Expression Of Aquaporin 9 And Resistin On Granulosa Cells In Polycystic Ovary Syndrome And Their Impact On The Outcome In IVF Cycles

BackgroundPolycystic ovary syndrome (PCOS) is the commonest cause of anovulatory infertility and affects 5%~10% of reproductive age women. It is a very heterogeneous syndrome both in its clinical presentation and laboratory manifestations. The majority of women with anovulation due to PCOS have menstrual irregularities, usually oligomenorrhea or amenorrhea, associated with clinical and/or biochemical evidence of hyperandrogenism and insulin resistance. Clomiphene citrate (CC) has long been the first line of treatment for those with absent or irregular ovulation. Ovulation is restored in approximately 80% but will result in pregnancy in only about 35-40% of patients who are given clomiphene. Additionally, around 20-25% of anovulatory women with PCOS will not respond at all to CC and are considered to be 'clomiphene resistant'. The clomephene resistant may be associated with obesity, insulin resistance and hyperandrogenism. Gonadotrophin therapy is usually the next step following failure with clomiphene. Gonadotrophin stimulation characteristically induces multiple follicle development, resulting in a high frequency of multiple pregnancies (34%) and OHSS (4.6%) because of the higher response to gonadotropin in PCOS patients.PCOS is usually associated with obesity and insulin resistance. Obesity andinsulin resistance affected the success of fertility treatment in PCOS patients. Ovulation induction with gonadotrophin in obese PCOS women required higher doses than in lean PCOS women, the rate of ovulatory cycles was lower and incidence of miscarriage was higher in obese women. However, some study reported that insulin resistance was thought neither to be related to hormone levels nor to the IVF outcome. Obesity, independent of insulin resistance, is associated with relative gonadotropin resistance.When concept still can't success with the available managements or combined with tubal and male factors, IVF-ET treatment is needed. Several problems may be encountered during the superovulation of patients with PCOS, undergoing IVF treatment: (1) the potential for reduced number and/or quality of eggs and embryos, (2) high baseline serum luteinizing hormone (LH) concentrations and the premature triggering of an LH surge, and (3) OHSS. We analysis the clinical outcomes of IVF-ET in the treatment of PCOS with infertility in retrospect and understand the responses to COH and effects of IVF-ET.ObjectiveWe evaluate the clinical outcomes of IVF-ET in treatment of polycystic ovarian syndrome (PCOS) with infertility in retrospect.MethodsThe groups of observation and the control were composed of 53 patients with PCOS and 408 with tubal infertility. General clinical parameters and basal hormone and fasting insulin levels, the duration of stimulation, amps of Y FSH, the level of serum E2n P on the day of hCG injection and follicular fluid, the number of oocytes retrieved, the rates of fertilization, cleavage, implantation and pregnancy, the incidence of OHSS and cancelled rate of ET were compared between the two groups.IVF stimulation protocol: GnRHa -based protocol (Triptorelin/ Gonal-F /hCG). A transvaginal ultrasound-guided oocyte retrieval was scheduled 34~36 h hCG later. Embryos were transferred 2-3 d after the oocyte retrieval. Luteal phasesupport was given by daily intramuscular injection of P in oil (80 mg). A pregnancy test was administered 14 d after the embryo transfer. If the test was positive, a transvaginal ultrasound study was performed 2-3 weeks later to confirm a clinical pregnancy.Results1. The clinical pregnancy rates /ET of PCOS (38.5%) is comparable to the control group (42%).2. The duration of stimulation and amps of Y FSH were not significantly different between the two groups.3. The numbers of oocyte retrieved and cleavaged embryos were significantly higher in PCOS. Fertilization rate was significantly lower in PCOS. The implantation, pregnancy and miscarriage rates per ET were not statistically significant.4. Multiple regression analysis showed that LH and fasting insulin levels in serum correlate positively with the duration of stimulation and amps of Y FSH.5. The concentration of E2, P in serum on the day of HCG injection was significantly higher, and FF P in PCOS was lower than that in control group.6. The OHSS rates and cancelled rates of ET were higher in PCOS.Conclusions1. The clinical pregnancy rates /ET of PCOS (38.5%) is comparable to the control group (42%), suggesting IVF-ET is an effective management of PCOS with infertility.2. Luteal phase deficiency may occur in PCOS patients during COH, and luteal support is needed after IVF-ET.3. The OHSS rates and cancelled rates of ET are higher in PCOS group after COH, suggesting application of frozen embryo transfer.BackgroundPolycystic ovary syndrome (PCOS) is a leading cause of anovulatory infertility and affects 5%~10% of reproductive age women. It is characterized by hyperandrogenemia and chronic anovulation and is associated with insulin resistance, obesity and increased risk for type 2 diabetes. Many women afflicted with PCOS exhibit insulin resistance and hyperinsulinaemia. Hyperinsulinaemia in PCOS is attributed to obese as well as to insulin resistance independent of body weight. Insulin resistance is thought to play an important role in aetiology of PCOS. In vitro and in vivo studies showed that the sensitivity of insulin to glucose metabolism was subnormal and hyperinsulinaemia in women with PCOS. The administration of insulin sensitizing agents, such