The efficacy of novel centrifugation-free sperm selection (Io-Lix) on sperm parameters and ICSI reproductive outcomes.

RESEARCH QUESTION: What is the effect of a novel non-centrifugation method (Io-Lix) of sperm selection on sperm parameters and intracytoplasmic sperm injection (ICSI) reproductive outcomes? DESIGN: This pilot study elevated the capacity of the Io-Lix sperm selection protocol to improve sperm parameters (concentration, motility and sperm DNA fragmentation) of the neat ejaculate. Once established, the reproductive outcomes of Io-Lix selected spermatozoa were used for autologous and donor oocyte ICSI programmes and their efficacy compared with those using conventional swim-up. RESULTS: Io-Lix sperm selection resulted in lower sperm concentration yield (P < 0.001) and sperm DNA fragmentation (P < 0.001) but higher sperm motility (P < 0.001) when compared with spermatozoa in the neat ejaculate. When compared with swim-up sperm selection the Io-Lix protocol resulted in a 14.7% (P = 0.028) increase in pregnancy rate and 16.3% (P = 0.047) reduction in miscarriages in the autologous ICSI programme. A similar comparison of sperm selection procedures employed for a donor oocyte ICSI programme showed no difference in terms of their respective reproductive outcomes. CONCLUSIONS: The Io-Lix sperm selection protocol resulted in improved pregnancy rate and reduction in miscarriage when applied to autologous ICSI, which was attributed to a reduction in the proportion of spermatozoa with DNA damage post-selection. A similar finding was not apparent in the donor oocyte programme, which may be associated with the capacity of the donor oocyte to repair sperm DNA post-syngamy.

Determining the effects of sperm activation in anuran cloaca on motility and DNA integrity in Epidalea calamita (Bufonidae).

The effect of time inside the animal's cloaca on sperm quality after hormone-induced spermiation is unknown. However, this knowledge is critical for the development of assisted reproductive biotechnologies in amphibians. Out-of-season spermatozoa were collected from Epidalea calamita for 4h after injection of 10IU g-1 human chorionic gonadotrophin either hourly (Group I (n =10); four samples per male) or every 2h (Group II (n =9); two samples per male). Sperm samples were assessed for motility and DNA integrity using the sperm chromatin dispersion (SCD) test and the sperm chromatin structure assay (SCSA). The collection strategy affected total motility (mean (±s.e.m.) 84.4±9.9% vs 73.6±16.7% in Group I and II respectively; P =0.014) and the sperm motility index (67.6±17.7% vs 57.6±16.3% in Group I and II respectively; P =0.034). There was a significant effect of the male in Group II, but not in Group I. In Group I, the quality of the first samples collected was lower than that of samples collected thereafter (P ≤ 0.032). No significant correlations were found between the results of the SCD test and SCSA, showing that these techniques provide different information in this species. In conclusion, collecting spermatozoa every hour resulted in better sperm quality and may be more efficient. However, the between-male differences were considerable and collection of spermatozoa at just 1h after hormone treatment produced lower-quality spermatozoa.

Antibiotic toxicity on human spermatozoa assessed using the sperm DNA fragmentation dynamic assay.

Sperm DNA fragmentation (SDF) dynamic assays were piloted on 4 fresh ejaculates to examine the possible sperm toxicity of three common antibiotics, ciprofloxacin, doxycycline and ampicillin, incubated at a concentration estimated to be reached in semen in vivo, and 100×, for 24 h. SDF was assessed in terms of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs). Low and high concentrations of ciprofloxacin and high concentration of doxycycline significantly increased the SDF rate, due to sperm containing SSBs. Ampicillin did not affect SDF dynamics at any dose. Based on these results, the effect of antibiotics on the global-SDF dynamics was further examined in 21 ejaculates assessed at 0, 4 and 6 h. Ciprofloxacin increased the rate of SDF at the low concentration in 17 from 21 subjects; the high concentration resulted in a stronger effect in all individuals. A significant increase in the rate of SDF in 17 ejaculates was also noted when spermatozoa were incubated with the high concentration of doxycycline. The dynamic SDF assay is a rapid and sensitive tool to evidence sperm toxicity. Ciprofloxacin should be avoided when it is necessary to preserve sperm quality for reproductive purposes and as additive in semen diluents.

DNA fragmentation of equine cumulus cells from Cumulus-Oocyte complexes submitted to vitrification and its relationship to the developmental competence of the oocyte.

