Background Subclinical hypothyroidism (SCH) remains largely unnoticed as a major cause of infertility due to asymptomatic. Polymorphisms of phosphodiesterase 8B gene (PDE8B) have been linked with various diseases, including female infertility. Hence, we aimed to study prevalence of SCH, in infertile females, explore associa- tion of PDE8B rs4704397 A/G and rs6885099 G/A polymorphisms with infertility in females suffering from SCH and genotype-phenotype correlation of the polymorphisms with thyroid stimulating hormone (TSH) levels in Gujarat population. Materials and Methods In this retrospective study, TSH level was estimated from plasma of 230 infertile and 100 control females by enzyme-linked fluorescence immunoassay (ELFA) to find out the prevalence of SCH. Further, based on TSH levels, thyroid function test (TFT) was performed in controls and infertile females with subclinical hypothyroidism (IF-SCH). PDE8B rs4704397 and rs6885099 polymorphisms were genotyped by PCR-RFLP and ARMS-PCR, respectively in 74 controls and 60 IF-SCH females. Results We observed i. significantly high prevalence of SCH (32%) in the infertile females, ii. significant- ly lower frequency of ‘G’ allele (P=0.006), while the frequency of ‘A’ allele (P < 0.0001) was higher in IF- SCH females, compared to the controls, for rs4704397 A/G SNP, iii. no significant difference in the genotype (P=0.214; OR=2.51; CI=0.74–8.42) and the allele frequency (P=0.129; OR=1.51; CI=0.92-2.47) of rs6885099 G/A SNP, iv) low linkage disequilibrium for the polymorphisms, v. significantly higher frequency of ‘AA’ hap- lotype (P=0.0001; OR=3.84; CI=1.86-8.01),while the ‘GG’ haplotype (P=0.0023; OR=0.33; CI=0.16-0.69) was significantly lower in IF-SCH females and vi. no significant difference in the TSH level of IF-SCH females with respect to the genotypes. Conclusion The present study reports an association of PDE8B rs4704397 polymorphism with infertility in SCH females. The study categorically shows a higher prevalence of SCH in infertile females of Gujarat and advocates the importance of screening for SCH in infertility management.
Apart from its multiple functions, thyroid hormones
play crucial role in reproduction. Hence, altered thyroid hormone levels can greatly affect reproductive
function (1). Thyroid diseases in women with reproductive age are very common due to the complex interplay of various hormones (2). Abnormal thyroid functions of hyper or hypothyroidisms are symptomatic and
they may have an adverse effect on the reproductive
health contributing to infertility (3-4). However, subclinical hypothyroidism (SCH) is silent and hence it
is often undiagnosed. It is a common thyroid disorder
often found to coexist with various other morbidities.
It is an asymptomatic condition where the patient has
a normal serum free T4 (fT4/thyroxin) levels, but high
thyroid stimulating hormone/thyrotropin (TSH) levels
(5). TSH is considered as a sensitive indicator of the
thyroid status and SCH. Normal TSH levels in serum
are finely regulated in humans. Nevertheless, serum
thyroid parameters show substantial inter- individual
variability (6), in which genetic variations are proved
as the major factors in several populations. It has been
shown that altered TSH levels are related to genetic
factors in up to 65% of the cases (7-9)
Different cohort studies reported phosphodiesterase 8B (PDE8B) as a genetic modulator of TSH
levels. PDE8B gene encodes a cyclic adenosine
monophosphate (cAMP) specifi c phosphodiesterase
(PDE) enzyme (10). PDE8B affects cAMP levels in
the thyroid gland resulting in changes in the levels of
thyroid hormones, which in turn affects the release of
TSH from the pituitary gland. PDE8B is mainly expressed in thyroid and brain (11, 12). Several single
nucleotide polymorphisms (SNPs) for PDE8B have
been demonstrated to associate with increased levels
of serum TSH. More than 360,000 SNPs were tested
for their associations with serum TSH levels with an
additive model. The obtained results revealed three
SNPs (i.e. rs4704397, rs6885099 and rs2046045)
with genome-wide significance (P<10-10). These
three SNPs were reported to be in strong linkage disequilibrium. Of the three SNPs, rs4704397 showed
strongest association and it could explain 2.3% of
the variations in TSH levels (13). PDE8B rs4704397
polymorphism has been found to associate with myocardial infarction,
height (14), pregnancy (15, 16),
recurrent miscarriage (17) and obesity in children
(18), apart from thyroid function. Another PDE8B
polymorphism, rs6885099 has also been shown to
increase TSH levels, but to a lesser extent, in different populations (13). The relevance of human reproduction to PDE has been well-documented (19-22).
