Analysis Of Pregnancy Outcomes In Obese-women With PCOS After IVF-ET And Effects Of Hypoxia-inducible Factor-1? On Endometrial Receptivity Of Them

Background Polycystic ovary syndrome(PCOS)is the most common gynecological endocrine disorder in females with prevalence between 15%and 20%.It is characterized by oligo-or anovulation,polycystic ovaries,infertility and is accompanied commonly by metabolic syndrome such as obesity or insulin resistance.The patients with PCOS have increased risk for cardiovascular health and endometrial cancer.Infertility is a major clinical problem in PCOS women of reproductive age.Evidences have shown that the rate of primary infertility accounts for 50%.As a result,the cumulative pregnancy rate is up to 50%through the treatment of controlled ovarian stimulation for six times,but some patients still have no pregnancy or higher miscarriage rate that might be associated with the metabolic disorders such as obesity or insulin resistance.With the changes of life-style and dietary habits,the incidence of obesity has increased gradually.Studies have shown that obesity is the cause of chronic diseases,such as hypertension,diabetes,cardiovascular disease.The prevalence of obesity in PCOS populations approaches 30%?60%,although it has not been clear about the role of obesity in the pathogenesis of PCOS.Obesity can further increase the insulin resistance and androgen production which might result in negative effect on reproductive potential of PCOS women.There are some argues about the quality of oocytes and embryos in PCOS women undergoing IVF/ICSI treatment.Some believe that the rates of mature oocytes,fertilization and high quality embryo are lower in PCOS women and that affect the outcome of IVF-ET.Obesity also has negative effects on the oocyte quality such as the increased aneuploidy,mitochondrial distribution and endoplasmic reticulum stress,which might impair the outcomes of IVF/ICSI in obese PCOS women.In contrast,some insist that the patients with PCOS have poorer ovarian response to gonadotropin,less retrieved oocytes with the increasement in body mass index,but no differences in the quality of oocytes and embryos were observed.Data concerning the effects of PCOS and obesity on the outcome of IVF were inconsistent.Therefore,the first part of our study was performed to analyze the outcomes of IVF-ET in PCOS infertile patients,and to assess the effects of obesity on them.Embryo quality and endometrial receptivity are vital to the successful implantation.In the last few decades,with the development of embryo culture and freezing technology in assisted reproductive technology,the rate of high quality embryos increased significantly,but the implantation rate of PCOS women remained low in clinical settings.Poor reproductive outcomes are might related to the decreased endometrial receptivity.Studies have shown that implantation,clinical pregnancy and live birth rates are significantly reduced with the increasement of body weight index.Obese patients are often accompanied by the insulin resistance that can inhibit insulin-like growth factor(IGF-1)receptor expression and impair the endometrial deciduas.As we all known,PCOS is an endocrine disorder accompanied by the metabolic syndrome such as obesity or insulin resistance.Therefore,the decreased endometrial receptivity of women with PCOS might be associated with the abnormal endocrine or metabolic environment.But the mechanism of it is unclear today.HIF-1 is an oxygen-sensitive transcription factor,which comprises ? and ?subunits.HIF-1? is regulated by oxygen concentration that determines HIF-I activity.Under normal oxygen concentration,HIF-1? is synthesized in cells but it is rapidly degraded by the ubiquitin-proteasome system.Under hypoxic conditions,the function of HIF-1? increases and its target genes as the vascular endothelial growth factor(VEGF)and glucose transportersl and 4(GLUT1,4)are activated.VEGF is an important mediator of angiogenesis,while GLUTs are responsible for glucose uptake and storage in the endometirum.In recent years,the study of HIF-1 in the reproductive field has been a hot topic.Evidence indicates that the micro-environment of the follicle is hypoxia,which stimulates the HIF-1? expression of granular cell and promotes the occurrence of ovulation.At the time of embryo implantation,cell proliferation and implantation of embryo requires an establishment of the vascular network.So we supposed that HIF-1? might play an important role in the embryo implantation and the establishment of endometrial receptivity.To date,there is no report about the regulation of HIF-1? on the endometrial receptivity of PCOS women.Based on the previous research,the second part of our study was to investigate the possible mechanism of the decreased endometrial receptivity of PCOS women by examining the expression pattern of HIF-1? and its target genes in the endometrium.Meanwhile,the effects of obesity on their expressions were also analyzed.Part ?:Analysis of pregnancy outcomes in obese-women with PCOS after IVF-ETObjective:To asess effects of PCOS and obesity on the outcomes of IVF/ICSI-ET through the analysis of clinical data in PCOS and non-PCOS patients.Methods:A retrospective analysis of data from Reproductive