Sustained Implantation Rates Research Articles

Does Recurrent Implantation Failure Exist? Prevalence and Outcomes of Five Consecutive Euploid Blastocyst Transfers in 123 987 Patients

(Abstracted from Hum Reprod 2024;39(5):974–980 Recurrent implant failure (RIF) has been defined as repeated failed implantations following multiple embryo transfers. A landmark paper on RIF reported the cumulative sustained implantation and live birth rates achieved with 3 consecutive single euploid blastocyst transfers and found a similar live birth rate across the first 3 consecutive transfers with a “true” unexplained RIF rate of <5% in the in vitro fertilization population.

P-056 NOA-SERA: combined autologous progenitor cells and platelet-derived growth factors for male factor infertility due to non-obstructive azoospermia and severe oligoasthenoteratozoospermia (OATS) – a case series

Abstract Study question Can NOA-SERA be utilizing autologous progenitor cells and platelet-derived growth factors,effectively address male factor infertility in non-obstructive azoospermia (NOA) and severeoligoasthenoteratozoospermia (OATS) Summary answer Intra-testicular NOA-SERA transplantation of autologous progenitor cells andgrowth factors shows promise in improving spermatogenesis and fertilization potential,presenting potential breakthrough for male factor infertility. What is known already Male infertility affects 10–15% of couples, with non-obstructive azoospermia (NOA)diagnosed in 60% of azoospermic men. Current interventions, including platelet-rich plasma and stem cells, show variable success. This study introduces a novel protocol, NOA-SERA, combining autologous mobilized progenitor cells and platelet-derived growth factors to restore spermatogenesis. Despite advancements in assisted reproduction, male factor infertility due to primary NOA remains challenging. This case series explores the safety and efficacy of NOA-SERA, avoiding in vitro breeding risks and emphasizing intratesticular transplantation’s safety and efficacy Study design, size, duration his case series (March 2019–November 2023) includes 7 male factor infertility patients,consisting of 4 non-obstructive azoospermic NOA, 1 cryptozoospermia and 2 severe oligoaesthenoteratospermic (OATS, strongly desirous of pregnancy with self-gamete. All patients received NOA-SERA following written consent. One-year follow-up included hormonal profiling, semen analysis, and testicular biopsy assessment Participants/materials, setting, methods Seven men, with normal karyotype, underwent NOA-SERA due to male factor infertility. This intervention involved intra-testicular transplantation of autologous CD34+/CD133+ enriched peripheral blood mononuclear stem cells (MPB-MNCs) combined with platelet derived growth factors. The procedure, avoiding in vitro breeding and manipulation, prioritized safety and efficacy. Follow-up for one year included hormonal profile, semen analysis, testicular biopsy assessments, clinical pregnancy (b-hCG), sustained implantation (>8 weeks), and live birth rates were followed. Main results and the role of chance Analysis of NOA-SERA treatment in this case series revealed positive outcomes in 70% of patients, showing increased testicular size, elevated testosterone, and reduced FSH levels. In our study, out of 4 NOA, 2 NOA remained NOA, 1 progressed to OATS, 1 progressed to normal semen analysis after 3 months. Couple conceived with intrauterine insemination (IUI) first pregnancy and they also had a subsequent natural conception. 1 Crypto progressed to OATS and 1 severe OATS remained the same and one progressed to OATS. Although no appearance of sperm in the ejaculate was observed, 2 patients displayed sperms in Testicular Sperm Aspiration (TESA) sufficient for ICSI. Couples undergoing IVF therapy post-biopsy achieved successful fertilization in two cases, leading to positive pregnancies and sustained implantation and livebirth of healthy full-term babies. Post-procedure FSH was slightly lower than pre-procedural levels, indicating hormonal improvement without observed therapy-related deterioration. The novelty lies in the intratesticular transplantation of mobilized purified autologous CD34+/CD133+ enriched MPBMNCs with platelet-derived growth factors, avoiding in vitro risks.” To summarise, 3 out of 7 achieved successful pregnancy, 2 NOA with ICSI, 1 NOA progressed to normal semen analysis and pregnancy with IUI Limitations, reasons for caution Despite interesting results, limitations include the self-controlled case series design and asmall patient cohort, lacking a comparison to a control group. Future studies should include more patients, stratified for age, aetiology, and other risk factors, to further elucidate this intervention’s mechanism of action Wider implications of the findings NOA-SERA demonstrates promise in enhancing spermatogenesis and fertilization potential, emphasizing necessity for larger randomized trials to ascertain clinical efficacy, particularly in subgroups of OATS&NOA patients. NOA-SERA emerges as safe, cost-effective adjuvant for optimizing spermatogenesis and improving IVF success and pregnancy outcomes in male factor infertility. Trial registration number Not applicable

A pilot study to investigate the clinically predictive values of copy number variations detected by next-generation sequencing of cell-free deoxyribonucleic acid in spent culture media

