Sperm Chromatin Fragmentation Research Articles

An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm

BackgroundIn vitro incubation of epididymal and vas deferens sperm with Mn2+ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DNAses and thus far only described in epididymal mouse sperm, can be triggered in ejaculated sperm is yet to be elucidated. The current study aimed to determine if exposure of pig ejaculated sperm to divalent ions (Mn2+ and Mg2+) activates SCF, and whether this has any impact on sperm function and survival. For this purpose, sperm DNA integrity was evaluated through the Comet assay and Pulsed Field Gel Electrophoresis (PFGE); sperm motility and agglutination were assessed with computer assisted sperm analysis (CASA); and sperm viability and levels of total reactive oxygen species (ROS) and superoxides were determined through flow cytometry.ResultsIncubation with Mn2+/Ca2+ activated SCF in a dose-dependent (P < 0.05) albeit not time-dependent manner (P > 0.05); in contrast, Mg2+/Ca2+ only triggered SCF at high concentrations (50 mM). The PFGE revealed that, when activated by Mn2+/Ca2+ or Mg2+/Ca2+, SCF generated DNA fragments of 33–194 Kb, compatible with the size of one or multiple toroids. Besides, Mn2+/Ca2+ affected sperm motility in a dose-dependent manner (P < 0.05), whereas Mg2+/Ca2+ only impaired this variable at high concentrations (P < 0.05). While this effect on motility was concomitant with an increase of agglutination, neither viability nor ROS levels were affected by Mn2+/Ca2+ or Mg2+/Ca2+ treatments.ConclusionMn2+/Ca2+ and Mn2+/Ca2+ were observed to induce SCF in ejaculated sperm, resulting in DNA cleavage at TLRs. The activation of this mechanism by an intracellular, non-oxidative factor sheds light on the events taking place during sperm cell death.

Low levels of mouse sperm chromatin fragmentation delay embryo development.

We previously demonstrated that MnCl2 induces double-stranded DNA breaks in sperm in a process that we term as sperm chromatin fragmentation. Here, we tested if the levels of double-stranded DNA breaks were corelated to the concentration of MnCl2, and we compared this to another agent that causes single-stranded DNA breaks, H2O2. We found that both methods have the advantage of inducing DNA breaks in a concentration-dependent manner. Mouse sperm were treated with varying concentrations of either H2O2 or MnCl2, and the DNA damage was assessed by pulse-field gel electrophoresis, and the alkaline and neutral comet assays. Oocytes were injected with either treated sperm and the resulting embryos analyzed with an embryoscope to detect subtle changes in embryonic development. We confirmed that H2O2 treatment induced primarily single-stranded DNA breaks and MnCl2 induced primarily double-stranded DNA breaks, indicating different mechanisms of damage. These sperm were injected into oocytes, and the development of the resulting embryos followed with an embryoscope equipped with time lapse recording. We found that aberrations in early embryonic development by day 2 with even the lowest levels of DNA damage and that the levels of embryonic aberrations correlated to the concentration of either H2O2 or MnCl2. Low levels of H2O2 caused significantly more aberrations in embryonic development than low levels of MnCl2 even though the levels of DNA damage as measured by comet assays were similar. These data demonstrate that even low levels of sperm DNA damage cause delays and arrests in embryonic development.

P-250 The exclusive role of sperm DNA fragmentation on the clinical outcome of a sibling donor oocyte cohort

Abstract Study question Can sperm chromatin fragmentation (SCF) overwhelm the oocyte repair mechanism of healthy, young oocytes and impair embryo development and clinical outcome? Summary answer Elevated SCF, with its double-stranded DNA (dsDNA) component, remarkably impairs clinical outcome, despite oocyte repair mechanisms present in a sibling donor oocyte cohort. What is known already Despite a normal semen analysis, approximately 10-20% of males of reproductive age still have a subtle infertility. Therefore, conventional semen analysis is insufficient to identify the specific issues related to sperm function and its embryo developmental competence. Indeed, SCF with its dsDNA break component, is known to be associated with poor clinical outcome such as poor embryo development and pregnancy loss, by possibly increasing structural chromosomal abnormalities. Although a healthy oocyte may address certain levels of SCF, these oocyte repair mechanisms are incapable of repairing dsDNA breaks. Study design, size, duration Thirty-one couples were divided according to the degree and type of SCF, whether normal or abnormal, and shared the same sibling donor oocytes. The male partners in both groups had comparable semen parameters. Total SCF and dsDNA fragmentation were assessed in all men. Fertilization, implantation, clinical pregnancy (CPR; +FHB), delivery, and pregnancy loss rates were compared between the two cohorts. Participants/materials, setting, methods A total of 31 infertile couples underwent ICSI cycles utilizing split sibling donor oocytes due to advanced maternal age or premature ovarian insufficiency. Oocytes from the same donor were equally allocated among 2 couples. Semen analyses were performed according to WHO 6th edition. Overall SCF was assessed by TUNEL with a ≤ 15% normal threshold. Double-strand DNA (dsDNA) fragmentation was assessed by neutral comet assay with ≤3% considered normal. Main results and the role of chance A total of 18 male partners (44.3 ± 7yrs) with a normal SCF of 8.9 ± 2% had their spermatozoa injected into sibling donor oocytes, while 13 male partners with abnormal SCF at 25.4% ± 11 (P&amp;lt;0.001) had their spermatozoa injected into the remaining half. In the latter, the average dsDNA fragmentation rate was 3.6%, while in the former it was 0.3% (P&amp;lt;0.001). Sibling donor oocytes injected with normal SCF resulted in a fertilization of 87.4% (146/167), implantation of 56.4% (22/39), +bHCG of 69.0% (20/29), CPR of 62.1% (18/29), and delivery rate of 51.7% (15/29). The other half of the sibling oocytes, injected with spermatozoa with elevated SCF, resulted in a fertilization of 70.8% (97/137) (P&amp;lt;0.001), implantation of 15.4% (4/26) (P&amp;lt;0.01), +bHCG of 42.1% (8/19), CPR of 21.1% (4/19) (P&amp;lt;0.01), and delivery rate of 10.5% (2/19) (P&amp;lt;0.01). The pregnancy loss in cycles utilizing the oocyte cohort injected with spermatozoa with normal SCF was 8.7% (2/23), while the pregnancy loss in those injected with spermatozoa with elevated SCF was 50.0% (2/4). Limitations, reasons for caution This preliminary study indicates that SCF and dsDNA fragmentation negatively affects clinical outcome, overwhelming oocyte repair mechanisms. Nonetheless, the study has a limited number of observations and needs to be validated in a larger cohort. Wider implications of the findings SCF, with its dsDNA fragmentation component, seems to contribute a higher incidence of chromosomal abnormalities. The compounded aneuploidy of male and female gametes may explain the compromised embryo development and higher pregnancy loss rate observed in infertile couples with apparently normal semen parameters and younger oocytes. Trial registration number not applicable

O-252 Genetic characterization of human sperm centrosome defects in infertile couples with complex embryo aneuploidy

