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Humans present remarkable diversity in their mitochondrial DNA (mtDNA) in terms of variants across individuals as well as across tissues and even cells within one person. We have investigated the timing of the first appearance of this variant-driven mosaicism. For this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 embryos present blastomeres that carry variants only detected in that cell, showing that mtDNA mosaicism arises very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across different samples. Recurring variants had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants unique to one oocyte or single embryonic cell. These differences were maintained through development, suggesting that the mtDNA mosaicism arising in the embryo is maintained into adulthood. We observed a decline in potentially pathogenic variants between day 3 and day 5 of development, suggesting early selection. We propose a model in which closely clustered mitochondria carrying specific mtDNA variants in the ooplasm are asymmetrically distributed throughout the cell divisions of the preimplantation embryo, resulting in the earliest form of mtDNA mosaicism in human development.

Children born using assisted reproductive technologies (ART) have an increased risk of a lower birth weight, the cause of which remains unclear. As a causative factor, we hypothesized that variants in the mitochondrial DNA (mtDNA) that are not associated with disease, may explain changes in birth weight. We deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or cycles with ovarian stimulation (OS). The mtDNA genotypes were compared across groups and logistic regression and discriminant analysis were used to study the impact of the different factors on birth weight percentile. ART individuals more frequently carried variants with higher heteroplasmic loads in protein and rRNA-coding regions. These differences in the mitochondrial genome were also predictive of the risk of a lower birth weight percentile, irrespective of the mode of conception but with a sex-dependent culture medium effect. The higher incidence of these variants in ART individuals results both from maternal transmission and de novomutagenesis, which we found not to be caused by OS but to be associated to maternal ageing. MtDNA variants in protein and rRNA coding regions are associated with a lower birth weight and are more frequently observed in ART children. We propose that these non-disease associated variants can result in a suboptimal mitochondrial function that impacts birth weight. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future.

We know that polarity and YAP1 play a key role in trophectoderm initiation in compacted human embryos, however we know little about the TEAD family of transcription factors that become activated by YAP1 and especially if they play a role during epiblast and primitive endoderm formation. Here we show that compaction occurs heterogeneously between the 8- and 16-cell stages. While 8-cell stage blastomeres are not yet polarized, polarized outer cells and non-polarized inner cells arise in compacted 16-cell stage embryos. While trophectoderm specifiers TEAD1, YAP1 and GATA3 mostly co-localise in the nuclei of polarized outer/trophectoderm cells, they are also found in some cells of compacting embryos before polarity is established indicating that differentiation into trophectoderm cells can be initiated independently of polarity. In the inner cell mass, TEAD1 and YAP1 also distinguish GATA4 positive cells in a salt-and-pepper distribution and in the sorted primitive endoderm cells. Our detailed roadmap on polarization, compaction, position and lineage segregation events during human preimplantation development paves the road for further functional studies. Fundamental knowledge of lineage segregation events will eventually explain how and why embryos fail to develop further before or during implantation.

BACKGROUND As in other domains of medicine, high-throughput sequencing methods have led to the identification of an ever-increasing number of gene variants in the fields of both male and female infertility. The increasing number of recently identified genes allows an accurate diagnosis for previously idiopathic cases of female infertility and more appropriate patient care. However, robust evidence of the gene–disease relationships (GDR) allowing the proper translation to clinical application is still missing in many cases. OBJECTIVE AND RATIONALE An evidence-based curation of currently identified genes involved in female infertility and differences in sex development (DSD) would significantly improve both diagnostic performance and genetic research. We therefore performed a systematic review to summarize current knowledge and assess the available GDR. SEARCH METHODS PRISMA guidelines were applied to curate all available information from PubMed and Web of Science on genetics of human female infertility and DSD leading to infertility, from 1 January 1988 to 1 November 2021. The reviewed pathologies include non-syndromic as well as syndromic female infertility, and endocrine and reproductive system disorders. The evidence that an identified phenotype is caused by pathogenic variants in a specific gene was assessed according to a standardized scoring system. A final score (no evidence, limited, moderate, strong, or definitive) was assigned to every GDR. OUTCOMES A total of 45 271 publications were identified and screened for inclusion of which 1078 were selected for gene and variant extraction. We have identified 395 genes and validated 466 GDRs covering all reported monogenic causes of female infertility and DSD. Furthermore, we present a genetic diagnostic flowchart including 105 genes with at least moderate evidence for female infertility and suggest recommendations for future research. The study did not take into account associated genetic risk factor(s) or oligogenic/polygenic causes of female infertility. WIDER IMPLICATIONS We have comprehensively reviewed the existing research on the genetics of female infertility and DSD, which will enable the development of diagnostic panels using validated genes. Whole genome analysis is shifting from predominantly research to clinical application, increasing its diagnostic potential. These new diagnostic possibilities will not only decrease the number of idiopathic cases but will also render genetic counselling more effective for infertile patients and their families.

BackgroundChildren born using assisted reproductive technologies (ART) have an increased risk of a lower birth weight, the cause of which remains unclear. As a causative factor, we hypothesized that variants in the mitochondrial DNA (mtDNA) that are not associated with disease, may explain changes in birth weight.MethodsWe deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or cycles with ovarian stimulation (OS). The mtDNA genotypes were compared across groups and logistic regression and discriminant analysis were used to study the impact of the different factors on birth weight percentile.ResultsART individuals more frequently carried variants with higher heteroplasmic loads in protein and rRNA-coding regions. These differences in the mitochondrial genome were also predictive of the risk of a lower birth weight percentile, irrespective of the mode of conception but with a sex-dependent culture medium effect. The higher incidence of these variants in ART individuals results both from maternal transmission and de novo mutagenesis, which we found not to be caused by OS but to be associated to maternal ageing.ConclusionsMtDNA variants in protein and rRNA coding regions are associated with a lower birth weight and are more frequently observed in ART children. We propose that these non-disease associated variants can result in a suboptimal mitochondrial function that impacts birth weight. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future.

Objective: What are the cost per live birth and the incremental cost of preventing a miscarriage with preimplantation genetic testing for aneuploidy (PGT-A) by polar body biopsy and array-based comprehensive genome hybridisation (aCGH) versus regular IVF/ICSI without PGT-A for infertility treatment in women 36–40 years of age?. Design: Decision tree model. Population: A randomised clinical trial on PGT-A (ESTEEM study). Methods: Two treatment strategies were compared: one cycle of IVF/ICSI with or without PGT-A. Costs and effects were analysed with this model for four different cost scenarios: high-, higher medium, lower medium and low-cost. Base case, sensitivity, threshold, and probabilistic sensitivity analyses were used to examine the cost-effectiveness implications of PGT-A. Results: PGT-A increased the cost per live birth by approximately 15% in the high-cost scenario to approximately 285% in the low-cost scenario. Threshold analysis revealed that PGT-A would need to be associated with an absolute increase in pregnancy rate by 6% to >39% or, alternatively, would need to be US$2,969 (high-cost scenario) to US$4,888 (low-cost scenario) cheaper. The incremental cost to prevent one miscarriage by PGT-A using the base case assumptions was calculated to be US$34,427 (high-cost scenario) to US$51,146 (low-cost scenario). A probabilistic sensitivity analysis showed cost-effectiveness for PGT-A from 1.9% (high-cost scenario) to 0.0% (low-cost scenario) of calculated samples. Conclusions: While avoiding unnecessary embryo transfers and miscarriages are important goals, patients and doctors need to be aware of the high-cost implications of applying PGT-A using aCGH on polar bodies. Tweetable abstract: PGT-A by polar body biopsy and comprehensive genome hybridisation increases cost per live birth and requires high financial spending per miscarriage averted.

