To prepare for embryo implantation and pregnancy, uterine endometrial stromal fibroblast [human(h)ESF] differentiate or “decidualize” in response to progesterone to become decidual cells (Evans et al., 2016). Decidualization is initiated immediately post-ovulation under the control of progesterone and involves the reprogramming of hESF such that different genes are expressed at different stages of differentiation (Popovici et al., 2000). During implantation extravillous trophoblast (EVT) invades into the decidualized endometrium (decidua) and upper third of the myometrium (Lunghi et al., 2007). The decidua produces factors which regulate trophoblast invasion (Dimitriadis et al., 2005; Lunghi et al., 2007; Burton et al., 2010; Menkhorst et al., 2012, 2015, 2019; Pollheimer et al., 2018) and protect the conceptus from the maternal immune system and oxidative stress (Evans et al., 2016; Okada et al., 2018). Poor pregnancy outcomes including recurrent pregnancy loss (RPL) and preeclampsia are associated with impaired decidualization (Founds et al., 2009; Salker et al., 2010; Dimitriadis et al., 2020).

Prednisolone is a corticosteroid which classically acts via the glucocorticoid receptor (GR) and has anti-inflammatory and immuno-modulatory effects (Frolkis et al., 2010). Prednisolone administration during the menstrual cycle and early pregnancy may affect endometrial stromal and trophoblast cells: GR are present in the glandular epithelium and stromal cells of the endometrium and 1st trimester decidua (Henderson et al., 2003) as well as trophoblast cells of the 1st trimester placenta (Yang et al., 2016; Kisanga et al., 2018). Murine models suggest that prednisolone or other corticosteroid administration during early pregnancy affects fertility and pregnancy outcome via actions on the uterus (Matejevic et al., 1995; Li et al., 2018; Kieffer et al., 2020), however, the precise impact of these drugs on endometrial cells specifically is unknown.

Prednisolone is used an off-label therapy for RPL to reduce uterine Natural Killer (uNK) cell numbers (Dimitriadis et al., 2020). The reported efficacy of prednisolone at preventing miscarriage in women with a history of idiopathic miscarriage is highly variable between studies (Tang et al., 2013; Gomaa et al., 2014; Dan et al., 2015; Cooper et al., 2019) however, the most recent systematic review and meta-analysis found that prednisolone administration did not improve miscarriage rates or pregnancy outcome (Woon et al., 2020). Concerningly, there is emerging evidence linking corticosteroid use to preeclampsia incidence in women (Boyd et al., 2015; Bandoli et al., 2017) and dexamethasone treatment in pregnant rats induces the development of PE features (Zhang et al., 2016, 2018), however, most studies investigating the role of prednisolone in RPL are not sufficiently powered to identify rare pregnancy outcomes such as preeclampsia.

We hypothesized that prednisolone treatment may have off-target actions on endometrial stromal fibroblasts, affecting decidualization, decidual regulation of trophoblast function and ultimately the formation of a healthy placenta. In this study, using primary and gestational-age appropriate tissue, we aimed to define the effect of prednisolone on hESF decidualization and determine whether hESF decidualized in the presence of prednisolone would differently regulate trophoblast function.

This study was conducted under approvals from The Royal Women’s Hospital and Monash Health Human Research and Ethics Committees (#90317B, #06014C, and #03066B). Written and informed consent was obtained from each patient before surgery. All experiments were performed in accordance with the NHMRC guidelines for ethical conduct in human research.

Endometrial biopsies were collected by dilatation and curettage from women (n = 15 women; age 36.4 ± 1.3 years; range 29–46 years). Women were fertile (n = 2/15), primary infertile (n = 3/15; unable to conceive for ≥12 months), secondary infertile (n = 9/15; unable to conceive for >6–12 months but who have had a previous successful pregnancy), or unknown fertility (n = 1/15; have not attempted to conceive). 4/15 women had polyps, 1/15 had endometriosis, 1/15 had PCOS, 2/15 had menorrhagia and the reminder (7/15) had no obvious endometrial pathology. The women had no hormonal treatment for ≥3 months before tissue collection, however, 2/15 were prescribed prednisolone.

