Julia Jauckus

Regulation of matrix metalloproteinases and their inhibitors in uterine endometrial cells of patients with and without endometriosis

OBJECTIVE To determine whether alterations in the secretion and regulation of matrix metalloproteinases (MMPs) and their inhibitors are present in uterine endometrial cells from endometriosis patients. STUDY DESIGN In an in vitro study, uterine endometrial cells from 19 regularly cycling women with and 32 without endometriosis were treated with diethyl stilbestrol, promegestone (R5020), interleukin-1 (IL-1) and tumor necrosis factor a (TNF-alpha). Culture supernatants were assayed for MMPs 1, 2, 3, and 9, and for tissue inhibitors of MMP (TIMP-1 and TIMP-2) by ELISA. RESULTS MMP-3 was secreted in high concentrations, moderate concentrations were seen for MMP-1 and MMP-2, and very low concentrations for MMP-9. Substantially more TIMP-1 than TIMP-2 was secreted. MMP-1 and MMP-3 were uniformly attenuated by R5020, while MMP-2 was not influenced by hormone treatment. MMP-3 was upregulated by TNF-alpha in all samples while IL-1 only increased secretion in cells from endometriosis patients. CONCLUSION The upregulation of MMP-3 by IL-1 may contribute to an increased invasiveness of uterine endometrial fragments in endometriosis patients.

Intravaginal and intracervical application of seminal plasma in in vitro fertilization or intracytoplasmic sperm injection treatment cycles—a double-blind, placebo-controlled, randomized pilot study

OBJECTIVE To evaluate whether intravaginal application of seminal plasma at the time of follicle aspiration in IVF or intracytoplasmic sperm injection treatment cycles has the potential to increase pregnancy rate. To calculate the number of patients needed to achieve significance in a multicenter trial. DESIGN Double-blind, placebo-controlled randomized pilot study. SETTING University department of gynecological endocrinology and reproductive medicine. PATIENT(S) One hundred sixty-eight patients undergoing IVF or intracytoplasmic sperm injection treatment. INTERVENTION(S) Cryopreserved seminal plasma from the patients partner or sodium chloride (placebo) was injected into the cervix and the posterior fornix of the vagina just after follicle aspiration. MAIN OUTCOME MEASURE(S) Clinical-pregnancy rate. RESULT(S) One hundred sixty-eight patients agreed to participate in the study. Participation was limited to one treatment cycle. Thirty-one patients (18%) were excluded from the study, mainly as a result of canceled embryo transfers. Seventy patients received placebo, and 67 received seminal plasma. The clinical-pregnancy rate was 25.7% (18/70) in the placebo group. The clinical-pregnancy rate in the seminal plasma group reached 37.3% (25/67), corresponding to a relative increase of 45%. CONCLUSION(S) Even though significance was not reached in this pilot study, the data suggest that seminal plasma has the potential to improve pregnancy rate. It is estimated that around 450 patients need to be recruited to reach significance in a multicenter study.

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.

Metformin modulates IL-8, IL-1β, 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 1β 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-1β (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.

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

BackgroundThe 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.MethodsLH-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.ResultsThe 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).ConclusionsWomen 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.

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.

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.

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

BackgroundReproductive 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.MethodsWe 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.ResultsCellular 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.

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.

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

ObjectivePolycystic 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/designTen 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.ResultsMetformin 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.ConclusionWhen 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.