Single Euploid Embryo Transfer Research Articles

COVID-19 mRNA vaccines have no effect on endometrial receptivity after euploid embryo transfer

Research questionDoes the COVID-19 vaccination affect endometrial receptivity after single euploid embryo transfer, measured by sustained implantation rate? DesignA retrospective cohort study analysing two groups of single euploid embryo transfers using own oocytes: one historical cohort of 3272 transfers 1 year before the pandemic; and one comprising 890 transfers in women previously vaccinated with mRNA vaccines against severe acute respiratory syndrome coronavirus 2. The main outcomes were clinical pregnancy rate (CPR) and sustained implantation rate (SIR) per embryo transfer. These outcomes were compared between non-vaccinated and vaccinated women, and women who had received one and two doses. Lastly, vaccinated women were divided into quartiles according to the time from last dose to embryo transfer. ResultsSimilar CPR and SIR were found between non-vaccinated and vaccinated women, and the odds ratio for both outcomes was not statistically significant after being controlled for potential confounders (OR 0.937, 95% CI 0.695 to 1.265 and OR 0.910, 95% CI 0.648 to 1.227 respectively). Within the vaccinated group, women who had received one or two doses also had similar outcomes. In addition, no differences were found according to the time interval from vaccination to embryo transfer. ConclusionThe administration of mRNA vaccines against COVID-19 had no effect on endometrial receptivity and embryo implantation, regardless of the number of doses and time interval from vaccination to embryo transfer. The potential negative effect of the vaccine on endometrial receptivity and reproductive outcomes is reassuring for patients in the process of undergoing assisted reproductive treatment.

Live birth rate following a euploid blastocyst transfer is not affected by double vitrification and warming at cleavage or blastocyst stage.

To compare reproductive outcomes following a euploid embryo transfer, between those embryos vitrified-warmed twice to those vitrified-warmed once. We retrospectively analysed 694 single euploid frozen embryo transfer cycles following preimplantation genetic testing for aneuploidy (PGT-A). For cycles in group 1 (N = 451), embryos were biopsied for PGT-A at blastocyst stage and vitrified. For cycles in group 2 (N = 146), embryos were vitrified at blastocyst stage, before being warmed and biopsied for PGT-A and vitrified again. For cycles in group 3 (N = 97), embryos were vitrified on day-3, before being warmed, cultured to day-5 and biopsied for PGT-A and re-vitrified. The pregnancy, clinical pregnancy and livebirth rate in group 2 were not statistically different to group 1 (pregnancy rate, adjusted OR 1.09, 95% CI 0.62-1.91; clinical pregnancy, aOR 0.89, 95% CI 0.58-1.37; live birth rate, aOR 0.85, 95% CI 0.56-1.28). There was also no significant difference between group 3 and group 1, with similar pregnancy rate (aOR 1.22, 95% CI 0.74-1.99), clinical pregnancy rate (aOR 1.21, 95% CI 0.75-1.96) and live birth rate (aOR 1.15, 95% CI, 0.73-1.80). There was no significant difference in miscarriage rates between all three groups. The age at the oocyte collection, embryo quality and day of biopsy were associated with pregnancy, clinical pregnancy and live birth rate. This study suggests that vitrifying and warming embryos twice at blastocyst or at cleavage and then blastocyst stage, can lead to similar reproductive outcomes to embryos vitrified-warmed once, after a single euploid embryo transfer.

Comparison of day 5 blastocyst with day 6 blastocyst: Evidence from NGS-based PGT-A results.

Aneuploidy is one genetic factor leading to the failure of embryo implantation. This study aimed to investigate the relationship between the day of embryo blastulation and the ploidy status of embryo, to aid in selecting embryos with the most likelihood of of beingeuploid in anoninvasive way. This retrospective study recruited women undergoing preimplantation genetic testing (PGT) for aneuploidy (PGT-A) with trophectoderm biopsy from January 2019 to December 2020. The ploidy status of embryos was determined by next-generation sequencing (NGS). Altogether, 2531 blastocysts from 839 PGT-A cycles were evaluated. The euploid rate of day 5 blastocysts was significantly higher than that of day 6 blastocysts, either from the same ovarian stimulation (OS) cycles (49.9% vs 35.7%, P < 0.001) or from different OS cycles (48.2% vs 27.8%, P < 0.001). This effect of increasing time to embryo blastulation significantly reducing the prevalence of euploidy was not seen in women at age 38 or older. However, after single euploid embryo transfer, the clinical outcomes of day 5 blastocysts were comparable to those of day 6 blastocysts. In non-PGT cycles, our data support the selection of day 5 blastocysts for transfer over day 6 blastocysts. Further, women with poor ovarian reserve incline to obtain only day 6 blastocysts, and PGT-A is valuable for identifying the ploidy status of embryos, especially for those with advanced age. However, the stage of blastocysts will not affect the clinical outcome after transfer when they are identified as euploid.

