Embryonic Development Research Articles

Venom gland organogenesis in the common house spider

Venom is a remarkable innovation found across the animal kingdom, yet the evolutionary origins of venom systems in various groups, including spiders, remain enigmatic. Here, we investigated the organogenesis of the venom apparatus in the common house spider, Parasteatoda tepidariorum. The venom apparatus consists of a pair of secretory glands, each connected to an opening at the fang tip by a duct that runs through the chelicerae. We performed bulk RNA-seq to identify venom gland-specific markers and assayed their expression using RNA in situ hybridisation experiments on whole-mount time-series. These revealed that the gland primordium emerges during embryonic stage 13 at the chelicera tip, progresses proximally by the end of embryonic development and extends into the prosoma post-eclosion. The initiation of expression of an important toxin component in late postembryos marks the activation of venom-secreting cells. Our selected markers also exhibited distinct expression patterns in adult venom glands: sage and the toxin marker were expressed in the secretory epithelium, forkhead and sum-1 in the surrounding muscle layer, while Distal-less was predominantly expressed at the gland extremities. Our study provides the first comprehensive analysis of venom gland morphogenesis in spiders, offering key insights into their evolution and development.

TFEB controls syncytiotrophoblast formation and hormone production in placenta.

TFEB, a bHLH-leucine zipper transcription factor belonging to the MiT/TFE family, globally modulates cell metabolism by regulating autophagy and lysosomal functions. Remarkably, loss of TFEB in mice causes embryonic lethality due to severe defects in placentation associated with aberrant vascularization and resulting hypoxia. However, the molecular mechanism underlying this phenotype has remained elusive. By integrating in vivo analyses with multi-omics approaches and functional assays, we have uncovered an unprecedented function for TFEB in promoting the formation of a functional syncytiotrophoblast in the placenta. Our findings demonstrate that constitutive loss of TFEB in knock-out mice is associated with defective formation of the syncytiotrophoblast layer. Indeed, using in vitro models of syncytialization, we demonstrated that TFEB translocates into the nucleus during syncytiotrophoblast formation and binds to the promoters of crucial placental genes, including genes encoding fusogenic proteins (Syncytin-1 and Syncytin-2) and enzymes involved in steroidogenic pathways, such as CYP19A1, the rate-limiting enzyme for the synthesis of 17β-Estradiol (E2). Conversely, TFEB depletion impairs both syncytial fusion and endocrine properties of syncytiotrophoblast, as demonstrated by a significant decrease in the secretion of placental hormones and E2 production. Notably, restoration of TFEB expression resets syncytiotrophoblast identity. Our findings identify that TFEB controls placental development and function by orchestrating both the transcriptional program underlying trophoblast fusion and the acquisition of endocrine function, which are crucial for the bioenergetic requirements of embryonic development.

GNA13 suppresses proliferation of ER+ breast cancer cells via ERα dependent upregulation of the MYC oncogene

GNA13 (Gα13) is one of two alpha subunit members of the G12/13 family of heterotrimeric G-proteins which mediate signaling downstream of GPCRs. It is known to be essential for embryonic development and vasculogenesis and has been increasingly shown to be involved in mediating several steps of cancer progression. Recent studies found that Gα13 can function as an oncogene and contributes to progression and metastasis of multiple tumor types, including ovarian, head and neck and prostate cancers. In most cases, Gα12 and Gα13, as closely related α-subunits in the subfamily, have similar cellular roles. However, in recent years their differences in signaling and function have started to emerge. We previously identified that Gα13 drives invasion of Triple Negative Breast Cancer (TNBC) cells in vitro. As a highly heterogenous disease with various well-defined molecular subtypes (ER+ /Her2−, ER+ /Her2+, Her2+, TNBC) and subtype associated outcomes, the function(s) of Gα13 beyond TNBC should be explored. Here, we report the finding that low expression of GNA13 is predictive of poorer survival in breast cancer, which challenges the conventional idea of Gα12/13 being universal oncogenes in solid tumors. Consistently, we found that Gα13 suppresses the proliferation in multiple ER+ breast cancer cell lines (MCF-7, ZR-75-1 and T47D). Loss of GNA13 expression drives cell proliferation, soft-agar colony formation and in vivo tumor formation in an orthotopic xenograft model. To evaluate the mechanism of Gα13 action, we performed RNA-sequencing analysis on these cell lines and found that loss of GNA13 results in the upregulation of MYC signaling pathways in ER+ breast cancer cells. Simultaneous silencing of MYC reversed the proliferative effect from the loss of GNA13, validating the role of MYC in Gα13 regulation of proliferation. Further, we found Gα13 regulates the expression of MYC, at both the transcript and protein level in an ERα dependent manner. Taken together, our study provides the first evidence for a tumor suppressive role for Gα13 in breast cancer cells and demonstrates for the first time the direct involvement of Gα13 in ER-dependent regulation of MYC signaling. With a few exceptions, elevated Gα13 levels are generally considered to be oncogenic, similar to Gα12. This study demonstrates an unexpected tumor suppressive role for Gα13 in ER+ breast cancer via regulation of MYC, suggesting that Gα13 can have subtype-dependent tumor suppressive roles in breast cancer.

Evaluation of temperature- and ethanol-related developmental degree variations by a new scoring system (FETAX-score) applicable to Frog Embryo Teratogenicity Assay: Xenopus

The aim of the present work is to propose a new quantitative assessment method (FETAX-score) for determining the degree of Xenopus laevis embryo development intended for use in embryotoxicity studies. Inspired by a similar scoring system used to evaluate developmental delays (young-for-age phenotypes) in rat embryos cultured in vitro, the FETAX-score was established by considering seven morphological features (head, naris, mouth, lower jaw, tentacles, intestine, anus) that are easily evaluable in tadpoles during the late stages of development at the conclusion of the test. Given that X. laevis development is temperature-dependent and that temperatures below 14°C and above 26°C are teratogenic, the FETAX-score was tested in embryos maintained at 17, 20, 23 and 26°C. No abnormalities were observed in any group, while the total score was temperature-related, suggesting that the FETAX-score is sensitive to moderate distress that does not influence general morphology. Intestine and anus were the least sensitive structures to temperature variations. To assess the applicability of the FETAX-score in developmental toxicological studies, we evaluated FETAX-score in tadpoles exposed during the morphogenetic period to Ethanol (Eth) at concentrations of 0, 0.25, 0.5, 1, 1.5, and 2 % v/v. Gross malformations were observed only in tadpoles from the Eth 2 % group. By contrast, data analysis of the other Eth groups showed dose-related reductions in the FETAX-score. Tentacles were the most sensitive structures to Eth-related delays. These results support the use of the FETAX-score to quantitatively assess developmental deviations in FETAX embryotoxicity studies.

