Oxidative stress status and sperm DNA fragmentation in fertile and infertile men

Evidence suggests that disturbing the balance between reactive oxygen species levels and antioxidant contents in seminal plasma leads to oxidative stress resulting in male infertility. This study was carried out to identifying clinical significance of seminal oxidative stress and sperm DNA fragmentation in treatment strategies of male infertility in southwest Iran. Sperm parameters, lipid peroxidation and activity of antioxidant enzymes were assessed in fertile (n = 105) and infertile (n = 112) men. Malondialdehyde (MDA) levels in seminal plasma were found to be higher significantly (p < .001) in patients. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in seminal plasma were significantly (p < .001) lower in infertile men. Significant negative correlations were observed between MDA levels and sperm motility and normal morphology. Spermatozoa with fragmented DNA were higher (p < .001) in infertile men and significantly correlated with MDA levels and SOD and GPx activities. MDA of 4.2 nmol/ml, SOD of 4.89 U/ml and GPx of 329.6 mU/ml were optimum cut‐off limits to discriminate infertile patients from fertile men. The results show the leading role of oxidative stress in aetiology of male infertility in southwest Iran and indicate that evaluation of seminal antioxidant status and DNA integrity can be helpful in men attending infertility clinics during fertility assessment.     

Relationship between sperm telomere length and sperm quality in infertile men

Telomeres, noncoding and repetitive DNA sequences play a significant function in chromatin integrity. Telomere length is age-dependent in somatic cells, while it increases in sperm cell with age. Therefore, we aimed to assess sperm chromatin, leucocyte and sperm telomere length (LTL, STL) in spermatozoon of 38 infertile and 19 fertile men aged between 20 and 50 years. Protamine deficiency (chromomycin A3 test), DNA fragmentation (TUNEL assay), lipid peroxidation (Bodipy probe) and telomere length (quantitative real-time PCR) were assessed. A significant decrease in mean of sperm concentration and motility and a significant increase in means of sperm abnormal morphology, DNA fragmentation, lipid peroxidation and protamine deficiency were observed in infertile compared with fertile men. In addition, the mean of LTL and STL were significantly shorter in infertile men compared with fertile individuals. We observed significant associations between telomere length with sperm concentration, DNA fragmentation and lipid peroxidation. We hypothesised that increased oxidative stress in spermatozoa of infertile men can result in abnormal packaging of chromatin, damage of DNA and shorter sperm telomere length. Together, these anomalies may account for fertility failure in these individuals.     

Sperm DNA damage and seminal antioxidant activity in subfertile men

Supraphysiological ROS levels can lead to apoptosis, lipid peroxidation, and DNA and protein damage. This pilot study aimed to investigate the sperm oxidative damage in subfertile men, to describe the relationship between the antioxidant system and ROS. Sixty-four semen samples were categorised according to the evaluated routine parameters (WHO, WHO laboratory manual for the examination and processing of human semen, 2010). Results were cross-referenced with the DNA damage [Comet (n = 53) and TUNEL (n = 49) assays], antioxidant enzyme activity [SOD (n = 51), CAT (n = 48) and GST (n = 48)], and content of total thiols (n = 36), lipid hydroperoxides (n = 35) and MDA (n = 31). Compared to pathospermic samples, normozoospermic presented 40%–45% fewer spermatozoa with fragmented DNA, 19% fewer hydroperoxides, and slightly higher total thiols and MDA levels. Asthenozoospermic/asthenoteratozoospermic samples had the lowest GST activity. SOD and CAT showed a similar trend. Our results evidenced significant positive correlations between DNA damage and immotile spermatozoa; SOD and CAT, GST and total thiols; CAT and GST; total thiols and sperm concentration; and MDA levels and head/midpiece abnormalities and hydroperoxides. This work contributes to the existing body of knowledge by showing that the oxidative status correlates with the classic sperm analysis parameters. Oxidative stress and DNA damage evaluation might be a valuable diagnostic and prognostic tool in cases of idiopathic male subfertility.     

