Vitrified-warmed Embryo Transfer Research Articles

Is the Human Chorionic Gonadotropin Level on Day 10 After Cleaved Embryo Transfer a Predictor of Perinatal Outcome?: A Retrospective Cohort Study.

This study aimed to determine whether human chorionic gonadotropin (hCG) levels at day 10 after day 2 cleaved embryo transfer can predict pregnancy and perinatal outcomes. Patients who underwent oocyte retrieval with minimal stimulation or natural cycles and fresh or vitrified-warmed transfer of a single, day 2 cleaved embryo at our clinic between November 2018 and December 2020 were included in this study. Patients were classified into four age groups for oocyte retrieval and into ten groups based on the hCG level on day 10 after embryo transfer; pregnancy complications and delivery and neonatal outcomes were examined. Of the 5,840 cycles, 3,722 (63.7%) and 2,118 (36.3%) were fresh-cleaved and vitrified-warmed embryo transfers, respectively. The mean hCG level was 24.8 mIU/mL and the clinical pregnancy and live birth rates per transfer were 29.6% and 23.4%, respectively. Maternal age at the time of oocyte retrieval, husband's age, treatment cycle, embryo type and grade, cell number, and hCG levels were correlated with pregnancy and delivery outcomes in the univariate analysis. Conversely, only maternal age and hCG levels were correlated with the outcomes in the multivariate analysis. hCG levels on day 10 post-transfer are a useful predictor of pregnancy and delivery outcomes after cleaved embryo transfer. Live birth rates vary with maternal age, even when hCG levels are the same, but they do not vary according to the treatment cycle or type of embryo transferred. Low hCG levels may be associated with vasa previa but did not affect delivery outcomes.

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.

A model for predicting low progesterone levels on the day of embryo transfer in hormonally prepared vitrified-warmed embryo transfer cycles: A secondary analysis of a prospective cohort study.

To develop a model that predicts low progesterone (P) levels on the day of embryo transfer (ET) based on patient and cycle characteristics, including serum estradiol (E2 ) concentration after vaginal administration of micronized E2 for endometrial preparation. A prospective cohort of 193 patients scheduled for cryopreserved blastocyst transfer during an artificial endometrial preparation cycle, using micronized E2 vaginally for first 4 days (4 mg/day), followed by oral administration (6 mg/day). Blood sampling for E2 was performed prospectively on day-5 of vaginal administration and analysis was performed retrospectively. On sixth day of P treatment (daily 300 mg of vaginal micronized P tablets), P levels were measured on ET day. Primary outcome measure was serum P levels after vaginal E2 administration. Patients with low P levels on the day of ET (<7.8 ng/mL, 25th percentile) were heavier (p < 0.001) and exhibited lower day 5 serum E2 levels (p < 0.001) compared with patients with adequate P levels. Multivariate linear regression analysis revealed that weight (p = 0.003) and day 5 E2 levels (p < 0.001) were independently associated with the P levels. The sensitivity, specificity, and positive and negative predictive values (%) were 71.4, 55.6, 35.4, and 85.1 for weight ≥ 65 kg; 71.4, 61.8, 38.9, and 86.4 for day 5 E2 ≤ 1615 pg/mL; 59.2, 83.3, 54.7, and 85.7 for the combination of these two variables; and 82.9, 62.5, 54.7, and 87.0 for the sequential inclusion of these variables, respectively. Low day 5 E2 levels following vaginal administration and high weight are independently associated with low P levels on the day of ET. Predictive performance is enhanced when these variables are considered sequentially or in combination.

Effect of exposed-to-air frequency of cryopreserved embryo on clinical outcomes of vitrified-warmed embryo transfer cycles: a retrospective analysis of 9,200 vitrified-warmed transfer cycles

BackgroundCryopreservation of embryos plays a major role in the in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. However, the storage condition of the cryopreserved embryo can change temporarily due to repeated retrieval of the embryo from the liquid nitrogen (LN2) tank during the practical application during cryopreservation. Whether the implantation potential of a cryopreserved embryo will be damaged when the cane containing it is temporarily exposed to air due to the transfer between the LN2 tank and LN2 container is yet to be elucidated. Also, whether the exposed-to-air frequency (EAF) of cryopreserved embryos influences the clinical outcomes is unclear.ObjectiveTo investigate whether the EAF of cryopreserved embryo affects the clinical outcomes of vitrified-warmed embryo transfer.MethodsA total of 9200 vitrified-warmed embryo transfer cycles were included in this study. All cycles were divided into five groups according to different EAFs (2, 4, 6, 8, or ≥ 10). Post-warming survival rates and clinical outcomes, including implantation, clinical pregnancy and live birth rates were investigated. Kruskal–Wallis test and Pearson’s chi-squared tests were used to compare the patient characteristics and clinical outcomes among the five groups. Furthermore, multivariate logistic regression analyses were conducted to investigate the association between EAF and clinical outcomes.ResultsNo significant differences were observed in the positive HCG rate, implantation rate and live birth rate (P > 0.05) among five EAF groups with respect to D3 embryo, D5 blastocyst and D6 blastocyst. Post-warmed survival rate of D3 embryos (P = 0.015) differed significantly among the five EAF groups, but it was not EAF-dependent. Although clinical pregnancy was different among the five groups with respect to D5 blastocyst (P = 0.042), multivariate logistic regression analysis adjusted for confounding variables suggested that EAF did not adversely affect clinical pregnancy or live birth.ConclusionThese findings indicated that human vitrified embryos in the open system could be repeatedly retrieved from the LN2 tank without affecting the implantation potential of the embryo.

