This study aimed to evaluate the effects of linseed supplementation on the in vitro production of embryos subjected to vitrification. Pantaneira cows supplemented with linseed or not (control) were evaluated. The best-quality embryos produced in vitro from both groups were vitrified. Oocyte quality and blastocyst rate did not differ between the groups. However, the rates of vitrifiable embryos and re-expansion at 3 h were higher in the linseed-supplemented group. In conclusion, linseed supplementation in Pantaneira cows improved the quality of embryos produced in vitro.

Artificial oocyte activation (AOA) with Calcium ionophore (Ca-I) application has been shown to improve fertilization rates in patients with poor reproductive outcomes and fertilization failure; however, the effect of Ca-I on embryomorphokinetic parameters remains unclear. The aim of this study was to evaluate the effect of AOA with Ca-I application on embryomorphokinetic parameters and clinical pregnancy outcomes. In this retrospective cohort study, a total of 412 cycles were involved and participants were classified into two groups as intracytoplasmic sperm injection ICSI + AOA (n = 225) and ICSI (n = 187). There were no significant differences between groups in terms of baseline parameters. When the duration from 2PN to tB (time to blastulation) was examined, t2, t3, t4, or t5 times were not significantly different between the groups; however, Ca ionophore application was found to extend time to start of blastulation (tsB) time and tB (p < 0.05). Optimal time-lapse parameters (t5, cc2, S2, and t5–t2) also did not differ between the two groups, and there were no significant differences in embryo grades determined by optimal time-lapse parameters. According to the fertilization and pregnancy results, no significant differences were found between the groups. In conclusion, Ca ionophore application does not seem to affect most embryomorphokinetic parameters except blastulation time, and the clinical importance of this finding needs further research.

Women with diabetes frequently experience ovarian dysfunction, which is a significant indication that diabetes adversely affects their reproductive health. This study investigates the efficacy of combined treatment with Pioglitazone and Cynara scolymus extract in improving ovarian function in streptozotocin-induced diabetic rats. A total of 35 female rats were divided into five groups: (1) Control (saline), (2) DM (diabetic control), (3) DM + Piog (30 mg/kg Pioglitazone), (4) DM + Cynara (400 mg/kg C. scolymus extract), and (5) DM + Piog + Cynara (combined treatment), and were administered orally for 30 consecutive days. Following treatment, histopathological and stereological analyses were performed to evaluate follicular development, corpus luteum volume, and oxidative stress markers. The results indicated that the combined treatment (DM + Piog + Cynara) led to significant improvements, including increased numbers of preantral, antral, and Graafian follicles, alongside an enhanced corpus luteum volume. Additionally, there was a notable reduction in atretic follicles and fasting blood glucose levels compared to the diabetic group. Diabetes resulted in elevated malondialdehyde (MDA) levels and decreased antioxidant enzyme activities. However, the combination therapy effectively modulated apoptotic pathways, enhancing Bcl-2 expression while reducing Caspase-9 activity. This study demonstrates that the combined therapy of C. scolymus extract and Pioglitazone mitigates diabetic-induced ovarian dysfunction by reducing oxidative stress, normalizing hormonal levels, and promoting healthy folliculogenesis. These findings suggest that this approach may serve as a promising complementary treatment for diabetes-related reproductive issues, potentially improving reproductive health.

Spermatogonial stem cells (SSCs) constitute the foundation of male sperm production and fertility. SSCs are characterised by their capacity for self-renewal and differentiation into spermatozoa throughout a male’s reproductive life, thereby transmitting genetic information to subsequent generations. During embryonic development, SSCs are derived from postnatal testicular gonocytes, which originate from long-lived primordial germ cells. A large proportion of men’s infertility is detected by sperm analysis. Unusual sperm parameters include sperm concentration, morphology and motility. Male infertility is mainly due to hormone deficits, physical causes, sexually transmitted diseases, environment, lifestyle and hereditary factors. The most severe form of male infertility is non-obstructive azoospermia (NOA), which is defined as no sperm in the ejaculate because of spermatogenesis failure. The first-line treatment for these individuals has been testicular sperm extraction under an operating microscope (micro-TESE). SSCs are also considered a promising pathway for regenerating impaired or damaged spermatogenesis. This review addresses SSC transplantation as a potential method for treating male infertility and restoring spermatogenesis in patients with degenerative diseases such as cancer, radiotherapy and chemotherapy.

