MBE Advance Access originally published online on January 11, 2007
Molecular Biology and Evolution 2007 24(3):868-874; doi:10.1093/molbev/msm004
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Research Articles |
No Evidence for an mtDNA Role in Sperm Motility: Data from Complete Sequencing of Asthenozoospermic Males
* Instituto de Patologia e Imunologia Molecular da Universidade do Porto, (IPATIMUP), Porto, Portugal
Medical Faculty, University of Porto, Portugal
Centro de Genética Humana Instituto Nacional de Saude Dr Ricardo Jorge, Lisboa, Portugal
Maternidade Dr Alfredo da Costa, Lisboa, Portugal
|| Institute of Integrative & Comparative Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
¶ Department of Statistics, University of Glasgow, Glasgow, United Kingdom
E-mail: lpereira{at}ipatimup.pt.
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Abstract |
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The first complete mitochondrial DNA (mtDNA) sequences (
16,569 bp) in 20 patients with asthenozoospermia and a comparison with 23 new complete mtDNA sequences in teratoasthenozoospermic individuals, confirmed no sharing of specific polymorphisms or specific mitochondrial lineages between these individuals. This is strong evidence against the accepted claim of a major role played by mtDNA in male fertility, once supported by haplogroup association studies based on the screening of hypervariable region I. The hypothesis of maternally driven selection acting in male reproductive success must thus be treated with caution.
Key Words: mtDNA • complete sequences • asthenozoospermia • association
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Several associations between complex traits and mitochondrial haplogroups have been described in recent years, such as haplogroup J and longevity (e.g., De Benedictis et al. 1999
) and haplogroup U/K and increased risk of macrodeletions associated with myopathies (Crimi et al. 2003) and, surprisingly, haplogroup T and asthenozoospermia and haplogroup H and nonasthenozoospermia (Ruiz-Pesini et al. 2000). Asthenozoospermia is a male subfertility phenotype characterized by low sperm motility, which accounts, together with oligozoospermia (low number of sperm cells), for 75% of untreatable male subfertility (see Moore and Reijo-Pera 2000). Because mitochondrial DNA is maternally inherited, the idea of a neutral haplogroup in females conditioning male reproductive success opened new avenues in the mechanisms of differential sex selection (Moore and Reijo-Pera 2000), the role of mitochondria in germ cell selection, and the very establishment of maternal transmission of mitochondria (Giannelli 2001).
Nevertheless, for most of those reported associations, conflicting results, or lack of confirmation have been described in other populations surveyed. Several explanations have been put forward to account for these contradictions, such as the following: 1) poorly matched case and control samples, a highly relevant factor due to the well-established population structure of mtDNA haplogroups (even at a microgeographic scale; Pereira, Goncalves, et al. 2005), 2) the low resolution at which the mtDNA phylogeny is often assayed (Carelli et al. 2006), and 3) the lack of power of the statistical methods employed to detect association, because the mtDNA haplogroup distribution is usually characterized by many very low frequency classes (Samuels et al. 2006). In this last work, the authors showed that the small P value (P = 0.0311, declared significant at the 5% level) obtained by Ruiz-Pesini et al. (2000), in a test of association of haplogroup T with asthenozoospermia, is a considerable underestimate of the exact type I error (false-positive) rate estimated via permutation (P = 0.1435 and hence nonsignificant). Samuels et al. (2006) also determined that to have a 90% power to detect a 10% change between cases and controls in the frequency of haplogroup H in a typical European population, approximately 6,000 cases and 6,000 controls would be required; even greater samples sizes would be needed for less common haplogroups or more subtle changes in haplogroup frequencies.
These results force us to rethink our methodologies for assessing the association of mtDNA haplogroups with complex traits. The huge sample sizes are, at present, unrealistic, indeed impossible to obtain in cases of rare diseases. Rather than assaying a small number of polymorphisms in large samples of cases and controls, another strategy for examining the mtDNA variation of individuals with certain phenotypes compared with well-matched controls is to sequence their whole-mtDNA genome (but in smaller numbers due to cost). This allows one to take a fully phylogenetic view of the data and guard against premature claims about the pathogenicity of haplogroup-defining polymorphisms, where frequency differences between cases and controls can well reflect poor matching (Bandelt et al. 2005; Kong et al. 2006). Of course, instead of a primary role, those haplogroup-defining polymorphisms can act in a synergistic way with other mtDNA mutations (as in the case of Leber hereditary optic neuropathy, LHON), but, in this case, studies with a phylogenetic focus would also be needed (Herrnstadt and Howell 2004). Further, a phylogenetic analysis allows homoplasy and association through shared history to be identified, both of which can confound the search for causative alleles.
