Subsequent Frozen Embryo Transfer Research Articles

P–626 A freeze-all strategy improves clinical pregnancy rate in patients with few available embryos

Abstract Study question Is elective frozen blastocyst transfer an advantageous strategy for all patients? Summary answer A freeze-all strategy improves the outcomes in patients with few available embryos. What is known already With the aim of defining the best moment to perform embryo transfer, in recent years, relevance has been given to the understanding of the implantation window, however oocyte and embryo quality are key factors that are not to be disregarded. It has been suggested that a freeze-all strategy and subsequent frozen embryo transfers improve pregnancy rates. However, it is unclear whether this strategy benefits all kind of patients (i.e. with or without surplus embryos, etc). In this study, we aim to provide an answer on which patients may benefit of a freeze-all policy and a subsequent frozen embryo transfer. Study design, size, duration This retrospective cohort study includes infertile patients aged 21 to 44 years old, without previous history of recurrent failure of ART (including recurrent miscarriages). Enrolments took place between January 2015 and November 2019 and cycles with oocyte donation and PGT were excluded. Embryo transfers were performed in: 1) a fresh cycle (ET) or 2) a deferred cycle with surplus frozen embryos (FET) or embryos that were frozen in a freeze-all policy (FET-FA). Participants/materials, setting, methods Patients with blastocysts transfer were included. PGT cycles were excluded. The number of cycles complying with the inclusion criteria were: 617 ICSI cycles. Fresh embryo transfers (ET) were performed in 396 cycles (43 with a subsequent Frozen embryo transfer, FET). Frozen embryo transfers following a freeze-all strategy (FET-FA) were performed in 221 cycles. Clinical pregnancy rates (CPR) and Cumulative clinical pregnancy rates (CCPR) were calculated and compared among those groups. Main results and the role of chance Mean age of patients was 36.1 ± 3.7 years old (mean ± SD). In average, 1.83 ± 0.41 (mean ± SD) embryos were transferred. Following the first transfer (either ET and FET-FA), CPR was 40.4% and 58.4% (ET and FET-FA, respectively). Following the subset analysis of 2 age groups (≤38 & >38 years-old); in the ≤38-group, CPR was 45.2% and 58.9% (ET and FET-FA, respectively), while in > 38-group, the rates were 30.8% and 54.8% (ET and FET-FA, respectively); p < 0.05. CCPR were also significantly better in the FET-FA group: 51.3% vs 66.8% and 33.8% vs 58.1% in the ≤38-group and >38-group, respectively. Additionally, CPR was analysed independently for patients with ≤2 usable embryos (1 attempt) or ≥ 3 usable embryos (surplus embryos after first attempt). When a single attempt was possible; in the ≤38-group, CPR was 36.1% and 56.9% (ET and FET-FA, respectively) while in the >38-group, the rates were 24.7% and 63.6% (ET and FET-FA, respectively); p < 0.05. When surplus embryos were available, no difference in CPR (or CCPR) between ET and FET-FA groups were observed. After first attempt CPR were 58.4% and 48.2% in the ≤38-group & >38-group, respectively; while CCPR were 69.8% and 57.1% in the ≤38-group & >38-group, respectively. Limitations, reasons for caution Although the authors consider that the patient population is of optimal size, a detailed analysis of the stimulation protocol and hormonal values (estradiol and progesterone) during treatment, and its potential relation to the outcomes, should follow. Wider implications of the findings: In our setting, the data suggests that freeze-all strategy (with subsequent frozen embryo transfer) over fresh transfer is advantageous for patients with few available embryos (1 or 2 embryos for a single attempt). This increases the chances to pregnancy in 30.3% in the ≤38-group and 77.9% in the >38-group. Trial registration number Not applicable

P-767 Cumulative live birth rate after IVF - trend over time and the impact of blastocyst culture and vitrification

Abstract Study question Has cumulative live birth rate (CLBR) improved over time and which factors are associated with such an improvement? Summary answer During 2007-2017, CLBR per oocyte aspiration increased significantly (27.0 % to 36.3 %), in parallel with an increase in blastocyst transfer and cryopreservation by vitrification. What is known already While it has been shown that live birth rate (LBR) per embryo transfer (ET) is higher for fresh blastocyst than for fresh cleavage stage embryo transfer, CLBR per oocyte aspiration, including one fresh ET and all subsequent frozen embryo transfers (FET), does not seem to differ between the two culture strategies. Study design, size, duration STUDY DESIGN, SIZE, DURATION: National register study including all oocyte aspirations performed in Sweden 2007-2017, n = 124 700. Donation cycles excluded. Participants/materials, setting, methods Data were retrieved from the Swedish National Registry of Assisted Reproduction (Q-IVF). CLBR was defined as the number of deliveries with at least one live birth resulting from one oocyte aspiration, including all fresh and/or frozen embryo transfers within one year. The delivery of a singleton, twin, or other multiples was registered as one delivery. Cryopreservation of cleavage stage embryos was performed by slow freezing and of blastocyst by vitrification. Main results and the role of chance Overall, the CLBR per oocyte aspiration increased significantly during the study period, from 27.0 % to 36.3 % (OR 1.039, 95% CI 1.035-1.043) and from 30.0 % to 43.3 % if at least one ET was performed (AOR 1.055, 95% CI 1.050-1.059). The increase in CLBR was independent of maternal age, number of oocytes retrieved and number of previous IVF live births. The CLBR for women < 35 years and ≥ 35 years both increased significantly, following the same pattern. During the study period a substantially increasing number of blastocyst transfers were performed, both in fresh and in FET cycles. An important contributor included in the blastocyst strategy, may be the extended culture of the total cohort of embryos, also embryos earlier discarded at early cleavage stages, in order to reach the blastocyst stage. These embryos may contribute to the total number of available blastocysts and thereby increase the chance of a live birth within that oocyte aspiration cycle. Other important predicting factors for live birth, such as number of embryos transferred, could not explain the improvement, on the contrary the single embryo transfer (SET) rate increased with time. Limitations, reasons for caution The retrospective design implicates that other confounders of importance for CLBR can not be ruled out. In addition, some FET cycles might be performed later than one year post oocyte aspiration for the last year (2017) and are thus not included in this study. Wider implications of the findings The results suggest that blastocyst transfer, particularly when used in FET cycles and in combination with vitrification, is an important contributor to the improved live birth rates over time. This gives a possibility for fewer oocyte aspirations needed to achieve a live birth and a shortened time to live birth. Trial registration number -

