Embryonic Developmental Arrest Research Articles

P-191 Pronuclear transfer overcomes embryonic developmental arrest in Padi6-deficient mice

Abstract Study question Can pronuclear transfer (PNT) overcome embryonic developmental arrest (EDA) observed in the created Padi6-/- mouse model? Summary answer Applying PNT significantly improved the blastocyst rate of Padi6-/- zygotes, indicating its potential in overcoming female-related infertility. What is known already Various components of the subcortical maternal complex (SCMC), including peptidyl arginine deiminase 6 (PADI6), play a crucial role in early embryonic development and female fertility. Recent studies have revealed that mutations in PADI6 can result in EDA and thus female infertility, which is hard to overcome by current available treatment methods. The PNT technique, designed to replace compromised cytoplasm with a healthy counterpart, has been proposed to overcome specific female-related infertility indications. The Padi6-/- mouse displaying EDA, offers a valuable model to investigate the efficacy of PNT to overcome female-related infertility. Study design, size, duration From April 2022 to December 2023, an experimental study was conducted with CRISPR/Cas9 created Padi6 knockout mice. For fertility evaluation, Padi6-/- and Padi6+/- females were paired with wildtype males. Next, 30 wildtype and 25 Padi6-/- oocytes were collected for parthenogenetic activation. A total of 81, 54 and 145 zygotes were collected from wildtype, Padi6+/- and Padi6-/- females, respectively, for evaluating embryonic development and the effectiveness of PNT in overcoming EDA. Participants/materials, setting, methods The C57BL/6J Padi6-/- mouse model was established by introducing Padi6 c.1577_1578insT with CRISPR/Cas9. Female mice aged 6-12 weeks underwent ovarian hyperstimulation and mated with wildtype males. Oocytes were parthenogenetically activated with SrCl2 in presence of Cytochalasin D, and checked for 2-cell and blastocyst rates. PNT treatment was applied by transferring the 2PN from Padi6-/- to wildtype enucleated zygotes using electrofusion, and the efficiency was evaluated in terms of embryonic developmental potential. Main results and the role of chance After mating Padi6-/- female mice with wild-type males, no offspring could be obtained. In contrast, Padi6+/- females exhibited normal fertility, yielding an average of 7.3 offspring per litter. In addition, Padi6-/- males are also fertile. Twenty-eight wildtype oocytes (n = 30) survived parthenogenetic activation, with 85.71% (24/28) reaching the 2-cell stage, and 75% (18/24) subsequently forming blastocysts. In contrast, all Padi6-/- oocytes (n = 25) survived after activation, but only 20% (5/25) reached the 2-cell stage (P < 0.0001), with none progressing to blastocyst (P = 0.0039). Regarding zygote development, 82.14% (46/56) of wildtype zygotes and 98.14% (53/54) of Padi6+/-embryos reached the 2-cell stage, showing blastocyst rates of 95.65% (44/46) and 100% (53/53) respectively. For zygotes obtained from Padi6-/- females, 72.50% (87/120) of them progressed to the 2-cell stage (P = 0.1908, vs wildtype), but only 4.60% (4/87) reached blastocysts (P< 0.0001, vs wildtype), exhibiting the EDA phenotype. Following PNT to wildtype cytoplasm (n = 25), 23 reconstructed PNT zygotes were obtained. Of these, 91.30% (21/23) reached the 2-cell stage (P = 0.4924, vs wildtype), and 85,71% (18/21) developed to the blastocyst stage (P = 0.3151, vs wildtype), which is equivalent to the wildtype zygotes. Limitations, reasons for caution This study is limited by the small sample size of the PNT, which will still be extended. Next to the blastocyst rate, additional assessment methods such as multi-omics analyses will be employed to assess the efficiency and safety of PNT treatment. Wider implications of the findings This study provides further insight into the potential of the nuclear transfer technology for female-related infertility issues due to cytoplasmic incompetence. For couples experiencing recurrent EDA after ICSI due to inferior oocyte quality, PNT still offers them the possibility of having genetically related children. Trial registration number Not applicable

Novel PLCZ1 mutation caused polyspermy during in vitro fertilization.

