Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation

Progestin-only long-acting reversible-contraceptive (pLARC)-exposed endometria displays decidualized human endometrial stromal cells (HESCs) and hyperdilated thin-walled fragile microvessels. The combination of fragile microvessels and enhanced tissue factor levels in decidualized HESCs generates excess thrombin, which contributes to abnormal uterine bleeding (AUB) by inducing inflammation, aberrant angiogenesis, and proteolysis. The- zinc finger and BTB domain containing 16 (ZBTB16) has been reported as an essential regulator of decidualization. Microarray studies have demonstrated that ZBTB16 levels are induced by medroxyprogesterone acetate (MPA) and etonogestrel (ETO) in cultured HESCs. We hypothesized that pLARC-induced ZBTB16 expression contributes to HESC decidualization, whereas prolonged enhancement of ZBTB16 levels triggers an inflammatory milieu by inducing pro-inflammatory gene expression and tissue-factor-mediated thrombin generation in decidualized HESCs. Thus, ZBTB16 immunostaining was performed in paired endometria from pre- and post-depo-MPA (DMPA)-administrated women and oophorectomized guinea pigs exposed to the vehicle, estradiol (E2), MPA, or E2 + MPA. The effect of progestins including MPA, ETO, and levonorgestrel (LNG) and estradiol + MPA + cyclic-AMP (E2 + MPA + cAMP) on ZBTB16 levels were measured in HESC cultures by qPCR and immunoblotting. The regulation of ZBTB16 levels by MPA was evaluated in glucocorticoid-receptor-silenced HESC cultures. ZBTB16 was overexpressed in cultured HESCs for 72 h followed by a ± 1 IU/mL thrombin treatment for 6 h. DMPA administration in women and MPA treatment in guinea pigs enhanced ZBTB16 immunostaining in endometrial stromal and glandular epithelial cells. The in vitro findings indicated that: (1) ZBTB16 levels were significantly elevated by all progestin treatments; (2) MPA exerted the greatest effect on ZBTB16 levels; (3) MPA-induced ZBTB16 expression was inhibited in glucocorticoid-receptor-silenced HESCs. Moreover, ZBTB16 overexpression in HESCs significantly enhanced prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP1), and tissue factor (F3) levels. Thrombin-induced interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels in control-vector-transfected HESCs were further increased by ZBTB16 overexpression. In conclusion, these results supported that ZBTB16 is enhanced during decidualization, and long-term induction of ZBTB16 expression by pLARCs contributes to thrombin generation through enhancing tissue factor expression and inflammation by enhancing IL-8 and PTGS2 levels in decidualized HESCs.


Introduction
Progestin-only long-acting reversible contraceptives (pLARCs), e.g., injectable form of depo-medroxyprogesterone acetate (DMPA), subdermal implants releasing etonogestrel (ETO), and intrauterine devices releasing levonorgestrel (LNG), are safe and highly effective in averting unwanted pregnancies, particularly among adolescents, and preterm births by we evaluated in vitro pLARC-mediated regulation of ZBTB16 in HESCs and human endometrial endothelial cells (HEECs), as well as the functional role of ZBTB16 in cultured HESCs to gain insight into the underlying mechanism of pLARC-induced inflammation and AUB.

Increased Endometrial ZBTB16 Levels in Women Receiving DMPA Treatment
The immunohistochemical analysis of paired endometrial sections (n = 7) obtained from women pre-and post-DMPA administration revealed that ZBTB16 is expressed by endometrial stromal, glandular epithelial, and endothelial cells ( Figure 1A). Moreover, strong ZBTB16 immunoreactivity was observed in post-DMPA-administered endometrial stromal and glandular cells, as well as superficial microvascular endothelial cells, whereas pre-DMPA endometria displayed significantly less ZBTB16 immunostaining, which was primarily localized to stromal cells ( Figure 1A). HSCORE analysis ( Figure 1B inflammatory gene expression and tissue-factor-mediated thrombin generation in decidualized HESCs. This hypothesis was tested by investigating the in situ regulation of ZBTB16 levels in endometrial sections obtained from pLARC-administrated women or guinea pigs. Subsequently, we evaluated in vitro pLARC-mediated regulation of ZBTB16 in HESCs and human endometrial endothelial cells (HEECs), as well as the functional role of ZBTB16 in cultured HESCs to gain insight into the underlying mechanism of pLARCinduced inflammation and AUB.

