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

Abstract: 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. Lay summary: 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.

Ascidian gastrulation and blebbing activity of isolated endoderm blastomeres.

Gastrulation is the first dynamic cell movement during embryogenesis. Endoderm and mesoderm cells are internalized into embryos during this process. Ascidian embryos provide a simple system for studying gastrulation in chordates. Gastrulation starts in spherical late 64-cell embryos with 10 endoderm blastomeres. The mechanisms of gastrulation in ascidians have been investigated, and a two-step model has been proposed. The first step involves apical constriction of endoderm cells, followed by apicobasal shortening in the second step. In this study, isolated ascidian endoderm progenitor cells displayed dynamic blebbing activity at the gastrula stage, although such a dynamic cell-shape change was not recognized in toto. Blebbing is often observed in migrating animal cells. In ascidians, endoderm cells displayed blebbing activity, while mesoderm and ectoderm cells did not. The timing of blebbing of isolated endoderm cells coincided with that of cell invagination. The constriction rate of apical surfaces correlated with the intensity of blebbing activity in each endoderm-lineage cell. Fibroblast growth factor (FGF) signaling was both necessary and sufficient for inducing blebbing activity, independent of cell fate specification. In contrast, the timing of initiation of blebbing and intensity of blebbing response to FGF signaling were controlled by intrinsic cellular factors. It is likely that the difference in intensity of blebbing activity between the anterior A-line and posterior B-line cells could account for the anteroposterior difference in the steepness of the archenteron wall. Inhibition of zygotic transcription, FGF signaling, and Rho kinase, all of which suppressed blebbing activity, resulted in incomplete apical constriction and failure of the eventual formation of cup-shaped gastrulae. Blebbing activity was involved in the progression and maintenance of apical constriction, but not in apicobasal shortening in whole embryos. Apical constriction is mediated by distinct blebbing-dependent and blebbing-independent mechanisms. Surface tension and consequent membrane contraction may not be the sole mechanical force for apical constriction and formation of cup-shaped gastrulae. The present study reveals the hidden cellular potential of endodermal cells during gastrulation and discusses the possible roles of blebbing in the invagination process.

Cellular and Molecular Nature of Fragmentation of Human Embryos.

Embryo fragmentation represents a phenomenon generally characterized by the presence of membrane-bound extracellular cytoplasm into the perivitelline space. Recent evidence supports the cellular and molecular heterogeneity of embryo fragments. In this narrative review, we described the different embryo fragment-like cellular structures in their morphology, molecular content, and supposed function and have reported the proposed theories on their origin over the years. We identified articles related to characterization of embryo fragmentation with a specific literature search string. The occurrence of embryo fragmentation has been related to various mechanisms, of which the most studied are apoptotic cell death, membrane compartmentalization of altered DNA, cytoskeletal disorders, and vesicle formation. These phenomena are thought to result in the extrusion of entire blastomeres, release of apoptotic bodies and other vesicles, and micronuclei formation. Different patterns of fragmentation may have different etiologies and effects on embryo competence. Removal of fragments from the embryo before embryo transfer with the aim to improve implantation potential should be reconsidered on the basis of the present observations.

Morphometric and morphokinetic differences in the sperm- and oocyte-originated pronuclei of male and female human zygotes: a time-lapse study.

PURPOSE: To study the morphometric and morphokinetic profiles of pronuclei (PN) between male and female human zygotes. METHOD(S): This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles leading to a singleton live birth. All oocytes were placed in the EmbryoScope + incubator post-sperm injection with all annotations performed retrospectively by one embryologist (L-SO). Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance, and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2), and 0 h before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement. RESULTS: Male zygotes had longer time intervals of tPB2_tSPNa than female zygotes (4.8 ± 0.2 vs 4.2 ± 0.1 h, OR = 1.442, 95% CI 1.009-2.061, p = 0.044). SPN increased in size from stage 1 through 2 to 3 (435.3 ± 7.2, 506.7 ± 8.0, and 556.3 ± 8.9 um2, p = 0.000) and OPN did similarly (399.0 ± 6.1, 464.3 ± 6.7, and 513.8 ± 6.5 um2, p = 0.000), with SPN being significantly larger than OPN at each stage (p < 0.05 respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (76.7% vs 51.0%, p = 0.010), stage 2 (97.7% vs 86.3%, p = 0.048), and stage 3 (97.7% vs 86.3%, p = 0.048). More OPN showed aligned NPBs than in SPN at stage 1 only (44.7% vs 28.7%, p = 0.023). CONCLUSION(S): Embryos with different sexes display different morphokinetic and morphometric features at the zygotic stage. Embryo selection using such parameters may lead to unbalanced sex ratio in resulting offspring.

