ABSTRACT
Sperm DNA integrity is important for fertility, however the incidence of high levels of DNA fragmentation (DNA fragmentation index (DFI) >30%) is not well described. In 2011, our clinics implemented guidelines for sperm DNA fragmentation (SDF) testing based on risk factors using the sperm chromatin structure assay (SCSA). The aim of this retrospective study is to characterise SDF and associated factors (age, semen parameters, smoking status and BMI) for sub-fertile males (n = 1082) and sperm donors (n = 234). The average DFI was 12.1 ± 9.8%. The distribution of men with low, moderate and high SDF (<15, 15–30 and >30%) was 74.8%, 19.4% and 5.8%, respectively. Men with high DFI were older (45 ± 9.5 vs 38 ± 6.7) and had lower percentage of motile sperm (38.8 ± 16.1% vs 55.3 ± 15.8%) than men with normal DFI. Over 17% of the men in the quartile with the highest age and lowest motility had a high DFI (>30%), compared to a high DFI rate of 2-4% for the other 3 quartiles. Repeat testing following lifestyle interventions was available for 29 couples where the men had high initial DFI (35 ± 9.5%). Of these men, 71.4% had a decrease of DFI into the moderate or low range. This study shows that SDF testing can be targeted based on age and sperm motility, thereby reducing unnecessary testing. Furthermore, we provide evidence that lifestyle modifications can reduce DNA fragmentation in men with high DFI.
Introduction
Male factor infertility affects approximately 50% of infertile couples and is one of the leading causes of subfertility amongst couples undergoing IVF treatment (O’Flynn Citation2014; Simon et al. Citation2014). A significant proportion of men with normal semen profiles are still considered infertile (Høst et al. Citation1999; Saleh et al. Citation2003) suggesting that other semen parameters contribute to unexplained infertility, such as genomic factors like DNA damage (Evenson and Wixon Citation2006), spermatozoal RNAs (Ostermeier et al. Citation2004; Rogenhofer et al. Citation2017; Gòdia et al. Citation2018), genome organization (Ioannou and Tempest Citation2018) and epigenetics (Barratt et al. Citation2010). Sperm DNA integrity can be compromised by fragmentation, through a single or double stranded DNA breaks (Osman et al. Citation2015). High levels of sperm DNA fragmentation (SDF) are associated with an increased time to conception, impaired fertilisation, slow early embryo development, higher miscarriage rates and recurrent pregnancy loss after ART (Evenson et al. Citation1980, Citation1999; Morris et al. Citation2002; Carrell et al. Citation2003; Zini et al. Citation2008). Thus, detection of SDF levels can serve as an additional marker of potential fertility and can aid in predicting the success of IVF.
There are many methods of testing SDF including the sperm chromatin structure assay (SCSA), which detects the percentage of denatured spermatozoa using flow cytometry, the TUNEL assay, wherein the ends of fragmented DNA are tagged, and the COMET assay, which utilizes single-cell gel electrophoresis to distinguish DNA fragments. Numerous studies have been conducted using these different assays, mostly finding an inverse relationship between pregnancy rates and high levels of SDF (Tomsu et al. Citation2002; Benchaib et al. Citation2003; Virro et al. Citation2004; Tomlinson et al. Citation2013). While there are benefits to each test, and debate about their relative effectiveness (Simon et al. Citation2014; Ribeiro et al. Citation2017), our practice uses the SCSA as it is the most well studied of the various assays.
Though a link between SDF and fertility has been extensively researched and reviewed, indications for testing are not well defined. Poor quality of evidence (Level C) indicates SDF testing is not recommended for routine use in the evaluation and treatment of the infertile couple (Practice Committee of the American Society for Reproductive Medicine Citation2013). Nonetheless, testing is advocated for several clinical presentations, including varicocele, unexplained infertility, recurrent pregnancy loss, and recurrent implantation failure (Cho et al. Citation2017), though the level of evidence does not rise above ‘C’ for these indications. Testing for patients who fail to conceive with assisted reproduction (ART) or who have unexplained poor embryo development are particularly attractive to help guide a decision regarding the use of donor gametes, when it is unclear whether to use donor sperm or oocytes.
Targeted and useful testing of sperm function beyond the WHO semen analysis remains a challenge. To assist with the application of testing, in 2011 our clinics instituted guidelines to offer SDF testing based on a patient’s medical and fertility history and lifestyle factors. Our clinic guidelines recommended testing for men age ≥ 50, men who smoke, men who have a BMI ≥ 30, men who have large varicoceles, or in cases where couples have experienced prolonged unexplained infertility or unexplained recurrent miscarriage.
