Transcriptome changes in maternal peripheral blood during term parturition mimic perturbations preceding spontaneous preterm birth†.

The complex physiologic process of parturition includes the onset of labor, which requires the orchestrated stimulation of a common pathway involving uterine contractility, cervical ripening, and chorioamniotic membrane activation. However, the labor-specific processes taking place in these tissues have limited use as predictive biomarkers unless they can be probed in non-invasive samples, such as the peripheral blood. Herein, we utilized a transcriptomic dataset to assess labor-specific changes in the peripheral blood of women who delivered at term. We identified a set of genes that were differentially expressed with labor and enriched for immunological processes, and these gene expression changes were strongly correlated with results from prior studies, providing in silico validation of our findings. We then identified significant correlations between labor-specific transcriptomic changes in the maternal circulation and those detected in the chorioamniotic membranes, myometrium, and cervix of women at term, demonstrating that tissue-specific labor signatures are partly mirrored in the peripheral blood. Finally, we demonstrated a significant overlap between the peripheral blood transcriptomic changes in term parturition and those observed in asymptomatic women, prior to the diagnosis of preterm prelabor rupture of the membranes, who ultimately delivered preterm. Collectively, we provide evidence that the normal process of labor at term is characterized by a unique immunological expression signature, which may serve as a useful tool for assessing labor status and for potentially identifying women at risk for preterm birth.

The morphofunctional pattern of neuronal biogenic amines in postpartum involution period-an in vivo study.

Postpartum uterine diseases are associated with significant imbalance in the levels of biogenic amines (BAs) in rat uterus. Mast cells (MCs) are the main suppliers of BAs such as serotonin, catecholamines, and histamine in uterus. There is limited evidence of the BA-positive elements involved in the physiological regulation of uterus during postpartum involution. The aim of present study is to determine the concentration and distribution of biogenic amines (BAs) such as histamine, serotonin, and catecholamines in the uterine endometrium, myometrium, and peritoneal fluid (PF) during the postpartum uterine involution. A total of 110 nulliparous outbred female nonpregnant Wistar rats of mature age were divided into eleven groups (n=10 per group) according to days of postpartum involution. Tissue specimens of uterine segments, PF were prepared. Serotonin, catecholamines, and histamine concentrations were examined by fluorescence-histochemical techniques. The fluorescence of the BA-positive elements was detected and analyzed by microspectrofluorimetry. Results were analyzed using the Kruskal-Wallis chi-squared test and pairwise Mann-Whitney-Wilcoxon tests with "Benjamini-Hochberg correction" in R 3.6.3. Mast cells in uterine segments, PF exhibited characteristic yellowish-green fluorescence. The highest MCs number was reported in corpus uteri on the 15th day of postpartum involution. Serotonin, catecholamines, and histamine levels were significantly higher in BA-positive elements in the initial days. BA content was dynamic and relies on the time elapsed after parturition. There was statistically significant difference in the levels of BAs in the cornu and cervix uteri. A single morphofunctional complex of BA supply was noticed in the reproductive system of the rats. The coupled interactions of intra- and extra-organic BA-positive elements was associated with anabolic/catabolic equilibrium in uterus through the metabolism of serotonin, catecholamines, and histamine during postpartum involution.

Evaluation of KI-67 expression in uterine leiomyoma and in healthy myometrium: a pilot study

SUMMARY OBJECTIVE Evaluate the expression of KI-67 in uterine leiomyomas and adjacent myometrial tissue and verify the existence of a correlation between clinical parameters and KI-67 expression in tumors. METHODS This is a cross-sectional, controlled, analytical study. Samples of leiomyomas and myometrium were obtained from patients who underwent hysterectomy. The samples were processed by immunohistochemistry using KI-67 antibody, and the expression was evaluated by two blinded observers. Student›s T-test was used for comparison of means, and Pearson›s P test for correlation with clinical parameters. RESULTS A total of 9 patients were included in the study. The mean age was 40.7 years, ranging from 35 to 44 years. The mean expression of KI-67 in myometrium was 1.63%, and, in leiomyomas, 5.96% (p <0.001). The highest expression of KI-67 was moderately related to the severity of anemia, bleeding, and pain level. CONCLUSION The expression of KI-67 in normal myometrium was significantly lower than in leiomyomas. The highest expression of KI-67 was moderately related to the severity of anemia, bleeding, and pain level in the patients of this study.

