Search

To determine what patient and embryo characteristics are correlated with the developmental potential of the peri-implantation embryo.Retrospective study.Research laboratory.Six hundred fifty-one cryopreserved human blastocysts donated for research with informed patient consent.Not applicable.Blastocyst attachment to fibronectin-coated plates, trophectoderm outgrowth area, epiblast cell number, total cell number, human chorionic gonadotropin secretion.Patients' body mass index, age, follicle-stimulating hormone: luteinizing hormone ratio on menstrual cycle day 3, antral follicle count on menstrual cycle day 3, antimüllerian hormone level on menstrual cycle day 3, and blastocyst morphological grade were correlated with peri-implantation development outcomes. After controlling for good-quality morphological grades, blastocysts from patients of advanced maternal age developed fewer epiblast cells than blastocysts from younger patients.Extended embryo culture during the peri-implantation period mirrors several disparities in fertility treatment outcome that we see clinically, including those from patients with advanced maternal age, high body mass index, and low ovarian reserve and from embryos with lower-quality morphological grades. This model system may be useful by providing an alternative or more sensitive endpoint assessment in studying patient, clinical, or laboratory factors that may influence preimplantation embryo developmental potential.

Single-cell RNA sequencing of cells from cultured human blastocysts has enabled us to define the transcriptomic landscape of placental trophoblast (TB) that surrounds the epiblast and associated embryonic tissues during the enigmatic day 8 (D8) to D12 peri-implantation period before the villous placenta forms. We analyzed the transcriptomes of 3 early placental cell types, cytoTB (CTB), syncytioTB (STB), and migratoryTB (MTB), picked manually from cultured embryos dissociated with trypsin and were able to follow sublineages that emerged from proliferating CTB at the periphery of the conceptus. A unique form of CTB with some features of STB was detectable at D8, while mature STB was at its zenith at D10. A form of MTB with a mixed MTB/CTB phenotype arose around D10. By D12, STB generation was in decline, CTB had entered a new phase of proliferation, and mature MTB cells had begun to move from the main body of the conceptus. Notably, the MTB transcriptome at D12 indicated enrichment of transcripts associated with IFN signaling, migration, and invasion and up-regulation of HLA-C, HLA-E, and HLA-G. The STB, which is distinct from the STB of later villous STB, had a phenotype consistent with intense protein export and placental hormone production, as well as migration and invasion. The studies show that TB associated with human embryos is in rapid developmental flux during peri-implantation period when it must invade, signal robustly to the mother to ensure that the pregnancy continues, and make first contact with the maternal immune system.

Research Question: In the early stages of the COVID-19 pandemic, IVF clinics stopped the majority of patient treatment cycles to minimize the risk of disease transmission. The risk of SARS-CoV-2 viral exposure and potential cross contamination within the IVF lab remains largely unclear. To that end, the objective of this study was to examine follicular fluid (FF), culture media (M) and vitrification solution (VS) for SARS-CoV-2 in an IVF lab. Design: Prospective clinical study. All females undergoing transvaginal oocyte retrieval (TVOR) were required to have a negative SARS-CoV-2 RNA test 3-4 days prior to the procedure. Male partners were not tested. All cases utilized intracytoplasmic sperm injection (ICSI). The first tube of FF aspirated during oocyte retrieval, M drops following removal of embryos on day 5, and VS after blastocyst cryopreservation were analyzed for SARS-CoV-2 RNA. Results: In total, M from 61 patients, VS from 200 patients, and FF from 300 patients were analyzed. All samples were negative for SARS-CoV-2 viral RNA. Conclusion(s): With stringent safety protocols in place, including female patient testing and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in FF, M or VS. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Invitro culture (IVC) systems fail to completely recapitulate the invivo environment, resulting in metabolic stress during pre-implantation development and reduced blastocyst quality. We hypothesised that IVC-induced metabolic dysregulation in bovine embryos is mediated by changes in expression and/or activity of protein biomarkers associated with key metabolic pathways. Our objectives were to determine (1) expression of enzymes involved in glycolysis (HK-2, PKM2, LDHA, B and C isoforms), entry into the tricarboxylic acid (TCA) cycle (PDH), energy sensing/fatty acid oxidation (AMPK), and the metabolic signalling pathways (AKT, ERK, STAT3, 4EBP1) at the 1-cell (1C), 8- to 16-cell (8–16 C), and Day 7 blastocyst (d7BL) stage; and (2) evaluate the functional activity of these proteins both invivo (superovulated and flushed) and invitro (IVM/IVF/IVC) produced embryos using capillary Western blot (Protein-Simple, JESS; n=1 embryo/stage; n=3 replicates). For each protein, expression was normalized with total protein abundance in the same capillary and functional activity was determine based on the ratio of phosphorylated (p) to total (t) protein abundance in each sample. Data were analysed using a two-sample t-test. Results demonstrated significantly (P