Cytoplasmic strings in human blastocysts: hypotheses of their role and implications for embryo selection.

STUDY QUESTION: What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts? SUMMARY ANSWER: Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality. WHAT IS KNOWN ALREADY: Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial. STUDY DESIGN, SIZE, DURATION: Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics. MAIN RESULTS AND THE ROLE OF CHANCE: About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700 µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10). LIMITATIONS, REASONS FOR CAUTION: Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Embryo multinucleation: detection, possible origins, and implications for treatment.

Cell cycle regulation is crucial to assure expansion of a cell population, while preserving genome integrity. This notion is especially relevant to fertilization and early embryo development, a time when the cell cycle transforms from meiotic into mitotic cycles. Zygote-to-embryo transition is acutely error-prone, causing major developmental perturbations, including cleavage delays, tri- and multi-chotomous cleavages, and cell fragmentation. Another such alteration is bi- and multinucleation, consisting of the simultaneous formation of two or more nuclei at interphase. Indeed, multinucleation affects a large proportion of early human embryos, typically at the two-cell stage. Mechanistically, several factors, including spindle dysfunction, failed cleavage, and cell fusion, may generate this cell anomaly. In assisted reproduction treatment, multinucleation is associated with reduced developmental rates and lower implantation rates in Days 2-3 embryo transfers. However, many multinucleated embryos can develop to the blastocyst stage. In blastocyst transfers, the current evidence does not suggest a major impact of a previous history of multinucleation on the odds of euploidy or successful treatment outcomes. Human embryo multinucleation remains a not-fully-understood but developmentally relevant and intriguing phenomenon which requires further research of its generative mechanisms and clinical implications.

A multi-faceted exploration of unmet needs in the continuing improvement and development of fertility care amidst a pandemic.

PURPOSE: The continuous improvement and development of fertility care, internationally, requires ongoing monitoring of current delivery processes and outcomes in clinical practice. This descriptive and exploratory mixed-methods study was conducted in eight countries (Brazil, China, France, Germany, Italy, Mexico, Spain and the United Kingdom) to assess the unmet needs of fertility patients (male and female), and existing challenges, barriers and educational gaps of physicians and laboratory specialists involved in human fertility care during the COVID-19 pandemic. MATERIALS AND METHODS: The study was deployed sequentially in two phases: 1) in-depth 45-minute semi-structured interviews (n=76), transcribed, coded and thematically analysed using an inductive reasoning approach, 2) an online survey (n=303) informed by the findings of the qualitative interviews, face validated by experts in reproductive medicine, and analysed using descriptive and inferential statistical methods. RESULTS: The integrated results of both phases indicated numerous areas of challenges, including: 1) investigating male-related infertility; 2) deciding appropriate treatment for men and selective use of assisted reproductive technology; and 3) maintaining access to high-quality fertility care during a pandemic. CONCLUSIONS: The paper presents a reflective piece on knowledge and skills that warrant ongoing monitoring and improvement amongst reproductive medicine healthcare professionals amidst future pandemics and unanticipated health system disruptions. Moreover, these findings suggest that there is an additional need to better understand the required changes in policies and organizational processes that would facilitate access to andrology services for male infertility and specialized care, as needed.

An expert opinion on rescuing atypically pronucleated human zygotes by molecular genetic fertilization checks in IVF.

IVF laboratories routinely adopt morphological pronuclear assessment at the zygote stage to identify abnormally fertilized embryos deemed unsuitable for clinical use. In essence, this is a pseudo-genetic test for ploidy motivated by the notion that biparental diploidy is required for normal human life and abnormal ploidy will lead to either failed implantation, miscarriage, or significant pregnancy complications, including molar pregnancy and chorionic carcinoma. Here, we review the literature associated with ploidy assessment of human embryos derived from zygotes displaying a pronuclear configuration other than the canonical two, and the related pregnancy outcome following transfer. We highlight that pronuclear assessment, although associated with aberrant ploidy outcomes, has a low specificity in the prediction of abnormal ploidy status in the developing embryo, while embryos deemed abnormally fertilized can yield healthy pregnancies. Therefore, this universal strategy of pronuclear assessment invariably leads to incorrect classification of over 50% of blastocysts derived from atypically pronucleated zygotes, and the systematic disposal of potentially viable embryos in IVF. To overcome this limitation of current practice, we discuss the new preimplantation genetic testing technologies that enable accurate identification of the ploidy status of preimplantation embryos and suggest a progress from morphology-based checks to molecular fertilization check as the new gold standard. This alternative molecular fertilization checking represents a possible non-incremental and controversy-free improvement to live birth rates in IVF as it adds to the pool of viable embryos available for transfer. This is especially important for the purposes of 'family building' or for poor-prognosis IVF patients where embryo numbers are often limited.

