京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22117号 / 医博第4530号 / 新制||医||1039(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 武藤 学, 教授 松原 和夫, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Einleitung: Reproduktionsstörungen sind die wichtigste Abgangsursache von Sauen. Nicht selten ist der Uterus erkrankt. Einige der Sauen mit Uteropathien sind klinisch auffällig, andere hingegen auch mittels konventioneller B-Mode-Ultrasonographie nicht zu erkennen. Bei solchen Sauen erscheint es sinnvoll, zusätzlich zum B-Mode dopplersonographisch zu untersuchen, um anhand der Uterusdurchblutung ergänzende diagnostische Informationen zu erhalten, wie es schon vormals bei Stuten und Kühen erfolgte. Zur Beurteilung pathologischer Perfusionsverhältnisse des Uterus beim Schwein bedarf es jedoch der Kenntnis des physiologischen Zustandes, die bisher fehlt. Ziele der Untersuchungen: Ziel dieser Studie war es, die Durchblutung des Uterus von Jungsauen mittels transabdominaler Dopplersonographie während des Sexualzyklus zu charakterisieren. Dabei sollte auch getestet werden, ob sich diese Methode intra vitam, d.h. bei der lebenden Sau eignet. Tiere, Material und Methoden: Insgesamt wurden 15 zyklussynchronisierte, gynäkologisch gesunde Jungsauen einbezogen und in dem darauffolgenden spontanen Sexualzyklus zwischen Proöstrus und Diöstrus in einem mobilen Kastenstand dopplersonographisch untersucht. Es fanden Color, Power und Pulse Wave Doppler Anwendung, um die uterine Perfusion anhand der Gefäßversorgung innerhalb und zwischen den Uterusschlingen zu charakterisieren. Durch Aufstallung der Jungsauen in einem mobilen eigens angefertigten Stand wurden Tierbewegungen soweit reduziert, dass Untersuchungen mit Color und Power, nicht aber Pulse Wave Doppler möglich waren. Zudem gelang es mittels Pulse Wave Doppler nicht, das Messtor (gate) aufgrund der geschlängelten Gefäßanatomie konstant auf uterinen Gefäßen zu platzieren. Im Vergleich zum Power Doppler überzeugte der Color Doppler durch geringere Empfindlichkeit für Bewegungsartefakte und höhere Bildaufbauraten. Er wurde deshalb zur Auswertung aufgezeichneter Einzelbilder und Videos genutzt. Dabei kam die Software PixelFlux® zur Anwendung, mit deren Hilfe eine in ihrer Größe standardisierte „Region of Interest“ (ROI) hinsichtlich Anzahl und Farbton der vorhandenen Pixel ausgewertet wurde, um Blutflussgeschwindigkeit, perfundierte Fläche, Blutflussintensität, sowie Resistenz- und Pulsatilitätsindex zu berechnen. Die statistische Auswertung der Ergebnisse erfolgte in SPSS. Da die Zyklusphasen der untersuchten Jungsauen in ihrer Länge variierten, wurden diese normalisiert und an der Ovulation als Tag 2 ausgerichtet. Die danach für jeden Zyklustag vorhandenen Werte/Parameter wurden entsprechend der Zyklusphasen gemittelt und nach Bonferroni-Korrektur mit dem Friedman- und Wilcoxontest analysiert. Zusammenhänge zwischen den Parametern wurden mit der Korrelationsanalyse nach Spearman oder Pearson untersucht. Ergebnisse: Uterine Durchblutung konnte jederzeit zwischen und gelegentlich auch innerhalb der Uterusschlingen dargestellt werden. Alle erhobenen Blutflussparameter zeigten im Verlauf des Zyklus einen charakteristischen Verlauf. Blutflussgeschwindigkeit, durchblutete Fläche und Blutflussintensität waren im Proöstrus hoch, sanken im Östrus, um im Met- und den meisten Teilen des Diöstrus niedrig zu bleiben und danach wieder anzusteigen. Alle drei Parameter korrelierten mittelstark bis stark positiv miteinander (r: 0,63-0,95; p<0,05). Nahezu reziprok verhielten sich Resistenz- und Pulsatilitätsindex mit den vorherigen Parametern. Sie korrelierten mit der Blutflussgeschwindigkeit, der durchbluteten Fläche und der Blutflussintensität mittelstark bis stark negativ (r: -0,52 – -0,88; p<0,05), miteinander jedoch mittelstark bis stark positiv (r: 0,6-0,85; p< 0,05). Schlussfolgerungen: In dieser Arbeit konnte demonstriert werden, dass die Dopplersonographie zur Charakterisierung der Durchblutung des Uterus bei im Stand immobilisierten Jungsauen prinzipiell möglich ist. Während Pulse Wave und Power Doppler nicht oder nur begrenzt anwendbar waren, konnten mittels Color Doppler charakteristische Blutflussveränderungen während des Sexualzyklus festgestellt werden. Die Ergebnisse dieser Arbeit ermutigen zu weiterführenden Untersuchungen unter anderem im Rahmen der Abklärung von Ursachen uterin-bedingter Sub- bzw. Infertilität.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 4 2.1 Sexualzyklus der Sau 4 2.2 Gefäßversorgung des Uterus 6 2.3 Dopplersonographie 8 2.3.1 Physikalische Grundlagen 8 2.3.2 Dopplermodi 9 2.3.3 Insonationswinkel 11 2.3.4 Artefakte 12 2.3.5 Auswertung von Doppleruntersuchungen 13 2.3.5.1 Qualitative Analyse 13 2.3.5.2 Semiquantitative Analyse 14 2.3.5.3 Quantitative Analyse 15 2.3.5.4 Pixelanalytische Methode 16 2.3.5.4.1 Vergleich pixelanalytischer Programme 17 2.3.6 Dopplersonographie am ingraviden Uterus in der Humanmedizin 18 2.3.7 Dopplersonographie am ingraviden Uterus in der Veterinärmedizin 19 3 Publikation 21 4 Diskussion 22 4.1 Immobilisation der Jungsau 23 4.2 Insonationswinkel 24 4.3 Uterusgefäße. 25 4.4 Analysen von Einzelbildern bzw. Videos. 27 5 Zusammenfassung 29 6 Summary 31 7 Literaturverzeichnis Anhang Danksagung