Association between clinical and IVF laboratory parameters and miscarriage after single euploid embryo transfers

BackgroundThe goal of this study was to investigate which factors, excluding embryo aneuploidies, are associated with miscarriage in patients who have undergone a single euploid blastocyst transfer.MethodsRetrospective, observational and multicenter study with 2832 patients undergoing preimplantational genetic testing for aneuploidies (PGT-A) due to repeated implantation failure, recurrent pregnancy loss, advanced maternal age or severe male factor were transferred one single euploid embryo.ResultsOne of the main findings was a significant relationship between body mass index (BMI) and miscarriage rates (13.4% in underweight women, 12.1% in normal weight, 14.5% in overweight, and 19.2% in obese women, odds ratio [OD] 1.04; 95% confidence interval [CI], 1.01–1.07 p = 0.006). Endometrial thickness (OD 0.65; 95%, 0.52–0.77 p = 0.04) and type of endometrial preparation (natural cycle or hormone replacement cycle) (OD 0.77; 95%, 0.52–0.77, p = 0.04) were also associated with miscarriage rates.ConclusionsBMI was strongly associated to miscarriage rates. We also observed a weaker association with endometrial thickness and with the type of endometrial preparation (natural cycle or hormone replacement cycle). None of the other studied variables (biopsy day, maternal and male age, duration of infertility, cycle length, previous miscarriages, previous live births, previous In Vitro Fertilization (IVF) cycles, endometrial pattern and/or diagnosis) were associated with miscarriage rates.

EFFECT OF RACE ON EUPLOIDY STATUS AND LIVE BIRTH RATES IN ART TREATMENTS

Racial disparities in outcome have been reported in fertility care, and in particular, in Assisted Reproductive Technology (ART) treatment. Existing studies have been limited by sample size and have relied upon outcomes from fresh IVF. To our knowledge, no study has evaluated the impact of race and ethnicity with regard embryo ploidy status and subsequent euploid embryo transfer outcomes. The aim of this study is to analyze the euploidy status of embryos using preimplantation genetic testing for aneuploidy (PGT-A) and live birth (LB) outcomes following single, euploid embryo transfer (SEET) among various racial groups. The study included all ovarian stimulation cycles in a single academic institution in which PGT-A was performed using Next Generation Sequencing from January 2016 to February 2021. Our primary outcome was embryo euploidy status; the secondary outcome was LB. Baseline demographics were obtained: age, body mass index (BMI), antimüllerian hormone (AMH), basal antral follicle count, basal follicle stimulating hormone, peak serum estradiol, endometrial thickness at transfer, and self-reported race. ANOVA, Kruskal-Wallis, chi-square, and multivariate logistic regression were used for analysis. A total of 6122 patients underwent an IVF cycle with ICSI & PGT-A: Of these, 3628 self-reported as white (59%), 336 as black (5%), 1122 as Asian (18%), 534 as Hispanic (8%), 150 other (2%), and 352 (6%) did not specify their ethnicity. Black women were significantly older (38.0 ±3.1 yrs, p=0.0006), and Black and Hispanic women had significantly higher BMI (27.9 and 26.6 kg/m2, respectively, p=0.001) compared to the other cohorts. All other cycle characteristics were comparable among groups. When analyzing embryo ploidy status, white women had higher number of euploid embryos/euploidy rate (3.4 ± 1.9/49.7±35.3%, p<.02) compared to the other groups. After adjusting for age, BMI and AMH, white women were more likely to have euploid embryos (aOR 1.13, CI 1.06-1.20, p= 0.0001) when compared to other races. SEET was performed for 4524 women. No statistical significance was observed in LB rates between white women and other races after adjusting for confounders (aOR 1.19, CI 95% 0.2-6.4, p=0.8). The higher rate of aneuploidy in non-white races may explain, in part, the poorer outcomes reported in prior studies of untested embryos in fresh embryo transfers. These results suggest differences in basic mechanisms governing meiosis and/or its repair mechanisms by self-reported race. However, LB outcomes following a SEET do not appear to differ among women of different races. This data may help reassure non-white patients that once a euploid embryo is transferred, the odds for a live birth is not affected by race.

SUPRAPHYSIOLOGIC SERUM ESTRADIOL NEGATIVELY IMPACTS EUPLOID EMBRYO IMPLANTATION

Euploid embryo transfer failure remains a frustrating clinical conundrum for patients and providers. Emerging research now focuses on optimizing the embryo (beyond euploidy) versus maternal environment, though many questions remain. This study evaluated how maternal estradiol level at time of embryo transfer influences the success of single euploid embryo transfer.

DOES THE MOSAIC EMBRYO RATIO OF PGT-A CYCLES IMPACT THE LIVE BIRTH CHANCE EVEN WHEN AN EUPLOID EMBRYO IS TRANSFERRED?