To investigate the positive predictive value and false positive risk of copy number variations (CNV's) detected in cell free deoxyribonucleic acid (DNA) from spent culture media for nonviable or aneuploid embryos. Diagnostic/prognostic accuracy study. Patients aged 35 and younger with an indication for IVF-ICSI and elective single frozen embryo transfer at a single, private IVF center. Embryo selection was performed according to the conventional grading, blinded to noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) results. After clinical outcomes were established, spent culture media samples were analyzed. Prognostic accuracy of CNVs according to niPGT-A results to predict nonviability or clinical aneuploidy. One hundred twenty patients completed the study. Interpretations of next-generation sequencing (NGS) profiles were as follows: 7.5% (n = 9) failed quality control; 62.5% (n = 75) no CNVs detected; and 30% (n = 36) abnormal copy number detected. Stratification of abnormal NGS profiles was as follows: 15% (n = 18) whole chromosome and 15% (n = 18) uncertain reproductive potential. An intermediate CNV was evident in 27.8% (n = 5) of the whole chromosome abnormalities. The negative predictive value for samples with no detected abnormality was 57.3% (43/75). Whole chromosome abnormality was associated with a positive predictive value of 94.4% (17/18), lower sustained implantation rate (5.6%, 1/18), and higher relative risk (RR) for nonviability compared with no detected abnormalities (RR 2.21, 95% CI: 1.66-2.94). No other CNVs were associated with significant differences in the sustained implantation or RRs for nonviability. Unequal sex chromosome proportions suggested that maternal contamination was not uncommon. A secondary descriptive analysis of 705 supernumerary embryos revealed proportions of NGS profile interpretations similar to the transferred cohort. Significant median absolute pairwise differences between certain subcategories of CNV abnormalities were apparent. Whole chromosome abnormalities were associated with a high positive predictive value and significant RR for nonviability. Embryos associated with other CNVs had sustained implantation rates similar to those with no abnormalities detected. Further studies are required to validate the clinical applicability of niPGT-A. clinicaltrials.gov (NCT04732013).

Assisted Reproductive Technologies (ART) Failures: Is It the Seed or the Soil?

Assisted reproductive technologies (ART) have accomplished spectacular progresses over the last few years, leading to sustained implantation rates (sIR) [Formula: see text]60% following euploid blastocyst transfers. These results raise new challenges for infertility specialists, notably the ability to care for infertile couples who fail ART, sometimes recurrently. ART failures may be due to disorders residing in the endometrium (the soil) or embryo (seed). To simplify the review of this dilemma and limit variables, we focused our research on the outcome of euploid blastocysts transfers conducted in E2 and i.m. progesterone replacement cycles. This methodological choice for our research should not be confused with a therapeutic recommendation in case of ART failures. The sum of the data collected indicates that if recurrent implantation failure (RIF) exists, it is extremely rare, affecting only [Formula: see text]5% of ART couples.

P-473 The reproductive performance of euploid blastocysts declines after each consecutive embryo transfer: The pendulum is swinging back?

Abstract Study question the impact of euploid blastocysts on reproductive performance by association between the number of previous failed IVF cycles and live birth rate per transfer Summary answer Our findings suggest a negative association between the number of previous failed IVF cycles per euploid transfers. implantation rates decreased following each consecutive FE-ET cycles What is known already In assisted reproductive technologies, implantation failure presents a challenging clinical dilemma since its underlying etiology is unknown. Implantation is a very complex process which involves a variety of factors related to the embryo, endometrium, and immune system (Fox et al. 2016). Therefore, implantation failure is also a complex pathological condition that involve both embryonic defects and/or endometrial dysfunctions (Das et al.2012). This situation poses a therapeutic dilemma. Hence implantation failure etiologies and treatments remain unresolved. which results in ongoing debate over whether embryonic chromosomal aneuploidy is a major contributor or within the endometrium itself. Study design, size, duration This observational cohort study included 387 patients undergoing euploid embryo transfer between January 2015 and until October 2022. Participants/materials, setting, methods Participants: Women with an anatomically normal uterus who underwent autologous vitrified-warmed euploid blastocyst transfer (Frozen Euploid Embryo Transfer: FE-ET) cycles were included in the study. Setting: Fertility center at a university-affiliated public hospital. Intervention(s): Trophectoderm biopsy and comprehensive preimplantation 24-chromosome analysis using Next Generation Sequencing (NGS). Main Outcome Measure(s): Cumulative outcomes from these cycles were analyzed. A logistic regression model was used to assess the differences in outcomes between the first, second, and third FE-ET. Main results and the role of chance The mean age of the patient population under investigation was 33.4 ± 5.3 years. Maternal age at oocyte retrieval was negatively associated with the mean euploidy rate per cohort of biopsied blastocysts from each patient (m-ER). The m-ER did not show any association with the number of previous failed IVF cycles (0/1-2/≥3) among different ranges of maternal age at oocyte retrieval (≤30/31-35/36-39/≥40). The transfer of the first single vitrified-warmed euploid blastocyst was associated with an implantation rate (IR) of 47%, a miscarriage rate of 17.9% and a live birth rate (LBR) of 33.9%. Miscarriage rate and LBR were sensitive to the number of previous failed IVF cycles, showing statistically significant differences between women with no previous and ≥3 failed IVF cycles (11.3% versus 27.8%, P = 0.04) and (40.7% versus 26.3%, P = 0.05) respectively. Sustained implantation rates of the first, second, and third FE-ET were 47%, 40.5%, and 30% per transfer, respectively. The sustained IR was sensitive to the number of consecutive euploid blastocyst transfers (P = 0.03). Limitations, reasons for caution The retrospective nature of the study. The data should also be verified using a multicenter approach, to increase the sample size. Wider implications of the findings After experiencing multiple unsuccessful IVF cycles, transferring euploid blastocysts leads to improved clinical outcomes, but these improvements eventually level off. Additional information beyond embryo euploidy is needed to further enhance clinical outcomes. Trial registration number not applicable

EMBRYOS DIAGNOSED AS PUTATIVE MOSAIC BY THE PGTSEQ PGT-A PLATFORM HAVE A SIMILAR SUSTAINED IMPLANTATION RATE AS THOSE NEGATIVE FOR PUTATIVE MOSAICISM: A BLINDED NON-SELECTION STUDY

The objective of the current study was to determine the sustained implantation rates (SIR) of mosaicism masked, whole chromosome aneuploid (WCA) negative embryo transfers that after unmasking of the raw data were putative mosaic, in comparison to transfers of WCA-negative embryos without any signs of mosaicism.

