{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T10:05:59Z","timestamp":1787997959142,"version":"build-2784847793"},"reference-count":59,"publisher":"Oxford University Press (OUP)","issue":"9","license":[{"start":{"date-parts":[[2023,6,28]],"date-time":"2023-06-28T00:00:00Z","timestamp":1687910400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/pages\/standard-publication-reuse-rights"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2023,9,5]]},"abstract":"Abstract<\/jats:title>\n \n STUDY QUESTION<\/jats:title>\n 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\u20137)?<\/jats:p>\n <\/jats:sec>\n \n SUMMARY ANSWER<\/jats:title>\n Increasing times to blastocyst formation are associated with a worse clinical outcome and perturbations in developmental patterns appear as early as the fertilization stage.<\/jats:p>\n <\/jats:sec>\n \n WHAT IS KNOWN ALREADY<\/jats:title>\n 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\u20137 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.<\/jats:p>\n <\/jats:sec>\n \n STUDY DESIGN, SIZE, DURATION<\/jats:title>\n This retrospective study involved time-lapse technology (TLT) monitoring of Day 4 (N\u2009=\u200970), Day 5 (N\u2009=\u20096147), Day 6 (N\u2009=\u20093243), and Day 7 (N\u2009=\u2009149) blastocysts generated in 9450 ICSI cycles. Oocyte retrievals were carried out after clomiphene citrate-based minimal ovarian stimulation, between January 2020 and April 2021.<\/jats:p>\n <\/jats:sec>\n \n PARTICIPANTS\/MATERIALS, SETTING, METHODS<\/jats:title>\n 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\u20137 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).<\/jats:p>\n <\/jats:sec>\n \n MAIN RESULTS AND THE ROLE OF CHANCE<\/jats:title>\n From 19\u00a0846 microinjected oocytes, 17\u00a0144 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\u2009\u00b1\u20090.4, 112.4\u2009\u00b1\u20090.1, 131.6\u2009\u00b1\u20090.1, and 151.2\u2009\u00b1\u20090.5\u2009h in the Day 4\u20137 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\u2009&lt;\u20090.0001). The differences in development times and intervals increased progressively until blastocyst expansion (P\u2009&lt;\u20090.0001 for all development times). Strikingly, such differences were already markedly evident as early as the time of pronuclear fading (tPNf) (20.6\u2009\u00b1\u20090.3, 22.5\u2009\u00b1\u20090.0, 24.0\u2009\u00b1\u20090.0, 25.5\u2009\u00b1\u20090.3; Days 4\u20137, respectively; P\u2009&lt;\u20090.0001). 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\u2009&lt;\u20090.0001), 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.<\/jats:p>\n <\/jats:sec>\n \n LIMITATIONS, REASONS FOR CAUTION<\/jats:title>\n The study is limited by its retrospective design. Having been obtained from a single centre, the data require independent validation.<\/jats:p>\n <\/jats:sec>\n \n WIDER IMPLICATIONS OF THE FINDINGS<\/jats:title>\n 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\u20137 blastocysts occur as early as the fertilization stage, possibly dictated by intrinsic gamete-derived factors.<\/jats:p>\n <\/jats:sec>\n \n STUDY FUNDING\/COMPETING INTEREST(S)<\/jats:title>\n This study was supported by the participating institutions. The authors have no conflict of interest to declare.<\/jats:p>\n <\/jats:sec>\n \n TRIAL REGISTRATION NUMBER<\/jats:title>\n N\/A.<\/jats:p>\n <\/jats:sec>","DOI":"10.1093\/humrep\/dead136","type":"journal-article","created":{"date-parts":[[2023,6,29]],"date-time":"2023-06-29T03:49:37Z","timestamp":1688010577000},"page":"1690-1699","source":"Crossref","is-referenced-by-count":36,"title":["The destinies of human embryos reaching blastocyst stage between Day 4 and Day 7 diverge as early as fertilization"],"prefix":"10.1093","volume":"38","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1635-9205","authenticated-orcid":false,"given":"Giovanni","family":"Coticchio","sequence":"first","affiliation":[{"name":"IVIRMA Global Research Alliance , 9.baby Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3778-2259","authenticated-orcid":false,"given":"Kenji","family":"Ezoe","sequence":"additional","affiliation":[{"name":"Kato Ladies Clinic , Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1663-1355","authenticated-orcid":false,"given":"Cristina","family":"Lagalla","sequence":"additional","affiliation":[{"name":"IVIRMA Global Research Alliance , 9.baby Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9017-6805","authenticated-orcid":false,"given":"Carlotta","family":"Zac\u00e0","sequence":"additional","affiliation":[{"name":"IVIRMA Global Research Alliance , 9.baby Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9049-0020","authenticated-orcid":false,"given":"Andrea","family":"Borini","sequence":"additional","affiliation":[{"name":"IVIRMA Global Research Alliance , 9.baby Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1394-4532","authenticated-orcid":false,"given":"Keiichi","family":"Kato","sequence":"additional","affiliation":[{"name":"Kato Ladies Clinic , Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2023,6,28]]},"reference":[{"key":"2023090508445524200_dead136-B1","doi-asserted-by":"crossref","first-page":"1270","DOI":"10.1093\/humrep\/der037","article-title":"The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting","volume":"26","author":"Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology","year":"2011","journal-title":"Hum Reprod"},{"key":"2023090508445524200_dead136-B2","doi-asserted-by":"crossref","first-page":"hoaa008","DOI":"10.1093\/hropen\/hoaa008","article-title":"Good practice recommendations for the use of time-lapse technology\u2020","volume":"2020","author":"Apter","year":"2020","journal-title":"Hum Reprod Open"},{"key":"2023090508445524200_dead136-B3","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1016\/j.rbmo.2020.11.013","article-title":"The freeze-all strategy after IVF: which indications?","volume":"42","author":"Bourdon","year":"2021","journal-title":"Reprod Biomed Online"},{"key":"2023090508445524200_dead136-B4","doi-asserted-by":"crossref","first-page":"1948","DOI":"10.1093\/humrep\/dez163","article-title":"Day 5 versus Day 6 blastocyst transfers: a systematic review and meta-analysis of clinical outcomes","volume":"34","author":"Bourdon","year":"2019","journal-title":"Hum Reprod"},{"key":"2023090508445524200_dead136-B5","doi-asserted-by":"crossref","first-page":"2860","DOI":"10.1016\/j.cell.2021.04.013","article-title":"Parental genome unification is highly error-prone in mammalian embryos","volume":"184","author":"Cavazza","year":"2021","journal-title":"Cell"},{"key":"2023090508445524200_dead136-B6","doi-asserted-by":"crossref","first-page":"1806","DOI":"10.3390\/jcm12051806","article-title":"Towards automation in IVF: pre-clinical validation of a deep learning-based embryo grading system during PGT-A cycles","volume":"12","author":"Cimadomo","year":"2023","journal-title":"J Clin Med"},{"key":"2023090508445524200_dead136-B7","doi-asserted-by":"crossref","first-page":"1134","DOI":"10.1093\/humrep\/deac080","article-title":"How slow is too slow? 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