Mol. Hum. Reprod. Advance Access originally published online on March 3, 2008
Molecular Human Reproduction 2008 14(4):245-249; doi:10.1093/molehr/gan012
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CAG repeat length variation in the polymerase gamma (POLG) gene: effect on semen quality
1Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Academic Medical Center, Meibergdreef 9, H4-205, 1105 AZ Amsterdam, The Netherlands 2Department of Clinical Epidemiology and Biostatistics, Academic Medical Center, Amsterdam, The Netherlands
3 Correspondence address. Tel: +31-20-5663458; Fax: +31-20-6963489; E-mail: g.h.westerveld{at}amc.uva.nl
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Abstract |
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Several case–control studies have investigated the effect of CAG repeat length variation in the POLG gene on male fertility and semen quality. Some described an association between the homozygous not10 CAG-repeat genotype and male subfertility and/or reduced semen quality, whereas others did not. The aim of our study was to investigate whether the not10/not10 variant is associated with spermatogenic failure. By direct sequencing methods, we determined the CAG repeat length of POLG in a cohort of 700 consecutive included men with variable degrees of spermatogenesis to investigate its effect on semen quality. The frequency of the not10/not10 variant in our cohort was 4.7%. There were no differences in semen quality between groups with various POLG genotypes. There was a significant difference in frequency of the three CAG-repeat genotypes between ethnic subgroups. In conclusion, the not10/not10 POLG variant is not associated with clinically significant decreases in semen quality, but its frequency is dependent on ethnic background.
Key words: CAG repeat/male subfertility/POLG/polymerase gamma/semen quality
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Introduction |
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Subfertility, i.e. the inability to conceive within 1 year of unprotected intercourse, is an increasing problem in the Western world. It is estimated that one in every eight couples experiences difficulties in conceiving naturally. In about half of these cases, the male partner has spermatogenic failure, defined as one or more semen parameters below the WHO threshold for normal semen quality (World Health Organization, 1992; de Kretser, 1997). Spermatogenic failure can result from exogenous factors, such as previous chemotherapy, or endogenous factors, such as Y-chromosome deletions (Gianotten et al., 2003; Noordam and Repping, 2006). Unfortunately, the cause of spermatogenic failure remains unknown in the majority of cases. Many case–control studies have attempted to determine whether certain genetic variants are associated with spermatogenic failure (Krausz and Giachini, 2007). One of these genetic variants studied is the not10/not10 CAG-repeat variant in the polymerase gamma (POLG) gene. The not10/not10 genetic variant describes a genotype in which both alleles in exon 1 of the POLG gene have a CAG repeat length with more or less than 10 repeats.
The human POLG gene is located on the long arm of chromosome 15 and has a function in the replication of human mitochondrial DNA (Lestienne, 1987). The first exon of POLG contains a potentially unstable CAG repeat. The length of the CAG repeat is polymorphic with the most frequent allele containing 10 repeats (Ropp and Copeland, 1996). Mouse models showed that knock-in mice that expressed a proofreading-deficient version of PolgA, the mouse homolog of human POLG, have increased levels of mtDNA mutations. These mutations are associated with reduced life span and premature onset of aging-related phenotypes such as reduced fertility and alopecia (Trifunovic et al., 2004; Kujoth et al., 2005).
In 2001, an association between the not10/not10 POLG genotype and male subfertility was claimed (Rovio et al., 2001). More precisely, men with reduced semen quality more often had a not10/not10 CAG-repeat genotype as compared to men with normal semen quality and male partners from subfertile couples (regardless of their semen quality) more often had this not10/not10 genotype than males partners from fertile couples.
Since this initial publication, five studies have assessed the association between the not10/not10 CAG-repeat variant in POLG and male subfertility and/or spermatogenic failure with conflicting results (Table I) (Jensen et al., 2004; Krausz et al., 2004; Aknin-Seifer et al., 2005; Brusco et al., 2006; Harris et al., 2006). These conflicting results may be due to the case–control design used in these studies, which is prone to selection bias. Patients and, especially, controls were defined differently in the various studies, and in most studies, controls were recruited from a different population than the patients. Given these conflicting results, the clinical significance of the not10/not10 POLG variant is at present unclear.
