Inner Cell Mass Grade Research Articles

P–232 A trophectoderm morphology can predict a live birth rate and gender imbalance

Abstract Study question Which morphology parameter is the most predictable in the live birth rate and can affect the sex ratio? Summary answer The trophectoderm grade (TE) can predict the live birth rate and skewed to male gender after single vitrified-warmed blastocyst transfers (SVBT). What is known already The Gardner and Schoolcraft grading system of blastocyst evaluation with morphology is the major predictor of the clinical outcome in ART. Inner cell mass (ICM) and trophectoderm (TE) morphology are strongly correlated between clinical pregnancy, live birth, and miscarriage. A greater degree of expansion of the transferred blastocysts showed a higher implantation rate. Therefore, it is essential to clarify which parameter is more predictable in clinical outcome during elective SVBT. However, SVBT has some potential limitations, including adverse effects such as a male-biased imbalance in the sex ratio. Study design, size, duration The retrospective analysis used 1138 cycles of SVBT in the Ojinmed IVF center, Mongolia, between May 2015 to January 2019. The morphology grade and blastocyst inner diameter compared with clinical pregnancy rate (CPR), live birth rate (LBR), and miscarriage. The sex ratio was estimated for all patients, excluding those who underwent PGT-A, donor oocytes, and monozygotic twins. Blastocyst quality was evaluated with Gardner and Schoolcraft grading system and measured inner diameter of the blastocyst. Participants/materials, setting, methods All patients underwent a clomiphene-based minimal ovarian stimulation protocol or drug-free natural cycle IVF treatment. On day 5 to 6, blastocysts that reached an inner diameter >160μm were immediately vitrified. Blastocyst morphology evaluated by ICM and TE grade. The CPR (with a confirmed gestational sac at 6–7 weeks of pregnancy) and the LBR (live birth at 22 weeks of pregnancy over) were estimated per embryo transfer procedure, followed by miscarriage rate. Main results and the role of chance The CPR was 44.69%, 38.97%, and 25.91% for A, B, C grades of ICM, respectively. And the LBR was 39.82%, 34.62%, and 19.1% for A, B, C grades of ICM, respectively. TE was strongly related to CPR (aOR=2.47, 95% CI 1.71–3.58, p < 0.01) and LBR (aOR=1.77, 95% CI 1.06–2.96, p = 0.028) in univariate and multivariable logistic regression analysis (A grade vs C grade). Also, CPR and LBR were increased with blastocyst inner diameter, proportionally. The A and B grade ICM blastocysts showed 2.8 - 2.9 times less miscarriage rate than the C grade of ICM in the univariate logistic regression analysis. The result of multivariable logistic regression analysis showed B grade of ICM had 2.3 times less than C grade of ICM (aOR 2.36, CI 95% 1.20–4.61, p = 0.012) and TE, patient age and blastocyst inner diameter were not significantly associated with miscarriage rate. The gender ratio was 56.8% (204/359) for male. The result of multivariable logistic regression analysis showed that A grade TE had a 2.3 times higher probability of male than C grade (aOR 2.31, CI 95% 1.22–4.37, p = 0.01). Neither fertilization method, ICM, expiation grade, nor fertility case was significantly associated with the sex ratio. Limitations, reasons for caution The result of the current retrospective study is limited to data from a single IVF center. Wider implications of the findings: Our study suggests that TE grade is the most predictable and ICM grade was associated with miscarriage. The high grade such as A-grade TE blastocyst transfer has more live birth rate, whereas it can affects at sex ratio in favor of male embryos after SVBT. Trial registration number Not applicable

O-222 An artificial intelligence model that was trained on pregnancy outcomes for embryo viability assessment is highly correlated with Gardner Score

Abstract Study question Do artificial intelligence (AI) models used to assess embryo viability (based on pregnancy outcomes) also correlate with known embryo quality measures such as Gardner score? Summary answer An AI for embryo viability assessment also correlated with Gardner score, further substantiating the use of AI for assessment and selection of good quality embryos. What is known already The Gardner score consists of three separate components of embryo morphology that are graded individually, then combined to give a final score describing Day 5 embryo (blastocyst) quality. Evidence suggests the Gardner score has some correlation with clinical pregnancy. We hypothesized that an AI model trained to evaluate likelihood of clinical pregnancy based on fetal heartbeat (in clinical use globally) would also correlate with components of the Gardner score itself. We also compared the ability of the AI and Gardner score to predict pregnancy outcomes. Study design, size, duration This study involved analysis of a prospectively collected dataset of single static Day 5 embryo images with associated Gardner scores and AI viability scores. The dataset comprised time-lapse images of 1,485 embryos (EmbryoScope) from 638 patients treated at a single in vitro fertilization (IVF) clinic between November 2019 and December 2020. The AI model was not trained on data from this clinic. Participants/materials, setting, methods Average patient age was 35.4 years. Embryologists manually graded each embryo using the Gardner method, then subsequently used the AI to obtain a score between 0 (predicted non-viable, unlikely to lead to a pregnancy) and 10 (predicted viable, likely to lead to a pregnancy). Correlation between the AI viability score and Gardner score was then assessed. Main results and the role of chance The average AI score was significantly correlated with the three components of the Gardner score: expansion grade, inner cell mass (ICM) grade, and trophectoderm grade. Average AI score generally increased with advancing blastocyst developmental stage. Blastocysts with expansion grades of ≥ 3 are generally considered suitable for transfer. This study showed that embryos with expansion grade 3 had lower AI scores than those with grades 4-6, consistent with a reduced pregnancy rate. AI correlation with trophectoderm grade was more significant than with ICM grade, consistent with studies demonstrating that trophectoderm grade is more important than ICM in determining clinical pregnancy likelihood. The AI predicted Gardner scores of ≥ 2BB with an accuracy of 71.7% (sensitivity 75.1%, specificity 45.9%), and an AUC of 0.68. However, when used to predict pregnancy outcome, the AI performed 27.9% better than the Gardner score (accuracies of 49.8% and 39.0% respectively). Even though the AI was highly correlated with the Gardner score, the improved efficacy for predicting pregnancy suggests that a) the AI provides an advantage in standardization of scoring over the manual and subjective Gardner method, and b) the AI is likely identifying and evaluating morphological features of embryo quality that are not captured by the Gardner method. Limitations, reasons for caution The Gardner score is not a linear score, creating challenges with setting a suitable threshold relating to the prediction of pregnancy. The 2BB treshold was chosen based on literature (Munné et al 2019) and verified by experienced embryologists. This correlative study may also require additional confirmatory studies on independent datasets. Wider implications of the findings The correlation between AI scores and known features of embryo quality (Gardner score) substantiates the use of the AI for embryo assessment. The AI score provides further insight into components of the Gardner score, and may detect morphological features related to clinical pregnancy beyond those evaluated by the Gardner method. Trial registration number Not applicable

