Adherence to Mediterranean diet impacts gastrointestinal microbial diversity throughout pregnancy.

BACKGROUND: Consumption of a diet with high adherence to a Mediterranean diet pattern (MDP) has been associated with a favorable gastrointestinal tract (GIT) microbiome. A healthy GIT microbiome in pregnancy, as defined by increased alpha diversity, is associated with lower chance of adverse perinatal outcomes. This study aimed to evaluate the impact of adherence to an MDP on GIT microbial diversity longitudinally throughout pregnancy. METHODS: Adherence to MDP was scored by the Alternate Mediterranean (aMED) Diet Quality Score, after being applied to a validated Food Frequency Questionnaire. Association of aMED Scores with GIT alpha diversity profiles were compared linearly and across time using a linear mixed model, including covariates of age, body mass index (BMI), ethnicity, and parity. RESULTS: Forty-one participants of Filipino, Japanese, Native Hawaiian, and Non-Hispanic White descent provided dietary information and microbiome samples during each trimester of pregnancy. Alpha diversity profiles changed over gestation, with decreased microbial diversity in the third trimester. aMED scores positively correlated with Chao1 Index and Observed Species Number (r = 0.244, p = 0.017, and r = 0.233, p = 0.023, respectively). The strongest association was detected in the third trimester (Chao 1: r = 0.43, p = 0.020, Observed Species Number: r = 0.41, p = 0.026). Participants with higher aMED scores had higher relative abundance of Acidaminoacaeae at the family level (p = 0.0169), as well as higher abundance of several species known to increase production of short chain fatty acids within the GIT. CONCLUSIONS: Adherence to MDP pattern is associated with increased maternal GIT microbial diversity, and promotes the abundance of bacteria that produce short chain fatty acids. Increased consumption of fruits, vegetables and legumes with low red meat consumption were key components driving this association. The effect of nutrition however, was less of an effect than pregnancy itself. Further studies are needed to determine if adherence to a Mediterranean diet translates not only into microbial health, but also into reduced risk of adverse pregnancy outcomes.

Mouse Y-Encoded Transcription Factor Zfy2 Is Essential for Sperm Formation and Function in Assisted Fertilization.

Spermatogenesis is a key developmental process allowing for a formation of a mature male gamete. During its final phase, spermiogenesis, haploid round spermatids undergo cellular differentiation into spermatozoa, which involves extensive restructuring of cell morphology, DNA, and epigenome. Using mouse models with abrogated Y chromosome gene complements and Y-derived transgene we identified Y chromosome encoded Zfy2 as the gene responsible for sperm formation and function. In the presence of a Zfy2 transgene, mice lacking the Y chromosome and transgenic for two other Y-derived genes, Sry driving sex determination and Eif2s3y initiating spermatogenesis, are capable of producing sperm which when injected into the oocytes yield live offspring. Therefore, only three Y chromosome genes, Sry, Eif2s3y and Zfy2, constitute the minimum Y chromosome complement compatible with successful intracytoplasmic sperm injection in the mouse.

Testicular abnormalities in mice with Y chromosome deficiencies.

We recently investigated mice with Y chromosome gene contribution limited to two, one, or no Y chromosome genes in respect to their ability to produce haploid round spermatids and live offspring following round spermatid injection. Here we explored the normalcy of germ cells and Sertoli cells within seminiferous tubules, and the interstitial tissue of the testis in these mice. We performed quantitative analysis of spermatogenesis and interstitial tissue on Periodic acid-Schiff and hematoxylin-stained mouse testis sections. The seminiferous epithelium of mice with limited Y gene contribution contained various cellular abnormalities, the total number of which was higher than in the males with an intact Y chromosome. The distribution of specific abnormality types varied among tested genotypes. The males with limited Y genes also had an increased population of testicular macrophages and internal vasculature structures. The data indicate that Y chromosome gene deficiencies in mice are associated with cellular abnormalities of the seminiferous epithelium and some changes within the testicular interstitium.

