Treatment with follicle-stimulating hormone (FSH) and testosterone (T2) and their combination have been observed to be influential on ovarian follicles of 1-day-old mice ovaries cultured for 8 days. Given that extension of the culture period could positively impact the development of follicles in cultured ovaries, the present study was conducted to evaluate the main and interaction effects of FSH by T2 on the development of ovarian follicles in 1-day-old mice ovaries cultured for 12 days. One-day-old mice ovaries were initially cultured with base medium for 4 days; thereafter, different hormonal treatments were added to the culture media, and the culture was continued for 8 additional days until day 12. Ovaries were collected for histological and molecular assessments on day 12. The greatest activation of primordial follicles and progression of activated follicles to the preantral stage was detected in ovaries treated with the combination of FSH and T2 (P < 0.05). This positive effect on the morphology of ovarian follicles was accompanied by upregulation of Pi3k, Gdf9, Bmp15, Cx37 and Fshr in the ovaries cultured with the combination of FSH and T2 (P < 0.05). Nonetheless, treatment with FSH and T2 led to a diminished proportion of intact follicles (P < 0.05), even though Bax/Bcl2 gene expression ratio, as an apoptotic index, was less in hormone-treated ovaries (P < 0.05). In conclusion, the combination of FSH and T2 could improve the activation of primordial follicles and the growth of activated follicles towards the preantral stage. This positive effect of FSH plus T2 appeared to be at least partly mediated through the upregulation of Pi3k and oocyte-derived growth factors including Gdf9 and Bmp15.

With the advancement in the embryo culture media, which supports nutrient requirements of embryos up to 5 to 6 days, there’s a chance to select more viable embryos, which are more likely to result in pregnancy, compared to earlier stages. Also, there is a controversy regarding the frozen embryo transfer compared to the fresh type. To compare the chemical pregnancy rates between fresh embryo transfer (ET), and frozen embryo transfer (FET), on day 3 (cleavage), and day 4 (morula) of development. In this retrospective study, data of 242 fresh and 758 frozen embryo transfer cycles were obtained in one infertility center in Isfahan, Iran. The study’s groups were assigned based on the day of fresh or frozen embryo transfer (day 3, or day 4 embryos) and the embryo grading. Chemical pregnancy was the main outcome measurement (implantation rates). The chemical pregnancy rate was higher in the good quality frozen embryo day 3 and transfer on day 4 group (40.1%). This rate was near the results of transferring the good quality frozen embryo on day 4 (39.2 %). There was no significant difference in the chemical pregnancy rate related to the number of transferred embryos (p = 0.55). The higher PRs, when the embryos were transferred on day 4, provided further support for the morula stage embryo transfer, possibly because of better synchrony with the endometrium. It is concluded that morula/compact embryos are good candidates for embryo transfer, which simultaneously reduces the number of transferred embryos.

This study aimed to evaluate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and inactivated virus vaccination on intracytoplasmic sperm injection (ICSI) outcomes in infertile couples. A retrospective case–control study was conducted at the Royan Institute from August 2020 to March 2022. The study included 90 couples in the COVID-19 infection phase and 31 in the vaccination phase. A total of 30 infected but unvaccinated couples were compared to a control group of 60 couples with no COVID-19 infection or vaccination history. Additionally, 31 couples underwent treatment before and after receiving the Sinopharm inactivated vaccine. Key variables analysed included sperm parameters (concentration, motility, progressive motility and morphology), ovarian parameters (antral follicle count, oocyte retrieval), embryological outcomes and pregnancy outcomes. SARS-CoV-2 infection significantly reduced sperm motility (P = 0.02) and progressive motility (P = 0.01) compared to controls. Sperm concentration and morphology showed non-significant declines. Post-vaccination analysis revealed similar but statistically insignificant changes in sperm parameters. Ovarian stimulation parameters and embryological outcomes remained unaffected by both infection and vaccination. Although biochemical, clinical pregnancy and live birth rates were lower among the infected group, these differences did not reach statistical significance (p = 0.16, 0.08 and 0.09). SARS-CoV-2 infection has been associated with impaired sperm progressive motility, which may negatively influence ICSI outcomes. In contrast, vaccination with an inactivated virus does not appear to impact fertility outcomes. These findings provide crucial guidance for physicians and infertile couples managing treatments during and after the pandemic, suggesting the need for extended recovery periods before ART procedures following COVID-19 infection.

