Objectives: The study aimed to explore the association of endometriosis risk factors with single nucleotide polymorphisms rs6166 and rs6165 (Asn680Ser and Ala307Thr) of follicle stimulating hormone receptor (FSHR) gene in Pakistani women.
Methods: This study was conducted from 2013 to 2016. The sampling and extraction of DNA was done in Department of Zoology GC University, Lahore, while the sequencing was performed at Yale University, USA. This case control study consisted of 364 subjects including 156 women diagnosed with endometriosis and 208 conveniently recruited controls. Subjects diagnosed at stage II-IV endometriosis with infertility were pooled for study. The women with adenomyosis, ovarian cancer and leiomyoma were excluded. The whole blood leukocytes were used for DNA extraction. Two important polymorphisms of exon 10 of FSHR gene were analyzed by direct DNA sequencing both in endometriosis and controls.
Results: Genetic variant SNP rs6166 in the affected endometriosis subjects exhibited high incidence of allele "A" (Asn/Asn) 68.3% as compared to controls 33.7% (OR= 4.240; P =0.001). Similarly, the allele "A" of SNP rs6165 (Thr/Thr) was more frequent in endometriosis 67.3% than in control subjects 37.5% (OR =3.430, P =0.001). The occurrence of haplotype AA (Asn/Thr) was 45.5% in endometriosis and 11 % in control subjects (P= 0.001). Remarkably, the incidence of haplotype GG (Ser/Ala) was contrary to previous observations, since only 9.9% occurred in endometriosis as opposed to 45.2% in controls (P= 0.001).
Conclusion: Investigation of FSHR gene polymorphisms rs6165and rs6166 (Ala307Thr and Asn680Ser) in the current study showed that haplotype AA (680Asn/307Thr) was associated with endometriosis in Pakistani women.
Keywords: Endometriosis, Receptors, FSH, Polymorphism, Single Nucleotide, Haplotypes, Linkage Disequilibrium. (JPMA 71: 1118; 2021)
Infertility is among the prevailing health issues plaguing young women worldwide. Endometriosis is one of the important factors to be considered in female back pain, pelvic pain and infertility. The global burden of endometriosis is almost 7-10% which affects women of all reproductive ages,1,2 is increasing day by day,3 resulting in high healthcare expenditures and loss of reproductivity.4
Endometriosis is a "sex hormone dependent" disease that reverts menopause and subsequently affects fertility of women.5 In this disorder, endometrial tissue grafts also develop out side the uterus and consequently affect ovarian function, fallopian tubes and uterus.6 Despite enormous research on endometriosis, consistent mechanism of its development is not fully understood. Similarly, pathogenesis leading to infertility also varies in different studies, although approximately 20-50% women with endometriosis showed variations in their fertility status.7 Many hypotheses were proposed to elucidate the exact cause of disease development; these include pelvic factors, alteration in peritoneal function, adhesions in pelvic anatomy, ovarian factors that alter oocytes and uterine factors.8 In previous decade, several studies involving various ethnicities evaluated association of SNPs of various genes with endometriosis.9,10 The investigations involving various genes and SNPs have reported different results.
The follicle stimulating hormone (FSH) plays a significant role in the development and maturation of follicles, growth of granulosa cells, steroid synthesis regulation and synthesis of aromatase enzyme that induces androgen conversion.11,12 FSH hormone acts by binding with a trans-membrane glycoprotein receptor encoded by FSHR gene at 2p16.3.13 However, the role of FSHR gene in signal transduction pathway is still not clear.14 The amino acids of this region are found in extracellular domain of FSH binding protein. This important region is responsible for signal transduction mechanism which affect hormone binding capacity of the gene.15,16 Many genetic variants (SNPs) are present in FSHR gene that have shown to affect the pathway of signal transduction.17
Very little work is available on the FSHR gene polymorphism in relation to endometriosis. However, in FSHR gene, more work is available on polymorphism rs6166 the rs6165 (Asn680Ser and Ala307Thr).18 The homozygous polymorphism GG (Ser/Ser) in SNP rs6166 of FSHR gene; boosts aromatase action at 680, which later induces estrogens synthesis motivating the propagation of endometriosis.19 Furthermore, in Taiwanese population endometriosis was studied with non-synonymous homozygous GG (Ser/Ser) SNP as well as heterozygous GA (Ser/Asn) at 680 of FSHR gene. To date, only four studies with conflicting results have described association of FSHR gene polymorphism with endometriosis.19-23 In observation of significant role of FSHR gene in endometriosis studies, we examined genomic variant associated with risk of endometriosis at FSHR gene in infertile Pakistani women.
Patients and Methods
This study was permitted by "Institutional Bioethics Committee" of GC University Lahore, Pakistan. This was a case control study. The sampling technique was convenient.
