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Protein glycosylation is a post-translational modification involved in wide range of biological processes. In mammalian spermatozoa this modification has been identified in numerous proteins, and membrane glycoproteins are involved in the fertilization process. The objective of the present study was to identify changes in protein glycosylation after acrosome reaction (AR) induction using the 4-Br-A23187 ionophore. Our results showed that treatment with 10 μM of 4-Br-A23187 for 20 min significantly increased the percentage of live acrosome-reacted spermatozoa compared to the control (69.8 ± 0.8 vs. 6.4 ± 0.5; mean % ± SEM, respectively). Also, we observed an increase in 32 kDa tyrosine-phosphorylated protein (p32) and a decrease in serine/threonine phosphorylation of the protein kinase A substrates (phospho-PKA-substrates) after ionophore treatment. Furthermore, changes in glycosylated proteins following AR induction were analyzed using different HRP-conjugated lectins (GNA, DSA, and SNA), revealing changes in mannose and sialic acid residues. Proteomic analysis of isolated proteins using GNA lectin revealed that 50 proteins exhibited significantly different abundance (q-value < 0.01). Subsequent analysis using Uniprot database identified 39 downregulated and 11 upregulated proteins in the presence of 4-Br-A23187. Notably, six of these proteins were classified as transmembrane proteins, namely LRRC37A/B like protein 1 C-terminal domain-containing protein, Membrane metalloendopeptidase like 1, VWFA domain-containing protein, Syndecan, Membrane spanning 4-domains A14 and Serine protease 54. This study shows a novel protocol to induce acrosome reaction in boar spermatozoa and identifies new transmembrane proteins containing mannose residues. Further work is needed to elucidate the role of these proteins in sperm-oocyte fusion., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Epigenetics refers to how gene expression and function are modulated without modifying the DNA sequence but through subtle molecular changes or interactions with it. As spermatogenesis progresses, male germ cells suffer plenty of epigenetic modifications, resulting in the definitive epigenome of spermatozoa conditioning its functionality, and this process can be altered by several internal and external factors. The paternal epigenome is crucial for sperm function, fertilization, embryo development, and offspring's health, and altered epigenetic states are associated with male infertility with or without altered semen parameters, embryo quality impairment, and worse ART outcomes together with the future offspring's health risks mainly through intergenerational transmission of epigenetic marks. Identifying epigenetic biomarkers may improve male factor diagnosis and the development of targeted therapies, not only to improve fertility but also to allow an early detection of risk and disease prevention in the progeny. While still there is much research to be done, hopefully in the near future, improvements in high-throughput technologies applied to epigenomes will permit our understanding of the underlying epigenetic mechanisms and the development of diagnostics and therapies leading to improved reproductive outcomes. In this review, we discuss the mechanisms of epigenetics in sperm and how epigenetics behave during spermatogenesis. Additionally, we elaborate on the relationship of sperm epigenetics with sperm parameters and male infertility, and highlight the impact of sperm epigenetic alterations on sperm parameters, embryo quality, ART outcomes, miscarriage rates and offspring's health. Furthermore, we provide insights into the future research of epigenetic alterations in male infertility.

Purpose: To describe the proteomic profiles in semen samples and define the differences in sperm proteomic profiles among samples that ultimately achieved pregnancy (P) via intracytoplasmic sperm injection (ICSI) in an oocyte donation program and those that were unsuccessful (NP)., Methods: Prospective, analytical, observational nested case and control study evaluating the proteomic profile of spermatozoa from patients' ejaculates where pregnancies were (group pregnant (P), n= 4) or were not (group non-pregnant (NP), n=4) achieved after ICSI in an oocyte donation program aiming to standardize female factor. Proteins were separated and analyzed by means of SWATH-MS) and compared between P/NP groups to identify sperm biomarkers of fertility/infertility. Proteins are available via ProteomeXchange., Results: We identified and quantified 2228 proteins, with 37 significantly higher in the P group and 16 higher in NP. Enrichment analysis revealed that the increased proteins in P group sperm were related to motility, anaerobic metabolism, and protein biosynthesis functions, while the increased proteins in the NP group were involved in protein biosynthesis, protein folding, aerobic metabolism, and signal transduction, all of which are functions not previously described as influencing sperm success. Some proteins identified (e.g., SLC2A3, or CD81) are located in the cell membrane and thus may be employed to select spermatozoa by magnetic-activated cell sorting (MACS)., Conclusion(s): This work revealed differences in the proteomic profiles of sperm samples successful in achieving pregnancy and those that were not, expanding our understanding of sperm function and infertility-related molecular markers, and enabling the future development of male fertility diagnostic tools and therapies.

Transcriptome analyses using high-throughput methodologies allow a deeper understanding of biological functions in different cell types/tissues. The present study provides an RNA-seq profiling of human sperm mRNAs and lncRNAs (messenger and long non-coding RNAs) in a well-characterized population of fertile individuals. Sperm RNA was extracted from twelve ejaculate samples under strict quality controls. Poly(A)-transcripts were sequenced and aligned to the human genome. mRNAs and lncRNAs were classified according to their mean expression values (FPKM: Fragments Per Kilobase of transcript per Million mapped reads) and integrity. Gene Ontology analysis of the Expressed and Highly Expressed mRNAs showed an involvement in diverse reproduction processes, while the Ubiquitously Expressed and Highly Stable mRNAs were mainly involved in spermatogenesis. Transcription factor enrichment analyses revealed that the Highly Expressed and Ubiquitously Expressed sperm mRNAs were primarily regulated by zinc-fingers and spermatogenesis-related proteins. Regarding the Expressed lncRNAs, only one-third of their potential targets corresponded to Expressed mRNAs and were enriched in cell-cycle regulation processes. The remaining two-thirds were absent in sperm and were enriched in embryogenesis-related processes. A significant amount of post-testicular sperm mRNAs and lncRNAs was also detected. Even though our study is solely directed to the poly-A fraction of sperm transcripts, results indicate that both sperm mRNAs and lncRNAs constitute a footprint of previous spermatogenesis events and are configured to affect the first stages of embryo development., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Research Question: Do donor spermatozoa improve IVF outcomes after first oocyte donation failure?, Design: Retrospective, multicentre study including couples undergoing oocyte donation cycles using autologous or donor spermatozoa after a failed first attempt. Male partners were further characterized as normozoospermic or oligoasthenoteratospermic, i.e. fewer than 5 million motile progressive spermatozoa in the ejaculate. The main outcomes measured were live birth rate (LBR) per embryo transfer, LBR per number of embryos transferred, and cumulative LBR (CLBR) considering oocytes consumed in the previous donation cycles., Results: Analysis comprised 6065 cycles of oocyte donation failure; among these, subgroup analyses by sperm quality comprised 4113 cycles with severe male factor and 1150 cycles with suboptimal/normal spermatozoa. Sperm replacement in the first cycle after failure increased LBR per embryo transfer (OR 2.21, 95% CI 1.7-2.8, P < 0.001) and per number of embryos transferred (OR 2.46, 95% CI 1.9-3.1, P < 0.001) for normospermic and oligoasthenoteratospermic men. Replacement by the third cycle after failure was less beneficial (LBR per embryo transfer: OR 1.35, 95% CI 0.9-2.1, P = 0.16; LBR per embryos transferred: OR 1.33, 95% CI 0.9-2.0, P = 0.186). Kaplan-Meier curves of CLBR per oocyte fertilized with autologous or donor spermatozoa were statistically different (P < 0.001) and demonstrate how each additional oocyte may affect success based on sperm source (donor/autologous)., Conclusions: Donor spermatozoa improved outcomes when used after an initial failed oocyte donation cycle. The CLBR curves can be used to determine the cumulative chances of live birth using either autologous or donor spermatozoa, providing guidance on when to replace spermatozoa., (Copyright © 2021 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Sperm fertility ability may be modulated by different molecular systems, such as the renin-angiotensin system (RAS). Although renin is one of its most relevant peptides, the presence and role of the (pro)renin receptor (PRR) is completely unknown. We have proved for the first time the existence of PRR and its transcript in human sperm by western blot and RT-PCR. Immunofluorescence studies showed that this receptor is mainly located in the apical region over the acrosome and in the postacrosomal region of the sperm head and along the sperm tail. In addition, this prospective cohort study also proves that semen samples with higher percentages of PRR-positive spermatozoa are associated with poor sperm motility, worse blastocyst development and no-viable blastocysts. Our results provide insight into how PRR play a negative role in sperm physiology that it may condition human embryo quality and development. An in-depth understanding of the role of PRR in sperm fertility can help elucidate its role in male infertility, as well as establish biomarkers for the diagnosis or selection of sperm to use during assisted reproductive techniques.

