Mosaic human preimplantation embryos and their developmental potential in a prospective, non-selection clinical trial.

Chromosome imbalance (aneuploidy) is the major cause of pregnancy loss and congenital disorders in humans. Analyses of small biopsies from human embryos suggest that aneuploidy commonly originates during early divisions, resulting in mosaicism. However, the developmental potential of mosaic embryos remains unclear. We followed the distribution of aneuploid chromosomes across 73 unselected preimplantation embryos and 365 biopsies, sampled from four multifocal trophectoderm (TE) samples and the inner cell mass (ICM). When mosaicism impacted fewer than 50% of cells in one TE biopsy (low-medium mosaicism), only 1% of aneuploidies affected other portions of the embryo. A double-blinded prospective non-selection trial (NCT03673592) showed equivalent live-birth rates and miscarriage rates across 484 euploid, 282 low-grade mosaic, and 131 medium-grade mosaic embryos. No instances of mosaicism or uniparental disomy were detected in the ensuing pregnancies or newborns, and obstetrical and neonatal outcomes were similar between the study groups. Thus, low-medium mosaicism in the trophectoderm mostly arises after TE and ICM differentiation, and such embryos have equivalent developmental potential as fully euploid ones.

Extracellular vesicles secreted by cumulus cells contain microRNAs that are potential regulatory factors of mouse oocyte developmental competence.

The role of cumulus cells (CCs) in the acquisition of oocyte developmental competence is not yet fully understood. In a previous study, we matured cumulus-denuded fully-grown mouse oocytes to metaphase II (MII) on a feeder layer of CCs (FL-CCs) isolated from developmentally competent (FL-SN-CCs) or incompetent (FL-NSN-CCs) SN (surrounded nucleolus) or NSN (not surrounding nucleolus) oocytes, respectively. We observed that oocytes cultured on the former could develop into blastocysts, while those matured on the latter arrested at the 2-cell stage. To investigate the CC factors contributing to oocyte developmental competence, here we focused on the CCs' release into the medium of extracellular vesicles (EVs) and on their miRNA content. We found that, during the 15-h transition to MII, both FL-SN-CCs and FL-NSN-CCs release EVs that can be detected, by confocal microscopy, inside the zona pellucida (ZP) or the ooplasm. The majority of EVs are <200 nm in size, which is compatible with their ability to cross the ZP. Next-generation sequencing of the miRNome of FL-SN-CC versus FL-NSN-CC EVs highlighted 74 differentially expressed miRNAs, with 43 up- and 31 down-regulated. Although most of these miRNAs do not have known roles in the ovary, in silico functional analysis showed that seven of these miRNAs regulate 71 target genes with specific roles in meiosis resumption (N = 24), follicle growth (N = 23), fertilization (N = 1), and the acquisition of oocyte developmental competence (N = 23). Overall, our results indicate CC EVs as emerging candidates of the CC-to-oocyte communication axis and uncover a group of miRNAs as potential regulatory factors.

Does recurrent implantation failure exist? Prevalence and outcomes of five consecutive euploid blastocyst transfers in 123 987 patients.

STUDY QUESTION: What are the clinical pregnancy and live birth rates in women who underwent up to two more euploid blastocyst transfers after three failures in the absence of another known factor that affects implantation? SUMMARY ANSWER: The fourth and fifth euploid blastocyst transfers resulted in similar live birth rates of 40% and 53.3%, respectively, culminating in a cumulative live birth rate of 98.1% (95% CI = 96.5-99.6%) after five euploid blastocyst transfers. WHAT IS KNOWN ALREADY: The first three euploid blastocysts have similar implantation and live birth rates and provide a cumulative live birth rate of 92.6%. STUDY DESIGN, SIZE, DURATION: An international multi-center retrospective study was conducted at 25 individual clinics. The study period spanned between January 2012 and December 2022. A total of 123 987 patients with a total of 64 572 euploid blastocyst transfers were screened for inclusion. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients with a history of any embryo transfer at another clinic, history of any unscreened embryo transfer at participating clinics, parental karyotype abnormalities, the use of donor oocytes or a gestational carrier, untreated intracavitary uterine pathology (e.g. polyp, leiomyoma), congenital uterine anomalies, adenomyosis, communicating hydrosalpinx, endometrial thickness <6 mm prior to initiating of progesterone, use of testicular sperm due to non-obstructive azoospermia in the male partner, transfer of an embryo with a reported intermediate chromosome copy number (i.e. mosaic), preimplantation genetic testing cycles for monogenic disorders, or structural chromosome rearrangements were excluded. Ovarian stimulation protocols and embryology laboratory procedures including trophectoderm biopsy followed the usual practice of each center. The ploidy status of blastocysts was determined with comprehensive chromosome screening. Endometrial preparation protocols followed the usual practice of participating centers and included programmed cycles, natural or modified natural cycles. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 105 (0.085% of the total population) patients met the criteria and underwent at least one additional euploid blastocyst transfer after failing to achieve a positive pregnancy test with three consecutive euploid blastocyst transfers. Outcomes of the fourth and fifth euploid blastocyst transfers were similar across participating centers. Overall, the live birth rate was similar with the fourth and fifth euploid blastocysts (40% vs 53.3%, relative risk = 1.33, 95% CI = 0.93-1.9, P value = 0.14). Sensitivity analyses excluding blastocysts biopsied on Day 7 postfertilization, women with a BMI >30 kg/m2, cycles using non-ejaculate or donor sperm, double-embryo transfer cycles, and cycles in which the day of embryo transfer was modified due to endometrial receptivity assay test result yielded similar results. Where data were available, the fourth euploid blastocyst had similar live birth rate with the first one (relative risk = 0.84, 95% CI = 0.58-1.21, P = 0.29). The cumulative live birth rate after five euploid blastocyst transfers was 98.1% (95% CI = 96.5-99.6%). LIMITATIONS, REASONS FOR CAUTION: Retrospective design has its own inherent limitations. Patients continuing with a further euploid embryo transfer and patients dropping out from treatment after three failed euploid transfers can be systematically different, perhaps with regard to ovarian reserve or economic status. WIDER IMPLICATION OF THE FINDINGS: Implantation failure seems to be mainly due to embryonic factors. Given the stable and high live birth rates up to five euploid blastocysts, unexplained recurrent implantation failure should have a prevalence of <2%. Proceeding with another embryo transfer can be the best next step once a known etiology for implantation failure is ruled out. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

