Are artificial intelligence-based embryo evaluation and selection algorithms equally effective for male and female embryos? Compared to female embryos, male embryos exhibited higher automatic scores, superior performance in KIDScore D5 v3, and poorer performance in iDAScore v2 in predicting implantation. It is known that the secondary sex ratio favors males in assisted reproductive techniques. The reason could be an unbalanced selection, as there is evidence that male embryos undergo faster development and have better morphology than female embryos. Based on this premise, artificial intelligence (AI) models currently used for embryo selection may not perform equally well for embryos of both sexes. We aimed to analyze the performance of two models for assessing male and female embryos: KIDScore D5 v3 and iDAScore v2. This is a retrospective cohort study involving 481 patients included in the preimplantation genetic testing program over a year and a half. The euploid embryos (n = 1,181) were divided into male (n = 577) and female (n = 604) groups and the automatic scores received by two artificial intelligence-based evaluation models (KIDScore D5 v3 and iDAScore v2) were considered with respect to implantation. All embryos underwent assisted hatching on day 3 of embryo development and trophoectoderm biopsy at the blastocyst stage (day 5 or 6). The embryos’ sex chromosomes were revealed using a highly validated method. Routine embryo assessment and selection were performed using conventional morphology according to ASEBIR criteria. Retrospectively, embryos were automatically scored from 1 to 9.9. Means, standard deviations, odds ratios (OR), and areas under the ROC curve (AUC) were calculated using IBMSPSS Statistics software. In general, male embryos exhibited a significantly higher iDAScore than female embryos (6.9±2.2 vs. 6.4±2.5)**; no differences for KIDScore (6.1±1.7 vs. 5.9±1.8). This indicates that if the selection were based on the iDAScore, more male embryos would be transferred. Male embryos: 323 male embryos were transferred with an implantation rate of 53.8%. Implanted male embryos had higher automatic score compared to non-implanted ones (6.7±1.6 vs. 6±1.7 for KIDScore** and 7.3±2 vs. 6.8±2.1 for iDAScore*). For KIDScore: OR = 1.3, 95% CI [1.1-1.5] and AUC 0.612 (0.545-0.678). For iDAScore: OR = 1.1, 95% CI [1-1.3] and AUC 0.575 (0.511-0.638). This suggests a higher performance of KIDScore in predicting implantation in male embryos. Female embryos: 318 female embryos were transferred with an implantation rate of 47.7%. Implanted female embryos had higher iDAScore compared to non-implanted ones (7±2.3 vs. 6.3±2.5)*; no differences for KIDScore (6.2±1.8 vs. 6±1.8). For KIDScore: OR = 1.1, 95% CI [0.9-1.2] and AUC 0.542 (0.472-0.612). For iDAScore: OR = 1.1 95% CI [1-1.2] and AUC 0.584 (0.520-0.648). This suggests a higher performance of iDAScore in predicting implantation in female embryos. *pvalue<0.05; **pvalue<0.01 The primary limitation of our study is the assisted hatching process performed on embryos on day 3 of development, which may impact late-stage development. This could be a possible reason for the considerably lower performance of the models. Our study revealed objective differences between male and female embryos, as well as distinct performance for two AI-based embryo selection models. These findings suggest that the employed embryo selection model may impact the secondary sex ratio. Not applicable

Are results showing limited clinical utility of mosaicism reporting in PGT from a blinded mosaic embryo transfer American study, confirmed in a European setting? Even between different patient populations and clinical settings, putative mosaicism reporting has limited clinical utility for embryo selection when co-evaluated with other clinical/embryological factors. Intermediate chromosomal copy number (ICN) results in NGS-based PGT-A are commonly interpreted as embryo mosaicism. A recent prospective, non-selection study in America showed that reporting mosaicism would not significantly improve embryo selection. At odds with this, results suggesting that embryo selection based on putative mosaicism can improve clinical outcomes continue to be reported, possibly reflecting limitations in accurate mosaicism identification or retrospective analysis biases. This study replicates the design of previously reported American study, using the same PGT-A assay, examining the predictive value of whole-chromosome ICN (wICN) and