| Chapter 1 High throughput sequencing profile of lncRNA and mRNA in ovarian granulosa cells of patients with PCOSObjective:Polycystic Ovary Syndrome(PCOS)is one of the most common reproductive endocrine disorders in women of reproductive age.According to previous studies,the incidence of PCOS has reached 5.6%among Han nationality women aged 19-45 in China.The clinical manifestations of PCOS are heterogeneous,including not only ovarian polycystic changes,hyperandrogenemia and ovulation dysfunction,but also accompanied by metabolic abnormalities such as insulin resistance,diabetes,obesity,hyperlipidemia.Moreover,PCOS patients are prone to developing long-term complications such as cardiovascular disease and endometrial carcinogenesis.Many factors were proved to be associated with PCOS,including genetic,environmental,dietary,etc.,but the specific causes and pathogenic mechanisms are not clear.Currently,research on the etiology of PCOS was mainly focused on regions with the function of encoding proteins,but relatively little on the role of non-coding RNAs in the etiology of PCOS.Non-coding RNA mainly includes long-chain non-coding RNA(lncRNA),miroRNA,snRNA,snoRNA,piRNA,siRNA,which regulate the expression of genes or proteins through direct or indirect regulation in cells or tissues.Wherein,lncRNA is a class of non-coding RNAs with a length of more than 200nt,which are expressed in cells or tissues in low amounts.By transcriptional regulation and post-transcriptional regulation,the lncRNA could regulate the expression of genes and play an important role in the proliferation,apoptosis,differentiation,and development of various diseases.At present,the role of lncRNA in the pathogenesis of PCOS is not fully understood.The objective of this study was to observe the abnormal expression of lncRNA in ovarian granule cells of PCOS patients by performing highthroughput sequencing of lncRNA in ovarian granulocytes of PCOS patients.Methods:A total of 6 PCOS patients and 6 control patients were included in this study.Their follicular fluid on the day of oocytes retrieval were collected and performed ovarian granulosa cells extraction immediately.High-throughput sequencing techniques were used to detect differentially expressed lncRNA and messenger RNA(mRNA)in both groups after extraction of total RNA from ovarian granulosa cells.After preliminary screening of the highthroughput sequencing results,each group used 30 samples of ovarian granulosa cells to validate the sequencing results by qRT-PCR.Results:Through the detection of high-throughput sequencing technology,a total of 1049 differentially expressed lncRNA and 3246 differentially expressed mRNA were detected.Through qRT-PCR verification,7 lncRNAs(NONHSAT101926.2,NONHSAT136825.2,NONHSAT227177.1,NONHSAT010538.2,NONHSAT191377.1,NONHSAT230904.1,ENST00000607307)and 3 mRNAs(YAP1,ENTPD6 and EREG)were expressed in accordance with the sequencing results.Then the correlation analysis found that 7 lncRNAs were associated with hormone levels and follicle counts,and 3 mRNAs were associated with lipid metabolism.The receiver operating characteristic analysis found that 7 lncRNAs had value in distinguishing PCOS patients from the control group.In addition,NONHSAT230904.1 and NONHS AT227177.1 have value in distinguishing whether the number of high-quality embryos exceeds 50%of the total number of embryos.Conclusion:This chapter exhibited the IncRNA and mRNA of ovarian granulocytes could be involved in the occurrence and development of PCOS,which helps to elucidate the etiology and pathogenesis of PCOS.Chapter 2 The role of LncRNA NONHSAT227177.1 in ovarian granulosa cells in Polycystic ovary syndromeObjective:Polycystic ovary syndrome(PCOS)is one of the most common endocrine disorders,affecting an increasing number of women in childbearing age.The ovaries play an important role in oocyte development and hormone secretion and ovarian granulosa cells are a critical component for the ovaries.The dynamic balance between proliferation and apoptosis in ovarian granulosa cells plays a key role in the development of follicles.One of the main features of the ovaries in PCOS patients is that the stagnation of follicle development occurs during follicle development,and lead to follicular atresia.Studies have shown that ovarian granulosa cells