Molecular Human Reproduction, Vol. 7, No. 1, 103-111,
January 2001
© 2001 European Society of Human Reproduction and Embryology
Reproductive genetics |
Men with oligoasthenoteratozoospermia harbour higher numbers of multiple mitochondrial DNA deletions in their spermatozoa, but individual deletions are not indicative of overall aetiology
1 Assisted Conception Unit, Birmingham Women's Hospital, Birmingham B15 2TG, 2 Reproductive Biology & Genetics Research Group, Department of Medicine, University of Birmingham, Birmingham B15 2TH and 3 Department of Obstetrics and Gynaecology, University of Sheffield, Sheffield, S3 7RE, UK
Abstract
It is believed that one cause of sperm dysfunction might arise through multiple mitochondrial DNA deletions (
mtDNA) resulting in the formation of an incomplete electron transport chain. This study investigates the incidence of multiple mtDNA in human spermatozoa prepared on Percoll gradients. Firstly, we investigated for the presence of two frequently analysed mtDNA, the 4977 and 7.4 kb deletions, using conventional polymerase chain reaction (PCR). These two deletions are characteristically flanked by direct repeats. We further analysed the incidence of one other deletion, the 15 bp deletion in the cytochrome c oxidase subunit III (COX III) of complex IV to determine whether other deletions flanked by direct repeats could be equally predictive. The incidence of these three deletions was not clearly associated with the diagnostic categorization of male infertility. However, the use of long PCR showed that samples harbouring high numbers of mtDNA were associated with the diagnostic categorization of male infertility. We propose that these deletions could arise through a free radical-driven event occurring at the spermatogonial cell stage resulting in the replication of mtDNA molecules at the expense of wild-type molecules. These anomalies in ejaculated sperm mtDNA could account for reproductive failure in some men.
electron transfer chain/long PCR/mitochondrial DNA deletions/reactive oxygen species/spermatozoa
Introduction
Mitochondria are the cell's major source of oxidative energy through their production of ATP. ATP is produced by the transfer of electrons along the electron transfer chain (ETC) and the subsequent reduction of oxygen to water. Mitochondria possess several copies of their own genome, each encoding for 13 of the proteins required for the ETC. Each mitochondrion or cell can be either homoplasmic (all wild-type or all mutated/deleted), or heteroplasmic (a combination of both wild-type and mutated/deleted).
Deletions in mtDNA (mtDNA) are `common' to many post-mitotic cell types. In particular, the 4977 bp deletion found in a number of tissues, e.g. brain, liver, heart and testis (Yen et al., 1991
; Corral-Debrinski et al., 1992; Soong et al., 1992; Katsumata et al., 1994; Lee et al., 1994), has been described as the `common' deletion. This `common' deletion, like the 7.4 kb deletion (another frequently analysed deletion) is flanked by direct repeats. It has been proposed that this characteristic of flanking direct repeats could result in polymerase misreading, large scale deletion of the mitochondrial genome and the loss of several vital genes from the ETC (Ozawa, 1995; Zhang et al., 1995). In many instances, these frequently analysed mtDNA have been analysed individually through conventional polymerase chain reaction (PCR) to assess the overall level of multiple deletions present in various tissues (Hayakawa et al., 1991, 1992; Corral-Debrinski et al., 1992).
To date, several studies have investigated the relationship between multiple mtDNA and sperm dysfunction. Two studies analysing the 4977 bp `common' deletion appeared to produce conflicting data: in one of these studies, a negative correlation was observed between the level of the 4977 bp deletion and the degree of motility in separated sperm populations (Kao et al., 1995). The other study analysed semen samples and found no correlation with patient pathology (Cummins et al., 1998).
