Infertility is an important reproductive health problem, and male infertility is especially important in more than half of infertility cases. Due to the importance of genetic factors in this condition, analysis of semen alone is not enough to recognize men with idiopathic infertility. A molecular non-invasive investigation is necessary to gain valuable information. Currently, microRNAs (miRNAs) are being used as non-invasive diagnostic biomarkers. miRNAs, single-stranded non-coding RNA molecules, act as post-transcriptional gene silencing regulators either by inhibition or repression of translation. Changes in the regulation of miRNAs have been investigated in several different types of male infertility, therefore the biological role of miRNA and gene targets has been defined. The purpose of this study was to review recent research on the altered expression of miRNA in semen, sperm, and testicular biopsy samples in infertile males with different types of unexplained infertility. Changes in miRNA regulation were investigated using microarray and the miRNA levels were confirmed by real-time qRT-PCR. This review explains why creating a non-invasive diagnostic method for male infertility is necessary and how changes in miRNA expression can be used as new diagnostic biomarkers in patients with differing spermatogenic and histopathologic injury.

Several studies have proposed that cell-free DNA (cfDNA) is a potential biomarker present in follicular fluid (FF) for oocyte quality. Recently we reported that mitochondria-derived cfDNA (mt-cfDNA) closely reflects the amount of cfDNA in FFs. The present study investigated the mechanism regulating mt-cfDNA secretion from porcine granulosa cells. Oocytes and cumulus cell complexes or granulosa cells (GCs) were cultured in maturation medium for 24 or 48 h respectively. Then, nuclear-derived cell-free DNA (n-cfDNA) or mt-cfDNA contents in the spent medium were examined using real-time polymerase chain reaction. When 10 μM of MG132, a proteasome inhibitor, was added to the culture medium, cellular viability of both COCs and GCs decreased and n-cfDNA significantly increased in the culture medium, whereas mt-cfDNA significantly decreased. Supplementation of the culture medium with GW4869, an inhibitor of intracellular vesicle formation, significantly decreased the mt-cfDNA, whereas no effect was observed on n-cfDNA in the medium of both COCs and GCs. Furthermore, the addition of bafilomycin, an inhibitor of autophagy to the culture medium significantly increased mt-cfDNA in the culture medium. After filtration (0.22 μm) and centrifugation (23,000 g), the mt-cfDNA content of the medium decreased significantly. In conclusion, the proteasomal mitochondrial quality control system is upstream of mt-cfDNA secretion and autophagy plays a role in cellular digestion of mitochondrial DNA in the cytoplasm. It is further suggested that dsDNA is enclosed in certain vesicles or associated with small molecules and secreted into the medium.

Vitrification is a highly efficient technique for the cryopreservation of the human embryo. The effect of delayed blastulation may be responsible for implantation failures and negatively affects in vitro fertilization (IVF) outcomes. The current literature displays discordant results; some studies have announced higher pregnancy rates after day 5 (D5) transfer compared with day 6 (D6) transfer, while others have shown equivalent outcomes. In the present study an investigation into the clinical implications of delayed blastulation (D5 versus D6) was carried out. We performed a retrospective study comparing clinical pregnancies and implantation rates following warmed single blastocyst transfer (WSBT). All patients coming for a programmed warmed transfer at Edinburgh Assisted Conception Programme, EFREC, Royal Infirmary of Edinburgh, were included in this study and divided in two groups according to the day of blastocyst vitrification: D5 (n = 1563) and D6 (n = 517). The overall survival rate was 95.0% (1976/2080) with no significant difference between the D5 and D6 groups: 95.3% (1489/1563) and 94.2% (487/517) respectively. WSBT of D6 blastocysts resulted in a lower implantation and clinical pregnancy compared with D5 embryos. The implantation rate (IPR) and clinical pregnancy rate (CPR) were respectively 49.4% and 42.6% for the D5 and 37.4% and 32.2% for the D6 embryos, which was statistically significant. The multiple pregnancy rate was 1.32% (1.14% for D5 vs 1.84% for D6). Although the transfer of D6 vitrified-warmed blastocyst remains a reasonable option, priority to a D5 embryo would reduce the time to successful pregnancy.

