Suturing method as a factor for uterine vascularity after laparoscopic myomectomy.

OBJECTIVE: To evaluate the vascularity of the myometrium after laparoscopic myomectomy sutured by two different methods using contrast-enhanced Magnetic Resonance Imaging. STUDY DESIGN: Twenty-eight women who had symptomatic leiomyomas and underwent laparoscopic myomectomy between June 2013 and July 2014 were included in the present study. In the first half period, continuous sutures were used in 12 patients, and in the latter half period, single interrupted sutures were used in 16 patients. Contrast-enhanced Magnetic Resonance Imaging was used 3 or 6 months after surgery to evaluate vascularity after laparoscopic myomectomy. We defined avascularity index as the percentage of avascular area after surgery to cross sectional area of myoma before surgery. The Wilcoxon rank-sum test was applied to compare avascularity indeces in the two study groups. RESULTS: At 3 months after surgery, avascularity index in continuous sutures group was significantly higher than that in single interrupted sutures group (median 5.0 vs.1.2, p<0.001), suggesting a poorer vascular recovery of the myometrium sutured continuously. CONCLUSION: Simple interrupted suturing might be superior to continuous suturing in terms of vascularity evaluated using contrast enhanced Magnetic Resonance Imaging.

Expression and regulation of transient receptor potential cation channel, subfamily M, member 2 (TRPM2) in human endometrium.

To identify estrogen-responsive genes, we previously isolated estrogen receptor (ER)-binding DNA fragments from human genomic DNA using a recombinant ER protein. Six DNA fragments, each including a perfect palindromic estrogen response element (ERE), were obtained. The nucleotide sequence of one of the six fragments (E1 fragment) showed that the ERE of the E1 fragment is located in the 3'-untranslated region (UTR) of transient receptor potential cation channel, subfamily M, member 2 (TRPM2). Here, we confirmed the estrogen-dependent enhancer activity of the ERE of the E1 fragment by chloramphenicol acetyltransferase assay. TRPM2 mRNA expression was investigated in human endometrium, cultured human endometrial stromal cells (ESCs), and cultured human endometrial epithelial cells (EECs) using RT-PCR. Quantitative RT-PCR revealed that TRPM2 mRNA expression in ESCs increased after 17ß-estradiol (E2) treatment. This study demonstrated for the first time that TRPM2 is an estrogen-responsive gene expressed in human endometrial cells.

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.

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.

Expression and regulation of cholesterol sulfotransferase (SULT2B1b) in human endometrium.

OBJECTIVE: To investigate the hormonal regulation of SULT2B1b in human endometrium. DESIGN: In vitro study with human endometrial tissues and cultured human endometrial cells. SETTING: University hospital. PATIENT(S): Thirty-seven women undergoing hysterectomy for benign disease. INTERVENTION(S): Human endometrial tissues were collected for in situ hybridization. Culture medium of human endometrial epithelial cells (EECs) was collected for determination of secretion of cholesterol sulfate (CS). Total RNAs were extracted from human endometrial tissues and cultured cells for real-time reverse transcriptase-polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURE(S): The expression of SULT2B1b mRNA in human endometrial tissues and cultured cells. RESULT(S): In situ hybridization studies and real-time RT-PCR showed that the amount of SULT2B1b mRNA in human endometrial tissues was significantly higher during the midluteal phase than during other phases of the menstrual cycle. The secretion of CS from EECs was confirmed using [(35)S]-phosphoadenosine phosphosulfate. The expression of SULT2B1b mRNA was induced by cAMP or P in human endometrial stromal cells (ESCs), whereas it was induced by cAMP or relaxin in EECs. The induction of SULT2B1b mRNA by P or relaxin was abolished by the specific protein kinase A (PKA) inhibitor, Rp-adenosine 3',5' cyclic monophosphothioate (Rp-cAMPS). CONCLUSION(S): The expression of SULT2B1b mRNA in ESCs is induced by P and that in EECs is induced by relaxin via the cAMP pathway.

Induction of early decidualization by cadmium, a major contaminant of cigarette smoke.

To investigate the effect of cadmium (Cd) contamination on endometrial function, human endometrial stromal cells were isolated and cultured with E(2) plus P in the presence of Cd, a major contaminant in cigarette smoke, and assayed for PRL concentrations and its messenger RNA expression. Cd significantly increased PRL concentrations in the culture media and significantly up-regulated PRL messenger RNA expression of the endometrial stromal cells, suggesting that Cd stimulates decidualization of the endometrium and may disrupt endometrial environment, causing early decidualization.

Inhibitory effects of cholesterol sulfate on progesterone production in human granulosa-like tumor cell line, KGN.

