Sperm DNA Fragmentation Testing Research Articles

O-210 Sperm DNA fragmentation (SDF) is not associated with adverse maternal and neonatal outcomes in IVF-ICSI cycles with autologous oocytes

Abstract Study question Does an elevated SDF (>15%) increase the odds of adverse maternal and neonatal outcomes in autologous oocyte IVF-ICSI cycles from unselected couples? Summary answer No adverse effects of high SDF on obstetric and neonatal outcomes have been found in couples with sperm fragmentation undergoing IVF-ICSI cycles with own eggs. What is known already Sperm chromatin integrity assessment has been implemented as an additional tool in the clinical evaluation of sperm quality in infertile patients undergoing an assisted reproduction treatment. All of the published reports to date appraise its effect on clinical outcomes, and how it impacts embryo quality and the pregnancy chances after IVF and ICSI cycles. Sperm DNA integrity has also been hypothesized to affect offspring health but not many studies have reported in humans if an elevated SDF raises the risks of obstetric, delivery and neonatal outcomes. Study design, size, duration Multicentric retrospective cohort study of all IVF-ICSI cycles using autologous oocytes between January 2000-March 2019 at Spain IVIRMA clinics of couples with a SDF test on their ejaculated semen. The sperm fragmentation index was measured in all men with TUNEL assay. The database included 228 couples which had a delivery with at least a newborn. Subjects were divided into two study groups according to their level of SDF: ≤15% (low SDF) or > 15% (high SDF). Participants/materials, setting, methods Patients with missed information on maternal and neonatal outcomes were not counted for the analysis. The obstetric outcomes were gestational age, gestational diabetes, preeclampsia (hypertension with proteinuria after 20 weeks of gestation) and type of delivery. Neonatal outcomes were sex, birth weight, length, head circumference, Apgar score at 1, 5, 10 minutes, and neonatal intensive care unit (NICU) admission. Student’s t-test and Fisher’s test were used for statistical analysis. A p-value<0.05 was considered statistically significant. Main results and the role of chance Maternal age mean was 37.4 years (95%CI 36.9-38.0) in ≤ 15%SDF group and 37.2 years (95%CI 36.1-38.4) in > 15%SDF group (p = 0.8). Similar gestational age was found, 41.8 weeks (95%CI 41.3-42.2) in ≤ 15%SDF and 41.3 weeks (95%CI 40.4-42.3) in > 15%SDF. Gestational diabetes incidence was higher in > 15%SDF compared to ≤ 15%SDF group (3.5% versus 1.7% (OR = 2.0 (95%CI 0.03-39.8), p = 0.5). Equally, the incidence of preeclampsia was 3.6% in patients with high SDF versus 1.7% in couples with low SDF, OR = 2.1 (95%CI 0.03-41.3), p = 0.5. Type of delivery frequency was in the ≤15%SDF group 61.9% vaginal and 38.1% cesarean, while in the >15%SDF group 62.1% vaginal and 37.9% cesarean (OR = 1.0 (95%CI 0.4-2.6), p = 1.0). The overall proportion of singleton pregnancies was 87.2% (95%CI 82.4-91.2) and twins 12.8% (95%CI 8.8-17.6). There were no statistically differences between groups in the rate of delivery of twins and in the sex ratio of the newborns. When comparing the newborns of ≤ 15%SDF with >15%SDF group, the average of weight was 3011.7g (95%CI 2912.2-3111.2) versus 2986.4g (95%CI 2753.1-3219.7), of length was 49.2cm (95%CI 48.3-50.0) versus 49.5cm (95%CI 49.2-49.9), of head circumference was 34.9cm (95%CI 34.6-35.2) versus 34.3cm (95%CI 33.4-35.2). No statistically differences were observed for Apgar punctuation and for NICU admission. Limitations, reasons for caution Due to the retrospective nature of the study we have missing data from the lack of follow-up of many patients after the confirmation of the ongoing pregnancy. Although pregnancies of couples with elevated SDF have a higher incidence of gestational diabetes and preeclampsia, the sample size evaluated is a limitation. Wider implications of the findings This is one of the first reports to evaluate the relationship between paternal DNA damage and obstetric risks and neonatal health in couples with high SDF who underwent IVF-ICSI in our centers. Despite SDF did not jeopardize the maternal and neonatal outcomes, more studies are needed to confirm this conclusion. Trial registration number NA

P–080 Higher sperm DNA fragmentation reduces the proportion of good quality embryos at day 5 on IVF and ICSI cycles from unselected males

