Sperm DNA Fragmentation Values Research Articles

One out of two idiopathic infertile men has pathologic sperm DNA fragmentation values: Potential implications for clinical practice.

To investigate the distribution of sperm DNA fragmentation (SDF) values and their association with clinical and seminal parameters in idiopathic infertile men. Data from 3224 primary infertile men (belonging to couples having failed to conceive a pregnancy within 12 months) who underwent a thorough diagnostic work-up were analysed. A SDF value ≥ 30% (according to Sperm Chromatin Structure Assay) was considered pathologic. We excluded: (1) men with genetic abnormalities; (2) men with history of cryptorchidism; (3) men with biochemical hypogonadism; (4) men with clinical varicocele; and (5) men with other possible known aetiological factors. Descriptive statistics and logistic regression analyses were used to describe the whole cohort. Of all, 792 (23%) men with at least one abnormal WHO semen parameter but without any identified aetiologic factor for infertility, were considered as idiopathic infertile men. Of 792, 418 (52.7%) men had SDF≥30%. Men with pathologic SDF were older (p = .02), had higher Follicle-stimulating hormone(FSH) (p = .04) but lower total testosterone (p = .03) values than those with SDF<30%. The homoeostatic model assessment index for insulin resistance (HOMA-IR) was higher in men with SDF≥30% (p = .01). Idiopathic infertile men with SDF≥30% presented with lower sperm concentration (p < .001) and lower progressive sperm motility (p < .01) than those with SDF < 30%. Logistic regression analysis revealed that older age (OR: 1.1, p = .02) and higher HOMA-IR score (OR: 1.8, p = .03) were associated with SDF ≥ 30%, after accounting for FSH and sperm concentration values. Approximately half of infertile men categorized as idiopathic had pathologic SDF values. Idiopathic infertile men with pathologic SDF showed worse clinical, hormonaland semen parameters than those with normal SDF values. These results suggest that including SDF testing could be clinically relevant over the real-life management work-up of infertile men.

P-018 Study of sperm DNA fragmentation in the infertile population before, during and after the SARS-CoV-2 pandemic: a nationwide retrospective cohort study

Abstract Study question Did the values of single- and double-strand sperm DNA breaks (SSB and DSB) increase during the SARS-CoV-2 pandemic? Summary answer During the SARS-CoV-2 pandemic (2020), the mean values of SSB and DSB in infertile patients were statistically higher compared to the periods 2018-2019 and 2021-2022. What is known already Some studies have found lower sperm concentration and motility in patients during and after SARS-CoV-2 infection (COVID-19). These effects appear to be reversible, with significant improvements being observed after three months post-infection (complete spermatogenesis cycle). On the contrary, vaccination against this virus does not seem to affect semen parameters. The objective of the study was to compare the values of SSB and DSB sperm DNA fragmentation in the periods before the pandemic (2018-2019), during (2020, highest number of infections and social confinement), and after (2021-2022, lower infection rate, end of social confinement and vaccination period). Study design, size, duration The average SSB and DSB sperm DNA fragmentation values of 11,215 patients referred from 74 fertility centres throughout Spain from January 2018 to December 2022 were retrospectively compared. Participants/materials, setting, methods Semen samples from infertile patients were analysed using the Comet assay in alkaline or neutral pH conditions to evaluate SSB or DSB, respectively. Three periods were compared (two years before, during 2020, and two years after). Mean SSB and DSB values were statistically compared among these periods using the Kruskal-Wallis and Mann-Whitney tests. Differences were established at the 99% confidence interval. Main results and the role of chance The average SSB values obtained were: 41.67%±15.33 in 2018-2019; 43.92%±17.84 in 2020, and 41.68%±18.58 in 2021-2022. The average DSB values obtained were: 65.64%±14.27 in 2018-2019; 67.77%±13.90 in 2020, and 65.23%±15.18 in 2021-2022. The SSB and DSB values were statistically higher in 2020 compared to the periods 2018-2019 and 2021-2022 (p &amp;lt; 0.001 in both cases). No differences were detected between the previous period (2018-2019) and the following period (2021-2022) (p = 0.101 and p = 0.235, respectively). Limitations, reasons for caution The observed increase in SSB and DSB values during 2020 cannot be directly attributed to the SARS-CoV-2 infection (COVID-19). It is possible that it is associated with other concurrent factors. The studied samples were only from infertile patients, not from the general male population. Wider implications of the findings Given that the SARS-CoV-2 virus still exists in 2021-2022 but the social situation has normalized, the increase in fragmentation observed during 2020 could be more related to other aspects of the pandemic, such as social confinement, stress, among others. Trial registration number Not applicable

The Association of Sperm DNA Fragmentation With Serum Lipid Profile Among Males With Abnormal Semen Parameters: An Analytical Cross-Sectional Study.