The metrics maze in science: navigating academic evaluation without journalistic pressures.

In recent years a troubling trend has emerged in the medical research field, notably in reproductive medicine, manifesting an increased emphasis on quantity over quality in articles published. The pressure to collect copious publication records risks compromising meticulous expertise and impactful contributions. This tendency is exemplified by the rise of 'hyper-prolific researchers' publishing at an extraordinary rate (i.e. every 5 days), prompting a deeper analysis of the reasons underlying this behaviour. Prioritizing rapid publication over Galileo Galilei's systematic scientific principles may lead to a superficial approach driven by quantitative targets. Thus, the overreliance on metrics to facilitate academic careers has shifted the focus to numerical quantification rather than the real scientific contribution, raising concerns about the effectiveness of the evaluation systems. The Hamletian question is: are we scientist or journalist? Addressing these issues could necessitate a crucial re-evaluation of the assessment criteria, emphasizing a balance between quantity and quality to foster an academic environment that values meaningful contributions and innovation.

GnRH agonist trigger in poor prognosis patients undergoing a multicycle approach through DuoStim or consecutive stimulations: a SWOT analysis.

PURPOSE OF REVIEW: Identify the most recent and significant evidence regarding the ovulation trigger within the framework of a multicycle approach through DuoStim, providing valuable insights for improving treatment strategies in patients with a poor prognosis. RECENT FINDINGS: The trigger method plays a pivotal role in optimizing in-vitro fertilization (IVF) stimulation, influencing oocyte retrieval and maturation rates, as well as follicle recruitment in consecutive ovarian stimulations such as double stimulation. Decision-making involves multiple factors and, while guidelines exist for conventional stimulation, specific recommendations for the multicycle approach are not well established. SUMMARY: The different methods for inducing oocyte maturation underscore the need for personalization of IVF protocols. The GnRH agonist trigger induces rapid luteolysis and establishes favorable hormonal conditions that do not adversely affect the recruitment of consecutive follicular waves in the context of DuoStim. It serves as a valid alternative to hCG in freeze-all cycles. This strategy might enhance the safety and flexibility of ovarian stimulations with no impact on oocyte competence and IVF efficacy.

Cryostorage management of reproductive cells and tissues in ART: status, needs, opportunities and potential new challenges.

Among the wide range of procedures performed by clinical embryologists, the cryopreservation of reproductive cells and tissues represents a fundamental task in the daily routine. Indeed, cryopreservation procedures can be considered a subspecialty of medically assisted reproductive technology (ART), having the same relevance as sperm injection or embryo biopsy for preimplantation genetic testing. However, although a great deal of care has been devoted to optimizing cryopreservation protocols, the same energy has only recently been spent on developing and implementing strategies for the safe and reliable storage and transport of reproductive specimens. Herein, we have summarized the content of the available guidelines, the risks, the needs and the future perspectives regarding the management of cryopreservation biorepositories used in ART.

Double stimulation for the management of poor-prognosis patients: where are we going?

