Abstract Study question Considering maternal influence or management of male factor, should only PGT-A be indicated to reduce miscarriage? Summary answer The finest cost-effective treatment for each patient to improve ART success, is a good, tailored, and individualized indication for choosing the best IVF technique. What is known already Advanced maternal age is related with fertility decline, oocyte quality and the increased risk of aneuploid embryos. Aneuploid embryos lead to a reduced implantation potential and higher miscarriage rate. PGT-A is a screening method to determine the chromosomal status of the embryos and it’s the most expanded technique used to reduce the miscarriage rates in most clinics. Common sperm capacitation techniques used in IVF laboratories could cause sperm DNA damage, leading to a smaller number of blastocysts obtained per cycle and a higher miscarriage rate. Microfluidics devices or sperm selection centrifugation-free (Swim-up modified techniques) could reduce sperm DNA fragmentation. Study design, size, duration This is a retrospective study from 2018-2020 including patients using their own oocytes fertilized by ICSI. No major differences on male factor were found between male patients. In all cycles at least one good quality blastocyst was obtained to biopsy on blastocyst stage in cycles with PGT-A or to transfer without PGT-A. Live-Birth Rate (LBR) per cycle is compared and analyzed by the t-Student and X2 test between groups. Participants/materials, setting, methods A total of 308 cycles were included in this study. Three groups of patients were formed: Group 1: 76 cycles with PGT-A; Group 2: 191 cycles without PGT-A and sperm samples processed by Swim-Up technique; and Group 3: 41 cycles without PGT-A and a centrifugation free sperm selection (IO-Lix) used for ICSI. The main indication for PGT-A was maternal age. No differences between male or other female infertility factors were found between groups. Main results and the role of chance When we compare the 3 groups, no statistically significant differences were obtained in fertilization rate (79.8%; 73.5%; 72.6%; p = 0.28); development to the blastocyst stage (48.7%; 43.1%; 44.9%; p = 0.11) and pregnancy rate per transfer (48.8%; 45.6%; 52.8%; p = 0.55). It seems that PGT-A doesn’t affect the viability, or the implantation potential of the biopsied embryos and a special sperm capacitation technique is useful for daily clinical use. If we include only patients with ages >38 years old to avoid age differences, comparing Group 1 (PGT-A) and Group 3 (no-PGT-A and IO-lix) (LBR=19.7% vs 50%), we obtain statistically significant differences between both groups (p = 0.01). No differences were found between Group 2 (LBR=26.4%; p = 0.25) and the Group 1. To summarize, the female age appears to be the most important factor for IVF success and the PGT-A can help to avoid a miscarriage, however when we compare women with advanced maternal age, the LBR is increased when PGT-A is non performed, and the sperm DNA is not damaged by the capacitation technique. Why not PGT-A to be complemented with efficient sperm selection? Limitations, reasons for caution Although sperm selection techniques to decrease sperm DNA damage could reduce miscarriage rates, PGT-A is the only used technique that allows us to ensure that euploid embryos are transferred. Wider implications of the findings A better understanding of male factor in IVF treatments can reduce miscarriage. Male factor assessment is a cost-effective technique to improve LBR in those patients without a clear indication for PGT-A, (advanced maternal age). Patient must be studied individually, and PGT-A shouldn’t be used indiscriminately to ensure a healthy new-born. Trial registration number Not Applicable