Towards a more sustainable balance between optimal live birth rate and supernumerary embryos in ART treatments.

PURPOSE: To assess the relation between number of inseminated oocytes and cumulative live birth rate (CLBR) in order to provide guidance for limiting the number of surplus blastocysts. METHODS: The study was a retrospective, single-center cohort analysis of 1223 ART complete cycles. Cycles were stratified according to female age (≤ 34, 35-38, and 39-42 years) and number of inseminated oocytes (1-5, 6-10, and > 10). Inclusion criteria were indication for IVF/ICSI with own spermatozoa and blastocyst culture up to day 6 of all embryos. RESULTS: In patients younger than 35 years, insemination of more than ten oocytes produced an increase in overall blastocyst number, CLBR (40.3%, 54.3%, and 75.8%, respectively, for each oocyte group) and surplus embryo rate (12.9%, 27.8%, and 49.7% of cases for each group). Instead, in the middle age group, the use of more than ten oocytes was solely associated with an increase in the rate of surplus embryos (1.25%, 21.33%, and 28.68% of cases after stratification for oocyte number). In older patients, neither CLBR (9.1%, 23.9%, and 24.7%, respectively) nor rate of surplus embryos (2.0%, 7.1%, and 13.4% of cases for each group) were higher in cycles with more than ten inseminated oocytes. CONCLUSION: In women up to 38 years, sustainable CLBR are achieved while limiting the number of inseminated oocytes and the resulting blastocysts remaining unused. Based on this notion, novel treatment strategies could pursue high outcome rates, while alleviating the problems derived from surplus stored embryos.

First case of dichorionic diamniotic triplet pregnancy after single blastocyst transfer.

Multiple pregnancies are associated with significant maternal, fetal, and neonatal risks, including prematurity, low birth weight, pre-eclampsia, anemia, postpartum hemorrhage, intrauterine growth restriction, neonatal morbidity, and increased neonatal and infant mortality rates. Assisted reproductive technology (ART) treatments should prioritize efforts to reduce such events, resisting patient demand for the transfer of multiple embryos at each transfer to increase success rates. Extended culture, embryo selection, and single blastocyst transfer can mitigate the risk of high-order multiple pregnancies. Intriguingly, elective single-embryo transfer (eSET) greatly reduces, but does not completely eliminate, the likelihood of multiple gestations. The occurrence of monozygotic twinning (MZT) gives rise to identical twins. It is more prevalent in women undergoing in vitro fertilization (IVF) compared with natural conception. In fact, the reported risks of monozygotic twinning in IVF and natural conception are 1.7 and 0.4%, respectively. The factors suspected to increase the risk of MZT in IVF are multiple embryo transfer, micromanipulation, and extended in vitro culture. Determining chorionicity and amnionicity is crucial in the assessment of multiple pregnancies during the first-trimester ultrasound examination. Dichorionic twins result from embryo splitting within 3 days after fertilization, while monochorionic twins occur when the splitting takes place between 4 and 8 days after fertilization. These timings are suggested by observations carried out in natural pregnancies. In ART, there is evidence of dichorionic twins derived from single embryo transfer (SET). Here, we report a case of dichorionic diamniotic triplets after a single blastocyst transfer occurred in our center. To our knowledge, this is the first case documented so far.

Discard or not discard, that is the question: an international survey across 117 embryologists on the clinical management of borderline quality blastocysts.

