STUDY QUESTION:Is patient consent to research associated with the distribution of population characteristics and study outcomes in ART cohort studies? SUMMARY ANSWER:The distribution of population characteristics in the patient consent subset differs from that in the non-consent subset and is not fully representative of the general ART population; thus, study results of population subsets requiring patient consent may be subject to bias. WHAT IS KNOWN ALREADY:Non-consent in epidemiological studies may bias study results if the consent subset differs systematically from the non-consent subset and is thus not representative of the full study population. ART cohort datasets may be biased if they require patients to consent to use their data. As an example, from September 2009 onwards, ART patients in the UK have been asked for specific 'consent to disclosure of identifying information' (CD) for research studies. STUDY DESIGN, SIZE, DURATION:This cohort study utilized an anonymized version of the Human Fertilisation and Embryology Authority (HFEA) dataset containing all CD and non-CD autologous ART treatment cycles (n = 819 512) conducted from 2004 to 2018 in the UK. A live birth (LB) subset of 155 986 singletons born during the same period was used to analyse child outcomes. Additionally, an aggregated version of the HFEA dataset was used to explore CD trends by clinic type (National Health Service [NHS], private, or both NHS and privately funded). PARTICIPANTS/MATERIALS, SETTING, METHODS:The dataset containing all gamete cycles was used to explore factors associated with giving CD and to compare LB outcome trends (number of LBs per yearly treatment cycles started) between CD and non-CD cycles. The LB subset was used to compare the birthweight outcomes (low birthweight (LBW = birthweight < 2500 g or otherwise) and macrosomia (birthweight ≥4000 g or otherwise)) between CD and non-CD cycles. Logistic regression models explored the association between CD and population characteristics and the impact of CD on birthweight outcomes over the calendar years. Each regression model was adjusted for potential confounders: for all models (maternal age, ethnicity, previous IVF cycles, previous pregnancies, previous LBs, causes of infertility (tubal, endometriosis, male factor, ovulatory, unknown), and embryo transfer type and stage); for LB and birthweight models (ICSI, elective single embryo transfer, and ovarian stimulation); and additionally for birthweight models (child sex and gestation). MAIN RESULTS AND THE ROLE OF CHANCE:During the study period, CD rates increased from 16% at its inception in 2009 to 64% in 2018. Fewer cycles from older patients (40-44 years old) and ethnic minorities (Black and Asian) gave CD. Cycles with previous ART treatments and LBs had lower rates of giving CD. CD was also associated with LB rates (higher in the CD group) and LBW (slightly more prevalent in the non-CD group). CD rates were consistently higher in NHS-only funded clinics than in clinics with partly or fully private funding. It may be possible to adjust for much of the post-2009 bias by weighting by the probability of inclusion derived from supplementary data. LIMITATIONS, REASONS FOR CAUTION:Important factors not provided or unavailable in the dataset included socio-economic and lifestyle factors. Additionally, the anonymized dataset provided to us had banded/categorized maternal age, gestation, and birthweight variables, possibly limiting our estimates' precision. WIDER IMPLICATIONS OF THE FINDINGS:This study shows that using only consented data in ART observational cohort studies may result in a sample that differs from the non-consented sample and general ART population. Specifically, our results show differences in the distribution of population characteristics, LB, and LBW outcomes between CD and non-CD groups in the UK HFEA ART register dataset. Careful attention is therefore required when analysing and interpreting these and similar cohort data; failure to consider the impact of consent will likely produce misleading results. In the HFEA register, this applies to research studies using CD data (including bespoke data requests and linkage studies) after the introduction of CD in October 2009. A potential solution weighting by the probability of consent is briefly introduced. 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.

[This corrects the article DOI: 10.1371/journal.pone.0299365.].