P–767 Cumulative live birth rate after IVF - trend over time and the impact of blastocyst culture and vitrification

Abstract Study question Has cumulative live birth rate (CLBR) improved over time and which factors are associated with such an improvement? Summary answer During 2007–2017, CLBR per oocyte aspiration increased significantly (27.0% to 36.3%), in parallel with an increase in blastocyst transfer and cryopreservation by vitrification. What is known already While it has been shown that live birth rate (LBR) per embryo transfer (ET) is higher for fresh blastocyst than for fresh cleavage stage embryo transfer, CLBR per oocyte aspiration, including one fresh ET and all subsequent frozen embryo transfers (FET), does not seem to differ between the two culture strategies. Study design, size, duration STUDY DESIGN, SIZE, DURATION: National register study including all oocyte aspirations performed in Sweden 2007–2017, n = 124 700. Donation cycles excluded. Participants/materials, setting, methods Data were retrieved from the Swedish National Registry of Assisted Reproduction (Q-IVF). CLBR was defined as the number of deliveries with at least one live birth resulting from one oocyte aspiration, including all fresh and/or frozen embryo transfers within one year. The delivery of a singleton, twin, or other multiples was registered as one delivery. Cryopreservation of cleavage stage embryos was performed by slow freezing and of blastocyst by vitrification. Main results and the role of chance Overall, the CLBR per oocyte aspiration increased significantly during the study period, from 27.0% to 36.3% (OR 1.039, 95% CI 1.035–1.043) and from 30.0% to 43.3% if at least one ET was performed (AOR 1.055, 95% CI 1.050–1.059). The increase in CLBR was independent of maternal age, number of oocytes retrieved and number of previous IVF live births. The CLBR for women <35 years and ≥ 35 years both increased significantly, following the same pattern. During the study period a substantially increasing number of blastocyst transfers were performed, both in fresh and in FET cycles. An important contributor included in the blastocyst strategy, may be the extended culture of the total cohort of embryos, also embryos earlier discarded at early cleavage stages, in order to reach the blastocyst stage. These embryos may contribute to the total number of available blastocysts and thereby increase the chance of a live birth within that oocyte aspiration cycle. Other important predicting factors for live birth, such as number of embryos transferred, could not explain the improvement, on the contrary the single embryo transfer (SET) rate increased with time. Limitations, reasons for caution The retrospective design implicates that other confounders of importance for CLBR can not be ruled out. In addition, some FET cycles might be performed later than one year post oocyte aspiration for the last year (2017) and are thus not included in this study. Wider implications of the findings: The results suggest that blastocyst transfer, particularly when used in FET cycles and in combination with vitrification, is an important contributor to the improved live birth rates over time. This gives a possibility for fewer oocyte aspirations needed to achieve a live birth and a shortened time to live birth. Trial registration number -

P–761 Can modified luteal support in fresh cycle after agonist trigger “rescue” the cumulative live birth rate (CLBR) in high responders

