Sperm DNA Testing Research Articles

P-088 The Paternal Clock: Shedding Light on the Relationship Between Paternal Age and Sperm DNA Fragmentation

Abstract Study question What is the impact of advanced paternal age (APA) on sperm DNA fragmentation index (DFI)? Summary answer Sperm DFI levels remain relatively stable until the age of 35 and increase progressively beyond that age. What is known already APA can have a negative impact on male fertility and the health of offspring. Previous studies have shown that APA is linked to poor conventional sperm parameters, including decreased semen volume, sperm count, motility, morphology, as well as poor sperm DNA integrity. Additionally, APA has been associated with reduced natural or assisted reproduction and perinatal outcomes and a higher risk of genetic and chromosomal abnormalities in the offspring. Study design, size, duration A retrospective cohort study of 4250 consecutive semen samples from men undergoing infertility evaluation at the OVO clinic, in Montreal, Canada, between April 2016 and December 2022. Participants were stratified into seven age groups: <26 (n = 36; 0,8%), 26-30 (n = 500; 11,8%), 31-35 (n = 1269; 29,9%), 36-40 (n = 1268; 29,8%), 41-45 (n = 732; 17,2%), 46-50 (n = 304; 7,2%), >50 years (n = 141; 3,3%). The mean age was 37.4 ± 6.4 years (range 18-71 years). Participants/materials, setting, methods The study was population-based and included male patients from all ages, ethnicities, and medical histories. Semen samples were collected after 2-3 days of abstinence. For patients who underwent more than one DFI testing, only the first sample was included, and any duplicates were excluded. DFI was evaluated by flow-cytometry based TUNEL assay using the APO-Direct Kit. Data were analyzed using one-way ANOVA and T3 Dunnett post-hoc multiple comparison test, as well as Pearson’s correlation coefficient. Main results and the role of chance The results show a significant positive correlation between %DFI and age (r = 0.19, p < 0.001). Mean %DFI levels were relatively stable in men aged <26 to 35 years (17.9%, 18.1% and 18.1% in men aged <26, 26-30 and 31-35, respectively), with %DFI increasing progressively beyond age 35 (20.7%, 22.5%, 25.7% and 27.9% in men aged 36-40, 41-45, 46-50 and >50, respectively). The mean %DFI in the 36-40 age group was significantly higher than in the 31-35 age group (p < 0,001). Our study has uncovered that %DFI follows an exponential curve starting at age 35, indicating that the %DFI accelerates significantly as men age beyond their mid-30s. Limitations, reasons for caution This retrospective analysis has inherent limitations that may introduce confounding variables. The patient clinical background, such as medical history and lifestyle factors was not assessed. Also, the studied cohort consisted of a population under investigation for infertility and may not be representative of the general male population. Wider implications of the findings The study demonstrates the age-related increase in sperm %DFI and suggests that there may be an age cut-off below which sperm %DFI is stable and beyond which sperm %DFI increases. This information may be useful to medical specialists that offer sperm DNA testing to infertile couples. Trial registration number not applicable

Recurrent MiscarriageGreen-top Guideline No. 17.