Long PCR is a modification of conventional PCR and can amplify all or part of the whole mitochondrial genome. This allows detection of both wild-type and multiple somatic mtDNA from one sample within one reaction. In one study, a male patient with Kearns–Sayre syndrome (a debilitating mtDNA neuropathy), presented with oligozoospermia and long PCR analysis confirmed the presence of multiple deletions in his spermatozoa (Lestienne et al., 1997). Two further studies have demonstrated that the number of multiple somatic mtDNA in men with oligoasthenozoospermia was higher than those of the fertile males (Kao et al., 1998; Reynier et al., 1998). However, none of the above mentioned studies has specifically extracted leukocytes and, consequently, because leukocytes are fast replicating cells that favour replication of wild-type molecules at the expense of deleted molecules (see Li et al., 1995), a mixed population of spermatozoa and leukocytes were analysed. Indeed, 90% of infertile men have leukocytes in their ejaculate (mean 133x103/ml; Tomlinson et al., 1993). Therefore, in the interests of accuracy in any analysis of sperm mtDNA, it is vital that leukocytes are removed.
This study investigates the impact that multiple mtDNA have on sperm quality. First, we analysed the presence of the 4977 and 7.4 kb deletions in both the high and low density Percoll fractions of spermatozoa from different patients, in order to determine whether these frequently analysed deletions are more prevalent in poor quality than in better quality spermatozoa. Second, we also analysed for the presence of the 15 bp deletion in the cytochrome c oxidase subunit III (COX III) of complex IV to ascertain whether other deletions, also flanked by direct repeats, could be equally associated with poor sperm quality. Finally, in order to determine whether multiple mtDNA were indicative of poor sperm quality we employed long PCR. For each of the studies, Percoll-separated leukocyte-extracted sperm samples were studied.
Materials and methods
Preparation of spermatozoa from semen samples
Semen samples were kindly provided by men of proven fertility and patients attending the Assisted Conception Unit, Birmingham Women's Hospital, Birmingham, and the Infertility Clinic at the Jessop Hospital for Women, Leavygreave Road, Sheffield, UK. Both centres are licensed by the Human Fertilisation and Embryology Authority (HFEA) (0119-Birmingham; 060-Sheffield). Recruitment of both patients and fertile men was in accordance with the HFEA Code of Practice. Patients in the study were those diagnosed with idiopathic infertility following physical examination and history taking. All men were aged <40 years.
The samples were produced by masturbation and were categorized according to the World Health Organization (WHO) guidelines (WHO, 1992). In the majority of cases, sperm concentration and motility were determined using a Hamilton–Thorn Motility (HTM) Analyser (Barratt et al., 1993). Motility I is classified as >25 µm/s and motility II as 
25 and >5 µm/s. In cases where it was not appropriate to use the HTM (e.g. samples <5x106/ml), concentration and motility analysis were performed using a standard manual protocol (WHO, 1992) where sperm motility grades I and II were equivalent to the HTM analysis (as above). Sperm morphology was assessed on Papanicolaou-stained smears at x1000 magnification using bright field illumination on an Olympus BX40 microscope. In our laboratory, samples with <15% normal morphology are assessed as abnormal (see Barratt et al., 1995) and classed as teratozoospermic.
In order to remove much of the debris from the ejaculate and separate spermatozoa according to motility, each sample underwent separation on Percoll (Pharmacia, UK) density centrifugation gradients of 45 and 90% as previously described (Kessopoulou et al., 1992). Each fraction was then washed in 3 ml Earle's balanced salt solution (EBSS; Gibco BRL, UK) by centrifuging at 500 g for 10 min.
Extraction of contaminating leukocytes
Following Percoll separation, the sperm fractions were purified from leukocyte contamination by using antibody (CD45) coated magnetic beads according to Kessopoulou et al. (1992). The removal of contaminating leukocytes was confirmed by viewing a 10 µl aliquot of the sample under phase contrast illumination at a final magnification of x200 on an Olympus BX40 microscope.
