Low-quality Blastocysts Research Articles

O-129 Live birth of Day 7 blastocyst transfers. Another chance to succeed

Abstract Study question Does the reproductive potential, especially live birth probability, of embryos decrease when blastocyst development takes longer than the traditionally accepted 5 and 6 days?. Summary answer The reproductive potential decreases with blastulation time. Day 7 had the worst live birth (LB), aneuploidy and miscarriage rates compared to Day 5 and 6. What is known already Ending culture on Day 6 is current practice in most clinics, it is important to elucidate whether or not this is a good practice. It has been reported that Day 7 blastocysts represent only 5% of useable blastocysts, although slower growing compared to Day 5 and 6 blastocysts.Day 7 blastocysts can be viable, they can be of top morphological grade, euploid and result in a LB. Furthermore, the effects of prolonged culture to Day 7 in regards to embryo development and their reproductive potential are not well established. Study design, size, duration Retrospective cohort study of patients from US RMA clinics attempting conception through ICSI and utilizing preimplantation genetic testing for aneuploidy screening (PGT-A) from January 2022 to January 2023. A total of 6,132 ICSI PGT-A cycles were included in the analysis; three thousand six hundred and eighty frozen single embryo transfer (SET) from those resulting ICSI cycles occurred during that period of time were included in the study. Participants/materials, setting, methods Patients that performed an ICSI cycle with autologous oocytes were included in the study. Patients with frozen oocytes, donor egg recipients, non PGT-A cases, and conventional insemination were excluded. The main outcome measures include euploidy rates, miscarriage rates and LB rates between Days 5, 6 and 7. Statistical tests were conducted through Chi-Square Test and logistic regression methods adjusted for maternal age at time of transfer and morphological quality of the blastocyst. Main results and the role of chance Euploidy rate was worse on D7 than D5 or D6, with 45.5%95%CI(43,46%-47,50%), 78.1% 95%CI(77,02%-79,13%), and 63.2%95%CI(62,42%-63,96%) respectively, p>0.0001. LB rate per ET (embryo transfer) was also worse on D7, 21.6%95%CI(15.05-28.09), than D5, 62.3%95%CI(59.73-64.95), and D6, 53.3%95%CI(51.23-55.40). We found D5 and D6 statistically significant vs D7, p<0.0001. When adjusted for potential confounders, and establishing D7 as reference, for LB AdjORD5=4.127,95%CI(2.671-6.378), and AdjORD6=3.241, 95%CI(2.145-4.898). Clinical miscarriage was 20.8%, 95%CI(9.84-31.67) on D7, and 11.2%, 95%CI(9.38-13.09) and 14.1%, 95%CI(12.41-15.75) for D5 and D6 respectively, with AdjORD5=0.478, 95%CI(0.230-0.994) and AdjORD6, 95%CI(0.619, 95%CI(0.310-1.239), being statistically different only for AdjORD5 (p=0.048). Finally, when we analyzed the quality of the inner cell mass (ICM) and the trophectoderm among Day 7 ETs, we found that blastocysts with an A quality ICM had a higher chance of LB (40%) when compared with B (25%) and C (7.1%) ICM. The adjusted OR comparing A vs C revealed significant difference (p=0.005), when A ICM is established as reference. Also, ET of blastocysts graded with an A trophectoderm had higher probability of a LB (44%) when compared with B and C (17.3% and17%, respectively). The adjusted OR, with A trophectoderm as reference, B and C trophectoderm revealed significant difference (p=0.012 and p=0.007, respectively). Limitations, reasons for caution The primary limitation is the low number of Day 7 blastocyst transfers. To determine whether Day 7 blastocysts are intrinsically worse from Day 5/6 embryos, prospective randomized studies with patients who have at least one embryo developed on day 5, 6 and 7 suitable for transfer are needed. Wider implications of the findings Expanding blastocyst culture increases the number of useable embryos per cycle and provides further opportunity of LB for patients, especially those with a few or low-quality blastocysts. This study demonstrates that Day 7 blastocysts can achieve live births and challenges the predominant practice of ending culture before Day 7. Trial registration number 2206-SFR-079-MC

Should we should consider day 3 blastomere number during single vitrified-warmed blastocyst transfer cycle? A retrospective study

The present study investigated whether day 3 blastomere number has an effect on the clinical outcomes during single vitrified-warmed blastocyst transfer cycles. A total of 3294 vitrified-warmed single day 5 blastocyst transferred cycles were analyzed in this retrospective study from January 2018 to December 2021. The cycles were divided into ≥ 7 and < 7 blastomere groups depending on the day 3 embryo blastomere number. The clinical outcomes were compared between the two groups, moreover multivariate logistic regression analysis was conducted to investigate the correlation between the number of day 3 blastomeres and clinical outcomes. The chi-square test demonstrated that the rates of clinical pregnancy and live birth were significantly higher in the ≥ 7 blastomere group compared to the < 7 blastomere group with respect to single high-quality blastocyst transfer cycles. Conversely, these rates were similar in the two groups with respect to single low-quality blastocyst transfer cycles. These results were confirmed by multivariate logistic regression analysis. However, the miscarriage rate was higher in the < 7 blastomere group than in ≥ 7 group during low-quality blastocyst transfer cycles. These results suggested that day 3 blastomere number should be considered during single vitrified-warmed blastocyst transfer cycles. Thus, blastocsyts derived from ≥ 7 blastomere embryos are preferred when choosing the same quality blastocysts.

