Müllerian cyst of the uterus treated with laparoscopy and diagnosed using immunohistology.

Among uterine cystic tumors, uterine cyst arising from secondary Müllerian epithelium is exceedingly rare. A 45-year-old woman presented with pelvic cystic mass, which was initially diagnosed as a paraovarian cyst by ultrasound and magnetic resonance imaging. At laparoscopy, the cyst proved to be a pedunculated uterine cyst, which was easily resected. Histologically, the cyst wall was lined by fallopian epithelium and positively stained for WT-1, estrogen receptor, and progesterone receptor. The final diagnosis was Müllerian cyst of the uterus. Preoperative diagnosis of uterine Müllerian cyst is usually impossible. Laparoscopy is useful as a minimally invasive treatment to diagnose and resect the cyst at the same time. Specific immunostaining is useful to make a definite diagnosis of Müllerian cyst of the uterus.

Cholesterol sulfate induces expression of the skin barrier protein filaggrin in normal human epidermal keratinocytes through induction of RORα.

Cholesterol sulfate is abundant in the human epidermis and is a putative natural ligand for retinoic acid receptor-related orphan receptor alpha (RORα). Although direct binding of cholesterol sulfate is expected to activate RORα, cholesterol sulfate can also induce RORα expression and increase RORα target gene expression. The purpose of this study was to determine whether cholesterol sulfate induces profilaggrin expression, a precursor of the barrier protein filaggrin in the epidermis, through activation of RORα by directly binding to RORα, or through increased RORα expression. Immunohistochemical and polymerase chain reaction (PCR) analyses showed that RORα was expressed in normal human epidermal keratinocytes (NHEKs) and that its expression increased during keratinocyte differentiation in parallel with that of profilaggrin and cholesterol sulfotransferase, which catalyzes the synthesis of cholesterol sulfate. Exogenous cholesterol sulfate significantly increased both RORα and profilaggrin expression in NHEKs, whereas no effect on profilaggrin expression was observed in cells in which RORα was knocked down with small interfering RNA (siRNA). Additionally, a luciferase reporter gene assay revealed that exogenous RORα dose-dependently increased the activity of the profilaggrin gene promoter even in the absence of cholesterol sulfate, and that this response involves activator protein-1. In conclusion, the results of this study indicate that cholesterol sulfate induces filaggrin expression through increased RORα expression. Further studies are required to fully elucidate the mechanisms involved.

Inhibition of proteases involved in embryo implantation by cholesterol sulfate.

BACKGROUND: Matrix metalloproteinases (MMPs) and the plasminogen activator (PA)/plasmin system are two major groups of proteases involved in the matrix degradation required for embryo implantation. We previously showed that the content of cholesterol sulfate (CS) in rabbit endometrium increases characteristically during the implantation period. Furthermore, CS has been reported to inhibit serine proteases. In this study, we investigated whether CS can regulate the activity of proteases in cultured human endometrial stromal cells. METHODS AND RESULTS: CS (1-30 microM) and plasminogen (precursor of plasmin) were added to the culture media of human endometrial stromal cells and incubated for 24 h. Culture media were collected for analysis of plasmin and MMP-2, -3 and -9 enzyme activities using fluorescence assays. Plasmin and MMP-3 activities were significantly reduced by CS in a dose-dependent manner (P < 0.001). Western blot analysis of the culture media revealed that CS inhibited the conversion by plasmin of MMP-3 from the precursor form to the active form. Fluorescence assay using a common substrate of MMP-2 and MMP-9 showed that enzymatic activity remains at approximately 50%, even at 30 microM CS. Gelatin zymography demonstrated that CS inhibited the activation of MMP-9 but not MMP-2 from the precursor, suggesting that the activation of MMP-2 may be independent of plasmin. CONCLUSIONS: CS inhibits not only plasmin activity but also MMP activities indirectly by inhibiting the plasmin-mediated process. These findings suggest that CS may be an important regulator of proteolysis during trophoblast invasion.

Identifying patients who can improve fertility with myomectomy.