Increased Endometrial ZBTB16 Levels in Women Receiving DMPA Treatment
The immunohistochemical analysis of paired endometrial sections (n = 7) obtained from women pre-and post-DMPA administration revealed that ZBTB16 is expressed by endometrial stromal, glandular epithelial, and endothelial cells ( Figure 1A). Moreover, strong ZBTB16 immunoreactivity was observed in post-DMPA-administered endometrial stromal and glandular cells, as well as superficial microvascular endothelial cells, whereas pre-DMPA endometria displayed significantly less ZBTB16 immunostaining, which was primarily localized to stromal cells ( Figure 1A). HSCORE analysis ( Figure 1B
Compared to their levels on Day 0, EMC treatment significantly induced both PRL and IGFBP1 mRNA levels by up to 2-fold on Day 3 with a further increase observed for both PRL (by 10-fold) and IGFBP1 (by 28-fold) during HESC decidualization on Day 6 ( Figure S1; p < 0.05).
fold at 3 and 6 days of decidualization, respectively, vs. Day 0 of induction ( Figure 3A; p < 0.001). In addition to verifying decidualization-related morphological changes of stromal fibroblasts to epithelioid-like decidual cells, the well-documented decidualization markers, prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1) levels [41], were also measured by qPCR to confirm in vitro decidualization. Compared to their levels on Day 0, EMC treatment significantly induced both PRL and IGFBP1 mRNA levels by up to 2-fold on Day 3 with a further increase observed for both PRL (by 10-fold) and IGFBP1 (by 28-fold) during HESC decidualization on Day 6 ( Figure S1; p < 0.05).
To determine the individual contribution of the EMC components to HESC ZBTB16 expression, parallel experiments were performed in HESCs treated with either the control, E2, cAMP, or MPA for 6 days. Compared to the control, neither E2 nor cAMP treatment induced ZBTB16 mRNA levels, whereas MPA treatment significantly enhanced ZBTB16 levels by ~200-fold ( Figure 3B; p < 0.05), confirming MPA as a primary EMC component that upregulates ZBTB16 mRNA in HESCs. Day 0 analyzed by one-way ANOVA followed by the Student-Newman-Keuls method. (B) MPA mediated upregulation of ZBTB16 mRNA levels in HESCs treated with vehicle (control; Cont) or E2 or MPA or cAMP for 6 days. Bars represent mean ± SEM; n = 4/each; * p < 0.05 vs. Cont or E2 or cAMP analyzed by one way ANOVA followed by Student-Newman-Keuls method.

In Vitro Comparison of Induction of ZBTB16 Levels by Different pLARCs
ZBTB16 mRNA and protein levels were measured by qPCR and immunoblotting, respectively, in HESCs treated with 10 −8 M E2 as the control ± various progestins used in different pLARC formulations including 10 −7 M ETO, LNG, and the mixed progestin/glucocorticoid agonist, MPA, as well as a pure glucocorticoid, dexamethasone (DEX), and a pure progestin, Organon 2058 (ORG), for 7 days. This analysis revealed that all treatments significantly increased ZBTB16 mRNA levels compared to the E2 treatment ( Figure 4A; p < 0.05). Among these treatments, DEX was the most-effective (~280 fold), whereas ORG was the least-effective (~8-fold) inducer of ZBTB16 levels in HESCs. However, based on the fold increases of ZBTB16 levels by individual progestins used in pLARCs, MPA was the strongest inducer, increasing ZBTB16 mRNA levels by 150-fold, followed by ETO and LNG treatment, which increased ZBTB16 by 47-and 15-fold, respectively ( Figure 4A; p < 0.05). Immunoblot analysis confirmed parallel changes in ZBTB16 protein levels. Thus, To determine the individual contribution of the EMC components to HESC ZBTB16 expression, parallel experiments were performed in HESCs treated with either the control, E 2 , cAMP, or MPA for 6 days. Compared to the control, neither E 2 nor cAMP treatment induced ZBTB16 mRNA levels, whereas MPA treatment significantly enhanced ZBTB16 levels by~200-fold ( Figure 3B; p < 0.05), confirming MPA as a primary EMC component that upregulates ZBTB16 mRNA in HESCs.

