Search Results (310)

The cryopreservation of pre-implantation embryos has become routine in medically assisted reproduction (MAR), and the proportion of frozen embryo transfers has steadily increased in recent years. Because cryopreservation through either slow-cooling protocols or ultra-rapid vitrification requires potentially cytotoxic cryoprotective agents to prevent uncontrolled and detrimental ice crystal formation, the safety of these procedures must be carefully considered. Evidence from human epidemiological studies, including retrospective and prospective controlled studies, and data from national patient registries indicate that children born after frozen embryo transfer have a higher birth weight than those born after spontaneous conception and have an increased risk of rare genomic imprinting disorders, such as Beckwith–Wiedemann, Silver–Russell, or Prader–Willi syndrome. Encompassing not only reversible DNA methylation patterns established during gametogenesis, but also the timed abundance and availability of transcripts and proteins required to establish or maintain epigenetic marks throughout development and differentiation, as well as persistent or transient post-translational histone modifications and non-coding RNAs, the epigenome may be particularly sensitive to cryopreservation. Importantly, epigenetic regulation is highly complex. Alterations of the epigenome at any developmental stage are often not monocausal, do not necessarily result in immediate disturbances in the pre-implantation embryo, and are unlikely to operate through simple all-or-nothing mechanisms; however, they may have long-lasting effects at later developmental stages. To make matters even more complex, differences between species in terms of epigenetic regulation or lineage differentiation are well known and translation from animal model systems to humans must be considered with caution. More recently, epigenetic regulation by non-coding RNAs has also come into focus, as these molecules are crucial, either directly or indirectly, for gene expression, translation, and protein biosynthesis during development. Therefore, assessing potential adverse effects of cryopreservation on the entire epigenome remains a major challenge, particularly because little is known about indirect factors, such as post-translational histone modifications and non-coding RNAs. In this review, we focus on the potential influence of the cryopreservation of pre-implantation embryos on the epigenetic profile in humans and animals. Specifically, we consider DNA methylation of imprinted genes and global DNA methylation; post-translational histone modifications; the abundance and availability of transcripts and proteins required to establish, maintain, or protect epigenetic patterns; and the presence of non-coding RNAs involved in epigenetic control. Full article

Epigenetic regulation is pivotal in reproductive processes, such as oocyte maturation and pre-implantation embryonic development, and it impacts gene expression without altering DNA sequence through mechanisms including DNA methylation, histone modifications, and non-coding RNAs. Primarily, microRNA-21 is involved in meiotic progression, apoptosis, and cumulus cell function, which are necessary for oocyte competency. miR-21 dysregulation can lead to improper oocyte maturation and poor embryonic development, ultimately causing developmental defects. During pre-implantation embryonic development, DNA methylation and histone modifications contribute to cellular reprogramming, ensuring proper gene activation and repression. Environmentally, endocrine disruptors affect miR-21 expression, potentially disrupting pathways involved in reproductive health and developmental programming. Overall, this review explores the correlation between epigenetics, miRNA regulation, and environmental factors, emphasizing the intricacies of oocyte maturation and pre-implantation embryonic development. This highlights the need for additional mechanistic and translational research in reproductive epigenetics. Full article

Background: The transfer of a nucleus from one oocyte to another offers patients harbouring high levels of mitochondrial DNA mutation and sufferers of frequent fertilisation failure or early embryonic arrest the potential to have healthy children. However, a small amount of mtDNA is carried over with the nucleus as the transfer takes place. Consequently, we still need to distinguish between the effects of the carryover and the transfer of a nucleus itself from a mature oocyte. Methods: To overcome this, we analysed a series of hatching stage blastocysts generated using metaphase II spindle transfer and mitochondrial supplementation. The latter approach also introduces a small amount of mtDNA into the oocyte as fertilisation takes place. For both manipulations, an autologous approach was used to overcome the effects of third-party transfer. Results: We then compared the changes in global gene expression between the two groups. We found that the nuclear transfer process affected a number of gene networks and pathways. These included metabolic, cell cycle, inflammatory and immune, and epigenetic responses. A comparison with earlier stage blastocysts did not suggest that the cause was due to developmental delay. Conclusions: Critically, these changes could affect offspring health and well-being as is the case following somatic cell nuclear transfer. 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