OBJECTIVE: To identify the characteristics of cases and fibroids that will indicate which patients should undergo myomectomy to improve fertility. MATERIALS AND METHODS: We recruited patients (n=101) who had undergone myomectomy to improve fertility and received follow-up care for at least six months by the hospital. Medical records were retrospectively reviewed to analyze the pregnancy rates after myomectomy and to identify clinical factors that correlate with pregnancy rates. Cumulative pregnancy rates were analyzed using the Kaplan-Meier method and the Log rank test. The patients were then divided into three groups according to the timing of the myomectomy. The analysis was performed for all patients, for patients in the post-superovulation and/or intrauterine insemination (post-SO/IUI) group and the post-assisted reproductive technology (post-ART) group combined, and for patients in the post-ART group. RESULTS: Sixty-three pregnancies were achieved by 58 patients (57.4%) who underwent myomectomy. The mean time period between surgery and conception was 9.8 months. Most pregnancies (91.5%) were achieved within two years after surgery. Pregnancy rates were higher in patients aged less than 36 years, with no male factors, and without severe endometriosis, in comparison with patients 36 years of age or older (p<0.05), with male factor (p<0.05), and severe endometriosis (p<0.05). In the analysis of the post-ART group, pregnancy rates were higher (p<0.05) in cases where enucleation had penetrated the endometrial cavity in comparison with patients where the cavity was not penetrated; however, fibroid characteristics did not correlate with the post-myomectomy pregnancy rate in the post-SO/IUI plus post-ART group. CONCLUSION: Post-myomectomy pregnancy rates were higher in women who did not have additional infertility factors. These results suggest that the removal of fibroids benefits especially patients who suffer from infertility of an otherwise unknown cause: surgery should be strongly recommended for these patients. Our study also shows the difficulty in identifying fibroids for removal to improve fertility. Further studies are needed to develop new diagnostic techniques for identifying patients who can improve fertility with myomectomy.

Expression of retinoic acid-related orphan receptor alpha and its responsive genes in human endometrium regulated by cholesterol sulfate.

Cholesterol sulfate (CS) is a major sterol sulfate in human plasma that is detected in the uterine endometrium. CS plays a role in steroidogenesis, cellular membrane stabilization, and regulation of the skin barrier. We previously reported that CS increased in rabbit endometrium during the implantation period. Recently, CS has been reported to be a ligand of retinoic acid receptor-related orphan receptor alpha (RORA). NR1D1 is one of the genes regulated by RORA. In the present study, we investigated the regulation of RORA and NR1D1 by CS in human endometrium. We determined the association-dissociation curves for the interaction of CS with RORA and the kinetic rates by surface plasmon resonance. Immunohistochemical staining and in situ hybridization revealed that RORA and NR1D1 were expressed in human endometrial stromal and epithelial cells. CS treatment significantly induced the mRNA expression of RORA and NR1D1 mRNA in ESCs. The results of a luciferase assay showed that RORA significantly activated the human NR1D1 promoter regardless of CS. Our results suggest that CS regulates the expression of RORA responsive genes in human endometrial cells but not as a ligand for RORA.

The progesterone-responsive gene 14-3-3τ enhances the transcriptional activity of progesterone receptor in uterine cells.

Members of the 14-3-3 family are intracellular dimeric phosphoserine-binding proteins that can associate with and modulate the activities of many proteins. In our efforts to isolate the genes regulated by progesterone (P(4)) using suppressive subtractive hybridization, we previously found that 14-3-3τ is one of the genes upregulated by P(4). In this study, we demonstrated by quantitative RT-PCR (qRT-PCR), western blot analyses, and immunohistochemistry that 14-3-3τ mRNA and protein levels were increased in the rat uterus after P(4) treatment. Furthermore, qRT-PCR indicated that P(4) increased 14-3-3τ mRNA levels in human endometrial epithelial cells and endometrial stromal cells (ESCs). Western blot and qRT-PCR analyses revealed that in vitro decidualization using cAMP and medroxyprogesterone 17-acetate increased levels of 14-3-3τ mRNA and protein in ESCs. We have shown by qRT-PCR and western blot analyses that P(4) increased the mRNA and protein levels of 14-3-3τ in Ishikawa cells that stably express P(4) receptor-B (PR-B). Immunocytochemistry revealed that 14-3-3τ colocalizes with PR and translocates from the cytoplasm to the nucleus in response to P(4). Moreover, by luciferase reporter assay, we demonstrated that 14-3-3τ enhances the transcriptional activity of PR-B. Taken together, we propose that 14-3-3τ is a P(4)-responsive gene in uterine cells that modulates P(4) signaling.

Inhibition of cell invasion and protease activity by cholesterol sulfate.

We demonstrated that cholesterol sulfate (CS) inhibits invasion of a trophoblast cell line and plasmin enzyme activity in a noncompetitive manner by binding to the enzyme itself, suggesting that CS can repress cell invasion by inhibiting proteinases such as those involved in the plasminogen activator/plasmin system. Considering these results, it is possible that CS may act as a signaling molecule between the trophoblast and endometrium, and may regulate the process of implantation.