Early symptoms of autism spectrum disorder (ASD) in 1-8 year old children with sex chromosome trisomies (XXX, XXY, XYY), and the predictive value of joint attention.

The objective of the present study is to investigate the impact of Sex Chromosome Trisomy (SCT; XXX, XXY, XYY) on the early appearance of Autism Spectrum Disorder (ASD) symptoms, and the predictive value of Joint Attention for symptoms of ASD. SCTs are specific genetic conditions that may serve as naturalistic 'at risk' models of neurodevelopment, as they are associated with increased risk for neurobehavioral vulnerabilities. A group of 82 children with SCT (aged 1-8 years) was included at baseline of this longitudinal study. Joint Attention was measured at baseline with structured behavior observations according to the Early Social Communication Scales. ASD symptoms were assessed with the Modified Checklist for Autism in Toddlers questionnaire and Autism Diagnostic Interview-Revised in a 1-year follow-up. Recruitment and assessment took place in the Netherlands and in the United States. The results demonstrate that ASD symptoms were substantially higher in children with SCT compared to the general population, with 22% of our cohort at clinical risk for ASD, especially in the domain of social interaction and communication. Second, a predictive value of Joint Attention was found for ASD symptoms at 1-year follow-up. In this cohort, no differences were found between karyotype-subtypes. In conclusion, from a very early age, SCT can be associated with an increased risk for vulnerabilities in adaptive social functioning. These findings show a neurodevelopmental impact of the extra X or Y chromosome on social adaptive development associated with risk for ASD already from early childhood onward. These findings advocate for close monitoring and early (preventive) support, aimed to optimize social development of young children with SCT.

Nanoparticle-mediated transgene expression of insulin-like growth factor 1 in the growth restricted guinea pig placenta increases placenta nutrient transporter expression and fetal glucose concentrations.

Fetal growth restriction (FGR) significantly contributes to neonatal and perinatal morbidity and mortality. Currently, there are no effective treatment options for FGR during pregnancy. We have developed a nanoparticle gene therapy targeting the placenta to increase expression of human insulin-like growth factor 1 (hIGF1) to correct fetal growth trajectories. Using the maternal nutrient restriction guinea pig model of FGR, an ultrasound-guided, intraplacental injection of nonviral, polymer-based hIGF1 nanoparticle containing plasmid with the hIGF1 gene and placenta-specific Cyp19a1 promotor was administered at mid-pregnancy. Sustained hIGF1 expression was confirmed in the placenta 5 days after treatment. Whilst increased hIGF1 did not change fetal weight, circulating fetal glucose concentration were 33%-67% higher. This was associated with increased expression of glucose and amino acid transporters in the placenta. Additionally, hIGF1 nanoparticle treatment increased the fetal capillary volume density in the placenta, and reduced interhaemal distance between maternal and fetal circulation. Overall, our findings, that trophoblast-specific increased expression of hIGF1 results in changes to glucose transporter expression and increases fetal glucose concentrations within a short time period, highlights the translational potential this treatment could have in correcting impaired placental nutrient transport in human pregnancies complicated by FGR.

Insulin-like growth factor 1 signaling in the placenta requires endothelial nitric oxide synthase to support trophoblast function and normal fetal growth.

Currently, there is no effective treatment for placental dysfunction in utero. In a ligated mouse model of fetal growth restriction (FGR), nanoparticle-mediated human insulin-like 1 growth factor (hIGF1) gene delivery (NP-Plac1-hIGF1) increased hIGF1 expression and maintained fetal growth. However, whether it can restore fetal growth remains to be determined. Using the endothelial nitric oxide synthase knockout (eNOS-/-) mouse model, a genetic model of FGR, we found that despite inducing expression of hIGF1 in the placentas treated with NP-Plac1-hIGF1 (P = 0.0425), FGR did not resolve. This was associated with no change to the number of fetal capillaries in the placental labyrinth; an outcome which was increased with NP-Plac1-hIGF1 treatment in the ligated mouse model, despite increased expression of angiopoietin 1 (P = 0.05), and suggested IGF1 signaling in the placenta requires eNOS to modulate placenta angiogenesis. To further assess this hypothesis, BeWo choriocarcinoma cell line and human placental explant cultures were treated with NP-Plac1-hIGF1, oxidative stress was induced with hydrogen peroxide (H2O2), and NOS activity was inhibited using the inhibitor NG-monomethyl-l-arginine (l-NMMA). In both BeWo cells and explants, the protective effect of NP-Plac1-hIGF1 treatment against H2O2-induced cell death/lactate dehydrogenase release was prevented by eNOS inhibition (P = 0.003 and P < 0.0001, respectively). This was associated with an increase in mRNA expression of oxidative stress markers hypoxia inducing factor 1α (HIF1α; P < 0.0001) and ADAM10 (P = 0.0002) in the NP-Plac1-hIGF1 + H2O2 + l-NMMA-treated BeWo cells. These findings show for the first time the requirement of eNOS/NOS in IGF1 signaling in placenta cells that may have implications for placental angiogenesis and fetal growth.

