Synergie entre biologie du développement et techniques d’assistance médicale à la procréation pour l’étude de l’embryon humain préimplantatoire

Since the beginning of Assisted Reproductive Technologies (ART) in the 80’s, techniques allowing embryo development and observation were optimized. Among them, Time-lapse incubators gave embryologists access to all embryo preimplantation development parameters. In the clinical part of this work, I focused on the contribution of morphokinetic parameters for the evaluation of embryo quality in ART. In parallel with these technological advances, fundamental knowledge concerning biology of pre- implantation human development remains largely incomplete. Omics tools, such as transcriptomics and proteomics, allowing high throughput and indepth analysis, as well as animal models, have helped to advance knowledge in developmental biology. However, it is essential to identify very precisely the stage of development and the location of each cell to decipher the mecanisms of cellular specification in human morula and blastocyst. As part of this research, I was first able to use my time-lapse expertise to accurately annotate the analyzed embryos. I was also able to use my skills in embryonic micro-manipulation to set up and implement human embryo dissection to separate the cells of different embryonic compartments.Depuis les débuts de l’Assistance Médicale à la Procréation (AMP), les technologies permettant la culture et l’observation embryonnaire ont beaucoup évolué. Les incubateurs de type time-lapse ont notamment permis aux embryologistes d’avoir accès à l’ensemble du développement préimplantatoire. Dans le cadre de la partie clinique de ce travail, j’ai étudié l’apport des paramètres morphocinétiques pour l’évaluation de la qualité embryonnaire en AMP. En parallèle à ces avancées technologiques en AMP clinique, les connaissances fondamentales en biologie du développement humain pré- implantatoire restent largement incomplètes. La mise en place d’outils Omics de plus en plus performants, tels que l’analyse transcriptomique ou protéomique, ainsi que les modèles animaux, ont permis de faire progresser la connaissance. Cependant, il est indispensable de connaitre très précisément le stade de développement et la localisation de chaque compartiment cellulaire / cellule pour comprendre les mécanismes de destin cellulaire. Dans le cadre de ce travail de recherche, j’ai pu tout d’abord mettre à profit mon expertise en time-lapse pour annoter précisément les embryons analysés. J’ai pu également appliquer mon expertise en micro- manipulation embryonnaire pour mettre au point dissection embryonnaire permettant de séparer les cellules constituant les différents compartiments embryonnaires

Can time-lapse parameters predict embryo ploidy? A systematic review

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Developmental-stage specific single-cell human embryo dissociation

Summary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Morphokinetic parameters in chromosomal translocation carriers undergoing preimplantation genetic testing

International audienceRESEARCH QUESTION: Can embryo morphokinetic parameters help identify unbalanced embryos in translocation carriers?DESIGN: This retrospective study was conducted in 67 translocation carriers undergoing 105 preimplantation genetic testing cycles for chromosomal structural rearrangements (PGT-SR) without aneuploidy screening (PGT-A). Using time-lapse imaging analysis, morphokinetic parameters of balanced and unbalanced embryos were compared, as well as the frequency of abnormal cellular events. The performance of a previously published prediction model of aneuploidy was also tested in this population.RESULTS: Significant differences were observed between balanced and unbalanced embryos for some morphokinetic parameters: t5 (P = 0.0067), t9+ (P = 0.0077), cc2 (P = 0.0144), s2 (P = 0.0003) and t5-t2 (P = 0.0028). Also, multinucleation at the two- or four-cell stages, abnormal division and cell exclusion at the morula stage were significantly (all P < 0.05) more frequent in unbalanced than in balanced embryos. None, however, could accurately predict embryo chromosomal status. A previously published morphokinetic prediction model for embryo aneuploidy did not adequately classify balanced and unbalanced embryos.CONCLUSIONS: No significant morphokinetic predictor of chromosomal status could be found. Time-lapse should not be used as a diagnostic tool for chromosomal status in translocation carriers

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

International audiencePurpose: 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

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

International audiencePurpose: 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