Are commercial warming kits interchangeable for vitrified human blastocysts? Further evidence for the adoption of a Universal Warming protocol.

PURPOSE: To study whether a new combination of different warming kits is clinically effective for vitrified human blastocysts. METHODS: This is a longitudinal cohort study analysing two hundred fifty-five blastocysts warming cycles performed between January and October 2018. Embryos were vitrified using only one brand of ready-to-use kits (Kitazato), whereas the warming procedure was performed with three of the most widely used vitrification/warming kits (Kitazato, Sage and Irvine) after patient stratification for oocyte source. The primary endpoint was survival rate, while the secondary endpoints were clinical pregnancy, live birth and miscarriage rates. RESULTS: We observed a comparable survival rate across all groups of 100% (47/47) in KK, 97.6% (49/50) in KS, 97.6% (41/42) in KI, 100% (38/38) in dKK, 100% (35/35) in dKS and 100% (43/43) in dKI. Clinical pregnancy rates were also comparable: 38.3% (18/47) in KK, 49% (24/49) in KS, 56.1% (23/ 41) in KI, 47.4% (18/38) in dKK, 31.4% (11/35) in dKS and 48.8% (21/ 43) in dKI. Finally, live birth rates were 29.8% (14/47) in KK, 36.7% (18/49) in KS, 46.3% (19/41) in KI, 36.8% (14/38) in dKK, 25.7% (9/35) in dKS and 41.9% (18/43) in dKI, showing no significant differences. CONCLUSION: This study confirmed the efficacy of applying a single warming protocol, despite what the "industry" has led us to believe, supporting the idea that it is time to proceed in the cryopreservation field and encouraging embryologists worldwide to come out and reveal that such a procedure is possible and safe.

Retrospective comparison of pregnancy outcomes of fresh and frozen-warmed single blastocyst transfer: a 5-year single-center experience.

PURPOSE: To assess whether live birth rates (LBR) and maternal/neonatal complications differed following single fresh and frozen-warmed blastocyst transfer. METHODS: The present retrospective observational study analyzed 4,613 single embryo transfers (SET) (646 fresh and 3,967 frozen) from January 1, 2014, to December 31, 2018. Fresh embryo transfer at blastocyst stage was considered according to the age of the patient and her prognosis. In case of the risk of ovarian hyperstimulation syndrome, premature progesterone rise, non-optimal endometrial growth, or supernumerary embryos, cryopreservation with subsequent frozen embryo transfer (FET) was indicated. RESULTS: No differences in LBR were recorded. Fresh embryo transfers yielded an increase both in neonatal complications OR 2.15 (95% CI 1.20-3.86, p 0.010), with a higher prevalence of singletons weighting below the 5th percentile (p 0.013) and of intrauterine growth retardation (p 0.015), as well as maternal complications, with a higher placenta previa occurrence OR 3.58 (95% CI 1.54-8.28, p 0.003), compared to FET. CONCLUSION: LBR appears not to be affected by the transfer procedure preferred. Fresh embryo transfer is associated with higher risk of neonatal complications (specifically a higher prevalence of singletons weighting below the 5th percentile and of intrauterine growth retardation) and placenta previa. Reflecting on the increased practice of ART procedures, it is imperative to understand whether a transfer procedure yields less complications than the other and if it is time to switch to a "freeze-all" procedure as standard practice. TRIAL REGISTRATION: Clinical Trial Registration Number: NCT04310761. Date of registration: March 17, 2020, retrospectively registered.

Yolk platelets impede nuclear expansion in Xenopus embryos.

