Nodal asymmetry and hedgehog signaling during vertebrate left-right symmetry breaking.

Development of visceral left-right asymmetry in bilateria is based on initial symmetry breaking followed by subsequent asymmetric molecular patterning. An important step is the left-sided expression of transcription factor pitx2 which is mediated by asymmetric expression of the nodal morphogen in the left lateral plate mesoderm of vertebrates. Processes leading to emergence of the asymmetric nodal domain differ depending on the mode of symmetry breaking. In Xenopus laevis and mouse embryos, the leftward fluid flow on the ventral surface of the left-right organizer leads through intermediate steps to enhanced activity of the nodal protein on the left side of the organizer and subsequent asymmetric nodal induction in the lateral plate mesoderm. In the chick embryo, asymmetric morphogenesis of axial organs leads to paraxial nodal asymmetry during the late gastrulation stage. Although it was shown that hedgehog signaling is required for initiation of the nodal expression, the mechanism of its asymmetry remains to be clarified. In this study, we established the activation of hedgehog signaling in early chick embryos to further study its role in the initiation of asymmetric nodal expression. Our data reveal that hedgehog signaling is sufficient to induce the nodal expression in competent domains of the chick embryo, while treatment of Xenopus embryos led to moderate nodal inhibition. We discuss the role of symmetry breaking and competence in the initiation of asymmetric gene expression.

Human blastocyst spontaneous collapse is associated with worse morphological quality and higher degeneration and aneuploidy rates: a comprehensive analysis standardized through artificial intelligence.

STUDY QUESTION: What are the factors associated with human blastocyst spontaneous collapse and the consequences of this event? SUMMARY ANSWER: Approximately 50% of blastocysts collapsed, especially when non-viable, morphologically poor and/or aneuploid. WHAT IS KNOWN ALREADY: Time-lapse microscopy (TLM) is a powerful tool to observe preimplantation development dynamics. Lately, artificial intelligence (AI) has been harnessed to automate and standardize such observations. Here, we adopted AI to comprehensively portray blastocyst spontaneous collapse, namely the phenomenon of reduction in size of the embryo accompanied by efflux of blastocoel fluid and the detachment of the trophectoderm (TE) from the zona pellucida (ZP). Although the underlying causes are unknown, blastocyst spontaneous collapse deserves attention as a possible marker of reduced competence. STUDY DESIGN, SIZE, DURATION: An observational study was carried out, including 2348 TLM videos recorded during preimplantation genetic testing for aneuploidies (PGT-A, n = 720) cycles performed between January 2013 and December 2020. All embryos in the analysis at least reached the time of starting blastulation (tSB), 1943 of them reached full expansion, and were biopsied and then vitrified. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI, blastocyst culture, TE biopsy without Day 3 ZP drilling, comprehensive chromosome testing and vitrification were performed. The AI software automatically registered tSB and time of expanding blastocyst (tEB), start and end time of each collapse, time between consecutive collapses, embryo proper area, percentage of shrinkage, embryo:ZP ratio at embryo collapse, time of biopsy (t-biopsy) and related area of the fully (re-)expanded blastocyst before biopsy, time between the last collapse and biopsy. Blastocyst morphological quality was defined according to both Gardner's criteria and an AI-generated implantation score. Euploidy rate per biopsied blastocyst and live birth rate (LBR) per euploid single embryo transfer (SET) were the main outcomes. All significant associations were confirmed through regression analyses. All couple, cycle and embryo main features were also investigated for possible associations with blastocyst spontaneous collapse. MAIN RESULTS AND THE ROLE OF CHANCE: At least one collapsing embryo (either viable or subsequently undergoing degeneration) was recorded in 559 cycles (77.6%) and in 498 cycles (69.2%) if considering only viable blastocysts. The prevalence of blastocyst spontaneous collapse after the tSB, but before the achievement of full expansion, was 50% (N = 1168/2348), irrespective of cycle and/or couple characteristics. Blastocyst degeneration was 13% among non-collapsing embryos, while it was 18%, 20%, 26% and 39% among embryos collapsing once, twice, three times or ≥4 times, respectively. The results showed that 47.3% (N = 918/1943) of the viable blastocysts experienced at least one spontaneous collapse (ranging from 1 up to 9). Although starting from similar tSB, the number of spontaneous collapses was associated with a delay in both tEB and time of biopsy. Of note, the worse the quality of a blastocyst, the more and the longer its spontaneous collapses. Blastocyst spontaneous collapse was significantly associated with lower euploidy rates (47% in non-collapsing and 38%, 32%, 31% and 20% in blastocysts collapsing once, twice, three times or ≥4 times, respectively; multivariate odds ratio 0.78, 95%CI 0.62-0.98, adjusted P = 0.03). The difference in the LBR after euploid vitrified-warmed SET was not significant (46% and 39% in non-collapsing and collapsing blastocysts, respectively). LIMITATIONS, REASONS FOR CAUTION: An association between chromosomal mosaicism and blastocyst collapse cannot be reliably assessed on a single TE biopsy. Gestational and perinatal outcomes were not evaluated. Other culture strategies and media should be tested for their association with blastocyst spontaneous collapse. Future studies with a larger sample size are needed to investigate putative impacts on clinical outcomes after euploid transfers. WIDER IMPLICATIONS OF THE FINDINGS: These results demonstrate the synergistic power of TLM and AI to increase the throughput of embryo preimplantation development observation. They also highlight the transition from compaction to full blastocyst as a delicate morphogenetic process. Blastocyst spontaneous collapse is common and associates with inherently lower competence, but additional data are required to deepen our knowledge on its causes and consequences. STUDY FUNDING/COMPETING INTEREST(S): There is no external funding to report. I.E., A.B.-M., I.H.-V. and B.K. are Fairtility employees. I.E. and B.K. also have stock or stock options of Fairtility. TRIAL REGISTRATION NUMBER: N/A.

