Human ovarian aging is characterized by oxidative damage and mitochondrial dysfunction.

STUDY QUESTION: Are human ovarian aging and the age-related female fertility decline caused by oxidative stress and mitochondrial dysfunction in oocytes? SUMMARY ANSWER: We found oxidative damage in oocytes of advanced maternal age, even at the primordial follicle stage, and confirmed mitochondrial dysfunction in such oocytes, which likely resulted in the use of alternative energy sources. WHAT IS KNOWN ALREADY: Signs of reactive oxygen species-induced damage and mitochondrial dysfunction have been observed in maturing follicles, and even in early stages of embryogenesis. However, although recent evidence indicates that also primordial follicles have metabolically active mitochondria, it is still often assumed that these follicles avoid oxidative phosphorylation to prevent oxidative damage in dictyate arrested oocytes. Data on the influence of ovarian aging on oocyte metabolism and mitochondrial function are still limited. STUDY DESIGN, SIZE, DURATION: A set of 39 formalin-fixed and paraffin-embedded ovarian tissue biopsies were divided into different age groups and used for immunofluorescence analysis of oxidative phosphorylation activity and oxidative damage to proteins, lipids, and DNA. Additionally, 150 immature oocytes (90 germinal vesicle oocytes and 60 metaphase I oocytes) and 15 cumulus cell samples were divided into different age groups and used for targeted metabolomics and lipidomics analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissues used for immunofluorescence microscopy were collected through PALGA, the nationwide network, and registry of histo- and cytopathology in The Netherlands. Comprehensive metabolomics and lipidomics were performed by liquid-liquid extraction and full-scan mass spectrometry, using oocytes and cumulus cells of women undergoing ICSI treatment based on male or tubal factor infertility, or fertility preservation for non-medical reasons. MAIN RESULTS AND THE ROLE OF CHANCE: Immunofluorescence imaging on human ovarian tissue indicated oxidative damage by protein and lipid (per)oxidation already at the primordial follicle stage. Metabolomics and lipidomics analysis of oocytes and cumulus cells in advanced maternal-age groups demonstrated a shift in the glutathione-to-oxiglutathione ratio and depletion of phospholipids. Age-related changes in polar metabolites suggested a decrease in mitochondrial function, as demonstrated by NAD+, purine, and pyrimidine depletion, while glycolysis substrates and glutamine accumulated, with age. Oocytes from women of advanced maternal age appeared to use alternative energy sources like glycolysis and the adenosine salvage pathway, and possibly ATP which showed increased production in cumulus cells. LIMITATIONS, REASONS FOR CAUTION: The immature oocytes used in this study were all subjected to ovarian stimulation with high doses of follicle-stimulating hormones, which might have concealed some age-related differences. WIDER IMPLICATIONS OF THE FINDINGS: Further studies on how to improve mitochondrial function, or lower oxidative damage, in oocytes from women of advanced maternal age, for instance by supplementation of NAD+ precursors to promote mitochondrial biogenesis, are warranted. In addition, supplementing the embryo medium of advanced maternal-age embryos with such compounds could be a treatment option worth exploring. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Amsterdam UMC. The authors declare to have no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Longevity pathways are associated with human ovarian ageing.

