Historically, the placenta has been considered an asexual organ, however the fertilized egg gives rise to both fetus and placenta, thus the placenta and fetus are the same sex [1,3,4]. Sex differences in fetal development and growth have long been established, with males having accelerated growth rates relative to females, which can be observed as early as the blastocyst stage [5–7]. Additionally, growth of the placental unit is also sex dependent. In humans, male placentas exhibit greater placental growth (mass) compared to females, which facilitates the increased growth demands of the male fetus [8]. Blood flow to the placenta also differs between sexes with male fetuses from normotensive pregnancies having increased blood flow over time compared to female fetuses [9]. Sex also impacts placental nutrient transfer. Female placentas exhibit increased fatty acid oxidation, purine degradation, and uric acid levels, indicating that females may be more susceptible to oxidative stress [10]. Additional disease states [11,12] or environmental insults [13,14] can further alter and exacerbate oxidative stress within the placenta.

Oxidants are produced through aerobic metabolism [15] and can impair cellular function through damage to proteins, lipids, and DNA [16]. To combat oxidants, the body produces antioxidants to maintain a homeostatic balance of these redox compounds [15]. This balance is especially important in early gestation as redox signaling is required for placentation [15]. A study of hexavalent chromium (a by-product of industrial processes involving stainless steel or metals containing chromium [17]) exposure during gestation in humans found that male placentas had increased oxidative stress markers compared to females and exhibited a greater reduction in antioxidant activity of the enzymes, catalase and superoxide dismutase (SOD) [18]. Hydroxyl radicals (HO^• and hydroperoxyl, HO₂^•) can result in lipid peroxidation and HO^• is thought to be formed via the interaction of H₂O₂ and free iron (Fe²⁺) [19]. The hydroperoxyl radical, HO₂^•, can yield H₂O₂ which can further react with metals, such as iron or copper, to further generate more HO^• [19]. To add to this complex system, COX, along with lipoxygenase, have been demonstrated to be minor endogenous sources of these oxidants [20–23]. The rat and human placenta have been shown to express increasing cyclooxygenase (COX)-1 and COX-2, with COX-2 expression towards parturition [24]. In a mouse model, oxidative stress increased close to term and the authors proposed that this played a physiologic role by increasing prostaglandin synthesis, some of which helped to induce parturition [25]. Nano-TiO₂ (a white powder used for its optical properties in paint or photocatalyst [26]) inhalation exposure during gestation increased maternal liver oxidant load and placentas from nano-TiO₂ exposed dams had greater H₂O₂ production capacity [2]. This indicates that nano-TiO₂ exposure during gestation may increase oxidant balance.

Maternal nano-TiO₂ inhalation exposure during gestation increased inflammatory signals from the maternal lung, such as interleukin (IL)-4, -5, and -13 [27]. We also previously demonstrated increased prostacyclin synthase and decreased prostacyclin receptor mRNA expression in the exposed dam lungs [28]. Pulmonary inflammatory signals may enter the circulation due to the interdependence of the pulmonary and cardiovascular systems and therefore act systemically throughout the body. Nano-TiO₂ inhalation exposure decreased endothelium-dependent relaxation in thoracic and femoral arteries [26], uterine radial arterioles [29], and increased uterine radial arteriole vasoconstriction in response to TXA₂ mimetic, U46619 [28]. Additionally, maternal nano-TiO₂ inhalation exposure during gestation increased maternal hepatic mass and H₂O₂ production capacity [2], and decreased GSH: GSSH ratio. Fetal female placental outflow [1] was decreased in response to cyclooxygenase metabolites after maternal nano-TiO₂ inhalation exposure during gestation. Hepatic oxidant production is linked to vascular oxidant production and atherosclerosis [30]. Changes in redox balance during gestation may underlie the hindered fetal development and increased cyclooxygenase vasoreactivity. Whole, placental samples had increased mass [31] and H₂O₂ production capacity [2] from nano-TiO₂ exposed dams (highlighted in Fig. 1). Due to the inflammatory changes within the lungs, microcirculatory adaptations in COX metabolites, and liver oxidant signaling changes, we sought to investigate the changes that occurred within distinct placental zones and how fetal sex impacted these changes. This led to our hypothesis that maternal nano-TiO₂ inhalation during gestation increased placental production of PGI₂ and TXA₂ through H₂O₂ in a sexually dimorphic manner.

Female Sprague-Dawley (SD) rats were purchased from Hilltop Laboratories (Scottdale, PA) and single-housed in an American Association for Accreditation of Laboratory Animal Care (AAALAC) approved facility at West Virginia University (WVU). Temperature (20–26 °C), relative humidity (30–70 %), and light-dark cycle (12:12 h) was maintained for housed rats. After 48–72 h for acclimation, rats were randomly assigned to either sham-control (N = 13) or nano-TiO₂ (N = 14) exposure groups. Rats had ad libitum access to standard chow (2918X; Envigo, Indianapolis, IN) and water throughout the acclimation and exposure periods. Rats were euthanized via thoracotomy and heart removal and then fetal distribution within the uterine horns and fetal sex were recorded. Fetal tissue was weighed and grouped according to fetal sex: placental junctional zone (endocrine production site), and placental labyrinth zone (nutrient-waste exchange), serum, liver, and lung were collected. Placental junctional zone (JZ) and labyrinth zone (LZ) were bluntly dissected [33], flash frozen in liquid nitrogen, and stored at −80 °C until use. All procedures were approved by the WVU Institutional Animal Care and Use Committee.