Abstract Study question Does sperm DNA fragmentation (SDF) reduce the ratio of good-quality embryos in day 3 (D3) and day 5 (D5) of embryonic development? Summary answer High sperm DNA fragmentation (SDF >15%) is associated with poor embryo quality at blastocyst-stage per cycle in unselected patients undergoing IVF and ICSI. What is known already It has been shown that the proportion of spermatozoa with DNA fragmentation is higher in infertile men than in semen from fertile men. However, the controversy regarding the impact that sperm genome damage can have on IVF or ICSI treatments is evident in the published literature. The effects of SDF would become evident after activation of the embryonic genome at 8-cell stage, compromising not only the quality of the embryos obtained, but also the reproductive outcomes, as reduced implantation rates, higher miscarriages rates and thus, a decreased chance of pregnancy. Study design, size, duration This multicentric observational retrospective study included 1339 couples who underwent 2759 IVF-ICSI cycles using autologous oocytes from January 2000 to March 2019. All men have an SDF test in their ejaculated spermatozoa by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling). The subjects were divided into two groups according to their sperm DNA integrity: low (≤15%) (n = 2287 cycles) or high (>15%) (n = 472) SDF. Participants/materials, setting, methods Embryo quality was assessed complying morphological standards at cleavage-stage on D3 and at blastocyst-stage on D5 (inner cell mass (ICM) and trophectoderm (TE) grade (A, B, C or D)) in according to ASEBIR’s embryo selection criteria, being embryos of good quality those categorized as A+B. The outcomes were calculated in relation to the total number of zygotes obtained. The results were compared by Student t test; p value <0.05 was considered significant. Main results and the role of chance The SDF average of the low group was 5.8% (95% CI 5.6–5.9) whereas in high group was 23.7% (95% CI 23.0–24.4). The female age was equal, 37.1 years (95%CI 37.0–37.2) and 37.1 years (95% CI 36.8–37.4) respectively. A total of 9796 embryos were evaluated. The optimal cleavage-stage embryo rate per cycle was 25.0% (95% CI 21.7–28.3) (8.0 average cells number, 1.5 embryo fragmentation average, symmetry 1, mononucleated cells) versus 26.7% (95%CI 19.1–34.2) (7.9 average cells number, 1.8 embryo fragmentation average, symmetry 1, mononucleated cells) when comparing between groups (p < 0.001). Blastocyst-stage arrival rate (number of embryos at D5) per cycle was 55.8% (95% CI 54.3–57.2) in ≤ 15% SDF group (embryo quality score was ICM A:12.1%, B:69.5%, C:8.8%, D:4.5%; TE A: 7.5%, B:42.2%, C:42.2%, D:8.1%) and 55.9% (95% CI 52.8–59.1) in the >15% SDF group (ICM A:12.0%, B:68.7%, C:10.6%, D: 5.2%; TE A:9.1%, B:44.8%, C:37.8%, D:8.3%) (p < 0.001). The good quality blastocyst rate per cycle was significantly higher in the group with SDF ≤15%, 27.7% (95%CI 26.5–29.0) versus SDF >15% (27.4% (95%CI 24.6–30.2)). Of the total number of blastocysts, the proportion of A+B blastocyst was 60.5% (95% CI 58.3–62.7) and 64.2% (95% CI 59.2–69.2) (p < 0.001), respectively. Limitations, reasons for caution The retrospective and multicenter nature of this study leads to uncontrolled biases derived from the clinical practice. Although the results were not adjusted for female’s age, it was not statistically different between groups. Embryo morphology evaluation was performed by senior embryologists, it still remains a subjective evaluation, though. Wider implications of the findings: In this study, a higher amount of data was compiled so that a large number of embryos were analyzed. The DNA integrity of the sperm may be an important consideration when poor quality embryos were obtained in the previous cycle when deciding on the next clinical strategy to apply. Trial registration number NA

P–058 The effect of sperm DNA fragmentation on intracytoplasmic sperm injection (ICSI) outcome

Abstract Study question Does the sperm DNA fragmentation (SDF) level impact the clinical outcome of couples undergoing intracytoplasmic sperm injection (ICSI)? Summary answer No significant effect was observed for SDF on the reproductive outcome of couples undergoing ICSI. What is known already Sperm DNA Fragmentation (SDF) has emerged as an important biomarker in the assessment of male fertility potential. It is currently being used as one of the advanced sperm function tests along with other conventional methods in male fertility evaluation. The impact of SDF on the reproductive outcomes of ICSI remains to be controversial. Evidence extracted from three meta-analyses have indicated that higher SDF is not associated with a negative impact on ICSI outcomes. On the contrary, another meta-analysis revealed that SDF can have a significant impact on the pregnancy rate of ICSI with an OR of 1.31. Study design, size, duration This is a retrospective cohort study carried out in the assisted conception unit of a tertiary medical center. The study duration was over a 5-year period from August 1st, 2014 to August 1st, 2019. The charts of 1922 patients who underwent ICSI were screened for inclusion in the study. Inclusion criteria were patients who underwent ICSI using ejaculate spermatozoa and had a recorded SDF test done within a week before ICSI (n = 390). Participants/materials, setting, methods Sperm chromatin dispersion was used to evaluate SDF utilizing the Halosperm G2 test kit (Halotech, Madrid, Spain). All patients performed the ICSI trial using ejaculated spermatozoa. Patients were divided according to the SDF level into 3 groups; SDF <20% (n = 148), SDF 20–30% (n = 133), and SDF >30% (n = 109). Female partner fertility status was recorded and couples were grouped into 2 groups based on age and AMH levels; (1) favorable female and (2) unfavorable female status. Main results and the role of chance Overall, clinical pregnancy occurred in 45% of cases, live birth rate was 33.60%, and 1.30% of patients had miscarriage. A significant negative correlation between SDF and sperm count (r–0.232), motility (r–0.469), progressive motility (r–0.312) and normal morphology (r–0.297) was detected (p < 0.001 for all). Fertilization rate, clinical pregnancy and live birth rate were greater in patients with lower SDF than those with higher SDF in both favorable and unfavorable groups, however the difference was not statistically significant (Table 1). Limitations, reasons for caution The main limitation of our study was the retrospective nature of the study where some data may be missing or incomplete. The data was also retrieved from one ART center, therefore our data lacked diversity within methodologies for IVF and SDF testing. Wider implications of the findings: SDF was found to be significantly correlated with conventional semen parameters highlighting its significance as a robust diagnostic test during male fertility evaluation. In this study, while patients with higher SDF values had worse reproductive outcomes with ICSI, the results did not reach statistical significance. Trial registration number NA