RESUMO OBJETIVO Avaliar a expressão do KI-67 em leiomiomas uterinos e tecido miometrial adjacente e verificar a existência de correlação entre parâmetros clínicos e expressão do KI-67 em tumores. MÉTODOS Estudo transversal, controlado e analítico. Amostras de leiomiomas e miométrio foram obtidas de pacientes que realizaram histerectomia. As amostras foram processadas por imuno-histoquímica utilizando anticorpo para KI-67 e a expressão avaliada por dois observadores cegos. O teste t de Student foi utilizado para comparação de médias e o teste P de Pearson para correlação com parâmetros clínicos. RESULTADOS Um total de 9 pacientes foi incluído no estudo. A idade média foi de 40,7 anos, variando de 35 a 44 anos. A expressão média do KI-67 no miométrio foi de 1,63% e nos leiomiomas de 5,96% (p <0,001). A maior expressão do KI-67 foi moderadamente relacionada com a gravidade da anemia, sangramento e nível de dor. CONCLUSÃO A expressão do KI-67 no miométrio normal foi significativamente menor que nos leiomiomas. A maior expressão do KI-67 foi moderadamente relacionada à gravidade da anemia, sangramento e nível de dor nos pacientes deste estudo.

Gestation age-associated dynamics of mitochondrial calcium uniporter subunits expression in feto-maternal complex at term and preterm delivery.

Calcium plays a role of universal cellular regulator in the living cell and one of the crucial regulators of proper fetal development during gestation. Mitochondria are important for intracellular calcium handling and signaling. Mitochondrial calcium uniporter (mtCU) is a multiprotein complex of the mitochondrial inner membrane responsible for the transport of calcium to the mitochondrial matrix. In the present study, we analyzed the expression level of mtCU components in two parts of the feto-maternal system - placenta and myometrium at full-term delivery and at preterm birth (PTB) on different stages: 22-27, 28-32, 33-36 weeks of gestation (n = 50). A gradual increase of mRNA expression and changes in protein content of MCU and MICU1 subunits were revealed in the placenta during gestation. We also observed slower depolarization rate of isolated placental mitochondria induced by Ca2+ titration at PTB. In myometrium at PTB relative gene expression level of MCU, MCUb and SMDT1 increased as compared to full-term pregnancy, but the tendency to gradual increase of MCU protein simultaneous with MCUb increase and MICU1 decline was shown in gestational dynamics. Changes observed in the present study might be considered both natural dynamics as well as possible pathological mechanisms underlying preterm birth.

Evaluation of KI-67 expression in uterine leiomyoma and in healthy myometrium: a pilot study.

OBJECTIVE: Evaluate the expression of KI-67 in uterine leiomyomas and adjacent myometrial tissue and verify the existence of a correlation between clinical parameters and KI-67 expression in tumors. METHODS: This is a cross-sectional, controlled, analytical study. Samples of leiomyomas and myometrium were obtained from patients who underwent hysterectomy. The samples were processed by immunohistochemistry using KI-67 antibody, and the expression was evaluated by two blinded observers. Student>s T-test was used for comparison of means, and Pearson>s P test for correlation with clinical parameters. RESULTS: A total of 9 patients were included in the study. The mean age was 40.7 years, ranging from 35 to 44 years. The mean expression of KI-67 in myometrium was 1.63%, and, in leiomyomas, 5.96% (p <0.001). The highest expression of KI-67 was moderately related to the severity of anemia, bleeding, and pain level. CONCLUSION: The expression of KI-67 in normal myometrium was significantly lower than in leiomyomas. The highest expression of KI-67 was moderately related to the severity of anemia, bleeding, and pain level in the patients of this study.

