P-1 Kolaviron Protects Primary Cultures of Rat Leydig Cells from Atrazine Induced Damages. S. Abarikwu, MS Ebenezer O. Farombi.

Redeemers University, Redemption City, Nigeria. BACKGROUND: Atrazine (ATZ) induced testicular impairment has been reported in vivo in rats. The current study examined the cytotoxic effect of ATZ and the protective potentials of kolaviron (KV) in vitro employing interstitial Leydig cells (ILC) in culture. ILC isolated from juvenile rats were co-cultured with 232mM ATZ in the presence or absence of KV (60mM). Expression of steroidogenic acute regulatory proteins (StAR), cytochrome P450scc, 3b-hydroxysteroid dehydrogenase (3b-HSD), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), glutathione reductase (GR), superoxide dismutase-1(SOD-1) and superoxide dismutase-2 (SOD-2) were monitored by real-time PCR following 6 h of treatment. Enzymatic activities of GR and GSH-Px and malondialdehyde (MDA) production were assessed after a 24 h culture. ILC viability was assessed after 24 and 48 h of treatment. OBJECTIVE(S): In this report we demonstrate that ATZ increased the transcripts levels of StAR, cytochrome P450scc and 3b-HSD. In addition to this, antioxidant genes expression of GSH-Px, GR, and GST was up-regulated whereas the mRNA expression of SOD-1 and SOD-2 was down-regulated in ILC. When both KV and ATZ were added to the culture medium, the transcript levels of StAR, P450scc, 3b-HSD and the antioxidant genes were restored to the control values. Furthermore, KV in the culture medium attenuates ATZ-induced increase in GSH-Px and GR activities and MDA production. When ILC were treated with KV alone in the absence of ATZ, the mRNA expression of StAR, P450scc, 3b-HSD, GSH-Px, GST, SOD-1 and SOD-2 were up-regulated. The increased GSH-Px expression aligned with an increase in activity at 24 h of culture period, indicating a similar mechanism of action at both the mRNA level and activity level. KV increased the viability of ILC at 10$60mM dose-dependently at 24 h culture. At 48 h culture, the lowest KV dose (10mM) increased the cell viability whereas the effects of the higher doses (30$90mM) on cell viability were not different from the control value. Compared with ATZ treatment alone, supplementation with KV prevented the time-dependent decrease in cell viability. CONCLUSION(S): These observations suggest that KV inhibits ATZinduced cell death, increase on antioxidant enzyme activities, ILC steroidogenesis and antioxidant gene expression. In addition, our study suggests a potential for KV to stimulate steroidogenesis gene expression in ILC.

icant difference between hyper and normo-stimulated patients in day 3 serum FSH, LH, estradiol, leptin or VEGF levels. However, the mean ( SD) AFC was higher in the hyper-stimulated group (21.64 3.20 versus 14.32 3.81; P<0.0002). Similarly, the mean ( SD) serum level of AMC was higher in the hyper-stimulated group (4.50 2.87 ng/ml versus 2.17 1.55; P<0.005). Receiver operator characteristic curves showed that the day 3 AFC was a better predictor of ovarian hyperstimulation than day 3 level of AMH (area under the curve 0.926 versus 0.771). Seven patients developed clinical OHSS, three were treated with IV albumin and one required aspiration of ascetic uid. CONCLUSION(S): Day 3 AFC is a better predictor of ovarian hyperstimulation than day 3 AMH level in PCOS patients treated with HMG. Day 3 FSH, LH, E2, leptin and VEGF are bad predictors. SUPPORT: Departmental funds

