MicroRNAs (miRNAs) are small non-encoding RNAs that actively regulate biological and physiological processes, and play an important role in regulating gene expression in all cells, especially in most animal cells, including oocytes and embryos. The expression of miRNAs at the right time and place is crucial for the oocyte’s maturation and the embryo’s subsequent development. Although assisted reproductive techniques (ART) have helped to solve many infertility problems, they cause changes in the expression of miRNA and genes in oocytes and preimplantation embryos, and the effect of these changes on the future of offspring is unknown, and has caused concerns. The relevant genomic alterations commonly imposed on embryos during cryopreservation may have potential epigenetic risks. Understanding the biological functions of miRNAs in frozen maturated oocytes may provide a better understanding of embryonic development and a comparison of fertility conservation in female mammals. With the development of new techniques for genomic evaluation of preimplantation embryos, it has been possible to better understand the effects of ART. The results of various articles have shown that freezing of oocytes and the cryopreservation method are effective for the expression of miRNAs and, in some cases, cause changes in the expression of miRNAs and epigenetic changes in the resulting embryo. This literature review study aimed to investigate the effects of oocyte cryopreservation in both pre-maturation and post-maturation stages, the cryopreservation method and the type of cryoprotectants (CPA) used on the expression of some epigenetic-related genes and miRNAs.

In recent years, the incidence of teratospermia has been increasing, and it has become a very important factor leading to male infertility. The research on the molecular mechanism of teratospermia is also progressing rapidly. This article briefly summarizes the clinical incidence of teratozoospermia, and makes a retrospective summary of related studies reported in recent years. Specifically discussing the relationship between gene status and spermatozoa, the review aims to provide the basis for the genetic diagnosis and gene therapy of teratozoospermia.

In zebrafish, a punctate band of F-actin is reported to develop in the external yolk syncytial layer (E-YSL) during the latter part of epiboly in zebrafish embryos. Here, electron microscopy (EM) and fluorescence confocal microscopy were conducted to investigate dynamic changes in the E-YSL membrane during epiboly. Using scanning EM, we report that the surface of the E-YSL is highly convoluted, consisting of a complex interwoven network of branching membrane surface microvilli-like protrusions. The region of membrane surface protrusions was relatively wide at 30% epiboly but narrowed as epiboly progressed. This narrowing was coincident with the formation of the punctate actin band. We also demonstrated using immunogold transmission EM that actin clusters were localized at the membrane surface mainly within the protrusions as well as in deeper locations of the E-YSL. Furthermore, during the latter part of epiboly, the punctate actin band was coincident with a region of highly dynamic endocytosis. Treatment with cytochalasin B led to the disruption of the punctate actin band and the membrane surface protrusions, as well as the attenuation of endocytosis. Together, our data suggest that, in the E-YSL, the region encompassing the membrane surface protrusions and its associated punctate actin band are likely to be an integral part of the localized endocytosis, which is important for the progression of epiboly in zebrafish embryos.

The success of an assisted reproduction cycle should be the achievement of a healthy singleton live birth following the replacement of one embryo. Therefore, one of the most critical points for embryologists has been the selection criteria and how to choose the best embryo to transfer with high implantation potential. In this vein, morphological evaluation has been historically the method applied. However, this practice relies on a limited number of single observations and is associated with high operator variability. Recently, a major innovation in embryo culture has been the introduction of a new type of incubator with integrated time-lapse monitoring, which enables the embryologist to analyze the dynamic events of embryo development, from fertilization to blastocyst formation. This novel practice is quickly growing and has been implemented in many IVF clinics worldwide. Therefore, the main aim of this review is to illustrate the benefits of time-lapse technology in a modern embryology laboratory. In particular, we discuss the blastocyst collapse(s) event and morphometric blastocyst assessment and analyse their association with embryo viability and implantation potential.

