1. Introduction
The placenta is a temporary fetal organ that attaches to the uterine lining during pregnancy. It delivers oxygen and nutrients to the developing fetus. Disruptions in placental homeostasis can have significant implications for maternal and fetal health, highlighting the vital role of the placenta in a healthy pregnancy. Oxytocin (OXT) is a neuropeptide produced by the hypothalamus that is subsequently released into the bloodstream by the anterior pituitary gland. OXT is well known for its role in uterine contractions during labor in addition to milk letdown during lactation. Importantly, however, OXT is a multifaceted regulator that maintains the placental microenvironment and optimizes placental function throughout pregnancy and promotes fetal development. Given the key role of oxytocin in several placental functions, it is necessary to determine whether OXT crosses the placenta during gestation [
1]. Malek et al. first noted the ability of OXT to cross the human placenta nearly 30 years ago. They determined that OXT crosses the placenta in both directions by simple diffusion, but the maternal-to-fetal direction was favored in their study [
2]. However, it remains unclear whether the fetus solely produces endogenous OXT [
3]. Understanding the sources of OXT during pregnancy is important because it is not only a signal for parturition but also impacts overall fetal growth and development, [
4]. vascular proliferation, and organogenesis [
5]. Notably, there is controversy surrounding the ability of OXT to cross the placenta and transfer to the fetus. Our study investigated the ability of OXT to cross the placenta and transfer to the fetus throughout gestation. Hence, we believe that our study may also reconcile the available literature regarding OXT’s ability to cross the placenta.
There is a reasonable correlation between human eye development and the morphological stages of fetal development throughout gestation [
6].
. OXT has been detected in both human fetuses and animal fetuses as early as 16 weeks of gestation and coincides with the retinal and retinal vessel formation that occurs during eye development. [
6]. The sharp increase in OXT levels, which occurs in the last trimester of pregnancy, is synonymous with rapid retinal development and vascular growth. This relationship suggests that OXT may play a role in regulating retinal and retinal angiogenesis [
1]. Development of the retina begins at 28 days of gestation, and the retina is completely vascularized by 40 weeks of gestation [
2]. In preterm infants, abnormal development and vascularization of the retina can occur, leading to retinopathy of prematurity (ROP), a significant cause of blindness in this population. ROP is a disease caused by a combination of factors, including the cessation of normal retinal vascular growth and the suppression of growth-promoting factors following premature birth. Smith et al. proposed that deficiencies in developmental hormones and vascular growth factors may contribute to retinopathy pathogenesis [
7]. Therefore, understanding the molecular action of OXT may provide a biomarker for retinal disorders or provide treatment options to prevent ROP.
In 1983, Gauquelin et al. first identified OXT in the human retina [
7]. Despite these landmark findings, the literature rarely describes the role of OXT in healthy neural retinal development [
5]. The retinal pigmented epithelium (RPE) is a vital component of the neural retina and is affected primarily by ROP. The RPE is a monolayer of polarized pigmented epithelial cells interposed between the neural retina and chorio-capillaries. It performs many vital functions to support the neural retina in maintaining normal and healthy vision, [
8,
9]. such as forming a blood‒retinal barrier, absorbing stray light, transporting nutrients and metabolites, performing retinoid recycling, and phagocytosing shed outer segments of photoreceptors (PRs). Although the RPE performs essential functions in healthy vision and OXT also regulates many other critical physiological processes, including neurophysiology (and much like the maternal–fetal barrier), it is unknown whether OXT crosses the blood‒retina barrier, altering physiology during health and disease. Another key yet unresolved aspect of the complex role of OXT in both fetuses and the potential role of OXT in retinal development has yet to be identified.
We previously identified oxytocin receptor (OXTR) mRNA and protein in the RPE of human retinas and rhesus monkey retinas and cultured human fetal RPE (HfRPE) cells [
10]. Additionally, we detected OXT in the rhesus monkey retina. These findings suggest that OXT plays a crucial role in the signaling mechanisms responsible for regulating vascular growth in the retina. We believe that it may also have the potential to control the abnormal retinal vascular growth often observed in ROP. Thus, our current study has two main hypotheses. The first is that maternal OXT crosses the placenta and affects OXT concentrations within the fetal circulation. Second, systemic OXT within fetal circulation affects the RPE transcriptome in cultured HfRPE cells, indicating its potential role in retinal development and visual function regulation.