International audience; Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is knownabout fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss offunction of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has beenlinked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves a1,2-, a1,3-, and a1,6-mannose residues. In this study,we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants inMAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomaliessuch as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemisphericcysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experimentswith isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstratethat MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize theinvolvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

About 8 out of 10 human embryos obtained in vitro harbour chromosomal abnormalities of either meiotic or mitotic origin. Abnormalities of mitotic origin lead to chromosomal mosaicism, a phenomenon that has sparked much debate lately as it confounds results obtained through preimplantation genetic testing for aneuploidy (PGT-A). PGT-A in itself is still highly debated, not only on the modalities of its execution but also on whether it should be offered to patients at all. We will focus on post-zygotic chromosomal abnormalities leading to mosaicism. First, we will summarize what is known about the rates of chromosomal abnormalities at different developmental stages. Next, based on the current understanding of the origin and cellular consequences of chromosomal abnormalities, which is largely based on studies on cancer cells and model organisms, we will offer a number of hypotheses on which mechanisms may be at work in early human development. Finally, and very briefly, we will touch upon the impact our current knowledge has on the practice of PGT-A. What is the level of abnormal cells that an embryo can tolerate before it loses its potential for full development? And is blastocyst biopsy as harmless as it seems?

Humans present remarkable mitochondrial DNA (mtDNA) variant mosaicism, not only across tissues but even across individual cells within one person. The timing of the first appearance of this mosaicism has not yet been established. In this study, we hypothesized it occurs during preimplantation development. To investigate this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 preimplantation embryos already present blastomeres that carry variants unique to that cell, showing that the first events of mtDNA mosaicism happen very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across sibling oocytes and embryos, and between single cells and samples from the same embryos or adult individuals. Variants that recurred across samples had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants that were unique to one oocyte or single embryonic cell. These differences were maintained through developmental stages, suggesting that the mtDNA mosaicism arising in preimplantation development is maintained into adulthood. Further, the results support a model in which close clustering of mitochondria carrying specific mtDNA variants in the ooplasm leads to asymmetric distribution of these mitochondria throughout the cell divisions of the preimplantation embryo, resulting in the appearance of the first form of mtDNA mosaicism in human development.

Study question Are aneuploid cells in human preimplantation embryos eliminated by apoptosis due to proteotoxic stress and autophagy-mediated apoptosis? Summary answer Proteotoxic stress, autophagy and apoptosis are differentially activated in aneuploid embryos, showing that aneuploid cells are eliminated by these mechanisms during early human embryogenesis. What is known already Aneuploidies are a common feature of human preimplantation embryos which could explain low success rates after in vitro fertilization (IVF). While most aneuploidies of meiotic origin are detrimental, transfer of euploid-aneuploid mosaic embryos can lead to healthy live-births. Moreover, the proportion of aneuploid cells are lower in blastocysts when compared to cleavage stage embryos. In the mouse, aneuploid cells are eliminated from the epiblast by autophagy-mediated apoptosis in a p53-dependent manner. We propose that in human embryos, aneuploidy causes chronic protein misfolding which leads to autophagy-induced apoptosis. Study design, size, duration Eighty-one blastocysts that were diagnosed by PGT as euploid (n = 49) or uniformly combined abnormal (CA, n = 32), i.e. 2 or more chromosomes were abnormal in every cell, were warmed. Sixty-seven were suitable for trophectoderm (TE) biopsy, 54 biopsies were successfully tubed and sent for RNA-sequencing while the remainder of the embryos was fixed for immunostaining. Thirty-three day-3 embryos were overnight incubated in 0.5µM reversine allowed to develop into blastocysts and treated as the PGT embryos. Participants/materials, setting, methods After TE biopsy, we live-stained the embryos with either Caspase-3/7 or 8 and subsequently fixed them. The biopsies underwent RNA-sequencing using the SMART-seqv4 and the fixed embryos were immunostained for LC3B, p62 (autophagy) and HSP70 (proteotoxic stress). Confocal imaging was performed using a Zeiss LSM800 confocal microscope and the presence of signal was quantified using the Zen Blue 2.0 and Arivis software. Main results and the role of chance Forty-two percent of the embryos in which we induced aneuploidies using reversine developed into blastocysts, which is comparable to untreated embryos. After immunostaining, we observed that CA and reversine-treated (RT) embryos contained less cells than euploid embryos (median number of nuclei: 43.5, 47, 90, respectively). This correlates with a higher expression of apoptotic markers Caspase-3/7 in CA embryos (p = 0.0199) and Caspase-8 in both aneuploid groups (CA: p = 0.0085 and RT: p = 0.0394). Aneuploid embryos showed significantly increased HSP70 levels (median intensity per cell: euploid=165, CA = 313, RT = 400), LC3B (median puncta per cell: euploid=3.07, CA = 10.10, RT = 19.62) and p62 (median puncta per cell: euploid=17.60, CA = 30.53), suggesting increased proteotoxic stress and autophagy. Preliminary analysis of the RNA-sequencing data reveals enrichment for pathways such as the p53-pathway, protein secretion, TNFA signaling via NFkB and apoptosis, supporting the hypothesis of a link between aneuploidy and apoptosis. Limitations, reasons for caution No functional tests e.g. with inhibitors of autophagy were carried out. RNA-sequencing was carried out on a small sample; we will expand this sample in the near future. Wider implications of the findings This study shows for the first time the mechanism by which aneuploid cells are eliminated from the human preimplantation embryo, explaining how mosaic embryos can still lead to a healthy and genetically normal live birth. Trial registration number not applicable

