Human Preimplantation Embryo Development Research Articles

P-073 Comparison of human preimplantation embryo development after sperm selection for ICSI using the microfluidic ZyMōt device versus density gradient centrifugation

Abstract Study question Does microfluidic sperm selection using ZyMōt device has a significant effect on blastocyst development, ploidy, and improves pregnancy outcomes when compared with density-gradient centrifugation. Summary answer The study demonstrated that sperm selection using a microfluidic device had no impact on euploid blastocyst rates or clinical pregnancy outcomes. What is known already Selecting competent spermatozoa with the highest genomic integrity for ICSI is a key to achieving normal embryo development and a healthy live birth. Several sperm preparation techniques have long been scrutinized with varied success. The novel microfluidic ZyMōt device appears promising for improving the quality of sperm used in Assisted Reproductive Technologies. Preliminary tests have shown that ZyMōt may increase sperm motility and considerably decrease DNA fragmentation, which could potentially enhance euploid blastocyst development rates. However, the impact of this sperm selection method on clinical outcomes has not been fully investigated. Study design, size, duration This is a retrospective cohort study performed in a single academic IVF centre (IRB # 16367). Data were collected from 1286 ICSI cycles in 2020-2021 (730 with ZyMōt and 556 with DGC), including 867 cycles with PGT-A outcomes. Morphological grading of 6640 blastocysts cultured in a time lapse imaging incubator (Embryoscope™) and ploidy of 4783 blastocysts using high resolution NGS (Illumina platform and Blue Gnome) were compared between the two study groups. Participants/materials, setting, methods All male partners were normo-spermic according to standard semen analysis. ICSI results for a total of 7277 mature oocytes injected with spermatozoa processed with ZyMōt device and 5977 oocytes injected with spermatozoa processed by gradient-density centrifugation were compared. For the analysis of 1478 euploid embryo transfer outcomes, all cases included a single blastocyst transferred in a frozen-thaw cycle. Associated patient characteristics, including age and fertility diagnosis were collected. Main results and the role of chance A significantly higher fertilization rate was observed with ZyMōt compared to the gradient prepared sperm (83.4% vs 81.0%, P = 0.0004, respectively). The blastocyst rates (61.3%, vs 60.4%, P = 0.34) as well as the proportion of high-quality blastocysts were similar in both groups (P = 0.20). Euploidy rates were not different between all female age groups, including young egg donors (≤ 30y of age) to patients with advanced maternal age (>40 y of age). Microfluidic sperm selection did not improve clinical pregnancy rates after euploid embryo transfer (42.9% vs 41.4%; P = 0.58). Limitations, reasons for caution Although this is the largest study to date evaluating application of the ZyMōt device, the retrospective nature and cohort design limited the interpretation of our results. The patient population did not include male factor cases and sperm DFI data was not available. Embryo transfer outcomes were limited to clinical pregnancies. Wider implications of the findings The ZyMōt device offers a convenient alternative to standard sperm processing by density gradient method. Further studies are needed to fully determined the impact of ZyMōt on embryo quality and clinical outcomes in different age and diagnosis groups of patients; particularly for those with high DFI and poor IVF/ICSI outcomes. Trial registration number not applicable

Evaluate the developmental competence of human 8-cell embryos by single-cell RNA sequencing.

The transition of maternal to zygotic gene expression regulation is critical for human preimplantation embryo development. In recent years, single-cell RNA sequencing (scRNA-seq) had been applied to detect the factors that regulate human oocyte maturation and early embryo development. Here, the evaluation of transcriptomes in single blastomere from the embryo collected from patients by scRNA-seq was performed. There were 20 blastomeres biopsied from 8-cell embryos of seven patients who received more than two ART cycles due to low embryo competence. Meanwhile, ten cells were collected from 8-cell embryos of four patients who received ART treatment due to male or tubal factors. The blastomeres were then evaluated using the previously established scRNA-seq method to determine the associations between their gene expression and developmental competence. The total number of genes detected in 8-cell embryos that failed to form blastocyst including maternal and zygotic mRNAs was reduced. There were 324 differently expressed genes detected among the 8-cell embryos including 65 genes that were significantly suppressed in the 8-cell embryos that failed to form blastocyst. Further analysis found these 8-cell embryos arrested at the cleavage stage due to the dysfunction of the cell cycle, DNA transcription activity, histone methylation, and cell division-related genes such as SMCO-1, ZNF271P,ZNF679, ASF1b, BEX3, DPPA2, and ORC4. The alterations of gene expression detected in human 8-cell embryos are tightly associated with its developmental competence and could be used as targets to enhance embryo development or parameters to predict the embryo's development outcomes. Many females are suffering infertility due to the failure of embryonic development at early stages due to unknown causes. At the very beginning of human embryo development, the embryos start to express its own genes, which should be achieved at 8-cell stage. In current research, we isolated one cell from 8-cell embryos and detected the gene expression at single-cell level. Then the remaining cells of these embryos were cultured to form blastocyst. Meanwhile, the data was analyzed according to the outcomes of embryo development. We detected 324 differently expressed genes between the 8-cell embryos that succeeded and failed to form blastocyst. Our research showed the association between the gene expression and the developmental competence of 8-cell embryos. The findings could be used to predict the embryo quality and potential therapy target to improve the efficiency of assisted reproductive techniques.

