1
Fetus 1 10 20 30
Stewart et al.
810
ABCC
28,38,55
Animal data
1,2,61,6466,108
Age (years)
R
i
s
k

o
f

l
e
u
k
e
m
i
a

p
e
r

1
0
6

p
e
r
s
o
n
s
f
o
l
l
o
w
i
n
g

0
.
0
1

t
o

0
.
0
2

G
y
40 50 60 70
Fig. 2. Risk of cancer from in-utero radiation. When Stewart rst reported the risks of
cancer in the offspring of pregnancies in which the mother had been exposed to
diagnostic radiological studies in the 1950s, the risk of leukemia was stated as one to
two orders of magnitude greater than the risk of cancer following similar exposures in
childhood. Children were believed to be slightly more vulnerable than adults. Animal
studies were inconsistent, but many of the animal studies were negative and many of
the studies did not expose the pregnant animals to doses of <0.10 Gy. ABCC, Atomic
Bomb Casualty Commission.
Table 1
Risk of specic and total childhood cancers in offspring of women undergoing prenatal diagnostic X-ray procedures: caseecontrol studies.
a
Reference, country, birth year No. of cases/no.
of controls
Type of
control
Source of X-ray
procedure
information
Controls with
abdominal X-ray
procedures (%)
Any X-ray
procedure
Estimated
RR (95% CI)
b
Abdominal
procedure
Pelvimetry
Acute lymphoblastic leukemia
Bithell and Stewart [24],
c
UK, 1943e1967
2007/8513 Population Interview,
medical records,
questionnaire
11.5 1.5 (1.3e1.8)
Van Steensel-Moll et al. [25],
The Netherlands, 1959e1980
517/509 Population Questionnaire 3.7 2.2 (1.2e3.8)
Shu et al. [26], Shanghai, China,
1960e1986
172/618 Population Interview 7.1 1.6 (0.9e2.8) 2.0 (0.7e3.8)
Magnani et al. [27], Turin, Italy
(years not provided)
142/307 Hospital Interview 5.5 11 (not provided)
Naumberg et al. [28], Sweden,
1973e1989
449/450 Population Medical records 9.8 1.0 (0.6e1.7) 1.1 (0.8e1.6)
Shu et al. [29], USA and
Canada, 1972e1992
1842/1986 Population Interview 6 (before 1980)
2.3 (1981e1986)
1.8 (after 1986)
1.2 (0.8e1.7)
(all ages)
2.4 (1.2e5.0)
(ages 11e14
years)
Acute myeloid leukemia
c
UK, 1943e1967
866/8513 Population Interview;
medical records;
questionnaire
11.5 1.5 (1.2e1.8)
1960e1986
92/618 Population Interview 7.1 1.4 (0.6e3.0) 0.6 (0.1e5.0)
Van Duijn et al. [30],
The Netherlands, 1969e1979
80/240 Population Questionnaire 3 2.4 (0.8e7.0)
All leukemias
Kaplan [31], California, USA,
1943e1967
150/150 Friends Interview 16 1.7 (1.1e2.7)
Polhemus and Koch [18],
Los Angeles, California, USA
(years not provided)
251/251 Hospital Questionnaire 23.1 1.2 (0.8e1.8)
Graham et al. [20], Baltimore,
Minneapolis, New York State,
USA (Tristate Study, 1969e1979)
313/854 Population Medical records 23.4 1.4 (0.9e2.3)
Salonen and Saxen [32], Finland,
1945e1968
Hirayama [33], Japan, 1969e1977 4607/5968 Other cancers Not provided 10.6 1.6 (1.4e1.8)
Monson and MacMahon (1984)
[34],
d
Northeast USA, 1947e1960
704/14 276 Hospital Medical records 9.4 1.5 (1.2e2.0)
e
1986e1991
166/166 Population Interview 7.1 2.4 (0.5e10.6)
Infante-Rivard et al. [36],
InfanteeRivard and Deadman [37],
Canada, 1980e1993
701/701 Population Interview 0.8 (0.6e1.3)
Rajaraman et al. [38], UK,
1976e1996
CNS tumors
c
UK,
1943e1967
medical records;
questionnaire
11.5 1.4 (1.2e1.7)
Salonen and Saxen [32], Finland,
1945e1968
Preston-Martin et al. [39],
Los Angeles, California, USA,
1948e1977
209/209 Friends,
neighborhood
Interview 15.0 1.3 (not provided)
Monson and MacMachon [34],
d
f
Bunin et al. [40], Greater Delaware
Valley, USA, 1980e1986
155 astro/32
166 PNET/321
Population Interview 1.1 (0.3e3.9)
0.8 (0.3e2.3)
Schuz et al. [41], Germany,
1988e1993
466/2458 Population Interview 0.8 (0.4e1.4)
Stalberg et al. [42], Sweden,
1975e1984
512/524 (total CNS)
105 PNET/524
191 astro/524
Population Medical records;
registry
9.1 1.1 (0.7e1.6)
1.7 (0.9e3.2)
0.8 (0.4e1.5)
1976e1996
Neuroblastoma
c
UK,
1943e1967
medical records;
questionnaire
11.5 1.5 (1.2e1.8)
Several associations of these data should be pointed out. In the
studies of Stewart and colleagues [7,10], there was a higher inci-
dence of previous abortion in the mothers receiving pelvimetry,
and the children in the pelvimetry group had a higher incidence of
upper respiratory infections before the development of leukemia.
Others reported that infants with a strong family history of allergy
are also more susceptible to radiation-induced leukemia when
exposed to diagnostic radiation in utero. The problem with these
data is that patients with an allergic history and no exposure to
radiation had a higher frequency of leukemia than did other groups
that had received radiation in utero [48].
Of the 86 persons exposed in utero at Nagasaki, none developed
leukemia [8]. These persons received considerably higher doses of
radiation than did those patients in the previous studies.
Shiono et al. [49] examined the potential risk of diagnostic X-
rays in the 44 908 pregnant patients studied in the Collaborative
Perinatal Project of the National Institute of Neurological and
Communicative Disorders and Stroke. These ndings were sur-
prising in that they reported a statistically increased relative risk for
malignancies in the offspring of mothers who were exposed before
pregnancy (preconception) [relative risk (RR): 2.61; 90% condence
limits (CL): 1.26e5.85]. However, there was no statistically signi-
cant increase in the risk of malignant or benign tumors in the
offspring of mothers who were exposed to radiation during preg-
nancy (RR: 1.09; 90% CL: 0.47e2.40 for malignant neoplasms; and
RR: 0.94; 90% CL: 0.46e1.82 for benign neoplasms). Court-Brown
et al. [50] evaluated the incidence of leukemia in 39 166 offspring
of mothers who had been irradiated in utero, and Salonen [51]
studied the relationship between pregnancy radiation exposure
and childhood cancers. Neither study could establish a statistically
signicant increase in leukemia (Table 2).
Although it is true that the population of offspring exposed in
utero in Hiroshima and Nagasaki did not have an increased
incidence of leukemia and other childhood cancers during the
childhood years, they have of course developed cancer as adults
(Fig. 3).
A larger number of cancers must accumulate before we can
reliably establish a risk of cancer in adults who were exposed in
utero. Whether there is an increased risk has been partially
answered by Preston et al. [67] One conclusion is certain: the risk of
developing cancer as an adult from in-utero radiation is below the
risks of childhood cancer that have been suggested by several in-
vestigators (Fig. 4) [7e10,17e19,21].
Hoshino et al. [68] reported no increase in leukemia in a study of
17 000 children of parents who had received radiation before
conception. The question arises as to what extent the same biases
that contribute to the increased risk of leukemia in the cases of
radiation exposure before conception also affect the in-utero ra-
diation cases. Graham et al. [20] pointed out that children of
mothers with a history of abortion or stillbirths also had children
with a higher risk of leukemia.
Miller [69] and others [56,70,71] do not believe that the risk of
prenatal radiation is as great as Stewart suggests. Miller writes:
It is surprising that in Stewarts studies minimal doses of x-rays
are equally oncogenic whether exposure occurred before
conception or during pregnancy, whether the neoplasm studied
was leukemia or any other major cancer of childhood, and
whether the study was based on interviews, which may be
biased, or from hospital records. Taken in aggregate, the simi-
larity of results, in the absence of a doseeresponse effect or of
supporting data from animal experimentation, raises a question
about biologic plausibility of a causal relationship.
Furthermore, Miller [69] points out that siblings of leukemic
children have a risk of childhood leukemia of 1 in 720 in the rst 10
Table 1 (continued)
Reference, country, birth year No. of cases/no.
of controls
Type of
control
Source of X-ray
procedure
information
Controls with
abdominal X-ray
procedures (%)
Any X-ray
procedure
Estimated
RR (95% CI)
b
Abdominal
procedure
Pelvimetry
Bone tumors
c
UK,
1943e1967
medical records;
questionnaire
11.5 1.1 (0.7e1.7)
Ewings sarcoma
Winn et al. (1992) [43], USA,
multicenter (years not provided)
204/204
191/191
Population
Siblings
Interview
Interview
27.5
17.3
0.8 (0.5e1.2)
1.5 (0.8e3.2)
Rhabdomyosarcoma
Gufferman et al. [44], USA,
multicenter, 1962e1988
319/319 Population Interview 6.8 1.4 (0.7e2.9)
Total childhood cancer
c
UK,
1943e1967
medical records;
questionnaire
11.5 1.5 (1.3e1.6)
MacMahon [45],
d
Northeast USA,
1947e1954
556/7230 Hospital Medical records 10.6 1.5 (1.2e1.8)
e
Monson and MacMahon [34],
d
e
1976e1996
RR, relative risk; CI, condence interval; CNS, central nervous system; PNET, primitive neuroectodermal tumour.
a
Adapted from Little. [46].
b
RR and CI values are presented to one decimal place (rounded), except that one CI value is presented as 0.95 (rather than 1.0).
c
Initial Oxford Survey of Childhood Cancer CaseeControl Study (Stewart et al.
10
) was extended to include 12 additional birth years and an increase to 8513 cancer cases
from 1299 in the original study (Bithell and Stewart [24]). A third publication from the same investigation included cases born during 1948 to 1978 who died during 1953 to
1979 (Knox et al. [47]).
d
Initial study of MacMahon [45] was extended by Monson and MacMahon [34] to include ve additional hospitals, six additional birth years, seven additional years of