in PCOS patients are prone to apoptosis when compared with non-PCOS patients.And the ratio of pro-apoptotic factors to anti-apoptotic factors in ovarian granulosa cells in PCOS patients is significantly imbalanced.In the human genome,most transcripts are non-coding RNAs,where long non-coding RNAs(lncRNAs)were involved in a variety of diseases.Many studies provided evidence that the lncRNA could regulate cellular functions such as cell proliferation and apoptosis,and affect many female reproductive diseases,such as early-onset ovarian insufficiency(POI)and endometriosis.However,the role of lncRNA in the etiology or pathogenesis of PCOS remain unclear.Therefore,we attempted to elucidate the role and possible mechanisms of differentially expressed lncRNA in ovarian granulosa cells of PCOS patients based on the chapter I.Methods:Through the results of Chapter 1,we obtained the result of a significant upregulation of the expression of NONHSAT227177.1 in the PCOS patient group,and we analyzed the correlation between NONHSAT227177.1 expression and the basic clinical characteristics of patients.In functional experiments,NONHSAT227177.1 was found to have a significant effect on apoptosis ability in the KGN cell line derived from ovarian granulosa cells,and subsequent molecular mechanism studies were carried out using techniques such as Luciferase Assay and RNA binding protein immunoprecipitation(RIP).Results:After expanded sample size validation of the initially screened lncRNAs,it was found that the expression of NONHSAT227177.1 in the PCOS patient group was significantly upregulated.Correlation analysis showed that NONHSAT227177.1 was positively correlated with basic FSH and basic LH.Cell function studies have found that NONHSAT227177.1 can increase the apoptosis of ovarian granulosa cells,block the G1/S phase of the cell cycle,and can reduce the expression of CHTOP(FOP),thereby affecting the expression of estrogen receptor α(ERα),and eventually cause follicle maturation and ovulation disorders and a significant increase in LH levels in blood circulation,and eventually participate in the development of PCOS.Conclusion:LncRNA NONHSAT227177.1 can promote apoptosis of ovarian granulosa cells,block the development of the cell cycle,and inhibit the expression of CHTOP(FOP),affect the expression of ERα,thereby affecting follicle maturation and normal ovulation,and eventually causing the occurrence of PCOS.Chapter 3 The role of LncRNA ENST00000607307 in ovarian granulosa cells in Polycystic ovary syndromeObjective:Ovarian granulosa cells are essential for follicular development and play a growing role in the etiology and pathogenesis of polycystic ovary syndrome(PCOS).Studies have shown that ovarian granulosa cells in PCOS patients tend to be prone to apoptosis,which affects the function of ovarian granulosa cells and causes the occurrence of disease.The role of mitochondria is also very important in maintaining the normal functioning of ovarian granulosa cells.Mitochondria play a key role in cellular energy metabolism and participating the signal transduction for cell proliferation.Mitochondria could also release reactive oxygen species(ROS),which have important impact on cell proliferation,metabolism,gene expression,and immune response.Studies have shown that long non-coding RNAs(lncRNAs)participated in disease development by regulating mitochondrial function,and mediate apoptosis by influencing mitochondrial function.However,the specific regulatory modalities in which lncRNAs participate in the development of PCOS by influencing the normal function of mitochondria are unclear.Therefore,based on the results of Chapter 1,we further elucidate the function of differentially expressed lncRNA ENST00000607307 to explore its role in the development of PCOS.Methods:The results of Chapter 1 showed that the expression of ENST00000607307 in the PCOS patient group had an upward trend,so we continued to expand the sample size validation of ENST00000607307,and then we analyzed the correlation between ENST00000607307 relative expression and the clinical characteristics of patients.In functional experiments,ENST00000607307 was found to have a significant effect on apoptosis ability in the KGN cell line derived from ovarian granules,and subsequent molecular mechanism studies were