Day Of Transfer Research Articles

Early graft-infiltrating lymphocytes are not associated with graft rejection in a mouse model of skin transplantation.

Graft-infiltrating lymphocytes (GILs) play an important role in promoting rejection after organ transplantation. We recently reported that GILs that accumulated up to 3 days post-transplantation did not promote rejection, whereas GILs present 3-5 days post-transplantation promoted rejection in a mouse heart transplantation model. However, the immunological behaviour of GILs in murine skin transplantation remains unclear. GILs were isolated on days 3, 5 or 7 post-transplantation from C57BL/6 (B6) allogeneic skin grafts transplanted onto BALB/c mice. BALB/c Rag2-/- γc-/- mice (BRGs) underwent B6 skin graft transplantation 10 weeks after adoptive transfer of day 3, 5, or 7 GILs. BRGs reconstituted with day 5 or 7 GILs completely rejected B6 grafts. However, when B6 grafts harvested from recipient BALB/c mice on day 5 or 7 were re-transplanted into BRGs, half of the re-transplanted day 5 grafts established long-term survival, although all re-transplanted day 7 grafts were rejected. BRGs reconstituted with day 3 GILs did not reject B6 grafts. Consistently, re-transplantation using day 3 skin grafts resulted in no rejection. Administration of anti-CD25 antibodies did not prevent the phenomenon observed for the day 3 skin grafts. Furthermore, BRGs reconstituted with splenocytes from naïve BALB/c mice immediately rejected the naïve B6 skin grafts and the re-transplanted day 3 B6 grafts, suggesting that day 3 GILs were unable to induce allograft rejection during the rejection process. In conclusion, the immunological role of GILs depends on the time since transplantation. Day 3 GILs had neither protective nor alloreactive effects in the skin transplant model.

O-040 Pro -better late than never

Abstract In IVF, blastocysts developed on Day5-6 are typically selected for transfer, cryopreservation, or biopsy, while embryos that do not reach this developmental stage before 144 hours post-insemination (hpi) are often discarded. Recently, there has been an increased adoption of blastocyst culture aimed at enhancing embryo selection potential and supporting a single embryo transfer approach. Consequently, the clinical significance of Day7 blastocysts (embryos reaching this stage beyond 144 hpi) has become a prominent topic of discussion, particularly in the context of treating patients with poor prognosis. Numerous studies worldwide have reported lower competence for these embryos, as evidenced by poorer morphological quality, higher aneuploidy rates, and lower implantation rates following both untested and euploid transfer. Nonetheless, a notable proportion of these blastocysts are euploid and result in healthy live births. While their clinical utilization may entail lower efficiency per transfer, it ultimately increases the cumulative live birth rate per cycle. However, most studies have encountered challenges due to diverse definitions of Day7, varying clinical settings, culture strategies, incubators, and local regulations. Presently in IVF, leveraging time-lapse technology (TLT), artificial intelligence (AI), and preimplantation genetic testing for aneuploidies (PGT-A) could standardize and enhance Day7 blastocyst characterization and clinical assessment. Consequently, we designed a study exclusively in freeze-all PGT-A cycles with vitrified-warmed euploid single embryo transfer after continuous culture in time-lapse incubators, trophectoderm biopsy without zona pellucida drilling on day3, comprehensive chromosome testing, confirming embryo morphological assessments through an AI-powered software, and utilizing this tool also to automate developmental timing annotations. Our analysis confirmed that all blastocysts were biopsied only after reaching an expansion level suitable for the procedure (approximately 24,000 µm²) and consistent across all embryos clustered every 12 hours in 6 groups (<120, 120-132, 132-144, 144-156, 156-168, and >168 hpi). Day7 blastocysts accounted for approximately 15% of all blastocysts obtained over 8 years, derived from older oocytes, exhibiting poorer quality according to both Gardner’s score and AI score, slower development from early stages, and accumulating delays across various intermediate processes like blastocyst expansion from tEB to the time of biopsy. They were less euploid and resulted in a lower live birth rate per euploid transfer. However, only the latter association was significant when adjusted for confounders (e.g., blastocyst quality). Comparing only embryos biopsied before 144 hpi versus embryos biopsied after this threshold, neither the euploidy nor the live birth rate were significantly different, supporting the extension of embryo culture beyond Day6. Overall, we estimated that Day7 embryos contribute to a relative increase of 4.4%, 13.7%, and 5.2% in the number of patients who achieved a live birth, who were not pregnant after the transfer of faster embryos, and who have supernumerary transferable embryos after a live birth, respectively. The main limitations of our investigation were its retrospective nature and the fact that retrospective associations do not provide evidence of causation. There might be a bias towards poor prognosis patients being subject to the transfer of Day 7 embryos, for instance. Lastly, more data are required regarding the putative impact of extended embryo culture including post-natal outcomes (reassuring to date), as well as the cost-effectiveness of this clinical choice. Considering the increasing personalization and patient-centeredness of IVF, where permitted by local regulations, the decision to culture embryos to Day7 should be based on a careful evaluation of couples’ reproductive history and future prognosis, and should be subject to a patient-centered decision-making process. In the future, AI can be harnessed to support IVF practitioners by objectifying the assessment of poor-quality and slow-growing embryos with measurable information and reliable predictions, thereby mitigating the inherent subjectivity of embryologists’ decisions.

P-110 Live birth of blastocyst culture in women with previous poor blastocyst formation: a cohort study

