A comprehensive analysis of spermatozoal RNA elements in idiopathic infertile males undergoing fertility treatment.

Current approaches to diagnosing male infertility inadequately assess the complexity of the male gamete. Beyond the paternal haploid genome, spermatozoa also deliver coding and non-coding RNAs to the oocyte. While sperm-borne RNAs have demonstrated potential involvement in embryo development, the underlying mechanisms remain unclear. In this study, 47 sperm samples from normozoospermic males undergoing fertility treatment using donor oocytes were sequenced and analyzed to evaluate associations between sperm RNA elements (exon-sized sequences) and blastocyst progression. A total of 366 RNA elements (REs) were significantly associated with blastocyst rate (padj < 0.05), some of which were linked to genes related to critical developmental processes, including mitotic spindle formation and both ectoderm and mesoderm specification. Of note, 27 RE-associated RNAs are predicted targets of our previously reported list of developmentally significant miRNAs. Inverse RE-miRNA expression patterns were consistent with miRNA-mediated down-regulation. This study provides a comprehensive set of REs which differ by the patient's ability to produce blastocysts. This knowledge can be leveraged to improve clinical screening of male infertility and ultimately reduce time to pregnancy.

Germ Cell-Specific Proteins AKAP4 and ASPX Facilitate Identification of Rare Spermatozoa in Non-Obstructive Azoospermia.

Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intracytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis-specific and germ cell-specific proteins would facilitate flow cytometry-assisted identification of rare spermatozoa in semen cell pellets of NOA patients, thus enabling non-invasive diagnostics prior to mTESE. Data mining, targeted proteomics, and immunofluorescent microscopy identified and verified a panel of highly testis-specific proteins expressed at the continuum of germ cell differentiation. Late germ cell-specific proteins AKAP4_HUMAN and ASPX_HUMAN (ACRV1 gene) revealed exclusive localization in spermatozoa tails and acrosomes, respectively. A multiplex imaging flow cytometry assay facilitated fast and unambiguous identification of rare but morphologically intact AKAP4+/ASPX+/Hoechst+ spermatozoa within debris-laden semen pellets of NOA patients. While the previously suggested markers for spermatozoa retrieval suffered from low diagnostic specificity, the multistep gating strategy and visualization of AKAP4+/ASPX+/Hoechst+ cells with elongated tails and acrosome-capped nuclei facilitated fast and unambiguous identification of the mature intact spermatozoa. AKAP4+/ASPX+/Hoechst+ assay may emerge as a noninvasive test to predict retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of severe forms of male infertility.

Assessing spermatozoal small ribonucleic acids and their relationship to blastocyst development in idiopathic infertile males.

Clinical testing strategies for diagnosing male factor infertility are limited. A deeper analysis of spermatozoa-derived factors could potentially diagnose some cases of 'unexplained infertility'. Spermatozoa carry a rich and dynamic profile of small RNAs, which have demonstrated potential developmental importance and association with fertility status. We used next-generation sequencing to correlate sperm small RNA profiles of normozoospermic males (n = 54) with differing blastocyst development rates, when using young donor oocytes. While ribosomal RNAs accounted for the highest number of sequencing reads, transfer RNA fragments of tRNAGly/GCC and tRNAVal-CAC were the most abundant sequences across all sperm samples. A total of 324 small RNAs were differentially expressed between samples with high (n = 18) and low (n = 14) blastocyst rates (p-adj < 0.05). Ninety three miRNAs were differentially expressed between these groups (p-adj < 0.05). Differentially expressed transfer RNA fragments included: 5'-tRF-Asp-GTC; 5'-tRF-Phe-GAA; and 3'-tRF-Ser-GCA. Differentially expressed miRNAs included: let-7f-2-5p; miR-4755-3p; and miR-92a-3p. This study provides the foundation on which to validate a clinical panel of fertility-related sperm small RNAs, as well as to pursue potential mechanisms through which they alter blastocyst development.

Staining-free, Automated Sperm Analysis for In Vitro Fertilization Lab Use.