A sexually dimorphic murine model of IUGR induced by embryo transfer.

Animal models are needed to develop interventions to prevent or treat intrauterine growth restriction (IUGR). Foetal growth rates and effects of in utero exposures differ between sexes, but little is known about sex-specific effects of increasing litter size. We established a murine IUGR model using pregnancies generated by multiple embryo transfers, and evaluated sex-specific responses to increasing litter size. CBAF1 embryos were collected at gestation day 0.5 (GD0.5) and 6, 8, 10 or 12 embryos were transferred into each uterine horn of pseudopregnant female CD1 mice (n = 32). Foetal and placental outcomes were measured at GD18.5. In the main experiment, foetuses were genotyped (Sry) for analysis of sex-specific outcomes. The number of implantation sites (P = 0.033) and litter size (number of foetuses, P = 0.008) correlated positively with the number of embryos transferred, while placental weight correlated negatively with litter size (both P < 0.01). The relationship between viable litter size and foetal weight differed between sexes (interaction P = 0.002), such that foetal weights of males (P = 0.002), but not females (P = 0.233), correlated negatively with litter size. Placental weight decreased with increasing litter size (P < 0.001) and was lower in females than males (P = 0.020). Our results suggest that male foetuses grow as fast as permitted by nutrient supply, whereas the female maintains placental reserve capacity. This strategy reflecting sex-specific gene expression is likely to place the male foetus at greater risk of death in the event of a 'second hit'.

Sexual dimorphisms in brain gene expression in the growth-restricted guinea pig can be modulated with intra-placental therapy.

BACKGROUND: Fetal responses to adverse pregnancy environments are sex-specific. In fetal guinea pigs (GPs), we assessed morphology and messenger RNA (mRNA) expression in fetal growth-restricted (FGR) tissues at midpregnancy. METHODS: Female GPs were assigned either an ad libitum diet (C) or 30% restricted diet (R) prior to pregnancy to midpregnancy. At midpregnancy, a subset of R females underwent ultrasound-guided nanoparticle (NP) injection to enhance placental function. Five days later, fetuses were sampled. Fetal brain, heart, and liver were assessed for morphology (hematoxylin and eosin), proliferation (Ki67), and vascularization (CD31), as well as expression of inflammatory markers. RESULTS: R fetuses were 19% lighter with reduced organ weights and evidence of brain sparing compared to controls. No increased necrosis, proliferation, or vascularization was found between C and R nor male or female fetal organs. Sexual dimorphism in mRNA expression of Tgfß and Ctgf was observed in R but not C fetal brains: increased expression in females. NP treatment increased fetal brain mRNA expression of Tgfß and Ctgf in R males, abolishing the significant difference observed in untreated R fetuses. CONCLUSIONS: Sex-specific differences in mRNA expression in the fetal brain with FGR could impart a potential survival bias and may be useful for the development of treatments for obstetric diseases. IMPACT: Male and female fetuses respond differently to adverse pregnancy environments. Under fetal growth restriction conditions, inflammatory marker mRNA expression in the fetal brain was higher in females compared to males. Differences in gene expression between males and females may confer a selective advantage/disadvantage under adverse conditions. Better characterization of sexual dimorphism in fetal development will aid better development of treatments for obstetric diseases.

Conditional Mutation of Hand1 in the Mouse Placenta Disrupts Placental Vascular Development Resulting in Fetal Loss in Both Early and Late Pregnancy.