PURPOSE OF REVIEW: The technical improvements in IVF allowed the implementation of nonconventional ovarian stimulation protocols for some specific patients. Where time is crucial, such as with oncologic patients, poor-prognosis patients, patients with low ovarian reserve, and those with advanced maternal age, access to IVF treatment is even more critical. Some of these protocols might start in the late follicular phase, luteal phase, or involve both stimulations within the same ovarian cycle. RECENT FINDINGS: Until now, published evidence showed that oocytes retrieved from unconventional protocol seem to be developmentally, genetically, and reproductively competent. Second stimulation in the same ovarian cycle after the conventional approach may represent a sound alternative to oocyte accumulation. This can be proposed in progress after careful counselling focused on the patients' chances of finding at least one euploid embryo on account of their age and of the number of blastocysts obtained after the conventional approach. SUMMARY: The adoption of these new strategies, known as double stimulation protocol, can be conceived as a real full-personalization of ovarian stimulation. Multicentre prospective RCTs are urgently needed to evaluate the efficacy, efficiency, and costs of double stimulation versus two consecutive conventional approaches with standard or mild stimulation and in a different IVF setting.

Metaphase-II oocyte competence is unlinked to the gonadotrophins used for ovarian stimulation: a matched case-control study in women of advanced maternal age.

PURPOSE: An impact of different gonadotrophins selection for ovarian stimulation (OS) on oocyte competence has yet to be defined. In this study, we asked whether an association exists between OS protocol and euploid blastocyst rate (EBR) per metaphase-II (MII) oocytes. METHODS: Cycles of first preimplantation genetic testing for aneuploidies conducted by women ≥ 35 years old with their own metaphase-II oocytes inseminated in the absence of severe male factor (years 2014-2018) were clustered based on whether recombinant FSH (rec-FSH) or human menopausal gonadotrophin (HMG) was used for OS, then matched for the number of fresh inseminated eggs. Four groups were outlined: rec-FSH (N = 57), rec-FSH plus rec-LH (N = 55), rec-FSH plus HMG (N = 112), and HMG-only (N = 127). Intracytoplasmic sperm injection, continuous blastocyst culture, comprehensive chromosome testing to assess full-chromosome non-mosaic aneuploidies and vitrified-warmed euploid single embryo transfers (SETs) were performed. The primary outcome was the EBR per cohort of MII oocytes. The secondary outcome was the live birth rate (LBR) per first SETs. RESULTS: Rec-FSH protocol was shorter and characterized by lower total gonadotrophin (Gn) dose. The linear regression model adjusted for maternal age showed no association between the Gn adopted for OS and EBR per cohort of MII oocytes. Similarly, no association was reported with the LBR per first SETs, even when adjusting for blastocyst quality and day of full blastulation. CONCLUSION: In view of enhanced personalization in OS, clinicians shall focus on different endpoints or quantitative effects related to Gn action towards follicle recruitment, development, and atresia. Here, LH and/or hCG was administered exclusively to women with expected sub/poor response; therefore, we cannot exclude that specific Gn formulations may impact patient prognosis in other populations.

Clinical and laboratory key performance indicators in IVF: A consensus between the Italian Society of Fertility and Sterility and Reproductive Medicine (SIFES-MR) and the Italian Society of Embryology, Reproduction and Research (SIERR).

PURPOSE: Infertility is increasing worldwide, and many couples seek IVF. Clinical management and laboratory work are fundamental in the IVF journey. Therefore, the definition of reliable key performance indicators (KPIs) based on clinical and laboratory parameters, is essential for internal quality control (IQC). Laboratory performance indicators have been identified and a first attempt to also determine clinical ones has been recently published. However, more detailed indicators are required. METHODS: An Italian group of experts in Reproductive Medicine from both public and private clinics on behalf of SIFES-MR and SIERR was established to define IVF indicators to monitor clinical performance. RESULTS: The working group built a consensus on a list of KPIs, performance indicators (PIs) and recommendation indicators (RIs). When deemed necessary, the reference population was stratified by woman age, response to ovarian stimulation and adoption of preimplantation genetic testing for aneuploidies (PGT-A). Each indicator was scored with a value from 1 to 5 and a weighted average formula - considering all the suggested parameters-was defined. This formula generates a center performance score, indicating low, average, good, or excellent performance. CONCLUSION: This study is intended to provide KPIs, PIs and RIs that encompass several essential aspects of a modern IVF clinic, including quality control and constant monitoring of clinical and embryological features. These indicators could be used to assess the quality of each center with the aim of improving efficacy and efficiency in IVF.

How slow is too slow? A comprehensive portrait of Day 7 blastocysts and their clinical value standardized through artificial intelligence.

