Intrauterine hypoxic condition increases the generation of reactive oxygen species and fetal oxidative stress. Multiple pregnancy always bears an additional oxidative stress condition with severe complications, such as prematurity, structural abnormalities, delayed development and low birthweight. The umbilical cord (UC) vessels, along with circulating fetal red blood cells (RBCs), highly determine the oxygenation status of fetus and regulate the feto-placental circulation. As UC lacks innervation, the activation of the endothelial nitric oxide synthase (NOS3) is fundamental for proper NO production. Therefore, we aimed to study the NOS3 activation pathways along with damages to macromolecules in the endothelium of UC vessels and RBCs of mature non-discordant twins, in connection to major differences in their birth weight. We provide evidence that, under severe hypoxic conditions such as twin pregnancy, the NOS3-related NO production pathways are altered both in UC vessels and RBCs; moreover, the extent of changes is highly birthweight-specific. Furthermore, macromolecular damages are prominent in the RBCs and arteries compared to the vein, with a similar increase in the Arginase1 level, which is believed to play a role in NOS3 functionality, resulting in endothelial dysfunctionality, which might have relevance to the major etiologies of cardiovascular diseases in later life.

The relationship between smoking and human health has been investigated mostly in adults, despite the fact that the chemicals originating from sustained maternal smoking disrupt the carefully orchestrated regulatory cascades in the developing fetus. In this study, we followed molecular alterations in the umbilical cord (UC) vessels and fetal red blood cells (RBCs), which faithfully reflect the in vivo status of the fetus. We showed evidence for the decreased level of DNA-PKcs-positive nuclei in samples with smoking origin, which is associated with the impaired DNA repair system. Furthermore, we pointed out the altered ratio of MMP-9 metalloproteinase and its endogenous inhibitor TIMP-1, which might be a possible explanation for the morphological abnormalities in the UC vessels. The presented in vivo dataset emphasizes the higher vulnerability of the veins, as the primary target for the toxic materials unfiltered by the placenta. All these events become amplified by the functionally impaired fetal RBC population via a crosstalk mechanism between the vessel endothelium and the circulating RBCs. In our ex vivo approach, we looked for the molecular explanation of metal-exposure-induced alterations, where expressions of the selected genes were upregulated in the control group, while samples with smoking origin showed a lack of response, indicative of prior long-term in utero exposure.

The increased rate of twinning has pointed out newer challenges in clinical practices related to gestational complications, intrauterine growth restriction, perinatal mortality, and comorbidities. As a twin pregnancy progresses, the increased demand for oxygen supply can easily disrupt the redox homeostasis balance and further impose a greater challenge for the developing fetuses. A substantial birth-weight difference acts as an indicator of a deficit in oxygenation or blood flow to one of the fetuses, which might be related to a low bioavailable nitric oxide level. Therefore, in this study, we focused on networks involved in the adjustment of oxygen supply, like the activation of inducible and endothelial nitric oxide synthase (NOS3) along with free radical and lipid peroxide formation in mature twin pairs with high birth-weight differences. The selected parameters were followed by immunofluorescence staining, fluorescence-activated cell sorting analysis, and biochemical measurements in the umbilical cord vessels and fetal red blood cells. Based on our data set, it is clear that the lower-weight siblings are markedly exposed to persistent intrauterine hypoxic conditions, which are connected to a decreased level in NOS3 activation. Furthermore, the increased level of peroxynitrite aggravates lipid peroxidation and induces morphological and functional damage and loss in redox homeostasis.

The red blood cell (RBC) membrane is unique and crucial for maintaining structural–functional relationships. Maternal smoking induces significant changes in the morphological, rheological, and functional parameters of both maternal and foetal RBCs, mainly due to the continuous generation of the free radicals. The major aim of this study was to follow the consequences of a secondary stressor, like fungal infection, on the already compromised RBC populations. The impact of Candida infection, a growing health concern, was investigated on four blood sample groups: mothers and their neonates originating from non-smoking versus smoking populations. Here, we searched for phenotypical and molecular markers that precisely reflected the effect of Candida infection on the RBC membrane; this included the level of hemolysis, appearance of morphological variants, formation of the lipid peroxidation marker 4-hydroxyl-nonenal, arrangement of the Band 3 molecules and activation of the Caspase 3. In most of the examined cases, the fungal infection increased the adverse symptoms induced by smoking, indicating a general stress response, likely due to an altered redox state of the cells. However, we were able to identify an atypical phenotype (clustered populations with shrinkage and membrane blebbing) in both the non-smoking and smoking populations, which might be a unique marker for Candida spp. infection.

