Lien Dhaenens

STUDY QUESTION: Is there evidence of a plateau in the cumulative live birth rate (cLBR) after a certain number of consecutive transfers of untested embryos? SUMMARY ANSWER: In our cohort of 11 463 women, the cLBR continues to increase with each additional transfer of an untested embryo, reaching 68.3% after six blastocyst transfers and 78.0% after 10 blastocyst transfers. WHAT IS KNOWN ALREADY: While cumulative success rates in ART are rising, implantation failure remains a persistent challenge. The actual frequency of recurrent implantation failure (RIF) and whether RIF surpasses the inherent implantation potential of transferred embryos remains a matter of debate. A recent study reported a cLBR of 98% after five euploid blastocyst transfers, suggesting that most implantation failures are likely embryonic rather than endometrial. However, it remains unclear how these findings can be extrapolated to patients who did not undergo preimplantation genetic testing for aneuploidy (PGT-A). While theoretical models estimate cumulative implantation probabilities based on published blastocyst euploidy rates by female age, real-world data on cumulative success in routine clinical practice remain limited. STUDY DESIGN, SIZE, DURATION: This non-interventional retrospective cohort study included records of all completed IVF/ICSI cycles (including thus fresh and frozen–thawed transfers of one oocyte retrieval cycle) in women who underwent IVF/ICSI at the Ghent University Hospital between January 2010 and December 2022. After excluding treatments involving PGT, oocyte donation and surrogacy, or a mix of cleavage stage and blastocyst stage transfers, our dataset consisted of 11 463 women who underwent a total of 19 378 IVF/ICSI cycles, resulting in a total of 31 478 embryo transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: The number of embryos transferred (‘time’) until achieving live birth (‘event’) was analysed using a Kaplan–Meier approach with inverse probability weighting (IPW). Additionally, logistic regression analysis was conducted to assess the predictive value of the number of previously transferred embryos on the live birth rates (LBRs) of the second and subsequent transfers, adjusting for female age, quality of previously transferred embryos, and stage of embryos transferred (cleavage stage versus blastocyst stage). MAIN RESULTS AND THE ROLE OF CHANCE: Kaplan–Meier estimates using an IPW approach showed cLBRs increasing from 51.1% (95% CI: 49.2–53.0%) after a third, up to 68.3% (95% CI: 64.6–72.0%) after a sixth and even as high as 78.0% (95% CI: 69.5–86.5%) after a tenth blastocyst transfer, respectively. As maternal age increases, higher numbers of blastocysts are required to achieve the same cLBR. Moreover, no age category achieves an 80% cLBR until after the transfer of eight blastocysts. Maternal age has a considerable effect, as illustrated by cLBR after the fourth blastocyst transfer of 68.9% (95% CI: 65.8–71.8%) for 42 years, respectively. In the adjusted logistic regression analysis, the odds for achieving live birth are estimated to decrease for each additional embryo transferred; however, this effect is not statistically significant (OR = 0.91; 95% CI: 0.86–1.07). Female age, as expected, was a significant predictor of implantation rate with subsequent transfers (OR = 0.92; 95% CI: 0.91–0.93). Additionally, being assigned to a blastocyst transfer strategy rather than to a cleavage stage transfer strategy was also a significant predictor (OR = 1.34; 95% CI: 1.20–1.51), as was proportion of embryos classified as excellent or good quality based on predefined morphological criteria out of the total number of embryos previously transferred (OR = 1.21; 95% CI: 1.06—1.38). Implantation rate is also correlated with the response to stimulation (P = 0.016) and the blastocyst formation rate (P < 0.0001). There was no significant difference in LBR after an equal number of previously unsuccessful blastocyst transfers, depending on how many oocyte collection cycles it took to reach that number of blastocysts. LIMITATIONS, REASONS FOR CAUTION: The results are limited by the observational retrospective design, and while regression analyses were adjusted for potential confounding factors, residual confounding may persist, particularly given the considerable