STUDY QUESTION: Do embryologists from different European countries agree on embryo disposition decisions ('use' or 'discard') about Day 7 (>144 h post-insemination) and/or low-quality blastocysts (LQB;

The destinies of human embryos reaching blastocyst stage between Day 4 and Day 7 diverge as early as fertilization.

STUDY QUESTION: What clinical and laboratory differences emerge from parallel direct comparison of embryos reaching the blastocyst stage between Days 4, 5, 6, and 7 (Days 4-7)? SUMMARY ANSWER: Increasing times to blastocyst formation are associated with a worse clinical outcome and perturbations in developmental patterns appear as early as the fertilization stage. WHAT IS KNOWN ALREADY: Previous evidence indicates that later times to blastocyst development are associated with a worse clinical outcome. However, the vast majority of these data concern Day 5 and Day 6 blastocysts, while Day 4 and Day 7 blastocysts remain less thoroughly investigated. In addition, studies comparing in parallel the developmental patterns and trajectories of Day 4-7 blastocysts are lacking. This leaves unanswered the question of when and how differences among such embryos emerge. Acquisition of such knowledge would significantly contribute to understanding the relative impact of intrinsic and extrinsic causes of embryo developmental kinetics and competence. STUDY DESIGN, SIZE, DURATION: This retrospective study involved time-lapse technology (TLT) monitoring of Day 4 (N = 70), Day 5 (N = 6147), Day 6 (N = 3243), and Day 7 (N = 149) blastocysts generated in 9450 ICSI cycles. Oocyte retrievals were carried out after clomiphene citrate-based minimal ovarian stimulation, between January 2020 and April 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Couples included in the study presented with different diagnoses, mainly male factor and unexplained infertility. Cases involving cryopreserved gametes or surgically retrieved sperm were excluded. Microinjected oocytes were assessed by a combined TLT-culture system. Day 4-7 blastocyst groups were compared in terms of morphokinetics (pronuclear dynamics, cleavage patterns and timings, and embryo quality) and clinical outcome. Clinically usable blastocysts were cryopreserved and transferred in single vitrified-warmed blastocyst transfers (SVBT). MAIN RESULTS AND THE ROLE OF CHANCE: From 19 846 microinjected oocytes, 17 144 zygotes (86.4%) were obtained. Overall, the blastocyst development rate was 56.0%. Rates of blastocysts formation on Days 4, 5, 6, and 7 were 0.7%, 64.0%, 33.8%, and 1.6%, respectively. The average expanded blastocyst development times were 98.4 ± 0.4, 112.4 ± 0.1, 131.6 ± 0.1, and 151.2 ± 0.5 h in the Day 4-7 groups, respectively. Female age was positively associated with longer times to blastocyst development. Rates of both inner cell mass (ICM) and trophectoderm (TE) morphological grade A blastocysts were negatively associated with the day of blastocyst development (P < 0.0001). The differences in development times and intervals increased progressively until blastocyst expansion (P < 0.0001 for all development times). Strikingly, such differences were already markedly evident as early as the time of pronuclear fading (tPNf) (20.6 ± 0.3, 22.5 ± 0.0, 24.0 ± 0.0, 25.5 ± 0.3; Days 4-7, respectively; P < 0.0001). Rates of cleavage anomalies (tri-/multi-chotomous mitosis or rapid cleavage) occurring at the first or second/third division cycles were also positively associated with longer times to blastocyst development. Implantation, ongoing pregnancy, and live birth rates were progressively reduced with increasing blastocyst development times (P < 0.0001), even after stratification for maternal age. When controlled for female age, male age, number of previous embryo transfer cycles, morphological grade of the ICM and TE, and progesterone supplementation, the probabilities of implantation, clinical, and ongoing pregnancy and live birth were significantly decreased in Day 6 blastocysts in comparison to Day 5 blastocysts. Follow-up data on birth length, weight, and malformations were comparable among the four blastocyst groups. LIMITATIONS, REASONS FOR CAUTION: The study is limited by its retrospective design. Having been obtained from a single centre, the data require independent validation. WIDER IMPLICATIONS OF THE FINDINGS: This study extends previous data on the relation between time of blastocyst formation and clinical outcome. It also indicates that differences in developmental times and patterns of Day 4-7 blastocysts occur as early as the fertilization stage, possibly dictated by intrinsic gamete-derived factors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the participating institutions. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Endometriosis affects the number of retrieved oocytes but not early embryonic development and live birth: a retrospective analysis of 716 IVF cycles.