P-108 Automated sperm selection software (SiD) provides similar results as human selection for ICSI

Abstract Study question Automated sperm selection or human eye-based sperm selection, does it make a difference on IVF lab outcomes and clinical outcomes? Summary answer Single sperm selection by the AI SiD was as proficient as highly experienced embryologists and embryo development may have improved for inexperienced embryologists What is known already The implementation of artificial intelligence in reproductive medicine has proven effective for a variety of applications. One application for this technology is single sperm selection during ICSI. The computer assisted program SiD, was designed to grade sperm, in real-time, based on motility parameters transforming qualitative sperm assessments into a quantitative score. SiD’s assistance can be used to select the most optimal sperm for ICSI. The application of sperm selection by AI is expected to bypass user bias, fatigue, limited training, while also optimising time. However, there are limited studies correlating the usage of AI for sperm selection with ICSI outcomes. Study design, size, duration 1 year period 608 sibling MII oocytes were randomly divided in two groups; 1-ICSI group (n = 301): ICSI performed with sperm selected by the embryologist and 2- SID-ICSI group (n = 307): ICSI performed with sperm selected using SiD software. Priority was given to sperm classified as “Best” by SID software for ICSI.. Morphology selection was performed before ICSI in both groups. Embryos were cultured during 6 days in the same conditions. Participants/materials, setting, methods Sperm preparation was performed using a microfluidic chamber (Zymot™). Sperm selection was performed as previously described, and ICSI was performed following routine procedures. Biological and clinical outcomes were analysed in both groups and compared including fertilization rate, cleavage rate, embryo and blastocyst development rate, top quality embryo rate, blastocyst development rate and pregnancy rates. Biological outcomes in both groups were also compared based on embryologist seniority. Embryo morphokinetics parameters were analysed using time-lapse system. Main results and the role of chance There was a non significant trend towards better outcomes in the SID-ICSI group for all biological outcomes including fertilization rate (83,1% vs 82,4%), cleavage rate (97,6% vs 97,2%), day 3 embryo development rate (72,9% vs 70,6%), blastocyst development rate at day 5 (49,0% vs 44,8 %), good quality blastocyst development rate at day 5 (45,1% vs 41,5%), and top quality blastocyst development rate at day 5 (25,9% vs 22,2%). Similarly, a non-significant increase was observed in all these outcomes when sperm selection is performed by a junior embryologist. Embryo development was monitored using a timelapse system. Our data show that 2nd polar body extrusion (tPB2) (4,0h vs 4,8 h [p = 0,005]) and early cleavage (t2) (27,3h vs 28,7h [p = 0,05]) happen significantly earlier when SiD is used for ICSI. No significant difference was observed for all other fertilization events (cytoplasmic wave,tPN1,tPN2,presence of cytoplasmic halo,tPNf,and cytoplasmic halo disappearance), and other cleavage timings (t3->t10,tM,tSC,tEC,tSB), and embryonic cell cycles (ECC1,ECC2,ECC3,s2,s3). Euploid rate obtained with in the 2 groups was comparable (53,3% in SID-ICSI vs 44,4% in ICSI). Although there was a trend toward higher cumulative early and clinical pregnancy rate after ICSI-SID, the difference did not reach significance (43,1%vs34,0% and 27,5%vs21,2%, respectively). Limitations, reasons for caution The ZymotTM microfluidic chamber was used for sperm preparation. This device optimises sperm selection, giving rise to an already optimised population of spermatozoa. Future investigations evaluating the effect of SiD automated sperm selection software on biological outcomes in samples prepared with a less effective technique would potentially yield conversing results. Wider implications of the findings This pilot study demonstrated that the usage of the automated sperm selection software SiD gives rise to similar biological outcomes and embryo morphokinetics, indicating its effectiveness in sperm selection. We expect this program would be useful in the presence of less experienced laboratory members helping by standardizing sperm selection. Trial registration number NA

O-077 Depletion of MFF from oocytes impairs mitochondrial dynamics, leading to inhibited oocyte maturation and early embryonic development in mice

Abstract Study question To investigate the effect of mitochondrial fission factor (MFF) on oocyte competence and female fertility using an oocyte-specific Mff knockout mouse model. Summary answer Oocyte-specific Mff deficiency hindered oocyte maturation and early embryonic development via regulation of mitochondrial dynamics, resulting in female subfertility. What is known already Emerging studies have demonstrated that mitochondrial dynamics play a vital role in oocyte maturation and early embryo development. MFF is one of the crucial proteins regulating mitochondrial dynamics. Our previous studies have indicated that Mff is necessary for oocyte competence and fertility using a global Mff knockout mouse model. However, further investigation is needed to determine whether the deletion of Mff in oocytes could affect oocyte competence and early embryonic development by regulating mitochondrial dynamics. Study design, size, duration The oocyte-specific Mff knockout (Mfffl/fl; Gdf9-Cre) mice were generated using the Cre-LoxP conditional knockout system. The 2-month-old female mice from each group (Mfffl/fl; Gdf9-Cre or Mfffl/fl, n = 6) were mated to assess the fecundity over 10 months. The maturation and mitochondrial dynamics of germinal vesicle (GV) oocytes were determined. The spindle and chromosome morphologies of ovulated oocytes were also analyzed. Additionally, the 2-cell embryos were collected and cultured in vitro to assess early embryonic development. Participants/materials, setting, methods Female mice from each group were mated with adult males to assess the fecundity. GV oocytes, ovulated oocytes, and 2-cell embryos were collected and cultured in vitro after superovulation, as indicated. Mitochondria, spindle, and chromosome were stained and then imaged using confocal microscopy. The microscopic morphology of mitochondria was assessed using electron microscopy. Main results and the role of chance The Mfffl/fl; Gdf9-Cre mice consistently exhibited reduced fertility with a significant decrease in litter size (3.00 vs. 5.42, p < 0.001), number of litters per female (4.33 vs. 7.50, p < 0.001), and number of pups per female (13.00 vs. 40.67, p < 0.001) compared to the Mfffl/fl mice. The numbers of GV oocytes (29.37 vs. 39.10, p < 0.05), ovulated oocytes (9.81 vs. 20.91, p < 0.001) and 2-cell embryos (3.50 vs. 10.17, p < 0.05) were significantly lower in the Mfffl/fl; Gdf9-Cre mice. The blastocyst (32.32% vs. 68.44%, p < 0.05) embryo development rate per 2-cell embryos was also significantly lower. In vitro maturation revealed that of Mfffl/fl; Gdf9-Cre GV oocytes had significantly lower rates of GV break-down (68.89% vs. 91.57%, p < 0.05) and polar body extrusion (42.76% vs. 81.87%, p < 0.05). The ratios of aberrant spindle (71.56% vs. 23.74%, p < 0.001) and misaligned chromosomes (71.82% vs. 23.14%, p < 0.01) in ovulated oocytes from Mfffl/fl; Gdf9-Cre mice were significantly increased. In terms of mitochondrial dynamics, the mitochondrial size (0.59 µm2 vs. 0.15µm2, p < 0.001) and the ratio (62.50% vs. 18.67%, p < 0.01) of abnormal mitochondrial distribution (unevenly aggregated) in Mfffl/fl; Gdf9-Cre oocytes significantly elevated. This study found that the Mff played a crucial role in oocyte maturation and early embryonic development. Limitations, reasons for caution Further research is needed to determine the downstream targets or pathways of Mff in oocytes. Wider implications of the findings These results shed light on the important role played by MFF in ensuring proper mitochondrial dynamics in oocytes and underscore the significance of this protein in female fertility. This study provides insights into potential therapeutic targets for infertility and related diseases in humans. Trial registration number not applicable

