Comparing endometrial preparation protocols and clinical outcomes: lessons learned from 3507 vitrified-warmed euploid transfers.

Study question Which ovarian stimulation and transfer features do impact on clinical, gestational, obstetric and neonatal outcomes after vitrified-warmed euploid single blastocyst transfers? Summary answer Artificial endometrial preparation involved slightly lower live-birth, higher miscarriage and risk for hypertensive gestational disorders, and larger newborns than modified-natural cycle. What is known already Enhancing IVF success requires a comprehensive evaluation of each stage along the treatment. While no conclusive evidence links ovarian stimulation features to clinical, gestational and neonatal outcomes per embryo-transfer, emerging data suggest that regimens of endometrial preparation may impact on miscarriage and live birth rates. Lately, it has been suggested that natural/modified-natural endometrial preparation may be protective against long-term obstetric complications, particularly hypertensive pregnancy disorders and large-for-gestational-age newborns. However, no robust evidence exist that demonstrated the superiority of any endometrial preparation protocol especially in the context of euploid blastocyst transfer. Study design, size, duration Retrospective observational study of 3698 vitrified-warmed euploid single blastocyst transfers conducted at a private IVF clinic (years:2013-2023) by 2361 couples. The following features were tested for associations with clinical outcomes after transfer, gestational, obstetric and neonatal outcomes per delivery: maternal age, BMI, cause of infertility, days between menses and oocyte-retrieval, duration of ovarian-stimulation, ovarian-stimulation protocol, type of hypothalamic suppression and trigger of ovulation, endometrial preparation protocol, blastocyst quality and day. Participants/materials, setting, methods Single euploid day5-7 blastocysts were transferred 2-3hours after warming. GnRH-antagonist/short-agonist/progestin-primed ovarian-stimulation were conducted, either conventional or DuoStim, with GnRH-analogues (agonist/antagonist)/progesterone hypothalamic suppression and hCG/GnRH-agonist ovulation induction. Endometrial preparation included: artificial-cycle (estradiol-valerate 6mg/daily followed by 600 mg/day micronized-progesterone) and modified-natural-cycle (10,000hCG administered when the leading follicle was >17 mm). Gestational outcomes included embryo twinning, diabetes, cholestasis, placental-abruption, uterine-atony, hypertensive-disorders, placenta-previa, premature-rupture-of-the-membranes, and intrauterine-growth-restriction. Obstetric outcomes included gestational-age and birthweight. Neonatal outcomes included mild and severe malformations. Main results and the role of chance Median maternal age was 37 and 38 years at oocyte-retrieval and embryo-transfer, respectively. The median BMI was 21.5Kg/m2. They underwent 11days (median) of ovarian-stimulation, and 14days (median) separated menses from oocyte-retrieval. Generalized-estimated-equations were used to confirm associations with all outcomes adjusting for multiple observations. Ovarian-stimulation and ovarian-cycle features were not associated with any outcome. Decreasing blastocyst Gardner-quality involved lower live-birth rate per transfer (LBR), and in case of <BB-grade also higher miscarriage rate per clinical-pregnancy (MR). Slower blastocyst development involved lower LBR. Increasing maternal age was associated with higher MR (OR:1.06,95%CI:1.02-1.11) and lower LBR (OR:0.98,95%CI:0.96-0.99). Although, no difference was reported for positive pregnancy test per transfer and biochemical pregnancy loss per positive pregnancy test, artificial rather than modified-natural cycle involved higher MR (N = 178/1100,16.2% versus 73/731,10%; OR:1.7,95%CI:1.27-2.31,p<0.001; Power=98%) and also lower LBR (N = 922/2261,40.8% versus N = 658/1437,45.8%; OR:0.83,95%CI:0.72-0.96,p=0.011; Power=91%). Additionally, artificial-cycle was associated with larger birthweight (median:3300g versus 3240g; unstandardized coefficient-B = +79g, 95%CI:+27g to 132g,p<0.001; Power=90%), higher prevalence of large-for-gestational-age and lower of small-for-gestational-age newborns (N = 139/836,16.6% versus N = 69/590,11.7% and N = 70/836,8.4% versus N = 73/590,12.4%; p < 0.001; Power=93%). Lastly, although the overall prevalence of gestational complication, and neonatal malformations was comparable, a higher prevalence of hypertensive disorders was reported after artificial-cycle endometrial preparation (N = 14/836,1.7% versus N = 3/590,0.5%; OR:3.3,95%CI:1.01-11.63,p=0.05; Power=60%). Limitations, reasons for caution Retrospective study. Only oocyte retrievals after ovarian-stimulation were included. Pure natural cycle for endometrial preparation was not conducted. Secondary outcomes are biased by limited sample size. All data were obtained by phone interviews and 10.3% of the deliveries lacked gestational and neonatal information (N = 164/1590). Wider implications of the findings No significant association was found between ovarian stimulation features and obstetric or neonatal outcomes. Conversely, modified natural-cycle preparation may involve better prognosis. While artificial-cycle remains essential for premature-ovarian-insufficiency, hypothalamic-amenorrhea, and resistance to ovulation-induction, (modified-)natural endometrial-preparation should be preferred, whenever possible. A randomized-controlled-trial in PGT-A cycles is warranted. Trial registration number No