childhood cancer deaths, an increase to 1 429 400 children from 734 243 in the original study.
e
Results for children who died from cancer before their 10th birthday.
f
Results for children who died from cancer before their 20th birthday; condence interval computed from data in Monson and MacMahon. [34].
years of their life, which is greater than the 1:2000 risk of leukemia
after pelvimetry exposure and the 1:3000 probability of leukemia
in the general population of children followed for 10 years (Table 3).
Stewart and Kneale [7] reinforces the contention that radiation may
not be the only etiologic factor responsible for the induction of
malignancy because of unirradiated siblings of the irradiated pa-
tient population with a higher incidence of leukemia also had an
incidence higher than in control siblings and in control patients.
This observation certainly would indicate that genetic or other
environmental factors may be important in the etiology of
leukemia.
At present, some investigators believe that in-utero exposure to
small amounts of radiation increased the risk of leukemia and other
malignancies, whereas others seriously question the contention
that the embryo is markedly more sensitive to the leukemogenic
effects of irradiation when compared with the child or adult. Until
the mechanism is understood, there will be doubt concerning the
magnitude of the role of in-utero diagnostic radiology studies in
leukemia induction. The increased incidence of cancer in children
exposed to in-utero diagnostic radiation should be claried in view
of the fact that much higher doses of radiation to animal embryos
and to the children exposed in utero at Hiroshima and Nagasaki
have not resulted in a marked increase in the incidence of cancers
from higher doses of radiation, which one would expect if the
embryo were as sensitive to the carcinogenic effects of radiation as
Stewart and colleagues suggest (Table 2) (Figs. 3 and 4) [74e77].
One cannot overemphasize either the importance of the multi-
plicity of factors or the difculties involved in identifying and con-
trolling for such factors. Even laboratory experiments concerned
with tumor production are difcult to interpret. For example, Ross
and Bras [48] reported that the incidence of spontaneous tumors
varied with the diet and weight of the animals. Heavier animals on
high-protein diets had a higher incidence of tumors than did the
lighter rats onlow-proteindiets. Hence, there are many unanswered
questions pertaining to the relationship between leukemia and
malignancy and in-utero radiation exposure.
Because of the introduction of new diagnostic techniques, such
as the use of ultrasound, and because of the concerns about the
risks of radiation, fewer pregnant patients will be exposed in the
future. Therefore it is unlikely that adequate numbers of exposed
patients will be available to evaluate the carcinogenic risks of in-
utero diagnostic radiation. MacMahon [21] in his editorial in the
Table 2
Cancer mortality rates in cohorts of children whose mothers underwent diagnostic
X-ray procedures during pregnancy.
a
Reference, location No. of cancer deaths;
no. of children
exposed in utero
b
Total cancer
[RR (95% CI)]
Leukemia
[RR (95% CI)]
Murray et al. [52]
(Rochester, NY)
3 (L); w6740
c
0.9 (0.3e3.1)
Court Brown et al. [50]
(Edinburgh)
d
9 (L); 39 166 0.9 (0.4e1.6)
Lewis [53] (London) 1 (L); 11 443 0.4 (0.1e2.6)
Griem et al. [54]
(Chicago)
4 (I L, 3 O); 982 1.2 (0.4e4.0) 0.4 (0.1e2.6)
Oppenheim et al.
[55,56] (Chicago)
e
1 (L); 393 0.7 (0.1e5.0)
Diamond et al. [22]
(Baltimore)
13 (6 L, 7 O); 19 889 1.1 (0.5e2.1) 1.6 (0.6e4.6)
Shiono et al. [49]
(USA, multicenter)
7; w5000
f
1.1 (0.5e2.4)
Golding et al. [57]
(UK, national)
12; w3000
g
1.2 (0.6e2.5)
Combined small
cohorts (ICRP [58])
7 4.6 (0.9e25.1)
Dempster [59] [0; 148]
Milis et al. [60] [0; 190]
Lejeune et al. [61] [2; 491]
Magnin [62] [1; 5353]
Nokkentved [63] [0; 152]
Hagstrom et al. [64],
h
[4; 649]
Ray et al. [65]
(Ontario, Canada)
4; 5590 0.7 (0.3e1.8)
a
Adapted from Doll and Wakeford [66] and ICRP. [58].
b
The number of leukemias (L) and other cancers (O) are given when available.
c
A total of 140 438 children aged <20 years were included; w6460 of the 6740
exposed mothers had pelvimetry or other abdominal X-ray procedures during
pregnancy.
d
Doll pointed out his concerns about the adequacy of the identication of irra-
diated women that arose when he tried to extend the Court Brown et al. [50] study.
Doll indicated that some of the ndings may therefore be unreliable (Doll and
Wakeford [66]).
e
Subsequent follow-up study of Griem et al. [54].
f
A total of 55 908 live births were studied; w10% of the mothers had abdominal
X-ray procedures during pregnancy.
g
A total of 16 193 live births were studied; w18% of the mothers had abdominal,
non-abdominal or dental X-ray procedures during pregnancy.
h
Exposure in Hagstrom et al. [64] was to radioactive iron (
59
Fe) administered
during pregnancy. The RR and 95% CI for this small cohort was 6.1 (1.7e15.8).
Fig. 3. Solid cancer risk patterns for in-utero and childhood exposures e atom bomb survivors. The graphs indicate that there is a clear and signicant difference in the risk of tumor
development in the embryo-irradiated and childhood-irradiated populations. During the course of the 55 year post-radiation period tumors are accumulating at a much lower rate
in the irradiated in-utero population. ERR, excess relative risk; PY, person-years; CI, condence interval. Reproduced with permission from Preston et al. [67].
New England Journal of Medicine wrote that It seems likely that the
question of the association between fetal irradiation and childhood
cancer will fade into medical history unresolved and remain a
source of more confusion than enlightenment.
There is little disagreement with the concept that radiation may
represent a carcinogenic risk to the embryo and adult and that
there may be different risks per rad at different stages of devel-
opment. The concept that is difcult to explain from a basic science
viewpoint is, Why would embryonic cells be orders of magnitude
more sensitive to radiation-induced cancer than cells of children or
adults?
Recent publications have provided added perspectives, data,
and interpretations of the prior publications [78e86]. Boice and
Miller [78] point out that numerous epidemiologic studies have
been performed. Positive associations for an increased incidence of
cancer after in-utero diagnostic radiation exposures have been
derived almost exclusively from caseecontrol studies (Tables 1e3),
whereas almost all of the cohort studies have found no association
(Figs. 3 and 4). It is of great interest that the in-utero atomic bomb
population did not demonstrate an increase in childhood leukemia
despite the fact that many in the in-utero population were exposed
to high doses of acute irradiation.
Because many of the positive associations have been derived
from caseecontrol studies, the questions of confounding factors
have been raised to explain the ndings. Twin studies have been
used to eliminate some of the confounding factors [87e89].
Although the reports of Mole [89] and Harvey et al. [87] were
positive, the Rodvall study [88] was not statistically signicant.
The most recent estimates of the carcinogenic risk of in-utero
radiation have moved from two extremes. The rst viewpoint
popularized by Stewart [8,10] suggested a risk of one or two orders
greater than the carcinogenic risk of postnatal exposure to children
and adults. Based on animal data and the Radiation Effects Research
Foundation (RERF) data, it appeared that the embryo and fetus may
actually have a lower risk than children exposed postnatally. Boice
and Miller [78] have concluded that the more recent data have
reduced the discrepancy between these two extreme viewpoints.
Based on the reports of Muirhead and Kneale [82] and Mole [89]
they believe that The risk estimate associated with intrauterine
radiation is not substantially greater than that seen following
childhood irradiation.
Doll and Wakeford [90] expressed their opinion about the
carcinogenic effect of intrauterine radiation and concluded that
Irradiation of the fetus in utero increases the risk of childhood
cancer, and increases the risk fromexposures of the order of 10 mGy,
and that in these circumstances the excess risk is w6% per Gy.
The atomic bomb survivors of in-utero irradiation have been
followed into adulthood, and the incidence of cancer in this pop-
ulation has been studied [86]. Although some of the exposures to
this population of pregnant women was considerably higher than
the exposure from the population exposed to radiation from
pelvimetry, there was only a small excess of adult tumors among
the atomic bomb survivors exposed in utero (Tables 2 and 3).
Boice and Miller [78] conclude:
Learned debate continues as to the causal nature of low level
intrauterine radiation and subsequent cancer risk. The associa-
tion is not questioned, but the etiologic signicance is. Different
scientists interpreting the same data have different opinions as
to the causal nature of the association and the possible level of
risk.
100
10
1
Fetus 1 10 20
Age (years)
Stewart et al.
810
ABCC
28,38,55
Animal data
1,2,61,6466,108
R
i
s
k

