Single Blastocyst Transfer Research Articles

LIVE BIRTH RATES ARE SIGNIFICANTLY DECREASED FOLLOWING TRANSFER OF A EUPLOID BLASTOCYST WITH DIRECT UNEQUAL CLEAVAGE

Embryos can have direct unequal cleavage (DUC) during the first and/or second divisions. Although embryos with DUC have decreased blastulation rates, some develop into high quality euploid blastocysts. The objective of this study is to compare live birth (LB) rates following transfer of single frozen euploid blastocysts that either did or did not display DUC. This was a retrospective chart review of patients who underwent single frozen euploid blastocyst transfers from November 2018 to March 2020. Uninterrupted embryo culture was done in the Embryoscope up to 6 days post retrieval. Embryos were evaluated on day 2 for presence/absence of DUC and graded on day 5 or 6 according to the Gardner system. Blastocyst selection for frozen embryo transfer was based on euploidy status, developmental day, morphological score, and sex preference. Information regarding presence/absence of DUC was not accessed during blastocyst selection. Patient characteristics and LB rates were compared between the groups with and without DUC. Chi-square and Student’s t-tests were used for data analysis. A total of 114 single euploid blastocyst transfers were included, resulting in an overall LB rate per transfer of 63.2%. Twenty-two of the transferred blastocysts displayed DUC, whereas 92 did not. The overall LB rate in the DUC group was significantly lower than in the non-DUC group (31.8% vs 70.7%, p<0.01). Subgrouping blastocysts by developmental day and morphology showed significantly lower LB rates for day 6 and fair quality DUC blastocysts, compared with non-DUC blastocysts (18.2% vs 63.6%, p<0.05) (35.7% vs 70.6%, p<0.05). Live birth rates following single frozen euploid blastocyst transfer are significantly lower when blastocysts display DUC compared with blastocysts without DUC. When available, embryo division patterns should be considered when selecting a euploid blastocyst for frozen embryo transfer.

ENDOMETRIAL THICKNESS AND OBSTETRICAL OUTCOME IN ASSISTED REPRODUCTIVE TECHNOLOGY

Mounting evidence suggests that assisted reproductive technology (ART) alters certain obstetrical parameters as compared to natural pregnancies. Endometrial thickness (EMT) is one possible parameter which could impact on the depth of placentation – either too shallow, or too deep. While the impact of EMT on actual pregnancy rates has been studied in ART, little is known on whether EMT affects the quality of placentation. Therefore, we studied the impact of EMT on obstetrical outcomes following fresh and frozen embryo transfers (ET). Retrospective cohort study including patients who obtained a live birth (LB) after autologous ART in our center, between January 2017 and December 2019. Single and double blastocyst transfers were included. Fresh and frozen ET with singleton and twin pregnancies were analyzed separately. We divided them into 4 groups according to the last measured EMT before ET : <7mm,7 to 9 mm; 9 to 12 mm; ≥ 12 mm. Primary outcome was pregnancy duration in days. Secondary outcomes were the birth weight, obstetrical complications and the delivery mode. Associations between the 4 EMT groups were assessed using ANOVA and Fisher tests. 120 patients obtained single LB after fresh ET and 19 patients obtained twin LB. After frozen ET 571 patients had single LB and 75 patients had twin LB. No significant difference was reported between the 4 EMT groups when compared for pregnancy duration in fresh or frozen ET, and in singleton or twin pregnancies. In fresh ET with singleton pregnancies, more obstetrical complications and medically induced deliveries before 41 gestational weeks (GW) were reported in the groups<7 mm and >12mm.Table 1Results for singleton pregnancies<7 mm7-9 mm9-12 mm≥12 mmpvalueFresh ETN=5N=25N=68N=22Pregnancy Duration256.7±20.3262.7±13.4260.7±18.5263.3±10.20.78Birth Weight3153±744.73263.7±407.13196.1±509.23128.2±507.70.83SpontaneousInduced<41GWInduced≥41GW1 (20)2 (40)2 (40)19 (76)5 (20)1 (4)49 (72)14 (21)5 (7)9 (41)10 (45)3 (14)0.01Complications4 (80)4 (16)20 (29)7 (32)0.04Frozen ETN=19N=324N=202N=26Pregnancy Duration256.7±20.3262.7±13.4260.7±18.5263.3±10.20.23Birth Weight3139.2±777.03326.7±535.73271.3±635.23422.7±469.50.29SpontaneousInduced<41GWInduced≥41GW9 (47)7 (37)3 (16)147 (45)126 (39)51 (16)89 (44)78 (39)35 (17)12 (46)10 (38)4 (15)0.99Complications4 (21)87 (27)49 (24)7 (27)0.88 Open table in a new tab Our findings report no difference in the pregnancy duration (in days) or in birth weight related to endometrial thickness at the time of fresh or frozen ET. This suggests that EMT does not impact the quality of placentation.

