Subsequent Frozen Embryo Transfer Cycle Research Articles

Transcriptomic Analysis of Vitrified-Warmed vs. Fresh Mouse Blastocysts: Cryo-Induced Physiological Mechanisms and Implantation Impact.

Blastocyst vitrification has significantly improved embryo transfer methods, leading to higher implantation success rates and better pregnancy outcomes in subsequent frozen embryo transfer cycles. This study aimed to simulate the transcriptional changes caused by vitrifying human blastocysts using mouse blastocysts as a model and to further investigate these changes' effects. Utilizing a human vitrification protocol, we implanted both vitrified and fresh embryos into mice. We observed the implantation success rates and performed transcriptomic analysis on the blastocysts. To validate the results from messenger RNA sequencing, we conducted reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of specific genes. Based on mRNA profiling, we predicted the microRNAs responsible for the regulation and used qPCR basic microRNA assays for validation. Our observations revealed a higher implantation success rate for vitrified embryos than fresh embryos. Transcriptomic analysis showed that vitrified-warmed blastocysts exhibited differentially expressed genes (DEGs) primarily associated with thermogenesis, chemical carcinogenesis-reactive oxygen species, oxidative phosphorylation, immune response, and MAPK-related signaling pathways. RT-qPCR confirmed increased expression of genes such as Cdk6 and Nfat2, and decreased expression of genes such as Dkk3 and Mapk10. Additionally, gene-microRNA interaction predictions and microRNA expression analysis identified twelve microRNAs with expression patterns consistent with the predicted results, suggesting potential roles in uterine epithelial cell adhesion, trophectoderm development, invasive capacity, and immune responses. Our findings suggest that vitrification induces transcriptomic changes in mouse blastocysts, and even small changes in gene expression can enhance implantation success. These results highlight the importance of understanding the molecular mechanisms underlying vitrification to optimize embryo transfer techniques and improve pregnancy outcomes.

Precise hourly personalized embryo transfer significantly improves clinical outcomes in patients with repeated implantation failure.

This study investigated whether RNA-Seq-based endometrial receptivity test (rsERT)-which provides precision for the optimal hour of the window of implantation (WOI)-can improve clinical outcomes of frozen embryo transfer (FET) cycles in patients with a history of repeated implantation failure (RIF). Patients with a history of RIF who received at least one autologous high-quality blastocyst during the subsequent FET cycle were retrospectively enrolled and divided into two groups: rsERT and FET, comprising patients who underwent rsERT-guided pET (n=115) and standard FET without rsERT (n=272), respectively. In the rsERT group, 39.1% (45/115) of patients were receptive. rsERT patients showed a higher probability of achieving both positive human chorionic gonadotropin (63.5% vs. 51.5%, P=0.03) and clinical pregnancy (54.8% vs. 38.6%, P=0.003) rates. In subgroup analysis, rsERT patients with non-receptive results had higher clinical pregnancy rates than patients undergoing FET (58.6% vs. 38.6%, P=0.003). rsERT patients with receptive results guided by rsERT with a precise WOI time had higher, although non-significant, clinical pregnancy rates (48.9% vs. 38.6%, P=0.192) than patients who underwent standard-time FET. Hourly precise rsERT can significantly improve the probability of achieving clinical pregnancy in patients with RIF, especially in those with non-receptive rsERT results.

P-262 Clinical viability of oocytes with micronuclei at fertilisation

Abstract Study question Does the presence of micronuclei at fertilisation impact on embryo potential? Summary answer Embryos displaying micronuclei at fertilisation exhibit decreased utilisation rates and lower euploidy rates, however are capable of resulting in clinical pregnancies. What is known already The appearance of two pronuclei defines normal fertilisation. With advances in imaging techniques, a range of pronuclei phenotypes can now be identified in more detail, including additional micronuclei. Micronuclei are smaller satellite structures appearing around the pronuclei. Previous studies have determined that embryos containing two primary pronuclei and one micronuclei are generally diploid and can develop to blastocyst stage. However, these studies were limited in sample size, did not have consistent imaging techniques and assessed only the presence or absence of micronuclei, not their size or number. Study design, size, duration Retrospective cohort analysis in a single, large IVF centre including all oocytes entering culture between January 2022 and December 2023 (n = 33,237). All embryos were cultured in Embryoscope+ using Vitrolife sequential media at 6% CO2 and 5% O2. ICSI and naturally inseminated oocytes were included. All embryos were cultured to day 5/6 before determining suitability for transfer, freezing and/or genetic testing. Participants/materials, setting, methods During routine daily annotation, embryologists assessed fertilised oocytes for the presence of micronuclei. This study re-examined all embryos annotated as displaying micronuclei and further assessed: number of micronuclei, their position (‘apposed’ or ‘distant’ from the pronuclei, and in the case of > 1 micronuclei ‘clustered’ or ‘spread’) along with the area (μm2) of the two largest micronuclei and area (μm2) of primary pronuclei using Embryoscope+ software. Clinical pregnancy was defined as presence of a gestational sac. Main results and the role of chance Micronuclei were observed in 0.45% (n = 149/33,237) of all oocytes assessed. The majority of these presented as one additional nuclei 92.60% (n = 138), 7.38% (n = 11) showed two additional nuclei and 0.67%(n = 1) had multiple (more than 2) micronuclei. All micronuclei appeared apposing a primary pronucleus. In the event of multiple micronuclei, 82% were clustered whereas 9% was spread throughout the around the primary pronuclei. The average area of the largest micronuclei in each oocyte was 80.33μm2 ± 26.81.The mean area of all micronuclei was 76.80μm2, approximately 16% of the size of primary pronuclei measured, 465.09μm2. The utilisation rates of embryos derived from micronuclei fertilisation were 28.18% (N = 42) compared to 49%, n = 4118 of 2PN derived embryos. All but one of these presented two primary pronuclei and one additional micronuclei, with the remaining embryo displaying two micronuclei. Twelve of these embryos were transferred (in a fresh stimulated cycle (n = 5) or a subsequent frozen embryo transfer cycle (n = 7), resulting in four clinical pregnancies, however only one ongoing clinical pregnancy. PGT testing was performed on 19 embryos, with only two, 6.67% returning a euploid results, 68.42% resulting an aneuploid, of the remaining four embryos, 2 achieved a mosaic result and 2 received a no result. Limitations, reasons for caution As the occurrence of micronuclei is rare, only a limited number of embryos were available for analysis. Furthermore, this study was conducted in a large single laboratory and can not account for differences in culture conditions between laboratories. Inter-operator variability among initial fertilisation annotations could not be excluded. Wider implications of the findings By creating a definition of the micronuclei measurement, it provides safeguard for micronuclei to be assessed in culture. The data presented here can help guide clinical decisions where micronuclei are present. Trial registration number not applicable