The reproductive efficiency of dairy cows decreases significantly in hot climates. Exposure to heat stress causes damage to different stages of the reproductive cycle including a decrease in the quality of oocytes. Antioxidant supplementation has been introduced as one of the main approaches to alleviate the effects of free radical damage associated with heat stress. Gamma-oryzanol (ORY), a component of rice bran oil, is introduced as a novel antioxidant. As a supplement of culture media for maturation, the effect of ORY on the development of heat-shocked bovine cumulus–oocyte complexes was evaluated in this study. At the end of maturation in vitro using the heat-shock model, a higher proportion of metaphase II oocytes (0.78 ± 0.03 vs 0.42 ± 0.03) and lower metaphase I and germinal vesicle breakdown (0.10 ± 0.02 vs 0.38 ± 0.03) were recorded for the treated group (N = 205) in comparison with the control (N = 203) (P < 0.05). Moreover, the treatment exerted upregulation of NRF2, SOD, CAT and GPX transcripts in matured oocytes and GPX in CCs, along with a considerable increase in the cleavage (0.52 ± 0.04 vs 0.33 ± 0.03) and total blastocyst rates (0.30 ± 0.03 vs 0.14 ± 0.02) (P < 0.05). These results showed that ORY increased the mRNA expression of the transcripts associated with antioxidant enzymes and enhanced the developmental potential of heat-shocked bovine oocytes and warranted further studies to explore this antioxidant’s influence on improving dairy cattle’s reproductive efficiency during heat stress.

Extracellular vesicles (EVs) are implicated in various functions within the complex mechanisms of intercellular communication. There are several subpopulations of EVs, including apoptotic bodies, microvesicles and exosomes. These nanovesicles are capable of transferring functional proteins and genetic information to alter the phenotype and function of recipient cells. In animal reproduction, numerous studies have demonstrated that EVs are actively involved in the regulation of different physiological events, modulating a variety of processes such as follicular development, spermatogenesis, oocyte maturation, fertilization and embryo development, with results indicating improved gamete quality, embryo development and cryotolerance. Additionally, EVs show therapeutic potential in restoring reproductive function and supporting maternal-embryonic communication in both domestic and wild species. Therefore, the present review aimed to describe the main studies conducted using EVs in the field of animal reproduction, highlighting their biological relevance, experimental applications and future prospects for clinical implementation in assisted reproductive technologies.

Globally, numerous infertile couples have been assisted by extensive research on mammalian fertilization and the rapid development of Assisted Reproductive Technology (ART). However, 5%–15% of the couples that are selected for in vitro fertilization (IVF) experience a total fertilization failure (TFF), where no zygotes develop despite oocytes and semen parameters appear to be normal. Notably, an essential early event in fertilization is the binding of spermatozoa to the oocyte’s external envelope, which followed by the spermatozoa-oocyte fusion. Meanwhile, oocyte activation is a crucial cellular process necessary to block polyspermy and start the development of the zygote. Improper membrane fusion of gametes has been demonstrated to be one of the mechanisms of TFF. Moreover, considering the large amount of research on sperm proteins in recent years, thus in this review, we characterize the role and molecular mechanisms of sperm proteins in the three key processes of gamete adhesion and fusion and oocyte activation, which would provide a comprehensive understanding of the role of sperm proteins in fertilization in mammals and a favourable reference for future studies in assisted reproduction due to FF.

Rainbow trout (Oncorhynchus mykiss) is a promising cultivable fish species with significant potential for expansion. As a cold-water fish belonging to the Salmonidae family, it requires an optimal temperature range of 10–15°C for optimal growth. This study explores a method for producing sterile rainbow trout with maximum survival rates by using heat shock treatment to enhance growth characteristics and improve aquaculture practices. A control group and four heat shock treatments were given at 26°C and 28°C for 10 min, applied 15 and 20 min after the mixing of eggs and milt, using a water bath. Among the treated groups, the highest fertilisation, hatching and yolk sac absorption rates were 90.3 ± 0.3%, 81.8 ± 0.8% and 83.9 ± 0.5%, respectively. The highest triploidy rate of 76.6 ± 3.3% was observed with a heat shock at 28°C, 20 min after fertilisation. In contrast, none of the fish from the control group were triploids. The control group demonstrated higher survival rates at fertilisation (93.1 ± 0.4%), hatching (84.2 ± 0.4%) and complete yolk sac absorption (86.2 ± 0.5%) compared to the heat-shocked groups. The diploid and triploid chromosome numbers in rainbow trout were determined to be 2n = 60 and 3n = 91, respectively. This study confirms that heat shock treatment can effectively induce triploidy in rainbow trout, with significant variations in triploidy rates depending on the temperature and timing of the shock. While heat shock can enhance the production of sterile fish, it is essential to balance the treatment parameters to maintain high survival rates. These findings contribute to the optimisation of triploidy induction techniques and support the advancement of aquaculture practices by improving the growth, management and survival rates of rainbow trout which could significantly benefit aquaculture efficiency and sustainability.

