Birth weight, growth indices, and seminal parameters in male offspring are resilient features to maternal pre-conceptional dietary manipulation in sheep.

Gestational diet manipulation can lead to inadequate fetal nutrient supply resulting in low birth weight, limited postnatal growth, and consequently, reduced reproductive performance in the progeny. However, effects of short-term maternal pre-conceptional dietary manipulation on postnatal growth and reproductive parameters of male offspring in large animals remains unexplored. To determine these consequences, female crossbred (Polypay x Dorset) sheep were allocated to three groups (n = 33/group) of dietary manipulation for 21 days prior to mating under the following conditions: (1) control at 100 % of maintenance energy requirements (40 Kcal of metabolizable energy/kg body weight [BW]), (2) undernutrition (UN) at 50 % of Control intake, and (3) overnutrition (ON) at 200 % of maintenance energy. Singleton ram lambs (UN:9; C:12; ON:6) were monitored from birth until 8 months of age, including birth weight, weekly weights, weight gain, body mass index (BMI), and circulating testosterone. After weaning, monthly scrotal circumference and subcutaneous fat depth were measured. Semen morphology and motility were evaluated at 7 and 8 months of age. Birth weight, weight gain, and BMI at birth and weaning were not significantly different among nutritional treatments. None of the pre-conceptional diets affected body weight change from weaning until 36 weeks of age, BMI, fat depth, or scrotal circumference across the experiment. A sustained rise in plasma testosterone concentrations was detected when ram lambs were, on average, 82 days old and 37 kg. Both testosterone concentrations and scrotal circumference were positively correlated to body weight regardless of treatment group. In addition, seminal parameters did not differ among treatments, but a transient increase in plasma testosterone at 18 weeks of age was observed in ON ram lambs compared to control rams. In conclusion, birth weight, growth indices, and seminal parameters in singleton rams are resilient features in the progeny upon maternal pre-conceptional dietary manipulation in sheep.

Preimplantation or gestation/lactation high-fat diet alters adult offspring metabolism and neurogenesis.

Poor maternal nutrition during pregnancy is known to impair fetal development. Moreover, the preimplantation period is vulnerable to adverse programming of disease. Here, we investigated the effect of a mouse maternal high-fat diet in healthy non-obese dams during preimplantation or throughout pregnancy and lactation on metabolism-related parameters and hippocampal neurogenesis in adult offspring. Female mice were fed from conception either a normal fat diet (normal fat diet group) or high-fat diet throughout gestation and lactation (high-fat diet group), or high-fat diet only during preimplantation (embryonic high-fat diet group, high-fat diet up to E3.5, normal fat diet thereafter). Maternal high-fat diet caused changes in the offspring, including increased systolic blood pressure, diurnal activity, respiratory quotient, and energy expenditure in high-fat diet females, and increased systolic blood pressure and respiratory quotient but decreased energy expenditure in high-fat diet males. High-fat diet males had a higher density of newborn neurons and a lower density of mature neurons in the dentate gyrus, indicating that exposure to a maternal high-fat diet may regulate adult neurogenesis. A maternal high-fat diet also increased the density of astrocytes and microglia in the hippocampus of high-fat diet males and females. Generally, a graded response (normal fat diet < embryonic high-fat < high-fat diet) was observed, with only 3 days of high-fat diet exposure altering offspring energy metabolism and hippocampal cell density. Thus, early maternal exposure to a fatty diet, well before neural differentiation begins and independently of maternal obesity, is sufficient to perturb offspring energy metabolism and brain physiology with lifetime consequences.

Environmental Exposures around Conception: Developmental Pathways Leading to Lifetime Disease Risk.

