1 School of Medicine, Obstetrics & Gynaecology, Queens University, Belfast BT12 6BJ, 2 Epidemiology and Public Health, Queens University Belfast, Belfast BT12 6BA and 3 Regional Fertility Centre, Royal-Jubilee Maternity Service, Belfast BT12 6BJ, UK S.E.M.Lewis and M.OConnell contributed equally to this work.
4 To whom correspondence should be addressed. e-mail: s.e.lewis{at}qub.ac.uk
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Abstract |
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Key words: fertilization/human testicular and ejaculated sperm/mitochondrial DNA/nuclear DNA/pregnancy
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Introduction |
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A useful assay for the detection of DNA damage in ejaculated and testicular sperm is the single-cell gel electrophoresis assay, also known as the alkaline Comet assay (Donnelly et al., 1999). The Comet assay has been used in vitro and in vivo to assess DNA damage and repair induced by various agents in a wide variety of mammalian cells (Singh et al., 1988
; Tice et al., 1990
; Olive et al., 1998
). It has become so useful that its applications range from studies of effects of UV radiation, carcinogens, radiotherapy and toxicology to tumour suppression (Fairbairn et al., 1995
). This is the first technique to allow measurement of nDNA damage in single cells. It does so rapidly (McKelvey-Martin et al., 1993
; Fairbairn et al., 1995
), reproducibly (Hughes et al., 1997
) and with higher sensitivity than alkaline elution or nick translation (Leroy et al., 1996
; Irvine et al., 2000
). One of its unique and powerful features is its ability to characterize the range of responses to DNA damage within a heterogeneous population of cells such as that of sperm by measuring damage within individual cells. The assay is simple, inexpensive and requires few cells for analysis.
The nucleus is not the sperms only source of DNA; the mitochondrion also has its own genome. During recent years, mitochondrial (mt) DNA mutations have been identified in numerous diseases (Wallace et al., 1994). mtDNA changes have also been identified as the origin of ageing (Shigenaga et al., 1994
), and more recently as a cause of male infertility (Cummins et al., 1994
; St John et al., 1997
). mtDNA is also an indicator of the health of the sperm. mtDNA deletions lead to deficient oxidative phosphorylation that, in turn, causes abnormal metabolism and inadequate sperm motility. Despite its importance, mtDNA has not yet been fully characterized in human sperm, nor has any potential relationship with successful assisted reproduction treatment (ART) outcome been determined.
In this study we used a long PCR (LPCR) (Lestienne et al., 1997) to determine the number and size of mtDNA deletions in testicular and ejaculated sperm and a modified alkaline Comet assay (Hughes et al., 1996
; Donnelly et al., 1999
) to assess nDNA. We then investigated the relationship between mtDNA, nDNA, and fertilization and pregnancy rates following ICSI with ejaculated and testicular (TICSI) sperm to assess the value of mtDNA and nDNA damage separately and in combination as prognostic tests for treatment outcomes.
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Materials and methods |
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Semen samples were obtained from male partners of couples attending the Regional Fertility Centre, Royal Maternity Hospital, Belfast for ICSI (n = 77). All men were the partners of women who had failed to conceive after at least 2 years of unprotected intercourse. Twenty men had asthenozoospermia, three had had failed fertilization in a previous cycle of IVF and the remainder had oligoasthenoteratozoospermia.
Samples were obtained on the day of oocyte retrieval following a recommended 4896 h of sexual abstinence and included in this study if there was a sufficient quantity of sperm left over after therapeutic insemination. Couples who were attending for ICSI using donor sperm or oocyte donation were not included. Informed written consent for participation was obtained. The project was approved by the Queens University Belfast Research and Ethics Committee and was in accordance with The Declaration of Helsinki as revised in 1983.
Testicular sperm retrieval and preparation (Steele et al., 2000)
Briefly, the spermatic cord was located and 10 ml of 0.5% bupivacaine injected around it. The epididymis was then located and a 21 G butterfly needle used to aspirate the sperm into BiggersWhittenWhittingham (BWW) (Biggers et al., 1971) media. This was spun at 110 g for 10 min to pellet the sperm. The supernatant was discarded and the pellet resuspended in BWW for analysis. A testicular biopsy was performed by passing a 14 G Trucut biopsy needle (Baxter Healthcare Ltd, Thetford, UK) into the testis after the skin over the site had been anaesthetized with 2 ml of 1% lignocaine. The biopsy was transferred into BWW medium.