O-207 Which infertile patients mostly deserve to have a sperm DNA fragmentation index done? Findings from a cross-sectional study

Abstract Study question Current scientific guidelines do not clearly suggest which patients would benefit the most from a sperm DNA fragmentation (SDF) test. Summary answer We aimed to investigate potential predictive factors for altered SDF in a homogenous cohort of white-European men presenting for primary couple’s infertility. What is known already High SDF has been associated with reduced fertilization rates, reduced chances of natural conception and an increased risk of early pregnancy loss. Study design, size, duration Data from 478 consecutive men with normal or altered SDF were analysed. Infertility was defined according to the WHO criteria. Semen analysis, SDF (according to SCSA) and serum hormones were measured in every patient. Health significant comorbidities were scored with the Charlson Comorbidity Index (CCI). Altered SDF was considered with a threshold of > 30%. Participants/materials, setting, methods Descriptive statistics compared the overall characteristics of patients with normal SDF and altered SDF. Logistic regression analysis tested potential predictors of altered SDF. ROC curve was used to test the accuracy of the model in predicting SDF alteration Main results and the role of chance Of 478 patients, 253 (57.7%) had altered SDF. Median (IQR) age and BMI of the whole cohort were 38 (35-42) years and 25.1 (23.3-27.1) kg/m2 respectively. Patients with altered SDF were older (median (IQR) age: 39 (36-43) vs. 37 (34-38) years, p < 0.0001), had lower sperm concentration (5 (1.1–18) vs. 17 x106/mL (6–38.8), p < 0.0001), testicular volume (15.1 (12 –20) vs. 16.8 (12 – 25) Prader, p = 0.0005), and total motile sperm count (TMSC) (1.8 (0.21–10.71) vs. 11.8x106 (2–37.26), p < 0.0001). Conversely, men with altered SDF had higher FSH (6.1 (3.85–9.7) vs. 4.8 (3.85 – 7.9) mIU/mL, p < 0.0001) and prolactin levels (9.8 (7.43–14.04) vs. 8.3 (6.6–11.3) pg/mL, p = 0.0004) than those with normal SDF. At multivariable logistic regression analysis, patients’ age >35 years (OR: 2.45, p = 0.0009), FSH > 8.0 mIU/mL (OR: 2.23, p < 0.0001) and lower TMSC (OR: 2.04, p = 0.002) were identified as indipendent predictors of altered SDF, after adjusting for testicular volume and CCI≥1. ROC curve (Figure 1) revealed that the model has a good predictive ability to identify patients with SDF alteration (AUC: 0.72, 95%CI: 0.67 - 0.77). Limitations, reasons for caution It is a retrospective analysis at a single, tertiary-referral academic centre, thus raising the possibility of selection biases. In spite of this, all patients have been consistently analysed over time with a rigorous follow-up, thus limiting potential heterogeneity in terms of data reporting Wider implications of the findings Primary infertile men older than 35 years, with high serum FSH and low TMSC at baseline are the ones who mostly deserve a SDF test over their diagnostic work-up and that would potentially benefit the most of certain treatments to improve SDF value, thus increasing chances of conceiving. Trial registration number Not applicable

P–009 A modified sperm chromatin dispersion test, LensHooke® R10, for quick and accurate determination of human sperm DNA fragmentation

