Mitochondrial DNA (mtDNA) diseases pose unique challenges for genetic counselling and require tailored approaches to address recurrence risks and reproductive options. The intricate dynamics of mtDNA segregation and heteroplasmy shift significantly impact the chances of having affected children. In addition to natural pregnancy, oocyte donation, and adoption, IVF-based approaches can reduce the risk of disease transmission. Prenatal diagnosis (PND) and preimplantation genetic testing (PGT) remain the standard methods for women carrying pathogenic mtDNA mutations; nevertheless, they are not suitable for every patient. Germline nuclear transfer (NT) has emerged as a novel therapeutic strategy, while mitochondrial gene editing has increasingly become a promising research area in the field. However, challenges and safety concerns associated with all these techniques remain, highlighting the need for long-term follow-up studies, an improved understanding of disease mechanisms, and personalized approaches to diagnosis and treatment. Given the inherent risks of adverse maternal and child outcomes, careful consideration of the balance between potential benefits and drawbacks is also warranted. This review will provide critical insights, identify knowledge gaps, and underscore the importance of advancing mitochondrial disease research in reproductive health.

PURPOSE OF THE REVIEW:Aneuploidy is a major cause of embryonic arrest. While meiotic aneuploidies, especially maternal, are a well-documented cause of embryo and fetal arrest, increasing evidence highlights the crucial role played by mitotic aneuploidies. This review explores the molecular and cellular pathways underlying these abnormalities, focusing on abnormal cleavage, chromatin cohesion, spindle stability, maternal effect genes, and mitochondria. RECENT FINDINGS:Approximately half of human embryos cease development in vitro or shortly after transfer to the uterus. Genetic investigation of these embryos has highlighted that 90% of these exhibit aneuploidies. Surprisingly, most of these arise from errors during the early mitotic divisions of preimplantation embryos. These findings strongly correlate with disruptions of early cleavage possibly due to faulty spindle assembly or mitochondrial dysfunction during the in-vitro development. Moreover, maternal effects, such as faulty meiotic recombination and variants in maternal effect genes involved in the subcortical maternal complex, may further predispose the embryo to high rates of chromosomal imbalance. SUMMARY:Meiotic and mitotic aneuploidies play a significant role in embryo arrest, yet their molecular and cellular origin are not well understood. Investigating these pathways may lead to interventions that could be developed to improve success rates with IVF or even fertility rates in general.

Abstract Study question Is the composition of the endometrial microbiome comparable to that of the vagina and can vaginal sampling provide sufficient information to characterize the uterine microbiome? Summary answer The compositions of the endometrial and vaginal microbiome are largely comparable and therefore, in most cases, vaginal sampling is sufficient to characterize the uterine microbiome. What is known already A dysbiotic endometrial microbiome (i.e., a shift from a predominance of beneficial bacteria to less favourable species) has been reported in association with adverse pregnancy outcomes in assisted reproductive treatment (ART). If dysbiosis is detected, antibiotics or probiotics can be administered to restore an optimal microbiome. However, characterization of the endometrial microbiome is challenging, involving invasive sampling, and technical difficulties due to the low microbial mass in the endometrium. In contrast, vaginal sampling is considered minimally invasive and microbial analysis is technically less complex. It is important to clarify whether the microbiomes of the endometrium and the vagina are comparable. Study design, size, duration A prospective multi-centre study including 60 ART patients was conducted from June 2022 to August 2023. During the luteal phase (on average day-19), paired vaginal swabs and endometrial biopsies were collected sequentially and stored in a preservation medium for further analysis. Patients and physicians were also asked to complete a questionnaire to identify any lifestyle or medical factors that may have differentially affected the microbial composition of the vagina and endometrium. Participants/materials, setting, methods A carefully validated qPCR methodology was used to accurately assess the presence and relative abundance of the four major lactobacillus species associated with eubiosis (crispatus, gasseri, iners, and jensenii) and the 15 most common bacterial species implicated in dysbiosis. Additionally, the relative abundance of microbial and human DNA was examined. Of note, endometrial analysis required additional tissue lysis. Furthermore, due to the low microbial DNA content in endometrial samples, preamplification was necessary prior to qPCR. Main results and the role of chance The proportion of microbial versus human DNA was approximately 90 times higher in vaginal samples compared to endometrial samples (p < 0.0001). However, the microbial compositions of both the endometrium and the vagina showed remarkable similarities. Lactobacillus species were predominant at both sites, accounting for an average of 86% and 89% of the bacterial population in the vagina and