o
f

l
e
u
k
e
m
i
a

p
e
r

1
0
6

p
e
r
s
o
n
s
f
o
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o
w
i
n
g

0
.
0
1

t
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0
.
0
2

G
y
incorrect
30 40 50 60 70
Fig. 4. Risk of leukemia in children following radiation exposure during pregnancy.
This graph illustrates the modication proposed for Fig. 2, which indicates that the risk
of leukemia from radiation of the embryo/fetus is not one to two orders greater than
for the child, but actually lower than the risk for the child.
Table 3
Risk of leukemia.
a
Group Risk Latency
Identical twin of a leukemic twin 1:5 Weeks to months
Radiation-induced polycythemia 1:6 10e15 years
Bloom syndrome 1:8 <10 years of age
Hiroshima survivors <1000 m
hypocenter
1:60 3e12 years
Down syndrome 1:95 Weeks to months
Radiation treatment of ankylosing
spondylitis
1:270 15 years
Siblings of a leukemic child 1:720 10 years
Combined background risk of
leukemia plus radiation risk
from Stewart
1:2000 10 years
Additional risk of in-utero
diagnostic radiation studies
(Stewart et al. [8])
1:6000 10 years
In-utero diagnostic radiation
(RERF) data and other cohort
studies
Risk the same for
exposure during
childhood but
actual risk is
uncertain (Miller
[72]; Brent [73])
Lifetime
US Caucasian aged <15 years 1:3000 10 years
RERF, Radiation Effects Research Foundation.
a
Modied from Miller et al. [69].
Box 1
Arguments supporting a causal association between prenatal
radiation and childhood leukemia and cancer
1. Consistency. Practically all studies are statistically
consistent, with relative risk of 1.40 for leukemia
[23,66,91].
2. Dose response. Risk of childhood cancer was found to
increase with number of X-ray lms [68].
3. Coherence. Apparent lower risk of childhood cancer in
birth cohorts born in years when dose per lm was lower
[66,92].
4. Recall bias is unlikely to be a major factor [45].
5. Confounding variables have been sought, but none has
been found [68,34].
6. Selection bias related to reason for radiographic exami-
nation is not supported by caseecontrol studies of twins
[87,89].
7. Risk estimates after intrauterine exposures are generally
comparable to risks after childhood exposures for leu-
kemia [82,91].
Boxes 1 and 2 summarize the opinion of John Boice and Robert
Miller regarding the controversy pertaining to the carcinogenic risk
of ionizing radiation exposure to the developing embryo following
the National Council on Radiation Protection and Measurements
(NCRP) annual meeting dealing with the developmental, repro-
ductive, carcinogenic and mutagenic risks of ionizing radiation
published in 1999 [78].
During the period from 1997 to 2013 there were other publi-
cations dealing with the carcinogenic risk of exposing the embryo
to ionizing radiation besides Boice and Miller [78], the RERF pro-
gram in Japan [67] and the publications of Wakeford
[66,90,100,101]. Wakeford continued his estimate of the risks of
cancer following in-utero radiation and persisted with the
conclusion that the embryo was more vulnerable to the carcino-
genic effects of ionizing radiation than the risks in exposed
children.
The most important recent publication dealing with the carci-
nogenic risks of in-utero radiation was that by Preston et al. [67]
ICRP 90 was published in 2003 and was titled Effects of prenatal
irradiation (embryo and fetus). In 2013 an update of NCRP Hand-
book 54 was published as NCRP Report 174, Preconception and
prenatal radiation exposure: health effects and protective guid-
ance. [102] Dr Martha Linet had nal responsibility for the section
on oncogenesis. Contributions and suggestions were submitted by
other committee members to Dr Linet. Dr Roy Shores critiques
from the RERF program in Japan were particularly helpful.
Before reviewing the Preston et al. [67] the data pertaining to
the caseecontrol and cohort studies are examined.
2.1. Caseecontrol and cohort studies
Alice Stewart and her colleagues were the rst to indicate that
diagnostic radiological studies of pregnant women could signi-
cantly increase the risk of cancer in the offspring [9,10]. Similar
caseecontrol studies were performed in eight other countries and
they are summarized in Table 1. Some investigators were skeptical
of Dr Stewarts conclusions because the results were partly based
on medical histories obtained fromthe mother rather than fromthe
medical records. However, later analyses utilized primarily medical
records and the increased RR > 1.0 persisted. Attempts to deter-
mine the actual fetal exposure of the pregnant women were not
successful, since the exposures were never measured on the
pregnant women whose offspring were part of the analysis.
There are 35 caseecontrol studies listed in Table 1 dealing with
childhood lymphoblastic leukemia, acute myeloid leukemia, all
leukemias, CNS tumors, neuroblastoma, bone tumors, Ewing sar-
coma, rhabdomyosarcoma and total childhood cancer. Twenty-ve
of the caseecontrol studies were not statistically signicant. Ten of
the studies with RR >1 were statistically signicant. The consensus
was that the caseecontrol studies supported a RR of 1.2e1.3 based
on a meta-analysis [101].
3. Epidemiological cohort studies (Table 2)
There were 17 cohort studies of the offspring of womenwho had
been exposed to radiation during their pregnancy. None of the
studies individually reported a statistically increased RR for cancer
in the offspring. Cohort investigations to assess childhood cancer
risks among those undergoing diagnostic X-ray procedures
involving in-utero exposure included radiation-exposed pop-
ulations ranging in size from <200 to nearly 40 000 children. The
largest cohort study in this group was the report of Court Brown
et al. [50] This article was published in 1960 just a few years after
Giles et al. [9] and Stewart et al. [10] had indicated that the embryo
may be much more vulnerable to the carcinogenic effects of radi-
ation. There were 39 166 exposed and more than 1.5 million un-
exposed. There were nine leukemia subjects in the radiated
population and 14 in the controls [RR: 0.9; 95% condence interval
(CI): 0.4e1.6]. None of the cohort studies was statistically signi-
cant. The problemof cohort studies is that very large populations of
exposed individuals are needed, which was not the case with most
of these cohort studies.
4. Environmental exposures: atomic bomb survivors (Preston
et al. [67])
Although there was no evidence of a dose-related increase in
cancer mortality at ages prior to 15 years of age among the w2500
persons who were in utero at the time of the bombings [103], as the
cohort has grown older, a statistically signicant excess relative risk
(ERR) of solid cancers became apparent (ERR: 2.1 Gye1; 90% CI:
0.2e6.0), based on 10 deaths among those with weighted uterine
doses >0.01 Gy [80]. In a follow-up of cancer mortality risks during
1950 to 1992 comparing risks among a subset of persons who were
in utero versus those who were 0 to <6 years of age at the time of
the bombings, there were only two deaths from leukemia (both
exposed to relatively low doses and none during childhood) in the
in-utero cohort versus 24 among children <6 years of age at
exposure (Fig. 3) [80].
Subsequently, Preston et al. [67] compared solid cancer inci-
dence risks among in-utero cohort members aged 12e55 years
during 1958 to 1999 (based on 94 cancers) with risks among sur-
vivors who were aged <6 years of age at the time of the bombings
(based on 649 cancers). The difference in ERRs and excess absolute
risks (EARs) between the two cohorts suggests that lifetime cancer
risks at age 50 years following in-utero exposure are lower than
risks for early childhood exposures. However, the investigators
state, Additional follow-up of this cohort is necessary before
denitive conclusions can be made about the nature of the risk for
those exposed in utero [67] (Figs. 3 and 4). The difference in ERRs
and EARs between the two cohorts suggests that lifetime cancer
risks at 50 years of age following in-utero exposure are lower than
risks for early childhood exposure.
However, the investigators state that additional follow-up of
this cohort is necessary before denitive conclusions can be made
about the nature of the risks for those exposed in utero.
67
The
Box 2
Grounds for uncertainty regarding the causal nature of the as-
sociation between prenatal radiation and childhood cancer
1. Atomic bomb in-utero study nds no excess of childhood
cancer deaths [93], whereas a lower limit of 5.2 extra
cancer deaths was predicted from the risk model based
on obstetric X-ray data [7]. The central estimate of excess
cancer deaths predicted was about 10.