SPERM DNA FRAGMENTATION AND FUNCTIONAL FEATURES OF HUMAN SPERM SELECTED BY MICROFLUIDIC SORTING FOR CLINICAL USE

High sperm DNA damage is associated with decreased normal fertilization, embryo development and pregnancy rates, and an increased miscarriage rate. Thus, selecting sperm with minimal DNA damage is important for assisted reproductive technology. A microfluidic sperm selection chamber (MSS, ZyMōt™; DxNow) is a device designed to collect sperm with high motility and chromatin integrity, which has recently been clinically used for ICSI/IVF. Here we aimed to comprehensively evaluate MSS compared to density-gradient centrifugation (DGC) in terms of (1) sperm DNA fragmentation by sperm chromatin dispersion (SCD) test and comet assay, (2) sperm motility and morphological features of motile sperm, and (3) blastocyst development after ICSI and clinical outcomes after vitrified-thawed single blastocyst transfers (vSBT). Sperm analysis was performed in 23 cases between October 2020 and February 2021. SCD test was optimized as a rapid procedure (≤30 min), with sperm showing a halo deemed normal, and those without a halo abnormal. Comet assay results were analyzed using CometScore 2.0, with comparison of %Tail DNA, Tail Length (TL), and Tail Moment (TM). Sperm motility parameters were investigated using the Sperm Motility Analysis System (SMAS, Dietect). To assess motile spermatozoa characteristics, middle piece morphology and active mitochondrial distribution were labeled by MitoTracker-Red. The reactive oxygen species (ROS) level was labeled using CM-H2DCFDA. ICSI outcomes (normal fertilization, blastocyst development, and pregnancy rates) were compared between DGC and MSS for 620 cycles between September 2019 and December 2020. In SCD test, MSS yielded a significantly lower abnormality rate than for raw sperm and DGC in all cases. The comet assay showed that %Tail DNA, TL, and TM were significantly lower with MSS than for raw sperm or DGC. Sperm motility rates were significantly higher with DGC and MSS than for raw sperm. Sperm motility analysis demonstrated significantly lower straightness, linearity, and beat-cross frequency (Hz) with MSS compared to DGC. The morphological abnormality rate of the sperm midpiece was significantly higher with MSS than DGC. Abnormal sperm midpieces exhibited significantly high ROS activity around mitochondria. Fertilization after ICSI and blastocyst rates were similar between MSS and DGC, while the vitrified blastocyst rate was significantly higher with MSS than DGC. Clinical pregnancy and miscarriage rates after vSBT did not significantly different between MSS and DGC. MSS-selected sperm had high DNA integrity and were homogeneous. The sperm had a higher motility rate, but the characteristic sperm motility pattern was meandering motion. Additionally, MSS did not exclude sperm that were morphologically abnormal in the middle piece with high ROS levels. MSS-ICSI yielded an improved vitrified blastocyst rate, and MSS is an effective device for sperm collection for ICSI.

PREDICTING WHICH BLASTOCYSTS WILL DEVELOP INTO FETUSES: TIME INTERVAL FROM MORULAE TO BLASTOCYSTS IS A NOVEL AND USEFUL INDICATOR FOR SUCCESSFUL PREGNANCY

The increased use of blastocyst transfer has emphasized the importance of selecting high-quality embryos. Analyses of embryos in a time-lapse incubator system showed that zygotes with the shortest intervals between the first and second or the second and third mitotic divisions were higher blastocyst rate. Additionally, embryos that developed relatively faster to the morulae and blastocysts had higher implantation rates. However, the useful indicators that predict which embryos will develop into fetuses without pregnancy loss have not yet been identified. Do time to morulae and interval between morulae and blastocysts predict which blastocysts will develop into fetuses? We analyzed 208 blastocysts derived from ICSI in 177 patients from September 2019 to September 2020. All embryos were monitored by a time-lapse system. The outcome of vitrified-thawed single blastocyst transfers was classified into pregnancy and non-pregnancy groups. The pregnancy group was divided into ongoing pregnancy and pregnancy loss. Data were analyzed using multiple logistics regressions. According to the logistic analysis of women age, the data were classified as less than or more than 39 years old. Time-lapse analyses provided data on time from pronuclear fading to morulae (tM), time to expanded blastocysts (tEB), interval between morulae and blastocysts (tB-tM), the number of equatorial trophectoderm cells (eTE), and the KIDScore D5 v3 (KS5). Data from pregnancy (hCG > 50 mIU/ml at 4 weeks) versus non-pregnancy were compared, as well those from ongoing pregnancy (a gestational sac at 12 weeks) versus pregnancy loss groups. In this study, 51.4% of total blastocyst transfers resulted in pregnancy. Significant differences were found between pregnancy and non-pregnancy groups: tM, 63.0 vs. 65.8 h; tEB, 92.4 vs. 95.5 h; tM-t8, 29.5 vs. 32.2 h; eTE, 6.5 vs. 5.5; and KS5, 6.7 vs. 5.8. The pregnancy rate in women less than 39 years old was significantly higher than in women over 39 years old (67.4% vs. 38.1%). Under 39-years-old, ongoing pregnancy rate was 79.7%, and pregnancy loss rate was 20.3%. A faster tM, a longer tB-tM, and a more eTE was found in the ongoing pregnancy group compared to the pregnancy loss group. The potential of tM, tB-tM and eTE as indicators for pregnancy was predicted with an AUC of 0.79. Over 39-years-old, ongoing pregnancy rate was 72.1%, and pregnancy loss rate was 27.9%. In ongoing pregnancy group, t2, t4 and tM were significantly different to the pregnancy loss group, which suggested delay of early embryonic development increases pregnancy loss risk. Under 39 years old, tM, tB-tM and eTE are effective predictors for selection of high-quality blastocysts that will be successful pregnant. The time interval tB-tM is a new indicator for identifying high-quality blastocysts. Over 39 years old, faster embryo development is a critical indicator for pregnancy.

SERUM HCG LEVEL MEASURED 5 DAYS AFTER VITRIFIED-WARMED BLASTOCYST TRANSFER IS PREDICTIVE OF OUTCOME REGARDLESS OF THE USE OF PRE-IMPLANTATION GENETIC TESTING

In typical viable natural conceptions, trophoblast invasion is well underway 10-11 days after ovulation, and delayed implantation evidenced by delayed elevation in human chorionic gonadotropin (hCG) levels is a reported predictor of early pregnancy loss. Similarly, serum hCG level on day 5 after thawed blastocyst transfer has been shown to be predictive of outcome. However, it has not previously been shown that this test is predictive of outcome of transfers of genetically screened blastocysts. The purpose of this study was to evaluate the effect of PGT on the predictive value of peri-implantation hCG levels for early pregnancy outcomes in vitrified-thawed single blastocyst transfers, including clinical pregnancy and ongoing pregnancy.

Live Birth Rate of Frozen-Thawed Single Blastocyst Transfer After 6 or 7 Days of Progesterone Administration in Hormone Replacement Therapy Cycles: A Propensity Score-Matched Cohort Study.

