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Cited by 44Endometriosis Research and TreatmentEstrogen and related hormone effectsPlatelet Disorders and Treatments

Platelets induce increased estrogen production through NF-κB and TGF-β1 signaling pathways in endometriotic stromal cells

Open access
Qi, Qiuming, Liu, Xishi, Zhang, Qi, Guo, Sun-Wei·2020·Scientific Reports
DOI 10.1038/s41598-020-57997-6PMC PMC6987096
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Abstract

Endometriosis is estrogen-dependent disorder. Two theories provide the explanations for the increased estrogen production. One is the feed-forward loop model linking inflammation and estrogen production. The more recent model evokes the tissue hypoxia resulting from endometrial debris detached and then regurgitated to the peritoneal cavity. Both models tacitly assume that everything occurs within the endometriotic stromal cells, seemingly without the need for exogenous factors. This study was undertaken to investigate as whether platelets may be responsible for local estrogen overproduction. We employed in vitro experimentation that evaluated the 17β-estradiol (E 2 ) levels in endometriotic stromal cells treated with activated platelets, and the genes and protein expression levels of StAR, HSD3B2, aromatase, and HSD17B1, as well as their upstream genes/proteins such as NF-κB, TGF-β1, HIF-1α, SF-1 and phosphorylated CREB. In addition, we conducted 2 animal experimentations using platelet depletion/infusion and also neutralization of NF-κB and TGF-β1, followed by immunohistochemistry analysis of involved in StAR, HSD3B2, aromatase, and HSD17B1, as well as SF-1 and p-CREB. We found that treatment of endometriotic stromal cells by activated platelets increase the E 2 production by 4.5 fold, and concomitant with increased gene and protein expression of StAR, HSD3B2, aromatase, and HSD17B1, the four genes/enzymes important to estrogen synthesis, along with their upstream genes HIF-1α, SF-1 and phosphorylated CREB. Moreover, platelets activate these genes through the activation of NF-κB and/or TGF-β1, and antagonism of either signaling pathway can abolish the induction of the 4 genes and thus increased estrogen production. The two animal experimentations confirmed these changes. Thus, platelets increase the E 2 production in endometriotic stromal cells through upregulation of StAR, HSD3B2, aromatase, and HSD17B1 via the activation of NF-κB and/or TGF-β1. These findings provide a yet another compelling piece of evidence that endometriotic lesions are indeed wounds undergoing repeated tissue injury and repair. They strongly indicate that non-hormonal therapeutics for endometriosis is theoretically viable, with anti-platelet therapy being one promising avenue.

AI snapshot

Written from the paper’s full text.

TL;DR

Activated platelets increased 17β-estradiol (E2) production in human endometriotic stromal cells by upregulating StAR, HSD3B2, aromatase, and HSD17B1, largely through NF-κB and/or TGF-β1 signaling. Mouse experiments using platelet infusion/depletion and NF-κB or TGF-β1 antagonism supported these findings and suggested anti-platelet or non-hormonal therapeutic avenues.

Key findings

  • Activated platelets increased E2 production in HESCs by 4.5-fold versus PBS (119.16 pg/mL vs. 26.32 pg/mL; p = 0.018), while platelets alone increased it 3.0-fold (79.15 pg/mL); thrombin alone did not (p = 0.87).
  • Activated platelets upregulated StAR, HSD3B2, aromatase, and HSD17B1 at mRNA and protein levels, with protein fold increases of 1.6, 1.3, 1.5, and 1.3, respectively (product 4.1, close to the 4.5-fold E2 increase); SF-1 and p-CREB protein were also significantly higher (all p = 0.012).
  • NF-κB neutralization with JSH-23 abolished platelet-induced E2 production and steroidogenic gene/protein expression; TGF-β1 neutralization with A83-01 abolished platelet-induced HIF-1α, COX-2, and VEGF and steroidogenic gene expression, though E2 remained higher than vehicle (p = 0.012) and StAR protein remained higher (p = 0.012).
  • In mice, platelet infusion increased total lesion weight by 52.4% (p = 0.025) and platelet depletion decreased it by 40.2% (p = 0.017); PI mice had higher and PD mice lower immunostaining for StAR, HSD3B2, aromatase, HSD17B1, SF-1, and p-CREB (all p’s < 0.032). Antagonism of TGF-β1 or NF-κB reduced lesion weight by 65.7% and 34.3%, respectively (p = 0.014 and p = 0.004), and reduced platelet aggregation and staining of the six markers (all p’s < 0.007).

Methods

  • In vitro: primary human endometriotic stromal cells (HESCs) from 15 premenopausal patients with ovarian endometriomas (mean age 32.2 ± 5.2 years) and an endometrial stromal cell line (ESCL) were co-cultured for 48 hours with PBS, platelets, activated platelets, or thrombin; some HESCs were pretreated with JSH-23 (NF-κB inhibitor) or A83-01 (TGF-β receptor inhibitor). Outcomes were measured by ELISA, real-time PCR, and Western blotting.
  • In vivo: 90 virgin female Balb/C mice were used in two experiments. One experiment used platelet infusion, platelet depletion, or non-immune antibody (n = 10 per group); the other used vehicle, SB431542 (TGF-β1 inhibitor), or JSH-23 (NF-κB inhibitor) (n = 10 per group). Lesions were assessed by hotplate latency, lesion weight, and immunohistochemistry for StAR, HSD3B2, aromatase, HSD17B1, SF-1, p-CREB, and CD41.
  • Statistics: paired Wilcoxon’s test for in vitro comparisons; Wilcoxon’s test, Kruskal’s test, Pearson’s correlation, and multiple linear regression for mouse data; p < 0.05 considered significant.

Limitations

  • The paper states it did not evaluate all genes possibly involved in estrogen biosynthesis, focusing mainly on StAR, aromatase, HSD3B2, and HSD17B1 plus upstream HIF-1α, SF-1, and CREB; other genes/enzymes such as AMPK were not assessed.
  • The paper states it did not fully elucidate the exact molecular mechanisms, nor exactly how platelets activate NF-κB and/or TGF-β1.
  • The paper states the in vitro study used only ovarian endometrioma tissue samples, raising the question of whether conclusions apply to all endometriosis subtypes; it also notes small sample sizes may have limited detection of other genes such as CYP11A1 and CYP17A1.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41598-020-57997-6

Cites

110 of 110 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 103 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In the absence of endometriotic or endometrial cells, E2 concentration in medium cultured with or without activated platelets for 48 hours was barely detectable in all groups.

    “we also evaluated, in the absence of endometriotic or endometrial cells, the E2 concentration in the medium that was cultured with or without activated platelets for 48 hours and only found barely detectable E2 in all groups (data not shown)”
    Quote in the paperSubstantial claim
  • Findingown data

    E2 concentrations were very low in normal endometrial stromal cell line (ESCL) irrespective of co-culture with or without activated platelets.

    “In contrast, the E2 concentrations were all very low in the normal endometrial stromal cell line (ESCL), irrespective co-culture with or without activated platelets (Fig. 1A, right panel).”
    Quote in the paperSubstantial claim
  • Findingown data

    Neutralization of NF-κB by JSH-23 nearly completely abolished the activated platelet-induced upregulation of HIF-1α, COX-2, and VEGF (all p≤0.036).

    “Neutralization of NF-κB by JSH-23 nearly completely abolished this upregulation (all p’s ≤ 0.036, Fig. 2C–E).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: JSH-23 specifically neutralizes NF-κB without off-target effects.
  • Findingown data

    With the exception of p-CREB protein levels, gene and protein expression levels of StAR, HSD3B2, aromatase, HSD17B1, SF-1, and p-CREB in HESCs co-cultured with platelets but pre-treated with JSH-23 were no different from controls (all p>0.05).

