PI3K/AKT pathway is altered in the endometriosis patient's endometrium and presents differences according to severity stage.

Based on the inflammatory nature and hormone-dependency of endometriosis, PI3K/AKT signaling appears to influence its progression. Could the endometriosis stages be linked to differential changes in PI3K/AKT pathway regulation? The objective is to evaluate the expression of PI3K, PTEN, AKT and p-AKT in endometrial human biopsies, according to the presence or absence of the disease, and to assess the underlying differences regarding the endometriosis stages. Biopsy specimens of the ectopic and eutopic endometrium were obtained from twenty women with untreated peritoneal endometriosis as well as endometrium biopsies from nine controls. Our study revealed an increased expression of PI3K in eutopic and ectopic endometrium from patients with endometriosis, and a reduced expression of PTEN and increased levels of AKT phosphorylation, compared to control endometrium. Both eutopic and ectopic endometrium from patients with minimal-mild endometriosis expressed a significant reduced PTEN level compared to the respective endometrium from patients with moderate-severe endometriosis. The ratio p-AKT/total AKT showed higher levels of AKT phosphorylation in endometriotic tissue from patients with minimal-mild endometriosis. This study has firmly confirmed the alteration in PI3K/AKT pathway regulation and demonstrated clear differences between the stages of endometriosis, emphasizing the importance of this pathway in the first stage of the disease.

Is cytochrome P450 3A4 regulated by menstrual cycle hormones in control endometrium and endometriosis?

The estrogen-metabolizing activities of cytochrome P450 (CYP) enzymes have been implicated in endometriosis. However, their regulation in various sources of endometrial tissue under different hormonal conditions has not been clarified. Our objective was to study the hormone regulation of a specific CYP enzyme, namely CYP3A4, in control (n = 15) and endometriosis patients (n = 42). To this end, we evaluated mRNA expression (using real-time PCR) of CYP3A4 in tissue samples classified according to the phase of menstrual cycle at which they were obtained as confirmed by the related circulating hormone levels. Protein expression was also evaluated by Western Blot. In order to further investigate the hormonal regulation of CYP3A4, stromal cells from ovarian endometriotic lesions were cultured with the prevailing hormones of the distinct phases of the menstrual cycle. We observed that all control and endometriosis tissues express CYP3A4. Nevertheless, changes in CYP3A4 gene expression related to cycle phase were only seen in the control eutopic endometrium and not in samples from endometriosis patients, with an increase in the luteal phase. Stromal cells isolated from ovarian endometriotic lesions expressed CYP3A4 and their exposure to luteal phase-mimicking hormones (estradiol + progesterone) reduced CYP3A4 mRNA in parallel with a diminished expression of the corresponding receptors, estrogen receptor alpha and progesterone receptor. Our findings suggest that steroid hormones are able to regulate CYP3A4 mRNA expression, although the circulating levels of these hormones can only regulate control endometrium and not endometriosis tissues, probably because of dysregulated local steroid concentration in these latter samples.

Deep Invasive Endometriosis Lesions of the Rectosigmoid May Be Related to Alterations in Cell Kinetics.

OBJECTIVES: The aim of this study was to analyze cell kinetics through expression and apoptosis of topoisomerase 2-α (TOP2A), p53, and c-erb2 in rectosigmoid endometriotic lesions and in healthy endometrial tissue and to establish correlations between such findings and clinical data in patients with rectosigmoid endometriosis. METHODS: Sixty patients with rectosigmoid endometriosis and 20 control women without endometriosis were included. Immunohistochemical assays were used to measure expression of TOP2A, p53, and c-erB-2. Apoptosis was quantified by directly counting the apoptotic bodies. FINDINGS: The number of lesions was positively correlated with expression of TOP2A in the lesion. There was also significant correlation between the lesions' size and number and cell turnover index. Apoptosis index (AI) was the same for endometriosis lesions and eutopic endometrium. Expression of TOP2A was significantly lower in the endometriosis group compared to the controls. CONCLUSIONS: Changes in cell proliferation but not in the AI in rectosigmoid endometriosis are indicative of an imbalance in cell kinetics that may lead to the development of the disease.

Unremitting cell proliferation in the secretory phase of eutopic endometriosis: involvement of pAkt and pGSK3ß.