Study question Does maternal body-mass-index (BMI) associate with blastocysts’ chromosomal constitution and clinical outcomes in infertile patients undergoing preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer A higher euploidy rate per biopsied blastocyst was reported among underweight women. Overweight women were instead subject to higher miscarriage (MR) and lower live-birth-rates (LBR). What is known already Different studies in the literature revealed an association between BMI and infertility, suggesting a J-shaped relationship: both underweight and overweight women can suffer from infertility issues. Even if IVF might increase the success rate in both these categories of patients, it seems insufficient per se to overcome the complex and multifactorial fertility impairment derived from unbalanced nutritional intakes. Miscarriage, in particular, is common in both underweight and overweight women. However, most of the literature is based on chromosomally-untested embryos. Study design, size, duration: Retrospective observational study. Only the first IVF cycle with ≥1 biopsied blastocyst from each woman was included. The primary outcome was the association between maternal BMI (underweight, BMI<18.5, n = 160; normal-weight, BMI=18–25, N = 1392; overweight, BMI>25, N = 259) and the mean euploidy rate per cohort of biopsied blastocysts (m-ER). The secondary outcomes were the association between maternal BMI with clinical (mainly MR and LBR), gestational and perinatal outcomes after first vitrified-warmed single euploid blastocyst transfers. Participants/materials, setting, methods We included 1811 women undergoing PGT-A at a private IVF center between April–2013 and March–2020. The secondary outcomes were investigated on 1125 first vitrified-warmed single euploid blastocyst transfers from all patients obtaining ≥1 transferable blastocyst. Only ICSI with ejaculated sperm and continuous culture in standard incubators were performed. Logistic regressions were conducted to identify putative confounders and adjust the results accordingly. Main results and the role of chance Except for a lower maternal age among underweight women (38.3±3.1 versus 38.9±3.4 yr, p < 0.01) and higher among overweight ones (39.3±3.6 yr, p = 0.04), no difference was reported with respect to normal-weight women in terms of duration of infertility, hormonal levels, main cause of infertility, sperm quality, and reproductive history. The mean number of biopsied blastocysts was ∼3 in all groups. The m-ER shows a decreasing trend as the maternal BMI increases between 17 and 22–23, to then plateau. In fact, a significant difference was reported between underweight (50.8%±36.4%) and normal-weight women (41.4%±37.5%, p < 0.01). A linear regression adjusted for maternal age confirmed this moderate association between increasing BMI and m-ER (unstandardized-coefficient-B –0.6%, 95%CI:–1.1% to –0.1%, p = 0.02). Morphological quality and day of full-blastulation among transferred euploid blastocysts was similar in the three groups. Overweight women showed higher MR per pregnancy (N = 20/75, 26.7%, 95%CI:17.4%–38.3% versus N = 67/461, 14.5%, 95%CI:11.5%–18.2%; OR 2.0, 95%CI:1.1–3.6, p = 0.01) and lower LBR per transfer (N = 55/154, 35.7%, 95%CI:28.3%–43.8% versus N = 388/859, 45.2%, 95%CI:41.8%–48.6%; OR adjusted for euploid blastocysts’ features 0.67, 95%CI:0.46–0.96, p = 0.03). Clinical outcomes were instead similar among underweight and normal-weight women. All gestational and perinatal outcomes were comparable in the tree groups. Limitations, reasons for caution Our study is limited by its retrospective nature, and the fact that maternal BMI was measured only before oocyte retrieval and not before embryo transfer. Moreover, the reduced sample size did not allow for further relevant sub-analyses among solely obese women. Wider implications of the findings: When possible nutritional/lifestyle modifications should be encouraged to adjust maternal BMI before IVF. Overweight patients should be especially informed of their higher risk for miscarriage. Yet, BMI is just a gross marker, future studies based on body fat localization and percentage (e.g. by bioelectrical impedance analyses) are desirable. Trial registration number None

Endometrial thickness: How thin is too thin?