Abstract Study question The performance and efficiency of the LensHooke® R10 test kit were evaluated by the clinical examination for precision, accuracy, and time. Summary answer The LensHooke® R10 based on sperm chromatin dispersion test offers not only quick testing for sperm DNA fragmentation but also reliable and accurate test results. What is known already Sperm chromatin dispersion (SCD) test, one of the most commonly used testing for sperm DNA fragmentation (SDF), can be conducted promptly and without the need for expensive laboratory instruments. However, the main disadvantage of the SCD test is inter-observer variability in categorizing the size of characteristics halos surrounding the core of sperm. Moreover, it takes more than one hour to accomplish whole assay procedures making this testing an inefficient diagnostic tool. These may hinder its broad availability among andrology laboratories or prevent it from being routinely used for the evaluation of male infertility. Study design, size, duration A total of 108 participants was included in this prospective study. Data was collected from the reproductive medicine center between June and December 2020. Participants/materials, setting, methods This study included 108 consecutive male partners of couples attending for assisted reproductive treatment. SDF was simultaneously tested by using LensHooke® R10 (R10) and Halosperm® G2 (G2) respectively. We evaluated the correlation and agreement between two SCD-based test kits. The repeatability and reproducibility of the SCD kits were assessed by intra-and inter-observer agreement experiments. The sensitivity, specificity, positive predictive value, negative predictive value for the R10 was determined by receiver operator characteristics (ROC) curve analysis. Main results and the role of chance The R10 produced more clear sperm core and dispersed chromatin, therefore highly recognizable images can be easily and accurately categorized when scoring of SDF. It took 50% less time for SDF testing by the R10 compared to the G2 (38.26 ± 9.85 minutes vs. 76.52 ± 19.7 minutes, P < 0.0001). The SDF% results showed a strong correlation for the R10 and G2 with Spearman’s coefficients of rank correlation (rho) above 0.8 (P < 0.0001, N = 108). The R10 showed 89.8% accuracy with 87.9% sensitivity, 90.8% specificity, 82.9% PPV, and 93.7% NPV on the measurement of SDF% at the threshold value of 22%. Intraclass correlation coefficients (ICC) >0.9 showed a strong agreement between two observers on the testing of SDF using the R10. ICC >0.9 showed a high intra-observer agreement within 4 repeated testing on SDF using the R10. The R10 showed an intra-observer’s precision of coefficient variation, CV < 10% for SDF%. In addition, SDF% test results obtained by the R10 for asthenospermic (31.8% ± 16.7%), teratospermic (22.9% ± 14.4%), and oligoasthenoteratozoospermic samples (36.6% ± 14.4%) were significantly higher than that observed in normozoospermic samples (15.3% ± 10.2%, p < 0.05), was comparable with the G2. Limitations, reasons for caution The sample size of 4 semen specimens used to evaluate the intra-and inter-observer agreement was a limitation. Besides, evaluating the relationship between the SDF and clinical outcome of ART is necessary for further study. Wider implications of the findings: The new in vitro diagnostics reagent, LensHooke® R10, is a simple and quick test kit that offers reliable and accurate test results of sperm DNA fragmentation, can be routinely used in male infertility evaluation. Trial registration number CS2–20012

The effect of sperm DNA fragmentation on intracytoplasmic sperm injection outcome.

Our study objective was to assess the effect of various sperm DNA fragmentation levels on clinical intracytoplasmic sperm injection outcome. This retrospective study included 392 patients who underwent ICSI and performed sperm DNA fragmentation testing before the procedure. Based on sperm DNA fragmentation cut-off values, the patients were differentiated into 3 groups as <20%, 20%-30% and >30%. According to the female status, patients were differentiated into favourable group (n=259) with female age <35years and anti-Mullerian hormone level ≥7.1pmol/L; and unfavourable group (n=133) with female age ≥35years and anti-Mullerian hormone level ≤7.1pmol/L. The patient's medical records were reviewed, and patient's demographic, laboratory data including semen analysis, sperm DNA fragmentation determined by means of sperm chromatin dispersion, hormonal profile and data regarding intracytoplasmic sperm injection cycle were collected. This cohort reported that the clinical reproductive outcomes of intracytoplasmic sperm injection showed no statistical significance with increase sperm DNA fragmentation levels. In sperm DNA fragmentation above 30%, favourable females had significantly higher clinical pregnancy rate and live birth rate than unfavourable females, while fertilisation rate and miscarriage rate showed no significance between the subgroups. High sperm DNA fragmentation is linked to poor semen parameters.

Evaluation of the sperm DNA fragmentation index in infertile Japanese men by in-house flow cytometric analysis.

Semen analysis has long been used to evaluate male fertility. Recently, several sperm function tests have been developed. Of those, the sperm DNA fragmentation index (DFI), which describes the status of the sperm DNA, is thought to be a suitable parameter for evaluating male fertility. However, there have been no large-scale studies on the sperm DFI of Japanese men. Therefore, we investigated the feasibility of using an in-house flow cytometry-based sperm DFI analysis based on the sperm DNA fragmentation test of sperm chromatin structure assay (SCSA) to assess male fertility in Japan. This study enrolled 743 infertile and 20 fertile Japanese men. To evaluate reproducibility, inter- and intraobserver precision was analyzed. A receiver operating characteristic curve analysis was used to set a cutoff value for the sperm DFI to identify men who could father children by timed intercourse or intrauterine insemination. The variability of the sperm DFI among fertile volunteers was determined. The relationship between semen parameters and the sperm DFI was assessed by Spearman's rho test. A precision analysis revealed good reproducibility of the sperm DFI. The cutoff value of sperm DNA fragmentation in infertile men was 24.0%. Semen volume had no relationship with the sperm DFI. Sperm concentration, sperm motility, total motile sperm count, and percentage of normal-shaped sperm were significantly and negatively correlated with the sperm DFI. The median sperm DFI was smaller in fertile volunteers (7.7%) than that in infertile men (19.4%). Sperm DNA fragmentation analysis can be used to assess sperm functions that cannot be evaluated by ordinary semen analysis.

