In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial

Reply of the Authors

Does euploidy on day 5 or day 6 matter for clinical outcome in frozen single blastocyst transfers?

Are serum progesterone (P4) levels on the embryo transfer (ET) day predictive of ongoing pregnancy (OP) following a single euploid blastocyst transfer in a natural cycle (NC) when luteal phase support is routinely given? In single euploid frozen ETs in NC, P4 levels on ET day are not predictive for OP, when luteal phase support (LPS) is routinely added after the ET. In an NC frozen embryo transfer (FET), P4 produced by the corpus luteum initiates secretory transformation of the endometrium and maintains pregnancy after implantation. There are ongoing controversies on the existence of a P4 cutoff level on the ET day, being predictive for the chance of OP as well as of the possible role of additional LPS after ET. Previous studies in NC FET cycles, evaluating and identifying P4 cutoff levels did not exclude embryo aneuploidy as a possible reason for failure. This retrospective study analyzed single, euploid FET in NC, conducted in a tertiary referral IVF centre between September 2019 and June 2022, for which measurement of P4 on the day of ET and the treatment outcomes were available. Patients were only included once into the analysis. Outcome was defined as OP (ongoing clinical pregnancy with heartbeat, >12 weeks) or no-OP (not pregnant, biochemical pregnancy, early miscarriage). Patients with an ovulatory cycle and a single euploid blastocyst in an NC FET cycle were included. Cycles were monitored by ultrasound and repeated measurement of serum LH, estradiol, and P4. LH surge was identified when a rise of 180% above the previous level occurred and P4 levels of ≥1.0 ng/ml were regarded as confirmation of ovulation. The ET was scheduled on the fifth day after P4 rise and vaginal micronized P4 was started on the day of ET after P4 measurement. Of 266 patients included, 159 (59.8%) patients had an OP. There was no significant difference between the OP- and no-OP-groups for age, BMI, and day of embryo biopsy/cryopreservation (Day 5 versus Day 6). Furthermore, P4 levels were not different between the groups of patients with OP (P4: 14.8 ng/ml (IQR: 12.0-18.5 ng/ml)) versus no-OP (P4: 16.0 ng/ml (IQR: 11.6-18.9 ng/ml)) (P = 0.483), and no differences between both groups, when P4 levels were stratified into categories of P4 levels of >5 to ≤10, >10 to ≤15, >15 to ≤20, and >20 ng/ml (P = 0.341). However, both groups were significantly different for the embryo quality (EQ), defined by inner cell mass/trophectoderm, as well as when stratified into three EQ groups (good, fair, and poor) (P = 0.001 and 0.002, respectively). Stratified EQ groups remained the only significant parameter influencing OP in the uni- and multivariate analyses (P = 0.002 and P = 0.004, respectively), including age, BMI, and P4 levels (each in categories) and embryo cryopreservation day. Receiver operator characteristic curve for the prediction of an OP revealed an AUC of 0.648 when age, BMI and EQ groups were included into the model. The inclusion of P4 measurement on ET day into the model did not add any benefit for OP prediction (AUC = 0.665). The retrospective design is a limitation. Monitoring serum P4 levels can be abandoned in NC FET cycles with routine LPS as they do not seem to be predictive of live birth. No external funding was used for this study. The authors state that they do not have any conflicts of interest. N/A.

