IUI

CONCLUSION: In vitro maturation outcome can be improved by reversible attenuation of the maturation process for 5 days culture with a combination of meiotic inhibitors. Further work is needed to determine the best combination of inhibitors.

P-456 Wednesday, October 19, 2011 A MODIFIED PROTOCOL FOR IN VITRO MATURATION OF MOUSE OOCYTES FROM SECONDARY PREANTRAL FOLLICLES. Y. Tang, W. Wang, L. Ni, H.-C. Liu. Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: To compare the efficiency of three culture media in preantral follicle maturation. DESIGN: Culturing preantral follicles (PFs) by hanging drop method (HDM) at early stage of culture may ease spatial restriction, prevent premature ovulation and synchronize development. At later stage, the medium is optimized to facilitate meiosis. MATERIALS AND METHODS: PFs from 15-day old ovaries were allocated into groups A,B and C. 3-5 follicles were pooled into a 25ml medium droplet and cultured for 6 days by HDM. Aggregates were transferred to 30ul medium droplets covered with oil at day 7. Media used are: A, Medicult +100IU/l FSH for 12 days, followed by 1.5IU/ml hCG on day13. B, SAGE+100IU/l FSH for 12 days, followed by hCG. C, a-MEM+ 10% FBS + ½ ITS + 100IU/l FSH (MIF) for 10 days. Additional 25mM Vitamin C and 2mM RA were added at day11. The medium was further added with 10ng/ml EGF and 1.5IU/ml hCG at day13. Prior to culture in CytoD, MII oocytes were treated with 5mM ionomycin for 20 min. Blastocyst formation of activated ooctyes was scored. RESULTS: TABLE 1. Comparison of different media in oocyte maturation from preantral follicles. <12-day <12-day culture culture

Experimental group

<12-day culture

<48hr after <48hr after <48hr after hCG (total) hCG (total) hCG (total)

Follicles Medium A (Medicult) B (SAGE) C (MIF)

collected

GV

GVBD

Mature (%)

GV

GVBD

Mature (%)

92 102 101

28 38 32

10 38 52

0 1 7

18 27 8

15 42 43

0 (0) 3 (3) 33 (33)

* the percentage was calculated based on the total number of follicles collected; Numbers of oocytes not listed in the talbe are those degenerated. Although optimized for human oocyte IVM, both Medicult and SAGE systems failed to produce mature mouse oocytes due to premature rupture. MIF is able to sustain expansion, ovulation, and meiotic maturation of the COCs with a 33% success rate.
20% of the MII oocytes developed into blastocysts by parthenogenesis, indicating a good ooctye competency. CONCLUSION: Our IVM system is effective to ensure follicle/oocyte maturation from early preantral stage.

OXIDATIVE STRESS P-457 Wednesday, October 19, 2011 ANALYSIS OF THE CORRELATION BETWEEN SEMEN PARAMETERS AND THE LEVELS OF RETINOL, TOCOPHEROL AND CAROTENOIDS IN HUMAN SEMINAL PLASMA AND BLOOD. S. Palini, S. De Stefani, S. Dott. Benedetti, D. C. Bulletti, F. Canestrari. Cervesi Hospital, Cattolica, Rimini, Italy; Universita di Urbino, Urbino, Pesaro-urbino, Italy. OBJECTIVE: Investigate the levels of liposoluble vitamins in both human seminal plasma and blood and draw correlations to sperm quality in terms of concentration, motility and morphology. Additionally, MDA (Malonyldialdeide) and AOPP (Advanced Oxidation Protein Products) levels were analyzed as indicators to the degree of oxidation.

