Growth of twins conceived using assisted reproductive treatments up to 5 years old: a national growth cohort.

STUDY QUESTION: Do twins conceived through assisted reproductive treatments (ART) grow differently from naturally conceived (NC) twins in early life? SUMMARY ANSWER: Assessments at 6-8 weeks old and at school entry show that ART twins conceived from frozen embryo transfer (FET) grow faster than both NC twins and ART twins conceived from fresh embryo transfer (ET). WHAT IS KNOWN ALREADY: Singletons born from fresh ET grow more slowly in utero and in the first few weeks of life but then show postnatal catch-up growth by school age, compared to NC and FET babies. Evidence on early child growth of ART twins relative to NC twins is inconsistent; most studies are small and do not distinguish FET from fresh ET cycles. STUDY DESIGN, SIZE, DURATION: This cohort study included 13 528 live-born twin babies conceived by ART (fresh ET: 2792, FET: 556) and NC (10 180) between 1991 and 2009 in Scotland. The data were obtained by linking Human Fertilisation and Embryology Authority ART register data to the Scottish Morbidity Record (SMR02) and Scottish child health programme datasets. Outcome data were collected at birth, 6-8 weeks (first assessment), and school entry (4-7 years old) assessments. The primary outcome was growth, measured by weight at the three assessment points. Secondary outcomes were length (at birth and 6-8 weeks) or height (at school entry), BMI, occipital circumference, gestational age at birth, newborn intensive care unit admission, and growth rates (between birth and 6-8 weeks and between 6-8 weeks and school entry). PARTICIPANTS/MATERIALS, SETTING, METHODS: All twins in the linked dataset (born between 1991 and 2009) with growth data were included in the analysis. To determine outcome differences between fresh ET, FET, and NC twins, linear mixed models (or analogous logistic regression models) were used to explore the outcomes of interest. All models were adjusted for available confounders: gestational age/child age, gender, maternal age and smoking, Scottish Index of Multiple Deprivation, year of treatment, parity, ICSI, and ET stage. MAIN RESULTS AND THE ROLE OF CHANCE: In the primary birth weight models, the average birth weight of fresh ET twins was lower [-35 g; 95% CI: (-53, -16)g] than NC controls, while FET twins were heavier [71 g; 95% CI (33, 110) g] than NC controls and heavier [106 g; 95% CI (65, 146) g] than fresh ET twins. However, the difference between FET and NC twins was not significant when considering only full-term twins (≥37 weeks gestation) [26 g; 95% CI (-30, 82) g], while it was significantly higher in preterm twins [126 g; 95% CI (73, 179) g]. Growth rates did not differ significantly for the three groups from birth to 6-8 weeks. However, FET twins grew significantly faster from 6 to 8 weeks than NC (by 2.2 g/week) and fresh ET twins (by 2.1 g/week). By school entry, FET twins were 614 g [95% CI (158, 1070) g] and 581 g [95% CI (100, 1063) g] heavier than NC and fresh ET twins, respectively. Length/height and occipital frontal circumference did not differ significantly at any time point. LIMITATIONS, REASONS FOR CAUTION: Although the differences between ART and NC reflect the true ART effects, these effects are likely to be mediated partly through the different prevalence of mono/dizygotic twins in the two groups. We could not explore the mediating effect of zygosity due to the unavailability of data. The confounding variables included in the study were limited to those available in the datasets. WIDER IMPLICATIONS OF THE FINDINGS: Live-born twins from FET cycles are heavier at birth, grow faster than their fresh ET and NC counterparts, and are still heavier at school entry. This differs from that observed in singletons from the same cohort, where babies in the three conception groups had similar weights by school entry age. The results are reassuring on known differences in FET versus fresh ET and NC twin outcomes. However, FET twins grow faster and are consistently larger, and more ART twins depict catch-up growth. These may lead to an increased risk profile for non-communicable diseases in later life. As such, these twin outcomes require careful evaluation using more recent and comprehensive cohorts. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the EU H2020 Marie Sklodowska-Curie Innovative Training Networks (ITN) grant Dohartnet (H2020-MSCA-ITN-2018-812660). The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

General health in a cohort of children conceived after assisted reproductive technology in the United Kingdom: a population-based record-linkage study.