The maternal physiological state during pregnancy have an excess demand for oxygen supply which can easily disrupt the redox homeostasis balance. There is a change in the maternal haemodynamic profile with an increased cardiac output, reduced systemic vascular resistance and blood pressure (de Haas et al., 2017). In response to this high oxygen demand red blood cell (RBC) mass level steadily increases by 20-30% from 8 to 10 weeks of the gestational period till the end of pregnancy (Foley, 2018). In connection to that, there are increasing evidences of enhanced oxidative insults during intrauterine development leading to severe abnormalities or pathological states like spontaneous miscarriage, preterm delivery, preeclampsia, ectopic pregnancy, intrauterine growth restriction (IUGR), placental abruption, perinatal death etc. Moreover, in cases of multiple/twin pregnancy there is an additional stress with the greater risks of miscarriage, anaemia, preterm delivery, gestational hypertensive disorders, IUGR, diabetes operative delivery and related postnatal and neonatal illnesses. Modern usage of in-vitro fertilization (IVF) therapies led to higher incidence of multiple pregnancies. There have been relevant data on the increasing foetal, neonatal and perinatal mortality rate of 3-6 times in twin and 5-15 times in other multiple pregnancies with comparison to the singleton pregnancies. During pregnancy, the connection between the mother and the foetus is provided by the placenta and the umbilical cord. The umbilical cord vascular system is the sole pathway of oxygen and nutrient transport to the foetus from the placenta. Therefore, the major obstetric complications are directly or indirectly connected to the placental or umbilical cord disorders causing intrauterine hypoxia and/or impaired blood flow to the developing foetus. The major part of umbilical cord lacks innervations and hence the vascular tone is mainly regulated by nitric oxide (NO), i.e. derived from the endothelial nitric oxide synthase (NOS3). The NO being a potent vasoactive agent causes vasodilation and increases the rate of perfusion, nutrient and oxygen supply. Usually intensive prenatal care and surveillances are done in high risk cases like multiple/twin pregnancy by the advanced non-invasive biomedical devices. The usage of high performance equipment like Blood oxygen level-dependent–magnetic resonance imaging was utilized to study changes in the placental oxygenation status in human pregnancies. It can also assess the placental perfusion and oxygen transport. Similarly, ultrasound and Doppler flow measuring techniques not only can visualize the umbilical cord but simultaneously can assess the foetal blood flow parameters through the umbilical cord vessels. These approaches can easily detect any abnormalities in the umbilical cord circulation, but lacks to highlight the underlying molecular mechanisms behind any kind of impaired functionality in the foetal vascular system that can highly influence the in-utero development. Further on this topic we elaborated in details.

This study investigates the toxic effect of harmful materials, unfiltered by the placenta, on neonatal umbilical cord (UC) vessels, focusing on stress-induced adaptations in transcriptional and translational processes. It aims to analyze changes in pathways related to mRNA condensate formation, transcriptional regulation, and DNA damage response under maternal smoking-induced stress. UC vessels from neonates born to smoking (Sm) and nonsmoking mothers (Ctr) were examined. Immunofluorescence staining and confocal microscopy assessed the localization of key markers, including Transcription Complex Subunit 1 (CNOT1) and the largest subunit of RNA polymerase II enzyme (RPB1). Additionally, markers of DNA damage response, such as Poly(ADP-ribose) polymerase-1, were evaluated. In Sm samples, dissolution of CNOT1 granules in UC vessels was observed, potentially aiding stalled translation and enhancing transcription via RPB1 assembly and translocation. Control vessels showed predominant cytoplasmic RPB1 localization. Despite adaptive responses, Sm endothelial cells exhibited significant damage, indicated by markers like Poly(ADP-ribose) polymerase-1. Ex vivo metal treatment on control vessels mirrored Sm sample alterations, emphasizing marker roles in cell survival under toxic exposure. Maternal smoking induces specific molecular adaptations in UC vessels, affecting mRNA condensate formation, transcriptional regulation, and DNA damage response pathways. Understanding these intricate molecular mechanisms could inform interventions to improve neonatal health outcomes and mitigate adverse effects of toxic exposure during pregnancy.