Search Results (242)

In vitro maturation (IVM) of oocytes remains less efficient than in vivo maturation, partly because conventional culture media cannot fully reproduce the follicular microenvironment. This study evaluated whether human amniotic membrane stem cell-conditioned medium (hAMSC-CM) improves IVM efficiency of mouse cumulus–oocyte complexes (COCs) recovered from ICR females primed with 0, 5, or 10 IU pregnant mare serum gonadotropin (PMSG). hAMSC-CM was added at 10% or 20% (v/v), and maturation, cleavage, blastocyst formation, and gene expression in MII oocytes, cumulus cells, and blastocysts were assessed. Supplementation with 20% hAMSC-CM significantly increased maturation rates across all PMSG groups, reaching 89.4 ± 1.4% versus 59.5 ± 1.3% in the 5 IU PMSG control (p < 0.05). With PMSG priming, 20% hAMSC-CM also enhanced cleavage and blastocyst formation. Molecular analyses showed suppression of CASPASE9 in MII oocytes, upregulation of AMH, BMP15, and GDF9 with downregulation of AMHR in cumulus cells, and increased IGF1 and BCL2 expression in blastocysts. These findings indicate that 20% hAMSC-CM, together with optimized PMSG priming, improves mouse oocyte IVM efficiency and embryo developmental competence. Full article

Background: Polycystic ovary syndrome (PCOS) is a leading cause of anovulatory infertility and a frequent indication for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). Multivariable regression estimates adjusted associations, whereas association rule mining (ARM) can describe frequently co-occurring attributes; however, ARM outputs are unadjusted and may be sensitive to analytic specification. Objectives: We used ARM alongside multivariable logistic regression and internal robustness analyses to describe combinations of clinical and treatment-related variables that co-occurred with clinical pregnancy failure among women with PCOS undergoing IVF/ICSI. Methods: We retrospectively analyzed 733 deidentified patients with PCOS from the Reproductive Hospital of Guangxi. The original one-hot-encoded analytic file was linked to a supplemental clinical record file containing continuous age, BMI, infertility duration, anti-Müllerian hormone, total gonadotropin dose, fertilization method, embryo-transfer day, number of embryos transferred, and embryo score. Embryo quality was derived from D3 cleavage-stage and blastocyst morphology records. Multivariable logistic regression was used to estimate adjusted associations. ARM results were assessed using Fisher’s exact test, Benjamini–Hochberg false discovery rate (FDR) correction, 1000 bootstrap resamples, fivefold cross-validation, and threshold/cutoff sensitivity analyses. Results: In the multivariable model (n = 730), age remained associated with clinical pregnancy failure (adjusted odds ratio [aOR] = 1.04 per year, 95% CI 1.00–1.08, p = 0.031), whereas BMI was not independently associated with failure (aOR = 1.00 per kg/m², 95% CI 0.96–1.05, p = 0.860). Transfer of two embryos (aOR = 0.55, 95% CI 0.35–0.85, p = 0.008), D5 blastocyst transfer (aOR = 0.49, 95% CI 0.32–0.75, p = 0.001), and transfer of at least one high-quality embryo (aOR = 0.63, 95% CI 0.43–0.93, p = 0.020) were associated with lower odds of failure. Under the primary host-factor specification, age > 35 years + BMI ≥ 24 kg/m² + pelvic adhesion defined 48 patients, 30 of whom had clinical pregnancy failure (support = 0.07; confidence = 0.63; lift = 1.38; FDR q = 0.098). In the secondary treatment-inclusive analysis, infertility duration > 5 years + no high-quality embryo transferred defined 43 patients with 30 failures (support = 0.06; confidence = 0.70; lift = 1.54; FDR q = 0.026). Lift represents the subgroup failure proportion divided by the overall failure proportion and is not an adjusted risk ratio. The rule composition and the number of FDR-supported rules changed under alternative cutoffs and confidence thresholds. Conclusions: ARM described transparent but specification-sensitive co-occurrence profiles that complement, rather than compete with, regression. Treatment-inclusive profiles combine baseline and downstream cycle characteristics and are therefore especially exploratory. These findings require prospective multicenter validation before any clinical decision-support use. Full article