P-190 Spontaneous blastocyst collapse during pre-vitrification equilibration is related to a lower live birth rate: a prospective cohort study

Abstract Study question This report provides updated data from a study investigating the association between the spontaneous collapse patterns of blastocysts during pre-vitrification equilibration and pregnancy success. Summary answer Live birth rates were lower for completely collapsed blastocysts, suggesting the possibility for including blastocyst collapse pattern as a criterion for selecting embryos for transfer. What is known already Previous time-lapse studies have found that blastocysts exhibiting strong contraction during culture have a low probability of hatching in animals and implanting in humans, suggesting blastocyst contraction has a negative impact on reproductive outcomes. Conversely, artificial shrinkage of human blastocysts before vitrification is considered to improve embryo survival and/or pregnancy rates by promoting cryoprotectant permeation inside the blastocoel. However, shrinkage is not induced in some vitrification protocols, including ours, as the viability is high; this demonstrates the potential of existing protocols to achieve sufficient cryoprotectant permeation. Moreover, the Alpha consensus meeting on cryopreservation did not issue recommendations regarding artificial shrinkage. Study design, size, duration This study included 798 patients who were undergoing their first autologous IVF/ICSI cycle followed by a freeze-all strategy, including blastocyst vitrification, at our clinic between June 2018 and November 2021. For patients with multiple vitrified blastocysts, embryos for transfer were selected hierarchically based only on morphological scoring during culture. To reduce bias, only data from a single blastocyst (day 5, Gardner score 4, excluding CC) transfer from each patient’s first warmed cycle was analyzed. Participants/materials, setting, methods Blastocysts were vitrified-warmed in in-house-prepared solutions using Rapid-i carriers. Prior to vitrification, intact blastocysts were equilibrated in 10% ethylene glycol (15 min, 37 °C). Their spontaneous collapse patterns were assessed under an inverted microscope before they were vitrified in 15% ethylene glycol + 15% dimethylsulfoxide + 0.5 M sucrose. Blastocyst collapse was defined as the separation of the trophectoderm cells from the zona pellucida. Collapsed blastocysts with/without a blastocoel cavity were defined as partially/completely collapsed blastocysts. Main results and the role of chance Non-collapsed (Nc), partially collapsed (Pc), and completely collapsed (Cc) blastocyst transfers (embryo survival rate, 99.6%) constituted 27.1%, 54.6%, and 18.3% of the 798 cycles, respectively. Live birth rates differed significantly between groups (Nc, 54.2%; Pc, 46.6%; Cc, 34.2%; P &amp;lt; 0.001). Regarding perinatal outcomes of 366 singletons, the female rate tended to differ between groups (Nc, 35.3%; Pc, 44.3%; Cc, 53.1%; P = 0.084). Significant differences in maternal age (years: Nc, 34.6 ± 4.0; Pc, 35.4 ± 3.7; Cc, 36.4 ± 4.0; P &amp;lt; 0.001), MII oocyte number (Nc, 13.0 ± 6.2; Pc, 12.0 ± 6.6; Cc, 10.8 ± 6.9; P = 0.009), blastocyst diameter (μM: Nc, 184 ± 18; Pc, 181 ± 20; Cc, 173 ± 23; P &amp;lt; 0.001), and good-quality (4AA/4AB/4BA) blastocyst percentage (Nc, 70.4%; Pc, 61.2%; Cc, 47.9%; P &amp;lt; 0.001) were found between groups. Upon logistic regression analysis including these potential confounders, only maternal age (odds ratio (OR), 0.89; 95% confidence interval (CI), 0.86–0.93; P &amp;lt; 0.001) and collapse pattern (Cc/Nc: OR, 0.59; 95% CI, 0.37–0.93; P = 0.023) significantly impacted the live birth rate, whereas blastocyst morphology (good/not: OR, 1.37; 95% CI, 0.97–1.94; P = 0.069) tended to impact it. Limitations, reasons for caution The cryoprotectant concentration of our pre-vitrification equilibration solution is relatively low. Thus, caution is required when assessing the collapse pattern, as the solution osmolarity can influence blastocyst shrinkage. Follow-up studies with more participants are warranted to confirm these results and the health of the children born after vitrified-warmed embryo transfers. Wider implications of the findings A negative relationship was found between spontaneous blastocyst collapse during pre-vitrification equilibration and live birth rate. Studying blastocyst collapse pattern may assist selection of the most viable blastocysts after vitrification in ethylene glycol and dimethylsulfoxide—a widely used permeable cryoprotectant combination—thus, increasing IVF success rates. Trial registration number Not applicable

P-230 Does laser-assisted hatching improve pregnancy rates : a retrospective analysis

Abstract Study question Is there any effect of laser-assisted zona drilling in improving pregnancy outcomes in women undergoing vitrified-warmed embryo transfers? Summary answer Our data suggests that there is evidence of possible improvement in pregnancy rates with laser-assisted hatching of embryos among women &amp;gt;40 years of age. What is known already Mammalian embryo is surrounded by a glycoprotein shell called zona pellucida. The embryo must hatch out of this shell for it to implant in the uterus. However, certain factors such as sub-optimal in vitro culture conditions, advanced maternal age, zona-hardening due to vitrification and warming, among others, negatively impact the ability of the embryo to escape from the matrix surrounding it. Laser-assisted hatching is the safest and easiest technique available to overcome the problem of hatching by artificially disrupting the zona, though there isn’t sufficient data available on whether this has any actual benefit or is just a fancy add-on. Study design, size, duration Retrospective cohort study comparing embryo transfer results of 88 women whose embryos underwent LAH prior to transfer with that of a control group of women. Data was collected from embryo transfers that took place between March 2021 to December 2022. Participants/materials, setting, methods Beta-hCG results of women (N = 88) whose embryos underwent LAH prior to transfer was compared with the results of transfer without LAH in women (N = 88) of similar age and clinical history. Patient age, clinical indications and embryo grades were comparable across both groups. LAH was performed immediately post-warming of blastocysts using a 1.48 µm diode to drill the zona at a point opposite the ICM. Data was analyzed using independent t test and chi square test. Main results and the role of chance Women from both test and control groups were further stratified into the following sub-groups based on age – A (25-29 years), B (30-34 years), C (35-39) and D ( &amp;gt;40 years). Beta-hCG results were assessed and was found to be lower in sub-groups A (57.1% vs. 78.5%) and B (46.4% vs. 67.7%) of the test group as compared to control. It was marginally higher in test group C as compared to that of control (50% vs. 47.3%). However, we found an increase in pregnancy outcomes of test sub-group D in comparison to control (83.3% vs. 20%). Limitations, reasons for caution This is a retrospective, preliminary study to assess initial results of effect of LAH on vitrified-warmed embryo transfer outcomes. A prospective study with a larger sample size is needed to confirm our results in both frozen and fresh embryos. Wider implications of the findings Our results indicate that laser-assisted hatching does not provide any advantage except possibly in older women above the age of 40 years. Thus we can select the exact group of patients who would benefit with this add-on and offer it to them routinely to improve their pregnancy rates. Trial registration number Not applicable