Abstract Study question Can we characterize the genetic aspects related to sperm centrosome dysfunction in couples with high embryo aneuploidy that cannot exclusively be attributed to the oocyte? Summary answer We characterized structural and genomic abnormalities responsible for sperm centrosome dysfunction in infertile couples with complex embryo aneuploidy not exclusively related to the female partner. What is known already Conventional semen analysis provides only limited information on the proper function and embryo developmental competence of the male gamete. Therefore, an ancillary test, such as sperm chromatin fragmentation (SCF) and particularly double-stranded DNA breaks, has been claimed responsible for structural chromosomal abnormalities. It remains puzzling the source of complex embryo aneuploidy and chaotic chromosomal mosaicism that cannot solely be attributed to the female partner. The availability of genomic analysis may shed light on the integrity and function of the sperm centrosome responsible for ordaining chromosomal distribution at the first mitotic division. Study design, size, duration In the past 4 years, we identified 13 couples with unexpected high poor embryo cleavage resulting in a higher number of embryos with complex aneuploidy not solely attributable to the female gamete, resulting in extremely poor embryo implantation. Therefore, ancillary testing on the spermatozoa of the male partners was carried out to determine if the couple’s reproductive failure was eventually due to a stealth male gamete deficiency. Participants/materials, setting, methods Thirteen couples with poor clinical outcomes, characterized by poor embryo cleavage, high and complex embryo aneuploidy not exclusively due to the oocyte, with poor embryo implantation in multiple IVF/ICSI cycles, were offered adjuvant male partner testing including centrosome assessment through immunofluorescence staining on centrin (normal ≥ 60%), sperm chromatin fragmentation testing through terminal deoxynucleotide dUTP transferase nick-end labeling (TUNEL, normal ≤ 15%), transmission electron microscopy (TEM), and whole exome sequencing (WES) on spermatozoal DNA. Main results and the role of chance In 13 couples with an average paternal age of 38.1±2 years, semen analysis revealed a volume of 22.2±1.4mL, concentration of 58.2±37x10^6/mL, motility of 35.2±18% and normal morphology of 2.6±1%. They underwent with female partners (maternal age of 37.2±2 years) a total of 16 ICSI cycles with aneuploidy testing. Only 11 cycles yielded embryos for transfer due to poor embryo development with high aneuploidy rate, and none of them achieved a pregnancy. Centrosome staining revealed that only 45.8±22% of the spermatozoa displayed this key organelle. An overall SCF was observed at 16.7±10%. TEM confirmed that approximately 70% of the spermatozoa had irregular proximal centrioles. Moreover, genomic analysis by WES revealed mutations on PLK4 gene responsible for centriole duplication, BRSK1 gene involved in centrosome duplication, TUBGCP6 gene responsible for centrosomal microtubule nucleation, MARK4 for centrosome localization, MAP1S for microtubule anchoring and DNA binding, RIC8A for chromosome distribution, and CALM3 for cytokinesis. Of this cohort, 3 couples that subsequently underwent 3 ICSI cycles had their spermatozoa processed by microfluidics, aiming at selecting male gametes with higher genomic integrity. A fertilization rate of 78.3% (18/23) was achieved generating euploid embryos that resulted in 3 successful pregnancies. Limitations, reasons for caution We identified sperm centrosome dysfunction responsible for the peculiar and recurrent embryo aneuploidy. Although we identified shared gene mutations in those couples, these data are still preliminary. With exception of improving spermatozoa selection, there is a lack of a single effective treatment option to address sperm centrosome abnormality. Wider implications of the findings Ancillary tests with semen analysis were able to assess the function and competence of the male gamete. Corroborative genomic study can help provide insights into the reason for poor embryo implantation particularly those with high complex aneuploidy. Trial registration number Not applicable

P-072 Efficacy of a microfluidic chip technology for sperm selection in intrauterine insemination cycles

Abstract Study question Is there an advantage of using a new sperm selection method by microfluidics (MFSS) over standard density gradient (DG) for couples undergoing intrauterine insemination (IUI)? Summary answer MFSS reduced sperm processing time and selected spermatozoa with greater progressive motility and superior genomic integrity, suggesting a better clinical outcome than DG. What is known already The most conservative, cost-effective reproductive treatments for infertile couples are timed intercourse and IUI. The latter is generally performed using DG to enrich and purify the most motile spermatozoa; it requires technician effort and skills as well adequate processing time. Moreover, during DG centrifugation, spermatozoa are exposed to potentially harmful silica gel particles and eventual reactive oxygen species (ROS). The recent availability of a microfluidic chamber has been proposed as a method to safely process spermatozoa in an expedited manner while reducing human intervention. Therefore, we decided to test MFSS in a laboratory that executes over 2,000 IUI cycles/year. Study design, size, duration Since September 2022, 93 couples underwent 103 IUI-MFSS. Post-processing semen parameters and clinical outcomes were compared with those of 103 age-matched couples who underwent 103 IUI-DG during the same period. To confirm findings, 45 couples undergoing their first IUI-MFSS had post-processing semen parameters and clinical outcomes compared with 45 first-time IUI-DG couples, serving as a matched control. Finally, within the same 66 couples, outcomes were compared between their initial IUI-DG cycle and a subsequent IUI-MFSS. Participants/materials, setting, methods Men had normal raw semen parameters according to the most recent WHO criteria. DG was performed according to WHO21 guidelines, while MFSS as per manufacturer protocol (ZyMōt® Multi 850µL). Stimulation protocols were similar between all patients. Before and after processing, volume, concentration, and motility were compared between the two processing methods. Sperm chromatin fragmentation (SCF) was assessed by TUNEL (In Situ Cell Death Detection Kit; normal threshold,≤15%). For all comparisons, +bHCG and clinical pregnancy (+FHB) were compared retrospectively. Main results and the role of chance Ninety-three couples (maternal age,37.3±4; paternal age,39.2±6) underwent 103 IUI-MFSS cycles and were matched for age and abstinence days (1–4d) to 103 IUI-DG patient/cycles. IUI-DG and IUI-MFSS yielded 0.5±0mL volume, 76.8±40x106/mL and 41.3±26x106/mL concentrations, and 89.4±3% and 98.3±1% motility, respectively (P&amp;lt;0.001). IUI-DG resulted in a 20.4% +bHCG (21/103) and 15.5% +FHB (16/103). IUI-MFSS yielded a 19.4% +bHCG (20/103) and 16.5% +FHB (17/103). Forty-five couples (maternal age,36.8±4; paternal age,37.8±6) who underwent their first IUI with MFSSwere matched to a first-time IUI-DG cohort with similar semen parameters and abstinence. IUI-DG led to a 0.5±0 mL volume, 77.3±43x106/mL concentration, and 90±2% motility. IUI-MFSS yielded a lower concentration (45.6±27x106/mL), but a remarkable increase in motility (98.3±1%) (P&amp;lt;0.001). IUI-DG yielded a 13.3% +bHCG (6/45) and an 8.9% +FHB (4/45). IUI-MFSS yielded a 15.6%(7/45) +bHCG and an 11.1% +FHB (5/45). Finally, we assessed 66 couples who underwent IUI-DG and subsequently IUI-MFSS. IUI-DG resulted in a 77.5±39x106/mL concentration and 89.0±3% motility, while IUI-MFSS resulted in a lower concentration (36.9±21x106/mL), but an enhanced motility (98.3±1%)(P&amp;lt;0.001). IUI-DG yielded a 15.2% +bHCG (10/66) and an 8.9% +FHB (5/66), while IUI-MFSS yielded a 15.2% +bHCG (10/66); all became clinically pregnant. In all comparisons, SCF was 6.5±3% in raw samples, became 4.7±2% after DG, and negligible after MFSS at 0.8±0.6%(P&amp;lt;0.001). Limitations, reasons for caution IUI-MFSS cycles yielded a higher number of progressively motile spermatozoa with superior genomic integrity and a comparable clinical outcome to IUI-DG. This study came at a higher cost due to the device and offered only preliminary results. Therefore, it needs to be repeated in a larger study population. Wider implications of the findings DG is time consuming, labor intensive, and its outcome is technician-dependent. While reducing exposure to chemicals and ROS, MFSS grants higher motility and superior genomic integrity. If a greater clinical outcome is confirmed in a larger cohort, MFSS may prove to be a superior sperm processing method for IUI. Trial registration number not applicable

Can a sperm selection technique improve embryo ploidy?