Center of Yantai Yuhuangding hospital was performed.A total of 1056 IVF cycles,including 275 PCOS patients and 781 non-PCOS patients(tubal factor)who underwent the IVF/ICSI-ET were studied.The diagnosis of PCOS was in accordance with the 2003 Rotterdam Diagnostic criteria.According to the Asia Pacific Population Obesity Standard,the PCOS patients were divided into normal weight(Al)and obese(A2)subgroups,the non-PCOS patients were divided into normal weight(B1)and obese(B2)subgroups.Clinical data including age,duration of infertility,body weight,the basal endocrine,and blood lipid were collected.The gonadotropin-releasing hormone antagonist(GnRH-ant)protocol was used for controlled ovarian hyperstimulation(COH)in all patients.Levels of estrogen(E2),progesterone(P)and endometrium thickness were recorded on the day of human chorionic gonadotropin(HCG)administration.Oocytes were retrieved 34-36 h after the administration of HCG.An evaluation of the egg maturation and fertilization was performed after the short-term insemination and the rate of M? oocytes was recorded.The quality score of embryo was performed according to the cell number and fragment of the cell on the D3.The rates of fertilization and high quality embryos were calculated.Luteal phase support was administrated on the day of oocyte retrieval and embryo transfer(ET)was performed three days later.Clinical pregnancy was defined as the presence of a gestationa sac by ultrasonography on the 34th day of ET.The differences of the implantation rate,clinical pregnancy rate and early abortion rate were compared.Results(1)In PCOS group,the primary infertility accounted for 64.4%,its average BMI was higher than the control group.The basal AMH,LH,E2,TT and fasting lipid profile(CHOL,LDL-C,TG)were significantly elevated(P<0.05).During the COH,the duration of gonadotropin(Gn),the level of E2 on HCG day and the number of oocytes were increased in PCOS group compared to the control group(P<0.05).No significant differences were observed in the dosage of Gn,endometrial thickness,M?oocytes rate,fertilization rate and high quality embryo rate(P>0.05)The implantation rate and clinical pregnancy rate in PCOS patients were significantly decreased,while the early miscarriage rate was highly increased(P<0.05).(2)In PCOS patients,the average BMI of A2 subgroup was significantly higher than that of A1 subgroup(P<0.05).The duration of infertility in A2 subgroup was prolonged,and the fasting lipid metabolism were significantly elevated(P<0.05).There were no significant difference in the basal level of FSH,LH,E2,AMH,T in two subgroups(P>0.05).During the treatment of IVF-ET,the total dosage and the duration of Gn were increased,but the E2 level on HCG day was decreased obviously in A2 subgroup when compared with A1 subgroup(P<0.05).No significant differences were observed in the endometrial thickness,M? oocytes rate,fertilization rate and high quality embryo rate between two subgroups(P>0.05).(3)In non-PCOS patients,the average BMI of B2 subgroup was significantly higher than that of B1 subgroup(P<0.05).The LDL-c and TG of blood lipids in B2 subgroup were significantly elevated,while the HDL-c was decreased obviously(P<0.05).No obvious difference was observed in basal reproductive endocrine of two subgroups(P>0.05).During the treatment of IVF-ET,the total dosage and the duration of Gn were increased,but the E2 level on HCG day was decreased obviously in B2 subgroup when compared with the B1 subgroup(P<0.05).No significant differences were observed in the endometrial thickness,M? oocytes rate,fertilization rate and high quality embryo rate between two subgroups(P>0.05).The miscarriage rate was significantly increased in B2 subgroup(P<0.05).But there were no significant difference in the implantation rate and clinical pregnancy rate between two subgroups(P>0.05).Conclusions1.The ratio of primary infertility and obesity was increased remarkably in women with PCOS.Obvious abnormal reproductive endocrine and lipid metabolic disorders were observed in them.2.No statistically significant differences were identified with respect to M? oocyte rate and high quality embryo rate between PCOS and non-PCOS group.The implantation rate and clinical pregnancy rate were lower significantly in PCOS group compared with those in control group.However,the miscarriage rate was increased highly in PCOS group.3.Abnormal lipid metabolism was showed in PCOS and non-PCOS patients with obesity.Obesity reduced the ovarian reactivity and E2 level on HCG day significantly.But the number of eggs retrieved,M? oocyte rate and the high quality embryo rate were not associated with the body weight.4.The clinical pregnancy rate decreased significantly in obese-patients with PCOS,while only the early miscarriage rate increased in obese-patients with non-PCOS.Part ?:Effects of hypoxia-inducible factor-1? on endometrial receptivity of women with PCOSObjective:The study was to investigate the possible mechanism of the decreased endometrial receptivity of women with PCOS by examining the expression pattern of HIF-1? in endometrial tissue.Meanwhile,the effects of body weight on their expression levels were also detected.Materials and Methods:According to the inclusive criteria,a total 81 