Abstract Study question Can modified luteal support in fresh cycle “rescue” the cumulative live birth rate (CLBR) in high responders who receive agonist trigger? Summary answer Live birth rate in high responders who had agonist trigger in fresh cycle was significantly reduced despite modified luteal support. What is known already Previous studies, including small randomised controlled trials, claimed that good live birth rate could be achieved at fresh transfer in “high responders” who had GnRHa trigger with modified luteal phase support. However, majority of these studies exclude the true high responders (patients with 20 and above oocytes) and average number of collected eggs reported in many of these studies in the range of 9 to 12. The data on outcome of fresh transfer in true high responders is very limited. Study design, size, duration A prospective observational study was conducted in 407 patients, aged 23–42 years who were expected to be at risk of high response (AFC>18, AMH>20 pmol/l) undergoing controlled ovarian stimulation between 2014–2019 triggered either with HCG or GnRH agonist. Live birth rate (LBR) in a fresh and subsequent 3 frozen transfers were compared in groups with different triggers and freeze all. Participants/materials, setting, methods Patients were stimulated in short antagonist protocol. The trigger was chosen based on the background characteristics, peak oestradiol and clinician discretion. Triggering was achieved either with 0.5 mg buserelin (GnRHa) 0.5mgin 230 patients (A) or with 250 mcg of hCG(H) in 177 patients. Modified luteal support included vaginal progesterone, oral oestrogen and 1500 iu of hCG on the day of egg retrieval. The later was omitted with more than 20 oocytes. Main results and the role of chance The mean age, AFC, number of previous cycles, number of embryos transferred were 33.3, 22.4, 0.26 and 1.2 respectively and did not have significant difference between different triggers. Whereas AMH (53 pmol/l (A) vs 43.1 pmol/l (H), P = 0.003), peak oestradiol (15140.74 (A) vs 9738.59 (H), P = 5.59X10–14), and number of oocytes collected (21 vs.17, P = 5.63X10–7) were significantly higher in GnRHa group. Seventeen patients in buserelin group had elective freeze all. Ovarian Hyperstimulation Syndrome (OHSS) rate was 3.9% in buserelin group (more then half of these cases had a bolus of hCG at egg collection; most were mild/moderate). On the other hand, hCG group had 2.5% of OHSS (all severe). Live birth rate in fresh cycle was 31% in hCG and 21% in GnRHa groups. If freeze all was undertaken in fresh cycle after GnRHa trigger, then LBR in the first frozen cycle of this group was 53% (P = 0.003, fresh vs frozen GnRHa group). CLBR was not different between GnRHa and hCG groups (51%). However, this was significantly lower than CLBR in GnRHa trigger freeze all group 76% (P = 0.03) Limitations, reasons for caution The limitation of this study is its non-randomised nature. However, since it is one of the biggest studies for high responders it has a power to minimise bias by adjusting for multiple variables. Wider implications of the findings: Proceeding with fresh transfer in high responders after GnRHa trigger with modified luteal support not only maintains the risk of OHSS (equivalent to hCG group) but also significantly impairs the LBR not compensated even after 3 subsequent frozen embryo transfers. Therefore, freeze-all approach should be preferred in this group. Trial registration number NA

P–276 Factors associated with first oocyte retrievals affecting time to live birth – a retrospective study

Abstract Study question Which are the main factors on first oocyte retrievals influencing the time to achieve a live birth? Summary answer The number of oocytes collected on the first retrieval is the most important factor to decrease time to live birth. What is known already The goal of infertile couples when they attend a fertility clinic is to obtain a healthy baby as soon as possible. Cumulative live birth rate is today considered the most reliable estimate of ART success. However, the time used to achieve such goal is still not clear both to health professionals and patients. Although there is a general idea of which factors predict ART success, both clinicians and embryologists are still not aware of which variables can effectively influence the time to a live birth. Study design, size, duration We analyzed retrospectively 333 couples who performed their first fresh IVF/ICSI cycles from January 2015 to December 2018, along with their eventual subsequent FET and/or IVF/ICSI cycles, leading to 146 live births. The aim of this study was to use “Time” as an additional measure of ART success, and to identify which variable of the first oocyte retrieval has a major influence on time to live birth. Participants/materials, setting, methods We included in total 430 oocyte retrievals and 147 FET cycles. Oocyte donation cycles were excluded. Data were studied cumulatively until the childbirth (couples at risk) or until the last treatment record (censored couples). Cox Regression Model for survival analysis was used to study the variables that may influence the time to live birth, in order to take both confounding and collinearity into account. Main results and the role of chance We considered the date of the first oocyte retrieval as the starting point and the date of the first childbirth as the ending point to determine the time to live birth. The mean age of the woman at the first oocyte retrieval was 36.8±4.67, the cumulative pregnancy rate 43.5% (95%CI:41.77%;45.23%) and the live birth rate 34.0%(95%CI:32.32%;35.63%). As variables for the Cox Regression Model we selected the woman’s age and number of collected oocytes from the first oocyte retrieval. We also considered the number of frozen embryo transfers (FET). We have found a positive association between the number of oocytes collected at the first oocyte retrieval and the period until obtaining a healthy singleton (HR = 1.20;95%CI:1.105–1.297;p>0.001). Conversely, a negative association concerning the number of FET and time to live birth (HR = 0.32;95%CI:0.18–0.562;p>0.001) was demonstrated. Importantly, woman’s age, was not found to have a significant effect on time to live birth. Our results indicated that the number of oocytes collected at the first cycle, but not woman’s age, mostly affect time to live birth. Poor prognosis patients were found to be associated with several embryo transfers. Limitations, reasons for caution This is a retrospective study with a small number of cycles and freeze-all procedures were not included. Additional inclusion of freeze-all procedures in a larger study will be needed to confirm our preliminary results. Wider implications of the findings: This study identified oocyte number at first collection as a major influence on time to live birth. Increased attention to the “Time” parameter will be helpful for health professionals and patients, to further personalize reproductive treatment procedures, potentially decreasing psychological burdens associated with ART treatments. Trial registration number Not applicable

THE ASSOCIATION OF ESTROGEN/PROGESTERONE RATIO AND IVF OUTCOMES IN FROZEN EMBRYO CYCLES

The number of frozen Embryo transfers (FET) in invitro fertilization cycles (IVF) cycles is increasing and has already surpassed in numbers of fresh stimulated IVF cycles. Increasing numbers of elective single ETs are also resulting in more frozen embryos available for subsequent FET cycles. Most commonly, FET cycles are medicated or hormone supplemented cycles (HC) in which the endometrium is prepared by exogenous estrogen and progesterone following pituitary downregulation with gonadotropin releasing hormone agonist (1,2).

