ARTICLE IN PRESS Reproductive BioMedicine Online (2016) ■■, ■■–■■
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Association between premature ovarian failure, polymorphisms in MTHFR and MTRR genes and serum homocysteine concentration
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Ningning Hou a,b, Songchang Chen a,c, Feng Chen a,b, Minmin Jiang d, Junyu Zhang c, Yanmei Yang a,b, Bo Zhu b, Xiaoxia Bai b, Yuting Hu c, Hefeng Huang a,c,*, Chenming Xu a,c,*
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Key Laboratory of Reproductive Genetics, Ministry of Education (Zhejiang University), Hangzhou 310006, China; Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China; c International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, China; d Institute of Public Administration, Zhejiang Normal University, Hangzhou 310012, China b
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* Corresponding authors.
E-mail addresses: [email protected] (H Huang), [email protected] (C Xu). Dr Ningning Hou obtained her MD at the School of Medicine, Zhejiang University, Hangzhou, China in 1999. She is studying for her PhD at the Key Laboratory of Reproductive Genetics (Ministry of Education), Women’s Hospital in the School of Medicine, Zhejiang University. Dr Hou has received training in both clinical and scientific research areas. Her research interests include reproductive genetics and endocrinology.
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This study investigated the association between premature ovarian failure (POF), MTHFR C677T/A1298C and MTRR A66G genotypes and serum homocysteine (Hcy) concentration. A prospective study was conducted in Chinese women, which included POF patients (n = 180) and controls (n = 195). Peripheral blood samples were used to determine MTHFR C677T/A1298C and MTRR A66G genotypes, and serum Hcy and sex hormone concentrations. Results showed that serum Hcy concentrations of POF patients were significantly higher than those of controls (P < 0.0001). In POF patients, serum Hcy concentrations were significantly correlated with oestradiol and FSH concentrations (r = −0.174, P = 0.037 and r = +0.238, P = 0.006, respectively). There were no significant differences in the distributions of MTHFR C677T/A1298C or MTRR A66G genotypes between the two groups. However, these genetic variants influenced serum Hcy concentrations in POF patients, especially for MTRR 66 AA/AG/GG genotypes, which were significantly correlated with the patients’ Hcy concentrations (τ = 0.166, P = 0.033). These results suggest that serum Hcy concentrations in Chinese POF patients are increased and correlated with serum oestradiol/FSH concentrations. In conclusion, MTHFR C667T/A1298C and MTRR A66G genotypes are not associated with POF development, but they affect the patients’ serum Hcy concentrations.
Abstract
© 2016 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
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KEYWORDS: gene polymorphism, homocysteine, MTHFR, MTRR, premature ovarian failure
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http://dx.doi.org/10.1016/j.rbmo.2016.01.009 1472-6483/© 2016 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
Please cite this article in press as: Ningning Hou, et al., Association between premature ovarian failure, polymorphisms in MTHFR and MTRR genes and serum homocysteine concentration, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.01.009
ARTICLE IN PRESS 2 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101
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N Hou et al.