During metazoan early embryogenesis, the intracellular properties of proteins and organelles change dynamically through rapid cleavage. In particular, a change in the nucleus size is known to contribute to embryonic development-dependent cell cycle and gene expression regulation. Here, we compared the nuclear sizes of various blastomeres from developing Xenopus embryos and analyzed the mechanisms that control the nuclear expansion dynamics by manipulating the amount of intracellular components in a cell-free system. Nuclear expansion was slower in blastomeres from vegetal hemispheres during a longer interphase than in those from animal hemispheres. Furthermore, upon recapitulating interphase events by manipulating the concentration of yolk platelets, which are originally rich in the vegetal blastomeres, in cell-free cytoplasmic extracts, nuclear expansion and DNA replication became slower than that in normal yolk-free conditions. Under these conditions, the supplemented yolk platelets accumulated around the nucleus in a microtubule-dependent manner and impeded the organization of the endoplasmic reticulum network. Overall, we propose that yolk platelets around the nucleus reduce membrane supply from the endoplasmic reticulum to the nucleus, resulting in slower nuclear expansion and cell cycle progression in the yolk-rich vegetal blastomeres.

Semi-automated versus manual embryo vitrification: inter-operator variability, time-saving, and clinical outcomes.

PURPOSE: Does semi-automated vitrification have lower inter-operator variability and better clinical outcomes than manual vitrification? METHODS: Retrospective analyses of 282 patients whose embryos had been cryopreserved, manually with Irvine®-CBS® (MV) or semi-automatically vitrified with the GAVI® method (AV) (from November 2017 to September 2020). Both techniques were performed during the same period by 5 operators. Inter-operator variability was statistically analyzed between operators who performed the vitrification and those who performed the warming process to compare the intact survival rate (% embryos with 100% intact blastomeres) and the positive survival rate (at least 50% intact blastomeres). Additionally, the complete vitrification time was assessed for the 2 techniques according to the number of vitrified embryos. RESULTS: Manual vitrification involved warming 338 embryos in 266 cycles for 181 couples compared to 212 embryos in 162 AV cycles for 101 patients. The positive survival rate was higher (p < 0.05) after MV (96%; 323/338) than after AV (90%; 191/212). The intact survival rate (86 vs 84%) and the clinical pregnancy rate (27 vs 22%) were not significantly different between MV and AV. Regarding the inter-operator variability, no significant difference in positive and intact survival rate was evident between the 5 technicians, neither by vitrification nor by warming steps with MV and AV. Concerning time-saving, the MV technique proved to be quicker than AV (minus 11 ± 9 min). CONCLUSIONS: Manual vitrification exhibited favorable total survival rates and was more time efficient, while both MV and AV cooling and warming treatments showed little operator variability.

Number of Blastomeres in Day-2 Embryos Affect the Rates of Blastocyst Formation and Clinical Pregnancy During In Vitro Fertilization Cycles.

In the current in vitro fertilization and embryo transfer protocol, the 8 blastomeres in the day-3 embryo are selected for transfer because these embryos can produce high rates of blastocyst formation and clinical pregnancy. However, the relationship between the blastomere number in day-2 embryos and the rate of blastocyst formation or clinical pregnancy remains unclear. The purpose of this retrospective study is to explore the relationship between the blastomere number in day-2 embryos and the rate of blastocyst formation or clinical pregnancy. From January 2015 to April 2020, we collected 8126 day-3 embryos (8 blastomeres) from 2282 patients. These embryos were classified into 8 groups (1 blastomere, 2 blastomeres, 3 blastomeres, 4 blastomeres, 5 blastomeres, 6 blastomeres, 7 blastomeres, and 8 blastomeres) based on their blastomeres number on day 2 after insemination. Of these groups, the 4 blastomeres group accounted for the largest proportion (74.44%). The 1 blastomere group accounted for the smallest proportion (0.22%). A total of 3554 day-3 embryos (8 blastomeres) from 1648 patients developed into blastocysts. The rate of blastocyst formation from the 4 blastomeres group was the highest (94.06%). Finally, 800 patients received single day-3 embryos (8 blastomeres) transfer. The rate of clinical pregnancy from 4 blastomeres group was the highest (51.98%). In conclusion, our data provide evidence that the number of blastomeres in day-2 embryos affects the rate of blastocyst formation and clinical pregnancy.

The use of a two-step removal protocol and optimized culture conditions improve development and quality of zona free mouse embryos.

