Articles de revues : « Vitrolite Division »

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Nowak, Agnieszka, Joanna Kochan, Barbara Kij-Mitka, Karolina Fryc et Wojciech Witarski. « The Use of Commercial Microvolume Techniques for Feline Oocyte Vitrification ». Animals 13, n^o 1 (22 décembre 2022) : 36. http://dx.doi.org/10.3390/ani13010036.

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This project aimed to compare the three most popular commercial oocyte vitrification techniques to determine their suitability for the vitrification of felid germlines in rescue and conservation programs. The present study aimed to determine the viability and developmental competence of feline oocytes after IVM and vitrification using a commercial vitrification method. In the first experiment, oocytes were vitrified after in vitro maturation (IVM) using the Kitazato, Cryotech, and Vitrolife methods. The oocytes were stained with fluorescein diacetate and ethidium bromide to evaluate their viability. The differences between Vitrolife and the control, Cryotech and Kitazato were statistically significant (p < 0.05), and between the control and Kitazato, were highly significant (p < 0.01). There were no significant differences between the control and Cryotech, Vitrolife and Cryotech, or Kitazato and Vitrolife. In the second part of the experiment, oocytes, after IVM and vitrification using three commercial methods, were subjected to fertilization. After vitrification, IVF was performed. We observed 35% of embryonic divisions in the group where Vitrolife and Kitazato media were used and 45% in the control group. In the presented experiment, vitrification with Vitrolife media gave slightly better results for survival and fertilization, while in the case of emergency protocol vitrification, all of the above methods may be useful to protect material derived from valuable wild felids.

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Quinn, Molly M., Philip Marsh, Salustiano Ribeiro, Rhodel K. Simbulan, Cristina Hickman, Jørgen Berntsen et Mitchell P. Rosen. « Aneuploidy rates and morphokinetic parameters of embryos cultured in distinct culture media : a sibling oocyte study ». Human Reproduction 37, n^o 2 (13 novembre 2021) : 226–34. http://dx.doi.org/10.1093/humrep/deab253.

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Abstract STUDY QUESTION Do embryos from sibling oocytes assigned to distinct single-step media culture systems demonstrate differences in early embryo development, morphokinectics or aneuploidy rates? SUMMARY ANSWER Embryo quality, morphokinetic parameters and aneuploidy rates from trophectoderm biopsy were similar between sibling embryos cultured in distinct media systems from the time of gamete isolation. WHAT IS KNOWN ALREADY Studies on the effect of commercially available embryo culture media systems have demonstrated inconsistent impact on human embryonic development, morphokinetics, aneuploidy rates and clinical outcomes. In addition, these studies have been primarily randomized at the level of the embryo or the patient to culture media. STUDY DESIGN, SIZE, DURATION Prospective sibling oocyte cohort derived from 200 subjects undergoing IVF at a tertiary academic medical center between February 2018 and November 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS Sibling oocytes were allocated to Global® or SAGE® media system based upon laterality of ovary from which they were retrieved. All embryos were cultured in a time-lapse incubator. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy using next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE One hundred twenty-seven subjects (n = 127) had paired blastocysts for biopsy in each culture media system. There was no difference in top quality blastocyst formation (47.1 ± 31.0 vs 48.1 ± 27.2%; P = 0.87) nor aneuploidy rate (62.3 ± 34.0 vs 56.1 ± 34.4%; P = 0.07) for sibling embryos cultured in Global versus SAGE media system. Embryo morphokinetic parameters including time to each cell division from two cells (t2) to eight cells (t8), time to morula stage (tM), time to blastocele formation (tSB), time to fully formed blastocyst (tB) and time to expansion of the blastocyst (tEB) were similar between paired blastocysts from each culture media system. LIMITATIONS, REASONS FOR CAUTION Pregnancy outcomes and offspring health data were not available for analysis. WIDER IMPLICATIONS OF THE FINDINGS Commercially available culture media may not have a differential impact on embryo development and blastocyst aneuploidy rate when patient and stimulation-related factors are held constant. STUDY FUNDING/COMPETING INTEREST(s) There was no external funding for this study. C.H. is owner of a consultancy company, IVF Professionals, Chief Scientific Officer at Apricity, Executive Director at TMRW and co-owner and shareholder of Aria Fertility. She has received speaker fees, consulting fees and travel support from Cooper Surgical and Vitrolife. J.B. is an employee and shareholder of vitrolife. TRIAL REGISTRATION NUMBER N/A.

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Tain, Diana Chieh Xing, Michelle Sheng Rong Lim, Bee Lian Ng, Elizabeth Hammond et Pak Seng Wong. « The Influence of Day 2 Blastomere Symmetry on Blastocyst Grade and Ploidy Status ». Fertility & ; Reproduction 01, n^o 02 (juin 2019) : 115–18. http://dx.doi.org/10.1142/s2661318219500117.

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Previous studies have suggested that aneuploidy rates are co-related with cell asymmetry at the cleavage stage. A retrospective study was carried out to determine the significance of blastomere symmetry at the 4-cell stage on blastocyst grade and ploidy status. 732 Day 5/6 blastocysts from 191 patients undergoing Pre-implantation Genetic Testing for Aneuploidy were analysed with time-lapse imaging (Embryoscope, Vitrolife) during 2017. Blastomere symmetry was measured at the first image of 4-cells on Day 2 by tabulating the mean diameter of 2 lines drawn perpendicularly on each blastomere. Symmetry was defined as the blastomere diameter difference of [Formula: see text] 25%. Trophectoderm (TE) biopsy was performed on Day 5/6 followed by chromosomal evaluation using Next Generation Sequencing (VeriSeq Protocol, Illumina). Blastocyst grade was classified as either “Good” (inner cell mass (ICM) and TE, AA respectively), “Fair/Good” (AB, BA), “Fair” (BB) and “Poor” (early blastocyst grade 2 or TE grading of C). The significance of blastomere symmetry on blastocyst grade and ploidy status was measured using chi-square tests. There was no significance difference in resulting blastocyst quality for symmetrical and asymmetrical embryos (Table 1: p [Formula: see text] 0.10). Furthermore, there was no significance difference in the euploid rate (42.5% vs. 45.3%) or mosaic rate (22.1% vs. 16.2%) between symmetrical and asymmetrical embryos (p [Formula: see text] 0.24). In conclusion, the presence of asymmetrical blastomeres at the 4-cell stage do not impact the good quality blastocyst formation rate and euploidy rate for embryos that progress into blastocysts. However, this study excludes embryos that do not develop to the blastocyst stage and those with erratic division patterns, direct cleavage and reverse cleavage on Day 2, both of which have potential to influence ploidy result. Asymmetrical 4-cell embryos have the potential for high quality euploid blastocyst progression and can be considered for day 2 embryo transfer in the absence of symmetrical 4-cell embryos.

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Gazzo, Eduardo, Fernando Peña, Federico Valdez, Arturo Chung, Mario Ascenzo, Marcelo Velit et Ernesto Escudero. « Las contracciones en blastocistos humanos se correlacionan con aneuploidía, menor implantación y mayor tiempo de llegada a blastocisto : estudio retrospectivo con incubadora TIME-LAPSE ». Revista Peruana de Ginecología y Obstetricia 65, n^o 2 (9 mai 2019) : 171–78. http://dx.doi.org/10.31403/rpgo.v65i2170.