Background: 1 in 39 UK babies are conceived using assisted reproductive technologies (ART). Pregnancy complications are more common following ART and may be further influenced by individual ART variables, with evidence linking suboptimal periconceptual/ intrauterine environment and adult cardiometabolic risk. However, high quality retrospective studies linking detailed ART variables and subsequent pregnancy outcomes are made more challenging by UK data-sharing restrictions and lack in-depth examination of maternofetal health. This study aimed to examine the feasibility of prospective observation of ART pregnancies and subsequent retrospective ART data linkage. Methods: The START clinic was a single-centre observational antenatal cohort study recruiting medically healthy participants with singleton pregnancies after IVF treatment, ovulation induction (OI) or spontaneous conception. Serial maternofetal assessments were made at 3-6 week intervals throughout pregnancy and at birth. Participants self-identified ART treatment centres and consented to fertility treatment data access, from which ART treatment details were extracted where possible. Recruitment, patient experience, uptake of consent for data access and optional maternofetal assessments, as well as the possibility of data access, were considered as part of the feasibility assessment. Results: 120 participants were recruited, 99 IVF-conceiving (36 fresh-ET, 62 frozen-ET, 1 unknown ET type) and 21 non-IVF-conceiving (7 OI, 14 spontaneous); 100% of participants were followed to delivery and 100% satisfaction reported. Consent to access fertility treatment data was obtained in 101/106 (95.3%) of ART-conceiving participants, and could be accessed from 3/16 fertility centres approached (one NHS, two private). Overall, ART treatment data could be linked to 57/101 (58%) of consenting study participants, and accuracy of participant-reported ART parameter data ranged from 98-100% when compared to ART treatment records. Conclusions: It is feasible and highly acceptable to patients to prospectively study ART pregnancies in the UK. Retrospective linkage to ART treatment records is possible but access is inconsistent; a prospective or participant-reported approach is recommended for future study designs. Such studies are needed to better understand the potential impacts of ART on mothers and fetuses, and to guide future ART practices, potentially reducing risk of future morbidity and mortality in the mother and offspring. Trial Registration: clinicaltrials.gov NCT04488861, registered on 23/7/2020

Demand for in vitro fertilization (IVF) treatment is growing; however, success rates remain low partly due to difficulty in selecting the best embryo to be transferred. Current manual assessments are subjective and may not take advantage of the most informative moments in embryo development. Here, we apply convolutional neural networks (CNNs) to identify key windows in pre-implantation human development that can be linked to embryo viability and are therefore suitable for the early grading of IVF embryos. We show how machine learning models trained at these developmental time points can be used to refine overall embryo viability assessment. Exploiting the well-known capabilities of transfer learning, we illustrate the performance of CNN models for very limited datasets, paving the way for the use on a clinic-by-clinic basis, catering for local data heterogeneity.

Sperm DNA contains strand breaks and base damage that can potentially affect reproductive health. This study aims to determine to what extent sperm DNA integrity and alkylation is associated with semen quality and assisted reproduction technology (ART) treatment outcomes, in particular fertilisation and cleavage rates. Male partners of couples attending for infertility treatment were recruited. DNA integrity (% tail DNA, sperm with either low (LDD) or high (HDD) damage levels) was measured by a neutral Comet assay and N7-methyldeoxyguanosine (N7-MedG) DNA levels by an immunoslotblot in sperm prepared by density gradient centrifugation. Associations between DNA damage, semen quality and ART treatment outcomes were assessed. N7-MedG levels were lower and the proportion of LDD sperm higher in prepared than in neat sperm samples. The proportion of HDD sperm and % tail DNA were significantly negatively associated and the proportion of LDD sperm positively associated with semen quality. Fertilisation, but not cleavage, rate nor live birth, was significantly negatively associated with N7-MedG levels, the proportion of HDD sperm and % tail DNA and was positively associated with the proportion of LDD sperm. These results confirm that DNA damage, even in prepared sperm, is associated with adverse semen quality and suggest that sperm DNA damage affects the early stages of embryo formation.

