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Sperm progressive motility has been reported to be one of the key factors influencing in vitro fertilization rates. However, recent studies have shown that sperm DNA fragmentation is a more robust predictor of assisted reproductive outcomes including reduced fertilization rates, embryo quality and pregnancy rates. This study aims to compare the usefulness of sperm progressive motility and DNA damage as predictive tools for in vitro fertilization rates. In this study, 136 couples provided 1767 eggs for the study with an overall fertilization rate of 64.2%. The fertilization rate in vitro correlated with both sperm progressive motility (r2 = 0.236; P = 0.002) and DNA fragmentation (r2 = -0.318; P < 0.001). The relative risk of poor fertilization rate was 9.5 times higher in sperm of men with high DNA fragmentation (>40%) compared with 2.6 times in sperm with poor motility (<40%). Further, sperm DNA fragmentation gave a higher specificity (93.3%) in predicting the fertilization rate than progressive motility (77.8%). Finally, the odds ratio to determine fertilization rate (>70%) was 4.81 (1.89-12.65) using progressive motility compared with 24.18 (5.21-154.51) using DNA fragmentation. This study shows that fertilization rates are directly dependent upon both sperm progressive motility and DNA fragmentation, but sperm DNA fragmentation is a stronger test.
One in six couples experiences fertility problems during their reproductive lives so assisted reproductive technologies (ART) have a major role in modern society. The success of ART varies depending on a range of male and female factors, but adequate structure and function of male and female gametes is essential (Varghese et al. 2009). The in vitro fertilization (IVF) outcomes are generally lower in couples with male infertility, due to lower fertilization rates and hence fewer embryos available for transfer (Imoedemhe et al. 1992). Sperm progressive motility is known to be important for the sperm to penetrate the zona pellucida both in vivo and in vitro, and thus this has been considered as an important factor determining fertilization rates (Chiu et al. 1987; Donnelly et al. 1998; Turner 2006).
The quality of motility assessed either manually or by computer aided sperm motion analysis has been considered a key parameter in diagnosing male infertility for many years (Lewis 2007). Fertilization is comprised of two major steps, the interaction between the sperm and oocyte, and fusion of male and female to form pronucleus (Wassarman 1999). Vigorous sperm motility is known to facilitate fertilization by enabling the sperm to penetrate the cumulus cell, corona radiata and finally the zona pellucida (Liu et al. 1991). Reduced motility is observed in infertile men and associated with increased mitochondrial abnormalities (Folger et al. 1993; Kao et al. 1998) and structural deformities in the flagella (Baccetti et al. 1993; Chemes et al. 1998). Impaired motility is also caused by an increase in oxidative stress in the seminal plasma (Urata et al. 2001; Kao et al. 2008), increased age (Kidd et al. 2001) and electromagnetic radiation (Yan et al. 2007).
The limitations of conventional WHO parameters such as motility to predict ART success have been highlighted (Lefievre et al. 2007; Lewis 2007) and novel tests such as sperm DNA damage advanced. In recent years many studies proposed have analysed the possible association between sperm DNA fragmentation and ART outcomes specifically with fertilization (Donnelly et al. 1998; Sun et al. 1997; Esterhuizen et al. 2000; Host et al. 2000; Tomlinson et al. 2001; Benchaib et al. 2003 & 2007; Henkel et al. 2003 & 2004; Saleh et al. 2003; Chohan et al. 2004; Gandini et al. 2004; Huang et al. 2005; Payne et al. 2005; Borini et al. 2006; Muriel et al. 2006; Bakos et al. 2007; Bungum et al. 2007; Frydman et al. 2008; Lin et al. 2008). Other studies report that sperm DNA damage does not affect fertilization rates (Tomlinson et al. 2001; Henkel et al. 2003; Chohan et al. 2004; Gandini et al. 2004; Benchaib et al. 2007; Bungum et al. 2007; Frydman et al. 2008; Lin et al. 2008). Thus, there is a controversy in the literature as to the impact o sperm DNA damage on this early fertility check point. To compare the power of motility with DNA damage as predictors of IVF fertilization, we determine both parameters in the same semen samples and correlated them with fertilization rates in vitro.