Paternal contributions to preimplantation embryo development are increasingly recognized as an important component of reproductive success, but the associations between male reproductive phenotype, early embryonic competence, and embryo molecular characteristics remain insufficiently defined. Using bovine in vitro embryo production as a translational large-mammalian model, this study evaluated whether paternal reproductive phenotype and oocyte origin were associated with embryo developmental performance and embryonic sirtuin expression. Oocytes obtained from mature cows or heifers were fertilized with semen from bulls differing in reproductive status, including bulls characterized by libido disturbances and irregular semen collection. Fertilization, cleavage, blastocyst formation, and hatching were assessed, while embryonic SIRT2, SIRT3, SIRT6, and SIRT7 expression was quantified by immunofluorescence. Early developmental endpoints were relatively preserved, whereas differences were more evident at later stages of preimplantation development. Cow-derived oocytes were associated with higher hatching rates than heifer-derived oocytes. Embryos generated using semen from bulls with an impaired reproductive phenotype showed lower developmental competence and lower SIRT6 and SIRT7 expression. SIRT6 expression showed the strongest positive correlation with hatching, and positive correlations were also observed among the analyzed sirtuins. These findings indicate that paternal reproductive phenotype and oocyte origin are associated with bovine embryo developmental competence and embryonic sirtuin profiles. Embryonic SIRT6 and SIRT7 expression may represent candidate molecular indicators of embryo competence; however, causal relationships cannot be inferred from the present study design. Full article

Sire-dependent variation in bovine in vitro fertilization (IVF) success is poorly explained by conventional semen parameters once samples meet standard quality thresholds. Sperm capacitation is required for acquisition of fertilization competence, yet its contribution to sire-dependent variation in bovine IVF outcomes remains unclear. Fifteen bulls (Bos taurus) were evaluated across five IVF replicates using 30 abattoir-derived oocytes per bull per replicate. Sperm were analyzed before (0 h) and after 3 h of in vitro capacitation using image-based flow cytometry to quantify zinc ion localization patterns (zinc signatures; Fluo-Zin 3 AM), acrosome remodeling (peanut agglutinin conjugated to Alexa Fluor 647; PNA-AF647), and plasma membrane integrity (propidium iodide). Motility parameters were assessed using computer-aided sperm analysis (CASA). Cleavage rate was assessed on Day 3 and blastocyst development on Day 7. Substantial bull-to-bull variation was observed for both cleavage (31.5–76.3%) and blastocyst development (7.3–40%). Bull effects on cleavage were significant (p < 0.0001). Predictive modeling revealed that CASA or biomarker variables alone explained limited variance in embryo development. In contrast, models incorporating capacitation-induced changes in zinc signatures and acrosome remodeling significantly improved prediction of IVF outcomes. Integration of biomarker and motility parameters across time points yielded the highest explanatory power, with zinc signature dynamics among the most consistently informative predictors. These findings demonstrate that sperm capacitation-associated zinc flux and acrosomal remodeling are biologically relevant determinants of bull-specific variation in bovine IVF embryo development and provide mechanistic biomarkers that improve prediction beyond conventional motility assessment alone. Full article

Objectives: Although blastocyst transfer may improve embryo selection and early pregnancy outcomes, its impact on live birth in women with recurrent implantation failure (RIF) remains uncertain. This study compared reproductive and perinatal outcomes between single blastocyst transfer (SBT) and double cleavage-stage embryo transfer (DET) among women younger than 38 years with RIF. Methods: Secondary observational analysis of a multicenter, randomized, double-blind, placebo-controlled clinical trial conducted across eight academic fertility centers in China. In the original trial, patients with RIF under 38 years of age were enrolled and randomized to receive either prednisone or placebo. For the present analysis, a total of 582 patients were categorized according to the embryo transfer strategy. The SBT-versus-DET comparison was not randomized, and the two strategies differed in both embryo developmental stage and number of embryos transferred. Results: No statistically significant difference in live birth rate was observed between the SBT and DET groups (41.8% vs. 38.4%; p = 0.471), despite a higher biochemical pregnancy rate in the SBT group (59.9% vs. 45.7%, p = 0.003). Pregnancy loss was more frequent in the SBT group than in the DET group (30.2% vs. 15.9%; p = 0.018), particularly biochemical pregnancy loss (15.5% vs. 5.8%; p = 0.036), whereas twin live birth was less frequent (0.7% vs. 9.9%; p < 0.001). After adjustment, no significant difference in live birth rate was observed between DET and SBT (adjusted OR 1.04, 95% CI 0.68–1.57). In addition, Day 5 SBT showed a higher live birth rate than Day 6 SBT, whereas pregnancy loss rates were similar between Day 5 and Day 6 SBT cycles. Other exploratory sensitivity and stratified analyses showed broadly consistent findings. Conclusions: Although SBT is generally preferred in clinical practice, our study found no statistically significant difference in live birth rates between SBT and DET among women younger than 38 years with RIF. Given that pregnancy loss was observed more frequently in the SBT group, whereas twin live births were less frequent, our findings support individualized counseling rather than uniform use of either transfer strategy. However, given the non-randomized nature of this comparison, these findings should be interpreted with caution. Full article