    “With the only exception of p-CREB protein levels, the gene and protein expression levels of these genes in HESCs co-cultured with platelets but pre-treated with JSH-23 were no different from controls (all p’s > 0.05, Fig. 3B,D–F).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: JSH-23 specifically neutralizes NF-κB without off-target effects.
  • Findingown data

    NF-κB neutralization by JSH-23 completely abolished platelet-induced expression of StAR, HSD3B2, aromatase, and HSD17B1 at both transcriptional and protein levels (all p≤0.025).

    “Consistently, NF-κB neutralization by pre-treatment with JSH-23 completely abolished platelets-induced expression of StAR, HSD3B2, aromatase and HSD17B1 at both transcriptional and protein levels (all p-values ≤ 0.025, Fig. 3B–D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: JSH-23 specifically neutralizes NF-κB without off-target effects.
  • May combine two claims. 43
  • Classified as probably not a substantial claim. 45

  • May combine two claims.

    Findingown data

    In HESCs, co-culture with platelets or activated platelets for 48 hours significantly elevated E2 concentration in supernatant compared with PBS (both p=0.018), while thrombin alone did not (p=0.87).

    “We found that, compared with those treated with PBS, the E2 concentration was significantly elevated in HESCs co-cultured with either platelets or activated platelets (both p-values = 0.018), but with thrombin alone (p = 0.87, Fig. 1A, left panel).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In HESCs co-cultured with activated platelets, but not thrombin alone, mRNA levels of StAR, HSD3B2, aromatase, and HSD17B1 were significantly higher than vehicle (all p≤0.028), while CYP11A1 and CYP17A1 were not.

    “the mRNA levels of StAR, HSD3B2, aromatase and HSD17B1, but not CYP11A1 or CYP17A1, in HESC cells co-cultured with activated platelets, but not thrombin alone (all p’s > 0.20), were all significantly higher than those treated with vehicle (all p’s ≤ 0.028, Fig. 1B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    TGF-β1 neutralization by A83-01 completely abolished platelet-induced expression of StAR, HSD3B2, aromatase, and HSD17B1 at both transcriptional and protein levels (all p≤0.025), with the exception of StAR protein levels which were still significantly higher than control (p=0.012).

    “Consistently, TGF-β1 neutralization by pre-treatment with A83-01 completely abolished platelets-induced expression of StAR, HSD3B2, aromatase and HSD17B1 at both transcriptional and protein levels (all p’s ≤ 0.025), with the only exception of the StAR protein levels which were still significantly higher than the control (p = 0.012, Fig. 5B–D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: A83-01 specifically neutralizes TGF-β1 without off-target effects.

  • May combine two claims.

    Findingown data

    In endometriotic lesions, StAR and aromatase staining were primarily localized in the cell cytoplasm of stromal cells, but not epithelial cells; HSD3B2 and HSD17B1 staining were seen both in epithelial and stromal cells and localized in cytoplasm; SF-1 and p-CREB staining were seen in both epithelial and stromal cells and localized in nucleus.

    “We found that StAR and aromatase staining were primarily localized in the cell cytoplasm of stromal cells, but not epithelial cells. HSD3B2 and HSD17B1 stain were seen both in epithelial cells and stromal cells and localized in the cell cytoplasm. SF-1 and p-CREB stain were seen in both epithelial and stromal cells and localized in the cell nucleus.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Pre-treatment of HESCs with JSH-23 significantly reduced E2 production in culture media compared with without pre-treatment (p=0.036) without reducing cell viability, and completely abolished E2 production induced by platelets (p=0.13).

    “We found that pre-treatment of HESCs with JSH-23 significantly reduced the E2 production in culture media as compared with that without the pre-treatment (p = 0.036) without reducing the cell viability (Supplementary Fig. S1), and, in fact, completely abolished the E2 production induced by platelets (p = 0.13, Fig. 3A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: JSH-23 specifically neutralizes NF-κB without off-target effects.

  • May combine two claims.

    Findingown data

    PI mice had significantly shorter hotplate latency (p=0.018), while PD mice had significantly longer latency (p=0.0055) than control NI mice.

    “In particular, PI mice had significantly shorter (p = 0.018), while PD mice had significantly longer (p = 0.0055), latency than the control mice injected with a matched non-immune (NI) antibody.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In mice with induced endometriosis, there was no difference in hotplate latency among the 3 groups 2 days prior to induction (p=0.98), but 12 days after induction the difference was highly significant (p=0.0001).

    “We found that, while there was no difference in hotplate latency among the 3 groups of mice 2 days prior to the induction of endometriosis (p = 0.98, Fig. 6A), but 12 days after the induction the difference was highly significant (p = 0.0001, Fig. 6A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Compared with NI mice, total lesion weight in PI mice increased by an average of 52.4% (p=0.025), while that of PD mice decreased by 40.2% (p=0.017).

    “In addition, compared with the NI mice that received a mock antibody, the total lesion weight in PI mice increased by an average of 52.4% (p = 0.025, Fig. 6B), while that of PD mice was decreased by 40.2% (p = 0.017, Fig. 6B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pre-treatment of HESCs with A83-01 significantly reduced E2 production compared with without pre-treatment (p=0.017) without reducing cell viability, but resultant E2 levels were still significantly higher than vehicle (p=0.012).

    “We found that pre-treatment of HESCs with A83-01 significantly reduced the E2 production in culture media as compared with that without the pre-treatment (p = 0.017) without reducing the cell viability (Supplementary Fig. S1), but the resultant E2 levels were still significantly higher than those pre-treated with vehicle (p = 0.012, Fig. 5A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: A83-01 specifically neutralizes TGF-β1 without off-target effects.

  • May combine two claims.

    Findingown data

    By multiple linear regression, immunostaining levels of StAR, HSD3B2, aromatase, HSD17B1, SF-1, and p-CREB were significantly higher in PI group but significantly lower in PD group compared with controls (all p<0.032, R2≥0.38).

    “we found, by multiple linear regression analysis, significantly higher immunostaining levels of StAR, HSD3B2, aromatase, HSD17B1, SF-1 and p-CREB in the PI group but significantly lower staining levels of all these proteins in the PD group as compared with controls (all p’s < 0.032, R2 ≧ 0.38, Fig. 6C–H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expressions of HIF-1α, COX-2, and VEGF were significantly elevated after co-culture with activated platelets at both transcriptional and protein levels (all p≤0.012), but the upregulation was completely abolished by TGF-β1 neutralization with A83-01 (all p≤0.012).

    “We found that, at both transcriptional and protein expression levels, the expressions of HIF-1α, COX-2 and VEGF were significantly elevated after co-culture with activated platelets (all p’s ≤ 0.012, Fig. 4A–C), but the upregulation was completely abolished by TGF-β1 neutralization with pre-treatment with A83-01 (all p’s ≤ 0.012, Fig. 4A–C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: A83-01 specifically neutralizes TGF-β1 without off-target effects.

  • May combine two claims.

    Findingown data

    In HESCs co-cultured with platelets or activated platelets, but not thrombin alone, protein levels of StAR, HSD3B2, aromatase, and HSD17B1 were significantly higher than vehicle (all p≤0.017).

