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Background: Cord-clamping strategies may modify blood pressure (BP) and cerebral tissue oxygen saturation (rStO 2 ) immediately after birth., Methods: We conducted a sub-study nested within the Baby-Directed Umbilical Cord-Clamping trial. Infants ≥32 +0 weeks' gestation assessed as requiring resuscitation were randomly allocated to either physiologically-based cord clamping (PBCC), where resuscitation commenced prior to umbilical cord clamping, or standard care where cord clamping occurred early (ECC). In this single-site sub-study, we obtained additional measurements of pre-ductal BP and rStO 2 . In a separate observational arm, non-randomised vigorous infants received 2 min of deferred cord clamping (DCC) and contributed data for reference percentiles., Results: Among 161 included infants, n = 55 were randomly allocated to PBCC (n = 30) or ECC (n = 25). The mean (SD) BP at 3-4 min after birth (primary outcome) in the PBCC group was 64 (10) mmHg compared to 62 (10) mmHg in the ECC group, mean difference 2 mmHg (95% confidence interval -3-8 mmHg, p = 0.42). BP and rStO 2 were similar across both randomised arms and the observational arm (n = 106)., Conclusion: We found no difference in BP or rStO 2 with the different cord clamping strategies. We report reference ranges for BP and rStO 2 for late-preterm and full-term infants receiving DCC., Impact: Among late-preterm and full-term infants receiving varying levels of resuscitation, blood pressure (BP, at 3-4 minutes and 6 min) and cerebral tissue oxygen saturation (rStO 2 ) are not influenced by timing of cord clamping in relation to establishment of ventilation. Infants in this study did not require advanced resuscitation, where cord clamping strategies may yet influence BP and rStO 2 . The reference ranges for BP and rStO 2 represent the first, to our knowledge, for vigorous late-preterm and full-term infants receiving deferred cord clamping. rStO 2  > 90% (~90 th percentile) may be used to define cerebral hyperoxia, for instance when studying oxygen supplementation after birth., (© 2024. The Author(s).)

Maternal malnutrition increases fetal and neonatal morbidity, partly by affecting placental function and morphology, but its impact on placental hemodynamics are unknown. Our objective was to define the impact of maternal malnutrition on placental oxygen reserve and perfusion in vivo in a rhesus macaque model of protein restriction (PR) using advanced imaging. Animals were fed control (CON, 26% protein), 33% PR diet (17% protein), or a 50% PR diet (13% protein, n = 8/group) preconception and throughout pregnancy. Animals underwent Doppler ultrasound and fetal biometry followed by MRI at gestational days 85 (G85) and 135 (G135; term is G168). Pregnancy loss rates were 0/8 in CON, 1/8 in 33% PR, and 3/8 in 50% PR animals. Fetuses of animals fed a 50% PR diet had a smaller abdominal circumference (G135, p < 0.01). On MRI, placental blood flow was decreased at G135 (p < 0.05) and placental oxygen reserve was reduced (G85, p = 0.05; G135, p = 0.01) in animals fed a 50% PR diet vs. CON. These data demonstrate that a 50% PR diet reduces maternal placental perfusion, decreases fetal oxygen availability, and increases fetal mortality. These alterations in placental hemodynamics may partly explain human growth restriction and stillbirth seen with severe PR diets in the developing world., (© 2023. The Author(s).)

Hypoxic-ischaemia renders the neonatal brain susceptible to early secondary injury from oxidative stress and impaired autoregulation. We aimed to describe cerebral oxygen kinetics and haemodynamics immediately following return of spontaneous circulation (ROSC) and evaluate non-invasive parameters to facilitate bedside monitoring. Near-term sheep fetuses [139 ± 2 (SD) days gestation, n = 16] were instrumented to measure carotid artery (CA) flow, pressure, right brachial arterial and jugular venous saturation (SaO 2 and SvO 2 , respectively). Cerebral oxygenation (crSO 2 ) was measured using near-infrared spectroscopy (NIRS). Following induction of severe asphyxia, lambs received cardiopulmonary resuscitation using 100% oxygen until ROSC, with oxygen subsequently weaned according to saturation nomograms as per current guidelines. We found that oxygen consumption did not rise following ROSC, but oxygen delivery was markedly elevated until 15 min after ROSC. CrSO 2 and heart rate each correlated with oxygen delivery. SaO 2 remained > 90% and was less useful for identifying trends in oxygen delivery. CrSO 2 correlated inversely with cerebral fractional oxygen extraction. In conclusion, ROSC from perinatal asphyxia is characterised by excess oxygen delivery that is driven by rapid increases in cerebrovascular pressure, flow, and oxygen saturation, and may be monitored non-invasively. Further work to describe and limit injury mediated by oxygen toxicity following ROSC is warranted.