This study consisted of 156 infertile women (clinically and laproscopically) diagnosed with endometriosis and 208 fertile women without any history of endometriosis. Endometriosis patients were screened from the outpatient's clinics of tertiary care government hospitals (Lady Willington hospital and Sir Ganga Ram hospital Lahore, Pakistan). All women (both with and without endometriosis) had normal levels of prolactin, thyroid and follicle stimulating hormone as well as both functional ovaries with normal morphology and ovulatory cycle of 25-35 days. The average age of women with endometriosis was 30.25 ± 5.46 years, while Body Mass Index (BMI) was < 25. All women of control group were fertile with average age of 30.59 ± 4.95 years and matched BMI of < 25. The detailed inclusion and exclusion criteria were according to published lliterature.24 Sample size calculated by formula (n = z2a/2 p (1- p)/e2) was 156.25
The protocol for DNA extraction, DNA sequencing, genotyping panel, primer design and PCR (Polymerase Chain Reaction Analysis) conditions were followed as described previously by Liaqat at el.24,26 PCR was performed in a reaction volume of 25 µl. PCR product was confirmed with 2% agarose gel electrophoresis. The PCR product was cleaned using Qiaquick PCR kit (Qiagen, Hilden Germany) and sent for direct sequencing with forward primer.
Hardy-Weinberg equilibrium was tested in both endometriosis and controls by chi-square test. The confidence interval and odd ratios were calculated by Sigma Plot 14.0. Data was tested for normality and Student t-test was performed to compare means of anthropometric parameters. The calculation of haplotype frequency, standard errors and linkage disequilibrium was performed by HAPLO.27 The significance level for statistical analysis was set at p<0.05.
The genotype, alleles and haplotype incidences of both polymorphisms i.e. rs6166 and rs6165 were in accordance to "Hardy-Weinberg equilibrium" in all subjects. The genotype and allele occurrence with odd ratios, confidence interval and level of significance are presented in Table-1. In rs6166 SNP (A>G), the genotype frequency of AA (Asn/Asn), GG (Ser/Ser) homozygous and AG (Ser/Asn) heterozygous were 46.2%, 9.6 % and 44.2% respectively in subjects with endometriosis and 11.5%, 44.2% and 44.2% respectively in control subjects. Whereas, in rs6166 SNP, a higher frequency of "allele A" (OR= 4.240, 95% CI. 2.37- 7.57, P =0.001) was observed in endometriosis subjects (68.3%) in contrast to control subjects (33.7%). In rs6165 SNP (A>G), frequencies of Asn/Asn, Ser/Asn and Ser/Ser were 46.2%, 42.3% and 11.5% in endometriosis cases, whereas 13.5%, 48.1% and 38.5% in controls, as illustrated in Table-1.
Both the haplotypes AA (Asn/Thr; 45.5% in endometriosis vs 16.3% in control) and GG (Ser/Ala; 9.9% in endometriosis vs 45.2% in control) exhibited distinct changes in both studied groups (Table-2).
The pairwise linkage disequilibrium was 0.76 for observed sites in endometriosis as is presented in Figure.
This LD value proposes that the two SNPs were in adjacent linkage disequilibrium.
The present study investigated polymorphism of FSHR gene as a risk of endometriosis development in selected Pakistani women. The genotype frequency of AA (Asn/Asn) of rs6166 SNP was observed more frequently in endometriosis (46.2%) as compared to control subjects (11.5%) (P =0.001). In addition, the combined allele haplotypes of Asn 680 and Thr 307 (AA) were more frequent in endometriosis as compared to controls. Similarly, haplotype GG was frequently found in controls as compared to endometriosis. The results of previous findings were conflicting from strong association to no link between FSHR polymorphism and endometriosis (Table-3).
The findings of Wang et al.19,20 showed no significant association between FSHR polymorphisms and endometriosis in Taiwanese population. His studies revealed that genotype AA (307Ala/Ala) and AG (307Ala/Thr) polymorphism, as well as, both GG genotype (680Ser/Ser) and GA genotype (680Ser/Asn) were linked with a significantly lesser risk of endometriosis. Similarly, FSHR polymorphism was studied in Turkish women with endometriosis and controls. The genotype frequencies showed no significant difference in Turkish women;22 results are contrary to the present study where strong association was observed at both (Ala307Thr and Asn680Ser) polymorphisms. A recent study on Brazilian infertile endometriosis women exhibited no relationship between FSHR gene polymorphism and onset of disease.21 Finally, a more recent (2018) study on Brazilian women exxibited no significant difference for both (Ala307Thr and Asn680Ser) polymorphisms between endometriosis and control group. However, when women were divided according to fertility status and stage of severity, a positive association was observed in fertile endometriosis women and FSHR gene polymorphism.23 This result seems similar with the current study where significant (P=0.001) association was observed between endometriosis and FSHR gene variants.
In the light of above discussion it is concluded that the Pakistani population showed diversity in their genetic makeup on the basis of significant association of FSHR gene with endometriosis.
Disclaimer: Article is part of PHD thesis research work.
Conflict of Interest: None
Funding Disclosure: None
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