Failure of oocyte activation, including polyspermy and defects in pronuclear (PN) formation, triggers early embryonic developmental arrest. Many studies have shown that phospholipase C zeta 1 ( PLCZ1 ) mutations cause failure of PN formation following intracytoplasmic sperm injection (ICSI); however, whether PLCZ1 mutation is associated with polyspermy during in vitro fertilization (IVF) remains unknown. Whole-exome sequencing (WES) was performed to identify candidate mutations in couples with primary infertility. Sanger sequencing was used to validate the mutations. Multiple PLCZ1 -mutated sperm were injected into human and mouse oocytes to explore whether PN formation was induced. Assisted oocyte activation (AOA) after ICSI was performed to overcome the failure of oocyte activation. We identified three PLCZ1 mutations in three patients who experienced polyspermy during IVF cycles, including a novel missense mutation c.1154C>T, p.R385Q. PN formation failure was observed during the ICSI cycle. However, injection of multiple PLCZ1- mutated sperm induced PN formation, suggesting that the Ca 2+ oscillations induced by the sperm exceeded the necessary threshold for PN formation. AOA after ICSI enabled normal fertilization, and all patients achieved successful pregnancies. These findings expand the mutational spectrum of PLCZ1 and suggest an important role for PLCZ1 in terms of blocking polyspermy. Furthermore, this study may benefit genetic diagnoses in cases of abnormal fertilization and provide potential appropriate therapeutic measures for these patients with sperm-derived polyspermy.

PADI6: What we know about the elusive fifth member of the peptidyl arginine deiminase family.

Peptidyl arginine deiminase 6 (PADI6) is a maternal factor that is vital for early embryonic development. Deletion and mutations of its encoding gene in female mice or women lead to early embryonic developmental arrest, female infertility, maternal imprinting defects and hyperproliferation of the trophoblast. PADI6 is the fifth and least well-characterized member of the peptidyl arginine deiminases (PADIs), which catalyse the post-translational conversion of arginine to citrulline. It is less conserved than the other PADIs, and currently has no reported catalytic activity. While there are many suggested functions of PADI6 in the early mouse embryo, including in embryonic genome activation, cytoplasmic lattice formation, maternal mRNA and ribosome regulation, and organelle distribution, the molecular mechanisms of its function remain unknown. In this review, we discuss what is known about the function of PADI6 and highlight key outstanding questions that must be answered if we are to understand the crucial role it plays in early embryo development and female fertility. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Short C-terminal Musashi-1 proteins regulate pluripotency states in embryonic stem cells

The RNA-binding protein Musashi-1 (MSI1) regulates the proliferation and differentiation of adult stem cells. However, its role in embryonic stem cells (ESCs) and early embryonic development remains poorly understood. Here, we report the presence of short C-terminal MSI1 (MSI1-C) proteins in early mouse embryos and mouse ESCs, but not in human ESCs, under conventional culture conditions. In mouse embryos and mESCs, deletion of MSI1-C together with full-length MSI1 causes early embryonic developmental arrest and pluripotency dissolution. MSI1-C is induced upon naive induction and facilitates hESC naive pluripotency acquisition, elevating the pluripotency of primed hESCs toward a formative-like state. MSI1-C proteins are nuclear localized and bind to RNAs involved in DNA-damage repair (including MLH1, BRCA1, and MSH2), conferring on hESCs better survival in human-mouse interspecies cell competition and prolonged ability to form blastoids. This study identifies MSI1-C as an essential regulator in ESC pluripotency states and early embryonic development.

A novel homozygous variant in PADI6 is associate with human cleavage-stage embryonic arrest.

Repeated absence of useable embryos is a difficult problem for infertility patients. Among them, embryonic developmental arrest is more common, but the genetic cause is not known. The embryos of a patient who came to our hospital three times could not develop beyond the four-cell stage. In addition to recording the developmental details of the embryos by daily photo-taking, the PADI6 R132C homozygous variants was further confirmed by whole-exome sequencing. Subsequently, PADI6 R132C was analyzed by bioinformatics methods for conservativeness across species. In addition, the possible impact of the pathogenic mutation on the structure of the protein PADI6 were also assessed. Generally, we identified a homozygous variants [NM_207421.4, c.394C>T(p.R132C] in the middle protein-arginine deiminase domain in PADI6 gene. The homozygous variant is highly conserved across species. Homozygous variant in PADI6 R132C could cause a human cleavage-stage embryonic arrest in female patients. These findings provide further evidence for the important roles of the homozygous PADI6R132C variant in embryonic development. Our findings contribute to a deeper understanding of the molecular genetic basis of female infertility.