Briefly, once dams were euthanized and pup count was recorded (previously published [1]), the uterus was surgically excised and placed into a dissection dish containing physiological salt solution (PSS, in mmol/L: 129.8 NaCl, 5.4 KCl, 0.5 NaH₂PO₄, 0.83 MgSO₄, 19.0 NaHCO₃, 1.8 CaCl₂, 5.5 glucose). The uterus was incised longitudinally, and amnionic sacs were opened to allow for quick identification of fetal sex. Fetal sex and position within the horn was recorded, then the first male and female nearest the cervix were removed with the placenta still attached. This unit was utilized for placental hemodynamic assessment and has been previously published [1]. The next set of male and female units were collected for H&E and IHC assessment and has been previously published [1]. The remaining males and females in the litter were utilized for serum and tissue collection for this study.

This study aimed to identify the relationship between H₂O₂ and the cyclooxygenase metabolites, PGI2 and TXA2, in male and female feto-placental units after maternal nano-TiO₂ inhalation exposure during gestation. We have previously demonstrated that maternal nano-TiO₂ inhalation exposure during gestation reduces fetal female mass, female placenta mass and area, as well as placenta immune cell invasion [1]. Liver and placental tissues from dams directly exposed to nano-TiO₂ exhibit increased indices of oxidant stress and an increased H₂O₂ abundance, indicating that these tissues have increased oxidant load [2]. As adults, females exposed to nano-TiO₂ in utero exhibited decreased circulating estrogen levels during gestation, decreased litter size, and decreased pup mass [2]. This suggests that maternal nano-TiO₂ inhalation exposure during gestation not only impacts redox balance in the dam but also impacts female feto-placental growth and development during gestation and later in life. Modifications in redox balance could potentially contribute to the reduced cyclooxygenase metabolite vasoreactivity in dam uterine radial arterioles [28] and/or decreased placental outflow in the presence of U46619, the stable TXA₂ mimetic, in exposed fetal females [1] previously shown. In this study, placentas from female pups whose mothers were exposed to nano-TiO₂ during gestation had increased XOR activity and decreased catalase activity, which indicates an oxidative stress/antioxidant imbalance. Taken together, placentas from female pups exposed in utero demonstrate redox and cyclooxygenase imbalance, impacts placental nutrient-waste exchange, and ultimately fetal growth and development.

It is important to note that toxicant exposure consistently increases oxidant production [37], decreases catalase activity [38], and increases prostacyclin metabolite production [39], in HUVECs or guinea pig alveolar cells. This implies that toxicants, such as nano-TiO₂, can modify the balance between oxidants and antioxidants, thus disrupting downstream signaling by molecules such as prostacyclin or thromboxane. Additionally, placental growth occurs in a hypoxic environment, with oxygen tension residing at 2–3 % (25–38 μM dissolved O₂) in the first trimester and beginning of the second [40]. This becomes important when considering that the low O₂ tension diminishes superoxide generation by xanthine oxidase (XO), thus elevates H₂O₂ production to nearly 90 % [41]. Our current study found that nano-TiO₂ exposed females had increased XDH activity. This will lead to increased oxidants and uric acid production, which may increase placental inflammation and insufficiency, both of which we have previously demonstrated in exposed fetal females [1]. This implies that increased XOR in these exposed fetal females could contribute to their increased placental resistance and modified placental structure, thus impacting fetal female growth and development.

XOR can generate superoxide, H₂O₂, and NADH when transferring electrons from xanthine to oxygen and NAD⁺ [42]. Due to the increased XOR activity found in exposed fetal female placentas, we examined H₂O₂ levels. No differences were observed in H₂O₂ levels across fetal sex or placental zones. This differs with previous results from our laboratory, which demonstrated increased placental capacity to produce H₂O₂ in collected tissue from nano-TiO₂ exposed dams [2]. In a mouse model of gestational exposure to hypoxia, placentas from hypoxic male fetuses were heavier than the hypoxia exposed fetal female placentas [43], and the hypoxic fetal female placentas demonstrated increased oxidative stress markers. This study highlights that oxidative stress is impacted by fetal sex. In homeostasis, redox balance is maintained via the action of small molecule antioxidants and enzymes such as catalase. Herein, we demonstrated that catalase activity in nano-TiO₂ females was significantly decreased within the LZ. Therefore, the decreased growth in female fetuses previously shown to occur due to maternal nano-TiO₂ inhalation exposure [1] may be explained, at least in part, by oxidative imbalance within the LZ, where nutrient-waste exchange occurs.

