High DNA Fragmentation Index Research Articles

Impact of sperm DNA fragmentation in couples with unexplained recurrent pregnancy loss: A cross-sectional study.

Recurrent pregnancy loss (RPL) has a multifactorial etiology, with a majority of cases remaining unexplained. To account for these unexplained cases, possible male factors are being explored. Conventional semen analysis lacks a qualitative assessment of sperms and information regarding sperm DNA integrity. Sperm DNA fragmentation (SDF) has diagnostic value in unexplained RPL, and it may account for a number of unexplained cases. Hence, we planned a study to explore and evaluate the impact of sperm DNA fragmentation in couples with unexplained recurrent pregnancy losses. Analytical cross-sectional study was conducted at a tertiary-level referral facility in India between August 2021 and July 2023. Participants (n = 70) were divided into two groups-male partners of couples with unexplained RPL (following spontaneous conceptions) (n = 35) and men with at least one previous live birth (spontaneous or following fertility treatments for female factor infertility such as ovulation induction or intrauterine insemination) as controls (n = 35). Neither of the two groups of couples recruited for this study had undergone ART as fertility treatment. Primary outcome assessed was mean DNA fragmentation index (DFI). Secondary outcomes included differences in semen parameters such as sperm concentration, progressive sperm motility and morphology, proportion of men with high (≥30%) and low DFI in the two groups, and the association between various semen parameters and DFI. Univariate logistic regression revealed that sperm DNA fragmentation was higher in men with unexplained RPL (30.0; IQR (interquartile range) 19.0, 46.0) as compared to controls (22.0; IQR 14.0, 30.0) although it was not statistically significant (OR, odds ratio, 1.02; 95% CI 1.0-1.1, p = 0.08). A higher proportion of men with unexplained RPL had DFI ≥30% compared to controls (54.2% vs. 25.7%; OR 3.43 (95% CI 1.2-9.4); p = 0.02). No statistically significant differences were observed in semen volume, sperm concentration, progressive motility, and morphology between the two groups. Sperm DNA fragmentation index also showed a weak but significant inverse relationship with sperm morphology (r = -0.336, p = 0.004). The current study did not show any significant difference in the mean sperm DNA fragmentation levels in male partners of couples with unexplained RPL compared to controls. However, a higher proportion of men with DFI ≥30% were observed in unexplained RPL population when compared to controls.

High sperm deoxyribonucleic acid fragmentation index is associated with an increased risk of preeclampsia following assisted reproduction treatment

To study the association between sperm DNA fragmentation index (DFI) and the odds of preeclampsia and other adverse perinatal outcomes after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment. A prospective cohort study including infertile couples undergoing conventional IVF or ICSI treatment and their children. Data regarding preeclampsia and perinatal outcomes were derived from the Swedish National Birth Register. 1594 infertile couples undergoing IVF or ICSI treatment and their 1660 children conceived by assisted reproduction. Sperm DNA fragmentation index measured by Sperm Chromatin Structure Assay. The primary outcome was preeclampsia. Secondary outcomes were preterm birth, low birth weight, low Apgar score, and small for gestational age. With DFI < 20% as a reference, the OR for preeclampsia was statistically significantly increased in the group with DFI ≥ 20% when IVF was used as fertilization method (OR 2.2; 95% CI 1.1 to 4.4; p = 0.02). Already at DFI levels ≥ 10%, in IVF pregnancies, preeclampsia odds were increased in a dose-response manner, from a prevalence of 3.1% in the reference group to more than 10% among those with DFI of 30% or higher. The DFI was not associated with preeclampsia odds in the ICSI group. In the entire cohort, DFI ≥ 20% was associated with an increased OR of preterm birth (OR 1.4; 95% CI 1.0 to 2.0; p = 0.03). High DNA fragmentation index was associated with increased odds of preterm birth and, in IVF pregnancies, also increased odds of preeclampsia.

P-086 Consequences of elevated levels of DNA fragmentation and its influence on pregnancy – A multicentric cohort analysis

Abstract Study question Does an abnormal DNA Fragmentation Index impact the clinical outcomes of patients undergoing IVF treatment? Summary answer Statistical differences were seen in biochemical, implantation, clinical, and ongoing pregnancy rate in sperms with high and low DFI scores with autologous and donated oocytes. What is known already Sperm chromatin integrity is associated with embryo development and clinical outcomes of an IVF cycle. DNA Fragmentation Index (DFI) is a diagnostic tool for the detection of spermatozoa with damaged chromosomes in the ejaculate. DNA fragmentation is a significant cause of day 3 embryo arrest and the development of poor-quality embryos. The interventions associated with elevated levels of DFI are ICSI, lifestyle changes and antioxidant therapies. However, the published data regarding the potential of embryo development with high DFI is limited to the sample size thus, encouraging a need for more research in this area. Study design, size, duration The study is population-based and included 768 male patients who underwent DFI score detection across 44 centers at Indira IVF Hospital Private Limited from January 2023 to September 2023. The abstinence period for all the patients was recorded during the DFI detection. After the DFI assessment patients were classified into normal DFI score ( &amp;lt; =15%) (n = 203) and abnormal DFI score (&amp;gt;15%) (n = 565). Participants/materials, setting, methods The retrospective cohort analysis was performed on our databank on the male patients regardless of their age (21-50), ethnicity, and medical history. DFI was calculated using a Sperm Chromatin Dispersion assay. Patients were categorized into two groups: Normal DFI ( &amp;lt; =15%) and Abnormal DFI (&amp;gt;15%). The characteristics of population along with clinical outcomes were evaluated after transferring embryos fertilized with normal DFI sperm and abnormal DFI sperm. The data was analyzed and estimated by SPSS software. Main results and the role of chance Our analyses discovered that embryos fertilized with sperms of normal DFI score have significantly enhanced the likelihood of implantation compared with embryos fertilized with sperms of abnormal DFI (50.27% v/s 44.40%; p = 0.047). Simultaneously, early pregnancy was evaluated by determining the values of beta-HCG in blood, also known as biochemical pregnancy rate (BPR). The embryos fertilized with sperms of normal DFI score significantly enhanced the BPR compared with embryos fertilised with sperms of abnormal DFI (67% v/s 59.1%; p = 0.048). Clinical Pregnancy Rate (CPR) defined as presence of a heartbeat of gestational sac observed during the 6-week scan. Furthermore, it was evaluated that the CPR showed a significant enhancement in embryos fertilized with sperms of normal DFI score in comparison with embryos fertilized with sperms of abnormal DFI (65.5% v/s 57.9%; p = 0.034). However, it was noticed that male DFI score had no impact on the miscarriage rate of embryos inseminated with normal DFI score and abnormal DFI score (7.52% v/s 7.34%; p = 0.687). Moreover, the pregnancy was tracked, and it was revealed that embryos inseminated with sperms of normal DFI score significantly enhanced the ongoing pregnancy till 12 weeks compared with embryos inseminated with sperms of abnormal DFI score (60.6% v/s 53.6%; p = 0.039). Limitations, reasons for caution Embryos are known to have a self-correct mechanism to correct the presence of mosaicism in the embryos. Hence, the oocyte quality also affects the outcomes of the IVF cycle. Therefore, using a high grade of the oocyte for the patients with abnormal DFI score can be useful. Wider implications of the findings Our data suggest that abnormal DFI have an impact on the clinical outcomes of an IVF cycle. Therefore, examining the levels of DNA Fragmentation is utmost imperative for a successful pregnancy. Trial registration number not applicable

