Human Chorionic Gonadotropin (hCG)

Finland

Part A

Introduction

The Expanding World of hCG

Robert O Hussa

Medical College of Wisconsin, Milwauke WI, Sunnyvale, CA, USA

More than four decades ago, in 1969, the door to the world of hCG opened for me when I joined Roland Pattillo's research team in the Department of Ob/Gyn at the Medical College of Wisconsin (MCW) in Milwaukee. Pattillo established the hCG-secreting BeWo choriocarcinoma cell line in 1966 in George Gey's laboratory at Johns Hopkins University [1]. Pattillo literally carried the cell line in a culture flask filled with culture fluid in his shirt pocket during his move from Baltimore to Milwaukee. Pattillo, along with Eleanor Delfs and Richard Mattingly, comprised the trophoblast disease clinical team that migrated from Johns Hopkins University to Milwaukee. Mattingly was chairman of the Department of Ob/Gyn at MCW and editor-in-chief of Obstetrics & Gynecology. From my desk in the laboratory (the room adjoining Pattillo's cell culture laboratory), I would observe Eleanor Delfs personally perform hCG extractions on serum samples from her hydatidiform mole and choriocarcinoma patients prior to injecting the samples into rats, per her uterine-weight bioassay—which was published the year I was born [2].

The emphasis of the Department of Ob/Gyn at MCW on treatment of patients with trophoblast disease and the study of hCG in patients, as well as in hCG-secreting cell lines, led to the need to quantitatively measure hCG in large numbers of samples. Pattillo's thriving cell culture laboratory soon established a second hCG-secreting trophoblast cell line, JAr [3]. In those years, the glycoprotein hormone field was filled with excitement, such as that engendered by the revelation that hCG, LH, FSH, and TSH all contained a common α-subunit and a hormone-specific β-subunit [4–6]. In addition, the technology of the new radioimmunoassays (RIAs) was making great strides, particularly with the β-subunit-specific RIA created by Vaitukaitis and colleagues in Griff Ross's laboratory at the National Institutes of Health [7]. Thus it was that our laboratory also evolved in the 1970s and 1980s and began to use RIAs to measure hCG and its α- and β-subunits in our research.

Another significant body of research emanated out of labs such as that of Om P. Bahl, where the carbohydrate structure of hCG was characterized utilizing digestion with specific exoglycosidases [8]. More excitement for our lab came when Pattillo's crackerjack tissue culture staff established the hCGβ-secreting CaSki and DoT cell lines from cervical carcinoma [9,10]. It needs to be emphasized that Roland Pattillo has provided a great service to science by willingly providing his trophoblastic and nontrophoblastic cell lines to all requesters over the past four decades.

It was into this 1979 setting that a young biochemistry student at MCW, Laurence A. Cole, began his PhD research in my laboratory. Larry actually occupied the same desk that I had used in my first years in the laboratory at MCW. Larry, a UK native, who greeted me each morning with a friendly Good morning, sire, was a hard-working, prodigiously productive, and insightful researcher, who occasionally found the time to bring special treats for the lab, such as fresh scones with clotted cream flown in from London, or some of his home-brewed beer.

Larry's dissertation on the characterization of the ectopic hCGβ secreted by the CaSki and DoT cervical carcinoma cell lines [11] led to his first publications [12–15] and established his early fascination with the oligosaccharide structure of hCG. This experience undoubtedly led to Larry Cole's great contributions toward a better understanding of the many variant forms of hCG in pregnancy and cancer, as described in this book. It is indeed fitting that Larry has taken on the monumental task—akin to that of herding cats—of gathering the work of so many experts in the various areas of hCG (most of whom who have been contributing to the field for decades) and compiling it in this comprehensive compendium of reviews on all aspects of hCG, from its molecular and oligosaccharide structure and biosynthesis to its applications as a clinical marker and use as a therapeutic agent.

A Web of Science search of this book's areas of emphasis was conducted at the end of 2009 (Table 1.1). Despite the fact that no effort was made to cull through the articles and citations to eliminate redundant and irrelevant publications, the results give an excellent broad perspective of how the world of hCG has expanded over time. The number of publications in a given area is an indicator of the amount of activity in that area. Thus, the number of times a reference is cited by peers should correlate with the publications that experts value as being the most significant.