Background: Autism spectrum disorder (ASD) has increased over tenfold over the past several decades and appears predominantly associated with paternal transmission. Although genetics is anticipated to be a component of ASD etiology, environmental epigenetics is now also thought to be an important factor. Epigenetic alterations, such as DNA methylation, have been correlated with ASD. The current study was designed to identify a DNA methylation signature in sperm as a potential biomarker to identify paternal offspring autism susceptibility., Methods and Results: Sperm samples were obtained from fathers that have children with or without autism, and the sperm then assessed for alterations in DNA methylation. A genome-wide analysis (> 90%) for differential DNA methylation regions (DMRs) was used to identify DMRs in the sperm of fathers (n = 13) with autistic children in comparison with those (n = 13) without ASD children. The 805 DMR genomic features such as chromosomal location, CpG density and length of the DMRs were characterized. Genes associated with the DMRs were identified and found to be linked to previously known ASD genes, as well as other neurobiology-related genes. The potential sperm DMR biomarkers/diagnostic was validated with blinded test sets (n = 8-10) of individuals with an approximately 90% accuracy., Conclusions: Observations demonstrate a highly significant set of 805 DMRs in sperm that can potentially act as a biomarker for paternal offspring autism susceptibility. Ancestral or early-life paternal exposures that alter germline epigenetics are anticipated to be a molecular component of ASD etiology.

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment., Competing Interests: The authors declare no conflict of interest.

Although assisted reproduction techniques involve the use of semen samples, there is little scientific methodology applied when selecting sperm. To select the most appropriate spermatozoa, first we need to define the optimal molecular characteristics. Sperm lipids may contribute to sperm function, thus our aim was to compare the lipidic profiles of sperm samples used in intracytoplasmic sperm injection cycles that ultimately led to a pregnancy with those that did not.Spermatozoa from infertile patients after intracytoplasmic sperm injection (group non-pregnant, n = 16; vs. group pregnant, n = 22) were analyzed for lipid composition using ultra-high performance liquid chromatography coupled to mass spectrometry, by means two platforms for measuring fatty acyls, bile-acids, lysoglycerophospholipids, glycerolipids, cholesteryl-esters, sphingolipids, and glycerophospholipids. Lipid levels were compared using a univariate test and multivariate analyses after logarithmic transformation.We detected 151 different lipids in the sperm samples, 10 of which were significantly increased in sperm samples from the NP group, ranging from 1.10- to 1.30-fold change. These were primarily ceramides, sphingomyelins and three glycerophospholipids, a lysophosphatidylcholine, and two plasmalogen species. Additionally, 2-Monoacylglycerophosphocholine were also found in higher levels in non-pregnant group.Our results describe the composition of sperm lipids linked to optimal sperm function, opening new possibilities for the development of male fertility diagnostic tools and culture media formulations to improve sperm quality and enhance reproductive results. Given that lipids compose the majority of the sperm plasma membrane, this information is also useful in designing new sperm selection tools that will allow for the selection of the best spermatozoa.

Angiotensin-converting enzyme functions in the male reproductive system, but the extent of its function in reproduction is not fully understood. The primary objective of this work was to investigate the relationship between the testicular isoform of angiotensin-converting enzyme present in human spermatozoa and semen parameters, human embryo quality, and assisted reproduction success. A total of 81 semen samples and 635 embryos from couples undergoing oocyte donation cycles at the IVI Bilbao Clinic were analyzed. Semen parameters, embryos quality, and blastocyst development were examined according to the World Health Organization standards and the Spanish Association of Reproduction Biology Studies criteria. The percentage of testicular angiotensin-converting enzyme-positive spermatozoa and the number of molecules per spermatozoon were analyzed by flow cytometry. Both parameters were inversely correlated with human sperm motility. Higher percentages of testicular angiotensin-converting enzyme-positive spermatozoa together with fewer enzyme molecules per spermatozoon were positively correlated with better embryo quality and development. Our results suggest that embryos with a higher implantation potential come from semen samples with higher percentages of testicular angiotensin-converting enzyme-positive cells and fewer enzyme molecules per spermatozoon. Based on these findings, we propose that testicular angiotensin-converting enzyme could be used to aid embryologists in selecting better semen samples for obtaining high-quality blastocysts during in vitro fertilization procedures., Competing Interests: All authors declared no competing interests

Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval.

Aim: To characterize the sperm methylome in semen samples from 19 donors with proven fertility., Materials & Methods: Bisulfite-converted sperm DNA was hybridized on the HumanMethylation450 Infinium BeadChip platform. CpG fluorescence intensities were extracted and converted to β-values., Results: The sperm methylome is highly homogeneous and hypomethylated. Genes with hypomethylated promoters are ontologically associated to biological functions related to spermatogenesis and embryogenesis. Sex chromosomes are the most hypomethylated chromosomes, supporting data that indicated their essential role in spermatogenesis. A total of 94 genes are resistant to demethylation, being strong candidates for transgenerational inheritance., Conclusion: Spermatozoa carry a homogeneous methylation profile that is a footprint of past events (spermatogenesis), is designed to facilitate future events (embryogenesis) and has a possible influence in the adult life (transgenerational effects).