endometrium, respectively (p = 0.6). Of the dysbiotic species, Gardnerella vaginalis was the most common, accounting for 8% (vagina) and 7% (endometrium) of the total bacterial population (p = 1). There was no difference in the average number of distinct microbial species between the two niches (1.8 vs. 2.1; p = 0.3). Direct comparison of paired samples highlighted the high similarity of microbial compositions between both sites and showed strong correlations of species abundances in individual patients (L.crispatus: r = 0.9, L.iners: r = 0.9, L.gasseri: r = 1, L.jensenii: r = 0.9, G.vaginalis: r = 0.7; p < 0.0001/all species). However, the microbial composition differed in four patients (6.6%), with three having a dysbiotic vaginal profile (≥50% dysbiotic species) but a eubiotic endometrial profile (≥50% lactobacillus species) and one showing the reverse pattern. The medical conditions of two patients, including chronic endometritis and use of antibiotics and probiotics in the preceding cycle, may account for the observed differences. Limitations, reasons for caution The species analysed were carefully selected to ensure a comprehensive analysis. Nevertheless, the potential presence of unevaluated species could impact the results. While the included patients displayed diversity in their reproductive histories, it is important to acknowledge that the observed results may still be influenced by underlying causes of infertility. Wider implications of the findings The close similarity between the microbiomes at the two sites investigated suggests that minimally invasive vaginal sampling may be sufficient to characterise the endometrial microbiome. The ease of vaginal sampling and processing is expected to improve patient recruitment in microbiome research and facilitate easier application of clinical microbiome tests. Trial registration number not applicable

Does vaginal microbiome have an impact on vaginal absorption of exogenous progesterone (P intake) in artificial cycles? Results suggest that the presence of lactobacillus in the vagina may positively impact the uptake of P. Low serum P levels on ET day decrease significantly live birth rates. About 20% of patients receiving micronized vaginal P show inadequate levels. It is of interest to find out which intrinsic factors might influence on the capacity of absorption (P intake) of vaginal P, and if they could be treated to prevent this situation. Vaginal microbiome hast been suggested as a possible factor, although this has not been addressed yet. The aim of this study was to explore if there is a correlation between vaginal microbiome and pH, and P intake. A prospective single-centre cohort pilot study including 92 ART patients was conducted from February 2022 to January 2023 in IVI-RMA Valencia. Embryo transfer was conducted in the context of a hormonal replacement therapy cycle with use of vaginal P (400mg/12h) for luteal phase support. “Progesterone intake” capacity was evaluated according to the vaginal microbiome status (lactobacillus dominant (LDM, >90%) or non-lactobacillus dominant (NLDM) and pH levels determinations (considered normal when moderately acidic ( < =4.8). Samples were taken twice: A) proliferative phase (day of initiation of exogenous P) and B) mid-luteal phase (day of ET, P + 5). Serum E2 and P levels as well as vaginal microbiome and pH were analysed. A validated qPCR methodology was used to accurately assess the presence and relative abundance of the four major lactobacillus species associated with eubiosis (crispatus, gasseri, iners, and jensenii) and the most common bacterial species implicated in dysbiosis. Distribution of LDM and NLDM profiles were comparable between Samples A and B. No significant difference between any of the potential confounding factors (age, BMI,baseline P) between the lactobacillus CST groups (CST-I,-II,-III,-V & lactobacillus) and the dysbiotic CST group (CST-IV) in Samples A and B were observed. Serum P levels on the ET day were significantly higher in LDM profiles both in Sample A (15.2 vs. 12.9ng/mL in NLDM;p=0.009) and B (15.0 vs. 12.8ng/mL;p=0.014). A positive significant correlation was found between serum P levels and Lactobacillus abundance in Samples A (r = 0.28; p = 0.008) and B (r = 0.30; p = 0.004). The relative abundance of Gardnerella vaginalis in Sample A was negatively related to serum P levels on the ET day (r=-0.24;p=0.02). Vaginal pH was significantly lower in LDM profiles both in Sample A (4.9 vs. 5.6 in NLDM; p = 0.002) and B (4.7 vs. 5.1;p<0.001). pH levels were negatively correlated with Lactobacillus abundance in Samples A (r=-0.34;p=0.0012) and B (r=-0.41;p<0.001). Additionally, serum P levels were negatively correlated with pH levels in Sample A (r=-0.14;p=0.191) and B (r=-0.27; p = 0.010). Indeed, when patients are grouped based on a cut-off of P = 8.8ng/mL, patients with a pH ≤ 4.8 have significantly higher P levels than patients with a pH > 4.8. This is a pilot study, thus further trials with larger sample sizes should be performed in order to confirm these results. Additionally, vaginal microbiome alone does not explain the total variability in serum P levels measured in artificial cycles when using MVP. We might have found one of the causes of the high heterogeneity on vaginal absorption of MVP in artificial cycles. This finding may help us with patient management when programming an ET in this type of cycles, improving personalised patient care and progressing on luteal phase support individualisation. 2110-VLC-095-EL

