Studies have shown that daily exposure to different products, whether chemical or natural, can cause irreversible damage to women’s reproductive health. Therefore it is necessary to use tests that evaluate the safety and efficacy of these products. Most reproductive toxicology tests are performed in vivo. However, in recent years, various cell culture methods, including embryonic stem cells and tissues have been developed with the aim of reducing the use of animals in toxicological tests. This is a major advance in the area of toxicology, as these systems have the potential to become a widely used tool compared with in vivo tests routinely used in reproductive biology and toxicology. The present review describes and highlights data on in vitro culture processes used to evaluate reproductive toxicity as an alternative to traditional methods using in vivo tests.

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.

The optimum oxygen tension for culturing mammalian embryos has been widely debated by the scientific community. While several laboratories have moved to using 5% as the value for oxygen tension, the majority of modern in vitro fertilization (IVF) laboratory programmes still use 20%. Several in vivo studies have shown the oxygen tension measured in the oviduct of mammals fluctuates between 2% and 8% and in cows and primates this values drops to <2% in the uterine milieu. In human IVF, a non-physiological level of 20% oxygen has been used in the past. However, several studies have shown that atmospheric oxygen introduces adverse effects to embryo development, not limited to numerous molecular and cellular physiology events. In addition, low oxygen tension plays a critical role in reducing the high level of detrimental reactive oxygen species within cells, influences embryonic gene expression, helps with embryo metabolism of glucose, and enhances embryo development to the blastocyst stage. Collectively, this improves embryo implantation potential. However, clinical studies have yielded contradictory results. In almost all reports, some level of improvement has been identified in embryo development or implantation, without any observed drawbacks. This review article will examine the recent literature and discusses ongoing efforts to understand the benefits that low oxygen tension can bring to mammal embryo development in vitro.

The aim of this study was to determine the capacity of the meiotic spindle (MS) to predict embryo implantation and live birth rates. For this purpose, we performed a broad systematic literature search. Of all publications retrieved, only those in which the implantation rates were related to some characteristics of the MS were evaluated. Despite the different methodology used in all the chosen studies, presence of the MS in oocytes was found to be positively associated with embryo implantation. Moreover, high retardance values, as well as strict criteria of normality in the MS structure, are significantly related to higher embryo implantation numbers and live birth rates.

Intraspecific and interspecific cloning via somatic cell nuclear transfer (iSCNT) is a biotechnique with great possibilities for wild mammals because it allows the maintenance of biodiversity by recovering species, nuclear reprogramming for the production of pluripotency-induced cells, and studies related to embryonic development. Nevertheless, many areas in cloning, especially those associated with wild mammals, are still in question because of the difficulty in obtaining cytoplasmic donor cells (or cytoplasts). Conversely, donor cell nuclei (or karyoplasts) are widely obtained from the skin of living or post-mortem individuals and often maintained in somatic cell banks. Moreover, the creation of karyoplast–cytoplast complexes by fusion followed by activation and embryo development is one of the most difficult steps that requires further clarification to avoid genetic failures. Although difficult, cloning different species, such as wild carnivores and ungulates, can be successful via iSCNT with embryo development and the birth of offspring. Thus, novel research in the area that contributes to the conservation of biodiversity and knowledge of the physiology of species continues. The present review presents the failures and successes that occurred with the application of the technique in wild mammals, with the goal of helping future work on cloning via iSCNT.

Spermatogenesis is a dynamic process that culminates in the production of mature spermatozoa in the seminiferous tubules of sexually mature animals. Although sperm leaving the testis are fully differentiated, they must further undergo two additional maturation steps before acquiring the capability to fertilize the egg. Such processes take place during the epididymal residency and transport in the seminal fluid during ejaculation and, after delivery into the female reproductive tract, during the journey aiming the encountering the egg in the oviduct. Throughout this trip, spermatozoa are exposed to different reproductive fluids whose molecular compositions regulate the progress towards obtaining a fertilized competent cell. This review summarizes the evidence obtained so far supporting the participation of male and female reproductive tract-derived proteins in the modulation of sperm fertilizing ability and discusses the mechanisms by which such regulation may be accomplished.