O-138 Covid-19 vaccine does not affect sustained implantation rates after single euploid embryo transfer, a retrospective study with 4868 cases

Study questionIs sustained implantation rate (SIR) of covid-19 vaccinated women diminished in assisted reproduction treatments due to endometrial receptivity harm?Summary answerSIR of euploid embryos remains constant regardless vaccination and doses applied, but impact of interval last dose-embryo transfer needs to be further evaluated.What is known alreadyLittle is known about the effects of both covid infection and vaccines on endometrial receptivity of women attempting motherhood.There is a generalized concern about potential secondary effects of covid vaccine on many health areas, and assisted reproduction is not an exception. Then, it is mandatory on the current epidemic context to evaluate if either infection or its preventive treatment may interfere reproductive physiology of infertile patients.ART offers a robust model to study this problem by controlling oocyte and embryo quality with the use of PGT-A, then enabling the study of endometrial independent contribution to reproductive success.Study design, size, durationRetrospective study analyzing two cohorts, historical cohort of PGT-A cases using own oocytes one year pre-pandemia, and post vaccination initiation from women already having received one or two doses.Patients undergoing single embryo transfers (ET) after PGT-A were included, to be able to discern purely endometrial factors of sustained implantation. Means and proportions with their corresponding 95%CI (within brackets) were calculated, and crude/adjusted odds ratios calculated for the main outcomes, SIR and clinical pregnancy rate (CPR)Participants/materials, setting, methodsA total of 4868 ET were included on this study, 3272 for the control, non vaccinated group, vs 890 from women already vaccinated with at last one dose at the time of ET.The main outcomes were CPR per embryo transfer (presence of a sac by ultrasonography on week 7th), and SIR (fetal heartbeat at week 12th). Crude and adjusted odds ratio were calculated, using logistic regression models to control for potentially confounding variables.Main results and the role of chanceMean age was 38.3 years 95%CI(38.2-38.4), BMI, 23.2kg/m2 95%CI(23.1 23.4), fresh oocytes on 80%, mixed 16.4% and vitrified on 3.6% of cases. Donor sperm used on 12.8% of treatments, and testicular retrieved sperm on 2.5% of them. Day of embryo transfer was D5 on 71.3% and D6 on 28.6% cases.CPR per ET was 70.6% 95%CI(69.3-71.9) in the control group and 70.4%95%CI(67.4-73.4) on vaccinated, OR 0.994 95%CI(0.849-1.163), and after adjustment by patient’s age, oocyte age, source of sperm, donor sperm use, day of ET, use of vitrified oocytes and BMI, AdjOR 1.039 95%CI(0.876-1.233), while SIR in the controls was 64.3% 95%CI(62.7-66.0) vs. 62.6% 95%CI(58.8-66.4) on vaccinated, with OR 0.929 95%CI(0.777-1.110) and AdjOR 0.981 95%CI(0.807-1.192), p > 0.05.Those patients having received only one dose or two doses by the time of ET, showed comparable results, on both CPR and SIR.Concerning data categorized per time quartiles (from vaccine to ET), while CPR was comparable, SIR, on the first quartile (Q1) was 66.5%, while Q2 was 68.0%, Q3 66.3%, and Q4 50.4%, and using Q1 as reference, ORQ2-Q1 1.073 95%CI(0.680-1.693), ORQ3-Q1 0.869 95%CI(0.545-1.385) and OR Q4-Q1 0.512 95%CI(0.321-0.818), p = 0.009 while after adjustment AdjORQ2-Q1 0.965 95%CI(0.585-1.594), AdjORQ3-Q1 0.834 95%CI(0.492-1.413) and AdjORQ4-Q1 0.533 95%CI(0.316-0.899), p = 0.018.Limitations, reasons for cautionThis is a retrospective study, and although controlled statistically, possible biases due to the nature of the work remain possible, and a cause-effect link cannot be purely drawn from it. Further prospective studies on the potential effect of covid vaccines on reproductive outcomes are still needed.Wider implications of the findingsOur results send a message of reassurance to patients in the process of assisted reproductive treatment regarding the potential negative impact of the vaccine on endometrial receptivity and reproductive outcomes. Aiming motherhood is no reason for delaying vaccination against covid-19.Trial registration numberNot applicable