P-630 Comparison of follitropin delta versus follitropin alfa in progestin-primed ovarian stimulation in IVF

Abstract Study question Is there a difference in a clinical outcome between using follitropin delta and follitropin alfa undergoing a progestin primed ovarian stimulation (PPOS) protocol? Summary answer There was no difference between follitropin delta and follitropin alfa in terms of pregnancy rates. What is known already Follitropin delta (REKOVELLE, Ferring Pharmaceuticals, Switzerland) is the first recombinant human follicle-stimulating hormone (FSH) using the algorithm-based individualized dosing regimen while reducing the risk of ovarian hyperstimulation syndrome (OHSS) as compared with conventional dosing strategies. The efficacy and safety of follitropin delta has been demonstrated in randomized controlled trials (RCTs). Study design, size, duration We performed a retrospective analysis of 645 patients who underwent ovarian stimulation by a PPOS protocol using follitropin delta or follitropin alfa between April 2022 and August 2022. (325 patients using follitropin delta and 320 patients using follitropin alfa, respectively.) Participants/materials, setting, methods 325 cycles of women who used follitropin delta were compared to 320 cycles of women who used follitropin alfa. The following outcomes were analysed; fertilized rate, 2 pronuclear embryo rate, good quality embryo rate, blastocyst rate, good quality blastocyst rate. A total of 499 women who underwent embryo transfer in cryo cycles using natural cycle or hormone replacement cycle were analysed about biochemical pregnancy rate, clinical pregnancy rate. Main results and the role of chance Baseline demographics for 325 women in follitropin delta group and 320 women in follitropin alfa were: age, 35.1±4.3 years and 35.2±4.3 years; number of previous IVF cycles, 1.6±1.6 and 1.7±1.7, respectively. The laboratory data were: fertilization rate, 81.4% and 80.1%, 2 pronuclear embryo rate, 73.9% and 71.5%, good quality cleavage stage embryo rate, 59.4% and 55.9%, blastocyst rate, 58.9% and 55.7%, good quality blastocyst rate, 41.3% and 38.7%, respectively. Only good quality cleavage stage embryo rate and good quality blastocyst rate were significantly higher (P &amp;lt; 0.05) in follitropin delta group. A vitrified-warmed embryo transfer was performed for 257/325 women (79.1%) and 242/320 women (75.6%) and subsequent positive β-human chorionic gonadotropin (βhCG) reported for 218/621 cycles (35.1%) and 194/570 cycles (34.0%), respectively. Ongoing pregnancy rate after embryo transfer were 19.2% (n = 119) and 19.6% (n = 112) and cumulative ongoing pregnancy rate were both 46.3%. Mild OHSS was reported for 131 women (40.3%) and 170 women (53.1%), respectively (P = 0.07). Limitations, reasons for caution This is a non-controlled, retrospective study. Wider implications of the findings The present study shows that in addition to reducing the OHSS risk, follitropin delta in its individualised fixed-dose regimen has the potential to improve the quality of embryos although there was no significant difference in clinical outcomes. Trial registration number not applicable

P-773 No impact of cleavage-stage or blastocyst-stage embryo biopsy on growth and health in children up to 2 years of age

Abstract Study question Does embryo biopsy for preimplantation genetic testing (PGT) followed by fresh or vitrified-warmed embryo transfer affect children’s health up to 2 years of age? Summary answer Cleavage- or blastocyst-stage embryo biopsy followed by fresh or vitrified-warmed-embryo transfer had neither impact on anthropometry at birth, infancy or childhood nor on health outcomes. What is known already Literature data regarding neonatal outcomes after embryo biopsy for PGT show mixed results due to small sample sizes and/or the heterogeneity in terms of embryo biopsy (cleavage- or blastocyst-stage) and type of embryo transfer (fresh or frozen-thawed). Even fewer data exist on the impact of embryo biopsy on children’s health beyond infancy, including growth. Study design, size, duration This single-center cohort-study compared outcomes in singletons conceived after cleavage- or blastocyst-stage embryo biopsy either in a fresh or vitrified-warmed transfer cycle with results after non-biopsied embryo transfer between 2014 and 2018. Pregnancies after IVM, oocyte vitrification or oocyte/embryo donation were excluded. Eligible singletons living in Belgium were invited for examination at 3-6 months and 2 years. Anthropometric measurements at birth, infancy and childhood and health outcomes including surgeries, medication use and hospitalisations are reported. Participants/materials, setting, methods Birth characteristics were available for 630 and 222 children after cleavage-stage and blastocyst-stage embryo biopsy and for 1532 children after transfer of a non-biopsied embryo. Follow-up data were available for 426, 131 and 662 children, respectively. The impact on children’s health following embryo biopsy in vitrification and fresh cycles was the primary outcome. Other outcomes were the impact of timing of biopsy and of vitrification. Subgroup analysis according to infertility background was additionally performed. Main results and the role of chance Regarding the impact of embryo biopsy, either at the cleavage or blastocyst stage, no differences in anthropometrics were found at birth, infancy or childhood in vitrified-warmed transfer cycles compared to outcomes in non-biopsied vitrified-warmed transfer cycles, even after adjustment for neonatal, treatment and maternal characteristics. Likewise, no impact of embryo biopsy (cleavage stage) was found in fresh transfer cycles. The timing (day 3 or day 5/6) of embryo biopsy did not affect anthropometrics at birth, infancy or childhood. However, children born after cleavage-stage embryo biopsy followed by a vitrified-warmed transfer cycle had larger birth sizes than children born after cleavage-stage biopsy followed by a fresh transfer. Weight and height gain from birth to infancy and from infancy to early childhood were comparable in all biopsied and non-biopsied groups. Reassuringly, comparable rates of major congenital malformations, (severe) developmental problems, hospital admissions, surgical interventions and of chronic medication use up to the age of 2 years were observed in biopsied and non-biopsied groups. Subgroup analysis in children born to parents with an infertility diagnosis showed that birth parameters were not different in children born after embryo biopsy. Limitations, reasons for caution As the majority of the PGT cycles in our center are performed for a genetic indication only, the number of children born after embryo biopsy in infertile parents is rather limited and the results should therefore be interpreted cautiously. Wider implications of the findings Our findings add reassurance that embryo biopsy does not adversely affect the health of offspring up to 2 years of age. This is of particular importance given the widespread shift to blastocyst-stage embryo biopsy in PGT. Trial registration number Not applicable

