1 Department of Growth and Reproduction, Copenhagen University Hospital (Rigshospitalet), DK-2100 Copenhagen, 2 Department of Biostatistics, University of Copenhagen, DK-2200 Copenhagen, 3 Fertility Clinic, Rigshospitalet, DK-2100 Copenhagen and 4 Department of Environmental Medicine, University of Southern Denmark, DK-4000 Odense, Denmark The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
5 To whom correspondence should be addressed at: Department of Growth and Reproduction, Section GR-5064, Copenhagen University Hospital (Rigshospitalet), Blegdamsvej 9, DK-2100 Copenhagen, Denmark. e-mail: erm{at}rh.dk
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Abstract |
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Key words: gene polymorphism/male infertility/mitochondrial DNA/sperm quality/POLG gene
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Introduction |
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Clinical assessment of male infertility/subfertility is primarily based on the analysis of semen quality. The most informative parameters are sperm concentration and total sperm count, followed by sperm motility and morphology (WHO, 1999). Sperm concentration exceeding 20 x 106/ml is usually considered normal, although values up to 40 x 106/ml have been associated with subfertility (Bonde et al., 1998
). There is also ongoing debate concerning the predictive value of other sperm parameters, such as motility and morphology, for the assessment of fertility potential (Comhaire and Vermeulen, 1995
; Zinaman et al., 2000
; Guzick et al., 2001
; Menkveld et al., 2001
).
Studies of sperm function, especially motility, turned the attention of researchers to the possible role of sperm mitochondria which produce large quantities of energy (Folgero et al., 1993). Mitochondria have their own genome (mtDNA), which codes for proteins involved in the respiratory chain and oxidative phosphorylation system. Numerous studies indicated an association between different polymorphisms, mutations or deletions in the mitochondrial genome and sperm dysfunction (Lestienne et al., 1997
; Ruiz-Pesini et al., 2000
; Holyoake et al., 2001
; St John et al., 2001
; Spiropoulos et al., 2002
). One of these studies (Ruiz-Pesini et al., 2000
) was particularly informative as it identified specific mtDNA haplogroups that are associated with asthenozoospermia. On the other hand, in one study no association of mtDNA with sperm dysfunction was found (Cummins et al., 1998
), and some recent reports showed an increased mtDNA content in infertile men (Díez-Sanchez et al., 2003
; May-Panloup et al., 2003
).
The key nuclear enzyme involved in the elongation and repair of mtDNA strands is DNA polymerase gamma (POLG), believed to be the only polymerase acting in the mitochondria (Bolden et al., 1977). The catalytic subunit of POLG is encoded by the POLG gene, which was mapped to chromosome 15q24 and includes a CAG repeat region (Ropp and Copeland, 1996
). A recent investigation of the frequency of different CAG repeat lengths in different European populations showed a high frequency (88%) of 10 codons (Rovio et al., 2001
), indicating that this common allele is maintained by selection. According to that study (Rovio et al., 2001
), the absence of the common allele on both chromosomes (x/x or
10/
10 according to our nomenclature) seems to be associated with male infertility, since it was observed in 3.59% of infertile males with impaired sperm quality (after exclusion of azoospermia and severe oligozoospermia), while none of the fertile control individuals was homozygous for the lack of the common allele. These intriguing findings prompted us to examine the polymorphism of the POLG gene among Danish men, whose average sperm counts appear to be low (Andersen et al., 2000
; Jørgensen et al., 2001
). In particular, we were interested in exploring possible associations between the POLG polymorphism and semen quality and fecundity. We report here the results of our analysis of a large series of well characterized patients and controls.
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Materials and methods |
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The fertile control group of 374 Danish subjects was invited to participate in research projects via their female partners. Their fatherhood was not genetically verified. However, these men were recruited to the study by their pregnant spouses, who knew that they would be subjected to genetic studies. Moreover, we have complete hormone and semen quality data on these individuals, and they are consistent with their good reproductive function. These subjects included: (A) 306 men recruited from the antenatal care unit for a study of reproductive health of Danish men (Jørgensen et al., 2001). Only couples that achieved the pregnancy without any fertility treatment were invited to participate. (B) Sixty-eight healthy men, selected from couples recruited for a prospective study of the association of time-to-pregnancy (TTP) with reproductive variables (Bonde et al., 1998
; Jensen et al., 2000
; 2001). Only men who achieved fatherhood during the study period (12 months) were included in the current investigation.
