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.

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.

Assisted reproduction involving gestational surrogacy: an analysis of the medical, psychosocial and legal issues: experience from a large surrogacy program.

STUDY QUESTION: What are the medical, psychosocial and legal aspects of gestational surrogacy (GS), including pregnancy outcomes and complications, in a large series? SUMMARY ANSWER: Meticulous multidisciplinary teamwork, involving medical, legal and psychosocial input for both the intended parent(s) (IP) and the gestational carrier (GC), is critical to achieve a successful GS program. WHAT IS KNOWN ALREADY: Small case series have described pregnancy rates of 17-50% for GS. There are no large case series and the medical, legal and psychological aspects of GS have not been addressed in most of these studies. To our knowledge, this is the largest reported GS case series. STUDY DESIGN, SIZE AND DURATION: A retrospective cohort study was performed. Data were collected from 333 consecutive GC cycles between 1998 and 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: There were 178 pregnancies achieved out of 333 stimulation cycles, including fresh and frozen transfers. The indications for a GC were divided into two groups. Those who have 'failed to carry', included women with recurrent implantation failure (RIF), recurrent pregnancy loss (RPL) and previous poor pregnancy outcome (n = 96; 132 cycles, pregnancy rate 50.0%). The second group consisted of those who 'cannot carry' including those with severe Asherman's syndrome, uterine malformations/uterine agenesis and maternal medical diseases (n = 108, 139 cycles, pregnancy rate 54.0%). A third group, of same-sex male couples and single men, were analyzed separately (n = 52, 62 cycles, pregnancy rate 59.7%). In 49.2% of cycles, autologous oocytes were used and 50.8% of cycles involved donor oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: The 'failed to carry' group consisted of 96 patients who underwent 132 cycles at a mean age of 40.3 years. There were 66 pregnancies (50.0%) with 17 miscarriages (25.8%) and 46 confirmed births (34.8%). The 'cannot carry pregnancy' group consisted of 108 patients who underwent 139 cycles at a mean age of 35.9 years. There were 75 pregnancies (54.0%) with 15 miscarriages (20.0%) and 56 confirmed births (40.3%). The pregnancy, miscarriage and live birth rates between the two groups were not significantly different (P = 0.54; 0.43; 0.38, respectively). Of the 178 pregnancies, 142 pregnancies were ongoing (surpassed 20 weeks) or had ended with a live birth and the other 36 pregnancies resulted in miscarriage (25.4%). Maternal (GS) complication rates were low, occurring in only 9.8% of pregnancies. Fetal anomalies occurred in only 1.8% of the babies born. LIMITATIONS, REASONS FOR CAUTION: Although it is a large series, the data are retrospective and conclusions must be drawn accordingly while considering bias, confounding and power. Due to the retrospective nature of this study, follow-up data on 6.3% of birth outcomes were incomplete. In addition, long-term follow-up data on GCs and IPs were not available to us at the time of publication. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the largest GS series published. We have included many details regarding not only the medical protocol but also the counseling and legal considerations, which are an inseparable part of the process. Data from this study can be included in discussions with future intended parents and gestational carriers regarding success rates and complications of GS.

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.

Sperm content of postacrosomal WW binding protein is related to fertilization outcomes in patients undergoing assisted reproductive technology.

OBJECTIVE: To determine the levels of postacrosomal WW binding protein (PAWP) in the spermatozoa of men that were used clinically for intracytoplasmic sperm injection (ICSI) and to correlate them with infertility treatment outcomes. DESIGN: Prospective clinical and laboratory study. SETTING: University-based laboratory and infertility clinic. PATIENT(S): Men undergoing ICSI for the treatment of couples' infertility (n=110). INTERVENTION(S): Quantitative analysis of sperm PAWP levels by flow cytometry and developmental analysis of PAWP expression by immunoblotting, immunofluorescence, and immunohistochemistry. MAIN OUTCOME MEASURE(S): PAWP flow-cytometric levels and immunolocalization in spermatozoa. RESULT(S): A strong positive correlation was found between PAWP expression levels and fertilization rates after ICSI, with high levels of PAWP being associated with higher fertilization rates; the positive correlation was independent of age, DNA fragmentation index, and other sperm parameters. PAWP expression levels were correlated with embryonic development, with high levels of PAWP being associated with a lower number of arrested embryos within 3-5 days post-ICSI. PAWP expression was detected during the late stages of human spermiogenesis in elongating spermatids, confirming previous findings in various animal models. CONCLUSION(S): Our clinical data from infertile couples demonstrate significant correlations between sperm PAWP levels and both fertilization rates and normal embryonic development after ICSI. Considering its proposed role in the initiation of oocyte activation, we suggest that PAWP could have potential applications in the diagnosis and treatment of infertility.

Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice.

Mammalian zygotic development is initiated by sperm-mediated intracellular calcium oscillations, followed by activation of metaphase II-arrested oocytes. Sperm postacrosomal WW binding protein (PAWP) fulfils the criteria set for an oocyte-activating factor by inducing oocyte activation and being stored in the perinuclear theca, the sperm compartment whose content is first released into oocyte cytoplasm during fertilization. However, proof that PAWP initiates mammalian zygotic development relies on demonstration that it acts upstream of oocyte calcium oscillations. Here, we show that PAWP triggers calcium oscillations and pronuclear formation in human and mouse oocytes similar to what is observed during intracytoplasmic sperm injection (ICSI). Most important, sperm-induced calcium oscillations are blocked by coinjection of a competitive inhibitor, derived from the WWI domain-binding motif of PAWP, implying the requirement of sperm PAWP and an oocyte-derived WWI domain protein substrate of PAWP for successful fertilization. Sperm-delivered PAWP is, therefore, a unique protein with a nonredundant role during human and mouse fertilization, required to trigger zygotic development. Presented data confirm our previous findings in nonmammalian models and suggest potential applications of PAWP in the diagnosis and treatment of infertility.-

Assisted reproduction in a cohort of same-sex male couples and single men.

To date, there is limited published data on same-sex male couples and single men using assisted reproduction treatment to build their families. The objective of this retrospective study was to better understand treatment considerations and outcomes for this population when using assisted reproduction treatment. A total of 37 same-sex male couples and eight single men (seven homosexual and one heterosexual) who attended the CReATe Fertility Centre for assisted reproduction services were studied. There was a 21-fold increase in the number of same-sex male couples and single men undergoing assisted reproduction treatment since 2003. The mean age was 46years (24-58). Twenty-eight couples (76%) chose to use spermatozoa from both partners to fertilize their donated oocytes. Most men (32 same-sex male couples and seven single men; 87%) obtained oocytes from an anonymous donor, whereas five couples and one single man (13%) had a known donor. Anonymous donors who were open to be contacted by the child after the age of 18 were selected by 67% of patients. Of all 25 deliveries, eight (32%) were sets of twins. All of the twins were half genetic siblings.

Evaluation of potential protein biomarkers in patients with high sperm DNA damage.

The laboratory evaluation of male infertility remains an essential area of research as 40-60% of infertility cases are attributable to male-related factors. Current sperm analysis methods add only partial information on sperm quality and fertility outcomes. The specific underlying cause of infertility in most cases is unknown, while a proportion of male infertility could be caused by molecular factors such as the absence or abnormal expression of some essential sperm proteins. The objective of this study was to screen for associations between sperm protein profiles and sperm concentration, motility, and DNA fragmentation index in patients undergoing fertility evaluation in a clinical setting. Based on those parameters, semen samples were categorized as either normal or abnormal. We screened 34 semen samples with various abnormal parameters and compared them to 24 normal control samples by using one dimensional (1-D) gel electrophoresis and mass-spectrometry. In this study, we anticipated to establish a normal sperm parameter profile which would be compared to abnormal sperm samples and reveal candidate proteins. Our preliminary results indicate that no normal uniform profile could be established, which affirms the complexity of male fertility and confirms the limitations of standard semen analysis. Four main protein groups were identified in correlation with abnormal DNA fragmentation and/or motility. The first group included sperm nuclear proteins such as the SPANX (sperm protein associated with the nucleus on the X chromosome) isoforms and several types of histones. The second group contained mitochondria-related functions and oxidative stress proteins including Mitochondrial Ferritin, Mitochondrial Single-Stranded DNA Binding Protein, and several isoforms of Peroxiredoxins. Two other protein groups were related to sperm motility such as microtubule-based flagellum and spindle microtubule as well as proteins related to the ubiquitin-proteasome pathway. Further research is required in order to characterize these potential biomarkers of male fertility potential.

In vitro fertilization-intracytoplasmic sperm injection outcome in patients with a markedly high DNA fragmentation index (>50%).