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. The study was conducted at the Regional Fertility Centre, Royal Jubilee Maternity Services, Belfast, Northern Ireland, UK during the period April, 2008 to December, 2009. Sperm samples for research were obtained after written consent from all patients. A total of 216 couples attending for IVF were recruited and of these 136 couples were included in this study when following criteria were met a) a minimum of five oocytes retrieved, b) female partners < 40 years. In addition, couples with failed fertilization and men with antisperm antibodies in their semen were excluded from the study.
Semen analysis and sperm preparation:
Semen samples were collected by masturbation from men after 2-5 days of recommended abstinence, on the day of IVF treatment. Semen analysis was performed according to WHO guidelines (WHO 1992). Semen analysis was performed within 1 hour of ejaculation, following a period of incubation at 37°C to allow for liquefaction. After liquefaction, routine semen analyses were performed and subsequently semen was density gradient centrifugation (DGC) using a two-step discontinuous Puresperm gradient (90% - 45%; Hunter Scientific Limited, UK). For each semen sample with a normozoospermic profile the whole sample was layered on top of 2 ml (90%) and 4 ml (45%) gradient and centrifuged at 250 x g for 20 minutes. For semen samples with less than normal WHO parameters, 1 ml of semen was layered on top of 1 ml (90%) and 1 ml (45%) gradient and centrifuged at 100 x g for 20 minutes. The resulting sperm pellets were washed twice with culture media (Vitrolife G5 sequential media series; Vitrolife Inc, Goteborg, Sweden) and concentrated by centrifugation at 250 and 100 x g respectively, for 10 minutes or resuspended in fresh culture media (2 ml). Hence, two populations of sperm were used to measure DNA damage using the alkaline Comet assay: that with the best fertilizing potential as used for clinical treatment (DGC), and the whole population (native semen) for each patient.
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 GnRH analogue. HCG was administered when there were at least three follicles of diameter >17 mm, 36 h before oocyte retrieval. Mature, metaphase II oocytes obtained by vaginal ultrasound-guided aspiration were cultured in media Vitrolife G5 sequential media series (Vitrolife Inc, Goteborg, Sweden) at 37Â°C with 6% CO2 in air. 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.
Alkaline Comet assay:
Sperm DNA damage was assessed using an alkaline single cell gel electrophoresis (Comet) assay as previously modified by our group (Hughes et al. 1997; Donnelly et al. 1999). Our previous study has reported an intra-assay coefficient variation of 6% for this assay (Hughes et al. 1997).
Data was analysed using the Statistical Package for the Social Sciences (SPSS 15) for Windows (SPSS Inc., Chicago, IL, USA). Data are presented as mean Â± standard error. The amount of fragmented DNA migrated in the Comet tail was expressed as percentage of damage for native semen and DGC sperm. Sperm DNA damage was categorised into three groups: low DNA damage (0-40%), moderate DNA damage (41-70%) and high DNA damage (71-100%). The fertilization rate for each couple was calculated as the percentage of oocytes fertilized. Fertilization rate were categorized into two groups (â‰¤70% and >70%) and three groups, poor (0-40%), moderate (41-70%) and good (71-100%) fertilization rate. Similarly, progressive motility was categorized into three groups: poor (0-40%), moderate (41-70%) and good (71-100%) motility. All tests were two-sided with a probability value of less than 0.05 to be regarded as significant.
Spearman's Rank correlation coefficient was used to analyse the relationship between semen parameters with sperm DNA fragmentation. Logistic regression was used to evaluate the effect of semen parameters, male age or female age on fertilization rate. Duncan's test for multi-group comparison was performed to analyse each categories of fertilization rate with semen parameters and age. Pearson correlation was used to find any association between the variables; progressive motility, sperm DNA fragmentation and fertilization rate. Chi-square analysis was performed comparing each of the three variables and their categories separately. We evaluated the effect of sperm DNA fragmentation on fertilization rate, in cases where all the standard semen parameters were normal and also one of the semen parameter was abnormal. Each semen parameters was categorised into a normal and abnormal category according to WHO guidelines, to which univalent and multivalent analyses were performed using sperm DNA damage as fixed variable and the odds ratio to obtain a fertilization rate (>70%) were determined. The odds ratios and their 95% CI, specificity, sensitivity, positive and negative predictive power and relative risk in predicting good fertilization rates (>70%) was then estimated for sperm progressive motility and DNA damage.