Our clinics continue to order SDF testing yet we do not know the effectiveness of our guidelines. Therefore, the aim of this study was to determine the incidence of high DFI after a targeted introduction of DNA fragmentation testing to male factor screening. In addition, we provide further data to characterise the incidence of high DFI and to identify some of its associated factors in a clinically relevant population.
Results
Study demographics
Sperm chromatin integrity of 1316 men was tested using SCSA. Age (41 ± 7.3 vs. 41 ± 0.5), DFI (12.0 ± 9.9 vs. 12.6 ± 9.4%) and total motile sperm (TMS; 71 ± 2.7 vs. 78 ± 6.0 million) were not different for subfertile patients and donors, respectively. This is in contrast to a prior study on donor vs. subfertile patients (Tvrdá et al. Citation2018). Since SDF is performed for all donors who at our center have similar baseline characteristics to subfertile patients, data from patients and donors were combined. The mean DFI was 12.1 ± 9.8%, which is categorized as the ‘low DFI’ category (). The average age was >40 years, reflecting the practice guideline to offer testing to men with advanced age. The population average for semen parameters were within the normal range according to the WHO criteria for concentration (>15M/mL), volume (>1.5 mL) and motility (>40%). BMI was available for 53% of the men tested and averaged 27.1 ± 4.4, while smoking status was known in 73% of participants, of which fewer than 10% were currently smoking and almost 90% had never smoked. According to practice guidelines, patients who presented with high DFI levels (≥30%) were recommended a minimum of 3 months of antioxidant therapy (e.g., Menevit). Of the study population, 104 men reported using antioxidants without providing specifics in terms of dosage and duration; anti-oxidant usage was not known for the other participants. These individuals may have made lifestyle changes and/or used antioxidants.
Table 1. Demographics of the study population.
DFI incidence and associations
High DFI levels (>30%) were found in 5.8% of men tested, with low DFI levels (≤ 15%) in 74.8% of men and moderate levels (15< DFI<30%) for the remaining 19.4%. Patient age increased while sperm motility decreased for each increasing DFI category (p < 0.001; ). This study found no significant correlation between DFI levels and smoking status, BMI, sperm concentration or semen volume.
Table 2. Relationship between age, semen parameters, BMI and DFI category.
In order to determine the incidence of high DFI relative to patient age and sperm motility, patients were allocated to four groups based on quartiles of age and sperm motility (). Note that classification to quartiles of two separate variables yields an uneven number of patients in each group. Patients in group 4 (who were the oldest and had the lowest sperm motility) had the highest incidence of DFI ≥30% (17%), while the remaining 3 groups were non-discriminatory, having similar incidence of high DFI (2-5%). In general, men >45 had lower DFI (11.2 vs 17.0%) and motility (49.4 vs 54.0%) than men ≤45.
Table 3. Average DFI and incidence of high DFI (≥30%) by age and motility quartiles.
Results for patients with repeat SCSA testing
A total of 29 patients with initial high to moderate DFI levels underwent repeat testing, with the majority of these patients having DFI >30% in their first test (n = 21). Of the men with high DFI at initial testing, 71% (15) had DFI < 30% at the subsequent test conducted between 3 to 8 months later. Where data was available, 40.0% (n = 6) reported using antioxidants and/or making lifestyle modifications such as losing weight, improving diet and exercising more frequently.
In this subgroup of 29 men with initial high to moderate DFI, female partners had an average age of 37.5 ± 4.6 and AMH of 9.6 ± 12.7 pM. The average DFI was high (35.6 ± 13.3%); however, all sperm parameters were within the normal range according to WHO classification, indicating an otherwise fertile subset of patients. Thirteen of these 29 men reported never having smoked, none claimed to be presently smoking and one had smoked in the past. The smoking status of the remaining 15 men was unknown.
For patients with repeat testing, the outcomes of ART treatments were also recorded. Of the 29 patients, 14 went on to have live births; 13 were after either IVF or ICSI treatment. Four patients were lost to follow-up. When considering just the patients with high DFI, 4 of the 6 patients who showed no improvement in DFI levels at repeat testing had live births compared to 8 of the 15 where DFI scores decreased.
Discussion
The incidence and impact of high DNA fragmentation is poorly understood, making it difficult to know which patients would benefit from being tested. We found that older men with low sperm motility had higher DFIs than younger men with normal sperm motility. We believe this is new information related to a general adoption of SDF and that this finding will help guide our practice, limiting the application of this test to cases where it has a good chance to identify an affected individual.
