Hydrogels from TEMPO-Oxidized Nanofibrillated Cellulose Support In Vitro Cultivation of Encapsulated Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are the most prominent type of adult stem cells for clinical applications. Three-dimensional (3D) cultivation of MSCs in biomimetic hydrogels provides a more physiologically relevant cultivation microenvironment for in vitro testing and modeling, thus overcoming the limitations of traditional planar cultivation methods. Cellulose nanofibers are an excellent candidate biomaterial for synthesis of hydrogels for this application, due to their biocompatibility, tunable properties, availability, and low cost. Herein, we demonstrate the capacity of hydrogels prepared from 2,2,6,6-tetramethylpiperidine-1-oxyl -oxidized and subsequently individualized cellulose-nanofibrils to support physiologically relevant 3D in vitro cultivation of human MSCs at low solid contents (0.2-0.5 wt %). Our results show that MSCs can spread, proliferate, and migrate inside the cellulose hydrogels, while the metabolic activity and proliferative capacity of the cells as well as their morphological characteristics benefit more in the lower bulk cellulose concentration hydrogels.

Sexual Function in Postmenopausal Women With Symptomatic Pelvic Organ Prolapse Treated Either with Locally Applied Estrogen or Placebo: Results of a Double-Masked, Placebo-Controlled, Multicenter Trial.

BACKGROUND: Local estrogen therapy (LET) has beneficial effects on genitourinary atrophy; however it is currently unclear if LET improves sexual function in postmenopausal women with pelvic organ prolapse (POP). AIM: To evaluate if LET vs placebo results in an improved sexual function in postmenopausal women with symptomatic POP. METHODS: We performed a secondary analysis of sexual outcomes of a previous randomized controlled trial comparing LET and placebo in 120 postmenopausal women (60/group) with symptomatic POP stage ≥3 and planned prolapse surgery. Women were randomly assigned to receive local estrogen or placebo cream 6 weeks preoperatively. The effect of therapy vs placebo was assessed with ANOVA with interaction effect of time*group and a multivariable linear regression model was built to assess the impact of different variables on sexual function before therapy. OUTCOMES: We evaluated the sexual function score in sexually active women of our study population using the German Pelvic Floor Questionnaire at recruitment time and again after 6 weeks of treatment. RESULTS: Among 120 randomized women, 66 sexually active women remained for final analysis. There was no significant difference in the change of the sexual function score over time between the treatment groups (difference in changes in score from baseline to 6 weeks for Estrogen group vs control group was -0.110 with 95% CI -0.364 to 0.144) Multivariable analysis showed that no independent risk factor for unsatisfying sexual function score could be identified. CLINICAL IMPLICATIONS: Based on our results, LET has no beneficial effect on sexual function in postmenopausal women with POP. STRENGTHS AND LIMITATIONS: Main strength of our study lies in the study design and in the use of a condition- specific questionnaire. As this is a secondary analysis, this study may be insufficiently powered to identify differences in sexual data between groups. CONCLUSION: LET had no impact on female sexuality in postmenopausal women with POP. Marschalek M-L, Bodner K, Kimberger O, et al. Sexual Function in Postmenopausal Women With Symptomatic Pelvic Organ Prolapse Treated Either with Locally Applied Estrogen or Placebo: Results of a Double-Masked, Placebo-Controlled, Multicenter Trial. J Sex Med 2022;19:1124-1130.

Prevalence of iron deficiency in pregnant women: A prospective cross-sectional Austrian study.