Study question Is it all about the embryo? Can a single euploid blastocyst transfer sustain reproductive outcomes in women with advanced reproductive age? Summary answer Despite euploid embryo transfer, endometrial senescence appears to play a crucial role in determining reproductive success, particularly in older patients undergoing in vitro fertilization (IVF). What is known already Advanced reproductive age significantly impairs oocyte and embryo quality. Effective crosstalk and synchronization between embryo and endometrium are pivotal for successful pregnancies, both in vivo and in vitro. Implantation failure remains a persistent challenge in IVF, exacerbated by delayed family planning trends. The transfer of euploid blastocysts through preimplantation genetic testing for aneuploidy (PGT-A) is a potential strategy to address implantation issues, decrease miscarriages, and shorten time-to-pregnancy. However, a significant proportion of IVF treatments remain unsuccessful, suggesting critical factors beyond the embryo. Study design, size, duration This retrospective study analyzed 457 single euploid blastocyst transfers performed by two doctors at a private IVF center from January to November 2023. Inclusion criteria encompassed cases with PGT-A indications (advanced maternal age, repeated implantation failure, recurrent pregnancy loss, etc.) or cases where embryo biopsy was patient-requested. The primary outcome was ongoing pregnancy rate (OPR), with secondary outcomes including pregnancy rate (PR) and clinical pregnancy rate (CPR). Participants/materials, setting, methods Participants were categorized into four groups: Group 1 (< 35 years old; N = 100), Group 2 (35-37 years old; N = 102), Group 3 (38-40 years old; N = 118), and Group 4 (> 41 years old; N = 87). A control group (oocyte donation; N = 50) with single euploid embryo transfers showed a mean recipient age of 43 years. Donors were always less than 32 years old. Endometrial preparation protocols were consistent across all groups. Chi-square test was used to compare groups. Main results and the role of chance Among the 457 transfers analyzed, with a mean female age of 37 years, the overall PR was 71.3%, CPR was 64.8%, and OPR was 60.2%. Groups showed no differences in demographic variables and cycle parameters. Group 1 demonstrated higher PR (83.3%), CPR (78.4%), and OPR (73.5%) compared to Group 2 (PR 70.9%, p = 0.034; CPR 66%, p = 0.047; OPR 62.1%, p = 0.081), Group 3 (PR 68.2%, p = 0.009; CPR 61.2%, p = 0.005; OPR 56.6%, p = 0.008), and Group 4 (PR 65%, p = 0.002; CPR 56.1%, p < 0.001; OPR 51.2%, p = 0.001). Furthermore, the control group (oocyte recipients) showed significantly lower PR (68%, p = 0.031), CPR (54%, p = 0.002), and OPR (46%, p < 0.001) compared to Group 1, but not with the other groups. These findings demonstrate that even with euploid embryos originating from young donors, older women exhibited lower pregnancy rates than young women transferring single euploid blastocysts from their own oocytes and similar rates compared to those with advanced reproductive age transferring their own euploid blastocysts, implying that endometrial ageing may disproportionately influence IVF treatment outcomes. Limitations, reasons for caution The retrospective design introduces inherent bias, notably selection bias and unmeasured confounders. This preliminary data did not account for male infertility factors and their impact on embryo quality. Wider implications of the findings Reproductive aging appears to be influenced by factors beyond the embryo itself, especially in women above 35 years. Further research is warranted to elucidate the causes and impact of reproductive aging on implantation potential, aiming to refine clinical practices and enhance the success of IVF treatments. Trial registration number not applicable

Response: how PGS/PGT-a laboratories succeeded in losing all credibility.

Study question Will using a hyaluronan-rich transfer medium increase the ongoing pregnancy rate and live birth rate when transferring euploid embryos cultivated in a dry incubator? Summary answer Transferring euploid embryos in a media with high concentrations of hyaluronic acid increases the ongoing pregnancy rate compared with media with no hyaluronic acid. What is known already Cochrane Review found that solutions with high concentrations of hyaluronic acid increase the chance of live birth by adding probably 1 additional live birth for every 14 embryos transferred in a high-concentration hyaluronic acid solution for patients aged 27 to 35. A study involving 85 frozen embryo transfer cycles showed a positive effect of high concentrations of hyaluronic acid on the results of transfer of morphologically poor euploid blastocysts. Previous studies were conducted in humidified incubators where hyaluronic acid-rich media was equilibrated without oil overlay. Our clinic uses dry incubators, necessitating oil overlay with all media, including hyaluronic acid-rich media. Study design, size, duration A randomized prospective study involving 255 patients aged 19-44 undergoing a transfer of a single euploid embryo between June 2023 and November 2023. Patients were randomly assigned to the study and control groups. Blastocysts were incubated after thawing for 3 hours either in a hyaluronic acid-enriched medium or in a medium without hyaluronic acid. Participants/materials, setting, methods Embryos underwent PGT-A at the blastocyst stage on either day 5 or day 6, using the EmbryoMap method. Trophectoderm biopsy was performed using the Flicking technique without a laser. 5-7 cells were removed for analysis. Blastocysts were vitrified following biopsy. Hyaluronic acid-enriched media was equilibrated for 16-18 hours in a 5%O2 6%CO2 atmosphere in a well of 4-well culture dish under oil overlay (600 microliters of media and 500 microliters of oil). Main results and the role of chance In this ongoing study, 127 transfers of euploid blastocysts in a hyaluronic acid-rich media resulted in 70 ongoing pregnancies, with a 55.1% ongoing pregnancy rate. In comparison, 128 transfers of euploid blastocysts in a media with no hyaluronic acid resulted in 65 ongoing pregnancies, with a 50.8% ongoing pregnancy rate. The relative improvement in the ongoing pregnancy rate is 8.5%. Our data suggests that using a hyaluronic acid-rich medium for the transfer of euploid blastocysts of good morphology can improve clinical pregnancy rates, although the effect is less dramatic than was shown in a 2023 study by Nakagawa et al. for morphologically poor euploid blastocysts, where more than twofold increase in clinical pregnancy rates was detected. Limitations, reasons for caution Specific media equilibration protocol is required for the incubation and transfer of embryos in a hyaluronan-rich transfer medium for IVF clinics utilizing dry incubators. Further research is required to confirm results and to establish whether transferring embryos in hyaluronic acid-enriched media increases the live birth rate after euploid embryo transfer. Wider implications of the findings Our results show that it is feasible to use a hyaluronic acid-enriched medium in a dry incubator with an oil overlay and that using it according to described protocol could increase the ongoing pregnancy rate after a single euploid blastocyst transfer. Trial registration number not applicable