Table 1.1 Web of science hCG search, 1975–2009.

In the years from 1975 to 1985 and 1986 to 2009, there were more publications and citations on clinical applications of hCG than in any other category; followed by receptor activity and biological functions of hCG. In contrast, there were no publications or citations prior to 1986 on hyperglycosylated hCG, degradation products of hCG, or diet/sports/HIV, and only two publications devoted to hCG standards and reference preparations. The lack of publications in these areas of emphasis underscores the timeliness and relevance of the reviews for each of these areas in this book.

After the 1980 reviews [16,17], various aspects of hCG have been reviewed more recently, including three-dimensional structure [18,19], gene expression and cloning [20,21], biosynthesis [22], glycosylation [23], immunochemistry [24], clinical measurement [25–28], receptors [29,30], and infertility [31]. The recently emerging category that has received the most attention (as measured by the 2698 publications since 1985, compared to no prior publications) is that of diet, sports, and HIV. Similarly, the Varki review on the biological roles of oligosaccharides was cited in an astonishing 2975 references [36]; the next most cited reference in hCG literature (738 citations) was on placental implantation, by Cross et al. [37]; the third most cited publication (602 citations) was on the crystal structure of hCG, by Lapthorn et al. [34].

The yearly trend of publications and citations provides interesting information on the dynamics of activity in each of the areas of emphasis described in this book. In the areas of hCG biosynthesis, hCG genes/mRNA, hCG structure, and hCG receptor/biological functions, there was a jump in research activity in 1991–1994 (Figures 1.1–1.4), with a corresponding increase in the number of citations starting in 1992 (Figures 1.5–1.8). In the area of hCG clinical applications, the publishing activity (Figure 1.9) has remained relatively constant (260–400 articles per year), whereas the number of citations has increased dramatically in linear fashion since 1991 (Figure 1.10). Very similar trends were observed for the search areas of hCG assays/tests and vaccines/cancer (not shown). The newly emerging emphasis areas of diet/sports/HIV reveal sporadic publishing activity starting in 1991 (Figure 1.11) and a surge of citations beginning around 1996 (Figure 1.12).

Figure 1.1 Web of Science search for biosynthesis of hCG; publications (ordinate) by year (abscissa).

Figure 1.2 Web of Science search for hCG genes and mRNA; publications (ordinate) by year (abscissa).

Figure 1.3 Web of Science search for hCG structure; publications (ordinate) by year (abscissa).

Figure 1.4 Web of Science search for hCG receptor/biological functions; publications (ordinate) by year (abscissa).

Figure 1.5 Web of Science search for biosynthesis of hCG; citations (ordinate) by year (abscissa).

Figure 1.6 Web of Science search for hCG genes and mRNA; citations (ordinate) by year (abscissa).

Figure 1.7 Web of Science search for hCG structure; citations (ordinate) by year (abscissa).

Figure 1.8 Web of Science search for hCG receptor/biological functions; citations (ordinate) by year (abscissa).

Figure 1.9 Web of Science search for hCG and clinical applications, infertility, pregnancy, and nontrophoblastic disease; publications (ordinate) by year (abscissa).

Figure 1.10 Web of Science search for hCG and clinical applications, infertility, pregnancy, and nontrophoblastic disease; citations (ordinate) by year (abscissa).

Figure 1.11 Web of Science search for hCG and diet/sports/HIV; publications (ordinate) by year (abscissa).

Figure 1.12 Web of Science search for hCG and diet/sports/HIV; citations (ordinate) by year (abscissa).

I hope the tapestry of hCG research trends from over the decades will provide an appropriate background for readers and guide them toward a favorite area of interest within the evolving world of hCG as covered in this comprehensive book. As mentioned earlier, several of these topics are being reviewed for the first time; others are continuously updated with new information emanating from the rapidly expanding field of hCG research. I am grateful and proud to have been part of this body of investigation since 1969.