Through the ages, infertility, affecting 8% to 12% of couples worldwide, has been a perturbing clinical problem. Approximately 40% to 50% of all infertility cases are due to 'male factor' infertility. Semen analysis is crucial in routinely evaluating idiopathic male infertility. Studies support the idea that semen parameters are associated with serum lipids and sperm DNA fragmentation (SDF). Therefore, it is possible to evaluate male infertility by serum lipid levels, especially before assisted reproduction technology, and modify it by bringing about lifestyle modifications. This study aimed to measure the correlation of SDF with levels of total cholesterol (TC), triglycerides (TG),very low-density lipoprotein (VLDL),low-density lipoprotein (LDL), and high-density lipoprotein (HDL) among males with abnormal semen parameters. A cross-sectional analytical study was conducted in the infertility clinic of a tertiary care hospital. A total of 106 infertile males with abnormal semen analysis as per the WHO criteria (2010) were enrolled in the study. After routine semen analysis, SDF was studied using the comet assay. The serum fasting lipid profile was analyzed using the spectrophotometric kit in the autoanalyzer. The relationship of SDF with serum lipid profile parameters was analyzed. Out of 106 infertile men, 52% (n = 55) had severe SDF. A modest positive correlation was observed between SDF (percentage of DNA in comet tail) and serum lipid values (serum TG, serum LDL, and serum VLDL). Our study is novel in its research on the correlation between SDF and serum lipid values. Based on the findings of our study, it can be concluded that a significant level of SDF was observed in men with high levels of serum TG, LDL, and VLDL. This provokes a potential relationship between sperm DNA integrity and serum lipid profile, which warrants further research.

Severe sperm DNA fragmentation may persist for up to 3 years after cytotoxic therapy in patients affected by Hodgkin lymphoma and non-Hodgkin lymphoma.

Does sperm DNA recover from damage in all men after 2 years from the end of cytotoxic treatments? The current indication of 2 years waiting time for seeking natural pregnancy after cytotoxic treatment may not be adequate for all men, since severe sperm DNA damage is present in a proportion of subjects even after this timeframe. Data in the literature on sperm DNA fragmentation (SDF) in lymphoma patients after cytotoxic treatments are scarce. The largest longitudinal study evaluated paired pre- and post-therapy (up to 24 months) semen samples from 34 patients while one study performed a longer follow-up (36 months) in 10 patients. The median/mean SDF values >24 months after therapy did not show significant differences but the studies did not explore the proportion of patients with severe DNA damage and the analysis was done on frozen-thawed samples. In this study, 53 Hodgkin lymphoma (HL) and 25 non-Hodgkin lymphoma (NHL) post-pubertal patients were included over a recruitment period of 10 years (2012-2022). Among them, 18 subjects provided paired semen samples for SDF analysis at the three time points. SDF was evaluated in patients before (T0) and after 2 (T2) and 3 years (T3) from the end of, cytotoxic treatments (chemotherapy alone or in combination with radiotherapy). A cohort of 79 healthy, fertile, and normozoospermic men >18 years old served as controls (recruited between 2016 and 2019). SDF was evaluated on fresh semen samples (i.e. spermatozoa potentially involved in natural conception) from patients and controls using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled with flow cytometry. SDF median values were compared between groups: (i) HL and NHL patients versus controls at the three time points; (ii) HL versus NHL patients at baseline; and (iii) patients at T0 versus T2 and T3. Severe DNA damage (SDD) was defined for SDF levels above the 95th percentile of controls (50%) and the proportion of patients with SDD at all time points was established. At T0, patients displayed higher median SDF than controls, reaching statistical significance in the NHL group: 40.5% [IQR: 31.3-52.6%] versus 28% [IQR: 22-38%], P < 0.05. Comparing SDF pre-treatment to that post-treatment, HL patients exhibited similar median values at the three time points, whereas NHL showed significantly lower values at T3 compared to T0: 29.2% [IQR: 22-38%] versus 40.5% [IQR: 31.3-52.6%], P < 0.05. The proportion with SDD in the entire cohort at T2 was 11.6% and 13.3% among HL and NHL patients, respectively. At T3, only one in 16 NHL patients presented SDD. TUNEL assay requires at least 5 million spermatozoa to be performed; hence, severe oligozoospermic men were not included in the study. Although our cohort represents the largest one in the literature, the relatively small number of patients does not allow us to establish precisely the frequency of SDD at T2 which in our study reached 11-13% of patients. Our data provide further insights into the long-term effects of cytotoxic treatments on the sperm genome. The persistent severe DNA damage after 2 years post-treatment observed in some patients suggests that there is an interindividual variation in restoring DNA integrity. We propose the use of SDF as a biomarker to monitor the treatment-induced genotoxic effects on sperm DNA in order to better personalize pre-conceptional counseling on whether to use fresh or cryopreserved spermatozoa. This work was supported by grants from the Istituto Toscano Tumori (ITT), Fondazione Ente Cassa di Risparmio di Firenze, the European Commission-Reproductive Biology Early Research Training (REPROTRAIN). C.K., G.F., V.R., and A.R.-E. belong to COST Action CA20119 (ANDRONET) which is supported by the European Cooperation in Science and Technology (www.cost.eu). The authors declare no conflict of interest. N/A.

