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Background: Endometriosis is associated with inflammatory autoimmune reactions; however, aetiopathogenesis of the disease is still poorly understood. While autoimmune disorders are often associated with particular HLA alleles, the possible involvement of HLA in the aetiopathogenesis of endometriosis is still a subject of controversy. The aim of the study was to examine the distribution of HLA-DRB1 alleles in women with endometriosis. To ensure homogeneity of the studied group, only women with ovarian endometrial cysts were included., Methods: The study included 65 Polish patients of Caucasian origin in whom ovarian endometriosis had been confirmed by laparoscopic and histopathological examinations. HLA-DRB1 alleles were typed using a reverse slot blot method. A frequency of particular HLA-DRB1 alleles in patients was compared with that of a control group of 700 unrelated ethnically matched individuals as well as 193 age-matched women without endometriosis., Results: No statistically significant differences were found in the distribution of HLA-DRB1 alleles in patients with ovarian endometriosis as compared with control populations., Conclusions: The results of the present study show that ovarian endometriosis is not associated with particular HLA-DRB1 allele(s). This may suggest that aetiology of this form of endometriosis may be not primarily associated with class II HLA-mediated autoimmune reactions.

We present a patient with acromesomelic chondrodysplasia and genital anomalies caused by a novel homozygous mutation in BMPR1B, the gene coding for bone morphogenetic protein receptor 1B. The 16 year old girl, the offspring of a multiconsanguinous family, showed a severe form of limb malformation consisting of aplasia of the fibula, severe brachydactyly, ulnar deviation of the hands, and fusion of carpal/tarsal bones. In addition, she presented with hypoplasia of the uterus and ovarian dysfunction resulting in hypergonadotrophic hypogonadism. Mutation analysis of BMPR1B revealed a homozygous 8 bp deletion (del359-366). This mutation is expected to result in a loss of function and is thus different from the heterozygous missense mutations in BMPR1B recently shown to cause brachydactyly type A2 through a dominant negative effect. The patient's skeletal phenotype shows an overlap with the clinical spectrum of the acromesomelic chondrodysplasias of the Grebe, Hunter-Thompson, and DuPan types caused by homozygous mutations in the gene coding for growth differentiation factor 5 (GDF5) which is a high-affinity ligand to BMPR1B. However, the phenotype described here differs from GDF5 associated chondrodysplasias because of the additional presence of genital anomalies and the distinct limb phenotype.

Background: The plasminogen activator (PA) and matrix metalloproteinase (MMP) systems are implicated in the establishment of endometriosis. The mechanisms by which these systems are involved in the pathogenesis of this disease are not well defined and controversial results have been published. The aim of this study was to analyse mRNA and protein levels of several components of the PA and MMP systems in endometriotic tissue and endometrium from women with and without endometriosis., Methods and Results: Real-time quantitative RT-PCR assays were developed to quantify mRNA levels of these components in 57 women with endometriosis and 32 controls. Endometrium of women with endometriosis showed higher mRNA and antigenic levels of urokinase type-PA (uPA) and MMP-3 than endometrium from controls. In these patients, ovarian endometriotic tissue had higher mRNA and antigenic levels of PA inhibitor type 1 (PAI-1) and MMP inhibitor type 1 (TIMP-1) than endometrium., Conclusions: The increase in mRNA and protein levels of uPA and MMP-3 observed in endometrium of women with endometriosis may facilitate the attachment of endometrial tissue to the peritoneum and ovarian surface, as well as the invasion of the extracellular matrix. This process would lead to the formation of early endometriotic lesions. Once the ovarian endometriotic cyst is developed, PAI-1 and TIMP-1 would increase which could explain the frequent clinical finding of an endometrioma without invasion of the adjacent ovarian tissue.

In hens, the ovarian follicles committed to ovulation are arranged in an ordered follicular hierarchy. In standard broiler breeders hens genetically selected for high growth rate the reproductive function is clearly dysfunctional. Feed restriction is needed during reproductive development to limit the formation of excessive numbers of ovarian yellow follicles arranged in multiple hierarchies. To determine whether leptin is involved in the nutritional and reproductive interactions controlling follicular hierarchy in hens, blood leptin levels and ovarian expression of the leptin receptor mRNA were determined during follicle maturation in three chicken lines; a slow growing broiler "Label" genotype without reproductive dysfunction, a fast growing "Standard" genotype fed ad libitum or restricted and a fast growing "Experimental" line with intermediate reproductive performance levels. Whereas expression of the leptin receptor mRNA did not change in the theca, it clearly decreased with follicular differentiation in the granulosa of slow growing hens. In fast growing standard hens fed ad libitum and presenting significant reproductive dysfunction, the decrease was disrupted and dramatic up-regulation of granulosa cell expression of the leptin receptor was observed. On the other hand, feed restriction decreased the overall level of expression of the leptin receptor mRNA and restored the decrease with follicular growth. The level of expression of the leptin receptor probably modulates the action of leptin on follicular differentiation. Since blood leptin and other metabolic factors were not affected by the genotype or by nutritional state, the factors involved in the regulation of leptin receptor gene expression remain to be determined. This study demonstrates the involvement of leptin in the nutritional control of reproduction in birds. Leptin action on the ovary probably controls follicular hierarchy through the regulation of steroidogenesis.

Study Objective: We report the case of a 7-year-old girl who underwent laparoscopic ovariopexy for a suspected ovarian torsion after a previous oophorectomy. We consider the role of elective ovariopexy of the contralateral ovary in the case of adnexal torsion., Design: Case study and review of the literature., Result: There was evidence to suggest a very recent adnexal torsion and an unusually long ovarian pedicle, with a possible familial linkage. The patient underwent laparoscopic ovariopexy for the remaining normal ovary, which was found to be loosely twisted at operation. After detorsion, ovariopexy was performed laparoscopically, by suturing the ovary to the back of uterus. There are no other descriptions in the literature of a familial linkage with ovarian torsion., Conclusion: The case presented reminds doctors of the strong possibility of ovarian torsion in young girls presenting with pelvic pain. Laparoscopic ovariopexy for the contralateral ovary should be considered in all women with evidence of torsion, including children and adolescents, as is standard for testicular torsion.