Cumulative live birth rate after IVF: trend over time and the impact of blastocyst culture and vitrification.

STUDY QUESTIONHas cumulative live birth rate (CLBR) improved over time and which factors are associated with such an improvement?SUMMARY ANSWERDuring an 11-year period, 2007–2017, CLBR per oocyte aspiration increased significantly, from 27.0% to 36.3%, in parallel with an increase in blastocyst transfer and cryopreservation by vitrification.WHAT IS KNOWN ALREADYWhile it has been shown that live birth rate (LBR) per embryo transfer (ET) is higher for fresh blastocyst than for fresh cleavage stage embryo transfer, CLBR per oocyte aspiration, including one fresh ET and all subsequent frozen embryo transfers (FET), does not seem to differ between the two culture strategies.STUDY DESIGN, SIZE, DURATIONA national register study including all oocyte aspirations performed in Sweden from 2007 to 2017 (n = 124 700 complete IVF treatment cycles) was carried out. Oocyte donation cycles were excluded.PARTICIPANTS/MATERIALS, SETTING, METHODSData were retrieved from the Swedish National Registry of Assisted Reproduction (Q-IVF) on all oocyte aspirations during the study period where autologous oocytes were used. CLBR was defined as the proportion of deliveries with at least one live birth per oocyte aspiration, including all fresh and/or frozen embryo transfers within 1 year, until one delivery with a live birth or until all embryos were used, whichever occurred first. The delivery of a singleton, twin, or other multiples was registered as one delivery. Cryopreservation of cleavage stage embryos was performed by slow freezing and of blastocyst by vitrification.MAIN RESULTS AND THE ROLE OF CHANCEIn total, 124 700 oocyte aspirations were performed (in 61 313 women), with 65 304 aspirations in women <35 years and 59 396 in women ≥ 35 years, resulting in 38 403 deliveries with live born children. Overall, the CLBR per oocyte aspiration increased significantly during the study period, from 27.0% to 36.3% (odds ratio (OR) 1.039, 95% CI 1.035–1.043) and from 30.0% to 43.3% if at least one ET was performed (adjusted OR 1.055, 95% CI 1.050–1.059). The increase in CLBR was independent of maternal age, number of oocytes retrieved and number of previous IVF live births. The CLBR for women <35 and ≥35 years both increased significantly, following the same pattern. During the study period, a substantially increasing number of blastocyst transfers was performed, both in fresh and in FET cycles. Other important predicting factors for live birth, such as number of embryos transferred, could not explain the improvement. An increased single embryo transfer rate was observed with time.LIMITATIONS, REASONS FOR CAUTIONThe retrospective design implicates that other confounders of importance for CLBR cannot be ruled out. In addition, some FET cycles might be performed later than 1 year post oocyte aspiration for the last year (2017) and are, thus, not included in this study. In addition, no data on ‘dropouts’, i.e. patients that do not continue their treatment despite having cryopreserved embryos, are available, or if this drop-out rate has changed over time.WIDER IMPLICATIONS OF THE FINDINGSThe results suggest that blastocyst transfer, particularly when used in FET cycles and in combination with vitrification, is an important contributor to the improved live birth rates over time. This gives a possibility for a lower number of oocyte aspirations needed to achieve a live birth and a shortened time to live birth.STUDY FUNDING/COMPETING INTERESTSThe study was financed by grants from the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement (ALFGBG-70940) and by Hjalmar Svensson’s research foundation. None of the authors declares any conflict of interest.

Affected-embryo-based SNP haplotyping with NGS for the preimplantation genetic testing of Marfan syndrome

ABSTRACT Marfan syndrome (MFS), an autosomal dominant heritable disease of the connective tissue, is characterized by broad clinical manifestations in the musculoskeletal, cardiovascular, pulmonary, and ocular systems. In this study, a male patient with MFS caused by a heterozygous mutation NM_000138.5(FBN1):c.6037 + 2 T > C in the fibrillin 1 gene (FBN1) underwent preimplantation genetic testing (PGT) by using affected-embryo-based single nucleotide polymorphism (SNP) haplotyping. Multiple displacement amplification was used for whole genome amplification of biopsied trophectoderm cells after controlled ovarian stimulation. Sanger sequencing and next-generation sequencing (NGS) were used to detect the state of FBN1 mutation. A total of 14 blastocysts formed after intracytoplasmic sperm injection were biopsied. After NGS, 60 informative polymorphic SNP markers located upstream and downstream of the FBN1 gene and its pathogenic mutation site were linked to individual alleles. Sanger sequencing further confirmed that 8 blastocysts carried the mutation NM_000138.5(FBN1):c.6037 + 2 T > C, while 6 did not. Four of the non-carriers were euploid verified by copy number variation results. A female infant without MFS was born at 37 weeks gestation after a subsequent frozen embryo transfer. In conclusion, the successful case indicates that SNP haplotyping using sibling embryos as a reference is applicable to PGT in monogenetic diseases. Abbreviations MFS: Marfan syndrome; PGT: preimplantation genetic testing; FBN1: fibrillin 1 gene; NGS: next-generation sequencing; SNP: single nucleotide polymorphism.