In this guideline, recurrent miscarriage has been defined as three or more first trimester miscarriages. However, clinicians are encouraged to use their clinical discretion to recommend extensive evaluation after two first trimester miscarriages, if there is a suspicion that the miscarriages are of pathological and not of sporadic nature. Women with recurrent miscarriage should be offered testing for acquired thrombophilia, particularly for lupus anticoagulant and anticardiolipin antibodies, prior to pregnancy. [Grade C] Women with second trimester miscarriage may be offered testing for Factor V Leiden, prothrombin gene mutation and protein S deficiency, ideally within a research context. [Grade C] Inherited thrombophilias have a weak association with recurrent miscarriage. Routine testing for protein C, antithrombin deficiency and methylenetetrahydrofolate reductase mutation is not recommended. [Grade C] Cytogenetic analysis should be offered on pregnancy tissue of the third and subsequent miscarriage(s) and in any second trimester miscarriage. [Grade D] Parental peripheral blood karyotyping should be offered for couples in whom testing of pregnancy tissue reports an unbalanced structural chromosomal abnormality [Grade D] or there is unsuccessful or no pregnancy tissue available for testing. [GPP] Women with recurrent miscarriage should be offered assessment for congenital uterine anomalies, ideally with 3D ultrasound. [Grade B] Women with recurrent miscarriage should be offered thyroid function tests and assessment for thyroid peroxidase (TPO) antibodies. [Grade C] Women with recurrent miscarriage should not be routinely offered immunological screening (such as HLA, cytokine and natural killer cell tests), infection screening or sperm DNA testing outside a research context. [Grade C] Women with recurrent miscarriage should be advised to maintain a BMI between 19 and 25 kg/m2 , smoking cessation, limit alcohol consumption and limit caffeine to less than 200 mg/day. [Grade D] For women diagnosed with antiphospholipid syndrome, aspirin and heparin should be offered from a positive test until at least 34 weeks of gestation, following discussion of potential benefits versus risks. [Grade B] Aspirin and/or heparin should not be given to women with unexplained recurrent miscarriage. [Grade B] There are currently insufficient data to support the routine use of PGT-A for couples with unexplained recurrent miscarriage, while the treatment may carry a significant cost and potential risk. [Grade C] Resection of a uterine septum should be considered for women with recurrent first or second trimester miscarriage, ideally within an appropriate audit or research context. [Grade C] Thyroxine supplementation is not routinely recommended for euthyroid women with TPO who have a history of miscarriage. [Grade A] Progestogen supplementation should be considered in women with recurrent miscarriage who present with bleeding in early pregnancy (for example 400 mg micronised vaginal progesterone twice daily at the time of bleeding until 16 weeks of gestation). [Grade B] Women with unexplained recurrent miscarriage should be offered supportive care, ideally in the setting of a dedicated recurrent miscarriage clinic. [Grade C].

P-099 The effect of sperm DNA quality on blastocyst and euploid rates

Abstract Study question Do sperm DNA quality measurements influence the blastocyst rate and/or euploid rates? Summary answer Sperm DNA fragmentation has a negative influence on the rate of blastulation. It does not impact on euploidy rates assessed by pre-implantation genetic testing (PGT-A). What is known already Some studies show that sperm DNA has no influence on embryo development and that its negative influence begins following implantation. Other studies show that every step of the fertility process from fertilization onwards is impacted by the male gamete as well as the female gamete. To date, data has been confounded by different sperm DNA tests and different clinical practices and parameters measured. Study design, size, duration This was a retrospective study using the sensitive Comet assay to analyse whether it’s three sperm DNA fragmentation scores influence embryo development or euploidy. Participants/materials, setting, methods Data from 110 cycles of intracytoplasmic sperm injection (ICSI) where the blastocyst rate (number of blastocysts/number of mature oocytes injected) was obtained from the patient database of a UK single centre. Inclusion criteria: Only outcomes from the first assisted reproductive cycle after a Comet assay had been performed were included. Included cycles were within one year of the test. Main results and the role of chance Blastocysts were ranked in 4 groups- 0.25-0.49, 0.5-0.74, 0.75-1.00. Three Comet scores were analysed: Average Comet Score (damage across the semen sample; ACS), Low Comet Score (proportion of sperm with good DNA; LCS) and High Comet Score ( proportion of sperm with high DNA damage; HCS). Clinical thresholds were calculated by comparison with fertile donor sperm. High ACS and LCS and low HCS were all associated with higher blastocyst rates (ANOVA, p = 0.013; Kruskal Wallis, p = 0.027, Post Hoc pairwise comparisons between blastocyst groups (p = 0.016-0.024). No relationship was found between euploid rate (number of euploid embryos/total embryo number) and sperm DNA damage p = 0.761). Limitations, reasons for caution Retrospective design requiring more data to clarify these associations coupled with the lack of randomisation and low power analysis. Wider implications of the findings This study reports the negative impact of sperm DNA fragmentation on blastocyst formation rates. Male partner diagnostic assessments such as sperm DNA quality testing are useful adjuncts to semen analysis. Further research to determine if there is a relationship between sperm DNA damage and euploid embryo outcomes is ongoing. Trial registration number IRB-001C06-01-22

The efficacy of add-ons: selected IVF "add-on" procedures and future directions.