BackgroundProgesterone administration before transfer in hormone replacement treatment (HRT) is crucial to pregnancy outcomes in frozen-thawed blastocyst transfer (FET), but the optimal progesterone duration is inconsistent. The objective of this study was to investigate live birth rate (LBR) of different progesterone duration before blastocyst transfer in HRT–FET cycles.MethodIn this retrospective cohort study, patients underwent first HRT–FET (including suppression HRT) from January 2016 to December 2019 were included. Logit-transformed propensity score matching (PSM) was performed to assess covariates. The primary outcome was live birth rate after 28 weeks’ gestation. Basing on different duration of progesterone before transfer, patients were classified into P6-protocol (blastocyst transfer performed on the sixth day), or P7-protocol (blastocyst transfer performed on the seventh day). Subgroup analyses were conducted as follows: age stratification (–35, 35–38, 38–), development days of blastocyst (D5 or D6), blastocyst quality (high-quality or poor-quality), and endometrial preparation protocols (HRT or suppression HRT).ResultAfter case matching with propensity score methods, a total of 1,400 patients were included finally: 700 with P6-protocol and 700 with P7-protocol. Significantly higher live birth rate (38.43% versus 31.57%, respectively, P = 0.01) and clinical pregnant rate (50.43% versus 44.14%, respectively, P = 0.02) were observed in P6-protocol than those of P7-protocol. First-trimester abortion rates (18.13% versus 20.71%, P = 0.40) and ectopic pregnancy rates (2.27% versus 1.94%, P = 0.77) were similar between P6- and P7-groups. Preterm birth rate, low birth weight rate, newborn sex proportion, neonatal malformation rate were comparable between groups. Significantly higher LBRs were observed in patients with: age under 35, D5 blastocyst transfer, high-quality blastocyst transfer, and undergoing HRT cycles combined P6-protocol.ConclusionFrozen-thawed blastocyst transfer on the sixth day of progesterone administration in first HRT cycle is related to higher live birth rate compared with transfer on the seventh day, especially among patients aged under 35, D5 blastocyst and/or high-quality blastocyst transfer.

P–237 The good, the fast and the slow: which is better? Live birth rate following single blastocyst frozen embryo transfer

Abstract Study question Are embryo quality and day of vitrification (Day 5, 6 or 7) associated with live birth rates (LBR) following single blastocyst transfer (SBT) in frozen embryo transfer cycle (FET)? Summary answer Both blastocyst quality and day of vitrification are significantly associated with LBRs, with very low LBR when poor quality embryos are frozen on day 6. What is known already Evidence suggests that chromosomal status (ploidy) is strongly associated with blastocyst morphology and good quality embryos are more likely to be euploid. Furthermore, previous studies have shown a relationship between the time that embryos need to reach blastocyst stage and their euploidy rate with slowly developing blastocysts showing higher rate of aneuploidy. Nonetheless, despite all this evidence little is known about the actual effect of the combination of blastocyst quality and day of its vitrification. The scope of this study was to quantify the actual effect of the embryo quality and day of vitrification on live birth rates following FET. Study design, size, duration Retrospective analysis of 1546 FET cycles with SBT conducted between 2017 and 2019 in the university-affiliated private clinic. The embryos used for FET were obtained from IVF/ICSI: with PGT (FET-PGT) or without PGT (FET0) or from donated oocytes (FET-DON). Participants/materials, setting, methods FET with natural, natural-modified and completely medicated cycles to prepare endometrial lining were included. Blastocysts were classified according to Spanish Association for the Study of Reproductive Biology (ASEBIR) classification, ranging from A (the highest) to D (the lowest). The impact on LBR of different subgroups, formed within FET-PGT, FET0, FET-DON groups due to different day of vitrification and blastocyst quality, was assessed, using logistic regression after adjusting for age, day of vitrification and embryo quality. Main results and the role of chance We included 1546 FET cycles. Of those, 543 (35%) corresponded to FET-PGT; 648 (42%) to FET0 and 355 (23%) to FET-DON cycles. Overall, 1051 (68%) embryos were frozen on day 5(D5), 472 (30.5%) on day 6(D6) and 23 (1.5%) on day 7(D7). As far as embryo quality was concerned, 215 (13.9%) grade A; 957 (61.9%) B; 371(24%) C and 3(0.2%) D blastocysts were transferred. LBRs were significantly different between different embryos frozen on D5 44.3%; on D6 28.8% and on D7 8.7%, p &amp;lt; 0.001. When blastocyst quality was considered, LBR were 48.4% for grade A; 42.5% for B; 25.1% for C and 0% for D, p &amp;lt; 0.001. After applying logistic regression analysis, the odds ratio (OR) for transferring D6-blastocyst was 1.08, 95% CI[0.45; 2.62] and blastocyst with grade B and C; 0.71, 95% CI[0.51; 1.00]; 0.57,95% CI[0.36; 0.88] respectively. However, after transferring D6-blastocyst graded as C, the OR was 0.33, 95% CI[0.12; 0.90]. Our predictive model showed that the impact of the embryo quality on LBR was sustained across three groups. Transfer of D5/D6 grade A blastocyst resulted in the highest, while D6-C in the lowest LBR in all the groups. In the latter case vitrification on D6 impaired additionally the outcome. Limitations, reasons for caution The study should be interpreted with caution due to its retrospective character and the assessment of blastocyst quality on the day of vitrification and not on the day its transfer. Wider implications of the findings: Our robust findings could be considered a useful tool for counselling couples who seek advice regarding their expected success rates in the setting of FET with SBT. The very low livebirth rates in low quality (C) slow developing (D6) embryos should be communicated to patients prior to planning a FET. Trial registration number Not applicable

P–519 Investigation of embryo chromosomal constitution and live birth rate after vitrified-warmed euploid single blastocyst transfer across ranges of maternal body-mass-index

