Oviductal Oxygen Homeostasis in Patients with Uterine Myoma: Correlation between Hypoxia and Telocytes.

Oxygen balance is crucial for angiogenesis, immunity, and tissue repair. The human oviduct is essential for reproductive function, and any imbalance in homeostasis leads to fertility disturbances and might be a reason for ectopic pregnancy development. Uterine myoma is a widespread benign tumour, which is often accompanied by infertility. Telocytes have been discussed in the contexts of motility, fibrosis development, and angiogenesis. We observed the oviducts from patients with and without uterine myoma, comparing the expression of HIF-1, HO, VEGF and its receptor, NOS, oestrogen, and progesterone receptors by immunolabeling. The myometrial and oviductal telocytes were also compared in both groups. Biochemical analyses were conducted for FSH, LH, AMH, sFlt, oestrogen, and progesterone in blood samples. Patients with uterine myoma have different expressions of sex steroid receptors and an increased number of telocytes. The decreasing VEFG expression was compensated by the rise in the HIF-1 and NOS expression. Blood biochemical analyses revealed a higher progesterone level and lower AMH in patients with uterine myoma. No differences in sFlt, FSH, and LF were observed. Uterine myoma impacts oviduct oxygen homeostasis and might cause fertility disturbances (uterine and oviductal infertility factors).

Lichong decoction inhibits micro-angiogenesis by reducing the expressions of hypoxia inducible factor-1α and vascular endothelial growth factor in hysteromyoma mouse model.

OBJECTIVE: To investigate the efficacy of Lichong decoction (LD) from Traditional Chinese Medicine, on micro-angiogenesis in a mouse model of hysteromyoma. METHODS: A mouse model of hysteromyoma was developed by orthotopic intrauterine injection of primary human myoma cells isolated from patients from the Beijing Obstetrics and Gynecology Hospital into CB-17 Scid mice. Mice were administered high-dose LD, low-dose LD, mifepristone or water (control) daily by gavage for 4 weeks. Uterine diameter and coefficient (uterine weight/body weight) were measured. Uterine morphology was assessed by light microscopy (hematoxylin and eosin) and transmission electron microscopy. Serum levels of estradiol, progesterone, follicle-stimulating hormone and luteinizing hormone (LH) were measured by enzyme-linked immunosorbent assay. Uterine protein expression of hypoxia inducible factor (HIF)-1α, CD31 and proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. VEGF and HIF-1α mRNAs were quantified by RT-PCR. RESULTS: High-dose LD, low-dose LD and mifepristone reduced uterine diameter and coefficient, and attenuated the morphologic abnormalities associated with hysteromyoma. High-dose LD, low-dose LD and mifepristone inhibited hysteromyoma-induced micro-angiogenesis, as evidenced by a decrease in the number of new microvessels co-immunostaining for CD31 and PCNA (P < 0.01). High-dose LD and mifepristone lowered serum levels of estradiol, progesterone and LH (P < 0.05). High-dose LD, low-dose LD and mifepristone down-regulated HIF-1α mRNA and protein expressions and VEGF mRNA expression (P < 0.01). CONCLUSION: The inhibition of hysteromyoma by LD may involve reductions in HIF-1α and VEGF expression and suppression of micro-angiogenesis.

Identification of the molecular relationship between intravenous leiomyomatosis and uterine myoma using RNA sequencing.

The purpose of this study was to explore the potential relationship between intravenous leiomyomatosis (IVL) and uterine myoma (UM) at the molecular level. RNA-sequencing was performed on IVL tumours, UM tumours, and adjacent normal uterine muscle. We compared the gene expression levels between IVL and normal uterine muscle, UM and normal uterine muscle, to identify differentially expressed genes (DEGs). Then we used Gene Ontology Enrichment Analysis to determine the functions of the DEGs and performed specimen cluster analysis. We obtained 98 DEGs between IVL and adjacent normal uterine muscle, and 61 DEGs between UM and adjacent normal uterine muscle. Functional enrichment of both IVL and UM DEGs showed that they are associated with hormone stimulus, extracellular matrix, and cell adhesion. Unsupervised clustering analysis showed that IVL and UM could not be separated completely. Among these dysregulated genes, we found that HOXA13 showed a distinct dysregulated status between IVL and UM. HOXA13 may therefore serves as a biomarker to distinguish IVL and UM. Our results showed that IVL and UM may have similar dysregulated gene networks. They may be closely related, and HOXA13 may serves as a biomarker to distinguish between IVL and UM.