The objective of this study was to compare embryonic morphokinetic parameters and clinical outcomes of two controlled ovarian stimulation (COS) protocols in women of advanced maternal age (AMA): Pergoveris (r-hFSH:r-hLH, 2:1 ratio) versus r-hFSH monotherapy (GONAL-f). This retrospective, non-interventional descriptive study was conducted at a private university–affiliated IVF center and included 136 ICSI cycles performed in AMA patients between March 2019 and May 2020. Patients were grouped by COS protocol (GONAL-f, n = 64; Pergoveris, n = 72), and embryo morphokinetics and ICSI outcomes were extracted from the clinic’s database; the main outcome was time to complete blastulation (tB, hours). Embryos from the Pergoveris group reached tB earlier than those from the GONAL-f group (mean 109.3 h vs 112.6 h), and all morphokinetic milestones occurred sooner with Pergoveris, alongside lower multinucleation rates at the 2-cell and 4-cell stages. Although blastocyst development was higher in the GONAL-f group, Pergoveris was associated with higher oocyte yield and maturity, higher implantation, clinical and ongoing pregnancy rates, and lower miscarriage and OHSS rates; Pergoveris cycles also required higher gonadotropin doses and longer stimulation. Overall, embryos from the r-hFSH:r-hLH group exhibited faster morphokinetic timings and improved implantation and pregnancy outcomes, and prospective studies are warranted to confirm these observations.

Aneuploidy in oocytes is a leading cause of implantation failure, miscarriage and congenital disorders. During meiosis, proper timing of chromosome segregation is regulated by the spindle assembly checkpoint (SAC) and the anaphase-promoting complex/cyclosome (APC/C). However, how pharmacological manipulation of these regulatory pathways affects aneuploidy remains incompletely understood. In this study, we investigated whether SAC inhibition by reversine induces aneuploidy in mouse oocytes and whether partial inhibition of APC/C by proTAME can alleviate these errors. Germinal vesicle (GV) oocytes were matured in vitro in the presence of various concentrations of reversine. To optimize the timing of treatment, oocytes were exposed to reversine for 0, 3, 5 or 7 h, followed by culture with or without proTAME. A proTAME-only group (2.5 nM) was also included. Chromosome spreads were analyzed at the metaphase II (MII) stage to determine aneuploidy rates. Reversine (5 nM) yielded an MII maturation rate of 80.5% but induced a high aneuploidy rate of 77.0%. Sequential treatment with 2.5 nM proTAME significantly reduced aneuploidy to 33.3%. In contrast, proTAME alone led to 79.0% aneuploidy, suggesting its effect is contingent upon prior SAC disruption. These results indicate that reversine compromises chromosomal integrity, while appropriately timed, low-dose proTAME can partially rescue segregation errors. Our findings underscore the potential of pharmacologically regulating APC/C activity to reduce aneuploidy and enhance oocyte quality, offering new avenues for improving outcomes in assisted reproductive technologies.

This study aimed to evaluate the effects of acetyl-L-carnitine on follicle survival and growth, stromal cell density and extracellular matrix, as well as on the expression of mRNA for nuclear factor erythroid 2-related factor (NRF2), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and peroxiredoxin 6 (PRDX6) in cultured bovine ovarian cortical tissues. Ovarian fragments (3 × 3 × 1 mm) were cultured for 6 days in α-MEM+ alone or supplemented with 10, 50 or 100 μM acetyl-L-carnitine at 38.5°C with 5% CO2 in humidified air. Before (non-cultured tissues) and after culture, the ovarian fragments were fixed in 4% paraformaldehyde for 12 h for histological analysis or stored at –80ºC for mRNA expression analysis of NRF2, SOD, CAT, PRDX6 and GPX1. The results showed that 100 μM acetyl-L-carnitine increased the percentages of morphologically normal follicles and stromal cell density in cultured ovarian tissues. On the other hand, acetyl-L-carnitine did not influence the percentage of collagen in ovarian tissue nor the expression of mRNAs for NRF2, SOD, CAT, PRDX6 and GPX1. In conclusion, 100 μM acetyl-L-carnitine increased follicle survival and stromal cell density in cultured bovine ovarian tissues but does not influence collagen fibre distribution or the expression of mRNAs for NRF2, SOD, CAT, PRDX6 and GPX1.

