International Journal of
Reproductive Biomedicine
Thu, Dec 5, 2024
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Volume 22, Issue 7 (July 2024)
IJRM 2024, 22(7): 539-552 |
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Ethics code: WH2018-2019_1
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Amin J, Alle N S, Prajapathi B, patel A, Makwana P, Gomedhikam J P et al . Significance of FSHR and LHCGR gene polymorphisms on clinical outcomes in gonadotropin-releasing hormone antagonist protocol with freeze-all strategy: A case-control study. IJRM 2024; 22 (7) :539-552
URL: http://ijrm.ir/article-1-3288-en.html
URL: http://ijrm.ir/article-1-3288-en.html
Jayesh Amin * 
1, Naga Sandhya Alle2 , Bansi Prajapathi2 , Ami Patel2 , Pareshkumar Makwana2 , Jaya Prakash Gomedhikam3 , Murali krishna Kota3
1, Naga Sandhya Alle2 , Bansi Prajapathi2 , Ami Patel2 , Pareshkumar Makwana2 , Jaya Prakash Gomedhikam3 , Murali krishna Kota3
1- Wings IVF Women’s Hospital, Ahmedabad, Gujarat, India. , drjayeshamin8@gmail.com
2- Wings IVF Women’s Hospital, Ahmedabad, Gujarat, India.
3- Life Fertility and Research Center, Collector Office Jn, Maharanipeta, Visakhapatnam, Andhra Pradesh, India.
2- Wings IVF Women’s Hospital, Ahmedabad, Gujarat, India.
3- Life Fertility and Research Center, Collector Office Jn, Maharanipeta, Visakhapatnam, Andhra Pradesh, India.
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1. Introduction
Infertility/subfertility is a significant concern worldwide for couples planning to have a child. It ranks the fifth highest global problem among all other health conditions (1). The prevalence of infertility is on the rise due to various factors, including the prolonged age of a couple to plan for a pregnancy, heightened stress levels, increased exposure to various environmental pollutants, and various health conditions resulting from unhealthy lifestyles (2). Fortunately, the assisted reproductive technique is critical for an infertile couple planning for parenthood. The success of these techniques depends on the specific protocol followed, whether it be antagonist or agonist, as well as optimizing the dosage of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) used for stimulation (3, 4). In recent years, there have been groundbreaking studies suggesting a pharmacogenomic approach to trailer stimulation protocols based on the genetic makeup of infertile women. This personalized approach aims to enhance the success rate of assisted reproductive technology (ART) (5-10).
The gonadotropin-releasing hormone plays a crucial role in regulating 2 essential hormones: FSH and LH (11). These 2 hormones apply themselves on their respective granulosa and theca cells through their receptors, namely follicle-stimulating hormone receptor (FSHR) and luteinizing hormone/choriogonadotropin receptor (LHCGR) (12) and help in the proliferation and maturation of follicles (13). The FSHR gene is present on chromosome 2, containing 10 exons; it has a common polymorphism on exon-10 known as N680S (rs6166 G>A), located in the intracellular domain. It encodes asparagine and serine amino acids, with the AAT codon coding for asparagine and the AGT codon coding for serine (14). Previous reports indicate that in regular in-vitro fertilization (IVF) cycles, individuals with FSHR680 homozygous SS exhibit higher sensitivity to FSH, elevated basal FSH, reduced estradiol production, and increased clinical pregnancy rates compared to individuals with other FSHR680 genotypes (14, 15).
The LHCGR gene contains 11 exons on chromosome 2 at cytogenic band 2p16.3 near the FSHR gene (7). Among the various polymorphisms identified in the LHCGR gene, the N312S polymorphism (rs2293275) found in exon 10 was widely studied (5, 7). N312S polymorphism substitutes asparagine with serine amino acid; the AAT codon codes the asparagine, and the AGT codon codes the serine.
Various research groups have investigated the impact of the LHCGRN312S polymorphic variant on controlled ovarian stimulation protocols (5, 7, 14). Previous studies have consistently shown that individuals homozygous to the serine allele have a higher chance of getting pregnant in regular IVF cycles (7, 14). Moreover, previous findings by Ga et al., showed that the homozygous serine group required higher doses of recombinant human luteinizing hormone compared to other genotypes in agonist protocol combined with a regular embryo transfer cycle (7).
To date, no research study has investigated the comparative clinical outcomes of recombinant follicle-stimulating hormone (rFSH) and human menopausal gonadotropin (hMG), and their respective combinations with gonadotropin-releasing hormone antagonistic protocol based on FSHR and LHCGR polymorphism, specifically in freeze-all embryo transfer cycles. In the current study, the objective was to assess the impact of FSHR680 and LHCGR312 polymorphisms on the clinical pregnancy rate and live birth rate in freeze-all cycles where the influence of hormonal stimulation on endometrial receptivity is absent due to superovulation by gonadotropins (FSH, LH).
2. Materials and Methods
2.1. Study design
This observational study was conducted at the Wings IVF Center, Ahmedabad, India, from March 2019 to April 2022. During this period, data collection and participant recruitment were done, utilizing the MediTEX IVF software, Regensburg, Germany for data retrieval.
A total of 421 women initially participated in the study. Inclusion criteria: age between 25 and 40 yr, a body mass index (BMI) of less than 30 kg/m2, a regular menstrual cycle lasting between 21 and 35 days, presence of bilateral ovaries, non-smoking status, and a quantifiable antral follicle count (AFC).
Exclusion criteria: Participants were excluded if they were recipients of oocyte donation, had a history of pelvic inflammatory disease, possessed only one ovary, had severe endometriosis, or showed serum anti-Müllerian hormone (AMH) levels below 1 ng/mL. Additionally, exclusion criteria extended to men with significant male factor infertility (defined as oligospermia with a sperm count below 2 million/ml or azoospermia), and couples who did not complete the follow-up.
Following the application of these criteria, the study was finalized with 306 women who met all the requirements necessary for inclusion in the research analysis.
2.2. Sample size estimation
The sample size was determined based on a power analysis. The criteria used were effect size of f = 0.25, a significance level of 0.05, and a desired power of 0.80 (indicating an 80% chance of detecting a true effect). This analysis indicates the necessity of a total 84 participants.