4. Discussion
This translational study revealed that OXT likely crosses the placenta and affects fetal OXT concentrations. Specifically, maternal exposure to OXT resulted in infant cord blood OXT levels 2–4 times higher than those in infants whose mothers were not exposed to synthetic OXT. These findings were statistically significant and strongly associated with the presence or absence of labor. Importantly, RNA sequencing revealed that 46 genes were differentially expressed in OXT-treated RPE cells. These up- and downregulated genes were related to vital metabolic and signaling pathways and critical cellular components.
Additionally, a complex cluster of co-expressed and predicted interaction networks was identified based on our query list of DEGs and the list of genes predicted by GeneMANIA, indicating that OXT also influences the RPE transcriptome. Many colocalizations occurred among the proteins of these genes as well. Our clinical and scientific findings suggest that OXT crosses the placenta and may have the ability to increase fetal OXT concentrations in addition to regulating retinal and retinal vascular growth. These findings suggest that OXT may be a potential early therapeutic intervention for ROP.
Notably, RPE cells undergo terminal differentiation early in development. However, they remain dormant and have little or no cell turnover throughout normal life [
24,
25]. Thus, maintaining structural, metabolic, and functional homeostasis among RPE cells is vital for decoding visual signals. In the present study, we revealed that OXT-regulated, transcribed genes function in metabolic pathways, cholesterol biosynthetic processes, phagosomes, focal adhesions, actin cytoskeleton regulation, PPAR signaling pathways, PI3K‒Akt signaling pathways, extracellular matrices, and ECM‒receptor interactions. These regulated pathways involve multiple cellular functions, including cell-membrane phagocytosis, cell survival and protection, and structural integrity of the ECM in RPE cells.
In this study, multiple GO and KEGG terms related to cholesterol biosynthesis were used to analyze the DEGs upregulated by OXT. Cholesterol is an essential constituent of the cell membrane. It regulates various cellular processes, including membrane trafficking, ligand binding, receptor recycling, and signal transduction [
26,
27]. The maintenance of cholesterol homeostasis is necessary for normal cell function and viability. These GO and KEGG terms included metabolic pathways, steroid biosynthesis, terpenoid backbone biosynthesis, metabolic processes, and cholesterol biosynthetic processes. Further analysis of those genes via the GeneMANIA network revealed a close cluster of genes involved in cholesterol biosynthetic pathways and regulation. Among this cluster of genes, eight are cholesterol biosynthetic enzymes: one is a receptor for the endocytosis of cholesterol, and the other is a transcription factor that suppresses cholesterol biosynthetic enzymes [
26,
27,
28,
29,
30,
31]. Our quantitative validation of the genes implicated in the synthesis, uptake, elimination, and regulation of cholesterol levels revealed significant downregulation. Most importantly,
HMGCR, a rate-limiting enzyme of cholesterol biosynthesis, and CYP27A1, which helps eliminate cholesterol from the retina, were downregulated at the mRNA level. Thus, our data suggest that OXT affects cholesterol biosynthesis and homeostasis in RPE cells by modulating the transcript expression of various genes. When cholesterol homeostasis in the RPE is impaired, vision is affected by macular degeneration [
32]. Zheng et al. demonstrated the presence of HMGCR in human and murine RPE cells via immunohistochemical analyses [
33,
34]. Ramachandra et al. studied the human induced pluripotent stem cell (iPSC)-derived RPE and reported active cholesterol biosynthesis [
35]. Biswas et al. found cholesterol in an immortalized human RPE-derived cell line (ARPE-19) via the use of radiolabeled acetate [
36].
We found that genes whose expression was upregulated by OXT treatment were involved in phagocytosis. These genes included those involved in focal adhesion, regulation of the actin cytoskeleton, phagosome formation, and calcium signaling. To understand the relevance of these findings, we reviewed the essential and functional components of the human retina. When photoreceptors are exposed to light, damaged proteins and lipids generate photooxidative radicals [
37,
38]. These toxic substances accumulate in the photoreceptor each day [
37,
38]. To maintain normal function, photoreceptors constantly remove these substances when their outer segments are renewed [
37,
38]. In this renewal process, a new portion of the photoreceptor outer segment (POS) is built, and the POS tip containing a high concentration of toxic substances is shed from every photoreceptor [
37,
38]. A critical role of the RPE is phagocytosis of the shed POS to maintain the structural and functional homeostasis of the photoreceptor [
8,
37]. Phagocytosis of the POS by the RPE is a sequence of events that includes binding of the POS to the RPE, ingestion of bound POS, maturation of the phagosome, enzymatic breakdown of macromolecules, and resolution of the RPE phagolysosome [
8,
37]. The α
vβ
5 integrin receptors are involved in the binding of POSs to the RPE [
8,
37,
39,
40] After binding, focal adhesion kinase (FAK) is activated, phosphorylating receptor tyrosine kinase c-mer (MerTK) and initiating subsequent events [
8,
37,
39,
40]. These events include the activation of intracellular inositol 1,4,5-trisphosphate (InsP3)/Ca
2+ and the rearrangement of F-actin, which leads to the ingestion of bound POS [
37,
41,
42,
43].