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The aim of the present study was therefore to assess the effect of CAG repeat length variation in POLG on semen quality by performing a genotype driven rather than phenotype driven approach. To do so, we studied a large cohort of consecutively included men with variable degrees of spermatogenesis and compared semen quality in men with different POLG genotypes.
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Materials and Methods |
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Study subjects
All men who attended the Center for Reproductive Medicine in the Academic Medical Center from January 2000 until September 2005 as part of a subfertile couple, defined as a couple failing to conceive within 1 year of unprotected intercourse, and who gave informed consent were included in our study. During the first visit, a medical history was taken to check for possible exclusion criteria. Primary exclusion criteria were a history of surgery of the vasa deferentia, bilateral orchiectomy, chemo- or radiotherapy and bilateral cryptorchidism. Men were also excluded if the fertility workup had identified obstructive azoospermia, retrograde ejaculation, numerical or structural chromosome abnormalities or Y-chromosome deletions. At least two semen analyses were performed for each patient according to WHO guidelines as part of standard patient care, and retrospectively linked to each included patient (World Health Organization, 1992). Ethnic background was registered for each patient. The Institutional Review Board of the Academic Medical Center approved this study.
Genetic analysis
DNA was extracted from peripheral blood leucocytes according to standard procedures.
The CAG repeat in exon 1 of the POLG gene was amplified by using sense (5’-CCAAAGCCAGGTGTTCTGAC) and antisense (5’-GAAGTGCTGGTCCAGGTTGT) primers, using the available POLG genomic sequence information on the web (NM_002693 [GenBank] ). PCR was carried out in a total volume of 25 µl and contained 250 ng DNA, 5.0 µl 5x betaine buffer [0.25 M Tris (pH 9.2), 70 mM NH4SO4, 10% DMSO and 1.5% Tween], 2.5 µl 5 M betaine, 0.2 mM dNTPs, 30 pmol forward and reverse primer, 2 mM MgCl2 and 1 U SuperTaq polymerase. We used a touchdown PCR program with a temperature range of 69–62°C with a 1°C decrement per cycle and one cycle increment per temperature step and a final amplification for 20 cycles at 94°C for 30 s, 50°C for 30 s and 72°C for 30 s with a final extension at 72°C for 5 min.
To determine the CAG repeat length, we used direct sequencing methods, using the same primers as those used for PCR, on an automated ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). All sequences were analyzed with the CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA).
Statistic analysis
A chi-square test was used to determine whether the observed frequencies of genotypes conformed to Hardy–Weinberg (H-W) expectations.
The average of all semen analyses for each individual patient was used in all statistical analyses. Semen parameters were tested for normal distribution using the Shapiro–Wilk test. Data are either presented as means ± standard deviation (SD) in case of normally distributed variables or as median with 25th and 75th percentiles in case of a non-normal distribution. Associations between the not10/not10 POLG variant and semen parameters were analyzed using a Kruskal–Wallis test. To investigate the possible effect of genetic heterogeneity on the association between the not10/not10 POLG variant and semen quality, all semen characteristics were analyzed using a (non-parametric) ANOVA model that included CAG repeat length, ethnicity and their interaction as predictors. All statistical analyses were carried out using SPSS (version 14.0). P-values <0.05 were considered significant.
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Results |
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We included 700 men with variable degrees of spermatogenesis. Cohort characteristics are shown in Table II. Forty-three (6.1%) men were azoospermic, 70 (10.0%) men had severe oligozoospermia (<5.0 x 106/ml), 87 (12.4%) men moderate oligozoospermia (5.0–20.0 x 106/ml) and 500 (71.4%) men were normospermic (>20.0 x 106/ml) according to the WHO criteria for normal sperm concentration. The frequency of the not10/not10 POLG variant was 4.7%. Distribution of genotypes was in H-W equilibrium.