P–772 Blastocyst quality and perinatal outcomes in women undergoing single blastocyst transfer in frozen cycles

Abstract Study question Is the morphological grading systems of a blastocyst associated with perinatal outcomes in women undergoing frozen-thawed single blastocyst transfer (SBT)? Summary answer Preferential transfer of a blastocyst based on their morphological grading systems appears to be supported by observed differences in perinatal outcomes. What is known already The transfer of a morphologically good quality blastocyst is associated with higher implantation and pregnancy rates as compared with a poor quality blastocyst. However to date, the association of the morphologic parameters of the blastocyst (developmental stage, inner cell mass (ICM), and trophectoderm (TE)) with the perinatal outcomes after blastocyst transfer remains unknown. Study design, size, duration A retrospective cohort study including 21,648 frozen-thawed SBT cycles from January 2013 to March 2019. Participants/materials, setting, methods 6037 women with singleton delivery in Peking University Third Hospital were eligible for analysis. Multivariate logistic regression was used to test the risk of factors with the expression of crude odds ratios (OR) and adjusted OR (aOR) with 95% confidence intervals (CI). Main results and the role of chance Transfer of a blastocyst with grading lower than 3BB was associated with a higher chance of female baby (49% vs 43%, aOR = 1.27 (1.12, 1.43)) and a higher rate of cesarian section (C-section) (69% vs 65%, aOR = 1.17 (1.03, 1.34)). Compared with stage 4 blastocyst, transfer of a stage 3 blastocyst was associated with a higher chance of preterm delivery (PTD) (aOR = 1.77 (1.08, 2.90)). Both stage 3 and stage 6 blastocyst transfer was associated with a lower chance of female baby (aOR = 0.68 (0.48, 0.97), 0.66 (0.47, 0.93), respectively). Compared with grade A ICM blastocyst transfer, Grade B ICM and grade C ICM blastocyst transfer was associated with a lower chance of a female baby (adjusted OR = 0.84 (0.73, 0.96), 0.63 (0.48, 0.83), respectively) and a higher risk of large for gestational age (LGA) (aOR = 1.20 (1.01, 1.42), 1.46 (1.07, 1.98), respectively). Grade C ICM blastocyst transfer was associated with an increased risk of macrosomia (aOR = 1.66 (1.14, 2.42)).Grade B TE and grade C TE blastocyst transfer had a lower risk of gestational diabetes mellitus (GDM) (aOR = 0.76 (0.60, 0.98), 0.69 (0.50, 0.94), respectively) than grade A TE blastocyst transfer. Limitations, reasons for caution The main limitations of this study were its retrospective nature and the relative subjectivity of blastocyst scoring. The follow-up was conducted through a phone call and some patients might not report their obstetrical and neonatal outcomes, leading to a relatively lower rate of several obstetrical outcomes. Wider implications of the findings: Transfer of a poor quality blastocyst is associated with a higher rate of C-section. The association between ICM grading and LGA and macrosomia would suggest that blastocysts with grade A ICM grading should be transferred preferentially and supports the use of current morphological grading systems for embryo prioritisation. Trial registration number N/A

P–147 The impact of blastocyst morphological parameters on live birth and singleton birthweight in single blastocyst transfer cycles

Abstract Study question Does blastocyst morphological parameters: blastocyst expansion degree(Expansion), inner cell mass (ICM), and trophectoderm (TE) grades affect live birth and singleton birthweight in single blastocyst transfer cyles? Summary answer The effects of blastocyst morphological parameters on live birth and singleton birthweight are different between biopsied blastocysts cycles and non-biopsied blastocysts cycles. What is known already It has been known that blastocysts with highest scores for three blastocyst morphological parameters achieve highest pregnancy rates, however, very few studies have comparatively analysed the effect of individual parameters on live birth and singleton birthweight about single blastocyst transfer cycles. Study design, size, duration This retrospective study involved all single blastocyst transfers cycles and their live birth outcome and singleton birthweight during the period from January 2014 to August 2019 at a tertiary care center. Participants/materials, setting, methods A total of 28515 single blastocyst transfer cycles were available for analysis and were divided into four groups: biopsied blastocysts cycles (BBC), thawed blastocysts cycles(TBC), blastocysts from thawed cleavage embryos cycles(BTCEC) and fresh blastocysts cycles(FBC). The primary outcome were live birth and singleton birthweight. Multiple logistics regression and linear regression analyses were respectively performed to investigate the effect of blastocyst morphological parameters on live birth and birthweight after adjusting potential confounders. Main results and the role of chance While analyzing the effect on live birth, we found that live birth of grade B ICM were lower than grade A ICM , live birth of grade C TE were lower than grade A TE and Expansion doesn’t matter. Those result were same in three kinds of non-biopsied cycles. While all three parameters were statistically independently significant in biopsied blastocysts cycles. While analyzing the effect on singleton birthweight, only Expansion was found to be statistically significant in biopsied blastocysts cycles, and birthweight of Expansion grade 5 was lower than Expansion grade 6(P = 0.005),with a mean difference of 57g(3375.12±527.91 versus 3318.42±510.33). Limitations, reasons for caution: Most blastocysts with poor grade, especially ICM grade C, were not transferred, then the effect of poor grade such as ICM grade C were still unknown. Wider implications of the findings: The study identified the association between blastocyst morphological parameters and live rate and compared the relative importance of three parameters in different kind of cycles through large size comparative analysis, which would help selecting high-quality embryos for transfer. Trial registration number Not applicable