Study question Can mitochondrial DNA (mtDNA) variants explain the differences in birthweight between ART and spontaneously conceived (SC) individuals and how do they originate? Summary answer Children born after ART carry more frequently a different mtDNA variant composition, both maternally inherited and de novo, which are predictive of their birthweight percentile. What is known already Children born after ART show an increased risk of lower birthweight and of developing a mild abnormal cardio-metabolic profile later in life. Variation in the mtDNA associates with overall health in the general population, including cardio-metabolic fitness, and can result in changes in mitochondrial function. We hypothesized that mitochondrial DNA variants could explain the differences in birthweight between ART and SC individuals and that these differences may result from maternal transmission and/or from the ovarian stimulation (OS) used in ART. Study design, size, duration We deep-sequenced the mtDNA of 472 individuals of who 283 ART and 189 SC, 182 mother-child pairs and 113 single oocytes from both natural menstrual cycles and OS cycles. The mtDNA was compared between groups and Fisher linear discriminant analysis was used as predictive model for the birthweight percentile. Participants/materials, setting, methods Mitochondrial DNA was enriched by long-range PCR and subsequently sequenced on an Illumina platform. mtDNA server and MuTect were used for variant calling for variants with a load higher than 1.5%, versus the reference NC_012920.1. An orthogonally rotated factor analysis was used to reduce the dimensionality of the studied dependent variables in the complex data of the heteroplasmic variants. Main results and the role of chance ART individuals carried more frequently haplogroup U4 (p = 0.004) and component analysis indicated that they carry a different mtDNA heteroplasmic variant composition than SC individuals (p = 0.01), driven by non-synonymous protein-coding and rRNA-coding variants. These differences were also predictive of the risk of a lower birthweight percentile, especially for the SC children, together with the absence of haplogroup T, the presence of homoplasmic tRNA-variants, pregnancy-induced hypertension and the embryo culture medium used. The differences in heteroplasmic variation observed in the ART children resulted from both maternal transmission (p = 0.03) and de novo mutagenesis (p = 0.02). Mothers of ART children showed a similar mtDNA genotype as their children and differed in the same variant composition when compared to the mothers of SC children (p = 0.03). Furthermore, the comparison of oocytes from the same donors retrieved in natural menstrual cycles and after one OS cycle showed that OS does not increase de novo mutagenesis. Additionally, clinical parameters such as the total dosage of FSH units, the number of oocytes retrieved, and maternal age did not show any correlation with the differences observed in ART individuals. Limitations, reasons for caution This study is observational with no functional tests being performed. Wider implications of the findings We demonstrate an association between a lower birthweight percentile and a mtDNA variant composition which is more frequently carried by ART children. These non-disease associated mtDNA variants could cause a suboptimal mitochondrial function affecting the birthweight. Long-term health consequences of these differences remain to be further elucidated. Trial registration number Not applicable

SummaryThough gains of chromosome 12p13.31 are highly recurrent in hPSC, their impact on differentiation is poorly understood. We identify a reduction in differentiation capacity towards all three germ layers and a subpopulation of residual pluripotent cells that appear during hepatic specification. These cells form as a result of the overexpression of NANOG and GDF3, whereby NANOG as the primary driver delays activation of WNT signaling, partly as a result of a direct physical interaction with TCF7. Entry into the residual state is determined by cell cycle position at the onset of differentiation and is maintained by a feedback loop between NANOG and GDF3. These findings highlight the ability of genetically abnormal hPSC to escape correct differentiation and to form residual pluripotent cells, an important risk in the safe clinical translation of hPSC. Our results further refine the molecular mechanisms that underpin the exit from pluripotency and onset of differentiation.

We identified a human embryonic stem cell subline that fails to respond to the differentiation cues needed to obtain endoderm derivatives, differentiating instead into extra-embryonic mesoderm. RNA-sequencing analysis showed that the subline has hyperactivation of the WNT and BMP4 signalling. Modulation of these pathways with small molecules confirmed them as the cause of the differentiation impairment. While activation of WNT and BMP4 in control cells resulted in a loss of endoderm differentiation and induction of extra-embryonic mesoderm markers, inhibition of these pathways in the subline restored its ability to differentiate. Karyotyping and exome sequencing analysis did not identify any changes in the genome that could account for the pathway deregulation. These findings add to the increasing evidence that different responses of stem cell lines to differentiation protocols are based on genetic and epigenetic factors, inherent to the line or acquired during cell culture.

Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology.

Low differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.

We identified a human embryonic stem cell subline that fails to respond to the differentiation cues needed to obtain mesendoderm derivatives, differentiating into trophoblast-like cells instead. RNA-sequencing analysis showed that the subline has hyperactivation of the WNT and BMP4 signalling. Modulation of these pathways with small molecules confirmed them as the cause of the differentiation impairment. While activation of WNT and BMP4 in control cells resulted in a loss of mesendoderm differentiation and induction of trophoblast markers, inhibition of these pathways in the subline restored its ability to differentiate. Karyotyping and exome sequencing analysis did not identify any changes in the genome that could account for the pathway deregulation. These findings add to the increasing evidence that different responses of stem cell lines to differentiation protocols are based on genetic and epigenetic factors, inherent to the line or acquired during cell culture.

Study question: How is the trophectoderm lineage specified in the human preimplantation embryo? Summary answer: Outer cells at the fully compacted embryo stage display characteristics of trophectoderm cells such as apical polar distribution of p-ERM and nuclear GATA3/YAP1/TEAD4 protein expression. What is known already: In mice, the first indication of inner cell mass (ICM) and trophectoderm (TE) segregation occurs three days post fertilisation (dpf3). After compaction, asymmetric divisions give rise to inner and outer blastomeres that ubiquitously express transcription factor TEAD4 and sequester polarity markers (p- ERM), and TE lineage specifiers (YAP1, GATA3) to outer blastomeres. This is considered the onset of TE lineage segregation in the mammalian embryo. Studies regarding this event in the human embryo are absent and extrapolations from the mouse model cannot be a surrogate for studies in the human embryo. Study design, size, duration: Human embryos used in this study, were donated to research as a surplus of IVF-ICSI treatment at our IVF-center after the cryo-storage expiration date of 5 years with informed consent. The project was approved by the institutional ethical committee and the federal committee for research on human embryos. Participants/materials, setting, methods: Cryopreserved human 8-cell stage embryos, donated to research, or zygotes created for research after informed consent were warmed (Vit Kit -Thaw, Irvine Scientific, USA) and cultured until dpf5 according to standard laboratory procedures of the associated IVF-clinic. Time course immunofluorescence was performed on fixed embryos at predefined stages for TEAD4, YAP1, GATA3, p-ERM as well as F-actin and Hoechst for imaging (LSM800, ZEISS) and the manual estimation of individual blastomere counts per embryo (ImageJ). Main results and the role of chance: The compaction process of the human embryo is interrupted by cleavage divisions this results in a fully compacted embryo at dpf4.These fully compacted embryos are comprised of a total of 16±3 cells, n=21, of which 76%, n=16/21 contain 1±1 inner blastomere in their core excluded from the exterior. The nuclei of the outer blastomeres are positive for TE-specifier YAP1 (69±22%, n=8) and begin to co-express TEtranscription factor GATA3 (15±22%, n=8). The apical membranes of the outer blastomeres appear polarised by p-ERM staining (100%, n=8) and TEAD4 is expressed ubiquitously (100%, n=8). While inner cells also express TEAD4 they remain apolar and rarely express YAP1. This inner/outer pattern is maintained in the blastocyst ICM and TE. All nuclei of TE cells of dpf5 blastocysts are positive for YAP1/GATA3 and TEAD4 and apical membranes of TE cells show polar distribution of p-ERM. Whereas the ICM remains apolar, TEAD4 positive, GATA3 negative, and very rarely contains YAP1 positive cells. Limitations, reasons for caution: In spite of available functional data on the interaction between YAP1 and TEAD4 and their effect on GATA3 expression and embryo development in the mouse and the bovine model, functional experiments in the human embryo still need to confirm the link between polarity, nuclear TEAD4/YAP1/GATA3, TE specification and blastocyst formation. Wider implications of the findings: The same molecular determinants are involved in the TE lineage segregation of mice and humans, but their expression differs according to the morphological development of the respective species. In contrast to the stepwise model o

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra-embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4-treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4-treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.