Abstract Study question Is it useful to avoid sperm centrifugation in laboratory routine work to improve sperm quality and reproductive outcome in Assisted Reproduction Techniques (ART)? Summary answer Exclusion of sperm centrifugation for sperm selection using neat sperm samples (IO-lix), increases sperm quality in the collected subpopulation decreasing miscarriage rate after using ICSI. What is known already Inclusion of sperm centrifugation in ART is an aggressive intervention for sperm selection with ineludible production iatrogenic damage affecting sperm integrity. The application of IMSI, PICSI or microfluidic devices avoid sperm centrifugation and may improve the quality of the subsample obtained. However, these methodologies may result time consuming, expensive or producing poor results when the quality of the sperm is limited. We have already shown that a modified swim-up avoiding centrifugation (called IO-lix) is a low-cost and efficient alternative to microfluidic devices, recovers 100 times more concentration and reduces sperm DNA fragmentation with no significant differences to other methodologies. Study design, size, duration This is a retrospective study from 2018 to 2020 which includes patients with an average of age of 38.2 years using their own oocytes with ICSI as fertilization technique. Two aleatory groups of patients were made: Group 1: 88 cycles with 503 fertilized oocytes and 206 blastocysts were obtained with sperm samples processed by IO-lix and Group 2: 303 cycles, 1451 fertilized oocytes and 591 blastocysts using a standard “swim up” technique to process sperm. Participants/materials, setting, methods A total of 391 ICSI cycles were included in this retrospective study. The male factor was similar in both groups and they showed altered SDF previously to the cycle. We compared data of the motility and SDF of sperm samples before and after applying IO-lix and we analyzed by X2 contingence test differences on miscarriage rates between groups 1 and 2. Main results and the role of chance General sperm parameter changes after IO-lix showed that averaged sperm concentration observed in neat ejaculated samples was 62M/SD=46.4. Values obtained after IO-lix in the same samples were 12.3M/SD8.0. Averaged sperm motility in neat samples was 54%/SD=9.3 and 70.9%/SD=13.2 after IO-lix. Finally, sperm DNA fragmentation in neat samples was 35.8%/SD17.3, while these values decreased to 9.2%/SD=3.9 after IO-lix. About reproductive outcome results, significant differences were not obtained on the development to blastocyst stage rate comparing both groups (X2=0.003; p value = 0.954; Alpha 0.05). In the case of IO-lix processed samples, the pregnancy rate was 59.42% in Group 1 and 44.72% in Group 2 (X2=0.651; p value =0.419; Alpha 0.05). A total of 9 miscarriages of 41 clinical pregnancies (21.95%) were observed after IO-lix, while this number increases to 59 out of 123 clinical pregnancies, which means the 47.96% of the embryo transfers, when “swim-up” was used. In this case significant differences were obtained (X2=3.935; p value = 0.0.047; Alpha 0.05). Limitations, reasons for caution Being a pilot study aimed to understand the results of IO-lix in ART, correlations have not been stablished between the levels of sperm improvement after IO-lix and paired results of ART. This study would be necessary, specially to identify the possible origin of miscarriage associated to the male factor. Wider implications of the findings: Elimination of sperm centrifugation using a combined strategy of gradients and “swim-up” for sperm isolation, reduce miscarriage rate and produce equivalent results of blastocyst development to those obtained with “swim-up”. Being a cost-effective and improving laboratory workload, its use for sperm selection is recommended. Trial registration number Not applicable

Horses are long-day seasonal breeding animals, however, with modern stallion reproductive management it is important for collection of semen during periods that are not part of the traditional breeding season. This study was conducted to examine variation in the seminal characteristics of individual stallions in Avila, Spain during 1 year with a particular emphasis on sperm DNA fragmentation. Semen was collected twice per season from a total of 20 stallions. There was a marked seasonal effect on all seminal characteristics, with the greatest on progressive motility, % membrane integrity and least for SDF in the spring months; there was also an interaction effect with respect to individual stallion, indicating that some stallions did not fit this generalised pattern for semen quality. Sperm DNA fragmentation was assessed both immediately after semen collection (T0) and following incubation of extended semen for 24 h (T24) to broadly mimic changes in SDF that might occur in the female reproductive tract. While SDF evaluated at T0 was also generally less in spring, the proportion of stallions with the least SDF values in spring increased from 45% to 60% when assessed at T24, therefore, being consistent with the importance of dynamic SDF assessment in detecting DNA damage that was not detected at T0 or cryptic DNA damage. The results from this study indicate there is individual seasonal variation among stallions in all aspects of seminal characteristics; such variation needs to be considered when prioritising stallions that are to be used for breeding.