Purpose: The purpose of this study is to assess outcomes after magnetic-activated cell sorting (MACS) technology on obstetric and perinatal outcomes compared with those achieved after swim up from randomized controlled trial., Methods: This is a two-arm, unicentric, prospective, randomized, and triple-blinded trial and has a total of 237 infertile couples, between October 2010 and January 2013. A total of 65 and 66 newborns from MACS and control group, respectively, were described., Results: MACS had no clinically relevant adverse effects on obstetric and perinatal outcomes. No differences were found for obstetric problems including premature rupture of membranes 6.1% (CI95% 0-12.8) vs. 5.9% (CI95% 0-12.4), 1st trimester bleeding 28.6% (CI95% 15.9-41.2) vs. 23.5% (CI95% 11.9-35.1), invasive procedures as amniocentesis 2.0% (CI95% 0-5.9) vs. 3.9% (CI95% 0-9.2), diabetes 14.3% (CI95% 4.5-24.1) vs. 9.8% (CI95% 1.6-17.9), anemia 6.1% (CI95% 0-12.8) vs. 5.9%(CI95% 0-12.4), 2nd and 3rd trimesters 10.2% (CI95% 1.7-18.7) vs. 5.9% (CI95% 0-12.4), urinary tract infection 8.2% (CI95% 0.5-15.9) vs. 3.9% (CI95% 0-9.2), pregnancy-induced hypertension 6.1% (CI95% 0-12.8) vs. 15.7% (CI95% 5.7-25.7), birth weight (g) 2684.10 (CI95% 2499.48-2868.72) vs. 2676.12 (CI95% 2499.02-2852.21), neonatal height (cm) 48.3 (CI95% 47.1-49.4) vs. 46.5 (CI95% 44.6-48.4), and gestational cholestasis 0%(CI95% 0-0) vs. 3.9% (CI95% 0-9.2), respectively, in MACS group compared with control group., Conclusions: Our data suggest that MACS technology does not increase or decrease Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation adverse obstetric and perinatal outcomes in children conceived when this technology was performed, being the largest randomized control trial with live birth reported results with MACS., Competing Interests: Compliance with ethical standards The study was approved by the Institutional Review Board of the Instituto Valenciano de Infertilidad, Valencia, Spain (0810-C-051-MM).

The influence of aberrant sperm DNA methylation on the reproductive capacity of couples has been postulated as a cause of infertility. This study compared the DNA methylation of spermatozoa of 19 fertile donors and 42 infertile patients using the Illumina 450K array. Clustering analysis of methylation data arranged fertile and infertile patients into two groups. Bivariate clustering analysis identified a differential distribution of samples according to the characteristics of seminogram and age, suggesting a possible link between these parameters and specific methylation profiles. The study identified 696 differentially methylated cytosine-guanine dinucleotides (CpG) associated with 501 genes between fertile donors and infertile patients. Ontological enrichment analysis revealed 13 processes related to spermatogenesis. Data filtering identified a set of 17 differentially methylated genes, some of which had functions relating to spermatogenesis. A significant association was identified between RPS6KA2 hypermethylation and advanced age (P = 0.016); APCS hypermethylation and oligozoospermia (P = 0.041); JAM3/NCAPD3 hypermethylation and numerical chromosome sperm anomalies (P = 0.048); and ANK2 hypermethylation and lower pregnancy rate (P = 0.040). This description of a set of differentially methylated genes provides a framework for further investigation into the influence of such variation in male fertility in larger patient cohorts., (Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Deciphering the underlying causes of idiopathic male infertility is one of the main challenges in reproductive medicine. This is especially relevant in infertile patients displaying normal seminal parameters and no urogenital or genetic abnormalities. In these cases, the search for additional sperm biomarkers is of high interest. This study was aimed to determine the implications of the sperm miRNA expression profiles in the reproductive capacity of normozoospermic infertile individuals. The expression level of 736 miRNAs was evaluated in spermatozoa from eight normozoospermic infertile males using TaqMan ® qRT-PCR. Results were contrasted with data from 10 control normozoospermic fertile individuals analyzed under the same conditions. Clustering analysis of miRNA expression data separated the individuals according to their fertility condition (fertile and infertile). Fifty-seven miRNAs were differentially expressed (DE-miRNAs) between populations; 20 of them was regulated by a host gene promoter that in three cases comprised genes involved in fertility. The predicted targets of the DE-miRNAs (n = 8,606) unveiled a significant enrichment of biological processes related to embryonic morphogenesis and chromatin modification. Normozoospermic infertile individuals exhibit a specific sperm miRNA expression profile clearly differentiated from normozoospermic fertile individuals. This miRNA cargo has potential implications in the individuals' reproductive competence., (© 2016 American Society of Andrology and European Academy of Andrology.)