EXPOSURE OF OVARIES TO COVID-19 VACCINATION DOES NOT IMPAIR FERTILITY

Misinformation regarding Covid vaccination has contributed to vaccine hesitancy. Initially, there were claims that immune cross reactivity between the SARS CoV-2 spike protein and syncytin-1 would prevent embryo implantation. We previously demonstrated no difference in implantation and sustained implantation rates between previously vaccinated or infected women compared to other women.1 More recently, misinterpretation of vaccine biodistribution data has led to a second claim that mRNA containing lipid nanoparticles are concentrated in the ovaries and spike protein produced there would also cause infertility. The purpose of this study is to determine whether prior in vivo ovarian exposure to lipid nanoparticle-mRNA vaccination against SARS-CoV2 spike protein reduces subsequent fertility in women. This is an ongoing observational study of women undergoing frozen embryo transfer with a single expanded blastocyst. This is an interim report (n =128) encompassing transfers between Jan 1 and Jul 02. All patients had serum analyzed prior to starting stimulation for egg retrieval to quantitatively determine the level of Anti-SARS-CoV-2 Spike IgG. Reactive (antibody positive) patients were questioned to determine a history of vaccination or infection. Patients were divided into three groups based on their status. Women who were vaccinated (n = 26); women who had previous infection with SARS-CoV-2 (n=11) and women without a history of either vaccination or infection (n=91). Only patients receiving the mRNA vaccines from BioNTech / Pfizer (BNT162b2) and Moderna (mRNA-1273) were analyzed. Outcome measure for the three groups were initial implantation rate (serum hCG level > 5 mIU/mL obtained 8 days after embryo transfer followed by a rising level two to three days later), sustained implantation rate (transvaginal ultrasound documented positive FHTs at two time points at least one week apart) and miscarriage rate (the difference between initial and sustained implantation rates). Baseline characteristics were analyzed using ANOVA. Chi square analysis was used to compare pregnancy rates. Tabled 1Non-reactiveVaccinationInfectionP ValueTotal transfers912611Implantation rate86.8% (n=79)84.6% (n=22)90.9% (n=10)0.19Sustained implantation rate63.7% (n=58)61.5% (n=16)54.6% (n=6)0.10Miscarriage rate23.1%23.1%36.3%0.15 Open table in a new tab Embryos produced from oocytes exposed in vivo to lipid nanoparticles containing mRNA for the SARS CoV-2 spike protein are not less likely to produce pregnancy or more likely to miscarry.

Effect of age and morphology on sustained implantation rate after euploid blastocyst transfer

Research questionWhat impact does maternal age and embryo morphology have on sustained implantation rates of euploid blastocysts? DesignThis was a retrospective analysis of sustained implantation rates of euploid blastocysts stratified by maternal age and morphology. The primary analysis included 208 embryo transfers with a total of 229 embryos transferred from January 2017 through August 2020. ResultsFor all ages the sustained implantation rates for day 5 good quality blastocysts were higher than for day 5 fair, day 5 poor and day 6 blastocysts. At a maternal age of 36 years the best-fit sustained implantation rates were 86% for day 5 good quality blastocysts, 64% for day 5 fair, 63% for day 5 poor, and 51% for all day 6 blastocysts analysed as one group. When controlling for morphology and day of biopsy, there were higher sustained implantation rates for euploid embryos of younger patients compared with older patients. The best-fit sustained implantation rates for age 33 compared to age 39 years were 86% versus 80% for day 5 good, 71% versus 62% for day 5 fair, 59% versus 55% for day 5 poor, and 81% versus 46% for all day 6. ConclusionsThere was a clinically significant higher sustained implantation rate at all ages for euploid day 5 good quality embryos compared with day 5 fair, day 5 poor and day 6 embryos.

SARS-CoV-2 spike protein seropositivity from vaccination or infection does not cause sterility

Several reports claim that the purported similarity between syncytin-1 and the SARS-CoV-2 spike protein may induce immune cross-reactivity resulting in female sterility. We used frozen embryo transfer as a model for comparing the implantation rates between SARS-CoV-2 vaccine seropositive, infection seropositive, and seronegative women. No difference was found in serum hCG documented implantation rates or sustained implantation rates between the three groups. Reports claiming that COVID-19 vaccines or illness cause female sterility are unfounded.

The Appraisal of Body Content (ABC) trial: Increased male or female adiposity does not significantly impact in vitro fertilization laboratory or clinical outcomes

To investigate the impact of obesity as determined by bioelectric impedance analysis (BIA) and body mass index (BMI) on invitro fertilization (IVF) laboratory and clinical outcomes. Prospective cohort study. Academic-affiliated private practice. A total of 1,889 infertile couples undergoing IVF from June 2016 to January2019. Female patients and male partners underwent BIA and BMI measurement at the time of oocyte retrieval. Embryology and clinical outcomes were prospectively tracked with comparison groups determined by percentage of body fat (%BF) and BMI categories. Fertilization rate, blastocyst formation rate, euploidy rate, miscarriage rate, sustained implantation rate, live birth rate, rates of low birth weight/very low birth weight, prematurity rates. Fertilization rates and euploidy rates were equivalent among all women. Blastocyst formation rates were slightly higher (55%) in women with an obese %BF compared with all other %BF categories (51%); however, this trend was not noted in women defined as obese by BMI. Miscarriage rates, sustained implantation rates, and live birth rates were equivalent among all women. The rate of very low birth weight was low but increased in obese women (3%) versus underweight, normal-weight, and overweight counterparts (0%-1.3%) as determined by %BF and BMI. Obesity in men did not significantly affect any embryologic or clinical outcomes. Although maternal obesity imposes a small but increased risk of very low birth weight infants, most embryology and pregnancy outcomes are equivalent to normal weight patients. Paternal obesity does not appear to affect IVF, pregnancy, or delivery outcomes.

Rate of true recurrent implantation failure is low: results of three successive frozen euploid single embryo transfers

To study the true prevalence of recurrent implantation failure. Retrospective cohort study. A private assisted reproductive technology center. Women (n = 4,429) with anatomically normal uterus who underwent up to three consecutive frozen euploid single embryo transfers (FE-SETs) were included in the study. Cycles with donor eggs or gestational carriers were excluded. None. Cumulative outcomes from these cycles were analyzed. A logistic regression model was used to assess the differences of outcomes between first, second, and third FE-SET and a Kaplan-Meier curve as used to analyze cumulative implantation rate. The mean age of the patients included in the study was of 35.4 years. The sustained implantation rates of the first, second, and third FE-SET were 69.9%, 59.8%, and 60.3% per transfer, respectively. The cumulative sustained implantation rate after up to three consecutive FE-SET was 95.2%. The live birth rates after the first, second, and third FE-SET were 64.8%, 54.4%, and 54.1% per transfer, respectively. The cumulative live birth rate after up to three consecutive FE-SET was 92.6%. The miscarriage rate after observing a positive heartbeat was not different between the first (7.2%), second (8.8%), and third (12.7%) FE-SET. Our findings suggest that true recurrent implantation failure is rare. For those patients with the ability to make euploid blastocysts, <5% would fail to achieve a clinical pregnancy with three embryos transferred. It remains to be further investigated whether this threshold identifies a truly recalcitrant group or simply a statistical certainty based on random variation.