The Effect of a Comprehensive Incentive Program on Patient Participation in Elective Single Blastocyst Transfers

Are pregnancy rates affected by day of blastocyst cryopreservation in single euploid frozen embryo transfer cycles?

Study question Does post-thaw morphological change in euploid blastocysts impact pregnancy rates in single frozen-thawed embryo transfers? Summary answer Morphological downgrades of ICM and TE or lack of expansion post-thaw significantly reduce pregnancy rates. Conversely, re-expanding and upgraded blastocysts show higher success. What is known already Euploid embryo implantation failure can result from various factors, including embryonic quality, uterine conditions, endometrial receptivity, and immune responses. Despite the euploid status, embryos may fail to implant due to suboptimal morphological quality or other non-embryonic factors. While many reasons contribute to implantation failure, this study aims to investigate whether morphological grading of euploid embryos —specifically changes in ICM, TE, and expansion—can be a key risk factor for pregnancy outcomes. Understanding these factors may help improve success rates in frozen-thawed embryo transfer cycles. Study design, size, duration This retrospective study analyzed 585 euploid blastocysts from January 2022 to December 2024. All blastocysts underwent preimplantation genetic testing for aneuploidy (PGT-A) using next-generation sequencing (NGS). Single euploid blastocyst transfers were performed during frozen-thawed cycles. The blastocysts were categorized based on ICM, TE grades (A, B, C), and expansion status (non-expansion, upgrade, downgrade). Pregnancy outcomes were assessed according to these morphological classifications to identify potential risk factors for implantation success. Participants/materials, setting, methods The study analyzed clinical pregnancy rates based on the morphological grading of blastocysts. blastocysts were grouped based on ICM, TE grades (A, B, C), and expansion status (non-expansion, upgrade, downgrade). Statistical analysis was conducted using t-tests and chi-square tests (p < 0.05) to compare pregnancy outcomes between the different morphological categories. The study was conducted in a clinical setting, focusing on single euploid blastocyst transfers in frozen-thawed cycles. Main results and the role of chance The pregnancy rate significantly decreased when ICM grade B blastocysts downgraded to grade C post-thaw (56.0% vs. 15.0%, p < 0.001). In contrast, ICM grade C blastocysts that improved to grade B showed a notable increase in pregnancy rate (42.4% vs. 66.7%, p < 0.008). Similarly, TE grade B blastocysts downgraded to grade C had a reduced pregnancy rate (61.1% vs. 39.7%, p < 0.001), while those with grade C improving to grade B saw a significant increase (46.3% vs. 61.1%, p < 0.001). Expansion status showed that upgraded blastocysts had significantly higher pregnancy rates compared to non-expanding ones (58.4% vs. 30.5%, p < 0.001). These findings indicate that post-thaw morphological changes, including downgrades in ICM, TE, and lack of expansion, are critical factors influencing pregnancy outcomes. In contrast, blastocysts that show improvement or re-expansion after thawing are more likely to result in successful pregnancies. The study highlights the importance of monitoring these changes to predict implantation success in frozen-thawed single blastocyst transfers. Limitations, reasons for caution This study’s findings may be limited by its relatively small sample size, which could affect generalizability. Non-embryonic factors influencing pregnancy outcomes were not considered. Further research with larger sample sizes and inclusion of additional variables is needed to confirm these findings and enhance their applicability. Wider implications of the findings This study suggests the importance of morphological changes in euploid blastocysts suggesting that post-thaw grading can be a key predictor of pregnancy success, potentially improving embryo selection in clinical practice. Further studies are needed to determine what clinical factors lead to changes in embryo grade after thawing in clinical practice. Trial registration number No