P-036 Female age affects the combined impact of DNA fragmentation and high DNA stainability on embryo development in intracytoplasmic sperm injection cycles

Abstract Study question To investigate the role of female age in regulating the co-effect of DNA fragmentation index (DFI) and high DNA stainability (HDS) on ICSI embryological outcomes. Summary answer When female age exceeded 35, co-exposure high DFI and middle HDS might delay embryonic development after ICSI treatment. What is known already Sperm DNA damage had a negative influence on embryonic development. Oocytes and embryos have adopted specific response and repair mechanisms to repair sperm DNA damage. However, the DNA repair capacity in the oocyte is limited and likely decline with increasing age. Study design, size, duration This historical cohort study was included 1334 couples underwent ICSI from January 2017 to December 2022. An unsupervised K-means clustering method was used to divide participants into three clusters base on DFI value and HDS value, as follow: cluster 1 with low DFI values and low HDS values (lDFI-lHDS group), cluster 2 with middle DFI values and high HDS values (mDFI-hHDS group), and cluster 3 with high DFI values and middle HDS values (hDFI-mHDS group). Participants/materials, setting, methods All participants were further stratified into female age&amp;lt;35 subgroup and female age≥35 subgroup. The univariate and multivariate linear regression model was used for statistical analysis. Multivariate model was adjusted for female factor (including age, AMH, basal FSH and the numbers of retrieved oocyte) and male age which were significantly correlated with embryo development in univariate models. The main outcome measure was D5 blastocyst development rate and D6 blastocyst development rate. Main results and the role of chance In the female age&amp;lt;35 subgroup, D5 blastocyst development rate in the mDFI-hHDS group (48.53% [25.00%, 66.67%] and the hDFI-mHDS group (50.00% [28.57%, 71.07%]) was significantly higher than that in the lDFI-lHDS group (40.00% [0.00%, 63.96%]). No significant differences were observed between the three groups regarding D6 blastocyst development rate. And univariate model and multivariate model showed that the mDFI-hHDS group (adjusted B value=0.36, 95% CI: 0.08∼0.64, P=0.010) and hDFI-mHDS group (adjusted B value=0.41, 95% CI: 0.12∼0.70, P=0.006) had higher D5 blastocyst development rate compared with lDFI-lHDS group. In the female age≥35 subgroup, D5 blastocyst development rate in the hDFI-mHDS (20.00% [0.00%, 50.00%]) group was significantly lower than that in the lDFI-lHDS group (36.93% [0.00%, 66.67%]) and the mDFI-hHDS group (41.43% [0.00%, 63.54%]). Accordingly, D6 blastocyst development rate in the hDFI-mHDS group (66.67% [33.33%, 100.00%]) was significantly higher than that in the lDFI-lHDS group (50.00% [16.67%, 80.00%]) and the mDFI-hHDS group (50.00% [33.33%, 66.67%]). And both model showed that hDFI-mHDS group (adjusted B value=0.73, 95% CI: 0.36∼1.11, P&amp;lt;0.001) had higher D6 blastocyst development rate compared with the lDFI-lHDS group. Although not statistically significant, hDFI-mHDS had negative correlation with D5 blastocyst development rate. Limitations, reasons for caution The main weakness of this study was the participants were not randomized. Wider implications of the findings hDFI-mHDS group were negatively associated with D5 blastocyst development rate and positively related with D6 blastocyst development rate in female age≥35 subgroup. These results suggested that co-exposure high DFI and middle HDS might be recommended as a predictor for the detection of delayed embryo development when female age exceeded 35. Trial registration number not applicable

P-107 Sperm DNA fragmentation impacts embryonic developmental potential but is not associated with PGT-A outcomes

Abstract Study question Does sperm DNA Fragmentation Index (DFI) affect embryonic development and influence Preimplantation Genetic Testing for Aneuploidy (PGT-A) outcomes in IVF? Summary answer DFI adversely affects early embryonic development, diminishing blastocyst potential. Nonetheless, PGT-A outcomes are unaffected, underscoring the importance of selecting optimal sperm for enhanced IVF results. What is known already Several major factors are thought to cause DFI. The relationship between sperm DFI and IVF results remains debated. Study design, size, duration This study was conducted from January 2018 to March 2023, included 56 married couples undergoing IVF after performing a sperm DFI test. The DFI was assessed at the first semen examination and categorized into three groups: low (≤15%), moderate (15-30%), and high (≥30%). Participants/materials, setting, methods Intracytoplasmic sperm injection (ICSI) was performed on 1006 mature oocytes, followed by blastocyst culture. A total of 278 blastocysts meeting freezing criteria were vitrified. Trophectoderm biopsies from 216 blastocysts underwent Next-Generation Sequencing for aneuploidy analysis. Statistical analysis utilized the Cochran-Armitage test to compare outcomes among the DFI groups. Main results and the role of chance The average age of the husbands and wives who underwent IVF after the DFI examination was 42.3±4.2 and 41.4±2.4 years old. Normal fertility rates after ICSI in low, moderate, and high DFI were 85.5%, 84.5%, and 83.1%, with no significant difference. Similarly, the blastocyst freezing rates were 35.4%, 26.3%, and 29.6%, and tended to decrease as the DFI value increased (p &amp;lt; 0.05). However, the euploid rates for PGT-A were 22.4%, 16.3%, and 18.2%. There were no statistically significant differences between the DFI groups in terms of euploidy. Limitations, reasons for caution Cases declining ICSI due to patient preference or historical medical reasons were excluded from this study. Wider implications of the findings This suggests that sperm DNA imperfections have a negative impact on the developmental process during the early stages of division, but during late stages of division, the ability to repair DNA works to differentiate into blastocysts with normal developmental potential. Trial registration number not applicable