Spermatozoa with the highest motility retain a superior genomic integrity, and elevated sperm chromatin fragmentation (SCF) has been linked to a lower ability of the conceptus to develop and implant. Therefore, the utilization of a sperm selection method, such as microfluidic sperm selection (MFSS), is capable of reducing the SCF by yielding the most motile fraction of spermatozoa with the highest embryo developmental competence. What remains unclear, however, is the causal mechanism that linksSCF to an impaired embryo development. To identify a relationship between SCF and an unexpectedly high proportion of embryo aneuploidy, while addressing treatment options. We identified couples with a high incidence of embryo aneuploidy in a previous intracytoplasmic sperm injection (ICSI) cycle with pre-implantation genetic testing for aneuploidy (PGT-A), utilizing spermatozoa selected by density gradient (DG). Terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) and neutral Comet assays were carried out on the semen specimens to assess total SCF and double-stranded DNA (dsDNA) fragmentation, respectively. These couples underwent subsequent ICSI/PGT-A cycles with MFSS. Total SCF and dsDNA fragmentation were compared between the two sperm selection methods. Embryo aneuploidy, implantation, clinical pregnancy, delivery,and pregnancy loss rates were compared between the couples' historical DG and subsequent MFSS cycles. In 57 couples undergoing 71 ICSI/PGT-A cycles, where DG sperm selection was carried out, a high incidence of aneuploid embryos (74.7%) resulted in poor implantation and no viable pregnancies. Testing for SCF, inclusive of dsDNA breaks, evidenced a SCF of 26.2% and dsDNA break of 3.6% in the raw specimen, that decreased to 18.0% (p < 0.001) and 3.1%, respectively, in the DG processed specimen. Following MFSS, total SCF and dsDNA fragmentation decreased to 1.9% and 0.3%, respectively (p<0.001). The embryo euploidy rate remarkable improved from 25.3% in the DG cycles to 42.9% in the MFSS cycles (p<0.001). The 6.7% implantation rate in the DG cycles increased to 65.5% in the MFSS cycles (p<0.001). Similarly, the clinical pregnancy rate rose from 10.5% (DG) to 64.6% (MFSS), resulting in a 62.5% delivery rate (p<0.001). In couples with a relatively young female partner with a negative infertility workup, and a male partner with semen parameters adequate for ICSI, presenting with a high rate of embryo aneuploidy, an additional subtle male factor component may be the culprit. Thus, it is crucial to assess the SCF and test for the dsDNA breaks, which can eventually contribute to embryo chromosomal abnormalities. Given the inverse relationship between SCF and motility, a selection of the most motile gamete by MFSS enhanced the proportion of spermatozoa with an intact genome, contributing to the generation of moreeuploid embryos that are capable of implanting and yielding increased term pregnancies.

Assessing male gamete genome integrity to ameliorate poor assisted reproductive technology clinical outcome

To assess the role of evaluating sperm chromatin fragmentation (SCF) as a tool to guide treatment in couples who achieved unexpectedly poor clinical outcomes after intracytoplasmic sperm injection (ICSI). We identified couples with an unexpectedly suboptimal clinical outcome after ICSI who were then screened for SCF. Consequently, the same couples were counseled to undergo a subsequent ICSI cycle using either ejaculates processed by microfluidic sperm selection (MFSS) or spermatozoa retrieved from the testis, and clinical outcomes were compared between history and treatment cycles. To confirm the sole effect of a compromised male gamete, we compared the ICSI outcome in cycles where male gametes with abnormal SCF were used to inseminate autologous and donor oocytes. Finally, to eliminate an eventual confounding female factor component, we compared the clinical outcome of ICSI cycles using sibling donor oocytes injected with spermatozoa with normal or abnormal SCF. Academic reproductive medicine center point of care. The patient population consisted of 76 couples with reproductively healthy and relatively young female partners and male partners with compromised semen parameters, but suitable for ICSI. In a subanalysis, we identified 67 couples with abnormal SCF who underwent ICSI cycle(s) with donor oocytes. Furthermore, we identified 29 couples, 12 with normal SCF and 17 with abnormal, uncorrected SCF, and 7 couples with abnormal, corrected SCF vs. a control, who used sibling donor oocytes for their ICSI cycle(s). For couples who resulted in surprisingly low clinical outcomes after ICSI, despite semen parameters adequate for ICSI and a normal female infertility evaluation, a SCF assessment was performed on the semen specimen using the terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labeling (TUNEL) assay. The couples then underwent a subsequent ICSI cycle with spermatozoa processed by MFSS or surgically retrieved. Moreover, cycles with donor oocytes were used to confirm the sole contribution of the male gamete. Clinical outcomes, such as fertilization, embryo implantation, clinical pregnancy, delivery, and pregnancy loss rates were compared between history and treatment cycle(s) using ejaculated spermatozoa selected by MFSS or from a testicular biopsy, taking into consideration the level of SCF. In a subanalysis, we reported the clinical outcomes of 67 patients who used donor oocytes and compared them with cycles where their own oocytes were used. Furthermore, we compared the ICSI clinical outcomes between cycles using sibling donor oocytes injected with low or high SCF with or without sperm intervention aimed at correcting, or alleviating the degree of SCF. In a total of 168 cycles, 76 couples had in a prior cycle a 67.1% fertilization rate, and clinical pregnancy and pregnancy loss rates of 16.6% and 52.3%, respectively. After testing for SCF, the DNA fragmentation rate was 21.6%. This led to a subsequent ICSI cycle with MFSS or testicular sperm extraction, resulting in clinical pregnancy and delivery rates of 39.2%, and 37.3%, respectively. The embryo implantation rate increased to 23.5%, whereas the pregnancy loss rate decreased to 5% in the treatment cycle. This was particularly significant in the moderate SCF group, reaching embryo implantation, clinical pregnancy, and delivery rates of 24.3%, 40.4%, and 36.2%, respectively, and reducing the pregnancy loss rate to 10.5% in post-sperm treatment cycles. In 67 patients with high SCF who used donor oocytes, a significantly higher fertilization rate of 78.1% and embryo implantation rate of 29.1% were reported, compared with those in couples also with an elevated SCF who used their own. Interestingly, the clinical pregnancy and delivery rates only increased slightly from 28.0%-36.1% and from 23.7%-29.2%, respectively. To further control for a female factor, we observed couples who shared sibling donor oocytes, 17 with normal SCF and 12 with abnormal (uncorrected) SCF. Interestingly, the abnormal SCF group had impaired fertilization (69.3%), embryo implantation (15.0%), and delivery (15.4%) rates. For an additional 15 couples who split their donor oocytes, 8 had normal SCF, and although 7 couples originally had abnormal SCF, 4 used microfluidic processing, 2 used testicular spermatozoa, and 1 used donor spermatozoa to alleviate the degree of SCF, resulting in comparable clinical outcomes with the normal SCF group. A superimposed male factor component may explain the disappointing ICSI outcome in some couples despite reproductively healthy female partners. Therefore, it may be useful to screen couples for SCF to guide treatment options and maximize chances of a successful pregnancy. The improved, but suboptimal pregnancy and delivery outcomes observed in couples using donor oocytes confirmed the exclusive detrimental role that the male gamete exerted on embryo development despite the presence of putative oocyte repair mechanisms.

Sperm degradation after vasectomy follows a sperm chromatin fragmentation-dependent mechanism causing DNA breaks in the toroid linker regions

Vasectomy is a widely used surgical technique creating an obstructive azoospermia. Although sperm cannot be ejaculated, the testis maintains sperm production in vasectomized males. The continuous accumulation of sperm deposited in the epididymis and the vas deferens fraction necessarily need to be degraded and eliminated. While the elimination process is carried out by granulomas that form after vasectomy, the detailed mechanisms of sperm degradation are still not known. The aim was to assess whether sperm chromatin fragmentation (SCF), a mechanism that degrades the entire sperm genome at the toroid linker regions (TLRs), is activated after vasectomy in sperm cells. We vasectomized mice and evaluated the presence of TLR-specific double-strand breaks through pulsed-field gel electrophoresis and the Comet assay at 1, 2 and 3 weeks after surgery. Results for DNA damage (Olive tail moment) at single-cell level showed an increase of double-strand breaks after vasectomy for vas deferens sperm after 1, 2 and 3 weeks postvasectomy (21.78 ± 2.29; 19.71 ± 1.79 and 32.59 ± 1.81, respectively), compared to mock surgery (7.04 ± 1.03; 10.10 ± 1.29 and 8.64 ± 0.85, respectively; P < 0.001). Similar findings were obtained for cauda epididymis sperm (P < 0.001), but not for caput epididymis (P > 0.05). Pulsed-field gel electrophoresis showed the presence of double-stranded breaks between 15 and 145 kb, indicating that DNA breaks were produced mainly in the sperm TLRs. Results presented here suggest that SCF is a mechanism activated in vas deferens after vasectomy to degrade sperm DNA when they cannot be ejaculated, preventing their function.