The objectives of this study were to evaluate the effect of vitrification on the DNA fragmentation rate of equine cumulus cells and to assess its relationship to oocyte in vitro maturation (IVM) after vitrification. Cumulus cells (CC) from 14 mares were recovered from COCs, previously submitted to vitrification (VIT) and IVM. The DNA fragmentation rate of the cumulus cells (CC-DF) was assessed using a chromatin dispersion test. CC-DF rates between vitrified and control COCs were statistically compared by Student's t-test. The rates of CC-DF from control COCs were lower than in vitrified COCs. The percentage of CC-DF was not significantly different (p > .05) between groups of COCs able to reach metaphase II (MII > 0) and those in which oocyte maturation was not achieved (MII = 0). In conclusion, vitrification has a deleterious effect on the DNA fragmentation of equine cumulus cells; however, this parameter cannot be used as a predictor for IVM success after COCs vitrification.

DNase activity in human seminal plasma and follicular fluid and its inhibition by follicular fluid chelating agents.

RESEARCH QUESTION: What is the mechanism by which human follicular fluid inhibits seminal plasma DNase activity? DESIGN: Human genomic DNA was incubated with human follicular fluid and seminal plasma (reaction mixture) under different experimental conditions; increasing volumes of human follicular fluid; proteinase K digested or heat inactivated human follicular fluid; and the addition of Ca2+ or Mg2+ to the reaction mixture. RESULTS: Increasing volume of human follicular fluid resulted in a dose-dependent inhibition of seminal plasma DNase activity. Inhibition was not caused by proteins in the human follicular fluid as digestion with proteinase K or heat inactivation of human follicular fluid failed to abolish its inhibitory effect. Addition of divalent cations resulted in a reversion of the inhibitory effect, providing evidence that human follicular fluid inhibition of seminal plasma DNase activity seems to be mediated by a compound with chelating activity. Furthermore, incubation of genomic DNA with human follicular fluid in the presence of divalent cations served to elicit the existence of DNase activity. CONCLUSIONS: Human follicular fluid seems to contain a molecule or molecules with chelating capacity that inhibits DNase activity of both follicular fluid and seminal plasma. Our findings provide new insight to understanding sperm preservation and the physiology of fertilization biology.

Associations between urinary concentrations of bisphenol A and sperm DNA fragmentation in young men.

BACKGROUND: Bisphenol A (BPA) is one of the most common endocrine disruptor compounds in our environment, promoting a xenoestrogenic state. Numerous studies have shown a relationship between exposure to BPA and male infertility problems. Spermatic DNA integrity is a critical factor for the correct transmission of paternal genetic material to the embryo. However, only a very few studies have investigated the association between urinary BPA concentrations and human sperm DNA fragmentation (SDF). METHOD: Cross-sectional study conducted with 158 healthy university students (18-23 years), recruited between 2010 and 2011 in the Region of Murcia (Spain). The subjects provided urine and semen samples on a single day. Urinary BPA concentrations were measured by dispersive liquid-liquid microextraction and ultrahigh performance liquid chromatography with tandem mass spectrometry detection, and SDF analysed using the Sperm Chromatin Dispersion test. Statistical analyses were made using linear regression adjusting for potential covariates and confounding factors. RESULTS: No association was found between urinary BPA concentrations and SDF index in the total group. However, in the subgroup of men with SDF index> 30%, significant positive associations across quartiles (p-trend=0.02) and as a continuous BPA levels were observed (ß = 0.055, 95%, CI: 0.002; 0.108). CONCLUSION: Our results show that, within the subgroup of men with relatively high SDF index, the higher the concentration of BPA the greater the SDF index. Nonetheless, more studies are required to confirm these results and draw conclusions in other male populations.

Free circulating DNA and DNase activity in the ejaculates of men with spinal cord injury.