BACKGROUND: Assisted reproductive technology use is increasing annually; however, data on long-term child health outcomes including hospital admissions are limited. OBJECTIVE: This study aimed to examine the potential effects of assisted reproductive technology on any and cause-specific hospital admissions unrelated to perinatal diagnoses. STUDY DESIGN: This was a population-based record-linkage study that included a previously established cohort of children born after assisted reproductive technology in the United Kingdom between 1997 and 2009 (n=63,877), their naturally conceived siblings (n=11,343), and matched naturally conceived population controls (n=127,544) linked to their postnatal health outcomes up to March 31, 2016 to provide robust risk estimates of the potential effects of assisted reproductive technology on any and cause-specific hospital admissions unrelated to perinatal diagnoses. In addition, comparison of hospital admissions by type of treatment was made. Cox regression was used to estimate the risk of hospital admission, and negative binomial regression was used to compare the number of hospital admissions per year. RESULTS: This study had 1.6 million person-years of follow-up (mean, 12.9 years; range, 0-19 years), and the mean age at the time of first hospital admission was 6.5 years (range, 0-19 years). Singletons born after assisted reproductive technology had increased risk of any hospital admission compared with naturally conceived population controls (hazard ratio, 1.08; 95% confidence interval, 1.05-1.10) but not naturally conceived siblings (hazard ratio, 1.01; 95% confidence interval, 0.94-1.09). We observed increased risk of diagnoses related to neoplasms and diseases of the respiratory, musculoskeletal, digestive, and genitourinary systems, and lower risk of injury, poisoning, and consequences of external causes compared with naturally conceived population controls. Children born after intracytoplasmic sperm injection had a lower risk of hospital admission compared with those born after in vitro fertilization, although no such differences were observed between children born after fresh embryo transfers and those born after frozen embryo transfers. CONCLUSION: Children born after assisted reproductive technology had greater numbers of hospital admissions compared with naturally conceived population controls. Attenuation of these differences in relation to their naturally conceived siblings suggested that this could be partially attributed to the influence of parental subfertility on child health, increased parental concerns, and an actual increase in morbidity in children born after assisted conception.

Trophectoderm differentiation to invasive syncytiotrophoblast is promoted by endometrial epithelial cells during human embryo implantation.

STUDY QUESTION: How does the human embryo breach the endometrial epithelium at implantation? SUMMARY ANSWER: Embryo attachment to the endometrial epithelium promotes the formation of multinuclear syncytiotrophoblast from trophectoderm, which goes on to breach the epithelial layer. WHAT IS KNOWN ALREADY: A significant proportion of natural conceptions and assisted reproduction treatments fail due to unsuccessful implantation. The trophectoderm lineage of the embryo attaches to the endometrial epithelium before breaching this barrier to implant into the endometrium. Trophectoderm-derived syncytiotrophoblast has been observed in recent in vitro cultures of peri-implantation embryos, and historical histology has shown invasive syncytiotrophoblast in embryos that have invaded beyond the epithelium, but the cell type mediating invasion of the epithelial layer at implantation is unknown. STUDY DESIGN, SIZE, DURATION: Fresh and frozen human blastocyst-stage embryos (n = 46) or human trophoblast stem cell (TSC) spheroids were co-cultured with confluent monolayers of the Ishikawa endometrial epithelial cell line to model the epithelial phase of implantation in vitro. Systems biology approaches with published transcriptomic datasets were used to model the epithelial phase of implantation in silico. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human embryos surplus to treatment requirements were consented for research. Day 6 blastocysts were co-cultured with Ishikawa cell layers until Day 8, and human TSC spheroids modelling blastocyst trophectoderm were co-cultured with Ishikawa cell layers for 48 h. Embryo and TSC morphology was assessed by immunofluorescence microscopy, and TSC differentiation by real-time quantitative PCR (RT-qPCR) and ELISA. Single-cell human blastocyst transcriptomes, and bulk transcriptomes of TSC and primary human endometrial epithelium were used to model the trophectoderm-epithelium interaction in silico. Hypernetworks, pathway analysis, random forest machine learning and RNA velocity were employed to identify gene networks associated with implantation. MAIN RESULTS AND THE ROLE OF CHANCE: The majority of embryos co-cultured with Ishikawa cell layers from Day 6 to 8 breached the epithelial layer (37/46), and syncytiotrophoblast was seen in all of these. Syncytiotrophoblast was observed at the embryo-epithelium interface before breaching, and syncytiotrophoblast mediated all pioneering breaching events observed (7/7 events). Multiple independent syncytiotrophoblast regions were seen in 26/46 embryos, suggesting derivation from different regions of trophectoderm. Human TSC spheroids co-cultured with Ishikawa layers also exhibited syncytiotrophoblast formation upon invasion into the epithelium. RT-qPCR comparison of TSC spheroids in isolated culture and co-culture demonstrated epithelium-induced upregulation of syncytiotrophoblast genes CGB (P = 0.03) and SDC1 (P = 0.008), and ELISA revealed the induction of hCGß secretion (P = 0.03). Secretory-phase primary endometrial epithelium surface transcriptomes were used to identify trophectoderm surface binding partners to model the embryo-epithelium interface. Hypernetwork analysis established a group of 25 epithelium-interacting trophectoderm genes that were highly connected to the rest of the trophectoderm transcriptome, and epithelium-coupled gene networks in cells of the polar region of the trophectoderm exhibited greater connectivity (P < 0.001) and more organized connections (P < 0.0001) than those in the mural region. Pathway analysis revealed a striking similarity with syncytiotrophoblast differentiation, as 4/6 most highly activated pathways upon TSC-syncytiotrophoblast differentiation (false discovery rate (FDR < 0.026)) were represented in the most enriched pathways of epithelium-coupled gene networks in both polar and mural trophectoderm (FDR < 0.001). Random forest machine learning also showed that 80% of the endometrial epithelium-interacting trophectoderm genes identified in the hypernetwork could be quantified as classifiers of TSC-syncytiotrophoblast differentiation. This multi-model approach suggests that invasive syncytiotrophoblast formation from both polar and mural trophectoderm is promoted by attachment to the endometrial epithelium to enable embryonic invasion. LARGE SCALE DATA: No omics datasets were generated in this study, and those used from previously published studies are cited. LIMITATIONS, REASONS FOR CAUTION: In vitro and in silico models may not recapitulate the dynamic embryo-endometrial interactions that occur in vivo. The influence of other cellular compartments in the endometrium, including decidual stromal cells and leukocytes, was not represented in these models. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the mechanism of human embryo breaching of the epithelium and the gene networks involved is crucial to improve implantation success rates after assisted reproduction. Moreover, early trophoblast lineages arising at the epithelial phase of implantation form the blueprint for the placenta and thus underpin foetal growth trajectories, pregnancy health and offspring health. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by grants from Wellbeing of Women, Diabetes UK, the NIHR Local Comprehensive Research Network and Manchester Clinical Research Facility, and the Department of Health Scientist Practitioner Training Scheme. None of the authors has any conflict of interest to declare.