2. All major cohort studies are negative [22,50,91].
3. Biological implausibility; the equality of relative risks
associated with obstetric X-rays for leukemia and solid
tumors is perplexing given the variability in tissue
radiosensitivity, dissimilar origins, and different inci-
dence patterns [94,95]. The extended MacMahon study
did not nd an increased risk for solid cancers [34].
4. Risk estimates appear greater for in-utero versus
newborn exposures, for solid cancers [91].
5. Twin cohorts have lower risk of childhood cancer than
singletons despite more frequent X-rays [91,96,97].
6. Supporting animal evidence is weak [95,98,99].
investigators also note that this study cannot provide information
on the effect of radiation on the incidence of childhood cancers
because comprehensive data on solid cancer incidence are un-
available for the period from 1945 to 1957. Mortality follow-up for
the in-utero cohort, however, was available from 1950 and indi-
cated no deaths from childhood leukemia [80]. Another limitation
is the small numbers of cancers in each dose category in the in-
utero cohort.
Nevertheless, this investigation is the only cohort study with
long-term, continuous, active follow-up of a population with in-
utero radiation exposure and high-quality estimated doses for
each subject.
5. Animal studies
Many animal studies were performed in which pregnant ani-
mals were irradiated with ionizing radiation and the risk of cancer
in the offspring was evaluated in the offspring. A major problem
with these studies is that very few of the protocols utilized expo-
sures in the diagnostic range of clinical X-ray studies (<0.10 Gy). So
very few of the studies were planned to answer the question of the
risk of diagnostic radiation to pregnant women.
Several studies have reported excess risks of various tumors in
mice after in-utero irradiation, mostly after whole-body doses
higher than 2 Gy. Offspring of BC3F1 mice who received whole-
body in-utero doses (17th day post coitus) ranging from 0.3 to
2.1 Gy (41e58 animals in each dose group) developed small in-
creases in liver tumor occurrence [104]. Offspring of B6C3F1 mice
exposed to a whole-body dose of 3.8 Gy in utero developed
increased risk of pituitary, ovarian, liver, and bone tumors; an in-
crease in lung tumors was statistically signicant after doses of 1.9,
3.8, and 5.7 Gy; and an elevation in malignant lymphoma, lym-
phocytic type, was statistically signicant after 5.7 Gy [105].
In-utero irradiation [0.3 and 1 Gy (X-rays, whole body)] during
day 10.5 postconception (PT HT F
1
) did not induce an increased
incidence of neoplasms in the offspring [106].
Studies assessing tumor risks in different strains of mice
demonstrate high susceptibility of the ovaries for radiation-related
tumor induction during the fetal period, with 0.25 Gy the lowest
dose associated with a statistically signicant increase
[105,107,108].
Other investigations found no excess cancer after in-utero
irradiation of mice with 3 or 2 Gy [99,109], although each of
these studies showed increased risks of cancer in the mice
following administration of similar doses postnatally. Although
investigators found no excess cancer in BC3F1 mice after
in-utero exposures to 0.3 Gy, increased risks were seen in mice
given the same dose postnatally [84].
Rugh et al. [110] in a very large study irradiated mice with 1 Gy
on each day post conception and observed the incidence of tumors
in the offspring as adults. There was no statistically signicant in-
crease in the incidence of tumors in adult animals from irradiation
in utero on any day. Brent and Bolden [6] exposed pregnant mice to
doses of 0.3, 0.6, and 0.9 Gy at 0.5, 7.5, 8.5, 12.5, and 16.5 days post
conception. They also did not observe an increase in the incidence
of tumors. However, the pre-sexually mature mouse was more
vulnerable than the adult mouse to the leukemogenic effect of
radiation.
Offspring of pregnant beagles treated with mean doses of 0.16 or
0.81 Gy at 8th, 28th, or 55th days post coitus (120 dogs in each dose
and treatment day group) experienced increases in mortality from
total cancers that were not statistically signicant, and statistically
signicant elevated mortality risks from lymphoma. Detailed
assessment revealed that the increased risk of fatal neoplasms was
most pronounced in beagles irradiated in the neonatal period [111].
These data suggest that irradiation in both the fetal and neonatal
periods are associated with an increase of early onset and lifetime
cancer risk. However, the lower-dose group (0.16 Gy) did not have
an increased incidence of tumors.
Warkany et al. [112] studied the interaction of ethylnitrosourea
and X-irradiation in rats. The original goal of the investigators was
to determine the effect of X-irradiation administered on the 16th
day post conception on the incidence of tumors following the
administration of ethylnitrosourea on the 20th day post conception.
Sixteen months after delivery 62.2% of the rats that had received
only the ethylnitrosourea during the fetal period had neurogenic
tumors. After fetal irradiation on the 16th day post conception,
followed by ethylnitrosourea 4 days later, 16.7% of the rats devel-
oped neurogenic tumors. The mechanisms of these unexpected
ndings, whereby irradiation before receiving an oncogenic drug
reduced the incidence of cancer, have not been determined.
Nakano et al. [113] irradiated mice at various stages of preg-
nancy with 1 or 2 Gy. Translocation frequencies in the peripheral
blood T-cells, spleen cells, and bone-marrowcells were determined
when the offspring were aged 20 weeks. The translocation fre-
quency was very low in the mice that were irradiated in utero
(0.8%). The mice irradiated during days to weeks after birth had
translocation frequencies of 5%. The authors suggested that the
abnormal cells in the fetus were replaced by normal fetal stem cells
during the postnatal growth of the animal. If this phenomenon
occurs in humans, it could explain why the fetus may be less
vulnerable to the oncogenic effect of radiation than the child.
Earlier research supporting the ndings of Nakano et al. [113] found
that X-irradiation of the rat embryo during early organogenesis
resulted in the production of hundreds of small growths that
resembled well-differentiated ependymomas or retinoblastomas
(Fig. 1) [1,2]. As the embryo developed, some of the tumors dedif-
ferentiated into more primitive growths. However, at term almost
all the cytogenetically abnormal cells had regressed esimilar to the
result reported by Nakano et al [113].
6. Counseling patients about the in-utero carcinogenic risks
of ionizing radiation
Although it is our opinion that a dose of <0.10 Gy to the
implanted embryo does not result in a signicant increase risk for
congenital malformations, intrauterine growth restriction, or fetal
death (deterministic effects), low-risk tumorigenic or genetic haz-
ards cannot be ruled out. Even if one believed that the tumorigenic
(leukemogenic) effects of low-level radiation were real, let us
examine how difcult it would be to use this information in
counseling a patient who has received a dose of perhaps 2 rad
(0.02 Gy) during her pregnancy. According to Stewart et al. [7,8,10],
the risk of leukemia after this exposure in utero is 1:2000 versus
1:3000 in unexposed controls over a 10-year period (Table 3). If one
were inclined to recommend therapeutic abortion for this preg-
nancy because the probability of developing leukemia is 50%
greater than controls, one would perform abortions in almost 2000
exposed non-leukemic subjects for every leukemic subject saved.
It is one thing to avoid radiation because of a potential or conjec-
tured hazard, but it is another matter to recommend therapeutic
abortion on this basis. If a physician were inclined to accept this
increased probability (1:2000) as a risk great enough to recom-
mend therapeutic abortion, he or she would be placed in a serious
dilemma because there are other epidemiologic situations in which
the risk of leukemia is greater. In fact, the hypothetical incremental
risk for 2 rad of in-utero radiation is 1:6000 over a 10-year period. It
is the combination of the control risk plus the incremental radiation
risk that results in a 1:2000 risk for these patients. From Table 1 it
should be clear that the risk of leukemia is greater in unirradiated
siblings of leukemics (1:720) than in patients subjected to diag-
nostic radiation (1:2000) if one uses Stewarts risk estimate.
Certainly, the position that all future pregnancies of parents
with one leukemic child should be aborted would be untenable.
One can carry this argument to its ridiculous extreme by advocating