We isolated and characterized Xenopus tropicalis hb4 flanking DNA and showed that the −3076/+29 sequence was able to drive stage-specific transcription in the developmental process. Transgenic reporter analysis indicated that green fluorescent protein was expressed in the ovaries of female frogs at 3 months of age and in both the ovaries and testis of frogs at 6 months of age. A series of experiments with deletion of the flanking sequence and a subsequent luciferase reporter assay revealed that there were two positive regulatory regions and that the most proximal sequence of the promoter region had a certain level of transcriptional activity in oocytes. Subsequently, we showed that a conserved sequence containing Nobox-binding element (NBE) was essential for transcriptional activation and that Nobox expressed in the ovary had a crucial role in hb4 transcription through the NBE sequence.

The effect of different dilution rates and equilibration times on the cryopreservation of Bactrian camel spermatozoa was evaluated in the current study. Semen samples from four healthy adult males were collected, processed and pooled. They were then subjected to a completely randomized 4×2 factorial design including four dilution rates (DR; 1:1, 1:2, 1:4 or 1:8; v:v with SHOTOR diluent) and two equilibration times (ET; 1 or 2 h at 5ºC). After freezing and thawing, sperm kinematic parameters as well as viability, plasma membrane integrity, abnormality and seminal malondialdehyde level were assessed. According to the results, four-fold diluted samples recorded significantly higher values (P < 0.05) for sperm total (39.58 vs 31.83 and 33.33,%) and progressive motility (19.50 vs 14.00 and 14.25,%), viability (55.37 vs 43.50 and 48.75,%) and plasma membrane integrity (46.75 vs 37.25 and 37.37,%) than those of both less (1:1) and high (1:8) concentrated samples, respectively. By contrast, the percentage of abnormal spermatozoa and the concentration of seminal malondialdehyde were comparable among all treated groups. Moreover, ET revealed that 1 h equilibration had significantly higher sperm motility (37.04 vs 33.33%), linearity (42.29 vs 32.26%), beat cross-frequency (13.15 vs 8.70 Hz), plasma membrane integrity (42.25 vs 39.75%) and viability (51.37 vs 48.12%) compared with 2 h of ET (P < 0.05). Taken together, a four-fold dilution along with 1 h equilibration can be an optimal procedure to cryopreserve Bactrian camel sperm.

Melatonin plays a critical role in several types of cells as an antioxidant to protect intracellular molecules from oxidative stress. The anti-oxidation effect of melatonin in yak embryos is largely unknown. We report that melatonin can protect the development of yak preimplantation embryos against oxidative stress induced by hydrogen peroxide (H2O2). Therefore, the quality of blastocysts developed from zygotes exposed to H2O2 was promoted. In addition, we observed that melatonin reduced H2O2-induced intracellular reactive oxygen species (ROS) levels and prevented mitochondrial dysfunction in zygotes. These phenomena revealed the effective antioxidant activity of melatonin to prevent oxidative stress in yak embryos. To determine the underlying mechanism, we further demonstrated that melatonin protected preimplantation embryos from oxidative damage by preserving antioxidative enzymes. Collectively, these results confirmed the anti-oxidation effect of melatonin in yak embryos that significantly improved the quantity and quality of blastocysts in the in vitro production of embryos in yaks.

The aim of the present study was to determine whether clinical or laboratory factors can influence the development of single pronucleated zygotes (1PN) and two polar bodies (PB) after ICSI. In total, 341 ICSI cycles performed at FertiClinic-Villa Margherita from January 2012 to December 2014 were enrolled in the study. Group A included 240 cycles with no 1PN−2PB while group B included 101 cycles with one or more 1PN−2PB. Age, stimulation protocol, infertility factor, amount of gonadotropin administered, duration of therapy, peak estradiol levels, number of follicles at maturation triggering, oocytes retrieved and mature oocytes, time between retrieval and injection and sperm characteristics were compared between groups. In opposition to previous results showing no relationship between 1PN occurrence and clinical or laboratory variables, we observed that 1PN−2PB zygote formation seems to be associated with a lower female age, higher level of E2 and higher number of follicles on day of oocyte maturation triggering, higher number of astenozoospermic male patients, more oocytes retrieved at pick-up, more mature oocytes (MII) and longer time to injection.

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.