Objective: To investigate the presence of angiotensin II type 2 receptor (AT2R) in human spermatozoa and its implication in sperm fertility status., Design: We carried out expression assays for AT2R by reverse transcription-polymerase chain reaction, Western blot, immunofluorescence, and flow cytometry techniques in human sperm cells. Percentage of AT2R-positive sperm cells was analyzed by flow cytometry., Setting: Assisted reproduction unit and academic research laboratory., Patient(s): Ninety-seven human semen samples from the Clínica IVI Bilbao., Intervention(s): All samples were examined and classified according to World Health Organization guidelines. Spermatozoa were isolated from semen on discontinuous colloidal silica gradient (45%-90%) and swim-up techniques., Main Outcome Measure(s): Presence and location of the AT2R in spermatozoa and percentage of AT2R-positive sperm cells measured by flow cytometry., Result(s): We demonstrated the existence of AT2R and its transcript in human sperm by Western blot and reverse transcription-polymerase chain reaction. Immunofluorescence studies showed that AT2R is mainly located at the equatorial segment of the sperm head. The AT2R levels were associated with sperm motility parameters. Particularly, we found a significant positive correlation between AT2R and spermatozoa with progressive motility grade and a significant negative correlation with immotile spermatozoa, both in fresh semen samples and in prepared sperm cells. Regarding pathologic studies, the levels of AT2R measured by flow cytometry were lower in spermatozoa of asthenozoospermic men than in normozoospermic controls., Conclusion(s): Angiotensin II type 2 receptor is present in human semen and may be involved in the control of sperm motility. In-depth understanding of the proteins involved in sperm motility can help to elucidate the role of these proteins in male infertility as well as to establish new biomarkers for male infertility., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: In natural conception only a few sperm cells reach the ampulla or the site of fertilization. This population is a selected group of cells since only motile cells can pass through cervical mucus and gain initial entry into the female reproductive tract. In animals, some studies indicate that the sperm selected by the reproductive tract and recovered from the uterus and the oviducts have higher fertilization rates but this is not a universal finding. Some species show less discrimination in sperm selection and abnormal sperm do arrive at the oviduct. In contrast, assisted reproductive technologies (ART) utilize a more random sperm population. In this review we contrast the journey of the spermatozoon in vivo and in vitro and discuss this in the context of developing new sperm preparation and selection techniques for ART., Methods: A review of the literature examining characteristics of the spermatozoa selected in vivo is compared with recent developments in in vitro selection and preparation methods. Contrasts and similarities are presented., Results and Conclusions: New technologies are being developed to aid in the diagnosis, preparation and selection of spermatozoa in ART. To date progress has been frustrating and these methods have provided variable benefits in improving outcomes after ART. It is more likely that examining the mechanisms enforced by nature will provide valuable information in regard to sperm selection and preparation techniques in vitro. Identifying the properties of those spermatozoa which do reach the oviduct will also be important for the development of more effective tests of semen quality. In this review we examine the value of sperm selection to see how much guidance for ART can be gleaned from the natural selection processes in vivo., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Objective: To compare the microRNA (miRNA) expression profile in spermatozoa from three infertile populations vs. a group of fertile men., Design: Evaluation of the expression level of 736 miRNAs in human spermatozoa using TaqMan quantitative reverse transcription-polymerase chain reaction., Setting: University research facility., Patient(s): Semen samples with a single seminal alteration were collected from infertile individuals: asthenozoospermic (n = 10), teratozoospermic (n = 10), and oligozoospermic (n = 10)., Intervention(s): None., Main Outcome Measure(s): Correlation of the expression level of each miRNA with seminal parameters, age, and chromosome instability; clustering of the individuals according to their miRNA expression profiles and influence of the seminogram, age, chromosome instability, and assisted reproductive technology outcome in the clustering; analysis of the differentially expressed miRNAs (DE-miRNAs) in each infertile population; genome annotation of these DE-miRNAs; and ontological analysis of their predicted targets., Result(s): The hsa-miR-34b-3p correlated with age, the hsa-miR-629-3p with sperm motility, and the hsa-miR-335-5p, hsa-miR-885-5p, and hsa-miR-152-3p with sperm concentration. The individuals clustered into two groups, and only the seminogram was differentially distributed. We identified 32 DE-miRNAs in the asthenozoospermic group, 19 in the teratozoospermic group, and 18 in the oligozoospermic group. The up-regulated miRNAs presented an enriched localization in introns, affecting relevant genes for spermatogenesis. The predicted targets of the DE-miRNAs contained critical genes associated to infertility, and their ontological analysis revealed significantly associated functions related to the seminal alterations of each group., Conclusion(s): Spermatozoa from patients with seminal alterations exhibit a differential miRNA profile. This provides new evidence that miRNAs have an essential role in spermatogenesis, contributing to the mechanisms involved in human fertility., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the effect of removing presumptive apoptotic sperm cells from samples from unselected males by means of magnetic activated cell sorting (MACS) on live-birth delivery rates after intracytoplasmic sperm injection (ICSI) in couples undergoing ovum donation (OD)., Design: Prospective, randomized, triple-blinded, and controlled study., Setting: Private university-affiliated IVF center., Patient(s): A total of 237 infertile couples undergoing ICSI as part of an OD program., Intervention(s): Semen specimens from the control group were prepared by swim-up. Samples from the study group were prepared by swim-up followed by MACS and incubation with annexin V-conjugated microbeads to remove annexin V-positive (AV+) sperm cells., Main Outcome Measure(s): Fertilization rates, morphological features of early embryo development, implantation rates, ongoing pregnancy rates, and live-birth rates., Result(s): Similar results were obtained between groups for all the parameters compared: fertilization rates of 75.3% (95% confidence interval [CI], 71.6-78.9) versus 72.1% (95% CI, 68.6-75.7); percentage of good-quality embryos on day 2 of 53.7% (95% CI, 50.3-57.1) versus 51.8% (95% CI, 48.3-55.3) and on day 3 of 54.2% (95% CI, 50.7-57.6) versus 48.9% (95% CI, 45.3-52.4); implantation rates of 42.2% (95% CI, 33.8-48.1) versus 40.1% (95% CI, 34.8-49.6); positive beta-hCG tests of 63.2% (95% CI, 54.7-71.6) versus 68.6% (95% CI, 60.2-76.9), and live-birth rates of 48.4% (95% CI, 39.6-57.1) versus 56.4% (95% CI, 47.3-65.5) in the MACS versus control group. None of the differences reached statistical significance., Conclusion(s): Applying MACS technology to remove AV+ sperm cells from unselected males does not improve the reproductive outcome of ICSI in OD., (Copyright © 2014. Published by Elsevier Inc.)

Objective: To characterize the microRNA (miRNA) expression profile in spermatozoa from human fertile individuals and their implications in human fertility., Design: The expression levels of 736 miRNAs were evaluated using TaqMan arrays. Ontologic analyses were performed to determine the presence of enriched biological processes among their targets., Setting: University research and clinical institutes., Patient(s): Ten individuals with normal seminogram, standard karyotype, and proven fertility., Intervention(s): None., Main Outcome Measure(s): Expression levels of 736 miRNAs, presence of enriched metabolic routes among their targets, homogeneity of the population, influence of demographic features in the results, presence of miRNA stable pairs, and best miRNA normalizing candidates., Result(s): A total of 221 miRNAs were consistently present in all individuals, 452 were only detected in some individuals, and 63 did not appear in any sample. The ontologic analysis of the 2,356 potential targets of the ubiquitous miRNAs showed an enrichment of processes related to cell differentiation, development, morphogenesis, and embryogenesis. None of the miRNAs were significantly correlated with age, semen volume, sperm concentration, motility, or morphology. Correlations between samples were statistically significant, indicating a high homogeneity of the population. A set of 48 miRNA pairs displayed a stable expression, a particular behavior that is discussed in relationship to their usefulness as fertility biomarkers. Hsa-miR-532-5p, hsa-miR-374b-5p, and hsa-miR-564 seemed to be the best normalizing miRNA candidates., Conclusion(s): Human sperm contain a stable population of miRNAs potentially related to embryogenesis and spermatogenesis., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The aim of this work is to review the need for new diagnostic tools for sperm, the relevance of sperm mRNA in reproductive success, and the potential of a state-of-the-art microarray-based diagnostic tool for improving reproductive results., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The topic of imprinting defects present in the sperm of infertile patients has been addressed by several reports in the last few years. However, whether methylation abnormalities at one or few CpGs within an imprinted locus are pathological is a matter of debate. Moreover, whether imprinting anomalies in sperm could interfere with fertility treatment outcomes is still unknown. In this report we analyze the sperm DNA methylation profile of H19-ICR, KvDMR, SNRPN-ICR, IG-DMR and MEG3-DMR by pyrosequencing in 107 infertile men series and a control population of 30 proven fertile males. DNA methylation was statistically evaluated from two points of view: first, the methylation of each CpG was analyzed in the control population and the mean, standard deviation and range were determined and compared with infertile population data; second, in order to define altered methylation patterns for each region, a hierarchical cluster analysis was performed by which individuals were grouped in different clusters according to the degree of similarity of their methylation pattern. Two pieces of data supported the results obtained in the multi-variate analysis: the classification of the vast majority of control individuals in clusters with normal methylation patterns and the significant differences in methylation levels found between individuals within the normal and abnormal clusters. Individuals included in normal and abnormal methylation clusters were compared according to seminal parameters as well as to the outcome of assisted reproduction.