To investigate differences in the post-thaw DNA stability of koala and wombat spermatozoa, protamine amino acid sequences were compared and it was found that there were three more arginine residues for the wombat. Koala and wombat spermatozoa, cryopreserved using identical protocols, were examined for changes in sperm DNA fragmentation (SDF) dynamics over 24h of post-thaw incubation. Following validation of a wombat sperm chromatin dispersion test, wombat DNA showed a rate of SDF that was 6-fold higher than for koala spermatozoa (P=0.038). Finally, we examined whether expected differences in chromatin compactness, associated with protamine sequence, had an effect on restriction site accessibility of sperm DNA. Thawed spermatozoa were exposed to Alu I and EcoR1 endonuclease restriction enzymes and the SDF dynamics were observed. Koala spermatozoa exposed to Alu I showed a greater rate of SDF (P=0.01), whereas wombat spermatozoa exposed to EcoR1 showed a greater rate of SDF (P=0.032). We conclude that restriction sites in these species are differentially present or exposed and potentially account for differences in SDF dynamics. Although differences in the arginine composition of protamine may explain relative differences in SDF following cryopreservation, they do not support the hypothesis that increased arginine composition increases DNA stability; rather, increased arginine composition in the wombat may reduce post-thaw chromatin swelling.

Artificial insemination programs for horses usually involve ex vivo handling and transporting of sperm. The present experiment was designed to: (i) assess the effect of transportation on sperm DNA integrity at different time post semen collection, and (ii) evaluate if sperm DNA quality deteriorates rapidly beyond 24 h of cooled storage. After collection, the ejaculates were extended using INRA 96 and semen was prepared for prompt analysis (A0) or 24 h/48 h cooled-shipping (B24 and C48 respectively). Each sample was assessed for sperm DNA fragmentation index (SDFI) at time 0 and after incubation for 2, 6 and 24 h at 37 °C. There was very little difference in SDFI between freshly extended (A0) and 24 h/48 h cooled-transported semen samples (B24/C48) at time 0. After 2 h of incubation at 37 °C, there was an increase in SDFI ranging from 2.7% to 7.5% per hour in freshly extended semen samples (A0: 5.1 ± 1.5), while cooled-transported semen samples had a much greater increase in SDFI, ranging from 5.0% to 20.5% (B24: 14.7 ± 5.6) and from 8.2% to 26.8% (C48: 18.3 ± 7.2) respectively. There were not marked differences in the sperm DNA integrity between 24 and 48 h for transported samples, thus there is the possibility of desirable fertility with use of stallion sperm after 48 h of cooled storage.

We investigated the association between progressive stages of cervical neoplasia and DNA damage in 1p36 DNA sequences of chromosome 1 in cervical epithelium using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). We used a hospital based unmatched case control study of 29 women that were grouped according to disease stage and selected according to histological diagnosis: 10 with low grade squamous intraepithelial lesions (LG-SILs), 10 with high grade SILs (HG-SILs) and nine with no cervical lesions; the 1pter sequence was used as internal control. We found a significant increase in the number of patients with HG-SIL compared to patients with LG-SILs or with no cervical lesions. 1p36 Genomic instability was validated by DBD-FISH using neutral comets. Genetic instability at specific gene loci, such as 1p36, might be characteristic of cervical cancer progression. DBD-FISH appears to be a useful approach for detecting and comparing damage to specific chromosomal regions related to the progression of cervical cancer.

Prostasomes are exosomes such as extracellular vesicles, produced in the prostatic epithelium and released into the seminal plasma, that play an important role enhancing male fertility. Although some studies have demonstrated that prostasomes have a rich proteomic content, it is still unclear if that proteomic content varies depending on the male fertility status. Prostasomes from 12 normozoospermic and 14 non-normozoospermic seminal samples were isolated by differential ultracentrifugation. Protein content was studied by quantitative mass spectrometry and compared between both cohorts. We identified 1282 proteins with 745 of them (57.8%) being present in all seven prostasome pools. Forty-seven of those commonly present proteins showed differential expression levels in both cohorts. Specifically, prostasomes from non-normozoospermic samples showed a pattern of protein underexpression for a group of proteins including several proteins from the spermatozoa's energy production pathways as well as some proteins directly implicated in sperm activity. Variations in prostasomal protein content levels may have a relevant correlation with male fertility and thus could be of great utility as a biomarker of fertility status.