PURPOSE: Computer-aided sperm analysis is typically used in andrology labs, not in in vitro fertilization labs, which requires staining for sperm morphology measurement. In in vitro fertilization labs, sperm analysis still relies on manual observation and suffers from subjectivity and inconsistency. We developed a system for automated measurement of sperm concentration, motility, and morphology without the need for sperm staining. The reproducibility and reliability of the system were evaluated. MATERIALS AND METHODS: Thirty-five fresh semen and 25 washed samples were obtained from male partners attending for fertility investigations. Sperm concentration, motility, and morphology were automatically measured simultaneously, leveraging robust sperm tracking for concentration and motility measurement and low contrast image segmentation for morphology measurement of live sperm. Reproducibility of sperm measurements was evaluated by intraclass correlation coefficients. Reliability of sperm measurement was evaluated by Passing and Bablok regression analysis and Bland-Altman analysis. RESULTS: Automated measurement of concentration, motility, and morphology had intraclass correlation coefficients higher than 0.97. The regression and Bland-Altman analysis indicated that automated measurement and off-line manual benchmarking with zoomed-in images were interchangeable. Further analysis on semen and washed samples and the measurement on progressive and nonprogressive motility also showed high reproducibility and reliability. CONCLUSIONS: Automated sperm analysis revealed high reproducibility and reliability. The system is designed for routine use in in vitro fertilization labs to perform quantitative sperm analysis on live samples.

Whole-genome sequencing of H3K4me3 and DNA methylation in human sperm reveals regions of overlap linked to fertility and development.

The paternal environment has been linked to infertility and negative outcomes. Such effects may be transmitted via sperm through histone modifications. To date, in-depth profiling of the sperm chromatin in men has been limited. Here, we use deep sequencing to characterize the sperm profiles of histone H3 lysine 4 tri-methylation (H3K4me3) and DNA methylation in a representative reference population of 37 men. Our analysis reveals that H3K4me3 is localized throughout the genome and at genes for fertility and development. Remarkably, enrichment is also found at regions that escape epigenetic reprogramming in primordial germ cells, embryonic enhancers, and short-interspersed nuclear elements (SINEs). There is significant overlap in H3K4me3 and DNA methylation throughout the genome, suggesting a potential interplay between these marks previously reported to be mutually exclusive in sperm. Comparisons made between H3K4me3 marked regions in sperm and the embryonic transcriptome suggest an influence of paternal chromatin on embryonic gene expression.

Advances in sperm analysis: techniques, discoveries and applications.

Infertility affects one in six couples worldwide, and fertility continues to deteriorate globally, partly owing to a decline in semen quality. Sperm analysis has a central role in diagnosing and treating male factor infertility. Many emerging techniques, such as digital holography, super-resolution microscopy and next-generation sequencing, have been developed that enable improved analysis of sperm motility, morphology and genetics to help overcome limitations in accuracy and consistency, and improve sperm selection for infertility treatment. These techniques have also improved our understanding of fundamental sperm physiology by enabling discoveries in sperm behaviour and molecular structures. Further progress in sperm analysis and integrating these techniques into laboratories and clinics requires multidisciplinary collaboration, which will increase discovery and improve clinical outcomes.

Is there an association between paternal age and aneuploidy? Evidence from young donor oocyte-derived embryos: a systematic review and individual patient data meta-analysis.

