Die Implantation einer Blastozyste im Uterus stellt für den maternalen Organismus eine große Herausforderung dar. Neben morphologischen Veränderungen des Endometriums sind Anpassungen auch im Immun- und Gefäßsystem notwendig. Kenntnisse über die verantwortlichen Faktoren für diese Prozesse sind bei Menschen und anderen Primaten noch immer unvollständig. Dabei sind Implantationsstörungen oftmals Ursache von Fruchtbarkeitsproblemen (SHARKEY und SMITH 2003). Besonders im Bereich der assistierten Reproduktion stellen implantationsbedingte Probleme eine Hauptursache für geringe Erfolgsraten dar (APLIN und KIMBER 2004). Das Ziel der Erforschung von Mechanismen im Zusammenhang mit der Implantation ist es, ein besseres Verständnis für die physiologischen und auch pathologischen Vorgänge zu erlangen. In dessen Folge die Erkenntnisse für die Behandlung von Implantationsstörungen genutzt werden könnten. Anhand des in der reproduktionsbiologischen Forschung etablierten Primatenmodells des Weißbüschelaffen (Callithrix jacchus) wurde in der vorliegenden Arbeit die Expression ausgewählter Faktoren in verschiedenen Zyklusphasen mit dem Schwerpunkt der Implantationsphase untersucht. Dafür wurden zeitlich genau terminierte Uterusproben von konzeptiven und nicht-konzeptiven Tieren entnommen und molekularbiologisch, histologisch und immunhistochemisch analysiert. Erstmals konnte im Uterus von Weißbüschelaffen die zeitliche und räumliche Regulierung von Choriongonadotropin (CG), Östradiolrezeptor-α (ERα), Östradiolrezeptor-ß (ERß), Progesteronrezeptor (PR), Aromatase (ARO), 17β-Hydroxysterois-Dehydrogenase Typ 7 (17β-HSD7) sowie Relaxin (RLN) und Relaxinrezeptor (RXFP1) auf Gen- und Proteinebene in verschiedenen Zyklusphasen in der vorliegenden Vollständigkeit dargestellt werden. Alle in dieser Arbeit untersuchten Faktoren zeigten eine rezeptorspezifische Veränderung der Expressionsmuster, auf räumlicher und zeitlicher Ebene im Verlauf des Implantationsprozesses. Es konnte dargestellt werden, dass die feto-maternale Kommunikation bereits vor dem direkten Kontakt der Blastozyste mit dem Endometrium beginnt. So wurde bei konzeptiven Tieren eine verstärkte Expression von CG, ERα, PR, 17β-HSD7 und RXFP1 in der Implantationsphase nachgewiesen. Diese Faktoren sind geeignete Kandidaten für weiterführende Untersuchungen und Evaluierung von Markern für die Präimplantationsphase und rezeptiven Uteri von Weißbüschelaffen. Die ebenfalls untersuchten Faktoren ERβ, ARO und RLN zeigten Expressionsmuster, welche erst nach der Implantation bei konzeptiven Tieren Unterschiede aufwiesen. Aus diesem Grund sind diese Faktoren nicht als frühe Marker der Implantationsphase geeignet, gleichwohl sie wichtige Funktionen in der Postimplantationsphase übernehmen. Die Ergebnisse dieser Arbeit verdeutlichen, dass nicht alleine ein einzelner Faktor eine herausragende Bedeutung im Implantationsprozess hat, sondern vielmehr ein äußerst komplexes Netzwerk von Signalen nötig ist, um eine erfolgreiche Gravidität zu gewährleisten. Die Aufgabe der Steroidrezeptoren ERα und PR liegt in der grundlegenden Bereitstellung eines rezeptiven Uterus während des Implantationszeitfensters. Gleichzeitig erfolgt eine sehr genau koordinierte Regulation dieser Steroidrezeptoren über die lokal produzierten Faktoren CG, ARO, 17β-HSD7, RLN und der Rezeptoren RXFP1 und ERβ. Neben den morphologischen Veränderungen des Endometriums, sind einzelne Faktoren auch bei der Angiogenese und Entwicklung der maternalen Immuntoleranz beteiligt. Es kann demnach davon ausgegangen werden, dass die präzise Regulation dieser Faktoren eine essentielle Voraussetzung für den Erfolg der Implantation und Gravidität ist.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

