Expression of miRNA from spent pre-implantation embryos culture media.

This study examines the expression of three microRNAs (hsa-miR-661, hsa-miR-21-5p, hsa-miR-372-5p) in spent pre-implantation embryos culture media to identify possible new non-invasive biomarkers of embryo competence, predictive of development to the blastocyst stage. A preliminary analysis on 16 patients undergoing IVF cycles was performed by collecting and stored spent culture media on the fifth/sixth day of embryo culture. Expression of miRNAs was evaluated according to the embryos' fate: 1) NE/DG: non-evolved or degenerate embryos; 2) BLOK: embryos developed to the blastocyst stage. Preliminary results revealed a higher miRNAs expression in NE/DG spent media. To elucidate the roles of these miRNAs, we employed a robust bioinformatics pipeline involving: 1) in-silico miRNA Target Prediction using RNAHybrid, which identified the most-likely gene targets; 2) Construction of a Protein-Protein Interaction network via GeneMania, linking genes with significant biological correlations; 3) application of modularity-based clustering with the gLay app in Cytoscape, resulting in three size-adapted subnets for focused analysis; 4) Enrichment Analysis to discern the biological pathways influenced by the miRNAs. Our bioinformatics analysis revealed that hsa-miR-661 was closely associated with pathways regulating cell shape and morphogenesis of the epithelial sheet. These data suggest the potential use of certain miRNAs to identify embryos with a higher likelihood of developing to the blastocyst stage. Further analysis will be necessary to explore the reproducibility of these findings and to understand if miRNAs here investigated can be used as biomarkers for embryo selection before implantation into the uterus or if they may be reliable predictors of IVF outcome.

Calprotectin as a novel diagnostic approach to screen male infertility risk: A pilot study.

RESEARCH QUESTION: Diagnosis of male infertility is essentially based on the evaluation of semen quality (sperm concentration, motility, viability, and morphology). However, there is a lack of knowledge about possible molecules used as candidates for the early identification of male infertility risk. Calprotectin is a biological marker for inflammation, measured prevalently in stool specimens, widely used to discriminate between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), and for the subsequent monitoring of gastrointestinal diseases' development. Would it be possible to use calprotectin determination to identify also male infertility risk? DESIGN: Cross-sectional pilot study investigated calprotectin concentration in the seminal fluid of 45 men (range: 23-51 yrs) that were under evaluation for semen quality at our Center for Reproductive Medicine. Calprotectin concentration was determined with a commercially available immuno-chromatographic test and successfully detected in 37 of the 45 analyzed men (age: 37.38 ± 6.59). A correlation with semen quality (concentration, motility, morphology) was assessed. RESULTS: Higher calprotectin concentration seemed to indicate a better quality of the seminal fluid. Normozoospermic subjects (Group A) had on average a calprotectin value of 0.215 ± 0.162 µg/ml (mean ± SD), while subjects with at least one of the semen parameters below reference values (Group B) showed lower calprotectin concentration (mean ± SD: 0.126 ± 0.068, p-value < 0.05). A significant difference was clearly evident between calprotectin concentration measured in seminal fluids with physiological sperm morphology (≥4%) as compared with teratozoospermic samples (<4%) (p-value < 0.05). Indeed, the developed ROC curves showed a good diagnostic accuracy (around 67 %) using calprotectin concentration (threshold value: 0.121 µg/ml) as a preliminary test to discriminate subjects with and without abnormal semen parameters, especially morphology. CONCLUSIONS: Calprotectin determination in the seminal fluid may be proposed as a biological marker for preliminary screening in male subjects at risk of infertility due to one or more alterations of semen quality.

Myeloperoxidase and lactoferrin expression in semen fluid: Novel markers of male infertility risk?

RESEARCH QUESTION: Infections and/or inflammation processes of male genital tract are highly prevalent and often associated with risk of infertility. These conditions represent a possible cause of leukocytospermia, which is still under debate. Leukocytes are key-factors to reactive oxygen species (ROS) production and the increase of ROS in semen fluid is associated with the worsening of semen parameters. At present, there are not appropriate andrological tests to identify asymptomatic inflammatory conditions when the amount of leukocytes is in the normal range. DESIGN: We studied the innate immunity profile of myeloperoxidase and lactoferrin (MPO/LAC) proteins expressed in the semen fluid of 39 men evaluated for couple infertility, in the absence of leukocytospermia. RESULTS: The presence of both MPO and LAC proteins was associated with a decrease of sperm concentration and of progressive/total motility, whereas the increase of MPO-/LAC + indicated a worse sperm morphology. It is worth to report the predictive potential of MPO+/LAC + pattern (above 4.36 %) as a biological marker to distinguish normozoospermic from pathological patients. CONCLUSION: Our findings indicate MPO/LAC analysis as a potential diagnostic tool to identify asymptomatic conditions eventually related to male infertility, even when the number of leukocytes in semen fluid is below 1 million/mL.