Abstract Study question Do cumulative live birth rates(CLBRs) in the second cycle differ between blastocyst-stage and cleavage-stage transfer in women with low blastocyst progression during initial IVF ? Summary answer The CLBRs following subsequent oocyte retrievals were comparable for extended culture with blastocyst-stage embryo versus cleavage-stage transfers. What is known already Fresh blastocyst-stage embryo transfer increases live birth rates when compared to cleavage- stage embryo transfer in women with good prognosis. However, it remains unclear whether a blastocyst-culture approach can still be considered for women with a history of failed or reduced blastocyst formation. Study design, size, duration A retrospective cohort study evaluating outcomes of the second IVF/ICSI in women who had a history of poor blastocyst development in their first-time retrievals at a single fertility center from September 2019 to December 2021. Women fulfilled embryo quality criteria of having at least two good-quality cleavage-stage embryos (graded 1-2) on day 3 were included for analysis. Participants/materials, setting, methods A total of 396 patients were analyzed and categorized into two groups. Group D5/6: Extending culture of all embryos to blastocyst-stage in vitro followed by blastocyst transfer; Group D3: transfer or cryopreservation of one or two of the highest quality cleavage-stage embryo(s) on Day3.The main outcome measure was the CLBR per oocyte retrieval.Multivariable logistic regression analyses were used to assess the association between two culture approaches and live birth outcomes after adjusting for possible confounders. Main results and the role of chance The CLBRs (57.01% versus 49.48%) were numerically higher in women in D5/6 group (N = 107) than those in D3 group (N = 289) although the difference was attenuated and did not reach statistical significance after controlling for confounders (aOR: 1.27, 95%CI 0.78, 2.07,P=0.345). All subgroup analyses showed similar conclusions as the main model, even restricting on women with a limited number of quality embryos 3 days post-fertilization. Among cycles with only 2 or 3 good-quality cleavage embryos, Day3 transfers were still associated with lower CLBR compared to all blastocyst culture and transfer (aOR 0.66, 95% CI 0.51-0.87). Limitations, reasons for caution The retrospective nature of the present study, alongside the selection bias towards blastocyst culture, may predispose to the risk of residual confounding. The cohort of women characterized by previous failed or reduced blastocyst formation was comparatively small. Larger prospective studies focusing on the unique group are still needed. Wider implications of the findings Even in poor prognosis IVF patients with a history of poor blastocyst formation and/ or blastulation failure, all blastocyst culture and transfer can still be considered when at least two good-quality embryos are available at day 3. These findings are clinically relevant and require additional investigation. Trial registration number Not Applicable

Diameter of Human Day Five Blastocysts and Birth Sex.

Background This study aimed to evaluate the offspring sex ratio, born through fresh and cryo-thawed single blastocyst (BL) transfers regarding a single morphological, static parameter, namely, BL diameter. Methodology This retrospective, observational study was conducted at an assisted reproductive technology (ART) center, Kinderwunschzentrum Niederrhein Germany. We conducted a statistical analysis of all births resulting from fresh and thawed in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles after a single embryo transfer (SET). The main outcome measure was the offspring sex ratio after SET of a day five BL in relation to the BL diameter measurement. Results There were more female than male babies born in our study. We observed a tendency for BL to have a higher diameter, resulting in female offspring, which was not statistically relevant. We also compared the BL diameter in the fresh embryo transfer (ET) group with that of the cryo-thawed ET group, showing a tendency toward a larger diameter in the fresh ET group. In the ICSI cycles, there was a higher tendency for a larger BL diameter when compared to IVF cycles. In the fresh ET cycles, BL leading to the male sex at birth had a tendency toward a larger diameter than the female BL. In the cryo-thaw ET cycles, BL leading to the female sex had a tendency toward a larger diameter than the male BL. Conclusions Our results showed a tendency in the sex of offspring toward the female sex and no significant difference in the BL diameter of BL leading to birth after ART and consecutive transfer of day five BL.

Trade-off between double cleavage-stage embryos transfer and single blastocyst-stage embryo transfer in patients with few good quality embryos in antagonist cycles: a retrospective study using a propensity score matching analysis

ObjectiveThis study aimed to compare the per OPU clinical outcomes for transfer of Day 3 double cleavage-stage embryos (DET) and Day 5 single blastocyst-stage (SBT) in patients with five or fewer good quality embryos on day 3 per occyte pick-up cycle (OPU) in antagonist cycles with consideration of blastocyst formation failure.MethodsThis was a retrospective, observational cohort study of 2,116 cases of OPU treated with antagonist protocol in the affiliated Chenggong Hospital of Xiamen University between January 2013 and December 2020. DET was performed in 1,811cycles and SBT was performed in 305 cycles. The DET group was matched to the SBT group by propensity score (PS) matching according to multiple maternal baseline covariates. After PS matching, there were 303 ET cycles in each group. The primary outcomes were the cumulative live birth rate (CLBR), cumulative multiple pregnancy rate(CMPR)per OPU and the number of ET to achieve live birth per OPU. Secondary outcomes were the percentage of clinical pregnancy(CPR), live birth rate(LBR), multiple pregnancy rate(MPR).ResultsFollowing PS mating, the CLBR was slightly higher (48.8% versus 40.3% ; P = 0.041) and the CMPR was significantly higher in the DET group compared to SBT group(44.2% versus 7.9%, P < 0.001). The CPR, LBR and MPR per fresh transfer were higher in DET group compared to SBT group(50.2% versus 28.7%; 41.3% versus 21.5%;29.6% versus 0%, P < 0.001). The number of ET to achieve live birth per OPU in SBT group was obiviously more than in DET group(1.48 ± 0.578 versus 1.22 ± 0.557 ,P < 0.001).ConclusionWith a marginal difference cumulative live birth rate, the lower live birth rate per fresh transfer and higher number of ET per OPU in the SBT group suggested that it might take longer time to achieve a live birth with single blastocyst strategy. A trade-off decision should be made between efficiency and safety.

P-511 Prolonging the time of vaginal progesterone supplementation does not improve pregnancy outcomes after day 6 blastocyst transfer