Deficiency of the multi-copy mouse Y gene Sly causes sperm DNA damage and abnormal chromatin packaging.

In mouse and man Y chromosome deletions are frequently associated with spermatogenic defects. Mice with extensive deletions of non-pairing Y chromosome long arm (NPYq) are infertile and produce sperm with grossly misshapen heads, abnormal chromatin packaging and DNA damage. The NPYq-encoded multi-copy gene Sly controls the expression of sex chromosome genes after meiosis and Sly deficiency results in a remarkable upregulation of sex chromosome genes. Sly deficiency has been shown to be the underlying cause of the sperm head anomalies and infertility associated with NPYq gene loss, but it was not known whether it recapitulates sperm DNA damage phenotype. We produced and examined mice with transgenically (RNAi) silenced Sly and demonstrated that these mice have increased incidence of sperm with DNA damage and poorly condensed and insufficiently protaminated chromatin. We also investigated the contribution of each of the two Sly-encoded transcript variants and noted that the phenotype was only observed when both variants were knocked down, and that the phenotype was intermediate in severity compared with mice with severe NPYq deficiency. Our data demonstrate that Sly deficiency is responsible for the sperm DNA damage/chromatin packaging defects observed in mice with NPYq deletions and point to SLY proteins involvement in chromatin reprogramming during spermiogenesis, probably through their effect on the post-meiotic expression of spermiogenic genes. Considering the importance of the sperm epigenome for embryonic and fetal development and the possibility of its inter-generational transmission, our results are important for future investigations of the molecular mechanisms of this biologically and clinically important process.

Temporal Investigation of the Maternal Origins of Fetal Gut Microbiota.

In utero colonization or deposition of beneficial microorganisms and their by-products likely occurs through various mechanisms, such as hematogenous spread or ascension from the reproductive tract. With high-throughput sequencing techniques, the identification of microbial components in first-pass neonatal meconium has been achieved. While these components are low-biomass and often not abundant enough to culture, the presence of microbial DNA signatures may promote fetal immune tolerance or epigenetic regulation prior to birth. The aim of this study was to investigate the maternal source of the neonatal first-pass meconium microbiome. Maternal vaginal and anal samples collected from twenty-one maternal-infant dyad pairs were compared via principal component analysis to first-pass neonatal meconium microbial compositions. Results demonstrated the greatest degree of similarity between the maternal gut microbiome in the second and third trimesters and vaginal microbiome samples across pregnancy, suggesting that the maternal gut microbiota may translocate to the fetal gut during pregnancy. This study sheds light on the origin and timing of the potential transfer of maternal microbial species to offspring in utero.

Effects of Dietary Quality on Vaginal Microbiome Composition Throughout Pregnancy in a Multi-Ethnic Cohort.

Background: Vaginal Lactobacillus predominance is associated with improved vaginal health and reduced pregnancy complications. Little is known about how dietary quality may improve vaginal microbial composition or about dietary interventions that may promote Lactobacillus abundance. To understand the host factors affecting vaginal microbiota during pregnancy in a multi-ethnic cohort in Hawai`i. We hypothesize that better diet quality improves vaginal microbial composition, as represented by Lactobacillus abundance and depletion of anaerobic organisms. Methods: We compared comprehensive diet quality, as measured by the Healthy Eating Index-2015 (HEI-2015), to taxonomic classifications of bacteria present within the vagina. Participants of the four predominant ethnic groups in Hawai'i (Japanese, Filipino, Non-Hispanic White and Native Hawaiian) completed Quantitative Food Frequency Questionnaires (QFFQs) and collected vaginal swabs during each trimester. 16s rRNA amplicon sequencing (V2-V9 regions) was performed on vaginal samples. HEI-2015 scores and macro- and micronutrient intake were compared with the predominant species present using the Mann-Whitney-U test, PERMANOVA, and the Pearson correlation coefficient. A mixed-effects logistics regression model was used to predict the depletion of Lactobacillus species while accounting for confounding demographic factors. Results: Matched, longitudinal data for 40 participants demonstrated three predominant Lactobacillus species: L. crispatus, L. iners, and L. gasseri, with another subset of samples with anaerobic abundance. (Primarily, Atopobium vaginae, Prevotella, and Gardnerella vaginalis.) Non-Hispanic White participants had lower amounts of Lactobacillus iners compared to other racial and ethnic groups. HEI scores correlated with the chao index and observed species number primarily in the first trimester (r = 0.25, p < 0.05). Greater carbohydrate intake was associated with a higher abundance of L. crispatus, while lower carbohydrate intake trended towards more L. iners (0.056) and anaerobic species. Conclusions: Increased carbohydrate consumption and improved diet quality may be associated with beneficial vaginal microbial composition in pregnancy.