IRF5 is increased in labouring myometrium and regulates pro-labour mediators.

Preterm birth continues to be the leading cause of neonatal mortality and morbidities that can extend into adult life. Few treatment options stem from our incomplete understanding of the mechanisms of human labour and delivery. Activation of the inflammatory response in gestational tissues by inflammation and/or infection leads to the production of pro-inflammatory and pro-labour mediators, thus preterm birth. Interferon regulatory factor 5 (IRF5) has recently emerged as an important pro-inflammatory transcription factor involved in acute and chronic inflammation. The aims of this study were to determine the expression of IRF5 in human myometrium from labouring and non-labouring women, and whether IRF5 is involved in the genesis of pro-inflammatory and pro-labour mediators induced by pro-inflammatory cytokines or toll-like receptor (TLR) ligands. IRF5 mRNA and protein expression was significantly higher in human myometrium after spontaneous term labour, compared to non-labouring tissues. IRF5 mRNA expression was also significantly higher in primary myometrial cells treated with the pro-inflammatory cytokines IL1B or TNF. In primary myometrial cells, IRF5 knockdown by siRNA (siIRF5) was associated with significantly decreased expression and or secretion of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL8, CCL2), adhesion molecules (ICAM1, VCAM1) and contraction-associated proteins PTGS2, PGF2α and PTGFR when in the presence of IL1B, TNF, fsl-1 (TLR2/6 ligand) or flagellin (TLR5 ligand). siIRF5-transfected cells also displayed decreased NF-κB RELA transcriptional activity in the presence of these preterm birth mediators. Our study suggests a novel role for IRF5 in the regulation of the inflammatory response in human myometrium.

Endometrioid endometrial carcinomas with microcystic, elongated, and fragmented (MELF) type of myoinvasion: role of immunohistochemistry in the detection of occult lymph node metastases and their clinical significance.

In endometrioid endometrial carcinomas (EECs), microcystic, elongated, and fragmented (MELF) myoinvasion is associated with easily overlooked lymph node metastases; however, the role of immunohistochemistry in their detection and their clinical significance have not been addressed. We identified MELF in 43 of 101 (43%) myoinvasive EECs. Nodes were removed in 49 (49%), 25 with MELF and 24 without MELF. Metastases were initially reported in 3 of the former (12%) and 2 of the latter (8%). All negative nodes were reviewed, and cytokeratin immunohistochemistry was performed. Three metastases were identified in the MELF group but none in the EECs without MELF. By immunohistochemistry, metastatic nodal isolated tumor cells (ITCs) were found in 6 of the remaining 19 MELF-positive cases. In contrast, lymph node metastases were detected in only 2 of the 22 EECs without MELF. MELF-positive cases had more lymph node metastases (P=.03) than myoinvasive EECs without MELF. At follow-up, all 6 patients with grade 1-2 EECs and nodal ITCs/micrometastases were alive (5 no evidence of disease and 1 with perineal disease). In contrast, 3 of 4 patients with grade 3 EECs and nodal ITCs/micrometastases died of disease, and the other patient was alive with tumor. In MELF, the frequency of ITCs/micrometastases in apparently negative lymph nodes is high. In patients with grade 1-2 EEC who had not received chemotherapy, the presence of nodal ITCs/micrometastases did not affect survival. In contrast, in high-grade tumors, ITCs/micrometastases were associated with unfavorable prognosis. Immunohistochemistry should be done in MELF-positive cases to detect occult lymph node metastases, especially in high-grade tumors.

[Microcystic, elongated and fragmented invasive pattern in endometrial adenocarcinoma: a clinicopathologic analysis of 72 cases].

