Changes in the expression of cancer- and metastasis-related genes and proteins after metformin treatment under different metabolic conditions in endometrial cancer cells.

Research question: Hyperinsulinemia and elevated estrogen levels are known risk factors for endometrial cancer (EC) development and are associated with obesity, type 2 diabetes mellitus (T2DM), insulin resistance, among others. Metformin, an insulin-sensitizing drug, displays anti-tumor effects in cancer patients, including EC, but the mechanism of action is still not completely understood. In the present study, the effects of metformin on gene and protein expression were investigated in pre- and postmenopausal EC in vitro models in order to identify candidates that are potentially involved in the drug's anti-cancer mechanism. Design: After treating the cells with metformin (0.1 and 1.0 mmol/L), changes in the expression of >160 cancer- and metastasis-related gene transcripts were evaluated with RNA arrays. A total of 19 genes and 7 proteins were selected for a follow-up expression analysis, including further treatment conditions, in order to evaluate the influence of hyperinsulinemia and hyperglycemia on metformin-induced effects. Results: Changes in the expression of BCL2L11, CDH1, CDKN1A, COL1A1, PTEN, MMP9 and TIMP2 were analyzed on gene and protein level. The consequences resulting from the detected expression changes as well as the influence of varying environmental influences are discussed in detail. With the presented data, we contribute to a better understanding of the direct anti-cancer activity of metformin as well as its underlying mechanism of action in EC cells. Conclusions: Although further research will be necessary to confirm the data, the influence of different environmental settings on metformin-induced effects could be highlighted with the presented data. Additionally, gene and protein regulation were not similar in the pre- and postmenopausal in vitro models.

Glucose and fatty acids catabolism during in vitro decidualization of human endometrial stromal cells.

The differentiation of endometrial stromal cells, named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption and its utilization by endometrial cells to produce energy. Besides glucose, fatty acids are another important energy source for living cells and it has been described that endometrial stromal cells rely on the proper function of the oxidation of fatty acids for the correct decidualization. It is, however, unknown whether the turn-over of fatty acid degradation is modified during decidualization. Furthermore, it is also unknown how the final products of glucose and fatty acid catabolism are related to the function of the tricarboxylic acid cycle for the efficient ATP production. In this study, we evaluated the content levels of different intermediate metabolites and the expression of the key enzymes related to the degradation of glucose and fatty acids during the in vitro decidualization of human endometrial stromal cells. Our results suggest that human endometrial stromal cells undergo energetic metabolic changes during decidualization and that decidualizing and non-decidualizing cells differ in the level of activation of different metabolic pathways and, probably, in the use of intermediate metabolites.

GRN, NOTCH3, FN1, and PINK1 expression in eutopic endometrium - potential biomarkers in the detection of endometriosis - a pilot study.

PURPOSE: Endometriosis (EM) is a common gynecological disease affecting 10-15% of women of reproductive age. However, molecular mechanisms and pathogenesis are still not completely understood. Furthermore, due to the absence of a reliable clinical biomarker, the only viable method for the often-delayed definitive diagnosis is laparoscopic surgery. Our objective was to analyze molecular differences of selected endometrial proteins and genes of women suffering from different stages of EM compared with healthy women to evaluate potential clinical biomarkers. METHODS: We analyzed eutopic endometrial tissue samples from women undergoing a laparoscopic surgery (n = 58). mRNA gene expression of progranulin (GRN), neurogenic locus notch homolog protein (NOTCH3), fibronectin (FN1), and PTEN-induced kinase 1 (PINK1) was analyzed using qRT-PCR. Protein expression was determined using ELISA and immunohistochemistry. RESULTS: Significant differences in gene expression between the different stages of the disease were noted for GRN, NOTCH3, FN1, and PINK1 (p < 0.05). The endometrium of women with minimal EM (ASRM I) showed the highest mRNA expression. Protein levels of GRN and FN1 on the other hand were significantly decreased in the endometrium of women with EM compared with those of healthy controls. Furthermore, for GRN and FN1, we could detect a correlation of protein expression with the severity of the disease. CONCLUSION: Our findings suggest a potential use of GRN and FN1 as clinical biomarkers to detect endometriosis. In addition, GRN, NOTCH3, FN1, and PINK1 could potentially be useful to differentiate between the underlying stages of the disease. However, a validation with a larger study population is needed.