Extraction of total DNA
Following leukocyte isolation, the sperm samples were then pelleted at 5000 g (9000 rpm in a Sanyo MSE Microcentaur centrifuge using 1.5 ml Eppendorf tubes) for 1 min, the supernatant was then removed and the spermatozoa resuspended in any residual supernatant. The DNA was then extracted according to the sperm DNA isolation protocol using the Puregene DNA Isolation Kit, (Flowgen, UK) supplemented with 1.5 µl of 20 mg/ml Proteinase K (Sigma, UK) and 12 µl of 1 mol/l dithiothreitol (Sigma) and incubated overnight at 55°C. The resultant DNA was recovered in 50 µl of autoclaved UltraPure water.
PCR amplification
PCR amplification of the 4977 bp, 7.4 kb and 15 bp deletions was conducted individually on DNA extracted from 45 and 90% Percoll-fractionated leukocyte extracted sperm samples (nos. 1 to 27) from fertile males (n = 9), asthenozoospermic males (n = 8), oligoasthenozoospermic males (n = 3) and normozoospermic males (n = 7). Six additional sperm samples (nos. 28 to 33) from men with oligoasthenozoospermia were also analysed, but for each of these samples the 45 and 90% Percoll fractions were pooled prior to leukocyte extraction. PCR amplification was performed in either a Perkin Elmer GeneAmp 2400 Thermal cycler or an MJ Research DNA Engine PTC200. All primers were those previously cited in the literature and checked for accuracy against the Anderson Sequence (Anderson et al., 1981).
Detection of the 4977 bp deletion
PCR using 2 U BioTaq Polymerase (Bioline, London, UK) was carried out in 50 µl volumes in 1x PCR buffer (Bioline), and 2.5 mmol/l MgCl2 (Bioline). The primer and nucleotide concentrations were 0.5 and 187.5 µmol/l deoxynucleoside triphosphate (dNTP) mix respectively. For detection of mtDNA molecules harbouring the 4977 bp deletion, primers MT1A (8224–8247: 5' AAT TCC CCT AAA ATC TTT GAA AT 3'; see Soong and Arnheim, 1996) and MT3 (13,580–13551: 5' GCG ATG AGA GTA ATA GAT AGG GCT CAG GCG 3'; see Soong and Arnheim, 1996) were employed for 25 cycles of first round PCR. This was followed by 25 cycles of second round PCR using primers MT1A and MT2 (13 501–13 477: 5' AAC CTG TGA GGA AAG GTA TTC CTG C 3'; see Soong and Arnheim, 1996) to produce a fragment of 303 bp. A reaction to detect wild-type mt DNA employed the same reaction conditions as for amplification of the deleted products but used primers MT1C (13 176–13 198: 5' AGG CGC TAT CAC CAC TCT GTT CG 3'; see Soong and Arnheim, 1996) and MT2 to amplify a fragment of 324 bp after 25 cycles. A semi-`hot start' technique was employed, i.e. the reaction tubes, with all the components present, were placed in the PCR machine at the start of the denaturation phase. Cycling conditions were: Initial denaturation at 92°C for 3 min followed by denaturation of 20 s and a combined annealing and extension phase of 20 s at 60°C. A final extension was at 72°C for 3 min.
Detection of the 7.4 kb deletion
Amplification of wild-type molecules
PCR using 2 U of BioTaq Polymerase (Bioline) was performed in 50 µl volumes in lx PCR buffer (Bioline) and 2 mmol/l MgCl2, (Bioline). The concentration for each primer was 0.5 µmol/l and the nucleotide concentration was 200 µmol/l dNTP mix (Bioline). Primers 12a (1077–1098: 5' TAG ATA CCC CAC TAT GCT TAG C 3') and 12b (1552–1572: 5' TAC CTT GTT ACG ACT TGT CTC 3') (Marin-Garcia et al., 1996), were employed for 25 cycles to amplify a product of 496 bp in length. Although this region of the genome, the 12s rRNA gene, is distant from the 7.4 kb deletion, it is a region less prone to deletion and is representative of wild-type mtDNA. The amplification conditions consisted of initial denaturation at 92°C for 3 min followed by 25 cycles of denaturation at 92°C for 20 s, and a combined annealing and extension phase at 61°C for 35 s. After the required number of cycles, an additional extension step at 72°C for 3 min was performed.