The aim of this study was to compare different concentrations of soy lecithin (LEC0.01%, LEC0.05% and LEC0.1%) with egg yolk (Control) in cooling extenders during the storage of semen at 5ºC for 5 days. Twelve dogs (n = 12) were selected, and semen was cooled and assessed after 2, 24, 48, 72, 96 or 120 h. At each time point, sperm were analyzed for kinetic patterns (using computer-assisted sperm analysis), mitochondrial activity (3′3- diaminobenzidine assay), lipid peroxidation (TBARS assay), DNA fragmentation (SCSA®) and plasma and acrosome membrane integrity (eosin/nigrosin and fast green/rose Bengal stains, respectively). The Control group (1814.4 ± 197.2) presented the highest rates of lipid peroxidation at 120 h. Conversely, progressive motility (42.8 ± 4%), linearity (45.4 ± 1%), and VAP (88 ± 3%) were higher in the Control group. In addition, there was lower mitochondrial activity in the Control group at 72 h. Therefore, our data show that lecithin used at these concentrations was not able to maintain sperm viability at as high qualities as would egg yolk. Moreover, the decrease in high mitochondrial activity and the persistence of sperm motility may indicate a compensatory mechanism in canine spermatozoa (i.e., glycolytic pathway). Furthermore, these higher lipid peroxidation indexes could indicate the necessity for future therapy using extenders and antioxidants over a long cooling time for dog sperm.

Direct swim-up procedure is widely used to separate the motile competent spermatozoa from the antioxidant-rich semen. Subsequently, spermatozoa become more vulnerable to reactive oxygen species (ROS) due to their cytological characteristics. The effect of vitamin C, a highly concentrated antioxidant in the semen, on direct swim-up-enriched sperm population is not fully investigated. Therefore, the aim of the present study was to assess the effect of vitamin C on sperm functional properties during direct swim-up procedure. Semen samples were collected from 22 participants. Each semen sample was divided into several aliquots. The first portion was overlaid with sperm medium without ascorbic acid (0 µM AA). The second and third fractions were overlaid with sperm medium supplemented with 300 µM and 600 µM AA; respectively. After 1 h of incubation, basic sperm parameters, intracellular ROS levels, acrosome reaction, chromatin integrity, and glucose uptake were assessed. Swim-up without AA significantly increased the percentage of ROS(+) spermatozoa compared with the raw semen (P<0.01). Interestingly, swim-up with 300 µM AA did not increase the percentage of ROS(+) sperm compared with the raw semen. In parallel, the percentage of sperm with altered chromatin integrity was significantly lower in the 300 µM AA group compared with that in the raw semen (P<0.05). These findings suggest that supplementation of vitamin C to sperm medium could be beneficial for direct swim-up-derived spermatozoa.

Endometrial injury is an important cause of intrauterine adhesion (IUA), amenorrhea and infertility in women, with limited effective therapies. Recently, stem cells have been used in animal experiments to repair and improve injured endometrium. To date, our understanding of adipose-derived stem cells (ADSCs) in endometrial injury repair and their further therapeutic mechanisms is incomplete. Here, we examined the benefit of ADSCs in restoration of injured endometrium by applying a rat endometrial injury model. The results revealed by immunofluorescence showed that green fluorescent protein (GFP)-labelled ADSCs can differentiate into endometrial epithelial cells in vivo. At 30 days after ADSCs transplantation, injured endometrium was significantly improved, with increased microvessel density, endometrial thickness and glands when compared with the model group. Furthermore, the fertility of rats with injured endometrium in ADSCs group was improved and had a higher conception rate (60% vs 20%, P = 0.014) compared with the control phosphate-buffered saline (PBS) group. However, there was no difference in the control group compared with the sham group. In addition, expression levels of the oestrogen receptor Eα/β (ERα, ERβ) and progesterone receptor (PR) detected by western blot and enzyme-linked immunosorbent assay (ELISA) were higher in the ADSCs group than in the PBS group. Taken together, these results suggested that ADSC transplantation could improve endometrial injury as a novel therapy for IUA.