Objective: To investigate the clinicopathologic features of microcystic, elongated and fragmented (MELF) pattern invasion of endometrial adenocarcinoma. Methods: HE and immunohistochemistry staining method were used to analysis morphologic features and immunophenotype of 72 patients of endometrial adenocarcinoma with MELF pattern invasion, and chi-square test was used to analysis the clinicopathologic features. Results: The mean age of 72 patients was 54 years (40 to 70 years). Thirty-two patients were pre-menopausal and 40 were post-menopausal. According to the FIGO staging system (2014), 32 cases(44.4%)were at stage Ⅰ, 22 cases(30.6%)at stage Ⅱ, 17 cases(23.6%)at stage Ⅲ and 1 case(1.4%) at stage Ⅳ. Microscopically, MELF invasion showed microcystic, elongated slit-like or fragmented glands in myometrium and their lining cells usually were cube or flat, as well as the single or clusters of eosinophilic tumor cells mimicking histocytes. In addition, a fibromyxoid or inflammatory stromal response was often present.Immunohistochemical staining showed that MELF invasion was positive for p16, CA125 and CA19-9, but negative for ER, PR and p53.Compared with non-MELF pattern invasion, significant differences were noted in menopause pausimenia, FIGO stages, deep invasion into myometrium, lymph metastasis, lymphovascular space invasion (LVSL), serum CA125 and CA19-9 in patients with MELF pattern invasion (all P<0.05). Conclusions: MELF pattern invasion of endometrial adenocarcinoma is characterized by advanced FIGO stage, deep myoinvasion, high metastasis rate to lymph node and LVSL. Pathologists should recognize the MELF invasion and evaluate the depth of myometrium of infiltration and LVSL with special attention to the presence of MELF invasion with necessary immunohistochemistry for more accurate pathological diagnosis.

Prolactin role in the bovine uterus during adenomyosis.

Adenomyosis is uterine dysfunction defined as the presence of endometrial glands within the myometrium. It is suggested that adenomyosis is estrogen-dependent pathology, and prolactin (PRL) also affects its development. In the uterus of ruminants, PRL stimulates gland proliferation and function. We hypothesized that in the bovine uterus, the expression of PRL and its receptors (PRLRs) during adenomyosis is disturbed and modulated by estradiol (E2). Uterine tissues were collected postmortem from cows; epithelial, stromal, and myometrial cells were isolated; and cultured and treated with E2. Material was divided into 2 groups: control (nonadenomyotic) and uteri with adenomyosis. In adenomyotic uterine tissue, PRL and its long-form receptor protein were increased, as determined by Western blotting. Immunohistostaining showed that during adenomyosis, PRL and its receptors are highly expressed in adenomyotic lesions. In cultured myometrial cells, protein expression of PRL and its receptors was increased during adenomyosis. Estradiol decreased PRLRs protein expression in nonadenomyotic stromal cells and in adenomyotic myometrial cells, and increased PRL secretion by adenomyotic myometrial cells. Moreover, PRL secretion was increased in untreated epithelial and stromal cells during adenomyosis. On the other hand, in stromal cells, PRLRs messenger RNA and protein expression was decreased, as determined by real-time PCR and Western blotting, respectively. Obtained results show that significant changes in PRL and PRLRs expression are observed in uterine tissue and cells during adenomyosis, which were also affected by E2. These data suggest involvement of PRL in adenomyosis development and the link between PRL and E2 actions during the dysfunction in cows.

May Sonic Hedgehog proteins be markers for malignancy in uterine smooth muscle tumors?