In our study, the effect size for the FSHR680 polymorphism was found to be 0.36. Given this effect size, 3 groups in the variable, a minimum sample size of 71 participants per group, and a significance level of 0.05, the power for detecting differences in total rFSH dose using one-way ANOVA was calculated to be 0.99. For LHCGR312 and total gonadotropins, the power was calculated to be 0.86 with an effect size of 0.34 and a minimum sample size of 33 participants per group.
2.3. Treatment protocol
Before initiating ovarian stimulation, all participants in the study were genotyped for FSH receptor (FSHR680) and LH/CG receptor (LHCGR312) polymorphisms. Stimulation commenced on either day 2 or 3 of the menstrual cycle using either rFSH alone or in combination with hMG. Dosages were initially set based on each woman's age, BMI, and AFC, with subsequent adjustments made between 150 IU and 450 IU of rFSH depending on individual responses assessed by transvaginal ultrasound scans of follicular size and serum estradiol levels, maintained below 450 pg/ml. While rFSH doses were not altered due to FSHR polymorphism results, women homozygous for LHCGR312S received an additional 75 IU of hMG from the first day of treatment. The protocol was set down by administering 10,000 IU of human chorionic gonadotropin as a trigger between days 10 and 12, based on estradiol levels and follicle size, followed by oocyte retrieval 36 hr later under transvaginal ultrasonography guidance.
2.4. Genotyping of FSHR and LHCGR
In brief, the genomic DNA from peripheral leukocytes was retrieved to determine the single nucleotide polymorphisms at amino acid position 680 (Asp680Ser) in the FSHR gene and amino acid position 312 (N312S) in the LHCGR gene. The LHCGR and FSHR genes were amplified using high-fidelity PCR in a total volume of 25 µL containing 1 µL of the forward primer (5′-ACGCACAGTCAGGTTTAGCC-3′ for LHCGR and 5′-CTCCTGTGCCAACCCCTTC-3′ for FSHR gene) and 1 µL of the reverse primer (5′-AACAGCTCCGTAACCAAG-3′ for LHCGR and 5′-TTAGATGAAATGTGTAGAAGCACTG-3′ for FSHR gene) along with 15 µL PCR master mix (Takara, USA) and 100 ng template DNA. The final volume was adjusted by using dH2O. DNA was denatured for 10 min at 95oC before the start of the amplification program. This was followed by 37 cycles of amplification, each of which included denaturation at 95oC for 1 min, annealing at 60oC for 30 sec, and elongation at 72oC for 3 min. The final elongation was carried out for 7 min at 72oC. The PCR product was purified and checked using 2% agarose gel before being directly sequenced on an 8-capillary applied biosystems sequencing apparatus (Applied Biosystems, USA). An advanced Big Dye Terminator 3.1V was utilized for sequencing. The collected data were then compared to the reference gene in the NCBI database to analyze the polymorphism.
2.5. Ethical considerations
Participants or their immediate relatives (in case of uneducated women who have difficulty in understanding scientific language) are provided informed consent to participate in the study. The study was approved by the Institutional Ethics Committee of Wings IVF Center, Ahmedabad, India (Code: 2019/003/31C/NSA). Ethical considerations were followed as per the Indian Council of Medical Research guidelines.
2.6. Statistical analysis
The statistical analysis of the data was performed using R Studio version 3.6.4. Mean and standard deviation values were calculated for numerical variables like age, BMI, AFC, marital life, daily rFSH dose, total rFSH, total hMG, stimulation days, and embryology parameters (no. of oocytes, methaphase [M] 2), M1, germinal vesicles (GV), and atretic oocytes, no. of blastocysts) among genotypes and between genotypes of 2 polymorphisms. The p-values were calculated using one-way ANOVA and two-way ANOVA. For categorical variables like type of infertility, clinical pregnancy rate, and live birth rate, percentages were calculated, and Chi-square was used to calculate the p-value. P-value < 0.05 was considered statistically significant.
3. Results
3.1. Genotypic distribution
The study includes 306 participants who underwent IVF treatment and received COS protocol based on functional, hormonal, and genetic biomarkers. The AA (NN) genotype distribution was 28.76% (n = 88), GA (NS) heterozygous was 48.04% (n = 147), and GG (SS) homozygous was 23.20% (n = 71) with respect to FSHRN680S polymorphism. For LHCGRN312S polymorphism, the AA (NN) genotype distribution was 10.78% (n = 33), AG (NS) heterozygous was 48.37% (n = 148), and GG (SS) homozygous was 40.85% (n = 125) (Table I). For FSHR680 polymorphism, the allele frequency of the G allele in the South Asian population was 48%, and that of the A allele was 52% (rs6166 RefSNP Report - dbSNP - NCBI [nih.gov], and in the current study, the frequency of the G allele was 46%, and that of the A allele was 54%. The allele frequency of the A allele in the South Asian population was 39%. The G allele was 61% for LHCGR polymorphism from the worldwide database (rs2293275 RefSNP Report - dbSNP - NCBI [nih.gov], and the data from the present study have shown the frequency of the A allele was 21%, and the G allele was 79% for LHCGR312 polymorphism (Table I).
3.2. FSHRN680S
The mean age, AFC, BMI, and marital life (yr) of study participants with FSHR polymorphism were similar, with no significant difference, among the 3 genotypes (NN, NS, and SS). No association was found for the type of infertility. Women with homozygous NN and heterozygous NS received a similar dose of mean daily rFSH compared to the homozygous SS group. A significant difference was observed in rFSH dose (I.U.) received per day (227.30 ± 19.2, 225.00 ± 14, and 284.50 ± 31.70 respectively, p ≤ 0.0001) and total mean rFSH dose (2786.44 ± 456.92, 2724.84 ± 410.40 and 3565.38 ± 556.91 respectively) with p ≤ 0.0001 among FSHRN680S genotypes (NN, NS, and SS).