The mechanisms causing luteolysis of the primate corpus luteum are unknown. There is an increase in expression of liver x receptor (LXR) target genes and reduced low density lipoprotein receptor (LDLR) during spontaneous luteolysis in primates. The LXRs belong to the nuclear receptor superfamily and increase cholesterol efflux by inducing transcription of their target genes. Uptake of cholesterol into primate luteal cells occurs primarily via LDL, and LDLR transcription is regulated by sterol regulatory element binding transcription factor 2 (SREBF2). Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) maintain luteal function by binding to the LH/CG receptor (LHCGR), which stimulates progesterone (P4) synthesis via protein kinase A (PKA). It has also been previously reported that there is an increase in 27-hydroxycholesterol (27OH) concentrations during spontaneous luteolysis in primates. Pregnenolone and P4 inhibit the enzyme activity of CYP27A1 (cytochrome p450, family 27, subfamily A, polypeptide 1), which converts cholesterol into 27OH, an oxysterol that is a natural LXR agonist and SREBF2 inhibitor. Therefore, the overall hypothesis is that LXR-induced cholesterol efflux and reduced LDL uptake via inhibition of SREBF2 activity mediate luteolysis of the human CL. The objective of study 1 is to determine the effects of LXR activation and SREBF2 inhibition on P4 production, cholesterol metabolism and gene expression; and how hCG signaling via PKA regulates these effects in human luteinized granulosa cells. Basal and hCG-stimulated P4 secretion were significantly decreased by the combined actions of the LXR agonist T0901317 (T09) and the SREBF2 inhibitor fatostatin, which was associated with alterations in cholesterol metabolism leading to reduced intracellular cholesterol storage. Expression of LXR target genes in the presence of T09 was significantly reduced by hCG, while hCG significantly increased LDLR expression. These effects of hCG were reversed by a specific PKA inhibitor. Chronic hCG exposure had similar effects on LXR target gene and LDLR expression without an exogenous LXR agonist. The objective of study 2 is to determine the effects of 27OH on P4 production and cholesterol metabolism; and to determine if inhibiting the conversion of cholesterol into pregnenolone increases LXR and decreases SREBF2 target gene expression via CYP27A1 in human luteinized granulosa cells. During luteolysis in primates and sheep, CYP27A1 expression significantly increased. 27OH significantly decreased hCG-stimulated P4 secretion and enhanced cholesterol efflux. Aminoglutethimide, which inhibits the conversion of cholesterol to pregnenolone, significantly increased ABCA1 and decreased LDLR. Knock-down of CYP27A1 resulted in a significant increase in P4 secretion, but did not prevent aminoglutethimide-induced effects on ABCA1 and LDLR. Knock-down of steroidogenic acute regulatory protein (STAR), which controls cholesterol transport into the mitochondria where CYP27A1 resides, significantly decreased LDLR transcription. Collectively, the data from study 1 support the hypothesis that LXR-induced cholesterol efflux and reduced LDL uptake via inhibition of SREBF2 activity mediates luteolysis in primates, which is reversed by hCG. Data from study 2 indicates that 27OH produced via CYP27A1 may contribute to reductions in P4 synthesis during luteolysis, partially by serving as a dual LXR agonist and SREBF2 inhibitor, although other oxysterols are also likely involved.

No description available.

