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Centre for Public Health, Institute of Clinical Sciences, Queen’s University Belfast, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland, UKPresent address: Department of Andrology, University of Utah Health Care, 675 Arapeen Dr. #205, SLC, UT 84108, USA
Sperm DNA damage has a negative impact on pregnancy rates following assisted reproduction treatment (ART). The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage. Following IVF, couples with <25% sperm DNA fragmentation had a live-birth rate of 33%; in contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13%. Following ICSI, no significant differences in sperm DNA damage were found between any groups of patients. Sperm DNA damage was also associated with low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men with idiopathic infertility have high sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF.
Sperm DNA damage has a negative impact on assisted reproduction treatment outcome, in particular, on pregnancy rates. The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage and treatment outcome. Following IVF, couples with <25% sperm DNA fragmentation had a live birth rate of 33%. In contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13% following IVF. Following ICSI, there were no significant differences in levels of sperm DNA damage between any groups of patients. Sperm DNA damage was also associated with the very low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men have high level of sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF.
). As a result of population ageing and adverse changes in lifestyle, infertility continues to increase but with only marginal improvement in pregnancy and birth rates after assisted reproduction treatment in the developed world (
). One reason for this is that little has been done to resolve the causes and potential therapies for male infertility at the molecular level. Furthermore, there are currently no routine pharmaceutical therapies for male infertility.
Sperm DNA damage has been identified as a major contributor to male infertility as well as poor outcomes following assisted reproduction treatment, including impaired embryo development, miscarriage and birth defects in the offspring (
Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications.
A position report. Hum. Reprod.2010; 25 (http://dx.doi.org/10.1093/humrep/dep465. Available from: http://humrep.oxfordjournals.org/cgi/content/abstract/25/4/824?etoc): 824-838
). The Comet assay is becoming a popular method to determine sperm DNA damage. It is recognized as the most sensitive of all methods and routinely used to assess genotoxic DNA damage in somatic cells (
). It is the only technique that allows quantification of DNA damage in individual cells, so it is particularly useful in a heterogeneous cell population like spermatozoa. The Comet assay measures both single- and double-strand breaks using an alkaline pH method (
The present study employed the alkaline Comet assay to examine sperm DNA damage in patients undergoing IVF and intracytoplasmic sperm injection (ICSI). The relationship between sperm DNA fragmentation and pregnancy, miscarriage and live-birth rates was examined. The second objective of this study was to assess the incidence of sperm DNA damage in couples and male partners with idiopathic infertility and determine if there was a relationship between their diagnosis and their treatment outcomes. Thirdly, this study determined the repeatability of the alkaline Comet assay by comparing data from three or two replicate slides for the same patient.
Materials and methods
This project was approved by the Office for Research Ethics Committees in Northern Ireland and the Royal Group of Hospitals Trust Clinical Governance Committee (ref. no. 06/NIR01/142, 9 January 2007). The study was conducted at the Regional Fertility Centre, Royal Jubilee Maternity Services, Belfast, Northern Ireland, UK. Sperm samples for research were obtained after written consent given by each couple.
Study population
A total of 203 couples undergoing IVF (age 35.6 ± 0.3 years), and 136 couples undergoing ICSI (age 34.9 ± 0.4 years) were recruited in the study after a minimum of 1 year of unprotected intercourse without pregnancy.
Of the 203 men undergoing IVF, a subset was identified as the unexplained group (n = 147), which consisted of those remaining after the exclusion of men diagnosed with infertility according to criteria from the World Health Organization (2010; low sperm count, reduction in motility and normal morphology) or with a known history of fertility problems including blockages, testicular cancer or abnormal development of reproductive organs. For couples with idiopathic infertility (n = 70), those with women with endometriosis, ovulation dysfunction, hormone imbalance, ovarian cysts or tumours were also excluded.
Semen analysis and sperm preparation
Semen samples were provided by masturbation by the male partner after 2–5 days of recommended abstinence. After liquefaction, routine semen analysis was performed according to guidelines from the
and subsequently semen was prepared using a two-step discontinuous Puresperm gradient (90–45%; Hunter Scientific, UK). Two populations of spermatozoa for each patient were used to measure DNA damage by Comet assay: the whole population (native semen) and those prepared by density-gradient centrifugation, typical of those used for clinical treatment.
