Is the CAG repeat of mitochondrial DNA polymerase gamma (POLG) associated with male infertility? A multi-centre French study

I.E. Aknin-Seifer1,2, R.L. Touraine2, H. Lejeune3, C. Jimenez4, J. Chouteau5, J.P. Siffroi6, K. McElreavey9, T. Bienvenu7, C. Patrat8 and R. Levy1,10,10

1 Laboratoire de Biologie de la Reproduction, 2 Service de Génétique Moléculaire, CHU-Hôpital Nord, Saint Etienne, 3 Département de Médecine de la Reproduction, Hôpital Edouard Herriot, 10 INSERM U418/INRA UMR 1245, Communications Cellulaires et Differenciation, Hopital Debrousse, Lyon, 4 Laboratoire de Biologie de la Reproduction, Maternité de l'Hôpital du Bocage, Dijon, 5 Clinilab, Saint Martin d'Hères, 6 Service d'Histologie, Biologie de la Reproduction et Cytogénétique (EA1533), Hôpital Tenon, AP-HP, Paris, 7 Laboratoire de Biochimie et Génétique Moléculaire Hôpital Cochin, AP-HP, Paris, 8 Laboratoire de Biologie de la Reproduction, Hôpital Cochin, AP-HP, Paris, 9 Reproduction, Fertility and Populations, Institut Pasteur, Paris, France

10 To whom correspondence should be addressed. Email: rachel.levy{at}chu-st-etienne.fr


    Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Recent data emphasized the implication of polymerase {gamma} (POLG) CAG repeats in infertility, making it a very attractive gene for study. A comparison of POLG CAG repeats in infertile and fertile men showed a clear association between the absence of the usual 10-CAG allele and male infertility, excluding azoospermia. It has also been suggested that the POLG gene polymorphism should be considered as a possible contributing factor in unexplained couple infertility where semen parameters are normal. In this study, we investigated the POLG CAG repeats, in a well-defined population of patients with severe male factor infertility. METHODS: We conducted a large study of POLG CAG repeats in 433 infertile and 91 fertile, normozoospermic and healthy males. In all subjects, phenotypic data, including semen parameters, hormonal status and clinical profiles, were available. RESULTS: Thirteen ‘homozygous mutants’ (3%) were found among the 433 idiopathic infertile patients. The follow-up of the 13 ‘homozygous mutant’ resulted in pregnancy for more than half of the couples, through assisted reproductive techniques or even spontaneously. In addition, one ‘homozygous mutant’ was identified in 91 fertile men (1.1%) CONCLUSION: Under our conditions, our study does not confirm any relationship between the polymorphic CAG repeat in the POLG gene and male infertility.

Key words: CAG repeats/male infertility/POLG


    Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
The human nuclear gene encoding the mitochondrial DNA polymerase {gamma} (POLG) (OMIM 174763), which is the sole polymerase for mitochondrial DNA (mtDNA), maps to 15q24–15q26. Missense mutations in the POLG gene have been associated with progressive external ophthalmoplegia or mitochondrial neurogastrointestinal encephalomyopathy, transmitted as either a recessive or a dominant trait (Van Goethem et al., 2001Go,2003Go). A motif (CAG)10CAACAGCAG coding for a stretch of 13 glutamines is located in the first coding exon of this gene. From previous studies in humans, it has been established that the length of this CAG repeat is polymorphic with a major allele at 10 repeats (Ropp and Copeland, 1996Go; Lecrenier and Foury, 2000Go). Various human pathologies have been ascribed to an expansion of a CAG repeat in some genes. Conversely, the polymorphic CAG repeat of the POLG gene has also been studied previously in syndromes with multiple mtDNA deletion or depletion, where it did not seem to play any role (Rovio et al., 1999Go).

Recent data suggested the implication of POLG CAG repeats in infertility (Rovio et al., 2001Go, 2004Go; Jensen et al., 2004Go; Trifunovic et al., 2004Go), but are debated (Krausz et al., 2004Go). First, comparing the POLG CAG repeats in infertile and fertile men, Rovio et al. (2001)Go concluded that there was a strong association between the absence of the usual 10-CAG allele and male infertility, excluding azoospermia. The authors suggested that 9% of male infertility cases, excluding azoospermia and extreme oligozoospermia, might be ascribed to this mutation of POLG, making it a very attractive gene for study. Recently, Jensen et al. (2004)Go suggested that the POLG gene polymorphism should be considered as a possible contributing factor in patients with unexplained couple infertility and normal semen parameters. Krausz et al. (2004)Go did not confirm any influence of the POLG CAG polymorphism on the efficiency of spermatogenesis and concluded that there was an absence of any clinical diagnostic value of the CAG repeat length. The current study takes place in this recent debate concerning the role of POLG polymorphism in male infertility. We investigated the POLG CAG repeats, in a well-defined population of patients with severe male factor infertility.


    Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
We conducted a large study of POLG CAG repeats in 433 infertile and 91 fertile well characterized, normozoospermic and healthy males from seven French infertility centres. None of our patients presented any neuromuscular symptoms, and in particular no ophthalmoplegia. In the fertile normozoospermic controls, the biological fecundity measured as waiting time to pregnancy (TTP) for the couples was not available. At least one sperm sample per individual, collected after a 3 day abstinence, was analysed according to WHO recommendations (World Health Organization, 1999Go). Phenotypic data, including semen parameters, hormonal status and clinical profiles, were available for all infertile men. Semen parameters for the 433 infertile men were as follow: 119 azoospermia, 154 extreme oligozoospermia (<1 x 106 spermatozoa/ml), 99 severe oligozoospermia (1–5 x 106 spermatozoa/ml), 26 moderate oligozoospermia (5–20 x 106 spermatozoa/ml) and 35 asthenozoospermia and/or teratozoospermia with normal sperm concentration. Most of our patients were Caucasian French with a minority from North Africa, with no difference between fertile and infertile men. All the patients exhibited a normal 46 XY karyotype and were assessed negative for Y chromosome microdeletion. Either buccal cells or blood cells were used for DNA extraction.

Using previously published conditions (mip51 and mip33 primers) with minor modifications, we correctly amplified the CAG repeat of the POLG gene (Rovio et al., 1999Go). The PCR fragments were run on a CEQ2000XL Beckman sequencing machine, to classify patients as (i) homozygous for the 10-CAG allele; (ii) ‘homozygous mutants’ when they did not have a 10-CAG allele, whether they had two different alleles (compound heterozygote: x/y) or a single allele (true homozygote: x/x); or (iii) heterozygote (10/not10) (Table I). The follow-up of the patients lacking the common POLG allele [including the outcome of the assisted reproductive techniques (ARTs)] was detailed (Table II). For statistical analysis, frequencies were compared using {chi}2 test or Fisher exact test when appropriate. A P-value < 0.05 was considered as significant.


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Table I. Review of genotype frequencies of the POLG gene in infertile and in normospermic men

 

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Table II. Phenotype (clinical and biological data), genotype and outcome of ART treatment in the 13 homozygous patients for the POLG gene polymorphism

 

    Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
One ‘homozygous mutant’ was identified among the 91 fertile men (1.1%) (Table I). In parallel, 13 ‘homozygous mutants’ with a normal karyotype were found among the 433 idiopathic infertile patients, corresponding to 3%. Seven of them were true homozygotes (one was 12/12, and six were 11/11) and six were compound heterozygotes (three were 11/12, two were 9/11 and one was 6/9) (Table II). Of the 13 ‘homozygous’ mutants, one was azoospermic, four cryptozoospermic and two presented extreme oligozoospermia (Table II). Surprisingly, Rovio et al. (2001)Go did not identify ‘homozygous mutants’ previously among men with azoospermia and extreme oligozoospermia. The frequency of the homozygous mutant genotype in azoospermic, extreme oligozoospermic, severe oligozoospermic, moderate oligozoospermic and isolated asthenoteratozoospermic groups was 2.5, 2.6, 4, 7.7 and 0%, respectively.

We noted a similar frequency of heterozygosity (10/not10) in idiopathic infertile men (26.1%, 113 out of 433) and in fertile normozoospermic men (26.4%, 24 out of 91) (Table I). Therefore, the frequency of ‘homozygous mutants’ was 3% in idiopathic infertile men (13 out of 433) and, more precisely, 3.6 and 3.4% in patients with oligozoospermia and asthenozoospermia, respectively.

Interestingly, among the 13 ‘homozygous mutants’, eight couples obtained at least one pregnancy: five after ICSI (five singletons), one after the first intra-uterine insemination (IUI, one healthy girl), one after IVF using a semen donor (one healthy child) and one without any ART (patient M) (two paternally genetically assessed spontaneous pregnancies resulting in two healthy children). This ‘homozygous mutant’ patient exhibiting a severe oligozoospermia (4 x 106 spermatozoa/ml) previously had naturally fathered one child, and went on to father a second one during the study (M, Table II). Another ‘homozygous mutant’ switched from severe to moderate oligozoospermic after varicocele embolization, and a healthy girl was conceived following the first IUI (B, Table II).


    Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
As found by Krausz et al. (2004)Go, our data suggest that the CAG repeat of the POLG gene does not play a significant role in men with severe male factor infertility. In fact, no significant difference was observed in the distribution of the three genotypes (‘homozygous mutant’, homozygote 10/10 and heterozygote) among either fertile or infertile patients (Table I). We only found 13 homozygous mutants out of 433 idiopathic infertile patients, which is in disagreement with the data from Rovio et al. (2001)Go, where nine ‘homozygous mutants’ were identified out of 99 infertile men. We hypothesize that the larger number of infertile patients studied here enabled sampling bias to be avoided. Along these lines, since we detected seven ‘homozygous mutants’ exhibiting either azoospermia, cryptozoospermia or extreme oligozoospermia, we consider that we cannot simply exclude this population as Rovio did previously (Rovio et al., 2001Go). Therefore, if we add the 817 various infertile (including azoospermia and extreme oligozoospermia) men from Finland, Germany and the UK in the population of Rovio et al. (2001)Go, the frequency of ‘homozygous mutants’ they observed falls from 9 to 3.7%, which is quite similar to our findings (see Table I, footnote c). In addition, among the infertile men, the observed frequency of ‘homozygous mutants’ (3%) was close to the expected frequency deduced from the frequency of the 10-CAG homozygotes, according to the Hardy–Weinberg law (2.5%) (Wellek, 2004Go). In our population of fertile normozoospermic men, the observed frequency of ‘homozygous mutants’ (1.1%) was slightly lower than the expected one (2.2%), but this difference is not significant and is probably related to our rather limited population. This remark is also valid for the population of 98 fertile males in the study of Rovio et al., where no ‘homozygous mutant’ was observed although one was to be expected. Jensen et al. (2004)Go claimed that POLG polymorphism contributed to ‘unexplained subfertility’ based on a high POLG homozygous frequency among men with >20 x 106 spermatozoa/ml (eight out of 104; 7.7%), in particular if of normal motility (seven out of 49; 14.3%). Table I of Jensen et al. (2004)Go indicated that, of the 104 ‘so-called subfertile’ men with >20 x 106 spermatozoa/ml, only 49 have normal motility (>50%): in this latter group, seven homozygous patients were found. This is in disagreement with Table II of Jensen et al. (2004)Go since, in fact, only one homozygous patient can be assessed as normozoospermic, considering a cut-off of 50% for normal motility value and >20 x 106 spermatozoa/ml (World Health Organization, 1999Go). Accordingly, one can recalculate the homozygous mutant frequency in subfertile patients with normozoospermia as follows: 1 out of 42, i.e. 2.38%. This recalculated frequency is similar to that of Krausz et al. (2004)Go or our value. This was noted previously by Krausz et al. (2004)Go, leading to the same conclusion.

As Krausz et al. (2004)Go found, the relatively high frequency in our moderate oligospermic group (7.7%) was not significantly different from the control group (1.1%). The number of moderate oligozoospermic men in our study and that of Krausz et al. (2004)Go seems to be relatively low. It would be of interest to join data on patients with >5 x 106 spermatozoa/ml and >20 x 106 spermatozoa/ml from Jensen et al. and Rovio et al. and calculate the overall frequency.

Furthermore, if the ‘homozygous mutant’ men do have a reduced fertility, a progressive reduction of this unfavourable genotype over the generations is to be expected, and, in the absence of any selective advantage for the heterozygous individuals, the ‘homozygous mutant’ genotype should be extremely rare today.

The follow-up of the 13 ‘homozygous mutants’ resulted in pregnancy for more than half of the couples, through ART or even spontaneously (Table II). These results are similar to Jensen's. Thus, if associated with infertility, the POLG gene polymorphism should be only considered as a minor possible contributing factor in infertile male patients with no impact on obtaining a pregnancy.

Finally, our data are in agreement with the apparent absence of severe deleterious effects on mitochondrial genome replication when the POLG CAG repeat is deleted experimentally (Spelbrink et al., 2000Go). As frequently observed in other genes, the glutamine motif is absent in the mouse and rat POLG proteins, whereas a shorter repeat can be found in gorillas (6-CAG) and chimpanzees (4- and 7-CAG) (GenBank accession nos NM017462, NM053528, AF415155, AF415156 and AF415157) (Hancock et al., 2001Go; Rovio et al., 2004Go). It is well known that CAG repeats are involved in various human pathologies, but all these diseases are ascribed to an expansion of the CAG repeat.

In conclusion, in France and in Italy (Krausz et al., 2004Go), there is no relationship between the polymorphic CAG repeat in the POLG gene and male infertility.

Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/OMIM/

GeneBank http://www.ncbi.nlm.nih.gov/entrez/. The accession no. for POLG mRNA is X08093 and for a genomic clone containing the POLG gene is AC005317.


    Acknowledgements
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Dr Philippe Durand and Dr Sophie Paulhac for their valuable contributions to this article.


    References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on July 13, 2004; resubmitted on November 2, 2004; accepted on November 11, 2004.