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Kolaviron Protects Primary Cultures of Rat Leydig Cells from AtrazineInduced Damages. S. Abarikwu, MS Ebenezer O. Farombi. Redeemer’sUniversity, Redemption City, Nigeria.

BACKGROUND: Atrazine (ATZ) induced testicular impairment has beenreported in vivo in rats. The current study examined the cytotoxic effect of ATZ and the protective potentials of kolaviron (KV) in vitro employinginterstitial Leydig cells (ILC) in culture. ILC isolated from juvenile ratswere co-cultured with 232mM ATZ in the presence or absence of KV(60mM). Expression of steroidogenic acute regulatory proteins (StAR), cyto-chrome P450scc, 3b-hydroxysteroid dehydrogenase (3b-HSD), glutathioneperoxidase (GSH-Px), glutathione-S-transferase (GST), glutathione reduc-tase (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)productionwere assessed after a 24 h culture. ILCviability wasassessed after24 and 48 h of treatment.

OBJECTIVE(S): In thisreport we demonstratethat ATZ increased the tran-scripts levels of StAR, cytochrome P450scc and 3b-HSD. In addition to this,antioxidant genes expression of GSH-Px, GR, and GST was up-regulatedwhereas the mRNA expression of SOD-1 and SOD-2 was down-regulatedin ILC. When both KV and ATZ were added to the culture medium, the tran-script levels of StAR, P450scc, 3b-HSD and the antioxidant genes wererestored to the control values. Furthermore, KV in the culture mediumattenuates ATZ-induced increase in GSH-Px and GR activities and MDAproduction. When ILC were treated with KV alone in the absence of ATZ,the mRNA expression of StAR, P450scc, 3b-HSD, GSH-Px, GST, SOD-1and SOD-2 were up-regulated. The increased GSH-Px expression alignedwith an increase in activity at 24 h of culture period, indicating a similarmechanismof action at both themRNAleveland activity level.KV increasedthe viability of ILC at 10$60mM dose-dependently at 24 h culture. At 48 hculture, the lowest KV dose (10mM) increased the cell viability whereasthe effects of the higher doses (30$90mM) on cell viabilitywere not differentfrom the control value. Compared with ATZ treatment alone, supplementa-tion with KV prevented the time-dependent decrease in cell viability.

CONCLUSION(S): These observations suggest that KV inhibits ATZ–in-ducedcell death,increase on antioxidantenzyme activities, ILC steroidogenesisand antioxidant gene expression. In addition, our study suggests a potential forKV to stimulate steroidogenesis gene expression in ILC.

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Prediction of Ovarian Hyperstimulation in Patients With PolycysticOvary Syndrome (PCOS) Treated With Human Menopausal Gonado-trophins. 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 Wom-en’s Health and Development; b Departments of Obstetricsand Gynaecology,University of Alexandria; c Department of clinical pathology, University of Alexandria, Alexandria, Egypt.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a rareiatrogenic complicationof ovarian stimulation.It is more commonin patientswith polycystic ovarian syndrome (PCOS) and is difficult to predict.

OBJECTIVE(S): The aim of this work was to evaluate some biochemicaland ultrasound indices as predictors of ovarian hyperstimulation in patientswith PCOS treated for ovulation induction with human menopausal gonado-trophins (HMG).

MATERIALS AND METHOD(S): A total of 33 anovulatory patients withPCOS (based on the Rotterdam consensus) were studied. Antral folliclecount (AFC) was measured on day 3 of a spontaneous or induced menstrualcycle. Day 3 serum levels of estradiol (E2), FSH, LH, AMH, leptin andVEGF were also determined. A dose of 2 HMG ampoules (150 IU) was ad-ministered IM daily starting on day 5 of the cycle. Monitoring was effectedby ultrasound and the dose was increased to 3 ampoules (225 IU) if the re-sponse was inadequate. HCG (5000 IU) was administered IM when the lead-ing follicle reached 18 mm in diameter. Serum E2 was measured again on theday of HCG administration and ovarian hyperstimulation was diagnosed if above 3000 pg/mL. The patient was seen 7 days later to determine the occur-rence 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 (Æ SD) on the day of HCG was 5965.82 pg/ mL (Æ 1591.99) pg/mL in the hyper-stimulated group compared to 2207.27(Æ 859.32) in the normo-stimulated group (P<0.0001). There was no signif-

