Protease secretions by the invading blastocyst induce calcium oscillations in endometrial epithelial cells via the protease-activated receptor 2.

BACKGROUND: Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive. METHODS: To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression. RESULTS: We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the 'Window of implantation' markers in human endometrial epithelial cells. CONCLUSIONS: These findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.

Predicting the chance on live birth per cycle at each step of the IVF journey: external validation and update of the van Loendersloot multivariable prognostic model.

OBJECTIVE: To study the performance of the 'van Loendersloot' prognostic model for our clinic's in vitro fertilisation (IVF) in its original version, the refitted version and in an adapted version replacing previous by current cycle IVF laboratory variables. METHODS: This retrospective cohort study in our academic tertiary fertility clinic analysed 1281 IVF cycles of 591 couples, who completed at least one 2nd-6th IVF cycle with own fresh gametes after a previous IVF cycle with the same partner in our clinic between 2010 and 2018. The outcome of interest was the chance on a live birth after one complete IVF cycle (including all fresh and frozen embryo transfers from the same episode of ovarian stimulation). Model performance was expressed in terms of discrimination (c-statistics) and calibration (calibration model, comparison of prognosis to observed ratios of five disjoint groups formed by the quintiles of the IVF prognoses and a calibration plot). RESULTS: A total of 344 live births were obtained (26.9%). External validation of the original van Loendersloot model showed a poor c-statistic of 0.64 (95% CI: 0.61 to 0.68) and an underestimation of IVF success. The refitted and the adapted models showed c-statistics of respectively 0.68 (95% CI: 0.65 to 0.71) and 0.74 (95% CI: 0.70 to 0.77). Similar c-statistics were found with cross-validation. Both models showed a good calibration model; refitted model: intercept=0.00 (95% CI: -0.23 to 0.23) and slope=1.00 (95% CI: 0.79 to 1.21); adapted model: intercept=0.00 (95% CI: -0.18 to 0.18) and slope=1.00 (95% CI: 0.83 to 1.17). Prognoses and observed success rates of the disjoint groups matched well for the refitted model and even better for the adapted model. CONCLUSION: External validation of the original van Loendersloot model indicated that model updating was recommended. The good performance of the refitted and adapted models allows informing couples about their IVF prognosis prior to an IVF cycle and at the time of embryo transfer. Whether this has an impact on couple's expected success rates, distress and IVF discontinuation can now be studied.

Intra- and interobserver analysis in the morphological assessment of early stage embryos during an IVF procedure: a multicentre study.

BACKGROUND: Quality control programs are necessary to maintain good clinical practice. Embryo grading has been described as one of the external quality assurance schemes. Although the evaluation of embryos is based on the assessment of morphological characteristics, considerable intra- and inter-observer variability has been described. In this multicentre study, the variability in the embryo evaluation has been evaluated using morphological characteristics on day 1, day 2 and day 3 of embryo development. METHODS: Five embryologists of four different IVF centers participated in this study. Multilevel images of embryos were presented on a website at different time points to evaluate intra-and inter-observer agreement in the assessment of embryo morphology. The embryos were evaluated on day 1, day 2 and day 3 of their development and each embryologist had to decide if the embryo had to be transferred, cryopreserved or discarded. RESULTS: Both intra-observer agreement and inter-observer agreement were good to excellent for the position of the pronuclei on day 1, the number of blastomeres on day 2 and day 3 and the clinical decision (transfer, cryopreservation, discard). For all other characteristics (size of pronuclei, presence of cytoplasomic halo, degree of fragmentation and size of blastomeres) the intra- and inter-observer agreement was moderate to very poor. CONCLUSIONS: Mono- or multicentre quality control on embryo scoring by morphological assessment can easily be performed through the design of a simple website. In the future the website design can be adapted to generate statistical feedback upon scoring and can even include a training module.

Intra- and inter-observer analysis in the morphological assessment of early-stage embryos.

BACKGROUND: The aim of this study was to determine the intra- and inter-observer variability in the evaluation of embryo quality. Multilevel images of embryos on day 1, day 2 and day 3, were analysed using different morphological parameters. METHODS: Multilevel images of embryos on day 1, day 2 and day 3, were analysed using a standard scoring system. The kappa coefficient was calculated to measure intra- and inter-observer variability before and after training sessions. RESULTS: Good to excellent intra-observer agreement was present for most parameters exceptions being scoring the position of pronuclei and the presence of a cytoplasmic halo on day 1, multinucleation on day 2 and the size of fragments on day 3. Inter-observer agreement was only good to excellent for the number of blastomeres on day 2 and day 3 and the orientation of the cleavage axes on day 2. Training sessions had a positive impact on inter-observer agreement. CONCLUSION: In conclusion, assessment of morphological characteristics of early stage embryos using multilevel images was marked by a high intra-observer and a moderate inter-observer agreement. Training sessions were useful to increase inter-observer agreement.