that all pregnancies should be aborted because of the risk of serious
malformations is w30 per 1000 deliveries (Table 3) and this does
not include the probability of postnatal diseases occurring in these
offspring. Some may interpret this as a facetious discussion, but the
clinician and the patient must recognize that spontaneous risks of
pregnancy are two orders of magnitude greater than the theoretical
risks of diagnostic radiation (Box 1).
7. Conclusions and counseling advice
The radiation risks determined from the A-bomb exposure to
the populations in Hiroshima and Nagasaki have been referred to as
the "gold standard" for determining radiation risks because the
study was a large cohort study with a major effort to determine the
actual exposure of each survivor. Yet we know that if individuals or
populations are exposed to x-rays or gamma rays of different
photon energy or length of exposure, the risks may deviate from
the risks determined for the A-bomb survivors. Furthermore, there
was a neutron component in the radiation from the A-bomb det-
onations. So one can assume that exposures to the embryo from an
IVP or uoroscopy in a pregnant mother may represent a different
risk to the embryo per mGy than the A-bomb data. So that is why it
is problematic to provide denitive carcinogenic risks for diag-
nostic radiologic studies utilizing x-rays or radionuclides. This
article has discussed the subject of the carcinogenic risk of ionizing
radiation to the embryo.
1. There is no doubt that if the exposure is high enough and of
short duration, the carcinogenic risks are increased. Protracted
continuous radiation of 0.02 Gy/day does not represent an
increased carcinogenic risk. Acute radiation of 1.0 Gy at mid-
gestation does represent an increased carcinogenic risk.
2. The embryo is less vulnerable to the carcinogenic effects of ra-
diation the earlier in gestation it is exposed.
3. There are at least three viewpoints on the carcinogenic risks of
<0.10 Gy embryonic radiation.
(a) There is the scholarly, conservative view of Martha Linet
who writes that the risk is very small and would not justify
canceling a radiological study in a pregnant woman if the
study is medically indicated. She also suggests that we wait
to determine whether the risk increases based on future
data from the Preston et al. study, which stated that addi-
tional follow-up of this cohort is necessary before denitive
conclusions can be made about the nature of the risks for
those exposed in utero. [67]
(b) Richard Wakeford has been interested in this subject for
decades. We rst met many years ago when we were de-
fense experts in litigation between the UK and Ireland
regarding the allegation that the Sellaeld Nuclear Facility
was discharging nuclear waste that was responsible for an
increase in cancer and birth defects in the inhabitants on the
East Coast of Ireland. The World Court deliberations ended
after 10 years with a defense verdict. Wakefords views have
changed with time. One of his most recent publications in-
dicates that he believes that 20% of childhood leukemia in
the UK is due to background radiation [114]. He still is the
proponent of the idea that the embryo is more vulnerable to
the carcinogenic effects of radiation than the child.
(c) I am not one who is reluctant to make predictions. I agree
with Martha Linet regarding the risks of embryonic ionizing
radiation. However, I would predict that in the next 20 years
we will learn that the risk of cancer from embryonic radia-
tion will be further reduced from the ndings of the Preston
et al. 2008 study. At my present age I will not be alive to
know the results. I believe that the omnipotential (stem)
cells protective effect that was present in the embryo at the
time of the radiation will continue to be manifested.
7.1. Counseling an individual patient
If a pregnant woman has had a diagnostic radiological proce-
dure that exposed her embryo or who has been scheduled for an X-
ray that will expose her embryo and is concerned about the
increased risk of cancer fromthe exposure, howshould a counselor
respond?
The majority of diagnostic radiological studies expose the
embryo to <0.10 Gy (<10 rad) which is a very low exposure. Based
on all the studies we have available, the risk of cancer to the embryo
is very low and possibly so low that we may never be able to
measure the risk. Therefore, diagnostic radiological studies that are
considered to be important for optimal patient care should be
performed. It is important to be aware of the background risk of
cancer for all individuals, which is 23% for potentially lethal can-
cers. Fortunately, each year the percentage of cancers that are cured
is increasing. The background risk of cancer is hundreds or more
times the theoretical risks of diagnostic radiological exposures to
the embryo.
None declared.
Funding sources
None.
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Review
New genetic testing in prenatal diagnosis
Natalia Babkina, John M. Graham Jr.
*
Medical Genetics Institute, CedarseSinai Medical Center and Division of Medical Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
Keywords:
Chromosomal microarray
Exome sequencing
Next-generation sequencing
Non-invasive prenatal screening
Preimplantation genetic testing
s u m m a r y
Determining a genetic diagnosis prenatally permits patients to make informed reproductive decisions
and to be counseled about possible fetal outcomes. Therefore, it is important for the provider to be aware
of the spectrum of genetic conditions and to use appropriate testing modality to obtain specic diag-
nosis. This article reviews genetic techniques available for prenatal diagnosis such as preimplantation
genetic testing, chromosomal microarray, non-invasive prenatal screening, and next-generation
sequencing. Chromosomal microarray has emerged as the rst diagnostic test for evaluation of multi-
ple congenital anomalies and developmental delay as most of the next-generation sequencing methods
do not detect copy-number variants (CNVs). Exome sequencing and whole genome sequencing are time-
consuming, so if this needs to be done to obtain an accurate genetic diagnosis, allow sufcient time.
1. Introduction
Prenatal diagnosis plays an essential role in contemporary
obstetrical care, and with proper planning, it can be available for
pregnancies at risk for chromosomal and single-gene disorders.
Standard chromosome testing has been available since the 1960s
[1] and has been commonly used in the prenatal setting, when a
fetus has abnormal ndings on ultrasound. Prenatal genetic testing
has now become much more sophisticated with an improved level
of resolution. Standard chromosome testing is being superseded by
the use of chromosomal microarrays. Most forms of prenatal
diagnosis require invasive procedures for fetal-sample collection
and therefore, although considered safe, these procedures involve a
risk of fetal loss. The presence of cell-free fetal DNA in maternal
circulation has been used as a basis for the development of
non-invasive prenatal testing [2]. Application of the latest tech-
nologies, such as next-generation sequencing, which features both
high sensitivity and accuracy, is allowing for more accurate pre-
conception counseling when there is a previously affected relative,
thereby broadening the scope of prenatal diagnosis to include
many diseases that are both paternally and maternally inherited.
Determining genetic diagnosis prenatally permits patients to
make informed reproductive decisions and to be counseled about
possible fetal outcomes, management options and recurrence
risks. Therefore, it is important for the physician to be aware of the
full spectrum of genetic conditions, and to use appropriate testing
and referrals to genetic healthcare providers in order to obtain a
specic diagnosis.
2. Preconception counseling
The prevalence of paternal and maternal conditions that are
relevant to pregnancy outcomes varies according to many factors,
such as parental age, ethnicity, medical history, and family history.
Family history plays a critical role in assessing the risk of
inherited medical conditions and single-gene disorders [3]. In
general practice, the family history can be obtained using a
questionnaire or a three-generation pedigree. A family history
screening allows stratication of the risk level. Also, the use of a
family history screening has been shown to increase the likelihood
of detecting a patient at high risk of developing an inherited
medical condition by 20%, compared with medical record review
alone [4]. Family history of developmental delay, congenital mal-
