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Is there any imbalance in the sex ratio at the blastocyst stage of human embryos? And what is the sex ratio in euploid, transferred, implanted blastocysts and at birth?Embryos from 646 women undergoing 921 preimplantation genetic testing for aneuploidy (PGT-A) cycles from September 2017 to February 2020 were included. Data from the chromosomal constitution of 2637 biopsied blastocysts were retrospectively analysed. Trophectoderm samples were analysed by next-generation sequencing. Embryos were categorized as euploid, mosaic or aneuploid. A total of 548 blastocysts diagnosed as euploid were warmed and transferred in a subsequent single-embryo transfer cycle.The blastocyst sex ratio was skewed in favour of male sex with 53.1% (1401/2637) of blastocysts diagnosed as male and 46.9% (1236/2637) as female (sex ratio 1.13, 95% confidence interval [CI] 1.05-1.22). Following biopsy and PGT-A, 41.2% (1086/2637) of blastocysts were classified as euploid, 7.7% (202/2637) as mosaic and 51.2% (1349/2637) as aneuploid. More chromosome euploidy was observed among female than male blastocysts (adjusted odds ratio 1.29, 95% CI 1.08-1.55) after adjusting for female age, male age and gonadotrophin dose. Euploid blastocysts were comparable between the sexes (sex ratio 0.99, 95% CI 0.88-1.11). No significant differences were observed between the sexes in implantation (sex ratio 0.86, 95% CI 0.68-1.08), miscarriage (sex ratio 1, 95% CI 0.51-1.97) or live birth rate (sex ratio 0.85, 95% CI 0.66-1.08).More male than female embryos develop to the blastocyst stage. Male blastocysts exhibit a higher aneuploidy rate. The capacity to implant and lead to a live birth is similar between the sexes.

Background: in 2020, SARS-CoV-2 and the COVID-19 pandemic had a huge impact on the access to and provision of ART treatments. Gradually, knowledge of the virus and its transmission has become available, allowing ART activities to resume. Still, questions on the impact of the virus on human gametes and fertility remain. Onjective and rationale: This article summarizes published data, aiming to clarify the impact of SARS-CoV-2 and the COVID-19 disease on human fertility and assisted reproduction, as well as the impact of vaccination, and from this, provide answers to questions that are relevant for people contemplating pregnancy and for health care professionals. Search/methods: PUBMED/MEDLINE and the WHO COVID-19 database were searched from inception to 5 October 2022 with search terms focusing on 'SARS-CoV-2' and gametes, embryos, reproductive function, fertility and ART. Non-English studies and papers published prior to 2020 were excluded, as well as reviews and non-peer reviewed publications. Full papers were assessed for relevance and quality, where feasible. Outcomes: from the 148 papers included, the following observations were made. The SARS-CoV-2-binding proteins, angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2), are expressed in the testis, but co-expression remains to be proven. There is some evidence of SARS-CoV-2 RNA in the ejaculate of COVID-19 patients with severe disease, but not in those with mild/moderate disease. SARS-CoV-2 infection can impair spermatogenesis, but this seems to resolve after one spermatogenic cycle. Testosterone levels seem to be lower during and after COVID-19, but long-term data are lacking; disease severity may be associated with testosterone levels. COVID-19 cannot be considered a sexually transmitted disease. There is no co-expression of ACE2 and TMPRSS2 in the myometrium, uterus, ovaries or fallopian tubes. Oocytes seem to have the receptors and protease machinery to be susceptible to SARS-CoV-2 infection; however, viral RNA in oocytes has not been detected so far. Women contemplating pregnancy following COVID-19 may benefit from screening for thyroid dysfunction. There is a possible (transient) impact of COVID-19 on menstrual patterns. Embryos, and particularly late blastocysts, seem to have the machinery to be susceptible to SARS-CoV-2 infection. Most studies have not reported a significant impact of COVID-19 on ovarian reserve, ovarian function or follicular fluid parameters. Previous asymptomatic or mild SARS-CoV-2 infection in females does not seem to negatively affect laboratory and clinical outcomes of ART. There are no data on the minimum required interval, if any, between COVID-19 recovery and ART. There is no evidence of a negative effect of SARS-CoV-2 vaccination on semen parameters or spermatogenesis, ovarian function, ovarian reserve or folliculogenesis. A transient effect on the menstrual cycle has been documented. Despite concerns, cross reactivity between anti-SARS-CoV-2 spike protein antibodies and Syncytin-1, an essential protein in human implantation, is absent. There is no influence of mRNA SARS-CoV-2 vaccine on patients' performance during their immediate subsequent ART cycle. Pregnancy rates post-vaccination are similar to those in unvaccinated patients. Wider implication: this review highlights existing knowledge on the impact of SARS-CoV-2 infection or COVID-19 on fertility and assisted reproduction, but also identifies gaps and offers suggestions for future research. The knowledge presented should help to provide evidence-based advice for practitioners and couples contemplating pregnancy alike, facilitating informed decision-making in an environment of significant emotional turmoil., There was no funding for the current paper, apart from technical support from ESHRE.