Abstract Study question Does maternal body-mass-index (BMI) associate with blastocysts’ chromosomal constitution and clinical outcomes in infertile patients undergoing preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer A higher euploidy rate per biopsied blastocyst was reported among underweight women. Overweight women were instead subject to higher miscarriage (MR) and lower live-birth-rates (LBR). What is known already Different studies in the literature revealed an association between BMI and infertility, suggesting a J-shaped relationship: both underweight and overweight women can suffer from infertility issues. Even if IVF might increase the success rate in both these categories of patients, it seems insufficient per se to overcome the complex and multifactorial fertility impairment derived from unbalanced nutritional intakes. Miscarriage, in particular, is common in both underweight and overweight women. However, most of the literature is based on chromosomally-untested embryos. Study design, size, duration: Retrospective observational study. Only the first IVF cycle with ≥1 biopsied blastocyst from each woman was included. The primary outcome was the association between maternal BMI (underweight, BMI&amp;lt;18.5, n = 160; normal-weight, BMI=18–25, N = 1392; overweight, BMI&amp;gt;25, N = 259) and the mean euploidy rate per cohort of biopsied blastocysts (m-ER). The secondary outcomes were the association between maternal BMI with clinical (mainly MR and LBR), gestational and perinatal outcomes after first vitrified-warmed single euploid blastocyst transfers. Participants/materials, setting, methods We included 1811 women undergoing PGT-A at a private IVF center between April–2013 and March–2020. The secondary outcomes were investigated on 1125 first vitrified-warmed single euploid blastocyst transfers from all patients obtaining ≥1 transferable blastocyst. Only ICSI with ejaculated sperm and continuous culture in standard incubators were performed. Logistic regressions were conducted to identify putative confounders and adjust the results accordingly. Main results and the role of chance Except for a lower maternal age among underweight women (38.3±3.1 versus 38.9±3.4 yr, p &amp;lt; 0.01) and higher among overweight ones (39.3±3.6 yr, p = 0.04), no difference was reported with respect to normal-weight women in terms of duration of infertility, hormonal levels, main cause of infertility, sperm quality, and reproductive history. The mean number of biopsied blastocysts was ∼3 in all groups. The m-ER shows a decreasing trend as the maternal BMI increases between 17 and 22–23, to then plateau. In fact, a significant difference was reported between underweight (50.8%±36.4%) and normal-weight women (41.4%±37.5%, p &amp;lt; 0.01). A linear regression adjusted for maternal age confirmed this moderate association between increasing BMI and m-ER (unstandardized-coefficient-B –0.6%, 95%CI:–1.1% to –0.1%, p = 0.02). Morphological quality and day of full-blastulation among transferred euploid blastocysts was similar in the three groups. Overweight women showed higher MR per pregnancy (N = 20/75, 26.7%, 95%CI:17.4%–38.3% versus N = 67/461, 14.5%, 95%CI:11.5%–18.2%; OR 2.0, 95%CI:1.1–3.6, p = 0.01) and lower LBR per transfer (N = 55/154, 35.7%, 95%CI:28.3%–43.8% versus N = 388/859, 45.2%, 95%CI:41.8%–48.6%; OR adjusted for euploid blastocysts’ features 0.67, 95%CI:0.46–0.96, p = 0.03). Clinical outcomes were instead similar among underweight and normal-weight women. All gestational and perinatal outcomes were comparable in the tree groups. Limitations, reasons for caution Our study is limited by its retrospective nature, and the fact that maternal BMI was measured only before oocyte retrieval and not before embryo transfer. Moreover, the reduced sample size did not allow for further relevant sub-analyses among solely obese women. Wider implications of the findings: When possible nutritional/lifestyle modifications should be encouraged to adjust maternal BMI before IVF. Overweight patients should be especially informed of their higher risk for miscarriage. Yet, BMI is just a gross marker, future studies based on body fat localization and percentage (e.g. by bioelectrical impedance analyses) are desirable. Trial registration number None

P–373 Initial β-hcg levels and the increase rates after 2 days are effective in predicting pregnancy outcomes in single blastocyst transfer in frozen-thawed or fresh cycles

Abstract Study question What is the role of the initial β-hCG value and the β-hCG rate of increase after 2 days in the predictability of the pregnancy outcomes? Summary answer The initial β-hCG values and β-hCG increase rates after 2 days are effective in predicting early pregnancy loss (EPL) and live birth rates (LBR). What is known already β-hCG is a glycoprotein-structured hormone secreted by the cyto syncytiotrophoblasts of the blastocyst and detected in the blood at the earliest 6–8 days after fertilization. The β-hCG level increases approximately by doubling in 48 hours in normal pregnancies. There are few studies conducted about the initial β-hCG values and the increase rates after 2 days on the pregnancy outcome of ART cycles and these studies indicated different threshold values and its level still remains unclear. Study design, size, duration This is a retrospective cohort study and was conducted at IVF and Reproductive Genetics Centre, Memorial Sisli Hospital, Istanbul, Turkey between January 2016 and December 2019. A total of 4605 including 3834 FET and 771 Fresh cycles resulted in a positive pregnancy test after a single blastocyst transfer was examined. Participants/materials, setting, methods The initial β-hCG test was applied 9 days after ET, when is the 14th day after ovulation. The latter β-hCG test was applied 2 days later. The cases with missing initial β-hCG tests or second β-hCG tests and missing follow-up during pregnancy were excluded. The mean β-hCG values and the increase rates after 2 days of the cases who had biochemical pregnancy loss (BPL), EPL and achieved live birth were reviewed. Main results and the role of chance The mean initial serum β-hCG level on 9 days after ET in the live birth group was 185.51 ±97.38 IU/L in FET cycles, which was significantly higher than the groups of BPL (62.75 ±50.70 IU/L) and EPL (133.93 ±95.10 IU/L). However, in fresh cycles, these levels in the live birth group was 167.70 ±114.05 IU/L which was significantly higher than the groups of BPL (50.13 ±27.49 IU/L) and EPL (106.55 ±71.80 IU/L). The mean β-hCG levels in FET cycles were significantly found higher than in fresh cycles (P &amp;lt; 0.005) regardless of pregnancy outcomes. The β-hCG threshold value predicting live birth for fresh cycle was found 108 IU/L (sensitivity 71.5%, specificity 70.8%, PPV 87.% and NPV 46.3%), while this value was found 101 IU/L (sensitivity 81.4%, specificity 55%, PPV 81.6% and NPV 53.8 %) for FET cycles. The β-hCG increase rate of threshold value predicting LBR for fresh cycle was 1.92 (sensitivity 90.6%, specificity 36.3%, PPV 80.1% and NPV 57%), while this rate was found 2.01 (sensitivity 90.1%, specificity 38%, PPV 78.4% and NPV 61.1%) for FET cycles. The β-hCG increase rate was not different between fresh and FET cycles. Limitations, reasons for caution Retrospective study Wider implications of the findings: The initial β-hCG values and the increases in β-hCG values after 2 days can be used as effective parameters in the diagnosis of pregnancy outcomes. Early prediction of pregnancy outcomes may help to the clinician to manage and follow-up high risk pregnancies. Trial registration number Not applicable

P–629 Is spontaneous ovulation better than induced ovulation for frozen-thawed embryo transfer cycle?