Congenital heart defects (CHD) affect approximately 1% of all live births, and often require complex surgeries at birth. We have previously demonstrated abnormal placental vascularization in human placentas from fetuses diagnosed with CHD. Hand1 has roles in both heart and placental development and is implicated in CHD development. We utilized two conditionally activated Hand1A126fs/+ murine mutant models to investigate the importance of cell-specific Hand1 on placental development in early (Nkx2-5Cre) and late (Cdh5Cre) pregnancy. Embryonic lethality occurred in Nkx2-5Cre/Hand1A126fs/+ embryos with marked fetal demise occurring after E10.5 due to a failure in placental labyrinth formation and therefore the inability to switch to hemotrophic nutrition or maintain sufficient oxygen transfer to the fetus. Labyrinthine vessels failed to develop appropriately and vessel density was significantly lower by day E12.5. In late pregnancy, the occurrence of Cdh5Cre+;Hand1A126fs/+ fetuses was reduced from 29% at E12.5 to 20% at E18.5 and remaining fetuses exhibited reduced fetal and placental weights, labyrinth vessel density and placenta angiogenic factor mRNA expression. Our results demonstrate for the first time the necessity of Hand1 in both establishment and remodeling of the exchange area beyond early pregnancy and in patterning vascularization of the placental labyrinth crucial for maintaining pregnancy and successful fetal growth.

Pre-pregnancy fast food and fruit intake is associated with time to pregnancy.

STUDY QUESTION: Is preconception dietary intake associated with reduced fecundity as measured by a longer time to pregnancy (TTP)? SUMMARY ANSWER: Lower intake of fruit and higher intake of fast food in the preconception period were both associated with a longer TTP. WHAT IS KNOWN ALREADY: Several lifestyle factors, such as smoking and obesity, have consistently been associated with a longer TTP or infertility, but the role of preconception diet in women remains poorly studied. Healthier foods or dietary patterns have been associated with improved fertility, however, these studies focused on women already diagnosed with or receiving treatments for infertility, rather than in the general population. STUDY DESIGN, SIZE, DURATION: This was a multi-center pregnancy-based cohort study of 5628 nulliparous women with low-risk singleton pregnancies who participated in the Screening for Pregnancy Endpoints (SCOPE) study. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 5598 women were included. Data on retrospectively reported TTP and preconception dietary intake were collected during the first antenatal study visit (14-16 weeks' gestation). Dietary information for the 1 month prior to conception was obtained from food frequency questions for fruit, green leafy vegetables, fish and fast foods, by a research midwife. Use of any fertility treatments associated with the current pregnancy was documented (yes, n = 340, no, n = 5258). Accelerated failure time models with log normal distribution were conducted to estimate time ratios (TR) and 95% CIs. The impact of differences in dietary intake on infertility (TTP >12 months) was compared using a generalized linear model (Poisson distribution) with robust variance estimates, with resulting relative risks (RR) and 95% CIs. All analyses were controlled for a range of maternal and paternal confounders. Sensitivity analyses were conducted to explore potential biases common to TTP studies. MAIN RESULTS AND THE ROLE OF CHANCE: Lower intakes of fruit and higher intakes of fast food were both associated with modest increases in TTP and infertility. Absolute differences between the lowest and highest categories of intake for fruit and fast food were in the order of 0.6-0.9 months for TTP and 4-8% for infertility. Compared with women who consumed fruit ≥3 times/day, the adjusted effects of consuming fruit ≥1-<3 times/day (TR = 1.06, 95% CI: 0.97-1.15), 1-6 times/week (TR = 1.11, 95% CI: 1.01-1.22) or <1-3 times/month (TR = 1.19, 95% CI: 1.03-1.36), corresponded to 6, 11 and 19% increases in the median TTP (Ptrend = 0.007). Similarly, compared with women who consumed fast food ≥4 times/week, the adjusted effects of consuming fast food ≥2-<4 times/week (TR = 0.89, 95% CI: 0.81-0.98), >0-<2 times/week (TR 0.79, 95% CI 0.69-0.89) or no fast food (TR = 0.76, 95% CI: 0.61-0.95), corresponded to an 11, 21 and 24% reduction in the median TTP (Ptrend <0.001). For infertility, compared with women who consumed fruit ≥3 times/day, the adjusted effects of consuming fruit ≥1-<3 times/day, 1-6 times/week or <1-3 times/month corresponded to a 7, 18 and 29% increase in risk of infertility (Ptrend = 0.043). Similarly, compared with women who consumed fast food ≥4 times/week, the adjusted effects of consuming fast food ≥2-<4 times/week, >0-<2 times/week, or no fast food, corresponded to an 18, 34 and 41% reduced risk of infertility (Ptrend <0.001). Pre-pregnancy intake of green leafy vegetables or fish were not associated with TTP or infertility. Estimates remained stable across a range of sensitivity analyses. LIMITATIONS, REASONS FOR CAUTION: Collection of dietary data relied on retrospective recall and evaluated a limited range of foods. Paternal dietary data was not collected and the potential for residual confounding cannot be eliminated. Compared to prospective TTP studies, retrospective TTP studies are prone to a number of potential sources of bias. WIDER IMPLICATIONS OF THE FINDINGS: These findings underscore the importance of considering preconception diet for fecundity outcomes and preconception guidance. Further research is needed assessing a broader range of foods and food groups in the preconception period. STUDY FUNDING/COMPETING INTEREST(S): The SCOPE database is provided and maintained by MedSciNet AB (http://medscinet.com). The Australian SCOPE study was funded by the Premier's Science and Research Fund, South Australian Government (http://www.dfeest.sa.gov.au/science-research/premiers-research-and-industry-fund). The New Zealand SCOPE study was funded by the New Enterprise Research Fund, Foundation for Research Science and Technology; Health Research Council (04/198); Evelyn Bond Fund, Auckland District Health Board Charitable Trust. The Irish SCOPE study was funded by the Health Research Board of Ireland (CSA/2007/2; http://www.hrb.ie). The UK SCOPE study was funded by National Health Service NEAT Grant (Neat Grant FSD025), Biotechnology and Biological Sciences Research council (www.bbsrc.ac.uk/funding; GT084) and University of Manchester Proof of Concept Funding (University of Manchester); Guy's and St. Thomas' Charity (King's College London) and Tommy's charity (http://www.tommys.org/; King's College London and University of Manchester); and Cerebra UK (www.cerebra.org.uk; University of Leeds). L.E.G. is supported by an Australian National Health and Medical Research Council (NHMRC) Early Career Fellowship (ID 1070421). L.J.M. is supported by a SACVRDP Fellowship; a program collaboratively funded by the National Heart Foundation, the South Australian Department of Health and the South Australian Health and Medical Research Institute. L.C.K. is supported by a Science Foundation Ireland Program Grant for INFANT (12/RC/2272). C.T.R. was supported by a National Health and Medical Research Council (NHMRC) Senior Research Fellowship (GNT1020749). There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Vitamin D levels in an Australian and New Zealand cohort and the association with pregnancy outcome.

