Patrick G. Groothuis

Increased Production of 17β-Estradiol in Endometriosis Lesions Is the Result of Impaired Metabolism

CONTEXT substantial evidence suggests that the expression of steroid metabolizing enzymes in endometriosis is altered, turning the ectopic endometrium into a source of 17beta-estradiol. However, whether these differences result in a net increase in local 17beta-estradiol production/activity has not been shown. SUBJECTS AND METHODS The activities of the most important steroidogenic enzymes synthesizing and inactivating 17beta-estradiol were determined by HPLC in matched eutopic and ectopic tissue from patients with endometriosis (n = 14) and in endometrium from controls (n = 20). RESULTS Aromatase activity is negligible in the ectopic endometrium, whereas the activity of estrogen sulfatase is high though not different between ectopic, eutopic and control endometrium. The activity of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) converting estrone into 17beta-estradiol is higher in the ectopic compared to the eutopic endometrium in patients. The activity of 17beta-HSDs converting 17beta-estradiol back to estrone is significantly lower in the ectopic compared to the eutopic endometrium of both patients and controls. To evaluate the net metabolic capacity of tissues to synthesize 17beta-estradiol, we calculated the activity ratio between 17beta-HSDs synthesizing versus 17beta-HSDs inactivating 17beta-estradiol. This ratio is significantly higher in the ectopic compared to the eutopic endometrium of patients and controls, indicating a high synthesis of 17beta-estradiol in the ectopic locations. This is further supported by the elevated mRNA levels of the estrogen-responsive gene TFF1 in all ectopic compared to eutopic endometria. CONCLUSION Endometriotic lesions have higher production of 17beta-estradiol than the eutopic endometrium of patients and controls. This is mostly the result of impaired metabolism.

Oestrogen-modulated gene expression in the human endometrium

Abstract.To identify key regulatory mechanisms in the growth and development of the human endometrium, microarray analysis was performed on uncultured human endometrium collected during menstruation (M) and the late-proliferative (LATE-P)-phase of the menstrual cycle, as well as after 24 h incubation in the presence of oestradiol (17β-E2). We demonstrate the expression of novel gene transcripts in the human endometrium. i.e. mucin-9, novel oestrogen-responsive gene transcripts, i.e. gelsolin and flotillin-1, and genes known to be expressed in human endometrium but not yet shown to be oestrogen responsive, i.e. connexin-37 and TFF1/pS2. Genes reported to be expressed during the implantation window and implicated in progesterone action, i.e. secretoglobin family 2A, member 2 (mammaglobin) and homeobox-containing proteins, were up-regulated in uncultured LATE-P-phase endometrium compared to M-phase endometrium. Some gene transcripts are regulated directly by 17β-E2 alone, others are influenced by the in vivo environment as well. These observations emphasise that the regulation of endometrium maturation by oestrogen entails more then just stimulation of cell proliferation.

Oestrogen and progestin responses in human endometrium

Our understanding of the mechanisms of the actions of oestrogens and progestins have evolved from the simple concept of nuclear receptor-mediated regulation of transcription to a highly sophisticated, finely tuned interplay between various coregulators, other signaling cascades and transcription factors. The net result of these complex regulatory mechanisms is a steroid-, cell-, or tissue-specific action of oestrogens and progestins, their antagonists or selective modulators of their receptors. In this review, we have attempted to shed some light on the regulation of the actions of oestrogens and progestins on the human endometrium.

Adhesion of human endometrial fragments to peritoneum in vitro

OBJECTIVE To evaluate the adhesion of endometrial fragments obtained during the proliferative phase of the menstrual cycle to fresh human peritoneum obtained during abdominal surgery. DESIGN A prospective, descriptive, morphologic and cell biologic study. SETTING Tertiary care university medical center. PATIENT(S) Six female volunteers. INTERVENTION(S) After endometrial biopsies performed during diagnostic laparoscopy, endometrial fragments were generated by enzymatic digestion and mechanical separation. Peritoneum was obtained during abdominal operations for benign indications. MAIN OUTCOME MEASURE(S) Adhesion of endometrial fragments was studied by histologic examination and scanning and transmission electron microscopy. RESULT(S) After incubation, the mesothelium was intact in some areas, whereas in other areas mesothelial cells were damaged or absent. Adhesion of endometrial fragments was observed only at locations where the basement membrane was exposed. In areas largely denuded of mesothelial cells, endometrial fragments spread over the basement membrane to form monolayers. CONCLUSION(S) Human peritoneum is suitable for studying the adhesion of endometrial fragments. Intact mesothelium prevents the adhesion of endometrial fragments, suggesting that trauma to the mesothelial lining is a prerequisite for endometrial cell adhesion.