Reliable preimplantation embryo sexing is relevant in cattle breeding; however, PCR-based Y-marker detection may be sensitive to limited DNA input and sample-type-specific properties of embryo-derived material. In this observational study, we examined assay-output patterns in a low-input multiplex PCR workflow targeting a male-specific Y-chromosome marker (MSY) together with an autosomal internal control targeting cyclin-dependent kinase 1 (CDK1). A total of 141 bovine embryo-derived samples were analyzed across three sample categories: whole embryos, cleavage-stage blastomere biopsies, and blastocyst-stage trophectoderm biopsies. Assay evaluability differed across sample categories, being highest in blastocyst-stage biopsy samples (90.6%, 29/32), intermediate in whole embryos (82.9%, 58/70), and lowest in blastomere-biopsy samples (64.1%, 25/39). Among technically evaluable CDK1-positive samples, apparent MSY-positive classification also differed across categories, with rates of 55.2% (32/58) in whole embryos, 52.0% (13/25) in blastomere-biopsy samples, and 6.9% (2/29) in blastocyst-stage biopsy samples. In a same-lysate repeat-amplification subset, overall evaluability concordance was 92.5% and overall apparent MSY-based classification concordance was 88.2%, although subgroup sizes were limited. Because no independent reference method for true embryo sex was available and sample category was structurally confounded with developmental stage, semen type, sire, and partly PCR-date structure, the findings should be interpreted as assay-output observations rather than measures of biological sex ratio or diagnostic sexing accuracy. Overall, embryo-derived sample categories showed differing assay evaluability and apparent MSY-classification patterns within this low-input PCR setting, supporting the need for sample-type-specific validation when low-cell embryo-derived samples are used. Full article

Background/Objectives: Oocytes retrieved from the pouch of Douglas during transvaginal oocyte pick-up (OPU) have historically been described mainly in isolated case reports and small series. Their developmental competence and clinical relevance remain incompletely characterized. This study aimed to evaluate the embryological and clinical outcomes of oocytes recovered from pouch of Douglas fluid and to compare developmental outcomes between ovarian-derived and pouch of Douglas-derived oocytes. Methods: This retrospective observational study was conducted at a tertiary referral university hospital IVF center. Clinical and embryological data collected between February 2023 and June 2025 were retrospectively analyzed. A total of 2423 OPU cycles were screened, and 49 cycles with ≥2 cm of free fluid in the pouch of Douglas before OPU met the inclusion criteria. Following completion of ovarian follicular aspiration, free peritoneal fluid from the pouch of Douglas was aspirated separately and assessed for the presence of oocytes. Cycles were categorized according to whether oocytes were identified in the pouch of Douglas. Baseline cycle characteristics, ovarian response parameters, embryological outcomes, and clinical outcomes were evaluated. Developmental outcomes of ovarian-derived and pouch of Douglas-derived oocytes were compared within the same cycles. Results: Oocytes were recovered from pouch of Douglas fluid in 30 of 49 cycles (61.2%). Baseline characteristics, trigger-day hormonal parameters, follicle numbers, and ovarian embryological outcomes were similar between cycles with and without pouch of Douglas oocytes. A total of 44 oocytes were retrieved from pouch of Douglas fluid, of which 36 (81.8%) were metaphase II (MII) oocytes. Twenty-six oocytes demonstrated normal fertilization (2PN), with subsequent cleavage-stage and blastocyst-stage embryo development observed. No significant differences were observed between ovarian-derived and pouch of Douglas-derived oocytes in fertilization or embryo development rates. The proportion of MII oocytes was significantly higher among pouch of Douglas-derived oocytes (81.8% vs. 66.2%, p = 0.021). Five embryo transfers involving embryos derived from pouch of Douglas oocytes resulted in clinical pregnancy and live birth, including three transfers exclusively involving pouch of Douglas-derived embryos. No procedure-related complications were observed. Conclusions: Oocytes recovered from pouch of Douglas fluid demonstrated fertilization capacity, embryo developmental potential, and the ability to contribute to clinical pregnancy and live birth. These findings suggest that free peritoneal fluid identified before OPU may contain developmentally competent oocytes that would otherwise remain unrecovered. Given the retrospective design, limited number of embryo transfers, and uncertainty regarding the precise origin of these oocytes, the findings should be considered exploratory and require confirmation in larger prospective studies. Full article

