Can adeno-associated viruses (AAVs) efficiently and safely transduce human primary granulosa cells and human ovarian stromal fibroblasts to modulate the expression of genes involved in follicle recruitment and ovarian microenvironment regulation?

Porcine in vitro embryo production (pIVP) would have a major impact on both livestock production and biomedical research if its efficiency-currently limited by suboptimal oocyte competence-could be improved. This study aimed to characterize transcriptional dynamics across oocyte in vitro maturation (IVM) and fertilization (IVF). Immature oocytes (IMO), mature oocytes (MO) and zygotes (ZY) produced in vitro were analysed by RNA sequencing followed by differential gene expression and functional enrichment analyses. IMO showed higher abundance of zona pellucida (ZP) transcripts (ZP2, ZP3, ZP4), whereas MO were enriched in genes related to cytoskeletal remodelling and calcium signalling (e.g., ITPR1, TGM2), consistent with the acquisition of cytoplasmic competence. After fertilization, ZY exhibited increased representation of transcripts involved in gamete interaction, vesicle trafficking, and maternal-effect complexes (e.g., IZUMO1R, NLRP5, PADI6), reflecting the transition to post-fertilization regulatory support. The chain of transcriptional shifts defines the molecular progression underlying maturation and fertilization competence in the porcine in vitro system.

The efficiency of AI in sheep depends partly on maintaining sperm function during storage. This study compared INRA 96 and a skim milk-based formulation for storing ram semen at 15°C for 6 h, evaluating sperm quality and IVF and field fertility outcomes. INRA 96 showed higher VCL, ALH, and sperm viability after 6 h of storage. However, cleavage rate, blastocyst rate, and blastocyst cell number after IVF, as well as field fertility and prolificacy, did not differ between extenders. These results indicate that the skim milk-based formulation preserved reproductive outcomes at levels comparable to INRA 96, supporting its suitability as a cost-effective alternative for routine AI programs.

Sperm capacitation, a crucial process for successful fertilization, is modulated by reactive oxygen species (ROS) and involves molecular changes including increased membrane fluidity, cholesterol depletion, and protein tyrosine phosphorylation. Although an effective equine in vitro fertilization protocol has recently been described, its requirement for prolonged capacitation limits applicability to high-quality ejaculates. Because stallion spermatozoa possess endogenous antioxidant systems that may mitigate ROS accumulation and delay ROS-mediated capacitation, this study investigated whether inhibition of catalase with 3-amino-1,2,4-triazole (3-AT), superoxide dismutase 1 with LCS1, γ-glutamylcysteine synthetase with buthionine sulfoximine (BSO), or their combination (INH), could enhance endogenous ROS and promote capacitation. Sperm were incubated for 24 h under control, capacitating, or inhibitor-treated capacitating conditions (3-AT, LCS1, BSO, or their combination [INH]). Samples were evaluated using CASA and flow cytometry. Incubation increased intracellular calcium in all samples, but sperm under capacitating conditions showed lower calcium levels than control after 22 h (100.8 ± 2.4 vs 112.7 ± 4.81 AU respectively; p ≤ 0.01), an effect further enhanced by 3-AT (p ≤ 0.05) and BSO (p ≤ 0.01). After 22 h, BSO concentrations above 500 μM increased membrane lipid disorder (CTR: 70.81 ± 5.2 vs 500 μM BSO: 104.85 ± 5.1 AU, p ≤ 0.01), and hyperactivation-associated motility kinematics (TM, VCL, ALH, BCF; all p ≤ 0.05). When data were pooled across time points, INH increased tyrosine phosphorylation compared with control (31.55 ± 2.1% vs 25.24 ± 1.8% respectively; p ≤ 0.01). In conclusion, inhibition of antioxidant systems-predominantly the glutathione system-may drive stallion sperm capacitation and reduce the time required for IVF protocols, although further studies are required to determine its impact on in vitro fertility.