Equus caballus papillomavirus type 2 (EcPV-2) is implicated in genital neoplasms in horses, including equine penile papillomas, penile intraepithelial neoplasia, and squamous cell carcinoma (SCC). This virus seldom regresses spontaneously and can result in the development of SCC, which may result in significant clinical damage and economic cost. However, the prevalence of this virus is unknown in clinically unaffected horses. The aim of this study was to determine the prevalence of EcPV-2 DNA in genital swabs from healthy horses in the Republic of Korea and to investigate genetic variability within EcPV-2. A total of 797 genital swab samples obtained from Thoroughbred horses (35 stallions and 762 mares) were tested for the presence of the EcPV-2 gene E1 using a polymerase chain reaction (PCR) assay. Positive results were confirmed by repeat PCR to detect the presence of E6 and amplicon sequencing. Equus caballus papillomavirus type 2 DNA was detected in 1% (8/762) of mare swabs but in none of stallion swabs (0/35). All EcPV-2–positive mares had been imported from the United States. Equus caballus papillomavirus type 2 E6 gene sequences were used for phylogenetic analysis and revealed three subdivisions related to the European strains, and three isolates were located on two separate branches. This study documents the molecular prevalence and phylogenetic analysis of EcPV-2 DNA in the Republic of Korea.

Static assessment of sperm DNA Fragmentation (SDF at the time of ejaculation or sperm thawing when cryopreserved) and the dynamic assessment of SDF (SDF assessed after T2hr, T6hr and T24hr of sperm thawing) were used to establish cut-off values associated with sperm donors when compared with closely related normozoospermic patients. Cryopreserved samples from donors revealed SDF levels two times lower in comparison with the patients. Donor sperm DNA exhibited a 2.5 times higher longevity when compared with the patients. Static values of SDF after thawing of approximately 11% identify the donors with a 71% of sensitivity and 84% specificity. With respect to the dynamic assessment, SDF increases of 2.3 per hr during the first 2hr of incubation identify the donors with 70% of sensitivity and 66% of specificity. Creating the Rate of Combined Damage (RCD) defined as the product of SDF-T-0 by the increase in the damage registered during the first 2hr of incubation (r-SDF-T0-2), an index of RCD=22.2 units has an identification capacity of donors with a 78% sensitivity and 77% specificity. Such cut-off values could be used to characterise donors with high chromatin resistance to damage when meeting the above-established criteria.

Zinc (Zn) is essential for the development and activity of sperm, although its cytotoxic effect on sperm has been little studied. This study evaluated the effect of organic Zn; that is, Zn-methionate (Zn-Met), on the DNA fragmentation of boar sperm. Domestic boars (York × Landrace, Sus scrofa domesticus; n = 15) were randomly allocated into 3 levels of Zn dietary concentrations: 25 (Control), 150, or 200 ppm. Sperm DNA fragmentation dynamics were evaluated over an 8-wk period after Zn-Met supplementation. The Sperm-Sus-Halomax® Kit (Halosperm SL, Madrid, Spain), a 1:1 mixture of SYBR I (10×; Invitrogen Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) in Vectashield Mounting Medium (Vector Laboratories Inc., Burlingame, CA, USA) for DNA staining, and fluorescence microscopy (Nikon Eclipse 80i; Nikon, Tokyo, Japan) were used to analyse DNA fragmentation dynamics; that is, sperm chromatin dispersion (SCD) test, of the boar sperm. Samples were diluted 2:10 (v:v) in either (a) Beltsville Thawing Solution (BTS) extender, or (b) PBS, to determine the effects, if any, of extender. Extended sperm were stored at 15°C for 8 days for daily SCD testing. Data were analysed as a completely randomised design with repeated-measures serially in time (SAS Institute Inc., Cary, NC, USA). Main effects of the variables (i.e. dietary Zn and extender) and their interaction were studied. Means were compared using Tukey´s test, with significance set at the <0.05 α-level. Supplementation of the diet with 200 ppm of Zn-Met had an adverse effect on the integrity of pig sperm DNA from the beginning of supplementation to the last day of the experiment (i.e. fresh ejaculate 0 h = 9.44% fragmented, motility = 79.6%). However, Control and 150 ppm Zn-Met dietary levels did not significantly affect sperm DNA integrity (i.e. fresh ejaculate 0 h = 1.43% and 1.73% fragmented and motility = 84.1% and 84.5%, respectively). With regard to the semen extenders BTS and PBS, there was no difference (P > 0.05) in sperm DNA fragmentation dynamics for the first 3 days in extender: Day 3 = 4.09 and 6.45%, respectively. However, the sperm DNA fragmentation index was different (P < 0.05) between extenders BTS and PBS based on extended sperm for Day 4: 4.28 and 7.28%, respectively, through Day 8 (5.52 and 10.02%). These results demonstrated the importance of providing the correct amount of organic Zn in boar diets and potential impacts on reproduction.