The unselected control group of 495 young Danish men from the general population with unknown fertility was recruited for prospective studies of reproductive health in Europe (Jørgensen et al., 2001; 2002) while attending a compulsory medical examination before being considered for military service.
Clinical analysis
All subjects underwent a thorough andrological examination, including a comprehensive analysis of reproductive hormones in serum (testosterone, inhibin B, LH, FSH, oestradiol and sex hormone-binding globulin) and semen analyses. Control subjects donated only one semen sample. The semen analysis was performed as previously described (Bonde et al., 1998; Andersen et al., 2000
; Jørgensen et al., 2001
; 2002). Three parameters of sperm quality, concentration, motility and morphology, were used for the statistical analysis of an association with the POLG polymorphism. Sperm concentration was divided into clinical categories defined as: azoospermia and severe oligozoospermia, <5 x 106/ml; moderate oligozoospemia, 520 x 106/ml; and normospermia, >20 x 106/ml. Motility was assessed according to the WHO guidelines (WHO, 1999
) with small modifications (Jørgensen et al., 2001
; 2002), and a cut-off value of 50% of motile sperm was used to define normal motility. Sperm morphology was assessed either according to the WHO (Rowe et al., 1993
; WHO, 1999
), or using the so-called strict criteria (Menkveld et al., 1990
; 2001). In order to compare the data, we chose different normal cut-off values, according to previous studies of the impact of sperm morphology on fecundity. For the subjects analysed according to the WHO, 21% of normal forms was the lower cut-off value (Menkveld et al., 2001
), 39% the upper threshold (Slama et al., 2002
), and 2238% was considered the uncertain zone. It was difficult to select the cut-off value for the strict criteria, because of some disagreement in the literature. We selected 8% of normal spermatozoa as the lower cut-off value (Guzick et al., 2001
), 19% as the upper threshold (Slama et al., 2002
), and 918% as the uncertain zone. In addition, we calculated the median values of spermatozoa with defects of mid-piece (neck) and tail in all studied subjects, which were 8 and 3% respectively, and assessed the distribution of subjects below and above these values.
Molecular analysis of the POLG gene
DNA was isolated from peripheral blood samples using a kit (Roche Diagnostics Gmbh, Mannheim, Germany). Two primers (GGTCCCTGCACCAACCATGA and CTTGCCCGAAGATTTGC TCGT), matching positions 267286 and 535553 respectively, of the POLG mRNA (accession number X98093) were used to amplify a 286 bp DNA fragment, using the Pfu DNA polymerase (Stratagene, San Diego, CA). PCR was performed in 30 µl of (final concentrations): 12 mmol/l TrisHCl, pH 8.8; 10 mmol/l KCl; 10 mmol/l (NH4)SO4, 2.0 mmol/l MgSO4; 0.1% Triton X-100; 0.1 mg/ml BSA; 250 µmol/l dNTP; 30 pmol of each primer and 2 IU Pfu polymerase. PCR conditions were: 98°C for 5 min, 40 cycles of 98°C for 30 s, 63°C for 1 min, 72°C for 1min 45 s and one cycle of 72°C for 5 min. DNA fragments were purified from 1% agarose gels and analysed first by applying a Cy5-labelled sequencing primer (CY5-CTGGATGTC CAATGGGTTGT, position 494513) under standard PCR conditions, resulting in a DNA fragment, which was sequenced on an ALF-express sequencer and analysed by the Fragment Analyser software (Amersham-Pharmacia Biotech, Uppsala, Sweden). Next, all bands that differed from a normal CAG 10/10 band (putative 10/
10) were re-analysed by direct sequencing of the fragment, using the same Cy5-labelled primer or a Cy5-labelled primer positioned on the other side of the CAG repeat (CY5-TCGGGCCAGGGCCGGTT, position 323339); each DNA fragment was sequenced twice. The positions with heterogeneous base call were determined and compared with what was expected from the presence of the different combinations of CAG repeat lengths. The two methods were equally sensitive and all those detected by one method were confirmed using the alternative method.
