Altered aquaporin expression in women with polycystic ovary syndrome: hyperandrogenism in follicular fluid inhibits aquaporin-9 in granulosa cells through the phosphatidylinositol 3-kinase pathway.

BACKGROUND: The present study was designed to evaluate whether the alteration of aquaporin-9 (AQP-9) expression in granulosa cells (GCs) of patients with polycystic ovary syndrome (PCOS) was associated with the hyperandrogenism in follicular fluid (FF). METHODS: We recruited infertile women with PCOS (n = 14) and infertile women with tubal blockage (controls, n = 31) for this study. We examined total testosterone (TT), free androgen index (FAI), sex hormone-binding globulin (SHBG), FSH, LH and estradiol in FF. Real-time PCR and western blotting were performed to assess AQP-9 expression in GCs, including effects of dihydrotestosterone (DHT) in vitro. RESULTS: AQP-9 protein was localized in the nucleus, cytoplasm and cell membrane of the human GCs. The TT, FAI and LH levels were all higher, and SHBG levels lower, in the FF of women with PCOS versus controls (P = 0.0145, 0.0001, 0.0191, 0.0001, respectively). AQP-9 mRNA level in GCs of patients with PCOS was tightly correlated with the TT, SHBG levels and FAI in FF (P = 0.0020, 0.0001, 0.0020, respectively). In vitro, DHT (10(-9) mol/l) decreased AQP-9 mRNA (lowest at 12 h) and protein levels in control GCs (P = 0.0005, 0.0247, respectively). The inhibitory effect of DHT on AQP-9 mRNA was attenuated by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor (P = 0.0013). Fifty micromolar 4-(hydroxymercuri) benzoic acid sodium salt (PMB) and 10(-9) mol/l DHT blunted the swelling of GCs in hypotonic medium, respectively (P = 0.0350, 0.0027). CONCLUSION: Hyperandrogenism in FF of women with PCOS inhibited AQP-9 in GCs through the PI3K pathway.

Association of serum and follicular fluid leptin concentrations with granulosa cell phosphorylated signal transducer and activator of transcription 3 expression in fertile patients with polycystic ovarian syndrome.

OBJECTIVE: Our objective was to evaluate whether polycystic ovarian syndrome (PCOS)-associated infertility is related to alterations of leptin, leptin receptor (Ob-R), and the phosphorylated signal transducer and activator of transcription 3 (p-STAT3)/suppressor of cytokine signal 3 (SOCS3) system in the ovary. DESIGN AND SETTING: A case-control study was conducted in a university hospital. PATIENTS: Thirty-one infertile PCOS women with oligoovulation plus polycystic ovarian morphology and 79 infertile women with tubal blockage (control) participated in the study. The subjects were stratified according to in vitro fertilization outcomes: successful and failed subgroups. METHODS: Serum and follicular fluid (FF) leptin levels were measured with ELISA. RT-PCR and Western blotting were performed to assess expression of mRNA encoding leptin and Ob-R and proteins of p-STAT3 and SOCS3 in granulosa cells (GCs). RESULTS: Leptin levels in serum and FF of PCOS women were significantly higher than those of control (P < 0.01). There were no significant differences in expression of leptin mRNA and short and long Ob-Rs between PCOS and control (P > 0.05). The p-STAT3 level was decreased in PCOS compared with control (P < 0.01), whereas SOCS3 remained significantly unchanged (P > 0.05). Further analysis showed that serum and FF leptin levels were significantly higher, whereas p-STAT3 in GCs was lower in the failed subgroup of PCOS than those in the successful subgroup of PCOS (P < 0.05). CONCLUSION: Hyperleptinemia and high FF leptin are important pathologies of PCOS with infertility. Lower levels of p-STAT3 in GCs may be related to ovarian leptin resistance and fecundity in PCOS women. Relatively high serum and FF leptin and low p-STAT3 in GCs may account for decreased fertilization, implantation, and pregnancy rates of in vitro fertilization in PCOS women.

[Expression of aquaporin 9 in granulosa cells of patients with polycystic ovary syndrome in IVF-cycles].