STUDY QUESTION: What is the clinical value of Day 7 blastocysts? SUMMARY ANSWER: Ending embryo culture at 144 hours post-insemination (h.p.i.; i.e. 6 days) would involve 7.3% and 4.4% relative reductions in the number of patients obtaining euploid blastocysts and live birth(s) (LBs), respectively. WHAT IS KNOWN ALREADY: Many studies showed that Day 7 blastocysts are clinically valuable, although less euploid and less competent than faster-growing embryos. Nevertheless, a large variability exists in: (i) the definition of 'Day 7'; (ii) the criteria to culture embryos to Day 7; (iii) the clinical setting; (iv) the local regulation; and/or (v) the culture strategies and incubators. Here, we aimed to iron out these differences and portray Day 7 blastocysts with the lowest possible risk of bias. To this end, we have also adopted an artificial intelligence (AI)-powered software to automatize developmental timings annotations and standardize embryo morphological assessment. STUDY DESIGN, SIZE AND DURATION: Observational study including 1966 blastocysts obtained from 681 patients cultured in a time-lapse incubator between January 2013 and December 2020 at a private Italian IVF center. PARTICIPANTS/MATERIALS, SETTING, METHODS: According to Italian Law 40/2004, embryos were not selected based on their morphology and culture to ≥168 h.p.i. is standard care at our center. ICSI, continuous culture with Day 5 media refresh, trophectoderm biopsy without assisted hatching and comprehensive chromosome testing (CCT) to diagnose full-chromosome non-mosaic aneuploidies, were all performed. Blastocysts were clustered in six groups based on the time of biopsy in h.p.i. at 12 hr intervals starting from <120 h.p.i. (set as control) up to >168 h.p.i. Blastocyst quality was assessed using Gardner's scheme and confirmed with AI-powered software. AI was also used to automatically annotate the time of expanding blastocyst (tEB) and the hours elapsing between this moment and the achievement of full expansion when blastocysts were biopsied and vitrified. Also, blastocyst area at tEB and at the time of biopsy was automatically assessed, as well as the hour of the working day when the procedure was performed. The main outcomes were the euploidy rate and the LB rate (LBR) per vitrified-warmed euploid single blastocyst transfer. The results were adjusted for confounders through multivariate logistic regressions. To increase their generalizability, the main outcomes were reported also based on a 144-h.p.i. cutoff (i.e. 6 exact days from ICSI). Based on this cutoff, all the main patient outcomes (i.e. number of patients obtaining blastocysts, euploid blastocysts, LBs, with supernumerary blastocysts without a LB and with surplus blastocysts after an LB) were also reported versus the standard care (>168 h.p.i.). All hypothetical relative reductions were calculated. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 14.6% of the blastocysts reached full expansion beyond 144 h.p.i. (5.9% in the range 144-156 h.p.i., 7.9% in the range 156-168 h.p.i. and 0.8% beyond 168 h.p.i.). Slower blastocysts were of a worse quality based on the evaluation of both embryologists and AI. Both later tEB and longer time between tEB and full blastocyst expansion concurred to Day 7 development, quite independently of blastocyst quality. Slower growing blastocysts were slightly larger than faster-growing ones at the time of biopsy, but no difference was reported in the risk of hatching, mainly because two dedicated slots have been set along the working day for these procedures. The lower euploidy rate among Day 7 blastocysts is due to their worse morphology and more advanced oocyte age, rather than to a slower development per se. Conversely, the lower LBR was significant even after adjusting for confounders, with a first relevant decrease for blastocysts biopsied in the range 132-144 h.p.i. (N = 76/208, 36.5% versus N = 114/215, 53.0% in the control, multivariate odds ratio 0.61, 95% CI 0.40-0.92, adjusted-P = 0.02), and a second step for blastocysts biopsied in the range 156-168 h.p.i. (N = 3/21, 14.3%, multivariate odds ratio: 0.24, 95% CI 0.07-0.88, adjusted-P = 0.03). Nevertheless, when the cutoff was set at 144 h.p.i., no significant difference was reported. In this patient population, ending embryo culture at 144 h.p.i. would have caused 10.6%, 7.3%, 4.4%, 13.7% and 5.2% relative reductions in the number of patients obtaining blastocysts, euploid blastocysts, LBs, supernumerary blastocysts without an LB and surplus blastocysts after an LB, respectively. LIMITATIONS, REASONS FOR CAUTION: Gestational and perinatal outcomes were not assessed, and a cost-effectiveness analysis is missing. Moreover, we encourage other groups to investigate this topic with different culture and biopsy protocols, as well as in different clinical settings and regulatory contexts. WIDER IMPLICATIONS OF THE FINDINGS: In view of the increasing personalization and patient-centeredness of IVF, whenever allowed from the local regulations, the choice to culture embryos to Day 7 should be grounded on the careful evaluation of couples' reproductive history. Patients should be aware that Day 7 blastocysts are less competent than faster-growing ones; still, poor prognosis couples, couples less compliant toward other attempts in case of a failure and couples wishing for more than one child, may benefit from them. AI tools can help improving the generalizability of the evidence worldwide. STUDY FUNDING/COMPETING INTEREST(S): This study did not receive any funding. I.E., A.B.M. and I.H.-V. are employees of Fairtility Ltd. TRIAL REGISTRATION NUMBER: N/A.

Clinical, obstetric and perinatal outcomes after vitrified-warmed euploid blastocyst transfer are independent of cryo-storage duration.