An overall fertilization rate of 64.2% was observed using the 1,767 eggs included in the study. Of the conventional semen parameters: volume, concentration, and morphology, male age and female age there was no correlation with fertilization rate (Table 1). In contrast, there was a positive correlation with progressive motility (r2 = 0.236; P = 0.002). A significant negative correlation was observed between fertilization rate and sperm DNA measured in both native semen (r2 = -0.318; P < 0.001) and DGC sperm (r2 = -0.261; P < 0.001). When the fertilization rate was categorized into two categories (<70% and â‰¥70%) then there was a significant difference in mean progressive motility (49.24Â±3.14 vs. 57.81Â±1.22; P = 0.006) and sperm DNA damage measured in the native semen (56.60Â±4.43 vs. 47.03Â±1.73; P = 0.010) and the DGC sperm (41.00Â±4.16 vs. 33.02Â±1.51; P = 0.044) between the two categories. There was also a negative correlation between sperm DNA fragmentation and sperm progressive motility (r2 = -0.214; P = 0.005) but no association was found with any other semen parameter and sperm DNA damage.
When progressive motility was divided into the three categories, high and moderate motility groups had good fertilization rates 71% and 67% respectively, while low motility was associated with a lower fertilization rate of 52% (Figure 1). As sperm DNA fragmentation in the native semen increased fertilization rate reduced, low DNA damage group (73%), moderate damage (62%) and high damage (51%) fertilization rate (Figure 2). The relationship was also true for DGC sperm. The Chi-square analysis showed a significant association among the three categories of fertilization rate with both progressive motility and sperm DNA fragmentation (Ï‡2 = 16.06 and 35.68) respectively, with four degrees of freedom.
In the univalent model, abnormal motility and high sperm DNA damage in both native and DGC sperm showed a significant decrease in fertilization rate (Table 2). The association became stronger when DNA fragmentation was included in the multivalent analysis. In the multivalent analyses, semen parameters (volume, concentration and normal morphology) showed no statistically significance in predicting fertilization rate. However, when sperm progressive motility and DNA damage were included in the model, the odds ratio to obtain good fertilization rate (>70%) when DNA damage <40% was 6.01 (CI: 1.57-24.78) and for DNA damage >70% was 2.03 (CI: 1.38-11.22), was significant.
The odds ratio to determine fertilization using sperm motility was 4.81 (1.89-12.65) while the odds ratio was 24.18 (5.21-154.51) using sperm DNA fragmentation. Sperm of men with high DNA fragmentation and low motility results in 9.5 times and 2.6 times the increased relative risk of lower fertilization (<40%), when compared with low DNA damage and poor motility categories. Similarly, sperm DNA damage showed a higher specificity (93.3%) in predicting fertilization rates than progressive motility (77.8%). Sperm with high progressive motility and low sperm DNA damage had a 96.0% probability of resulting in >70% fertilization rate (Table 3). Sperm with good progressive motility and low sperm DNA damage had 96% probability of resulting in good fertilization rate (>70%).
Evaluation of semen parameters is still the gold standard in diagnosing male infertility and selecting whether IVF or ICSI will be performed (Repping et al. 2002; van Weert et al. 2004). Semen parameters are used not only to determine assisted male fertility potential with ART but also in determining the chance of a spontaneous pregnancy (Wald 2008). However, this conventional method of infertility diagnosis analysis has been shown to be of limited use as it lacks the predictive power to determine male infertility and assisted reproductive outcomes (reviewed by Tomlinson et al. 1999; Lewis 2007). Microscopic analysis is the first stage of testing but molecular testing may provide more information (Lopes et al. 1998; Tomlinson et al. 2001).
Over the past decade, sperm DNA damage been associated with many indicators of reproductive health (Frydman et al. 2008) and has been highlighted as a promising test. Some studies have shown relationships between semen parameters and sperm DNA damage (Tomlinson et al. 2001; Larson-Cook et al. 2003; Virro et al. 2004). However, our study supports those of Frydman et al. (2008) and Greco et al. (2005) in showing few correlations between conventional semen parameters and sperm DNA fragmentation as we observed a significant negative correlation only between sperm DNA and sperm motility.