formations, or other constellation of clinical ndings suggestive of
a genetic condition requires thorough evaluation. If a disorder in
the individuals family has been identied as having a genetic
cause, it may be possible to test parents to determine the risk for
having an affected child. For example, a family history of known
genetic conditions, such as TayeSachs or cystic brosis, should
prompt testing for at least the person with an affected relative. It is
paramount to identify the mutation in an affected individual, so
prenatal diagnosis or preimplantation genetic diagnosis in cases of
in-vitro fertilization (IVF) can be performed in order to test spe-
cically for an identied familial mutation and decrease the risk of
affected pregnancy. A history of recurrent pregnancy loss should
* Corresponding author. Address: 8700 Beverly Blvd, PACT Suite 400, Los Angeles,
CA 90048, USA. Tel.: 1 310 423 9909; fax: 1 310 423 2080.
E-mail address: john.graham@cshs.org (J.M. Graham).
prompt testing of both parents for chromosomal translocations. A
parent with a balanced translocation identied by standard
chromosome analysis can produce normal, balanced or unbal-
anced gametes that result in normal, balanced or unbalanced
fetus. Recent data show that apparently balanced chromosomal
rearrangements are associated with an abnormal phenotype in
6.7% of cases that may be due to cryptic genomic imbalances or to
the disruption of genes at the breakpoints [5].
Whereas some genetic conditions that affect pregnancy out-
comes are easily identied early in life, others are not and may
require additional diagnostic testing. Up until about 10 years ago,
w65e70% of patients with congenital malformations remained
undiagnosed, including many patients with multifactorial defects
(e.g. non-syndromic cleft lip with or without cleft palate or neural
tube defects) or polygenic defects (e.g. Hirschsprung disease or
hypospadias), 15e25% were thought to be genetic or genomic, and
10% were thought to be environmentally determined [6].
Previously, the diagnostic yield for children with intellectual
disability varied between 50% and 80%, with 17e47% having a ge-
netic/genomic cause and 20e50% remaining undiagnosed [7]. With
the advent of high-resolution chromosomal microarrays, an addi-
tional 10e15% of such patients receive a genomic diagnosis (which
is often a sporadic occurrence in an otherwise normal family) [8,9].
As the use of exome sequencing has increased over the last 2 years,
most laboratories are reporting an additional 25e40% success rate
in obtaining a genetic diagnosis (some of which are also sporadic).
The practical implication of all these recent developments is that
with modern genomic/genetic diagnostic techniques, up to 70e80%
of patients can receive a genetic/genomic diagnosis, with an addi-
tional 10% attributed to environmental inuences and maternal
conditions. Multifactoral defects such as isolated congenital heart
defects or orofacial clefts have had a large number of genetic and
environmental inuences identied, but genetic diagnosis for
multifactoral defects is not currently available for clinical use.
Fortunately, recurrence risks are relatively low, in the 3e5% range,
and this recurrence risk can be reduced by up to 50% through use of
folic acid prior to conception and during early pregnancy (covered
elsewhere in this volume). Exome sequencing can be quite useful
for polygeneic or conditions manifesting genetic heterogeneity.
Ancestry inuences the probability of being a carrier of many
disorders that affect pregnancy. Typically, there is no family history
of the condition as the carriers are asymptomatic. For example,
using the current recommended mutation panel, negative carrier
testing for cystic brosis in a Caucasian couple would result in a
different chance of having an affected child than negative testing in
an African-American couple since the carrier rate and the type of
mutations are different in these two populations.
Parental age is an important risk factor for adverse pregnancy
outcomes. The risk of chromosomal non-disjunction increases with
increasing maternal age that translates into higher risk of trisomy
21, 13 and 18. Recent data emphasize the importance of paternal
age as a risk factor for newdominant mutations. The male germline
accumulates point mutations due to replication errors and reduced
activity of repair enzymes, strands mispairing of short tandem re-
peats and longer exposure to environmental mutagens [10]. In
addition, in human sperm DNA is more methylated than oocyte
DNA, which may account for the greater number of paternally
derived point mutations occurring within a CpG dinucleotide [11].
Because of the large number of cell divisions during spermato-
genesis, the mutation rate for base substitutions is much higher in
men than in women, and increases with paternal age. The risk for
de-novo autosomal dominant mutations calculated by Friedman
was 0.3e0.5% among the offspring of fathers aged >40 years [12].
Recently, with the use of whole-genome sequencing the increase in
the rate of de-novo mutations has been estimated to be two
mutations per year [13]. The conditions most strongly associated
with advanced paternal age are those caused by point mutations in
the FGFR2, FGFR3 and RET genes and include Pfeiffer syndrome,
Crouzon syndrome, Apert syndrome, achondroplasia as well as
multiple endocrine neoplasia type 2A (MEN2A) and multiple
endocrine neoplasia type 2B (MEN2B) [14]. Certain dominant
conditions such as neurobromatosis type 1 (NF-1) caused by point
mutations or small deletions show a lesser association with
paternal age. Some genetic conditions such as Noonan syndrome,
Apert syndrome and MEN2B demonstrate germline mosaicism as a
result of selection in male germline stem cells that explains an
association of these conditions with advanced paternal age [15].
There is also a growing body of evidence that advanced paternal
age is associated with an increased risk for complex disorders such
as certain congenital anomalies, schizophrenia, and autism spec-
trum disorders [16,17]. For autosomes and sex chromosomes, there
is no strong evidence that aneuploidy is signicantly increased in
newborns as paternal age increases but two possible exceptions are
trisomy 21 and Klinefelter syndrome as recent data suggest a
paternal effect, either acting alone or in combination with a
maternal age effect [18].
Therefore, genetic risk stratication based on family history,
ethnicity and parental age is paramount for preconception
counseling. Increased awareness of the importance of using family
history as a screening tool, and of the value of preventive measures
and increased surveillance, can improve the outcomes. The avail-
ability of advanced technology allows for the identication of ge-
netic etiologies in many conditions for which specic diagnostic
testing is currently available.
3. Preimplantation genetic testing
Preimplantation genetic testing comprises all types of genetic
testing performed on the embryos obtained from an IVF cycle and
was rst described by Handyside et al. [19] when the sex of the
embryo was determined in two cases with a history X-linked ge-
netic disorders. Preimplantation genetic testing is divided into two
categories: preimplantation genetic screening (PGS) and preim-
plantation genetic diagnosis (PGD).
PGS is performed on the embryos obtained from the parents
with presumed normal karyotypes. Chromosomal aneuploidy is a
major factor in implantation failure and spontaneous abortions. It
was demonstrated that half of the embryos produced in vitro had
chromosomal abnormalities that signicantly decreased the im-
plantation rate [20]. PGS is used as an embryo screening method for
aneuploidy, as the morphological analysis is not reliable for aneu-
ploidy prediction. The most common method used for PGS is
analysis of the embryonic cells on day 3 after fertilization with
uorescent in-situ hybridization (FISH). Fast turnaround times and
high accuracy are the advantages of PGS with FISH but the limited
number of chromosomes that can be evaluated is a signicant
limitation. Prospective trials of PGS by FISH have not demonstrated
any improvement in pregnancy rates [21]. Comprehensive aneu-
ploidy screening using whole-genome array showed that aneu-
ploidy may occur in any of the 23 pairs of chromosomes. Recent
retrospective studies showed that PGS using genome-wide
approach and testing for all 23 pairs of chromosomes could
improve the pregnancy outcome in certain groups or patients.
Specically, in women aged >35 years with a history of recurrent
pregnancy loss, PGS is associated with reduced rst-trimester