D6 high-quality expanded blastocysts and D5 expanded blastocysts have similar pregnancy and perinatal outcomes following single frozen blastocyst transfer.

We compared the pregnancy and perinatal outcomes between expanded blastocysts vitrified on D5 versus D6 following single frozen blastocyst transfer. Clinical data on 7,606 cycles of frozen-thawed blastocyst implantations were retrospectively analyzed. Depending on whether blastocysts were vitrified on D5 or D6 and the transferred blastocysts, the blastocysts were divided into 6 groups: HQB-D5, HQB-D6, 4XC-D5, 4XC-D6, 4CX-D5, and 4CX-D6 groups. The differences in clinical pregnancy rate, live birth rate, first trimester abortion rate, preterm birth rate, gestational age, birth weight, and sex ratio at birth among the groups were compared. Our study showed that there was no difference in pregnancy and perinatal outcomes between the delayed formation of D6 high-quality expanded blastocysts and D5 expanded blastocysts, whether they were high-quality blastocysts or not. For low-quality blastocysts, the clinical pregnancy rate of D5 was higher than that of D6, and D5 was also better than D6 in live birth rate for those with inner cell mass rating B or above, while there was no difference between D5 and D6 for those with inner cell mass rating C. Based on our research, we suggest that when we are developing the implantation strategy, we give priority to the selection of high-quality expanded blastocysts, regardless of D5 and D6, whose clinical outcomes are not different. For low-quality blastocysts, D5 expanded blastocysts are preferred for transfer.

The Comparison of Two Whole-Genome Amplification Approaches for Noninvasive Preimplantation Genetic Testing (ni-PGT) and the Application Scenario of ni-PGT during the Fresh Cycle

Recently, noninvasive preimplantation genetic testing (ni-PGT) using degenerate oligonucleotide primer PCR (DOP-PCR) and multiple annealing and looping-based amplification cycle (MALBAC)-based whole-genome amplification (WGA) methods has demonstrated predictable results in embryo testing. However, a considerable heterogeneity of results has been reported in numerous studies on these two WGA methods. Our aim was to evaluate the current WGA method for ni-PGT while further clarifying the applicable scenarios of ni-PGT in the fresh cycle. A total of 173 embryos were tested with trophectoderm biopsy and ni-PGT. In the whole preimplantation genetic testing, the clinical concordance rates of the detection results of DOP-PCR and MALBAC with the corresponding trophectoderm biopsy results were 64.12% (84/131) and 68.99% (89/129), respectively (P=0.405). However, in the detection of abnormal embryos, the detection efficiency of ni-PGT is significantly improved [MALBAC: 96.55% versus 68.99% (P<0.001); and DOP-PCR: 89.09% versus 64.12% (P<0.001)]. In addition, the diagnostic efficiency of ni-PGT in low-quality blastocysts was significantly higher than that in high-quality blastocysts [MALBAC: 95.24% versus 51.85% (P=0.001); and DOP-PCR: 91.30% versus 48.15% (P=0.001)]. These results contribute to further understanding ni-PGT and to clarifying its application scenario in the fresh cycle.

Discard or not discard, that is the question: an international survey across 117 embryologists on the clinical management of borderline quality blastocysts.

Do embryologists from different European countries agree on embryo disposition decisions ('use' or 'discard') about Day 7 (>144 h post-insemination) and/or low-quality blastocysts (LQB; <Gardner's BB) (i.e. borderline quality blastocysts)? The prevalence of 'discard' answers was 38.7%; nevertheless, embryologists' agreement was overall just fair (Fleiss-k = 0.26). The utilization of LQBs and adoption of culture beyond 144 h post-insemination is increasing worldwide. Although morphology and morphokinetics are associated with embryo developmental competence, previous studies demonstrated significant interobserver variability among embryologists regarding embryo quality assessment and disposition decisions for borderline quality blastocysts. An anonymous survey was run in a large network of IVF centers. A total of 117 embryologists from 6 European countries and 29 IVF centers filled in the survey. Randomly selected anonymous time-lapse videos of 50 Day 7 and/or LQB whole embryo preimplantation development were assessed by the embryologists. The key information on patients/cycles was provided along with each video. All cycles entailed preimplantation genetic testing for aneuploidies. Each embryologist specified whether he/she would have discarded or used ('transfer-fresh'/'cryopreserve'/'biopsy') any embryo. Inter-rater agreement was measured with Fleiss-k. Examiners were asked about their years of experience, center location, average number of cycles and average maternal age, number of colleagues, and use of time-lapse incubators at their centers. All participants were blinded to artificial intelligence (AI) scores generated by two commercially available software packages, chromosomal diagnosis (all blastocysts were tested for aneuploidies), and clinical outcomes after vitrified-warmed euploid single blastocyst transfer. These data were known only by one embryologist not involved in the survey. Participants were Italian (40%, N = 47), Spanish (24%, N = 28), Portuguese (5%, N = 6), Czech (5%, N = 6), Swedish (23%, N = 27), and Icelandic (3%, N = 3). In total, 2263 (38.7%) 'discard' and 3587 (61.3%) 'use' decisions were recorded. Czech, Portuguese, and Italian embryologists expressed lower 'discard' decision rates (mean ± SD 17 ± 7%, range 8-24%; 23 ± 14% range 4-46%; and 27 ± 18% range 2-72%, respectively), while Spanish gave intermediate (37 ± 16% range 4-66%) and Nordic gave higher (67 ± 11% range 40-90%) rates. The prevalence of 'discard' answers was 38.7% out of 5850 choices (mean per embryologist: 39 ± 23% range 2-90%). Only embryologists' country and IVF group were associated with this rate. Overall agreement among embryologists was fair (Fleiss-k = 0.26). The prevalence of 'discard' responses per embryo was 37 ± 24% (range 2-87%). Only the number of sibling blastocysts influenced this rate (i.e. the larger the cohort, the higher the inclination to 'discard'). No difference was shown for the two scores between euploid and aneuploid borderline quality blastocysts, while the embryologists were, by chance, more prone to 'discard' the latter (28.3 ± 21% range 9-71% versus 41.6 ± 24.8% range 2-87%, respectively). The survey included only private IVF clinics located in Europe. Moreover, a key variable is missing, namely patients' access to care. Indeed, all embryologists involved in the survey were part of the same network of private IVF clinics, while the embryo disposition decisions might be different in a public setting. Decision-making by European embryologists regarding Day 7 embryos or LQBs is inconsistent with putative clinical consequences, especially in patients with low prognosis. Although the embryologists could make decisions independent from their local regulations, their mindset and clinical background influenced their choices. In the future, AI tools should be trained to assess borderline quality embryos and empowered with cost-effectiveness information to support embryologists' decisions with more objective assessments. No external funding was obtained for this study. The authors have no conflict of interest to declare. N/A.