The present study has shown that the outcome of in vitro fertilization is not influenced by the semen volume, sperm concentration or morphology. However, the chance of fertilization rates increased with the progressive motile population of sperm in the sample. Several studies have shown fertilization failure when sperm motility is less than 30% and a reduction of fertilization rate in semen with motility < 50% (van Uem et al. 1985; Hirsch et al. 1986). Similarly, our results show a good fertilization rate in semen with both good (> 70%) and moderate (40-70%) motility but not with motility < 40% (Figure 1). Our findings (also reported in another study; Simon et al. 2010) conflict with several older studies reporting a decrease in fertilization rate with decrease in sperm concentration, percentage motility or normal morphology (Battin et al. 1985; Hirsch et al. 1986; Matson et al. 1986). Mahadevan et al. (1984) showed an influence of sperm concentration on fertilization rate when the count fell below a level of 10 million/ml, whereas variations in sperm concentration above this level have no influence on fertilization rate. However, in support of this study, sperm motility alone was shown to be the only semen parameter to influence fertilization rate (Amann 1989; (Bongso et al. 1989). Donnelly et al. 1998). Further, Battin et al. (1985) showed sperm motility after swim-up was also associated with fertilization rates. Abnormalities of sperm motility include flagellar abnormalities, deficient mitochondrial metabolism, failure of sperm recognition of the zona pellucida, and an inability to complete sperm-oocyte fusion (Kao et al. 2008), any or all of these could the the cause of reduced fertilization. Also in agreement with our study, Bartoov et al. (1993) and Robinson et al. (1994) have shown that the rate of fertilization is not affected by an increase in abnormal morphology of the sperm.
The most important finding in this study is that we report DNA fragmentation measured by the alkaline Comet assay has a stronger prognostic ability to predict in vitro fertilization than progressive motility. The existing data regarding the relationship between sperm DNA fragmentation and fertilization rates are conflicting (Morris et al. 2002; Chohan et al. 2004; Henkel et al. 2004; Borini et al. 2006; Bakos et al. 2007; Bungum et al. 2007; Lin et al. 2008). Our data show a strong relationship between sperm DNA fragmentation and fertilization rates in IVF both in native semen (r2 = -0.318; P < 0.001) and DGC sperm (r2 = -0.261; P < 0.001). When sperm DNA fragmentation > 40%, we found a significant negative relationship with fertilization rate. Again, this is in agreement with Benchaib et al. (2003) although they used a threshold value of 10% when measuring damage by the TUNEL assay. This is due to the sensitivity of the alkaline Comet assay where all double and single strand breaks are measured throughout the entirety of relaxed chromatin, in contrast to other assays where perhaps only peripheral DNA damage is measurable. The correlation between sperm DNA damage and fertilization may be explained that, when the sperm DNA fragmentation is low, the oocytes are able to repair the DNA damage (Sakkas et al. 1996; Ahmadi and Ng 1999). However, their ability to repair is limited and as the level of sperm DNA fragmentation increases. It is also postulated that sperm with DNA fragmentation may not be able to develop into the pronuclear stage by failing to decondense resulting in fertilization failure (Sakkas et al. 1996).
The odds ratio to obtain a good (> 70%) fertilization was higher using sperm DNA fragmentation then the conventional sperm motility (24.18 vs. 4.81, respectively). Further although both progressive motility and DNA damage were significant to determine the fertilization rate, measurement of sperm DNA damage showed greater sensitivity (66%) and specificity (93%) in predicting good fertilization rate than progressive motility (sensitivity 58% and sensitivity 78%). Here, sperm of men with high DNA fragmentation had a relative risk of 9.5 times to fertilization rate â‰¤ 40% and the positive predictive value for sperm DNA fragmentation was 97.44%, proving again its prognostic value. In semen samples having both high motility and low sperm DNA fragmentation, the probability of obtaining good fertilization rate was > 96.0%. In conclusion, measurement of sperm DNA fragmentation by the alkaline Comet assay has a markedly greater relative risk and specificity than the conventional measurement of sperm progressive motility in predicting in vitro fertilization rates.