Transfer Of Vitrified Blastocysts Research Articles

P-128 Non-invasive morphometric assessment of embryo quality: Effectiveness of the number of trophectoderm cells at the equatorial region of expanded blastocysts

Abstract Study question Does the number of trophectoderm cells at the equatorial region of expanded blastocysts (eTEs) affects pregnancy rate in single vitrified blastocyst transfer (SVBT)? Summary answer The number of eTEs highly correlated with the total number of TEs. The greater number of eTEs was higher pregnancy rate became. What is known already The increased use of blastocyst transfer has emphasized the importance of selecting high-quality embryos. Morphological grading: Gardner’s criteria, or time-lapse imaging of embryo morpho kinetics is established a tool for selection of high-quality blastocysts. The Gardner’s criteria is one of the most used evaluation according to grading scores of developmental stage, trophectoderm (TE) and inner cell mass (ICM). Although, it cannot completely exclude subjectivity, and it varies from observer to observer. A quantitative evaluation method for blastocyst selection is necessary to solve these problems and to improve the pregnancy rate of SVBT. Study design, size, duration First, we checked the correlation between the number of eTEs and the total number of TEs using 23 discarded blastocysts by immunostaining of CDX2. Then, we analyzed 1103 vitrified-warmed blastocysts from July 2017 to June 2021 for pregnancy rates. Expanded blastocysts which reached the blastocoel cavity of 160 µm or more with obvious ICM were vitrified and cryopreserved. Relationship between the number of eTEs and pregnancy rate after SVBT was analyzed by logistic regression. Participants/materials, setting, methods CDX2-positive cells were defined as TE, and the difference between total cells by Hoechst staining and CDX2-positive cells as ICM. The correlation between the number of eTEs and the total number of TEs was examined. In SVBT, all embryos were monitored by a time-lapse system. eTEs were counted in the time-lapse image just before cryopreservation. Pregnancy and abortion rates were examined for each eTEs. Also, logistic regression analysis was performed on eTEs and pregnancy. Main results and the role of chance There was a strong positive correlation between the number of eTEs and the total number of TEs (CDX2-positive cells) in the whole blastocysts (r = 0.945). In addition, there was a significant correlation between the number of eTEs and the total cell number of blastocysts (r = 0.73). On the other hand, there was no correlation between the number of eTEs and the cell number of ICM (r = 0.008). The number of eTEs was presented as quartiles: Pregnancy rates for <5, 5-6, 6-7 and ≥7 eTEs were 25.9, 29.4, 38.4, and 64.2% (P < 0.05), respectively, and the miscarriage rates were 50.0, 28.3, 14.1 and 19.2% (P < 0.05), respectively. Pregnancy rate for ≥7 eTEs was significantly higher than that for the other groups (P < 0.05), and miscarriage rate was lower than that for <6 eTEs. That is, increased eTEs significantly increased the pregnancy rate and decreased the miscarriage rate. Significant relationship between the number of eTEs and pregnancy was shown by logistic regression analysis (P < 0.0001), with an AUC of 0.68 and a cutoff value of 6.5. Limitations, reasons for caution In this study, expanded blastocysts at 160 µm or more in diameter were analyzed and cryopreserved. The different size of blastocysts may be different number of eTEs, and the relationship between the number of eTEs and pregnancy rate will be needed to confirm in individual clinic. Wider implications of the findings Quantitative analysis of eTEs makes it possible to select highly fertile blastocysts. This assessment is simply and easy. It can be the additional possible evaluation before cryopreservation to allows for quantitative treatment of continuous variables by classifying them in more detail than Gardner’s criteria. Trial registration number N/A

P-193 Does the day of laser-assisted hatching affects the clinical outcomes of single vitrified blastocyst transfer cycles? A propensity score–matched study

Abstract Study question What is the impact of different days of laser-assisted hatching on the clinical outcomes of single vitrified blastocyst transfer cycles? Summary answer Clinical pregnancy of single vitrified blastocyst were similar, irrespective of day of laser-assisted hatching. What is known already Assisted hatching aims to create a weakness in the ZP to aid embryo hatching and implantation. Day 5 assisted hatching is typically performed shortly before the embryo transfer while day 3 assisted hatching is performed before blastocyst culture expansion and vitrification. Despite over 25 years of use, there is still no standardized protocol for AH, and published data on its efficacy are controversial and inconclusive. While efforts have been made to study the link between AH techniques and pregnancy outcomes, there is limited consensus for optimizing AH protocols like the day of performance. Study design, size, duration This was a retrospective study based on data collected between January 2022 and October 2023. Propensity score matching (PSM) was performed in a 1:1 matching ratio. 123 patients of day 3 laser assisted hatching group (D3 LAH group) were successfully matched to 123 cycles of day 5 laser assisted hatching group (D5 LAH group). The D5 LAH group was from March 2023 to October 2023, and the D3 LAH from January 2022 to February 2023. Participants/materials, setting, methods Single vitrified day 5 embryo transfer, embryos survived after warming, and patient agreed to receive LAH were include in this study. We excluded donation cycles, missing data, severe female factors. Primary outcome is clinical pregnancy. Comparisons between groups were performed with chi-square test, Fisher’s exact test, Mann–Whitney U test, and Student’s t-test where appropriate, with a P value <0.05 considered statistically significant. A multivariable analysis in the GEE model was adjusted for the confounding factors. Main results and the role of chance After propensity score matching, there was no significant difference in patient characteristics between the groups, creating similar cohorts. For example, the frequency of primary infertility was significantly different between groups initially (61% vs 46.7% in D5 LAH vs D3 LAH respectively, P = 0.004). Following propensity score matching the rate of primary infertility was similar in both groups (with SMD=0.08). Standardized mean difference (SMD) < 0.1 suggests adequate variable balance after propensity matching. Clinical pregnancy rate were similar between two groups: 66/123 (53.7%) D3 LAH cycles and 75/123 (61%) D5 LAH cycles. When generalized estimating equation were performed: only TE grade, but not the day of laser assisted hatching, was noted to be an independent predictor of clinical pregnancy (OR = 0.068; 95% CI: 0.005-0.977; p = 0.048). Poor TE grade had a weak negative relationship with clinical pregnancy of single blastocyst cycles. Limitations, reasons for caution The main limitation of the study is its retrospective design and small sample size. Wider implications of the findings Clinical outcomes are comparable between day 3 laser assisted hatching and day 5 laser assisted hatching, although clinical pregnancy can be slightly improved with higher TE grade. Trial registration number not applicable