P-272 The impact of CHLOE-EQ and embryologist seniority on the ability and confidence to predict ploidy

Abstract Study question Can embryologists and CHLOE-EQ predict ploidy? Does their confidence and ability to predict vary with embryologist seniority? Summary answer High inter-observer variability between embryologists on prediction of ploidy. CHLOE-EQ provided a consistent prediction of ploidy. What is known already Previous studies have demonstrated the value of using Artificial Intelligence (AI)-based tools, such as CHLOE-EQ (Fairtility), to support, quantify and standardize embryo assessment. CHLOE-EQ uses AI-based algorithms to predict implantation. Recent studies have demonstrated that the algorithms also have ploidy predictive capabilities. Little is known about the ability of human embryologists to predict ploidy of blastocysts deemed suitable for biopsy, and whether this ability to predict varies with seniority or confidence level. Study design, size, duration Cohort study including 141 patients treated in a Memorial Sisli Hospital ART and Reproductive Genetics Center between January 2020-August 2022, with at least 4 blastocysts with different PGT-A results per cycle, leading to a total of 734 embryos. The same blastocysts were blindly assessed by CHLOE-EQ and by a senior and a junior embryologists working in the same clinic. Intraobserver variance of senior embryologist was also evaluated. Participants/materials, setting, methods Embryologists were asked to predict whether a blastocyst was euploid or aneuploid, their confidence of this determination (confident, neutral, not confident), the rank of the embryos based on chance of being euploid. The same embryos were assessed using CHLOE-EQ, scoring embryos from 0 to 10. The extent of mosaicism (from 35-70%) was quantified. Trophectoderm biopsy was performed to embryos at least 3BB, four low quality blastocysts (Aneuploid:5AC,4AC,6CB; Euploid:2AA) were also included into the study. Main results and the role of chance The average patient age was 33 ± 4 years (ranging from 22 to 39 years). Embryologists agreed on euploid prediction in a minority of blastocysts(48%,324/670). Agreement of the senior embryologist on same embryos in a different time was higher (Kappa = 0.42, moderate) than with the junior embryologist [Kappa = 0.19(slight)/0.26(fair)], suggesting seniority affected prediction consistency. CHLOE-EQ ranking had fair agreement with the embryologists (Kappa = 0.22,0.22,0.23), bringing consistency to prediction irrespective of seniority. Confidence was not affected by seniority (senior vs junior: ‘Confident’/’Neutral’/’Not confident’:60/26/14% vs 57/27/16% vs 66/24/10%,NS). Efficacy of prediction of ploidy reduced with junior embryologist (senior: AUC = 0.58,0.57 vs junior:AUC = 0.55). The senior embryologist was able to predict ploidy with a greater accuracy when ‘confident’ (AUC = 0.60, n = 441, p&amp;lt;0.001) compared with ‘not confident’ (AUC = 0.50, n = 107, NS). This was not the case with the junior embryologist (‘Confident’ AUC = 0.56,NS vs ‘Not Confident’ AUC = 0.54,NS). Euploidy rate was greater in high scoring embryos(CHLOE-EQ 5.1-10) than low(0-5) scoring (60%, 272/457 vs 48%,135/280;p&amp;lt;0.005,AUC = 0.58). This was maintained in blastocysts where the senior embryologist was ‘confident’(64% vs 54%) and ‘not confident’ (57% vs 36%), showing consistency in assessment irrespective of confidence. CHLOE-EQ score reduced with increasing degree of mosaicism (Euploid 6.3 ± 2; Mosaic&amp;lt;35% 5.98 ± 2.87; Mosaic&amp;gt;50% 4.31 ± 2.80; Mosaic&amp;gt;70% 2.85 ± 4;p = 0.04). Euploid embryos had a higher CHLOE-EQ score than aneuploid/mosaic embryos (6.3 ± 2, n = 396 vs 5.78 ± 2,n = 319;p = 0.008). Limitations, reasons for caution Mostly optimal quality blastocysts were included in this study. There is a need to better understand the role of Artificial Intelligence in improving consistency of selection of blastocysts for biopsy, and to extend this study for viable lower quality blastocysts to be included rather than discarded. Wider implications of the findings Effective prediction of ploidy can (i) improve biopsy criteria so that viable embryos are not discarded, (ii) reduce the cost of PGT-A by prioritizing embryos with increased chances of being euploid. CHLOE-EQ’s ploidy prediction improved consistency can support both PGT-A programs, and cycles where PGT-A is not an option. Trial registration number Not applicable