INTRODUÇÃO: A definição de fatores preditivos de lesão morfológica e funcional da tuba uterina poderia colaborar na escolha do tratamento de pacientes com gestação ectópica. O objetivo deste estudo foi verificar o comportamento do tecido trofoblástico em relação à sua penetração na parede da tuba uterina em gestações ampulares, relacionar a profundidade dessa penetração com idade gestacional, concentração de beta-hCG, tipo de imagem ultra-sonográfica e dimensão da massa ectópica à ultra-sonografia e avaliar a possibilidade de predição dessa invasão pelos parâmetros estudados. MÉTODOS: realizou-se estudo retrospectivo, entre 1° de janeiro de 2000 a 31 de março de 2004, com 105 pacientes com gestação tubária ampular submetidas à salpingectomia. As imagens ectópicas foram classificadas pelo aspecto ultra-sonográfico em anel tubário, massa complexa e embrião com atividade cardíaca e sua dimensão foi obtida pela medida do maior eixo. Histologicamente a invasão trofoblástica na parede tubária foi classificada em grau I: quando limitada à mucosa da tuba uterina; grau II: até a camada muscular; grau III: invasão de toda a espessura da tuba uterina. RESULTADOS: 29 pacientes tiveram infiltração tubária grau I, 30 pacientes infiltração grau II e 46 pacientes infiltração grau III. Os graus de invasão trofoblástica não estiveram associados à idade gestacional (p = 0,53) nem ao maior diâmetro da imagem à ultra-sonografia (p = 0,43). Os diferentes graus de invasão trofoblástica apresentaram diferença significativa da beta-hCG (p < 0,001). O grau I apresentou valores menores que os graus II e III (p < 0,05) e o grau II valores menores que o grau III (p < 0,05). Houve associação entre o grau de invasão trofoblástica e a descrição do tipo de imagem identificada à ultra-sonografia (p = 0,001). Embrião com atividade cardíaca foi mais prevalente nos casos de invasão grau III. O valor de 2 400 mUI/ml apresentou sensibilidade de 82,8%, especificidade de 85,5%, valor preditivo positivo de 68,6% e valor preditivo negativo de 92,7% (acurácia de 84,8%) para determinar invasão trofoblástica grau I. beta-hCG de 5 990 mUI/ml foi o melhor ponto de corte para predição de invasão trofoblástica grau III: sensibilidade de 82,6%, especificidade de 74,6%, valor preditivo positivo de 71,7% e valor preditivo negativo de 84,6% (acurácia de 78,1%). CONCLUSÕES: Em gestações ampulares, o tecido trofoblástico se desenvolve a partir de sua penetração na parede tubária, a profundidade da penetração do trofoblasto na tuba uterina relaciona-se às concentrações séricas de beta-hCG e ao tipo de imagem ultra-sonográfica, sendo que a concentração sérica da beta-hCG é a melhor preditora da profundidade da invasão na tuba uterina. / INTRODUCTION: The definition of predictive factors of morphologic and functional damage to the Fallopian tube may help in the choice of treatment for patients with ectopic pregnancy. The objective of the present study was to verify the presence of trophoblastic invasion into the tubal wall in ampular pregnancies, correlate the depth of penetration of trophoblastic tissue into the tubal wall with gestational age, beta-hCG concentration, type of ultrasonographic image and dimension of the ectopic mass upon ultrasound, and to evaluate the possible prediction of this invasion based on the parameters studied. METHODS: A retrospective study was conducted on 105 patients with ampular pregnancy submitted to salpingectomy between January 1, 2000 and March 31, 2004. Ectopic images were classified based on ultrasonographic findings in tubal ring, complex mass and embryonic heart activity. The dimension of the mass was determined by measuring the major axis. Histologically, trophoblastic invasion into the tubal wall was classified as grade I when limited to the tubal mucosa, grade II when reaching the muscle layer, and grade III when comprising the full thickness of the Fallopian tube. RESULTS: Twenty-nine patients had tubal infiltration grade I, 30 had grade II and 46 had grade III. The level of trophoblastic invasion was associated neither with gestational age (p = 0.53) nor with a greater diameter of the ultrasound image (p = 0.43). The different levels of trophoblastic invasion were significantly associated with beta-hCG concentration (p < 0.001), with lower concentrations being observed for grade I compared to grades II and III (p < 0.05) and for grade II compared to grade III (p < 0.05). There was an association between the level of trophoblastic invasion and the type of ultrasonographic image (p = 0.001). Embryos with heart activity were more prevalent in cases of grade III invasion. beta-hCG levels of 2 400 mIU/ml showed 82.8% sensitivity, 85.5% specificity, a positive predictive value of 68.6% and a negative predictive value of 92.7% (84.8% accuracy) for the diagnosis of grade I trophoblastic invasion. A beta-hCG titer of 5990 mIU/ml was the best cut-off for the prediction of grade III trophoblastic invasion: 82.6% sensitivity, 74.6% specificity, positive predictive value of 71.7% and negative predictive value of 84.6% (78.1% accuracy). CONCLUSIONS: trophoblastic tissue penetrate tubal wall in ampular pregnancies, the depth of penetration of trophoblastic tissue is correlated with beta-hCG concentration and type of ultrasonographic image and beta-hCG titer is the best predictor of the depth of penetration into tubal wall.

