Prostaglandins are integral mediators in reproductive processes but their exact role in uterine function is still not clear. In addition, ethical restraints have limited the availability of human tissue to investigate uterine prostanoid receptor populations. The aim of this thesis was to characterise the prostanoid receptors on the human and rat myometrium in order to evaluate the potential of the rat as an animal model of human uterine function and disease. For functional analysis of myometrial prostanoid receptors the immersion technique was utilised. LC-ESI-MS/MS was also used to measure the ex vivo myometrial release of prostanoid metabolites. The results show that both the rat and human uterus displays cyclical changes in uterine motility, with myogenicity greatest in the follicular and oestrus stages. The data also indicate that whilst the human uterus is responsive to EP3, EP2, TP, FP and IP receptor agonists, a functional population of only EP3, EP2 and FP receptors is present on the rat uterus, although the TP receptor appears to be upregulated at gestation and post-partum. The results also show that myometrial prostanoid release in the human uterus is cyclically regulated, with the greatest amount of prostaglandins being released during the late follicular stage. In conclusion, although similarities do exist with regard to the ovarian regulation of uterine motility in both the rat and human uterus, the differences in the apparent functional prostaglandin receptor populations between the two species suggest further work is required before the rat can be used as a model of human uterine function.

Studies indicate an association between both term labour (TL) and preterm labour (PTL) and the presence of uterine inflammatory cytokines and leukocyte infiltration. We hypothesized that peripheral leukocytes are recruited to uterine tissues by locally produced cytokines where they contribute to the initiation of TL and PTL. The cytokine expression profile was analyzed using an in vivo mouse model of gestation and two PTL models (Lipopolysaccharide- and RU486-induced). Myometrial neutrophil and macrophage infiltration was also studied. My results demonstrate that macrophage infiltration precedes neutrophil infiltration during late gestation and that both leukocyte subsets increase during PTL and further increase post partum. These changes in leukocyte numbers are associated with significant changes in multiple myometrial cytokines with TL and RU486-induced PTL showing similar cytokine profiles. Importantly, post partum involution, the process by which the uterus completes the reproductive cycle and returns to its pre-pregnant state, appears similar in all three models.

Studies indicate an association between both term labour (TL) and preterm labour (PTL) and the presence of uterine inflammatory cytokines and leukocyte infiltration. We hypothesized that peripheral leukocytes are recruited to uterine tissues by locally produced cytokines where they contribute to the initiation of TL and PTL. The cytokine expression profile was analyzed using an in vivo mouse model of gestation and two PTL models (Lipopolysaccharide- and RU486-induced). Myometrial neutrophil and macrophage infiltration was also studied. My results demonstrate that macrophage infiltration precedes neutrophil infiltration during late gestation and that both leukocyte subsets increase during PTL and further increase post partum. These changes in leukocyte numbers are associated with significant changes in multiple myometrial cytokines with TL and RU486-induced PTL showing similar cytokine profiles. Importantly, post partum involution, the process by which the uterus completes the reproductive cycle and returns to its pre-pregnant state, appears similar in all three models.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 4 uppsatser.

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.

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.

Prostaglandins are integral mediators in reproductive processes but their exact role in uterine function is still not clear. In addition, ethical restraints have limited the availability of human tissue to investigate uterine prostanoid receptor populations. The aim of this thesis was to characterise the prostanoid receptors on the human and rat myometrium in order to evaluate the potential of the rat as an animal model of human uterine function and disease. For functional analysis of myometrial prostanoid receptors the immersion technique was utilised. LC-ESI-MS/MS was also used to measure the ex vivo myometrial release of prostanoid metabolites. The results show that both the rat and human uterus displays cyclical changes in uterine motility, with myogenicity greatest in the follicular and oestrus stages. The data also indicate that whilst the human uterus is responsive to EP3, EP2, TP, FP and IP receptor agonists, a functional population of only EP3, EP2 and FP receptors is present on the rat uterus, although the TP receptor appears to be upregulated at gestation and post-partum. The results also show that myometrial prostanoid release in the human uterus is cyclically regulated, with the greatest amount of prostaglandins being released during the late follicular stage. In conclusion, although similarities do exist with regard to the ovarian regulation of uterine motility in both the rat and human uterus, the differences in the apparent functional prostaglandin receptor populations between the two species suggest further work is required before the rat can be used as a model of human uterine function. / Allergan Inc

In the human uterus prostaglandins (PG) PGE2, PGD2, PGI2, PGF2¿ and Thromboxane A2 (TXA2), also termed prostanoids, are synthesised and deactivated to 15-keto PGE2, J2 metabolites, 6-keto-PGF1¿, 15-keto PGF2¿ 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 PGF2¿ than myometrium in vitro (p<0.05). Fifteen prostanoids were detected in human myometrium. Production of 6-keto-PGF1¿, PGE1 and PGF1¿ 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-1¿ (MIP-1¿) and Monocyte Chemotactic Protein-1 (MCP-1) (p<0.05). Endometrial TXB2, PGE2 and PGF2¿ 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-1¿ and IL-6 could mark a short interval to labour onset. / Allergan Inc.

Lipids havepreviously been considered primarily as building blocks of the cell membrane, but are now also recognized as important cell signaling molecules. Lysophosphatidic acid (LPA) is a glycerophospholipid consisting of a phosphate head group, a linker region, and a lipophilic tail. LPA has earlier been shown to exert a diversity of cellular effects such as aggregation, apoptosis, contraction, migration, and proliferation. The effects of LPA are elicited by activation of its cognate G protein-coupled receptors LPA1, LPA2, and LPA3. In the present study we have used cultures of human smooth muscle cells (SMCs) and erythroleukemia cells (HEL), and isolated human platelets to characterize physiological effects of LPA compared with adrenaline and noradrenaline as well as structure-activity relationships of LPA. SMCs were isolated from biopsies of human myometrium obtained at cesarean sections. We show that cultured myometrial SMCs express multiple LPA and α2-adrenergic receptor subtypes. Treatment of SMCs with LPA and noradrenaline resulted in increases in proliferation. However, LPA elicits a much more pronounced stimulatory effect than noradrenaline. The ability to increase calcium might be one explanation why LPA is more effective. Further studies indicated that several pathways mediate the growth stimulatory effect of LPA where transactivation of epidermal growth factor receptors through matrix metalloproteinases as well as calcium/calmodulin-dependent protein kinases appears to be important. LPA enantiomers and LPA analogues were synthesized and characterized due to their capacity to increase calcium in HEL cells. Our study is the first to show that both natural (R) and unnatural (S) LPA enantiomers are capable of stimulating cells, suggesting LPA receptors are not stereoselective. Moreover, we have synthesized a LPA analogue with higher maximal effect than LPA by reducing the hydrocarbon chain length. In platelets we demonstrated that LPA is a weak calciumelevating compound which failed to stimulate aggregation. However, in combination with adrenaline, another weak platelet agonist, a complete aggregatory response was obtained in blood from some healthy individuals. These results are important since platelet activation is a key step in distinguishing normal from pathological hemostasis. Since LPA is present at high concentrations in atherosclerotic lesions, the synergistic effect of LPA and adrenaline might be a new risk factor for arterial thrombosis. / On the day of the public defence the status of the article IV was: Submitted for publication.

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