Epigenetic Changes in the HTR8 and 3A-sub E placental Cell Lines Exposed to Bisphenol A and Benzyl Butyl Phthalate.

OBJECTIVE: Bisphenol A and phthalate are known endocrine disruptors and capable of inducing epigenetic changes in the human population. However, their impact on the placenta is less well studied. Our objective was to measure the effect of exposure to bisphenol A and benzyl butyl phthalate in first-trimester HTR8-SVneo and third-trimester 3A-sub E trophoblast cells by profiling the DNA methylation pattern of the imprinting control region of the IGF2 (insulin-like growth factor) and H19 genes. METHODS: Human placental HTR8-SVneo and 3A-sub E cell lines were treated with two sub-lethal concentrations of bisphenol A and benzyl butyl phthalate. Demethylating agent, 5-azacytidine, was used as a positive control. Cells were harvested on post-treatment days 1 and 4. The methylation profile of six CpG dinucleotide sites, part of the CTCF 6 binding site of the IGF2/H19 imprinting control region, was determined by pyrosequencing. RESULTS: In the first-trimester HTR8-SVneo cell line, we observed a significant increased methylation of the CpG sites 3, 4 when treated with a high concentration of bisphenol A or benzyl butyl phthalate while increased methylation at site 6 for both high and low dose treatment on day 4. Demethylation of the CpG sites 1, 4, and 6 was observed when treated with 5-azacytidine on day 4. In the third-trimester 3A-sub E cell line, no significant changes in the methylation profile were observed under any treatment conditions. CONCLUSIONS: The results of this study demonstrate the capability of epigenetic changes in human placenta cells induced by bisphenol A and benzyl butyl phthalate. The observed methylation changes only in the first-trimester HTR8-SVneo cells phthalate may reflect a window of epigenetic susceptibility related to these environmental toxicants.

Impact of Maternal Mediterranean-Type Diet Adherence on Microbiota Composition and Epigenetic Programming of Offspring.

Understanding how maternal diet affects in utero neonatal gut microbiota and epigenetic regulation may provide insight into disease origins and long-term health. The impact of Mediterranean diet pattern adherence (MDA) on fetal gut microbiome and epigenetic regulation was assessed in 33 pregnant women. Participants completed a validated food frequency questionnaire in each trimester of pregnancy; the alternate Mediterranean diet (aMED) score was applied. Umbilical cord blood, placental tissue, and neonatal meconium were collected from offspring. DNA methylation patterns were probed using the Illumnia EPICarray Methylation Chip in parturients with high versus low MDA. Meconium microbial abundance in the first 24 h after birth was identified using 16s rRNA sequencing and compared among neonates born to mothers with high and low aMED scores. Twenty-one mothers were classified as low MDA and 12 as high MDA. Pasteurellaceae and Bacteroidaceae trended towards greater abundance in the high-MDA group, as well as other short-chain fatty acid-producing species. Several differentially methylated regions varied between groups and overlapped gene regions including NCK2, SNED1, MTERF4, TNXB, HLA-DPB, BAG6, and LMO3. We identified a beneficial effect of adherence to a Mediterranean diet on fetal in utero development. This highlights the importance of dietary counseling for mothers and can be used as a guide for future studies of meconium and immuno-epigenetic modulation.

