STUDY QUESTION Do perturbations of embryo morphogenesis at compaction affect blastocyst development and clinical outcomes in assisted reproduction cycles? SUMMARY ANSWER Cell exclusion and extrusion, i.e. cell disposal occurring respectively before or during morula compaction, affect blastocyst yield and quality, as well as rates of pregnancy and live birth. WHAT IS KNOWN ALREADY Despite its pivotal role in morphogenesis for blastocyst organisation and cell fate determination, compaction at the morula stage has received little attention in clinical embryology. Time lapse technology (TLT) allows detailed morphokinetic analysis of this developmental stage. However, even in the vast majority of previous TLT studies, compaction was investigated without a specific focus. Recently, we reported that compaction may be affected by two clearly-distinct patterns of cell disposal, exclusion and extrusion, occurring prior to and during compaction, respectively. However, the crucial question of the specific relevance of partial compaction for embryo development and competence in ART has remained unanswered until now. STUDY DESIGN, SIZE, DURATION This study involved the assessment of laboratory and clinical outcomes of 2,059 morula stage embryos associated with 1,117 ICSI patients, who were treated with minimal stimulation and single vitrified-warmed blastocyst transfer (SVBT) from April 2017 to March 2018. Patterns of morula compaction were assessed and analyzed in relation to embryonic and clinical outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS Following ICSI, time-lapse videos were analysed to annotate morphokinetic parameters relevant to both pre- and post-compaction stages. According to their morphokinetic history, morulae were classified as: (I) fully compacted morulae (FCM); (II) partially compacted morulae (PCM), showing cells (a) excluded from the compaction process from the outset (Exc-PCM), (b) extruded from an already compacted morula (Ext-PCM), or (c) showing non-compacted cells arisen from both patterns (Exc/Ext-PCM). The number of excluded/extruded cells was also annotated. Possible correlations of compaction patterns with 13 morphokinetic parameters, abnormal cleavage, blastocyst yield and morphological grade, clinical and ongoing pregnancy rates, and live birth rate were evaluated. Other factors, such as patient and cycle characteristics, possibly associated with compaction patterns and their outcomes, were investigated. MAIN RESULTS AND THE ROLE OF CHANCE Full compaction was observed in 39.0% of all embryos. However, partially compacted morulae (PCM) showing excluded (Exc-PCM), extruded (Ext-PCM) cells, or indeed both phenotypes (Exc/Ext-PCM) were frequently detected (24.8%, 16.6%, and 19.6%, respectively) and collectively (61%) exceeded fully compacted morulae. Blastomere exclusion or extrusion affected one or several cells, in different proportions. In comparison to FCM, the developmental pace of the three PCM groups, observed at 13 developmental stages starting from pronuclear fading, was progressively slower (P < 0.0001). Developmental delay at post-compaction stages was more pronounced in the group showing both patterns of partial compaction. Blastomere exclusion and/or extrusion had a large negative impact on blastocyst development. In particular, rates of blastocyst formation and cryopreservation were very low in the Ext-PCM and Exc/Ext-PCM groups (P < 0.0001). Rates of blastocysts with ICM or TE of highest quality (Grade A) were severely affected in all PCM groups (P < 0.0001). In 1,083 SVBTs, blastocysts derived from all PCM groups produced much lower clinical pregnancy, ongoing pregnancy, and live birth rates (P < 0.0001). All three patterns of partial compaction emerged as factors independently associated with live birth rate, even after multivariate logistic regression analysis including maternal/paternal age, female BMI, and number of previous embryo transfers as possible confounding factors. LIMITATIONS, REASONS FOR CAUTION The retrospective design of the study represents a general limitation. WIDER IMPLICATIONS OF THE FINDINGS This large-scale study represents a further important demonstration of embryo plasticity and above all indicates new robust morphokinetic parameters for improved algorithms of embryo selection. STUDY FUNDING/COMPETING INTEREST(S) This study was exclusively supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER NA.

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.