Environment around conception can influence the developmental programme with lasting effects on gestational and postnatal phenotype and with consequences for adult health and disease risk. Peri-conception exposure comprises a crucial part of the 'Developmental Origins of Health and Disease' (DOHaD) concept. In this review, we consider the effects of maternal undernutrition experienced during the peri-conception period in select human models and in a mouse experimental model of protein restriction. Human datasets indicate that macronutrient deprivation around conception affect the epigenome, with enduring effects on cardiometabolic and neurological health. The mouse model, comprising maternal low protein diet exclusively during the peri-conception period, has revealed a stepwise progression in altered developmental programming following induction through maternal metabolite deficiency. This progression includes differential effects in extra-embryonic and embryonic cell lineages and tissues, leading to maladaptation in the growth trajectory and increased chronic disease comorbidities. The timeline embraces an array of mechanisms across nutrient sensing and signalling, cellular, metabolic, epigenetic and physiological processes with a coordinating role for mTORC1 signalling proposed. Early embryos appear active participants in environmental sensing to optimise the developmental programme for survival but with the trade-off of later disease. Similar adverse health outcomes may derive from other peri-conception environmental experiences, including maternal overnutrition, micronutrient availability, pollutant exposure and assisted reproductive treatments (ART) and support the need for preconception health before pregnancy.

In Utero Programming of Testicular Cancer.

It is well established that the intrauterine biological environment plays important roles in fetal development. In this review, we re-visit the hypothesis that testicular germ cell cancer (TGCC), especially in adolescents and young adults, has been programmed in utero. The origin for extreme in utero environments is mostly maternal driven and may be due to nutritional, physical and psychological stressful conditions that alter the optimal molecular and biophysical in utero environments. Moreover, precursors for TGCC may originate as early as during fertilization or implantation of the blastocyst. Further investigations of human developmental biology, both in vivo and in vitro, are needed in order to establish better understanding of in utero programming of future wellbeing or diseases.

Effect of a dietary and exercise intervention in women with overweight and obesity undergoing fertility treatments: protocol for a randomized controlled trial.

BACKGROUND: Distinct molecular, inflammatory, and metabolic signatures are present in oocytes and follicular fluid derived from women with obesity when compared to those derived from normal weight women, which suggest existing signals that may program future offspring for metabolic diseases. This study aims to assess the feasibility and efficacy of a peri-conception nutrition and exercise intervention on mitigating obesity-associated changes in oocyte gene expression profiles and follicular fluid metabolites. METHODS: This single blinded randomized control trial will include 120 women with a BMI of 25-45 kg/m2, ≥21 years of age, and undergoing in vitro fertilization (IVF) treatments. Participants will be randomized to standard of care (N = 60) or an intervention group (N = 60) in a block design by polycystic ovary syndrome status. The intervention will combine a dietary component (Mediterranean meal plan) with exercise prescription following the Physical Activity Guidelines for Americans. Participants will be assessed pre- and post-intervention. The standard of care group will be offered to join the intervention group if the IVF treatments are unsuccessful as a cross over design. Recruitment is anticipated to start in July of 2021. Primary outcomes will include single oocyte gene expression profiles and follicular fluid metabolites. Mann-Whitney U nonparametric tests will be used to assess potential differences for each stratum. Follicular fluid and serum metabolites will be analyzed using a one-factor Analysis of Covariance (ANCOVA) at four levels, pair-wise comparisons using Tukey-Kramer post-hoc tests will be used to identify groups whose means differ significantly while retaining the family-wise error rate at 5%. When the design is balanced, two-way Analysis of Variance (ANOVA), or non-parametric Friedman test will be used in data analysis. Additionally, general linear models and ANCOVA may be used to control for covariates. Significance will be set at p < 0.05. Findings will be disseminated via peer-reviewed manuscripts and presentations at scientific conferences. DISCUSSION: This study will provide novel data and key information on the impact of a dietary and exercise intervention on oocyte gene expression and follicular fluid content. Results will demonstrate the potential of such intervention in mitigating obesity-induced changes in oocyte gene expression and follicular fluid metabolites. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT04273048 ): submitted November 13, 2019; posted February 17, 2020.

Folic Acid Supplementation in Chinese Peri-conceptional Population: Results from the SPCC Study.