segmental ICN (sICN) following concurrent copy-number and genotyping-PGT in a European setting. A multisite double-blinded study involving 14 European IVF clinics was conducted from April 2022 to July 2023, including 3365 patients and 4293 single embryo transfers. Embryos, reported as negative for uniform aneuploidies, including those with wICN or sICN, were chosen for transfer based solely on standard developmental and morphological evaluation. Primary outcome measure was live birth rate defined as any pregnancy reaching the 24th week of gestation (LBR). Trophectoderm biopsies were analysed using a validated targeted-NGS assay with approximately 5000 loci across the genome, providing both quantitative and genotyping information to support aneuploidy classification. Putative mosaicism was inferred from ICN deviations from the expected two copies for autosomes and partly supported by corresponding SNP B-allele frequency patterns. Related clinical and embryological variables were analysed in a multivariate analysis and a Random Forrest regression used to model the clinical utility of putative mosaicism. Of the embryos transferred, 6.8% (293/4293) were wICN only; 6.2% (269/4293) were sICN only and 0.5% (23/4293) were a combination of wICN and sICN. The detected ICN ranged from 23%-84% with no overrepresentation of any individual chromosomes. The LBR of embryos in the control group (without ICN), sICN and wICN were 46.8% (1736/3708; 95%CI:45.2-48.4%), 36.4% (98/269; 95%CI:30.9-42.3%) and 39.2% (115/293; 95%CI:33.8-44.9%) respectively. The miscarriage rate for control, sICN and wICN were 12.9% (257/1993; 95%CI:11.5-14-4%), 19.7% (24/122; 95%CI:13.6-27-6%) and 16.7% (23/138; 95%CI:11.4-23.8%), respectively. Logistic regression (P = 0.05) showed that the presence of sICN (OR: 0.7; 95%CI: 0.54-0.9) and wICN (OR: 0.62; 95%CI: 0.36-1.07) were modestly but significantly associated with LBR along with other embryological and clinical factors including embryo morphology (OR: 0.89; 95%CI: 0.84-0.96), day of biopsy (OR: 0.65;95%CI: 0.56-0.75), previous ET failures (OR: 0.79; 95%CI: 0.71-0.88) that were more strongly associated. No significant effect was observed of any specific intermediate CN cut-off on the association with LBR. Random Forrest regression, considering all correlating variables, showed a LBR prediction (AUC) equal to 0.578 without putative mosaicism and AUC of 0.583 with putative mosaicism included, a negligible increase considering the low incidence of mosaicism in our clinical setting along with its modest predictive value. Follow-up prenatal and post-natal data at this time was not available, hence conclusions about these outcomes wasn’t possible. Despite the overall large sample size, the analysis of specific effects at different ICN values lacked sufficient power. Furthermore, this study’s data is platform-specific and cannot be translated to other PGT-A assays. Although putative mosaicism was associated with modestly impaired pregnancy rate, two independent studies now confirm that reporting mosaicism has limited impact when prospectively selecting embryos for transfer based on standard embryological parameters. Considering the established downsides, including potential embryo disposal, the practice of mosaicism reporting in PGT is questionable. Not applicable

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 What is the clinical utility and associated outcomes of mosaic whole chromosome or segmental aneuploidies detected using concurrent copy-number and genotyping analysis in PGT-A cycles? Summary answer Although high-level whole-chromosome mosaicism is linked to reduced sustained implantation, it has limited clinical significance in PGT-A cycles when co-evaluated with other clinical/ embryological factors What is known already NGS-based PGT-A can detect intermediate chromosomal copy number (CN), commonly interpreted as mosaic chromosomal aneuploidies in embryos. A prospective non-selection approach is the most effective way to assess the clinical utility of reporting putative mosaicism findings in PGT-A, wherein the presence of mosaicism is not disclosed and does not influence embryo selection. Conflicting results have been reported previously, possibly due to technological limitations in mosaicism assessment or retrospective analysis methods. This study reports the results of the largest multisite prospective non-selection clinical study examining the predictive value of whole-chromosome and segmental mosaicism, assessed