Fertility preservation is one of the most important issues in assisted reproductive technology. Previous studies have shown that cytokines and growth factors can improve follicle growth. The endometrial stromal cells secrete various factors that are involved in maintaining the integrity of uterine and epithelial secretory function. The platelet-rich plasma contains a large assembly of platelets suspended in plasma that successfully improves the viability and growth of various cell lines. This work aimed to investigate the influences of conditioned medium (CM) and platelet-rich plasma (PRP) on the development of ovarian follicles in infertile mice due to cyclophosphamide (CYC) exposure. In this study, 65 healthy BALB/c female mice (∼28–30 g and 6-8 weeks old) in five groups were studied. Immunohistochemistry (IHC) was used to detect growth differentiation factor 9 (GDF9)-positive cells. The mRNA expression levels of SMAD1, SMAD2, and BMP15 was assessed using reverse transcription-polymerase chain reaction (RT-PCR) method. The expression levels of SMAD1, GDF9, BMP15, and SMAD2 in the CM+PRP group was significantly more than in the CM and PRP groups. In addition, live birth occurred in the CM+PRP group. Treatment with CM+PRP in infertile mice due to Cy exposure increased fertility and live-birth rate. In general, our study suggested that the CM and PRP combination could improve the growth of mice ovarian follicles in vivo.

Preantral to early antral follicles transition is a complex process regulated by endocrine and paracrine factors, as well as by a precise interaction among oocyte, granulosa cells and theca cells. Understanding the mechanisms that regulate this step of folliculogenesis is important to improve in vitro culture systems, and opens new perspectives to use oocytes from preantral follicles for assisted reproductive technologies. Therefore, this review aims to discuss the endocrine and paracrine mechanisms that control granulosa cell proliferation and differentiation, formation of the antral cavity, estradiol production, atresia, and follicular fluid production during the transition from preantral to early antral follicles. The strategies that promote in vitro growth of preantral follicles are also discussed.

The aim of this study was to assess the consequences of treatment with pentoxifylline (PTX), an inducer of sperm motility, on sperm DNA fragmentation (SDF) and clinical characteristics in non-obstructive azoospermia (NOA) patients. The pilot study included 15 NOA patients. Half of each sperm sample before and after rapid freezing, was treated with PTX (3.6 mM /l, 30 min) as the PTX group and the remaining samples were considered as the control. SDF and sperm motility were assessed in each group. The clinical study comprised 30 fresh testicular sperm extractions (TESE) and 22 post-thawed TESE intracytoplasmic sperm injection cycles. Half of the mature oocytes from each patient were injected with PTX-treated spermatozoa and the remaining oocytes were injected with non-treated spermatozoa. Fertilization was assessed at 16 h post injection. Embryo transfer was carried out on day 2 after fertilization. Chemical pregnancy was assessed 2 weeks after transfer. PTX was found to significantly increase (P < 0.05) sperm motility. There was an insignificant difference in SDF rates between the groups (P > 0.05). In patient ovaries given fresh TESE, there was not any significant difference in clinical characteristics (P > 0.05). In patient ovaries given post-thawed TESE, there was a significant difference in the number of 2PN and in embryo formation (P < 0.05). Differences in the results of chemical pregnancy were insignificant (P > 0.05) between the groups. In addition, there was not any correlation between DNA fragmentation index and sperm motility and laboratory outcomes. Therefore, obtaining viable spermatozoa using PTX was more effective in post-thawed TESE regime patients in terms of 2PN and in embryo formation, deprived of damaging effects on sperm DNA integrity.

Since the birth of the first baby by in vitro fertilization in 1978, more than 9 million children have been born worldwide using medically assisted reproductive treatments. Fertilization naturally takes place in the maternal oviduct where unique physiological conditions enable the early healthy development of the embryo. During this dynamic period of early development major waves of epigenetic reprogramming, crucial for the normal fate of the embryo, take place. Increasingly, over the past 20 years concerns relating to the increased incidence of epigenetic anomalies in general, and genomic-imprinting disorders in particular, have been raised following assisted reproduction technology (ART) treatments. Epigenetic reprogramming is particularly susceptible to environmental conditions during the periconceptional period and non-physiological conditions such as ovarian stimulation, in vitro fertilization and embryo culture, as well as cryopreservation procedure, might have the potential to independently or collectively contribute to epigenetic dysregulation. Therefore, this narrative review offers a critical reappraisal of the evidence relating to the association between embryo cryopreservation and potential epigenetic regulation and the consequences on gene expression together with long-term consequences for offspring health and wellbeing. Current literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, in terms of osmotic shock, temperature and pH changes, and toxicity of cryoprotectants, it is therefore, critical to have a more comprehensive understanding and recognition of potential unanticipated iatrogenic-induced perturbations of epigenetic modifications that may or may not be a consequence of vitrification.