Assisted oocyte activation does not overcome recurrent embryo developmental problems.

Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. NCT03354013.

P-253 Shorter duration of compaction during human in-vitro preimplantation embryo development is associated with a higher clinical pregnancy rate

Abstract Study question Is the duration of compaction, KID- (Known Implantation Data) and iDAScore (Intelligent Data Analysis for embryo evaluation) associated to clinical pregnancy rate? Summary answer Duration of compaction, KID- or iDAScore can be used to select the best embryo for transfer to increase clinical pregnancy rate. What is known already The development of human preimplantation embryos follows a programmed timeline in which a series of critical events occur. Compaction is a typical event at 3 to 4 days post fertilisation that is characterised by flattening of the blastomeres and the formation of tight junctions between the blastomeres. It is considered the first morphological event in the differentiation process of the embryo. Time-lapse technology introduced continuous monitoring of the embryo throughout development in the IVF laboratory. Evaluation of the developmental events combined with calculating KID- and iDAScore can optimise the selection of the most competent embryos for transfer and/or cryopreservation. Study design, size, duration This single-centre retrospective observational study included 158 IVF/ICSI cycles with fresh single embryo transfer (SET) was performed between December 2018 and November 2021. Embryos were cultured during 5 days in cleavage/blastocyst medium (Coopersurgical) in the EmbryoScope + (Vitrolife). Transferred embryos were evaluated for start of compaction, time to full compaction and duration of compaction. Embryo quality was calculated using KID- and iDAScore. These parameters were compared between the clinically pregnant and non-pregnant group (primary outcome). Participants/materials, setting, methods Only IVF/ICSI cycles with ejaculated sperm and fresh SET on day 5 were included. MNC, IVM and PGT cycles were excluded. Time zero was the start of injection or insemination. Pregnancy was confirmed by hCG and clinical pregnancy was defined by gestational sac visualisation at ultrasound. GraphPad Prism and R-studio were used for statistical analysis. For prediction of clinical pregnancy, univariate logistic regression was used. Other significant differences were determined using t-test. Main results and the role of chance Out of 158 fresh ET, 101 (63.9%) had a positive hCG, of which 88 (55.6%) achieved clinical pregnancy. All 158 transferred blastocysts were annotated to calculate KID- and iDAScore. There was no statistical difference in age between the two groups (34.7 years vs 35.0 years; p = 0.69). Start of compaction was heterogeneous (between 50.9 and 98.3 hours post injection/insemination; mean=76.5±7.7), as well as the blastomere number at its initiation (between 4 and 16 blastomeres; mean=11.8±2.1). Univariate logistic regression showed that each individual parameter, i.e. duration of compaction (p = 0.02), KID-score (p = 0.001) and iDAScore (p = 0.0006) was different between the clinically pregnant and non-pregnant group. The total duration of compaction was significantly shorter in the clinical pregnant group (mean=8.6±3.4 hours vs 10.2±4.7 hours; p = 0.01; t-test). In the pregnant group the KIDscore (mean=7.7±1.4 vs 6.7±2.3; p = 0.0007) and iDAScore (mean=8.9±0.7 vs 8.3±1.3; p = 0.0002) were significantly higher. During partial compaction, cells were rather excluded (93%) than extruded from the process; 17 embryos underwent this process, 10 of which resulted in a clinical pregnancy. Limitations, reasons for caution As this is a retrospective study, the influence of uncontrolled variables cannot be excluded. In the future, different models will be applied that can combine duration of compaction, KID- and iDAScore in a larger study. Wider implications of the findings Our analysis confirms previous findings that KID- and iDAScore are good predictors of clinical pregnancy. We also show that duration of compaction can be used as a potential predictor for pregnancy, especially in IVF clinics that have no access to KID- or iDAScore. Trial registration number not applicable