Background Recently, great efforts have been made to design protocols for obtaining ocular cells from human stem cells to model diseases or for regenerative purposes. Current protocols generally focus on isolating retinal cells, retinal pigment epithelium (RPE), or corneal cells and fail to recapitulate the complexity of the tissue during eye development. Here, the generation of more advanced in vitro multiocular organoids from human induced pluripotent stem cells (hiPSCs) is demonstrated. Methods A 2-step method was established to first obtain self-organized multizone ocular progenitor cells (mzOPCs) from 2D hiPSC cultures within three weeks. Then, after the cells were manually isolated and grown in suspension, 3D multiocular organoids were generated to model important cellular features of developing eyes. Results In the 2D culture, self-formed mzOPCs spanned the neuroectoderm, surface ectoderm, neural crest, and RPE, mimicking early stages of eye development. After lifting, mzOPCs developed into different 3D multiocular organoids composed of multiple cell lineages including RPE, retina, and cornea, and interactions between the different cell types and regions of the eye system were observed. Within these organoids, the retinal regions exhibited correct layering and contained all major retinal cell subtypes as well as retinal morphological cues, whereas the corneal regions closely resembled the transparent ocular-surface epithelium and contained of corneal, limbal, and conjunctival epithelial cells. The arrangement of RPE cells also formed organoids composed of polarized pigmented epithelial cells at the surface that were completely filled with collagen matrix. Conclusions This approach clearly demonstrated the advantages of the combined 2D-3D construction tissue model as it provided a more ocular native-like cellular environment than that of previous models. In this complex preparations, multiocular organoids may be used to model the crosstalk between different cell types in eye development and disease. Graphical abstract

BackgroundThe appearance of bilateral vestibular schwannomas (VS) is one of the most characteristic features of NF2-related schwannomatosis (NF2-related SWN), an autosomal dominant syndrome that predisposes to the development of tumours of the nervous system. VS are caused by the bi-allelic inactivation of the NF2 gene in a cell of the Schwann cell lineage. Our current understanding of VS initiation and progression as well as the development of new effective therapies is hampered by the absence of human non-perishable cell-based models.Principal FindingsWe generated and characterized induced pluripotent stem cell (iPSC) lines with single or bi-allelic inactivation ofNF2by combining the direct reprogramming of VS cells with the use of CRISPR/Cas9 editing. Despite the difficulty of maintaining merlin-deficient iPSCs, we were able to differentiate them into neural crest (NC) cells. At this stage, these cells showed spontaneous expression of the SC marker S100B and the impossibility of generating Schwann cells in 2D cultures. Nevertheless, by applying a 3D Schwann cell differentiation protocol, we successfully generatedNF2(+/-) andNF2(-/-) spheroids homogeneously expressing classical markers of the NC-SC axis.ConclusionsOur results show a critical function ofNF2for both reprograming and maintaining a stable pluripotent state. In addition, merlin-deficient cultures also denoted an altered differentiation capacity of merlin-deficient cells towards the NC-SC axis, in thein vitroconditions used. Finally, the generatedNF2(+/-) andNF2(-/-) spheroids show potential as a genuine in vitro model of NF2-related tumours.

To assess patients' and embryonic characteristics that may have an influence on the decision to transfer a mosaic embryo.Single centre retrospective cohort study including 1247 PGT-A cycles. Demographic and clinical factors associated with a decision to transfer a mosaic embryo were studied. Female age, number of previous cycles, previous availability of euploid embryos, history of miscarriages and parity as well as percentage of mosaicism, type of anomaly and chromosome risk were studied in relation to decision-making. Outcomes after mosaic embryo transfer were assessed.To date, in 7.9% of cycles (99/1247), patients have had to make a decision on the fate of their mosaic embryos. In 23.2% of cycles (23/99), patients decided to transfer. In most cases (79.8%; 79/99), patients underwent genetic counselling before the decision. None of the variables analysed were associated with the patients' decision, although parity and the high-degree mosaicism (50%) seemed to be negatively associated with the decision to transfer (18.2% vs. 29.8%, p = 0.294; 10% vs. 32.2%, p = 0.052).Neither reproductive history nor information on mosaic embryo characteristics through counselling seems to be determinative for patients when deciding to transfer a mosaic embryo. Promising and increasing data on clinical outcomes after mosaic embryo transfer will be of utmost importance to soften risk perception regarding mosaic embryos and give a better, simplified and more evidence-based counselling.

Study question: what is the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the outcome of a pregnancy after medically assisted reproduction (MAR)?. Summary answer: our results suggest that MAR pregnancies are not differentially affected by SARS-CoV-2 infection compared to spontaneous pregnancies. What is known already: information on the effects of coronavirus disease 2019 (COVID-19) on pregnancy after MAR is scarce when women get infected during MAR or early pregnancy, even though such information is vital for informing women seeking pregnancy. Study design, size, duration: data from SARS-CoV-2 affected MAR pregnancies were collected between May 2020 and June 2021 through a voluntary data collection, organised by the European Society of Human Reproduction and Embryology (ESHRE). Participants/materials, setting, methods: all ESHRE members were invited to participate to an online data collection for SARS-CoV-2-infected MAR pregnancies. Main results and the role of chance: the dataset includes 80 cases from 32 countries, including 67 live births, 10 miscarriages, 2 stillbirths and 1 maternal death. An additional 25pregnancies were ongoing at the time of writing. Limitations, reasons for caution: an international data registry based on voluntary contribution can be subject to selective reporting with possible risks of over- or under-estimation. Wider implications of the findings: the current data can be used to guide clinical decisions in the care of women pregnant after MAR, in the context of the COVID-19 pandemic., NA

BACKGROUND The embryo implantation process is crucial for the correct establishment and progress of pregnancy. During implantation, the blastocyst trophectoderm cells attach to the epithelium of the endometrium, triggering intense cell-to-cell crosstalk that leads to trophoblast outgrowth, invasion of the endometrial tissue, and formation of the placenta. However, this process, which is vital for embryo and foetal development in utero, is still elusive to experimentation because of its inaccessibility. Experimental implantation is cumbersome and impractical in adult animal models and is inconceivable in humans. OBJECTIVE AND RATIONALE A number of custom experimental solutions have been proposed to recreate different stages of the implantation process in vitro, by combining a human embryo (or a human embryo surrogate) and endometrial cells (or a surrogate for the endometrial tissue). In vitro models allow rapid high-throughput interrogation of embryos and cells, and efficient screening of molecules, such as cytokines, drugs, or transcription factors, that control embryo implantation and the receptivity of the endometrium. However, the broad selection of available in vitro systems makes it complicated to decide which system best fits the needs of a specific experiment or scientific question. To orient the reader, this review will explore the experimental options proposed in the literature, and classify them into amenable categories based on the embryo/cell pairs employed. The goal is to give an overview of the tools available to study the complex process of human embryo implantation, and explain the differences between them, including the advantages and disadvantages of each system. SEARCH METHODS We performed a comprehensive review of the literature to come up with different categories that mimic the different stages of embryo implantation in vitro, ranging from initial blastocyst apposition to later stages of trophoblast invasion or gastrulation. We will also review recent breakthrough advances on stem cells and organoids, assembling embryo-like structures and endometrial tissues. OUTCOMES We highlight the most relevant systems and describe the most significant experiments. We focus on in vitro systems that have contributed to the study of human reproduction by discovering molecules that control implantation, including hormones, signalling molecules, transcription factors and cytokines. WIDER IMPLICATIONS The momentum of this field is growing thanks to the use of stem cells to build embryo-like structures and endometrial tissues, and the use of bioengineering to extend the life of embryos in culture. We propose to merge bioengineering methods derived from the fields of stem cells and reproduction to develop new systems covering a wider window of the implantation process.