Products of conception were collected from first trimester pregnancies (n = 9; amenorrhea 5–13 weeks) following elective termination of pregnancy by evacuation for psychosocial reasons.

This is the first study to investigate the effect of prednisolone treatment on decidualization and decidual-trophoblast interactions. Prednisolone treatment during in vitro decidualization did not alter production of the classic decidualization markers PRL or IGFBP1 but altered hESF cytokine gene expression and decidualized hESF cellular protein. Intriguingly, trophoblast-decidual interactions were altered following hESF decidualization in the presence of prednisolone: we found prednisolone treatment enhanced trophoblast outgrowth, elevated EVT PLCG1 production and reduced EVT PGF production.

To our knowledge the direct effect of prednisolone on decidualization in women has never been investigated. Although prednisolone has been shown to downregulate GR production in HeLa cells (Shimojo et al., 1995) here we saw no effect on GR production in non-decidualized or decidualized hESF. Our data suggests that prednisolone suppresses hESF pro-inflammatory cytokine production as has previously been shown in other cell types (Karagiannidis et al., 2004; Andersson et al., 2005; Shmarina et al., 2017). It is interesting that prednisolone suppressed hESF production of IL11, LIF, and LIFR which we previously showed enhanced progesterone-induced decidualization (Dimitriadis et al., 2003; Shyua et al., 2011). Inhibition of IL11 expression by methylprednisolone has previously been show in bronchial epithelium (Chakir et al., 2003), but there is no previous investigation of the effect of prednisolone on LIF production. As IL11 and LIF are only two of many pathways altered during decidualization (Gellersen and Brosens, 2014; Evans et al., 2016) it is unsurprising that suppression of IL11 and LIF did not lead to altered production of PRL or IGFBP1, however, dysregulation of these factors may still impact hESF decidualization. It must be noted that the absolute levels of PRL and IGFBP1 secretion were different between hESF cultured fresh vs. those which were frozen before thawing for culture experiments. As most of the fresh hESF were from women with primary infertility and most of the frozen hESF were from women with secondary infertility this difference could also reflect the clinical characteristics of the women. Regardless, prednisolone had no effect on PRL or IGFBP1 production by hESF.

Since we found no effect of prednisolone treatment on gene expression or secretion of the classical markers of decidualization, we performed mass spectrometry to identify whether hESF proteins were altered by prednisolone treatment during decidualization. Of the 176 hESF proteins significantly regulated by prednisolone, 27 had previously been identified in decidua, including factors which promote decidualization [including GNA11/GNAQ (De Oliveira et al., 2019), CDK6 (Tan et al., 2002), and SCRIB (Whitby et al., 2018; Yuan et al., 2019)], or which are upregulated during decidualization [including CTTN (Paule et al., 2011), CTNNA1 (Patterson et al., 2017), SPTLC2 (Ding et al., 2018), and ALDH1A1 (Tomari et al., 2020)]. Decidualization itself is associated with substantial post-translational modification (Díaz-Gimeno et al., 2014) and here we found that prednisolone stimulated the production of proteins associated with acetylation, however, the effect of altered acetylation in decidualized hESF is unknown. Prednisolone treatment also altered hESF proteins associated with mitochondria, including increased production of factors associated with ATP generation and transport (e.g., UQCRC2, VDAC1/2; ATP5 synthases; NDUF enzymes; SLC25A mitochondrial carrier proteins). Again, the precise effect of altered hESF mitochondrial function is unknown. It is likely that proteins regulated by prednisolone will alter decidual cell interactions with other cells in the uterus, including trophoblast as demonstrated here.