The zona pellucida (ZP) plays an important role in both the fertilization and embryonic development. For the successful handling of early stage blastomeres for differentiation analysis, the production of identical twins or quadruplets, nuclear transfer or gene introduction requires the removal of the ZP (ZPR). Although single use of either acidic Tyrode's solution or pronase are commonly used for ZPR, long-term exposure to these agents can result in the inhibition of development with the collapse of the three-dimensional blastomere structure. Here, we demonstrate the benefits of using a two-step combined ZPR method, which relies upon a customized well-of-well (cWOW) system with smaller well size, on developmental competence and the quality of the zona free (ZF) mouse embryos. We first isolated 2-cell embryos using acid Tyrode's solution and then cultured these embryos using either commercially available or cWOW, which had a smaller microwell size. The rate of blastocyst was significantly increased by use of cWOW when compared to other culture systems. Then we evaluated the use of a two-step ZPR protocol, relying on acid Tyrode's solution and proteinase K, and subsequent culture in the cWOW system. Although acid Tyrode's solution treatment alone reduced ZPR time, blastomere morphology became wrinkled, significant decrease in blastocyst rate associated with increased number of apoptotic cells and increased expression of apoptosis-related genes were observed. Using proteinase K alone increased ZPR time and significantly decreased the blastocyst rate, but did not induce an increase in apoptotic cell number or apoptosis-related gene expression. In contrast, two-step method significantly reduced ZPR time and improved blastocyst rate by increasing the total number of cells in these wells an reducing the number of apoptotic cells in these experiments. These results suggest that the two-step ZPR protocol is beneficial for reducing the toxic effects of zona removal on ZF embryo development and quality when combined with a suitable culture system.

Tauroursodeoxycholic acid/TGR5 signaling promotes survival and early development of glucose-stressed porcine embryos†.

Conditions of impaired energy and nutrient homeostasis, such as diabetes and obesity, are associated with infertility. Hyperglycemia increases endoplasmic reticulum stress as well as oxidative stress and reduces embryo development and quality. Oxidative stress also causes deoxyribonucleic acid damage, which impairs embryo quality and development. The natural bile acid tauroursodeoxycholic acid reduces endoplasmic reticulum stress and rescues developmentally incompetent late-cleaving embryos, as well as embryos subjected to nuclear stress, suggesting the endoplasmic reticulum stress response, or unfolded protein response, and the genome damage response are linked. Tauroursodeoxycholic acid acts via the Takeda-G-protein-receptor-5 to alleviate nuclear stress in embryos. To evaluate the role of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling in embryo unfolded protein response, we used a model of glucose-induced endoplasmic reticulum stress. Embryo development was impaired by direct injection of tauroursodeoxycholic acid into parthenogenetically activated oocytes, whereas it was improved when tauroursodeoxycholic acid was added to the culture medium. Attenuation of the Takeda-G-protein-receptor-5 precluded the positive effect of tauroursodeoxycholic acid supplementation on development of parthenogenetically activated and fertilized embryos cultured under standard conditions and parthenogenetically activated embryos cultured with excess glucose. Moreover, attenuation of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling induced endoplasmic reticulum stress, oxidative stress and cell survival genes, but decreased expression of pluripotency genes in parthenogenetically activated embryos cultured under excess glucose conditions. These data suggest that Takeda-G-protein-receptor-5 signaling pathways link the unfolded protein response and genome damage response. Furthermore, this study identifies Takeda-G-protein-receptor-5 signaling as a potential target for mitigating fertility issues caused by nutrient excess-associated blastomere stress and embryo death.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

Physical constraints on early blastomere packings.

At very early embryonic stages, when embryos are composed of just a few cells, establishing the correct packing arrangements (contacts) between cells is essential for the proper development of the organism. As early as the 4-cell stage, the observed cellular packings in different species are distinct and, in many cases, differ from the equilibrium packings expected for simple adherent and deformable particles. It is unclear what are the specific roles that different physical parameters, such as the forces between blastomeres, their division times, orientation of cell division and embryonic confinement, play in the control of these packing configurations. Here we simulate the non-equilibrium dynamics of cells in early embryos and systematically study how these different parameters affect embryonic packings at the 4-cell stage. In the absence of embryo confinement, we find that cellular packings are not robust, with multiple packing configurations simultaneously possible and very sensitive to parameter changes. Our results indicate that the geometry of the embryo confinement determines the packing configurations at the 4-cell stage, removing degeneracy in the possible packing configurations and overriding division rules in most cases. Overall, these results indicate that physical confinement of the embryo is essential to robustly specify proper cellular arrangements at very early developmental stages.

What Are the Live Birth and Multiple Pregnancy Rates When 1 Versus 2 Low-Quality Blastocysts Are Transferred in a Cryopreserved Cycle? a Retrospective Cohort Study, Stratified for Age, Embryo Quality, and Oocyte Donor Cycles.