RESEARCH QUESTION: The study aimed to retrospectively evaluate the impact of cryo-storage duration on clinical, obstetric and perinatal outcomes after vitrified-warmed euploid blastocyst transfer. DESIGN: This was an observational study including 2688 vitrified-warmed euploid single blastocyst transfers that was conducted at a private IVF centre between May 2013 and March 2020. It included a total of 1884 women (age 38 ± 3 years) undergoing at least one transfer after preimplantation genetic testing for aneuploidies. The euploid blastocysts transferred were clustered into seven groups according to the cryo-storage duration between vitrification and warming: ≤60 days (n = 646; control group), 61-90 days (n = 599), 91-180 days (n = 679), 181-360 days (n = 405), 361-720 days (n = 144), 721-1080 days (n = 118) and >1080 days (n = 97). The primary outcome was the live birth rate (LBR) per transfer. The secondary outcomes were miscarriage rate, obstetric and perinatal issues. The data were adjusted for confounders through logistic or linear regressions. RESULTS: A significantly lower LBR was reported for transfers performed within 91-180 days (n = 291/679, 42.9%; P = 0.017), 181-360 days (n = 169/405, 41.7%; P = 0.016) and 361-720 days (n = 57/144, 39.6%; P = 0.034) versus ≤60 days (n = 319/646, 49.4%). However, this was mainly due to top-quality embryos being transferred first when more euploid blastocysts were available, thereby leaving lower quality ones for subsequent procedures. Indeed, the multivariate odds ratios adjusted for confounders showed similar results across all cryo-storage duration clusters. No difference was reported also for all secondary outcomes. CONCLUSIONS: Cryo-storage duration even beyond 3 years from blastocyst vitrification does not affect clinical, obstetric and perinatal outcomes.

Second stimulation in the same ovarian cycle: an option to fully-personalize the treatment in poor prognosis patients undergoing PGT-A.

PURPOSE: Our primary objective was to assess whether immediately undergoing a second stimulation in the same ovarian cycle (DuoStim) for advanced-maternal-age and/or poor-ovarian-reserve (AMA/POR) patients obtaining ≤ 3 blastocysts for preimplantation-genetic-testing-for-aneuploidies (PGT-A) is more efficient than the conventional-approach. METHODS: All AMA/POR patients obtaining ≤ 3 blastocysts after conventional-stimulation between 2017 and 2019 were proposed DuoStim, and 143 couples accepted (DuoStim-group) and were matched for the main confounders to 143 couples who did not accept (conventional-group). GnRH-antagonist protocol with recombinant-gonadotrophins and agonist trigger, intra-cytoplasmatic-sperm-injection (ICSI) with ejaculated sperm, PGT-A and vitrified-warmed euploid single-blastocyst-transfer(s) were performed. The primary outcome was the cumulative-live-birth-delivery-rate per intention-to-treat (CLBdR per ITT) within 1 year. If not delivering, the conventional-group had 1 year to undergo another conventional-stimulation. A cost-effectiveness analysis was also conducted. RESULTS: The CLBdR was 10.5% in the conventional-group after the first attempt. Only 12 of the 128 non-pregnant patients returned (165 ± 95 days later; drop-out = 116/128,90.6%), and 3 delivered. Thus, the 1-year CLBdR was 12.6% (N = 18/143). In the DuoStim-group, the CLBdR was 24.5% (N = 35/143; p = 0.01), 2 women delivered twice and 13 patients have other euploid blastocysts after a LB (0 and 2 in the conventional-group). DuoStim resulted in an incremental-cost-effectiveness-ratio of 23,303€. DuoStim was costlier and more effective in 98.7% of the 1000 pseudo-replicates generated through bootstrapping, and the cost-effectiveness acceptability curves unveiled that DuoStim would be more cost-effective than the conventional-approach at a willingness-to-pay threshold of 23,100€. CONCLUSIONS: During PGT-A treatments in AMA/POR women, DuoStim can be suggested in progress to rescue poor blastocyst yields after conventional-stimulation. It might indeed prevent drop-out or further aging between attempts.

Leave the past behind: women's reproductive history shows no association with blastocysts' euploidy and limited association with live birth rates after euploid embryo transfers.

STUDY QUESTION: 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? SUMMARY ANSWER: 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). WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): No funding was received and there are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Recombinant human luteinizing hormone co-treatment in ovarian stimulation for assisted reproductive technology in women of advanced reproductive age: a systematic review and meta-analysis of randomized controlled trials.