In Vitro Comparison of Induction of ZBTB16 Levels by Different pLARCs
ZBTB16 mRNA and protein levels were measured by qPCR and immunoblotting, respectively, in HESCs treated with 10 −8 M E 2 as the control ± various progestins used in different pLARC formulations including 10 −7 M ETO, LNG, and the mixed progestin/glucocorticoid agonist, MPA, as well as a pure glucocorticoid, dexamethasone (DEX), and a pure progestin, Organon 2058 (ORG), for 7 days. This analysis revealed that all treatments significantly increased ZBTB16 mRNA levels compared to the E 2 treatment ( Figure 4A; p < 0.05). Among these treatments, DEX was the most-effective (~280 fold), whereas ORG was the leasteffective (~8-fold) inducer of ZBTB16 levels in HESCs. However, based on the fold increases of ZBTB16 levels by individual progestins used in pLARCs, MPA was the strongest inducer, increasing ZBTB16 mRNA levels by 150-fold, followed by ETO and LNG treatment, which increased ZBTB16 by 47-and 15-fold, respectively ( Figure 4A; p < 0.05). Immunoblot analysis confirmed parallel changes in ZBTB16 protein levels. Thus, compared to the E 2 -treated control, each treatment significantly elevated immunoreactive ZBTBP16 levels in HESCs, with DEX-treated HESCs displaying the greatest induction, followed by MPA, then ETO, LNG, and ORG by~17, 9, 6, and 4-fold, respectively ( Figure 4B; p < 0.05).
In addition to HESCs, cultured HEECs were also treated with E2 alone ± ORG, or ETO, or LNG, or MPA, or DEX to further confirm glucocorticoid-receptor-mediated ZBTB16 upregulation in endothelial cells since HEECs express the glucocorticoid receptor [42], but not the progesterone receptor [43,44]. The evaluation of ZBTB16 levels in HEECs by qPCR showed that only MPA and DEX significantly induced ZBTB16 mRNA levels by ~28-and 80-fold, respectively, compared to the E2-treated control group ( Figure S2; p < 0.05), whereas ETO-or LNG-induced changes did not attain statistical significance, and ORG as a pure progestin did not alter ZBTB16 expression in HEEC cultures ( Figure S2).  In addition to HESCs, cultured HEECs were also treated with E 2 alone ± ORG, or ETO, or LNG, or MPA, or DEX to further confirm glucocorticoid-receptor-mediated ZBTB16 upregulation in endothelial cells since HEECs express the glucocorticoid receptor [42], but not the progesterone receptor [43,44]. The evaluation of ZBTB16 levels in HEECs by qPCR showed that only MPA and DEX significantly induced ZBTB16 mRNA levels by~28and 80-fold, respectively, compared to the E 2 -treated control group ( Figure S2; p < 0.05), whereas ETO-or LNG-induced changes did not attain statistical significance, and ORG as a pure progestin did not alter ZBTB16 expression in HEEC cultures ( Figure S2).

ZBTB16 Overexpression Increases Expression of Decidualization Markers in HESCs
To investigate the functional role of ZBTB16 on pLARC-induced decidualization, HESCs were transiently transfected with either the ZBTB16 expression vector or an empty vector as the control. Following transfection, the cultures were incubated with or without EMC for 3 days. As shown in Figure S3, the qPCR results confirmed that, compared to control vector transfection, the ZBTB16 levels were significantly increased in ZBTB16transfected HESCs. However, EMC did not significantly increase ZBTB16 in transfected cells (p = 0.19; Figure S3A).

ZBTB16 Overexpression Increases Expression of Decidualization Markers in HESCs
To investigate the functional role of ZBTB16 on pLARC-induced decidualization, HESCs were transiently transfected with either the ZBTB16 expression vector or an empty vector as the control. Following transfection, the cultures were incubated with or without EMC for 3 days. As shown in Figure S3, the qPCR results confirmed that, compared to control vector transfection, the ZBTB16 levels were significantly increased in ZBTB16transfected HESCs. However, EMC did not significantly increase ZBTB16 in transfected cells (p = 0.19; Figure S3A).