Early mammalian embryos are highly sensitive to environmental, metabolic, hormonal, and genomic stress, yet embryo assessment during In Vitro Fertilization (IVF) relies largely on morphology and ploidy for embryo assessment, but these tests incompletely predict miscarriage. We present a transcriptomics based framework to classify and quantify lineage-specific stress in early embryos by benchmarking human preimplantation embryos against dose-, time-, and quality-dependent stress programs defined in Embryonic and placental Trophoblast Stem Cells (ESCs, TSCs) from the implanting blastocyst. Human embryos and stressed ESCs and TSCs are screened using transcriptomic markers from eleven biologically distinct stress Gene Ontology (GO) groups that define functional stress states and enable quantification of pathway presence and upregulation, pathway activity, and downstream outcomes. This framework determines whether the Integrated Stress Response (ISR), once initiated, resolves to enable the Developmentally Associated Stress Response (DASR). High-throughput screening (HTS) titrates stress to define increasingly risky yet biologically equivalent doses for levels of diminished stem cell growth across mechanistically diverse stressors. Then bulk RNA seq derives lineage specific transcriptomic markers putatively respond to common levels of diminished growth and that distinguish weak vs. strong stress and resolved vs. unresolved ISR. These stem cell transcriptomic signatures are applied to bulk RNA seq data from IVF embryos graded for morphology or adhesion, enabling quantitative inference of stress burden, lineage vulnerability, and developmental trajectory. Full article

Background and Objectives: Endometriosis is a multifactorial gynecological condition associated with impaired fertility; however, its impact on embryo competence remains incompletely understood. This study aimed to evaluate embryo competence through a stepwise analysis of IVF outcomes across the developmental continuum, while also comparing patients with endometriosis and controls. Materials and Methods: A retrospective observational study was conducted, including 160 patients undergoing IVF, comprising 55 patients with endometriosis and 105 controls. Clinical and embryological data were analyzed sequentially across key developmental stages, including oocyte retrieval, metaphase II (MII) oocyte formation, fertilization (2PN), embryo development, and euploidy in a subgroup undergoing preimplantation genetic testing for aneuploidy (PGT-A). Stage-specific efficiency rates were calculated, and correlations between early- and late-developmental parameters were assessed. In addition, comparative analysis between groups was performed. Results: A progressive decline in developmental efficiency was observed across the IVF continuum, with approximately one-quarter of retrieved oocytes reaching the embryo stage and only a small proportion ultimately resulting in euploid Blastocysts. Strong positive correlations were identified among early-stage parameters, particularly retrieved oocytes, MII oocytes, and embryo yield (r = 0.77–0.96, p < 0.001), indicating that ovarian response and oocyte maturity significantly influence downstream outcomes. However, efficiency-based parameters showed limited predictive value for chromosomal competence. A moderate association was observed between MII oocytes and euploid Blastocysts (r = 0.58), whereas the relationship between embryo number and euploidy remained weak. Comparative analysis revealed no statistically significant differences between the endometriosis and control groups across the evaluated embryological parameters (p > 0.05 for all comparisons), suggesting that sequential analyses may provide complementary insight beyond direct comparisons. Conclusions: IVF outcomes follow a sequential developmental trajectory with a progressive decline in efficiency across stages. In endometriosis, early developmental competence appears to be affected, while chromosomal competence remains relatively preserved. Full article

Heat shock proteins (HSPs) fulfil protective tasks in the whole organism; in pregnant dogs, they are expressed in the ovary, placenta and preimplantation embryo. Our objective was to compare the expression of HSP60 and -70, along with indicators of proliferation and apoptosis, in the non-pregnant and pregnant uterus/placenta and ovaries. Tissues were obtained after ovariohysterectomy and examined by means of immunohistochemistry. There were differences between pregnant and non-pregnant tissues: the expression level of HSP70 during preimplantation in superficial cells was significantly lower than that in early diestrus, with similar results observed for Ki67. The immunosignal for HSP70 was significantly decreased during the postimplantation stage in almost all cell types, whilst the number of HSP60-positive cells did not change. In pregnant animals, the number of Ki67-positive cells significantly increased until the postimplantation stage. In the placenta and trophoblast, the expression of HSP60 and -70 was strong, while no HSP70 signal was detected in endometrial epithelial cells. The caspase 3 immunosignal in the uterus and placenta was generally weak. In the corpora lutea, HSP60, HSP70 and caspase 3 were mainly detected in theca lutein cells, while no signal for KI67 was seen. In follicles, caspase 3 and KI67 expression was low, except in granulosa cells of tertiary follicles and oocytes. We conclude that the different expression of HSPs in pregnant and non-pregnant animals may point towards different regulatory and/or protective tasks. Full article