Empty follicle syndrome (EFS) is characterized by a lack of retrieved oocytes in the presence of multiple follicle development, in both natural and stimulated cycles. The aim of the present case report is to point out the possibility of genetic factors that could be responsible for some occurrences of EFS. Two sisters with moderate deafness underwent controlled ovarian hyperstimulation and IVF/ICSI cycles at the same centre. During all three cycles there were normal follicular development, estradiol levels and bio-available hCG plasma levels, but no oocytes and cumulus-corona complexes were retrieved, despite second hCG injections. These cases may represent an inherited condition of EFS with hearing loss with genetic factors affecting both the aetiology of EFS and the hearing loss.

Study Objectives: To investigate the pathophysiology of human endometriosis, we examined by morphological and molecular biological methods., Methods: Samples of ovarian endometriosis and normal ovarian tissues were obtained laparoscopically after informed consent. A morphological study by toluidine blue staining, immunohistochemistry of c-kit and electron microscopy demonstrated the localization of mast cells in the stromal lesions of endometriosis. Oligonucleotide microarrays were used for gene expression analysis., Results: Infiltration of numerous mast cells and development of fibrosis was observed throughout the stromal lesions. Gene expression analysis by oligonucleotide microarrays indicated inflammatory immunoreactions in the lesions. Expressions of the FCER1G and PGDS, which are considered to be mast cell-specific genes, were upregulated in the ovarian endometriotic lesions as compared to the normal ovarian tissues. Furthermore, expressions of genes associated with immunological inflammation, such as IL-8, GRO1, GRO2, CXCR4, MCP1, and those related to tissue remodeling (MMP, COL4A2, and COL5A2) were also higher in endometriotic lesions than in the normal ovarian tissue., Conclusions: Thus it is likely that mast cells and their related inflammatory immunoreactions via chemokines play important roles in producing fibrosis and adhesions in endometriotic lesions.

To determine the role of each estrogen receptor (ER) form (ERalpha, ERbeta) in mediating the estrogen actions necessary to maintain proper function of the hypothalamic-pituitary-gonadal axis, we have characterized the hypothalamic-pituitary-gonadal axis in female ER knockout (ERKO) mice. Evaluation of pituitary function included gene expression assays for Gnrhr, Cga, Lhb, Fshb, and Prl. Evaluation of ovarian steroidogenic capacity included gene expression assays for the components necessary for estradiol synthesis: i.e. Star, Cyp11a, Cyp17, Cyp19, Hsd3b1, and Hsd17b1. These data were corroborated by assessing plasma levels of the respective peptide and steroid hormones. alphaERKO and alphabetaERKO females exhibited increased pituitary Cga and Lhb expression and increased plasma LH levels, whereas both were normal in betaERKO. Pituitary Fshb expression and plasma FSH were normal in all three ERKOs. In the ovary, all three ERKOs exhibited normal expression of Star, Cyp11a, and Hsd3b1. In contrast, Cyp17 and Cyp19 expression were elevated in alphaERKO but normal in betaERKO and alphabetaERKO. Plasma steroid levels in each ERKO mirrored the steroidogenic enzyme expression, with only the alphaERKO exhibiting elevated androstenedione and estradiol. Elevated plasma testosterone in alphaERKO and alphabetaERKO females was attributable to aberrant expression of Hsd17b3 in the ovary, representing a form of endocrine sex reversal, as this enzyme is unique to the testes. Enhanced steroidogenic capacity in alphaERKO ovaries was erased by treatment with a GnRH antagonist, indicating these phenotypes to be the indirect result of excess LH stimulation that follows the loss of ERalpha in the hypothalamic-pituitary axis. Overall, these findings indicate that ERalpha, but not ERbeta, is indispensable to the negative-feedback effects of estradiol that maintain proper LH secretion from the pituitary. The subsequent hypergonadism is illustrated as increased Cyp17, Cyp19, Hsd17b1, and ectopic Hsd17b3 expression in the ovary.

The frequency of variant LHbeta containing two point mutations (T(986)-C and T(1008)-C) and its relationship to reproductive disorders differ widely between ethnic groups. In a Japanese population, variant luteinizing hormone (LH) correlates with ovulatory disorders. Here we examined the relationship between two missense mutations and five silent mutations (C(894)-T, G(1018)-C, C(1036)-A, C(1098)-T and C(1423)-T) in the LHbeta gene, and ovulatory disorders. We studied 43 patients with ovulatory disorders, 79 patients with normal ovulatory cycles, and 23 healthy men who agreed to join our DNA analysis. PCR-amplified LHbeta-subunit gene sequences were compared with a base sequence of wild-type LH reported after direct sequencing. The highest frequency (0.945) of novel allele was observed at the position of the C(1036)-A transition. No homozygotes for wild-type LHbeta (C(1036)) were identified. The frequency of novel allele in patients with polycystic ovary syndrome, endometriosis, premature ovarian failure and luteal insufficiency was significantly different from that of healthy women. The frequencies of novel alleles (C(894)-T, C(1098)-T and C(1423)-T) in patients with ovulatory disorders were significantly higher than those with normal ovulatory cycles. The mean incidence of point mutation in patients with ovulatory disorders was higher than in those with normal ovulatory cycles. Among patients with variant LH, five silent mutations were identified in 87.5% of patients with ovulatory disorders, whereas only a few silent mutations were identified in patients with normal ovulatory cycles. In a Japanese population, five silent mutations of variant LH could have influenced two missense mutations and/or other unknown missense mutations, causing ovulatory disorders.

