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.

Incidence, Origin, and Predictive Model for the Detection and Clinical Management of Segmental Aneuploidies in Human Embryos.

Despite next-generation sequencing, which now allows for the accurate detection of segmental aneuploidies from in vitro fertilization embryo biopsies, the origin and characteristics of these aneuploidies are still relatively unknown. Using a multifocal biopsy approach (four trophectoderms [TEs] and one inner cell mass [ICM] analyzed per blastocyst; n = 390), we determine the origin of the aneuploidy and the diagnostic predictive value of segmental aneuploidy detection in TE biopsies toward the ICM's chromosomal constitution. Contrary to the prevalent meiotic origin of whole-chromosome aneuploidies, we show that sub-chromosomal abnormalities in human blastocysts arise from mitotic errors in around 70% of cases. As a consequence, the positive-predictive value toward ICM configuration was significantly lower for segmental as compared to whole-chromosome aneuploidies (70.8% versus 97.18%, respectively). In order to enhance the clinical utility of reporting segmental findings in clinical TE biopsies, we have developed and clinically verified a risk stratification model based on a second TE biopsy confirmation and segmental length; this model can significantly improve the prediction of aneuploidy risk in the ICM in over 86% of clinical cases enrolled. In conclusion, we provide evidence of the predominant mitotic origin of segmental aneuploidies in preimplantation embryos and develop a risk stratification model that can help post-test genetic counseling and that facilitates the decision-making process on clinical utilization of these embryos.

The first mitotic division: a perilous bridge connecting the zygote and the early embryo.

Human embryos are very frequently affected by maternally inherited aneuploidies, which in the vast majority of cases determine developmental failure at pre- or post-implantation stages. However, recent evidence, generated by the alliance between diverse technologies now routinely employed in the IVF laboratory, has revealed a broader, more complex scenario. Aberrant patterns occurring at the cellular or molecular level can impact at multiple stages of the trajectory of development to blastocyst. In this context, fertilization is an extremely delicate phase, as it marks the transition between gametic and embryonic life. Centrosomes, essential for mitosis, are assembled ex novo from components of both parents. Very large and initially distant nuclei (the pronuclei) are brought together and positioned centrally. The overall cell arrangement is converted from being asymmetric to symmetric. The maternal and paternal chromosome sets, initially separate and scattered within their respective pronuclei, become clustered where the pronuclei juxtapose, to facilitate their assembly in the mitotic spindle. The meiotic spindle is replaced by a segregation machinery that may form as a transient or persistent dual mitotic spindle. Maternal proteins assist the decay of maternal mRNAs to allow the translation of newly synthesized zygotic transcripts. The diversity and complexity of these events, regulated in a precise temporal order and occurring in narrow time windows, make fertilization a highly error-prone process. As a consequence, at the first mitotic division, cellular or genomic integrity may be lost, with fatal consequences for embryonic development.

Discard or not discard, that is the question: an international survey across 117 embryologists on the clinical management of borderline quality blastocysts.

STUDY QUESTION: Do embryologists from different European countries agree on embryo disposition decisions ('use' or 'discard') about Day 7 (>144 h post-insemination) and/or low-quality blastocysts (LQB;

Maternal age affects pronuclear and chromatin dynamics, morula compaction and cell polarity, and blastulation of human embryos.