Non-invasive embryo assessment strategies are increasingly being explored in in vitro fertilization (IVF) to complement conventional morphological evaluation and improve embryo selection. This narrative review provides a comprehensive overview of current and emerging approaches, with a focus on time-lapse monitoring (TLM), artificial intelligence (AI), omics-based technologies and non-invasive genomic testing, and critically evaluates their clinical utility and limitations. Recent literature investigating morphokinetic parameters, AI-based embryo evaluation, transcriptomic, metabolomic and secretomic biomarkers, and non-invasive preimplantation genetic testing was reviewed, including relevant clinical studies, systematic reviews and guideline-based recommendations. TLM enables continuous monitoring of embryo development and provides morphokinetic information associated with developmental competence; however, randomized clinical evidence has not demonstrated a consistent improvement in clinically meaningful outcomes when TLM-based embryo selection is used in isolation. AI-based models may improve the objectivity and reproducibility of embryo evaluation, but heterogeneous predictive performance, limited external validation and insufficient evidence of improved live birth outcomes currently support their use primarily as decision-support tools. Omics-based approaches have identified potential non-invasive biomarkers associated with embryo competence and implantation, although methodological variability, lack of standardization and limited prospective validation continue to restrict clinical translation. Among non-invasive genomic approaches, cfDNA-based non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) has shown promising potential, but variable concordance with trophectoderm biopsy and insufficient clinical validation currently preclude its use as a replacement for conventional PGT-A. More comprehensive genomic applications of embryo-derived cfDNA remain exploratory. Overall, embryo assessment is evolving toward integrative, multidimensional strategies combining morphological, developmental, molecular and computational information. Despite substantial technological advances, most emerging non-invasive approaches remain investigational or experimental, and rigorous standardization, multicentre prospective validation and demonstration of improvements in clinically meaningful outcomes are required before routine clinical implementation.

Growth disorders are common in in vitro fertilization (IVF)-conceived twins, but risk factors specific to dichorionic-diamniotic twins remain incompletely characterized. This study aimed to assess these risk factors. Data from in vitro fertilization pregnancies (December 2020 - November 2024) were retrospectively analyzed. Small for gestational age was defined as estimated fetal weight below the 10th percentile. Intertwin discordance greater than 15% was considered significant. Change in uterine artery pulsatility index was calculated by subtracting first-trimester from second-trimester values. Of 169 dichorionic-diamniotic twin pregnancies, 30 (17.8%) were from fresh embryo transfer and 139 (82.2%) from frozen embryo transfer. Mean uterine artery pulsatility index was higher in the fresh group in both trimesters (first: 1.72 vs. 1.30; second: 0.91 vs. 0.78; both p = .001). Small for gestational age incidence was similar between groups (56.7% vs. 50.3%, p = .441), as was significant discordance (36.6% vs. 25.9%, p = .081). Logistic regression identified advanced maternal age (odds ratio: 1.07, 95% CI [1.00, 1.14]; p = .024), higher first-trimester uterine artery pulsatility index (OR 2.19, 95% CI [1.27, 3.76]; p = .002), and preeclampsia (OR 3.74, 95% CI [1.19, 11.67]; p = .023) as predictors of small for gestational age. For significant discordance, predictors were second-trimester intertwin estimated fetal weight difference (OR 1.03, 95% CI [1.01, 1.04]; p = .001) and change in uterine artery pulsatility index (odds ratio: 2.51, 95% CI [1.01, 6.22]; p = .047). Uterine artery pulsatility index and second-trimester biometric differences independently predict small for gestational age and intertwin discordance in IVF-conceived dichorionic-diamniotic twin pregnancies.

This systematic review and meta-analysis compared reproductive, perinatal and neonatal outcomes between letrozole-based endometrial preparation, with or without adjuvant gonadotrophins, and programmed-cycle endometrial preparation for frozen embryo transfer (FET) in women with polyendocrine metabolic ovarian syndrome (PMOS), including only randomized controlled trials (RCTs). MEDLINE, Embase and the Cochrane Central Register of Controlled Trials were searched from inception through September 10, 2025. Four RCTs met inclusion criteria (n = 891). Random-effects models were used to pool risk ratios, risk of bias was assessed with RoB-2 and certainty of evidence was evaluated using Grading of Recommendations, Assessment, Development and Evaluations. No significant difference in live birth rate was observed between the two protocols (RR 0.92, 95% CI 0.79 to 1.07). Similarly, no significant differences were observed in clinical pregnancy, miscarriage, hypertensive disorders of pregnancy or cycle cancellation rates. A lower risk of gestational diabetes mellitus (GDM) was observed with programmed-cycle FET in a meta-analysis of two RCTs (RR 0.33, 95% CI 0.14 to 0.82, P = 0.02); however, this hypothesis-generating finding should be interpreted cautiously because of the unexpectedly high GDM rates and requires confirmation in larger studies. Other perinatal and neonatal outcomes did not differ significantly, although evidence was underpowered. In contrast to observational studies, current RCT evidence does not demonstrate superiority of letrozole-based over programmed-cycle endometrial preparation for reproductive outcomes in women with PMOS.