The mean dose of rhMG and the number of days antagonists were administered were similar across all genotypes. Although not statistically significant, the SS group required slightly more days for ovarian stimulation (12.41 ± 1.60) and had higher mean numbers of oocytes (15.12 ± 9.70) and M2 oocytes (10.42 ± 7.06) compared to the NN and NS groups. An increasing trend was observed in the number of blastocysts formed and vitrified in the SS group, but these differences were not significant.
The SS genotype group had higher implantation (47.87%), clinical pregnancy (71.83%), and live birth rates (66.20%) compared to the NN and NS groups, but these differences were not statistically significant. Although there were no statistically significant differences in the no. of embryos transferred, an increasing trend was observed in the clinical pregnancy and live birth rates, with insignificant p-values (0.15 and 0.25), respectively (Table I).
3.3. LHCGRN312S
Similar to FSHRN680S polymorphism, no significant difference was observed in the mean female age, AFC, BMI, and marital life among the 3 genotypes of LHCGRN312S polymorphism. An association was found between the type of infertility and the N312S genotype; the number of women with primary infertility was higher in the SS genotype among the LHCGRN312S genotypes (69.70%, 77.03%, 85.60%, p = 0.04).
No significant difference was observed in the daily rFSH dose among the genotypes. In the present study, the homozygous NN group required a higher total rFSH dose compared to LHCGR312 NS and SS. In LHCGRN312S polymorphism, subjects in the SS group received 75 units of rhMG from day 1. The mean total number of days required for rFSH stimulation in women of the NN genotype group is significantly more (12.64 ± 1.42) when compared to women who received rFSH in NS (12.03 ± 1.42) and SS (11.99 ± 1.30) genotypes with p = 0.048.
No significant difference was observed in the mean no. of oocytes retrieved (14.78 ± 8.87) and M2 oocytes (9.98 ± 6.53) among 3 genotypes of LHCGRN312S. However, the mean number of oocytes retrieved in women of the NN genotype (15.87 ± 9.90) was higher compared to NS (14.47 ± 8.05), and SS (14.84 ± 9.72) women. No difference was observed in the number of M1, GV, and atretic oocytes obtained. No statistically significant difference was observed in the number and quality of blastocysts formed or vitrified among genotypes of LHCGRN312S polymorphism.
No significant difference was observed in the implantation rates. Also, no significant difference was observed in the clinical pregnancy rate and live birth rate among LHCGRN312S genotypes in freeze-all cycles (Table I).
3.4. Combined assessment of FSHR and LHCGR on clinical outcomes
The combined polymorphism analysis of FSHR and LHCGR showed significant interaction concerning the total rFSH dose given to study participants in the controlled ovarian stimulation protocol. The higher dose requirement of total rFSH was observed when LHCGR312 is combined with genotypes of FSHR680 (NN: NN-2758 ± 520, NN: NS-3031 ± 764, NN: SS-4013 ± 391; NS: NN-2700 ± 333, NS: NS-2713 ± 389, NS: SS-3610 ± 611; SS: NN-2829 ± 508, SS: NS-2672 ± 324, SS: SS-3402 ± 497), showing a strong association between these 2 polymorphisms in total rFSH dose required for controlled ovarian stimulation (p = 0.02).
Though statistically not significant, the mean number of days of antagonist supplemented was on the higher side to downregulate the pituitary gland during COS when LHCGR genotypes were combined with FSHR genotypes (NN: NN-5.53 ± 0.87, NN: NS-5.75 ± 0.75, NN: NS-6.00 ± 0.75; NS: NN-5.57 ± 0.70, NS: NS-5.59 ± 0.86, NS: NS-5.61 ± 0.98; SS: NN-5.38 ± 0.88, SS: NS-5.52 ± 0.70, SS: SS-5.53 ± 0.91). The increasing trend in the mean no. of ovarian stimulation days was observed when LHCGR312 NN and NS compared with FSHR genotypes, but no such pattern was observed when LHCGR312 SS compared with FSHR genotypes.
The mean no. of GV and atretic oocytes were increased in the combined assessment of LHCGR312 (NN and SS) and FSHR680 genotypes (NN: NS: SS); however, no such pattern was observed with the LHCGR312 NS group.
The mean number of Grade-I blastocysts formed and vitrified increased when LHCGR312 NS and SS were combined with FSHR680 genotypes. However, these observations were not identified with LHCGR312 NN.
Though statistically insignificant, a vital interaction was found concerning the clinical pregnancy rate (p = 0.2) and live birth rate (p = 0.25) between the 2 polymorphisms. The present data shows that increased S alleles in FSHR680 and LHCGR312 gene polymorphisms were associated with increased clinical outcomes. The observed upward change in clinical pregnancy rate and the live birth rate was not consistent when LHCGR312 NN was combined with genotypes of FSHR680 (NN: NS: SS). However, a consistent increase was observed clinical pregnancy rate and live birth rate (Table II) when LHCGR312 NS and SS were combined with genotypes of FSHR680.
No statistically significant interaction or specific upward or down trend was observed between polymorphisms concerning female age, marital life, BMI, type of infertility, mean no. of AFC, IVF failures, the daily dose of rFSH, total hMG dose, mean no. of oocytes, M2, M1, rate of fertilization, mean no. blastocysts formed, and Grade-II and III blastocysts and implantation rate among all the genotype combinations.
4. Discussion
In this study, we investigated the relationship between FSHR680 and LHCGR312 genotypes on the response to controlled ovarian stimulation and clinical outcomes in women undergoing in vitro fertilization in freeze-all cycles. G protein-coupled receptors, FSHR and LHCGR, are activated by their receptive hormones, FSH and LH (16). These receptors express on their target cells and signal the follicular development through the cAMP/protein kinase A pathway (17). These 2 receptors play an essential role in reproductive biology, and variants in these receptors can produce altered physiological and signaling mechanisms, thus resulting in unsuccessful pregnancy (14, 18). In the present study, we identified a significant difference in the total dose of rFSH between the different genotype groups of FSHR680 and LHCGR312 (p = 0.02).