Dynamic systems for in vitro embryo production (IVEP) have long been sought to allow a higher resemblance to the physiological conditions of the oviduct and uterus. Although significant progress has been demonstrated in the microfluidic field, some challenges are still present regarding the microfluidic devices’ sterilization protocol. This complication is related to the leached components from the poly(dimethylsiloxane) (PDMS). The PDMS is one of the safest materials used in soft lithography for embryo production. However, the use of moulds required for soft lithography can compromise embryo culture. This limitation arises from the restricted range of biocompatible resins available for 3D-printing of moulds. While the replacement of the biomaterial for mould production is not a straightforward option, we hypothesized that PDMS devices fabricated from such moulds could be rendered biocompatible through an optimized cleaning protocol. In this current study, we evaluated the effectiveness of sequential washes, sonication, and extended incubation in washing solutions. Our results demonstrate that a prior incubation of the PDMS devices in PBS supplemented with antibiotics and antifungal (at 4°C for 48 hours), in addition to the incubation in IVC medium (at 38.5°C for 48 hours), enables bovine embryo development within PDMS devices fabricated with moulds from a 3D printer. This optimized protocol supported IVEP at rates equivalent to standard conditions, with no increase in apoptosis or oxidative stress, validating its suitability for artificial reproductive techniques applications.

Infertility aetiology has been shown to be related to reproductive outcomes in the wide reproductive age range. The aim of this study was to examine young women in vitro fertilization (IVF) cycle outcomes according to age and infertility cause. This retrospective cohort study was comprised of 305 couples with a female partner aged 17–25 years with either male or female infertility that underwent a fresh IVF cycle between 2003 and 2020. Comparison according to age groups 17–22 years (n = 117) and 23–25 years (n = 188) showed similar outcomes, except for higher rates of 3 pronuclei (PN) embryos in the younger group. However, allocation to groups by infertility aetiology - male factor (MF) (n = 241) vs. female factor (FF) (n = 64) demonstrated lower fertilization rate in the FF vs. MF group (58% vs. 63%, p = 0.049) and higher percent of 1PN and 3PN embryos (0.7% vs. 0.2% and 0.7% vs. 0.1%, p < 0.001 for both). Chemical pregnancy and live-birth rate were significantly higher in the MF vs. FF group (48.9% vs. 29.5%, p = 0.02 and 41.9% vs. 11.0%, p = 0.038). The only parameter associated with live birth rate in a logistic regression analysis was male infertility aetiology (OR = 2.17, p = 0.038). Young couples undergoing IVF cycles due to female factor infertility have poorer reproductive outcomes compared to male factor-related infertility and higher rates of 1PN and 3PN embryos.

The role of Polyvinylpyrrolidone (PVP) on human gametogenesis and embryonic development has been investigated in clinical setting. PVP is routinely used in sperm handling during intracytoplasmic sperm injection (ICSI) procedure. However, the controversies about its harmful effects on embryonic cells has been debated in recent years. Some reported that PVP decreases the membrane integrity and chromatin stability and strongly affects the fine structure of spermatozoa, such as axonemal tubules, fibrous sheath, and accessory fibres. However, others claimed that PVP delayed the onset of calcium oscillations in the oocyte in ICSI setting. Therefore, PVP affects the decondensation of male pronuclear formation after sperm injection. Also, it has been reported that after injection of conventional 10%PVP to embryos, PVP solution remained in the embryos and suppressed development of these embryos. Recent studies indicated that with a decrease in PVP concentration, embryo development increased and morphokinetics improved in ICSI cycles. Also, low concentration of PVP decreased the rate of apoptosis when compared to 10%PVP. In this review, the irreversible effects of PVP on sperm and embryonic chromosomal aberrant were reported. In conclusion, it is recommended to use the low concentration of PVP in conventional ICSI programme to limit its damage on fertilization processes as well as embryo development.