P-112 Utilizing surgical sperm retrieval to improve clinical outcomes for men with high sperm chromatin fragmentation in their ejaculates

Abstract Study question In men with elevated sperm chromatin fragmentation (SCF), can surgical sperm retrieval from the proximal area of the male genital tract improve clinical outcomes? Summary answer Spermatozoa retrieved from the epididymis or testes demonstrate higher genomic integrity and are associated with enhanced embryo implantation, clinical pregnancy, and delivery rates. What is known already Sperm genomic integrity is a key factor in achieving a successful pregnancy. During spermiogenesis, alteration of DNA topology involving nuclease and ligase mechanisms is required for supercoiling and DNA compaction. If a proper DNA repair mechanism fails during this intricate process, spermatozoa with DNA fragmentation should be picked up by the epididymis and phagocytized. When this mechanism fails, along with the presence of superimposed reactive oxygen species in the male genital tract, spermatozoa with DNA fragmentation may reach the ejaculates. We have previously demonstrated that retrieving spermatozoa directly from the epididymis and testes yields gametes with higher conserved genomic integrity. Study design, size, duration In a nine-year timespan, we identified over 1,000 men with high SCF in their ejaculates; 144 patients consented to undergo surgical retrieval of spermatozoa. In a preliminary assessment, their clinical outcomes were compared to those from men with normal SCF (n = 539). Of these men, 53 consented to undergo subsequent ICSI cycles with their female partners using surgically retrieved spermatozoa from the epididymis (n = 15) or testes (n = 38). Fertilization and clinical outcomes were compared. Participants/materials, setting, methods Ejaculated, epididymal, and testicular specimens were screened by terminal deoxynucleotidyl dUTP nick-end labeling (TUNEL) to assess SCF using a commercially available kit. A total of 500 spermatozoa/sample were screened with a normal threshold of 15%. Surgical samples from the epididymis and testes were cryopreserved in multiple vials for subsequent ICSI cycles. Main results and the role of chance In the preliminary assessment, ICSI outcomes utilizing ejaculated spermatozoa were compared between men with normal (n = 539, 9.3±3%) and abnormal (n = 144, 28.2±13%) SCF. Although fertilization did not differ, implantation (24.1% vs. 7.8%) and clinical pregnancy rates (CPR, 35.8% vs. 14.6%) were significantly impaired (P &amp;lt; 0.001) in men with elevated SCF. SCF assessment on surgically retrieved spermatozoa revealed a decreasing trend from 28.2±13% in the ejaculate to 16.6±9.2% in the epididymis and 10.1±5.7% in the testes. A total of 53 men underwent 79 subsequent ICSI cycles using surgically retrieved spermatozoa. Compared to their historical cycles using ejaculates (n = 65), ICSI with surgically retrieved sperm substantially improved implantation (18.8% vs. 2.1%; P &amp;lt; 0.001), CPR (36.5% vs. 5.3%; P &amp;lt; 0.0001), and ongoing/delivery rates (33.8% vs. 4.1%; P &amp;lt; 0.0001). Among these couples, 15 underwent 21 ICSI cycles utilizing epididymal spermatozoa with a mean SCF of 16.6±9%. Implantation (26.5% vs. 3.7%; P &amp;lt; 0.05), CPR (68.4% vs. 4.2%; P &amp;lt; 0.0001), and ongoing/delivery rates (57.1% vs. 4.2%; P &amp;lt; 0.0001) were significantly improved compared to the historical cycles. The remaining 38 patients underwent ICSI with testicular spermatozoa with an average SCF of 10.1±6%. Implantation (15.0% vs. 2.6%; P &amp;lt; 0.01), CPR (25.5% vs. 6.1%; P &amp;lt; 0.01), and ongoing/delivery rates (23.6% vs. 3.0%; P &amp;lt; 0.05) were higher when compared to historical cycles. Limitations, reasons for caution Although surgically retrieved spermatozoa can be used to enhance clinical outcomes in couples with high SCF, epididymal spermatozoa yielded higher pregnancy rates despite the higher level of SCF in these specimens compared to testicular spermatozoa. This can be explained by the corrective action of the ICSI procedure itself. Wider implications of the findings Our study demonstrated that sperm DNA integrity progressively increases through the journey of the male genital tract. In couples with a compromised sperm genome, surgically retrieved spermatozoa can be beneficial. Therefore, assessing SCF might be used a routine tool to evaluate the male gamete. Trial registration number N/A

P-071 Assessing the integrity of the male gamete genome to improve ART clinical outcomes

Abstract Study question Does screening for sperm chromatin fragmentation (SCF) benefit and help guide the treatment of patients with subtle male factor infertility? Summary answer In patients with abnormal SCF, an enhanced sperm selection or surgical sampling procedures improved clinical outcomes, demonstrating the value of assessing the male genome. What is known already Standard infertility workups include an assessment of the female genital tract, ovarian reserve, ovulation, and a semen analysis. Nevertheless, pregnancy failure is still encountered in couples with a young female partner with patent tubes and normal ovulation profile, and a male partner with normal semen analysis. SCF has previously been described as responsible for recurrent miscarriages and persistent ART failure. Therefore, the observed presence and degree of SCF may help guide treatment utilizing microfluidic sperm selection (MFSS) or Intracytoplasmic Sperm Injection (ICSI) with testicular spermatozoa, where in both cases, spermatozoa with higher genomic integrity were isolated, yielding improved clinical outcomes. Study design, size, duration From 2010 to 2021, we included 76 couples who had disappointing clinical outcomes; the female partners were relatively young with negative infertility workups, and the male partners had adequate semen parameters and were screened for SCF. The couples were then counseled to undergo a subsequent cycle utilizing either surgically retrieved spermatozoa or semen specimens processed by microfluidics. Clinical outcomes were measured and compared between history and post-treatment cycle(s). Participants/materials, setting, methods A total of 76 couples with poor clinical outcomes were included. Semen parameters were deemed adequate for ICSI with an average concentration of 33x106/mL and 33% motility. A minimum of 500 spermatozoa per sample were assessed by TUNEL assay with a 15% threshold. Fertilization, implantation, clinical pregnancy, delivery, and pregnancy loss rates were compared between history and post treatment cycle(s). Paired t and Chi-square tests were used to compare semen parameter and clinical outcomes, respectively. Main results and the role of chance In 168 cycles, 76 couples had poor clinical outcome results with a 67.1% fertilization rate and an 8.5% implantation rate, leading to a 16.6% clinical pregnancy rate and a 52.3% pregnancy loss rate. In these couples, testing for SCF resulted in an average DNA fragmentation of 21.6%. In efforts to reduce DNA fragmentation, 63 couples underwent microfluidics and 13 underwent surgical sperm retrieval. In an immediate post-treatment cycle, there was improvement in implantation (23.5%) (P &amp;lt; 0.001), clinical pregnancy (39.2%) (P &amp;lt; 0.01), and ongoing/delivery (37.3%) (P &amp;lt; 0.001), with a concurrent reduction in the pregnancy loss rate from 52.3% to 5.0% (P &amp;lt; 0.01). Patients were stratified according to their level of SCF. In the moderate category (15-30%, n = 60), there was meaningful improvement in implantation (24.3%) (P &amp;lt; 0.001), clinical pregnancy (40.4%) (P &amp;lt; 0.01), and ongoing/delivery rates (36.2%) (P &amp;lt; 0.001), that yielded a reduced pregnancy loss rate of 10.5% (P &amp;lt; 0.01) compared to the history cycle. In the severe SCF category (&amp;gt;30%, n = 16), there was also a meaningful improvement in implantation (15.4%), clinical pregnancy (21.7%), and ongoing/delivery rates (21.7%), as well as a reduction in the pregnancy loss rate (0%), albeit not significant. Limitations, reasons for caution In this cohort, we were able to identify a subtle male factor that was missed in the initial semen evaluation to guide treatment. However, this cohort was arbitrarily selected by the initial unexpected outcome and is a relatively small sample size. Wider implications of the findings The presence of a subtle male factor may explain disappointing clinical outcomes in some couples who have otherwise negative infertility evaluations. In these cases, screening for SCF can be a beneficial tool to help guide treatment and maximize the chances of a successful pregnancy. Trial registration number not applicable

