Ulrika Ottander

Oocyte-Specific Deletion of Pten in Mice Reveals a Stage-Specific Function of PTEN/PI3K Signaling in Oocytes in Controlling Follicular Activation

Immature ovarian primordial follicles are essential for maintenance of the reproductive lifespan of female mammals. Recently, it was found that overactivation of the phosphatidylinositol 3-kinase (PI3K) signaling in oocytes of primordial follicles by an oocyte-specific deletion of Pten (phosphatase and tensin homolog deleted on chromosome ten), the gene encoding PI3K negative regulator PTEN, results in premature activation of the entire pool of primordial follicles, indicating that activation of the PI3K pathway in oocytes is important for control of follicular activation. To investigate whether PI3K signaling in oocytes of primary and further developed follicles also plays a role at later stages in follicular development and ovulation, we conditionally deleted the Pten gene from oocytes of primary and further developed follicles by using transgenic mice expressing zona pellucida 3 (Zp3) promoter-mediated Cre recombinase. Our results show that Pten was efficiently deleted from oocytes of primary and further developed follicles, as indicated by the elevated phosphorylation of the major PI3K downstream component Akt. However, follicular development was not altered and oocyte maturation was also normal, which led to normal fertility with unaltered litter size in the mutant mice. Our data indicate that properly controlled PTEN/PI3K-Akt signaling in oocytes is essential for control of the development of primordial follicles whereas overactivation of PI3K signaling in oocytes does not appear to affect the development of growing follicles. This suggests that there is a stage-specific function of PTEN/PI3K signaling in mouse oocytes that controls follicular activation.

Allopregnanolone and pregnanolone are produced by the human corpus luteum.

Using a dispersed human luteal cell culture model, progesterone, allopregnanolone and pregnanolone release following treatment by incremental doses of human chorionic gonadotrophin (hCG) were evaluated. Corpus luteum tissues, obtained from 48 healthy women scheduled for benign surgery, were grouped according to luteal age and tissue concentration of allopregnanolone and pregnanolone was determined. The mRNA expression of 5alpha-, and 5beta-reductase and 3alpha-HSOR mRNA expressions were evaluated in corpora lutea from the late luteal phase. Allopregnanolone concentrations in corpus luteum tissue were consistently about three- to four-fold higher than pregnanolone levels. Allopregnanolone tissue concentrations significantly decreased between early- and late-luteal phase, p<0.05. When exposed to hCG, progesterone output from freshly obtained human corpora lutea cells was two- three-fold increased compared to control levels. With 0.1U/ml hCG a two-fold increase in allopregnanolone levels were noted, whereas pregnanolone levels were increased by approximately 40%. Furthermore, the mRNA of 5alpha-, 5beta-reductase and 3alpha-HSOR mRNA were all expressed in human corpus luteum. In conclusion, the neurosteroids allopregnanolone and pregnanolone are produced in the human corpus luteum and their release is stimulated by trophic hormone.

A Putative Stimulatory Role of Progesterone Acting via Progesterone Receptors in the Steroidogenic Cells of the Human Corpus Luteum

Abstract To further explore the proposed auto-regulatory role of progesterone action in the human corpus luteum (CL), the expression and functional roles of progesterone receptor (PR) isoforms A and B during the luteal phase (LP) of the menstrual cycle were investigated. A total of 27 otherwise healthy patients previously scheduled for surgery were recruited after informed consent. An LH rise was detected, and CL were grouped according to age (Days 2–5 post-LH-rise, early LP; Days 6–10, mid LP; Days 11–14, late LP). Using a semiquantitative reverse transcription-polymerase chain reaction assay, the PR-B mRNA levels, which were 100- to 1000-fold lower than PR-A/B mRNA, were 46% lower (P < 0.05, n = 24) in mid LP, compared to early and late LP. CL tissue levels of progesterone and PR-A/B protein levels were inversely correlated to increasing CL age; i.e., significantly reduced levels were observed in the late LP (r2 = 0.34, P < 0.01, n = 23). Expression of PR-A/B mRNA as well as PR-A/B protein were detected by in situ hybridization and immunohistochemistry, respectively. Both methods revealed a clear and distinct localization to cells in the steroidogenic layer of the CL. Freshly obtained mid-luteal CL cells were cultured in vitro, and media were analyzed for progesterone concentrations after treatment by incremental doses of hCG and the stable PR antagonist mifepristone, alone or in combination. Mifepristone did not per se alter progesterone synthesis, but when it was added in conjunction with hCG, a dose-related inhibitory response was seen, with a maximal 47% reduction in progesterone output at a 10 μM addition (P < 0.05, n = 3). Collectively, these data implicate a stimulatory role of progesterone receptor-mediated action in the steroidogenic cells of the human CL, which may serve as an important pathway for maintaining functional homeostasis during early pregnancy.