Abstract Study question Does the administration of vaginal progesterone for 6 days improve pregnancy outcomes when transferring day 6 blastocysts, as opposed to 5 days of progesterone treatment? Summary answer We observed similar pregnancy outcomes after 5 and 6 days of vaginal progesterone supplementation following day 6 blastocyst transfer in oocyte recipient cycles. What is known already Progesterone administration promotes the final phase of endometrial preparation for embryo transfer. However, the optimal duration of progesterone transformation still remains unknown. The implantation potential of blastocysts formed at different timepoints is also contentious, as blastocysts on day 6 have been linked to poorer clinical outcomes compared to their day 5 counterparts. It is unclear whether this effect stems from an intrinsic impaired implantation capacity of slow-developing blastocysts, or a displaced window of implantation (WOI). It has been hypothesized that 6 days of progesterone administration may improve clinical outcomes after frozen day 6 blastocyst transfer. Study design, size, duration Retrospective cohort study of oocyte recipients undergoing single day 6 blastocyst transfer, between January 2020 and December 2022. This study compared two groups: day 6 blastocysts transferred after 5 days of progesterone administration (n = 284) and day 6 embryo transfers performed after 6 days of progesterone administration (n = 69). Participants/materials, setting, methods Our study included oocyte recipients who underwent a single embryo transfer of a day 6 blastocyst and hormonal replacement treatment for endometrial preparation with vaginal external progesterone administration (800mg/24h) for either 5 or 6 days prior to embryo transfer. Outcomes were analyzed using unpaired t-test or Fisher’s test and logistic regression. Multivariate models were adjusted for oral or transdermal estrogen administration and endometrial thickness on the day of embryo transfer. P-values &amp;lt;0.05 were considered significant. Main results and the role of chance Mean maternal age was 43.5 years, while patients had a mean BMI of 25.1. Mean days of estrogen therapy prior to embryo transfer were 22.4, with 75.3% of patients using transdermal estrogen administration, and achieving a mean endometrial thickness of 9.6 mm on the day of transfer. Mean progesterone levels on the day of transfer were 11.9 ng/ml. Thirty-seven percent of the day 6 blastocysts were of high quality. None of the demographic or cycle characteristics were significantly different between the study groups. When considering outcomes of day 6 blastocyst transfers, we found no significant differences in biochemical (26.33% vs 20.13%, p = 0.343), clinical (22.06% vs 17.46%, p = 0.49) and ongoing (14.64% vs 9.52%, p = 0.41) pregnancy rates between 5 and 6 days of progesterone administration. Our adjusted analysis further corroborated these results. Limitations, reasons for caution The retrospective nature of the study and low number of transfers performed after 6 days of progesterone treatment warrant careful interpretation. This study only evaluated endometrial preparation by vaginal progesterone administration in oocyte recipients and cannot be extrapolated to other routes of progesterone administration or other patient populations. Wider implications of the findings Poorer reproductive outcomes associated with the transfer of day 6 blastocysts appear to be related to a reduced implantation capacity of slow-developing embryos rather than a displaced WOI due to rising progesterone. WOI appears wider than assumed, as prolonged progesterone transformation times had no effect on pregnancy outcomes. Trial registration number Not applicable

P-125 Day 5 endometrium, Is the pregnancy rate following day 5 or day 6 vitrified/thawed blastocysts transfer affected?

Abstract Study question Are ongoing pregnancy rates of embryos vitrified on day 5 and day 6 when transferred into a day 5 endometrium comparable? Summary answer Day 5 blastocysts implant better in day 5.5 endometrium than day 6, whether they were vitrified on day 6 or 5 and cultured 24 hours. What is known already Preparation of the endometrium for transfer of cryopreserved embryos is usually done so that transfer takes place on endometrial day 5, although embryos may be at day 5 or 6 of development, so there may be asynchrony between the endometrium and the embryo if a day 6 embryo is transferred. Fresh transfer of day 5 blastocysts has been reported to have better results than day 6 blastocysts, predictably due to this endometrial asynchrony. However, data from frozen blastocyst transfers are less clear, possibly due to heterogeneity of the patient population and/or embryo quality. Study design, size, duration In this study we aim to analyse whether ongoing pregnancy after vitrified/thawed embryo transfers is affected if day 5 or day 6 embryos are transferred into day 5 endometrium. For this purpose, we examined 2266 cryotransfers performed in three Next Fertility clinics in Spain between 2016 and 2022. Day 5 (n = 1583) and 6 (n = 519) blastocyst transfers and day 5 embryos that devitrified one day before transfer (n = 164) were retrospectively compared. Participants/materials, setting, methods In this work blastocyst transfers were performed from donor or autologous oocytes, with or without PGT-A. Embryos were vitrified on day 5 or 6. Endometria were prepared with hormone replacement therapy or in natural cycle. All FET were performed after 5.5 days of progesterone exposure, regardless of embryo stage (day 5 or 6). The results were analysed considering all the above groups. Main results and the role of chance Ongoing pregnancy are superior with day 5 blastocysts versus day 6 and day 5 cultured for 24 hours prior to transfer. However, day 6 blastocysts were similar to cultured ones. Own oocytes: Day 5 44.98% (n = 538) vs Day 6 35.75% (n = 207) (p &amp;lt; 0.03); Day 5 44.98% vs Day 5 cultured 32.58% (n = 89) (p &amp;lt; 0.03); Day 6 35.75% vs Day 5 cultured 32.58% (p &amp;gt; 0.05). Donated Oocytes: Day 5 45.86% (n = 726) vs Day 6 28.51% (n = 221) (p &amp;lt; 0.00001); Day 5 44.98% vs Day 5 cultured 39.34% (n = 61) (p &amp;gt; 0.05); Day 6 28.51% vs Day 5 cultured 39.34% (p &amp;gt; 0.05). PGT-A Own oocytes: Day 5 51.72% (n = 203) vs Day 6 25.39% (n = 63) (p &amp;lt; 0.003); Day 5 51.72% vs Day 5 cultured 40.00% (n = 10) (p &amp;gt; 0.05); Day 6 25.39% vs Day 5 cultured 40.00% (p &amp;gt; 0.05). PGT-A Donated Oocytes: Day 5 42.24% (n = 116) vs Day 6 35.71% (n = 28) (p &amp;gt; 0,05); Day 5 42.24% vs Day 5 cultured 40.00% (n = 4) (p &amp;gt; 0.05); Day 6 35.71% vs Day 5 cultured 50.00% (p &amp;gt; 0.05). Miscarriage rates were similar across all groups. Limitations, reasons for caution Not all cohorts in the study were equally large, so some results have less statistical power, especially in abortion rates. Wider implications of the findings Day 5 blastocysts should always be transferred into a 5.5-day endometrium on the day they are devitrified, as this is when they are most synchronous with the endometrium. Further studies are needed to assess whether day 6 embryos implant better in a 6.5-day endometrium than in a 5.5-day endometrium. Trial registration number not applicable

MP43-05 TESTICULAR SPERM ASPIRATION (TESA) VS. MICROFLUIDIC SPERM SEPARATION (MSS) IN COUPLES WITH HIGH SPERM DNA FRAGMENTATION UNDERGOING ICSI: WHICH APPROACH IS BETTER?