BACKGROUND: Pregnant women are at increased susceptibility to vitamin D deficiency. Hence, there is continuing interest in determining how vitamin D influences pregnancy health. We aimed to compare vitamin D status in two distinct populations of pregnant women in Australia and New Zealand and to investigate the relationship between vitamin D status and pregnancy outcome. This included evaluating possible effect measure modifications according to fetal sex. METHODS: Serum 25-hydroxy vitamin D (25(OH)D) was measured at 15 ± 1 weeks' gestation in 2800 women from Adelaide and Auckland who participated in the multi-centre, prospective cohort SCreening fOr Pregnancy Endpoints (SCOPE) study. RESULTS: Mean serum 25(OH)D in all women was 68.1 ± 27.1 nmol/L and 28% (n = 772) were considered vitamin D deficient (< 50 nmol/L). Serum 25(OH)D was lower in the women recruited in Adelaide when compared to the women recruited in Auckland and remained lower after adjusting for covariates including maternal body mass index and socioeconomic index (Adelaide: 58.4 ± 50.3 vs. Auckland: 70.2 ± 54.5 nmol/L, P < 0.001). A 53% decreased risk for gestational diabetes mellitus (GDM) was observed with high (> 81 nmol/L) "standardised" vitamin D status when compared to moderate-high (63-81 nmol/L, aRR, 0.47; 95% CI: 0.23, 0.96). Marginal sex-specific differences occurred between vitamin D status and GDM: women carrying a female fetus had a 56% decreased risk for GDM in those with low-moderate levels of standardised vitamin D (44-63 nmol/L) compared to moderate-high levels (aRR: 0.44; 95% CI: 0.20, 0.97), whilst in women carrying a male fetus, a 55% decreased risk of GDM was found with high standardised vitamin D when compared to moderately-high vitamin D, but this was not statistically significant (aRR: 0.45; 95% CI: 0.15, 1.38). CONCLUSIONS: High serum 25(OH)D at 15 ± 1 weeks' gestation was shown to be protective against the development of GDM. A possible association between fetal sex, vitamin D status and GDM provides further questions and encourages continual research and discussion into the role of vitamin D in pregnancy, particularly in vitamin D replete populations.

Identification of Regions in the Receiver Domain of the ETHYLENE RESPONSE1 Ethylene Receptor of Arabidopsis Important for Functional Divergence.