In vitro maturation of immature oocytes from ovarian tissue prior to shipment to a cryobank.

PURPOSE: Female fertility preservation prior to gonadotoxic therapies can be achieved by the cryopreservation of ovarian cortical tissue. Immature oocytes may be recovered during the preparation, matured in vitro and lead to live births, thereby providing an additional option for fertility preservation. The purpose of this study was to test the feasibility of this approach in a setting with unilateral biopsy of a small piece of ovarian tissue and minimal tissue preparation prior to shipment to an external cryobank. METHODS: A prospective observational clinical study in an academic center was performed from January 2018 through December 2019. Ovarian tissue was obtained laparoscopically. Immature oocytes were recovered by minimal preparation of the tissue before shipment to an external cryobank for cryopreservation. In vitro maturation was performed on recovered immature oocytes. RESULTS: Twelve patients were enrolled. Immature oocytes could be recovered for all. The maturation rate was 38.9% (n = 14/36). Metaphase II (MII) were either directly used for intracytoplasmic sperm injection (ICSI) with a fertilization rate of 66.6% (n = 4/6) or vitrified (n = 8). PNs were cryopreserved (n = 4). Vitrified MII were warmed with a post-warming vitality rate of 75.0% (n = 3/4) and used for ICSI with a fertilization rate of 33.3% (n = 1/3). CONCLUSIONS: Immature oocytes can be successfully retrieved from ovarian tissue through minimal tissue preparation prior to shipment to a cryobank, matured in vitro, fertilized and cryopreserved for potential future fertility treatments. The total number of oocytes available for fertility preservation can be increased even without controlled ovarian stimulation in a situation where only ovarian biopsy for cryopreservation is performed. TRIAL REGISTRATION: German Clinical Trials Register (DRKS), DRKS00013170. Registered 11 December 2017, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013170 .

Endometrial expression of receptivity markers subject to ovulation induction agents.

PURPOSE: Implantation rates differ according to ovulation induction agents in ART. This study investigates the different local endometrial effects of LH- versus hCG-induced ovulation. METHODS: Endometrial stromal cells from healthy patients were cultured with hCG or LH in different concentrations, supplemented with 250 ng/mL hCG and progesterone after 2 and 5 days. In addition after decidualization induction, cells were treated with hCG (50 or 250 ng/mL) or LH (10 or 50 ng/mL) for 3 days. Receptivity markers expression was evaluated by real-time quantitative PCR on day 3 and 6. RESULTS: On day 3, non-decidualized cells treated with LH showed an increased expression of IGFBP1, IL-8 and CXCL12 compared to hCG. The expression pattern changed on day 6, where cells treated with hCG showed higher expression of implantation markers compared to LH-treated cells. Furthermore, on day 3, decidualized cells treated with hCG250 showed an increased IL8 and CXCL12 expression compared to LH10. CONCLUSIONS: LH seems to modulate the local endometrial expression of receptivity markers earlier compared to hCG; however, the effect is not sustained over time in cells without prior decidualization. Though, in decidualized cells, pattern changed and an earlier positive effect of hCG was shown on IL-8 and CXCL12.

Cytokines in relation to hCG are significantly altered in asymptomatic women with miscarriage - a pilot study.