Since the advent of ART, technology has continuously evolved to improve embryology and pregnancy outcomes. However, not all technologies that are integrated into practice have convincing evidence of clinical effectiveness, and they often increase the financial burden of fertility care. We discuss here a selection of commonly utilized IVF "add-ons" and discuss the existing evidence for their utility. The procedures included in this review are time-lapse imaging of embryos, assisted hatching, EmbryoGlue, sperm DNA testing, egg activation with calcium ionophore, endometrial receptivity array, and physiological intracytoplasmic sperm injection (PICSI). While there is rather limited supporting evidence for nearly all IVF add-ons that we reviewed, there is strong demand from patients, physicians, and the biotechnology industry to continue further research and development in this arena. We propose that all add-on procedures should provide true efficacy for the patient, and reproductive endocrinologists should inform patients of the costs and benefits of utilizing various technologies before they undergo treatment. In the future, add-ons that show clear evidence of efficacy and justifiable cost should be incorporated into routine practice, while others that do not meet these criteria should be phased out entirely.

Clinical implications of sperm DNA damage in IVF and ICSI: updated systematic review and meta-analysis.

The clinical effect of sperm DNA damage in assisted reproduction has been a controversial topic during recent decades, leading to a variety of clinical practice recommendations. While the latest European Society of Human Reproduction and Embryology (ESHRE) position report concluded that DNA damage negatively affects assisted reproduction outcomes, the Practice Committee of the American Society for Reproductive Medicine (ASRM) does not recommend the routine testing of DNA damage for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Herein, our aim was to perform a systematic review and meta-analysis of studies investigating whether sperm DNA damage affects clinical outcomes in IVF and ICSI, in order to contribute objectively to a consistent clinical recommendation. A comprehensive systematic search was conducted according to PRISMA guidelines from the earliest available online indexing year until March 2020, using the MEDLINE-PubMed and EMBASE databases. We included studies analysing IVF and/or ICSI treatments performed in infertile couples in which sperm DNA damage was well defined and assessed. Studies also had to include information about pregnancy, implantation or live birth rates as primary outcomes. The NHLBI-NIH quality assessment tool was used to assess the quality of each study. Meta-analyses were conducted using the Mantel-Haenszel method with random-effects models to evaluate the Risk Ratio (RR) between high-DNA-damage and control groups, taking into account the 95% confidence intervals. Heterogeneity among studies was evaluated using the I2 statistic. We also conducted sensitivity analyses and post-hoc subgroup analyses according to different DNA fragmentation assessment techniques. We identified 78 articles that met our inclusion and quality criteria and were included in the qualitative analysis, representing a total of 25639 IVF/ICSI cycles. Of these, 32 articles had sufficient data to be included in the meta-analysis, comprising 12380 IVF/ICSI cycles. Meta-analysis revealed that, considering IVF and ICSI results together, implantation rate (RR=0.74; 95% CI=0.61-0.91; I2 = 69) and pregnancy rate (RR=0.83; 0.73-0.94; I2 = 58) are negatively influenced by sperm DNA damage, although after adjustment for publication bias the relationship for pregnancy rate was no longer significant. The results showed a non-significant but detrimental tendency (RR=0.78; 0.58-1.06; I2 = 72) on live birth rate. Meta-analysis also showed that IVF outcomes are negatively influenced by sperm DNA damage, with a statistically significant impact on implantation (RR=0.68; 0.52-0.89; I2 = 50) and pregnancy rates (RR=0.72; 0.55-0.95; I2 = 72), although the latter was no longer significant after correction for publication bias. While it did not quite meet our threshold for significance, a negative trend was also observed for live birth rate (RR=0.48; 0.22-1.02; I2 = 79). In the case of ICSI, non-significant trends were observed for implantation (RR=0.79; 0.60-1.04; I2 = 72) or pregnancy rates (RR=0.89; 0.78-1.02; I2 = 44), and live birth rate (RR=0.92; 0.67-1.27; I2 = 70). The current review provides the largest evidence to date supporting a negative association between sperm DNA damage and conventional IVF treatments, significantly reducing implantation and pregnancy rates. The routine use of sperm DNA testing is therefore justified, since it may help improve the outcomes of IVF treatments and/or allow a given couple to be advised on the most suitable treatment. Further well-designed controlled studies on a larger number of patients are required to allow us to reach more precise conclusions, especially in the case of ICSI treatments.