Age, sexual abstinence duration, sperm morphology, and motility are predictors of sperm DNA fragmentation.

Sperm DNA fragmentation (SDF) has recently received attention as a cause of male infertility. However, SDF cannot be fully assessed using conventional semen parameter evaluations alone. Therefore, the authors aimed to elucidate the relationship between SDF and sperm parameters via computer-assisted sperm analysis (CASA) to improve treatment strategies in reproductive medicine. This retrospective observational study analyzed the relationship between sperm parameters assessed by CASA and SDF values determined by the TUNEL assay in 359 patients who visited the Mie University Hospital for infertility treatment. The methodology involved semen analyses covering concentration, motility, and morphology, followed by SDF quantification using the flow cytometry. Statistical analysis revealed significant correlations between SDF and various factors, including age, sexual abstinence period, and specific CASA-measured parameters. Notably, lower sperm motility rates and abnormal head dimensions were associated with higher SDF values, indicating that these parameters were predictive of SDF. This study highlights the importance of sperm motility and head morphology as indicators of SDF, suggesting their usefulness in assessing male fertility. These findings demonstrate the efficacy of detailed sperm analysis, potentially increasing the success rate of assisted reproductive technologies by improving sperm selection criteria.

#242 : Correlation of Sperm DNA Fragmentation with Laboratory Performance in Good and Poor Prognosis Patient Groups at Morula IVF Jakarta

BACKGROUND AND AIMS: Infertility in males contributes to about 40% of cases of infertility in couples, either as the main factor or along with female factors. Fragmentation of sperm DNA is known as a cause of male infertility, which impacts the failure of couples to undergo assisted reproductive programs, such as embryo development and recurrent miscarriage. However, some literature studies are still debating this. This research aims to determine the correlation between sperm DNA fragmentation and IVF laboratory performance. METHODS: Infertile couples undergoing an in vitro fertilization program at Morula IVF Jakarta in 2022 are the samples for this study. We divided them into two groups: good prognosis (maternal age [Formula: see text]38 years, AFC[Formula: see text]7; AMH[Formula: see text]1.1) and poor prognosis (maternal age &gt; 38 years, AFC &lt;7, AMH&lt;1.1). Sperm DNA fragmentation was examined using the sperm chromatin dispersion (SCD) assay. Correlation statistical analysis using the Spearman test (p-value &lt;0.05). RESULTS: Based on receiver operating characteristic (ROC) curve analysis, it was found that the sperm DNA fragmentation rate &gt;14.9% had a significant negative correlation with IVF laboratory performance, particularly in the fifth-day embryo quality and blastocyst rate in the good prognosis group. Whereas in the group with poor prognosis, all laboratory performance parameters were not affected by high sperm DNA fragmentation values. CONCLUSIONS: Sperm DNA fragmentation rate &gt; 14.9% correlated with fifth-day embryo quality and a lower blastocyst rate in the good prognosis group.

Long-term effect of cytotoxic treatments on sperm DNA fragmentation in patients affected by testicular germ cell tumor.

Testicular germ cell tumor (TGCT) is the most frequent neoplasia in men of reproductive age, with a 5-year survival rate of 95%. Antineoplastic treatments induce sperm DNA fragmentation (SDF), especially within the first year post-therapy. Data in the literature are heterogeneous concerning longer follow-up periods and the large majority is limited to 2 years. To define the timing for the recovery of sperm DNA damage and the proportion of patients with severe DNA damage (SDD) at 2 and 3 years from the end of therapy. SDF was evaluated in 115 TGCT patients using TUNEL assay coupled with flow cytometry before (T0 ) and 2 (T2 ) and 3 (T3 ) years post-treatment. Patients were divided based on the type of treatment: carboplatin (CP), bleomycin-etoposide-cisplatin (BEP), radiotherapy (RT). For 24 patients paired SDF data were available at all time-points (T0 -T2 -T3 ). 79 cancer-free, fertile normozoospermic men served as controls. SDD was defined as the 95th percentile in controls (SDF = 50%). Comparing patients versus controls we observed: i) no differences at T0 and T3 ; ii) significantly higher SDF levels (p<0.05) at T2 in all treatment groups. Comparing pre and post-therapy in the 115 patients, the median SDF values were higher in all groups at T2 , reaching significance (p<0.05) only in the CP group. While the median SDF values were higher also in the strictly paired cohort at T2 , about 50% of patients returned to baseline. The proportion of SDD in the entire cohort was 23.4% and 4.8% of patients at T2 and T3 , respectively. Currently, TGCT patients are advised to wait two years post-therapy before seeking natural pregnancy. Our results suggest that this period may not be sufficient for all patients. The analysis of SDF may represent a useful biomarker for pre-conception counselling following cancer treatment. This article is protected by copyright. All rights reserved.