STUDY DESIGN: Retrospective descriptive study. OBJECTIVES: To study the presence of cell-free DNA (cfDNA) and DNase activity in males with spinal cord injury (SCI) with elevated sperm DNA fragmentation. SETTING: Hospital in Toledo, Spain; University-based Genetics laboratory in Madrid, Spain. METHODS: Semen was collected from 15 males with spinal cord injury and elevated sperm DNA fragmentation (SDF). The presence and concentration of cfDNA was assessed using standard gel electrophoresis and microfluidic electrophoresis. DNase activity was evaluated in seminal plasma and under the presence of EDTA and EGTA to control the response of enzyme activity. cfDNA fragments were mapped on the sperm genome using FISH. All results were compared to 15 normozoospermic fertile donors. RESULTS: Standard gel electrophoresis revealed a cfDNA band of ~150 bp in all samples from males with SCI; this band was ocasionally accompanied by another band of ~90 bp. These bands were not observed in normozoospermic donors. Microfluidid electrophoresis only identified the equivalent band of 150 bp. No correlation was observed between the intensity of the two bands and the level of SDF in males with SCI. Although DNase activity was present in the seminal plasma of both normozoospermic donors and men with SCI it did not digest cfDNA. cfDNA fragments were found to be hybridized all over the sperm genome. CONCLUSIONS: SCI patients with elevated sperm DNA fragmentation showed a 150 bp DNA band of cfDNA in the seminal plasma, which appeared resistant to DNase activity.

Microencapsulation of human spermatozoa increases membrane stability and DNA longevity.

The microencapsulation of spermatozoa offers potential benefits for maintaining sperm survival in vitro. The technique has also resulted in the production of offspring in several domestic animal species, but as yet, it has not been successfully applied in human reproductive medicine. This study examined the effect of alginic acid microencapsulation on human sperm membrane integrity (viability) and sperm DNA fragmentation (SDF) following storage for 24 hr at 37°C. The cumulative sperm viability (Log-rank, Mantel-Cox; Chi-square = 114.95, p = .000) and cumulative sperm DNA fragmentation (Log-rank, Mantel-Cox; Chi-square = 187.86, p = .000) of encapsulated spermatozoa were substantially improved when compared to control spermatozoa. Significant differences in the dynamic behaviour of different individuals were only apparent for sperm viability in microencapsulated samples (p = .021) while no significant differences were observed in control spermatozoa (p = .245); the equivalent comparison for SDF showed no differences (control p = .320; microencapsulated p = .432). We present potential scenarios for the use of microencapsulated human spermatozoa in reproductive medicine.

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

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

Quick assessment of DNA damage in cervical epithelial cells using a chromatin dispersion test.

PURPOSE: This study was aimed to quantify genomic DNA breakages in the cervical epithelium cells of patients diagnosed with different grades of cervical lesions using a quick test based on chromatin dispersion after controlled protein depletion. The association between the progressive stages of cervical dysplasia and the levels of DNA damage, taking into account the presence of papillomavirus human (HPV) infection, was investigated. METHODS: A hospital-based unmatched case-control study was conducted during 2018 with a sample of 78 women grouped according to histological diagnosis as follows: 23 women with low grade-squamous intraepithelial lesion (LG-SIL), 34 women with high grade- squamous intraepithelial lesion (HG-SIL), and three women with cervical carcinoma (CC). In parallel, 15 women without cervical lesions were included as a Control cohort. DNA damage levels in cervical epithelial cells were assessed using the chromatin dispersion test (CDT) and controlled in parallel with DNA breakage detection coupled with florescent in situ hybridization (DBD‒FISH) using whole genomic DNA probes. RESULTS: CDT produces different morphotypes in the cervical epithelium that can be associated with the level of DNA breakage revealed with DBD‒FISH. A significant increase of DNA damage was correlated with the histological progression of the patients and human papillomavirus (HPV) infection. CONCLUSION: The CDT is a simple, accurate and inexpensive morphological bioassay to identify different levels DNA damage that can be associated with the level of abnormal cells present in the cervical epithelium in patients who commonly present HPV infection.

Dynamic assessment of human sperm DNA damage III: the effect of sperm freezing techniques.