Abstract Study question Between spontaneous ovulation (SPO) and induced ovulation (INO) comparing clinical pregnancy rate and ongoing pregnancy rate for frozen-thawed embryo transfer (FET) cycle, which is better? Summary answer Both spontaneous ovulation and induced ovulation protocols showed no significant difference in clinical pregnancy rates and ongoing pregnancy rates. What is known already Recent practice worldwide is moving towards elective freezing of all embryos and subsequent frozen-thawed transfer, both for a perceived higher pregnancy rate as well as the significant reduction of ovarian hyperstimulation. The timing of FET can be determined by either detecting the spontaneous Luteinizing Hormone surge (SPO group) or by the administration of hCG (INO group). There is still an ongoing debate to determine which is the best protocol for frozen-thawed embryo transfer in the non-hormone replacement therapy (non-HRT) cycle. Study design, size, duration This retrospective study included 500 FET cycles for patients who had regular menses between June 2017 and June 2020. The FET cycles were grouped by type as follows: SPO (n = 281) and INO (n = 219). The primary outcome was the clinical pregnancy rate and the secondary outcome was ongoing pregnancy rate. Ongoing pregnancy is defined as a viable intrauterine pregnancy at 12 weeks of gestation confirmed on an ultrasound scan. Participants/materials, setting, methods This study was conducted in a single IVF centre. Vitrification was used as the cryopreservation method. To standardize outcome measures, only patients having single blastocyst transfer and aged under 38 years old were included. The average age of the patient was 32.9. Gamete donation, embryo donation, pre-implantation testing and assisted hatching cycles were also excluded from the analysis. Categorical data were analysed using Chi-square test SPSS version 25. Main results and the role of chance Clinical pregnancy rate for SPO group was 54.8% (154/281) versus 52.9% (116/219) in INO group. Even though clinical pregnancy rate was higher in SPO group as compared to INO group, it did not reach significance level (ꭓ2 = 0.17, p = 0.68). As all patients had single blastocyst transferred, the implantation rate was the same as clinical pregnancy rate. Ongoing pregnancy rate was also found higher in SPO group as compared to INO group (135/281, 48.0% and 97/219, 44.3% respectively) but again failed to reach significance level (ꭓ2 = 0.70, p = 0.40). Limitations, reasons for caution The retrospective nature of the study and therefore, the analysis was not adjusted for confounding factors such as blastocyst grading, etiology of infertility, and ethnicity of patients. Wider implications of the findings: In natural cycle, both spontaneous ovulation and induced ovulation protocols had the same pregnancy outcomes for frozen-thawed embryo transfer. However, induced ovulation can facilitate in scheduling FET timing to avoid weekends and public holidays, if necessary. Trial registration number Not applicable

P–266 Morphometric assessment of Ratio of inner-cell-mass diameter to blastocyst diameter is an effective criterion to select best embryo for successful outcomes in single blastocyst transfer-cycles

Abstract Study question Can semi-quantitative measurement of ICM diameter vis-à-vis blastocyst diameter, calculated from photographic images, be a significant predictor of implantation potential in single blastocyst transfer cycles? Summary answer A simple, non-invasive measurement of ICM:Blastocyst diameter ratio is a potentially effective predictor of high developmental potential, implantation, live-birth rates in single blastocyst transfer cycles. What is known already: Correlationship between quantitative measurements of blastocyst morphology such as size and shape of ICM and its implantation potential were first reported by Richter et al. Widely used Gardner and Schoolcraft’s qualitative scoring assessment is based on three major variables: expansion stage of blastocyst, cohesiveness of the inner cell mass (ICM), and consistency of trophectodermal (TE) cells. A top quality single blastocyst transfer yields implantation rate nearing 70% and live-birth rate about 50%. If an additional morphological but vital parameter evaluating ICM:Blastocyst diameter ratio is introduced to the Gardner’s blastocyst gradation system, it may help enhance IVF success rates still further. Study design, size, duration A prospective observational cohort study of women (n = 244) undergoing IVF treatment at our private fertility clinic from April 2018 until March 2020. Women with their autologous fertilized oocytes undergoing extended culture and transfer of a single fresh, at least fully expanded (Grade 3) blastocyst with a measurable ICM diameter and blastocyst inner diameter on day 5/6, irrespective of age, cycle number and indication for treatment, were included. Cryopreservation cycles were excluded. Participants/materials, setting, methods Each blastocyst was evaluated for expansion grade, ICM and TE according to Gardner et al. conventional method. Additionally, ratio of ICM diameter w.r.t. blastocyst inner diameter was also calculated from the photographic images on screen using the Hamilton-Thorne software package embedded with their laser system. Measurements were done on blastocysts where expansion had occurred between 114 and 120 hours after insemination. Implantation rates and live-birth rates were the major end-points. Main results and the role of chance A total of 244 blastocyst transfers were performed in same number of women, and 130 clinical pregnancies were achieved (53.3%). The average age of the women was 32.5 ± 4.2 years. The blastocysts that implanted successfully had an average ICM:blastocyst diameter ratio of 0. 469±0.082), whereas blastocysts that did not implant had a significantly lower ratio of 0.325±0.09 (P &amp;lt;.0001). No statistical significant difference was found in the TE grade between the blastocysts that implanted successfully and those that did not. Out of the 130 pregnancies achieved, 89 (68%) resulted in the delivery of a healthy baby. After ROC analysis, a cutoff value for the ICM/blastocyst ratio showed equal rank for sensitivity (0.81) and specificity (0.72). The resultant positive predictive value was 78%, and the negative predictive value was 74%. In our study, the conditional probability of achieving pregnancy upon transfer of a single blastocyst with an ICM:blastocyst ratio higher than the cutoff value of 0.4255 was 76%. This was significantly higher than the probability of pregnancy from blastocysts graded by conventional morphometry. Limitations, reasons for caution Our study is limited by the small sample size. Hence more multicentric studies are warranted to observe similar conclusion. Wider implications of the findings: The strengths of the study are the performance of single blastocyst transfers and using live births as the measurement endpoint. It is important to mention that embryo grading is inevitably subject to intra- and interobserver variations. However, with simple measurements of image data recording eliminates a degree of these variations. 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–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–515 Clinical outcome of mosaic-blastocyst transfer versus euploid-blastocyst transfer in single frozen blastocyst transfer cycles

