**3. Concepts on endometrial peculiarities, as a steroid hormones target in endometriosis. Estrogen dominance. Progesterone resistance. Progesterone attenuance**

The efficacy of endometriosis therapies may be improved by dissecting the unique molecular properties of eutopic and ectopic endometria compared to normal endometrium. At first glance, the terms "estrogen dominance" and "progesterone resistance" appear to describe opposite sides of the same coin [6]. Endometrial Progesterone Resistance is described since many years [33] as there are other hormones resistance, being first named as "Pseudocorpus Luteum Insufficiency" [34] meaning that endometrium is not able to respond to bioavailable P4 plasma levels inbetween normal limits. In non-ill women E2 induces epithelial proliferation to build endometrial thickness during the proliferative phase of the menstrual cycle, then P4 inhibits E2-induced proliferation and allows stromal cells to begin decidualization during the secretory phase [35] in order to prepare the stroma to become receptive to blastocyst invasion during "window of receptivity/implantation" in normal, fertile women [36].

#### **3.1 Dysregulation of endometrial steroid hormones receptors in endometriosis**

The molecular mechanism triggered by ovarian steroid hormones in endometrium is well known: steroid hormones link to specific nuclear (ERα, ERβ; PRA, PR-B, with specific ratios between them), cytoplasmic (ERβ), and membrane receptors (non- canonical G protein-coupled estrogen receptor (GPER) [37] and PGRMC-1 and PGRMC-2), which are able to bind to the promoter of the target genes, being up- or down- regulated by their co-activators (SRC-1, SRC-2, SRC-3 for estrogen receptors- ER) or downstream effectors (TGFβ, Dickkopf-1, retinoic acid, c-*myc*, *etc.* for progesterone receptors- PRs) [28] and influence endometrial cells proliferation and differentiation [38]. Endometrial repair after menstruation, proliferation and/or differentiation processes are dysregulated in endometriosis when menstrual reflux with endometrial stromal and epithelial glands cells with their exosomes migrate, adhere and invade peritoneal surface and/or other ectopic sites, associated to waves of inflammation (by systemic and local reactive responses to the presence of endometrial debris), neuro- angiogenesis and neuroinflammation, and aberant scar formation through fibrosis and adherences (possible a self-protection mechanism for ectopic lesion, as in chronic infections) at every ovarian cycle (different from normal endometrium restoration after each menstrual cycle). All these events are genetic controlled, epigenetic changed by hyper (for PR promoter) or hypomethylation (for ERβ) of DNA steroid hormones receptors promoters [39–41]. When the tightly regulated balance of epithelial- stromal P4 and E2 signaling is lost, P4 resistance and E2 dominance are prone to ensue, as in endometriosis [6]. P4 resistance may lead to both increased lesion growth and a non-receptive endometrium, and actually one speaks also about progesterone attenuated response in eutopic endometrium, which is associated to infertilty/ subfertility. Estrogen dominance, progesterone resistance and progesterone attenuance in the ectopic and eutopic endometrium are explained by the dysregulation of estrogen and progesterone receptors their genes (*ESR1* and *ESR2*) [32]; a single gene- *PRG* for PRs) [42] with their micro-RNA (miRNA) –the cells' critical regulators for development, and physiology, their mis-expression being associated to pathology [43].

When dysregulation condition persists it leads to both increased lesion growth and progression to a superior stage, and a non-receptive endometrium. P4 acts on stromal cells of the normal endometrium, inducing paracrine factor(s), and they induce the expression of the enzyme 17β-hydroxysteriod dehydrogenase type 2 (17β-HSD-2) for metabolization of E2 to estrone (E1), in the epithelial cells, and cell proliferation arrest. Excess E2 with Estrogen Dominance, and Progesterone Resistance are well documented in ectopic endometrium with aberrant levels of ERs and changed ER-α/ ER β *ratio*, where the genes analyses have proved to become an acquired property, through their migration and exposure process to

#### *Potential Therapeutic Options and Perspectives for Alleviation of Endometrial Estrogen… DOI: http://dx.doi.org/10.5772/intechopen.100039*

peritoneal environement [5]. ER-α and ER- β have essential roles in establishment and progression of ectopic lesions [32]. ERβ is excessively expressed in stromal cells of the ectopic lesions, versus non-ill women, fact due to hypomethylation of ERβ promoter, which contributes to low ER-α expression in ectopic endometrium [41, 44]. ERβ protein net -work together with SRC-1 coactivator isoform with which it cooperates, may have a cytoplasmic, non-genomic action in endometriosis as Han SJ, Jung SY, Wu SP, et al. discovered [44] (**Figure 1**).