BACKGROUND: Delayed parenthood, by both women and men, has become more common in developed countries. The adverse effect of advanced maternal age on embryo aneuploidy and reproductive outcomes is well known. However, whether there is an association between paternal age (PA) and embryonic chromosomal aberrations remains controversial. Oocyte donation (OD) is often utilized to minimize maternal age effects on oocyte and embryo aneuploidy, thus providing an optimal model to assess the effect of PA. Several studies have revealed a higher than expected rate of aneuploidy in embryos derived from young oocyte donors, which warrants examination as to whether this may be attributed to advanced PA (APA). OBJECTIVE AND RATIONALE: The objective of this systematic review and individual patient data (IPD) meta-analysis is to evaluate existing evidence regarding an association between PA and chromosomal aberrations in an OD model. SEARCH METHODS: This review was conducted according to PRISMA guidelines for systematic reviews and meta-analyses. Medline, Embase and Cochrane databases were searched from inception through March 2020 using the (MeSH) terms: chromosome aberrations, preimplantation genetic screening and IVF. Original research articles, reporting on the types and/or frequency of chromosomal aberrations in embryos derived from donor oocytes, including data regarding PA, were included. Studies reporting results of IVF cycles using only autologous oocytes were excluded. Quality appraisal of included studies was conducted independently by two reviewers using a modified Newcastle-Ottawa Assessment Scale. A one-stage IPD meta-analysis was performed to evaluate whether an association exists between PA and aneuploidy. Meta-analysis was performed using a generalized linear mixed model to account for clustering of embryos within patients and clustering of patients within studies. OUTCOMES: The search identified 13 032 references, independently screened by 2 reviewers, yielding 6 studies encompassing a total of 2637 IVF-OD cycles (n = 20 024 embryos). Two 'low' quality studies using FISH to screen 12 chromosomes on Day 3 embryos (n = 649) reported higher total aneuploidy rates and specifically higher rates of trisomy 21, 18 and 13 in men ≥50 years. One 'moderate' and three 'high' quality studies, which used 24-chromosome screening, found no association between PA and aneuploidy in Day 5/6 embryos (n = 12 559). The IPD meta-analysis, which included three 'high' quality studies (n = 10 830 Day 5/6 embryos), found no significant effect of PA on the rate of aneuploidy (odds ratio (OR) 0.97 per decade of age, 95% CI 0.91-1.03), which was robust to sensitivity analyses. There was no association between PA and individual chromosome aneuploidy or segmental aberrations, including for chromosomes X and Y (OR 1.06 per decade of age, 95% CI 0.92-1.21). Monosomy was most frequent for chromosome 16 (217/10802, 2.01%, 95% CI 1.76-2.29%) and trisomy was also most frequent for chromosome 16 (194/10802, 1.80%, 95% CI 1.56-2.06%). WIDER IMPLICATIONS: We conclude, based on the available evidence, that APA is not associated with higher rates of aneuploidy in embryos derived from OD. These results will help fertility practitioners when providing preconception counselling, particularly to older men who desire to have a child.

Is there a correlation between paternal age and aneuploidy rate? An analysis of 3,118 embryos derived from young egg donors.

OBJECTIVE: To investigate a possible correlation between chromosomal aberrations and paternal age, analyzing embryos derived from young oocyte donors, with available preimplantation genetic testing for aneuploidy results from day 5/6 trophectoderm biopsy obtained by next-generation sequencing for all 24 chromosomes. DESIGN: Retrospective cohort study. SETTING: Canadian fertility centre. PATIENT(S): A total of 3,118 embryos from 407 male patients, allocated into three paternal age groups: group A, ≤39 years (n = 203); group B, 40-49 years (n = 161); group C, ≥50 years (n = 43). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The primary outcomes were aneuploidy, euploidy, mosaicism, and blastocyst formation rates. Secondary endpoints were comparison of specific chromosome aneuploidy, segmental and complex (involving two chromosomes + mosaicism >50%) aneuploidy, and analysis of overall percentage of chromosomal gains and losses within each group. RESULT(S): The study included 437 in vitro fertilization (IVF) antagonist cycles using 302 oocyte donors in which preimplantation genetic testing for aneuploidy was performed. Overall, 70.04% of embryos were euploid, 13.9% were aneuploid, and 16.06% were mosaic. No significant differences among paternal age groups A, B, and C were found in euploidy rates (69.2%, 70.6%, 71.4%, respectively), aneuploidy rates (14.7%, 12.8%, 13.9%, respectively) or mosaicism rates (16.1%, 16.6%, 13.6%; respectively). The fertilization rate was lower in group C compared with group B (76.35% vs. 80.09%). No difference was found in blastocyst formation rate between the study groups (median 52% [interquartile range, 41%, 67%] vs. 53% [42%, 65%] vs. 52% [42%, 64%], respectively). A generalized linear mixed model regression analysis for embryo ploidy rates found older oocyte donor age to be independently associated with embryo aneuploidy (odds ratio = 1.041; 95% CI, 1.009-1.074). The rate of segmental aneuploidies was significantly higher in the older versus younger paternal age group (36.6% vs. 19.4%). CONCLUSION(S): No association was found between paternal age and aneuploidy rates in embryos derived from IVF cycles using young oocyte donors, after adjusting for donor, sperm, and IVF cycle characteristics. Advanced paternal age ≥ 50, compared with younger paternal ages, was associated with a lower fertilization rate and increased rate of segmental aberrations.