The aim of the study was to determine, for the first time, in a prospective cross-sectional multicenter study, the prevalence of iron deficiency (ID) in an Austrian pregnant population. A cohort of 425 pregnant women was classified into four groups of different weeks of gestation. Group 1 was monitored longitudinally, while groups 2-4, iron status, were sampled only once. Evaluation of the prevalence of ID was performed by comparing the diagnostic criteria of the WHO to the cutoff proposed by Achebe MM and Gafter-Gvili A (Achebe) and the Austrian Nutrition Report (ANR). In comparison with the ANR, the prevalence of ID was lower in group 1 and higher in groups 2-4 (17.2% vs. 12.17%, 25.84%, 35.29%, and 41.76%, respectively) (p-values < .01 except group 1). According to WHO, the prevalence in group 1 was 12.17% at inclusion, 2 months later 31.7%, and further 2 months later 65.71%, respectively. According to Achebe, the number of cases doubled; for group 1, the number of cases rose from 13 to 42 (115 patients total); for groups 2-4, we observed an increase from 112 to 230 (340 patients total). This study reported a prevalence of around 12% at the beginning of pregnancy, which increased during pregnancy up to 65%. ID can have a massive impact on quality of life, justifying screening, as iron deficiency would be easy to diagnose and treat.

Surgical Assessment of Tissue Quality during Pelvic Organ Prolapse Repair in Postmenopausal Women Pre-Treated Either with Locally Applied Estrogen or Placebo: Results of a Double-Masked, Placebo-Controlled, Multicenter Trial.

The aim of this prospective randomized, double-masked, placebo-controlled, multicenter study was to analyze the surgeon's individual assessment of tissue quality during pelvic floor surgery in postmenopausal women pre-treated with local estrogen therapy (LET) or placebo cream. Secondary outcomes included intraoperative and early postoperative course of the two study groups. Surgeons, blinded to patient's preoperative treatment, completed an 8-item questionnaire after each prolapse surgery to assess tissue quality as well as surgical conditions. Our hypothesis was that there is no significant difference in individual surgical assessment of tissue quality between local estrogen or placebo pre-treatment. Multivariate logistic regression analysis was performed to identify independent risk factors for intra- or early postoperative complications. Out of 120 randomized women, 103 (86%) remained for final analysis. Surgeons assessed the tissue quality similarity in cases with or without LET, representing no statistically significant differences concerning tissue perfusion, tissue atrophy, tissue consistency, difficulty of dissection and regular pelvic anatomy. Regarding pre-treatment, the rating of the surgeon correlated significantly with LET (r = 0.043), meaning a correct assumption of the surgeon. Operative time, intraoperative blood loss, occurrence of intraoperative complications, total length of stay, frequent use of analgesics and rate of readmission did not significantly differ between LET and placebo pre-treatment. The rate of defined postoperative complications and use of antibiotics was significantly more frequent in patients without LET (p = 0.045 and p = 0.003). Tissue quality was similarly assessed in cases with or without local estrogen pre-treatment, but it seems that LET prior to prolapse surgery may improve vaginal health as well as tissue-healing processes, protecting these patients from early postoperative complications.

Relaxin and gonadal steroid receptors in uterosacral ligaments of women with and without pelvic organ prolapse.

INTRODUCTION AND HYPOTHESIS: This study evaluates the expression of estrogen receptor isoforms alpha (ERα) and beta (ERß), progesterone receptor (PR), and relaxin receptor isoforms 1 and 2 (LGR7, LGR8) in uterosacral ligament (USL) tissue of women with pelvic organ prolapse and controls. METHODS: Tissue samples of USL from women with and without pelvic organ prolapse (POP) were subjected to immunohistochemistry against ERα, ERß, PR, and LGR7 proteins. The respective mRNA expression as well as of LGR8 was assessed by quantitative real-time polymerase chain reaction. RESULTS: The cellular distribution of the receptor proteins was different due to cell types, independent of POP: ERα and PR were found in smooth muscle cells, but not in endothelial cells, whereas ERß was found in endothelial cells, but not in connective tissue. ERα, ERß, PR, and LGR7 mRNAs could be detected in all patients of both groups. ERα mRNA expression was significantly and ERß mRNA borderline significantly higher in USL of patients with POP: ERα: p < 0.001, ERß: p = 0.057. CONCLUSIONS: Enhanced effects of estrogen via altered mRNA expression patterns of ERα and ERß--but not those of progesterone--may exist in USL of patients affected by POP. A local effect of relaxin needs to be further clarified because of this first report of prevalent ligamental expression of LGR7.