Statistical analysis
Differences between frequencies were quantified using odds ratios (ORs) and compared using Fishers exact test. Confidence intervals (CIs) for prevalences and ORs were exact, i.e. based on the binomial and the non-central hypergeometric distributions respectively. Testing a tendency towards higher prevalences among the groups, taking advantage of their ordinal structure (i.e. increasing fertility), was done with Goodmans gamma test (Goodman and Kruskal, 1979). A CI for the predictive value of homozygosity for the POLG gene polymorphism was obtained by first expressing it in terms of the ratio of the prevalences of the homozygosity among the fertile and infertile men, and, second, inserting an exact CI for the corresponding OR. The result was confirmed by a profile likelihood approach. Distributions of TTP were compared using a t-test and confirmed by a MannWhitney test and a discrete time Cox model.
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Results |
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Is there an association between the POLG gene polymorphism and spermatogenesis and semen quality?
The subjects were stratified according to sperm concentration and motility (Table I). Only one patient with POLGs CAG 10/
10 was found in the subgroup with azoospermia or severe oligozoospermia (his mean sperm concentration was 2 x 106/ml), and only three in the subgroup with moderate oligozoospermia. The proportion of heterozygotes was relatively higher among the infertile/subfertile patients with azoospermia or severe oligozoospermia in comparison with the controls. These low percentages among the controls may be due to chance because of the very small numbers of subjects with severe oligozoospermiaas is indicated by the very broad CIs.
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We did not observe an association of the POLG polymorphism with decreased sperm motility; the majority of subjects either heterozygous or homozygous for the absence of the common allele had reasonably good motility (Table I). Sperm morphology had to be analysed separately, because two different methods of assessment were used, therefore the data are not included in Table I. We had to analyse the data using different thresholds for different morphological forms. Regardless of how we defined the morphology thresholds, we found no association between the POLG polymorphism and sperm morphology.
The analysis of the reproductive hormone profile was consistent with the status of spermatogenesis and normal testicularpituitary axis function in all patients and controls. There was no association between the hormone profile and the POLG polymorphism (data not shown).
Analysis of association between the POLG gene polymorphism and fecundity
We then addressed the question whether or not the POLG polymorphism has any association with fertility or fecundity. Most of our fertile controls were young men recruited when they awaited their first child; therefore, it was not possible to associate the distribution of subjects with the POLG polymorphism with the number of children. For all subjects in our fertile control group B we had recorded the waiting TTP during a previous prospective study of factors affecting couple fecundity (Bonde et al., 1998). In addition, a large proportion of men in the control group A provided retrospective information on TTP in a questionnaire. We used those data to analyse a possible association of TTP with the POLG polymorphism, especially the heterozygotes, since we found only three homozygotes among the fertile controls. The three fertile
10/
10 subjects had normal sperm parameters and reproductive hormones. In the fertile control group A, the male partners with 10/10 reported an average TTP of 3.9 ± 0.4 (SE) months, whereas the 10/
10 men reported a mean TTP of 6.3 ± 1.9 months (P = 0.21, not significant). In the fertile control group B, those with 10/10 had a mean TTP of 3.4 ± 0.27 months, those with 10/
10, 2.5 ± 0.42 months (P = 0.13, not significant). For both fertile subgroups combined, the mean TTP values were 3.8 ± 0.4 months (median 2 months) for the 10/10 subjects versus 5.6 ± 1.6 months (median 2 months) for the 10/
10 subjects (P = 0.26, not significant). Thus, no significant association between the POLG gene polymorphism and TTP was found.
Eleven of the 12 patients homozygous for the POLG polymorphism were treated by assisted reproductive techniques (ART). In four cases a contributing female factor was present, with one exception (case 10, partial tubal occlusion) the problems were negligible. The outcome of the treatment is listed in Table II. Three cases underwent standard IVF. In one couple, three of four oocytes cleaved to an embryo, whereas only one of nine and one of five cleaved in the two other couples. One patient conceived but miscarriaged, two remaining patients conceived after ICSI, but one aborted. Nine of the 11 ART-treated 10/
10 patients underwent ICSI, either as the first treatment or after failed IVF. Apart from a single case, where none of 16 oocytes were fertilized after ICSI (case 4, Table II), all others had fertilization and cleavage. Seven partners conceived and delivered eight healthy children; one couple (case 1) subsequently underwent a second ICSI procedure which resulted in another child.