Expert consensus on the morphological evaluation of human cleavage-stage embryos and blastocysts.

Expert consensus on the morphological evaluation of human cleavage-stage embryos and blastocysts.

P-255 Double vitrification and warming does not impact clinical pregnancy rate in single blastocyst transfer cycles

Abstract Study question Does double vitrification and thawing impact clinical pregnancy rate after a single blastocyst transfer? Summary answer The clinical pregnancy rate obtained after double vitrification was comparable to that obtained after single vitrification. What is known already Double vitrification-warming (DVW) is commonly practiced to accommodate surplus viable embryos suitable for transfer, allow retesting of inconclusive-diagnosed blastocysts for PGT and circumvent limitations associated with national policies on embryo culture in certain countries. Despite its popularity, the evidence concerning the impact of DVW on IVF/ICSI outcomes is limited and lacking credibility. Biopsied blastocysts have comparable chance of clinical pregnancy following double and single round of vitrification. However, our study is the first to report clinical pregnancy outcomes following DVW in the absence of biopsy and in the case where the first round of vitrification occurred at the zygote stage. Study design, size, duration This is a retrospective observational analysis of n = 452 single blastocyst transfers that were either vitrified-warmed once (SVW, n = 349) or twice (DVW, n = 103) between January 2017 and December 2021. Participants/materials, setting, methods In the SVW group, blastocysts were vitrified on day 5/6 and warmed on the day of embryo transfer (ET). In the DVW group, zygotes (2PN) were first vitrified-warmed and then vitrified again on day 5/6 and warmed on the day of ET. Exclusion criteria were ETs from PGT and vitrified-warmed oocyte cycles. All ETs were performed at the University Hospital of Zurich in Switzerland following a spontaneous or artificial endometrial preparation. Main results and the role of chance Mean maternal age at oocyte pick-up (OPU) and at ET did not differ between the two groups: at OPU: 35.1±4.4 and 35.9±4.1 years for DVW and SVW groups respectively (p = 0.106), at ET: 36.6±4.4 and 36.5±4.4 years for DVW and SVW respectively (p = 0.73). The causes of infertility did not differ between the groups (p = 0.87): male factor infertility was most common: 46.6% and 50.1% of cases for DVW and SVW respectively while other causes included idiopathic infertility, anovulation, endometriosis and polycystic ovarian syndrome. The rate of fertilisation method utilised was similar between the groups (p = 0.98): DVW had 71.8% ICSI and 29% IVF while SVW had 71.9% ICSI and 28% IVF. The quality of blastocysts at ET was equal in the two groups (p = 0.09): DVW had 33.9% top, 30.9% medium and 35.9% low quality blastocyst while SVW had 34.3% top, 31.2% medium and 34.3% low quality blastocysts. The blastocyst expansion grade at ET was similar between the groups (p = 0.087): DVW had 64.9% 3-4 expanded, 32% hatching and 2.9% hatched blastocysts while SVW had 75.8% 3-4 expanded, 21.7% hatching and 2.2% hatched blastocysts. The clinical pregnancy rate was comparable between the groups (p = 0.54): for DVW it was 46.6% and for SVW it was 43.2%. Limitations, reasons for caution The study is limited by its retrospective nature and rather small cohort. Caution should be taken concerning interpretation of these findings in the case that double vitrification-warming occurs at different stages of embryo development. Wider implications of the findings The result of the present study on double vitrification-warming procedure provides a framework for counselling couples on their chance of clinical pregnancy per warming cycle. It additionally provides confidence and reassurance to laboratory professionals in certain countries where national policies limit embryo culture strategies making DVW inevitable. Trial registration number N/A

A deep dive into the morphokinetics and ploidy of low-quality blastocysts

ObjectiveTo describe morphokinetic parameters and ploidy among low-quality blastocysts not meeting the criteria for clinical use.DesignProspective cohort study.SettingAcademic medical center.Patient(s)Two hundred patients undergoing in vitro fertilization between February 2018 and November 2019.Intervention(s)All embryos were cultured in a time-lapse incubator. All expanded blastocysts underwent preimplantation genetic testing for aneuploidy using next-generation sequencing.Main Outcome Measure(s)Static blastocyst morphology grading; morphokinetic parameters, including time to each cell division (2-cell formation to 8-cell formation); time to morula formation; time to the start of blastulation; time to blastocyst formation; and preimplantation genetic testing for aneuploidy results.Result(s)A total of 1,306 embryos progressed to the expanded blastocyst stage; of these, 935 embryos met the criteria for clinical use and were designated as high quality, whereas 371 embryos were graded as low quality and did not meet the criteria for use. In morphokinetic evaluation, low-quality embryos developed more quickly to 5-cell formation (t5) 48.4 [42.4–48.7) vs 50.2 [46.3–50.1] hours, but progressed more slowly thereafter with tM 91.5 [85.9–92.3] vs 88.3 [82.1–88.3] and tB 114.0 [106.4–113.9] vs 106.9 [101.3–107.4] hours. Among the low-quality embryos, 75.5% were aneuploid, 22.4% were euploid, and 2.2% had undetermined chromosome copy number results. Morphokinetic parameters did not differ between the euploid and aneuploid low-quality embryos.Conclusion(s)Morphokinetic analysis did not distinguish between euploid and aneuploid low-quality embryos.

