Transfer of Cleavage-Stage Embryos May Benefit Normal Responders With Low Rate of Morphologically Good Embryos Formation On Day 3: A Comparison Study

Background Do morphologically good (MG) embryos from patients with high and low rate of MG embryos on day 3 (RMD3) show similar developmental potential (DP)? Methods This respective study finally included a total of 916 fresh cycles and related 1074 FET cycles from Jan 2017 to May 2020 in our reproductive center. Cycles with high RMD3 were defined as the H group, while cycles with low RMD3 were defined as the L group. The basic characteristics of patients and fresh cycles, blastulation rate, and clinical outcomes were compared between the H and L groups in either ET cycles with MG day 3 cleavage embryos (ETC group) or ET cycles with MG blastocysts (ETB group). Results The overall characteristics of patients and cycles were grossly comparable between the H and L groups either in ETC or ETB groups. In ETB group, useable blastocysts formation rate, implantation rate and live birth rate was significantly reduced in the L group, compared to the H group;In ETC group, useable blastocysts formation rate was significantly reduced in the L group. However, implantation rate and livebirth rate was similar between the L and H groups. Conclusion The in vitro DP of MG day 3 embryos and in vivo DP of MG blastocysts were reduced significantly, while a similar in vivo DP of MG day 3 embryos was observed in patients with low RMD3 as compared to patients with high RMD3. It seems that direct transfer of day 3 MG embryos instead of extended culture may benefit patients with RMD3.

Change in the Strategy of Embryo Selection with Time-Lapse System Implementation—Impact on Clinical Pregnancy Rates

Time-lapse systems (TLS) and associated algorithms are interesting tools to improve embryo selection. This study aimed to evaluate how TLS and KIDScore™ algorithm changed our practices of embryo selection, as compared to a conventional morphological evaluation, and improved clinical pregnancy rates (CPR). In the study group (year 2020, n = 303 transfers), embryos were cultured in an EmbryoScope+ time-lapse incubator. A first team observed embryos conventionally once a day, while a second team selected the embryos for transfer based on time-lapse recordings. In the control group (year 2019, n = 279 transfers), embryos were selected using the conventional method, and CPR were recorded. In 2020, disagreement between TLS and the conventional method occurred in 32.1% of transfers, more often for early embryos (34.7%) than for blastocysts (20.5%). Irregular morphokinetic events (direct or reverse cleavage, multinucleation, abnormal pronuclei) were detected in 54.9% of the discordant embryos. When it was available, KIDScore™ was decreased for 73.2% of the deselected embryos. Discordant blastocysts mainly corresponded with a decrease in KIDScore™ (90.9%), whereas discordant Day 3 embryos resulted from a decreased KIDScore™ and/or an irregular morphokinetic event. CPR was significantly improved in the TLS group (2020), as compared to the conventional group (2019) (32.3% vs. 21.9%, p = 0.005), even after multivariate analysis. In conclusion, TLS is useful to highlight some embryo development abnormalities and identify embryos with the highest potential for pregnancy.

Comparison of the developmental potential of the morphological good embryos between normal-responders with low and high rate of MG day 3 embryos formation

Background To determine whether the morphological good (MG) day 3 embryos or blastocysts from normal responders with the low rate of MG day 3 embryos formation (LRTD3) and high rate of MG day 3 embryos formation (HRTD3) have similar developmental potential. Methods Oocyte retrieval cycles were ranked by the rate of MG day 3 embryos (MG day 3 embryos/ 2PN zygotes), cycles in the bottom25th percentile was defined as low potential group (L) while cycles in the top 25th percentile was defined as high potential group (H). The basic characteristics of patients and cycles, blastocyst formation rate, and clinical outcomes of transfer of the MG blastocysts or day 3 embryos were compared between the H and L groups. Results The overall characteristics of patients and cycles were grossly comparable between the H and L group in patients with MG day 3 embryo transfer or blastocysts transfer. In patients with transfer of MG day 3 embryos, patients with LRTD3 showed decreased rate of blastocyst formation and similar clinical outcomes of direct transfer of MG day 3 embryos, compared to patients with HRTD3. In patients with transfer of MG blastocysts, patients with LRTD3 showed decreased rate of blastocyst formation, as well as decreased rate of implantation and livebirth of transfer of MG blastocysts. compared to patients with HRTD3. Conclusion We concluded that the quality of MG day 3 embryos or MG blastocysts from patients with LRTD3 was reduced, compared to patients with HRTD3. However, it seems that direct transfer of MG day 3 embryos effectively avoid the flaws of poor quality of MG day 3 embryos from patients with LRTD3. Therefore, our study support the strategy of transfer of MG day 3 embryos rather than expended culture for patients with LRTD3.