Fertilization failure (FF) and zygotic arrest after ICSI have a huge effect on both patients and clinicians, but both problems are usually unexpected and cannot be properly diagnosed. Fortunately, in recent years, gene sequencing has allowed the identification of multiple genetic variants underlying failed ICSI outcomes, but the use of this approach is still far from routine in the fertility clinic. In this systematic review, the genetic variants associated with FF, abnormal fertilization and/or zygotic arrest after ICSI are compiled and analyzed. Forty-seven studies were included. Data from 141 patients carrying 121 genetic variants affecting 16 genes were recorded and analyzed. In total, 27 variants in PLCZ1 (in 50 men) and 26 variants in WEE2 (in 24 women) are two of the factors related to oocyte activation failure that could explain a high percentage of male-related and female-related FF. Additional variants identified were reported in WBP2NL, ACTL9, ACTLA7, and DNAH17 (in men), and TUBB8, PATL2, TLE6, PADI6, TRIP13, BGT4, NLRP5, NLRP7, CDC20 and ZAR1 (in women). Most of these variants are pathogenic or potentially pathogenic (89/121, 72.9%), as demonstrated by experimental and/or in silico approaches. Most individuals carried bi-allelic variants (89/141, 63.1%), but pathogenic variants in heterozygosity have been identified for PLCZ1 and TUBB8. Clinical treatment options for affected individuals, such as chemical-assisted oocyte activation (AOA) or PLCZ1 cRNA injection in the oocyte, are still experimental. In conclusion, a genetic study of known pathogenic variants may help in diagnosing recurrent FF and zygotic arrest and guide patient counselling and future research perspectives.

Although the role of myo-inositol (MYO) in promoting the oocyte quality of PCOS patients has been documented in human studies; the cellular effects of this supplement on oocytes have not been directly examined due to ethical limitations. In the first phase of this study, MYO dosimetry was carried out simultaneously with the PCOS model development. An effective dose was obtained following the assessment of fasting insulin and testosterone levels using ELISA and ovarian morphology appraisal by histopathology. In the second phase, following the continuous administration of the effective dose of MYO and dehydroepiandrosterone (DHEA), cellular evaluation was performed. The quality of oocytes from superovulation was analyzed by examining maturity and normal morphology percentage using a stereomicroscope, intracellular reactive oxygen species (ROS) and glutathione (GSH) levels using fluorometry, and ATP count evaluation using ELISA. The results revealed that, among the four different MYO concentrations, the 0.36 mg/g dose compared with the DHEA group reduced testosterone levels and large atretic antral follicles (LAtAnF) diameter. This dose also increased the corpus luteum count and the granulosa:theca (G/T)layer thickness ratio in antral follicles. Furthermore, this dose increased mature oocytes and normal morphology percentage, ATP count, and GSH levels; however, it decreased ROS levels in mature oocytes. Our findings provide the grounds for further cellular and molecular studies on the PCOS mouse model, suggesting that the improvement in mitochondrial function and its antioxidant properties is probably one of the mechanisms by which MYO increases oocyte quality.

Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, has been demonstrated to ameliorate the histopathological characteristics of liver damage. Nonetheless, the systemic safety profile of OCA with regard to reproduction and development remains poorly understood. In the present study, we conducted a dose–response experiment by administering OCA at doses of 5 mg/kg, 10 mg/kg, or 20 mg/kg through tube feeding to investigate its effect on reproductive development and fertilization rate in both male and female mice. Furthermore, we evaluated the levels of protein and mitochondrial function in the placenta through western blot, qPCR, and scanning electron microscopy. The results showed that 10 mg/kg and 20 mg/kg OCA doses significantly reduced the rate of placental implantation (P < 0.05). Also, OCA increased maternal body weight. In addition, OCA increased levels of FXR and TGR5 and produced changes in oxidative stress levels (P < 0.05). Mitochondrial activity result found that 10 mg/kg and 20 mg/kg of OCA significantly reduced the mitophagy autosomes/nucleus compared with the normal control group (P < 0.05). What is more, there was no significant difference in sperm count after OCA intervention in either C57BL/10 mice or BALB/c mice. Overall, we demonstrated that OCA treatment protected against placental implantation by suppressing placental oxidative stress and mitochondrial activity.