Ein wichtiges Merkmal der normalen Schwangerschaft ist die Toleranz des mütterlichen Immunsystems gegenüber den fremden fetalen Antigenen. Regulatorische T-Zellen (Treg-Zellen) steigen während der frühen Schwangerschaft an und leisten einen entscheidenden Beitrag zur fetalen Toleranz. Allerdings sind die genauen Mechanismen ihres Anstieges und ihrer Wirkungsweise noch weitgehend ungeklärt. Erste Untersuchungen zeigten, dass die Anzahl an Treg-Zellen in der empfänglichen Phase des Estruszyklus in der Maus am höchsten war. Es kann vermutet werden, dass die erhöhte Anzahl an Treg-Zellen zum Zeitpunkt der Insemination eine frühe Erkennung von väterlichen Antigenen begünstigt und somit zur Vorbereitung des mütterlichen Immunsystems auf die Implantation des Embryos beiträgt. Weiterhin konnte nachgewiesen werden, dass Alloantigene im Ejakulat den frühen Anstieg der Treg-Zellen in der Schwangerschaft bedingen und dass das in der Samenblasenflüssigkeit enthaltene Zytokin TGF-beta die Expansion der Treg-Zellen unterstützt. Diese Ergebnisse befürworten eine Alloantigen-vermittelte Expansion der Treg-Zellen während der frühen Schwangerschaft, die darüber hinaus durch TGF-beta begünstigt wird. Im Rahmen dieser Arbeit konnte ebenfalls belegt werden, dass das Schwangerschaftshormon hCG einen entscheidenden Einfluss auf die Treg-Zellen im Verlauf der Schwangerschaft ausübt. Untersuchungen unter Verwendung von menschlichem Probenmaterial konnten deutlich zeigen, dass hCG die Migration von Treg-Zellen zu Trophoblasten vermittelt und zur Konvertierung von konventionellen T-Zellen in Treg-Zellen beiträgt. Darüber hinaus konnten Versuche im Mausabortmodell zeigen, dass die Applikation von hCG die Expansion und Funktion der Treg-Zellen entscheidend beeinflusst, wodurch das Auftreten von Aborten in Abortweibchen verhindert werden konnte. HCG vermittelt demnach die Generierung, Migration und Funktion der Treg-Zellen und trägt entscheidend zum erfolgreichen Verlauf der Schwangerschaft bei. / Normal pregnancy is characterized by the generation of maternal immune tolerance towards the foreign fetal antigens. Regulatory T cells (Treg cells) have been shown to increase in number during early pregnancy stages and to be essential for the establishment of fetal tolerance. However, the mechanisms and factors supporting their increase and function are not well defined. First investigations showed that Treg cells accumulated in the sexually receptive phase of the murine estrous cycle. Thus, it can be assumed that the accumulation of Treg cells around the time of insemination favours early recognition of paternal antigens and thereby prepares the maternal immune system for the implantation of the blastocyst into the maternal endometrium. Experiments investigating the increase of Treg cells during early pregnancy suggest that alloantigens present in the ejaculate mediated early Treg cell augmentation. Moreover TGF-beta, which represents a major component in the seminal fluid, provoked the proliferation of Treg cells. These results suggest that the early expansion of Treg cells is alloantigen-mediated and that seminal fluid-derived TGF-beta is involved in this expansion. Additionally it has been shown that the pregnancy hormone human Chorionic Gonadotropin (hCG) has an important impact on Treg cells during pregnancy. By using human samples it has been proven that hCG mediates the migration of Treg cells to trophoblast cells and supports the conversion of naïve T cells into Treg cells directly at the fetal-maternal interface. In mice, the application of hCG resulted in an increase in the number and function of Treg cells and thereby prevented abortion in a mouse abortion-prone model. Thus, hCG mediates the generation, migration and function of Treg cells and contributes to a successful pregnancy outcome.