STUDY QUESTION: Are genes known to be involved in somatic cell ageing, particularly related to longevity pathways, associated with the accelerated ageing process of the ovary? SUMMARY ANSWER: Growth, metabolism, and cell-cycle progression-related pathways that are involved in somatic cell ageing are also associated with ovarian ageing. WHAT IS KNOWN ALREADY: Ovarian ageing is characterized by a gradual decline in ovarian follicle quantity, a decline in oocyte quality, and lower chances of pregnancy. Genetic pathways modulating the rate of somatic cell ageing have been researched intensively. Ovarian ageing does not follow the same timeline as somatic cell ageing, as signs of ovarian ageing occur at a younger female age, while the somatic cells are still relatively young. It is not known whether the generally recognized somatic cell longevity genes also play a role during ovarian ageing. Looking at somatic cell longevity genes can lead to new hypotheses and possible treatment options for subfertility caused by ovarian ageing. STUDY DESIGN SIZE DURATION: In this observational study, we analysed a dataset of individual gene expression profiles of 38 germinal vesicle (GV) oocytes from 38 women aged between 25 and 43 years. We correlated female age (calendar age in years) and biological age (factors known to be associated with ovarian ageing such as dosage of FSH needed for ovarian hyperstimulation, and antral follicle count (AFC)) with gene expression signatures of longevity pathways. PARTICIPANTS/MATERIALS SETTING METHODS: Transcripts of 38 GV oocytes were used for individual gene expression analysis. R version 3.5.1 was used to process and analyse data. The GeneAge database (build 19) was used to obtain mouse ageing-related genes. Human to mouse orthologues were obtained using the R package biomaRt. Correlations and significance between gene expression data and age were tested for using Pearson's product moment correlation coefficient using ranked expression data. Distributions were compared with an ANOVA, and the Tukey Honest Significant Difference method was used to control for the Type I error rate across multiple comparisons. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 136 genes in the GeneAge database, the expression of 15 anti-longevity genes identified in oocytes showed a positive correlation with female calendar age and FSH dosage administered during ICSI treatment, and a negative correlation with AFC. Expression of 32 pro-longevity genes was negatively correlated with calendar age and FSH dosage, and positively correlated with AFC. In general, anti- and pro-longevity genes changed in opposing directions with advancing maternal age in oocytes. Notably, the anti-longevity genes include many 'growth'-related genes involved in the mechanistic target of rapamycin (mTOR) Complex 1 pathway, such as EIF5A2, EIF3H, EIF4E, and mTOR. The pro-longevity genes include many cell-cycle progression-related genes involved in DNA damage repair (e.g. XRCC6, ERCC2, and MSH2) or cell-cycle checkpoint regulation genes (e.g. ATM, BRCA1, TP53, TP63, TP73, and BUB1B). LIMITATIONS REASONS FOR CAUTION: Using mature oocytes instead of GV-stage oocytes discarded from ICSI treatments may provide different results. No correction for multiple testing was carried out on individual genes because a small set of longevity-related genes was selected a priori for the analysis. The global trend was corrected for multiple testing and remained significant. This work was an observational study and, as no additional experimental work was performed, the associations described do not directly demonstrate the involvement of such genes in oocyte ageing. WIDER IMPLICATIONS OF THE FINDINGS: Growth, metabolism, and cell-cycle progression-related pathways that are known to be involved in somatic cell ageing were associated with ovarian ageing. If experimental data are obtained to support these associations, we suggest that interventions known to modulate these processes could benefit women suffering from ovarian ageing. STUDY FUNDING/COMPETING INTERESTS: G.E.J. is supported by a VENI grant from ZonMw (https://www.zonmw.nl). Work in the Houtkooper group is financially supported by an ERC Starting grant (No. 638290), a VIDI grant from ZonMw (No. 91715305), and the Velux Stiftung (No. 1063). M.G. declares several research and educational grants from Guerbet, Merck and Ferring (all location VUmc), outside the scope of the submitted work. The other authors report no competing interest. TRIAL REGISTRATION NUMBER: N/A.

Preimplantation genetic testing for aneuploidies (abnormal number of chromosomes) in in vitro fertilisation.