OBJECTIVE: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. DESIGN: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3ß), and phosphorylated glycogen synthase kinase 3 beta (pGSK3ß), throughout the menstrual cycle. RESULTS: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3ß was identical in all samples and cycle phases, whereas pAkt and pGSK3ß, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. CONCLUSION: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways.

RHOC: a key gene for endometriosis.

Considerable efforts have been invested in elucidating the potential mechanisms involved in the physiopathology of endometriosis. The aims of our study were to investigate whether RHOC expression is differentially altered in the endometrium and in endometriotic lesions. A total of 40 patients diagnosed with endometriosis and 15 healthy fertile women were selected for the study. Paired biopsies of endometrial tissue (eutopic endometrium) and endometriotic lesions (ectopic endometrium) were obtained from the patients with endometriosis. Endometrium from women without endometriosis was used as a control. Expression of the RHOC gene was analyzed by real-time polymerase chain reaction in autologous endometrial tissues of women with endometriosis and in the endometrium of control women. Increased RHOC expression was detected in endometriotic lesions compared to the eutopic endometrium of women with endometriosis and control women. RHOC changes may be among the key elements involved in the origin and the maintenance of endometriosis.

Increased circulating MMP-2 levels in infertile patients with moderate and severe pelvic endometriosis.

The current study compares the levels of matrix metalloproteinase (MMP)-2 and MMP-9 in the follicular fluid (FF) of infertile patients with and without endometriosis submitted to ovarian stimulation for in vitro fertilization and the levels of MMP-2 in the serum of the same patients. We also evaluated whether the severity of endometriosis can influence serum and/or FF concentration of these metalloproteinases. A cross-sectional study was conducted on 30 patients: stage I/II endometriosis (n = 10), stage III/IV endometriosis (n = 10), and control (infertility due to tubal and/or male factor; n = 10). Blood samples for the analysis of MMP-2 levels were obtained during the early follicular phase of the menstrual cycle. The FF samples for the analysis of MMP-2 and MMP-9 were obtained on the day of oocyte retrieval. The concentrations of MMP-2 and MMP-9 were determined by zymography. No intragroup or intergroup difference was observed in MMP-2 or MMP-9 levels in FF. Significantly higher MMP-2 levels were detected in the serum of infertile women with stage III/IV endometriosis compared to women with stage I/II endometriosis. In conclusion, no differences were observed in the follicular levels of MMP-2 and MMP-9 between infertile patients with and without endometriosis. However, the levels of MMP-2 were significantly higher in the serum of infertile women with advanced stages of endometriosis. Taken together, the present results demonstrate that advanced pelvic endometriosis severity is related to higher serum MMP-2 levels but does not influence follicular MMP-2 or MMP-9 levels in periovulatory follicles obtained from stimulated cycles.

Evaluation of N-acetilglucosaminidase and myeloperoxidase activity in patients with endometriosis-related infertility undergoing intracytoplasmic sperm injection.

AIM: Inflammation is as an important factor in ovulation with the active participation of leucocytes and their inflammatory mediators. The present study was performed to compare the activity of the inflammatory enzymes myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) in patients with endometriosis-related infertility and in normally ovulating women undergoing intracytoplasmic sperm injection (ICSI). MATERIAL AND METHODS: This prospective study included infertile women undergoing ICSI treatment. These women were divided into two groups: endometriosis anovulation (n = 18) and normally ovulating (n = 20). NAG and MPO activity was evaluated colorimetrically in serum and in follicular fluids obtained at the time of oocyte retrieval. RESULTS: There was a significant correlation between the serum and follicular fluid activities of NAG and MPO (τ = 0.256, P = 0.025; and τ = -0.234, P = 0.041; respectively). Both serum and follicular fluid NAG activities were higher in patients with endometriosis compared to the control group (P < 0.001). MPO follicular fluid activity was lower in patients with endometriosis compared to normally ovulating women (P = 0.016). CONCLUSION: Infertile patients with endometriosis show a distinct pattern of serum and follicular fluid macrophage/neutrophil activation compared to normally ovulating women undergoing ICSI, which may reflect the role of immune and inflammatory alterations in endometriosis-related infertility.

Single-nucleotide polymorphisms in the lysyl oxidase-like protein 4 and complement component 3 genes are associated with increased risk for endometriosis and endometriosis-associated infertility.