BACKGROUND: Spontaneous abortion is one of the most common complications in early pregnancy. A preventive test to identify women who will experience a miscarriage, even before first symptoms occur, is not established. Activation of maternal immunological tolerance seems to be essential for early fetal development and various cytokines have been described in different stages of pregnancy. Therefore, we aimed to investigate if chemokine levels at the time of pregnancy testing relative to human Choriogonadotropin (hCG) are altered in patients who will experience a miscarriage in this pregnancy. METHODS: We obtained blood samples from 39 women. Dependent on the follow-up, patients with a positive pregnancy test were subsequently divided in two groups: ongoing pregnancy (n = 22) and miscarriage (n = 17) in this pregnancy. Immunological and endocrine profiling of maternal plasma at the time of pregnancy testing (5th week of gestation) was performed for each group at the time of pregnancy test using Multiplex and ELISA analysis. RESULTS: hCG was significantly decreased in patients with abortion whereas levels of IL-1ra, MIP-1a and TNF-alpha were significantly increased. GCSF/ IL-1ra-ratio was 1.66-fold increased in patients with ongoing pregnancy. TGF-beta /MIP1a-ratio was significantly 3.45-times higher in patients with miscarriage. Comparing patients with ongoing pregnancy to patients experiencing a miscarriage, we could demonstrate significant alterations of the ratios MIP1a/hCG, IL-1ra/hCG, TNFalpha/hCG, MCP1/hCG, IL-6/hCG, TPO/hCG and TGF-beta1/hCG. The strongest effects were seen for the ratio MIP1a/hCG, IL-1ra/hCG and TNFalpha/hCG. CONCLUSIONS: We have shown that cytokines in relation to hCG after 4 weeks of gestation are significantly altered in women with miscarriage, promising potential as a prognostic biomarker.

Expression of semaphorin class 3 is higher in the proliferative phase on the human endometrium.

PURPOSE: The semaphorins are related to angiogenesis and cell proliferation depending on the tissue. The purpose of this study was to assess gene expression of class 3 semaphorin (SEMA3A-F) and protein expression of semaphorin 3A (SEMA3A) within human endometrium throughout the menstrual cycle. METHODS: Gene expression of SEMA3A-F was analyzed by real-time PCR (qRT-PCR) and protein expression of SEMA3A was analyzed by ELISA in endometrial biopsies in the proliferative and secretory phase of the menstrual cycle. RESULTS: Gene expression of SEMA3A, SEMA3C, SEMA3D, and SEMA3E was statistically significant decreased in secretory compared to proliferative phase endometrium (p < 0.05). Accordingly, SEMA3A protein expression in the secretory phase was lower than protein expression in proliferative phase endometrium (p ≤ 0.05). CONCLUSION: SEMA3A, 3C, 3D, and 3E are possibly related to cell proliferation in the endometrium, being more expressed in the proliferative phase of the cycle. This finding may stimulate studies of class 3 semaphorins as a possible target for treatment of endometrial pathologies.

Altered miRNA-profile dependent on ART outcome in early pregnancy targets Wnt-pathway.

Main goal of this study is to detect the possible alterations in microRNA (miRNA) expression and the pathway targeted in plasma at the time of embryo transfer and pregnancy testing dependent on the assisted reproductive treatment (ART) outcome after ovarian hyperstimulation for in vitro fertilization. Changes in miRNA expression in plasma of women, who became pregnant (n = 6) vs women who failed implantation (n = 6) following day 5 embryo transfer (ET), were investigated at the day of ET and pregnancy testing (PT). Protein expression to validate the finding was performed with a sample size of n = 20 (10 per group) using enzyme-linked immunosorbent assay. Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using DIANA-miRPath, v3.0 software based on predicted targets by DIANA-microT-CDS. 4 miRNAs could be identified as possible biomarkers for implantation success. The 11 miRNAs showing the highest significant alterations were all associated with the regulation of WNT3 and WNT7a. While WNT7a presented with a significant decrease between ET and PT in case of ongoing pregnancy, women with implantation failure showed unaltered concentrations. WNT3 presented with a significant decrease in both groups. However, the loss of WNT3 between ET and PT was significantly higher in patients who became pregnant. Main limitation of this prospective study is its small sample size, defining it as a pilot analysis. To conclude, we could demonstrate a significant change in miRNA profile dependent on the ART outcome affecting Wnt pathway. Our findings indicate a possible prospective use of miRNA as biomarkers for implantation success.