How Well Do Semen Analysis Parameters Correlate with Sperm DNA Fragmentation? A Retrospective Study from 2567 Semen Samples Analyzed by the Halosperm Test.

Sperm DNA fragmentation (SDF) levels have been measured in the workup for in vitro fertilization (IVF) at PIVET since 2007, with the Halosperm test having replaced the previous sperm chromatin structure assay (SCSA) since 2013. Of 2624 semen samples analyzed for the Halosperm test, 57 were excluded as the sperm concentration was <5 million/mL, a level too low for accurate testing, leaving 2567 samples for assessment within this study. The SDF rates were categorized in 5 sperm DNA fragmentation indices (DFI), ranging from <5% to levels >30%, and these categories were correlated with the respective semen analysis profiles and two clinical parameters, namely the age of the male and the ejaculatory abstinence period prior to the sample. The results showed a significant correlation with male age (r = 0.088; p < 0.0001), the abstinence period (r = 0.076; p = 0.0001), and the semen volume (r 0.063; p = 0.001), meaning an adversely high SDF was associated with advanced age, prolonged abstinence, and raised semen volume parameters. There was a significant negative correlation with sperm morphology (r = -0.074; p = 0.0001), progressive motility (r = -0.257; p < 0.0001), and semen pH (r = -0.066; p < 0.001), meaning these semen anomalies were associated with high SDF values. With respect to abnormal morphology, sperm tail defects had a positive correlation (r = 0.096; p < 0.0001) while midpiece defects showed a negative correlation (r = -0.057; p = 0.004), meaning that tail defects are most likely to associate with adverse DFI values. With respect to motility patterns, the poorer patterns showed a positive correlation with increased DFI, namely C pattern (r = 0.055; p = 0.005) and D pattern (r = 0.253; p < 0.0001). These results imply that raised DFI reflects poor sperm quality and should be investigated in clinical trials involving IVF and the consideration of intracytoplasmic sperm injection (ICSI).

The definition of idiopathic male infertility should include sperm DNA fragmentation values: Findings from a cross-sectional study

The definition of idiopathic male infertility should include sperm DNA fragmentation values: Findings from a cross-sectional study

Low Birth Weight is Associated with Sperm DNA Fragmentation and Assisted Reproductive Technology Outcomes in Primary Infertile Men: Results of a Cross-Sectional Study.

To assess the relationship between clinical and semen characteristics and assisted reproductive technology (ART) outcomes with different birth weight (BW) categories in a cohort of infertile men. Data from 1,063 infertile men were analyzed. Patients with BW ≤2,500, 2,500-4,000, and ≥4,000 g were considered as having low BW (LBW), normal BW (NBW), and high BW (HBW), respectively. Testicular volume (TV) was assessed with a Prader orchidometer. Serum hormones were measured in all cases. Semen analyses were categorized based on 2021 World Health Organization reference criteria. Sperm DNA fragmentation (SDF) was tested in every patient and considered pathological for SDF >30%. ART outcomes were available for 282 (26.5%) patients. Descriptive statistics and logistic regression analyses detailed the association between semen parameters and clinical characteristics and the defined BW categories. Of all, LBW, NBW, and HBW categories were found in 79 (7.5%), 807 (76.0%), and 177 (16.5%) men, respectively. LBW men had smaller TV, presented higher follicle-stimulating hormone (FSH) but lower total testosterone levels compared to other groups (all p<0.01). Sperm progressive motility (p=0.01) and normal morphology (p<0.01) were lower and SDF values were higher (all p<0.01) in LBW compared to other groups. ART pregnancy outcomes were lower in LBW compared to both NBW and HBW categories (26.1% vs. 34.5% vs. 34.5%, p=0.01). At multivariable logistic regression analysis, LBW was associated with SDF >30% (odd ratio [OR] 3.7; p<0.001), after accounting for age, Charlson Comorbidity Index (CCI), FSH, and TV. Similarly, LBW (OR 2.2; p<0.001), SDF >30% (OR 2.9; p<0.001) and partner's age (OR 1.3; p=0.001) were associated with negative ART outcomes, after accounting for the same predictors. LBW was associated with impaired clinical and semen characteristics in infertile men compared to both NBW and HBW. SDF and ART outcomes were significantly worse in the LBW group.

Variability in Sperm DNA Fragmentation in Men with Mild/Unexplained Subfertility in a Prospective Longitudinal Intrauterine Insemination Trial.