Clinical and pregnancy outcome following ectopic pregnancy; a prospective study comparing expectancy, surgery and systemic methotrexate treatment

Background. The improved possibility of an early diagnosis of ectopic pregnancy by use of serial quantitative beta‐subunit human chorionic gonadotropin hormone levels together with transvaginal ultrasound has opened up options for conservative treatment. Systemic methotrexate treatment of unruptured ectopic pregnancy has emerged as a safe and effective alternative to surgical procedures. The aim of the present study was to investigate the effectiveness of methotrexate treatment in routine clinical practice, but also to assess pregnancy outcome during a 2.5‐year follow‐up period.

Chlamydia trachomatis, Mycoplasma genitalium, Neisseria gonorrhoeae, human papillomavirus, and polyomavirus are not detectable in human tissue with epithelial ovarian cancer, borderline tumor, or benign conditions

OBJECTIVE We sought to analyze the presence of the microorganisms Chlamydia trachomatis, Mycoplasma genitalium, Neisseria gonorrhoeae, human papillomavirus (HPV), and the polyomaviruses BK virus (BKV) and JC virus (JCV) in ovarian tissues of women with ovarian carcinomas, borderline tumors, and benign conditions. STUDY DESIGN Ovarian tissue, snap-frozen and stored at -80 degrees C, from 186 women with benign conditions, borderline tumors, and epithelial ovarian cancer, as well as tissue from the contralateral ovary of 126 of these women, were analyzed regarding presence of C trachomatis and N gonorrhoeae (transcription mediated amplification), M genitalium (real-time polymerase chain reaction [PCR]), HPV (PCR), and BKV and JCV (PCR). RESULTS All the tissue samples studied were found negative for the microorganisms analyzed. CONCLUSION C trachomatis, M genitalium, N gonorrhoeae, HPV, and the polyomaviruses BKV and JCV are not detectable in ovarian tissues either from women with benign conditions and borderline tumors or from women with ovarian cancer.

Chlamydia trachomatis and Mycoplasma genitalium plasma antibodies in relation to epithelial ovarian tumors.

Objective. To assess associations of Chlamydia trachomatis and Mycoplasma genitalium antibodies with epithelial ovarian tumors. Methods. Plasma samples from 291 women, undergoing surgery due to suspected ovarian pathology, were analyzed with respect to C. trachomatis IgG and IgA, chlamydial Heat Shock Protein 60-1 (cHSP60-1) IgG and M. genitalium IgG antibodies. Women with borderline tumors (n = 12), ovarian carcinoma (n = 45), or other pelvic malignancies (n = 11) were matched to four healthy controls each. Results. Overall, there were no associations of antibodies with EOC. However, chlamydial HSP60-1 IgG antibodies were associated with type II ovarian cancer (P = .002) in women with plasma samples obtained >1 year prior to diagnosis (n = 7). M. genitalium IgG antibodies were associated with borderline ovarian tumors (P = .01). Conclusion. Chlamydial HSP60-1 IgG and M. genitalium IgG antibodies are in this study associated with epithelial ovarian tumors in some subsets, which support the hypothesis linking upper-genital tract infections and ovarian tumor development.

Assessment of cytokine mRNA expression profiles in tumor microenvironment and peripheral blood mononuclear cells of patients with high-grade serous carcinoma of the ovary

Objective: Tumor establishment, metastatic spreading and poor survival in ovarian cancer is strongly associated with progressive derangement of the patient’s immune system. Accumulating evidence suggests that immune impairment is influenced by the production and presence of cytokines in the tumor microenvironment. Methods: Cytokine mRNA profiles in tumor tissue and peripheral blood mononuclear cells (PBMC) were analyzed in patients with high grade serous carcinoma (HGSC) of the ovary and compared it to patients with benign ovarian conditions and controls with normal ovaries. Cytokine assessment was done by real-time quantitative RT-PCR and specific primers and probes for 12 cytokines-IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, TNF-α, TNF-β/LTA, TGF-β1, and GM-CSF chosen to distinguish between cytotoxic Th1, humoral Th2, regulatory Th3/Tr1 and inflammatory responses. Results: The cytokine mRNA response in the HGSC patients was significantly up regulated compared to patients with benign ovarian conditions and normal ovary controls confirming the immunogenicity of HGSC and implying immune recognition and reaction locally in the tumor microenvironment and systemically in the peripheral blood.There was an up-regulation of inflammatory and inhibitory cytokine mRNA promoting tumor progression, T-regulatory cell priming and T-regulatory cell-mediated immune suppression. In contrast, there was an inability to mount the crucially important IFN gamma response needed for upregulation of the cytotoxic anti-tumor response in the local microenvironment. In addition, systemic IL-4- mediated Th2 response prevailed in the peripheral blood deviating the systemic defense towards humoral immunity. Conclusions: Taken together, these results suggest local and systemic cytokine cooperation promoting tumor survival, progression and immune escape. Our study confirms and extends previous investigations and contributes to the evaluation of potential cytokine candidates for diagnostic cytokine mRNA profiles and for future therapeutic interventions based on cytokine inhibition.

