Phosphodiesterase 8B Polymorphism rs4704397 Is Associated with Infertility in Subclinical Hypothyroid Females: A Case-Control Study

The present retrospective study is a matched, casecontrol study. Two hundered and therty infertile females were recruited from Dr. Mahesh Pandya’s Ghanshyam Clinic (a fertility management center; Vadodara, India) along with 100 control females recruited from various health check-up camps. Random sampling method was followed for selection of the groups. The study protocol was explained and informed consent was obtained from all participants of the study. Seventy four out of 230 infertile females were found to have (IF) for the TSH level with the inclusion criteria of primary infertility diagnosis and duration of more than one year of unprotected intercourse without pregnancy, while 76 out of 100 controls were found to be euthyroid (with normal thyroid hormone levels). Exclusion criteria were male factor infertility, any tubal anomaly congenital or urogenital tract anomaly and history of thyroid disease/medication/surgery.

For this study, IF-SCH females/case group are defined as the infertile females who have subclinical hypothyroidism with no other clinical difficulty. In addition, they should not be under any type of medication, including thyroid disorder. Whereas, the control group includes fertile, perous, healthy euthyroid females with no medical history for thyroid or any other disorder. Control group does not include any subclinical hypothyroid female.

Sample size for the present study was calculated using G-Power software with Alpha 0.05 and effect size of 0.9. The effect size was calculated based on the observed genotype frequencies (34).

Thyroid function test Five ml blood samples was collected by venous puncture from fasting individuals and serum was separated for thyroid function test (TFT). Estimation of serum TSH, free T₃ (fT₃) and fT₄ were carried out by enzyme-linked fluorescence immunoassay (ELFA) on mini VIDAS® immuno-analyzer (BioMérieux India Pvt. Ltd., India). Females having TSH values between 3.5 and 10 μIU/ml with normal fT₄, along with an opinion from gynecologist and endocrinologist were considered as IF-SCH females. Fertile females having TSH values within the normal/euthyroid range (i.e. 0.35-3.5 μIU/ml) and fT₄ levels within the normal range were included as controls in the present study. The reference range for serum thyroid hormones (fT₃ and fT₄) and TSH levels for different conditions are shown in Table S 1 (See Supplementary Online Information at www.celljournal.org). The confounding variables such as age, body mass index (BMI), smoking and hemoglobin (Hb) levels showed no significant difference between control and IF-SCH females ( Table S2, See Supplementary Online Information at www.celljournal.org).

DNA was extracted from peripheral blood mononuclear cells (PBMCs) using ‘IAamp DNA Blood Kit (QIAGEN Inc., USA) as per manufacturer’s instructions. PDE8B rs4704397 A/G genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) while PDE8B rs6885099 (G/A) genotyping was done by amplification refractory mutation system (ARMS)-PCR. Amplification was performed using Mastercycler Gradient PCR (Eppendorf, Germany) according to the following protocol: initial denaturation at 94°C for 10 minutes, followed by 30 cycles of denaturation at 94°Cfor 45 seconds, annealing at 60°C for 45 seconds and 72°C for 1 minute. The amplified products were analyzed by electrophoresis in a 2.0% agarose gel stained with ethidium bromide. The respective primers and restriction enzyme (RE) used for genotyping are shown in Table S3. 15 μl of the amplified products was digested for 16 hours at 37°C, using 1 U restriction enzyme. For PCR-RFLP based genotyping, the digested products (300 bp and 219 bp) with 100 bp DNA ladder (Bioron, Germany) were loaded in 3.5% agarose gels stained with ethidium bromide and visualized under UV transilluminator. Furthermore, genotyping of PDE8B rs6885099 G/A was done by Amplification refractory mutation system (ARMS-PCR) in 60 IF-SCH females and 76 control females. Human growth hormone (HGH) was used, as a reaction control in the ARMS-PCR (35). Amplification was performed using Mastercycler Gradient PCR according to the following protocol: initial denaturation at 94°C for 10 minutes, followed by 35 cycles of 94°C for 30 seconds, primer dependent annealing for 30 seconds and 60°C for 1 minute. The amplified products were analyzed by electrophoresis in a 3.5% agarose gel stained with ethidium bromide using 100 bp DNA ladder.