P-527 Low sperm motility and elevated sperm DNA fragmentation index (DFI) are associated with increased rates of embryo aneuploidy in patients with advanced reproductive age

Abstract Study question Is there a correlation between the paternal age, sperm characteristics, and the prevalence of chromosomal abnormalities in embryos generated through in vitro fertilization (IVF)? Summary answer A correlation exists between advanced paternal age, suboptimal sperm parameters, and elevated rates of embryo aneuploidy in individuals of advanced reproductive age. What is known already The exact mechanisms underlying the relationship between paternal age, sperm quality and embryo ploidy, although a subject of considerable research in assisted reproduction, are not fully elucidated. Some reports, including our group’s, suggest that paternal age may influence sperm quality and segmental embryo aneuploidy, while others suggest a potential association between high DFI(DNA Fragmentation Index) and an increased likelihood of embryo aneuploidy. Studies also show that sperm with optimal motility and fertilization competence are more likely to contribute to the development of euploid embryos, while data on the effect of abnormal sperm parameters on embryo ploidy is scarce and conflicting. Study design, size, duration This was a retrospective cohort study performed in the Reproductive Genetics Department, CReATe Fertility Centre, Toronto, Canada, between January 2019-December 2023. A total of 7628 blastocysts from 1512 cycles (1392 patients) were analyzed using high resolution NGS PGT-A. Sperm motility, concentration and DFI data, as well as, IVF cycle parameter data were collected. In addition, age of gamete providers and any relevant clinical, laboratory and demographic data were obtained for statistical analysis. Participants/materials, setting, methods Embryos were allocated into two groups by oocyte-provider age: OP-≤33 years and OP-≥34 years. Each group was divided into two sub-groups by paternal age: SP-≤39 years (n = 4570); SP-≥40 years (n = 3058). Multiple logistic/ordinal regressions were conducted to evaluate Segmental abnormalities and Segmental mosaicism between these age groups in relation to sperm DFI (GOOD ≤15%; FAIR 15-30%; POOR ≥30%), Motility (HIGH &amp;gt;40%; LOW &amp;lt;40%) and Count (LOW &amp;lt;5 mil/ml; MEDIUM 5-15 mil/ml; HIGH &amp;gt;15 mil/ml). Main results and the role of chance PGT-A analysis of 7628 embryos showed 51.5% euploidy, 36.7% aneuploidy and 11.8% mosaicism. In the OP-≤33 group, there were no significant differences between the two paternal age groups for euploidy, aneuploidy or mosaicism rates. In the OP-≥34 group, decreasing euploidy rates (p &amp;lt; 0.0001 and increasing aneuploidy rates (p &amp;lt; 0.0001) were observed between the two paternal age groups, while mosaicism rates were not different, an expected oocyte age-effect result. No significant differences in aneuploidy rates were observed for each of the sperm parameters (DFI, motility, count) in the groups of the OP-≤33 with SP-≤39, OP-≤33 with SP-≥40 and OP-≥34 with SP-≤39. In the group of OP-≥34 with SP-≥40, in patients with LOW motility vs. HIGH motility the aneuploidy rates were higher (56.8% vs.50.6%, p = 0.005) and euploidy rates were lower (33.8% vs.38.4%, p = 0.03). Each sperm parameter in each age group was further analyzed for chromosomal segmental abnormalities and segmental mosaicism. Higher rates of segmental abnormalities were seen in LOW motility groups with POOR-DFI compared to GOOD DFI in OP-≤33 with SP-≤39 (3.2% vs. 7.4%, p = 0.03). Limitations, reasons for caution The main limitation was the retrospective nature of this study; however, multiple regression analyses were conducted to limit potential bias by adjusting for sperm and oocyte characteristics. In addition, PGT-A analysis cannot provide information on origin of the aneuploidy and all conclusions are deduced from indirect associations to paternal age. Wider implications of the findings The identified associations could stimulate further research into exploring the molecular/cellular processes involved in fertilization, early embryonic development, and the potential impact of sperm-related factors on the chromosomal stability of developing embryos. The broader implications of these findings extend to clinical practice, patient counseling, technological advancements, and ethical considerations. Trial registration number not applicable

Correlation of Sperm DNA Fragmentation Index With Semen Parameters, Lifestyle and Clinical Pregnancy Outcome After Intracytoplasmic Injection.

This study aimed to measure the correlation of sperm DNA fragmentation with semen parameters, lifestyle, and fertility outcomes after intracytoplasmic injection (ICSI). The partners who were candidates for ICSI with a history of one In vitro fertilization (IVF) failure or male factor were recruited in the study. Semen parameters including sperm count, motility, and morphology as well as DNA fragmentation index (DFI) (that were divided into 2 groups as high (>15%), and low (≤15%) fragmentation scales) were evaluated either. The correlation of DFI with semen parameters, lifestyle, and clinical pregnancy after ICSI were compared between groups. In 120 included couples, 59 men (49.2%) had DFIs ≤ 15% and 61 (50.8%) cases had DFIs >15%. In the group with higher DFI, abnormal morphology (p=0.010) was higher whereas, progressive motility (p=0.001), total motility (p<0.001), and total count (p<0.001) of sperm were significantly lower. In addition, the DFI was significantly higher in the subgroup of male infertility (0.012). Logistic regression showed that a lower risk of DFI>15% was associated with higher values of progressive motility (OR=0.97, p=0.001), total motility (OR=0.96, p=<0.001), count (OR=0.96, p=<0.001) and even clinical pregnancy (OR=0.27, p=0.011). However, a history of testicular surgery was associated with a higher risk of DFI>15% (OR=3.37, p=0.046). Although no correlation was found between male age and lifestyle components with DFI, the number of embryos was lower in DFI≥15% (p<0.001). DFI provide a clinically important measurement of sperm quality and have an impact on IVF outcomes; however, lifestyle components may not correlate with DFI.