    “The protein levels of StAR, HSD3B2, aromatase and HSD17B1 in HESCs co-cultured with platelets or activated platelets, but not thrombin alone, were also significantly higher than that that were co-cultured with vehicle (all p’s ≤ 0.017, Fig. 1C,D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The changes of StAR, HSD3B2, aromatase, HSD17B1, SF-1, and p-CREB in NI group were more than PD group, but less than PI group.

    “The changes of StAR, HSD3B2, aromatase, HSD17B1, SF-1 and p-CREB in NI group were obviously more as compared with PD group, but obviously less than in PI group.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Activated platelets increased protein expression levels of StAR, HSD3B2, aromatase, and HSD17B1 by an average of 1.6, 1.3, 1.5, and 1.3 fold, respectively.

    “the activated platelets increased the protein expression levels of StAR, HSD3B2, aromatase, and HSD17B1 by an average of 1.6, 1.3, 1.5, and 1.3 fold, respectively (Fig. 1C,D)”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    The result of 4.5 fold increase in estrogen production in endometriotic stromal cells stimulated with activated platelets vs. those unstimulated is remarkably close to the reported fold increase found between testosterone-added endometriotic and endometrial stromal cells (4.1 for follicular phase and 4.5 for luteal phase).

    “The result of 4.5 fold increase in estrogen production in endometriotic stromal cells stimulated with activated platelets vs. those unstimulated is remarkably close to the reported fold increase found between testosterone-added endometriotic and endometrial stromal cells (4.1 for follicular phase and 4.5 for luteal phase)41.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Izawa, M. et al. An epigenetic disorder may cause aberrant expression of aromatase gene in endometriotic stromal cells. Fertil. Steril. 89, 1390–1396, https://doi.org/10.1016/j.fertnstert.2007.03.078 (2008). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Compared with vehicle-treated mice, mice treated with either SB431542 or JSH-23 had significantly reduced platelet aggregation and immunostaining levels of StAR, HSD3B2, aromatase, HSD17B1, SF-1, and p-CREB in the stromal component of endometriotic lesions (all p<0.007).

    “We can see that, compared with mice treated with vehicle, mice treated with either SB431542 or JSH-23 had significantly reduced platelet aggregation, and immunostaining levels of StAR, HSD3B2, aromatase, HSD17B1, SF-1 and p-CREB in the stromal component of endometriotic lesions (all p’s < 0.007, Fig. 7C–I).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: JSH-23 specifically neutralizes NF-κB without off-target effects.
    • Rests on an assumption: SB431542 specifically inhibits TGF-β1 without off-target effects.

  • May combine two claims.

    Findingown data

    Platelets induce HIF-1α, SF-1 and p-CREB.

    “Platelets also induce HIF-1α, SF-1 and p-CREB”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Total lesion weight in mice treated with inhibitors of either TGF-β1 or NF-κB was reduced by an average of 65.7% and 34.3%, respectively (p=0.014 and 0.004), compared with vehicle.

    “In addition, the total lesion weight in mice treated with inhibitors of either TGF-β1 or NF-κB was reduced by an average of 65.7% and 34.3%, respectively (p = 0.014 and 0.004, respectively, Fig. 7B) as compared with that in those treated with vehicle.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: JSH-23 specifically neutralizes NF-κB without off-target effects.
    • Rests on an assumption: SB431542 specifically inhibits TGF-β1 without off-target effects.

  • May combine two claims.

    Findingown data

    In the second mouse experiment, there was no difference in hotplate latency among the 3 groups 2 days prior to induction (p=0.67), but the difference was highly significant 12 days after induction (p=0.0046).

    “we found that, while there was no difference in hotplate latency among the 3 groups of mice 2 days prior to the induction of endometriosis, the difference was highly significant 12 days after the induction (p = 0.67 and p = 0.0046, respectively, Fig. 7A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Activated platelets can directly induce a hypoxia state in endometriotic lesions, which subsequently cause further phenotypic changes.

    “Third, our study provides evidence that activated platelets can directly induce a hypoxia state in endometriotic lesions, which subsequently cause further phenotypic changes39.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [39] Wu, M. H., Hsiao, K. Y. & Tsai, S. J. Hypoxia: The force of endometriosis. J Obstet Gynaecol Res, https://doi.org/10.1111/jog.13900 (2019). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In a gene expression profiling study of wound tissues from young and elder men, 78% of differentially expressed genes were estrogen-regulated and only 3% were age-related.

    “One gene expression profiling study of wound tissues from young and elder men found that among genes that were differentially expressed, 78% of them being estrogen-regulated and only 3% being age-related34”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Hardman, M. J. & Ashcroft, G. S. Estrogen, not intrinsic aging, is the major regulator of delayed human wound healing in the elderly. Genome Biol. 9, R80, https://doi.org/10.1186/gb-2008-9-5-r80 (2008). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the feed-forward model, increased E2 further induces ERβ, yielding further induction of COX-2.

    “The increased E2 further induces estrogen receptor β (ERβ), yielding further induction of COX-2.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Bulun, S. E. et al. Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. Pharmacol. Rev. 57, 359–383, https://doi.org/10.1124/pr.57.3.6 (2005). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Platelets can induce HIF-1α expression, effectively inducing a hypoxic state in both endometriotic and endometrial stromal cells.

    “we recent found that platelets can induce hypoxia-inducible factor 1α (HIF-1α) expression, effectively inducing a hypoxic sate in both endometriotic and endometrial stromal cells40.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [40] Qi, Q., Guo, S.-W. & Liu, X. Activated Platelets Induce Hypoxia-Inducible Factor-1α Expression Likely through Transforming Growth Factor-β1 in Human Endometrial Stromal Cells. Reprod Dev Med In press. (2019). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    PGE2 activates PKA signaling via raising intracellular cAMP, which can enhance binding of SF-1 to promoters of steroidogenic genes and induce phosphorylation of CREB.

    “PGE2 activates the protein kinase A (PKA) signaling pathway via raising the intracellular levels of cyclic adenosine 3′,5′-monophosphate (cAMP)12–14, which can enhance the binding of steroidogenic factor-1 (SF-1) to promoters of these steroidogenic genes15, and induce phosphorylation of the transcriptional activator cAMP-response element-binding protein (CREB)16.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [12] Noble, L. S. et al. Prostaglandin E2 stimulates aromatase expression in endometriosis-derived stromal cells. J. Clin. Endocrinol. Metab. 82, 600–606, https://doi.org/10.1210/jcem.82.2.3783 (1997). source

      Not checked in the free run.

    • cites [13] Yang, S. et al. Regulation of aromatase P450 expression in endometriotic and endometrial stromal cells by CCAAT/enhancer binding proteins (C/EBPs): decreased C/EBPbeta in endometriosis is associated with overexpression of aromatase. J. Clin. Endocrinol. Metab. 87, 2336–2345, https://doi.org/10.1210/jcem.87.5.8486 (2002). source

      Not checked in the free run.

    • cites [14] Breyer, R. M., Bagdassarian, C. K., Myers, S. A. & Breyer, M. D. Prostanoid receptors: subtypes and signaling. Annu. Rev. pharmacology Toxicol. 41, 661–690, https://doi.org/10.1146/annurev.pharmtox.41.1.661 (2001). source

      Not checked in the free run.

    • cites [15] Bulun, S. E. et al. Steroidogenic factor-1 and endometriosis. Mol. Cell Endocrinol. 300, 104–108, https://doi.org/10.1016/j.mce.2008.12.012 (2009). source

      Not checked in the free run.