Hardy-Weinberg equilibrium (HWE) test was evaluated for the polymorphisms using chi-square test equating the observed and expected genotype frequencies. The genotype and allele risk associations were calculated by chisquare test using Prism 5 software (GraphPad Software Inc, USA; 2007). For genetic analysis, Bonferroni's correction was applied and statistical significance was considered at P-value less than 0.025. The linkage disequilibrium (LD) and haplotype analysis were carried out using http://analysis.bio-x.cn/myAnalysis.php (36). Levels of TSH and thyroid hormones were analyzed by non-parametric unpaired t-test and one-way ANOVA using Prism 5 software (GraphPad Software Inc.; 2007).

Web-based in-silico prediction tool HaploReg v4.1 (https://www.pubs.broadinstitute.org/mammals/haploreg/ haploreg.php) was employed to predict the effect of noncoding rs4704397 polymorphism. Tissue specific effect of rs4704397 was assessed by an eQTL database-GTeX portal (https://www.gtexportal.org).

It was ensured that the study design complies with the ethical standards of the Institutional Ethical Committee for Human Research (IECHR), Faculty of Science, The Maharaja Sayajirao University of Baroda,Vadodara, Gujarat, India (FS/IECHR/BC/PR/1) and with the 1964 Hel inki declaration.

Analysis of TSH, fT₃ and fT₄ levels in the studied subjects revealed that among 230 females with primary infertility, 58% (n=133) were euthyroid, 32% (n=74) were SCH, 6% (n=14) were overt hypothyroid and the rest 4% (n=9) females were hyperthyroidism (Fig .1 A, Table S3) (See Supplementary Online Information at www. celljournal.org). IF-SCH females had significantly higher (P<0.0001; Fig.1B) TSH levels (mean ± SEM: 5.34 ± 0.21 μIU/ml) compared to the control females (mean ± SEM: 1.91 ± 0.08 μIU/ml) and they had no significant difference in fT3 levels (P=0.1159, mean ± SEM: 3.036 ± 0.0462pg/ml; Fig.1C) compared to the controls (mean ± SEM: 2.935 ± 0.0436). There was no significant difference between fT4 levels (P=0.0741, mean ± SEM: 1.22 ± 0.0249) in IF-SCH females compared to controls (mean ± SEM: 1.195±0.0318 ng/dl).

Genotyping PDE8B rs4704397 polymorphism was carried out in 60 IF-SCH females and 76 healthy fertile females (Fig .2A). Other variables such as age (P=0.419), BMI (P=0.309), smokers (0%) and Hb (P=0.117) levels were not significantly different between the subjects of each genotypes ( Table S4). The observed genotype frequencies of PDE8B rs4704397 SNP in IF-SCH females were slightly deviated from HWE (P=0.049; Table 1), whereas the control population was under HWE (P=0.062; Table 1). Ancestral allele ‘A' and genotype ‘AA’ were considered as the reference allele and genotype respectively. The frequency of AG and GG genotypes were significantly lower in IF-SCH females, compared to controls (P=<0.0001 and P=0.006 respectively; Table 1). The frequency of ‘G’ allele was also significantly lower in IFSCH females, compared to the control females (23% vs. 47%, P<0.0001, OR=0.34). Hence, “G” allele was identified to have a protective effect and ‘A’ allele was identified as the risk allele for SCH and infertility in females.

Genotyping of PDE8B rs6885099 polymorphism was carried out in 60 IF-SCH and 76 control females (Fig .2B). The observed genotype frequencies of PDE8B rs6885099 polymorphism among the control and IF-SCH females were in accordance with HWE (P=0.248 and P=0.134 respectively; Table 2). Distribution of genotype as well as allele frequencies revealed no significant difference among the IF-SCH and control females (Table 2).

TSH levels in IF-SCH females were analyzed with respect to the genotypes of PDE8B rs4704397 A/G and rs6885099 G/A. No significant difference in TSH levels was observed with respect to genotypes of the both SNPs (Fig .3).

Analysis of functional consequences of PDE8B rs4704397 by HaploReg v4.1 predicted that PDE8B rs4704397 could alter heat shock factor-type (HSF) motif and enhancer state by H3K27 acetylation (H3K27ac) in inferior temporal lobe of brain (https:// www.pubs.broadinstitute.org/mammals/haploreg/detail_ v4.1.php?query=&id=rs4704397).eQTL database GTEx portal showed significantly elevated PDE8B transcripts in thyroid tissue of individuals carrying ‘A’ allele, compared to ‘G’ allele (https://www.gtexportal.org/home/snp/ rs4704397).