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Study Question: Are the primary cell cultures and cell lines used in endometriosis research of sufficient quality?, Summary Answer: Primary cells used in endometriosis research lack purity and phenotypic characterisation, and cell lines are not genotypically authenticated., What Is Known Already: The poor reproducibility of in vitro research and the lack of authenticity of the cell lines used represent reasons of concern in the field of reproductive biology and endometriosis research., Study Design, Size, Duration: In the present study, past in vitro research in the field of endometriosis was systematically reviewed to determine whether the appropriate quality controls were considered. In addition, we explored the performance of Paired Box 2 (Pax2) as an endometrium specific marker in endometrial and endometriotic primary cell cultures; we also characterised the most diffused endometriosis cell lines with respect to important markers including the short tandem repeat (STR) profile., Participants/materials, Setting, Methods: Literature review part: almost 300 published protocols describing the isolation and creation of primary cell cultures from endometriosis were reviewed. Wet-lab part: primary cells isolated from 13 endometriosis patients were analysed by immunohistochemistry, immunofluorescence and FACS for the expression of Pax2. Cell lines Z11 and Z12, the most diffused endometriosis cell lines, were characterised with respect to the expression of Pax2, steroid hormone receptors and STR profile., Main Results and the Role of Chance: From the literature review work, we underscored the lack of sufficient cell purity and phenotypic characterisation of primary cell cultures, which present high risk of contaminations from surrounding non-endometriotic tissues. Past work based on the use of cell lines was reviewed as well, and it emerged that cell line authentication was never performed.In an effort to address these weaknesses for future research, we present data on the performance of Pax2, a suitable marker to exclude ovarian (and other non-endometrial) cell contaminations from primary cell cultures; STR profiles of cell lines Z11 and Z12 were analysed and indicated that the cells were authentic. These profiles are now available for authentication purposes to researchers wishing to perform experiments with these cells.A quality control pipeline to assure sufficient quality of in vitro research in the field of reproductive biology and endometriosis is proposed. We encourage scientists, research institutes, journal reviewers, editors and funding bodies to raise awareness of the problem and adopt appropriate policies to solve it in the future., Large-Scale Data: STR profiles of cell lines Z11 and Z12 are deposited at the Cellosaurus database-web.expasy.org., Limitations, Reasons for Caution: There may be additional markers suitable to assess cell quality., Wider Implications of the Findings: Future in vitro research in endometriosis and the reliability of outcomes can be improved by using the recommendations presented in this study., Study Funding/competing Interest(s): The study was partly financed by the 'Stichting Fertility Foundation' (The Netherlands). The authors declare no existing conflict of interest., Trial Registration Number: Non-applicable., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Context: Endometriosis affects 10% of the women before menopause and has important personal, professional, and societal economic burdens. Because current medical treatments are aimed at reducing the symptoms only, novel therapeutic targets should be identified. Endometriosis is estrogen dependent and in some patients the endometriosis tissue is able to produce estrogens in an autocrine/paracrine manner. In a number of patients, this is the consequence of the high local activity of the 17β-hydroxysteroid-dehydrogenases (17β-HSDs), enzymes able to generate active estrogens from precursors with low activity., Objective: The objective of the study was to identify the 17β-HSD(s) responsible for the high local generation of estrogens in endometriosis and test the possibility to inhibit these enzymes for therapeutic purposes., Design: The expression of different 17β-HSDs involved in the estrogen metabolism was assessed by real-time PCR in eutopic and ectopic tissue from endometriosis patients (n=14). These biopsies had previously confirmed unbalanced local 17β-HSD activity, which caused high estrogen generation. The possibility to block the synthesis of estrogens by one inhibitor specific for type 1 17β-HSD was assessed by HPLC in tissue lysates from endometriosis tissues (n=27)., Results: In all but one of the patients, a high type 1 17β-HSD level is associated with the unbalanced metabolism of estrogens, leading to higher estrogen synthesis in endometriosis than in the endometrium inside the uterus. Inhibition of type 1 17β-HSD restores to various extents, depending on the patient, the correct metabolism. In 19 of 27 patients analyzed (70%), the 17β-HSD type 1 inhibitor decreased the generation of 17β-estradiol by greater than 85%., Conclusions: Inhibition of 17β-HSD type 1 can be a potential future treatment option aimed at restoring the correct metabolic balance of estrogens in endometriosis patients with increased local 17β-HSD type 1 enzyme activity.

