| Part ⅠSubtype Analysis in Polycystic Ovary SyndromeBackground:Polycystic ovary syndrome (PCOS) is one of the most common endocrinal metabolic syndrome affecting6-8%women of reproductive age. It is not a single pathophysiological disorder, but a group of manifestations, which presents extremely heterogeneous consequently. The main symptoms include irregular menstrual cycle, hirsutism, obesity, hyper-luteinizing hormone, hyperinsulinemia, insulin resistance, dyslipidemia, etc., of which oligo-/anovulation (OA), hyperandrogenism (HA), and polycystic ovary (PCO) are considered as the diagnostic phenotypes. According to the consensus of European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine (ESHRE/ASRM) in Rotterdam conference in2003, the diagnosis will be established when two of the above phenotypes existed after excluding for other known disease causing ovulatory dysfunction and hyperandrogenism. Based on Rotterdam consensus, there are four subtypes of the syndrome:a) subtype A presents both OA and HA, but with normal ovary morphology (classical type Ⅰ); b) subtype B presents all of the three phenotypes (classical type Ⅱ); c) subtype C presents both HA and PCO, the ovulatory cycle of whom is regular; d) subtype D presents OA and PCO, with neither biological nor clinical sign of androgen excess. It has been evidenced that there are differences among subtypes in endocrinal and metabolic characteristics. Metabolic disturbances are more severe in PCOS patients without PCO, and relatively milder in patients with normal androgen. The prevalence of the complications, such as obesity, diabetes and cardiovascular disease are demonstrated to be increased in classical types (subtype A and subtype B). Highly heterogeneity brings a huge obstacle in etiological and clinical researches of the syndrome.Identifying the clinical characteristics of every subtype exactly and evaluating the reproductive and metabolic changes mean a lot to explore the mechanism of the syndrome and choose more suitable diagnostic criteria and reasonable therapy in clinical work.Objective:The aim of the present study is to analyze the clinical data of large-scale patients, identify the clinical characteristics of every subtype, and provide theoretical basis to further fundamental researches and clinical trials.Methods:A total of5287PCOS patients diagnosed by Rotterdam criteria and2016normal control subjects were enrolled. The patients were divided into four groups according their subtypes:subtype A (OA+HA), subtype B (OA+HA+PCO), subtype C (HA+PCO), and subtype D (OA+PCO). The parameters of anthropometry, endocrine, and metabolism were compared among four groups of patients and normal controls.Results:a) Basic characteristics:BMI was similar among four groups of patients, which was significantly higher than controls (P<0.01). The m-FG score of subtype D is lower than other subtypes with hyperandrogenism (subtype A, B, C. P<0.01). No significance was found among the latter three groups.b) Endocrinal characteristics:LH and LH/FSH were elevated in every subtype of patients compared with controls (P<0.01). In the cohort of patients, subjects presented all of the three phenotypes (subtype B) have the highest LH level and LH/FSH (P<0.01), no significance were found among the other three subtypes. Testosterone was gradient descent from hyperandrogenic subtypes (subtype A, B, and C) to subtype D and then to controls (P<0.01). No significance was found in PRL (P=0.17).c) Glucose metabolism:INSO in subtype B and D were higher than controls (P<0.01). The highest HOMA-IR was found in subtype D, and then in subtype B. Subtypes A and D as well as controls had the lowest HOMA-IR (all P<0.01). All above results were independent of BMI.d) Lipid metabolism:Levels of CHOL among each group were similar. Compared with controls, TG in all patients groups were elevated and HDL-C were decreased (all P<0.01). In the cohort of patients, level of LDL-C was highest in subtype D (OA+PCO), and lowest in subtype C (HA+PCO). Suptype B (OA+HA+PCO) was in the middle (all P<0.01). Nevertheless it should be noticed that the level of LDL-C of subtype D (OA+PCO) was close to controls, which was also higher than subtype C (HA+PCO)(P<0.01). All results remained significance adjusting for BMI except for TG (P=0.721).Conclusion:The reproductive and metabolic manifestations were different among the four subtypes of PCOS. Endocrinal disturbance and hyperandorgenism were identified in all subtypes, among which subtype B (OA+HA+PCO) was the most severe, while