We present a commentary on the article published in the Zygote FirstView: ‘Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection’ by Fumiaki Itoi and colleagues. We comment on the importance of providing the microscope setup details whenever sperm morphology visualization is discussed. The claim of ×6000–10,000 magnification is misleading as such levels of magnification are impossible to achieve.

Kisspeptin is characterized as a neuropeptide with a pivotal function in female and male infertility, and its antioxidant properties have been demonstrated. In this study, the effects of kisspeptin on the improvement of the vitrification and thawing results of human ovarian tissues were investigated. In this work, 12 ovaries from patients who underwent hysterectomy were collected laparoscopically, and then 32 samples from each of their tissues were taken. Haematoxylin and eosin (H&E) staining was performed to check the normality of the ovarian tissue and, subsequently, the samples were allocated randomly into four groups, including: (1) fresh (control), (2) vitrification, (3) vitrified + 1 μM kisspeptin, and (4) vitrified + 10 μM kisspeptin groups. After vitrification, thawing, and tissue culture processes, H&E staining for tissue quality assessment, terminal deoxynucleotidyl transferase dUTP nick end labelling assay for apoptosis evaluation, and malondialdehyde (MDA), superoxide dismutase (SOD), and ferric reducing ability of plasma tests for oxidative stress appraisal were carried out. Our histological results showed incoherency of ovarian tissue morphology in the vitrification group compared with other groups. Other findings implicated increased apoptosis rate and MDA concentration and reduced SOD activity and total antioxidant capacity (TAC) in the vitrification group compared with the control group (P < 0.05). Moreover, decreased apoptosis rate and MDA concentration, and increased TAC and SOD function were observed in the vitrification with kisspeptin groups (1 μM and 10 μM) compared with the vitrified group (P < 0.05). Our reports express that kisspeptin is an effective agent to overcome the negative effects of vitrification by regulating reactive oxygen species-related apoptotic processes.

Induction of puberty in cattle breeds that attain puberty in later stages, such as Gir, allows the earlier beginning of reproductive life and it might increase oocyte quality. Here, the ovulatory capacity of prepuberal Gir heifers was studied and its relationship to follicular growth, luteinizing hormone (LH) secretion and oocyte quality was evaluated. Peripubertal Gir heifers were treated with a progesterone-based protocol and according to ovulatory response were separated into groups: not-ovulated (N-OV) and ovulated (OV). Serial blood samples were taken 24 h after estradiol treatment on day 12 to evaluate LH secretion. Cumulus–oocyte complexes (COCs) were collected using ovum pick-up and assessed for brilliant cresyl blue (BCB) staining rate, IVF-grade oocytes rate, and mean oocyte diameter, in comparison with cow oocytes. Gene expression of developmental competence markers (ZAR1, MATER, and IGF2R) was also analyzed. The largest follicle diameters were similar between N-OV and OV groups on the day of estradiol treatment (d12) and the next day and decreased (P = 0.04) in the N-OV group thereafter. LH pulse secretion was different between groups (N-OV = 3.61 ± 0.34 vs OV = 2.83 ± 0.21 ng/ ml; P = 0.04). COC assessment showed that the number of recovered oocytes, BCB+ rate, IVF-grade oocytes and oocyte size was similar (P > 0.05) among groups, resembling adult cow patterns. ZAR1, MATER and IGF2R gene expression in oocytes were also similar (P > 0.05) in N-OV and OV groups. In conclusion, our results demonstrate a lower LH secretion profile in peripubertal Gir heifers prone to ovulate after induction protocol, and that oocyte quality is not affected on a short-term basis by ovulation itself.