Expression of VEGF isoforms and their receptors in uterine myomas.

OBJECTIVES: The aim of the study was to determine the expression of VEGF (vascular endothelial growth factor) isoforms and their receptors in uterine myomas. MATERIAL AND METHODS: The study included 40 women with myomas of reproductive age and 40 perimenopausal women (the study group). Myometrial samples (the control group) were taken from 10 women undergoing hysterectomy for ovarian tumors and 10 older women undergoing hysterectomy for uterine prolapse. RESULTS: A significantly increased expression of VEGF-A has been found in myomas, both small and large, in the younger women, which may by a sign of increased angiogenesis and intensive tumor growth. In perimenopausal women, the increase of VEGF expression was observed only in the endothelium and vascular smooth muscle. CONCLUSION: An important conclusion of this study is that angiogenesis is independent of myoma size, which may suggest intensive tumor growth and the related increased angiogenesis. High expression of VEGF-A and VEGF-R1 receptors in large myomas can probably cause malignant transformation and more extensive growth, regardless of patient age.

Effect of Lichong decoction on expression of Bcl-2 and Bcl-2-associated X protein mRNAs in hysteromyoma model rat.

OBJECTIVE: To study on effects of Lichong decoction on expression of apoptosis-controlling genes, Bcl-2 and Bcl-2-associated X protein (Bax) mRNAs in hysteromyoma tissue of the hysteromyoma model rat. METHODS: Fifty Wistar female rats were randomly divided into a normal group, a model group, a Lichong decoction group, a Guizifuling capsule group and a Mifepristone group. The hysteromyoma rat model was established by intraperitoneal injection of exogenous estrin and progestogens. Pathological examination of uterine tissue, uterine coefficient and uterine transverse diameter were made under optic microscope and expressions of Bcl-2 and Bax mRNAs in uterine tissue in the groups were detected with real-time fluorescent quantitative polymerase chain reaction (PCR) technique. RESULTS: After treatment, under microscope it was found that in the Lichong decoction group myometrium thinned, muscle fiber slightly overgrowth or long and thin, regular arrangement, inserting phenomenon of inner circular muscle and external longitudinal muscle was occasionally or not seen in the Lichong decoction group. The uterine coefficient and the uterine transverse diameter significantly decreased (P < 0.01), and Bcl-2 mRNA expression significantly decreased (P < 0.01) and Bax mRNA expression significantly increased in hysteromyoma tissue (P < 0.01) in the Lichong decoction group as compared with the model group. CONCLUSION: Therapeutic effects of Lichong decoction on hysteromyoma is related with decrease of Bcl-2 mRNA expression and increase of Bax mRNA expression.

Immunohistochemical localization of selected pro-inflammatory factors in uterine myomas and myometrium in women of various ages.

Uterine myomas represent one of the most frequently manifested benign tumors in women. They originate from smooth muscle cells of myometrium or its blood vessels. Many studies suggest that inflammation and pro-inflammatory factors may play a role in the carcinogenesis with an involvement of the transcription factor NF-kappaB which activity can be controlled by various environmental factors, including many cytokines. The aim of the study was to investigate the expression of NF-B, interleukin-1ß (IL-1ß), tumor necrosis factor a (TNF-α), cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) in myometrium and uterine myomas of women of various age. The expression of NF-kappaB, selected cytokines and enzymes was estimated in women of reproductive or perimenopausal age by semiquantitative immunohistochemistry. The expression of the examined proteins was higher in myomas than in control myometrium and was dependent on the size of myomas and the age of women. However, the expression of the cytoplasmic NF-kappaB observed in uterine myomas was independent on the size of myomas and no significant differences were observed in the number of stained nuclei between control and myoma groups. Thus, the expression of proinflammatory factors in myomas was not accompanied by the nuclear activation of NF-kappaB p65. The results of our study indicate that the examined factors may be involved in the pathogenesis of benign tumors and not only malignant diseases.