heterogeneity in treatments. We did not exclude patients with factors associated with less favourable reproductive outcomes because our goal was to evaluate actual clinical practice. WIDER IMPLICATIONS OF THE FINDINGS: Our data emphasize the potential for successful live birth even after multiple unsuccessful transfers. Factors such as age, embryo quality, response to ovarian stimulation, and rate of blastocyst formation influence outcomes. By addressing these multifaceted influences, our research provides valuable insights and a hopeful outlook for patients undergoing fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): L.D. received a grant from the Agency for Innovation through Science (IWT SB-141441). The funder did not have any role in the study design; data collection, data analysis, and interpretation of data; the writing of the report; nor the decision to submit the paper for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

BACKGROUND: Trophectoderm (TE) biopsy and next generation sequencing (NGS) are currently the preferred techniques for preimplantation genetic testing for aneuploidies (PGT-A). Although this approach delivered important improvements over previous testing strategies, increased sensitivity has also prompted a rise in diagnoses of uncertain clinical significance. This includes reports of chromosomal mosaicism, suggesting the presence of karyotypically distinct cells within a single TE biopsy. Given that PGT-A relies on the chromosomal constitution of the biopsied cells being representative of the entire embryo, the prevalence and clinical implications of blastocyst mosaicism continue to generate considerable controversy. OBJECTIVE AND RATIONALE: The objective of this review was to evaluate existing scientific evidence regarding the prevalence and impact of chromosomal mosaicism in human blastocysts. We discuss insights from a biological, technical and clinical perspective to examine the implications of this diagnostic dilemma for PGT-A. SEARCH METHODS: The PubMed and Google Scholar databases were used to search peer-reviewed publications using the following terms: ‘chromosomal mosaicism’, ‘human’, ‘embryo’, ‘blastocyst’, ‘implantation’, ‘next generation sequencing’ and ‘clinical management’ in combination with other keywords related to the subject area. Relevant articles in the English language, published until October 2019 were critically discussed. OUTCOMES: Chromosomal mosaicism predominately results from errors in mitosis following fertilization. Although it appears to be less pervasive at later developmental stages, establishing the true prevalence of mosaicism in human blastocysts remains exceedingly challenging. In a clinical context, blastocyst mosaicism can only be reported based on a single TE biopsy and has been ascribed to 2–13% of embryos tested using NGS. Conversely, data from NGS studies disaggregating whole embryos suggests that mosaicism may be present in up to ~50% of blastocysts. However, differences in testing and reporting strategies, analysis platforms and the number of cells sampled inherently overshadow current data, while added uncertainties emanate from technical artefacts. Moreover, laboratory factors and aspects of in vitro culture generate further variability. Outcome data following the transfer of blastocysts diagnosed as mosaic remain limited. Current studies suggest that the transfer of putative mosaic embryos may lead to healthy live births, but also results in significantly reduced ongoing pregnancy rates compared to the transfer of euploid blastocysts. Observations that a subset of mosaic blastocysts has the capacity to develop normally have sparked discussions regarding the ability of embryos to self-correct. However, there is currently no direct evidence to support this assumption. Nevertheless, the exclusion of mosaic blastocysts results in fewer embryos available for transfer, which may inevitably compromise treatment outcomes. WIDER IMPLICATIONS: Chromosomal mosaicism in human blastocysts remains a perpetual diagnostic and clinical dilemma in the context of PGT-A. This review offers an important scientific resource, informing about the challenges, risks and value of diagnosing mosaicism. Elucidating these uncertainties will ultimately pave the way towards improved clinical and patient management.