Objective: To study the pathophysiological effect of the expression of the apoptosis-regulating protein Bcl-2 and Bax in endometrosis., Method: Samples were obtained from 16 patients with adenomyosis, 12 ovarian endometriosis, and 17 normal endometrum. Bcl-2 and Bax expression were examined by immunohistochemical ABC staining with specific monoclonal antibodies., Results: 1. The expression of Bcl-2 or Bax in the eutopic endometrium was the same as the normal endometrium and their immunoreactivity was found predominately in the glandular epithelial cells, showing obvious cyclic changes. 2. The Bcl-2 and Bax expression in adenomyosis was not the cyclic change in ectopic glandular cells. 3. Less expression of Bcl-2 and Bax was found in the ovarian endometriosis., Conclusions: 1. The Bcl-2 and Bax cyclic change may play an important role in the proliferation and physiologic death of normal and eutopic endometrial glandular epithelial cells. 2. Bcl-2 may play an important role in forming adenomyosis. 3. Bcl-2 may be the cause of forming chocolate cyst by prompting apoptosis.

Using a cDNA microarray consisting of 23,040 genes, we analyzed gene-expression profiles of ovarian endometrial cysts from 23 patients in order to identify genes involved in endometriosis. By comparing expression patterns between endometriotic tissues and corresponding eutopic endometria, we identified 15 genes that were commonly up-regulated in the endometrial cysts during both proliferative and secretory phases of the menstrual cycle, 42 that were up-regulated only in the proliferative phase, and 40 that were up-regulated only in the secretory phase. The up-regulated elements included genes encoding some HLA antigens, complement factors, ribosomal proteins, and TGFBI. On the other hand, 337 genes were commonly down-regulated throughout the menstrual cycle, 144 only in the proliferative phase, and 835 only in the secretory phase. The down-regulated elements included the tumor suppressor TP53, genes related to apoptosis such as GADD34, GADD45A, GADD45B and PIG11, and the gene encoding OVGP1, a protein involved in maintenance of early pregnancy. Semi-quantitative RT-PCR experiments supported the results of our microarray analysis. These data should provide useful information for finding candidate genes whose products might serve as molecular targets for diagnosis or treatment of endometriosis.

Turner syndrome, resulting from a complete or partial absence of one X chromosome, is the most commonly occurring chromosomal abnormality in females. Patients have traditionally been carefully followed in paediatric practice during childhood, but were often discharged to primary care on reaching adulthood. Adults with Turner syndrome are thought to have a reduced life expectancy, mainly due to excess cardiovascular risk, but they may also have multiple comorbidities including hypothyroidism, deafness, osteoporosis and the attendant problems of oestrogen deficiency and infertility. A multidisciplinary approach to focused adult care is needed, with consideration of how to optimise surveillance strategies in these women., (Copyright 2003 S. Karger AG, Basel)

Malignant transformation of endometriosis, an uncommon phenomenon, can occur in gonadal and extragonadal sites and results in a wide histological range of tumors. Published series reporting malignant transformation of endometriosis have largely been confined to clinical and histopathological discussions with no studies reporting oncoprotein expression and genetic alterations. We report three cases of carcinomas arising in ovarian endometriosis: a serous cystadenocarcinoma, an endometrioid carcinoma with squamous differentiation, and a pure squamous cell carcinoma. Each tumor was analyzed immunohistochemically to compare oncoprotein expression (p53, bcl2, cyclin D1, and c-erb B2) between the tumors and the endometriotic tissue as well as with comparative genomic hybridization (CGH) to compare genetic alterations. All three tumors expressed nuclear p53, in contrast to the endometriotic tissue in which no p53 expression was found. Both endometrial and tumor tissue expressed bcl-2. No expression of cyclin D1 or c-erb B2 was detected in endometriotic or tumoral tissues. The CGH analysis revealed one or two chromosomal aberrations in each of the three tumors with gains on chromosomes 1q, 8q, and 13q, and losses on chromosome 10p. The endometriotic tissue, as expected, showed a normal genetic profile. These results suggest that p53 protein abnormalities and chromosomal aberrations may be involved in malignant transformation of endometriosis in the ovary. However, our results are limited by the number of cases examined and a definite conclusion on the pathogenesis of this process should be followed by future studies with a larger number of cases.

Day 3 thymectomy (D3Tx) results in a loss of peripheral tolerance mediated by CD4(+)CD25(+) T cells and the development of autoimmune ovarian dysgenesis (AOD) in A/J and (C57BL/6J x A/J)F(1) (B6AF(1)) hybrids but not in C57BL/6J mice. Quantitative trait loci (QTL) linkage analysis using a B6AF(1) x C57BL/6J backcross population verified Aod1 and Aod2 that were previously mapped as qualitative traits. Additionally, three new QTL intervals, Aod3, Aod4, and Aod5, on chromosomes 1, 2, and 7, respectively, influencing specific subphenotypes of AOD were identified. QTL linkage analysis using the A x B and B x A recombinant inbred lines verified Aod3 and confirmed linkage to H2. Aod5 colocalized with Mater, an ovarian-specific autoantigen recognized by anti-ovarian autoantibodies in the sera of D3Tx mice. Sequence analysis of Mater identified allelic, strain-specific splice variants between A/J and C57BL/6J mice making it an attractive candidate gene for Aod5. Interaction analysis revealed significant epistatic effects between Aod1-5 and Gasa2, a locus associated with susceptibility to D3Tx-induced autoimmune gastritis, as well as with H2. These results indicate that the QTL controlling D3Tx-induced autoimmune phenomenon are both organ specific and more generalized in their effects with respect to the genesis and activity of the immunoregulatory mechanisms maintaining peripheral tolerance.

Massive ovarian edema is a rare tumor-like condition found predominantly in young women. Patients usually present with abdominal pain and/or abdominal mass. Pre-operative diagnosis is often difficult. Awareness of this rare and benign lesion in young women may allow conservative management and prevention of oophorectomy in some patients.