Abstract Study question What is the occurrence of contamination in embryo biopsy samples and could this lead to incorrect interpretation of preimplantation genetic testing for aneuploidy (PGT-A) results? Summary answer On average, contamination affects 0.4% of biopsy samples, but can be significantly more common in some clinics. Misdiagnosis can occur when contamination is not detected. What is known already Until recently, most commercially available platforms for PGT-A have utilised whole genome amplification followed by sequencing of a random selection of DNA fragments scattered across the genome using next generation sequencing (NGS). However, the simple quantitative measurements of DNA fragments derived from each chromosome, provided by such methods, cannot reveal when a biopsy sample is contaminated with non-embryonic DNA. Negative controls are seldom used during PGT-A and are inadequate as they do not evaluate contamination in the actual tube containing the biopsy specimen. The extent to which failure to detect DNA contamination is a problem for PGT-A is unknown. Study design, size, duration This was a retrospective study involving analysis of 49,287 trophectoderm biopsy samples that underwent PGT-A over a three-year period. Embryos found to have a contaminated biopsy specimen typically underwent a second biopsy. In such cases, results from the two samples were compared to ascertain whether the contaminated sample would have been misdiagnosed if the analysis had been restricted to examining only the relative chromosome copy number, as is the case for most NGS-based PGT-A methods. Participants/materials, setting, methods All trophectoderm biopsies underwent targeted DNA amplification and next generation sequencing using a highly validated PGT-A method that evaluates the relative chromosomal copy number, similar to traditional PGT-A methods, but combines this with analysis of variations in DNA sequence (single nucleotide polymorphisms - SNPs). The genotype of each SNP, and the relative quantity of DNA fragments containing each of the different alleles, allows detection of otherwise invisible states, such as triploidy, haploidy, and contamination. Main results and the role of chance From the 49,287 TE biopsies analysed, contamination with non-embryonic DNA was detected in 218 (0.44%). There was variation in the rates of contamination between the 25 clinics that provided samples, ranging between 0% and 1.5%. Additionally, one clinic had a contamination rate of 7.7%, but the number of biopsies derived from that site was considered too small for reliable evaluation (n = 26). 156 of the embryos with a contaminated biopsy specimen underwent secondary biopsy (71.5%), allowing the relative chromosome copy number result from the contaminated specimen to be compared to that obtained from an uncontaminated sample. The results were split into three categories: 1) no change in interpretation between the first (contaminated) and second biopsy specimens; 2) false positive – the contaminated sample was euploid but would have been wrongly interpreted as triploid and would have been erroneously discarded, potentially impacting the patients chances of achieving a pregnancy; 3) false negative – the contaminated sample was fully aneuploid but would have been incorrectly classified mosaic or euploid and could have been eligible for transfer, potentially leading to implantation failure or abnormal pregnancy. 19% of contaminated samples gave a false negative result, while 24% gave a false positive, appearing to be triploid. Limitations, reasons for caution It is not possible to determine the origin of contaminants with certainty without having DNA from the contamination source for comparison. Additionally, we were unable to conclude whether contamination is more likely to occur in IVF or ICSI cycles as only 3% of samples were fertilized using IVF. Wider implications of the findings Contamination detection during PGT-A is important to prevent misdiagnosis of embryos. Misclassification due to undetected contamination can lead to discard of potentially viable embryos. It can also lead to the transfer aneuploid embryos, wrongly classified as mosaic, which could lead to increased rates of implantation failure, miscarriage and aneuploid pregnancy. Trial registration number Not applicable

Abstract Study question Following transfer of the meiotic spindle from a patient’s oocyte into an enucleated donor oocyte, do relative levels of patient and donor mtDNA remain stable? Summary answer mtDNA transferred along with the patient’s spindle typically remain at a low level. However, disproportionate expansion of the patient’s mtDNA can occur in some cases. What is known already Mitochondrial DNA disorders are caused by mutations in the mitochondrial genome, disrupting ATP production. They have few treatment options and no cure. All mitochondria are derived from the oocyte, therefore mtDNA disorders are maternally inherited. It has been proposed that disease transmission could be avoided if female mtDNA mutation carriers underwent meiotic spindle transfer (MST), removing the chromosomes (on the spindle) from an affected oocyte and transferring them into the healthy cytoplasm of a donor oocyte. However, research using embryonic stem cells has suggested that the small population of mitochondria transferred along with the spindle can sometimes undergo dramatic expansion. Study design, size, duration 25 infertile couples