Acknowledgments

I thank Jack Black, the senior librarian at El Camino Hospital Health Library and Resource Center, Mountain View, California, for his expert assistance with the literature searches on Web of Science, provided by Thomson Reuters.

References

[1]. RA Pattillo, GO Gey. The establishment of a cell line of human hormone-synthesizing trophoblastic cells in vitro. Cancer Res, 28 : 1968, 1231 – 1236.

[2]. E Delfs. An assay method for human chorionic gonadotropin. Endocrinology, 28 : 1941, 196 – 202.

[3]. RA Pattillo, A Ruckert, R Hussa, R Bernstein, E Delfs. The JAr cell line–continuous human multi-hormone production and controls. In Vitro, 6 (Abstract): 1971, 398 – 399.

[4]. N Swaminathan, OP Bahl. Dissociation and recombination of the subunits of human chorionic gonadotropin. Biochem Biophys Res Commun, 40 : 1970, 422 – 427.

[5]. JG Pierce. Eli Lilly Lecture: the subunits of pituitary thyrotropin–their relationships to other glycoprotein hormones. Endocrinol, 89 : 1971, 1331 – 1344.

[6]. OP Bahl, RB Carlsen, R Bellisario, N Swaminathan. Human chorionic gonadotropin: amino acid sequence of the α and β subunits. Biochem Biophys Res Commun, 48 : 1972, 416 – 422.

[7]. JL Vaitukaitis, GD Braunstein, GT Ross. A radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone. Am J Obstet Gynecol, 113 : 1972, 751 – 758.

[8]. OP Bahl, L Marz, MJ Kessler. Isolation and characterization of N- and O-glycosidic carbohydrate units of human chorionic gonadotropin. Biochem Biophys Res Commun, 84 : 1978, 667 – 676.

[9]. RA Pattillo, RO Hussa, MT Story, ACF Ruckert, MR Shalaby, RF Mattingly. Tumor antigen and human chorionic gonadotropin in CaSki cells: a new epidermoid cervical cancer cell line. Science, 196 : 1977, 1456 – 1458.

[10]. RA Pattillo, RO Hussa, ACF Ruckert, MT Story, RF Mattingly. hCG-beta production by epidermoid carcinoma of the cervix. Endocrinol(Suppl), 100 (Abstract): 1977, 179.

[11]. Cole LA. Studies with ectopic hCGβ: synthesis, glycosylation, isolation and characterization. PhD Dissertation, Milwaukee: Medical College of Wisconsin; 1982.

[12]. LA Cole, RO Hussa, CV Rao. Discordant synthesis and secretion of human chorionic gonadotropin and subunits by cervical carcinoma cells. Cancer Res, 41 : 1981, 1615 – 1619.

[13]. LA Cole, RO Hussa. Use of glycosidase digested human chorionic gonadotropin and β-subunit to explain the partial binding of ectopic glycoprotein hormones to Con A. Endocrinol, 109 : 1981, 2276 – 2278.

[14]. MT Story, LA Cole, RO Hussa. A procedure using immobilized antibody for the isolation of the β-subunit of human chorionic gonadotropin from the culture fluid of a β-subunit-secreting nontrophoblastic cell line. J Clin Endocrinol Metab, 53 : 1981, 1090 – 1095.

[15]. LA Cole, S Birken, S Sutphen, RO Hussa, RA Pattillo. Absence of the COOH-terminal peptide on ectopic human chorionic gonadotropin β-subunit (hCG β). Endocrinol, 110 : 1982, 2198 – 2200.

[16]. RO Hussa. Biosynthesis of human chorionicgonadotropin. Endocr Rev, 1 : 1980, 268 – 294.

[17]. RO Hussa. The clinical marker hCG, 1987, Praeger Publishers, New York, NY.

[18]. JW Lustbader, DL Yarmush, S Birken, D Puett, RE Canfield. The application of chemical studies of human chorionic gonadotropin to visualize its three-dimensional structure. Endocr Rev, 14 : 1993, 291 – 311.

[19]. H Wu, JW Lustbader, Y Liu, RE Canfield, WA Hendrickson. Structure of human chorionic gonadotropin at 2.6 å resolution from MAD analysis of the selenomethionyl protein. Structure, 2 : 1994, 545 – 558.