The effect of an extended culture period on birth weight among singletons born after single or double vitrified embryo transfer.

To evaluate the effect of an extended culture period on birth weight among singletons born after vitrified-warmed embryo transfer. A retrospective cohort study was performed among 12400 women who gave birth to 1015, 1027, 687, and 9671 singletons after single blastocyst transfer, single cleavage-stage embryo transfer, double blastocyst transfer, and double cleavage-stage embryo transfer, respectively. The unadjusted birth weight of singletons born after vitrified blastocyst transfer were heavier than those born after cleavage-stage transfer (β=30.28, SE=13.17, P=0.022), as were the adjusted birth weights (β=0.09, SE=0.03, P=0.007). In addition, there was a 37% increased odd of having an infant with high birth weight after vitrified blastocyst transfer compared with vitrified cleavage stage transfer (OR=1.37, 95% CI:1.07-1.77). The unadjusted and adjusted birth weight and odds of having an infant with high birth weight significantly increased after blastocyst transfer compared with cleavage-stage embryo transfer in vitrified-warmed cycles.

Effect of repeated vitrification of human embryos on pregnancy and neonatal outcomes

BackgroundRepeated cryopreservation of embryos should occasionally be considered when embryos were not suitable for transfer. The effect of re-cryopreservation on embryos remains contentious.MethodsThis retrospective cohort study aimed to evaluate the pregnancy and neonatal outcomes of twice vitrificated blastocyst derived from once vitrified embryos. Total 410 vitrified-warmed blastocyst transfer cycles were divided into two groups according to the times of embryo vitrification: (1) vitrified blastocysts derived from fresh blastocysts (control group, n = 337); (2) twice vitrified blastocysts derived from once vitrified embryos (n = 73). The primary outcome was live birth rate. Multivariable logistic or linear regression analysis model was performed to describe the association between embryo cryopreservation times and clinical outcomes.ResultsNo difference was observed in female age at retrieval and transfer, infertility period, body mass index (BMI), infertility type, endometrial thickness, and embryo transfer numbers between the two groups. The pregnancy outcomes of embryos in repeated cryopreservation group were comparable to those of embryos in control group, including implantation rate, clinical pregnancy rate, and live birth rate. In multivariate logistic regression analysis, the cryopreservation times did not affect the outcomes of biochemical pregnancy, clinical pregnancy, and live birth. Moreover, there was no difference in gestational age, birthweight and sex ratio of singleton newborns between groups. After correcting several possible confounding variables, no significant association was observed between cryopreservation times and neonatal birthweight.ConclusionIn conclusion, pregnancy and neonatal outcomes achieved with twice vitrified blastocyst transfer were comparable to those achieved with vitrified blastocyst transfer in control group.

Early pregnancy bleeding after assisted reproductive technology: a systematic review and secondary data analysis from 320 patients undergoing hormone replacement therapy frozen embryo transfer.

Early pregnancy bleeding after assisted reproductive technology: a systematic review and secondary data analysis from 320 patients undergoing hormone replacement therapy frozen embryo transfer.

Factors affecting the ongoing pregnancy rate in women with repeated implantation failure undergoing an endometrial receptivity array.

In this retrospective study, we analyzed factors influencing the ongoing pregnancy rate (PR) in women with repeated implantation failure (RIF) undergoing embryo transfer with endometrial receptivity array (ERA). Eighty-three consecutive personalized embryo transfers (pETs) with ERA, from 54 women with RIF, were selected from June 2020 to April 2022. Vitrified blastocyst transfer was timed based on ERA results. The ongoing PR per pET was 33.7%. Using ERA, the endometrium was identified as pre-receptive in 26 cycles, early receptive in 25 cycles, receptive in 31 cycles, and late receptive in one cycle. With cycles categorized into three receptivity phases (pre-receptive, early receptive, or receptive), no significant differences were found in the clinical PR (27.3%, 55.6%, and 40%, respectively) or ongoing PR (9.1%, 55.6%, and 40%, respectively) after a single blastocyst transfer. Similarly, no significant differences were observed in the clinical PR or ongoing PR after the transfer of two or more blastocysts. Among women with ongoing pregnancy relative to those without, age at first pET was significantly lower (35 years vs. 39 years, p=0.001), while blastocyst score (23 vs. 18, p=0.012) and the proportion of blastocyst scores >18 (71.4% vs. 38.9%, p=0.005) were significantly higher. In multiple logistic regression analysis, the woman's age (odds ratio [OR], 0.814; 95% confidence interval [CI], 0.706 to 0.940; p=0.005) and blastocyst score >18 (OR, 3.052; 95% CI, 1.075 to 8.665; p=0.036) were identified as significant factors influencing ongoing pregnancy. In pET with ERA, ongoing pregnancy was closely associated with woman's age and blastocyst quality.

