The overall success of human reproduction, either spontaneously or after IVF, is highly dependent upon maternal age. The main reasons for age-related infertility include reduced ovarian reserve and decreased oocyte/embryo competence due to aging insults, especially concerning an increased incidence of aneuploidies and possibly decreased mitochondrial activity. Age-related chromosomal abnormalities mainly arise because of meiotic impairments during oogenesis, following flawed chromosome segregation patterns such as non-disjunction, premature separation of sister chromatids, or the recent reverse segregation. In this review, we briefly discuss the main mechanisms putatively impaired by aging in the oocytes and the deriving embryos. We also report the main strategies proposed to improve the management of advanced maternal age women in IVF: fertility preservation through oocyte cryopreservation to prevent aging; optimization of the ovarian stimulation and enhancement of embryo selection to limit its effects; and oocyte donation to circumvent its consequences.

How should recurrent implantation failure (RIF) in patients undergoing ART be defined and managed? This is the first ESHRE good practice recommendations paper providing a definition for RIF together with recommendations on how to investigate causes and contributing factors, and how to improve the chances of a pregnancy. RIF is a challenge in the ART clinic, with a multitude of investigations and interventions offered and applied in clinical practice, often without biological rationale or with unequivocal evidence of benefit. This document was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, if available, and the results of a previously published survey on clinical practice in RIF and the expertise of the working group. A literature search was performed in PubMed and Cochrane focussing on ‘recurrent reproductive failure’, ‘recurrent implantation failure’, and ‘repeated implantation failure’. The ESHRE Working Group on Recurrent Implantation Failure included eight members representing the ESHRE Special Interest Groups for Implantation and Early Pregnancy, Reproductive Endocrinology, and Embryology, with an independent chair and an expert in statistics. The recommendations for clinical practice were formulated based on the expert opinion of the working group, while taking into consideration the published data and results of the survey on uptake in clinical practice. The draft document was then open to ESHRE members for online peer review and was revised in light of the comments received. The working group recommends considering RIF as a secondary phenomenon of ART, as it can only be observed in patients undergoing IVF, and that the following description of RIF be adopted: ‘RIF describes the scenario in which the transfer of embryos considered to be viable has failed to result in a positive pregnancy test sufficiently often in a specific patient to warrant consideration of further investigations and/or interventions'. It was agreed that the recommended threshold for the cumulative predicted chance of implantation to identify RIF for the purposes of initiating further investigation is 60%. When a couple have not had a successful implantation by a certain number of embryo transfers and the cumulative predicted chance of implantation associated with that number is greater than 60%, then they should be counselled on further investigation and/or treatment options. This term defines clinical RIF for which further actions should be considered. Nineteen recommendations were formulated on investigations when RIF is suspected, and 13 on interventions. Recommendations were colour-coded based on whether the investigations/interventions were recommended (green), to be considered (orange), or not recommended, i.e. not to be offered routinely (red). While awaiting the results of further studies and trials, the ESHRE Working Group on Recurrent Implantation Failure recommends identifying RIF based on the chance of successful implantation for the individual patient or couple and to restrict investigations and treatments to those supported by a clear rationale and data indicating their likely benefit. This article provides not only good practice advice but also highlights the investigations and interventions that need further research. This research, when well-conducted, will be key to making progress in the clinical management of RIF. The meetings and technical support for this project were funded by ESHRE. N.M. declared consulting fees from ArtPRED (The Netherlands) and Freya Biosciences (Denmark); Honoraria for lectures from Gedeon Richter, Merck, Abbott, and IBSA; being co-founder of Verso Biosense. He is Co-Chief Editor of Reproductive Biomedicine Online (RBMO). D.C. declared being an Associate Editor of Human Reproduction Update, and declared honoraria for lectures from Merck, Organon, IBSA, and Fairtility; support for attending meetings from Cooper Surgical, Fujifilm Irvine Scientific. G.G. declared that he or his institution received financial or non-financial support for research, lectures, workshops, advisory roles, or travelling from Ferring, Merck, Gedeon-Richter, PregLem, Abbott, Vifor, Organon, MSD, Coopersurgical, ObsEVA, and ReprodWissen. He is an Editor of the journals Archives of Obstetrics and Gynecology and Reproductive Biomedicine Online, and Editor in Chief of Journal Gynäkologische Endokrinologie. He is involved in guideline developments and quality control on national and international level. G.L. declared he or his institution received honoraria for lectures from Merck, Ferring, Vianex/Organon, and MSD. He is an Associate Editor of Human Reproduction Update, immediate past Coordinator of Special Interest Group for Reproductive Endocrinology of ESHRE and has been involved in Guideline Development Groups of ESHRE and national fertility authorities. D.J.M. declared being an Associate Editor for Human Reproduction Open and statistical Advisor for Reproductive Biomedicine Online. B.T. declared being shareholder of Reprognostics and she or her institution received financial or non-financial support for research, clinical trials, lectures, workshops, advisory roles or travelling from support for attending meetings from Ferring, MSD, Exeltis, Merck Serono, Bayer, Teva, Theramex and Novartis, Astropharm, Ferring. The other authors had nothing to disclose. This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation. ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type. Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE.