Enhanced Th1 and inflammatory mRNA responses upregulate NK cell cytotoxicity and NKG2D ligand expression in human pre-eclamptic placenta and target it for NK cell attack

Pre‐eclampsia (PE), a severe human pregnancy disorder, is associated with exaggerated systemic inflammation, enhanced cytokine production, and increased shedding of microvesicles leading to endothelial dysfunction, coagulopathy, and extensive placenta destruction. The cause of PE is still unclear. Evidence suggests that its origin lies in the placenta and that the maternal immune system is involved. A shift in cytokine production in PE pregnancy promotes NK cell activation, suggested to be important in PE pathogenesis. In line with this suggestion, we studied NK cell cytotoxicity in peripheral blood of PE patients and controls and the mRNA expression of cytokines and of the NKG2D receptor and its ligands MICA/B and ULBP1‐3 in PE‐ and normal placenta.

Abstract C140: RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and ovarian cancer.

Background: Recent studies have shown that RNA isolated from exosomes and other microvesicles (exoRNA) contain diagnostically relevant transcripts of tumor origin. In this study we sought to identify specific biomarkers of glioblastoma multiforme (GBM) and malignant ovarian cancer in the ribonucleic acid fraction extracted from microvesicles (exoRNA) isolated from the serum of affected patients. Methods: All samples were collected following informed consent in accordance with the appropriate protocols approved by the MGH Institutional Review Board (GBM) and the Umea University ethics committee (ovarian tumors). Patients with primary GBM (N=9), normal controls (N=7); and pathologically proven benign (N=16), borderline (N=19), and malignant (N=29, stage I-IV, grade 1–3) neoplastic transformation of the ovaries were included. ExoRNA isolated from patient serum was amplified and analyzed by microarray analysis on Agilent 4×44K arrays (GBM) and Agilent 8×60K arrays (ovarian tumors). Differential expressions between control and GBM samples were validated by qRT-PCR in a separate set of samples (N=10 in both groups). Results: Array analysis of the amplified exoRNA yielded significant signals from at least 10,000 genes on the array for all samples. The expression profiles of the exoRNA from GBM patients were shown to be different from those of normal healthy volunteers. The most significant expression differences observed in the array analysis pertained to down-regulated genes (121 genes >2-fold down) in the GBM patient exoRNA, which was validated by qRT-PCR on several genes. Gene ontology analysis of the down-regulated genes indicated these are primarily mRNAs coding for ribosomal proteins and other genes related to ribosome production. Supervised analysis showed no significant differential gene expression between the benign, borderline, and malignant ovarian tumors. However, unsupervised Consensus Clustering yielded a clear segregation of the patients into two new groups (100% resampling consensus for 61 samples). Gene Set Enrichment Analysis (GSEA) showed a strong association of one of the two groups with genes linked to translation initiation (FDR q-value 0.04), and mRNA processing (FDR q-value 0.032). GSEA also confirmed a strong overlap of the genes separating the ovarian cancer samples into two groups with those discriminating GBM from healthy control individuals. Conclusions: Microvesicle isolated RNA from patients with GBM is significantly different from that of normal control individuals. The same genes support a binary classification of ovarian tumors suggesting that common biological processes may be in effect for these different tumor types. The common signature is hallmarked by messenger RNAs coding for ribosome production, which are significantly downregulated in GBM exoRNA and in one of two novel subgroups of ovarian tumors. While the biological role of this signature remains to be elucidated, we conclude that exosomal RNA expression profiling has the potential to serve as a clinically relevant diagnostic source of information about the biology, malignancy, and state of tumors. This underlines the diagnostic potential of exosomes not only in early diagnosis, but also in directing therapies, evaluating response and in situations where biopsy samples are difficult to access. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C140.