Effect of Ginger Extract on Semen Parameters and Sperm DNA Fragmentation during the Liquid Storage at 4°C in Native Roosters (Research Note)

The goal of the current study was to determine the effect of ginger extract on some quality parameters of rooster spermatozoa during liquid storage. A total of 10 healthy adult native roosters were selected and sampled twice a week for three weeks. Treatment groups were made up of basal extender supplemented with various concentrations of ginger extract including 0, 20, 40, 60, and 80 μg/mL. Sperm plasma membrane integrity, viability, and motility were assessed at 0, 24, 48, and 72 hours after semen collection. However, the sperm DNA fragmentation test was determined only after 48 hours of storage. The sperm membrane integrity was high at 24 (81.66%) and 48 (58.66%) hours in control and 60 µg/mL of ginger extract, respectively. The highest sperm viability (70.50%) and motility (76.50%) were obtained in 20 μg/mL ginger extract at 48h. The lowest sperm DNA fragmentation (32%) was observed in the 20 μg/mL of ginger extract. According to the results, the ginger extract showed a protective effect against time-associated harmful changes in semen quality of roosters during liquid storage.

Predictive Significance of Sperm DNA Fragmentation Testing in Early Pregnancy Loss in Infertile Couples Undergoing Intracytoplasmic Sperm Injection.

ObjectiveThis study aimed to determine the role of sperm DNA fragmentation as a marker that could predict early pregnancy loss (EPL), either independently or in combination with another marker or markers, after intracytoplasmic sperm injection (ICSI) cycles.MethodsThis prospective descriptive cohort study retrieved data from 162 couples who underwent their first ICSI cycles at the Center for Reproductive Endocrinology and Infertility of Hue University Hospital in Vietnam from May 2018 to December 2019. General characteristics, semen parameters, sperm DNA fragmentation index (DFI), fertilization, embryo development, pregnancy rates, and EPL were assessed. The receiver-operating characteristic (ROC) method was performed to identify the threshold of DFI in EPL. Multivariate analysis was used to demonstrate the relationship between the sperm DNA fragmentation level and EPL.ResultsOf 162 ICSI cycles, 23 (14.2%) involved EPL. There was no significant difference between the sperm DNA fragmentation rate and the overall rate of pregnancy loss, although the negative pregnancy outcome group had a median DFI that was higher than that of the positive pregnancy outcome group (20% vs 17.8%). The ROC analysis indicated that a sperm DNA fragmentation rate of 16.6% was the priority cut-off that could be used to distinguish EPL with a sensitivity of 73.9% (95% confidence interval [CI], 67.15–80.67) and specificity of 47.48% (95% CI, 39.79–55.17). The multivariate analysis confirmed that in female factors such as age, body mass index (BMI), and sperm DNA fragmentation level affected the EPL rate. However, a combination of the sperm DNA fragmentation level and female age or female BMI could not sufficiently predict EPL.ConclusionEPL can be affected by multiple factors including sperm DNA fragmentation; however, there is no sufficient evidence indicating that sperm DNA fragmentation, both as a single marker and combined with other markers, is a good predictor of EPL.

Study of Sperm Dna Fragmentation Index in Patients from Rural Areas of Wardha Region

Background: Sperm DNA fragmentation test is an advanced test for proper sperm function with respectto semen analysis, as semen analysis is used for motility and morphology assessment, it cannot explainsperm at molecular level and thus SDF test has been introduced to evaluate male fertility. The integrity ofgenetic material in sperm is crucial for successful fertilization &amp; normal embryo development as this willlead to proper transmission of genetic information. Patients having varicocele, recurrent failure of ARTtecniques and improper lifestyle (radiation, smoking, alcohol, BMI, ageing, etc) are considered for spermDNA fragmentation test. SDF test is useful in selecting patients for the ART procedures i.e. IVF/ICSI/IUIand highest pregnancy and live birth outcomes.Objectives:· To predict male infertility with normal semen parameters &amp; its diagnostic values.· To investigate the clinical factors related to sperm DNA fragmentation in male with infertility.· To investigate the rate of sperm DNA fragmentation in male related to improper fertilization, embryodevelopment and recurrent pregnancy loss.Methods: The participants (n=40) with male fertility problems will be included in randomised control trial.All 40 participants will be randomly selected to evaluate their sperm DNA integrity from their respectivesemen samples using sperm chromatin dispersion test.Discussion: Efficiency of the Sperm DNA Fragmentation test is assessed by performing sperm chromatindispersion test on respective processed semen sample using Halosperm G2 test kit.

Sperm selection during ICSI treatments reduces single- but not double-strand DNA break values compared to the semen sample.