FERTILITY & STERILITYÒ

DESIGN: A total of 43 blood and semen samples (31 samples from patients with male infertility + 12 samples from subjects who had children in the last six months) were collected from males. Liposoluble vitamins were evaluated in every sample. MATERIALS AND METHODS: Semen analisys according with WHO 2009. The plasma from the blood and semen samples were then analyzed for the vitamins and MDA using a standard method of HPLC (High Performance Liquid Chromatography) with UV detection. Correlation was calculated using linear regression analysis. RESULTS: Levels of antioxidant vitamins in non pathological blood samples were generally higher than in pathological blood samples. In seminal plasma samples, infertile males had higher MDA values while non pathological patients had higher levels of antioxidants. Also, we demonstrated that levels of MDA correlate negatively with morphology (R ¼ -0.27741 P¼0.09308) and motility (R ¼ -0.36893 P¼0.1761), while levels of antioxidants correlate positively with seminal parameters. Interestingly, antioxidant profiles in blood and semen also correlate positively. CONCLUSION: These results are consistent with a correlation between the antioxidant profile in plasma and semen. Also, pathological subjects (n ¼ 31), compared to controls (n ¼ 12), have higher levels of lipid and protein oxidation products in semen while exhibiting lower concentrations of liposoluble antioxidants. In addition, the levels of alpha, delta, and gamma tocopherol in plasma from blood correlated positively with semen values. Supported by: These results offer an additional tool in the diagnosis of male infertility, and could improve a possible intervention based on specific vitamin supplementation.

P-458 Wednesday, October 19, 2011 THE RELATIONSHIP BETWEEN PREGNANCY AND OXIDATIVE STRESS MARKERS ON PATIENTS UNDERGOING IVF/IUI. A. Younis, C. Clower, D. Nelsen, W. Butler, M. Garelnabi. Department of Obstetrics and Gynecology, Mercer University School of Medicine, Macon, GA; Dept of Clin Lab and Nutritional Sci, School of Health and Environment, Lowell, MA. OBJECTIVE: The effects of oxidative stress (OS) on female reproductive functions are not clear; however OS is believed to play an important role in endometriosis, PCOS, and unexplained infertility. The aim of this study was to investigate the impact of reproductive hormones on OS markers on women undergoing hormonal stimulation for IVF or IUI cycles. DESIGN: This is a prospective IRB approved study. MATERIALS AND METHODS: Blood samples obtained from 15 patients undergoing ovarian stimulation with rFSH or hMG were divided into two groups. Grp-1 was baseline blood collected on day-2-3 of women cycle. Grp-2 is blood collected at the end of FSH/hMG injection. Serum was collected and stored in LN2 tanks at -196oC until analysis. Standard IVF and IUI procedures were followed. The serum levels of Superoxide Dismutases (SOD), and Interleukin-6 (IL-6) were measured by ELISA kits (Cayman Chemical); the Glutathione Peroxidase (GPx) was spectrophotometically measured (ZeptoMetrix Co.). Student’s t-test was used for statistical analysis. RESULTS: The mean age of patients was (33.3  3.4 yrs) and etiology of infertility was endometriosis (n ¼ 7), PCOS (n ¼ 2), and unexplained (n ¼ 6). Results in Table-1 show a significant positive correlation between levels of E2 and that of SOD, GPx and IL-6. Pregnancies were recorded among 7 of the 15 couples. Interleukin-6 was significantly (P¼0.02) higher in pregnant than non-pregnant group. SOD and GPx levels was not different between pregnant and non-pregnant groups. Oxidative Stress Markers & Estradiol levels in Serum of Women Before & After Hormonal Stimulations (Mean  SEM)

Parameters SOD (U/mL) GPx mU/mL IL-6 (pg/ml) E2

Before

After Stimulation

P value

141.0  36.0 464.5  140 3.4  2.3 28.2  10.1

178.6  102.6 604.1  110 15.5  3.8 907.2  430

0.043 0.022 0.029

CONCLUSION: The results of our study indicated that ovarian stimulation has an impact on the production of SOD, GPx and IL-6. The increased