O-152 Pre-implantation genetic testing for aneuploidies (PGT-A) improves reproductive outcomes in advanced maternal age patients undergoing IVF/ICSI: a multicentre retrospective cohort study with propensity score matching

Abstract Study question In patients > 37 years undergoing IVF/ICSI, does PGT-A improve live birth rates (LBR) and cumulative live birth rates (CLBR)? Summary answer In advanced maternal age women with ≥1 embryo to transfer, PGT-A improves the live birth and cumulative live birth rates. What is known already The age-related rate of aneuploidy remains a major cause of IVF failure. Therefore, PGT-A has been proposed as a method to select embryos with the highest implantation potential. Previous studies have shown that women younger than 37 years old do not benefit from PGT-A given the comparable outcomes in terms of implantation, clinical pregnancy, and live-birth rates. Moreover, PGT-A may also be inherently ineffective given the risk of mitotic mosaicism, sampling errors and misinterpretation of results. However, we are currently lacking solid scientific evidence to support whether this approach benefits advanced maternal age women. Study design, size, duration A multicenter retrospective cohort study was conducted including 9328 patients >37y undergoing their first IVF/ICSI cycle with an oocyte retrieval performed between 1/01/2013-31/07/2021 in a multinational private fertility clinic. The study group (n = 4664) included patients who performed PGT-A. The control group included patients without PGT-A (n = 4664) and was selected by propensity score matching adjusted for age at oocyte retrieval, number of oocytes retrieved and year of oocyte retrieval. Participants/materials, setting, methods The primary outcome was CLBR (delivery of at least one live birth per started cycle, including the fresh and subsequent frozen embryo transfer cycles). The secondary outcomes were LBR and time to pregnancy. The analysis was conducted including all patients with an initiated cycle of ovarian stimulation and also per embryo transfer. Comparisons are presented as adjusted odd-ratios (adjOR) and 95% confidence intervals (95%CI) following multivariable logistic regression (LBR) or cox regression (CLBR) analysis. Main results and the role of chance Patients in the PGT-A group had more oocytes retrieved (11.03±6.82 vs 9.24±6.15, p < 0.001), and a higher number of available blastocysts (2.82±2.21 vs 1.22±2.00, p < 0.001). In the PGT-A group, 13.153 embryos were analysed, with an euploidy rate of 29.30% (n = 3854). In total, 49.83% (n = 2324) patients did not undergo embryo transfer due to unavailable euploid embryos. Considering all patients that initiated ovarian stimulation, the PGT-A group presented a higher unadjusted CLBR per started cycle (27.38% vs 21.81%, p < 0.001). Considering only patients who underwent embryo transfer, a total of 5934 embryo transfers were analysed (n = 3166 in the control group and n = 2768 in the PGT-A group). Following multivariable logistic regression analysis, LBR and CLBR were significantly higher in the PGT-A group (respectively, adjOR 1.56 [95% CI 1.38-1.77], adjOR 1.53 [95% CI 1.39-1.68]). Following the transfer of 5 embryos, the CLBR was approximately 100% in the PGT-A group and 74.51% in the control group. Survival analysis was also perfomed to assess the impact of PGT-A on time to pregnancy. Despite the fact that from 6 months onwards the CLBR was higher in the PGT-A group, the median time to pregnancy was higher in the PGT-A group (3.27 (95%CI 3.16-3.40) vs 2.74 months (95%CI 2.57-3.03). Limitations, reasons for caution This study is limited by its retrospective design and low number of women with more than 3 embryos transferred. Also, the fact that only the first IVF/ICSI cycle was analysed provides a proper patient counselling in this specific context but does not provide data on repeated cycle outcomes. Wider implications of the findings Our findings suggest that, in women older than 37 years with at least one available embryo to biopsy, performing PGT-A may improve the reproductive outcomes in terms of LBR and CLBR, providing a useful clinical tool for embryo selection. Trial registration number not applicable

Antibiotic cured chronic endometritis remains a risk factor for early pregnancy loss in the subsequent frozen euploid embryo transfer

Research questionDo patients with antibiotic-cured chronic endometritis (CCE) have a comparable pregnancy outcome to those with non-chronic endometritis (NCE) in the subsequent frozen embryo transfer (FET) cycle? DesignA retrospective cohort analysis included 833 patients in their first FET cycles with single euploid embryo transfer. Chronic endometritis (≥5 CD138+ plasma cells per high-power field [CD138+/HPF]) was treated with standard antibiotic therapy. Patients were classified into two groups: the NCE group (n = 611, <5 CD138+/HPF) and the CCE group (n = 222, ≥5 CD138+/HPF and cured after antibiotic treatment). Pregnancy outcomes were compared. NCE group was divided into subgroup 1 (CD138+/HPF = 0) and subgroup 2 (CD138+/HPF = 1–4) for further analysis. ResultsThe rate of early pregnancy loss (EPL), incorporating all losses before 10 weeks’ gestation, was significantly higher in the CCE group than the NCE group (21.2% versus 14.2%, P = 0.016), and the difference was statistically significant (adjusted odds ratio [AOR] 1.68, 95% confidence interval [CI] 1.11–2.55). No significant differences were observed between the two groups with regard to other pregnancy outcomes. In the subgroup analysis, the EPL rate and biochemical pregnancy rate were significantly higher in subgroup 2 than subgroup 1 (17.2% versus 9.4%, AOR 2.21, 95% CI 1.30–3.74; 12.2% versus 6.9%, AOR 2.01, 95% CI 1.09–3.68). ConclusionsChronic endometritis cured by standard antibiotic therapy remains a risk factor for EPL in FET cycles, although no differences were found in live birth rates between patients with CCE or with NCE.