P–242 Gene expression profiles of SARS-CoV–2-associated receptors and proteases in human early embryonic development and follicular cells

Study question Are the oocytes, embryos, granulosa and cumulus cells, used during ART, susceptible to the SARS-CoV–2 infection? Summary answer Transcriptomic analyses of SARS-CoV–2-associated receptors and proteases strongly suggest that blastocysts are most permissive to SARS-CoV–2 compared with mature oocytes and day 3 embryos. What is known already Very few studies analyzed the gene expression profiles of SARS-CoV–2-associated receptors and proteases, mainly focusing on ACE2 and TMPRSS2 expression, resulting in partial knowledge in different specimens from female genital tract. To date, the gene expression profile of SARS-CoV–2 host entry candidates in the entire preimplantation embryos is scarcely available. Moreover, reports on oocyte and granulosa cells susceptibilityto SARS-CoV–2 are very sparse. Study design, size, duration To address this question, we retrospectively examined the gene expression profiles of SARS-CoV–2-associated receptors and proteases in human granulosa cells (GCs), cumulus cells (CCs), mature oocytes, day 3 embryos, blastocysts and trophectoderm cells obtained from our previously described Affymetrix microarray data. Participants/materials, setting, methods Human GCs and CCs (n = 17), mature oocytes (n = 6), and preimplantation embryos (n = 20) were analyzed. The comparison of gene expression levels of receptors and proteases closely related to SARS-CoV–2 infection. For each gene, the number of samples with the probe set ‘present’, based on the detection call, was analyzed. Each probe set was classified according to the signal intensity value median, as low (<100), medium (100–200) or high expression level (>200). Main results and the role of chance ACE2, BSG, CTSL, CTSA were detectable at high expression level in all mature oocyte samples, while only CTSL was strongly expressed in all day 3 embryos. The most representative dual co-expression of SARS-CoV–2-associated receptor and protease (60% of samples) during the embryonic genome activation stage (EGA) was ACE2-CTSL and BSG-CTSL. In blastocysts, ACE2, BSG, CTSL, CTSA and FURIN were detectable in the entire cohort at high expression level, and the prevalence of the different dual co-expression of SARS-CoV–2-associated proteases and receptors was optimal (100% of samples). Interestingly, only CTSL was detectable in all trophectoderm samples and a prevalence of 60% was found for the BSG-CTSL co-expression. ACE2, BSG, CTSL and CTSA were present at high expression level in CCs samples. In contrast, ACE2 and BSG expression was very low while CTSL and CTSA showed a high expression level in GCs.A prevalence of 100% was reported for ACE2-CTSL, ACE2-CTSA co-expression for both cell types. In addition, BSG-CTSL and BSG-CTSA co-expression were also present in all CCs against ∼70% in GCs samples. This data suggest a potential risks of SARS-CoV–2 infection either GC or early embryo development. Limitations, reasons for caution Analyses of Affymetrix microarray gene expression data were performed in non-COVID–19 patients. Whether the SARS-CoV–2 infection change the gene expression profile of SARS-CoV–2-associated receptors and proteases is under investigation. Wider implications of the findings: Specimens from female genital tract may be considered as potential targets for SARS-CoV–2. Trial registration number Not applicable

O-125 Development of an artificial intelligence embryo witnessing system to accurately track and identify patient specific embryos in a human IVF laboratory