In vitro maturation (IVM) is a critical step affecting the efficiency of bovine in vitro embryo production; however, oxidative stress during in vitro culture can impair oocyte quality and subsequent developmental competence. This study investigated the effects of cysteine supplementation on bovine oocyte IVM, redox homeostasis, mitochondrial status, and transcriptomic changes. Bovine cumulus-oocyte complexes were cultured in IVM medium supplemented with 0, 25, 50, 75, 100, or 125 μM cysteine, and 75 μM was identified as the optimal concentration. Compared with the control group, 75 μM cysteine increased the first polar body extrusion rate from approximately 78% to 81% and improved the fertilization/cleavage rate from approximately 74% to 82%. It also significantly increased the proportions of 2-cell, 4-cell, and 8-cell embryos, whereas morula and blastocyst rates were not significantly affected. At the cellular level, 75 μM cysteine significantly reduced ROS levels and increased GSH content, as indicated by changes in relative fluorescence intensity. JC-1 staining showed that the JC-1 monomer signal decreased from approximately 16.0 to 13.5, whereas the JC-1 aggregate signal increased from approximately 13.2 to 14.8, indicating improved mitochondrial membrane potential status. In addition, lipid droplet fluorescence intensity increased from approximately 11.8 to 13.4, mitochondrial fluorescence intensity increased from approximately 6.0 to 7.0, and cytoskeletal fluorescence intensity showed no significant difference between groups. Smart-seq2 transcriptomic analysis identified 1935 differentially expressed genes, including 1778 upregulated and 157 downregulated genes, which were mainly enriched in translation, ribosomal structural components, RNA binding, oxidative phosphorylation, and metabolism-related pathways. qRT-PCR further confirmed the upregulation of key genes, including NDUFS2, VDAC3, ANXA2, MTHFD1L, and SCD. Overall, 75 μM cysteine improves bovine oocyte IVM quality by enhancing antioxidant capacity, improving mitochondrial membrane potential, increasing lipid-derived energy substrate storage, and regulating genes related to energy metabolism and developmental competence. Full article

Advanced maternal age is a significant social and clinical issue associated with the natural decline in a woman’s ovarian reserve. This prospective, single-center study included women with primary infertility who presented to the Gyncentrum Clinic in Katowice and analyzed 77 ovarian follicles. The study group consisted of patients of advanced reproductive age with diminished ovarian reserve, who underwent hormonal stimulation in preparation for oocyte retrieval. Each metaphase II (MII) oocyte was fertilized in vitro and cultured individually in a time-lapse incubator. Follicular fluid obtained during oocyte retrieval was collected separately from each follicle and used for non-invasive biochemical analysis of prognostic factors using a Multiplex assay. The concentrations of interleukin 10 (IL-10), granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), and C-type natriuretic peptide (CNP) were evaluated. The analysis showed that lower concentrations of GM-CSF and CNP were associated with an increased probability of oocyte fertilization, whereas higher levels of IL-10 and G-CSF had the greatest impact on blastocyst formation. This model was supported by continuous embryo monitoring. An eightfold increase in the likelihood of blastocyst formation was observed when early embryo cleavage occurred between 25 and 27 h after insemination. Furthermore, prolonged duration of the first cytokinesis reduced the probability of blastocyst development, while an extended cell cycle at the two-blastomere stage significantly affected further embryo development. These findings may support non-invasive embryo selection strategies. Full article