A prospective study was performed to assess the impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes. The study population included 178 couples (62 cycles of IVF, 116 of intracytoplasmic sperm injection (ICSI)) with own (n=77) and donor (n=101) oocytes. DNA fragmentation was evaluated by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Correlation between DNA damage to oocyte fertilization, embryo quality and clinical pregnancy, implantation and miscarriage rates was evaluated. DNA fragmentation was not related to fertilization rates in either IVF (r=0.08) or ICSI (r=-0.04) cycles. DNA fragmentation was similar in patients with <50% embryo utilization rate compared with ≥50%, in cancelled and in embryo transfer cycles and in miscarriages and in successful deliveries. Moreover, DNA fragmentation was similar in pregnant and non-pregnant women as well as in IVF with own or donor oocytes. In the multivariable analysis, the odds ratio of DNA after controlling by age was 1.0. Using a 36% sperm fragmentation threshold, results did not vary. It is concluded that DNA damage was not related to outcomes of IVF or ICSI with own or donor oocytes., (Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: To demonstrate that the use of donor sperm leads to varying outcome rates and that its use has evolved., Design: Retrospective observational cohort study., Setting: University-affiliated private IVF setting., Patient(s): Women (2,934) undergoing donor insemination (DI) or IVF with donor sperm (IVF-D)., Intervention(s): None., Main Outcome Measurement(s): We evaluated the distribution of the clinical indications for the use of donated sperm, studying the reproductive outcome., Result(s): A total of 1,663 DI (57%) and 1,271 IVF-D (43%) were performed. There were significant differences in the indications for the use of donated sperm (DI vs. IVF-D). Regarding pregnancy rates (PR), cases of nonobstructive azoospermia presented the highest rate (29.1%), whereas cases of intracytoplasmic sperm injection (ICSI) failures and single women showed rates of 27.6% and 22.6%, respectively. Meanwhile, patients with ICSI failures achieved the highest PRs in IVF cycles (48.7%), whereas nonobstructive azoospermia and single women showed rates of 42.0% and 38.2%, respectively. There have been significant increases in the use of donated sperm in single women., Conclusion(s): Single women, which also represented the oldest group, show a lower probability of achieving pregnancy, and thus represent a subfertile population. Associated factors could include advanced maternal age., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To quantify the effect of sperm DNA fragmentation (SDF) on reproductive outcome by evaluating the most statistically significant bias factors using logistic regression., Design: Prospective blind observational cohort study., Setting: University affiliated private IVF unit., Patient(s): Two hundred ten male partners of couples undergoing in vitro fertilization (IVF) or first intracytoplasmic sperm injection (ICSI) cycles with fresh or thawed sperm with the women's own or donated oocytes., Intervention(s): None., Main Outcome Measure(s): SDF determined before and after swim-up (n=420), odds ratio calculated of the effect of an increase of one unit of SDF on pregnancy, and stratified regression analysis performed to evaluate the confusion effect of oocyte quality, sperm origin, and the fertilization procedure., Result(s): The effect of SDF on pregnancy was not affected by sperm origin (fresh or thawed) or fertilization procedure when measured both before and after swim-up. When oocytes from infertile patients were employed, SDF had a statistically significant negative impact on chance of pregnancy. For every 10% increase in SDF, the probability of not achieving pregnancy increased by 1.31. When donated oocytes were employed, SDF did not have a statistically significant effect., Conclusion(s): The effect of SDF on the probability of pregnancy can be calculated independent of the fertilization procedure or sperm origin. Oocyte quality conditions the extent of the negative impact of SDF on pregnancy; this can be overcome when good quality oocytes are employed., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Basic sperm analysis is limited as a method of estimating pregnancy. This study’s objective was use of microarray technology to differentiate the gene expressions of spermatozoa that achieved pregnancy in an intracytoplasmic sperm injection (ICSI)cycle in an oocyte donation programme with those that did not achieve pregnancy. A study of nested cases and controls was designed to evaluate fresh and frozen spermatozoa from infertile males undergoing ICSI with donor oocytes. The global genome expression of pooled samples from each group (achieving pregnancy versus those that didn’t, from fresh or frozen spermatozoa)was compared using microarray analysis. The level of expression of some of the transcripts from fresh spermatozoa was shown to differ for those that achieved pregnancy versus those that didn’t. Additionally, exclusively expressed transcripts were identified for both outcome groups. Analysis of frozen spermatozoa didn’t reveal differential expression, but exclusively expressed transcripts were detected. Lists of the transcripts were systematically analysed using different databases in order to provide information about them and their relationship with male fertility. The results revealed profound differences between the expression profiles of spermatozoa that resulted in pregnancy versus those that didn’t. These differences may explain ICSI failure associated with male factor infertility., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: To differentiate transcripts' expression in the sperm from patients who achieved pregnancy in their first IUI cycle from those who did not. Basic sperm analysis is limited to forecasting pregnancies by means of assisted reproduction. New assays, such as microarray analysis, are potential predictive tools for this purpose., Design: Nested case-control study., Setting: University-affiliated private setting., Patient(s): Twenty sperm samples were obtained from infertile males undergoing their first IUI cycle with healthy partners. Sperm samples with which pregnancy was achieved (P; n=10) and those with which it was not achieved (NP; n=10) were identified and their respective messenger RNA expression profiles were compared., Intervention(s): None., Main Outcome Measure(s): Using microarrays, global genome expression was compared in pooled samples from each group. Results were evaluated to detect differentially expressed transcripts (TDEs; FC>2; P<0.05) and to identify those transcripts that were expressed in only one of the groups (exclusive transcripts [ETs])., Result(s): In group P, 756 TDEs presented increased expression, whereas 194 in group NP were found to be overexpressed. Furthermore, we found 741 ETs that were expressed only in group P and 976 that were expressed only in group NP., Conclusion(s): Results reveal profound differences between expression profiles of sperm samples that impregnate successfully and those that do not. These differences might improve the predictive power of sperm evaluation to estimate IUI success by complementing the basic sperm analysis., (Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the relevance of sperm DNA oxidation caused by free radicals in samples obtained via testicular biopsies by means of flow cytometry by correlating the measurements of 8-hydroxy-2'-deoxyguanosine (8-OHdG) with embryo features and pregnancy achievement., Design: Prospective cross-sectional study., Setting: Private University-affiliated setting., Patient(s): Fifty-seven azoospermic patients undergoing testicular sperm extraction (TESE) were analyzed in their corresponding assisted reporductive technology cycles using ovum donation to standardize female's characteristics., Intervention(s): None., Main Outcome Measure(s): Quantification of the adduct 8-OHdG in testicular tissue samples, and its effect on markers of embryo quality and reproductive success, and its relevance as marker of TESE sperm quality., Result(s): We found the status of sperm DNA oxidation to have very little clinical relevance for several parameters of embryo quality, fertilization rates, early (days 2-3) and late (days 5-6) development, and achievement of pregnancy., Conclusion(s): The TESE obtained cells from azoospermic males do not possess a DNA oxidation status of significant importance in the success of assisted reproduction treatments, as determined by 8-OHdG measurement of each category of cell ploidy., (Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the effects of tobacco consumption on the oxidative defenses of sperm, the glutathione system (GS), and sperm DNA oxidation., Design: Double-blind experimental study., Setting: Andrology laboratory in a university-affiliated private setting., Patient(s): One hundred seventeen semen samples from infertile males., Intervention(s): None., Main Outcome Measure(s): (a) sperm GS enzymatic activity with respect to glutathione peroxidase isoforms GPx-1 and GPx-4, glutathione reductase (GR), and cellular glutathione (GSH) content (n = 29); (b) GPx-1, GPx-4, and GR mRNA expression analysis (n = 33); (c) oxidative DNA damage quantification using OXIDNA assay kit (n = 55). Two groups were established: nonsmoking and smoking males. The t tests were employed to detect significant differences between groups., Result(s): We identified a significant decrease in sperm GPx-4 activity but not in GPx-1 and GSH activity in smokers compared with nonsmokers. A significant decrease was also observed in GPx-1, GPx-4, and GR mRNA expression in the former group. Interestingly, we did not observe any significant variation in the percentage of cells with oxidative damage of the DNA or in the average level of oxidation of affected cells with respect to the smoking condition of the male., Conclusion(s): We demonstrate that smoking has a negative impact on intracellular antioxidant enzymes but that effect does not increase oxidative DNA damage. Thus, the effects of reduced oxidative defenses in sperm as a result of cigarette smoking are yet to be elucidated., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: The aim of the present study was to evaluate the implication of male factor, in terms of sperm DNA oxidation and fragmentation, and Y chromosome microdeletions in recurrent spontaneous abortion (RSA) of unknown origin in a strictly selected cohort., Methods: A prospective cohort study was carried out in a private university-affiliated setting. Three groups, each comprised of 30 males, were compared. The first was formed by healthy and fertile sperm donors (SD) with normal sperm parameters (control group), the second by men presenting severe oligozoospermia (SO) without RSA history, and the third by men from couples who had experienced idiopathic RSA. Frequency of Y chromosome microdeletions and mean sperm DNA fragmentation and oxidation were determined., Results: Y chromosome microdeletions were not detected in any of the males enrolled in the study. Moreover, sperm DNA oxidation measurements were not demonstrated to be relevant to RSA. Interestingly, sperm DNA fragmentation was higher in the SO group than in the RSA and the SD groups, and also higher in the RSA group compared with the SD group, but lacked an adequate predictive power to be employed as a discriminative test of RSA condition., Conclusions: Sperm DNA features and Y chromosome microdeletions do not seem to be related to RSA of unknown origin. Other molecular features of sperm should be studied to determine their possible influence on RSA. Clinicaltrials.gov reference: NCT00447395.