Ovulation is considered an inflammatory, cytokine-mediated event. Cytokines, which are recognized as growth factors with immunoregulatory properties, are involved in many cellular processes at the ovarian level. In this sense, cytokines affect fertility and are involved in the development of different ovarian disorders such as bovine cystic ovarian disease (COD). Because it has been previously demonstrated that ovarian cells represent both sources and targets of cytokines, the aim of this study was to examine the expression of several cytokines, including IL-1β, IL-1RA, IL-1RI, IL-1RII, IL-4 and IL-8, in ovarian follicular structures from cows with spontaneous COD. The protein expression of these cytokines was evaluated by immunohistochemistry. Additionally, IL-1β, IL-4 and IL-8 concentrations in follicular fluid (FF) and serum were determined by enzyme-linked immunosorbent assay (ELISA). In granulosa and theca cells, IL-1RI, IL-1RII, IL-1RA and IL-4 expression levels were higher in cystic follicles than in the control dominant follicles. The serum and FF concentrations of IL-1β and IL-4 showed no differences between groups, whereas IL-8 concentration was detected only in FF of cysts from cows with COD. The FF and serum concentrations of IL-1β and IL-8 showed no significant differences, whereas IL-4 concentration was higher in FF than in serum in both the control and COD groups. These results evidenced an altered expression of cytokines in ovaries of cows with COD that could contribute to the pathogenesis of this disease.

Intracytoplasmic sperm injection (ICSI) is performed in cases of infertility by injecting a motile and morphologically normal sperm cell under a routine ×400 magnification at which is hard to distinguish morphologically healthy sperm. Recently, the use of high-powered differential interference contrast optics gave the opportunity to select a sperm under ultra-high magnification of ×10,160. The aim of the present study was to evaluate the efficacy of the intracytoplasmic morphologically selected sperm injection (IMSI) technique in different infertility populations undergoing ICSI. Main outcome measures of routine ICSI were compared with IMSI in three different groups of patients (1, non-selected; 2, male infertility; and 3, repeated implantation failure group). Results were analysed to evaluate the effects of the IMSI procedure and to find the most suitable group of patients who may benefit from the procedure. IMSI caused a significant increase in the fertilization and top quality embryo rates in the male infertility group and a significant increase in fertilization and pregnancy rates in the repeated implantation failure group, whereas no effect was observed in the non-selected group with patients of various indications. A positive effect of IMSI on the outcome of male factor infertility and repeated implantation failure patients was observed. Data observed confirmed that the application of IMSI was beneficial for a selected group of patients with male factor infertility and repeated implantation failure.

Male germ cell development is a critical period during which epigenetic patterns are established and maintained. The progression from diploid spermatogonia to haploid spermatozoa involves the incorporation of testis-specific histone variants, mitotic and meiotic divisions, haploid gene expression, histone–protamine transitions and massive epigenetic reprogramming. Understanding the protein players and the epigenetic mark network involved in the setting of the epigenetic programme in spermatogenesis is an exciting new clue in the field of reproductive biology with translational outcomes. As information in the existing literature regarding cross-talk between DNA methylation and histone hyperacetylation in the advanced stages of murine spermatogenesis is still scarce and controversial we have investigated the effect of a DNA-methyltransferase inhibitor, 5-aza-2′-deoxycytidine, at the cytological and molecular level (by transmission electron microscopy, immunocytochemistry and immunoprecipitation methods). Our results revealed an important role for regulation of DNA methylation in controlling histone hyperacetylation and chromatin remodelling during spermatogenesis.

We compare the efficiency of mechanical or enzymatic methods, and their combination, for the isolation of ovarian preantral follicles (PFs) from collared peccaries. The ovaries from six females were subjected to the different methods investigated here. For the enzymatic method, ovary fragments were exposed to collagenase type IV in TCM-HEPES medium; the mechanical procedure was based on ovarian cortex dissociation by using a scalpel blade. The residual solution obtained after the mechanical isolation was subjected to the enzymatic procedure. The number of isolated PFs was quantified and classified as primordial, primary, or secondary; their viability was assessed using trypan blue dye assay. To confirm the results, PFs derived from the most efficient method were evaluated for integrity using scanning electron microscopy (SEM) and subjected to a 24 h in vitro culture for subsequent evaluation of viability by using fluorescent probes. A higher number of PFs (P < 0.05) was obtained from the enzymatic method (961.7 ± 132.9) in comparison with the mechanical method (434.3 ± 88.9), but no difference was observed between the two methods and their combination (743.2 ± 92.8). The trypan blue assay showed that the enzymatic method (98.7 ± 0.6%) provided the highest percentage of viable follicles (P < 0.05). Furthermore, SEM confirmed the ultrastructural integrity of the surface architecture of peccary PFs isolated by the enzymatic procedure; epifluorescence microscopy was used to confirm their viability (86.0%). In conclusion, we suggest that the enzymatic method investigated here is useful for the isolation of viable ovarian PFs from collared peccaries.