Stretch magnitude- and frequency-dependent cyclooxygenase 2 and prostaglandin E2 up-regulation in human endometrial stromal cells: possible implications in endometriosis.

Endometriosis, with a prevalence rate ranging from 6% to 10%, is the major contributor to pelvic pain and subfertility, and considerably reduces the quality of life in affected women. However, the pathogenesis of this disease remains largely unknown. The present study aimed to uncover the role of hyperperistalsis in the pathogenesis of endometriosis, by exploring the response of human endometrial stromal cells (ESCs) to the cyclic stretch in vitro. ESCs isolated from 18 different endometrium biopsies undergoing hysterectomy for myoma were subjected to uniaxial cyclic stretches with different magnitude and frequency using the Uniaxial Tension System. Expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) in stretched and unstretched ESCs were assessed by realtime quantitative polymerase chain reaction and Western blot. Production of prostaglandin E2 (PGE(2)) in the culture medium was measured by enzyme-linked immunosorbent assay. The cyclic stretch mimicking hyperperistalsis in endometriosis (5% elongation at 4 cycles/min) stimulated quick up-regulations of COX-2 and mPGES-1 simultaneously on both transcriptional and translational levels, and delayed PGE(2) overproduction was also noted in ESCs. As the stretch magnitude or frequency increased, so did overexpression of COX-2 and PGE(2) (P < 0.05). By contrast, the cyclic stretch mimicking physiological peristalsis (3% elongation at 2 cycles/min) did not induce significant COX-2, mPGES-1 or PGE(2) production within 12 h. Both COX-2 and mPEGS-1 are PGE(2) synthases, and the aberrant COX-2 and PGE(2) production play important roles in the pathogenesis of endometriosis. Therefore, the present findings revealed that increased stretch stimuli from the hyperperistalsis of endometriosis were capable of causing the aberrant COX-2 and PGE(2) expression in the endometrium by mechanotransduction, in a magnitude and frequency-dependent manner. It implied possible roles of hyperperistalsis in the pathogenesis of endometriosis, particularly in the view of COX-2 and PGE(2).

PTEN, tau-AP-3, thymidylate synthase immunohistochemistry scoring expression in patients with uterine leiomyomas, uterine smooth muscle tumors of uncertain malignancy potential and uterine leiomyosarcomas.

Uterine smooth muscle tumors are frequently classified as benign and malignant. However, an assortment of mitotic counts and nuclear atypia can be indecisive between uncertain malignant potential, and malignant uterine smooth muscle tumors. We applied three immunohistochemical parameters to distinguish between cases of benign, malignant, and those with uncertain malignant histology.

Processes of apoptosis and cell proliferation in uterine myomas originating from reproductive and perimenopausal women.

We studied uterine myomas originating from females of reproductive age and from females of perimenopausal age. Uterine myomas represent benign tumors of the myometrium, and they develop frequently in women of reproductive age. The frequency of uterine myomas increases with age until women reach the menopause. The study included patients with a myomatous uterus, in the reproductive age or peri-menopausal age, independently evaluating small and large myomas. Myometrial alterations in their direct vicinity were evaluated independently of the myomas. The study included evaluation of immunolocalization of two index proteins which participate in myoma cells growth control: Ki-67 nuclear antigen and caspase 3. In women of reproductive age, both in small and large myomas, elevated immunostaining of Ki-67 was noted in parallel to low levels of caspase 3 staining, which indicated the ongoing process of proliferation. In women of peri-menopausal age with small or large myomas, no Ki-67 immunostaining was detected, while staining of caspase 3 manifested low levels. Proliferation in reproductive age women myomas is higher than in the peri-menopausal age.

Influence of antibiotic therapy on the level of selected angiogenic factors in patients with benign gynecologic tumors--preliminary report.