P-203 The Effect of c-Abl Tyrosine Kinase Inhibition in Cell Fate Specification and Morphogenesis During Preimplantation Mouse Embryo

Abstract Study question What are the mechanisms governing the regulation of c-Abl tyrosine kinase in preimplantation embryo development? Summary answer Inhibition of c-Abl impairs the fate determination of TE and ICM cells during mouse preimplantation embryo development, preventing cell specilization after compaction. What is known already The successful completion of polarization, compaction and lineage specification stages is required for the embryo to reach the blastocyst stage from the zygote stage during preimplantation embryonic development. c-Abl is a tyrosine kinase localized in cell nucleus and cytoplasm and also capable of nuclear-cytoplasmic shuttling. c-Abl is activated when DNA damage occurs and plays a role in the DNA repairment mechanism. The small-molecule inhibitor imatinib is classified as type II inhibitors which targets the inactive conformation of the kinase domain and specifically inhibit c-Abl. Study design, size, duration C57BL6 female mice at 6-8-wk old were superovulated with 7,5 IU PMSG and after 48 hr 7,5 IU hCG, then mated. 20 hours after hCG enjection, zygotes were collected and cultured for 96h at 5% CO2, 5% O2 at 37oC. We designed 5 experimental groups: control, DMSO, 1 µM, 5 µM and 10 µM (in KSOM+AA) imatinib group. To determine how altering the imatinib might affect embryonic growth and development, morphokinetic parameters were evaluated. Participants/materials, setting, methods Immunofluorescence staining for embryos was performed after 4% paraformaldehyde fixation. Then embryos incubated c-Abl, YAP, TEAD4, PARD6, E-cadherin and CDX2. DAPI mounting medium used for nucleus staining. Imaging was performed under a confocal microscope. Fluorescent intensity analysis was performed with ImageJ. We detected c-Abl, Yap, Tead, Cdx2, Oct4 and Nanog mRNA expression levels using the qRT-PCR. For the statistical analysis we used GraphPad Prism (2-Way ANOVA and and One-way ANOVA (used Geisser-Greenhouse correction)). Main results and the role of chance Treatment of zygotes with imatinib resulted in embryonic developmental arrest (control, 81%; 1 µM imatinib, 58.5%; 5 µM imatinib, 54%, 10 µM imatinib 0%) and led to a significant decrease in the rate of blastocyst formation. We detected that the expression level of c-Abl was decreased in the imatinib groups compared to the control group. We determined that YAP and TEAD, which should be localized in the nuclei of TE cells, lost their nuclear expression patterns and were expressed in the cytoplasm and their expression levels decreased. We detected the PARD6 pattern, which was evident at the cell borders in the control group, distributed throughout the cytoplasm in the imatinib groups. While cytoplasmic and decreased localization of CDX2, which should be nuclear in TE cells, was determined in imatinib groups, we also defined that E-cadherin distribution was impaired at cell borders compared to the control group. c-Abl mRNA level was decreased in the 1 and 5 µm imatinib groups, Yap mRNA level was decreased in the 5 µm imatinib group. Cdx2 and Nanog mRNA levels were decreased in the 1 and 5 µm imatinib group (*p<0.1, **p <0.01, ***p <0.001, ****p <0.0001). Limitations, reasons for caution All animals used in this study were obtained from Yeditepe University Faculty of Medicine Experimental Research Center (YUDETAM) and all the experimental procedures have been approved by Yeditepe University Ethical Committee. Wider implications of the findings Imatinib significantly inhibited the development of eight-cell embryos to the blastocyst stage compared to controls a concentration-dependent manner, furthermore, c-Abl inhibition affected linegae specification by changing the localization of YAP/TEAD, markers of lineage differentiation. Therefore, c-Abl can regulate cell specification and cell fate determination during mouse preimplantation embryonic development. Trial registration number YAP-AP-SAB-21019

A novel compound heterozygous mutation in TUBB8 causing early embryonic developmental arrest.

