Association of Rs13405728, Rs12478601, and Rs2479106 single nucleotide polymorphisms and in vitro fertilization and embryo transfer efficacy in patients with polycystic ovarian syndrome: A case control genome-wide association study.

Initially, we analyzed the baseline characteristics of all the study subjects included. As shown in Table 2 , age, infertility duration, WHR, BMI, and family history of diabetes in the PCOS and control groups had no significant difference ( P  > 0.05). These results suggested that the PCOS and control groups could be used for comparison for study purposes. Comparison of baseline characteristics of study subjects in the PCOS and control groups Abbreviations: BMI, body mass index; PCOS, polycystic ovarian syndrome; WHR, waist‐to‐hip ratio. Frequency distributions of genotypes of rs13405728, rs12478601, and rs2479106 in the PCOS and control groups were analyzed as illustrated in Table 3 . Both actual values and theoretical values accorded with Hardy‐Weinberg genetic equilibrium with goodness of fit ( P  > 0.05). Genetic model analysis indicated that the dominant, recessive and addictive models in each SNP sites were statistically significant ( P  < 0.05). All 3 SNP sites were related to PCOS within which difference of the TT genotype frequency of rs13405728 in the two groups was significant, which indicated that T allele might be a predisposing gene of PCOS; the CC genotype frequency of rs12478601 was significantly higher in the PCOS group than in the control group, which meant that patients carrying the CC genotype had an increased risk of suffering from PCOS; AG genotype frequency of rs2479106 was also remarkably higher in the PCOS group than in the control group, which indicated that patient carrying AG genotype had an increased risk of suffering from PCOS. Patients with the CC genotype or the AG genotype of THADA rs12478601 had a increased risk of suffering from PCOS Abbreviations: CI, confidence interval; OR, odds ratio; PCOS, polycystic ovarian syndrome; SNP, single nucleotide polymorphism. Shesis software was adopted to analyze the haplotype of SNP sites. Haplotypes with frequency less than 3% were ignored and failed to be counted. Because DENND1A rs2479106 was in different chromosomes with rs13405728 THADA and THADA rs12478601, they were also excluded from analysis (Table 4 ). Frequency of the TG haplotype in the PCOS group was significantly higher than in the control group, and frequency of the CT and TT haplotypes in the control group was markedly higher than in the PCOS group ( P   0.05). Taken together, frequency of the TG haplotype was higher, while frequency of the CT and TT haplotypes were lower in PCOS. Frequency of the TG haplotype was higher, while frequencies of the CT and TT haplotypes were lower in PCOS Abbreviations: χ2, Chi‐square test; CI, confidence interval; OR, odds ratio; PCOS, polycystic ovarian syndrome. We performed regression analysis, with PCOS or not as the dependent variable and rs13405728, rs12478601, rs2479106, age, infertility duration, WHR, BMI, and family history of DM as independent variables. As it is shown in Supporting Information Table S1 , PCOS was closely related with rs13405728, rs12478601, and family history of DM ( P  < 0.05). Subsequently, we explored that whether different genotypes of THADA rs12478601 could influence IVF‐ET efficacy. Supporting Information Table S2 shows outcome indexes after IVF‐ET in the PCOS and control groups. At the day of HCG injection, the PCOS group had greater E 2 value and number of oocytes retrieved, increased rate of abortion but decreased rate of fertilization and clinical gestation as well as incidence of OHSS when compared with the control group ( P  < 0.05). According to Supporting Information Table S2 , 10 indexes valuable for analysis were selected to correlate SNP and IVF‐ET therapeutic outcomes (Supporting Information Table S3 ). Compared with the TC + CC genotype, the TT genotype in rs13405728 significantly increased rate of abortion (30.46 ± 9.79 vs 20.26 ± 4.12, P  < 0.05) but remarkably decreased rate of high quality embryo and clinical gestation (27.29 ± 6.08 vs 33.09 ± 8.72, P  < 0.05). Compared with the CT + TT genotype, the CC genotype in rs12478601 increased rate of abortion and number of oocytes retrieved but decreased rate of clinical gestation ( P  < 0.05). Compared with the AG + GG genotype, the AA genotype in rs2479106 significantly increased number of oocytes retrieved, rate of abortion as well as incidence of GDM ( P  < 0.05). In order to investigate the influencing factors for efficacy of IVF‐ET, we performed regression analysis, with whether gestation existed or not after PCOS patients received IVF‐ET as the dependent variable and rs13405728, rs12478601, rs2479106, serum E 2 value, number of oocytes retrieved, fertility rate, rate of abortion, and incidence of OHSS as independent variables. As it is shown in Supporting Information Table S4 , whether gestation existed or not after PCOS patients received IVF‐ET was related with rs13405728, rs12478601, serum E 2 value, and fertility rate, ( P  < 0.05).

