Blastomere Cells Research Articles

Identifying key predictive features for live birth rate in advanced maternal age patients undergoing single vitrified-warmed blastocyst transfer

BackgroundInfertility affects one in six couples worldwide, with advanced maternal age (AMA) posing unique challenges due to diminished ovarian reserve and reduced oocyte quality. Single vitrified-warmed blastocyst transfer (SVBT) has shown promise in assisted reproductive technology (ART), but success rates in AMA patients remain suboptimal. This study aimed to identify and refine predictive factors for live birth following SVBT in AMA patients, with the goal of enhancing clinical decision-making and enabling personalized treatment strategies.MethodsThis retrospective cohort study analyzed 1,168 SVBT cycles conducted between June 2016 and December 2022 at the First Affiliated Hospital of Guangxi Medical University and Nanning Maternity and Child Health Hospital. Nineteen machine-learning models were applied to identify key predictive factors for live birth. Feature selection and 10-fold cross-validation were employed to validate the models.ResultsThe most significant predictors of live birth included inner cell mass quality, trophectoderm quality, number of oocytes retrieved, endometrial thickness, and the presence of 8-cell blastomeres on day 3. The stacking model demonstrated the best predictive performance (AUC: 0.791), followed by Extra Trees (AUC: 0.784) and Random Forest (AUC: 0.768). These models outperformed traditional methods, achieving superior accuracy, sensitivity, and specificity.ConclusionLeveraging advanced machine-learning models and identifying critical predictive factors can improve the accuracy of live birth outcome predictions for AMA patients undergoing SVBT. These findings offer valuable insights for enhancing clinical decision-making and managing patient expectations. Further research is needed to validate these results in larger, multi-center cohorts and to explore additional factors, including fresh embryo transfers, to broaden the applicability of these models in clinical practice.

Effect of blastomere cell number on ART outcome of fresh single day 3 embryo transfer

PurposeExplore the effect of blastomere cell number on ART outcome of fresh embryo transfer on day 3.MethodsRetrospective analysis of 540 fresh single day 3 embryo transfer cycles at the Reproductive Center of the Third Affiliated Hospital of Guangzhou Medical University from January 1, 2018 to October 31, 2022. Patients were divided into 5–6 cell group (n = 55), 7–9 cell group (n = 457), and ≥ 10 cell group(n = 28) based on the number of blastomeres. Single factor analysis of variance and Pearson's chi square test were used to compare the basic data, cycle information, pregnancy outcome and neonatal outcome. Univariate logistic regression was used to correct for confounding factors and analyze the influencing factors of pregnancy outcome.ResultsThe positive HCG rate were 20%, 43%, 25% for the 5–6-cell, 7–9 cell and ≥ 10 cell groups respectively, with statistically significant differences (P < 0.001). The clinical pregnancy rate was 18%, 42%,21%, respectively (P < 0.001). The live birth rates were 13%, 34%,21% with P-value less than 0.05 which is statistically significant. In order to exclude the influence of confounding factors, multivariable logistic regression analysis was performed, and the outcomes were consistent with previous findings. There were no significant differences found in neonatal outcome between groups (P > 0.05).ConclusionThe results suggested that intermediate cleaving embryos (7–9 cell) still presents the highest clinical potential. Fast and slow cleaving embryos are not conducive to the ART outcome.

Proteome asymmetry in mouse and human embryos before fate specification.