BACKGROUND: In in vitro fertilisation (IVF) with or without intracytoplasmic sperm injection (ICSI), selection of the most competent embryo(s) for transfer is based on morphological criteria. However, many women do not achieve a pregnancy even after 'good quality' embryo transfer. One of the presumed causes is that such morphologically normal embryos have an abnormal number of chromosomes (aneuploidies). Preimplantation genetic testing for aneuploidies (PGT-A), formerly known as preimplantation genetic screening (PGS), was therefore developed as an alternative method to select embryos for transfer in IVF. In PGT-A, the polar body or one or a few cells of the embryo are obtained by biopsy and tested. Only polar bodies and embryos that show a normal number of chromosomes are transferred. The first generation of PGT-A, using cleavage-stage biopsy and fluorescence in situ hybridisation (FISH) for the genetic analysis, was demonstrated to be ineffective in improving live birth rates. Since then, new PGT-A methodologies have been developed that perform the biopsy procedure at other stages of development and use different methods for genetic analysis. Whether or not PGT-A improves IVF outcomes and is beneficial to patients has remained controversial. OBJECTIVES: To evaluate the effectiveness and safety of PGT-A in women undergoing an IVF treatment. SEARCH METHODS: We searched the Cochrane Gynaecology and Fertility (CGF) Group Trials Register, CENTRAL, MEDLINE, Embase, PsycINFO, CINAHL, and two trials registers in September 2019 and checked the references of appropriate papers. SELECTION CRITERIA: All randomised controlled trials (RCTs) reporting data on clinical outcomes in participants undergoing IVF with PGT-A versus IVF without PGT-A were eligible for inclusion. DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies for inclusion, assessed risk of bias, and extracted study data. The primary outcome was the cumulative live birth rate (cLBR). Secondary outcomes were live birth rate (LBR) after the first embryo transfer, miscarriage rate, ongoing pregnancy rate, clinical pregnancy rate, multiple pregnancy rate, proportion of women reaching an embryo transfer, and mean number of embryos per transfer. MAIN RESULTS: We included 13 trials involving 2794 women. The quality of the evidence ranged from low to moderate. The main limitations were imprecision, inconsistency, and risk of publication bias. IVF with PGT-A versus IVF without PGT-A with the use of genome-wide analyses Polar body biopsy One trial used polar body biopsy with array comparative genomic hybridisation (aCGH). It is uncertain whether the addition of PGT-A by polar body biopsy increases the cLBR compared to IVF without PGT-A (odds ratio (OR) 1.05, 95% confidence interval (CI) 0.66 to 1.66, 1 RCT, N = 396, low-quality evidence). The evidence suggests that for the observed cLBR of 24% in the control group, the chance of live birth following the results of one IVF cycle with PGT-A is between 17% and 34%. It is uncertain whether the LBR after the first embryo transfer improves with PGT-A by polar body biopsy (OR 1.10, 95% CI 0.68 to 1.79, 1 RCT, N = 396, low-quality evidence). PGT-A with polar body biopsy may reduce miscarriage rate (OR 0.45, 95% CI 0.23 to 0.88, 1 RCT, N = 396, low-quality evidence). No data on ongoing pregnancy rate were available. The effect of PGT-A by polar body biopsy on improving clinical pregnancy rate is uncertain (OR 0.77, 95% CI 0.50 to 1.16, 1 RCT, N = 396, low-quality evidence). Blastocyst stage biopsy One trial used blastocyst stage biopsy with next-generation sequencing. It is uncertain whether IVF with the addition of PGT-A by blastocyst stage biopsy increases cLBR compared to IVF without PGT-A, since no data were available. It is uncertain if LBR after the first embryo transfer improves with PGT-A with blastocyst stage biopsy (OR 0.93, 95% CI 0.69 to 1.27, 1 RCT, N = 661, low-quality evidence). It is uncertain whether PGT-A with blastocyst stage biopsy reduces miscarriage rate (OR 0.89, 95% CI 0.52 to 1.54, 1 RCT, N = 661, low-quality evidence). No data on ongoing pregnancy rate or clinical pregnancy rate were available. IVF with PGT-A versus IVF without PGT-A with the use of FISH for the genetic analysis Eleven trials were included in this comparison. It is uncertain whether IVF with addition of PGT-A increases cLBR (OR 0.59, 95% CI 0.35 to 1.01, 1 RCT, N = 408, low-quality evidence). The evidence suggests that for the observed average cLBR of 29% in the control group, the chance of live birth following the results of one IVF cycle with PGT-A is between 12% and 29%. PGT-A performed with FISH probably reduces live births after the first transfer compared to the control group (OR 0.62, 95% CI 0.43 to 0.91, 10 RCTs, N = 1680, I² = 54%, moderate-quality evidence). The evidence suggests that for the observed average LBR per first transfer of 31% in the control group, the chance of live birth after the first embryo transfer with PGT-A is between 16% and 29%. There is probably little or no difference in miscarriage rate between PGT-A and the control group (OR 1.03, 95%, CI 0.75 to 1.41; 10 RCTs, N = 1680, I² = 16%; moderate-quality evidence). The addition of PGT-A may reduce ongoing pregnancy rate (OR 0.68, 95% CI 0.51 to 0.90, 5 RCTs, N = 1121, I² = 60%, low-quality evidence) and probably reduces clinical pregnancies (OR 0.60, 95% CI 0.45 to 0.81, 5 RCTs, N = 1131; I² = 0%, moderate-quality evidence). AUTHORS' CONCLUSIONS: There is insufficient good-quality evidence of a difference in cumulative live birth rate, live birth rate after the first embryo transfer, or miscarriage rate between IVF with and IVF without PGT-A as currently performed. No data were available on ongoing pregnancy rates. The effect of PGT-A on clinical pregnancy rate is uncertain. Women need to be aware that it is uncertain whether PGT-A with the use of genome-wide analyses is an effective addition to IVF, especially in view of the invasiveness and costs involved in PGT-A. PGT-A using FISH for the genetic analysis is probably harmful. The currently available evidence is insufficient to support PGT-A in routine clinical practice.