The human female prostate-immunohistochemical study with prostate-specific antigen, prostate-specific alkaline phosphatase, and androgen receptor and 3-D remodeling.

INTRODUCTION: The constitution of glands surrounding the human female urethra has been under debate; especially regarding as to what extent they equal the male prostate. Defining their composition may help to understand the development of neoplasms arising from this tissue. AIMS: The aim of this study was to define the existence, structure, and arrangement of a possible human female prostate. METHODS: Urethras of 25 women were investigated by immunohistochemistry and stained with specific monoclonal antibodies against prostate-specific antigen (PSA, mono- and polyclonal antibody), prostate specific alkaline phosphatase (PSAP), and androgen receptor (AR). From two urethras, which underwent a totally serial work up with PSA-staining, a three-dimensional model of the urethra and the prostatic glands was created to enable 3D-perception of the results. MAIN OUTCOME MEASURE: The main outcome measures used in this study were identifying glandular structures in hematoxylin-eosin-staining, positive staining with the respective antibodies, and 3-D orientation of described glands. RESULTS: Fourteen of 25 patients had glandular structures encircling the urethra. Twelve of 14 showed positive staining for PSA, PSAP, and AR in gland acini, while the excretory ducts, the urethra, and the surrounding stroma did not express those proteins. The strongest PSA and PSAP expression was found in apical cytoplasm of the glandular cells, and AR was confined to cell nuclei. Prostatic glands were located laterally to the distal half of the urethra. CONCLUSION: A female prostate was found in every second woman in this study and can be discriminated from other urethral caverns and immature paraurethral ducts. Possible neoplasms of this source tissue expressing the prostate-specific markers may therefore be denominated as female prostate tumors.

Anti-Mullerian hormone levels decline under hormonal suppression: a prospective analysis in fertile women after delivery.

BACKGROUND: AMH's reported stability during periods of hormonal change makes it a practical tool in assessing ovarian reserve. However, AMH declines with age and age-specific cut-offs remain to be established in women with proven fertility. This study aims to determine age-specific ranges of AMH in women with proven fertility. METHODS: Two hundred-ten fertile women, aged 18-40 years, were prospectively recruited for AMH measurements within 14 days after delivery and age stratified into 3 groups (18-30, 31-36 and 37-40 years). Eligibility required spontaneous conception within a maximal period of six months. Autoimmune diseases, chemotherapy, radiation, ovarian surgery and polycystic ovary syndrome precluded inclusion. RESULTS: 95% confidence intervals of AMH declined with advancing female age from 0.9-1.1 to 0.6-0.9 and 0.2-0.4 ng/mL (P < 0.001). AMH levels were not statistically associated with day of blood draw after delivery or pregnancy characteristics. Neither were they predictive of resumption of menses. They, however, at all ages were lower than reported in the literature for infertile patients. CONCLUSIONS: Like infertile populations, fertile women demonstrate declining AMH with advancing age. Uniformly lower levels than in infertile women suggest that AMH levels do not appear as stable under all hormonal influences as previously reported.

Testosterone dependent androgen receptor stabilization and activation of cell proliferation in primary human myometrial microvascular endothelial cells.