Assisted reproduction treatment
All IVF cycles were performed according to routine procedures (
). Briefly, ovulation induction was achieved with recombinant FSH following a long protocol of pituitary desensitization with a gonadotrophin-releasing hormone analogue. Human chorionic gonadotrophin was administered when there were at least four follicles of >17 mm diameter, 36 h before oocyte retrieval. Mature, metaphase-II oocytes were obtained by vaginal ultrasound-guided aspiration and cultured in media (G5 sequential media series; Vitrolife, Goteborg, Sweden) at 37°C with 6% CO2 in air. The ICSI procedure has been described in detail previously (
). In brief, a suspension of washed spermatozoa was placed in polyvinylpyrrolidone (Vitrolife) and a free, motile spermatozoon was immobilized. The spermatozoon was aspirated into the injection pipette tail-first and injected into an oocyte. Fertilization was recorded 12–16 h after injection. In each case, one or two embryos were transferred into the uterine cavity after an additional 24–48 h. Luteal-phase support was provided by vaginally administered progesterone. An intrauterine pregnancy with fetal heart beat was confirmed by ultrasound 5 weeks after embryo transfer.
Alkaline Comet assay
Sperm DNA fragmentation was assessed using single-cell gel electrophoresis (Comet) assay which has been previously optimized for human spermatozoa (
). Briefly, fully frosted slides were covered with 150 μl of 1% normal-melting-point agarose and left to solidify for 15 min at room temperature. Sperm samples were diluted to a final concentration 2 × 106 cells/ml with phosphate-buffered saline and 10 μl cell suspension were mixed with 75 μl of 0.5% low-melting-point agarose and placed on slides. After 15 min, slides were immersed in cold lysis solution (2.5 mol/l NaCl, 10 mmol/l Na2EDTA, 10 mmol/l Tris and 1% Triton X-100) and incubated for 1 h. Following lysis, slides were incubated with 10 mmol/l dithiothreitol for 30 min at 4°C followed by incubation with 4 mmol/l lithium diiodosalicylate for 90 min at room temperature. Slides were incubated in cold electrophoresis buffer (300 mmol/l NaON, 1 mmol/l EDTA, pH 13) for 20 min to allow DNA to unwind. Electrophoresis was carried out at 25 V, 300 mA for 10 min. Slides were flooded with three changes of neutralization buffer (0.4 M Tris) for 5 min. Slides were stained with 20 μg/ml ethidium bromide and visualized using Komet version 5.5 (Andor Technology, Belfast, UK). Fifty comets were analysed for each slide.
Statistical analysis
Since some of the groups contained a small number of patients, residuals were tested with Statistical Package for the Social Sciences for Windows version 15 (SPSS, Chicago, IL, USA) to assure normality of distribution so that t-testing was appropriate. Having confirmed that residuals followed a normal distribution, P-values were calculated using an unpaired two-tailed t-test (GraphPad Prism for Windows version 5.01; GraphPad Software, San Diego, California, USA). Data are presented as mean ± standard error.
The chi-squared test was used to assess whether there was a linear trend between the level of sperm DNA damage and pregnancy or live-birth rates in IVF and ICSI patients as well as men and couples with idiopathic infertility. Pregnancy and live-birth rates were organized in the frequency tables according to the level of sperm DNA damage and chi-squared and P-values were obtained for each analysis.
Repeatability assessment
Analysis of repeatability was performed according to the guidelines of
to determine the repeatability and reproducibility of a standard measurement method. Fifty-two patient samples were prepared in duplicate (28/52) or in triplicate (24/52) (128 measurements in total). Five comets were scored on 10 different areas of each slide (50 comets per slide). Data from 50 comets for each measurement were used to calculate means and standard errors. Residual values were calculated to identify and remove outliers using Dixon’s test. The test statistic r22 was calculated using the equation
(1)
and compared with the critical value for r22 at a significance level of 0.05. The repeatability (r) was defined as
(2)
where is the repeatability variance or average of the within-laboratory variances and calculated as
(3)
The values were calculated separately for duplicate and triplicate measurements based on 75 degrees of freedom.
Results
Demographic characteristics of patients undergoing IVF
Patients receiving IVF were divided in three groups according to the treatment outcome: (i) clinical pregnancy that resulted in miscarriage; (ii) clinical pregnancy that resulted in a live birth; and (iii) a nonpregnant group. Each group was compared with the live-birth group and confidence intervals and P-values were calculated for each comparison.