We previously generated mice lacking aromatase activity by targeted disruption of Cyp19 (ArKO mice), and reported phenotypes of the female mice, showing hemorrhage formation and follicular depletion in the ovary, diminution in uterine size, and bone loss. In the present study, we examined the influence of dietary bisphenol A (BPA), a monomer used for the production of polycarbonate and known to have estrogenic activity, on these phenotypes of the ArKO mice. When ArKO mice were fed chow diets supplemented with 0.1% or 1% (w/w) BPA for 5 months, they were protected from ovarian degeneration, uterine diminution and bone loss in a dose-dependent manner. Northern blot analyses of ovarian RNA of ArKO mice showed differences in the expression levels of insulin-like growth factor (IGF)-I, IGF-I receptor, growth differentiation factor 9 and bone morphogenetic protein 15 as compared with those in the ovaries of wild-type mice. The differences in the expression levels were restored by dietary BPA. In the ArKO uteri, expression of progesterone receptor and vascular endothelial growth factor mRNAs was diminished, and was restored by BPA to the levels in wild-type mice. In contrast, BPA had little effect on the ovarian, uterine and skeletal structures of wild-type mice. In conclusion, estrogenic effects of BPA on the reproductive tract as well as skeletal tissue were evident in adult female ArKO mice. These results suggest that the ArKO mouse is an animal model suitable for studying effects of estrogenic chemicals as well as estrogen in vivo.

In the ovary, primordial follicles have to pass different stages in order to become preovulatory follicles. In the past few years, new genes and therefore new proteins have been recognized as major players in folliculogenesis. Atm, kit ligand and its receptor c-kit are necessary for the maintenance of ovarian follicle pool. GDF-9, BMP15, originating from the oocyte play a major role in early folliculogenesis. Pro and antiapoptotic proteins such as Bax and Bcl2 complete in granulosa cells, in order to maintain or not the follicle alive. FSH receptor is necessary for final follicular maturation, from the preantral stage and beyond. LH receptor is necessary for follicle ovulation. However, new genes and their regulation need to be identified as many ovarian diseases such as premature ovarian failure are not yet clarified.

Objective: To investigate the malignant potential of ovarian atypical endometriosis and ovarian endometrioid adenocarcinoma., Methods: 12 cases of ovarian endometroid adenocarcinoma with associated endometriosis were reviewed retrospectively. Microsatellite markers were used to detect the loss of heterozygosity on chromosome 10q23 in ovarian carcinoma and their coexisting endometriosis., Results: Among the 12 cases, benign eosinophilic metaplasia was observed in 8 cases, 5 of which (41.7%) with atypia. In these 5 cases, a spectrum of benign-atypical endometriosis with a transition to endometrioid adenocarcinoma was identified, and common genetic lesions were also detected in 2 of the 5 atypical endometriosis and their coexisting carcinomas., Conclusion: Histological observations as well as the molecular genetic evidence indicate that ovarian atypical endometriosis may possess a precancerous potential or play a role in the pathogenesis of certain malignant ovarian cancers.

Inherited BRCA2 mutations predispose individuals to breast cancer and increase risk at other sites. Recent studies have suggested a role for the APC I1307K allele as a low-penetrance breast cancer susceptibility gene that enhances the phenotypic effects of BRCA1 and BRCA2 mutations. To model the consequences of inheriting mutant alleles of the BRCA2 and APC tumor suppressor genes, we examined tumor outcome in C57BL/6 mice with mutations in the Brca2 and Apc genes. We hypothesized that if the Brca2 and Apc genes were interacting to influence mammary tumor susceptibility, then mammary tumor incidence and/or multiplicity would be altered in mice that had inherited mutations in both genes. Female and male offspring treated with a single IP injection of 50 mg/kg N-ethyl-N-nitrosourea (ENU) at 35 days of age developed mammary adenoacanthomas by 100 days of age. The female Apc-mutant and Brca2/Apc double-mutant progeny had mean mammary tumor multiplicities of 6.7+/-2.8 and 7.2+/-2.7, respectively, compared to wild-type and Brca2-mutant females, which had mean mammary tumor multiplicities of 0.1+/-0.4 and 0.3+/-0.5, respectively. Female ENU-treated Apc-mutant and Brca2/Apc double heterozygotes were also susceptible to premature ovarian failure. Thus, the inheritance of an Apc mutation predisposes ENU-treated female and male mice to mammary tumors and, in the case of female mice, to ovarian failure. These results indicate that mammary tumor development in Apc-mutant mice can progress independently of ovarian hormones. The Apc mutation-driven phenotypes were not modified by mutation of Brca2, perhaps because Brca2 acts in a hormonally dependent pathway of mammary carcinogenesis.

Objective: To determine whether the known inactivating FSH receptor gene mutations are present in Japanese women with secondary amenorrhea because of premature ovarian failure (POF) and polycystic ovary syndrome (PCOS)., Design: Clinical and molecular studies., Setting: An outpatient clinic in a university hospital., Patient(s): Fifteen women with idiopathic POF, 38 women with PCOS, and three normal controls., Intervention(s): Extraction of DNA from blood samples for subsequent polymerase chain reaction (PCR)., Main Outcome Measure(s): PCR fragments digested with MunI, BsmI, and HhaI were compared in patients and controls. PCR fragments were also analyzed by denaturing gradient gel electrophoresis (DGGE) and direct sequencing., Result(s): No inactivating mutations reported thus far in exons 6, 7, 9, and 10 of the FSH receptor gene were identified in Japanese women with POF and PCOS. DGGE analysis of PCR fragments of exon 10 also revealed no FSH receptor gene mutations in this region., Conclusion(s): Although we cannot exclude the presence of point mutations in other regions of the FSH receptor gene, the described FSH receptor mutations may be uncommon in Japanese patients with POF and PCOS.

It has been reported that cases of ovarian endometriosis those with epithelial cytological atypia have potential for malignant transformation. This study was planned to determine the incidence of atypical endometriosis and its cytological criteria, to evaluate the malignant potential of atypical endometriosis via immunohistochemical methods (p53). In this study we evaluated 140 samples obtained from 120 cases of ovarian endometriosis and 10 ovarian endometrioid carcinomas that have been previously diagnosed histopathologically. We re-evaluated endometriosis cases with respect to their epithelial and stromal features, existence of acute or chronic inflammatory cells in endometriotic epithelium or stroma and other accompanying histological findings. We observed atypia in 7 (5.8%) cases; reactive atypia in 37 (30.8%) cases, no atypia in 76 (63.4%) cases. We evaluated immunohistochemical p53 expression in 7 atypical cases, 37 reactive atypical cases, and in 10 of those without atypia and in 10 endometrioid carcinoma cases. We noted no staining in cases with atypia, reactive atypia and without atypia while 3 cases of endometrioid carcinoma had positive staining for p53. We concluded that prominent nucleolus and angulation of nuclear contour could be added to criteria of atypia that were mentioned before in the literature. In our study, even though p53 expression could not be shown with immunohistochemical methods in atypical endometriotic cases; it can not be determined that atypical endometriosis lesions are not premalignant. Still, endometriosis cases should be evaluated carefully by the pathologist for foci of cytological atypia and it should be kept in mind that malignant transformation might occur in these atypical endometriosis cases.