Advanced maternal age (AMA; >35 year) is associated with a decline in both ovarian reserve and oocyte competence. At present, no remedies are available to counteract the aging-related fertility decay, however different therapeutic approaches can be offered to women older than 35 year undergoing IVF. This review summarizes the main current strategies proposed for the treatment of AMA: (i) oocyte cryopreservation to conduct fertility preservation for medical reasons or “social freezing” for non-medical reasons, (ii) personalized controlled ovarian stimulation to maximize the exploitation of the ovarian reserve in each patient, (iii) enhancement of embryo selection via blastocyst-stage preimplantation genetic testing for aneuploidies and frozen single embryo transfer, or (iv) oocyte donation in case of minimal/null residual chance of pregnancy. Future strategies and tools are in the pipeline that might minimize the risks of AMA through non-invasive approaches for embryo selection (e.g., molecular analyses of leftover products of IVF, such as spent culture media). These are yet challenging but potentially ground-breaking perspectives promising a lower clinical workload with a higher cost-effectiveness. We also reviewed emerging experimental therapeutic approaches to attempt at restoring maternal reproductive potential, e.g., spindle-chromosomal complex, pronuclear or mitochondrial transfer, and chromosome therapy. In vitro generation of gametes is also an intriguing challenge for the future. Lastly, since infertility is a social issue, social campaigns, and education among future generations are desirable to promote the awareness of the impact of age and lifestyle habits upon fertility. This should be a duty of the clinical operators in this field.

Folliculogenesis occurs in the highly dynamic environment of the ovary. Follicle cyclic recruitment, neo-angiogenesis, spatial displacement, follicle atresia and ovulation stand out as major events resulting from the interplay between mechanical forces and molecular signals. Morphological and functional changes to the growing follicle and to the surrounding tissue are required to produce oocytes capable of supporting preimplantation development to the blastocyst stage. This review will summarize the ovarian morphological and functional context that contributes to follicle recruitment, growth and ovulation, as well as to the acquisition of oocyte developmental competence. We will describe the changes occurring during folliculogenesis to the ovarian extracellular matrix (ECM) and to the vasculature, their influence on the mechanical properties of the ovarian tissue, and, in turn, their influence on the regulation of signal transduction. Also, we will outline how their dysregulation might be associated with pathologies such as polycystic ovary syndrome (PCOS), endometriosis or premature ovarian insufficiency (POI). Finally, for each of these three pathologies, we will highlight therapeutic strategies attempting to correct the altered biomechanical context in order to restore fertility. For each area discussed, a systematic bibliographical search was performed, without temporal limits, using PubMed Central, Web of Science and Scopus search engines employing the keywords extracellular matrix, mechanobiology, biomechanics, vasculature, angiogenesis or signalling pathway in combination with: ovary, oogenesis, oocyte, folliculogenesis, ovarian follicle, theca, granulosa, cumulus, follicular fluid, corpus luteum, meiosis, oocyte developmental competence, preimplantation, polycystic ovary syndrome, premature ovarian insufficiency or endometriosis. Through search engines queries, we yielded a total of 37 368 papers that were further selected based on our focus on mammals and, specifically, on rodents, bovine, equine, ovine, primates and human, and also were trimmed around each specific topic of the review. After the elimination of duplicates, this selection process resulted in 628 papers, of which 287 were cited in the manuscript. Among these, 89.2% were published in the past 22 years, while the remaining 8.0%, 2.4% or 0.3% were published during the 1990s, 1980s or before, respectively. During folliculogenesis, changes occur to the ovarian ECM composition and organization that, together with vasculature modelling around the growing follicle, are aimed to sustain its recruitment and growth, and the maturation of the enclosed oocyte. These events define the scenario in which mechanical forces are key to the regulation of cascades of molecular signals. Alterations to this context determine impaired folliculogenesis and decreased oocyte developmental potential, as observed in pathological conditions which are causes of infertility, such as PCOS, endometriosis or POI. The knowledge of these mechanisms and the rules that govern them lay a sound basis to explain how follicles recruitment and growth are modulated, and stimulate insights to develop, in clinical practice, strategies to improve follicular recruitment and oocyte competence, particularly for pathologies like PCOS, endometriosis and POI. Alterations of the ovarian ECM, vasculature and biomechanical properties have implications for fertility.

Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential.