Among the currently available strategies for sperm freezing, vitrification may be considered as the leading alternative to conventional cryopreservation. Nevertheless, a direct comparison of both techniques with respect to the iatrogenic sperm DNA damage has not been performed yet. As such, this study was focused to assess the static and dynamic behavior of human sperm DNA damage following thawing of cryopreserved or vitrified spermatozoa. Semen samples were obtained from fifty donors with a normal spermiogram, and divided into four aliquots. The first aliquot represented the neat sample. In the second aliquot the seminal plasma was discarded, and the resulting sperm pellet was resuspended in PBS. The third fraction was used for slow freezing and the fourth fraction was subjected to vitrification. Each set of samples was incubated at 37 °C for 24 h and sperm DNA damage (SDF) was assessed using the chromatin-dispersion test following 0 h, 2 h, 4 h and 24 h of incubation. When comparing the rate of DNA fragmentation (r-SDF) at 2 h, significant differences were observed between the PBS group, cryopreserved (p .000) or vitrified semen (p .015). Furthermore, the sperm longevity comparison using Kaplan-Meier survival curves revealed significant differences between cryopreservation and vitrification (p .000). Our data suggest that exposure of spermatozoa to low temperatures, independently of the chosen freezing protocol, leads to a higher susceptibility of sperm DNA towards damage. This damage is nevertheless lower following vitrification in comparison to traditional cryopreservation. As vitrification leads to a smaller proportion of spermatozoa with DNA damage, we may recommend its use in reproductive techniques which rely on a longer sperm survival, such as artificial insemination.

Assessment of avian sperm DNA fragmentation using the sperm chromatin dispersion assay.

Herein we report a simple method for assessing avian sperm DNA fragmentation (SDF) using the sperm chromatin dispersion test (SCDt). The presence of sperm DNA damage was confirmed indirectly by correlating results of the SCDt determined in three bird species with results of a corresponding neutral comet assay (r=0.99; P<0.005). Frozen-thawed spermatozoa of each species were also incubated at 37°C for 5h and the within- and between-species variation of SDF, as an indicator of sperm DNA longevity, examined. The dynamic assessment of SDF using the SCDt revealed species and individual bird (rooster and turkey) differences in sperm DNA longevity.

The incidence and etiology of sperm DNA fragmentation in the ejaculates of males with spinal cord injuries.

STUDY DESIGN: Retrospective descriptive study. OBJECTIVES: To determine the incidence and probable etiology of sperm DNA fragmentation (SDF) in a sample of males with spinal cord injury (SCI). SETTING: Hospital in Toledo, Spain; University-based Genetics laboratory in Madrid, Spain. METHODS: Semen collected by vibro-stimulation from 27 males with various levels of spinal cord injury. Classical semen parameters, SDF, leukocytospermia and pro-oxidant capacity were assessed and compared with a cohort of normozoospermic fertile donors (n = 10). RESULTS: Males with SCI presented with lower semen quality compared with normozoospermic donors with respect to progressive motility (p = 0.0002), SDF (p < 0.00005), pro-oxidant capacity (p = 0.0191) and leukocytospermia (p < 0.00005). Although there was no significant correlation between semen quality and time since the lesion occurred, the period of abstinence appeared to be positively correlated with SDF (r = 0.486; p = 0.041). When the semen parameters of males with SCI were categorized based on those with cervical and thoracic lesions, sperm concentration was higher for those with cervical damage (p = 0.0257). Males with complete lesions (AIS A) had ejaculates that were lower in progressive motility (p = 0.0040) than those with incomplete injuries (AIS B-D). CONCLUSIONS: Ejaculates of males with SCI have excessively elevated SDF when compared with normozoospermic donors, which is likely to be associated with coincident high levels of leucocytospermia and pro-oxidant capacity. We propose that these phenomena are caused by the accumulation and degeneration of spermatozoa in the cauda epididymidis.

Co-incubation of spermatozoa with human follicular fluid reduces sperm DNA fragmentation by mitigating DNase activity in the seminal plasma.

PURPOSE: To examine the effect of co-incubating spermatozoa with human follicular fluid (HFF) on the rate of sperm DNA fragmentation. METHODS: This prospective study used semen (n = 23) and HFF from oocyte donors (n = 23). Liquified semen was divided into four aliquots: (1) neat semen (NEAT), (2) seminal plasma removed and replaced with sperm media (HTF) containing 0% (FF0), (3) 20% (FF20), or (4) 50% (FF50) HFF. Sperm motility and DNA fragmentation (SDF) were assessed following 24 h of incubation at 37 °C. Pro-oxidant capacity of HFF and seminal plasma and the effect of HFF on seminal plasma DNase activity was assessed in a sub-sample of 10 ejaculates. RESULTS: Sperm motility was higher after 3 h of incubation in media that contained HFF compared to the NEAT sample or when sperm was diluted in media without HFF. r-SDF (increase of SDF per time unit) values after 24 h of incubation for NEAT, FF0, FF20 and FF50 were 0.91, 0.69, 0.25 and 0.36, respectively. While pro-oxidant capacity of seminal plasma samples showed large variation (mean: 94.6 colour units; SD 65.4), it was lower and more homogeneous in FF samples (mean: 29.9 colour units; SD: 6.3). Addition of HFF to seminal plasma appeared to inhibit DNase activity. CONCLUSION: While differences exist in the pro-oxidant capacity of seminal plasma of patients, sperm DNA integrity was preserved with addition of HFF to sperm media, irrespective of the level of pro-oxidant capacity. DNase activity in the original seminal plasma was abolished after HFF co-incubation.