Deep Inception-ResNet: A Novel Approach for Personalized Prediction of Cumulative Pregnancy Outcomes in Vitro Fertilization Treatment (IVF).

Infertility is one of the major causes of socioeconomic stress worldwide due to social stigma and stressful lifestyles. Despite technological advances, couples still undergo several IVF cycles for conceiving without knowing their true prognosis which is causing a huge social and medical impact, and the live birth rate continues to be relatively low (~ 25%). A prediction model that predicts IVF prognosis accurately considering the pre-treatment parameters before starting the IVF cycle will help clinicians and patients to make better-informed choices. In this study, clinical details of 2268 patients with 79 features who underwent IVF/ICSI procedure from January 2018 to December 2020, at the Center of IVF and Human Reproduction, Sir Ganga Ram Hospital were retrospectively collected. The machine learning model was developed considering features such as maternal age, number of IVF cycle, type of infertility, duration of infertility, AMH, indication for IVF, sperm type, BMI, embryo transfer, and β-hCG value at the end of a fresh cycle and/or one subsequent frozen embryo transfer cycle was selected as the measure of outcome. Compared to other classifiers, for an 80:20 train-test split with feature selection, the proposed Deep Inception-Residual Network architecture-based neural network gave the best accuracy (76%) and ROC-AUC score of 0.80. For tabular datasets, the applied approach has remained unexplored in previously made studies for reproductive health. This model is the starting point for providing a personalized prediction of a successful outcome for an infertile couple before they enter the IVF procedure. The online version contains supplementary material available at 10.1007/s13224-023-01773-9.

O-297 Contribution of Frozen Embryo Transfer Cycles to ICSI Clinical Outcomes in Male Factor Infertility

Abstract Study question What is the benefit of adding frozen embryo transfer (FET) cycles to ICSI clinical outcomes in male factor infertility? Summary answer FET carried out on spare embryos that reached blastocyst stage remarkably contributed to additional clinical pregnancies in ICSI cycles for male factor infertility. What is known already Current trends in reproductive medicine lean toward full-preimplantation development and eventually to PGT-A to select a single euploid embryo for transfer. The utilization of this approach, while beneficial in most couples, is not ideal for male factor infertility due to the tendency of being characterized by impaired embryo development. So, we wonder if the utilization in FET cycles of leftover embryos that reached to blastocyst stage or those that eventually reached day 5 for aneuploidy testing contributed to the clinical outcomes. Study design, size, duration In the past 7 years, we included 22,289 couples who underwent ICSI while the large majority (84.8%) received a fresh embryo transfer, of which 70.9% were transferred at day 3. Leftover embryos, together with those euploid after PGT-A at blastocyst stage, were replaced in subsequent FET cycles. The clinical outcomes including clinical pregnancy rate (CPR) and deliveries were compared between the fresh embryo transfer and those after FET in total and after PGT-A. Participants/materials, setting, methods Couples with male factor underwent ICSI in standard fashion using exclusively ejaculated sample. For fresh embryo transfer cycles, embryos were transferred either at day 3 or at day 5. For FET cycles, embryos were cultured up to exclusively blastocyst stage and cryopreserved by vitrification. For aneuploidy, NGS was carried out for PGT-A. FET was carried out in natural or programmed cycles. Main results and the role of chance In the cohort underwent fresh embryo transfer, 18,896 couples underwent 37,751 ICSI cycles, where 322,916 oocytes were injected and 243,768 (75.5%) fertilized. Additionally, 3,393 patients received 4,712 FET in total with a fertilization of 69.8% (46,163/66,171) that did not differ from the fresh transfer group. The number of average embryos transferred in fresh cycles was 2.4±2 while FET was carried out exclusively on single embryo. Fresh transfer yielded 37.3% (14,087/37,751) CPR, while overall FET cycles achieved a higher CPR at 52.2% (2,462/4,712, P&amp;lt;0.0001). Similarly, the delivery rate in fresh cycles was 31.8% (12004/37751) and became 45.5% (2145/4712) in the FET (P&amp;lt;0.0001). To identify the advantage of selecting a single euploidy embryo, we compared FET on leftover unscreened blastocyst to those that were planned for PGT-A. Spare embryo transfers involved 1774 patients in 2282 cycles with a fertilization rate of 67.7% (18,905/27,937). For the PGT-A cycles, 1619 couples in 2430 cycles achieved a comparable fertilization rate of 71.3% (27,258/38,234), In this comparison, the FET on spare unscreened embryos achieved a CPR at 47.0% (1,072/2,282) while in the PGT-A group reached CPR at 57.2% (1390/2430, P&amp;lt;0.0001). Similarly, the delivery rate was 37.6% (857/2,282) in the FET and PGT-A was 53.0% (1,288/2,430) (P&amp;lt;0.0001). Limitations, reasons for caution The comparison is retrospective and is carried out on male factor infertility where day 3 embryo transfer were performed almost exclusively on fresh cycle to overcome poor embryo development. Nonetheless, those spare embryos that reached blastocyst stage and those that electively underwent aneuploidy testing significantly contributed to enhance clinical outcomes. Wider implications of the findings ICSI can overcome most of male infertility; however, the risk of impaired embryo development proposes a transfer at cleavage stage. The advanced embryo culture condition together with time-lapse allowed us to monitor embryos up to the blastocyst stage that once transferred, improving clinical outcomes, especially for embryos with confirmed euploidy. Trial registration number N/A