OBJECTIVE: To determine the prevalence and determinants of folic acid (FA) supplementation in Chinese couples planning for pregnancy and in women during early pregnancy. METHODS: This was a cross-sectional study based on the Shanghai PreConception Cohort (SPCC) study. Data on FA supplementation and socio-demographic features were collected using questionnaires. Couples visiting clinics for pre-pregnancy examination and pregnant women at < 14 gestational weeks were recruited in Shanghai, China, between March 2016 and September 2018. RESULTS: Among the pregnancy planners, 42.4% (4,710/11,099) women and 17.1% (1,377/8,045) men used FA supplements, while 93.4% (14,585/15,615) of the pregnant women used FA supplements. FA supplement use was higher in female pregnancy planners who were older ( RR: 1.13, 95% CI: 1.08-1.18), had higher education ( RR: 1.71, 95% CI: 1.53-1.92), and were residing in urban districts ( RR: 1.06, 95% CI: 1.01-1.11) of FA supplementation; female pregnancy planners with alcohol consumption ( RR: 0.95, 95% CI: 0.90-0.99) had lower odds of FA supplementation. In early pregnancy, women with higher educational level ( RR: 1.04, 95% CI: 1.03-1.06), who underwent pre-pregnancy examination ( RR: 1.02, 95% CI: 1.01-1.03) had higher odds of using an FA supplement; older aged ( RR: 0.99, 95% CI: 0.98-0.99), and multigravida ( RR: 0.97, 95% CI: 0.96-0.98) had lower odds of FA supplementation. CONCLUSION: Although the majority of pregnant women took FA supplements, more than half of the women planning for pregnancy did not. Urgent strategies are needed to improve pre-conception FA supplementation.

Impact of an integrated nutrition, health, water sanitation and hygiene, psychosocial care and support intervention package delivered during the pre- and peri-conception period and/or during pregnancy and early childhood on linear growth of infants in the first two years of life, birth outcomes and nutritional status of mothers: study protocol of a factorial, individually randomized controlled trial in India.

BACKGROUND: The period from conception to two years of life denotes a critical window of opportunity for promoting optimal growth and development of children. Poor nutrition and health in women of reproductive age and during pregnancy can negatively impact birth outcomes and subsequent infant survival, health and growth. Studies to improve birth outcomes and to achieve optimal growth and development in young children have usually tested the effect of standalone interventions in pregnancy and/or the postnatal period. It is not clearly known whether evidence-based interventions in the different domains such as health, nutrition, water sanitation and hygiene (WASH) and psychosocial care, when delivered together have a synergistic effect. Further, the effect of delivery of an intervention package in the pre and peri-conception period is not fully understood. This study was conceived with an aim to understand the impact of an integrated intervention package, delivered across the pre and peri-conception period, through pregnancy and till 24 months of child age on birth outcomes, growth and development in children. METHODS: An individually randomized controlled trial with factorial design is being conducted in urban and peri-urban low- to mid-socioeconomic neighbourhoods in South Delhi, India. 13,500 married women aged 18 to 30 years will be enrolled and randomized to receive either the pre and peri-conception intervention package or routine care (first randomization). Interventions will be delivered until women are confirmed to be pregnant or complete 18 months of follow up. Once pregnancy is confirmed, women are randomized again (second randomization) to receive either the intervention package for pregnancy and postnatal period or to routine care. Newborns will be followed up till 24 months of age. The interventions are delivered through different study teams. Outcome data are collected by an independent outcome ascertainment team. DISCUSSION: This study will demonstrate the improvement that can be achieved when key factors known to limit child growth and development are addressed together, throughout the continuum from pre and peri-conception until early childhood. The findings will increase our scientific understanding and provide guidance to nutrition programs in low- and middle-income settings. TRIAL REGISTRATION: Clinical Trial Registry - India #CTRI/2017/06/008908; Registered 23 June 2017, http://ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=19339&EncHid=&userName=society%20for%20applied%20studies.

Folic Acid Supplementation in Chinese Peri-conceptional Population: Results from the SPCC Study / 生物医学与环境科学（英文）

