One of the major healthcare issues found almost worldwide, especially in the United States, is the obesity epidemic. Obesity is known to have deleterious effects on many body/organ systems. C1q TNF-related protein-3 (CTRP3) is effective at preventing high-fat diet-induced fatty liver. With these two factors taken into consideration, this study explores the possible effects of a high caloric diet on the muscle wall of the uterus, i.e., the myometrium, and how over-expression of CTRP3 may modify those effects. We hypothesize that consumption of excessive amounts of fat and sugar will have detrimental effects on the dual layers of the mouse myometrium. For this study, 17 mice were divided into 4 treatment groups: wild type/low fat diet, wild type/high fat diet, CTRP3-overexpressing/low fat diet, and CTRP3-overexpressing/high fat diet. The mice were placed on their respective diets at 7 weeks of age with a feeding duration of 12 weeks. At the conclusion of the feeding protocol, the female reproductive tissues were harvested and fixed in 4% paraformaldehyde and subsequently paraffin embedded. The uterine horns of each mouse were painstakingly paraffin embedded in a vertical position so that cross sections of the uterus could be obtained and measured. These 4µ sections were stained using standard H&E staining techniques and visualized under light microscopy. A randomization grid was utilized to determine measurement locations on the tissue. For each animal, 15 measurements were made of the outer longitudinal layer of the uterine horn, as well as 15 measurements of the inner circular layer, and 15 measurements of the thickness of the two layers combined. Two-way ANOVA was used to determine if any changes seen were statistically significant. At the time of the writing of this abstract, no appreciable differences have been found between the treatment groups, although there will be more data and final statistics completed before the presentation of our findings.

This study comprises the ramifications of a diet high in fat and sugar contents that manifest in the uterus. What a clinical setting deems excessive is unfortunately a societal norm in terms of diet. For this reason, it is of a critical nature to investigate the consequences of diets high in sugar and fat contents for the sake of women’s health. Our hypothesis is that consumption of excessive amounts of fat and sugar will have detrimental effects on the muscle layers of the uterus, the myometrium. Since much of the research concerning uterine tissue behavior involves aspects such as pregnancy and fertility assays, this study does not include such facets. The uterine horns of nine high sugar/high fat diet fed mice were collected and transversely sectioned in order to measure the thickness of each uterine layer. At the time of this submission measurements were still being taken and statistical analyses has not yet been completed.

As scientific discovery and human presence push further into space, it is necessary to investigate the effects of spaceflight on physiological systems. Research into the effects of the space flight environment on the human body is still in its relative infancy. Although initial studies have indicated harmful effects of spaceflight environments on certain body systems, this phenomenon still needs illumination with regards to the female reproductive system. Better understanding of these consequences can change the way society views space travel and colonization of other planets. The spaceflight environment consists of at least two major factors that could confer negative effects on physiology, namely radiation and microgravity. In this experiment, uterine smooth muscle, or the myometrium, was analyzed in 6-month old female C57BL/6 mice that were exposed to 21 days of low dose/low dose rate whole-body radiation with γ-radiation using 57Co plates (0.04 Gy at 0.01 cGy/h) and/or simulated microgravity (via hind limb unloading). Tissue samples were harvested 4 months after the 21-day simulated spaceflight period. Following embedding, sectioning, and hematoxylin and eosin staining (H&E), the tissues were examined, and the average thicknesses of the myometrial layers were measured. Three types of measurements were made 1 – outer longitudinal layer, 2 – inner circular layer, and 3 –total muscle layer thickness (outer and inner combined). Two-way ANOVA statistical tests were used to compare the thicknesses of the myometrial muscle layers between the various treatment groups. A statistical difference was found between the thicknesses in the outer longitudinal layer of smooth muscle between the control animals and the unloaded animals (P: 0.051).