To investigate the potential effect of endometriosis on embryo development and clinical outcomes, a retrospective analysis of 716 women undergoing their first standard in vitro fertilization (sIVF) cycles (205 endometriosis and 511 with tubal factor infertility) was performed. The endometriosis group included women with an ultrasonographic or surgical diagnosis. Control subjects were women diagnosed with tubal factor infertility by laparoscopy or hysterosalpingogram. The primary outcome of the study was live birth. Cumulative live birth was also assessed in a subgroups analysis. After adjusting for confounders we found no significant difference in fertilization rate, blastulation, top-quality blastocyst, live birth, cumulative live birth (subgroups analysis) and miscarriage rate. In the endometriosis group, the number of retrieved oocytes was smaller (6.94 ± 4.06 Vs 7.50 ± 4.6, adjusted p < 0.05). We observed a statistically significant difference in the percentage of day-3 embryos with ≥8 blastomeres (33.12 ± 22.72 endometriosis vs, 40.77 ± 27.62 tubal factor, adjusted p < 0.01) and a negative correlation between the presence of endometriomas and a number of retrieved oocytes [B coefficient =-1.41, 95%CI (-2.31-0.51), adjusted p = 0.002]. Our results suggest that endometriosis affects the number of retrieved oocytes but not embryo development and live birth.

Fertility preservation in women with benign gynaecological conditions.

Although a wealth of data has been published regarding fertility preservation (FP) in women with malignant diseases who receive gonadotoxic treatment, the role of FP in non-malignant conditions has been studied to a much lesser extent. These include benign haematological, autoimmune, and genetic disorders, as well as a multitude of benign gynaecological conditions (BGCs) that may compromise ovarian reserve and/or reproductive potential due to pathogenic mechanisms or as a result of medical or surgical treatments. Alongside accumulating data that document the reproductive potential of cryopreserved oocytes and ovarian tissue, there is potential interest in FP for women with BGCs at risk of infertility; however, there are currently insufficient data about FP in women with BGCs to develop guidelines for clinical practice. The purpose of this article is to appraise the available evidence regarding FP for BGC and discuss potential strategies for FP based on estimated ovarian impairment and on short-term and long-term reproductive goals of patients. Cost-effectiveness considerations and patients' perspectives will also be discussed.

Humidified atmosphere in a time-lapse embryo culture system does not improve ongoing pregnancy rate: a retrospective propensity score model study derived from 496 first ICSI cycles.

PURPOSE: To investigate whether high relative humidity conditions (HC), when using a time-lapse system (TLS) with sequential culture media, are beneficial to embryo culture, improving ongoing pregnancy rates. METHODS: We included patients undergoing their first ICSI cycle treatment from April 2021 to May 2022. Patients assigned to dry conditions (DC) or HC were 278 and 218, respectively. We used a GERI TLS, three chambers configured in humidity conditions and three in dry conditions. The effect of HC on ongoing pregnancy rate was assessed by the propensity matched sample, to reduce potential differences between women undergoing either HC or DC and reduce biased estimation of treatment effect. RESULT: After adjusting for several confounding variables and applying the propensity score (PS), no significant differences were observed in the rates of normal (2PN) and abnormal (1PN and 3PN) fertilization, blastulation, top-quality blastocysts, frozen blastocysts, ongoing pregnancies, and miscarriages. The 2-cell (t2) and 4-cell (t4) stages and cell divisions between such stages occurred earlier and were more synchronous in the in DC. CONCLUSION: These results suggest that HC conditions do not improve the rate of ongoing pregnancy and several embryological outcomes, under the conditions used in this study based on a time-lapse system and sequential culture with day 3 medium change-over.