P-472 Endometrial stimulation with culture medium containing granulocyte macrophage colony-stimulating factor promotes embryo implantation after vitrified-warmed blastocyst transfer

Abstract Study question Does endometrial stimulation with culture medium containing granulocyte macrophage colony-stimulating factor (GM-CSF) promote implantation potential of vitrified-warmed blastocysts? Summary answer Endometrial stimulation with culture medium containing GM-CSF prior to transfer of vitrified-warmed blastocysts may promote embryo implantation and improve pregnancy continuation. What is known already Human embryonic development and implantation in vivo is precisely regulated by various cytokines, including growth factors. GM-CSF is a cytokine that plays an important role in reproductive function. We reported the possibility that the use of culture medium containing GM-CSF in cleavage stage vitrified-warmed embryo transfers (ETs) promotes embryo implantation (ESHRE2018). However, it has been reported that endometrial stimulation with culture supernatant up to blastocyst stage (SEET method) improves embryo implantation, but the effect of endometrial stimulation with culture medium containing GM-CSF (G-SEET method) on embryo implantation is not clear. Study design, size, duration This study was conducted at a single in vitro fertilization (IVF) center from June 2020 to February 2022. The G-SEET procedure was performed at each patient’s request. All study participants provided informed consent, and the study design was approved by the ethics committee of IVF Nagata Clinic, Fukuoka, Japan. Participants/materials, setting, methods We examined 702 cycles of vitrified-warmed blastocyst transfer. Warmed blastocysts were incubated in recovery culture for several hours and transferred on day 5 (control group). For the G-SEET method, only culture medium containing GM-CSF was injected into the uterus two days before ET, which was performed as in the control group (G-SEET group). The human chorionic gonadotropin (hCG) positive, clinical pregnancy, miscarriage, and ongoing pregnancy rates were compared between the two groups. Main results and the role of chance Of the 702 cycles of single vitrified-warmed blastocyst transfers, the control and G-SEET groups had 459 and 243 cycles, respectively. The G-SEET group had a higher patient age (37.5±4.2 vs. 36.2±3.8, p &amp;lt; 0.01), a thinner endometrial thickness (10.3±1.9 vs. 10.9±2.0, p &amp;lt; 0.01), a lower percentage of good blastocyst transfers (65.4% vs. 79.5%, p &amp;lt; 0.01), and a higher cumulative number of implantation failures (3.6±2.5 vs. 1.9±1.9, p &amp;lt; 0.01) than the control group. The G-SEET method was performed at the patient’s request, so the number of cycles and patient background varied. Multivariate analysis was performed using female age, male age, number of miscarriages, endometrial thickness, presence of good blastocysts, and cumulative number of implantation failures as confounding factors. Compared with the control group, the G-SEET group exhibited the following: hCG positivity rate [odds ratio (OR): 1.14, 95% confidence interval (CI): 0.79–1.64, p = 0.481], clinical pregnancy rate (OR: 1.81, 95% CI: 1.21–2.71, p = 0.003), miscarriage rate (OR: 0.735, 95% CI: 0.30–1.76, p = 0.491), and ongoing pregnancy rate (OR: 1.79, 95% CI: 1.20–2.69, p = 0.004). Clinical pregnancy and ongoing pregnancy rates were significantly higher in the G-SEET group compared with the control group. Limitations, reasons for caution The study was limited by the study size and lack of data about live birth rates after ET. In addition, intrauterine infusion of GM-CSF-free culture medium should be used as a control, but this was difficult in the private clinic setting. Wider implications of the findings This study suggests that endometrial stimulation with culture media containing GM-CSF prior to transfer of vitrified-warmed blastocysts promotes embryo implantation and enhances the ongoing pregnancy. The G-SEET method may be clinically useful as a new ET method. Trial registration number not applicable

Opening the black box: why do euploid blastocysts fail to implant? A systematic review and meta-analysis.