In the specific case of asthenozoospermia, to have a 90% power to detect a change of 10% frequency in haplogroup T, the number of patients and controls should be 40,000 each (derived from fig. 2D in Samuels et al. 2006 for haplogroup J, which has a similar frequency to haplogroup T in their representative European population), and this is unfeasible at present. In fact, although reduced sperm motility is observed in many subfertile males, it is usually associated with other sperm parameters, such as a low number of spermatozoa or abnormal sperm head morphology, which per se are also fertility conditioning, so that purely asthenozoospermic males have a rather low frequency. Because of this, we decided to take a different approach to the problem. We performed complete mtDNA sequencing in a sample of 20 (pure) asthenozoospermic males from Portugal, as well as in 23 teratoasthenozoospermic (defined by fewer than 30% spermatozoa with normal head morphology and reduced sperm motility) as another group for comparison. Patients were selected from the same infertility unit (Maternidade Dr Alfredo da Costa) so that geographical matching criteria were addressed (given the mtDNA microstructure observed in Portugal; Pereira, Goncalves, et al. 2005).
So far as we know, this study is the first to address the following questions surrounding the issue of mtDNA association with asthenozoospermia: Is any particular mtDNA mutation present in asthenozoospermic males? Do asthenozoospermic males carry more replacement or tRNA mutations than teratoasthenozoospermic males? Do asthenozoospermic males belonging to certain mtDNA haplogroups present any differences from controls in other populations sharing the same ancestry?
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Samples
A total of 20 asthenozoospermic and 23 teratoasthenozoospermic males were selected from a database of infertile males from Portugal. The infertile patients included in the database were picked up in a hierarchical 2-step survey. After 1) andrological and hormonal evaluation, only patients with idiopathic infertility, that is, absence of history of hypospadias, urinary infection, epididymitis, orchitis, varicocele, prostatic abnormalities, testis tumors, and testicular maldescent proceeded to 2) further chromosomal analyses, and only the patients having a 46, XY normal karyotype, and no AZFa (markers DBY and DFFRY), AZFb (markers RBMY1, sY1224, sY134, sY143, and sY119), and AZFc (markers sY283, RRM3, and sY254) microdeletions were included in the database. Sperm analysis was performed according to World Health Organization's guidelines (WHO 1992
). Each patient's semen sample was classified into one of the following categories: oligozoospermia, if sperm concentration is less than 20 million/ml but more than 0.0; asthenozoospermia, fewer than 50% spermatozoa with forward progression (categories A and B) or fewer than 25% spermatozoa with rapid linear progression; teratozoospermia, fewer than 30% spermatozoa with normal head morphology; and oligoteratoasthenozoospermia, if all 3 variables were abnormal; combination of only 2 prefixes were also used according to abnormal sperm variables. The samples analyzed in this work were either pure asthenozoospermic or teratoasthenozoospermic, with sperm counts between 20 and 312 million/ml. Appropriate informed consent was obtained from all subjects.
Complete mtDNA Sequencing
mtDNA was amplified using 32 overlapping fragments basically as described elsewhere (Maca-Meyer et al. 2001). After purification, the forward primers were used for the sequencing, and in some cases, also the reverse primers (in the presence of poly-C stretches, when polymorphisms A574C and T16189C occur). Sequencing was performed on a 3100 DNA Analyzer (AB, Applied Biosystems, Foster City, CA), and the resulting sequences were analyzed with SeqScape (AB, Applied Biosystems) and BioEdit version 7.0.4.1
[EC]
(Hall et al. 1999), by 2 independent investigators. In cases of ambiguous base calling, polymerase chain reaction and sequencing reactions were repeated. Furthermore, the norms of rechecking both the haplogroup-defining polymorphisms, known from the literature, as well as the private ones (Bandelt et al. 2005) were followed. Mutations were scored relative to the reference sequence (rCRS; Andrews et al. 1999). The 43 complete mtDNA sequences have been deposited in GenBank (accession numbers EF177405–EF177447).