A MULTI-CENTER, PROSPECTIVE, BLINDED, NON-SELECTION STUDY EVALUATING THE PREDICTIVE VALUE (PV) OF AN ANEUPLOID DIAGNOSIS WITH PGT-A AND THE IMPACT OF BIOPSY

Two concerns regarding PGT-A are: 1) embryos labeled aneuploid may be reproductively competent and thus may be wrongfully discarded, and 2) trophectoderm (TE) biopsy may have an adverse effect on embryo reproductive potential. This study addresses these concerns by 1) directly measuring the predictive value (PV) of an aneuploid PGT-A diagnosis, and 2) comparing sustained implantation rates (SIR) of the study group to a control group, in which biopsy/ PGT-A were not utilized.

Varying levels of serum estradiol do not alter the timing of the early endometrial secretory transformation.

Do supraphysiologic estradiol (E2) levels in the ranges attained during normal and high response superovulation cycles modify the onset of endometrial secretory transformation? Highly supraphysiologic levels of E2 do not alter the ability of physiologic levels of progesterone (P4) to induce secretory transformation. Previous studies have demonstrated that premature P4 elevations during IVF cycles are associated with a decrement in clinical pregnancy rates after fresh embryo transfer due to shifts in the window of implantation (WOI). However, alterations in the onset of secretory transformation may not apply uniformly to all patients. High responders with supraphysiologic E2 levels accompanied by similar subtle increases in P4 have not been shown to have decreased sustained implantation rates. This prospective investigation in which whole-genome transcriptomic and methylomic analysis of the endometrium is performed for individual patients under a range of E2 concentrations brings clarity to a long-debated issue. A randomized, prospective and paired trial was conducted in which 10 participants were enrolled and randomized to the order in which they completed three distinct uterine stimulation cycles, each at a specific E2 concentration: physiologic (∼180 pg/ml), moderately supraphysiologic (600-800 pg/ml) or supraphysiologic (2000 pg/ml). Target E2 ranges were selected to mimic those seen in natural, controlled ovarian stimulation and IVF cycles. E2 valerate was administered in order to maintain stable E2 levels for 12 days followed by intramuscular P4 in oil 10 mg/day for two doses, after which an endometrial biopsy was performed. A total of 30 endometrial biopsies were included in a whole-genome transcriptomic and methylomic analysis. Healthy volunteers without a history of infertility were included in this study at a single large infertility center. DNA was isolated from the endometrial biopsy specimens and bisulfite sequencing was performed to construct a methylation array. Differential methylation analysis was conducted based on differences in M-values of individuals across treatment groups for each probe as well as carrying out t-tests. RNA was isolated for RNA-Seq analysis and gene expression values were compared using DESeq2. All analyses were performed in a pairwise fashion to compare among the three stimulation cycles within individuals and secondarily to compare all participants in each of the cycles. The mean peak E2 and P4 levels were 275 pg/ml and 4.17 ng/ml in the physiologic group, 910 pg/ml and 2.69 ng/ml in the moderate group was, and 2043 pg/ml and 2.64 ng/ml in the supraphysiologic group, respectively. Principal component analysis of 834913 CpG sites was performed on M-values of individuals within the low, moderate and supraphysiologic conditions in a paired approach. There were no differences in genome-wide methylation within participants across E2 groups. A paired analysis revealed that gene expression profiles did not differ within the same individual at each of the three E2 levels. No significant alterations in gene expression as related to endometrial physiology were identified between the low, moderate and supraphysiologic groups in an inter-participant analysis. Although each participant completed a physiologic cycle in which E2 levels were maintained in a range that would simulate a natural cycle, our findings are limited by lack of an unmedicated control to assess if there was a potential effect from E2V. Additionally, our results were obtained in fertile individuals, who may have a different endometrial response compared to an infertile population. Despite the whole genomic endometrial assessment and rigorous, paired study design, the sample size was limited. Given that the endometrial response to P4 is unaffected by E2 levels in the supraphysiologic range, diminutions in implantation seen in stimulated cycles may result from embryonic-endometrial dyssynchrony following early P4 elevations or slowly blastulating embryos, which occur independently of the magnitude of the E2 rise. This study was funded by the Foundation for Embryonic Competence, Basking Ridge, NJ, USA. Dr E.S. reports consultancy work for The Foundation for Embryonic Competence, Basking Ridge, NJ, USA. The other authors declare no conflict of interests related to this topic. NCT02458404.

Evaluation of fertilization, usable blastocyst development and sustained implantation rates according to intracytoplasmic sperm injection operator experience.