INTRODUÇÃO: A definição de fatores preditivos de lesão morfológica e funcional da tuba uterina poderia colaborar na escolha do tratamento de pacientes com gestação ectópica. O objetivo deste estudo foi verificar o comportamento do tecido trofoblástico em relação à sua penetração na parede da tuba uterina em gestações ampulares, relacionar a profundidade dessa penetração com idade gestacional, concentração de beta-hCG, tipo de imagem ultra-sonográfica e dimensão da massa ectópica à ultra-sonografia e avaliar a possibilidade de predição dessa invasão pelos parâmetros estudados. MÉTODOS: realizou-se estudo retrospectivo, entre 1° de janeiro de 2000 a 31 de março de 2004, com 105 pacientes com gestação tubária ampular submetidas à salpingectomia. As imagens ectópicas foram classificadas pelo aspecto ultra-sonográfico em anel tubário, massa complexa e embrião com atividade cardíaca e sua dimensão foi obtida pela medida do maior eixo. Histologicamente a invasão trofoblástica na parede tubária foi classificada em grau I: quando limitada à mucosa da tuba uterina; grau II: até a camada muscular; grau III: invasão de toda a espessura da tuba uterina. RESULTADOS: 29 pacientes tiveram infiltração tubária grau I, 30 pacientes infiltração grau II e 46 pacientes infiltração grau III. Os graus de invasão trofoblástica não estiveram associados à idade gestacional (p = 0,53) nem ao maior diâmetro da imagem à ultra-sonografia (p = 0,43). Os diferentes graus de invasão trofoblástica apresentaram diferença significativa da beta-hCG (p < 0,001). O grau I apresentou valores menores que os graus II e III (p < 0,05) e o grau II valores menores que o grau III (p < 0,05). Houve associação entre o grau de invasão trofoblástica e a descrição do tipo de imagem identificada à ultra-sonografia (p = 0,001). Embrião com atividade cardíaca foi mais prevalente nos casos de invasão grau III. O valor de 2 400 mUI/ml apresentou sensibilidade de 82,8%, especificidade de 85,5%, valor preditivo positivo de 68,6% e valor preditivo negativo de 92,7% (acurácia de 84,8%) para determinar invasão trofoblástica grau I. beta-hCG de 5 990 mUI/ml foi o melhor ponto de corte para predição de invasão trofoblástica grau III: sensibilidade de 82,6%, especificidade de 74,6%, valor preditivo positivo de 71,7% e valor preditivo negativo de 84,6% (acurácia de 78,1%). CONCLUSÕES: Em gestações ampulares, o tecido trofoblástico se desenvolve a partir de sua penetração na parede tubária, a profundidade da penetração do trofoblasto na tuba uterina relaciona-se às concentrações séricas de beta-hCG e ao tipo de imagem ultra-sonográfica, sendo que a concentração sérica da beta-hCG é a melhor preditora da profundidade da invasão na tuba uterina. / INTRODUCTION: The definition of predictive factors of morphologic and functional damage to the Fallopian tube may help in the choice of treatment for patients with ectopic pregnancy. The objective of the present study was to verify the presence of trophoblastic invasion into the tubal wall in ampular pregnancies, correlate the depth of penetration of trophoblastic tissue into the tubal wall with gestational age, beta-hCG concentration, type of ultrasonographic image and dimension of the ectopic mass upon ultrasound, and to evaluate the possible prediction of this invasion based on the parameters studied. METHODS: A retrospective study was conducted on 105 patients with ampular pregnancy submitted to salpingectomy between January 1, 2000 and March 31, 2004. Ectopic images were classified based on ultrasonographic findings in tubal ring, complex mass and embryonic heart activity. The dimension of the mass was determined by measuring the major axis. Histologically, trophoblastic invasion into the tubal wall was classified as grade I when limited to the tubal mucosa, grade II when reaching the muscle layer, and grade III when comprising the full thickness of the Fallopian tube. RESULTS: Twenty-nine