Temporal trends in microplastic accumulation in placentas from pregnancies in Hawai'i.

Microplastics are created for commercial use, are shed from textiles, or result from the breakdown of larger plastic items. Recent reports have shown that microplastics accumulate in human tissues and may have adverse health consequences. Currently, there are no standardized environmental monitoring systems to track microplastic accumulation within human tissues. Using Raman spectroscopy, we investigated the temporal exposures to plastic pollution in Hawai'i and noted a significant increase in the accumulation of microplastics in discarded placentas over the past 15 years, with changes in the size and chemical composition of the polymers. These findings provide a rare insight into the vulnerability and sensitivity of Pacific Island residents to plastic pollution and illustrate how discarded human tissues can be used as an innovative environmental plastic pollution monitoring system.

Short-term storage of human spermatozoa in electrolyte-free medium without freezing maintains sperm chromatin integrity better than cryopreservation.

Previous attempts to maintain human spermatozoa without freezing were based on short-term storage in component-rich medium and led to fast decline in motility and increased incidence of chromosome breaks. Here we report a new method in which sperm are maintained without freezing in an electrolyte-free medium (EFM) composed of glucose and bovine serum albumin. Human sperm were stored in EFM or human tubal fluid medium (HTFM) or were cryopreserved, and their motility, viability, and DNA integrity were examined at different intervals. Cryopreservation led to significant decline in sperm motility and viability and induced DNA fragmentation. Sperm stored in EFM maintained motility and viability for up to 4 and 7 wk, respectively, much longer than sperm stored in HTFM (<2 and <4 wk, respectively). DNA integrity, assessed with comet assay, was also maintained significantly better in EFM than in HTFM. One-week storage in EFM yielded motility and viability similar to that of cryopreserved sperm, but DNA integrity was significantly higher, resembling that of fresh sperm. After several weeks of storage in EFM, sperm were able to activate oocytes, undergo chromatin remodeling, and form normal zygotic chromosomes after intracytoplasmic sperm injection. This study demonstrated that human spermatozoa can be stored in EFM without freezing for several weeks while maintaining motility, viability, and chromatin integrity and that 1-wk storage in EFM offers better protection of sperm DNA integrity than cryopreservation. Sperm storage in EFM may become a viable option for the physicians working in assisted reproduction technology clinics, which would avoid cryodamage.

Pregnancy environment, and not preconception, leads to fetal growth restriction and congenital abnormalities associated with diabetes.

Maternal diabetes can lead to pregnancy complications and impaired fetal development. The goal of this study was to use a mouse model of reciprocal embryo transfer to distinguish between the preconception and gestational effects of diabetes. To induce diabetes female mice were injected with a single high dose of streptozotocin and 3 weeks thereafter used as oocyte donors for in vitro fertilization (IVF) and as recipients for embryo transfer. Following IVF embryos were cultured to the blastocyst stage in vitro or transferred to diabetic and non-diabetic recipients. Diabetic and non-diabetic females did not differ in regard to the number of oocytes obtained after ovarian stimulation, oocytes ability to become fertilized, and embryo development in vitro. However, diabetic females displayed impaired responsiveness to superovulation. Reciprocal embryo transfer resulted in similar incidence of live fetuses and abortions, and no changes in placental size. However, fetuses carried by diabetic recipients were smaller compared to those carried by non-diabetic recipients, regardless hyperglycemia status of oocyte donors. Congenital abnormalities were observed only among the fetuses carried by diabetic recipients. The findings support that the diabetic status during pregnancy, and not the preconception effect of diabetes on oogenesis, leads to fetal growth restriction and congenital deformities.

Live offspring from mice lacking the Y chromosome long arm gene complement.

