Susanne Kunze

Expression of the proliferation marker Ki-67 and of p53 tumor protein in trophoblastic tissue of preeclamptic, HELLP, and intrauterine growth-restricted pregnancies.

Summary: The human placenta owns the biochemical machinery to proliferate throughout gestation. The aim of this study was to investigate the expression of the proliferation marker Ki-67 in trophoblastic tissue of intrauterine growth retarded (IUGR) placentas, preeclamptic, HELLP, and in normal trophoblastic tissue. Slides of paraffin-embedded trophoblastic tissue of patients with IUGR, preeclamptic patients, HELLP patients, and normal term placentas were incubated with monoclonal antibodies against Ki-67 and p53. Staining reaction was performed with the ABC reagent. Intensity of immunohistochemical reaction on the slides was analyzed using a semiquantitative score. Identification of Ki-67-expressing cells was done by immunofluorescence double staining with Ki-67 and cytokeratin antibodies. Expression of Ki-67 and p53 are significantly elevated in cytotrophoblastic cells of placentas with HELLP as investigated by immunohistochemistry and double immunofluorescence. However, preeclamptic cytotrophoblastic tissue on the other hand showed no significantly different expression intensity of Ki-67 compared with normal placental tissue controls and no changes in p53 expression compared with controls. In IUGR cytotrophoblastic cells, we found no statistically significant change in Ki-67 expression but a statistically significant down-regulation of p53. An elevated proliferation of cytotrophoblastic cells seems to be related to HELLP, and this enhanced proliferation seems to be controlled by p53.

Inhibin-alpha subunit is an independent prognostic parameter in human endometrial carcinomas: Analysis of inhibin/activin-alpha, -betaA and -betaB subunits in 302 cases

Inhibins are dimeric glycoproteins, composed of an alpha-subunit (inhibin-alpha) and one of two possible beta-subunits (betaA or betaB), with substantial roles in human reproduction and in endocrine-responsive tumours. The aims of this study were to determine the distribution of inhibin-alpha, -betaA and -betaB subunits in malignant human endometrial tissue and the assessment of an association with specific clinicopathologic tumour features and clinical outcome. A series of 302 endometrial cancer tissue samples were immunohistochemically analysed with monoclonal antibodies against inhibin subunits. The inhibin-alpha subunit showed a significant association with histological grading, surgical staging, lymph node status and diabetes in patients with endometrial cancer. Interestingly, loss of inhibin-alpha expression resulted in a poorer survival of endometrial cancer patients. Additionally, survival analysis demonstrated that inhibin-alpha immunoreactivity was an independent prognostic factor for progression-free survival, cause-specific survival as well as for overall survival. In contrast, although inhibin-betaA- and -betaB subunits showed a significant association between endometrial histological subtypes and histological grading, both subunits were not found to be associated with survival in endometrial cancer patients. Therefore, inhibin-alpha immunostaining might be used as a simple and efficient marker to identify high-risk patients leading to the selection of patients for an aggressive adjuvant therapy.

Expression of inhibin/activin subunits alpha (-alpha), beta A (-beta (A)) and beta B (-beta (B)) in placental tissue of normal and intrauterine growth restricted (IUGR) pregnancies.

During human pregnancy the placenta produces a variety of proteins like steroid hormones and their receptors that are responsible for the establishment and ongoing of the feto-placental unit. Inhibins are dimeric glycoproteins, composed of an α-subunit and one of two possible β-subunits (βA or βB). Aims of the present study were the determination of the frequency and tissue distribution patterns of the inhibin/activin subunits in human placental tissue of normal pregnancies and pregnancies complicated with fetal growth restriction (IUGR). Slides of paraffin embedded placental tissue were obtained after delivery from patients diagnosed with IUGR (n = 6) and normal term placentas (n = 8). Tissue samples were fixed and incubated with monoclonal antibodies inhibin/activin-subunits -α, -βA, -βB. Intensity of immunohistochemical reaction on the slides was analysed using a semi-quantitative score and statistical analysis was performed (P<0.05). A significant lower expression of the inhibin-α subunit in IUGR extravillous trophoblast compared to normal pregnancies was observed, while the inhibin-α immunostaining was significantly upregulated in syncytiotrophoblast. Additionally, a significant down-regulation of inhibin-βB subunit in extravillous trophoblast cells in IUGR syncytiotrophoblast cells was demonstrated. A co-localisation of inhibin-α and the β-subunits was also observed, suggesting a production and secretion of intact inhibin A and inhibin B. Although the precise role of these inhibin/activin subunits in human placenta and IUGR pregnancies is still unclear, they could be involved in autocrine/paracrine signalling, contributing to several aspects like angiogenesis and tissue remodelling.

Peroxisome proliferator-activated receptor-gamma in normal human pregnancy and miscarriage.