When the miscarriage group was compared with the live-birth group, the only parameter that differed significantly was the total embryo cumulative score (Table 1). Between nonpregnant couples and the live-birth groups, there were significant differences in female age (35.9 ± 0.3 versus 34.5 ± 0.7 years, P = 0.04), male age (37.5 ± 0.4 versus 35.5 ± 0.9 years, P = 0.02), transferred embryo cumulative score (35.8 ± 1.7 versus 43.0 ± 2.7, P = 0.01) and the number of fertilized oocytes (4.4 ± 0.3 versus 6.9 ± 0.6, P < 0.0001).
Table 1Demographic characteristics of IVF patients.
Demographic characteristics of patients undergoing ICSI
Patients undergoing ICSI were divided into three groups similarly to those undergoing IVF (Table 2) and each group was compared with the live-birth group. No statistically significant difference was detected between the miscarriage and live-birth groups in any parameters studied. However, there was a significant decrease in number of fertilized oocytes in nonpregnant couples compared with the live-birth group (4.9 ± 0.3 versus 6.1 ± 0.6, P = 0.04).
Table 2Demographic characteristics of ICSI patients.
Sperm DNA fragmentation and treatment outcomes following IVF and ICSI
The level of sperm DNA damage was significantly higher for groups undergoing IVF in the nonpregnant group compared with the live-birth group both in terms of native semen and those prepared by density-gradient centrifugation (P < 0.01 and P < 0.05, respectively, Figure 1). In contrast, no significant difference was found in DNA damage between any of the groups undergoing ICSI in both native and prepared spermatozoa (Figure 1).
Figure 1Sperm DNA damage in patients undergoing IVF or intracytoplasmic sperm injection (ICSI) as assessed by the alkaline Comet assay. Each group was compared with the live-birth group and confidence intervals and P-values were calculated for each comparison. Bar = mean ± SE. ∗P < 0.05; ∗∗P < 0.01.
Patients undergoing IVF were divided into three groups depending on the level of sperm DNA damage (0–24%, 25–20% and over 50%) in accordance with previously reported clinical thresholds for achieving successful pregnancy after natural conception, IVF and ICSI, respectively (
). This previous study reported that if men had <25% sperm DNA damage, they were more likely to achieve a pregnancy without assisted reproduction treatment. If the men had 25–50% sperm DNA damage, their chances of success following IVF were high. However, if the men had >50% sperm DNA damage, their best chance of success was with ICSI. The proportions of pregnant couples, couples with miscarriages and couples whose pregnancies resulted in live birth were also calculated for each group (Table 3). The highest proportion of pregnant couples (39.4%) was recorded in the group of patients with <25% sperm DNA damage. More importantly, 11 out of the 13 pregnancies in this group of patients (33.3% of the whole IVF group) resulted in live births. When the sperm DNA fragmentation threshold increased to 25–50%, the proportions of pregnancies and live births declined to 29.6% and 23.9%, respectively. Statistically significant reductions in pregnancy rate (chi-squared = 8.05, P = 0.005) and live-birth rate (chi-squared = 7.20, P = 0.007) were also recorded in patients with >50% sperm DNA damage (16.2 and 13.1%, respectively).
Table 3Pregnancy, miscarriage and live-birth rates for men, categorized by sperm DNA damage in native semen.
Analysis of pregnancy and live-birth rates in patients undergoing ICSI showed that there was no relationship between sperm DNA fragmentation and either pregnancies or live births (Table 3). Further, no relationship was found between sperm DNA damage and gender or the number of singletons compared with twins (data not shown).
Sperm DNA fragmentation in men with normal semen analysis (idiopathic infertility)
To gain further insight into the relationship between different types of infertility and treatment outcomes, this study assessed DNA fragmentation levels in couples undergoing IVF according to diagnosis. Patients were divided into two groups depending on infertility diagnosis: (i) idiopathic men (couples with a diagnosed female factor but no detectable problems in the male partners); and (ii) idiopathic couples (no detectable male or female factors). Sperm DNA fragmentation was assessed in each group and related to pregnancy, miscarriage and live-birth rates. Sperm DNA damage in the idiopathic men group (n = 147) was 46.9 ± 1.8% in native semen and 32.8 ± 1.6% in prepared spermatozoa (Table 4). The overall pregnancy and live-birth rates in this patient group was 21.1% (31 out of 147 patients) and 17.0% (25 out of 147 couples), respectively (Table 5).
