Leila Unkila-Kallio

The FOXL2 C134W mutation is characteristic of adult granulosa cell tumors of the ovary.

Granulosa cell tumors of the ovary represent ∼5% of malignant ovarian cancers. It has recently been reported that 95–97% of adult granulosa cell tumors carry a unique somatic mutation in the FOXL2 gene. We undertook this study to verify the presence of the FOXL2 Cys134Trp mutation in two geographically independent cohorts of granulosa cell tumors and to examine the expression pattern of FOXL2 in these tumors. A total of 56 tumors with the histological diagnosis of adult granulosa cell tumor from two centers, Melbourne and Helsinki, were examined for the presence of the mutation using direct sequence analysis. Two granulosa cell tumor-derived cell lines, COV434 and KGN, three juvenile granulosa cell tumors and control tissues were also examined. The expression of the FOXL2 gene was determined using quantitative RT-PCR and/or immunohistochemistry. We found that 52 of the 56 adult granulosa cell tumors harbor the mutation, of which three were hemi/homozygous. Of the four cases with wild-type FOXL2 sequence, reappraisal suggests that three may have been misclassified at primary diagnosis. The KGN cells were heterozygous for the mutation, whereas the COV434 cells had a wild-type FOXL2 genotype. The expression levels of FOXL2 were similar across the adult granulosa cell tumors and the normal ovary controls; one mutation-negative granulosa cell tumor had high FOXL2 mRNA levels, whereas the COV434 cells and two of the three juvenile granulosa cell tumors lacked the expression of FOXL2. Our data provide confirmation of the frequent presence of the FOXL2 C134W mutation in adult granulosa cell tumors and demonstrate that the mutation is not associated with altered FOXL2 expression. The mutation analysis may be a useful tool to differentiate particularly between cell-rich diffuse granulosa cell tumors and mitotically active sex cord-stromal tumors. This unique FOXL2 mutation appears to be characteristic of adult granulosa cell tumors.

Screening for coeliac disease in women with a history of recurrent miscarriage or infertility

Because subclinical coeliac disease may decrease fertility or complicate pregnancy, we screened women with recurrent miscarriage of unknown aetiology (n= 63), unexplained infertility (n= 47) and infertility with a known cause (n= 82), for anti‐endomysium antibodies in serum to find undiagnosed coeliac disease. One woman (1.6%) with recurrent miscarriage, another woman (2.1%) with unexplained infertility and one woman (2.0%) in the control group (n= 51), were considered to have coeliac disease. We could not demonstrate a higher frequency of coeliac disease in women with infertility or recurrent miscarriage, but suggest that undiagnosed coeliac disease is common in women.

GATA-4 Regulates Bcl-2 Expression in Ovarian Granulosa Cell Tumors

Excessive cell proliferation and decreased apoptosis have been implicated in the pathogenesis of ovarian granulosa cell tumors (GCTs). We hypothesized that transcription factor GATA-4 controls expression of the antiapoptotic factor Bcl-2 and the cell cycle regulator cyclin D2 in normal and neoplastic granulosa cells. To test this hypothesis, a tissue microarray based on 80 GCTs was subjected to immunohistochemistry for GATA-4, Bcl-2, and cyclin D2, and the data were correlated to clinical and histopathological parameters. In addition, quantitative RT-PCR for GATA-4, Bcl-2, and cyclin D2 was performed on 21 human GCTs. A mouse GCT model was used to complement these studies. The role of GATA-4 in the regulation of Bcl2 and ccdn2 (coding for cyclin D2) was studied by transactivation assays, and by disrupting GATA-4 function with dominant negative approaches in mouse and human GCT cell lines. We found that GATA-4 expression correlated with Bcl-2 and cyclin D2 expression in human and murine GCTs. Moreover, GATA-4 enhanced Bcl-2 and cyclin D2 promoter activity in murine GCT cells. Whereas GATA-4 overexpression up-regulated and dominant negative GATA-4 suppressed Bcl-2 expression in human GCT cells, the effects on cyclin D2 were negligible. Our results reveal a previously unknown relationship between GATA-4 and Bcl-2 in mammalian granulosa cells and GCTs, and suggest that GATA-4 influences granulosa cell fate by transactivating Bcl-2.

Fear of childbirth and pregnancy-related anxiety in women conceiving with assisted reproduction.

