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Abstract It has long been speculated that the mechanical properties of the human oocyte can be an indicator for oocyte viability. Recent studies have demonstrated that embryo implantation rates, following Intra-Cytoplasmic Sperm Injection (ICSI) procedures, may be increased if the shear modulus value of the oocyte Zona Pellucida (ZP) is taken into consideration during embryo transfer. The shear modulus was determined by an iterative oocyte specific finite element (FE) analysis based on the clinical ICSI data. Nevertheless, the results obtained from the computational analysis may depend on the choice of the constitutive model used for the ZP. In the current study, three different hyper-elastic strain energy density functions (SEDF’s) are considered. The Neo-Hookean (NH), Mooney-Rivlin (MR) and Ogden models (OG) were used to determine the ZP shear modulus from ICSI clinical data. The sensitivity of each SEDF in extracting the oocyte specific shear modulus is examined, for the first time. It is demonstrated that the NH and MR models are more sensitive than the OG model for determining the ZP shear modulus from clinical ICSI data. It is also demonstrated that the accuracy of ZP shear modulus identification process greatly depends on the value of suction pressure applied during the ICSI procedure.

Abstract In vitro fertilization is typically associated with high failure rates per transfer, leading to an acute need for the identification of embryos with high developmental potential. Current methods are tailored to specific times after fertilization, often require expert inspection, and have low predictive power. Automatic methods are challenged by ambiguous labels, clinical heterogeneity, and the inability to utilize multiple developmental points. In this work, we propose a novel method that trains a classifier conditioned on the time since fertilization. This classifier is then integrated over time and its output is used to assign soft labels to pairs of samples. The classifier obtained by training on these soft labels presents a significant improvement in accuracy, even as early as 30 h post-fertilization. By integrating the classification scores, the predictive power is further improved. Our results are superior to previously reported methods, including the commercial KIDScore-D3 system, and a group of eight senior professionals, in classifying multiple groups of favorable embryos into groups defined as less favorable based on implantation outcomes, expert decisions based on developmental trajectories, and/or genetic tests.

Background: Sperm quality has decreased over the last decades worldwide. It is affected, among others, by season and heat. This study aimed to address the association between ambient temperature and sperm quality by assessing its shape using flexible multivariate models and identifying distinct time-dynamic patterns of temperature change based on unsupervised analysis., Material and Methods: A retrospective population-based study has been conducted, including all samples of males attending the Fertility and In-Vitro-Fertilization unit at a single medical center during 2016-2022. Flexible generalized models were fitted to characterize the relations between sperm quality and temperature while accounting for patients characteristics, and to identify temperature levels that correspond with the optimal sperm quality. This information was then used to estimate adjusted slope coefficients at specified time-windows., Results: In total, 4555 sperm samples were provided by 3229 individuals. Sperm concentration, motility and progressive motility were higher by 8 %, 11 % and 16 %, respectively, during the spring versus the fall season. Furthermore, their quality during early spermatogenesis improved with temperature, until a certain optimum around 23 °C-24 °C. Increasing temperature at later developmental stages was associated with lower sperm concentration and higher motility. Sperm concentration and motility were highest following a period of moderate gradual warming. Motility was higher and sperm concentration was lower, following a period with heatwaves or summer., Conclusions: This study assessed temperature role in sperm production quality by considering both average and time-dynamic temperatures. It identified several temperature change patterns over time and stratified the analysis by them. The differences in the relations across stages of spermatogenesis were addressed. Several mechanisms may explain the associations found, including heat-induced apoptosis of the sperm cells, and destruction of sperm cells DNA integrity by over-production of reactive oxygen species. The gradual global warming necessitates exploration of individual response to outdoor temperature in relations to genetic predisposition, lifestyle, and other health characteristics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To investigate whether morphokinetic parameters differ between male and female embryos in IVF embryos resulting in live births, a retrospective cohort study was undertaken. Files of all live births resulting from a single embryo transfer (SET) cultured in time-lapse incubators between 2013 and 2019 in two tertiary care centres were reviewed. The study group consisted of 187 SETs resulted in 187 live births, of which 100 were females (53.5%) and 87 were males (46.5%). Embryo selection for transfer was based on the known implantation data (KID) score provided by the Embryoscope and morphological assessment by experienced embryologists. Neonatal sex was confirmed through live birth documentation. Morphokinetic parameters and day 3 and day 5 KID scores of male and female embryos were compared. Maternal baseline and treatment characteristics were similar between groups. Morphokinetic time-lapse parameters of male and female embryos including: pronuclei fading; cleavage timings (t2-t9); second and third cell cycle durations; synchrony of the second and third cleavages; late morphokinetic parameters and KID scores did not differ between groups. In conclusion, time-lapse morphokinetic parameters and embryo selection methods do not seem to differ between male and female embryos, and their utilization does not bias towards any neonatal sex.