Comparative analysis of tests used to assess sperm chromatin integrity and DNA fragmentation

Male infertility has a complex etiology, and many times, the cause is unknown. While routine semen analysis provides an overview of basic semen parameters, such as sperm concentration, motility, viability and morphology, a significant overlap of these parameters has been reported in fertile and infertile men. Moreover, conventional semen parameters do not reveal the cellular or molecular mechanisms of sperm dysfunctions leading to infertility. Therefore, sperm functional parameters, including sperm chromatin integrity, are evaluated to provide information on subtle sperm defects that are not routinely identified. Incomplete or defective sperm chromatin condensation increases the susceptibility of the sperm DNA to oxidative damage or other factors. To evaluate sperm chromatin integrity, different methods with varying degrees of diagnostic and prognostic capabilities are available. Among these assays, SCSA, TUNEL and SCD assays are most commonly used. While these assays rather evaluate the DNA directly for damages, the aniline blue and chromomycin A3 stains test for the quality of chromatin condensation. Thus, this review discusses and compares different methods used to evaluate sperm chromatin integrity and condensation, and their inclusion in the routine evaluation of the male infertility.     

Relationship between chromatin condensation, DNA integrity and quality of ejaculated spermatozoa from infertile men

Normal chromatin condensation is important for sperm fertilising ability. However, routine semen analysis does not identify defects in sperm chromatin structure. This study aimed to investigate the condensation of chromatin and DNA integrity in spermatozoa of infertile men and deduce the relationship with sperm quality, as measured by conventional semen parameters. Semen analysis was carried out to assess sperm quality according to World Health Organization criteria. The remaining aliquot of each sample was processed for transmission electron microscopy, chromomycin A3 (CMA3) and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays. The ultrastructural analysis of spermatozoa from infertile men showed heterogeneity in sperm nuclear morphology. Some spermatozoa displayed a round nucleus with incomplete chromatin condensation. Immunoreactivity with antitransitional protein and antiprotamine antibodies indicated nuclear maturation defects in the spermatozoa of infertile men. Spearman's correlation analysis indicated a positive correlation between the percentages of CMA3- and TUNEL-positive spermatozoa. In addition, these two parameters were negatively correlated with sperm concentration, motility and normal morphology. This study demonstrated that men with morphologically normal spermatozoa of <30% have greater degree of protamine deficiency and DNA damage than men with morphologically normal spermatozoa of >30%. Evaluation of chromatin integrity appears to be a useful tool for assessing male fertility.     

Association between seminal granulysin and malondialdehyde in infertile men with varicocele and the potential effect of varicocelectomy

This study assessed the seminal plasma granulysin and malondialdehyde (MDA) levels in patients suffering from varicocele-associated infertility prior to and after varicocelectomy. This study was conducted on 34 infertile men with varicocele (group A) and same patients after varicocelectomy (group B) and 32 fertile normozoospermic males (group C). A detailed history taking, clinical examination, scrotal doppler ultrasound for varicocele diagnosis and grading, semen analysis and estimation of seminal granulysin and MDA before and after varicocelectomy were done to all participants. The mean (SD) granulysin and MDA levels in patients with varicocele were higher than in controls with highly significant differences. Post-operatively, there was a significant reduction in mean (SD) granulysin and in MDA level. Basal seminal granulysin positively correlated with basal seminal MDA, abnormal forms and negatively correlated with basal sperm count, concentration, and progressive motility. The receiver operating characteristic curve of seminal granulysin and MDA levels were conducted for discrimination between infertility cases with varicocele and control groups. Excellent AUCs were found for both markers (AUC = 0.971, 0.991 respectively). We concluded that high levels of granulysin and MDA in the semen of infertile males with varicocele negatively impact their spermatogenesis. Varicocelectomy leads to the improvement of semen parameters and significantly decreases seminal plasma granulysin and MDA levels. Hence, seminal granulysin and MDA could be used as a prognostic test in infertile patients with varicocele.     