For amplification of deleted molecules
Primers 7.4a (8150–8166: 5' CCG GGG GTA TAC TAC GG 3') and 7.4b (16142–16159: 5' GTA CTA CAG GTG GTC AAG 3') (Marin-Garcia et al., 1996) were employed for 25 cycles of first round PCR. The second round PCR consisted of 25 cycles with primers MT1A and 7.4b to amplify a product of 488 bp in length. Other reaction components and concentrations were as described for wild-type amplification. Cycling conditions for the first round of PCR were initial denaturation at 94°C for 3 min followed by 25 cycles of denaturation at 92°C for 20 s; annealing at 55°C for 30 s and an extension phase at 72°C for 35 s. A final extension step of 72°C for 3 min was performed. First round PCR used the same quantity of total DNA as the wild-type reaction. Second round PCR employed 2 µl from the first round reaction with the following cycling conditions: initial denaturation at 94°C for 3 min followed by 17–25 cycles of denaturation at 94°C for 30 s; annealing at 56°C for 30 s and an extension phase at 72°C for 30 s. The final extension step was run at 72°C for 3 min.
Detection of the 15 bp deletion in the COX III subunit
Each reaction was performed using the same reaction components and concentrations as for the 4977 bp deletion. However, COX IIIa: (9440 to 9463: 5' CCT TCG ATA CGG GAT AAT CCT ATT 3') and COX IIIb (9525 to 9502: 5' CTA GGC TGG AGT GGT AAA AGC CTC 3') were the primers used. The cycling conditions consisted of initial denaturation at 94°C for 3 min, followed by 32 cycles of denaturation at 94°C for 1 min; annealing at 60°C for 30 s; and extension at 72°C for 30 s. A final extension phase of 72°C for 3 min completed the cycling. Primers COX IIIa and COX IIIb produced a fragment of 85 and 70 bp for wild-type and deleted molecules respectively. We used primer shift PCR (Lee et al., 1994) to confirm the presence of the sought after amplicons by using a further primer that is 50bp upstream from COX IIIa, namely COX IIIc (9390 to 9413: 5' ACA CGA GAA AGC ACA TAC CAA GGC 3') in combination with COX IIIb. This then produces amplicons of 135 and 120 bp for wild-type and deleted molecules respectively.
Second round PCR was performed as described for the 4977 bp and 7.4 kb deletions using the same primers as for primer shift PCR (COX IIIc and COX IIIb) for 25 cycles of first round PCR. This was followed by an additional 25 cycles of second round PCR with primers COX IIIa and COX IIIb.
Restriction enzyme digest
For confirmation of the amplification of the 4977 bp and 7.4 kb deleted and wild-type amplicons, wild-type product was digested with RsaI (New England Biolabs, UK) to produce fragments of 174 and 150 bp (4977 bp) and 232, 170 and 94 bp (7.4 kb). Deleted products were digested with DdeI (New England Biolabs) to produce fragments of 217 and 86 bp (4977 bp) and 86, 206 and 196 bp (7.4 kb). Each restriction enzyme digest was performed in 15 µl volumes, consisting of 5–10 U of restriction enzyme, 1x buffer (New England Biolabs) and 12.5 µl of PCR product.
Visualization of conventional PCR and restriction enzyme digest products
Following both conventional PCR and restriction enzyme digest, 15 µl of the products were resolved on 3% (w/v) agarose gels and visualized.