What human sperm RNA-Seq tells us about the microbiome.

PURPOSE: The study was designed to assess the capacity of human sperm RNA-seq data to gauge the diversity of the associated microbiome within the ejaculate. METHODS: Semen samples were collected, and semen parameters evaluated at time of collection. Sperm RNA was isolated and subjected to RNA-seq. Microbial composition was determined by aligning sequencing reads not mapped to the human genome to the NCBI RefSeq bacterial, viral and archaeal genomes following RNA-Seq. Analysis of microbial assignments utilized phyloseq and vegan. RESULTS: Microbial composition within each sample was characterized as a function of microbial associated RNAs. Bacteria known to be associated with the male reproductive tract were present at similar levels in all samples representing 11 genera from four phyla with one exception, an outlier. Shannon diversity index (p < 0.001) and beta diversity (unweighted UniFrac distances, p = 9.99e-4; beta dispersion, p = 0.006) indicated the outlier was significantly different from all other samples. The outlier sample exhibited a dramatic increase in Streptococcus. Multiple testing indicated two operational taxonomic units, S. agalactiae and S. dysgalactiae (p = 0.009), were present. CONCLUSION: These results provide a first look at the microbiome as a component of human sperm RNA sequencing that has sufficient sensitivity to identify contamination or potential pathogenic bacterial colonization at least among the known contributors.

Customized MethylC-Capture Sequencing to Evaluate Variation in the Human Sperm DNA Methylome Representative of Altered Folate Metabolism.

BACKGROUND: The sperm DNA methylation landscape is unique and critical for offspring health. If gamete-derived DNA methylation escapes reprograming in early embryos, epigenetic defects in sperm may be transmitted to the next generation. Current techniques to assess sperm DNA methylation show bias toward CpG-dense regions and do not target areas of dynamic methylation, those predicted to be environmentally sensitive and tunable regulatory elements. OBJECTIVES: Our goal was to assess variation in human sperm DNA methylation and design a targeted capture panel to interrogate the human sperm methylome. METHODS: To characterize variation in sperm DNA methylation, we performed whole genome bisulfite sequencing (WGBS) on an equimolar pool of sperm DNA from a wide cross section of 30 men varying in age, fertility status, methylenetetrahydrofolate reductase (MTHFR) genotype, and exposures. With our targeted capture panel, in individual samples, we examined the effect of MTHFR genotype ([Formula: see text] 677CC, [Formula: see text] 677TT), as well as high-dose folic acid supplementation ([Formula: see text], per genotype, before and after supplementation). RESULTS: Through WGBS we discovered nearly 1 million CpGs possessing intermediate methylation levels (20-80%), termed dynamic sperm CpGs. These dynamic CpGs, along with 2 million commonly assessed CpGs, were used to customize a capture panel for targeted interrogation of the human sperm methylome and test its ability to detect effects of altered folate metabolism. As compared with MTHFR 677CC men, those with the 677TT genotype (50% decreased MTHFR activity) had both hyper- and hypomethylation in their sperm. High-dose folic acid supplement treatment exacerbated hypomethylation in MTHFR 677TT men compared with 677CC. In both cases, [Formula: see text] of altered methylation was found in dynamic sperm CpGs, uniquely measured by our assay. DISCUSSION: Our sperm panel allowed the discovery of differential methylation following conditions affecting folate metabolism in novel dynamic sperm CpGs. Improved ability to examine variation in sperm DNA methylation can facilitate comprehensive studies of environment-epigenome interactions. https://doi.org/10.1289/EHP4812.

Male Oxidative Stress Infertility (MOSI): Proposed Terminology and Clinical Practice Guidelines for Management of Idiopathic Male Infertility.

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm's potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause.

Robotic Immobilization of Motile Sperm for Clinical Intracytoplasmic Sperm Injection.