Catalase (CAT), the key enzyme for high H2O2 elimination, constitutes, along with superoxide dismutase (SOD) and gluthatione peroxidase (GPX), the main enzymatic antioxidant system. Catalase enzyme plays an important role in seminal antioxidant defence and is present in seminal plasma at high concentrations. Low concentrations of catalase enzyme in seminal plasma have been related with male infertility. The objective of this study was to evaluate the correlation between the CAT C-262T polymorphism and male infertility. Semen samples from 400 infertile patients and 80 donors with proven fertility were included in the study. Standart semen parameters, DNA integrity and oxidative stress were evaluated. Genotype frequencies for the CAT C-262T polymorphism were determined by PCR-RFLP. Catalase concentration and activity in seminal plasma were evaluated by western-blot and activity gels respectively while catalase mRNA levels in mature spermatozoa were evaluated by qPCR. The CC genotype was associated with an increased risk of male infertility (OR = 1.976; CI = 1.241-3.216; P = 0.006) while the CT genotype was associated with a protective effect against infertility (OR = 0.479; CI = 0.293-0.783; P = 0.003). Functionally, the CT genotype showed higher concentrations of the enzyme in seminal plasma as well as 2.70 times higher activity levels than the CC genotype. Also, the CT genotype showed 3.5 higher levels of CAT mRNA in the mature spermatozoa than the CC genotype. This suggests that the CAT-262CT genotype correlates with a lower risk of suffering male infertility.

Herein we report a method of assessing DNA fragmentation in the saltwater crocodile using the sperm chromatin dispersion test (SCDt) after including frozen-thawed spermatozoa in a microgel (Halomax; Halotech DNA, Madrid, Spain). Following controlled protein depletion, which included a reducing agent, sperm nuclei with fragmented DNA showed a homogeneous and larger halo of chromatin dispersion with a corresponding reduced nucleoid core compared with sperm with non-fragmented DNA. The presence of DNA damage was confirmed directly by incorporation of modified nucleotides using in situ nick translation (ISNT) and indirectly by studying the correlation of the SCDt with the results of DNA damage visualisation using a two-tailed comet assay (r=0.90; P=0.037). Results of the SCDt immediately following thawing and after 5h incubation at 37°C in order to induce a range of DNA damage revealed individual crocodile differences in both the baseline level of DNA damage and DNA longevity.

This study was conducted to evaluate the effect of amino acid addition to semen on post-thaw quality of donkey spermatozoa. Eighteen ejaculates were pooled and divided into aliquots which were cryopreserved in Gent A® containing 1% ethylene glycol (Gent-EG) and supplemented with 0 (as control), 20, 40, or 60 mM of glutamine, proline, or taurine. The greatest concentration (60 mM) of glutamine and taurine resulted in greater (P < 0.001) post-thaw sperm motility. Amino acid supplementation did not improve (P > 0.05) sperm morphology and membrane plasma integrity compared with the control samples. Whereas, improvement (P < 0.05) of acrosome integrity was observed with use of 60 mM glutamine. After thawing, there were no differences (P > 0.05) in the sperm DNA fragmentation index (sDFI) among treatments. The 60 mM glutamine and 40 mM taurine treatments, however, resulted in a reduction (P < 0.05) in sDFI values in the first 6 h of semen incubation, compared with the control samples. At 24 h, the sDFI values were less (P < 0.05) in all supplemented as compared with control samples, except for the 20 mM proline treatment group. In conclusion, supplementation of the Gent-EG extender with glutamine or taurine at 60 mM improved post-thaw donkey sperm quality. The addition of proline to the freezing extender, however, did not provide any significant enhancement in sperm quality, compared with the control group.