Calcium signaling mediates phagosome maturation, which requires the fusion of phagosomes with endosomes and lysosomes [
41]. FAK is the key kinase in focal adhesion. It mediates the POS ingestion process via the regulation and rearrangement of actin [
37,
39,
44]. Phagosomes (or cellular compartments) are needed to remove toxic radicals and macromolecules from POSs [
37,
45]. Furthermore, RPE phagocytosis has circadian rhythms [
46]. Calcium signaling mediates phagosome maturation and may be the driving force for POS-shedding rhythms [
46]. Interestingly, OXT was found to regulate Tetrahymena phagocytosis [
47].
This study revealed multiple enriched pathway terms with a protective function in the RPE from OXT-upregulated DEGs. These terms included the PI3K-Akt signaling pathway, the PPAR signaling pathway, and the cAMP signaling pathway. Previous studies have demonstrated that the PI3K-Akt and PPAR signaling pathways each mediate the protective actions of various factors and pharmacological inhibitors of oxidative stress in RPE cells, ultimately promoting cell survival [
48,
49,
50,
51,
52]. Notably, RPE cells undergo constant oxidative stress from multiple sources.
53-55 The RPE is located close to the choriocapillaris and receives high rates of blood flow that are well saturated with oxygen [
53,
54]. Long-term light exposure increases the interaction of light with endogenous chromophores in the RPE, causing excitation that results in a highly reactive state.53,54 Highly reactive chromophores rapidly interact with other molecules, including oxygen, generating reactive oxygen species (ROS) [
53,
54] Furthermore, a robust amount of metabolic activity occurs in the mitochondria of the RPE to meet its high-energy needs, which also generates a high concentration of ROS.53,54 These various sources of oxidative stress can elicit harmful effects on biomolecules and the RPE53-55 mitochondrial network. Thus, it is essential to protect the RPE from oxidative damage to preserve its integrity and function. In addition, mature human RPE cells undergo minimal proliferation and have a limited proliferative response to injury. Pathologic processes generally accompany marked proliferation in the human RPE [
24,
25] For this reason, controlling the proliferation of RPE cells is essential. Hecquet et al. reported that the cAMP signaling pathway inhibits RPE proliferation via the mitogen-activated protein kinase pathway [
56]. Furthermore, the cAMP signaling pathway also plays a role in transducing the protective function of klotho, an aging-suppressor gene, in RPE cells [
57].
Our study enriched GO and KEGG terms related to the extracellular matrix (ECM) from the upregulated genes. These terms included ECM-receptor interaction, extracellular matrix, and extracellular matrix binding. One of the critical functions of the RPE is transporting nutrients and metabolic waste products from photoreceptors across Bruch’s membrane (BrM). BrM houses RPE cells and is located between the RPE and choriocapillaris [
8,
9] Thus, maintaining both a stable interaction between the RPE and BrM and a stable or appropriate composition within BrM is essential [
8,
9,
58]. Otherwise, alterations in these properties can lead to ocular pathogenesis [
9,
58] RPE cells produce ECM components within BrM and express the receptors that bind to the ECM [
59]. Sugino et al. demonstrated that cell-deposited matrices promote the survival of the RPE on specimens of aged human BrM [
60]. Several aspects of this fundamental study demonstrate that OXT may modulate BrM and the RPE and, in turn, the RPE-regulated interaction between BrM and the RPE.
Among those downregulated genes, the enriched terms included prostate cancer, EGFR tyrosine kinase inhibitor resistance, and the MAPK signaling pathway. Activating pathways included in these terms can lead to cellular proliferation, migration, and invasion. The MAPK signaling pathway is an essential pathway that mediates the proliferation of the RPE [
56]. Downregulation of this pathway may have a protective effect on RPE cells [
24,
25]. OXT treatment activates, suppresses, or modulates various pathways that perform diverse functions. The findings from this study, similar to those of previous studies, indicate that OXT-regulated pathways mediate essential RPE functions, including cholesterol biosynthesis and homeostasis, phagocytosis, RPE protection, and Bruch’s membrane integrity (
Figure 8).