Telomere length and sperm DNA fragmentation were determined in sperm samples from 27 patients, using a quantitative PCR (Q-PCR) assay and the Sperm Chromatin Dispersion (SCD) test, respectively. Comparisons of the samples before and after swim-up processing demonstrated that this procedure selects a sperm population with longer average telomere size and lower frequency of sperm cells with fragmented DNA., (Copyright 2010 Elsevier B.V. All rights reserved.)

Purpose: To catalogue Gene Ontology terms in the sperm of infertile human males vs. donors of proven fertility by analyzing five samples from each of the two groups (five aliquots from fresh sperm and five post-swim-up)., Methods: Microarray technology was employed to study the mRNA profile of both fresh and post-swim-up pooled samples from infertile males and donors., Results: Genes that were differentially expressed in the two populations and expressed in only one of two were analyzed to determine the gene products in terms of their associated Gene Ontology terms. Each group presented a different number and pattern of Gene Ontology terms., Conclusions: We found differences in Gene Ontology terms between the two groups. These differences could potentially be employed to establish markers of fertility success and to identify cellular processes and complex systems related with male infertility.

Deoxyribonucleic acid fragmentation and oxidative DNA damage were simultaneously determined in the same sperm cell, incubating with an 8-oxoguanine DNA probe on human spermatozoa processed by the sperm chromatin dispersion test. The assay was validated by incubation with agents that induce DNA fragmentation with or without oxidative base damage. In all samples examined, increased levels of 8-oxoguanine were present only in those spermatozoa with fragmented DNA, suggesting a link between both DNA damage types., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To analyze sperm DNA fragmentation (SDF) in testicular sperm samples from patients with azoospermia either from spermatogenic failure or from duct obstruction. Several technologies can be applied in the evaluation of SDF, but given the ease and low costs, the sperm chromatin dispersion test (SCD) has emerged as a promising standard., Design: Prospective blind observational cohort study., Setting: University-affiliated private IVF setting., Patient(s): Azoospermic patients from couples undergoing intracytoplasmic sperm injection cycles., Intervention(s): Testicular sperm extraction (TESE)., Main Outcome Measurement(s): We determined testicular SDF, and a basic comparison between nonobstructive (n = 22) and obstructive azoospermia (n = 40) was performed. We also correlated SDF with embryo quality and pregnancy outcome., Result(s): SDF in the testicular sperm of patients with nonobstructive azoospermia was significantly higher, 46.92% (SEM = 4.47), than that of patients with obstructive azoospermia, 35.96% (SEM = 2.63). A moderate relationship between embryo morphology and testicular SDF was detected. Logistic regression analysis of the effect of testicular SDF on pregnancy outcome revealed no significant effect (odds ratio = 1.015)., Conclusion(s): Ours is the first report of SDF analysis in testicular sperm by using SCD in azoospermia. This result suggests that spermatogenesis failure may result in a severe affectation of sperm DNA integrity. The degree of DNA fragmentation using the SCD test is not reflected in pregnancy chances, and the explanation could be that embryos have been selected.

Human immunodeficiency virus and hepatitis C infections are sexually transmitted diseases that require sperm samples to be pretreated to eliminate the viral presence before their safe use in assisted reproduction treatments. In this report we describe our experience with sperm washing protocols applied to sperm cells from testicular biopsies as well as the results obtained in subsequent assisted reproduction treatments on seropositive males that are also azoospermic.

Sperm analysis following World Health Organization guidelines is unable to explain the molecular causes of male infertility when basic sperm parameters are within a normal range and women do not present gynecologic pathology. Consequently, there is a need for accurate diagnostic tools in this area, and microarray technology emerges as promising. We present, for the first time, preliminary results of a comparison of sperm mRNA expression profiles between fertile and infertile men with normal semen parameters, discovering profound discrepancies between groups, with potential diagnostic and therapeutic possibilities.