Long PCR amplification
In this experiment, one semen sample from each of 39 men (numbered 34 to 72; Table II) was examined. Long PCR amplification was performed on DNA extracted from spermatozoa pooled from both the 45 and 90% Percoll fractions. The 45 and 90% fractions were pooled for each patient to provide sufficient DNA for long PCR amplification and to provide an overall picture of the number of deletions present in a sperm sample. Leukocytes were extracted from each pooled sample. The diagnostic categorization of these men was as follows: normozoospermia (n = 9); asthenozoospermia (n = 2), asthenoteratozoospermia (n = 5), oligozoospermia (n = 4), oligoteratozoospermia (n = 2) oligoasthenoteratozoospermia (n = 11) and teratozoospermia (n = 6).
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In all, 8.7 kb out of the 16.6 kb of the mitochondrial genome was amplified from whole sperm samples. This region encompasses the most heavily deleted region of the genome and is the site of the 4977 bp and 7.4 kb deletions. The region amplified incorporates the following genes: complex I (the NADH dehydrogenase genes ND6, ND5, ND4, ND4L and ND3); complex III (xytochrome b); complex IV (the cytochrome c oxidase gene, COXIII); and complex V, the ATPase synthase genes ATPase 6 and 8.
Long PCR using Bio-X-Act (Bioline) was performed in 50 µl volumes. Each reaction contained 1x Optiform PCR Buffer (Bioline), 0.25 mmol/l dNTPs, 500 ng DNA template, 1.5 mmol/l MgCl2, 2 IU of Bio-X-Act (Bioline), and 0.5 µmol/l each primer (D6: 5' TCT AGA GCC CAC TGT AAA G 3' and R10: 5' AGT GCA TAC CGC CAA AAG A 3'; see Lestienne et al., 1997). Reaction conditions were: initial denaturation at 94°C for 2 min, followed by 34 cycles of denaturation at 94°C for 10 s, annealing at 52°C for 30 s and extension at 68°C for 10 min. The semi `hot start' technique was employed.
Results
Detection of mtDNA deletions in spermatozoa by PCR
The presence of the mitochondrial 4977 bp deletion would result in the following genes being lost: NADH dehydrogenase (ND ND5, ND4, ND4L, ND3), COXIII and ATPase 6 and 8 (see Figure 1). To test the hypothesis that poor spermatozoa with consequently poor motility would have a higher incidence of the 4977 bp deletion, we analysed the 45 and 90% Percoll fractions from sperm samples of fertile men (n = 9). As a consequence of Percoll density centrifugation, proportionally more motile spermatozoa migrate to the denser 90% fraction while the less motile move into the 45% fraction (Ord et al., 1990). To ensure that a pure population of spermatozoa was analysed, contaminating leukocytes were removed (Li et al., 1995). Interestingly, six of the nine fertile men (67%) possessed the 4977 bp deletion in their 45% fractions whilst two (22%) harboured the deletion in their 90% fractions (Table I). Only one fertile man harboured the deletion in both fractions whilst one other fertile man showed multiple banding following PCR analysis in the 45% fraction (Figure 2a). These fragments are probably indicative of multiple deletions found within the 4977 bp region (see Zhang et al., 1992; Pallotti et al., 1996; Ferlin et al., 1997). Some samples also harboured a single smaller deletion within the 4977 bp region (e.g. Figure 2a, lanes 2 and 10).
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X-174 RF DNA-HincII digest (Pharmacia); N = 100 bp ladder (Pharmacia).A higher incidence of deleted mtDNA might be expected in the 45% fractions of asthenozoospermic and oligoasthenozoospermic patients when compared with the 90% fractions. We detected and determined the levels of the 4977 bp deletion in a pure population of spermatozoa for a range of patients: astheno- (n = 8), oligoastheno- (n = 9) and normozoospermic (n = 7). Due to sample size in six of the oligoasthenozoospermic patients, only pooled 45 and 90% leukocyte-free samples were analysed. Table I
shows that there was not a clear distinction between the incidence of the 4977 bp deletion in these various groups. As in the fertile men, some samples harboured multiple deletions within the 4977 bp region (Figure 2b), though interestingly this was not evident in men with normozoospermia.
