Introduction
The anti-Müllerian hormone (AMH), also known as Müllerian-inhibiting substance, belongs to the transforming growth factor-beta (TGF-β) superfamily of growth and differentiation factors [
1]. AMH is synthesized by granulosa cells of preantral and small antral follicles [
2], and its level strongly correlates with the size of primordial follicle pool and the number of antral follicles [
3], which has made AMH an ideal marker of the ovarian reserve [
4].
AMH plays a key role in the regulation of primordial follicle recruitment and cyclic selection. Through modulating the threshold of follicle-Stimulating Hormone (FSH) sensitivity, AMH could inhibit FSH-induced antral follicle growth and limit the transition of follicles from the primordial to primary stage [
5,
6]. AMH exerts its specific biological function mainly through the AMH type II receptor (AMHR2), which is expressed on granulosa and theca cells [
7].
Considering the potential role of AMH in affecting ovarian response to stimulation, it has been proposed that variation in the genes encoding the AMH signaling pathway may influence the ovarian response during controlled ovarian stimulation (COS). The AMH gene is located on the short arm of chromosome 19 and consists of 5 exons [
8,
9]. The gene of AMHR2 is located on chromosome 12 and is comprised of 11 exons [
10]. Several polymorphisms related to these two genes have been studied. The polymorphisms AMH c.146G > T, p.Ile49Ser (rs10407022) and AMHR2 -482A > G (rs2002555) have drawn the most attention. The AMH rs10407022 polymorphism rests in the promoter region. This polymorphism leads to the replacement of serine from isoleucine in the position 49 of AMH protein, and it can affect AMH bioactivity [
11]. The AMHR2 rs2002555 polymorphism is located in the non-coding region of the promoter, and it can affect the transcription process of AMHR2. Several studies have focused on these two polymorphisms and have suggested that these two polymorphisms are associated with elevated follicular phase estradiol levels in normo-ovulatory women [
12], unexplained infertility [
13], follicle number, and androgen levels in polycystic ovary syndrome (PCOS) [
14]. Some studies have also investigated the effects of these two polymorphisms during COS in assisted reproduction technology (ART) treatment [
15‐
21]. However, the results of these studies were inconsistent. A meta-analysis of the polymorphism AMH (rs10407022) has been published [
22]. However, this study only explored the association between AMH polymorphisms and reproductive outcomes in the Caucasian population. Since then, several new studies on SNPs of the AMH/AMHR2 pathway have been published. Considering this, we feel that it is clinically important to conduct a meta-analysis to comprehensively evaluate the role of AMH (rs10407022) and AMHR2 (rs2002555) in the ovarian response and the outcomes of in vitro fertilization (IVF) during the process of ovarian stimulation.
Materials and methods
We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines to design and report this systematic review and meta-analysis [
23].
Search strategy
Studies were searched from PubMed, Web of Science, Embase, and Cochrane Central Register of Controlled Trials databases published without language restriction from inception to December 2019. The search strategies used a combination of terms “polymorphism,” “pharmacogenetics,” “AMH,” “AMHR,” and “controlled ovarian stimulation.” The detailed search strategies are provided in the supplemental material (Appendix 1). Reference lists of relevant reviews and articles were manually searched.
Eligibility criteria
The criteria of the inclusion of studies were as follows: (1) participants underwent IVF/ intracytoplasmic sperm injection (ICSI); (2) single nucleotide polymorphisms (SNPs) of AMH and AMHR2 were detected in some or all of the participants; (3) COS outcomes based on the gene polymorphisms were available.
Study selection
After removing duplicates, titles and abstracts were screened by two individual reviewers. Disagreements were discussed and resolved by consensus. Only trials published in peer-reviewed journals were included. Case reports, case series, conference abstracts, reviews, editorials, and gray literature were excluded.
Data were extracted independently from all eligible articles by two reviewers, and they included the first author, publication year, region, SNPs reported, sample size, treatment protocol, study design, and outcomes. If the median and percentile values rather than the mean and standard deviation (SD) were provided, the data were converted to mean and SD through the method described elsewhere [
24]. Two subgroups (e.g. AA vs. AB, AA vs. BB) were combined into one group (e.g. AA vs. BB/AB) by referring to the method described in the Cochrane Handbook for Systematic Reviews of Interventions if necessary [
25]. Consensus was reached to resolve the discrepancies.
Quality assessment
Two independent reviewers assessed the quality of included studies by the Newcastle-Ottawa scale (NOS) scores. The judgment of NOS scores was based on the following three domains: selection of the study group, comparability between groups, and ascertainment of exposed/not exposed cohorts [
26].
Outcomes of interest
The primary outcome was defined as the number of retrieved oocytes. The secondary outcomes included stimulation duration, Gn dosage, the number of metaphase II (MII) oocytes, and pregnancy rate. Pregnancy was defined as at least one gestational sac with a fetal heart activity under ultrasonographic visualization.