OBJECTIVE: In clinical intracytoplasmic sperm injection (ICSI), a motile sperm must be immobilized before insertion into an oocyte. This paper aims to develop a robotic system for automated tracking, orientation control, and immobilization of motile sperms for clinical ICSI applications. METHODS: We adapt the probabilistic data association filter by adding sperm head orientation into state variables for robustly tracking the sperm head and estimating sperm tail positions under interfering conditions. The robotic system also utilizes a motorized rotational microscopy stage and a new visual servo control strategy that predicts and compensates for sperm movements to actively adjust sperm orientation for immobilizing a sperm swimming in any direction. RESULTS: The system robustly tracked sperm head with a tracking success rate of 96.0% and estimated sperm tail position with an accuracy of 1.08 µm under clinical conditions where the occlusion of the target sperm and interference from other sperms occur. Experimental results from robotic immobilization of 400 sperms confirmed that the system achieved a consistent immobilization success rate of 94.5%, independent of sperm velocity or swimming direction. CONCLUSION: Our adapted tracking algorithm effectively distinguishes the target sperm from interfering sperms. Predicting and compensating for sperm movements significantly reduce the positioning error during sperm orientation control. These features make the robotic system suitable for automated sperm immobilization. SIGNIFICANCE: The robotic system eliminates stringent skill requirements in manual sperm immobilization. It is capable of manipulating sperms swimming in an arbitrary direction with a high success rate.

Automated Non-Invasive Measurement of Single Sperm's Motility and Morphology.

Measuring cell motility and morphology is important for revealing their functional characteristics. This paper presents automation techniques that enable automated, non-invasive measurement of motility and morphology parameters of single sperm. Compared to the status quo of qualitative estimation of single sperm's motility and morphology manually, the automation techniques provide quantitative data for embryologists to select a single sperm for intracytoplasmic sperm injection. An adapted joint probabilistic data association filter was used for multi-sperm tracking and tackled challenges of identifying sperms that intersect or have small spatial distances. Since the standard differential interference contrast (DIC) imaging method has side illumination effect which causes inherent inhomogeneous image intensity and poses difficulties for accurate sperm morphology measurement, we integrated total variation norm into the quadratic cost function method, which together effectively removed inhomogeneous image intensity and retained sperm's subcellular structures after DIC image reconstruction. In order to relocate the same sperm of interest identified under low magnification after switching to high magnification, coordinate transformation was conducted to handle the changes in the field of view caused by magnification switch. The sperm's position after magnification switch was accurately predicted by accounting for the sperm's swimming motion during magnification switch. Experimental results demonstrated an accuracy of 95.6% in sperm motility measurement and an error <10% in morphology measurement.

In vitro generation of Sertoli-like and haploid spermatid-like cells from human umbilical cord perivascular cells.

BACKGROUND: First trimester (FTM) and term human umbilical cord-derived perivascular cells (HUCPVCs), which are rich sources of mesenchymal stem cells (MSCs), can give rise to Sertoli cell (SC)-like as well as haploid germ cell (GC)-like cells in vitro using culture conditions that recapitulate the testicular niche. Gamete-like cells have been produced ex vivo using pluripotent stem cells as well as MSCs. However, the production of functional gametes from human stem cells has yet to be achieved. METHODS: Three independent lines of FTM and term HUCPVCs were cultured using a novel 5-week step-wise in vitro differentiation protocol recapitulating key physiological signals involved in testicular development. SC- and GC-associated phenotypical properties were assessed by real-time polymerase chain reaction (RT-PCR), quantitative PCR immunocytochemistry, flow cytometry, and fluorescence in-situ hybridization (FISH). Functional spermatogonial stem cell-like properties were assessed using a xenotranplantation assay. RESULTS: Within 3 weeks of differentiation, two morphologically distinct cell types emerged including large adherent cells and semi-attached round cells. Both early GC-associated markers (VASA, DAZL, GPR125, GFR1α) and SC-associated markers (FSHR, SOX9, AMH) were upregulated, and 5.7 ± 1.2% of these cells engrafted near the inner basal membrane in a xenograft assay. After 5 weeks in culture, 10-30% of the cells were haploid, had adopted a spermatid-like morphology, and expressed PRM1, Acrosin, and ODF2. Undifferentiated HUCPVCs secreted key factors known to regulate spermatogenesis (LIF, GDNF, BMP4, bFGF) and 10-20% of HUCPVCs co-expressed SSEA4, CD9, CD90, and CD49f. We hypothesize that the paracrine properties and cellular heterogeneity of HUCPVCs may explain their dual capacity to differentiate to both SC- and GC-like cells. CONCLUSIONS: HUCPVCs recapitulate elements of the testicular niche including their ability to differentiate into cells with Sertoli-like and haploid spermatid-like properties in vitro. Our study supports the importance of generating a niche-like environment under ex vivo conditions aiming at creating mature GC, and highlights the plasticity of HUCPVCs. This could have future applications for the treatment of some cases of male infertility.