S241

level of Interleukin-6 activity among pregnant patients is suggestive of its role in enhancing implantation. P-459 Wednesday, October 19, 2011 CAN ANTIOXIDANTS SUPPLEMENTATION IMPROVE ICSI/IVF OUTCOMES IN WOMEN UNDERGOING IVF/ICSI TREATMENT CYCLES? RANDOMISED CONTROLLED STUDY. I. Aboulfoutouh, M. Youssef, S. Khattab. Egyptian International Fertility IVF Center (EIFC-IVF), Cairo, Egypt. OBJECTIVE: To evalute the role of antioxidants supplementation in improving the oocyte maturity and clinical pregnancy rate in infertile women undergoing IVF/ICSI treatment cycle. DESIGN: Randomized controlled trial using computer generated list and closed opaque envelops compared the effect of antioxidants supplementation for women undergoing IVF/ICSI. MATERIALS AND METHODS: 170 women with different indications for IVF/ICSI have been randomized into 2 groups. Study group (n ¼ 85) has been supplemented with antioxidant supplementation daily from the cycle preceding IVF/ICSI cycle and the control group (n ¼ 85) did not. RESULTS: There was no significant difference between both group as regards the age, BMI, basal FSH and type of infertility. There was no evidence of statistically significant difference between both groups as regards the clinical pregnancy rate (20/66 (30%) VS 17/57 (29.8%) OR: 1.02; 95% CI: 0.47 2.22), No. retrieved oocytes (13.6  9.22 vs. 15.3  9.12.P¼0.3, 95% CI:-5.2 – 1.97), No. MII oocytes (10.8  8.16 VS 12.1  7.9, P¼0.4, 95% CI:-4.4 1.86), No. embryos obtained (5.52  1.2 VS 6.0  3.68, P¼0.5, 95% CI: -2.0 – 1.09) and the total units of FSH used (4391  2373.0 vs. 4553.8  2725.7, P¼0.7, 95 % CI:-11173.9 – 849.6). CONCLUSION: Antioxidants supplementation for women undergoing IVF/ICSI they did not improve clinical or laboratory outcomes.

FERTILIZATION

CONCLUSION: 3PN zygotes resulting from ICSI vs. INSEM initially divide more often into 2 blastomeres. Regardless of fertilization method, BF occurred only from zygotes that initially cleaved into 2 BM. Parental origin of the 3rd pronucleus and resulting differences in mitotic spindle formation may explain this difference in developmental phenotypes. TLM provides insight into early embryo development by allowing more precise observation of cleavage events than traditional spot-check analysis. Supported by: NIH R-01 grant.

P-461 Wednesday, October 19, 2011 DETERMINATION OF OOCYTE MATURATION BY BIREFRINGENT IMAGING (BI): ONE STEP CLOSER TO EFFECTIVELY CHOOSING THE BEST EMBRYO FOR TRANSFER IN WOMEN UNDERGOING IVF-ICSI. C. Mullin, K. Melzer, L. Lu, J. Lee, P. LaBella, N. Noyes. NYU Fertility Center, New York University School of Medicine, New York, NY. OBJECTIVE: Using BI, we examined whether oocyte maturation [telophase (TI) vs. metaphase (MII)] at the time of ICSI affects fertilization (fert) and/or embryo development DESIGN: Prospective cohort study. MATERIALS AND METHODS: From 5/2010-4/2011, 47 women
P-460 Wednesday, October 19, 2011 PHENOTYPIC OUTCOMES OF TRIPRONUCLEAR (3PN) ZYGOTES RESULTING FROM INTRACYTOPLASMIC SPERM INJECTION (ICSI) VS. CONVENTIONAL INSEMINATION (INSEM) USING TIME-LAPSE MICROSCOPY (TLM). R. S. Weinerman, M. E. Fino, Y. Kramer, K. Gunsalus, C. McCaffrey, N. Noyes. NYU Fertility Center, New York University School of Medicine, New York, NY; Center for Genomics and Systems Biology, New York University, New York, NY. OBJECTIVE: To compare developmental outcome of 3PN zygotes resulting from ICSI compared to INSEM using TLM. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: 3PN zygotes from ICSI and INSEM cycles performed from 10/2007-12/2009 were eligible for inclusion after research consent had been obtained. 3PN zygotes resulting from ICSI (n ¼ 10) and INSEM (n ¼ 59) cycles were identified at fertilization check (
18 h post-sperm exposure) and cultured in a stage-top incubator for 96 hours. Developmental image-capture was performed every 240-420 seconds using TLM; images were then converted to digital recordings for analysis. Outcome measures included rates of initial day 2 (D2) cleavage, D2 cleavage to 2 vs. 3 blastomeres (BM) and blastocyst formation (BF). RESULTS: Overall, D2 cleavage rates were 98.6% and BF rates were 16.9%. Statistically significant differences in D2 cleavage rates to 2 vs. 3 BM were observed between the two groups [Table]. In both groups, BF was observed only in zygotes that initially divided into 2 BM. The rate of BF was not statistically different between the two groups.