Nomenclature
For haplogroup and subhaplogroup affiliation of the samples, the most recent phylogenetic data were followed: Achilli et al. (2004) for H; Behar et al. (2006) for K; Palanichamy et al. (2004) for J, L2, R1, T, and V; Achilli et al. (2005) and Palanichamy et al. (2004) for U; Kivisild et al. (2006) for I and M1; and Reidla et al. (2003) for X. Numbers 1–16569 refer to the position of the mutation in the rCRS.
Statistical Analyses
Analysis of molecular variance (AMOVA) and nucleotide diversity estimates were calculated by using the software Arlequin version 2.0 (Schneider et al. 2000). Fisher's exact tests were applied to 2 x 2 contingency tables in DnaSP 3.51 (Rozas J and Rozas R 1999) and to haplogroup distributions in asthenozoospermic versus teratoasthenozoospermic individuals.
To construct the mtDNA phylogeny, only the more slowly evolving coding region of the molecule between nucleotide positions 577 and 16023 was considered. A preliminary network analysis (Bandelt et al. 1995) led to a suggested branching order for the tree. Assuming the HKY85 mutation model with gamma-distributed rates (approximated by a discrete distribution with 32 categories), PAML 3.13 (Yang 1997) was used to estimate branch lengths and parameters of the mutation model. In addition, it was used to check the molecular clock hypothesis, via a likelihood ratio test.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Haplogroup representation (fig. 1) was highly diverse both in asthenozoospermic and teratoasthenozoospermic males, ranging from the common European haplogroups to rarer ones (I1a, M1, and R1) and the sub-Saharan L2a1, which does appear in a higher frequency in Portugal than in the rest of Europe (Pereira et al. 2004
). In the JT (super-)haplogroup, both subhaplogroups of T (T1 and T2) were observed, whereas only J1c was detected within J. J1c was recently reported to be associated with the LHON 11778/ND4 mutation (Carelli et al. 2006), and considerations on the possible interaction between the 2 replacement substitutions in the cytochrome b gene and the 11778 mutation are explored therein. In haplogroup H, rare subhaplogroups in Iberia (Pereira, Richards, et al. 2005) were observed and a lower frequency of the rather common H3 subhaplogroup, in both asthenozoospermic and teratoasthenozoospermic males. The haplogroup distributions in the 2 geographically matched groups were not different (Fisher's exact test P = 0.170).
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A total of 287 substitution events in the coding region (positions 577–16023) were observed in the 43 individuals sequenced. A total of 17 positions showed homoplasy (table 1) and, of these, 4 of the 7 replacement substitutions involved the threonine codon, which has been shown to be implicated in 5 out of 6 replacement substitutions with recurrence over 4 times in a worldwide mtDNA phylogeny (Kivisild et al. 2006
). All those recurrent mutations were shared between asthenozoospermic and teratoasthenozoospermic males and were already described in the literature. The haplogroup T and R1 defining substitution 4917 (replacement in ND2) was present in sample AT19; whereas the substitution at position 15928 (in tRNAThr), shared between T samples and the asthenozoospermic AT03 from K1c1, was also observed in one H3 (Herrnstadt et al. 2002) and one R8 (Palanichamy et al. 2004) sample.
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The sample TA19 does not have the defining control polymorphism at position 16223, making this lineage a peculiar one inside M1. Other M1 samples matching TA19 in the first hypervariable segment have been observed: 1 in Portugal (Pereira et al. 2004
), 2 in Azores (Brehm et al. 2003) and 1 in Spain (Crespillo et al. 2000). The sample AT11 displays the haplogroup-defining polymorphisms of K1b1a (Behar et al. 2006), except for the substitution at position 13967. More data will be needed to ascertain if this substitution is really basal to K1b1a or only specific to a subgroup within it (so far, it is present in 3 out of 4 samples). A minor proportion of the substitutions observed (20 out of 287, counting recurrences; table 1 in Supplementary Material online) have, so far, not been described elsewhere (in a database consisting of around 1,500 published complete or coding-region sequences). Each of these substitutions occurs only once in this data set; that is, no peculiar new substitution was common to more than 1 asthenozoospermic male. All of the 8 new replacement substitutions, except 2, involved threonine and valine codons, a bias observed before (Kivisild et al. 2006).
