Transcriptome comparisons of trophoblasts from regenerative cell models with peri-implantation human embryos.

Mechanisms controlling trophoblast proliferation and differentiation during embryo implantation are poorly understood. Human trophoblast stem cells (TSC) and BMP4/A83-01/PD173074-treated pluripotent stem cell-derived trophoblast cells (BAP) are two widely employed, contemporary models to study trophoblast development and function, but how faithfully they mimic early trophoblast cells has not been fully examined. We evaluated the transcriptomes of trophoblast cells from BAP and TSC and directly compared them with those from peri-implantation human embryos during extended embryo culture (EEC) between embryonic day 8 to 12. The BAP and TSC grouped closely with trophoblast cells from EEC within each trophoblast sublineage following dimensional analysis and unsupervised hierarchical clustering. However, subtle differences in transcriptional programs existed within each trophoblast sublineage. We also validated the presence of six genes in peri-implantation human embryos by immunolocalization. Our analysis reveals that both BAP and TSC models have features of peri-implantation trophoblasts, while maintaining minor transcriptomic differences, and thus serve as valuable tools for studying implantation in lieu of human embryos.

Estrogen signaling encourages blastocyst development and implantation potential.

PURPOSE: Estrogen is well-known for preparing uterine receptivity. However, its roles in regulating embryo development and implantation are unclear. Our objective was to characterize estrogen receptor 1 (ESR1) in human and mouse embryos and determine the effect of estradiol (E2) supplementation on pre- and peri-implantation blastocyst development. METHODS: Mouse embryos, 8-cell through hatched blastocyst stages, and human embryonic days 5-7 blastocysts were stained for ESR1 and imaged using confocal microscopy. We then treated 8-cell mouse embryos with 8 nM E2 during in vitro culture (IVC) and examined embryo morphokinetics, blastocyst development, and cell allocation into the inner cell mass (ICM) and trophectoderm (TE). Finally, we disrupted ESR1, using ICI 182,780, and evaluated peri-implantation development. RESULTS: ESR1 exhibits nuclear localization in early blastocysts followed by aggregation, predominantly in the TE of hatching and hatched blastocysts, in human and mouse embryos. During IVC, most E2 was absorbed by the mineral oil, and no effect on embryo development was found. When IVC was performed without an oil overlay, embryos treated with E2 exhibited increased blastocyst development and ICM:TE ratio. Additionally, embryos treated with ICI 182,780 had significantly decreased trophoblast outgrowth during extended embryo culture. CONCLUSION: Similar ESR1 localization in mouse and human blastocysts suggests a conserved role in blastocyst development. These mechanisms may be underappreciated due to the use of mineral oil during conventional IVC. This work provides important context for how estrogenic toxicants may impact reproductive health and offers an avenue to further optimize human-assisted reproductive technology (ART) to treat infertility.

Dynamics of trophoblast differentiation in peri-implantation-stage human embryos.

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.

Absence of SARS-CoV-2 (COVID-19 virus) within the IVF laboratory using strict patient screening and safety criteria.

RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. 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.

Egg cylinder development during in vitro extended embryo culture predicts the post transfer developmental potential of mouse blastocysts.

PURPOSE: To establish parameters during mouse extended embryo culture that accurately predict fetal developmental potential of a blastocyst without performing embryo transfer. METHODS: Embryos of three varying qualities were produced: poor quality embryos produced from in vitro matured oocytes (IVM), intermediate quality embryos produced from in vivo matured oocytes followed by in vitro fertilization and embryo culture (IVF); high quality embryos developed in vivo (VIVO). Embryonic day (E) 3.5 embryos from each group with similar morphologies were used for surgical embryo transfer to assess implantation and fetal developmental potential, in addition to placing these embryos into extended culture until E 8.5 to examine outgrowth area, egg cylinder volume, epiblast cell number, and outgrowth morphologies by immunofluorescence and 3D confocal microscopy. RESULTS: The proportional differences in epiblast cell number are strikingly similar to fetal development following embryo transfer, suggesting that this parameter may be indicative of the potential of an embryo to successfully develop into a fetus. CONCLUSION: Extended embryo culture provides more accurate information regarding developmental potential than blastocyst morphological assessment. Specifically, epiblast cell number is an accurate and valuable predictor of fetal developmental potential. This work sets the stage for routine evaluation of embryo quality past the time embryos would normally be transferred. The ability to determine post implantation potential without embryo transfer may greatly improve efforts to culture higher quality embryos in vitro for human IVF, as well as reducing animal use and eliminating confounding maternal factors associated with embryo transfer experiments in research.

Maternal physiology and blastocyst morphology are correlated with an inherent difference in peri-implantation human embryo development.

OBJECTIVE: To determine what patient and embryo characteristics are correlated with the developmental potential of the peri-implantation embryo. DESIGN: Retrospective study. SETTING: Research laboratory. PATIENTS: Six hundred fifty-one cryopreserved human blastocysts donated for research with informed patient consent. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Blastocyst attachment to fibronectin-coated plates, trophectoderm outgrowth area, epiblast cell number, total cell number, human chorionic gonadotropin secretion. RESULTS: 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. CONCLUSIONS: 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.

Fatty acids present in commercial albumin preparations differentially affect development of murine embryos before and during implantation.