A case‐control analysis of infertile women who had underwent conventional IVF‐ET in our hospital between September 2013 and September 2015 was performed. A total of 163 PCOS patients (52 cycles) were selected as the PCOS group and 171 patients with tubal factor infertility without PCOS (68 cycles) were selected as the control group. Patients in the PCOS group were between 21 and 34 years old, had body mass index (BMI) between 16.31 and 31.54 without taking any hormone therapies 3 months before being selected for this study. The diagnosis criteria were in accordance with the 2003 Rotterdam Consensus Criteria 18 and patients with at least two of the following three criteria were enrolled: (a) oligoovulation and anovulation; (b) hyperandrogenism or hyperandrogenemia as the clinical or biochemical indexes; and (c) pathological changes of PCOS with numbers of 2 to 9 mm follicles more than or equal to 12 or with an ovarian volume larger than or equal to 10 mL checked by B‐ultrasonography. The exclusive criteria were as below: (a) hyperprolactinemia or thyroid diseases; (b) late‐onset congenital adrenal hyperplasia (atypia CAH), Cushing's syndrome, hypophysoma and polycystic ovaries; (c) known chromosome abnormalities of couples and husband had abnormal sperm. Patients in the control group underwent IVF‐ET treatment in our hospital at the same time with patients in the PCOS group and no significant difference in age, BMI, waist‐to‐hip ratio (WHR) and infertility duration existed between the two groups ( P  > 0.05). Patients in the control group were sterile because of oviduct factors and had normal menstrual cycle and basic indices of endocrine without clinical and biochemical performance of hyperandrogenism, polycystic ovaries under B‐ultrasonography, endometriosis, past failures in gestation and parturition or history of ovarian surgery. Bilateral fallopian tube obstruction or tubal resection detected by hysterosalpingography and laparoscopy were used to diagnose the oviduct factors. On the 2 to 3 days of the menstrual period, the detailed medical history of all patients were collected and they underwent physical examination; the family history of related metabolic diseases (including DM, hypertension and cardiovascular diseases), age, BMI [calculation formula: weight (kg)/height 2 (m 2 )], and WHR were recorded. This experimental plan was approved by the Medical Ethics Committee of our hospital (No. 201308003) and in accordance with the Declaration of Helsinki with written informed consents of patients and their family members. Long protocol for controlled ovarian hyperstimulation was performed for all patients. At the midluteal phase of menstrual cycle 1 month before treatment, subcutaneous injection of long‐acting gonadotropin releasing hormone agonist (GnRH‐a, IPSEN, Paris, France) was applied to all patients at 0.5 to 1 dose each day (0.1 mg/dose). At the 3 days of menstrual cycle, patients were checked of whether reaching the pituitary down‐regulation standards as the following: endometria thickness ≤ 5 mm, diameter of the maximal follicle <10 mm, luteal hormone (LH) ≤ 5 mlU/ml and estradiol ( E 2 ) < 50 pg/mL. When reaching standards, patients were injected with gonadotropin and follicle‐stimulating hormone (FSH, IBSA Institut Biochimique SA, Massagno, Switzerland) every day. Besides, patients underwent transvaginal ultrasound and tests of indexes such as LH, E 2 , and progesterone ( p ) in blood regularly to monitor conditions of follicles' development and uterus. When diameter of one of the dominant follicles reached 18 mm or two reached 17 mm through transvaginal ultrasound or the E 2 level in peripheral blood (PB) reached 200 to 300 pg/mL of dominant follicles, 5000 to 10 000 U human chorionic gonadotrophin (HCG, Harbin Pharmaceutical Group Co., Ltd., Harbin, Heilongjiang, China) was injected into patients at 20:00 to 22:00 very day. After 36 to 38 hours HCG injection, follicle puncture was conducted via transvaginal ultrasound. After the fertilized egg was taken out, the oocyte corona cumulus complex (OCCS) which had already cultured for 3 to 4 hours was transferred into the fertilized nutrient solution and then injected with IVF/intra‐cytoplasmic sperm injection (ICSI). The fertilized condition was assessed 16 to 20 hours after. The fertilized egg was transferred into nutrient solution covered with