Sperm DNA damage in men from infertile couples

Aim： To investigate the prevalence of high levels of sperm DNA damage among men from infertile couples with both normal and abnormal standard semen parameters. Methods： A total of 350 men from infertile couples were assessed. Standard semen analysis and sperm chromatin structure assay （SCSA） were carried out. Results： Ninety-seven men （28% of the whole study group） had a DNA fragmentation index （DFI） 〉 20%, and 43 men （12%） had a DFI 〉 30%. In the group of men with abnormal semen parameters （n = 224）, 35% had a DFI 〉 20%, and 16% had a DFI 〉 30%, whereas these numbers were 15% and 5%, respectively, in the group of men with normal semen parameters （n = 126）. Men with low sperm motility and abnormal morphology had significantly higher odds ratios （ORs） for having a DFI 〉 20% （4.0 for motility and 1.9 for morphology） and DFI 〉 30% （6.2 for motility and 2.8 for morphology） compared with men with normal sperm motility and morphology. Conclusion： In almost one-third of unselected men from infertile couples, the DFI exceeded the level of 20% above which, according to previous studies, the in vivo fertility is reduced. A significant proportion of men with otherwise normal semen parameters also had high sperm DNA damage levels. Thus, the SCSA test could add to explaining causes of infertility in cases where semen analysis has not shown any deviation from the norm. We also recommend running the SCSA test to choose the appropriate assisted reproductive technique （ART）.     

Obesity leads to higher risk of sperm DNA damage in infertile patients

There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be altered in case of high body mass index （BMI）. A few studies assessing the impact of BMI on sperm DNA integrity have been published, but they did not lead to a strong consensus. Our objective was to explore further the relationship between sperm DNA integrity and BMI, through a 3-year multicentre study. Three hundred and thirty male partners in subfertile couples were included. Using the terminal uridine nick-end labelling （TUNEL） assay, we observed an increased rate of sDerm DNA damage in obese men （odds ratio （95% confidence interval）： 2.5 （1.2-5.1））.     

Novel association between sperm deformity index and oxidative stress-induced DNA damage in infertile male patients

Aim: To investigate the impact of abnormal sperm morphology using the sperm deformity index (SDI) on reactive oxygen species (ROS) production and its correlation with sperm DNA damage. Methods: Semen samples were collected from men undergoing infertility screening (n=7) and healthy donors (n=6). Mature spermatozoa were isolated and incubated with 5mmol/L β-nicotinamide adenine dinucleotide phosphate (NADPH) for up to 24h to induce ROS. Sperm morphology was evaluated using strict Tygerberg‘s criteria and the SDI. ROS levels and DNA damage were assessed using chemiluminescence and terminal deoxynucleotidyl transferase-mediated fluoresceindUTP nick end labeling (TUNEL) assays, respectively. Results: SDI values (median [interquartiles]) were higher in patients than donors (2 [1.8,2.1] vs. 1.53 [1.52, 1.58], P=0.008). Aliquots treated with NADPH showed higher ROS levels (1.22 [0.30, 1.87] vs. 0.39 [0.10, 0.57], P=0.03) and higher incidence of DNA damage than those not treated (10 [4.69, 24.85] vs. 3.85 [2.58, 5.10], P=0.008). Higher DNA damage was also seen following 24h of incubation in patients compared to donors. SDI correlated with the percentage increase in sperm DNA damage following incubation for 24h in samples treated with NADPH (r=0.7, P=0.008) and controls (r=0.58, P=0.04).Conclusion: SDI may be a useful tool in identifying potential infertile males with abnormal prevalence of oxidative stress (OS)-induced DNA damage. NADPH plays a role in ROS-mediated sperm DNA damage, which appears to be more evident in infertile patients with semen samples containing a high incidence of morphologically abnormalspermatozoa.     

Evaluation of sperm DNA fragmentation index,Zinc concentration and seminal parameters from infertile men with varicocele

The aim of this study was to investigate the effect of varicocele on DNA fragmentation index (DFI), zinc concentration and seminal parameters in infertile patients. In this prospective study, 179 men with at least 1‐year history of infertility and varicocele were examined for semen quality at Hanoi Medical University Hospital (HMUH), Hanoi, Vietnam. In addition, an inverse correlation between zinc concentration and the degree of sperm DNA fragmentation in patients with clinical varicocele was found. The difference in mean values of sperm DNA fragmentation index in patients with various grades of varicoceles can be neglected, whereas most patients with varicocele of grades II and III had DFI >30%. Varicocele is associated with high levels of DNA damage in spermatozoa and reduced zinc levels that correlate with different grades of disease. Therefore, DNA fragmentation index and zinc concentration can be used as essential additional diagnostic test for patients with clinical varicocele. A study should be conducted to evaluate the benefits of zinc supplementation to improve seminal parameters in patients with varicocele.     