P-3 HMG-Only Protocol, a Practical Option for Developing Countries. H. Sallam,a,b F. Ezzeldin,b N. Sallam,b A. Agameya,a,b A. Farrag.b a Department of Obstetrics and Gynaecology, Alexandria University; b Alexandria Fertility Center, Alexandria, Egypt. BACKGROUND: GnRH down-regulation prior to FSH administration is now the standard protocol in developed countries to achieve controlled ovarian hypertimulation prior to IVF or ICSI. However, this protocol is expensive and requires a longer stimulation time. OBJECTIVE(S): The aim of this work was to evaluate the HMG-only stimulation regimen as an alternative protocol suitable for developing countries. MATERIALS AND METHOD(S): Retrospective analysis of data from all patients attending our Assisted Reproduction Clinic was conducted. A total of 608 cycles of IVF and ICSI were performed from the rst of January to the end of December 2009. Of those, 558 cycles were stimulated using an HMGonly protocol and 50 cycles were stimulated by a standard GnRH long protocol. Monitoring was conducted by serial ultrasound scanning of the follicles. There were no statistically signicant differences between both groups regarding the age of the couple, the duration of pregnancy or the indication of IVF or ICSI. RESULT(S): There were no signicant difference in the number of ampoules used, the number of oocytes, metaphase II oocytes, premature or postmature oocytes retrieved between both groups of patients. Similarly, there were no signicant differences in the number of grade I and grade II embryos obtained or the number of embryos replaced. A total of 165 patients became pregnant with an overall pregnancy rate of 27.06%. In the HMG-only group, 151 patients became pregnant (27.13%) compared to 14 in the GnRH/FSH group (28%) and this difference is not statistically signicant (P0.982). The mean (SD) number of sacs was 1.9 (1.2) in the HMG-only group compared to 2.5 (1.7) and this difference is also not signicant (P0.552). The mean cost of the HMG-only cycle was US$ 1101.82 compared to US$ 1558.18 for the GnRH/FSH cycle. The mean number of ultrasound examinations was 3.5 in the HMG group and 5.2 in the GnRH/ FHS group. Two patients developed ovarian hyperstimulation in the GnRH/FSH group (4%) compared to seven in the HMG-only protocol (1.25%) (P0.353). CONCLUSION(S): HMG-only protocol is a simple and cheap protocol for IVF and ICSI and offers a practical alternative for infertility specialists in developing countries compared to the classical long stimulation protocol. SUPPORT: None.

P-2 Prediction of Ovarian Hyperstimulation in Patients With Polycystic Ovary Syndrome (PCOS) Treated With Human Menopausal Gonadotrophins. H. Sallam,a,b D. El-Kaffash,a,c M. Abou-Heif,b M. El-Abd,b A. Sallam,b A. Agameya.a,b a Suzanne Mubarak Regional Center for Womens Health and Development; b Departments of Obstetrics and Gynaecology, University of Alexandria; c Department of clinical pathology, University of Alexandria, Alexandria, Egypt. BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a rare iatrogenic complication of ovarian stimulation. It is more common in patients with polycystic ovarian syndrome (PCOS) and is difcult to predict. OBJECTIVE(S): The aim of this work was to evaluate some biochemical and ultrasound indices as predictors of ovarian hyperstimulation in patients with PCOS treated for ovulation induction with human menopausal gonadotrophins (HMG). MATERIALS AND METHOD(S): A total of 33 anovulatory patients with PCOS (based on the Rotterdam consensus) were studied. Antral follicle count (AFC) was measured on day 3 of a spontaneous or induced menstrual cycle. Day 3 serum levels of estradiol (E2), FSH, LH, AMH, leptin and VEGF were also determined. A dose of 2 HMG ampoules (150 IU) was administered IM daily starting on day 5 of the cycle. Monitoring was effected by ultrasound and the dose was increased to 3 ampoules (225 IU) if the response was inadequate. HCG (5000 IU) was administered IM when the leading follicle reached 18 mm in diameter. Serum E2 was measured again on the day of HCG administration and ovarian hyperstimulation was diagnosed if above 3000 pg/mL. The patient was seen 7 days later to determine the occurrence or otherwise of clinical OHSS and managed accordingly. RESULT(S): A total of 11 patients had ovarian hyperstimulation (33.3%). The mean serum estradiol level ( SD) on the day of HCG was 5965.82 pg/ mL ( 1591.99) pg/mL in the hyper-stimulated group compared to 2207.27 ( 859.32) in the normo-stimulated group (P<0.0001). There was no signif-

P-4 Angiopoitien 2, Interleukin 1b and Vascular Endothelial Growth Factor (Vegf) Concentrations in Peritoneal Fluid and Plasma, a Possible Role in Endometriosis. Fady S. Moiety, M.D.a,b Abdel Fattah A. Agameya, M.D.a,b a Department of Obstetrics and Gynecology, Faculty of Medicine, Alexandria University, Egypt; b The Suzanne Mubarak Regional Centre for Womens Health and Development. OBJECTIVE(S): To investigate the serum and peritoneal uid (PF) concentrations of Angiopoitien 2 (Ang-2), Interleukin 1b (IL 1b), and Vascular Endothelial Growth Factor (VEGF), aiming to point out any predictive role in endometriosis. DESIGN: Prospective, Case control study. SETTING: University hospital.