OBJECTIVE: To investigate differences in fertilization, clinical pregnancy, and miscarriage rates between men with a markedly high sperm DNA fragmentation index (DFI) (>50%) and those with low DFI (≤ 15%) in couples matched by female partner age and ovarian reserve as determined by antimüllerian hormone (AMH) level. DESIGN: Retrospective cohort study. SETTING: University-affiliated fertility center. PATIENT(S): Men undergoing intracytoplasmic sperm injection (ICSI) cycles who had low (n = 114) or markedly high (n = 36) DNA damage. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm DNA damage evaluated by acridine orange flow cytometry and expressed as the DFI, with the potential confounders of ovarian reserve and age controlled for by multivariable logistic regression analysis. RESULT(S): The fertilization and clinical pregnancy rates were not different between the two groups. We observed a trend toward a higher miscarriage rate with the high DFI group, but it did not reach statistical significance. CONCLUSION(S): Intracytoplasmic sperm injection in men with a high DFI with sperm selected by movement and morphology characteristics resulted in a similar pregnancy rate compared with the controls with a normal DFI. However, the trend observed of an increase in miscarriages suggests that any potential negative impact may appear later in development. Future studies involving a larger cohort may determine if the miscarriage trend reaches statistical significance.

The three-dimensional image analysis of the chromocenter in motile and immotile human sperm.

Chromosomes in human spermatozoa are arranged non-randomly with the centromeres of non-homologous chromosomes forming a chromocenter. We have compared motile and immotile sperm populations in normozoospermic patients to determine if there is any dissimilarity in the formation of the chromocenter and the nuclear position of chromosome 17. Based on the differences between motile and immotile populations, we propose for the 'optimal' nuclear organization to be defined as containing 1 to 3 chromocenter(s) with central radial and median longitudinal position for the centromere of chromosome 17. By this definition, 42% of motile spermatozoa had 'optima' nuclei, in comparison to 25% of immotile spermatozoa (P < 0.05). Immotile spermatozoa exhibited a greater disruption in the formation of the chromocenter, altered position of the centromere of chromosome 17, and were more prone to chemical decondensation, resulting in higher nuclear and chromocenter volumes. The altered topology of the chromosomes might lead to the disruption of the sequence of events involved in fertilization and early embryonic development.

Methods of sperm vitality assessment.

Sperm vitality is a reflection of the proportion of live, membrane-intact spermatozoa determined by either dye exclusion or osmoregulatory capacity under hypo-osmotic conditions. In this chapter we address the two most common methods of sperm vitality assessment: eosin-nigrosin staining and the hypo-osmotic swelling test, both utilized in clinical Andrology laboratories.

A comparison of ejaculated and testicular spermatozoa aneuploidy rates in patients with high sperm DNA damage.

Testicular spermatozoa are utilized to achieve pregnancy in couples with severe male factor infertility. Several studies suggest that aneuploidy rates in spermatozoa are elevated at the testicular level in infertile patients compared to ejaculates of normal controls. However, essential data regarding aneuploidy rates between ejaculated and testicular spermatozoa in the same individuals is lacking. The purpose of our study was to compare aneuploidy rates at the testicular and post-testicular level from the same patients with persistently high sperm DNA damage. Ejaculates and testicular biopsies were obtained from eight patients with persistently high DNA damage (>30%). Both ejaculated and testicular samples were analyzed for sperm DNA damage and sperm aneuploidy for chromosomes 13, 18, 21, X, and Y. In addition, semen samples from ten normozoospermic men presenting for fertility evaluation served as a control group. A strong correlation between the alteration of spermatogenesis and chromatin deterioration was observed in our study. In the same individuals, testicular samples showed a significantly lower DNA damage compared to ejaculated spermatozoa (14.9% ± 5.0 vs. 40.6% ± 14.8, P<0.05), but significantly higher aneuploidy rates for the five analyzed chromosomes (12.41% ± 3.7 vs. 5.77% ± 1.2, P<0.05). While testicular spermatozoa appear favourable for ICSI in terms of lower DNA damage, this potential advantage could be offset by the higher aneuploidy rates in testicular spermatozoa.

The effects of low-level laser light exposure on sperm motion characteristics and DNA damage.