External validation of a time-lapse prediction model

International audienceOBJECTIVE:To study the performance of a previously published implantation prediction model based on morphokinetics in a different setting, in an unselected population and with various embryo transfer strategies.DESIGN:Retrospective monocentric study.SETTING:University-based assisted reproduction technology (ART) center.PATIENT(S):450 unselected couples undergoing intracytoplasmic sperm injection (ICSI) cycle with embryo culture in the EmbryoScope (Unisense Fertilitech), corresponding to 528 embryos with known implantation.INTERVENTION(S):None.MAIN OUTCOME MEASURE(S):Implantation rates (IR) in embryo categories defined by the model in the overall population and in subgroups according to the day of embryo transfer.RESULT(S):The distribution of IR among detailed morphokinetic categories in the overall population and in subgroups according to the day of embryo transfer was more heterogeneous than expected according to the published model. The distribution corresponded better to the original when a simplified version of the model was used, although it worked better in the cleavage-stage group than in the blastocyst-stage group.CONCLUSION(S):This study was unsuccessful in replicating the sensitivity of the previously published model for predicting implantation rate of embryos ranked according to morphokinetic categories. Further work is required to assess the utility of the model for embryo selection. Each team using time-lapse technology should build a center-specific prediction model based on its own data and transfer policy

Does sperm origin affect embryo morphokinetic parameters?

International audiencePurpose The purpose of our study was to use time-lapse in order to evaluate the impact of sperm origin (fresh ejaculate or surgically retrieved) on embryo morphokinetic parameters and clinical outcome in intracytoplasmic sperm injection (ICSI) cycles.Methods This retrospective monocentric study was conducted in 485 unselected couples undergoing 604 ICSI cycles with embryo culture in the Embryoscope®. Among them, 445 couples underwent ICSI cycle with fresh ejaculated sperm and 40 with surgically retrieved sperm (26 with testicular sperm and 14 with epididymal sperm). Embryo morphokinetic parameters and clinical cycle outcome were compared between fresh ejaculated sperm and surgically retrieved sperm. A subgroup analysis was also conducted between testicular and epididymal sperm ICSI cycles.Results Clinical outcome was comparable between groups according to sperm origin. Although most early morphokinetic parameters were comparable between ejaculated and surgical sperm groups, a few parameters were significantly different between both groups, but with a considerable overlap in their distribution. Late cellular events occurred significantly later in the surgical sperm group than in the ejaculated sperm group. Conclusions Morphokinetic analysis did not allow us to identify clinically relevant differences between fresh ejaculate and surgically retrieved sperm groups. Further studies are needed, especially concerning the relationship between sperm origin and late morphokinetic parameters, such as blastocyst development

Performance of Day 5 KIDScore™ morphokinetic prediction models of implantation and live birth after single blastocyst transfer

International audiencePURPOSE : While several studies reported the association between morphokinetic parameters and implantation, few predictive models were developed to predict implantation after day 5 embryo transfer, generally without external validation. The objective of this study was to evaluate the respective performance of 2 commercially available morphokinetic-based models (KIDScore™ Day 5 versions 1 and 2) for the prediction of implantation and live birth after day 5 single blastocyst transfer.METHODS : This monocentric retrospective study was conducted on 210 ICSI cycles with single day 5 embryo transfer performed with a time-lapse imaging (TLI) system between 2013 and 2016. The association between both KIDScore™ and the observed implantation and live birth rates was calculated, as well as the agreement between embryologist's choice for transfer and embryo ranking by the models.RESULTS : Implantation and live birth rate were both 35.7%. A significant positive correlation was found between both models and implantation rate (r = 0.96 and r = 0.90, p = 0.01) respectively. Both models had statistically significant but limited predictive power for implantation (AUC 0.60). There was a fair agreement between the embryologists' choice and both models (78% and 61% respectively), with minor differences in case of discrepancies.CONCLUSIONS : KIDScore™ Day 5 predictive models are significantly associated with implantation rates after day 5 single blastocyst transfer. However, their predictive performance remains perfectible. The use of these predictive models holds promises as decision-making tools to help the embryologist select the best embryo, ultimately facilitating the implementation of SET policy. However, embryologists' expertise remains absolutely necessary to make the final decision