patients were enrolled for IVF/ICSI.They were divided into PCOS(n=40)and Control(n=41)group,both groups were further divided into obese and normal-weight subgroups based on the body weight.Clinical data,including age,duration of infertility and basal serum reproductive hormone,endometrial thickness,number of oocytes,fertilization rate,the evaluation of oocytes and embryos were recorded.Endometrial tissues were collected from the whole embryo freezing patients on day 5 following the retrieval of oocyte.Expressions of HIF-1?,vascular endothelial growth factor(VEGF)and glucose transporter protein 1,4(GLUT1,4)were detected by quantitative real-time RCR and immunohistochemistry.Results:1.Clinical and COH characteristics of patients.There were no significant difference in the average age,E2,P level,endometrial thickness on HCG day,and the number of oocytes,high-quality embryo rate between the PCOS and Control groups(P>0.05).Serum levels of bLH,TT,fasting insulin,fasting glucose and HOMA-IR in PCOS patients were significantly higher than those of the controls(P<0.05).The PCOS patients with obesity exhibited higher BMI,WHR,fasting insulin and HOMA-IR compared with normal-weight PCOS(P<0.05).In the control group,only BMI and WHR were significantly different between the normal weight and obese subgroups(P<0.05).No significant difference were observed in the serum levels of hormones,endometrial thickness,number of oocytes and high-quality embryo rate between two subgroups(P>0.05).2.Endometrial mRNA expression levels of HIF-1?,VEGF,GLUT1 and GLUT4.(1)In the PCOS and the control groups,the mRNA expressions of HIF-1? were the highest.(2)Compared to the control group,the mRNA expression levels of HIF-1? and VEGF in PCOS patient were significantly decreased(P<0.05).GLUT4 mRNA expression levels were very low in both groups and there was no statistically difference with respect to GLUT]and GLUT4 mRNA expression between the two groups(P>0.05).(3)In the PCOS group,mRNA expression levels of HIF-1? and VEGF in obesity subgroup were significantly lower than in the normal-weight subgroup(P<0.05).There were no significant difference in the mRNA expression levels of GLUT1 and GLUT4 between two subgroups(P>0.05).(4)In the Control group,mRNA expression levels of HIF-1?,VEGF and GLUT1 in obesity subgroup were significantly lower than in the normal-weight subgroup(P<0.05),but no difference in GLUT4 expression level was identified between two subgroups(P>0.05).(5)The mRNA expression levels of HIF-1? in PCOS patients with obesity was significantly lower than that of non-PCOS patients with obesity(P<0.05),while the mRNA expression levels of GLUT1 was obviously increased in PCOS patients with obesity(P<0.05).3.Endometrial histology and protein expression levels of HIF-1?,VEGF,GLUT1 and GLUT4(1)The histological stage of endometrium in PCOS and control groups was in secretary phase.(2)In the PCOS and control groups,strong immunostaining for HIF-1? and VEGF were observed in the nuclei of epithelial and stroma cells.Positive nuclei immunostaining of epithelial cells for GLUT1 and GLUT4 was detected in the PCOS group,while they were mainly localized in the membrane of cells in the Control group.(3)The protein expression levels of HIF-1? and VEGF were significantly lower than those of the Control group(P<0.05).No significant inter-group differences were detected with respect to the expression levels of GLUT1 and GLUT4(P>0.05).(4)In the PCOS group,the protein expression levels of HIF-1? and VEGF in obese patients were lower than those of normal-weight patients(P<0.05),but no significant difference in GLUT1 and GLUT4 protein expression levels between two subgroups(P>0.05).(5)In the Control group,compared with the normal-weight patients,the protein expression levels of HIF-1?,VEGF and GLUT1 decreased remarkably in obese patients(P<0.05).There was no significant difference in the protein expression level of GLUT4 between two subgroups(P>0.05).(6)Compared with the non-PCOS patients with obesity,the protein expression levels of VEGF was decreased significantly in PCOS patients with obesity(P<0.05).No obvious differences were observed in the protein expression levels of HIF-la,GLUT1 and GLUT4 between two subgroups(P>0.05).Conclusions1.The mRNA and protein expressions of HIF-1? were strongly detected in the endometrium during the implantation window,which suggested that the endometrial microenvironment was hypoxic.2.The mRNA and protein expression levels of HIF-1? and VEGF were significantly decreased in patients with PCOS,which indicated that the hypoxic endometrial microenvironment and the establishment of the endometrial vascular network of them might be impaired.3.No significant difference was observed in the mRNA and protein expression levels of GLUT1 and GLUT4 in patients with PCOS.However,the immune localization of them had changed in patients with PCOS,which probably impaired glucose uptake of the cells and affected the decidualization of the endometrium.4.The mRNA and protein expressions of HIF-1? and VEGF decreased significantly in obese patients.It seemed that obesity was oppositively correlated with the outcome of IVF by influencing the establishment of endometrial receptivity especially in the PCOS patients with obesity.