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13514号 / 論医博第2264号 / 新制||医||1061(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 湊谷 謙司, 教授 中島 貴子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The aim of this study was to determine the contractile responses of normal virgin rat uterine smooth muscle to the ⍺₂ adrenergic agonist, xylazine HCl, in the presence or absence of the selective ⍺₂ adrenoceptor blocker, idazoxan HCl. Sections of full thickness uterus measuring 5 x 1 x 1 mm taken from mature, virgin Sprague-Dawley rats were used in isolated tissue baths containing 37°C Krebs-bicarbonate solution, and continually aerated with 95% O₂ and 5% CO₂. Following stabilization of spontaneous contractions, the tissues were exposed to either no idazoxan (control), 10⁻⁵ M idazoxan (low), 10⁻⁴ M idazoxan (medium), or 10⁻³ M idazoxan (high). Five minutes later, xylazine was added to all baths in a cumulative manner at quarter log increments from 1 x 10⁻⁵ through 1 x 10⁻³ M. The % response in peak developed tension and effective concentration resulting in a 50% response (EC₅₀) for the four treatment groups were examined. Results indicated that xylazine alone, at a concentrations greater than 1 x 10⁻⁴ M, caused a significant negative inotropic response. Pre-treatment with idazoxan at a concentration greater than 10⁻⁴ M enhanced the negative inotropic effect of xylazine in a dose-dependent manner. The mechanism of this synergism is unknown but is proposed to be a local anesthetic action due to sodium channel blockade. / Master of Science

Esta investigação experimental teve como finalidade de avaliar os efeitos do ultra-som pulsado de baixa intensidade sobre a regeneração do miométrio e do endométrio de ratas submetidas à uterotomia. Foram utilizadas neste experimento 20 ratas adultas da raça Wistar, submetidas a uma uterotomia mediante incisão longitudinal de 1,5 cm total de uma das paredes do corno uterino. As ratas foram divididas em 2 grupos de 10 animais cada, sendo que um grupo foi submetido à estimulação ultra-sônica por 10 dias durante 15 minutos diários e outro grupo, chamado controle, foi submetido à estimulação fictícia pelo mesmo tempo e período. Seis horas antes do sacrifício, os animais receberam via intramuscular 0,10 mg de colchicina para cada 100 g de peso corporal com a finalidade de bloquear as mitoses em metáfase. A avaliação dos tecidos baseou-se na análise comparativa entre a contagem de células em mitose do endométrio e do miométrio de animais estimulados com o de animais controle. Os achados morfológicos foram analisados estatisticamente e são sugestivos de que o U.S.P. estimula a regeneração do miométrio bem como do endométrio de ratas. / This experimental investigation had the purpose to evaluate the low-intensity pulsed ultrasound effects under the regeneration of the myometrium and the endometrium of the rats that were submitted to uterotomia. It was used, in this experiment, twenty adult rats of the Wistar race, submitted to uterotomia beyond the longitudinal incision of total 1, 5 cm of one of the uterine horn walls. The rats were divided in two groups of ten animals each, the first group went trough fifteen daily minutes ultra-sound stimulation during ten days, and the second group, called control, went through fictional stimulation by the same time and period of the first group. Six hours before the sacrifice, the animals received, by intra muscular, 0, 10 mg of colchicine for each 100 g of body weight, with the intention to block the mitosis in metaphase. The evaluation of the tissue was based on the comparative analysis between the mitosis cell count of the stimulated animals\' endometric tissue and the control animals\' one. The morphologic findings were statistically analyzed.

In the human uterus prostaglandins (PG) PGE2, PGD2, PGI2, PGF2α and Thromboxane A2 (TXA2), also termed prostanoids, are synthesised and deactivated to 15-keto PGE2, J2 metabolites, 6-keto-PGF1α, 15-keto PGF2α and TXB2 respectively. However, not all metabolites have been analysed simultaneously within the same tissue. The primary objective of this thesis was to determine full uterine prostanoid profiles in human non-pregnancy, pregnancy and parturition, to better understand these processes and find suitable tocolytic targets. In addition, ten cytokines in human cervico-vaginal fluid (CVF) were measured according to interval to labour to test their suitability as labour onset predictors, with a view to developing a test to determine women at risk of preterm labour. Prostanoid analysis was carried out in endometrium (n=9) and myometrium (n=15- 16) donated by non-pregnant women and lower segment myometrium obtained from pregnant women (before (n=14) and after labour onset (n=7)) by liquid chromatography coupled with electrospray ionisation mass spectrometry (LC/ESIMS/ MS). Cytokines produced by CVF collected from pregnant donors (20-41 weeks gestation, n=2-10) were investigated using Enzyme-Linked Immunosorbent Assay (ELISA) or Luminex®. Human endometrium produced greater concentrations of TXB2, PGE2 and PGF2α than myometrium in vitro (p<0.05). Fifteen prostanoids were detected in human myometrium. Production of 6-keto-PGF1α, PGE1 and PGF1α increased whilst 15- keto PGE2 and PGJ2 decreased at term pregnancy (37-41 weeks gestation) versus non-pregnancy (p<0.05). Myometrium from parturient donors synthesised TXB2 and PGE2 more abundantly than the non-labouring equivalent. Cytokine concentration was greatest in CVF sampled the week before labour, in particular Interleukin-6 (IL-6), Macrophage Inflammatory Protein-1α (MIP-1α) and Monocyte Chemotactic Protein-1 (MCP-1) (p<0.05). Endometrial TXB2, PGE2 and PGF2α could aid in proliferation of glandular epithelium prior to ovulation. Prostacyclin may facilitate prolongation of pregnancy to term and thromboxane could contribute to uterine stimulation during labour. Cervical dilation may be influenced by PGE2 in lower segment myometrium. MCP- 1, MIP-1α and IL-6 could mark a short interval to labour onset.