were enrolled in a prospective pilot study evaluating MST as a treatment for infertility. The female participants had a mean age of 37.2, and an average of 6.4 previous unsuccessful IVF cycles (range 3-11) characterised by extremely poor embryo development and without any previous pregnancy. Importantly, none of the patients were carriers of a mtDNA disorder. Oocyte donors with previous successful IVF outcomes were matched with patients according to standard practice. Participants/materials, setting, methods Metaphase-II-spindles from patient oocytes were transferred into enucleated donor oocytes. Reconstructed oocytes underwent ICSI and the resulting embryos were transferred at the blastocyst stage. Mitochondrial genomes were sequenced to identify polymorphisms differing between the patient and oocyte donor. These variations were quantified in the embryos (blastocyst biopsies), during pregnancy (amniocentesis), in newborns (cord blood, cord tissue, urine), at 3-6 months and at one year (saliva, urine, blood), revealing the relative amounts of each mitochondrial type. Main results and the role of chance This MST pilot study resulted in the birth of six children, indicating that the procedure is compatible with the production of viable embryos, capable of producing healthy live births. The patient’s mtDNA was shown to represent <1% of the total in all blastocysts produced, confirming that MST is highly reproducible and that relatively few mitochondria are transferred along with the spindle. For five of the six children, the proportion of the total mtDNA attributable to the patient appeared to be stable, remaining at very low levels in all of the samples from later developmental stages. However, in one child the small quantity of mtDNA transferred along with the spindle increased disproportionately with respect to the mtDNA of the oocyte donor, ultimately representing 30-60% of the total at birth, depending on the tissue tested. The precise timing of the expansion of one type of mtDNA at the expense of the other is not known but occurred sometime between the blastocyst stage and birth. By the time of birth, the levels of donor and patient mtDNA appeared to have stabilised (no further increases were seen at 6 months and one year). All of the children born remain developmentally normal and healthy. Limitations, reasons for caution After using MST, several pregnancies were achieved for patients with a long history of unsuccessful IVF attempts, associated with poor oocyte quality and a failure to produce blastocysts. However, this small pilot study lacked the controls necessary for a definitive evaluation of MST as a tool for infertility treatment. Wider implications of the findings All children born following MST were healthy. However, our results clearly demonstrate that a substantial degree of mtDNA ‘reversal’ is possible. Consequently, mitochondrial replacement therapies used for avoidance of mtDNA disorders might not always be successful, even when initial levels of mutant mtDNA in reconstructed oocytes are very low. Trial registration number ISRCTN11455145

Abstract Study question Are Lactobacillus-dominant vaginal microbial compositions advantageous for clinical outcomes and is the specific Lactobacillus species relevant? Summary answer Favourable clinical outcomes are increased in patients with a Lactobacillus-dominant vaginal microbiome, particularly in those patients with microbial communities dominated by L.crispatus and L.jensenii. What is known already The clinical relevance of the urogenital microbiome is emerging as a topic in female reproductive health. Recent evidence suggests that the composition of the vaginal microbiome may have an impact on clinical outcomes in patients undergoing assisted reproductive treatment. A Lactobacillus-dominant microbiome (LDM), typically composed of L.crispatus, L.gasseri, L.iners or L.jensenii, is considered to be associated with favourable outcomes. In contrast, a non-Lactobacillus-dominated microbiome (NLDM), consisting predominantly of anaerobe species, may be linked to poor outcomes. The evidence surrounding this relationship is controversial and the importance of relative Lactobacillus abundance in the vagina in relation to clinical outcomes remains unclear. Study design, size, duration A single-centre prospective pilot study included 81 patients (mean age: 38.2) undergoing frozen embryo transfer using their own (n = 52) or donated (n = 29) oocytes. A vaginal swab was taken on the embryo transfer day and the microbiome analysed. Implantation, ongoing pregnancy and early miscarriage rates were compared between different microbiome compositions. To minimise the confounding effect of aneuploidy on clinical outcomes, a patient sub-group (n = 54) receiving donor oocytes or a euploid embryo transfer was also considered. Participants/materials, setting, methods Quantitative PCR was utilised to determine the relative abundance of the four predominant Lactobacillus species as well as 15 species associated with vaginal dysbiosis. Microbiome compositions were grouped according to the relative Lactobacillus abundance and patients divided into an LDM (>80%) or NLDM (<80%) group. Patients were further sub-divided into community state types (CSTs) according to the dominant species present in their sample (CST-I: L.crispatus, CST-II: L.gasseri, CST-III: L.iners, CST-V: L.jensenii and CST-IV: dysbiotic species). Main results and the role of chance Relative abundance of Lactobacillus was