Are intrinsic or extrinsic factors associated with embryo mosaicism prevalence in IVF cycles?Retrospective cohort study of preimplantation genetic testing for aneuploidy (PGT-A) cycles carried out at a university-affiliated IVF clinic between October 2017 and October 2019. Trophectoderm biopsies were analysed by next generation sequencing. Mosaicism prevalence, type of anomaly and the chromosomes involved were analysed. Intrinsic and extrinsic factors potentially inducing mosaicism were studied: maternal and paternal age, antral follicle count, cumulus-oocyte complexes retrieved, female body mass index, PGT-A indication, sperm concentration, total dosage of gonadotrophins, embryo quality and day of blastocyst formation, single-step commercial media used and biopsy operator.Overall prevalence of mosaicism in our PGT-A setting was 13.9%. In segmental mosaicism, larger chromosomes tended to be more affected, which was not observed in whole-chromosome mosaicism. Additionally, segmental mosaicism was mostly observed in monosomy (69.6%; P0.01) compared with whole-chromosome mosaicism (49.7% monosomies versus 50.3% trisomies; P = 0.83). Although a high inter-patient variability was observed, only paternal age showed a positive association with mosaicism (adjusted OR 1.26, 95% CI 1.02 to 1.54) among the analysed variables.Our results suggest remarkable differences in the mechanisms generating segmental and whole-chromosome mosaicism, indicating that they may deserve different consideration when studying them and when prioritizing them for transfer. Male factor seems to be associated with mosaicism and may be worthy of specific assessment in future studies.

Purpose: Microgravity has severe effects on cellular and molecular structures as well as on metabolic interactions. The aim of this study is to investigate the effects of microgravity (µg) exposure on human frozen sperm samples. Methods: Sibling samples from 15 normozoospermic healthy donors were frozen using glycerol as cryoprotectant and analyzed under microgravity and ground conditions. Microgravity was obtained by parabolic flights using a CAP10B plane. The plane executed 20 parabolic maneuvers with a mean of 8.5 seconds of microgravity for each parabola. Results: Frozen sperm samples preserved in cryostraws and stored in a secure and specific nitrogen vapor cryoshipper do not suffer significant alterations after µg exposure. Comparing the study group (µg) and the control group (1g), similar results were obtained in the main parameters studied: Sperm motility (M/ml) 13.72 ± 12.57 vs 13.03±12.13 (-0.69 95% CI [-2.9;1.52]); Progressive a+b sperm motility (%) 13 21.83±11.69 vs 22.54±12.83 (0.03 95% CI [-0.08;0.15]); Sperm vitality (%) 46.42±10.81 vs 44.62±9.34 14 (-0.04 95% CI [-0.13;0.05]); Morphologically normal spermatozoa (%) 7.03±2.61 vs 8.09±3.61 (0.12 15 95% CI [0.01;0.24]); DNA sperm fragmentation by SCD (%) 13.33±5.12 vs 13.88±6.14 (0.03 95% CI [- 16 0.09;0.16]); Apoptotic spermatozoa by MACS (%) 15.47±15.04 vs 23.80±23.63 (-0.21 95% CI [- 17 0.66;1.05]). Conclusion: The lack of differences obtained between frozen samples exposed to µg and those maintained in ground conditions provides the possibility of considering the safe transport of human male gametes to space. Nevertheless, further research is needed to validate the results and to consider the possibility of creating a human sperm bank outside the Earth.

SummaryThe aim of this study was to provide a more comprehensive understanding of 1PN intracytoplasmic sperm injection (ICSI) zygotes. To achieve this objective, we assessed whether all 1PN-derived embryos showed a similar morphokinetic pattern, and if the morphokinetic behaviour of 1PN-derived embryos was comparable with that of 2PN-derived embryos. In total, 149 1PN ICSI zygotes (study group) and 195 2PN ICSI zygotes (control group) were included in the study. Embryo development potential was evaluated in terms of blastocyst rate. Morphokinetic parameters, including the pronucleus diameter and kinetics of in vitro development, were also analyzed. Embryos derived from 1PN ICSI zygotes showed impaired development compared with 2PN-derived embryos, with blastocyst rates of 28.9% and 67.2%, respectively. The diameter of the pronucleus of 1PN zygotes was larger than that of 2PN zygotes. When compared with 2PN-derived embryos, those derived from 1PN zygotes had a visible pronucleus for a shorter time, in addition to a longer syngamy time and slower kinetic behaviour from two to nine cells. When 1PN-derived blastocysts and 2PN-derived blastocysts were compared, the developmental kinetics were similar in both groups, except for a delayed and longer duration of the compaction phase in 1PN-derived embryos. In conclusion, monopronucleated ICSI zygotes present differences in developmental capacity and morphokinetic behaviour compared with 2PN ICSI zygotes, showing particular morphokinetic parameters related to pronucleus formation. Only the 1PN ICSI-derived embryos that reached the blastocyst stage have similar morphokinetic development to blastocysts from 2PN zygotes.