The clinical in vitro fertilisation (IVF) need for optimal culture conditions and improved selection of human embryos for transfer led to the development of time-lapse systems built into incubators to allow a stable, well-defined and undisturbed culture environment with continual monitoring of embryo development. Clinical data suggest that both undisturbed culture of embryos and selection algorithms based on time-lapse morphometric parameters can potentially improve embryo development and live birth rates. However, there remains a need to validate and understand the mechanisms underlying the potential benefits of the time-lapse technology in IVF. In this study, we compared the gene expression of human embryos grown in an undisturbed time-lapse system with a conventional incubator and showed that there is no fundamental difference in the developmental program of the undisturbed embryos, which provides important reassurance concerning the time-lapse technology. We then performed a network-based integrative analysis based on the undisturbed blastocyst transcriptomes and identified time-lapse parameter correlated genes. These showed cross talk with identified embryo development gene functional modules, suggesting potential molecular mechanisms underlying the clinical predictive capabilities of embryo time-lapse morphokinetic parameters for subsequent live birth. This study suggests methodologies for assessing the impact of additional predictive correlates of value for optimal embryo development and understanding their mechanisms of action. ### Competing Interest Statement The authors have declared no competing interest.

Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycemia on these processes remains poorly understood. Here, we show that physiological levels of glucose (5 mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17 mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in cocultures at 17 mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17 mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarized epithelium. Two-dimensional (2-D) and three-dimensional (3-D) morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of Rho (ras homologous)A-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5 mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signaling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes.

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 TGFβ 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.

Objective To compare the risk of hospitalisation for conditions originating in the perinatal period between children conceived via assisted reproductive technology and those that are naturally conceived, differentiating by treatment type.Study design, setting and participants Population-based record-linkage study of children born after assisted reproduction in the UK between 2002 and 2009 (n=44 618), their naturally conceived siblings (n=8462) and matched naturally conceived population (n=89 072) controls linked to their hospital inpatient records up to 31 March 2016.Primary and secondary outcome measures Robust estimates of the overall and cause-specific risk of hospital admission for adverse perinatal events and the comparison of outcomes by type of treatment.Results Over the study period, 17 132 (38.40%) children conceived via assisted reproduction and 30 306 (34.02%) and 1738 (20.54%) naturally conceived population and sibling controls, respectively, were admitted to the hospital for severe perinatal events. Compared with the population controls, singletons (Risk ratio (95% CI 1.30 (1.26, 1.34))) and twins (1.01 (0.99, 1.03)) conceived via assisted reproduction exhibited a higher risk of hospitalisation for any adverse perinatal event. However, no such increase was observed in the within-sibling analysis (0.97 (0.84, 1.12)). Similar patterns were seen for diagnoses related to length of gestation and fetal growth (vs population controls: 1.37 (1.29, 1.46); vs siblings: 1.17 (0.86, 1.60)); birth trauma (vs population controls: 1.23 (1.04, 1.44); vs siblings: 0.78 (0.47, 1.30)); respiratory and cardiovascular disorders (vs population controls: 1.28 (1.20, 1.38); vs siblings: 0.72 (0.53, 0.98)); infections (vs population controls: 1.30 (1.06, 1.59); vs siblings: 0,68 (0.24, 1.90)) and several other conditions. Associations were similar when comparing in vitro fertilisation to intracytoplasmic sperm injection and were higher when comparing fresh to frozen embryo transfers.Conclusion Children conceived via assisted reproduction showed modest increases in the risk of hospitalisations for severe perinatal events when compared with population controls, although these findings were attenuated in the sibling analyses. The imprecision of within-sibling analyses highlights the need for larger studies to explore potential causal effects.