Preimplantation genetic testing for aneuploidy (PGT-A) has the potential to detect mosaicism in trophectoderm biopsies with Next-Generation Sequencing (NGS). Many couples who opted for PGT-A have both euploid and mosaic embryos in their cohort of tested embryos. However, little is known about how live birth outcomes are affected by mosaic embryo ratios of PGT-A tested embryos even though euploid embryos are transferred. This retrospective study was based on 636 PGT-A tested frozen-thawed single euploid embryo transfer cycles of which live birth outcome is known. The inclusion criteria were having at least one euploid and one mosaic embryos. Mosaicism was reported for PGT-A outcomes deviated for 20-80% from 2 chromosome copy number. Mosaic embryo ratio was defined as the number of mosaic embryos divided by the total number of blastocysts tested. This data included embryo transfers between January 2017 and March 2020 in a single ART clinic. PGT-A was done by NGS ReproSeq on IonTorrent S5 (Thermo Fisher) following trophectoderm biopsy. For the categorical data Chi-Square test was used. For continuous variables, Mann-Whitney Test was applied. Pearson correlation coefficient was used for testing how two variables are related to each other. The live birth rate of the study group was 55.8%. When data was evaluated according to the live birth status (+(n=355) or -(n=281)), no significant differences were observed for the number of oocytes collected, mature oocytes, blastocysts, biopsied blastocysts, number of euploid and mosaic embryos per cycle. Female age at the time of transfer and mosaic embryo ratio were statistically significant (p=0.015 and p=0.040, respectively). Female age for women who had reached live-birth was 0.9 years less and mosaic embryo ratio was 2.3% less when compared to women who did not reach live-birth. As only female age and mosaic embryo ratio were found to be statistically significant between the groups, their relationship with each other was tested and found to be inversely correlated, eg. with increasing female age, the mosaic embryo ratio was decreasing (r= -0.07, -0.14 to -0.01 95% CI, p=0.021). The median value for mosaic embryo ratio was 28.6%. When the study group was divided into two with 28.6% as a cut-off, the live birth rate was 10.1% higher for cycles with lower mosaic embryo ratio (62.4% vs 52.3%) (p=0.160). The live-birth rate for euploid FET with at least 1 mosaic embryo was not statistically different when compared to 1193 euploid FET of PGT-A cycles with no mosaic embryo (55.8% vs 55.3%, respectively) (p=0.839). Diverse molecular mechanisms including altered cell cycle checkpoints, aberrations of the centrosome and failed chromatid cohesion are known to contribute to mitotic errors (McCoy, 2017). Although euploid, some embryos from the same PGT-A tested cohort may be affected by the molecular pathways contributing to embryonic mosaicism.

MORPHOLOGIC PARAMETERS OF EUPLOID BLASTOCYSTS THAT PREDICT PROGRESSION TO CLINICAL PREGNANCY

Pregnancy loss after a single euploid embryo transfer remains one of the greatest conundrums in the field of assisted reproduction. Expansion (Exp), inner cell mass (ICM) grade, and trophectoderm (TE) grade all are associated with ongoing pregnancy/live birth (OP/LB) rate, but the factors that govern initial implantation vs. progression to ongoing pregnancy are less clear.1 Our study aims to determine which morphologic parameters are associated with progression to clinical pregnancy once implantation has begun.

THE HYPOTHESIS OF PREFERENCIAL LOSS OF FEMALE EMBRYOS: EMBRYONIC SEX IS NOT ASSOCIATED WITH INCREASED ODDS OF EARLY PREGNANCY LOSS

The human sex ratio has long interested developmental biologists, statisticians, and other scientists. Primary Sex Ratio (PSR) in humans has been thought to be male-biased. Previously published studies about PSR have suggested this bias is due to a preferential loss of female conceptuses during the earliest stages of pregnancy1,2. To date, the dynamics of PSR from conception to birth are poorly understood. Literature about PSR is currently based on subjective estimates, extrapolations from large historical cohorts, or based on data sourced from natural conceptions3. Yet, the use of data from patients who undergo ART treatment presents an opportunity to observe embryonic sex prior to transfer through the application of PGT. This study aims to analyze whether the selection of female embryos prior to frozen embryo transfer (FET) are at higher risk of suffering early pregnancy loss as compared to the selection of male embryos. The study included patients who underwent a single euploid FET cycle from 2016-2021. Gender selection for family balancing cases were excluded from the analysis. PGT-A with NGS was performed for all cases. Cohorts were segregated into groups based on embryonic sex prior to transfer selection. Only top morphology embryos with a grade ≥4AA (Modified Gardner’s) were included in the study. Patient demographics and IVF cycle outcomes were recorded. Biochemical pregnancy loss (BPL) was defined as detectible serum ß-hCG and embryonic arrest prior to the development of a clinical pregnancy. Clinical pregnancy loss (CPL) was defined as the loss of a pregnancy diagnosed by ultrasonographic visualization of one or more gestational sacs. Comparative statistics and an adjusted mixed model with a GEE were utilized for statistical analyses. A sample size of 906 single, euploid FETs per cohort was calculated to ensure an 80% power to detect a difference of 5% on clinical pregnancy loss rates (α=0.05). 1,363 male were compared to 1,045 female FETs. No differences were found in patient’s age, BMI, AMH, endometrial thickness and other demographic variables between cohorts. Also, no differences were found in implantation, clinical pregnancy, ongoing pregnancy, BPL and CPL. After adjusting for age, BMI, AMH, day of biopsy and endometrial thickness, no association was found between embryo sex and increased odds of BPL (OR 0.50 CI95% 0.11-2.27, p=0.37); or CPL (OR 0.28 CI95% 0.07-1.16, p=0.08). By analyzing a large cohort of single euploid embryo transfers, we can observe the proportion of pregnancy losses that are not associated with aneuploidy, thus assessing the differences on early pregnancy loss rates based on embryonic sex while adjusting for other important covariates. For patients who undergo IVF with subsequent PGT-A and FET, there does not appear to be increased odds of pregnancy loss in selecting a single, euploid female embryo compared to male embryo counterparts.