significantly higher in samples from patients achieving an ongoing pregnancy (80.7% vs. 61.7%; p = 0.05). Implantation rates were comparable between LDM and NLDM patients (66.0% and 64.3%), but the ongoing pregnancy rate showed an apparent increase in LDM patients (58.5% vs. 39.3%; p = 0.11), concomitant with a significant decrease in the miscarriage rate (11.4% vs. 38.9%; p = 0.03). Limiting the analysis to patients at low risk of aneuploidy (young donor and PGT-A cycles), Lactobacillus abundance appeared higher in patients with ongoing pregnancies (74.8% vs. 58.0%; p = 0.34). Likewise, LDM patients showed higher ongoing pregnancy (61.3% vs. 43.5%; p = 0.27) and lower miscarriage (5.0% vs. 33.3%; p = 0.06) rates, although significance was not achieved in this restricted sample. Comparison of CST groups indicated better outcomes in CST-I and CST-V compared to other groups. The ongoing pregnancy rate was significantly higher (65.7% vs. 41.3%; p = 0.04), alongside a lower miscarriage rate (8.0% vs. 32.1%; p = 0.04). When only considering patients with low aneuploidy risk, similar results were obtained (implantation rate: 78.9% vs. 57.1%, p = 0.14; ongoing pregnancy rate: 73.7% vs. 42.9%, p = 0.04; miscarriage rate: 6.7% vs. 25.0%, p = 0.21). Of note, there were no differences in baseline characteristics (age, ethnicity & BMI) or embryo morphologies between groups. Limitations, reasons for caution All patients were from a single fertility clinic and the study population was predominantly composed of white women. Consequently, results may not be applicable to women of different ethnicities. Patients with a low risk of aneuploid embryo transfer represented a small subgroup where meaningful statistical analysis was not always possible. Wider implications of the findings This study identifies a correlation between Lactobacillus species colonisation in the vagina and successful clinical outcomes, suggesting that the vaginal microbiome modulates the chances of IVF success. The results are promising, providing motivation for further, larger studies, involving more diverse populations in order to draw definitive conclusions. Trial registration number not applicable

Abstract Study question It is possible to identify a characteristic pattern of endometrial gene expression indicative of implantation failure, which is independent of implantation window displacements? Summary answer An endometrial transcriptomic signature was able to identify patients with a > 3-fold increased risk of implantation/pregnancy failure with 95% accuracy. What is known already Implantation failure of endometrial origin is a complex and multifactorial symptom with diverse causes, diagnosed in IVF patients after repeated failures with good quality embryos. A generation of gene expression tools have assumed that Window of implantation (WOI) displacement is the principal cause of this condition, but strategies seeking to counteract this problem by adjusting the day of embryo transfer have not yielded convincing improvements in outcomes. However, it is conceivable that other forms of endometrial disruptions, relevant to embryo implantation, could exist. New endometrial diagnostic strategies are needed to understand, diagnose and potentially treat patients affected with such problems. Study design, size, duration A prospective multicenter study between January 2018 and December 2021 recruited 281 Caucasian IVF patients (mean age of 39.4±4.8 years and BMI of 22.9±3.5 kg/m2) undergoing hormone replacement therapy and encompassing 114.5±7.2 h of progesterone administration at time of endometrial biopsy. Following experimental quality controls and clinical follow up, 186 patients who had a good quality embryo transferred in the cycle after endometrial biopsy collection were included for gene discovery and prediction performance. Participants/materials, setting, methods The expression of 404 genes selected for their potential to distinguish endometrial timing and/or endometrial disruption was measured. Transcriptomic variation related to progression of the menstrual cycle was removed using transcriptomic endometrial dating (TED) and linear models. Study groups were established according to clinical and gene expression parameters through a semi-supervised artificial intelligence procedure. Gene signature discovery and a cross-validation processes were undertaken to define predictive expression patterns. Reproductive outcomes were compared between prognosis profiles. Main results and the role of chance We developed a procedure called Clinically Acute Transcriptomic Stratification (CATranS), combining clinical parameters and deep transcriptomic molecular characterisation to stratify patients according to endometrial prognosis: ‘poor’ (n = 137) or ‘good’ (n = 49). These two transcriptomic profiles were associated with differing reproductive outcomes in the single embryo transfer following biopsy: pregnancy rate (45.1% vs 79.6%, poor vs good prognosis, respectively, p = 3.8E-5); live birth rate (56.4% vs 97.5%, p = 3.0E-06); clinical miscarriage rate (27.9% vs 2.6%, p = 0.0020); biochemical pregnancy rate (20.4% vs 0%, p = 0.0023). Patients with a poor prognosis profile had 3.3-times higher relative risk of a transferred embryo failing to implant or a pregnancy not being sustained to term, compared with good prognosis patients. Initially, a reference dataset was used to build a prototype for diagnosing endometrial failure, revealing that a gene expression signature