The mouse Y chromosome long arm (Yq) comprises approximately 70 Mb of repetitive, male-specific DNA together with a short (0.7-Mb) pseudoautosomal region (PAR). The repetitive non-PAR region (NPYq) encodes genes whose deficiency leads to subfertility and infertility, resulting from impaired spermiogenesis. In XSxr(a)Y*(X) mice, the only Y-specific material is provided by the Y chromosome short arm-derived sex reversal factor Sxr(a), which is attached to the X chromosome PAR; these males (NPYq- males) produce sperm with severely malformed heads and are infertile. In the present study, we investigated sperm function in these mice in the context of intracytoplasmic sperm injection (ICSI). Of 261 oocytes injected, 103 reached the 2-cell stage, and 46 developed to liveborn offspring. Using Xist RT-PCR genotyping as well as gamete and somatic cell karyotyping, all six predicted genotypes were identified among ICSI-derived progeny. The sex chromosome constitution of NPYq- males does not allow production of offspring with the same genotype, but one of the expected offspring genotypes is XY*(X)Sxr(a) (NPYq-(2)), which has the same Y gene complement as NPYq-. Analysis of NPYq-(2) males revealed they had normal-sized testes with ongoing spermatogenesis. Like NPYq- males, these males were infertile, and their sperm had malformed heads that nevertheless fertilized eggs via ICSI. In vitro fertilization (IVF), however, was unsuccessful. Overall, we demonstrated that a lack of NPYq-encoded genes does not interfere with the ability of sperm to fertilize oocytes via ICSI but does prevent fertilization via IVF. Thus, NPYq-encoded gene functions are not required after the sperm have entered the oocyte. The present work also led to development of a new mouse model lacking NPYq gene complement that will facilitate future studies of Y-encoded gene function.

Rescue of Sly Expression Is Not Sufficient to Rescue Spermiogenic Phenotype of Mice with Deletions of Y Chromosome Long Arm.

Mice with deletions of the Y-specific (non-PAR) region of the mouse Y chromosome long arm (NPYq) have sperm defects and fertility problems that increase proportionally to deletion size. Mice with abrogated function of NPYq-encoded gene Sly (sh367 Sly-KD) display a phenotype similar to that of NPYq deletion mutants but less severe. The milder phenotype can be due to insufficient Sly knockdown, involvement of another NPYq gene, or both. To address this question and to further elucidate the role of Sly in the infertile phenotype of mice with NPYq deletions, we developed an anti-SLY antibody specifically recognizing SLY1 and SLY2 protein isoforms and used it to characterize SLY expression in NPYq- and Sly-deficient mice. We also carried out transgene rescue by adding Sly1/2 transgenes to mice with NPYq deletions. We demonstrated that SLY1/2 expression in mutant mice decreased proportionally to deletion size, with ~12% of SLY1/2 retained in shSLY sh367 testes. The addition of Sly1/2 transgenes to mice with NPYq deletions rescued SLY1/2 expression but did not ameliorate fertility and testicular/spermiogenic defects. Together, the data suggest that Sly deficiency is not the sole underlying cause of the infertile phenotype of mice with NPYq deletions and imply the involvement of another NPYq gene.

Two genes substitute for the mouse Y chromosome for spermatogenesis and reproduction.

The mammalian Y chromosome is considered a symbol of maleness, as it encodes a gene driving male sex determination, Sry, as well as a battery of other genes important for male reproduction. We previously demonstrated in the mouse that successful assisted reproduction can be achieved when the Y gene contribution is limited to only two genes, Sry and spermatogonial proliferation factor Eif2s3y. Here, we replaced Sry by transgenic activation of its downstream target Sox9, and Eif2s3y, by transgenic overexpression of its X chromosome-encoded homolog Eif2s3x. The resulting males with no Y chromosome genes produced haploid male gametes and sired offspring after assisted reproduction. Our findings support the existence of functional redundancy between the Y chromosome genes and their homologs encoded on other chromosomes.

Two Y genes can replace the entire Y chromosome for assisted reproduction in the mouse.