Pre-Vitrification and Post-Warming Variables of Vitrified-Warmed Blastocysts That Are Predictable for Implantation.

Human IVF embryos that are not used for fresh transfer are cryopreserved by vitrification for later embryo transfers. This study evaluates pre-vitrification and post-warming embryo characteristics that are suitable to predict the chance of clinical pregnancy in single vitrified blastocyst transfer (SVBT) cycles. In a multicenter observational trial (IMBOS trial), embryos were cultured in a time-lapse system before and after vitrification. Associations between clinical pregnancy, morphokinetic parameters, blastocyst collapse, KIDScore D5, pre-vitrification and post-warming Gardner scores, post-warming blastocyst size and re-expansion rates before SVBT were analyzed in 182 SVBTs which resulted in 89 clinical pregnancies. No association was found between clinical pregnancy after SVBT and the number of collapses or the maximal collapse size before vitrification. The multifactorial analysis of pre-vitrification Gardner scores showed a significant association with clinical pregnancy for trophectoderm grading but not for expansion/hatching status and inner cell mass grading. A significant association with clinical pregnancy was found for the time to reach a blastocyst after pronuclear fading (tB-tPNf), KIDScore D5 and post-warming size but not the rate of expansion or maximal expansion size. The selection of blastocysts for SVBT could benefit from using pre-vitrification parameters like tB-tPNf, trophectoderm grading and post-warming blastocyst size.

P-232 Beyond 12 months, blastocyst cryostorage duration in close system affects live birth rate: a monocentric retrospective study

Abstract Study question In order to evaluate the effect of blastocyst cryopreservation duration on live birth rate (LBR). Summary answer Blastocyst cryopreservation duration negatively affects live birth rate when blastocysts are vitrified in complete close system for more than 12 months. What is known already With the development of embryo vitrification, frozen-thawed program embryo transfer has been widely used in the past decade. However, the association between cryopreservation duration and frozen blastocyst transfer outcomes are limited. So far, very few studies have provided evidence on the relationship between blastocyst storage duration and LBR. Study design, size, duration This monocentric retrospective study included 1267 women (n = 1462 cycles) who underwent single frozen blastocyst transfer. Duration of vitrification in close system was categorized into five groups included 2144 frozen blastocysts (Day 5/6) : <3 months (n = 417; mean female age = 32.6 ± 4.74y), 3-6 months (n = 596; mean female age = 33.2 ± 4.96y), 6-12 months (n = 440; mean female age = 33.4 ± 4.83y), 12-36 months (n = 512; mean female age = 32.6 ± 4.75y), and >36 months (n = 179; mean female age = 31.1 ± 3.90y). Participants/materials, setting, methods Clinical pregnancy and live birth outcomes were compared between the cohorts stratified according to blastocyst cryostorage duration. A binary multivariate logistic regression was performed, adjusting for confounders: female age at vitrification, male age, ART technique (IVF or ICSI) and endometrial preparation protocol (spontaneous, substituted and stimulated FET cycle). Multivariable analysis was used to assess the association between crystorage duration of vitrified blastocyst and LBR. All frozen blastocyst transfers were realized in spontaneous, substituted or stimulated cycles. Main results and the role of chance After multivariable analysis, live birth rate was significantly different depending on blastocyst cryostorage duration (all groups combined) (p = 0.033): live birth rate was significantly lower when blastocyst cryostorage duration was between 12-36 months (OR 0.740 IC95%[0.558; 0.981] p = 0.036) and beyond 36 months (OR 0.674 IC95%[0.456; 0.986] p = 0.044) compared to less than 3 months of blastocyst cryostorage duration. However, no significant impact of blastocyst cryostorage duration was observed on clinical pregnancy rate (p = 0.52). Limitations, reasons for caution Our study was limited by its retrospective design with restricted samples. Wider implications of the findings This study provides new findings on the effect of blastocyst cryostorage duration with a significant impact on LBR beyond 12 months of vitrification in close system. However, it’s crucial to confirm that cryostorage duration doesn’t lead to adverse obstetric and perinatal outcomes after vitrified blastocyst transfer. Trial registration number not applicable

Comparison of the efficacy of subcutaneous versus vaginal progesterone using a rescue protocol in vitrified blastocyst transfer cycles