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Objetivos. Estudiar los patrones de contracciones en blastocistos humanos mediante el uso de una incubadora time-lapse y correlacionarlos con su estado de ploidía por análisis PGT-A, el tiempo para alcanzar el estado blastocisto, la tasa de implantación y de embarazo clínico. Diseño. Estudio de cohortes retrospectivo. Intervenciones. Entre octubre 2016 y mayo 2018, se evaluó 270 pacientes; se hizo cultivo extendido de 5 a 6 días a 912 embriones en la incubadora time-lapse (Embryoscope, Vitrolife), y a 778 se les estudió para aneuploidía usando una plataforma NGS en un laboratorio de referencia. Hubo posterior vitrificación, según resultado del desarrollo embrionario y en espera del resultado del NGS, seguido de desvitrificación y transferencia de embrión único. Se determinó las contracciones del blastocisto (CTB) mediante la herramienta de dibujo del embrión EmbryoViewer (EmbryoViewer drawing tools), de manera de obtener el área, porcentaje de contracción y los diferentes tipos de contracciones, y se comparó los embriones con el resultado del estudio genético mediante NGS. Se transfirió 182 embriones en pacientes de 30,4 años promedio, rango entre 24 y 39 años. Finalmente, se correlacionó la tasa de implantación y embarazo clínico de los embriones euploides que fueron transferidos, en el programa de reproducción asistida. Resultados. Se separó los embriones en dos grupos de acuerdo a las contracciones durante su desarrollo, en aquellos que las tuvieron (CT) y aquellos que no, denominados ‘solo expanding’ (SE). Los embriones SE fueron euploides en 58,3%, mientras los embriones CT fueron aneuploides en 53,6%, con significancia estadística (p=0,012). Ello indica que la mayoría de los embriones euploides hacen ‘solo expanding’ durante su desarrollo, mientras que la mayoría de los embriones aneuploides (53,9%) hacen contracciones durante su desarrollo (p=0,029). Del mismo modo, la tasa de embarazo clínico de los embriones SE euploides fue 63,1% frente a 46,7% de los embriones CT, p=0.012. Finalmente, los embriones euploides CT tardaron más en convertirse en blastocistos tempranos que los embriones SE, p=0.004. La edad de la mujer no representó un factor para contracción embrionaria. Conclusiones. Los resultados obtenidos en este estudio muestran que los embriones que muestran contracciones, sin importar que tan intensas sean, están relacionados con mayor probabilidad de aneuploidías, menor tasa de implantación y ritmos de división lentos. El simple hecho de observar contracciones en un embrión podría ser útil para decidir transferir otro embrión que no las haya tenido. Se requiere más estudios para comprobar estos hallazgos.

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Velez, D. A., H. Atashi, J. Dewulf, K. Smits et A. Van Soom. « 29 Time-lapse analysis of bovine embryos derived after invitro fertilization from vitrified and fresh oocytes ». Reproduction, Fertility and Development 32, n^o 2 (2020) : 140. http://dx.doi.org/10.1071/rdv32n2ab29.

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Oocyte vitrification enables the long-term conservation of female genetic resources. However, obtaining viable bovine embryos from vitrified oocytes has proven to be difficult. Embryo development is a dynamic process, and critical stages can go unnoticed with the use of traditional morphologic assessments. Therefore, the aim was to evaluate morphokinetic parameters in embryos derived from fresh versus vitrified oocytes. After 22h of IVM, oocytes were divided into (1) oocytes surrounded by multiple layers of cumulus cells (COCs; n=275) and (2) oocytes partially denuded, leaving only corona radiata (CR; n=178). Then, two-thirds of the CR oocytes were subjected to one of two vitrification protocols as follows: high concentration of cryoprotectants (CR-H; n=171; Ortiz-Escribano et al. 2016 Theriogenology 86, 635-641) and low concentration of cryoprotectants (CR-L; n=163; Ishii et al. 2018 J. Reprod. Develop. 6). After warming, vitrified oocytes were incubated ~2h in maturation medium. Invitro fertilization and culture were performed simultaneously for all groups. Four to eight zygotes from each group were assigned randomly to time lapse (Primo Vision; Vitrolife), and group culture was performed in leftovers as a control. Differences between groups in survival (intact zygotes after IVF), cleavage, and blastocyst rates were evaluated by logistic regression. Morphokinetic data (time to reach first (1-2 cells), second (3-4 cells), third (5-8), fourth (9-16 cells), fifth (>16 cells), cleavage, and blastocyst stage, and time in lag phase) were investigated. Survival rates in COCs (98%) and CR (96%) groups were not different; CR-H (87%) showed lower survival than COCs but similar survival to that of CR. Group CR-L (82%) had a lower survival rate than the rest of the groups (P<0.05). Higher cleavage rate (83%) was found in COCs compared with the rest of the groups. The CR and CR-H groups showed similar cleavage rate (65 and 55%, respectively), whereas CR-L had a lower cleavage rate (42%) than other groups (P<0.05). As expected, both vitrified groups showed lower blastocyst rates (4% for CR-H and 10% for CR-L) than fresh COCs (44%; P<0.05). Morphokinetics measures were affected by the treatments: time to reach first cleavage was similar for COCs (35.3h), CR (38.4h), and CR-L (34.8h), whereas CR-H was slower (42.4h). However, the fourth division was reached earlier by CR-H (51.7h), which was significantly faster than for CR-L (88.8 h; P<0.05) but similar for COCs (71.3h) and CR (60.7h). In conclusion, more morphokinetic data are needed to compare different vitrification methods and confirm whether time-lapse analysis can be used to predict blastocyst outcome after oocyte vitrification.

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Tomida, M., S. Watanabe, S. Suzuki, Y. Matsuda, K. Yoshikai, E. Nakano et T. Sawada. « P-123 Early irregular division of human embryos changes in style between the first and second divisions ». Human Reproduction 38, Supplement_1 (1 juin 2023). http://dx.doi.org/10.1093/humrep/dead093.487.