O-234 The guinea pig embryo: a potential new model for human development

Abstract Study question Is the guinea pig a good model for human preimplantation embryo development? Summary answer Preimplantation guinea pig embryos better recapitulate the human embryo compared to the mouse, offering a promising new model to study preimplantation, naive pluripotency and infertility. What is known already Mouse embryos have historically been used to model reproduction, but caution is warranted as several discrepancies with the human exist during preimplantation. Guinea pigs have been a long standing model of human development and are known to better recapitulate placentation and brain development compared to the mouse. We now speculate that guinea pigs may also represent a superior animal model to study preimplantation development. However, to date, guinea pig preimplantation embryos remain to be investigated in great detail. We now aim to assess the developmental dynamics of the guinea pig preimplantation embryo with cross-species comparisons to both human and mouse. Study design, size, duration In vivo guinea pig embryos were flushed 3-6 days post-fertilization (N = 10-12/ time point). Embryos (embryonic day (E)3-6 were collected and processed for downstream experiments. Participants/materials, setting, methods Immunofluorescence of Sox2 (epiblast (EPI) marker), Sox17 and Gata6 (primitive endoderm (PE) marker) and Cdx2 and Gata3 (trophectoderm (TE)) was performed. Cells were counted and the dynamics of lineage marker expression and blastocyst formation were determined. In parallel, scRNA-seq (Smartseq2) of guinea pig embryos was performed. Main results and the role of chance At the compacted morula stage (late E4.25-E4.75), similar to the human and in contrast to the mouse, we observe co-expression Sox2, Gata6 and Gata3, with no detection of Sox17 and Cdx2. Similar to the human, blastocyst formation begins between E5.0-E5.25. By mid-blastocysts (E5.25-E5.5), cells are poised for lineage specification with a high number of cells co-expressing Sox2/Sox17, and no cells co-expressing markers of all three lineages. By late blastocyst, E5.5-E5.75, specification of all three lineages is achieved and no co-expression Sox2/Sox17/Cdx2 or Sox2/Gata6/Gata3 is observed. Further, similar to the human and in contrast to the mouse, there is a disconnect between morphology and lineage specification in the early blastocyst. These data suggest that signaling pathways governing lineage specification in the human and guinea pig differ compared to the mouse. Finally, cross-species analysis of scRNA-seq revealed conserved pluripotency gene signatures between the human and guinea pig embryos, suggesting that the guinea pig may also contain naive stem cells and that plasticity of these lineages may be more similar to the human compared to the mouse. Limitations, reasons for caution Our model only explores similarities/differences between the human and guinea pig preimplantation embryo with regards to timing of development and lineage segregation. Wider implications of the findings In addition to helping us understand human preimplantation development, this new model may serve to examine the longer-term consequences of preimplantation exposures. Further, insights from this study have broad application in the fields of Reproduction, Development, ART and Stem cell biology. Trial registration number NA

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

Biallelic variants in ZFP36L2 cause female infertility characterised by recurrent preimplantation embryo arrest

BackgroundRecurrent preimplantation embryo developmental arrest (RPEA) is the most common cause of assisted reproductive technology treatment failure associated with identified genetic abnormalities. Variants in known maternal genes can only account...