High-quality human preimplantation embryos stimulate endometrial stromal cell migration via secretion of microRNA hsa-miR-320a.

STUDY QUESTION: How do high-quality human preimplantation embryos influence the endometrium to promote their own implantation? SUMMARY ANSWER: High-quality human preimplantation embryos secrete a specific microRNA (miRNA), hsa-miR-320a, which promotes migration of human endometrial stromal cells (hESCs). WHAT IS KNOWN ALREADY: We have previously shown that high-quality human preimplantation embryos excrete unknown factors that influence migration of hESCs. STUDY DESIGN, SIZE, DURATION: Embryo excreted miRNAs, specifically those excreted by high-quality embryos, were identified and their effect on hESCs was determined by measuring the migration capacity and gene expression patterns of primary isolated hESCs. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryo conditioned medium (ECM) from routine ICSI procedures was used to identify embryo excreted miRNAs. miRNome analyses were performed on ECM from individually cultured embryos with high morphological quality, with low morphological quality or empty control medium. MiRNA mimics and inhibitors were then used to further study the effect of miRNAs of interest on migration and gene expression of hESCs. Migration assays were performed using hESCs that were obtained from endometrial biopsies performed on hysterectomy specimens from women that received surgery for spotting due to a niche in a cesarean section scar. MAIN RESULTS AND THE ROLE OF CHANCE: By using miRNA mimics and inhibitors, we showed that hsa-miR-320a alone can stimulate migration of decidualized hESCs, accurately resembling the response typically triggered only by high-quality embryos. Transcriptome analysis further demonstrated that this effect is very likely mediated via altered expression of genes involved in cell adhesion and cytoskeleton organization. LIMITATIONS, REASONS FOR CAUTION: The effect of hsa-miR-320a on hESCs was measured in vitro. Further studies on the in vivo effect of hsa-miR-320a are warranted. WIDER IMPLICATIONS OF THE FINDINGS: Implantation failure is one of the major success limiting factors in human reproduction. By secreting hsa-miR-320a, high-quality human preimplantation embryos directly influence hESCs, most likely to prime the endometrium at the implantation site for successful implantation. Together, our results indicate that hsa-miR-320a may be a promising target to further increase success rates in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Amsterdam University Medical Centers and the Amsterdam Reproduction & Development Research Institute. R.P.B., G.H. and S.M. have a patent on the use of hsa-miR-320a in assisted reproduction treatments pending. TRIAL REGISTRATION NUMBER: N/A.

Premature expression of the decidualization marker prolactin is associated with repeated implantation failure.