P–132 Centralized versus local embryologists scoring of blastocyst quality obtained in a large European multicenter clinical trial

Abstract Study question Does blastocyst quality scoring by central assessment deviate from local assessment and potentially lead to the selection of a different single blastocyst for transfer? Summary answer Central and local assessment provided the same quality classification (poor / good / top) in 69% of all blastocysts and 63% of all transferred blastocysts. What is known already Blastocyst quality is scored most frequently by three morphological parameters, namely expansion and hatching (EH) status, inner cell mass (ICM) grading and trophectoderm (TE) grading. The score is used to define the quality classification (poor / good / top) which determines which embryo is to be transferred or cryopreserved. Blastocyst scoring and grading can be highly subjective, which does influence the choice for transfer and cryopreservation. Time-lapse imaging technology captures additional input about embryo development as well as enables centralized data storage and sharing for independent central assessments. Study design, size, duration Pooled embryo analysis from a prospective, randomized, multicenter trial (RAINBOW) of 619 women undergoing ovarian stimulation with an individualized dose of follitropin delta in a long GnRH agonist protocol between May 2018 and January 2020. Blastocysts were centrally assessed using time-lapse images by two independent assessors and one adjudicator . Selection of the blastocyst for transfer by local assessment was based on morphological scoring and not on morphokinetic time-lapse parameters. Participants/materials, setting, methods Oocytes were fertilized by ICSI and cultured in the Embryoscopeâ (Vitrolife) up to day 5 for transfer or day 5/6 for cryopreservation. Embryos were assessed as either non-blastocyst or blastocyst. Blastocysts were graded centrally and locally at 116 hrs of development, based on EH status (1–6), ICM (A-D) and TE grading (A-D). Central assessors were blinded to local assessment and embryo transfer selection. Main results and the role of chance In total 4282 embryos were assessed centrally, of which 2046 day 5 embryos (48%) were adjudicated due to a scoring difference of at least one parameter between the two central assessors. In total 38% of day 5 embryos were judged as non-blastocysts and 62% as blastocysts of which 61% (i.e. 38% of all embryos) were determined to be of good or top quality. Identical results in terms of quality classification (poor / good / top) were obtained for 69% of blastocysts between local and central assessment and in 78%, between the two central assessors. Moreover, central and local scoring were identical in 62% for EH status, 53% for ICM grading and 57% for TE grading. For all transferred blastocysts (n = 508), central and local quality assessment was aligned for 63%. The ongoing pregnancy rate following single blastocyst transfer (SBT) was 41% (202/489), and similar to when considering only the transfers for which the central assessment had the same or a higher classification than the local assessment (166/411=40%). In 16% of all SBT, central quality assessment gave a lower score for the transferred blastocyst than the central assessment. This discrepancy could potentially have led to transfer of a different blastocyst. Limitations, reasons for caution This trial included assessments made by embryologists from 20 IVF centres. Some centres has limited experience with time-lapse technology for morphological blastocyst scoring. Scoring could therefore have been affected by differences in focal planes, magnification and contrast compared to inverted microscopy, with potential influence on blastocyst scores and quality classification. Wider implications of the findings: Local and central blastocyst quality classification based on morphology aligns well but remains subjective. Embryo assessment may benefit from using tools like artificial intelligence-based algorithms for a more objective analysis. Trial registration number NCT03564509

P–138 When is low quality really low? Should we transfer low-grade blastocysts?

Abstract Study question What is the live birth rate after single, low-grade blastocyst (LGB) transfer? Summary answer The live birth rate for LGBs is 28%, ranging between 15–31% for the different inner cell mass (ICM) and trophectoderm (TE) subgroups of LGBs. What is known already Live birth rates following LGB transfer are varied and have been reported to be in the range of 5–39%. However, these estimates are inaccurate as studies investigating live birth rates following LGB transfer are inherently limited by sample size (n = 10–440 for LGB transfers) due to LGBs being ranked last for transfer. Further, these studies are heterogenous with varied LGB definitions and design. Collating LGB live birth data from multiple clinics is warranted to obtain sufficient numbers of LGB transfers to establish reliable live birth rates, and to allow for delineation of different LGB subgroups, including blastocyst age and female age. Study design, size, duration We performed a multicentre, multinational retrospective cohort study in 9 IVF centres in China and New Zealand from 2012 to 2019. We studied the outcome of 6966 single blastocyst transfer cycles on days 5–7 (fresh and frozen) according to blastocyst grade, including 875 transfers from LGBs (<3bb, this being the threshold typically applied to LGB studies). Blastocysts with expansion stage 1 or 2 (early blastocysts) were excluded. Participants/materials, setting, methods The main outcome measured was live birth rate. Blastocysts were grouped according to quality grade: good-grade blastocysts (GGBs; n = 3849, aa, ab and ba), moderate-grade blastocysts (MGBs; n = 2242, bb) and LGBs (n = 875, ac, ca, bc, cb and cc) and live birth rates compared using the Pearson Chi-squared test. A logistic regression analysis explored the relationship between blastocyst grade and live birth after adjustment for the confounders: clinic, female age, expansion stage, and blastocyst age. Main results and the role of chance The live birth rates for GGBs, MGBs and LGBs were 45%, 36% and 28% respectively (p < 0.0001). Within the LGB group, the highest live birth rates were for grade c TE (30%) and the lowest were for grade c ICM (19%). The lowest combined grade (cc) maintained a 15% live birth rate (n = 7/48). After accounting for confounding factors, including female age and blastocyst characteristics, the odds of live birth were 2.33 (95% CI = 1.88–2.89) for GGBs compared to LGBs and 1.56 (95% CI = 1.28–1.92) for MGBs compared to LGBs following fresh and frozen blastocyst transfers (p < 0.0001, odds ratios confirmed in exclusively frozen blastocyst transfer cycles). When stratified by individual ICM and TE grade, the odds of live birth according to ICM grade were 1.31 (a versus b; 95% CI = 1.15–1.48), 2.82 (a versus c; 95% CI = 1.91–4.18) and 2.16 (b versus c; 95% CI = 1.48–3.16; all p < 0.0001). The odds of live birth according to TE grade were 1.33 (a versus b; 95% CI = 1.17–1.50, p < 0.0001), 1.85 (a versus c; 95% CI = 1.45–2.34, p < 0.0001) and 1.39 (b versus c; 95% CI = 1.12–1.73, p = 0.0024). Limitations, reasons for caution Despite the large multicentre design of the study, analyses of transfers occurring within the smallest subsets of the LGB group were limited by sample size. The study was not randomised and had a retrospective character. Wider implications of the findings: LGBs maintain satisfactory live birth rates (averaging 28%) in the general IVF population. Even those in the lowest grading tier maintain modest live birth rates (15%; cc). It is recommended that LGBs not be universally discarded, and instead considered for subsequent frozen embryo transfer to maximize cumulative live birth rates. Trial registration number Not applicable