Neutrophils infiltrate myometrium and decidual tissue prior to parturition. Activated neutrophils release reactive oxygen species (ROS) and tumor necrosis factor α (TNFα), which might increase expression of pro-labor genes such as matrix metalloproteinase-1 (MMP-1), progesterone receptor (PR) A/B ratio, and cause demethylation of DNA. These changes might cause labor. Decidual tissue was obtained from consented, healthy women at term (37+ weeks of gestation) not in labor (no contractions, without cervical effacement), term labor and preterm labor (under 37 weeks of pregnancy). Decidual and myometrial cells in culture were treated with (1) ROS, (2) TNFα, or (3) 5-aza-2’-deoxycytidine. Total RNA was extracted, converted to cDNA and evaluated by qRT-PCR for MMP-1, PR-A+B and PR-B. TNFα increased MMP-1 by 17 fold in decidual cells and more than 12 fold in myometrial cells. PR-A/B was increased by 5.6 fold in decidua. ROS up-regulated MMP-1 by 6 fold and elevated the PR-A/B ratio by 4.5 fold in decidual tissue. DNA demethylation increased MMP-1 by about 4 and 11 fold in decidual and myometrium, respectively. The PR-A/B ratio was increased by 4 fold in decidua and the PR-B was decreased by 40% in the myometrium due to DNA demethylation. Decidual tissue in preterm labor showed a 7-fold increase in MMP-1 over term laboring and over a 15-fold increase over term not in labor tissue. In conclusion, MMP-1 expression and PR-A/B ratio was increased by neutrophil products possibly through a mechanism of DNA methylation in decidua and myometrium. Preterm decidua showed a dramatic increase in MMP-1 over normal labor tissue. TNFα and ROS increased expression of MMP-1 to possibly initiate parturition. These data might help explain mechanisms responsible for preterm labor unrelated to infection or premature rupture of membranes.

Da die Kontrolle der Kontraktion am Myometrium in der klinischen Praxis eine bedeutende Rolle spielt, sind Kenntnisse über die Physiologie zur Blockierung der Wehentätigkeit und Vermeidung von Frühgeburtlichkeit unverzichtbar. Der Einfuß des sympathischen Nervensystems mit Alpha und Beta-Rezeptoren ist gut untersucht, weshalb wir in der vorliegenden Arbeit eine Untersuchung zur Verteilung der muscarinischen Rezeptoren M1-M5 im schwangeren und nicht schwangeren Myometrium durchführten. Diese G-Protein gesteuerten parasympathischen Rezeptoren sind beispielsweise an Kontraktionsvorgängen in Blasen-, Magen-Darm- und Bronchialmuskulatur beteiligt und könnten so auch im Myometrium eine Rolle spielen. Mittels RT-PCR wurden 21 Myometriumbiopsien analysiert, wovon 11 Myometriumproben von Patientinnen in der 40. Schwangerschaftswoche, sowie jeweils 3 Proben von Patientinnen in der 32. Schwangerschaftswoche und von Patientinnen in der 25. Schwangerschaftswoche stammten, desweiteren eine Probe einer in der 10. Schwangerschaftswoche durch Hysterektomie abgebrochenen Schwangerschaft. Die drei letzten Proben stammten von nicht schwangeren Patientinnen nach vaginaler Hysterektomie. Dabei konnte gezeigt werden, dass sowohl zu verschiedenen Zeitpunkten in der Schwangerschaft, als auch am nicht schwangeren Myometrium jeweils m-RNA für M2, M3 und M5 muscarinische Rezeptoren existiert. Die durch die hier angewendeten Verfahren zur Gewebepräparation, Gewinnung von RNA und Anwendung der RT-PCR vorliegenden Ergebnisse sollten auf Proteinebene bestätigt werden, die Durchführung funktioneller Studien wäre wünschenswert. / Contraction in human myometrium plays an important role concerning preterm labour. As the influence of sympathetic nervous system with alpha- and beta-receptors is well known, we were searching for muscarinic receptors M1-M5 in pregnant and non pregnant human myometrium. These G-protein coupled parasympathetic receptors induce contraction in tissues like bladder, gastrointestinal and bronchial smooth muscle. Via RT-PCR we analysed 21 tissue biopsies, 11 from 40th week of pregnancy, three probes from 32th and 25th week and one from 11th week. Last 3 tissues were from non pregnant myometrium after vaginal hysterectomy. We were able to show that mRNA for the muscarinic receptors M2, M3 and M5 exists at different points in pregnancy and in the non pregnant myometrium. We hope that your methods for tissue preparation, mRNA isolation and processing RT-PCR and the results will help to prove the existence of muscarinic receptors on protein level and in functional studies.