Testicular sperm were retrieved from the seminiferous tubules by milking the tubular contents with size 5 jewellers forceps into BWW, under a dissecting microscope. The contents of the seminiferous tubules were then centrifuged at 1000 g for 10 min to remove debris and the sperm pellet resuspended in BWW (300 µl).
ICSI procedure
All ICSI cycles were performed according to routine procedures. Ovulation induction was achieved with Metrodin HP (Serono, Welwyn Garden City, UK) following a long protocol of pituitary desensitization with buserelin acetate (Suprefact; Hoechst, Hounslow, UK or Synarel, Syntex Pharmaceuticals Ltd, Maidenhead, UK). HCG (Pregynl, Organon, UK) was administered when there were at least four follicles of diameter >17 mm, 36 h before oocyte retrieval. Mature, metaphase II oocytes obtained by vaginal ultrasound-guided aspiration were cultured in universal IVF medium, Medicult 10310500 (Biotipp; Cahir, Co., Tipperary, Ireland) at 37°C, 5% CO2 in air. The injection procedure has been described in detail previously (Van Steirteghem et al., 1993). In brief, the injection pipette was filled with a small amount of polyvinylpyrrolidone (PVP) (Sigma, Poole, UK) and a free, motile sperm was aspirated into the pipette, head-first from 5 µl of universal IVF medium. The single sperm was then placed, without any cell debris, in another droplet of PVP and immobilized. The sperm was aspirated again into the injection pipette tail-first and injected into an oocyte. Fertilization was recorded 1216 h after injection if two pronuclei were detected. In each case, two embryos were transferred into the uterine cavity after an additional 24 h. Luteal support was provided by Progynova (8 mg) and Cyclogest by vaginal pessary twice daily (400 mg). An intrauterine pregnancy with fetal heart beat was confirmed by ultrasound at 6 weeks.
Preparation of ejaculated semen using Puresperm
In the ejaculated sperm group, each patient produced a split ejaculate in order to collect the majority of the sperm in the first container without dilution by seminal fluid. The sperm were prepared for oocyte injection using a discontinuous (95.047.5%) PureSpermTM (Hunter Scientific Ltd, Saffron Walden, UK) gradient. Semen (1 ml) was layered on top of the gradient and centrifuged at 500 g for 20 min, followed by resuspension in fresh media (2 ml) and a further concentration step by centrifugation at 250 g for 10 min. The final sperm preparation was suspended in a suitable volume of Earles medium to a concentration of 1 x 105/ml before injection of the oocyte. An aliquot of this sample was reserved for determination of both mtDNA and nDNA integrity.
Determination of mtDNA mutations by LPCR
Sperm DNA isolation. A minimum of 104 sperm were added to a 1.5 ml tube. This was centrifuged at 13 000 g for 3 min to pellet the cells and the supernatant removed, leaving behind 2040 µl residual liquid. The tube was vortexed vigorously to resuspend the cells in the residual supernatant: this facilitates cell lysis. Cell lysis solution (300 µl; Flowgen, Lichfield, UK) was then added to the resuspended cells and pipetted up and down to mix throughly. Dithiothreitol (DTT; 200µl of 1 M; Sigma) was then added, followed by 1.5 µl proteinase K (20 mg/ml; Sigma). This mix was inverted 25 times and incubated at 55°C overnight, until the cells had completely lysed. RNase A solution (1.5 µl; Flowgen) was added to the cell lysate and the sample inverted 25 times to aid mixing before incubation at 37°C for 60 min. The sample was cooled to room temperature (22°C) and 100 µl of protein precipitate solution (Flowgen) added to the RNase A-treated cell lysate. This was then vortexed to mix the sample. The sample was placed on ice for 5 min and then centrifuged at 13 000 g for 7 min. The precipitated proteins formed a tight pellet: the supernatant containing the DNA was poured into a 1.5 ml Eppendorf tube containing 300 µl isopropanol (100%; Sigma). This was centrifuged at 2000 g for 3 min prior to mixing. The supernatant was poured off and the Eppendorf tube drained on clean absorbent paper. Ethanol 300 µl (70%; Sigma), was added to the Eppendorf to wash the DNA pellet before being centrifuged at 2000 g for 3 min. The ethanol was poured off so as not to disturb the DNA. The Eppendorf tube was inverted and allowed to air dry for 15 min. Finally, DNA hydration solution (50 µl; Flowgen) were added to the DNA. This was heated at 65°C for 1 h, to rehydrate.