Effects of a hyperandrogenaemic state on the proliferation and decidualization potential in human endometrial stromal cells.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women, involving hyperandrogenaemia and insulin resistance. Treatment options include dexamethasone, as well as the off-label use of metformin. To evaluate the impact of those drugs on cyclic changes in endometrial development, we tested possible effects of metformin and dexamethasone on endometrial stromal cells decidualisation, proliferation, and gene regulation in a hyperandrogenaemic microenvironment in vitro. METHODS/DESIGN: Ten endometrial biopsies (of which five were decidualized in vitro) were used from regularly cycling women. Cells were treated with testosterone, dexamethasone, and metformin in different concentrations. Thereafter, cells were assessed for proliferation and decidualization capacity, as well as mTor and MMP-2 gene regulation. RESULTS: Metformin showed a dose-dependent negative effect on prolactin secretion, a known decidualization marker. This effect was stronger in a hyperandrogenaemic condition and could not be compensated by dexamethasone. Testosterone had a dose dependent negative effect on proliferation in decidualized endometrial stromal cells. Dexamethasone slightly compensated the negative proliferative effect only in low-dose testosterone. High-dose metformin also showed a dose-dependent reduction in endometrial stromal cell proliferation without a major impact by testosterone or dexamethasone in decidualized and non-decidualized cells. High-dose metformin significantly reduced the expression of matrix metalloproteinase-2 (MMP-2) and mechanistic Target of Rapamycin (mTor), regardless of the concentration of dexamethasone and testosterone. The strongest effect could be observed for the combination with high-dose dexamethasone. CONCLUSION: When therapies, such as metformin and dexamethasone, are used to normalize peripheral androgen levels in patients with PCOS, their effect on the endometrial microenvironment should be taken into consideration as well, especially metformin has to be used with caution because of its dose dependent, possibly inhibiting effect at the endometrial proliferation.

Proliferation and metastatic potential of endometrial cancer cells in response to metformin treatment in a high versus normal glucose environment.

In order to improve our understanding of the potential preventive and therapeutic role of metformin, the present study aimed to investigate the capability of low-dose metformin in the efficient inhibition of cancer development and the reduction of the metastasis of endometrial adenocarcinoma type I and primary endometrial epithelial cells (eEPs), with the drug acting as a treatment in a hyperinsulinemic environment exposed to high and normal glucose conditions. The Ishikawa endometrial adenocarcinoma cell line and primary eEPs were exposed to an environment with high (17 mM) or normal glucose (5 mM) and treated with insulin, low-dose metformin (0.1 mM) or a combined treatment. Metastatic potential was assessed by migration and invasion assays, and relative cell proliferation was determined. Metformin at a low dose potently inhibited the insulin action, decreasing the ability of the endometrial cancer (EC) cell line to migrate and invade in a high and normal glucose environment, and decreasing the migration ability of the primary eEPs. In the EC cell line, the insulin treatment increased the proliferation, without any subsequent reduction of proliferation by the addition of 0.1 mM metformin; however, relative cell proliferation sensitivity to metformin was observed in the range between 1 and 5 mM regardless of the glucose concentration present. Overall, metformin at 0.1 mM is not efficient enough to decrease the proliferation in an EC cell line. However, at this concentration, metformin can inhibit the insulin action in endometrial epithelial cancer cells, demonstrating an anti-metastatic effect in high and normal glucose environments.

Timing of ovarian stimulation in patients prior to gonadotoxic therapy: an analysis of 684 stimulations.

OBJECTIVE: Time to therapy initiation in patients requiring gonadotoxic therapy is crucial. This article evaluates the efficiency of random start ovarian stimulation in affected women. STUDY DESIGN: Retrospective anonymous registry data analysis from 85 university and non-university fertility centres participating in the international network FertiPROTEKT. The study comprised 684 women undergoing ovarian stimulation for fertility preservation from 2007 to 2013. According to the time of stimulation initiation, days of ovarian stimulation, total dose of gonadotropins used, gonadotropin dose used per day, number of oocytes retrieved and incidence of ovarian hyperstimulation syndrome were analysed. Statistical analysis was performed using analysis of variance in case of continuous outcome variables and chi-square tests in case of categorical variables. RESULTS: Among 684 women who underwent ovarian stimulation prior to gonadotoxic therapy 472 (69.0%) started ovarian stimulation between menstrual cycle day 1-5 (group A), 109 (15.9%) between day 6-14 (group B) and 103 (15.1%) after day 14 (group C). The days of stimulation (A: 10.8±2.4, B: 10.6±2.7, C: 11.5±2.2) and total dose of gonadotropins (A: 2496IU±980, B: 2529IU±940, C: 2970IU±1145) were significantly increased in group C. Numbers of obtained oocytes (Group A: 11.6±7.7, B: 13.9±9.1, C: 13.6±7.9) were significantly increased in group B and C, while the overall incidence of ovarian hyperstimulation syndrome III° was 0.15%. CONCLUSION: The outcome of ovarian stimulation is similar after stimulation initiation during any phase of the menstrual cycles, supporting the concept of random-start ovarian stimulation before gonadotoxic therapy without disadvantage for the patient concerning later fertility preservation.