The 4977 bp deletion is flanked on either side by direct repeats. To further test the hypothesis that those deletions flanked by direct repeats are indicators of overall mtDNA damage and are correlated with reduced motility, we also investigated the incidence of the 7.4 kb `common' deletion and the 15 bp microdeletion found in the COX III subunit of complex IV in the same range of samples. Amplification of the 7.4 kb deletion showed a similar trend to the 4977 bp deletion (Table I) together with the presence of multiple deletions within the region in many of the samples. However, the incidence of the 15 bp microdeletion was very low, being restricted to two fertile men and one patient (Table I). For the two fertile men, the deletion could be detected in the 45% fraction for one and both the 45 and 90% fractions of the other. The deletion was also detected in both the 45 and 90% fractions of a patient with normozoospermia.
In order to determine whether the overall incidence of the 15 bp microdeletion was indeed low in spermatozoa, we analysed pooled 45 and 90% leukocyte-extracted sperm samples from a further three fertile men and 17 other patients. The deletion was detected in two out of three fertile men (67%), one of the six asthenozoospermic men (17%) and two out of the five (40%) teratozoospermic patients. To account for the very low levels of deletion present, we also performed nested PCR though this provided no further evidence of the deletion in those samples that were previously found to be negative. To confirm that the correct fragments were being amplified, restriction enzyme digestion was performed on the products from the 4977 bp and 7.4 kb wild-type and deleted reactions using RsaI and DdeI respectively. Primer shift PCR confirmed the presence of the 15 bp deletion (data not shown).
Detection of multiple deletions in sperm samples through long PCR
In order to determine whether particular deletions were indicative of overall patient aetiology, we analysed a further 39 sperm samples (samples 34 to 72) for multiple deletions. These samples were obtained from normozoospermic (n = 9), asthenozoospermic (n = 2), asthenoteratozoospermic (n = 5), oligozoospermic (n = 4), oligoteratozoospermic (n = 2) oligoasthenoteratozoospermic (n = 11) and teratozoospermic (n = 6) patients. In each reaction, one blood sample was run as a positive control to determine whether mispriming of the multi-enzyme system had taken place, along with the sperm sample employed to determine the optimal starting template amount and cycle number. Table II shows the number of deletions present for each patient.
As can be seen from Table II, six of the nine (67%) men analysed with normozoospermia, presented with either wild-type molecules and no deletions or one deletion (Figure 3a). A further three normozoospermic men (33%) harboured three, five, or six deletions. Figure 3b is a representative gel of the mtDNA deletions observed in asthenozoospermic and oligoasthenoteratozoospermic patients. Of the two men with asthenozoospermia, one presented with one deletion whilst the other harboured two. The asthenoteratozoospermic, oligozoospermic and teratozoospermic patients possessed a range of deletions. However, the four oligozoospermic patients possessed a greater number of deletions, namely seven, six, four and one each (see Table II). Patients with a combination of oligo- and teratozoospermia (oligoteratozoospermia) on the whole had a higher incidence of deleted molecules compared with the asthenozoospermic and asthenoteratozoospermic men. One of these patients demonstrated no wild-type molecules but harboured multiple deletions (more than seven), whilst the other possessed four deletions and wild-type mtDNA. Interestingly, of the 11 patients with oligoasthenoteratozoospermia, four (36%) had multiple deletions (more than seven) and no wild-type mtDNA whilst two patients had five deletions and two others had four deletions. Additionally, one patient harboured no deletions yet his pathology was the severest of all (see sample 71). All of the spermatozoa from this patient were non-viable (dye exclusion assay; data not shown).