To detect a possible bias in sperm DNA fragmentation (SDF) testing when performed on semen samples or on those few spermatozoa selected for Intracytoplasmic Sperm Injection (ICSI) treatments. A multimethodological analysis of Single- and Double-Strand DNA Breaks (SSB and DSB, respectively) was performed through the Neutral Comet, the Alkaline Comet, the Sperm Chromatin Dispersion (SCD) and the Terminal deoxynucleotidyl transferase dUTP Nick End Labelling (TUNEL) assays. SDF was evaluated in (i) semen samples from 23 infertile patients (not achieving pregnancy or suffering recurrent miscarriage); (ii) samples after a Swim-up and (iii) spermatozoa microselected for ICSI (ICSI-S). The analysis of 3217 ICSI-S revealed a significant reduction of SSB values compared to the Ejaculate and the Swim-up samples. On the contrary, DSB values were not reduced after any sperm selection method. The No-pregnancy group presented poorer semen parameters and higher SSB values. The Recurrent miscarriage group presented better semen parameters but also higher DSB values. The analysis of SDF on semen samples may not be fully representative of those few spermatozoa selected for ICSI. Since oxidative stress impairs sperm motility and causes SSB, selecting a motile sperm may intrinsically imply choosing a sperm not affected by this damage. DSB have an enzymatic origin which does not affect motility, making it difficult to select a sperm without this damage. Therefore, ICSI treatments could be effective in patients presenting high SSB values. Patients presenting high DSB values should expect bad ICSI results if this damage is not reduced through other specific methods.

The Prevalence and Components of Metabolic Syndrome in Men from Infertile Couples and Its Relation on Semen Analysis.

PurposeMetabolic syndrome (MetS) has been reported as a deleterious factor in male fertility potential, associated with hypogonadism, impaired spermatogenesis, decreased sperm concentration and motility, and increased sperm DNA damage. This study aimed to determine the prevalence of MetS in men from infertile couples and evaluate its effect on semen analysis (SA).Participants and MethodsA cross-sectional descriptive study was performed in men from infertile couples diagnosed based on the World Health Organization 2010 criteria and treated at the Hue Center for Reproductive Endocrinology and Infertility, Vietnam. General information included medical history, lifestyle, MetS factors, SA, and sperm DNA fragmentation test were collected. Based on the diagnostic criteria of the American Heart Association and the National Heart, Lung, and Blood Institute for Asian men, the study population was divided into two groups: MetS and non-MetS groups. The outcomes were analyzed for any relationship between MetS and the SA index and the DNA fragmentation index (DFI).ResultsA total of 534 men from infertile couples were included in this study. The prevalence of MetS was 23.4%, and abnormal semen analysis accounted for 93.8%. Age, body mass index (BMI), waist-to-hip ratio (WHR), waist-to-height ratio (WHtR), hepatitis B and total cholesterol were related to the occurrence of MetS in infertile men (p <0.05). MetS did not reveal any impact on the parameters of SA. There was a positive correlation between waist circumference (WC), WHR, WHtR, and systolic blood pressure (BP) with abnormal sperm head and DFI (p <0.05).ConclusionAlthough the prevalence of MetS was remarkable in men from infertile couples, there was no association between MetS and semen quality. However, WC, WHR, WHtR, and systolic BP were found to be significantly associated with abnormal sperm head and DFI.

European Association of Urology Guidelines Panel on Male Sexual and Reproductive Health: A Clinical Consultation Guide on the Indications for Performing Sperm DNA Fragmentation Testing in Men with Infertility and Testicular Sperm Extraction in Nonazoospermic Men

Accumulating evidence has highlighted the contribution of oxidative stress and sperm DNA fragmentation (SDF) in the pathophysiology of male infertility. SDF has emerged as a novel biomarker of risk stratification for patients undergoing assisted reproductive technologies. Studies have also supported the use of testicular over ejaculated sperm at the time of intracytoplasmic sperm injection, as testicular sperm may have lower SDF than ejaculated samples. The European Association of Urology Working Panel on Male Sexual and Reproductive Health provides an evidence-based consultation guide on the indications for SDF testing in male infertility and also for testicular sperm extraction (TESE) in nonazoospermic men. We present the limitations and advantages of SDF testing and a framework to ensure that it is appropriately utilised in clinical practice. Furthermore, we critically appraise the current literature advocating the use of TESE in nonazoospermic men. Patient summaryThis article reviews the evidence supporting the use of sperm DNA fragmentation testing in the assessment of male infertility and testicular sperm extraction in nonazoospermic men.

