The morphological and functional ultrastructure of cells in pre-implantation embryos.

Cells of pre-implantation embryos are equipped with many morphological and functional systems through which they can synthesize specific proteins and effectively ensure the protection of early embryonic development. Here we present evidence for the existence of these systems in morphologically normal and abnormal bovine blastocyst stage embryos in vivo at the ultrastructural and actin cytoskeleton levels. The appearance of organelles in the trophectoderm (TE) and inner cell mass (ICM) cells, responsible for their synthetic activities and their role in the development of early bovine embryos are described. We point out the importance of endocytic processes and the participation of extracellular vesicles in the formation of intercellular contacts and homeostasis of the embryo microenvironment. Several changes in the ultrastructural morphology of embryos produced by different methods (ICSI, parthenogenetic AC/DC electrical activation, IVF with separated sperm) and freezing/thawed embryos are described. We also show alterations occurred in the organelles after viral contamination of embryos with BHV-1 and BVDV viruses, and in embryos from over-conditioned cows. Recorded changes in organelles and appearance of cellular autophagic structures (vesicles, multivesicular bodies and autophagolysosomes) may negatively affect embryo metabolism and lead to the emergence of pathological processes in TE and ICM cells of preimplantation embryos.

Bisphenol Exposure Disrupts Cytoskeletal Organization and Development of Pre-Implantation Embryos.

The endocrine disrupting activity of bisphenol compounds is well documented, but less is known regarding their impact on cell division and early embryo formation. Here, we tested the effects of acute in vitro exposure to bisphenol A (BPA) and its common substitute, bisphenol F (BPF), during critical stages of mouse pre-implantation embryo development, including the first mitotic division, cell polarization, as well as morula and blastocyst formation. Timing of initial cleavage was determined by live-cell imaging, while subsequent divisions, cytoskeletal organization and lineage marker labeling were assessed by high-resolution fluorescence microscopy. Our analysis reveals that brief culture with BPA or BPF impeded cell division and disrupted embryo development at all stages tested. Surprisingly, BPF was more detrimental to the early embryo than BPA. Notably, poor embryo development was associated with cytoskeletal disruptions of the actomyosin network, apical domain formation during cell polarization, actin ring zippering for embryo sealing and altered cell lineage marker profiles. These results underscore that bisphenols can disrupt cytoskeletal integrity and remodeling that is vital for early embryo development and raise concerns regarding the use of BPF as a 'safe' BPA substitute.

Viability assessment using fluorescent markers and ultrastructure of human biopsied embryos vitrified in open and closed systems.

RESEARCH QUESTION: Are there any differences in viability and ultrastructure amongst embryos biopsied on Day 5 versus Day 3 following vitrification in open and closed systems and compared to fresh embryos? DESIGN: One hundred human embryos (40 blastocysts biopsied on Day 5 and subsequently vitrified in open or closed systems and 60 Day 3 biopsied embryos that developed to blastocysts but were rejected for transfer following preimplantation genetic testing for monogenic/single gene defects and for aneuploidies were either treated fresh [n = 20] or vitrified [n = 40] in open or closed systems) and following warming and culture for 4 h were subjected to viability staining with carboxyfluorescein-diacetate succinimidylester/propidium iodide or processed for transmission electron microscopy. RESULTS: No statistically significant differences were observed in the viability of human biopsied embryos following vitrification in open and closed systems. Compared to fresh embryos, vitrified ones had a higher incidence of damage (propidium iodide-stained cells) irrespective of the vitrification method (P = 0.005). These damaged cells were more prominent in Day 5 biopsied blastocysts and mainly located at the position of cutting. Characteristic lipofuscin droplets (representative of apoptosis) and a higher number of vacuoles and distension of mitochondria were also more evident in vitrified embryos, although this was not statistically assessed. CONCLUSIONS: Vitrification in open and closed systems does not adversely affect the viability and ultrastructure of Day 5 and Day 3 biopsied embryos as revealed by the minimal yet statistically significant cell damage observed. This damage may be compensated by the embryos, which in their attempt to fully recover following vitrification, potentially enable 'rescue' processes to eliminate it.