OBJECTIVE: To compare the prevalence and predictors of severe fear of childbirth and pregnancy-related anxiety in groups of assisted reproduction treatment (ART) and spontaneously conceiving women with singleton pregnancies. METHODS: The ART group (n = 367, nulliparous 260) represented a cohort from five Finnish infertility clinics in 1999. The control group (n = 379, nulliparous 135) was enrolled in this study by consecutive sampling the same year. Fear of childbirth was assessed by means of the revised version of the Fear-of-Childbirth Questionnaire and pregnancy-related anxiety by means of the Pregnancy Anxiety Scale at gestational week 20 ± 3.2 (mean±standard deviation). RESULTS: The frequency of severe fear of childbirth and anxiety (classified as total scores in the 90th percentile or higher in the revised Fear of Childbirth Questionnaire and Pregnancy Anxiety Scale) did not differ between the groups. Nulliparity was associated with more frequent severe anxiety only in the controls. In nulliparous participants, a partnership of more than 5 years decreased the risk of severe fear of childbirth (odds ratio 0.3, 95% confidence interval 0.2–0.7). In the nulliparous ART group, a long duration of infertility (7 or more years) increased the risk of severe fear of childbirth (odds ratio 4.4, 95% confidence interval 1.2–16.9). CONCLUSION: Women conceiving after ART do not experience severe fear of childbirth or pregnancy-related axiety more often than spontaneously conceiving controls. However, a long duration of infertility is an independent risk factor regarding severe fear of childbirth. LEVEL OF EVIDENCE II-2

BRCA1 and BRCA2 mutations among 233 unselected Finnish ovarian carcinoma patients.

Germline mutations of BRCA1 and BRCA2 predispose to hereditary breast-ovarian cancer syndrome. In Finland, 20 different BRCA1/2 mutations have been identified, and 13 of them are founder mutations that account for the vast majority of Finnish BRCA1/2 families. The purpose of our study was to determine the prevalence of BRCA1/2 mutations in unselected Finnish ovarian carcinoma patients and to evaluate the relationship between mutation carrier status and personal/family history of cancer. Two hundred and thirty-three patients were screened for all the 20 BRCA1/2 mutations known in the Finnish population. Additionally, a subgroup of patients with personal history of breast cancer and/or family history of breast and/or ovarian cancer was screened for novel BRCA1/2 mutations. Thirteen patients (5.6%) had mutations: eleven in BRCA1 and two in BRCA2. All the mutation-positive patients were carriers of the previously known Finnish BRCA1/2 mutations, and seven recurrent founder mutations accounted for 12 of the 13 mutations detected. A logistic regression analysis was used to determine the odds of mutation for ovarian carcinoma patients. The most significant predictor of a mutation was the presence of both breast and ovarian cancer in the same woman, but family history of breast cancer was also strongly related to mutation carrier status. Although BRCA1/2 mutation testing is not warranted in the general Finnish ovarian cancer patient population, patients who have also been diagnosed with breast cancer or have family history of breast or breast and ovarian cancer could benefit from referral to genetic counselling and mutation testing.

Surgical treatment of vulvar vestibulitis: a review

Vulvar vestibulitis syndrome, a subset of vulvodynia, is a complex pain syndrome. It causes severe dyspareunia and affects mainly young women. The etiology is unknown and no uniformly effective treatment exists. Surgery has been considered as ‘the last resort’ in the management of patients not responding to conservative treatment modalities. For this review, all studies of surgical treatment of vulvar vestibulitis were evaluated. We describe the evolution of vestibulectomy techniques through the years. Our aim was also to find out whether any surgical technique is better than others providing better patient satisfaction and lower complication rates. We conclude that surgical technique as such plays a relatively small role. Surgery seems to be effective. However, lack of randomized trials and insufficient data on complication rates must be emphasized.

FOXL2, GATA4, and SMAD3 co-operatively modulate gene expression, cell viability and apoptosis in ovarian granulosa cell tumor cells.

Aberrant ovarian granulosa cell proliferation and apoptosis may lead to granulosa cell tumors (GCT), the pathogenesis of which involves transcription factors GATA4, FOXL2, and SMAD3. FOXL2 gene harbors a point mutation (C134W) in a vast majority of GCTs. GATA4 is abundantly expressed in GCTs and its expression correlates with poor prognosis. The TGF-β mediator SMAD3 promotes GCT cell survival through NF-κB activation, and interacts with FOXL2. Here, we find that the expression patterns of these factors overlap in the normal human ovary and 90 GCTs, and positively correlate with each other and with their mutual target gene CCND2, which is a key factor for granulosa cell proliferation. We have explored the molecular interactions of FOXL2, GATA4, and SMAD3 and their roles in the regulation of CCND2 using co-immunoprecipitation, promoter transactivation, and cell viability assays in human GCT cells. We found that not only SMAD3, but also GATA4 physically interact with both wild type and C134W-mutated FOXL2. GATA4 and SMAD3 synergistically induce a 8-fold increase in CCND2 promoter transactivation, which is 50% reduced by both FOXL2 types. We confirmed that wild type FOXL2 significantly decreases cell viability. Interestingly, GATA4 and SMAD3 caused a marked reduction of GCT cell apoptosis induced by wild type FOXL2. Thus, the effects of GATA4 and SMAD3 on both cell viability and apoptosis are distinct from those of wild type FOXL2; a perturbation of this balance due to the oncogenic FOXL2 mutation is likely to contribute to GCT pathogenesis.