Impact of bull age, sperm processing, and microclimatic conditions on the viability and DNA integrity of cryopreserved bovine sperm.

Context Seasonal microclimatic fluctuations can cause changes in sperm quality even in dairy bulls bred under temperate climate. These changes can vary between sires of different age and affect sperm freezability. Aims We aimed to evaluate the modulating effect of bull age and equilibration time before freezing on the seasonal pattern of sperm viability and DNA integrity post-thaw. Methods In the frame of systematic sperm quality control, we assessed the integrity of sperm plasma membrane and acrosome (PMAI) in 15,496 cryopreserved bovine batches, and the percentage of sperm with high DNA fragmentation index (%DFI) after 0h and 3h incubation at 38°C post-thaw (3h) in 3422 batches. Semen was equilibrated for 24h before freezing if collected on Monday or Wednesday and 72h if produced on Friday. We investigated the effect of season, bull age, equilibration, and temperature-humidity index (THI) on the day of semen collection on sperm traits using mixed-effects linear models. Key results PMAI and %DFI (0h and 3h) deteriorated with increasing THI. The effect of THI on %DFI was detected with a 30-day time lag. Seasonal fluctuations of sperm quality were similar between young, mature, and older sires. Prolonged equilibration did not affect PMAI but was linked to elevated %DFI (3h) in summer. Conclusions Extending equilibration from 24 to 72h is compatible with commercial standards of bovine sperm quality post-thaw; however, it could interfere with the seasonal pattern of the latter. Implications Systematic monitoring of bovine sperm quality enables the prompt detection of stress factors related to microclimate and semen processing.

Maternal intake of paracetamol during pregnancy and biomarkers of male fecundity in young adult sons

Paracetamol is suggested to have endocrine disrupting properties possibly affecting fetal programming of reproductive health that might lead to impaired semen quality and changes in reproductive hormones. In this longitudinal study, we included 1058 young adult men born 1998–2000 into the Danish National Birth Cohort with follow-up at 18–21 years of age. The exposure, maternal intake of paracetamol, was modelled in three ways: dichotomized, trimester-specific, and as duration of exposure categorized into: short (1–2 weeks), medium (3–9 weeks) or long duration (>9 weeks) vs. no intake. Outcomes included semen characteristics, self-measured testis volume, and reproductive hormone levels. We used negative binominal regression to estimate the percentage difference and 95% confidence interval (CI) for each outcome. In total, 547 (48%) sons were prenatally exposed to paracetamol due to maternal intake at least once. Maternal intake of paracetamol during pregnancy was not associated with any of the biomarkers in the dichotomized or trimester-specific exposure models. For duration of exposure, sons of mothers with long duration of maternal intake of paracetamol showed tendencies towards lower semen concentration (-14% [95% CI: -31%; 8%]), a higher proportion of nonprogressive and immotile spermatozoa (8% [95% CI: -4%; 21%]) and higher DNA Fragmentation Index (16% [95% CI: -1%; 36%]) compared to son of mothers with no intake. Maternal intake of paracetamol during pregnancy was not clearly associated with biomarkers of male fecundity in adult sons. However, it cannot be ruled out that long duration of maternal intake of paracetamol might be associated with impaired semen characteristics.

ICSI using testicular spermatozoa after failure of ICSI with ejaculated spermatozoa could be a good choice: A propensity score-matched cohort study.

Ejaculated spermatozoa are considered to possess a higher fertilisation potential than testicular spermatozoa. In selected cases, the use of testicular spermatozoa from non-azoospermic infertile men resulted in a higher implantation and pregnancy rate than the use of ejaculated spermatozoa. The primary objective was to compare the live birth rate and cumulative live birth rate between couples with failed intracytoplasmic sperm injection procedure using ejaculated spermatozoa who subsequently had an intracytoplasmic sperm injection cycle with testicular spermatozoa and those who subsequently had an intracytoplasmic sperm injection cycle with ejaculated spermatozoa. The secondary objective was to determine the indications for the use of testicular spermatozoa after intracytoplasmic sperm injection failure with ejaculated spermatozoa. A retrospective study of matched couples using propensity score matching analysis was performed. After an intracytoplasmic sperm injection failure (cycle_1), intracytoplasmic sperm injection with either ejaculated spermatozoa (ejaculated sperm group), or testicular spermatozoa (testicular sperm group), was performed (cycle_2). The matching was on intracytoplasmic sperm injection performed in cycle_1 according to spermatozoa used (testicular or ejaculated) in cycle_2. Logistic regression was used to evaluate the influence of sperm origin on cumulative live birth rate. Univariate analysis on parameters of cycle_1 was used to identify the prognostic factors to propose an intracytoplasmic sperm injection with testicular spermatozoa in case of cycle_1 failure. The study outcomes were live birth rate and cumulative live birth rate. Among the 6034 couples available, 63 were selected to constitute the testicular sperm group and 63 were selected by propensity score matching to constitute the ejaculated sperm group. After matching, the DNA fragmentation index was higher in the testicular sperm group (13.43%±9.65% vs. 8.93%±4.47%, p=0.013); no significant difference was observed for the fertilisation rate, the number of obtained embryos, blastulation rate and frozen embryo rate. In cycle_2, the live birth rate was higher in the testicular group (22.2% vs. 0.0%, p<0.001), as was the cumulative live birth rate (25.4% vs. 6.3%, p=0.065). The prognostic factors identified for the proposal of intracytoplasmic sperm injection procedure with testicular spermatozoa after intracytoplasmic sperm injection failure with ejaculated spermatozoa were: teratozoospermia, cryptozoospermia and high DNA fragmentation index. According to the present study and current knowledge, the use of testicular spermatozoa after failed intracytoplasmic sperm injection procedure in non-azoospermic men could be proposed instead of sperm donation in case of high sperm DNA fragmentation index, cryptozoospermia and teratozoospermia. A good oocyte response to ovarian stimulation during the previous assisted reproductive technology attempt will increase the chance of success. Although the main limitation of the current study is its retrospective nature, the use of the propensity score matching to perform causal inference study increases its reliability. The present study supports that the use of testicular spermatozoa outside the classical indication of azoospermia is a good option when the indication is well established. However, before proposing a testicular biopsy, an improvement in sperm characteristics should be considered by treating the causes of sperm alteration.