CONSEQUENCES OF THE ASSISTANT REPRODUCTIVE TECHNOLOGY PROGRAM IN WOMEN WITH HYPERPLASTIC PROCESSES OF ENDOMETRIUM

In women of reproductive age, reproductive dysfunction is associated with uterine factor, which ranges from 24% to 62% in the population, especially in the case of endocrine and metabolic disorders.&#x0D; Literature sources show a significant decrease in fertility in such patients, 38.6% of women report infertility, 11.9% - recurrent miscarriage.&#x0D; The aim was to study the effectiveness and consequences of in vitro fertilization programs in patients with infertility and hyperplastic processes of endometrium.&#x0D; Materials and methods. A prospective cohort research included 75 patients with history of hyperplastic processes of endometrium and reduced reproductive potential (infertility and history of fetal loss syndrome) - the main group, as well as 40 women without uterine infertility and without menstrual irregularities (comparison group).&#x0D; Results. In the main group, the average age of women was significantly higher and 1.4 times - the average body mass index against the data of the comparison group (p &lt;0,05). The share of comorbidity of somatic pathology and proliferative processes of the reproductive system (fibroids, adenomyosis etc.) was significantly higher in the main group (p &lt;0.05). Assessment of reproductive function and obstetric history allowed to establish a significantly higher number of pregnancies, miscarriages and intrauterine interventions in the main group (p &lt;0,05). The results of indicators of hormonal homeostasis showed a tendency to reduce the level of antimullerian hormone, although it corresponded to the reference values, which can be explained by the older age group of patients in the main group and a high concentration of testosterone (p&lt;0.05), that required a longer period of superovulation stimulation and a higher dose of gonadotropins, while chorionic gonadotropin was more often used as an ovulation trigger. Women of the main group had smaller number of mature oocytes, more oocytes with cytoplasmic and extracytoplasmic dysmorphism and low-quality blastocysts versus data in the comparison group. The incidence of biochemical and clinical pregnancies did not differ significantly between groups, whereas the proportion of spontaneous abortions was 6.4 times higher in the case of uterine infertility (history of endometrial hyperplasia and polyposis), which was accompanied by a lower percentage of live births in this category of patients compared to the comparison group (p &lt;0.05).&#x0D; Conclusions. The research found a reduced chance of prolonging clinical pregnancy and live birth in patients with history of hyperplastic processes of endometrium, as well as a decrease in the effectiveness of assisted reproductive technology programs due to the weaker response of the ovaries to superovulation stimulation. Patients with history of hyperplastic processes of endometrium had a higher BMI, a high proportion of uterine fibroids, adenomyosis and comorbidity of somatic pathology, which is naturally associated with age and pathogenesis of hyperplastic processes of the reproductive system. Patients of the main group were found to have higher androgenic rates and such following features of superovulation stimulation: appointment of combined drugs FSH / LH, high total doses of gonadotropins and chorionic gonadotropin as a trigger of ovulation, also an increase in the duration of superovulation stimulation with increasing BMI.

Cycloleucine negatively regulates porcine oocyte maturation and embryo development by modulating N6-methyladenosine and histone modifications

Maturation of oocytes and early embryo development are regulated precisely by numerous factors at transcriptional and posttranslational levels through precise mechanisms. N6-methyladenosine (m6A) is the most common modification in mRNA which regulates RNA metabolism and gene expression. However, the role of RNA m6A on porcine oocyte maturation and early embryogenesis is largely unknown. Here, we found that oocytes treated with cycloleucine (CL), an RNA m6A inhibitor, express impaired cumulus expansion, increased production of reactive oxygen species (ROS) in the mitochondria, and delayed maturation of oocytes by disrupting spindle organization and chromosome alignment. Also, CL halted the development of embryos at the 4-cell stage and resulted in low-quality blastocysts. Furthermore, CL treatment decreased the RNA m6A, H3K4me3, and H3K9me3 levels, but increased the acetylation level of H4K16 during parthenogenetic embryonic development in pigs. Single-cell RNA-seq (scRNA-seq) analysis further revealed that CL treatment dramatically up-regulated the expression of metabolism-related genes (SLC16A1, and MAIG3 etc.) and maternal related genes, including BTG4, WEE2, and BMP15 among others, at the blastocyst stage. Taken together, inhibition of RNA m6A by CL impaired meiosis of oocytes and early embryonic development of porcine via RNA m6A methylation, histone modifications, and altering the expression of metabolism-related genes in blastocysts.