OBJECTIVE: To clarify, whether uterine endothelial proliferation could be regulated via an autocrine estrogen producing mechanism or direct actions of testosterone. DESIGN: In vitro study. SETTING: Tertiary care facility. PATIENT(S): Human myometrial tissue obtained from 40 women undergoing hysterectomy without further intrauterine pathology. INTERVENTION(S): Cell culture, proliferation assay and CYP19 activity assay on human myometrial endothelial cells treated with testosterone, estradiol, letrozole, flutamide, PD98059, MG-132 alone or in combination. MAIN OUTCOME MEASURE(S): We analyzed whether aromatase is expressed in human myometrial microvascular endothelial cells (HMMECs) and whether it affects proliferation and converts androgens to estrogens. In addition, we aimed to define whether or not T could have a direct capability to affect HMMEC proliferation. RESULT(S): Using quantitative real-time PCR and Western analysis, primary passage four HMMECs were shown to express low levels of aromatase mRNA and protein, respectively. However, HMMECs were unable to convert radioactively labeled 3∗H-1ß-androstenedione to estrogen. Pharmacologic doses of T (10(-6) and 10(-4) M) increased HMMEC proliferation, assessed through a bromodeoxyuridine ELISA. This effect of T on proliferation could not be blocked after pretreatment of cells with the aromatase inhibitor letrozole. In addition, HMMECs were found to express androgen receptors (ARs), and the AR antagonist flutamide abolished T-dependent proliferation. T was shown to increase AR protein levels, which was due to T-dependent receptor stabilization and not activation of gene transcription. CONCLUSION(S): We conclude that myometrial endothelial proliferation is not regulated through myometrial endothelial estrogen production. However, pharmacologic doses of T increase myometrial endothelial proliferation through a receptor-dependent and -stabilizing mechanism.

Multiple site sampling does not increase the sensitivity of Chlamydia trachomatis detection in infertility patients.

OBJECTIVE: Persistent Chlamydia trachomatis infections are associated with tubal pathology. We studied whether sampling from multiple sites would increase the identification of the infections. DESIGN: Prospective cohort study. SETTING: Tertiary care facility. PATIENT(S): Two hundred two infertile women. INTERVENTION(S): Smears were taken from the cervix, urethra, high vagina, fimbriae and the Douglas cavity. Blood samples were collected and tubal patency was assessed by pertubation with lipiodol and methylene blue. MAIN OUTCOME MEASURE(S): Detection of C. trachomatis DNA, detection of IgA and IgG antibodies against C. trachomatis, and antibodies against chlamydial heat-shock protein 60, tubal patency. RESULT(S): Chlamydia trachomatis was detected in 2 of 202 patients, for an overall prevalence of 1%. In both patients PCR results were positive in the cervical, vaginal, and urethral specimens. Chlamydia trachomatis IgG, IgA, and chlamydial heat-shock protein 60 IgG were significantly more prevalent in women with distal tubal pathology than in those without (26/40 [65.0%] vs. 16/162 [9.9%], 9/40 [22.5%] vs. 7/162 [4.3%], and 34/40 [85.0%] vs. 34/162 [21.0%]). Bacterial colonization was found in 1 of 202 samples from the Douglas cavity. CONCLUSION(S): Routine DNA testing for C. trachomatis should be confined to cervical sampling. The association between tubal pathology and seropositivity of IgG, IgA, and cHSP60 IgG was confirmed but did not add clinically valuable information during the diagnostic workup of infertility patients.

Estrogen receptor-beta is the predominant estrogen receptor subtype in normal human synovia.

OBJECTIVE: Joint pain increases after menopause with more than 50% of woman suffering from arthralgies. Since pain and inflammation of joints originate from synovial tissue, we aimed to discover whether estrogen receptors are present in the human synovia. METHODS: This in vitro study was performed on samples of human synovial tissue, obtained from pre- (n = 8) and postmenopausal woman (n = 11) and men (n = 5) following surgery due to traumatic lesions. Fresh synovial tissue specimens were assessed for the localization as well as the presence of estrogen receptor-alpha (ER alpha) and estrogen receptor-beta (ER beta) by means of immunohistochemistry, as well as Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: ER beta protein and mRNA were found to be equally and highly expressed in synovial stroma and lining cells of all explants independent of sex or menopausal status. In contrast, weak ER alpha staining was localized in the synovial lining cells in only three of 24 explants. ER alpha protein was found to be weakly expressed in three of ten explants. ER alpha mRNA was found with highly variable amounts in seven of ten explants. CONCLUSION: In view of our observation that ER beta but not ER alpha is expressed regularly in normal human synovia in high amounts, we propose that estrogen could play a significant role in synovial membrane function in women and men, operating preferably via the ER beta isoform.

