The present case-control study evaluated the association of a polymorphic variant rs2293275 of LHCGR p.S312N with the PCOS risk and its correlation with the clinical and biochemical indices. We found a significant association of rs2293275 with the PCOS risk and linearly increased LH levels in the subjects harboring heterozygous (GA and the mutant (AA) genotype when compared to the wild-type (GG) genotype carriers.
Our results demonstrated a significant difference in genotypic as well as allelic frequencies of rs2293275 LHCGR gene between PCOS women and controls, indicating that women with GA and AA genotypes are at higher risk for developing PCOS. The higher frequency of the A allele found in PCOS cases revealed a > 2-fold increased risk of PCOS in our study. These findings are in agreement with the earlier studies (Capalbo, Sagnella et al. 2012, Bassiouny, Rabie et al. 2014, Ha, Shi et al. 2015, El-Shal, Zidan et al. 2016),reporting a positive association between various ethnicities. However, no significant association of this variant with the risk of PCOS was reported in Caucasian and Bahraini populations respectively(Valkenburg, Uitterlinden et al. 2009, Almawi, Hubail et al. 2015).On the contrary, Thathapudi et al. revealed that the GG(major allele) genotype, rather than AA, conferred a significant risk of developing PCOS in South Indian women(3.36-fold)(Thathapudi, Kodati et al. 2015), while a recent meta-analysis reported a 4.1 risk increase of developing PCOS for carriers of the AA (minor allele) genotype of LHCGR(Zou, Wu et al. 2019). These conflicting results among the studies might be explained by differences in sample size, non-uniform diagnostic criteria, ethnic background, and study design.
LH is an associated member of the glycoprotein family that stimulates follicular development, steroid biogenesis, and the formation of the corpus luteum(Dufau 1998),and ovulation(Ascoli, Fanelli et al. 2002)acts by binding with its high-affinity receptor, LHCGR)(Dufau 1998)and transducing luteinizing hormone-mediated signals that play a vital role in the ovulation process(Qiao and Han 2019).LHCGR gene is one of the few candidate genes recognized susceptibility loci consistently associated with the risk of PCOS in diverse ethnicities.Abnormal LH signaling is believed to play a crucial role in augmenting ovarian androgen production in PCOS and leading to anovulation(Balen 1993, Norman, Dewailly et al. 2007). Evidence from the study conducted by Zhihuaet al showed that mutation in LHCGR causes abnormal LHCGR glycosylation, decreased LHCGR protein level, effects on subcellular localization, and reduced cellular ATP consumption, which indicate the signal transduction may be affected and leads to the cause of abnormal ovulation(Zhang, Wu et al. 2020).Reports have shown that enhanced expression or overactivation of LHCGR might contribute to the development of PCOS(Kanamarlapudi, Gordon et al. 2016). In addition to the two-cell, two-gonadotrophin theory, LH modulates multiple genes' mRNA levels in granulose cells through the LHCGR receptor, which can aid in the growth of follicles(Sasson, Rimon et al. 2004, Lindeberg, Carlström et al. 2007).The secretion of androgen hormones by ovarian theca cells promotes by LH, which may result in follicular maturation arrest.Consequently, the variation that occurred at the LH level may potentially influence the reproductive process that it leads to and is associated with menstruation dysfunction and infertility,thereby orchestrating the risk of PCOS(Laven, Imani et al. 2002).The genetic variants of LHCGR p.S312N, which falls within exon 10 of the LHCGR gene, and is next to the glycosylation signals of the protein, might affect the trafficking and stability of the receptor, resulting in an increased risk of developing polycystic ovary syndrome (PCOS) in women (Thathapudi, Kodati et al. 2015). An earlier study reported that mutant homozygous or heterozygous inactivating gene variants of the LHCGR cause gonadal resistance to LH thereby increasing the LH level and subsequent feedback to the pituitary resulting in the further elevation of LH levels leads to the anovulation(Segaloff 2009).Moreover, a recent study showed a strong association of LHCGR rs2293275 polymorphism with high LH levels and LH/FSH ratio in PCOS women contributes to enhancing the risk of PCOS development. The study suggested that high serum LH levels in PCOS subjects are important for PCOS diagnosis and may be useful as a molecular marker for early detection of high risk for PCOS(Atoum, Alajlouni et al. 2022).Given the important pivotal role of LH in androgen metabolism and ovulation, can be a plausible explanation for the enhanced PCOS risk in the women that harbored the variant genotype of LHCGR in our study.However,further mechanistic studies are warranted to elucidate LHCGR(rs2293275) mediated PCOS etiology. On stratification analysis, similar to earlier reports, we found a significantly increased serum LH level in subjects with PCOS who harbored variant genotypes when compared to healthy controls(Piersma, Berns et al. 2006, El-Shal, Zidan et al. 2016).