Is there an association between patients' reproductive history and the mean euploidy rates per biopsied blastocysts (m-ER) or the live birth rates (LBRs) per first single vitrified-warmed euploid blastocyst transfers? Patients' reproductive history (as annotated during counselling) showed no association with the m-ER, but a lower LBR was reported after euploid blastocyst transfer in women with a history of repeated implantation failure (RIF). Several studies have investigated the association between the m-ER and (i) patients' basal characteristics, (ii) ovarian stimulation strategy and dosage, (iii) culture media and conditions, and (iv) embryo morphology and day of full blastocyst development. Conversely, the expected m-ER due to women's reproductive history (previous live births (LBs), miscarriages, failed IVF cycles and transfers, and lack of euploid blastocysts among prior cohorts of biopsied embryos) still needs investigations. Yet, this information is critical to counsel new patients about a first cycle with preimplantation genetic testing for aneuploidy (PGT-A), but even more so after former adverse outcomes to prevent treatment drop-out. This observational study included all patients undergoing a comprehensive chromosome testing (CCT)-based PGT-A cycle with at least one biopsied blastocyst in the period April 2013-December 2019 at a private IVF clinic (n = 2676 patients undergoing 2676 treatments and producing and 8151 blastocysts). m-ER were investigated according to women's reproductive history of LBs: no/≥1, miscarriages: no/1/>1; failed IVF cycles: no/1/2/>2, and implantation failures after previous transfers: no/1/2/>2. Among the 2676 patients included in this study, 440 (16%) had already undergone PGT-A before the study period; the data from these patients were further clustered according to the presence or absence of euploid embryo(s) in their previous cohort of biopsied blastocysts. The clinical outcomes per first single vitrified-warmed euploid blastocyst transfers (n =1580) were investigated according to the number of patients' previous miscarriages and implantation failures. The procedures involved in this study included ICSI, blastocyst culture, trophectoderm biopsy without hatching in Day 3, CCT-based PGT-A without reporting segmental and/or putative mitotic (or mosaic) aneuploidies and single vitrified-warmed euploid blastocyst transfer. For statistical analysis, Mann-Whitney U or Kruskal-Wallis tests, as well as linear regressions and generalised linear models among ranges of maternal age at oocyte retrieval were performed to identify significant differences for continuous variables. Fisher's exact tests and multivariate logistic regression analyses were instead used for categorical variables. Maternal age at oocyte retrieval was the only variable significantly associated with the m-ER. We defined five clusters (<35 years: 66 ± 31%; 35-37 years: 58 ± 33%; 38-40 years: 43 ± 35%; 40-42 years: 28 ± 34%; and >42 years: 17 ± 31%) and all analyses were conducted among them. The m-ER did not show any association with the number of previous LBs, miscarriages, failed IVF cycles or implantation failures. Among patients who had already undergone PGT-A before the study period, the m-ER did not associate with the absence (or presence) of euploid blastocysts in their former cohort of biopsied embryos. Regarding clinical outcomes of the first single vitrified-warmed euploid blastocyst transfer, the implantation rate was 51%, the miscarriage rate was 14% and the LBR was 44%. This LBR was independent of the number of previous miscarriages, but showed a decreasing trend depending on the number of previous implantation failures, reaching statistical significance when comparing patients with >2 failures and patients with no prior failure (36% versus 47%, P < 0.01; multivariate-OR adjusted for embryo quality and day of full blastocyst development: 0.64, 95% CI 0.48-0.86, P < 0.01). No such differences were shown for previous miscarriage rates. The sample size for treatments following a former completed PGT-A cycle should be larger in future studies. The data should be confirmed from a multicentre perspective. The analysis should be performed also in non-PGT cycles and/or including patients who did not produce blastocysts, in order to investigate a putative association between women's reproductive history with outcomes other than euploidy and LBRs. These data are critical to counsel infertile couples before, during and after a PGT-A cycle, especially to prevent treatment discontinuation due to previous adverse reproductive events. Beyond the 'maternal age effect', the causes of idiopathic recurrent pregnancy loss (RPL) and RIF are likely to be endometrial receptivity and selectivity issues; transferring euploid blastocysts might reduce the risk of a further miscarriage, but more information beyond euploidy are required to improve the prognosis in case of RIF. No funding was received and there are no competing interests. N/A.