Mutations in the β-tubulin isotype, TUBB8, can cause female infertility. Although several mutations of TUBB8 have been reported, the full spectrum for guiding genetics counseling still needs to be further explored. Here, we sought to identify novel variants in TUBB8 and their phenotypic effects on microtubule network structure in vitro. Whole-exome sequence analysis was performed in two families with infertility to detect pathogenic variants, with validation by Sanger sequencing. All gene variants and protein structures were predicted in silico. Cells were transfected with wild-type and mutants, and immunofluorescence analysis was performed to visualize microtubule network changes. We detected a novel compound heterozygous mutation, c.915_916delCC (p.Arg306Serfs*21) and c.82C > T (p.His28Tyr), and a benign heterozygous variant c.1286C > T (p.Thr429Met) in TUBB8 in the two families. Female patients with p.Arg306Serfs*21 and p.His28Tyr were infertile with early embryonic developmental arrest. The female patient with p.Thr429Met gave birth to a healthy baby in the second in vitro fertilization frozen embryo transfer cycle. The p.Arg306Serfs*21 mutation was predicted to cause large structural alteration in the TUBB8 protein and was confirmed to produce a truncated and trace protein by western blot analysis. Immunofluorescence analysis of transfected HeLa cells showed that p.Arg306Serfs*21 significantly disrupted microtubule structure. Our findings expand the known mutational spectrum of TUBB8 associated with early embryonic developmental arrest and female infertility.

Knockdown of Csnk1a1 results in preimplantation developmental arrest in mice

Casein kinase 1, alpha 1 (CSNK1A1), is a member of the highly conserved serine/threonine protein kinase family. This study was established to analyze the expression and localization of CSNK1A1 and its function in early embryonic development in mice. Csnk1a1 mRNA and protein are expressed in multiple mouse tissues including the ovary. After ovulation and fertilization, Csnk1a1 mRNA and protein were detected in preimplantation embryos and their expression was highest in two-cell-stage embryos. CSNK1A1 protein was also mainly localized in the cytoplasm of preimplantation embryos. Moreover, knockdown of Csnk1a1 in zygotes led to a significant decrease in the rate of blastocyst formation. Furthermore, treatment of zygotes with the CSNK1A1-specific inhibitor D4476 also resulted in embryonic developmental arrest. These results provide the first evidence for a novel function of CSNK1A1 in early embryonic development in mice.

Profiling the transcriptomic signatures and identifying the patterns of zygotic genome activation – a comparative analysis between early porcine embryos and their counterparts in other three mammalian species

BackgroundThe transcriptional changes around zygotic genome activation (ZGA) in preimplantation embryos are critical for studying mechanisms of embryonic developmental arrest and searching for key transcription factors. However, studies on the transcription profile of porcine ZGA are limited.ResultsIn this study, we performed RNA sequencing in porcine in vivo developed (IVV) and somatic cell nuclear transfer (SCNT) embryo at different stages and compared the transcriptional activity of porcine embryos with mouse, bovine and human embryos. The results showed that the transcriptome map of the early porcine embryos was significantly changed at the 4-cell stage, and 5821 differentially expressed genes (DEGs) in SCNT embryos failed to be reprogrammed or activated during ZGA, which mainly enrichment to metabolic pathways. c-MYC was identified as the highest expressed transcription factor during ZGA. By treating with 10,058-F4, an inhibitor of c-MYC, the cleavage rate (38.33 ± 3.4%) and blastocyst rate (23.33 ± 4.3%) of porcine embryos were significantly lower than those of the control group (50.82 ± 2.7% and 34.43 ± 1.9%). Cross-species analysis of transcriptome during ZGA showed that pigs and bovines had the highest similarity coefficient in biological processes. KEGG pathway analysis indicated that there were 10 co-shared pathways in the four species.ConclusionsOur results reveal that embryos with impaired developmental competence may be arrested at an early stage of development. c-MYC helps promote ZGA and preimplantation embryonic development in pigs. Pigs and bovines have the highest coefficient of similarity in biological processes during ZGA. This study provides an important reference for further studying the reprogramming regulatory mechanism of porcine embryos during ZGA.

Genetics of Oocyte Maturation Defects and Early Embryo Development Arrest.

Various pathogenic factors can lead to oogenesis failure and seriously affect both female reproductive health and fertility. Genetic factors play an important role in folliculogenesis and oocyte maturation but still need to be clarified. Oocyte maturation is a well-organized complex process, regulated by a large number of genes. Pathogenic variants in these genes as well as aneuploidy, defects in mitochondrial genome, and other genetic and epigenetic factors can result in unexplained infertility, early pregnancy loss, and recurrent failures of IVF/ICSI programs due to poor ovarian response to stimulation, oocyte maturation arrest, poor gamete quality, fertilization failure, or early embryonic developmental arrest. In this paper, we review the main genes, as well as provide a description of the defects in the mitochondrial genome, associated with female infertility.