Ethylene influences the growth and development of Arabidopsis (Arabidopsis thaliana) via five receptor isoforms. However, the ETHYLENE RESPONSE1 (ETR1) ethylene receptor has unique, and sometimes contrasting, roles from the other receptor isoforms. Prior research indicates that the receiver domain of ETR1 is important for some of these noncanonical roles. We determined that the ETR1 receiver domain is not needed for ETR1's predominant role in mediating responses to the ethylene antagonist, silver. To understand the structure-function relationship underlying the unique roles of the ETR1 receiver domain in the control of specific traits, we performed alanine-scanning mutagenesis. We chose amino acids that are poorly conserved and are in regions predicted to have altered tertiary structure compared with the receiver domains of the other two receptors that contain a receiver domain, ETR2 and ETHYLENE INSENSITIVE4. The effects of these mutants on various phenotypes were examined in transgenic, receptor-deficient Arabidopsis plants. Some traits, such as growth in air and growth recovery after the removal of ethylene, were unaffected by these mutations. By contrast, three mutations on one surface of the receiver domain rendered the transgene unable to rescue ethylene-stimulated nutations. Additionally, several mutations on another surface altered germination on salt. Some of these mutations conferred hyperfunctionality to ETR1 in the context of seed germination on salt, but not for other traits, that correlated with increased responsiveness to abscisic acid. Thus, the ETR1 receiver domain has multiple functions where different surfaces are involved in the control of different traits. Models are discussed for these observations.

The Ethylene Receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE2 Have Contrasting Roles in Seed Germination of Arabidopsis during Salt Stress.

In Arabidopsis (Arabidopsis thaliana), ethylene responses are mediated by a family of five receptors that have both overlapping and nonoverlapping roles. In this study, we used loss-of-function mutants for each receptor isoform to determine the role of individual isoforms in seed germination under salt stress. From this analysis, we found subfunctionalization of the receptors in the control of seed germination during salt stress. Specifically, loss of ETHYLENE RESPONSE1 (ETR1) or ETHYLENE INSENSITIVE4 (EIN4) leads to accelerated germination, loss of ETR2 delays germination, and loss of either ETHYLENE RESPONSE SENSOR1 (ERS1) or ERS2 has no measurable effect on germination. Epistasis analysis indicates that ETR1 and EIN4 function additively with ETR2 to control this trait. Interestingly, regulation of germination by ETR1 requires the full-length receptor. The differences in germination between etr1 and etr2 loss-of-function mutants under salt stress could not be explained by differences in the production of or sensitivity to ethylene, gibberellin, or cytokinin. Instead, etr1 loss-of-function mutants have reduced sensitivity to abscisic acid (ABA) and germinate earlier than the wild type, whereas etr2 loss-of-function mutants have increased sensitivity to ABA and germinate slower than the wild type. Additionally, the differences in seed germination on salt between the two mutants and the wild type are eliminated by the ABA biosynthetic inhibitor norflurazon. These data suggest that ETR1 and ETR2 have roles independent of ethylene signaling that affect ABA signaling and result in altered germination during salt stress.

ERECTA family genes regulate auxin transport in the shoot apical meristem and forming leaf primordia.

Leaves are produced postembryonically at the flanks of the shoot apical meristem. Their initiation is induced by a positive feedback loop between auxin and its transporter PIN-FORMED1 (PIN1). The expression and polarity of PIN1 in the shoot apical meristem is thought to be regulated primarily by auxin concentration and flow. The formation of an auxin maximum in the L1 layer of the meristem is the first sign of leaf initiation and is promptly followed by auxin flow into the inner tissues, formation of the midvein, and appearance of the primordium bulge. The ERECTA family genes (ERfs) encode leucine-rich repeat receptor-like kinases, and in Arabidopsis (Arabidopsis thaliana), this gene family consists of ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERL2. Here, we show that ERfs regulate auxin transport during leaf initiation. The shoot apical meristem of the er erl1 erl2 triple mutant produces leaf primordia at a significantly reduced rate and with altered phyllotaxy. This phenotype is likely due to deficiencies in auxin transport in the shoot apex, as judged by altered expression of PIN1, the auxin reporter DR5rev::GFP, and the auxin-inducible genes MONOPTEROS, INDOLE-3-ACETIC ACID INDUCIBLE1 (IAA1), and IAA19. In er erl1 erl2, auxin presumably accumulates in the L1 layer of the meristem, unable to flow into the vasculature of a hypocotyl. Our data demonstrate that ERfs are essential for PIN1 expression in the forming midvein of future leaf primordia and in the vasculature of emerging leaves.

Mid-pregnancy placental transcriptome in a model of placental insufficiency with and without novel intervention.