Introduction
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Gene polymorphisms of the enzymes in the one-carbon metabolic pathway have been shown to be linked to female reproduction. Women who carry these related gene variants may be susceptible to infertility, implantation failure and pregnancy loss (Laanpere et al., 2010, 2011). The enzymes 5–10 methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) are two of the key enzymes for the remethylation of Hcy to methionine in the one-carbon metabolic pathway (Fowler, 2005; Watkins and Rosenblatt, 2012). It has been reported that Hcy concentrations in follicular fluid are associated with ovarian reserve, response to ovarian stimulation and the quality of oocytes and early embryos in women undergoing assisted reproduction (Berker et al., 2009; Ocal et al., 2012). An increased risk of pregnancy loss with raised Hcy concentrations in mothers’ blood has been reported (Haggarty et al., 2006), which might be related to defective chorionic villous vascularization (Nelen et al., 2000). MTHFR 677C>T (rs1801133), 1298A>C (rs1801131) and MTRR 66A>G (rs1801394) are the common polymorphisms studied extensively in the coding region of the MTHFR and MTRR genes. Variants of the genes coding for these enzymes may reduce enzyme activity and consequently increase blood Hcy concentration, which is associated with several health risks such as cardiovascular disease and bone loss (Kim et al., 2013; Veeranna et al., 2011). Premature ovarian failure (POF) accounts for a large proportion of premature menopause before the age of 40 and is accompanied by deficient sex steroid hormones and elevated gonadotrophin concentrations. POF increases health risks for approximately 1% of women, including infertility (Cartwright et al., 2012) and psychosocial issues (Deeks et al., 2011), in addition to the long-term effects of cardiovascular disease (Yorgun et al., 2013) and osteoporosis (Gallagher, 2007). Most cases of POF are idiopathic, but the underlying mechanism remains uncertain. Observation of familial cases with idiopathic POF (Lin and Yang, 2006) and the analysis of twin-pair data (Gosden et al., 2007) confirm that the timing of menopause has a heritable component and the role of genetic aberration is involved in the pathogenesis of idiopathic POF. Several studies have screened candidate genes in affected women and have found not only genetic defects in the X chromosome but also autosomal abnormalities to be involved in this complex disorder (Goswami and Conway, 2005). Autosomal region mutations are thought to play more important roles in ovarian development than traditionally believed (Qin et al., 2012). Little is known about the distribution of the gene polymorphisms of enzymes in the one-carbon metabolic pathway among women with idiopathic POF. It was reported that a MTHFR A1298C polymorphism may be associated with higher basal FSH concentrations and a lower response to ovarian stimulation (Rosen et al., 2007). A Korean study suggested that the MTHFR 677T allele may increase the risk for idiopathic POF and could be a novel genetic marker for predicting risk (Rah et al., 2012). More proof is needed from other populations regarding the genetic effects of MTHFR and MTRR polymorphisms and the respective changes of Hcy concentrations
in POF patients. Thus, the goal of this study was to investigate the distribution of three gene polymorphisms, MTHFR C677T/A1298C and MTRR A66G, and serum Hcy concentrations in Chinese idiopathic POF patients and to determine the association between these gene polymorphisms, POF and serum Hcy concentration.
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Materials and methods
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Participants
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This study was approved by the Women’s Hospital Ethics Committee on 25 December 2014 (no. 20140056), Zhejiang University. In total, 375 Chinese women aged from 20 to 45 were recruited from the outpatient clinic during the period 2013 to 2015 and were divided into two groups: the POF patient group and the control group. The control group consisted of 195 women who had regular menstrual cycles and were diagnosed with infertility caused by obstruction of the Fallopian tube and/or male problems. The POF patient group consisted of 180 women who were diagnosed by idiopathic menopause before the age of 40 and serum FSH concentrations >40 IU/l, without known chromosomal abnormalities, previous ovarian surgery, chemotherapy or radiotherapy (Conway, 2000). In this study MTHFR C677T/A1298C and MTRR A66G genotypes were determined in all the participants, including 195 controls and 180 POF patients. Peripheral blood samples were obtained on the second or third day of menstrual cycle after overnight fasting for serum Hcy and sex hormone determination for all 195 controls and 125 POF patients.
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Basal sex hormone assays The basal serum sex hormone concentrations were measured using electrochemiluminescence immunoassay (ECLIA) on a cobas 6000 analyzer (Roche Diagnostics, USA), following the manufacturer’s instructions. The hormones measured included FSH, LH, oestradiol, progesterone, testosterone and prolactin.
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Serum Hcy concentration quantification Total serum Hcy concentrations were measured using a Hcy assay kit (Beijing Strong Biotechnologies, China) by an enzymatic cycle method on an ARCHITECT c16000 automatic analyser (Abbott Diagnostics, USA) as per the manufacturer’s recommended protocol.