Sperm Motility is the Best Semen Parameter to Predict Sperm DNA Fragmentation

Purpose: Sperm DNA fragmentation (SDF) is associated with male infertility and abnormal semen parameters. However, the effect of SDF on each parameter may differ. In this study, we evaluated the correlation between different semen parameters and SDF to identify the most suitable predictor for abnormal SDF. Materials and Methods: We conducted a retrospective review from a prospective database. Enrollees who underwent conventional semen analysis and an SDF test for medical purposes or elective examinations were enrolled. SDF ≥20% was regarded as abnormal. Spearman correlation coefficient was used to determine the correlations. Area under the receiver operating characteristic curve area under the curve (AUC) was calculated to determine the predictive value. Youden index was used to determine the optimal cutoff value of conventional semen parameters to predict abnormal SDF. Results: In total, 90 men were enrolled, of whom 44 (48.89%) visited for infertility and 51 (56.67%) had abnormal semen analysis. Immotile sperm (IM) and nonprogressive sperm (NPS, NPS = nonprogressive motility + IM) were significantly correlated with SDF (r = 0.50, P&lt; 0.001 for NPS; r = 0.49, P&lt; 0.001 for IM) and were the most predictive for abnormal SDF (AUC = 0.77 for NPS; AUC = 0.78 for IM). By using Youden index, the cutoff values for the prediction of abnormal SDF were 66.37% for NPS and 48.73% for IM. Conclusion: Sperm motility is the most predictive and relevant parameter for the prediction of abnormal SDF. Suboptimal sperm motility should be considered an indication for SDF testing.

Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations.

We herein summarise the evidence concerning the impact of sperm DNA fragmentation in various clinical infertility scenarios and the advances on sperm DNA fragmentation tests. The collected evidence was used to formulate 41 recommendations. Of these, 13 recommendations concern technical aspects of sperm DNA fragmentation testing, including pre‐analytical information, clinical thresholds and interpretation of results. The remaining 28 recommendations relate to indications for sperm DNA fragmentation testing and clinical management. Clinical scenarios like varicocele, unexplained infertility, idiopathic infertility, recurrent pregnancy loss, intrauterine insemination, in vitro fertilisation/intracytoplasmic sperm injection, fertility counselling for men with infertility risk factors and sperm cryopreservation have been contemplated. The bulk evidence supporting the recommendations has increased in recent years, but it is still of moderate to low quality. This guideline provides clinicians with advice on best practices in sperm DNA fragmentation testing. Also, recommendations are provided on possible management strategies to overcome infertility related to sperm DNA fragmentation, based on the best available evidence. Lastly, we identified gaps in knowledge and opportunities for research and elaborated a list of recommendations to stimulate further investigation.

Effect of sperm DNA fragmentation on ICSI outcome: A prospective study

Aim and objectives: The primary aim was to measure the sperm DNA damage and to study the magnitude of sperm DNA damage. Secondary objective was to study the effect of sperm DNA fragmentation on Day 5 Blastocyst expansion (graded 1-5). Results: There is an increase in sperm DNA fragmentation with an increase in age. Increased sperm DNA fragmentation is also associated with abnormal motility and morphology in semen samples. However, there is no reduction in expansion or grade of blastocyst. Conclusion: Sperm DNA fragmentation testing is a useful investigation in unexplained infertility. However, Sperm DNA fragmentation has no significant association with Day 5 embryo grade in ICSI cycles. Thesis work of Fellowship in Reproductive Medicine student: Dr. Ramya Harish

Sperm Cell Damage Induced by Urogenital Tract Infections

Male infertility has traditionally been diagnosed by microscopic assessment of concentration, motility and morphology of sperm in the ejaculate. These tests are essential to provide the fundamental information on sperm quality. However, evidence based medicine shows that sperm DNA fragmentation (SDF) tests can also differentiate fertile from infertile males.

IMPACT OF SPERM DNA FRAGMENTATION ON PRE- AND POST-WASH SEMEN SAMPLES AND PREGNANCY OUTCOME WITH INTRACYTOPLASMIC SPERM INJECTION

The spermatozoa nuclear component especially it’s DNA integrity is essential for normal fertilization, pregnancy & embryo development. Currently sperm DNA fragmentation (SDF) testing is one advanced sperm function test used along other conventional tests to evaluate male infertility.We aim to assess the impact of SDF on pre- & post-wash semen parameters and to investigate effect of various SDF cut-off values on pregnancy outcome with Intracytoplasmic sperm injection (ICSI). Retrospective, Chart review. The charts of 2277 patients who underwent ICSI were reviewed. 481 infertile men who had SDF testing were included. Patients demographic & laboratory data along with ICSI cycle outcome were included. Semen samples & SDF were analyzed according to WHO 5thedition guidelines & sperm chromatin dispersion (Halosperm kit, Halotech, Madrid, Spain) respectively. Patients were divided according to SDF cut-offs; <20, 20-30 & >30%. Variables were reported as mean ± SE and compared using Kruskal Wallis Test. P value of <0.05 was considered statistically significant The mean patient age was 36.88 ± 0.362, while mean SDF was 25.41± 0.72. The pre & post wash semen parameters in patients with SDF cut-offs <20, 20-30 & >30% were compared (Table 1). Significantly worse results were obtained with increasing SDF cut-offs for pre- & post-wash sperm concentration, total motility & post-wash normal morphology. SDF was significantly inversely correlated with pre wash parameters (concentration: r -.140, p=o.oo3; total Motility: r -.282, p<0.001; normal morphology: r -.143, p=0.003) and post wash parameters (concentration: r -.156, p=o.oo1; total Motility: r -.218, p<0.00; normal morphology: r -.140, p=0.002) The clinical pregnancy rate decreased with increasing SDF cut-offs (44.4% with SDF <20%, 31.4% with SDF 20-30% and 24.3% with SDF >30), though the result was not statistically significant SDF negatively impacts pre-& post-processing semen parameters for patients undergoing ICSI. This may influence ICSI pregnancy rate, though the impact was not statistically significant in this cohort.Tabled 1ParametersTotalSDF <20SDF 20-30SDF >30P ValuePre wash Concentration (M/ml)38.94 ± 1.6945.24 ± 2.7935.82 ± 2.6130.57 ± 2.870.016Pre wash Total Motility %38.61 ± 0.944.18 ± 1.2837.82 ± 1.6229.26 ± 1.68<0.001Pre wash Normal Morphology %12.49 ± 0.6112.01 ± 0.7814.53 ± 1.3511.18 ± 1.270.121Post Wash Concentration (M/ml)13.93 ± 0.6315.96 ± 0.9513.854 ± 1.129.95 ± 1.16<0.001Post Wash Total Motility %4.21 ± 0.275.24 ± 0.453.99 ± 0.432.45 ± 0.47<0.001Post Wash normal Morphology %12.31 ± 0.6512.12 ± 0.8913.36 ± 1.3111.53 ± 1.370.010 Open table in a new tab