P-117 A novel method to select spermatozoa with the best morphokinetic characteristics and superior genomic integrity

Abstract Study question Can an automated device based on membrane electrophoresis be used to select spermatozoa with superior morphokinetic characteristics and lower sperm chromatin fragmentation (SCF)? Summary answer Albeit at an expense of concentration, a membrane-electrophoretic device was able to isolate a proportion of spermatozoa with the highest motility, morphology, and genomic integrity. What is known already Conventional sperm selection based on density gradient centrifugation (DGC) can enhance the proportion of progressively motile spermatozoa; however, DGC methods are limited in their ability to select spermatozoa with higher genomic integrity. Membrane-based microfluidic technologies (MFSS) have already been used clinically to select for spermatozoa with a superior chromatin status; however, these methods are based on the intrinsic characteristics of motile spermatozoa capable of selecting themselves. By incorporating an extrinsic electrophoretic drive, membrane electrophoresis would overcome a limitation of progressive motility in the semen sample and be able to isolate viable gametes with better morphology and reduced SCF. Study design, size, duration From August 2020 to December 2021, semen samples from 68 men were evaluated by standard semen analysis and simultaneously processed by DGC or a novel membrane-electrophoretic sperm sorter (EPSS) to select for progressively motile spermatozoa. Concentration, motility, progressive motility, morphology, and SCF were measured and compared between raw, DGC-, and EPSS-processed specimens. Participants/materials, setting, methods Fresh ejaculates were evaluated by standard semen analysis according to WHO 2021 criteria. Following complete liquefaction, specimens were divided into two equal aliquots for DGC and EPSS sorting. SCF was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay under fluorescent microscopy; at least 500 spermatozoa were evaluated for each specimen with a normal threshold of ≤ 15%. Main results and the role of chance A total of 68 men with an average age of 38.1±6 years had the following average semen parameters: volume of 3.1±1 mL, concentration of 58.2±33 x106/mL, 43.3±9% motility, 40.5±9% forward progression motility, and a normal morphology of 2.9±0.8%. When comparing the final parameters between the EPSS and DGC methods, despite a reduced sperm concentration (7.3±9 x106/mL vs 45.0±36 x106/mL, P&amp;lt;0.0001, respectively), EPSS was highly selective for motile spermatozoa, yielding significantly higher motility (93.1±16% vs. 86.8±15% P&amp;lt;0.0001) and forward progressive motility (92.3±17% vs 85.2±15%, P&amp;lt;0.0001). Despite an overall lower sperm recovery rate (21.4±25% vs 75.4±17%, P&amp;lt;0.0001), normal morphology improved to 3.4±0.8% (P&amp;lt;0.0001) after EPSS but remained unchanged in the DGC-processed sample. Although both EPSS and DGC improved SCF from 12.2±9% in the raw specimen to 4.8±6% and 6.6±7% (P &amp;lt; 0.0001), respectively, EPSS outperformed DGC (P &amp;lt; 0.05). Moreover, the DGC method took up to 60 min to process, whereas the EPSS technique took a total of 6 min. Limitations, reasons for caution The selection of spermatozoa by EPSS is a promising technique to isolate progressively motile spermatozoa with enhanced morphology and superior chromatin integrity, albeit at a lower concentration. This is a preliminary study; the benefit and safety of this method must be further demonstrated by insemination or IVF treatment. Wider implications of the findings A membrane electrophoresis device may be a viable alternative method to MFSS to identify spermatozoa with superior morphokinetic characteristics and intact chromatin. Moreover, incorporating an automated device can reduce gamete processing time while minimizing labor costs and inter-operator errors. Trial registration number N/A

O-251 Defining the exclusive role of male genome integrity on conceptus development

Abstract Study question To determine the effect of sperm DNA fragmentation on embryo development by ruling out a female factor component. Summary answer By utilizing healthy donor oocytes, it is possible to quantify the sole deleterious effect of sperm DNA fragmentation and explore the ooplasmic repair mechanism. What is known already In approximately 50% of couples with unexplained ART failure, a subtle male factor is present that is missed in a conventional semen analysis assessing concentration, motility, and morphology. Additional information on male gamete competence can be acquired by assessing Sperm Chromatin Fragmentation (SCF). Indeed, a fragmented male genome can give rise to poorly developing embryos, leading to impaired implantation, lower pregnancy, and higher miscarriage rates. It has been previously seen that an oocyte, according to its age, can repair the damaged DNA contributed by the male gamete. Study design, size, duration In last decade, 316 couples, who have an elevated SCF, underwent ICSI cycles and resulted in disappointing clinical outcomes. To exclude an eventual confounding female factor, couples who utilized donor oocytes were identified and clinical outcome was compared to a control. To measure the role of an impaired sperm genome and a concurrent oocyte repair mechanism, we compared clinical outcomes between cycles in the same couples who used their own and subsequently donor oocytes. Participants/materials, setting, methods We included 381 couples screened for SCF; 65 underwent ICSI cycles with donor oocytes; of these, 46 underwent a previous ICSI cycle with their own oocytes. Fertilization, implantation, clinical pregnancy, and delivery rates were compared between above-mentioned groups. A TUNEL assay was used to measure SCF (≥500 spermatozoa were assessed/sample at a 15% threshold). Paired t and Chi-square tests were used to compare ages and clinical outcomes, respectively. Main results and the role of chance We included 381 couples (maternal age, 37.8 ± 4.2; paternal age, 41.8 ± 8.2), whose male partners had the following semen parameters: average volume of 2.5 mL, concentration of 25.4x106/mL, 33% motility, and normal morphology of 1.7. Of these, 316 had elevated average DNA fragmentation of 25.5% and utilized their own oocytes in 683 ICSI cycles, resulting in a 67.5% fertilization, 12.2% implantation, 27.9% clinical pregnancy, and a 22.5% ongoing/delivery. Of the 381 couples, 65 underwent ICSI utilizing donor oocytes (SCF of 24.3%) resulting in a significantly higher fertilization (78.4%) (P &amp;lt; 0.00001) and embryo implantation rates (34.2%) (P &amp;lt; 0.0001). Similarly, albeit not statistically significant, the clinical pregnancy and ongoing/delivery rates increased from 27.9% to 37.1% and from 22.5% to 30.0%, respectively. To further explore the exclusive role of the male gamete in embryo development, we identified a cohort of patients (n = 46), with an elevated average DNA fragmentation of 23.6%, who underwent an ICSI cycle using their own oocytes and a subsequent cycle using donor oocytes. Compared to cycles where couples used their own oocytes, fertilization, embryo implantation, clinical pregnancy, and ongoing/delivery rates rose from 67.5% to 76.6% (P &amp;lt; 0.0001), 2.8% to 24.7% (P &amp;lt; 0.00001), 8.4% to 38.8% (P &amp;lt; 0.0001), and 1.2% to 28.6% (P &amp;lt; 0.00001), respectively. Limitations, reasons for caution Although this study attempts to control for a concurrent confounding female factor, it cannot be completely excluded. It is difficult to assess the extent and contribution of the ooplasmic repair mechanisms on the male genome. Wider implications of the findings Ooplasmic repair mechanisms of healthy female gametes appear to repair or improve the deleterious impact of sperm DNA fragmentation on ART outcomes. Trial registration number not applicable

P-111 Spermiogenic maturation in relation to sperm genomic integrity throughout the epididymis