#204 : First Recorded Normal Live Birth After ICSI with Electrophoretically Isolated Spermatozoa Using the Felix™ System

Background and Aims: This report describes the first live birth with sperm isolated using the Felix™ system. Methods: Thirty-three couples undergoing ovarian stimulation were recruited based on high raw semen DNA Fragmentation Index (DFI) of &gt;20%, as assessed by the sperm chromatin structure assay. The Felix™ system, which isolates sperm based on charge and size was used as the sperm preparation method, over a six-minute interval. Mature oocytes were injected using Intracytoplasmic Sperm Injection (ICSI). Normally fertilised zygotes were cultured until day three or five. Suitable embryos were vitrified and subsequently transferred in a frozen embryo cycle. Biochemical pregnancy was determined by plasma human chorionic gonadotrophin level of &gt;20 IU and clinical pregnancy was determined by the presence of fetal heat and/or sac on ultrasound. Miscarriage and live births rates were also recorded. Results: Raw semen concentration, progressive motility and total motility levels were in the normal range according to 6[Formula: see text] World Health Organisation criteria. However, sperm DFI was relatively high (range 20% to 80%). The average age of females was 30 years (range 21 to 44). A total of 374 oocytes were collected from the 33 females, with 266 mature oocytes then injected by ICSI. This resulted in 83% fertilisation rate and embryo utilisation of 65%. Four females underwent single embryo transfer, and 29 females underwent double embryo transfer. Of those, 19 (58%) biochemical pregnancies were achieved resulting in 14 (42%) ongoing clinical pregnancies. Three miscarriages and two normal live births have been reported to date. Interestingly, the two live births resulted from men of DFI of 80% and 33%. Conclusion: This report describes the first birth after sperm isolated using the Felix™ system followed by ICSI. The Felix™ system is an efficient procedure to prepare sperm and has the potential to be advantageous for patients with high DFI.

#353 : Microfluidic Preparation of Spermatozoa: A Potential Approach in Assisted Reproductive Technology

Background and Aims: While density gradient centrifugation (DGC) is a conventional method in sperm preparation, this method can induce reactive oxygen species (ROS) which cause sperm dysfunction, sperm DNA damage, a recent advancement in sperm processing using microfluidic system is believed to reduce damage and to select the good quality sperm. The aim of this study was to evaluate the effectiveness of sperm preparation between DGC method and microfluidic system. Method: 30 semen samples were separated into 2 equal small samples, each sample was processed by DGC methods (control) and microfluidic (intervention) respectively. Pre- and post processing parameters was evaluated according to WHO-2010 and the DNA fragmentation index (DFI) was determined by flow cytometry technique. Results: The result showed that DFI values from the microfluidic preparation (0.67 ± 0.53%) was significantly lower than that of the DCG group (1.18 ± 13.73%). In particular, for semen samples with high DFI (&gt;15%), the microfluidic system showed a noticeable reduction in DFI compared to the results from the DGC method. The progressive motile sperm recovery rate was higher in the intervention group (39.75 ± 19.20 vs 32.14 ± 18.14). Our data also showed that the vitality and mobility of sperm in the microfluidic group was higher than those of the DGC group (97.9 ± 2.21 vs 96.56 ± 4.11%, 96.36 ± 2.60 vs 95.06 ± 2.35%, respectively). However, there was no difference in sperm progressive motility and sperm morphology between two groups. Conclusions: There were significant improvement in sperm DFI and progressive motility in the microfluidic system. With this result, microfluidic system is a potential approach to reduce DNA fragmentation for ICSI procedures.

High sperm DNA fragmentation: do we have robust evidence to support antioxidants and testicular sperm extraction to improve fertility outcomes? a narrative review.

To date, infertility affects 10% to 15% of couples worldwide. A male factor is estimated to account for up to 50% of cases. Oral supplementation with antioxidants could be helpful to improve sperm quality by reducing oxidative damage. At the same time, there is a growing interest in the literature on the use of testicular sperm in patients with high DNA fragmentation index (DFI). This narrative review aims to evaluate the effectiveness of supplementation of oral antioxidants in infertile men with high DFI compared to testicular sperm retrieval. The current evidence is non-conclusive because of serious risk of bias due to small sample sizes and statistical methods. Further large well-designed randomised placebo-controlled trials are still required to clarify the exact role of these to different therapeutic approaches.

The impact of female BMI on sperm DNA damage repair ability of oocytes and early embryonic development potential in intracytoplasmic sperm injection cycles.

Obesity adversely influences the quality of oocytes and embryos and can affect DNA repair in embryos, leading to reproductive issues. However, the effects of body mass index (BMI) on DNA repair ability in oocytes during intracytoplasmic sperm injection (ICSI) cycles have not yet been investigated. Therefore, this retrospective study aimed to analyze the influence of sperm DNA damage on embryo development and reproductive outcomes in overweight/obese and normal-weight women in ICSI cycles. A total of 1,141 patients who received the first fresh ICSI cycle treatments were recruited from July 2017 to July 2021. Based on the BMI of the women, all patients were divided into normal weight (18.5≤BMI<25 kg/m2; n=824; 72.22%) and overweight/obese (BMI≥25 kg/m2; n=317; 27.78%) groups. Furthermore, according to the sperm DNA fragmentation index (DFI), these two groups were subdivided into two subgroups: DFI<30% and DFI≥30%. In the normal-weight women group, the embryonic development and reproductive outcomes of ICSI cycles were not statistically different between the two subgroups (DFI<30% and DFI≥30%). However, in the overweight/obese women group, couples with a sperm DFI≥30% had a significantly lower fertilization rate (76% vs. 72.7%; p=0.027), cleavage rate (98.7% vs. 97.2%; p=0.006), and high-quality embryo rate (67.8% vs. 62.6%; p=0.006) than couples with a sperm DFI<30%. When injected sperm with high DFI into the oocytes of overweight/obese women, resulting in lower fertilization, cleavage, and high-quality embryo rates in ICSI cycles, and the decreased early developmental potential of embryos from overweight/obese patients may be caused by the diminished capacity of oocytes to repair sperm DNA damage.