Effect of smoking on seminal plasma ascorbic acid in infertile and fertile males

This work aimed to assess the relationship of seminal ascorbic acid levels with smoking in infertile males. One hundred and seventy men were divided into four groups: nonobstructive azoospermia [NOA: smokers (n = 20), nonsmokers (n = 20)]; oligoasthenozoospermia [smokers (n = 30), nonsmokers (n = 20)]; asthenozoospermia [smokers (n = 20), nonsmokers (n = 20)] and normozoospermic fertile men [smokers (n = 20), nonsmokers (n = 20)]. The patients underwent medical history, clinical examination, conventional semen analysis and estimation of ascorbic acid in the seminal plasma calorimetrically. There was a significant decrease in the mean seminal plasma ascorbic acid levels in smokers versus nonsmokers in all groups (mean +/- SD; 6.03 +/- 2.18 versus 6.62 +/- 1.29, 7.81 +/- 1.98 versus 9.44 +/- 2.15, 8.09 +/- 1.98 versus 9.95 +/- 2.03, 11.32 +/- 2.15 versus 12.98 +/- 12.19 mg dl(-1) respectively). Fertile subjects, smokers or not, demonstrated significant higher seminal ascorbic acid levels than any infertile group. Seminal plasma ascorbic acid in smokers and nonsmokers was correlated significantly with sperm concentration (r = 0.59, 0.60, P < 0.001), sperm motility (r = 0.65, 0.55, P < 0.001) and negatively with sperm abnormal forms per cent (r = -0.53, -0.50, P < 0.001). Nonsignificant correlations were elicited with semen volume (r = 0.2, 0.09) or liquefaction time (r = 0.03, 0.06). It is concluded that seminal plasma ascorbic acid decreased significantly in smokers and infertile men versus nonsmokers and fertile men, and is significantly correlated with the main sperm parameters: count, motility and normal morphology. Also, cigarette smoking is associated with reduced semen main parameters that could worsen the male fertilizing potential, especially in borderline cases.     

Lycopene and resveratrol improve post‐thaw bull sperm parameters: sperm motility,mitochondrial activity and DNA integrity

We focussed on evaluating the protective effect of lycopene and resveratrol on post‐thaw bull sperm and oxidative stress parameters. Nine ejaculates for each bull were used in the study. Each ejaculate, splitted into three equal aliquots and diluted at 37 °C with base extenders containing lycopene (1 × 10^?3 g ml^?1) and resveratrol (1 mm ), and no antioxidant (control), was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. The supplementation of the semen extender with lycopene and resveratrol increased the percentages of post‐thawed computer‐assisted sperm analysis (CASA) motility (55.8 ± 3.8 and 61.9 ± 4.0%) and progressive motility (38 ± 2.4 and 37 ± 8.8), compared with the controls (50.7 ± 2.65 and 33.3 ± 3.74%, respectively, P < 0.05). Resveratrol provided a higher ALH (4.3 ± 0.1), in comparison with the control (3.9 ± 0.3, P < 0.05). The supplementation of the semen extender with lycopene and resveratrol produced a higher mitochondrial activity (24.6 ± 2.9 and 30.1 ± 6.5% respectively), compared with that of the control (11.8 ± 9.5%, P < 0.05). It was determined that both antioxidants resulted in a lower percentage of sperm with damaged DNA than that of the control (P < 0.05). Sperm motion characteristics except for ALH, acrosome integrity, sperm viability and oxidative stress parameters were not affected by the adding of lycopene and resveratrol.     

Novel insights into the pathophysiology of varicocele and its association with reactive oxygen species and sperm DNA fragmentation

Varicocele has been associated with reduced male reproductive potential. With the advances in biomolecular techniques, it has been possible to better understand the mechanisms involved in testicular damage provoked by varicocele. Current evidence suggests the central role of reactive oxygen species (ROS) and the resultant oxidative stress (OS) in the pathogenesis of varicocele-associated male subfertility although the mechanisms have not yet been fully described and it is likely to be multifactorial. Excessive ROS is associated with sperm DNA fragmentation, which may mediate the clinical manifestation of poor sperm function and fertilization outcome related to varicocele. Testing of ROS/OS and DNA fragmentation has the potential to provide additional diagnostic and prognostic information compared to conventional semen analysis and may guide therapeutic management strategies in individual patient.     