    • cites [16] Hsu, C. C., Lu, C. W., Huang, B. M., Wu, M. H. & Tsai, S. J. Cyclic adenosine 3′,5′-monophosphate response element-binding protein and CCAAT/enhancer-binding protein mediate prostaglandin E2-induced steroidogenic acute regulatory protein expression in endometriotic stromal cells. Am. J. Pathol. 173, 433–441, https://doi.org/10.2353/ajpath.2008.080199 (2008). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Binding of SF-1 and CREB to promoters of steroidogenic genes is responsible for inducing expression and activity of these enzymes, thus promoting estrogen biosynthesis in endometriotic stromal cells.

    “The binding of SF-1 and CREB to the promoters of steroidogenic genes is responsible for inducing the expression levels and activity of these enzymes, thus promoting the estrogen biosynthesis in endometriotic stromal cells10,17,18.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [10] Bulun, S. E. et al. Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. Pharmacol. Rev. 57, 359–383, https://doi.org/10.1124/pr.57.3.6 (2005). source

      Not checked in the free run.

    • cites [17] Sun, H. S., Hsiao, K. Y., Hsu, C. C., Wu, M. H. & Tsai, S. J. Transactivation of steroidogenic acute regulatory protein in human endometriotic stromalcells is mediated by the prostaglandin EP2 receptor. Endocrinol. 144, 3934–3942, https://doi.org/10.1210/en.2003-0289 (2003). source

      Not checked in the free run.

    • cites [18] Zeitoun, K. M. & Bulun, S. E. Aromatase: a key molecule in the pathophysiology of endometriosis and a therapeutic target. Fertil. Steril. 72, 961–969, https://doi.org/10.1016/s0015-0282(99)00393-3 (1999). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Estrogen is important to wound healing, and its deficiency delays or impairs wound healing.

    “Numerous studies have shown that estrogen is important to wound healing, and its deficiency delays or impairs wound healing29–33.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [29] Ashcroft, G. S. et al. Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels. Nat. Med. 3, 1209–1215 (1997). source

      Not checked in the free run.

    • cites [30] Ashcroft, G. S. et al. Estrogen modulates cutaneous wound healing by downregulating macrophage migration inhibitory factor. J. Clin. Invest. 111, 1309–1318, https://doi.org/10.1172/JCI16288 (2003). source

      Not checked in the free run.

    • cites [31] Hardman, M. J., Emmerson, E., Campbell, L. & Ashcroft, G. S. Selective estrogen receptor modulators accelerate cutaneous wound healing in ovariectomized female mice. Endocrinol. 149, 551–557, https://doi.org/10.1210/en.2007-1042 (2008). source

      Not checked in the free run.

    • cites [32] Pepe, G. et al. Self-renewal and phenotypic conversion are the main physiological responses of macrophages to the endogenous estrogen surge. Sci. Rep. 7, 44270, https://doi.org/10.1038/srep44270 (2017). source

      Not checked in the free run.

    • cites [33] Mukai, K. et al. 17beta-Estradiol administration promotes delayed cutaneous wound healing in 40-week ovariectomised female mice. Int. wound J. 13, 636–644, https://doi.org/10.1111/iwj.12336 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Estrogen is involved in all phases of wound healing.

    “In fact, estrogen is found to be involved in all phases of wound healing27.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Horng, H. C. et al. Estrogen Effects on Wound Healing. Int J Mol Sci 18, https://doi.org/10.3390/ijms18112325 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The feed-forward model proposed by Bulun et al. posits that proinflammatory cytokines activate COX-2, resulting in increased PGE2, which stimulates StAR, aromatase, and HSD17B1, resulting in elevated E2.

    “the feed-forward model proposed by Bulun et al.10, in which proinflammatory cytokines activate cyclooxygenase-2 (COX-2), resulting in increased production of prostaglandin E2 (PGE2), which, in turn, stimulates some key genes involved in the production of E2, such as steroidogenic acute regulatory protein (StAR), aromatase and 17β-hydroxysteroid dehydrogenase type-1 (HSD17B1), resulting in elevated”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Bulun, S. E. et al. Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. Pharmacol. Rev. 57, 359–383, https://doi.org/10.1124/pr.57.3.6 (2005). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Endometriosis is a pelvic inflammatory condition featuring increased production of proinflammatory cytokines and chemokines.

    “It is also commonly viewed as a pelvic inflammatory condition, featuring increased production of proinflammatory cytokines and chemokines11.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Vercellini, P., Vigano, P., Somigliana, E. & Fedele, L. Endometriosis: pathogenesis and treatment. Nat. Rev. Endocrinol. 10, 261–275, https://doi.org/10.1038/nrendo.2013.255 (2014). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Endometriosis features estrogen-dependent growth and maintenance of ectopic endometrium and increased local production of estrogens due to molecular aberrations in steroidogenesis.

    “Endometriosis has been viewed as a ultimate hormonal disease9, featuring estrogen-dependent growth and maintenance of ectopic endometrium as well as increased local production of estrogens due to molecular aberrations in steroidogenesis10.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [9] Gurates, B. & Bulun, S. E. Endometriosis: the ultimate hormonal disease. Semin. Reprod. Med. 21, 125–134, https://doi.org/10.1055/s-2003-41319 (2003). source

      Not checked in the free run.

    • cites [10] Bulun, S. E. et al. Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. Pharmacol. Rev. 57, 359–383, https://doi.org/10.1124/pr.57.3.6 (2005). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ERβ plays a critical role in wound healing.

    “ERβ has been shown to play a critical role in wound healing37,38.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] Merlo, S., Frasca, G., Canonico, P. L. & Sortino, M. A. Differential involvement of estrogen receptor alpha and estrogen receptor beta in the healing promoting effect of estrogen in human keratinocytes. J. Endocrinol. 200, 189–197, https://doi.org/10.1677/JOE-08-0442 (2009). source

      Not checked in the free run.

    • cites [38] Campbell, L. et al. Estrogen promotes cutaneous wound healing via estrogen receptor beta independent of its antiinflammatory activities. J. Exp. Med. 207, 1825–1833, https://doi.org/10.1084/jem.20100500 (2010). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The difference in E2 production between HESCs and ESCLs is consistent with what has been documented previously.

    “The difference in E2 production between HESCs and ESCLs is consistent with what have been documented previously41–45.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [41] Izawa, M. et al. An epigenetic disorder may cause aberrant expression of aromatase gene in endometriotic stromal cells. Fertil. Steril. 89, 1390–1396, https://doi.org/10.1016/j.fertnstert.2007.03.078 (2008). source

      Not checked in the free run.

    • cites [42] Izawa, M. et al. Demethylation of a nonpromoter cytosine-phosphate-guanine island in the aromatase gene may cause the aberrant up-regulation in endometriotic tissues. Fertil. Steril. 95, 33–39, https://doi.org/10.1016/j.fertnstert.2010.06.024 (2011). source

      Not checked in the free run.

    • cites [43] Zeitoun, K. et al. Deficient 17beta-hydroxysteroid dehydrogenase type 2 expression in endometriosis: failure to metabolize 17beta-estradiol. J. Clin. Endocrinol. Metab. 83, 4474–4480, https://doi.org/10.1210/jcem.83.12.5301 (1998). source

      Not checked in the free run.

    • cites [44] Xue, Q. et al. Transcriptional activation of steroidogenic factor-1 by hypomethylation of the 5′ CpG island in endometriosis. J. Clin. Endocrinol. Metab. 92, 3261–3267, https://doi.org/10.1210/jc.2007-0494 (2007). source

      Not checked in the free run.