Effects of dietary polyunsaturated fatty acid enriched diets on breeding, embryonic development and organogenesis of Rohu carp, Labeo rohita (Hamilton 1822)

There is a considerable interest in supplementing fish diets with bio-functional substances such as polyunsaturated fatty acids (PUFAs), and research is being conducted to evaluate their effects on fish physiology, particularly on breeding performances. The objective of this study was to find out how squid-derived lipids (PUFAs) influence the Rohu carp, Labeo rohita, in terms of reproduction, and embryonic and larval growth. The experimental fish were provided with formulated diet for a duration of 90 days, containing 1 % squid-extracted lipids as a primary source of polyunsaturated fatty acids (PUFAs). To determine the accuracy of the formulation, a proximate analysis of the feed ingredients and the formulated feeds were conducted. The results demonstrated remarkable reproductive outcomes during the spawning trial. Observations in fish treated with PUFAs revealed a significant improvement (p < 0.001) in sperm quality and viability, despite spawning occurring late in the breeding season. In addition, the fertilization rate (92.27 ± 1.74), hatching rate (89.33 ± 1.76), and survival rate (85.83 ± 1.74) of the offspring exhibited a statistically significant increase (p < 0.05) when subjected to PUFAs treatment in L. rohita. Consequently, the treated fish exhibited enhanced early embryonic and larval development in comparison to the control group. Hence, the current study demonstrates that the inclusion of PUFAs in the diet of Rohu carp leads to improved maturation, breeding, and developmental outcomes, specifically in terms of embryonic development and organogenesis.

P-115 GATAD2B is required for pre-implantation embryo development by delaying zygotic genome activation

Abstract Study question Major zygotic genome activation (ZGA) involving the activation of thousands of genes occurs at the late-2cell stage, but the regulatory mechanisms governing ZGA remain unclear. Summary answer Our results demonstrate that GATAD2B is essential for early embryonic development, in part through facilitating zygotic genome activation (ZGA). What is known already Acetylation modifications of histone tails promote transcriptional activation, and the maternal deletion of H4K16ac leads to failure in ZGA. GATAD2B is one of the core subunits of the Nucleosome remodeling and histone deacetylation complex(NuRD). Study design, size, duration Using mouse embryos as the research model, the study was conducted over a period of approximately two years to ensure a relatively small-scale investigation. Participants/materials, setting, methods We examined the dynamic expression of NURD complex components, including GATAD2B, during pre-implantation embryonic stages. GATAD2B expression was specifically reduced via microinjection of GATAD2B-siRNA during the zygote stage, revealing its impact on early embryonic development in mice. Zygotic genome activation (ZGA), morula compaction, and blastocyst lineage differentiation were investigated using immunofluorescence, western blot, and other techniques. Additionally, we employed RNA-seq, protein immunoprecipitation, and mass spectrometry to uncover underlying molecular mechanisms. Main results and the role of chance Our research has shown that GATAD2B exhibits specific nucleus localization and high protein expression from the late-2-cell to 8-cell. This intriguing phenomenon prompted us to investigate the relationship between GATAD2B and the ZGA. We discovered a distinctive pattern of GATAD2B, starting from the late 2-cell stage with nuclear localization. GATAD2B depletion resulted in defective embryonic development, including increased DNA damage at morula, decreased blastocyst formation rate and abnormal differentiation of ICM/TE lineages. Consistent with the delay during the cleavage stage, the transcriptome analysis of the 2-cell revealed inhibition of the cell cycle G2/M phase transition pathway. Furthermore, the GATAD2B proteomics data provided clear evidence of a certain association between GATAD2B and molecules involved in the cell cycle pathway. As hypothesized, GATAD2B-deficient 2-cell embryos exhibited abnormalities in ZGA during the maternal-to-embryonic transition, with lower expression of the major ZGA mark MERVL. Limitations, reasons for caution Given the limitations of antibody effectiveness and specificity required for the experiments, the exploration of molecular mechanisms is still ongoing. Wider implications of the findings Our findings have elucidated the impact of GATAD2B on early embryonic development through the regulation of zygotic genome activation (ZGA), providing a novel genetic explanation for clinical defects in early embryonic development. Trial registration number not applicable

P-117 Extracellular vesicles from fallopian tubal fluid benefit human embryo development in vitro