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PATIENTS AND METHOD(S): Serum and peritoneal uid samples were taken from 112 women undergoing laparoscopy for infertility, pelvic pain, and/or adnexal masses. Sixty one were diagnosed with endometriosis and 51 were controls. No cases were excluded. MAIN OUTCOME MEASURE(S): Primary outcome was the estimate of the serum and PF concentrations of (Ang 2), (IL 1b) and (VEGF), secondarily, was to correlate these concentrations to the disease stage, presentation and thus assuming their diagnostic potential. RESULT(S): A signicant difference was found between patients and controls serum and PF concentration of all studied markers except IL 1b. Only PF VEGF showed a signicantly higher level in more advanced stages of endometriosis with a positive correlation with the stage of the disease, (spearman coefcient0.442*) Serum or PF concentrations of any of the studied markers did not show any specic pattern of stage-related level changes. Diagnostic potential of the serum and PF concentrations of the 3 markers was assessed by the ROC curve. PF and serum VEGF proved an equal and best diagnostic performance, whereas, PF Ang-2 was the least efcient. It was also possible, based on our results, to suggest preliminary threshold values for these markers to use as diagnostic or follow-up landmarks with relatively acceptable sensitivity, specicity, positive and negative predictive values. CONCLUSION(S): A non-invasive predictive tool for endometriosis is becoming closer. Serum Ang-2, IL 1b, and VEGF, could be reasonable predictors -independently or in combination- with the estimated threshold values. Furthermore, a minimally invasive prediction via peritoneal uid laparoscopic sampling for markers assay especially VEGF would be an addition to the diagnostic potential in endometriosis. SUPPORT:

P-6 A Case of Acquired Nonobstructive Azoospermia. K. OLeary,a R. Lathi,a P. Turek,b L. Westphal.a a Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA; b The Turek Clinic, San Francisco, CA. BACKGROUND: Nonobstructive azoospermia (NOA) is a challenging condition to diagnose and manage. Most NOA cases are either idiopathic or genetically determined, but acquired causes are also possible due to bacterial or viral infections (mumps), vascular injury, or trauma. OBJECTIVE(S): To describe a unique case of acquired nonobstructive azoospermia. DESIGN: Case report. MATERIALS AND METHOD(S): A 25 year-old male and his 26 year-old G0 anc ee presented for infertility evaluation. The male partner had a history of a massive pelvic fracture (hip and sacrum) and complete urethral disruption after being hit by a train in Paris one year prior. He had had multiple orthopedic procedures to stabilize the pelvis and to allow weight bearing activity. He presented with a suprapubic catheter in place and was taking heavy doses of chronic opiates for nerve pain. Due to erectile dysfunction and anejaculation, in vitro fertilization with testicular sperm extraction (TESE) with ICSI was planned. On the day of retrieval, ve biopsies were performed with no sperm found; all oocytes were frozen. On more thorough urologic evaluation, the patient was found to have an unremarkable urologic examination with normal testis volume and palpable vasa deferentia. Testosterone was 370 ng/mL, and LH, FSH, prolactin, and estradiol were normal. For possible mild hypogonadotropic hypogonadism due to chronic opiate use, the patient eliminated opiates and began clomiphene citrate 25 mg daily with planned bilateral microdissection TESE six months later. At the time of bilateral microdissection TESE, no sperm were found. In addition, a diffuse lmy layer of scar tissue was observed throughout the testis. Additional history revealed that the patient had at least 20 pelvic CT scans and 30 plain lm X-rays (about 520 mGy to the gonads) over a 6 month period. The cause of azoospermia was thought to be due to ionizing radiation that induced germ cell eradication or arrest. Recommendation: semen analysis and possible ne needle aspiration map in three years; meanwhile, the couple conceived with a natural cycle intrauterine insemination using directed donor sperm. CONCLUSION(S): Germ cell loss and disruption of Sertoli cell function from ionizing radiation is a rare cause of acquired NOA. Doses as low 300 mGy may transiently suppress spermatogenesis, while doses above 6000 mGy usually cause irreversible azoospermia and infertility. The reversibility of radiation exposure in this case is unclear but should become evident with follow-up.