    • cites [45] Attar, E. et al. Prostaglandin E2 via steroidogenic factor-1 coordinately regulates transcription of steroidogenic genes necessary for estrogen synthesis in endometriosis. J. Clin. Endocrinol. Metab. 94, 623–631, https://doi.org/10.1210/jc.2008-1180 (2009). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ERβ is overexpressed in endometriotic lesions.

    “in striking similarity to endometriotic lesions in which ERβ is shown to be overexpressed35,36”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [35] Brandenberger, A. W. et al. Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal endometrial and endometriosis-derived stromal cells. Mol. Hum. Reprod. 5, 651–655, https://doi.org/10.1093/molehr/5.7.651 (1999). source

      Not checked in the free run.

    • cites [36] Fujimoto, J., Hirose, R., Sakaguchi, H. & Tamaya, T. Expression of oestrogen receptor-alpha and -beta in ovarian endometriomata. Mol. Hum. Reprod. 5, 742–747, https://doi.org/10.1093/molehr/5.8.742 (1999). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Activated platelets induce the activation of the NF-κB signaling pathway and TGF-β1/Smad3 signaling pathway in endometriotic lesions.

    “Activated platelets induce the activation of the NF-κB signaling pathway and TGF-β1/Smad3 signaling pathway in endometriotic lesions21.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Zhang, Q., Duan, J., Liu, X. & Guo, S. W. Platelets drive smooth muscle metaplasia and fibrogenesis in endometriosis through epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation. Mol. Cell Endocrinol. 428, 1–16, https://doi.org/10.1016/j.mce.2016.03.015 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The two signaling pathways regulate several genes coding for hypoxia and inflammatory molecules, such as HIF-1α, COX-2 and VEGF, which are upregulated after platelets treatment in HESCs.

    “The two signaling pathways regulate several genes coding for hypoxia and inflammatory molecules, such as HIF-1α, COX-2 and VEGF99–101, which are upregulated after platelets treatment in HESCs.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [99] Konstantinopoulos, P. A. et al. EGF-R is expressed and AP-1 and NF-kappaB are activated in stromal myofibroblasts surrounding colon adenocarcinomas paralleling expression of COX-2 and VEGF. Cell. oncology: Off. J. Int. Soc. Cell. Oncol. 29, 477–482, https://doi.org/10.1155/2007/831416 (2007). source

      Not checked in the free run.

    • cites [100] van Uden, P., Kenneth, N. S. & Rocha, S. Regulation of hypoxia-inducible factor-1alpha by NF-kappaB. Biochemical J. 412, 477–484, https://doi.org/10.1042/BJ20080476 (2008). source

      Not checked in the free run.

    • cites [101] Chang, H. J. et al. Transforming growth factor (TGF)-beta1-induced human endometrial stromal cell decidualization through extracellular signal-regulated kinase and Smad activation in vitro: peroxisome proliferator-activated receptor gamma acts as a negative regulator of TGF-beta1. Fertil. Steril. 90, 1357–1365, https://doi.org/10.1016/j.fertnstert.2007.09.010 (2008). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Normal endometrial stromal cells only produce negligible levels of estrogens likely due to a silenced aromatase because of hypermethylation.

    “While normal endometrial stromal cells only produce negligible levels of estrogens41 likely due to a silenced aromatase because of hypermethylation41,42”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [41] Izawa, M. et al. An epigenetic disorder may cause aberrant expression of aromatase gene in endometriotic stromal cells. Fertil. Steril. 89, 1390–1396, https://doi.org/10.1016/j.fertnstert.2007.03.078 (2008). source

      Not checked in the free run.

    • cites [42] Izawa, M. et al. Demethylation of a nonpromoter cytosine-phosphate-guanine island in the aromatase gene may cause the aberrant up-regulation in endometriotic tissues. Fertil. Steril. 95, 33–39, https://doi.org/10.1016/j.fertnstert.2010.06.024 (2011). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    Other genes, such as CYP11A1 and CYP17A1, that are involved in the synthesis of E2 may also be involved but were not detected due to either small fold increase or lack of adequate statistical power due to small sample sizes.

    “it suggests that other genes, such as CYP11A1 and CYP17A1 (Fig. 1), that are involved in the synthesis of E2 may also be involved but we were not able to detect them simply because of either the fold increase is small or there is lack of adequate statistical power due to small sample sizes used in our study.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Epidermal ERβ expression declines with age in both sexes concomitant with declining wound healing ability.

    “Concomitant with declining wound healing ability, epidermal ERβ expression declines with age in both sexes72.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [72] Inoue, T. et al. The role of estrogen-metabolizing enzymes and estrogen receptors in human epidermis. Mol. Cell Endocrinol. 344, 35–40, https://doi.org/10.1016/j.mce.2011.06.015 (2011). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    TGF-β1/Smad3 signaling pathway is involved in the transcriptional regulation of aromatase in several cell types, and particularly in endometriotic stromal cells.

    “Previous studies have reported that the TGF-β1/Smad3 signaling pathway is involved in the transcriptional regulation of aromatase in several cell types56–58, and particularly in endometriotic stromal cells59.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [56] Fiorelli, G. et al. Aromatase expression and activity in the human leukaemic cell line FLG 29.1. J. Steroid Biochem. Mol. Biol. 66, 105–112, https://doi.org/10.1016/s0960-0760(98)00050-8 (1998). source

      Not checked in the free run.

    • cites [57] Shozu, M., Zhao, Y. & Simpson, E. R. TGF-beta1 stimulates expression of the aromatase (CYP19) gene in human osteoblast-like cells and THP-1 cells. Mol. Cell Endocrinol. 160, 123–133, https://doi.org/10.1016/S0303-7207(99)00233-6 (2000). source

      Not checked in the free run.

    • cites [58] Liu, Y. et al. Effects of Smad3 on the proliferation and steroidogenesis in human ovarian luteinized granulosa cells. IUBMB life 66, 424–437, https://doi.org/10.1002/iub.1280 (2014). source

      Not checked in the free run.

    • cites [59] Qu, J. et al. Smad3/4 Binding to Promoter II of P450arom So As to Regulate Aromatase Expression in Endometriosis. Reprod. Sci. 24, 1187–1194, https://doi.org/10.1177/1933719116681517 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Increased HIF-1α accumulation results in DUSP2 down-regulation, which results in prolonged activation of ERK and p38 MAPK and increased COX-2 expression in endometriotic stromal cells, as well as increased ERβ expression which can also be induced by activated platelets.

    “Increased HIF-1α accumulation results in DUSP2 down-regulation, which results in prolonged activation of ERK and p38 MAPK and increased COX-2 expression in endometriotic stromal cells49, as well as increased ERβ expression66 which can also be induced by activated platelets.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [49] Wu, M. H., Lin, S. C., Hsiao, K. Y. & Tsai, S. J. Hypoxia-inhibited dual-specificity phosphatase-2 expression in endometriotic cells regulates cyclooxygenase-2 expression. J. Pathol. 225, 390–400, https://doi.org/10.1002/path.2963 (2011). source

      Not checked in the free run.

    • cites [66] Wu, M. H., Lu, C. W., Chang, F. M. & Tsai, S. J. Estrogen receptor expression affected by hypoxia inducible factor-1alpha in stromal cells from patients with endometriosis. Taiwan. J. Obstet. Gynecol. 51, 50–54, https://doi.org/10.1016/j.tjog.2012.01.010 (2012). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Peripheral tissues produce estrogens, and estrogens play a vital role in wound healing.