Decreased AKAP4/PKA signaling pathway in high DFI sperm affects sperm capacitation.

The sperm DNA fragmentation index (DFI) is a metric used to assess DNA fragmentation within sperm. During in vitro fertilization-embryo transfer (IVF-ET), high sperm DFI can lead to a low fertilization rate, poor embryo development, early miscarriage, etc. A kinase anchoring protein (AKAP) is a scaffold protein that can bind protein kinase A (PKA) to subcellular sites of specific substrates and protects the biophosphorylation reaction. Sperm protein antigen 17 (SPA17) can also bind to AKAP. This study intends to explore the reason for the decreased fertilization rate observed in high sperm DFI (H-DFI) patients during IVF-ET. In addition, the study investigates the expression of AKAP, protein kinase A regulatory subunit (PKARII), and SPA17 between H-DFI and low sperm DFI (L-DFI) patients. SPA17 at the transcriptional level is abnormal, the translational level increases in H-DFI patients, and the expression of AKAP4/PKARII protein decreases. H 2 O 2 has been used to simulate oxidative stress damage to spermatozoa during the formation of sperm DFI. It indicates that H 2 O 2 increases the expression of sperm SPA17 protein and suppresses AKAP4/PKARII protein expression. These processes inhibit sperm capacitation and reduce acrosomal reactions. Embryo culture data and IVF outcomes have been documented. The H-DFI group has a lower fertilization rate. Therefore, the results indicate that the possible causes for the decreased fertilization rate in the H-DFI patients have included loss of sperm AKAP4/PKARII proteins, blocked sperm capacitation, and reduced occurrence of acrosome reaction.

DNA Integrity in Absolute Teratozoospermia Patients and its Impact on Assisted Reproductive Technology Outcome

Numerous studies showed that Sperm DNA Fragmentation (SDF) is inversely correlated to semen parameters and that high SDF is a major contributor to failure of conception. In the present study, the severity of Sperm DNA Fragmentation in absolute teratoozoospermia patients and its impact on ICSI outcome compared to males with normal semen was examined. A prospective study was performed including 36 absolute teratozoospermia and 30 controls. The mean of DNA Fragmentation Index (DFI) using Sperm Chromatin Dispersion (SCD) test in the absolute teratozoospermia was 42.9 ± 13.2, compared to 24.59 ± 6.9 for the controls. The percentage of patients with high DFI (&gt;30) was higher in the absolute teratozoospermia group 78% than in the control group 30%. Moderate DFI (15-30%) was more common in controls 60% than in absolute teratozoospermia 22%. Normal DFI (≤ 15%) was completely absent in absolute teratozoospermia. The pregnancy rate following Intracytoplasmic Sperm Injection (ICSI) in the control group was 66.6% and 50% in the absolute teratozoospermia. The miscarriage rate was 26% in the control group compared to 53% in the absolute teratozoospermia. Live birth was significantly lower in absolute teratozoospermia compared to fertile men; 19% and 52% respectively. High levels of SDF are predominant in absolute teratozoospermia patients consequently affecting the outcome of ICSI.

P-019 Short ejaculatory abstinence time is associated with higher pregnancy and live birth rates and improved DNA fragmentation index (DFI): A Systematic Review

Abstract Study question Does short ejaculatory abstinence time (EA) improve DNA fragmentation and reproductive outcomes compared with long EA following assisted reproductive technology (ART)? Summary answer Shorter EA is associated with improved pregnancy and live birth rates following ART and lower DFI compared with longer EA at different cut-off points. What is known already When spermatozoa pass the epididymal tract, they are exposed to reactive oxygen species (ROS). ROS is known to cause DNA fragmentation. Testicular sperm has a lower DNA fragmentation when compared to ejaculated sperm, which indicates that DNA fragmentation is increased during the transition and storage in the epididymal duct. There are studies that suggest that a high DFI is associated with poor pregnancy and live birth rates following ART. WHO recommends 2-7 days of EA prior to semen collection; however, several recent studies have reported a correlation between shorter EA and improved reproductive outcomes and DFI. Study design, size, duration A systematic review was conducted according to the PRISMA guidelines and registered in PROSPERO (CRD42022379039). The electronic databases PubMed, Embase and Cochrane were searched for eligible studies by a research librarian in April 2022. Additional articles were manually retrieved after reviewing the reference lists from relevant publications. Participants/materials, setting, methods Two authors independently screened studies based on the eligibility criteria; studies including men referred to fertility treatment undergoing short and long EA reporting data on pregnancy rate, live birth rate and DFI. Study demographics, population characteristics and data on study outcomes including statistical analyses were extracted. Risk of bias in each study was evaluated according to The Scottish Intercollegiate Guidelines Network (SIGN). Any disagreements were resolved by a third author. Main results and the role of chance Out of 1237 studies initially identified, 24 of them met eligibility criteria and were included in the review with a total of 14.173 cases. The studies included in this review were conducted in Asia, North and South America and Europe. The cause of referral to fertility treatment includes both male, female, mixed and unexplained infertility. The EA varied from less than a one hour to as long as 15-20 days. Nine of the 13 studies investigated the influence of varying EA on pregnancy rate, found a significantly higher pregnancy rate with short EA compared with long EA, five with EA as short as one day or less. Three studies evaluated live birth rates and found a significantly higher live birth rate when comparing short EA with long EA. Eleven of the 15 studies that reported DFI found significantly lower DFI with short EA compared with long EA. EA of one day or less were associated with the lowest rates of DFI in the studies. In general, pregnancy rate, live birth rate and DNA fragmentation are likely to be improved with short EA compared to long EA. Limitations, reasons for caution Many of the included studies used different EA intervals, leading to heterogenous data, and therefore, only allowed limited conclusion to be formulated regarding the ideal timeframe of EA. Four studies scored poorly in SIGN methodology checklist due to confounding and population size, suggesting their findings to be treated with caution. Wider implications of the findings Even though it is difficult to make a clear recommendation on the ideal timeframe for EA because of the heterogenous abstinence periods, this review suggest decreasing the EA limits of 2-7 days as recommended by WHO to improve pregnancy rate and decrease DNA fragmentation in semen, followed by ART. Trial registration number not applicable