The subcortical maternal complex: emerging roles and novel perspectives.

Since its recent discovery, the subcortical maternal complex (SCMC) is emerging as a maternally inherited and crucial biological structure for the initial stages of embryogenesis in mammals. Uniquely expressed in oocytes and preimplantation embryos, where it localizes to the cell subcortex, this multiprotein complex is essential for early embryo development in the mouse and is functionally conserved across mammalian species, including humans. The complex has been linked to key processes leading the transition from oocyte to embryo, including meiotic spindle formation and positioning, regulation of translation, organelle redistribution, and epigenetic reprogramming. Yet, the underlying molecular mechanisms for these diverse functions are just beginning to be understood, hindered by unresolved interplay of SCMC components and variations in early lethal phenotypes. Here we review recent advances confirming involvement of the SCMC in human infertility, revealing an unexpected relationship with offspring health. Moreover, SCMC organization is being further revealed in terms of novel components and interactions with additional cell constituents. Collectively, this evidence prompts new avenues of investigation into possible roles during the process of oogenesis and the regulation of maternal transcript turnover during the oocyte to embryo transition.

Morphology-based selection from available euploid blastocysts induces male-skewed sex proportion in the offspring.

PURPOSE: To determine whether blastocyst morphology has an impact on sex proportion at pre-implantation and birth in PGT-A and non-PGT-A cycles. METHODS: A total of 1254 biopsied blastocysts from 466 PGT-A cycles were analyzed for sex proportion, day of biopsy, degree of expansion, inner cell mass (ICM), and trophectoderm (TE) morphology. From these, 197 frozen single embryo transfers (SET) were assessed for clinical outcomes and sex proportion of ongoing pregnancies and deliveries. In addition, we evaluated the day of vitrification/embryo transfer, degree of expansion, and TE morphology in a group of 229 births (217 cycles) from frozen or fresh transfers of non-biopsied blastocysts. RESULTS: Sex proportion was impacted by day of biopsy and TE morphology, but not by ICM morphology, in PGT-A cycles. Therefore, biopsy on day 5 and TE "A" shifted the sex proportion towards males. Interestingly, we noted that our morphology-based embryo selection for SET of euploid blastocysts has favored the choice for XY embryos, generating a 54.3% XY proportion at transfer and 56.1% XY proportion at ongoing pregnancy/delivery. Our models indicate a weaker association between blastocyst morphology parameters and sex proportion of babies in non-PGT-A cycles. CONCLUSION: Blastocyst features associated with a skewed sex proportion towards XY embryos, such as biopsy on day 5 and top quality TE, are also parameters used for selecting euploid embryos for SET. Therefore, our data suggest that morphology-based embryo selection represents a strong factor responsible for a skewed male sex proportion at birth in PGT-A cycles.

The presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart.

PURPOSE: Is the presence of cytoplasmic strings (CS) in human blastocysts associated with the probability of clinical pregnancy with fetal heart (CPFH) after transfer. METHODS: This case-control study involved 300 single blastocyst transfers. 150 of these resulted in a CPFH (cases) while 150 did not (controls). All embryos were cultured in Embryoscope+ and AI software (IVY) was used to select the blastocyst with the highest score from the cohort for transfer. An embryologist, blind to the transfer outcome, recorded the CS number, location, and duration of their activity. RESULTS: There was a significant difference in the number of blastocysts that contained CS, with 97.3% of women's blastocysts resulting in +CPFH containing the CS compared to 88.7% of blastocysts in women who did not have a pregnancy (p = 0.007, OR; 4.67, CI 95% 1.5-14.2). CS appeared 2.4 h earlier in embryo development in the +CPFH group compared to their negative counterparts (p = 0.007). There was a significant difference in the average number of CS/blastocyst with a higher number being present in those that achieved a clinical pregnancy (mean: 6.2, SD 2.9) compared to those that did not (mean: 4.6, SD 3.0) (p ≤ 0.0001). There was a significant increase in the number of vesicles seen traveling along the CS with more seen in the blastocysts resulting in a +CPFH (mean: 4.3 SD 2.1) compared to those in the -CPFH group (mean: 3.1, SD 2.1). CONCLUSION: This study has shown that the presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart.