You have accessJournal of UrologyCME1 Apr 2023MP43-05 TESTICULAR SPERM ASPIRATION (TESA) VS. MICROFLUIDIC SPERM SEPARATION (MSS) IN COUPLES WITH HIGH SPERM DNA FRAGMENTATION UNDERGOING ICSI: WHICH APPROACH IS BETTER? Rabea Akram, Karen Kteily, Michael Maalouf, Ahmad Alshammari, Simon Phillips, and Armand Zini Rabea AkramRabea Akram More articles by this author , Karen KteilyKaren Kteily More articles by this author , Michael MaaloufMichael Maalouf More articles by this author , Ahmad AlshammariAhmad Alshammari More articles by this author , Simon PhillipsSimon Phillips More articles by this author , and Armand ZiniArmand Zini More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003289.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Testicular sperm aspiration (TESA) and microfluidic sperm separation (MSS) are modalities designed to select the best sperm quality for intra-cytoplasmic sperm injection (ICSI) in men with poor sperm DNA integrity. We sought to investigate and compare reproductive outcomes in couples with a high sperm DNA fragmentation index (DFI) using TESA-ICSI or MSS-ICSI. METHODS: We conducted a retrospective study of consecutive infertile couples with high DFI who underwent ICSI using TESA, or MSS. Couples were divided into 3 subgroups based on sperm DFI results (20-29%, ≥30-39% and ≥40% DFI). Sperm DFI was determined by flow cytometry based TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling). We utilized the ZyMōt® microfluidic sperm selection device. We excluded cycles with advanced maternal age (>40 years) and included cycles with transfer of day 5 blastocysts. RESULTS: After excluding cycles with advanced maternal age and those that did not proceed to day 5 blastocyst transfer, we identified 66 couples in the TESA cohort and 73 couples in the MSS cohort. Maternal age was not significantly different in the TESA vs. the MSS cycles in any of the DFI subgroups. However, paternal age was significantly higher in the TESA than in the MSS cycles within the 20-29% and 30-39% DFI subgroups (Table 1). There were no significant differences in pregnancy rates per embryo transfer in any of the DFI subgroups, However, live birth rates were higher in the MSS vs. the TESA cycles in the 20-29% DFI group. A trend toward higher live birth rates in the TESA vs. MSS cycles in the ≥40% DFI subgroup was noted (Table 1). CONCLUSIONS: TESA-ICSI and MSS-ICSI offer similar pregnancy rates per embryo transfer in couples with high DFI although MSS may be a better option than TESA in terms of live birth rates in couples with DFI in the 20-29% range. Larger studies are needed to define the optimal use of these sperm selection modalities in the treatment of couples with high DFI. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e602 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rabea Akram More articles by this author Karen Kteily More articles by this author Michael Maalouf More articles by this author Ahmad Alshammari More articles by this author Simon Phillips More articles by this author Armand Zini More articles by this author Expand All Advertisement PDF downloadLoading ...

Cloning horses by somatic cell nuclear transfer: Effects of oocyte source on development to foaling

The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abattoir-sourced ovaries and from live mares by OPU. A total of 1,128 oocytes were obtained, of which 668 were abattoir-derived and 460 were OPU-derived. The methods used for in vitro maturation and SCNT were identical for both oocyte groups, and the embryos were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham medium supplemented with 10% fetal calf serum. Embryo development in vitro was assessed, and Day 7 blastocysts were transferred to recipient mares. The embryos were transferred fresh when possible, and a cohort of vitrified-thawed OPU-derived blastocysts was also transferred. Pregnancy outcomes were recorded at Days 14, 42 and 90 of gestation and at foaling. The rates of cleavage (68.7 ± 3.9% vs 62.4 ± 4.7%) and development to the blastocyst stage (34.6 ± 3.3% vs 25.6 ± 2.0%) were superior for OPU-derived embryos compared with abattoir-derived embryos (P < 0.05). Following transfer of Day 7 blastocysts to a total of 77 recipient mares, the pregnancy rates at Days 14 and 42 of gestation were 37.7% and 27.3%, respectively. Beyond Day 42, the percentages of recipient mares that still had a viable conceptus at Day 90 (84.6% vs 37.5%) and gave birth to a healthy foal (61.5% vs 12.5%) were greater for the OPU group compared with the abattoir group (P < 0.05). Surprisingly, more favourable pregnancy outcomes were achieved when blastocysts were vitrified for later transfer, probably because the uterine receptivity of the recipient mares was more ideal. A total of 12 cloned foals were born, 9 of which were viable. Given the differences observed between the two oocyte groups, the use of OPU-harvested oocytes for generating cloned foals is clearly advantageous. Continued research is essential to better understand the oocyte deficiencies and increase the efficiency of equine cloning.

A Trend analysis of frozen embryo transfer at Alhikmah fertility center, Addis Ababa, Ethiopia

Background: In recent years, the use of frozen embryo transfers in Artificial reproductive technology has gradually increased, owing to improvements in laboratory techniques such as vitrification and blastocyst culture making pregnancy rates approaching those of fresh transfer cycles. Objective: The objective of this study is to determine the pregnancy rate after frozen embryo transfer, analyze the clinical factors that can potentially influence the result of pregnancy and identify patients who could benefit from this approach. Methods: A retrospective cross sectional study design was used to select patients that have undergone frozen embryo transfer from January 1, 2019- March 31, 2022 at Alhikmah fertility center, Addis Ababa, Ethiopia. Baseline data and pregnancy rate was collected, computed with SPSS version 24 for statistical associations and presented with tables. The primary outcome was a positive pregnancy test defined as serum ?-HCG level of &gt;10mIU/ml. Result: A total of 180 frozen embryo transfers were done for 128 women over the study period and the pregnancy rate was 26.6%. The mean age of the patients was 31.88(± 5.04). Majority (45.3%) had tubal factor infertility. ? 13(median) oocytes were collected for 53.9%. A freeze-all technique was used in 46% of the patients and the rest had extra embryos. Age of the patient &lt;35 and retrieval of oocytes above median had relation with positive pregnancy outcome. More importantly, freezing for risk of hyperstimulation and transfer of day 5embryos had a significant association with pregnancy with p-value &lt;0.05. Conclusion: From this study, we’ve seen promising pregnancy rate after frozen embryo transfer in patients with age group &lt;35 and those having ?13 oocyte. In addition, we can conclude that freezing for women at risk of hyperstimulation and transferring day 5embryos leads to a positive pregnancy result. However, since this is a retrospective study, we suggest a prospective comparative study to be done in the future for better recommendation. Key words: frozen embryo transfer, pregnancy rate, outcome predictors, assisted reproductive technology