Objective@#To determine the prevalence and determinants of folic acid (FA) supplementation in Chinese couples planning for pregnancy and in women during early pregnancy.@*Methods@#This was a cross-sectional study based on the Shanghai PreConception Cohort (SPCC) study. Data on FA supplementation and socio-demographic features were collected using questionnaires. Couples visiting clinics for pre-pregnancy examination and pregnant women at < 14 gestational weeks were recruited in Shanghai, China, between March 2016 and September 2018.@*Results@#Among the pregnancy planners, 42.4% (4,710/11,099) women and 17.1% (1,377/8,045) men used FA supplements, while 93.4% (14,585/15,615) of the pregnant women used FA supplements. FA supplement use was higher in female pregnancy planners who were older ( : 1.13, 95% : 1.08-1.18), had higher education ( : 1.71, 95% : 1.53-1.92), and were residing in urban districts ( : 1.06, 95% : 1.01-1.11) of FA supplementation; female pregnancy planners with alcohol consumption ( : 0.95, 95% : 0.90-0.99) had lower odds of FA supplementation. In early pregnancy, women with higher educational level ( : 1.04, 95% : 1.03-1.06), who underwent pre-pregnancy examination ( : 1.02, 95% : 1.01-1.03) had higher odds of using an FA supplement; older aged ( : 0.99, 95% : 0.98-0.99), and multigravida ( : 0.97, 95% : 0.96-0.98) had lower odds of FA supplementation.@*Conclusion@#Although the majority of pregnant women took FA supplements, more than half of the women planning for pregnancy did not. Urgent strategies are needed to improve pre-conception FA supplementation.

Association between peri-conceptional bisphenol A exposure in women and men and time to pregnancy-The HOPE study.

BACKGROUND: Bisphenol A (BPA) is a non-persistent endocrine-disrupting chemical with nearly ubiquitous, involuntary exposure. Previous studies have shown that BPA causes reproductive dysfunction in animal models, but there are limited data regarding the effects of BPA exposure on time to pregnancy (TTP) in humans. OBJECTIVE: To evaluate whether peri-conceptional BPA exposure of women and men is associated with couples' TTP. METHODS: A total of 164 heterosexual couples (164 women; 163 men) who have available BPA information as well as time to pregnancy from the Home Observation of Peri-conceptional Exposures (HOPE) Study were included and were followed up to 12 months. Women collected first-morning urine samples starting at the beginning of the fertile window and continued until the onset of menses or 18 days after the estimated day of ovulation (EDO+18 days). The time to pregnancy (TTP) after the enrolment was self-reported and used for the analysis. Discrete-time Cox proportional hazards models were performed to generate fecundability odds ratio (FOR) between BPA and TTP after adjusting for education and age, accounting for right censoring and prior number of cycles trying to conceive. RESULTS: Among 164 couples, 125 couples became pregnant during the study. There was no association between TTP and peri-conceptional BPA exposure for both men (FOR 1.02, 95% CI 0.72, 1.47) and women (FOR 1.07, 95% CI 0.75, 1.53) after adjusting for education and age. CONCLUSIONS: No association was found between peri-conceptional BPA exposure and fecundability in this preconception cohort of relatively young, healthy pregnancy planners.

Placental 11 ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) expression very early during human pregnancy.

Maternal physiologic stress during gestation has been reported to be associated with negative developmental outcomes, including intra-uterine growth restriction and reduced birth weight, which can impact postnatal development, behavior and health. The human fetus is partially protected from elevated cortisol exposure by placental 11 ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), which oxidizes bioactive cortisol into bio-inactive cortisone. Importantly, despite the critical protective role hypothesized for 11ß-HSD2, the onset of its placental expression has yet to be clearly established. To this aim, we present immunocytochemical analysis of placentas collected 3-6 weeks post-conception. 11ß-HSD2 was present as early as 3 weeks post-conception in syncytiotrophoblasts, where most maternal-fetal exchange occurs, and in columnar epithelial cells encircling uterine endometrial glands, which provide early histiopathic nutrition to the embryo. 11ß-HSD2 expression in these critical maternal-fetal exchange areas is consistent with its hypothesized protective role. Future studies should investigate the mechanisms that may modulate embryonic glucocorticoid exposure earlier, immediately post-conception.