Study question Does glucose concentration in culture medium have an impact on the DNA methylome of the early human embryo? Summary answer Glucose concentration is associated with changes in gene expression, global DNA methylation, methylation levels at CpG islands and at key histone modifications in human blastocysts. What is known already Preimplantation human embryos are highly sensitive to their local environment, and this may have long term implications for the health of the developing embryo, fetus and offspring. Glucose is a standard component of human embryo culture media, due to its importance as a nutrient. However, concentrations of glucose differ widely between different commercially available types. The present study was designed to determine whether changes in glucose concentration could influence global methylation and gene expression in the human preimplantation embryo. Study design, size, duration Human embryos were cultured in clinically relevant concentrations of glucose and global DNA methylation analysis was performed. The effect of glucose concentration on the embryo epigenome, specifically DNA methylation, was analysed. Participants/materials, setting, methods Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Embryos were cultured to the blastocyst stage in Vitrolife G-TL™ medium, either at 0.9 mM or 3.5 mM glucose, separated via immunosurgery into Inner Cell Mass (ICM) and trophectoderm (TE) samples, and compared for both DNA methylation and gene expression. This allowed us to evaluate the association between DNA methylation and previously importantly identified biological pathways. Main results and the role of chance The concentration of glucose in human embryo culture medium was associated with changes in gene expression and global DNA methylation in both ICM and TE, and methylation levels at CpG islands and key histone modifications. These results are significant because glucose is a major nutrient metabolised by human embryos in culture, and yet we know relatively little of its downstream effects on the genome and epigenome. Wider implications of the findings Commercially available embryo culture media with varying glucose levels have also been associated with altered fetal growth, birthweight and postnatal development of IVF offspring. Our findings may have important ramifications for potential clinical markers of embryo quality and pregnancy initiation, and improve understanding of the mechanisms underlying the impact of the early environment on the long term health of ART offspring. Study funding/competing interest(s) This work was funded by the National Council for Science and Technology of Mexico (CONACyT), an NIHR pre-doctoral fellowship (PCAF) to MM, the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility, the University of Manchester and Manchester University NHS Foundation Trust. None of the authors has any conflict of interest to declare. ### Competing Interest Statement The authors have declared no competing interest.

Low-cost personal exposure monitors (PEMs) to measure personal exposure to air pollution are potentially promising tools for health research. However, their adoption requires robust validation. This study evaluated the performance of twenty-one Plume Lab Flow2s (PLFs) by comparing its air pollutant measurements, particulate matter with a diameter of 2.5 μm or less (PM2.5), 10 μm or less (PM10), and nitrogen dioxide (NO2), against several high-quality air pollution monitors under field conditions (at indoor, outdoor, and roadside locations). Correlation and regression analysis were used to evaluate measurements obtained by different PLFs against reference instrumentation. For all measured pollutants, the overall correlation coefficient between the PLFs and the reference instruments was often weak (r < 0.4). Moderate correlation was observed for one PLF unit at the indoor location and two units at the roadside location when measuring PM2.5, but not for PM10 and NO2 concentration. During periods of particularly higher pollution, 11 PLF tools showed stronger regression results (R2 values > 0.5) with one-hour and 9 PLF units with one-minute time interval. Results show that the PLF cannot be used robustly to determine high and low exposure to poor air. Therefore, the use of PLFs in research studies should be approached with caution if data quality is important to the research outputs.

Almost 40 years ago David Barker made his observation that poor in utero growth increased cardiovascular disease risk in the offspring. A few years prior to this, the first baby to be conceived through IVF was born. Since then, an estimated 8 million babies worldwide have been born via one form or another of Assisted Reproductive Technology (ART). However, data from experimental animal and human clinical studies have highlighted the period around conception as being particularly sensitive to sub-optimal environmental conditions. Furthermore, there is growing concern that aspects of the ART procedures themselves may alter fetal and neonatal growth and increase the incidence of cardiovascular and metabolic diseases, cancer, asthma and neurodevelopmental issues in the children. However, a large degree of confounding factors including parental infertility, disparity in ART culture media and methods, and even the design of the follow-up studies, mean that further investigations are required in the definition of causal relationships between ART and child health.

