Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts

The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.

Prevention of postmenopausal bone loss using tibolone or conventional peroral or transdermal hormone replacement therapy with 17beta-estradiol and dydrogesterone

Postmenopausal bone loss can be prevented by continuous or intermittent estradiol (E2) administration. Concomitant progestogen therapy is mandatory in nonhysterectomized women to curtail the risk of endometrial hyperplasia or cancer. However, the recurrence of vaginal bleeding induced by sequential progestogen therapy in addition to continuous estrogen administration is one of the reasons for noncompliance to hormone replacement therapy (HRT). Tibolone, a synthetic steroid with simultaneous weak estrogenic, androgenic, and progestational activity, which does not stimulate endometrial proliferation, has recently been proposed for the treatment of climacteric symptoms. To compare the efficacy of conventional oral and transdermal HRT with that of tibolone in the prevention of postmenopausal bone loss, 140 postmenopausal women (age, 52 +/- 0.6 years; median duration of menopause, 3 years) were enrolled in an open 2-year study. Volunteers had been offered a choice between HRT and no therapy (control group, CO). Patients selecting HRT were randomly allocated to one of the following three treatment groups: TIB, tibolone, 2.5 mg/day continuously, orally; PO, peroral E2, 2 mg/day continuously, plus sequential oral dydrogesterone (DYD), 10 mg/day, for 14 days of a 28-day cycle; TTS, transdermal E2 by patch releasing 50 microg/day, plus DYD as above. Bone densitometry of the lumbar spine, upper femur, and whole body was performed using dual-energy X-ray absorptiometry at baseline, and then 6, 12, 18, and 24 months after initiation of therapy. One hundred and fifteen women (82%) completed the 2 years of the study. The dropout rate was similar in each group. Over 2 years, bone preservation was observed in all three treatment groups as compared with controls, without significant differences among treatment regimens. In conclusion, tibolone can be regarded as an alternative to conventional HRT to prevent postmenopausal bone loss.

Enhanced inflammatory activity of endometriotic lesions from the rectovaginal septum.

Endometriosis is characterised by the growth of ectopic lesions at multiple locations outside the uterine cavity and may be considered a collection of distinct but related conditions. The exact aetiology of endometriosis is still not clear although a role for inflammation is increasingly accepted. We therefore investigated the inflammatory activity of eutopic tissue and that of the matching ectopic lesions from different locations by measuring the genetic expression of inflammatory chemokines and cytokines. The gene expression in matching eutopic and ectopic tissue was compared, as was the gene expression in lesions from different locations. A significantly higher mRNA expression of the chemokines ENA-78 and RANTES and the cytokines IL-6 and TNF α was observed in endometriotic lesions of the rectovaginal septum (RVS) compared to that of matching eutopic tissue. Comparisons across lesion locations showed a significantly higher expression of IL-6 and TNF α in the RVS compared to lesions from either the ovaries or the peritoneum. These results show that the production of some inflammatory chemokines and cytokines is significantly increased in the ectopic endometrial tissue compared to matching eutopic tissue. Furthermore, IL-6 and TNF α are produced in significantly higher quantities in RVS lesions compared to other lesions.

Cell-type specific expression and regulation of apolipoprotein D and E in human endometrium.

OBJECTIVE To assess the expression and regulation of antilipoprotein D (ApoD) and antilipoprotein E (ApoE) in human endometrium. STUDY DESIGN Endometrial biopsies from healthy, regularly cycling women were collected during the late proliferative and mid-secretory phase. mRNA gene expression of ApoD and ApoE was determined using real-time PCR in whole tissue, in isolated stromal (ESC), epithelial (EEC) and CD45(+) leukocytes (EIC), as well as after hormonal stimulation of ESC and EEC in vitro. Protein expression was analyzed using immunohistochemistry. RESULTS ApoD and ApoE mRNA was expressed in all cell types examined. A rise in ApoD mRNA expression was seen in whole endometrium, ESC, and EEC in the secretory phase, as well as after hormonal stimulation of ESC and EEC in vitro. ApoE mRNA was significantly upregulated in whole endometrium of secretory phase biopsies, while its expression was not altered by progesterone in vitro. Immunohistochemistry of whole endometrial tissue localized ApoD mainly in ESC and EEC. While ApoE was localized slightly in ESC, it was particularly noted on the surface of secretory phase endothelial cells. CONCLUSION We demonstrate for the first time the cell-type and cycle dependent expression of ApoD and ApoE within human endometrium, suggesting their role in endometrial modulation.