Semen analysis, as stated by the World Health Organization, is the only accepted tool to assess male fertility. However its predictive value to assess male capacity to initiate a pregnancy is limited. With the introduction of IVF (especially via intracytoplasmic sperm injection), infertility caused by diminished sperm production is frequently solved, but knowledge of sperm physiology remains very poor. Moreover, a percentage of males with apparently normal semen are unable to impregnate healthy women. Therefore, improvements in the diagnostic tools to assess male fertility potential are necessary. The aim of this review is to describe sperm molecular factors implicated in male fertility, demonstrated by their role in sperm physiology, the molecular differences found between fertile and infertile males, or by their influence on the results obtained in assisted reproduction treatments in terms of embryo quality and pregnancy achievement. From a search and objective evaluation of the currently available evidence, it is concluded that there is no unique factor able to predict male fertility, but several molecular factors are involved in sperm function and can potentially be considered as fertility markers. In this context, a complex molecular tool designed to analyse a battery of parameters seems to be necessary.

MUC1 is a cell surface glycoprotein with a previously described antiadhesive role involved in different aspects of reproductive function. We found MUC1 expressed in male germ cell line and within the ejaculated sperm, but its presence in mature sperm does not seem to be related to male fertility. This was confirmed after analysis of results from assisted reproduction techniques with oocyte donation related with MUC1, although higher MUC1 expression is related to sperm recovery after preparation.

The percentage of spermatozoa with fragmented DNA from cancer patients before surgery, chemotherapy, or radiotherapy treatments was compared with infertile male patients in an assisted reproduction program and with sperm donors of proven fertility. The percentages of DNA fragmentation were 34.3% in cancer patients, 30.9% in infertile men whose partners did not become pregnant, 28.8% in men who partners became pregnant, and 10.8% in fertile sperm donors. The DNA fragmentation of sperm donors was statistically significantly lower compared the other groups. No statistically significant differences were found in the levels of DNA fragmentation when comparing cancer types, including those of testicular origin.

Objective: One byproduct resulting from free radical damage is the DNA hydroxylation also known as DNA oxidation. Our aim with this work was to determine the relevance of sperm DNA oxidation on embryo quality in oocyte donation cycles., Design: We prospectively studied pairs of oocyte donation cycles, i.e., the same oocyte donors, donating to two recipients, where the only difference between the two treatments was the use of a different sperm sample., Setting: University-affiliated private IVF setting., Patient(s): Infertile male partners from couples undergoing oocyte donation cycles (n = 38): 76 semen aliquots analyzed before and after semen processing by swim up., Intervention(s): None., Main Outcome Measure(s): We measured sperm DNA oxidation by flow cytometry using the OxiDNA assay and correlated it with embryo quality parameters, implantation, and pregnancy outcome., Result(s): A positive correlation was seen between embryo fragmentation and DNA oxidation of capacitated samples at 48 hours and 72 hours after fertilization. However, when we analyzed the differences in the IVF outcome parameters of the couples who shared the oocyte cohort (same donor) with the differences in the OxiDNA values, we observed increased and further relationships with cell embryo division 48 hours after fertilization. A negative association with blastocyst formation was also detected., Conclusion(s): Oxidative damage in the DNA is clearly increased in samples with lower sperm motility. An association between early and late embryo quality and sperm DNA oxidation supports the relevance of the hydroxylation of 8-oxoguanine as a biomarker of sperm quality reflecting the free radical damage in human sperm.

Toxic habits and their relationship with male factor infertility have been a matter of investigation in recent years, and smoking is one of the most common lifestyle toxic exposures to harmful substances. The analysis of sperm deoxyribonucleic acid fragmentation after capacitation detected a detrimental effect produced by tobacco, and this deleterious effect alters the sperm swim-up selection process in smokers, although the molecular and cellular basis of this phenomenon remain to be elucidated.

The equilibrium of the creation and scavenging of free radicals is mandatory in the spermatozoa to fertilize and initiate a full-term pregnancy. The glutathione (GSH) enzymatic system studies have discovered its relationship with oxidative stress in the ejaculate and new strategies to regulate its activity in the semen could be developed. Intracellular sperm GSH system components are altered in infertile men, and these alterations seem to be linked to sperm morphology. We have been able to correlate embryo morphology on 8 cell embryos with the sperm expression of GPx family members; this relationship appears quite promising for discovery of molecular causes of male infertility. Oxidative stress imbalance potentially leads to damage of the structure of plasma membrane. The freezing and subsequent thawing of sperm is a physically stressful process carried out during routine procedures in assisted reproduction, which results in a highly variable and unpredictable reduction of motile sperm. Subsequently, oxidative status can positively or negatively affect the motility, viability, and fertilizing capacity of thawed sperm. A reserve of glutathione, together with GPx expression, is necessary to eliminate free radicals using GSH or GPx-4 like structural protein and seems to be essential for a good post thaw recovery.

Objective: To prospectively determine the impact of concrete components of the sperm oxidative glutathione stress system in terms of enzymatic activity and mitochondrial RNA (mRNA) expression on embryo quality and reproductive outcome. Human spermatozoa use the glutathione system to inactivate reactive oxygen metabolites, and there is a close correlation between some components of the glutathione system and male fertility. However, very few data are published regarding this system in sperm cells and its effect on fertilization ability and embryo development in human beings., Design: An oocyte-donation model, used to homogenize the female factor., Setting: University-affiliated private IVF setting., Patient(s): Semen samples from infertile males (n = 43) of couples undergoing oocyte-donation cycles (n = 43)., Intervention(s): None., Main Outcome Measure(s): Gene expression and activity of glutathione peroxidases (GPXs) 1 and 4, glutathione reductase, and intracellular glutathione (GSH) by fluorescent quantitative polymerase chain reaction and spectrophotometry, respectively., Result(s): Fertilization rate, pronuclear number, asymmetry, and pronuclear body distribution were not correlated with any sperm glutathione parameters that were considered. When day 3 embryo parameters were evaluated, only GPX4 mRNA expression in sperm cells was statistically significantly lower when asymmetric embryos were observed. Also, worst embryo development and morphology on day 5 was statistically significantly correlated with lower sperm GPX1 activity (101.07 vs. 258.8 IU/mg protein). Glutathione system analysis in fresh sperm was not statistically significantly different in patients achieving pregnancy compared with those who not, and we did not find any correlation with implantation rate., Conclusion(s): We have been able to correlate embryo morphology on day 3 with the sperm expression of GPX family members. The results indicate that sperm-derived mRNA may condition human embryo quality and persist even to blastocyst stage. The correlation of the sperm GPX family mRNA expression with embryo health appears quite promising for discovery of molecular causes of male infertility.

Men who are seropositive for human immunodeficiency virus (HIV) or hepatitis C virus now can father children safely, thanks to sperm washing and nested polymerase chain-reaction techniques, followed by intracytoplasmic sperm injection. However, in a small percentage of such patients, it is impossible to recover spermatozoa after said procedures because of their highly impaired spermatogenesis. We have established that less rigorous methods, such as repeated centrifugation, yield nested polymerase chain-reaction HIV- and hepatitis C virus-negative specimens, even in sperm samples from men with severe oligoasthenozoospermia.