oil and cultured in a 5% CO 2 incubator at 37°C, and the next day the development of embryo was observed. At the 3 days after fertilization, two embryos with relatively high‐grade morphology were selected to transplant and other embryos were cryopreserved for subsequent uses. Since the day of taking eggs, patients were given luteal support by progesterone injection or combined with oral dydrogesterone tablets (Solvay Pharmaceuticals B.V., Weesp, Holland, the Netherlands) for 17 days. At the 14 days after IVF‐ET, HCG level was measured to diagnose gestation, and if HCG was positive, the gestation was determined to be biochemical so that the luteal support was continued. Intrauterine gestational sac discovered by B‐ultrasonography 35 days later determined that the gestation was a clinical one. According to genome wide association studies (GWAS), 3 PCOS predisposing gene SNP sites: rs13405728 (LHCGR gene), rs2479106 (DENND1A gene), and rs12478601 (THADA gene) were chosen. Their primers were designed by Primer 5.0 software and synthesized by Sangon Biotech Shang‐hai Co., Ltd., Shanghai, China; Table 1 ). A total of 5 mL PB was extracted from patients on an empty stomach at the 2 to 3 days of natural menstrual cycle or at menostasis, added with ethylenediaminetetraacetic acid (EDTA) and preserved at −80°C refrigerator. Genomic DNA was extracted by a PB DNA genome extraction kit (Tiangen, Beijing, China). Spectrophotometer (GENESYS 10S, Thermo Fisher Scientific, Boston, Massachusetts) was used to measure absorbance ( A ) at the wavelength of 260 nm and 280 nm as well as calculating A 260 nm / A 280 nm . If the obtained value was greater than 1.8, the DNA specimen was considered to be in a greater purity and could be used for further experiments. Polymerase chain reaction was adopted to amplify genomic DNA with a volume of 30 μL reaction system: 10 × Pfu buffer 3 μL, dNTP Mix (10 mm) 0.6 μL, forward primer F (10 mM) 0.6 μL, reverse primer R (10 mM) 0.6 μL, template 0.6 μL, Pfu polymerase (2.5 U/μL) 0.3 μL and ddH 2 O 24.3 μL. The PCR amplification conditions were as below: predenaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 seconds; anneal at 58°C for 30 seconds, extension at 72°C for 45 seconds and overall elongation at 72°C for 7 minutes. Fluorescence dissolution curve was used to analyze the amplification process and direct‐sequence was adopted for genotyping. Primer sequences of SNPs (rs13405728, rs12478601, and rs2479106) Abbreviations: F, forward; SNP, single nucleotide polymorphism; R, reverse. E 2 value on the day of HCG injection, the number of oocytes retrieved and the fertility rate 24 hours after egg retrieval were recorded. The embryos were observed at 72 hours, among which those with 6 to 8 divided segmentation‐spheres and less than 20% fragment were diagnosed as high quality embryos and the rate of high quality embryo was then recorded. Subsequently, IVF‐ET was performed. Embryo implantation rate was recorded 2 weeks after transfer. Embryos with positive urine HCG test were viewed as biochemical gestation with the rates kept recorded of. B‐ultrasonography was used 35 days after transfer to check intrauterine gestational sac which was used to diagnose clinical gestation. Rates of clinical gestation and abortion, incidences of ovarian hyper‐stimulation syndrome (OHSS), gestational DM and gestation induced hypertension (PIH) syndrome were recorded. SPSS 17.0 (SPSS Inc., Chicago, IL) was used to analyze the data. Measurement data were represented by mean and SD, and t test was adopted for comparisons between groups of measurement data. Enumeration data were represented by percentage, and analyses of enumeration data were carried out by chi‐square test; a P value less than 0.05 was considered to be statistically significant difference. The PLINK software ( http://pngu.mgh.harvard.edu/purcell/plink ) was applied to analyze frequency distribution of genotypes at both the control and PCOS groups which was further verified by Hardy‐Weinberg genetic equilibrium. The Genetic Power Calculator software ( http://statgen.iop.kcl.ac.uk/gpc /) was adopted for performing genetic power calculations, Shesis analysis software was used to analyze SNP haplotype and logistic regression to risk ratio (OR) and 95% credibility interval (CI) of PCOS. The bilateral P  < 0.05 means the statistically significant difference.