STUDY QUESTION: Does maternal ageing impact early and late morphokinetic and cellular processes of human blastocyst formation? SUMMARY ANSWER: Maternal ageing significantly affects pronuclear size and intra- and extra-nuclear dynamics during fertilization, dysregulates cell polarity during compaction, and reduces blastocoel expansion. WHAT IS KNOWN ALREADY: In ART, advanced maternal age (AMA) affects oocyte yield, fertilization, and overall developmental competence. However, with the exception of chromosome segregation errors occurring during oocyte meiosis, the molecular and biochemical mechanisms responsible for AMA-related subfertility and reduced embryo developmental competence remain unclear. In particular, studies reporting morphokinetics and cellular alterations during the fertilization and pre-implantation period in women of AMA remain limited. STUDY DESIGN, SIZE, DURATION: A total of 2058 fertilized oocytes were stratified by maternal age according to the Society for Assisted Reproductive Technology classification (<35, 35-37, 38-40, 41-42, and >42 years) and retrospectively analysed. AMA effects were assessed in relation to: embryo morphokinetics and morphological alterations; and the presence and distribution of cell polarity markers-Yes-associated protein (YAP) and protein kinase C-ζ (PKC-ζ)-involved in blastocyst morphogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1050 cycles from 1050 patients met the inclusion criteria and were analysed. Microinjected oocytes were assessed using a time-lapse culture system. Immature oocytes at oocyte retrieval and mature oocytes not suitable for time-lapse monitoring, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared among groups. Furthermore, 20 human embryos donated for research by consenting couples were used for immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: Static microscopic observation revealed that blastocyst formation and expansion were impaired in the 41-42 and >42-year groups (P < 0.0001). The morphological grades of the inner cell mass and trophectoderm were poorer in the >42-year group than those in the <35-year group (P = 0.0022 and P < 0.0001, respectively). Time-lapse microscopic observation revealed a reduction in nucleolus precursor body alignment in female pronuclei in the 41-42 and >42-year groups (P = 0.0010). Female pronuclear area decreased and asynchronous pronuclear breakdown increased in the >42-year group (P = 0.0027 and P < 0.0122, respectively). Developmental speed at cleavage stage, incidence of irregularity of first cleavage, type and duration of blastomere movement, and number of multinucleated cells were comparable among age groups. Delayed embryonic compaction and an increased number of extruded blastomeres were observed in the >42-year group (P = 0.0002 and P = 0.0047, respectively). Blastulation and blastocyst expansion were also delayed in the 41-42 and >42-year groups (P < 0.0001 for both). YAP positivity rate in the outer cells of morulae and embryo PKC-ζ immunoflourescence decreased in the >42-year group (P < 0.0001 for both). LIMITATIONS, REASONS FOR CAUTION: At the cellular level, the investigation was limited to cell polarity markers. Cell components of other developmental pathways should be studied in relation to AMA. WIDER IMPLICATIONS OF THE FINDINGS: The study indicates that maternal ageing affects the key functions of embryo morphogenesis, irrespective of the well-established influence on the fidelity of oocyte meiosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

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.

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.

Humidified atmosphere in a time-lapse embryo culture system does not improve ongoing pregnancy rate: a retrospective propensity score model study derived from 496 first ICSI cycles.

PURPOSE: To investigate whether high relative humidity conditions (HC), when using a time-lapse system (TLS) with sequential culture media, are beneficial to embryo culture, improving ongoing pregnancy rates. METHODS: We included patients undergoing their first ICSI cycle treatment from April 2021 to May 2022. Patients assigned to dry conditions (DC) or HC were 278 and 218, respectively. We used a GERI TLS, three chambers configured in humidity conditions and three in dry conditions. The effect of HC on ongoing pregnancy rate was assessed by the propensity matched sample, to reduce potential differences between women undergoing either HC or DC and reduce biased estimation of treatment effect. RESULT: After adjusting for several confounding variables and applying the propensity score (PS), no significant differences were observed in the rates of normal (2PN) and abnormal (1PN and 3PN) fertilization, blastulation, top-quality blastocysts, frozen blastocysts, ongoing pregnancies, and miscarriages. The 2-cell (t2) and 4-cell (t4) stages and cell divisions between such stages occurred earlier and were more synchronous in the in DC. CONCLUSION: These results suggest that HC conditions do not improve the rate of ongoing pregnancy and several embryological outcomes, under the conditions used in this study based on a time-lapse system and sequential culture with day 3 medium change-over.

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.

Human blastocyst spontaneous collapse is associated with worse morphological quality and higher degeneration and aneuploidy rates: a comprehensive analysis standardized through artificial intelligence.