KNOCKDOWN OF Dnmt1 AND Dnmt3a ENZYMES DISRUPTS PREIMPLANTATION EMBRYO DEVELOPMENT BY AFFECTING GLOBAL DNA METHYLATION

DNA methylation is one of the epigenetic mechanisms that play critical roles in preimplantation embryo development executed by DNA methyltransferase (Dnmt) enzymes. Dnmt1, responsible for the maintenance of global DNA methylation and Dnmt3a for the de novo methylation, are gradually erased from zygote to blastocyst stage by a demethylation process, and then reestablished by de novo methylation around blastocyst stage. This important differentiation mechanism indicates pivotal roles of global DNA methylation during the course of preimplantation embryo development. So, a fundamental question which also served as the aim of the current study was to address the spatio-temporal expression of Dnmt1 and Dnmt3a and thus the establishment of DNA methylation could affect the embryo development. We set up four groups as control, Dnmt1 siRNA, Dnmt3a siRNA, and non-targeted siRNA. In vivo developed Balb/c mouse zygotes were collected, divided into groups and then cultured for 96 h to reach blastocyst stage. Zygotes were transfected with 50 nM Dnmt1 and Dnmt3a-spesific small interfering RNA (siRNA) duplexes vs non-targeting control siRNA duplexes for 96 h using DharmaFECT as a transfection reagent. Nontargeting siRNA duplex served as the negative control. Knockdown of Dnmt genes using siRNA was confirmed by analyzing the proteins using immunofluorescence and Western blot techniques after 96 h in culture. Following knockdown of Dnmt1 and Dnmt3a, we analyzed the developmental competence of embryos. We also evaluated global DNA methylation levels by 5 methylcytosine staining in blastocyst stage. We found that both Dnmt genes were successfully knocked-down (approximately 80%) in 2-4 cell embryos. In Dnmt1 siRNA group, embryo arrest rates were 29.5% in 2-cell, 15.7 in %4-cell, and 9.8% in morula stages. Only 16.7% embryos reached to blastocyst stage in this group. In Dnmt3a siRNA group, embryos arrested in 26.2% 2-cell, 21.9% 4-cell, 9.2% morula and 29.5% embryos reached to blastocyst stage. Degenerated embryo rates were 28.2% in Dnmt1 siRNA, and 13.1% in Dnmt3a siRNA group. Interestingly, we noted that global DNA methylation level significantly decreased in Dnmt1 knockdown group while increased in Dnmt3a knockdown group (P<0.001). These results suggested that temporal Dnmt enzymes could contribute to the process of early embryonic development and thus play vital roles in the mouse embryos. Therefore it seems evident that these enzymes should also be elucidated in human to check whether they play any major role in the human preimplantation embryo development.

Inferring simple but precise quantitative models of human oocyte and early embryo development.

Macroscopic, phenomenological models are useful as concise framings of our understandings in fields from statistical physics to finance to biology. Constructing a phenomenological model for development would provide a framework for understanding the complicated, regulatory nature of oogenesis and embryogenesis. Here, we use a data-driven approach to infer quantitative, precise models of human oocyte maturation and pre-implantation embryo development, by analysing clinical in-vitro fertilization (IVF) data on 7399 IVF cycles resulting in 57 827 embryos. Surprisingly, we find that both oocyte maturation and early embryo development are quantitatively described by simple models with minimal interactions. This simplicity suggests that oogenesis and embryogenesis are composed of modular processes that are relatively siloed from one another. In particular, our analysis provides strong evidence that (i) pre-antral follicles produce anti-Müllerian hormone independently of effects from other follicles, (ii) oocytes mature to metaphase-II independently of the woman's age, her BMI and other factors, (iii) early embryo development is memoryless for the variables assessed here, in that the probability of an embryo transitioning from its current developmental stage to the next is independent of its previous stage. Our results both provide insight into the fundamentals of oogenesis and embryogenesis and have implications for the clinical IVF.

Time-lapse imaging of human embryos fertilized with testicular sperm reveals an impact on the first embryonic cell cycle.

Testicular sperm is increasingly used during in vitro fertilization treatment. Testicular sperm has the ability to fertilize the oocyte after intracytoplasmic sperm injection (ICSI), but they have not undergone maturation during epididymal transport. Testicular sperm differs from ejaculated sperm in terms of chromatin maturity, incidence of DNA damage, and RNA content. It is not fully understood what the biological impact is of using testicular sperm, on fertilization, preimplantation embryo development, and postimplantation development. Our goal was to investigate differences in human preimplantation embryo development after ICSI using testicular sperm (TESE-ICSI) and ejaculated sperm. We used time-lapse embryo culture to study these possible differences. Embryos (n = 639) originating from 208 couples undergoing TESE-ICSI treatment were studied and compared to embryos (n = 866) originating from 243 couples undergoing ICSI treatment with ejaculated sperm. Using statistical analysis with linear mixed models, we observed that pronuclei appeared 0.55 h earlier in TESE-ICSI embryos, after which the pronuclear stage lasted 0.55 h longer. Also, significantly more TESE-ICSI embryos showed direct unequal cleavage from the 1-cell stage to the 3-cell stage. TESE-ICSI embryos proceeded faster through the cleavage divisions to the 5- and the 6-cell stage, but this effect disappeared when we adjusted our model for maternal factors. In conclusion, sperm origin affects embryo development during the first embryonic cell cycle, but not developmental kinetics to the 8-cell stage. Our results provide insight into the biological differences between testicular and ejaculated sperm and their impact during human fertilization.