Peroxisome proliferator-activated receptors (PPAR) belong to the superfamily of nuclear hormone receptors. Recent investigations emphasize a possible involvement of PPAR in obstetric and gynaecologic disorders like polycystic ovarian syndrome, endometriosis and preeclampsia. The aim of this study was to determine the frequency and distribution of peroxisome proliferator-activated receptor-gamma (PPARgamma) in normal human pregnancy and miscarriage. Placental tissue was obtained from normal human pregnancy and miscarriage during the first trimester of pregnancy. PPARgamma localisation was investigated by immunohistochemical methods. Immediate immunoreactivity of PPARgamma was observed in villous and extravillous trophoblast nuclei in normal first trimester pregnancy. A significantly enhanced labelling of PPARgamma was identified in extravillous trophoblast of miscarriage patients. This enhanced immunopositivity was also found in nuclei of villous trophoblast cells of miscarriage patients but without statistical significance. Because trophoblast invasion is negatively correlated to PPARgamma stimulation and blocking of PPARgamma leads to increased trophoblast invasion, our findings may suggest that enhanced expression of PPARgamma in abortive extravillous trophoblasts may be one factor linked to miscarriage.

Inhibin/activin-betaC subunit does not represent a prognostic parameter in human endometrial cancer.

IntroductionInhibins, dimeric peptide hormones composed of an α subunit and one of two possible β subunits (betaA or betaB), exhibit substantial roles in human reproduction and in endocrine-responsive tumors. Recently, two novel inhibin-beta subunits, defined as betaC and betaE, have been identified in humans. However, the prognostic significance and clinical implications of the novel inhibin-betaC subunit in endometrial cancers is still quite unclear.Materials and methodsA series of 296 uterine endometrial carcinomas were immunohistochemically analyzed with specific antibody against the inhibin-betaC subunit. The staining reactions were correlated with several clinicopathological characteristics and the clinical outcome.ResultsEndometrial cancer tissue demonstrated an immunolabelling against the inhibin-betaC subunit. The inhibin-betaC expression in endometrial carcinoma samples revealed a significant association with hemangiosis. However, the expression of this inhibin subunit did not affect patients’ progression-free, cause-specific and overall survival.ConclusionOverall, inhibin-betaC subunit was demonstrated in endometrial cancer tissue. This novel betaC subunit demonstrated a significant association with hemangiosis although without any impact on the patients’ survival. Moreover, the inhibin-betaC subunits did not constitute an independent prognostic parameter in endometrial cancer patients. Therefore, the isolated analysis of this subunit might be of minor prognostic value in identifying high-risk patients.

Erythropoietin and erythropoietin receptor expression in normal and disturbed pregnancy.

OBJECTIVE Erythropoietin (Epo) is known to regulate the number of circulating erythrocytes. Epo receptor (Epo-R) expression is limited to few organs including the uterus. We investigated differences in Epo and Epo-R expression in normal and disturbed first trimester human pregnancy. STUDY DESIGN Placental tissue was obtained from normal human pregnancy, abortion and hydatidiform mole during the first trimester of pregnancy. Epo and Epo-R expression was investigated by Immunohistochemistry and real time PCR (TaqMan). The intensity and distribution patterns of Epo and Epo-R expression were evaluated by using a semi-quantitative method (immunoreactive score) as previously described. RESULTS Epo-R expression was upregulated in the villous trophoblast (VT) of abortive tissue (p=0.002) as compared to normal pregnancy. This was further confirmed on mRNA level. With regard to mole pregnancy, Epo-R expression was also significantly increased in the VT (p<0.001). In extravillous trophoblasts (EVT), only Epo, not Epo-R expression was significantly increased in abortive tissue (p=0.006) as well as in hydatidiform mole (p=0.041) in comparison to normal pregnancy. Identification of EVT as Epo-and Epo-R-positive cells was obtained by double immunofluorescence with CK7 and Epo/ Epo-R double staining. Both mole pregnancy and abortion were accompanied by an upregulation of Epo (p=0.041; p=0.018) and Epo-R expression (p=0.007; p=0.015) in decidual tissue as compared to normal pregnancy. CONCLUSION Within our study we were able to demonstrate increased expression of Epo and Epo-R in abortive tissue and hydatidiform mole. Whether upregulation of Epo and Epo-R is secondary to placental hypoxia as part of the abortion process or a risk factor of its own, needs to be further investigated.