Modulation of the IGF system and proliferation in human endometrial stromal cells by metformin: a dose-dependent effect.

PURPOSE: To assess the metformin effect on endometrial stromal cell decidualization, proliferation, gene and protein expression of IGFBPs, IGFs and their receptors. METHODS: Human endometrial stromal cells (hESCs) were cultured from endometrial biopsies of 11 women undergoing surgery for benign reasons. hESCs were decidualized with and without metformin in increasing doses. Supernatant and cells were harvested after decidualization for 12-14 days, followed by real-time PCR of IGFBP 1-6, IGF I, IGF II and their receptors. Prolactin, and IGFBP-1, -3, and -6 were additionally analyzed in supernatant by ELISA. Proliferation of hESCs and decidualization of hESCs were assessed under the influence of metformin. Data were analyzed using the paired t test with p < 0.05 considered significant. RESULTS: While lower concentrations of metformin (10(-4), 10(-5 )M) did not influence the decidualization and proliferation capacity of hESCs, higher concentrations (10(-3), 10(-2 )M metformin) significantly (p < 0.05) diminished decidualization, as well as stromal cell proliferation in a dose-dependent manner. Higher concentrations of metformin lead to a significant (p < 0.05) dose-dependent attenuation of the progesterone effect with regard to IGFBP-1, -3, -5, -6, as well as IGF I receptor, while it did not change the expression of IGFBP-2 and -4, IGF I and II and the IGF II receptor. This was confirmed on the protein level for IGFBP-1, -3, and -6. CONCLUSION: We were able to demonstrate for the first time a dose-dependent local effect of metformin within hESCs. Metformin might therefore influence locally the endometrial proliferation and maturation, and could open up new treatment options for gynecological diseases by vaginal application of metformin.

Endometrial beta3 integrin profile reflects endometrial receptivity defects in women with unexplained recurrent pregnancy loss.

BACKGROUND: The pathophysiology of recurrent pregnancy loss (RPL) is still unknown in 50% of the cases. Herein we measure the expression of beta3 integrin subunit, a well-known implantation marker, in women with or without RPL and correlate it with the histological dating of the endometrial tissue. METHODS: LH-timed endometrial biopsies were obtained from cases (RPL; n = 21, age 33.9+/-4.7) and healthy controls (n = 29; age 29.8+/-4.1) during the mid-secretory phase (post ovulatory day: 8 to 10). Endometrial samples were timed histologically according to Noyes' criteria and underwent immunohistochemical staining for beta3 integrin expression. For statistical analysis the semi-quantitative HSCORE was assessed. Type I (beta3 negative in an out-of-phase endometrium) and Type II defect (beta3 negative in an in-phase endometrium) were also analysed. Statistical analysis was done with Student t-test, Mann Whitney U test, ANCOVA and chi square for trend. Significance was set as P < 0.05. RESULTS: The mean (SD) age in controls was lower compared to cases [(29.8 (4.1) vs. 33.9 (4.7) - P = 0.001; Student t-test)]. The median (range) expression of beta3 integrin in controls and cases was 1.94 (0 to 3.5) vs. 0.82 (0 to 3.6), respectively (P = 0.001; Mann Whitney U test). Significance was still significant after adjusting for age (P = 0.03;ANCOVA). The normal positive staining > =0.7 of beta3 integrin subunit and in-phase endometrium was seen in 24 out of 29 (82.8%) controls, but in only 6 out of 21 (28.6%) of cases with RPL; Type I and II defects were seen in 10.3 and 6.9% of controls, while present in 52.4 and 19.1% of cases, respectively (P = 0.0005; chi-square). CONCLUSIONS: Women with unexplained RPL had significantly reduced integrin expression compared to controls. Our findings underline the need for further molecular analysis of endometrial tissue in affected women.