Expression of Retroelements in Mammalian Gametes and Embryos.

Retroelements are genetic mobile elements, expressed during male and female gamete differentiation. Retrotransposons are normally regulated by the methylation machinery, chromatin modifications, non-coding RNAs, and transcription factors, while retrotransposition control is of vital importance in cellular proliferation and differentiation process. Retrotransposition requires a transcription step, by a cellular RNA polymerase, followed by reverse transcription of an RNA intermediate to cDNA and its integration into a new genomic locus. Long interspersed elements (LINEs), human endogenous retroviruses (HERVs), short interspersed elements (SINEs) and SINE-VNTR-Alu elements (SVAs) constitute about half of the human genome, play a crucial role in genome organization, structure and function and interfere with several biological procedures. In this mini review, we discuss recent data regarding retroelement expression (LINE-1, HERVK-10, SVA and VL30) and retrotransposition events in mammalian oocytes and spermatozoa, as well as the importance of their impact on human and mouse preimplantation embryo development.

Cytoplasmic halo characteristics during fertilization and their implications for human preimplantation embryo development and pregnancy outcome.

Is the spatiotemporal phenomenology of the cytoplasmic halo during fertilization related to embryonic competence? Time-lapse images from 1009 zygotes were retrospectively analysed from 560 patients who underwent IVF with minimal stimulation and single vitrified-warmed blastocyst transfer between April 2017 and March 2018. Halo presence and morphokinetics were monitored and compared relative to embryo quality, blastocyst expansion and ongoing pregnancy. Halo was observed in 88% of fertilized oocytes. Embryos derived from zygotes without halo had significantly higher rates of rapid cleavage (P = 0.0004), cell fusion (P = 0.0028) and asymmetrical division (P = 0.0002) compared with those derived from zygotes with halo. Multivariate logistic regression analysis had significantly higher developmental rates compared with the expanded blastocyst stage in embryos displaying a halo, regardless of its distribution (adjusted odds ratio 0.435; P = 0.0004). Prolonged halo time intervals were significantly correlated with increased asymmetrical division at first cell division (P = 0.0412, P = 0.0088, respectively) and decreased developmental rates to expanded blastocyst stage (P = 0.0062, P = 0.0020, respectively). Additionally, prolonged presence of the cytoplasmic halo was associated with a decreased ongoing pregnancy rate (adjusted odds ratio 0.871; P = 0.006). Poor sperm quality and decreased oocyte diameter were correlated with absence of the cytoplasmic halo (P = 0.0477, P < 0.0001, respectively) or prolonged halo presence (P = 0.0139, P = 0.0002, respectively). Halo presence and morphokinetics are associated with cleavage patterns, development to blastocyst stage and ongoing pregnancy rate after single blastocyst transfer. Halo morphokinetics seems to reflect sperm and oocyte quality. Cytoplasmic halo might be valuable predictor for refining selection of more developmentally competent blastocysts.

Effects of thyroid hormone on mitochondria and metabolism of human preimplantation embryos.

Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10−7 M triiodothyronine (T3) till blastocyst stage. Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0v2018 JUN platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes—10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes—0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.

C-kit signaling promotes human pre-implantation 3PN embryonic development and blastocyst formation