PURPOSE: To determine whether embryo mosaicism prevalence in preimplantation genetic testing for aneuploidy (PGT-A) cycles is associated with the trophectoderm biopsy technique used (a. number of laser pulses or b. the use of flicking or pulling) or the time to tubing. METHODS: Prospective observational study performed in a single IVF-PGT-A setting from May 2019 to May 2021. Trophectoderm biopsies were analysed by next-generation sequencing. Mosaicism was analysed in relation to the biopsy methodology (number of laser pulses and pulling vs flicking), time elapsed from biopsy to tubing (min), and time of sample cryostorage from tubing to amplification (days). As a secondary objective, the number of laser pulses and biopsy methodology were studied in relation to clinical outcomes of transferred euploid blastocysts. RESULTS: None of the analysed variables were associated to mosaicism prevalence. Multivariable regression analysis demonstrated that mosaicism prevalence was comparable either when > 3 laser pulses were used as compared to ≤ 3 (13.9% vs 13.8%, aOR = 0.8726 [0.60–1.28]) and pulling compared to flicking (13.1% vs 14.0%, aOR = 0.86 [0.60–1.23]). Moreover, neither the number of laser pulses during biopsy (> 3 vs ≤ 3) nor the technique used (pulling vs flicking) were associated with clinical pregnancy after the transfer of frozen-thawed euploid blastocysts (54.9% vs 55.2%, aOR = 1.05 [0.53–2.09]; 61.1% vs 52.9%, aOR = 1.11 [0.55–2.25], respectively). CONCLUSION: Our results suggest that, as long as the biopsy and tubing procedures are performed following standardized high quality procedures, no specific approach would increase the generation of artefactual mosaicism as a result of trophectoderm biopsy. Trophectoderm biopsies should be performed regardless of the methodology but always aiming on minimising blastocyst manipulation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-022-02453-9.

STUDY QUESTION Three years after the start of the ESHRE ART Centre Certification (ARTCC) programme, what is the current state of the system, in terms of the interest expressed in it and experiences during the assessment of ART services? SUMMARY ANSWER As of 1 December 2021, 25 European ART centres have been involved in the various stages of certification and the most common recommendations from inspectors were the need for documented training, verification of competencies for all staff members, verification of laboratory and clinical performance indicators, implementation of a quality management system and avoidance of overusing ICSI and add-ons. WHAT IS KNOWN ALREADY European Union (EU) legislation has included ART activities in the EU Tissue and Cells Directives (EUTCDs). Following inspections by national EUTCD authorities, many details regarding documentation, laboratory environment, handling of reproductive cells and tissues, traceability, coding and patient testing have become standardized. However, the EUTCDs do not cover all ART-specific aspects. For this reason, the ARTCC was established to focus on peculiar areas, including relevant staff qualifications, training, continuing professional development, workload, equipment suitability, (non)-evidence-based laboratory and clinical methods used, treatment approaches according to ESHRE guidelines, recommendations and laboratory and clinical key performance indicators. STUDY DESIGN, SIZE, DURATION The article reviews the state-of-the-art of the ESHRE certification of ART centres for good clinical and laboratory practice over an initial 3-year period of operation, including the number of ART centres involved in the different stages of certification and the most common recommendations by inspectors. PARTICIPANTS/MATERIALS, SETTING, METHODS In 2016, the ARTCC working group began to establish a new ESHRE ARTCC programme. Since then, the working group has organized 4 preparatory courses and appointed 37 inspectors (19 clinicians, 17 embryologists and one paramedical). A tool to verify compliance with ESHRE recommendations for good laboratory and clinical practice was developed. The ARTCC has been open for applications since September 2018. In Step 1, the applicant enters basic information about the ART centre, staff and ART activities into the application platform. After review and approval, the applicant is given the opportunity to enter Step 2 and provide detailed online checklists on general, laboratory, clinical services and clinical outcomes. Two inspectors (one clinician and one embryologist) independently evaluate the submitted checklists. The condition to proceed to evaluation is a positive mean score (at least 66%) from each of the four checklists. In Step 3, a live site visit (or virtual owing to the coronavirus disease 2019 (COVID-19) pandemic) is organized and the inspectors prepare a final report with appropriate recommendations. The application may be rejected at any time if the criteria required to advance to the next stage are not met. The ARTCC programme is currently available for European countries listed in ESHRE internal rules, available on the ESHRE website. The certificate is valid for 3 years, after which an application for renewal can be submitted. MAIN RESULTS AND THE ROLE OF CHANCE Over a 3-year period (until 1 December 2021), 63 ART centres from 25 countries started applying through an online platform. So far, 38 applications did not progress owing to lack of completion of the initial application within a 1-year period or because applications came from non-European countries. Of the remaining 25 applications, 8 centres have been inspected and 7 centres have been certified. The most common recommendations given by inspectors to assessed centres were the need for documented training, verification of competencies, skills and continuing professional development for all staff members, verification of laboratory and clinical performance indicators and implementation of a quality management system. The inspectors identified some recurring areas of medically assisted reproduction that deviate from good practice: the overuse of ICSI, preimplantation genetic testing for aneuploidies, freeze-all and other add-ons. They often reported that the clinical outcomes could not be objectively assessed because of non-inclusion of the started cycles or the frequent use of freeze-all cycles. LIMITATIONS, REASONS FOR CAUTION No major modifications have been made to the application platform and checklists since the early stages of the certification programme. However, in this short time, quite a few changes in clinical practice have occurred, especially concerning the more frequent use of the ‘freeze-all’ strategy. As a result, problems arose in the evaluation of clinical outcomes. In addition, because of the COVID-19 pandemic, site visits were substituted by the implementation of virtual visits. While this enabled the certification programme to continue, it is possible that certain critical details that would have been noticed during a traditional site visit may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS Regular monitoring of the observations of ARTCC inspectors and analysis of their reports is certainly useful to harmonize inspectors’ criteria in the assessment process and to identify chronic deficiencies in clinical and laboratory practice. Non-conformities can be addressed by ESHRE through guidelines and recommendations, as well as through discussion with EU institutions and competent authorities. STUDY FUNDING/COMPETING INTEREST(S) The ARTCC programme was developed and funded by ESHRE, covering expenses associated with the meetings. The Steering Committee members who are the authors of this article did not receive payments for the completion of this study. The inspectors were remunerated for their work with an honorarium. The authors have no conflicts of interest to declare.