Fresh embryo transfer versus freeze-only after in vitro maturation with a pre-maturation step in women with high antral follicle count: a randomized controlled pilot study.

In vitro maturation (IVM) is an alternative to in vitro fertilization (IVF) for women at high risk of developing ovarian hyperstimulation syndrome (OHSS). This study determined the effectiveness and safety of a freeze-only strategy versus fresh embryo transfer (ET) after IVM with a pre-maturation step (CAPA-IVM) in women with a high antral follicle count (AFC). This randomized, controlled pilot study (NCT04297553) was conducted between March and November 2020. Forty women aged 18-37 years with a high AFC (≥24 follicles in both ovaries) undergoing one cycle of CAPA-IVM were randomized to a freeze-only strategy with subsequent frozen ET (n = 20) or to fresh ET (n = 20). The primary endpoint was ongoing pregnancy resulting in live birth after the first ET of the started treatment cycle. The ongoing pregnancy rate in the freeze-only group (65%) was significantly higher than that in the fresh ET group (25%; p = 0.03), as was the live birth rate (60% versus 20%; p = 0.02). Clinical pregnancy rate was numerically, but not significantly, higher after frozen versus fresh ET (70% versus 35%; p = 0.06), while the number of day 3 or good quality embryos, endometrial thickness on the day of oocyte pick-up, implantation rate, and positive pregnancy test rate did not differ significantly between groups. No cases of OHSS were observed, and miscarriage and multiple pregnancy rates were similar in the two groups. These findings suggest that the effectiveness of CAPA-IVM could be improved considerably by using a freeze-only strategy followed by frozen ET in subsequent cycles. NCT04297553 ( www.clinicaltrials.gov ).

Endometrial laminin subunit beta-3 expression associates with reproductive outcome in patients with repeated implantation failure.

Endometrial laminin subunit beta-3 (LAMB3) is a candidate gene whose expression distinguishes the endometrial window of receptivity (WOR) in human. This study aims to examine endometrial LAMB3 levels in patients with repeated implantation failure (RIF), in order to assess the ability of LAMB3 to predict pregnancy outcome. Endometrial biopsies were taken during the WOR from 21 healthy volunteers in natural menstrual cycles and from 50 RIF patients in mock cycles prior to frozen embryo transfer (FET) cycles. Immunohistochemistry (IHC) staining of LAMB3 was performed, and the H-score was correlated with the pregnancy outcome in subsequent FETs. In healthy volunteers, endometrial LAMB3 was demonstrated to be highly expressed during the WOR with the staining exclusively in the cytoplasm of the epithelial cells. In a discovery set of RIF patients, the LAMB3 expression level was found to be significantly higher in those who conceived compared to those who did not in subsequent FETs. A receiving operator characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.7818 (95% confidence interval 59.92-96.44%) with an H-score cutoff of 4.129 to differentiate cases with positive or negative pregnancy outcomes. This cutoff achieved an accuracy of 75% in pregnancy prediction in a following validation set of RIF patients, in which the pregnancy rate in subsequent FETs was three-fold higher when the mock cycle LAMB3 H-score was ≥ 4.129 compared to < 4.129. IHC measurement of endometrial LAMB3 expression could be a promising prognostic method to predict pregnancy outcome for RIF patients undergoing FETs.

Individualised luteal phase support in artificially prepared frozen embryo transfer cycles based on serum progesterone levels: a prospective cohort study.

