Journal of Genetic Disorders & Genetic Reports ISSN: 2327-5790

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Research Article, J Genet Disor Genet Rep Vol: 3 Issue: 1

Are Antimullerian Hormone and its Receptor Genes Associated with Low Ovarian Response?

Chelsi Goodman1, Hosam Zaki2, Larry Fischel3, Hisham Greiss3 and Carolyn Coulam3,4*
1Chicago Medical School, Rosalind Franklin University of Medicine and Science,North Chicago, IL, USA
2Ganin Fertility Center, Cairo, Egypt
3Fertility and Cryogenics Laboratory, Downers Grove, IL, USA
4Reproductive Medicine Institute, Evanston, IL, USA
Corresponding author : Dr. Carolyn Coulam
Reproductive Medicine Institute, 2500 Ridge Ave, Suite 200, Evanston, IL 60201, USA
Tel: 847 869 7777; Fax: 847 869 7782;
E-mail: [email protected]
Received: February 03, 2014 Accepted: March 06, 2014 Published: March 12, 2014
Citation: Goodmana C, Zakib H, Fischelc L, Greissc H, Coulam C (2014) Are Antimullerian Hormone and its Receptor Genes Associated with Low Ovarian Response?. J Genet Disor Genet Rep 3:1. doi:10.4172/2327-5790.1000112

Abstract

Are Antimullerian Hormone and its Receptor Genes Associated with Low Ovarian Response?

One of the most frustrating problems in the treatment of infertility is that of poor ovarian response to stimulation. It therefore, would be advantageous to have a genetic marker that could predict low ovarian reserve at a young age before the impact of low ovarian reserve affects a woman’s fertility. Antimullerian hormone (AMH) has emerged as the the most accurate measure of ovarian reserve. The purpose of the present study is to investigate the correlation between AMH and AMH Receptor II (AMHRII) polymorphisms and low ovarian reserve.

Keywords: AMH gene polymorphisms; AMH receptor gene II polymorphisms; Low ovarian reserve

Keywords

AMH gene polymorphisms; AMH receptor gene II polymorphisms; Low ovarian reserve

Introduction

One of the most frustrating problems in the treatment of infertility, including in vitro fertilization (IVF), is that of poor ovarian response. This event has been called poor ovarian reserve, low ovarian reserve, diminished ovarian reserve, premature ovarian aging, and premature ovarian insufficiency. The number of oocytes retrieved directly influences success rates after IVF and embryo transfer (ET) [1]. Poor ovarian response reduces the number of embryos generated and results in decreased pregnancy rates in both index and subsequent IVF cycles [2]. Decreased pregnancy rates per cycle of IVF lead to more cycles being performed in an attempt to accomplish the goal of desired family. Poor response to gonadotropin is a significant problem in assisted conception occurring in 9% to 24% of patients and can precede the diagnosis of premature ovarian failure by months to years [3]. Thus, the economic impact of low ovarian reserve is significant. Identification of young women with a potential of low ovarian reserve would allow consideration of more cost efficient and desirable proactive management of infertile patients before low ovarian reserve impacted pregnancy outcome. Recent animal studies indicate oocytespecific genes play important roles in regulation of oogenesis and folliculogenesis [4].
Serum concentrations of antimullerian hormone (AMH) have emerged as the most accurate measure of ovarian reserve [4-6]. Genetic variants of AMH and AMH receptor II (AMHR2) have been associated with unexplained infertility [6]. Allelic frequencies of AMH2I -482A>G, IVS1+149 T>A, IVS5-6 C>T, IVS 10+77 A>G and AMH 146 T>G- polymorphisms have been reported to be significantly different in infertile women when compared with fertile women [7]. The purpose of the present study is to investigate the correlation between AMH and AMHR2 polymorphisms and low ovarian reserve to see if these genetic markers can serve as predictors of low ovarian reserve.

