Maternal and obstetric outcomes following the transfer of embryos warmed with fatty acid-supplemented solutions.

BACKGROUND: Vitrification procedures decrease intracytoplasmic lipid content and impair developmental competence. Adding fatty acids (FAs) to the warming solution has been shown to recover the lipid content of the cytoplasm and improve developmental competence and pregnancy outcomes. However, the influence of the FA supplementation on live birth rates after embryo transfers and perinatal outcomes remains unknown. In the present study, we examined the influence of FA-supplemented warming solutions on live birth rates, pregnancy complications, and neonatal outcomes after single vitrified-warmed cleavage-stage embryo transfers (SVCTs). METHODS: The clinical records of 701 treatment cycles in 701 women who underwent SVCTs were retrospectively analyzed. Vitrified embryos were warmed using solutions (from April 2022 to June 2022, control group) or FA-supplemented solutions (from July 2022 to September 2022, FA group). The live birth rate, pregnancy complications, and perinatal outcomes were compared between the control and FA groups. RESULTS: The live birth rate per transfer was significantly higher in the FA group than in the control group. Multivariate logistic regression analysis further demonstrated a higher probability of live births in the FA group than in the control group. Miscarriage rates, the incidence and types of pregnancy complications, the cesarean section rate, gestational age, incidence of preterm delivery, birth length and weight, incidence of low birth weight, infant sex, and incidence of birth defects were all comparable between the control and FA groups. Multivariate logistic regression analysis further demonstrated no adverse effects of FA-supplemented warming solutions. CONCLUSIONS: FA-supplemented warming solutions improved live birth rates after SVCTs without exerting any adverse effects on maternal and obstetric outcomes. Therefore, FA-supplemented solutions can be considered safe and effective for improving clinical outcomes and reducing patient burden.

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.

Fatty acid supplementation into warming solutions improves pregnancy outcomes after single vitrified-warmed cleavage stage embryo transfers.

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.

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.

Association between a deep learning-based scoring system with morphokinetics and morphological alterations in human embryos.

RESEARCH QUESTION: What is the association between the deep learning-based scoring system, iDAScore, and biological events during the pre-implantation period? DESIGN: 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. RESULTS: The duration of the cytoplasmic halo was significantly prolonged in low-scoring blastocysts (P < 0.0001). The timing of female and male pronuclei breakdown was significantly delayed in low-scoring blastocysts compared with high-scoring blastocysts (P < 0.0001 in both). Embryos with either trichotomous, multi-chotomous, rapid or reverse cleavage or asymmetric division had a lower score than embryos with normal cleavage (P < 0.0001-0.0098). The cell number and amount of blastomere fragmentation on days 2 and 3 were significantly associated with iDAScore (P < 0.0001-0.0008). Delayed compaction, blastulation and blastocyst expansion were observed in low-scoring embryos (P < 0.0001 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 (P < 0.0001). Multiple linear regression analysis revealed that, during the transformation to blastocyst stage, morphokinetic and morphological events were strongly associated with iDAScore (P < 0.0001-0.0116). CONCLUSIONS: 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.

Fatty acid supplementation during warming improves pregnancy outcomes after frozen blastocyst transfers: a propensity score-matched study.

This study aimed to examine the viability of human blastocysts after warming with fatty acids (FAs) using an in vitro outgrowth model and to assess pregnancy outcomes after a single vitrified-warmed blastocyst transfer (SVBT). For the experimental study, we used 446 discarded vitrified human blastocysts donated for research purposes by consenting couples. The blastocysts were warmed using FA-supplemented (FA group) or non-FA-supplemented (control group) solutions. The outgrowth area was significantly larger in the FA group (P = 0.0428), despite comparable blastocyst adhesion rates between the groups. Furthermore, the incidence of outgrowth degeneration was significantly lower in the FA group than in the control group (P = 0.0158). For the clinical study, we retrospectively analyzed the treatment records of women who underwent SVBT in natural cycles between January and August 2022. Multiple covariates that affected the outcomes were used for propensity score matching as follows: 1342 patients in the FA group were matched to 2316 patients in the control group. Pregnancy outcomes were compared between the groups. The rates of implantation, clinical pregnancy, and ongoing pregnancy significantly increased in the FA group after SVBTs (P = 0.0091-0.0266). These results indicate that warming solutions supplemented with FAs improve blastocyst outgrowth and pregnancy outcomes after SVBTs.