through combined CN and genotyping data analysis. Study design, size, duration A multisite study involving seven IVF clinics was conducted from Feb 2020 to Oct 2022, including 6951 patients and 9828 single embryo transfers. The study involved a prospective non-selection approach, where embryos suspected of having whole chromosomal or segmental mosaicism were reported as negative for non-mosaic aneuploidies. Embryos were chosen for transfer based solely on standard morphological features. The primary outcome was sustained implantation rate (SIR) defined as pregnancy continuing beyond 8 weeks of gestation. Participants/materials, setting, methods In this study, the trophectoderm biopsies were analyzed using a custom, targeted NGS assay that examined approximately 5000 loci across the genome, providing genotyping information to support aneuploidy classification. Mosaicism was identified by any copy number deviation from the expected two copies (LogR plots) and confirmed by corresponding SNP B-allele frequency (BAF) patterns. Confounding factors, such as clinical and embryological variables, were controlled for in the multivariate analysis. Main results and the role of chance The average female age in this cohort was 34.9 years (SD = 4.1), with aneuploidy rate of 30% in embryos and SIR of 61.2%. Of the embryos transferred, 6.5% (636/9828) were whole chromosomal mosaic (WCM) only; 9.6% (947/9828) were segmental mosaic (SM) only and 1% (83/9829) were a combination of WCM and SM. The rate of putative mosaicism ranged from 15%-89%. The SIR of embryos in the control group (non-mosaic), SM and WCM were 62% (5190/8328; 95%CI), 58% (549/947;95%CI) and 50.3% (320/636; 95%CI P < 0.01) respectively. A logistic model found that the level of WCM was associated to SIR, along with other embryological and clinical factors. In particular, WCM with a CN difference >50% as well as poor embryo morphology were associated with lower SIR (OR = 0.5; 95% CI:0.32-0.76), but low-level (<50%) mosaicism was not significant (NS). Notably, female age (OR = 0.98; 95% CI:0.97-0.99 per year), BMI (0.98; 95% CI:0.98-0.99) and previous ET failures (OR = 0.58; 95% CI:0.5-0.68) were strongly associated with SIR. A predictive model, taking into account all relevant variables, yielded a significant stratification of SIR, from 43% to 68%. Given the low incidence of WCM in our clinical setting, the multivariate SIR prediction (AUC) was 0.580 without WCM and 0.585 with mosaicism included. Limitations, reasons for caution This study did not have prenatal and post-natal data available at the time of the abstract's writing, hence conclusions about these outcomes wasn’t possible. Despite the large sample size, chromosome specific analysis was not feasible. Furthermore, this study’s data is platform-specific and cannot be translated to other PGT-A assays. Wider implications of the findings In this non-selection study, WCM of > 50% variation was associated with lower SIR. However, high-level WCM has a minimal overall impact on SIR when co-evaluating with other clinical/ embryological parameters. Decisions on reporting criteria for these findings must weigh the risk of discarding potentially viable embryos with substantial reproductive potential. Trial registration number Not applicable

Abstract Study question Embryo selection methods based upon morphological or morphokinetic evaluation assume that male and female embryos have identical rates of preimplantation development, but is this true? Summary answer The distribution of trophectoderm grades differs for male and female blastocysts. Furthermore, male and female embryos of identical grade may have different probabilities of viability. What is known already Previous studies have suggested that male and female embryos may have subtle differences in their rates of preimplantation development. However, this possibility remains controversial. Apart from being of scientific interest, the question of whether the sex of an embryo can affect its growth trajectory is of clinical importance. Morphological grading, the primary method used by most IVF clinics when deciding which embryo to prioritise for transfer, assumes that developmental rates are independent of sex. Similarly, morphokinetic strategies for embryo evaluation, using data gathered from time-lapse incubators, are likely to be compromised if male and female embryos have differing developmental behaviour. Study design, size, duration 1,241 blastocysts underwent PGT-A and were shown