A normal chromosomal constitution defined through PGT-A assessing all chromosomes on trophectoderm (TE) biopsies represents the strongest predictor of embryo implantation. Yet, its positive predictive value is not higher than 50-60%. This gap of knowledge on the causes of euploid blastocysts' reproductive failure is known as 'the black box of implantation'. Several embryonic, maternal, paternal, clinical, and IVF laboratory features were scrutinized for their putative association with reproductive success or implantation failure of euploid blastocysts. A systematic bibliographical search was conducted without temporal limits up to August 2021. The keywords were '(blastocyst OR day5 embryo OR day6 embryo OR day7 embryo) AND (euploid OR chromosomally normal OR preimplantation genetic testing) AND (implantation OR implantation failure OR miscarriage OR abortion OR live birth OR biochemical pregnancy OR recurrent implantation failure)'. Overall, 1608 items were identified and screened. We included all prospective or retrospective clinical studies and randomized-controlled-trials (RCTs) that assessed any feature associated with live-birth rates (LBR) and/or miscarriage rates (MR) among non-mosaic euploid blastocyst transfer after TE biopsy and PGT-A. In total, 41 reviews and 372 papers were selected, clustered according to a common focus, and thoroughly reviewed. The PRISMA guideline was followed, the PICO model was adopted, and ROBINS-I and ROB 2.0 scoring were used to assess putative bias. Bias across studies regarding the LBR was also assessed using visual inspection of funnel plots and the trim and fill method. Categorical data were combined with a pooled-OR. The random-effect model was used to conduct the meta-analysis. Between-study heterogeneity was addressed using I2. Whenever not suitable for the meta-analysis, the included studies were simply described for their results. The study protocol was registered at http://www.crd.york.ac.uk/PROSPERO/ (registration number CRD42021275329). We included 372 original papers (335 retrospective studies, 30 prospective studies and 7 RCTs) and 41 reviews. However, most of the studies were retrospective, or characterized by small sample sizes, thus prone to bias, which reduces the quality of the evidence to low or very low. Reduced inner cell mass (7 studies, OR: 0.37, 95% CI: 0.27-0.52, I2 = 53%), or TE quality (9 studies, OR: 0.53, 95% CI: 0.43-0.67, I2 = 70%), overall blastocyst quality worse than Gardner's BB-grade (8 studies, OR: 0.40, 95% CI: 0.24-0.67, I2 = 83%), developmental delay (18 studies, OR: 0.56, 95% CI: 0.49-0.63, I2 = 47%), and (by qualitative analysis) some morphodynamic abnormalities pinpointed through time-lapse microscopy (abnormal cleavage patterns, spontaneous blastocyst collapse, longer time of morula formation I, time of blastulation (tB), and duration of blastulation) were all associated with poorer reproductive outcomes. Slightly lower LBR, even in the context of PGT-A, was reported among women ≥38 years (7 studies, OR: 0.87, 95% CI: 0.75-1.00, I2 = 31%), while obesity was associated with both lower LBR (2 studies, OR: 0.66, 95% CI: 0.55-0.79, I2 = 0%) and higher MR (2 studies, OR: 1.8, 95% CI: 1.08-2.99, I2 = 52%). The experience of previous repeated implantation failures (RIF) was also associated with lower LBR (3 studies, OR: 0.72, 95% CI: 0.55-0.93, I2 = 0%). By qualitative analysis, among hormonal assessments, only abnormal progesterone levels prior to transfer were associated with LBR and MR after PGT-A. Among the clinical protocols used, vitrified-warmed embryo transfer was more effective than fresh transfer (2 studies, OR: 1.56, 95% CI: 1.05-2.33, I2 = 23%) after PGT-A. Lastly, multiple vitrification-warming cycles (2 studies, OR: 0.41, 95% CI: 0.22-0.77, I2 = 50%) or (by qualitative analysis) a high number of cells biopsied may slightly reduce the LBR, while simultaneous zona-pellucida opening and TE biopsy allowed better results than the Day 3 hatching-based protocol (3 studies, OR: 1.41, 95% CI: 1.18-1.69, I2 = 0%). Embryo selection aims at shortening the time-to-pregnancy, while minimizing the reproductive risks. Knowing which features are associated with the reproductive competence of euploid blastocysts is therefore critical to define, implement, and validate safer and more efficient clinical workflows. Future research should be directed towards: (i) systematic investigations of the mechanisms involved in reproductive aging beyond de novo chromosomal abnormalities, and how lifestyle and nutrition may accelerate or exacerbate their consequences; (ii) improved evaluation of the uterine and blastocyst-endometrial dialogue, both of which represent black boxes themselves; (iii) standardization/automation of embryo assessment and IVF protocols; (iv) additional invasive or preferably non-invasive tools for embryo selection. Only by filling these gaps we may finally crack the riddle behind 'the black box of implantation'.

Agonist long protocol improves outcomes of vitrified-warmed embryo transfer in repeatedly thin endometrium

Research questionDoes follicular stimulation using human menopausal gonadotrophin (HMG) after pituitary down-regulation by a GnRH agonist improve endometrial thickness (EMT) and clinical outcomes of frozen-thawed embryo transfer (FET; using vitrified-warmed embryos) in women with thin endometrium after intensified oestrogen administration (IOA)? DesignThis was a retrospective study. A total of 627 patients attempted 683 FET cycles with at least one previous history of thin endometrium. None of the cycles reached over 7 mm EMT after using oral and vaginal oestradiol for more than 21 days (IOA protocol). A total of 129 cycles proceeded with FET, 305 cycles were cancelled, and 249 cycles involved administration of HMG following GnRH agonist pituitary down-regulation (GnRH agonist + HMG protocol) for further endometrial preparation. ResultsEMT became significantly greater (7.18 ± 1.14 mm versus 6.13 ± 0.63 mm, P < 0.001) using GnRH agonist + HMG compared with previous IOA cycles, but this was not related to serum oestrogen concentrations. A total of 213 cycles after the GnRH agonist + HMG protocol proceeded with FET, showing a significantly increased clinical pregnancy rate, implantation rate and live birth rate compared with those after IOA. ConclusionsThe GnRH agonist + HMG protocol for endometrial preparation in FET cycles improves EMT in women with a thin endometrium after IOA and showed significantly better clinical outcomes than IOA. The authors suggest that the GnRH agonist + HMG protocol should be used for EMT that is less than 7 mm after there has been no optimal response to IOA.