Around 60–80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus–oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

Mammalian Pou5f1 encodes the POU family class V (POU-V) transcription factor which is essential for the pluripotency of embryonic cells and germ cells. In vertebrates, various POU-V family genes have been identified and classified into the POU5F1 family or its paralogous POU5F3 family. In this study, we cloned two cDNAs named CpPou5f1 and CpPou5f3, which encode POU-V family proteins of the Japanese red bellied newt Cynops pyrrhogaster. In the predicted amino acid sequence encoded by CpPou5f1, the typical MAGH sequence at the N-terminus and deletion of arginine at the fifth position of POU-homeodomain were recognized, but not in the sequence encoded by CpPou5f3. Phylogenetic analysis using Clustal Omega software indicated that CpPou5f1 and CpPou5f3 are classified into the clade of the POU5F1 and POU5F3 families, respectively. In a real-time polymerase chain reaction (RT-PCR) analysis, the marked gene expression of CpPou5f1 was observed during oogenesis and early development up to the tail-bud stage, whereas weak gene expression of CpPou5f3 was detected only in the early stages of oogenesis and gastrula. In adult organs, CpPou5f1 was expressed only in the ovary, while gene expression of CpPou5f3 was recognized in various organs. A regeneration experiment using larval forelimb revealed that transient gene expression of CpPou5f1 occurred at the time of wound healing, followed by gene activation of CpPou5f3 during the period of blastema formation. These results suggest that CpPou5f1 and CpPou5f3 might play different roles in embryogenesis and limb regeneration.

The aim of this study was to evaluate the effects of different timing for frozen–thawed bovine ampullary epithelial cell (BAEC) and bovine oviductal epithelial cell (BOEC) co-culture on the development and quality of bovine embryos produced in vitro. Embryo development was assessed by day 8 blastocyst yield, whereas embryo quality was determined using blastocyst differential cell count, cryotolerance and the expression of selected genes related to embryo quality. The results showed that the presence of BAECs during the last 6 h of in vitro maturation (IVM) increased blastocyst yield and survival of the vitrified-warmed blastocysts. In addition, embryos produced in the presence of BAECs during the last 6 h of IVM or in the presence of BOECs during the first 4 days of in vitro culture (IVC) showed a greater number of trophectoderm cells and a greater inner cell mass. In terms of gene expression, IFN-T was downregulated and PLAC8, AQP3 and ATP1A1 were upregulated in the presence of the BAECs during the last 6 h of the IVM and/or in the presence of BOECs during the first 4 days of IVC. In conclusion, co-culturing bovine oocytes with a frozen–thawed ampullary cell monolayer during the last 6 h of maturation increased blastocyst yield and quality.

The aim of this study was to investigate if there is an adverse effect of multiple controlled ovarian stimulation (COS) on the maturity of oocytes (MI and MII), fertilization rate, embryo developmental qualities and clinical pregnancy rates in donation cycles. In total, 65 patients undergoing oocyte donation cycles multiple times were included in this study. Patients were grouped as group A that consisted of donors with ≤2 stimulation cycles while B consisted of donors with ≥3 stimulation cycles; and group C included donors who had ≤15 oocytes, while group D had donors with ≥16 oocytes. Numbers of oocytes obtained, MI and MII oocytes, fertilization, embryo quality and clinical pregnancy outcomes were compared. Significant statistical differences were observed in total number of oocytes obtained, maturity of oocytes (MI and MII), fertilization rate, embryo qualities and clinical pregnancy outcomes of donors in groups A–D. Donors with ≤2 ovarian stimulation cycles had lower numbers of immature oocytes than donors with three or more stimulation cycles. However, donors with ≥3 stimulation cycles had higher numbers of mature oocytes, zygotes, with better day 3 embryo qualities and higher clinical pregnancy rates than donors with ≤2 stimulation cycles. Repeated COS does not seem to have any adverse effect on ovarian response to higher dose of artificial gonadotropin, as quality of oocytes collected and their embryological developmental potential were not affected by the number of successive stimulation cycles. The effect of multiple COS on the health of the oocyte donor needs to be assessed for future purpose.

Activated pERK1/2 and pAKT are key players in supporting cell survival and proliferation pathways. Translocation of pERK1/2 into the nucleus, where it interacts with transcription factors and DNA itself, is instrumental in exerting an anti-apoptotic effect. In this study, pAKT levels, pERK1/2 nuclear localization and DNA fragmentation index (DFI) in cumulus cells of single cumulus–oocyte complexes of patients undergoing in vitro fertilization programmes were evaluated and correlated with the clinical outcome of the related embryos. For a positive clinical outcome of blastocyst development, pERK1/2 nuclear localization and DFI value had a significant inverse relationship, whereas the former and the intracellular accumulation of pAKT had a significant direct relationship. This relationship was not observed for the negative clinical outcome of the arrested embryos. Moreover, intracellular accumulation of pAKT and DFI value had a significant inverse relationship in all samples examined. The obtained data suggest that the intranuclear relocation of pERK1/2, along with an enhanced intracellular accumulation of pAKT, may exert a survival effect and increase cell viability, therefore providing a novel marker tool to choose the best oocyte to be fertilized and submitted to an intracytoplasmic sperm injection cycle.