Centromeres are chromosomal loci essential for the correct segregation of genetic material during cell division. Defects in centromere function can lead to aneuploidy and cancer. During early embryonic development in mammals, prior to the first cell division, male and female genomes are separated in pronuclei located at the centre of the zygote. Parental chromatin clusters at the interface between the two pronuclei and this clustering step is critical to avoid aneuploidy in human and bovine zygotes. Yet, despite their essential function in chromosome segregation, the position and spatial organization of centromeres during the first cell cycle in mammals is mostly unknown. Previous studies conducted in bovine embryos derived from in vitro fertilization (IVF) showed that cell cycle progression impacts on the success rate of blastocyst formation. Specifically, embryos that entered earliest into S-phase or the earliest cleaving embryos were more likely to develop into blastocysts. To determine the precise timing of these events we performed a detailed characterization of key phases of the first cell cycle in bovine zygotes derived from IVF. In parallel we examined the spatial positioning of centromeres. We identify 20 h post insemination (hpi) as the timepoint when male and female pronuclei are juxtaposed and are completing S-phase. At this timepoint, we show that centromeres are positioned distal to the pronuclear interface and use super resolution microscopy to demonstrate extensive centromere clustering into chromocentres. Our results identify distinct nuclear features observed at 20 hpi, which may serve as cell cycle markers in determining successful bovine IVF.

This study aimed to compare the clinical outcomes of using refrigerated versus pre-warmed media for preparing time-lapse dishes in in vitro fertilization (IVF). Patients undergoing their first IVF/ICSI cycle were divided into two groups. The control group used pre-warmed culture media, while the experimental group used refrigerated culture media. The osmotic pressure of the culture droplets in both groups was tested. No statistical differences were found between the two groups’ basic data. The proportion of air microbubbles affecting imaging significantly decreased (4.55% vs. 37.97%, P < 0.001) when using pre-warmed media. However, the blastocyst formation rate (56.62% vs. 49.70%, P = 0.046) and total high-quality embryo rate (22.26% vs. 17.06%, P = 0.047) were significantly higher in the refrigerated media group compared to the pre-warmed media group. The higher rate of high-quality embryos in the refrigerated media group might result in a higher single embryo transfer rate (45.10% vs. 18.52%, P = 0.020) and implantation rate (58.23% vs. 34.69%, P = 0.010). From day –1 to day 1, osmolality increased, with the P-3.5 group showing a significant elevation compared to the other three groups. After 5 days of incubation, the osmotic pressure of group R-4.0 was significantly lower than that of groups P-3.5, P-4.0 and P-3.5. In conclusion, refrigerated culture media dishes helped stabilize the osmotic pressure of the culture microenvironment and reduce water evaporation. The refrigerated group showed a higher rate of high-quality embryos and live births, although pre-warmed culture media effectively reduced the occurrence of air microbubbles that affect embryo imaging in the next day’s dishes.

Introduction: Long non-coding RNAs (lncRNAs) are a subset of RNA molecules that have been shown to be involved in gene regulation. A lot of different pathways are involved during gametogenesis and any disturbance to these pathways may have a derogatory impact on producing a haploid gamete and thus a euploid embryo. Steroidogenesis pathway plays a crucial role in gametogenesis. The purpose of this work was to quantify the levels of lnc-CYP11A1-1 and RP11573D15.8 expression levels in aneuploid and euploid embryos. Materials and methods: A total of 20 surplus human embryos, of which 10 euploid and ten aneuploid embryos, were collected from an IVF centre. The expression levels of two lncRNAs, which have been hypothesized to regulate expression of CYP11A1, were evaluated in these embryos. RNA was extracted and used to synthesize cDNA for the experiments. Real-time polymerase chain reaction was performed to evaluate the expression levels of each lncRNA in aneuploid and euploid embryos, respectively. Results and discussion: This study shows that lnc-CYP11A1-1 was more expressed in aneuploid than in euploid embryos. RP11-573D15.8 is expressed more in aneuploid embryos than in euploid ones. The results for RP11-573D15.8 were statistically significant with a p-value of 0.02 (less than the standard threshold of p 0.05), whereas the results for lnc-CYP11A1-1 were not statistically significant with a p-value of 0.07 (greater than the standard threshold of p 0.05). Thus, the result of this study demonstrates that lncRNAs may have a role in gametogenesis and formation of aneuploid gametes.

