Mark Grossmann

Spermatozoa from patients with seminal alterations exhibit a differential micro-ribonucleic acid profile

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.

Semen samples showing an increased rate of spermatozoa with imprinting errors have a negligible effect in the outcome of assisted reproduction techniques

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.

Lack of association of MTHFR rs1801133 polymorphism and CTCFL mutations with sperm methylation errors in infertile patients

PurposeTo find out whether the MTHFR rs1801133 polymorphism is a risk factor for male infertility in the Spanish population. To determine if a pattern of sperm DNA hypomethylation at the paternally imprinted loci H19-ICR and/or IG-DMR is related to the MTHFR rs1801133 polymorphism and/or CTCFL mutations.MethodsOne hundred and seven samples from individuals who sought consultation for fertility problems and twenty-five semen samples from sperm donors were analyzed. The MTHFR rs1801133 SNP was analyzed in all samples by the PCR-RFLP method. We compared the distribution of the genotypes between control and infertile populations and among the groups of patients with altered seminal parameters. In those patients with the most severe hypomethylation pattern (n = 12) we also analyzed the CTCFL protein-coding exons by sequencing.ResultsThere were no significant differences in the distribution of the genotypes among the control and infertile populations. Moreover, none of the genotypes were associated, neither to the characteristics of the seminogram, nor to the presence of sperm DNA hypomethylation. We did not identify frameshift, nonsense or missense mutations of the CTCFL gene.ConclusionsThe MTHFR rs1801133 polymorphism is not associated with male infertility in the Spanish population. Neither the MTHFR polymorphism, nor CTCFL mutations explain a pattern of sperm hypomethylation at paternally imprinting loci.

The decision to cancel a preimplantation genetic diagnosis cycle.

It has been suggested that a minimum number (six) of cumulus‐oocyte complexes (COCs) should be retrieved for fertilization to offer enough chances to ensure a pregnancy after a preimplantation genetic diagnosis (PGD) procedure. Therefore a decision to cancel a PGD cycle should be adequately weighted to offer the patients the highest chances to obtain a pregnancy. We describe a case where, after retrieving only three COCs suitable for fertilization, a triplet pregnancy was obtained. This case suggests that, although low numbers of COCs can reduce the effectiveness of the PGD procedure, other factors are involved in its final result. Thus, the opportunity of routinely cancelling such cycles should be reconsidered. In addition, this is, to our knowledge, the first case where sex selection was carried out to prevent the birth of carriers of the abnormal gene, and not of affected offspring. Copyright

Spermatozoa from infertile patients exhibit differences of DNA methylation associated with spermatogenesis-related processes: an array-based analysis

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.

Plasma membrane and cytocortex alterations in frozen/thawed mouse embryos deprived of the zona pellucida

We analyzed the morphological and ultrastructural abnormalities present in two-cell mouse embryos frozen/thawed without the zona pellucida (ZP). Transmission electron microscopy revealed that these embryos had plasma membrane abnormalities that were not observed in the embryos frozen/thawed with an intact ZP. The most frequent anomalies were a decreased number of microvilli with a nonhomogeneous distribution and showing an abnormal morphology and the presence of an increased number of vesicles in the periphery of the cell. The distribution of actin filaments, studied by fluorescence microscopy revealed alterations in both embryos, frozen/ thawed with ZP and embryos frozen/thawed without ZP: an increased staining in some regions of the peripheral actin band, discontinuities or gaps in the band, or the presence of a second actin band connected to the peripheral one.