Seasonal Changes of Nuclear DNA Fragmentation in Boar Spermatozoa in Spain.

Simple SummaryArtificial insemination is widely used in pig production and currently a boar performs several thousand matings per year. Traditionally sperm quality is focused on the number of spermatozoa, their motility and morphology. However, the quality of sperm DNA, which contains genetic information, is also related to fertility problems. The aim of this research was to study the effect of natural light hours and age of the boar on the status of the sperm DNA. After a powerful statistical analysis, it was found that the percentage of spermatozoa with fragmented DNA decreases within the observed age range as the boar gets older. On the other hand, the amount of spermatozoa with fragmented DNA was the lowest in autumn while it was the highest in summer. This study demonstrates the remaining seasonality of boars in Spain and highlights the importance of controlling the environmental conditions in the farms. Sperm DNA testing provides a basis for improving the selection of AI boars by excluding males with higher DNA fragmentation due to their very young reproductive age that may pose a potential subfertility.There are numerous cases when conventional spermiogram parameters are all within an acceptable range but boar subfertility persists. The total sperm nuclear DNA fragmentation index (tDFI) is a trait related to fertility and prolificacy problems that is not routinely evaluated in commercial AI boars. The aim of this research was to study the effect of the photoperiod, season and reproductive age of the boar on tDFI (measured by SCSA) of 1279 ejaculates from 372 different boars belonging to 6 different breeds located in 6 AI studs in Spain. tDFI data ranged from 0.018% to 20.1%. Although there was a significant single boar effect in the tDFI occurrence, a negative correlation between the tDFI and the age of the boar was found (p < 0.001). tDFI would decrease due to aging of the boar 0.66% each year old within the observed age range. After including age as a covariate in the ANCOVA, no differences were found in tDFI between photoperiods when the sperm collection date was evaluated. However, when the date of the production of semen in the testis was evaluated, the total percentage of spermatozoa with fragmented nuclear DNA was 1.46% higher in the increasing photoperiod in comparison to the decreasing photoperiod (p < 0.0001). On the other hand, for both dates, the lowest tDFI values corresponded to minimum day length for decreasing photoperiod phase (autumn), while the highest tDFI values were found in summer (maximum day length for decreasing photoperiod phase).

IMPACT OF SINGLE AND MULTIPLE SPERM ABNORMALITIES AND LOW-LEVEL LEUKOCYTOSPERMIA ON SPERM DNA

&#x0D; Background and objective&#x0D; The aim of the present study was to identify the impact of defective standard sperm parameters individually and in combination on DNA damage in a large cohort of infertile men.&#x0D; Material and methods&#x0D; Retrospective analysis of semen characteristics was conducted on 436 patients. DNA fragmentation analysis was performed by using the terminal deoxynucleotidyl transferase (TdT)-mediated fluoresce-in-dUTP nick end labeling (TUNEL) assay. Sperm parameters were arranged into different categories such as normospermia asthenospermic, teratospermic, asthenoteratospermic, and oligoasthenoteratospermic. GraphPad Prism version 7 software was used for data analysis.&#x0D; Results&#x0D; Our results suggest that the mean percentage of DNA damage was proportionally higher than the semen abnormalities. Sperm with 3 abnormal parameters showed significantly higher DNA damage, suggesting that sperm having more than 2 abnormalities are more likely to have higher DNA damage.&#x0D; Conclusion&#x0D; Sperm motility had significant correlation and is supposed to be a predictor for these tests, while morphology was the second standard sperm parameter inversely correlated with sperm DNA damage. Patients demonstrating low levels of leukocytospermia should be advised sperm DNA testing before assisted reproductive technology treatment. However, there is a clear need for more research studies to further address these issues.&#x0D; &#x0D; &#x0D;

CASA in the medical laboratory: CASA in diagnostic andrology and assisted conception.