to be euploid. Chromosome analysis also revealed the sex of the embryos, although this was not disclosed to patients. Standard morphological grading was carried out blindly with respect to the sex of the embryo. Information on clinical outcomes following embryo transfer was available for a subset of 336 embryos. Data was evaluated using various statistical methods to reveal any associations between embryo sex, morphology and clinical outcome. Participants/materials, setting, methods Embryos included in this study were derived from patients undergoing routine IVF with PGT-A. The only embryos excluded were those derived from patients carrying a monogenic disease mutation or a chromosome rearrangement. Embryos underwent PGT-A at the blastocyst stage on either day-5 or day-6, using a highly validated method. The euploid embryos were divided into male and female groups and blastocyst morphological grades were considered with respect to rates of implantation, miscarriage and ongoing pregnancy/birth. Main results and the role of chance The proportion of embryos biopsied on day-5 versus day-6 was identical for males and females (68% day-5 for both). No difference was observed in blastocyst expansion or inner cell mass grading on day-5. However, a highly significant difference was noted in the distribution of trophectoderm grades (A, B, C, D according to the system of Gardner and Schoolcraft, 1999) (P < 0.0001). This was characterised by a disproportionate representation of the highest grade amongst male embryos. 21.6% of male blastocysts had trophectoderm graded ‘A’, compared to 14.9% of females. Interestingly, euploid male embryos with a ‘B’ grade trophectoderm were associated with significantly higher implantation rates than females of the same morphological grade (82.1% vs. 58.7%; P = 0.0002), but the males of this grade also experienced a higher incidence of biochemical losses (16.7% vs. 4.2%; P = 0.017). As a group, male embryos do not have greater viability than female embryos, which implies that the greater proportion of high trophectoderm grades amongst these embryos is not an indicator of superior potential. In turn, this suggests that different criteria should be used for grading trophectoderm in males and females. Limitations, reasons for caution It may be desirable to introduce parallel morphological grading systems, one for male embryos and another for female. However, this would only be applicable in cycles involving PGT-A. The observation that embryos of equal grade can have different outcomes depending on their sex should be confirmed in a prospective study. Wider implications of the findings These results suggest the development of sex-specific morphological grading strategies might provide more reliable insights into embryonic potential, increasing the likelihood of selecting a viable embryo for transfer. Consideration of differences in the development of male and female embryos will also be important when developing morphokinetic algorithms for embryo selection. Trial registration number Not applicable

Abstract Study question PGT-M often involves diagnoses based upon the analysis of polymorphisms linked to the mutant gene. Are such methods sufficiently reliable to be used alone? Summary answer Several problems can lead to misdiagnosis when linkage analysis is used in isolation. Therefore, PGT-M should also include direct mutation testing (DMT) whenever possible. What is known already Many PGT-M strategies involve analysis of DNA sequence polymorphisms in close proximity to the mutant gene, which have specific alleles that are inherited along with the disease. Unlike diagnostics that focus on detection of specific mutations, which can be unique to individual families, PGT-M protocols using linked polymorphisms can usually be reused for multiple families. Indeed, strategies such as karyomapping, which assess thousands of polymorphisms across the genome, provide a single method applicable to numerous diseases. Such methods are attractive since the work-up required for individual cases is minimal, reducing costs and patient waiting times. However, are such methods safe? Study design, size, duration Over a period of three years, we carried out 261 PGT-M cases covering 312 different mutations in 116 genes. Each couple requesting PGT-M provided blood samples from which DNA was extracted. The patients underwent IVF and embryos that reached the blastocyst stage were subjected to trophectoderm biopsy. All PGT-M cases involved the