Cholesterol sulfate (CS) is a component of cell membranes that plays a role in stabilizing the cell membrane. We previously reported that CS increased in the endometrium during implantation, suggesting that CS plays an important role in reproduction. It has been reported that CS regulates progesterone and pregnenolone production in the placenta, adrenal glands and ovary. The regulatory mechanisms of steroid hormone production by CS, however, are still unknown. In the present study, we investigated the effect of CS on the expression of progesterone production-related genes in KGN cells, derived from human granulosa-like tumor. KGN cells were cultured with CS (10 muM) or cholesterol (10 muM) in the presence of 8-bromo-cAMP (1 mM). Progesterone levels in the culture media were measured by enzyme linked fluorescent assay at 24 h after treatment of CS and cAMP. Total RNAs were extracted for quantitative real time RT-PCR with specific primer of StAR protein, P450scc, HSD3B2, ferredoxin and ferredoxin reductase. Whole cell lysates were extracted for western blot analysis with antibody for StAR protein. Progesterone concentration in the culture medium increased to 38-fold by treatment of cAMP. CS significantly reduced progesterone concentration by 30% compared with those of cAMP treatment (p<0.05), while cholesterol did not change the progesterone concentration. CS treatment down-regulated the expression of StAR mRNA and P450scc mRNA was to 54% and 60%, respectively (p<0.05). Western blot analysis revealed that the amount of StAR protein was also reduced by CS treatment. The expression of HSD3B2 mRNA was up-regulated to 3.4-fold by treatment of cAMP. The expression of ferredoxin and ferredoxin reductase mRNA was not affected by CS treatment. These data implied that CS has an inhibitory effect on progesterone production by regulating the expression of StAR and P450scc gene expression.

Expression and regulation of periostin/OSF-2 gene in rat uterus and human endometrium.

Periostin/OSF2 is a ligand for alphavbeta3 and alphavbeta5 integrins and activates the Akt/PKB pathway. Recent reports of periostin/OSF2 gene disrupted mice indicate that periostin/OSF-2 plays an important role in implantation. Quantitative RT-PCR revealed that the expression of periostin/OSF-2 mRNA in rat uteri was reduced to approximately 10% at 12 h after 17beta-estradiol (E2) injection, but was not changed after progesterone (P) injection. RT-PCR revealed the expression of periostin/OSF-2 in human endometrium, cultured human endometrial stromal cells (ESCs), and cultured human endometrial epithelial cells. In ESCs, the expression of periostin/OSF-2 mRNA was reduced to approximately 50% at 6 h after E2 treatment. The amount of periostin/OSF2 mRNA in human endometrium significantly increased during mid-proliferative and early secretory phases of menstrual cycle, and decreased during late-proliferative, mid-secretory and late secretory phases. The expression of periostin/OSF2 mRNA significantly decreased in ESCs decidualized by treatment with E2 and P for 7 and 11 days. By immunohistochemistry, the expression of periostin/OSF-2 was strongly detected in endometrial stromal cells during early proliferative, mid-proliferative and early secretory phases, and was strongly detected in endometrial epithelial cells during late secretory phase. This study demonstrated that the expression of periostin/OSF-2 is regulated by ovarian steroid hormones in rat uterus and human endometrium.

Evidence for the expression of alcohol dehydrogenase class I gene in rat uterus and its up-regulation by progesterone.

The endometrium is one of the target tissues of the ovarian steroid hormones, estrogen and progesterone. In order to elucidate the mechanism of gene regulation in the endometrium, suppressive subtraction hybridization was performed to isolate the candidate genes controlled by progesterone in rat uterus. Alcohol dehydrogenase (ADH) class I gene was one of the candidate genes. Here we investigated the expression and regulation of ADH class I gene in rat uterus. The mRNA of ADH class I was detected in uterus by RT-PCR using specific primers. Using specific probe for ADH class I, in situ hybridization was performed to investigate localization in rat uterus. Positive signals were detected in the endometrial stromal cells of rat uterus by in situ hybridization and were not detected in endometrial epithelial cells and myometrium in rat uterus. Ovariectomized rats were treated with 17-beta estradiol and progesterone and the uteri of these rats were used for Northern blot analysis and assay of the ADH activity. Northern blot analysis revealed that the expression of ADH class I mRNA in rat uteri was up-regulated approximately two-fold after progesterone treatment, but not estrogen. Likewise, ADH activity was approximately two-fold higher in progesterone-treated rat uteri compared with controls. This study demonstrated that ADH class I gene is progesterone-responsive in the uterus. This implies that progesterone might be involved with retinoic acid synthesis in the uterus, since ADH is the key enzyme for retinoic acid synthesis.