Rescuing monopronucleated-derived human blastocysts: a model to study chromosomal topography and fingerprinting.

OBJECTIVE: To quantify the percentage of monopronuclear-derived blastocysts (MNBs) that are potentially useful for reproductive purposes using classic and state-of-the-art chromosome analysis approaches, and to study chromosomal distribution in the inner cell mass (ICM) and trophectoderm (TE) for intertissue/intratissue concordance comparison. DESIGN: Prospective experimental study. SETTING: Single-center in vitro fertilization clinic and reproductive genetics laboratory. PATIENT(S): A total of 1,128 monopronuclear zygotes were obtained between June 2016 and December 2018. INTERVENTION(S): MNBs were whole-fixed or biopsied to obtain a portion of ICM and 2 TE portions (TE1 and TE2) and were subsequently analyzed by fluorescence in situ hybridization, new whole-genome sequencing, and fingerprinting by single-nucleotide polymorphism array-based techniques (a-SNP). MAIN OUTCOME MEASURE(S): We assessed MNB rate, ploidy rate, and chromosomal constitution by new whole-genome sequencing, and parental composition by comparative a-SNP, performed in a "trio"-format (embryo/parents). The 24-chromosome distribution was compared between the TE and the ICM and within the TE. RESULT(S): A total of 18.4% of monopronuclear zygotes progressed to blastocysts; 77.6% of MNBs were diploid; 20% of MNBs were male and euploid, which might be reproductively useful. Seventy-five percent of MNBs were biparental and half of them were euploid, indicating that 40% might be reproductively useful. Intratissue concordance (TE1/TE2) was established for 93.3% and 73.3% for chromosome matching. Intertissue concordance (TE/ICM) was established for 78.8%, but 57.6% for chromosome matching. When segmental aneuploidy was not considered, intratissue concordance and chromosome matching increased to 100% and 80%, respectively, and intertissue concordance and chromosome matching increased to 84.8% and 75.8%, respectively. CONCLUSION(S): The a-SNP-trio strategy provides information about ploidy, euploidy, and parental origin in a single biopsy. This approach enabled us to identify 40% of MNBs with reproductive potential, which can have a significant effect in the clinical setting. Additionally, segmental aneuploidy is relevant for mismatched preimplantation genetic testing of aneuploidies, both within and between MNB tissues. Repeat biopsy might clarify whether segmental aneuploidy is a prone genetic character.

SA-Net: A scale-attention network for medical image segmentation.

Semantic segmentation of medical images provides an important cornerstone for subsequent tasks of image analysis and understanding. With rapid advancements in deep learning methods, conventional U-Net segmentation networks have been applied in many fields. Based on exploratory experiments, features at multiple scales have been found to be of great importance for the segmentation of medical images. In this paper, we propose a scale-attention deep learning network (SA-Net), which extracts features of different scales in a residual module and uses an attention module to enforce the scale-attention capability. SA-Net can better learn the multi-scale features and achieve more accurate segmentation for different medical image. In addition, this work validates the proposed method across multiple datasets. The experiment results show SA-Net achieves excellent performances in the applications of vessel detection in retinal images, lung segmentation, artery/vein(A/V) classification in retinal images and blastocyst segmentation. To facilitate SA-Net utilization by the scientific community, the code implementation will be made publicly available.

Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1.