spontaneous abortion rate [22]. However, prospective random-
ized trials have not demonstrated a denitive benet of PGS and
therefore, currently, PGS is not recommended for routine use.
PGD involves testing the embryos for a specic genetic disorder.
PGD requires prior identication of the genetic cause of the
N. Babkina, J.M. Graham Jr. / Seminars in Fetal & Neonatal Medicine 19 (2014) 214e219 215
condition in the family. Multiple conditions can be tested for,
including cystic brosis [23], Huntington disease [24], achondro-
plasia [25] and many others. PGDwas initially developed to identify
the embryos that carried genes for serious, childhood-onset dis-
eases. Recently, the American Society for Reproductive Medicine
reported that PGD for adult-onset conditions is ethically justiable
when the condition is serious and there is no known intervention
for the condition, or the available interventions are either inade-
quately effective or signicantly burdensome, thus justifying
testing for Huntington disease, Alzheimer disease, and cancer
susceptibility genes (e.g. BRCA1 and -2) [26].
Depending on the nature of the condition, different methods
can be used, such as FISH and chromosomal microarrays for CNVs
and DNA sequencing for single-gene disorders with known muta-
tion in the parent. Most commonly, the biopsy of the embryo is
performed on day 3 or day 5 after fertilization. Blastomere stage
biopsy on day 3 is associated with higher risk of damaging the
embryo and misdiagnosis secondary to possible mosaicism [27],
whereas trophectoderm biopsy performed at the blasctocyst stage
on day 5 is associated with better results and more accurate diag-
nosis [22,27]. Although the risk of mosaicism at blastocyst stage is
lower than at the blastomere stage, there still might be a discrep-
ancy between results in the trophectoderm that further develops
into placental tissue and the inner cell mass that becomes a fetus
(e.g. conned placental mosaicism). Therefore, even with the
availability of advanced techniques, the risk of embryo misdiag-
nosis exists, and it is currently recommended to offer chorionic
villus sampling (CVS) or amniocentesis to rule out genetic diagnosis
in the fetus, especially when there is a proven family history of a
genetic condition.
4. Standard karyotype and chromosomal microarray
Routine microscopic analysis of chromosomes (standard kar-
yotype) has been used for more than 50 years to detect large
chromosomal abnormalities such as translocations, deletions or
duplications, but it cannot identify genomic imbalances
<5 Mb. Submicroscopic deletions and duplications are identied in
15e20% of cases of autism and intellectual disability, thus chro-
mosomal microarray became a rst-line diagnostic test for the
evaluation of congenital anomalies and intellectual disability
postnatally [9]. Genomic microarrays provide a genome-wide
screen for genomic imbalances at a high resolution, allowing for
the detection of all known recurrent microdeletion and micro-
duplication syndromes when using resolution of 400 kb for older
arrays versus 7.7 kb for single nucleotide polymorphism (SNP) ar-
rays. Current microarray technology also allows genome-wide
detection of microscopic and submicroscopic copy number
changes <100 kb. The use of DNA-based microarrays eliminates the
need to use cell culture, decreases overall turnout time, and in
general is less labor intensive [28,29].
Microarray technology is based on hybridization of labeled pa-
tient DNA with specic probes with known genomic coordinates.
The measurement of signal intensity ratio of patient DNA to refer-
ence DNA from individual probes allows identication of gains or
losses of chromosomal material. Proof of principle for the use of
microarray in prenatal diagnosis was rst described by Rickman
et al. [28] DNA extracted from uncultured amniotic uid cells was
used for comparative genomic hybridization (CGH). Fetal DNA from
CVS specimens has also been shown to be suitable for performing
CGH. The feasibility of microarray analysis for prenatal detection of
CNVs was demonstrated using different oligonucleotide array CGH
platforms, as well as SNP arrays [30,31]. SNP arrays have additional
oligonucleotide probes in order to provide more even coverage of
the genome and improved detection of CNVs. The additional probes
for SNP array also allow genotyping based on allele frequency and
detection of copy-neutral runs of homozygosity, chromosomal
mosaicism, and uniparental disomy. Although SNP arrays can
detect uniparental isodisomy, parental parenatal samples are
required for the detection of uniparental heterodisomy [31]. By
design the microarrays can be further subdivided into targeted and
whole-genome arrays. Targeted arrays have the most dense probe
coverage in regions of the genome associated with known micro-
deletion/duplications syndromes or genes known to have clinical
implications for human disease. Whole-genome arrays have evenly
distributed probe coverage and a higher overall resolution.
A number of prospective and retrospective studies testing the
efcacy of microarray technique in detection of clinically signicant
chromosomal gains or losses during prenatal diagnosis have been
performed. The additional diagnostic yield for clinically signicant
CNVs has ranged from 1.3% to 5% [29,30,32]. Most of the studies are
small and include women at risk of having a fetus with chromo-
somal abnormalities based on evidence of structural defects
detected on ultrasound. The largest prospective study includes
4406 women who underwent prenatal diagnosis secondary to
advanced maternal age, abnormal screening results or abnormal
ndings on prenatal ultrasound. In samples with a normal karyo-
type, microarray analysis revealed clinically relevant deletions or
duplications in 6% with a structural anomaly and 1.7% of those
whose indications were advanced maternal age or positive
screening results [33].
However, microarrays provide no information about the struc-
ture of the genome, so structural rearrangements that are copy
number neutral, such as balanced translocations or inversions,
cannot be detected by array and require the use of other techniques.
Although inability to detect balanced rearrangements with array is
considered a limitation, truly balanced translocations without gain
or loss of material are less likely to be associated with a clinically
signicant phenotype in a neonate. However, apparently balanced
rearrangements identied by chromosomal analysis are associated
with a 6.7% risk of abnormal phenotype in a neonate that are
usually caused by a genomic gain or loss at breakpoints, that are not
detected with standard cytogenetic analysis and may be due to
cryptic genomic imbalances or to the disruption of genes at the
breakpoint. Array is useful not only in identifying the genomic
imbalances at the breakpoints, but also in detecting unexpectedly
complex rearrangements in other chromosomes. Schluth-Bolard
et al. used next-generation sequencing to locate breakpoints at
the molecular level in four patients with multiple congenital
anomalies and/or intellectual deciency, who were carrying
apparently balanced chromosomal rearrangements and genomic
imbalance excluded by microarray [5]. Thus, array CGH combined
with next-generation sequencing is a powerful tool allowing rapid
breakpoint cloning of apparently balanced chromosomal rear-
rangements at the molecular level. Systematic studies of apparently
balanced chromosomal rearrangements with abnormal phenotype
by array CGH have showed that the phenotype might occur due to
genomic imbalances near or far from breakpoints. A recent meta-
analysis estimated that 37% of two-breakpoint rearrangements
are unbalanced [34]. Polyploidies such as triploidy or tetrapolidy
cannot be detected by array CGH secondary to correction of DNA
concentration; however, additional allelic information provided in
SNP arrays improves polyploidy detection. High-level mosaicism
can be diagnosed with arrays, but detection of low-level mosaicism
is a potential limitation of microarrays. The capacity of array to
detect low-level mosaicism depends on standard deviation and
dynamic range. Several reports from the studies focused on post-
natal evaluation suggested that array was comparable to conven-
tional cytogenetic analysis in the detection rate of chromosomal
mosaicism.
Concern for the potential risk for detection of the variants of
unknown clinical signicance that might result in parental anxiety
is one of the limiting factors for the use of array technology pre-
natally. The reported incidence of variants of unknown signicance
varies from 0.5% to 2% depending on the array design and the