Modulation of tumor necrosis factor-stimulated gene-6 (TSG-6) expression in human endometrium.

INTRODUCTION: The human endometrium undergoes repetitive, cyclical changes under the influence of endocrine signals (estrogen and progesterone). In particular, the extensive tissue remodeling, angiogenesis and leukocyte infiltration that occur during decidualization of the endometrium give rise to an environment that is permissive to blastocyst attachment. However, it is now well established that crosstalk (via paracrine signals) between the trophoblast and the endometrium is also a key for successful implantation. Microarray studies have identified TSG-6 as a gene with elevated expression in endometrial stromal cells following the exposure to trophoblast and immune cell products. TSG-6 is an inflammation-associated protein which acts as a potent inhibitor of neutrophil extravasation and also plays important roles in matrix remodeling, e.g., by promoting hyaluronan (HA) cross-linking and down-regulating the protease network. Female TSG-6 (-/-) mice are infertile and this has been attributed to their inability to ovulate; however, the importance of TSG-6 in implantation has not been directly investigated. MATERIAL AND METHODS: Real-time PCR, as well as immunofluorescence staining was performed on endometrial biopsies of proliferative and secretory phase samples. In addition stromal cell cultures of human endometrium were used to assess the influence of different stimulating factors on the TSG-6 gene and protein expression via real-time PCR and ELISA. RESULTS: Herein demonstrate that TSG-6 mRNA is expressed by the endometrium during the proliferative and secretory phases of the menstrual cycle. We also show that conditioned media from placental tissues induce rapid upregulation of TSG-6 mRNA expression and sustained protein secretion, with evidence that TNF is an important factor in this effect. Furthermore, we demonstrate changes in protein expression in the mid-secretory phase in women affected by recurrent abortions. CONCLUSION: These data suggest that TSG-6 expression might be essential in endometrial matrix organization and feto-maternal communication during the implantation process.

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.

Does metformin influence the insulin-, IGF I- and IGF II-receptor gene expression and Akt phosphorylation in human decidualized endometrial stromal cells?

OBJECTIVE: To assess the effects of metformin on insulin-, IGF I-, and IGF II-receptor gene expression and Akt phosphorylation in decidualized human endometrial stromal cells (ESC) after stimulation with insulin, IGF I and II. STUDY DESIGN: ESC were isolated from healthy, regularly cycling women and after two passages decidualized with estrogen/progesterone±metformin. Cells were incubated with insulin, IGF I or IGF II for 1, 5, and 10 min to assess Akt phosphorylation by Western blot. To investigate the insulin-, IGF I- and IGF II-receptor gene expression ESC were incubated with insulin, IGF I or IGF II for 6 and 24h. RESULTS: Insulin- and IGF I-receptor gene expression in ESC changed significantly after incubation with insulin, IGF I or IGF II. This was further augmented in metformin pretreated cells, while IGF II-receptor gene expression changed particularly after pretreatment with metformin. Akt phosphorylation peaked after 5 min insulin, IGF I and IGF II stimulation in ESC in both control (control 0.08 ± 0.03 vs. insulin 0.74 ± 0.19, IGF I 0.68 ± 0.22, IGF II 0.53 ± 0.13, p<0.05) and metformin pretreated cells (control 0.03 ± 0.01 vs. insulin 0.75 ± 0.11, IGF I 0.74 ± 0.15, IGF II 0.67 ± 0.09, p<0.005). However, there was no significant difference between the control and metformin pretreated group. CONCLUSION: Insulin, IGF I and IGF II lead to changes in their receptor gene expression and induced Akt phosphorylation in ESC. These effects were further highlighted in the presence of metformin.

Metformin modulates IL-8, IL-1ß, ICAM and IGFBP-1 expression in human endometrial stromal cells.