Elevated ZBTB16 in HESCs Induces Tissue Factor Expression
We previously reported that bleeding versus non-bleeding sites of pLARC-exposed endometrium display elevated tissue factor (F3) expression, which causes excess thrombin generation [15]. Therefore, to investigate the impact of ZBTB16 alone or in combination with thrombin on F3 expression, cultured HESCs transfected with either the ZBTB16 or control vector were incubated with or without EMC for 72 h, then treated with 1 U/mL of thrombin for 6 h. Subsequent qPCR analysis revealed that F3 expression was higher in ZBTB16-transfected HESCs compared to control transfection (1.5 ± 0.03 vs. 1.0 ± 0.001; p < 0.05; Figure 5B). Moreover, thrombin significantly elevated F3 mRNA levels in control-transfected HESCs ( Figure 5B; p < 0.05), and thrombin-induced F3 levels were further increased in ZBTB16-transfected HESCs vs. control-transfected HESCs treated with thrombin (2.9 ± 0.17 vs. 2.0 ± 0.04; p < 0.05; Figure 5, upper panel), but not in EMC-treated HESCs ( Figure 5B, lower panel). Note that thrombin treatment in HESCs with or without EMC exposure did not change ZBTB16 levels in either control-or ZBTB16-vector-transfected cells ( Figure S3B).