Abstract Study question Which portion of the epididymis yields spermatozoa with the highest chromatin integrity and embryo developmental competence? Summary answer Spermatozoa retrieved from the distal portion of the epididymis retains the highest chromatin integrity, characterized by higher fertilization, implantation, and clinical pregnancy rates. What is known already Surgical sperm retrieval from the proximal male reproductive tract has proven to be an effective treatment for couples with recurrent pregnancy loss due to high DNA fragmentation in the ejaculate. Although spermatozoa retrieved directly from the germinal epithelium displays the highest genomic integrity, epididymal spermatozoa maintain a similar DNA integrity profile but yield higher fertilization and pregnancy rates. Although the caput is the preferred site for sperm aspiration procedures in the epididymis, it is unclear which epididymal region yields the best-quality gametes. Study design, size, duration Over a 10-year period, we identified 51 obstructive azoospermic (OA) men who underwent surgical sperm retrieval. These men were grouped according to the different epididymal regions from which their spermatozoa were obtained. Sperm chromatin fragmentation (SCF) was assessed and compared among these groups. The epididymal spermatozoa were then used for ICSI cycles. Fertilization and clinical outcomes were compared among the epididymal regions. All female partners (≤37 years old) had negative infertility workups. Participants/materials, setting, methods Epididymal spermatozoa were retrieved from the caput (n = 39), corpus (n = 8), and cauda (n = 4) regions. SCF was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, with a normal threshold of ≤ 15%. Unpaired t and Fisher’s exact tests were used to compare the SCF as well as ICSI and pregnancy outcomes according to the epididymal sperm source. P &amp;lt; 0.05 was considered statistically significant. Main results and the role of chance A total of 51 men were categorized based on the site of spermatozoa retrieval: caput, corpus, and cauda. Maternal age was comparable among all three groups (37.9±5, 37.3±4, and 38±3, respectively). Sperm concentration was 23.9±30x106/ml (caput), 24.3±30x106/ml (corpus), and 29.3±36x106/ml (cauda), with 10.8±15% (caput), 14.3±20%(corpus), and 15.2±13% motility (cauda). Average oocytes injected per group was 13.3, 12.3, and 13.2, respectively. Average SCF scores were 19.8%, 9.2% (P &amp;lt; 0.05), and 8.4% (P &amp;lt; 0.05), respectively. The fertilization rates rose from 69.0% in the caput to 78.6% in the corpus and 86.2% (P &amp;lt; 0.05) in the cauda. Implantation rates followed a similar trend: 35.3% in the caput and 44.4% and 50.1% in the corpus and cauda, respectively. In cycles using caput epididymal spermatozoa, there was a 52.9% delivery rate with a 5.5% pregnancy loss rate. Cycles using corpus epididymal sperm showed a 75.0% delivery rate with no pregnancy loss. Finally, cycles using spermatozoa from the cauda resulted in a 100% delivery rate. Limitations, reasons for caution Although spermatozoa retrieved from the cauda epididymis has superior genomic integrity and yielded satisfactory clinical outcomes, this approach may be restricted to patients with obstructive azoospermia. While we attempted to control for an eventual confounding female factor, it cannot be entirely ruled out. Wider implications of the findings The epididymis plays an important role in sperm maturation; epididymal spermatozoa retain adequate chromatin integrity as demonstrated by the fertilization and pregnancy rates achieved with sperm retrieved from the cauda region. Confirmation of these findings in larger studies may help determine the most favorable site for epididymal sperm retrieval. Trial registration number Not applicable

P-028 Microfluidics as a methodology for sperm selection. A prospective functional study

Abstract Study question Is microfluidics an optimal technique to improve the sperm selection process in comparison with standard techniques like Density Gradient Centrifugation or Swim-up? Summary answer A significant increase in sperm quality was obtained when microfluidics was compared to density gradient centrifugation but improvement evidence regarding swim-up is not yet demonstrated. What is known already Assisted reproduction clinics for in vitro fertilization treatments have developed several techniques to perform sperm selection, being density gradient centrifugation (DGC) and swim-up the most widely used. However, sperm selection is a procedure that requires bulky and expensive equipment, long waiting times and gamete manipulation, which results in cellular stress. The SwimCount Harvester is a microfluidic-based device capable of performing sperm selection and overcoming the problems of other sorting systems. In our study, we analyzed several sperm quality parameters between these three sperm selection techniques. Study design, size, duration This was a prospective, cohort and observational study including 111 semen samples from patients and donors (mean age 33,7±9,3 years) between February 2021 and January 2022. The semen sample from each patient or donor was divided into two volumes, one part, the sperm selection was performed using the SwimCount Harvester and the other part using DGC or Swim-up. These sperm selection techniques were used to isolate sperm based on fluid dynamics and cell motility. Participants/materials, setting, methods Fresh ejaculate and sperm selected samples from each patient were analyzed according to the 2010 WHO-criteria to assess concentration, motility, morphology and vitality, using automatic image analysis. The excessive histone retention indicating defective chromatin compaction was assessed using aniline blue staining. Sperm chromatin fragmentation (SCF) was assessed by TUNEL on at least 20.000 sperm using flow cytometry. Kruskal-Wallis test was performed in order to assess statistical differences of the variables between the sperm selection methods. Main results and the role of chance The SwimCount Harvester was compared to DGC (n = 95). Ejaculated sperm yielded an average concentration of 53,2±34,2x106/mL. After DGC and SwimCount Harvester, the sperm concentration was 11,1±8,8 and 12,5±11,2x106/mL, respectively. The motility of fresh sperm sample improved from 41,9±10,4% to 71,6±10,6% after DGC and 76,8±10,0% after SwimCount Harvester (P &amp;lt; 0,05). The percentage of normal sperm increased from 2,1±1,2%, for the fresh samples, to 3,5±1,4% and 4,2±1,6% for the samples processed by DGC and microfluidics, respectively (P &amp;lt; 0,05). The percentage of live sperm increased from 74,0±8,1% and 77,5±8,7% in fresh sperm and after DGC, respectively to 85,9±9,0% after using microfluidics (P &amp;lt; 0,05). In the same way, the normal sperm chromatine structure percentage increased from 67,4±7,5% to 75,4±7,9% for the sperm samples selected by DGC and 77,7±8,9% when the SwimCount Harvester was used (P &amp;lt; 0,05). A decrease in SCF was observed from 12,9±8,4% in samples selected by DGC to 10,4±5,1% in raw samples (P &amp;gt; 0,05). However, after sperm selection using SwimCount Harvester, SCF fell to 4,6±4,1%, showing significant differences between both sperm selection methods (P &amp;lt; 0,05). Similar results were obtained for oligozoospermic samples (n = 6). When the SwimCount Harvester was compared with the Swim-up (n = 10), non-significant improvements were observed for all the parameters studied due to the reduced sample size. Limitations, reasons for caution The database of samples processed using swim-up and oligozoospermic samples is too small to draw reliable conclusions. Although significantly better results are obtained in sperm samples selected by the SwimCount Harvester with respect to DGC, a clinical study using the microfluidic device in assisted reproduction cycles has to be performed. Wider implications of the findings The SwimCount Harvester, in addition to significantly improving sperm selection and quality, is a reliable alternative to integrate numerous laboratory steps into a single automated procedure, reducing workload, the amount of culture media and equipment used, gamete handling and the stress that produces. Moreover, microfluidics may eliminate inter-laboratory variability. Trial registration number This project has received funding from the Eurostars-2 joint program with co-funding from CDTI and the European Union's Horizon 2020 Research and Innovation Framework Program E! 113740/ CIIP-20201009.

Cryoprotectant-free vitrification of llama spermatozoa: cryoloop vs sphere method, warmed rapidly or ultra-rapidly

Our objective was to determine the effect of vitrification using both cryoloop and sphere methods, combined with two warming protocols, on the percentage of motility, membrane integrity and DNA fragmentation/condensation of llama spermatozoa. Raw semen (n = 5; r = 3) was incubated with collagenase (0.1 % in Tyrode's Albumin Lactate Pyruvate, TALP, solution), centrifuged and diluted in TALP with or without 5% dimethylformamide (DMF). Two spermatozoa concentrations were used: 20 × 106 sperm/mL (cryoloops) and 5 × 106 sperm/mL (spheres). Two warming procedures were evaluated: a rapid method (30 s at 37 °C) and an ultra-rapid method (7 s at 75 °C, followed by 30 s at 37 °C). Percentages of sperm total motility, viability, membrane function, chromatin condensation and chromatin fragmentation were evaluated before and after vitrification and analyzed using Friedman’s test. The only seminal parameters maintained following vitrification were: 1) chromatin condensation, in all evaluated protocols and 2) DNA integrity, with the spheres method using both warming protocols without cryoprotectants and warming rapidly with cryoprotectants. Spermatozoa vitrification is a simple and inexpensive cryopreservation method that was able to preserve llama sperm chromatin condensation and integrity, especially using the spheres method without cryoprotectants combined with warming at 37 °C for 30 s. However it failed to preserve spermatozoa motility, membranes and viability.