The results indicate that the total rFSH was significantly higher in the SS genotype group of FSHR680 and the NN genotype group of LHCGR312 in antagonist cycles (Table II). These findings suggest that women with FSHR60 SS and LHCGR312 NN require a higher rFSH dose than other genotype combinations (Table II). Women with homozygous SS in LHCGR312 were supplemented with LH in the form of hMG, along with an rFSH dose. The total dose of gonadotropins (rFSH+hMG) was higher in the SS genotype group for both FSHR680 and LHCGR312 compared to the NN and NS groups. These findings suggest that individuals with the SS genotype of FSHR and LHCGR may require higher doses of gonadotropins in antagonist cycles, in line with previous studies that have reported a similar association between the 2 genotypes (14).
The reason for supplementation of LH in homozygous for SS in LHCGR312 was due to poor ovarian response and higher IVF failure in previous cycles performed elsewhere, and observations from previous studies also suggested the addition of LH instead of increasing the FSH dose (7). Even though the supplementation of LH has resulted in a smaller number of oocytes in LHCGR312 SS, the proportion of GV and atretic oocytes were reduced in this group compared to other LHCGR312 genotypes generating equal mean number of mature oocytes. These results suggest that LH supplementation may particularly benefit individuals with the LHCGR312 SS genotype undergoing controlled ovarian stimulation.
Despite LH supplementation in the LHCGR312 homozygous serine group, the total number of blastocysts formed was comparatively lower than that of the N312 homozygous, though statistically insignificant (Table I). The two-way ANOVA reveals an increasing trend in the mean number of blastocysts formed when combining LHCGR312 SS with FSHR680 genotypes; these results suggest that SS in each polymorphism was advantageous in forming blastocysts (Table II). The mean number of Grade-1 blastocysts increased among LHCGR312 genotypes (Table I), and LHCGR312 NS and SS showed some interaction with FSHR680 genotypes with an increasing trend (Table II).
In the present retrospective study, we identified that women homozygous for SS in both polymorphisms have a 15% higher chance of getting pregnant compared with women NN in FSHR680 and LHCGR312 in freeze-all cycles. The results were in concordance with previous studies that there is a higher chance of conception in the serine group (7, 14). The results show that the clinical pregnancy and live birth rates vary across different genotypes of both genes, but the association was not statistically significant. For the FSHR680 and LHCGR312, the SS genotype group had the highest clinical pregnancy and live birth rate. However, the p-value for the clinical pregnancy rate was 0.15, indicating a borderline significance. Analyzing FSHR680 and LHCGR312 in combination reveals an increasing trend with the rise in number of S alleles in both polymorphisms. This increase might be attributed to physiological changes in the receptors resulting from the formation of hetero/homo dimers and oligomers, as described previously, rather than the individual receptor alone (18, 19). The decreased pregnancy rate in the presence of the Asn group on both receptors also might be due to decreased cAMP activity (15). The trend in FSHR680 might be due to the increased signaling capacity of the S680 variant in the presence of FSH in the normal physiological condition as described previously (14). One important observation from the current study is that in freeze-all cycles under the absence of hormonal influence (possible superovulation effect on endometrium receptivity), the clinical pregnancy and live birth rates were significantly lower in the 312SS:680NN group compared to the other 2 groups (312SS:680NS, SS). This suggests that even though the 312SS has an added advantage of getting more pregnancy rates as observed by previous studies, the subgroup analysis revealed that the presence of asparagine in the FSHR680 had a negative effect in the embryological as well as clinical outcome irrespective of hormone dose (FSH and LH). This study proves that these polymorphisms not only play a role in fresh embryo transfer cycles (6, 7, 14) but also play a significant role in freeze-all cycles in the absence of high levels of hormonal influence concerning clinical outcomes.
A Tang et al., meta-analysis stated that the FSH dose and pregnancy rates were not influenced by FSHR680 polymorphism. However, the present study observed that the total rFSH dose in antagonist protocol and pregnancy rates were dependent on FSHR polymorphism and further influenced by the addition of serine in LHCGR (20, 21). The current study investigated the benefit of individualizing the treatment options in infertile women to improve the outcome of ART procedures based on FSHR680 and LHCGR312 variants. The results show a promising relationship between genetic variants and the requirement of FSH, LH, and clinical outcomes. The current study findings explain the benefit of genotypes, as suggested by previous authors, along with functional biomarkers like AFC (22, 23, 24). However, the studies using these biomarkers were able to understand the variability in ovarian response to some extent (25). These genetic biomarkers help predict the ovarian response better than hormonal and functional biomarkers, leading to successful COS outcomes. Women with adequate AFC and sufficient hormonal levels show poor response during stimulation (26).
The current findings demonstrated a significant strength for a beneficial effect on clinical outcomes. They provided novel insights into the role of FSHR680 and LHCGR312 polymorphisms in freeze-all cycles. This study is the first to investigate the impact of these specific genetic variations on reproductive outcomes, in the absence of any detrimental effects caused by ovarian stimulation on the endometrium during the freeze all embryo transfer cycles. However, a weakness of the paper is the need for a comparative analysis of clinical outcomes between fresh and frozen cycles. Overall, this study contributes significantly to the existing literature by elucidating the role of these specific gene polymorphisms in freeze-all cycle.
5. Conclusion
Incorporating the pharmacogenomic approach in the COS regimen helps predict the women with adequate ovarian reserves, but shows a poor response during COS. The current study found that women with homozygous serine in FSHR polymorphism require, higher doses of rFSH to develop an adequate number of oocytes in the antagonist cycle. Second, in freeze-all cycles, both FSHR680 and LHCGR312 polymorphisms significantly influenced the clinical pregnancy and live birth rates, eliminating the impact of hormonal influences due to superovulation. This highlights the importance of consideration of these genetic variations as predictive markers for reproductive outcomes. Integrating genetic biomarkers along with functional and hormonal biomarkers in COS treatment protocols can help to improve the outcomes of assisted reproduction technologies, which can reduce the socioeconomic stress for the infertile couple planning to have offspring.
Data availibility
Data supporting the findings of this study are available upon reasonable request from the corresponding author.