O-087 Embryos with higher mitochondrial DNA ratios show better clinical outcomes in single euploid embryo transfer

Abstract Study question To assess whether there is a relationship between mitochondrial DNA content and implantation result. Summary answer The embryos with a higher mitochondrial DNA ratio increase pregnancy rate and implantation rate in single euploid embryo transfer. What is known already Mitochondria is an important organelle that generates energy during embryonic development. Recent literature points out that mitochondrial content and function may be related to implantation success and embryo viability. Some studies have linked increased ratios of mitochondrial DNA to aneuploidy, advanced maternal age, and euploid blastocyst with implantation failure, while others have failed to demonstrate similar findings. Study design, size, duration This study is a retrospective cohort study from 2016 to 2019, including 1465 single embryo transfer cycles. Participants/materials, setting, methods The involved embryos were biopsied on Day 5 or 6 and the mitochondrial DNA ratio of 1465 embryos was examined undergoing PGS/NGS. The mitochondrial DNA ratios were normalized for technical batch-to-batch variation. The mitochondrial DNA ratio between the implantation group and non-implantation group was statistically analyzed. Data were analyzed by the student’s t-test for continuous variables and Chi-square test for categorical variables. Main results and the role of chance The mitochondrial DNA ratio of embryos was no significant difference between different age spans ( p = 0.772) and ploidy (p = 0.224). D5 biopsied embryos, however, contained a significantly higher mitochondrial DNA ratio than D6 biopsied embryos (p &amp;lt; 0.0001). All of the single embryo transferred embryos were classified into two groups; implanted and non-implanted embryos. Results from 1465 transferred embryos show that the mitochondrial DNA ratio of implanted embryos was statistically significantly higher than non-implanted embryos (p = 0.0053). Besides, the cut-off values were established, dividing the transferred embryos into high and low mitochondrial DNA ratio groups. The pregnancy rate and implantation rate of the high mitochondrial DNA ratio group was higher than the low mitochondrial DNA ratio group: [Pregnancy rate] 74% vs. 63.5% (p = 0.0209); [Implantation rate] 57.3% vs. 50.8% (p = 0.1907). Limitations, reasons for caution The mitochondrial DNA ratios were analyzed by bioinformatics processing in Miseq reporter software (Illumina) files in the BAM and FASTQ format. Not sure if there is reproducibility in different sequencing platforms. Wider implications of the findings There still remains a lack of clarity regarding the relationship between mitochondrial function and transfer outcome. This retrospective study links an association between increased mtDNA content and increased implantation. Trial registration number not applicable

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

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

P–390 The intrauterus administration of peripheral blood mononuclear cells increase the pregnancy rates for the patients with advanced maternal age after single euploid embryo transfer

Abstract Study question Does the intrauterine administration of peripheral blood mononuclear cells (PBMCs) effect the outcome of IVF for patients with advanced maternal age when the euploid embryos after PGT-A are transferred? Summary answer The implantation rates were significantly higher after the intrauterine application of PBMCs in patients with advanced maternal age (AMA) before transfer of the euploid embryo. What is known already The aneuploidy rates of blastocysts in IVF is in range 45–70% depending on different factors. Besides that, the endometrium plays an important role in achieving optimal outcomes of assisted reproductive technologies. It has been proposed that intrauterine administration of peripheral blood mononuclear cells modulates maternal immune response to favor implantation. Study design, size, duration The effect of the intrauterine application of PBMCs to improve the implantation rates in the group of patients with advanced maternal age was studied. Two group of patients (PBMCs-group and non-PBMCs-group_ were formed. Single euploid embryo was transferred for each patient. Participants/materials, setting, methods: The ploidy status of 373 blastocysts from 82 AMA-patients was analyzed by the method of next generation sequencing (NGS). PBMCs were applied for 39 women with the mean age 39.2±3.2 y.o. before embryo transfer (Group 1). For 43 patients with the mean age 38.2±2.1 y.o. single euploid embryo transfers were performed without PBMCs administration (Group 2). Chi-squared test was used for data analysis. The study’s protocol was approved by the Center’s IRB. Main results and the role of chance Totally the rate of euploid embryos was 27.1% (101 blastocysts). In the mentioned study 55.0% of examined blastocysts were aneuploid (205 embryos) and 17.9% of blastocysts were detected as mosaic (67 embryos). Single euploid embryo was transferred in each case in the patients of both experimental groups. The implantation rate was significantly higher in Group 1 with PBMCs application comparing with non-PBMCs experimental Group 2 (38.5% (15 pregnancies) vs. 23.3% (10 pregnancies), df = 1, χ2 = 5.487, χ2critical = 3.841, P = 0.020). Limitations, reasons for caution The embryo biopsy was performed only for blastocyst with top-quality morphology. Wider implications of the findings: The implantation rates were significantly higher when the intrauterine application of PBMCs in patients with advanced maternal age before the transfer of the euploid embryo (P = 0.020). The randomized studies to improve our knowledge in immunogenic therapy in reproductive medicine should be performed. Trial registration number -