Prolonging the time of progesterone supplementation to improve the pregnancy outcomes of single day 6 blastocyst transfer in frozen-thawed cycles: study protocol for a randomized controlled trial

BackgroundInfertility is one of the most important and underappreciated reproductive health problems in developing countries. Currently, in vitro fertilization and embryo transfer is the most effective treatment strategy for infertility. In a frozen-thawed cycle, single-blastocyst transfer can not only ensure relatively higher pregnancy and live birth rates but also effectively reduce the risk of maternal and neonatal complications. In frozen-thawed cycles, progesterone is initiated to promote the final phase of endometrial preparation prior to embryo transfer. However, the optimal duration of exposure to progesterone has remained inconclusive. Therefore, we designed a randomized controlled trial (RCT) to compare the effects of different prolonged progesterone transformation times (P+6 and P+7) on the pregnancy outcomes of D6 single blastocyst transfer in a frozen-thawed cycle.MethodsThis is a single-center, prospective, randomized controlled clinical trial involving 900 patients with single blastocyst transfer in the frozen-thawed cycle, aged from 20 to 38 years, with less than three transfers, and with HRT-cycle single D6 blastocyst transfer in the current cycle. Participants will be randomly assigned (1:1) into two parallel groups: the transfer of day 6 blastocysts on the 7th day of progesterone supplementation and the transfer of day 6 blastocysts on the 6th day of progesterone supplementation. The primary outcome measure is the clinical pregnancy rate. Secondary outcome measures include the miscarriage rate and live birth rate.DiscussionThis is the first randomized controlled trial to compare the transfer of day 6 blastocysts on the 6th and 7th day of progesterone supplementation. The results of this study will provide evidence for whether to prolong the duration of exposure to progesterone prior to embryo transfer.Trial registrationClinicalTrials.gov, ID: NCT04938011. Registered on 19 June 2021.

Insulin-like growth factor system components expressed at the conceptus-maternal interface during the establishment of equine pregnancy.

In many species, the insulin-like growth factors (IGF1 and IGF2), their receptors and IGF binding proteins play important roles in preparing the endometrium for implantation, and regulating conceptus growth and development. To determine whether the IGF system may contribute to conceptus-maternal interaction during equine pre-implantation development, we evaluated mRNA expression for IGF system components in conceptuses, and endometrium recovered from pregnant and cycling mares, on days 7, 14, 21 and 28 after ovulation. We also investigated expression of IGF1, IGF2 and their receptors 6 and 11 days after transfer of day 8 embryos to synchronous (day 8) or asynchronous (day 3) recipient mares. Expression of IGF1 and IGF2, IGF1R, IGF2R, INSR and IGFBPs 1, 2, 4 and 5 was evident in endometrium and conceptus membranes during days 7–28. Endometrial IGF2, INSR, IGFBP1 and IGFBP2 expression increased between days 7 and 28 of pregnancy. In conceptus membranes, expression of all IGF system components increased with developmental stage. Immunohistochemistry revealed strong expression of IGF1, IGF2 and IGF1R in both endometrium and conceptus membranes, whereas INSR was highly expressed in endometrium but barely detectable in the conceptus. Finally, a negatively asynchronous uterine environment retarded IGF1, IGF2 and INSR expression in the conceptus, whereas in the endometrium only INSR expression was altered by asynchrony. The presence of IGFs, their receptors and IGFBPs in the endometrium and conceptus during early equine pregnancy, and down-regulation in the conceptus following asynchronous embryo transfer, suggest a role in conceptus-maternal communication during the preparation for implantation.

P-206 Could day 7 embryo culture become standard practice to improve patients’ treatment and outcome?

Abstract Study question What are the clinical outcomes following transfer of day 7 embryos; could day 7 embryo culture become part of standard embryology practice? Summary answer Although clinical outcomes remain low, success has been seen following transfer of day 7 embryos. Extended culture plays an important role in standard embryology practice. What is known already In vitro culture conditions and cryopreservation techniques have improved greatly in recent years, allowing for extended culture, and freezing of embryos at the blastocyst stage. Optimally, embryos should develop to the blastocyst stage on day 5, however deviation from normal development can sometimes occur due to problems in either the embryonic intrinsic factors or imprinting timings. Live births have been reported from slower developing day 7 embryos. Extended culture to day 6 is standard practice in most IVF laboratories, however the aim of this study is to investigate the outcomes following prolonged embryo culture to day 7. Study design, size, duration This is a retrospective analysis study of 47 patients who underwent frozen embryo transfers (FETs) with a cryopreserved day 7 embryo at a UK licensed centre between 2017-2021. Patients were split into two groups: those transferring untested (non-PGT-A) day 7 embryos, and those transferring euploid PGT-A tested day 7 embryos. Clinical pregnancy (CPR) was analysed to study the clinical outcome of day 7 embryo culture followed by a frozen transfer. Participants/materials, setting, methods All patients who had FET with a day 7 embryo between 2017-2021 were included in this study. A total of 49 FETs (48 eSET,1 DET) involving 47 patients took place: 38 with untested embryos and 11 with euploid embryos. Clinical pregnancy rate (CPR) and live birth (LBR) was analysed to study the efficacy in transfer of day 7 embryos and the two groups of patients (untested and PGT-A tested) were compared. Main results and the role of chance The overall CPR per embryo transfer was 8% (47 patients, 49 ETs, 50 embryos transferred) and the LBR per embryo transfer was 4%. In the PGT-A group, a total of 201 day 7 embryos were biopsied over the 5-year study period; 28 were euploid following testing (14% euploidy rate). Of these, 11 have been transferred; 4 resulted in a pregnancy and 2 in a live birth. (LBR 18%) There is a significant difference in CPR between the PGT-A group (11 patients, 11 FETs; 11 embryos transferred, 4 foetal hearts seen, CPR=36%) and the untested group (36 patients, 38 ETs; 39 embryos transferred, CPR=0%) Chi square test performed and confirmed (p &amp;lt; 0.001). The mean age of the patient at the time of embryo transfer was 38.6 ± 0.5 and the mean age of the embryo transferred was 35.6± 0.5 (this was the patient’s age at the time of embryo freezing). Limitations, reasons for caution Limiting factors include the retrospective study design and the small sample size. It is also worth noting the high number of day 7 embryos required to undergo PGT-A testing to result in a low number of euploid embryos suitable for embryo transfer. Wider implications of the findings This study indicates the value of day 7 embryo culture as it has resulted in some patients achieving an ongoing pregnancy and a live birth. However, care must be taken to counsel patients appropriately around the chance of success when transferring day 7 embryos, particularly patients using untested embryos. Trial registration number Not Applicable