INTRODUCTION: Several studies suggest that luteinizing hormone (LH) could improve IVF outcome in women of advanced reproductive age by optimizing androgen production. In this review, we assessed the role of recombinant-human LH (r-hLH) and recombinant human follicle stimulating hormone (r-hFSH) co-treatment in ovarian stimulation for assisted reproductive technology in women of advanced reproductive age candidates for assisted reproduction. MATERIAL AND METHODS: Using a preregistered protocol we systematically searched Medline/PubMed, Scopus and the ISI Web of Science databases to identify randomized controlled trials in which r-hFSH monotherapy protocols were compared with r-hFSH/r-hLH co-treatment in women ≥35 years undergoing fresh IVF cycles. We calculated the pooled odds ratio (OR) for dichotomous data and the weight mean difference (WMD) for continuous data with an associated 95% confidence interval (CI). The meta-analyses were conducted using the random-effect model. P values < 0.05 were considered statistically significant. Subgroup analyses of all primary and secondary outcomes were performed only in women aged 35-40 years. RESULTS: Twelve studies were identified. In women aged between 35 and 40 years, r-hFSH/r-hLH co-treatment was associated with higher clinical pregnancy rates (OR 1.45, CI 95% 1.05-2.00, I2 = 0%, P = 0.03) and implantation rates (OR 1.49, CI 95% 1.10-2.01, I2 = 13%, P = 0.01) versus r-hFSH monotherapy. Fewer oocytes were retrieved in r-hFSH/r-hLH-treated patients than in r-hFSH-treated patients both in women aged ≥35 years (WMD -0.82 CI 95% -1.40 to - 0.24, I2 = 88%, P = 0.005) and in those aged between 35 and 40 years (WMD -1.03, CI - 1.89 to - 0.17, I2 = 0%, P = 0.02). The number of metaphase II oocytes, miscarriage rates and live birth rates did not differ between the two groups of women overall or in subgroup analysis. CONCLUSION: Although more oocytes were retrieved in patients who underwent r-hFSH monotherapy, this meta-analysis suggests that r-hFSH/r-hLH co-treatment improves clinical pregnancy and implantation rates in women between 35 and 40 years of age undergoing ovarian stimulation for assisted reproduction technology. However, more RCTs using narrower age ranges in advanced age women are warranted to corroborate these findings.

Measuring success in IVF is a complex multidisciplinary task: time for a consensus?

The goal of an IVF cycle is the birth of at least one baby per intention to treat. However, IVF cannot confer competence on an embryo, but only can provide each couple with a safe treatment to meet a predetermined chance of success. This commentary highlights how clinical, financial and patient-centred perspectives should be included in the definition of success in IVF. The primary outcome, which is the cumulative live birth delivery rate per intention to treat, must always be complemented by analyses of risks, costs and time invested, as well as by measures of patient satisfaction. Finally, it is essential, whenever clinical conditions exist, to limit treatment discontinuation after failed attempts. Constant monitoring of the data is pivotal and must be adjusted for patient characteristics and compared with national and international registers. The authors aimed to review all these aspects and highlight the points that are still open for discussion. Is it time for a consensus?

Perinatal and obstetric outcomes in singleton pregnancies following fresh versus cryopreserved blastocyst transfer: a meta-analysis.

The transfer of cryopreserved blastocysts is increasing in IVF centres. However, little is known about the perinatal and obstetric outcomes of this procedure. In an attempt to further elucidate these issues, a systematic review and meta-analysis was conducted to compare cryopreserved transfer with fresh blastocyst embryo transfer. The results show that the risk of both preterm (odds ratio [OR] 0.89, 95% confidence interval [CI] 0.80-0.99, P = 0.04) and low birthweight births (OR 0.82, 95% CI 0.68-0.99, P = 0.04) was significantly lower after cryopreserved blastocyst transfer than after fresh blastocyst transfer. The rate of large for gestational age births was significantly higher (OR 1.68, 95% CI 1.55-1.82, P < 0.00001) and the rate of small for gestational age births significantly lower (OR 0.59, 95% CI 0.54-0.65, P < 0.00001) after cryopreserved blastocyst transfer. The transfer of cryopreserved blastocysts was associated with a significantly lower risk of placental abruption (OR 0.58, 95% CI 0.40-0.83, P = 0.003) but a significantly higher risk of Caesarean section (OR 1.21, 95% CI 1.01-1.43, P = 0.03). In conclusion, the perinatal and obstetric outcomes associated with the transfer of cryopreserved blastocysts differ from those associated with fresh blastocyst transfer.

Maternal body mass index associates with blastocyst euploidy and live birth rates: the tip of an iceberg?

RESEARCH QUESTION: Does maternal preconceptional body mass index (BMI) associate with mean blastocyst euploidy rate (m-ER) per patient and live birth rate (LBR) after vitrified-warmed euploid single embryo transfer (SET)? DESIGN: Observational study conducted between April 2013 and March 2020 at a private IVF clinic, involving 1811 Caucasian women undergoing trophectoderm biopsy and comprehensive chromosome testing. The outcomes of 1125 first vitrified-warmed euploid SET were also analysed. Patients were clustered as normal weight (BMI 18.5-25; n = 1392 performing 859 SET), underweight (BMI <18.5; n = 160 performing 112 SET) and overweight (BMI >25; n = 259 performing 154 SET). m-ER per patient was the primary outcome. The secondary outcomes were all clinical outcomes per euploid SET. All data were adjusted for confounders through regression analyses. RESULTS: The m-ER per patient decreases as maternal BMI increases from 17 up to 22-23 before reaching a plateau. A linear regression adjusted for maternal age confirmed this moderate association (unstandardized coefficient B: -0.6%, 95% confidence interval [CI]: -1.1 to -0.1%, P = 0.02). All clinical outcomes were similar between normal weight and underweight women. Overweight women, instead, showed higher miscarriage rate per clinical pregnancy (n = 20/75, 26.7% versus n = 67/461, 14.5%; odds ratio [OR] adjusted for blastocyst quality and day of full blastulation: 2.0, 95% CI: 1.1-3.6, P = 0.01) and lower LBR per SET (n = 55/154, 35.7% versus n = 388/859, 45.2%; OR adjusted for blastocyst quality and day of full blastulation: 0.67, 95% CI: 0.46-0.96, P = 0.03). CONCLUSION: These data indicate a need for future research on more sensitive metrics to assess body fat mass and distribution, as well as on the mechanisms leading to lipotoxicity, thereby impairing embryo competence and/or endometrial receptivity. Overweight women should be informed of their higher risk for miscarriage and, whenever possible, encouraged to lose weight, especially before transfer.