P–359 Blastocyst quality, transfer difficulty and endometrial thickness affect clinical pregnancy after frozen embryo transfer (FET) of euploid blastocysts in the upper uterine cavity

Abstract Study question Which factors affect the clinical pregnancy rate (CPR) after single euploid frozen embryo transfers (FET), when the blastocyst is transferred in the upper uterine cavity area? Summary answer Blastocyst quality, embryo transfer difficulty and endometrial thickness affect the CPR in FET. What is known already There is a limited understanding of the factors affecting success rates after FET. The most important factors influencing implantation rates are patient characteristics, type of endometrial preparation, embryo quality and transfer difficulty. It has been shown that the position of the euploid blastocyst, measured as distance from the fundus (DFF) of the uterine cavity (mm), affects the implantation potential. Although the ideal location within the uterine cavity is still being debated in very heterogeneous patient populations, most studies have found that the highest pregnancy rates are obtained when the embryo is placed in the upper area of the uterine cavity. Study design, size, duration This single center retrospective cohort study included a total of 603 single euploid FET cycles, in the upper half of the uterine cavity, between January 2019 and November 2020 in ART Fertility Clinic Abu Dhabi, UAE. Participants/materials, setting, methods Trophectoderm biopsy samples were subjected to Next Generation Sequencing to screen the ploidy state. Vitrification and warming were performed using the Cryotop method (Kitazato, Biopharma). The full length of the uterine cavity and the longitudinal distance between the fundal endometrial surface and the air bubble after transfer were measured. Main results and the role of chance The patients were on average 33.9 (19–46) years old. The FET was performed in a natural cycle (NC) (n = 278) or hormone replacement therapy (HRT) (n = 325). Of the 603 transfers which had been performed in the upper half of the uterus, 412 (68.3%) resulted in a pregnancy and 311 (51.5%) in a clinical pregnancy. After bivariate analysis, the clinical pregnancy rate was significantly higher for high quality blastocysts (grade 1–2 versus 3–4) (p &amp;lt; 0.001), after easy embryo transfers (p = 0.001) and for higher endometrial thickness (p = 0.027). After performing a multivariate logistic regression analysis to consider the effect of all explanatory variables (age, Anti Müllerian hormone, body mass index, endometrial thickness, quality of the blastocyst, difficulty of the transfer [requirement of additional instrumentation], presence of mucus or blood on the transfer catheter, day 5 or day 6 biopsy, FET endometrial preparation), the clinical pregnancy was affected by the endometrial thickness: OR 1.20 [1.05–1.37], p = 0.007; transfer difficulty: OR 0.44 [0.25–0.79], p = 0.006; blastocyst quality 3: OR 0.38 [0.18–0.79], p = 0.01 and blastocyst quality 4: OR 0.15 [0.06–0.37], p &amp;lt; 0.0001. Age did not affect the clinical pregnancy after transferring a single euploid blastocyst: OR 1.03 [1.00–1.06], p = 0.052. Limitations, reasons for caution The limitation of this study was its retrospective nature and the small sample size. Other parameters may be important in live birth outcomes. Wider implications of the findings: Optimization of clinical pregnancy outcomes after FET depends on multiple factors. Even after transfer of euploid blastocysts in the upper uterine cavity, the endometrial thickness, transfer difficulty and blastocyst quality will still affect the clinical pregnancy outcomes. Trial registration number NA

P–520 Live birth rate with a euploid embryo is the same irrespective of the number of oocyte retrievals undertaken to produce a euploid embryo