Does PGT-A improve assisted reproduction treatment success rates: what can the UK Register data tell us?

PURPOSE: To show how naïve analyses of aggregated UK ART Register data held by the Human Fertilisation and Embryology Authority to estimate the effects of PGT-A can be severely misleading and to indicate how it may be possible to do a more credible analysis. Given the limitations of the Register, we consider the extent to which such an analysis has the potential to answer questions about the real-world effectiveness of PGT-A. METHODS: We utilise the publicly available Register datasets and construct logistic regression models for live birth events (LBE) which adjust for confounding. We compare all PGT-A cycles to control groups of cycles that could have had PGT-A, excluding cycles that did not progress to having embryos for biopsy. RESULTS: The primary model gives an odds ratio for LBE of 0.82 (95% CI 0.68-1.00) suggesting PGT-A may be detrimental rather than beneficial. However, due to limitations in the availability of important variables in the public dataset, this cannot be considered a definitive estimate. We outline the steps required to enable a credible analysis of the Register data. CONCLUSION: If we compare like with like groups, we obtain estimates of the effect of PGT-A that suggest an overall modest reduction in treatment success rates. These are in direct contrast to an invalid comparison of crude success rates. A detailed analysis of a fuller dataset is warranted, but it remains to be demonstrated whether the UK Register data can provide useful estimates of the impact of PGT-A when used as a treatment add-on.

The expression and activity of Toll-like receptors in the preimplantation human embryo suggest a new role for innate immunity.

STUDY QUESTION: Is the innate immunity system active in early human embryo development? SUMMARY ANSWER: The pattern recognition receptors and innate immunity Toll-like receptor (TLR) genes are widely expressed in preimplantation human embryos and the pathway appears to be active in response to TLR ligands. WHAT IS KNOWN ALREADY: Early human embryos are highly sensitive to their local environment, however relatively little is known about how embryos detect and respond to specific environmental cues. While the maternal immune response is known to be key to the establishment of pregnancy at implantation, the ability of human embryos to detect and signal the presence of pathogens is unknown. STUDY DESIGN, SIZE, DURATION: Expression of TLR family and related genes in human embryos was assessed by analysis of published transcriptome data (n = 40). Day 5 (D-5) human embryos (n = 25) were cultured in the presence of known TLR ligands and gene expression and cytokine production measured compared to controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Embryos were cultured to Day 6 (D-6) in the presence of the TLR3 and TLR5 ligands Poly (I: C) and flagellin, with gene expression measured by quantitative PCR and cytokine release into medium measured using cytometric bead arrays. MAIN RESULTS AND THE ROLE OF CHANCE: TLR and related genes, including downstream signalling molecules, were expressed variably at all human embryo developmental stages. Results showed the strongest expression in the blastocyst for TLRs 9 and 5, and throughout development for TLRs 9, 5, 2, 6 and 7. Stimulation of Day 5 blastocysts with TLR3 and TLR5 ligands Poly (I: C) and flagellin produced changes in mRNA expression levels of TLR genes, including the hyaluronan-mediated motility receptor (HMMR), TLR5, TLR7, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and monocyte chemoattractant Protein-1 (MCP-1) (P < 0.05, P < 0.001 compared to unstimulated controls), and release into culture medium of cytokines and chemokines, notably IL8 (P = 0.00005 and 0.01277 for flagellin and Poly (I: C), respectively). LIMITATIONS, REASONS FOR CAUTION: This was a descriptive and experimental study which suggests that the TLR system is active in human embryos and capable of function, but does not confirm any particular role. Although we identified embryonic transcripts for a range of TLR genes, the expression patterns were not always consistent across published studies and expression levels of some genes were low, leaving open the possibility that these were expressed from the maternal rather than embryonic genome. WIDER IMPLICATIONS OF THE FINDINGS: This is the first report of the expression and activity of a number of components of the innate immunity TLR system in human embryos. Understanding the role of TLRs during preimplantation human development may be important to reveal immunological mechanisms and potential clinical markers of embryo quality and pregnancy initiation during natural conception and in ART. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Ministry of Higher Education, The State of Libya, the UK Medical Research Council, and the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility and the European Union's Horizon 2020 Research and Innovation Programmes under the Marie Sklodowska-Curie Grant Agreement No. 812660 (DohART-NET). In accordance with H2020 rules, no new human embryos were sacrificed for research activities performed from the EU funding, which concerned only in silico analyses of recorded time-lapse and transcriptomics datasets. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: n/a.