Summary In this study, we deep-sequenced the mtDNA of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) and their source cells and found that the majority of variants pre-existed in the cells used to establish the lines. Early-passage hESCs carried few and low-load heteroplasmic variants, similar to those identified in oocytes and inner cell masses. The number and heteroplasmic loads of these variants increased with prolonged cell culture. The study of 120 individual cells of early- and late-passage hESCs revealed a significant diversity in mtDNA heteroplasmic variants at the single-cell level and that the variants that increase during time in culture are always passenger to the appearance of chromosomal abnormalities. We found that early-passage hiPSCs carry much higher loads of mtDNA variants than hESCs, which single-fibroblast sequencing proved pre-existed in the source cells. Finally, we show that these variants are stably transmitted during short-term differentiation., Graphical Abstract, Highlights • Early-passage hESCs carry mainly mtDNA variants inherited from the embryos • Clonal culture takeover results in increased variant load and pathogenic potential • hESCs and somatic cell cultures are mosaic in terms of mtDNA genotypes • hiPSCs carry high levels of variants due to clonal reprogramming of a mosaic source, In this work, Spits and colleagues present an in-depth study of mtDNA variants in hPSCs. Early-passage hESCs carry few low-frequency variants, while higher variant loads are observed in late-passage hESCs and hiPSCs. The presence of variants is determined by the somatic or embryonic origin of the line and modulated by random drift, spontaneous mutagenesis, and clonal culture takeover.

CTG repeat expansions in DMPK cause myotonic dystrophy (DM1) with a continuum of severity and ages of onset. Congenital DM1 (CDM1), the most severe form, presents distinct clinical features, large expansions, and almost exclusive maternal transmission. The correlation between CDM1 and expansion size is not absolute, suggesting contributions of other factors. We determined CpG methylation flanking the CTG repeat in 79 blood samples from 20 CDM1-affected individuals; 21, 27, and 11 individuals with DM1 but not CDM1 (henceforth non-CDM1) with maternal, paternal, and unknown inheritance; and collections of maternally and paternally derived chorionic villus samples (7 CVSs) and human embryonic stem cells (4 hESCs). All but two CDM1-affected individuals showed high levels of methylation upstream and downstream of the repeat, greater than non-CDM1 individuals (p = 7.04958 × 10 −12 ). Most non-CDM1 individuals were devoid of methylation, where one in six showed downstream methylation. Only two non-CDM1 individuals showed upstream methylation, and these were maternally derived childhood onset, suggesting a continuum of methylation with age of onset. Only maternally derived hESCs and CVSs showed upstream methylation. In contrast, paternally derived samples (27 blood samples, 3 CVSs, and 2 hESCs) never showed upstream methylation. CTG tract length did not strictly correlate with CDM1 or methylation. Thus, methylation patterns flanking the CTG repeat are stronger indicators of CDM1 than repeat size. Spermatogonia with upstream methylation may not survive due to methylation-induced reduced expression of the adjacent SIX5 , thereby protecting DM1-affected fathers from having CDM1-affected children. Thus, DMPK methylation may account for the maternal bias for CDM1 transmission, larger maternal CTG expansions, age of onset, and clinical continuum, and may serve as a diagnostic indicator.

Human pluripotent stem cells (hPSCs) have significant levels of low-grade genetic mosaicism, which commonly used techniques fail to detect in bulk DNA. These copy number variations remain a hurdle for the clinical translation of hPSC, as their effect in vivo ranges from unknown to dangerous, and the ability to detect them will be necessary as the field advances. As such there is need for techniques which can efficiently analyse genetic content in single cells with higher throughput and lower costs. We report here on the use of the Fluidigm C1 single cell WGA platform in combination with shallow whole genome sequencing to analyse the genetic content of single hPSCs. From a hPSC line carrying an isochromosome 20, 56 single cells were analysed and found to carry a total of 50 aberrations, across 23% of cells, which could not be detected by bulk analysis. Aberrations were predominantly segmental gains, with a fewer number of segmental losses and aneuploidies. Interestingly, 40% of the breakpoints seen here correspond to known DNA fragile sites. Our results therefore demonstrate the feasibility of single cell shallow sequencing of hPSC and further expand upon the biological importance and frequency of single cell mosaicism in hPSC.

Summary Gain of 20q11.21 is one of the most common recurrent genomic aberrations in human pluripotent stem cells. Although it is known that overexpression of the antiapoptotic gene Bcl-xL confers a survival advantage to the abnormal cells, their differentiation capacity has not been fully investigated. RNA sequencing of mutant and control hESC lines, and a line transgenically overexpressing Bcl-xL, shows that overexpression of Bcl-xL is sufficient to cause most transcriptional changes induced by the gain of 20q11.21. Moreover, the differentially expressed genes in mutant and Bcl-xL overexpressing lines are enriched for genes involved in TGF-β- and SMAD-mediated signaling, and neuron differentiation. Finally, we show that this altered signaling has a dramatic negative effect on neuroectodermal differentiation, while the cells maintain their ability to differentiate to mesendoderm derivatives. These findings stress the importance of thorough genetic testing of the lines before their use in research or the clinic., Highlights • Bcl-xL overexpression drives the transcriptomic profile of 20q11.21 mutant lines • 20q11.21 mutant lines downregulate CHCHD2, a known TGF-β pathway modulator • Mutant lines differentially express genes involved in TGF-β and SMAD signaling • Mutant lines show impaired ectoderm commitment due to TGF-β signaling deregulation, In this article Spits and colleagues show that Bcl-xL overexpression is the main driver of the distinct transcriptomic profile of human embryonic stem cells with a gain of 20q11.21. The cells have an abnormal TGF-β- and SMAD-mediated signaling, which impairs commitment to neuroectoderm while differentiation to the other lineages remains unaffected.

Detecting heteroplasmies in the mitochondrial DNA (mtDNA) has been a challenge for many years. In the past, Sanger sequencing was the main option to perform this analysis, however, this method could not detect low frequency heteroplasmies. Massive Parallel Sequencing (MPS) provides the opportunity to study the mtDNA in depth, but a controlled pipeline is necessary to reliably retrieve and quantify the low frequency variants. It has been shown that differences in methods can significantly affect the number and frequency of the retrieved variants. In this protocol, we present a method involving both wet lab and bioinformatics that allows identifying and quantifying single nucleotide variants in the full mtDNA sequence, down to a heteroplasmic load of 1.5%. For this, we set up a PCR-based amplification of the mtDNA, followed by MPS using Illumina chemistry, and variant calling with two different algorithms, mtDNA server and Mutect. The PCR amplification is used to enrich the mitochondrial fraction, while the bioinformatic processing with two algorithms is used to discriminate the true heteroplasmies from background noise. The protocol described here allows for deep sequencing of the mitochondrial DNA in bulk DNA samples as well as single cells (both large cells such as human oocytes, and small-sized single cells such as human embryonic stem cells) with minor modifications to the protocol.