Repeated implantation failure (RIF) is a poorly understood reproductive pathology defined by the inability to achieve a clinical pregnancy in at least three consecutive IVF cycles. In this study, we investigated whether the onset of decidualization, marked by prolactin (PRL) expression, is associated with RIF. We performed a retrospective cohort study using endometrial biopsies from women with idiopathic subfertility, that conceived naturally during the same cycle in which the biopsy was taken (group 1; n = 15) conceived naturally within three months after the biopsy was taken (group 2; n = 20), or unsuccessfully underwent six IUI cycles and three IVF cycles with transfer of at least one high-quality embryo (group 3, RIF; n = 20). Our results demonstrated that immunohistochemical PRL-staining was present in 8/15 women from group 1 (53.3%), in 1/20 women from group 2 (5.0%), and in 11/20 women from group 3 (55.0%). Increased proliferation, analyzed by Ki67 expression, was seen in women that were pregnant during the biopsy, compared to all women combined that were not pregnant (p≤.01). In conclusion, our study demonstrates that premature expression of the decidualization marker PRL during the luteal phase is associated with RIF.

Female subfertility.

Subfertility is common and affects one in six couples, half of whom lack an explanation for their delay in conceiving. Developments in the diagnosis and treatment of subfertility over the past 50 years have been truly remarkable. Indeed, current generations of couples with subfertility are more fortunate than previous generations, as they have many more opportunities to become parents. The timely access to effective treatment for subfertility is important as many couples have a narrow window of opportunity before the age-related effects of subfertility limit the likelihood of success. Assisted reproduction can overcome the barriers to fertility caused by tubal disease and low sperm count, but little progress has been made in reducing the effect of increasing age on ovarian function. The next 5-10 years will likely see further increases in birth rates in women with subfertility, a greater awareness of lifestyle factors and a possible refinement of current assisted reproduction techniques and the development of new ones. Such progress will bring challenging questions regarding the potential benefits and harms of treatments involving germ cell manipulation, artificial gametes, genetic screening of embryos and gene editing of embryos. We hope to see a major increase in fertility awareness, access to safe and cost-effective fertility care in low-income countries and a reduction in the current disparity of access to fertility care.

The influence of retinoic acid-induced differentiation on the radiation response of male germline stem cells.

Lifelong mammalian male fertility is maintained through an intricate balance between spermatogonial proliferation and differentiation. DNA damage in spermatogonia, for instance caused by chemo- or radiotherapy, can induce cell cycle arrest or germ cell apoptosis, possibly resulting in male infertility. Spermatogonia are generally more radiosensitive and prone to undergo apoptosis than somatic cells. Among spermatogonial subtypes the response to DNA damage is differentially modulated; undifferentiated spermatogonia, including the spermatogonial stem cells (SSCs), are relatively radio-resistant, whereas differentiating spermatogonia are very radiosensitive. To investigate the molecular mechanisms underlying this difference, we used an in vitro system consisting of mouse male germline stem (GS) cells that can be induced to differentiate. Using RNA-sequencing analysis, we analyzed the response of undifferentiated and differentiating GS cells to ionizing radiation (IR). At the RNA expression level, both undifferentiated and differentiating GS cells showed a very similar response to IR. Protein localization of several genes found to be involved in either spermatogonial differentiation or radiation response was investigated using mouse testis sections. For instance, we found that the transcription factor PDX1 was specifically expressed in undifferentiated spermatogonia and thus may be a novel marker for these cells. Interestingly, also at the protein level, undifferentiated GS cells showed a more pronounced upregulation of p53 in response to IR than differentiating GS cells. The higher p53 protein level in undifferentiated spermatogonia may preferentially induce cell cycle arrest, thereby giving these cells more time to repair inflicted DNA damage and increase their radio-resistance.

Developmental outcome of 9-year-old children born after PGS: follow-up of a randomized trial.