Is intracytoplasmic sperm injection (ICSI) operator experience associated with fertilization, usable blastocyst development and sustained implantation rates (SIR) when at least two embryologists carry out ICSI for a single cohort of oocytes? A retrospective cohort study of all IVF/ICSI cycles at a single large infertility centre between 2008 and 2018. Cycles were included if a cohort of oocytes was split between two embryologists for ICSI. The embryologist's experience of ICSI was used to evaluate laboratory and clinical outcomes overall and by pairs of inseminating embryologists. Logistic regression, analysis of variance and Kruskal-Wallis testing were used where appropriate. Analysis of 14,362 ICSI procedures showed an association between least ICSI experience and lower mean fertilization rates (P < 0.0001), higher odds of failed fertilization (adjusted OR 4.3; P < 0.0001) and lower number of fertilization 'wins' per cohort (P < 0.0001). Usable blastocyst development rates (number of usable blastocysts/number of two pronuclear zygotes) were not associated with ICSI embryologist experience (P = 0.44), but the odds of obtaining no usable blastocysts were higher (adjusted OR 1.4; P < 0.0001) and the proportion of usable blastocyst 'wins' was lower (P = 0.0001) when embryologists with the least experience carried out ICSI. Increased ICSI experience was associated with higher mean SIR (P < 0.0001). Laboratory and clinical outcomes were similar among embryologists once 1000 ICSI cycles and above were carried out. Increased ICSI operator experience is associated with higher fertilization rates, SIR and a lower likelihood of failed fertilization and usable blastocyst development. Splitting a single oocyte cohort between more than one embryologist for ICSI is a quality-control measure that can be implemented.

The Rate of True Recurrent Implantation Failure (RIF) Is Low: Results of Three Successive Frozen Euploid Single Embryo Transfers (FE-SET)

OBJECTIVE: To determine the true prevalence of recurrent implantation failure (RIF). DESIGN Retrospective cohort study METHODS: Women (n=4,429) with anatomically normal uterus who underwent up to three consecutive euploid frozen SETs were included in the study. Cycles with donor eggs or gestational carriers were excluded. Cumulative outcomes from these cycles were analyzed. A logistic regression model was employed to assess the differences of outcomes between first, second, and third euploid SET and a Kaplan-Meier curve as utilized to analyze cumulative implantation rate. RESULTS: The mean age of the patients included in the study was of 35.4±4.2 years. The sustained implantation rates (SIR) of the 1 st , 2 nd and 3 rd FE-SET were 69.9%, 59.8%, and 60.3% per transfer, respectively. The cumulative SIR after up to 3 consecutive FE-SET was 95.2% (95% CI: 94.0%-96.2%). The live birth rates (LBRs) after the 1 st , 2 nd and 3 rd FE-SET were 64.8%, 54.4%, and 54.1% per transfer, respectively. The cumulative LBR after up to 3 consecutive FE-SET was 92.6% (95% CI: 91.2%-93.9%). The miscarriage rate after observing a positive heartbeat was not different between the 1 st (7.2%, 95%CI: 6.4%-8.2%), 2 nd (8.8%, 95% CI:6.3%-11.7%) and 3 rd (12.7%, 95%CI: 6.2%-22.0%) FE-SET. CONCLUSIONS: Our findings suggest that true RIF is rare. For those patients with the ability to make euploid blastocysts, less than 5% would fail to achieve a clinical pregnancy with 3 embryos transferred. It remains to be further investigated whether this threshold identifies a truly recalcitrant group, or simply a statistical certainty based on random variation. FUNDING STATEMENT: All funding was internal from one program – RMA New Jersey, Basking Ridge, US. No outside funding was provided, thus no commercial interest for anyone involved. RMA New Jersey does not collect any money for the performance of PGT-A and thus does not resale or profit from it. Patients were not charged for PGT-A screening. DECLARATION OF INTERESTS: E.S. is a consultant for and receives research funding from the Foundation for Embryonic Competence; he is co-founder and a shareholder of ACIS. All other authors have no competing interests to disclose. ETHICS APPROVAL STATEMENT: No embryos were biopsied specifically for the purpose of this study. Given the retrospective nature of this study, the access and processing of patient data was approved by the Advarra Institutional Review Board under a protocol for retrospective studies - protocol number Pro00027158.

Erratum. Worth the wait? Day 7 blastocysts have lower euploidy rates but similar sustained implantation rates as Day 5 and Day 6 blastocysts.