Transcriptional profiling of embryo cryotolerance.

The cryosurvival of embryos is a complex process involving dynamic and integrated morphological, functional, and molecular changes. Here, we evaluated the transcriptional profiling of bovine embryos possessing high and low cryotolerance (HC and LC, respectively) by assessing the resumption of development. Embryos were produced in vitro (N = 1137) and cryopreserved (N = 894). Blastocysts samples possessed pronounced group individualization at RNA sequencing. A total of 114 genes were differentially expressed, and 27 and 84 genes were upregulated in HC and LC, respectively. Among the over-represented biological functions, cellular growth and proliferation, cell death and survival, and organismal survival were predicted to be activated, while cellular movement and cell-to-cell signaling were predicted to be inhibited in HC embryos. Enriched canonical pathways and upstream regulators related to cellular proliferation and survival (HC), inflammatory processes, and cell death (LC) were predicted to represent two embryonic molecular profiles present during the resumption of development after cryopreservation. The marked contrast in transcriptional profiles between HC and LC strongly suggests the influence of embryonic competence after cryopreservation on its respective transcriptome and indicated that HC and LC presented two different molecular strategies to overcome cryopreservation-related stress and resume postcryopreservation development.

Embryo's Natural Motion (enMotion): a paired randomized controlled trial evaluating a dynamic embryo culture system.

OBJECTIVE: To determine if a dynamic embryo culture system affects the reproductive potential of human embryos resulting from in vitro fertilization (IVF). DESIGN: Paired randomized controlled trial (RCT). SETTING: IVF center. PATIENT(S): IVF patients with normal ovarian reserve eligible for two-embryo transfer. INTERVENTION: IVF care was routine until fertilization was confirmed. Two-pronuclear embryos (2PNs) were then randomized: One-half of each patient's 2PNs were cultured in dynamic culture and one-half in static culture. Preimplantation genetic testing for embryonic aneuploidy was used to control for aneuploidy and allow for DNA fingerprinting. The best euploid blastocyst from each culture system was selected and patients underwent a frozen two-embryo transfer. If a singleton gestation resulted, DNA-fingerprinting was used to determine which of the two blastocysts implanted. The dynamic platform used was the NSSB-300 (Nepagene). MAIN OUTCOME MEASURE(S): The primary outcome was the proportion of usable blastocysts obtained. The secondary outcome was sustained implantation rate (SIR). RESULT(S): One hundred participants completed oocyte retrieval and blastocyst vitrification for frozen-thawed embryo transfer; 609 dynamic 2PNs and 615 static 2PNs were followed; and 304 blastocysts developed in dynamic culture and 333 blastocysts developed in static culture. In the paired analysis, the rate of usable blastulation was similar between dynamic and static culture (58.3% vs. 57.1%). In addition, there was no difference in the rate of aneuploidy (20.0% vs. 33.3%) or SIR (67.1% vs. 63.1%) between groups. CONCLUSION(S): In this paired RCT, dynamic culture did not improve usable blastulation rate or SIR. CLINICAL TRIAL REGISTRATION NUMBER: NCT02467725.