Background: Sperm DNA integrity has been used as a new marker of sperm quality in the prediction of pregnancy. Nevertheless, no previous study has been performed by analysing the same samples that were employed in assisted reproduction. The main objective of this work was to correlate sperm chromatin dispersion (SCD), measured by the SCD test, with semen parameters and pregnancy outcome in intrauterine insemination (IUI)., Methods: A total of 100 semen samples obtained from males of couples undergoing IUI were analysed by the SCD test before and after swim-up, and the results were correlated with semen parameters and pregnancy outcome., Results: SCD was negatively correlated with sperm motility in both ejaculated and processed semen. Sperm recovered by swim-up did not show a significant improvement in DNA integrity. No correlation was found between SCD and pregnancy outcome in IUI., Conclusions: DNA dispersion, as measured by the SCD test, is not correlated with pregnancy outcome in IUI.

Objective: Oncologic treatments can destroy spermatogenic dividing cells and cause azoospermia which could be irreversible. Sperm banking is the best option to preserve male fertility after these treatments. It is easy, inexpensive, and safe. To date, few clinical data are available about large series of cancer patients. Our objective was to determine the usefulness of these preventive sperm freezing protocols., Design: Prospective study., Setting: University-affiliated private fertility center., Patient(s): One hundred eighty-six cancer patients who banked sperm samples at our center before surgery or chemo- or radiotherapy treatments from 1991 to 2004., Intervention(s): Conjugal status, age, type of cancer, treatment, and future use (if any) of the cryopreserved sperm samples for assisted reproduction technology (ART), and cycle results were recorded, analyzed, and compared with a control group., Main Outcome Measure(s): Basic sperm analysis of semen samples from cancer patients prior to freezing, after thawing, and after capacitation for ART., Result(s): A total of 320 semen samples were frozen before antineoplasic treatment. Six months later, 27% of the males recovered normal sperm production. From all frozen samples, 8.7% were discarded; the reasons were pregnancy achievement (55%), normal sperm production (28%), and patient death (18%). Finally, 5 IUI cycles and 30 ICSI cycles were done from frozen samples, with 1 and 15 pregnancies, respectively; results were comparable with those obtained in a control group., Conclusion(s): A significant number of males who cryopreserved semen samples before receiving antitumoral treatments have employed them. The results obtained showed that this is the strategy of choice, aiming to preserve fertility for the future, because the cost/benefit ratio is favorable. Patients should be counseled accordingly.

Objective: To determine the prognostic value of sperm DNA fragmentation levels, as measured by the sperm chromatin dispersion (SCD) test, in predicting IVF and ICSI outcome., Design: Double-blind prospective study., Setting: University-affiliated private IVF setting., Patient(s): A total of 85 couples undergoing infertility treatment with IVF/ICSI., Intervention(s): Analysis of DNA fragmentation by the SCD test in 170 aliquots obtained from the ejaculate and from the processed semen used for assisted reproductive technologies (ART)., Main Outcome Measure(s): Percentage of spermatozoa with fragmented DNA was statistically correlated with embryo quality and reproductive success., Result(s): Fertilization rate was inversely correlated with DNA fragmentation (r = -0.245 P = .045). Higher DNA fragmentation rate gave an increased proportion of zygotes showing asynchrony between the nucleolar precursor bodies of zygote pronuclei (73.8% vs. 28.8% P < .001). In addition, the slower embryo development and worst morphology on day 6 was correlated with higher sperm DNA fragmentation (47.7% vs. 29.4% P = .044). We also observed a negative correlation between DNA fragmentation and the implantation rate (r = -0.250 P = .042). However, SCD test values were not statistically different in cycles that resulted in a pregnancy compared with those that did not (33.2 vs. 28.2 and 32.4 vs. 34.7)., Conclusion(s): This is the first report that describes a correlation between sperm DNA integrity, as measured by the SCD test, and fertilization rate, embryo quality, and implantation rate in IVF/ICSI. The degree of DNA fragmentation was inversely correlated with fertilization rate, synchrony of the nucleolar precursor bodies' pattern in pronuclei, embryo ability to achieve blastocyst stage, and embryo morphological quality. Because SCD test values were correlated with embryo quality and blastocyst rate, the lack of correlation between sperm DNA fragmentation and pregnancy outcome in IVF might be due to embryo selection before transfer. The ability of the SCD test to predict the blastocyst rate after IVF/ICSI warrants further study.

Background: Human immunodeficiency virus (HIV)/hepatitis C virus (HCV)-seropositive males can now father children safely, avoiding transmission risks to the mother and the children using sperm washing and nested PCR (nPCR) techniques. Nevertheless, we still lack enough data to determine the reasons why approximately 10% of the performed sperm washes remain positive, thus forcing the repetition of the treatment. Semen quality in infected males is also essential to these procedures. We aimed to determine the predictive value of the semen parameters, sperm washing procedure and the infection status for the post-wash viral positivity, as well as the correlation between the semen and the disease features., Methods: Semen characteristics were evaluated in 136 samples provided from 125 males. We also included a control group of 125 males matched by age and length of sexual abstinence. At the time of semen retrieval, 70 of them were infected with HIV (45 also with HCV, 64.3%), and 55 of them with HCV alone. nPCR for viral detection was performed in each sample., Results: Thirteen out of 136 (9.5%) of the samples were positive for one or more viral detections (HIV RNA, HIV DNA and HCV RNA, when needed). From a total of 240 nPCR viral analyses, 16 were positive (6.6%). None of the seminal parameters were adequate to predict post-wash results, nor was a positive result dependent on the volumes used in the semen wash. A positive correlation was found between post-wash progressive motility and CD4 blood levels, as well as a negative correlation between progressive motility and time of evolution of the disease in HIV-infected males., Conclusions: Semen analysis, according to the World Health Organization criteria, of HIV- and HCV-affected patients showed no differences from that of non-infected males. Moreover, low CD4 blood levels, and a long evolution of the disease do not negatively affect sperm motility.

Background: Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) serodiscordant couples with the male infected can be helped to have children minimizing the transmission risk. Our aim was to evaluate the results of our assisted reproduction programme for these couples and to develop adequate strategies for their management., Methods: Members of serodiscordant couples: (i) HIV positive males attending our centre for sperm wash and assisted reproduction and (ii) HCV positive males needing assisted reproduction for infertility provided 134 semen samples for sperm wash. Before ICSI treatment, semen was confirmed to be negative for viral presence by reverse transcription and nested PCR after the sperm wash., Results: Sperm washes were effective in 90% of the samples. Regardless of the type of infection, no differences were found in semen quality, embryos obtained and pregnancy rates (40-48% per cycle). To date, 41 pregnancies and 23 newborns were obtained. Fertilization rates were lower in HCV than in HIV serodiscordant couples (59.3+/-5.3% versus 72.0+/-8.1%), probably because they were infertile couples for whom we recommended sperm wash and PCR. No seroconversion was detected in the patients' follow-up., Conclusions: To date, sperm wash, nested PCR and ICSI is a safe and effective procedure that avoids HIV and HCV transmission with reasonable pregnancy rates, and is cost-effective.