In cattle, maternal metabolic health has been suggested to influence oocyte and embryo quality. Here, we examined whether maternal liver abnormalities affected oocyte maturation by screening meiotic maturation, spindle morphology, actin filaments, and lysosomes. In oocytes from the abnormal liver group, the maturation rate (80.2%) was significantly lower compared to a control group with healthy livers (90.8%; P < 0.05). Mean spindle area in oocytes of the abnormal group (50.4 ± 3.4 μm) was significantly larger than in the control (40.8 ± 1.6 μm; P < 0.05). Likewise, mean spindle width in the abnormal group (8.8 ± 0.3 μm) was significantly larger than in the control group (7.8 ± 0.2 μm; P < 0.05). The proportion of cells with correctly aligned chromosomes in the abnormal group (48.0%) was significantly lower than in the control (78.3%; P < 0.05). The number of cortical actin filaments in mature oocytes of the abnormal group (299.3 ± 3.7) was significantly lower than in the control (314.7 ± 3.2; P < 0.05). The number of lysosomes in mature oocytes of the abnormal group (1363.6 ± 39.0) was significantly higher than in the control (1123.4 ± 26.3; P < 0.05). In conclusion, our findings indicate that the quality of matured oocytes is lower in cattle with liver abnormalities than in healthy cattle.

Recently, the World Health Organization recommendation for abstinence time for semen analysis has been challenged in some studies and many of them have supported the advantages of a second short abstinence ejaculation. More evidence is needed to approve this for clinical use. This study aimed to compare the average routine abstinence time (2-7 days) with the short time (1-2 h) on sperm quality based on functional parameters in a population of oligo-astheno-teratozoospermia (OAT) men. The semen samples were retrieved from 50 men with OAT two times: one standard 2-7 days (long ejaculation) and short duration trimming (1-2 hours later the first ejaculation). All semen parameters as well as sperm DNA integrity were compared between groups. Results showed that mean sperm concentration (10.40 vs. 8.76), total sperm count (28.53 vs. 12.24) and mean semen volume (2.69 vs. 1.40) were higher in the first ejaculation (2-7 days of abstinence), while progressive motility (20.52 vs. 13.32), non-progressive motility (53.46 vs. 48.86), morphology (2.46 vs. 1.46) and viability (83.90 vs. 77.96) were significantly higher in the second ejaculation ( < 0.05). The second sample also showed lower immotile (26.82 vs. 38.02) and DNA fragmentation (19.5 vs. 26.96) ( < 0.05). Taking all data into account, an additional short abstinence period (AP) may be a simple and helpful strategy to obtain better sperm quality in couples with male infertility causes, especially in OAT patients. The recommended current guidelines regarding the AP may need to be revisited in severe male factors.

Substance use refers to the consumption of drugs that have varying degrees of impact on a persons' physical, mental and emotional well-being. While the adverse health effects of drugs have been extensively documented, further research is needed to understand their impact on fertility. Studies have indicated that substance use affects both the male and female reproductive systems. As substance use is more prevalent among young adults compared with the elderly, it appears that individuals of reproductive age are particularly vulnerable to the reproductive impairments associated with substance use. Although numerous studies have reported detrimental effects of substance use on pregnant women and their foetus during the post-implantation stages, there are limited studies on critical pre-implantation period and gamete stages. In this narrative review, we aimed to focus on the most significant evidence regarding the impact of substances on gametes and pre-implantation embryos.

Human oocyte maturation is a lengthy process that takes place over the course of which oocytes gain the inherent ability to support the next developmental stages in a progressive manner. This process includes intricate and distinct events related to nuclear and cytoplasmic maturation. Nuclear maturation includes mostly chromosome segregation, whereas rearrangement of organelles, storage of mRNAs and transcription factors occur during cytoplasmic maturation.Human oocyte maturation, both in vivo and in vitro, occurs through a process that is not yet fully understood. However, it is believed that the second messenger, cyclic adenosine monophosphate (cAMP), plays a pivotal role in the upkeep of the meiotic blocking of the human oocyte. Relatively high levels of cAMP in the human oocyte are required to maintain meiosis blocked, whereas lower levels of cAMP in the oocyte enable meiosis to resume. Oocyte cAMP concentration is controlled by a balance between adenylate cyclase and phosphodiesterases, the enzymes responsible for cAMP generation and breakdown.In addition to nuclear maturation, the female gamete requires a number of complicated structural and biochemical modifications in the cytoplasmic compartment to be able to fertilize normally. According to ultrastructural studies, during the transition from the germinal vesicle stage to metaphase II (MII), several organelles reorganize their positions. The cytoskeletal microfilaments and microtubules found in the cytoplasm facilitate these movements and regulate chromosomal segregation.The aim of this review is to focus on the nuclear and cytoplasmic maturation by investigating the changes that take place in the process of oocytes being competent for development.