GATA4 protects granulosa cell tumors from TRAIL-induced apoptosis

Disturbances in granulosa cell apoptosis have been implicated in the pathogenesis of human granulosa cell tumors (GCTs). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent cytokine that induces apoptosis in a variety of malignancies without toxic effects on benign cells. The aim of this study was to investigate the expression and functionality of the TRAIL receptors DR4 and DR5 in human GCTs. Additionally, we examined the role of GATA4, a transcription factor expressed in normal and malignant granulosa cells, in TRAIL-induced GCT apoptosis. For this purpose, a tissue microarray of 80 primary and 12 recurrent GCTs was subjected to immunohistochemistry for DR4 and DR5, and freshly isolated primary GCT cultures were utilized to evaluate the functional effects of TRAIL on GCT cells. To clarify the role of GATA4 in the regulation of TRAIL-induced apoptosis, a human GCT-derived cell line (KGN) was transduced with lentiviral vectors expressing small hairpin RNAs targeting GATA4 or transfected with adenovirus expressing either wild-type or dominant negative mutant GATA4. We found that receptors DR4 and DR5 are expressed in a vast majority of GCTs as well as in primary GCT cultures, and that TRAIL induces apoptosis in the primary GCT cultures. Moreover, we showed that overexpressing GATA4 protects GCTs from TRAIL-induced apoptosis in vitro, whereas disrupting GATA4 function induces apoptosis and potentiates the apoptotic effect of TRAIL administration. Our results demonstrate that the TRAIL pathway is functional in GCT cells, and suggest that transcription factor GATA4 may function as a survival factor in this ovarian malignancy.

Prenatal Expectations in Transition to Parenthood: Former Infertility and Family Dynamic Considerations

Prenatal expectations are important for the future parent-child relationship. The authors examined how maternal and paternal prenatal expectations of the relationship with the child predicted 1st-year parenting stress and whether these expectations were violated over the transition to parenthood. They further examined how former infertility affected these associations. The participants were 745 Finnish couples, 367 having undergone a successful assisted reproductive treatment and 378 conceiving spontaneously. Couples completed a questionnaire of family representations during pregnancy and when the child was 2 and 12 months old and Abidins Parenting Stress Index at 2 and 12 months postpartum. The hypothesis of moderately high expectations predicting the lowest level of parenting stress was substantiated only concerning paternal expectations of own autonomy with the child. Generally, however, negative expectations of own and spouses relationship with the child were linearly associated with higher parenting stress. Postnatal representations were more positive or equal to expectations, except for negative violation occurring in maternal expectation of the father-child relationship, especially among normative mothers. The results are discussed in relation to family dynamic considerations and special features of formerly infertile couples.

Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 are highly expressed in ovarian granulosa cell tumors

OBJECTIVE Ovarian granulosa cell tumors (GCTs) are hormonally active sex cord stromal tumors accounting for 3-5% of all ovarian cancers. These tumors are generally diagnosed at an early stage but there is a high risk of recurrence, associated with high mortality. Treatment of recurrent GCTs is difficult, and biologically targeted treatment modalities are lacking. GCTs are highly vascularized, and angiogenic factors most probably play a role in their pathology. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis, but in GCTs, the role of VEGF and its receptors VEGFR-1 (FLT1) and VEGFR-2 (KDR) remains largely unknown. Our objective is to study the expression of VEGF and its receptors in human GCTs. METHODS We analyzed GCTs from 106 patients for the expressions of VEGF and its receptors utilizing tumor tissue microarray, tumor mRNA, and patient serum samples. RESULTS We found that VEGF and its main biologically active receptor VEGFR-2 were highly expressed in primary and recurrent GCTs, when compared with normal granulosa-lutein cells. The expression of VEGF correlated positively to tumor microvessel density and to VEGFR-2 expression at the protein (P<0.05) and mRNA (P<0.05) levels. In contrast to VEGFR-2, the expression of VEGFR-1 was weak. Tumor VEGF protein expression was not prognostic for recurrence, however, we found high levels of circulating VEGF in the serum of patients with primary GCT. CONCLUSIONS The results suggest an important role of VEGF and VEGFR-2 in GCT pathology and support the possibility of applying novel VEGF- or VEGFR-2-targeted treatments to patients with GCT.