UTILIZING SPERMATOZOA WITH THE HIGHEST GENOMIC INTEGRITY ENHANCES ICSI OUTCOME

To demonstrate that selecting spermatozoa with the highest genomic integrity enhances ICSI outcomes. Over the last 4 years, consenting men undergoing ART provided ejaculates for sperm chromatin fragmentation (SCF) screening before and after density gradient centrifugation (DGC). In a subsequent cycle, this assay was carried out before and after microfluidics sperm selection (MFSS). SCF was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL); at least 500 spermatozoa were assessed per sample with a normal threshold of ≤15%. Ejaculated specimens were analyzed in the standard fashion and used for ICSI after selection by DGC or MFSS. Fertilization and clinical outcome were recorded and compared between sperm-processing methods. A total of 21 men (43.3±8 years) had an average SCF in their raw semen of 22.1±10%, which decreased to 19.1±7% after DGC. These men underwent 39 ICSI cycles with their female partners (38.0±4 years), achieving a fertilization rate of 57.0% (167/293), an implantation rate of only 5.1% (4/78), and a clinical pregnancy rate (CPR) of 11.8% (4/34), with half resulting in pregnancy loss (2/4). Subsequently, these couples underwent 26 ICSI cycles with MFSS. The SCF after selection was 1.2±1%, remarkably lower than in the raw specimen (P<0.05). In these cycles, there was an improved fertilization rate of 69.3% (167/241; P<0.05). The implantation rate rose to 30.8% (16/52; P<0.0001) and the CPR increased to 53.8% (14/26; P<0.001); all are ongoing or have delivered (P<0.0001). Based on these results, 55 men (42.3±8 years) with elevated SCF in their ejaculate solely selected by MFSS were treated in 69 ICSI cycles with their female partners (38.3±5 years). The SCF in their raw samples was 22.3±10%, which fell to 3.0±4% following MFSS (P<0.0001). These cycles led to a 75.9% (456/605) fertilization rate, with an implantation rate of 24.8% (36/145), and a CPR of 47.8% (32/67), with 41.8% (28/67) ongoing or delivered pregnancies. Compared to the more conventional DGC, MFSS is capable of selecting the most progressively motile spermatozoa with the highest genomic integrity. Treatment by ICSI with MFSS for men with high sperm DNA fragmentation enhances fertilization, embryo development, and clinical pregnancies.

A SPERM SELECTION TECHNIQUE TO IMPROVE EMBRYO PLOIDY

To assess the role of an enhanced sperm selection method in mitigating paternal contributions to embryo aneuploidy. Over the last 4 years, 57 couples underwent ICSI with sperm selected by density gradient centrifugation (DGC), resulting in few frozen embryo transfers (FETs) due to consistent embryo aneuploidy following preimplantation genetic testing for aneuploidy (PGT-A). These men consented to sperm chromatin fragmentation (SCF) assessment, inclusive of double-stranded DNA breaks (dsDNA) in their raw semen, as well as post-DGC and post-microfluidic sperm selection (MFSS). These couples underwent subsequent ICSI cycles with MFSS. Outcomes of cycles processed by DGC and MFSS were analyzed and compared. SCF was assessed by terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) on ≥500 spermatozoa per patient, with a normal threshold of ≤15%. A neutral Comet assay was used to assess dsDNA on ≥200 spermatozoa, utilizing a modified in-house protocol and a normal threshold of ≤3%. A total of 57 men had the following semen parameters: concentration of 40.0±32x106/mL, 37.1±11% motility, and 2.2±1% normal morphology. After selection by DGC or MFSS, the concentrations were 3.3±3.4 and 8.0±13x106/mL, with 58.0±29% and 96.9±9% motility, respectively (P<0.0001). The SCF decreased from 21±14% in raw specimens to 18±6% following DGC and to 1.9±1% following MFSS (P<0.001). The dsDNA fell from 3.6±2% in raw specimens to 3.1±1% after DGC and to 0.3±0.2% after MFSS (P<0.001). These men (40.9±6 years) underwent DGC selection for 71 ICSI cycles with their female partners (36.5±5 years), achieving a fertilization rate of 58.4% (403/690) and a blastocyst euploidy rate of 28.5% (47/165). Only 19 FET cycles were performed, with a 6.7% implantation rate (2/30) and two clinical pregnancies resulting in miscarriage. Subsequently, these men had their specimens selected by MFSS in 71 ICSI cycles, resulting in a higher fertilization rate of 75.9% (647/852; P<0.0001) and a much improved (P<0.0001) blastocyst euploidy rate of 48.9% by PGT-A (192/389). In 48 FET cycles, 51 embryos were replaced with an increased implantation rate of 60.8% (31/51; P<0.0001), a CPR of 64.6% (31/48; P<0.001), and an ongoing/delivery rate of 62.5% (30/48; P<0.0001). With its dsDNA component, SCF tangibly contributes to embryo structural chromosomal abnormalities. An enhanced spermatozoa selection method for ICSI appears to remarkably increase the proportion of euploid blastocysts with consequent successful clinical outcomes.

P–119 The correlation between sperm chromatin fragmentation and intrauterine insemination outcome

Abstract Study question Does sperm genomic integrity affect the intrauterine insemination (IUI) outcomes in couples with unexplained infertility and young maternal age? Summary answer Spermatozoa with higher genomic integrity are correlated with higher clinical pregnancy rates in couples with unexplained infertility undergoing IUI. What is known already It is known that elevated sperm chromatin fragmentation (SCF) on the male gamete affects embryo development and implantation. This is particularly relevant in IVF as well as programmed intercourse and IUI. By complementing the standard semen analysis with an SCF assay, we can assess the competence of the male gamete and its ability to generate euploid embryos and healthy offspring. Elevated SCF has been used as a way to identify subtle male factor infertility in couples undergoing IUI with poor pregnancy outcomes in order to plan for further treatments. Study design, size, duration This is a retrospective cohort study of IUI outcomes of couples with young maternal age and a negative infertility workup treated at our center from 2016–2020. Terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) assay was used to assess sperm genomic integrity. Couples were grouped based on SCF level: normal (≤15%) or abnormal (&amp;gt;15%). Rates of clinical pregnancy, defined as the presence of a fetal heartbeat, were compared between the groups following IUI. Participants/materials, setting, methods A total of 189 consenting couples, in which the female partner had a normal uterine cavity and patent fallopian tubes, underwent 454 IUI attempts. Only women ≤37 years old were included to control for age-related confounding factors. At least 500 spermatozoa were assessed per patient, and a threshold of ≤ 15% was considered normal. Women were either untreated for natural cycle IUI or stimulated with clomiphene citrate, gonadotropins, or Letrozole. Main results and the role of chance A total of 454 IUI cycles were reported at our center; 302 of these were carried out in 132 couples in which the male partner had normal SCF averaged at 9.29%. The average maternal age was 34.1±3 years, and the average paternal age was 37.1±5 years. These men had the following semen parameters: a concentration of 46.2±5x106/mL, 43.8±3% motility, and an average SCF of 9.3±3%. There were 45 documented clinical pregnancies (45/302, 14.9%) as confirmed by the presence of at least one fetal heartbeat detected by ultrasound; 26 delivered, 9 are ongoing, 5 were spontaneous abortions, and 5 were lost to follow-up. A total of 57 couples in which the male partner (37.2±5.9 years) had abnormal SCF underwent 152 IUI cycles (maternal age, 34.0±2.7 years). The men had the following semen parameters: an average SCF of 23.8±10 (p &amp;lt; 0.0001), a concentration of 26.0±10 x106/ml, and 40.1±4% motility. These IUI attempts yielded a clinical pregnancy rate of only 4.6% (7/152; P &amp;lt; 0.0001); 4 delivered and 3 were spontaneous abortions. Limitations, reasons for caution This study is a retrospective cohort analysis of a relatively small number of patients. Furthermore, most patients were screened for SCF due to at least one prior IUI failure. A prospective, randomized trial, in which men are concurrently screened for SCF levels at the first IUI attempt, would be ideal. Wider implications of the findings: Assessment of SCF at the initial male infertility screening can be a useful tool to investigate the competence of the male gamete. Screening couples with idiopathic infertility for a subtle male factor would guide those with higher SCF toward alternative reproductive treatments to avoid unnecessary IUI treatments. Trial registration number Not applicable