In vitro embryo production systems are limited by their inability to consistently produce embryos with the competency to develop to the blastocyst stage, survive cryopreservation, and establish a pregnancy. Previous work identified a combination of three cytokines [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1)], called FLI, that we hypothesize improve preimplantation development of bovine embryos in vitro. To test this hypothesis, FLI was supplemented into oocyte maturation or embryo culture medium. Embryos were produced in vitro using abattoir-derived oocytes and fertilized with sperm from a single bull known to have high fertility. After an 18-20 h fertilization period, putative zygotes were cultured in synthetic oviductal fluid (SOF) for 8 days. The addition of FLI to the oocyte maturation medium increased (P < 0.05) the dissociation of transzonal projections at 12, 18, and 24 h of maturation, as well as, the proportion of oocytes that reached the metaphase II stage of meiosis. Additionally, lipid content was decreased (P < 0.05) in the blastocyst stage embryo. The addition of FLI during the culture period increased development to the blastocyst stage, cytoskeleton integrity, and survival following slow freezing, as well as, decreased post thaw cell apoptosis (P < 0.05). In conclusion, the supplementation of these cytokines in vitro has the potential to alleviate some of the challenges associated with the cryo-survival of in vitro produced bovine embryos through improving embryo development and embryo quality.

Microfabricated Device for High-Resolution Imaging of Preimplantation Embryos.

The mouse preimplantation embryo is an excellent system for studying how mammalian cells organize dynamically into increasingly complex structures. Accessible to experimental and genetic manipulations, its normal or perturbed development can be scrutinized ex vivo by real-time imaging from fertilization to late blastocyst stage. High-resolution imaging of multiple embryos at the same time can be compromised by embryos displacement during imaging. We have developed an inexpensive and easy-to-produce imaging device that facilitates greatly the imaging of preimplantation embryo. In this chapter, we describe the different steps of production and storage of the imaging device as well as its use for live imaging of mouse preimplantation embryos expressing fluorescent reporters from genetically modified alleles or after in vitro transcribed mRNA transfer by microinjection or electroporation.

In Vitro Culture of Mouse Blastocysts to the Egg Cylinder Stage via Mural Trophectoderm Excision.

The developmental transition from the blastocyst to the egg cylinder stage is associated with stark changes in the overall shape of the embryo, as well as with reorganization of the transcriptional network and epigenetic landscape in the pluripotent and the supportive extraembryonic lineages. To directly analyze this pre- to postimplantation switch, culture conditions are needed that can support mouse embryogenesis beyond the blastocyst stage without maternal input. Here we provide a step-by-step protocol describing an experimental pipeline for isolating late blastocysts, excising (manually or via laser assistance) the mural trophectoderm, and, finally, culturing the embryo to the egg cylinder stage.

Mapping of Chromosome Territories by 3D-Chromosome Painting During Early Mouse Development.

Following fertilization in mammals, the chromatin landscape inherited from the two parental genomes and the nuclear organization are extensively reprogrammed. A tight regulation of nuclear organization is important for developmental success. One main nuclear feature is the organization of the chromosomes in discrete and individual nuclear spaces known as chromosome territories (CTs). In culture cells, their arrangements can be constrained depending on their genomic content (e.g., gene density or repeats) or by specific nuclear constrains such as the periphery or the nucleolus. However, during the early steps of mouse embryonic development, much less is known, specifically regarding how and when the two parental genomes intermingle. Here, we describe a three-dimensional fluorescence in situ hybridization (3D-FISH) for chromosome painting (3D-ChromoPaint) optimized to gain understanding in nuclear organization of specific CTs following fertilization. Our approach preserves the nuclear structure, and the acquired images allow full spatial analysis of interphase chromosome positioning and morphology across the cell cycle and during early development. This method will be useful in understanding the dynamics of chromosome repositioning during development as well as the alteration of chromosome territories upon changes in transcriptional status during key developmental steps. This protocol can be adapted to any other species or organoids in culture.

Embryonic MTHFR contributes to blastocyst development.