Chemical signals from eggs facilitate cryptic female choice in humans.

Mate choice can continue after mating via chemical communication between the female reproductive system and sperm. While there is a growing appreciation that females can bias sperm use and paternity by exerting cryptic female choice for preferred males, we know surprisingly little about the mechanisms underlying these post-mating choices. In particular, whether chemical signals released from eggs (chemoattractants) allow females to exert cryptic female choice to favour sperm from specific males remains an open question, particularly in species (including humans) where adults exercise pre-mating mate choice. Here, we adapt a classic dichotomous mate choice assay to the microscopic scale to assess gamete-mediated mate choice in humans. We examined how sperm respond to follicular fluid, a source of human sperm chemoattractants, from either their partner or a non-partner female when experiencing a simultaneous or non-simultaneous choice between follicular fluids. We report robust evidence under these two distinct experimental conditions that follicular fluid from different females consistently and differentially attracts sperm from specific males. This chemoattractant-moderated choice of sperm offers eggs an avenue to exercise independent mate preference. Indeed, gamete-mediated mate choice did not reinforce pre-mating human mate choice decisions. Our results demonstrate that chemoattractants facilitate gamete-mediated mate choice in humans, which offers females the opportunity to exert cryptic female choice for sperm from specific males.

Associations of IVF singleton birthweight and gestation with clinical treatment and laboratory factors: a multicentre cohort study.

STUDY QUESTION: Do IVF treatment and laboratory factors affect singleton birthweight (BW)? SUMMARY ANSWER: BWs of IVF-conceived singleton babies are increasing with time, but we cannot identify the specific treatment factors responsible. WHAT IS KNOWN ALREADY: IVF-conceived singleton babies from fresh transfers have slightly lower BW than those conceived naturally, whilst those from frozen embryo transfer (FET) cycles are heavier and comparable to naturally conceived offspring. Our recent studies have shown that BW varies significantly between different IVF centres, and in a single centre, is also increasing with time, without a corresponding change in BWs of naturally conceived infants. Although it is likely that factors in the IVF treatment cycle, such as hormonal stimulation or embryo laboratory culture conditions, are associated with BW differences, our previous study designs were not able to confirm this. STUDY DESIGN, SIZE, DURATION: Data relating to BW outcomes, IVF treatment and laboratory parameters were collated from pre-existing electronic records in five participating centres for all singleton babies conceived between August 2007 and December 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Seven thousand, five hundred and eighty-eight births, 6207 from fresh and 1381 from FET. Infants with severe congenital abnormalities were excluded. The primary outcome of gestation-adjusted BW and secondary outcomes of unadjusted BW and gestation were analysed using multivariable regression models with robust standard errors to allow for the correlation between infants with the same mother. The models tested treatment factors allowing for confounding by centre, time and patient characteristics. A similar matched analysis of a subgroup of 379 sibling pairs was also performed. MAIN RESULTS AND THE ROLE OF CHANCE: No significant associations of birth outcomes with IVF embryo culture parameters were seen independent of clinic or time, including embryo culture medium, incubator type or oxygen level, although small differences cannot be ruled out. We did not detect any significant differences associated with hormonal stimulation in fresh cycles or hormonal synchronization in FET cycles. Gestation-adjusted BW increased by 13.4 (95% CI 0.6-26.1) g per year over the period of the study, and babies born following FET were 92 (95% CI 57-128) g heavier on average than those from the fresh transfer. LIMITATIONS, REASONS FOR CAUTION: Although no specific relationships have been identified independent of clinic and time, the confidence intervals remain large and do not exclude clinically relevant effect sizes. As this is an observational study, residual confounding may still be present. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrates the potential for large scale analysis of routine data to address critical questions concerning the long-term implications of IVF treatment, in accordance with the Developmental Origins of Health and Disease hypothesis. However, much larger studies, at a national scale with sufficiently detailed data, are required to identify the treatment parameters associated with differences in BW or other relevant outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the EU FP7 project grant, EpiHealthNet (FP7-PEOPLE-2012-ITN-317146). No competing interests were identified. TRIAL REGISTRATION NUMBER: N/A.

Glucose concentration during equine in vitro maturation alters mitochondrial function.

The use of in vitro embryo production in the horse is increasing in clinical and research settings; however, protocols are yet to be optimised. Notably, the two most commonly used base media for in vitro maturation (IVM) supply glucose at markedly different concentrations: physiological (5.6 mM, M199) or supraphysiological (17 mM, DMEM/F-12). Exposure to high glucose has detrimental effects on oocytes and early embryos in many mammalian species, but the impact has not yet been examined in the horse. To address this, we compared the energy metabolism of equine COCs matured in M199-based maturation medium containing either 5.6 or 17 mM glucose, as well as expression of key genes in oocytes and cumulus cells. Oocytes were fertilised by ICSI and cultured. Analysis of spent medium revealed that COC glucose consumption and production of lactate and pyruvate were similar between treatments. However, the glycolytic index was decreased at 17 mM and analysis of mitochondrial function of COCs revealed that IVM in 17 mM glucose was associated with decreased ATP-coupled respiration and increased non-mitochondrial respiration compared to that for 5.6 mM glucose. We also found that the metabolic enzyme lactate dehydrogenase-A (LDHA) was downregulated in cumulus cells of oocytes that completed IVM in 17 mM glucose. There was no difference in maturation or blastocyst rates. These data indicate that COC mitochondrial function and gene expression are altered by high glucose concentration during IVM. Further work is needed to determine if these changes are associated with developmental changes in the resulting offspring.