P–120 Selecting spermatozoa with the highest genomic integrity in order to enhance clinical outcomes in men with high DNA fragmentation levels

Abstract Study question What are the best methods of selecting spermatozoa with the highest genomic integrity in order to improve embryo implantation and term pregnancy rates with ICSI? Summary answer Testicular or ejaculate spermatozoa isolated by microfluidic sperm selection (MFSS) were characterized by superior genomic integrity with improved clinical pregnancy and delivery rates. What is known already In couples with unexplained infertility, a subtle male factor can often be identified. Both single-strand (ss) and double-strand (ds) DNA nicks and breaks hinder the ability of the male gamete to support embryonic development. Surgical retrieval of spermatozoa from the proximal male genital tract can prevent their exposure to oxidative stress. Moreover, use of membrane-based microfluidics chips has been shown to allow for selection of the most progressively motile spermatozoa with higher genomic integrity. Study design, size, duration Over the course of 48 months, 86 consenting men presenting with high sperm chromatin fragmentation (SCF) in their ejaculate with prior ART failure underwent a subsequent cycle with specimens retrieved by testicular biopsy or ejaculate processed by MFSS. A concurrent TUNEL assay was performed on samples collected or selected by each method. Sperm specimens of both origins were utilized for ICSI cycles. Semen parameters, chromatin integrity, and pregnancy outcomes were compared between the two methods. Participants/materials, setting, methods Fresh ejaculates from consenting men were collected for standard semen analysis (WHO 2010). Testicular biopsy and MFSS were used to isolate spermatozoa with a higher genomic integrity after previous ART failure. SCF was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) on at least 500 spermatozoa under a fluorescent microscope with a threshold of ≥ 15%. MFSS was carried out by Zymot® chips. ICSI was performed in the standard fashion. Main results and the role of chance A total of 86 men (36.5±5 years) had the following semen parameters: volume of 2.6 ±1mL, concentration of 27.0±33 x 106/mL, 35.6±15% motility, and high SCF (24.1±10%). They underwent 146 ICSI cycles with their partners (maternal age, 33.7±3) resulting in a high incidence of pregnancy loss (100%; 13/13). Of those who failed to conceive, 22 couples used surgically retrieved spermatozoa (SRS) with a concentration of 1.8 ± 4 x 106/mL (P &amp;lt; 0.01), 5.0±11% motility (P &amp;lt; 0.01), and an SCF of 12.6 ± 6% (P &amp;lt; 0.0001). SRS was used in 37 ICSI cycles, yielding a fertilization rate of 61.6% (204/331, P &amp;lt; 0.01), an implantation rate of 10.6% (9/85, P &amp;lt; 0.01), a CPR of 23.5% (8/34, P &amp;lt; 0.01), and a delivery rate of 17.6% (6/34, P &amp;lt; 0.01). Another 24 couples underwent ICSI cycles with ejaculated spermatozoa processed by MFSS with a concentration of 1.8±3 x 106/mL (P &amp;lt; 0.01), but an increased motility of 99±1% (P &amp;lt; 0.01) and an SCF of 1.2 ±1%, lower than both the raw and testicular specimens (P &amp;lt; 0.0001). MFSS-processed specimens resulted in a fertilization rate of 76% (335/441, P &amp;lt; 0.01), an implantation rate of 26.3% (15/57, P &amp;lt; 0.05), and a CPR of 67.9% (19/28, P &amp;lt; 0.01), of which 15 patients delivered and 2 pregnancies are ongoing (89.5%; P &amp;lt; 0.01). Limitations, reasons for caution This is a preliminary study on a small number of subjects. A randomized prospective study conducted on a larger cohort would be required to confirm our findings. Wider implications of the findings: SCF severely affects pregnancy by impairing embryonic development, consequently promoting implantation failure. While retrieving spermatozoa from the germinal epithelium is a viable option, MFSS provides an alternative. Although MFSS requires an adequate number of sperm with good kinetic characteristics, it provides a more palatable option, reducing surgical risk and costs. Trial registration number Not applicable

P–118 A sperm selection technique to mitigate paternal contributions to embryo aneuploidy

Abstract Study question Can microfluidic sperm selection (MFSS) select male gametes without sperm chromatin fragmentation (SCF) and double-stranded DNA breaks (dsDNA) in order to generate euploid conceptuses? Summary answer Couples treated by ICSI with MFSS had significantly improved embryo ploidy rates and pregnancy outcomes, demonstrating the efficacy of this novel selection method. What is known already SCF has been linked to infertility, specifically to embryo developmental and implantation failure. This damage can be both single-stranded (ssDNA) or double-stranded (dsDNA). Recent studies have shown that dsDNA in particular causes chromosomal aberrations and contributes to embryo aneuploidy, which leads to implantation failure. Study design, size, duration Consenting couples treated at our center by intracytoplasmic sperm injection (ICSI) with spermatozoa selected by MFSS were included. The majority of these couples had a medical history significant for elevated SCF, recurrent implantation failure, and embryo aneuploidy. ICSI clinical outcome, as well as preimplantation genetic testing for aneuploidy (PGT-A) and frozen embryo transfer (FET), was recorded and compared to the couples’ historical treatments following sperm selection by density gradient centrifugation (DGC). Participants/materials, setting, methods From 2016 to 2020, 51 consenting men had their ejaculates screened for SCF levels by terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) using a commercially available kit. At least 500 spermatozoa were assessed per patient, with a normal threshold of ≤ 15%. To screen for dsDNA, neutral Comet using a modified in-house protocol was also performed in a pilot study. At least 200 spermatozoa were assessed per patient, with a normal threshold of ≤ 3%. Main results and the role of chance A total of 51 men (average age, 41.0±8 years) had mean sperm concentrations of 39.0±33x106/mL, 38.4±12% motility, and 2.1±1% normal morphology. Following DGC and MFSS, the concentrations were 4.7±8 and 4.3±8x106/mL and the motility was 64.0±33 and 98.0±3%, respectively (P &amp;lt; 0.0001). The average SCF decreased from 20.1±18% in the ejaculate to 16±3% following DGC, but was 2.9±4% after MFSS. The dsDNA fell from 3.4±3% in raw specimens to 2.9±1% after DGC, and to only 0.5±0.7% following MFSS (P &amp;lt; 0.0001). These men underwent ICSI with their female partners (average age, 37.3±4 years), with sperm selected by DGC; they achieved a fertilization rate of 56.4% (337/597) with 26.0% euploid embryos (36/139). FET cycles from these embryos yielded an implantation rate of 8.3% (2/24) and a clinical pregnancy rate (CPR) of 15.4% (2/13), but both miscarried. These couples then underwent ICSI with MFSS, with a fertilization rate of 78.0% (588/754; P &amp;lt; 0.0001) and 50.0% (172/344; P &amp;lt; 0.0001) euploid embryos after PGT-A. A total of 37 embryos have been replaced, with an implantation rate of 67.6% (25/37; P &amp;lt; 0.0001) and a CPR of 73.5% (25/34; P &amp;lt; 0.001), with an ongoing/delivery rate of 70.6% (24/34; P &amp;lt; 0.0001). Limitations, reasons for caution While the oocyte contribution cannot be discounted, MFSS was able to yield spermatozoa that had the highest motility and ability to produce euploid embryos following ICSI. Wider implications of the findings: The genome and epigenome of the spermatozoon, and their contribution to reproductive outcomes, are being vigorously explored and scrutinized. Alternative approaches to gamete selection, such as MFSS, in couples with elevated SCF and dsDNA provide the best chances for future pregnancies by mitigating embryo aneuploidy. Trial registration number Not applicable