CASA has been used in reproductive medicine and pathology laboratories for over 25 years, yet the 'fertility industry' generally remains sceptical and has avoided automation, despite clear weaknesses in manual semen analysis. Early implementers had difficulty in validating CASA-Mot instruments against recommended manual methods (haemocytometer) due to the interference of seminal debris and non-sperm cells, which also affects the accuracy of grading motility. Both the inability to provide accurate sperm counts and a lack of consensus as to the value of sperm kinematic parameters appear to have continued to have a negative effect on CASA-Mot's reputation. One positive interpretation from earlier work is that at least one or more measures of sperm velocity adds clinical value to the semen analysis, and these are clearly more objective than any manual motility analysis. Moreover, recent CASA-Mot systems offer simple solutions to earlier problems in eliminating artefacts and have been successfully validated for sperm concentration; as a result, they should be viewed with more confidence in relation to motility grading. Sperm morphology and DNA testing both require an evidence-based consensus and a well-validated (reliable, reproducible) assay to be developed before automation of either can be of real clinical benefit.

Evaluation of sperm DNA quality in men presenting with testicular cancer and lymphoma using alkaline and neutral Comet assays.

Despite more cancers in young men over the past two decades, improvements in therapies give a greater chance to live full lives following treatment. Sperm genomic quality is variable following cancer diagnosis, so its assessment is important if sperm cryopreservation is being considered. Here, we evaluated DNA damage using two DNA damage assays: an alkaline and for the first time, a neutral Comet assays in men presenting with testicular cancer (n=19 for alkaline and 13 for neutral group) and lymphoma (n=13 for alkaline and 09 for neutral group) compared with fertile donors (n=20 for alkaline and 14 for neutral group). No significant differences were observed in any semen analysis parameters. In contrast, sperm DNA damage was higher in men with testicular cancer than in donors as assessed by both the alkaline (12.4% vs. 37.4%, p<0.001) and neutral (7.5% vs. 13.4%; p<0.05) Comet assays. Similar trends were observed in men with lymphoma. Here, sperm DNA damage was higher using both the alkaline (35.0% vs. 12.4%) and neutral (10.7% against 7.5% (p<0.05) Comet assays. Moreover, the DNA strand breaks (particularly double-strand breaks) were significantly more prominent in men with cancer having abnormal seminal parameters than normozoospermic ones. This study showed that sperm DNA testing using alkaline and neutral Comet assays is more sensitive than semen analysis in detecting impaired sperm quality in men presenting with cancer. It may provide a useful adjunct when considering storage prior to cancer investigations and assisted reproductive techniques (ART)-based treatment.

The benefits and limitations of sperm DNA testing in clinical practice.

Over the past 40 years, numerous studies on the composition, organization and function of the sperm chromatin have allowed us to better understand the role of this structure in maintaining DNA integrity, and, in fertilization and embryonic development.

Commentary: sperm DNA fragmentation testing in action.

The expert panel proposed a guideline on the clinical utility of sperm DNA fragmentation testing (1). Rapid advancement in the field of sperm DNA testing over the last two decades was summarized and structurally presented by the four clinical scenarios in an evidence-based approach.

Commentary: the value of testing sperm DNA fragmentation in infertile men.

Over the past 20 years, the necessity and usefulness of clinically testing for DNA fragmentation in spermatozoa has been increasingly researched and debated. Studies have shown that sperm with impaired DNA integrity are related to prolonged time to pregnancy, significantly lower conception rates both naturally and after intrauterine insemination (IUI), as well as increased pregnancy loss. High levels of sperm DNA damage have also been linked to poor integrity of the embryonic genome and impaired embryo development. However, both scientists and clinicians remain divided as to whether the potential information that the measurement of immature chromatin or fragmented DNA in spermatozoa may offer, in addition to that provided by routine semen analysis, is actually relevant in a clinical setting. In a recent paper, a select team of authorities in the field of male infertility recommended a practice guideline pertaining to the clinical value of sperm DNA fragmentation assessment (1). The group employed an evidence-based approach to describe clinical scenarios that most warranted sperm DNA testing as well as the management of patients with high sperm DNA fragmentation.