Table 4Sperm DNA fragmentation in men and couples with idiopathic infertility.
) 23 (15.6%) patients had <25% DNA damage in native semen. Sixty-four men (43.5%) had 25–50% DNA fragmentation while 60 men (40.8%) had >50% sperm DNA damage. The pregnancy rate for men with <25% sperm DNA fragmentation was 26.1%. All couples in this group had successful pregnancies. While a slight decrease in pregnancy rate was detected for the group of patients with 25–50% sperm DNA fragmentation (25.0%), the live-birth rate dropped markedly to 18.8%. The pregnancy rate in patients with >50% sperm DNA fragmentation was 15.0% while the live-birth rate in this group was 11.7%. This was more than a 2-fold reduction compared with the live-birth rates in the <25% sperm DNA damage group.
Sperm DNA fragmentation in couples diagnosed with idiopathic infertility
The same analysis was performed for 70 couples with idiopathic infertility (no detectable cause of male or female infertility). The DNA damage levels in native and prepared spermatozoa did not differ significantly from that of the group of men with normal semen parameters and were 45.8 ± 2.6 and 32.7 ± 2.4, respectively (Table 4). The same proportion of patients had <25% sperm DNA damage (15.7%) and 25–50% sperm DNA fragmentation (45.7%, Table 5). Slightly lower numbers of couples with idiopathic infertility (38.6%) had >50% sperm DNA damage compared with men with idiopathic infertility whose female partners had detectable causes.
Lower overall pregnancy and live-birth rates were recorded in this group (20.0% and 14.3%, respectively). The pregnancy and live-birth rates in the group with <25% sperm DNA damage were both 27.3% (Table 5). The pregnancy rates for the other two groups of patients decreased to 21.9% and 14.8% for 25–50% sperm DNA damage and >50% sperm DNA damage, respectively. Surprisingly low live-birth rates (12.5% and 11.1%) with a concomitant increase in miscarriage rates were recorded for both groups compared with patients with <25% sperm DNA fragmentation; however, the miscarriage trend was not statistically significant (chi-squared = 1.24). Subdividing patients into subgroups with 50–60%, 60–70%, 70–80% and 80–90% sperm DNA damage showed a similar distribution pattern between the groups (data not shown).
Repeatability of the Comet assay
Repeatability refers to within-laboratory agreement between replicate observations of the same test performed by the same operator under similar conditions. Repeatability of Comet assay was assessed through comparison of duplicate or triplicate results from the same sample. One-way ANOVA was used to calculate variance. Based on this value, the standard errors of precision for single, duplicate and triplicate measurements were measured. To conduct the repeatability assessment of the alkaline Comet assay, DNA damage data was obtained from five random comets scored on 10 different areas on each slide (50 comets per slide) on two or three replicate slides for all 52 samples. Data from 50 comets were used to calculate mean and standard deviation, which provided 128 means and 76 degrees of freedom. After plotting residual values for mean DNA damage, three potential outliers were detected in this dataset and Dixon’s test was used for identification and rejection of the outliers (Figure 2). One of them was classified as an outlier (P = 0.05) after applying Dixon’s test and was excluded from further analysis, thus decreasing the degrees of freedom to 75.
Figure 2Normal Q–Q plot of the sperm DNA damage residual. Residuals were plotted for all means.
The value for a single DNA damage measurement was found to be 3.73% while it decreased to 2.65% and 2.16% for duplicates and triplicates, respectively. According to the standard error of precision, the likely range of the true value for a patient with 60% sperm DNA damage will be narrow, at 55.7–64.3%.
Discussion
Sperm quality is routinely assessed by measuring concentration, motility and morphology (
). While conventional semen analysis can be useful as initial evaluation of a patient’s fertility status, there is growing evidence that it has limited value in diagnosing male infertility or predicting success of assisted reproduction (
). Although currently not in the battery of routine clinical tests offered to patients, sperm DNA damage has been demonstrated to be a promising tool in determining the male patient’s fertility status and to predict successful treatment in terms of the short-term goals of fertilization and pregnancy and the long-term goal of healthy offspring.