Modelo animal; Medicina regenerativa; Retina Model animal; Medicina regenerativa; Retina Animal model; Regenerative medicine; Retina Background: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). Methods: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. Results: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. Conclusions: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA. This work was supported by Spanish Ministry of Science and Innovation (MICINN, RTI2018-095377-B-100), Instituto de Salud Carlos III ISCIII/FEDER (PRB2 PT13/0001/0041; TerCel RD16/0011/0024 and Oftared-RETICS RD16/0008), ERA-NET EuroNanoMed III/ISCIII (AC19/00080), the Catalan Government, AGAUR (2017-SGR-899), CERCA Programme/Generalitat de Catalunya, and by Fundació Carmen i Mª José Godó (Fundació CMJ Godó 2017).

Background: The mammalian eye is a complex organ, comprising different highly specialized tissues derived from various cell linages, including neural ectoderm, surface ectoderm, neural crest and the periocular mesenchyme. Great efforts have been made to design protocols for obtaining ocular cells from human stem cells to model diseases or for regenerative purposes, but the complex crosstalk between ocular cell types driving self-organizing growth is usually limited by restricted experimental designs. Current protocols are overall focused on the isolation of retinal, retinal pigment epithelium (RPE) or corneal cells. Here, we obtained multi-ocular organoids from human induced pluripotent stem cells. Methods: We sought to establish a simple method to induce eye field-commitment in 2D hiPSC cultures with the aim of obtaining self-organized multi-zone ocular progenitor cells in 3 weeks. From this starting point, we generated 3D multi-ocular organoids from the same culture, recapitulating important cellular features of the developing eye. Results: Self-formed multi-zone ocular progenitors in 2D culture spanned the neuroectoderm, surface ectoderm, neural crest and RPE. After manual isolation and growth in suspension, they develop into different 3D multi-ocular organoids composed of multiple cell lineages: retinal pigment epithelium, retina and cornea which could be also generated individually. Within these organoids, retinal regions display correct layering and harbor all major retinal cell subtypes as well as retinal morphological cues, whereas corneal regions closely resemble the transparent ocular-surface epithelium with characteristics of corneal, limbal and conjunctival epithelial cells. RPE also arranged to form organoids composed of polarized pigmented epithelial cells at the surface, full-filled with collagen matrix. Conclusions: The multi-ocular organoids offer a new platform to study early human eye development and disease, and provide a source of human ocular cells from the same individual.

Study question Is there any imbalance in the sex ratio (SR) and in the aneuploidy rate of male and female human blastocysts from a PGT-A programme? Summary answer Although SR in human blastocysts is significantly male-biased, more aneuploidies are observed among male blastocysts, resulting in comparable euploid male and female embryos available. What is known already More boys than girls are born worldwide, meaning that the SR at birth is biased towards males. Differences in the SR of children born after ART have been also reported. Factors such as the insemination technique or the day of embryo transfer have been shown to be related to the SR at birth, but whether the SR is shifted during the preimplantation and/or postimplantation development remains unknown. Study design, size, duration: Embryos from patients undergoing 921 PGT-A cycles from September 2017 to February 2020 were included in the study. Data from the chromosomal constitution of 2637 biopsied blastocysts was retrospectively analysed. Participants/materials, setting, methods Embryos were cultured in time-lapse incubators with low oxygen tension (5%) (Embryoscope®; Geri®) using single-step medium (Global®, LifeGlobal®; GTL™, Vitrolife). Blastocyst biopsy was performed between D5-D7 followed by immediate vitrification (Cryotop®, Kitazato). Trophectoderm samples were analysed by NGS. Embryos were categorized as euploid, aneuploid or mosaic. Embryos were called as mosaic when the deviation from the normal copy number was ≥30% and Main results and the role of chance Overall biopsies from 2637 blastocysts were analysed, 1320 on day 5 (50.1%), 1169 on day 6 (44.3%) and 148 on day 7 (5.6%). Sex distribution among the embryos analysed was skewed in favor of male sex with 1401 diagnosed as male (53.1%) and 1236 were female (46.9%), [OR (95%CI):1.13(1.05–1.22)]. As a consequence of this biased SR, more male embryos reached the blastocyst stage and were biopsied both on day 5/6 (708/1320, 53.6% on day 5 and 619/1169, 53% on day 6). Embryos biopsied on day 7 were balanced between sexes with 50% being male and 50% being female. Following biopsy and PGT-A, 1086 (41.2%) of the embryos were classified as euploid, 1349 (51.16%) as aneuploid, and 202 (7.7%) as mosaic embryos. More chromosomal anomalies were observed among male blastocysts when compared to the female ones, 738 (52.7%) vs 611 (49.4%). Similarly, mosaicism was more frequents in male as compared with female blastocysts, 123 (8.8%) vs 79 (6.4%). (P = 0.000). As more aneuploidies are observed among male blastocysts, the final number of available euploid blastocysts for embryo transfer was comparable between sexes (540 male/546 female), [OR (95%CI): 0.99 (0.87–1.11)]. Limitations, reasons for caution This is a retrospective study. Only embryos at the blastocyst stage have been analyzed. Potential confounding factors such as sperm quality or the female age have not been analyzed. No data regarding the SR at birth have been analyzed in these study. Wider implications of the findings: In our study, more male embryos develop to the blastocyst when compared to female ones. It can be hypothesized that female embryos can be more affected by an early arrest at cleavage stages. SR at birth would be expected to be similar as more aneuploidy is observed in male embryos. Trial registration number Not applicable