P-787 Factors associated with live birth rate (LBR) and multiple live birth rate (mLBR): UK vs France registries

Abstract Study question Do the associations between age, previous attempts, frozen and day-5 transfers relative to LBR and mLBR differ between national registries (UK vs France)? Summary answer Both registries agree age negatively correlates with LBR and mLBR; disagree on effects of frozen transfers, previous attempts and day-5 transfers on LBR and mLBR. What is known already Due to the high risks of multiple pregnancy, the focus of ART success was shifted from having a high number of live birth rate (LBR) to one full-term, healthy baby and a low multiple live birth rate (mLBR). Elective single embryo transfer (eSET) has been an effective strategy for reducing the risk of mLBR but in most guidelines, it is recommended for young patients (&amp;lt;35 years) only. There is extensive evidence from large-scale studies characterising how factors, seen in eSET policies, affect LBR. However, only a few studies focus on mLBR. Study design, size, duration A retrospective analysis of two national registry datasets, HFEA (UK) (N = 442042; 2014-2018) and ABM (France) (N = 305142; 2014-2018), evaluating how age (&amp;lt;35 yrs, 35-37 yrs, 38-40 yrs, &amp;gt;41 yrs), fresh/frozen embryo transfer (ET), previous attempts (0, 1, 2, 3, 4, 5 attempts), and embryo stage (day-5 or day-3 transfers) impact LBR and mLBR following eSET (control group) and multiple embryo transfer (MET) (treatment group). Participants/materials, setting, methods Statistical analyses of the datasets were conducted using adjusted two-sided odds ratios from chi-squared tests with 95% confidence intervals (α = 0.05) with Bonferroni correction. Main results and the role of chance Two registries agreed on the negative correlation between age and LBR (HFEA: eSET OR:1.00-0.79-0.73-0.57, p &amp;lt; 0.001; MET OR:1.00-0.85-0.74-0.55, p &amp;lt; 0.001; ABM: eSET OR:1.00-0.75-0.71-0.52, p &amp;lt; 0.001; MET OR:1.00-0.80-0.69-0.50, p &amp;lt; 0.001) and mLBR following MET (HFEA OR:1.00-0.79-0.70-0.71, p &amp;lt; 0.001; ABM OR:1.00-0.74-0.74-0.59, p &amp;lt; 0.001). mLBR following eSET remained constant with increasing age in both datasets (HFEA OR:1.00-0.91-0.92-1.34, NS; ABM OR:1.00-0.87-1.02-1.13, NS). LBR was significantly higher for fresh vs frozen eSET in both datasets (HFEA OR:1.36, p &amp;lt; 0.001; ABM OR:1.83, p &amp;lt; 0.001). However, HFEA showed no significant difference in LBR between fresh and frozen MET (OR:0.98, NS), whereas LBR in fresh MET was significantly higher in ABM (OR:1.28, p &amp;lt; 0.001). There was disagreement on fresh vs frozen ET mLBR (HFEA: eSET OR:1.10, NS; MET OR:0.97, NS; ABM: eSET OR:0.81, p &amp;lt; 0.05; MET OR:1.36, p &amp;lt; 0.001). Registry results differed regarding the impact of previous attempts on LBR and mLBR.Day-5 ETs had significantly higher LBR compared to day-3 (HFEA eSET OR:1.29, p &amp;lt; 0.001; MET OR:1.37, p &amp;lt; 0.001; ABM: eSET OR:1.50, p &amp;lt; 0.001; MET OR:1.35, p &amp;lt; 0.001). mLBR was significantly higher following day-5 MET (HFEA OR:1.59, p &amp;lt; 0.001; ABM OR:1.27, p &amp;lt; 0.001) but only significantly higher following eSET in the French registry (HFEA OR:0.64, NS; ABM OR:1.40, p &amp;lt; 0.01). Limitations, reasons for caution Limitations included selection bias due to the retrospective design of the study, as a result, the populations include predominantly caucasian, European populations. The conclusions drawn relate to the practices from particular countries, so it is important to evaluate multiple, more diverse datasets. Wider implications of the findings It is encouraging that both registries agreed on the effects of age, whilst remaining factors were not generalizable and require further assessment. This study demonstrated that even large, national registries contain inherent biases and policymakers should not draw policies based on one dataset, especially when prospective trial is not feasible. Trial registration number N/A

P-251 Cumulus cell analysis as a non-invasive oocyte selection strategy to reduce the number of oocytes/embryos cultured and increase pregnancy rates