Detection of DNA damage in pigeon erythrocytes using a chromatin dispersion assay.

The monitoring of environmental genotoxicity requires the selection of model organisms as 'sentinels' as well as the development of sensitive and reliable tests for the assessment of DNA damage. The aims of this study were to quantify genomic DNA strand breakage in the erythrocytes of Columba livia induced by thermal stress using the modified chromatin dispersion test and to validate the results by alkaline comet assay and DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). The chromatin dispersion test allowed for clear visualization of erythrocyte cells with DNA damage and of cells with no DNA damage. DNA damage increased significantly with increase in temperature. Additionally, we observed nuclear abnormalities associated with apoptosis, such as karyorrhexis (nuclear disintegration) and karyolysis (nuclear dissolution). These results were validated by alkaline comet assay and DBD-FISH. In conclusion, this procedure is a reliable, precise, and inexpensive morphological bioassay for routine quantitative analysis of DNA breakage in pigeon erythrocytes induced by thermal stress. This method could also be useful as a practical screening tool for genotoxicity testing in environmental care.

Association of polymorphisms in genes coding for antioxidant enzymes and human male infertility.

PURPOSE: Although oxidative stress is thought to be an important cause of male infertility, primarily due to DNA and cell membrane damage, little is known about the genetic causes underlying suboptimal function of the seminal enzymatic antioxidant system. The aim of this study was to investigate the relationship of four potentially functional polymorphisms associated with oxidative stress pathway genes (superoxide dismutase-SOD2 lle58Thr and SOD2 rs4880, catalase-CAT C-262T, glutathione peroxidase 1-GPX1 Pro200Leu) and two null variants of the glutathione S transferase (GSTT and GSTM) genes and infertility risk. METHODS: A case control study was conducted on 313 infertile patients and 80 fertile donors. Each ejaculate was subjected to a seminal analysis that included the classical parameters seminal volume, sperm concentration, sperm motility, and sperm morphology, as well as sperm DNA fragmentation (patients only). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR multiplex methods were carried out for genotyping. RESULTS: Statistically significant differences were found between fertile donors and infertile patients for SNP CAT C-262T; the CC genotype was related with a twofold increased risk of infertility (odds ratio [OR] = 2.262; 95% confidence interval [CI] = 1.369-3.733; P = 0.001), whereas the CT genotype was associated with a protective effect (OR = 0.401; 95% CI = 0.241-0.667; P = 0.001). Surprisingly, the SOD2 Ile58ssThr SNP was not represented in the sample population, so its frequency in the current population frequenting fertility clinics in Madrid may be very low. CONCLUSIONS: Our results suggest that the CAT SNP C-262T is potentially associated with an increased risk of male infertility.

Dynamic assessment of human sperm DNA damage II: the effect of sperm concentration adjustment during processing.

PURPOSE: To evaluate the effect of sperm concentration adjustment in human ejaculates on the sperm DNA quality and longevity. METHODS: Semen samples were obtained from 30 donors with a normal spermiogram. Following centrifugation, the sperm pellet was resuspended in PBS, and the sperm concentration adjusted to 200, 100, 50, 25, 12, and 6 × 106/mL. Each set of samples was incubated at 37 °C for 24 h, and the sperm DNA damage was assessed using the chromatin-dispersion test following 0 h, 2 h, 6 h, and 24 h of incubation. RESULTS: Sperm DNA fragmentation (SDF) did not differ between the selected experimental conditions at T0; however, Kaplan-Meier estimates for survival showed significant differences with respect to the dilution and time (all P values were smaller than .001). DNA fragmentation in semen samples adjusted to 200 × 106/mL was approximately 3.3 times higher when compared to samples containing 25 × 106/mL and 3.9 higher in comparison with samples adjusted to 12 × 106/mL following 2 h of in vitro incubation. Although there was evidence of individual variation in SDF during the incubation period, the general finding was that lower sperm concentrations resulted in a slower rate of DNA fragmentation. CONCLUSIONS: Incubation of spermatozoa for ART purposes should be done following a concentration adjustment below 25 × 106/mL in order to avoid a higher susceptibility of the sperm DNA molecule towards fragmentation.