Background The KIDScore™ Day 5 (KS-D5) model, version 3, is a general morphokinetic prediction model (Vitrolife, Sweden) for fetal heartbeat prediction after embryo transfer that was developed based on a large data set that included implantation results from a range of clinics with different patient populations, culture conditions and clinical practices. However, there was no study to comparing their pregnancy and live birth prediction ability among different maternal age. The aim of this study is to analyze the performance of KS-D5 in predicting pregnancy and live birth in various maternal age groups after single vitrified-warmed blastocyst transfer (SVBT). Methods A total of 2486 single vitrified-warmed blastocyst transfer (SVBT) cycles were analyzed retrospectively. Confirmed fetal heartbeat positive (FHB+) and live birth (LB+) rates were stratified by Society for Assisted Reproductive Technology (SART) maternal age criteria (< 35, 35–37, 38–40, 41–42 and ≥ 43 years of age). Within each age group, the performance of the prediction model was calculated using the AUC, and the results were compared across the age groups. Results In all age groups, the FHB+ rates decreased as the KIDScore decreased (P <  0.05). Conversely, the AUCs increased as the maternal age increased. The AUC of the < 35 age group (0.589) was significantly lower than the AUCs of the 41–42 age group (0.673) and the ≥43 age group (0.737), respectively (P <  0.05). In all age groups, the LB+ rates decreased as the KIDScore decreased (P <  0.05). Conversely, the AUCs increased as the maternal age increased. The AUC of the ≥43 age group (0.768) was significantly higher than the AUCs of other age groups (P <  0.05; < 35 age group = 0.596, 35–37 age group = 0.640, 38–40 age group = 0.646, 41–42 age group = 0.679). Conclusions In the present study, we determined that the KIDScore model worked well for prediction of pregnancy and live birth outcomes in advanced age patients.

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 (&lt;35, 35–37, 38–40, 41–42, and &gt;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 &gt;42-year groups (P &lt; 0.0001). The morphological grades of the inner cell mass and trophectoderm were poorer in the &gt;42-year group than those in the &lt;35-year group (P = 0.0022 and P &lt; 0.0001, respectively). Time-lapse microscopic observation revealed a reduction in nucleolus precursor body alignment in female pronuclei in the 41–42 and &gt;42-year groups (P = 0.0010). Female pronuclear area decreased and asynchronous pronuclear breakdown increased in the &gt;42-year group (P = 0.0027 and P &lt; 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 &gt;42-year group (P = 0.0002 and P = 0.0047, respectively). Blastulation and blastocyst expansion were also delayed in the 41–42 and &gt;42-year groups (P &lt; 0.0001 for both). YAP positivity rate in the outer cells of morulae and embryo PKC-ζ immunoflourescence decreased in the &gt;42-year group (P &lt; 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.

Purpose This study aimed to examine the embryonic development of human 4‐cell stage embryos after warming with fatty acids (FAs) and to assess the pregnancy outcomes after single vitrified‐warmed cleavage stage embryo transfers (SVCTs). Methods Experimental study: A total of 217 discarded, vitrified human 4‐cell stage embryos donated for research by consenting couples were used. The embryos were warmed using the fatty acid (FA)‐supplemented solutions (FA group) or nonsupplemented solutions (control group). The developmental rate, morphokinetics, and outgrowth competence were analyzed. Clinical study: The treatment records of women undergoing SVCT in natural cycles between April and September 2022 were retrospectively analyzed (April–June 2022, control group; July–September 2022, FA group). Results Experimental study: The rate of morphologically good blastocysts was significantly higher in the FA group than in the control group ( p = 0.0302). The morphokinetics during cleavage, morula, and blastocyst stages were comparable between the groups. The outgrowth was significantly increased in the FA group ( p = 0.0438). Clinical study: The rates of implantation, clinical pregnancy, and ongoing pregnancy after SVCTs were significantly increased in the FA group ( p = 0.0223–0.0281). Conclusions Fatty acid‐supplemented warming solutions effectively improve embryo development to the blastocyst stage and pregnancy outcomes after SVCTs.