The first mitotic division: a perilous bridge connecting the zygote and the early embryo.

Human embryos are very frequently affected by maternally inherited aneuploidies, which in the vast majority of cases determine developmental failure at pre- or post-implantation stages. However, recent evidence, generated by the alliance between diverse technologies now routinely employed in the IVF laboratory, has revealed a broader, more complex scenario. Aberrant patterns occurring at the cellular or molecular level can impact at multiple stages of the trajectory of development to blastocyst. In this context, fertilization is an extremely delicate phase, as it marks the transition between gametic and embryonic life. Centrosomes, essential for mitosis, are assembled ex novo from components of both parents. Very large and initially distant nuclei (the pronuclei) are brought together and positioned centrally. The overall cell arrangement is converted from being asymmetric to symmetric. The maternal and paternal chromosome sets, initially separate and scattered within their respective pronuclei, become clustered where the pronuclei juxtapose, to facilitate their assembly in the mitotic spindle. The meiotic spindle is replaced by a segregation machinery that may form as a transient or persistent dual mitotic spindle. Maternal proteins assist the decay of maternal mRNAs to allow the translation of newly synthesized zygotic transcripts. The diversity and complexity of these events, regulated in a precise temporal order and occurring in narrow time windows, make fertilization a highly error-prone process. As a consequence, at the first mitotic division, cellular or genomic integrity may be lost, with fatal consequences for embryonic development.

The time has come for harmonized international ART registration.

For more than two decades, the European IVF-Monitoring Consortium has collected data on IVF in Europe with the aim of monitoring the quality and safety of assisted reproductive technology (ART) treatments, to ensure the highest performance with the lowest risk for patients and their offspring. Likewise, the Society for Assisted Reproductive Technology in the USA and the Australia/New Zealand Assisted Reproduction Database collect, process and publish data in their regions. The better the legal framework for ART surveillance, the more complete and reliable are the datasets. Worldwide, the landscape of ART regulation is fragmented, and until there is a legal obligation to report ART data in all countries, with an appropriate quality control of the data collected, the reported outcomes should be interpreted with caution. Once uniform and harmonized data are achieved, consensus reports based on collective findings can begin to address key topics such as cycle segmentation and complications. Improved registration systems and datasets allowing optimized surveillance should be developed in collaboration with patient representatives to consider patients' needs, especially aiming to provide higher transparency around ART services. Support from national and international reproductive medicine societies will also be essential to the future evolution of ART registries.

Towards Automation in IVF: Pre-Clinical Validation of a Deep Learning-Based Embryo Grading System during PGT-A Cycles.

Preimplantation genetic testing for aneuploidies (PGT-A) is arguably the most effective embryo selection strategy. Nevertheless, it requires greater workload, costs, and expertise. Therefore, a quest towards user-friendly, non-invasive strategies is ongoing. Although insufficient to replace PGT-A, embryo morphological evaluation is significantly associated with embryonic competence, but scarcely reproducible. Recently, artificial intelligence-powered analyses have been proposed to objectify and automate image evaluations. iDAScore v1.0 is a deep-learning model based on a 3D convolutional neural network trained on time-lapse videos from implanted and non-implanted blastocysts. It is a decision support system for ranking blastocysts without manual input. This retrospective, pre-clinical, external validation included 3604 blastocysts and 808 euploid transfers from 1232 cycles. All blastocysts were retrospectively assessed through the iDAScore v1.0; therefore, it did not influence embryologists' decision-making process. iDAScore v1.0 was significantly associated with embryo morphology and competence, although AUCs for euploidy and live-birth prediction were 0.60 and 0.66, respectively, which is rather comparable to embryologists' performance. Nevertheless, iDAScore v1.0 is objective and reproducible, while embryologists' evaluations are not. In a retrospective simulation, iDAScore v1.0 would have ranked euploid blastocysts as top quality in 63% of cases with one or more euploid and aneuploid blastocysts, and it would have questioned embryologists' ranking in 48% of cases with two or more euploid blastocysts and one or more live birth. Therefore, iDAScore v1.0 may objectify embryologists' evaluations, but randomized controlled trials are required to assess its clinical value.