The biological variability of semen and sperm DNA fragmentation (SDF) parameters in a longitudinal intrauterine insemination (IUI) trial over multiple IUI cycles was investigated. A TUNEL assay was used for SDF testing, both before and after density gradient centrifugation. A significant age effect was observed: while semen parameters deteriorated with advancing age, on average, higher SDF values were observed for older males. There was quite some variability observed for both semen and SDF variables. Using fertile threshold values, three patient categories were distinguished: those with a high SDF in all samples, those with low SDF in all samples and those who fluctuated between high and low during the whole IUI trial. Density gradient centrifugation increases SDF. However, the three patient categories react differently after semen processing. A large percentage of those with high SDF retain their high SDF even after gradient centrifugation. The SDF fluctuaters react with a high SDF after gradient centrifugation. The low SDF category, on the contrary, distributes itself evenly between the three categories after gradient centrifugation. SDF testing after semen processing might be indispensable for therapeutic purposes, probably influencing medical decision-making. In order to isolate fluctuaters, a second SDF testing might be advocated in certain cases. SDF after semen processing is indispensable for therapeutic management.

Clinically Meaningful Improvements in Sperm DNA Fragmentation Severity in Infertile Men Treated with Superoxide Dismutase Supplementation: A Single-Center Experience.

Antioxidants are commonly used for the treatment of idiopathic male infertility. Previous studies have shown that antioxidants are able to improve sperm quality, but little is known about their impact on sperm DNA fragmentation (SDF). Preliminary findings showed that superoxide-dismutase (SOD)-based antioxidant plus hydroxytyrosol and carnosol (FertiPlus® SOD) therapy was associated with SDF improvement in a small cohort of infertile men. Therefore, we aimed to assess rates of and predictors of semen parameters and SDF improvements in infertile men treated with FertiPlus® SOD therapy (SOD+) or with other antioxidants without SOD (SOD-) in the real-life setting. Data from 60 consecutive infertile men with baseline SDF ≥ 30% and treated with SOD+ or SOD- for at least three months were analyzed. Clinical parameters and serum hormones were collected. Sperm parameters and SDF were requested at baseline and after SOD+ or SOD- treatment. Clinically meaningful SDF change after treatment was defined as SDF improvement &gt;20% compared to baseline. Propensity score matching was performed to adjust for baseline differences between groups. Descriptive statistics were used to compare clinical and hormonal characteristics between SOD+ and SOD- groups. Semen characteristics were compared before and after treatment. Logistic regression models investigated the association between clinical variables and SDF improvement. Groups were similar in terms of clinical, serum hormones and semen parameters at baseline after matching. Compared to baseline, sperm progressive motility (17 (10-36)% vs. 27 (12-41)%) and normal morphology (2 (1-6)% vs. 4 (2-6)%) significantly improved after SOD+ treatment (all p &lt; 0.01), but not after SOD-. SDF values significantly improved after treatment in both groups, compared to the baseline evaluation (all p &lt; 0.01). However, SDF values were lower after SOD+ than SOD- treatment (30 (22-36)% vs. 37 (31-42)%, p = 0.01). Similarly, a clinically meaningful improvement in SDF at follow-up was more frequently found after SOD+ than SOD- treatment (76.7% vs. 20.0%, p = 0.001). Multivariable logistic regression analysis showed that SOD+ treatment (OR 5.4, p &lt; 0.001) was an independent predictor of clinically meaningful SDF improvement, after accounting for age and baseline FSH values. This cross-sectional study showed that, in a cohort of primary infertile men with SDF ≥ 30%, SOD-based treatment was significantly effective in improving SDF compared to antioxidants without SOD. Approximately 80% of men treated with SOD+ achieved clinically meaningful improvement in SDF after three months of treatment. Sperm progressive motility and normal morphology also improved after SOD+ therapy but not after SOD-. These results suggest that SOD+ treatment could be considered an effective option for the management of idiopathic infertile men with elevated SDF.

O-016 Variability of sperm DNA fragmentation in a longitudinal trial of intrauterine inseminations