Impact of elevated body mass index on cumulative live birth rate and obstetric safety in women undergoing assisted reproductive technology

This study evaluated the impact of elevated body mass index (BMI) on short- and long-term outcomes of in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatments. A total of 7229 patients undergoing IVF/ICSI fresh cycles and subsequent frozen embryo transfer cycles from 2014 to 2020 were divided into normal (18.5–24.9 kg/m2) and high BMI (≥ 25 kg/m2) groups. Ovarian response, pregnancy outcomes, and safety of both mother and fetus were the main outcome measures. Furthermore, multivariate analysis was used to determine whether BMI was associated with cumulative live birth rate (CLBR). Results showed that for younger women (< 38 year), CLBR was significantly reduced in the high BMI group compared with the normal BMI control and was accompanied by fewer retrieved oocytes and available embryos. Additionally, the incidence of hypertensive disorders of pregnancy, fetal macrosomia, and cleft lip and palate birth defects resulting from cumulative live births was significantly higher compared with the normal BMI group. No differences were observed among older women (≥ 38 year). Multivariate analysis revealed that high BMI was a risk factor for CLBR. Our study suggested that elevated BMI has a greater adverse impact on younger women.

Multicentre study on rates and reasons for treatment discontinuation in patients with remaining cryopreserved embryos

Research questionWhat is the discontinuation rate among patients with remaining cryopreserved embryos in Belgium and what are the reasons for discontinuation? DesignMulticentre, cross-sectional study across 11 Belgian fertility clinics. Patients were eligible (n = 1917) if they had previously undergone an unsuccessful fresh embryo transfer (fresh group) or frozen embryo transfer (FET) (in-between group) and did not start a subsequent FET cycle within 1 year despite having remaining cryopreserved embryos. The denominator was all patients with embryos cryopreserved during the same period (2012–2017) (n = 21,329). Data were collected through an online anonymous questionnaire. ResultsThe discontinuation rate for patients with remaining cryopreserved embryos was 9% (1917/21329). For the final analysis, 304 completed questionnaires were included. The most important reasons for discontinuing FET cycles were psychological (50%) and physical (43%) burden, effect on work (29%), woman's age (25%) and effect on the relationship (25%). In 69% of cases, the patient themselves made the decision to delay FET treatment. In 16% of respondents, the decision to delay FET was determined by external factors: treating physician (9%), social environment (4%), close family (3%) and society (3%). Suggested improvements were psychological support before (41%), during (51%) and after (51%) treatment, as well as lifestyle counselling (44%) and receiving digital information (43%). ConclusionsThe discontinuation rate is remarkably high in patients with remaining cryopreserved embryos who have a good prognosis. Respondents stressed the need to improve the integration of psychological and patient-tailored care into daily assisted reproductive technology practice.

P-424 Endometrial receptivity as determined by Endometrial Receptivity Analysis (ERA) can change after a successful live birth from a frozen embryo transfer (FET)

Abstract Study question Endometrial receptivity as determined by Endometrial Receptivity Analysis (ERA) can change after a successful live birth from a frozen a frozen embryo transfer (FET) Summary answer 27 of 33 patients with a previous live birth from FET had an altered endometrial receptivity profile by ERA after a subsequent unsuccessful FET. What is known already The endometrial window of implantation is approximately 24 hours wide with a complex interaction of autocrine, paracrine, and endocrine factors. Successful embryo implantation involves a 3-step process of apposition, adhesion, and invasion of an embryo into a receptive endometrium. The ERA was developed using the expression profile of 248 genes using Next Generation Sequencing to determine if the endometrium is receptive, early receptive, late receptive, pre-receptive or post-receptive to objectively determine the optimal timing of progesterone exposure prior to embryo transfer. Some studies have shown statistically significant improvements in live birth rates while other studies have shown no difference. Study design, size, duration Case series of 33 patients with successful live birth following euploid FET using 5 days of progesterone (P + 5) with subsequent unsuccessful FET using the same protocol from January 2018 – December 2020. 27 of 33 patients who repeated their ERA were found to be non-receptive using P + 5. The new ERA profile results were used for their next FET. Implantation and live birth rates were calculated. Participants/materials, setting, methods A single private practice fertility clinic performed 340 ERA cycles from January 2018 – December 2020 with 276 subsequent FET cycles. An observational study of 33 patients with a previous live birth using P + 5 elected to repeat their ERA cycle after an unsuccessful FET. Implantation and live birth rates were calculated for their subsequent FET after completing the repeat ERA. Main results and the role of chance Repeat ERA result for patients with a previous live birth using P + 5 revealed that 6 patients still needed P + 5 (18.2%) for a receptive endometrial profile, 8 patients (24.2%) needed an extra 12 hours (P + 5.5), 18 patients (54.5%) needed an extra 24 hours (P + 6) and only one patient (3.0%) needed an extra 48 hours (P + 7). No patient that repeated their ERA after a previous live birth had a post-receptive profile (P + 4 or P + 4.5) and one patient had successful live births after using 3 different ERA profiles (P + 5, P + 6 and P + 7). All patients except one had repeat ERA results that were pre-receptive with only one patient changing from P + 6 to P + 5. FET cycles using P + 5 and P + 7 had a 100% implantation and pregnancy rate, while patients using P + 5.5 had the lowest implantation rate (62.5%) and live birth rate (37.5%). Patients with an ERA profile of P + 6 had an implantation rate of 94.1% and live birth rate of 88.2%. The small number of patients in this case series make a type I error possible because of the low number of patients in each ERA profile category with insufficient power to reach statistical significance. Limitations, reasons for caution Implantation rate for the clinic during the study period was 75% and live birth rate of 68% using euploid embryos. Our high baseline implantation and live birth rates, coupled with low numbers of patients in this observational study, make these results less generalizable to the IVF population at large. Wider implications of the findings It is widely accepted that ERA results are reproducible and do not change during a woman’s reproductive life. These results suggest that some women may have an altered window of receptivity after both successful and unsuccessful FET cycles. It may be reasonable to repeat ERA cycles for carefully selected patients. Trial registration number N/A

