The placenta is an autonomous and transient organ that is critical for the maintenance of pregnancy and materno‐fetal exchanges. Aberrant formation and function of the placenta is associated with pregnancy disorders such as preeclampsia and intrauterine growth restriction (Gupta et al., 2015). The syncytium is critical in forming the materno‐fetal interface. In vivo, formation of the syncytium is dependent upon the differentiation and fusion of the underlying stem cells, the cytotrophoblasts. Cytotrophoblast differentiation is characterized by biochemical and morphological changes, including upregulation and expression of hPL, hCGβ, and production of estrogen and progesterone (Gude et al., 2004; Gupta et al., 2015; Wice, Menton, Geuze, & Schwartz, 1990). As the placenta is an organ with high endocrine activity, hormone production and release are vital aspects of a placental/trophoblast model. hCG is a secreted peptide hormone that mediates important physiological events such as progesterone production (Weedon‐Fekjær & Taskén, 2012), placental vascularization (Cole, 2010), immune protection for mother and fetus (Wan et al., 2007), fetal organogenesis (Cole, 2010), and promotes the continuous differentiation of cytotrophoblasts to syncytiotrophoblasts throughout gestation (Cole, 2010; Pidoux et al., 2007; Shi, Lei, Rao, & Lin, 1992).

To evaluate whether THC treatment impacted these critical trophoblast functions, we used the BeWo cells as a model to study proliferation, differentiation, and syncytialization, as they are the only cell line which can be induced to differentiate and syncytialize in a protein kinase A‐dependent process (Knerr et al., 2005); emulating what is observed in trophoblasts in vivo, in humans (Knerr et al., 2005; Pidoux, 2015). While there are acknowledged differences between isolated human primary trophoblast cells and cultured trophoblast cell lines, the use of established cell lines have been advocated for by many researchers (Bode et al., 2008; Orendi et al., 2010; Rothbauer et al., 2017) and reviewed by Sullivan (2004).

In the present study, we assessed the effect of THC exposure on the process of BeWo fusion by quantifying (a) markers of syncytialization and indicators of trophoblast fusion; (b) hCG secretion; and (c) the observation of multinucleated syncytia via immunofluorescent staining. We demonstrate that exposure of BeWo cells to THC results in a significant reduction in CG gene transcription mediated by both CB1 and CB2 and attenuated the release of hCGβ from the STs. Furthermore, THC exposure also resulted in reductions in the transcriptional markers of syncytialization largely mediated via CB1 binding, and a reduction in the percentage of fused trophoblasts. Aberrant syncytialization has been implicated in preeclampsia (Roland et al., 2016), and several reports have indicated that the master players in syncytialization, including GCM1 and the syncytins (Vargas et al., 2009), are also downregulated in preeclampsia (Langbein et al., 2008; Orendi et al., 2010; Roland et al., 2016). In this way, THC may interfere with the mechanisms involved in trophoblast fusion and, thus, be implicated in poor pregnancy outcomes.

One of the primary functions of the human placenta is to produce hormones and other mediators which are critical for pregnancy success. These include hPL and IGFs, molecules which are implicated in fetal growth and development (Constância et al., 2002; Forbes, Westwood, Baker, & Aplin, 2008; Mcintyre et al., 2000). Treatment of trophoblasts with THC for 48 hr led to reduced transcriptional expression of both markers, which further suggests perturbed syncytial function. Various studies demonstrate that infants that are born small for their gestational age (SGA) were exposed to decreased levels of hPL and IGFs when compared to infants of normal pregnancies (Koutsaki et al., 2011; Mcintyre et al., 2000; Mirlesse et al., 1993). These growth factors were measured in maternal serum, plasma, and term placental tissue, respectively. hPL is a polypeptide hormone mainly secreted by syncytiotrophoblasts and its overall function is that of regulation of fetal and maternal lipid and carbohydrate metabolism (Costa, 2016b). Furthermore, hPL and IGFs are also potent stimulators of tissue growth and regulate the metabolic status of both mother and fetus (Forbes et al., 2008). IGF1 and IGF2 are expressed by fetal tissues and placenta; however, reduced fetal growth in mice lacking the placental‐specific transcript of IGF2 (Constância et al., 2002) is associated with reduced placental mass (Sibley et al., 2004).

Birth weight is indicative of future risk of metabolic disorders including obesity, diabetes, and cardiovascular disease (Freemark, 2010). Healthy fetal growth and weight gain is dependent upon maternal metabolism, and placental development and function (Anthony, Pratt, Liang, & Holland, 1995; Freemark, 2010). The possibility that THC‐mediated changes in the placenta can restrict fetal growth is supported by the recent finding of Natale et al. (2020). Using a rat model of pregnancy, Natale et al. (2020) reported symmetrical fetal growth restriction following in utero exposure to THC (Natale et al., 2020). Furthermore, this group also demonstrated an increase in the labyrinth region of the placenta, leading to an overall greater mass for placentae from dams exposed to THC. Changes in placental vascularization, resulting from the development of narrower vascular networks, has also been observed in women who smoked cannabis without the confounding coexposure of alcohol or tobacco (Chang et al., 2018). Thus, the effect of THC on the reduced expression of hPL and IGF2 in our in vitro system may contribute to the better understanding of mechanisms linking exposure of the syncytium to THC and outcomes reported in SGA fetuses.