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DNA isolation and genotyping of MTHFR and MTRR Genomic DNA was extracted from peripheral blood using a QIAamp DNA Mini Kit (Qiagen) according to the manufactur- Q3 er’s instructions. A folate metabolism-related genetic polymorphisms test kit (Xiamen Zeesan Biotech, China) was used
Please cite this article in press as: Ningning Hou, et al., Association between premature ovarian failure, polymorphisms in MTHFR and MTRR genes and serum homocysteine concentration, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.01.009
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ARTICLE IN PRESS Premature ovarian failure, MTHFR/MTRR gene polymorphisms and homocysteine concentration 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193
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to determine three polymorphisms in two genes: MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131) and MTRR A66G (rs1801394), by fluorescence PCR melting curve analysis. Immediately after real-time PCR, melting peak analysis was performed on the same LightCycler 4802 System (Roche Diagnostics).
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Statistical analysis
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The parameters were summarized and the statistical analysis was conducted using the Statistical Package for Social Sciences version 15.0 (SPSS, USA). A comparison of serum sex hormone concentrations and Hcy concentrations between the POF patient group and the control group was performed using Student’s t-test. Pearson correlation coefficient r was utilized to evaluate the association between serum oestradiol/FSH concentrations along with serum Hcy concentrations in each of the two groups. Differences in the MTHFR C677T/A1298C and MTRR A66G genotypic and allelic frequencies and differences in the MTHFR C677T/A1298C haplotype frequencies between the two groups were compared using a χ2-test. Odds ratios (OR) and 95% confidence intervals (CI) were used as measures of the strength of the association between these genotypes/alleles/haplotypes and the risk of POF. Distributions of the genotypes were checked with a Hardy-Weinberg equilibrium test. Both the HardyWeinberg equilibrium test and haplotype association were analysed by the SHEsis platform (Li et al., 2009). Serum Hcy concentrations were summarized according to the genotypes in each polymorphism and were compared using the ANOVA F-test. The Kendall rank correlation coefficient τ was taken to evaluate the correlation between serum Hcy concentrations and the three genotypes in each polymorphism. P-values of
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Association between premature ovarian failure, polymorphisms in MTHFR and MTRR genes and serum homocysteine concentration
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Ningning Hou a,b, Songchang Chen a,c, Feng Chen a,b, Minmin Jiang d, Junyu Zhang c, Yanmei Yang a,b, Bo Zhu b, Xiaoxia Bai b, Yuting Hu c, Hefeng Huang a,c,*, Chenming Xu a,c,*
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Key Laboratory of Reproductive Genetics, Ministry of Education (Zhejiang University), Hangzhou 310006, China; Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China; c International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, China; d Institute of Public Administration, Zhejiang Normal University, Hangzhou 310012, China b
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* Corresponding authors.
E-mail addresses: [email protected] (H Huang), [email protected] (C Xu). Dr Ningning Hou obtained her MD at the School of Medicine, Zhejiang University, Hangzhou, China in 1999. She is studying for her PhD at the Key Laboratory of Reproductive Genetics (Ministry of Education), Women’s Hospital in the School of Medicine, Zhejiang University. Dr Hou has received training in both clinical and scientific research areas. Her research interests include reproductive genetics and endocrinology.
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This study investigated the association between premature ovarian failure (POF), MTHFR C677T/A1298C and MTRR A66G genotypes and serum homocysteine (Hcy) concentration. A prospective study was conducted in Chinese women, which included POF patients (n = 180) and controls (n = 195). Peripheral blood samples were used to determine MTHFR C677T/A1298C and MTRR A66G genotypes, and serum Hcy and sex hormone concentrations. Results showed that serum Hcy concentrations of POF patients were significantly higher than those of controls (P < 0.0001). In POF patients, serum Hcy concentrations were significantly correlated with oestradiol and FSH concentrations (r = −0.174, P = 0.037 and r = +0.238, P = 0.006, respectively). There were no significant differences in the distributions of MTHFR C677T/A1298C or MTRR A66G genotypes between the two groups. However, these genetic variants influenced serum Hcy concentrations in POF patients, especially for MTRR 66 AA/AG/GG genotypes, which were significantly correlated with the patients’ Hcy concentrations (τ = 0.166, P = 0.033). These results suggest that serum Hcy concentrations in Chinese POF patients are increased and correlated with serum oestradiol/FSH concentrations. In conclusion, MTHFR C667T/A1298C and MTRR A66G genotypes are not associated with POF development, but they affect the patients’ serum Hcy concentrations.