Association between birth defects and special risk exposures during peri-conception period / 西安交通大学学报(医学版)

Objective To analyze the effect of special risk exposures during periconception period on birth defects of newborns.Methods From Jul.to Dec.2013,the multi-stage stratified random sampling method was adopted.Women of childbearing age between 15 and 49 who were pregnant during 2010 to 2013 in Shaanxi Province were selected as study subjects for investigation on special risk factors exposed during periconception period.The Logistic regression model was adopted to analyze the association between newborns' birth defects and special risk exposures.Results The study included 30 010 women of childbearing age and 29 550 newborns with 572 (193.57/ 10 000) cases of birth defects.After adjusting for demographic factors,the risk factors for birth defects were drinking [OR=2.29,95％ CI (1.22,4.29)] and passive smoking [OR=1.25,95％ CI (1.02,1.53)] during periconception.There was a higher risk of birth defects when exposure to medicine [OR =1.64,95％ CI (1.04,2.61)],pesticides [OR =2.41,95％ CI (1.09,5.35)],biological risk factors [OR-1.64,95％ CI (1.05,2.56)],physical risk factors [OR=1.15,95％ CI (1.13,2.34)] and chemical risk factors [OR =2.36,95％ CI (1.36,4.11)] 3 months both before and after pregnancy.Similarly,after adjusting for demographic factors and behaviors,we found that birth defects were related to antibiotics,salicylates,and antitussive,which could increase the risk of birth defects (P＜0.05).Conclusion Exposure to passive smoking and drinking during periconception and exposure to medicines and pesticides,as well as biological,physical and chemical risk factors 3 months before and after pregnancy could increase the risk of birth defects in newborns.

PrEP as Peri-conception HIV Prevention for Women and Men.

Daily oral tenofovir (TDF)-based pre-exposure prophylaxis (PrEP) is an effective HIV prevention strategy and recommended for men and women with substantial risk of HIV acquisition. The peri-conception period, the stage prior to pregnancy when condom use is necessarily reduced, has elevated HIV risk that can be mitigated by PrEP use. Data from a randomized trial suggest that peri-conception PrEP use by HIV-seronegative women does not increase the risk of pregnancy loss, birth defects or congenital anomalies, preterm birth, or infant growth faltering. Women considering PrEP use throughout pregnancy must weigh the known increased risk of HIV acquisition with unknown risks of drug effects on infant growth. PrEP has been used safely by HIV-seronegative men with HIV-seropositive female partners who have become pregnant. As an effective user-controlled HIV prevention strategy, PrEP offers autonomy and empowerment for HIV prevention and can be recommended alongside antiretroviral therapy, fertility screening, vaginal self-insemination, intercourse timed to peak fertility, medically assisted reproduction, and other safer conception strategies to provide multiple options. The integration of PrEP into safer conception programs is warranted and will safely reduce HIV transmission to women, men, and children during the peri-conception period.

Does epigenetic dysregulation of pancreatic islets contribute to impaired insulin secretion and type 2 diabetes?

ß cell dysfunction is central to the development and progression of type 2 diabetes (T2D). T2D develops when ß cells are not able to compensate for the increasing demand for insulin caused by insulin resistance. Epigenetic modifications play an important role in establishing and maintaining ß cell identity and function in physiological conditions. On the other hand, epigenetic dysregulation can cause a loss of ß cell identity, which is characterized by reduced expression of genes that are important for ß cell function, ectopic expression of genes that are not supposed to be expressed in ß cells, and loss of genetic imprinting. Consequently, this may lead to ß cell dysfunction and impaired insulin secretion. Risk factors that can cause epigenetic dysregulation include parental obesity, an adverse intrauterine environment, hyperglycemia, lipotoxicity, aging, physical inactivity, and mitochondrial dysfunction. These risk factors can affect the epigenome at different time points throughout the lifetime of an individual and even before an individual is conceived. The plasticity of the epigenome enables it to change in response to environmental factors such as diet and exercise, and also makes the epigenome a good target for epigenetic drugs that may be used to enhance insulin secretion and potentially treat diabetes.

The phenotype of an IVF child is associated with peri-conception measures of follicular characteristics and embryo quality.