icant difference between hyper and normo-stimulated patients in day 3 serumFSH, LH, estradiol, leptin or VEGF levels. However, the mean (Æ SD) AFCwas higher in the hyper-stimulated group (21.64Æ 3.20 versus 14.32Æ 3.81;P<0.0002). Similarly, the mean (Æ SD) serum level of AMC was higher inthe hyper-stimulated group (4.50 Æ 2.87 ng/ml versus 2.17 Æ 1.55;P<0.005). Receiver operator characteristic curves showed that the day 3AFC was a better predictor of ovarian hyperstimulation than day 3 level of AMH (area under the curve 0.926 versus 0.771). Seven patients developedclinical OHSS, three were treated with IV albumin and one required aspira-tion of ascetic fluid.

CONCLUSION(S): Day 3 AFC is a better predictor of ovarian hyperstim-ulation 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

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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 Depart-ment of Obstetrics and Gynaecology, Alexandria University; b AlexandriaFertility Center, Alexandria, Egypt.

BACKGROUND: GnRH down-regulation prior to FSH administration isnow the standard protocol in developed countries to achieve controlled ovar-ian hypertimulation prior to IVF or ICSI. However, this protocol is expensiveand requires a longer stimulation time.

OBJECTIVE(S): The aim of this work was to evaluate the HMG-only stim-ulation 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 totalof 608 cycles of IVF and ICSI were performed from the first of January to theend of December 2009. Of those, 558 cycles were stimulated using an HMG-only protocol and 50 cycles were stimulated by a standard GnRH longprotocol. Monitoring was conducted by serial ultrasound scanning of thefollicles. There were no statistically significant differences between bothgroups regarding the age of the couple, the duration of pregnancy or theindication of IVF or ICSI.

RESULT(S): There were no significant difference in the number of am-poules used, the number of oocytes, metaphase II oocytes, premature or post-mature oocytes retrieved between both groups of patients. Similarly, therewere no significant differences in the number of grade I and grade II embryosobtained 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/FSHgroup (28%) and this difference is not statistically significant (P¼0.982).The mean (ÆSD) number of sacs was 1.9 (Æ1.2) in the HMG-onlygroup compared to 2.5 (Æ1.7) and this difference is also not significant(P¼0.552). The mean cost of the HMG-only cycle was US$ 1101.82 com-pared 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 theGnRH/FSH group (4%) compared to seven in the HMG-only protocol(1.25%) (P¼0.353).

CONCLUSION(S): HMG-only protocol is a simpleand cheap protocol forIVF and ICSI and offers a practical alternative for infertility specialists indeveloping countries compared to the classical long stimulation protocol.

SUPPORT: None.

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Angiopoitien 2, Interleukin 1b and Vascular Endothelial Growth Factor(Vegf)’ Concentrations in Peritoneal Fluidand Plasma, a PossibleRole inEndometriosis. Fady S. Moiety, M.D.a,b Abdel Fattah A. Agameya, M.D.a,ba Department of Obstetrics and Gynecology, Faculty of Medicine, Alexan-dria University, Egypt; b The Suzanne Mubarak Regional Centre forWomen’s Health and Development.

OBJECTIVE(S): To investigate the serum and peritoneal fluid (PF) con-centrations of Angiopoitien 2 (Ang-2), Interleukin 1b (IL 1b), and VascularEndothelial GrowthFactor (VEGF), aimingto point out anypredictive role inendometriosis.

DESIGN: Prospective, Case control study.SETTING: University hospital.

FERTILITY & STERILITYÒ S9

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PATIENTS AND METHOD(S): Serum and peritoneal fluid samples weretaken from 112 women undergoing laparoscopy for infertility, pelvic pain,and/or adnexal masses. Sixty one were diagnosed with endometriosis and51 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), secondar-ily, was to correlate these concentrations to the disease stage, presentationand thus assuming their diagnostic potential.