Endometriosis is a disease of unknown ethology; there are several theories that are unproven. It is known that women with such disease present with infertility, and that recurrence is very high, and apparently not depending of estrogenic stimulus. This phenomenon has been contributed to other growth factors and some oncogens. There are few studies about oncogens or other hormonal receptors; so, this study tries to evaluate them in eutopic tissue and in ectopic one of women with previous diagnosis of endometriosis. Our results show that myc as PRLr are expressed differently in the different tissues; and neu is expressed in the same way in both tissues. So, it was concluded that the cells of the endometriosis focus, have a differential status relating to expression of some of their genes, which target on their development and maintenance in a hormonal environment which differs from the uterine cavity.

The present study was conducted to investigate whether GnRH-receptor (GnRH-R) gene is expressed in endometriosis ovarian implants and whether a GnRH-analogue (GnRH-a) may exert an effect on endometriosis cell proliferation in vitro. The presence of GnRH-R transcripts in ovarian endometriosis cells was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and further confirmed by Southern blot analysis. GnRH-R mRNA was detected in all the 13 samples examined. In contrast, GnRH-R transcripts were not detectable in endometriosis-free peritoneal tissue. In the second part of the study, endometriosis cells were cultured for 9 days with different doses of leuprolide acetate (ranging from 0 to 10(-5) M). In 4 out of 13 cases, a significant anti-proliferative effect was observed at doses of leuprolide acetate ranging from 10(-9) to 10(-5) M. In one case, a significant inhibition of cell proliferation was observed only at 10(-5) M leuprolide acetate concentration. In contrast, the GnRH-a did not affect cell growth, regardless of the expression of GnRH-R transcripts and the given doses, in the remaining 8 experiments. To date, this is the first evidence indicating that GnRH-R mRNA is expressed in human ovarian endometriomas. Moreover, the inhibition of endometriosis cell proliferation induced by the GnRH-a in vitro suggests that, at least in some cases, this compound might exert a direct effect on endometriosis lesions.

To identify potential prognostic indicators of ovarian cancer and identify targets for therapeutic strategies, mRNA differential display was used to analyze gene expression differences in normal, benign, and cancerous ovarian tissue. One cDNA isolated by this technique, Op18/stathmin, is a highly conserved gene that is reported to have many different functions within a cell, including signal transduction, control of the cell cycle, and the regulation of microtubules. Quantitative Northern blot analysis of 12 malignant ovarian samples, 8 benign ovarian tumors, and 10 normal ovarian tissue samples demonstrated overexpression of Op18/stathmin mRNA in the malignant cancers. Immunohistochemistry showed an apparent overexpression of Op18/stathmin protein level and an association with proliferating cells.

The functions of the hypothalamic-pituitary-ovarian and -adrenal axes are intertwined, and molecular defects in either axis may cause ovarian dysfunction. Advances in molecular genetics have allowed new insights into the pathophysiology of ovarian disorders. Specific gene mutations causing delayed puberty and/or ovarian failure, and heterosexual or isosexual precocious puberty have recently been described. The molecular insights gained into ovarian dysfunction have already led to rational therapies for some of these conditions.

Background: Ovarian dysplasia, a potential precursor to ovarian carcinoma, has been described in ovarian tissue obtained by prophylactic oophorectomy and also adjacent to ovarian carcinoma. Women with a family history of ovarian carcinoma, especially those of Jewish Ashkenazi descent, often test positive for BRCA mutant genes. Prophylactically removed ovaries, generally described as normal on macroscopic examination, can exhibit a "preneoplastic phenotype" and unsuspected neoplasm., Methods: Histologic slides of ovarian tissue from 54 Ashkenazi Jewish women were reviewed. All had a family history of ovarian carcinoma and all were tested for BRCA mutations. Forty-four women tested positive. Thirty-one women underwent prophylactic oophorectomy and 23 underwent oophorectomy for ovarian carcinoma. Normal, dysplastic, and ovarian carcinoma epithelial cells were analyzed morphometrically combining nuclear area measurements with chromatin texture assessment using a novel method based on the computation of autocorrelation coefficients and a derived parameter (Beta). Discriminant analysis between classificatory algorithms was used to obtain results., Results: Ovarian dysplasia was identified in 77.6% of the prophylactic oophorectomy specimens. An unsuspected ovarian carcinoma was diagnosed in one prophylactic oophorectomy specimen. Of 10 women who underwent prophylactic oophorectomy and were negative for BRCA mutations, three had ovarian dysplasia. The average nuclear measurements of the dysplastic cells were similar to those published previously. The new autocorrelation-based method evaluating nuclear texture, as revealed by tridimensional surface plots, demonstrated high discriminatory potential. Discriminant analysis based on nuclear area and nuclear texture information resulted in the correct classification of nearly all the cases in the three diagnostic categories., Conclusions: Ovaries removed by prophylactic oophorectomy examined in their entirety often reveal ovarian dysplasia and occasionally ovarian carcinoma. The new morphometric method used was highly discriminatory in the evaluation of nuclear texture. Ovarian dysplasia in women with risk factors for ovarian carcinoma is significant in early ovarian carcinogenesis., (Copyright 1999 American Cancer Society.)