The Y chromosome is thought to be important for male reproduction. We have previously shown that, with the use of assisted reproduction, live offspring can be obtained from mice lacking the entire Y chromosome long arm. Here, we demonstrate that live mouse progeny can also be generated by using germ cells from males with the Y chromosome contribution limited to only two genes, the testis determinant factor Sry and the spermatogonial proliferation factor Eif2s3y. Sry is believed to function primarily in sex determination during fetal life. Eif2s3y may be the only Y chromosome gene required to drive mouse spermatogenesis, allowing formation of haploid germ cells that are functional in assisted reproduction. Our findings are relevant, but not directly translatable, to human male infertility cases.

Paternal DNA damage resulting from various sperm treatments persists after fertilization and is similar before and after DNA replication.

In spite of its highly condensed state, sperm DNA is vulnerable to damage that can originate from oxidative stress, the activity of sperm-specific nucleases, or both. After fertilization, in the oocyte, paternal chromatin undergoes dramatic changes, and during this extensive remodeling, it can be both repaired and degraded, and these processes can be linked to DNA synthesis. Here, we analyzed sperm response to damage-inducing treatments both before and after fertilization and before or after zygotic DNA replication. Epididymal mouse spermatozoa were either frozen without cryoprotection (FT) or treated with detergent Triton X-100 coupled with dithiothreitol (TX+DTT) to induce DNA damage. Fresh, untreated sperm served as control. Immediately after preparation, spermatozoa from 3 groups were taken for comet assay, or for intracytoplasmic sperm injection into prometaphase I oocytes to visualize prematurely condensed single-chromatid chromosomes, or into mature metaphase II oocytes to visualize chromosomes after DNA replication. Comet assay revealed increased DNA fragmentation in treated sperm when compared with control, with FT sperm more severely affected. Chromosome analysis demonstrated paternal DNA damage in oocytes injected with treated, but not with fresh, sperm, with FT and TX+DTT groups now yielding similar damage. There were no differences in the incidence of abnormal paternal karyoplates before and after DNA synthesis in all examined groups. This study provides evidence that subjecting sperm to DNA damage-inducing treatments results in degradation of highly condensed sperm chromatin when it is still packed within the sperm head, and that this DNA damage persists after fertilization. The difference in DNA damage in sperm subjected to 2 treatments was ameliorated in the fertilized oocytes, suggesting that some chromatin repair might have occurred. This process, however, was independent of DNA synthesis and took place during oocyte maturation.

Deficiency in mouse Y chromosome long arm gene complement is associated with sperm DNA damage.

BACKGROUND: Mice with severe non-PAR Y chromosome long arm (NPYq) deficiencies are infertile in vivo and in vitro. We have previously shown that sperm from these males, although having grossly malformed heads, were able to fertilize oocytes via intracytoplasmic sperm injection (ICSI) and yield live offspring. However, in continuing ICSI trials we noted a reduced efficiency when cryopreserved sperm were used and with epididymal sperm as compared to testicular sperm. In the present study we tested if NPYq deficiency is associated with sperm DNA damage - a known cause of poor ICSI success. RESULTS: We observed that epididymal sperm from mice with severe NPYq deficiency (that is, deletion of nine-tenths or the entire NPYq gene complement) are impaired in oocyte activation ability following ICSI and there is an increased incidence of oocyte arrest and paternal chromosome breaks. Comet assays revealed increased DNA damage in both epididymal and testicular sperm from these mice, with epididymal sperm more severely affected. In all mice the level of DNA damage was increased by freezing. Epididymal sperm from mice with severe NPYq deficiencies also suffered from impaired membrane integrity and abnormal chromatin condensation and suboptimal chromatin protamination. It is therefore likely that the increased DNA damage associated with NPYq deficiency is a consequence of disturbed chromatin remodeling. CONCLUSIONS: This study provides the first evidence of DNA damage in sperm from mice with NPYq deficiencies and indicates that NPYq-encoded gene/s may play a role in processes regulating chromatin remodeling and thus in maintaining DNA integrity in sperm.