How fixed versus variable gonadotropin dose during controlled ovarian stimulation could influence the management of infertility patients undergoing IVF treatment: a national Delphi consensus.

AIM: Define how and when fixed starting gonadotropin doses can be used in current clinical ART practices in Italy. METHODS: A Delphi conference consisting of three rounds was performed in order to define the ideal clinical conditions in which fixed-gonadotropin-dose during COS should be applied. During the conference, 19 statements about the current ART practice were provided to a panel of twenty-nine national experts. Median score was 5 (IQ:4-6) in all Delphi rounds. RESULTS: Eleven statements (57.9%) were classified as shareable with high-degree of convergence, 2 (10.5%) as shareable with low convergence and 6 (31.6%) as un-shareable with high convergence. The panel reached high consensus regarding some statements: (i) fixed FSH-dose in normoresponders and poor-responder, (ii) importance of predicting ovarian response before COS, considering multiple markers to select the right stimulation protocol for each patient, (iii) importance of therapy simplification and standardization to improve efficiency during COS. Moreover, a low-convergence was reached about use of GnRH antagonist as first treatment line and drug storage at room temperature. However from these findings, the debate remains open regarding some other statements: (a) usefulness of Bologna-criteria to define poor-responders; (b) efficacy to change always stimulation protocol after a failure IVF; (c) utility of AMH-dosed with standardized automatic mode to define normo-responder patients; (d) usefulness to modify the dosage of 12.5 IU/die during COS to improve stimulation effectiveness. CONCLUSION: Controlled ovarian stimulation remains a challenging clinical step in Assisted Reproductive Technique, especially in some specific patient groups for which no clinical consensus is available. This study is the first attempt to describe the shared clinical opinion regarding the fixed versus variable gonadotropin dose in the real IVF practice.

Oocyte competence is independent of the ovulation trigger adopted: a large observational study in a setting that entails vitrified-warmed single euploid blastocyst transfer.

PURPOSE: To assess whether the GnRH-agonist or urinary-hCG ovulation triggers affect oocyte competence in a setting entailing vitrified-warmed euploid blastocyst transfer. METHODS: Observational study (April 2013-July 2018) including 2104 patients (1015 and 1089 in the GnRH-a and u-hCG group, respectively) collecting ≥1 cumulus-oocyte-complex (COC) and undergoing ICSI with ejaculated sperm, blastocyst culture, trophectoderm biopsy, comprehensive-chromosome-testing, and vitrified-warmed transfers at a private clinic. The primary outcome measure was the euploid-blastocyst-rate per inseminated oocytes. The secondary outcome measure was the maturation-rate per COCs. Also, the live-birth-rate (LBR) per transfer and the cumulative-live-birth-delivery-rate (CLBdR) among completed cycles were investigated. All data were adjusted for confounders. RESULTS: The generalized-linear-model adjusted for maternal age highlighted no difference in the mean euploid-blastocyst-rate per inseminated oocytes in either group. The LBR per transfer was similar: 44% (n=403/915) and 46% (n=280/608) in GnRH-a and hCG, respectively. On the other hand, a difference was reported regarding the CLBdR per oocyte retrieval among completed cycles, with 42% (n=374/898) and 25% (n=258/1034) in the GnRh-a and u-hCG groups, respectively. Nevertheless, this variance was due to a lower maternal age and higher number of inseminated oocytes in the GnRH-a group, and not imputable to the ovulation trigger itself (multivariate-OR=1.3, 95%CI: 0.9-1.6, adjusted p-value=0.1). CONCLUSION: GnRH-a trigger is a valid alternative to u-hCG in freeze-all cycles, not only for patients at high risk for OHSS. Such strategy might increase the safety and flexibility of controlled-ovarian-stimulation with no impact on oocyte competence and IVF efficacy.

Definition of a clinical strategy to enhance the efficacy, efficiency and safety of egg donation cycles with imported vitrified oocytes.