Maternal age affects pronuclear and chromatin dynamics, morula compaction and cell polarity, and blastulation of human embryos.

STUDY QUESTION: Does maternal ageing impact early and late morphokinetic and cellular processes of human blastocyst formation? SUMMARY ANSWER: Maternal ageing significantly affects pronuclear size and intra- and extra-nuclear dynamics during fertilization, dysregulates cell polarity during compaction, and reduces blastocoel expansion. WHAT IS KNOWN ALREADY: In ART, advanced maternal age (AMA) affects oocyte yield, fertilization, and overall developmental competence. However, with the exception of chromosome segregation errors occurring during oocyte meiosis, the molecular and biochemical mechanisms responsible for AMA-related subfertility and reduced embryo developmental competence remain unclear. In particular, studies reporting morphokinetics and cellular alterations during the fertilization and pre-implantation period in women of AMA remain limited. STUDY DESIGN, SIZE, DURATION: A total of 2058 fertilized oocytes were stratified by maternal age according to the Society for Assisted Reproductive Technology classification (<35, 35-37, 38-40, 41-42, and >42 years) and retrospectively analysed. AMA effects were assessed in relation to: embryo morphokinetics and morphological alterations; and the presence and distribution of cell polarity markers-Yes-associated protein (YAP) and protein kinase C-ζ (PKC-ζ)-involved in blastocyst morphogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1050 cycles from 1050 patients met the inclusion criteria and were analysed. Microinjected oocytes were assessed using a time-lapse culture system. Immature oocytes at oocyte retrieval and mature oocytes not suitable for time-lapse monitoring, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared among groups. Furthermore, 20 human embryos donated for research by consenting couples were used for immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: Static microscopic observation revealed that blastocyst formation and expansion were impaired in the 41-42 and >42-year groups (P < 0.0001). The morphological grades of the inner cell mass and trophectoderm were poorer in the >42-year group than those in the <35-year group (P = 0.0022 and P < 0.0001, respectively). Time-lapse microscopic observation revealed a reduction in nucleolus precursor body alignment in female pronuclei in the 41-42 and >42-year groups (P = 0.0010). Female pronuclear area decreased and asynchronous pronuclear breakdown increased in the >42-year group (P = 0.0027 and P < 0.0122, respectively). Developmental speed at cleavage stage, incidence of irregularity of first cleavage, type and duration of blastomere movement, and number of multinucleated cells were comparable among age groups. Delayed embryonic compaction and an increased number of extruded blastomeres were observed in the >42-year group (P = 0.0002 and P = 0.0047, respectively). Blastulation and blastocyst expansion were also delayed in the 41-42 and >42-year groups (P < 0.0001 for both). YAP positivity rate in the outer cells of morulae and embryo PKC-ζ immunoflourescence decreased in the >42-year group (P < 0.0001 for both). LIMITATIONS, REASONS FOR CAUTION: At the cellular level, the investigation was limited to cell polarity markers. Cell components of other developmental pathways should be studied in relation to AMA. WIDER IMPLICATIONS OF THE FINDINGS: The study indicates that maternal ageing affects the key functions of embryo morphogenesis, irrespective of the well-established influence on the fidelity of oocyte meiosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

ESHRE survey results and good practice recommendations on managing chromosomal mosaicism.