Statistical analysis
The meta-analysis was performed with the Review Manager software (Revman), version 5.3.5. Relative risk (RR) was used for categorical data. Mean difference (MD) was used for continuous data. All of the outcomes were calculated with 95% confidence intervals (CIs). Four genetic models were used in this study (dominant model: AA vs Aa/aa; homozygote model: AA vs aa; heterozygote model AA vs Aa; and recessive model: aa vs AA/Aa). A random effects model was used as the clinical heterogeneity existed among studies. We evaluated the heterogeneity between studies using Cochran’s Q statistic with associated
P-value [
27]. The degree of heterogeneity was quantified by measuring I
2. I
2 > 50% and
P < 0.05 indicated substantial heterogeneity. Subgroup analysis was performed to explore the source of heterogeneity. Sensitivity analysis by sequentially removing an individual study was also conducted to investigate the source of heterogeneity and the stability of the results. Statistical significance was set at
P < 0.05.
Discussion
There is growing evidence supporting that SNP may contribute to the differences in complex characteristics between individuals. Previous studies on the AMH/AMHR2 signaling pathway have revealed that polymorphisms of AMH and AMHR2 may associate with the ovarian response. A meta-analysis by Pabalan et al. [
22] investigated the association of AMH rs10407022 and AMHRII rs2002555 with reproductive outcomes and PCOS. They found no evidence of significant associations of the two polymorphisms with reproductive outcomes and PCOS, and they also found that AMH rs10407022 could increase the risk of PCOS up to 1.5-fold in Caucasians.
Therefore, we focused on the association between the AMH/AMHR2 gene polymorphisms and ovarian stimulation outcomes, and then we provided a more comprehensive evaluation of the outcomes of assisted reproductive technology (ART)herapy.
The primary outcome of our study was the number of retrieved oocytes. It is closely related to the success rate of ART therapy-the more the number of oocytes retrieved, the higher the cumulative delivery rate [
28]. In this study, AMH (rs10407022) polymorphism had no association with the number of oocytes retrieved, even though in the subgroup analysis based on the regions, the number of oocytes retrieved in TT homozygotes was significantly lower than that retrieved in GG/GT carriers in the Asian region. In addition, when the Peluso [
19] study was omitted in these four genetic models, all of the results turned significant, and the T allele carriers had obviously fewer retrieved oocytes than the G allele carriers. The AMHR2 (rs2002555) seemed to have no effect on the oocytes retrieved, although the results became significant after removing the study by Cerra [
15] in the heterozygote model.
In the sensitivity analysis, we found that eliminating the studies by Peluso [
19] or Cerra [
15] could change the significance of the results of the oocytes retrieved. One potential reason could be the region or ethnicity difference among the studies since different ethnicities may have different allelic frequencies [
19,
21]. Another reason may be different genotyping techniques used in the study because only Cerra [
15] and Peluso [
19] used TaqMan and other researchers used PCR. The differences in the age of the participants and treatment protocols could also have contributed to the high heterogeneity.
The previous meta-analysis only reported that AMH (rs10407022) reduced the risk in reproductive outcomes and increased the risk of PCOS among Caucasian population. In our study, in the European region, we did not find a significant association between AMH (rs10407022) and the number of oocytes retrieved. Indeed, we found that the number of oocytes was smaller in TT carriers in the dominant model in the Asian region. Different results may have arisen from different populations focused on. The previous meta-analysis mainly targeted the PCOS patients, while in our study, we included not only the PCOS patients but also the healthy patients. Among the seven studies included in this analysis, three studies [
17,
19,
20] mentioned that they excluded participants with PCOS, Lazaros [
16] only reported the included participants without the sign of hyperandrogenism, while the other two studies [
15,
18] also included the PCOS population. Different inclusion and exclusion criteria may have led to this difference and high heterogeneity. However, in the subgroup analysis on the PCOS population, we found no significant difference between the two subgroups (data not shown). Therefore, excluding PCOS patients or not has a limited effect on the result and is not the main source of heterogeneity.
In the analysis of MII oocytes, we found that even though the T allele carriers of AMH rs10407022 had significantly fewer oocytes and they tended to have more MII oocytes. Some studies have found that high follicular FSH levels could interfere with the meiotic division and increase aneuploidy rates of oocytes during IVF treatment [
29,
30]. Consistent with these findings, we found that the T allele carriers of AMH rs10407022 polymorphism tended to have lower basal FSH (data not shown). Based on this finding, we speculated that the function of AMH protein translated from G mutation may somehow be impaired; it could lead to high FSH in the circulation and follicle liquid, and then disturb the maturation of oocytes and eventually cause less MII oocytes. With respect to the Gn dosage, stimulation duration, and the pregnancy rate, our analysis showed that these two polymorphisms barely affected these outcomes.
In conclusion, we think that SNPs of the AMH/AMHR2 pathway, especially AMH rs10407022, could affect the number of retrieved oocytes and MII oocytes, but the specific mechanism needs further exploration.
This study indicated that the polymorphisms of AMH/AMHR2 could affect the outcomes of COS; however several limitations need to be addressed. First, inherent heterogeneity, such as the baseline characteristics of patients, ovarian stimulation protocol, and study design, existed among studies. Second, as all the included studies were observational, some unknown confounders could not be excluded, which could have caused extra bias in our estimates. Third, the number of the studies and the sample size of the included studies in our analysis were relatively small. Ideally, the COH outcomes should have been corrected with the AMH level. However, we were unable to do so due to the lack of AMH level. Moreover, a region-based subgroup analysis was limited in explaining the source of heterogeneity, as the number of studies from North American and Asian regions were relatively small.
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