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Discussion |
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In a previous analysis of the POLG polymorphism, Rovio et al. (Rovio et al., 2001) found a substandard motility of spermatozoa in a substantial subset of infertile patients, and hypothesized that the variant POLG might cause some defects in, for example, mtDNA repair or replication, leading to a reduced energy metabolism and impaired motility of spermatozoa. However, we could not corroborate this attractive hypothesis: the surprising finding of our study was that the majority (18 of 23) of the subjects homozygous for the POLG polymorphism in our series had sperm motility within the normal range. Depletion and rearrangements of mtDNA, including those caused by mutations in other regions of the POLG gene, are often manifested as serious diseases in humans, e.g. progressive external ophtalmoplegia (Van Goethem et al., 2001
; Elpeleg et al., 2002
), whereas variants of POLG limited to the CAG repeats were not associated with any systemic disorders and seem to cause solely an impairment in male fertility (Rovio et al., 1999
; 2001; this study). The molecular mechanism of this impairment remains to be elucidated.
We found an association between sperm concentration and the POLG polymorphism, but in the opposite direction than we had expected. Nearly all 10/
10 homozygotes had reasonable sperm counts; most of them, in fact, exceeding the current normal cut-off value of 20 x 106 sperm/ml. Such an association was not observed among the heterozygotes, who were more or less evenly distributed across the sperm concentration strata, except the controls, who were less represented in the subgroup with the lowest sperm counts, most probably because these groups were very small. Rovio et al. (2001
) observed a low number of morphologically normal spermatozoa in some patients with
10/
10, but always in conjunction with either poor motility or decreased sperm concentration. We could not confirm this in our study, and we found no correlation between the POLG polymorphism and sperm motility or morphology. This discrepancy, however, could simply be caused by technical differences between the laboratories. The assessment of these variables, especially morphology, is subjective and not very sensitive, thus discrete differences between the groups cannot be excluded and will require further studies using other methods and parameters of semen quality.
We investigated a possibility that the POLG polymorphism could impair the post-ejaculatory functions of spermatozoa, which include oocyte penetration and fertilization. We had recorded an in vitro sperm penetration test in some patients but we had too few polymorphic subjects to observe any associations. Another marker of biological fecundity is the TTP. We found a weak trend to longer TTPs in the control subjects heterozygous for the POLG polymorphism, but could not substantiate this observation in a more conclusive manner, because of the low number of subjects in this category. However, we had data on the outcome of ART in nearly all homozygous 10/
10 patients. In three cases treated with IVF, cleavage of embryos was obtained, but the oocyte fertilization rate tended to be low, thus we cannot exclude some impairment of the
10/
10 sperm ability to penetrate or fertilize the oocyte. When ICSI was used, fertilization of oocytes did not seem to be impaired, as nearly all couples conceived. So far, all children appear to be healthy.
Finally, this study found a similar distribution of the normal and polymorphic POLG alleles in the Danish population to that reported in several other populations, including Finland (Rovio et al., 1999; 2001). In Finland, semen quality appears to be better than in Denmark and the incidences of genital malformations and testicular cancer are low (Jørgensen et al., 2001
; 2002; Toppari et al., 2001
). The differences in the male reproductive health between the two countries cannot be explained by the clustering of the
10/
10 genotype in Denmark; however, it is theoretically possible that the POLG polymorphism may render spermatozoa more sensitive to certain adverse environmental or lifestyle factors to which the Danish population is exposed.
In conclusion, our study confirmed an association between the POLG gene polymorphism and male subfertility. The 10/
10 patients had better average semen parameters than infertile men in general, thus the POLG gene polymorphism should be considered as a possible contributing factor in cases of idiopathic subfertility with normal spermiograms. Molecular mechanisms leading to the impairment of fertility in patients with this polymorphism remain to be elucidated, but we found that spermatozoa of
10/
10 patients may have a somewhat impaired ability to penetrate or fertilize intact oocytes. This problem can be solved by ICSI and the clinical results of this treatment are promising.
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Acknowledgements |
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References |
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Submitted on August 4, 2003; accepted on September 23, 2003.