While the underlying mechanism regulating oocyte
maturation is not clearly known yet, the second messenger cyclic adenosine monophosphate (cAMP)
role in oocyte maturation is well known (23) and
thus research investigating the role of rs4704397 in
the oocyte maturation might give an insight to primary infertility caused by hypothyroidism.
Numerous studies have reported the importance of
screening for SCH, and the worldwide prevalence of
SCH in infertile-females has been reported to be as high
as 26.7% in various populations (24-27). In India, prevalence of SCH is high and reported to be 25% (28-33).
However, there is no study on the status of SCH per se or
its prevalence amongst infertile females in western part
of India. Furthermore, there is no report on the role of
PDE8B polymorphisms in female infertility. We therefore, aimed to estimate the prevalence of SCH in infertile
females and explore association of PDE8B rs4704397
and rs6885099 polymorphisms in infertile females of Gujarat population.
Materials and Methods
Study subjects
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).
Genotyping PDE8B rs4704397 and rs6885099 polymorphisms
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.
Statistical analysis
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).
In-silico analysis
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).
Ethical consideration
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.
Results
Estimation of thyroid stimulating hormone, free T3
and free T4 levels
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).
PDE8B rs4704397 SNP in infertile females with subclinical hypothyroidism females
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.
PDE8B rs6885099 SNP in infertile females with
subclinical hypothyroidism
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).
Estimation of TSH and thyroid hormone levels. A. Prevalence of thyroid dysfunction among the infertile females. B. TSH level in controls and IF-SCH
females. C. fT3 levels in the controls and IF-SCH females. D. fT4 levels in controls and IF-SCH females. TSH; Thyroid stimulating hormone, IF-SCH; Infertile
females with subclinical hypothyroidism, fT3; Free T3, fT4; and Free T4.
Distribution of genotype and allele frequencies for PDE8B rs4704397 A/G polymorphism
Genotype or allele
IF-SCH females(Freq. %)
Control females(Freq. %)
P value
Odds Ratio
95% CI
P value HWE
Genotype
n= 60
n=76
AA
38 (63%)
17 (22%)
R
1
0.07-0.35
0.062 (C)
AG
16 (27%)
46 (61%)
<0.0001a
0.16
0.07-0.63
GG
06 (10%)
13 (17%)
0.006a
0.21
0.049 (P)
Allele
A
92 (77%)
80 (53%)
R
1
-
G
28 (23%)
72 (47%)
<0.0001b
0.34
0.19-0.57
n; number of IF-SCH females/control females, R; reference group, Freq.; Frequency, CI; Confidence interval, P; IF-SCH females, C; Control females, a IF-SCH female vs. control females
(genotype) using chi-squared test with 2×2 contingency table, and b IF-SCH females vs. control females (allele) using chi-squared test with 2×2 contingency table, and IF-SCH; Infertile
females with subclinical hypothyroidism.
Distribution of genotypes and alleles for PDE8B rs6885099 G/A polymorphism
Genotype or allele
IF-SCH females(Freq. %)
Control females(Freq. %)
P value
Odds Ratio
95% CI
P value HWE
Genotype
n= 60
n=76
GG
17 (28%)
32 (42%)
R
1
-
-
GA
35(58%)
38 (50%)
0.1914a
1.73
0.82-3.65
0.248 (C)
AA
08 (13%)
06 (8%)
0.2145a
2.51
0.74-8.42
Allele
0.134 (P)
A
69 (58%)
102 (67%)
R
1
-
G
51 (42%)
50 (33%)
0.1292b
1.51
0.92-2.47
F-SCH; Infertile females with subclinical hypothyroidism; n; number of IF-SCH females/Control females, R; reference group, Freq.; Frequency, CI; Confidence interval, P; IF-SCH females
and C; Control females, a IF-SCH female vs. control females (genotype) using chi-squared test with 2×2 contingency table, and b IF-SCH females vs. control females (allele) using
chisquared test with 2×2 contingency table.