The role of Trp53 in the mouse embryonic response to DNA damage.

Apoptosis occurs primarily in the blastocyst inner cell mass, cells of which go on to form the foetus. Apoptosis is likely to play a role in ensuring the genetic integrity of the foetus, yet little is known about its regulation. In this study, the role of the mouse gene, transformation-related protein 53 (Trp53) in the response of embryos to in vitro culture and environmentally induced DNA damage was investigated using embryos from a Trp53 knockout mouse model. In vivo-derived blastocysts were compared to control embryos X-irradiated at the two-cell stage and cultured to Day 5. An analysis of DNA by comet assay demonstrated that 1.5 Gy X-irradiation directly induced damage in cultured two-cell mouse embryos; this was correlated with retarded development to blastocyst stage and increased apoptosis at the blastocyst stage but not prior to this. Trp53 null embryos developed to blastocysts at a higher frequency and with higher cell numbers than wild-type embryos. Trp53 also mediates apoptosis in conditions of low levels of DNA damage, in vivo or in vitro in the absence of irradiation. However, following DNA damage induced by X-irradiation, apoptosis is induced by Trp53 independent as well as dependent mechanisms. These data suggest that Trp53 and apoptosis play important roles in normal mouse embryonic development both in vitro and in vivo and in response to DNA damage. Therefore, clinical ART practices that alter apoptosis in human embryos and/or select embryos for transfer, which potentially lack a functional Trp53 gene, need to be carefully considered.

The impact of IVF on birthweight from 1991 to 2015: a cross-sectional study.

STUDY QUESTION: Has birthweight (BW) changed over time among IVF-conceived singletons? SUMMARY ANSWER: Singleton BW has increased markedly over the past 25 years. WHAT IS KNOWN ALREADY: IVF conceived singletons have had a higher incidence of low BW compared to spontaneously conceived singletons, and this has raised concerns over long-term increased risks of cardio-metabolic disease. However, few causal links between IVF procedures and BW have been robustly established, and few studies have examined whether BW has changed over time as IVF techniques have developed. STUDY DESIGN, SIZE, DURATION: A total of 2780 live born singletons conceived via IVF or ICSI treated in the reproductive medicine department of a single publicly funded tertiary care centre between 1991 and 2015 were included in this retrospective study. The primary outcome measure was singleton BW adjusted for gestational age, maternal parity and child gender. Multivariable linear regression models were used to estimate the associations between patient prognostic factors and IVF treatment procedures with adjusted BW. PARTICIPANTS/MATERIALS, SETTING, METHODS: All singletons conceived at the centre following IVF/ICSI using the mother's own oocytes, and non-donated fresh or frozen/thawed embryos with complete electronic data records, were investigated. Available electronic records were retrieved from the Human Fertilization and Embryology Authority for dataset collation. Multiple linear regression analysis was used to evaluate associations between IVF treatment parameters and BW, after adjusting for the year of treatment and patient characteristics and pregnancy factors. MAIN RESULTS AND THE ROLE OF CHANCE: In the primary multivariable model, singleton BW increased by 7.4 g per year (95% CI: 3.2-11.6 g, P = 0.001), an increase of close to 180 g throughout the 25-year period after accounting for gestational age, maternal parity, child gender, IVF treatment parameters, patient prognostic characteristics and pregnancy factors. Fresh and frozen embryo transfer-conceived singletons showed a similar increase in BW. Frozen/thawed embryo transfer conceived singletons were on average 53 g heavier than their fresh embryo conceived counterparts (95% CI: 3.7-103.3 g, P = 0.035). LIMITATIONS, REASONS FOR CAUTION: The independent variables included in the study were limited to those that have been consistently recorded and stored electronically over the past two decades. WIDER IMPLICATIONS OF THE FINDINGS: There has been a progressive BW increase in IVF singletons over time in one large centre with consistent treatment eligibility criteria. Such a change is not seen in the general population of live born singletons in the UK or other developed countries, and seems to be specific to this IVF population. This may be a reflection of changes in practice such as undisturbed extended embryo culture to the blastocyst stage, optimized commercial culture media composition, single embryo transfer and ICSI. Moreover, singletons conceived from frozen/thawed embryos had higher birth weights when compared to their fresh embryo transfer counterparts. The causal pathway is unknown; however, it could be due to the impact on embryos of the freeze/thaw process, self-selection of embryos from couples who produce a surplus of embryos, and/or embryo replacement into a more receptive maternal environment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the EU FP7 project grant, EpiHealthNet (FP7-PEOPLE-2012-ITN-317146). The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Temperature of embryo culture for assisted reproduction.