In the current study, SDF testing was guided by doctor preference and clinic policy, with an incidence of high DFI (>30%) consistent with other reports. Our result of 5.8% is similar to studies of presumably fertile couples with DFI >30% of 6.1% (Evenson et al. Citation1999) and 3.7% (Giwercman et al. Citation2010) as well as to couples with recurrent miscarriage (6.5%; Leach et al. Citation2015), and couples with unexplained infertility (8.4%; Oleszczuk et al. Citation2013). In contrast, a recent meta-analysis of subfertile patients (those presenting for fertility treatment) found an incidence of high DFI (either >27% or >30% with SCSA) of 20.9% (Zhang et al. Citation2015) which is similar to results from another study where the incidence of DFI>30% was 20.7% for couples with unexplained infertility (Giwercman et al. Citation2010). The variable incidence of high DFI in the literature may be population specific and the low rates of elevated DFI reported here may not apply to other patient populations. Furthermore, our findings that some men with persistent high DFI were able to conceive, albeit in a small subset of patients, is consistent with previous reports (Dar et al. Citation2013).
Reliable diagnosis of definitive male factor infertility remains elusive, particularly when semen parameters are in the normal range (per WHO). Targeted testing with advanced methods, such as SDF, spermatozoal RNA (Ostermeier et al. Citation2004; Rogenhofer et al. Citation2017; Gòdia et al. Citation2018) or sperm aneuploidy (Zidi-Jrah et al. Citation2016) provide evidence that targeted diagnostic testing for genomic integrity has promise. Nonetheless, the standard of care continues to be limited to a routine semen analysis, a test that fails to detect underlying pathologies associated with the paternal genome. The most common advanced test studied, DNA fragmentation, is a genetic structural test that has gained relatively widespread acceptance, even though its clinical relevance is uncertain (Agarwal et al., Citation2016; Practice Committee of the American Society for Reproductive Medicine Citation2013; Cho et al. Citation2017). Given this uncertainty and its significant cost, the test is typically offered to a subset of patients with risk factors associated with oxidative damage, such as smoking, age and obesity (Cohen-Bacrie et al. Citation2009; Belloc et al. Citation2014b) . Furthermore, the test is offered to couples with unexplained infertility (Oleszczuk et al. Citation2013) and recurrent pregnancy loss (Leach et al. Citation2015). While many targeted studies are available on the amount of DNA fragmentation in subsets of patients, the incidence of high DNA fragmentation is poorly understood in the general sub-fertile population. Our goal here was to determine the rate of elevated DNA fragmentation in men presenting to a fertility clinic as patients or as donors. Notably, donors in our program are often recruited by patients, resulting in a donor population that, based on age and semen parameters, could be considered subfertile. Thus results are in contrast to a study that compared clinic recruited young sperm donors to subfertile patients (Tvrdá et al. Citation2018).
Whilst it is known that maternal age negatively impacts fertilization rates and is associated with increased genetic risks, the effect of increasing paternal age on fertility is less pronounced (Schwartz et al. Citation1982; Ford et al. Citation2000). As more men are attempting to have children at later stages in their life, a better understanding of the effects of male age on fertility is needed. In this study, the average age of males seeking treatment was over 40 years, which is significantly older than the reported average age of new fathers in New Zealand (32 years; Statistics NZ, 2010), but close to the average age (39 years) of men in couples presenting with infertility in New Zealand. Recent studies have shown that as paternal age increases, there is a concurrent increase in DNA fragmentation levels and risk of adverse IVF outcomes including fewer high-quality embryos, higher rates of miscarriage and decreased pregnancy rates (Klonoff-Cohen and Natarajan Citation2004; Moskovtsev et al. Citation2006; Belloc et al. Citation2014a; Wu et al. Citation2016; Kaarouch et al. Citation2018).
Many studies have investigated the effect of male age on semen parameters. Although the results are inconsistent, the existing literature generally supports that sperm motility and morphology decrease with age whilst changes in sperm concentration and semen volume are less significant (Schwartz et al. Citation1983; Jacques et al. Citation1995; Fisch et al. Citation1996; Andolz et al. Citation1999; Sloter et al. Citation2006; Harris et al. Citation2011; Belloc et al. Citation2014a). The results of our study are consistent with these findings as it was found that older men were more likely to have higher DFI levels and lower sperm motility. Sperm motility is acquired over a period of several weeks during sperm transit through the epididymis, and thus is more likely to be vulnerable to increased ROS production as a result of aging and other lifestyle factors such as an unhealthy diet and inadequate physical activity (Aitken et al. Citation2007; Sakkas and Alvarez Citation2010). Indeed, studies have linked aging and lifestyle factors such as smoking and alcohol consumption with an increase in testicular ROS production which can have negative impacts on fertility (Aboulmaouahib et al. Citation2018; Shi et al. Citation2018).