Noninvasive preimplantation genetic testing for aneuploidy in spent culture medium as a substitute for trophectoderm biopsy

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

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

Study question Could the presence, quantity, and type of cytoplasmic strings (Cyt-S) serve as biomarkers for the implantation competence of euploid blastocysts? Summary answer The absence of Cyt-S during blastulation dynamics is associated with a lower live birth rate (LBR) per transfer. What is known already Cyt-S are dynamic cellular projections observed in most human embryos, connecting the inner cell mass (ICM) to the trophectoderm during blastulation. Their presence, number, and type correlate with embryo viability, morphological quality, and blastocyst expansion, though their exact function remains unclear and only hypothesized from animal models in the 90s. Preliminary data suggest Cyt-S may also serve as biomarkers of implantation competence. This study evaluated Cyt-S presence, number, and type in euploid blastocysts, investigating their association with clinical, gestational, and neonatal outcomes following embryo transfer, aiming to clarify their potential role in predicting implantation success and overall reproductive outcomes. Study design, size, duration Retrospective observational study (2015-2020) of 539 transferred euploid blastocysts. Timings from tSB (start of blastulation) to t-biopsy (time-of-biopsy) in hours-post-insemination (hpi) were recorded along with Cyt-S presence, number, and type [bridges (B), threads (T), or B transitioning to T (B-T)], and timings of appearance and fading. Associations between Cyt-S and positive pregnancy test (PPT) and LBR per transfer, biochemical pregnancy loss per PPT, miscarriage rate (MR) per clinical pregnancy, gestational and neonatal outcomes were assessed. Participants/materials, setting, methods ICSI, continuous blastocyst culture (days 5-7) in Embryoscope incubator, trophectoderm biopsy on fully-expanded blastocyst without previous zona-pellucida drilling and comprehensive-chromosome-testing were performed. Blastocyst quality was classified according to Gardner’s criteria as excellent (AA), good (AB,BA), average (BB,AC,CA) and low (CC,BC,CB). Only vitrified-warmed single euploid blastocyst transfers were performed, with endometrial preparation using modified natural or artificial cycles. Regression analyses were conducted adjusting for confounders and using Generalized Estimating Equations (GEE). Main results and the role of chance 369 patients (age at retrieval:38±3.2 years; age at transfer:38±3.3 years; BMI:22±3.8 kg/m²) underwent 539 transfers. 63% of all euploid blastocysts analyzed (N = 341/539) had ≥1 Cyt-S (mean:2.4±1.5, range:1-9, mostly T or B-T [96%], the first appearing at 111.5±10.8 hpi, the last disappearing at 116.3±10.6 hpi). Euploid blastocysts without Cyt-S (Cyt-S-) were 3-4 hours slower than control (Cyt-S+) at both tEB and t-biopsy, and of lower morphological quality (AA:59.1% versus 69.5% and CC/BC/CB:20.7% versus 12.3%, p = 0.025). In Cyt-S- versus Cyt-S+, PPT was lower (43.9% versus 62.5%, p &amp;lt; 0.001), and BPL was higher (25.3% versus 9.9%, p &amp;lt; 0.001). Indeed, lower LBR was reported in the absence of Cyt-S (N = 50/198, 25.3%, 95%CI:19.7-31.8 versus N = 163/341, 47.8%, 95%CI:42.6-53.1; OR adjusted for morphological quality and t-biopsy=2.5, 95%CI: 1.7-3.7, p &amp;lt; 0.001, power=99.9%). Number and type of Cyt-S were not associated. No difference was reported in Cyt-S- versus Cyt-S+ for gestational age (36.2 versus 37.9 weeks), birthweight (3136 versus 3251 g), normal for gestational age rate (82% versus 79%), prevalence of gestational complications (12% versus 6%) and of minor/major neonatal malformations (2% versus 1.3%). Also Cyt-S number and type showed no associations. All data were confirmed by GEE, and among first embryo transfers conducted by each patient. Limitations, reasons for caution Retrospective single center study. Cyt-s were assessed manually. Larger datasets are needed to validate the associations between Cyt-S and clinical outcomes. Cyt-S were not used to rank embryos for transfer, though their absence was linked to lower blastocyst quality and slower development. Wider implications of the findings Emerging evidence suggests Cyt-S presence is linked to euploid blastocyst implantation, independent of morphology and timing, warranting consideration in embryo prioritization. However, Cyt-S function remains unclear. Further research should explore their role by comparing species and investigating the effects of interfering with Cyt-S dynamics. Trial registration number No