Higher Doses of FSH Used for Superovulation Do Not Adversely Affect Embryonic Ploidy: A Randomized Controlled Trial (STimulation Resulting in Embryonic Aneuploidy using Menopur (STREAM) Trial)

Research Question: Does the dose of gonadotropin used for superovulation in IVF affect the proportion of euploid blastocysts obtained after fertilization? Study Design: Multicentre randomized controlled trial recruiting 57 women who were treated with ovarian stimulation using either 150 or 300 IU Menopur per day. Both groups received GnRH antagonist from day 5 of ovarian stimulation and final oocyte maturation was induced using a leuprolide GnRH (gonadotropin releasing hormone) agonist trigger when three or more follicles reached 17 mm diameter. Oocyte collection was scheduled 36–38 hours post trigger. In vitro fertilization (IVF) or Intracytoplasmic Sperm Injection (ICSI) were performed according to individual unit protocol and embryos were cultured to blastocyst stage. A trophectoderm biopsy was performed on day 5 of embryo culture and used for preimplantation genetic testing for aneuploidy. Euploid embryos were transferred in subsequent frozen embryo transfer cycles with appropriate endometrial preparation. Results: The number of oocytes obtained from women randomized to 150 IU Menopur was between 3 and 17 (mean = 9), whereas the number of oocytes obtained from women randomized to 300 IU Menopur was between 3 and 24 (mean = 11). There was a positive linear relationship between serum AMH concentration and oocyte yield in both the 150 and 300 IU Menopur groups ([Formula: see text] = 0.3359, [Formula: see text] = 0.1129 and [Formula: see text] = 0.3741, [Formula: see text] = 0.1399). The percentage of euploid to aneuploid embryos in the 150 IU Menopur group was 63% and in the 300 IU Menopur group, the proportion was 75%, which was not significantly different ([Formula: see text] = 0.17). Conclusion: The higher dose ovarian stimulation protocol did not significantly increase the number of oocytes retrieved, nor did the higher dose protocol reduce the proportion of euploid embryos created. This study does not support the hypothesis that use of higher doses of gonadotropin for ovarian stimulation results in a reduction in the proportion of euploid embryos obtained after IVF.

CLINICAL VALUE OF HISTOLOGIC ENDOMETRIAL DATING IN WOMEN WITH INADEQUATE ENDOMETRIUM DURING FROZEN-THAWED BLASTOCYST- STAGE EMBRYO TRANSFER: A RETROSPECTIVE PILOT STUDY

Inadequate endometrium (IE) in preparation for a frozen embryo transfer (FET) is a challenging occurrence in assisted reproduction. While there is no accepted consensus on how to define IE a conservative approach is a measurement of less than or equal to 8mm. When the endometrium is inadequate, patients and physicians face a decision of whether to proceed with the embryo transfer. A recent study evaluating the impact of IE on FET reported a decline in pregnancy and live birth rates with each millimeter decline in endometrial thickness below 7mm (1). Histologic examination of the endometrium has been used to determine the adequacy of endometrial proliferation. To potentially reduce cycle cancellation, we evaluated the pregnancy outcome of FET cycles from patients with IE but “in-phase” histologic endometrial dating. This is a retrospective pilot study of patients between the years of 2014 and 2020 who underwent a standard hormonal endometrial preparation for FET that had been canceled due to IE defined as a maximum thickness ≤ 8mm following up to 35 days of estradiol supplementation. Endometrial biopsy was performed for histologic dating using the criteria of Noyes et al after 9 to 11 days of progesterone supplementation. Patient outcomes were also stratified based on the number and quality of embryos transferred. All study patients were determined to have “in-phase” endometrium who underwent FET cycle with the transfer of at least one good-quality blastocyst. Descriptive statistics and Pearson Correlation was calculated using SPSS software v23. A total of 21 women mean age was 38.2 ± 6.7 years and IE underwent an endometrial biopsy after estradiol proliferation and 9-11 days of progesterone supplementation. The mean endometrial thickness was 6.4 ± 0.5 mm. Seventeen women (81%) demonstrated endometria that were “in-phase” and underwent a subsequent FET cycle. The mean number of embryos transferred was 1.47 ± 0.6, the clinical pregnancy rate was 47% (8/17); miscarriage rate was 11.8% (2/17); and the live birth rate was 35.3% (6/17). There was no correlation between age and endometrial thickness (Pearson Correlation Coefficient R = 0.08, P = 0.7). Histologic dating in women with a prior canceled FET due to IE is a potentially reliable method to allow the opportunity for a successful FET with comparable outcomes to the National Average (SART National Summary Report for 2018). Studies with a larger sample size are necessary to corroborate this finding.

Do endometrial natural killer and regulatory T cells differ in infertile and clinical pregnancy patients? An analysis in patients undergoing frozen embryo transfer cycles.