Abstract Study question Do extracellular vesicles (EVs) from Fallopian tubes benefit human embryo development, and how do they affect preimplantation embryo development? Summary answer The Fallopian tubal EVs can be internalized by human embryos, and they contain miR-21-5p that downregulated P53 in blastocysts. What is known already EVs in the Fallopian tubes are identified as key mediators in embryo-oviduct interactions that contribute to a successful pregnancy in vivo. We previously showed Fallopian tubal EVs can benefit mouse embryo development in vitro. However, if a similar phenomenon can be observed in human embryos and what is the key component in Fallopian tubal EVs remains elusive. Study design, size, duration The EVs were isolated from the luminal fluid of human Fallopian tubes (n = 23). Immature human oocytes were collected and in vitro matured and fertilized, and 2-pronuclei embryos were included in this study and randomly divided (n = 87). We cocultured these zygotes with Fallopian tubal EVs until the blastocyst stage. Further, two-cell mouse embryos were used to explore the expression levels of miRNAs in Fallopian tubal EVs. The effects of miR-21-5p on mouse embryos were analyzed. Participants/materials, setting, methods EVs were isolated using ultracentrifugation. The uptake of labeled EVs by human embryos was analyzed using confocal microscopy. The fragmentation of the day-3 embryos and the blastocyst rate were compared between the control and the EV-treatment group. Then the stage-specific expression of miRNAs was compared in mouse embryos that were either developed in vivo or cultured in vitro. miR-21-5p were supplemented to the culture medium and the p53-related apoptosis in embryos was evaluated. Main results and the role of chance EVs were measured less than 200 nm in diameter with a typical bi-layer membrane structure and expressed protein markers Heat shock protein 70 (HSP70) and CD9. Most of the fluorescence-labeled EVs were attached to the zona pellucida within 2 h of co-culture. However, human embryos internalized EV within 4 h, with fluorescence signals observed in embryonic cells. The fragmentation rate was decreased and the blastocyst rate was increased by EVs. miR-21-5p, which was highly enriched in EVs, was found to be expressed at a significantly lower level in in-vitro-cultured mouse embryos compared to the in-vivo-developed embryos, through the two-cell stage to the eight-cell stage. miR-21-5p agomir supplementation to the culture medium was found to increase the mouse blastocyst formation rate, decrease apoptosis, and downregulate the P53 expression level in mouse blastocysts. Limitations, reasons for caution Though we observed that Fallopian tubal EVs could be internalized by human embryos and improve embryo development morphologically, the subsequent mechanism exploration was conducted using mouse embryos. Whether the stage-specific expression level of miR-21-5p and the effects of miR-21-5p are the same in human embryos remains to be determined. Wider implications of the findings This study showed miRNAs in Fallopian tubal EVs can act as embryonic survival signals, which opens the insight into further explorations to demonstrate the molecular mechanisms during early embryo development, as well as holds the potential to optimize the culture medium used in ART in the future. Trial registration number 2022YFC2702503

P-797 Assessment of embryo development following fertilization with de novo male gametes generated in a three-dimensional culture

Abstract Study question Can de novo male gamete from embryonic stem cell contribute to the full pre-implantation development? Summary answer Gametes generated after 29 days of differentiation in our three-dimensional (3D) culture system can support consistent fertilization and normal embryo development to the blastocyst stage. What is known already In regenerative medicine, several 3D culture systems have been proposed and are capable of producing functional tissue implants. In reproductive biology, recent studies have reported preliminary success in generating functional de novo gametes through soft-agar culture and testicular organoids from mouse embryonic stem cells. However, in order to achieve successful fertilization and satisfactory embryo development, an heterologous transplantation in a host seminiferous tubule is required to allow proper spermiogenesis to obtain functional gametes. Here we attempt to perform neogametogensis in a novel 3D niche to generate de novo gamete ready to be used for ICSI. Study design, size, duration Mouse ESCs were first cultured on a gelatin coated 6-well plate with fibroblasts in monolayer and later spherified using sodium alginate. Spheres were submerged in specifically designed conditioned media to encourage differentiation of the mESCs into germ-like cells. Over the course of differentiation, cells were assessed for germ cell differentiation biomarkers. Considering that a normal spermatogenesis occur in 30 days, utilization of the de novo gametes was planned for day-15, 22, 29 and 36. Participants/materials, setting, methods Mouse ESCs were differentiated by submerging the spheres in EpiLC medium containing Activin A, bFGF and KSR for 3 days followed by PGCLC medium containing BMP4, LIF, SCF and EGF for up to 36 days. Differentiation was assessed for markers DAZL (spermatogonium), VASA (spermatocyte), BOULE (post-meiotic stage) and acrosin (spermatid). Differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored in a time-lapse incubator. Main results and the role of chance The evaluation of germline-specific markers through immunofluorescence revealed consistent levels of Vasa expression, a ubiquitous germline marker, with percentages at 15% on day-3, 18% on day-10, 17% on day-15, 19% on day-22, 20% on day-29, and 13% on day-36. Dazl, a specific marker for spermatogonia, exhibited a peak expression of 45% on day-10 in spherified cells. Boule, expressed by secondary spermatocytes, reached its highest level of 10% on day-15 but progressively decreased to 8% on day-22. Acrosin, a marker for spermatids, initially manifested at day-10 at 2% positivity and increased to 5% on day-15, reaching 7% on day-22, and peaked at 20% on day-29. According to spermatogenic marker expression, the neogametes follows a maturational profile similar to in vivo mouse spermatogenesis. To prove neogametes competence, we inject the neogametes into mature oocytes. The control ICSI cohort achieved 89.2% fertilization and 77.8% blastocyst rates. Neogametes generated on day-15, 22, 29 and 36, achieved fertilization rates of 35.0%, 61.1%, 81.8% and 75.0%, respectively, and yielded blastulation rates of 5.0%, 16.7%, 36.4% and 8.3%, respectively. Resulting embryos had morphokinesis comparable to control up to the 8-cell stage, but a delay was observed from compaction to blastocyst hatching. Limitations, reasons for caution In spite of the ability to fertilize normally and support blastulation, efficiency rate remained suboptimal. More importantly, the ability to generate live offspring still has to be proven. Wider implications of the findings This novel 3D differentiation model is capable of generating competent gametes and obviate the need for allo-/xeno-geneic transplantation. Once reproducibility and ability to obtain healthy offspring are confirmed, this method may represent a novel treatment option for azoospermic men Trial registration number N/A

P-611 Quantity or quality? Comparative study on the effectiveness of recombinant, purified and mixed gonadotropins on the outcome of ICSI treatments from an embryologist perspective