Does administration of subcutaneous (s.c.) progesterone support ongoing pregnancy rates (OPR) similar to vaginal progesterone using a rescue protocol in hormone replacement therapy frozen embryo transfer cycles? Retrospective cohort study. Two sequential cohorts - vaginal progesterone gel (December 2019-October 2021; n=474) and s.c. progesterone (November 2021-November 2022; n=249) -were compared. Following oestrogen priming, s.c. progesterone 25 mg twice daily (b.d.) or vaginal progesterone gel 90 mg b.d. was administered. Serum progesterone was measured 1 day prior to warmed blastocyst transfer (i.e. day 5 of progesterone administration). In patients with serum progesterone concentrations <8.75 ng/ml, additional s.c. progesterone (rescue protocol; 25 mg) was provided. In the vaginal progesterone gel group, 15.8% of patients had serum progesterone <8.75 ng/ml and received the rescue protocol, whereas no patients in the s.c. progesterone group received the rescue protocol. OPR, along with positive pregnancy and clinical pregnancy rates, were comparable between the s.c. progesterone group without the rescue protocol and the vaginal progesterone gel group with the rescue protocol. After the rescue protocol, the route of progesterone administration was not a significant predictor of ongoing pregnancy. The impact of different serum progesterone concentrations on reproductive outcomes was evaluated by percentile (<10th, 10-49th, 50-90th and >90th percentiles), taking the >90th percentile as the reference subgroup. In both the vaginal progesterone gel group and the s.c. progesterone group, all serum progesterone percentile subgroups had similar OPR. Subcutaneous progesterone 25 mg b.d. secures serum progesterone >8.75 ng/ml, whereas additional exogenous progesterone (rescue protocol) was needed in 15.8% of patients who received vaginal progesterone. The s.c. and vaginal progesterone routes, with the rescue protocol if needed, yield comparable OPR.

Polycystic ovary syndrome increases the rate of early spontaneous miscarriage in women who have undergone single vitrified euploid blastocyst transfer

Research questionDoes polycystic ovary syndrome (PCOS) independently influence the risk of early spontaneous miscarriage in patients undergoing single euploid vitrified blastocyst transfer? DesignThis observational cohort study retrospectively analysed 1498 patients undergoing their first single euploid blastocyst frozen transfer cycles between October 2016 and December 2021. Patients were divided into PCOS and non-PCOS groups according to the Rotterdam criteria. Logistic regression analysis was conducted to study the independent effect of maternal PCOS status on early spontaneous miscarriage after single euploid embryo transfer after adjusting for confounding factors. ResultsNo statistically significant differences were identified in the rates of positive pregnancy test (68.95% versus 64.86%, P = 0.196) or clinical pregnancy (59.93% versus 57.33%, P = 0.429) between the PCOS and non-PCOS groups after single euploid embryo transfer. Early spontaneous miscarriage occurred more frequently in women with PCOS compared with controls (18.67% versus 12.00%, P = 0.023). In single euploid embryo transfer cycles, PCOS significantly increased the incidence of early spontaneous miscarriage after adjusting for some potential confounders (adjusted odds ratio 1.649, 95% CI 1.032 to 2.635, P = 0.036). ConclusionsAlthough no significant difference was observed in clinical pregnancy rates, PCOS status increased the risk of early spontaneous miscarriage after single vitrified euploid blastocyst transfer, suggesting an additional role of endometrial dysfunction affected by endocrine disorders. Further studies are needed to investigate the specific mechanisms and effective intervention strategies.

Live birth rates in day 5 fresh versus vitrified single blastocyst transfer cycles: A cross-sectional analysis.

The use of frozen embryo transfers (FET) in assisted reproduction has increased worldwide. Controlled ovarian hyperstimulation in a fresh transfer may impair endometrial-embryo synchronicity. However, there is conflicting evidence on live birth rates (LBR) and clinical pregnancy rates (CPR). To compare LBRs and CPRs between single autologous day 5 fresh vs. vitrified blastocyst transfer cycles, to investigate the impact of controlled ovarian hyperstimulation on embryo-endometrium asynchrony. A large cross-sectional analysis of 6002 embryo transfers (ET) comprised 3774 fresh and 2228 FET cycles from 2016 to 2019. Multivariate and subgroup analysis were performed for high responders ( 20 oocytes). Univariate analysis showed no difference in LBR (28.3% vs. 27.4%, p = 0.43) and CPR (32.2% vs. 30.9%, p = 0.30); however, multivariate analysis demonstrated significantly lower LBR (OR 0.864, p = 0.046, 95% CI 0.749-0.997) and CPR (OR 0.852, p = 0.024, 95% CI 0.742-0.979) in FET compared to fresh ETs. Younger participant age, previous in vitro fertilization pregnancy, advanced blastocyst expansion, higher trophectoderm quality, and lower cumulative number of ETs all improved the odds of LBR and CPR. Conventional in vitro fertilization, rather than intracytoplasmic sperm injection, improved CPR but not LBR. Body mass index affected neither LBR nor CPR. In the subgroup, multivariate analysis of high responders showed no difference in LBR or CPR. This study demonstrates relatively higher LBR and CPR of nearly 14% for fresh ETs compared to FETs, in multivariate analysis. A universal freeze-all strategy, without appropriate indication, may lead to suboptimal outcomes. In high responders, freeze-all cycles may be beneficial, as outcomes appear similar.

Single vitrified blastocyst transfers using an aseptic microdroplet vitrification-warming system: live birth rate is determined by expansion rate on day 5 and influenced by morphological quality

Background and Aim: Selection of embryos on day-5/6 for transfer or cryopreservation is based on scoring systems including blastocyst expansion and morphology of the inner cell mass and trophectoderm. In this study we evaluated the performance of single vitrified blastocyst transfers (SVBT). Outcome parameters were positive hCG (hCG), miscarriage rate (MR), live birth rate (LBR) and neonatal outcome.