Abstract Study question What is the clinical outcome of transferring a mosaic blastocyst versus a euploid blastocyst in single frozen blastocyst transfer (sFBT) cycles? Summary answer Single mosaic blastocyst transfer has similar clinical outcome to single euploid blastocyst transfer. What is known already Embryonic mosaicism occurs when there are two or more distinct cell lines found in preimplantation embryos derived from IVF. Data from recent studies show that mosaic blastocysts have the potential to implant and can result in healthy live births. As a result, patients now have the option of transferring mosaic blastocyst when they do not have any euploid blastocyst available for transfer. However, the clinical outcome of transferring mosaic blastocyst has not been definitively reported. Thus, a retrospective study was conducted to compare the clinical outcome of mosaic sFBT and euploid sFBT. Study design, size, duration A total of 602 patients underwent frozen blastocyst transfer in Alpha IVF from January to October 2019 and had their blastocysts screened for aneuploidy. These patients were divided into 2 groups: 26 patients with mosaic blastocysts transferred (Group A, age ranged 19–44), and 576 patients with euploid blastocysts transferred (Group B, age ranged 21–44). The mean age of patients from Group A and B were 34.0 and 32.8 respectively (p &amp;gt; 0.05). Participants/materials, setting, methods All samples had their DNA libraries constructed for sequencing using Next Generation Sequencing according to manufacturer’s specification (IonTorrent, USA). All blastocysts were frozen for subsequent sFBT cycle (Cryotech, Japan). All thawed blastocysts for sFBT survived with morphologically intact inner cell mass and trophectoderm cells. The importance of antenatal confirmation of the fetal chromosome status was emphasized in patients from Group A. The clinical outcomes of both groups were analysed and compared. Main results and the role of chance No significant differences were seen in the clinical pregnancy and implantation rate of Group A and B (65.4% vs 63.0%; p &amp;gt; 0.05). The miscarriage rate of Group A and B were 23.5% and 14.0% respectively. Albeit the higher miscarriage rate in Group A, there was no statistical significance between these two groups (p &amp;gt; 0.05). Group A was further divided into two subgroups, Subgroup A1: low risk mosaic blastocyst transfer; Subgroup A2: high risk mosaic blastocyst transfer. In the comparison of Group A subgroups, the clinical pregnancy and implantation of Group A1 is higher than Group A2 (76.9% vs 44.4%). In addition, the miscarriage rate of Group A1 and A2 were 23.1% and 0.0% respectively. Interestingly, there was no statistical significance in clinical pregnancy rate, implantation rate and miscarriage rate between these two subgroups. Limitations, reasons for caution This is a retrospective study and the sample size was comparatively smaller in the mosaic blastocyst transfer group than the euploid blastocyst transfer group. Further studies with a larger sample size should be carried out to ascertain the clinical outcome. Wider implications of the findings: Single mosaic blastocyst transfer has similar clinical outcome to single euploid blastocyst transfer. Thus, mosaic blastocyst can be considered for transfer when no euploid blastocyst are available. Nevertheless, stringent antenatal surveillance for chromosomal abnormalities to confirm the chromosomal status of the fetus must be followed. Trial registration number Not applicable

P–415 Clinical and In Vitro Fertilization laboratory parameters that contribute to clinical miscarriage after single euploid embryo transfer

Abstract Study question To investigate which factors, excluding embryo aneuploidies, are associated with miscarriage in patients who have undergone a single euploid blastocyst transfer. Summary answer Miscarriage was related to the body mass index (BMI), the type of cycle and the thickness of the endometrium. What is known already Preimplantation genetic testing for aneuploidies (PGT-A) is widely used in-vitro fertilization (IVF) to select euploid embryos. Several studies have shown that embryo aneuploidy is the main contributing factor for IVF failure, reinforcing the relevance of PGT-A as a method to select chromosomally normal embryos. A recent meta-analysis confirmed that patients undergoing PGT-A have a lower miscarriage rate than women that conceived naturally (9% vs 28%, respectively). Even though most of the studies show that PGT-A significantly reduces miscarriage rate, still some women do lose their pregnancies. We investigated which other reasons may be related to this early pregnancy loss. Study design, size, duration Retrospective, observational, and multicenter study of 6910 patients undergoing single euploid blastocyst transfer after PGT-A from January 2017 to December 2019 in our institution. Several laboratory and clinic variables were analyzed to study the effect of these variables on the miscarriage rate Participants/materials, setting, methods Indications for PGT-A were advanced maternal age, implantation failure, recurrent pregnancy loss and male factor. Embryos were cultured 5% O2 concentration and 6.5% CO2 concentration. Trophectoderm biopsy was performed on day 5/6 of development and analyzed through Next Generation Sequencing (NGS); embryos were vitrified until transfer was performed. Single euploid embryo transfer was performed in all cases. We performed a multivariate regression analysis to compare the different variables and search for there are significant differences. Main results and the role of chance We studied a total of 6910 patients undergoing PGT-A to describe which factors, excluding embryo aneuploidies, were correlated with miscarriage in patients who underwent single thawed euploid embryo transfer. When considering embryo morphology (embryo grading, quality of inner cell and quality of trophectoderm), we did not find differences in miscarriage rate among groups (high quality= 15.9%; normal quality= 14.3%; low quality= 15.0%; poor quality= 14.8%) p = 0.833. BMI was significantly associated with miscarriage rate (odds ratio [OD] 1.04; 95% confidence interval [CI], 1.012–1–076 p = 0.006) and miscarriage rate. We observed a weak association between endometrial thickness and miscarriage rate ([OD] 0.65; 95%, 0.528–0.778 p = 0.04) and also between type of endometrial preparation (natural cycle or hormone replacement cycle) ([OD] 0.77; 95%, 0.528–0.778) p = 0.04. Body mass index, according to our findings, was the main variable correlated with miscarriage rate. We did not find any association with the other variables studied (biopsy day, maternal age, male age, duration infertility, cycle length, previous miscarriage, previous live birth, previous cycles IVF, endometrial pattern and diagnosis). Limitations, reasons for caution The retrospective study design limits the generalization of our results but offers a good insight to be validated in prospective trials. Wider implications of the findings: According to our findings, BMI, endometrial thickness the day of the embryo transfer, and the type of endometrial preparation should be considered when transferring an euploid blastocyst. Trial registration number NO APLICA

P–241 Construction of a Machine Learning algorithm based on early morphokinetics for human blastocyst development prediction: a retrospective analysis of 575 cleavage-stage embryos