Abstract Study question Are oocyte retrieval and embryo development affected by the origin of the gonadotropins used for controlled ovarian stimulation (COS), when controlling fertilization and culture conditions? Summary answer Patients stimulated with recombinant gonadotropins obtained similar number of oocytes than those using purified hormones, but more blastocysts and better embryo morphology, needing lower doses. What is known already Many authors have questioned the safety of purified urinary gonadotropins used for COS prior to an IVF treatment, and have demonstrated the presence of impurities and a high heterogeneity between batches. Another strategy involves using recombinant follicle-stimulating hormone with/without luteinizing hormone (rFSH+/-rLH) synthetized in vitro. Many studies have compared the cost-effectiveness of these drugs; however, a conclusion has not been reached. These studies have varying inclusion criteria and focus on final endpoints such as pregnancy and live birth, sometimes lacking control over the many variables introduced during the fertilization and culture procedures, which affect embryo development and potential. Study design, size, duration Preliminary retrospective observational study performed over 453 patients subjected to COS prior to autologous ICSI treatments in a private clinic over 3 years, using Time-lapse incubator. The total dose employed, the number and maturity rate of the oocytes retrieved of patients subjected to stimulation with recombinant (RH; n = 46) or mixed hormones (MH; n = 332) were compared with those using purified hormones (PH; n = 75). After ICSI, fertilization and embryo development were also compared between the three groups. Participants/materials, setting, methods The stimulation protocol was adjusted for each patient following the standard protocols of the clinic. Fertilization was performed by ICSI and embryo culture was carried out in Geri time-lapse incubators using Geri Medium (Genea Biomedx). When available (1,097 embryos), Eeva-Xtend Test was applied. Statistical analysis was performed by generalized estimating equations (significance: P≤0.05). Considered confounders include age, body mass index, days of stimulation, down-regulation protocol, and other variables relevant to embryo development. Main results and the role of chance Proper follicle stimulation was achieved with lower doses of RH (1,606.26±1,110.52 IU) than PH (2,941.73±1,011.14 IU) and MH (3,629.68±1,080.79 IU). No association was found between the use of different gonadotropins and the number of oocytes collected (Odds ratio (OR, RH vs PH)=2.51, P=0.411; OR (MH vs PH)=1.26, P=0.754) or their maturity rate (OR (RH vs PH)=2.38, P=0.328; OR (MH vs PH)=1.01, P=0.988). No significant association was found between the odds of a correct fertilization and the type of hormone used. However, the odds of reaching blastocyst stage were statistically higher for embryos from patients treated with RH: OR (RH vs PH)=1.72, P=0.006; OR (MH vs PH)=1.45, P=0.005; blastocyst rate=68.7% RH, 61.9% MH and 54.6% PH. The mean morphological classification by ASEBIR criteria of the embryos was significantly better in the RH group than in the PH group (OR = 1.55, P=0.032), but no association was found in the MH-PH comparison (OR = 1.26, P=0.092). No association was found between the type of hormone and the distribution of the Eeva-Xtend scores. Significantly lower values of the morphokinetic parameter s2 (synchrony between the second and third cell divisions) were also associated with the use of RH (OR (RH vs PH)=0.311, P=0.004). Limitations, reasons for caution The reduced sample size and the heterogeneous number of patients in the three groups limit the statistical power of the comparisons. Different hormones and brands were included in each group. Wider implications of the findings Although not altering the number of oocytes retrieved, the use of RH may improve the chances of success of ICSI cycles by increasing the number of available blastocysts. The positive outcomes paired with the lower dose needed, could make the use of recombinant hormones a cost-effective strategy. Trial registration number Not applicable

P-796 A novel method of somatic cell nuclear transfer to generate artificial oocytes

Abstract Study question Can we identify an alternative method to Sendai-Virus fusion for somatic cell nuclear transfer (SCNT) aiming at generating artificial oocyte? Summary answer The utilization lipofectamine proved itself as a valid alternative method to virus-mediated SCNT, allowing successful membrane fusion and supporting proper embryo development. What is known already SCNT involves transferring a diploid cell’s nucleus into an enucleated oocyte. Early attempts of SCNT involves direct injection and electrofusion but both approaches can affect oolemma integrity. To avoid mechanical damage to oolemma, fusogenic agents have been employed for SCNT including polyethylene glycol (PEG) and more efficient Hemagglutinating Virus of Japan Envelope (HVJ-E). However, despite its high efficiency, HVJ-E is difficult to be used in human because it is a virus derivative, and a recent shortage of this compound made it not readily available. On the other hand, cationic phospholipid is fusogenic and is widely utilized in transfection experiments. Study design, size, duration In the past 4 months, SCNT was carried out through PEG-mediated, HVJ-E-mediated, or novel lipofectamine-mediated fusion using cumulus cells as nuclear donor. Post-SCNT oocytes were inseminated and cultured up to the blastocyst stage. Embryo development and morphokinetics were assessed and compared among the three SCNT experimental cohorts. In some cases, blastocysts were transferred into pseudo-pregnant mice, post-natal development were monitored. Fertilization, embryo development, and offspring wellbeing were monitored. Participants/materials, setting, methods Oocytes from B6D2F1 mice were enucleated by micromanipulation. The ooplasts were allocated to 3 cohorts: 1) exposure to 45% PEG, 2) subzonal transfer of donor cell with HVJ-E, or 3) subzonal transfer the donor cell exposed to 1.75% lipofectamine. Reconstructed oocytes were monitored for fusion 30 minutes after exposure to fusogens and inseminated by spermatozoa from B6-EGFP mice to confirm paternal genome provenance. Fertilization and embryo development were monitored in a time-lapse system. Main results and the role of chance A total of 165 oocytes were enucleated and 160 (97.0%) survived and allocated to PEG-fusion (n = 50), Sendai-virus-fusion (n = 41) and lipofecamine-fusion (n = 80) cohorts. Fusion rate was 56.0% (28/50) for the PEG-fusion cohort, while Sendai-virus and lipofectamine-fusion cohorts yielded higher fusion rate at 92.7%(38/41) and 86.3%(69/80), respectively (P&amp;lt;0.0001). Control oocytes yielded 96.7% (145/150) fertilization rate and developed to 2-cell stage at 93.8% (136/145), and yielded an eventual blastocyst rate at 80.7% (117/145). Despite a lower oocyte reconstruction rate, PEG-fusion cohort fertilized at 89.3% (25/28), but cleavage rate to 2-cell stage was lower at 48.0% (12/25, P&amp;lt;0.0001) and did not yield any blastocyst. The HVJ-E cohort yielded a fertilization rate of 94.7% (36/38) and cleavage rate at 97.2% (35/36) similar to control. The blastocyst rate was observed at 22.9% (8/35, P&amp;lt;0.0001) when comparing to control. Lipofectamine-fusion cohort yielded 91.3% (63/69) fertilization rate and subsequent cleavage rate at 95.2% (60/63). Albeit at a lower blastocyst development rate comparing to control, the lipofectamine cohort yielded similar full-preimplantation rate at 19.0% (12/63) when comparing to HVJ-E cohort. All blastocysts (n = 20) were transferred into 2 pseudo-pregnant mice and 3 healthy female offspring were yielded to date. Limitations, reasons for caution HVJ-E mediated membrane fusion has been well established with proven safety record; however, its recent shortage has led to a temporary cessation of its utilization. Our lipofectamine-mediated cell fusion technique yielded satisfactory fusion, fertilization, blastocyst formation and some live offspring, however the safety and efficacy still remain to be validated. Wider implications of the findings This novel lipofectamine-mediated cell fusion has broader implications for advancing cloning technologies. Lipofection is a non-viral approach, overcomes shortage of HVJ-E and therefore propose itself as a more appealing cell fusion method for human SCNT. Trial registration number N/A

P-210 A new insight into the effects of systemic lupus erythematosus on oocyte and embryo development and female fertility