Fetal growth restriction (FGR) affects between 5-10% of all live births. Placental insufficiency is a leading cause of FGR, resulting in reduced nutrient and oxygen delivery to the fetus. Currently, there are no effective in utero treatment options for FGR, or placental insufficiency. We have developed a gene therapy to deliver, via a non-viral nanoparticle, human insulin-like 1 growth factor ( hIGF1 ) to the placenta as potential treatment of placenta insufficiency and FGR. Using a guinea pig maternal nutrient restriction (MNR) model of FGR, we aimed to understand the transcriptional changes within the placenta associated with placental insufficiency that occur prior to/at initiation of FGR, and the impact of short-term hIGF1 nanoparticle treatment. Using RNAsequencing, we analyzed protein coding genes of three experimental groups: Control and MNR dams receiving a sham treatment, and MNR dams receiving hIGF1 nanoparticle treatment. Pathway enrichment analysis comparing differentially expressed genelists in sham-treated MNR placentas to Control revealed upregulation of pathways associated with degradation and repair of genetic information and downregulation of pathways associated with transmembrane transport. When compared to sham-treated MNR placentas, MNR + hIGF1 placentas demonstrated changes to genelists associated with transmembrane transporter activity including ion, vitamin and solute carrier transport. Overall, this study identifies the key signaling and metabolic changes occurring in the placenta contributing to placental insufficiency prior to/at initiation of FGR, and increases our understanding of the pathways that our nanoparticle-mediated gene therapy intervention regulates. Statements and Declarations: Competing Interests: Authors declare no conflicts of interest.

Maternal-fetal interfaces transcriptome changes associated with placental insufficiency and a novel gene therapy intervention.

The etiology of fetal growth restriction (FGR) is multifactorial, although many cases often involve placental insufficiency. Placental insufficiency is associated with inadequate trophoblast invasion resulting in high resistance to blood flow, decreased availability of nutrients, and increased hypoxia. We have developed a non-viral, polymer-based nanoparticle that facilitates delivery and transient gene expression of human insulin-like 1 growth factor ( hIGF1 ) in placental trophoblast for the treatment of placenta insufficiency and FGR. Using the established guinea pig maternal nutrient restriction (MNR) model of placental insufficiency and FGR, the aim of the study was to identify novel pathways in the sub-placenta/decidua that provide insight into the underlying mechanism driving placental insufficiency, and may be corrected with hIGF1 nanoparticle treatment. Pregnant guinea pigs underwent ultrasound-guided sham or hIGF1 nanoparticle treatment at mid-pregnancy, and sub-placenta/decidua tissue was collected 5 days later. Transcriptome analysis was performed using RNA Sequencing on the Illumina platform. The MNR sub-placenta/decidua demonstrated fewer maternal spiral arteries lined by trophoblast, shallower trophoblast invasion and downregulation of genelists involved in the regulation of cell migration. hIGF1 nanoparticle treatment resulted in marked changes to transporter activity in the MNR + hIGF1 sub-placenta/decidua when compared to sham MNR. Under normal growth conditions however, hIGF1 nanoparticle treatment decreased genelists enriched for kinase signaling pathways and increased genelists enriched for proteolysis indicative of homeostasis. Overall, this study identified changes to the sub-placenta/decidua transcriptome that likely result in inadequate trophoblast invasion and increases our understanding of pathways that hIGF1 nanoparticle treatment acts on in order to restore or maintain appropriate placenta function.

Placental Nanoparticle-mediated IGF1 Gene Therapy Corrects Fetal Growth Restriction in a Guinea Pig Model.

Fetal growth restriction (FGR) caused by placental insufficiency is a major contributor to neonatal morbidity and mortality. There is currently no in utero treatment for placental insufficiency or FGR. The placenta serves as the vital communication, supply, exchange, and defense organ for the developing fetus and offers an excellent opportunity for therapeutic interventions. Here we show efficacy of repeated treatments of trophoblast-specific human insulin-like 1 growth factor ( IGF1 ) gene therapy delivered in a non-viral, polymer nanoparticle to the placenta for the treatment of FGR. Using the guinea pig maternal nutrient restriction model of FGR, nanoparticle-mediated IGF1 treatment was delivered to the placenta via ultrasound guidance across the second half of pregnancy, after establishment of FGR. This treatment resulted in correction of fetal weight in MNR animals compared to control, improved fetal physiology and no negative maternal side-effects. Overall, we show for the first time a therapy capable of improving the entire pregnancy environment: maternal, placental, and fetal. This combined with our previous studies using this therapy at both mid pregnancy and in numerous cell and animal models demonstrate the plausibility of this therapy for future human translation to improve health outcomes of neonates and decrease numerous morbidities associated with the developmental origins of disease.