Abstract Study question Can morphokinetic features included into Machine Learning (ML) algorithms identify cleavage-stage embryos with the best chance to reach the expanded blastocyst stage on day 5? Summary answer A ML algorithm based on early morphokinetic features can identify cleaving embryos that will reach the expanded blastocyst stage on day 5. What is known already To date, the conventional morphology assessment of cleaving human embryos has a limited predictive power on further embryo developmental potential. The morphokinetic analysis using Time-Lapse systems (TLS) was introduced in order to provide a new tool to identify dynamic biomarkers of embryo quality. More recently, ML approach has been applied for the analysis of specific embryo-related features, aiming at developing predictive algorithms to assess the embryo development potential. Study design, size, duration We retrospectively analysed 575 embryos obtained from 80 women aged 25–42 years, with normal BMI, AFC≥8, day 3 FSH&amp;lt;12 IU/l, AMH&amp;gt;2.5 ng/ml, no diagnosis of polycystic ovary syndrome or endometriosis. These patients underwent IVF at our IVF Unit between March 2018 and March 2020; their embryos were cultured using the Geri plus® TLS and a single blastocyst transfer was performed. Participants/materials, setting, methods A total number of 29 morphological and morphokinetic parameters were considered to build six different ML algorithms. The performance to assess which was the best-fitting algorithm was calculated using the ROC curve considering accuracy (% of embryos correctly classified by the algorithm), Cohen-kappa coefficient (measurement of the agreement among features), mean number of TP (embryos correctly classified as undergoing developmental arrest), mean number of TN (embryos uncorrectly classified as undergoing developmental arrest). Main results and the role of chance Overall, 210 embryos progressed to the expanded blastocyst stage on day 5 (BL group), whereas 365 displayed developmental delay or arrest at any stage (nBL group). Among the six different algorithms, the best-fitting algorithm was obtained using the Kbest features selection approach combined with a Random Forrest evaluation strategy. This algorithm was based on 7 variables: embryo morphological score on day 2, pronuclear fading time (tPNf), completion time of cleavage to two, four and eight cells (t2, t4, and t8 respectively), time intervals t4-t3 and t8-t4. The algorithm showed an AUC of 0.78, with an accuracy of 0.73, a Cohen-kappa of 0.41, a mean TP number of 302/365 embryos in the nBL group and a mean TN number of 120/210 embryos in the BL group. Mean false positive (FP) and false negative (FN) numbers were of 63 and 90.2, respectively. Limitations, reasons for caution The results obtained in this study may not be generalizable to patients with other clinical characteristics, to other time-lapse systems or different laboratory settings. The predictive power of the algorithm should be validated prospectively on a larger number of embryos. Wider implications of the findings: The current study represents a preliminary analysis for the development of hierarchical predictive models for embryo assessment based on their developmental potential, that embryologists will be able to apply as a support for decision-making. Trial registration number Not applicable

P–304 The endometrial preparation protocol does not affect the live-birth-rate after vitrified-warmed euploid single blastocyst transfers: an analysis of 1884 procedures

Abstract Study question Is the live-birth-rate (LBR) different when comparing artificial (AC) and modified-natural (M-NC) cycle for endometrial preparation to vitrified-warmed euploid blastocyst transfer? Summary answer The LBR after vitrified-warmed euploid blastocyst transfer seem independent of the endometrial preparation administered. What is known already Only the transfer of a competent embryo on a receptive endometrium might result in successful implantation. Three main protocols for endometrial preparation to vitrified-warmed embryo transfer exist: NC, M-NC, and AC. None among them, though, has been shown more appropriate than the others to date, especially since, only in a few studies, the analysis was restricted to single euploid blastocyst transfers to limit the impact of embryonic issues on implantation. In conclusion, no clear consensus exists and the choice is still largely based on menstrual/ovarian cycle characteristics and patient’s needs. Study design, size, duration All first vitrified-warmed single euploid blastocyst transfers performed between April–2013 and March–2020 were included in the analysis. Endometrial preparation was conducted with either an AC (N = 1211) or a M-NC (N = 673). The protocol was chosen based on patients’ logistical reasons. The primary outcome was the LBR per transfer. Sub-analyses based on blastocyst quality and day of development were conducted. Birthweight, gestational age, gestational and perinatal issues were secondary outcomes. Participants/materials, setting, methods AC: oral estradiol-valerate 3-times/day from day2–3 of the cycle until the endometrial thickness reached ≥7mm, then 600 mg/day of micronized progesterone. The transfer was conducted on day6 of progesterone administration. M-NC: an intramuscular dose of 10,000IU hCG was administrated when the leading follicle was &amp;gt;17 mm and the endometrium was thicker than 7mm and trilaminar, plus 400 mg/day of micronized-progesterone as luteal phase support starting 36–40hr post-hCG. The transfer was conducted on day7 after trigger. Main results and the role of chance The two groups were similar for maternal age at retrieval (38.0±3.3yr) and transfer (38.3±3.3yr), reproductive history, embryological outcomes of the IVF cycle, body-mass-index, basal hormonal levels, and blastocyst features (Gardner’s classification: AA = 73%, AB/BA=11%, BB/AC/CA=8%, CC/BC/CB=8%; day5=48%, day6=47%, day7=5%). The LBR was 46.7% (N = 565/1211) and 49.9% (N = 336/673) after AC and M-NC, respectively, resulting in an odds-ratio 1.14, 95%CI:0.94–1.37. The absence of significant differences was confirmed also when adjusted for blastocyst quality and day of full-development (1.16, 95%CI:0.96–1.41). Among the 565 and 336 deliveries, the birthweight was similar (3290.3±470.7 versus 3251.7±521.5 g, Mann-Whitney-U-test=0.5), the gestational age was similar (38.5±1.7 versus 38.4±1.9 weeks, Mann-Whitney-U-test=0.5). Also, the rates of newborns who were normal (81% versus 82%), large (8% versus 9%), and small (11% versus 9%) for gestational age were similar (Chi-squared-test=0.5). The rates of patients experiencing gestational (6% versus 7%) and/or perinatal issues (3% versus 3%) were also similar (Fisher’s-exact-tests=0.4). Limitations, reasons for caution This is a retrospective study conducted in poor prognosis patients indicated to preimplantation genetic testing for aneuploidies. Future randomized controlled trials and cost-effectiveness analysis are desirable, as well as studies in different patient populations. Lastly, each gestational/perinatal issue shall be analyzed per se (e.g. different placentation disorders). Wider implications of the findings: The absence of clinical and perinatal differences between the two protocols for endometrial preparation supports the adoption, whenever needed, of AC. This approach, in fact, allows a higher flexibility in patients’ and daily workload management. Trial registration number None

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–226 Failure of blastocoele expansion within the first two hours post thawing could halve the chances of implantation