Inhibin-betaC subunit expression in normal and pathological human placental tissues

Inhibins and activins are important regulators of the female reproductive system. Recently, a novel inhibin betaC subunit has been identified. However, only limited data on the expression of this novel inhibin-betaC subunit in normal and pathological human placentas exist. Tissue specimens of normal, preeclamptic, hemolysis, elevated liver enzymes, low platelets (HELLP), and intrauterine growth restriction (IUGR) pregnancies (n = 24) were obtained at the conclusion of a cesarean section. Normal and pathological placental tissues were analyzed by an immunohistochemical staining reaction with a specific antibody against this novel inhibin-betaC subunit. Overall, expression of the inhibin-betaC subunit could be demonstrated in normal and pathological placental tissue. The immunoreactive score (IRS) for inhibin-betaC did not show any significant differences between normal, preeclamptic, HELLP, and IUGR tissue in extravillous trophoblast and syncytiotrophoblast cells. Immunolabelling of this novel inhibin-βC protein in normal and pathological placental tissue was demonstrated, although no differences in the staining intensity could be observed. Therefore, the inhibin-βC isoform might not primarily be involved in the pathogenesis of these pregnancy-associated disorders. The functional role of this novel inhibin-betaC subunit in normal and pathological human placenta is still quite unclear and should thus be further investigated.

Evidence of inhibin/activin subunit betaC and betaE synthesis in normal human endometrial tissue

BackgroundInhibins are important regulators of the female reproductive system. Recently, two new inhibin subunits betaC and betaE have been described, although it is unclear if they are synthesized in normal human endometrium.MethodsSamples of human endometrium were obtained from 82 premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Endometrium samples were classified according to anamnestic and histological dating into proliferative (day 1-14, n = 46), early secretory (day 15-22, n = 18) and late secretory phase (day 23-28, n = 18). Immunohistochemical analyses were performed with specific antibodies against inhibin alpha (n = 81) as well as inhibin betaA (n = 82), betaB (n = 82), betaC (n = 74) and betaE (n = 76) subunits. RT-PCR was performed for all inhibin subunits. Correlation was assessed with the Spearman factor to assess the relationship of inhibin-subunits expression within the different endometrial samples.ResultsThe novel inhibin betaC and betaE subunits were found in normal human endometrium by immunohistochemical and molecular techniques. Inhibin alpha, betaA, betaB and betaE subunits showed a circadian expression pattern, being more abundant during the late secretory phase than during the proliferative phase. Additionally, a significant correlation between inhibin alpha and all inhibin beta subunits was observed.ConclusionsThe differential expression pattern of the betaC- and betaE-subunits in normal human endometrial tissue suggests that they function in endometrial maturation and blastocyst implantation. However, the precise role of these novel inhibin/activin subunits in human endometrium is unclear and warrants further investigation.

Immunolabeling of the inhibin-βA and -βB subunit in normal and malignant human cervical tissue and cervical cancer cell lines.

Objectives: Inhibins, dimeric peptide hormones composed of an &agr;-subunit and 1 of 2 possible &bgr; subunits (&bgr;A or &bgr;B), exhibit substantial roles in human reproduction and in endocrine-responsive tumors. However, it is still unclear whether normal and cancerous cervical tissues as well as cervical cancer cell lines express the inhibin-&bgr;A and -&bgr;B subunits. Materials and Methods: Normal human uterine cervical tissue was obtained from 4 premenopausal nonpregnant patients. In addition, a total of 32 specimens of cervical intraepithelial neoplasia (CIN) of different stages were obtained (CIN 1 = 10, CIN 2 = 9, and CIN 3 = 13). Moreover, 30 squamous cervical cancer samples of well-differentiated (grade 1; n = 10), moderate differentiated (grade 2; n = 10), and poorly differentiated (grade 3; n = 10) grading were analyzed. Results: An immunohistochemical staining reaction for inhibin-&bgr;A and -&bgr;B subunits could be observed in normal and malignant cervical tissue as well as in cervical cancer cell lines. Regarding inhibin-&bgr;A significant differences were observed between normal tissue and CIN 1 and CIN 3. Moreover, the immunohistochemical staining reaction for inhibin-&bgr;A was significantly higher in CIN 3 compared with that in cervical carcinoma grades 1 and 2. The inhibin-&bgr;B expression was higher in CIN and cervical cancer compared with that in normal cervical tissue. Inhibin-&bgr;B was significantly higher in CIN 2 and CIN 3 compared with cancer tissues of histological grade 1. In addition, a significant increase of the staining intensity was observed between cervical cancer grades 1 and 2 as well as grade 3. Conclusions: Both inhibin-&bgr; subunits demonstrated a differential expression in CIN and squamous cancer, suggesting important roles in cervical carcinogenesis. Inhibin-&bgr;A might be important during progression of CIN, whereas the inhibin-&bgr;B subunit could exert a substantial function during differentiation of cervical carcinomas. Moreover, the synthesis of this subunit in cervical carcinoma cell lines also allows the use of this cell line to elucidates their functions in cervical cancer pathogenesis.