This study was conducted to assess genetic associations with endometriosis in a Puerto Rican population. Statistically significant differences in the allelic frequencies and genotype distribution of genetic variants in lysyl oxidase-like protein 4 (LOXL4) and complement component 3 (C3) were documented in patients with endometriosis-associated infertility versus controls, and in patients with endometriosis versus controls, respectively. In women who have the risk genotype at both single-nucleotide polymorphisms, the estimated risk for endometriosis nearly doubled.

Differential immunohistochemical expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in cow uteri with adenomyosis during follicular phase.

The present investigation was intended to show a different immunohistochemical profile of matrix metalloproteinase-2 and Tissue inhibitor metalloproteinase-2 in bovine uteri with adenomyosis during follicular phase. Uterine samples of 32 cows in reproductive age were taken from the medial third of one of the uterine horns and grouped according to the adenomyosis degree (superficial and deep). Tissue sections (4 µm) were incubated overnight at 4°C with monoclonal antibody for matrix metalloproteinase-2 and Tissue inhibitor metalloproteinase-2. Staining intensities were evaluated in the luminal epithelium, ectopic and dystopic endometrial tissue (stroma, capillaries and glands), endometrial-myometrial border, myometrium, myometrial vessels (middle tunic and endothelium). The matrix metalloproteinase-2 expression was higher for deep adenomyosis samples, showing a differential mean reactivity in superficial endometrium, myometrial vessels, myometrium adjacent to adenomyotic focus and endometrial-myometrial border (P < 0.05). Moreover, matrix metalloproteinase-2 expression was higher in deep adenomyosis samples than that of Tissue inhibitor metalloproteinase-2 in almost all uterine structures analyzed (except for the endometrial and myometrial vessels and endometrial-myometrial border). The opposite was observed in the follicular phase, for both normal specimens and with superficial adenomyosis, where Tissue inhibitor metalloproteinase-2 expression was higher than that of matrix metalloproteinase-2. In conclusion, a differential pattern of matrix metalloproteinase-2 and Tissue inhibitor metalloproteinase-2 was observed in cow uteri with adenomyosis.

RsaI polymorphism of the ERß gene in women with endometriosis.

We examined the frequency of RsaI polymorphism of the ERß gene in 54 patients diagnosed with endometriosis and 46 controls. Peripheral blood was collected from women undergoing laparoscopy with a confirmed diagnosis of endometriosis. Polymorphisms of the ERß gene and p53 were assessed by PCR and analyzed on 2% agarose gel stained with ethidium bromide. The AG polymorphism genotype frequency in patients with endometriosis was 59.3%, with 40.7% GG. In the control group, the frequency of AG was 6.5%, with 93.5% GG. The frequency of heterozygous AG was nine times higher in patients with endometriosis than in the control group (P < 0.0001).

The effect of oral contraceptives on aromatase expression in the eutopic endometrium of patients with endometriosis.

OBJECTIVE: To determine the effect of oral contraceptives containing gestodene on aromatase expression in the endometrium of patients diagnosed with endometriosis. PATIENTS AND METHODS: Endometrial biopsies were taken at the time of laparoscopy in 40 patients with endometriosis, 16 of whom were using an oral contraceptive containing gestodene at the time of laparoscopy. The remaining 24 patients were receiving no form of treatment for endometriosis. Endometrial biopsies taken from 23 patients with normal echographic signs and no symptoms were used as controls. Aromatase expression was evaluated in endometrial samples using immunohistochemistry. RESULTS: In the untreated, symptomatic endometriosis patients, aromatase expression was detected during the proliferative phase in 92% of cases, while in the symptom-free control patients aromatase was expressed in only 9% of cases. In patients with endometriosis who were using oral contraceptives, there were significantly fewer cases of positive endometria compared with the untreated patients with endometriosis (6%). CONCLUSION: Oral contraceptives containing gestodene are effective in decreasing aromatase expression in the eutopic endometrium of patients with endometriosis.

Effect of the menstrual cycle and oral contraceptives on aromatase and cyclooxygenase-2 expression in adenomyosis.