Correlation of Blastocyst Quality and Pregnancy Outcome during Freeze - Thaw Cycle.

To explore the relationship between the blastocyst quality and biochemical pregnancy or early embryonic cessation of development during the freeze-thaw cycle of in vitro fertilisation embryo transfer (IVF-ET). Descriptive study. Reproductive Centre of Baoding Maternal and Child Health Hospital, from January 2014 to August 2019. Eight hundred and twenty-nine embryos, treated by frozen-thawed blastocyst transfer, were analysed, retrospectively. These included 232 embryos in inner cell mass (ICM) Grade C, 272 embryos with trophoderm (TE) Grade C, and 325 embryos with Grades excluding C; ICM Grade and TE Grade were A or B. The pregnancy rate, rate of early embryonic cessation of development, and biochemical pregnancy rate were compared among the three groups after transfer. Compared with embryos with Grades excluding C in the score (with 55.7% in clinical pregnancy rate, 6.5% in biochemical pregnancy rate, and 5.2% in early embryonic development arrest rate), the embryos with ICM Grade C has lower clinical pregnancy rate (43.5%), higher biochemical pregnancy rate (15.1%), and rate of early embryonic cessation of development (19.8%), while the embryos with TE Grade C has lower pregnancy rate (41.2%) and higher biochemical pregnancy rate (14.3%). The differences were statistically significant (all p ＜0.05). There was no significant difference about the above indicators between the ICM Grade C and TE Grade C groups (p >0.05). Embryos with Grades excluding C in the score had better developmental potential and better prognosis. The rate of early embryonic development arrest in the ICM Grade C group was higher than that in the TE Grade C group. Key Words: Embryo quality and score, Blastocyst, Biochemical pregnancy, Early embryonic cessation of development.

The Morphology of Inner Cell Mass Is the Strongest Predictor of Live Birth After a Frozen-Thawed Single Embryo Transfer.

BackgroundThe scoring system for human blastocysts is traditionally based on morphology; however, there are controversies on the effect of morphology parameters on pregnancy outcomes. The aim of this study is to evaluate the predicting value of each morphology parameter on pregnancy outcomes in a setting of single embryo transfer.MethodsThis is a retrospective cohort study on patients undergoing frozen-thawed single blastocyst transfer at our center, between Jan. 2009 and Dec. 2018. A total of 10,482 cycles were analyzed. The blastocysts were scored according to the expansion and hatching status, morphology of inner cell mass (ICM), and cells of trophectoderm (TE). The primary outcome measure was live birth rate. One-way analysis of variance, chi-square test, and multiple logistic regression were used for statistical analysis.ResultsThe clinical pregnancy rate was lower in the blastocysts of stage 3 (48.15%), compared with those of stage 4 (56.15%), stage 5 (54.91%), and stage 6 (53.37%). The live birth rate was lower in the blastocysts of stage 3 (37.07%), compared with those of stage 4 (44.21%) and stage 5 (41.67%). The rates of clinical pregnancy (A: 66.60%, B: 53.25%, C: 39.33%) and live birth (A: 54.62%, B: 41.29%, C: 28.45%) were both decreased with decreasing grade of ICM morphology, and these differences were pairwise significant. The miscarriage rate of blastocysts with ICM grade A was lower, compared with ICM grade C (17.53 vs. 27.66%). Blastocysts with TE morphology of C had lower rates of clinical pregnancy (43.53%) and live birth (32.57%), compared with those with TE morphology of A and B (clinical pregnancy rate: 64.26% for A, 58.11% for B; live birth rate: 52.74% for A, 45.64% for B). There were no significant differences in rates of clinical pregnancy, live birth, and miscarriage between the blastocysts with TE grade A and B.ConclusionsThe blastocyst expansion stage, ICM grade, and TE grade are all associated with pregnancy outcomes. ICM grade is the strongest predictor of live birth. A blastocyst with stage 4–5, ICM grade A, and TE grade A/B should be given priority for single embryo transfer.

Embryo Selection Based on Morphological Parameters in a Single Vitrified-Warmed Blastocyst Transfer Cycle

The process of selecting a good quality embryo to improve the pregnancy outcomes is very important. The aim of our study was to elaborate the embryo selection process in a single vitrified-warmed blastocyst transfer (VBT) cycle by analyzing pre-vitrified and post-warmed blastocyst morphological factors to improve pregnancy outcomes. In this retrospective cohort study, we performed 329 single VBT cycles. The pre-vitrified and post-warmed morphological factors of all blastocysts were analyzed. Logistic regression analysis was conducted to select the independent morphological factor associated with ongoing pregnancy. The expansion of blastocoel (mid blastocoel; aOR 2.27, 95% CI.0.80-6.42, p = 0.12, expanded blastocoel; aOR 3.15, 95% CI.1.18-8.44, p = 0.02) in a pre-vitrified blastocyst and the grade of inner cell mass (ICM) (grade B; aOR 0.47, 95% CI.0.27-0.83, p = 0.01, grade C; aOR 0.22, 95% CI 0.09-0.56 p < 0.01) in post-warmed blastocysts significantly predicted the ongoing pregnancy. After fertilization, the embryo developed as a blastocyst on day 5 (day 5) showed a higher ongoing pregnancy than that on day 6 (day 6) (aOR 0.50, 95% CI.0.26-0.94, p = 0.03). The results suggest that while selecting a vitrified-warmed blastocyst in a single VBT cycle, the day 5 vitrified blastocyst should be considered, and a higher expansion grade in the pre-vitrified blastocyst should be selected. Our study has shown that post-warmed ICM grade tends to be a predictive indicator for the selection of the best blastocyst and allows for successful pregnancy, with ongoing pregnancy in a single blastocyst transfer.