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Abstract Study question Is there a difference in irregular division kinetics between first and second division of human embryos? Summary answer The incidence of rapid cleavage was reduced in the second division, but the developmental potential of the blastomeres by this dynamics was unexpectedly low. What is known already The styles of irregular division of early human embryos are known: direct cleavage (DC), in which one cell becomes three (or more) cells without a two-cell phase, and rapid cleavage (RaC), in which one (or both) cells dividing rapidly after two-cell phase, both of which reduces the embryonic developmental potential. However, there are no reports that have examined in detail whether these division styles occur similarly in the first and second divisions, and how these dynamics affect the potential. Study design, size, duration This is a retrospective study of 635 embryos collected in 2020 at our clinic and cultured for at least 5 days. The embryos were time-lapse monitored by EmbryoScope (Vitrolife, Sweden) to observe the first and second divisions, and the blastomeres of each embryo were classified according to their style of division. Participants/materials, setting, methods It was observed whether there was a DC (division into 3 cells without a 2-cell phase) or RaC (One or two blastomeres divide within 5 hours after the 2-cell phase) at the first division. First dividing normal embryos were observed for the presence of a DC or RaC at the second division. Blastomeres by DC or RaC were observed for subsequent development and whether they participated in blastocysts or not was recorded. Main results and the role of chance Among the subject embryos, 63 embryos had DC and 199 embryos had RaC in the first division, and their blastomeres were classified into the DC1 and RaC1 groups, respectively. Of the 373 first division normal embryos, 39 had DC and 23 had RaC (there were 4 embryos with both DC and RaC) in the second division, and their blastomeres were classified into DC2 and RaC2 groups. The blastomeres blastocyst participation rate was 4.4% in the DC1 group and 19.5% in the RaC1 group, respectively, the former was significantly lower (P < 0.01), however, in the DC2 and RaC2 groups, 23.3% and 4.2%, respectively, the latter was significantly lower (P < 0.01). The incidence of DC was similar in the first (9.9%) and second (10.5%) divisions, but the incidence of RaC was 31.3% in the first division and 6.2% in the second division, the latter was significantly lower (P < 0.01). The development rate of good blastocysts (≥4BC in Gardner grade) was 4.8% in embryos with DC1 and 19.5% in embryos with RaC1, with the former significantly lower (P < 0.01), 37.0% in embryos with DC2, and 26.3% in embryos with RaC2, with no significant difference. Limitations, reasons for caution As of 2020, the clinical use of PGT-A is not approved in principle in Japan, and the subject embryos have not been chromosomally analyzed. Wider implications of the findings RaC would lead to inadequate DNA proliferation, but the incidence of this and the prognosis of the blastomeres were different in the first and second divisions. In human embryos, the mechanisms that control blastomeres from dividing may change as development progresses, but further studies are needed to elucidate this. Trial registration number not applicable

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van Duijn, Linette, Melek Rousian, Charlotte S. Kramer, Eva S. van Marion, Sten P. Willemsen, Jeroen P. Speksnijder, Joop S. E. Laven, Régine P. M. Steegers-Theunissen et Esther B. Baart. « The Impact of Culture Medium on Morphokinetics of Cleavage Stage Embryos : An Observational Study ». Reproductive Sciences, 9 mai 2022. http://dx.doi.org/10.1007/s43032-022-00962-7.

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AbstractTo study the impact of culture media on preimplantation morphokinetics used for predicting clinical outcomes. All IVF and ICSI cycles performed between 2012 and 2017 with time-lapse information available were included. In November 2014, culture medium was changed from Vitrolife G-1 PLUS to SAGE 1-Step. Each embryo was retrospectively assigned a morphokinetic-based KIDScore for prediction of implantation. Clinical outcomes were retrieved from medical records. Linear mixed models were used to study differences in morphokinetic parameters, a proportional odds model for KIDScore ranking and logistic regression for differences in clinical outcomes. All analyses were adjusted for patient and treatment characteristics. In 253 (63.1%) cycles, embryos (n = 671) were cultured in Vitrolife, and in 148 (36.9%) cycles, embryos (n = 517) were cultured in SAGE. All cleavage divisions occurred earlier for SAGE embryos than for Vitrolife embryos (2-cell: -2.28 (95%CI: -3.66, -0.89), 3-cell: -2.34 (95%CI: -4.00, -0.64), 4-cell: -2.41 (95%CI: -4.11, -0.71), 5-cell: -2.54 (95%CI: -4.90, -0.18), 6-cell: -3.58 (95%CI: -6.08, -1.08), 7-cell: -5.62 (95%CI: -8.80, -2.45) and 8-cell: -5.32 (95%CI: -9.21, -1.42) hours, respectively). Significantly more embryos cultured in SAGE classified for the highest KIDScore compared to embryos cultured in Vitrolife (p < 0.001). No differences were observed in clinical outcomes. Our results demonstrate an impact of culture medium on preimplantation embryo developmental kinetics, which affects classification within the KIDScore algorithm, while pregnancy outcomes were comparable between the groups. This study underscores the need to include the type of culture medium in the development of morphokinetic-based embryo selection tools.

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Urich, Michael, Muhammet Rasit Ugur, Fang Li, F. Nicholas Shamma, Ahmad Hammoud, Hanh N. Cottrell et Sule Dogan. « Comparison of two culture media on morphokinetics and ploidy status of sibling embryos ». Zygote, 9 décembre 2021, 1–6. http://dx.doi.org/10.1017/s0967199421000927.

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Summary To investigate the effects of culture media with different lactate concentrations on early embryonic development, data collected from our patients undergoing preimplantation genetic testing (PGT) were assessed using the EmbryoScope™ time-lapse culturing system. After intracytoplasmic sperm injection (ICSI), sibling oocytes were cultured in the same EmbryoScope (Vitrolife) slides including two different commercially available media. The patients with fewer than five mature oocytes were not included in the analyses. All embryos were hatched on day 3, and trophectoderm biopsies (n = 212) were performed accordingly. PGT for aneuploidy (PGT-A) on biopsied materials was carried out using next generation sequencing. Morphokinetic parameters, fertilization, irregular division, degeneration, blastulation, euploidy, and pregnancy rates of embryos cultured in LifeGlobal Global Total medium (LGGT) and Continuous Single Culture-NX Complete medium (CSCM-NXC) were compared. There were no differences observed in time to pronuclear fade, or in time spent as 2-cell (cc2) and 3-cell (s2), to 4-cell, 5-cell, morula and blastocyst stages (P > 0.05). Embryos reached the 2-cell (t2) and 3-cell (t3) stages significantly faster in LGGT (P < 0.05), whereas embryos grown in CSCM-NXC with lower lactate reached starting blastulation significantly sooner (P = 0.026). However, there were no statistical differences observed in fertilization, blastulation, degeneration, irregular division euploidy, and pregnancy rates between the two groups (P > 0.05). Even though pregnancy and fertilization rates did not indicate statistical differences, results are significant to provide better insight on potential roles of lactate in embryo development. These finding will advance the fundamental knowledge of human embryo development and assisted reproductive technologies.

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Watanabe, S., M. Tomida, S. Suzuki, Y. Matsuda, K. Yoshikai, E. Nakano et T. Sawada. « P-117 The reason why direct cleavage and rapid cleavage should be differentiated ». Human Reproduction 38, Supplement_1 (1 juin 2023). http://dx.doi.org/10.1093/humrep/dead093.481.