OBJECTIVE: To investigate aquaporin 9 (AQP9) mRNA and protein expression in antrum follicle and luteinizing granulosa cells of polycystic ovarian syndrome (PCOS) ovary, and its relation to follicular fluid steroids hormone levels during IVF cycles. METHODS: AQP9 mRNA expression on luteinizing granulosa cells in IVF cycles was detected by RT-PCR. AQP9 protein expression in antrum follicles of PCOS ovary and luteinizing granulosa cells was measured by immunohistochemistry. The concentrations of estradiol (E2), progesterone (P) and testerone (T) in follicular fluid were measured by radioimmunoassay (RIA). RESULT: The expression of AQP9 mRNA in luteinizing granulosa cells during IVF cycles was positive by RT-PCR. No significant differences in AQP9 mRNA levels in granulosa cells between PCOS and control group were found during IVF cycles. The expression level of AQP9 mRNA in large follicles was higher than that in small follicles, but not significantly. The immunoreactivity for AQP9 was localized in membrane and cytoplast of granulosa cells in antrum follicles from PCOS ovary and luteinizing granulosa cells during IVF cycles. Multiple regression analysis showed that AQP9 mRNA levels on granulosa cells were not correlated with E2, P and T levels in follicular fluid during IVF cycles. CONCLUSION: AQP9 may play an important role in the follicle development and antrum formation through water transport and AQP9 may be involved in the mechanism of follicle development in PCOS.

[Cathepsin D expression in ovaries from polycystic ovarian syndrome patients].

OBJECTIVE: To investigate the expression of cathepsin D in ovary of patients with polycystic ovarian syndrome (PCOS). METHODS: Western blot was performed to detect the expression of cathepsin D and immunohistochemistry was used to detect the protein distribution in ovarian tissue. RESULT: Semi-quantity values of cathepsin D expression in PCOS and control group were 2.06 +/- 0.39 and 4.76 +/- 1.43 (P<0.05), respectively. Immunostaining for cathepsin D was obvious in both follicles and stromal cells, and the strongest immunostaining was seen in granulosa cells of follicles. Immunochemical study showed the protein was mainly located on the cytoplasm and cell membrane. CONCLUSION: Cathepsin D expression is down-regulated in ovaries of PCOS patients, which may provide a clue for the abnormality of follicle development in PCOS.

[Women with poor response to ovarian stimulation have increased follicular bone morphogenetic protein-15 levels].

OBJECTIVE: To evaluate the levels of bone morphogenetic protein-15 (BMP-15) in human follicular fluid (FF) and its association with response to ovarian stimulation. METHODS: Western blotting was performed to determine the levels of BMP-15 in FF obtained from follicle aspirates in 70 patients undergoing IVF treatment. According to the response to ovarian stimulation the patients were divided into poor responder group and normal responder group. RESULT: BMP-15 levels in FF of poor responders were significantly higher than those in normal responders (1.01 +/- 0.34 vs 0.77 +/- 0.24, P<0.01). CONCLUSION: Increased levels of BMP-15 in FF may be associated with poor response to ovarian stimulation.

[Outcome of in vitro fertilization-embryo transfer in treatment of polycystic ovarian syndrome].

OBJECTIVE: To evaluate the outcome of in vitro fertilization embryo transfer (IVF-ET) in treatment of polycystic ovarian syndrome (PCOS) with infertility. METHODS: A retrospective analysis was performed from 52 patients with PCOS (PCOS group) and 408 cases with tubal infertility (control group). Both groups underwent IVF-ET treatment from 2001 to 2004. The duration of stimulation, amps of gammaFSH, the level of serum E2, P on the day of HCG injection, the number of oocytes retrieved, the rates of fertilization, cleavage, implantation and pregnancy, the incidence of ovarian hyperstimulation syndrome (OHSS) and cancelled rate of ET were compared between the two groups. RESULT: The duration of stimulation and amps of gammaFSH were not significantly different between the two groups. The concentration of serum E2, P on the day of HCG injection, the numbers of oocytes retrieved and cleavaged embryos were significantly higher in PCOS group (P <0.01, <0.05). Fertilization rate was significantly lower in PCOS group (P <0.01). The implantation, pregnancy and miscarriage rates per ET were not statistically significant. The OHSS rates and cancelled rates of ET were higher in PCOS group (P <0.01). CONCLUSION: Women with PCOS have a lower fertilization rate compared with those with tubal-factor fertility during IVF-ET. However, more oocytes are recovered and the preimplanted embryo has a normal chance of implantation leading to similar pregnancy rates. The OHSS rates and cancelled rates of ET are higher in PCOS because of a greater number of oocytes developed and a higher level of E2.