PURPOSE: Reduction in methylenetetrahydrofolate reductase (MTHFR) activity due to genetic variations in the MTHFR gene has been controversially implicated in subfertility in human in vitro fertilization. However, there is no direct gene-knockdown study of embryonic MTHFR to assess its involvement in mammalian preimplantation development. The purpose of this study is to investigate expression profiles and functional roles of MTHFR in bovine preimplantation development. METHODS: Reverse transcription-quantitative PCR (RT-qPCR) and analysis of publicly available RNA-seq data were performed to reveal expression levels of MTHFR during bovine preimplantation development. We knocked down MTHFR by siRNA-mediated RNA interference from the 8- to 16-cell stage and assessed the effects on preimplantation development. RESULTS: The RT-qPCR analysis showed relatively high MTHFR expression at the GV oocyte stage, which was decreased toward the 8- to 16-cell stage and then slightly restored at the blastocyst stage. Public data-based analysis also showed the similar pattern of expression with substantial embryonic expression at the blastocyst stage. MTHFR knockdown reduced the blastocyst rate (P < 0.01) and the numbers of total (P < 0.0001), trophectoderm (P < 0.0001), and inner cell mass (P < 0.001) cells. CONCLUSION: The results indicate that embryonic MTHFR is indispensable for normal blastocyst development. The findings provide insight into the debatable roles of MTHFR in fertility and may be applicable for the improvement of care for early embryos via modulation of surrounding folate-related nutritional conditions in vitro and/or in utero, depending on the parental and embryonic MTHFR genotype.

Non-invasive imaging of mouse embryo metabolism in response to induced hypoxia.

PURPOSE: This study used noninvasive, fluorescence lifetime imaging microscopy (FLIM)-based imaging of NADH and FAD to characterize the metabolic response of mouse embryos to short-term oxygen deprivation. We investigated the response to hypoxia at various preimplantation stages. METHODS: Mouse oocytes and embryos were exposed to transient hypoxia by dropping the oxygen concentration in media from 5-0% over the course of ~1.5 h, then 5% O2 was restored. During this time, FLIM-based metabolic imaging measurements of oocyte/embryo cohorts were taken every 3 minutes. Experiments were performed in triplicate for oocytes and embryos at the 1- to 8-cell, morula, and blastocyst stages. Maximum hypoxia response for each of eight measured quantitative FLIM parameters was taken from the time points immediately before oxygen restoration. RESULTS: Metabolic profiles showed significant changes in response to hypoxia for all stages of embryo development. The response of the eight measured FLIM parameters to hypoxia was highly stage-dependent. Of the eight FLIM parameters measured, NADH and FAD intensity showed the most dramatic metabolic responses in early developmental stages. At later stages, however, other parameters, such as NADH fraction engaged and FAD lifetimes, showed greater changes. Metabolic parameter values generally returned to baseline with the restoration of 5% oxygen. CONCLUSIONS: Quantitative FLIM-based metabolic imaging was highly sensitive to metabolic changes induced by hypoxia. Metabolic response profiles to oxygen deprivation were distinct at different stages, reflecting differences in metabolic plasticity as preimplantation embryos develop.

Selenoprotein I is essential for murine embryogenesis.

Selenoprotein I (SELENOI) is an ethanolamine phosphotransferase that catalyzes the third reaction of the Kennedy pathway for the synthesis of phosphatidylethanolamine. Since the role of SELENOI in murine embryogenesis has not been investigated, SELENOI-/+ mating pairs were used to generate global KO offspring. Of 323 weanling pups, no homozygous KO genotypes were found. E6.5-E18.5 embryos (165 total) were genotyped, and only two E18.5 KO embryos were detected with no discernable anatomical defects. To screen embryos prior to uterine implantation that occurs ~ E6, blastocyst embryos (E3.5-E4.4) were flushed from uteruses of pregnant females and analyzed for morphology and genotype. KO embryos were detected in 5 of 6 pregnant females, and 7 of the 32 genotyped embryos were found to be SELENOI KO that exhibited no overt pathological features. Overall, these results demonstrate that, except for rare cases (2/490 = 0.4%), global SELENOI deletion leads to early embryonic lethality.

Morphokinetic analysis of cleavage stage embryos and assessment of specific gene expression in cumulus cells independently predict human embryo development to expanded blastocyst: a preliminary study.

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage.