To evaluate the effects of metformin on endometrial stromal cell gene expression and on the decidualization process, endometrial biopsies were collected from five healthy, regularly cycling women. Stromal cell culture was performed and decidualized with oestrogen/progesterone in the presence or absence of metformin and thereafter stimulated with insulin. The effect of metformin on decidualization was analysed by prolactin determination in the cell culture supernatant. Gene expression of insulin-like growth factor binding protein 1 (IGFBP-1), interleukin (IL) 8 and 1ß and intercellular adhesion molecule (ICAM) was analysed by real-time PCR. Decidualization was significantly diminished in cells incubated with metformin (P<0.05) accompanied by a significant reduction of prolactin secretion in the supernatant (day 10: 2.2 fold, P<0.05; day 15: 3.1 fold, P<0.05). IGFBP-1 gene expression was reduced after long-term metformin exposure (7.7 fold, P<0.05). The negative effect of insulin on IL-8 (4.8 fold) and IL-1ß (9.3 fold) gene expression was similarly found in cells incubated with metformin. As far as is known, this is the first demonstration of a change in endometrial gene and protein expression after in-vitro stimulation with metformin, including a diminished decidualization process and changes in genes relevant to implantation.

Fertility preservation in >1,000 patients: patient's characteristics, spectrum, efficacy and risks of applied preservation techniques.

INTRODUCTION: Data on the characteristics of female patients counselled for fertility preservation and the efficacy and risk of the applied procedures are still poor. We therefore analysed the registry of a network of 70 infertility centers which are involved in fertility preservation in Germany, Switzerland and Austria, called FertiPROTEKT ( hhtp://www.fertiprotekt.eu ). MATERIALS AND METHODS: 1,280 counselled patients (15-40 years) were analysed regarding characteristics and different fertility preservation treatments before cytotoxic therapy in 2007-2009. RESULTS: 34.8% of the counselled patients were diagnosed with breast cancer, 30.5% with Hodgkin's lymphoma, 25.4% with other malignancies and 9.3% with non-malignant diseases. 89.6% of the treated breast cancer patients were 25-40 years of age, and 87.5% of the lymphoma patients were 15-30 years of age. At the time of counselling, 85.3% of the breast cancer patients and 92.7% of the lymphoma patients were childless. 1,080 patients received a single or combined therapy such as GnRH agonists (n = 823), cryopreservation of ovarian tissue (n = 500), ovarian stimulation (n = 221) and transposition of the ovaries (n = 24). Only one severe complication, requiring postponement of the chemotherapy, was documented. In stimulated patients, 2,417 oocytes (mean n = 11.6, SD ± 7.7) were received. Fertilisation rate per received oocyte was 61.3%. CONCLUSIONS: Fertility preservation programmes mainly involve women without children, diagnosed with breast cancer or Hodgkin's lymphoma. Fertility preservation techniques can be applied with low risk. The limited and age-dependant success rate of the different therapies require individualised approaches of single or combined fertility preservation techniques.

Efficacy and safety of ovarian stimulation before chemotherapy in 205 cases.

Ovarian stimulation and cryopreservation of fertilized oocytes before cancer therapy is the best established and efficient fertility preservation technique and should still be considered before chemotherapy. Within a short time frame of 2 weeks, between 8.6 (18-25 y) and 5.1 (36-40 y) fertilized oocytes can be cryopreserved.

Changes in cell proliferation, but not in vascularisation are characteristic for human endometrium in different reproductive failures--a pilot study.

BACKGROUND: Reproductive failure, determined as recurrent spontaneous abortions (RSA) or recurrent implantation failure (RIF) in women is not well understood. Several factors, including embryo quality, and cellular and molecular changes in endometrium may contribute to the insufficient feto-maternal interaction resulting in reproductive failure. Prior clinical studies suggest an inadequate endometrial growth and development of the endometrium, leading to a lesser endometrial thickness. METHODS: We therefore aimed to determine the cellular proliferation using Ki67, and the expression of markers of vascularisation, such as factor VIII (a marker of endothelial cells) and smooth muscle cell actin (SMCA; a marker of pericytes and smooth muscle cells) in endometrium of healthy women and women with RSA or RIF. LH-dated mid-secretory endometrial biopsies of seven healthy women and twenty women with reproductive failure were examined via immunohistochemistry followed by image analysis. RESULTS: Cellular proliferation but not expression of factor VIII or SMCA was decreased (P < 0.0004) in endometrium of women with RSA and RIF compared to healthy controls. CONCLUSION: Our data indicate that reproductive failure is due to insufficient cell proliferation/tissue growth rather than inadequate vascularisation in the endometrium.