No methods are currently available to measure DNA damage in spermatozoa that allow the sperm to be subsequently used clinically. However, of the methods currently available to determine sperm DNA damage, the Comet assay is considered the most sensitive (
Evaluation of three methods for the detection of DNA single-strand breaks in human lymphocytes: alkaline elution, nick translation, and single-cell gel electrophoresis.
). It is the only test that quantifies DNA fragmentation in an individual spermatozoon. In addition, it requires a small number of cells, therefore making it suitable to assess DNA damage in semen samples supplied for routine analysis without the need of a further sample dedicated to DNA testing. Most importantly, robust clinical threshold values have now been established for this test (
). Some have suggested that the low number of cells (50 spermatozoa in duplicate or triplicate) counted in the Comet assay gives it low repeatability. In order for it to be fully accepted by the clinicians and scientific community, there must be tight regulations on the protocol used. Furthermore, to be approved as a standard analytical tool in clinical laboratories, the Comet assay must be shown to have single laboratory validation, the process that includes optimization, standardization, establishing of thresholds, measuring diagnostic specificity and sensitivity and calculating repeatability and reproducibility. This study group has reported results for optimization and standardization of the Comet assay together with clinical thresholds, diagnostic specificity and sensitivity (
). The current study evaluated within-laboratory variance (repeatability) of the assay as recommended in ISO 5725 and found results highly repeatable and consistent between replicate slides. The standard error of precision for duplicate slides was found to be 2.65%. The greatest source of error in detecting tail DNA in the Comet assay is the electrophoresis step, in particular the drop in voltage across the gel (
). To account for that, the current study routinely analysed five comets on 10 different areas of the slide, thus increasing sample representativeness, and this was also reflected in extremely low variability between the duplicate or triplicate slides.
This study observed a close negative relationship between nonpregnant and the live-birth groups in some parameters of treatment outcome, in particular total embryo cumulative score and transferred embryo score and a failure to achieve a pregnancy following IVF. This is consistent with previous reports from this study group (
Embryo quality is more important for younger women whereas age is more important for older women with regard to in vitro fertilization outcome and multiple pregnancy.
). Both the total embryo cumulative score and transferred embryo cumulative score were considerably reduced in the nonpregnant group compared with patients having live births after IVF. In contrast, in ICSI patients, no relationship was detected in either total or transferred embryo cumulative score and live births. These results are in agreement with other studies that have shown a strong correlation between embryo quality (
The significance of sperm DNA oxidation in embryo development and reproductive outcome in an oocyte donation program: a new model to study a male infertility prognostic factor.
There is a growing body of research showing a link between high sperm DNA damage and poor ART outcomes, including fertilization rate, pre- and post-implantation embryo development and implantation rate (
The significance of sperm DNA oxidation in embryo development and reproductive outcome in an oocyte donation program: a new model to study a male infertility prognostic factor.
The main focus of the present study was to determine, as far as is known for the first time using the Comet assay, if there was an association between levels of sperm DNA damage in male patients and live-birth rates following IVF and ICSI. As with this study group’s earlier results of total embryo cumulative score, transferred embryo cumulative score and number of oocytes fertilized (
), the inverse relationship between sperm DNA damage and outcome was consistent for both native semen and prepared spermatozoa. Furthermore, analysis of pregnancy rates and sperm DNA damage in IVF patients showed a strong negative relationship, with pregnancy rates declining from 39.4% in men with <25% sperm DNA damage to 16.2% in patients with >50% sperm DNA fragmentation. This, again, is well supported by this study group (
Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line.
). In the current study, the relationships between sperm DNA fragmentation and outcomes, including live-birth rates in couples with idiopathic infertility (all couples with either male or female factors were excluded) undergoing IVF, were also assessed. Elevated levels of sperm DNA damage in both native and prepared spermatozoa in comparison to the low levels of sperm DNA damage (12.47 ± 1.67%) reported in native spermatozoa of fertile donors (
), they declined to 21% for both men and couples with idiopathic infertility. Since it is well recognized that pregnancy rates after IVF and ICSI are much lower compared with pregnancy rates after natural conception and need to be improved (
), a further reduction in pregnancy rates in groups of patients with idiopathic infertility is an unacceptable and worrying fact. Obviously, efforts should be concentrated on elucidating the causes of idiopathic infertility in order to prescribe appropriate and effective treatment for these patients.