The role of sperm DNA testing on male infertility.

Male factor is responsible for nearly 50% of all causes of infertility. Approximately 30–40% of the cases are deemed idiopathic, as there are no identifiable factors to explain the abnormal semen analysis results. Semen analysis (sperm concentration, motility and morphology) is still used for routine male infertility assessment; however, these parameters have shown to be limited as surrogate markers of male fertility. In fact, nearly 15% of patients who are suffering from male infertility have a normal semen analysis.

1H Magnetic Resonance Spectroscopy of live human sperm.

STUDY QUESTIONCan 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa?SUMMARY ANSWERPercoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface.WHAT IS KNOWN ALREADYCurrent methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm.STUDY DESIGN, SAMPLES/MATERIALS, METHODSSperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet (‘80%’ sperm) and the 40/80 interface (‘40%’ sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of ‘40%’ and ‘80%’ sperm.MAIN RESULTS AND THE ROLE OF CHANCEDGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65–0.67, lactate/lipid ROC AUC = 0.86–0.87.LIMITATIONS, REASONS FOR CAUTIONOnly 3–4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the ‘40%’ and ‘80%’ sperm.WIDER IMPLICATIONS OF THE FINDINGS 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility.STUDY FUNDING AND COMPETING INTEREST(S)This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.

Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment

Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)? Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study. Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown. Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013. Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated. Histone retention was associated with sperm DNA damage (P < 0.001), reduced embryo quality (P = 0.005) and clinical pregnancies (P < 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P < 0.001) and alkaline Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P < 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 ± 1.46 versus 14.93 ± 1.65; P = 0.379). Of the protamine parameters analysed, only the P1/P2 ratio was associated with sperm count (P = 0.013), men's age (P = 0.037), maturity (P = 0.049) and blastocyst quality (P = 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P < 0.05), embryo quality (P < 0.05) and implantation rate (P < 0.05). A potential drawback of this study is that it is cross-sectional. Generally in such studies there is more than one variable that could cause the effect. Analysing sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART outcomes. Therefore, given the large and well-established role of female factors in infertility, normal sperm DNA integrity and protamination do not necessarily ensure clinical pregnancy in ART. Thus, female factors can reduce the prognostic value of sperm DNA tests. Further, our use of native semen instead of prepared sperm may have iatrogenically increased the DNA damage. Alteration in sperm nuclear protein affects sperm DNA integrity. Further, with the current dataset, TUNEL and Comet assays appeared more predictive of ART success than FCCE. No personal or direct financial support has been received for any of this work. The authors declare no competing interests. N/A.

High prevalence of isolated sperm DNA damage in infertile men with advanced paternal age

Sperm DNA damage is associated with male infertility, lower pregnancy rates and pregnancy loss. The primary aim of our study was to evaluate the prevalence of sperm DNA damage in younger and older men with normozoospermia. We obtained semen from 277 consecutive non-azoospermic men presenting for sperm DNA testing. The main outcome measures included sperm % DNA fragmentation index (%DFI, using sperm chromatin structure assay), sperm concentration, motility and morphology, and, paternal age. Sperm % DFI was positively correlated with paternal age (r = 0.20, P < 0.001) and inversely correlated % progressive motility (r = -0.16, P = 0.01). Sperm %DFI was significantly higher in older (≥40 years) compared to younger (<40 years) normozoospermic men (17 ± 13 vs. 12 ± 8, respectively P = 0.008), whereas, sperm concentration, progressive motility and morphology were not significantly different in these two groups. Moreover, the prevalence of high levels of sperm DNA damage (>30 % DFI) was significantly higher in older compared to younger normozoospermic men (17 % vs. 3 %, respectively, P < 0.001). The data indicate that a conventional semen analysis can often fail to detect a defect in spermatogenesis (high %DFI) in older men and suggest that infertile couples with advanced paternal age, including those with normal semen parameters, should consider sperm DNA testing as part of the couple evaluation.