consisting of 135 genes was the most predictive. Prediction performance was estimated using a 5-fold 100-times cross-validation process, resulting in a median accuracy of 0.92, median sensitivity of 0.96, and median specificity of 0.84. From these 135 predictive genes, 122 were differentially expressed (FDR<0.05) in endometrial poor prognosis, 59 up- and 63 down-regulated, most involved in functional processes such as regulation (17%), metabolism (8.4%), immune response and inflammation (7.8%). Limitations, reasons for caution We describe a potential new strategy for evaluating endometrial competence. However, to confirm predictive value, validation using additional samples from patients independent of signature discovery set is required. Further research to identify potential treatments for patients classified as poor prognosis is needed, providing a tailored clinical pathway for these patients. Wider implications of the findings The prototype described is a novel concept, potentially leading to development of a new generation of tools for diagnosis of fertility problems related to endometrial factors. The ‘poor’ prognosis profile is not caused by asynchronies in menstrual cycle progression, opening the possibility of finding new treatment pathways for these patients. Trial registration number NA

Abstract Study question Can accurate mitochondrial DNA (mtDNA) quantification of trophectoderm (TE) biopsy specimens provide insights into the biology and viability of blastocyst-stage human embryos? Summary answer mtDNA quantity in TE cells is correlated with embryo morphology and shows alterations associated with aneuploidy. However, measurement does not significantly improve embryo viability assessment. What is known already Mitochondria are essential organelles, responsible for producing ATP. Changes in the amount of mtDNA in blastocysts biopsy specimens have been reported in association with embryo implantation potential, leading to proposals that mtDNA quantification might serve as a useful biomarker of embryo viability. However, results from clinical studies to explore this possibility have yielded contradictory data, due in part to deficiencies of the molecular methods used for mtDNA measurement. We sought to clarify what quantification of mtDNA can tell us about embryo biology and viability by developing and applying a method that we believe to be the most accurate ever devised. Study design, size, duration This study involved the analysis of samples collected during the course of routine preimplantation genetic testing for aneuploidy (PGT-A). The IVF treatments and embryo biopsies were undertaken at two different clinics, while chromosomal analyses were carried out at a single reference laboratory. Mitochondrial data was subsequently analysed in a university setting. The embryos analysed were derived from a broad population of patients referred for PGT-A (average age 38.7 years; range 25-47). Participants/materials, setting, methods 651 blastocysts from 133 couples underwent trophectoderm biopsy on day-5 or day-6. The specimens were analysed using a highly validated real-time PCR method, which was used to quantify three distinct sites in the mtDNA and 198 loci in the nuclear genome. The measurement of multiple independent loci provided outstanding sensitivity and accuracy. The nuclear loci were used to normalise the mtDNA data, adjusting for differences in the number of cells in the biopsy specimens. Main results and the role of chance The method developed displayed extraordinary sensitivity and accuracy when quantifying mtDNA. Lower mtDNA quantities were associated with day-6 biopsy (p < 0.0001), extent of blastocyst expansion (p < 0.0001), and superior TE morphology, although the latter was not statistically significant (p = 0.09). mtDNA levels were higher in aneuploid embryos (p < 0.0001), independent on patient age. However, the difference was not sufficient to be considered diagnostic. There was no correlation between mtDNA level and the chances of blastocyst implantation in this dataset. Lower mtDNA levels, previously reported to be associated with higher probabilities of embryo implantation, were most often observed in embryos of excellent morphological grade and likely reflect the increased TE cell numbers of such embryos. Little if any mtDNA replication occurs during preimplantation development and consequently the mtDNA content is divided amongst an ever-growing number of cells, meaning less mtDNA per cell. In this context, mtDNA quantification of blastocyst biopsy specimens provides a highly sensitive measure of TE cellularity, but probably provides little additional benefit for embryo selection beyond conventional morphological grading. However, the fact that higher mtDNA quantities were observed in aneuploid embryos, may indicate that subtle differences in TE cellularity exist in abnormal embryos, which are not fully captured by traditional morphological assessment. Limitations, reasons for caution Previous studies suggested that some blastocysts have greatly elevated mtDNA levels and that such embryos are not viable. In this study, only 5% of embryos were considered outliers in terms of mtDNA quantity. Unfortunately, none of these embryos were transferred, so the potential of these embryos could not be assessed. Wider implications of the findings The quantification of mtDNA in trophectoderm biopsies has sometimes been used for the prioritisation of embryos for transfer. While our results confirm existence of biologically interesting associations between mtDNA and aneuploidy, and a relationship with certain aspects of embryo morphology, measurement of mtDNA seems unlikely to significantly improve embryo selection. Trial registration number not applicable