Of couples undergoing IVF, 72% of men and 34.5% had idiopathic infertility. Over 40% of men (60 out of 147) and nearly 39% of couples (27 out of 70) with idiopathic infertility had sperm DNA damage >50%. If these levels of damage are compared with those reported in spermatozoa of donor men with proven fertility by this study group (
) showed that 50% sperm DNA damage is the clinical threshold above which men are less likely to achieve a pregnancy by IVF. The live-birth rates are depressingly low (11.7% and 11.1%). Furthermore, if these couples or men are removed from the patients receiving IVF, the success rates in terms of pregnancies and live-birth rates will increase enormously; they would be expected to have high success rates since no anomalies can be detected.
It is now clear that many men with normal semen parameters have high levels of sperm DNA damage. They are often mistakenly classified as having idiopathic infertility or even being fertile individuals which, in turn, can lead to offering intrauterine insemination or IVF to such couples with little chance of success. This fact is reflected in disappointedly low effectiveness of IVF, which is measured as the cumulative incidence of live delivery after commencing IVF (
Since many women will not return after a poor response to IVF, this value may overestimate the cumulative live-birth rate, so that the actual live-birth rates following IVF are even lower. Yet, it falls to an unprecedented low of 11.7% and 11.1% in men and couples diagnosed with idiopathic infertility who have >50% sperm DNA damage, respectively. According to sperm DNA fragmentation analysis these patients should be directed straight to ICSI, thus avoiding loss of valuable biological time, cost of failed cycles and heartache after repeatedly unsuccessful cycles of IVF. This once again underscores the importance of DNA fragmentation analysis in guiding the patients to the appropriate treatment and necessity of including it in routine clinical testing (
), this study found that spermatozoa from patients having ICSI that did not result in clinical pregnancy tended to have a higher level of DNA fragmentation but this was not significantly greater than that of the live-birth group irrespective of type of spermatozoa (native or prepared) used in the assay. It has been reported in the literature that sperm DNA damage assessment may be less informative in patients undergoing ICSI since this technique bypasses all natural selection barriers such as binding to the zona pellucida, acrosomal reaction and fusion to the ooplasm, and therefore fertilization with highly DNA fragmented spermatozoa is still possible (
There is a striking decrease in the live-birth rates following IVF in couples with high sperm DNA damage and to a lesser degree after ICSI. Live-birth rates decreased by more than 2-fold, from 33.3% to 13.1% (Table 3) in IVF patients with high (>50%) sperm DNA fragmentation compared with <25% sperm DNA damage. Not surprisingly, the number of ICSI patients with <25% sperm DNA damage was very small (only nine out of 136 patients), so the group was too small to determine any associations between levels of sperm DNA damage and live-birth rates. However, it is worth noting that both pregnancies in this group of patients resulted in live births. In agreement with a previous report from this group (
), this study observed a shift towards a higher level of sperm DNA damage as well as an increase in average sperm DNA fragmentation in ICSI patients compared with IVF patients. Since ICSI is a more efficient treatment, in particular for patients with higher sperm DNA fragmentation levels, this resulted in slightly greater live-birth rates after ICSI compared with IVF for groups with the same levels of sperm DNA damage. A strong decline in live-birth rates, from 32.4% to 20.4%, was detected in ISCI patients with 25–50% sperm DNA damage compared with those with >50% sperm DNA damage. This contradicts the finding of
that delivery rates after ICSI rose from 35.3% to 42.4% for patients with DNA fragmentation <30% and >30%, respectively. This may be attributed to a different technique used in the study, since the sperm chromatin structure assay measures the proportion of spermatozoa with fragmented DNA in the ejaculate rather than DNA damage per spermatozoon, as assessed here by the alkaline Comet assay. One question that comes to mind is why do spermatozoa with DNA damage not reduce success following ICSI? There are at least four hypotheses as to why this is, as discussed below.
First, up to 30% of women having ICSI have no detectable problems. They may be fertile and their oocytes may have more capacity to repair DNA damage even if the injected spermatozoon is of poor quality. This is supported by the findings of
showed that significantly fewer good-quality embryos were available for transfer from women older than 40 years compared with the younger group, reflecting better DNA repair capacity of oocytes from young women. This is relevant in that women undergoing ICSI are often younger than those undergoing IVF as their cause of infertility has been diagnosed earlier and they have not spent time undergoing failed intrauterine insemination or IVF before embarking on a cycle of ICSI.
