| Background Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrinopathies, affecting5-10%of women of reproductive age. The characteristic clinical features of PCOS include menstrual irregularity such as oligomenorrhoea, signs of hyperandrogenaemia including hirsutism, acne, and/or obesity, and polycystic morphology on ovarian ultrasound. More recent studies have highlighted the link between PCOS and metabolic syndrome, insulin resistance and compensatory hyperinsulinemia appear to be a prominent feature of this phenotype. The pathophysiology of PCOS still remains largely uncharacterized and it has puzzled gynecologists and endocrinologists for many years and proven very difficult to define. Anovulation is the common cause of infertility in PCOS patients, the vast majority of women with PCOS will ovulate and conceive following ovulation induction therapy. Those who fail to be pregnancy often have an additional factor, such as male factor, fallopian tube factor or endometriosis. They should seek in-vitro fertilization&embryo transfer (IVF-ET) or intracytoplasmic sperm injection (ICSI) as a treatment for infertility. Controlled ovarian hyperstimulation (COH) is the key processor in assisted reproductive technology (ART). Women with PCOS will almost yield more oocytes than those without PCOS. However, the fertilization rate of the oocytes from PCOS is almost inevitably lower. A meta-analysis demonstrated an increased cycle cancellation rate, but more oocytes retrieved per retrieval and a lower fertilization rate in PCOS patients undergoing IVF. Although the regulatory mechanisms determining the extent of the sensitivity of individual antral follicles to follicle stimulating hormone (FSH) remains to be elucidated, the appropriate response of antral follicles to FSH and a high quality of oocytes may result in a good outcome after IVF/ICSI.There is no marker that can predict both ovarian response and oocyte competence. To evaluate follicular responsiveness to exogenous FSH, the use of the Follicular Output RaTe (FORT) as an innovative term has been suggested. FORT is assessed by the ratio of the pre-ovulatory follicle (16-22mm) counts (PFC) obtained in response to FSH administration on the day of hCG to the small antral follicle (3-10mm) count (AFC) observed after the complete suppression of endogenous gonadotropins by gonadotropin-releasing hormone agonist (GnRHa)(FORT=PFC/AFC). Gallot found that FORT might be a qualitative reflector of ovarian follicular competence only in patients with regular menstrual cycles. The values of FORT as a predictor of IVF/ICSI outcome in PCOS and non-PCOS patients were unknown.In cumulus-oocyte complex (COCs), cumulus cells are the specialized granulosa cells that directly surround the oocyte during follicular development. Importantly, these cells accompany the oocyte throughout the development from an immature to a fully mature ovulated gamete, as well as beyond, as it awaits fertilization in the fallopian tube. Not surprisingly, cumulus cells thus serve a pivotal role in supporting the oocyte, whether in vivo or in vitro.Adiponectin is the most abundant adipocytokine and it may have insulin-sensitizing property. Clinical evidences demonstrate that the reduction of plasma or serum adiponectin levels is commonly observed in the patients with insulin resistance in comparison with healthy control subjects. Similarly, hypoadiponectinemia is evident in PCOS, and adiponectin levels correlated inversely with insulin resistance. Our previous study confirmed that SNPs rs2241766+45G15(T/G) and rs1501299+276(G/T) in the ADIPOQ gene were strongly associated with PCOS among Han Chinese women. The susceptibility of these two polymorphisms was mainly conferred in insulin resistance and insulin action, which suggested that adiponectin genetic variations might play a common role in the pathogenesis of PCOS. Recently, few reports have summarized the effects of adiponectin on the reproductive organs, either at a molecular level or for clinical relevance. Studies have showed that adiponectin can modulate not only follicle growth but also embryo development in mice and humans. But, the role of adiponectin in embryo development of PCOS patients was unknown.Therefore, the present study was divided into two parts. The first part was to assess the true accuracy of FORT as a prognostic indicator of the response to FSH and the reproductive competence reflected by the outcomes of oocytes and embryos after IVF/ICSI treatment, especially in PCOS patients. The second one was to identify the mRNA and protein expressions of adiponectin and adiponectin receptors in cumulus cells, and the role of adiponectin in embryo development of PCOS patients after IVF treatment.PartⅠPrediction of IVF/ICSI outcome based on the values of the Follicular Output RaTe (FORT) in PCOS patientsObjective To assess the true accuracy of the Follicular Output RaTe (FORT) as a prognostic indicator of the response to FSH and reproductive competence after IVF/ICSI treatment, especially in PCOS patients.Method A total of1643IVF/ICSI cycles, including140PCOS patients who underwent ovarian stimulation, were studied.140cases were diagnosed as PCOS based on the criteria of the Rotterdam ESHRE/ASRM-sponsored PCOS Consensus Workshop Group. The median age was32in both the PCOS and non-PCOS groups. Among the patients with PCOS, there were79cases (56.43%) with oligo-and/or anovulation+hyperandrogemism+PCO,46cases (32.86%) with oligo-and/or anovulation+PCO,11cases (7.86%) with oligo-and/or anovulation+ hyperandrogemism and4cases (2.86%) with hyperandrogemism+PCO. All patients underwent standard pituitary down-regulation protocol. FORT was calculated as the ratio of the pre-ovulatory follicle (16-22mm in diameter) count (PFC) on the day of hCG/small antral follicle (3-10mm in diameter) count (AFC) at baseline (the first day of rFSH use). Low, middle and high FORT groups were divided according to tertile values. Trans-vaginal oocyte retrieval was performed34-36hours after the