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Introduction: Uterine fibroids are the most common benign tumours of female genital tract with the peak incidence in the 4th and 5th decennium. The aetiology of uterine fibroids still remains poorly understood. Genetic factors play undisputed role in the onset of uterine fibroids. Up to date numerous gene mutations were identified in certain percentage of patients with uterine fibroids. One of the candidate genes is Fumarate hydratase gene (FH). Heterozygous germiline mutations of FH cause two hereditary syndromes: Multiple smooth muscle tumours of the skin and uterus (MCUL1)/ Hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC) characterised by leiomyomata of the skin, early onset uterine fibroids between 20-30 years of age and renal papillary carcinoma. The aim of our thesis was to identify the frequency of FH mutations in patients with early onset sporadic uterine fibroids. Methods: Patients with the diagnosis of uterine fibroids up to the age of 30 years were enrolled in the study. Control group consisted of patients with absence of uterine fibroids. Activities of Fumarate hydratase and control protein Citrate synthase were measured in lymphocytes and compared to the results obtained from the healthy controls. Mutation analysis of FH gene was performed. Activity of Fumarate...

Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryodecidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5′‐nucleotidase (5′‐NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno‐derivatized and quantitated by high‐performance liquid chromatography‐fluorescence detection (0.03 pmol/mg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmol/mg protein) and day 5 (0.59 nmol/mg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmol/mg protein) and day 7 (0.10 nmol/mg protein). The periimplantation adenosine surge coincided with uterine expression of 5′‐NT, an enzyme which catalyzes the irreversible dephos‐phorylation of 5′‐AMP to adenosine. 5′‐NT expression was shown by Northern blot analysis to peak in the embryo‐decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5′‐NT activity was localized to the primary decidual zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5′‐NT appeared on giant trophoblast (days 7–13) and the metrial gland (days 11–13). Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S‐adenosylhomocysteine hydrolase) were not detected in the embryo‐decidual unit suggesting that the net flux of utero‐placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation. The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6–11), secondary giant cells (days 7–13), and spongiotrophoblasts (days 8–13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5′‐NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities. Purine nucleoside phosphorylase and xanthine oxidase, which are two catabolic enzymes acting subsequent to 5′‐NT and ADA in the sequential degradation of AMP to xanthine, remained low and constant in the tissues examined suggesting that the catabolic pathway is geared toward regulation of adenosine levels. These results suggest the establishment of an adenosine gradient across the developing antimesometrium. It is proposed that the source of adenosine is AMP released during uterine cell death, and that adenosine, in turn, serves as a regulatory signal to coordinate early postimplantation morphogenetic events with the progression of cell death at the uterine‐embryo interface.