DNA calibration. Deionized and distilled water (490 µl) was added to a 0.5 ml quartz cuvette (Sigma) and mixed with 10 µl of the hydrated DNA sample. The DNA quantity was calculated at 260 nm using an Ultrospec II, LKB Biochrom spectrophotometer.
8.7 kb LPCR amplification. LPCR products were amplified in a Hybaid TouchDown thermal cycling system (Hybaid Ltd, Ashford, UK). LPCR using Bio-X-Act (Bioline, London, UK) was performed in 50 µl volumes. Each reaction contained 1x Optiform PCR buffer (Bioline), 0.25 mM dNTPs, 500 ng DNA template, 1.5 mM MgCl2, 2 IU of Bio-X-Act polymerize (Bioline) and 0.5 µM of each primer (D6: 5'-TCT AGA GCC CAC TGT AAA G-3', L strand sequence, position 82868304; and R10: 5'-AGT GCA TAC CGC CAA AAG A-3', L strand sequence position 421403; Lestienne et al., 1997). In brief, the steps consisted of 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 technique was employed, i.e. the reaction tubes, with all the components present, were placed in the thermal-cycler (Px2 thermal-cycler; Thermo Hybaid) at the start of the denaturation phase. Half (8.7 kb out of the 16.6 kb) of the mitochondrial genome was amplified from each sperm sample. This region was chosen as it encompasses the most heavily deleted region of the genome. The region amplified incorporates the following genes: complex I, the NADH dehydrogenase genes; complex III, cytochrome b; complex IV, the cytochrome c oxidase gene and CO III; and complex V, with the ATP synthase genes and up to nine of the tRNA genes (Lestienne et al., 1997
).
In each reaction, one negative control and one positive blood control sample were run to determine whether mis-priming of the multi-enzyme system had taken place. LPCR was repeated in duplicate samples to ensure reproducibility of the proportions of deleted mtDNA molecules and identical mutations were found. The reaction products were electrophoresed on a 0.8% agarose (Sigma) and 10x Tris-acetateEDTA buffer (Gibco-BRL Life Technologies, Paisley, UK) gel containing ethidium bromide (1 µg/ml) at 120 V for 60 min. Electrophoresed products were photographed using Kodak gel documentation system 120 (Scientific Imaging Systems, New Haven, CT, USA).
The number of mitochondrial deletions detected was calculated as the total number of bands detected from LPCR amplification for each patient. The size of each deletion was determined by noting its position on the gel and reading its size from the ladder marker in the adjacent lane.
The mean size of the deletions was calculated using the formula: sum of all deletion sizes divided by deletion number for each patient.
Determination of DNA integrity by modified alkaline single-cell gel electrophoresis Comet assay
The following procedure (adapted from Hughes et al., 1997 and Donnelly et al., 1999
) was performed under yellow light to prevent induced DNA damage.
Embedding of sperm in agarose gel. Fully frosted microscope slides (Richardson Supply Co. Ltd, London, UK) were gently heated, covered with 100 µl of 0.5% normal melting point agarose in Ca2+- and Mg2+-free phosphate buffered saline (Sigma) at <45°C and immediately covered with a large (22 x 50 mm) coverslip. The slides were placed in a chilled metal tray and left at 4°C for at least 30 min to allow the agarose to solidify. The coverslips were then removed and a minimum of 104 sperm in 10 µl BWW were mixed with 75 µl of 0.5% low melting point agarose at 37°C. This cell suspension was rapidly pipetted on top of the first agarose layer, covered with a coverslip and allowed to solidify at room temperature.
Lysing of cells and decondensation of DNA. The coverslips were removed and the slides immersed in a Coplin jar containing freshly prepared cold lysing solution [2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10, with 1% Triton X-100 (Sigma) added just before use] for 1 h at 4°C. Subsequently, slides were incubated for 30 min at 4°C with 10 mM DTT followed by a 90 min incubation at 20°C with 4 mM lithium diiodosalicyclate (LIS; Sigma) (Robbins et al., 1993).