as metformin, may increase insulin sensitivity and thus induce ovulation.The hormone resistin, a 12.5 kDa cysteine-rich protein, is secreted by adipocytes. Resistin, a novel signaling molecule isolated in mice, has been suggested to be the putative hormone thought to link obesity with type-2 diabetes. Serum resistin levels are significantly increased in insulin-resistant mice and genetically (ob/ob, db/db) or diet-induced obese mice. In addition, administration of resistin in mice impaired both glucose tolerance and insulin action, In addition, neutralization of endogenous resistin with antibodies significantly suppresses hyperglycaemia in diet-induced obese mice by increasing insulin sensitivity. Furthermore, resistin gene expression in genetically or diet-induced obese mice is down-regulated by thiazolidinedione compounds a new class of antidiabetic drugs with an insulin-sensitizing action. These results suggest that resistin may be an important link between increased fat mass and insulin resistance.Several reports discussed the association between PCOS and resistin. Seow et alreported no difference in serum resistin levels between obese women with PCOS and normal-weight controls. In their study, no association of serum resistin levels with body mass index was observed. Variation in resistin gene promoter was not associated with PCOS. These findings suggest that resistin is unlikely to be a major determining factor in the pathogenesis of PCOS with insulin resistance. However, Panidis et al found significantly higher levels of resistin in the group of PCOS women with BMI>25 kg/m2, compared with normal weight women with PCOS and BMI<25 kg/m2 and control. A significant correlation between resistin and BMI had also been demonstrated. Resistin mRNA levels in adipocytes were increased 2-fold in obese PCOS patients, suggesting that the resistin gene may be a local determining factor in the pathogenesis of PCOS.Thus, insulin resistance is the typical character of PCOS patients, but the mechanism is still illegibility. No distinction was made in previous studies between PCOS and insulin resistance because of difference subjects and methods. No reports about the association between PCOS and the expression of resistin on granulosa cell have been discussed. For further study, we explore the resistin expression on ovary tissue and abdominal fat, and the relation between expression levels of resistin on luteinizing granulosa cells and the hormonal and metabolic parameters of IVF. No previous studies of resistin expression on granulosa cells in women with PCOS were found in the literature.Objective1. To characterize resistin mRNA and protein expression in ovary tissue, abdominal fat tissue and luteinizing granolosa cells from PCOS patients;2. To measure serum and follicular fluid resistin, steroids hormone levels in women with PCOS;3. To assess possible correlations of resistin levels to the hormonal and metabolic parameters;4. To analyze possible correlations of resistin levels to the clinical outcomes of IVF-ET in women with PCOS and tubal infertility.Material & MethodsSubjectsPart 1: The little ovary and abdominal fat tissues were collected from PCOS and control group during laporoscopy or other benignant ovarian cyst operations. Part 2: refer part one.Sample collection:Part 1: a little ovary tissue and abdominal fat tissue from PCOS and control groups.Part 2: Blood samples were collected: basal, between 8:00~10:00 am on the day of hCG. The follicular fluid (FF) (F=1.8 cm) was taken from 20 PCOS and 20 control group subjects and centrifuged at 1000 g for 10 min immediately. Granulosa cells were collected during oocyte pick-up.Serum and tissue samples were stored at - 80 °C until assayed. Methods:1. Resistin mRNA and protein expression levels were investigated in ovary, fat tissue and luteinizing granolosa cells by RT-PCR and Western-blot.2. The resistin staining in ovary and abdominal fat tissue was measured by immunhistochemistry.3. The concentrations of FSH, LH, oestradiol (E2), and progesterone (P) were measured by radioimmunoassay (RIA).4. Insulin levels were assayed using chemiluminescent immunoassay (CL1A).5. Resistin was determined by enzyme-linked immunoassay (ELISA)Results1. The expressions of resistin in mRNA and protein levels in ovary tissue and granulosa cells were negative by RT-PCR and Western-blot.2. The resistin staining in abdominal fat tissue was positive by immunhistochemistry.3. Resistin levels in serum were significantly higher than that in follicular fluid in both groups during IVF cycles.4. No significant differences in resistin levels of either serum or follicular fluid between PCOS and control group were found during IVF cycles.5. Multiple regression analysis showed that resistin levels in serum did notcorrelate with BMI, estradiol, LH and insulin level in fasting blood. Conclusions1. The expression of resistin in mRNA and protein levels in ovary tissue were negative, suggesting that human resistin gene don't express in ovary tissue or expression is at very low levels.2. Resistin levels in serum were significantly higher than that in follicular fluid in both groups during IVF cycles, and the expressions of resistin in mRNA and protein levels on granulosa cells were negative, suggesting that it is unlikely to regulate microenvironment of follicle development in PCOS.BackgroundPolycystic ovary syndrome (PCOS) is a heterogenous condition with a broad clinical and pathologic spectrum. It is a leading cause of anovulatory infertility and affects 5%~10% of reproductive age women. It is characterized by hyperandrogenemia and chronic