Antioxidants are thought to improve sperm quality by reducing oxidative stress (Ross et al. Citation2010; Showell et al. Citation2014; Gual-Frau et al. Citation2015; Martínez-Soto et al. Citation2016). Thus, men use antioxidants with the goal to improve their fertility because antioxidants are relatively non-invasive, readily available and inexpensive compared to other fertility treatments. Of those men with high DFI who retested and lowered their DFI, more than half reported taking antioxidants or making lifestyle changes such as healthy eating and losing weight. These results support the theory that it is possible to reduce DFI levels with antioxidant use, though larger well-controlled trials are needed to confirm this finding.
There has been a lot of debate regarding the use of routine semen analysis to predict DNA integrity; however, most studies find no or poor correlation between semen parameters and DNA fragmentation (Evenson et al. Citation2002; Cohen-Bacrie et al. Citation2009; Boushaba and Belaaloui Citation2015). In this study only sperm motility was significantly associated with SDF, which was also reported by Belloc et al (Belloc et al. Citation2014a). Thus, semen analysis on its own is unlikely to be informative of how much DNA damage has occurred, but instead could serve as a guide to clinicians indicating who might benefit most from sperm DNA testing. If SCSA and other sperm DNA fragmentation tests are to have important clinical value, their use should be limited to patients who present with clear indications.
Evidence based medicine should be a priority for this field and thus, it is important to assess the benefits of adjunct diagnostic tests such as DNA fragmentation testing. In the absence of informative randomised controlled trials (RCTs) one of the challenges faced by fertility clinics is to determine who should be screened for DNA fragmentation (Practice Committee of the American Society for Reproductive Medicine., Citation2013; Harper et al. Citation2017). Diagnostic testing should be applied in a cost-effective manner. Given the low incidence of elevated DNA fragmentation seen in this and other studies (Oleszczuk et al. Citation2013; Leach et al. Citation2015), targeted testing is recommended. Based on the findings of this study, testing could be offered to patients over the age of 45 with sperm motility <40%, or those with recurrent implantation failure. Evidence for other indications for testing such as smoking or high BMI remain controversial but may be indicated on a case by case basis.
Methods
This retrospective analysis included males who underwent SDF testing between April 2012 and April 2016 when presenting for either infertility treatment (n = 1082) or sperm donation (n = 234). Sperm donors were mostly patient-recruited and SDF testing was required. Case notes from the 1316 males (and their partners, where appropriate) were reviewed to identify DNA fragmentation levels and when possible, obtain patient characteristics such as age, sperm motility, semen volume, sperm concentration, smoking status and BMI.
Additional information including female age, AMH levels and ART treatment strategies and outcomes were obtained for those patients who had moderate to high DFI levels and underwent repeat testing (n = 29). The number of patients who achieved live births was also recorded for this subset.
Institutional review and informed consent were not required as per NZ Health and Disability Ethics Committee policy.
Sperm processing and SCSA
Frozen-thawed aliquots of semen samples obtained from four clinic locations were used for evaluation of DNA fragmentation. In brief, semen samples were collected by masturbation into sterile cups after a recommended 2–4 days of sexual abstinence. A routine semen analysis was performed, unless done recently, and a portion of the semen sample was snap frozen in liquid nitrogen. Frozen aliquots were shipped in a dry shipper to a reference laboratory for SCSA analysis as previously described (Evenson et al. Citation1999).
Statistical analysis
Statistical analysis was carried out with JMP software. Impact of semen parameters, male age, BMI and smoking status on DFI were determined with multivariate analysis of variance. Mean DFIs among categories were compared using Tukey-Kramer Multiple Comparisons Test.
Categorical data were expressed as percentages whilst numerical data as mean ± SD. A p-value less than 0.05 was considered statistically significant.
Author contributions
Conceived and designed the experiments: DEM, LC, JP; Performed the experiments: CV; Analyzed the data: DEM; Wrote the manuscript: CV. Edited the manuscript: DEM, LC, JP.
Correction Statement
This article has been republished with minor changes. These changes do not impact the academic content of the article.
Acknowledgments
The authors thank Elizabeth Hammond, PhD, for her assistance with editing the manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
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