The objective of this study was to determine the effects of low-level laser light exposure on the motility of spermatozoa and on DNA damage. Thirty-three semen samples were collected for routine analysis and were classified as normospermic, oligospermic, or asthenospermic. After routine semen analysis was performed, residual semen was divided into treated and control aliquots. Treated samples were exposed to a 30-second infrared laser pulse of 50 mW/cm(2) at 905 nm, a wavelength thought to increase light-sensitive cytochrome c oxidase in the mitochondrial electron transport chain. Samples were then incubated at 37°C, and aliquots were analyzed at 30 minutes and 2 hours using computerassisted semen analysis. After incubation, 250 µL of each sample was frozen at 280°C until DNA fragmentation analysis by flow cytometry. A significant increase in motility, most prominent in oligospermic and asthenospermic samples (85% increase), was observed 30 minutes after the treatment (P < .0001). No significant increase in DNA damage compared with control samples was observed. Significant changes in sperm motion kinetics were observed. Low-level laser light exposure appears to have a positive short-term effect on the motility of treated spermatozoa and did not cause any increase in DNA damage measured at 2 hours. We conclude that some cases of asthenospermia may be related to mitochondrial dysfunction. The implications of this study in terms of future clinical applications needs further investigation.

Disruption of telomere-telomere interactions associated with DNA damage in human spermatozoa.

Telomeres play a fundamental role in the organization of the sperm nucleus resulting in the looped chromosome configuration and non-random positioning of chromosomes. Telomeres localize in the nuclear periphery and interact dynamically by forming dimers and tetramers. The purpose of this study was to evaluate the relationship of telomere interactions to DNA damage, a factor known to adversely influence male fertility. Telomeres were localized by fluorescence in situ hybridization (FISH) using human chromosome pan-telomeric probe in ten samples with low and ten samples with high sperm DNA damage. The samples with a low DNA fragmentation index (DFI) had a mean number of telomere signals of 21.7±1.9 compared to a mean of 26.5±3.4 signals in the samples with a high DFI (p<.005). The percentage of cells with a typical telomere distribution of ≤23 telomere-telomere dimers was observed in 70.8%±15.6 samples with a low DFI compared to 44.2%±22.4 in samples with a high DFI (p<.05). These results suggest that sperm DNA damage is associated with disruption of the normal telomere-telomere interactions leading to possible loss of the looped chromosome configuration. Improperly packed and organized sperm chromatin might have a high probability of disrupting the extremely structured sequence of sperm chromosome deposition, activation, and processing by the oocyte at the time of fertilization. These results might provide additional information on the nature of sperm DNA damage and the role of such damage on fertilization and development of the zygote.

Frequency and severity of sperm DNA damage in patients with confirmed cases of male infertility of different aetiologies.

Men with high sperm DNA damage have a reduced fertility potential. Correlation of specific clinical factors to the degree of sperm DNA damage remains unclear. This retrospective study evaluated the frequency and severity of sperm DNA damage in men with different aetiologies of male factor infertility. Patients with male factor infertility (n=288) underwent flow cytometry-based sperm DNA damage assessment and results were correlated with the major aetiologies of male infertility: varicocele, bacteriospermia and idiopathic infertility. Sperm DNA damage was significantly correlated to the patient's age, sperm motility, normal morphology and vitality (P < 0.001). High sperm DNA damage (30%) was most frequently found in the group with bacteriospermia (48%), compared with 30% of the men with varicoceles and 22% of the men with idiopathic infertility (P < 0.02). While some tendency was observed for a correlation of increasing sperm DNA damage in patients with grade III and bilateral varicoceles, this difference did not reach statistical significance. The data support the importance of proper physical and laboratory investigations of the fertility status in men to correctly diagnose and treat male infertility.

Testicular spermatozoa have statistically significantly lower DNA damage compared with ejaculated spermatozoa in patients with unsuccessful oral antioxidant treatment.

OBJECTIVE: To compare DNA damage in ejaculated and testicular spermatozoa in patients with previously unsuccessful oral antioxidant treatment. DESIGN: Prospective clinical study. SETTING: University-affiliated teaching hospital. PATIENT(S): Twelve men with persistently high sperm DNA damage. INTERVENTION(S): Evaluation of DNA damage of ejaculated and testicular spermatozoa by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. MAIN OUTCOME MEASURE(S): The DNA damage of ejaculated spermatozoa compared with that of testicular spermatozoa, both samples collected on the day of intracytoplasmic sperm injection. RESULT(S): Ejaculated spermatozoa showed a threefold higher DNA damage when compared with testicular samples (39.7% +/- 14.8 vs. 13.3% +/- 7.3). CONCLUSION(S): Our results indicated that in patients with previously unsuccessful oral antioxidant treatment the retrieved testicular spermatozoa had a lower degree of DNA damage compared with ejaculated sperm collected on the same day.