patients had tubal infiltration grade I, 30 had grade II and 46 had grade III. The level of trophoblastic invasion was associated neither with gestational age (p = 0.53) nor with a greater diameter of the ultrasound image (p = 0.43). The different levels of trophoblastic invasion were significantly associated with beta-hCG concentration (p < 0.001), with lower concentrations being observed for grade I compared to grades II and III (p < 0.05) and for grade II compared to grade III (p < 0.05). There was an association between the level of trophoblastic invasion and the type of ultrasonographic image (p = 0.001). Embryos with heart activity were more prevalent in cases of grade III invasion. beta-hCG levels of 2 400 mIU/ml showed 82.8% sensitivity, 85.5% specificity, a positive predictive value of 68.6% and a negative predictive value of 92.7% (84.8% accuracy) for the diagnosis of grade I trophoblastic invasion. A beta-hCG titer of 5990 mIU/ml was the best cut-off for the prediction of grade III trophoblastic invasion: 82.6% sensitivity, 74.6% specificity, positive predictive value of 71.7% and negative predictive value of 84.6% (78.1% accuracy). CONCLUSIONS: trophoblastic tissue penetrate tubal wall in ampular pregnancies, the depth of penetration of trophoblastic tissue is correlated with beta-hCG concentration and type of ultrasonographic image and beta-hCG titer is the best predictor of the depth of penetration into tubal wall.

INTRODUÇÃO: A definição de fatores preditivos de lesão morfológica e funcional da tuba uterina poderia colaborar na escolha do tratamento de pacientes com gestação ectópica. O objetivo deste estudo foi relacionar a penetração do tecido trofoblástico na parede tubária acometida por gestação ampular com a concentração sérica materna de VEGF, avaliar a possibilidade do VEGF predizer a profundidade da invasão do tecido trofoblástico, comparando o desempenho do VEGF com o desempenho das concentrações séricas de beta-hCG na predição da profundidade da invasão do trofoblasto. MÉTODOS: realizou-se estudo prospectivo, entre 21 de dezembro de 2006 a 30 de setembro de 2007, com 30 pacientes com gestação tubária ampular submetidas à salpingectomia. Foram dosadas as concentrações séricas maternas de VEGF após confirmação do diagnóstico de gestação tubária e antes da realização da salpingectomia. Histologicamente a invasão trofoblástica na parede tubária foi classificada em grau I: quando limitada à mucosa da tuba uterina; grau II: até a camada muscular; grau III: invasão de toda a espessura da tuba uterina. RESULTADOS: 10 pacientes tiveram infiltração tubária grau I, 9 pacientes infiltração grau II e 11 pacientes infiltração grau III. Os diferentes graus de invasão trofoblástica apresentaram diferença significativa das concentrações séricas de VEGF (p< 0,001). O título sérico de VEGF de 305 pg/mL apresentou sensibilidade de 100,0%, especificidade de 85,0%, valor preditivo positivo de 76,9% e valor preditivo negativo de 100,0% para determinar invasão trofoblástica grau I. Título sérico de 425,9 pg/mL foi o melhor ponto de corte para predição de invasão trofoblástica grau III: sensibilidade de 81,8%, especificidade de 94,7%, valor preditivo positivo de 90,0% e valor preditivo negativo de 90,0%. Regressão logística selecionou a concentração sérica de VEGF como fator de melhor desempenho na predição da invasão trofoblástica quando comparado com título sérico de beta-hCG. CONCLUSÕES: em gestações ampulares, a profundidade da penetração do tecido trofoblástico na parede tubária acometida por gestação ectópica se relaciona com a concentração sérica de VEGF, a concentração sérica de VEGF é preditora da profundidade da invasão do tecido trofoblasto na parede tubária acometida por GE e a concentração sérica de VEGF apresenta melhor desempenho que a concentração sérica de beta-hCG como preditora da profundidade da invasão do trofoblasto na parede da tuba uterina acometida