Does an individualised luteal phase support (iLPS), according to serum progesterone (P4) level the day prior to euploid frozen embryo transfer (FET), improve pregnancy outcomes when started on the day previous to embryo transfer? Patients with low serum P4 the day prior to euploid FET can benefit from the addition of daily subcutaneous P4 injections (Psc), when started the day prior to FET, and achieve similar reproductive outcomes compared to those with initial adequate P4 levels. The ratio between FET/IVF has spectacularly increased in the last years mainly thanks to the pursuit of an ovarian hyperstimulation syndrome free clinic and the development of preimplantation genetic testing (PGT). There is currently a big concern regarding the endometrial preparation for FET, especially in relation to serum P4 levels around the time of embryo transfer. Several studies have described impaired pregnancy outcomes in those patients with low P4 levels around the time of FET, considering 10 ng/ml as one of the most accepted reference values. To date, no prospective study has been designed to compare the reproductive outcomes between patients with adequate P4 the day previous to euploid FET and those with low, but restored P4 levels on the transfer day after iLPS through daily Psc started on the day previous to FET. A prospective observational study was conducted at a university-affiliated fertility centre between November 2018 and January 2020 in patients undergoing PGT for aneuploidies (PGT-A) IVF cycles and a subsequent FET under hormone replacement treatment (HRT). A total of 574 cycles (453 patients) were analysed: 348 cycles (leading to 342 euploid FET) with adequate P4 on the day previous to FET, and 226 cycles (leading to 220 euploid FET) under iLPS after low P4 on the previous day to FET, but restored P4 levels on the transfer day. Overall we included 574 HRT FET cycles (453 patients). Standard HRT was used for endometrial preparation. P4 levels were measured the day previous to euploid FET. P4 > 10.6 ng/ml was considered as adequate and euploid FET was performed on the following day (FET Group 1). P4 < 10.6 ng/ml was considered as low, iLPS was added in the form of daily Psc injections, and a new P4 analysis was performed on the following day. FET was only performed on the same day when a restored P4 > 10.6 ng/ml was achieved (98.2% of cases) (FET Group 2). Patient's demographics and cycle parameters were comparable between both euploid FET groups (FET Group 1 and FET Group 2) in terms of age, weight, oestradiol and P4 levels and number of embryos transferred. No statistically significant differences were found in terms of clinical pregnancy rate (56.4% vs 59.1%: rate difference (RD) -2.7%, 95% CI [-11.4; 6.0]), ongoing pregnancy rate (49.4% vs 53.6%: RD -4.2%, 95% CI [-13.1; 4.7]) or live birth rate (49.1% vs 52.3%: RD -3.2%, 95% CI [-12; 5.7]). No significant differences were also found according to miscarriage rate (12.4% vs 9.2%: RD 3.2%, 95% CI [-4.3; 10.7]). Only iLPS through daily Psc was evaluated. The time for Psc injection was not stated and no serum P4 determinations were performed once the pregnancy was achieved. Our study provides information regarding an 'opportunity window' for improved ongoing pregnancy rates and miscarriage rates through a daily Psc injection in cases of inadequate P4 levels the day previous to FET (P4 < 10.6 ng/ml) and restored values the day of FET (P4 > 10.6 ng/ml). Only euploid FET under HRT were considered, avoiding one of the main reasons of miscarriage and implantation failure and overcoming confounding factors such as female age, embryo quality or ovarian stimulation protocols. No external funding was received. B.C. reports personal fees from MSD, Merck Serono, Ferring Pharmaceuticals, IBSA and Gedeon Richter outside the submitted work. N.P. reports grants and personal fees from MSD, Merck Serono, Ferring Pharmaceuticals, Theramex and Besins International and personal fees from IBSA and Gedeon Richter outside the submitted work. The remaining authors have no conflicts of interest to declare. NCT03740568.

Oocyte or embryo number needed to optimize live birth and cumulative live birth rates in mild stimulation IVF cycles

Research questionHow many oocytes or embryos are needed to optimize the live birth rate (LBR) per cycle and cumulative LBR (CLBR) following mild stimulation IVF (MS-IVF) in women with uncompromised ovarian reserve? DesignRetrospective analysis of a 4-year database of five fertility centres. The study population included women with normal/high ovarian reserve, who underwent autologous MS-IVF (daily ≤150 IU gonadotrophin) with fresh and subsequent frozen embryo transfer(s) (FET) from surplus embryos. Only the first cycle of each patient was included. Cycles with >150 IU daily average of gonadotrophin were excluded. ‘Freeze-all embryo’ (FAE) cycles were analysed separately. ResultsA total of 862 consecutive cycles fulfilled the inclusion criteria; 592 were eligible for fresh embryo transfer, 239 had non-elective 'freeze-all' cycles. Median age (25–75th percentile) of women who had fresh embryo transfer was 35 (32–37) years, median antral follicle count 19 (14–28) and anti-Müllerian hormone 19.2 (13–28.9) pmol/l. LBR/fresh cycle and CLBR inclusive of FAE cycles in the <35, 35–37, 38–39 and 40–42 year age groups were 37.8% and 45.1%, 36.0% and 41.6%, 18.4% and 29.1%, and 8.9% and 18.1%, respectively. The LBR following fresh embryo transfer plateaued after nine oocytes (40.3%) or four embryos (40.8%). The CLBR optimized when 12 oocytes (42.9%) or nine embryos (53.8%) were obtained. The LBR per oocyte peaked in women under 35 years when <5 oocytes were retrieved (11.4%), then declined with age and with higher oocyte yield. There were no cases of severe ovarian hyperstimulation syndrome (OHSS). ConclusionNine oocytes, or four embryos, can optimize fresh transfer cycle LBR in MS-IVF. The CLBR are optimized with 12 oocytes, or nine embryos in predicted normal responders, while safeguarding against OHSS.

Does shipping vitrified embryos to a long-term storage bank affect outcomes?