Sperm capacitation involves biochemical and functional changes necessary for fertilization. The ubiquitin–proteasome system (UPS) regulates protein turnover, but the roles of proteasomal receptors during capacitation remain unclear. This study investigates the regulation and functional impact of proteasomal subunits RPN13 (ADRM1) and RPN2 (PSMD1) during in vitro capacitation of boar sperm. Semen samples (n = 6) were assigned to four groups: non-capacitating control (Non-Cap, 4 h in medium lacking bovine serum albumin (BSA)), capacitating control (Cap, 4 h with BSA) and groups treated with vehicle (CM + dimethyl sulfoxide (DMSO)) or the RPN13 inhibitor RA190. We measured RPN2/RPN13 levels, RPN13-associated ubiquitin signal and RPN13–RPN2 interactions (Western blot, Co-IP) and assessed sperm kinematics, intracellular Ca2+, protein tyrosine phosphorylation (pTyr), mitochondrial membrane potential (ΔΨm), ATP, ZP binding and early embryo cleavage after IVF. The Cap group showed successful capacitation, confirmed by increases in pTyr, sperm hyperactivation and Ca2+-associated Fluo-4 fluorescence. During capacitation, RPN2 and RPN13 abundance decreased, while RPN2–RPN13 association increased. RA190 treatment increased RPN13 levels, inhibited RPN13 turnover and decreased its interaction with RPN2 and the associated ubiquitin signal. Functionally, RA190 attenuated sperm hyperactivation, Ca2+-associated Fluo-4 fluorescence, protein tyrosine phosphorylation, mitochondrial membrane potential and ATP levels and was associated with reduced fertilization-related outcomes, including ZP binding and early embryo cleavage after IVF. These findings highlight the role of RPN13 in sperm capacitation and fertilization.

Currently, the research on the key factors which affect clinical and non-clinical pregnancy in high-quality single blastocyst transfer cycles remains relatively limited. This is particularly true for FET cycles, where the relationship between the transfer of high-quality single blastocysts and pregnancy outcomes has not been fully explored. This study aimed to identify key factors influencing clinical pregnancy outcomes in high-quality single blastocyst frozen-thawed transfer cycles to optimize assisted reproductive technology (ART). Patients under 38 years old who underwent high-quality single blastocyst frozen-thawed embryo transfer were included. Based on clinical pregnancy outcomes, they were divided into clinical pregnancy (Group A) and non-clinical pregnancy (Group B) groups. Key influencing factors were analyzed to guide the selection of blastocysts with the highest pregnancy potential.The result showed that Group B showed significantly higher age and infertility duration, but lower AMH levels, antral follicle count, and endometrial thickness on the day of transfer compared to Group A (P < 0.01). Infertility type also differed significantly (P < 0.01). Blastocyst grading differed between groups (P < 0.01), while E2, LH, P levels, embryo age, and D3 cleavage-stage cell count showed no significant differences (P > 0.05). Multivariate analysis revealed that infertility type, age, infertility duration, and endometrial thickness significantly impacted clinical pregnancy outcomes (P< 0.05), while AMH, antral follicle count, and blastocyst grading had no significant effect. All in all, clinical pregnancy outcomes are significantly influenced by age, infertility type, infertility duration, and endometrial thickness. Early treatment, optimized endometrial conditions, and selecting high-quality blastocysts are recommended to improve pregnancy rates.

Trafficking Protein Particle Complex Subunit 4 (TRAPPC4) is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus. It plays a crucial role in tethering COPII vesicles and assembling SNARE complexes, ensuring precise protein delivery, which is vital for cellular communication and growth. Mutations in TRAPPC4 and other TRAPP subunits are associated with a group of human diseases collectively known as TRAPPopathies, often manifesting as neurological disorders. This study investigates the role of TRAPPC4 during early embryonic development by analysing the phenotypic consequences when the gene is knocked out in the mouse. Our results show that TRAPPC4 knockout leads to early embryonic lethality, where TRAPPC4 homozygous mutant embryos form morphologically normal blastocysts but fail to implant in the uterus and are not present at embryonic day 7.5. We show that mutant embryos can hatch from the zona pellucida, but do not form proper inner cell mass (ICM) outgrowths. Unlike the tightly compressed ICM colonies in control littermates, the mutant ICM appears necrotic with cells that are not adherent. This dispersion suggests that ICM cells may begin deteriorating at peri-implantation stages, underscoring TRAPPC4’s pivotal role in maintaining cellular viability during early embryonic development.

This study investigated how hormonal induction, female presence, and production system affect sperm quality in Astyanax lacustris across three experiments. In Experiment 1, males and females were kept together in the same recirculating aquaculture system (RAS) before testing. Hormonal induction consistently boosted initial motility and prolonged sperm activity, while female presence offered only a modest benefit to non-induced males and no measurable effect when males were induced. Sperm concentration remained similar across treatments. Experiment 2 evaluated the same factors using broodstock originating from different rearing environments. When males and females came from separate RAS units, hormonal induction again sustained higher motility, and induced males paired with females showed higher sperm concentration than some non-induced groups. In the RAS–biofloc technology (BFT) pairing, hormonal induction maintained motility regardless of female presence; however, in the absence of hormonal induction, both sperm motility and concentration were modulated by the rearing system of females, with non-induced males paired with BFT females exhibiting lower reproductive performance. Overall, hormonal induction proved to be the most reliable strategy for ensuring high semen quality in A. lacustris. Nonetheless, when induction was not applied, male reproductive performance became more sensitive to female origin and rearing environment, highlighting the importance of broodstock compatibility and production system history in reproductive management protocols.