Blastocyst selection is primarily based on morphological scoring systems and morphokinetic data. These methods involve subjective grading and time-consuming techniques. Artificial intelligence allows for objective and quick blastocyst selection. In this study, 608 blastocysts were selected for transfer using morphokinetics and Gardner criteria. Retrospectively, morphometric parameters of blastocyst size, inner cell mass (ICM) size, ICM-to-blastocyst size ratio, and ICM shape were automatically measured by a semantic segmentation neural network model. The model was trained on 1506 videos with 102 videos for validation with no overlap between the ICM and trophectoderm models. Univariable logistic analysis found blastocyst size and ICM-to-blastocyst size ratio to be significantly associated with implantation potential. Multivariable regression analysis, adjusted for woman age, found blastocyst size to be significantly associated with implantation potential. The odds of implantation increased by 1.74 for embryos with a blastocyst size greater than the mean (147 ± 19.1 μm). The performance of the algorithm was represented by an area under the curve of 0.70 (p < 0.01). In conclusion, this study supports the association of a large blastocyst size with higher implantation potential and suggests that automatically measured blastocyst morphometrics can be used as a precise, consistent, and time-saving tool for improving blastocyst selection., (© 2023. Springer Nature Limited.)

Purpose: Our objective was to design an automated deep learning model that extracts the morphokinetic events of embryos that were recorded by time-lapse incubators. Using automated annotation, we set out to characterize the temporal heterogeneity of preimplantation development across a large number of embryos., Methods: To perform a retrospective study, we used a dataset of video files of 67,707 embryos from four IVF clinics. A convolutional neural network (CNN) model was trained to assess the developmental states that appear in single frames from 20,253 manually-annotated embryos. Probability-weighted superposition of multiple predicted states was permitted, thus accounting for visual uncertainties. Superimposed embryo states were collapsed onto discrete series of morphokinetic events via monotonic regression of whole-embryo profiles. Unsupervised K-means clustering was applied to define subpopulations of embryos of distinctive morphokinetic profiles., Results: We perform automated assessment of single-frame embryo states with 97% accuracy and demonstrate whole-embryo morphokinetic annotation with R-square 0.994. High quality embryos that had been valid candidates for transfer were clustered into nine subpopulations, as characterized by distinctive developmental dynamics. Retrospective comparative analysis of transfer versus implantation rates reveals differences between embryo clusters as marked by poor synchronization of the third mitotic cell-cleavage cycle., Conclusions: By demonstrating fully automated, accurate, and standardized morphokinetic annotation of time-lapse embryo recordings from IVF clinics, we provide practical means to overcome current limitations that hinder the implementation of morphokinetic decision-support tools within clinical IVF settings due to inter-observer and intra-observer manual annotation variations and workload constrains. Furthermore, our work provides a platform to address embryo heterogeneity using dimensionality-reduced morphokinetic descriptions of preimplantation development., (© 2023. The Author(s).)

Purpose: First trimester miscarriage is a major concern in IVF-ET treatments, accounting for one out of nine clinical pregnancies and for up to one out of three recognized pregnancies. To develop a machine learning classifier for predicting the risk of cleavage-stage embryos to undergo first trimester miscarriage based on time-lapse images of preimplantation development., Methods: Retrospective study of a 4-year multi-center cohort of 391 women undergoing intra-cytoplasmatic sperm injection (ICSI) and fresh single or double embryo transfers. The study included embryos with positive indication of clinical implantation based on gestational sac visualization either with first trimester miscarriage or live-birth outcome. Miscarriage was determined based on negative fetal heartbeat indication during the first trimester. Data were recorded and obtained in hospital setting and research was performed in university setting., Results: A minimal subset of six non-redundant morphodynamic features were screened that maintained high prediction capacity. Features that account for the distribution of the nucleolus precursor bodies within the small pronucleus and pronuclei dynamics were highly predictive of miscarriage outcome as evaluated using the SHapley Additive exPlanations (SHAP) methodology. Using this feature subset, XGBoost and random forest models were trained following a 100-fold Monte-Carlo cross validation scheme. Miscarriage was predicted with AUC 0.68 to 0.69., Conclusion: We report the development of a decision-support tool for identifying the embryos with high risk of miscarriage. Prioritizing embryos for transfer based on their predicted risk of miscarriage in combination with their predicted implantation potential is expected to improve live-birth rates and shorten time-to-pregnancy., (© 2022. The Author(s).)