Blasts from the past: is morphology useful in PGT-A tested and untested frozen embryo transfers?

Research questionDay of cryopreservation, inner cell mass (ICM) grade, trophectoderm grade and blastocyst expansion grade have been associated with differences in live birth rate in frozen embryo transfer (FET) cycles. This study sought to examine the likelihood of live birth and whether the morphological grade of the blastocyst is more or equally useful in FET cycles among preimplantation genetic testing for aneuploidies (PGT-A) tested and untested blastocysts. DesignThis was a retrospective cohort study of 6271 vitrified-warmed, autologous, single-embryo transfer cycles among patients undergoing IVF from July 2013 to December 2017 at a single, university-affiliated infertility practice. The primary outcome was live birth, calculated by generalized estimating equations. ResultsAmong PGT-A tested embryos, inferior ICM grade was associated with a lower chance of live birth (ICM grade B versus A: adjusted risk ratio [aRR] 0.91, 95% confidence interval [CI] 0.84–0.99). Among untested blastocysts there was a lower live birth rate in blastocysts cryopreserved on day 6 versus day 5 (aRR 0.87, 95% CI 0.78–0.96), and those with inferior pre-vitrification trophectoderm grade (trophectoderm grade B versus A: aRR 0.86, 95% CI 0.79–0.94). Blastocysts with a higher pre-vitrification expansion grade (pre-vitrification expansion grade 5 versus 4: aRR 1.1, 95% CI 1.01–1.2) were associated, but ICM grade was not associated (ICM grade B versus A: aRR 0.93, 95% CI 0.86–1.02), with chance of live birth. ConclusionsAmong PGT-A untested blastocysts, assessing embryo quality by day of cryopreservation, trophectoderm grade and expansion grade may help to identify embryos with the highest likelihood of live birth. Identifying euploid embryos by PGT-A appears to homogenize the cohort, making blastocyst morphological grade and day of cryopreservation less important.

Prognostic value of blastocyst grade after frozen euploid embryo transfer in patients with recurrent pregnancy loss

ObjectiveTo determine whether trophectoderm (TE) grade or inner cell mass (ICM) grade have predictive value after euploid frozen embryo transfer (euFET) among recurrent pregnancy loss (RPL) patients.DesignRetrospective cohort study.SettingSingle fertility center.Patient(s)Women with ≥2 prior pregnancy losses with ≥1 euploid embryo for transfer undergoing preimplantation genetic testing for aneuploidy.Intervention(s)Intracytoplasmic sperm injection, TE biopsy, blastocyst grading and vitrification, and single euFET, with first transfer outcome recorded.Main Outcome Measure(s)Live birth and clinical miscarriage rates.Result(s)The study included 660 euFET cycles. In a binomial logistic regression analysis accounting for age, body mass index, antimüllerian hormone level, and day of blastocyst biopsy, or ICM grade C was not significantly associated with odds of live birth, miscarriage, or biochemical pregnancy loss. TE grade C was significantly associated with odds of live birth and was not associated with odds of miscarriage or biochemical pregnancy loss. Blastocyst grade CC had significantly lower live birth rate compared with all other blastocyst grades.Conclusion(s)Embryo grade CC and TE grade C are associated with decreased odds of live birth after euFET in RPL patients. Embryo grade is not associated with odds of clinical miscarriage in this cohort of RPL patients, suggesting that additional embryonic or uterine factors may influence the risk of pregnancy loss.

Automatic grading of human blastocysts from time-lapse imaging

Background:Blastocyst morphology is a predictive marker for implantation success of in vitro fertilized human embryos. Morphology grading is therefore commonly used to select the embryo with the highest implantation potential. One of the challenges, however, is that morphology grading can be highly subjective when performed manually by embryologists. Grading systems generally discretize a continuous scale of low to high score, resulting in floating and unclear boundaries between grading categories. Manual annotations therefore suffer from large inter-and intra-observer variances. Method:In this paper, we propose a method based on deep learning to automatically grade the morphological appearance of human blastocysts from time-lapse imaging. A convolutional neural network is trained to jointly predict inner cell mass (ICM) and trophectoderm (TE) grades from a single image frame, and a recurrent neural network is applied on top to incorporate temporal information of the expanding blastocysts from multiple frames. Results:Results showed that the method achieved above human-level accuracies when evaluated on majority votes from an independent test set labeled by multiple embryologists. Furthermore, when evaluating implantation rates for embryos grouped by morphology grades, human embryologists and our method had a similar correlation between predicted embryo quality and pregnancy outcome. Conclusions:The proposed method has shown improved performance of predicting ICM and TE grades on human blastocysts when utilizing temporal information available with time-lapse imaging. The algorithm is considered at least on par with human embryologists on quality estimation, as it performed better than the average human embryologist at ICM and TE prediction and provided a slightly better correlation between predicted embryo quality and implantability than human embryologists.

Association between blastocyst morphology and pregnancy and perinatal outcomes following fresh and cryopreserved embryo transfer.