For the clinical purpose of our study, we investigated whether OXT is transferred across the placenta. Our study included 74 near-term infants. Compared with term deliveries, preterm births were associated with more frequent episodes of spontaneous labor, high blood pressure (HBP)/preeclampsia, diabetes, and antenatal-steroid use. No significant associations were found between cord-blood OXT concentrations and gestational age or birth weight. However, the administration of OXT during labor resulted in significantly higher median cord blood OXT concentrations than did the absence of exogenous OXT. This finding suggests that OXT crosses the placenta and equilibrates between the mother and fetus. When both labor and OXT were present, the median cord blood OXT concentration was 2.53–3.50 times higher than when only one or none were present. Hence, it is likely that the administration of synthetic OXT during labor increases fetal OXT levels, regardless of gestational age. The presence of labor also showed a positive association with increased fetal OXT concentrations. This translational study represents a critical step in understanding the role of OXT in human gestation.
This aspect of our study is unprecedented: monitoring the transfer of OXT across the maternal–fetal barrier
in vivo. Previous studies have shown that OXT crosses the placenta in in vitro models. Malek et al. investigated the transport of OXT and inulin in both directions across human placental tissues
in vitro. He reported similar transfer rates across each placental circuit and concluded that OXT likely crosses the placenta via simple diffusion, with the permeability of both compounds being greater in the maternal-to-fetal direction than in the fetal-to-maternal transport direction [
2].. Our results demonstrated that fetal OXT concentrations were significantly greater among mothers who received OXT before delivery. This finding strongly implies that OXT crosses the maternal–fetal barrier during human gestation.
Preterm and term neonates whose mothers underwent labor and received OXT before delivery had significantly higher OXT concentrations than those whose mothers did not undergo or receive OXT before delivery. This statistical significance was especially true among the preterm group, highlighting that many preterm neonates are likely born due to maternal preterm labor [
61]. Furthermore, our findings imply that maternal OXT concentrations not only increase in the presence of labor or at birth but also likely cross the maternal–fetal barrier and contribute to surges in fetal plasma OXT levels. Additional findings included associations between preterm delivery and maternal illness. The frequency of deliveries involving diabetes and HBP/preeclampsia among preterm births was greater than that among term deliveries when maternal and infant birth characteristics were reviewed. Preterm births were also associated with the administration of antenatal steroids and the need for oxygen or advanced respiratory support during resuscitation.
The clinical arm of our study is the first to monitor OXT transfer across the maternal–fetal barrier in vivo and at several time points throughout human gestation (23–42 weeks). Research using in vitro models has shown that OXT can cross the placenta via simple diffusion [
2]. Our study, which was conducted on the maternal–infant dyad in ‘real-time’, is consistent with these models and demonstrated significantly higher fetal OXT concentrations among mothers who received OXT before delivery (especially in the presence of labor). This is a significant strength of our study, and these findings strongly support the theory that maternal OXT not only crosses the placenta but also contributes to surges in fetal OXT levels.
Additionally, we aimed to achieve accurate and effective OXT extraction via an advanced protocol. While prior studies have only measured OXT via an in situ approach (to conserve blood volume), we successfully extracted OXT from whole blood volumes as small as 50 microliters [
62,
63]. This novel technique uses organic solvents and HPLC columns (solid-phase extraction) to increase the ability of ELISA kits to detect and extract highly volatile molecules such as OXT from their natural biological matrix (plasma).
Our study had several limitations. A primary factor is the considerable variation in fetal OXT concentrations. Although our findings strongly suggest that OXT crosses the placenta, our novel extraction protocol in this pilot study was likely subject to procedural error and varying concentrations. A more efficient protocol is needed for future studies to ensure that optimal OXT extraction occurs. Another study limitation was the small and unequal sample size between term and preterm infants. Our statistical methods of log transformation helped improve the symmetry between the 2 cohorts and allowed for improved statistical power. However, our study was not initially powered with equal control arms or an ideal number of study participants to identify the transplacental effects of OXT. We also recognize that RNA-seq-based approaches are accepted in the scientific community; technological improvements are seen to overcome some gene expression analysis shortfalls and do not account for the contributions of noncoding sequences.
This translational study, however, shows promise for further investigation of OXT as a gestational hormone essential for the normal development of the retina. As OXT likely transfers from mother to fetus, it is possible that OXT concentrations continually increase throughout human gestation while the retina matures, surging just before birth and in the presence of labor. If this effect is valid, OXT could affect certain vascular growth factors that are expressed during the pathologic and vasoproliferative stages of ROP. Future studies will need larger cohorts with infants at varying gestational ages to assess the specific trajectory of OXT among the maternal-infant dyad throughout human gestation. It may also be worthwhile to examine whether delivery methods play a role in transferring OXT across the maternal-fetal barrier.