Background: Paired-box 2 (Pax2) is involved in the development of the female genital tract and has been associated with endometrial pathologies. The expression of Pax2 is induced by epidermal growth factor (EGF) and estrogens. In the present study, Pax2 expression and regulation were investigated in endometriosis., Methods and Results: Pax2 protein expression was assessed by immunohistochemistry in the eutopic (i.e. inside the uterus) and ectopic tissue (endometriosis) from 11 patients. Immunoreactivity was high in the endometrium, with strong epithelial and weaker stromal staining. Similar expression patterns of Pax2 were observed in the endometrium of women without endometriosis (n = 12). The mRNA level of Pax2 was assessed by real-time PCR in the eutopic and ectopic endometria of 14 patients and in the endometrium from women without endometriosis (n = 20). Pax2 expression was lower in endometriotic lesions than that in the eutopic endometrium of patients (P< 0.001) and controls (P= 0.007). Three possible mechanisms determining low Pax2 expression were investigated: EGF signalling, CpG DNA methylation of the Pax2 promoter and steroid response. The mRNA level of the EGF receptor (EGFR1) was assessed in the samples used for Pax2 mRNA assessment. A significant correlation between EGFR1 and Pax2 in both eutopic and ectopic tissues was observed (R = 0.58; slope regression line, 0.81; 95% CI: 0.09-1.52 and R = 0.54; slope regression line, 2.51; 95% CI: 0.02-4.99, respectively). CpG DNA methylation was analyzed by methyl-specific PCR in two regions of the Pax2 promoter but they were unmethylated in all samples. Steroid responsiveness was assessed using endometrial explant cultures and Pax2 was not regulated by either 17β-estradiol or progesterone., Conclusions: In endometriosis patients, Pax2 is down-regulated in the lesions compared with the eutopic tissue, possibly due to low EGF signalling.

Context: The local interconversions between estrone (low activity) and 17β-estradiol (potent compound) by 17β-hydroxysteroid dehydrogenases (17β-HSDs) can lead to high 17β-estradiol generation in endometrial cancer (EC)., Objective: Examine the balance between the 17β-HSDs reducing estrone to 17β-estradiol (types 1, 5, 12, and 7) and those oxidizing 17β-estradiol to estrone (2, 4, and 8), in EC., Patients and Methods: Reducing and oxidizing 17β-HSD activities (HPLC) and mRNA level (RT-PCR) were assessed in normal post-menopausal (n = 16), peritumoral endometrium (normal tissue beside cancer, n = 13), and 58 EC (29 grade 1, 18 grade 2, 11 grade 3)., Results: Grade 1 EC displayed a shifted estrone reduction/17β-estradiol oxidation balance in favor of 17β-estradiol compared with controls. This was more pronounced among estrogen receptor-α (ER-α)-positive biopsies. Type 1 17β-HSD mRNA (HSD17B1 gene expression, real time PCR) and protein levels (immunohistochemistry) were higher in ER-α-positive grade 1 EC than controls. The mRNA coding for types 4, 5, 7, 8, and 12 17β-HSD did not vary, whereas that coding for type 2 17β-HSD was increased in high-grade lesions compared with controls. Three-dimensional ex vivo EC explant cultures demonstrated that 17β-HSD type 1 generated 17β-estradiol from estrone and increased tumor cell proliferation. Additional in vitro studies using EC cells confirmed that in the presence of 17β-HSD type 1, estrone induced estrogen signaling activation similarly to 17β-estradiol. Therefore, estrone was reduced to 17β-estradiol., Conclusions: Type 1 17β-HSD increases 17β-estradiol exposure in grade 1 EC, thus supporting tumor growth. This enzyme represents a potential therapeutic target.

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.

Background: Estradiol (E(2)) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent., Methods: In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17beta-hydroxysteroid dehydrogenases (17beta-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)] in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures., Results: Aromatase and 17beta-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17beta-HSD, EST and STS were readily detectable. Only 17beta-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17beta-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies., Conclusions: In endometriosis lesions, the balance is tilted in favor of enzymes producing E(2). This is due to a suppression of types 2 and 4 17beta-HSD, and an increased expression of aromatase and type 1 17beta-HSD in ectopic endometrium.

We have developed a liquid chromatography-isotope dilution mass spectrometry procedure to quantify total cholesterol in serum. A particle-beam interface was used for coupling the liquid chromatograph and the mass spectrometer. After electron impact ionization the ions m/z = 386 and m/z = 389 were used for selective ion monitoring of cholesterol and the internal standard [25,26,27-(13)C]cholesterol. The sample preparation steps required for serum materials are alkaline hydrolysis and an extraction of the cholesterol into the cyclohexane phase. Imprecision for the determination of cholesterol in control materials is typically <1.0%. The deviation from the certified reference values was <0.75% for all control materials tested. A method comparison of the results obtained by this method with those obtained by gas chromatography-isotope dilution mass spectrometry for n = 28 pooled human sera derived from samples analyzed in our routine laboratory did not show differences >2.5%.