The contribution of oestrogen receptor (ER) isoforms, ER-alpha and ER-beta, in oestrogen-dependent development and growth of ovarian endometriomata, is unknown. Therefore, we examined the expression of ER-alpha and ER-beta in ovarian endometriomata and normal uterine endometrium. ER-alpha and ER-beta were shown to be dominantly expressed in the nuclei of the epithelial lining cells of ovarian endometrioma and of the glandular cells of normal uterine endometrium. ER-beta was expressed at a much lower level than ER-alpha in the glandular cells of normal uterine endometrium, while ER-beta was expressed at a slightly lower level than ER-alpha in the epithelial lining cells of ovarian endometrioma. In normal uterine endometrium, ER-beta mRNA was expressed at a much lower level than ER-alpha mRNA, and the expression pattern of ER-beta mRNA during the menstrual cycle was similar to that of ER-alpha mRNA. On the other hand, ER-beta mRNA expression was significantly higher and over a much greater range in ovarian endometriomata (P < 0.05) than in normal uterine endometrium during the menstrual cycle, while ER-alpha mRNA expression was relatively lower and more random. Therefore, in ovarian endometriomata, oestrogen action via ER-alpha cascades seems to be partially damaged, as the expression of ER-alpha mRNA does not respond to endocrinological alterations during the menstrual cycle, while the relative over-expression of ER-beta might be related to a unique oestrogen-dependent growth and spreading of ovarian endometriomata.

Background: The p73 gene is structurally related to the tumor suppressor gene p53. The role of p73 in tumor development is still unclear and no data on ovarian cancer are so far available. For this reason we have analyzed, in a panel of ovarian cancers, the allelic distribution and expression of p73., Patients and Methods: Fifty-one samples from ovarian cancers and five human ovarian cancer cell lines growing in culture were analyzed. Allelic origin was analyzed by PCR after digestion with the restriction enzyme Sty I. Heterozygous, informative cases were selected for studies aimed at evaluating allelic expression of p73., Results: We found an allelic distribution similar to that previously reported. LOH was found in two patients with ovarian cancer. In one case in which normal ovarian tissue was available biallelic expression of p73 was found., Conclusion: In comparisons of ovarian cancers and borderline tumors, no differences in allelic distribution and/or expression were found, suggesting that p73 does not play an important role in the pathogenesis and development of ovarian cancer.

An increase in androgen receptor (AR) caused by estrogen is recognized as one of the biological phenomena related to estrogen-induced growth in uterine endometrium. The A/B region of AR gene in X chromosome involves the cytosine, adenine, and guanine (CAG) repeats. Random X chromosome inactivation with AR alleles in individual cells occurs in females. Therefore, approximately either paternal or maternal single dominant polymorphic AR mRNA must be expressed in neoplastic tissue originated from monoclone. This prompted us to determine deviated number of CAG repeats in AR mRNA to understand clonality in ovarian endometriosis. In all cases of heterozygous AR alleles, although paternal and maternal AR mRNAs from normal eutopic uterine endometrium were consistently expressed as AR alleles, either paternal or maternal single dominant AR mRNA expression was found in an individual ovarian endometrioma. Therefore, an individual ovarian endometrioma might be formed from an independent monoclonal ovarian endometriotic endometrial cell after inactivation of either AR allele in X chromosome.

Objective: To determine whether mutations in the FSH receptor gene are associated with premature ovarian failure (POF) or resistant ovary syndrome (ROS) in women in the UK. To determine whether an allelic variant of the FSH receptor gene affects fertility parameters in women with polycystic ovary syndrome (PCOS)., Design: A mutation screen using DNA from women with POF and ROS. Restriction digest of amplified DNA from women with POF, ROS, PCOS and controls to determine allelic variant status. Fertility parameters were compared between allelic variant subgroups of women with PCOS., Patients: The study population comprised 49 women with POF, 5 with ROS, 93 with PCOS and 51 controls., Measurements: In women with PCOS, fertility and menstrual status was recorded and serum FSH and ovarian volume were measured., Results: No mutation of the FSH receptor gene was found in women with POF or ROS. The allelic variant Thr307/Ser680 was found to be similarly prevalent in all study groups. The Thr307/Ser680 variant was found to have no phenotype in terms of fertility parameters in women with PCOS., Conclusions: Mutations of the FSH receptor gene are rare in women with premature ovarian failure or resistant ovary syndrome in the UK. Polymorphisms of the FSH receptor gene do not appear to have pathophysiological significance with regard to ovarian function.

We studied five groups of women with ovarian dysfunction for the CGG expansion in FMR1 and a (TA)n polymorphism in the estrogen receptor gene: a) poor responders to ovarian stimulation as part of in vitro fertilization (n = 13); b) women with familial premature ovarian failure (POF) (n = 7); c) sporadic cases with POF (n = 16); d) FRAXA premutation carriers with POF (n = 7); and e) FRAXA premutation carriers without POF (n = 9). FRAXA premutation was found in one woman with familial POF. A significant association of familial POF and FRAXA premutation carriers with POF having low copy of the (TA)n polymorphism as compared to controls was observed. Our preliminary data suggest a potential role of the estrogen receptor in POF, and it may influence the variable age of menopause of the FRAXA premutation carriers.

This study was designed to examine the biological implications of progesterone receptor form A (PR-A) and B (PR-B) mRNA expressions in human ovarian endometriosis (ectopic endometrium). A high ratio of PR-B to PR-AB (PR-A+PR-B) mRNA expression was found in 8 of 14 cases of endometriosis, compared with the ratio in eutopic endometrium. The mean ratio in ectopic endometria was significantly (p < 0.01) higher than in eutopic endometria. The ratio in eutopic and ectopic endometria showed no significant change during the menstrual cycle. The mean ratio in ectopic endometria in the proliferative and secretory phases of the endometrium was significantly (p < 0.01) higher than in eutopic endometria. In conclusion, PR-B mRNA was relatively highly expressed in some endometriomas, which might lead to aberrations in the control of progestational effects involving responsiveness to sex steroidal growth regulation., (Copyright 2000 S. Karger AG, Basel)

We demonstrated the expression of various exon-deleted progesterone receptor (PR) variant mRNAs in human uterine endometrium and ovarian endometriosis using the reverse transcription-polymerase chain reaction-DNA sequencing analyses. In addition to PR wild-type mRNA, various exon-deleted PR variant mRNAs were identified in all samples analyzed. The sequence of these variants showed a perfect junction between exons surrouding the deletion area. PR wild-type, exon 6-deleted, exon 4-deleted, exon 5, 6-deleted and exon 4, 5, 6-deleted PR variant mRNAs were observed in all samples analyzed. Exon 4, 6-deleted PR mRNA was observed only in ovarian endometriosis. This is the first study to demonstrate the coexpression of various PR exon-deleted variant mRNAs with the wild-type in uterine endometrium and ovarian endometriosis. All resulting variant proteins might indicate functional diversity and modify the progestational action of wild-type PR, and thus be involved in the pathophysiology of ovarian endometriosis., (Copyright 1998 Academic Press.)