Foram utilizados 15 úteros de macacos Rhesus (Macaca mulatta), sendo 5 fêmeas nulíparas, 5 primíparas e 5 pluríparas. Todas as fêmeas eram maduras sexualmente, tinham de 38 a 87 meses de idade e pesavam entre 4,9 a 7,39 Kg. Em todos os animais foram feitos o estudo topográfico, biometria dos órgãos, microscopia de luz e quantificação de colágeno no miométrio. O útero se situa na pelve, sobre a vagina, entre a bexiga urinária e o intestino reto. O útero é do tipo simples, caracterizado por um fundo globóide, localizado sobre a junção com as tubas uterinas, oco, alongado e com contorno piriforme. Pode ser dividido macroscopicamente em quatro regiões: corpo, fundo, istmo e cérvix uterina. Não foram observadas diferenças macroscópicas e de topografia entre as fêmeas nulíparas, primíparas e pluríparas. O comprimento total do útero nas fêmeas nulíparas tiveram média de 4,12 ± 0,2 cm, nas primíparas 4,32 ± 0,26 cm e nas pluríparas 5,14 ± 0,71 cm. Comparando as mensurações entre as três categorias, observou-se que as fêmeas pluríparas possuem as maiores medidas. O endométro é formado por um epitélio prismático simples e uma lâmina própria que contém as glândulas endometriais, que também possui epitélio prismático simples. Em todos os animais estudados, as glândulas endometriais eram tubulares simples e retas. Em relação à distribuição do colágeno no miométrio, o método de Picrossirius Red, permitiu observar uma ampla distribuição do colágeno por todo o compartimento. A cérvix é constituída por um epitélio prismático simples, possui poucas fibras musculares lisas e consiste principalmente em tecido conjuntivo denso. A porcentagem de colágeno encontrado no miométrio das fêmeas nulíparas teve uma média de 26,32 ± 0,82, nas primíparas 29,07 ± 1,01 e nas pluríparas 38,93 ± 1,07, pelo método de coloração de Picrosirius Red. / Fifteen uterus of rhesus macaques (Macaca mulatta) were used, collected from 5 nulliparous, 5 primaparous and 5 pluriparous individuals. All females were sexually mature, they were 38-87 months of age and weighed between 4,9 to 7,39 Kg. All animals were submitted to study topography, biometry of the organs, light microscopy and quantification of collagen in the myometrium. The uterus is located in the pelvis on the vagina between the bladder and rectum. The uterus has a simple type, characterized by a globular background, located on the junction of the fallopian tubes, hollow, elongated and pear-shaped contour. Grossly it can be divided into four parts: body, bottom, isthmus and cervix. No differences were observed macroscopic and topography between nulliparous, rimiparous and pluriparous. The total length of the uterus in nulliparous females had a mean of 4,12 ± 0,2 cm in primiparous 4,32 ± 0,26 cm and 5,14 ± 0,71 cm in pluriparous. Comparing the measurements among the three categories, it was observed that the sows have greater measures. The endometrial epithelium is formed by a simple prismatic and a lamina propria containing the endometrial glands, wich also has a simple prismatic epithelium. In all studied animals the endometrial glands were simple and straight tubular. Regarding the distribution of collagen around the bay. The cervix is composed of a simple prismatic epithelium, has a few smooth muscle fibers and consists mainly of dense connective tissue. The percentage of collagen found in the myometrium of the nulliparous females had an average of 26, 32 ± 0, 82, 29, 07 ± 1, 01 in primiparous and pluriparous 38,93 ± 1,07, through the staining method of Picrosirius Red.