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Abstract Study question Direct cleavage (DC) and Rapid cleavage (RaC) both appear to be 1 cell divided into 3 (or more) cells, should they be distinguished? Summary answer Since blastomeres by RaC had higher developmental potential than blastomeres by DC, and since many RaC embryos had “normal” blastomere, the two should be distinguished. What is known already DC, in which one cell divides into three (or more) cells without the two-cell stage, and RaC, in which one or two cells divide rapidly after the short two-cell stage (The former lead to segregation of chromosomes into three cells, while the latter leads to inadequate DNA replication), are both often recorded as “DC”, but when classified in detail, there are reports that the blastocyst development rate is higher in RaC embryos. However, the reason for this has not been clarified. Study design, size, duration This was a retrospective study of 643 embryos collected and cultured for at least 5 days in 2020 at our clinic. The embryos were time-lapse monitored by EmbryoScope (Vitrolife, Sweden) and classified into three groups according to the style of first division. Participants/materials, setting, methods The embryos were classified as follows: DC group: embryos that have divided into three (or more) cells without a two-cell stage; RaC group: embryos that rapidly (<5 hours) divided one (or both) cells after the 2-cell stage; Normal cleavage (NC) group: embryos that had a 2-cell stage for more than 5 hours. The subsequent development of blastomeres of all embryos was observed and whether they participated in blastocysts or not was recorded. Main results and the role of chance Of the embryos, 63 were in the DC group and 199 were in the RaC group (173 of which had one rapidly dividing blastomere and 26 of which had two). The blastocyst participation rate of blastomeres was 4.4% in the DC group, 19.5% of rapidly dividing blastomeres and 61.6% of normal blastomeres in the RaC group, with significant differences between all groups (p < 0.01). The good blastocyst (4BC or higher in Gardner grade) development rate was 4.8% (3/63) in the DC group, 40.5% (70/173) in the RaC group with one rapidly dividing blastomere (RaC1 group), 19.2% (5/26) in the RaC group with two such blastomeres (RaC2 group), showing a significant difference between the DC and RaC1 groups (P < 0.01). The live birth rate for single blastocyst transfer was 50% (1/2) in the DC group, 27.8% (5/18) in the RaC1 group, and 100% (1/1) in the RaC2 group. (Note that there were 381 in the NC group, and the above rates were 76.5%, 63.3% (241/381), and 29.3% (22/75), respectively) Limitations, reasons for caution This study examined only the style of first division and did not consider second and subsequent divisions. In addition, as of 2020, the clinical use of PGT-A is not approved in principle in Japan, and the subject embryos have not undergone chromosome analysis. Wider implications of the findings The reason for the higher blastocyst development rate in RaC embryos was indicated by the difference in blastocyst participation rates of the respective blastomeres and the presence of normal blastomeres, but since live births were obtained in both embryos, so these irregular divisions would not completely compromise the blastocyst euploidy. Trial registration number not applicable

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Jung, N. H., S. H. Jeong, M. J. Kim, H. J. Jeong, M. H. Kim, H. S. Lee et M. K. Chung. « P-264 Analysis of embryo development based on time of pronuclei appearance (tPNa) and aspects of overcoming delayed pronuclei appearance through morphokinetic patterns ». Human Reproduction 39, Supplement_1 (1 juillet 2024). http://dx.doi.org/10.1093/humrep/deae108.634.

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Abstract Study question How much delay in the appearance of 2PN can be considered normal when predicting embryo transfer potential? Summary answer Embryos with tPNa <13hpi post-insemination develop into good-quality blastocysts; embryos with tPNa ≥13hpi develop blastocysts by shortening the time of tPNf-tPNa. What is known already Oocytes generally exhibit 2PNs at 17–20 hours, with pronuclei fading (time of pronuclei fading; tPNf) at 23–25 hours and developing into two cells (time of 2-cell division; t2) at 25–33 hours. Fertilization confirmation is typically performed 17–18 hours after insemination. However, a few oocytes with no visible PN (0PN) at the time of fertilization confirmation develop morphologically normal blastocysts, ultimately leading to pregnancy. The failure to identify PNs can be attributed to two scenarios: rapid fading or delayed appearance. Notably, there is still a lack of research on the normal range for delayed PN appearance. Study design, size, duration This study was conducted with 2153 embryos obtained from 390 intracytoplasmic sperm injection cycles (August 2021 to June 2023). All embryos were incubated for 5 days using a time-lapse system (EmbryoScopeTM+, Vitrolife). The blastocyst development rate and morphokinetic parameters according to the tPNa of embryos were analyzed using KIDScore D5 v3 and iDAScore v2.0 (VTH server+, Vitrolife). Clinical pregnancy was also analyzed. Participants/materials, setting, methods Morphokinetic parameters were analyzed from time of second polar body (tPB2) ∼ time of expanded blastocyst (tEB). The times taken for PN to appear after the second polar body release (tPNa–tPB2), for PN to fade (tPNf–tPNa) and for 2-cell division (t2–tPNf) were calculated and compared. Blastocysts were graded using the Gardner system, a grade of BB or higher divided into good quality blastocysts (GQ-BL). Main results and the role of chance tPNa was observed as 8.09±2.11hpi [1.93hpi∼32.46hpi; <5hpi (n = 49), 5∼6hpi (n = 474), 7∼8hpi (n = 1220), 9∼10hpi (n = 266), 11∼12hpi (n = 91), 13∼14hpi (n = 29), 15∼20hpi (n = 18) and >20hpi (n = 6)]. The rate of blastocysts was highest at 5∼6hpi (64.98%) and significantly lower at 9∼10hpi (54.14%), 11∼12hpi (42.86%) and 13∼14hpi (31.03%) (p < 0.005). Similarly, the rate of GQ-BL was also highest at 5∼6hpi (29.11%) and significantly lower at 9∼10hpi (19.17%), 11∼12hpi (10.99%) and 13∼14hpi (3.45%) (p < 0.005). No embryos developed into GQ-BL at 15∼20hpi, and no embryos developed into blastocysts at > 20hpi. The iDAScore was significantly different at < 13hpi and ≥13hpi (6.00±1.86 vs. 4.24±2.26, p < 0.005). Similarly, KIDScore D5 showed the same patterns (6.33±1.93 vs. 4.05±1.66, p < 0.005). At ≥ 13hpi, no blastocysts led to pregnancy. Morphokinetic parameters were analyzed to identify the factors influencing the development of blastocysts. The analysis revealed that tPNf-tPNa tended to gradually become shorter with delayed tPNa. Notably, there were significant differences in tPNf-tPNa between <13hpi and ≥13hpi (16.37±4.10 vs. 13.72±6.28, p < 0.005). At ≥ 13hpi, tPNf-tPNa was shorter in blastocysts than in cases of cleavage arrest (10.68±5.74 vs. 15.28±5.97, p < 0.05). It was particularly observed that tPNf-tPNa of GQ-BL in ≥ 13hpi was 7.90hpi, and t2 was not significantly different from blastocysts and GQ-BL in < 13hpi (25.06 vs. 25.66±3.18 vs. 25.19±2.97). Limitations, reasons for caution More data are needed for conclusive pregnancy results due to the small number of samples. Additionally, the maturation of oocytes had yet to be considered; further detailed studies related to oocyte maturity are needed. Wider implications of the findings Embryos with a tPNa of < 13hpi develop into GQ-BL. Embryos with delayed tPNa tend to overcome this by shortening the tPNf-tPNa, resulting in develop into blastocysts. Predicting blastocyst development can be achieved by considering factors such as tPNa∼t2. This can aid in improving the selection of embryos for cleavage-stage ET. Trial registration number N/A

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Park, H., O. Yhr, A. Khatibi, M. Gülich, H. Habte Selassie, K. Lundin et J. Hreinsson. « P-145 Direct-ICSI results in good embryological outcomes. A time-lapse study ». Human Reproduction 38, Supplement_1 (1 juin 2023). http://dx.doi.org/10.1093/humrep/dead093.509.

