Developmental-stage specific single-cell human embryo dissociation.

Isolation of individual cells ensures detailed analysis of human embryos and promotes our understanding of molecular mechanisms driving embryo development and cell specification. Here, we present a protocol for the processing of human embryos for single-cell analysis. We describe steps for growing embryos and individualizing cells from the polar and the mural parts of trophectoderm at the blastocyst stage using laser dissection. We then detail embryo dissociation followed by steps to pick, wash, and dispense cells in plates.

What is the best strategy for slowly developing blastocysts?

Day 5 fresh blastocyst transfers results in higher clinical pregnancy and live birth rates than day 6 fresh blastocyst transfer. This study aimed to identify the strategy to adopt with slowly developing blastocysts. Should not fully expanded blastocyst on day 5 be transferred on day 5, or when expanded on day 6, or be frozen? 1093 single blastocyst transfer cycles performed between January 2016 and December 2018 were divided in 4 groups: day 5 fresh transfers of full or expanded blastocyst (≥B3), day 5 fresh transfers of slowly developing blastocysts (B1 or B2), day 6 fresh transfers of expanded blastocysts (≥B4), day 6 frozen-thawed single blastocyst transfer cycles. Clinical pregnancy rate and live birth rate were significantly higher with fresh expanded blastocyst transfer on day 5 than in any other group. No statistical difference could be found between the other 3 groups. Slowly developing day 5 blastocysts have poorer implantation potential than expanded day 5 blastocysts but can be fresh transferred on day 5 rather than being cultured until day 6 for transfer or freezing when no expanded blastocyst is available on day 5.

Human blastoids model blastocyst development and implantation.

One week after fertilization, human embryos implant into the uterus. This event requires the embryo to form a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form a blastocyst model that we called a blastoid1. Here we show that naive human pluripotent stem cells cultured in PXGL medium2 and triply inhibited for the Hippo, TGF-ß and ERK pathways efficiently (with more than 70% efficiency) form blastoids generating blastocyst-stage analogues of the three founding lineages (more than 97% trophectoderm, epiblast and primitive endoderm) according to the sequence and timing of blastocyst development. Blastoids spontaneously form the first axis, and we observe that the epiblast induces the local maturation of the polar trophectoderm, thereby endowing blastoids with the capacity to directionally attach to hormonally stimulated endometrial cells, as during implantation. Thus, we propose that such a human blastoid is a faithful, scalable and ethical model for investigating human implantation and development3,4.

Integrated pseudotime analysis of human pre-implantation embryo single-cell transcriptomes reveals the dynamics of lineage specification.

Understanding lineage specification during human pre-implantation development is a gateway to improving assisted reproductive technologies and stem cell research. Here we employ pseudotime analysis of single-cell RNA sequencing (scRNA-seq) data to reconstruct early mouse and human embryo development. Using time-lapse imaging of annotated embryos, we provide an integrated, ordered, and continuous analysis of transcriptomics changes throughout human development. We reveal that human trophectoderm/inner cell mass transcriptomes diverge at the transition from the B2 to the B3 blastocyst stage, just before blastocyst expansion. We explore the dynamics of the fate markers IFI16 and GATA4 and show that they gradually become mutually exclusive upon establishment of epiblast and primitive endoderm fates, respectively. We also provide evidence that NR2F2 marks trophectoderm maturation, initiating from the polar side, and subsequently spreads to all cells after implantation. Our study pinpoints the precise timing of lineage specification events in the human embryo and identifies transcriptomics hallmarks and cell fate markers.

Comparison of two automated sperm analyzers using 2 different detection methods versus manual semen assessment.

PURPOSE: The exploration of male infertility is mainly based on semen analysis, but its evaluation might be affected by the operator's competence and subjectivity. This led to the development of automated semen analyzing systems. Despite continuous improvement, the precision and correlation of these automated systems with manual sperm assessment performed strictly according to WHO guidelines remains variable in the literature, and their role in daily practice is debated. METHODS: In this double blind prospective study, we compared the results provided by 2 automated systems based on different concepts (CASA and electro-optical signal) with manual sperm assessment. Sperm concentration, motility and morphology were performed simultaneously and independently by different operators, blinded to each other. RESULTS: A total of 102 unselected men attending the andrology department for routine sperm analysis were included in the study. We found no significant difference between each automated method and manual assessment for all sperm parameters, except for sperm morphology assessment where the electro-optical system gave higher results and performed slightly poorer than CASA. Correlation was moderate to high between manual assessment and each automated methods for all sperm parameters, with randomly distributed differences. CONCLUSIONS: Overall, these results show that both types of automated systems can be implemented in andrology laboratory for routine sperm analysis.

Time-lapse technology improves total cumulative live birth rate and shortens time to live birth as compared to conventional incubation system in couples undergoing ICSI.