Removing the zona pellucida can decrease cytoplasmic fragmentations in human embryos: a pilot study using 3PN embryos and time-lapse cinematography.

PURPOSE: The aim of this study was to establish a new method of decreasing cytoplasmic fragmentation in early-stage human embryos. METHODS: The zona pellucida (ZP) of abnormally-fertilized oocytes (zygotes with three pronuclei (3PN)), which were donated by patients, was removed at the pronuclear stage. ZP-free embryos were observed in a time-lapse imaging and culturing system in order to examine developmental morphology and embryonic quality. RESULTS: Based on a modification of Veeck's criteria, 47 of 69 ZP-free 3PN embryos (68.1%) showed fragmentation of less than 20% of the total volume of cytoplasm at the first cleavage (grades 1 and 2), 17 (24.6%) showed 20-40% cytoplasmic fragments (grade 3), and only 5 (7.2%) showed more than 40% fragments (grade 4). These results suggest that the rate of fragmentation is decreased by ZP removal before the first cleavage, compared with normal (ZP-intact) 3PN and 2-pronuclear/2-polar body embryos. CONCLUSIONS: This study revealed that the ZP is not always necessary for normal development after the pronuclear stage because the ZP-free embryos studied herein developed normally, maintained their cell adhesion well, and showed a decreased rate of fragmentation. This innovative culture system might provide the major breakthrough needed for patients who have difficulty obtaining good-quality embryos.

Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers.

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 µl droplets of culture media, and 50 µl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential.

Blastulation of a zygote to a hatched blastocyst without any clear cell division: an observational finding in a time-lapse system after in vitro fertilization.

PURPOSE: To describe an interesting not previously described morphokinetic finding. METHODS: Retrospective case report of a couple undergoing controlled ovarian stimulation (COS) followed by in vitro fertilization and blastocyst transfer. RESULTS: We identified a unique finding of blastulation of a fertilized human zygote after conventional in vitro fertilization. The fertilized zygote did not show any clear cytokinesis until approximately 107 h post insemination, when it started dividing into a blastocyst. By 113 h post insemination, inner cell mass and trophectoderm cells could be clearly distinguished and the blastocyst was completely hatched by 136 h post insemination. CONCLUSION: Time-lapse systems offer more detailed observations of embryonic development. Here, we report an atypical development of an embryo that was not described previously. We hope to become an insightful discussion among peers and incentive the publication of such findings in the future.

Oocyte meiotic spindle morphology is a predictive marker of blastocyst ploidy-a prospective cohort study.

OBJECTIVE: To evaluate oocyte meiotic spindle (OMS) morphology at intracytoplasmic sperm injection (ICSI) as a predictor of blastocyst ploidy and whether OMS morphology could aid standard morphology-based blastocyst selection. DESIGN: Prospective cohort study. SETTING: In vitro fertilization clinic. PATIENT(S): Patients undergoing ICSI cycles with an intention to perform preimplantation genetic testing for aneuploidy (PGT-A) from October 2014 to December 2017. INTERVENTION(S): The OMS was visualized with the use of polarized light microscopy at the time of ICSI and the morphology classified as normal, dysmorphic, translucent, not visible, or in telophase. Blastocyst biopsy for PGT-A was performed on embryos with suitable development. MAIN OUTCOME MEASURE(S): The association of OMS morphology with the resulting blastocyst ploidy was evaluated on an "intention-to-treat" (ITT) and an "as-treated analysis" (ATA) basis. RESULT(S): The morphology of 2,056 OMSs were classified. A strong association of OMS morphology with fertilization, cleavage to at least 6 cells on day 3, and good/top-quality blastocyst formation was present. Normal OMS was positively associated with blastocyst euploidy compared with all other OMS types combined, per either ITT or ATA. Even after controlling for female age, blastocyst quality, and developmental stage, the presence of a normal OMS was strongly associated with the probability of blastocyst euploidy. CONCLUSION(S): OMS morphology is a predictive marker of blastocyst ploidy and can potentially aid standard morphology-based blastocyst selection.