What is the accuracy of preimplantation genetic testing for aneuploidies (PGT-A) when considering human peri-implantation outcomes in vitro? The probability of accurately diagnosing an embryo as abnormal was 100%, while the proportion of euploid embryos classified as clinically suitable was 61.9%, yet if structural and mosaic abnormalities were not considered accuracy increased to 100%, with a 0% false positive and false negative rate. Embryo aneuploidy is associated with implantation failure and early pregnancy loss. However, a proportion of blastocysts are mosaic, containing chromosomally distinct cell populations. Diagnosing chromosomal mosaicism remains a significant challenge for PGT-A. Although mosaic embryos may lead to healthy live births, they are also associated with poorer clinical outcomes. Moreover, the direct effects of mosaicism on early pregnancy remain unknown. Recently, developed in vitro systems allow extended embryo culture for up to 14 days providing a unique opportunity for modelling chromosomal instability during human peri-implantation development. A total of 80 embryos were cultured to either 8 (n = 7) or 12 days post-fertilisation (dpf; n = 73). Of these, 54 were PGT-A blastocysts, donated to research following an abnormal (n = 37) or mosaic (n = 17) diagnosis. The remaining 26 were supernumerary blastocysts, obtained from standard assisted reproductive technology (ART) cycles. These embryos underwent trophectoderm (TE) biopsy prior to extended culture. We applied established culture protocols to generate embryo outgrowths. Outgrowth viability was assessed based on careful morphological evaluation. Nine outgrowths were further separated into two or more portions corresponding to inner cell mass (ICM) and TE-derived lineages. A total of 45 embryos were selected for next generation sequencing (NGS) at 8 or 12 dpf. We correlated TE biopsy profiles to both culture outcomes and the chromosomal status of the embryos during later development. Of the 73 embryos cultured to 12 dpf, 51% remained viable, while 49% detached between 8 and 12 dpf. Viable, Day 12 outgrowths were predominately generated from euploid blastocysts and those diagnosed with trisomies, duplications or mosaic aberrations. Conversely, monosomies, deletions and more complex chromosomal constitutions significantly impaired in vitro development to 12 dpf (10% vs. 77%, P < 0.0001). When compared to the original biopsy, we determined 100% concordance for uniform numerical aneuploidies, both in whole outgrowths and in the ICM and TE-derived outgrowth portions. However, uniform structural variants were not always confirmed later in development. Moreover, a high proportion of embryos originally diagnosed as mosaic remained viable at 12 dpf (58%). Of these, 71% were euploid, with normal profiles observed in both ICM and TE-derived lineages. Based on our validation data, we determine a 0% false negative and 18.5% false positive error rate when diagnosing mosaicism. Overall, our findings demonstrate a diagnostic accuracy of 80% in the context of PGT-A. Nevertheless, if structural and mosaic abnormalities are not considered, accuracy increases to 100%, with a 0% false positive and false negative rate. The inherent limitations of extended in vitro culture, particularly when modelling critical developmental milestones, warrant careful interpretation. Our findings echo current prenatal testing data and support the high clinical predictive value of PGT-A for diagnosing uniform numerical aneuploidies, as well as euploid chromosomal constitutions. However, distinguishing technical bias from biological variability will remain a challenge, inherently limiting the accuracy of a single TE biopsy for diagnosing mosaicism. This research is funded by the Ghent University Special Research Fund (BOF01D08114) awarded to M.P., the Research Foundation-Flanders (FWO.KAN.0005.01) research grant awarded to B.H. and De Snoo-van't Hoogerhuijs Stichting awarded to S.M.C.d.S.L. We thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. N/A.

To what extent does a trophectoderm (TE) biopsy reliably reflect the chromosomal constitution of the inner cell mass (ICM) in human blastocysts? Concordance between TE and ICM was established in 62.1% of the embryos analysed. Next generation sequencing (NGS) platforms have recently been optimised for preimplantation genetic testing for aneuploidies (PGT-A). However, higher sensitivity has led to an increase in reports of chromosomal mosaicism within a single TE biopsy. This has raised substantial controversy surrounding the prevalence of mosaicism in human blastocysts and the clinical implications of heterogeneity between the TE and ICM. To define the distribution and rate of mosaicism in human blastocysts, we assessed chromosomal profiles of the ICM and multiple TE portions obtained from the same embryo. We evaluated donated embryos with an unknown chromosomal profile (n = 34), as well as PGT-A blastocysts, previously diagnosed as abnormal or mosaic (n = 24). Our intra-embryo comparison included a total of 232 samples, obtained from 58 embryos. Four embryo samples, including the ICM and three distinct TE portions, were acquired from good quality blastocysts by micromanipulation. Whole genome amplification (WGA), followed by NGS was performed on all embryo segments. Profiles were compared between samples from the same embryo, while the results from pretested blastocysts were further correlated to the original report. The embryos investigated in our untested group were obtained