P-5 Development of a Novel Protocol for Isolation and Purication Of Human Granulosa Cells. R. A. Chilvers, Y. H. Bodenburg, L. A. Denner, R. J. Urban. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of Texas Medical Branch, Galveston, TX. Department of Internal Medicine, Division of Endocrinology, University of Texas Medical Branch, Galveston, TX. OBJECTIVE(S): To develop an optimal method of purication of human granulosa cells from heterogenous follicular uid and a method to account for red blood cell (RBC) contamination in samples for accurate comparison of results between individual patients. DESIGN: Prospective experimental study. MATERIALS AND METHOD(S): Follicular uid was collected from patients undergoing oocyte retrieval for IVF. A series of experimental granulosa isolation and purifying techniques was performed with added complexity after determination of results from former sample collections. The techniques include: 1) density gradient (DG) centrifugation with 50% solution of colloidal silica, 2) DG centrifugation with a polymer of sucrose, 3) indirect, negative selection of RBCs using primary antibody (Ab) to glycophorin A and secondary Ab connected to spherical, paramagnetic, epoxy beads, 4) positive selection of granulosa cells using primary Ab to mullerian inhibiting substance receptor II (MISRII) and secondary Ab coupled to irregularly-shaped, superparamagnetic (SPM) silanized microparticles, 5) positive selection of granulosa cells using primary Ab to MISRII and secondary Ab coupled to biodegradable, SPM nanoparticles, 6) dilution of follicular uid samples with buffer before DG centrifugation. In addition, Western blotting was used to analyze samples for relative amounts of hemoglobin a (Hgba) as compared to a standard curve of blood samples. RESULT(S): The procedure that resulted in the largest increase in purity of granulosa cells as well as resulted in the largest ratio of live to dead granulosa cells was dilution of the follicular uid with buffer, then use of DG centrifugation using sucrose polymer, then positive selection of granulosa cells using MISRII and secondary Ab coupled to irregularly-shaped, superparamagnetic, silanized iron oxide beads. Western blotting with staining of granulosa cells for Hgba provided visible quantication of RBCs in patient samples. CONCLUSION(S): A novel protocol for granulosa cell isolation and purication has been developed yielding granulosa cell samples that are largely free of nondesirable cells. This protocol provides a solution for patient samples that have heavy RBC contamination and uniquely involves a primary Ab against the MISRII. Western blotting with Hbga antibody is an effective means of comparing patient sample purity from RBCs. SUPPORT: NIH RO1 HD044566-01.

P-7 PGRMC1 And PGRMC2 Expression is Signicantly Decreased in the Endometrium of Women With Endometriosis. K. Bunch,a D. Tinnemore,b S. Huff,c Z. Hoffer,c R. Burney,a,b J. Stallings.b a Department of Obstetrics/Gynecology, Madigan Army Medical Center, Tacoma, WA; b Department of Clinical Investigation, Madigan Army Medical Center, Tacoma, WA; c Department of Pathology, Madigan Army Medical Center, Tacoma, WA. BACKGROUND: Endometriosis is a hormone dependent inammatory condition associated with pain and infertility. Compelling clinical and molecular evidence demonstrate attenuated secretory-phase progesterone responsiveness in affected women, which may contribute to infertility. OBJECTIVE(S): To compare gene expression of progesterone receptor membrane components (PGRMC) 1 and 2 in endometria of women with and without endometriosis. METHOD(S): Endometrial pipelle biopsies were obtained from 11 women with histologically proven stage III/IV endometriosis and 23 surgically documented disease-free women. Endometrial cycle phase was determined using a combination of self-report cycle day, serum hormone prole, and endometrial histologic dating. Specimens were dually processed for total RNA and protein immunohistochemistry (IHC). Real time quantitative polymerase chain reaction (RT-PCR) and IHC measured PGRMC gene and protein expression levels in both groups. RESULT(S): PGRMC1 (fold change 3.3; P<0.001) and PGRMC2 (fold change 8.8; P<0.01) gene expression were signicantly downregulated in secretory phase endometrium in women with endometriosis. Immunohistochemistry showed that, in these women, downregulated gene expression resulted in decreased PGRMC1 and 2 protein production.

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Abstracts

Vol. 95, No. 4, Supplement, March 15, 2011