[20]. JF Habener. Molecular cloning of hormonegenes, 1987, The Humana Press, Clifton, NJ.

[21]. JL Jameson, AN Hollenberg. Regulation of chorionic gonadotropin gene expression. Endocr Rev, 14 : 1993, 203 – 221.

[22]. WE Merz. Biosynthesis of human chorionic gonadotropin: a review. Eur J Endocr, 135 : 1996, 269 – 284.

[23]. P Van den Steen, PM Rudd, RA Dwek, P Opdenakker. Concepts and principles of O-linked glycosylation. Crit Rev Biochem Mol Biol, 33 : 1998, 151 – 208.

[24]. JF O'Connor, S Birken, JW Lustbader, A Krichevsky, Y Chen, RE Canfield. Recent advances in the chemistry and immunochemistry of human chorionic gonadotropin: impact on clinical measurements. Endocr Rev, 15 : 1994, 650 – 683.

[25]. T Chard. Pregnancy tests: a review. Hum Reprod, 7 : 1992, 701 – 710.

[26]. MJ Duffy. Clinical uses of tumor markers: a critical review. Crit Rev Clin Lab Sci, 38 : 2001, 225 – 262.

[27]. FM Reis, D D'Antona, F Petraglia. Predictive value of hormone measurements in maternal and fetal complications of pregnancy. Endocr Rev, 23 : 2002, 230 – 257.

[28]. UH Stenman, H Alfthan, K Hotakainen. Review. Human chorionic gonadotropin in cancer. Clin Biochem, 37 : 2004, 549 – 561.

[29]. M Ascoli, F Fanelli, DL Segaloff. The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocr Rev, 23 : 2002, 141 – 174.

[30]. G Kleinan, G Krause. Thyrotropin and homologous glycoprotein hormone receptors: structural and functional aspects of extracellular signaling mechanisms. Endocr Rev, 30 : 2009, 133 – 151.

[31]. NS Macklon, RL Stouffer, LC Giudice. Fauser BCJM. The science behind 25 years of ovarian stimulation for in vitro fertilization. Endocr Rev, 27 : 2006, 170 – 207.

[32]. MM Elliott, A Kardana, JW Lustbader, LA Cole. Carbohydrate and peptide structure of the α- and β-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma. Endocrine, 7 : 1997, 15 – 32.

[33]. TR Kumar, Y Wang, N Lu, MM Matzuk. Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nature Genetics, 15 : 1997, 201 – 204.

[34]. AJ Lapthorn, DC Harris, A Littlejohn, JW Lustbader, RE Canfield, KJ Machin, et al. Crystal structure of human chorionic gonadotropin. Nature, 369 : 1994, 455 – 461.

[35]. S Rotmensch, LA Cole. False diagnosis and needless therapy of presumed malignant disease in women with false-positive human chorionic gonadotropin concentrations. Lancet, 355 : 2000, 712 – 715.

[36]. A Varki. Biological roles of oligosaccharides—all of the theories are correct. Glycobiol, 3 : 1993, 97 – 130.

[37]. JC Cross, Z Werb, SJ Fisher. Implantation and the placenta—key pieces of the development puzzle. Science, 266 : 1994, 1508 – 1518.

[38]. JE Hansen, O Lund, N Tolstrup, AA Gooley, KL Williams, S Brunak. NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility. Glycoconj J, 15 : 1998, 115 – 130.

[39]. GM Mead, SP Stenning, P Cook, International Germ Cell Cancer Consensus Group. International germ cell consensus classification: a prognostic factor-based staging system for metastatic germ cell cancers. J Clin Oncol, 15 : 1997, 594 – 603.

[40]. SD Jayasena. Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem, 45 : 1999, 1628 – 1650.

[41]. LA Cole, A Kardana. Discordant results in human chorionic gonadotropin assays. Clin Chem, 38 : 1992, 263 – 270.