    “Indeed, peripheral tissues produce estrogens71, and estrogens play a vital role in wound healing27,28.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [71] Labrie, F. All sex steroids are made intracellularly in peripheral tissues by the mechanisms of intracrinology after menopause. J. Steroid Biochem. Mol. Biol. 145, 133–138, https://doi.org/10.1016/j.jsbmb.2014.06.001 (2015). source

      Not checked in the free run.

    • cites [27] Horng, H. C. et al. Estrogen Effects on Wound Healing. Int J Mol Sci 18, https://doi.org/10.3390/ijms18112325 (2017). source

      Not checked in the free run.

    • cites [28] Wilkinson, H. N. & Hardman, M. J. The role of estrogen in cutaneous ageing and repair. Maturitas 103, 60–64, https://doi.org/10.1016/j.maturitas.2017.06.026 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Many other cells in its microenvironment. Platelets, neutrophils, macrophages, and recently nerve fibers all have been identified to be accessories to aid and abet the lesional development.

    “Platelets21,23, neutrophils69, macrophages19,24, and recently nerve fibers25,26 all have been identified to be accessories to aid and abet the lesional development.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [21] Zhang, Q., Duan, J., Liu, X. & Guo, S. W. Platelets drive smooth muscle metaplasia and fibrogenesis in endometriosis through epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation. Mol. Cell Endocrinol. 428, 1–16, https://doi.org/10.1016/j.mce.2016.03.015 (2016). source

      Not checked in the free run.

    • cites [23] Ding, D., Liu, X., Duan, J. & Guo, S. W. Platelets are an unindicted culprit in the development of endometriosis: clinical and experimental evidence. Hum. Reprod. 30, 812–832, https://doi.org/10.1093/humrep/dev025 (2015). source

      Not checked in the free run.

    • cites [69] Takamura, M. et al. Neutrophil depletion reduces endometriotic lesion formation in mice. Am. J. Reprod. Immunol. 76, 193–198, https://doi.org/10.1111/aji.12540 (2016). source

      Not checked in the free run.

    • cites [19] Duan, J., Liu, X. & Guo, S.-W. The M2a macrophage subset may be critically involved in fibrogenesis of endometriosis in mouse. Reprod. Biomed. Online 37, 254–268, https://doi.org/10.1016/j.rbmo.2018.05.017. (2018). source

      Not checked in the free run.

    • cites [24] Bacci, M. et al. Macrophages are alternatively activated in patients with endometriosis and required for growth and vascularization of lesions in a mouse model of disease. Am. J. Pathol. 175, 547–556, https://doi.org/10.2353/ajpath.2009.081011 (2009). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Estrogen promotes wound healing mainly through ERβ, not ERα.

    “it has been reported that estrogen promotes wound healing mainly through ERβ, not ERα38.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Campbell, L. et al. Estrogen promotes cutaneous wound healing via estrogen receptor beta independent of its antiinflammatory activities. J. Exp. Med. 207, 1825–1833, https://doi.org/10.1084/jem.20100500 (2010). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    These cells are known to be irreplaceable partners in a successful wound healing.

    “Indeed, these cells are known to be irreplaceable partners in a successful wound healing70.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [70] Ellis, S., Lin, E. J. & Tartar, D. Immunology of Wound Healing. Curr. dermatology Rep. 7, 350–358, https://doi.org/10.1007/s13671-018-0234-9 (2018). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Estrogen is important to wound healing.

    “estrogen is very important to wound healing27,28”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [27] Horng, H. C. et al. Estrogen Effects on Wound Healing. Int J Mol Sci 18, https://doi.org/10.3390/ijms18112325 (2017). source

      Not checked in the free run.

    • cites [28] Wilkinson, H. N. & Hardman, M. J. The role of estrogen in cutaneous ageing and repair. Maturitas 103, 60–64, https://doi.org/10.1016/j.maturitas.2017.06.026 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Activation of HIF-1α and thus hypoxia can lead to increased estrogen production through depressing DUSP2 expression and thus COX-2 overexpression in endometriotic stromal cells, as well as increased ERβ expression.

    “Our results are consistent with the finding that activation of HIF-1α and thus hypoxia can lead to increased estrogen production through depressing DUSP2 expression and thus COX-2 overexpression in endometriotic stromal cells49, as well as increased ERβ expression66.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [49] Wu, M. H., Lin, S. C., Hsiao, K. Y. & Tsai, S. J. Hypoxia-inhibited dual-specificity phosphatase-2 expression in endometriotic cells regulates cyclooxygenase-2 expression. J. Pathol. 225, 390–400, https://doi.org/10.1002/path.2963 (2011). source

      Not checked in the free run.

    • cites [66] Wu, M. H., Lu, C. W., Chang, F. M. & Tsai, S. J. Estrogen receptor expression affected by hypoxia inducible factor-1alpha in stromal cells from patients with endometriosis. Taiwan. J. Obstet. Gynecol. 51, 50–54, https://doi.org/10.1016/j.tjog.2012.01.010 (2012). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisereasoning

    ERβ, but not ERα, is overexpressed in endometriosis.

    “this may explain as why ERβ, but not ERα, is overexpressed in endometriosis35,36.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [35] Brandenberger, A. W. et al. Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal endometrial and endometriosis-derived stromal cells. Mol. Hum. Reprod. 5, 651–655, https://doi.org/10.1093/molehr/5.7.651 (1999). source

      Not checked in the free run.

    • cites [36] Fujimoto, J., Hirose, R., Sakaguchi, H. & Tamaya, T. Expression of oestrogen receptor-alpha and -beta in ovarian endometriomata. Mol. Hum. Reprod. 5, 742–747, https://doi.org/10.1093/molehr/5.8.742 (1999). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The absence or over-abundance of any of these cell types would derail the normal healing process, leading healing astray and causing pathological healing such as fibrosis.

    “The absence or over-abundance of any of these cell types would derail the normal healing process, leading healing astray and causing pathological healing such as fibrosis70.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [70] Ellis, S., Lin, E. J. & Tartar, D. Immunology of Wound Healing. Curr. dermatology Rep. 7, 350–358, https://doi.org/10.1007/s13671-018-0234-9 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Endometriotic stromal cells produce a much higher level of estrogens, due to demethylated aromatase and overexpression of StAR, HSD3B2 and HSD17B1.

    “endometriotic stromal cells, in contrast, do produce a much higher level of estrogens, due to demethylated aromatase42, and overexpression of StAR, HSD3B2 and HSD17B112,43,45,47.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [42] Izawa, M. et al. Demethylation of a nonpromoter cytosine-phosphate-guanine island in the aromatase gene may cause the aberrant up-regulation in endometriotic tissues. Fertil. Steril. 95, 33–39, https://doi.org/10.1016/j.fertnstert.2010.06.024 (2011). source

      Not checked in the free run.

    • cites [12] Noble, L. S. et al. Prostaglandin E2 stimulates aromatase expression in endometriosis-derived stromal cells. J. Clin. Endocrinol. Metab. 82, 600–606, https://doi.org/10.1210/jcem.82.2.3783 (1997). source

      Not checked in the free run.

    • cites [43] Zeitoun, K. et al. Deficient 17beta-hydroxysteroid dehydrogenase type 2 expression in endometriosis: failure to metabolize 17beta-estradiol. J. Clin. Endocrinol. Metab. 83, 4474–4480, https://doi.org/10.1210/jcem.83.12.5301 (1998). source

      Not checked in the free run.

    • cites [45] Attar, E. et al. Prostaglandin E2 via steroidogenic factor-1 coordinately regulates transcription of steroidogenic genes necessary for estrogen synthesis in endometriosis. J. Clin. Endocrinol. Metab. 94, 623–631, https://doi.org/10.1210/jc.2008-1180 (2009). source

      Not checked in the free run.