Abstract
© 2016 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
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KEYWORDS: gene polymorphism, homocysteine, MTHFR, MTRR, premature ovarian failure
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http://dx.doi.org/10.1016/j.rbmo.2016.01.009 1472-6483/© 2016 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
Please cite this article in press as: Ningning Hou, et al., Association between premature ovarian failure, polymorphisms in MTHFR and MTRR genes and serum homocysteine concentration, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.01.009
ARTICLE IN PRESS 2 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101
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N Hou et al.
Introduction
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Gene polymorphisms of the enzymes in the one-carbon metabolic pathway have been shown to be linked to female reproduction. Women who carry these related gene variants may be susceptible to infertility, implantation failure and pregnancy loss (Laanpere et al., 2010, 2011). The enzymes 5–10 methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) are two of the key enzymes for the remethylation of Hcy to methionine in the one-carbon metabolic pathway (Fowler, 2005; Watkins and Rosenblatt, 2012). It has been reported that Hcy concentrations in follicular fluid are associated with ovarian reserve, response to ovarian stimulation and the quality of oocytes and early embryos in women undergoing assisted reproduction (Berker et al., 2009; Ocal et al., 2012). An increased risk of pregnancy loss with raised Hcy concentrations in mothers’ blood has been reported (Haggarty et al., 2006), which might be related to defective chorionic villous vascularization (Nelen et al., 2000). MTHFR 677C>T (rs1801133), 1298A>C (rs1801131) and MTRR 66A>G (rs1801394) are the common polymorphisms studied extensively in the coding region of the MTHFR and MTRR genes. Variants of the genes coding for these enzymes may reduce enzyme activity and consequently increase blood Hcy concentration, which is associated with several health risks such as cardiovascular disease and bone loss (Kim et al., 2013; Veeranna et al., 2011). Premature ovarian failure (POF) accounts for a large proportion of premature menopause before the age of 40 and is accompanied by deficient sex steroid hormones and elevated gonadotrophin concentrations. POF increases health risks for approximately 1% of women, including infertility (Cartwright et al., 2012) and psychosocial issues (Deeks et al., 2011), in addition to the long-term effects of cardiovascular disease (Yorgun et al., 2013) and osteoporosis (Gallagher, 2007). Most cases of POF are idiopathic, but the underlying mechanism remains uncertain. Observation of familial cases with idiopathic POF (Lin and Yang, 2006) and the analysis of twin-pair data (Gosden et al., 2007) confirm that the timing of menopause has a heritable component and the role of genetic aberration is involved in the pathogenesis of idiopathic POF. Several studies have screened candidate genes in affected women and have found not only genetic defects in the X chromosome but also autosomal abnormalities to be involved in this complex disorder (Goswami and Conway, 2005). Autosomal region mutations are thought to play more important roles in ovarian development than traditionally believed (Qin et al., 2012). Little is known about the distribution of the gene polymorphisms of enzymes in the one-carbon metabolic pathway among women with idiopathic POF. It was reported that a MTHFR A1298C polymorphism may be associated with higher basal FSH concentrations and a lower response to ovarian stimulation (Rosen et al., 2007). A Korean study suggested that the MTHFR 677T allele may increase the risk for idiopathic POF and could be a novel genetic marker for predicting risk (Rah et al., 2012). More proof is needed from other populations regarding the genetic effects of MTHFR and MTRR polymorphisms and the respective changes of Hcy concentrations
in POF patients. Thus, the goal of this study was to investigate the distribution of three gene polymorphisms, MTHFR C677T/A1298C and MTRR A66G, and serum Hcy concentrations in Chinese idiopathic POF patients and to determine the association between these gene polymorphisms, POF and serum Hcy concentration.