BACKGROUND: 'Infertility' is defined as the failure to achieve pregnancy after 12 months or more of regular unprotected sexual intercourse. One in six couples experience a delay in becoming pregnant. In vitro fertilisation (IVF) is one of the assisted reproductive techniques used to enable couples to achieve a live birth. One of the processes involved in IVF is embryo culture in an incubator, where a stable environment is created and maintained. The incubators are set at approximately 37°C, which is based on the human core body temperature, although several studies have shown that this temperature may in fact be lower in the female reproductive tract and that this could be beneficial. In this review we have included randomised controlled trials which compared different temperatures of embryo culture. OBJECTIVES: To assess different temperatures of embryo culture for human assisted reproduction, which may lead to higher live birth rates. SEARCH METHODS: We searched the following databases and trial registers: the Cochrane Gynaecology and Fertility (CGF) Group Specialised Register of Controlled Trials, the Cochrane Central Register of Studies Online, MEDLINE, Embase, PsycINFO, CINAHL, clinicaltrials.gov, The World Health Organization International Trials Registry Platform search portal, DARE, Web of Knowledge, OpenGrey, LILACS database, PubMed and Google Scholar. Furthermore, we manually searched the references of relevant articles and contacted experts in the field to obtain additional data. We did not restrict the search by language or publication status. We performed the last search on 6 March 2019. SELECTION CRITERIA: Two review authors independently screened the titles and abstracts of articles retrieved by the search. Full texts of potentially eligible randomised controlled trials (RCTs) were obtained and screened. We included all RCTs which compared different temperatures of embryo culture in IVF or intracytoplasmic sperm injection (ICSI), with a minimum difference in temperature between the two incubators of ≥ 0.5°C. The search process is shown in the PRISMA flow chart. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial eligibility and risk of bias and extracted data from the included studies; the third review author resolved any disagreements. We contacted trial authors to provide additional data. The primary review outcomes were live birth and miscarriage. Clinical pregnancy, ongoing pregnancy, multiple pregnancy and adverse events were secondary outcomes. All extracted data were dichotomous outcomes, and odds ratios (OR) were calculated with 95% confidence intervals (CIs) on an intention-to-treat basis. We assessed the overall quality of the evidence for the main comparisons using GRADE methods. MAIN RESULTS: We included three RCTs, with a total of 563 women, that compared incubation of embryos at 37.0°C or 37.1°C with a lower incubator temperature (37.0°C versus 36.6°C, 37.1°C versus 36.0°C, 37.0° versus 36.5°C). Live birth, miscarriage, clinical pregnancy, ongoing pregnancy and multiple pregnancy were reported. After additional information from the authors, we confirmed one study as having no adverse events; the other two studies did not report adverse events. We did not perform a meta-analysis as there were not enough studies included per outcome. Live birth was not graded since there were no data of interest available. The evidence for the primary outcome, miscarriage, was of very low quality. The evidence for the secondary outcomes, clinical pregnancy, ongoing pregnancy and multiple pregnancy was also of very low quality. We downgraded the evidence because of high risk of bias (for performance bias) and imprecision due to limited included studies and wide CIs.Only one study reported the primary outcome, live birth (n = 52). They performed randomisation at the level of oocytes and not per woman, and used a paired design whereby two embryos, one from 36.0°C and one from 37.0°C, were transferred. The data from this study were not interpretable in a meaningful way and therefore not presented. Only one study reported miscarriage. We are uncertain whether incubation at a lower temperature decreases the miscarriage (odds ratio (OR) 0.90, 95% CI 0.52 to 1.55; 1 study, N = 412; very low-quality evidence).Of the two studies that reported clinical pregnancy, only one of them performed randomisation per woman. We are uncertain whether a lower temperature improves clinical pregnancy compared to 37°C for embryo incubation (OR 1.08, 95% CI 0.73 to 1.60; 1 study, N = 412; very low-quality evidence). For the outcome, ongoing pregnancy, we are uncertain if a lower temperature is better than 37°C (OR 1.10, 95% CI 0.75 to 1.62; 1 study, N = 412; very low quality-evidence). Multiple pregnancy was reported by two studies, one of which used a paired design, which made it impossible to report the data per temperature. We are uncertain if a temperature lower than 37°C reduces multiple pregnancy (OR 0.80, 95% CI 0.31 to 2.07; 1 study, N = 412; very low-quality evidence). There was insufficient evidence to make a conclusion regarding adverse events, as no studies reported data suitable for analysis. AUTHORS' CONCLUSIONS: This review evaluated different temperatures for embryo culture during IVF. There is a lack of evidence for the majority of outcomes in this review. Based on very low-quality evidence, we are uncertain if incubating at a lower temperature than 37°C improves pregnancy outcomes. More RCTs are needed for comparing different temperatures of embryo culture which require reporting of clinical outcomes as live birth, miscarriage, clinical pregnancy and adverse events.

The growth of assisted reproductive treatment-conceived children from birth to 5 years: a national cohort study.