STUDY QUESTION: What are the factors associated with human blastocyst spontaneous collapse and the consequences of this event? SUMMARY ANSWER: Approximately 50% of blastocysts collapsed, especially when non-viable, morphologically poor and/or aneuploid. WHAT IS KNOWN ALREADY: Time-lapse microscopy (TLM) is a powerful tool to observe preimplantation development dynamics. Lately, artificial intelligence (AI) has been harnessed to automate and standardize such observations. Here, we adopted AI to comprehensively portray blastocyst spontaneous collapse, namely the phenomenon of reduction in size of the embryo accompanied by efflux of blastocoel fluid and the detachment of the trophectoderm (TE) from the zona pellucida (ZP). Although the underlying causes are unknown, blastocyst spontaneous collapse deserves attention as a possible marker of reduced competence. STUDY DESIGN, SIZE, DURATION: An observational study was carried out, including 2348 TLM videos recorded during preimplantation genetic testing for aneuploidies (PGT-A, n = 720) cycles performed between January 2013 and December 2020. All embryos in the analysis at least reached the time of starting blastulation (tSB), 1943 of them reached full expansion, and were biopsied and then vitrified. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI, blastocyst culture, TE biopsy without Day 3 ZP drilling, comprehensive chromosome testing and vitrification were performed. The AI software automatically registered tSB and time of expanding blastocyst (tEB), start and end time of each collapse, time between consecutive collapses, embryo proper area, percentage of shrinkage, embryo:ZP ratio at embryo collapse, time of biopsy (t-biopsy) and related area of the fully (re-)expanded blastocyst before biopsy, time between the last collapse and biopsy. Blastocyst morphological quality was defined according to both Gardner's criteria and an AI-generated implantation score. Euploidy rate per biopsied blastocyst and live birth rate (LBR) per euploid single embryo transfer (SET) were the main outcomes. All significant associations were confirmed through regression analyses. All couple, cycle and embryo main features were also investigated for possible associations with blastocyst spontaneous collapse. MAIN RESULTS AND THE ROLE OF CHANCE: At least one collapsing embryo (either viable or subsequently undergoing degeneration) was recorded in 559 cycles (77.6%) and in 498 cycles (69.2%) if considering only viable blastocysts. The prevalence of blastocyst spontaneous collapse after the tSB, but before the achievement of full expansion, was 50% (N = 1168/2348), irrespective of cycle and/or couple characteristics. Blastocyst degeneration was 13% among non-collapsing embryos, while it was 18%, 20%, 26% and 39% among embryos collapsing once, twice, three times or ≥4 times, respectively. The results showed that 47.3% (N = 918/1943) of the viable blastocysts experienced at least one spontaneous collapse (ranging from 1 up to 9). Although starting from similar tSB, the number of spontaneous collapses was associated with a delay in both tEB and time of biopsy. Of note, the worse the quality of a blastocyst, the more and the longer its spontaneous collapses. Blastocyst spontaneous collapse was significantly associated with lower euploidy rates (47% in non-collapsing and 38%, 32%, 31% and 20% in blastocysts collapsing once, twice, three times or ≥4 times, respectively; multivariate odds ratio 0.78, 95%CI 0.62-0.98, adjusted P = 0.03). The difference in the LBR after euploid vitrified-warmed SET was not significant (46% and 39% in non-collapsing and collapsing blastocysts, respectively). LIMITATIONS, REASONS FOR CAUTION: An association between chromosomal mosaicism and blastocyst collapse cannot be reliably assessed on a single TE biopsy. Gestational and perinatal outcomes were not evaluated. Other culture strategies and media should be tested for their association with blastocyst spontaneous collapse. Future studies with a larger sample size are needed to investigate putative impacts on clinical outcomes after euploid transfers. WIDER IMPLICATIONS OF THE FINDINGS: These results demonstrate the synergistic power of TLM and AI to increase the throughput of embryo preimplantation development observation. They also highlight the transition from compaction to full blastocyst as a delicate morphogenetic process. Blastocyst spontaneous collapse is common and associates with inherently lower competence, but additional data are required to deepen our knowledge on its causes and consequences. STUDY FUNDING/COMPETING INTEREST(S): There is no external funding to report. I.E., A.B.-M., I.H.-V. and B.K. are Fairtility employees. I.E. and B.K. also have stock or stock options of Fairtility. TRIAL REGISTRATION NUMBER: N/A.

Inter-centre reliability in embryo grading across several IVF clinics is limited: implications for embryo selection.

RESEARCH QUESTION: What is the intra- and inter-centre reliability in embryo grading performed according to the Istanbul Consensus across several IVF clinics? DESIGN: Forty Day 3 embryos and 40 blastocysts were photographed on three focal planes. Senior and junior embryologists from 65 clinics were invited to grade them according to the Istanbul Consensus (Study Phase I). All participants then attended interactive training where a panel of experts graded the same embryos (Study Phase II). Finally, a second set of pictures was sent to both embryologists and experts for a blinded evaluation (Study Phase III). Intra-centre reliability was reported for Study Phase I as Cohen's kappa between senior and junior embryologists; inter-centre reliability was instead calculated between senior/junior embryologists and experts in Study Phase I versus III to outline improvements after training (i.e. upgrade of Cohen's kappa category according to Landis and Koch). RESULTS: Thirty-six embryologists from 18 centres participated (28% participation rate). The intra-centre reliability was (i) substantial (0.63) for blastomere symmetry (range -0.02 to 1.0), (ii) substantial (0.72) for fragmentation (range 0.29-1.0), (iii) substantial (0.66) for blastocyst expansion (range 0.19-1.0), (iv) moderate (0.59) for inner cell mass quality (range 0.07-0.92), (v) moderate (0.56) for trophectoderm quality (range 0.01-0.97). The inter-centre reliability showed an overall improvement from Study Phase I to III, from fair (0.21-0.4) to moderate (0.41-0.6) for all parameters under analysis, except for blastomere fragmentation among senior embryologists, which was already moderate before training. CONCLUSIONS: Intra-centre reliability was generally moderate/substantial, while inter-centre reliability was just fair. The interactive training improved it to moderate, hence this workflow was deemed helpful. The establishment of external quality assessment services (e.g. UK NEQAS) and the avant-garde of artificial intelligence might further improve the reliability of this key practice for embryo selection.