The place of sperm DNA fragmentation testing in current day fertility management

Abstract In this debate article, I am going to set out the case that sperm DNA fragmentation testing is essential in current day fertility management because- • Our current semen analysis testing is unfit for purpose • Sperm DNA damage testing has strong associations with every fertility check point • Sperm DNA damage testing has strong associations with miscarriage • Sperm DNA testing can explain ‘unexplained’ infertility • There are reasons why sperm with poor DNA are successful in ICSI • There are no non-invasive sperm function tests that provide the same information • We need to take a fresh look at the ‘evidence’ against sperm DNA testing • We have no reason to wait. There are benefits for clinics and couples alike.

Correlation between DNA defect and sperm-head morphology

The utility of sperm DNA testing remains controversial. However, it may be helpful in couples with unexplained failures of multiple assisted reproductive techniques and/or recurrent abortions. This study analysed 10,400 spermatozoa of 26 patients for sperm-head morphology with high-magnification microscopy, DNA fragmentation and sperm chromatin decondensation. A significant negative correlation was demonstrated between sperm-parameters and abnormal sperm-head morphology as assessed by high magnification (score 0 according to this study’s classification): concentration ( r = −0.41; P = 0.03), motility ( r = −0.42; P = 0.03), morphology ( r = −0.63; P = 0.0008). No correlation was found with DNA fragmentation. However, the sperm chromatin-decondensation rate of score-0 spermatozoa was twice as high as the controls (19.5% versus 10.1%; P < 0.0001). This observation suggests that score-0 spermatozoa should not be selected for intracytoplasmic sperm injection. We analysed 10,400 spermatozoa of 26 patients for sperm head morphology at high magnification, DNA fragmentation and sperm chromatin decondensation. We demonstrated a significant negative correlation between sperm parameters: concentration ( r = –0.41; P = 0.03), motility ( r = −0.42; P = 0.03), morphology ( r = –0.63; P = 0.0008) and abnormal sperm head morphology as assessed by high magnification (score 0 according our classification). No correlation was found with DNA fragmentation. The sperm chromatin decondensation rate of score-0 spermatozoa was twice as high as than in controls (19.5% versus 10.1%; P < 0.0001). No significant relationship with sperm DNA fragmentation was observed. We suggest that high-magnification sperm selection could be an important step and a new tool for the clinician, who could decide to discard score-0 spermatozoa with a high risk of abnormal chromatin and select the best sperm cells for intracytoplasmic sperm injection.

Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success

To evaluate sperm DNA fragmentation and semen parameters to diagnose male factor infertility and predict pregnancy after IVF. Prospective study. Academic research laboratory. Seventy-five couples undergoing IVF and 28 fertile donors. Sperm DNA fragmentation was measured by the alkaline Comet assay in semen and sperm after density gradient centrifugation (DGC). Binary logistic regression was used to analyze odds ratios (OR) and relative risks (RR) for IVF outcomes. Semen parameters and sperm DNA fragmentation in semen and DGC sperm compared with fertilization rates, embryo quality, and pregnancy. Men with sperm DNA fragmentation at more than a diagnostic threshold of 25% had a high risk of infertility (OR: 117.33, 95% confidence interval [CI]: 12.72-2,731.84, RR: 8.75). Fertilization rates and embryo quality decreased as sperm DNA fragmentation increased in semen and DGC sperm. The risk of failure to achieve a pregnancy increased when sperm DNA fragmentation exceeded a prognostic threshold value of 52% for semen (OR: 76.00, CI: 8.69-1,714.44, RR: 4.75) and 42% for DGC sperm (OR: 24.18, CI: 2.89-522.34, RR: 2.16). Sperm DNA testing by the alkaline Comet assay is useful for both diagnosis of male factor infertility and prediction of IVF outcome.

Session 16: New Approaches for Sperm DNA Testing

Session 16: New Approaches for Sperm DNA Testing