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Abstracts

Oocyte maturation stage (n ¼ imaged oocytes) 2PN fertilization Good-quality day 3 embryos/2pn fert Usable day 5 embryos/2pn fert ET/2pn fert

MII(n ¼ 499)

TI(n ¼ 49)

RR(95% CI)

356/499(71%) 245/356(69%)

29/49(59%) 10/29(34%)

0.83(0.72-0.91) 0.49(0.37-0.62)

212/356(60%)

10/29(34%)

0.50(0.43-0.69)

92/356(26%)

5/29(17%)

0.65(0.44-0.75)

Oocytes with a visible MS were more likely to fert and progress to good quality day 3 & usable day 5 embryos; MII did significantly better than TI oocytes (Table) although absolute MS and zona measures did not correlate with these outcomes. 37% of ETs resulted in clinical pregnancy (CP). 5 ETs included embryos from both a MII+TI derived-oocyte, 2 of which resulted in CP. No transfer was solely TI oocyte-derived thus CP could not be directly attributed to a TI oocyte. CONCLUSION: Determination of MS and oocyte maturation by BI appears to help predict fert and embryo competence which can aid in selecting both the superior embryo for transfer and the best supernumerary embryos for cryopreservation as IVF moves toward single embryo transfer.

P-462 Wednesday, October 19, 2011

Outcome measures of 3PN zygotes from ICSI vs. INSEM

D2 cleavage rate D2 cleavage: 2 BM D2 cleavage: 3 BM D2 cleavage: >3 BM BF rate BF: 2 BM at D2 cleavage

MII and T1 fert and subsequent development [expressed as n (% of 2PN fert)]

ICSI

INSEM

P (Fisher’s)

9/10 (90%) 9/10 (90%) 0/10 (0%) 0/10 (0%) 3/10 (30%) 3/9 (33%)

59/59 (100%) 23/59 (39%) 34/59 (58%) 2/59 (3%) 7/59 (12%) 7/23 (30%)

NS 0.0043 0.0009 NS NS NS

RECOMBINANT HUMAN PHOSPHOLIPASE C ZETA 1 AS BIOLOGICAL SOLUTION IN ARTIFICIAL EGG ACTIVATION INDUCES INTRACELLULAR CALCIUM OSCILLATIONS AND EGG ACTIVATION IN MOUSE AND HUMAN EGGS. S.-Y. Yoon, Y. S. Kim, J. E. Han, W. S. Lee, D. R. Lee, T. K. Yoon. Fertility Center of CHA Gangnam Medical Center, CHA University, Seoul, Korea. OBJECTIVE: Egg activation is a crucial step that comprises the release of the egg from meiotic arrest, pronuclear formation, and subsequent embryo

Vol. 96., No. 3, Supplement, September 2011