Distribution of haplotype frequencies for PDE8B rs4704397 and rs6885099 polymorphisms
Haplotype[rs4704397(A/G):rs6885099 (G/A)]
IF-SCH FemaleFreq. (%)
Control femalesFreq. (%)
P value for association
P value (Global)
Odds Ratio [95% CI]
AG
48 (46%)
49 (21%)
0.4434
7.5 × 10-5
1.230 [0.72-2.09]
AA
31 (30%)
12 (10%)
0.0001
3.84 [1.86-8.01]
GG
12 (12%)
34 (28%)
0.0023
0.33 [0.160-0.69]
GA
13 (12%)
25 (21)
0.0876
0.53 [0.25-11.10]
Freq.; Frequency, CI; Confidence interval (Frequency <0.03 in both control and case has been dropped and it was ignored in the analysis), and IF-SCH; Infertile females with subclinical
hypothyroidism
Representative gel images for PDE8B rs4704397 and rs6885099
genotyping. A. PCR-RFLP analysis of PDE8B rs4704397 SNP on 3.5%
agarose gel. Lane 1 shows 100 bp ladder, lane 2 shows homozygous
(AA) genotype, lanes 3, 4 and 7 show heterozygous (AG) genotypes,
lanes 5, 6, 8 and 9 show heterozygous (GG) genotypes. B. ARMS-PCR
analysis of PDE8B rs6885099 SNP on 3.5% agarose gel. Lanes 1 and 2
show homozygous (GA); lane 4, 5, 6 and 7 show homozygous (GG) genotypes and lane 3 shows 100 bp ladder,
lanes 8 and 9 show heterozygous (GA) genotypes. PCR-RFLP; Polymerase chain reaction-restriction
fragment length polymorphism.
Linkage disequilibrium and haplotype analysis
Linkage disequilibrium (LD) analysis revealed that
two investigated PDE8B polymorphisms (i.e. rs4704397
and rs6885099) were in low LD association (D’=0.060,
r2=0.003). Haplotype analysis revealed that the frequency of ‘AA’ haplotype was significantly higher in the patients and risk of IF-SCH females was increased by 3.84
fold (P=0.0001, OR=3.84; CI=1.86-8.01; Table 3). The
frequency of ‘GG’ haplotype was significantly lower in
IF-SCH females, compared to the controls suggesting
its protective effect (P=0.0023, OR=0.33; CI=0.16-0.69;
Table 3).
Genotype-phenotype correlation analysis
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).
Correlation of PDE8B rs4704397 and rs6885099 with TSH levels in
IF-SCH females. No significant difference of TSH levels was observed with
respect to PDE8B polymorphisms A. rs4704397 and B. rs6885099. TSH;
Thyroid stimulating hormone, IF-SCH; Infertile females with subclinical
hypothyroidism
In-silico analysis
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).
Discussion
The present study shows a high prevalence rate of
SCH in infertile females (32%) in comparison with
the healthy controls (Table S1) and the association of
rs4704397 SNP with infertility in IF-SCH females of
Gujarat region. In developing countries, one among
four couples suffers from infertility and in these couples, hypothyroidism is one of the key perpetrators.
In a study performed by Verma et al. (28), out of 394
infertile women, 23.9% were hypothyroid (TSH>4.2
μIU/ml). An intervention to rectify the hypothyroidism resulted in 76.6% of the conceived infertile women. Primary health caregivers most often pick up overt
hypothyroidism easily; however, SCH with its subtle
symptoms most often goes unnoticed. The prevalence
of SCH amongst infertile females is common, but there
is a scarcity on available data. However, there are a
few studies reporting the prevalence of hypothyroidism, ranging from 15-25% in Indian population (28-
33). As SCH is largely asymptomatic, it goes undiagnosed, resulting in infertility. It is essential to include
evaluation of thyroid related hormones as a standard
practice along with other tests to ascertain the causes
of infertility.
SCH occurs due to multiple factors. Some of them
include congenital agenesis, defect in synthesis due to
iodine deficiency or anti-thyroid drugs, autoimmune
diseases, post-surgery, hypopituitarism, TSH deficiency, environmental pollutants, mutations and SNPs
(37). Of these factors, the present study focuses on the
SNPs. To evaluate possible correlation between the
polymorphisms associated with increased TSH levels
and infertility, two SNPs (rs4704397 and rs6885099)
of the PDE8B were studied in healthy controls and
IF-SCH females. Higher frequency of the “A” allele
for PDE8B rs4704397 polymorphism in SCH related
infertile patients which revealed “A” as a risk allele
for infertility in IF-SCH females. However, PDE8B
rs6885099 was not associated with infertility. Earlier, PDE8B rs4704397 was also found to associate
with recurrent miscarriage (17). PDE8B is found in
the thyroid but not pituitary. In addition, given the
importance of cAMP activity in TSH signaling, it is
suggested that the PDE8B rs4704397 polymorphism
could reduce cAMP levels in the thyroid resulting in a
decreased response of thyroid gland to TSH stimulation, which leads to an increase of TSH set point for
the same free T3 and T4 levels (18). Polymorphism in
PDE8B, rs4704397 results in an increase in PDE8B
enzyme expression. We propose that this could result
in a faster degradation of cAMP, which decreases the
synthesis and release of T3 and T4. In such a scenario,
the negative inhibition of Thyrotropin-releasing hormone (TRH) will not take place and this will result
in increased levels of TRH and hence TSH. As a consequence, T3 and T4 levels become normal. The increased level of TSH results in development of SCH.