Several studies have demonstrated that the Sonic Hedgehog signaling pathway (SHH) plays an important role in tumorigenesis and cellular differentiation. We analyzed the protein expression of SHH pathway components and evaluated whether their profile could be useful for the diagnosis, prognosis, or prediction of the risk of malignancy for uterine smooth muscle tumors (USMTs). A total of 176 samples (20 myometrium, 119 variants of leiomyoma, and 37 leiomyosarcoma) were evaluated for the protein expression of the SHH signaling components, HHIP1 (SHH inhibitor), and BMP4 (SHH target) by immunohistochemistry. Western blot analysis was performed to verify the specificity of the antibodies. We grouped leiomyoma samples into conventional leiomyomas and unusual leiomyomas that comprise atypical, cellular, mitotically active leiomyomas and uterine smooth muscle tumors of uncertain malignant potential. Immunohistochemical analysis showed that SMO, SUFU, GLI1, GLI3, and BMP4 expression gradually increased depending on to the histologic tissue type. The protein expression of SMO, SUFU, and GLI1 was increased in unusual leiomyoma and leiomyosarcoma samples compared to normal myometrium. The inhibitor HHIP1 showed higher expression in myometrium, whereas only negative or basal expression of SMO, SUFU, GLI1, and GLI3 was detected in these samples. Strong expression of SHH was associated with poorer overall survival. Our data suggest that the expression of SHH proteins can be useful for evaluating the potential risk of malignancy for USMTs. Moreover, GLI1 and SMO may serve as future therapeutic targets for women with USMTs.

Immunoexpression of Steroid Hormone Receptors and Proliferation Markers in Uterine Leiomyoma and Normal Myometrial Tissues from the Miniature Pig, Sus scrofa.

Uterine leiomyomas in miniature pet pigs occur similarly to those in women with regard to frequency, age, parity, and cycling. Clinical signs, gross, and histologic features of the porcine tumors closely resemble uterine leiomyomas (fibroids) in women. Although fibroids are hormonally responsive in women, the roles of estrogen and progesterone have not been fully elucidated. In this study, immunohistochemistry was used to assess the expression of the steroid hormone receptors, estrogen receptor alpha (ER-α), estrogen receptor beta (ER-ß) and progesterone receptor (PR), and cell proliferation markers, proliferating cell nuclear antigen (PCNA) and Ki-67 in tumor and matched myometrial tissues sampled from miniature pigs. A "quickscore" method was used to determine receptor expression and labeling indices were calculated for the markers. ER-α/ß and PR were localized to the nuclei of smooth muscle cells in both tissues. PR expression was intense and diffuse throughout all tissues, with correlation between tumors and matched myometria. Conversely, ER-α expression was variable between the myometrial and tumor tissues, as well as between animals. ER-ß expression was low. PCNA and Ki-67 were localized to the nucleus and expression varied among tumors; however, normal tissues were overall negative. These findings support further investigation into the use of the miniature pig as a model of fibroids in women.

Ca(2+)-dependent regulation of the Ca(2+) concentration in the myometrium mitochondria. I. Trifluoperazine effects on mitochondria membranes polarization and [Ca(2+)](m).

Са2+-dependent regulation of Ca2+ exchange in mitochondria is carried out with participation of calmodulin. We have shown previously that calmodulin antagonists reduced the level of mitochondrial membrane polarization and induced increase of the ionized Са concentration in both the mitochondrial matrix and cell cytoplasm. The concentration-dependent influence of trifluoperazine on the level of polarization of mitochondrial membranes has been shown in this work. The coordinates of the Hill graphs were used to calculate the constant K0.5 and the Hill coefficient. K0.5 was 24.4 ± 5 µM (n = 10). The Hill coefficient was 2.0 ± 0.2, indicating the presence of two centers of the trifluoperazine binding. We have also studied [Ca2+]m changes, when incubating mitochondria in mediums of different composition: without ATP and ions of Mg (0-medium), in the presence of 3 mM Mg (Mg-medium) and 3 mM Mg + 3 mM ATP (Mg,ATP-medium). It was shown that the composition of the incubation medium affected the [Ca2+]m values in the absence of exogenous Ca2+ and did not affect them in the presence of the latter. Preincubation of mitochondria in mediums of different composition with 25 µM trifluoperazine did not affect the [Ca2+]m values both before and after the addition of 100 µÐœ Са2+ to the incubation medium. It was concluded, that trifluoperazine depolarized myometrial mitochondria membranes in concentration-dependent manner. However, mitochondria preincubation with 25 µM trifluope­razine accompanied by 50% decrease in membrane polarization did not affect the [Ca2+]m values.