Relationship between DNA fragmentation of equine granulosa cells and oocyte meiotic competence after in vitro maturation.

The acquisition of equine oocyte developmental capacity is ensured by the follicular environment, such as granulosa cells, which could reflect the meiotic development potential of immature oocytes. This study evaluated the relationship between DNA fragmentation of granulosa cells, using the chromatin dispersion test, and equine oocyte meiotic development after in vitro maturation. Granulosa cells and cumulus-oocytes complexes (n = 50) were recovered from slaughterhouse-derived ovaries. Oocytes were in vitro matured, stained and evaluated under fluorescence microscopy. Maturation rates were classified into outstanding, medium and poor levels of maturation using 25th and 75th percentiles as thresholds. For DNA assessment, each sample was processed with the Ovoselect® kit (Halotech DNA). High, low and total DNA fragmentation percentages were compared among levels of maturation rates by ANOVA, followed by Duncan test. Results were expressed as mean ± SE. Total and high DNA fragmentation rates of granulosa cells were significantly higher (p < 0.05) in follicles whose oocytes had reached outstanding maturation level than those originating from follicles whose oocytes had reached poor maturation level. In conclusion, the DNA fragmentation analysis of equine granulosa cells can be a valuable test to identify equine oocytes showing the best meiotic competence after in vitro maturation.

Effect of permeable cryoprotectant-free vitrification on DNA fragmentation of equine oocyte-cumulus cells.

DNA fragmentation of cumulus cells could be used as an indicator of oocyte vitrification success as an indirect indicator of the quality of the oocyte. This study was designed to compare the DNA fragmentation of post-mortem equine cumulus cells before or after vitrification in the absence of permeable cryoprotectant agents. Cumulus-oocyte complexes (COCs; n = 56) were recovered from slaughterhouse ovaries and subjected to in vitro maturation (42 hr/38.2°C/5%CO2 ) before (control group) or after a permeable cryoprotectant-free vitrification method using 1 M sucrose (vitrification group). After in vitro maturation, COCs were denuded, and cumulus cells were washed and stored at -80°C until thawing. Cumulus cell samples were processed with the chromatin dispersion test (Ovoselect, Halotech DNA, Spain). Low, high and total DNA fragmentation percentages of cumulus cells were recorded and compared between the two groups by Student's t test. Results were expressed as mean ± SEM. The vitrified group resulted in significantly higher (p < 0.05) percentages for low (16.81 ± 1.62 vs. 6.63 ± 0.77) and total (21.14 ± 1.84 vs. 12.76 ± 1.48) DNA fragmentation of cumulus cells. There were no significant differences between groups for high DNA fragmentation of cumulus cells. In conclusion, permeable cryoprotectant-free vitrification of equine oocytes increased the total DNA fragmentation rate of cumulus cells but protected them against high DNA fragmentation rates. Further studies are needed to examine the relationship between DNA fragmentation of cumulus cells and the developmental competence of equine oocytes.

DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development.

Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi's demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa. In this work, we applied this technique to ram epididymal spermatozoa, checking the correlation between DNA integrity and embryo development following ICSI. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, and Trolox media. To evaluate DNA damage and fragmentation after rehydration, samples were processed for Sperm Chromatin Dispersion test (SCD), Two-Tailed Comet Assay, and were used for ICSI. Ram #2 had a higher rate of spermatozoa with intact DNA compared with rams #1, #3, and #4 (28% vs. 3.8%, 2.8%, and 5%, respectively) and the lowest rate of Single-Strand Breaks (SSBs) (70% vs. 95.9%, 92.6%, and 93% respectively). Ram #3 had a higher level of Double-Strand Breaks (DSBs) compared to Ram #1 (4.6% vs. 0.33%, respectively). Embryo development to the blastocyst stage following ICSI was only reached from rams whose sperm had higher level of intact DNA - Rams #2 and #4 (6%, 5/147 and 6.3%, 4/64, respectively). Definitively, the impact of sperm DNA damage on embryonic development depends on the balance between sperm DNA fragmentation extent, fragmentation type (SSBs or DSBs), and the oocyte's repair capacity.