Abstract Study question Does SLE affect oocyte and embryo development and female fertility in female patients? Summary answer Patients with SLE present worse outcomes in oocyte and embryonic development, thus yielding compromised female fertility and clinical pregnancy. What is known already Systemic lupus erythematosus (SLE) is often associated with adverse reproductive outcomes. However, it’s currently unclear regarding the role of SLE in oocyte and embryonic development. Also, it’s controversial whether SLE hurts fertility. There is a lack of comprehensive understanding and assessment of fertility in patients with SLE. Study design, size, duration This study retrospectively investigated oocyte and embryo development, ovarian reserve, and clinical outcomes in SLE patients who underwent in vitro fertilization (IVF) treatment from January 2013 to September 2022 at the University Hospital. Participants/materials, setting, methods In this study, we collected data from 34 SLE patients who were previously diagnosed and in remission for a total of 44 IVF cycles and matched 102 infertile women with a total of 148 IVF cycles by Propensity Score Matching (PSM) of 1:3 ratio. We then evaluated baseline characteristics, ovarian reserve, IVF laboratory outcomes, and clinical outcomes between the two groups. Main results and the role of chance After PSM matching, baseline characteristics including age, infertility types, and duration, as well as infertility causes overall coincided between the two groups. Anti-müllerian hormone (AMH) was significantly lower in the SLE group vs comparison (1.9 vs. 3.3 ng/mL, P=0.001). The SLE group performed a significant reduction in available embryo rate (76.6% vs. 86.0%, P=0.001), good-quality blastocyst formation rate (35.1% vs. 47.0%, P=0.003), and blastocyst formation rate (51.0% vs. 67.7%, P=0.001) compared to the comparison. As for clinical outcomes, the implantation rate in the SLE group was notably lower (37.9% vs. 54.9%, P=0.022). The CLBR following every embryo-transfer procedure was distinctly lower (41.2% vs 68.6%, P=0.016) in the SLE group vs comparison. Also, the conservative and optimal CLBRs following every complete cycle procedure were significantly reduced in the SLE group vs the comparison (P=0.001, both) Limitations, reasons for caution It’s a retrospective and single-center study, which is relevant to an inevitable risk of bias. Due to its special population selection, the sample size of this study is quite small. Wider implications of the findings This study provides data support to explore further the impact of SLE or other autoimmune diseases on female fertility, and it also calls for more attention to be given to these special patients by reproductive physicians. Comprehensive fertility assessment and individualized fertility guidance are recommended for patients with SLE. Trial registration number TJ-IRB20211280

P-025 Mitochondrial activity in sperm is associated to blastocyst development: a double-donation study

Abstract Study question Do oxidative parameters in sperm affect oocyte fertilization or blastocyst development in double-donation ICSI cycles? Summary answer While intracellular oxidative stress in sperm does not suppose an impact on fertilization or embryo development, mitochondrial activity is related to increased blastocyst development rates What is known already Reactive oxygen species(ROS) are molecules causing a detrimental effect on sperm quality and function, especially when they are imbalanced with respect to the sperm and seminal plasma antioxidant capacity. Previous research have extensively demonstrated that sperm affected by oxidative stress(OS) show reduced fertilizing capacity, and lead to embryos with impaired development after conventional IVF. The role of OS in sperm in the case of ICSI is, however, controversial because many factors may cause a bias. Not only could this explain the inconsistent results reported thus far, but would also support the need of testing healthy donor gametes to reduce variability. Study design, size, duration This research was devised in the context of a collaboration between a sperm bank and a public university, and was set as a blinded, retrospective study that included samples from 45 sperm donors used in a total of 55 oocyte donation cycles. Intracellular levels of total ROS and of superoxides (O2•-), oxidative stress and mitochondrial activity (MA) were evaluated in frozen-thawed sperm donor samples, and related to the embryo outcomes obtained from the same samples. Participants/materials, setting, methods Samples from selected sperm donors were used to fertilize oocytes through ICSI in oocyte donation programs, and fertilization and blastocyst rates were recorded. Sperm viability and intracellular levels of total ROS and superoxides were determined through a combined assay (2’,7’-Dichlorofluorescin diacetate/Hydroethidine/Sytox Red); oxidative stress and sperm viability were assessed through co-staining with CellRox Far Red and propidium iodide (PI); and MA (mitochondrial membrane potential) and sperm viability were evaluated after staining with DiOC6(3) and PI. Main results and the role of chance The average and standard deviation for fertilization and blastocyst rates were 76.8%±17.3% and 67.5%±22.8%, respectively. Taking the viable sperm population into account, percentages of sperm with high ROS and low O2•- were 17.6%±15.9%, those with high ROS and high O2•- were 0.5%±0.4%, percentages of sperm with low ROS and low O2•- were 25.9%±9.2%, and those with low ROS and high O2•- were 54.5%±21.1%. The evaluation of oxidative stress with CellRox revealed that the percentage of viable sperm with high OS was 63.1%±16.5%. Neither intracellular levels of ROS and superoxides, nor oxidative stress in viable sperm were found to be correlated to fertilization or blastocyst rates (P&amp;gt;0.05). In spite of this, the percentage of viable sperm with high MA, which was 20.7%±10.5%, was observed to be positively correlated to blastocyst rates (Rs=0.276; P=0.04). While sperm mitochondria do not participate in further embryo development, a greater mitochondrial activity in post-thawed donor sperm could indicate a healthier sperm function. Mitochondrial activity could, therefore, be used as a predictive biomarker for embryo development. Limitations, reasons for caution The sample size is the main limitation of this study, despite the efforts made to include a high number of sperm donors. Wider implications of the findings Results obtained in the present study show that ICSI evades the detrimental effects of sperm oxidative stress on fertilization and embryo development. Yet, sperm mitochondrial activity could be used as a biomarker to test the ability of a sperm donor to generate healthy embryos that develop up to blastocyst stage. Trial registration number Not applicable

P-804 Smchd1 is a key regulator of epigenetic cellular memory induced by morphine in mouse embryonic stem cells (mESC)