Discussion
A variety of contraceptive agents including progestin-only or combined oral contraceptive pills, DMPA injections, subdermal implants, or intrauterine devices are used by women to prevent unintended pregnancies during their reproductive lifespan [45]. AUB accompanying pLARCs is a common side effect and the most-cited cause for discontinuation of these otherwise safe and effective agents. Each year, more than one-million unintended pregnancies occur because of the discontinuation or misuse of these contraceptives [46]. The bleeding sites of pLARC-treated endometria display decidualized stomal cells, as well as irregularly distributed hyperdilated thin-walled superficial vessels associated with deficiency in both basement membranes and perivascular smooth muscle cells or pericytes [8,9,39]. Clotting factor leakage from these impaired vessels interacts with the decidualized stromal cell tissue factor induced by pLARCs, thereby generating excess thrombin, which cause dysfunctional regulation of cellular pathways and/or mediators that play crucial roles in endometrial hemostasis, angiogenesis, and inflammation ( Figure 6). In support of these findings, we previously showed that excess thrombin disrupts newly formed vessels [47]. The current study revealed that ZBTB16 may indirectly contribute to pLARC-associated AUB by inducing endometrial pro-inflammatory molecules IL8 and PTGS2, as well as decidualization-associated F3 levels. The integration of our in situ findings from endometria of human DMPA users and MPA-treated guinea pigs with in vitro results from ZBTB16 overexpressing HESCs supports the potential role of enhanced ZBTB16 levels in pLARC-associated AUB.
Previous studies demonstrated that ZBTB16 is widely expressed in various tissues and displays diverse biological functions such as cell growth, self-renewal, and differentiation through the regulation of tissue-specific target genes [25,28]. In addition to the documented cellular and molecular roles of ZBTB16, in the female reproductive system, Fahnenstich et al. [35] initially reported elevated ZBTB16 expression in endometrial stromal and myometrial cells during the secretary vs. proliferative phase of the menstrual cycle, thereby revealing hormonal regulation of ZBTB16 in the human endometrium. Subsequently, Szwarc et al. [49] reported that ZBTB16 plays a crucial role during HESC decidualization by acting as a potential mediator of progesterone receptor actions. The current study expanded the potential role of ZBTB16 in the endometrium by investigating whether ZBTB16 contributes to pLARC-induced tissue-factor-mediated thrombin generation and the inflammatory milieu. Therefore, we initially evaluated ZBTB16 expression in pre-and post-DMPA-administered women and guinea pigs as a highly relevant model [9,11]. Consequentially, the results provided the first evidence that DMPA administration induces ZBTB16 expression in situ in endometrial stromal, glandular epithelial, as well as endothelial cells in both women and guinea pigs.  [8,9] and induces HESC decidualization, which increases tissue factor [15,23] and ZBTB16 levels. Increased ZBTB16 levels induce excess tissue factor expression, which generates thrombin. The resulting excess thrombin binds to protease-activated receptors (PARs), which increase the expression of several inflammatory factors and angiogenic factors, such as VEGF or IL-8 [19,20,48]. These factors promote excess angiogenesis which results in abnormal uterine bleeding. pLARC-induced excess ZBTB16 exacerbates thrombininduced endometrial angiogenesis and inflammation by increasing IL8 and PTGS2 levels in HESCs Previous studies demonstrated that ZBTB16 is widely expressed in various tissues and displays diverse biological functions such as cell growth, self-renewal, and differentiation through the regulation of tissue-specific target genes [25,28]. In addition to the documented cellular and molecular roles of ZBTB16, in the female reproductive system, Fahnenstich et al. [35] initially reported elevated ZBTB16 expression in endometrial stromal and myometrial cells during the secretary vs. proliferative phase of the menstrual cycle, thereby revealing hormonal regulation of ZBTB16 in the human endometrium. Subsequently, Szwarc et al. [49] reported that ZBTB16 plays a crucial role during HESC decidualization by acting as a potential mediator of progesterone receptor actions. The current study expanded the potential role of ZBTB16 in the endometrium by investigating whether ZBTB16 contributes to pLARC-induced tissue-factor-mediated thrombin generation and the inflammatory milieu. Therefore, we initially evaluated ZBTB16 expression in pre-and post-DMPA-administered women and guinea pigs as a highly relevant model [9,11]. Consequentially, the results provided the first evidence that DMPA administration induces ZBTB16 expression in situ in endometrial stromal, glandular epithelial, as well as endothelial cells in both women and guinea pigs.
Decidualization represents the cellular transformation of fibroblast-like endometrial stromal cells into epithelial-like decidual cells and is essential for the establishment of a successful pregnancy [50]. Decidualization results from complex interactions of several key factors including steroid hormones, growth factors, signaling molecules, transcription  [8,9] and induces HESC decidualization, which increases tissue factor [15,23] and ZBTB16 levels. Increased ZBTB16 levels induce excess tissue factor expression, which generates thrombin. The resulting excess thrombin binds to protease-activated receptors (PARs), which increase the expression of several inflammatory factors and angiogenic factors, such as VEGF or IL-8 [19,20,48]. These factors promote excess angiogenesis, which results in abnormal uterine bleeding. pLARC-induced excess ZBTB16 exacerbates thrombininduced endometrial angiogenesis and inflammation by increasing IL8 and PTGS2 levels in HESCs.