anovulation and is associated with insulin resistance, obesity and increased risk for type 2 diabetes. The common entity is a polycystic ovary. Such an ovary contains preantral follicles, few antral follicles, many atretic follicles, and follicular and degenerative cysts. The follicles lack a sufficient number of mature granulosa cells to produce enough estrogens. On the other hand, there is a hypertrophy of stromal and thecal tissue continuously producing androgens. Many studies had reports that such small PCOS follicles also are prematurely advanced in development, as evidenced by cell culture studies showing that granulosa cells from such follicles prematurely respond to luteinizing hormone (LH) with progesterone (P4) hypersecretion and overexpress LH receptors. Now it is thought that hyperandrogenemia and insulin resistance results in PCOS granulosa-luteal cells dysfunction and premature follicle atresia.Antrum formation, after gonadotropin stimulation, is a very rapid process, with the rate of follicle expansion approximately 50-fold higher than during the preantral phase of follicle growth. This dramatic increase in size necessitates a rapid and massive transport of water. The water transport mechanism is through proteinaceous water channels, termed aquaporins (AQPs), which are able to support a large volume of water flow. AQPs are members of the major intrinsic protein (MIP) family ofintegral, channel-forming proteins of which >80 have been identified in many different organisms ranging from bacteria to humans. Currently, 13 AQPs have been identified in mammalian tissues. All members of the AQP family contain six transmembrane segments and two "hemi-channels" that fold together into an hourglass conformation to mediate water movement. Most AQPs possess a cysteine residue on the extracellular side of the membrane between the fifth transmembrane segment and the second hemi-channel. This cysteine residue has been shown to bind Hg2+, which sterically blocks the flow of water through the channel. Thus Hg2+ acts as an effective and general inhibitor of most AQPs.Flow cytometry demonstrated the presence of Aqps-7, -8, and -9, thus identifying candidate AQP potentially mediating water movement into antral follicles. These results suggest that water permeability of antral follicles occurs primarily through transcellular mechanisms, which may be mediated by AQPs -7, -8, and/or -9 in granulosa cells. Jablonski's study indicated that AQP-mediated water loss was important for the apoptosis volome decrease (AVD) and downstream apoptotic events, that the water permeability of the plasma membrane could control the rate of apoptosis. Overexpression of AQP-1 in CHO cells enhances AVD and apoptosis.Several AQPs express in mouse uterine myometrium and /or stromal cells. Treatment of adult ovariectomized mice with replacement steroids demonstrates steroid hormones regulated the expression levels, suggesting that AQP play a role in uterine fluid imbibition. AQP5, 8 and 9 mRNA had identified by RT-PCR in rat oviductal epithelium. Staining of AQP5, -8, and -9 was lost after ovariectomy, and only AQP9 immunoreactivity was restored after estradiol and/or progesterone treatments. These results indicate that steroid hormones regulate AQP expression at the mRNA and protein level and might control water transport in the uterium and oviductal lumen.As to now, the mechanism about many antrum follicles in the PCOS ovary and anovulation and atresia is unclear. No reports about the association between PCOS and AQPs and the effects of AQP expression in granulosa cell on the outcomes of IVF have been discussed.Objective1. To characterize AQP9 mRNA and protein expression in antrum follicle in PCOS ovary and luteinizing granulosa cells in IVF cycles.2. To study the relation between AQP9 expression on granulosa cells and follicular fluid steroids hormone levels in women during IVF cycles.3. To analyze possible correlations of AQP9 to the clinical outcomes of in vitro fertilization-embryo transfer (IVF-ET) in women with PCOS and tubal infertility.Material & Methods Materials: refer part 1 and 2. Methods:1. AQP9 mRNA expression levels on luteinizing granulosa cells in IVF cycles were investigated by RT-PCR.2. AQP9 protein stainings in antrum follicles of PCOS ovary and luteinizing granulosa cells were measured by immunhistochemistry.3. The concentrations of oestradiol (E2), progesterone (P) and testerone (T) in follicular fluid in IVF cycles were measured by radioimmunoassay (RIA).Results1. 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 was found during IVF cycles. The expression level of AQP9 mRNA in large follicles was higher than that in small follicle, but not significantly.2. 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.3. Multiple regression analysis showed that AQP9 mRNA levels on granulosa cells did not correlate with E2, P levels in follicular fluid during IVF cycles.4. No correlations were found between AQP9 mRNA levels on granulosa cells and the clinical outcomes of IVF-ET.Conclusions1. AQP9 expresses on granulosa cells in antrum follicles from PCOS ovary and luteinizing granulosa cells during IVF cycles, suggesting that AQP9 may play an important role in the follicle development and antrum formation through water transport.2. The expression level of AQP9 mRNA in large follicles was higher than that in small follicle, but not significantly, suggesting that AQP9 may involve in the mechanism of follicle development in PCOS.3. Expression levels of AQP9 on granulosa cells don't affect steroidogenesis, growth & maturation of oocytes and the outcomes of IVF-ET.