Nanoparticle-mediated delivery of placental gene therapy via uterine artery catheterization in a pregnant rhesus macaque.

Nanoparticles offer promise as a mechanism to non-invasively deliver targeted placental therapeutics. Our previous studies utilizing intraplacental administration demonstrate efficient nanoparticle uptake into placental trophoblast cells and overexpression of human IGF1 ( hIGF1 ). Nanoparticle-mediated placental overexpression of hIGF1 in small animal models of placental insufficiency and fetal growth restriction improved nutrient transport and restored fetal growth. The objective of this pilot study was to extend these studies to the pregnant nonhuman primate and develop a method for local delivery of nanoparticles to the placenta via maternal blood flow from the uterine artery. Nanoparticles containing hIGF1 plasmid driven by the placenta-specific PLAC1 promoter were delivered to a mid-gestation pregnant rhesus macaque via a catheterization approach that is clinically used for uterine artery embolization. Maternal-fetal interface, fetal and maternal tissues were collected four days post-treatment to evaluate the efficacy of hIGF1 treatment in the placenta. The uterine artery catheterization procedure and nanoparticle treatment was well tolerated by the dam and fetus through the four-day study period following catheterization. Nanoparticles were taken up by the placenta from maternal blood as plasmid-specific hIGF1 expression was detected in multiple regions of the placenta via in situ hybridization and qPCR. The uterine artery catheterization approach enabled successful delivery of nanoparticles to maternal circulation in close proximity to the placenta with no concerns to maternal or fetal health in this short-term feasibility study. In the future, this delivery approach can be used for preclinical evaluation of the long-term safety and efficacy of nanoparticle-mediated placental therapies in a rhesus macaque model. Highlights: Novel method to deliver therapeutics to maternal-fetal interfaceDelivery of nanoparticles to the placenta via maternal catheterization.

A comparative study of ethylene growth response kinetics in eudicots and monocots reveals a role for gibberellin in growth inhibition and recovery.

Time-lapse imaging of dark-grown Arabidopsis (Arabidopsis thaliana) hypocotyls has revealed new aspects about ethylene signaling. This study expands upon these results by examining ethylene growth response kinetics of seedlings of several plant species. Although the response kinetics varied between the eudicots studied, all had prolonged growth inhibition for as long as ethylene was present. In contrast, with continued application of ethylene, white millet (Panicum miliaceum) seedlings had a rapid and transient growth inhibition response, rice (Oryza sativa 'Nipponbare') seedlings had a slow onset of growth stimulation, and barley (Hordeum vulgare) had a transient growth inhibition response followed, after a delay, by a prolonged inhibition response. Growth stimulation in rice correlated with a decrease in the levels of rice ETHYLENE INSENSTIVE3-LIKE2 (OsEIL2) and an increase in rice F-BOX DOMAIN AND LRR CONTAINING PROTEIN7 transcripts. The gibberellin (GA) biosynthesis inhibitor paclobutrazol caused millet seedlings to have a prolonged growth inhibition response when ethylene was applied. A transient ethylene growth inhibition response has previously been reported for Arabidopsis ethylene insensitive3-1 (ein3-1) eil1-1 double mutants. Paclobutrazol caused these mutants to have a prolonged response to ethylene, whereas constitutive GA signaling in this background eliminated ethylene responses. Sensitivity to paclobutrazol inversely correlated with the levels of EIN3 in Arabidopsis. Wild-type Arabidopsis seedlings treated with paclobutrazol and mutants deficient in GA levels or signaling had a delayed growth recovery after ethylene removal. It is interesting to note that ethylene caused alterations in gene expression that are predicted to increase GA levels in the ein3-1 eil1-1 seedlings. These results indicate that ethylene affects GA levels leading to modulation of ethylene growth inhibition kinetics.

Placental nanoparticle gene therapy normalizes gene expression changes in the fetal liver associated with fetal growth restriction in a fetal sex-specific manner.