BACKGROUND: Birth weight and early child growth are important predictors of long-term cardiometabolic disease risk, in line with the Developmental Origins of Health and Disease hypothesis. As human assisted reproductive technologies (ARTs) occur during the sensitive periconceptional window of development, it has recently become a matter of urgency to investigate risk in ART-conceived children. METHODS: We have conducted the first large-scale, national cohort study of early growth in ART children from birth to school age, linking the register of ART, held by the UK's Human Fertilisation and Embryology Authority, to Scottish maternity and child health databases. RESULTS: In this study of 5200 ART and 20,800 naturally conceived (NC) control children, linear regression analysis revealed the birthweight of babies born from fresh embryo transfer cycles is 93.7 g [95% CI (76.6, 110.6)g] less than NC controls, whereas babies born from frozen embryo transfer (FET) cycles are 57.5 g [95% CI (30.7, 86.5)g] heavier. Fresh ART babies grew faster from birth (by 7.2 g/week) but remained lighter (by 171 g), at 6-8 weeks, than NC babies and 133 g smaller than FET babies; FET and NC babies were similar. Length and occipital-frontal circumference followed the same pattern. By school entry (4-7 years), weight, length and BMI in boys and girls conceived by fresh ART and FET were similar to those in NC children. CONCLUSIONS: ART babies born from fresh embryo transfer grow more slowly in utero and in the first few weeks of life, but then show postnatal catch up growth by school age, compared to NC and FET babies. As low birth weight and postnatal catch-up are independent risk factors for cardiometabolic disease over the life-course, we suggest that further studies in this area are now warranted.

Osmotic stress induces JNK-dependent embryo invasion in a model of implantation.

In vitro culture during assisted reproduction technologies (ART) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2h in medium with osmolarity raised by 400mOsm induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation, and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety.

Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

STUDY QUESTION: How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model? SUMMARY ANSWER: Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium. WHAT IS KNOWN ALREADY: In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models. STUDY DESIGN, SIZE, DURATION: This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P < 0.05, P < 0.01 and P < 0.001. MAIN RESULTS AND THE ROLE OF CHANCE: Hatched E4.5 mouse blastocysts exhibited weak attachment to confluent Ishikawa cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5.5 also resulted in intermediate and stable attachment from E5.5 + 4 h; however, these embryos did not go on to breach the Ishikawa cell layer, even when co-culture was extended to E7.5 (P < 0.01). Blastocysts cultured from E4.5 in permeable transwell inserts above Ishikawa cells before transfer to direct co-culture at E5.5 went on to attach but failed to breach the Ishikawa cell layer by E6.5 (P < 0.01). Gene expression analysis at E5.5 demonstrated that direct co-culture with Ishikawa cells from E4.5 resulted in downregulation of trophectoderm transcription factors Cdx2 (P < 0.05) and Gata3 (P < 0.05) and upregulation of the TGC transcription factor Hand1 (P < 0.05). Co-culture with non-endometrial human fibroblasts did not alter the expression of these genes. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The in vitro model used here combines human carcinoma-derived endometrial cells with mouse embryos, in which the cellular interactions observed may not fully recapitulate those in vivo. The data gleaned from such models can be regarded as hypothesis-generating, and research is now needed to develop more sophisticated models of human implantation combining multiple primary endometrial cell types with surrogate and real human embryos. WIDER IMPLICATIONS OF THE FINDINGS: This study implicates blastocyst apposition to endometrial epithelial cells as a critical step in trophoblast differentiation required for implantation. Understanding this maternal regulation of the embryonic developmental programme may lead to novel treatments for infertility. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for SCB from the Anatomical Society. No conflict of interest is declared.

No common denominator: a review of outcome measures in IVF RCTs.

STUDY QUESTION: Which outcome measures are reported in RCTs for IVF? SUMMARY ANSWER: Many combinations of numerator and denominator are in use, and are often employed in a manner that compromises the validity of the study. WHAT IS KNOWN ALREADY: The choice of numerator and denominator governs the meaning, relevance and statistical integrity of a study's results. RCTs only provide reliable evidence when outcomes are assessed in the cohort of randomised participants, rather than in the subgroup of patients who completed treatment. STUDY DESIGN, SIZE, DURATION: Review of outcome measures reported in 142 IVF RCTs published in 2013 or 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Trials were identified by searching the Cochrane Gynaecology and Fertility Specialised Register. English-language publications of RCTs reporting clinical or preclinical outcomes in peer-reviewed journals in the period 1 January 2013 to 31 December 2014 were eligible. Reported numerators and denominators were extracted. Where they were reported, we checked to see if live birth rates were calculated correctly using the entire randomised cohort or a later denominator. MAIN RESULTS AND THE ROLE OF CHANCE: Over 800 combinations of numerator and denominator were identified (613 in no more than one study). No single outcome measure appeared in the majority of trials. Only 22 (43%) studies reporting live birth presented a calculation including all randomised participants or only excluding protocol violators. A variety of definitions were used for key clinical numerators: for example, a consensus regarding what should constitute an ongoing pregnancy does not appear to exist at present. LIMITATIONS, REASONS FOR CAUTION: Several of the included articles may have been secondary publications. Our categorisation scheme was essentially arbitrary, so the frequencies we present should be interpreted with this in mind. The analysis of live birth denominators was post hoc. WIDER IMPLICATIONS OF THE FINDINGS: There is massive diversity in numerator and denominator selection in IVF trials due to its multistage nature, and this causes methodological frailty in the evidence base. The twin spectres of outcome reporting bias and analysis of non-randomised comparisons do not appear to be widely recognised. Initiatives to standardise outcome reporting, such as requiring all effectiveness studies to report live birth or cumulative live birth, are welcome. However, there is a need to recognise that early outcomes of treatment, such as stimulation response or embryo quality, may be appropriate choices of primary outcome for early phase studies. STUDY FUNDING/COMPETING INTERESTS: J.W. is funded by a Doctoral Research Fellowship from the National Institute for Health Research. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health. J.W. also declares that publishing research is beneficial to his career. J.W. and A.V. are statistical editors, and M.S. is Information Specialist, for the Cochrane Gynaecology and Fertility Group, although the views expressed here are not necessarily those of the group. D.R.B. is funded by the NHS as Scientific Director of a clinical IVF service. The authors declare no other conflicts of interest.