Clinical significance of endometrial and peripheral blood natural killer (NK) and regulatory T cells (Tregs) during frozen embryo transfer (FET) cycles has not been well characterized. Retrospective cohort study. Endometrial tissue was collected from infertility patients prior to a frozen embryo transfer cycle as part of an endometrial receptivity analysis (ERA® ) biopsy or endometrial scratch test. Uterine NK (uNK) and Treg cell density was compared based on pregnancy status in the subsequent frozen embryo transfer cycle. Peripheral blood was also collected from a separate cohort of patients undergoing frozen embryo transfer. Treg cell density was compared by the presence or the absence of a clinical pregnancy in each phase of the cycle. In the 33 luteal phase biopsies there were more endometrial Tregs, similar uNK and a trend toward lower CD16+ uNK cells in women with a future ongoing clinical pregnancy compared to non-pregnant women. There were no differences in uNK and Treg density in natural scratch cycles vs programmed cycles or in non-receptive vs receptive endometrium (ERA® cycles). In the peripheral blood analysis, the pregnant group had higher peripheral blood Tregs on the day of serum β-hCG time point when compared to the non-pregnant group. Higher levels of endometrial Tregs and lower levels of CD16+ uNK cells are positive prognostic factors for infertile women prior to frozen embryo transfer. Our work on phenotypic and proportional analyses of endometrial immune cells may complement the ERA® in predicting improved pregnancy rates in patients with implantation failure.

Case Report: Successful removal of Candida parapsilosis from human embryos with the application of laser and micromanipulation techniques

Objective To describe a micromanipulation method for removal of yeast from contaminated embryos to rescue their development Design Review of 2 concurring IVF/ICSI cases resulting in blastocysts colonized by Candida parapsilosis. Method of yeast removal, isolation and identification of yeast species, and cycle outcome is reviewed. Materials and methods Mature oocytes retrieved from patients were injected with their partner's sperm and cultured under standard conditions. At fertilization, zygotes from two patients showed a contaminating mass in the culture droplets. Embryos from contaminated drops were rinsed multiple times and cultured in freshly equilibrated culture media. The following day culture media had no visual contamination. However, one patient's embryos had hyphae-like masses attached to their zonae pellucidae. Under an inverted microscope attached to a micromanipulator, embryos were held under suction. Hyphae-like masses were removed by applying suction through an assisted hatching pipette and firing a laser to facilitate the detachment of contaminating masses. Cleaned embryos were rinsed and cultured through day 6. All freezable quality blastocysts were vitrified on either day 5/6. Samples of visually contaminated culture drops (Day 1), post-rinse culture drops at the time of embryo vitrification (day 5/6), and stock solutions were sent to a reference lab for microbiological analysis. Patient whose embryos had hyphae mechanically removed underwent subsequent frozen embryo transfer cycle. Results Growth media stock solutions tested positive for Candida parapsilosis. Conclusion Candida parapsilosis hyphae were successfully removed from growing embryos using laser and micromanipulation techniques. After yeast contamination removal, normally developed blastocyst(s) may be transferred only after patient's consent. Disclosures None Funding None To describe a micromanipulation method for removal of yeast from contaminated embryos to rescue their development Review of 2 concurring IVF/ICSI cases resulting in blastocysts colonized by Candida parapsilosis. Method of yeast removal, isolation and identification of yeast species, and cycle outcome is reviewed. Mature oocytes retrieved from patients were injected with their partner's sperm and cultured under standard conditions. At fertilization, zygotes from two patients showed a contaminating mass in the culture droplets. Embryos from contaminated drops were rinsed multiple times and cultured in freshly equilibrated culture media. The following day culture media had no visual contamination. However, one patient's embryos had hyphae-like masses attached to their zonae pellucidae. Under an inverted microscope attached to a micromanipulator, embryos were held under suction. Hyphae-like masses were removed by applying suction through an assisted hatching pipette and firing a laser to facilitate the detachment of contaminating masses. Cleaned embryos were rinsed and cultured through day 6. All freezable quality blastocysts were vitrified on either day 5/6. Samples of visually contaminated culture drops (Day 1), post-rinse culture drops at the time of embryo vitrification (day 5/6), and stock solutions were sent to a reference lab for microbiological analysis. Patient whose embryos had hyphae mechanically removed underwent subsequent frozen embryo transfer cycle. Growth media stock solutions tested positive for Candida parapsilosis. Candida parapsilosis hyphae were successfully removed from growing embryos using laser and micromanipulation techniques. After yeast contamination removal, normally developed blastocyst(s) may be transferred only after patient's consent.

DO ENDOMETRIAL NATURAL KILLER AND REGULATORY T CELLS DIFFER IN INFERTILE AND CLINICAL PREGNANCY PATIENTS? AN ANALYSIS IN PATIENTS UNDERGOING FROZEN EMBRYO TRANSFER CYCLES

Problem Clinical significance of endometrial and peripheral blood natural killer (NK) and regulatory T cells (Tregs) during frozen embryo transfer (FET) cycles has not been well characterized. Design Retrospective cohort study METHOD OF STUDY: Endometrial tissue was collected from infertility patients prior to a frozen embryo transfer cycle as part of an endometrial receptivity analysis (ERA® ) biopsy or endometrial scratch test. Uterine NK (uNK) and Treg cell density was compared based on pregnancy status in the subsequent frozen embryo transfer cycle. Peripheral blood was also collected from a separate cohort of patients undergoing frozen embryo transfer. Treg cell density was compared by the presence or the absence of a clinical pregnancy in each phase of the cycle. Results In the 33 luteal phase biopsies there were more endometrial Tregs, similar uNK and a trend toward lower CD16+ uNK cells in women with a future ongoing clinical pregnancy compared to non-pregnant women. There were no differences in uNK and Treg density in natural scratch cycles vs programmed cycles or in non-receptive vs receptive endometrium (ERA® cycles). In the peripheral blood analysis, the pregnant group had higher peripheral blood Tregs on the day of serum β-hCG time point when compared to the non-pregnant group. Conclusion Higher levels of endometrial Tregs and lower levels of CD16+ uNK cells are positive prognostic factors for infertile women prior to frozen embryo transfer. Our work on phenotypic and proportional analyses of endometrial immune cells may complement the ERA® in predicting improved pregnancy rates in patients with implantation failure.