use of DMT to interrogate the mutation site(s). The DMT result was supplemented by analysis of multiple informative linked polymorphisms, as described below. Participants/materials, setting, methods Embryo biopsy samples were subjected to multiple displacement amplification (MDA). Mutation site(s) were amplified from MDA products using PCR and mutations were revealed using minisequencing, Sanger sequencing, or DNA fragment size analysis. Parental and embryo samples were also analysed using karyomapping, involving the genotyping ∼300,000 polymorphisms scattered across the genome with a microarray. Where possible, samples from additional family members were also tested, allowing determination of which alleles of linked polymorphisms accompanied mutant gene copies. Main results and the role of chance Multiple PGT-M cases were identified where DMT prevented potentially serious errors. In five cases, diagnostic reports provided to the PGT laboratory were incorrect. These reports are vital for defining the genetic status of an individual, allowing specific alleles of linked polymorphisms to be correctly associated with mutant or normal gene copies. In three cases, DMT carried out on patient samples during the initial work-up revealed that, contrary to the report, the patient did not carry a mutation. Therefore, PGT-M was not indicated, saving patients from the stress and expense of an unnecessary PGT-M cycle and avoiding discard of healthy embryos. In the other two cases, errors in the reports would have inverted all results based upon linkage analysis, leading to transfer of affected embryos and discard of unaffected embryos, potentially causing a serious misdiagnosis. Thirteen more cases had recombination events extremely close to the mutation site, preventing determination of the status of the embryos based on analysis of nearby polymorphisms. Two further cases displayed consanguinity (undisclosed by the patients), leading to large areas of homozygosity in the genome, precluding use of linked polymorphisms for diagnosis. In all these cases DMT unequivocally confirmed the true status of patients and their embryos. Limitations, reasons for caution In ∼8% of PGT-M cases at least one embryo could not be accurately diagnosed without DMT, while in ∼1% of cases our routine use of DMT averted a serious misdiagnosis. Nonetheless, even when DMT and linkage analysis are combined, it must be acknowledged that misdiagnoses remain possible (although extremely rare). Wider implications of the findings PGT-M is a valuable reproductive strategy for patients at high-risk of transmitting a single gene disorder. Linkage analysis is a valid strategy for PGT-M, but misdiagnoses are possible when testing relies entirely on such methods. These errors can be virtually eliminated by including direct testing of the mutation site. Trial registration number Not applicable

Abstract Study question Is it beneficial to culture 1PN embryos for clinical use? Summary answer Culturing 1PN embryos to blastocyst for potential clinical use is beneficial to all patients, especially those with low prognosis. What is known already 1PN embryos are those with only a single pronucleus visible at the time of fertilisation check. It is generally assumed that such embryos are haploid, having only 23 chromosomes in each of their cells. Given that haploid embryos are not viable, current guidelines recommend only transfering embryos where two pronuclei are visualised. Embryo biopsy at the blastocyst stage, followed by testing with next-generation sequencing (NGS), combined with analysis of DNA polymorphisms, can reveal whether 1PN embryos are truly haploid, or whether normal fertilsation has occurred. Previous studies have shown that in some cases a diploid chromosome number may be present. Study design, size, duration A new clinical policy was put into practice to culture all 1PN embryos to day 5/6. This is an ongoing study initiated in April 2022. The data reported are from an eight month period, up until December 2022. During this time, we cultured 288 1PN embryos from 203 patients. Trophectoderm biopsy specimens were shipped to a specialist genetics laboratory where they underwent PGT-A using a method that assesses chromosome copy number and polymorphisms in tandem. Participants/materials, setting, methods If embryos were assessed at fertilisation check and found to be 1PN they were not discarded, but rather they were maintained in culture. If any such embryos successfully produced a blastocyst the patients received counselling from their fertility consultant and an embryologist, giving them the options to either go ahead with transfer (only IVF derived), have the embryos genetic status clarified using PGT-A, freeze without testing, or to discard the embryo. Main results and the role of chance Of the 288 1PN embryos cultured (IVF and ICSI derived) 85 displayed signs of blastulation (29.5%). 