Fetal growth restriction (FGR) is associated with increased risk of developing non-communicable diseases. We have a placenta-specific nanoparticle gene therapy protocol that increases placental expression of human insulin-like growth factor 1 (hIGF1), for the treatment of FGR in utero. We aimed to characterize the effects of FGR on hepatic gluconeogenesis pathways during early stages of FGR establishment, and determine whether placental nanoparticle-mediated hIGF1 therapy treatment could resolve differences in the FGR fetus. Female Hartley guinea pigs (dams) were fed either a Control or Maternal Nutrient Restriction (MNR) diet using established protocols. At GD30-33, dams underwent ultrasound guided, transcutaneous, intraplacental injection of hIGF1 nanoparticle or PBS (sham) and were sacrificed 5 days post-injection. Fetal liver tissue was fixed and snap frozen for morphology and gene expression analysis. In female and male fetuses, liver weight as a percentage of body weight was reduced by MNR, and not changed with hIGF1 nanoparticle treatment. In female fetal livers, expression of hypoxia inducible factor 1 (Hif1α) and tumor necrosis factor (Tnfα) were increased in MNR compared to Control, but reduced in MNR + hIGF1 compared to MNR. In male fetal liver, MNR increased expression of Igf1 and decreased expression of Igf2 compared to Control. Igf1 and Igf2 expression was restored to Control levels in the MNR + hIGF1 group. This data provides further insight into the sex-specific mechanistic adaptations seen in FGR fetuses and demonstrates that disruption to fetal developmental mechanisms may be returned to normal by treatment of the placenta.

HAND1 knockdown disrupts trophoblast global gene expression.

Congenital heart disease (CHD) affects nearly 1% of births annually, and CHD pregnancies carry increased risk of developing pathologies of abnormal placentation. We previously reported significant developmental impacts of disrupting Hand1, a gene associated with CHD, expression in placenta trophoblast and endothelial cells in multiple mouse models. In this study, we aimed to build upon this knowledge and characterize the mechanistic impacts of disrupting HAND1 on human placenta trophoblast and vascular endothelial cell gene expression. HAND1 gene expression was silenced in BeWo cells, a choriocarcinoma model of human cytotrophoblasts, (n = 3-9 passages) and isolated human placental microvascular endothelial cells (HPMVEC; n = 3 passages), with HAND1 siRNA for 96 h. Cells were harvested, mRNA isolated and RNA sequencing performed using the Illumina NextSeq 550 platform. Normalization and differential gene expression analyses were conducted using general linear modeling in edgeR packages. Statistical significance was determined using a log2 fold change of >1.0 or < -1.0 and unadjusted p-value ≤0.05. Panther DB was used for overrepresentation analysis, and String DB for protein association network analysis. There was downregulation of 664 genes, and upregulation of 59 genes in BeWo cells with direct HAND1 knockdown. Overrepresentation analysis identified disruption to pathways including cell differentiation, localization, and cell projection organization. In contrast, only seven genes were changed with direct HAND1 knockdown in HPMVECs. Disruption to HAND1 expression significantly alters gene expression profile in trophoblast but not endothelial cells. This data provides further evidence that future studies on genetic perturbations in CHDs should consider the extra-embryonic tissue in addition to the fetal heart.

Breast Reconstruction Outcomes with and without Strattice: Long-Term Outcomes of a Multicenter Study Comparing Strattice Immediate Implant Breast Reconstruction with Submuscular Implant Reconstruction.

BACKGROUND: Over half of immediate implant-based breast reconstructions (IBBR) are performed with an acellular dermal matrix, despite limited long-term outcome data. METHODS: The Breast Reconstruction Outcomes with and without Strattice, or BROWSE, study was a retrospective multicenter cohort study comparing consecutive patients who had undergone immediate Strattice IBBR with those who had undergone immediate IBBR with a submuscular technique between January of 2009 and December of 2015. RESULTS: This study compared 553 Strattice reconstructions with 242 submuscular reconstructions, with a median follow-up of 4.3 years (range, 2 to 9.3 years) and 5.7 years (range, 2 to 8.1 years), respectively, demonstrating an equivalent total complication rate [Strattice, n = 204 (36.9%); submuscular, n = 77 (31.8%); P = 0.17] and implant loss rate (8.5% versus 5.4%, respectively; P = 0.12). Infection rates and wound dehiscence rates were higher in the Strattice cohort [ n = 114 (20.6%) versus n = 31 (12.8%), P = 0.009; and n = 90 (16.3%) versus n = 25 (10.4%), P = 0.03, respectively]. Overall revision rates were comparable [ n = 226 (46.7%) versus n = 79 (41.1%); P = 0.2], but significantly fewer Strattice reconstructions required revision surgery for capsular contracture (5.3% versus 15.6%; P < 0.001). CONCLUSION: Although the risk of complications associated with Strattice reconstruction is numerically higher than that for submuscular coverage, the difference is small and not statistically significant, and likely outweighed by the clear reduced rate of revision surgery because of capsular contracture when Strattice is used. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.