Impaired galactose metabolism has been proposed as a risk factor for ovarian cancer and endometriosis, which is a putative precursor of endometrioid and clear cell histological sub-types of ovarian cancer. The prevalence of the most common galactose-I-phosphate uridyl transferase gene mutations, Q188R and N314D, was assessed in 206 women with ovarian cancer, 78 women with endometriosis and 248 controls. No Q188R mutations were found in any of the groups. A statistically significant increase in the frequency of N314D mutations was observed in women with serous and undifferentiated histological sub-types of ovarian cancer, but not mucinous, endometrioid or clear cell sub-types. There were no significant differences observed in the N314D mutation frequency between women with endometriosis (18%) and controls (17%). Our results support previous reports of an association of impaired galactose metabolism with serous and undifferentiated ovarian cancers but contradict previous findings of increased N314D mutation frequencies among women with endometriosis and endometrioid and clear cell sub-types ovarian cancer.

We evaluated the immunohistochemical expression of cadherins and CD44 variants in 20 endometriomas, 20 cystadenomas, 20 borderline ovarian tumours as well as 20 ovarian carcinomas, and the serological and cystic fluid concentrations of soluble E-cadherin and soluble CD44 standard (sCD44sdt) in 20 endometriomas, 20 cystadenomas, six borderline and 11 carcinomas of the ovary. In endometriomas, immunostaining of E- and N-cadherin was negative (20 and 30% respectively). CD44 H, v3 and v6 immunostaining were detected in 63, 10 and 40% respectively. A difference in immunostaining for E-cadherin was found between endometriomas and cystadenomas (P < 0.001) and for N-cadherin between endometriomas and carcinomas (P < 0.001). A difference in CD44H immunostaining was observed between endometriomas and cystadenomas (P < 0.035) but not with borderline ovarian tumours and carcinomas. No difference in serum concentrations of soluble E-cadherins and CD44 standard was found between the four groups of tumours. Cystic fluid concentrations of E-cadherin were lower in endometriomas than in borderline tumours and ovarian carcinomas (P < 0.001). High concentrations of soluble CD44 standard cystic fluid were found in endometriomas than in other ovarian cysts. Endometriomas and borderline tumours share alterations of cadherins and CD44 isoforms which may help in the understanding of the aggressive and invasive potentials of endometriotic cells.

Cloning and characterization of all three human galactose-metabolic genes (GALK, GALT and GALE) has led to the identification of a number of mutations which are generally of the missense type in patients with galactosemia, an inborn error of metabolism. The predominance of missense mutations is interesting, considering the general importance of galactose metabolism for cellular energy production and proper modification of glycoproteins and glycolipids. Abnormalities in both of these macromolecules have been described in transferase-deficiency galactosemia, the most common and best-studied form of galactosemia. Thus, the parallel biochemical and molecular genetic analyses of human galactose metabolism are shedding light on this under-appreciated metabolic pathway that is critical for cellular energy production, modification of cellular macromolecules and normal human development.

Objective: To investigate the expression of sex hormone-binding globulin (SHBG) exon VII splicing variant messenger RNA (mRNA) in human ovarian endometriosis., Design: The expression of SHBG variant mRNA in normal uterine endometrium and endometriotic tissue was determined., Setting: Department of Obstetrics and Gynecology, Gifu University Hospital., Patient(s): Fourteen women with endometriosis and 18 women without endometriosis., Intervention(s): Normal uterine endometrial and ovarian endometriotic tissues from patients who had undergone gynecological surgery were studied., Main Outcome Measure(s): Levels of SHBG wild-type and variant mRNAs were determined using the quantitative reverse transcription-polymerase chain reaction., Results(s): Analysis of the missing base pairs proved that they corresponded to the entire exon VII. There was no significant difference between the levels of SHBG wild-type mRNA in normal endometria and in endometriotic endometria, although the levels of SHBG variant mRNA in endometriotic endometria were significantly higher than that in normal endometria. The ratio of SHBG variant to wild-type mRNA levels was significantly higher in endometriotic endometria than in normal endometria., Conclusion(s): This study demonstrates the coexpression of SHBG exon VII splicing variant mRNA with its wild-type and the dominant expression of the variant in ovarian endometriosis. These results might be involved in the cellular estrogenic interaction, plausibly assisting in the development and growth of ovarian endometriosis.

Mutation within the Mitf gene causes, in microphthalmic Mitf(mi)/Mitf(mi) (mi/mi) mice, multiple defects, including white coat color and functional defects in macrophages and osteoclasts. Our previous mating experiments have demonstrated that the mi mutation reduces the numbers of newborns and induces uterine inversion at delivery. The present study was designed to determine the causes of these pregnancy defects. The histology and number of F4/80-positive macrophages were not different between the ovaries of 23-day-old mi/mi and +/+ mice given eCG 48 h earlier. When ovulation was induced in these mice by hCG, the number of ovulated ova was significantly smaller in mi/mi mice than in wild-type (+/+) mice (p < 0.05). When bone marrow cells from +/+ mice were transplanted i.p. into 42 mi/mi female newborns, successful transplantation was observed in 16 of them at 20 days after birth. In one of these, the upper incisors had erupted. The mean number of tubal ova in mi/mi mice significantly increased after transplantation (p < 0.05) and was almost equal to that of +/+ mice. No uterine inversion occurred at 6 deliveries in 5 mi/mi females after bone marrow transplantation, while it occurred at 4 of 5 deliveries in mi/mi females during the same observation period (p < 0.05). These results indicate that bone marrow-derived cells, defective in mi/mi mice, are necessary for normal ovulation and delivery; the findings are consistent with the notion that macrophages play major roles in ovulation.