Abstract Study question Does a euploid embryo from one ovarian stimulation lead to the same live birth rate as a euploid embryo arising from multiple ovarian stimulations? Summary answer The live birth rate of a euploid embryo transferred is comparable irrespective of the number of ovarian stimulations required. What is known already Embryo transfer of a euploid embryo leads to a high live birth rate. Women with low ovarian reserve or poor responders may not have a euploid embryo from one cycle of ovarian stimulation and can be discouraged from undergoing preimplantation genetic testing for aneuploidy (PGT-A). Embryo batching from multiple cycles offers such patients a potential solution to increase their chance of achieving a euploid embryo. Study design, size, duration A retrospective analysis of 506 cycles of single euploid frozen embryo transfers (FET) from January 2015 to March 2020 was carried out. The indication for PGT-A was advanced maternal age, recurrent miscarriages or repetitive IVF failures. Only the first single euploid FETs per patient were included. Participants/materials, setting, methods Group A (N = 323) included women who had a normal ovarian reserve and only one cycle of ovarian stimulation before the FET, whilst Group B (N = 183) had low ovarian reserve or previous poor ovarian response requiring 2 or more cycles of ovarian stimulation. All embryos were biopsied at the blastocyst stage and subjected to a-CGH or NGS. Univariate statistical analysis with Chi-square or Wilcox Man U as required and multivariate logistic regression was performed with SPSS. Main results and the role of chance Group A and Group B were comparable in terms of BMI (average 22.3 vs 22.6), sperm origin, number of blastocysts biopsied (median N = 6, range 4–8), day 5 vs day 6 biopsy (day 5, 81.1% vs 75.4%, p = 0.130) and whether only one euploid embryo was available (42% vs 34%; p = 0.103). There was a significant difference in the number of eggs retrieved per cycle between the two groups (median 15 vs 9, p &amp;lt; 0.001), the total number of eggs retrieved (median 15 vs 20, p &amp;lt; 0.001) and whether a top-quality embryo was transferred (38% vs 25%, p = 0.026). Pregnancy rate, live birth rate and pregnancy loss was equivalent for both groups: 69.3% (224/323) vs 63.9% (178/183) (p = 0.212), 57.6% (186/323) vs 57.4% (105/183) (p = 0.096) and 17.0% (38/224) vs 10.3% (12/117) (p = 0.964), respectively. Multivariate logistic regression was performed to ascertain the effect of the treatment variables on the live birth rate. The number of oocyte collections was not a significant predictive factor (OR 1.19, 95% CI 0.72 - 1.96, p = 0.491), whilst an embryo biopsy performed on day 5 vs day 6, increased significantly the live birth rate (OR 2.58, 95% CI 1.61 - 4.13, p &amp;lt; 0.001). Limitations, reasons for caution The main limitation of this study is that it is retrospective, single centre and therefore vulnerable to confounding factors and bias. Wider implications of the findings: These results can be used to counsel and reassure women with poor response embarking on embryo batching and PGT-A that should a euploid embryo become available, their chance of success is unaffected by the number of cycles they undertake albeit the physical, emotional and financial burden of multiple ovarian stimulations Trial registration number Not applicable

P–500 Euploid embryo-transfer reduces advanced maternal age patients’ anxiety in the waiting period before the pregnancy-test

Abstract Study question Can PGT-A reduce the anxiety generally experienced by infertile women undergoing IVF in the waiting period between embryo transfer and the pregnancy test? Summary answer PGT-A reduces anxiety in infertile women after embryo transfer, probably due to a gain of confidence in their treatment route. What is known already The waiting period, i.e. the time between embryo-transfer and the pregnancy-test, is considered unpredictable and unmanageable, thus figuring amongst the most stressful steps of an IVF treatment. This is mainly imputable to women’s lost sense of control over the outcome. Uncertainty is in fact a source of fear and elevated distress. PGT-A has been shown to improve live birth rate per embryo transfer and reduce miscarriage rate per clinical pregnancy across several trials and observational studies worldwide, especially in advanced maternal age (AMA) women. Here, we investigated if euploid embryo transfer does involve also lower emotional burden over untested one. Study design, size, duration Prospective observational study evaluating the level of anxiety in the waiting period among women undergoing euploid or untested embryo transfer. Data were collected between September 2019 and September 2020 in a public hospital. A total of 48 infertile women were recruited: 25 undergoing euploid single embryo transfer after trophectoderm biopsy and NGS, and 23 undergoing untested single embryo transfer. Participants/materials, setting, methods To measure the level of anxiety, the two groups completed the STAI (State Trait Anxiety Inventory) questionnaire at two time points: before starting the ovarian stimulation (T0), and at day 8 after embryo transfer (T1). The chosen questionnaire has been previously validated to capture the level of patients’ anxiety during the waiting period. Outcomes of T0 were used to control for individual level state of anxiety at T1. Main results and the role of chance The two groups showed similar reproductive history and sociodemographic characteristics except for female age, which was higher in the PGT-A group (37.7±3.2 yr versus 32.3±2.2 yr in the control). This is due to AMA (maternal age &amp;gt;35 yr) being the main indication to PGT-A. Conversely, the duration of infertility was similar in the two groups (3.8±2.2 yr versus 3.7±1.9 in the control). At T0 all patients showed similar levels of anxiety (46.4 points versus 49.9 in the control, 95%CI of the difference: from –9.97 to 3.03 points, p = 0.3). Remarkably, at T1 instead, the women undergoing euploid embryo transfer showed a significantly decreased level of anxiety with respect to the control (39.9 points versus 53.4; 95% CI of the difference: from –18.26 to –8.69, p &amp;lt; 0.01). This difference remained significant also after controlling for the baseline value at T0, and adjusting for potential confounding factors in a multivariate analysis (adjusted p-value&amp;lt;0.01). Limitations, reasons for caution The sample size is small, yet the study resulted powered enough to reveal the considerable advantage of PGT-A toward the primary outcome. We analysed only the waiting period here. Therefore, data will be collected in the future at subsequent gestational stages, such as when prenatal genetic diagnosis is usually conducted. Wider implications of the findings: Women undergoing PGT-A seem reassured by the technique. This is probably due to the gain of confidence and control derived from an increased expectation of success. From this perspective, assessing women’s wellbeing and attitude towards all different clinical procedures should become a critical part of their treatment. Trial registration number None

Maternal body mass index associates with blastocyst euploidy and live birth rates: the tip of an iceberg?