Low-cost personal exposure monitors (PEMs) to measure personal exposure to air pollution are potentially promising tools for health research. However, their adoption requires robust validation. This study evaluated the performance of twenty-one Plume Lab Flow2 (PLF) by comparing its air pollutant measurements, particulate matter with a diameter of 2.5 μm or less (PM2.5), 10 μm or less (PM10), and nitrogen dioxide (NO2), against several high-quality air pollution monitors under field conditions (at indoor, outdoor, and roadside locations). Correlation and regression analysis were used to evaluate measurements obtained by different PLFs against reference instrumentation. For all measured pollutants, the overall correlation coefficient between the PLFs and the reference instruments was often weak (r<0.4). Moderate correlation was observed for one PLF unit at indoor location and two units at roadside location, when measuring PM2.5, but not for PM10 and NO2 concentration. During periods of particularly higher pollution, 11 PLF tools showed stronger regression results (R2 values > 0.5) with one-hour and 9 PLF units with one-minute time interval. Results show that the PLF cannot be used robustly to determine high and low exposure to poor air. Therefore, the use of PLFs in research studies should be approached with caution if data quality is important to the research outputs.

Abstract Purpose This study aims to determine to what extent sperm DNA damage is associated with semen quality and assisted reproduction technology (ART) treatment outcomes. Methods 105 male partners of couples attending for infertility treatment were recruited and DNA integrity was measured by a neutral Comet assay and N7-methyldeoxyguanosine (N7-MedG) DNA levels by an immunoslotblot in sperm prepared by density gradient centrifugation for treatment use. Associations between measures of DNA damage (%tail DNA, proportion of sperm with either low (LDD) or high (HDD) levels of DNA damage, and N7-methylguanine levels), semen quality (concentration and motility) and ART outcomes (% oocytes fertilised, % embryo fragmentation, % cleavage, and birth outcome) were assessed. Results In the prepared sperm samples, DNA damage was significantly associated with semen quality and was lower than that in the original neat sample. % fertilisation was significantly negatively associated with N7-MedG levels, %HDD and % tail DNA and was positively associated with %LDD. % cleavage and live birth in fresh cycles were not associated with DNA damage but there was evidence that %LDD was lower, and %HDD higher, in couples with live births after a frozen embryo transfer cycle. Conclusion DNA damage can negatively impact on semen quality and fertilization rate but not embryo cleavage or live birth rate. These results suggest that the impact of sperm DNA damage on pregnancy outcomes appears in the early stage of embryo development.

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 (DEGs) were compared in four to seven 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 hinder 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. Lay summary The endometrium lines the inner wall of the uterus and is the site where the fertilised egg implants to establish pregnancy. Disorders of the endometrium cause infertility and chronic pain. Techniques to measure genetic activity, termed transcriptomics, have been applied to better understand the endometrium in health and disease. We collated all studies, totalling 74, that describe transcriptomics of endometrial samples from non-pregnant women and compared study designs and genetic activity measurements. The studies generally looked at small numbers of samples, with most focussing on fertility rather than endometrial disorders. Study designs were variable, comparing women under different definitions of fertility and disease, and under different treatments. Additionally, key participant factors such as BMI were mostly not reported. These and other limitations produced genetic activity measurements that were inconsistent, especially in cases of infertility and endometrial disorders. Addressing these limitations could improve how transcriptomic approaches are used to advance endometrial health.