from good prognosis patients (n = 25), with maternal age ranging from 23 to 39 years. For the pretested embryo group, maternal age ranged from 23 to 40 years (n = 18). We uncover chromosomal mosaicism, involving both numerical and structural aberrations, in up to 37.9% of the blastocysts analysed. Within the untested group, the overall concordance between the ICM and all TE portions was 55.9%. A normal ICM was detected in 20.6% of blastocysts for which at least one TE portion showed a chromosomal aberration. Conversely, 17.6% of embryos presented with mosaic or uniform abnormalities within the ICM, while showing normal or mosaic TE profiles. For the pretested blastocysts, the overall concordance between the ICM and all TE samples was 70.8%. However, 50% of embryos previously diagnosed with mosaicism did not confirm the original diagnosis. Notably, 31.3% of embryos with a mosaic aberration reported in the original TE biopsy, revealed a euploid profile in the ICM and all three TE samples. Taken together, concordance between the ICM and all TE portions was established in 62.1% of blastocysts, across both embryo groups. Finally, we could not observe a significant effect of age on embryo mosaicism (P = 0.101 untested group; P = 0.7309 pretested group). Similarly, ICM and TE quality were not found to affect the occurrence of chromosomal mosaicism (P = 0.718 and P = 0.462 untested group; P = 1.000 and P = 0.2885 pretested group). All data that support the findings of this study are available online in Vivar (http://cmgg.be/vivar) upon request. Evaluating biological variation in some instances remains challenging. The technological limitations of sampling mitotic errors that lead to mosaicism, as well as WGA artefacts, warrant careful interpretation. Our results highlight the complex nature of genetic (in)stability during early ontogenesis and indicate that blastocysts harbour a higher rate of chromosomal mosaicism than may have been anticipated. Moreover, our findings reveal an overall high diagnostic sensitivity and relatively low specificity in the context of PGT-A. This suggests that a considerable proportion of embryos are potentially being classified as clinically unsuitable. Ultimately, more precise quantification will benefit the clinical management of embryo mosaicism. M.P. is supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF01D08114). J.T. and L.D. are supported by the agency for innovation through science (131673, 141441). B.H. and this research are supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF15/GOA/011). The authors declare no competing interests. Not applicable.

Can human oocyte calcium analysis predict fertilization success after assisted oocyte activation (AOA) in patients experiencing fertilization failure after ICSI? ICSI-AOA restores the fertilization rate only in patients displaying abnormal Ca2+ oscillations during human oocyte activation. Patients capable of activating mouse oocytes and who showed abnormal Ca2+ profiles after mouse oocyte Ca2+ analysis (M-OCA), have variable responses to ICSI-AOA. It remains unsettled whether human oocyte Ca2+ analysis (H-OCA) would yield an improved accuracy to predict fertilization success after ICSI-AOA. Sperm activation potential was first evaluated by MOAT. Subsequently, Ca2+ oscillatory patterns were determined with sperm from patients showing moderate to normal activation potential based on the capacity of human sperm to generate Ca2+ responses upon microinjection in mouse and human oocytes. Altogether, this study includes a total of 255 mouse and 122 human oocytes. M-OCA was performed with 16 different sperm samples before undergoing ICSI-AOA treatment. H-OCA was performed for 11 patients who finally underwent ICSI-AOA treatment. The diagnostic accuracy to predict fertilization success was calculated based on the response to ICSI-AOA. Patients experiencing low or total failed fertilization after conventional ICSI were included in the study. All participants showed moderate to high rates of activation after MOAT. Metaphase II (MII) oocytes from B6D2F1 mice were used for M-OCA. Control fertile sperm samples were used to obtain a reference Ca2+ oscillation profile elicited in human oocytes. Donated human oocytes, non-suitable for IVF treatments, were collected and vitrified at MII stage for further analysis by H-OCA. M-OCA and H-OCA predicted the response to ICSI-AOA in 8 out of 11 (73%) patients. Compared to M-OCA, H-OCA detected the presence of sperm activation deficiencies with greater sensitivity (75 vs 100%, respectively). ICSI-AOA never showed benefit to overcome fertilization failure in patients showing normal capacity to generate Ca2+ oscillations in H-OCA and was likely to be beneficial in cases displaying abnormal H-OCA Ca2+ oscillations patterns. The scarce availability of human oocytes donated for research purposes is a limiting factor to perform H-OCA. Ca2+ imaging requires specific equipment to monitor fluorescence changes over time. H-OCA is a sensitive test to diagnose gamete-linked fertilization failure. H-OCA allows treatment counseling for couples experiencing ICSI failures to either undergo ICSI-AOA or to participate in gamete donation programs. The present data provide an important template of the Ca2+ signature observed during human fertilization in cases with normal, low and failed fertilization after conventional ICSI. This work was supported by the Flemish fund for scientific research (FWO-Vlaanderen, G060615N). The authors have no conflict of interest to declare.