Research questionDoes maternal preconceptional body mass index (BMI) associate with mean blastocyst euploidy rate (m-ER) per patient and live birth rate (LBR) after vitrified–warmed euploid single embryo transfer (SET)? DesignObservational study conducted between April 2013 and March 2020 at a private IVF clinic, involving 1811 Caucasian women undergoing trophectoderm biopsy and comprehensive chromosome testing. The outcomes of 1125 first vitrified–warmed euploid SET were also analysed. Patients were clustered as normal weight (BMI 18.5–25; n = 1392 performing 859 SET), underweight (BMI <18.5; n = 160 performing 112 SET) and overweight (BMI >25; n = 259 performing 154 SET). m-ER per patient was the primary outcome. The secondary outcomes were all clinical outcomes per euploid SET. All data were adjusted for confounders through regression analyses. ResultsThe m-ER per patient decreases as maternal BMI increases from 17 up to 22–23 before reaching a plateau. A linear regression adjusted for maternal age confirmed this moderate association (unstandardized coefficient B: –0.6%, 95% confidence interval [CI]: –1.1 to –0.1%, P = 0.02). All clinical outcomes were similar between normal weight and underweight women. Overweight women, instead, showed higher miscarriage rate per clinical pregnancy (n = 20/75, 26.7% versus n = 67/461, 14.5%; odds ratio [OR] adjusted for blastocyst quality and day of full blastulation: 2.0, 95% CI: 1.1–3.6, P = 0.01) and lower LBR per SET (n = 55/154, 35.7% versus n = 388/859, 45.2%; OR adjusted for blastocyst quality and day of full blastulation: 0.67, 95% CI: 0.46–0.96, P = 0.03). ConclusionThese data indicate a need for future research on more sensitive metrics to assess body fat mass and distribution, as well as on the mechanisms leading to lipotoxicity, thereby impairing embryo competence and/or endometrial receptivity. Overweight women should be informed of their higher risk for miscarriage and, whenever possible, encouraged to lose weight, especially before transfer.

Methylenetetrahydrofolate reductase gene polymorphisms are not associated with embryo chromosomal abnormalities and IVF outcomes

ABSTRACT The aim of our study was to investigate the effect of maternal and embryo MTHFR C677T and A1298C polymorphisms on embryo aneuploidies and mosaicism and the correlation between these genetic variants in transferred euploid embryos and IVF outcomes. MTHFR genotype was analyzed in 77 women who performed an IVF/ICSI cycle with PGT-A. Moreover, to evaluate the effect of embryo MTHFR polymorphisms on embryo aneuploidies and mosaicism, the MTHFR genotype was analyzed in 191 biopsied embryos from the PGT-A cycles of these patients. Additionally, 218 DNA samples from trophectoderm biopsies belonging to a different group of patients were also genotyped. MTHFR polymorphisms were analyzed in a total amount of 409 trophectoderm samples. The main parameters analyzed were embryo aneuploidy and mosaicism rates. Finally, the IVF outcomes of 241 single euploid embryo transfers were assessed and compared between different MTHFR embryo genotypes. The aneuploidy rates were similar in embryos from homozygous normal women and women with at least one mutated allele (54.7% vs. 30.2% in 677C>T and 37.8% vs. 42.7% in 1298A>C). Furthermore, no differences were observed in the mosaicism rate (24.0% vs. 13.8% in 677C>T and 17.1% vs. 17.3% in 1298A>C). A similar analysis was performed, taking into account the embryo genotype results. No differences in aneuploidy rate were observed between the study groups. The only significant difference was the mosaicism rate among 677C>T genotype (13.5% in 677CC group vs. 5.4% in 677CT/TT; p = 0.019). Implantation rate, biochemical and clinical miscarriage rates, and ongoing pregnancy rate were compared between different embryo genotypes, and no statistically significant differences were found. In conclusion, the maternal MTHFR genotype did not influence embryo chromosomal abnormalities. Moreover, the embryo MTHFR genotype was not associated with embryo aneuploidy or IVF outcomes such as implantation, pregnancy loss, and ongoing pregnancy when euploid embryos were transferred. Abbreviations: MTHFR: methylenetetrahydrofolate reductase; IVF: in vitro fertilization; PGT-A: preimplantation genetic testing for aneuploidies; SAM: S-adenosyl methionine; SNP: single nucleotide polymorphism; SPSS: Statistical Package for Social Sciences; RIF: recurrent implantation failure; RPL: recurrent pregnancy loss; hCG: human chorionic gonadotropin; PBS: phosphate buffered saline; CGH: comparative genomic hybridization; NGS: next generation sequencing

The effect of endometrial thickness on live birth outcomes in women undergoing hormone-replaced frozen embryo transfer