The secondary metabolites of several plant species, particularly sesquiterpenic lactones (SLs) have been studied by different research groups for over 30 years. This group of metabolites presents numerous biological activities such as antibacterial, antiviral, antiulcer, cell proliferation inhibitor, and oocyte activator with participation in exocytosis processes. This study aims to assess some sperm parameters in epididymal gametes of exposed to increasing concentrations (0 to 2 mM) of DhL for various incubation times from 10 to 40 minutes. We determined the participation of different cell signalling pathways in the induced acrosome reaction. Our results showed an alteration in the progressive motility pattern and cell viability depending on DhL concentration and exposure time of gametes. When analyzing acrosomal status, higher percentages than the negative control were obtained in all tested doses. Both isolated and joint inhibition tests of PKA and phospholipases (PLC and PLA) showed a greater participation of PI-PLC. This is the first report concerning the effects of this lactone on the medium of sperm incubation. Consequently, further studies will be necessary to determine the molecular implications of this lactone on the fertilizing potential of the sperm.

production of porcine embryos is a complicated process that includes maturation (IVM), fertilization (IVF) and culture (IVC). Insufficient cytoplasmic maturation, slow zona reaction and improper embryo culture conditions will compromise the efficiency of porcine embryo production . Previous studies have shown that insulin-transferrin-selenium (ITS) in IVM or IVC medium could improve porcine oocyte maturation, decrease polyspermy fertilization and promote subsequent embryonic development . However, the effect of ITS both in IVM and IVC media on porcine embryo production hasn't been elucidated. In this study, we found that 1.0% ITS supplementation in IVM/IVC media promoted the expansion of cumulus cells, raised mitochondrial membrane potential, increased ATP content and reduced ROS level in matured oocytes, improved blastocyst rate and the cell number of blastocyst, simultaneously. In conclusion, the IVM/IVC media supplemented with 1.0% ITS can improve the efficiency of porcine embryo production .

This study aimed to demonstrate the utilization value of 1PN embryos. The 1PN zygotes collected from December 2021 to September 2022 were included in this study. The embryo development, the pronuclear characteristics, and the genetic constitutions were investigated. The overall blastocyst formation and good-quality blastocyst rates in 1PN zygotes were 22.94 and 16.24%, significantly lower than those of 2PN zygotes (63.25 and 50.23%, respectively, = 0.000). The pronuclear characteristics were found to be correlated with the developmental potential. When comparing 1PN zygotes that developed into blastocysts to those that arrested, the former exhibited a significantly larger area (749.49 ± 142.77 vs. 634.00 ± 119.05, = 0.000), a longer diameter of pronuclear (29.81 ± 3.08 vs. 27.30 ± 3.00, = 0.000), and a greater number of nucleolar precursor body (NPB) (11.56 ± 3.84 vs. 7.19 ± 2.73, = 0.000). Among the tested embryos, the diploidy euploidy rate was significantly higher in blastocysts in comparison with the arrested embryos (66.67 vs. 11.76%, = 0.000), which was also significantly higher in IVF-1PN blastocysts than in ICSI-1PN blastocysts (75.44 vs. 25.00%, = 0.001). However, the pronuclear characteristics were not found to be linked to the chromosomal ploidy once they formed blastocysts.In summary, while the developmental potential of 1PN zygotes is reduced, our study shows that, in addition to the reported pronuclear area and diameter, the number of NPB is also associated with their developmental potential. The 1PN blastocysts exhibit a high diploidy euploidy rate, are recommend to be clinically used post genetic testing, especially for patients who do not have other 2PN embryos available.