Objective To assess the pregnancy rate associated with an aggressive policy of moving vitrified embryos to off-site storage. Design Retrospective comparative study Materials and methods Frozen embryo transfers (FETs) in 2019 were reviewed. Only embryos vitrified from autologous patients at the blastocyst stage were included in this evaluation. FETs were grouped into No Shipping (Group A) and Shipping (Group B) categories. The Group A embryos always remained on site, while Group B embryos were shipped to one of two off-site storage facilities and then returned to us when the patient was ready to undergo FET. Shipping included both courier transport and UPS transport. Shipping tanks were unloaded following a procedure developed and validated in our laboratory. Only patients with ongoing pregnancies are included. Results The shipping of vitrified embryos to a long-term storage bank did not decrease pregnancy rates when compared to embryos which remain on site. The increase in pregnancy rate in the shipping group is logical. The patients with embryos shipped off-site were clinically pregnant and not planning on using them soon, while the embryos that were never shipped off-site were patients that were utilizing their frozen embryos. Nearly 80% of patients that had embryos shipped off-site for storage achieved a pregnancy when the embryos were returned to the clinic. Conclusions Shipping vitrified embryos to off-site storage is not detrimental to outcome with subsequent FET. This is a successful strategy for IVF biolaboratories to pursue. Outsourcing storage can ultimately decrease clinic liability and the conundrum of what to do with abandoned embryos. The physical space needed for storage can be greatly reduced. Disclosures Nothing to disclose Funding None

THE CLINICAL USE OF ENDOMETRIAL RECEPTIVITY ANALYSIS (ERA®) AND THE EFFECTS ON IMPLANTATION RATES IN WOMEN WITH PRIOR FAILED TRANSFERS

Endometrial receptivity is thought to be an important cause of implantation success or failure. ERA® (Endometrial Receptivity Analysis) analyzes the transcription profile of luteal phase endometrium (in a natural or programmed cycle) to determine its receptivity. ERA® results purport to allow physicians to personalize frozen embryo transfer (FET) protocols to transfer embryos during the “receptive” window, and thus increase implantation (IR) and pregnancy (PR) rates. In this study, we aimed to (1) evaluate the utilization of ERA® in a single large private ART practice, (2) for patients who had “non-receptive” ERA® results, determine if customized modifications of FET protocols according to ERA® results have led to changes in IR and PR in subsequent FETs. A retrospective cohort analysis. We reviewed records of patients who had an ERA® between 2014 to 2019 at our IVF center. All ERA® cycles were programmed cycles with an average progesterone duration of 133 (98-168) hours before the endometrial biopsy. Patient demographics, previous FET data, ERA® results, and outcome of subsequent FETs were analyzed. Data analysis was performed using Excel and MedCalc.org and SciStat.com. 173 patients completed an ERA®. The age was 39±4.781 years, with 82% of patients having had at least one prior FET (0-7). 8.6% had a chart diagnosis of uterine factor, 7.5% with endometriosis, 11% with adenomyosis, and 15% of patients had fibroids. The average peak endometrial thickness was 9.8±2.0 mm for the ERA® cycle and 9.9±2.1 mm during the first subsequent FET. Of all ERA®s completed, 40% (N=69) patients were receptive, 43% (N=74) pre-receptive (recommended transfer 24 hrs later), 14.45% (N=25) early receptive (12 hrs later), 2.3% (N=4) late receptive (12 hrs earlier), and one patient was post-receptive. 10% completed a second ERA® due to an invalid biopsy sample, and only one patient completed three ERA®s. Overall FET PR in this cohort of patients pre-ERA® was only 5% (16/320). The overall PR post-ERA® was 36% (63/171) with an odds ratio of 11.1 (95% CI 6.1-20.0). Pre-ERA® IR of autologous euploid embryos was 6.3% (11/176). The post-ERA® IR of autologous euploid embryos was 42% (47/112) with an odds ratio of 10.8 (95% CI 5.3-22.2). For the non-receptive groups (both pre- and post-receptive), the pre-ERA® PR was 5.5% (10/180) and post-ERA® PR was 36% (35/97) with an odds ratio of 7.9 (95% CI 3.3-18.8). The pre-ERA® IR of autologous euploid embryos was 8.7% (9/104). The post-ERA® IR was 42% (24/56) with an odds ratio of 9.6 (95% CI 4.5-20.53). For the receptive groups, the pre-ERA® PR was 4.3% (6/140), and post-ERA® PR was 37% (28/74) with an odds ratio of 13.6 (95% CI 5.3-34.9). The receptive group had a pre-ERA® IR of autologous euploid embryos of 2.8% (2/71), and a post-ERA® IR of 41% (23/55) with an odds ratio of 24.8 (95% CI 5.5-111.6). 60% of patients had non-receptive ERA®s. While non-receptive patients had increased implantation and pregnancy rates post-ERA®, similar improvements were also seen in receptive patients making it difficult to draw the conclusion that ERA® was clinically beneficial.

Cumulative Live Birth Rates According to Maternal Body Mass Index After First Ovarian Stimulation for in vitro Fertilization: A Single Center Analysis of 14,782 Patients.

Objective: To investigate the cumulative live birth rates (CLBR) according to body mass index (BMI) in women undergoing their first in vitro fertilization (IVF).Design: Retrospective cohort analysis.Setting: An IVF clinic in a public hospital.Patients: This is a retrospective study of 14,782 patients undergoing their first fresh IVF cycles and subsequent frozen embryo transfers in our clinic from January 2014 to January 2017. The follow-up for CLBR continued until January 2019. Patients with a BMI <18.5 kg/m2 were considered to be underweight and those with a BMI > 24 kg/m2 were considered to be overweight. Patients with a BMI ≥ 28 kg/m2 were considered to be obese.Intervention(s): None.Primary Outcome Measure: The primary outcome was cumulative live birth rate (CLBR).Result(s): This study illustrated the “inverted U shape” associations between body weight and IVF outcome (CLBR). The turning points in threshold analysis, as found by an automatic search, were BMIs of 18.5 and 30.4 kg/m2. The main finding of this retrospective data analysis is that the CLBR increased in underweight women, plateaued for normal weight and overweight women with a BMI between 18.5 and 30.4 kg/m2, and decreased in obese women.Conclusion(s): The data suggested an “inverted U shape” association between BMI and CLBR. The CLBR increases in underweight women, plateaus in normal weight and overweight women, and then decreases in obese women.