Decidualization represents the cellular transformation of fibroblast-like endometrial stromal cells into epithelial-like decidual cells and is essential for the establishment of a successful pregnancy [50]. Decidualization results from complex interactions of several key factors including steroid hormones, growth factors, signaling molecules, transcription factors, etc., [50][51][52]. Here, we confirmed that both morphologically and functionally differentiated decidual cells display significantly elevated ZBTB16 levels during in vitro decidualization, as previously shown by Kommagani et al. [36]. Our investigation of the individual effect of in vitro decidualization stimuli by using E 2 , or MPA, or cAMP alone identified MPA (progesterone/glucocorticoid) as the responsible agent for the robust increase in ZBTB16 levels in decidualized HESCs, suggesting that ZBTB16 is both a progestinand glucocorticoid-induced transcription factor. This result was consistent with previous studies demonstrating ZBTB16 upregulation in a variety of cell types by progesterone [35], corticosteroids [53], testosterone [54], and aldosterone [55]. Taken together, these results indicate that ZBTB16 is a steroid-responsive transcription factor.
Our current results obtained from in the situ analysis of human and guinea pig endometria, as well as in vitro HESC decidualization led us to analyze ZBTB16 expression in HESCs treated with various progestins including ORG, ETO, LNG, and MPA, which are used in pLARC formulations or DEX (a pure glucocorticoid). Although all these compounds elevated ZBTB16 levels, robust induction was observed in HESCs following either DEX or MPA exposure, confirming that glucocorticoids display a stronger effect on the upregulation of ZBTB16 mRNA and protein levels than progestins. Among progestins, ETO displayed the greatest induction in ZBTB16 levels, indicating that ETO exhibits both glucocorticoid and progestogenic effects [56]. Depending on their derivative molecules, several synthetic progestins display mixed progestin-glucocorticoid actions by binding to the progesterone and glucocorticoid receptors in target cells [57]. Previously, Kommagani et al. [36] reported that progestin-mediated ZBTB16 induction in HESCs requires a progesterone receptor. However, the results of the current study indicated that this effect is primarily mediated by the glucocorticoid receptor rather than the progesterone receptor because the MPA-induced ZBTB16 levels were significantly diminished in glucocorticoid-receptor-silenced HESCs. This was further supported by a moderate induction of ZBTB16 by pure progestin ORG.
Our current study also confirmed MPA-mediated ZBTB16 upregulation in HEECs, which express the glucocorticoid, but not the progesterone receptor [42][43][44]. Our findings that ZBTB16 levels were significantly upregulated in HEECs following either DEX or MPA exposure, but not by ORG, ETO, or LNG treatment, further support glucocorticoidreceptor-mediated regulation of ZBTB16 expression. Several studies reported that increased levels of ZBTB16 expression induce apoptosis and inhibit VEGF-induced proliferation, thereby acting as an anti-angiogenic factor in human umbilical cord and corneal endothelial cell cultures [58][59][60]. Thus, pLARC-induced endothelial ZBTB16 expression may impair endometrial angiogenesis to contribute to the mechanism of pLARC-induced AUB.
The current study provided the first demonstration that ZBTB16 overexpression in cultured HESCs without decidual stimuli (EMC treatment) significantly increases the generation of tissue factor, which is specifically enhanced at bleeding sites vs. non-bleeding sites in the endometrium of pLARC users [14]. Moreover, thrombin-treated HESCs display increased levels of tissue factor, which is further elevated in ZBTB16-overexpressed HESCs, suggesting the mechanism whereby tissue factor elicits excess local thrombin generation, which stimulates VEGF expression, thereby exacerbating abnormal endometrial angiogenesis observed in the endometrium of pLARC users [19]. Moreover, the endometrium of pLARC users contains a higher number of neutrophils and NK cells [61,62] and displays elevated IL-8 levels [20]. In support, our results revealed that thrombin further increases IL8 and COX2 (PTGS2) levels in ZBTB16 overexpressed HESCs, indicating that elevated ZBTB16 levels promote thrombin-induced inflammation in pLARC users ( Figure 6) and providing the first demonstration of the combined inflammatory effect of thrombin and ZBTB16 in HESCs. Previous studies reported that thrombin induces the expression of several cytokines and chemokines in a variety of cell types [48,[63][64][65]. Moreover, ZBTB16 has been shown to display a wide range of cell-type-specific functions including cell proliferation, cell cycle regulation, apoptosis, stem cell renewal, and differentiation [66][67][68][69][70][71][72]. These wide-ranging ZBTB16-induced effects result from either an activator or repressor effect of ZBTB16 binding to either DNA-specific consensus sequences or other transcription factors, resulting in upregulation or downregulation of gene expression. Mao et al. [70] showed that ZBTB16 indirectly regulates the secretion of several cytokines such as IL-4, -13, -17, or IFNγ by binding to multiple T-helper-specific transcription factors in NKT cells. ZBTB16 can interfere with glucocorticoid-mediated transcription [71,73]. In the current study, the amplification of thrombin-induced IL8 and PTGS2 expression in ZBTB16-overexpressed HESCs supported the function of ZBTB16 to interfere with glucocorticoid-receptor-mediated suppression of inflammatory gene expression. However, further studies are required to verify this effect in HESCs. In summary, ZBTB16 displays diverse functions as a transcription factor and/or chromatin remodeler. Thus, it activates and/or represses target gene expression depending on physiological or pathological conditions. The current study uncovered the novel roles played by ZBTB16 by revealing that: (1) pLARC-induced ZBTB16 expression contributes to HESC decidualization; (2) increased ZBTB16 expression in the endometrium of pLARC-treated women is primarily glucocorticoid receptor mediated; (3) elevated ZBTB16 levels induce tissue factor, which elicits excess local thrombin generation, thereby contributing to AUB; and (4) thrombin treatment in ZBTB16-overexpressed HESCs further increases PTGS2 and IL-8 levels, thereby promoting endometrial inflammation in pLARC users. The limitation of our study is that most of our results were obtained from in vitro findings. Therefore, further in situ study is needed to test the role of ZBTB16 in pLARC-induced inflammation and AUB in higher primates.