MACHINE LEARNING-BASED ASSESSMENT MODEL AS A PREDICTIVE TOOL FOR BLASTOCYST GRADING UTILIZING MORPHOKINETIC PARAMETERS PRIOR TO THE THIRD CLEAVAGE DIVISION

Can a machine learning model, based on morphokinetic data, predict blastocyst grading quality using data from time of ICSI to 6 cell stage? Retrospective cohort analysis of time-lapse data of 1111 embryos up to 65hrs post fertilization to predict blastocyst formation in an IVF center from January 2019 to March 2020. 561 were graded as good- and 550 as poor-quality blastocysts retrospectively. Random Forest, an ensemble learning, AI method was trained on binary prediction of blastocyst quality, based on time-lapse data exported to day 3. Feature importance was also evaluated with respect to successful blastocyst grading prediction. The model was trained using morphokinetic data up to day 3. Annotations included cell division times, multi-nucleation values, cell size, irregular cleavages, PN status and maternal age. The predictive power of the model was determined using the average area under the receiver operating characteristic curve (AUC), as well as the accuracy, recall, precision and confusion matrices over 5-fold cross-validation, ensuring model robustness. SHAP values were also used for depicting feature importance. The data set was divided into training (80%) and testing set (20%) and a 5-fold k-fold cross validation scheme was selected. The machine learning model was able to classify blastocysts with an average AUC of 0.80 and an average accuracy of 0.71 in the 5 folds created. The average recall was equal to 0.71 while average precision and F1 score were 0.78 and 0.71 respectively. The effect of feature value magnitude and blastocyst quality is explained and analyzed using SHAP values. According to that, the importance of the top 10 features out of the 27 total features used in a decreasing order is: Morphological grade, Fragmentation value, Age, Blastomere size value, t5-t4, t3-t2, t6-t2, t4-t3, t6-t5 and Multi-nucleation value. A machine learning framework utilizing morphokinetic data from day1 to day3 of embryo development could predict and distinguish between high- and low-quality blastocyst formation. The main limitation resides in the fact that all data are obtained from a single clinic. A slight decline in performance of the model is expected with the increase of data variance across different clinics.

Adding a low-quality blastocyst to a high-quality blastocyst for a double embryo transfer does not decrease pregnancy and live birth rate.

The effect of embryo quality on clinical outcomes of assisted reproductive technology following a double transfer is not well defined, with some studies suggesting that a low-quality embryo transferred with a high-quality embryo decreases the live birth rate (LBR), compared with transferring a single high-quality embryo. Our study examined whether the quality of a second blastocyst transferred affects the outcome, controlling for the number of the available high-quality blastocysts (HQB). A historical cohort study of 2346 fresh blastocyst transfers in a single fertility clinic between 2013 and 2019. The main outcomes were pregnancy, miscarriage, live birth, and multiple gestation rates. Outcomes were compared between single embryo transfers with a high-quality blastocyst (SET-H), double embryo transfers with two HQBs (DET-HH), and transfers with one high-quality and one low-quality blastocyst (DET-HL). Outcomes were also assessed between SET and DET when only low-quality blastocysts were available. With one HQB available, DET-HL increased LBR (adjusted odds ratio [OR] 1.65, 95% CI 1.09-2.49) compared with SET-H, but increased multiple gestation rate (aOR 23.1, 95% CI 3.0-177.6). With two HQBs available, DET-HH was associated with a higher LBR (aOR 1.62, 95% CI 1.28-2.04) and lower miscarriage rate (aOR 0.56, 95% CI 0.40-0.80), but very high twin rate (aOR 49.8, 95% CI 24.3-102.1) compared with SET-H. A SET-H with at least one or more HQB available to freeze, compared with a SET-H with no other HQB available, had a higher LBR (aOR 1.69, 95% CI 1.17-2.45). When there were no HQBs available, compared with SET-L, a DET-LL had a higher live birth (aOR 3.20, 95% CI 1.78-7.703) and twin rate (aOR 3.72×1010 ) and a lower miscarriage rate (aOR 0.24, 95% CI 0.10-0.58). When there is one HQB available, transferring an additional low-quality blastocyst would only slightly increase the LBR, but significantly increase the twin rate, therefore SET should be recommended. When two or more HQBs are available, SET-H would have a reasonably good chance of success without the very high twin rate associated with DET-HH. DET-LL when compared with SET-L, would increase the LBR, but increase the risk of multiple gestation.

The Association of Kinetic Variables with Blastocyst Development and Ploidy Status.

Background:Despite a plethora of studies conducted so far, a debate is still unresolved as to whether TLM can identify predictive kinetic biomarkers or algorithms universally applicable. Therefore, this study aimed to elucidate if there is a relationship between kinetic variables and ploidy status of human embryos or blastocyst developmental potential.Methods:For conducting this retrospective cohort study, the normal distribution of data was verified using Kolmogorov-Smirnov test with the Lilliefors’ amendment and the Shapiro-Wilk test. Kinetic variables were expressed as median and quartiles (Q1, Q2, Q3, Q4). Mann-Whitney U-test was used to compare the median values of parameters. Univariate and multiple logistic regression models were used to assess relationship between blastocyst developmental potential or ploidy status and kinetics. Several confounding factors were also assessed.Results:Blastocyst developmental potential was positively correlated with the t4-t3 interval (s2) (OR=1.417, 95% CI of 1.288–1.560). s2 median value was significantly different between high- and low-quality blastocysts (0.50 and 1.33 hours post-insemination, hpi, respectively; p=0.003). In addition, timing of pronuclear appearance (tPNa) (OR=1.287; 95% CI of 1.131–1.463) had a significant relationship with ploidy changes. The median value of tPNa was statistically different (p=0.03) between euploid and aneuploid blastocysts (Euploid blastocysts=8.9 hpi; aneuploid blastocysts=10.3 hpi).Conclusion:The present findings are in line with the study hypothesis that kinetic analysis may reveal associations between cleavage patterns and embryo development to the blastocyst stage and ploidy status.