The use of the Petri net method in the simulation modeling of mitochondrial swelling.

Using photon correlation spectroscopy, which allows investigating changes in the hydrodynamic dia­meter of the particles in suspension, it was shown that ultrahigh concentrations of Ca2+ (over 10 mM) induce swelling of isolated mitochondria. An increase in hydrodynamic diameter was caused by an increase of non-specific mitochondrial membrane permeability to Ca ions, matrix Ca2+ overload, activation of ATP- and Ca2+-sensitive K+-channels, as well as activation of cyclosporin-sensitive permeability transition pore. To formalize the experimental data and to assess conformity of experimental results with theoretical predictions we developed a simulation model using the hybrid functional Petri net method.

Calix[4]arene C-90 and its analogs activate ATPase of the myometrium myosin subfragment-1.

Numerous female reproductive abnormalities are consequences of disorders in uterus smooth muscle (myometrium) contractile function. In this work, we described activators of ATPase, which could be used for development of effective treatments for correcting this dysfunction. Myosin ATPase localized in the catalytic domain of myosin subfragment-1 transforms a chemical energy deposited in macroergic bonds of ATP into mechanical movement. It was shown that сalix[4]arene C-90 and its structural analogs functionalized at the upper rim of macrocycle with four or at least two N-phenylsulfonуltrifluoroacetamidine groups, are able to activate ATP hydrolysis catalyzed by myometrium myosin subfragment-1. It was shown with the method of computer modeling that N-phenylsulfonуltrifluoroacetamidine groups of calix[4]arene C-90 interact with responsible for binding, coordination and the hydrolysis of ATP amino acid residues of myosin subfragment-1. The results can be used for further research aimed at using calix[4]arene C-90 and its analogs as pharmacological compounds that can effectively normalize myometrium contractile hypofunction.

Ultramicro-trauma in the endometrial-myometrial junctional zone and pale cell migration in adenomyosis.

OBJECTIVE: To determine if ultrastructural tissue trauma occurs in the junctional zone in uteri in adenomyosis. DESIGN: A case-control experimental study. SETTING: Endometriosis research center. PATIENT(S): Twelve uteri with adenomyosis, and 9 uteri without adenomyosis, were gained during laparoscopy-assisted vaginal hysterectomy. INTERVENTION(S): Transmission electron microscopic study of the junctional zone, as well as immunohistochemical staining for epithelial cadherin, and van Gieson staining and immunofluorescence for CD45 and CD68. MAIN OUTCOME MEASURE(S): Analysis of the electron microscopy photos and the immunoreactive scores of the staining. RESULT(S): The inner myometrial muscle fibers were diversely arranged in adenomyosis; they were parallel to the basal endometrial glands in nonadenomyosis. Nuclear membrane infolding of the basal glandular epithelium and the disruption of the interface between basal endometrium and inner myometrium in adenomyosis (but not in nonadenomyosis) were evident. Intraepithelial pale cells were seen in the basal endometrial glands in both groups, but they lacked CD45 and CD68 expression. They were seen actively migrating into the stroma in adenomyosis only. CONCLUSION(S): The myofiber disarray in the inner myometrium, and the nuclear membrane irregularities in adenomyosis, are evidence for ultramicro-trauma in adenomyosis. The migrating nonleukocytic pale cells may be involved in pathogenesis of adenomyosis.

Stro-1/CD44 as putative human myometrial and fibroid stem cell markers.