This study investigated the effect of the flavonoid-based compound isorhamnetin (ISO) on maturation and developmental competence in oxidative stress-exposed porcine oocytes in vitro. Treatment with 2 μM ISO (2 ISO) increases the developmental rate of oxidative stress-exposed porcine oocytes during in vitro maturation (IVM). The glutathione level and mRNA expression of antioxidant-related genes (NFE2L2 and SOD2) were increased in the 2 ISO-treated group, whereas the reactive oxygen species level was decreased. Treatment with 2 ISO increased mRNA expression of a cumulus cell expansion-related gene (SHAS2) and improved chromosomal alignment. mRNA expression of maternal genes (CCNB1, MOS, BMP15 and GDF9) and mitogen activated protein kinase (MAPK) activity were increased in the 2 ISO-treated group. The total cell number per blastocyst and percentage of apoptotic cells were increased and decreased in the 2 ISO-treated group, respectively. Treatment with 2 ISO increased mRNA expression of development-related genes (SOX2, NANOG, and POU5F1) and anti-apoptotic genes (BCL2L1 and BIRC5) and decreased that of pro-apoptotic genes (CASP3 and FAS). These results demonstrate that 2 ISO improves the quality of porcine oocytes by protecting them against oxidative stress during IVM and enhances subsequent embryo development in vitro. Therefore, we propose that ISO is a useful supplement for IVM of porcine oocytes.

The aim of the present study was to evaluate the physiological and morphological parameters of pregnant does for early prediction of prenatal litter size. In total, 33 does were screened using ultrasonography and further categorized into three groups based on does bearing twins (n = 12), a single fetus (n = 12), or non-pregnant does (n = 9). The rectal temperature °F (RT) and respiration rate (RR) as physiological parameters, while abdominal girth in cm (AG) and udder circumference in cm (UC) as morphological parameters were recorded at different gestation times, i.e. 118, 125, 132 and 140 days. In addition to this, age (years) and weight at service (kg) were also used. The statistical analyses included analysis of variance (ANOVA) and linear discriminant analysis (LDA). The results indicated that groups had significant (P < 0.05) differences among morphological parameters at each gestation time, with higher AG and UC in does bearing twins followed by a single fetus and non-pregnant does. However, both physiological parameters were non-significantly (P > 0.05) associated with litter size groups. It was also revealed that the studied parameters showed increasing trends over gestation time in single and twin fetus categories, but they were on par among non-pregnant does. The results of the LDA revealed that estimated function based on age, weight at service, RR, RT, AG and UC had greater (ranging from 75.00 to 91.70%) accuracy, sensitivity and specificity at different gestation times. It was concluded that using an estimated function, future pregnant does may be identified in advance for single or twin litter size, with greater accuracy.

One of the prominent peculiarities of nanoparticles (NPs) is their ability to cross biological barriers. Therefore, the development of NPs with different properties has great therapeutic potential in the area of reproduction because the association of drugs, hormones and other compounds with NPs represents an alternative for delivering substances directly at a specific site and for treatment of reproductive problems. Additionally, lipid-based NPs can be taken up by the tissues of patients with ovarian failure, deep endometriosis, testicular dysfunctions, etc., opening up new perspectives for the treatment of these diseases. The development of nanomaterials with specific size, shape, ligand density and charge certainly will contribute to the next generation of therapies to solve fertility problems in humans. Therefore, this review discusses the potential of NPs to treat reproductive disorders, as well as to regulate the levels of the associated hormones. The possible limitations of the clinical use of NPs are also highlighted.