To assess the importance of each blastocyst morphological criteria with pregnancy and perinatal outcomes. This single-center retrospective cohort study included blastocyst single embryo transfers (SET) performed between 1/2012-2/2018. Poisson regression was used to evaluate pregnancy outcomes following fresh and cryopreserved embryo transfer (CET) for association with blastocyst expansion, inner cell mass (ICM) quality, and trophectoderm (TE) quality. Among cycles resulting in live birth, associations with preterm birth, small for gestational age (SGA) and large for gestational age (LGA), were evaluated using logistic regression. A total of 1023 fresh and 1222 CET cycles were included, of which 465 (45.1%) fresh and 600 (48.5%) CET cycles resulted in singleton live birth. Clinical pregnancy rates increased with increasing expansion among fresh transfers (p for trend = 0.001) but not CET (p = 0.221), and with TE quality for both fresh and CET cycles (p = 0.005 and < 0.0001, respectively). Live birth rates increased with increasing expansion (fresh p = 0.005, CET p = 0.018) and TE quality (fresh p = 0.028, CET p = 0.023). ICM grade was not associated with pregnancy outcomes; however, higher ICM quality among CET cycles was associated with increased chance of preterm birth (p =0.005). In blastocyst SET, blastocyst expansion and TE quality were each associated with clinical pregnancy and live birth. While higher ICM quality was associated with increased chance of preterm birth among CET, no other associations with perinatal outcomes were identified. Clinicians can be reassured that pregnancies from blastocysts with lower expansion, ICM, or TE qualities are not more likely to result in adverse perinatal outcomes.

Fine-tuning blastocyst selection based on morphology: a multicentre analysis of 2461 single blastocyst transfers.

Which blastocyst morphology parameter is associated with live birth after controlling for female age and endometrial receptivity? Retrospective study including fresh single blastocyst transfers (n = 2461) where the value of serum progesterone on day of human chorionic gonadotrophin trigger (PdHCG) was available. Generalized estimating equation regression models evaluated the independent effects of developmental stage (DevSt), inner cell mass (ICM) and trophectoderm grade on live birth rates while controlling for the confounding effects of female age and PdHCG. DevSt was strongly associated with the probability of live birth (P < 0.0001) independently of female age (odds ratio [OR] 0.89, 95% confidence interval [CI] 0.87-0.91) and PdHCG (OR 0.80, 95% CI 0.74-0.87). For full blastocysts, expanded blastocysts and hatching blastocysts, addition of ICM and trophectoderm grading in the multivariable analysis suggested that besides female age (OR 0.92, 95% CI 0.90-0.94) and PdHCG (OR 0.80, 95% CI 0.73-0.87), only DevSt (P = 0.001) and trophectoderm quality (P = 0.004) were independent predictors of live birth, while the predictive capacity of ICM was no longer significant. The mean probability of live birth was highest for AA blastocysts (35.0%), followed by BA blastocysts (31.2%) and AB blastocysts (27.7%). This large study analyses for the first time the independent role of blastocyst morphology in predicting live birth while controlling for female age and PdHCG. Its findings suggest that DevSt and then trophectoderm grade are stronger predictors of live birth over ICM grade when selecting a single blastocyst for transfer.

Why looks matter: morphology predicts success in embryo banking (EB) with preimplantation genetic testing for aneuploidy (PGT-A)

EB is increasingly common as more couples delay childbearing. Moreover, performing PGT-A before EB provides confidence that stored embryos are genetically competent and have a high chance of live birth (LB). We aimed to assess the specifics of blastocyst (BL) morphology in determining LB after euploid embryo (EE) transfer and develop counseling tools for patients electing EB+PGT-A. Retrospective cohort at a large university-based program. We reviewed all IVF cycles from 1/2014 - 2/2018 where fresh oocytes served as the female gamete and a single cryopreserved EE was transferred. We assessed age at retrieval, BL expansion (BE), inner cell mass (ICM) and trophectoderm (TE). BE, ICM and TE grades were based on Gardner’s criteria. Outcomes were ongoing pregnancy (>12 wks gestation) / live birth rate (LBR), implantation rate (IR) and spontaneous abortion rate (SABR). Fischer’s exact test was used. See Table. We reviewed 1641 transfers (142 donor+1499 autologous; median age at retrieval 37y, IQR 33-40y, range 21-46y). Overall IR was 66%, SABR was 12%, and LBR was 57%. There were 12 pregnancy terminations (TOP) and 7 ectopics. When stratified by BE, no differences in IR, SABR, or LBR were noted. When stratified by ICM grade, EE with ICM-C had a lower IR than with ICM-A and B (35% vs. 71% and 68%, p<0.01) and EE with ICM-A had a lower SABR than with ICM-B (6% vs. 13%, p=0.02); notably, EE with ICM-A had a higher LBR than with ICM-B and C (66% vs. 58% and 34%, p<0.03) and EE with ICM-B had a higher LBR than with ICM-C (58% vs. 34%, p<0.01). When stratified by TE grade, EE with TE-C had a lower IR than with TE-A and B (51% vs. 80% and 69%, p<0.01) and EE with TE-A had a lower SABR than with TE-C (3% vs. 18%, p=0.03); notably, EE with TE-C had a lower LBR than with TE-A and B (41% vs. 73% and 60%, p<0.01). Outcome differences re: ICM and TE were maternal-age independent. When comparing LBRs re: ICM+TE grade combinations, ICM-A+TE-A= 19/26 (73%), ICM-A+TE-B= 125/195 (64%), ICM-B+TE-A= 13/19 (68%), ICM-B+TE-B= 658/1101 (60%), ICM-B+TE-C= 91/205 (44%), ICM-C+TE-B= 23/57 (40%), and ICM-C+TE-C= 8/36 (22%). Only one EE had ICM-A+TE-C and one ICM-C+TE-A (both LBs). Euploidy is not the sole determinant when selecting embryos. Our data suggest that ICM and TE each play individual and critical roles related to IR, SABR and LBR - even with EE. In our study, EE with the best morphology had a LBR of 73% while EE with the worst had a LBR of 22%. These data add to realistic counseling for patients using EB+PGT-A for future pregnancy. Further, they help elucidate the biology of implantation and pregnancy, suggesting TE (placenta precursor) is as important as ICM (future fetus).Tabled 1Embryo morphologic parameters and outcome dataEmbryo ParameterIRSABRLBRTOP + Ectopic PregnanciesBE: <2 (n=110)73/110 (66%)14/73 (19%)58/110 (53%)1+0/110 (1%)BE: 3 (n=1389)928/1389 (67%)104/928 (11%)808/1389 (58%)9+7/1389 (1%)BE:4 (n=63)40/63 (63%)6/40 (15%)32/63 (51%)2+0/63 (3%)BE: >5 (n=79)47/79 (59%)6/47 (13%)41/79 (52%)0+0/79 (0%)ICM: A (n=221)157/221(71%)10/157 (6%)145/221 (66%)1+1/221(1%)ICM: B (n=1325)898/1325 (68%)119/898 (13%)762/1325 (58%)11+6/1325 (1%)ICM: C (n=95)33/95 (35%)1/33 (3%)32/95 (34%)0+0/95 (0%)TE: A (n=45)36/45 (80%)1/36 (3%)33/45 (73%)0+2/45 (4%)TE: B (n=1353)928/1353 (69%)107/928 (12%)806/1353 (60%)10+5/1353 (1%)TE: C (n=243)124/243 (51%)22/124(18%)100/243 (41%)2+0/243 (1%) Open table in a new tab