Resistin levels of serum and follicular fluid in non-obese patients with polycystic ovary syndrome during IVF cycles.

OBJECTIVES: To measure serum and follicular resistin, steroids hormone levels in women with PCOS (polycystic ovary syndrome) (BMI (body mass index)<25 kg/m(2)), to assess possible correlations of resistin to hormonal and metabolic parameters and to analyze the clinical outcomes of in vitro fertilization-embryo transfer (IVF-ET) in women with PCOS and tubal infertility. STUDY DESIGN: We analyzed the clinical outcomes of IVF-ET in women with PCOS (BMI<25 kg/m(2)) and tubal infertility during the years 2002 to 2004 and compared the serum and follicular fluid resistin levels, estradiol (E(2)), progesterone (P), testosterone (T) levels in 20 PCOS and 20 healthy, age-matched women without PCOS during IVF-stimulated cycles. The correlations between the resistin levels and the outcomes of IVF-ET were evaluated. RESULTS: No significant differences in resistin levels of either serum or follicular fluid between PCOS and control group were found. However, resistin levels in serum were higher than that in follicular fluid in both groups. Multiple regression analysis showed that resistin levels in serum did not correlate with BMI, estradiol, LH (luteinizing hormone) and insulin level in fasting blood. No significant correlations were found between follicular fluid reisistin levels and fertilization rate, implantation rate, clinical pregnancy rate or early miscarriage rate in both PCOS and control groups. CONCLUSION: Our results show that resistin does not have correlation with the hormonal and metabolic parameters as well as the outcomes of IVF. These data suggest that resistin is unlikely to be a local determinant factor in steroidogenesis and growth and maturation of oocytes during IVF-ET in lean women with PCOS.

High maternal serum estradiol environment in the first trimester is associated with the increased risk of small-for-gestational-age birth.

CONTEXT: There are increasing concerns that a disrupted endocrine environment may disturb the growth of the fetus. Assisted reproductive technology (ART) situates gamete/embryo in a supraphysiological estradiol (E2) environment and, thus, provides an ideal model to investigate this problem. OBJECTIVE: Our objective was to investigate whether the maternal high-E2 environment in the first trimester increases the risks of low birth weight (LBW) and small-for-gestational-age (SGA) birth. METHODS: In total, 8869 singletons born after fresh embryo transfer (ET) (n = 2610), frozen ET (n = 1039), and natural conception (NC) (n = 5220) and their mothers were included. Birth weight, LBW, SGA, and maternal serum E2 levels were investigated. RESULTS: The mean serum E2 levels of women undergoing fresh ET at 4 and 8 weeks of gestation were significantly higher than those of the women undergoing frozen ET and the women with NC (P < .01). Serum E2 levels of women undergoing fresh ET at 4 and 8 weeks of gestation were positively correlated to those on the day of human chorionic gonadotropin (hCG) administration (r = 0.5 and r = 0.4, respectively; P < 0.01). The birth weight after fresh ET was significantly lower than that after frozen ET and NC (P < 0.01), with increased incidence of LBW and SGA (P < .05). Furthermore, in the fresh ET group, singletons of mothers with high E2 levels (≥10460 pmol/L on the day of hCG administration) had higher risks of LBW (P < .01) and SGA (P < .01) than those with low E2 levels, and maternal serum E2 level on the day of hCG administration negatively correlated with the birth weight (P < .01). CONCLUSIONS: The maternal high-E2 environment in the first trimester is correlated with increased risks of LBW and SGA. Evaluation of serum E2 before ET should be adopted to reduce the possibility of high E2 exposure to gamete/embryo.

High bone morphogenetic protein-15 level in follicular fluid is associated with high quality oocyte and subsequent embryonic development.