Is vaginal hyaluronic acid as effective as vaginal estriol for vaginal dryness relief?

In a multicenter, randomized, controlled, open-label, parallel- group trial hyaluronic acid vaginal gel (Hyalofemme) was compared to estriol vaginal cream (Ovestin) in women with vaginal dryness due to various causes. A total of 144 supposedly postmenopausal women below age 70 years were randomized in a 1:1 ratio to either receive hyaluronic acid vaginal gel (5 g per application) or estriol vaginal cream (0.5 g cream per application = 0.5 mg estriol) every 3 days for a total of ten applications, respectively. Exclusion criteria included vaginal infections, conventional contraindications to estrogens, use of vaginal products other than the investigational compounds, being unmarried, pregnant, or breastfeeding. The aim of the study was to test for non-inferiority of hyaluronic acid vaginal gel compared to estriol vaginal cream. The primary efficacy end point was the percentage (%) improvement in vaginal dryness, with the secondary end points being the percentage (%) improvements in vaginal itching, burning, and dyspareunia. Efficacy was assessed by using a visual analog scale (VAS) (0-10; 0 = absent, 10 = intolerable) at baseline (V0), during telephone contact after the third administration (V1), and at the final visit after the tenth administration (V2). Safety parameters included vaginal pH, endometrial thickness, and a vaginal smear for vaginal microecosystem assessment. Adverse events were recorded according to international guidelines. 133 women completed the study. At baseline, participants' characteristics did not differ significantly. Mean age was 54 years, time since menopause was 5 years on average, and cause of menopause was mostly natural. However, mean menstrual cycle days were also reported, although according to inclusion criteria only postmenopausal women were eligible for the study. At V1, an improvement in vaginal dryness was reported by about 49 % of women using hyaluronic acid vaginal gel, and by 53 % of women using estriol vaginal cream (p = 0.31). At V2, the percentage improvement rates were 84 and 89 % (p = 0.13), respectively. Improvement rates for vaginal itching, burning, and dyspareunia at V2 were about 86, 85, and 57 % for hyaluronic acid vaginal gel, and 82, 87, and 62 % for estriol vaginal cream (p[0.05), respectively. After treatment, vaginal pH was significantly lower in estriol-treated women compared to those having received hyaluronic acid. Endometrial thickness did not differ between groups. In the majority of women, the vaginal microenvironment remained unaffected by treatment. However, the proportion of women whose abnormal vaginal microecological results became normal was higher in women using estriol vaginal cream. Adverse events (suspected to be) related to the investigational compounds were minor and included vaginal infection and genital itching. The authors concluded that hyaluronic acid vaginal gel was not inferior to estriol vaginal cream in women presenting with vaginal dryness. They suggest using hyaluronic acid vaginal gel not only as an alternative treatment to vaginal estrogens, but also to consider its general use in women presenting with vaginal dryness of any cause.

Intrauterine instillation of diluted seminal plasma at oocyte pick-up does not increase the IVF pregnancy rate: a double-blind, placebo controlled, randomized study.