OBJECTIVES: To determine whether aromatase expression in the eutopic endometrium and adenomyotic foci is affected by previous use of oral contraceptives containing gestodene, and to determine whether changes in cyclooxygenase-2 (COX-2) expression occur in adenomyosis during the menstrual cycle. PATIENT AND METHODS: This was a retrospective cohort study carried out in paraffin-embedded endometrial tissue obtained from patients with a histological diagnosis of adenomyosis obtained during the proliferative (n = 25) and luteal (n = 10) phases of the menstrual cycle and following the use of continuous oral contraception with gestodene/ethinyl estradiol (n = 7). COX-2 and aromatase expression were measured in both eutopic endometrium and adenomyotic foci using immunohistochemical methods. RESULTS: Aromatase expression was detected in 80% of the endometrial slices by immunohistochemistry. In positive cases, aromatase was mainly detected in the stromal cells of the eutopic endometrium, whereas in the adenomyotic foci this expression was negative in the majority of the cases. Oral contraceptives containing gestodene, on the other hand, were effective in suppressing aromatase expression in both eutopic and ectopic endometrium. COX-2 expression was detected by immunohistochemistry in the glandular epithelium of both eutopic endometrium and adenomyotic foci and there were no significant changes in its intensity throughout the menstrual cycle. CONCLUSION: Aromatase expression in the eutopic endometrium and adenomyotic foci is suppressed by oral contraceptives containing gestodene. Increased aromatase activity may be responsible for the persistent COX-2 expression during the luteal phase.

Reduced aromatase activity in granulosa cells of women with endometriosis undergoing assisted reproduction techniques.

The aim of the present study was to measure the in vitro aromatase activity in granulosa cells of women with endometriosis submitted to assisted reproduction techniques. A case-control study was conducted on eight patients with endometriosis and eight with other infertility causes submitted to in vitro fertilization or intracytoplasmic sperm injection. Granulosa cells were obtained from pre-ovulatory follicles during oocyte retrieval and cultured for 24 h in the presence or absence of testosterone (2 x 10(-6) and 2 x 10(-5) M), follicle-stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I) (both at 50 ng/ml). Estradiol (radioimmunoassay) was measured in the obtained culture fluids. The basal production of estradiol and its production under testosterone addition to the culture (aromatase activity) were analyzed. Reduced aromatase activity was detected in cultured granulosa cells in endometriosis cases, compared with controls, when testosterone was added at the concentration at 2 x 10(-6) M (p = 0.0303). The basal production of estradiol was also reduced in endometriosis patients (p = 0.0390). The effect of addition of FSH and IGF-I did not differ between groups. In conclusion, the in vitro basal production of estradiol and aromatase activity in granulosa cells were reduced in women with endometriosis submitted to assisted reproduction techniques, compared with the control group.

Effects of aromatase inhibitors on proliferation and apoptosis in eutopic endometrial cell cultures from patients with endometriosis.

OBJECTIVE: To study the effect of letrozole (Let) and anastrozole (Anas) on apoptosis and cell proliferation in epithelial endometrial cells (EEC) from patients with endometriosis (EDT). DESIGN: Prospective study. SETTING: Research institute and clinical fertility center. PATIENT(S): Eighteen women with untreated EDT. INTERVENTION(S): Biopsy specimens of eutopic endometrium were obtained from all subjects. Apoptosis and cell proliferation were examined in EEC after incubation with Let or Anas. MAIN OUTCOME MEASURE(S): Percentage of apoptotic cells (ApC) was evaluated by the acridine orange-ethidium bromide technique; cell proliferation was assessed by 3H-thymidine incorporation. RESULT(S): Treatment with Let 10 nM and Let 100 nM enhanced values of ApC in cultures from EDT patients. Epithelial endometrial cells treated with Anas 100 nM or Anas 500 nM showed a statistically significant induction on apoptosis levels. Cultures treated with Let 1 nM or Anas 50 nM did not show any significant differences in ApC levels compared with basal conditions. 3H-Thymidine uptake was down regulated by Let 10 nM and Let 100 nM. Similarly, Anas 100 nM and Anas 500 nM showed a significantly lower degree of cell proliferation in EEC. Lower concentrations of Let and Anas did not induce any significant change in cell proliferation rates. CONCLUSION(S): Our results show that Let and Anas produced a significant and positive effect on apoptosis and cell proliferation on EEC from EDT patients. These findings support the further investigation of aromatase inhibitors as a treatment option in EDT.