Author contributions
Jayesh Amin and Naga Sandhya Alle had complete access to all the data in the study and takes responsibility for both the integrity of the data and the accuracy of the data analysis, including the conceptualization and design of the study. Ami Patel, Bansi Prajapathi, and Paresh Makwana were responsible for the acquisition, analysis, and interpretation of the data. Kota Murali Krishna drafted the manuscript. Jaya Prakash and Kota Murali Krishna conducted statistical analysis. All authors contributed to the critical revision of the manuscript for significant intellectual content. Ami Patel and Kota Murali Krishna took care of the overall coordination and follow-up of the study.
Acknowledgments
The present study received the funding with grant number: 0031C, from the R&D division, Wings IVF Center, Ahmedabad, India. English language grammar tool was used for content revision of the manuscript for language corrections (Grammarly in).
Conflict of Interest
The authors declare that there is no conflict of interest.
Full-Text: (18 Views)
1. Introduction
Infertility/subfertility is a significant concern worldwide for couples planning to have a child. It ranks the fifth highest global problem among all other health conditions (1). The prevalence of infertility is on the rise due to various factors, including the prolonged age of a couple to plan for a pregnancy, heightened stress levels, increased exposure to various environmental pollutants, and various health conditions resulting from unhealthy lifestyles (2). Fortunately, the assisted reproductive technique is critical for an infertile couple planning for parenthood. The success of these techniques depends on the specific protocol followed, whether it be antagonist or agonist, as well as optimizing the dosage of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) used for stimulation (3, 4). In recent years, there have been groundbreaking studies suggesting a pharmacogenomic approach to trailer stimulation protocols based on the genetic makeup of infertile women. This personalized approach aims to enhance the success rate of assisted reproductive technology (ART) (5-10).
The gonadotropin-releasing hormone plays a crucial role in regulating 2 essential hormones: FSH and LH (11). These 2 hormones apply themselves on their respective granulosa and theca cells through their receptors, namely follicle-stimulating hormone receptor (FSHR) and luteinizing hormone/choriogonadotropin receptor (LHCGR) (12) and help in the proliferation and maturation of follicles (13). The FSHR gene is present on chromosome 2, containing 10 exons; it has a common polymorphism on exon-10 known as N680S (rs6166 G>A), located in the intracellular domain. It encodes asparagine and serine amino acids, with the AAT codon coding for asparagine and the AGT codon coding for serine (14). Previous reports indicate that in regular in-vitro fertilization (IVF) cycles, individuals with FSHR680 homozygous SS exhibit higher sensitivity to FSH, elevated basal FSH, reduced estradiol production, and increased clinical pregnancy rates compared to individuals with other FSHR680 genotypes (14, 15).
The LHCGR gene contains 11 exons on chromosome 2 at cytogenic band 2p16.3 near the FSHR gene (7). Among the various polymorphisms identified in the LHCGR gene, the N312S polymorphism (rs2293275) found in exon 10 was widely studied (5, 7). N312S polymorphism substitutes asparagine with serine amino acid; the AAT codon codes the asparagine, and the AGT codon codes the serine.
Various research groups have investigated the impact of the LHCGRN312S polymorphic variant on controlled ovarian stimulation protocols (5, 7, 14). Previous studies have consistently shown that individuals homozygous to the serine allele have a higher chance of getting pregnant in regular IVF cycles (7, 14). Moreover, previous findings by Ga et al., showed that the homozygous serine group required higher doses of recombinant human luteinizing hormone compared to other genotypes in agonist protocol combined with a regular embryo transfer cycle (7).
To date, no research study has investigated the comparative clinical outcomes of recombinant follicle-stimulating hormone (rFSH) and human menopausal gonadotropin (hMG), and their respective combinations with gonadotropin-releasing hormone antagonistic protocol based on FSHR and LHCGR polymorphism, specifically in freeze-all embryo transfer cycles. In the current study, the objective was to assess the impact of FSHR680 and LHCGR312 polymorphisms on the clinical pregnancy rate and live birth rate in freeze-all cycles where the influence of hormonal stimulation on endometrial receptivity is absent due to superovulation by gonadotropins (FSH, LH).
2. Materials and Methods
2.1. Study design
This observational study was conducted at the Wings IVF Center, Ahmedabad, India, from March 2019 to April 2022. During this period, data collection and participant recruitment were done, utilizing the MediTEX IVF software, Regensburg, Germany for data retrieval.
A total of 421 women initially participated in the study. Inclusion criteria: age between 25 and 40 yr, a body mass index (BMI) of less than 30 kg/m2, a regular menstrual cycle lasting between 21 and 35 days, presence of bilateral ovaries, non-smoking status, and a quantifiable antral follicle count (AFC).
Exclusion criteria: Participants were excluded if they were recipients of oocyte donation, had a history of pelvic inflammatory disease, possessed only one ovary, had severe endometriosis, or showed serum anti-Müllerian hormone (AMH) levels below 1 ng/mL. Additionally, exclusion criteria extended to men with significant male factor infertility (defined as oligospermia with a sperm count below 2 million/ml or azoospermia), and couples who did not complete the follow-up.
Following the application of these criteria, the study was finalized with 306 women who met all the requirements necessary for inclusion in the research analysis.
2.2. Sample size estimation
The sample size was determined based on a power analysis. The criteria used were effect size of f = 0.25, a significance level of 0.05, and a desired power of 0.80 (indicating an 80% chance of detecting a true effect). This analysis indicates the necessity of a total 84 participants.
In our study, the effect size for the FSHR680 polymorphism was found to be 0.36. Given this effect size, 3 groups in the variable, a minimum sample size of 71 participants per group, and a significance level of 0.05, the power for detecting differences in total rFSH dose using one-way ANOVA was calculated to be 0.99. For LHCGR312 and total gonadotropins, the power was calculated to be 0.86 with an effect size of 0.34 and a minimum sample size of 33 participants per group.