Embryos being treated using assisted reproductive technology (ART) are unavoidably exposed to physical stressors, thus producing reactive oxygen species (ROS) which trigger mitophagy to support embryonic development. However, the mechanisms underlying the regulation of mitophagy in early embryonic development remain largely unexplored. Here, we found that Mucin 1 (MUC1) exhibited a uniform distribution in both mouse and human oocytes, and its expression peaked at the blastocyst stage. Further analysis revealed that Muc1 knockout impairs blastocyst formation in vitro. Correspondingly, Muc1 knockout led to the accumulation of mitochondrial reactive oxygen species (mtROS) and a reduction in phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)/Parkinson protein 2 (PARK2/Parkin)-dependent mitophagy. Stimulation of mitophagy via low-dose carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment rescued the blastocyst formation defect in Muc1-null embryos. Vitamin C supplementation effectively scavenged mtROS and restored developmental competence. Together, our findings establish that MUC1 safeguards early embryonic development by promoting mitophagy to decrease mtROS levels in vitro. Moreover, vitamin C could compensate for Muc1 deficiency by eliminating mtROS. This study not only identified a new function of MUC1 in protecting early embryonic development in vitro, but also revealed a novel mechanism of mitophagy regulation in early embryos, which has potential applications for ART. Full article

Preimplantation embryo genomic selection (eGS) enables selection prior to implantation and could accelerate genetic gain in cattle. A major hurdle is the limited DNA from embryo biopsies, requiring efficient whole-genome amplification (WGA) for accurate genomic analyses. However, alternative WGA methods and genotyping strategies have not been systematically compared in cattle. This study evaluated different methods for WGA (multiple displacement amplification (MDA) or multiple annealing and looping-based amplification cycles (MALBAC)) and for genotyping (single nucleotide polymorphism array (SNP-array), genotyping by targeted sequencing (GBTS), or whole-genome sequencing (WGS)) using 3-, 6-, and 9-cell bovine samples. MDA consistently outperformed MALBAC across various performance metrics, including amplification length, call rates, genome coverage (93.43–94.40% vs. 53.01–67.08%), and genotyping concordance (0.89–0.98 vs. 0.75–0.92). GBTS achieved the highest call rates, while SNP-array and GBTS showed excellent concordance and low error rates. WGS provided genome-wide data for precise aneuploidy detection. We further validated the workflow in trophectoderm biopsies and arrested embryos, generating reliable data for genomic evaluation, sex determination, and aneuploidy screening. MDA from ≥6 cells combined with GBTS or SNP-array showed a favorable balance of efficiency and accuracy for bovine eGS. This framework may facilitate the application of eGS in cattle breeding by enhancing selection intensity and accelerating genetic improvement. Full article

Serotonin is a critical morphogen in early development, yet the mechanisms regulating its homeostasis in the preimplantation embryo remain unclear, particularly under conditions of maternal antidepressant exposure. Here, we investigated embryonic serotonergic autonomy using mouse models of pharmacological transport blockade (maternal fluoxetine treatment) and in vitro treatment with the monoamine oxidase inhibitor pargyline. We employed immunofluorescence, RT-qPCR, and live-cell imaging to assess metabolic flux, gene expression, and physiological health. We demonstrate that monoamine oxidase functions as a metabolic firewall, progressively maturing from zygote to blastocyst to degrade excess amines. Paradoxically, maternal serotonin transporter blockade triggered significant intracellular serotonin hyper-accumulation in blastocysts, associated with a trend toward a compensatory upregulation of the biosynthetic gene Ddc. While this serotonin overload did not compromise morphology, mitochondrial function, or pluripotency marker expression, it induced a robust epigenetic response. Excess serotonin promoted elevated H3Q5ser immunoreactivity in both nuclear and cytoplasmic compartments via a transglutaminase-dependent mechanism. These findings reveal that the preimplantation embryo possesses a resilient, autonomous serotonergic system capable of compensatory synthesis. However, environmental fluctuations are chemically recorded via transglutaminase-mediated serotonylation, representing an epigenetic mark that warrants further long-term study within the Developmental Origins of Health and Disease (DOHaD) framework. Full article