Study question Can a protocol combining letrozole-induced ovulation, vaginal estradiol,and a flexible hCG trigger improve clinical outcomes in frozen-thawed euploid blastocyst transfers compared to hormonal-replacement therapy (HRT)? Summary answer The letrozole+hCG protocol significantly improved clinical pregnancy and ongoing pregnancy rates compared to hormonal replacement therapy while also reducing luteal progesterone deficiencies before embryo transfer. What is known already Determining the optimal frozen-thawed embryo transfer (FET) protocol is crucial for improving IVF clinical and obstetric outcomes. The absence of a corpus luteum in artificial cycles has been linked to adverse obstetric outcomes. Letrozole, an aromatase inhibitor, has been associated with enhanced integrin expression, faster endometrial proliferation, and improved uterine blood flow. Letrozole-induced ovulation in FET cycles could enhance implantation and pregnancy rates. However, an asynchrony between follicular growth and endometrial thickness may impact cycle scheduling. The addition of vaginal estradiol after follicular dominance may optimize endometrial preparation and improve synchronization between ovulation and endometrial priming. Study design, size, duration This retrospective cohort study analyzed 879 FET cycles conducted between April 2022 and November 2024 in women aged &amp;lt;45 years with regular menstrual cycles. Patients underwent single euploid blastocyst transfers and were categorized into HRT (Control, n = 369) and Letrozole (Study, n = 510) groups. Primary outcomes included pregnancy rate (PR), clinical pregnancy rate (CPR), and ongoing pregnancy rate (OPR). Multivariate analyses were performed to identify independent associations between endometrial preparation protocols and clinical outcomes. Participants/materials, setting, methods Women with regular cycles undergoing single euploid blastocyst FET were included. HRT patients received estradiol valerate for endometrial priming and vaginal micronized progesterone plus oral dydrogesterone for luteal support. The study group received Letrozole 5mg/day (menstrual cycle days 3-7), vaginal estradiol (day 8 onward), and an hCG trigger when follicles reached ≥13mm and endometrium ≥7mm. Serum progesterone was measured the day before FET, and additional intramuscular progesterone was administered if serum progesterone levels were inadequate. Main results and the role of chance Both groups were comparable in maternal age, BMI, ovarian reserve, and pre-FET endometrial thickness. The letrozole+hCG protocol demonstrated significantly superior outcomes. Adjusted PR was 79.1% (Letrozole) vs. 66% (HRT) (RR 1.20, 95% CI: 1.10-1.31, P &amp;lt; 0.0001). CPR was 73.5% (Letrozole) vs. 62.6% (HRT) (RR 1.17, 95% CI: 1.07-1.29, P = 0.0008). OPR was 68% (Letrozole) vs. 58% (HRT) (RR 1.17, 95% CI: 1.06-1.30, P = 0.0029). In the HRT group, 30% of patients required additional luteal progesterone, versus only 3% in the Letrozole group. Multivariate analysis confirmed that endometrial preparation protocol significantly influenced clinical outcomes, while hCG-FET timing, follicle diameter, and serum estradiol levels did not. Limitations, reasons for caution The retrospective design introduces selection bias and the absence of randomization limits generalizability. Further randomized controlled trials are required to confirm findings. Despite this, results suggest letrozole+hCG enhances FET outcomes and may reduce the need for additional luteal progesterone, offering scheduling flexibility without compromising success rates. Wider implications of the findings This study suggests that letrozole+hCG FET protocols improve outcomes while providing greater scheduling flexibility. The findings challenge traditional FET cycle synchronization criteria in natural/induced endometrial priming, advocating for further research to refine follicular size thresholds for triggering ovulation and optimize embryo transfer timing in personalized IVF protocols. Trial registration number No