2.3. Treatment protocol
Before initiating ovarian stimulation, all participants in the study were genotyped for FSH receptor (FSHR680) and LH/CG receptor (LHCGR312) polymorphisms. Stimulation commenced on either day 2 or 3 of the menstrual cycle using either rFSH alone or in combination with hMG. Dosages were initially set based on each woman's age, BMI, and AFC, with subsequent adjustments made between 150 IU and 450 IU of rFSH depending on individual responses assessed by transvaginal ultrasound scans of follicular size and serum estradiol levels, maintained below 450 pg/ml. While rFSH doses were not altered due to FSHR polymorphism results, women homozygous for LHCGR312S received an additional 75 IU of hMG from the first day of treatment. The protocol was set down by administering 10,000 IU of human chorionic gonadotropin as a trigger between days 10 and 12, based on estradiol levels and follicle size, followed by oocyte retrieval 36 hr later under transvaginal ultrasonography guidance.
2.4. Genotyping of FSHR and LHCGR
In brief, the genomic DNA from peripheral leukocytes was retrieved to determine the single nucleotide polymorphisms at amino acid position 680 (Asp680Ser) in the FSHR gene and amino acid position 312 (N312S) in the LHCGR gene. The LHCGR and FSHR genes were amplified using high-fidelity PCR in a total volume of 25 µL containing 1 µL of the forward primer (5′-ACGCACAGTCAGGTTTAGCC-3′ for LHCGR and 5′-CTCCTGTGCCAACCCCTTC-3′ for FSHR gene) and 1 µL of the reverse primer (5′-AACAGCTCCGTAACCAAG-3′ for LHCGR and 5′-TTAGATGAAATGTGTAGAAGCACTG-3′ for FSHR gene) along with 15 µL PCR master mix (Takara, USA) and 100 ng template DNA. The final volume was adjusted by using dH2O. DNA was denatured for 10 min at 95oC before the start of the amplification program. This was followed by 37 cycles of amplification, each of which included denaturation at 95oC for 1 min, annealing at 60oC for 30 sec, and elongation at 72oC for 3 min. The final elongation was carried out for 7 min at 72oC. The PCR product was purified and checked using 2% agarose gel before being directly sequenced on an 8-capillary applied biosystems sequencing apparatus (Applied Biosystems, USA). An advanced Big Dye Terminator 3.1V was utilized for sequencing. The collected data were then compared to the reference gene in the NCBI database to analyze the polymorphism.
2.5. Ethical considerations
Participants or their immediate relatives (in case of uneducated women who have difficulty in understanding scientific language) are provided informed consent to participate in the study. The study was approved by the Institutional Ethics Committee of Wings IVF Center, Ahmedabad, India (Code: 2019/003/31C/NSA). Ethical considerations were followed as per the Indian Council of Medical Research guidelines.
2.6. Statistical analysis
The statistical analysis of the data was performed using R Studio version 3.6.4. Mean and standard deviation values were calculated for numerical variables like age, BMI, AFC, marital life, daily rFSH dose, total rFSH, total hMG, stimulation days, and embryology parameters (no. of oocytes, methaphase [M] 2), M1, germinal vesicles (GV), and atretic oocytes, no. of blastocysts) among genotypes and between genotypes of 2 polymorphisms. The p-values were calculated using one-way ANOVA and two-way ANOVA. For categorical variables like type of infertility, clinical pregnancy rate, and live birth rate, percentages were calculated, and Chi-square was used to calculate the p-value. P-value < 0.05 was considered statistically significant.
3. Results
3.1. Genotypic distribution
The study includes 306 participants who underwent IVF treatment and received COS protocol based on functional, hormonal, and genetic biomarkers. The AA (NN) genotype distribution was 28.76% (n = 88), GA (NS) heterozygous was 48.04% (n = 147), and GG (SS) homozygous was 23.20% (n = 71) with respect to FSHRN680S polymorphism. For LHCGRN312S polymorphism, the AA (NN) genotype distribution was 10.78% (n = 33), AG (NS) heterozygous was 48.37% (n = 148), and GG (SS) homozygous was 40.85% (n = 125) (Table I). For FSHR680 polymorphism, the allele frequency of the G allele in the South Asian population was 48%, and that of the A allele was 52% (rs6166 RefSNP Report - dbSNP - NCBI [nih.gov], and in the current study, the frequency of the G allele was 46%, and that of the A allele was 54%. The allele frequency of the A allele in the South Asian population was 39%. The G allele was 61% for LHCGR polymorphism from the worldwide database (rs2293275 RefSNP Report - dbSNP - NCBI [nih.gov], and the data from the present study have shown the frequency of the A allele was 21%, and the G allele was 79% for LHCGR312 polymorphism (Table I).
3.2. FSHRN680S
The mean age, AFC, BMI, and marital life (yr) of study participants with FSHR polymorphism were similar, with no significant difference, among the 3 genotypes (NN, NS, and SS). No association was found for the type of infertility. Women with homozygous NN and heterozygous NS received a similar dose of mean daily rFSH compared to the homozygous SS group. A significant difference was observed in rFSH dose (I.U.) received per day (227.30 ± 19.2, 225.00 ± 14, and 284.50 ± 31.70 respectively, p ≤ 0.0001) and total mean rFSH dose (2786.44 ± 456.92, 2724.84 ± 410.40 and 3565.38 ± 556.91 respectively) with p ≤ 0.0001 among FSHRN680S genotypes (NN, NS, and SS).
The mean dose of rhMG and the number of days antagonists were administered were similar across all genotypes. Although not statistically significant, the SS group required slightly more days for ovarian stimulation (12.41 ± 1.60) and had higher mean numbers of oocytes (15.12 ± 9.70) and M2 oocytes (10.42 ± 7.06) compared to the NN and NS groups. An increasing trend was observed in the number of blastocysts formed and vitrified in the SS group, but these differences were not significant.
The SS genotype group had higher implantation (47.87%), clinical pregnancy (71.83%), and live birth rates (66.20%) compared to the NN and NS groups, but these differences were not statistically significant. Although there were no statistically significant differences in the no. of embryos transferred, an increasing trend was observed in the clinical pregnancy and live birth rates, with insignificant p-values (0.15 and 0.25), respectively (Table I).