Study question Can convolutional neural networks (CNN) be used as a witnessing system to accurately track and identify patient specific embryos at the cleavage stage of development? Summary answer We developed the first artificial intelligence driven witnessing system to accurately track cleavage and blastocyst stage embryos in a human ART laboratory. What is known already There are reports of human errors in embryo tracking that have led to the births of children with different genetic makeup than their birth parents. Clinical practices rely on manual identification, barcodes or radio-frequency identification technology to track embryos. These systems are designed to track culture dishes but are unable to monitor developing embryos within the dish to help ensure an error-free patient match. Previously, we developed an AI witnessing system to track blastocysts with 100% accuracy. The goal of this study was to determine whether an AI witnessing system could be developed that accurately tracks cleavage stage embryos. Study design, size, duration A pre-developed deep neural network technology was first trained and tested on 4944 embryos images. The algorithm processed embryo images for each patient and produced a unique key that was associated with the patient ID at 60 hpi, which formed our library. When the algorithm evaluated embryos at 64 hpi it generated another key that was matched with the patient’s unique key available in the library. Participants/materials, setting, methods A total of 3068 embryos from 412 patients were examined by the CNN at both 60 hpi and 64 hpi. These timepoints were chosen as they reflect the time our laboratory evaluates Day 3 embryos (60 hpi) and the time we move them to another dish and prepare them for transfer (64 hpi). The patient cohorts ranged from 3-12 embryos per patient. Main results and the role of chance The accuracy of the CNN in correctly matching the patient identification with the patient embryo cohort was 100% (CI: 99.1% to 100.0%, n = 412). Limitations, reasons for caution Limitations of this study include that all embryos were imaged under identical conditions and within the same EmbryoScope. Additionally, this study only examined fresh Day 3 embryos cultured over a span of 4 hours. Future studies should include images of fresh and frozen/thawed embryos captured using different imaging systems. Wider implications of the findings This study describes the first artificial intelligence-based approach for cleavage stage embryo tracking and patient specimen identification in the IVF laboratory. This technology offers a robust witnessing step based on unique morphological features that are specific to each individual embryo. Trial registration number This work was partially supported by the Brigham Precision Medicine Developmental Award (Brigham Precision Medicine Program, Brigham and Women’s Hospital), Partners Innovation Discovery Grant (Partners Healthcare), and R01AI118502, and R01AI138800.

A Rapid NGS-Based Preimplantation Genetic Testing for Chromosomal Abnormalities in Day-3 Blastomere Biopsy Allows Embryo Transfer Within the Same Treatment Cycle

Nowadays, most of the preimplantation genetic testing (PGT) is performed with a strategy of comprehensive chromosome screening and trophectoderm biopsy. Nevertheless, patients with ovarian insufficiency may not have competent blastocysts. In the present study, we aimed to establish the value of multiple annealing and looping-based amplification cycle (MALBAC)-based next-generation sequencing (NGS) for PGT in day-3 embryos. A total of 94.3% (1168/1239) of embryos yielded informative results, and the overall embryo euploid rate was 21.9% (256/1168). Overall, 225 embryos were transferred in 169 cycles with a clinical pregnancy rate of 49.1% (83/169). The live birth and implantation rates were 47.3% (80/169) and 44.4% (100/225), respectively. Double embryos transfer showed higher clinical pregnancy and live birth rates compared with single embryo transfer, but the implantation rates were similar (44.2% vs. 44.6%, P > 0.05). The euploid rate for reciprocal translocations (16.1%) was significantly lower than that for Robertsonian translocations (28.0%, P < 0.01) and inversions (28.0%, P < 0.01). However, higher percentages of embryos with de novo abnormalities were observed with Robertsonian translocations (23.3%, P < 0.01) and inversions (30.5%, P < 0.01) than with reciprocal translocations (11.6%). We demonstrated that NGS for PGT on day-3 embryos is an effective clinical application, particularly for patients with a diminished ovarian reserve and limited embryos.