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Abstract Study question Is there a correlation between the method of sperm injection and morphokinetic and morphologic characteristics? Summary answer Results were comparable between the groups whereas the rate of utilizable embryos was significantly higher using Direct-ICSI. This suggests safety and efficacy using this technique. What is known already Maintaining high fertilization- and embryo development rates are key objectives of the IVF-laboratory. Little effort has been put into studying the effects of different manipulation techniques when performing manual microinjections. Specifically, we wanted to compare two methods for achieving cell-membrane puncture: Traditional cytoplasm aspiration technique versus membrane stretching and injection without aspiration –the so-called Direct ICSI method. This method has been proposed to give higher fertilization rates and is being implemented in IVF-laboratories to try to increase ICSI-results. As far as we are aware, no detailed comparison between the two methods on embryo development using time-lapse documentation has been published. Study design, size, duration The study included 44 ICSI-treatments from April 2020 to October 2021 with 119 oocytes injected with Direct-ICSI and 349 sibling oocytes using the conventional aspiration-ICSI technique. In this way, each patient contributed oocytes for both test- and control groups. However, as this was a pilot study, typically only a minority of the oocytes for each patient was injected using the Direct-ICSI method. All fertilized oocytes were included in the assessments up to the blastocyst stage. Participants/materials, setting, methods The center is a public funded University clinic performing > 500 ICSI cycles annually. Inclusion criteria were treatment cycles using fresh own oocytes with ejaculated sperm and at least 5 injectable oocytes after denudation. Time lapse culture was performed using the Embryoscope+ with GTL culture media, both supplied by Vitrolife. Clinical and laboratory procedures were performed according to standard practice. Fisheŕs exact test (categorical variables) and Mann-Whitney U test (continuous variables) were used for statistical analysis. Main results and the role of chance In total, 314 oocytes (67,1%) were fertilized and included in the morphology and morphokinetic assessments up to the blastocyst stage (day 5 or day 6 post-fertilization). Time-lapse parameters such as timing of cell division events and time to morula, time to blastocyst and similar were not significantly different between the groups. Rates of categorical classification of embryological anomalies such as degree of fragmentation, rate of multinucleated cell events, uneven cell size and irregular cell division events also did not differ significantly between the groups. Interestingly however, although fertilization rates did not differ between the two groups (68,1% in the test group vs 67,6% in the control group), the rate of utilizable embryos per injected oocyte was significantly higher in the Direct-ICSI group (40,3% vs 29,5%, p < 0.03), whereas the pregnancy rates did not differ between the groups. Limitations, reasons for caution The present study is a small pilot study with a limited number of cycles, however with 468 oocytes included. The study utilized internal (sibling) controls although oocytes were not truly randomized between the groups. To fully test the efficiency of Direct-ICSI a larger randomized study should be performed. Wider implications of the findings Our results indicate that Direct-ICSI is a safe method with no visible indication of irregularities in preimplantation cell division events using time-lapse monitoring. Aspirating an excess amount of cytoplasm during ICSI might impact embryo development, possibly explaining the increase of utilizable embryos in the Direct-ICSI group, where aspiration is avoided. Trial registration number Not applicable

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Browne, A., L. Kourouniadou et J. Leitch. « O-287 Prognostic value of perivitelline-thread-associated kinetic activity of the plasma membrane of the pronuclear human zygote : a retrospective, observational study ». Human Reproduction 37, Supplement_1 (29 juin 2022). http://dx.doi.org/10.1093/humrep/deac106.080.

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Abstract Study question Is perivitelline-thread-associated kinetic activity of the zygote membrane prior to pronuclear fading (tPNf) indicative of the developmental potential of human embryos in vitro? Summary answer Embryos displaying kinetic activity of the zygote membrane had a significantly decreased clinical utilization rate, compared to those without. What is known already Morphology (pronuclei, cytoplasm etc.) of the pronuclear human zygote has been shown to correlate with ongoing embryo development and implantation potential. Further morphokinetic observations have been noted during this dynamic period of preimplantation development with the use of time-lapse imaging. The hypothesized mechanical actions of perivitelline threads (PVT) have been associated with blastomere fragmentation during early cleavage divisions and subsequent poor embryo development. Their presence and activity prior to tPNf and prognostic value with regard to treatment outcome, have not been studied in detail. Study design, size, duration This retrospective study assessed the development of 1047 two pronuclear (2PN) zygotes post IVF/ICSI until culture day 5, from a total of 172 patients which underwent time-lapse imaging monitoring with the EmbryoScope + (Vitrolife) between 01/06/2019 and 20/03/2020. Participants/materials, setting, methods Time-lapse videos of embryo development were analyzed manually for the detection and characterization of the ‘event’ of interest: an evident wave of membrane displacement coordinated by PVT prior to tPNf and first cleavage division. Assessed embryos were then categorized into two groups: an event group and a non-event group. The primary outcome of embryo utilization rate was compared between the two groups. Live birth and miscarriage rates per blastocyst transferred were assessed as secondary outcomes. Main results and the role of chance Morphokinetic analysis of the event established a mean commencement time at 22.5 ± 9.6 hours, cessation at 27.3 ± 4.7 hours, followed by tPNf at 27.9 ± 5.1 hours post fertilization. A total of 157 embryos in the event group and 890 in the non-event group were assessed (event occurrence rate of 15.0%). The average age of oocyte utilized was not significantly different between the event and non-event group at 34.6 years versus 34.5 years, respectively. The embryo utilization rate in the non-event group was significantly higher than that observed in the event group; 50.6% versus 29.9%, respectively (p < 0.001). The odds of an embryo not being utilized were higher in embryos with an observed event, compared to those without (OR = 2.3; 95% CI = 1.66, 3.45). Live birth rates for fresh blastocyst transfers were 42.9% (6/14) and 29.8% (42/141) for the event and non-event groups, respectively (p > 0.05). Miscarriage rates for fresh blastocyst transfers were 33.3% (3/9) and 35.4% (23/65) for the event and non-event groups, respectively (p > 0.05). Overall, these results indicate that event observation is associated with a significantly decreased embryo utilization rate due to subsequent poor embryo development in extended culture conditions. Limitations, reasons for caution A number of events occurring outside the plane of focus prevented accurate assessment, resulting in data exclusion. High levels of variability between the degree of kinetic activity, and transient nature of the PVT were observed. Differences in treatment outcomes were not powered for statistical analysis. Wider implications of the findings Notably, embryos displaying kinetic activity of the zygote membrane prior to tPNf are less likely to develop into embryos which can be utilized for transfer or cryopreservation. Observation of the described event may aid with clinical decision-making and managing patient expectations during the period of in vitro monitoring and development. Trial registration number Not applicable

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Barrie, R., A. Barrie et A. Campbell. « P-222 Can embryo morphokinetics act as early warning key performance indicators in relation to consumable batching ». Human Reproduction 39, Supplement_1 (1 juillet 2024). http://dx.doi.org/10.1093/humrep/deae108.592.