Maternal family history is considered a risk factor for PCOS in daughters. PCOS is thought to be a heritable disorder based on familial case clustering(Rosenfield and Ehrmann 2016). The significant frequency of PCOS or its clinical manifestations, such as hyperandrogenism, hirsutism, infertility, and polycystic ovaries, among first-degree relatives suggests that genetic and familial factors play a role in the disorder(Bruni, Capozzi et al. 2021).We found an enhanced risk in the subjects who had a family history of T2DM or hirsutism and harbored the variant genotype of rs2293275 suggesting the heritability associated with the later onset in PCOS women.Although a direct correlation of LHCGR genotypes with a positive family history of T2DM has not been evaluated as before, a positive family history of T2DM has been previously associated with the development of PCOS(Kulshreshtha, Singh et al. 2013, Yilmaz, Vellanki et al. 2018).Vrbikovaet al. reported that defective early beta cell function was characteristic of only patients with PCOS and a positive family history of T2DM(Vrbikova, Bendlova et al. 2009),also reported a significant difference in glucose and lipid metabolism between PCOS patients with and without a family history of T2DM(Wang, Gao et al. 2021). A literature survey suggests that T2DM appears to be an important factor in predicting the risks of metabolic abnormalities in women with PCOS(Ehrmann, Kasza et al. 2005, Vrbíková, Grimmichová et al. 2008, Lerchbaum, Schwetz et al. 2014). However, further replicative, and mechanistic studies are required to validate and unveil the underlying role.
In the present study, we found an enhanced risk of PCOS in subjects with PCOS who had alopecia, acne, or Acanthosis nigricans compared to healthy controls and harbored variant genotypes of LHCGR (rs2293275).Hyperandrogenism is a major characteristic in women with PCOS, the hallmark feature of PCOS 58–82% of hyperandrogenic women have PCOS(Pinola, Puukka et al. 2017).Elevated LH levels or increased testosterone production from polycystic ovaries may cause hyperandrogenaemia(Ashraf, Nabi et al. 2019).Elevated insulin levels may also trigger increased testosterone levels in women and thus modulate the risk of PCOS(Nestler, Jakubowicz et al. 1998).Consequently, the resulting Androgen excess(hyperandrogenism) acts as the main promoting factor inducing anovulation and follicular arrest, suggesting decreased oocyte development and maturation(Qiao and Feng 2011).
Obesity is a common finding in PCOS that worsens its phenotype and is considered one of the most crucial pathophysiological features in PCOS. It also aggravates menstrual irregularity and increases serum total testosterone levels(Xita and Tsatsoulis 2006, Baldani, Skrgatić et al. 2013).This excessive amount of androgen in turn can affect the follicle growth and metabolic process and also trigger insulin levels, which further enhances the risk of PCOS development in obese women.Furthermore, a recent study that showed increased testosterone level promotes visceral fat accumulation and insulin resistance by inhibiting lipolysis and promoting lipogenesis(Rosenfield and Ehrmann 2016), which in turnis associatedwith suppressed ovulation and high LH levels(Roth, Allshouse et al. 2014).In this study,We found a synergistic effect on the PCOS risk in the subjects,carrying the LHCGR variant genotype(GA + AA) having BMI greater than ≥ 24 in subjects with PCOS women, albeit with wider CI’s due to low numbers in the model. Our findings are consistent with previous studies that found BMI to be statistically significant and highlight the contribution of LHCGR polymorphism to PCOS phenotypes, particularly BMI(Thathapudi, Kodati et al. 2015, Atoum, Alajlouni et al. 2022)While as the study was statistically powered to detect any associations, however, the low number in the subsequent stratification analysis might be a concern of the present study.