Background/Objectives: In the last decade, non-invasive methods for aneuploidy detection have been explored. The most successful approach involves analyzing the cell-free DNA (cfDNA) released by the embryo into the culture medium. The main objective of this study is to examine the technical feasibility of this new approach called non-invasive PGT-A or niPGT-A. In addition, as an exploratory objective, the impact of the niPGT-A results on clinic outcomes will be assessed. Methods: This was a multicenter, international study that included 716 patients and 2539 blastocysts (ClinicalTrials.gov: NCT03520933). Each embryo was cultured following a specific protocol for niPGT-A. Individual spent blastocyst medium (SBM) and trophectoderm (TE) biopsy were obtained, analyzed, and compared to assess concordance. In a subset of embryos, the comparison also included an inner cell mass (ICM) biopsy. Clinical outcomes from the embryo transfers performed (all based on the TE result) were registered, and results were analyzed blindly regarding the impact of aneuploidies in the culture medium. Results: The concordance rate between SBM and TE was 79.1% (range: 74.1–82.1; cycles with autologous oocytes). This value increased to 87.0% when comparing SBM and ICM. Applying an adapted embryo culture protocol to collect the SBM for niPGT-A did not affect blastocyst quality. Analysis of the embryo transfers performed (n = 265) revealed a trend towards lower miscarriage rate in blastocysts where both TE and SBM were concordant and euploid (13.0%), compared to blastocysts with a euploid TE and an aneuploid SBM (22.2%). Conclusions: The results obtained show a high concordance between the SBM and TE biopsies. Although additional refinement of the technique would further increase niPGT-A’s performance, the results obtained support the potential use of this non-invasive approach for aneuploidy detection. The high concordance of the cfDNA present in the SBM with the corresponding ICM biopsy and the miscarriage rate observed in cases with an aneuploid SBM, despite the euploid TE results, also support niPGT-A’s capacity to assess embryo aneuploidies and its potential as a prioritization system for selecting blastocysts to transfer. This approach could hold special interest in patients with no PGT-A indications, couples that prefer not to biopsy their embryos or those who do not have access to invasive PGT-A. Full article

Objective: To investigate the relationship between the number of previous implantation failures (IFs) and embryo ploidy status, as well as subsequent clinical outcomes, in women with recurrent implantation failure (RIF) undergoing preimplantation genetic testing for aneuploidy (PGT-A). Methods: This retrospective cohort study included 422 women with RIF who underwent their first PGT-A cycle between 2017 and 2022. Participants were stratified by maternal age (<38 years, n = 292; ≥38 years, n = 130) and by the number of previous IFs, categorized as 3, 4, or ≥5. The primary outcomes were embryo ploidy rates (euploidy, aneuploidy, and mosaicism). Secondary outcomes included reproductive outcomes after single euploid blastocyst transfer (biochemical pregnancy, clinical pregnancy, ongoing pregnancy, live birth, and pregnancy loss) and neonatal birth weight. Results: Women aged ≥38 years had a significantly lower euploidy rate than those <38 years (24.8% vs. 47.3%, p < 0.001). Ploidy distribution did not differ significantly across IF categories. Among women aged <38 years with ≥5 IFs, a greater number of previous embryo transfer attempts was independently associated with higher odds of live birth after euploid embryo transfer (adjusted OR = 1.258, 95% CI: 1.051–1.505; p = 0.012). Neonatal weight did not differ significantly across IF categories. Conclusions: The number of previous IFs was not independently associated with embryo ploidy or clinical outcomes after euploid transfer, whereas advanced maternal age was strongly associated with a lower likelihood of obtaining euploid embryos. In younger women with ≥5 IFs, a greater number of previous embryo transfer attempts was associated with live birth after euploid transfer; however, this exploratory subgroup finding should be interpreted cautiously and requires prospective validation. Because this study did not directly evaluate therapeutic strategies, any potential role for individualized endometrial evaluation or optimization should be considered as hypothesis-generating rather than supported by the present data. Full article

Background/Objectives: Early embryonic arrest during the cleavage stage (days 2–4) accounts for a substantial proportion of developmental failure in in vitro fertilization. This phenomenon remains poorly understood at the molecular level, even in chromosomally normal embryos identified by preimplantation genetic testing. This review aims to redefine cleavage-stage arrest from a passive energy deficit to a checkpoint-regulated endpoint caused by inadequate coordination among metabolism, transcriptome integrity, and stress-response pathways. Methods: We integrate evidence from long-read transcriptomics, metabolomics, epigenetics, and immunobiology relevant to pre-blastocyst development. These data are assembled into a unifying mechanistic framework and a clinically oriented stratification model, together with candidate multimodal readouts for early classification. Results: We propose a three-axis model linking: (i) metabolic–epigenetic insufficiency, including defective histone lactylation and impaired alpha-ketoglutarate-dependent DNA demethylation; (ii) isoform-level abnormalities, including intron retention and retrotransposon activation within a hidden transcriptomic landscape better resolved by long-read sequencing; and (iii) stress-related immune signaling, in which NLRP7 links alternative splicing and DNA-damage-response dysfunction with mitochondrial stress and p53-associated arrest. Within this framework, we distinguish three molecular arrest states: an early transition failure marked by defective maternal-to-embryonic reprogramming and severe splicing disruption; a metabolically quiescent state that may retain a limited rescue window; and a later stress-associated state characterized by senescence-like features, oxidative stress, and broad transcriptomic and genomic instability. Conclusions: Early embryo arrest should no longer be viewed as a nonspecific developmental failure, but as a mechanistically stratifiable condition with distinct metabolic, transcriptomic, and stress-associated trajectories. A diagnostic platform combining fluorescence lifetime imaging microscopy, long-read sequencing, and digital polymerase chain reaction may improve early mechanistic classification, help identify embryos with possible reversibility, and reduce uncertainty in embryo selection during in vitro fertilization. Full article