Unwinding of DNA. Slides were removed from the lysis solution, DTT + LIS, and carefully drained of any remaining liquid. A horizontal gel electrophoresis tank was filled with fresh alkaline electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH 13.0; Sigma) at 1215°C. The slides were placed into this tank side by side, with the agarose end facing the anode, and covered with electrophoresis buffer to a level of 0.25 cm above the slide surface. The slides were left in this high pH buffer for 20 min to allow DNA in the cells to unwind.
Separation of DNA fragments by electrophoresis. Electrophoresis was conducted for 10 min at 25 V (0.714 V/cm) adjusted to 300 mA by raising or lowering the buffer level in the tank. After electrophoresis the slides were drained, placed on a tray and flooded with three changes of neutralization buffer (0.4 M Tris, pH 7.5; Sigma) for 5 min each. This removed any remaining alkali and detergents that would interfere with ethidium bromide staining. The slides were then drained and stained with 50 µl of 20 µg/ml ethidium bromide (Sigma) and covered with a large coverslip. The slides were viewed using a Nikon E600 epifluorescence microscope that was equipped with an excitation filter of 515560 nm from a 100 W mercury lamp and a barrier filter of 590 nm. Fifty images were captured and analysed by an image analysis system using the programme Komet 3.1 (Kinetic Imaging Ltd, Liverpool, UK).
Statistical analysis
Mitochondrial and nDNA results were analysed using SPSS version 11.0 (www.SPSS.com). Students t-test for independent samples was employed to determine the differences between testicular or ejaculated samples for successful and unsuccessful cycles. Logistic regression was used to determine the relationships between nDNA and mtDNA.
Initially, two separate databases were set up. One looked at the effects of using ejaculated sperm on assisted conception outcome. The second looked at the effects of using testicular sperm on assisted conception outcome. It was questionable whether the two databases could be combined. This was tested by adding an explanatory dichotomous variable to identify the source of the sample. Sperm source was not a significant factor in this model, indicating that source is only important insofar as better quality sperm (see independent samples t-tests presented in Table I) tends to be obtained from testicular sperm, and consequently the combination of sources increased the range of each of the sperm quality variables. Once these factors were taken into consideration there was no further contribution from the source of sperm. Three sperm quality variables were thought to be related to a positive pregnancy outcome. These were nDNA fragmentation, the number of mtDNA deletions and the size of the deletions (if present).
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The result (see Table II) was a logistic regression model with nDNA fragmentation, mitochondrial deletion number and deletion size as independently significant factors in the odds of a successful outcome.
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Results |
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Relationship between the number and size of sperm mtDNA deletions and fertilization rates
There is no relationship between the number or size of mtDNA deletions in either testicular or ejaculated sperm and fertilization rates in ICSI. The data from each group are combined in Figure 2, showing that neither deletion number (r = 0.05) nor size (r = 0.17) haS a significant relationship with fertilization rates.
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The relationships between mtDNA deletion number, size and ICSI outcome for testicular and ejaculated sperm
There were a significantly higher number of mtDNA deletions in both testicular and ejaculated sperm of men whose partners did not become pregnant (Table I). These deletions were also of a larger scale (Table I). There were also significantly more mtDNA deletions of larger size in ejaculated sperm compared with testicular sperm (Table I).
Comparison of the incidence of mtDNA deletions in testicular sperm in successful and unsuccessful TICSI cycles
Of the patients who became pregnant, only 26% had partners with wild type (WT) sperm mtDNA. Single deletions were observed in 37% of partners sperm, double deletions in 18.5% and multiple (i.e. more than two deletions) in the remaining 18.5% of the group. In contrast, in those patients who did not become pregnant, only 6.5% had partners with WT sperm mtDNA and a much higher proportion (65%) had multiple deletions (Figure 3).
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Discussion |
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Traditionally, pregnancy rates have been associated primarily with the female partner. However, since ICSI facilitates fertilization by sperm that nature would reject, it is important to assess the quality of paternal genetic material and to establish criteria by which to choose appropriate cohorts of sperm. Assisted reproduction is expensive financially and emotionally, highly invasive and the long-term consequences remain unknown; therefore, couples justifiably want to know the likelihood of success before embarking on a treatment cycle. These patients have a right to the fullest information that we can provide. Although this was a retrospective analysis with relatively small numbers, we believe we have a test that would appear to give the couple and their clinician this information. We suggest that both nuclear and mitochondrial DNA quality of the sperm should be assessed before their cohort is used clinically, thereby giving the couple an accurate prediction of the likelihood of pregnancy with that sample.