Genital tract dysbiosis in infertile women with a history of repeated implantation failure and pilot study for reproductive outcomes following oral enteric coating lactoferrin supplementation

We prospectively investigated if oral enteric coating lactoferrin supplementation improves the reproductive outcomes in infertile women with a history of repeated implantation failure (RIF) and non-Lactobacillus-dominant (Lactobacillus rate < 90%) microbiota (NLDM) in vaginal secretions (VS)/endometrial fluid (EF). Paired VS/EF samples were obtained from RIF women and control infertile women (non-RIF group) for microbiome analysis. Chronic endometritis (CE) was diagnosed histopathologically and hysteroscopically. In a pilot study, oral enteric coating lactoferrin (700mg/day, at least 28 consecutive days) was administered to eligible patients with NLDM in VS/EF. Their reproductive outcomes in the subsequent vitrified-warmed embryo transfer cycles were followed up. While CE was more prevalent (OR 2.41, 95% CI 1.02-5.63, p = 0.042) in the RIF group (29.1%, n = 117) than in the non-RIF group (14.5%, n = 55), The NLDM rate was similar between the two groups (44.4 vs 52.7%). Lactoferrin supplementation improved NLDM in 43.2% of RIF women (n = 37). Within the RIF group, the live birth rate in the subsequent cycles was higher (OR 10.67, 95% CI 1.03 - 110.0, p = 0.046) in women with improved microbiota (57.1%, n = 14) than in those with unimproved microbiota (11.1%, n = 9). Unlike CE, NLDM was not unique to RIF but was common in infertile women. Although the therapeutic effect of the oral lactoferrin supplementation on NLDM was limited in a pilot study, the reproductive outcomes were better in RIF women who overcame NLDM than in those who failed. Randomized controlled trials are required to confirm the results. TRIAL REGISTRATION NUMBER AND DATE FOR PROSPECTIVELY REGISTERED TRIALS: UMIN-CTR 000036990, June 7, 2019.

Positive Association of the Number of Oocyte Retrieved with the Maternal hCG Concentration in Early Pregnancy Achieved by Vitrified-Warmed Embryo Transfer after In Vitro Maturation

Background: Human chorionic gonadotropin (hCG) is synthesized by trophoblast cells and play a critical effect in early pregnancy. The serum hCG concentration is significantly higher in early gestation period achieved through in vitro maturation (IVM) than that by traditional IVF. Since IVM does not involve ovarian hyperstimulation, we wonder whether the number of oocyte retrieved affect the hCG concentration in early pregnancy achieved by IVM. Methods: The retrospective analysis involved 51 women who delivered singletons were analyzed. The number of oocyte retrieved and the maternal hCG concentration were collected and analyzed. Results: 51 women had delivered singletons through IVM. The mean number of oocytes retrieved was 16.78 ± 11.23, and the hCG concentration was 614.47 ± 445.50 IU/L and 10930.20 ± 7223.17 IU/L at day 14 and 21, respectively. After confounding factors adjusted, the concentration of serum hCG at day 14 and 21 associated significantly with the number of oocyte retrieved. The result of crude linear regression analysis exhibited a positive relationship between the number of oocyte retrieved and the hCG concentration in early gestation. Conclusions: Our results suggested that the number of oocyte retrieved associated positively with the serum hCG level in female with early pregnancies that underwent IVM.

No difference in cumulative live birth rates between cleavage versus blastocyst transfer in patients with four or fewer zygotes: results from a retrospective study

STUDY QUESTIONIs the cumulative live birth rate (CLBR) per oocyte collection cycle (OCC) comparable after cleavage-stage or blastocyst-stage transfer in combination with supernumerary blastocyst vitrification on Day 5 (D5) in patients with four or fewer zygotes on Day 1?SUMMARY ANSWERThe CLBR in a fresh blastocyst-transfer or cleavage-stage transfer policy followed by vitrification on D5 is comparable in patients with four or fewer zygotes.WHAT IS KNOWN ALREADYBlastocyst transfer enhances the self-selection of the embryo and shortens the time to pregnancy in patients with normal or high ovarian response. Whether these advantages are also present in patients with a low ovarian response and/or a limited number of available zygotes is a continuous debate.STUDY DESIGN, SIZE, DURATIONThis was a retrospective, observational cohort study of 2359 consecutive OCCs between January 2014 and December 2018. According to a shift in transfer policy in our center, 571 OCCs had been scheduled for a fresh transfer on Day 3 (D3) and 1788 on D5. The D5 group was matched to the D3 group by propensity score (PS) matching according to multiple maternal baseline covariates. After PS matching, there were 571 OCCs in each group.PARTICIPANTS/MATERIALS, SETTING, METHODSOCCs scheduled for a D3 transfer (n = 571) or for a D5 transfer (n = 1788) were matched by PS matching in a 1:1 ratio accounting for potential confounding factors associated with CLBR. The model included patient characteristics, such as maternal age and cycle rank, as well as treatment characteristics such as GnRH analog regimen and ovarian response. Embryological variables included the number of zygotes and the number of 6- to 7- and 8-cell embryos on D3. The delivery outcomes of the fresh treatment cycle and the consecutive vitrified-warmed embryo transfers were analyzed up to the first live birth. The primary endpoint of this study was CLBR per OCC. Secondary outcomes were live birth rate per fresh transfer and embryo implantation rate per transferred embryo.MAIN RESULTS AND THE ROLE OF CHANCEThe CLBR per OCC was comparable between the D5 and D3 groups (16.8% versus 17.7%, respectively, P = 0.600). Live birth rates per OCC did not differ between a cleavage-stage transfer and blastocyst-stage transfer policy (15.2% versus 12.4%, respectively, P = 0.160). In the D5 group, 201 cycles did not result in a blastocyst to perform an embryo transfer or cryopreservation; in the D3 group, only 59 cycles did not have an embryo transfer because of poor embryo quality (35.2% versus 10.3%, respectively; P < 0.001). A significantly higher number of fresh double embryo transfers were performed in the D3 group compared to D5 (23.8% versus 7.0%, respectively, P < 0.001).LIMITATIONS, REASONS FOR CAUTIONAlthough adjusted for important confounders in the PS matching, BMI and embryo quality of the transferred embryo(s) were not taken into account. This study is limited by its retrospective design and is a single-center study, which may limit the generalizability of our findings.WIDER IMPLICATIONS OF THE FINDINGSThe CLBR in a fresh blastocyst-transfer or cleavage-stage transfer policy followed by vitrification on D5 is comparable. A fresh embryo transfer on D3 can still be considered in patients with a poor ovarian response and/or limited number of zygotes when combined with blastocyst vitrification without impacting the overall CLBR of the cycle.STUDY FUNDING/COMPETING INTEREST(S)No external funding was obtained for this study. There are no conflicts of interest to declare.TRIAL REGISTRATION NUMBERThis retrospective study was approved by the local ethical committee at Ghent University Hospital (B 670201731234).