Materials and Methods

Patients
One hundred and one women, of which 50 have a diagnosis of low ovarian reserve and 51 are fertile controls, were included in the study. All infertile patients experienced regular menstrual cycles (mean 25-35 days), had no relevant systemic disease, severe endometriosis, or uterine or ovarian abnormalities and underwent IVF for treatment of infertility. A diagnosis of low ovarian reserve was based on secondary amenorrhea for >6 months in women <40 years of age, FSH >20 mIU/ml, and no antral follicles on transvaginal scanning, and/or serum AMH concentrations less than 1.05 ng/ml [8]. Fifty-one fertile women who have a history of at least one live birth and no history of infertility constitute the control group. The ethnicity of the patents and controls were mixed with 40% of Arabic descents and 50% Caucasians and equal numbers with patients and controls.
This study was approved by the institutional review board (IRB). All the couples and control women gave written informed consent.

DNA Extraction

All women had their cheeks swabbed with a cotton swab to collect cells for DNA analysis. DNA was extracted from buccal swabs using a Promega Maxwell 16 instrument, with the Promega DNA IQ Casework Pro Kit. All procedures are performed according to the manufacturer’s instructions. Purified DNA was eluted into a final volume of 30 μL H2O.

Polymerase Chain Reaction

DNA samples were first mixed with the appropriate reagents for the desired polymerase chain reaction assay, including oligodeoxynucleotide primers specific for each mutation site being analyzed. Thermal cycling was conducted in a GeneMate Genius Thermal Cycler per the manufacturer’s instructions, using 0.2 mL PCR tubes (Axygen). Following PCR, the PCR products were purified using a Qiagen QIAquick PCR Purification Kit and the DNA sequenced to reveal the mutation regions. DNA sequencing was performed by the Genomics Core Facility at Northwestern University. The primer sequences are listed in Table 1.
Table 1: Description of primers used in this study (FOR= forward and REV= reverse).
Statistical Analysis
The frequencies of AMHR2 -482A>G, IVS1+149 T>A, IVS5-6 C>T, IVS 10+77 A>G and AMH 146 T>G polymorphisms of infertile women who had a diagnosis of low ovarian reserve were compared with those of fertile control women using ANOVA one way analysis of variance. Significance is defined as p<0.05.
Power analysis was performed for comparisons of each of the polymorphisms: AMHR2 -482A>G, IVS1+149 T>A, IVS5-6 C>T, IVS 10+77 A>G and AMH 146 T>G using Statistical Solutions, LLC, Wisconsin, USA, calculator. Calculations were based on the following formula:
where μ1 = mean of group 1
μ1 = mean of group 1
σ2 = common error variance
A 2-sided test was performed with a 2-sided test with α – 0.05 and samle size of 101 subjects. Power β= between 0.92 and 0.71 for each of the polymorphisms studied.

Results

The frequencies of AMHR2 -482A>G, IVS1+149 T>A, IVS5-6 C>T, IVS 10+77 A>G and AMH 146 T>G polymorphisms among infertile women who had a diagnosis of low ovarian reserve were compared with those of fertile control women and are shown in Figure 1.
Figure 1: Percentage of controls and patients who were wild type, heterozygous, and homozygous for polymorphisms of AMHRII -482 (A>G), IVS1+149 (T>A), IVS5(-6) (C>T), IVS 10+77 (A>G) and AMH 146 T>G.
In addition, Table 2 displays odds ratios, 95% confidence intervals and P values of the polymorphisms among these populations.
Table 2: Low molecular weight PAI-1 antagonists.
No significant differences in the percentages of wild type, herterozygous and homozygous polymorphisms of AMHR2 -482A>G, IVS1+149 T>A, IVS5-6 C>T, IVS 10+77 A>G and AMH 146 T>G were observed.