Fibromuscular differentiation in deeply infiltrating endometriosis is a reaction of resident fibroblasts to the presence of ectopic endometrium

BACKGROUND In this study, we characterized the fibromuscular (FM) tissue, typical of deeply infiltrating endometriosis, investigated which cells are responsible for the FM reaction and evaluated whether transforming growth factor-beta (TGF-beta) signaling is involved in this process. METHODS FM differentiation and TGF-beta signaling were assessed in deeply infiltrating endometriosis lesions (n = 20) and a nude mouse model of endometriosis 1, 2, 3 and 4 weeks post-transplantation. The FM reaction was evaluated by immunohistochemistry using different markers of FM and smooth muscle cell differentiation (vimentin, desmin, alpha-smooth muscle actin, smooth muscle myosin heavy chain). TGF-beta signaling was assessed by immunostaining for its receptors and phosphorylated Smad. RESULTS Deeply infiltrating endometriosis lesions contain myofibroblast-like cells that express multiple markers of FM differentiation. Expression of TGF-beta receptors and phospho-Smad was more pronounced in the endometrial component of the lesions than in the FM component. In the nude mouse model, alpha-smooth muscle actin expression was observed in murine fibroblasts surrounding the lesion, but not in human endometrial stroma. CONCLUSIONS FM differentiation in deeply infiltrating endometriosis is the result of a reaction of the local environment to the presence of ectopic endometrium. It shares characteristics with pathological wound healing, but cannot be explained by TGF-beta signaling alone.

Aberrations in the progesterone receptor gene and the risk of recurrent endometrial carcinoma

A case–control study was performed in order to determine whether expression of the progesterone receptor (PR) and/or aberrations of the PR gene contribute to the development of recurrent endometrial carcinoma. Primary tumours from 44 patients with recurrence of stage I endometrial carcinoma (patients) within 3 years after initial treatment were compared with tumours from 44 matched patients who were free of recurrence for a minimum of 3 years (controls). Paraffin wax‐embedded primary tumours (n = 88) and recurrent tumours (n = 32) were analysed immunohistochemically for PR expression. A staining index (SI = 0–9) based on the staining intensity and the number of stained cells was calculated. DNA extracted from paraffin wax‐embedded tissues was subjected to PCR–restriction fragment length polymorphism analysis (PCR–RFLP) for determination of the PROGINS DNA sequence alterations and the +331G/A‐promoter polymorphism. Low PR expression (SI < 1.0) was observed in 7% of primary tumours derived from controls, 25% of primary tumours from patients with recurrence, and 38% of recurrent tumours. The expression of PR was significantly lower in primary tumours from patients with recurrence (SI = 4.0 ± 0.5) than in the tumours in the control group (SI = 5.6 ± 0.5) (T‐test for paired analysis, p < 0.05). The PROGINS and +331G/A‐promoter polymorphism were not related to age at diagnosis, tumour grade or myometrial invasion. The +331G/A‐promoter polymorphism was present in 14% of primary tumours from patients without recurrence, compared with 17% of patients with recurrence. The PROGINS polymorphism was observed in 16% of primary tumours from patients without, and in 34% of patients with, recurrence (OR 2.6; 95% CI: 0.9–7.6). Most interestingly, patients who carried the PROGINS variant and in whom a PR‐expressing tumour was diagnosed were at significantly enhanced risk of relapse (OR 4.7; 95% CI: 1.3–17.1). In conclusion, low PR expression tended to be associated with recurrent disease, and PR expression in tumours from patients carrying the PROGINS allele was predictive of the risk of recurrence. Copyright

Matrix metalloproteinases and their tissue inhibitors in antegradely shed menstruum and peritoneal fluid

OBJECTIVE To investigate the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in antegradely shed menstruum and peritoneal fluid. DESIGN A cell biological and immunohistochemical study. SETTING Tertiary care university medical center. INTERVENTION(S) Immunohistochemistry was performed on cryostat sections and cultures of menstrual endometrium. Zymography was used to characterize MMP activity in peritoneal fluid, in menstrual serum, and in conditioned medium. Western blot analysis was used to further identify the MMPs in these fluids. MAIN OUTCOME MEASURE(S) Staining of MMPs and TIMPs in cryostat sections and cultures and MMP expression and activity in peritoneal fluid and menstrual blood serum. RESULT(S) Strong staining for MMP-1 and MMP-3 was observed in stroma and for MMP-7 in epithelium. Matrix metalloproteinase-2 and MMP-9 were weakly expressed in stroma. Both TIMP-1 and TIMP-2 were expressed in menstrual endometrium. Menstrual serum showed a pattern of MMP activity on zymography different from peritoneal fluid. Western blot analysis showed the presence of MMP-7 and MMP-9 in menstrual serum. CONCLUSION(S) Antegradely shed menstrual endometrium expresses several MMPs and TIMPs, even after culturing for 24 hours. MMP activity in menstrual serum is different from and more intense than MMP activity in peritoneal fluid. These enzymes may be involved in the early invasion of menstrual endometrium into the extracellular matrix of the peritoneum.

Deoxyribonucleic acid methyltransferases and methyl-CpG-binding domain proteins in human endometrium and endometriosis.