Abstract Study question The aim of this study was to evaluate the influence of blastocoele re-expansion time of warmed vitrified blastocysts on clinical pregnancy outcome. Summary answer Clinical pregnancy rate was significantly higher after transfer of warmed vitrified blastocysts that were fully expanded within 2 hours post thaw. What is known already The number of blastocysts being vitrified worldwide has increased dramatically over recent years. A combination of factors has led to this including the introduction of vitrification, an increase in freeze-all policies, single embryo transfer and an increase in preimplantation genetic testing. Currently, blastocyst re-expansion after thawing is used to indicate the survival status of the blastocyst and when combined with the morphology of blastocyst can predict its reproductive potential. While time taken for blastocoele re-expansion has been proposed to be a biomarker of viability, its value in clinical practice remains unclear. Study design, size, duration This retrospective study analysed outcomes in patients who had frozen embryo transfers between June-December 2020. 233 embryos were reviewed with time-lapse to assess their blastocoele expansion post-warming and three groups were identified. The first included fully expanded blastocysts post-warming. The second group included partially expanded blastocysts and the third non-expanded blastocysts. In addition, the groups were subcategorised into two further categories depending on whether they took less or more than 2 hours to complete expansion. Participants/materials, setting, methods 233 vitrified/warmed embryos from 216 patients were analysed using time-lapse incubators. The first group included 134 blastocysts, of which 70 were fully expanded within 2 hours and 64 after 2 hours post thaw. The second group had 70 embryos of which 45 expanded partially within 2 hours and 25 after 2 hours. The third had 28 embryos that had no expansion within the first 2 hours (n = 20) or after 2 hours (n = 8). Main results and the role of chance Blastocysts were collapsed by laser prior to vitrification. Single blastocyst transfer was performed for all patients. The mean transferred embryo age was 32.1± 5.5 and the recipient’s was 37.5± 5.9. Fully expanded blastocysts (n = 70) within 2 hours demonstrated a clinical pregnancy rate (CPR) of 57% compared with 38% from those that expanded fully after 2 hours (n = 64) (p = 0.02). Blastocysts with some form of expansion (full or partial) within 2 hours post-warming (n = 115) were associated a significantly higher CPR compared to those expanding after 2 hours (n = 89). The CPR was 55% and 39% respectively (p = 0.02). Embryos that showed no expansion (n = 20) within the first 2 hours post thaw resulted in CPR of 28%. Interestingly, embryos that showed no expansion after 2 hours resulted in no pregnancy. When combining morphology as a selection criterion, expansion within 2 hours of thawing was associated with a CPR of 62.5% for ≥4AB embryos, 50% for BB embryos and 45% for poorer embryos ≤CB.In conclusion, failure of blastocoele expansion post 2 hours reduced by half the chances of clinical pregnancy (p = 0.03). Combination of the degree of re-expansion and embryo morphology is an important predictor tool to improve clinical outcomes in frozen embryo transfers. Limitations, reasons for caution This study uses a small sample size of patients. The data are observational and were retrospectively analysed so unknown confounders could not be assessed. The addition of more cycles and further multivariate analysis, is essential for confirmation of the findings. However, initial results are very reassuring. Wider implications of the findings: The degree of speed of re-expansion post warming should be used as a predictor for prioritisation of embryos for transfer. Owing to these preliminary findings there is rationale for a larger scale study combining other morphological indicators that could further assess implantation indicators and assist patient counselling Trial registration number Not applicable

P–785 Double versus sequential single blastocyst transfer in freeze all cycles?

Abstract Study question Is double blastocysts transfer (DET) better than sequential single blastocyst transfer (seq-SET) in freeze all cycles? Summary answer Sequential single blastocyst transfer provides a higher live birth rate (LBR) per cycle initiated and eliminates multiple births in freeze-all cycles. What is known already Improvements in cryopreservation technology helped freeze-all strategy gain much popularity. The new debate is whether guidance for single embryo transfer should also be applied to frozen-thawed embryo transfers in freeze-all cycles. Study design, size, duration We performed a retrospective cohort analysis of 860 women in whom the entire cohort of embryos frozen at the blastocyst stage for various indications. All women aged 19–43 years, who had at least two blastocysts frozen and subsequently thawed and transferred were included. Preimplantation genetic testing cycles were excluded.The study period ranged from January 2016 to May 2019. Participants/materials, setting, methods Data regarding female age, number of embryos transferred, multiple pregnancy and live birth rates (LBR) were extracted from the electronic database. Women were categorized based on their age and the mode of embryo transfer. Primary outcome was live birth rate LBR per cycle initiated. Secondary outcomes were LBR per embryo transfer and multiple birth rate. Groups were compared using Fisher’s test, generalized estimating equation model and logistic regression analysis to adjust for confounding factors. Main results and the role of chance The study group comprised of 666 women (371 Seq-SET and 295 DET) who underwent 837 embryo transfer cycles. Second embryo transfer was affected in 46.1% of women in the Seq-SET group. Age, indication for freeze-all, and mode of transfer were related with the LBR. For women ≤ 35 (n = 370), LBRs per embryo transfer were similar in single and double embryo transfers (53.9% versus 64.2% respectively, p = 0.006, aOR=0.65, 95% CI:0.41–1.01). However, LBR per cycle initiated was significantly higher in Seq-SET group (78.9% versus 64.2% respectively, p = 0.004, aHR=2.09, 95% CI:1.28–3.41). While only one monochorionic twin delivery was observed with Seq-SET (0.5%), 19 out of 70 (27.1%) live births after DET were twins. For women &amp;gt;35 of age (n = 296) the likelihood of a live birth per embryo transfer was lower in single compared to double embryo transfers (33.2% versus 46.2%, respectively, p = 0.012, aOR=0.58 95% CI:0.38–0.88). Although LBR per cycle initiated was higher in Seq-SET (58.2%) than DET (46.2%), the difference did not reach statistical significance (p = 0.054, aHR=1.62, 95% CI:1.00–2.60). While no twin delivery was observed with Seq-SET, 8 out of 86 (9.3%) live births with DET were twins. Limitations, reasons for caution This was a retrospective study with small sample size performed at a single fertility center, which may limit the generalizability of our findings. Cost-efficiency was not studied. Wider implications of the findings: Seq-SET is associated with a comparable or higher likelihood of live birth per cycle initiated and a very low risk of twins when compared to DET. However, half of SET cases had to undergo two transfer cycles. Trial registration number NA