Comparison of Clinical Pregnancy from Humidified and Dry Incubation of Embryos in a Tropical Region

The laboratory induced cellular stress on zygotes and embryos in their microenvironment could negatively influence the clinical outcome. One of the core components of an IVF lab is the culture incubator. Incubators can provide a stable and appropriate-culture environment by regulating optimal conditions on parameters such as temperature, gas levels and humidity. Clinical studies which compare incubator characteristics may provide insight to their efficacy. In humid conditions of the tropical climate, the incubators without humidifiers can also be used. Hence this study was done to identify the role of a humidifier in a tropical country. In this multicentre retrospective study, embryos from a total of 787 patients were cultured as two groups -A and B in two different types of incubators- humidified and dry, respectively. 647 patients in group A and 140 patients in group B were examined for the developmental parameters. The embryos were frozen at the blastocyst stage and replaced in subsequent frozen embryo transfer cycles. The resulted pregnancy, miscarriage and the clinical pregnancy outcomes were compared. The data was subjected to statistical validation. There was no significant difference observed in the clinical pregnancy rates between the groups. This study validates the possible use of dry incubators for the in vitro culture of human embryos in tropical climate without aiding any humidification.

Isthmocele and ovarian stimulation for IVF: considerations for a reproductive medicine specialist.

What is the risk of developing intracavitary fluid (ICF) during ovarian stimulation in patients with an isthmocele after previous caesarean section (CS) delivery? In patients with an existing isthmocele, the risk of developing ICF during hormonal stimulation for IVF is almost 40%; therefore, special attention has to be paid to exclude fluid accumulation during stimulation and particularly at the time of transfer, in which case the reproductive outcomes of frozen embryo transfer (FET) cycles appear to be uncompromised. Lately, there is an increasing focus on the long-term impact of CS delivery on the health and future fertility of the mother. Development of an isthmocele is one of the sequelae of a CS delivery. The presence of ICF in combination with an isthmocele has been described previously, and the adverse effect of endometrial fluid on implantation is well recognised by reproductive medicine specialists. Accumulation of ICF has been previously described in patients with hydrosalpinx, less commonly in patients with polycystic ovary syndrome undergoing ovarian stimulation for IVF/ICSI, and even in some patients without any identifiable reason. Assisted reproductive techniques (ARTs) are a means to overcome infertility. Reproductive medicine specialists commonly see patients with secondary infertility with a history of having had one or more previous CS and with ultrasound confirmation of an isthmocele. However, the available data pertaining to the prevalence of intracavitary fluid during ovarian stimulation in patients with ultrasound confirmation of an isthmocele is limited. Furthermore, data on the influence of ICF in a stimulated cycle on the ART outcome of a subsequent FET cycle is scarce and merits further studies. A prospective observational exploratory study was performed in IVI Middle East Fertility Clinic, Abu Dhabi, from June 2018 to March 2019, and retrospective analysis of the reproductive outcomes was performed until July 2019. Patients with secondary infertility, defined as a minimum of 1year of infertility after a previous successful pregnancy, undergoing ovarian stimulation for IVF/ICSI and having a history of one or more previous CS with ultrasonographic visible isthmocele, were included (n= 103). Patients were monitored as a clinical routine with vaginal ultrasound examinations during ovarian stimulation for IVF/ICSI treatment. All patients included in the study were asked to complete a questionnaire regarding their previous obstetric history. Development of ICF was recorded as well as changes in the measurements of the isthmocele during the course of ovarian stimulation. Reproductive outcomes of FET cycles of the patients with an isthmocele were retrospectively compared to those of patients with infertility and without isthmocele in our clinic during the same time period. Patients with an existing isthmocele after previous CS have a risk of ~40% of developing ultrasonographic visible fluid in the endometrial cavity during the course of ovarian stimulation. Development of ICF was significantly correlated with the depth of the isthmocele on Day 2/3 (P= 0.038) and on the day of trigger (-1/-2days) (P= 0.049), circumference of the isthmocele on the day of trigger (-1/-2days) (P= 0.040), distance from the C-scar to the external os (P= 0.036), number of children delivered (P= 0.047) and number of previous CS (P= 0.035). There was a statistically significant increase in the parameters related to the size of the isthmocele during ovarian stimulation. No significant differences in the reproductive outcome (pregnancy rate and rates of biochemical and ectopic pregnancies, miscarriages and ongoing/delivered pregnancies) after FET were found between the patients with and without an isthmocele, when ICF was excluded prior to embryo transfer procedure. NA. This study was not primarily designed to investigate the causes of ICF during ovarian stimulation or to evaluate the reproductive outcomes. Further, the small number of reported reproductive outcomes may be seen as a limitation. The data highlights the need for an increased awareness on the part of reproductive medicine specialists towards the potentially adverse impact of an isthmocele on ART treatment, as there is a potential to develop intracavitary fluid during ovarian stimulation for IVF. The increase in the circumference of the isthmocele may increase embryo transfer difficulty. No funding of the study has to be reported. The authors have no competing interests. This prospective study was registered with clinicaltrials.gov. under the number NCT03518385.

Optimising the Outcome of Embryo Transfer

In vitro fertilisation (IVF) is a complex procedure, the success of which is dependent on several factors at every step of the process. Despite major advances, successful implantation rates in IVF remain low. Aside from the status of the embryo and endometrium, embryo transfer (ET) plays a major role in implantation. There are numerous variables in ET that are causative factors for IVF success. In this article, the authors discuss whether the stage at which (cleavage versus blastocyst) ET occurs; a fresh or frozen ET; and the technique of ET affects the results of an assisted reproductive technology cycle. Blastocysts had higher implantation potential than cleavage-stage embryos and it was also observed that extended embryo culture was not related to increased adverse obstetric and perinatal outcome. Though freezing has several advantages over fresh cycles, one must remember that evidence is still lacking for its use in all patients. Elective cryopreservation of all embryos with transfer in subsequent frozen ET cycles may be requited in cases at risk of developing ovarian hyperstimulation syndrome, women undergoing preimplantation genetic screening or preimplantation genetic diagnosis for genetic analysis, polycystic ovarian syndrome patients, and those who have high progesterone levels on the day of human chorionic gonadotropin, but to date it is debatable whether a freeze-all strategy will benefit normal and poor responders. For an optimal ET technique, the use of soft catheters and performing the process under ultrasound guidance will improve results by making it less traumatic, standardised across centres, and more technically precise.