Investigating the impact of the timing of blastulation on implantation: management of embryo-endometrial synchrony improves outcomes.

STUDY QUESTION: Do embryos with delayed blastulation have inferior reproductive potential or poorer outcomes due in part to embryo and endometrial synchrony? SUMMARY ANSWER: Diminished outcomes in embryos with delayed blastulation undergoing fresh embryo transfer (ET) may be attributed to a loss of embryonic-endometrial synchrony. WHAT IS KNOWN ALREADY: Embryos that blastulate slowly have lower sustained implantation rates (SIR) than those which blastulate normally on Day 5 (D5). Traditionally this has been attributed to reduced embryo quality; however, dyssynchrony with the endometrium is also a possibility and has not been fully described. This convenient cohort composed of groups that resulted from a practice wide change in laboratory protocol allows for evaluation of embryo and endometrial synchrony as it related to blastocyst expansion. STUDY DESIGN SIZE DURATION: A retrospective cohort analysis was carried out from January 2009 to February 2013. Three cohorts were identified: D5 ET, D6 ET and frozen ET that comprised 822 patients, 763 patients and 718 patients, respectively. Each of these cohorts had slowly blastulating and normally blastulating embryos identified. PARTICIPANTS/MATERIALS SETTING METHODS: The study setting was academic affiliated private practice. All first fresh or cryopreserved ETs from 2010 to 2013 were studied. Non-biopsied embryos were classified into two groups on D5: slowly blastulating (Morula-Gardner 1) or normally blastulating (Gardner 2-6). Only single ETs or transfer of two embryos within the same classification group were included. Outcomes were compared between classification groups in embryos undergoing transfer on D5, D6, or after cryopreservation. This assesses the impact of transfer timing in fresh cycles as well as isolating a pure embryonic factor in frozen ET cycles. Sustained implantation was defined as heart beat detection at discharge sonogram at 8 weeks gestation. SIR was defined as the number of embryos transferred meeting criteria for sustained implantation divided by the total number of embryos transferred. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 3391 embryos were transferred to 1966 patients. On D5, SIRs were significantly lower with slowly blastulating embryos (44% versus 64% in women <35 years of age ( P < 0.001) and 18% versus 56% in women ≥35 years of age ( P < 0.001)). Fresh D6 ETs also had significantly lower SIRs for embryos that were slowly blastulating on D5 (52% versus 63% in <35 years of age (P < 0.05) and 32% versus 48% in ≥35 years of age (P < 0.005)) despite continued development to full blastocysts and being morphologically equivalent at the time of ET, suggesting dyssynchrony. However, when slowly blastulating embryos underwent vitrification and then ET, they had SIRs which were equivalent to their normally blastulating counterparts (57% versus 60% in <35 years of age (P = 0.5) and 37% versus 42% in ≥35 years of age (P = 0.3)). An intraclass correlation and a generalized estimating equation corrected for patient age was performed which confirmed these findings. The normalization in cryopreserved ETs indicates that dyssynchrony may be a major adverse factor limiting outcomes with late blastulating embryos in fresh cycles. LIMITATIONS REASONS FOR CAUTION: This is a retrospective study comprising cohorts from a convenient sample chosen due to a uniform change in embryology laboratory protocol regarding ET day, however, this was done independent of the management of embryo and endometrial synchrony. Although strict ultrasound and serum progesterone criteria were utilized to make endometrial receptivity uniform, pathologic states of pre-receptive and post-receptive endometrium cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS: The data surrounding embryo and endometrial synchrony should be considered in patients undergoing IVF and attention to the variations in blastulation rates can be applied to any patient undergoing extended embryo culture. STUDY FUNDING/COMPETING INTERESTS: None.