RESULT(S): A significant difference was found between patients andcontrols’ serum and PF concentration of all studied markers except IL 1b.Only PF VEGF showed a significantly higher level in more advanced stagesof endometriosis with a positive correlation with the stage of the disease,

(spearman coefficient¼0.442*) Serum or PF concentrations of any of thestudied markers did not show any specific pattern of stage-related levelchanges. Diagnostic potential of the serum and PF concentrations of the 3markers was assessed by the ROC curve. PF and serum VEGF proved anequal and best diagnostic performance, whereas, PF Ang-2 was the least ef-ficient. It was also possible, based on our results, to suggest preliminarythreshold values for these markers to use as diagnostic or follow-up land-marks with relatively acceptable sensitivity, specificity, positive and negativepredictive values.

CONCLUSION(S): A ‘‘non-invasive’’ predictive tool for endometriosis isbecoming closer. Serum Ang-2, IL 1b, and VEGF, could be reasonable pre-dictors -independently or in combination- with the estimated thresholdvalues. Furthermore, a ‘‘minimally invasive’’ prediction via peritoneal fluidlaparoscopic sampling for markers assay especially VEGF would be anaddition to the diagnostic potential in endometriosis.

SUPPORT:

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Development of a Novel Protocol for Isolation and Purification Of Hu-man 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 MedicalBranch, Galveston, TX. Department of Internal Medicine, Division of Endocrinology, University of Texas Medical Branch, Galveston, TX.

OBJECTIVE(S): To develop an optimal method of purification of humangranulosa cells from heterogenous follicular fluid and a method to accountfor red blood cell (RBC) contamination in samples for accurate comparison

of results between individual patients.DESIGN: Prospective experimental study.MATERIALS AND METHOD(S): Follicular fluid was collected from pa-

tients undergoing oocyte retrieval for IVF. A series of experimental granulosaisolation and purifying techniques was performed with added complexity af-ter determination of results from former sample collections. The techniquesinclude: 1) density gradient (DG) centrifugation with 50% solution of colloi-dal silica, 2) DG centrifugation with a polymer of sucrose, 3) indirect, neg-ative selection of RBCs using primary antibody (Ab) to glycophorin A andsecondary Ab connected to spherical, paramagnetic, epoxy beads, 4) positiveselection of granulosa cells using primary Ab to mullerian inhibiting sub-stance 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 tobiodegradable, SPM nanoparticles, 6) dilution of follicular fluid sampleswith buffer before DG centrifugation. In addition, Western blotting was

used to analyze samples for relative amounts of hemoglobin a (Hgba) ascompared to a standard curve of blood samples.

RESULT(S): The procedurethat resulted in thelargest increase in purityof granulosa cells as well as resulted in the largest ratio of live to dead granulosacells was dilution of the follicular fluid with buffer, then use of DG centrifu-gation using sucrose polymer, then positive selection of granulosa cells usingMISRII and secondary Ab coupled to irregularly-shaped, superparamag-netic, silanized iron oxide beads. Western blotting with staining of granulosacells for Hgba provided visible quantification of RBCs in patient samples.

CONCLUSION(S): A novel protocol for granulosa cell isolation and pu-rification has been developed yielding granulosa cell samples that are largelyfree of nondesirable cells. This protocol provides a solution for patient sam-ples that have heavy RBCcontamination and uniquely involves a primary Abagainst the MISRII. Western blotting with Hbga antibody is an effectivemeans of comparing patient sample purity from RBCs.

SUPPORT: NIH RO1 HD044566-01.

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A Case of Acquired Nonobstructive Azoospermia. K. O’Leary,a R. Lathi,a

P. Turek,b L. Westphal.a a Division of Reproductive Endocrinology andInfertility, Department of Obstetrics and Gynecology, Stanford Univer-sity School of Medicine, Stanford, CA; b The Turek Clinic, San Fran-cisco, CA.

BACKGROUND: Nonobstructive azoospermia (NOA) is a challengingcondition to diagnose and manage. Most NOA cases are either idiopathicor genetically determined, but acquired causes are also possible due to bac-terial or viral infections (mumps), vascular injury, or trauma.