BACKGROUND: Bone morphogenetic protein-15 (BMP-15) has been shown to influence oocyte maturation and quality. However, no relationship has been established between BMP-15 and oocyte quality/embryonic development in humans. The aim of this study is to investigate BMP-15 level in human follicular fluid (FF) and its possible role in determining oocyte quality and developmental potential. METHODS: A total of 79 oocytes and their corresponding FF from 79 women undergoing ICSI were examined. Individual oocytes were inseminated and subsequently assessed on the basis of their fertilization, cleavage and preimplantation development. BMP-15, FSH, estradiol (E(2)) and progesterone levels of FF were also analysed via the techniques of western blot or radioimmunoassay. RESULTS: Higher FF BMP-15 levels were observed in the fertilized and cleaved groups versus the unfertilized and uncleaved groups, respectively (P < 0.05). The best (Grade I) embryo morphology was associated with higher FF BMP-15 levels than Grade II or III embryos (P < 0.01). A significant positive correlation was found between BMP-15 and E(2) levels in the same follicle. CONCLUSION: The present study demonstrates that the BMP-15 level in FF appears to be a potential factor in predicting oocyte quality and subsequent embryo development, and is correlated with E(2) level, which may additionally be a valuable predictor of oocyte fertilization.

Poor ovarian response to gonadotropin stimulation is associated with low expression of follicle-stimulating hormone receptor in granulosa cells.

OBJECTIVE: To explore the importance of follicle-stimulating hormone receptor (FSHR) in granulosa cells in the ovarian response to gonadotropin stimulation. DESIGN: Prospective study. SETTING: A women's hospital in China. PATIENT(S): One hundred infertile women undergoing ovarian stimulation with recombinant follicle-stimulating hormone (rFSH). INTERVENTION(S): These women were divided into three groups: poor, moderate, and high responders, according to the number of follicles with diameter >/=14 mm. The FSHR expression at both mRNA and protein levels was determined by either reverse transcription-polymerase chain reaction or Western blot in granulosa cells. E(2) concentrations in serum and FSH levels in serum/follicular fluid (FF) were measured by electrochemiluminescence immunoassay. MAIN OUTCOME MEASURE(S): Relative expression of mRNA and protein of FSHR in granulosa cells, serum E(2), FSH level in serum and FF, and the number of mature follicles. RESULT(S): The expression of FSHR, at both the mRNA and protein levels, was significantly different among the three groups, with the lowest expression in the poor responders. The level of FSHR protein was positively correlated with the peak level of serum E(2) and the number of mature oocytes. FSH levels in FF and the dosage of rFSH used were significantly different among the three groups, with the highest values in the poor responders. CONCLUSION(S): Different levels of FSHR expression in granulosa cells result in different ovarian response, and lower expression of FSHR may account for poor ovarian response to gonadotropin stimulation, which suggests the critical role of FSHR in the ovarian response to gonadotropin stimulation.

Vascular endothelial growth factor and matrix metalloproteinase-2 expedite formation of endometriosis in the early stage ICR mouse model.

OBJECTIVE: To establish a mouse model for endometriosis and to evaluate roles of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in the formation of disease. DESIGN: Experimental laboratory study. SETTING: A women's hospital in China. PATIENT(S) AND ANIMAL(S): Ten women with endometriosis and 10 control women, as well as ICR mice. INTERVENTION(S): Endometrial fragments were transplanted in the peritoneal cavities of mice at minilaparotomy. Transplants were observed and then removed for the assessment of morphology and immunohistochemical staining of VEGF and MMP-2. MAIN OUTCOME MEASURE(S): Observation of transplants, expression of VEGF and MMP-2. RESULT(S): On days 1 and 2, glandular and stromal cells were viable at the margins of transplants. On day 3, the transplants were surrounded by mesothelial cells, and the endometrial glands and stromal cells were clearly viable at the interface. The scores of VEGF and MMP-2 of viable glandular cells of transplants were increased compared with the ones before transplantation. The scores of VEGF and MMP-2 of transplants from women with endometriosis were higher than those of control women. CONCLUSION(S): Endometrial transplants from the patients with endometriosis express more VEGF and MMP-2 than endometrium in control women, suggesting that VEGF and MMP-2 may expedite the formation of endometriosis in its early stage.