A successful pregnancy and live birth after defragmented low quality blastocysts transfer (review of 20 clinical cases)

A successful pregnancy and live birth after defragmented low quality blastocysts transfer (review of 20 clinical cases)

What Are the Live Birth and Multiple Pregnancy Rates When 1 Versus 2 Low-Quality Blastocysts Are Transferred in a Cryopreserved Cycle? a Retrospective Cohort Study, Stratified for Age, Embryo Quality, and Oocyte Donor Cycles.

Outcomes among women who transferred only Gardner's grade BB or lower quality frozen embryos transferred (FET) are not well known. Our objective is to study whether transferring 2 versus 1 frozen low-quality blastocysts will increase the live birth rate (LBR) and the multiple pregnancy rate (MPR). This is a retrospective cohort study including 1104 FET cycles. Only day 5-6 blastocysts of grade BB or lower quality were included. Clinical pregnancy rate (CPR), MPR, and LBR per cycle were compared between single embryo transfer (SET) (n = 969) and double embryo transfer (DET) (n = 135). CPR and MPR were compared between SET and DET in grade BB, BC, CB, and CC individually. Among SET, BB blastocysts had higher CPR 34% (P = 0.0001) and a sub-significant increase in LBR 19% (P = 0.059) in comparison to other grade SET. Among all BB, MPR was significantly higher when transferring two versus one (5.9 vs. 1.9, P = 0.009). If age at egg collection ≥ 40years (n= 97), no difference was found in CPR (11.1 vs. 11.7, P= 0.9), MPR (0 vs. 0), and LBR (6.3 vs. 0,P= 0.13) when SET or DET was performed. If age was < 40years (n= 818), the MPR was significantly higher in DET than SET (6.7 vs. 1.63, P= 0.004). In egg donor cycles (n= 189), there was no difference in CPR, MPR, and LBR between SET and DET. Single embryo transfer should be offered even in women ≥ 40years of age or transferring lower quality embryos since transferring more did not increase outcomes in this group, and SET is likely the safest path.

High-quality Cleavage Embryo versus Low-quality Blastocyst in Frozen-thawed Cycles: Comparison of Clinical Outcomes.

This study compared the clinical outcomes of the frozen-thawed cycles of high-quality cleavage embryos with low-quality blastocysts to provide a reference for the choice of frozen-thawed embryo transfer schemes and to improve clinical pregnancy rates. A retrospective analysis was performed on the clinical data of patients undergoing frozen-thawed embryo transfer at the Reproductive Medicine Center of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology from 2016 to 2017. In total, 845 cases were divided into a high-quality cleavage embryo group (group A) and a low-quality blastocyst group (group B). Each group was further divided into subgroups based on the number of transplants. Group A was categorized into two subgroups comprising of 94 cases in subgroup A1 (1 high-quality 8-cell group) and 201 cases in subgroup A2 (2 high-quality 8-cell group). Group B was divided into four subgroups consisting of 73 cases in subgroup B1 (D53BC group), 65 cases in subgroup B2 (D54BC group), 110 cases in subgroup B3 (D63BC group), and 282 cases in subgroup B4 (D64BC group). The pregnancy outcomes and neonatal outcomes between the groups were compared. The clinical pregnancy rates (56.72% and 60.00%) and live birth rates (47.76% and 46.15%) in subgroups A2 and B2 showed no significant differences, but these rates were significantly higher in subgroups A2 and B2 than in the rest subgroups (P<0.05). The multiple birth rate (26.32%) in the subgroup A2 was significantly higher than that in the rest subgroups (P<0.05). There were no statistically significant differences in the abortion rates among all groups (P>0.05). In terms of neonatal outcomes, there were no statistically significant differences in the proportion of premature births, sex ratios, and birth defects among the low-weight and gigantic infants (P>0.05). Transplanting two high-quality cleavage embryos during the frozen-thawed embryo transfer cycles could significantly increase clinical pregnancy rates and live birth rates, but at the same time, it also increased the risks of multiple births and complications to mothers and infants. The D54BC subgroup had the most significant advantages among all groups (P<0.05). The rest low-quality blastocysts had clinical outcomes similar to the single high-quality cleavage embryo group.

The effect of Day 3 cell number on pregnancy outcomes in vitrified-thawed single blastocyst transfer cycles.