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 , , , and 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 / 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 and oocyte-derived growth factors including and .

Rainbow trout () 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.

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 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 -value of 0.02 (less than the standard threshold of 0.05), whereas the results for lnc-CYP11A1-1 were not statistically significant with a -value of 0.07 (greater than the standard threshold of 0.05). Thus, the result of this study demonstrates that lncRNAs may have a role in gametogenesis and formation of aneuploid gametes.

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.

Growth differentiation factor 9 ( is an oocyte-specific paracrine factor involved in bidirectional communication, which plays an important role in oocyte developmental competence. In spite of its vital role in reproduction, there is insufficient information about exact transcriptional control mechanism of GDF9. Hence, present study was undertaken with the aim to study the expression of basic helix-loop-helix (bHLH) transcription factors (TFs) such as the factor in the germline alpha (FIGLA), twist-related protein 1 (TWIST1) and upstream stimulating factor 1 and 2 (USF1 and USF2), and nuclear receptor (NR) superfamily TFs like germ cell nuclear factor (GCNF) and oestrogen receptor 2 (ESR2) under three different maturation (IVM) groups [follicle-stimulating hormone (FSH), insulin-like growth factor-1 (IGF1) and oestradiol)] along with all supplementation group as positive control, to understand their role in regulation of GDF9 expression. Buffalo cumulus-oocyte complexes were aspirated from abattoir-derived ovaries and matured in different IVM groups. Following maturation, TFs expression was studied at 8 h of maturation in all four different IVM groups and correlated with GDF9 expression. USF1 displayed positive whereas GCNF, TWIST1 and ESR2 revealed negative correlation with GDF9 expression. TWIST1 & ESR2 revealing negative correlation with GDF9 expression were found to be positively correlated amongst themselves also. GCNF & USF1 revealing highly significant correlation with GDF9 expression in an opposite manner were found to be negatively correlated. The present study concludes that the expression of GDF9 in buffalo oocytes remains under control through the involvement of NR and bHLH TFs.

EXOSC10 is an exosome-associated ribonuclease that degrades and processes a wide range of transcripts in the nucleus. The initial segment (IS) of the epididymis is crucial for sperm transport and maturation in mice by affecting the absorption and secretion that is required for male fertility. However, the role of EXOSC10 ribonuclease-mediated RNA metabolism within the IS in the regulation of gene expression and sperm maturation remains unknown. Herein, we established an conditional knockout ( cKO) mouse model by crossing mice with mice which expressed recombinase in the principal cells of IS as early as post-natal day 17. Morphological and histological analyses revealed that cKO males had normal spermatogenesis and development of IS. Moreover, the sperm concentration, morphology, motility, and frequency of acrosome reactions in the cauda epididymides of cKO mice were comparable with those of control mice. Thus, cKO males had normal fertility. Collectively, our genetic mouse model and findings demonstrate that loss of EXOSC10 in the IS of epididymis is dispensable for sperm maturation and male fertility.

Boric acid (BA) is an important mineral for plants, animals and humans that assists metabolic function and has both positive and negative effects on biological systems. The present study aimed to investigate the effects of different concentrations of BA added to the culture media, the quality and development potential of mouse embryos. Superovulated C57Bl6/6j female mice were sacrificed ∼18 hours after human chorionic gonadotropin (hCG) injection. Single-cell-stage embryos were collected from the oviduct, divided into experiment groups and cultured in embryo medium with supplemented BA+ in 5% CO at 37 °C until 96 hours at the blastocyst stage. The blastocyst development rates of 0, 1.62 × 10, 1.62 × 10, 1.62 × 10 and 1.62 × 10 µM BA were 51.52%, 73.47%, 77.36% and 81.13%, respectively. The development rates were significantly higher in the 1.62 × 10 () and 1.62 × 10 µM BA groups than in the control group (). These results indicated that low BA doses influenced embryo development by positively affecting development rates, embryo cell numbers, biochemical parameters and development at the molecular level by pluripotent and antioxidant genes. Therefore, BA seems to play an important role on embryo development.