Pre-patterning of the embryo, driven by spatially localized factors, is a common feature across several non-mammalian species 1-4 . However, mammals display regulative development and thus it was thought that blastomeres of the embryo do not show such pre-patterning, contributing randomly to the three lineages of the blastocyst: the epiblast, primitive endoderm and trophectoderm that will generate the new organism, the yolk sac and placenta respectively 4-6 . Unexpectedly, early blastomeres of mouse and human embryos have been reported to have distinct developmental fates, potential and heterogeneous abundance of certain transcripts 7-12 . Nevertheless, the extent of the earliest intra-embryo differences remains unclear and controversial. Here, by utilizing multiplexed and label-free single-cell proteomics by mass-spectrometry 13 , we show that 2-cell mouse and human embryos contain an alpha and a beta blastomere as defined by differential abundance of hundreds of proteins exhibiting strong functional enrichment for protein synthesis, transport, and degradation. Such asymmetrically distributed proteins include Gps1 and Nedd8, depletion or overexpression of which in one blastomere of the 2-cell embryo impacts lineage segregation. These protein asymmetries increase at 4-cell stage. Intriguingly, halved mouse zygotes display asymmetric protein abundance that resembles alpha and beta blastomeres, suggesting differential proteome localization already within zygotes. We find that beta blastomeres give rise to a blastocyst with a higher proportion of epiblast cells than alpha blastomeres and that vegetal blastomeres, which are known to have a reduced developmental potential, are more likely to be alpha. Human 2-cell blastomeres also partition into two clusters sharing strong concordance with clusters found in mouse, in terms of differentially abundant proteins and functional enrichment. To our knowledge, this is the first demonstration of intra-zygotic and inter-blastomere proteomic asymmetry in mammals that has a role in lineage segregation.

BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells

ABSTRACT Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.

Capturing totipotency in human cells through spliceosomal repression

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures invitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine invitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of invitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve invitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

Blastomere size in the human 2-cell embryo predicts the division order that leads to imbalanced lineage contribution to the future body.

Retrospective tracing of somatic mutations predicted that most cells in the human body could be traced back to a single cell of the 2-cell stage embryo. Accordingly, a recent prospective study of the developmental trajectory of blastomeres in human embryos confirmed that progeny of the first 2-cell stage blastomere to divide generates more epiblast cells (future body). How the 2-cell blastomeres differ is unknown. Here, we show that 2-cell stage blastomeres in human embryos are asymmetric; they differ in size and the bigger blastomere divides first to 4-cell stage. We propose that this asymmetry might originate differences in cell fate.

Blastomeres of 8-cell mouse embryos differ in their ability to generate embryonic stem cells and produce lines with different transcriptional signatures.

Embryonic stem cell (ESC) derivation from single blastomeres of 8-cell mouse embryos results in lower derivation rates than that from whole blastocysts, raising a biological question about the developmental potential of sister blastomeres. We aimed to assess the ability of 8-cell blastomeres to produce epiblast cells and ESC lines after isolation, and the properties of the resulting lines. Our results revealed unequal competence among sister blastomeres to produce ESC lines. At least half of the blastomeres possess a lower potential to generate ESCs, although culture conditions and blastomeres plasticity can redirect their non-pluripotent fate towards the epiblast lineage, allowing us to generate up to seven lines from the same embryo. Lines originated from the same embryo segregated into two groups according to their transcriptional signatures. While the expression of genes related to pluripotency and development was higher in one group, no differences were found in their trilineage differentiation ability. These results may help to improve our understanding of the ESC derivation process from single blastomeres and cell fate determination in the preimplantation mouse embryos.

Tris(1,3-dichloro-2-propyl) Phosphate Inhibits Early Embryonic Development by Binding to Gsk-3β Protein in Zebrafish

Recently, several studies have reported that exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) results in abnormal development of zebrafish embryos in blastocyst and gastrula stages, but molecular mechanisms are still not clear. This lacking strongly affects the interspecific extrapolation of embryonic toxicity induced by TDCIPP and hazard evaluation. In this study, zebrafish embryos were exposed to 100, 500 or 1000 μg/L TDCIPP, and 6-bromoindirubin-3′-oxime (BIO, 35.62 μg/L) was used as a positive control. Results demonstrated that treatment with TDCIPP or BIO caused an abnormal stacking of blastomere cells in mid blastula transition (MBT) stage, and subsequently resulted in epiboly delay of zebrafish embryos. TDCIPP and BIO up-regulated the expression of β-catenin protein and increased its accumulation in nuclei of embryonic cells. This accumulation was considered as a driver for early embryonic developmental toxicity of TDCIPP. Furthermore, TDCIPP and BIO partly shared the same modes of action, and both of them could bind to Gsk-3β protein, and then decreased the phosphorylation level of Gsk-3β in TYR·216 site and lastly inhibited the activity of Gsk-3β kinase, which was responsible for the increased concentrations of β-catenin protein in embryonic cells and accumulation in nuclei. Our findings provide new mechanisms for clarifying the early embryonic developmental toxicity of TDCIPP in zebrafish.