Otosclerosis is a common cause of conductive hearing loss thought to result from dysregulated bone remodeling in the embryonic tissues of the globuli interossei. Both familial and sporadic cases have been reported. To date, 10 published OTSC loci and four genes (FOXL1 (OTSC11), SMARCA4 (OTSC12), MEPE, SERPINF1) have been identified in autosomal dominant families. Using a combined genetic and genomics approach in five affected siblings, we identified a nonsense mutation in Karyopherin subunit α7 (KPNA7, c.49C>T, p.R17X), the newest of the importin-α family of nuclear transporters. KPNA7 is a key maternal factor involved in the classical transport of NLS-containing cargo proteins, active during early embryonic cleavage events and zygotic genome activation. So far, 377 cargo proteins associated with KPNA7 have been identified. Recessive KPNA7 variants cause skeletal abnormalities, epilepsy, intellectual disabilities and preimplantation embryo arrest (PREMBA). A closer look at the OTSC genes reveals their involvement in endochondral ossification signaling pathways. We explore how KPNA7 haploinsufficiency in the embryonic tissues of the otic capsule may cause dysregulated bone remodeling. This study expands the phenotypic spectrum of KPNA7 and provides new insights into the pathobiology of otosclerosis. Full article

Background: The sperm centrosome is a key organelle composed of two centrioles surrounded by pericentriolar material and is essential for sperm aster formation and first mitotic spindle formation after sperm entry into the oocyte, thereby supporting early embryonic development. Methods: This systematic review evaluated 83 publications published between 1986 and 2026 to provide an updated overview of the human sperm centrosome, including its structural organization, protein composition, role in reproductive success, and association with embryonic abnormalities. Results: The available evidence indicates that centrosome-associated proteins such as centrin, γ-tubulin, and pericentrin are critical for spindle assembly, microtubule nucleation, and centriole duplication, and that alterations in their expression or localization are associated with poor sperm quality and reduced fertilization potential. Centrosomal dysfunction has also been linked to fertilization failure despite intracytoplasmic sperm injection, abnormal embryo cleavage, and possible embryonic aneuploidy. Although diagnostic approaches have improved, targeted treatments remain limited. Conclusions: These findings highlight the clinical relevance of sperm centrosomal integrity and support the need for further research into its diagnostic and therapeutic implications. Full article

While early embryonic loss affects the conception rate in lactating cows, the underlying mechanisms remain unknown. Here, we examined whether the developmental morphokinetics and transcriptomic profiles of cleaved embryos are associated with developmental competence. Developing bovine embryos were produced in vitro, and their morphokinetics were monitored through 190 h using a time-lapse system. The proportion of embryos that developed to the blastocyst stage was lower following abnormal cleavage, i.e., reverse, direct, or unequal, relative to normally cleaved embryos (p < 0.05). In a second set of experiments, exploratory RNA-seq analysis was performed on first-cleaved embryos, which were individually collected immediately after the first division; embryos were defined by the time-lapse system as normally, directly, or unequally cleaved (n = 6 per group), or reverse-cleaved (n = 5). Analysis revealed 672 genes that were differentially expressed between normally and abnormally cleaved embryos (adjusted p < 0.05). Abnormally cleaved embryos differed in pathways associated with energy production and metabolism. The most profound difference in gene expression (n = 632) was between unequally and normally cleaved embryos, mainly in genes affiliated with the oxidative phosphorylation pathway. These findings support the concept that the morphokinetic pattern of early embryonic cleavage is associated with developmental competence. We therefore suggest that the observed differential expression profiles in abnormally cleaved embryos might be involved in the mechanism underlying early embryonic loss. Full article