Background/Objectives: Laser-assisted zona pellucida (ZP) drilling on day 4 embryos is routinely performed in IVF laboratories to facilitate trophectoderm (TE) herniation for blastocyst biopsy. Nevertheless, inner cell mass (ICM) herniation through the initial ZP opening occasionally occurs and may interfere with standard TE biopsy. Methods: This retrospective study assessed the clinical and obstetric safety of a double ZP drilling strategy for TE biopsy in preimplantation genetic testing for aneuploidy (PGT-A) cycles. A total of 560 single euploid embryo transfer cycles were analyzed. Blastocysts were categorized (Groups 1–6) based on ICM/TE herniation patterns and the corresponding biopsy approach. Clinical outcomes were compared between cycles undergoing TE biopsy through a single ZP opening (TE hatching with ICM remaining within the ZP) and cycles requiring a second opening to relocate the biopsy site when the ICM herniated through the original opening or was positioned externally. Results: The single-opening approach of Group 1 accounted for 295 cycles (52.7%), with implantation, miscarriage, and live birth rates of 65.4%, 14.0%, and 56.3%, respectively. The double-opening approach of Group 3 was applied in 21 cycles (3.8%), yielding implantation, miscarriage, and live birth rates of 66.7%, 0%, and 66.7%, respectively. No significant differences were observed between the two strategies in implantation, miscarriage, or live birth rates. Obstetric and neonatal outcomes, including gestational age, birth weight, and monozygotic twinning incidence, were comparable. Fifteen healthy infants were delivered following TE biopsy using the double-opening strategy. Conclusions: These data support incorporating ICM position into TE biopsy decision-making and suggest that creating a second ZP opening to reposition the biopsy site is clinically feasible and does not compromise reproductive or obstetric outcomes in PGT-A cycles. Full article

Background/Objectives: Dynamic remodeling of the zona pellucida (ZP) is a fundamental biochemical and structural process during human preimplantation development; however, its quantitative characterization and clinical relevance remain incompletely defined. The objective of this study was to evaluate dynamic ZP thinning as a functional marker of embryo developmental competence and to examine its relationship with follicular fluid (FF) biomarkers and clinical pregnancy. Methods: This prospective observational study included 47 IVF cycles performed at a single center, yielding 64 transferred blastocysts with complete time-lapse data. ZP thickness was measured from fertilization to 120 h post-fertilization using time-lapse imaging. Two quantitative parameters were derived: the relative thinning ratio (Δrel) and the linear thinning rate (slope). FF concentrations of growth differentiation factor 9 (GDF-9), hyaluronic acid (HA), and syndecan-4 (Syn4) were quantified by ELISA. Embryo-level associations with spontaneous blastocyst hatching were assessed using logistic regression and multivariate analyses, while patient-level models evaluated predictors of clinical pregnancy. Results: Embryos that underwent spontaneous hatching exhibited significantly greater Δrel than non-hatching embryos (p < 0.001). Δrel remained the strongest predictor of hatching in multivariable models (AUC = 0.91). Among FF biomarkers, only GDF-9 showed a positive association with spontaneous hatching. At the patient level, higher Δrel values of transferred embryos were associated with clinical pregnancy (OR 3.65, p = 0.009), whereas FF biomarkers and assisted hatching showed no significant association. Conclusions: Dynamic ZP thinning quantified by Δrel represents a promising indicator of embryo developmental competence. The concordance between embryo-level hatching behavior and patient-level clinical pregnancy suggests potential clinical relevance of ZP dynamics as an integrative embryological marker, warranting validation in larger cohorts. Full article