This is also the first study, to our knowledge, to investigate a close inverse relationship between the testicular sperm pregnancy rates in ICSI, and the level of DNA fragmentation. In agreement, we observed a similar significant negative correlation for ejaculated sperm. This confirms the view that a sperm nDNA fragmentation test is a valuable addition to current prognostic tests, irrespective of the sperm source and the sperms level of maturity. We have shown that testicular sperm head size is substantially larger than that of ejaculated sperm (Steele et al., 2000). By Tygerberg criteria, testicular sperm heads would be classified as large and amorphous and, in addition, they often have a cone shape of cytoplasm around the midpiece. Rather than being abnormal, we believe that this simply indicates a lack of maturity from incomplete chromatin compaction. The protamination that gives sperm DNA such protection is not completed until S-S bonds form between protamine molecules during epididymal transit. Thus, it is interesting to note that DNA fragmentation is also prognostic of ART success in these immature sperm. Our data support those of Sun et al. (1997)
and Ahmadi and Ng (1999)
, who found a negative association between embryo cleavage rates and DNA fragmentation measured by the TUNEL assay. They are also in agreement with data obtained using the sperm chromatin structure assay, where an increased susceptibility to nDNA denaturation reduced the chances of a pregnancy following ART (Evenson et al., 1999
). In first-pregnancy planners, with no knowledge of their fertility capability, time to pregnancy increased as a function of the proportion of sperm with abnormal chromatin (Spano et al., 2000
). Evenson et al. (1999)
have further showed that the miscarriage rate was higher in apparently fertile couples where the partners sperm had poor chromatin quality. Furthermore, assessment of nuclear packaging by ethidium bromide binding (Filatov et al., 1999
) indicated that abnormalities in chromatin packing reduced rates of embryo cleavage, again showing a link between sperm DNA and male fertility potential. For this reason, Sakkas et al. (2000)
have suggested the use of blastocyst culture to avoid the inheritance of an abnormal paternal genome after ICSI. Extended culture will give a test by which to choose only viable embryos, which may well reflect the quality of the gametes from which they were derived (Behr et al., 1999
).
Using sperm with damaged DNA may have far-reaching consequences on the health of offspring. One example of this is that fathers with DNA-damaged sperm due to smoking are more likely to have children with cancer (Sorahan et al., 1997). Animal studies have shown that damaged DNA can lead to increased post-implantation loss and are transmissible to the next generation (Hales et al., 1992
). Other concerns include indications that intracellular signalling is modified with the ICSI procedure (Tesarik et al., 1994
), and that first mitotic divisions may be disturbed (Hewitson et al., 1999
). Furthermore, it has been postulated that ICSI with surgically retrieved sperm could interfere with the genomic imprinting of the sperm genome (Tycko et al., 1997
).
The high level of DNA fragmentation observed in these studies may be due to environmental toxins and/or oxidative damage. As already mentioned, smoking leads to an increase in fragmented sperm DNA (Sun et al., 1997). Increased exposure to environmental estrogen or endocrine disrupters has also been shown to produce developmental, structural, functional and teratogenic changes in the male genital tract (Sharpe, 1998
). Various types of DNA damage have been found to be caused by exogenous and endogenous estrogens (Liehlr, 2001
). Our group has also observed DNA damage in sperm exposed to high levels of dietary phytoestrogens (unpublished observations). Another culprit leading to DNA damage is oxidative stress. Sperm are particularly susceptible to damage from reactive oxygen species (ROS) due to their unsaturated fatty acid membranes and lack of repair mechanisms. Many studies have reported a connection between such stress and DNA damage (Cummins et al., 1994
; Twigg et al., 1998
; Donnelly et al., 1999
). At high levels of oxidative stress, multiple sperm functions are compromised (Aitken et al., 1998
). The number of patients (26%) in this study with WT mtDNA is lower than in some of our previous studies (
45%) (OConnell et al., 2002
). One explanation for this may lie in the differing aetiologies of the obstruction. Obstructive azoospermia may occur for a number of reasons. The most common causes are vasectomy, coincidental ligation or resection of the vas during scrotal or hernia surgery, infection (gonorrhoea, chlamydia or tuberculosis) or congenital bilateral absence of the vas deferens (CBAVD). The degree of compromise of testicular sperm mtDNA in these infertile men may depend on the cause of the blockage. mtDNA may be affected by a range of conditions, from temperature fluctuations to transit times, as well as exposure to hydrolysing enzymes such as hyaluronidase and acrosin from dead and dying sperm acrosomes (Cummins et al., 1994
). Depending on the site of the obstruction, testicular sperm may be damaged by oxidative stresses within the testis caused by stasis or from a build-up of localized pressure resulting from the obstruction (Hess, 1998
). It is also possible that in these patients, this increased pressure delays the release of sperm from the seminiferous tubules, leading to premature ageing of the sperm, the effects of which are displayed as accumulated mtDNA deletions. Another example is in patients whose obstruction is caused by infection with its associated temperature increase. They may experience a greater deterioration in their sperm mtDNA quality than those with CBAVD from birth. Further studies are required to relate mtDNA status to aetiology and, in the case of vasectomy, the length of time since the surgically induced obstruction.