Mosaic results after preimplantation genetic testing for aneuploidy may be accompanied by changes in global gene expression.

Aneuploidy in preimplantation embryos is a major cause of human reproductive failure. Unlike uniformly aneuploid embryos, embryos diagnosed as diploid-aneuploid mosaics after preimplantation genetic testing for aneuploidy (PGT-A) can develop into healthy infants. However, the reason why these embryos achieve full reproductive competence needs further research. Current RNA sequencing techniques allow for the investigation of the human preimplantation transcriptome, providing new insights into the molecular mechanisms of embryo development. In this prospective study, using euploid embryo gene expression as a control, we compared the transcriptome profiles of inner cell mass and trophectoderm samples from blastocysts with different levels of chromosomal mosaicism. A total of 25 samples were analyzed from 14 blastocysts with previous PGT-A diagnosis, including five low-level mosaic embryos and four high-level mosaic embryos. Global gene expression profiles visualized in cluster heatmaps were correlated with the original PGT-A diagnosis. In addition, gene expression distance based on the number of differentially expressed genes increased with the mosaic level, compared to euploid controls. Pathways involving apoptosis, mitosis, protein degradation, metabolism, and mitochondrial energy production were among the most deregulated within mosaic embryos. Retrospective analysis of the duration of blastomere cell cycles in mosaic embryos revealed several mitotic delays compared to euploid controls, providing additional evidence of the mosaic status. Overall, these findings suggest that embryos with mosaic results are not simply a misdiagnosis by-product, but may also have a genuine molecular identity that is compatible with their reproductive potential.

Paracrine interactions through FGFR1 and FGFR2 receptors regulate the development of preimplantation mouse chimaeric embryo.

The preimplantation mammalian embryo has the potential to self-organize, allowing the formation of a correctly patterned embryo despite experimental perturbation. To better understand the mechanisms controlling the developmental plasticity of the early mouse embryo, we used chimaeras composed of an embryonic day (E)3.5 or E4.5 inner cell mass (ICM) and cleaving 8-cell embryo. We revealed that the restricted potential of the ICM can be compensated for by uncommitted 8-cell embryo-derived blastomeres, thus leading to the formation of a normal chimaeric blastocyst that can undergo full development. However, whether such chimaeras maintain developmental competence depends on the presence or specific orientation of the polarized primitive endoderm layer in the ICM component. We also demonstrated that downregulated FGFR1 and FGFR2 expression in 8-cell embryos disturbs intercellular interactions between both components and results in an inverse proportion of primitive endoderm and epiblast within the resulting ICM and abnormal embryo development. This finding suggests that FGF signalling is a key part of the regulatory mechanism that assigns cells to a given lineage and ensures the proper composition of the blastocyst, which is a prerequisite for its successful implantation in the uterus and for further development.

The aryl hydrocarbon receptor directs the differentiation of murine progenitor blastomeres

Key regulatory decisions during cleavage divisions in mammalian embryogenesis determine the fate of preimplantation embryonic cells. Single-cell RNA sequencing of early-stage—2-cell, 4-cell, and 8-cell—blastomeres show that the aryl hydrocarbon receptor (AHR), traditionally considered as an environmental sensor, directs blastomere differentiation. Disruption of AHR functions in Ahr knockout embryos or in embryos from dams exposed to dioxin, the prototypic xenobiotic AHR agonist, significantly impairs blastocyst formation, causing repression and loss of transcriptional heterogeneity of OCT4 and CDX2 and incidence of nonspecific downregulation of pluripotency. Trajectory—the path of differentiation—and gene variability analyses further confirm that deregulation of OCT4 functions and changes of transcriptional heterogeneity resulting from disruption of AHR functions restrict the emergence of differentiating blastomeres in 4-cell embryos. It appears that AHR directs the differentiation of progenitor blastomeres and that disruption of preimplantation AHR functions may significantly perturb embryogenesis leading to long-lasting conditions at the heart of disease in offspring’s adulthood.

Bioactive supplements influencing bovine in vitro embryo development.