PURPOSE: To determine the developmental competence of fast-cleaving D3 embryos. METHODS: Retrospective study including 4028 embryos from 513 PGT-A cycles performed between July 2014 and June 2017. Embryos were cultured in time-lapse incubators and biopsied at blastocyst stage. Embryos were classified in groups according to the number of cells on D3 (from 2-cell to ≥13 -cell and compacted). A generalized linear mixed model adjusted for confounding factors was performed to assess the chance to give rise to an euploid blastocyst in each group compared with the chance of 8-cell embryos. Implantation and live birth rates were also analyzed. RESULTS: The statistical analysis showed that embryos with 9 to 11 cells had a slightly lower euploid blastocyst rate than 8-cell embryos (OR (95% CI) 0.77 (0.61–0.96)) while embryos with more than 11 cells were found to be just as likely to give rise to an euploid blastocyst as the 8-cell embryos (OR (95% CI) 1.20 (0.92–1.56)). Conversely, slow-cleaving embryos had a significantly lower euploid blastocyst rate than 8-cell embryos (OR (95% CI) 0.31 (0.24–0.39)). Moreover, euploid blastocysts derived from fast-cleaving embryos and from 8-cell embryos exhibit similar live birth rates. No significant differences were found in the chance to give rise a live birth between 8-cell and 9- to 11-cell embryos (OR (95% CI) 1.23 (0.70–2.15)) and > 11-cell embryos (OR (95% CI) 1.09 (0.57–2.09)). CONCLUSIONS: Embryos with more than 11 cells exhibit similar developmental competence to 8-cell embryos. Their poor prognosis should be reconsidered. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01574-y) contains supplementary material, which is available to authorized users.

Photoreceptor loss is the principal cause of blindness in retinal degenerative diseases (RDDs). Whereas some therapies exist for early stages of RDDs, no effective treatment is currently available for later stages, and once photoreceptors are lost, the only option to rescue vision is cell transplantation. With the use of the Royal College of Surgeons (RCS) rat model of retinal degeneration, we sought to determine whether combined transplantation of human-induced pluripotent stem cell (hiPSC)-derived retinal precursor cells (RPCs) and retinal pigment epithelial (RPE) cells was superior to RPE or RPC transplantation alone in preserving retinal from degeneration. hiPSC-derived RPCs and RPE cells expressing (GFP) were transplanted into the subretinal space of rats. In vivo monitoring showed that grafted cells survived 12 weeks in the subretinal space, and rats treated with RPE + RPC therapy exhibited better conservation of the outer nuclear layer (ONL) and visual response than RPE-treated or RPC-treated rats. Transplanted RPE cells integrated in the host RPE layer, whereas RPC mostly remained in the subretinal space, although a limited number of cells integrated in the ONL. In conclusion, the combined transplantation of hiPSC-derived RPE and RPCs is a potentially superior therapeutic approach to protect retina from degeneration in RDDs., Graphical Abstract, In this article, Anna Duarri and colleagues aim to develop a novel cell-based therapeutic approach to preserve retinal structure and function in retinal degenerative diseases caused by dysfunctional retinal pigment epithelium (RPE). Giving to the eye support of healthy RPE together with healthy retinal cells protects the retina from degeneration

Decellularization; Ocular progenitors; Retina Descelularización; Progenitores oculares; Retina Descel·lularització; Progenitors oculars; Retina The retinal extracellular matrix (ECM) provides architectural support, adhesion and signal guidance that controls retinal development. Decellularization of the ECM affords great potential to tissue engineering; however, how structural retinal ECM affects in vitro development, differentiation and maturation of ocular cells remains to be elucidated. Here, mouse and porcine retinas were decellularized and the protein profile analyzed. Acellular retinal ECM (arECM) scaffolds were then repopulated with human iPSC-derived retinal pigment epithelial (RPE) cells or ocular progenitor cells (OPC) to assess their integration, proliferation and organization. 3837 and 2612 unique proteins were identified in mouse and porcine arECM, respectively, of which 93 and 116 proteins belong to the matrisome. GO analysis shows that matrisome-related proteins were associated with the extracellular region and cell junction and KEGG pathways related to signalling transduction, nervous and endocrine systems and cell junctions were enriched. Interestingly, mouse and porcine arECMs were successfully repopulated with both RPE and OPC, the latter exhibiting cell lineage-specific clusters. Retinal cells organized into different layers containing well-defined areas with pigmented cells, photoreceptors, Müller glia, astrocytes, and ganglion cells, whereas in other areas, conjunctival/limbal, corneal and lens cells re-arranged in cell-specific self-organized areas. In conclusion, our results demonstrated that decellularization of both mouse and porcine retinas retains common native ECM components that upon cell repopulation could guide similar ocular cell adhesion, migration and organization. This work was supported by La Marató de TV3 Foundation (484/C/2012); ERA-NET EuroNanoMed III-AC19/00080/ISCIII (CELLUX); Instituto de Salud Carlos III (CA18/00045 and PI18/00219); and the European Social Fund, the Ministerio de Ciencia, Innovación y Universidades, which is part of the Agencia Estatal de Investigación (PTA2018-016371-I). A.D. was supported by PT13/0001/0041 PRB2-ISCIII-SGEFI-FEDER-PE I+D+i 2013–2016 and ISCIII-FEDER RETICS (Oftared; RD16/0008). We thank the CERCA Programme/Generalitat de Catalunya for institutional support.