The estrogen metabolite 17beta-dihydroequilenin counteracts interleukin-1alpha induced expression of inflammatory mediators in human endothelial cells in vitro via NF-kappaB pathway.

In most studies showing cardio- and vasculoprotective effects of estrogens, 17beta-estradiol was used and little information on possible effects of different estrogen metabolites is yet available. We investigated whether particular estrogen metabolites are effective in counteracting inflammatory activation of human endothelium. Human endothelial cells were incubated with 17alpha-dihydroequilenin, 17beta-dihydroequilenin, delta-8,9-dehydroestrone, estrone and 17beta-estradiol and stimulated with interleukin (IL)-1alpha. The expression of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) was determined. 17beta-dihydroequilenin and 17beta-estradiol at a concentration of 1 microM reduced IL-1alpha-induced up regulation of IL-6, IL-8 and MCP-1 close to control levels. When both compounds were used in combination an additive effect was observed. 17alpha-dihydroequilenin and delta-8,9-dehydroestrone showed a similar anti-inflammatory effect only when used at 10 microM whereas estrone had no effect. The effect of 17beta-dihydroequilenin on IL-1alpha-induced production of IL-6, IL-8 and MCP-1 was reversed by the estrogen receptor antagonist ICI 182,780. 17beta-dihydroequilenin also inhibited IL-1alpha-induced translocation of p50 and p65 to the nucleus of the cells. We have identified the estrogen metabolite 17beta-dihydroequilenin, as an inhibitor of inflammatory activation of human endothelial cells. Characterization of specific estrogens--as shown in our study--could provide the basis for tailored therapies, which might be able to achieve vasoprotection without adverse side effects.

Expression of estrogen receptors in human corpus cavernosum and male urethra.

Estrogen, largely produced in testis and adrenal gland, may play important roles in male reproduction. Most of the effects of estrogens are mediated by binding of estrogen to one or both of the two estrogen receptor (ER) subtypes alpha and beta. Recently, they have been described in testis, prostate, and efferent ducts, mostly in rodents. The goal of this study was to prove the evidence of ERs in human corpus cavernosum and male urethra, exploring the protein expression of these receptors by immunohistochemistry. Corpus cavernosum and corpus spongiosum smooth muscle was immunoreactive for the androgen receptor (AR), ER alpha, and strongly for ER beta. Endothelial cells were negative for AR, sporadically positive for ER alpha, and positive for ER beta. Urethral epithelium showed strong nuclear expression of AR, predominantly in the basal cell layer, and nuclear expression of ER alpha in the intermediate cells. ER beta was highly expressed in almost all urethral nuclei and, much more weakly, in cytoplasm. Progesterone receptor (PGR) was negative in all cases and all tissues. These results represent the first report that ER alpha and particularly ER beta are regularly expressed in human penile tissue.

Differential regulation of proteasome-dependent estrogen receptor alpha and beta turnover in cultured human uterine artery endothelial cells.

Estrogen-induced loss of estrogen receptor (ER) alpha expression limits estrogen responsiveness in many target cells. However, whether such a mechanism contributes to changes in vascular endothelial ER alpha and/or ER beta levels is unclear. Using RT-PCR assays, we did not find any regulation of ER alpha or ER beta mRNA expression in human uterine artery endothelial cell (HUAEC) nuclear extracts on stimulation with 17 beta-estradiol for 1 or 2 h. By contrast, Western analysis on HUAEC extracts revealed that 17 beta-estradiol was capable of down-regulating both ER alpha and ER beta protein starting 1 h after treatment, an effect that can be blocked by pretreatment with tamoxifen as well as with the proteasome inhibitor lactacystin. The proteolysis inhibitors insulin, cycloheximide, and puromycin impede ER alpha, but not ER beta, turnover. Ubiquitin, but not its competitive inhibitor methyl-ubiquitin, induces rapid turnover of both ERs in a cell-free system of MCF-7 and HUAEC extracts. We, thus, propose the existence of estrogen-induced ER degradation that serves to control physiological responses in an estrogen target tissue, i.e. human vascular endothelium, by down- regulating ER alpha as well as ER beta through different proteasomal uptake mechanisms.