STUDY QUESTION Does intrauterine application of diluted seminal plasma (SP) at the time of ovum pick-up improve the pregnancy rate by ≥14% in IVF treatment? SUMMARY ANSWER Intrauterine instillation of diluted SP at the time of ovum pick-up is unlikely to increase the pregnancy rate by ≥14% in IVF. WHAT IS KNOWN ALREADY SP modulates endometrial function, and sexual intercourse around the time of embryo transfer has been suggested to increase the likelihood of pregnancy. A previous randomized double-blind pilot study demonstrated a strong trend towards increased pregnancy rates following the intracervical application of undiluted SP. As this study was not conclusive and as the finding could have been confounded by sexual intercourse, the intrauterine application of diluted SP was investigated in the present trial. STUDY DESIGN, SIZE, DURATION A single-centre, prospective, double-blind, placebo-controlled, randomized, superiority trial on women undergoing IVF was conducted from April 2007 until February 2012 at the University Department of Gynaecological Endocrinology and Reproductive Medicine, Heidelberg, Germany. PARTICIPANTS/MATERIALS, SETTING, METHODS The study was powered to detect an 14% increase in the clinical pregnancy rate and two sequential tests were planned using the Pocock spending function. At the first interim analysis, 279 women had been randomly assigned to intrauterine diluted SP (20% SP in saline from the patients' partner) (n = 138) or placebo (n = 141) at the time of ovum pick-up. MAIN RESULTS AND THE ROLE OF CHANCE The clinical pregnancy rate per randomized patient was 37/138 (26.8%) in the SP group and 41/141 (29.1%) in the placebo group (difference: -2.3%, 95% confidence interval of the difference: -12.7 to +8.2%; P = 0.69). The live birth rate per randomized patient was 28/138 (20.3%) in the SP group and 33/141 (23.4%) in the placebo group (difference: -3.1%, 95% confidence interval of the difference: -12.7 to +6.6%; P = 0.56). It was decided to terminate the trial due to futility at the first interim analysis, at a conditional power of 62%. LIMITATIONS, REASONS FOR CAUTION The confidence interval of the difference remains wide, thus clinically relevant differences cannot reliably be excluded based on this single study. WIDER IMPLICATIONS OF THE FINDINGS The results of this study cast doubt on the validity of the concept that SP increases endometrial receptivity and thus implantation in humans. STUDY FUNDING/COMPETING INTEREST(S) Funding was provided by the department's own research facilities. TRIAL REGISTRATION NUMBER DRKS00004615.

Inflammation and nerve fiber interaction in endometriotic pain.

Endometriosis is an extremely prevalent estrogen-dependent condition characterized by the growth of ectopic endometrial tissue outside the uterine cavity, and is often presented with severe pain. Although the relationship between lesion and pain remains unclear, nerve fibers found in close proximity to endometriotic lesions may be related to pain. Also, women with endometriosis pain develop central sensitization. Endometriosis creates an inflammatory environment and recent research is beginning to elucidate the role of inflammation in stimulating peripheral nerve sensitization. In this review, we discuss endometriosis-associated inflammation, peripheral nerve fibers, and assess their potential mechanism of interaction. We propose that an interaction between lesions and nerve fibers, mediated by inflammation, may be important in endometriosis-associated pain.

Detection of the pan neuronal marker PGP9.5 by immuno-histochemistry and quantitative PCR in eutopic endometrium from women with and without endometriosis.

PURPOSE To assess endometrial gene as well as protein expression of neuroendocrine and supposedly endometriosis-associated product PGP9.5 and pain symptoms in women with endometriosis and controls undergoing laparoscopy, using molecular biological and immuno-histochemical approaches in the same patients. METHODS Biopsy of eutopic endometrium from 29 patients by sharp curettage, and preparation of paraffin blocks. Determination of PGP9.5 gene expression and protein abundance using qPCR and immuno-histochemistry. RESULTS qPCR; The PGP9.5 mRNA expression level between women with (N = 16) and without (N = 13) endometriosis was not different, regardless of pain symptoms or menstrual cycle phase. PGP9.5 expression was higher in women who reported pain compared to those who did not; however, this association was not statistically significant. The expression of PGP9.5 mRNA was higher in women with endometriosis and pain during the proliferative than in the secretory phase (P = 0.03). Furthermore, in the first half of the cycle, the abundance of the PGP9.5 transcript was also significantly higher in endometriosis patients compared to those without (P = 0.03). Immuno-histochemistry; Thirteen of the 16 endometriosis patients showed positive PGP9.5 immuno-reactivity in the endometrium, whereas no such signal was observed in women without endometriosis. The absolute number of nerve fibres per mm(2) in women with endometriosis was similar, regardless of the pain symptoms. CONCLUSIONS PGP9.5 mRNA expression is increased in the proliferative phase of endometriotic women with pain. The presence of nerve fibres was demonstrated by a PGP9.5 protein signal in immuno-histochemistry and restricted to patients with endometriosis. Based on these results, however, there did not appear to be a direct association between the gene expression and protein abundance in women with and without endometriosis or those that experienced pain.

Cryopreservation and transplantation of ovarian tissue exclusively to postpone menopause: technically possible but endocrinologically doubtful.