The present study investigated if the presence of encircling granulosa cells protected against di(2-ethylhexyl)phthalate (DEHP)-induced oxidative stress in rat oocytes cultured in vitro. Denuded oocytes and cumulus–oocyte complexes (COCs) were treated with or without various doses of DEHP (0.0, 25.0, 50.0, 100, 200, 400 and 800 μM) in vitro. Morphological apoptotic changes, levels of oxidative stress and reactive oxygen species (ROS), mitochondrial membrane potential, and expression levels of apoptotic markers (Bcl2, Bax, cytochrome c) were analyzed. Our results showed that DEHP induced morphological apoptotic changes in a dose-dependent manner in denuded oocytes cultured in vitro. The effective dose of DEHP (400 µg) significantly (P>0.05) increased oxidative stress by elevating ROS levels and the mitochondrial membrane potential with higher mRNA expression and protein levels of apoptotic markers (Bax, cytochrome c). Encircling granulosa cells protected oocytes from DEHP-induced morphological changes, increased oxidative stress and ROS levels, as well as increased expression of apoptotic markers. Taken together our data suggested that encircling granulosa cells protected oocytes against DEHP-induced apoptosis and that the presence of granulosa cells could act positively towards the survival of oocytes under in vitro culture conditions and may be helpful during assisted reproductive technique programmes.

Isolated gametes can be used to investigate fertilization mechanisms, and probe distant hybridization between different species. Pollen grains of wheat and Setaria viridis are tricellular, containing sperm cells at anthesis. Sperm from these plants were isolated by breaking open pollen grains in a osmotic solution. Wheat ovules were digested in an enzyme solution for 20 min, and then transferred to an isolation solution without enzymes to separate egg cells from ovules. The fusion of wheat egg cells with wheat and S. viridis sperm was conducted using an electro-fusion apparatus. Under suitable osmotic pressure (10% mannitol), calcium concentration of 0.001% (CaCl2·2H2O), and a 30–35 V alternating electric field for 15 s, egg cells and sperm adhered to each other and became arranged in a line. Electroporation of the plasma membrane of egg cells and sperm using a 300–500 V direct-current electric field (45 µs amplitude pulse) caused them to fuse.

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.

Germ plasm-related structures (GPRS) are known to accompany meiotic cell differentiation but their dynamics are still poorly understood. In this study, we analyzed the ultrastructural mechanisms of GPRS transformation during oogenesis and spermatogenesis of the bivalve mollusc Ruditapes philippinarum (Manila clam), exploring patterns of GPRS activity occurring at meiosis onset, sex-specific difference/similarity of such patterns, and the involvement of mitochondria during GPRS-assigned events. In the two sexes, the zygotene–pachytene stage of meiosis is anticipated by three shared steps. First, the dispersion of germ plasm granules containing the germ line determinant VASA occurs. Second, the VASA protein deriving from germ plasm granules enters neighbouring mitochondria and appears to induce mitochondrial matter release, as supported by cytochrome B localization outside the mitochondria. Third, intranuclear VASA entrance occurs and the protein appears involved in chromatin reorganization, as supported by VASA localization in synaptonemal complexes. In spermatogenesis, these three steps are sufficient for the normal course of meiosis. In oogenesis, these are followed by the action of ‘germ plasm granule formation complex’, a novel type of structure that appears alternative to the Balbiani body. The possibility of germ plasm involvement in reproductive technologies is also suggested.

In assisted reproductive technology (ART) programmes, approximately 10% of infertile patients have at least two or three repeated implantation failures (RIFs) after an in vitro fertilization (IVF) protocol. Successful implantation mainly depends on local immune tolerance mechanisms involving a spectrum of cytokines, interleukins and growth factors. The latter have played pivotal roles in the recruitment of immune cells (and notably T-lymphocyte cells). In total, 250 couples participating in frozen–thawed embryo transfer programme were incorporated in a randomized clinical trial (peripheral blood mononuclear cells (PBMC) subgroup: n=122; control subgroup: n=128). In the PBMC group, a blood sample was collected 5 days before the scheduled frozen–thawed embryo transfer; PBMCs were isolated using Ficoll separation and then cultured for 72 h. Two days prior to embryo transfer, 0.4 ml of cultured PBMCs were transferred into the patient’s uterus. Although the clinical pregnancy rate was higher in the PBMC group (34.4%) than in the control group (23.4%), this difference was not statistically significant (P=0.05 in a chi-squared test). Nevertheless, when we limited the analysis to patients with ≥3 RIFs (n=138), there was a significant difference in the clinical pregnancy rate between the PBMC group (38.6%) and the control group (19.7%; P=0.01). Our results imply that PBMC transfer can be part of effective fertility treatment for patients with RIF.