Eutopic endometrium may prove the same endometrial dominance and progesterone resistance, but after some years of disesease evolution, being described cases with initial progesterone attenuance in eutopic endometrium and progesterone resistance in the ectopic sites, usually in deep endometriosis [28]. It is documented a local increased synthesis of estrogen [45, 46] not a systemic high level [47] through the presence of the enzymens (aromatase, and 17β-hydroxysteroid dehydrogenase-1, 17β-HSD-1) [48, 49] and a blunted response to progesterone in eutopic and ectopic endometrium, P4 serum levels being similar to non- ill women [45]. In human and experimental mice the ER-α content may be normal in eutopic endometrium, as in non ill women, but ER β are increased in both epithelial and stromal endometrial cells of eutopic and ectopic sites, as PRs levels are reduced in eutopic endometrium and PRs are lost in ectopic sites [44]. A recent analyses of ERs in deep endometriosis revealed that ER-α is a subtype for this condition [50] ER-α levels being predictive for symptoms severity, and for responses to treatment [51]. Progesterone attenuance of eutopic endometrium is connected to altered endometrial receptivity in the "window of receptivity/implantation" and to a significant reduction of implantation rate in ill-patients trying *in vitro* fertilization, due to stromal cells impair decidualization proved by a reduction of nearly 2-fold in IGFBP-1, and of leukemia inhibitory factor (LIF) [52] and to reduction/ dysregulation of P4 target genes during the "window of receptivity/implantation", time when normally, the endometrium is exposed to the highest levels of P4 [53] as it is strikingly down-regulated *glycodelin*- the prototype progesterone-responsive gene, in eutopic endometrium of ill-women compared to non-ill [53, 54]. LIF low levels are an intrisic glandular dysfunction, induced by the gland-specific transcription factor Forkhead box A2 (FOXA2) low level [55, 56] also a P4 gene target. There are contradictions on the status of ERs and PRs in ectopic endometrium – an ERs increased expression, or others show reduced expression of both ERα and ERβ

#### **Figure 1.**

*Role of ER-*β *in endometriosis. microRNA levels of steroids in primary stromal cells isolated from endometrium and endometriosis (n = 8 patients in each category). Comparable in vivo differences were also observed between whole tissues of endometrium and endometriosis Legend: ER: estrogen receptor; PR: progesterone receptor; real time-PCR: polymerase chain reaction (adapted from Bulun et al. [45]: open acces).*

[57–59] and lower levels of PRs [5, 60]. The eutopic endometrium has an attenuate answer to P4, the isoform PR- B is not expressed in patients' endometrium, being only the isoform PR-A, because progesterone-responsive genes are not deleted in eutopic endometrium in comparison to normal women in the early secretory phase of cycle [28–32], a normal PR-A/PR-B *ratio* is very important in endometrial function. One may consider that the relative differences between studies regarding the PR isoforms loss in ectopic and eutopic endometrium may be explained by stage of disease, type of lesion and cells, and method of analysis.