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In 2018, 1 in every 10 infants born in the United States was born preterm. The majority of neonatal deaths and nearly a third of infant deaths that occur are linked to preterm birth. Preterm birth is initiated when the quiescent state of the uterus ends prematurely, leading to contractions and parturition beginning as early as 32 weeks, though the origins are not well understood. Tocolytics are pharmaceuticals utilized to postpone preterm labor, but currently only manage to prolong pregnancy for up to 48 hours and have not proven effective in completely preventing preterm delivery. To enable research and discovery of therapeutics with potential to better address preterm birth, the capability to study isolated cell processes of pregnant uterine tissue in vitro is needed. Our development of an in vitro model of the myometrium utilizing uterine myocytes - uterine smooth muscle cells (uSMCs) responsible for contractions - provides a platform to examine the cellular mechanisms of late-stage pregnancy potentially involved in preterm birth. In this thesis, we discuss the optimized culture of uterine SMCs on a flexible polydimethylsiloxane (PDMS) substrate functionalized using a cationic solution, Poly-L-lysine (PLL), followed by extracellular matrix (ECM) protein coating. Using the model we developed, we then exposed this elastic substrate with uterine SMCs to different strain rates in order to investigate the impact of mechanical strain parameters on uterine SMC hypertrophy in the uterus during late-stage pregnancy. It was found that PLL and ECM protein coatings significantly impact cell morphology and density in unstrained substrates. It was also observed that when exposed to strain conditions, strain significantly increased hypertrophic morphological traits in select conditions. These results indicate that both surface and mechanical properties of in vitro systems impact uterine SMC phenotype, offering further understanding of cellular pathways involved in the uterus under mechanical load. / 2024-05-31T00:00:00Z

Placenta accreta spectrum disorder (PASD) is the adherence of the placenta caused by an abnormal trophoblast invasion into the myometrium. It is classified as placenta accreta, placenta increta, and placenta percreta depending on the extent of the invasion. Placenta accreta, defined as the superficial invasion of the placenta to the myometrium, accounts for 75% of PASD. Placenta increta is characterized by chorionic villi invasion deep into the myometrium. Placenta percreta involves placental invasion through the uterus and serosa and into the peritoneal cavity or surrounding viscera. Maternal morbidity and mortality can occur secondary to hemorrhage, disseminated intravascular coagulation, risks associated with blood transfusion, and pelvic and abdominal viscera injury. The standard of care in a known diagnosis of PASD is a cesarean delivery followed by hysterectomy with the placenta in situ. We report a case in which the diagnosis of focal PASD was not known antenatally but suspected after vaginal delivery. The patient subsequently underwent conservative management with uterine preservation and did not require laparotomy.

Although prostaglandins (PG) and oxytocin are crucial mediators of uterine contractility, their receptor-mediated effects during the menstrual cycle, pregnancy and labour are not fully understood. The aim of this thesis was to elucidate the functional expression of EP, FP, TP and oxytocin receptors in isolated human myometrium relative to myocyte mRNA and signal transduction pathways. Myometrial samples were obtained from consenting non-pregnant and pregnant donors. Functional techniques were used to determine isometric muscle contractions. Primary uterine myocytes and fibroblasts were cultured at term to identify stimulated changes in calcium (Ca2+), cyclic adenosine monophosphate (cAMP) and mRNA. Myometrial strips exhibited spontaneous contractions, which were most active midcycle under oestrogenic conditions. At this time intrinsic contractility and responsiveness to uterotonins decreased towards the fundus. PGE2 produced bellshaped responses with predominant utero-relaxant effects mediated via the EP2 subtype. Although activity was partially restored by PGE2 through EP3/1 receptors, tissue excitation was more pronounced at FP, TP and oxytocin receptors. Despite high FP mRNA expression, the lower segment uterus was particularly responsive to U46619 and oxytocin at term pregnancy. Even so, Ca2+ mobilisation by oxytocin was greater via principal release from intracellular stores. Incubations with atosiban, progesterone and a rho-kinase inhibitor reduced oxytocin-stimulated Ca2+ transients. EP2 also attenuated oxytocic effects but this appeared to be mediated through cAMP rather than Ca2+ signalling pathways. With advancing labour, intrinsic myogenic activity declined in parallel with oxytocin desensitisation. However, TP-induced contractions were continued in the lower parturient uterus. These findings demonstrate that PG and oxytocin receptor expression are regulated in a hormone-dependent temporal and spatial manner. EP2-mediated cAMP formation appears to promote uterine quiescence, whilst TP receptors may control muscle tonus during parturition. These receptors and their messenger systems represent effective tocolytic targets for uterine hypercontractile disorders, such as dysmenorrhoea and preterm labour. / Allergan Inc.