Ovum pickup and in vitro production (IVP) of bovine embryos are replacing traditional multiple ovulation embryo transfer (MOET) as the primary means for generating transferable embryos from genetically elite sires and dams. However, inefficiencies in the IVP process limit the opportunities to produce large numbers of transferable embryos. Also, the post-transfer competency of IVP embryos is inferior to embryos produced by artificial insemination or MOET. Numerous maternal, paternal, embryonic, and culture-related factors can have adverse effects on IVP success. This review will explore the various efforts made on describing how IVP embryo development and post-transfer competency may be improved by supplementing hormones, growth factors, cytokines, steroids and other bioactive factors found in the oviduct and uterus during early pregnancy. More than 40 of these factors, collectively termed as embryokines, are reviewed here. Several embryokines contain abilities to promote embryo development, including improving embryo survivability, improving blastomere cell numbers, and altering the distribution of blastomere cell types in blastocysts. A select few embryokines also can benefit pregnancy retention after IVP embryo transfer and improve neonatal calf health and performance, although very few embryokine-supplemented embryo transfer studies have been completed. Also, supplementing several embryokines at the same time holds promise for improving IVP embryo development and competency. However, more work is needed to explore the post-transfer consequences of adding these putative embryokines for any adverse outcomes, such as large offspring syndrome and poor postnatal health, and to specify the specific embryokine combinations that will best represent the ideal conditions found in the oviduct and uterus.

BET proteins are essential for the specification and maintenance of the epiblast lineage in mouse preimplantation embryos

BackgroundDuring mammalian preimplantation development, as the fertilized egg develops and differentiates, three cell lineages become specified: trophectoderm (TE), epiblast, and primitive endoderm (PrE). Through two steps of cell fate decisions, 16-cell blastomeres develop into TE and an inner cell mass (ICM), and thereafter, the latter differentiates into pluripotent epiblast and PrE. Although bromodomain and extra-terminal domain (BET) proteins, such as BRD4, are necessary for the transcriptional activation of genes involved in the maintenance of mouse embryonic stem cells by occupying their enhancers, their roles in the development of mouse preimplantation are unknown.ResultsTo evaluate the effect of BET protein deficiency on cell lineage formation, we cultured preimplantation embryos in the presence of JQ1, which blocks the binding of BET bromodomains to acetylated-histones. We found BET inhibition blocked the transcriptional activation of genes, such as Nanog, Otx2, and Sox2, important for the formation of the epiblast lineage in blastocysts. Expression studies with lineage-specific markers in morulae and blastocysts revealed BET proteins were essential for the specification and maintenance of the epiblast lineage but were dispensable for the formation of primarily extraembryonic TE and PrE lineages. Additional Ingenuity Pathway Analysis and expression studies with a transcriptionally active form of signal transducer and activator of the transcription 3 (STAT3) suggested BET-dependent activation was partly associated with the STAT3-dependent pathway to maintain the epiblast lineage. To identify BET proteins involved in the formation of the epiblast lineage, we analyzed mutant embryos deficient in Brd4, Brd2, and double mutants. Abolishment of NANOG-positive epiblast cells was only evident in Brd4/Brd2 double-deficient morulae. Thus, the phenotype of JQ1-treated embryos is reproduced not by a Brd4- or Brd2-single deficiency, but only Brd4/Brd2-double deficiency, demonstrating the redundant roles of BRD2 and BRD4 in the specification of the epiblast lineage.ConclusionsBET proteins are essential to the specification and maintenance of the epiblast lineage by activating lineage-specific core transcription factors during mouse preimplantation development. Among BET proteins, BRD4 plays a central role and BRD2 a complementary role in the specification and maintenance of epiblast lineages. Additionally, BET-dependent maintenance of the epiblast lineage may be partly associated with the STAT3-dependent pathway.

Efficient generation of embryonic stem cells from single blastomeres of cryopreserved mouse embryos in the presence of signalling modulators.