Abstract Study question Can non-invasive gene expression analysis of cumulus cells (CC) improve efficiency in ART by prioritizing oocytes for further culture and fresh single embryo transfer? Summary answer CC analysis can be used for the selective processing of oocytes. This may reduce culture work and improve the outcome in ICSI elective SETs (eSET). What is known already In an interventional, blinded, prospective cohort study (Van Vaerenbergh et al. 2021), 113 patients underwent a fresh Day3 eSET with embryos ranked and transferred based on morphology and CC gene expression (Aurora Test), while 520 control patients underwent a Day3 eSET without the Aurora Test. This resulted in a significant higher clinical pregnancy of 61% in the patients with eSET based on CC ranking applied on good morphology embryos, compared to 29% in the controls with eSET based on embryo morphology only. Live birth rate was also significantly increased, while time-to-pregnancy was significantly reduced with 3 transfer cycles. Study design, size, duration In a retrospective analysis, in a subset of patients with at least 6 growing follicles and at least five 2PN oocytes (n = 80), it was investigated whether the Aurora Test, used to select transferrable Day3-embryos, could also be applied to select oocytes on Day0/1. The effect of processing only the three highest ranked oocytes (based on the Aurora Test) on embryo development and clinical pregnancy was studied compared to processing all oocytes. Participants/materials, setting, methods Patients included in this single centre study had their first or second GnRH-antagonist ICSI cycle, were younger than 40y, had normal BMI, were stimulated with HP-hMG and scheduled for Day3 eSET. Two-sided statistical analysis (p &amp;lt; 0,05) was performed between a strategy of processing only the top 3 Aurora ranked oocytes, according to CC gene expression, and a strategy of processing all available oocytes. Main results and the role of chance On average, 8 MII oocytes were obtained per patient and the average fertilization rate was 83%. In total, 407 good quality embryos (GQE) on Day3 were generated from these 80 patients when utilising all 639 oocytes. Processing the three top-ranked oocytes only (240/639 oocytes) would have reduced the number of embryos to 169 GQE and would have resulted in 2.1 GQE on average on Day3 per patient; 75/80 (94%) patients would have had a fresh Day3 transfer resulting in a 63% clinical pregnancy rate. Processing all 639 available 2PN oocytes (standard of care) resulted in a fresh Day3 transfer in all 80 patients and a similar 64% clinical pregnancy rate (ns). However, 399 more oocytes would need to be processed. The strategy of restricting the number of oocytes to be processed would not have compromised cumulative cycle outcome. Considering all subsequent freeze/thawing cycles the cumulative clinical pregnancy rate calculated per all 80 patients would increase to 90%. Limitations, reasons for caution The limitation of this approach is that the Aurora Test requires individual oocyte denudation and individual oocyte vitrification. Secondly, this new strategy should be validated in a prospective study. Wider implications of the findings By applying this oocyte selection strategy patients would benefit from a high pregnancy rate in the fresh transfer cycle, while the lab would see reduction in embryo culture work, because freeze/thawing cycles and culture of embryos with lower competence would be prevented. Trial registration number NA

Transfer of Day 11 ruptured embryos can prevent luteolysis in recipient mares

Previous studies transferring Day 11 embryos to Day 11 or 12 recipient mares and then manually crushing the transferred embryo 24 hours later resulted in 83% (5/6) and 100% (6/6) mares, respectively, entering a period of luteostasis. Undertaking manual reduction 12 hours post transfer resulted in 100% (5/5) of Day 11 recipient mares becoming luteostatic. This work suggested that the mare could recognise the presence of an embryo within the uterus after only a short period of time. The present experiment aimed to shorten the window of the presence of an intact embryo still further by evaluating the ability of ruptured or damaged Day 11 embryos transferred to recipient mares to initiate a period of luteostasis. A total of sixteen Day 11 embryos were flushed from the uteri of donor mares. Embryos were moved to a petri dish containing 5mL of Vigro Hold Plus Solution where they were measured and graded. The intact embryos were ruptured using two 100µm diameter acupuncture needles, ensuring the blastocoele fluid was released into the medium. The ruptured embryo and the medium were then transferred to recipient mares at Day 11 (n = 8) or 12 (n = 8) post ovulation using a standard embryo transfer technique. Control Day 11 (n = 8) and 12 (n = 8) recipient mares received 5mL of Vigro Hold Plus. All recipient mares were monitored on alternate days post transfer using a combination of ultrasonography,including assessment of the corpus luteum using colour Doppler, and serial blood serum samples assayed for progesterone in peripheral blood to determine if, and when, they returned to oestrus. Mares with a functional corpus luteum with blood flow and progesterone levels >1ng/mL up to 25 post ovulation were deemed to have entered a period of luteostasis and at this time point luteolysis was achieved by administration of an i.m injection of prostaglandin. Transfer of the ruptured embryo and medium resulted in significantly more Day 12 recipients entering a period of luteostasis compared to Day 11 recipients (75% vs. 12.5%; 6/8 vs. 1/8; P= 0.04, respectively). Day 11 and 12 control recipient mares that received only medium had an incidence of luteostasis of 12.5% (1/8) in each group. Hence the presence of medium containing blastocoele fluid, a ruptured capsule and trophoblast within the uterus can prevent luteolysis around the time of maternal recognition of pregnancy (MRP) in the mare. If all or only some of the components of the ruptured Day 11 embryo are required to initiate luteostasis needs further study. If the mechanisms underlying luteostasis with this model mirror those of the MRP signal in pregnant mares this study also raises questions about the duration of the MRP signal and how long embryo migration within the uterus is needed to prevent luteolysis.

The effect of embryo reduction and transfer on luteostasis in the mare

This study investigated the effects of embryo reduction and transfer of Day 11 embryos, with or without subsequent reduction, on luteostasis in the mare. In Experiment 1, reduction of embryos at Days 10 (n = 15), 11 (n = 47), 12 (n = 36), 13 (n = 27), 14 (n = 5) and 16 (n = 2) of pregnancy resulted in luteostasis in 13%, 47%, 78%, 89%, 80% and 100% mares. Mares undergoing > 1 embryo reduction showed consistency in when luteostasis occurred. In Experiment 2, transfer of Day 11 embryos to recipient mares 10 (n = 9), 11 (n = 8), 12 (n = 9) and 13 (n = 8) days post ovulation resulted in luteostasis in 78%, 87.5%, 78% and 37.5% of mares. Only 22%, 37%, 0% and 12%, respectively, of these mares remained pregnant. In the Day 10, 11 and 12 recipients luteostasis occurred on at least one occasion when an embryo was detected at 24 h but not at 48 h post transfer. In the Day 12 recipients luteostasis occurred on three occasions (3/9;33%) when the transferred embryo was not detected at 24 h. In Experiment 3 reduction of a Day 11 embryo 24 h after transfer to a Day 10 (n = 4), 11 (n = 6), 12 (n = 6) or 13 (n = 6) recipient resulted in luteostasis in 100%, 83%, 100%, and 83% of mares. All five Day 11 recipients that had an embryo reduced 12 h post transfer became luteostatic. These results suggest there is plasticity overall, but individual rigidity, in the timing of maternal recognition of pregnancy. Furthermore, an intact embryo need only be present in the uterus for 12 h to cause luteostasis.