A Cross-Sectional Analysis of Live Birth Rates in Day 5 Fresh Versus Vitrified Single Blastocyst Transfer Cycles

Background: The use of frozen embryo transfers (FETs) in assisted reproduction has increased worldwide. Proponents of FETs suggest that controlled ovarian hyperstimulation (COH) in a fresh transfer impairs endometrial-embryo synchronicity, however there has been conflicting evidence on live birth rate (LBR) and clinical pregnancy rate (CPR) outcomes. Aim: To compare LBRs and CPRs between single autologous day 5 fresh versus vitrified blastocyst transfer cycles, in order to investigate the impact of COH on embryo-endometrium asynchrony. Method: A large cross-sectional analysis of 6,002 embryo transfers (ETs), comprising of 3774 fresh and 2228 FET cycles from 2016-2019. Inclusion criteria: first two stimulation cycles, single ET, and no pre-implantation genetic testing for aneuploidy (PGT-A). Multivariate analysis performed, also sub-group analysis for high-responders (&gt;20 oocytes collected). Results: Univariate analysis showed no absolute difference in LBR (28.3% vs 27.4%, p=0.43) and CPR (32.2% vs 30.9%, p=0.30), however multivariate analysis demonstrated significantly lower LBR (OR 0.864, p&lt;0.05, 95% CI 0.749-0.997) and CPR (OR 0.852, p&lt;0.05, 95% CI 0.742-0.979) in FET compared to fresh ETs. Younger patient age, previous IVF pregnancy, advanced blastocyst expansion, higher trophectoderm quality and lower cumulative number of ETs all improved the odds of both LBR and CPR (p&lt;0.001). Conventional IVF, rather than ICSI, improved CPR (p&lt;0.05) but not LBR (p=0.138). BMI affected neither LBR (p=0.492) nor CPR (p=0.359). In sub-group multivariate analysis of high-responders, no significant difference in LBR (p=0.439) or CPR (p=0.450) noted. Conclusion: This study demonstrates a relative higher LBR and CPR for fresh ETs compared to FETs, in multivariate analysis. Having a FET decreased the odds of LBR by nearly 14% compared to fresh ET, when adjusted for confounders. Our results suggest that universal freeze-all strategy, without appropriate indication, may lead to suboptimal outcomes. In high-responders, freeze-all cycles may be beneficial, as adjusted outcomes appear to be similar.

P-143 The initial blastocyst size after warming is a strong predictor for clinical pregnancy in single vitrified blastocyst transfer

Abstract Study question Which morphokinetic and morphological factors during embryo development before vitrification and after warming predict the chance of clinical pregnancy in single vitrified blastocyst transfer (SVBT)? Summary answer Time to reach blastocyst stage, numeric Gardner score before vitrification and blastocyst size after warming correlate with clinical pregnancy but not post-warming expansion rate. What is known already Several studies have evaluated pre-vitrification and post-warming characteristics of vitrified-warmed blastocysts to identify parameters that correlate with implantation. New pre-vitrification and post-warming morphokinetic parameters were identified by culturing embryos in time-lapse incubation systems before vitrification as well as after warming. Among others, the number of collapses before vitrification, the size after warming in combination with the post-warming expansion rate and the maximal expansion size was reported to have a significant correlation with implantation. To our knowledge these findings were so far not re-evaluated in another, independent study. Study design, size, duration Analysis of 549 treatment/cryo cycles in 349 patients participating in a multicenter observational trial (IMBOS trial). Subjects received one controlled ovarian stimulation with follitropin delta (Rekovelle). Of 377 transfers, 195 were fresh day 5 single blastocyst transfers and 182 were SVBTs from blastocysts that were vitrified on day 5. Both fresh and vitrified-warmed embryos were cultured in a time-lapse system. Various morphokinetic parameters, including Gardner score, were assessed in the fresh cycle and after vitrification-warming. Participants/materials, setting, methods Associations between clinical pregnancy (positive fetal heartbeat), morphokinetic parameters, blastocyst collapse, KIDScore D5, Gardner score before vitrification, blastocyst size after warming, re-expansion rate, and Gardner score before SVBT were investigated. Mixed logistic regression models were used with clinical pregnancy as dependent variable and different morphokinetic/morphological parameters as factors or covariates. Repeated transfers were accounted for by including subject as a random factor in models, with the log-odds for different subjects assumed to be normally distributed. Main results and the role of chance A total of 182 SVBT resulted in 89 clinical pregnancies. For pre-vitrification parameters no clear association was found between the number of collapses (P = 0.6095) or the maximal collapse size (P = 0.9122) before vitrification and clinical pregnancy. Multifactorial analysis of Gardner score before vitrification only showed significant association with clinical pregnancy for trophectoderm grading (P = 0.0247), and not for expansion and hatching status and inner cell mass. There was no significant association for any of the Gardner score parameters when assessed immediately before SVBT. However, when the Gardner score was transformed into a total numerical score, significant associations with clinical pregnancy was noted both when assessed before vitrification (P = 0.0003) and before cryotransfer (P = 0.0005). From the analysis of time-lapse parameters, the time to reach blastocyst (tB-tPNf; P = 0.0288) and KIDScore D5 (P = 0.0035) were significantly associated with clinical pregnancy. Multifactorial analysis showed that size after warming was significantly associated with clinical pregnancy (P = 0.0257), but not rate of expansion (P = 0.4241) or maximal expansion size (P = 0.1789). The prognostic potential of trophectoderm grading and KIDScore D5 was previously reported. Further analysis showed a positive correlation between numeric Gardner score before vitrification and size after warming, but not between time to reach blastocyst (tB-tPNf) and size after warming. Limitations, reasons for caution Blastocysts for SVBT were selected by each laboratorýs policy, which may constitute a selection bias. The proposed factors were derived by statistical analysis of a fixed dataset; offset thresholds for any of the parameters were not defined. Any potential clinical application must be tested and validated on an independent dataset. Wider implications of the findings Selection for which vitrified blastocyst to warm first may benefit from using tB-tPNf rather than Gardner score. Size after warming does constitute an early post-warming parameter if a decision is needed to eventually warm another blastocyst. Trial registration number NCT03697031