The spectrum of transversions also followed the usual bias toward higher levels to A and very low ones to G, a pattern that has been used to identify problematic data sets (Bandelt et al. 2006). We observed the following transversion spectrum: 6 to A, 3 to C, 2 to T, and 1 to G.
When we compared the ratio of synonymous to replacement substitutions (table 2) in the haplogroup-defining with nonhaplogroup-defining branches in the tree, no statistically significant differences were observed whether considering all the samples together (one-tailed Fisher's exact test P = 0.185), only the asthenozoospermic (P = 0.228), or only the teratoasthenozoospermic (P = 0.284). Our classification of sequences was rather detailed, so that even quite recent branches were classified as haplogroup defining in this study. Due to that finer classification, the results obtained here show a tendency to a more even distribution of the synonymous and replacement substitutions between the haplogroup-defining and nonhaplogroup-defining ones than other studies (e.g., Elson et al. 2006). But it is clear that the younger branches of the tree bear more replacement substitutions than the internal ones (Kivisild et al. 2006), both in asthenozoospermic and teratoasthenozoospermic individuals.
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Because Ruiz-Pesini et al. (2000)
obtained lower levels of complex I and IV activities (although not significant), we determined the ratios of synonymous and replacement substitutions per complex in the asthenozoospermic males versus teratoasthenozoospermic (table 3). No significantly divergent values were observed for each complex individually.
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Most of the mtDNA substitutions for which a pathogenic role has been convincingly demonstrated are located in tRNAs (McFarland et al. 2004
), presumably because mutations there usually have more serious consequences, affecting mitochondrial protein translation. All the tRNA substitutions observed here (table 2 in, Supplementary Material online) are not private and most are haplogroup defining. As reported before (Kivisild et al. 2006), tRNAThr bears significantly more substitutions than the other tRNAs. Compared with 31 mammalian species (Helm et al. 2000), 6 of the tRNA substitutions occurred in naturally variable sites (3 in sites where the human base is different from the mammalian consensus), 5 in sites with conservation between 50% and 90%, 2 in sites with conservation between 90% and 100%, and 5 in highly conserved (100%) ones. Interestingly, in this last class, the 4435 mutation in tRNAMet observed in sample TA07 is listed in Mitomap as a tRNA mutation having a provisional role as a LHON modulator. A search of the human worldwide database showed that besides our J1c sample, it appears in 1 D1 and in 1 J2a sample (Herrnstadt et al. 2002). The 12308 substitution in tRNALeu(CUN), although haplogroup defining of U, is still displayed in Mitomap as having an unclear role in CPEO/Stroke/CM. None of the tRNA substitutions with confirmed status as pathogenic were observed in our data set. The hypothesis of a uniform molecular clock is rejected (P = 0.013) when analyzing all of our data set, showing that some branches of the phylogenetic tree do seem to bear more or less substitutions than expected. However, for the individual analyses of asthenozoospermic and teratoasthenozoospermic individuals no departure from the molecular clock could be detected (P = 0.086 and P = 0.227, respectively). Possibly contributing to the rejection of the clock hypothesis when both sets are considered together is the fact that the asthenozoospermic group has slightly more diversity than the teratoasthenozoospermic one. Nucleotide diversities were 0.00137 ± 0.00072 and 0.00100 ± 0.00053 in asthenozoospermic and in teratoasthenozoospermic samples, respectively, for the coding region, and 0.00167 ± 0.00087 and 0.00128 ± 0.00067, respectively, for the complete sequence. Nonetheless, the estimated percentage of variation between the asthenozoospermic and teratoasthenozoospermic populations is not significantly different from zero in an AMOVA (P value = 0.10 for coding and 0.20 for complete sequence).
