Transforming growth factorβ1 level in follicular fluid undergoing ovarian stimulation and in vitro fertilization correlates to oocyte maturation and pregnancy

tiation similar to human ES cells. Ultimately, use of a long-acting hFSH analogue for COH in women undergoing IVF may be beneficial by requiring fewer injections, thereby increasing comfort and compliance.

P-487 Brain-derived neurotrophin factor activates mitogen-activated protein kinase in human granulosa-luteal cells. Shiling Chen, Juan Fu, Fuqi Xing. Dept.OB/GYN, Nanfang Hospital, The First Military Medical Univ, Guangzhou, China; Dept. OB/GYN, Nanfang Hospital,The First Military Medical Univ, Guangzhou, China. Objective: Brain-derived neurotrophin factor (BDNF) was produced by human cumulus granulosa-luteal cells (hCGLCs) in the preovulatory follicle. Our data showed that BDNF regulated steroidogenesis in human mural granulosa-luteal cells (hMGLCs) (unpublished). The present studies were to investigate the effect of BDNF on activation of Mitogen-Activated Protein Kinase (MAPK) signaling pathway in hMGLCs. Further, the role of BDNFinduced MARK activation in steroidogenesis was investigated. Design: The effect of BDNF in activating MAPK signaling pathway was determined in cultured hMGLCs. The effect of MARK inhibition on BDNFinduced influence on estrodial and progestone production was studied. Materials and Methods: hMGLCs were obtained from women undergoing in vitro fertilization-embryo transfer. For time-course experiments, hMGLCs were treated with BDNF at 100 ng/ml for 10, 20, 30 min. For dose-course experiments, hMGLCs were treated with BDNF at 0, 50, 100, 200 ng/ml for 20 min. Phosphorylated forms of p38 MAPK and p44 MAPK were examined by Western Blot. To determine the intracellular signaling pathway subsequent to BDNF treatment and the role of MARK in steroidogenesis, hMGLCs were treated with BDNF at 100 ng/ml for 24h with or without pre-treatment of SB203580 (an inhibitor for p38) at 10 ␮M or PD098059 (an inhibitor for p44) at 50 ␮M for 30 min. Enzyme imunoassay was used to determine the level of the estradiol and progesterone in culture medium of hMGLCs. Data were analyzed by Student-Newman-keuls tests. Results: BDNF up-regulated p38 MAPK activity and down-regulated p44 MAPK activity in a dose- and time- dependent manner in hMGLCs. Treatment with BDNF at 0 ng/ml or 100 ng/ml for 24 hours, E2 in culture medium of hMGLCs was 15142.33⫾2152.46 pg/ml, 18861.63⫾3765.16 pg/ml and P4 was 163.15⫾29.78ng/ml, 116.70⫾23.29 ng/ml, respectively. Treatment of BDNF at 100ng/ml with pre-treatment of SB203580 or PD098059, E2 in culture medium of hMGLCs was 16609.07⫾3020.17 pg/ml, 20118.90⫾3125.36pg/ml and P4 was 149.02⫾29.03 ng/ml, 106.78⫾17.78 ng/ml, respectively. Treatment of hMGLCs with p38 inhibitor significantly inhibited the E2 production (P0.05). Conclusion: Our data demonstrated that BDNF activated p38 MAPK signaling pathway in hMGLCs. And this activation was associated with changes in estradiol and progesterone production. It is suggested MARK signaling pathway is involved in mediating BDNF effect in the human ovary.

P-488 Transforming growth factor␤1 level in follicular fluid undergoing ovarian stimulation and in vitro fertilization correlates to oocyte maturation and pregnancy. Shiling Chen, Mingxiao Ge, Fuqi Xing. Dept.OB/GYN, Nanfang Hosp, The First Military Medical Univ, Guangzhou, China; Dept. OB/GYN, Nanfang Hospital,The First Military Medical Univ, Guangzhou, China. Objective: The follicular fluid plays an important biological role in folliculogenesis, oocyte maturation and ovulation. In the human follicle, TGF ␤1 had been found to be produced in both theca and granulosa cells in culture. We therefore hypothesized that TGF ␤1 might be involved in the regulation of follicular growth and oocyte maturation together with sex hormones, and be correlated to IVF outcome. Our purpose is to investigate the role of transforming growth factor1(TGF␤1) and reproductive hormones in the preovulatory follicular fluid during ovulation stimulation cycles. Design: Enzyme immunoassay was used to determine TGF␤1, estrodial (E2), progesterone(P) and luteinizing hormone(LH) concentrations in follicular fluid. The maturity and fertilization of the corresponding oocyte and clinical pregnancy were analyzed. Materials and Methods: Follicular fluids (FF) and their matched oocytes