P-193 Using blastocyst re-expansion rate in the decision-making when to warm a new blastocyst for optimal single vitrified-warmed blastocyst transfer

Abstract Study question Can post-warmed blastocyst re-expansion rate be used to identify embryos with low likelihood of live birth (LB) in single vitrified blastocyst transfer (SVBT)? Summary answer A statistical model using blastocyst re-expansion rate and Gardner score can be used to identify blastocysts that do not result in live birth. What is known already The ability to identify the most viable embryo with greatest chance of implantation and LB is crucial. As embryo development is a dynamic process, research has recently focused on finding the most predictive morphokinetic parameters to incorporate in the assessment of which embryos to select for transfer. Time-lapse systems allow continuous monitoring of the embryo while cultured uninterruptedly. Several studies have found that the degree of blastocoele expansion and re-expansion are significant parameters for prediction of LB. Furthermore, re-expansion rate has been demonstrated to correlate with pregnancy outcomes in SVBT. Study design, size, duration This study is a retrospective cohort study including a total of 931 SVBT´s at the Fertility Clinic at Horsens Regional Hospital in a 2-year period from 2019-2020. Prior to vitrification, laser assisted collapsing was performed on all blastocysts with an expansion grade ≤ 4, and grade 5 after individual assessment. Only blastocysts with data on both pre-vitrification Gardner score, LB outcome and at least two hours of post-warmed time-lapse data were included in the analysis. Participants/materials, setting, methods Using a time lapse system, both fresh and post-vitrification embryo developmental parameters were evaluated during culturing in an EmbryoScope (Vitrolife, Denmark). Re-expansion rate was calculated as the change in blastocyst area within the first two hours. Logistic regression models were used to estimate the relation between LB and pre- and post-vitrification variables. Based on model predictions, the diagnostic performance was evaluated at thresholds corresponding to a 1%, 2% and 5% false negative rate (FNR). Main results and the role of chance Of the 931 SVBT´s, a total of 388 resulted in a LB (41.7%). Using the initial blastocyst size and the two-hour re-expansion rate yielded a model with an area under the ROC curve (AUC) of 0.61 (95% bootstrap CI: 0.57-0.65) Adding pre-vitrification Gardner score to the model increased the AUC to 0.62 (95% bootstrap CI: 0.59-0.66). This model was used to calculate the true negative rate (TNR), being the proportion of transferred blastocysts that would not result in LB at each threshold. At a FNR threshold of 2%, the model predicts that out of 50 discarded blastocysts, 7 would have resulted in a LB and 43 would not have resulted in LB. In summary, the ratio of erroneously discarded embryos vs. correctly discarded embryos would be approximately 1:6 using a 2% FNR. Limitations, reasons for caution The statistical model was developed and tested on the same dataset with a risk of overfitting. Any potential clinical application of the model requires testing and validation on an independent dataset. Furthermore, clinical procedures and in particular laser collapse may bias the results. Wider implications of the findings Being able to identify blastocysts with a low likelihood of a LB allows for the possibility to thaw a new blastocyst. In a clinical setting, this could lead to a reduced time to pregnancy and LB in women who have multiple vitrified blastocysts. Trial registration number 1-16-02-583-20

P-757 Clinical and neonatal outcome of single vitrified-warmed blastocyst transfer with complete zona pellucida removal using laser-assisted hatching