por gestação ampular / INTRODUCTION: The definition of predictive factors of morphologic and functional damage to the Fallopian tube may help in the choice of treatment for patients with ectopic pregnancy. The objective of the present study was to correlate the depth of penetration of trophoblastic tissue into the tubal wall with maternal serum VEGF concentrations, to evaluate the prediction of this invasion based on these concentrations and to compare the performances of VEGF and beta-hCG as predictors of trophoblastic invasion. METHODS: A prospective study was conducted on 30 patients with ampular pregnancy submitted to salpingectomy between December 21st, 2006 and September 30th, 2007. Maternal serum VEGF concentrations were measured after the diagnosis confirmation and before salpingectomy was performed. Histologically, trophoblastic invasion into the tubal wall was classified as grade I when limited to the tubal mucosa, grade II when reaching the muscle layer, and grade III when comprising the full thickness of the Fallopian tube. RESULTS: ten patients had tubal infiltration grade I, nine had grade II and eleven had grade III. The different levels of trophoblastic invasion were significantly associated with VEGF concentrations (p< 0.001). VEGF levels of 305.0 pg/mL showed 100.0% sensitivity, 85.0% specificity, a positive predictive value of 76.9% and a negative predictive value of 100.0% for the diagnosis of grade I trophoblastic invasion. A VEGF titer of 425.9 pg/mL was the best cut-off for the prediction of grade III trophoblastic invasion: 81.8% sensitivity, 94.7% specificity, positive predictive value of 90.0% and negative predictive value of 90.0%. Logistic regression showed that VEGF presented higher performance as a predictor of trophoblastic invasion than beta-hCG. CONCLUSIONS: in ampullary pregnancies, the depth of penetration of trophoblastic tissue into the tubal wall is correlated with serum VEGF concentrations, serum VEGF titer is predictor of the depth of penetration into tubal wall and VEGF concentrations present higher performance than beta-hCG as predictor of the trophoblastic invasion

INTRODUÇÃO: A definição de fatores preditivos de lesão morfológica e funcional da tuba uterina poderia colaborar na escolha do tratamento de pacientes com gestação ectópica. O objetivo deste estudo foi relacionar a penetração do tecido trofoblástico na parede tubária acometida por gestação ampular com a concentração sérica materna de VEGF, avaliar a possibilidade do VEGF predizer a profundidade da invasão do tecido trofoblástico, comparando o desempenho do VEGF com o desempenho das concentrações séricas de beta-hCG na predição da profundidade da invasão do trofoblasto. MÉTODOS: realizou-se estudo prospectivo, entre 21 de dezembro de 2006 a 30 de setembro de 2007, com 30 pacientes com gestação tubária ampular submetidas à salpingectomia. Foram dosadas as concentrações séricas maternas de VEGF após confirmação do diagnóstico de gestação tubária e antes da realização da salpingectomia. Histologicamente a invasão trofoblástica na parede tubária foi classificada em grau I: quando limitada à mucosa da tuba uterina; grau II: até a camada muscular; grau III: invasão de toda a espessura da tuba uterina. RESULTADOS: 10 pacientes tiveram infiltração tubária grau I, 9 pacientes infiltração grau II e 11 pacientes infiltração grau III. Os diferentes graus de invasão trofoblástica apresentaram diferença significativa das concentrações séricas de VEGF (p< 0,001). O título sérico de VEGF de 305 pg/mL apresentou sensibilidade de 100,0%, especificidade de 85,0%, valor preditivo positivo de 76,9% e valor preditivo negativo de 100,0% para determinar invasão trofoblástica grau I. Título sérico de 425,9 pg/mL foi o melhor ponto de corte para predição de invasão trofoblástica grau III: sensibilidade de 81,8%, especificidade de 94,7%, valor preditivo positivo de 90,0% e valor preditivo negativo