OBJECTIVE(S): To describe a unique case of acquired nonobstructiveazoospermia.

DESIGN: Case report.MATERIALS AND METHOD(S): A 25 year-old male and his 26 year-old

G0 fiancee presentedfor infertility evaluation. The male partner had a historyof a massive pelvic fracture (hip and sacrum) and complete urethral disrup-tion after being hit by a train in Paris one year prior. He had had multiple or-thopedic procedures to stabilize the pelvis and to allow weight bearingactivity. He presented with a suprapubic catheter in place and was takingheavy doses of chronic opiates for nerve pain. Due to erectile dysfunctionand anejaculation, in vitro fertilization with testicular sperm extraction(TESE) with ICSI was planned. On the day of retrieval, five biopsies wereperformed with no sperm found; all oocytes were frozen. On more thoroughurologic evaluation, the patient was found to have an unremarkable urologicexamination with normal testis volume and palpable vasa deferentia. Testos-terone was 370 ng/mL, and LH, FSH, prolactin, and estradiol were normal.For possible mild hypogonadotropic hypogonadism due to chronic opiateuse, the patient eliminated opiates and began clomiphene citrate 25 mg dailywith planned bilateral microdissection TESE six months later. At the time of bilateral microdissection TESE, no sperm were found. In addition, a diffuse’’filmy’’ layer of scar tissue was observed throughout the testis. Additionalhistory revealed that the patient had at least 20 pelvic CT scans and 30 plainfilm X-rays (about 520 mGy to the gonads) over a 6 month period. The causeof azoospermia wasthought to be due to ionizing radiation that induced germcell eradication or arrest. Recommendation: semen analysis and possible fineneedle aspiration map in three years; meanwhile, the couple conceived witha natural cycle intrauterine insemination using directed donor sperm.

CONCLUSION(S): Germcell lossand disruptionof Sertoli cellfunctionfromionizing radiation is a rare cause of acquired NOA. Doses as low 300 mGy maytransiently suppress spermatogenesis, while doses above 6000 mGy usuallycauseirreversible azoospermia and infertility. The reversibility of radiation expo-sure in this case is unclear but should become evident with follow-up.

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PGRMC1 And PGRMC2 Expression is Significantly Decreased in theEndometrium of  Women With Endometriosis. K. Bunch,a

D. Tinnemore,b S. Huff,c Z. Hoffer,c R. Burney,a,b J. Stallings.b a Departmentof Obstetrics/Gynecology, Madigan Army Medical Center, Tacoma, WA;b Department of Clinical Investigation, Madigan Army Medical Center, Ta-coma, WA; c Department of Pathology, Madigan Army Medical Center, Ta-coma, WA.

BACKGROUND: Endometriosis is a hormone dependent inflammatorycondition associated with pain and infertility.Compelling clinical and molec-ular evidence demonstrate attenuated secretory-phase progesterone respon-siveness in affected women, which may contribute to infertility.

OBJECTIVE(S): To compare gene expression of progesterone receptormembrane components (PGRMC) 1 and 2 in endometria of women withand without endometriosis.

METHOD(S): Endometrial pipelle biopsies were obtained from 11women with histologically proven stage III/IV endometriosis and 23 surgi-cally documented disease-free women. Endometrial cycle phase was deter-mined using a combination of self-report cycle day, serum hormoneprofile, and endometrial histologic dating. Specimens were dually processedfor total RNA and protein immunohistochemistry (IHC). Real time quantita-tive polymerase chain reaction (RT-PCR) and IHC measured PGRMC geneand protein expression levels in both groups.

RESULT(S): PGRMC1 (fold change 3.3; P<0.001) and PGRMC2 (foldchange 8.8; P<0.01) gene expression were significantly downregulated in se-cretory phase endometrium in women with endometriosis.Immunohistochem-istry showed that, in these women, downregulated gene expression resulted indecreased PGRMC1 and 2 protein production.

S10 Abstracts Vol. 95, No. 4, Supplement, March 15, 2011