Abstract Study question Complete zona pellucida (ZP) removal using laser assisted hatching can improve clinical and neonatal outcome in single vitrified-warmed blastocyst transfer (SVBT) cycle? Summary answer Implantation rate, clinical pregnancy rate and live birth rate can be improved by complete removal of zona pellucida, whereas no impact on neonatal outcomes. What is known already Assisted hatching (AH) technique widely used in assisted reproductive technology for improving clinical outcome in frozen embryo transfer (FET), which could impair successful embryo hatching out of the zona pellucida (ZP) due to ZP hardening during vitrification-warming. The zona thinning and drilling methods of AH doesn’t affect clinical, live births outcomes and it could increase monozygotic twin birth, risk of placenta associated diseases. But recent studies showed that the complete removal of ZP may increase the chance of blastocyst adhesion and outgrowth afterwards, which required to be proven. Study design, size, duration Retrospective study data collected from July 2015 to November 2019 included SVBT in the Ojinmed IVF center. We divided into 2 groups as follows: case group 160 (randomly selected complete removal of ZP) and control group 160. The clinical outcomes were implantation rate (IR), clinical pregnancy rate (CPR), live birth rate (LBR), miscarriage and neonatal outcomes including monozygotic twin, birth baby sex, birth weight, height and gestational weeks. All parameters were compared between two groups. Participants/materials, setting, methods 320 vitrified blastocyst transfer cycles in 215 patients underwent a clomiphene-based minimal ovarian stimulation protocol or drug-free natural cycles. All embryos were vitrified-warmed by Cryotop method (Kitazato, Japan). Complete removal of ZP selected an survived blastocysts after warming and used an immediate laser drilling (Laser: 1.8-2.2 ms, Octax NaviLaseTM) in perivitelline space. And gently glass pipetting done for complete removal of ZP. Statistical analysis performed by Chi-square test and logistic regression analysis using STATA 12. Main results and the role of chance A total 320 SVBT cycles, including ZP completely removed 160 cycles and control 160 cycles. In analysis of the patient's age, fertility causes and fertilization methods were not significant (p &amp;lt; 0.05) between two groups. The IR (55.2 % and 66.2 %, p &amp;lt; 0.01), CPR (39.3 % and 52.5 %, p = 0.01) and LBR (29.3 % and 43.7 %, p &amp;lt; 0.01) are highly correlated between the control and the completely removal of ZP group, respectively. In multivariable logistic regression analysis showed the completely removal of ZP group had significance for IR (aOR 0.54, CI 95 % 0.31-0.93, p = 0.02), CPR (aOR 0.56, CI 95 % 0.32-0.96, p = 0.03) and LBR (aOR 0.56, CI 95 % 0.32-0.99, p = 0.048), which adjusted by patient age, blastocyst grade and vitrification day. Miscarriage rate didn’t show significance (23.8% and 21.4%, p = 0.73) between groups. The neonatal outcomes including monozygotic twin, sex of the births, average gestation week and birthweight were non significant (p &amp;lt; 0.05) between two groups (49 for control group and 66 for case group) children. 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 complete removal of ZP using laser assisted can improve clinical outcomes including implantation, clinical pregnancy and live birth after SVBT. And it may doesn’t affect to miscarriage rate and neonatal outcome. Trial registration number not applicable

Natural cycles achieve better pregnancy outcomes than artificial cycles in non-PCOS women undergoing vitrified single-blastocyst transfer: a retrospective cohort study of 6840 cycles.

To identify the optimal method for endometrial preparation in vitrified single-blastocyst transfer (VSBT) cycles. This was a retrospective cohort study for non-PCOS patients who underwent VSBT cycles from March 2015 to November 2019 in an academic reproductive medical center. A total of 6840 VSBT cycles were enrolled and classified into two groups according to different endometrial preparation methods. The non-PCOS patients who underwent VSBT showed a significantly higher clinical pregnancy rate (61.96% vs 56.85%, p < 0.001) and live birth rate (49.09% vs 39.86%, p < 0.001), as well as a statistically lower early miscarriage rate (12.02% vs 18.08%, p < 0.001) in the natural cycle (NC) group compared with the artificial cycle (AC) group. Multivariable analysis further confirmed that NC was associated with an increased likelihood of clinical pregnancy (adjusted odds ratio(aOR) 0.852, 95% confidence interval (CI) 0.765-0.949, p = 0.004) and live birth (aOR0.746, 95% CI 0.669-0.832, p < 0 .001), but decreased early miscarriage occurrence (aOR 1.447, 95% CI 1.215-1.724, p < 0.001) compared to AC. Our study demonstrated that non-PCOS patients could benefit from NC in vitrified blastocyst transfer. Increased clinical pregnancy rate and decreased early miscarriage rate led to a significantly higher live birth rate in NC patients compared with AC with our present protocol.

The vitrification system may affect preterm and cesarean delivery rates after single vitrified blastocyst transfer

ABSTRACT The purpose of this study was to investigate the possible effects of different vitrification systems on single vitrified blastocyst transfer cycles. The clinical and birth outcomes of 412 patients who underwent single vitrified blastocyst transfer between January 2018 and June 2020 were retrospectively analyzed and compared between patients who underwent blastocyst vitrification with kit A (group A, 196 patients) and those who underwent blastocyst vitrification with kit B (group B, 216 patients). Clinical outcomes, including the clinical pregnancy rate, ongoing pregnancy rate, early miscarriage rate, late miscarriage rate, ectopic pregnancy rate, twin pregnancy rate, and induced labor rate due to fetal malformation, were not significantly different between the two groups (P > 0.05). The preterm delivery rate among singleton newborns (11.57% vs. 3.23%, P < 0.05) and the cesarean delivery rate were significantly higher in group B than in group A (70.25% vs. 57.26%, P < 0.05). Birth outcomes, including the male-to-female ratio, low-birth-weight rate, macrosomia rate, birth defect rate, newborn gestational age, neonatal body weight, and singleton neonatal body length, were not significantly different (P > 0.05). Our findings suggest that different vitrification systems might differentially affect birth outcomes. Such disparity could reflect differences in kit composition and/or protocol. ABBREVIATIONS: DMSO: dimethyl sulfoxide; ES: equilibration solution; VS: vitrification solution; BMI: body mass index; ICSI: intracytoplasmic sperm injection; OR: odds ratio; CI: confidence interval

Successful birth after ICSI with testicular immotile spermatozoa from a patient with total MMAF in the ejaculates: a case report.