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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To date, the association between mtDNA haplogroup T and asthenozoospermia remains one of the few that has not been tested in another population. This is probably due to the work of Ruiz-Pesini et al. (2000)
being based on a sizable number of samples (by the standards of most work) and to the supporting evidence that asthenozoospermic males bearing haplogroup T did show a statistically significant lower sperm motility (and haplogroup H, the opposite), the same trend observed for complex I and IV activities (although in this latter case, the values were not significant). Very recently, the same group (Montiel-Soza et al. 2006) used a different strategy to assess mtDNA involvement in sperm motility, by measuring this parameter in males belonging to different lineages inside one haplogroup. The curious thing was that the chosen haplogroup was not T but U. Haplogroup U, according to this group, bears mtDNA substitutions that are adaptations to cold climate, and hence, via mitochondrial oxidative phosphorylation (OXPHOS) partial uncoupling, potentially affecting the sperm motility of most of U lineages. They listed the differences in sperm motility and vitality between U lineages in the following decreasing order: U5b, U5a, and U with 1811 substitutions (and inside this, Uk, U1811rest, and U4). In addition, haplogroup H levels were lower than U5b, and U4 lower than T. Astonishingly, they showed that U5b was the only lineage attaining a percentage of 50.1 ± 25.0 progressive spermatozoa, implying that all the males belonging to the other U lineages (especially those with the 1811 substitution, common to all lineages except U5) are asthenozoospermic by definition. Note, however, that the standard errors were very large. If some lineages from other haplogroups also share this low sperm motility, such as haplogroup T, as the first study demonstrated, the population frequency of asthenozoospermic males (sensu stricto) would turn out extremely high. No indication about fertility is given in that work for the males reported with low sperm motility.
In our study, the samples from the infertile males with less than 50% progressive spermatozoa (the asthenozoospermic ones) belonged to diverse lineages of several haplogroups: L2a1, I1a, X2b, both T1 and T2, J1c, U5a, K1 (K1*, K1a4, K1b1a, and K1c1), V1a, H*, and H1. Note that the first example of a sub-Saharan L2a lineage in asthenozoospermic males can hardly be reconciled with the driving selective force of cold adaptation advanced in (Montiel-Soza et al. 2006). No peculiar mtDNA mutation was shared between the asthenozoospermic males and was also absent in teratoasthenozoospermic males. The pattern of replacement substitutions, as well as those in tRNAs and their location in the genome, is indistinguishable from the pattern typically observed in population surveys at a European (Elson et al. 2004) or a worldwide level (Kivisild et al. 2006), as well as in the controls studied here. No systematic differences were observed between haplotypes from asthenozoospermic samples and those from population surveys. In summary, the first 20 complete mtDNA sequences in asthenozoospermic males did not allow the "isolation of well-defined genetic backgrounds that are positively or negatively associated with asthenozoospermia," as hoped by Ruiz-Pesini et al. (2000).
So, where do we stand at this moment on the role of mtDNA in sperm motility? Clearly, no firm association with any mtDNA haplogroup remains after the reanalysis of Samuels et al. (2006). These authors also present data that do undermine the design of any haplogroup association study for asthenozoospermia. The not so frequent pure asthenozoospermia phenotype in conjugation with the restrictions of population structure for mtDNA make it well-nigh impossible to attain the numbers of patients needed for a powerful association study.
The lack of power to detect a positive association between a mtDNA haplogroup and asthenozoospermia is not necessarily incompatible with detecting a role of mtDNA in asthenozoospermia. A recurrent mutation, present exclusively in asthenozoospermic males, could be responsible for this phenotype, as happens for the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokeline episodes syndrome) substitution 3243 (Torroni et al. 2003). The first complete mtDNA sequences in 20 patients reported here show, however, that this is not the case.