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Abstract Study question Can morphokinetics be used as an early warning indicator of a batch-related effect of oil currently in use in the laboratory on implantation rate? Summary answer Where morphokinetic timings were slower for a batch of oil, implantation rate was also reduced, suggesting they could be used as an early warning system. What is known already The commercialisation of oil means that there are strict manufacturing guidelines leading to consistency between batches. However, this does not mean that minor differences do not exist. Furthermore, oil can undergo peroxidation if stored incorrectly, causing it to become toxic to embryos. The introduction of timelapse incubation has allowed extensive research into several morphokinetic parameters. This research has established time-frames within which each developmental milestone should occur to indicate the likelihood of implantation. Implantation rate (IR) is often used as a key performance indicator in the laboratory however, potential systemic issues may be identified sooner if morphokinetic parameters were used. Study design, size, duration The following morphokinetic parameters were compared to IR to determine if a batch of oil was affecting embryo development; time of division to two cells (t2), five cells (t5), eight cells (t8), time to start of blastulation (tSB) and cell synchronicity between two and eight cells (CS2-8). Data from January 2019 to October 2019 from a single centre were used, and included data from 2008 embryos and 333 patients. Participants/materials, setting, methods Data was exported from the EmbryoScope + [Vitrolife, Sweden]. Data from RIWitness Laboratory Manager [CooperSurgical, Denmark] was utilised to identify when different batches of oil (Ovoil™, Vitrolife) were used within the specified time period. The data from the EmbryoScope+ was then separated by date to signify when a new batch of oil was open. A Kruskal-Wallis test was then used to identify whether the morphokinetic timings differed between batches of oil. Main results and the role of chance There was no significant difference in patient age (p = 0.178) or the number of oocytes collected (p = 0.097) between batches of oil used. There was no significant difference between the morphokinetic timings for each batch of oil used. However, the morphokinetic timings follow the same trend line as the IR for both t2 and t8; where t2 and t8 are higher, the IR for the corresponding batch of oil is reduced. The trend can also be seen in CS2-8. The batch of oil with the lowest IR (25.56%) also had the highest mean time to t2, t8 and tSB compared to the other batches of oil. This corresponds with previous research that identified a correlation between slower embryo development and reduced IR (Meseguer et al., 2011). A clear difference could be seen between the average time taken to reach each developmental milestone and each batch of oil used, perhaps suggesting oil can have an effect, however small, on embryo development. This study suggests that morphokinetic timings could be used as an early warning indicator that a new batch of oil may be affecting development allowing a rapid response by the laboratory team and possible cessation of use pending further investigation. Limitations, reasons for caution Different batches of culture medium were also used throughout this time period and were not introduced at the same time as oil batches. Therefore, the results of this investigation cannot be associated entirely to the introduction of a different batch of oil. Wider implications of the findings This information could be incorporated into an early warning algorithm that could be assessed after a batch of oil was introduced. The use of the batch could then be ceased immediately without waiting for a decrease in IR by which time it would have been used for many more embryos. Trial registration number Not applicable

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Watanabe, S., K. Yoshikai, M. Tomida, S. Suzuki, Y. Matsuda, S. Miyai, E. Nakano, H. Kurahashi et T. Sawada. « P-131 The fate of irregularly divided blastomeres : why does “Direct cleavage” reduce blastocyst development rate but not blastocyst euploid rate ? » Human Reproduction 37, Supplement_1 (29 juin 2022). http://dx.doi.org/10.1093/humrep/deac107.126.

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Abstract Study question How do the blastomeres formed by direct cleavage (dynamics of one cell dividing into three or more cells) subsequently develop? Summary answer About half of the blastomeres by direct cleavage did not form blastocysts. What is known already There are many reports that embryos with direct cleavage in the early development have a lower blastocyst development rate because direct cleavage produces chromosomal abnormal cells. However, when such embryos develop into blastocysts, there have been some reports that the transfer pregnancy rate and euploid rate did not decrease, but the reasons for this have not been clarified. Study design, size, duration This is a retrospective study of 89 blastocysts obtained during 2013-18. These embryos were those that patients requested to be discarded and consented to be used in this study. All target embryos were time-lapse monitored by EmbryoScope (Vitrolife, Sweden), and several trophectoderms were biopsied and examined for euploidy. Participants/materials, setting, methods The target embryos were classified into three groups: embryos with normal first and second cleavage (NC group), embryos with irregular division (one cell dividing into three or more cells) called direct cleavage at the first cleavage (DC1 group), and embryos with direct cleavage of one blastomere at the second cleavage (DC2 group). It was recorded whether the blastomeres of the embryos subsequently developed into blastocysts or not. NGS analysis was performed on the embryos. Main results and the role of chance The target embryos were classified as 48 in the NC group, 32 in the DC1 group, and 9 in the DC2 group. Whether the blastomeres in the target embryos subsequently formed blastocysts or not was recorded one by one by time-lapse images, resulting in the blastomeres’ blastocyst formation rate was 95.1% in the NC group and 55.9% in the DC1 group, which was significantly lower in the DC1 group (P < 0.01). In the DC2 group, blastomeres formed by normal division and those by direct cleavage at the second cleavage were recorded separately, and the blastocyst formation rate was 90.8% for normal cleavage blastomeres and 46.0% for direct cleavage blastomeres, with significantly lower rates for direct cleavage blastomeres (P < 0.01). Therefore, about half of the blastomeres generated by direct cleavage at the first or second cleavage did not form blastocysts. The results of NGS analysis were as follows: NC group: 35.4% euploid, 45.8% aneuploid, and 18.8% mosaic; DC1 group: 37.5%, 53.1%, and 9.4%, respectively; and DC2 group: 55.6%, 33.3%, and 11.1%, respectively. There was no significant difference in any of the items, suggesting that direct cleavage does not affect the euploidy of blastocysts. Limitations, reasons for caution For the purpose of NGS analysis, all the target embryos in this study were blastocysts, but if all the cultured embryos were included, arrested embryos would be included, which would probably result in more blastomeres formed by direct cleavage not developing into blastocysts. Wider implications of the findings The blastomeres generated by direct cleavage were often excluded from blastocyst formation. This may be an exclusion of chromosomally abnormal cells and may be one of the reasons why direct cleavage decreases blastocyst development rate but does not decrease blastocyst euploid rate. Trial registration number not applicable

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Pirastu, G., I. Listorti, R. Manzo, M. Barberi, M. Musella, V. Zazzaro, A. Colasante et al. « P-260 Study of kinetic parameters using KIDscoreTMDay5 version 3.0 in euploid, mosaic and aneuploid blastocysts ». Human Reproduction 37, Supplement_1 (29 juin 2022). http://dx.doi.org/10.1093/humrep/deac107.250.