PCOS affecting 6 to 8% Asian women of childbearing age has been identified as a significant cardiovascular risk factor to disorders such as vascular endothelial dysfunction and dyslipidemia and has detriment influence on female health. 19 In this study, genotype and allele frequency distributions of PCOS patients were analyzed to explore the association of different genotypes of LHCGR rs13405728, THADA rs12478601, and DENND1A rs2479106 with the efficacy of IVF‐ET in treating PCOS. The results showed that the LHCGR rs13405728, THADA rs12478601, and DENND1A rs2479106 were related to the efficacy of IVF‐ET in treating PCOS. Firstly, our results demonstrated that patients with the CC genotype or the AG genotype of THADA rs12478601 had an increased risk of suffering from PCOS and that frequency of the TG haplotype was higher, while frequencies of the CT and TT haplotypes were lower in PCOS. Genetic factors are likely to exert effect on the metabolic features of PCOS; low‐density lipoprotein cholesterol exhibited a notable elevation in CC genotype group than in the TC + TT genotype group at rs12478601 of THADA gene. 20 According to a previous study, THADA, one of the PCOS susceptibility genes, was found to carry risk alleles correlated with endocrine in addition to metabolic disturbances in PCOS patients of Han Chinese ancestry. 14 Secondly, rs13405728, rs12478601, and family history of DM were found to be influencing factors for the occurrence of PCOS. The significant association between increased risk of PCOS and rs12478601 has already been validated before. 21 As a previous genome‐wide association study demonstrated, the SNP variants rs12478601 and rs13405728 in the THADA, DENND1A as well as LHCGR genes were found to be independently correlated with PCOS. 14 Moreover, Bindu et al. reported that a family history of DM is able to affect the phenotype of PCOS, which is in consistency with our study. 22 Furthermore, we found that the rate of abortion of patients with the THADA rs13405728 TT genotype increased but the rate of high quality embryo and the rate of clinical gestation significantly decreased. These results indicated that PCOS patients with the THADA rs13405728 TT genotype had worse IVF‐ET efficacy. Additionally, rs13405728, rs12478601, serum E 2 value as well as fertility rate were detected to be influencing factors for IVF‐ET efficacy by logistic regression analysis. It has been reported that PCOS patients with the CC genotype had increased serum LDL level even after adjusting difference in age and BMI, and high LDL level as a significant component of metabolic syndrome as well as a risk factor of cardiovascular disease would also result in hyperlipemia and other severe complication. 14 SNPs of THADA are correlated with type 2 DM. 23 SNPs like rs2479106 located in introns of the DENND1A gene influence DENND1A expression through interacting with upstream and downstream chromosomal regions. 24 It is also reported that the same as PCOS rs2479106 associated with endometrioid adenocarcinoma also encountered with obesity and DM together with endometrial anomalous proliferation. 17 Peak serum E 2 level can serve as a potential marker of in IVF outcomes, 25 consistent with our results. Gene polymorphisms have been reported to affect efficacy of IVE, such as BDNF Val66Met polymorphism. 26 However, the specific mechanism by which rs13405728 and rs12478601 affect the IVF‐ET efficacy requires a further study. In conclusion, through analysis of several SNPs this study proved that PCOS patients with the LHCGR rs13405728 TT genotype, THADA rs12478601 CC genotype and DENND1A rs2479106 AG + GG genotype had worse IVF‐ET efficacy. The results may set a theoretical base for patients undergoing IVF‐ET to select a more suitable treatment according to their specific genotypes and thus have better therapeutic outcomes. However, the sample size included was limited, thus we plan to enlarge the sample size in the future study.