Human Endometrial Tissue Collection
Banked paraffin sections of paired endometrial tissues obtained from women prior to DMPA injection (pre-DMPA, n = 7) and three months post-DMPA administration (n = 7) were used for immunohistochemical studies. Endometrial biopsies were obtained from women with regular menstrual cycles during the secretory phase pre-DMPA or post-DPMA administration. All samples were de-identified after receiving written informed consent at New York University under Institutional Review Board approval (#H6023) and later approval of the University of South Florida (#Pro00019480). Endometrial biopsies for primary cultures of HESCs and HEECs were obtained from healthy, reproductive-age women not receiving any hormonal/steroid treatment undergoing hysterectomy for benign disease. Endometrial tissues were collected after receiving written informed consent under the approval of the Yale University and the University of South Florida Human Investigation Committees.

Guinea Pig Endometrial Tissue Collection
Nulliparous female guinea pigs (aged 2-6 months) after bilateral ovariectomy were administrated subcutaneous implants (based on a 21-day time release) of placebo as the control (n = 5), or 5 mg estradiol (E 2 ; n = 4), or 50 mg MPA (n = 5), or in combination of 5 mg E 2 with 50 mg MPA (n = 6) pellets purchased from Innovative Research of America (Sarasota, FL, USA). After three weeks, a hysterectomy was performed, and the right uterine horn was formally fixed for immunohistochemical studies. These studies were performed under the approval of the Yale Institutional Animal Care and Use Committee (IACUC#2006-11002).

Immunohistochemistry
ZBTB16 immunostaining was performed on human endometrial sections obtained from women before (pre-) and post-DMPA administration, as well as endometrial tissues obtained from guinea pigs administered with placebo, E 2 , MPA, or E 2 + MPA, as described [11]. Briefly, 5 µm-thick paraffin embedded sections were deparaffinized in xylene and rehydrated in a descending ethanol series. Antigen retrieval was performed in 10 mM citric acid solution (pH 6.0) by boiling in a microwave for 20 min. Subsequently, slides were rinsed in Tris-buffered saline with 0.1% tween-20 (TBS-T) and incubated in 3% hydrogen peroxide for 12 min at room temperature for endogenous peroxidase quenching. Thereafter, sections were blocked for 30 min at room temperature with 5% normal goat serum (Vector Labs, Burlingame, CA, USA) in TBS-T, then incubated with rabbit polyclonal anti-ZBTB16 antibody (1/150 dilution; Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 • C. After the washing steps in TBS-T, the slides were incubated with biotinylated goat anti-rabbit IgG (1/400 dilution, Vector Labs) in TBS-T for 30 min, then with streptavidin-conjugated peroxidase complex (Vector Labs) for 30 min. Following several TBT-T rinses, immunoreactivity was developed using 3,3 -diaminobenzidine kits (Vector Labs), and the slides were counterstained with hematoxylin. As a negative control, non-immune rabbit IgG was used at the same concentration as the primary antibody. ZBTB16 immunoreactivity was assessed by histologic score (HSCORE) analysis, a semiquantitative method that evaluates the intensity and number of immunostained cells, as described [23], by two independent investigators (S.A., N.S.) blinded to the source of the samples.