STUDY QUESTION: We wanted to probe the opinions and current practices on preimplantation genetic screening (PGS), and more specifically on PGS in its newest form: PGS 2.0? STUDY FINDING: Consensus is lacking on which patient groups, if any at all, can benefit from PGS 2.0 and, a fortiori, whether all IVF patients should be offered PGS. WHAT IS KNOWN ALREADY: It is clear from all experts that PGS 2.0 can be defined as biopsy at the blastocyst stage followed by comprehensive chromosome screening and possibly combined with vitrification. Most agree that mosaicism is less of an issue at the blastocyst stage than at the cleavage stage but whether mosaicism is no issue at all at the blastocyst stage is currently called into question. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A questionnaire was developed on the three major aspects of PGS 2.0: the Why, with general questions such as PGS 2.0 indications; the How, specifically on genetic analysis methods; the When, on the ideal method and timing of embryo biopsy. Thirty-five colleagues have been selected to address these questions on the basis of their experience with PGS, and demonstrated by peer-reviewed publications, presentations at meetings and participation in the discussion. The first group of experts who were asked about 'The Why' comprised fertility experts, the second group of molecular biologists were asked about 'The How' and the third group of embryologists were asked about 'The When'. Furthermore, the geographical distribution of the experts has been taken into account. Thirty have filled in the questionnaire as well as actively participated in the redaction of the current paper. MAIN RESULTS AND THE ROLE OF CHANCE: The 30 participants were from Europe (Belgium, Germany, Greece, Italy, Netherlands, Spain, UK) and the USA. Array comparative genome hybridization is the most widely used method amongst the participants, but it is slowly being replaced by massive parallel sequencing. Most participants offering PGS 2.0 to their patients prefer blastocyst biopsy. The high efficiency of vitrification of blastocysts has added a layer of complexity to the discussion, and it is not clear whether PGS in combination with vitrification, PGS alone, or vitrification alone, followed by serial thawing and eSET will be the favoured approach. The opinions range from in favour of the introduction of PGS 2.0 for all IVF patients, over the proposal to use PGS as a tool to rank embryos according to their implantation potential, to scepticism towards PGS pending a positive outcome of robust, reliable and large-scale RCTs in distinct patient groups. LIMITATIONS, REASONS FOR CAUTION: Care was taken to obtain a wide spectrum of views from carefully chosen experts. However, not all invited experts agreed to participate, which explains a lack of geographical coverage in some areas, for example China. This paper is a collation of current practices and opinions, and it was outside the scope of this study to bring a scientific, once-and-for-all solution to the ongoing debate. WIDER IMPLICATIONS OF THE FINDINGS: This paper is unique in that it brings together opinions on PGS 2.0 from all different perspectives and gives an overview of currently applied technologies as well as potential future developments. It will be a useful reference for fertility specialists with an expertise outside reproductive genetics. LARGE SCALE DATA: none. STUDY FUNDING AND COMPETING INTERESTS: No specific funding was obtained to conduct this questionnaire.

During the last few years a new generation of preimplantation genetic screening (PGS) has been introduced. In this paper, an overview of the different aspects of this so-called PGS 2.0 with respect to the why (what are the indications), the when (which developmental stage, i.e. which material should be studied) and the how (which molecular technique should be used) is given. With respect to the aims it is clear that PGS 2.0 can be used for a variety of indications. However, the beneficial effect of PGS 2.0 has not been proved yet in RCTs. It is clear that cleavage stage is not the optimal stage for biopsy. Almost all advocates of PGS 2.0 prefer trophectoderm biopsy. There are many new methods that allow the study of complete aneuploidy with respect to one or more of the 24 chromosomes. Because of the improved vitrification methods, selection of fresh embryos for transfer is more and more often replaced by frozen embryo transfer. The main goal of PGS has always been the improvement of IVF success. However, success is defined by different authors in many different ways. This makes it very difficult to compare the outcomes of different studies. In conclusion, the introduction of PGS 2.0 will depend on the success of the new biopsy strategies in combination with the analysis of all 24 chromosomes. It remains to be seen which approach will be the most successful and for which specific groups of patients.

Summary Human embryonic stem cells (hESC) show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term) impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem., Graphical Abstract, Highlights • Increased culture density induces DNA damage and genomic alterations in hESC • Medium acidification due to lactic acid accumulation is the main driver • More frequent medium refreshments rescues genomic integrity in high-density culture • Laminin-521 reduces DNA damage but has no clear effect on genomic instability, In this article, Spits and colleagues show that the culture density of hESC is directly correlated to increased DNA damage and genomic instability. This is caused by medium acidification and can be countered by more frequent medium refreshments. When cultured on laminin-521, hESC show less DNA damage than in the feeder-based system, but no clear difference in genomic instability was found.

BACKGROUND: With the recent development of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing technology, the possibility to genetically manipulate the human germline (gametes and embryos) has become a distinct technical possibility. Although many technical challenges still need to be overcome in order to achieve adequate efficiency and precision of the technology in human embryos, the path leading to genome editing has never been simpler, more affordable, and widespread. OBJECTIVE AND RATIONALE: In this narrative review we seek to understand the possible impact of CRISR/Cas9 technology on human reproduction from the technical and ethical point of view, and suggest a course of action for the scientific community. SEARCH METHODS: This non-systematic review was carried out using Medline articles in English, as well as technical documents from the Human Fertilisation and Embryology Authority and reports in the media. The technical possibilities of the CRISPR/Cas9 technology with regard to human reproduction are analysed based on results obtained in model systems such as large animals and laboratory rodents. Further, the possibility of CRISPR/Cas9 use in the context of human reproduction, to modify embryos, germline cells, and pluripotent stem cells is reviewed based on the authors' expert opinion. Finally, the possible uses and consequences of CRISPR/cas9 gene editing in reproduction are analysed from the ethical point of view. OUTCOMES: We identify critical technical and ethical issues that should deter from employing CRISPR/Cas9 based technologies in human reproduction until they are clarified. WIDER IMPLICATIONS: Overcoming the numerous technical limitations currently associated with CRISPR/Cas9 mediated editing of the human germline will depend on intensive research that needs to be transparent and widely disseminated. Rather than a call to a generalized moratorium, or banning, of this type of research, efforts should be placed on establishing an open, international, collaborative and regulated research framework. Equally important, a societal discussion on the risks, benefits, and preferred applications of the new technology, including all relevant stakeholders, is urgently needed and should be promoted, and ultimately guide research priorities in this area. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Chemotherapy resistance is responsible for high mortality rates in neuroblastoma. MYCN, an oncogenic driver in neuroblastoma, controls pluripotency genes including LIN28B. We hypothesized that enhanced embryonic stem cell (ESC) gene regulatory programs could mark tumors with high pluripotency capacity and subsequently increased risk for therapy failure. An ESC miRNA signature was established based on publicly available data. In addition, an ESC mRNA signature was generated including the 500 protein coding genes with the highest positive expression correlation with the ESC miRNA signature score in 200 neuroblastomas. High ESC m(i)RNA expression signature scores were significantly correlated with poor neuroblastoma patient outcome specifically in the subgroup of MYCN amplified tumors and stage 4 nonamplified tumors. Further data-mining identified FOXM1, as the major predicted driver of this ESC signature, controlling a large set of genes implicated in cell cycle control and DNA damage response. Of further interest, re-analysis of published data showed that MYCN transcriptionally activates FOXM1 in neuroblastoma cells. In conclusion, a novel ESC m(i)RNA signature stratifies neuroblastomas with poor prognosis, enabling the identification of therapy-resistant tumors. The finding that this signature is strongly FOXM1 driven, warrants for drug design targeted at FOXM1 or key components controlling this pathway.