STUDY QUESTION Does the reproductive potential of embryos change when blastocyst development takes longer than the traditionally accepted 5 days when accounting for aneuploidy and endometrial-embryo asynchrony? SUMMARY ANSWER Aneuploidy increases with increasing duration of blastulation, but if blastocyst morphologic quality and endometrial-embryo asynchrony are controlled for, euploid Day 7 embryos have similar sustained implantation as compared to Days 5 and 6 euploid blastocysts. WHAT IS KNOWN ALREADY The relative contributions of diminished embryo quality versus endometrial and embryo asynchrony to poor outcomes associated with embryos cultured past Day 6 are not clear. Asynchrony can be eliminated by embryo vitrification with transfer in a subsequent month after retrieval. STUDY DESIGN, SIZE, DURATION Retrospective cohort study of patients from a single center attempting conception through ICSI and utilizing preimplantation genetic testing for aneuploidy screening (PGT-A) from January 2017 to September 2018. Cycles were excluded if they utilized surgical sperm or preimplantation genetic testing for monogenetic/single gene defects. ICSI cycle outcomes from 2586 patients were evaluated for ploidy status of embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS Only patients undergoing single, euploid frozen embryo transfer were included when analyzing cycle outcomes by day of blastocyst expansion of the transferred embryo (n = 2130). Ploidy rates by the day upon which an embryo was considered to be usable (denoted, 'usable blastulation day') were determined so as to assess the contribution of aneuploidy to slow embryo development. Outcomes of euploid frozen single embryo transfers (SET) of Day 7 embryos were evaluated to assess the reproductive potential associated with embryos that were slowly developing for reasons other than aneuploidy. Analyses were adjusted by maternal age and blastocyst morphology. MAIN RESULTS AND THE ROLE OF CHANCE Overall, 67.7% (n = 3508) of usable Day 5 blastocysts were euploid, 52.1% (n = 5560) of usable Day 6 blastocysts were euploid and 43.1% (n = 229) of usable Day 7 embryos were euploid (Day 5 versus Day 6: odds ratio (OR) 0.7 (95% CI, 0.64-0.76), P < 0.001; Day 5 versus Day 7: OR 0.56 (95% CI, 0.46-0.69), P < 0.001; Day 6 versus Day 7: OR 0.81 (95% CI, 0.67-0.99), P = 0.036). Stratified by Society for Assisted Reproductive Technology maternal age groups, a reduction in the prevalence of euploidy by increasing time to embryo blastulation was still seen. The sustained implantation rate (SIR) was similar after euploid SET of Days 5 and 6 embryos (overall, 68.9% (95% CI, 66.0-71.6) and 66.8% (95% CI, 63.8-69.7), respectively; P = 0.81). SIR after euploid Day 7 SET appeared slightly lower than that of Days 5 and 6 embryos (52.6% (95% CI, 35.8-69.0); (Day 5 versus Day 7: OR, 0.67 (95% CI, 0.32-1.41), P = 0.29; Day 6 versus Day 7: OR 0.58 (95% CI, 0.28-1.2), P = 0.14)) but did not achieve statistical significance. LIMITATIONS, REASONS FOR CAUTION The primary limitation is the low number of Day 7 blastocyst transfers that limits statistical power. Additionally, the retrospective nature of this study may prevent full elucidation of potential biases with respect to culture, morphologic assessment and selection of Day 7 embryos for transfer. WIDER IMPLICATIONS OF THE FINDINGS Routine culture through Day 7 may successfully increase the pool of transferrable embryos for patients who would otherwise have no usable embryos if culture terminated on Day 6. This is particularly true for older patients (i.e. greater than 35 years of age), whose embryos take longer to blastulate and, therefore, are more susceptible to cycle cancelation. Additionally, as evidenced by an adequate overall SIR of 52.6% after euploid SET of Day 7 blastocysts, embryos developing to a usable blastocyst on Day 7 are likely within the 'window of blastulation.' STUDY FUNDING/COMPETING INTEREST(S) None.

Embryonic aneuploidy rates are equivalent in natural cycles and gonadotropin-stimulated cycles

To determine if natural selection and follicular stimulation produces a lower risk for embryonic aneuploidy than that attained following superovulation with exogenous gonadotropins. Prospective observational with historical control group. Large academically affiliated private practice. All patients presenting for their evaluation for infertility were offered participation in the study. All participants in the natural cycle group underwent an unstimulated invitro fertilization (IVF) cycle. A subsequent frozen embryo transfer was performed if a euploid blastocyst was attained. Rates of embryonic aneuploidy attained in unstimulated IVF cycles were compared to those observed in age-controlled historical cohort undergoing conventional stimulated IVF cycles with exogenous gonadotropins. Aneuploidy rates were equivalent in unstimulated and stimulated IVF cycles. The prevalence of aneuploidy in natural cycles increased with the age of the female partner in a manner identical to that seen in stimulated IVF cycles. Finally, sustained implantation rates of euploid blastocysts were equivalent in natural and stimulated IVF cycles. Rates of embryonic aneuploidy are not impacted by follicular stimulation with exogenous gonadotropins. Prior concerns of inducing a higher risk of embryonic aneuploidy are not supported by this data. NCT01866618.

Worth the wait? Day 7 blastocysts have lower euploidy rates but similar sustained implantation rates as Day 5 and Day 6 blastocysts

Does the reproductive potential of embryos change when blastocyst development takes longer than the traditionally accepted 5 days when accounting for aneuploidy and endometrial-embryo asynchrony? Aneuploidy increases with increasing duration of blastulation, but if blastocyst morphologic quality and endometrial-embryo asynchrony are controlled for, euploid Day 7 embryos have similar sustained implantation as compared to Days 5 and 6 euploid blastocysts. The relative contributions of diminished embryo quality versus endometrial and embryo asynchrony to poor outcomes associated with embryos cultured past Day 6 are not clear. Asynchrony can be eliminated by embryo vitrification with transfer in a subsequent month after retrieval. Retrospective cohort study of patients from a single center attempting conception through ICSI and utilizing preimplantation genetic testing for aneuploidy screening (PGT-A) from January 2017 to September 2018. Cycles were excluded if they utilized surgical sperm or preimplantation genetic testing for monogenetic/single gene defects. ICSI cycle outcomes from 2586 patients were evaluated for ploidy status of embryos. Only patients undergoing single, euploid frozen embryo transfer were included when analyzing cycle outcomes by day of blastocyst expansion of the transferred embryo (n = 2130). Ploidy rates by the day upon which an embryo was considered to be usable (denoted, 'usable blastulation day') were determined so as to assess the contribution of aneuploidy to slow embryo development. Outcomes of euploid frozen single embryo transfers (SET) of Day 7 embryos were evaluated to assess the reproductive potential associated with embryos that were slowly developing for reasons other than aneuploidy. Analyses were adjusted by maternal age and blastocyst morphology. Overall, 67.7% (n = 3508) of usable Day 5 blastocysts were euploid, 52.1% (n =5560) of usable Day 6 blastocysts were euploid and 43.1% (n =229) of usable Day 7 embryos were euploid (Day 5 versus Day 6: odds ratio (OR) 0.7 (95% CI, 0.64-0.76), P < 0.001; Day 5 versus Day 7: OR 0.56 (95% CI, 0.46-0.69), P < 0.001; Day 6 versus Day 7: OR 0.81 (95% CI, 0.67-0.99), P = 0.036). Stratified by Society for Assisted Reproductive Technology maternal age groups, a reduction in the prevalence of euploidy by increasing time to embryo blastulation was still seen. The sustained implantation rate (SIR) was similar after euploid SET of Days 5 and 6 embryos (overall, 68.9% (95% CI, 66.0-71.6) and 66.8% (95% CI, 63.8-69.7), respectively; P = 0.81). SIR after euploid Day 7 SET appeared slightly lower than that of Days 5 and 6 embryos (52.6% (95% CI, 35.8-69.0); (Day 5 versus Day 7: OR, 0.67 (95% CI, 0.32-1.41), P = 0.29; Day 6 versus Day 7: OR 0.58 (95% CI, 0.28-1.2), P = 0.14)) but did not achieve statistical significance. The primary limitation is the low number of Day 7 blastocyst transfers that limits statistical power. Additionally, the retrospective nature of this study may prevent full elucidation of potential biases with respect to culture, morphologic assessment and selection of Day 7 embryos for transfer. Routine culture through Day 7 may successfully increase the pool of transferrable embryos for patients who would otherwise have no usable embryos if culture terminated on Day 6. This is particularly true for older patients (i.e. greater than 35years of age), whose embryos take longer to blastulate and, therefore, are more susceptible to cycle cancelation. Additionally, as evidenced by an adequate overall SIR of 52.6% after euploid SET of Day 7 blastocysts, embryos developing to a usable blastocyst on Day 7 are likely within the 'window of blastulation.' None.