OBJECTIVE: To identify and characterize myometrial/fibroid stem cells by specific stem cell markers in human myometrium, and to better understand the stem cell contribution in the development of uterine fibroids. DESIGN: Prospective, experimental human and animal study. SETTING: University research laboratory. PATIENT(S)/ANIMAL(S): Women undergoing hysterectomy for treatment of symptomatic uterine fibroids and female NOD/SCID/IL-2Rγ(null) mice. INTERVENTION(S): Identification and isolation of stem cells from human fibroids and adjacent myometrium tissues using Stro-1/CD44-specific surface markers. MAIN OUTCOME MEASURE(S): Flow cytometry, semiquantitative polymerase chain reaction, clonogenicity assays, cell culture, molecular analysis, immunocyto-histochemistry, in vitro differentiation, and xenotransplantation assays. RESULT(S): Using Stro-1/CD44 surface markers, we were able to isolate stem cells from adjacent myometrium and human fibroid tissues. The undifferentiated status of isolated cells was confirmed by the expression of ABCG2 transporter, as well as additional stem cell markers OCT4, NANOG, and GDB3, and the low expression of steroid receptors ERα and PR-A/PR-B. Mesodermal cell origin was established by the presence of typical mesenchymal markers (CD90, CD105, and CD73) and absence of hematopoietic stem cell markers (CD34, CD45), and confirmed by the ability of these cells to differentiate in vitro into adipocytes, osteocytes, and chondrocytes. Finally, their functional capability to form fibroid-like lesions was established in a xenotransplantation mouse model. The injected cells labeled with superparamagnetic iron oxide were tracked by both magnetic resonance imaging and fluorescence imaging, thus demonstrating the regenerative potential of putative fibroid stem cells in vivo. CONCLUSION(S): We have demonstrated that Stro-1/CD44 can be used as specific surface markers to enrich a subpopulation of myometrial/fibroids cells, exhibiting key features of stem/progenitor cells. These findings offer a useful tool to better understand the initiation of uterine fibroids, and may lead to the establishment of effective therapeutic options.

PROTECTIVE EFFECTS OF POTASSIUM TRANSPORT IN MITOCHONDRIA FROM RAT MYOMETRIUM UNDER ACTIVATION OF MITOCHONDRIAL PERMEABILITY TRANSITION PORE.

We demonstrated using PBFI K(+)-sensitive fluorescent probe an enhancement of both components of K(+)-cycle--the ATP-sensitive K(+)-uptake and quinine-sensitive K+/H(+)-exchange--under the Ca2+ induced opening of mitochondrial permeability transition pore (MPTP) in rat myometrium mitochondria. Addition of CaCl2 (100 µM to K(+)-free medium results in the enhancement of reactive oxygen species (ROS) production, which was eliminated by cyclosporine A. Addition of CaCl2 to K(+)-rich medium did not increase the rate of ROS production, but blocking of mitoK+(ATP)-channels with glybenclamide (10 mcM increased production of ROS. We conclude that K(+)-cycle exerts a protective influence in mitochondria from rat myometrium by regulation of matrix volume and rate of ROS production under the condition of Ca(2+)-induced MPTP.

Potential diagnostic biomarkers: differential expression of LMP2/ß1i and cyclin B1 in human uterine leiomyosarcoma.