Post-warming degree of blastocoel re-expansion is the best prognosticator for live birth rates in vitrified-warmed single blastocyst transfer cycles

Recent studies have presented contradictory results regarding significance of Inner-cell-mass (ICM)/Trophectoderm (TE) gradation and degree of blastocoel expansion/re-expansion for increased live-birth rates. Also, few studies have reported combined results from fresh/frozen and single/double transfer cycles. This study aimed at individually assessing the relevance of grades of ICM, TE and degree of post-warm blastocoel re-expansion on live-birth rates exclusively in vitrified-warmed single-blastocyst transfer cycles. Retrospective study (n=216) vitrified-warmed elective single blastocyst transfer cycles. Oocyte donation, Embryo-donation, assisted hatching and preimplantation genetic diagnosis cycles were excluded. All blastocysts were graded as per Gardner and Schoolcraft method of classification as 1-6 for degree of expansion and grades A/B/C for ICM and TE. Natural-cycle endometrial preparation with hormone supplementation followed by luteal-phase support with micronized progesterone. Endometrial response was noted by ultrasound. Single blastocyst-transfer was done 3 hours after warming of vitrified blastocyst. Warmed blastocyst was compared with pre-vitrified blastocyst for degree of expansion, gradation of ICM/TE. β-hCG level measured on day8 of transfer indicated pregnancy. Live-birth was the primary outcome measure. Women were classified into Live-birth (LB; n=74) and non-pregnant (NP; n=142) groups. Age, BMI, infertility period, number of oocytes retrieved, rate of formation of good quality cleavage-stage and blastocyst-stage embryos and survival rates post-warming did not differ significantly between the two groups. Same brand of embryo-transfer catheter and same brand and volume of media was used for transfer. However, degree of re-expansion was significantly higher in LB group than in NP group (Mean ± SD: 3.6 ± 1.0 v 3.2 ± 0.94, P=0.0068). The Fisher-Exact test odds-ratio for achieving a live-birth with 3-4 degree of re-expansion was 2.78 (p=0.0016) whereas the odds ratio was much lower (0.36) with any other degree of re-expansion. Although ICM grade was higher/better in Live-birth group than in non-pregnant group, the difference remained statistically non-significant (Odds ratio 1.25, p=0.82). No significant difference was observed in TE grades between the two study groups (p=0.17). A notable difference was also observed in endometrial echopattern (p=0.03) although the endometrial thickness remained comparable between the groups. Thus, post-warming degree of re-expansion is the single most promising predictive factor for live birth rates in such cycles. Re-expansion of blastocoel to an expanded grade 3/4 is superior to ICM and TE cell gradation for better live-birth rates in vitrified-warmed single blastocyst transfer cycles. However, larger multicentric trials may be required to establish it as robust predictor of live-birth.

Inner cell mass and trophectoderm morphology impact the likelihood of achieving sustained implantation among expanded, euploid blastocysts

Preimplantation for genetic testing for aneuploidy (PGT-A) has been prospectively demonstrated to increase implantation rates over morphologic assessment alone. However, it is unclear whether morphology retains additional predictive power once an embryo is diagnosed as euploid with PGT-A. We sought to evaluate the influence of morphologic assessment by comparing pregnancy outcomes among expanded, euploid blastocysts (blasts). Retrospective cohort. All autologous IVF cycles between 2012-2016 were reviewed for inclusion. Only transfer of expanded blasts were performed during this time period (at least Grade 4 by Gardner criteria) and only single embryo transfers were included. The subjects were separated into 3 groups (good, fair, poor) based on the morphology of the inner cell mass (ICM) and trophectoderm (TE). Good quality blasts were defined as having any combination of A or B grades for both ICM and TE. Fair quality blasts were those with only 1 C grade assigned to either ICM or TE grade. Blasts with C grades in both ICM and TE were categorized as poor. The primary outcome was sustained implantation rate (SIR), defined as presence of fetal cardiac activity at 8 weeks. Logistic regression was performed to control for female age, size of the blast cohort, and day of transfer (5 or 6). Secondary outcome included pregnancy loss rate following a positive serum beta human chorionic gonadotropin (bHCG). A total of 2949 transfers were included. Blasts graded as good, fair, and poor quality demonstrated SIR of 70.5%, 56.9%, and 47.5%, respectively. Compared to the good quality cohort, and after controlling for age, cohort size, and day of transfer, the adjusted odds of sustained implantation were significantly reduced for the fair (aOR 0.56, 95% CI 0.44-0.71, p<0.001) and the poor-quality group (aOR 0.35, 95% 0.22-0.55) (p<0.001). Pregnancy loss rate was significantly lower among good quality blasts (p<0.001). Even among fully expanded, euploid blasts, ICM and TE morphology influence the likelihood of achieving a sustained implantation. The letter C grade in either or both ICM and TE is also associated with an increased risk of miscarriage. These data indicate that morphologic assessments should be considered when counseling patients on anticipated success - even among euploid blasts. This information may be particularly useful to patients who are planning to bank multiple euploid embryos to achieve their desired family size.Tabled 1Study CharacteristicsGoodFairPoorP-valueN251235780Age35.8 +/- 2.436.7 +/- 2.936.7 +/- 2.80.14AMH3.4 (2.1-5.7)2.6 (1.8-5.3)2.4 (0.9-4.1)<0.01Initial serum beta-HCG179.5 (140-202)117.4 (103-186)100.2 (93-163)<0.001% Serum Positive beta-HCG83.4% (2094/2512)79% (282/357)70% (56/80)<0.01Sustained Implantation Rate70.5% (1772/2512)56.9% (203/557)47.5% (38/80)<0.001Pregnancy Loss Rate after Positive beta-HCG15.4% (322/2094)38.9% (79/203)32.1% (18/56)<0.001 Open table in a new tab

Euploidy in relation to blastocyst sex and morphology.