These are the first data to study associations between mtDNA and assisted conception outcome. As with nDNA, we observed no relationship between the number or size of mtDNA mutations of either testicular or ejaculated sperm and fertilization rates inTICSI. In contrast, in those patients who became pregnant following ICSI, their partners had significantly higher levels of non-mutated (WT) sperm mtDNA and a much lower proportion of multiple deletions. This was true of both testicular and ejaculated sperm, although the association was stronger in the former. The data suggest that the quality of mtDNA, as well as nDNA, is a good prognostic factor for successful outcome. The quality of sperm mtDNA is important for two reasons. Sperm metabolism is dependent, at least in part, on energy derived from oxidative phosphorylation in the inner mitochondrial membrane (Wallace et al., 1994). If the mitochondria are dysfunctional, they may be incapable of providing sufficient ATP for optimal motility. In this study we probed the mitochondrial genome in the region located between the D loop and Cox II gene. This region contains the complexes involved in oxidative phosphorylation such as cytochrome c oxidase, ATPase, NADH dehydrogenase, cytochrome b and up to nine tRNAs (Lestienne et al., 1997
). Thus, large or multiple deletions here may indicate major disruption to the electron transport chain (ETC), which would reduce ATP output and impair metabolism
Functional membrane potentials are important to mitochondria and are achieved by the pumping of protons across the inner mitochondrial membrane as a result of oxidative phosphorylation (Donnelly et al., 2000). However, the mitochondrial respiratory system is also the major intracellular source of ROS. These are generated as by-products during the transfer of electrons in the ETC and complexes to molecular oxygen within the inner mitochondrial membrane. This increase in ROS may well result in an increase in mtDNA deletions. The most immediate effect of oxidative assault will be observed in the mitochondria, since the matrix of the mitochondrion is in closest proximity with ROS. Sperm mtDNA and nDNA are particularly vulnerable to damage induced by endogenous ROS due to the cells absence of significant repair mechanisms and high content of membrane polyunsaturated fatty acids. This exposure to ROS can progress to plasma membrane damage through an accumulation of lipid peroxides (Selley et al., 1991
). The production of excessive ROS, due to either increased generation or reduced antioxidant protection (Lewis et al., 1995
), is now thought to underlie many aspects of human male infertility, where sperm are rendered dysfunctional by lipid peroxidation and altered membrane function, together with impaired metabolism and motility (Cummins et al., 1994
).
The proportion of patients with WT sperm mtDNA in ejaculated sperm was extremely low in both those who did (8%) and those who did not (2%) achieve a pregnancy. The majority of partners sperm had multiple deletions. It is surprising to see such a high incidence of deletionsmuch higher than those commonly observed in somatic cells (Lestienne, 1992). It may be that in the evolution of the species, if paternal mtDNA is to be expelled from the embryo at an early stage (Sutovsky et al., 1999
), some of the mechanisms that are present in the oocyte to protect mtDNA have become obsolete during sperm differentiation (Reynier et al., 1998
), allowing mtDNA mutations to accumulate in sperm. However, the importance of non-mutated mtDNA in sperm cannot be disregarded. Recent findings by St John et al. (2000)
and St John and DeJonge (2000)
contrast with Sutovskys study (Sutovsky et al., 1999
), and provide compelling evidence for the persistence of paternal mtDNA well beyond the 8-cell stage and into the blastocyst stage of normal and abnormal embryos created by IVF and ICSI techniques. While it is reassuring to note from the studies of Torroni et al. (1998)
and Danan et al. (1999)
that the ICSI procedure did not alter the uniparental pattern of mtDNA inheritance, these studies were performed on children conceived using mature, i.e. ejaculated, sperm. As yet there are no studies to confirm that mechanisms for paternal mtDNA expulsion are not compromised when immature sperm are used, and this is another reason why characterization of mtDNA used for clinical purposes is important.