    • cites [47] Tsai, S. J., Wu, M. H., Lin, C. C., Sun, H. S. & Chen, H. M. Regulation of steroidogenic acute regulatory protein expression and progesterone production in endometriotic stromal cells. J. Clin. Endocrinol. Metab. 86, 5765–5773, https://doi.org/10.1210/jcem.86.12.8082 (2001). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Activated platelets activate NF-κB in cancer cells.

    “Our results are also consistent with the report that activated platelets activate NF-κB in cancer cells60.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [60] Labelle, M., Begum, S. & Hynes, R. O. Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell 20, 576–590, https://doi.org/10.1016/j.ccr.2011.09.009 (2011). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ERβ is important in wound healing.

    “and so is ERβ37,38 in wound healing”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] Merlo, S., Frasca, G., Canonico, P. L. & Sortino, M. A. Differential involvement of estrogen receptor alpha and estrogen receptor beta in the healing promoting effect of estrogen in human keratinocytes. J. Endocrinol. 200, 189–197, https://doi.org/10.1677/JOE-08-0442 (2009). source

      Not checked in the free run.

    • cites [38] Campbell, L. et al. Estrogen promotes cutaneous wound healing via estrogen receptor beta independent of its antiinflammatory activities. J. Exp. Med. 207, 1825–1833, https://doi.org/10.1084/jem.20100500 (2010). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Our results are consistent with our previous report that platelets induce the TGF-β1/Smad3 signaling pathway in endometriotic cells.

    “Our results are consistent with our previous report that platelets induce the TGF-β1/Smad3 signaling pathway in endometriotic cells21.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Zhang, Q., Duan, J., Liu, X. & Guo, S. W. Platelets drive smooth muscle metaplasia and fibrogenesis in endometriosis through epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation. Mol. Cell Endocrinol. 428, 1–16, https://doi.org/10.1016/j.mce.2016.03.015 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    We did not fully elucidate the exact molecular mechanisms in more detail, nor did we demonstrate just exactly how platelets activate NF-κB and/or TGF-β1.

    “Second, while we have identified NF-κB and TGF-β1 signaling pathways that are involved in lesional estrogen overproduction, we did not fully elucidate the exact molecular mechanisms in more detail, nor did we demonstrate just exactly how platelets activate NF-κB and/or TGF-β1.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The expression values were normalized to the geometric mean of GAPDH measurements.

    “The expression values were normalized to the geometric mean of GAPDH measurements and the quantification of mRNA abundance was made using the method as described in87.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [87] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408, https://doi.org/10.1006/meth.2001.1262 (2001). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

    Interpretationown data

    This study provides yet another piece of evidence that endometriotic lesions are indeed wounds undergoing ReTIAR.

    “this study provides yet another piece of evidence that endometriotic lesions are indeed wounds undergoing ReTIAR.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    To minimize any individual variation, the endometrial tissue fragments from two donor mice were mixed together and then divided into three or four parts, each injected i.p. to one mouse each from one of the three or four groups.

    “To minimize any individual variation, the endometrial tissue fragments from two donor mice were mixed together and then divided into three or four parts, each injected i.p. to one mouse each from one of the three or four groups, depending on the experiment design.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The mice in the first experiment were randomly divided into three groups of 10: PI, PD and NI.

    “The mice were randomly divided into three groups of 10: PI, PD and NI.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    JSH-23 suppresses NF-κB signaling and SB-431542 suppresses TGF-β1/Smad3 signaling.

    “JSH-23 and SB-431542 can suppress NF-κB and TGF-β1/Smad3 signaling pathways respectively and thus might inhibit platelet aggregation in endometriosis91,92.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [91] Shin, H. M. et al. Inhibitory action of novel aromatic diamine compound on lipopolysaccharide-induced nuclear translocation of NF-kappaB without affecting IkappaB degradation. FEBS Lett. 571, 50–54, https://doi.org/10.1016/j.febslet.2004.06.056 (2004). source

      Not checked in the free run.

    • cites [92] Halder, S. K., Beauchamp, R. D. & Datta, P. K. A specific inhibitor of TGF-beta receptor kinase, SB-431542, as a potent antitumor agent for human cancers. Neoplasia 7, 509–521, https://doi.org/10.1593/neo.04640 (2005). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    All existing drugs for treating endometriosis are hormonal, with the aim of reducing estrogen production through induction of state of pseudo-pregnancy, pseudo-menopause, or chronic anovulation, thus creating an acyclic, hypoestrogenic environment.

    “Currently, all existing drugs for treating endometriosis are hormonal74, with the aim of reducing estrogen production through induction of state of pseudo-pregnancy, pseudo-menopause, or chronic anovulation, thus creating an acyclic, hypoestrogenic environment75.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [74] Groothuis, P. G. & Guo, S. W. Drug Development in Endometriosis and Adenomyosis: It Takes More Than Just Good Science. Reprod. Sci. 25, 1318–1329, https://doi.org/10.1177/1933719118785767 (2018). source

      Not checked in the free run.

    • cites [75] Olive, D. L. & Pritts, E. A. Treatment of endometriosis. N. Engl. J. Med. 345, 266–275, https://doi.org/10.1056/NEJM200107263450407 (2001). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    If either assumption (small inter-individual variation in treatment window or response) is violated, this notion is unlikely to be therapeutically feasible.

    “If either assumption is violated, this notion is unlikely to be therapeutically feasible.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: The inter-individual variation in the treatment window is small.
    • The authors state this as an assumption: The inter-individual variation in response to drug treatment is small.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    In the second experiment, 30 mice were randomly divided into three groups of equal size: control group (CT), NF-κB antagonism group (NA), and TGF-β1 antagonism group (TA).

    “In the second experiment, 30 mice were randomly divided into three groups of equal size: control group (CT), NF-κB antagonism group (NA), and TGF-β1 antagonism group (TA).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    A series of 4-5 images randomly selected from several sections per tissue sample were taken for each immunostaining parameter to obtain a mean value.

    “A series of 4–5 images randomly selected from several sections per tissue sample were taken for each immunostaining parameter to obtain a mean value.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Through the use of both in vitro and in vivo studies, we have provided convincing evidence that platelets are an unindicted culprit responsible for increased local production of estrogen, and that this is mainly through the induction of NF-κB and TGF-β1 signaling pathways.

    “Second, through the use of both in vitro and in vivo studies, we have provided convincing evidence that platelets are an unindicted culprit responsible for increased local production of estrogen, and that this is mainly through the induction of NF-κB and TGF-β1 signaling pathways.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    To ensure that the real-time PCR and Western blot analysis results refer to HESCs but not platelets, the HESCs co-cultured with platelets were washed with sterile PBS three times to remove the platelets.

    “To ensure that the real-time PCR and Western blot analysis results refer to HESCs but not platelets, we washed the HESCs that co-cultured with platelets with sterile PBS three times to remove the platelets as reported previously86.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [86] Tsujii, M. et al. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93, 705–716, https://doi.org/10.1016/s0092-8674(00)81433-6 (1998). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Immunostaining results were evaluated blind to which group the slide belonged to.

    “to count the number or the intensity of positive stain, blind to which group the slide belonged to.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    Our findings strongly indicate that non-hormonal therapeutics for endometriosis is theoretically viable, with anti-platelet therapy being one promising avenue.

    “Our findings strongly indicate that non-hormonal therapeutics for endometriosis is theoretically viable, with anti-platelet therapy being one promising avenue.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ERβ expression can also be upregulated by activated platelets.