Derivation of embryonic stem cells (ESC) from single blastomeres is an interesting alternative to the use of whole blastocysts, but derivation rates are lower and the requirements for successful ESC obtention are still poorly defined. To investigate the effects of embryo cryopreservation and of signalling modulators present during embryo culture and/or ESC establishment on ESC derivation efficiency from single 8-cell mouse blastomeres. Fresh and cryopreserved 2-cell embryos were cultured and biopsied at the 8-cell stage. Single blastomeres were cultured in the presence of 2i or R2i cocktails, with or without adrenocorticotropic hormone (ACTH). We analysed ESC derivation efficiencies and characterised pluripotency genes expression and karyotype integrity of the resulting lines. We also evaluated the impact of embryo preculture with R2i on epiblast cell numbers and derivation rates. The ESC generation was not compromised by embryo cryopreservation and ACTH was dispensable under most of the conditions tested. While 2i and R2i were similarly effective for ESC derivation, R2i provided higher karyotype integrity. Embryo preculture with R2i yielded increased numbers of epiblast cells but did not lead to increased ESC generation. Our findings help to define a simplified and efficient procedure for the establishment of mouse ESC from single 8-cell blastomeres. This study will contribute to improving the potential of this experimental procedure, providing a tool to investigate the developmental potential of blastomeres isolated from different embryonic stages and to reduce the number of embryos needed for ESC derivation.

Duplication of spiralian-specific TALE genes and evolution of the blastomere specification mechanism in the bivalve lineage

BackgroundDespite the conserved pattern of the cell-fate map among spiralians, bivalves display several modified characteristics during their early development, including early specification of the D blastomere by the cytoplasmic content, as well as the distinctive fate of the 2d blastomere. However, it is unclear what changes in gene regulatory mechanisms led to such changes in cell specification patterns. Spiralian-TALE (SPILE) genes are a group of spiralian-specific transcription factors that play a role in specifying blastomere cell fates during early development in limpets. We hypothesised that the expansion of SPILE gene repertoires influenced the evolution of the specification pattern of blastomere cell fates.ResultsWe performed a transcriptome analysis of early development in the purplish bifurcate mussel and identified 13 SPILE genes. Phylogenetic analysis of the SPILE gene in molluscs suggested that duplications of SPILE genes occurred in the bivalve lineage. We examined the expression patterns of the SPILE gene in mussels and found that some SPILE genes were expressed in quartet-specific patterns, as observed in limpets. Furthermore, we found that several SPILE genes that had undergone gene duplication were specifically expressed in the D quadrant, C and D quadrants or the 2d blastomere. These expression patterns were distinct from the expression patterns of SPILE in their limpet counterparts.ConclusionsThese results suggest that, in addition to their ancestral role in quartet specification, certain SPILE genes in mussels contribute to the specification of the C and D quadrants. We suggest that the expansion of SPILE genes in the bivalve lineage contributed to the evolution of a unique cell fate specification pattern in bivalves.

Evaluation of quality and gene expression of goat embryos produced in vivo and in vitro after cryopreservation

In the present study, we aimed to identify morphological and molecular changes of in vivo and in vitro-produced goat embryos submitted to cryopreservation. In vivo embryos were recovered by transcervical technique from superovulated goats, whereas in vitro produced embryos were produced from ovaries collected at a slaughterhouse. Embryos were frozen by two-steps slow freezing method, which is defined as freezing to −32 °C followed by transfer to liquid nitrogen. Morphological evaluation of embryos was carried out by assessing blastocoel re-expansion rate and the total number of blastomeres. The expression profile of candidate genes related to thermal and oxidative stress, apoptosis, epigenetic, and implantation control was measured using RT-qPCR based SYBR Green system. In silico analyses were performed to identify conserved genes in goat species and protein-protein interaction networks were created. In vivo-produced embryos showed greater blastocoel re-expansion and more blastomere cells (P < 0.05). The expression level of CTP2 and HSP90 genes from in vitro cryopreserved embryos was higher than their in vivo counterparts. Unlikely, no significant difference was observed in the transcription level of SOD gene between groups. The high similarity of CPT2 and HSP90 proteins to their orthologs among mammals indicates that they share conserved functions. In summary, cryopreservation negatively affects the morphology and viability of goat embryos produced in vitro and changes the CPT2 and HSP90 gene expression likely in response to the in vitro production process.

Pluripotency-associated genes reposition during early embryonic developmental stages in pigs.