A novel variant in TLE6 is associated with embryonic developmental arrest (EDA) in familial female infertility

This study aims to identify genetic causes of familial female infertility characterized by embryonic developmental arrest (EDA) and repeated implantation failure (RIF) with oocyte donation IVF cycle. We used Whole-exome sequencing and Sanger validation to find causative genes in an Iranian consanguineous family that had 3 infertile daughters, 4 fertile daughters, and 2 fertile sons. All patients in this consanguineous family exhibited typical manifestations of unexplained RIF and EDA. Genetic analysis identified a homozygous missense variant (c.G1054C:p.G352R) in exon 13 of the TLE6 gene that cosegregated with the EDA phenotype in an autosomal recessive pattern. Other members of the family, the gene carriers, remain clinically asymptomatic and fertile. Our findings identify a novel nonsynonymous variant, c.G1054C:p.G352R, in the TLE6 gene within a consanguineous Iranian family with autosomal-recessive female infertility and broaden the genetic spectrum of TLE6-associated EDA.

Linker histone H1FOO is required for bovine preimplantation development by regulating lineage specification and chromatin structure†.

Linker histone H1 binds to the nucleosome and is implicated in the regulation of the chromatin structure and function. The H1 variant H1FOO is heavily expressed in oocytes and early embryos. However, given the poor homology of H1FOO among mammals, the functional role of H1FOO during preimplantation embryonic development remains largely unknown, especially in domestic animals. Here, we find that H1FOO is not only expressed in oocytes and preimplantation embryos but granulosa cells and spermatids in cattle. We then demonstrate that the interference of H1FOO results in preimplantation embryonic developmental arrest in cattle using either RNA editing or Trim-Away approach. H1FOO depletion leads to a compromised expression of critical lineage-specific genes at the morula stage and affects the establishment of cell polarity. Interestingly, H1FOO depletion causes a significant increase in the expression of genes encoding other linker H1 and core histones. Concurrently, there is an increase of H3K9me3 and H3K27me3, two markers of repressive chromatin and a decrease of H4K16ac, a marker of open chromatin. Importantly, overexpression of bovine H1FOO results in severe embryonic developmental defects. In sum, we propose that H1FOO controls the proper chromatin structure that is crucial for the fidelity of cell polarization and lineage specification during bovine preimplantation development.

In vivo toxicity assessment of four types of graphene quantum dots (GQDs) using mRNA sequencing

GQDs show great potential in drug carriers, bioimaging, biosensors, theranostics, and are recently reported as promising therapeutic agents to treat amyloid-related diseases such as Parkinson’s disease and inflammations such as colitis. However, current toxicity data about GQDs based on in vivo toxicity assessments remain scarce. In the study, we examined the mRNA expression changes of zebrafish embryos exposed to four types of GQDs, including raw graphene quantum dots (R-GQDs), graphene oxide quantum dots (GOQDs), carboxyl GQDs (C-GQDs), and aminated GQDs (A-GQDs). Firstly, we treated embryos with the four GQDs at three concentrations (50, 100, and 200 μg/mL), and found that only A-GQDs caused embryonic developmental arrest at 100 and 200 μg/mL with significantly decreased survival rates and heartbeat rates, as well as the elevated malformation rates. Next, we analyzed the mRNA sequencing data acquired from zebrafish embryos exposed to the four GQDs for 7 days at 100 μg/mL, and found that all GQDs can act on potassium (K+) and calcium (Ca2+) channels, and spliceosomes with varying degrees of regulatory effects. Compared to other GQDs, A-GQDs can strongly perturb the anticoagulant protein C (PC) pathway via activating most genes associated with complement and coagulation system, cell adhesion molecules (CAMs), and MAPK. In conclusion, this study provided substantial transcriptomic data underlying the common signaling pathways induced by various types of GQDs and pointed out the specific toxicity of A-GQDs on hemostatic system.