STUDY QUESTION: Are childhood measures of phenotype associated with peri-conception parental, IVF treatment and/or embryonic characteristics of IVF children? SUMMARY ANSWER: Birthweight, childhood body mass index (BMI) and height of pre-pubertal IVF children were strongly associated with peri-conception factors, including follicular and embryonic characteristics. WHAT IS KNOWN ALREADY: A growing number of studies have identified a range of phenotypic differences between IVF and naturally conceived pre-pubertal children; for example, birthweights are lower following a fresh compared with a thawed embryo transfer. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included IVF children (n = 96) born at term (>37 weeks) after a singleton pregnancy from the transfer of either fresh or thawed embryos in New Zealand. Between March 2004 and November 2008, these children were subjected to clinical assessment before puberty. PARTICIPANTS/MATERIALS, SETTING, METHODS: Clinical assessment provided anthropometric measures of children aged 3.5-11 years old. Peri-conception factors (n = 36) derived retrospectively from parental, treatment, laboratory and embryonic variables (n = 69) were analysed using multiple stepwise regression with respect to standard deviation scores (SDSs) of the birthweight, mid-parental corrected BMI and height of the IVF children. Data from children conceived from fresh (n = 60) or thawed (n = 36) embryos, that met inclusion criteria and had high-quality data with >90% completeness, were analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Embryo treatment at transfer was identified as a predictor of birthweight with thawed embryos resulting in heavier birthweights than fresh embryos [P = 0.02, 95% confidence interval (CI) fresh minus thawed: -1.047 to -0.006]. Birthweight SDS was positively associated with mid-parental corrected BMI SDS (P = 0.003, slope 0.339 ± 0.100). Four factors were related (P < 0.05) to mid-parental corrected height SDS. In particular, child height was inversely associated with the diameter of lead follicles at oocyte retrieval (P < 0.0001, slope -0.144 ± 0.040) and with the quality score of embryos at transfer (P = 0.0008, slope -0.425 ± 0.157), and directly associated with the number of follicles retrieved (P = 0.05, slope 1.011 ± 0.497). Child height was also positively associated with the transfer of a fresh as opposed to thawed embryo (P < 0.001, 95% CI 0.275-0.750). LIMITATIONS, REASONS FOR CAUTION: More than one embryo was transferred in most cycles so mean development and quality data were used. The large number of variables measured was on a relatively small sample size. Large cohorts from multiple clinics using a variety of treatment protocols and embryology methods are needed to confirm the associations identified and ultimately to test these factors as possible predictors of phenotype. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to directly associate peri-conception measures of IVF treatment with a pre-pubertal child's phenotype. Demonstration that peri-conception measures relate to a pre-pubertal child's phenotype extends the range of factors that may influence growth and development. These findings, if corroborated by larger studies, would provide invaluable information for practitioners, who may want to consider the impact of ovarian stimulation protocols as well as the quality of the embryo transferred on a child's growth and development, in addition to their impact on pregnancy rate. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from the National Research Centre of Growth and Development New Zealand (grant 3682065) and the Australasian Paediatric Endocrine Group (APEG; grant 3621994), as well as a fellowship from Fertility Associates New Zealand awarded to M.P.G. In terms of competing interest, J.C.P is a shareholder of Fertility Associates. M.P.G. currently holds the position of Merck Serono Lecturer in Reproductive Biology. W.S.C. and P.L.H. have also received grants and non-financial support from Novo Nordisk, as well as personal fees from Pfizer that are unrelated to the current study. The other authors have no conflict of interest to declare.

Reduced oocyte and embryo quality in response to elevated non-esterified fatty acid concentrations: a possible pathway to subfertility?

Reproductive performance is increasingly considered as a 'barometer' of general well-being of the mother. A normal maternal 'metabolic health' status is essential to safeguard successful ovulation, conception and further embryo development. When alterations in serum metabolites are reflected in the oocyte and embryonic micro-environment, these metabolic changes can affect follicle health, oocyte development and even subsequent embryo physiology. The search continues for signals that may be critically affecting the early developmental stages in life. Years of expertise in animal in vitro embryo culture models contribute to the awareness on the influence of elevated non-esterified fatty acid (NEFA) concentrations on follicle cells, oocyte and embryo quality. High NEFA concentrations in the blood are known to alter the follicular micro-environment. The latter alterations in NEFA concentrations have been associated with a disappointing fertility outcome through disabled ovarian cell function and reduced oocyte's developmental competence. Even more, elevated NEFA concentrations during bovine oocyte maturation influence the subsequent embryo characteristics. This review provides a cross-species overview on the consequences of elevated NEFA concentrations, originating from maternal lipolytic conditions, on female fertility, with particular focus on the early stages in life. Thereby, we will describe to what extent elevated serum NEFA concentrations are a potential threat around the period of conception.