Natural pigment lycopene (LYC), a carotenoid, possesses antioxidant, anti-apoptotic, anticancer, and immunoenhancing properties. During in vitro culture, this substance protects oocytes and early embryos from damage caused by reactive oxygen species (ROS), thereby enhancing the in vitro maturation (IVM) rate of oocytes and the developmental competence of early embryos. This study aimed to investigate the effects of supplementing different concentrations of LYC (0, 5, 10, and 15 μM) during in vitro culture of sheep oocytes and early embryos on their developmental competence. In contrast to the control group, the 5 μM LYC treatment group displayed a marked increase in the first polar body extrusion rate and the extent of cumulus cell expansion, as well as a significantly higher proportion of normal spindle assembly in sheep oocytes, but 15 μM LYC appeared to negatively affect oocyte maturation. Relative to all other experimental groups, the 5 μM LYC treatment group displayed significantly elevated rates of cleavage and blastocyst rate during early in vitro embryonic development. The levels of ROS in mature oocytes and early embryos were significantly decreased, whereas the GSH level was significantly elevated. Furthermore, LYC treatment significantly enhanced mitochondrial activity and markedly elevated the mitochondrial membrane potential (MMP) in mature oocytes and early embryos. Moreover, the total cell number of blastocysts was significantly increased. Moreover, in early embryos, the transcript levels of genes associated with both oxidative stress and apoptosis were favorably regulated. In conclusion, LYC supplementation boosted the rates of oocyte maturation and blastocyst formation in sheep, while elevating the developmental capacity of early embryos. Full article

Protein synthesis is a crucial biosynthetic process in all organisms, including plants. The integrity of the translational machinery, especially ribosomes, can be compromised during rapid cell division in ontogenesis or in response to environmental stress. In this study, Northern blotting was employed to analyze total RNA from various angiosperms, focusing on small 5′- and 3′-terminal 18S rRNA fragments. Stem-loop array RT-PCR was employed to map the cleavage sites within the target regions. Severe stress, such as extreme drought, induced the accumulation of three distinct 18S rRNA fragments across diverse angiosperm taxa, indicating that this phenomenon is likely universal. In rapidly dividing cells, such as those found in in vitro callus cultures and germinating wheat embryos, high levels of discrete 5′-terminal fragments were observed, while 3′-terminal fragments were absent. The stem-loop array RT-PCR mapping identified specific sites of 18S rRNA strand breaks. Structural annotation of the 3D model of the plant 40S subunit revealed spatial clustering of these sites in proximity to the RPS6 binding region. Notably, wheat cultivars that are tolerant to osmotic stress exhibited significantly higher levels of 18S rRNA fragmentation than sensitive cultivars. This suggests a regulatory mechanism rather than a mere byproduct of apoptotic-like regulated cell death. Additionally, fragmented ribosomes were gradually eliminated during embryo maturation, indicating a process of programmed functional ribophagy. Our findings suggest that a potential inability of plant tissues to selectively retain functional ribosomes might contribute to a decline in generative potential. Monitoring the integrity of the translational machinery could improve breeding efficiency and aid in preserving long-term stored germplasm. Full article

With the continuous advancement in reproductive biology, oocyte vitrification has become a critical technology for preserving female germplasm and protecting it from environmental disruptions. This technique also eliminates temporal and spatial constraints in animal embryo engineering research. However, during the vitrification of animal oocytes, exposure to low temperatures and high concentrations of cryoprotectants can cause various forms of damage, including cytoskeletal disruption, spindle abnormalities, mitochondrial dysfunction, apoptosis, oxidative stress and epigenetic modifications. These issues are now understood to severely restrict the subsequent developmental competence of oocytes, resulting in lower cleavage and blastocyst formation rates than those of fresh oocytes. Currently, the mechanisms of cryodamage in vitrified oocytes remain poorly understood, and standardized strategies to enhance vitrification efficiency have yet to be firmly established. This review provides a formal overview of the physiological factors underlying oocyte sensitivity to vitrification, alongside the mechanisms of cryodamage and the variables influencing post-thaw survival and reproductive success. It evaluates strategies for mitigating vitrification-induced stress, compares interspecies differences, and addresses current research limitations. By identifying future directions, this review offers new insights for optimizing mammalian oocyte cryopreservation techniques. Full article