Pregnancy Outcome Of Women With Polycystic Ovary Syndrome And Different Protein Expression Patterns In Their Follicular Fluid During Controlled Ovarian Hyperstimulation Under The Conventional Long Protocol

Polycystic ovary syndrome (PCOS), which is a common cause of female infertility, is one of the most common endocrine disorders in females of reproductive age with prevalence ranging between5and10%. Clinically, characterized by hyperandrogenism, anovulatory infertility and insulin resistance, PCOS mainly manifests oligomenorrhea, amenorrhea, irregular metrorrhagia, infertility, obesity, hirsutism, endometrial hyperplasia and malignant change, bilateral or unilateral polycystic ovaries and increased serum luteinizing hormone levels, etc. Currently, most of the domestic and overseas reproductive centers are using conventional long down-regulation to implement controlled ovarian hyperstimulation (COH) to PCOS patients, followed by IVF-ET or ICSI-ET treatment. However, there is still a debate on the influence of the conventional long protocol COH on the pregnancy outcome of PCOS patients, and its mechanism of PCOS is largely unknown.In order to study the influence of the conventional long protocol COH on PCOS patients'pregnancy outcome during IVF/ICSI-ET, and to explore its possible mechanism, the study has carried out the following four experiments:(1) By using a retrospective analysis, an analysis was made of630PCOS patients643cycles, who had received the conventional long protocol of IVF/ICSI-ET treatment from January2009to December2010in the Reproductive Center in CITIC Xiangya Reproductive and Genetic Hospital, and of8217normal patients8617cycles. A contrastive study was made between PCOS and normal patients in terms of related clinical indicators such as gonadotropin dosage, number of oocytes retrieved, fertilization rate, good quality embryo rate, number of embryos transferred, cancellation rate, clinical pregnancy rate, abortion rate, take-home baby rate, moderate to severe OHSS occurance and so on.(2) Using follicular fluid as experimental materials, through adjusting and optimizing2-DE technical conditions, we will establish relatively stable follicular fluid2-DE and MALDI-TOF-TOF mass spectrometry identification techniques.(3) Taking follicular fluid as a target material, and combining2-DE with mass spectrometry technique, we explored the differently expressed proteins in the follicular fluid of PCOS patients and normal patients with infertility caused by male factors during controlled ovarian hyperstimulation under the conventional long protocol, which may be related to PCOS follicular development. And the function of these proteins was analyzed by bioinformatics methods.(4) By using Western blot method, the expressions of some identified proteins as Alpha-1-antitrypsin, Apolipoprotein A-â… , Transferrin, etc. in follicular fluid were analyzed; by using RT-PCR method, the expressions of the mRNA of some proteins as Serine/threonine-protein kinase MAK, DNA damage-regulated autophagy modulator protein2, Apolipoprotein A-â…£,interleukin12A subunit alpha, Serine palmitoyltransferase2protein, etc. in granulosa cells were detected. The results are as follows:(1) Compared with normal patients, during the process of IVF/ICSI-ET treatment, PCOS patients under conventional long protocol COH are able to obtain more oocytes. There were no significant differences in fertilization rate, clinical pregnancy rate and cancellation rate, etc. However, the abortion rate and moderate to severe OHSS occurance of PCOS patients are significantly higher than the control, while its take-home baby rate is significantly lower than the control.(2) By optimizing the2-DE experimental conditions, the relatively stable technology platform of2-DE and MALDI-TOF-TOF mass spectrometry identification of follicular fluid protein was constructed.(3) By using2-DE and MALDI-TOF-TOF mass spectrometry,32protein spots that showed significant different expressions were screened in the follicular fluid of PCOS patients, among which20unique proteins were identified. Among them, there are13proteins up-regulated in follicular fluid components of PCOS patients as follows:Alpha-1-antitrypsin precursor, Apolipoprotein A-â…  precursor, Transthyretin precursor, Vitamin D-binding protein precursor, Haptoglobin precursor, Apolipoprotein A-â…£ precursor, Isoform3of NAD-dependent deacetylase sirtuin-2, Fibrinogen gamma chain precursor, Fibrinogen beta chain precursor, Plasma retinol-binding protein precursor, Alpha-1B-glycoprotein precursor, Tether containing UBX domain for GLUT4and Transferrin. There are7proteins down-regulated as follows:Keratin, type â…  cytoskeletal9, Serine palmitoyltransferase2, Keratin, type â…¡ cytoskeletal1, Serine/threonine-protein kinase MAK, DNA damage-regulated autophagy modulator protein2, interleukin12A subunit alpha, and Zinc-alpha-2-glycoprotein precursor. Subsequently, we analyzed the functions of the identified20proteins, and found that these proteins are related to the body's insulin resistance, fat metabolism, inflammatory, and cell proliferation, apoptosis and other physiological processes, directly or indirectly affecting the individual follicular development and pregnancy process.(4) Western blot showed that the three proteins Alpha-1-antitrypsin, Apolipoprotein A-â…  and Transferrin are up-regulated in follicular fluid of PCOS patients; RT-PCR results showed that mRNA of the three proteins serine/threonine-protein kinase a MAK, DNA damage-regulated autophagy in modulator protein2and serine palmitoyltransferase2is down-regulated in the granulosa cells of PCOS patients, suggesting that these three follicular fluid proteins originate from secretion of granule cells, which is consistent with the results of mass spectrometry. However, the mRNA of Apolipoprotein A-IV and interleukin12A subunit alpha have not been found expressed in the granulosa cells, indicating that these two proteins may have other origins.These results showed that the PCOS patients under conventional long protocol COH followed by IVF/of ICSI-ET treatment had the same pregnancy rate as the control, while the abortion rate and OHSS occurance had increased significantly, and the take-home baby rate had significantly decreased. Its possible mechanisms maybe related to the expression abnormalities of the identified20follicular fluid-proteins. These proteins may lead to relatively higher abortion rate and lower take-home baby rate of PCOS patients during IVF/ICSI-ET through directly or indirectly affecting their oocyte or embryo quality, and endometrial receptivity. A further study of these proteins may contribute to the effective ovulation treatment. This study has shed light on molecular mechanism of PCOS, and will provide valuable experimental evidence for the diagnosis and treatment of PCOS.