carried out using the Luciferase Assay technique.Results:ENST00000607307 in the PCOS patient group was found to be significantly upregulated after continuing to expand the sample size validation.Correlation analysis showed that ENST00000607307 was positively correlated with the thickness of the endometrium on the trigger day and negatively correlated with the albumin/globulin ratio,which reflected its potential role in metabolic processes.Cell function studies have found that increased ENST00000607307 expression of MRPL20,which contributed to the apoptosis of ovarian granule cells and blocks the G1/S phase of the cell cycle and could be associated with the occurrence and development of PCOS.Conclusion:ENST00000607307,highly expressed in the PCOS patient group,could increase the apoptosis of ovarian granule cells by increasing the expression of MRPL20,and was involved in the occurrence and development of PCOS.Chapter 4 Ovarian sensitivity decreased significantly in insulin resistant patients undergoing IVF-ETObjective:Insulin resistance(IR)refers to the biological response of target tissues to impaired insulin stimulation,mainly including liver,muscle,and adipose tissue.The steadystate model insulin resistance index HOMA-IR≥2.57 is generally considered to be IR.IR could result to metabolic diseases such as obesity,cardiovascular disease,and non-alcoholic fatty liver disease,as well as reproductive endocrine disorders such as polycystic ovary syndrome(PCOS).For PCOS patients receiving assisted reproductive therapy,in addition to the classic ovulation induction treatment methods of drugs such as clomiphene and letrozole,in vitro fertilization and embryo transfer(IVF-ET)technology is also a commonly used treatment.During IVF-ET treatment,the sensitivity of the ovaries to exogenous gonadotropins(Gn)plays an important role in follicle development and pregnancy outcomes.Therefore,the formulation of an appropriate dose of Gn according to the specific situation of the patient is of great significance for the outcome of the patient’s ovulation induction treatment and the safety of treatment.The ovarian sensitivity index(OSI)is calculated from the total number of oocytes obtained and the total dose of Gn,which can reflect the dose of Gn required for each oocyte.Studies have shown that IR not only impairs the early development of embryos,but also reduced the rate of embryo implantation in IVF-ET.However,the effect of IR on ovarian sensitivity is unclear,so we elucidate the effect of IR on ovarian sensitivity through OSI.Methods:We collected the basic clinical characteristics of the enrolled patients and the details of the IVF-ET treatment process,and divided the patients into PCOS group,Control group,PCOS-IR group,Control-IR group,IR group,and non-IR group,and compared the OSI levels in each group.We also analyzed the correlation of OSI with patient clinical characters and analyzed the role of OSI in pregnancy outcomes in IR patients and the choice of controlled ovarian stimulation(COS)protocols.The OSI was calculated by applying the following formula:(number of retrieved oocytes/total gonadotropin dose)× 1000.The rate of dominant follicle was calculated by the following formula:the dominant follicle count on trigger day/the follicle count on trigger day.Results:We found that OSI decreased significantly in the IR group,and that OSI was significantly positively correlated with anti-Mullerian hormone(AMH),antral follicle count(AFC),basal LH/FSH ratio,follicle count on trigger day,dominant follicle count on trigger day,retrieved oocytes,embryo count,and high-quality embryo count,and OSI was significantly negatively correlated with age,BMI,basal FSH level,starting dose of Gn,and total dose of Gn.In addition,the OSI value was significantly increased in patients with positive biochemical pregnancy and patients with positive clinical pregnancy.Patients with IR can achieve a higher dominant follicle rate with standard long protocol compared to the GnRH antagonist protocol.Conclusion:Ovarian sensitivity is significantly reduced in patients with IR,OSI is significantly associated with traditional markers of ovarian response and may be a good predictor of biochemical pregnancy and clinical pregnancy positivity rates and controlled ovulation induction regimens in IR patients. |