Abstract Study question Do current methods of open vitrification (OV) and closed vitrification (CV) impact on oocyte mitochondria function and transcriptome? Summary answer Oocyte vitrification confers unique mitochondria distribution patterns and alters the expression of stress response genes and mitochondria function relative to control (fresh) oocytes. What is known already Mitochondria are essential for maintenance of metabolic function and developmental competence at physiological conditions. Conversely, open and closed methods of oocyte vitrification are associated with physical, chemical and non-physiological changes, and exposures to cryoprotecting agents which could be harmful at extremely high and low temperatures. Study design, size, duration Ninety-four unfertilised Metaphase II (MII) oocytes were donated to research between May 2018 and August 2021. Fifty-seven oocytes were randomly allocated to the different study groups. Similarly, thirty-seven oocytes were allocated to the different study control groups. Participants/materials, setting, methods Fourteen women underdoing IVF/ICSI treatment cycles were involved in the study. cDNA was obtained for qPCR and the Smart-Seq2 protocol and single-cell RNA-Seq was performed (illumina NextSeq2000). Mitochondrial staining on live-oocytes was performed using Mito-tracker Deep Red stain, and images evaluated using fluorescence microscopy. Differentially expressed genes were identified using the R package edgeR and functional gene ontology was performed using an over-representation analysis via WebGestalt. Correction for multiple testing was calculated using Benjamini-Hochberg method Main results and the role of chance Mean mitochondrial intensity/density showed similarities between open (OV) and closed vitrification (CV) and control (fresh oocytes FO): 25.8 (12.7-44.9, standard deviation [SD] 10.2) for FO oocytes (n = 15), 30.3 (16.2-44.8, SD 10.1) for OV oocytes (n = 11), 26.6 (13.3-64.6, SD 15.3) for CV oocytes (n = 12) (p = 0.617). Distinct mitochondria distribution patterns (cytoplasmic and peripheral) were associated with OV and CV oocytes. Twice as many peripheral to cytoplasmic patterns were observed in FO and OV oocytes in contrast to CV oocytes (p = 037). Mean expression of the gene SDHB, which is associated with the TCA cycle and the mitochondrial electron transport chain was reduced in vitrified oocytes (n = 13) (0.0879 ± 0.0216 vs 0.0593 ± 0.0479), as was the anti-apoptotic gene BCL2 (p = 0.0061), relative to control oocytes (n = 13), (p = 0.01). Conversely, genes associated with apoptosis and stress response (BAX (p = 0.002) and BCL2L1 (p = 0.016)) were significantly enriched relative to control oocytes. RNAseq transcriptome analysis revealed a total of 71 differentially expressed genes following vitrification relative to control oocytes. Genes associated with critical metabolic processes (death domain binding, BCL-2 homology 3 (BH3) domains, and exopolyphosphatatse activities) were enriched in vitrified oocytes relative to the control group (FDR>0.05). Limitations, reasons for caution The use of unfertilised metaphase II (MII) oocytes on day 1 as a preclinical application screening model for vitrification may not reflect the molecular status and integrity of clinical grade MII oocytes on day 0. Wider implications of the findings This study emphasizes the inherent impact of vitrification on oocyte mitochondria function and transcriptome relative to control oocytes. Importantly, unintended perturbations may be detrimental to oocyte health and developmental competence, suggesting the need for a continuous safety screening and monitoring of ART methods of cryopreservation and offspring long-term health. Trial registration number National Institute for Health Research (NIHR). Grant Reference: ICA-CDRF-2015-01-068

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.

Introduction The use of pesticides poses a potential serious health threat to occupationally exposed populations. However, there is limited information on acute health effects that may follow pesticide exposure among farmers in Malaysia. Therefore, this study was conducted to determine the prevalence of self- reported acute health symptoms occurring 48 hours after a spraying event and identify potential risk factors associated with these symptoms. Methods A study was conducted in farmers from the Bachok and Kota Bharu districts, Kelantan state, Malaysia between September 2018 and February 2019. Both demographic and use of pesticide information was collected. A symptom diary was used to collect data on self-reported ill-health symptoms for seven days after use of a pesticide. The symptoms were categorised into pyrexial and respiratory factors. Results 150 farmers growing either rice (n=83) or vegetables/fruits (n=67) participated. More than half of the farmers used two or more types of pesticide. This can be observed especially among rice farmers. 32.7% of farmers reported on at least one occasion a health symptom within first 48hours after spraying. Symptoms were more common among the rice farmers (p=0.04) and the most common symptoms were feeling unusually tired which was reported by 16% of farmers, followed by feeling sweaty (13.3%) and having a headache (13%). 18.7% of the farmers were identified as having a pyrexial factor. The farmers who wore face shields had an increased risk (OR 2.9; 95% CI: 1.01, 8.71) and the farmers who wore chemically resistant boots had a reduced risk of having the pyrexial factor (OR 0.23; 95%CI: 0.08, 0.70). Conclusion Acute health symptoms were observed among the farmers following pesticide exposure. However, the symptoms were not specific to pesticides and might be due to other causes. The results will provide baseline data on pesticide exposure and health effects in Malaysia.