STUDY QUESTION: Does Day-3 cleavage-stage PGS affect neurodevelopment of 9-year-old IVF offspring? SUMMARY ANSWER: We did not find evidence of adverse consequences of Day-3 cleavage-stage PGS on neurodevelopment of 9-year-old IVF offspring, although children born after IVF with or without PGS often had a non-optimal neurological condition. WHAT IS KNOWN ALREADY: Knowledge on long-term sequelae for development and health of children born following PGS is lacking. This is striking as evidence accumulates that IVF itself is associated with increased risk for impaired health and development in the offspring. STUDY DESIGN SIZE, DURATION: This prospective, assessor-blinded, multicentre, follow-up study evaluated development and health of 9-year-old IVF children born to women who were randomly assigned to IVF with PGS (PGS group) or without PGS (control group). The follow-up examination at 9 years took place between March 2014 and May 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 408 women were included and randomly assigned to IVF with or without Day-3 cleavage-stage PGS. This resulted in 52 ongoing pregnancies in the PGS group and 74 in the control group. In the PGS group, 59 children were born alive; in the control group, 85 children were born alive. At the age of 9 years, 43 children born after PGS and 56 control children participated in the study. Our primary outcome was the neurological optimality score, a sensitive measure of neurological condition assessed with a standardized, age-specific test (Touwen test). Secondary outcomes were adverse neurological condition (neurologically abnormal and the complex form of minor neurological dysfunction), cognitive development (intelligence quotient and specific domains), behaviour (parental and teacher's questionnaires), blood pressure and anthropometrics. MAIN RESULTS AND THE ROLE OF CHANCE: Neurodevelopmental outcome of PGS children did not differ from that of controls; the neurological optimality scores (mean values [(95% CI]: PGS children 51.5 [49.3; 53.7], control children 53.1 [50.5; 55.7]) were not significantly different. The prevalences of adverse neurological outcome (in all but one child implying the presence of the complex form of minor neurological dysfunction) did not differ between the groups (PGS group 17/43 [40%], control group 19/56 [34%]), although the prevalence of complex minor neurological dysfunction in both groups was rather high. Also intelligence quotient scores of the two groups were not significantly different (PGS group 114 [108; 120]); control group 117 [109; 125]), and the behaviour, blood pressure and anthropometrics of both groups did not differ. Mean blood pressures of both groups were above the 60th percentile. LIMITATIONS REASONS FOR CAUTION: The power analysis of the study was not based on the number of children needed for the follow-up study, but on the number of women who were needed to detect an increase in ongoing pregnancy rates after PGS. In addition, our study evaluated embryo biopsy in the form of PGS at cleavage stage (Day-3 embryo biopsy), while currently PGS at blastocyst stage (Day-5 embryo biopsy) is recommended and increasingly being used. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that PGS in cleavage stage embryos is not associated with adverse effects on neurological, cognitive and behavioural development, blood pressure and anthropometrics of offspring at 9 years. This is a reassuring finding as embryo biopsy in the forms of PGS and PGD is increasingly applied. However, both groups of IVF offspring showed high prevalences of the clinically relevant form of minor neurological dysfunction, which is a point of concern for the IVF community. In addition, our study confirms findings of others that IVF offspring may be at risk of an unfavourable cardiovascular outcome. These findings are alarming and highlight the importance of research on the underlying mechanisms of unfavourable neurodevelopmental and cardiovascular outcomes in IVF offspring. STUDY FUNDING/COMPETING INTEREST(S): The randomized controlled trial was financially supported by the Organization for Health Research and Development (ZonMw), The Netherlands (Grant number 945-03-013). The follow-up was financially supported by the University Medical Center Groningen (Grant number: 754510), the Cornelia Foundation, and the graduate schools BCN and Share, Groningen, The Netherlands. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation or writing of the report. There are no conflicts of interest. TRIAL REGISTRATION NUMBER: ISRCTN76355836.

Trivial role for NSMCE2 during in vitro proliferation and differentiation of male germline stem cells.

Spermatogenesis, starting with spermatogonial differentiation, is characterized by ongoing and dramatic alterations in composition and function of chromatin. Failure to maintain proper chromatin dynamics during spermatogenesis may lead to mutations, chromosomal aberrations or aneuploidies. When transmitted to the offspring, these can cause infertility or congenital malformations. The structural maintenance of chromosomes (SMC) 5/6 protein complex has recently been described to function in chromatin modeling and genomic integrity maintenance during spermatogonial differentiation and meiosis. Among the subunits of the SMC5/6 complex, non-SMC element 2 (NSMCE2) is an important small ubiquitin-related modifier (SUMO) ligase. NSMCE2 has been reported to be essential for mouse development, prevention of cancer and aging in adult mice and topological stress relief in human somatic cells. By using in vitro cultured primary mouse spermatogonial stem cells (SSCs), referred to as male germline stem (GS) cells, we investigated the function of NSMCE2 during spermatogonial proliferation and differentiation. We first optimized a protocol to generate genetically modified GS cell lines using CRISPR-Cas9 and generated an Nsmce2-/- GS cell line. Using this Nsmce2-/- GS cell line, we found that NSMCE2 was dispensable for proliferation, differentiation and topological stress relief in mouse GS cells. Moreover, RNA sequencing analysis demonstrated that the transcriptome was only minimally affected by the absence of NSMCE2. Only differential expression of Sgsm1 appeared highly significant, but with SGSM1 protein levels being unaffected without NSMCE2. Hence, despite the essential roles of NSMCE2 in somatic cells, chromatin integrity maintenance seems differentially regulated in the germline.