2. CLINICAL COMPARISON OF TWO PGT-A PLATFORMS UTILIZING DIFFERENT THRESHOLDS TO DETERMINE PLOIDY STATUS

Introduction Unlike previous technologies that lack the diagnostic performance to reliably detect mosaicism for preimplantation genetic testing for aneuploidy (PGT-A), next generation sequencing (NGS) based platforms now allow for the detection of lower-level mosaicism. Due to this increased sensitivity, thresholds need to be put in place to classify embryos as euploid, aneuploid, or mosaic. The lower cutoff for mosaic samples ranges from 20 % to 30 %, and the upper cutoff for mosaic samples ranges from 70%-80% in most commercial PGT laboratories in the field today. These varying cutoffs will lead to varying percentages of mosaic embryos reported, suggested to be somewhere between 20 %. A more stringent threshold could result in greater pregnancy rates. Here we compare clinical outcomes and embryo reporting between two different PGT-A reference laboratories that use different testing platforms and different mosaic cutoffs. Materials and Methods Retrospective analysis of PGT-A results (n=623) and frozen embryo transfer (FET) cycles (n=99) using embryos diagnosed by either Cooper Genomics or Igenomix between January 2018 - January 2019. Cooper Genomics uses Illumina's MiSeq platform with Bluefuse software and thresholds of 20% to 80 % (lower to upper cutoff) while Igenomix uses ThermoFisher's Ion Torrent platform with Ion Reporter software and thresholds of 30% to 70 %. Patients with a history of recurrent miscarriage were excluded from this study. Results Fifty six (56) cycles included embryos analyzed by Cooper Genomics and 43 cycles included embryos analyzed by Igenomix as a PGT-A reference laboratory. There was no differences in maternal age or number of embryos replaced per transfer between the two cohorts. IVF outcome was similar between the two groups: The pregnancy (+FHB) observed in cycles that transferred embryos diagnosed as euploid by Cooper Genomics was 67.9% (38/56) compared to 69.8% (30/43) with Igenomix. Miscarriage rate was also similar, 8.9% (5/56) and 6.9% (3/43) respectively. Cooper Genomics reported a much higher rate of mosaicism (17.0%) compared to Igenomix (4.9%) (p Conclusion Retrospective analysis of PGT-A+FET cycles revealed there was no differences in pregnancy or miscarriage rates indicating a similar diagnostic efficacy achieved by the two PGT-A platforms. Although the embryos diagnosed as euploid by the two platforms appeared to have the same potential to generate a pregnancy, the methodology and higher threshold utilized by Igenomix resulted in significantly less mosaic embryos and a slightly greater percentage of euploid embryos available for transfer. It is assumed that cumulative pregnancy rates between the two reference laboratories would vary based on the finding that there were more euploid embryos available from Igenomix. Results from subsequent FET cycles will be collected and further examined. NGS has now created three categories of embryos that exist after PGT-A, the interpretation of the millions of data points generated by this technology still remains a challenge and it is important that the diagnostic methodology used does not overestimate the degree of mosaicism.

The combined impact of maternal age and body mass index on cumulative live birth following in vitro fertilization

It is critical to evaluate the combined impact of age and body mass index on the cumulative likelihood of live birth following invitro fertilization, as achieving a lower body mass index before infertility treatment often is recommended for women with overweight and obesity. It is important to consider whether achieving a particular body mass index, thus resulting in an older age at invitro fertilization cycle start, is beneficial or harmful to the likelihood of live birth. To evaluate the combined impact of age and body mass index on the cumulative live birth rate following invitro fertilization to inform when delaying invitro fertilization treatment to achieve a lower body mass index may be beneficial or detrimental to the likelihood of live birth. This is a retrospective study using linked fresh and cryopreserved/frozen cycles from January 2014 to December 2015 from the Society for Reproductive Technology Clinic Outcome Reporting System, representing >90% of invitro fertilization cycles performed in the United States. The primary outcome was live birth as measured by cumulative live birth rate. Secondary outcomes included implantation rate, clinical pregnancy rate, and miscarriage rate. Poisson and logistic regression were used to calculate risk and odds ratios with 95% confidence intervals to determine differences in implantation, clinical pregnancy, and miscarriage, as appropriate, among first fresh invitro fertilization cycles compared across age (years) and body mass index (kg/m2) categories. Cox regression was used to calculate hazard ratios with 95% confidence intervals to determine differences in the cumulative live birth rate using fresh plus linked frozen embryo transfer cycles. There were 51,959 first fresh cycles using autologous eggs and 16,067 subsequent frozen embryo transfer cycles. There were 21,395 live births, for an overall cumulative live birth rate of 41.2% per cycle start. The implantation rate, clinical pregnancy rate, and cumulative live birth rate decreased with increasing body mass index and age, and the miscarriage rate increased with increasing body mass index and age (linear trend P<.001 for all). Body mass index had a greater influence on live birth at younger ages as compared with older ages. Age-related decline in fertility has a greater impact than body mass index on the cumulative live birth rate at older ages, suggesting that taking time to achieve lower body mass index before invitro fertilization may be detrimental for older women with overweight or obesity. Delaying conception to lose weight before invitro fertilization should be informed by the combination of age and body mass index.