3.3. LHCGRN312S
Similar to FSHRN680S polymorphism, no significant difference was observed in the mean female age, AFC, BMI, and marital life among the 3 genotypes of LHCGRN312S polymorphism. An association was found between the type of infertility and the N312S genotype; the number of women with primary infertility was higher in the SS genotype among the LHCGRN312S genotypes (69.70%, 77.03%, 85.60%, p = 0.04).
No significant difference was observed in the daily rFSH dose among the genotypes. In the present study, the homozygous NN group required a higher total rFSH dose compared to LHCGR312 NS and SS. In LHCGRN312S polymorphism, subjects in the SS group received 75 units of rhMG from day 1. The mean total number of days required for rFSH stimulation in women of the NN genotype group is significantly more (12.64 ± 1.42) when compared to women who received rFSH in NS (12.03 ± 1.42) and SS (11.99 ± 1.30) genotypes with p = 0.048.
No significant difference was observed in the mean no. of oocytes retrieved (14.78 ± 8.87) and M2 oocytes (9.98 ± 6.53) among 3 genotypes of LHCGRN312S. However, the mean number of oocytes retrieved in women of the NN genotype (15.87 ± 9.90) was higher compared to NS (14.47 ± 8.05), and SS (14.84 ± 9.72) women. No difference was observed in the number of M1, GV, and atretic oocytes obtained. No statistically significant difference was observed in the number and quality of blastocysts formed or vitrified among genotypes of LHCGRN312S polymorphism.
No significant difference was observed in the implantation rates. Also, no significant difference was observed in the clinical pregnancy rate and live birth rate among LHCGRN312S genotypes in freeze-all cycles (Table I).
3.4. Combined assessment of FSHR and LHCGR on clinical outcomes
The combined polymorphism analysis of FSHR and LHCGR showed significant interaction concerning the total rFSH dose given to study participants in the controlled ovarian stimulation protocol. The higher dose requirement of total rFSH was observed when LHCGR312 is combined with genotypes of FSHR680 (NN: NN-2758 ± 520, NN: NS-3031 ± 764, NN: SS-4013 ± 391; NS: NN-2700 ± 333, NS: NS-2713 ± 389, NS: SS-3610 ± 611; SS: NN-2829 ± 508, SS: NS-2672 ± 324, SS: SS-3402 ± 497), showing a strong association between these 2 polymorphisms in total rFSH dose required for controlled ovarian stimulation (p = 0.02).
Though statistically not significant, the mean number of days of antagonist supplemented was on the higher side to downregulate the pituitary gland during COS when LHCGR genotypes were combined with FSHR genotypes (NN: NN-5.53 ± 0.87, NN: NS-5.75 ± 0.75, NN: NS-6.00 ± 0.75; NS: NN-5.57 ± 0.70, NS: NS-5.59 ± 0.86, NS: NS-5.61 ± 0.98; SS: NN-5.38 ± 0.88, SS: NS-5.52 ± 0.70, SS: SS-5.53 ± 0.91). The increasing trend in the mean no. of ovarian stimulation days was observed when LHCGR312 NN and NS compared with FSHR genotypes, but no such pattern was observed when LHCGR312 SS compared with FSHR genotypes.
The mean no. of GV and atretic oocytes were increased in the combined assessment of LHCGR312 (NN and SS) and FSHR680 genotypes (NN: NS: SS); however, no such pattern was observed with the LHCGR312 NS group.
The mean number of Grade-I blastocysts formed and vitrified increased when LHCGR312 NS and SS were combined with FSHR680 genotypes. However, these observations were not identified with LHCGR312 NN.
Though statistically insignificant, a vital interaction was found concerning the clinical pregnancy rate (p = 0.2) and live birth rate (p = 0.25) between the 2 polymorphisms. The present data shows that increased S alleles in FSHR680 and LHCGR312 gene polymorphisms were associated with increased clinical outcomes. The observed upward change in clinical pregnancy rate and the live birth rate was not consistent when LHCGR312 NN was combined with genotypes of FSHR680 (NN: NS: SS). However, a consistent increase was observed clinical pregnancy rate and live birth rate (Table II) when LHCGR312 NS and SS were combined with genotypes of FSHR680.
No statistically significant interaction or specific upward or down trend was observed between polymorphisms concerning female age, marital life, BMI, type of infertility, mean no. of AFC, IVF failures, the daily dose of rFSH, total hMG dose, mean no. of oocytes, M2, M1, rate of fertilization, mean no. blastocysts formed, and Grade-II and III blastocysts and implantation rate among all the genotype combinations.
4. Discussion
In this study, we investigated the relationship between FSHR680 and LHCGR312 genotypes on the response to controlled ovarian stimulation and clinical outcomes in women undergoing in vitro fertilization in freeze-all cycles. G protein-coupled receptors, FSHR and LHCGR, are activated by their receptive hormones, FSH and LH (16). These receptors express on their target cells and signal the follicular development through the cAMP/protein kinase A pathway (17). These 2 receptors play an essential role in reproductive biology, and variants in these receptors can produce altered physiological and signaling mechanisms, thus resulting in unsuccessful pregnancy (14, 18). In the present study, we identified a significant difference in the total dose of rFSH between the different genotype groups of FSHR680 and LHCGR312 (p = 0.02).
The results indicate that the total rFSH was significantly higher in the SS genotype group of FSHR680 and the NN genotype group of LHCGR312 in antagonist cycles (Table II). These findings suggest that women with FSHR60 SS and LHCGR312 NN require a higher rFSH dose than other genotype combinations (Table II). Women with homozygous SS in LHCGR312 were supplemented with LH in the form of hMG, along with an rFSH dose. The total dose of gonadotropins (rFSH+hMG) was higher in the SS genotype group for both FSHR680 and LHCGR312 compared to the NN and NS groups. These findings suggest that individuals with the SS genotype of FSHR and LHCGR may require higher doses of gonadotropins in antagonist cycles, in line with previous studies that have reported a similar association between the 2 genotypes (14).