Investigating the impact of the timing of blastulation on implantation: management of embryo-endometrial synchrony improves outcomes.

STUDY QUESTIONDo embryos with delayed blastulation have inferior reproductive potential or poorer outcomes due in part to embryo and endometrial synchrony?SUMMARY ANSWERDiminished outcomes in embryos with delayed blastulation undergoing fresh embryo transfer (ET) may be attributed to a loss of embryonic-endometrial synchrony.WHAT IS KNOWN ALREADYEmbryos that blastulate slowly have lower sustained implantation rates (SIR) than those which blastulate normally on Day 5 (D5). Traditionally this has been attributed to reduced embryo quality; however, dyssynchrony with the endometrium is also a possibility and has not been fully described. This convenient cohort composed of groups that resulted from a practice wide change in laboratory protocol allows for evaluation of embryo and endometrial synchrony as it related to blastocyst expansion.STUDY DESIGN, SIZE, DURATIONA retrospective cohort analysis was carried out from January 2009 to February 2013. Three cohorts were identified: D5 ET, D6 ET and frozen ET that comprised 822 patients, 763 patients and 718 patients, respectively. Each of these cohorts had slowly blastulating and normally blastulating embryos identified.PARTICIPANTS/MATERIALS, SETTING, METHODSThe study setting was academic affiliated private practice. All first fresh or cryopreserved ETs from 2010 to 2013 were studied. Non-biopsied embryos were classified into two groups on D5: slowly blastulating (Morula-Gardner 1) or normally blastulating (Gardner 2–6). Only single ETs or transfer of two embryos within the same classification group were included. Outcomes were compared between classification groups in embryos undergoing transfer on D5, D6, or after cryopreservation. This assesses the impact of transfer timing in fresh cycles as well as isolating a pure embryonic factor in frozen ET cycles. Sustained implantation was defined as heart beat detection at discharge sonogram at 8 weeks gestation. SIR was defined as the number of embryos transferred meeting criteria for sustained implantation divided by the total number of embryos transferred.MAIN RESULTS AND THE ROLE OF CHANCEIn total, 3391 embryos were transferred to 1966 patients. On D5, SIRs were significantly lower with slowly blastulating embryos (44% versus 64% in women <35 years of age (P < 0.001) and 18% versus 56% in women ≥35 years of age (P < 0.001)). Fresh D6 ETs also had significantly lower SIRs for embryos that were slowly blastulating on D5 (52% versus 63% in <35 years of age (P < 0.05) and 32% versus 48% in ≥35 years of age (P < 0.005)) despite continued development to full blastocysts and being morphologically equivalent at the time of ET, suggesting dyssynchrony. However, when slowly blastulating embryos underwent vitrification and then ET, they had SIRs which were equivalent to their normally blastulating counterparts (57% versus 60% in <35 years of age (P = 0.5) and 37% versus 42% in ≥35 years of age (P = 0.3)). An intraclass correlation and a generalized estimating equation corrected for patient age was performed which confirmed these findings. The normalization in cryopreserved ETs indicates that dyssynchrony may be a major adverse factor limiting outcomes with late blastulating embryos in fresh cycles.LIMITATIONS, REASONS FOR CAUTIONThis is a retrospective study comprising cohorts from a convenient sample chosen due to a uniform change in embryology laboratory protocol regarding ET day, however, this was done independent of the management of embryo and endometrial synchrony. Although strict ultrasound and serum progesterone criteria were utilized to make endometrial receptivity uniform, pathologic states of pre-receptive and post-receptive endometrium cannot be ruled out.WIDER IMPLICATIONS OF THE FINDINGSThe data surrounding embryo and endometrial synchrony should be considered in patients undergoing IVF and attention to the variations in blastulation rates can be applied to any patient undergoing extended embryo culture.STUDY FUNDING/COMPETING INTERESTSNone.