OBJECTIVE To determine [1] expression levels of both DNA methyltransferases (DNMTs) and methyl-CpG-binding domain proteins (MBDs) in human endometrium throughout the menstrual cycle and in eutopic and ectopic endometrium of patients with endometriosis and [2] hormone responsiveness of DNMT and MBD expression in explant cultures of proliferative phase endometrium. DESIGN In vitro study. SETTING Academic medical center. PATIENT(S) Premenopausal women with and without endometriosis. INTERVENTION(S) Explant cultures of proliferative phase endometrium were treated with vehicle, 17β-E(2), or a combination of E(2) and P (E(2) + P) for 24 hours. MAIN OUTCOME MEASURE(S) Expression levels of DNMT1, DNMT2, and DNMT3B and MBD1, MBD2, and MeCP2 with use of real-time quantitative polymerase chain reaction. RESULT(S) Expression levels of DNMT1 and MBD2 were significantly higher in secretory-phase endometrium compared with proliferative endometrium and menstrual endometrium. In explant cultures, treatment with E(2) + P resulted in significant up-regulation of DNMT1 and MBD2. Expression levels of several DNMTs and MBDs were significantly lower in endometriotic lesions compared with eutopic endometrium of women with endometriosis and disease-free controls. CONCLUSION(S) These findings suggest a role for DNMTs and MBDs in the growth and differentiation of the human endometrium and support the notion that endometriosis may be an epigenetic disease.

Haemoglobin expression in human endometrium

BACKGROUND The general concept that haemoglobin is only a carrier protein for oxygen and carbon dioxide is challenged since recent studies have shown haemoglobin expression in non-erythroid cells and the protection of haemoglobin against oxidative and nitrosative stress. Using microarrays, we previously showed expression of haemoglobins alpha, beta, delta and gamma and the haeme metabolizing enzyme, haeme oxygenase (HO)-1 in human endometrium. METHODS Using real-time quantitative PCR, haemoglobin alpha, beta, delta and gamma, and HO-1 mRNA levels were assessed throughout the menstrual cycle (n = 30 women). Haemoglobin and HO-1 protein levels in the human endometrium were assessed with immunohistochemistry. For steroid responsiveness, menstrual and late proliferative-phase endometrial explants were cultured for 24 h in the presence of vehicle (0.1% ethanol), estradiol (17beta-E(2,) 1 nM), progestin (Org 2058, 1 nM) or 17beta-E(2)+Org 2058 (1 nM each). RESULTS All haemoglobins and the HO-1 were expressed in normal human endometrium. Haemoglobin mRNA and protein expression did not vary significantly during the menstrual cycle. Explant culture with Org 2058 or 17beta-E(2)+Org 2058 increased haemoglobin gamma mRNA expression (P < 0.05). HO-1 mRNA levels, and not protein levels, were significantly higher during the menstrual (M)-phase of the cycle (P < 0.05), and were down-regulated by Org 2058 in M-phase explants and by 17beta-E(2)+Org 2058 in LP-phase explants, versus control (P < 0.05). CONCLUSIONS The haemoglobin-HO-1 system may be required to ensure adequate regulation of the bioavailability of haeme, iron and oxygen in human endometrium.

Menstruum Induces Changes in Mesothelial Cell Morphology

In previous studies, we have shown that menstrual endometrium preferentially adheres to the subepithelial lining of the peritoneum. It remains to be elucidated, however, whether this damage is preexisting or inflicted by the menstrual tissue itself. We hypothesized that the menstrual tissue itself damages the peritoneum. To investigate this, the viability of menstrual endometrial tissue in peritoneal fluid (PF) was evaluated and the morphologic changes in the mesothelial cells were studied by in vitro cocultures of menstruum with mesothelial cell monolayers. Menstruum was collected with a menstrual cup. Endometrial tissue was isolated from the menstruum, resuspended in culture medium or in the cell-free fraction of PF and cultured for 24, 48 or 72 h. A 3(4,5-dimethylthia-zolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to obtain a relative measure of viable adhered endometrial cells. Mesothelial cells isolated from human omental tissue were cultured on Matrigel or uncoated plastic. At confluence, overnight cocultures were performed and scanning electron microscopy was used to evaluate the morphologic changes. The viability of endometrial fragments was 84% (n = 36, p < 0.05), 82% (n = 27, not significant) and 104% (n = 14, not significant) when cultured in the cell-free fraction of PF for 24, 48 and 72 h, respectively, when compared to medium with 10% fetal calf serum. Menstrual endometrial fragments or menstrual serum added to and cocultured with mesothelial cells induced severe morphologic alterations of the latter, including retraction, shrinking and gap formation. Similar morphologic changes were observed when mesothelial cells were cocultured with menstrual endometrial fragments in PF or in culture inserts. Incubation with conditioned medium from cultured menstrual endometrium induced similar but less pronounced changes in morphology. In conclusion, menstrual endometrial fragments remain viable in PF in vitro for at least 72 h. Antegradely shed menstruum induces changes in mesothelial cell morphology, including retraction and shrinking with exposure of the underlying surface. These findings suggest that menstruum is harmful to the peritoneal lining. Therefore, by local destruction of the mesothelial layer, menstrual endometrium is able to create sites for adhesion.