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Abstract Study question Do kinetic parameters change among euploid, mosaic and aneuploid blastocysts? Is the KIDscoreTMDay5 version 3.0 (KS-5.3) correlated to preimplantation genetic testing for aneuploidies (PGT-A) results? Summary answer The KS-5.3 differs in embryo ploidy classes. The analysis of the kinetic variables showed that the aneuploid embryos were significantly slower than euploid and mosaic. What is known already Chromosomal abnormalities affect more than 50% of embryos in women with >35 years of age and PGT-A is the best way to predict embryo’s ploidy status decreasing implantation failure and miscarriage. However, this procedure is not always possible due to social or moral issues. So, the use of the non-invasive time lapse monitoring could be helpful to determine the morphokinetic characteristics in the different ploidy classes. KS-5.3 (vitrolife,Sweden) is a scoring model based on morphokinetic data, developed to predict the pregnancy rate of day-5 blastocysts. Recent publications showed differences in kinetic parameters between euploid and aneuploid embryos. Study design, size, duration This retrospective study analyzed 728 blastocysts with PGT-A results obtained at Villa Mafalda Clinic from May 2020 to June 2021. Embryos were cultured in EmbryoScope+ time-lapse system (Vitrolife) at 37 °C, 6%CO2, and 5% O2. The PGT-A was performed using next-generation sequence (NGS) technology on the trophectoderm biopsy sample on day 5/6/7. Automatic annotations for division times and quality gradings were performed by senior embryologists and all kinetic values were reported in hours post microinjection. Participants/materials, setting, methods 728 blastocysts were classified in: (E) euploid (n = 172), (M) mosaicism (n = 171) and (A) aneuploid (n = 385). In this study, they were considered KS-5.3 and the following kinetic variables: the time to reach 2 cells (t2), 3 cells (t3), 4 cells (t4), 5 cells (t5), and the blastocyst formation (TB). Continuous variables were reported as the median and interquartile range (IQR). For the statistical analysis, nonparametric tests were performed and p < 0.05 was considered statistically significant. Main results and the role of chance KS5.3 was significantly different between groups [E = 6.6(4.6-7.9) vs M = 5.3(2.9-7.2) vs A = 4.0(2.5-6.6), p < 0.0001]. It was significantly higher in euploid than in mosaic and aneuploid (EvsM p = 0.0007, EvsA p <0.0001, MvsA p = 0.0077). A significant delay in t2,t3,t4 and tb was showed in aneuploid embryos compared to euploid and mosaic, whereas there was no significant difference between euploid and mosaic: [t2: E = 25.80 (24.56-28.09), M = 25.99 (24.49-28.91), A = 27.02 (25.30-29.47), EvsA p <0.0001, AvsM p = 0.03, EvsM p = 0.32]; [t3: E = 37.08 (34.74-39.34), M = 36.69 (34.55-40.02), E = 38.45 (35.93-41.14), EvsA p = 0.0003, MvsA p = 0.002, EvsM p >0.99]; [t4: E = 38.28 (35.63-41.19), M = 38.49 (35.47-42.13), A 39.72 (37.25-43.31), EvsA p = 0.0001, MvsA p = 0.02, EvsM p = 0.65]; [tb: E = 107.70 (102.20-114.30), M = 110.10 (103.60-116.80), A = 113.7 (106.80-122.70), EvsA p <0.0001, MvsA p <0.0001, EvsM p = 0.42]. As for t5, there were no differences among the groups. Longer cell cycles in aneuploid embryos could be associated with activated DNA repair mechanism or during chromosome segregation. Instead, regarding the mosaics, there was a significant difference with euploid embryos only in KS5.3. The age was similar between euploid and mosaic [E = 36.29 (33.42-39.00) vs M = 36.71 (34.00-39.33) p = 0.99], whereas that was significantly higher in aneuploid embryos [A = 39.11(36.01-42.27), EvsA/EvsM p <0.0001]. Limitations, reasons for caution All these findings have to be validated in a larger sample size. Furthermore, for the retrospective nature of this study, there were some confounding factors, such as protocol of stimulation, female age, and malefactor. This research did not consider the importance of every single kinetic parameter. Wider implications of the findings A further study is needed to verify if there is a correlation between morphology and ploidy status. This could clarify the difference in KS-5.3 between euploid and mosaic. In order to decrease age bias, we should enlarge the sample size to analyze a subgroup of patients with higher maternal age. Trial registration number not applicable

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Sayme, N., T. Krebs, M. Kasoha, D. H. A. Maas, E. F. Solomayer et M. Kljajic. « P–233 The spatial arrangement of blastomeres and time of cavitation forming as predictors of blastocyst quality ». Human Reproduction 36, Supplement_1 (1 juillet 2021). http://dx.doi.org/10.1093/humrep/deab130.232.

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Abstract Study question Does the spatial arrangement of blastomeres and the start of blastulation affect blastocyst quality? Summary answer Better blastocyst quality is associated with the spatial arrangement of the embryo and the shorter time frame of blastulation (cavitation). What is known already The ability to select the human embryo with the highest implantation potential remains one of the greatest challenges in the management of In Vitro Fertilization patients. Several publications have proposed that additional morphological evaluations of blastomere arrangement and the dynamics of late-stage embryonic divisions might be a useful non-invasive way for embryo selection. In the last decade, the introduction of time-lapse technology enables continuous monitoring of embryo development, which leads to better outcomes than a selection based on the traditional morphology assessment. Study design, size, duration The spatial arrangement was defined as tetrahedrally if the cleavage planes were perpendicularly orientated, while embryos with rather parallelly orientated cleavage axes were considered as non-tetrahedral embryos. The injection time of ICSI was designated as “time zero” (t0), and EmbryoViewer software was used to calculate the time duration between injection and start of blastulation (cavitation). Obtained results were later correlated with the embryo’s capability to form a blastocyst as well as with blastocyst quality. Participants/materials, setting, methods A total of 195 oocytes from 40 patients undergoing the antagonist cycle for ICSI treatment were evaluated. All blastocysts were cultured in Embryoscope™ according to the manufacturer’s specifications (Vitrolife, Sweden). The Gardner and Schoolcraft scoring system was used to describe blastocyst quality. Statistical analyses were performed using IBM SPSS version 24. Data were reported as median and range. Differences between groups were tested using the Mann-Whitney U test. Statistical significance was defined as p < 0.05. Main results and the role of chance Obtained data showed that 83.6% (61/73) of embryos with tetrahedral arrangement formed blastocysts compared to 42.4% (50/116) of embryos with the non-tetrahedral arrangement (p < 0,001). Moreover, tetrahedral embryos more frequently formed good quality blastocyst compare to the non-tetrahedral [59% (36/61) vs 18 (9/50)% respectively; p < 0,001]. In addition, we found that good quality blastocyst had a significantly shorter time frame between injection and blastulation start, compared with blastocysts which did not reach good quality [95.00h (84–118) vs 102h (77–121) respectively; p = 0,006]. Limitations, reasons for caution The limitation of the present study was that due to the double-embryo transfer correlation between those morphokinetic parameters and pregnancy rate can not be calculated. Further research should link these morphokinetic parameters with pregnancy rate and live birth rate as well. Wider implications of the findings: The potential of our findings is considerable, especially for countries with strict Embryo Law Regulation. Obtained results might be highly useful for selecting embryos with high implantation potential. In addition, the present work illustrates the possibility of additional information that can potentially be incorporated into an embryo classification model. Trial registration number Not applicable

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Sugiura, T., M. Shioya, T. Kobayashi, M. Okabe, Y. Okada, M. Fujita et K. Takahashi. « P-192 Blastocyst collapse during embryonic development is a marker of low pregnancy potential, independent of other blastocyst evaluation criteria ». Human Reproduction 38, Supplement_1 (1 juin 2023). http://dx.doi.org/10.1093/humrep/dead093.552.