Maternal exome analysis for the diagnosis of oocyte maturation defects and early embryonic developmental arrest

Can a methodology be developed for case selection and whole-exome sequencing (WES) analysis of women who are infertile owing to recurrent oocyte maturation defects (OOMD) and/or preimplantation embryo lethality (PREMBL)? Data were collected from IVF patients attending the Istanbul Memorial Hospital (2015-2021). A statistical methodology to identify infertile endophenotypes (recurrent low oocyte maturation rate, low fertilization rate and preimplantation developmental arrest) was developed using a large IVF dataset (11,221 couples). Twenty-eight infertile women with OOMD/PREMBL were subsequently enrolled for WES on their genomic DNA. Pathogenic variants were prioritized using a custom-made bioinformatic pipeline set to minimize false-positive discoveries through resampling in control cohorts (the Human Genome Diversity Project and 1343 whole-exome sequences from oocyte donors). Individual single-cell RNA sequencing data from 18 human metaphase II (MII) oocytes and antral granulosa cells was used for genome-wide validation. WES and bioinformatics were performed at Igenomix and the National Research Council, Italy. Variant prioritization analysis identified 265 unique variants in 248 genes (average 22.4 per sample). Of the genes harbouring high-impact variants 78% were expressed by MII oocytes and/or antral granulosa cells, significantly higher than for random sample of controls (odds ratio = 5, Fisher's exact P = 0.0004). Seven of the 28 women (25%) were homozygous carriers of missense pathogenic variants in known candidate genes for OOMD/PREMBL, including PATL2, NLRP5 (n = 2),TLE6, PADI6, TUBB8 and TRIP13. Furthermore, novel gene-disease associations were identified. In fact, one woman with a low oocyte maturation rate was a homozygous carrier of high-impact variants in ENSA, an essential gene for prophase I meiotic transition in mice. This analytical framework could reveal known and new genes associated with isolated recurrent OOMD/PREMBL, providing essential indications for scaling this strategy to larger studies.

Insufficient pyruvate in culture medium arrests mouse embryos at the first cleavage stage associated with abnormal epigenetic modifications

Energy is essential for early embryogenesis, and fertilized eggs can successfully develop to blastocyst in in vitro culture medium with an appropriate energy supply. Conversely, embryonic development is negatively affected by a suboptimal energy supply. We previously observed that a low level of pyruvate greatly arrests mouse embryos at the 2-cell stage. However, how methylation modifications are affected at this specific stage remains unknown. In this study, we found that mouse embryos could timely develop to the 4-cell stage in K+simplex optimized medium (KSOM) with control level of pyruvate, but embryos were significantly arrested at the 2-cell stage when pyruvate was reduced to 0.2-fold of the control level. Moreover, the fluorescence intensities of 5 mC, H3K4me2, H3K9me2 and H3K27me2 in the 2-cell stage embryos of the 0.2-fold pyruvate group were notedly lower than those of the control group, but N6-methyladenosine (m6A) fluorescence intensity was higher, suggesting that global genomic DNA, histone and m6A methylation modifications are disrupted with low levels of pyruvate. Consistently, the mRNA levels of genes related to DNA methylation, histone methylation and m6A modifications were also disturbed in the 2-cell stage embryos cultured with low levels of pyruvate. In summary, our findings demonstrate that insufficient pyruvate in culture medium results in mouse embryonic developmental arrest, at least in part due to defects in methylation modifications.

The impact of using culture media containing granulocyte-macrophage colony-stimulating factor on live birth rates in patients with a history of embryonic developmental arrest in previous in vitro fertilization cycles

Objective:To investigate the effect of using culture media containing granulocyte-macrophage colony-stimulating factor (GM-CSF) on embryological data and reproductive outcomes in patients with early embryonic developmental arrest.Material and Methods:Retrospective case-control study. A total of 39 patients, whose embryos were incubated with culture media containing GM-CSF due to embryonic developmental arrest in two previous in vitro fertilization (IVF) cycles in-between January 2016 and November 2017 at Hacettepe University IVF Center, were enrolled. Control group was generated among patients with first IVF attempts due to tubal factor in the same time period. All embryos in the control group were incubated with single step culture medium (without GM-CSF). For the control group selection, matching was done 1:2 ratio considering female age, body mass index, number of M-II oocyte retrieved, and number of embryo transferred (n=80).Results:Demographic features and embryological data were comparable between two groups. Number of fertilized oocytes (2-pronuclear) was 3.7±2.0 in GM-CSF group and 3.9±2.5 in the control (p=0.576). Overall, number of embryos transferred (1.3±0.5 vs 1.3±0.5, respectively) and blastocyst transfer rate (67.6% vs 59.2%, respectively; p=0.401) were similar. For the reproductive outcomes, implantation rate (32.3% vs 33.1%, respectively; p=0.937), clinical pregnancy rate (33.3% vs 32.5%, respectively; p=0.770), and live birth rate (25.2% vs 26.2%, respectively; p=0.943) were similar.Conclusion:Using GM-CSF-containing culture media in patients with two previous failed IVF attempts due to embryonic developmental arrest might rectify embryological data and reproductive outcomes. To make solid conclusion further randomized controlled trials are warranted.