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Materials and methods
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Participants
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This study was approved by the Women’s Hospital Ethics Committee on 25 December 2014 (no. 20140056), Zhejiang University. In total, 375 Chinese women aged from 20 to 45 were recruited from the outpatient clinic during the period 2013 to 2015 and were divided into two groups: the POF patient group and the control group. The control group consisted of 195 women who had regular menstrual cycles and were diagnosed with infertility caused by obstruction of the Fallopian tube and/or male problems. The POF patient group consisted of 180 women who were diagnosed by idiopathic menopause before the age of 40 and serum FSH concentrations >40 IU/l, without known chromosomal abnormalities, previous ovarian surgery, chemotherapy or radiotherapy (Conway, 2000). In this study MTHFR C677T/A1298C and MTRR A66G genotypes were determined in all the participants, including 195 controls and 180 POF patients. Peripheral blood samples were obtained on the second or third day of menstrual cycle after overnight fasting for serum Hcy and sex hormone determination for all 195 controls and 125 POF patients.
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Basal sex hormone assays The basal serum sex hormone concentrations were measured using electrochemiluminescence immunoassay (ECLIA) on a cobas 6000 analyzer (Roche Diagnostics, USA), following the manufacturer’s instructions. The hormones measured included FSH, LH, oestradiol, progesterone, testosterone and prolactin.
135 136 137 138 139 140 141 142 143
Serum Hcy concentration quantification Total serum Hcy concentrations were measured using a Hcy assay kit (Beijing Strong Biotechnologies, China) by an enzymatic cycle method on an ARCHITECT c16000 automatic analyser (Abbott Diagnostics, USA) as per the manufacturer’s recommended protocol.
144 145 146 147 148 149 150 151
DNA isolation and genotyping of MTHFR and MTRR Genomic DNA was extracted from peripheral blood using a QIAamp DNA Mini Kit (Qiagen) according to the manufactur- Q3 er’s instructions. A folate metabolism-related genetic polymorphisms test kit (Xiamen Zeesan Biotech, China) was used
Please cite this article in press as: Ningning Hou, et al., Association between premature ovarian failure, polymorphisms in MTHFR and MTRR genes and serum homocysteine concentration, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.01.009
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ARTICLE IN PRESS Premature ovarian failure, MTHFR/MTRR gene polymorphisms and homocysteine concentration 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193
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to determine three polymorphisms in two genes: MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131) and MTRR A66G (rs1801394), by fluorescence PCR melting curve analysis. Immediately after real-time PCR, melting peak analysis was performed on the same LightCycler 4802 System (Roche Diagnostics).
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Statistical analysis
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The parameters were summarized and the statistical analysis was conducted using the Statistical Package for Social Sciences version 15.0 (SPSS, USA). A comparison of serum sex hormone concentrations and Hcy concentrations between the POF patient group and the control group was performed using Student’s t-test. Pearson correlation coefficient r was utilized to evaluate the association between serum oestradiol/FSH concentrations along with serum Hcy concentrations in each of the two groups. Differences in the MTHFR C677T/A1298C and MTRR A66G genotypic and allelic frequencies and differences in the MTHFR C677T/A1298C haplotype frequencies between the two groups were compared using a χ2-test. Odds ratios (OR) and 95% confidence intervals (CI) were used as measures of the strength of the association between these genotypes/alleles/haplotypes and the risk of POF. Distributions of the genotypes were checked with a Hardy-Weinberg equilibrium test. Both the HardyWeinberg equilibrium test and haplotype association were analysed by the SHEsis platform (Li et al., 2009). Serum Hcy concentrations were summarized according to the genotypes in each polymorphism and were compared using the ANOVA F-test. The Kendall rank correlation coefficient τ was taken to evaluate the correlation between serum Hcy concentrations and the three genotypes in each polymorphism. P-values of