Abstract Study question Does variability in sperm DNA fragmentation (SDF) follow that observed in standard semen parameters and is this linked to outcome results? Summary answer Variability in SDF and in semen parameters is comparable but live birth rate tends to be lower in men with repetitive high SDF. What is known already Environmental, technical and biological factors are implicated in the intra-individual variability observed in routine semen parameters. This variation is not observed in SDF testing using the sperm chromatin structure assay. Most laboratories undertake SDF testing on one semen sample, assuming that one single measurement represents the patient’s condition and is both stable and associated with a good diagnostic value. Most variability observed in SDF tests relies essentially on paired observations with a short or long time interval between test and retest. Study design, size, duration A monocentric, prospective cohort study was conducted October 2017 - October 2020. Couples with a mild or unexplained male infertility initiated a natural cycle IUI protocol until pregnancy was achieved for a maximum of four cycles. 313 semen samples from 112 men were analyzed for SDF before and after semen preparation. This work was supported by the Fonds Wetenschappelijk Onderzoek (FWO), Research Foundation Flanders (grant number T007016N). Participants/materials, setting, methods Semen samples were analyzed and prepared by density gradient centrifugation. SDF was assessed via TUNEL assay before and after preparation. Clinical pregnancies and live births were registered. Intraclass Correlation Coefficients (ICC) were used to estimate the intra- and inter-variability. Main results and the role of chance The ICC for SDF (0.33 in the ejaculate, 0.54 after density gradient) and semen variables (0.42-0.62) were comparable. These values mean considerable intra-individual variation, both for semen variables and for SDF variables. Using fertile threshold SDF values (13% before and 8% after density gradient), 3 patient groups were distinguished: 13.4% of men showed high SDF in all ejaculates, 58.0% always showed low SDF and 28.6% fluctuated between the two during the trial. After density gradient more patients were found in the High group (42.3%) and less patients in the Low group (26.1%), while the proportion of Fluctuaters remained constant (31.5%). Most (66.7%) men with high SDF retain their high SDF after gradient, and also 56.3% of the Fluctuaters react with a high SDF after gradient. The Low SDF category, on the contrary, distributes itself evenly between the three categories after gradient. In the 112 couples, 26 (23.2%) clinical pregnancies were obtained with 21 (18.8%) live births. The live birth rate for the High, Fluctuaters and Low groups was 6.7%, 18.8% and 20.0% for ejaculate SDF and 12.8%, 20.0% and 27.6% for SDF after density gradient. These differences however did not reach significance (chi-square testing, p &amp;gt; 0.05). Limitations, reasons for caution Our results need to be substantiated and opportunities created to explore populations with an extreme male factor and the clinical implications in different assisted reproductive techniques. Wider implications of the findings SDF testing provides additional information and helps identify causes otherwise missed with traditional semen analysis alone. One out of 3 patients fluctuate in their SDF values. A second SDF testing is advocated to isolate these fluctuaters. The detection of DNA damage after semen preparation is indispensable for therapeutic purposes. Trial registration number NCT03319654

P-053 Sperm chromatin integrity in men from infertile couples with teratozoospermia and normozoospermia

Abstract Study question What is the relationship between sperm morphology and sperm chromatin integrity (SCI) in patients with normal sperm concentration (SC) and motility (SM)? Summary answer SCI violations in form of sperm DNA fragmentation (SDF) and incomplete protamination (IP) are more common in teratozoospermia, even when SC and SM are normal What is known already Deterioration of sperm chromatin integrity in the form of SDF and IP are the significant factors affecting male reproductive potential (WHO, 2021). Study design, size, duration This multicenter retrospective cross-sectional study included 479 men aged 18-45 years who were observed for infertility for more than 1 year. Participants/materials, setting, methods Only men: 1) with sperm concentration and motility that were not lower than the 5% centile according to WHO-2021 (16 million/ml and 30%, respectively), 2) without any inflammatory processes in genitourinary organs, 3) with obtained tests for SCI were included in the study. The men were divided into 3 groups (gr.) depending on the percentage of morphologically abnormal forms: I - 86-95% (n = 229), II - 96-99% (n = 228), III - 100% (n = 22). Main results and the role of chance The mean SDF in groups I, II, III were 9.0% (6-15), 12.0% (8-16) and 16.5% (13-20) (pI-II = 0.009; pII-III = 0.002; pI-III = 0.001), respectively. SDF index (Halosperm® Assay) in groups I, II, and III were 1.3 (1.2-1.5), 1.4 (1.3-1.5), and 1.5 (1.4-1.7), respectively (pI-II = 0.004; pII-III = 0.001; pI-III = 0.009). In 23% (I), 27% (II), and 55% of cases (III) the SDF value exceeded the threshold value (&amp;gt;15%) (pI-II &amp;gt; 0.05; pII-III &amp;lt;0.02; pI-III &amp;lt;0.005). Histone-positive sperm were revealed in 17% (12-23), 19.5% (13-27), and 31% (19-42) of cases in groups I, II, and III, respectively (pI-II &amp;gt; 0.05; pII-III = 0.014; pI-III = 0.02); The 30% of SDF threshold value was exceeded in groups I, II, and III, in 12%, 20%, and 52% of cases (pI-II &amp;lt; 0.05; pII-III &amp;lt; 0.005; pI-III &amp;lt; 0.001). Limitations, reasons for caution The reference values of SDF and the proportion of histone-positive sperm have not yet been determined properly, even in WHO 2021, therefore, the prevalence of SCI abnormalities in various spermiological diseases needs to be clarified in the future. Wider implications of the findings SCI and IP can be applied more widely in the investigation of men from infertile couples, especially, men with teratozoospermia. It should also be considered in men from infertile couples with normal semen parameters. Trial registration number Not applicable