134 of these were derived from ICSI with 20 forming blastocysts (14.9%). 154 were derived from IVF with 65 forming blastocysts (42.2%). Three IVF derived 1PN embryos have been transferred. Currently one is an ongoing singleton pregnancy, one is an ongoing twin pregnancy and the third has resulted in a live birth. 35 embryos were biopsied and underwent genetic testing to confirm their ploidy status. Six of these were ICSI derived, four of which were found to be haploid and two diploid. The other 29 tested embryos were derived from IVF. Of these, 27 were found to be diploid (93.1%) and two were shown to be triploid (6.9%). None were confirmed as haploid. Traditionally, PGT-A methods using NGS examine relative chromosome copy number, but in cases of haploidy/triploidy all of the chromosomes are decreased/increased in number, meaning there is no change in the relative number of individual chromosomes. We established embryo ploidy status using an advanced PGT method, genotyping thousands of polymorphisms scattered across the genome, which are essential for accurate diagnosis of haploidy/triploidy. Limitations, reasons for caution The sample size for genetically tested ICSI derived 1PN’s is currently too small to clearly determine whether culture and testing of these embryos is beneficial to the patient. Patients who decided to transfer an embryo categorised as 1PN were made fully aware of the potential risks in doing so. Wider implications of the findings These findings show that the culture of 1PN embryos is clinically beneficial to a large number of patients, especially those with poor prognosis who would otherwise have had a failed cycle. Genetic testing demonstrates that IVF derived 1PN embryos that reach the blastocyst stage are likely to have fertilised normally. Trial registration number *

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.

To investigate the incidence, origin and clinical significance of segmental aneuploidy in human oocytes and preimplantation embryos. Retrospective analysis of preimplantation genetic screening (PGS) data from 4,104 oocytes and embryos of 690 IVF patients. 452 human oocytes, 1,762 cleavage stage and 1,890 blastocyst stage embryos were investigated. Comprehensive cytogenetic analysis, involving microarray comparative genomic hybridisation (aCGH) or next generation sequencing (NGS), was applied to polar bodies or cells biopsied from embryos. The incidence of segmental aneuploidy differed according to the stage at which the analysis was performed, affecting 10.39% (47/452) of oocytes, 24.29% (428/1762) of cleavage stage embryos and 15.59% (207/1327) of blastocysts. Hence, the frequency increased significantly after fertilisation (p<0.0001) but declined between the cleavage and blastocyst stages (p<0.0001). This suggests that most segmental aneuploidies spontaneously arise during the first few mitotic divisions. Patient age had no correlation with segmental aneuploidy. Analysis of the sites of chromosome breakage revealed hotspots occurring in specific regions, some corresponding to known fragile sites. The use of NGS for the analysis of 563 trophectoderm biopsies provided an insight into mosaicism. Mosaic segmental abnormality was detected in 122/563 biopsy specimens, higher than the incidence of such abnormalities affecting all of the biopsied cells (43/563) (p<0.0001). Transfer of embryos with mosaic segmental aneuploidy (8/13) was associated with a greater chance of ongoing pregnancy compared with embryos mosaic for whole chromosomal aneuploidy (4/31) (p=0.0021). Segmental aneuploidies are common in human embryos, yet the understanding of their genesis and clinical relevance is poor and appropriate clinical management is unclear. Should embryos with segmental aneuploidy be discarded or transferred? Our results show that the site of chromosomal breakage is predictive of the stage at which the abnormality arose and that this in turn influences the likelihood that the abnormality is mosaic. Importantly, this is of relevance to embryo viability and the potential to produce a healthy child.