AIMS AND BACKGROUND: Whilst most uterine smooth muscle neoplasms are benign, uterine leiomyosarcoma (Ut-LMS) is extremely malignant with a high incidence of metastasis and recurrence. Gynecological tumors are often associated with female hormone secretion, but no strong link has been detected between human Ut-LMS and the hormonal environment. In fact, the risk factors for Ut-LMS are poorly understood. In addition, no diagnostic biomarkers for differentiating between leiomyoma, a benign tumor, and malignant Ut-LMS have been found. Interestingly, mice that were homozygously deficient for LMP2/ß1i were found to spontaneously develop Ut-LMS and exhibited a Ut-LMS prevalence of ~40% by 14 months of age. Thus, analyzing potential risk factors for Ut-LMS (such as LMP2/ß1i) might aid the development of diagnostic biomarkers and clinical treatments for the condition. METHODS AND STUDY DESIGN: Fifty-seven patients (age range: 32-83 years) who had been diagnosed with uterine mesenchymal tumors were chosen from a pathological archive. Tissue samples from these patients were fixed in 10% buffered formalin, incubated in 4% paraformaldehyde for 8 hours, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin for standard histological examination or were subjected to further processing for immunohistochemical (IHC) examination. Serial Ut-LMS, bizarre leiomyoma, leiomyoma, and myometrium sections were subjected to IHC staining of ß-smooth muscle actin, estrogen receptor, cyclin B1, LMP2/ß1i, calponin h1, ki-67, tumor protein p53, and progesterone receptor. RESULTS: The Ut-LMS samples were positive for cyclin B1 and negative for LMP2/ß1i, while the opposite result was obtained for bizarre leiomyoma, leiomyoma, and myometrium samples. CONCLUSIONS: The expression pattern of LMP2/ß1i and cyclin B1 might be a diagnostic biomarker for human Ut-LMS. Studies of the biological roles of LMP2/ß1i and/or cyclin B1 could lead to the elucidation of new targets for therapies against Ut-LMS.

C-Met expression pattern in uterine leiomyoma.

AIM: Growth factors take place in the formation and growth of uterine leiomyomas (LMs). Transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and insulin-like growth factor (IGF) contribute to the pathophysiology of LMs when they bind with a specific membrane receptor and transmit a signal into the cell. Little is known about hepatocyte growth factor (HGF) and its receptor system c-Met in formation and growth of uterine LMs. The aim of this study was to evaluate the c-Met receptor expression on human myometrium and uterine LMs. MATERIALS AND METHODS: The study was performed on human myometrium and uterine LMs. Expression of c-Met receptor was evaluated by immunohistochemical analysis. RESULTS: Overexpression of c-Met was found in all LM cases and in none of normal myometrium samples c-Met overexpression was seen. CONCLUSION: HGF and c-Met receptor complex seem to have role in development of uterine LMs.

Differential expression of AP-1 proteins in human myometrium after spontaneous term labour onset.

OBJECTIVES: The aims of this study were (i) to determine the localisation of activator protein (AP)-1 family members (cFos, FosB, cJun, JunB and JunD) in human myometrium; and (ii) to determine the effect of human term labour on the expression of AP-1 family of transcription factors in myometrium. STUDY DESIGN: This localised the AP-1 family members cFos, FosB, cJun, JunB and JunD in human myometrium was performed by immunohistochemistry. The effect of term labour on the expression of these family members at the mRNA and protein level was assessed by qRT-PCR and Western blotting, respectively. The effect of pro-inflammatory stimuli on AP-1 transcriptional activity was assessed using a luciferase assay in primary human myometrial cells. RESULTS: Immunohistochemical expression of cFos, FosB, cJun, JunB and JunD were all present in human myometrial tissue and displayed cytoplasmic staining. FosB and JunD also displayed nuclear staining. Term labour was associated with an increase in cFos and JunB mRNA and protein expression. On the other hand, JunD mRNA and protein expression was decreased with labour. FosB mRNA was increased with labour, but there was no change at the protein level. There was no change in cJun mRNA or protein expression. AP-1 transcriptional activity was increased in human myometrial cells by the pro-inflammatory cytokine TNF-α. There was, however, no effect of the bacterial products lipopolysaccharide (LPS; TLR4 ligand), iE-DAP (NOD1 ligand), MDP (NOD2 ligand), FSL-1 (TLR2 ligand) or flagellin (TLR5 ligand) on AP-1 transcriptional activity. CONCLUSION: This study shows that human labour is associated with changes in AP-1 family members. Further studies are required to determine the exact role of the AP-1 family members in myometrium.