Background iPSC (induced pluripotent stem cells) banks of iPSC lines with homozygous HLA (human leukocyte antigen) haplotypes (haplobanks) are proposed as an affordable and off-the-shelf approach to allogeneic transplantation of iPSC derived cell therapies. Cord blood banks offer an extensive source of HLA-typed cells suitable for reprogramming to iPSC. Several initiatives worldwide have been undertaken to create national and international iPSC haplobanks that match a significant part of a population. Methods To create an iPSC haplobank that serves the Spanish population (IPS-PANIA), we have searched the Spanish Bone Marrow Donor Registry (REDMO) to identify the most frequently estimated haplotypes. From the top ten donors identified, we estimated the population coverage using the criteria of zero mismatches in HLA-A, HLA-B, and HLA-DRB1 with different stringencies: high resolution, low resolution, and beneficial mismatch. Results We have calculated that ten cord blood units from homozygous donors stored at the Spanish cord blood banks can provide HLA-A, HLA-B, and HLA-DRB1 matching for 28.23% of the population. Conclusion We confirm the feasibility of using banked cord blood units to create an iPSC haplobank that will cover a significant percentage of the Spanish and international population for future advanced therapy replacement strategies.

Skin fibroblasts were obtained from four patients with Williams-Beuren syndrome (WBS) carrying the typical 1.5 Mb or 1.8 Mb deletion at the 7q11.23 genomic region. Induced pluripotent stem cells (iPSCs) were generated by retroviral infection of fibroblasts with polycystronic vectors. The generated iPSC clones ESi059A, ESi060B and ESi068A had the 1.5 Mb deletion of 7q11.23 and ESi069A the 1.8 Mb, with no novel additional genomic alterations, stable karyotype, expressed pluripotency markers and could differentiate towards the three germ layers in vitro via embryoid body formation and in vivo by teratoma formation. WBS patient's lines are a valuable resource for in vitro modelling of WBS. This work was supported by the grant from Fundación Ramón Areces, the marathon “Todos Somos Raros, Todos Somos Únicos” P52, Spanish RETOS “RTI2018-101960-A-I00”, CERCA Programme / Generalitat de Catalunya; Red de Terapia Celular, TerCel, founded by Retics, RD12/0019/0034; Plataforma de Recursos Biomoleculares y Bioinformaticos, PRB2, PT13/0001/0041; both founded by Instituto de Salud Carlos III-ISCIII/FEDER. RC was recipient of Juan de la Cierva JCI-2012-14404 and Marie Skłodowska-Curie Actions (656359, H2020) and Ramón y Cajal (RYC-2017-21636) fellowships.

The Spanish National Stem Cell Bank (Banco Nacional de Líneas Celulares, BNLC) was established in 2006 thanks to a change in the legislative framework in Spain. The Law 14/2006 updated the previous Assisted Reproduction Techniques Law (Law 45/2003) allowing the use of the surplus frozen embryos following IVF for research. The BNLC has a network structure with 3 nodes: the Regenerative Medicine Program (IDIBELL), the Principe Felipe Research Center (CIPF) in Valencia and the Andalusian Public Health System Biobank (SSPA Biobank) in Granada. The aim of the BNLC is to guarantee throughout the national territory the availability of human stem cell lines for biomedical research. At present time, there are 40 human embryonic stem cell lines (hESC) and 171 human induced pluripotent stem cell lines (hiPSC) registered in the BNLC. These lines are fully characterized and available in the context of research projects approved by the Technical Committee of the BNLC. The National Spanish Stem Cell Bank is supported by the Plataforma de Proteomica, Genotipado y Líneas Celulares (PT1770019/0015) (PRB3), Instituto de Salud Carlos III. Sí

The aim of this study was to provide a more comprehensive understanding of 1PN intracytoplasmic sperm injection (ICSI) zygotes. To achieve this objective, we assessed whether all 1PN-derived embryos showed a similar morphokinetic pattern, and if the morphokinetic behaviour of 1PN-derived embryos was comparable with that of 2PN-derived embryos. In total, 149 1PN ICSI zygotes (study group) and 195 2PN ICSI zygotes (control group) were included in the study. Embryo development potential was evaluated in terms of blastocyst rate. Morphokinetic parameters, including the pronucleus diameter and kinetics of in vitro development, were also analyzed. Embryos derived from 1PN ICSI zygotes showed impaired development compared with 2PN-derived embryos, with blastocyst rates of 28.9% and 67.2%, respectively. The diameter of the pronucleus of 1PN zygotes was larger than that of 2PN zygotes. When compared with 2PN-derived embryos, those derived from 1PN zygotes had a visible pronucleus for a shorter time, in addition to a longer syngamy time and slower kinetic behaviour from two to nine cells. When 1PN-derived blastocysts and 2PN-derived blastocysts were compared, the developmental kinetics were similar in both groups, except for a delayed and longer duration of the compaction phase in 1PN-derived embryos. In conclusion, monopronucleated ICSI zygotes present differences in developmental capacity and morphokinetic behaviour compared with 2PN ICSI zygotes, showing particular morphokinetic parameters related to pronucleus formation. Only the 1PN ICSI-derived embryos that reached the blastocyst stage have similar morphokinetic development to blastocysts from 2PN zygotes.

SummaryShortly after the implementation of comprehensive chromosome screening (CCS) techniques for preimplantation genetic testing for aneuploidies (PGT-A), the discussion about the transition from day 3 to blastocyst stage biopsy was initiated. Trophectoderm biopsy with CCS is meant to overcome the limitations of cleavage-stage biopsy and single-cell analysis. The aim of this study was to assess the results obtained in our PGT-A programme after the implementation of this new strategy. Comparisons between the results obtained in 179 PGT-A cycles with day 3 biopsy (D+3) and fresh embryo transfer, and 204 cycles with trophectoderm biopsy and deferred (frozen–thawed) embryo transfer were established. Fewer embryos were biopsied and a higher euploidy rate was observed in the trophectoderm biopsy group. No differences in implantation (50.3% vs. 61.4%) and clinical pregnancy rate per transfer (56.1% vs. 65.3%) were found. Although the mean number of euploid embryos per cycle did not differ between groups (1.5 ± 1.7 vs. 1.7 ± 1.8), the final number of euploid blastocysts available for transfer per cycle was significantly higher in the trophectoderm biopsy group (1.1 ± 1.3 vs. 1.7 ± 1.8). This factor led to an increased cumulative live birth rate in this last group (34.1% vs. 44.6%). Although both strategies can offer good results, trophectoderm biopsy offers a more robust diagnosis and the intervention is less harmful for the embryos so more euploid blastocysts are finally available for transfer and/or vitrification.