Does cell number on Day 3 have an impact on pregnancy outcomes in vitrified-thawed single blastocyst transfer cycles? A low Day 3 cell number (≤5 cells) was independently associated with decreased live birth rate (LBR) during single blastocyst transfer cycles in young women. Day 3 cell number is an effective predictor of IVF success rates when transferring cleavage stage embryos. However, the association between Day 3 blastomere number and pregnancy outcomes after blastocyst transfer is still unknown. A retrospective cohort study of 3543 patients who underwent frozen-thawed single blastocyst transfers from January 2013 to June 2018 at a tertiary-care academic medical center. Patients were grouped into six groups according to the Day 3 cell number: ≤4 cells, 5 cells, 6 cells, 7 cells, 8 cells and >8 cells. The primary outcome measure was LBR. A logistic regression analysis was performed to explore the independent association between Day 3 blastomere number and LBR after adjustment for some potential confounders. In women <35 years old, the LBR varied significantly according to Day 3 cell number, with the rate of 31.2%, 34.4%, 41.9%, 45.1%, 48.1% and 48.2% for the ≤4-cell, 5-cell, 6-cell, 7-cell, 8-cell and >8-cell groups, respectively (P < 0.001). This significant difference was also observed in the high- and low-quality blastocyst subgroups of young women. However, for women ≥35 years old, the rate of live birth was similar between groups. Furthermore, after accounting for confounding factors, the LBR was significantly decreased in the ≤4-cell (adjusted odds ratio (aOR): 0.62, 95% CI: 0.48-0.80, P < 0.001) and 5-cell (aOR: 0.73, 95% CI: 0.57-0.92, P = 0.009) groups as compared to the 8-cell group. Likewise, the blastocysts arising from ≤4-cell (aOR: 0.73, 95% CI: 0.57-0.93, P = 0.010) or 5-cell (aOR: 0.77, 95% CI: 0.61-0.97, P = 0.024) embryos were associated with lower clinical pregnancy rate than those from 8-cell embryos. No significant differences were observed in biochemical pregnancy rate and miscarriage rate. A limitation of the current study was its retrospective design. Future prospective studies are needed to confirm our findings. Our observations suggested that a low Day 3 cell number was related to decreased LBR after blastocyst transfer in young women, which provided vital information for clinicians in selecting blastocyst during IVF treatment. This study was supported by the National Natural Science Foundation of China (NSFC) (31770989 to Y.W.; 81671520 to Q.C.) and the Shanghai Ninth People's Hospital Foundation of China (JYLJ030 to Y.W.). The authors have no conflicts of interest to declare. N/A.

Levels of caspase-3 and histidine-rich glycoprotein in the embryo secretome as biomarkers of good-quality day-2 embryos and high-quality blastocysts.

Morphological assessment at defined developmental stages is the most important method to select viable embryos for transfer and cryopreservation. Timing of different developmental stages in embryo development has been shown to correlate with its potential to develop into a blastocyst. However, improvements in pregnancy rates by using time-lapse techniques have been difficult to validate scientifically. Therefore, there is a need for new methods, preferably non-invasive methods based on metabolomics, genomics and proteomics, to improve the evaluation of embryo quality even further. The aim of this study was to investigate if different levels of caspase-3 and histidine-rich glycoprotein (HRG), secreted by the embryo into the culture media, can be used as biomarkers of embryo quality. In this study, a total of 334 samples of culture media were collected from in vitro fertilization (IVF) treatments at three different clinics. Protein analysis of the culture media was performed using multiplex proximity extension protein analysis to detect levels of caspase-3 and HRG in the embryo secretome. Protein levels were compared in secretome samples from high- and low-quality blastocysts and embryos that became arrested during development. Correlation between protein levels and time to morula formation was also analyzed. Furthermore, protein levels in secretomes from day-2 cultured embryos were compared on the basis of whether or not pregnancy was achieved. The results showed that caspase-3 levels were lower in secretomes from high-quality vs. low-quality blastocysts and those that became arrested (p ≤ 0.05 for both). In addition, higher HRG levels correlated with a shorter time to morula formation (p ≤ 0.001). Caspase-3 levels were also lower in secretomes from day-2 cultured embryos resulting in a pregnancy vs. those that did not (p ≤ 0.05). Furthermore, it was shown that caspase-3 might be used as a marker for predicting potential success rate after transfer of day-2 cultured embryos, where a caspase-3 cutoff level of 0.02 gave a prediction probability of 68% (p = 0.038). In conclusion, in future prediction models, levels of caspase-3 and HRG might be used as potential markers of embryo quality, and secreted caspase-3 levels could to some extent predict the outcome after transfer of day-2 cultured embryos.

Differences in secretome in culture media when comparing blastocysts and arrested embryos using multiplex proximity assay

Objectives: The aim of this study was to assess different patterns of the human embryo secretome analysed as protein levels in culture media. Furthermore, analyses to correlate protein levels with quality and timing to development of human embryos were performed.Material and methods: Human day-2 cryopreserved embryos were cultured for four days in an EmbryoScope® with a time-lapse camera, and embryo quality was evaluated retrospectively. After culture, the media were collected and relative levels of secreted proteins were analysed using Proseek Multiplex Assays. Protein levels were evaluated in relation to timing to development and the ability to form a blastocyst.Results: Specific patterns of timing of development of blastocysts were found, where a difference in time to start of cavitation was found between high- and low-quality blastocysts. There appeared to be a correlation between specific protein patterns and successful formation of morulae and blastocysts. Embryos developing into blastocysts had higher levels of EMMPRIN than arrested embryos, and levels of caspase-3 were lower in high- versus low-quality blastocysts. Also, higher levels of VEGF-A, IL-6, and EMMPRIN correlated with shorter times to morula formation.Conclusions: The secretome and timing to development differ in embryos forming blastocysts and those that become arrested, and in high- versus low-quality blastocysts. The levels of certain proteins also correlate to specific times to development.