Blastocyst versus cleavage embryo transfer improves cumulative live birth rates, time and cost in oocyte recipients: a randomized controlled trial

Research questionDoes embryo transfer day (day 5 versus day 3) affect cumulative live birth rates (CLBR), time to live birth (TLB) and cost per live birth (CPL) in recipients of donated oocytes? Study designA single-centre RCT conducted between April 2017 and August 2018. Recipients of donated oocytes were randomized to cleavage-stage (day 3) or to blastocyst-stage (day 5) embryo transfer. Eligible recipients were aged 18–50 years and in their first or second synchronous cycle. Primary outcome was CLBR (12 months from first embryo transfer), and fresh and subsequent cryopreserved transfers were considered; TLB and CPL were also analysed. ResultsRecipients (n = 134) were randomized to the day-3 group (n = 69) or to the day-5 group (n = 65). Day-5 transfer resulted in a 15.9% relative increase in CLBR and a significant shorter TLB compared with day-3 transfer. To reach a 50% CLBR, the day-3 group required 6 months more than the day-5 group (15.3 versus 8.9 months, respectively). The average CPL in the day-3 strategy cost 24% more than the day-5 strategy (€14817.10 versus €10959.20). Clinical pregnancy rate was 25% less in the day-3 group. The trial was prematurely stopped after poor initial results in the day-3 arm led to unplanned interim analysis. ConclusionsThe transfer of blastocyst-stage embryos in recipients of donated oocytes is preferred as it leads to a higher clinical pregnancy rate, live birth rate, shorter time to pregnancy and lower costs to achieve live birth, compared with cleavage-stage embryo transfer.

Clinical outcomes for Day 3 double cleavage-stage embryo transfers versus Day 5 or 6 single blastocyst transfer in frozen-thawed cycles: a retrospective comparative analysis.

ObjectiveThis study aimed to compare the clinical outcomes for transfer of Day 3 (D3) double cleavage-stage embryos and Day 5/6 (D5/6) single blastocysts in the frozen embryo transfer (FET) cycle to formulate a more appropriate embryo transplantation strategy.MethodsWe retrospectively analyzed 609 FET cycles from 518 women from April 2017 to March 2021. All FETs were assigned to the D3-DET group (transfer of a Day 3 double cleavage-stage embryo), D5-SBT group (transfer of a Day 5 single blastocyst), or D6-SBT group (transfer of a Day 6 single blastocyst). Clinical outcomes were comparatively analyzed.ResultsThere were no significant differences in the biochemical pregnancy rate, clinical pregnancy rate, or ongoing pregnancy rate between the D3-DET and D5-SBT groups, but these rates in the two groups were all significantly higher compared with those in the D6-SBT group. The implantation rate in the D5-SBT group was significantly higher than that in the D3-DET group. The twin pregnancy rate in the D5-SBT and D6-SBT groups was significantly lower than that in the D3-DET group.ConclusionThis study suggests that D5-SBT is the preferred option for transplantation. D6-SBT reduces the pregnancy rate, making it a more cautious choice for transfer of such embryos.

O-213 Slow day 5 development affects implantation potential of fresh transferred embryos but not birthweight once pregnancy occurs: A multi-center retrospective cohort study

Abstract Study question Does slow development of fresh transferred day 5 embryos lead to decreased implantation potential and birthweight? Summary answer Slow day 5 development was associated with reduced implantation potential when transferred fresh but the subsequent birthweight of the resulting baby was not impacted. What is known already Slow development of in vitro cultured cleavage stage embryos is associated with reduced blastocyst development and implantation rates. There is no current consensus regarding whether to transfer fresh slow developing day 5 embryos or to extend culture for a subsequent day with potential for cryopreservation. It is therefore important to understand the true prognosis of fresh transferred day 5 embryos at less advanced developmental stages. This would provide evidence based guidelines for the decision making process in regard to embryo transfer. Study design, size, duration This is a retrospective multi-center cohort study, including 1213 consecutive patients undergoing autologous oocyte in vitro fertilization (IVF) treatment during 2016-2019,with fresh transfer of a single day 5 embryo (selection based on developmental stage and inner cell mass and trophectoderm morphology if blastocyst was at the ≥expanding stage). Cycle data were collected from 4 associated private clinics, with repeat cycles of same patients excluded to avoid clustering effect at statistical analysis. Participants/materials, setting, methods Live birth and birthweight were followed up in all 1213 fresh day 5 SETs. Multiple regression (logistic or linear) was performed to investigate association between slow day 5 development (defined as ≤ early blastocyst) and (a)live birth, (b) birthweight, and (c) gestation-adjusted birthweight (Z score) to account for gestational age, gender and compared to embryos at ≥ expanded stage. Results were expressed as adjusted odds ratio (aOR) with 95% confidence interval (CI)or coefficients (β). Main results and the role of chance No implantation was achieved following single fresh transfer of day 5 embryos that failed to reach early blastocyst stage (n = 76) and were transferred as ≤ morula stage. Live birth rate was significantly lower following single day 5 fresh transfer of an early blastocyst (n = 237, 16%), in comparison to expanding (n = 329, 27%, P = 0.001), expanded(n = 392, 41%, P = 0.000), and hatching/hatched blastocysts (n = 169, 44%, P = 0.000). After adjusting for potential confounding factors including; maternal age, hours post insemination at day 5 assessment, number of oocytes collected, number of 2PN embryos, and number of embryos frozen; multiple logistic regression showed significantly reduced likelihood of live birth resulting from early blastocysts in reference to those at the expanding (aOR=0.584, 0.371-0.917, P = 0.020), expanded (aOR=0.322, 0.208-0.501, P = 0.000), or hatching/hatched stages (aOR=0.255, 0.147-0.443, P = 0.000). However, multivariate linear regression indicated that early blastocysts resulting in a live birth (n = 39) did not lead to altered birthweight (β=-9.091, P = 0.904; β=-34.960, P = 0.343; β=-26.074, P = 0.414; respectively) or Z score (β = 0.045, P = 0.706; β=-0.051, P = 0.426; β=-0.028, P = 0.506; respectively) in reference to the expanding (n = 90), expanded (n = 160), or hatching/hatched stages (n = 75). Limitations, reasons for caution The retrospective nature of this study does not allow controlling of unknown confounders. The 4 participating clinics are associated within the same network with shared protocols, therefore, results may not be generalized to other clinics with different settings. Wider implications of the findings The findings suggest no clinical value of fresh day 5 transfer of embryos ≤morula stage. Although early blastocysts implant at reduced rate, assuring birthweight outcomes suggest clinical value. Future studies intend to investigate slow growing day 5 fresh transfers versus embryos that were slow growing but transferred after day 6. Trial registration number NA