Study question When does germ cell loss and fibrosis occur in patients with Klinefelter syndrome (KS)? Summary answer In KS, germ cell loss is not observed in testicular tissue from fetuses in the second semester of pregnancy but present at a prepubertal age when the testicular architecture is still normal, while fibrosis is highly present at an adolescent age. What is known already Most KS patients are azoospermic at adult age because of a massive germ cell loss. However, the timing when this germ cell loss starts is not known. It is assumed that germ cell loss increases at puberty. Therefore, testicular sperm extraction (TESE) at an adolescent age has been suggested to increase the chances of sperm retrieval at onset of spermatogenesis. However, recent data indicate that testicular biopsies from peripubertal KS patients contain only a few germ cells. Study design, size, duration In this study, we give an update on fertility preservation in adolescent KS patients and evaluate whether fertility preservation would be beneficial at prepubertal age. The possibility of retrieving testicular spermatozoa by TESE was evaluated in adolescent and adult KS men. The presence of spermatogonia and the degree of fibrosis were also analysed in testicular biopsies from KS patients at different ages. The patients were divided into four age groups: foetal (n = 5), prepubertal (aged 4-7 years; n = 4), peripubertal (aged 12-16 years; n = 20) and adult (aged 18-41 years; n = 27) KS patients. Participants/materials, setting, methods In peripubertal and adult KS patients, retrieval of spermatozoa was attempted by semen analysis after masturbation, vibrostimulation, electroejaculation or by TESE. MAGE-A4 immunohistochemistry was performed to evaluate the presence of germ cells in testicular biopsies from foetal, prepubertal, peripubertal and adult KS patients. Tissue morphology was evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Main results and the role of chance Testicular spermatozoa were collected by TESE in 48.1% of the adult KS patients, while spermatozoa were recovered after TESE in only one peripubertal patient (5.0%). Germ cells were detectable in testicular biopsies from 21% of adult men for whom no spermatozoa could be retrieved by TESE and in 31.5% of peripubertal KS boys. Very small numbers of spermatogonia (0.03-0.06 spermatogonia/tubule) were detected in three out of four (75%) prepubertal patients. At a foetal age, the number of germ cells was similar for KS and control samples. Increased signs of fibrosis were not present at foetal and prepubertal ages, but peripubertal and adult KS patients showed high levels of fibrosis. Large scale data N/A. Limitations, reasons for caution Only four prepubertal biopsies were included in this study, but they all showed a very low germ cell number. A high variability in the number of spermatogonia per mm2 was observed in the limited (n = 5) number of foetal biopsies. However, testicular biopsies from prepubertal and foetal Klinefelter patients are difficult to obtain. Wider implications of the findings Testicular tissue banking at a prepubertal age has been suggested as a potential method for fertility preservation in early diagnosed KS boys. However, our results show that a reduction in germ cell number has already taken place in childhood. Therefore, offering testicular tissue banking in young KS boys to prevent subsequent sterility might be a questionable strategy. However, this should be confirmed in a larger study population. Study funding/competing interest(s) This project was funded by the scientific Fund Willy Gepts from the UZ Brussel (D.V.S., J.D.S.), grants from the Vrije Universiteit Brussel (E.G.) and a Methusalem grant (K.S.). D.V.S is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

The derivation of gametes from patient-specific pluripotent stem cells may provide new perspectives for genetic parenthood for patients currently facing sterility. We use current data to assess the gamete differentiation potential of patient-specific pluripotent stem cells and to determine which reprogramming strategy holds the greatest promise for future clinical applications. First, we compare the two best established somatic cell reprogramming strategies: the production of induced pluripotent stem cells (iPSC) and somatic cell nuclear transfer followed by embryonic stem cell derivation (SCNT-ESC). Recent reports have indicated that these stem cells, though displaying a similar pluripotency potential, show important differences at the epigenomic level, which may have repercussions on their applicability. By comparing data on the genetic and epigenetic stability of these cell types during derivation and in-vitro culture, we assess the reprogramming efficiency of both technologies and possible effects on the subsequent differentiation potential of these cells. Moreover, we discuss possible implications of mitochondrial heteroplasmy. We also address the ethical aspects of both cell types, as well as the safety considerations associated with clinical applications using these cells, e.g. the known genomic instability of human PSCs during long-term culture. Secondly, we discuss the role of the stem cell pluripotency state in germ cell differentiation. In mice, success in germ cell development from pluripotent stem cells could only be achieved when starting from a naive state of pluripotency. It remains to be investigated if the naive state is also crucial for germ cell differentiation in human cells and to what extent human naive pluripotency resembles the naive state in mouse.