PDE8B rs4704397 polymorphism might induce phosphodiesterase activity in PDE8B, thereby reducing
the ability of thyroid gland to generate free T4 when
stimulated by TSH. This results in SCH, which can be
the cause of infertility in IF-SCH patients. Arnaud et
al. in a GWAS study reported that PDE8B rs4704397
could affect plasma TSH levels. Each copy of the minor allele “A” may lead to a mean increase of 0.13
mU/l TSH levels (13). However, we did not observe
significant correlation of the PDE8B rs4704397 SNP
with circulating TSH levels. This might be due to
the limited sample size in the present study. PDE8B
rs4704397 SNP was also found to be associated with
various conditions like cardiovascular, body height,
pregnancy, recurrent miscarriage, obesity in children,
etc. (14-18). Though the exact underlying mechanism
of PDE8B rs4704397 SNP affecting TSH levels is not
clear, in-silico tools predicted that this variation might
lead to enhancement of PDE8B expression by influencing epigenetic level. The role of PDE8B in human
placenta and ovaries is still to be understood, while
human reproduction relevance to PDE has been proposed (19-22). The underlying mechanism of regulating oocyte maturation is not clearly documented yet,
but the second messenger cAMP role in oocyte maturation is well known (23). Thus, investigating the role
of rs4704397 in the oocyte maturation could be an interesting area of research as far as female infertility is
concerned.
On the other hand, medications given to alter the
levels of reproductive hormones have serious repercussions on the health of females with long-term
implications (38). Treatment of infertility is usually
done by direct targeting the reproductive system, instead of looking for the involvement of other factors,
such as genetic polymorphisms, as a cause of infertility. This genetic approach could be used to identify IF-SCH patients and treat infertility with greater
success and fewer side-effects without disturbing the
reproductive system. Since, small sample size was
a limiting factor for the present study, we suggest
investigating larger number of infertile females in
different populations. This might provide a significant insight into understanding the role of PDE8B in
infertility.
Conclusion
The present study establishes an association of PDE8B
rs4704397 with infertility in IF-SCH females and reiterates the importance of screening SCH, as a diagnostic tool
in infertility management.
Mansuri, T., Jadeja, S. D., Singh, M., Begum, R., & Robin, P. (2020). Phosphodiesterase 8B Polymorphism rs4704397 Is Associated with Infertility in Subclinical Hypothyroid Females: A Case-Control Study. International Journal of Fertility and Sterility, 14(2), 122-129. doi: 10.22074/ijfs.2020.6015
MLA
Tabassum Mansuri; Shahnawaz D. Jadeja; Mala Singh; Rasheedunnisa Begum; Pushpa Robin. "Phosphodiesterase 8B Polymorphism rs4704397 Is Associated with Infertility in Subclinical Hypothyroid Females: A Case-Control Study". International Journal of Fertility and Sterility, 14, 2, 2020, 122-129. doi: 10.22074/ijfs.2020.6015
HARVARD
Mansuri, T., Jadeja, S. D., Singh, M., Begum, R., Robin, P. (2020). 'Phosphodiesterase 8B Polymorphism rs4704397 Is Associated with Infertility in Subclinical Hypothyroid Females: A Case-Control Study', International Journal of Fertility and Sterility, 14(2), pp. 122-129. doi: 10.22074/ijfs.2020.6015
VANCOUVER
Mansuri, T., Jadeja, S. D., Singh, M., Begum, R., Robin, P. Phosphodiesterase 8B Polymorphism rs4704397 Is Associated with Infertility in Subclinical Hypothyroid Females: A Case-Control Study. International Journal of Fertility and Sterility, 2020; 14(2): 122-129. doi: 10.22074/ijfs.2020.6015