History and Introduction to Human Chorionic Gonadotropin (hCG)

One Name for at Least Three Independent Molecules

Laurence A. Cole

USA hCG Reference Service, Albuquerque, NM, USA

In 1919, Japanese scientist Toyoichi Hirose was the first to demonstrate the effects of implanting fragments of human placenta on the ovaries and uteruses of rabbits [1,2]. Hirose's work showed that there was a clear hormonal link between the placenta and the uterus. These findings were confirmed by Murata and Adashi in 1927 [3]. Around this time, the name human chorionic gonadotropin (hCG) was conceived: chorion is Latin for placenta and the hormone is produced by the placenta, hence chorionic; gonadotropin because the hormone is tropic, acting on female gonad tissue (ovaries), promoting steroid-induced actions. In 1927, Ascheim and Zondek demonstrated that the blood and urine of pregnant women contained a gonad-stimulating substance [4]. They showed that injecting this substance subcutaneously into intact immature female mice produced follicular maturation, luteinization, and hemorrhage into the ovarian stroma. These findings were confirmed by others [5,6] and the first hCG/pregnancy test was born [4–6]. These early tests primarily used urine to promote ovulation in rabbits [4–25], and were commonly referred to as the rabbit or Friedman test (Table 2.1).

Table 2.1 History of the Laboratory Pregnancy Test (the hCG and hCGβ assay).

Over the next four decades, bioassays like the rabbit test were the only practical way to detect pregnancy or measure hCG [4–10]. In 1960, we saw the first antibody-based pregnancy test. The first antibody-based tests examined hemagglutination inhibition and latex agglutination [11,12]. These were insensitive slide tests that detected hCG at a concentration of 1000 mIU/ml or greater. In 1964, the competitive hCG radioimmunoassay (RIA) was invented [13–17] and revolutionized pregnancy testing. At last a test was available that could measure hCG as low as 5 mIU/ml and measure pregnancy as early as the day of a missed period. The invention of the RIA led to readily available pregnancy testing/hCG measurement at clinical laboratories throughout the world [13–17].

The initial RIAs were problematic because they used an antibody against hCG dimer and detected both hCG and luteinizing hormone (LH). The problem was that the α-subunit of hCG was identical to the α-subunit of LH, and the β-subunit of hCG was 80% homologous with the β-subunit of LH. Hence, the early hCG dimer RIA detected both hCG and LH and could only show pregnancy hCG and exclude LH by demonstrating a continual increase in hormone levels. In 1973, Vaitukaitis et al. introduced the hCGβ test [18], a RIA pregnancy test using an antibody against the β-subunit of hCG. The hCGβ test was the first hCG-specific RIA. Unlike its predecessor, which detected both hCG and LH, the hCGβ test measured hCG alone and did not detect LH [18]. This was an important distinction because LH has no relationship to pregnancy and need not be measured when testing for pregnancy. The hCGβ test RIA became the world standard for the next 20 years. Even today, in the age of immunometric assays, both physicians and textbooks still describe hCG tests as hCGβ tests.

The discovery of monoclonal antibodies in 1975 was paramount to the development of modern immunometric hCG tests [19]. Modern 2- or 3-antibody immunometric hCG assays were developed in 1981. With these assays came the concept of antibody enzyme labeling and high-sensitivity fluorimetric and spectrometric detection [20–25]. The advent of chemiluminescent and europium labeling, automation, and sensitive detection led to the rapid high-sensitivity hCG tests that are used today. As described later in this book (Chapter 19) at least 13 automated platforms use cartridges that rapidly and accurately measure serum hCG and other molecules via chemiluminescent methods. The history of the pregnancy test and the development of variations on the original rabbit test are outlined in Table 2.1.

The principle behind the modern immunometric test begins with one or two antibodies (called the capture antibodies) binding to one or more antigen sites on hCG and its free β-subunit. This binding immobilizes the hCG and free β-subunit. A second antibody (called a tracer antibody) is labeled with a chemiluminescent labeling agent, and binds to a distant antigen site on hCG or its free β-subunit. In so doing, the immobilized complex becomes what is labeled the capture antibody–hCG-tracer antibody complex. This complex can then be quantified; the amount of tracer antibody is linearly proportional to the amount of complex and the concentration of hCG. Dual-antibody immunometric technologies are the principle of most modern physicians' office point-of-care (POC) rapid pregnancy tests and home/over-the-counter (OTC) rapid pregnancy tests [25]. These tests use one antibody immobilized in the result window on the nitrocellulose device, and one antibody (labeled with a blue or gold dye) mixed with the serum or urine. A positive result is indicated by a line formed in the plastic window by the immobilized antibody–hCG-dye antibody complex.