An even greater role of mtDNA in sperm motility could still be hypothesized, as the data from Montiel-Sosa et al. (2006) seems to suggest. Instead of 1 haplogroup being implicated in sperm motility, several lineages could display lower sperm motilities. How could this be explained? It is known that younger branches of the mtDNA phylogenetic tree bear more replacement substitutions, potentially mildly deleterious, which would ultimately likely be removed from the population (Ruiz-Pesini et al. 2004; Kivisild et al. 2006). This seems to be a pattern shared by all mtDNA haplogroups (Kivisild et al. 2006) and can be so strong that differential rates between lineages inside haplogroup L2 have been reported (Torroni et al. 2001; Howell et al. 2004). These mildly deleterious substitutions potentially uncouple slightly the OXPHOS activity (Ruiz-Pesini et al. 2004), being of greater limitation to cells with a lower mtDNA content, such as spermatozoa. Our data show that the asthenozoospermic pool presented slightly more substitutions than the teratoasthenozoospermic one, but statistical significance was not reached and the balance between synonymous and replacement substitutions maintained. One way of ascertaining the importance of this higher proportion of substitutions would be to do an in vitro time series evaluation of sperm motility in sperm presenting defects in the mitochondrial gamma-polymerase exonuclease domain. This effect on sperm motility was, unfortunately, not evaluated in knockin mice that express a proofreading-deficient version of polymerase gamma (Trifunovic et al. 2004), but an effect on male fertility was ascertained with testes considerably smaller from 12 weeks of age onward; reduced sperm content of the epididymis and severe testicular tubular degeneration with complete absence of sperm in 40-week-old mutator mice.
However, in recent years, independent experimental data have been providing evidence against a major role for mtDNA in sperm motility. It has been shown that 2 processes contribute to producing the energy necessary for sperm motility: 1) the OXPHOS performed by mitochondria localized in the sperm midpiece and 2) a local glycolysis in the sperm principal piece. It appears that the first process is far less important than the second (see revision in Luconi et al. 2006). In fact, knockout mice for the sperm-specific glycolytic enzyme (glyceraldehyde 3-phosphatase dehydrogenase S), showing unaffected OXPHOS, produced only 10.4% of the adenosine triphosphate necessary for sperm motility compared with wild mice (Miki et al. 2004). Additionally, mito-mice carrying intermediate levels of the common deletion (
4,696 bp) displayed oligospermia and asthenozoospermia with abnormalities in midpiece and nucleus (Nakada et al. 2006). The authors concluded that the asthenozoospermia in mito-mice was caused by morphological abnormalities rather than mitochondrial respiration deficiency in sperm.
One thing is clear, asthenozoospermia is a complex phenotype and mtDNA is only one (at most) of the factors contributing to it. Factors as divergent as calcium intake, osmotic balance, and protein phosphorylation (Luconi et al. 2006) are major conditions for sperm motility efficiency, and because they act at an ultrastructural level, they are not discernable in current diagnosis. At this stage, a better subcharacterization of the complex category "asthenozoospermic" is vital for any further evaluation on the contribution of mitochondria to sperm motility. Without question, asthenozoospermia must be moved from the group of strong positive mtDNA association to the group of complex traits where associations with mtDNA are not at all clear-cut.
In general, although the demonstration of the lack of power to detect mtDNA associations with complex traits seems to take us backwards in the seemingly promising field of mitochondrial genetics, we are slowly advancing onto much more solid ground. Getting rid of superfluous background noise due to false-positive associations and claims of pathogenicity for neutral polymorphisms is essential for the consolidation of the field. We are now at a stage of collecting and refining mtDNA phylogenies for complex traits, which will lead (if we are optimistic) to the emergence of particular signals in certain branches of the phylogeny. This may test the patience in the case of really complex phenotypes such as Alzheimer's disease (Elson et al. 2006), but it seems already promising in the only clear-cut association, between LHON mutations and haplogroup J background (Carelli et al. 2006). Better diagnosis strategies are demanded for faster and more secure conclusions in the field of male fertility.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary tables 1 and 2 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We wish to thank all the patients who agreed to participate in this study. We thank Hans-Jürgen Bandelt for further checking our complete sequences. L.P. had a grant support from Fundação Calouste Gulbenkian. Instituto de Patologia e Imunologia Molecular da Universidade do Porto is supported by Programa Operacional Ciência, Tecnologia e Inovação (POCTI) and Quadro Comunitario de Apoio III. This work was partially supported by Fundação para a Ciência e a Tecnologia—Programa de Financiamento Plirianual do Centro de Investigação de Genética Molecular Humana and by Research Project POCTI/SAU/97/2001 from Fundação para a Ciência e a Tecnologia.
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Footnotes |
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Connie Mulligan, Associate Editor
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References |
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