This study aimed to evaluate the ploidy and survival of larvae resulting from crosses between tetraploid females and diploid males of yellowtail tetra Astyanax altiparanae, both females (three diploids and three tetraploids) and males (n = 3 diploids). Breeders were subjected to hormonal induction with pituitary gland extract from common carp fish (Cyprinus carpio). Females received two doses at concentrations of 0.3 and 3.0 mg/kg −1 body weight and at intervals of 6 h. Males were induced with a single dose of 3.0 mg/kg −1 applied simultaneously with the second dose in females. Oocytes from each diploid and tetraploid female were fertilized with semen from the same male, resulting in two crosses: cross 1 (diploid male and diploid female) and cross 2 (diploid male and tetraploid female). The procedures were performed with separate females (diploid and tetraploid) and diploid males for each repetition (n = 3). For ploidy determination, 60 larvae from each treatment were analyzed using flow cytometry and cytogenetic analyses. As expected, flow cytometry analysis showed that progenies from crosses 1 and 2 presented diploid and triploid individuals, respectively, with a 100% success rate. The same results were confirmed in the cytogenetic analysis, in which the larvae resulting from cross 1 had 50 metaphase chromosomes and those from cross 2 had 75 chromosomes. The oocytes have a slightly ovoid shape at the time of extrusion. Diploid oocytes had a size of 559 ± 20.62 μm and tetraploid of 1025.33 ± 30.91 μm. Statistical differences were observed between eggs from crosses 1 and 2 (P = 0.0130). No significant differences between treatments were observed for survival at the 2-cell stage (P = 0.6174), blastula (P = 0.9717), gastrula (P = 0.5301), somite (P = 0.3811), and hatching (P = 0.0984) stages. In conclusion, our results showed that tetraploid females of the yellowtail tetra A. altiparanae are fertile, present viable gametes after stripping and fertilization using the ‘dry method’, and may be used for mass production of triploids. This is the first report of these procedures within neotropical characins, and which can be applied in other related species of economic importance.

The in vitro culture of domestic cat embryos without the zona pellucida affects their implantation capacity. MicroRNAs (miRNAs) have an important role in embryo–maternal communication and implantation. The objective of this study was to evaluate the expression of specific miRNAs in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were done: (1) domestic cat embryos cultured with the zona pellucida (zona intact control group, ZI); and (2) cultured without the zona pellucida (zona free group, ZF). The cleavage, morula and blastocyst rates were evaluated. The blastocysts and their spent medium were used for miRNA expression analysis using RT-qPCR (miR-21, miR-24, mi25, miR-29, miR-96, miR-98, miR-103, miR-191, miR-196, miR-199, miR-130, miR-155 and miR-302). The pre-mature microRNAs (pre-miRNAs) and miRNAs were evaluated in the blastocysts and only miRNAs were evaluated in the spent medium. No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups (P > 0.05). For miRNAs analysis, miR-103 and miR-191 had the most stable expression and were selected as internal controls. ZF blastocysts had a higher expression of miR-21, miR-25, miR-29 and miR-199 and a lower expression of miR-96 than their ZI counterparts (P < 0.05). Furthermore, higher levels of miR-21, miR-25 and miR-98 were detected in the spent medium of ZF blastocysts (P < 0.05). In conclusion, in vitro culture of domestic cat embryos without the zona pellucida modifies the expression of miR-21, miR-25, miR-29, miR-199 and miR-96 at the blastocyst stage and the release of miR-21, miR-25 and miR-98.

Regarding the low number of embryos that reach the blastocyst stage when cultured in vitro, this study aimed to evaluate the effects of quercetin on pre-implantation mouse (Mus musculus) embryos obtained using in vitro fertilization, especially during the passage from morula to blastocyst. Furthermore, we studied whether quercetin also affected the expression of hypoxia-inducible factor 1α (HIF-1α). The culture medium for the embryos was supplemented with quercetin, for long or short periods of time, and then the development potential, total cell number, apoptosis rates and expression of HIF-1α were studied to determine the effect of quercetin. Embryos failed to develop when cultured for long periods of time with quercetin, implying the possible toxic effects of this, alternatively antioxidant, compound. However, a short culture from morula to blastocyst significantly improved the development potential of in vitro produced embryos, increasing the final total cell number and reducing the apoptosis rate, observing similar results to those embryos cultured in low-oxygen concentrations or developed in utero. Furthermore, in embryos treated with quercetin for 2 or 4 h we found an increase in HIF-1α compared with untreated embryos. This work could imply a way to use quercetin in fertility clinics to improve the production of healthy blastocysts and, consequently, increase the success rates in assisted reproduction techniques.