P-064 Clinical outcomes of 127 patients with recurrent implantation failure treated with testicular sperm aspiration (TESA)

Abstract Study question Are the embryological, clinical and newborn outcomes using aspirated testicular sperm improved in cases with recurrent implantation failure previously treated with ejaculated sperm? Summary answer Aspirated testicular sperm enabled to obtain significant higher embryological, clinical and newborn outcomes in cases with recurrent implantation failure previously treated with ejaculated sperm. What is known already High levels of sperm DNA fragmentation (SDF) were associated to poor clinical outcomes (1-Simon et al., 2017). Testicular sperm display lower SDF than ejaculated sperm (2-Sakas and Alvarez, 2010), improving clinical outcomes in cases with abnormal semen parameters (3-Awaga et al., 2018; 4-Kang et al., 2018), recurrent implantation failure (RIF) and pregnancy loss (RPL) (5-Esteves et al., 2017), and elevated SDF (6-Ambar et al., 2021). As only a few studies are specifically dedicated to RIF, we expanded the number of cases and first provided full demographic, stimulation, embryological, clinical and newborn outcomes. References 1-(https://doi.org/10.4103/1008-682X.182822); 2-(https://doi.org/10.1016/j.fertnstert.2009.10.046); 3-(https://doi.org/10.1016/j.rbmo.2018.08.017); 4-(https://doi.org/10.1038/s41598-018-26280-0); 5-(https://doi.org/10.1016/j.fertnstert.2017.06.018); 6-(https://doi.org/10.5534/wjmh.200084 Study design, size, duration We retrospectively evaluated during consecutive years (2010-2020) 63 patients with recurrent implantation failure, which accepted to perform testicular sperm aspiration (TESA) as an alternative treatment. These patients presented a long history of failed treatments (153 cycles) using ejaculated sperm. From these cycles, no pregnancy ensued. The present study compares 127 treatment cycles, 80 with testicular sperm (17 cases repeated TESA) and 47 with ejaculated sperm from the same patients performed at the present IVF clinic. Participants/materials, setting, methods Patients were screened for karyotype abnormalities, for Y-chromosome microdeletions (7-Gonçalves et al., 2016), and for SDF with the TUNEL assay (8-Sá et al., 2015). Conventional semen analysis was performed according to World Health Organization guidelines (9-WHO, 2010). Male evaluation and TESA was performed by the same experienced urologist (LF) according to established protocols (10-Madureira et al 2014). The procedure was performed entirely on an outpatient basis, with no complications reported. References 7-(https://doi.org/10.4103/1008-682X.172827); 8-(https://doi.org/10.1016/j.rbmo.2015.06.019); 9-(https://apps.who.int/iris/handle/10665/44261); 10-(https://doi.org/10.1111/j.2047-2927.2014.00231.x). Main results and the role of chance The mean ages were 35.5±3.4 (26-42)-female and 38.1±5.7 (29-59)-male. There were 4 abnormal karyotypes (3-female, 1-male), all without known relevance. Most cases had asthenozoospermia and teratozoospermia (65.1%), or oligoasthenoteratozoospermia (41.8%). Of the 19 cases with &amp;lt;5M/ml, none presented Y-chromosome microdeletions. Although we do not routinely perform SDF testing, 15 patients had previous SDF values (12, &amp;gt;20%; 8, &amp;gt;36%). Female basal characteristics and testicular evaluation were under normal values. The TESA procedure took about 15-20 min, and the time of laboratorial search around 30-60 min. Cases using testicular sperm showed significant higher rates of fertilization (64% vs 73%-p=0.005), blastocyst development (47% vs 62%-p=0.010), implantation (6% vs 27%-p=0.000), clinical pregnancy (10% vs 39%-p=0.001), live birth delivery (5% vs 28%-p=0.005) and newborn (5% vs 32%-p=0.000) than ejaculated sperm. No significant differences were observed regarding the rates of embryo cleavage (95% vs 94.8%) and high quality embryos (89.4% vs 94%), in the mean number of transferred embryos (1.8±0.4 vs 1.9±0.4), or in the abortion rate (2 cases-50% vs 7 cases-25.9%). Cases using testicular sperm had 22 frozen-thawed embryo transfer cycles, enabling per initiated cycle a cumulative pregnancy rate of 45%, live birth delivery rate of 31.3% and newborn rate of 37.5% (32 newborn). Limitations, reasons for caution Although presenting the higher number of cycles using TESA in the treatment of RIF, this number needs to be increased for drawing more definitive conclusions, as these women present a diversity of conditions, rendering subgrouping difficult. In the future, it would also be important to evaluate SDF in all cases. Wider implications of the findings In conclusion, the present results gave further evidence for the superiority of using testicular sperm instead of ejaculated sperm in cases with recurrent implantation failure. Data also evidences the security of using testicular sperm aspiration, as there were no pregnancy or delivery complications, or congenital anomalies among the 32 newborn. Trial registration number Not Applicable