The objective of our study is to assess the relationship of embryo ploidy status in relation to embryo sex, morphological characteristics, and transfer parameters. This is a retrospective cohort study at an academic medical center of patients who underwent in vitro fertilization with preimplantation genetic screening (PGS) from 2010 to 2015. Embryos were screened with 24-chromosome preimplantation genetic screening with day 5/6 trophectoderm biopsy. We investigated embryo euploidy in relation to morphology (expansion, inner cell mass, trophectoderm), embryo sex, biopsy day, and blastocyst cohort size. We used multivariate logistic regression to calculate odds ratios of euploidy in relation to these parameters. A total of 1559 embryos from 316 cycles and 233 patients (mean maternal age = 37.8 ± 4.2years) were included in the analysis. Six hundred and twenty-eight blastocysts (40.3%) were found to be euploid. Expansion (p < 0.001), inner cell mass (ICM) (p < 0.01), and trophectoderm grade (p < 0.001) were significantly associated with embryo ploidy in bivariate models controlling for maternal age, while embryo sex, biopsy day, and blastocyst cohort size were not associated with embryo ploidy. In a multivariate model, we found that maternal age (p < 0.001), higher grade of expansion (p < 0.01), and better quality trophectoderm (p < 0.001 for A compared to C grade) remained significantly associated with increased embryo euploidy, but ICM grade was no longer significant. Embryo sex was not associated with ploidy status, though male embryos were found to be associated with higher trophectoderm scores (p < 0.02). This is the largest study to date to investigate PGS-tested embryo sex and ploidy status. While maternal age and some morphological parameters (expansion, trophectoderm grade) are associated with euploidy in our cohort, other parameters such as embryo sex, biopsy day, and cohort size are not. Though embryo sex was not associated with euploidy, male embryos were found to be associated with higher trophectoderm grades. Additional investigation in larger studies is warranted.

Inner cell mass incarceration in 8-shaped blastocysts does not increase monozygotic twinning in preimplantation genetic diagnosis and screening patients.

BackgroundThe use of assisted reproductive technology (ART) has been reported to increase the incidence of monozygotic twinning (MZT) compared with the incidence following natural conception. It has been hypothesized that splitting of the inner cell mass (ICM) through a small zona hole may result in MZT. In this study, using a cohort of patients undergoing preimplantation genetic diagnosis/screening (PGD/PGS), we compared the clinical and neonatal outcomes of human 8-shaped blastocysts hatching with ICM incarceration with partially or fully hatched blastocysts, and attempted to verify whether this phenomenon increases the incidence of MZT pregnancy or negatively impact newborns.MethodsThis retrospective study included 2059 patients undergoing PGD/PGS between March 1, 2013, and December 31, 2015. Clinical and neonatal outcomes were only collected from patients who received a single blastocyst transfer after PGD/PGS (n = 992). A 25- to 30-μm hole was made in the zona of day 3 embryos by laser. The blastocysts were biopsied and vitrified on day 6. The biopsied trophectoderm (TE) cells were analyzed using different genetic methods. One tested blastocyst was thawed and transferred to each patient in the subsequent frozen embryo transfer cycle. All the biopsied blastocysts were divided into three types: 8-shaped with ICM incarceration (type I), partially hatched without ICM incarceration (type II), and fully hatched (type III). ICM/TE grading, clinical and neonatal outcomes were compared between the groups.ResultsThe percentage of grade A ICMs in type I blastocysts (22.2%) was comparable to that in type III blastocysts (20.1%) but higher than that in type II blastocysts (4.5%). The percentage of grade A TEs in type I blastocysts (4.2%) was comparable to that in type II (3.6%) but lower than that in type III (13.5%). There were no significant differences in clinical pregnancy, MZT pregnancy, miscarriage, live birth, MZT births, and neonatal outcomes between the groups.ConclusionsCompared to partially and fully hatched blastocysts, 8-shaped blastocysts with ICM incarceration showed relatively higher ICM and lower TE grades. ICM incarceration in 8-shaped blastocysts does not increase the incidence of MZT and has no negative effects on newborns in PGD/PGS patients.

Grade of the inner cell mass, but not trophectoderm, predicts live birth in fresh blastocyst single transfers

Debate continues over which morphological parameter is most important in selecting blastocysts for transfer. We aimed to investigate which parameter more accurately predicts the occurrence of a live birth by designing a retrospective cohort study of 1084 fresh elective single blastocyst transfers. Primary outcome was live birth rate (LBR) and secondary outcomes were implantation, clinical pregnancy and early pregnancy loss rates. Blastocyst expansion and inner cell mass (ICM), but not trophoectoderm, were associated with LBR in the definitive multivariable regression analysis. When ICM grade dropped from A to C the likelihood of achieving a live birth was reduced by 55% (OR= 0.45, 95% CI 0.26-0.79, p = .005). These results were similar for clinical pregnancy rates. Early pregnancy loss rates of embryos with ICM grade C were more than double (38.0%) compared to those of grades A (15.95%) and B (17.17%, p = .002). The transfer of an embryo with an optimal inner cell mass reduces early pregnancy loss and increases the likelihood of a live birth. We did not find any significant association between trophectoderm and LBR in the multivariable analysis in contrast with recent studies.