Purpose: To assess the impact of post-thawing embryo culture on frozen embryo transfer (FET) outcomes., Methods: A retrospective cohort study including 678 consecutive FET cycles performed between the years 2004 and 2017 was conducted. Patients older than 45 years old were excluded. FET cycles were stratified as follows: (1) two-day (2d) embryos thawed and cultured to three-day (3d) versus 3d embryos thawed and transferred; (2) 2d or 3d embryos thawed and cultured to blastocysts versus blastocysts thawed and transferred. A p -value <.05 was considered statistically significant., Results: Maternal age, BMI, smoking, and basal FSH of the 2d and 3d cultured embryo group ( n  = 110) and the 3d non-cultured embryo group ( n  = 189) were comparable. Endometrium preparation protocols and the achieved endometrial thickness did not differ between groups. Pregnancy rate, implantation rate, clinical pregnancy, live birth rate, abortions, multiple pregnancies, perinatal outcomes, and birth weight were comparable.The 2d and 3d embryos cultured to blastocyst ( n  = 41) compared to non-cultured blastocyst ( n  = 338) showed that the non-cultured blastocyst patients had higher smoking rates and longer follicular phase. Endometrial thickness was comparable. The 2d and 3d embryos cultured to blastocyst stage had higher multiple pregnancies rate compared to the blastocyst non-cultured group, whereas pregnancy rate, implantation rate, live birth rate, miscarriages, perinatal outcomes, and birth weight were comparable., Conclusion: We could not demonstrate that the post-thaw culture had a significant impact on the outcome of FET cycles.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A time-lapse monitoring system provides a complete picture of the dynamic embryonic development process and simultaneously supplies extensive morphokinetic data. The objective of this study was to investigate whether the use of the morphokinetic parameter of time of starting blastulation (tSB) can improve the implantation rate of day-5 transferred blastocyst selected based on morphological parameters. In this retrospective study we analyzed the morphokinetics of 196 day-5 transferred blastocysts, selected solely based on morphological parameters. The interval time from intracytoplasmic sperm injection (ICSI) to time of starting blastocyst formation (tSB) was calculated for each embryo. The overall implantation rate of transferred blastocyst, selected based only on morphological parameters, was 49.2%. Implantation rate, determined retrospectively, was significantly higher (58.8% versus 42.6%, P = 0.02) for embryos with a short interval time to tSB (78-95.9 h) compared with embryos with a longer timeframe (96-114 h). Time of expanded blastocyst (tEB) post-ICSI was also significantly associated with implantation; however, this parameter was not available for all the embryos at time of transfer. When we tested only high ranked KIDScore day-3 sub-group embryos, the implantation rate was significantly higher in short interval time embryos compared with longer interval time embryos (62.2% vs. 45.5%, respectively, P = 0.02).These observations emphasize the importance of the timing of starting blastulation over blastocyst morphological parameters and may provide a preferable criterion for good morphology day-5 blastocyst selection.