An increased fibrin level enhances the activity of proangiogenic factors and may contribute to tumor formation. Formation of new blood vessels during angiogenesis leads to neoplasm development through interaction with factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and interleukins. The aim of this study was to investigate the influence of perioperative antibiotic therapy in women with benign gynecological tumors with regard to basic fibroblast growth factor level, fibrinogen concentration and fibrin viscosity. The influence of clindamycin plus metronidazole therapy (group I) and cephazolin therapy (group II) on fibrinogen concentration, level of bFGF and fibrin viscosity was studied in women diagnosed with nonmalignant myomas and cysts. In patients with benign gynecologic tumors, higher bFGF levels (51.40 +/- 13.72 pg/ml), fibrinogen concentration (348.26 +/- 164.74 mg/dl) and fibrin viscosity (2.63 +/- 0.36 mPa) were observed, as compared with healthy women. There were strong indications that antiangiogenic activity occurred with both clindamycin plus metronidazole and cephazolin, although the response to these particular antibiotic therapies was different. The use of various drug therapies in groups I and II resulted in faster and delayed antiangiogenic effects, respectively. Further research is essential to provide more detailed information about the mechanisms of the induction of antiangiogenic activity by perioperative adjuvant antibiotic treatment.

The peritoneal leptin, MCP-1 and TNF-α in the pathogenesis of endometriosis-associated infertility.

PROBLEM: To explore the roles of leptin, monocyte chemotactic protein (MCP)-1, and tumour necrosis factor (TNF)-α in the peritoneal fluid (PF) in the pathogenesis of endometriosis-associated infertility. METHOD OF STUDY: Leptin, MCP-1, and TNF-α levels in the PF from 28 infertile women with endometriosis (study group), 23 women with fallopian-associated infertility (controls), and 24 women with myoma (controls) were determined by performing enzyme-linked immunosorbent assay (ELISA). RESULT: Leptin and TNF-α levels in the PF showed no significant difference among three groups. The MCP-1 level in patients with endometriosis was higher than those in fallopian-associated infertility group and myoma group (P < 0.01). There was a positive correlation between leptin and MCP-1 levels in the PF of patients with endometriosis (P < 0.05). CONCLUSION: Peritoneal leptin and MCP-1 play important roles in the pathogenesis of infertility in the early stage of endometriosis.

The TRAF2 and TRAF6 expression in myomas and myometrium of women in reproduction and perimenopausal age.

Uterine myomas represent one of the most common female diseases. Uterine myomas or fibromas are benign, hormone-responding tumours of, respectively, smooth muscles and fibroblasts and their aetiology induces a significant interest. In myomas the presence of aromatase was detected and, in addition, oestrogen was found to be synthesized in myoma cells. The studies were performed on myoma patients of generative age and those in peri-menopausal age. Expression of TRAF2 and TRAF6 proteins was examined using immunohistochemistry and Western blot approach in small and large uterine myomas isolated from women of various age. In addition, the evaluation was conducted at the periphery of every myoma. We indicated that the level of both tested proteins in myomas is higher than in control. TRAF2 level in myometrium was lower than in myomas but higher than in control. In the case of TRAF6 those changes were ambiguous. Age didn't have influence the level of expression in both tested TRAF in studied structures.

Antioxidant enzymes and lipid peroxidation in endometrium of patients with polyps, myoma, hyperplasia and adenocarcinoma.

BACKGROUND: Oxidative stress and impaired antioxidant system have been proposed as a potential factors involved in the pathophysiology of diverse disease states, including carcinogenesis. In this study, we explored the lipid peroxidation levels and antioxidant enzyme activities in women diagnosed with different forms of gynecological diseases in order to evaluate the antioxidant status in endometrium of such patients. METHODS: Endometrial tissues of gynecological patients with different diagnoses were collected and subjected to assays for superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and lipid hydroperoxides. RESULTS: Superoxide dismutase activity was significantly decreased (50% in average) in hyperplastic and adenocarcinoma patients. Activities of both glutathione peroxidase and glutathione reductase were increased 60% and 100% on average, in hyperplastic patients, while in adenocarcinoma patients only glutathione reductase activity was elevated 100%. Catalase activity was significantly decreased in adenocarcinoma patients (47%). Lipid hydroperoxides level was negatively correlated to superoxide dismutase and catalase activities, and positively correlated to glutathione peroxidase and glutathione reductase activities. CONCLUSIONS: This study provided the first comparison of antioxidant status and lipid peroxidation in endometrial tissues of patients with polyps, myoma, hyperplasia and adenocarcinoma. The results showed that patients with premalignant (hyperplastic) and malignant (adenocarcinoma) lesions had enhanced lipid peroxidation and altered uterine antioxidant enzyme activities than patients with benign uterine diseases, polyps and myoma, although the extent of disturbance varied with the diagnosis. Further investigation is needed to clarify the mechanisms responsible for the observed alterations and whether lipid hydroperoxide levels and antioxidant enzyme activities in uterus of gynecological patients might be used as additional parameter in clinical evaluation of gynecological disorders.