Outcomes among women who transferred only Gardner's grade BB or lower quality frozen embryos transferred (FET) are not well known. Our objective is to study whether transferring 2 versus 1 frozen low-quality blastocysts will increase the live birth rate (LBR) and the multiple pregnancy rate (MPR). This is a retrospective cohort study including 1104 FET cycles. Only day 5-6 blastocysts of grade BB or lower quality were included. Clinical pregnancy rate (CPR), MPR, and LBR per cycle were compared between single embryo transfer (SET) (n = 969) and double embryo transfer (DET) (n = 135). CPR and MPR were compared between SET and DET in grade BB, BC, CB, and CC individually. Among SET, BB blastocysts had higher CPR 34% (P = 0.0001) and a sub-significant increase in LBR 19% (P = 0.059) in comparison to other grade SET. Among all BB, MPR was significantly higher when transferring two versus one (5.9 vs. 1.9, P = 0.009). If age at egg collection ≥ 40 years (n = 97), no difference was found in CPR (11.1 vs. 11.7, P = 0.9), MPR (0 vs. 0), and LBR (6.3 vs. 0,P = 0.13) when SET or DET was performed. If age was < 40 years (n = 818), the MPR was significantly higher in DET than SET (6.7 vs. 1.63, P = 0.004). In egg donor cycles (n = 189), there was no difference in CPR, MPR, and LBR between SET and DET. Single embryo transfer should be offered even in women ≥ 40 years of age or transferring lower quality embryos since transferring more did not increase outcomes in this group, and SET is likely the safest path.

A New Day 4 Grading System to Assess Embryo Quality in Frozen Embryo Transfer Cycles.

To present a new day 4 (D4) embryo grading system to assess embryos in frozen-thawed embryo transfer (FET) cycles. A new grading system (grades A-E) was developed from the 2011 ESHRE Istanbul Consensus for D4 embryos in FET cycles. Embryos with complete compaction were classified as grade A; those with partial compaction were assigned as grade B; and those without compaction were classified as grades C, D, and E according to their different blastomere number ratio (BNR; number of embryo blastomeres on D4/number of embryo blastomeres on D3, D4/D3). Embryos with a BNR of ≥ 1.5 were defined as grade C, those with a BNR of ≥ 1.2 and < 1.5 were defined as grade D, and those with a BNR of ≥ 1.0 and < 1.2 were defined as grade E. Using this proposed grading model, 5460 embryos with known implantation data were retrospectively analyzed after D4 transfer in FET cycles. The transferred embryos exhibited a similar declining trend in implantation and live birth rates from the top grade A to the lowest grade E. The in vitro fertilization group showed increased implantation rates of grade B and E embryos compared with the intracytoplasmic sperm injection group (grade B: 41.99%, 34.63%, χ2 = 5.84, p < 0.05 and grade E: 18.98%, 14.08, χ2 = 75.62, p < 0.01). Receiver-operating characteristic analysis revealed that our proposed model predicted the implantation outcomes and live birth rates of all embryos (area under the curve = 0.65; 95% confidence interval [CI],0.63-0.66; p < 0.01 and AUC = 0.73; 95%CI, 0.65-0.84; p < 0.001, respectively). This study demonstrates that the new grading system provided by us can be a useful tool for assisting embryo selection via changes in embryo morphology. D4 embryo transfer provides a simple and applicable method for FET cycles in daily practice.

The inclusion of blastomeres into the inner cell mass in early-stage human embryos depends on the sequence of cell cleavages during the fourth division.

The fate of the ICM in humans is still unknown, due to the ethical difficulties surrounding experimentation in this field. In this study we have explored the existing time-lapse recording data of embryos in the early stages of development, taking advantage of the large refractile bodies (RBs) within blastomeres as cellular markers. Our study found that the cellular composition of the ICM in humans is largely determined at the time of the fourth division and blastomeres which cleave first to fourth, during the fourth division from 8 cells to 16 cells, have the potential to be incorporated in the ICM.

Timing day-3 vitrification for PGT-M embryos: pre- or post-blastomere biopsy?