Objective: To study cardiovascular response to minimally invasive surfactant therapy in preterm infants with and without foetal growth restriction (FGR)., Design: Poractant alfa was administered and echocardiograms were performed before and 30 min after. FGR infants were compared with those appropriate for gestational age (AGA)., Results: Ten FGR infants were compared with 20 AGA infants (gestation [weeks], 28.9 ± 2 vs. 28.6 ± 1, p = 0.55 and birthweight [g], 813 ± 157 vs. 1141 ± 257, p = 0.01, respectively). The change in echocardiographic parameters was more prominent in AGA infants ([global contractility] fractional area change [FAC, %], FGR, 24.7 ± 2.2 to 27.9 ± 0.4, p = 0.08 vs. AGA, 26.6 ± 3 to 30.5 ± 1, p < 0.01, and [longitudinal contractility] tricuspid annular plane systolic excursion [mm], FGR, 3.9 ± 0.3 to 4.6 ± 0.5, p = 0.003 vs. AGA, 4.6 ± 0.3 to 5.5 ± 0.4, p = 0.0001). Significant difference was noted for change in FAC (%), FGR 2.1 ± 1.7 vs. AGA 4.1 ± 1.2, p = 0.02., Conclusions: Differential cardiovascular response to minimally invasive surfactant therapy amongst FGR infants may reflect an in-utero maladaptive state.

Polycystic ovary syndrome (PCOS) is a major reproductive disorder that is responsible for 80% of anovulatory infertility and that is associated with hyperandrogenemia, increased risk of obesity, and white adipose tissue (WAT) dysfunction. We have previously demonstrated that the combination of chronic testosterone (T) treatment and an obesogenic Western-style diet (WSD) exerts synergistic functional effects on WAT, leading to increased lipid accumulation in visceral adipocytes by an unknown mechanism. In this study, we examined the whole-genome transcriptional response in visceral WAT to T and WSD, alone and in combination. We observed a synergistic effect of T and WSD on gene expression, resulting in upregulation of lipid storage genes concomitant with adipocyte hypertrophy. Because DNA methylation is known to be associated with body fat distribution and the etiology of PCOS, we conducted whole-genome DNA methylation analysis of visceral WAT. While only a fraction of differentially expressed genes also exhibited differential DNA methylation, in silico analysis showed that differentially methylated regions were enriched in transcription factor binding motifs, suggesting a potential gene regulatory role for these regions. In summary, this study demonstrates that hyperandrogenemia alone does not induce global transcriptional and epigenetic response in young female macaques unless combined with an obesogenic diet.

Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package ( https://bioconductor.org/packages/release/bioc/html/msgbsR.html ).

Zinc is an essential micronutrient in pregnancy and zinc deficiency impairs fetal growth. We used a mouse model of moderate zinc deficiency to investigate the physiological mechanisms by which zinc is important to placental morphogenesis and the maternal blood pressure changes during pregnancy. A 26% reduction in circulating zinc (P = 0.005) was exhibited in mice fed a moderately zinc-deficient diet. Zinc deficiency in pregnancy resulted in an 8% reduction in both near term fetal and placental weights (both P < 0.0001) indicative of disrupted placental development and function. Detailed morphological analysis confirmed changes to the placental labyrinth microstructure. Continuous monitoring of maternal mean arterial pressure (MAP) revealed a late gestation decrease in the zinc-deficient dams. Differential expression of a number of regulatory genes within maternal kidneys supported observations on MAP changes in gestation. Increased MAP late in gestation is required to maintain perfusion of multiple placentas within rodent pregnancies. Decreased MAP within the zinc-deficient dams implies reduced blood flow and nutrient delivery to the placenta. These findings show that adequate zinc status is required for correct placental morphogenesis and appropriate maternal blood pressure adaptations to pregnancy. We conclude that insufficient maternal zinc intake from before and during pregnancy is likely to impact in utero programming of offspring growth and development largely through effects to the placenta and maternal cardiovascular system.

Homeobox genes regulate embryonic and placental development, and are widely expressed in the human placenta, but their regulatory control by DNA methylation is unclear. DNA methylation analysis was performed on human placentae from first, second and third trimesters to determine methylation patterns of homeobox gene promoters across gestation. Most homeobox genes were hypo-methylated throughout gestation, suggesting that DNA methylation is not the primary mechanism involved in regulating HOX genes expression in the placenta. Nevertheless, several genes showed variable methylation patterns across gestation, with a general trend towards an increase in methylation over gestation. Three genes (TLX1, HOXA10 and DLX5) showed inverse gains of methylation with decreasing mRNA expression throughout pregnancy, supporting a role for DNA methylation in their regulation. Proteins encoded by these genes were primarily localised to the syncytiotrophoblast layer, and showed decreased expression later in gestation. siRNA mediated downregulation of DLX5, TLX1 and HOXA10 in primary term villous cytotrophoblast resulted in decreased proliferation and increased expression of differentiation markers, including ERVW-1. Our data suggest that loss of DLX5, TLX1 and HOXA10 expression in late gestation is required for proper placental differentiation and function.

