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STUDY QUESTIONDoes maternal ageing impact early and late morphokinetic and cellular processes of human blastocyst formation?SUMMARY ANSWERMaternal 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 ALREADYIn 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, DURATIONA total of 2058 fertilized oocytes were stratified by maternal age according to the Society for Assisted Reproductive Technology classification (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, METHODSA 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 CHANCEStatic microscopic observation revealed that blastocyst formation and expansion were impaired in the 41–42 and >42-year groups (P 42-year group than those in the 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 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 42-year group (P LIMITATIONS, REASONS FOR CAUTIONAt 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 FINDINGSThe 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 NUMBERN/A.

What is the association between the deep learning-based scoring system, iDAScore, and biological events during the pre-implantation period?Retrospective observational study of patients (n = 925) who underwent oocyte retrieval in a clomiphene citrate-based minimal stimulation cycle and obtained expanded blastocysts between October 2019 and December 2020. The association between iDAScore with morphokinetics and morphological alteration during fertilization, cleavage stage, compaction and blastocyst stage was analysed.The duration of the cytoplasmic halo was significantly prolonged in low-scoring blastocysts (P0.0001). The timing of female and male pronuclei breakdown was significantly delayed in low-scoring blastocysts compared with high-scoring blastocysts (P0.0001 in both). Embryos with either trichotomous, multi-chotomous, rapid or reverse cleavage or asymmetric division had a lower score than embryos with normal cleavage (P0.0001-0.0098). The cell number and amount of blastomere fragmentation on days 2 and 3 were significantly associated with iDAScore (P0.0001-0.0008). Delayed compaction, blastulation and blastocyst expansion were observed in low-scoring embryos (P0.0001 in all). The incidence of blastomere exclusion and extrusion during embryonic compaction was significantly higher in low-scoring embryos than in high-scoring embryos (P ≤ 0.0001 in both). Blastocyst morphology was significantly associated with iDAScore (P0.0001). Multiple linear regression analysis revealed that, during the transformation to blastocyst stage, morphokinetic and morphological events were strongly associated with iDAScore (P0.0001-0.0116).iDAScore was significantly correlated with morphokinetics and morphological alterations of pre-implantation embryos, especially during the late pre-implantation period. Our findings contribute to research on deep learning model-based embryo selection, which may provide patients with a compelling explanation of blastocyst selection.

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 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.