DNA sperm damage correlates with nuclear ultrastructural sperm defects in teratozoospermic men

Sperm morphology has consistently been the best indicator of male fertility. Transmission electron microscopy currently provides the most information on the subcellular details of sperm structure. Recently, assessment of sperm DNA damage has been employed to assess fertility potential. The purpose of this work was to link sperm DNA damage, evaluated by an intercalated fluorescent dye, with the structural characteristics of sperm. Conventional semen analysis was performed on samples from men undergoing fertility evaluation. Thirty men were evaluated and assigned to three subgroups based on strict criteria for sperm morphology: normal morphology (>14% normal forms), intermediate morphology (5-14% normal forms), and poor morphology (<5% normal forms). By quantifying acridine orange-positive cells and ultrastructural sperm defects, we found that the poor morphology pattern group showed a positive association between sperm carrying damaged DNA and the percentage of sperm nucleus with vacuoles (P = 0.01). No statistically significant correlations were established in other ultrastructural characteristics of sperm, including immature chromatin, lytic changes, or abnormal sperm tails. These results suggest that zones without chromatin in the sperm nucleus reflect underlying chromosomal or DNA defects in severe teratozoospermic men. This association should be considered in the evaluation of male fertility.     

不育男性精子染色质结构与精液常规参数的关系

目的 研究不育男性精子染色质结构参数与精液常规参数的关系.方法 采集36例男性不育患者和18例健康对照者的精液标本,用TUNEL法检测精子DNA碎片化,CMA3染色法检测精子染色质包装质量,按照世界卫生组织标准(1999)检测精液常规参数.结果 不育组的精子TUNEL阳性率和CMA3阳性率均显著高于健康对照组,差异具有统计学意义(P＜0.05),精液常规参数正常的不育男性精子TUNEL阳性率和CMA3阳性率也显著高于健康对照组,差异具有统计学意义(P＜0.05),精子TUNEL阳性率和CMA3阳性率与精液常规参数之间具有显著的相关性(P＜0.05).结论 不育男性的精子染色质结构检测结果与健康男性存在显著差异,精子染色质结构检测可提供反映男性生育能力的信息.     

反复自然流产与精子DNA完整性的相关性研究

目的 探讨反复自然流产与精子DNA完整性的相关性。方法 按纳入标准将研究对象分成反复自然流产患者丈夫组（n=58）和正常生育男性对照组（n=50），采用吖啶橙实验（AOT）对研究对象精子DNA完整性进行检测，并对研究对象精液进行常规分析。砖果两组研究对象年龄、吸烟史、饮酒史无统计学差异（P〉0．05）；反复自然流产患者丈夫组精液的精子密度、精子活力显著低于正常生育对照组（P〈O．05）；反复自然流产患者丈夫组精子DNA的完整性低于正常生育对照组，差异有统计学意义（P〈O．05）。砖论精子DNA完整性损伤是反复自然流产的可能原因，精子DNA完整性损伤导致反复自然流产的致病机理尚需进一步研究。     

Best laboratory practices and therapeutic interventions to reduce sperm DNA damage

Conventional semen analysis is considered the cornerstone investigation for infertile men. Nonetheless, this routine test does not provide information on important sperm functions like sperm DNA fragmentation (SDF). Abnormalities of human spermatozoal nucleus and chromatin have a detrimental impact on both natural and assisted reproductive outcomes. In vivo, SDF results from abnormalities in chromatin compaction, abortive apoptosis and oxidative stress, while in vitro, a number of factors may be implicated. Various SDF testing methods are available, and the most commonly utilised assays include terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), sperm chromatin dispersion (SCD) test, sperm chromatin structure assay (SCSA) and Comet assay. SDF testing has shown beneficial effects on treatment decision-making; however, its routine use in the initial evaluation of infertile men is still not recommended. One of the treatment options to reduce sperm DNA damage is the use of antioxidants. Despite the documented improvement in semen parameters and sperm DNA integrity following antioxidant therapy, no definitive recommendation is reached due to lack of large, well-designed, randomised, placebo-controlled trials assessing their exact role in male factor infertility. The objectives of this review article are to illustrate the aetiologies of SDF, to describe the effects of SDF on male factor fertility, to explore the common techniques utilised in SDF testing, to review the clinical indications for SDF testing and to review the effect of antioxidant therapy as a method to alleviate SDF.     