What Is the Optimal Timing of Thawing for Transferring Vitrified-warmed Cleavage Stage of Slow-growing Embryos? A Cohort Retrospective Study

PurposeTo evaluate optimal thawing time, the early thawing or the routine thawing time, for transferring vitrified-warmed, and cultured overnight cleavage stage of the slow-growing embryos on Day 3 in frozen embryo transfer (FET) cycle. MethodsThis was a retrospective cohort study from January 2017 to July 2018, a total of 705 slow-growing embryos FET cycles in which the patients were aged <40. Thawing cleavage stage slow-growing Day 3 embryos on either the 2nd or 3rd day after ovulation in natural cycle or the equivalent timing of the artificial cycles. ResultsFor slow growing embryos, the clinical pregnancy rate of early thawing group (152/468 (32.5%)) was significantly higher than that of routine thawing group (55/235 (23.4%)) (OR 1.39(CI 1.06-1.81), p=0.01), while there was no statistically significant difference in pregnancy loss in early thawing group (39/170 (22.9%)) versus in routine thawing group (16/62 (25.8%)) (OR 0.89 (CI 0.53-1.47), p =0.65). ConclusionFor slow-growing embryos, higher pregnancy outcomes were shown in early thawing strategy as compared to the routine thawing, which suggested that the improvement of endometrium-embryo synchronism may correct the time difference brought by the slow-growing embryos.

What is the optimal timing of thawing for transferring vitrified-warmed cleavage stage of slow-growing embryos? A cohort retrospective study

Background To evaluate optimal thawing time, the early thawing or the routine thawing time, for transferring vitrified-warmed, and cultured overnight cleavage stage of the slow-growing embryos on Day 3 in frozen embryo transfer (FET) cycle. Methods This was a retrospective cohort study from January 2017 to July 2018, a total of 705 slow-growing embryos FET cycles in which the patients were aged < 40. Thawing cleavage stage slow-growing Day 3 embryos on either the 2nd or 3rd day after ovulation in natural cycle or the equivalent timing of the artificial cycles. Results For slow growing embryos, the clinical pregnancy rate of early thawing group (152/468 (32.5%)) was significantly higher than that of routine thawing group (55/235 (23.4%)) (OR 1.39(CI 1.06–1.81), p = 0.01), while there was no statistically significant difference in pregnancy loss in early thawing group (39/170 (22.9%)) versus in routine thawing group (16/62 (25.8%)) (OR 0.89 (CI 0.53–1.47), p = 0.65). Conclusion For slow-growing embryos, higher pregnancy outcomes were shown in early thawing strategy as compared to the routine thawing, which suggested that the improvement of endometrium-embryo synchronism may correct the time difference brought by the slow-growing embryos.

Optimal numbers of mature oocytes to produce at least one or multiple top-quality day-3 embryos in normal responders

Objective: We attempted to identify the optimal cutoff numbers of mature oocytes that would produce at least one or multiple top-quality (grade A) day-3 embryos in normal responders undergoing stimulated in vitro fertilization (IVF) cycles.Methods: We selected 210 fresh IVF cycles performed in 170 infertile women at a single center from January 2014 to November 2019. Four to 14 (total) oocytes were obtained in all cycles after conventional ovarian stimulation. A receiver operating characteristic curve analysis was performed to find the moderate and extreme cutoff numbers of mature oocytes that would produce ≥ 1, ≥ 2, ≥ 3, ≥ 4, and ≥ 5 top-quality embryos.Results: The cutoff number of mature oocytes was significantly correlated with the number of top-quality embryos (r = 0.467, p= 0.000). The moderate cutoff number of mature oocytes was ≥ 3, ≥ 5, ≥ 5, ≥ 6, and ≥ 6 for obtaining ≥ 1, ≥ 2, ≥ 3, ≥ 4, and ≥ 5 top-quality embryos, respectively. The extreme cutoff number of mature oocytes was ≥ 9, ≥ 9, ≥ 10, ≥ 10, and ≥ 11 for obtaining ≥ 1, ≥ 2, ≥ 3, ≥ 4, and ≥ 5 top-quality embryos, respectively.Conclusion: We present the optimal cutoff numbers of mature oocytes that would yield ≥ 1, ≥ 2, ≥ 3, ≥ 4, and ≥ 5 top-quality embryos with 95% specificity. Our findings could help infertility clinicians to set target mature oocyte numbers in women undergoing stimulated IVF cycles.