Polycystic ovary syndrome (PCOS) initially described by Stein and Leventhal in 1935 means a multi‐phenotypic disorder identified by menstrual acyclicity and androgen excess with obvious biochemical aberrations. 1 , 2 PCOS and obesity are two frequently happening metabolic disorders associated with subfertility. 3 PCOS is the most frequent female endocrine disorder firstly identified during early reproductive years with prevalence between 6% and 10%. 4 Insulin resistance and hyperinsulinemia have been identified of being related to the pathogenesis of PCOS and about 80% women with PCOS suffer from hyperinsulinemia because of insulin resistance. 5 Anovulation, oligo‐ovulation, presence of polycystic ovaries are several common clinical expressions of PCOS. 4 Subfertility of patients with PCOS would be aggravated by influence of abnormalities on oocyte quality, endometrial receptivity, ovulatory function as well as body mass index. 5 Therefore, therapies which include lifestyle modification such as weight loss, combined oral contraceptive pills, insulin‐lowering medications and androgen receptor antagonists become necessary in order to improve cosmetic appearance and to induce fertility and ovulatory cycles. 2 During the past decades assisted reproductive technologies (ARTs) have been applied in many families in order to have healthy offspring; as one of ARTs in vitro fertilization and embryo transfer (IVF‐ET) has been popular. 6 The most significant factors influencing outcomes of IVF‐ET remain largely unknown in spite of increasing couples of infertility seeking for the help of IVF‐ET. 7 Although IVF‐ET artificially offers supports such as selecting high‐quality embryos the implantation rate of patients with PCOS remains low which urges deeper investigation on that therapy. 8 Uterine receptivity and embryo quality have been reported as two main factors to bring a successful IVF‐ET cycle. 9 It has been also claimed that female's age was a main cause resulting in poor reproductive outcome of IVF‐ET. 6 The correlation between IVF‐ET outcome and endometrial thickness or pattern has been assessed before. 10 With the development of DNA technology and boosting of genomic knowledge, genetic factors involving in failed implantation can also be explored, particularly some single nucleotide polymorphisms (SNPs) such as thrombophilic SNPs. 7 , 11 LHCGR which is expressed in granulosa cells of ovary to influence ovulation via encoding luteinizing hormone/human chorionic gonadotropin receptor is mapped to chromosome 2p16.3. 12 The SNPs site LHCGR rs13405728 as a glycoprotein hormone receptor belongs to the G‐protein coupled receptor family. 13 THADA which is located on chromosome 2p21, was identified first by Rippe et al. as a target gene in the thyroid benign tumor; at present, THADA has been reported as a novel type 2 diabetes mellitus (DM)‐associated gene affecting pancreatic functions of beta‐cell, and 2 SNPs rs13429458 and rs12478601 located in it were identified to be associated with PCOS. 14 , 15 rs2479106 is located in DENND1A gene which encodes a protein charging a domain differentially expressed in normal and neoplastic cells (DENN) that play a role as nucleotide exchange factors for the GTPase Rab35. 16 It has been reported that rs2479106 in DENND1A, rs13405728 in LHCGR and rs13429458 in THADA all showed strong relation with PCOS. 17 Based on those previous studies, we aims at exploring the association of SNPs rs13405728, rs12478601, and rs2479106 with IVF‐ET efficacy in treating PCOS.

The authors have no conflicts of interest to report.

Supplemental Table S1 rs13405728, rs12478601, and family history of DM were influencing factors for the occurrence of PCOS Supplemental Table S2 PCOS group had greater E 2 value and number of oocytes retrieved, increased rate of abortion but decreased rate of fertilization and clinical gestation as well as incidence of OHSS after IVF‐ET Supplemental Table S3 PCOS patients with the THADA rs13405728 TT genotype had worse IVF‐ET efficacy Supplemental Table S4 rs13405728, rs12478601, serum E 2 value as well as fertility rate were influencing factors for efficacy of IVF‐ET