P–429 Calcium analysis and embryonic development of in vitro matured oocytes from transgender men

Abstract Study question Can oocytes isolated from transgender men after oophorectomy support embryonic development? Summary answer Embryo developmental arrest at 4–8cell stage (day 3 embryos) indicates poor quality of in vitro matured oocytes from transgender men. What is known already Gender affirming surgery for transgender men leads to permanent infertility, as it involves bilateral oophorectomy. Current approaches for fertility preservation, such as oocyte freezing following ovarian hyperstimulation, may interfere with the wanted masculine characteristics and enhance gender dysphoria. In vitro matured oocytes (IVM) isolated following oophorectomy have been proposed as a source of potential gametes to ensure fertility preservation for transgender men with a child wish. From previous studies, it has been shown that these oocytes are able to undergo maturation and display normal spindles, but their competence to be fertilized and support embryonic development has not been addressed yet. Study design, size, duration We evaluated the quality of in vitro matured oocytes isolated from ovaries of transgender men by applying calcium imaging and monitoring fertilization and embryonic development following intracytoplasmic sperm injection (ICSI). Ovaries were collected in cold (4oC) or warm (37oC) medium, to investigate the best collection procedure. So far, results from four transgender men have been included. Participants/materials, setting, methods: Ovaries from four transgender men undergoing testosterone treatment were collected after oophorectomy in cold or warm medium. Cumulus oocyte complexes (COCs) were isolated and cultured in maturation medium for 48hrs. Mature oocytes were injected with donated sperm and assessed either by calcium imaging, measuring the total calcium release following injection, or following embryonic development. Donated in vitro matured oocytes, germinal vesicle(GV) or metaphase I(MI) origin, from other patients undergoing IVF treatment were used as controls. Main results and the role of chance In total, 179 COCs were collected from ovaries (n = 8) of four transgender men. From the COCs collected in warm medium, 73/105(69%) survived and 33/73(45%) reached metaphase II (MII). Of 21 MII injected with sperm, 13/21(62%) fertilized, 9/21(43%) formed 2 pronuclei (PN), 8/9(89%) reached the 2-cell stage, 3/9(33%) reached 4–8cell stage but arrested. From 74 COCs isolated in cold medium, 57/74(77%) survived and 28/57(49%) matured. Of the 11 MII injected with sperm, 7/11(64%) fertilized, 6/11(54%) formed 2PN, 6/6(100%) reached the 2-cell stage, 4/6(67%) reached 4–8cell but arrested. In the control group, 10/13 oocytes injected with the same sperm sample, were normally fertilized (77%), 8/10(80%) reached the 2-cell stage, 7/10(70%) reached the 4–8cell stage and 4/10(40%) became blastocysts. From the warm, cold and control conditions, respectively 12,14 and 17 MII oocytes were used for calcium imaging. The product of amplitude and frequency of calcium peaks, representing total calcium release, was calculated. Oocytes showed an average release of 0.66AU and 1.69AU for the warm and cold condition, respectively. The average value for control oocytes was 2.19AU. Limitations, reasons for caution One major limitation of our study is the lack of ovaries from cis women as control group. Our control oocytes originated from women undergoing IVF treatment and have undergone ovarian stimulation. Furthermore, the number of oocytes analysed and number of patients per group was limited and is being increased. Wider implications of the findings: Our data indicate that in vitro matured oocytes from transgender men ovaries display poor quality, as demonstrated by the poor embryonic development. In the future, we will apply nuclear transfer technology as a mean to overcome embryonic developmental arrest in this group of oocytes. Trial registration number Not applicable

Embryonic developmental arrest in the annual killifish Austrolebias charrua: A proteomic approach to diapause III.