HOXA-10 expression in the mid-secretory endometrium of infertile patients with either endometriosis, uterine fibromas or unexplained infertility.

BACKGROUND: The aim of this study was to investigate HOXA-10 expression in endometrium from infertile patients with different forms of endometriosis; with uterine fibromas, or with unexplained infertility and from normal fertile women. METHODS: Expression levels of HOXA-10 mRNA and protein in endometrium were measured during the mid-secretory phase. This study utilized laser capture microdissection, real-time RT-PCR and immunohistochemistry. RESULTS: HOXA-10 mRNA and protein expression levels in endometrial stromal cells were significantly lower in infertile patients with different types of endometriosis (deep infiltrating endometriosis, ovarian endometriosis and superficial peritoneal endometriosis), with uterine myoma, and unexplained infertility patients as compared with healthy fertile controls. HOXA-10 mRNA expression levels of microdissected glandular epithelial cells were significantly lower than those of microdissected stromal cells, without significant differences among the different groups. No protein expression was detected in glandular epithelial cells. The percentage of patients with altered protein expression of HOXA-10 in stromal cells were significantly higher in patients with only superficial peritoneal endometriosis (100%, 20/20, P < 0.05) compared with the other infertile groups (deep infiltrating endometriosis: 72.7%, 16/22; ovarian endometriosis: 70.0%, 14/20; uterine myoma: 68.8%, 11/16; unexplained infertility: 55.6%, 5/9). CONCLUSION: The present findings suggested that altered expression of HOXA-10 in endometrial stromal cells during the window of implantation may be one of the potential molecular mechanisms of infertility in infertile patients, particularly in patients with only superficial peritoneal endometriosis. One of the underlying causes of infertility in patients with only superficial endometriosis may be altered expression of HOXA-10 in endometrial stromal cells.

The relationship between urokinase plasminogen activator/plasminogen activator inhibitor type-1 expression in myoma/myometrium and mechanism of uterine artery occlusion by laparoscopy for uterine myoma treatment.

The objective of the study was to investigate the pattern of expression of plasminogen activator/plasminogen activator inhibitor (PAI) system between myoma and myometrium and its correlation between outcome and laparoscopic uterine artery occlusion prior to myomectomy in the treatment of myoma. mRNA expression of PAI type-1 (PAI-1) and urokinase plasminogen activator (uPA) was detected with real-time PCR in the myoma and myometrium cells primary cultured in vitro, and uPA and PAI-I protein expression was detected with cellular immunity histochemistry. First, the expression of uPA mRNA was 0.123 +/- 0.189 in myoma, which was significantly lower than 0.331 +/- 0.306 in myometrium (P < 0.05); however, the expression of PAI-I mRNA was 0.091 +/- 0.036 in myoma, which was significantly higher than 0.016 +/- 0.020 in myometrium (P < 0.05). Second, the expression of uPA protein was 8.805 +/- 1.645 in myoma cells, which was lower than 22.173 +/- 4.381 in myometrium (P < 0.05); the expression of PAI-I protein was 44.765 +/- 1.090 in myoma cells, which was significantly higher than 35.928 +/- 5.351 in myometrium (P < 0.05). The distinct expression pattern of uPA/PAI in myoma and myometrium might be correlated to the low recurrence rate after uterine artery occlusion prior to myomectomy in the treatment of myoma.

The aromatase expression in myomas and myometriums of women in reproduction and perimenopausal age.