O-299 Sperm DNA fragmentation by TUNEL influences live birth in egg recipients after euploid embryo transfer

Abstract Study question Does sperm DNA fragmentation (SDF) by TUNEL affect reproductive success measured as live birth rate (LBR) in egg recipients after euploid embryo transfer? Summary answer SDF increases miscarriage rates in egg recipients with PGT-A. Therefore, live birth rates depends on sperm DNA damage in egg recipients after euploid embryo transfer. What is known already Sperm DNA integrity is important for optimal fertilisation, implantation and pregnancy. Although controversial, several studies have shown that sperm DNA fragmentation correlates with adverse pregnancy outcomes in infertile men. Ooplasmic sperm DNA fragmentation repair mechanisms may occur in female gametes, mainly in young women. However, research on the impact of DNA fragmentation on LBR are limited. Also, chromosomally normal embryos analysed by PGT-A offers opportunity to isolate female factor and study male factor. The aim of this study is to assess whether high level of semen DNA damage has an impact on reproductive outcomes in terms of live birth rate. Study design, size, duration This is a retrospective study of 467 PGT-A cycles in egg recipients. The study population consists of infertile couples for euploid embryo transfer (February 2017-November 2022). Semen samples were obtained to measure DNA damage from 447 men. PGT-A was performed in 1831 blastocysts. Trophectoderm biopsies (d5/d6 blastocysts) were analysed by NGS (Veriseq Illumina). Embryos were frozen and 568 blastocysts transferred in the subsequent cycle. Conventional hormone replacement was used for endometrial preparation in recipient patients. Participants/materials, setting, methods Cohort study with 447 infertile couples attending to private fertility clinic undergoing ART with their autologous semen, donated oocytes and PGT-A. SDF was measured by Terminal-deoxyribonucleotidyl-ARTSferase-mediated dUTP Nodule End Labelling (TUNEL) assay using the FITC-labelled-dUTP in situ cell death detection kit (Roche). The cohort was divided into two groups according the DNA fragmentation index (DFI): high SDF group (DFI&amp;gt;20%) and normal SDF group (DFI≤20%). To evaluate reproductive outcomes Chi-square and Linear regression tests were used. Main results and the role of chance Of the 447 couples included (average paternal age 41.29 + 7.32 years), 66.5% of male partners had normal semen parameters. We reported high sperm DNA fragmentation in 8% of patients. Overall, the embryo aneuploidy rate was 31.7%. For the further statistical analysis confounding factors such as, female and male age, embryo quality, day of embryo biopsy were included. PGT-A analysis showed an aneuploidy rate of 32.1% in the high DNA fragmentation group and 30.3% in the normal DNA fragmentation group (p = 0.453). Regarding clinical data, the overall implantation rate was 34.9%. There was no significant difference between elevate DNA fragmentation and normal DNA fragmentation with respect to biochemical miscarriage rate (4.3% vs 7.6%, p = 0.799). Although not significant differences between groups, a lower implantation rate near to reach significance was found in the high SDF group (20.0% vs 34.4%, p = 0.050). Pregnancy and clinical pregnancy rates were significantly lower in the high DNA fragmentation group (26.1% vs 44.9%, p = 0.042; 21.7% vs 31.3%, p = 0.044;), respectively. Also, miscarriage rate was significantly higher in the high SDF group (40% vs 12.5%, p = 0.014). Therefore, the live birth rate was significantly lower in the high SDF group (13.0% vs 32.6%, p = 0.024). Limitations, reasons for caution Larger studies including a higher number of samples are needed to confirm the correlation observed between LBR in eggs recipients with semen DNA fragmentation. Those patients with a DFI higher than control values should be treated with antioxidants. The pregnancy outcomes should be analyzed after treatment of DNA fragmentation. Wider implications of the findings Our findings suggest that autologous semen DNA fragmentation influence in live birth rate in egg recipients. Our data show that live birth rates decreased in patients with a high DFI after PGT-A. This study confirms published studies in the literature showing that ART outcomes are affected by sperm DNA fragmentation. Trial registration number Not Applicable

P-063 Microfluidic technology is highly effective in selecting a sperm population with high progressive motility and low DNA fragmentation index