Response to Comment on "Absence of sperm RNA elements correlates with idiopathic male infertility".

RNAs from other cell types have minimal impact on male fecundity-associated sperm RNA elements.

Absence of sperm RNA elements correlates with idiopathic male infertility.

Semen parameters are typically used to diagnose male infertility and specify clinical interventions. In idiopathic infertile couples, an unknown male factor could be the cause of infertility even when the semen parameters are normal. Next-generation sequencing of spermatozoal RNAs can provide an objective measure of the paternal contribution and may help guide the care of these couples. We assessed spermatozoal RNAs from 96 couples presenting with idiopathic infertility and identified the final reproductive outcome and sperm RNA elements (SREs) reflective of fecundity status. The absence of required SREs reduced the probability of achieving live birth by timed intercourse or intrauterine insemination from 73 to 27%. However, the absence of these same SREs does not appear to be critical when using assisted reproductive technologies such as in vitro fertilization with or without intracytoplasmic sperm injection. About 30% of the idiopathic infertile couples presented an incomplete set of required SREs, suggesting a male component as the cause of their infertility. Conversely, analysis of couples that failed to achieve a live birth despite presenting with a complete set of SREs suggested that a female factor may have been involved, and this was confirmed by their diagnosis. The data in this study suggest that SRE analysis has the potential to predict the individual success rate of different fertility treatments and reduce the time to achieve live birth.

Chromatin and extracellular vesicle associated sperm RNAs.

A diverse pool of RNAs remain encapsulated within the transcriptionally silent spermatozoon despite the dramatic reduction in cellular and nuclear volume following cytoplasm/nucleoplasm expulsion. The impact of this pronounced restructuring on the distribution of transcripts inside the sperm essentially remains unknown. To define their compartmentalization, total RNA >100 nt was extracted from sonicated (SS) mouse spermatozoa and detergent demembranated sucrose gradient fractionated (Cs/Tx) sperm heads. Sperm RNAs predominately localized toward the periphery. The corresponding distribution of transcripts and thus localization and complexity were then inferred by RNA-seq. Interestingly, the number of annotated RNAs in the CsTx sperm heads exhibiting reduced peripheral enrichment was restricted. However this included Cabyr, the calcium-binding tyrosine phosphorylation-regulated protein encoded transcript. It is present in murine zygotes prior to the maternal to the zygotic transition yet absent in oocytes, consistent with the delivery of internally positioned sperm-borne RNAs to the embryo. In comparison, transcripts enriched in sonicated sperm contributed to the mitochondria and exosomes along with several nuclear transcripts including the metastasis associated lung adenocarcinoma transcript 1 (Malat1) and several small nucleolar RNAs. Their preferential peripheral localization suggests that chromatin remodeling during spermiogenesis is not limited to nucleoproteins as part of the nucleoprotein exchange.

Vitrification of a small number of spermatozoa in normozoospermic and severely oligozoospermic samples.

Despite broad utilization of sperm cryopreservation, little progress has been made to modify freezing protocols or to improve rates of sperm survival. Vitrification is an alternative method for freezing human spermatozoa without toxic permeable cryoprotectants (CPAs). The purpose of our study was to optimize the vitrification and post thaw recovery of a small number of spermatozoa using only nonpermeating CPAs in a closed straw system in normozoospermic and severely oligozoospermic samples. Individual motile spermatozoa (n = 295) were selected from semen samples of 15 normozoospermic and 10 severe oligozoospermia patients. Overall sperm recovery after vitrification was 80% (n = 236) with 80% (n = 189) viability and 41.5% (n = 98) retained post-warming motility. Two different loading techniques were compared to transfer selected spermatozoa into straws in preparation for vitrification: by spontaneous capillary action (CA) and with the aid of a polar body biopsy (PBB) pipette. There was evidence that the PBB loading technique increases the odds of spermatozoa recovery in both subsets (p = 0.01 and p = 0.04) in the normal and abnormal subsamples, respectively.