Transplantation of cryopreserved ovarian tissue has been shown to induce pregnancies and puberty successfully. Therefore, using cryopreserved ovarian tissue to postpone menopause (tissue hormone therapy [THT]) seems to be an interesting option to avoid conventional menopause hormone therapy (MHT). Pregnancy induction and replacing MHT by THT, however, are completely different topics as different requirements need to be met. First, MHT requires long-lasting and continuous hormone production. It still needs to be proven if the transplanted tissue is active for at least 5 years with a continuous follicle growth to avoid phases with low oestrogen production, which would otherwise cause menopausal symptoms and could reduce the postulated benefit for women's health. Second, the advantage of a physiological hormone production over a non-physiological MHT is still hypothetical. Third, women who have undergone hysterectomies who do not need progesterone for endometrial protection would only require oestrogens, imposing more health benefits (cardiovascular system, mammary gland) than oestrogen and progesterone production or replacement. Therefore, transplanting ovarian tissue exclusively to postpone menopause is endocrinologically doubtful and should only be carried out within clinical trials.

H19 lncRNA alters stromal cell growth via IGF signaling in the endometrium of women with endometriosis

Endometriosis affects approximately 15% of reproductive aged women and is associated with chronic pelvic pain and infertility. However, the molecular mechanisms by which endometriosis impacts fertility are poorly understood. The developmentally regulated, imprinted H19 long noncoding RNA (lncRNA) functions to reduce the bioavailability of microRNA let-7 by acting as a molecular sponge. Here we report that H19 expression is significantly decreased in the eutopic endometrium of women with endometriosis as compared to normal controls. We show that decreased H19 increases let-7 activity, which in turn inhibits Igf1r expression at the post-transcriptional level, thereby contributing to reduced proliferation of endometrial stromal cells. We propose that perturbation of this newly identified H19/Let-7/IGF1R regulatory pathway may contribute to impaired endometrial preparation and receptivity for pregnancy in women with endometriosis. Our finding represents the first example of a lncRNA-based mechanism in endometriosis and its associated infertility, thus holding potential in the development of novel therapeutics for women with endometriosis and infertility.

Comparison of ovarian cancer markers in endometriosis favours HE4 over CA125

Endometriosis is a gynaecological condition with an associated chronic inflammatory response. The ectopic growth of 'lesions', consisting of endometrial cells outside the uterine cavity, stimulates an inflammatory response initiating the activation of macrophages, and resulting in increased cytokine and growth factor concentrations in the peritoneal fluid (PF). Endometriosis‑associated inflammation is chronic and long lasting. In patients with endometriosis, the risk of developing ovarian cancer within 10 years, particularly of the endometrioid or clear cell subtype, is increased 2.5‑4 times. Endometriosis creates a peritoneal environment that exposes the affected endometriotic and the normal ovarian surface epithelial cells to agents that have been suggested to be involved in the pathogenesis of cancer. Concentrations of several cytokines and growth factors were increased in the PF of patients with endometriosis. The ovarian cancer marker, CA125, was one such growth factor; however, this remains to be confirmed. Human epididymis protein 4 (HE4) was detected at high concentrations in patients with ovarian cancer and was identified as the best biomarker for the detection of ovarian cancer. The present study determined the levels of HE4 and CA125 in the peritoneal fluid of 258 patients with and 100 control individuals without endometriosis attending the Department of Obstetrics and Gynaecology, University of Berne (Berne, Switzerland) between 2007 and 2014. The cases were subdivided into groups without hormonal treatment (n=107), or treated with combined oral contraceptives (n=45), continuous gestagens (n=56) or GnRH agonists (n=50). Both of these markers were significantly increased in the non‑treated endometriosis samples compared with the control group. Hormone treatment with either of the three agents mentioned resulted in the concentration of CA125 returning to the control levels and the concentration of HE4 decreasing to below the control levels. CA125, however not HE4, significantly differed between the proliferative and secretory cycle phases. Since HE4 is sensitive to hormonal treatment and robust towards menstrual cycle variation, HE4 is potentially superior to CA125 as an endometriosis marker and therefore has greater potential as a marker for the identification of women at risk of developing ovarian cancer.

Ambulatory management of heavy menstrual bleeding.