availability of parental samples for comparison. In the studies that
enrolled the fetuses with major malformations detected via pre-
natal ultrasound, the rate of the variants of unknown signicance
was reported as high as 12.2% [35]. A large prospective study that
included women with advanced maternal age, abnormal screening
results and/or abnormal ndings on ultrasound reported an inci-
dence of variants of unknown clinical signicance of 3.4% among
karyotypically normal cases [33]. However, with additional expe-
rience acquired over more recent years, only 1.5% cases remained in
the category of variants of unknown clinical signicance.
5. Non-invasive prenatal screening
Non-invasive prenatal screening using fetal cell-free DNA from
maternal blood became available with advancement of genomic
technology and development of next-generation sequencing. The
phenomenon of fetus-derived cell-free DNA circulating in a preg-
nant womans blood was rst described in 1997 [2]. At present, it is
widely accepted that apoptosis of the trophoblastic cells is the
primary source of cell-free fetal DNA, which explains the high
turnover of circulating DNA and its rapid clearance after delivery. In
maternal serum up to 10% of DNA is of fetal origin [36]. Fetal cell-
free DNA is present in maternal circulation in a quantity sufcient
for testing from10 weeks of gestation. Next-generation sequencing
technology allows massive parallel sequencing of millions of
amplied genetic fragments, and by using sophisticated bioinfor-
matics analysis, it is possible to isolate fetal sequences from
maternal sequence and to determine the dosage difference be-
tween fetal and reference sequences.
Several studies demonstrated high sensitivity, high specicity,
and low false-positive rates for detection of trisomy 13, 18, and 21
[37]. The detection rate of sex chromosome abnormalities is also
high [38]. Overall, the detection rates are substantially higher than
with maternal serum analytes, and are accompanied by a much
lower (<1% false-positive rate) [39]. Recently, it has been shown
that fetal RhD genotyping can be very accurately determined in all
three trimesters using circulating cell-free fetal DNA in the
maternal circulation [40]. Diagnosis of single-gene disorders is
being validated for translation into clinical practice. In pregnancies
at risk for an autosomal dominant disorder of paternal origin, the
study of the paternal mutation or alleles in the maternal plasma
could detect the fetal condition with regard to the pathology.
Huntington disease [41], achondroplasia [42], and myotonic dys-
trophy [43] are autosomal dominant conditions that have been
diagnosed through non-invasive prenatal testing. In couples with
different mutations for a recessive disorder, the absence of the
paternal mutation or detection of the normal paternal allele in
maternal plasma can rule out the possibility of the fetus inheriting
the disease. Detection of the paternal mutation increases the risk to
the fetus from 1 in 4 to 1 in 2. Recent advances in the development
of new technologies have detected both paternal and maternal
mutations, and a genome-wide genetic map of the fetus has been
constructed for the paternal and maternal haplotypes. Haplotype
analysis in conjunction with targeted next-generation sequencing
allowed non-invasive prenatal diagnosis for alpha- and beta-
thalassemia by detecting paternally inherited alleles in the
maternal plasma [44]. Other autosomal recessive disorders suc-
cessfully diagnosed with non-invasive prenatal testing have
included cystic brosis [45], propionic acidemia [46], congenital
adrenal hyperplasia [47], and Leber congenital amaurosis [48].
Although the studies are promising, there are certain limitations
for non-invasive prenatal screening. Sequence information identi-
ed through non-invasive prenatal screening is derived from the
placenta, which raises the possibility of a false-positive result due to
conned placental mosaicism, and similar to CVS, placental DNA
may not reect the true fetal DNA. The majority of studies on
non-invasive prenatal screening include women aged >35 years
who are at higher risk for aneuploidy. The data concerning non-
invasive prenatal screening in low-risk populations are limited,
but a decreased incidence of aneuploidy may be associated with
lower positive predictive value. Also, prior knowledge about the
prevalence of aneuploidies in the samples may have affected an
analysts decisions about how to classify ambiguous test results.
Current technology does not allow detection of chromosomal rear-
rangements and has not been validated for the majority of single-
gene disorders and CNVs. In cases of abnormal ndings detected
via ultrasound, invasive testing may still be necessary. Non-invasive
prenatal testing does not substitute for routine rst- and second-
trimester screening, as it does not detect neural tube defects and
has limitations in evaluation of structural abnormalities detected via
ultrasound and in multiple gestations. As the clinical utility of cell-
free DNA testing in the general population is unproven,
professional organizations such as the American College of Obste-
tricians and Gynecologists, the Society for MaternaleFetal Medicine
and the National Society of Genetic Counselors currently recom-
mend cell-free DNA testing only for high-risk pregnancies and they
suggest that positive results be conrmed through invasive testing.
6. Whole-exome and whole-genome sequencing
Next-generation sequencing has been used to identify causative
genes in many Mendelian disorders. About 85% of known disease-
causing mutations occur within the 1% of the genome encoding for
proteins. Therefore, whole exome sequencing represents a power-
ful method to cost-effectively capture the majority of protein-
coding regions to identify single nucleotide variants (SNVs), and
small insertion/deletions. It is now possible to non-invasively
identify the fetal genome with the shotgun sequencing of
maternal plasma DNA. Also, molecular counting of parental hap-
lotypes and fetal exome capture has allowed exome screening of
clinically relevant and deleterious alleles that were paternally
inherited or were de novo [49]. There are data concerning the
clinical diagnosis established by whole-genome sequencing of a
prenatal sample e for example, a 13-day sequence and analysis
pipeline detection of a disruption of CHD7 in a fetus with a sus-
pected diagnosis of CHARGE syndrome who had multiple anoma-
lies detected on ultrasound [50].
However, although comprehensive non-invasive whole-exome
or whole-genome sequencing has signicant potential, it also
brings multiple concerns such as technical complexity, prolonged
turnaround time, difculty with bioinformatic interpretation, and
ethical issues that limit its clinical use. Whole-exome or whole-
genome sequencing is still considered impractical for routine pre-
natal care secondary to cost and turnaround time. Eventually,
decreasing costs may allow personal genome sequencing
to become available for routine analysis, but annotation and
interpretation of variant information are essential to provide in-
formation that can be used to better manage an individuals con-
dition. The expanded use of non-invasive fetal exome or genome
sequencing would present several advantages and challenges.
Broader use might lead to the improved detection of Mendelian
disorders in families who would not otherwise have been offered
prenatal testing, as well as in families who might have refused
invasive testing because of risk of fetal loss. However, the concern is
that it will identify variants that are beyond the scope of
conventional prenatal screening and diagnosis, specically variants
that indicate increased risk for developing adult-onset conditions.
The relevance of such information is controversial for a current
pregnancy, but it may have signicant implications for the health of
the parent. Therefore, the broader implementation of non-invasive
fetal genome sequencing will lead to wider application of the re-
sults and increased availability.
7. Conclusions
Determining accurate genetic diagnosis prenatally permits
families to make informed reproductive decisions. Therefore, it is
important for the provider to be aware of the spectrum of potential
genetic conditions, and to use appropriate testing modalities to
obtain specic diagnosis.
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The birth of a child with intellectual disabilities and/or
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Counseling women and men regarding exposures to reproductive

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