A normal chromosomal constitution defined through PGT-A assessing all chromosomes on trophectoderm (TE) biopsies represents the strongest predictor of embryo implantation. Yet, its positive predictive value is not higher than 50–60%. This gap of knowledge on the causes of euploid blastocysts’ reproductive failure is known as ‘the black box of implantation’. Several embryonic, maternal, paternal, clinical, and IVF laboratory features were scrutinized for their putative association with reproductive success or implantation failure of euploid blastocysts. A systematic bibliographical search was conducted without temporal limits up to August 2021. The keywords were ‘(blastocyst OR day5 embryo OR day6 embryo OR day7 embryo) AND (euploid OR chromosomally normal OR preimplantation genetic testing) AND (implantation OR implantation failure OR miscarriage OR abortion OR live birth OR biochemical pregnancy OR recurrent implantation failure)’. Overall, 1608 items were identified and screened. We included all prospective or retrospective clinical studies and randomized-controlled-trials (RCTs) that assessed any feature associated with live-birth rates (LBR) and/or miscarriage rates (MR) among non-mosaic euploid blastocyst transfer after TE biopsy and PGT-A. In total, 41 reviews and 372 papers were selected, clustered according to a common focus, and thoroughly reviewed. The PRISMA guideline was followed, the PICO model was adopted, and ROBINS-I and ROB 2.0 scoring were used to assess putative bias. Bias across studies regarding the LBR was also assessed using visual inspection of funnel plots and the trim and fill method. Categorical data were combined with a pooled-OR. The random-effect model was used to conduct the meta-analysis. Between-study heterogeneity was addressed using I². Whenever not suitable for the meta-analysis, the included studies were simply described for their results. The study protocol was registered at http://www.crd.york.ac.uk/PROSPERO/ (registration number CRD42021275329). We included 372 original papers (335 retrospective studies, 30 prospective studies and 7 RCTs) and 41 reviews. However, most of the studies were retrospective, or characterized by small sample sizes, thus prone to bias, which reduces the quality of the evidence to low or very low. Reduced inner cell mass (7 studies, OR: 0.37, 95% CI: 0.27–0.52, I² = 53%), or TE quality (9 studies, OR: 0.53, 95% CI: 0.43–0.67, I² = 70%), overall blastocyst quality worse than Gardner’s BB-grade (8 studies, OR: 0.40, 95% CI: 0.24–0.67, I² = 83%), developmental delay (18 studies, OR: 0.56, 95% CI: 0.49–0.63, I² = 47%), and (by qualitative analysis) some morphodynamic abnormalities pinpointed through time-lapse microscopy (abnormal cleavage patterns, spontaneous blastocyst collapse, longer time of morula formation I, time of blastulation (tB), and duration of blastulation) were all associated with poorer reproductive outcomes. Slightly lower LBR, even in the context of PGT-A, was reported among women ≥38 years (7 studies, OR: 0.87, 95% CI: 0.75–1.00, I² = 31%), while obesity was associated with both lower LBR (2 studies, OR: 0.66, 95% CI: 0.55–0.79, I² = 0%) and higher MR (2 studies, OR: 1.8, 95% CI: 1.08–2.99, I² = 52%). The experience of previous repeated implantation failures (RIF) was also associated with lower LBR (3 studies, OR: 0.72, 95% CI: 0.55–0.93, I² = 0%). By qualitative analysis, among hormonal assessments, only abnormal progesterone levels prior to transfer were associated with LBR and MR after PGT-A. Among the clinical protocols used, vitrified-warmed embryo transfer was more effective than fresh transfer (2 studies, OR: 1.56, 95% CI: 1.05–2.33, I² = 23%) after PGT-A. Lastly, multiple vitrification-warming cycles (2 studies, OR: 0.41, 95% CI: 0.22–0.77, I² = 50%) or (by qualitative analysis) a high number of cells biopsied may slightly reduce the LBR, while simultaneous zona-pellucida opening and TE biopsy allowed better results than the Day 3 hatching-based protocol (3 studies, OR: 1.41, 95% CI: 1.18–1.69, I² = 0%). Embryo selection aims at shortening the time-to-pregnancy, while minimizing the reproductive risks. Knowing which features are associated with the reproductive competence of euploid blastocysts is therefore critical to define, implement, and validate safer and more efficient clinical workflows. Future research should be directed towards: (i) systematic investigations of the mechanisms involved in reproductive aging beyond de novo chromosomal abnormalities, and how lifestyle and nutrition may accelerate or exacerbate their consequences; (ii) improved evaluation of the uterine and blastocyst-endometrial dialogue, both of which represent black boxes themselves; (iii) standardization/automation of embryo assessment and IVF protocols; (iv) additional invasive or preferably non-invasive tools for embryo selection. Only by filling these gaps we may finally crack the riddle behind ‘the black box of implantation’. Opening the black box of implantation: low blastocyst quality and maternal aging, obesity or repeated implantation failures (RIF), as well as poor or excessive embryo manipulations may reduce the live birth rate per euploid blastocyst transfer.