A nomogram to assist blastocyst selection in vitrified-warmed embryo transfer cycles.

The purpose of the study was to establish a mathematical model to help rank the order of blastocysts and assist selection of which blastocysts to warm in vitrified-thawed embryo transfer cycles. A total of 2862 women who underwent first vitrified-thawed single blastocyst transfer (SBT) between July 2015 and July 2019 were retrospectively recruited and randomized into a training set (n=2289) and testing set (n=573). Least absolute shrinkage and selection operator (LASSO) regression was used to screen the factors critical to live birth (LB). Subsequently, a nomogram model was established to convert the effect of each factor on LB into a measurable score. The efficacy of the model was then evaluated by the receiver operating characteristic and calibration curve. The performance of the model was also internally tested in the testing set. Maternal age, endometrial thickness, oocyte number, day-3 embryo quality, blastocyst morphology, and blastulation day were selected as the critical predictors of LB in the vitrified-thawed SBT cycle and fitted into a nomogram model. The area under the curve (AUC) of the model was 0.67, and the AUC in the testing set was 0.64, which indicates moderate discrimination. The calibration curve showed good concordance between prediction and observation. Importantly, the score of each variable in the nomogram helped to rank the order of the blastocysts resulting in LB. The nomogram model can provide guidance for embryo selection in vitrified-thawed blastocyst transfer cycles, which may help to optimize the LB rate.

Pregnancy outcomes after fresh versus vitrified-warmed embryo transfer in women with adenomyosis: a retrospective cohort study

Research questionIs the singleton live birth rate superior for vitrified-warmed versus fresh embryo transfer in women with adenomyosis? DesignThis cohort study retrospectively analysed data from the Reproductive Hospital Affiliated to Shandong University between January 2013 and December 2018. A total of 612 women diagnosed with adenomyosis, with 322 fresh embryo transfer cycles and 290 vitrified-warmed embryo transfer cycles, were included in this study. The primary outcome was singleton live birth. Outcomes were adjusted using multivariable logistic regression analysis. ResultsVitrified-warmed embryo transfer was associated with a higher rate of singleton live birth than fresh embryo transfer (25.9% versus 17.4%; P = 0.011). Although there was a trend towards a lower miscarriage rate after vitrified-warmed embryo transfer, the difference did not reach statistical significance (31.3% versus 40.6%; P = 0.111). The clinical pregnancy rate was comparable in the two groups (44.1% versus 44.4%; P = 0.946). Vitrified-warmed embryo transfer also resulted in a lower risk of preterm birth than fresh embryo transfer (7.0% versus 17.5%; P = 0.010). ConclusionsVitrified-warmed embryo transfer may be associated with better pregnancy outcomes than fresh embryo transfer among women with adenomyosis. It seems that vitrified-warmed embryo transfer is more appropriate for specific populations.

Neonatal and clinical outcomes after transfer of a mosaic embryo identified by preimplantation genetic testing for aneuploidies

Research questionDo clinical and neonatal outcomes differ between mosaic embryo transfers (MET) and euploid embryo transfers (EET)? DesignThis retrospective cohort study compared the implantation rate, live birth rate (LBR) and miscarriage rate between 513 euploid embryos and 118 mosaic embryos (72 whole chromosome mosaic [WCM], 40 segmental mosaic and six complex mosaic). Blastocysts were analysed using preimplantation genetic testing for aneuploidies with next-generation sequencing, followed by a single vitrified-warmed embryo transfer. Trophectoderm biopsies were classified as mosaic if they had 20–80% abnormal cells. ResultsOverall, EET resulted in a significantly higher implantation rate (47.0%) and LBR (40.7%) than MET (implantation rate 39.0%, P = 0.005; LBR 28.8%, P = 0.008) and WCM embryos (implantation rate 37.5%, P = 0.01; LBR 22.2%, P = 0.007) after covariate adjustment. Segmental mosaic embryos had an implantation rate (47.5%) and LBR (45.0%) comparable to those of euploid embryos. Mosaic embryos with a high percentage of aneuploid cells (≥60%) showed a significantly lower LBR (10.5% versus 40.7%, P = 0.03) than euploid embryos after covariate adjustment, with three of the five implantations of mosaic embryos resulting in miscarriage. Neonatal outcomes did not differ significantly between the mosaic and euploid groups. Of the 34 women with a live birth after MET, 13 had a prenatal or postnatal genetic testing result, and no abnormalities were found. ConclusionsMosaic embryos were associated with a lower LBR, while segmental mosaic embryos had similar clinical outcomes to euploid embryos. Mosaic embryos with a high aneuploidy percentage (≥60%) should be assigned a low transfer priority. Neonatal outcomes did not differ significantly between the euploid and mosaic groups.

BLASTOCYST CULTURE IN IN-VITRO FERTILIZATION IN WOMEN WITH POLYCYSTIC OVARY SYNDROME

Objective: This study aimed to evaluation of formation and development of blastocyst in in-vitro fertilization in women with polycystic ovary syndrome. Methods: Retrospective study on 53 women with PCOS and 52 women with non-PCOS, who underwent in-vitro fertilization with blastocyst culture at Center for Reproductive Endocrinology and Infertility, Hue University of Medicine and Pharmacy Hospital since January 2019 until December 2020. Results: In women with PCOS, ovarian stimulation resulted in a greater number of retrieved oocyte and blastocyst embryos. Study group with PCOS had a statistically significant lower fertilization rate and day-5 embryo rate of 74.8% and 54% compared with those in the control group (84% and 61.2%, respectively). No statistically significant differences were observed in the mature oocyte rate, the good cleavage embryo rate and the pregnancy rate in the group with PCOS compared with the control group. Conclusion: Women with PCOS have higher number of retrieved oocytes after stimulation. The ability to fertilization and quality of blastocyst was lower in the group with PCOS but the potential for implantation and pregnancy rate were similar to the control group after vitrified-warmed embryo transfer. Key words: Polycystic Ovary Syndrome (PCOS), blastocyst, in vitro fertilization, infertility