Discussion

Even though antimullerian hormone (AMH) serum concentrations remain an appropriate marker of low ovarian reserve, no significant differences in the following polymorphisms of the AMH gene and its receptor AMH R2 were found when infertile women undergoing IVF with the diagnosis of low ovarian reserve were compared with fertile women: AMHR2 -482 A>G, IVS1+149 T>A, IVS5-6 C>T, IVS 10+77 A>G and AMH 146 T>G. This observation is important because it suggests that while AMH is a marker for low ovarian reserve, AMH and its receptor genes included in this study are not directly related low ovarian reserve.
AMH is a homodimeric disulfide-linked glycoprotein member of the transforming growth factor-beta superfamily with a molecular weight of 140 kDa [9]. In females, AMH is secreted by granulosa cells of growing follicles measuring 4-6 mm in diameter [10] or follicles that have undergone recruitment from the primordial follicle pool but have not as yet been selected for dominance. The main physiologic role of AMH appears to be limitation of transition from primordial into growing follicles [11] as well as to diminish the responsiveness of growing follicles to follicle stimulating hormone (FSH) [12]. AMH secretion is stimulated in response to activation of the AMH gene [13,14], and AMH activity is expressed through interaction with its receptor AMHR2 [15]. Polymorphisms in the AMH and AMHR2 genes have been associated with follicular phase estradiol serum levels, [16] since both AMH and its receptor polymorphisms are not associated with serum AMH or FSH concentrations, the elevated estradiol levels have been postulated to reflect a role for AMH in the regulation of FSH sensitivity in the human ovary [16]. Functional AMH polymorphism has been associated with flooicle number and androgen levels in women with polycyctic ovary syndrome [17]. Furthermore, AMHR2 −482 A > G polymorphism has been associated with natural age at menopause which suggests a role for AMH signaling in the usage of the primordial follicle pool in women [18].
AMH acting with its receptor signals through a bone morphogenetic protein (BMP)-like pathway with downstream signaling of SMAD proteins [15]. These proteins bind to the common SMAD4 protein, resulting in translocation of the complex into the nucleus and binding directly to the DNA to regulate gene expression or by interacting with other DNA-binding proteins [15]. While serum AMH concentration reflects ovarian reserve, neither the particular polymorphisms of AMH nor its receptor (AMHR2) analyzed here are related to low ovarian reserve (Figure 1, Table 2). Since the gene polymorphisms are not directly associated with low AMH expression, the diminished response to gonadotropin is not the result of AMH or its receptor gene expression, but rather the low AMH is a response to upstream signals. Thus, AMH or its receptor genes are not the determinants but rather the respondents of ovarian reserve. The challenge is to find the regulator of AMH gene expression that is associated with low ovarian reserve. Finding such a genetic marker would allow young women to be screened before such time that her low ovarian reserve would impact her fertility. She, then, could make the decision to have her family first and her career second or to freeze her eggs for future use [19].
Recent animal studies indicate oocyte-specific genes play important roles in regulation of oogenesis and folliculogenesis [20,21]. Genetic studies in mice demonstrated critical roles of two key oocyte-derived growth factors belonging to the transforming growth factor-β (TGF-β) superfamily, growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), in ovarian function [22,23]. The BMP-15 gene, also named GDF9B, is encoded by two exons and maps to the X chromosome in Xp11.2 [24]. The GDF9 gene is also encoded by two exons and maps to chromosome 5 in 5q23.3 [25]. The identification of BMP-15 and GDF-9 gene mutations as the causal mechanism underlying infertility in several sheep strains in a dosagesensitive manner also highlight the crucial role these two genes play in ovarian function [26]. Furthermore mutations in the GDF9 and BMP15 genes have been identified in women with premature ovarian failure [27-30]. While both BMP-15 and GDF-9 gene mutations have been reported in women with premature ovarian failure, only BMP-15 has been shown to significantly induce AMH expression in human granulosa cells [31]. Future investigation into potential regulators of AMH expression should included the BMP-15 mutations.

Acknowledgments

The authors thank Ferring Pharmaceutical Company for their generous financial support of this study.

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