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.

Why Do Euploid Embryos Fail to Implant? The Role of CD138 and Chronic Endometritis

The objective of this prospective cohort study was to investigate the relationship between chronic endometritis (CE) and its treatment on pregnancy outcomes in patients undergoing frozen embryo transfers (FETs) from in vitro fertilization (IVF) with preimplantation genetic testing (PGT) who had previously failed ≥ 1 euploid FET. The diagnosis of CE was made using CD138 as a marker. This study occurred at a single, high volume academic based fertility center. 305 patients were included with a history of ≥ 1 failed euploid transfers. 59 patients underwent endometrial biopsies (EMB) prior to subsequent FET (Tested), 246 patients did not undergo EMB prior to subsequent transfer (Untested). Patients in the Tested group had an EMB prior to subsequent FET, whereas the patients in the Untested group did not have an EMB prior to next FET. Patients tested and found positive for CE were treated with antibiotics and underwent test-of-cure (TOC) EMBs prior to next FET to assess for adequate treatment. Patients without CE on EMB proceeded to next FET without antibiotic treatment. Main outcome measures included positive Beta-hCG (βhCG) on cycle day 28, implantation rate (IR), and subsequent ongoing pregnancy/live birth rates (OPR/LBR). Our results showed that 51% of patients (30/59) were diagnosed with CE and treated with antibiotics (Tested, CE treated); negative TOC biopsies were obtained prior to next FET. 49% of patients (29/59) tested negative for CE (Tested, No CE) and were not treated prior to next FET. Tested patients without CE (CE treated and No CE) had significantly higher incidences of positive βhCG, IR and OPR/LBR than patients who were not tested. Those who were biopsied and treated for CE had significantly higher OPR/LBR compared to patients who were biopsied and negative for CE (80% vs. 55%). We conclude that CE is common, with a prevalence of 51% in patients who have failed euploid FETs. Testing for CE may provide a benefit to patients, assuring higher incidences for positive βhCG, implantation, and subsequent ongoing/live births. CE is a treatable condition warranting investigation in patients with poor pregnancy outcomes.

A Slower Age-Related Decline in Treatment Outcomes After the First Ovarian Stimulation for in vitro Fertilization in Women With Polycystic Ovary Syndrome.

Background: Polycystic ovary syndrome (PCOS) patients have a better ovarian reserve and age-related improvement in endocrine disturbances than non-PCOS patients. The effects of age on in vitro fertilization (IVF) treatment outcomes associated with cumulative live birth rate (CLBR) remain unclear.Objectives: To study the effect of age on CLBR after the first ovarian stimulation in IVF in PCOS patients.Method: This retrospective cohort study included 3,502 PCOS patients and 18,596 patients with tubal factor infertility, who underwent their first IVF cycles and subsequent frozen embryo transfer (ET) attempts. The primary outcome was CLBR associated with a single stimulation cycle and secondary outcomes included the implantation rate, clinical pregnancy rate, live birth rate (LBR), large for gestational age (LGA) rate, small for gestational age (SGA) rate, and preterm birth (PTB) rate of fresh ET cycles.Results: PCOS patients over 40 years had a higher implantation rate (27.8 vs. 15.7%, P < 0.05), clinical pregnancy rate (51.4 vs. 26.1%, P < 0.05), LBR (42.3 vs. 18.2%, P < 0.05), and CLBR (50.0 vs. 21.5%, P < 0.05) than non-PCOS patients over 40 years. These rates were comparable between PCOS patients aged 35 to 40 years and those aged over 40 years (P = 0.263, 0.385, and 0.112, respectively). The changes in the implantation rate, clinical pregnancy rate, and CLBR by age were slower for PCOS patients than for non-PCOS patients (all P < 0.05). Among PCOS patients less than 35 years, BMI was negatively associated with CLBR [aOR: 0.961 (0.939–0.985); P < 0.05]; however, among PCOS patients over 35 years, instead of BMI (P = 0.353), age [aOR: 0.891 (0.803–0.990); P < 0.05] and the number of oocytes retrieved [aOR: 1.093 (1.002–1.078); P < 0.05] were significantly associated with CLBR. No significant differences in LGA, LGA, or PTB were detected between PCOS and non-PCOS patients over 35 years (all P > 0.05).Conclusions: The declines in treatment outcomes with age are slower for PCOS patients than for non-PCOS patients. For patients over 40 years, PCOS patients have reproductive advantages over non-PCOS patients. In contrast to younger PCOS patients (<35 years), older PCOS patients (≥35 years) may benefit less from taking time to lose weight before IVF treatment, and the immediate initiation of assisted reproductive treatment is essential.

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.

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.