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were obtained from follicles of 96 women undergoing ovulation stimulation and IVF treatment. TGF-␤1, E2, P, and LH concentrations in follicular fluid samples that had been collected during transvaginal oocyte retrieval were measured using an enzyme immunoassay. The maturity and fertilization of the corresponding oocyte were followed. Clinical pregnancies were confirmed by ultrasound at 4 weeks after embryo transfer. Results: It was found TGF ␤1, P and LH concentrations were significantly higher at mature oocytes group compared to immature group (2824.4⫾1414.4pg/ml, 3837.1⫾942.8 ng/ml and 0.71⫾0.35 mIU/ml versus 2015.0⫾1191.7pg/ml, 3188.1⫾617.4 ng/ml and 0.54⫾0.21 mIU/ml, respectively, P⬍0.05). The mean concentration of TGF ␤1, E2 and P in fertilized group are higher than in non-fertilized group (2845.7⫾1476.7 pg/ml, 3832.0⫾995.2 ng/ml and 0.73⫾0.35 mIU/ml verses 2044.4⫾1111.1 pg/ml, 3401.6⫾607.6 ng/ml and 0.53⫾0.21 mIU/ml respectively, P⬍0.05). The mean concentrations of TGF ␤1 and LH in FF in pregnancy group was significantly higher than that in non-pregnancy group (3631.4⫾1426.3 pg/ml and 0.74⫾0.25miu/ml versus 2189.2⫾1180.4 pg/ml and 0.52⫾0.29miu/ml respectively,P⬍0.05). No significant difference of level of E2 and P in the follicular fluid were found between the pregnant and non-pregnant groups. Conclusion: Increase of TGF␤1 and LH in the follicular fluid appears to be associated with a positive outcome in IVF treatment; TGF␤1 in FF maybe related to the oocyte quality and development potential.

P-489 ID gene family exhibits a differential expression in mouse uterus and preimplantation embryos. HeeYoung Nah, SeokHo Hong, JiYoon Lee, ChungHoon Kim. Asan medical Ctr, Seoul, Republic of Korea. Objective: The helix-loop-helix (HLH) protein class of transcription factors is important regulators of cellular proliferation and differentiation in a number of cell types. The Id proteins are a family of related mammalian HLH protein, which are hypothesized to act negative regulator of DNAbinding bHLH proteins. Recently, Chaudhary and colleagues have been reported that Id genes may be involved in the differentiation and hormonal regulation of sertoli cell. Design: The purpose of this study was to identify Ids (Id-1,-2,-3 and -4) genes and proteins differentially expressed at the sites of implantation compared to interimplantation on day 4.5 of pregnancy and at the preimplantation embryos, oocytes in mouse. Materials and Methods: The oocytes were obtained after priming sixweek-old female ICR mice with PMSG and the embryos were recovered after mating superovulated female mice with the same strain. To obtain pregnant mice, adult female mice (6-8 weeks old) were placed with fertile males of the same strain, and the day that a vaginal plug was found was considered as day 1 of pregnancy. On the evening of day 4 at the time of blastocyst attachment, implantation sites were visualized by intravenous injection of Chicago Blue B solution (1%) before killing the mice. Implantation segments containing implanting embryo were finely separated from nonimplantation segments. We confirmed the expression patterns of Ids in the uteri obtained from sixteen pregnant females and in the preimplatnation mouse embryos and oocytes by reverse transcription-polymerase chain reaction (RT-PCR). Localization of Ids was examined by immunocytochemistry and immunohistochemistry. Results: The expression level of Id-2 mRNA was similar to that of Id-4 mRNA was almost never detected in the implantation sites. Interestingly, the expression patterns of Id-1 and -3 mRNAs were shown to upregulate at the sites of implantation compared to interimplantation sites in all mice. In normal uteri of adult mice, expression level of Id-1 and -3 proteins were also differently localized, but Id-4 protein was never detected. Whereas Id-1 protein was predominantly localized in luminal and glandular epithelial cells, Id-3 was in two layers of smooth muscle cells. We examined the temporal expression patterns of Id genes during mouse preimplantation embryo development. RT-PCR showed that Id-1, -2 and -3 mRNAs were detected in germinal vesicle (GV) oocyte and the highest level at blastocyst stage. The presence of Id-1 and -3 proteins were also localized in preimplantation embryos using fluorescence immunocytochemistry. Exceptionally, Id-4 mRNA was almost never detected in preimplantation embryos. Conclusion: Id-1 and -3 genes were up-regulated at the sites of implantation compared to interimplantation sites on day 4.5 of pregnancy and the presence of Id-1 and -3 proteins were detected in preimplantation embryos and uterus. This study is the first report describing the expression patterns

Vol. 80, Suppl. 3, September 2003