Abstract Study question Can morphine induce stable epigenetic changes that persist mitotically in mESC? Summary answer SMCHD1 is a key regulator of epigenetic cellular memory induced by morphine, whose deregulation can be memorized by mESC and upon embryo development. What is known already Epigenetic cellular memory induced by environmental factors implies the existence of mitotically heritable changes that influence gene expression without altering DNA sequence, leading to the propagation of heritable changes in phenotype. X chromosome inactivation (XCI) is an important epigenetic process, which takes places during embryo development and is maintained in the adulthood. Since XCI is part of normal development, changes induced by environmental factors might affect embryo development and lead to health problems. As morphine can easily pass through the placental barrier and reach the embryo, our aim is to elucidate whether morphine causes epigenetic regulation on XCI process. Study design, size, duration Epigenetic cellular memory induced by environmental factors implies the existence of mitotically heritable changes that influence gene expression without altering DNA sequence, leading to the propagation of heritable changes in phenotype. To evaluate if morphine can induce cellular epigenetic memory, we analyzed the dynamic epigenetic changes over the time in the absence of morphine. For that purpose, OCT4-reported mESCs were chronically treated with morphine during 24h (10-5 mM) Participants/materials, setting, methods • Transcriptomic analysis by RNA-Sequencing and gene expression validation by qRT-PCR. • Epigenetic analysis by H3K27me3 ChIP-Sequencing and WGBS-Sequencing. • Smchd1 gene silencing by Crisper-Cas9. Main results and the role of chance Morphine induced cellular epigenetic memory upon embryo development. mESCs can memorize morphine exposure and induces transcriptional changes that can persist over the time, even in the absence of morphine. RNA-seq has shown that morphine chronic treatment produces a transcriptional deregulation of 932 genes in P1 just after the treatment and this amount increased after treatment withdrawal (1196 genes, P2 and 2138 in P3). Integrative analyses between P1, P2 and P3 identifies Smchd1 gene as a potential regulator of epigenetic cellular memory induced by morphine. Crisper-Cas9 silencing analyses confirmed that Structural maintenance of chromosomes flexible hinge domain-containing 1 (Smchd1), an architectural factor critical for X-chromosome inactivation (XCI), is a morphine-sensitive target gene. Morphine reduced the gene expression of Smchd1 in mESC at different time points, which is consistent with an increase of H3K27me3 and DNA methylation at promoter. Morphine alters normal X chromosome wide silencing. Our results identified Smchd1 a key regulator of epigenetic cellular memory induced by morphine, whose deregulation can be memorized by the cell, providing an epigenetic mechanism that is maintained into cell-to-cell memory and cell differentiation process, which might cause diseases or health problems during the adulthood. Limitations, reasons for caution To perform the in-vitro analysis in mouse Wider implications of the findings Our results provide insights into the epigenetic mechanisms induced by morphine and establish the bases to understand how environmental factors can cause epigenetic changes that cause health problems or diseases, since a skewed XCI pattern is behind the higher incidence on different neurological diseases such as Alzheimer or Autism Trial registration number not applicable

P-186 Embryos developing within the optimal morphokinetic range have a 5% higher chance of leading to a Live birth (LB)

Abstract Study question Is there an optimal range of morphokinetic parameters that have a higher LB rate, if so what is the percentage of LB improvement? Summary answer Optimal embryo development range in morphokinetic parameters was identified in t6, t8, tB, t8-t4 and t8-t2 leading to a 5% improvement in LB. What is known already Achieving LB is the main objective in IVF. Traditional embryo assessment based on morphology has drawbacks, with variability among embryologists and limited predictive ability for a single-day assessment. The emergence of AI in IVF, exemplified by CHLOE-EQ, an embryologist’s tool to register embryo morphokinetics and other parameters, presents a solution. This study investigates whether AI-detected morphokinetic parameters can significantly improve LB rates, providing a more precise and predictive approach to embryo assessment. This study investigates if embryos that lead to LB develop under specific morphokinetic optimal ranges. Study design, size, duration Conducted across multiple centres within a private clinic, this retrospective study investigated 7156 embryos with known birth outcome between January 2021 and January 2023. Participants/materials, setting, methods Absolute (tPNf to tEB) and interim morphokinetic parameters that have been previously been found to impact embryo development (CC1, CC2, CC3, S2, S3, t5-t2, t8-t4, t8-t2, tEB-tSB) were automatically annotated by an AI model (CHLOE-EQ, Fairtility Ltd). The interquartile range (Q1-Q3) of embryos that resulted in live birth was analyzed to identify the optimal range. Chi-square analysis compared live birth rates between embryos within and outside this range. Main results and the role of chance A significant Live birth optimal range was found for t6 (47.8-57.3), t8 (51.8-65.8), tB (100.9-113.6), t8-t4 (15.2-25.5), t8-t2 (26.9-39.2), p &amp;lt; 0.05. The following morphokinetic parameters were associated with higher LB rate when the embryo was within the optimal range hours as compared to outside the optimal range: t6 (38%, n = 1012 vs 35% n = 1007, p &amp;lt; 0.05), t8 (38%, n = 947 vs 35%, n = 941, p &amp;lt; 0.05), tB (33%, n = 548 vs 30%, n = 533, p &amp;lt; 0.05), t8-t4 (38%, n = 926 vs 34%, n = 943, p &amp;lt; 0.05), t8-t2 (38%, n = 938 vs 35% n = 936, p &amp;lt; 0.05). No significant LB rate differences were observed for other morphokinetics. Overall, LB rates improved by 3-5% in embryos developing at the optimal rate, with a slightly higher significance in absolute morphokinetic values over interim ones. Limitations, reasons for caution The study’s retrospective nature entails inherent biases. Additionally, this analysis does not account for oocyte origin and male factor as bias elements, which will be considered in future research. Thus results may not be transferrable to clinics that have other clinical practices and different patient demographics. Wider implications of the findings Certain morphokinetic parameters can be added to the already existing range of available LB evaluation methods. Future studies should explore diverse morphokinetic ranges to identify additional optimal intervals for each parameter. AI tools can facilitate automatic annotation and measurement of embryo morphokinetics, enhancing precision in assessment. Trial registration number Not applicable

Zinc oxide nanoparticles induces cell death and consequently leading to incomplete neural tube closure through oxidative stress during embryogenesis

The implementation of Zinc oxide nanoparticles (ZnO NPs) raises concerns regarding their potential toxic effects on human health. Although more and more researches have confirmed the toxic effects of ZnO NPs, limited attention has been given to their impact on the early embryonic nervous system. This study aimed to explore the impact of exposure to ZnO NPs on early neurogenesis and explore its underlying mechanisms. We conducted experiments here to confirm the hypothesis that exposure to ZnO NPs causes neural tube defects in early embryonic development. We first used mouse and chicken embryos to confirm that ZnO NPs and the Zn2+ they release are able to penetrate the placental barrier, influence fetal growth and result in incomplete neural tube closure. Using SH-SY5Y cells, we determined that ZnO NPs-induced incomplete neural tube closure was caused by activation of various cell death modes, including ferroptosis, apoptosis and autophagy. Moreover, dissolved Zn2+ played a role in triggering widespread cell death. ZnO NPs were accumulated within mitochondria after entering cells, damaging mitochondrial function and resulting in the over production of reactive oxygen species, ultimately inducing cellular oxidative stress. The N-acetylcysteine (NAC) exhibits significant efficacy in mitigating cellular oxidative stress, thereby alleviating the cytotoxicity and neurotoxicity brought about by ZnO NPs. These findings indicated that the exposure of ZnO NPs in early embryonic development can induce cell death through oxidative stress, resulting in a reduced number of cells involved in early neural tube closure and ultimately resulting in incomplete neural tube closure during embryo development. The findings of this study could raise public awareness regarding the potential risks associated with the exposure and use of ZnO NPs in early pregnancy.Graphical