Oxidative stress and mitochondrial dysfunction in the placenta have been previously associated with SGA offspring (Mandò et al., 2014). In fact, the regulation of oxidative stress is a critical parameter for normal progression of pregnancy (Holland et al., 2017) and excessive placental oxidative stress is characteristic of several gestational disorders, such as intrauterine growth restriction and preeclampsia (Holland et al., 2017). Mitochondria are known to contribute to placental oxidative stress (Myatt & Cui, 2004) and have also been shown to be negatively impacted by THC (Costa et al., 2015). Recent work by Lojpur et al. (2019) suggested that 15–30 µM THC triggered reduced mitochondrial oxidative phosphorylation and increased endoplasmic reticulum (ER) stress in unfused BeWo cytotrophoblasts. However, mitochondrial function and signals play a wide range of roles in the placenta including the regulation of syncytialization (Maloyan et al., 2012). To advance the understanding of how THC may reduce mitochondrial function and increase syncytiotrophoblast stress, we investigated the consequences of THC exposure on syncytiotrophoblast mitochondrial respiratory function, dynamics, antioxidant enzyme expression, as well as cellular ROS production and markers of cellular stress.

The MTS and LDH assays measure different aspects of cellular function. The MTS assay quantifies mitochondrial metabolic activity and indirectly reflects cellular proliferation. Conversion of MTS to its purple formazan precipitate is mediated by mitochondrial dehydrogenases. This precipitate, measured spectrophotometrically upon solubilization, is directly proportional to the number of viable cells (Rai et al., 2018), while the LDH assay allows for the assessment of plasma membrane integrity (Moghimi et al., 2005). We demonstrate that at 20 µM THC, mitochondrial metabolic activity was impaired while leaving the plasma membrane unperturbed. Furthermore, we show that THC, partly mediated via canonical CB1, also perturbs mitochondrial dynamics in trophoblasts as indicated by the increase in the markers of fission, DRP1, and the decrease in the markers of fusion, MFNs and OPA1. Interestingly, antagonism of CB2 did not normalize the THC‐mediated reduction in MFN2, OPA1, and DRP1. A healthy mitochondrial reticulum is fundamental for ATP synthesis, regulation of apoptosis, calcium signaling, and ROS production (Poidatz, Dos Santos, Loı, et al., 2015). Healthy mitochondrial reticulum is also an important indicator of overall cellular health and changes in its morphology is indicative of cellular stress (Eisner, Picard, & Hajnóczky, 2018; Zemirli, Morel, & Molino, 2018). We therefore assessed common indicators of oxidative and cellular stress. We observed elevated levels of cellular ROS, increased expression of HSP60 and HSP70 as well as SOD1 and SOD2. Normally, ROS generation by the mitochondria is tightly controlled by SOD2 in mitochondria and SOD1 in the cytosol (Wang & Walsh, 1998). In our study, the BeWo cells demonstrate the capacity to compensate for the THC‐induced increase in ROS generation by increasing the synthesis of SOD2 and SOD1 transcripts. Because intracellular ROS may indirectly regulate DRP1 in physiological and pathological processes (Gan et al., 2014; Guo, Sesaki, & Qi, 2014), our results raise the question whether increased DRP1 expression leads to trophoblast dysfunction driven by oxidative stress in our in vitro model.

Decreased mitochondrial membrane potential, a signal characteristic of dysfunctional mitochondria, can trigger mitophagy or mitochondrial recycling (Twig et al., 2008; Twig & Shirihai, 2011). This process represents the targeted degradation of mitochondria and a mechanism by which a cell can maintain a healthy mitochondrial pool. Fission precedes mitophagy as this ensures that defective mitochondria are small enough to be engulfed by autophagosomes for subsequent breakdown in the autolysosome (Osellame, Blacker, & Duchen, 2012). A number of factors can contribute to mitochondrial stress and mitophagy, such as increased ROS and loss of mitochondrial membrane potential (Twig et al., 2008). We also demonstrate that similar markers of mitochondrial stress are triggered by THC treatment of BeWo cells and associated with markers of mitophagy. While mitochondria are replenished via division of the existing mitochondrial pool, deficits in this process may shift the equilibrium to a state where the cell contains defective mitochondria, perpetuating insufficient respiratory activity, and oxidative stress/damage (Osellame et al., 2012; Twig et al., 2008). Twig et al. (2008) demonstrated that mitochondria undergoing fission are more likely to undergo mitophagy and that this pool of mitochondria has a significantly lower mitochondrial membrane potential than the cellular pool of mitochondria that is fusion competent.