Fungal metabolites are known to have potent and diverse properties such as antiviral, antidiabetic, antitumour, antioxidant, free radical scavenging, and antibacterial effects which can be utilized to treat diseases. In this study, we investigated the functional activity of stereumamide A (StA) derived from a culture broth of during the fertilization (IVF) of pig oocytes, to determine its effects on sperm penetration. Oocytes matured were fertilized in the absence or presence of varying concentrations of StA (0-50 μg/ml StA). When StA was directly added into the IVF medium, significantly lower fertilization rates were seen with the 20 or 50 μg/ml StA (2.0-17.5%) treatments compared with those of 10 μg/ml StA or the controls (60.9-62.3%), whereas StA had no influence on the survival of oocytes and spermatozoa throughout the IVF process. For evaluating the control of sperm entry, mature oocytes were pre-incubated in a medium containing 20 μg/ml StA for 1 h, and then IVF was subsequently performed. The incidence of polyspermy was significantly reduced when oocytes were pre-incubated with StA (15.0% 50.4-57.5% in controls). In conclusion, sperm penetration was inhibited in the medium in the presence of StA during IVF, while StA did not affect sperm motility and fertility competence. Fertilization was controlled when mature oocytes were incubated with StA prior to IVF, suggesting the possible use of the fungal metabolite in assisted reproductive technology for humans and animals.

Until a few years ago, it was assumed that oocyte renewal did not take place in the ovary of adult organisms; however, the existence of germline progenitor cells (GPCs), which renew the ovarian follicular reserve, has now been documented in mammals. Specifically, in the adult ovary of bats, the presence of cells located in the cortical region with characteristics similar to GPCs, called adult cortical germ cells (ACGC), has been observed. One of the requirements that a GPC must fulfil is to be able to proliferate mitotically, so the evaluation of cell proliferation in ACGC is of utmost importance in order to be able to relate them to a parental lineage. Currently, there are several methods to determine cell proliferation, including BrdU labelling or the use of endogenous proliferation markers. Thus, the aim of this work was to evaluate the proliferative activity of ACGC in the adult ovary of the bat , using different proliferation markers and correlating these with the protein expression of the transcription factor Oct4 and the germ line marker Ddx4. We found that the expression pattern of the proliferation markers BrdU, PCNA, Ki-67 and pH3 occurs at different times of the cell cycle, so co-localization of two or more of these markers allows us to identify proliferating cells. This allowed us to identify ACGC with proliferative capacity in the adult ovary of , suggesting that GPCs renew the follicle reserve during the adult life of the organism.

The study was conducted on indigenous Tharparkar cow () to evaluate FSH stimulation on follicular attributes, oocyte recovery and morpho-molecular developmental competence parameters concerning oocyte quality. A total of 20 OPU sessions were performed, which included 10 sessions in each FSH stimulated at the dose of 130 µg divided into four sub-doses and non-stimulated. Findings on the size of follicles having ≥6 mm showed a significantly higher, however an opposite trend was observed in the case of smaller sized follicle (<6 mm) between stimulated and non-stimulated respectively. The stimulated cows had a significantly higher number as well as the percentage of oocytes of Grade A, having a diameter ≥120 µm and BCB as compared to the non-stimulated cows. The relative mRNA expression profile of GDF9, BMP15, PCNA and BCL-2 genes was higher and BAX was lower in the FSH-stimulated cow. These results indicated that FSH stimulation before OPU in cows has a significant impact on follicle size, oocyte yield, recovery, and their quality with respect to COC's, diameter and BCB oocytes. Further, a significant increase in the relative mRNA expression levels of GDF9, BMP15 and PCNA genes in the FSH-stimulated group suggests that FSH plays a key role in modulating the expression of these important candidate genes and thus influencing oocyte quality. The higher mRNA expression of BCL-2 genes and concomitantly lower expression of BAX gene in FSH Stimulated cows indicates the protective role of these genes and preventing programmed cell death and thus promoting cell survival, quality and embryo development.