Previous studies [32] demonstrated in experimental mice that high levels of ERα are driving proliferation, adhesion and angiogenesis in ectopic tissue, and also modulate inflammation, and ERβ prevents apoptosis and enhance invasion, proliferation, adhesion, and inflammation to stimulate the growth of ectopic lesion, so both isoforms might sinergistically contribute to regulation of proliferation, adhesion, and inflammation in endometriotic lesion. These discrepant findings are explained by the differences in study design, patient selection criteria, cycle stage, and endometriosis type and stage. Similar to these contradictory results on endometrial steroid hormones receptors is the situation with miRNA indentification by real-time quantitative reverse transcription-polymerase chain reaction (real time qRT-PCR) in normal endometrium, eutopic and ectopic sites, connected to mi-RNA upregulation (over expression) or down regulation (under expression) in eutopic and in different ectopic areas (peritoneal, ovarian), and some are "mis-expressed" endometriosis [61]. There are differences between authors, with conflicting reports on whether or not miRNA expression was influenced by the menstrual cycle phases, endometrial cell type, miRNA type, level in ectopic/eutopic tissue, and stage of disease.

### **3.2 High micro-heterogeneity of endometrial steroid hormones receptors signaling in endometriosis**

Normal endometrium is containing large quantities of distinct stromal cells with abundant estrogen-induced PRs, which influence glandular epithelial cell proliferation and differentiation, and protect against carcinogenic transformation, when PRA/PRB are in a proper *ratio* to ensure normal P4 response. PRA and PRB are members of a superfamily of almost 50 ligand-activated nuclear transcription factors [62]. *In-vitro* studies suggest that the two PR isoforms differ functionally, and that their relative expression in a target cell may determine the nature and magnitude of response to P4. The two isoforms are in comparable levels expressed in proliferative phase, but in the mid- secretory only PR- B is present in the epithelial glands, PR-A is predominantly present in the stroma throughout the cycle. It is a homogeneity in the relative expression in PR-A and PR-B in adjacent cells within the same tissue compartment, and a heterogeneity between glands, observed under some circumstances in the endometrium functionalis, suggesting that PR isoforms down-regulation by P4 is asynchronous, and between the glands of the basalis and functionalis of the endometrium implying region specific responses to hormonal stimuli [63].

A recent European study on deep endometriosis [64] showed a high variability of ERα and PR distribution in the same gland, among distinct glands of the same sample, and among distinct patients receiving the same treatment. Luminal epithelial height variability was primarily due to epithelial cells heterogeneity in a gland, secondarily to the glands randomaly evaluated on the same section, and tertiary to the patient category. The heterogeneity of ERα and PR distribution in the same women could explain why endocrine treatments are unable to cure deep endometrios. The cause of heterogeneity in endometriotic tissues is difficult to be ascertain, one hypothesis being the DNA methylation of the steroid hormones [65] or of their promoters [39, 40] or

#### *Potential Therapeutic Options and Perspectives for Alleviation of Endometrial Estrogen… DOI: http://dx.doi.org/10.5772/intechopen.100039*

an abnormal proteolysis of steroid hormones chaperons [66], the co-chaperons are required by PR for signaling uterine cycles and implantation [67].

Mice induced endometriosis demonstrated a high inter-animal variation in the levels of ERs, PRs, in ectopic endometrium vs. controls; with variable levels by almost 100-fold within the same lesion, and with differences between two lesions from the same animal [68] aspect called "micro-heterogeneity". The changes are tissue intrinsic, and some researchers propose that the variable outcomes in hormonal therapy for endometriosis could be possibly due to heterogeneity or polymorphism in the expression of steroid hormone receptors in the ectopic endometrium [68] or in all endometrial locations- eutopic, and ectopic, being known the heterogeneity of PRs in glands, and the homogeneity of PR isoforms in stroma of normal human endometrium [63] making the magnitude in response, the attenuance, and the resistance in response to P4, independently to the serum levels of P4 [69]. This intrinsic biologic alteration of eutopic endometrium explains the missing differences in endometrial thickness, and histology –respectively luteal differentiation, or epithelial integrin expression at the lower mid-luteal serum progesterone level (as 3–4 ng/ml) in programmed cycles of physiological and subphysiological exogenous progesterone replacement in GnRH agonist-suppressed healthy volunteers. One may consider that these results are an answer to the questions whether the abnormalities of eutopic endometrium in early secretory phase suggestive of attenuated progesterone response in the transition from proliferative to secretory phase are due to lower level of circulation or local bioavailable progesterone or to the changes in endometrial transcriptome, with dysregulation of progesterone- regulated genes.