RAISED SPERM DNA FRAGMENTATION –RANDOMISED CONTROLLED TRIAL OF MACS VS TESA TO OPTIMISE REPRODUCTIVE OUTCOMES

In Individuals with raised Sperm DNA Fragmentation (SDF), randomization with magnetic activated cell sorting (MACS) or surgical retrieval of testicular sperms (TESA) will it reduce SDF and optimize the reproductive outcomes? This is an ongoing Randomized Control Trial (RCT) with prior approval from institutional Ethical Committee (IEC) conducted at a Private Teaching ART Clinic. This is preliminary data of the pilot study. Study duration 7months (January – July 2019). Couples undergoing IVF stimulation with raised SDF were randomized into MACS group (n=30) and TESA group (n=30) for sperm selection. Couples with history of one failed IVF cycle were offered testing for SDF. Individuals with SDF > 30% were included in the study. SDF testing was done with SCSA method and couples randomized using software into respective intervention groups. TESA was done as per our clinic’s Standard Operating Protocol (SOP) and MACS was done as per the instruction from the manufacturer. Intra Cytoplasmic Sperm Injection (ICSI) was the method of insemination in all cases. Extended embryo culture till blastocyst stage was done and a freeze all policy was opted. Two Blastocysts that showed 100% survival were transferred in a Frozen Embryo transfer (FET) cycle. Mean of Implantation Rates (IR) and Clinical Pregnancy Rates (CPR), Miscarriage Rates (MR) & Live Birth Rates (LBR) were compared between both groups. Mean Reproductive Outcomes for MACS & TESA groups were as follows: Blastocyst formation rates – 38% Vs 31% (P=0.5717) CPR – 50% Vs 83% (P= 0.0072) IR – 43% Vs 72% (P= 0.0243) MR- 33% Vs 16% (P= 0.1290) LBR – 17% Vs 67% (P= 0.0001) Blastocyst formation was comparable between the groups; However, TESA group had significantly higher CPR, IR, LBR and lesser MR. Testicular sperm seem to have better DNA quality than ejaculated sperm. In couples with failed IVF attempts and raised SDF, we can offer TESA as an active intervention to optimize reproductive outcomes. Poor outcomes in MACS group needs further research. LIMITATIONS, REASONS FOR CAUTION: Small sample size. TESA is a surgical intervention. TESA seems like a beneficial intervention to optimize sperm selection and reproductive outcomes for Individuals with raised sperm DFI.

Relationship of Seminal Oxidation-Reduction Potential with Sperm DNA Integrity and pH in Idiopathic Infertile Patients

Seminal oxidative stress (OS) is one of the most promising factors to describe the causes of idiopathic male infertility. Redox balance is essential in several biological processes related to fertility, so alterations such as high reactive oxygen species (ROS) levels or low antioxidant agent levels can compromise it. MiOXSYS has been developed to evaluate the seminal static oxidation-reduction potential (sORP) and it has been proposed as an effective diagnostic biomarker. However, its relationship with parameters like sperm DNA fragmentation (SDF), chromatin compaction status or seminal pH requires further analysis, making it the object of this study. Semen and sORP analysis were performed for all samples. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) and Comet assay were used to assess SDF and chromomycin a3 (CMA3) test to assess sperm chromatin compaction. Regarding sORP measures, it was found that alkaline pH has an effect on sample reproducibility. To our knowledge, this unexpected effect has not been previously described. A statistical analysis showed that sORP correlated negatively with CMA3 positive cells and sperm motility, but not with SDF. As redox dysregulation, which occurs mainly at the testicular and epididymal level, causes chromatin compaction problems and leaves DNA exposed to damage, an excess of ROS could be counterbalanced further by a seminal supply of antioxidant molecules, explaining the negative correlation with CMA3 positive cells but no correlation with SDF. Our results show that the study of idiopathic infertility would benefit from a combined approach comprising OS analysis, SDF and chromatin compaction analysis.