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Abstract Study question Is evaluation of blastocyst collapse during embryonic development an independent marker for ongoing pregnancy in single frozen-thawed blastocyst transfer cycles? Summary answer Collapse of blastocyst more than 20.4% of the area may be a marker of low ongoing pregnancy potential, independent of other evaluation criteria. What is known already Time-lapse monitoring revealed that a section of the extended trophectoderm (TE) is ruptured during development. This TE rupture causes an efflux of blastocoel fluid and a reduction in embryonic volume. This phenomenon is described as collapse. Several studies have reported that the collapse results in a low clinical and ongoing pregnancy potential for the blastocyst. However, the collapsed blastocysts were associated with poor morphological quality. Previous studies have not evaluated whether collapse is an independent predictor of pregnancy based on other blastocyst evaluation criteria. Therefore, it is necessary to determine whether blastocyst collapse is an independent predictor of ongoing pregnancy. Study design, size, duration This retrospective, single-center study was conducted at the TAKAHASHI WOMEN'S CLINIC from January, 2018 to December, 2020. All blastocysts were derived from the ICSI cycles. Time-lapse monitoring using Embryoscope + (Vitrolife) was used to observe the presence and degree of collapse. The degree of collapse was calculated from the embryonic volume at the maximum expansion and the lowest volume after collapse. We analyzed the relationship between 904 cycles of single vitrified-warmed blastocyst transfer and ongoing pregnancy. Participants/materials, setting, methods The ongoing pregnancy rates of collapsed and non collapsed blastocysts were compared using chi-squared test. The cut-off value of the degree of collapse for ongoing pregnancy was calculated using receiver operating characteristic (ROC) curve analysis. The effect of collapse above the cut-off value on ongoing pregnancy was examined using multivariate logistic regression analysis, including confounding factors (patient age, body mass index, inner cell mass (ICM) grade, TE grade, day of blastocyst formation, and expansion stage). Main results and the role of chance During the study period, 329 of the 904 transferred blastocysts exhibited collapse during time-lapse monitoring, and the degree of collapse ranged from 2.10% to 75.90%, with a median of 30.70%. In total, 575 transferred blastocysts were determined to be non collapsed. The mean patient age ( ± standard division) was 36.89 ± 4.77 years in the collapsed blastocyst group and 36.52 ± 4.42 years in the non collapsed blastocyst group (p = 0.151). The proportion of blastocysts with poor morphological grade (<BB ) was higher in the collapsed blastocysts group than in the non collapsed blastocysts group (32.5% vs. 17.91%, p < 0.0001). The ongoing pregnancy rate was significantly lower in the collapsed blastocysts group than in the non collapsed blastocysts group (45.4% vs. 34.0%, p = 0.0009). ROC curve analysis showed that the cut-off value for the degree of collapse for low pregnancy potential was 20.4%. Multivariate logistic analysis with confounding factors revealed that blastocyst collapse above the cut-off value (≥20.4%) had an effect on ongoing pregnancy (adjusted odds ratio: 0.53, 95% confidence interval: 0.34–0.82, p = 0.005) independent of ICM and TE grades, day of blastocyst formation, and expansion stage. Limitations, reasons for caution This retrospective study was conducted at a single fertility center. In addition, the analyzed blastocysts were derived only from the ICSI cycles. Therefore, it is necessary to analyze the effects of collapse in blastocysts derived from C-IVF cycles. Wider implications of the findings Our results demonstrate that ≥20.4% collapse may be an additional marker to consider when selecting blastocysts for transfer. If the blastocysts are of the same grade, it is better to select non collapsed embryos. Trial registration number not affect

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Erberelli, R., M. Nicolielo Barreto, C. K. Jacobs, E. L. A. Motta, J. R. Alegretti et A. R. Lorenzon. « O-100 Incidence, morphology, ploidy and developmental arrest in direct cleavage embryos : what are they teaching us ? Morphokinetic analysis of 15.081 embryos ». Human Reproduction 37, Supplement_1 (29 juin 2022). http://dx.doi.org/10.1093/humrep/deac105.123.

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Abstract Study question What is the impact of direct cleavage (DC) occurrence during embryo/blastocyst development regarding morphology grade, embryo arrest and ploidy status? Summary answer Blastocyst formation and morphology grade are significantly reduced in DC when compared to non-DC embryos; however, there is no difference in blastocyst ploidy status. What is known already Chromosome instability leading to aneuploidy during early cleavage is well known in humans. DC is defined as the abrupt cleavage of one into three or more daughter blastomeres as the result from an irregular mitosis. Three or more mitosis poles can impair early embryo development; however, how some embryos with these irregular divisions might be able to develop into expanded blastocysts is still unknown. Time-lapse system provides an uninterrupted evaluation of embryo morphological and dynamic parameters, allowing our understanding in such events and if those embryos may be associated with a self-correction mechanism to reach blastocyst stage. Study design, size, duration Retrospective cohort study from 15.081 embryos, 3.962 cycles and 3.106 patients that were undergoing in vitro fertilization (IVF) treatment according to medical referral in a single private ART center from December 2018 to November 2021. Only two-pronuclear (2PN) embryos cultured in time-lapse incubators (EmbryoScope, Vitrolife, Sweden) were included in the study. Direct cleavage occurance was annotated when a single blastomere directly cleaved into three or more daughter blastomeres. Participants/materials, setting, methods DC embryos were also classified in DC 1-3: abnormal cleavage occurred in zygote (1-cell) resulting in 3–4 blastomeres and in DC 2+: abnormal cleavage occurred at the 2-cell stage resulting in 5 or 6 blastomeres. Embryo arrest, blastocyst formation rate and morphology grade (Gardner and Schoolcraft, 1999) were annotated. Blastocyst biopsy reports for ploidy status were also considered for analysis. Mann-Whitney, chi-squared and Fisher tests were applied for statistical analysis. p < 0,05 was considered significant. Main results and the role of chance There were 1168 DC embryos (7,7%) from 896 cycles/841 patients; 502 DC1-3 (3,3%) from 391 cycles/366 patients and 666 DC2+ embryos (4,4%) from 475 patients/505 cycles. In DC group, around 21% of patients have the incidence of more than 1 DC embryo per cycle. Maternal age was lower in DC groups: 38,0±3,61 (DC1-3) and 38,60±3,21 (DC2+) years old versus not-DC (39,02±3,01, p < 0,0001). Embryo arrest was higher (DC1-3:85,5% and DC2+:56,8% versus not-DC:35,4%, p < 0,0001) and blastocyst formation rate was lower (14,5% and 43,2% versus 64,6%, p < 0,0001) in both DC groups and also between DC groups (p < 0,0001). There were 4124 biopsied not-DC blastocysts, 24 in DC1-3 and 154 in DC2+. There was no difference in ploidy between not-DC (57,9%, 40,2%, 1,9%, aneuploid, euploid and mosaic respectively) and DC groups (54,2%, 41,7%, 4,2% and 61%, 37% and 1,9% respectively, p = 0.86) and neither between DC-groups (p = 0.69). Inner cell mass morphology grades were distinct in all groups (not-DC A:45,8%, B:45,2% C:9,0%, DC1-3 A:35,6%, B:33,9%, C:30,5% and DC2+ A:23,1%, B:57,0% C:19,9%, p < 0,0001) and between DC-groups (p < 0,0001). Trophoectoderm morphology grades were also higher for not-DC group (not-DC A:30,5%, B:48,9% C:20,6%, DC1-3 A:15,3%, B:47,5%, C:37,3% and DC2+ A:13,6%, B:44,4% C:42,0%, p < 0,0001), however there was no difference between DC-groups (p = 0.80). Limitations, reasons for caution Limited data are available on the implantation potential of embryos with DC, as they are not prioritize for transfer, although our study did not demonstrated a higher aneyploidy rate. Patient parameters, with the exception of maternal age, were not considered in this study. Wider implications of the findings Although the morphology grades are lower and embryo arrest is higher in DC embryos, probably due to unequal cleavage, if they reach blastocyst stage, the ploidy status of these embryos are similar to not-DC embryos, speculative for a self-correction mechanism. Future steps should focus on their performance in pregnancy/live-birth. Trial registration number Not Applicable

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