Uterine myomas represent one of the most common female pathologies. Uterine smooth muscle myomas or fibromas are benign tumours which respond to hormones and their etiology induces wide interest. The myomas were found to contain aromatase and, in addition, cells of the myomas were found to synthesize estrogen. This study was conducted on patients with the myomas, in either generative age or in the perimenopausal period. Expression of aromatase was detected in patients of various age, with large or small uterine myomas, using an immunohistochemical technique. In addition expression of the enzyme was examined at the periphery of every myoma.

[The morphofunctional characteristics of proteins of nucleolar organizers in uterine smooth muscle tumors].

The authors have shown the activity of argyrophylic nucleolar organizer region proteins (Ag-NOR-proteins) in the uterine leiomyomas of varying histological structure and determined their relationship to the growth rate, size, and site of a node and endometrial pathology. The investigation indicat yomas with significant sclerosis, 4.32 +/- 0.1 in simple leiomyomas, and 6.2 +/- 0.2 in cell leiomyomas (r = 0.58). In menopause, myomas with significant sclerosis and hyalinosis were characterized with a small number of Ag-NOR (1.44 +/- 0.04). Thus, the organizers numbered 12.1 +/- 0.5 and 21.7 +/- 1.0 in high- and low-grade sarcomas, respectively, which may be used in the differential diagnosis of cellular, bizarre, mitotically active leiomyomas and leiomyosarcomas.

Differential elastin and tenascin immunolabeling in the uterosacral ligaments in postmenopausal women with and without pelvic organ prolapse.

Connective tissue, consisting mainly of collagen and structural glycoproteins, is an important part of the supportive structures of the genitourinary region. Relatively few data have been published with respect to the role of elastin and glycoproteins in pelvic organ prolapse (POP). Connective tissue of the uterosacral ligament in postmenopausal women with and without genital prolapse was compared. Fifty-nine consecutive women referred for hysterectomy were included in the study. The patients had POP or benign gynecological disease (e.g. myoma of the uterus). Tissue samples from the uterosacral ligament were investigated for localization and distribution of tenascin and elastin using immunofluorescence microscopy. Tissue samples of women with prolapse showed a significantly (p<0.001) weaker immunofluorescent labeling of tenascin compared to samples taken from women without prolapse. Tenascin was detectable in tissues of all women with POP, whereas its immunolabeling was decreased in the uterosacral ligament in women without POP. Intact elastin fibers were observed in tissues of all women without POP, whereas elastin was undetectable or sometimes fragmented in the uterosacral ligament in women with POP. Greater amounts of tenascin and lesser amounts of elastin were therefore found in patients with POP. These results suggest that an altered turnover of connective tissue in the uterosacral ligament might be responsible for the presence of pelvic floor relaxation in postmenopausal women. These data indicate a complex architecture of the extracellular matrix in the uterosacral ligaments, with marked differences in tenascin and elastin expression between postmenopausal women with or without POP.

Studies on leptin and leptin receptor gene expression in myometrium and uterine myomas of gnRH analogue-treated women.

AIM: To test if treatment with GnRH analogue, which leads to a significant reduction in myoma volume, changes expression of leptin genes and gene coding leptin receptor isoforms in uterine myomas and in the surrounding unaltered myometrium. METHODS: Using RT-PCR, expression of leptin genes and leptin receptor genes was studied in myomas and in the surrounding myometrium in women with uterine myomas, untreated or treated with GnRH analogue. In the randomly selected cases presence of leptin protein and of leptin receptor proteins was examined also by Western blotting. RESULTS: Expression of leptin genes was demonstrated both in myomas and in the surrounding myometrium, and a similar pattern of expression was found for leptin receptor isoforms. The results of RT-PCR were confirmed by Western blotting, which documented the identical distribution of leptin proteins and leptin receptor proteins in studied tissues. Treatment with GnRH analogue had no effect on the expression pattern of studied genes. CONCLUSION: The results of the present study on the administration of GnRH analogue to females with myomas suggest that no direct or immediate inter-relationship exists between expression of leptin genes in uterine myomas on one hand and estrogen, progesterone and leptin levels in the blood on the other. Expression seems to be of a more durable nature but factors that induce such expression remain unknown.