Zika virus (ZIKV) infection has emerged as a global health threat and infection of pregnant women causes intrauterine growth restriction, spontaneous abortion and microcephaly in newborns. Here we show using biologically relevant cells of neural and placental origin that following ZIKV infection, there is attenuation of the cellular innate response characterised by reduced expression of IFN-β and associated interferon stimulated genes (ISGs). One such ISG is viperin that has well documented antiviral activity against a wide range of viruses. Expression of viperin in cultured cells resulted in significant impairment of ZIKV replication, while MEFs derived from CRISPR/Cas9 derived viperin -/- mice replicated ZIKV to higher titers compared to their WT counterparts. These results suggest that ZIKV can attenuate ISG expression to avoid the cellular antiviral innate response, thus allowing the virus to replicate unchecked. Moreover, we have identified that the ISG viperin has significant anti-ZIKV activity. Further understanding of how ZIKV perturbs the ISG response and the molecular mechanisms utilised by viperin to suppress ZIKV replication will aid in our understanding of ZIKV biology, pathogenesis and possible design of novel antiviral strategies.

There are fetal sex-specific differences in the balance between angiotensin (Ang) II and Ang-(1-7) in the maternal circulation during pregnancy. To determine whether at 15 weeks' gestation plasma levels of Ang II and Ang-(1-7), as well as levels of prorenin and Ang-converting enzyme (ACE), predicted the development of gestational hypertension (GH) or preeclampsia (PreE) and were associated with estimates of fetal and maternal health, women who later developed GH (n=50) or PreE (n=50) were compared with body mass index-matched controls (n=100). Women who subsequently developed PreE or GH had increased Ang-(1-7) levels at 15 weeks' gestation compared with women with normal pregnancies. When separated by fetal sex, this difference was seen only in women carrying a female fetus. Prorenin and ACE concentrations were not useful biomarkers for the prediction of either PreE or GH at 15 weeks' gestation. Women with a male fetus who developed PreE and women who subsequently developed GH had increased blood pressures at 15 weeks' gestation compared with women with normal pregnancies, suggesting that these women were on an early trajectory for the development of hypertension. We propose that measurement of Ang-(1-7) during early gestation could be useful in predicting, those women who will go on to develop new-onset hypertension in pregnancy.

Kaposi sarcoma (KS), a multifocal neoplasm of the skin that can spread to visceral organs, is the most prevalent malignant tumor in acquired immuno deficiency syndrome (AIDS) patients. KS-associated herpesvirus (KSHV or HHV8) is considered the primary etiological factor of this malignancy, as well as of primary effusion lymphoma and multicentric Castleman's disease. KS lesions are characterized by proliferating spindle cells of endothelial cell (EC) origin. The action of the insulin-like growth factor (IGF) system has been implicated in many malignancies, and recent data have demonstrated that the IGF-I receptor (IGF-IR) is required for in vitro growth of the KS-derived KSIMM cell line. To examine whether the IGF pathway is also involved in KSHV-mediated transformation of ECs, we examined the expression and function of the IGF system in KSHV-infected, immortalized dermal microvascular EC (E-DMVEC). The expression of the insulin receptor (IR) was strongly induced in latently infected E-DMVEC, whereas the expression levels of the IGF-IR remained unchanged. Gene knockdown of IR, but not IGF-IR, prevented the characteristic focus formation seen in KSHV-infected E-DMVEC. Similarly, treatment with the IR-specific small-molecule inhibitor HNMPA-(AM(3)) inhibited postconfluent growth. These data suggest a role for the IR, but not the IGF-IR, in KSHV-induced transformation of vascular ECs.

The EWS family of genes is involved in numerous chromosomal translocations that are characteristic of a variety of sarcomas. A recently described member of this group is desmoplastic small round cell tumor (DSRCT), which is characterized by a recurrent t(11;22)(p13;q12) translocation that fuses the 5' exons of the EWS gene to the 3' exons of the WT1 gene. The originally described chimera comprises exons 1-7 of EWS and exons 8-10 of WT1. We have previously reported that the WT1 protein represses the expression of the IGF-I receptor gene, whereas the EWS(1-7)-WT1(8-10) fusion protein activates IGF-I receptor gene expression. It has recently become apparent that EWS-WT1 chimeras produced in DSCRT are heterogeneous as a result of fusions of different regions of the EWS gene to the WT1 gene. We have recently characterized additional EWS-WT1 translocations that involve the juxtaposition of EWS exons 7 or 8 to WT1 exon 8, and an EWS-WT1 chimera that lacks EWS exon 6. The chimeric transcription factors encoded by these various translocations differ in their DNA-binding characteristics and their ability to transactivate the IGF-I receptor promoter. These data suggest that the molecular pathology of DSRCT is more complex than previously appreciated, and that this diversity may provide the foundation for predictive genotype-phenotype correlations in the future.