BackgroundAlthough in vitro culture system has been optimized in the past few decades, the problem of few or no high quality embryos has been still not completely solved. Accordingly, fully understanding the regulatory mechanism of pre-implantation embryonic development would be beneficial to further optimize the in vitro embryo culture system. Recent studies have found the expression of c-kit in mouse embryo and its promotion effects on mouse embryonic development. However, it is unclear the expression, the role and the related molecular regulatory mechanism of c-kit in human pre-implantation embryo development. Therefore, the present study is to determine whether c-kit is expressed in human pre-implantation embryos, and to investigate the possible regulatory mechanism of c-kit signaling in the process of embryonic development.MethodsThe present study includes human immature oocytes and three pronucleus (3PN) embryos collected from 768 women (28–32 ages) undergoing IVF, and normal 2PN embryos collected from ICR mice. Samples were distributed randomly into three different experimental groups: SCF group: G-1™ (medium for culture of embryos from the pro-nucleate stage to day 3) or G-2™ (medium for culture of embryos from day3 to blastocyst stage) + HSA (Human serum album) solution + rhSCF; SCF + imanitib (c-kit inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + imanitib; SCF + U0126 (MEK/ERK inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + U0126; Control group: G-1™ or G-2™ + HSA solution + PBS; The rate of good quality embryos at day 3, blastulation at day 6 and good quality blastulation at day 6 were analysis. RT-PCR, western blot and immunofluorescence staining were applied to detect the target genes and proteins in samples collected from human or mice, respectively.Resultsc-kit was expressed ubiquitously in all human immature oocytes, 3PN embryos and 3PN blastocysts. In the experiment of human 3PN embryos, compared with other groups, SCF group showed obviously higher rate of good quality at day 3, better rate of blastocyst formation at day 6 and higher rate of good quality blastocyst formation at day 6. Furthermore, we observed a higher ETV5 expression in SCF group than that in other groups. Similar results were also found in animal experiment. Interestingly, we also found a higher phosphorylation level of MEK/ERK signal molecule in mice embryos from SCF group than those from other groups. Moreover, inhibition of MEK/ERK signaling would remarkably impeded the mice embryonic development, which might be due to the reduced ETV5 expression.ConclusionsThe present study firstly revealed that c-kit signaling might promote the human pre-implantation embryonic development and blastocyst formation by up-regulating the expression of ETV5 via MEK/ERK pathway. Our findings provide a new idea for optimizing the in vitro embryo culture condition during ART program, which is beneficial to obtain high quality embryos for infertile patients.

Elevated Exogenous Pyruvate Potentiates Mesodermal Differentiation through Metabolic Modulation and AMPK/mTOR Pathway in Human Embryonic Stem Cells.

SummaryPyruvate is a key metabolite in glycolysis and the tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell-fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance toward oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA-cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism but also modulates signaling pathways in hESC differentiation.

Genome editing in human embryos: ethical concerns and practical applications

As genome editing techniques are developed, they have been applied to somatic cells in clinical use. Regarding the adaptation of these techniques in human zygotes, advances in single-cell analysis technology are expected to enhance knowledge at the molecular level, even in human preimplantation embryo development, and the application of genome editing technology for molecular functional analysis is expected. However, the use of genetic modification techniques on germlines, including zygotes, spreads to all cells of an individual and is subsequently passed down through generations. Here we explore the possibilities and challenges of applying genome editing techniques to better understand the scientific aspects of the human germline.

MiR-21 in extracellular vesicles contributes to the growth of fertilized eggs and embryo development in mice.

Human preimplantation embryo development is susceptible to high rates of early embryo wastage. We determined the miR-21 expression of extracellular vesicles (EVs) in fertilized eggs and embryos of varying stages and their response to miR-21 microinjection. Sexually mature female and male mice were mated. Next, the expression and immunohistochemistry intensity of surface markers (CD9 and CD63) of EVs were detected in pregnant and non-pregnant mice. Exosomes were co-cultured with embryos for detection of blastocyst formation rate, and embryo apoptosis. Moreover, the expressions of Bcl-2 associated X protein (Bax), B cell lymphoma 2 (Bcl-2), and octamer-binding transcription factor-4 (Oct4) were determined. Finally, we detected miR-21 expression in EVs of uterus in pregnant mice, in embryos after embryo implantation and after embryo co-cultured with exosomes in uterine luminal fluid. MiR-21 was up-regulated in EVs of uterus, and higher immunohistochemistry intensity of CD9 and CD63, suggesting more EVs secreted in uterine luminal fluid in pregnant mice. After microinjection, miR-21 inhibitor suppresses embryo development of mice. Moreover, embryos co-cultured with exosomes display higher blastocyst formation rate, reduced apoptotic rate of embryos in pregnant mice. In addition, miR-21 was down-regulated with the development of embryos after embryo implantation, while miR-21 expression in embryos was up-regulated by exosomes in uterine luminal fluid in the pregnant mice. Increased miR-21 expression in EVs of uterus and increased miR-21 expression after implantation, which indicate the key role in the growth of fertilized eggs and embryo development in mice.

Transcriptome Profiling of &lt;em&gt;In-Vivo&lt;/em&gt; Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Characterization and target genes of nine human PRD-like homeobox domain genes expressed exclusively in early embryos.

PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development.