Previous results from our laboratory demonstrated significantly decreased circulating TXB₂ levels in nano-TiO₂ exposed dams [28]. Based on these results, both 6-keto-PGF₁α and TXB₂ are important vasoactive compounds, thus it was necessary to measure placental zone and fetal serum levels of 6-keto-PGF₁α and TXB₂. Increased circulating levels of 6-keto-PGF₁α were found in the fetal females from nano-TiO₂ exposed dams, which could be indicative of an elevated immune response. Complications during gestation, such as asthma and gestational diabetes, have been associated with increased placental expression of inflammatory immune markers such as, TNF-α, IL-1β, IL-6, and IL-8 [44,45]. Further, Hofbauer cells, which are fetal derived macrophages, have increased production of pro-inflammatory cytokines in gestational diabetes [45]. Maternal nano-TiO₂ exposure significantly increased female placenta Hofbauer cell invasion [1] and maternal circulating IL-1β and TNFα [27]. IL-1β is reported to induce COX-2 expression [46–48], which is the enzyme that catalyzes the production of PGI₂ [48], and could promote this increased 6-keto-PGF₁α levels in fetal females from nano-TiO₂ exposed dams. Placental tissue, specifically the LZ, exhibited significantly increased 6-keto-PGF₁α and TXB₂ levels from fetal females that were exposed in utero. Human pregnancies with intervillous blood flow below the normal mean (less than 130 mL/min) exhibit increased placental TXA₂ production [49]. In a preeclampsia mouse model that simulated modified blood flow to the fetoplacental unit, preeclampsia mice placentas had elevated TXA₂ synthase and plasma TXA₂, but not prostacyclin levels [50]. This indicates that the balance of TXA₂ and PGI₂ levels is critical in regulating blood flow to the feto-placental unit. A rat intrauterine growth restriction (IUGR) study found that IUGR male umbilical arteries had decreased maximal contraction in response to U46619, the TXA₂ mimetic, compared to IUGR females [51]. This highlights that changes in placental production of TXA₂, and PGI₂ to a lesser extent, impact blood flow and further impact fetal growth and development in a sex-based manner. It is possible that the redox imbalance, specifically the increased XOR-catalyzed peroxide production and decreased catalase activity within the LZ, leads to the overall increase in PGI₂ and TXA₂ levels seen in the LZ of exposed fetal females. This would ultimately result in modified uteroplacental blood flow [1], as we have observed previously, and impair fetal growth and development due to modified redox homeostasis.

However, with all this information, there are a few study limitations that need to be addressed. The first limitation is that we do not know where the XO is coming from. A way to address this concern would be the incorporation of an XO-tissue specific knock-out mouse model. We would need to run a few small studies on wild-type mice first, to verify that the pregnant mice would respond to nano-TiO₂ inhalation exposure during gestation similarly to our current rat model. Further, we do not have any febuxostat data in this study, to verify that XO could be driving these changes seen in H₂O₂ and catalase activity after maternal nano-TiO₂ inhalation exposure during gestation. This is already an on-going study in our laboratory. Another limitation that should be addressed is the difference of H₂O₂ levels seen in this study and a previous one from our laboratory [2]. The differences are most likely caused by a few things. First, herein, the placentas were split into the two main zones, JZ (the endocrine production site) and the LZ (nutrient-waste exchange site), previously the whole placenta was assessed. Second, this study assessed the impacts of fetal sex on placental tissue, which fetal sex does matter in relation to placental and fetal tissue assessment. The sex of the fetus was not accounted for in the previous study. Lastly, herein, we utilized an entirely different assay, and measurement for H₂O₂. In this study, we utilized an Amplex Red H₂O₂ kit, which measured the amount of H₂O₂. Whereas the previous study used a Coumarin boronic acid (CBA) assay, which measures the production rate of H₂O₂. These limitations are important to note and are points that are planned to be addressed in future experiments in our laboratory.

In conclusion, this study sought to determine if maternal nano-TiO₂ inhalation exposure resulted in modified redox homeostasis that altered production of PGI₂/TXA₂ within the placental zones in a sexually dimorphic manner. Our exposure paradigm has provided evidence that maternal nano-TiO₂ inhalation exposure modified redox homeostasis within placental zones and impacted fetal female growth and mass, placental development, and hemodynamics [1]. These modifications likely impact COX metabolite production, ultimately modifying fetal growth in a sexually dimorphic manner, with the greatest growth impacts on exposed females (Fig. 6). These adaptations in the exposed fetal female likely ensures survival throughout gestation but could have lasting negative health impacts on females later in life. Future studies should focus on if treatment with an XO inhibitor, such as febuxostat, during gestation can prevent diminished fetal female growth and development during maternal nano-TiO₂ inhalation exposure. This will help elucidate if dysregulated redox homeostasis underlies the fetal and placental deficits observed after exposure and provide a possible treatment option to women who have imbalanced redox and COX metabolites.