The hCGbeta gene family contains six genes linked in tandem on chromosome 19 and labeled beta genes 7, 8, 5, 1, 2, and 3. Previous studies on a small number of placentas have indicated that beta gene 5 was the most highly expressed gene during the first trimester of pregnancy, followed by genes 3 and 8. Beta genes 7, 1, and 2 were expressed at very low levels. The purpose of this study was to determine 1) whether this pattern of expression was typical during normal pregnancy by sampling a large number of first trimester placentas, and 2) whether there was a correlation between gestational age and the pattern of hCGbeta gene expression. Total RNA from 27 first trimester placentas varying in age from 6-16 weeks was reverse transcribed into complementary DNA. The complementary DNA was amplified by PCR, and the amount of DNA representative of each hCGbeta gene was quantified by Genescan analysis. In 14 of the 27 placentas, hCGbeta gene 5 accounted for 50% or more of the total beta messenger RNA expressed. Beta gene 3 was expressed at levels ranging from 1-42% of the total, and beta gene 8 expression ranged from 12-32% of the total. Gene 7 expression was less than 3% of the total beta expression in all 27 placentas. Although there appeared to be a trend toward lower expression of beta gene 3 in placentas beyond 10 weeks gestational age, there was no correlation of the pattern of beta expression with placental age. Beta gene expression was also examined in two blighted ova, a spontaneous abortion sample, and a hydatidiform mole as well as in cultured JAR choriocarcinoma cells. With the exception of JAR cells, these abnormal tissues had low levels of gene 3 expression, but these levels were within the range of the patterns observed in normal placentas. These data suggest that it is the total amount of hCGbeta gene expression rather than the expression of individual beta genes that is important for the maintenance of normal pregnancy.

Intraovarian cytokines play a pivotal role in the normal growth and development of the ovarian follicle. The purpose of this study was to investigate the pattern of cytokine mRNA expression in ovarian endometriomata. A total of 10 patients with histologically confirmed endometriomata undergoing surgery formed the study group while nine patients undergoing sterilization with no evidence of a cyst in the ovary formed the control group. Biopsies of the ovary were obtained at surgery and stored in liquid nitrogen until processed by reverse transcription-polymerase chain reaction amplification to identify the presence of mRNA for interleukin (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, tumour necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma). IL-6 and IL-10 mRNA were expressed by nine and seven patients respectively in the endometriosis group compared with three and one patients in the control group; this difference was significant (P < 0.05). IL-1 alpha mRNA was expressed by seven of 10 patients with endometriosis but by only one of the control group; this was again significantly different (P < 0.04). Ovarian IL-2 and IL-4 mRNA were not expressed in either group. There was no significant difference in the expression of IL-8, IL-13, IFN-gamma and TNF-alpha mRNA in the two groups. These findings suggest that abnormal local expression of certain cytokines may contribute to the development of endometriomata.

Ovarian cancer has been described in association with three autosomal dominant syndromes: familial site-specific ovarian cancer, familial breast and ovarian cancer, and the hereditary nonpolyposis colon cancer syndrome. It appears that most breast-ovarian and site-specific ovarian cancer families are explained by mutations in the BRCA1 tumor suppressor gene. Other genes associated with inherited susceptibility to ovarian cancer include BRCA2, p53, and the DNA mismatch repair genes.

Insulin resistance confers increased susceptibility to NIDDM, atherosclerotic cardiovascular disease, ovarian hyperandrogenism, and possibly hypertension. Insulin resistance is largely inherited, in rare cases as a monogenic disorder or more commonly as a complex trait. The search for insulin resistance genes relies mainly on two complementary approaches: 1) positional cloning using random DNA markers present throughout the genome; and 2) the analysis of specific candidate genes. This report briefly summarizes the candidate gene approach to insulin resistance. Progress related to the analysis of genes encoding molecules that participate in insulin action is reviewed. In addition, the spectrum of potential genetic defects that might contribute to insulin resistance, both at the level of the target cell and secondarily (e.g., obesity genes), is discussed.

In genetically susceptible strains of mice, such as A/J and (C57BL/6J x A/J)F1 hybrids, neonatal thymectomy-induced autoimmune ovarian dysgenesis (AOD) is characterized by the development of antiovarian autoantibodies, oophoritis, and atrophy. Temporally, atrophy may be observed during and after the regression of inflammatory infiltrates from the ovary. Histologically, lesions appear as areas devoid of ovarian follicles in all stages of development that have been replaced by luteinized interstitial cells. We report here the mapping of Aod2, the locus that controls this phenotype, to mouse chromosomes 3 within a region encoding Il2 and Fgfb. Most significant, however, is the co-localization of Aod2 to Idd3, a susceptibility gene that plays a role in autoimmune insulin-dependent type 1 diabetes mellitus in the nonobese diabetic mouse.

Mice thymectomized at three days of age (D3Tx) develop during adulthood a variety of organ-specific autoimmune diseases, including autoimmune ovarian dysgenesis (AOD). The phenotypic spectrum of AOD is characterized by the development of anti-ovarian autoantibodies, oophoritis, and atrophy. The D3Tx model of AOD is unique in that disease induction depends exclusively on perturbation of the normal developing immune system, is T-cell-mediated, and is strain specific. For example, D3Tx A/J mice are highly susceptible to AOD, whereas C57BL/6J mice are resistant. After D3Tx, self ovarian antigens, expressed at physiological levels, trigger an autoimmune response capable of eliciting disease. The D3Tx model provides, therefore, the opportunity to focus on the mechanisms of self-tolerance that are relevant to disease pathogenesis. Previous studies indicate that the principal mechanisms involved in AOD susceptibility are genetically controlled and govern developmental processes associated with the induction and maintenance of peripheral tolerance. We report here the mapping of the Aod1 locus to mouse chromosome 16 within a region encoding several loci of immunologic relevance, including scid, Igl1, VpreB, Igll, Igl1r, Mtv6 (Mls-3), Ly-7, Ifnar, and Ifgt.