PURPOSE: The improvement of clinical outcome provided by time-lapse technology (TLT) in IVF over conventional incubation (CI) still remains controversial. This study aimed at evaluating whether the exclusive use of time-lapse technology (TLT) during whole IVF care improves total cumulative live birth rate (TCLBR) and shortens time to live birth (TTLB) as compared to the use of CI in couples undergoing ICSI. METHODS: This retrospective cohort study was conducted in couples with male infertility undergoing their first ICSI cycle in 2014-2015 and for whom embryo culture system remained the same during their whole IVF care, i.e., TLT or CI. Couples were followed up up to 2020, including all following frozen-embryo transfers and ICSI cycles (if any). Survival analysis was used to compare clinical outcome and time-related endpoints between both groups. RESULTS: A total of 151 and 250 couples underwent their whole IVF care with the exclusive use of TLT and CI, respectively. Survival analysis showed that TCLBR after whole IVF care was significantly higher in TLT than in CI group (66.9 vs 56.4%, p=0.02, log-rank test). Median live birth time was significantly shorter in TLT than CI group (464 vs 596 days, p=0.01). CONCLUSIONS: We found that TCLBR and TTLB were significantly improved with TLT over CI in couples undergoing ICSI for male factor. This study fuels the debate on the clinical benefit of using TLT. The use of time-related endpoints adds important information for both patients and practitioners.

Modification of late human embryo development after blastomere removal on day 3 for preimplantation genetic testing.

The purpose of our study was to use a time-lapse monitoring (TLM) system to determine if day 3 blastomere biopsy for preimplantation genetic testing (PGT) had an impact on subsequent morphokinetic parameters at the morula and blastocyst stages. In this retrospective monocentric study conducted between May 2013 and August 2017, we compared late morphokinetic parameters in embryos undergoing day 3 blastomere biopsy for PGT and in control non-biopsied embryos obtained in intracytoplasmic sperm injection (ICSI) cycles for male infertility. All embryos in both groups were cultured in a TLM system. The biopsy group was composed of 1691 embryos (386 PGT cycles). The control group was composed of 2578 embryos (786 ICSI cycles). Early morphokinetic parameters up to day 3 were similar in both groups. Concerning late morphokinetic parameters, the onset of compaction (tSC), fully-compacted morula stage (tM), onset of cavitation/early blastulation (tSB), and blastocyst stages (tB and tEB) appeared significantly earlier in the biopsy group than in the control group. We found that late morphokinetic events at the morula and the blastocyst stages occurred significantly earlier in biopsied embryos than in control non-biopsied-embryos. The mechanisms underlying these modifications of embryo development after biopsy should be investigated in order to determine precisely, and this phenomenon could be associated with embryo, fetal, and offspring development.Abbreviations: TLM: time-lapse monitoring; PGT: preimplantation genetic testing; ICSI: intracytoplasmic sperm injection; tSC: the onset of compaction; tM: fully-compacted morula stage; tSB: onset of cavitation/early blastulation; tB and tEB: blastocyst stages; OHSS: ovarian hyperstimulation syndrome.

Induction of Human Trophoblast Stem Cells from Somatic Cells and Pluripotent Stem Cells.

Human trophoblast stem cells (hTSCs) derived from blastocysts and first-trimester cytotrophoblasts offer an unprecedented opportunity to study the placenta. However, access to human embryos and first-trimester placentas is limited, thus preventing the establishment of hTSCs from diverse genetic backgrounds associated with placental disorders. Here, we show that hTSCs can be generated from numerous genetic backgrounds using post-natal cells via two alternative methods: (1) somatic cell reprogramming of adult fibroblasts with OCT4, SOX2, KLF4, MYC (OSKM) and (2) cell fate conversion of naive and extended pluripotent stem cells. The resulting induced/converted hTSCs recapitulated hallmarks of hTSCs including long-term self-renewal, expression of specific transcription factors, transcriptomic signature, and the potential to differentiate into syncytiotrophoblast and extravillous trophoblast cells. We also clarified the developmental stage of hTSCs and show that these cells resemble day 8 cytotrophoblasts. Altogether, hTSC lines of diverse genetic origins open the possibility to model both placental development and diseases in a dish.

Initiation of a conserved trophectoderm program in human, cow and mouse embryos.

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2-5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.

Identification of cryptic Candida species by MALDI-TOF mass spectrometry, not all MALDI-TOF systems are the same: focus on the C. parapsilosis species complex.

Data about the performance of MALDI-TOF mass spectrometry against cryptic Candida species are limited. According to our findings within the C. parapsilosis species complex, microbiologists should be aware that the choice of the instrument is critical for accurate species identification due to the risk of misidentification in the clinical setting.