ObjectiveTo determine the impact of endometrial thickness on live birth outcomes and obstetric complication rate after hormone-replaced frozen embryo transfer.DesignRetrospective cohort study.SettingLarge, urban, academic fertility center.Patient(s)All patients with a singleton live birth after single euploid embryo transfer (by array comparative genomic hybridization or next-generation sequencing) in a hormone-replaced frozen embryo transfer cycle between January 2017 and December 2018 were reviewed.Intervention(s)None.Main Outcome measure(s)The primary outcomes were birth weight and obstetric complication rate.Result(s)A total of 492 patients were included. The median endometrial thickness was 8.60 mm (range, 6.0–20.0). The median gestational age at live birth was 39.4 weeks with a median birth weight of 3,345.2 g. Endometrial thickness was significantly correlated with birth weight. When patients were dichotomized into groups (those with an endometrial thickness of <7 mm and those with an endometrial thickness of >7 mm), neonates born from endometria with a thickness of <7 mm were born earlier (37.3 vs. 39.4 weeks and born with lower birth weights (2,749.9 vs. 3,345.2 g). It should be noted that only seven patients had an endometrium measuring <7 mm. Moreover, 7.1% (n = 35) of patients had an obstetric complication. Endometrial thickness was not significantly associated with obstetric complications, even with adjustments for age and medical history.Conclusion(s)Endometrial thickness may be a valuable predictor of placental health and birth weight. Further study is required to examine the relationship with individual obstetric complications, as our study may not have been powered to observe differences in obstetric complication rate, as well as the relationship between endometrial thickness and outcomes in natural frozen embryo transfer cycles.

Recurrent implantation failure: how common is it?

To clarify a lingering issue, the true incidence of repeated implantation failures (RIF) in women undergoing successive frozen euploid single embryo transfers (FE-SET). As not all Assisted reproductive techinique (ART) attempts are crowned by success, it has been questioned since incept of ART whether failures resulted from an embryonic or endometrial cause. RIF has received no precise definition but a trend has existed toward setting a more stringent definition, as reproductive biology has become more effective and ART success rates improved. No scientific society has yet convened on a universally accepted definition. The advent of effective and well tolerated pregestational testing of embryos for aneuploidy (PGT-A) has allowed to not transfer aneuploid embryos, which are bound not to succeed. This, therefore, justify revisiting the concept of RIF when only euploid embryos are transferred. Contrary to lingering beliefs, the results of our study indicate that RIF following three successive euploid embryo transfers in a morphologically normal endometrium is a rare occurrence (<5%). This supports the concept that ART failures mainly result from embryonic causes. Our data also propose a new - functional - definition of RIF being an ART failure following 3 successive FE-SET attempts. Our findings, therefore seriously question the soundness of prescribing the often complex and expensive endometrial testing procedures that largely publicized for treating RIF.

The association between prior cesarean delivery and subsequent in vitro fertilization outcomes in women undergoing autologous, frozen-thawed single euploid embryo transfer

The rates of cesarean deliveries continue to increase worldwide. Previous work suggests an association between a previous cesarean delivery and reduced fertility in natural conception and invitro fertilization treatment cycles. To our knowledge, there is no published research that explored the relationship between a previous cesarean delivery and the clinical outcomes after invitro fertilization and the subsequent transfer of a single frozen-thawed euploid embryo. This study aimed to investigate the relationship between the previous mode of delivery and subsequent pregnancy outcomes in patients undergoing a single frozen-thawed euploid embryo transfer after invitro fertilization. A retrospective cohort study was performed at a single academic fertility center from January 2012 to April 2020. All women with a history of a live birth undergoing autologous, frozen-thawed single euploid embryo transfers were identified. Cases included patients with a single previous cesarean delivery; controls included patients with a single previous vaginal delivery. Only the first embryo transfer cycle was included. The primary outcome was the implantation rate. Secondary outcomes included ongoing pregnancy and live birth rates, biochemical pregnancy rate, and clinical miscarriage rate. A total of 525 patients met the inclusion criteria and were included in the analysis. Patients with a previous cesarean delivery had a higher body mass index (24.5±4.5 vs 23.4±4.1; P=.004) than those in the vaginal delivery cohort; the rest of the demographic data were otherwise similar. In a univariate analysis, the implantation rate was significantly lower in patients with a previous cesarean delivery (111/200 [55.5%] vs 221/325 [68.0%]; P=.004). After adjusting for the relevant covariates, a previous cesarean delivery was associated with a 48% reduction in the odds of implantation (adjusted odds ratio, 0.52; 95% confidence interval, 0.34-0.78; P=.002). In addition, after adjusting for the same covariates, a previous cesarean delivery was significantly associated with a 39% reduction in the odds of an ongoing pregnancy and live birth (adjusted odds ratio, 0.61; 95% confidence interval, 0.41-0.90; P=.01). There were no differences in the biochemical pregnancy rates or clinical miscarriage rates. This study demonstrated a marked reduction in implantation and ongoing pregnancy and live birth associated with a previous cesarean delivery in patients undergoing a single euploid embryo transfer. Our work stresses the importance of reducing the primary cesarean delivery rates at a national level and elucidating the mechanisms behind the substantially lower implantation rates after a cesarean delivery.