The why, the how and the when of PGS 2.0: current practices and expert opinions of fertility specialists, molecular biologists, and embryologists.

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.

Preimplantation genetic screening: back to the future.

All agree that in hindsight the rapid adoption of preimplantation genetic screening (PGS) using cleavage stage biopsy and fluorescence in situ hybridization (FISH) in routine clinical practice without proper evaluation of (cost-)effectiveness basically resulted in couples paying more money for a less effective treatment. Now, almost 20 years later, we are on the verge of a new era of PGS. But have things really changed or are we simply going back to the future?

Limitations of embryo selection methods.

In in vitro fertilization (IVF), the selection of embryos for transfer is generally based on the morphology of the available embryos. However, not all embryos with good morphology implant and on average only one in four treatments are successful. This has driven a quest for alternative selection methods. The best known alternative selection method is preimplantation genetic screening (PGS), which has been used for over a decade before it was shown to be inferior to morphological selection. Now, new forms of PGS (performing biopsy at another stage of development and new methods for analysis) are emerging, just like alternative noninvasive embryo selection methods. However, the concept that better selection will lead to improved IVF results is not so certain anymore. Evidence is accumulating that all available embryos in an IVF cycle can be cryopreserved and transferred in subsequent cycles without impairing pregnancy rates or maybe even with an improvement in pregnancy rates. Embryo selection will then no longer be able to improve the live birth rate in IVF; it could even lower the live birth rate. Embryo selection will only be able to improve the time to pregnancy, if embryos with the highest implantation potential are transferred first.

Morphologic abnormalities in 2-year-old children born after in vitro fertilization/intracytoplasmic sperm injection with preimplantation genetic screening: follow-up of a randomized controlled trial.

OBJECTIVE: To evaluate the effect of preimplantation genetic screening (PGS) on morphologic outcome in children. DESIGN: Follow-up of a randomized controlled trial (RCT). SETTING: University hospital. PATIENT(S): Two-year-old children born to mothers who participated in an RCT on the efficacy of PGS: 50 children born after in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) with PGS (intervention group; PGS+) and 72 children born after IVF/ICSI only (control group; PGS-). Sixty-six age-matched children conceived without any form of assisted reproduction were recruited separately in a local public health service center (reference group). INTERVENTION(S): PGS. MAIN OUTCOME MEASURE(S): Body surface examination and anthropometry. The evaluation of morphologic abnormalities allowed assessment of children's phenotype in detail. Morphologic abnormalities were classified as major abnormalities (abnormal development in organogenesis, deformations, disruptions, or dysplasia) and minor anomalies (deviations in phenogenesis). RESULT(S): The percentage of children with ≥ 1 major abnormality was 28% in the PGS+ and 35% in the PGS- group [difference -7%, 95% CI -23% to 10%]. The percentage of children with ≥ 1 minor anomaly was 64% in the PGS+ and 67% in the PGS- group [difference -3%, 95% CI -15% to 20%]. In the reference group 30% of the children had ≥ 1 major abnormality [95% CI 20% to 43%] and 74% had ≥ 1 minor anomaly [95% CI 62% to 84%]. CONCLUSION(S): No statistically significant differences were found in minor anomalies between children conceived after IVF/ICSI with or without PGS. There is < 2.5% chance of ≥ 10% more major abnormalities in children born after PGS.