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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: CAG repeats/male infertility/POLG
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent data suggested the implication of POLG CAG repeats in infertility (Rovio et al., 2001, 2004
; Jensen et al., 2004
; Trifunovic et al., 2004
), but are debated (Krausz et al., 2004
). First, comparing the POLG CAG repeats in infertile and fertile men, Rovio et al. (2001)
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)
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)
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Using previously published conditions (mip51 and mip33 primers) with minor modifications, we correctly amplified the CAG repeat of the POLG gene (Rovio et al., 1999). 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
2 test or Fisher exact test when appropriate. A P-value < 0.05 was considered as significant.
|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As Krausz et al. (2004) 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)
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., 2000). 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., 2001
; Rovio et al., 2004
). 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., 2004), 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Jensen M, Leffers H, Petersen JH, Nyboe Andersen A, Jorgensen N, Carlsen E, Jensen TK, Skakkebaek NE and Rajpert-De Meyts E (2004) Frequent polymorphism of the mitochondrial DNA polymerase gamma gene (POLG) in patients with normal spermiograms and unexplained subfertility. Hum Reprod 19, 6570.
Krausz C, Guarducci E, Becherini L, degl'Innocenti S, Gerace L, Balercia G and Forti G (2004) The clinical significance of the POLG gene polymorphism in male infertility. J Clin Endocrinol Metab 89, 42924297.
Lecrenier N and Foury F (2000) New features of mitochondrial DNA replication system in yeast and man. Gene 246, 3748.[CrossRef][ISI][Medline]
Ropp PA and Copeland WC (1996) Cloning and characterization of the human mitochondrial DNA polymerase, DNA polymerase gamma. Genomics 36, 449458.[CrossRef][ISI][Medline]
Rovio A, Tiranti V, Bednarz AL, Suomalainen A, Spelbrink JN, Lecrenier N, Melberg A, Zeviani M, Poulton J, Foury F et al. (1999) Analysis of the trinucleotide CAG repeat from the human mitochondrial DNA polymerase gene in healthy and diseased individuals. Eur J Hum Genet 7, 140146.[ISI][Medline]
Rovio AT, Marchington DR, Donat S, Schuppe HC, Abel J, Fritsche E, Elliott DJ, Laippala P, Ahola AL, McNay D et al. (2001) Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility. Nat Genet 29, 261262.[CrossRef][ISI][Medline]
Rovio AT, Abel J, Ahola AL, Andres AM, Bertranpetit J, Blancher A, Bontrop RE, Chemnick LG, Cooke HJ, Cummins JM et al. (2004) A prevalent POLG CAG microsatellite length allele in humans and African great apes. Mamm Genome 15, 492502.[CrossRef][ISI][Medline]
Spelbrink JN, Toivonen JM, Hakkaart GA, Kurkela JM, Cooper HM, Lehtinen SK, Lecrenier N, Back JW, Speijer D, Foury F et al. (2000) In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells. J Biol Chem 275, 2481824828.
Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly- YM, Gidlof S, Oldfors A, Wibom R et al. (2004) Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417423.[ISI]
Van Goethem G, Dermaut B, Lofgren A, Martin JJ and Van Broeckhoven C (2001) Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat Genet 28, 211212.[CrossRef][ISI][Medline]
Van Goethem G, Martin JJ and Van Broeckhoven C (2003) Progressive external ophthalmoplegia characterized by multiple deletions of mitochondrial DNA: unraveling the pathogenesis of human mitochondrial DNA instability and the initiation of a genetic classification. Neuromol Med 3, 129146.[CrossRef][ISI]
Wellek S (2004) Tests for establishing compatibility of an observed genotype distribution with HardyWeinberg equilibrium in the case of a biallelic locus. Biometrics 60, 694703.[CrossRef][ISI][Medline]
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interactions, 4th edn. Cambridge University Press.
Submitted on July 13, 2004; resubmitted on November 2, 2004; accepted on November 11, 2004.