Heavy menstrual bleeding (HMB) has significant adverse effects on the quality of life of many women, placing an economic burden on both health services and society at large. Thus, it is essential that all women with HMB have easy access to the proper diagnostic and therapeutic work-up in an outpatient fashion, avoiding the more time-consuming inpatient management. This new outpatient approach for HMB is one of the latest development of gynecological practice and can offer both diagnostic and therapeutic procedures. This manuscript aims to show the current possibilities of the modern management of HMB, which can be safely and effectively accomplished in the outpatient setting: global and directed endometrial biopsy, levonorgestrel intrauterine system insertion as well as minimally invasive surgical procedures (encompassing a variety of operative hysteroscopic procedures and second-generation endometrial ablation) are described below.

Does dienogest influence the inflammatory response of endometriotic cells? A systematic review.

OBJECTIVE AND DESIGN A systematic review of all literature was done to assess the ability of the progestin dienogest (DNG) to influence the inflammatory response of endometriotic cells. MAIN OUTCOME MEASURES In vitro and in vivo studies report an influence of DNG on the inflammatory response in eutopic or ectopic endometrial tissue (animal or human). RESULTS After strict inclusion criteria were satisfied, 15 studies were identified that reported a DNG influence on the inflammatory response in endometrial tissue. These studies identified a modulation of prostaglandin (PG) production and metabolism (PGE2, PGE2 synthase, cyclo-oxygenase-2 and microsomal PGE synthase-1), pro-inflammatory cytokine and chemokine production [interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α, monocyte chemoattractant protein-1 and stromal cell-derived factor-1], growth factor biosynthesis (vascular endothelial growth factor and nerve growth factor) and signaling kinases, responsible for the control of inflammation. Evidence supports a progesterone receptor-mediated inhibition of the inflammatory response in PR-expressing epithelial cells. It also indicated that DNG inhibited the inflammatory response in stromal cells, however, whether this was via a PR-mediated mechanism is not clear. CONCLUSIONS DNG has a significant effect on the inflammatory microenvironment of endometriotic lesions that may contribute to its clinical efficacy. A better understanding of the specific anti-inflammatory activity of DNG and whether this contributes to its clinical efficacy can help develop treatments that focus on the inhibition of inflammation while minimizing hormonal modulation.

Kinase signalling pathways in endometriosis: potential targets for non-hormonal therapeutics.

BACKGROUND Endometriosis, the growth of endometrial tissue outside the uterine cavity, is associated with chronic pelvic pain, subfertility and an increased risk of ovarian cancer. Current treatments include the surgical removal of the lesions or the induction of a hypoestrogenic state. However, a reappearance of the lesion after surgery is common and a hypoestrogenic state is less than optimal for women of reproductive age. Additional approaches are required. Endometriosis lesions exist in a unique microenvironment characterized by increased concentrations of hormones, inflammation, oxidative stress and iron. This environment influences cell survival through the binding of membrane receptors and a subsequent cascading activation of intracellular kinases that stimulate a cellular response. Many of these kinase signalling pathways are constitutively activated in endometriosis. These pathways are being investigated as therapeutic targets in other diseases and thus may also represent a target for endometriosis treatment. METHODS To identify relevant English language studies published up to 2015 on kinase signalling pathways in endometriosis, we searched the Pubmed database using the following search terms in various combinations; 'endometriosis', 'inflammation', 'oxidative stress', 'iron', 'kinase', 'NF kappa', 'mTOR', 'MAPK' 'p38', 'JNK', 'ERK' 'estrogen' and progesterone'. Further citing references were identified using the Scopus database and finally current clinical trials were searched on the clinicaltrials.gov trial registry. RESULTS The current literature on intracellular kinases activated by the endometriotic environment can be summarized into three main pathways that could be targeted for treatments: the canonical IKKβ/NFκB pathway, the MAPK pathways (ERK1/2, p38 and JNK) and the PI3K/AKT/mTOR pathway. A number of pharmaceutical compounds that target these pathways have been successfully trialled in in vitro and animal models of endometriosis, although they have not yet proceeded to clinical trials. The current generation of kinase inhibitors carry a potential for adverse side effects. CONCLUSIONS Kinase signalling pathways represent viable targets for endometriosis treatment. At present, however, further improvements in clinical efficacy and the profile of adverse effects are required before these compounds can be useful for long-term endometriosis treatment. A better understanding of the molecular activity of these kinases, including the specific extracellular compounds that lead to their activation in endometriotic cells specifically should facilitate their improvement and could potentially lead to new, non-hormonal treatments of endometriosis.