We previously observed that CM from decidualized hESF enhanced trophoblast outgrowth when compared to non-decidualized hESF (Menkhorst et al., 2019). Despite prednisolone having no effect on classic markers of decidualization we observed that CM from hESF decidualized in the presence of prednisolone enhanced trophoblast outgrowth, suggesting that prednisolone altered hESF release of factors which regulate trophoblast function. Future studies are required to elucidate how prednisolone alters hESF CM and thus decidual regulation of trophoblast invasion, however, the data presented here suggests hESF CM may alter trophoblast motility genes including PLCG1. PLCG1 has not previously been identified in trophoblast or the placenta, however, it has well established roles in tumor metastasis where it promotes cell invasion (Kunze et al., 2014; Jang et al., 2018; Tang et al., 2019), potentially via its interactions with MMP2 (Zhang et al., 2019) or EGFR signaling (Jang et al., 2018). The precise effect that increased PLCG1 production by EVTs would have on trophoblast invasion and whether hESF CM regulates other trophoblast functions including viability or proliferation remains to be experimentally determined.

There is emerging evidence linking corticosteroid use to preeclampsia incidence in women (Bandoli et al., 2017) and the development of PE features in rodents (Zhang et al., 2016, 2018). The Danish National Cohort study identified corticosteroid medication use for inflammatory bowel disease had a strong and significant association with preeclampsia (Boyd et al., 2015). Diseases for which prednisolone is a common treatment are also conditions with elevated risk of preeclampsia, including Antiphospholipid syndrome (APS) (when prescribed in addition to aspirin and heparin) (Empson and Al, 2005), chronic kidney disease (10-fold increased risk of preeclampsia) (Wiles et al., 2020) and systemic lupus erythematosus (14% increased risk of preeclampsia) (Chen et al., 2019), however, the contribution of prednisolone vs. the effect of the disease itself to preeclampsia risk has not been established.

The contribution of decidual deficiency in the etiology of preeclampsia is emerging (Garrido-Gómez et al., 2020). Here we found that EVT treated with CM from hESF decidualized in the presence of prednisolone had reduced PGF production. Serum PGF is reduced in early pregnancy serum of women who develop PE and is a biomarker used in the 1st trimester screening test for preterm preeclampsia (Akolekar et al., 2013). In this study hESF also had altered production of factors previously identified to be increased in the decidua of women with preeclampsia, including COL4A1 (Yong et al., 2014, 2015), LNPEP (Yong et al., 2014), TM9SF2 (Garrido-Gomez et al., 2017) and COTL1 (Garrido-Gomez et al., 2017). The impact of prednisolone treatment on the decidua and decidual function could be a novel mechanism by which prednisolone or other corticosteroid use increases preeclampsia risk.

Overall, this study demonstrates that prednisolone alters decidualized hESF and altered decidual-trophoblast interactions. The clinical consequences of these changes are unknown, however, as all available data suggests that corticosteroid administration has no beneficial effect for IVF (Kaye et al., 2017; Mohammadi Yeganeh et al., 2018), RPL (Tang et al., 2013; Cooper et al., 2019; Woon et al., 2020), or repeated implantation failure (Siristatidis et al., 2018) and the emerging evidence that corticosteroid use during pregnancy may be associated with poor obstetrical outcomes (Boyd et al., 2015; Bandoli et al., 2017), off-label use of corticosteroids, in particular prednisolone, during the period of decidualization (secretory phase of the menstrual cycle and the 1st trimester), should be carefully considered.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

The studies involving human participants were reviewed and approved by Royal Women’s Hospital Human Research Ethics Committee Monash Health Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.

EG, EM, and ED wrote the main manuscript text. EG prepared Figures 1–4 and Supplementary Tables 2–4. TS prepared Figures 2–4 and Supplementary Tables 2, 3. SV and NW prepared Figure 3 and Supplementary Tables 2, 3. EM prepared Figures 1–4. All authors reviewed the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.