STUDY QUESTION:What are the key considerations for developing an enhanced transcriptomic method for secretory endometrial tissue dating? SUMMARY ANSWER:Multiple gene expression signature combinations can serve as biomarkers for endometrial dating, but their predictive performance is variable and depends on the number and identity of the genes included in the prediction model, the dataset characteristics and the technology employed for measuring gene expression. WHAT IS KNOWN ALREADY:Among the new generation of transcriptomic endometrial dating (TED) tools developed in the last decade, there exists variation in the technology used for measuring gene expression, the gene makeup and the prediction model design. A detailed study, comparing prediction performance across signatures for understanding signature behaviour and discrepancies in gene content between them, is lacking. STUDY DESIGN, SIZE, DURATION:A multicentre prospective study was performed between July 2018 and October 2020 at five different centres from the same group of clinics (Spain). This study recruited 281 patients and finally included in the gene expression analysis 225 Caucasian patients who underwent IVF treatment. After preprocessing and batch effect filtering, gene expression measurements from 217 patients were combined with artificial intelligence algorithms (support vector machine, random forest and k-nearest neighbours) allowing evaluation of different prediction models. In addition, secretory-phase endometrial transcriptomes from gene expression omnibus (GEO) datasets were analysed for 137 women, to study the endometrial dating capacity of genes independently and grouped by signatures. This provided data on the consistency of prediction across different gene expression technologies and datasets. PARTICIPANTS/MATERIALS, SETTING, METHODS:Endometrial biopsies were analysed using a targeted TruSeq (Illumina) custom RNA expression panel called the endometrial dating panel (ED panel). This panel included 301 genes previously considered relevant for endometrial dating as well as new genes selected for their anticipated value in detecting the secretory phase. Final samples (n = 217) were divided into a training set for signature discovery and an independent testing set for evaluation of predictive performance of the new signature. In addition, secretory-phase endometrial transcriptomes from GEO were analysed for 137 women to study endometrial dating capacity of genes independently and grouped by signatures. Predictive performance among these signatures was compared according to signature gene set size. MAIN RESULTS AND THE ROLE OF CHANCE:Testing of the ED panel allowed development of a model based on a new signature of 73 genes, which we termed 'TED' and delivers an enhanced tool for the consistent dating of the secretory phase progression, especially during the mid-secretory endometrium (3-8 days after progesterone (P) administration (P + 3-P + 8) in a hormone replacement therapy cycle). This new model showed the best predictive capacity in an independent test set for staging the endometrial tissue in the secretory phase, especially in the expected window of implantation (average of 114.5 ± 7.2 h of progesterone administered; range in our patient population of 82-172 h). Published sets of genes, in current use for endometrial dating and the new TED genes, were evaluated in parallel in whole-transcriptome datasets and in the ED panel dataset. TED signature performance was consistently excellent for all datasets assessed, frequently outperforming previously published sets of genes with a smaller number of genes for dating the endometrium in the secretory phase. Thus, this optimized set exhibited prediction consistency across datasets. LARGE SCALE DATA:The data used in this study is partially available at GEO database. GEO identifiers GSE4888, GSE29981, GSE58144, GSE98386. LIMITATIONS, REASONS FOR CAUTION:Although dating the endometrial biopsy is crucial for investigating endometrial progression and the receptivity process, further studies are needed to confirm whether or not endometrial dating methods in general are clinically useful and to guide the specific use of TED in the clinical setting. WIDER IMPLICATIONS OF THE FINDINGS:Multiple gene signature combinations provide adequate endometrial dating, but their predictive performance depends on the identity of the genes included, the gene expression platform, the algorithms used and dataset characteristics. TED is a next-generation endometrial assessment tool based on gene expression for accurate endometrial progression dating especially during the mid-secretory. STUDY FUNDING/COMPETING INTEREST(S):Research funded by IVI Foundation (1810-FIVI-066-PD). P.D.-G. visiting scientist fellowship at Oxford University (BEFPI/2010/032) and Josefa Maria Sanchez-Reyes' predoctoral fellowship (ACIF/2018/072) were supported by a program from the Generalitat Valenciana funded by the Spanish government. A.D.-P. is supported by the FPU/15/01398 predoctoral fellowship from the Ministry of Science, Innovation and Universities (Spanish Government). D.W. received support from the NIHR Oxford Biomedical Research Centre. The authors do not have any competing interests to declare.

