Follicle-stimulating hormone receptor gene polymorphism in chronic anovulatory women, with or without polycystic ovary syndrome: a cross-sectional study

The study was approved by the Ethics Committee of the Faculty of Medicine, Chiang Mai University (No. 012/2011, dated January 7, 2011). Written informed consent, previously approved by the Institutional Review Board, was read and signed by all participants. The study population consisted of women with chronic anovulation (n = 121) and PCOS (n = 133), who attended either the gynecologic outpatient clinic or gynecologic endocrine clinic, Maharaj Nakorn Chiang Mai University Hospital from January 2012 to August 2013. The controls were 132 pregnant women, who first visited the antenatal care clinic in their first trimesters during the same time period as women with chronic anovulation.

In the anovulation group, women were included if they were Thai, aged 20 to 40 years, had both ovaries present on ultrasound scan, had irregular menstrual cycles, and oligomenorrhea (>35 day intervals between periods), or amenorrhea (absence of vaginal bleeding for at least 6 months or 3 cycles of normal periods), which was persistent for at least 1 year. They were excluded if they: 1) used oral contraceptive pills, hormonal intrauterine devices (IUDs) or other hormonal therapy within 3 months prior to the study; 2) had other endocrine disorders, such as diabetes mellitus, thyroid diseases, premature ovarian failure, hyperprolactinemia or other known causes of androgen excess disorders; 3) had language or other communication barriers; 4) had previous history of ovarian surgery; 5) had previous exposure to cytotoxic drugs or pelvic radiation therapy; and 6) did not consent to participate in the study. Anovulatory women were classified as having PCOS if they fulfilled the Rotterdam criteria (1); otherwise they were classified as having chronic anovulation without PCOS.

The controls consisted of 132 pregnant women who fulfilled the following eligibility criteria: Thai women, aged 20 to 40 years, first attendance at the antenatal care clinic, had regular menstrual cycles with intervals of 21 to 35 days, and spontaneous pregnancy within 12 months of attempt. They were excluded if they: 1) had a history of infertility or any infertility treatment in the past; 2) had language or other communication barriers; 3) had a gestational age of ≥10 weeks; and 4) did not consent to participate in the study.

Anovulatory subjects underwent routine examinations, which included a detailed history, physical and pelvic examination, transvaginal ultrasound, and other laboratory studies as deemed necessary by the attending gynecologists. In addition, blood samples were obtained for hormonal studies and DNA analysis. Plasma concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E₂), prolactin (PRL), testosterone (T), and sex hormone binding globulin (SHBG) were measured with electrochemiluminescence immunoassays (Elecsys®, Roche Diagnostics, Indianapolis, USA). Plasma concentrations of androstenedione (ADD), 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone sulfate (DHEAS) were analyzed using Enzyme-linked Immunosorbent Assay (ELISA, NovaTec Immundiagnostica, GmbH, Germany). Intra- and inter-assay coefficients of variation of all hormonal assays were <4.5% and <6.5%, respectively. Any patient having 17-OHP levels higher than 4.0 ng/mL received ACTH stimulation test to exclude late-onset congenital adrenal hyperplasia.

Subjects in the control group had routine physical and obstetrical examinations, and blood tests as usual for those attending the antenatal clinic for the first time. In addition, three milliliters of blood were obtained in tubes containing ethylenediaminetetraacetic acid for DNA analysis.

DNA was extracted from peripheral blood leukocytes, using QIAamp DNA kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions, and kept frozen at -20°C until use. Genotyping was performed without knowledge of the clinical status of the subjects.

A part of exon 10, from nucleotides 1624 to 2143 of the FSHR gene, was amplified by Polymerase Chain Reaction (PCR), using forward and reverse primers as follows: (primer-1) 5′-TTTGTGGTCATCTGTGGCTGC-3′ and (primer-2) 5′-CAA AGGCAAGGACTGAATTATCATT-3′. The PCR reactions were performed in an Eppendorf Personal Cycler Thermocycler (Eppendorf, Germany) with 5 uL Taq polymerase PCR Master Mix (Invitrogen, USA), 20 pmols of each primer and 100 ng of DNA in a final volume of 10 μL. The cycling conditions were as follows: initial denaturation at 94°C for 5 minutes, 30 cycles consisting of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension and final elongation at 72°C for 7 minutes. The PCR products were digested with BsrI (Promega, USA), an endonuclease that recognized the A to G transition sites. The resulting products were run on 2% agarose gel electrophoresis, stained with ethidium bromide and photographed. Three possible patterns were expected (Figure 1): 1) a single band of undigested products of 520 base pairs in size, indicating homozygous Asn680Asn (NN); 2) a single band of digested products of 413 base pairs, indicating homozygous Ser680Ser (SS), and two bands of 520 and 413 base pairs, representing heterozygous state for Asn680Ser (NS). Ten percent of the samples were randomly selected for DNA sequencing to confirm the presence of polymorphisms shown by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis.

Detection of the Thr307Ala variant was performed by a nested PCR-RFLP method. In this case, a second round of PCR, using a second set of primers that bound to a secondary target within the first run products, was necessary to reduce non-specific products generated by the first run of PCR. The region of exon 10, containing nucleotides 931 to 1587 of the FSHR gene, was first amplified using forward and reverse primers as follows: (primer-3) 5′-TCTGAGCTTCATCCAATTTGCA-3′; and (primer-4) 5′-GGGAAAGAGGGCAGCTGCAA-3′. The PCR reactions were performed in an Eppendorf Personal Cycler Thermocycler with 5 uL Taq polymerase PCR Master Mix, 20 pmols of each primer and 100 ng of DNA in a final volume of 10 μL. The cycling conditions were as follows: initial denaturation at 94°C for 5 minutes, followed by 10 cycles of denaturation at 94°C for 1 minute, annealing at 62°C for 1 minute, extension at 72°C for 1 minute, and a final elongation at 72°C for 7 minutes. The PCR products (657 base pairs of DNA fragments) after the first round (2 μL) were further amplified by a second set of primers as follows: (primer-5) 5′-CAAATCTATTTTAAGGCAAGAAGTTGATTATATGCC TCAG-3′ and (primer-6) 5′-GTAGATTCCAATGCAGAGATCA-3′. The PCR cycle was carried out for 1 minute at 94°C for initial denaturation, followed by 25 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, and a final elongation step at 72°C for 7 minutes. The final PCR products were digested with Bsu36I restriction enzyme, which recognized the transition sites of T to C. Restriction endonuclease digestion products were visualized on 2% agarose gel, stained with ethidium bromide, and photographed. Three different patterns were possible (Figure 2): 1) a single band of undigested products of 364 base pairs in size, indicating a homozygous state for Thr307Thr (TT); 2) a single band of digested products of 324 base pairs in size, indicating a homozygous state for Ala307Ala (AA); and 3) two bands of 364 and 324 base pairs, indicating a heterozygous state for Thr307Ala (TA). Ten percent of samples were randomly selected for DNA sequencing to confirm the results obtained from PCR-RFLP analysis.

In a previous study in Japanese women [9], the prevalence of NS allele in PCOS women was significantly higher than that in the ovulating group (66.7% versus 43.5%, P < 0.05). Given this difference, and a type I error of 0.05 (two-tailed) and a power of 80%, we calculated that a sample size of 71 women would be required per group. To ensure a sufficient sample size, we planned to recruit at least 100 subjects with PCOS and 100 controls.