Study question Does a natural endometrial preparation for single euploid frozen embryo transfer increase the odds of live birth (LB) in obese patients? Summary answer Compared to an artificial approach, a natural endometrial preparation for single euploid frozen embryo transfer improves the odds of LB in obese patients. What is known already Several protocols to prepare the endometrium for frozen embryo transfer have been described with comparable clinical outcomes, and no individual protocol has been demonstrated to be superior. Likewise, no specific patient group has been defined that can substantially benefit from a natural endometrial preparation. Despite obesity being linked to adverse clinical outcomes, observational studies and clinical trials have traditionally included participants with normal weight when comparing protocols, resulting in obese patients being underrepresented in clinical research. Therefore, studies focusing on endometrial preparation protocols for frozen embryo transfer that include obese patients are required. Study design, size, duration This study involved the analysis of a retrospective dataset including 975 single euploid frozen embryo transfer cycles performed at two tertiary referral centers between March 2017 and November 2019. The primary outcome was LB after natural and artificial endometrial preparation. Participants were stratified according to the World Health Organization Body mass index (BMI) classification into three groups (G): G1: Normal-weight ≤24.9 (n = 390), G2: Overweight 25 to 29.9 (n = 332), and G3: Obese ≥30 (n = 253). Participants/materials, setting, methods Cycles included infertility patients attempting a single euploid frozen blastocyst transfer under natural (n = 324) or artificial (n = 651) endometrial preparation. PGT-A was performed after trophectoderm biopsy using Next-Generation Sequencing. Spontaneous LH rise confirmed ovulation for natural endometrial preparation, and embryo transfer occurred five days after initial progesterone elevation. Hormones were measured from blood samples. For artificial preparation, embryo transfer was performed after oral estradiol and 120-hour vaginal progesterone exposure. All participants received luteal phase support. Main results and the role of chance Within each BMI group, no statistically significant differences in age were observed in patients between a natural and artificial endometrial preparation: G1 (33.5±5.1 vs. 32.7±5.2 years; p = 0.13), G2 (34.5±4.9 vs. 33.9±5.8 years; p = 0.39), and G3 (35.6±4.9 vs. 34.3±5.6 years; p = 0.1). Similarly, no statistically significant differences were observed regarding implantation rates: G1 (60% vs 57.1%; p = 0.57), G2 (54.8% vs 55.7%; p = 0.88), and G3 (64.3% vs 51.4%; p = 0.07). LB rates remained constant across all BMI groups when a natural preparation was conducted: G1 [56.66% (n = 85/150)], G2 [49.03% (n = 51/104)], and G3 [58.57% (n = 41/70)]. We observed a decrease in LB rates in G3 when an artificial endometrial preparation was performed: G1 [47.5% (n = 114/240)], G2 [44.73% (n = 102/228)], and G3 [34.42% (n = 63/183)]. The positive impact of a natural preparation on LB rates was more significant in G3, showing a 24% difference of effect between the natural and artificial endometrial preparations. Univariate logistic regression analysis showed a statistically significant difference in the primary outcome (LB) when a natural endometrial preparation was conducted in G3 (OR 2.69, 95% CI 1.53-4.74; p = 0.001), with no differences found in G1 (OR 1.45, 95% CI 0.96-2.18; p = 0.08) and G2 (OR 1.19, 95% CI 0.75-1.89; p = 0.73). Limitations, reasons for caution This study could be subject to bias due to its retrospective nature. Sperm quality was not considered for the present study. Wider implications of the findings Obese women appear to be the patient group that can obtain benefit from a natural endometrial preparation for frozen embryo transfer by increasing the odds of LB. Future prospective studies should confirm this hypothesis. A natural approach might be unfeasible in a subgroup of obese patients due to anovulatory cycles. Trial registration number not applicable

Do children born after vitrified-thawed embryo transfers (ETs) using donated oocytes have worse perinatal outcomes when compared with fresh ET? No significant difference in birthweight and prematurity rates between fresh or frozen embryo transfers (FETs) in newborns after oocyte donation was found. Autologous singletons born after fresh ET have been previously associated with higher rates of preterm birth and low birthweight, while FETs seem to confer a higher risk of hypertensive disorders during pregnancy and macrosomia. However, studies comparing these outcomes using autologous oocytes are unable to adequately disentangle the putative detrimental consequences of embryo vitrification from the possible effects that ovarian stimulation and endometrial preparation may have on endometrial receptivity prior to ET. The oocyte donation model is, for this reason, a more appropriate setting to study these hypotheses; however so far, the information available regarding neonatal outcomes in this patient population is limited to either small and/or heterogeneous studies. We performed a multicentre retrospective cohort study including 5848 singletons born between 2009 and February 2020 following oocyte donation and single blastocyst transfer, subdivided according to whether a fresh ET or FET was performed. We also performed two additional sensitivity analyses, subgrouping the sample according to the type of endometrial preparation (natural versus artificial) and whether the donated oocytes had previously been vitrified or not. Patients with a first singleton livebirth after single blastocyst transfer were compared using multivariable regression analysis to account for potential confounding factors. The primary outcome was birthweight. Secondary outcomes were birthweight z-scores and percentiles, small/large for gestational age, gestational age at delivery, gender, prematurity (<37 weeks and <32 weeks), neonatal morbidity (Apgar scores and need for neonatal intensive care) and maternal morbidity (gestational hypertensive disorders, gestational diabetes and caesarean delivery). There was no significant difference between the fresh ET and FET groups in terms of mean birthweight (3215 g versus 3200 g) and birthweight z-scores (0.03 versus 0.1), in both the unadjusted and confounder-adjusted models. However, artificial endometrial preparation was associated with a higher birthweight (3220 g versus 3105 g) and birthweight z-scores (0.06 versus -0.13) when compared with a transfer in a natural cycle. Although a 1-day statistically significant difference in gestational age at birth (275 versus 274 days) was detected, premature birth rates (<37 weeks) did not vary significantly between groups (9.9% and 11.2% for fresh ET and FET, respectively). No other statistically significant differences were found in the remaining neonatal and maternal outcomes studies between the fresh ET and FET groups. This study is limited by its retrospective design and lack of information regarding congenital malformations. Moreover, the sample selection criteria that were used may limit the generalizability of our results. Perinatal outcomes did not seem to be affected significantly by the embryo vitrification process in an oocyte donation model. Hence, other factors may contribute to the hindered perinatal outcomes described in ART, particularly the potential effect that ovarian stimulation and endometrial preparation may have on endometrial receptivity. No specific funding was obtained for this study. All authors have no conflicts to declare. N/A.