Biological optimization, the Goldilocks principle, and how much is lagom in the preimplantation embryo.

The quiet embryo hypothesis postulates that early embryo viability is associated with a relatively low metabolism (Leese, 2002 BioEssays 24: 845-849). This proposal is re-visited here using retrospective and prospective data on the metabolic activity and kinetics of preimplantation development alongside the concept that an optimal range of such indices and of energetic efficiency influences embryogenesis. It is concluded that these considerations may be rationalized by proposing the existence of a "Goldilocks zone," or as it is known in Sweden, of lagom-meaning "just the right amount"-within which embryos with maximum developmental potential can be categorized. Mol. Reprod. Dev. 83: 748-754, 2016 © 2016 Wiley Periodicals, Inc.

Factors affecting embryo viability and uterine receptivity: insights from an analysis of the UK registry data.

Many studies have identified prognostic factors for IVF treatment outcome; however, little information is available on the mechanism of their action. Embryo-uterus models have the potential to distinguish between factors acting on the embryo directly and those acting through the uterine environment. Here we apply embryo-uterus models to comprehensive UK registry data from two periods, 2000-2005 and 2007-2011, containing 139,444 and 226,542 embryo transfer cycles, respectively. Given this large dataset, the embryo-uterus model is capable of distinguishing between uterine and embryo effects. Maternal age is the predominant predictor of live birth and acts on both the embryo and uterine components, but with larger effects on the embryo. Prolonged embryo culture is associated with greater embryo viability, reflecting the greater degree of selection, but is also associated with greater uterine receptivity. Cryopreserved embryos are less viable and were associated with poorer uterine receptivity. This work suggests that, in addition to the direct effects of in-vitro culture on the embryonic environment during the first few days of the embryo's life, the delay in transfer after extended culture or cryopreservation can lead to an altered uterine environment for the embryo after transfer.

A secreted proteomic footprint for stem cell pluripotency.

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFß deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

How should we assess the safety of IVF technologies?

Clinical IVF treatment was established over 30 years ago through pioneering work by Edwards and Steptoe and other teams around the world and is now considered routine treatment. However, the pace of scientific and technological advances means that IVF practitioners can now access an increasing array of new and invasive technologies. The examples are many but include: extended embryo culture, development of media to include growth factors, developments in genetic screening, use of time-lapse technology and the advent of vitrification of embryos and oocytes. In parallel, wider scientific and medical advances are raising our awareness of the potential impact of assisted reproduction technology on areas such as embryonic development, gene expression and genomic imprinting and the developmental origins of health and disease. A recently suggested paradigm for assessing new technologies in IVF includes development in animal models such as rodents and large animals, preclinical research with human gametes and embryos donated to research, prospective clinical trials in IVF and, finally, follow-up studies of IVF children. In this paper, we describe efforts to address key areas of this pathway, namely preclinical research using human gametes/embryos and long-term, follow-up studies of the health of assisted reproduction children.

A systematic review of transcriptomic studies of the human endometrium reveals inconsistently reported differentially expressed genes.

Genome-wide analysis of gene expression has been widely applied to study the endometrium, although to our knowledge no systematic reviews have been performed. Here, we identified 74 studies that described transcriptomes from whole (unprocessed) endometrium samples and found that these fitted into three broad investigative categories; endometrium across the menstrual cycle, endometrium in pathology, and endometrium during hormone treatment. Notably, key participant information such as menstrual cycle length and body mass index was often not reported. Fertility status was frequently not defined and fertility-related pathologies, such as recurrent implantation failure (RIF) and recurrent pregnancy loss, were variably defined, while hormone treatments differed between almost every study. A range of 1307-3637 reported differentially expressed genes (DEG) were compared in 4-7 studies in five sub-categories; (i) secretory vs proliferative stage endometrium, (ii) mid-secretory vs early secretory stage endometrium, (iii) mid-secretory endometrium from ovarian stimulation-treated participants vs controls, (iv) mid-secretory endometrium from RIF patients vs controls, and (v) mid-secretory eutopic endometrium from endometriosis patients vs controls. Only the first two sub-categories yielded consistently reported DEG between ≥3 studies, albeit in small numbers (<40), and these were enriched in developmental process and immune response annotations. This systematic review, though not PROSPERO registered, reveals that limited demographic detail, variable fertility definitions and differing hormone treatments in endometrial transcriptomic studies hinders their comparison, and that the large majority of reported DEG do not advance the identification of underlying biological mechanisms. Future studies should apply network biology approaches and experimental validation to establish causal gene expression signatures.