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Men with the not10/not10 POLG variant did not have a significant reduction in semen quality as compared to men with the 10/not10 or 10/10 genotype (Fig. 1).
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There was a significant difference in frequency of the three POLG CAG-repeat genotypes between ethnic subgroups (P = 0.015). The wild-type genotype (10/10) varied from 52% in African men to 79% in Turkish men. Similarly, the frequency of the not10/not10 genotype varied from 2.3% in Asian men to 11% in African men (Table III). An ANOVA prediction model that included CAG repeat length, ethnicity and their interaction showed a significant interaction between ethnicity, CAG repeat length and semen concentration (P = 0.012), as well as between ethnicity, CAG repeat length and total motile count (TMC) (P = 0.042) (Table IV). We therefore performed a subgroup analysis in the two largest ethnic groups, which suggested a different effect of the POLG genotypes on concentration and TMC for Dutch and African men (Fig. 2). In Dutch men, the POLG genotype had no influence on semen quality, whereas in men from African origin, the heterozygous genotype (10/not10) had a positive effect on concentration (P = 0.015) and TMC (P = 0.040).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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In this cohort of 700 men, we found no differences in semen quality between men with or without the not10/not10 POLG genotype. The frequency of the three POLG genotypes differed between ethnic subgroups. Subgroup analysis showed that the POLG genotype had no influence on semen quality in Dutch men, whereas in men from African origin, the heterozygous genotype (10/not10) had a positive effect on semen quality.
One of the limitations of our study is that we included only 700 men. In our cohort, the mean concentration of men without the not10/not10 POLG variant was 57 x 106/ml and in men with this homozygous variant 48 x 106/ml. Given the population size of 700 and an observed POLG not10 allele frequency of 0.2, we could detect a minimal difference of 13 x 106/ml between carriers and non-carriers of the POLG not10 allele, or in case of a recessive genetic model, a minimal difference of 27 x 106/ml between homozygous not10/not10 patients and the other variants.
We included men with variable degrees of spermatogenesis, because of uncertainty about the phenotype associated with the not10/not10 variant. Our cohort design allows for comparison of the average semen quality of men with a certain (genetic) risk factor (such as in this case the not10/not10 POLG variant) to the semen quality of men without this risk factor and avoids phenotype based bias.
We found differences in frequency of the three POLG genotypes between ethnic subgroups, which have been described earlier in 12 ethnic subgroups from North Eurasia (Malyarchuk et al., 2005). Although subgroup analysis showed a different effect of the POLG genotypes on spermatogenesis in Dutch men compared with men from African origin, this is most likely due to small numbers of the three genotypic subgroups in the African subgroup. Moreover, it is biologically not very plausible that only the heterozygous genotype has an effect on the phenotype. These differences in frequency between (ethnic) groups illustrate the importance of proper study design when performing genetic association studies. In contrast to candidate gene screening in which the purpose of the study is to find a direct cause-effect relationship, i.e. finding a gene mutation that causes spermatogenic failure, association studies aim to find an association between a genetic locus and a certain disease. Association studies are thus genetic mapping studies in which the genetic variant in itself is not the direct or only cause of the phenotype. Consequently, these variants (also known as polymorphisms) also occur in controls, but, in case of an existing association, at a significantly lower frequency. Thus, association studies are more sensitive to selection bias than candidate gene screening. In our opinion, a cohort design is the preferred design for association studies, whereas a case–control design is a more useful design for candidate gene screening.
In conclusion, the not10/not10 POLG variant is not associated with clinically significant decreases in semen quality. We therefore advise not to screen for POLG CAG repeat length in men from subfertile couples.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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We thank S.K. van Daalen for technical assistance.
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References |
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Submitted on January 10, 2008; resubmitted on February 12, 2008; accepted on February 19, 2008.
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