Reflections on preimplantation genetic testing for aneuploidy and mosaicism: how did we get here, and what does it mean clinically?

Can preimplantation genetic testing for aneuploidy (PGT-A) improve the live birth rate and reduce the miscarriage rate in patients with recurrent pregnancy loss (RPL) caused by an abnormal embryonic karyotype and recurrent implantation failure (RIF)? PGT-A could not improve the live births per patient nor reduce the rate of miscarriage, in both groups. PGT-A use has steadily increased worldwide. However, only a few limited studies have shown that it improves the live birth rate in selected populations in that the prognosis has been good. Such studies have excluded patients with RPL and RIF. In addition, several studies have failed to demonstrate any benefit at all. PGT-A was reported to be without advantage in patients with unexplained RPL whose embryonic karyotype had not been analysed. The efficacy of PGT-A should be examined by focusing on patients whose previous products of conception (POC) have been aneuploid, because the frequencies of abnormal and normal embryonic karyotypes have been reported as 40-50% and 5-25% in patients with RPL, respectively. A multi-centre, prospective pilot study was conducted from January 2017 to June 2018. A total of 171 patients were recruited for the study: an RPL group, including 41 and 38 patients treated respectively with and without PGT-A, and an RIF group, including 42 and 50 patients treated respectively with and without PGT-A. At least 10 women in each age group (35-36, 37-38, 39-40 or 41-42years) were selected for PGT-A groups. All patients and controls had received IVF-ET for infertility. Patients in the RPL group had had two or more miscarriages, and at least one case of aneuploidy had been ascertained through prior POC testing. No pregnancies had occurred in the RIF group, even after at least three embryo transfers. Trophectoderm biopsy and array comparative genomic hybridisation (aCGH) were used for PGT-A. The live birth rate of PGT-A and non-PGT-A patients was compared after the development of blastocysts from up to two oocyte retrievals and a single blastocyst transfer. The miscarriage rate and the frequency of euploidy, trisomy and monosomy in the blastocysts were noted. There were no significant differences in the live birth rates per patient given or not given PGT-A: 26.8 versus 21.1% in the RPL group and 35.7 versus 26.0% in the RIF group, respectively. There were also no differences in the miscarriage rates per clinical pregnancies given or not given PGT-A: 14.3 versus 20.0% in the RPL group and 11.8 versus 0% in the RIF group, respectively. However, PGT-A improved the live birth rate per embryo transfer procedure in both the RPL (52.4 vs 21.6%, adjusted OR 3.89; 95% CI 1.16-13.1) and RIF groups (62.5 vs 31.7%, adjusted OR 3.75; 95% CI 1.28-10.95). Additionally, PGT-A was shown to reduce biochemical pregnancy loss per biochemical pregnancy: 12.5 and 45.0%, adjusted OR 0.14; 95% CI 0.02-0.85 in the RPL group and 10.5 and 40.9%, adjusted OR 0.17; 95% CI 0.03-0.92 in the RIF group. There was no difference in the distribution of genetic abnormalities between RPL and RIF patients, although double trisomy tended to be more frequent in RPL patients. The sample size was too small to find any significant advantage for improving the live birth rate and reducing the clinical miscarriage rate per patient. Further study is necessary. A large portion of pregnancy losses in the RPL group might be due to aneuploidy, since PGT-A reduced the overall incidence of pregnancy loss in these patients. Although PGT-A did not improve the live birth rate per patient, it did have the advantage of reducing the number of embryo transfers required to achieve a similar number live births compared with those not undergoing PGT-A. This study was supported by the Japan Society of Obstetrics and Gynecology and grants from the Japanese Ministry of Education, Science, and Technology. There are no conflicts of interest to declare. N/A.