STUDY QUESTION: How should ART/preimplantation genetic testing (PGT) centres manage the detection of chromosomal mosaicism following PGT? SUMMARY ANSWER: Thirty good practice recommendations were formulated that can be used by ART/PGT centres as a basis for their own policy with regards to the management of 'mosaic' embryos. WHAT IS KNOWN ALREADY: The use of comprehensive chromosome screening technologies has provided a variety of data on the incidence of chromosomal mosaicism at the preimplantation stage of development and evidence is accumulating that clarifies the clinical outcomes after transfer of embryos with putative mosaic results, with regards to implantation, miscarriage and live birth rates, and neonatal outcomes. STUDY DESIGN SIZE DURATION: This document was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, a large survey evaluating current practice and published guidance documents. The literature search was performed using PubMed and focused on studies published between 2010 and 2022. The survey was performed through a web-based questionnaire distributed to members of the ESHRE special interest groups (SIG) Reproductive Genetics and Embryology, and the ESHRE PGT Consortium members. It included questions on ART and PGT, reporting, embryo transfer policy and follow-up of transfers. The final dataset represents 239 centres. PARTICIPANTS/MATERIALS SETTING METHODS: The working group (WG) included 16 members with expertise on the ART/PGT process and chromosomal mosaicism. The recommendations for clinical practice were formulated based on the expert opinion of the WG, while taking into consideration the published data and results of the survey. MAIN RESULTS AND THE ROLE OF CHANCE: Eighty percent of centres that biopsy three or more cells report mosaicism, even though only 66.9% of all centres have validated their technology and only 61.8% of these have validated specifically for the calling of chromosomal mosaicism. The criteria for designating mosaicism, reporting and transfer policies vary significantly across the centres replying to the survey. The WG formulated recommendations on how to manage the detection of chromosomal mosaicism in clinical practice, considering validation, risk assessment, designating and reporting mosaicism, embryo transfer policies, prenatal testing and follow-up. Guidance is also provided on the essential elements that should constitute the consent forms and the genetic report, and that should be covered in genetic counselling. As there are several unknowns in chromosomal mosaicism, it is recommended that PGT centres monitor emerging data on the topic and adapt or refine their policy whenever new insights are available from evidence. LIMITATIONS REASONS FOR CAUTION: Rather than providing instant standardized advice, the recommendations should help ART/PGT centres in developing their own policy towards the management of putative mosaic embryos in clinical practice. WIDER IMPLICATIONS OF THE FINDINGS: This document will help facilitate a more knowledge-based approach for dealing with chromosomal mosaicism in different centres. In addition to recommendations for clinical practice, recommendations for future research were formulated. Following up on these will direct research towards existing research gaps with direct translation to clinical practice. Emerging data will help in improving guidance, and a more evidence-based approach of managing chromosomal mosaicism. STUDY FUNDING/COMPETING INTERESTS: The WG received technical support from ESHRE. M.D.R. participated in the EQA special advisory group, outside the submitted work, and is the chair of the PGT WG of the Belgian society for human genetics. D.W. declared receiving salary from Juno Genetics, UK. A.C. is an employee of Igenomix, Italy and C.R. is an employee of Igenomix, Spain. C.S. received a research grant from FWO, Belgium, not related to the submitted work. I.S. declared being a Co-founder of IVFvision Ltd, UK. J.R.V. declared patents related to 'Methods for haplotyping single-cells' and 'Haplotyping and copy number typing using polymorphic variant allelic frequencies', and being a board member of Preimplantation Genetic Diagnosis International Society (PGDIS) and International Society for Prenatal Diagnosis (ISPD). K.S. reported being Chair-elect of ESHRE. The other authors had nothing to disclose. DISCLAIMER: This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation.  ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type.  Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE.

Human 1PN and 3PN zygotes recapitulate all morphokinetic events of normal fertilization but reveal novel developmental errors.