subtype D (OA+PCO) was the mildest. However, the prevalence of the latter is highest in Chinese patients. And they also manifest worse in metabolism. There might be different mechanism underlying each subtype. Part ⅡGeno-phenotype correlation analysis of PCOS susceptibility genes in PCOS patientsBackground:Polycystic ovary syndrome (PCOS) is a heterogeneous endocrinopathy characterized by oligo-anovulation (OA), hyperandrogenism (HA) and polycystic ovary (PCO). Both genetic and evironmental factors are well accepted as the main causes of the syndrome. The vast molecular genetic researches have identified many PCOS related genes. However, as a complex hereditary disease, polygenetic pathopoiesis and the interaction between gene and environment make the single gene association study less effective in screening the candidate genes of PCOS. Genome-wide association studies (GWAS) based on gene chip technology, brings an efficient way for detection and comparison of all or most of the individual genes, and therefore opens a door to study complex diseases. It determines all the variant allele frequencies in genome-wide by comparing the SNPs between cases and controls. Compared with candidate gene strategy which needs to presuppose disease-causing genes beforehand, GWAS can identify the genetic susceptibility of the disease in more systematic and comprehensive way. The previous GWAS on PCOS has studied tens of thousands of patients and controls, and found15susceptibility SNPs (PCOS-SNPs) at11loci. In first phase of GWAS,5SNPs of LHCGR (rs13405728), THADA (rs13429458and rsl2478601), and DENND1A (rs2479106and rs10818854) genes at2p16.3,2p21, and9q33.3respectively were proved to be associated to PCOS. Then the second one confirmed the three loci above and identified eight new ones in addition. Allele frequencies were found significantly different between patients and controls at rs4385527and rs3802457in C9orf3gene, rs1894116in YAP1gene, rs705702in RAB5B gene, rs2272046in HMGA2gene, rs4784165in TOX3gene, rs2059807in INSR gene, rs6022786in SUMO1P1gene, as well as rs2268361and rs2349415in FSHR gene. In these candidate genes, LHCGR, FSHR and INSR, as acting in the important pathways of follicular genesis or insulin metabolism, have been evidenced in the previous single gene screening earlier. But other genes were not mentioned in PCOS related reports before. According to the previous basic researches, it can be speculated that diabetes susceptibility genes THADA and HMGA2, may play a role in insulin metabolism; YAP and HMGA2genes were associated with cell proliferation, and may consequently participate in the mechanism of ovarian morphology changes; LHCGR and RAB5B genes were found to participate in some of the cellular activities such as endocytosis and calcium transportation, and may thus affect the function of some effect cells. But there is still little data about the association of these candidate genes to the phenotypes of PCOS, which is important for the exploration of gene function and the etiology of the syndrome. Therefore, more studies are expected on the geno-phenotype correlation.Obejective:The aim of this study was to identify additional correlations between the phenotypes and genotypes of the15PCOS-SNPs described in the previous studies.Methods:Clinical data of patients enrolled in the previous GWAS were collected. Appropriate genetic models were adapted according to the genotypes frequencies and served as group criteria. Endocrinal and metabolic characteristics were compared between different subgroups.Results:a) Rs13429458in THADA was associated with increased LH (P<0.01) and testosterone (T)(P=0.02) levels in subjects with PCOS. The LH/FSH ratio was also higher in the AA group (P<0.01). After adjusting for age and BMI, there remained significance (Padjust<0.01, Padjust=0.02, and Padjust<0.01, respectively)b) Rs12478601in THADA was associated with increased levels of LDL (P=0.02), which was independent of age and BMI (Padjust=0.02).c) In the dominant model, rs2479106in DENND1A was associated with elevated serum insulin levels2hours after a glucose load in the patients with PCOS (P=0.02). The comparisons was adjusted for age and BMI(Padjust=0.02).d) In dominant model of rs2349415(FSHR), patients carrying T allele had lower serum T level (P<0.01). After adjusting for age and BMI, there remained significance (Padjust<0.01).e) As for rs4385527in C9orf3gene, patients carrying A allele had higher BMI and serum LDL-C, as well as lower HDL-C (P<0.01in all variables). Differences in