Study question Which clinical and biological factors contribute to the risk of pregnancy loss and the likelihood of live birth in pre-implantation genetic testing for aneuploidy (PGT-A)? Summary answer Adenomyosis and delayed embryo development significantly affect live birth rate (LBR) following euploid blastocyst transfer, while artificial endometrial preparation is associated with increased pregnancy loss. What is known already Embryo selection by PGT-A is intended to improve reproductive outcomes per transfer. While selecting euploid blastocysts does not improve cumulative LBR, PGT-A aims to reduce the time to pregnancy and lower the risk of pregnancy loss. However, despite genetic selection, many euploid embryos still fail to implant or result in non-viable pregnancies. Multiple factors, including maternal and paternal characteristics, as well as clinical and laboratory variables contribute to these outcomes. The relative importance of these factors may differ depending on the clinical indication for PGT-A. Study design, size, duration This retrospective cohort study includes patients who underwent frozen embryo transfer (FET) of a euploid embryo following intracytoplasmic sperm injection (ICSI) with trophectoderm biopsy (day 5/6) and PGT-A in a single tertiary university hospital between 2017 and 2023. Patients with additional testing for monogenic diseases or structural rearrangements were excluded. Participants/materials, setting, methods A total of 375 patients undergoing 533 FET cycles following PGT-A for specific clinical indications, including recurrent pregnancy loss (RPL), recurrent implantation failure (RIF) and advanced maternal age. Transfers were performed in either a natural (n = 248) or artificial cycle (n = 285). Cycle monitoring included serial hormonal and ultrasound assessments. Ultrasound-guided single embryo transfer was performed once endometrial thickness was deemed sufficient. Ovulation triggering and/or luteal phase support was prescribed at the discretion of the treating physician. Main results and the role of chance Following euploid blastocyst transfer, biochemical pregnancy rate was 69.0%, LBR was 51.2% and early pregnancy loss (EPL) rate was 18.5%. When considering only the first FET per patient (n = 375), biochemical pregnancy rate was 73.3%, LBR reached 56.3% and EPL rate was 17.3%. Univariate and multivariate analyses were conducted with GEE to adjust for multiple transfers per patient, with a sub-analysis performed on the first cycle of each patient. Multivariate logistic regression identified delayed embryo development, reflected in day 6 transfers, as significantly associated with lower LBR compared to day 5 (OR 0.55, 95%CI 0.34-0.90, p = 0.017). Additionally, the presence of adenomyosis was linked to reduced LBR (OR 0.42, 95% CI 0.22-0.79, p = 0.007). Importantly, the type of endometrial preparation (natural versus artificial) did not significantly influence LBR. Conversely, considering only first embryo transfer cycles, the rate of pregnancy loss per clinical pregnancy was significantly lower for natural cycle compared to artificial cycle FET (10% [11/114] vs. 24% [33/140]; p = 0.003, OR 2.3, 95% CI 1.3-4.2). A history of ≥ 2 pregnancy losses did not significantly influence live birth outcomes (p = 0.49), nor did we observe an association between LBR and the number of pregnancy losses when treated as a continuous variable. Limitations, reasons for caution The primary limitation is the retrospective design, which carries an inherent risk of bias. Patient heterogeneity and varying indications for PGT-A may have influenced both treatment decisions and success rates. Additionally, greater standardization in defining RIF and RPL would have enhanced data consistency. Wider implications of the findings Our findings highlight the impact of adenomyosis on live birth rates, emphasizing the need for subtype classification and refined treatment strategies. Additionally, the association between endometrial preparation and EPL provides clinicians with essential insights for optimizing FET management. Trial registration number No

We evaluated whether preimplantation genetic testing for aneuploidy (PGT-A) could increase the cumulative live birth rate (CLBR) in patients with recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL). The clinical records of 7,668 patients who underwent oocyte retrieval (OR) with or without PGT-A were reviewed for 365days and retrospectively analyzed. Using propensity score matching, 579 patients in the PGT-A group were matched one-to-one with 7,089 patients in the non-PGT-A (control) group. Their pregnancy and perinatal outcomes and CLBRs were statistically compared. The live birth rate per single vitrified-warmed blastocyst transfers (SVBTs) significantly improved in the PGT-A group in all age groups (P < 0.0002, all). Obstetric and perinatal outcomes were comparable between both groups regarding both RIF and RPL cases. Cox regression analysis demonstrated that in the RIF cases, the risk ratio per OR was significantly lower in the PGT-A group than in the control group (P = 0.0480), particularly in women aged < 40years (P = 0.0364). However, the ratio was comparable between the groups in RPL cases. The risk ratio per treatment period was improved in the PGT-A group in both RIF and RPL cases only in women aged 40-42years (P = 0.0234 and P = 0.0084, respectively). Increased CLBR per treatment period was detected only in women aged 40-42years in both RIF and RPL cases, suggesting that PGT-A is inappropriate to improve CLBR per treatment period in all RIF and RPL cases.