Heme oxygenase and angiogenic activity of endothelial cells: stimulation by carbon monoxide and inhibition by tin protoporphyrin-IX.

The activity of heme oxygenase enzymes (HOs) is responsible for the endogenous source of carbon monoxide (CO). Their activities can be inhibited by tin protoporphyrin-IX (SnPPIX). Recent data indicate the involvement of HOs in the regulation of angiogenesis. Here, we investigated the role of the HO pathway in the production and angiogenic activity of vascular endothelial growth factor (VEGF) in endothelial cells treated with SnPPIX, or cultured in the presence of a CO-releasing molecule (CO-RM). Addition of CO-RM or induction of HO-1 by hemin resulted in a threefold elevation in CO production in culture medium (up to 20.3 microg/L) and was associated with a 30% increase in VEGF synthesis. Much higher levels of CO (up to 60 microg/L) and a further increase in VEGF production (by 277%) were measured in cells treated with prostaglandin-J(2), a potent activator of HO-1. SnPPIX prevented the induction of CO generation and inhibited VEGF synthesis. Moreover, SnPPIX reduced the VEGF-elicited angiogenic activities of endothelial cells by decreasing their proliferation (by 26%), migration (by 46%), formation of tubes on Matrigel (by 48%), and outgrowth of capillaries from endothelial spheroids (by 30%). In contrast, overexpression of HO-1 or incubation of cells with CO-RM led to an increase in capillary sprouting. Thus, HO activity up-regulates VEGF production and augments the capability of endothelial cells to respond to exogenous stimulation.

Identification of estrogen and progesterone receptor mRNA expression in the conjunctiva of premenopausal women.

PURPOSE: The purpose of the present study was to identify the expression of estrogen and progesterone receptor mRNA and of estrogen and progesterone receptor protein in the conjunctiva of healthy women. METHODS: Specimens of conjunctival tissue of 10 premenopausal women (age range, 13-38 years) were obtained during ophthalmic surgery in patients under general anesthesia. Specimens of approximately 4 mm(2) were taken from superior, nasal, or temporal bulbar conjunctiva adjacent to the bulbus and were immediately deep frozen with liquid nitrogen. Four women underwent strabismus surgery, two had phacoemulsification, and four had vitrectomy. Only three women were taking oral contraceptives. The expression of estrogen receptor (ER)-alpha, ERbeta, and progesterone receptor (PR) mRNA was analyzed by RT-PCR. Western blot analysis on nuclear extracts was performed with the anti-ERalpha mouse monoclonal antibody AB-15, the anti-ERbeta mouse monoclonal antibody 6B12, and the anti-PR mouse monoclonal antibody PgR 636. RESULTS: In two samples, ERalpha, ERbeta, and PR mRNAs were not accessible because of highly degraded RNA. In the remaining eight samples, an appearance rate of 100% was obtained for all three mRNAs. Similarly, an appearance rate of 100% was obtained for ERalpha, ERbeta, and PR protein in nine tissue samples accessible for analysis; one sample could not be analyzed due to a low amount of tissue. CONCLUSIONS: This study confirms the existence of estrogen and progesterone receptors in the human conjunctiva of premenopausal females. Because the proteins of estrogen and progesterone were also found in this study's specimens, the data indicate that the conjunctiva is a target site for sex steroids. Future studies are needed to elucidate the role of these receptors in ocular diseases involving the conjunctiva.