STUDY QUESTION: Does preimplantation genetic testing for aneuploidy (PGT-A) by comprehensive chromosome screening (CCS) of the first and second polar body to select embryos for transfer increase the likelihood of a live birth within 1 year in advanced maternal age women aged 36-40 years planning an ICSI cycle, compared to ICSI without chromosome analysis? SUMMARY ANSWER: PGT-A by CCS in the first and second polar body to select euploid embryos for transfer does not substantially increase the live birth rate in women aged 36-40 years. WHAT IS KNOWN ALREADY: PGT-A has been used widely to select embryos for transfer in ICSI treatment, with the aim of improving treatment effectiveness. Whether PGT-A improves ICSI outcomes and is beneficial to the patients has remained controversial. STUDY DESIGN, SIZE, DURATION: This is a multinational, multicentre, pragmatic, randomized clinical trial with intention-to-treat analysis. Of 396 women enroled between June 2012 and December 2016, 205 were allocated to CCS of the first and second polar body (study group) as part of their ICSI treatment cycle and 191 were allocated to ICSI treatment without chromosome screening (control group). Block randomization was performed stratified for centre and age group. Participants and clinicians were blinded at the time of enrolment until the day after intervention. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile couples in which the female partner was 36-40 years old and who were scheduled to undergo ICSI treatment were eligible. In those assigned to PGT-A, array comparative genomic hybridization (aCGH) analysis of the first and second polar bodies of the fertilized oocytes was performed using the 24sure array of Illumina. If in the first treatment cycle all oocytes were aneuploid, a second treatment with PB array CGH was offered. Participants in the control arm were planned for ICSI without PGT-A. Main exclusion criteria were three or more previous unsuccessful IVF or ICSI cycles, three or more clinical miscarriages, poor response or low ovarian reserve. The primary outcome was the cumulative live birth rate after fresh or frozen embryo transfer recorded over 1 year after the start of the intervention. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 205 participants in the chromosome screening group, 50 (24%) had a live birth with intervention within 1 year, compared to 45 of the 191 in the group without intervention (24%), a difference of 0.83% (95% CI: -7.60 to 9.18%). There were significantly fewer participants in the chromosome screening group with a transfer (relative risk (RR) = 0.81; 95% CI: 0.74-0.89) and fewer with a miscarriage (RR = 0.48; 95% CI: 0.26-0.90). LIMITATIONS, REASONS FOR CAUTION: The targeted sample size was not reached because of suboptimal recruitment; however, the included sample allowed a 90% power to detect the targeted increase. Cumulative outcome data were limited to 1 year. Only 11 patients out of 32 with exclusively aneuploid results underwent a second treatment cycle in the chromosome screening group. WIDER IMPLICATIONS OF THE FINDINGS: The observation that the similarity in birth rates was achieved with fewer transfers, less cryopreservation and fewer miscarriages points to a clinical benefit of PGT-A, and this form of embryo selection may, therefore, be considered to minimize the number of interventions while producing comparable outcomes. Whether these benefits outweigh drawbacks such as the cost for the patient, the higher workload for the IVF lab and the potential effect on the children born after prolonged culture and/or cryopreservation remains to be shown. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the European Society of Human Reproduction and Embryology. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. M.B.'s institution (UZBrussel) has received educational grants from IBSA, Ferring, Organon, Schering-Plough, Merck and Merck Belgium. M.B. has received consultancy and speakers' fees from Organon, Serono Symposia and Merck. G.G. has received personal fees and non-financial support from MSD, Ferring, Merck-Serono, Finox, TEVA, IBSA, Glycotope, Abbott and Gedeon-Richter as well as personal fees from VitroLife, NMC Healthcare, ReprodWissen, BioSilu and ZIVA. W.V., C.S., P.M.B., V.G., G.A., M.D.,T.E.G., L.G., G.Ka., G.Ko., J. L., M.C.M., M.P., A. S., M. T., K.V., J.G. and K.S. declare no conflict of interest. TRIAL REGISTRATION NUMBER: NCT01532284. TRIAL REGISTRATION DATE: 7 February 2012. DATE OF FIRST PATIENT'S ENROLMENT: 25 June 2012.

STUDY QUESTION Are there effective and clinically validated stem cell-based therapies for reproductive diseases? SUMMARY ANSWER At the moment, clinically validated stem cell treatments for reproductive diseases and alterations are not available. WHAT IS KNOWN ALREADY Research in stem cells and regenerative medicine is growing in scope, and its translation to the clinic is heralded by the recent initiation of controlled clinical trials with pluripotent derived cells. Unfortunately, stem cell 'treatments' are currently offered to patients outside of the controlled framework of scientifically sound research and regulated clinical trials. Both physicians and patients in reproductive medicine are often unsure about stem cells therapeutic options. STUDY DESIGN, SIZE, DURATION An international working group was assembled to review critically the available scientific literature in both the human species and animal models. PARTICIPANTS/MATERIALS, SETTING, METHODS This review includes work published in English until December 2014, and available through Pubmed. MAIN RESULTS AND THE ROLE OF CHANCE A few areas of research in stem cell and reproductive medicine were identified: in vitro gamete production, endometrial regeneration, erectile dysfunction amelioration, vaginal reconstruction. The stem cells studied range from pluripotent (embryonic stem cells and induced pluripotent stem cells) to monopotent stem cells, such as spermatogonial stem cells or mesenchymal stem cells. The vast majority of studies have been carried out in animal models, with data that are preliminary at best. LIMITATIONS, REASONS FOR CAUTION This review was not conducted in a systematic fashion, and reports in publications not indexed in Pubmed were not analyzed. WIDER IMPLICATIONS OF THE FINDINGS A much broader clinical knowledge will have to be acquired before translation to the clinic of stem cell therapies in reproductive medicine; patients and physicians should be wary of unfounded claims of improvement of existing medical conditions; at the moment, effective stem cell treatment for reproductive diseases and alterations is not available. STUDY FUNDING/COMPETING INTERESTS None. TRIAL REGISTRATION NUMBER NA.

Background Human pluripotent stem cell (hPSC) lines are known to have a bias in their differentiation. This gives individual cell lines a propensity to preferentially differentiate towards one germ layer or cell type over others. Chromosomal aberrations, mitochondrial mutations, genetic diversity and epigenetic variance are the main drivers of this phenomenon, and can lead to a wide range of phenotypes. Objective and rationale Our aim is to provide a comprehensive overview of the different factors which influence differentiation propensity. Specifically, we sought to highlight known genetic variances and their mechanisms, in addition to more general observations from larger abnormalities. Furthermore, we wanted to provide an up-to-date list of a growing number of predictive indicators which are able to identify differentiation propensity before the initiation of differentiation. As differentiation propensity can lead to difficulties in both research as well as clinical translation, our thorough overview could be a useful tool. Search methods Combinations of the following key words were applied as search criteria in the PubMed database: embryonic stem cells, induced pluripotent stem cells, differentiation propensity (also: potential, efficiency, capacity, bias, variability), epigenetics, chromosomal abnormalities, genetic aberrations, X chromosome inactivation, mitochondrial function, mitochondrial metabolism, genetic diversity, reprogramming, predictive marker, residual stem cell, clinic. Only studies in English were included, ranging from 2000 to 2017, with a majority ranging from 2010 to 1017. Further manuscripts were added from cross-references. Outcomes Differentiation propensity is affected by a wide variety of (epi)genetic factors. These factors clearly lead to a loss of differentiation capacity, preference towards certain cell types and oftentimes, phenotypes which begin to resemble cancer. Broad changes in (epi)genetics, such as aneuploidies or wide-ranging modifications to the epigenetic landscape tend to lead to extensive, less definite changes in differentiation capacity, whereas more specific abnormalities often have precise ramifications in which certain cell types become more preferential. Furthermore, there appears to be a greater, though often less considered, contribution to differentiation propensity by factors such as mitochondria and inherent genetic diversity. Varied differentiation capacity can also lead to potential consequences in the clinical translation of hPSC, including the occurrence of residual undifferentiated stem cells, and the transplantation of potentially transformed cells. Wider implications As hPSC continue to advance towards the clinic, our understanding of them progresses as well. As a result, the challenges faced become more numerous, but also more clear. If the transition to the clinic is to be achieved with a minimum number of potential setbacks, thorough evaluation of the cells will be an absolute necessity. Altered differentiation propensity represents at least one such hurdle, for which researchers and eventually clinicians will need to find solutions. Already, steps are being taken to tackle the issue, though further research will be required to evaluate any long-term risks it poses.

Massive parallel sequencing (MPS) can accurately quantify mitochondrial DNA (mtDNA) single nucleotide variants (SNVs), but no MPS methods are currently validated to simultaneously and accurately establish the breakpoints and frequency of large deletions at low heteroplasmic loads. Here we present the thorough validation of an MPS protocol to quantify the load of very low frequency, large mtDNA deletions in bulk DNA and single cells, along with SNV calling by standard methods. We used a set of well-characterized DNA samples, DNA mixes and single cells to thoroughly control the study. We developed a custom script for the detection of mtDNA rearrangements that proved to be more accurate in detecting and quantifying deletions than pre-existing tools. We also show that PCR conditions and primersets must be carefully chosen to avoid biases in the retrieved variants and an increase in background noise, and established a lower detection limit of 0.5% heteroplasmic load for large deletions, and 1.5 and 2% for SNVs, for bulk DNA and single cells, respectively. Finally, the analysis of different single cells provided novel insights into mtDNA cellular mosaicism.European Journal of Human Genetics advance online publication, 23 August 2017; doi:10.1038/ejhg.2017.129.