P-271 Deep sequencing of spermatozoal accessible chromatin reveals complex arrangements underlying spermatozoal chromatin packaging and fertilization

Abstract Study question Could structural aspects of sperm DNA packaging contribute to fertilization and early embryo development? Summary answer Structural aspects of sperm DNA include complex patterns of repetitive-DNA and developmental gene packaging that could delineate a paternal impact on fertilization and early development. What is known already Genetics, epigenetics and transcriptomics of ejaculate human spermatozoa could have important effects on the establishment of embryonic chromatin architecture that may impact early fetal development. Spermatozoa are transcriptionally silent; however, a small fraction (∼5%-15%) of their chromatin escapes repackaging by protamines and remains in a more open nucleosomal conformation that may be poised for transcription. The sensitivity of this fraction to progressive salt/detergent disruption is likely assayed in sperm chromatin dispersion (SCD) or halo tests that are commonly used in assessments of sperm DNA fragmentation. Study design, size, duration Eleven healthy male donors and 9 women undergoing IVF treatment agreed to participate in this study. Eleven normozoospermic semen samples were donated by male donors, aged 25-39 years, who abstained from sexual activity for 3 days. Seven MII oocytes and 5 pronuclear zygotes were donated from 5 and 4 women, respectively, undergoing IVF treatment. The samples were collected within a time frame of 6 months. Female donors provided either MII oocytes or pronuclear zygotes. Participants/materials, setting, methods Sperm chromatin was processed by differential salt extraction tailored with endonuclease or micrococcal nuclease treatment and RNA isolated using established protocols. Oocytes and pronuclear zygotes were analyzed at the single cell level. DNA library construction employed the NEBNext Ultra approach (NEB) and RNA libraries the Ovation Single Cell (NuGEN) for Illumina. Bioinformatics analysis included FastQC, HISAT2, StringTie, GREAT, edgeR and other source-software. Public datasets were employed to further increase the statistical power. Main results and the role of chance The aim of our approach was to dissect the accessible sperm chromatin fraction, identifying regions of great importance that could contribute towards the formation of the zygote and/or early embryonic development. Differing extraction methods revealed complementary accessible and resistant fractions in sperm, with accessible regions being significantly enriched for chromatin important in very early embryonic development. These chromatin fractions contained various genomic features including promoter sequences, CpG-islands and CTCF-binding sites that could attract transcription binding factors in the paternally-derived chromatin. Accessible sperm chromatin also contains high levels of specific DNA repeat classes of retroviral origin, known to impact early gene expression and gene expression programs. Accessible sperm chromatin was strongly associated with developmental and regulatory signatures with Bonferroni P values ranging between 2.07x10-4 and 1.03x10-20, potentially revealing a role for paternal chromatin in the initial start-up of early zygotic development. We further investigated paternally- and maternally- derived transcripts and identified mono-allelically transcribed genes present in the zygotes, that were entirely absent from all seven oocyte transcriptomes, suggesting a paternal origin, and potentially important roles following oocyte fertilization. Limitations, reasons for caution Due to the restraints of obtaining human material, the study relied on a relatively small sample size. However, sequencing results showed quite homogeneous clustering in both DNA/RNA samples, with at least 5 biological replicates from at least 4 donors per group, potentially reducing the impact of low sample size. Wider implications of the findings A better understanding of differentially accessible sperm chromatin and residual RNA, could help support the discovery of biomarkers that can be used to select more competent spermatozoa for IVF cycles, resulting in higher success rates in couples undergoing ART. Trial registration number not applicable

Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos.

P-146 The importance of cytoplasmic strings between inner cell mass and trophectoderm after blastulation on embryonic development and pregnancy outcome

Abstract Study question Whether the cytoplasmic strings between inner cell mass and trophectoderm during blastocyst expansion have a significant impact on embryo development and pregnancy outcomes? Summary answer The presence of cytoplasmic strings is related to morphological quality, but it has no impact on embryo ploidy or clinical outcome after euploid embryo transfer. What is known already Cytoplasmic strings are a dynamic cellular structure, and their width and length vary with the degree of embryonic expansion. The presence of cytoplasmic strings is considered a characteristic of embryos with poor vitality. In recent years, researchers have found the presence of cytoplasmic strings may indicate a higher embryonic developmental potential. However, the specific function of cytoplasmic strings remains unclear, and their impact on embryonic development is still controversial. Study design, size, duration A retrospective cohort analysis was conducted on clinical data of 530 patients who underwent single blastocyst transfer after pre-implantation genetic testing for aneuploidy (PGT-A) in our center from June 2019 to December 2021. Two embryologists annotated the presence of cytoplasmic strings, the morphological quality of blastocyst. Participants/materials, setting, methods A total of 2132 biopsied blastocysts were obtained from the 530 patients, with 534 blastocysts having cytoplasmic strings (+) and 1598 blastocysts having cytoplasmic strings (−). The quality and ploidy differences between the two groups were compared. After PGT-A, single euploid blastocysts were transferred, and the difference in pregnancy outcomes between cytoplasmic strings (+) (n = 115) and cytoplasmic strings (−) (n = 415) cycles was compared. Main results and the role of chance There was no significant difference in the euploidy rate between the two groups (44.6% vs. 45.3%). As the morphological quality of the embryos decreased, the rate of cytoplasmic strings (+) significantly decreased (Good-quality vs. Average-quality: 30.19% vs. 24.62%, P = 0.008; Average-quality vs. Low-quality: 24.62 vs. 12.63, P = 0.000). After adjusting for patient age, anti-Müllerian hormone, and gonadotropin dose, multivariate regression analysis showed that cytoplasmic strings were associated with embryo quality, but not with embryo ploidy. There were no significant differences in implantation rate (72.17% vs. 66.02%, P = 0.213), miscarriage rate (10.43% vs. 8.43%, P = 0.504), and live birth rate (61.74% vs. 57.59%, P = 0.424) between blastocysts with cytoplasmic strings (+) and without cytoplasmic strings (−). Limitations, reasons for caution Larger datasets are required to confirm putative associations between cytoplasmic strings and blastocyst ploidy, and the pregnancy outcome. Wider implications of the findings The presence of cytoplasmic strings is related to the embryological quality of the blastocyst, but it has no impact on embryo ploidy or clinical outcome after euploid embryo transfer. Trial registration number not applicable