Diapause is a reversible developmental arrest faced by many organisms in harsh environments. Annual killifish present this mechanism in three possible stages of development. Killifish are freshwater teleosts from Africa and America that live in ephemeral ponds, which dry up in the dry season. The juvenile and adult populations die, and the embryos remain buried in the bottom mud until the next rainy season. Thus, species survival is entirely embryo-dependent, and they are perhaps the most remarkable extremophile organisms among vertebrates. The aim of the present study was to gather information about embryonic diapauses with the use of a “shotgun” proteomics approach in diapause III and prehatching Austrolebias charrua embryos. Our results provide insight into the molecular mechanisms of diapause III. Data are available via ProteomeXchange with identifier PXD025196. We detected a diapause-dependent change in a large group of proteins involved in different functions, such as metabolic pathways and stress tolerance, as well as proteins related to DNA repair and epigenetic modifications. Furthermore, we observed a diapause-associated switch in cytoskeletal proteins. This first glance into global protein expression differences between prehatching and diapause III could provide clues regarding the induction/maintenance of this developmental arrest in A. charrua embryos. There appears to be no single mechanism underlying diapause and the present data expand our knowledge of the molecular basis of diapause regulation. This information will be useful for future comparative approaches among different diapauses in annual killifish and/or other organisms that experience developmental arrest.

Two novel mutations in PADI6 and TLE6 genes cause female infertility due to arrest in embryonic development

PurposeThis study aims to identify genetic causes of female infertility associated with recurrent failure of assisted reproductive technology (ART) characterized by embryonic developmental arrest.MethodsWe recruited infertile patients from two consanguineous families from the Reproductive Medicine Center of Guizhou Provincial People’s Hospital. Peripheral blood was collected for genomic DNA extraction. Two affected individuals and their family members were performed with whole-exome sequencing and Sanger validation in order to identify possible causative genes. For further analyzing the effect of splicing mutation on mRNA integrity in vivo, TLE6 cDNA from the peripheral blood lymphocyte of the affected individual was sequenced. In addition, the possible impact of the pathogenic mutation on the structure and function of the protein were also assessed.ResultsTwo novel homozygous mutations in the peptidylarginine deiminase type VI (PADI6) and the transducin-like enhancer of split 6 (TLE6) genes were identified in the two families. One patient carried the frameshift deletion mutation c.831_832del:p.S278Pfs*59 of the PADI6 gene and the other patient carried the splicing mutation c.1245-2 A>G of the TLE6 gene. The analysis of the mRNA from the proband’s peripheral blood leukocytes confirmed aberrant splicing.ConclusionsOur findings expand the mutational spectrum of PADI6 and TLE6 associated with embryonic developmental arrest and deepen our understanding of the genetic causes of infertility with recurrent ART failure.

Maternal UHRF1 Is Essential for Transcription Landscapes and Repression of Repetitive Elements During the Maternal-to-Zygotic Transition.

Maternal factors that modulate maternal-to-zygotic transition (MZT) are essential for the growth from specialized oocytes to totipotent embryos. Despite several studies, the mechanisms regulating epigenetic reprogramming during MZT remain largely elusive. UHRF1 plays a role in maintaining GC methylation in oocytes and early embryos. However, little is known about its role in mouse MZT. Here, we explored the function of maternal UHRF1 in zygotic genome regulation during early embryonic development in mice. We showed that the conditional knockout (cKO) of UHRF1 in either primordial or growing oocytes causes infertility but differentially affects early embryonic development. UHRF1 deficiency in primordial oocytes led to early embryonic developmental arrest at the two-cell stage, accompanied by significant alterations in global DNA and H3K4me3 methylation patterns. In comparison, UHRF1 ablation in growing oocytes significantly reduced developmental competence from two-cell embryos to blastocysts. At the transcriptional level, the absence of maternal UHRF1 led to aberrant transcriptional regulation of the zygotic genome during MZT at the two-cell stage. Furthermore, we observed that retrotransposable elements in UHRF1-deficient oocytes and embryos were not silenced properly; in particular, the LINE-1 and long terminal repeat (LTR) subfamily were activated abnormally. Collectively, the findings of our study reveal that maternal UHRF1 plays a critical role in establishing the correct epigenetic chromatin reprogramming of early embryos, regulating essential genes during MZT, and preserving genome integrity that drives early embryonic development in mice.