INTRODUCTION: Preimplantation genetic diagnosis (PGD) was introduced as an alternative to prenatal diagnosis: embryos cultured in vitro were analysed for a monogenic disease and only disease-free embryos were transferred to the mother, to avoid the termination of pregnancy with an affected foetus. It soon transpired that human embryos show a great deal of acquired chromosomal abnormalities, thought to explain the low success rate of IVF - hence preimplantation genetic testing for aneuploidy (PGT-A) was developed to select euploid embryos for transfer. Areas covered: PGD has followed the tremendous evolution in genetic analysis, with only a slight delay due to adaptations for diagnosis on small samples. Currently, next generation sequencing combining chromosome with single-base pair analysis is on the verge of becoming the golden standard in PGD and PGT-A. Papers highlighting the different steps in the evolution of PGD/PGT-A were selected. Expert commentary: Different methodologies used in PGD/PGT-A with theirpros and cons are discussed.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Study question: What is the incidence of aneuploidy and mosaicism in all cells of top-quality Day-4 embryos analysed by array-based comparative genomic hybridization (array CGH)? Summary answer: Our data show extensive abnormalities in Day-4 embryos. What is known already: Numerous studies on human embryos at Day 3 and Day 5 of development show that they frequently contain aneuploid cells and are mosaic, although Day-5 embryos contain proportionally more normal cells than at Day 3. In contrast, only limited data exist on Day 4 of preimplantation development, despite the fact that it is the suggested stage for the initiation of the process of self-correction. Study design, size, duration: Thirteen embryos were analysed: four fresh good-quality preimplantation genetic diagnosis (PGD) embryos and nine good-quality surplus embryos cryopreserved on Day 3 and donated for research. On Day 4, following removal of the zona pellucida, all blastomeres were disaggregated and collected. Participants/materials,setting, methods: The genomic DNA of 283 single blastomeres from disaggregated embryos was amplified. Array CGH was carried out using 24SureTM Cytochip microarrays. After scanning of the microarray slides, the images were analysed using BlueFuse Software (BlueGnome). Combined with selective microsatellite analysis, hypothetical reconstructions of embryo chromosome complements were made following each of the first four cleavage divisions. Main results and the role of chance: No chromosome imbalance was detected for one PGD embryo, the other three were mosaic containing between 16 and 75% abnormal cells. All nine frozen - thawed embryos were abnormal. Six were mosaic with between 30 and 100% abnormal cells; three had abnormalities of meiotic origin, two of which displayed mitotic abnormalities. Evidence was also found of mitotic unbalanced structural chromosome rearrangements. The higher rate of abnormality of frozen - thawed embryos is based on a small number of embryos and cannot be tested statistically. The aneuploidy can mostly be explained by anaphase lag and non-disjunction. In some cases, we hypothesize endoreduplication followed by a cellular division with multipolar spindles to explain the results. Limitations, reasons for caution: Array CGH technology determines relative quanti?cation of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. The division trees presented are hypothetical models projecting back in time that try to explain observations in single blastomeres of Day 4 embryos. The limited number of embryos analysed does not allow drawing ?rm conclusions, but nevertheless provides valuable data on the origin of aneuploidy in human embryos. Wider implications of the findings: Our data show extensive abnormalities in Day-4 embryos. We found no evidence of self-correction at this stage of development, suggesting that this process may start at a later stage of development. Study funding/competing interest (s): This research was supported by the Instituut voor de aanmoediging van innovatie door Wetenschap en Technologie in Vlaanderen (IWT-Vlaanderen). C.S. is a postdoctoral fellow at the Flemish Fund for Scienti?c Research [Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen]. There are no competing interests.

Transplantation of pancreatic progenitors derived from human embryonic stem cells (hESCs) is a promising way to treat diabetes. Strategies to obtain the required cell mass would rely on the up scaling of current differentiation protocols, or the proliferation of committed progenitors. We aimed at finding conditions that maintain a proliferating pancreatic progenitor pool and we assessed the role of BMP4 signaling in this process. hESCs were differentiated into PDX1 positive pancreatic progenitor stage following our established protocol with few modifications, and then the progenitor cells were passaged in a defined proliferation medium (PM). During passage, the effect of BMP4 signaling on the differentiation and proliferation of pancreatic progenitors was examined by RT-PCR and immunofluorescence analysis. We found that PDX1 positive pancreatic progenitors proliferated and gained NKX6.1 expression in the PM, whereas they failed to express NKX6.1 if BMP signaling was inhibited with Noggin. In this latter condition, part of the progenitors rather generated pro-endocrine cells denoted by NGN3 and synaptophysin expression. On the contrary, addition of BMP4 to the PM promoted the early derivation of PDX1 and NKX6.1 coexpressing pancreatic progenitors. Our findings are in line with mouse pancreas development, and indicate that BMP4 signaling is required for the derivation and maintenance of hESC-derived PDX1+NKX6.1+ pancreatic progenitors. These results are instructive for guiding the development of an efficient pancreas differentiation protocol in view of diabetes cell replacement therapy.

In March 2005, a group of experts from the European Society of Human Genetics and European Society of Human Reproduction and Embryology met to discuss the interface between genetics and assisted reproductive technology (ART), and published an extended background paper, recommendations and two Editorials. Seven years later, in March 2012, a follow-up interdisciplinary workshop was held, involving representatives of both professional societies, including experts from the European Union Eurogentest2 Coordination Action Project. The main goal of this meeting was to discuss developments at the interface between clinical genetics and ARTs. As more genetic causes of reproductive failure are now recognised and an increasing number of patients undergo testing of their genome before conception, either in regular health care or in the context of direct-to-consumer testing, the need for genetic counselling and preimplantation genetic diagnosis (PGD) may increase. Preimplantation genetic screening (PGS) thus far does not have evidence from randomised clinical trials to substantiate that the technique is both effective and efficient. Whole-genome sequencing may create greater challenges both in the technological and interpretational domains, and requires further reflection about the ethics of genetic testing in ART and PGD/PGS. Diagnostic laboratories should be reporting their results according to internationally accepted accreditation standards (International Standards Organisation - ISO 15189). Further studies are needed in order to address issues related to the impact of ART on epigenetic reprogramming of the early embryo. The legal landscape regarding assisted reproduction is evolving but still remains very heterogeneous and often contradictory. The lack of legal harmonisation and uneven access to infertility treatment and PGD/PGS fosters considerable cross-border reproductive care in Europe and beyond. The aim of this paper is to complement previous publications and provide an update of selected topics that have evolved since 2005. ispartof: European Journal of Human Genetics vol:21 issue:Suppl 2 pages:S1-S21 ispartof: location:England status: published