administration of hCG. Oocytes were fertilized either via conventional insemination or ICSI based on the couple’s history. Fertilization was assessed16-18hours after IVF or ICSI. Embryo transfers were performed3days after oocyte retrieval. No more than three embryos per patient were transferred; surplus embryos were cryopreserved. Progesterone vaginal tablets were administered as luteal support from the day of the oocyte retrieval. Clinical pregnancy was defined as the presence of a gestational sac confirmed5weeks after embryo transfer by ultrasonography.Result Among the140PCOS cases, the clinical pregnancy rate was47.14%(66/140). Women with PCOS who achieved clinical pregnancy exhibited significantly lower FORT values (0.56±0.21vs.0.66±0.29, P<0.05) than those without clinical pregnancy.The mean value of FORT was0.65. Low FORT (n=45), middle FORT (n=56) and high FORT (n=39) referred to FORT values below the33rd percentile (FORT<0.5), between the33rd and67th percentiles (FORT0.5～0.73) and above the67th percentile (FORT>0.73), respectively. In PCOS patients, there were no differences in embryo implantation or clinical pregnancy rates among the three groups according to FORT. Interestingly, although the number of retrieved oocytes was highest in the high FORT group (17.21±7.66vs.15.63±7.08vs.12.04±7.54, P<0.05), the rates of fertilization (69.49%vs.63.47%vs.60.66%, P<0.01) and good-quality embryos (72.71%vs.68.06%vs.64.99%, P<0.05) were significantly higher in middle FORT group. Furthermore, a better IVF/ICSI outcome was achieved in PCOS patients with middle FORT values.Among the1503non-PCOS cases, the clinical pregnancy rate was52.43%(788/1503). FORT was similar in patients who were pregnant and in those who were not pregnant (0.66±0.25vs.0.63±0.26). The rates of2PN fertilization (69.20%vs.64.79%, P<0.001) and good-quality embryos (75.77%vs.55.90%, P<0.001) were significantly higher in the patients who achieved clinical pregnancy.Low FORT (n=402), middle FORT (n=632) and high FORT (n-469) referred to FORT values, respectively. PFC (5.49±2.85vs.8.41±3.27vs.11.54±4.33, P<0.001), the number of retrieved oocytes (8.45±5.64vs.11.52±6.37vs.13.30±6.34, P<0.001) and the total number of embryos available (4.94±3.22vs.6.37±3.69vs.7.33±3.89, P<0.001) increased progressively from the low to high FORT groups. Meanwhile, the rates of good-quality embryos, embryo implantation and clinical pregnancy increased dramatically in accordance with the FORT values (P<0.05); the fertilization rate remained steady.FORT was correlated with AFC (r=-0.162, P<0.001), PFC (r=0.607, P<0.001), serum basal FSH levels (r=-0.123, P<0.001), total number of embryos that could be transferred (r=0.259, P<0.001), and the number of good-quality embryos (r=0.197, P<0.001). Multiple linear regression analysis was performed to study the major independent factors for the number of good-quality embryos, which was used as the dependent variable. Among all of the independent variables, the FORT value (t=1.982, P<0.05) and number of retrieved oocytes (t=17.246, P<0.01) were influential factors but not the AFC and PFC.Conclusion Data from the present study suggest that middle FORT values in PCOS patients and high FORT values in non-PCOS patients may predict better outcomes for IVF/ICSI. To improve the outcome of IVF/ICSI, we should modulate FORT according to the heterogeneity of follicle competence in PCOS patients. Part IIThe role of adiponectin and its receptors on early embryo development in PCOS patientsObjective To identify the mRNA and protein expressions of adiponectin and adiponectin receptors (AdipoRl, AdipoR2and T-cadherin) in cumulus cells isolated from oocytes according to blastocyst formation and to evaluate the role of adiponectin and its receptors on early embryo development in PCOS patients.Method A total of55patients (27PCOS patients and28controls) were studied in this study. PCOS patients were diagnosed based on the presence of two out of three criteria of the Rotterdam ESHRE/ASRM-sponsored PCOS Consensus Workshop Group. In the control group, the patients were non-PCOS, who sought for IVF for the tubal factor. All the patients were divided according to the BMI index. The CCs surrounding a single oocyte were removed using a sharp needle. Oocytes were further inseminated and only mature and normal fertilized oocytes (2PN) were included in this research. CCs from the oocytes yielding blastocyst after5/6days in vitro culture was classified as "B+" group and CCs from oocytes could not develop to blastocyst stage after Day6were divided into "B-" groups. CCs according to the oocytes from the same type of embryos were collected into one pool, and at least three pools were analyzed for one group.8-15CCs per group were collected for RNA or protein extraction. Real time RT-PCR and western blot were performed to identify the mRNA and protein expressions of adiponectin and adiponectin receptors (AdipoRl, AdipoR2and T-cadherin) in cumulus cells.Result mRNA and proteins of adiponectin and adiponectin receptors were expressed in cumulus cells of PCOS and non-PCOS patients by RT-PCR analysis and western blot. Adiponectin and receptor T-cadherin were weakly expressed in cumulus cells of human. The AdipoR2mRNA levels in cumulus cells were higher statistically in PB+group than in PB-group in non-obese or obese PCOS patients respectively (0.1647± 0.0161vs.0.0783±0.0385, P<0.05;0.1948±0.0307vs.0.1057±0.0236, P<0.05). And only in PCOS patients of obese, the AdipoRl mRNA levels in cumulus cells were higher statistically in PB`group than in PB-group (0.5162±0.0371vs.0.2448±0.0333, P<0.01).Conclusion Our studies support the recent reports adiponectin can modulate embryo development in human. The development of early embryo was associated with the up-regulation of AdipoRl and AdipoR2expression in cumulus cells of PCOS patients. Further investigations should be done to clarify the mechanisms of the association of adiponectin and embryo development. |
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