The successful freezing of rat spermatozoa and the development of innovative cryoprotectant formulations are of great importance in the field of reproductive biotechnology. In the present study, 4 mg/mL Ascorbic acid and 20 nM Biotin were added to cryopreservation medium containing 8% lactose-monohydrate, 23% egg yolk, and 10% tris-aminomethane to investigate the freezability of rat sperm. After cryopreservation, motility, viable spermatozoon ratio, plasma membrane integrity, abnormal acrosome ratio, and mRNA levels of certain heat shock proteins that play a critical role in the sperm cryopreservation process were evaluated. After thawing, the highest motility rate was found in the ascorbic acid group (17.50 ± 3.27), which was significantly higher compared with the control group (10.13 ± 1.16) (p < 0.05). Likewise, the highest viability rate was found in the ascorbic acid group (41.75 ± 3.69), while it was found to be significantly higher compared with the control group (19.63 ± 1.44) (p < 0.05). When the cells were evaluated in terms of plasma membrane integrity and abnormal acrosome rate, no statistical difference was found between the groups (p > 0.05). The expression levels of heat shock protein-related genes were determined using the RT-qPCR technique. Addition of biotin and ascorbic acid significantly reduced Hsp40, Hsp60, and Hsp70 mRNA levels compared with the control group (p < 0.05). In conclusion, based on the evaluated parameters, it was revealed that the addition of the antioxidant Ascorbic acid and biotin to rat sperm extender provides effective protection against cryodamage during the cryopreservation process. These findings provide a valuable foundation for research into the development of next-generation mediums for sperm cryopreservation from a molecular biotechnology perspective.

This study investigated the expression profile of the Toll-like receptor 2 (TLR2) gene in goats across three reproductive groups – twin-bearing (n = 12), single-bearing (n = 10) and non-pregnant (n = 8) – using quantitative real-time PCR (qRT-PCR). Descriptive statistics revealed that twin-bearing goats were significantly heavier at service and kidding, with higher litter weights compared to other groups (p < 0.05). No breed-wise differences were observed, allowing pooled analysis for gene expression. Melt curve and amplification plot analyses confirmed primer specificity and efficient amplification. TLR2 expression was significantly upregulated in twin-bearing goats (ΔCt = −2.14 ± 0.89), with a mean 4.5-fold increase (p < 0.001) compared to non-pregnant controls, suggesting enhanced immune activation. Single-bearing goats showed negligible and non-significant changes in expression (ΔCt = −0.01 ± 1.45; p = 0.984). Statistical comparisons showed a significant difference in TLR2 expression between twin- and single-bearing groups (mean difference: 3.39; p < 0.001). These results reveals a significant association of twin pregnancies with elevated expression level of TLR2 gene in resource population. While the functional implications require further investigation, this upregulation may be associated with immune adaptations to increased gestational load. Overall, this study identifies TLR2 as a candidate gene expression marker strongly correlated with twin pregnancy in goats. Further validation through longitudinal studies on larger cohorts and investigation of tissue-specific expression is necessary to assess its potential predictive value for reproductive performance in small ruminants.

This study aimed to investigate the involvement of the Wnt signalling pathway in the pathogenesis of non-obstructive azoospermia (NOA) and to identify potential diagnostic and therapeutic targets. We obtained the gene expression profiles of NOA patients from the GEO database, screened for differentially expressed genes (DEGs) and NOA-associated co-expressing gene modules, then intersected the DEGs with related genes in the Wnt signalling pathway and identified key genes. LASSO regression analysis was used to find hub genes, and qRT-PCR and WB methods were used to verify the expression of key genes in NOA patients. The immune infiltration and GSEA were conducted in order to investigate the relationship between hub genes and immune cells infiltration and NOA-associated pathways; furthermore, we built the miRNA-mRNA-TF regulatory network and predicted possible small molecules drug targets of the hub gene. Four candidate genes were related to NOA: CSNK1G2, GNG3, H2AFB1 and PARD6A. The key hub gene was CSNK1G2 based on the results of the LASSO regression analysis, and its expression level was significantly down-regulated in NOA patients. The immune infiltration analysis and GSEA results showed that the expression level of CSNK1G2 had a close relationship with immune cells infiltration and NOA-related pathways. Furthermore, we built a miRNA-mRNA-TF regulatory network and predicted the possible small molecule drugs of CSNK1G2. Our findings suggest that the Wnt signalling pathway, particularly CSNK1G2, plays a crucial role in the pathogenesis of NOA. CSNK1G2 may be a novel target for the diagnosis and treatment of NOA.