lipid parameters were all independent of BMI(Padjust<0.01in all variables).f) Similarly, in rs2272046of HMGA2gene, LDL-C level was also elevated in subjects carrying C allele(P<0.01), which was independent of BMI(Padjust<0.01).g) Rs4784165of TOX3gene was associated with hirsutism. PCOS women with GG genotype had lowest m-FG score of three subgroups (P<0.01).h) Serum insulin level2hours after OGTT was found increased in AA subgroup of rs2059807(INSR). There remained statistically significance after adjusting for age and BMI (Padjust=0.042).i) Independent association with hyperandrogenemia was also found in rs6022786(SUMO1P1). Subjects carrying G allele have higher T (P<0.01).Conclusion:The PCOS susceptibility genes THADA, FSHR, TOX3, and SUMO1P1are associated with hyperandorgenism, and THADA as well as DENND1A, C9orf3, HMGA2, and INSR carry risk alleles that are associated with metabolic disturbances in PCOS patients of Han Chinese. These genes might play an important role in the pathophysiological mechanism of the syndrome. Part ⅢGeno-phenotype correlation analysis of PCOS susceptibility genes in single phenotype subjectsBackground:Polycystic ovary syndrome (PCOS) is a complex genetic disorder with significant ethnic diversity and clinical heterogeneous. Two previous GWAS have found15PCOS susceptibility SNPs (PCOS-SNPs) in11locus.2p16.3(rs13405728),2p21(rsl3429458, rs12478601), and9q33.3(rs10818854, rs2479106) were identified in GWAS I, and confirmed in European and American Caucasians. LHCGR, THADA, and DENND1A genes were the suggested candidate genes. But as PCOS is such a complex syndrome affecting so many systems, the casual genes should not be only that three. Therefore GWAS Ⅱ were performed next and provided eight new locus:9q22.32(rs4385527, rs3802457),11q22.1(rs1894116),12q13.2(rs705702),12q14.3(rs2272046),16q12.1(rs4784165),19p13.3(rs2059807),20q13.2(rs6022786), and2p16.3(rs2268361, rs2349415). FSHR, C9orf3, YAP1, RAB5B, HMGA2, TOX3, INSR, and SUMO1P1were the suggested candidate genes.As mentioned above, the privous studies have evidenced the familial aggregation of PCOS phenotypes. The prevalence of insulin resistance, impaired glucose tolerance, and diabetes were significantly elevated in the first-degree relatives of the patients. The levels of andorgen in mothers and sisters of PCOS women were also higher than normal controls, while the LH response was lower. It is reported that compared to age-matched normal women, the daughters of the patients had higher serum insulin, LH, T, and17-OHP levels and larger ovary volume before puberty and would exist thoughout the whole puberty. Thus, it can be seen that genetic factor might play an important role in the etiology of PCOS phenotypes. However, few evidences were found about the direct correction between susceptibility genes and phenotype genesis. It is also unclear by which gene the specific phenotype was regulated. Therefore, in the present study, three cohorts of women presented only one of the key features, including aligo-/anovulation (OA), hyperandrogenism (HA), and polycystic ovary (PCO), were enrolled as cases and compared with strict controls without any of the above phenotypes in order to determine the genetic contributions of these PCOS susceptibility genes to its phenotypes.Methods:Three independent cohorts of women presenting single characteristic phenotype were recruited as cases (746subjects with OA,278subjects with HA, and536subjects with PCO). And a total of1790healthy women without any above pathological phenotypes were enrolled as controls.Results:a) After adjusted for age and body mass index (BMI), variants in LHCGR (rs13405728), C9orf3(rs4385527), and INSR (rs2059807) were strongly associated with OA (Padjust=9.47E-03,4.40E-09, and3.18E-02).b) Positive association were also observed between C9orf3(rs4385527) variation and HA which is independent of age and BMI (Padjust=1.77E-04).c) In addition, there was significant evidence for association of variants in THADA (rs13429458and rs12478601), DENND1A (rs10818854), and C9orf3(rs4385527) with PCO. The difference remained significant after adjusting for age and BMI.(Padjust=1.47E-03,6.38E-04,1.73E-02and2.20E-07).Conclusion:The SNPs in some candidate genes of PCOS () are directly associated to the main phenotypes (LHCGR and INSR with OA; THADA and DENND1A with PCO; C9orf3with all three phenotypes) and therefore likely to be important in their etiology. The results provide indications for further function. |
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