TUNEL assay: Establishing a sperm DNA fragmentation cut‐off value for Egyptian infertile men

Male factor infertility is responsible for half of all infertility cases. Conventional semen analysis is inadequate to evaluate male fertility. Sperm DNA fragmentation (SDF) test can be done by: direct methods such as Terminal deoxynucleotidyl transferase dUTP Nick‐End Labeling (TUNEL) and Comet assay, or indirect like Sperm Chromatin Structure Assay (SCSA) and Sperm Chromatin Dispersion (SCD). TUNEL assay measures both single‐ and double‐strand breaks and is technically less demanding, while SCSA tests for the susceptibility for nuclear DNA denaturation and samples should be sent to the reference lab. Studies showed that a single cut‐off value does not fit all. Therefore, this study aimed at establishing a cut‐off value to discriminate between fertile and infertile Egyptian men. We enrolled 354 infertile men and 40 proven fertile volunteers.TUNEL assay was performed using Apo‐Direct kit and bench top flow cytometer.The calculated SDF cut‐off value was 20.3% with a sensitivity of 96.6% and specificity of 87.5%, and the overall accuracy of the test was 95.7%. Sperm DNA fragmentation Test using TUNEL assay is valuable tool for male infertility evaluation, and it assists in offering the best treatment options based on it's results.     

Clinical correlates of the biological variation of sperm DNA fragmentation in infertile men attending an andrology outpatient clinic

Determination of sperm DNA fragmentation, as assessed by the sperm chromatin structure assay (SCSA), has become an important tool for the evaluation of semen quality. The aim of the present study was to describe the biological variation of sperm DNA fragmentation in men attending an andrology clinic and to identify clinical correlates of the biological variation of sperm DNA fragmentation. For this study, two consecutive semen samples from 100 patients attending our andrology outpatient clinic were subjected to semen analysis, performed in parallel according to WHO guidelines and by SCSA. A good agreement between pairs of samples was found for SCSA-derived variables, as indicated by a significantly lower median coefficient of variation (CV) of the DNA Fragmentation Index (DFI) and the high DNA stainability (HDS) compared with WHO semen parameters. In half of the men attending our andrology clinic, however, the individual biological variation of DFI and HDS, expressed as CV of two samples, exceeded 10%. Dysregulation of spermatogenesis, as seen as testicular insufficiency or varicocele, was not associated with increased variability of DFI or HDS. A backward multiple linear regression analysis, however, indicated that the biological variation of DFI may be more profound in men with characteristics of normal spermatogenesis. In conclusion, we confirm previous reports that sperm DNA fragmentation has a lower biological variability than classical semen parameters. We hypothesize that the sperm chromatin structure may be more influenced in patients with normal spermatogenesis, whereas in men with disturbed spermatogenesis, the chromatin structure may be already so impaired that the effect of unidentified factors leading to variability of sperm DNA fragmentation in time may not be as profound.     

Impact of weight loss on sperm DNA integrity in obese men

The objective of the study was to determine whether weight loss in obese men improves their fertility with respect to DNA fragmentation index and morphology. Collected fertility parameters included DFI and morphology. Body mass index (BMI) was calculated for all patients with comparisons to their fertility parameters before and after weight loss using paired t test and chi‐square tests. The mean BMI was significantly higher in group 1, before weight loss (33.18 kg/m²), than in group 2, after weight loss (30.43 kg/m²). Overall, 53.3% of men had DFI <20% while 43.8% had a DFI between 20% and 40%, and 2.9% of men had DFI >40%. The mean DFI of participants was higher before weight loss (20.2%) and had improved significantly after weight loss (17.5%) (p = <.001). The weight loss had significant positive correlation with percentage of DFI. There was a significant improvement in morphology after weight loss (p = <.05). In one of the largest cohorts of male fertility and obesity, DFI and morphology demonstrated significant relationship with adiposity, possibly contributing to subfertility in this population.