To our knowledge, we are the first group to demonstrate a relationship between nDNA fragmentation and the number of mtDNA deletions in both testicular and ejaculated sperm. In somatic cells there is a bi-directional flow of information between nucleus and mitochondrion. However, in somatic cells there are no physical barriers between the cytoplasm and the nucleus and the genomes interact in the synthesis and assembly of mitochondrial proteins, particularly for oxidative phosphorylation (Poyton and McEwen, 1996). In contrast, in post-spermiogenetic sperm, the nuclear genome is largely inactive and the nuclear and mitochondrial compartments are physically isolated by the cytoskeleton, making exchange of information much more difficult. Such cross-talk has been shown to be unimportant for metabolic pathways in the head, midpiece and flagellar compartments. The relationship between mtDNA deletions and nDNA fragmentation in sperm suggests that some interaction may still exist between the genomes, in that malfunction of one is observed in the other. Alternatively, the assault on both mtDNA and nDNA may be so great that both respond similarly to their environment despite differences in how each genome is protected, because in each the defence is inadequate. The most probable mechanism to explain this correlation is that DNA damage in sperm is caused, to a great extent, by ROS, gonadotoxicants and/or xenobiotics.
In contrast to the relationship with pregnancy rates, we found no relationship between DNA fragmentation and fertilization rates in ICSI using ejaculated or testicular sperm. These results are in agreement with others (Twigg et al., 1998; Host et al., 2000
) who showed that damaged DNA did not prevent fertilization or pronucleus formation (Twigg et al., 1998
) after ICSI, suggesting that the functional competence of the sperm is not essential for these processes. This may be expected, since the nature of the ICSI technique facilitates fertilization without the processes of capacitation, acrosome reaction or penetration of the zona pellucida.
It has also been suggested that the paternal genome does not exert its influence until around the 8-cell stage of the embryo (Braude et al., 1988), although a recent study has shown a paternal influence in the first cell cycle after ICSI (Tesarik et al., 2002
). In addition, the maintenance, and indeed survival, of somatic cells depends on their DNA integrity, although sperm appear to function without significant nuclear control. Even in IVF, where sperm competence is a prerequisite, there is conflicting evidence about the relationship between sperm nDNA fragmentation and fertilization rates. The presence of damaged DNA (regardless of the degree of damage) was reported to have no effect on fertilization in one study (Ahmadi and Ng, 1999
), but was shown to have a significant inverse relationship in others (Sun et al., 1997
; Host et al., 2000
) and to contribute to a failure of fertilization even in ISCI in a fourth study (Lopes et al., 1998
).
In conclusion, this study suggests that combining data about DNA quality from both sources within sperm provides a potentially valuable prognostic test. The information has been incorporated into a simple scoring system for clinicians to use to counsel patients more appropriately. For the first time, by using this algorithm, the clinician may be able to give couples a more quantitative estimate of their chances of a pregnancy from their treatment. A large prospective study is now ongoing to test the validity of the algorithm.
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Acknowledgements |
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Appendix |
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Determination of score
1. Nuclear component
Correct the nuclear DNA fragmentation to the nearest percent and subtract from 90 = (A)
2. Mitochondrial component
(a) Deletion number
(i) If the mitochondrial deletion number is 0, (A) is your final value
(ii) If the deletion number is not 0, remove 8 for each deletion = (B)
(b) Deletion size
(i) If mean deletion size is less than 4, subtract the value from 4 and multiply by 7 = (C)
(ii) If mean deletion size is greater than 4, subtract 4 from the value and multiply by 7 = (C)
3. The final score is (A) + (B) + (C) = (D)
If the score is <20, the couple has a low chance of pregnancy and should be counselled accordingly. As the score increases, the couples chance of a pregnancy increases proportionately. If the score is >80, there is a very high probability that a pregnancy will ensue.
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References |
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Submitted on December 12, 2003; accepted on February 13, 2004.