P450 arom y microambiente estrogénico en endometrios eutópicos de mujeres con endometriosis / P450Arom and estrogenic microenvironment of eutopic endometria in endometriosis

Background: Endometriosis, a common gynecologic disorder characterized by endometrial glands and stroma outside the uterus, is diagnosed by direct visualization of peritoneal and ovarian implants during laparoscopy. Aim: To study the estrogenic microenvironment in eutopic endometria of women with and without endometriosis. Patients and methods: Eutopic endometria, obtained during laparoscopy from 23 women with endometriosis and 20 fertile cyclic women undergoing tubal sterilization, was studied. P450Arom mRNA expression (RT-PCR) was measured. Also, P450Arom activity was assessed measuring testosterone conversion to estradiol and the concentration of this last hormone in cultured endometrial explants. Results: Age and body mass index was similar in both groups studied. Seventy nine percent of endometria from women with endometriosis and in 29.4 percent from control group expressed P450Arom mRNA (p <0.01). The intensity of the band was higher in secretory endometria from women with endometriosis when compared to controls (p <0.01), but it was similar during the proliferative phase. Estradiol secretion to the culture media by proliferative endometria explants from women with endometriosis was 3-fold higher than secretory endometria (p <0.01) and endometria from control women in both phases. P450Arom activity, in the presence of testosterone, was 7-fold higher in endometrial cultures from women with endometriosis, when compare with the basal culture (p <0.01). However, in endometrial explant cultures from control women, this activity was not statistical different. Conclusions: These results indicate that in women with endometriosis, the microenvironment in the endometria is estrogenic as a consequence of an increased expression and activity of the P450 Arom.

[P450Arom and estrogenic microenvironment of eutopic endometria in endometriosis]. / P450Arom y microambiente estrogénico en endometrios eutópicos de mujeres con endometriosis.

BACKGROUND: Endometriosis, a common gynecologic disorder characterized by endometrial glands and stroma outside the uterus, is diagnosed by direct visualization of peritoneal and ovarian implants during laparoscopy. AIM: To study the estrogenic microenvironment in eutopic endometria of women with and without endometriosis. PATIENTS AND METHODS: Eutopic endometria, obtained during laparoscopy from 23 women with endometriosis and 20 fertile cyclic women undergoing tubal sterilization, was studied. P450Arom mRNA expression (RT-PCR) was measured. Also, P450Arom activity was assessed measuring testosterone conversion to estradiol and the concentration of this last hormone in cultured endometrial explants. RESULTS: Age and body mass index was similar in both groups studied. Seventy nine percent of endometria from women with endometriosis and in 29.4% from control group expressed P450Arom mRNA (p <0.01). The intensity of the band was higher in secretory endometria from women with endometriosis when compared to controls (p <0.01), but it was similar during the proliferative phase. Estradiol secretion to the culture media by proliferative endometria explants from women with endometriosis was 3-fold higher than secretory endometria (p <0.01) and endometria from control women in both phases. P450Arom activity, in the presence of testosterone, was 7-fold higher in endometrial cultures from women with endometriosis, when compare with the basal culture (p <0.01). However, in endometrial explant cultures from control women, this activity was not statistical different. CONCLUSIONS: These results indicate that in women with endometriosis, the microenvironment in the endometria is estrogenic as a consequence of an increased expression and activity of the P450Arom.

Glycosidases in the peritoneal fluid from infertile women with and without endometriosis.

OBJECTIVES: To investigate the activity and biochemical properties of glycosidases in the peritoneal fluid of infertile patients with or without endometriosis in comparison with fertile women. DESIGN AND METHODS: Peritoneal fluid was collected from 29 women undergoing a laparoscopy. The sample was separated into the following groups: fertile women (n = 11), infertile with endometriosis (n = 11), and infertile without endometriosis (n = 7). The activity of glycosidases was determined with specific p-nitrophenylglycosides as colorimetric substrates. RESULTS: The activity of alpha-fucosidase, alpha-glucosidase, alpha-mannosidase, beta-galactosidase, beta-glucuronidase, and beta-N-acetylhexosaminidase was investigated. Enzymatic activities of alpha-fucosidase and beta-N-acetylglucosaminidase were detected in higher amounts than other glycosidases. The activities of alpha-fucosidase and N-acetylglucosaminidase were increased in the case of infertile patients without endometriosis, while beta-galactosidase was increased in endometriosis patients. Enzyme properties as pH optimum, pH stability, thermal stability and inhibition by specific carbohydrates were similar for both control and infertile samples. Analysis of kinetic parameters indicate that Vmax values of glycosidases were higher for infertile samples than their respective controls. CONCLUSIONS: The results indicate that higher amounts of glycosidases are present in the peritoneal fluid from infertile patients. The elevated activity of these hydrolytic enzymes suggest possible deleterious effect on gametes, and could explain some cases of infertility.