Circadian rhythms are intrinsic, endogenously generated cycles that regulate various physiological processes, including reproductive functions. These rhythms are orchestrated by a network of core clock genes and are influenced by external environmental cues, primarily the light–dark cycle. Disruptions in circadian rhythms can have profound effects on fertility in both males and females, impacting processes such as the estrous cycle, ovulation, sperm production, implantation and pregnancy maintenance. This review comprehensively explores the molecular mechanisms underlying circadian rhythms and their influence on reproductive health, integrating evidence from both animal models and human studies. We delve into the intricate interplay between circadian genes, hormonal regulation and environmental factors, underscoring the critical importance of circadian integrity for optimal reproductive outcomes. The potential therapeutic implications of maintaining circadian rhythms are also discussed, highlighting novel avenues for enhancing reproductive health.

This study aimed to investigate the relationship between oocyte meiotic and developmental competences and antral follicle diameter on random days of estrous in naturally cycling zebu females (Nellore - a tropical Bos indicus breed). Immature oocytes recovered from early antral follicles (diameter of 0.5 to 2 mm) exhibited a higher percentage of intact transzonal projections, but lower viability, smaller diameter and a higher proportion of chromatin decondensation when compared to those recovered from mid- (diameter > 2 to 6 mm) and late antral follicles (diameter > 6 mm), demonstrating that oocytes recovered from early antral follicles lack meiotic and developmental competence. Immature oocytes recovered from mid- or late antral follicles were homogeneous in terms of morphology and size, in addition to exhibiting a high proportion of a pattern of chromatin configuration compatible with high developmental competence, which may help explain the better oocyte quality and improved performance of zebu donors in IVP programs. At the end of the in vitro maturation culture, the percentage of oocytes that completed meiotic maturation was higher when they were recovered from late antral follicles compared to mid antral follicles, although the rate of embryonic development to the blastocyst stage was similar. Our results demonstrated a relationship between the acquisition of competences during the final stages of oocyte growth and morphofunctional events, including the chromatin structure remodeling, but further research is needed to better characterize the functional differences in ovarian physiology between bovine breeds that may impact the oocyte developmental potential.

This study explores the efficacy of a novel microfluidic device in isolating rheotactic sperm and assesses their advantages compared with other motile sperm. Two microfluidic devices were used in this study: the microfluidic device we designed to separate sperm based on rheotaxis and a simple passive microfluidic device. We compared the results with the density gradient centrifugation technique. Sperm attributes including concentration, morphology, viability and motility were assessed using related procedures. Statistical analyses were conducted using one-way analysis of variance. Results showed differences in sperm concentration, motility, morphology and vitality using different sperm separation techniques. The sperms separated using our microfluidic device demonstrated the highest motilities, normal morphology percentages and higher sperm vitality but significantly lower sperm concentrations. These findings suggest the potential of our microfluidic design in enhancing sperm quality. Our findings are in agreement with previous research, emphasizing the capability of microfluidics in enhancing sperm quality. Specifically, our designed microfluidic device exhibited exceptional efficacy in isolating highly motile sperm, a critical factor for successful fertilization.

Kisspeptin, encoded by Kiss1 gene, regulates reproduction via the hypothalamic-pituitary-gonadal axis. While kisspeptin treatment promotes follicular development in Tan sheep, its direct action on ovarian granulosa cells remains unclear. For this, we first detected the expression of Kiss1 and its receptor Kiss1r in primary ovarian granulosa cells of Tan sheep. Second, the effect of kisspeptin on steroid hormone secretion, proliferation and apoptosis of ovarian granulosa cells was investigated. Third, the signaling pathway of kisspeptin regulating steroid hormone secretion was revealed by western bolting in ovarian granulosa cells. The results showed that the Kiss1 and Kiss1r genes were present in ovarian granulosa cells of Tan-sheep, and 500 nM dose of kisspeptin significantly stimulated steroids hormone secretion (P < 0.05), and up-regulated StAR, HSD17B2, CPY19A1, FSHR, LHR, ERβ, PGR and p-ERK1/2 proteins expression (P < 0.05). Moreover, this treatment significantly promoted cell proliferation and increased the proportion of cells in S phase (P < 0.05), and significantly suppressed granulosa cell apoptosis (P < 0.05). Additionally, the stimulatory effects of kisspeptin on estradiol and progesterone secretion were blocked by inhibitors of the Mitogen-activated Protein Kinase (MAPK) signaling pathway (including PKA inhibitor, PLC inhibitor, PKC inhibitor, and Ca2+inhibitor). Western blot analysis confirmed that kisspeptin regulates steroid hormone secretion primarily through the MAPK-ERK signaling pathway. Our results demonstrate that kisspeptin can directly act on ovarian granulosa cells to promote steroidogenesis, proliferation, and inhibit apoptosis, providing a foundational basis for developing novel kisspeptin-mediated techniques to regulate reproduction in Tan sheep.