PD09-01 LOW BIRTH WEIGHT IS ASSOCIATED WITH SPERM DNA FRAGMENTATION AND ASSISTED REPRODUCTIVE TECHNOLOGY OUTCOMES IN PRIMARY INFERTILE MEN—RESULTS OF A CROSS-SECTIONAL STUDY

PD09-01 LOW BIRTH WEIGHT IS ASSOCIATED WITH SPERM DNA FRAGMENTATION AND ASSISTED REPRODUCTIVE TECHNOLOGY OUTCOMES IN PRIMARY INFERTILE MEN—RESULTS OF A CROSS-SECTIONAL STUDY

PRM1 Gene Expression and Its Protein Abundance in Frozen-Thawed Spermatozoa as Potential Fertility Markers in Breeding Bulls.

Functional genes and proteins in sperm play an essential role in bulls’ reproductive processes. They are more accurate in determining bull fertility than conventional semen quality tests. Protamine-1 (PRM1) is a gene or protein crucial for packaging and protecting sperm DNA until fertilization affects normal sperm function. This study analyzes the genes and proteins potential from PRM1 as fertility markers for different breeds of bulls utilized in the artificial insemination programs, expected to be an accurate tool in interpreting bull fertility in Indonesia. This study used Limousin, Holstein, and Ongole Grade bulls divided into two groups based on fertility, high-fertility (HF) and low fertility (LF). The semen quality assessment included progressive motility (computer-assisted semen analysis), viability (eosin-nigrosine), and plasma membrane integrity (HOS test). Sperm DNA fragmentation (SDF) was assessed using the acridine orange staining and the Halomax test. Sperm PRM deficiency was evaluated with the chromomycin A3 method. Moreover, PRM1 gene expression was measured using qRT-PCR, and the PRM1 protein abundance was measured with the enzyme immunoassay method. Semen quality values, relative expression of PRM1 gene, and quantity of PRM1 protein were significantly higher (p < 0.05) in HF bulls than in LF bulls. The SDF and PRM deficiency values in LF bulls were significantly higher (p < 0.05) than HF bulls. Additionally, PRM1 at the gene and protein levels correlated significantly (p < 0.01) with fertility. Therefore, PRM1 is a potential candidate for fertility markers in bulls in Indonesia.

CAN MICROFLUIDIC SPERM SORTING HELP SEPARATION OF SPERMS WITH GOOD QUALITY DNA?

It is evident that raised sperm DNA Fragmentation (SDF) negatively affects the reproductive outcomes. Management for raised sperm DFI to optimize reproductive outcomes is still elusive. Microfluidic sperm sorting and Magnetic cell sorting are few of the newer methods employed to obtain sperms with good DNA and optimize reproductive outcomes. However, there is further need for clinical validation of these newer interventions. In Individuals with raised SDF, will sperm sorting with Microfluidics principle help in obtaining sperms with good quality DNA? This was an observational pilot study performed at our private fertility unit from August 2020 to March 2021. Couples with raised SDF (>15%) were recruited and offered microfluidic sorting (n=34). Couples with history of one failed IVF cycle were offered testing for SDF. Individuals with SDF > 15% were included in the study. SDF testing was done with SCSA flow cytometer method. On the day of oocyte retrieval male partners were advised to come with 48hrs abstinence and semen sample was obtained by masturbation. Small aliquot of ejaculated neat sample was sent for SDF testing. Zymot multi ZMH0850 Microfluidic chamber was used for this study. Protocol for microfluidic sorting was done as per the manufacturer’s instruction. Post microfluidic processing the sample was sent to SDF testing again. SDF values of neat and processed samples were compared. Appropriate consents were obtained from the couples that were recruited in this study after thorough counseling. Mean of SDF of neat samples was 24%. Mean of SDF of microfluidic-sorted samples was 3.1%. SDF values of all samples after microfluidic sorting were less than 10%. Paired “t” test was done and the outcomes were statistically significant. There seemed a significant reduction in SDF values with microfluidic sperm sorting. Microfluidics Sperm Sorting seems to be a promising intervention to obtain sperms with good DNA quality. Microfluidics Sperm Sorting seems to be a beneficial intervention to optimize sperm selection with good DNA quality for Individuals with raised sperm DFI.

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 &amp;lt;20% (n = 148), SDF 20–30% (n = 133), and SDF &amp;gt;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 &amp;lt; 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

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.