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Discussion
The initial aim of this investigation was to test the hypothesis that sperm samples with lower levels of motility would have a higher incidence of deletions flanked by direct repeats. As a result, the 4977 bp and 7.4 kb `common' deletions and the 15 bp deletion in the COX III subunit of complex IV were analysed. None of the three deletions analysed was closely associated with a particular type of semen characteristic. These results are in broad agreement with those of a previous study (Cummins et al., 1998) which found no direct correlation between the various groups of patients and fertile men for the 4977 bp deletion. Indeed, both this investigation and that of Cummins et al. (1998) contradict an earlier study (Kao et al., 1995) which detected the 4977 bp deletion in semen samples from men of varying ages and fertility and found a negative correlation between sperm motility and the proportion of the 4977 bp deletion.
However, in this study, we have included one important addition to the experimental protocol. Our study used a magnetic bead antibody extraction technique to eliminate leukocytes. Unfortunately, both the previous investigations (Kao et al., 1995; Cummins et al., 1998) may not have performed analysis on a population of pure spermatozoa, as it is possible that their samples were contaminated by other cells, e.g. leukocytes. It is probable that failure to eliminate contaminating cells could have increased the proportion of wild-type mtDNA present to the detriment of deleted molecules, as leukocytes favour the elimination of deleted mtDNA (Li et al., 1995). Indeed, in ejaculates from azoospermic men, the Cummins' study showed the presence of the wild-type region, but not the 4977 bp deletion, indicating that mtDNA from other cell types was actually being amplified.
The use of nested PCR, due to its increased sensitivity, has allowed detection of other fragments within the deleted regions. Several other reports have also detected similar products that are representative of shorter deleted molecules, e.g. in muscle and brain tissue (Ikebe et al., 1990; Ozawa et al., 1990; Simonetti et al., 1992; Zhang et al., 1992; Ferlin et al., 1997). Further evidence for the presence of these fragments is provided by a few of our samples also harbouring the smaller 15 bp microdeletion in the COX III subunit. This deletion has been previously detected in muscle (Keightley et al., 1996) and, in this instance, is non-specific and possibly a random event just like the 4977 bp and 7.4 kb deletions. This supports the hypothesis that, individually, these multiple mtDNA are poor predictors of the overall incidence of deletion (Khrapko et al., 1999; Zhang et al., 1999).
Indeed, in this study, we have identified multiple deletions within a region of the mitochondrial genome that is most susceptible to deletion through the use of long PCR. Furthermore, the multiple mtDNA observed are associated with poor sperm quality. The most significant of these aetiologies is oligoasthenozoospermia. Individually, oligozoospermia, teratozoospermia and asthenozoospermia have varying numbers of multiple mtDNA present (see Table II). However, a combination of all three aetiologies suggests that when multiple mtDNA are present then spermatozoa could be rendered non-viable. Two previous reports using long PCR have identified multiple deletions in oligoasthenozoospermic patients (Kao et al., 1998; Reynier et al., 1998). The former report did, however, identify the presence of two types of 7.4 kb deletions and quantified their presence through conventional PCR and observed that there was a higher incidence of these deletions in those patients with primary infertility and oligoasthenozoospermia. The presence of the 7.4 kb deletion is further confirmed by our study, but our results indicate that it is not specific to any particular patient group. Previous studies undertaken with long PCR are (as is this study), qualitative. In a sample of purified spermatozoa, it would appear that no single deletion is indicative of poor sperm quality. The relevance of the predictive nature of multiple mtDNA is important when one considers the composition of patients attending an infertility clinic due to sperm dysfunction. Of male patients, ~15% attending infertility clinics present with oligoasthenoteratozoospermia (Irvine, 1998), and it is this group of patients that appears to harbour the greatest number of mtDNA.
As random deletions of varying size appear to be far more indicative of sperm quality (Reynier et al., 1998), the extension of the long PCR protocol to quantify such deletions would appear to be a useful approach. However, this approach is fraught with difficulties, as it is clear from our preliminary studies using 
DNA that long PCR enzyme systems preferentially amplify shorter molecules. A simple weighting system would not suffice, as we are so far uncertain of the processivity of the PCR system and its ability to be competitive and to faithfully amplify the full amplicon (see Kajander et al., 1999).