Deficiency in the multicopy Sycp3-like X-linked genes Slx and Slxl1 causes major defects in spermatid differentiation.

The human and mouse sex chromosomes are enriched in multicopy genes required for postmeiotic differentiation of round spermatids into sperm. The gene Sly is present in multiple copies on the mouse Y chromosome and encodes a protein that is required for the epigenetic regulation of postmeiotic sex chromosome expression. The X chromosome carries two multicopy genes related to Sly: Slx and Slxl1. Here we investigate the role of Slx/Slxl1 using transgenically-delivered small interfering RNAs to disrupt their function. We show that Slx and Slxl1 are important for normal sperm differentiation and male fertility. Slx/Slxl1 deficiency leads to delay in spermatid elongation and sperm release. A high proportion of delayed spermatids are eliminated via apoptosis, with a consequent reduced sperm count. The remaining spermatozoa are abnormal with impaired motility and fertilizing abilities. Microarray analyses reveal that Slx/Slxl1 deficiency affects the metabolic processes occurring in the spermatid cytoplasm but does not lead to a global perturbation of sex chromosome expression; this is in contrast with the effect of Sly deficiency which leads to an up-regulation of X and Y chromosome genes. This difference may be due to the fact that SLX/SLXL1 are cytoplasmic while SLY is found in the nucleus and cytoplasm of spermatids.

Freezing-free preservation of human spermatozoa--a pilot study.

This study tested a method for maintaining human spermatozoa without freezing for subsequent use in intracytoplasmic sperm injection (ICSI). We demonstrated that human sperm stored in electrolyte-free solution maintain their motility and viability for at least 4 and 6 weeks, respectively. We also have shown that preserved spermatozoa are fully functional in ICSI. Sperm chromosome analysis after injection of human sperm into mouse oocytes revealed that two weeks of storage does not negatively affect sperm DNA integrity. A mouse model was used to analyze the ability of preserved sperm to participate in normal embryogenesis. Mouse sperm preserved in electrolyte-free solution in a similar manner as human sperm maintained motility for up to 3 weeks. When mouse spermatozoa stored for 1 week were injected into the oocytes by ICSI, they yielded normal blastoctysts and normal viable fetuses. The results of the study bear significance for human assisted reproduction technologies (ART) and provide clinicians and infertile patients with a new method that can simplify sperm preparation for ICSI, assisting men who are unable to provide semen on the day of assisted fertilization.

Ejaculated and epididymal mouse spermatozoa are different in their susceptibility to nuclease-dependent DNA damage and in their nuclease activity.

Ejaculated mouse sperm retrieved from the uteri are more susceptible to DNA damage during freeze-drying and freezing without cryoprotection than epididymal sperm. This prompted us to speculate that a factor present in the uterus after mating, either male or female derived, was responsible for increased susceptibility of ejaculated sperm to DNA damage during preservation and that the differences between epididymal and ejaculated mouse sperm in response to stress originated from varying nuclease activity. We first exposed epididymal sperm to the uterine content from females mated to vasectomized males (UCSP), to the uterine content from unmated females in estrus (UC), and to the seminal vesicle fluid (SVF) and examined sperm chromosomes after intracytoplasmic sperm injection (ICSI). We found an increased incidence of chromosome breaks and extremely severe DNA breakage after exposure to UCSP and SVF, respectively, but the chromosomes were normal in sperm exposed to UC. Comet assay results verified that DNA damage after exposure to SVF was present in sperm before fertilization. Next, we examined nuclease activity in sperm and their associated components with a plasmid digestion assay. Nuclease activity was detected in isolated epididymal and ejaculated sperm, as well as in epididymal fluid and seminal plasma, and was much more pronounced in all samples originating from ejaculate. The combined results from the present study imply that there are intrinsic differences between the epididymal and ejaculated mouse sperm preparations in their susceptibility to nuclease-dependent DNA damage that originates from their nuclease activity. This nuclease activity was detected both in the sperm-free fraction of preparations and isolated sperm.