PURPOSE: To assess the efficacy and clinical outcomes of preimplantation genetic testing for monogenic diseases (PGT-M), following blastomere biopsy prior or following vitrification. METHODS: A cohort-historical study of all consecutive patients admitted to IVF in a large tertiary center for PGT-M and PCR cycle from September 2016 to March 2020. Patients were divided into 4 groups: Group A1 consisted of patients undergoing day-3 embryos biopsy followed by a fresh transfer of unaffected embryos. Group A2 consisted of Group A1 patients that their surplus unaffected embryos were vitrified, thawed, and transferred in a subsequent FET cycle. Group B1 consisted of patients that their day-3 embryos were vitrified intact (without biopsy) for a subsequent FET cycle. Later embryos were thawed and underwent blastomere biopsies, and the unaffected embryos were transferred, while the surplus unaffected embryos were re-vitrified for a subsequent FET cycle. Group B2 consisted of Group B1 patients that their surplus unaffected embryos were re-vitrified, thawed, and transferred in a subsequent FET cycle. The laboratory data and clinical results were collected and compared between groups. RESULTS: A total of 368 patients underwent 529 PGT-M cycles in our center: 347 with day-3 embryos biopsied before undergoing vitrification (Group A1) and 182 following vitrification and thawing (Group B1). There were no between group differences in embryo survival rate post-thawing, nor the ongoing implantation and pregnancy rates. CONCLUSION: In PGT-M cycles, the timing of embryos vitrification, whether prior or following blastomere biopsy, has no detrimental effect on post-thawing embryo survival rate, nor their potential ongoing implantation and pregnancy rates.

The impact of embryo binucleation depends upon its origin.

Summary: Preimplantation embryos frequently contain binucleated cells, but reports differ as to whether binucleation affects development and whether such embryos should be used clinically. In this Point Of View article, we propose a possible explanation for this disparity: binucleation can arise by distinct routes, one that produces healthy blastomeres and one that directly threatens embryo viability.

Next-generation sequencing analysis of each blastomere in good-quality embryos: insights into the origins and mechanisms of embryonic aneuploidy in cleavage-stage embryos.

PURPOSE: To explore the whole-chromosome status, origins, and mechanisms of chromosomal abnormalities in good-quality cleavage embryos using multiple annealing and looping-based amplification cycle (MALBAC) sequencing. METHODS: The embryos studied came from7 patients (maternal aged 26-35) who had healthy birth from the same IVF cycles. These 21 frozen day 3 good-quality embryos were thawed and disaggregated into individual blastomere. Each blastomere was collected and analyzed by MALBAC sequencing. RESULTS: Conclusive results were obtained from a high percentage of blastomeres (95.3%). A total of 46.6% of blastomeres were diploid, 53.4% were abnormal, and 28.0% had complex aneuploidy. Out of 21 embryos, 3 (14.3%) were normal and 18 (85.7%) were mosaics, showing the occurrence of mitotic errors; aneuploidy was confirmed in all cells of 4 of the 18 embryos, which showed the coexistence of meiotic errors. Conclusive results were obtained from all blastomeres of 15 embryos (71.4%, 15/21), which enabled us to reconstruct the cell lineage on the basis of the chromosomal content of the blastomeres in each division. There were 9 mitotic errors (8.7%, 9/103): nondisjunction accounted for 88.9% (8/9), and endoreplication accounted for 11.1% (1/9). CONCLUSIONS: In good-quality embryos, there was a high rate and diverse array of chromosomal abnormalities. Morphological evaluation does not appear to assist in the reduction in meiotic errors from parental origins. Mitotic errors were common, and nondisjunction was found to be the main mechanism causing malsegregation during the cleavage divisions.

Femtosecond laser-induced blastomere fusion results in embryo tetraploidy by common metaphase plate formation.

The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found within natural physiological conditions, e.g., placentation, and in pathophysiological conditions, such as cancer and early pregnancy failure in humans. Here we investigate the mechanism of tetraploidization with help of femtosecond laser-induced mouse blastomere fusion by the means of Hoechst staining, GFP, BODIPY dyes and fluorescent species generated intracellularly by a femtosecond laser. We establish diffusive mixing of cytosol, whereas the large components of a cytoplasm (organelles, cytoskeleton) are poorly diffusible and are not completely mixed after cell fusion and a subsequent division. We show that mechanisms which are responsible for the formation of a common metaphase plate triggered tetraploidization in fused mouse embryos and could be a significant factor in polyploidy formation in vivo. Thus, our results suggest that microtubules play a critical role in tetraploidization.