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.

Maternal effect factors that contribute to oocytes developmental competence: an update.

Oocyte developmental competence is defined as the capacity of the female gamete to be fertilized and sustain development to the blastocyst stage. Epigenetic reprogramming, a correct cell division pattern, and an efficient DNA damage response are all critical events that, before embryonic genome activation, are governed by maternally inherited factors such as maternal-effect gene (MEG) products. Although these molecules are stored inside the oocyte until ovulation and exert their main role during fertilization and preimplantation development, some of them are already functioning during folliculogenesis and oocyte meiosis resumption. This mini review summarizes the crucial roles played by MEGs during oocyte maturation, fertilization, and preimplantation development with a direct/indirect effect on the acquisition or maintenance of oocyte competence. Our aim is to inspire future research on a topic with potential clinical perspectives for the prediction and treatment of female infertility.

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.

Educational needs of fertility healthcare professionals using ART: a multi-country mixed-methods study.

RESEARCH QUESTION: What are the most pressing educational needs of fertility healthcare professionals using assisted reproductive technologies (ART)? DESIGN: This mixed-methods study combined qualitative interviews with quantitative surveys. Participants included physicians and nurses specialized in reproductive endocrinology or in obstetrics/gynaecology, and laboratory specialists, with a minimum of 3 years of experience, practising in Australia, Brazil, Canada, China, France, Germany, India, Italy, Japan, Mexico, Spain or the UK. Maximum variation purposive sampling was used to ensure a mix of experience and settings. Interviews were transcribed and coded through thematic analysis. Quantitative data were analysed using frequency tables, cross-tabulations and chi-squared tests to compare results by reimbursement context. RESULTS: A total of 535 participants were included (273 physicians, 145 nurses and 117 laboratory specialists). Knowledge gaps, skills gaps and attitude issues were identified in relation to: (i) ovarian stimulation (e.g. knowledge of treatments and instruction protocols for ovarian stimulation), (ii) embryo culture and cryopreservation/vitrification (e.g. diverging opinions on embryo freezing, (iii) embryo assessment (e.g. performing genetic testing), (iv) support of luteal phase and optimizing pregnancy outcomes (e.g. knowledge of assessment methods for endometrial receptivity), and (v) communication with patients (e.g. reluctance to address emotional distress). CONCLUSIONS: This descriptive, exploratory study corroborates previously reported gaps in fertility care and identifies potential causes of these gaps. Findings provide evidence to inform educational programmes for healthcare professionals who use ART in their practice and calls for the development of case-based education and interprofessional training programmes to improve care for patients with fertility issues.

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?

Blastulation rates of sibling oocytes in two IVF culture media: an evidence-based workflow to implement newly commercialized products.

RESEARCH QUESTION: An evidence-based novel commercially available continuous IVF culture medium in compliance with an efficient quality-management system is proposed. DESIGN: Non-interventional study on sibling oocytes. Intracytoplasmic sperm injection cycles among women aged 42 years or younger that used ejaculated spermatozoa and retrieved four to eight oocytes were included. Sibling oocytes were randomized for culture in the novel Geri-medium or continuous single culture medium (CSCM). Primary outcome measure was blastulation rate per cohort of inseminated oocytes; 1182 oocytes were required to outline down to a 7% difference (power = 80%). RESULTS: A total of 181 cohorts of sibling oocytes were included. Geri-medium (n = 631 oocytes) and CSCM (n = 643 oocytes) resulted in similar blastulation rates (mean ± SD: 42.8% ± 30.1% versus 43.1% ± 29.0%; Wilcoxon signed rank test = 0.77). Blastocysts cultured in the former (n = 275 versus n = 277) showed longer timings during preimplantation development (P < 0.01) and were poorer quality (26% versus 18%; P = 0.03). Euploidy rate was no different in cycles that underwent preimplantation genetic testing for aneuploidy (n = 113) (117/237 [49%] versus 117/249 blastocysts [47%]; P = 0.6). Ongoing implantation rate was comparable in the study arms after euploid (29/47 [63%] versus 14/ 34 [41%]; P = 0.1) or untested (12/31 [39%] versus 7/18 [39%]; P = 0.3) transfers. CONCLUSION: Blastulation rate among cohorts of sibling oocytes cultured in the same incubator is a fast, reliable and comprehensive performance indicator to validate novel commercially available culture medium. The media tested were considered similarly efficient. The differences in blastocyst morphology and developmental timings warrant further investigation, although euploidy and ongoing implantation rates were similar.