Free radicals have been shown to have a severe affect on sperm function and survival (de Lamirande et al., 1997). Furthermore, a strong link has been drawn between free radical activity and mtDNA in the testis (Cummins et al., 1994). To this extent, free radicals have been proposed to be the major cause of mtDNA damage and to initiate the vicious cycle resulting in cell loss (Ozawa, 1995). However, the presence of deleted molecules may also be explained by intrinsic anomalies that exist at the origin of spermatogenesis, and a `clonal expansion' hypothesis has been proposed (Tengan et al., 1997). This incorporates the concept of recognized breakpoints, characterized by direct flanking repeats, and is based on the observation of a positive correlation existing between age and `common' deletion levels in controls (r = 0.80) and patients (r = 0.69) following analysis on single muscle fibres from patients with inclusion body myositis and late onset mitochondrial myopathy. Indeed, this hypothesis is supported by other authors (Khrapko et al., 1999) who have shown in single cell analysis of human heart that a significant fraction of myocytes have clonally expanded deletions. This clonal hypothesis clearly complements the concept of the founder molecule theory (Marchington et al., 1997) involving a restriction/amplification event which selects for several mtDNAs, or a single mitochondrion and their subsequent accumulation in number in the oocyte. In relation to spermatogenesis, the deletions would be present in the stem cells and those testes with low sperm numbers and high levels of deletions would produce spermatozoa with a high ratio of deleted molecules. The high number of deleted molecules could arise as a result of the spermatogonial (stem) cell nuclear background preferentially favouring the amplification of deleted molecules (see St John et al., 2000 for review). Investigations in somatic cell lines have substantiated this (Dunbar et al., 1995; Holt et al., 1997). Furthermore, it is evident that a selection process does take place at the spermatocyte/elongating spermatid stage with the number of mitochondria being considerably reduced (Hecht and Liem, 1984) due to loss of transcriptional factor activity (Larsson et al., 1996, 1997). However, of those men who presented with normozoospermia and were analysed using long PCR, three harboured a higher number of deletions of three, five and six. In this instance, it is likely that those stem cells with heteroplasmy would, dependent on selection mechanisms, harbour a higher number of deleted molecules than the other normozoospermic patients or proportionally more stem cells would have a higher number of deleted copies. Nevertheless, these men would not harbour as many deleted copies as observed for those diagnosed with oligoasthenoteratozoospermia. These deleted molecules would then be preferentially selected, but sufficient intact molecules would still be generated to account for the patients with normal characteristics.
The relationship between deleted mtDNA and its impact on sperm mitochondrial function requires considerable investigation (St John et al., 2000). Indeed, the role that the electron transfer chain has in generating ATP to propagate sperm motility tends to be species specific (Storey, 1980). However, in those patients with mtDNA disease, there appears to be a clear loss of mitochondrial function in the associated tissue and also in sperm motility and sperm mitochondrial morphology (Folgero et al., 1993). A loss of mtDNA integrity is also associated with oligozoospermia (Lestienne et al, 1997). To this extent, cell models systems are required to determine how these important relationships function (St. John et al., 2000).
In conclusion, this study has demonstrated that no one particular mtDNA deletion was associated with poor quality semen characteristics. However, the use of long PCR clearly indicated higher numbers of mtDNA in patients with OAT. This is a further indication that in these patients who are candidates for intracytoplasmic sperm injection (ICSI), these spermatozoa may harbour genetic defects.
Acknowledgments
This study was supported by a NHS core grant to the Assisted Conception Unit (ACU) at Birmingham Women's Hospital and the Infertility Research Trust (IRT) at the University of Sheffield. The authors also wish to thank Dr Mathew Tomlinson (ACU) and Mrs Anne White (IRT) for assistance in providing samples.
Notes
4 To whom correspondence should be addressed. E-mail: j.stjohn{at}bham.ac.uk 
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Submitted on April 27, 2000; accepted on November 3, 2000.
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