The aim of the present study was to develop a protocol for the successful cryopreservation of Saltwater crocodile spermatozoa. Sperm cells were frozen above liquid nitrogen vapour in phosphate-buffered saline (PBS) containing either 0.3M trehalose, 0.3M raffinose or 0.3M sucrose and compared with glycerol (0.3-2.7M). Although the highest levels of mean post-thaw motility were observed following cryopreservation in 0.3M trehalose (7.6%) and 0.3M sucrose (7.3%), plasma membrane integrity (PI) was best following cryopreservation in 2.7M glycerol (52.5%). A pilot study then assessed the cytotoxicity of glycerol and sucrose prior to cryopreservation and revealed no loss of survival when spermatozoa were diluted in 0.68M glycerol or 0.2-0.3M sucrose once cryoprotectants were washed out with PBS or Biggers, Whitten and Whittingham medium containing sperm capacitation agents (BWWCAP). A final study refined the combined use of permeating (0.68 or 1.35M glycerol) and non-permeating (0.2 or 0.3M sucrose) cryoprotectants. Spermatozoa were cryopreserved in liquid nitrogen vapour at rates of approximately -21°Cmin-1 (fast freeze) or -6.0°Cmin-1 (slow freeze). Post-thaw survival was highest with a combination of 0.2M sucrose and 0.68M glycerol and when these cryoprotectants were washed out with BWWCAP, regardless of whether spermatozoa were frozen using a fast (motility 14.2±4.7%; PI 20.7±2.0%) or slow (motility 12.0±2.7%; PI 22±4%) cryopreservation rate.

It remains unknown whether human papillomaviruses (HPVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen samples of 22 normozoospermic patients undergoing infertility treatment, nine fertile donors and seven fertile men with a risk of HPV infection (genital warts or condylomas) were included in the study. The samples were examined by an INNO-LiPA test PCR-based reverse hybridisation array that identifies 28 types of HPVs as simple or multiple infections. Sperm DNA integrity was determined by sperm chromatin dispersion assay (SCD). Our preliminary findings demonstrate an increase in HPV infection in infertile men with respect to fertile men. However, the sperm DNA fragmentation index was not increased in semen containing these viruses.

There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. Adye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P < 0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

The aims of this study were to: 1) develop a new method for stallion sperm selection using a modified swim-up procedure through a colloid and 2) evaluate its impact in good quality ejaculates from bad freezers in comparison to methods involving centrifugation such as single layer centrifugation and sperm washing. Ejaculates were processed before freezing using three different procedures: sperm washing (SW), colloid single layer centrifugation (SLC) and a modified colloid swim-up (SU). After semen processing, sperm recovery rates were measured and sperm were frozen. Post-thaw sperm motility (assessed by computer-assisted sperm analysis), normal forms and plasma membrane integrity (evaluated under bright-field and fluorescence microscopy respectively), and DNA fragmentation (assessed by the Sperm-Halomax kit) were compared between treatments. Sperm recovery rates were similar between SU and SLC but lower than SW. Sperm motility after thawing was lower in SU in comparison to SLC and SW, maybe due to the incomplete removal of seminal plasma before freezing. Sperm DNA fragmentation was lower in SU and SLC selection methods, particularly in SLC selected samples during the first 6h of incubation. The remaining sperm parameters assessed were similar among treatments. In conclusion, SLC is more suitable than SW and SU to process stallion semen prior to freezing, in particular when sperm DNA damage is suspected. Further studies are needed in order to determine the potential benefits of SU in samples where centrifugation is not necessary, such as epididymal sperm, ejaculate fractioning or post-thaw semen samples.