    “we have shown previously that ERβ expression can also be upregulated by activated platelets73.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [73] Zhang, Q., Ding, D., Liu, X. & Guo, S. W. Activated Platelets Induce Estrogen Receptor beta Expression in Endometriotic Stromal Cells. Gynecol. Obstet. Invest. 80, 187–192, https://doi.org/10.1159/000377629 (2015). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    In the first experiment, 15 mice were randomly selected as donors.

    “Among the 45 mice, 15 were randomly selected as donors, and the remaining 30 were recipients who received endometrial tissues from donor mice.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    The findings from this study justify the use of non-hormonal drugs for therapeutic purpose.

    “However, the findings from this study justify the use of non-hormonal drugs for therapeutic purpose.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    We only employed ovarian endometrioma tissue samples in the in vitro study.

    “Lastly, we only employed ovarian endometrioma tissue samples in the in vitro study.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Just by suppression of platelet activation either through anti-platelet treatment or neutralization of NF-κB or of TGF-β1, the lesional production of estrogen can be substantially curtailed or even minimized to achieve the desired therapeutic effect without messing up the hormonal system.

    “They essentially show that, just by suppression of platelet activation either through anti-platelet treatment or neutralization of NF-κB or of TGF-β1, the lesional production of estrogen can be substantially curtailed or even minimized to achieve the desired therapeutic effect without messing up the hormonal system.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The first resultant slide was stained with hematoxylin and eosin (H&E) to confirm pathologic diagnosis.

    “with the first resultant slide was stained with hematoxylin and eosin (H&E) to confirm pathologic diagnosis”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    We did not evaluate all genes that are possibly involved in estrogen biosynthesis in endometriotic stromal cells, and focused mainly on StAR, aromatase, HSD3B2 and HSD17B1, in which the product of their fold increase seemingly accounted for most of the variation in estrogen production in endometriosis.

    “First, we did not evaluate all genes that are possibly involved in estrogen biosynthesis in endometriotic stromal cells, and focused mainly on StAR, aromatase, HSD3B2 and HSD17B1, in which the product of their fold increase seemingly accounted for most of the variation in estrogen production in endometriosis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Increased local production of estrogen is common to all subtypes of endometriosis and even to adenomyosis.

    “Moreoever, increased local production of estrogen is common to all subtypes of endometriosis83 and even to adenomyosis84.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [83] Bulun, S. E. Endometriosis. N. Engl. J. Med. 360, 268–279, https://doi.org/10.1056/NEJMra0804690 (2009). source

      Not checked in the free run.

    • cites [84] Yamamoto, T., Noguchi, T., Tamura, T., Kitawaki, J. & Okada, H. Evidence for estrogen synthesis in adenomyotic tissues. Am. J. Obstet. Gynecol. 169, 734–738, https://doi.org/10.1016/0002-9378(93)90654-2 (1993). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S1 (asserted).

    Premisecitation

    There is a hypothesis of treatment window, that pharmacologically reduces the estrogen level to a certain level so that the growth of ectopic endometrium is curtailed or contained but the level is not too low to cause any detrimental side-effects due to hypoestrogenism.

    “there is a hypothesis of treatment window, that pharmacologically reduces the estrogen level to a certain level so that the growth of ectopic endometrium is curtailed or contained but the level is not too low to cause any detrimental side-effects due to hypoestrogenism76.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [76] Barbieri, R. L. Hormone treatment of endometriosis: the estrogen threshold hypothesis. Am. J. Obstet. Gynecol. 166, 740–745, https://doi.org/10.1016/0002-9378(92)91706-g (1992). source

      Not checked in the free run.

75 of 75

  • The paper relies on this without support.

    JSH-23 specifically neutralizes NF-κB without off-target effects.

    “Neutralization of NF-κB by JSH-23 nearly completely abolished this upregulation”

  • The paper relies on this without support.

    A83-01 specifically neutralizes TGF-β1 without off-target effects.

    “TGF-β1 neutralization with pre-treatment with A83-01”

  • The paper relies on this without support.

    SB431542 specifically inhibits TGF-β1 without off-target effects.

    “mice treated with either SB431542 (a TGF-β1 inhibitor) or JSH-23 (an NF-κB inhibitor)”

  • The authors state this as an assumption.

    The inter-individual variation in the treatment window is small.

    “the inter-individual variation in the treatment window is small”

  • The authors state this as an assumption.

    The inter-individual variation in response to drug treatment is small.

    “the inter-individual variation in response to drug treatment also is small”

  • The authors state this as an assumption.

    Platelets are activated due to ReTIAR and platelet activating factors secreted by endometriotic stromal cells.

    “our study made a simpler and more natural assumption that platelets that are activated due to ReTIAR and platelet activating factors secreted by endometriotic stromal cells67.”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Activated platelets can upregulate StAR, SF-1, and aromatase, increasing estrogen production significantly in endometriotic stromal cells.

    “In fact, activated platelets can also upregulate the gene expression of StAR, SF-1 and aromatase, increasing the estrogen production significantly in endometriotic stromal cells [98].”

    From The Pathogenesis of Adenomyosis vis-à-vis Endometriosis (2020) · cites this paper as [98]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Claim that either inhibitor abolishes increased E2 conflicts with residual elevation

    Major

    The abstract says antagonism of either pathway abolishes increased estrogen production. In the TGF-β1 inhibition experiment, however, E2 remained significantly higher than in the vehicle-treated control (p = 0.012).

  • NF-κB and TGF-β1 inhibitor groups are assigned the opposite drugs

    Minor

    The methods define NA as NF-κB antagonism and TA as TGF-β1 antagonism, but then assign the TGF-β1 inhibitor SB-431542 to NA and the NF-κB inhibitor JSH-23 to TA. The Figure 7 caption identifies the drugs with the opposite, expected pathways, leaving the reported group assignments inconsistent.

2 of 2

Cites

Flagged first, then by citations

2 of the 112 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. Beshay, V. E. & Carr, B. R. In Williams Gynecology. (eds B., Hoffman et al.) Ch. 10, 281–303. (McGraw-Hill Education, 2012).

Cited by

Sorted by citations

Related

Authors
Qi, Q., Liu, X., Zhang, Q., & Guo, S.-W.
Affiliations
Shanghai Medical College of Fudan University (CN)
Corresponding author
Guo, Sun-Wei
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-01-28
Volume
10
Issue
1
Pages
1281-1281
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-020-57997-6
PMC
PMC6987096
OpenAlex
W3003511937
Semantic Scholar
95449075365a93082d887fd7342c05b460601c84
Cited by
44
References
112
Citations by year
2026: 7 · 2025: 7 · 2024: 4 · 2023: 4 · 2022: 10 · 2021: 5 · 2020: 6 · 2019: 1
FWCI
5.50
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
National Natural Science Foundation of China (81671436, 81871144, 81771553)
Topics
  • Health Sciences › Medicine › Reproductive Medicine › Endometriosis Research and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Estrogen and related hormone effects
  • Health Sciences › Medicine › Hematology › Platelet Disorders and Treatments
Keywords
endometriosis, platelets, estrogen synthesis, estradiol, endometriotic stromal cells, NF-κB signaling, TGF-β1 signaling, aromatase, stars, HSD3B2, HSD17B1, SF-1, CREB phosphorylation, HIF-1α, platelet depletion
MeSH terms
Blood Platelets, Endometriosis, Endometrium, Estrogens, Female, Humans, Signal Transduction, NF-kappa B, Stromal Cells, Transforming Growth Factor beta1

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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