The reason for supplementation of LH in homozygous for SS in LHCGR312 was due to poor ovarian response and higher IVF failure in previous cycles performed elsewhere, and observations from previous studies also suggested the addition of LH instead of increasing the FSH dose (7). Even though the supplementation of LH has resulted in a smaller number of oocytes in LHCGR312 SS, the proportion of GV and atretic oocytes were reduced in this group compared to other LHCGR312 genotypes generating equal mean number of mature oocytes. These results suggest that LH supplementation may particularly benefit individuals with the LHCGR312 SS genotype undergoing controlled ovarian stimulation.
Despite LH supplementation in the LHCGR312 homozygous serine group, the total number of blastocysts formed was comparatively lower than that of the N312 homozygous, though statistically insignificant (Table I). The two-way ANOVA reveals an increasing trend in the mean number of blastocysts formed when combining LHCGR312 SS with FSHR680 genotypes; these results suggest that SS in each polymorphism was advantageous in forming blastocysts (Table II). The mean number of Grade-1 blastocysts increased among LHCGR312 genotypes (Table I), and LHCGR312 NS and SS showed some interaction with FSHR680 genotypes with an increasing trend (Table II).
In the present retrospective study, we identified that women homozygous for SS in both polymorphisms have a 15% higher chance of getting pregnant compared with women NN in FSHR680 and LHCGR312 in freeze-all cycles. The results were in concordance with previous studies that there is a higher chance of conception in the serine group (7, 14). The results show that the clinical pregnancy and live birth rates vary across different genotypes of both genes, but the association was not statistically significant. For the FSHR680 and LHCGR312, the SS genotype group had the highest clinical pregnancy and live birth rate. However, the p-value for the clinical pregnancy rate was 0.15, indicating a borderline significance. Analyzing FSHR680 and LHCGR312 in combination reveals an increasing trend with the rise in number of S alleles in both polymorphisms. This increase might be attributed to physiological changes in the receptors resulting from the formation of hetero/homo dimers and oligomers, as described previously, rather than the individual receptor alone (18, 19). The decreased pregnancy rate in the presence of the Asn group on both receptors also might be due to decreased cAMP activity (15). The trend in FSHR680 might be due to the increased signaling capacity of the S680 variant in the presence of FSH in the normal physiological condition as described previously (14). One important observation from the current study is that in freeze-all cycles under the absence of hormonal influence (possible superovulation effect on endometrium receptivity), the clinical pregnancy and live birth rates were significantly lower in the 312SS:680NN group compared to the other 2 groups (312SS:680NS, SS). This suggests that even though the 312SS has an added advantage of getting more pregnancy rates as observed by previous studies, the subgroup analysis revealed that the presence of asparagine in the FSHR680 had a negative effect in the embryological as well as clinical outcome irrespective of hormone dose (FSH and LH). This study proves that these polymorphisms not only play a role in fresh embryo transfer cycles (6, 7, 14) but also play a significant role in freeze-all cycles in the absence of high levels of hormonal influence concerning clinical outcomes.
A Tang et al., meta-analysis stated that the FSH dose and pregnancy rates were not influenced by FSHR680 polymorphism. However, the present study observed that the total rFSH dose in antagonist protocol and pregnancy rates were dependent on FSHR polymorphism and further influenced by the addition of serine in LHCGR (20, 21). The current study investigated the benefit of individualizing the treatment options in infertile women to improve the outcome of ART procedures based on FSHR680 and LHCGR312 variants. The results show a promising relationship between genetic variants and the requirement of FSH, LH, and clinical outcomes. The current study findings explain the benefit of genotypes, as suggested by previous authors, along with functional biomarkers like AFC (22, 23, 24). However, the studies using these biomarkers were able to understand the variability in ovarian response to some extent (25). These genetic biomarkers help predict the ovarian response better than hormonal and functional biomarkers, leading to successful COS outcomes. Women with adequate AFC and sufficient hormonal levels show poor response during stimulation (26).
The current findings demonstrated a significant strength for a beneficial effect on clinical outcomes. They provided novel insights into the role of FSHR680 and LHCGR312 polymorphisms in freeze-all cycles. This study is the first to investigate the impact of these specific genetic variations on reproductive outcomes, in the absence of any detrimental effects caused by ovarian stimulation on the endometrium during the freeze all embryo transfer cycles. However, a weakness of the paper is the need for a comparative analysis of clinical outcomes between fresh and frozen cycles. Overall, this study contributes significantly to the existing literature by elucidating the role of these specific gene polymorphisms in freeze-all cycle.
5. Conclusion
Incorporating the pharmacogenomic approach in the COS regimen helps predict the women with adequate ovarian reserves, but shows a poor response during COS. The current study found that women with homozygous serine in FSHR polymorphism require, higher doses of rFSH to develop an adequate number of oocytes in the antagonist cycle. Second, in freeze-all cycles, both FSHR680 and LHCGR312 polymorphisms significantly influenced the clinical pregnancy and live birth rates, eliminating the impact of hormonal influences due to superovulation. This highlights the importance of consideration of these genetic variations as predictive markers for reproductive outcomes. Integrating genetic biomarkers along with functional and hormonal biomarkers in COS treatment protocols can help to improve the outcomes of assisted reproduction technologies, which can reduce the socioeconomic stress for the infertile couple planning to have offspring.
Data availibility
Data supporting the findings of this study are available upon reasonable request from the corresponding author.
Author contributions
Jayesh Amin and Naga Sandhya Alle had complete access to all the data in the study and takes responsibility for both the integrity of the data and the accuracy of the data analysis, including the conceptualization and design of the study. Ami Patel, Bansi Prajapathi, and Paresh Makwana were responsible for the acquisition, analysis, and interpretation of the data. Kota Murali Krishna drafted the manuscript. Jaya Prakash and Kota Murali Krishna conducted statistical analysis. All authors contributed to the critical revision of the manuscript for significant intellectual content. Ami Patel and Kota Murali Krishna took care of the overall coordination and follow-up of the study.
Acknowledgments
The present study received the funding with grant number: 0031C, from the R&D division, Wings IVF Center, Ahmedabad, India. English language grammar tool was used for content revision of the manuscript for language corrections (Grammarly in).
Conflict of Interest
The authors declare that there is no conflict of interest.
Type of Study: Original Article |
Subject:
Reproductive Genetics
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