BackgroundNatural cycle frozen embryo transfer (NC-FET) lowers obstetric risks by preserving ovulation and corpus luteum but limits scheduling flexibility. Natural proliferative phase FET (NPP-FET) offers a scheduling-friendly alternative, assuming ovulation is maintained after flexible progesterone (P4) initiation during the follicular phase. Only three peer-reviewed studies have investigated NPP-FET protocols, yet none verified spontaneous ovulation, characterized hormonal dynamics, or evaluated whether variation in P4 initiation timing influences clinical outcomes. Preserving spontaneous ovulation is essential for NPP-FET to replicate the physiologic benefits of NC-FET; confirming its consistency is critical to validating NPP-FET as a viable protocol. To our knowledge, this is the first study to comprehensively address these gaps, providing novel evidence to support NPP-FET’s clinical feasibility.MethodsThis retrospective cohort study included 196 first-time NPP-FET cycles with single euploid blastocyst transfers between January 2023 and October 2024. Dydrogesterone (40 mg/day) was initiated upon meeting the following criteria: leading follicle ≥ 14 mm, endometrial thickness ≥ 7 mm, serum estradiol > 150 pg/mL, and P4 < 1.5 ng/mL. Ultrasound and hormonal monitoring continued until ultrasound-documented ovulation (UDO), followed by three days of hormone assessments. Ovulation was confirmed by UDO and serum P4 > 3.0 ng/mL. Embryo transfer occurred on day 6 of dydrogesterone exposure. Multivariable logistic regression evaluated associations between pregnancy outcomes and P4 timing–related variables, including follicular phase duration, estradiol and follicular diameter at P4 initiation, P4 start-to-UDO interval, UDO-to-FET interval, and serum P4 on FET day.ResultsSpontaneous ovulation was confirmed in all participants. Median follicular diameter one day before UDO was 18.6 mm. UDO occurred within 1–2 days in 96.4% and 92.2% of cases based on two LH surge criteria. Peri-ovulatory hormone profiles resembled natural cycles. Clinical pregnancy, ongoing pregnancy, and clinical loss rates were 66.3%, 58.7%, and 11.5%, respectively. Embryo morphology and biopsy day predicted pregnancy outcomes, while P4 timing–related variables showed no association.ConclusionsFlexible dydrogesterone initiation at follicular diameters ≥ 14 mm, based on predefined criteria, preserves spontaneous ovulation and natural hormonal dynamics. Pregnancy outcomes were consistent across P4 initiation timings, supporting NPP-FET as a clinically viable, physiologically grounded, and scheduling-friendly protocol.

YOUNG EGGS IN AN OLD BASKET – IS THERE A MATERNAL AGE LIMIT FOR EMBRYO TRANSFER?

A review of factors influencing the implantation of euploid blastocysts after in vitro fertilization

BackgroundTo investigate whether the endometrial thickness change ratio from the progesterone administration day to the blastocyst transfer day is associated with pregnancy outcomes in a single frozen-thawed euploid blastocyst transfer cycle.MethodsAll patients used natural cycles with luteal support for endometrial preparation and selected a single euploid blastocyst for transfer after a biopsy for preimplantation genetic testing. The endometrial thickness was measured by transvaginal ultrasound on the progesterone administration day and the transfer day, the change in endometrial thickness was measured, and the endometrial thickness change ratio was calculated. According to the change rate of endometrial thickness, the patients were divided into three groups: the endometrial thickness compaction group, endometrial thickness non-change group and endometrial thickness expansion group. Among them, the endometrial thickness non-change and expansion groups were combined into the endometrial thickness noncompaction group.ResultsUltrasound images of the endometrium in 219 frozen-thawed euploid blastocyst transfer cycles were evaluated. The clinical pregnancy rate increased with the increase in endometrial thickness change ratio, while the miscarriage rate and live birth rate were comparable among the groups. The multiple logistic regression results showed that in the fully adjusted model a higher endometrial thickness change ratio (per 10%) was associated with a higher clinical pregnancy rate (adjusted odds ratio [aOR] 1.29; 95% confidence interval [CI], 1.01–1.64; P = .040). Similarly, when the patients were divided into three groups according to the change rate of endometrial thickness, the endometrial thickness noncompaction group had a significant positive effect on the clinical pregnancy rate compared with the endometrial thickness compaction group after adjusting for all covariates.ConclusionsIn frozen-thawed euploid blastocyst transfer cycles in which the endometrium was prepared by natural cycles with luteal support, the clinical pregnancy rate was higher in cycles without endometrial compaction after progesterone administration.