OBJECTIVE: To characterize fatty acid (FA) profile of commercially available albumin products and determine their effect on embryonic development. DESIGN: Research study. SETTING: Private research facility. ANIMAL(S): Outbred mice aged 4-8 weeks. INTERVENTION(S): Gas chromatography-mass spectrometry was used to quantify the FA content of 15 commercial albumins. Embryos were produced in media containing different albumin products, with or without carnitine or exogenous FA supplementation, to determine their effect on embryo development in vitro. MAIN OUTCOME MEASURE(S): Total micrograms of FA per milligram of albumin for the 15 albumin products, blastocyst development, cell number, allocation to the trophectoderm (TE) or inner cell mass (ICM), and evaluation of morphology during implantation. RESULT(S): The albumin products contained 0.07-16.77 µg total FA/mg albumin. Compared to media with with >1.4 µg FA/mg albumin, media with <0.5 µg FA/mg albumin supported improved blastocyst development, and addition of carnitine mitigated this difference. Addition of palmitoleic acid or oleic acid individually did not improve blastocyst development and decreased ICM:TE ratio. However, in the presence of carnitine, there was improved blastocyst development and maintenance of the ICM:TE ratio. Embryos cultured in Vitrolife human serum albumin with supplementation of carnitine, palmitoleic acid, and oleic acid were more likely to develop cells positive for POU5F1 in an extended embryo culture than embryos cultured in Origio serum protein substitute. CONCLUSION(S): Commercial albumin products contain FAs, which vary in abundance. These FAs have different effects on embryo development and quality before and during the implantation stage. Several of these albumin preparations are routinely used for human-assisted reproductive technologies; therefore, serious consideration is warranted when selecting a product for clinical use.

Human eggs, zygotes, and embryos express the receptor angiotensin 1-converting enzyme 2 and transmembrane serine protease 2 protein necessary for severe acute respiratory syndrome coronavirus 2 infection.

OBJECTIVE: To study messenger ribonucleic acid (mRNA) and protein expressions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry receptors (angiotensin 1-converting enzyme 2 [ACE2] and CD147) and proteases (transmembrane serine protease 2 [TMPRSS2] and cathepsin L [CTSL]) in human oocytes, embryos, and cumulus (CCs) and granulosa cells (GCs). DESIGN: Research study. SETTING: Clinical in vitro fertilization (IVF) treatment center. PATIENTS: Patients undergoing IVF were treated at the Colorado Center for Reproductive Medicine. INTERVENTIONS: Oocytes (germinal vesicle and metaphase II [MII]) and embryos (1-cell [1C] and blastocyst [BL]) were donated for research at the disposition by the patients undergoing IVF. Follicular cells (CC and GC) were collected from women undergoing egg retrieval after ovarian stimulation without an ovulatory trigger for in vitro maturation/IVF treatment cycles. MAIN OUTCOME MEASURES: Presence or absence of ACE2, CD147, TMPRSS2, and CTSL mRNAs detected using quantitative reverse transcription polymerase chain reaction and proteins detected using capillary Western blotting in human oocytes, embryos, and ovarian follicular cells. RESULTS: The quantitative reverse transcription polymerase chain reaction analysis revealed high abundance of ACE2 gene transcripts in germinal vesicle and MII oocytes than in CC, GC, and BL. ACE2 protein was present only in the MII oocytes, and 1C and BL embryos, but other ACE2 protein variants were observed in all the samples. TMPRSS2 protein was present in all the samples, whereas mRNA was observed only in the BL stage. All the samples were positive for CD147 and CTSL mRNA expressions. However, CCs and GCs were the only samples that showed coexpression of both CD147 and CTSL proteins in low abundance. CONCLUSIONS: CCs and GCs are the least susceptible to SARS-CoV-2 infection because of lack of the required combination of receptors and proteases (ACE2/TMPRSS2 or CD147/CTSL) in high abundance. The coexpression of ACE2 and TMPRSS2 proteins in the MII oocytes, zygotes, and BLs demonstrated that these gametes and embryos have the cellular machinery required and, thus, are potentially susceptible to SARS-CoV-2 infection if exposed to the virus. However, we do not know whether the infection occurs in vivo or in vitro in an assisted reproductive technology setting yet.

Evaluation of the TMRW vapor phase cryostorage platform using reproductive specimens and in vitro extended human embryo culture.

OBJECTIVE: To assess the impact of shipment and storage of sperm, oocytes, and blastocysts in vapor phase nitrogen compared with static storage in liquid phase nitrogen. DESIGN: Prospective cohort-matched study. SETTING: Multiple in vitro fertilization laboratories in an in vitro fertilization network. PATIENT(S): Fifty-eight human embryos, 32 human oocytes, 15 units of bovine semen. INTERVENTION(S): Vapor vs. liquid nitrogen. MAIN OUTCOME MEASURE(S): The postwarming survival of oocytes, sperm, and blastocysts, and the developmental potential of blastocysts during in vitro extended culture. RESULT(S): Custom-designed labware, for use with the TMRW platform, enables continuous temperature monitoring during shipment and/or storage in the vapor phase robotic storage system. The highest temperature recorded for specimens shipped to a domestic laboratory was -180.2 °C with a mean ± SD of -190.4 ± 0.5 °C during shipment and -181.1 ± 0.6 °C during storage. Likewise, specimens shipped internationally had a high of -180.2 °C with a mean ± SD of -193.5 ± 0.6 °C during shipment and -181.2 ± 0.7 °C during storage. Results from the extended culture assays have revealed no deleterious effect of shipment and storage in nitrogen vapor. The viability of mammalian gametes and embryos was equivalent between the vapor phase and liquid phase storage. CONCLUSION(S): The evaluated system did not have any deleterious effects on the postwarming survival of sperm, oocytes, and blastocysts. The postwarming developmental potential of human blastocysts during in vitro extended culture was unaffected by storage and handling in the vapor phase nitrogen TMRW platform when compared with static liquid phase nitrogen storage. Our results suggest that the vapor phase cryostorage platform is a safe system to handle and store reproductive specimens for human assisted reproductive technology.

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos.

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.