Quantitative Real-Time PCR
Total RNA obtained from cultured HESCs or HEECs was isolated using the RNeasy mini kit according to the manufacturer's instructions (Qiagen, Germantown, MD, USA). Then 500 ng of total RNA was used for reverse transcription, which was performed using the RETROscript kit (Ambion, Austin, TX, USA) using random decamer primers at 42 • C for 1 h, followed by 92 • C for 10 min for enzyme inactivation. The expression of target genes including ZBTB16, PRL, IGFBP1, F3, IL8, and PTGS2 was determined by using a specific TaqMan gene expression assay (Life Technologies; see Table S1). Amplification used 40 cycles of PCR in an ABI 7500 System (Applied Biosystems, Foster City, CA, USA), with the following program: initial denaturation at 95 • C for 10 min, followed by 40 cycles at 95 • C for 15 s and 60 • C for 60 s. Each reaction was performed in triplicate and the average used for each sample. The relative expression for each target gene was normalized with β-Actin as a reference gene and calculated using the comparative Ct method (2 −∆∆Ct ).

Immunoblotting Analyses
Total protein was isolated from cell lysates obtained from HESCs incubated with E 2 , or E 2 + ORG, or ETO, or LNG, or MPA, or DEX for 24 h using lysis buffer (Cell Signaling Inc., Danvers, MA, USA) containing a protease inhibitor cocktail. The protein concentration was determined by a detergent-compatible protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Then, 20 µg of total protein was diluted in 2× sample buffer (Bio-Rad) and then boiled for 5 min. Samples were then separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Bio-Rad) and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked overnight with 10% non-fat dry milk in TBS-T and then incubated overnight at 4 • C with mouse anti-ZBTB16 monoclonal antibody (ThermoFisher, Waltham, MA, USA). Following washing in TBS-T, the membrane was incubated with peroxidase-conjugated anti-horse IgG at a 1/5000 dilution (Vector Labs). The signals were developed using a chemiluminescence kit (Amersham; GE Healthcare, Piscataway, NJ, USA). The membrane was then sequentially stripped and re-probed with peroxidase-conjugated anti-β-actin rabbit monoclonal antibody at 1/1000 dilution (Cell Signaling Inc.).

Transient Transfection of ZBTB16 Overexpression
To overexpress ZBTB16 in HESCs, the expression vector containing the human fulllength ZBTB16 gene open reading frame (pCMV6-ZBTB16) was purchased from Origene Inc. (Rockville, MD, USA). Cultured HESCs seeded in 6-well plates were transiently transfected with either the ZBTB16 or empty (pCMV6 vector as the control) vector using the Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA, USA) in Opti-MEM serum reduced medium (Invitrogen) according to the manufacturer's instructions. After transfection for 4 h, HESCs were incubated with/without EMC media for 72 h. In a parallel set, ZBTB16or control-vector-transfected HESCs cultures ± EMC were treated with 1 U/mL of thrombin (American Diagnostic, Greenwich, CT, USA) for 6 h and stored at −80 • C for RNA isolation. The transfection efficiency for ZBTB16 overexpression was confirmed by qPCR and immunoblotting.

siRNA-Mediated Silencing of Glucocorticoid Receptor
To silence the expression of the glucocorticoid-receptor-encoded NR3C1 gene, HESCs were transfected with either NR3C1-specific siRNA or non-specific siRNA (Invitrogen) as the control using the RNAiMax Reagent according to the manufacturer's instructions (Invitrogen). Four hours after transfection, cultured HESCs were incubated with EMC media for 72 h. The transfection efficiency for glucocorticoid receptor silencing was confirmed by qPCR by measuring the mRNA levels of the NR3C1 and FKBP5 genes as a glucocorticoid-receptor-regulated gene.

Statistical Analysis
The results from ZBTB16 immunostaining in guinea pig endometria, or immunoblotting, or qPCR were each normally distributed as determined by the Kolmogorov-Smirnov test and analyzed by one-way ANOVA, followed by the post hoc Holm-Sidak method or Student-Newman-Keuls method. HSCOREs obtained from the immunostaining of human endometrial tissues from pre-and post-DMPA administration were compared using a t-test. p < 0.05 was considered statistically significant. The statistical calculations used the SigmaStat Version 3.0 software (Systat Software, San Jose, CA, USA).  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data supporting the results of the current study are available from the corresponding author upon request.

Conflicts of Interest:
The authors declare that no conflicts of interest exist.