The aim of this study was to investigate the chemical composition of Lippia sidoides essential oil (LSEO) and the influence of different concentrations of LSEO on activation and survival of primordial follicles, ovarian stromal cell density, collagen area in the extracellular matrix (ECM), as well as on thiol levels, messenger RNA (mRNA) of CAT, SOD1, GPX1 and PRDX6 and activity of antioxidant enzymes CAT, SOD1 and GPX1. To this end, the chemical composition of the oil was analysed using the gas chromatography (GC) method. The bovine ovaries were collected from a local slaughterhouse. In the laboratory, fragments of the ovarian cortex were then cultured in control medium alone or in the presence of 400, 800, 1600 and 3200 µg/mL LSEO. After six days of culture, the tissues were fixed and sent for histological analysis. Other fragments were used in molecular tests to determine the redox profile and to quantify mRNA levels. The results showed that a concentration of 400 μg/mL LSEO promoted follicular activation and growth, as well as increased stromal cell density and the mRNA levels of SOD1, CAT, and GPX1. Additionally, the LSEO at all tested concentrations reduced CAT activity in cultured bovine ovarian tissues.

The present study aimed to evaluate the effect of N-acetylcysteine (NAC) on in vitro maturation (IVM) and in vitro embryo production in sheep. Cumulus–oocyte complexes (COCs) were collected and matured in vitro in a control medium or supplemented with different concentrations of NAC (1, 1.5 and 2.0 mM). After maturation, a subset of oocytes was used for morphological assessment and chromatin configuration analysis, as well as for evaluating intracellular levels of reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and DNA fragmentation using the TUNEL assay. Another subgroup of oocytes was subjected to in vitro fertilization (IVF), and the presumed zygotes were cultured in in vitro culture (IVC) medium to assess cleavage rates. Data were statistically analysed, considering significance at P < 0.05. The concentration of 1 mM NAC resulted in lower ROS levels compared to the control and 1.5 mM groups, in addition to reducing DNA fragmentation. No significant differences (P > 0.05) were observed in GSH levels or mitochondrial activity between treated and control groups. Furthermore, oocytes treated with 1 mM NAC showed higher cleavage rates compared to the other experimental groups. In conclusion, supplementation with 1 mM NAC improves oocyte quality and early embryonic development, as demonstrated by reduced oxidative stress and DNA fragmentation, resulting in increased cleavage rates.

We evaluated the efficiency of two-dimensional (2D) and three-dimensional (3D) systems for in vitro culture of preantral follicles from the bovine species. Ovarian cortex fragments were obtained from a local slaughterhouse and randomly distributed into four groups: i) standard culture, directly on a culture plate (2D), ii) supported culture, on a gel monolayer (2D), iii) immersed culture on agarose gel (plating coated) covered in a medium (3D) and iv) Millicell–Biopore culture (3D), tested at 6 or 14 days. The following parameters were evaluated: follicular morphology and morphometry, reactive oxygen species production and granulosa cell proliferation. After 6 days of culture, follicles cultured on a gel monolayer (2D) showed a higher proportion of intact follicles than those cultured using other methods (P < 0.05). After 14 days of growth, standard culture on a culture plate (2D) and supported culture on a gel monolayer (2D) had a higher percentage of morphologically intact follicles (P < 0.05) than the 3D culture. An assessment of the redox status revealed that superoxide anion levels and antioxidant capacity of the samples cultured using the four methods were lower than those of the control group (non-cultured; P < 0.05). Taken together, these findings suggest that the agarose gel support system offers distinct advantages for bovine preantral follicle culture by maintaining morphological integrity and stabilising the redox environment, although further studies incorporating functional endpoints are warranted to fully confirm these observations.