Abstract Study question Which sperm preparation procedure is able to select the best population of sperm? Summary answer Microfluidic sperm sorting using Fertile Plus device isolates sperm with the highest motility and lowest levels of DNA fragmentation. What is known already The sperm selection in Medically Assisted Reproduction should lead to fully functional spermatozoa able to reach the fertilization site and allow optimal fertilization and embryo development. These abilities are dependent on specific sperm parameters such as, but not limited to, motility, morphology, Acrosome Index (AI), and DNA fragmentation Index (DFI). At present, the routine sperm selection is suboptimal, does not replicates the physiological processes observed in nature and consequently may induce sperm damage. The new Microfluidic Sperm Sorting technology allows a physiological selection of sperm. However, no comparison with other selection procedures regarding all the above-mentioned parameters was reported. Study design, size, duration Prospective-observational study performed at an University Hospital-IVF Centre between April-November 2022 comparing the effect of four different sperm selection procedures on various semen parameters. Fifty-two samples with minimum 20x106 sperm/ml and progressive motility ≥30% were included. Descriptive statistics reported the mean±SD [minimum-maximum] values for each variable. Interclass correlation coefficient (ICC) was calculated for the absolute agreement in between the four different procedures, where DGC was considered the standard-of-care. An ICC-value &amp;lt;0.4 indicates a low agreement. Participants/materials, setting, methods Unused raw semen after routine diagnostic analysis were individually split in 4 fractions and processed immediately by different sperm preparation methods: 1) sperm wash (SW), 2) Density Gradient Centrifugation (DGC), 3) Magnetic Activated Cell Sorting (MACS, (Miltenyi Biotec)), and 4) Microfluidic Sperm Sorting (MSS) using Fertile Plus Device (Koek Biotechnology). Each fraction was analyzed for progressive motility, morphology, Acrosome Index (AI) and DNA fragmentation Index (DFI; TUNEL assay protocol). Main results and the role of chance The mean age of the patients was 35.9 years (SD ± 6.8). The mean value of days of abstinence was 4.1 (SD ± 6.8). Mean progressive motility rates were: 54.3±10.6% [23-86%], 74.3±11.8% [38-90%], 77.2±12.5% [37-92%] and 88.6±4.2% [73-96%] for SW, DGC, MACS, and MSS, respectively. Mean percentages of normal morphology were: 3.3±2.9% [0-13%], 4.1±3.1% [0-13%], 4.2±3.7% [0-18%], and 5.1±3.9% [0-16%] for SW, DGC, MACS, and MSS, respectively. Mean AI were: 8.5±4.9% [1-20%], 9.7±6.0% [1-30%], 8.7±4.9% [0-19%], and 10.8±6.8% [1-30%] for SW, DGC, MACS, and MSS, respectively. Mean DFI were: 6.2±4.6% [0.8-26.1%], 2.7±3.2% [ 0.2-14%], 2.1±4.3% [0.9-20.8%], and 0.2±0.4% [0.0-2.3%] for SW, DGC, MACS, and MSS, respectively. Values referring to ICC absolute agreement between the DGC and the other methods indicate MSS as the only procedure in strong disagreement regarding progressive motility [0.29, 95%CI (-0.2-0.58)] and DFI [0.17; 95%CI (-0.19-0.45)]. No disagreement regarding morphology and AI was observed between all techniques. A further paired comparison confirmed the strong disagreement between MSS and MACS [0.2; 95%CI (-0.17-0.49) and 0.21; 95%CI (-0.25-0.52)] and between MSS and SW [0.06; 95%CI (-0.04-0.25) and 0.06; 95%CI (-0.15-0.28)] regarding progressive motility and DFI, respectively. Limitations, reasons for caution Differences in DNA-damage may have been smaller between techniques if abstinence period was shorter. Due to the design of the experiment, data on clinical outcomes are not available. Wider implications of the findings The selection of a population of highly motile spermatozoa with undamaged DNA from unprocessed semen is ideally performed with MSS using Fertile Plus. Question remains how these results compare with a shorter abstinence period and whether this improves the embryological outcomes in the IVF laboratory. Trial registration number NCT05383599

P-038 Correlation between sperm DNA fragmentation index, semen parameters and Human Papillomavirus: an analysis conducted under World Health Organization 2021 guidelines

Abstract Study question Following 6thWHO (2021), we analysed the correlation between DNA fragmentation index (DFI), Human papillomavirus (HPV), and seminal parameters, highlighting slow and rapid progressive motility alterations. Summary answer DFI rates and seminal parameters correlated with rapid, slow, and progressive motility. However, HPV-positivity caused the loss of association between DFI and slow progressive motility. What is known already HPV detection in semen samples has long opened an investigation into its influence on male infertility. Some studies indicate that HPV can affect sperm quality and DFI, while others have failed to find any correlation. With reference to 2010 WHO guidelines, our latest work highlighted how HPV positivity significantly impairs progressive motility, morphology, and immotile sperm rate. Since the latest 2021 WHO guidelines included the evaluation of slow and rapid progressive motility and DFI, we analysed if these new parameters and the other conventional parameters could be altered by HPV infection. Study design, size, duration From August 2021 to December 2022, 121 semen samples were collected from male partners of HPV-positive women attending in vitro fertilization (IVF). Every specimen underwent DFI evaluation, analysis of seminal parameters, and HPV test. Participants/materials, setting, methods Seminal samples were collected by masturbation after 3-5 days of sexual abstinence. The inclusion criteria were as follows: no other sexually transmitted infections, no genetic diseases, and no inflammatory disorders. Sperm concentration, morphology, non-progressive and immotile sperms, and both slow and rapid progressive motility were evaluated according to WHO 2021 guidelines. DFI analysis was assessed by sperm chromatin dispersion test (SCD), while HPV-DNA detection was performed using InnoLipa HPV Genotyping Extra II (Fujirebio, Tokyo, Japan). Main results and the role of chance Of the 121 semen samples tested, 60 (49.6%) were HPV-positive and 61 (50.4%) were HPV-negative. DFI rates showed a significant negative correlation with rapid progressive motility in both groups and a positive correlation with slow progressive motility in the HPV-negative group. Conversely, the significance of the correlation between DFI and slow progressive motility was completely lost in HPV-positive patients. Sperm concentration, normal forms and immotile spermatozoa percentages were correlated with both motility parameters in the HPV-negative group. Similar results were observed in HPV-positive samples, except for the normal form rate, which was not associated with slow progressive motility. In addition, the same samples displayed a negative correlation between non-progressive motility and rapid progressive motility, absent in HPV-negative samples. Significant associations were found also for the derived parameter of progressive motility, which was correlated with DFI, sperm concentration, immotile sperm, and normal forms rate in both groups. The results suggest how high DFI rates, in the presence or absence of HPV infection, could affect reproductive health through a consistent impairment of spermatozoa motility. In particular, the distinction of slow and rapid progressive motility by WHO 2021 allows a deeper understanding of the possible correlations between DFI, semen parameters and HPV infection. Limitations, reasons for caution This is a preliminary study characterized by a small number of samples. Therefore, confirmation of these findings requires the enlargement of the patient cohort, which is already taking place. Wider implications of the findings Our results highlight how the introduction of the new WHO 2021 evaluation criteria, i.e. DFI, and slow and rapid progressive motility, provides additional information about sperm quality and the impact of HPV infection on it. Trial registration number Not applicable