The aim of this study was to validate the Spindle Transfer (ST) technique in human donor oocytes and explore its feasibility for clinical application in the treatment of infertility associated with poor oocyte quality. Experiments were licensed by the Greek National Authority of Assisted Reproduction and approved by the IRB of IASO Maternity Hospital. Informed consent was obtained from the 20 donors participating in the study. We first aimed to compare the efficiency of two fusion protocols. In a second set of experiments, we evaluated an optimized protocol using donor fresh or vitrified oocytes with different morphological/developmental characteristics. Micromanipulation was performed on an inverted microscope (Olympus-IX73) equipped with polarized light. Karyoplast-cytoplast fusion was induced by exposure of the reconstructed oocytes to either a chemical solution or an inactivated protein extract (HVJ-E). The same donor's sperm sample was used in all experiments. Embryos underwent continuous culture (Embryoscope+, Vitrolife) in single medium (LifeGlobal) and were biopsied for assessing aneuploidy and mitochondrial DNA (mtDNA) carryover. Statistical significance was assessed by Students t-test or Fisher's exact test. We initially compared two fusion protocols using 63 MII donor oocytes. HVJ-E-mediated fusion rates were significantly higher (98.1%) than those obtained in the chemical method (76.8%, p<0.01), while fertilization and blastocyst formation rates were similar (p>0.05) between the control (70.2%-78.7%), HVJ-E (75.5%-76.3%) and chemical-fusion (60.5%-60.2%) groups. In the second set of experiments, ST was performed in 118 donor oocytes. Overall, results varied greatly depending on the quality of the recipient cytoplasm. When spindles were transferred from in vitro matured or morphologically "abnormal" oocytes into good quality cytoplasts, individual cohorts showed fertilization (66.7%-71.4%) and blastocyst rates (75.0%-60.0%) significantly improved (p<0.05) compared to non-manipulated controls (50.0%-33.3% and 0.0%-0.0%, respectively) or reciprocally reconstructed (25.0%-0.0% and 37.5%-33.3%, respectively) oocytes. From a total of 36 blastocysts analysed, aneuploidy rates were statistically equivalent (p=0.53) between controls (41.2%, n=17) and ST (52.6%, n=19) embryos. mtDNA carryover levels were estimated to be <1%. This study shows that cytoplasm replacement by ST can enhance the potential of developmentally compromised oocytes to develop up to the blastocyst stage without compromising euploidy rates. This opens up the possibility of providing new treatment options for patients with certain forms of infertility refractory to current clinical strategies.

STUDY QUESTION:What is the incidence, origin and clinical significance of segmental aneuploidy in human oocytes and preimplantation embryos?SUMMARY ANSWER:Segmental aneuploidy occurs at a considerable frequency in preimplantation embryos with a majority being mitotic in origin.WHAT IS KNOWN ALREADY:In recent years, accurate techniques for the detection of aneuploidy in single cells have been developed. Research using such methods has confirmed that aneuploidy is a common feature of human oocytes and preimplantation embryos. However, thus far research has mainly focused on loss or gain of whole chromosomes. We utilized sensitive molecular methods to study another important form of cytogenetic abnormality at the earliest stages of human development, namely segmental aneuploidy.STUDY DESIGN, SIZE, DURATION:Chromosomal copy number data was obtained from oocytes and embryos of 635 IVF patients, who requested chromosome screening for various reasons, most commonly for advanced maternal age or previously unsuccessful IVF treatments. A total of 3541 samples comprising of 452 human oocytes, 1762 cleavage stage and 1327 blastocyst stage embryos were investigated in the present study.PARTICIPANTS/MATERIALS, SETTING, METHODS:Whole genome amplification (Sureplex, Illumina) was performed on cells biopsied from oocytes and embryos of IVF patients who requested chromosome screening. The samples were subsequently processed and analyzed for their chromosome complement using microarray comparative genomic hybridization (aCGH), (Illumina, Cambridge, UK).MAIN RESULTS AND THE ROLE OF CHANCE:Segmental abnormalities, involving loss or gain of chromosomal fragments in excess of 15 Mb, were found to occur at a high frequency. The incidence of such abnormalities was 10.4% in oocytes, but this increased dramatically during the first 3 days of embryonic development (24.3%), before starting to decline as embryos reached the final (blastocyst) stage of preimplantation development (15.6%). While some segmental errors were clearly of meiotic origin, most appear to arise during the first few mitoses following fertilization. The reduction in frequency at the blastocyst stage suggests that many cells/embryos affected by segmental abnormalities are eliminated (e.g. via arrest of the affected embryos or apoptosis of abnormal cells). Interestingly, sites of chromosome breakage associated with segmental aneuploidy were not entirely random but tended to occur within distinct chromosomal regions. Some of the identified hotspots correspond to known fragile sites while others may be considered novel and may be specific to gametogenesis and/or embryogenesis.LIMITATIONS REASONS FOR CAUTION:The cytogenetic analysis was performed on biopsies of embryos, which might not be representative of the true incidence of mosaic segmental aneuploidy of the entire embryo.WIDER IMPLICATIONS OF THE FINDINGS:The findings of this study are valuable for understanding the origin of subchromosomal duplications and deletions, a clinically important class of abnormalities that are a common cause of congenital abnormalities and miscarriage. Furthermore, the results provide additional evidence that control of the cell cycle is more relaxed during the first few mitotic divisions following fertilization, permitting DNA double-strand breaks to occur and persist through cell division. The data are also of great relevance for preimplantation genetic testing, where the detection of segmental aneuploidy is currently considered problematic for embryo diagnosis and patient counseling.STUDY FUNDING/COMPETING INTEREST(S):This study was supported by institutional funding (Reprogenetics UK). Additionally, DW is supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. DB was supported by the University of Oxford's Clarendon funding. No conflict of interests to declare.