There has been no report on the outcome of vitrified blastocyst transfer from a vitrified oocyte injected with immotile testicular spermatozoa with only multiple morphological abnormalities of the sperm flagella (MMAF). A couple diagnosed with MMAF returned to the clinic to attempt pregnancy using their vitrified oocytes. Testicular spermatozoa were injected intracytoplasmically, and the following intracytoplasmic sperm injection results were observed. In the second cycle, surplus vitrified oocytes and testicular retrieved sperm were used, but no pregnancy ensued. In the third cycle, a surplus vitrified blastocyst was transferred, and a healthy female child was delivered, with a birth weight of 3050 g and a birth length of 53 cm. In this report we describe a successful pregnancy achieved in a patient presenting MMAF. The successful pregnancy was obtained from vitrified oocytes microinjected with testicular retrieved sperm in a vitrified blastocyst transfer.

P–221 Prospective randomized sibling study on gamete preparation, insemination and subsequent culture of human oocytes in a time-lapse system using media systems with and without antioxidants

Abstract Study question Does the addition of antioxidants for gamete preparation, insemination and embryo culture lead to differences in embryo development and clinical outcome Summary answer Using an antioxidant-containing media system for sperm preparation, insemination and embryo culture imparts significantly higher good-quality blastocyst rates and improved clinical outcome in elderly patients. What is known already A previous study showed that adding combined antioxidants for sequential embryo culture in conventional incubators (interrupted culture) improves embryo viability and clinical outcome, especially for elderly patients. Here we investigated the combined effect of three antioxidants Acetyl-L-Carnitine (10 µM), N-Acetyl-L-Cysteine (10 µM), and α-Lipoic Acid (5 µM) during sperm preparation, insemination, and time-lapse culture in a single step medium on human embryo development and clinical outcome. Study design, size, duration Prospective randomized single center study including 143 couples for IVF/ICSI between August 2018 and December 2019. Inclusion required at least eight cumulus-oocyte-complexes (COCs) after retrieval. Cycles involving PGT, split IVF/ICSI, and surgically retrieved sperm were excluded. Immediately after retrieval oocytes were randomly distributed to a study or control media system with or without antioxidants (Vitrolife). Similarly, ejaculates were split and prepared with and without antioxidants. Participants/materials, setting, methods Sibling oocytes were inseminated in the respective group with accordingly prepared sperm. Single step embryo culture was conducted in medium with (Gx-TL) and without (G-TL) antioxidants in the EmbryoScope+. Embryo quality and clinical outcome were assessed in relation to maternal age (&amp;lt;35/&amp;gt;35 years). Good-quality embryos on day 3 were defined as 8- to 10-cells with even cells and low fragmentation; good-quality blastocysts as &amp;gt; 3BB. Clinical outcome was assessed after single vitrified blastocyst transfer (SVBT). Main results and the role of chance From 143 participants (female age, 34.7±3.2 years), a total of 2424 COCs were collected; 1180 COCs/916 metaphase-II (MII) oocytes were allocated to Gx-TL media and 1244 COCs/981 MII oocytes to G-TL media. Age-related analysis in Gx-TL compared with G-TL in relation to allocated MII oocytes revealed a trend for higher fertilization rates in Gx-TL for both age groups (&amp;lt;35: 72.1% vs. 66.9%; &amp;gt;35: 70.7% vs. 64.9%, P &amp;lt; 0.1). Good-quality day 3 embryo development/MII oocytes was higher, albeit not significant, in the elderly patients in Gx-TL (&amp;lt;35: 35.9% vs. 34.4%; &amp;gt;35: 31.1% vs. 27.9%). Overall day 5/6 blastocyst rate was similar for both media (&amp;lt;35: 48.2% vs. 49.9%; &amp;gt;35: 42.3% vs. 39.5%). Day 5/6 GQB rate was comparable for younger patients (&amp;lt;35: 23.8% for Gx-TL vs. 26.0% for G-TL) but significantly higher in Gx-TL in elderly patients (&amp;gt;35: 20.7% vs. 14.4%; P &amp;lt; 0.05). A total of 200 SVBT were performed; 99 in the Gx-TL- and 101 in the G-TL-arm. We noted almost similar implantation and ongoing pregnancy rates between Gx-TL vs G-TL in the younger (&amp;lt;35) age group (50.0% vs. 55.4%; 50.0% vs. 55.6%) but higher albeit not significant rates for Gx-TL in older (&amp;gt;35) patients (44.1% vs. 33.3%; 44.1% vs. 33.3%). Limitations, reasons for caution In almost 95% of the cycles, oocytes were inseminated by ICSI; thus results may not equally apply for cycles with IVF. The use of a closed time-lapse system may have prevented from some environmental oxidative stress. Therefore results may come out different with a similar study using standard incubation. Wider implications of the findings: Supplementation of antioxidants to media for gamete isolation and preparation, as well as subsequent single step time-lapse culture may improve GQE/B rates and clinical outcomes in certain age groups, plausibly through the reduction of oxidative stress. Further studies in selected sub-groups (severe OAT syndrome / testicular cases) may be indicated. Trial registration number UMIN000034482