STUDY QUESTION: Does mono- (1PN) and tri-pronuclear (3PN) fertilization recapitulate the morphokinetic changes of normal bi-pronuclear (2PN) fertilization? SUMMARY ANSWER: Abnormal fertilization retraces the overall choreography of normal fertilization but reveals novel morphokinetic phenomena and raises scientifically and clinically relevant questions. WHAT IS KNOWN ALREADY: ART has allowed the extracorporeal observation of early human development. Time-lapse technology (TLT) has revealed the complexity of the morphokinetic changes underpinning fertilization and the importance of this process for the genetic and cellular integrity of the embryo. Abnormal fertilization has remained neglected, despite its relevance to the physiology and pathology of early human development. STUDY DESIGN, SIZE, DURATION: This retrospective study involved TLT observation of normally (2PN, N = 2517) and abnormally (1PN, N = 41; 3PN, N = 27) fertilized oocytes generated in ICSI cycles performed between October 2019 and December 2020. Oocyte retrieval was carried out after clomiphene citrate-based minimal ovarian stimulation. Oocytes of patients with different diagnoses of infertility were included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included 1231 couples treated for diverse infertility causes. The fraction of male factor cases was substantial (36.1%). Microinjected oocytes were assessed by a combined TLT-culture system. Oocytes not suitable for TLT assessment, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared between groups. MAIN RESULTS AND THE ROLE OF CHANCE: Extrusion of the second polar body (PBII) was observed in almost all 2PN/1PN (99.9% and 100.0%, respectively) and in a vast majority of 3PN zygotes (92.1%). Rates of PBII fusion with the ooplasm were much higher in 1PN and 3PN zygotes (P < 0.0001 versus 2PN). The cytoplasmic wave was observed not only in 2PN and 3PN but also in 1PN zygotes (positivity rates of 99.8% and 100% and 82.9%, respectively; P < 0.0001). More rarely, 2PN and 1PN zygotes emitted a third polar body (PBIII). The average times of this event were comparable. The presence and position of the cytoplasmic halo were comparable among the three classes of zygotes. In the 1PN group, the single PN was maternally or paternally derived in 17 and 24 zygotes, respectively, while in the vast majority of 3PN zygotes (121/127) the supernumerary PN was of maternal origin. Average times of maternal PN appearance were comparable, while average times of paternal PN appearance were delayed in 3PN zygotes (P = 0.0127). Compared with the control group, the area of the maternal PN was larger in 1PN zygotes, but smaller in 3PN zygotes (P < 0.0001). The paternal PNs displayed the same trend (P < 0.0001), although such values were consistently smaller than maternal PNs. The area of the third PN in the 3PN group was on average more than 50% smaller than those of maternal and paternal PNs. In maternal PNs of 3PN zygotes, nucleolus precursor bodies (NPBs) aligned along the area of PN juxtaposition at a lower rate compared with the 2PN group. The rate of NPB alignment was ∼50% smaller in 1PN zygotes (P = 0.0001). In paternal PNs, the rates of NPB alignment were not statistically different among the three groups. Asynchronous PN breakdown was increased in 3PN compared with 2PN zygotes (P = 0.0026). In 1PN zygotes, a developmental delay was observed starting from the disappearance of the cytoplasmic halo, reaching 9 h at the time of the first cleavage (P < 0.0001). Higher rates of abnormal cleavage patterns and blastomere fragmentation (P < 0.0001) were observed in 1PN compared to 2N and 3PN zygotes. Cleavage progression was increasingly affected after abnormal fertilization, especially 1PN, finally resulting in blastocyst formation rates of 70.2%, 12.2% and 53.5% in 2PN, 1PN and 3PN embryos, respectively (P < 0.0001). Both maternal and paternal ages were higher in cases involving 3PN fertilization. LIMITATIONS, REASONS FOR CAUTION: The study data were obtained from ICSI, but not standard IVF, treatments carried out in a single centre. The study findings therefore require independent verification. WIDER IMPLICATIONS OF THE FINDINGS: This study reports the first detailed morphokinetic map of human abnormal fertilization. Collectively, this evidence prompts new scientific hypotheses and raises clinical questions relevant to the aetiology and the treatment of abnormal fertilization. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

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.

Fertilization signatures as biomarkers of embryo quality.

Fertilization underpins the vital transition from gametic meiosis to embryonic mitosis. For decades, in human IVF, microscopic observation at a single time point has limited our appreciation of the morphokinetic complexity of this process. More recently, the introduction of time lapse technology-also enhanced by combination with artificial intelligence-has revealed the finest morphokinetic details of the beginning of human development. Overall, a picture has finally emerged in which the precise timing, morphology and geometry of several fertilization events offer clues to predict the fate of the embryo-a key aspect of assisted reproduction. In this scenario, correct unfolding of intra- and interpronuclear rearrangements emerge as a crucial factor to create a platform able to preserve genetic and cellular integrity at the first mitotic cleavage.