Unambiguous cell line authentication is essential to avoid loss of association between data and cells. The risk for loss of references increases with the rapidity that new human pluripotent stem cell (hPSC) lines are generated, exchanged, and implemented. Ideally, a single name should be used as a generally applied reference for each cell line to access and unify cell-related information across publications, cell banks, cell registries, and databases and to ensure scientific reproducibility. We discuss the needs and requirements for such a unique identifier and implement a standard nomenclature for hPSCs, which can be automatically generated and registered by the human pluripotent stem cell registry (hPSCreg). To avoid ambiguities in PSC-line referencing, we strongly urge publishers to demand registration and use of the standard name when publishing research based on hPSC lines., In this article Kurtz, Seltmann and colleagues propose a standard nomenclature and registry for human pluripotent stem cells. The nomenclature is based on fixed name elements and provides a unique identifier for PSC lines suitable for referencing over diverse data sources, registries, publications, and cell banks. Rigorous application is needed to reduce risks from misidentification and false referencing of lines and data.

Multiple sclerosis (MS) is considered a chronic autoimmune disease of the central nervous system that leads to gliosis, demyelination, axonal damage and neuronal death. The MS disease aetiology is unknown, though a polymorphism of the TNFRSF1A gene, rs1800693, is known to confer an increased risk for MS. Using retroviral delivery of reprogramming transgenes, we generated six MS patient-specific iPSC lines with two distinct genotypes, CC or TT, of the polymorphism rs1800693. iPSC lines had normal karyotype, expressed pluripotency genes and differentiated into the three germ layers. These lines offer a good tool to study MS pathomechanisms and for drug testing.

Resumen El presente estudio describe la implementacion de un test de cribado de portadores de enfermedades geneticas autosomicas recesivas y enfermedades ligadas al cromosoma X en un programa de donacion de ovocitos (donantes de ovocitos y parejas masculinas de las receptoras). El test empleado basado en tecnologia Next-Generation Sequencing (NGS), cubria 200 genes (68 mediante analisis completo de secuencia codificante y 132 por estrategia dirigida de analisis) asociados a 314 enfermedades (277 enfermedades autosomicas recesivas y 22 ligadas al cromosoma X). El resultado obtenido tras 2,5 anos mostro un alto grado de aceptacion (implementacion > 80%). Se identificaron un 56,4% de individuos (761/1.350) portadores de al menos una mutacion patogenica. Asimismo se identifico que el 1,9% de las candidatas a donantes eran portadoras de una enfermedad ligada al cromosoma X por lo que se excluyeron del programa de Donacion de Ovocitos. La carga mutacional media fue de 0,84 mutaciones por muestra. Se identificaron un 3,4% de preasignaciones donante-receptor con alto riesgo reproductivo para alguna enfermedad genetica recesiva (fibrosis quistica, hiperplasia suprarrenal congenita, sordera congenita no-sindromica, alfa-talasemia, fiebre mediterranea familiar, enfermedad de Niemann-Pick, dishormonogenesis tiroidea tipo 6 e hipoplasia de cartilago-pelo). La asignacion definitiva se realizo teniendo en cuenta los resultados de los estudios geneticos. El estado de portador heterocigoto para una enfermedad autosomica recesiva no fue motivo de exclusion del programa de Donacion de Ovocitos, pero implico la seleccion de una receptora cuya pareja masculina no fuera portadora de la misma enfermedad. El asesoramiento genetico de donantes y receptores en las diferentes etapas del proceso clinico de la donacion de ovocitos fue esencial para disminuir la ansiedad, conseguir una buena comprension de los resultados y poder otorgar las recomendaciones pertinentes sobre el riesgo reproductivo propio o de sus familiares, cuando fue necesario.

Resumen Introduccion La multinucleacion es un fenomeno frecuente en los embriones obtenidos por fecundacion in vitro y suele asociarse a un pobre pronostico reproductivo. La monitorizacion embrionaria en sistemas de cultivo time-lapse permite la observacion detallada y continua del desarrollo embrionario. En nuestro trabajo se ha evaluado el impacto de la presencia de multinucleacion en la morfocinetica, la capacidad de desarrollo y la implantacion embrionaria. Material y metodos Estudio retrospectivo de cohorte. Se han incluido 5.540 embriones cultivados en TL (Embryoscope®, Unisense Fertilitech®) procedentes de 763 ciclos de FIV-ICSI realizados durante el periodo 2012-2014. La presencia de multinucleacion se evaluo en todas las etapas del desarrollo. La transferencia embrionaria se realizo en D + 3, y los embriones multinucleados se cultivaron hasta alcanzar el estadio de blastocisto. Resultados Un 23,6% de los embriones mostraron multinucleacion en algun momento del desarrollo embrionario. Los valores de los parametros morfocineticos de los embriones multinucleados y no multinucleados mostraron diferencias estadisticamente significativas. El 18% de los embriones multinucleados alcanzaron el estadio de blastocisto y fueron congelados. No se observo efecto ni del tipo de multinucleacion ni del porcentaje de celulas multinucleadas sobre la tasa de blastocisto. La tasa de implantacion de los blastocistos procedentes de embriones multinucleados fue del 39,1%. Discusion La monitorizacion embrionaria continua permite una mejor deteccion de la multinucleacion. La presencia de multinucleacion embrionaria se asocia a una morfocinetica alterada y una capacidad de desarrollo reducida. No obstante, los embriones multinucleados que alcanzan el estadio de blastocisto muestran una elevada capacidad de implantacion.

A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community.eTOCThe report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.