Hydrogen sulfide (HS) has been shown to play a significant role in oxidative stress across various tissues and cells; however, its role in sperm function remains poorly understood. This study aimed to investigate the protective effect of GYY4137, a slow-releasing HS compound, on sperm damage induced by HO. We assessed the effects of GYY4137 on motility, viability, lipid peroxidation and caspase-3 activity in human spermatozoa in vitro following oxidative damage mediated by HO. Spermatozoa from 25 healthy men were selected using a density gradient centrifugation method and cultured in the presence or absence of 10 μM HO, followed by incubation with varying concentrations of GYY4137 (0.625-2.5 μM). After 24 h of incubation, sperm motility, viability, lipid peroxidation, and caspase-3 activity were evaluated. The results indicated that HO adversely affected sperm parameters, reducing motility and viability, while increasing oxidative stress, as evidenced by elevated lipid peroxidation and caspase-3 activity. GYY4137 provided dose-dependent protection against HO-induced oxidative stress (OS). We concluded that supplementation with GYY4137 may offer antioxidant protection during in vitro sperm preparation for assisted reproductive technology.

Polycystic ovary syndrome (PCOS) is a complex reproductive and endocrine disorder affecting 5-10% of women of reproductive age, but the pathophysiology of PCOS still remains unknown. Here, the aim of our study was to analyze the effects of rapamycin treatment that may regulate impaired hormonal levels and folliculogenesis in dehydroepiandrosterone (DHEA)-treated PCOS mouse. We hypothesized that rapamycin may ameliorate the negative effects of PCOS in DHEA-induced PCOS mouse model. The target of rapamycin (TOR) gene product is a serine/threonine kinase that has been implicated in the control of cell growth, proliferation and autophagy, and rapamycin is a potent inhibitor of mTORC1 pathway. In this study, for the first time, mTORC1 and activation products are presented at protein and mRNA levels after rapamycin treatment in DHEA-induced PCOS mouse ovary. We showed that rapamycin treatment may regulate follicular development, hormonal levels and provide ovulation in DHEA-induced PCOS mouse. Additionally, we assessed decreased primordial follicle reserve, increased number of primary and secondary follicles, corpus luteum structure forms again after 10 days of rapamycin treatment. This study presented here suggests rapamycin treatment regulates hormonal phenotype and folliculogenesis in the ovary and also mTOR signalling pathway in granulosa cells of DHEA-induced PCOS mouse ovary which may have potential to attenuate understanding the mechanism of dominant follicle selection and anovulatory infertility.

L-carnitine has an important role in the control of oxidative stress and lipid β-oxidation during culture and cryopreservation of ovarian follicles, oocytes and embryos. This substance balances the acetyl-CoA/CoA ratio, maintains glucose metabolism and increases energy production in mitochondria. It also plays a key role in reducing endoplasmic reticulum stress, by transferring palmitate to mitochondria or eliminating it to avoid toxicity. By eliminating reactive oxygen species, L-carnitine increases the percentages of mature oocytes with uniform mitochondrial distribution and improves embryo post-thaw cryotolerance. Therefore, L-carnitine controls lipid β-oxidation and oxidative stress during culture of ovarian follicles, oocyte maturation, embryonic development and cryopreservation.

In Assisted Reproductive Technologies (ART), efficient sperm preparation is vital for successful fertilization, with washing media enhancing the process. This pilot study examines the molecular-level impact of a new serotonin-containing sperm-washing medium (Prototype) on sperm motility and ROS metabolism, comparing it with commercially available media (Origio and Irvine). Semen samples from thirty-one individuals underwent preparation using the swim-up method post-semen analysis. Each sample was separately washed with the Prototype, Origio and Irvine mediums. ROS formation was determined through flow cytometric, and AT2R and PRDX2 protein levels, associated with sperm motility, were assessed via Western blot. Statistical evaluation compared the findings among the three outlined media. Significant differences were found among three washing media in terms of total and progressive motility. The Prototype medium showed the highest increase in both total (66%) and progressive motility (59%), while the control group exhibited the lowest increases (41% and 27.7%, respectively). Regarding ROS levels, the prototype (11.5%) and Origio (10.7%) groups demonstrated a notable decrease, contrasting with Irvine (25.8%). Molecular assessment revealed a significant elevation in AT2R protein levels in the prototype medium (59%), compared to other media. Additionally, an increase in PRDX2 protein levels was observed in the prototype medium, although this didn't reach statistical significance. Serotonin-activated washing media for sperm preparation can be a suitable choice for selecting high-quality sperm in ART. A broader molecular analysis with a larger sample size is required to explore the mechanisms and effectiveness of using a serotonin-containing sperm-washing medium in routine ART.