Study Question: What is the clinical value of Day 7 blastocysts?, Summary Answer: Ending embryo culture at 144 hours post-insemination (h.p.i.; i.e. 6 days) would involve 7.3% and 4.4% relative reductions in the number of patients obtaining euploid blastocysts and live birth(s) (LBs), respectively., What Is Known Already: Many studies showed that Day 7 blastocysts are clinically valuable, although less euploid and less competent than faster-growing embryos. Nevertheless, a large variability exists in: (i) the definition of 'Day 7'; (ii) the criteria to culture embryos to Day 7; (iii) the clinical setting; (iv) the local regulation; and/or (v) the culture strategies and incubators. Here, we aimed to iron out these differences and portray Day 7 blastocysts with the lowest possible risk of bias. To this end, we have also adopted an artificial intelligence (AI)-powered software to automatize developmental timings annotations and standardize embryo morphological assessment., Study Design, Size and Duration: Observational study including 1966 blastocysts obtained from 681 patients cultured in a time-lapse incubator between January 2013 and December 2020 at a private Italian IVF center., Participants/materials, Setting, Methods: According to Italian Law 40/2004, embryos were not selected based on their morphology and culture to ≥168 h.p.i. is standard care at our center. ICSI, continuous culture with Day 5 media refresh, trophectoderm biopsy without assisted hatching and comprehensive chromosome testing (CCT) to diagnose full-chromosome non-mosaic aneuploidies, were all performed. Blastocysts were clustered in six groups based on the time of biopsy in h.p.i. at 12 hr intervals starting from <120 h.p.i. (set as control) up to >168 h.p.i. Blastocyst quality was assessed using Gardner's scheme and confirmed with AI-powered software. AI was also used to automatically annotate the time of expanding blastocyst (tEB) and the hours elapsing between this moment and the achievement of full expansion when blastocysts were biopsied and vitrified. Also, blastocyst area at tEB and at the time of biopsy was automatically assessed, as well as the hour of the working day when the procedure was performed. The main outcomes were the euploidy rate and the LB rate (LBR) per vitrified-warmed euploid single blastocyst transfer. The results were adjusted for confounders through multivariate logistic regressions. To increase their generalizability, the main outcomes were reported also based on a 144-h.p.i. cutoff (i.e. 6 exact days from ICSI). Based on this cutoff, all the main patient outcomes (i.e. number of patients obtaining blastocysts, euploid blastocysts, LBs, with supernumerary blastocysts without a LB and with surplus blastocysts after an LB) were also reported versus the standard care (>168 h.p.i.). All hypothetical relative reductions were calculated., Main Results and the Role of Chance: A total of 14.6% of the blastocysts reached full expansion beyond 144 h.p.i. (5.9% in the range 144-156 h.p.i., 7.9% in the range 156-168 h.p.i. and 0.8% beyond 168 h.p.i.). Slower blastocysts were of a worse quality based on the evaluation of both embryologists and AI. Both later tEB and longer time between tEB and full blastocyst expansion concurred to Day 7 development, quite independently of blastocyst quality. Slower growing blastocysts were slightly larger than faster-growing ones at the time of biopsy, but no difference was reported in the risk of hatching, mainly because two dedicated slots have been set along the working day for these procedures. The lower euploidy rate among Day 7 blastocysts is due to their worse morphology and more advanced oocyte age, rather than to a slower development per se. Conversely, the lower LBR was significant even after adjusting for confounders, with a first relevant decrease for blastocysts biopsied in the range 132-144 h.p.i. (N = 76/208, 36.5% versus N = 114/215, 53.0% in the control, multivariate odds ratio 0.61, 95% CI 0.40-0.92, adjusted-P = 0.02), and a second step for blastocysts biopsied in the range 156-168 h.p.i. (N = 3/21, 14.3%, multivariate odds ratio: 0.24, 95% CI 0.07-0.88, adjusted-P = 0.03). Nevertheless, when the cutoff was set at 144 h.p.i., no significant difference was reported. In this patient population, ending embryo culture at 144 h.p.i. would have caused 10.6%, 7.3%, 4.4%, 13.7% and 5.2% relative reductions in the number of patients obtaining blastocysts, euploid blastocysts, LBs, supernumerary blastocysts without an LB and surplus blastocysts after an LB, respectively., Limitations, Reasons for Caution: Gestational and perinatal outcomes were not assessed, and a cost-effectiveness analysis is missing. Moreover, we encourage other groups to investigate this topic with different culture and biopsy protocols, as well as in different clinical settings and regulatory contexts., Wider Implications of the Findings: In view of the increasing personalization and patient-centeredness of IVF, whenever allowed from the local regulations, the choice to culture embryos to Day 7 should be grounded on the careful evaluation of couples' reproductive history. Patients should be aware that Day 7 blastocysts are less competent than faster-growing ones; still, poor prognosis couples, couples less compliant toward other attempts in case of a failure and couples wishing for more than one child, may benefit from them. AI tools can help improving the generalizability of the evidence worldwide., Study Funding/competing Interest(s): This study did not receive any funding. I.E., A.B.M. and I.H.-V. are employees of Fairtility Ltd., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)