NEONATAL FOLLOW-UP OF BABY BORN DERIVED FROM A TWO PRONUCLEI PLUS ONE MICRO PN OOCYTE FOLLOWING INTRACYTOPLASMIC SPERM INJECTION

A micro PN is very small and can be overlooked when observed under a microscope. Furthermore, it is difficult to see when it overlaps with the normal nucleus. Out of 5,026 oocytes derived by intracytoplasmic sperm injection, 0.7% showed micro PN. After performing chromosome and ploidy analysis on embryos that have grown to blastocysts, micro PN-derived balanced-diploid blastocysts have been transferred into three patients and childbirth has been reported in two patients. (Capalbo et al, Fertility and Sterility, 2017) However, there are no reported cases of developmental follow-up of babies born from micro PN oocytes. Therefore, we report on the neonatal follow-up of baby born derived from a two pronuclei plus one micro PN oocyte. Patient seeking infertility treatment in a well-established private IVF clinic. The birth weight and the height and weight at one and a half and three years old of one case in which a micro-PN-derived blastocyst was transferred were compared with the results of the 2010 Infant Physical Growth Survey by the Ministry of Health, Labor and Welfare (MHLW) of Japan. The patient was a nulliparous woman and treatment started at the age of 31. Therapy started in August 2009, and AIH was performed 6 times in total upto February 2011, but pregnancy was unsuccessful. After that, 19 oocytes were collected by controlled ovarian stimulation in April 2014. As a result of insemination of the collected oocytes, microPN (size: 9 μm) appeared in one of the ICSI-derived embryos, which was about one-third the size of the male and female pronuclei. Afterwards singleton vitrified-warmed embryo transfer in hormone replacement cycle was performed twice by July 2014. As a result of transferring micro PN-derived blastocyst (Gardner’s score 3AB) at the time of the second embryo transfer, a girl was born by Caesarean section at 38 weeks, and the weight was 3.02 kg, which was within the normal range of the growth curve of data from children in MHLW database. The height at one and a half years was 74.8 cm and the weight was 9.45 kg, which was within the normal range of the growth curve of the children by MHLW. At the age of three, she was 87 cm tall and weighed 13 kg. The height was slightly below the growth curve, but there were no other abnormalities in the child. Follow up from birth to age 3 years did not report any abnormalities or severe growth retardation in the infant.

P–651 Strict embryo-endometrial synchrony does not contribute to the successful pregnancy during vitrified-warmed embryo transfer with hormone replacement cycles

Abstract Study question Does strict embryo-endometrium synchronization relate to pregnancy during vitrified-warmed embryo transfer (ET) with hormone replacement (HRT) cycles? Summary answer A 12-hour delay in the embryo-endometrial synchrony was acceptable, and this delay was not an independent predictor of pregnancy during vitrified-warmed ET with HRT cycles. What is known already Embryo-endometrium synchrony is considered to be necessary for successful pregnancy in both fresh and frozen-thawed cycles. Until now, the date of ET has been determined by the synchronization of the embryo developmental stage and the endometrium on a daily basis. To date, with the advent of the time-lapse incubator, it is possible to observe the embryo development from fertilization over time and to calculate the exact time from fertilization of the transferred embryo. However, there are very few studies on the extent to which increases the accuracy of synchronization between embryo and endometrium contributes to a successful pregnancy. Study design, size, duration This retrospective cohort study included 319 consecutive cycles during vitrified-warmed ET with HRT between August 2016 and August 2018. This study was conducted in an academically affiliated private practice. Participants/materials, setting, methods We analyzed 319 vitrified-warmed single-blastocyst transfer cycles. All frozen expanded blastocysts were inseminated by intracytoplasmic sperm injection (ICSI) and cultured in a time-lapse incubator. We calculated time for the in vitro culture of the embryo after ICSI (t1) and time for progesterone-priming (t2) up to ET. The difference between t1 and t2 (delta-t) was used as an indicator of embryo-endometrium synchrony. We examined the relationship between delta-t and treatment outcomes using multivariate logistic analysis. Main results and the role of chance The mean patient’s age at oocyte retrieval was 35.7 (SD 4.3). The number of pregnant cycles was 157 in all treatment cycles (pregnancy rate, 49.2%). The mean value of delta-t was 9.9 h (SD 2.6) in all cycles. There was no significant difference of delta-t in pregnant (mean, SD: 10.0 h, 2.8 h) and non-pregnant cycles (mean, SD: 10.0 h, 2.3 h). Treatment cycles were classified according to the quartile of delta-t, and we examined the percentages of pregnant cycles in each group. There were no significant differences in pregnancy rates among the groups (p = 0.75). On multivariate logistic analysis, patient’s age (adjusted odds ratio [aOR]: 0.94, 95% confidence interval [CI]: 0.89–0.99), previous treatment cycles (aOR: 0.74, 95% CI: 0.56–0.99), endometrial thickness at ET (aOR: 1.19, 1.04–1.36), and good quality blastocysts (&amp;gt;3BB according to Gardner’s classification) at vitrification (aOR: 2.49, 95% CI: 1.23–5.05) were independent predictive factors for pregnancy. On the other hand, delta-t did not contribute to pregnancy (aOR: 1.00, 95% CI: 0.99–1.00). Limitations, reasons for caution We did not examine the effects of embryo-endometrium synchrony during vitrified-warmed ET in a natural cycle. Therefore, careful interpretation of the significance of embryo-endometrium synchrony during the vitrified-warmed ET should be taken. Wider implications of the findings: We showed the embryo-endometrium synchrony did not contribute to the pregnancy during vitrified-warmed ET with HRT cycles. These results cast doubt on the existence of an optimal implantation window by changing the timing of ET with the results of gene expression testing of the endometrium. Trial registration number Not applicable