The piracanjuba (Brycon orbignyanus) is a species threatened with extinction due to human activities, highlighting the need for genebanking and alternative propagation methods for its conservation. The objective of this study was to obtain a completely sterile yellowtail tetra (Astyanax altiparanae) and to transplant the stem spermatogonia (SSCs) of B. orbignyanus. Complete depletion of spermatogenesis in adult diploid males was successfully achieved through five applications of busulfan, using dosages of 15 mg kg−1 and 40 mg kg−1 at a high temperature of 35°C. One month after completing the busulfan treatment, spermatogenesis returned, occurring more rapidly in the 15 mg kg−1 treatment, with spermatozoa visible in the testicular lumen. In the treatment with 40 mg kg−1, only SSCs and some spermatid cysts were observed. Depletion of spermatogenesis in triploid juveniles was observed after two applications of busulfan (50 mg kg−1). One month after the end of the treatment, only a few SSCs cells were observed in the testes. The B. orbignyanus SSCs were isolated using a Percoll density gradient and identified by alkaline phosphatase activity, resulting in a cell suspension of 2.6 × 106 SSCs/mL with 93% cell viability. The cells were labeled with PKH26 and transplanted via the urogenital papilla into recipients (triploid juvenile males) previously treated with busulfan. PKH26-positive cells were visualized up to 80 days after transplantation. The combination of two sterilization methods appears to be more effective for cell transplantation after the endogenous germ cells have been eliminated from the recipient testes of triploid juveniles.

Recurrent pregnancy loss (RPL) is a clinically important condition in women undergoing assisted reproductive technologies (ART). This study evaluated embryo morphology and morphokinetic parameters using time-lapse monitoring (TLM) and assessed embryo ploidy by preimplantation genetic testing for aneuploidy (PGT-A) in women with RPL compared with unexplained infertility (UEI) controls. A total of 190 patients (100 RPL, 90 UEI) and 1169 embryos (634 RPL, 535 UEI) cultured under TLM were analyzed. Clinical characteristics, embryo morphology, morphokinetics and ploidy status were compared between groups, and logistic regression was used to identify predictors of aneuploidy. The euploidy rate was significantly lower in the RPL group than in controls (43.5% vs. 52.3%, p = 0.018). Group-wise analysis of all embryos revealed differences in selected morphokinetic parameters (t9 and tSC; p < 0.05). However, morphokinetic timing of euploid embryos did not differ between groups, suggesting that accelerated development alone was not associated with chromosomal abnormality. Embryo morphology was the strongest predictor of aneuploidy in both cohorts. Notably, direct uneven cleavage was associated with a 3.4-fold increased risk of aneuploidy specifically in the RPL group. Although embryos from RPL patients showed relatively faster developmental kinetics, morphokinetic speed alone did not predict chromosomal competence. Instead, embryo quality remained the key determinant of aneuploidy, while the association between direct cleavage and aneuploidy highlights the potential clinical value of TLM when combined with PGT-A. These findings support a complementary role for TLM in embryo assessment in RPL patients.

Manipulation of the zona pellucida, including laser-assisted hatching (LAH), has been associated with an increased risk of monozygotic multiple pregnancy (MMP). A leading hypothesis suggests that embryonic splitting could be triggered by the division of the inner cell mass as the blastocyst herniates through the zona pellucida. The present study aimed to investigate whether the size of the zona pellucida opening created by LAH influences the prevalence of MMP in frozen-thawed single blastocyst transfer (SBT) cycles. This retrospective observational study included 9452 pregnancies resulting from frozen-thawed SBT, of 13,271 clinical pregnancies between January 2020 and October 2024. MMP was defined as the presence of ≥2 gestational sacs and/or ≥2 fetuses after SBT. Between January and May 2021, the LAH opening size was enlarged from 30° to 90°. Prevalence of MMP was compared between 30° and 90° zona openings. Among the 9452 pregnancies analysed, 175 (1.85%) were MMP cases, including 169 twin and 6 triplet pregnancies. No significant differences in maternal age, fertilization method, blastocyst grade, culture duration or embryo biopsy rate were observed between singleton and MMP cases. In univariate analysis, a small zona opening was associated with an increased risk of MMP (odds ratio [OR] 1.51, 95% confidence interval [CI] 1.01–2.29, p = 0.047); however, this association was not significant after adjustment for confounders (OR 1.47, 95% CI 0.98–2.22, p = 0.06). A small zona pellucida opening created by LAH showed a trend towards an increased risk of MMP and warrants further investigation.