Critical for the maintenance of the mitochondrial membrane potential is the transfer of electrons through the ETC which results in the phosphorylation of ADP to form ATP via complex V–ATP synthase. Measurements of mitochondrial OCR in syncytiotrophoblasts allowed for direct quantification of the effect of THC on various aspects of the electron transport chain function. While we did not observe significant differences in basal respiration, ATP production was significantly reduced in THC‐treated cells. Interestingly, both the proton leak and maximal respiration demonstrated an increase following exposure to THC. Elevated proton leak may be a sign of mitochondrial inner membrane damage (Eisner et al., 2018) and may be connected to the decrease in membrane potential observed in THC‐treated cells. Furthermore, the reduced nonmitochondrial oxygen consumption observed in this study can be attributed to nonmitochondrial processes, such as enzymatic activity of oxygenases (Brand & Nicholls, 2011; Manfredi, Ang, Peker, Dagda, & Mcfarlane, 2019). Regarded as an indicator of bioenergetic health, nonmitochondrial OCR varies (Wagner, Venkataraman, & Buettner, 2011) and may decrease in the presence of stressors (Decleer et al., 2018) like ROS. It is well established that mitochondria are a target for the deleterious effects of reactive oxygen intermediate. Indeed, our group have demonstrated that 10 nM rotenone over 48 hr attenuated complex I activity in BeWo cells and led to increased ROS production concomitant with perturbed syncytialization (Walker et al., 2020).

Maximal respiratory rate is determined by several factors, including the functional capacity of the electron transport chain. The improved maximal respiration in the syncytiotrophoblasts concomitant with increased proton leak and decreased ATP production may be suggestive of a compensatory mechanism to preserve maximum oxygen consumption in the syncytiotrophoblasts. In fact, due to various perturbations, several mitochondrial compensatory responses have been reported in the literature (Haylett et al., 2016; Ireland, Maloyan, & Myatt, 2018; Manfredi et al., 2019) and references therein). These compensatory changes, along with the increase in SOD1 and SOD2 reported herein, may impart a protective effect on the placenta and the developing fetus in response to additional stressors, such as maternal obesity or preeclampsia. Lojpur et al. (2019) demonstrated that treatment of undifferentiated BeWo cells for 24 hr with 15 µM THC resulted in reduced state 3 respiration and this was associated with reduced expression of mitochondrial electron transport chain proteins. Lojpur et al. (2019) observed a reduction in basal respiration upon 24 hr of 15 µM THC exposure in undifferentiated BeWo cells (Lojpur et al., 2019), while we report no significant change in basal respiration in the differentiated BeWo cells. It may be important to consider that there have been reports of ultrastructural differences in the cristae structure of mitochondria from cytotrophoblasts and syncytiotrophoblasts (Castillo et al., 2011; Martinez, Kiriakidou, & Stauss, 1997). Syncytial mitochondria are thought to have lower ATP capacity and have smaller cristae structure to more effectively facilitate steroidogenesis (Castillo et al., 2011).

Mitochondrial dysfunction and the resulting oxidative stress are often associated with ER stress (Burton, Yung, & Murray, 2017). While we did not evaluate ER stress, it has been recently shown that in CTs, THC‐mediated reduction in mitochondrial function was associated with ER stress (Lojpur et al., 2019). There are numerous reports (Lojpur et al., 2019; Mizuuchi et al., 2016; Muralimanoharan et al., 2012; Natale et al., 2020; Shi et al., 2013; Walker et al., 2020) that have associated mitochondrial dysfunction and/or ER stress to adverse trophoblast function and birth outcomes. While we did not evaluate ER stress, it has been recently shown that in cytotrophoblasts, THC‐mediated reduction in mitochondrial function, as assessed by reduced OCR and impaired expression of ETC proteins, was associated with ER stress (Lojpur et al., 2019). Indeed, OXPHOS is, in part, regulated by crosstalk with intracellular calcium, such that efflux of calcium from the ER into the mitochondrial matrix signals an increase in energy demands. When mitochondrial calcium influx is excessive and prolonged, particularly when concomitant with oxidative stress, this may lead to various pathological changes, such as the release of apoptotic proteins from the matrix. Mitochondrial health is intimately linked to cellular viability and, when impaired, becomes associated with various disease states (Osellame et al., 2012).

Taken together, our findings demonstrate that THC attenuates the process of syncytialization and reduces the expression of hPL and IGF2, two important growth hormones connected to pregnancy success. Given the association between THC and reduced fetal growth, it is important to more clearly understand the mechanisms that underpin this association. Our report demonstrates that the observed mitochondrial dysfunction in syncytialized trophoblasts also contributes to increases in the markers of oxidative stress and changes to the dynamics of the mitochondrial reticulum. While it is important to verify these observations in primary trophoblasts, our data advance our understanding of the adverse effects of THC on the materno‐fetal interface.