STUDY QUESTION: Which is the most suitable clinical strategy in egg donation IVF cycles conducted with imported donated vitrified oocytes? SUMMARY ANSWER: The importation, and allocation, of at least eight vitrified eggs per couple during an egg donation cycle is associated with a high cumulative live birth delivery rate per cycle, as well as the confident adoption of a single blastocyst transfer strategy to minimize the risk of multiple pregnancies. WHAT IS KNOWN ALREADY: IVF using donor eggs is commonly used worldwide to treat women who are unable to conceive with their own oocytes. In 2014, the Constitutional Court (n.162/2014) gave permission for gamete donation to be allowed for ART in Italy. Initially recommended as a therapeutic approach for premature ovarian insufficiency, the use of donated oocytes has become more and more common. In countries such as Italy, fresh oocyte donation is theoretically possible, but practically impossible due to the lack of donors. In fact, the Italian law does not allow reimbursement to the young women, who can only voluntarily donate their eggs. Therefore, Italian IVF centers have established several collaborations with international oocyte cryo-banks. The most popular workflow involves the importation of donated oocytes that have been vitrified. However, recent evidence has questioned the overall efficacy of such an approach. This is because detrimental effects arising from oocyte vitrification and warming might reduce the number of eggs available for insemination, with a consequential reduction in the achievable live birth rate per cycle. STUDY DESIGN, SIZE, DURATION: This was a longitudinal cohort study, conducted between October 2015 and December 2018 at two private IVF centers. Overall, 273 couples were treated (mean maternal age: 42.5 ± 3.5 years, range: 31-50 years; mean donor age: 25.7 ± 4.2, 20-35 years) with oocytes purchased from three different Spanish egg banks. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed an overall analysis, as well as several sub-analyses clustering the data according to the year of treatment (2015-2016, 2017 or 2018), the number of warmed (6, 7, 8 or 9) and surviving oocytes (≤4, 5, 6, 7, 8 or 9) and the cycle strategy adopted (cleavage stage embryo transfer and vitrification, cleavage stage embryo transfer and blastocyst vitrification, blastocyst stage embryo transfer and vitrification). This study aimed to create a workflow to maximize IVF efficacy, efficiency, and safety, during egg donation cycles with imported vitrified oocytes. The primary outcome was the cumulative live birth delivery rate among completed cycles (i.e. cycles where at least a delivery of a live birth was achieved, or no embryo was produced/left to transfer). All cycles, along with their embryological, obstetric and neonatal outcomes, were registered and inspected. MAIN RESULTS AND THE ROLE OF CHANCE: The survival rate after warming was 86 ± 16%. When 6, 7, 8 and 9 oocytes were warmed, 94, 100, 72 and 70% of cycles were completed, resulting in 35, 44, 69 and 59% cumulative live birth delivery rates per completed cycle, respectively. When ≤4, 5, 6, 7, 8 and 9 oocytes survived, 98, 94, 85, 84, 66 and 68% of cycles were completed, resulting in 16, 46, 50, 61, 76 and 60% cumulative live birth delivery rates per completed cycle, respectively. When correcting for donor age, and oocyte bank, in a multivariate logistic regression analysis, warming eight to nine oocytes resulted in an odds ratio (OR) of 2.5 (95% CI: 1.07-6.03, P = 0.03) for the cumulative live birth delivery rate per completed cycle with respect to six to seven oocytes. Similarly, when seven to nine oocytes survived warming, the OR was 2.7 (95% CI: 1.28-5.71, P < 0.01) with respect to ≤6 oocytes. When cleavage stage embryos were transferred, a single embryo transfer strategy was adopted in 17% of cases (N = 28/162); the live birth delivery rate per transfer was 26% (n = 43/162), but among the pregnancies to term, 28% involved twins (n = 12/43). Conversely, when blastocysts were transferred, a single embryo transfer strategy was adopted in 96% of cases (n = 224/234) with a 30% live birth delivery rate per transfer (N = 70/234), and the pregnancies to term were all singleton (n = 70/70). During the study period, 125 babies were born from 113 patients. When comparing the obstetric outcomes for the cleavage and blastocyst stage transfer strategies, the only significant difference was the prevalence of low birthweight: 34 versus 5%, respectively (P < 0.01). However, several significant differences were identified when comparing singleton with twin pregnancies; in fact, the latter resulted in a generally lower birthweight (mean ± SD: 3048 ± 566 g versus 2271 ± 247 g, P < 0.01), a significantly shorter gestation (38 ± 2 versus 36 ± 2 weeks, P < 0.01), solely Caesarean sections (72 versus 100%, P = 0.02), a higher prevalence of low birthweight (8 versus 86%, P < 0.01), small newborns for gestational age (24 versus 57%, P = 0.02) and preterm births (25 versus 86%, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This retrospective study should now be confirmed across several IVF centers and with a greater sample size in order to improve the accuracy of the sub-analyses. WIDER IMPLICATIONS OF THE FINDINGS: Single blastocyst transfer is the most suitable approach to achieve high success rates per procedure, thereby also limiting the obstetric complications that arise from twin pregnancies in oocyte donation programs. In this regard, the larger the cohort of imported donated vitrified oocytes, the more efficient the management of each cycle. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: None.