We examined the allelic expression and positioning of two pluripotency‐associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4‐ and 8‐cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi‐allelically increased from 45% at the 4‐cell stage to 60% at the 8‐cell stage. Moreover, in 8‐cell embryos, SOX2 was expressed bi‐allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4‐ to 8‐cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4‐ or 8‐cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency‐associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.

Embryo fragmentation and its relationship with aneuploidy

Objective: Selecting embryos is an important step in the in vitro fertilization process before transferring them to the uterus. There are some invasive methods for choosing a good quality embryo, such as embryo grading. This method evaluates the equality and fragmentation of an embryo. However, this method does not adequately evaluate the chromosomal status of the embryos, which is often necessary for high-risk embryos. Here, we evaluated embryo fragmentation and chromosomal numbers using next-generation sequencing. Materials and Methods: Each embryo was biopsied on the 3rd or 5th culture day to obtain a single blastomere cell. DNA was then extracted from each blastomere and whole-genome amplification was carried out. Amplification products were then sequenced to obtain a ploidy number. Results: Among the 30 embryos that were evaluated, 19 embryos had no fragments, 10 embryos had small fragments, and 1 embryo had moderate fragments. However, 12 of 19 embryos, 57.9% with no fragments were detected to have chromosomal abnormality. Aneuploidy was increased in 7 of 10 embryos (70%) with mild fragments. One moderately fragmented embryo included was surprisingly found to have normal ploidy (100%). Gamma correlation test showed that there was no correlation between fragmentation and the incidence rate of aneuploidy (P > 0.05). Although there was no correlation, the study's result exemplifies that aneuploidy rate increased along with higher fragmentation. Conclusion: This research concluded that embryo fragmentation was not correlated with aneuploidy.

69. IMPROVED IVF SUCCESS OF COMBINED PGT-M AND PGT-A APPLICATIONS

Introduction Preimplantation Genetic Testing (PGT) has been performed since 1989 and utilized in the diagnosis of all genetic conditions at preimplantation stage ( 1 ). In the previous years, only a disorder could be diagnosed with the use of multiplex nested PCR in PGT applications. Increased use of trophectoderm biopsy and vitrification together with Whole Genome Amplification (WGA) technology enabled the detection of single gene disorders together with 24 chromosome screening. This approach allowed us to select healthy and chromosomally euploid embryos. Even though single gene disorder carrier patients are mostly young and fertile, they still have high risk of spontaneous miscarriage. In the recent years, it has been reported that when the embryos are screened for aneuploidy (PGT-A) in addition to single gene disorder testing (PGT-M), pregnancy rates can be increased from 45% to 68% and spontaneous miscarriage rates decreased from 15% to 5%. 34 single gene disorder patients, whose embryos were found to be healthy in terms of single gene disorder, were included in this study and these embryos were further screened for aneuploidy using Next Generation Sequencing (NGS) technology ( 2 ). Materials & Method On the 5th and 6th days of embryonic development, WGA was performed using SurePlex DNA Amplification System (Illumina, USA) and DOPlify (PerkinElmer Health Sciences - Australia). Following multiplex nested PCR on WGA samples, fragment analysis for haplotyping and sequence analysis for mutation detection were performed. Embryos found to be suitable for transfer regarding the single gene disorder have further been screened for aneuploidy using NGS technology with VeriSeq PGS Kit (Illumina, USA). Results This study was conducted for single gene disoder and 24 chromosome screening in 44 patients with an average age of 34. 280 embryos derived from these patients were included in PGT-M. Among 153 embryos that were found to be suitable for transfer after PGT-M, PGT-A was performed on 121 embryos upon patients' requests and 49% of these embryos were euploid. Embryo transfer could not be performed in 12 patients due to the absence of euploid embryos. According to the data we acquired, 64% pregnancy was achieved in the rest of the patients. Conclusion While pregnancy ratio can be improved with this approach, Allele drop out (ADO) and contamination errors are still major limitation which can lead to misdiagnosis of embryos. ADO ratio was demonstrated to be 25% in PGT for blastomere cells, whereas this ratio drops down to 5% when 5-7 cells obtained by trophectoderm biopsy are tested. Thus trophectoderm cells are more appropriate for PGT-M applications combined with PGT-A. Since stable and homogenous amplification of the entire genome is a crucial step, optimised WGA technologies will be a key factor in these applications.