This book starts with a three-chapter introduction to hCG: Chapter 1 by Robert Hussa; Chapters 2 and 3 by Laurence Cole. In this book, we present articles describing every aspect of hCG assays, hCG antibodies, laboratory hCG tests, POC and OTC hCG tests, false-positive hCG tests, hCG assay specificity, and hCG standards. Chapter 18, Antibodies for intact hCG, total hCG, free subunits, glycosylation variants, and hCG fragments, by Laurence Cole; Chapter 19, Quantitative hCG assays, by Laurence Cole; Chapter 20, False-positive hCG assays, by Laurence Cole; Chapter 21, Specificity of different hCG assays, by Laurence Cole; Chapter 22, Point-of-care pregnancy tests, by Laurence Cole; Chapter 23, Over-the-counter pregancy tests, by Laurence Cole, and Chapter 24, hCG standards, by Ulf Stenman.

In this book, we examine all possible applications of hCG-related molecule assays. We explore the advantages and disadvantages of different commercial hCG assays, and the advantages and disadvantages of different hCG standards; examine the applications of hCG, hyperglycosylated hCG (hCG-H), and free β tests in detecting pregnancies and monitoring the likelihood of pregnancy failures; examine the use of hCG and hCG-H in predicting Down Syndrome pregnancies and cases of maternal pre-eclampsia; and investigate the relationship of hCG-H and gestational trophoblastic disease, and of free β and nontrophoblastic malignancies. Background hCG, by Laurence Cole; Chapter 26, Pregnancy Testing, by Laurence Cole; Chapter 27, Predicting Spontaneously Aborted (SAB) Pregnancies, by Laurence Cole; Chapter 28, hCG, hyperglycosylated hCG, and free β-subunit in predicting Down syndrome pregnancies and preeclampsia, by Laurence Cole; Chapter 29, hCG in Monitoring Gestational Trophoblastic Diseases, by Laurence Cole; Chapter 30, Use of Hyperglycosylated hCG as a Unique Marker of Gestational Trophoblastic Neoplasms, by Laurence Cole and Carolyn Muller; Chapter 31, Pituitary hCG and Familial hCG, by Laurence Cole, and Chapter 32, hCG, free β-subunit, and β-core fragment as markers of malignancies, by Laurence Cole.

Hirose's 1919 discoveries about the interaction of placental and ovarian tissues started a long chain of investigations into the molecule we now know as hCG [1,2]. Today, we look at hCG not as one hormone, but as a mixture of two hormones and two autocrines. The likely activities of hCG, pituitary hCG (hCGp), hCG-H, and hCG-H free β-subunit (hCG-Hβ) are illustrated in Figure 2.1. These discoveries have been slowly unearthed over many years of development in the laboratory. The real understanding of hCG has emerged from the combined efforts of numerous individuals and multiple research centers.

Figure 2.1 Illustration of (A) endocrine action of hCGp, (B) cytokine or autocrine activities of cancer cell hCG-Hβ, (C) endocrine action of hCG, and (D) the cytokine or autocrine actions of hCG-H.

Multiple variants of hCG have been discovered. A variant of hCG with double-sized sugar side-chains (hCG-H) is made by cytotrophoblast cells [16] and promotes invasion during implantation of pregnancy and malignancy as occurs in choriocarcinoma [17–19]. A free β-subunit of hCG-H is made by nontrophoblastic neoplasms. This variant also has a role in cancer cell growth and malignancy [20–24]. Finally, there is hCGp, a sulfated form of hCG produced by the pituitary gland during the menstrual cycle [26]. hCGp supplements LH in promoting ovulation and progesterone production [26]. Thus, there are four separate forms of hCG: regular hCG, hCG-H, hCGp, and free β hCG. Each is an individual molecule with a unique structure and independent