Second, in ICSI, the gametes are not subjected to prolonged culture, so the spermatozoa may have less damage than those exposed to culture media overnight, as in IVF. The recent major study from
shows that even the birthweight of IVF babies can be markedly influenced by minor differences in culture conditions. In contrast to IVF, ICSI spermatozoa are injected into the optimal environment of the oocyte within a few hours of ejaculation. This may protect them from laboratory-induced damage.
Third, much sperm DNA damage is caused by oxidative stress (
) and so these spermatozoa are producing reactive oxygen species. If they are used in IVF, the oocyte may be exposed to oxidative assault during the overnight incubation from these ∼0.5 million spermatozoa. In ICSI, the oocyte is protected from this attack and can use its energies to repair the damage in the spermatozoon immediately following fertilization.
Fourth, there is now evidence that embryos created from spermatozoa with high DNA damage are associated with early pregnancy loss (reviewed by
Robinson, L., Gallos, I.D., Conner, S.J., Rajkhowa, M., Miller, D., Lewis, S., Kirkman-Brown, J., Coomarasamy, A., 2012. The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis. Hum. Reprod. 2012. (epub ahead of print).
) so ICSI success rates are sometimes affected adversely by sperm DNA damage, but at a later stage.
In conclusion, this study found a strong negative association of sperm DNA fragmentation levels and live-birth rates in IVF patients. The relationship was more pronounced in patients with idiopathic infertility, suggesting that a possible cause of idiopathic infertility might be a high degree of sperm DNA fragmentation in such patients. In addition, this study demonstrates an importance and feasibility of sperm DNA damage testing to not only assess the treatment outcomes in general, but also to specifically evaluate live-birth rates. Lastly, determination of sperm DNA fragmentation is highly repeatable and robust. It provides additional information to help clinicians to tailor assisted reproduction treatment for men with sperm DNA damage to that particular couple’s needs.
Acknowledgements
The authors gratefully acknowledge the funding of Hamilton Thorne Biosciences Ltd. and Ulster Garden Villages.
References
Aitken R.J.
Curry B.J.
Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line.
Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications.
A position report. Hum. Reprod.2010; 25 (http://dx.doi.org/10.1093/humrep/dep465. Available from: http://humrep.oxfordjournals.org/cgi/content/abstract/25/4/824?etoc): 824-838
Embryo quality is more important for younger women whereas age is more important for older women with regard to in vitro fertilization outcome and multiple pregnancy.
Evaluation of three methods for the detection of DNA single-strand breaks in human lymphocytes: alkaline elution, nick translation, and single-cell gel electrophoresis.
The significance of sperm DNA oxidation in embryo development and reproductive outcome in an oocyte donation program: a new model to study a male infertility prognostic factor.
Robinson, L., Gallos, I.D., Conner, S.J., Rajkhowa, M., Miller, D., Lewis, S., Kirkman-Brown, J., Coomarasamy, A., 2012. The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis. Hum. Reprod. 2012. (epub ahead of print).
Declaration: I.P. is an employee and a shareholder of Lewis Fertility Testing, a spin-out company of Queen’s University Belfast, which is now marketing the Comet test. S.E.M.L. is the Chief Executive Officer and a shareholder of Lewis Fertility Testing. The other authors report no financial or commercial conflicts of interest.
Footnotes
Luke Simon graduated in biotechnology from the University of Madras, India in 2005 with a specialization in DNA fingerprinting. From 2005 to 2007, he was an instructor and research assistant at the University of Agricultural Sciences, Bangalore, India, during which he explored various types of DNA markers to identify inter- and intra-species variations. In 2007, he joined the Centre for Public Health at Queen’s University Belfast as a doctoral research fellow. He is now a postdoctoral researcher at the School of Medicine, University of Utah, USA. His principal interests focus on sperm DNA damage, protamine and the effects of oxidative stress on male fertility.
Luke Simon graduated in biotechnology from the University of Madras, India in 2005 with a specialization in DNA fingerprinting. From 2005 to 2007, he was an instructor and research assistant at the University of Agricultural Sciences, Bangalore, India, during which he explored various types of DNA markers to identify inter- and intra-species variations. In 2007, he joined the Centre for Public Health at Queen’s University Belfast as a doctoral research fellow. He is now a postdoctoral researcher at the School of Medicine, University of Utah, USA. His principal interests focus on sperm DNA damage, protamine and the effects of oxidative stress on male fertility.
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