Study question Can TE-biopsy practitioners impact pre-implantation genetic test for aneuploidy (PGT-A) results, pregnancy, and live-birth outcomes in a single center that has nineteen biopsy practitioners? Summary answer Yes. Trophectoderm (TE)-biopsy practitioners, especially micromanipulation experience, can impact PGT-A results but do not impact pregnancy or live-birth outcomes after single euploid blastocyst transfer (SeBT). What is known already Recently, PGT-A has become important in in vitro fertilization treatment. However, several factors can impact the accuracy and interpretation of PGT-A results. Several studies have suggested that TE-biopsy practitioners can impact PGT-A results. However, these findings are controversial because these previous studies used data from a low number of TE-biopsy practitioners and from multiple centers. There is, therefore, still limited knowledge of the impact of TE-biopsy practitioners on PGT-A results and their clinical outcomes. Study design, size, duration A total of 3,589 PGT-A cycles (average maternal age, 40.9±3.2; 1,168 patients; February 2020 to November 2022) were retrospectively analyzed. Furthermore, 779 single frozen euploid blastocyst transfer (SeBT) cycles were analyzed. First, we investigated the association between TE-biopsy practitioners and transferable blastocysts that included euploid and low-level mosaic blastocysts (LLM, 20 to 40% abnormal cells). Second, we analyzed the impact of TE-biopsy practitioners on pregnancy and live-birth outcomes after SeBT. Participants/materials, setting, methods The nineteen practitioners (Years of intracytoplasmic sperm injection (ICSI) experience: 2 to 20 years) who received the same training performed a total of 5,613 TE-biopsies for PGT-A. The same practitioner performed both the biopsy and the tubing procedures for each blastocyst they biopsied. The Wald test for multivariable logistic regression analysis (mLR) was used to determine the impact of TE-biopsy practitioners. The confounding factors were selected by univariable logistic regression analysis before mLR. Main results and the role of chance TE-biopsy was carried out by the flicking method with laser assistance and it was taken five to seven TE cells. Overall, 22.9% of blastocysts were transferable blastocysts (euploid: 21.1%, LLM: 1.8%). The results were inconclusive for 0.1% of the blastocysts. Maternal/paternal age, number of previous egg retrieval/embryo transfers, anti-Müllerian hormone, sperm characteristics, indication for PGT-A, number of previous deliveries/chemical abortions, blastocyst quality (the day of vitrification, inner diameters of blastocysts, and Gardner grading) were used as confounding factors on the mLR. TE-biopsy practitioners were significantly associated with both ensuring transferable blastocysts and LLM in transferable blastocysts (p &amp;lt; 0.05). Additionally, the practitioner characteristics that impact ensuring transferable blastocysts or LLM in transferable blastocysts were assessed in a sub-analysis. Years of ICSI experience (p &amp;lt; 0.05), number of ICSI performed during the last years (p &amp;lt; 0.05), and number of TE-biopsies at that point (p &amp;lt; 0.05) were associated with ensuring transferable blastocysts and LLM in transferable blastocysts. On the other hand, TE-biopsy practitioners did not have a significant impact on biological pregnancy, chemical abortion, pregnancy, or live-birth rates after SeBT. Limitations, reasons for caution This study did not determine the influence of TE biopsy itself and the laser assistance on PGT-A and clinical results. And this study was based on a minimal stimulation cycle. Moreover, the patient cohort in this study only included cases involving recurrent implantation failure or repeated pregnancy loss. Wider implications of the findings This study showed that TE-biopsy practitioners could impact decreasing transferable blastocysts, and this depends on their micromanipulation experience. This result suggests TE-biopsy training should be started when embryologists have gained several years of experience with ICSI procedures. This could prevent the decline in the efficacy of PGT-A for clinical results. Trial registration number not applicable

Prospective evaluation of the optimal time for selecting a single embryo for transfer: day 3 versus day 5