de 90,0%. Regressão logística selecionou a concentração sérica de VEGF como fator de melhor desempenho na predição da invasão trofoblástica quando comparado com título sérico de beta-hCG. CONCLUSÕES: em gestações ampulares, a profundidade da penetração do tecido trofoblástico na parede tubária acometida por gestação ectópica se relaciona com a concentração sérica de VEGF, a concentração sérica de VEGF é preditora da profundidade da invasão do tecido trofoblasto na parede tubária acometida por GE e a concentração sérica de VEGF apresenta melhor desempenho que a concentração sérica de beta-hCG como preditora da profundidade da invasão do trofoblasto na parede da tuba uterina acometida por gestação ampular / INTRODUCTION: The definition of predictive factors of morphologic and functional damage to the Fallopian tube may help in the choice of treatment for patients with ectopic pregnancy. The objective of the present study was to correlate the depth of penetration of trophoblastic tissue into the tubal wall with maternal serum VEGF concentrations, to evaluate the prediction of this invasion based on these concentrations and to compare the performances of VEGF and beta-hCG as predictors of trophoblastic invasion. METHODS: A prospective study was conducted on 30 patients with ampular pregnancy submitted to salpingectomy between December 21st, 2006 and September 30th, 2007. Maternal serum VEGF concentrations were measured after the diagnosis confirmation and before salpingectomy was performed. Histologically, trophoblastic invasion into the tubal wall was classified as grade I when limited to the tubal mucosa, grade II when reaching the muscle layer, and grade III when comprising the full thickness of the Fallopian tube. RESULTS: ten patients had tubal infiltration grade I, nine had grade II and eleven had grade III. The different levels of trophoblastic invasion were significantly associated with VEGF concentrations (p< 0.001). VEGF levels of 305.0 pg/mL showed 100.0% sensitivity, 85.0% specificity, a positive predictive value of 76.9% and a negative predictive value of 100.0% for the diagnosis of grade I trophoblastic invasion. A VEGF titer of 425.9 pg/mL was the best cut-off for the prediction of grade III trophoblastic invasion: 81.8% sensitivity, 94.7% specificity, positive predictive value of 90.0% and negative predictive value of 90.0%. Logistic regression showed that VEGF presented higher performance as a predictor of trophoblastic invasion than beta-hCG. CONCLUSIONS: in ampullary pregnancies, the depth of penetration of trophoblastic tissue into the tubal wall is correlated with serum VEGF concentrations, serum VEGF titer is predictor of the depth of penetration into tubal wall and VEGF concentrations present higher performance than beta-hCG as predictor of the trophoblastic invasion

The glycoprotein hormone family comprising of Luteinizing Hormone (LH), Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) plays important role in reproduction and overall physiology of the organism. These hormones are heterodimeric molecules consisting of an identical α subunit non-covalently associated with the hormone-specific β subunit. Both subunits of all these hormones are N-glycosylated. In addition, hCGβ subunit also has four O-linked oligosaccharides located at the C-terminus of the polypeptide(1). The α and β subunits of all these hormones contain five and six disulfide bonds respectively and the crystal structures of hCG and hFSH indicate that both subunits of the hormones belong to the cystine knot family of proteins(2-4). Although the β subunits are hormone specific, there are distinct similarities in these subunits with the 12 cysteines conserved in all these subunits (1). These hormones, because of their unique structural features have proved to be important models for structure–function relationship studies of complex dimeric glycoproteins. Folding of subunits during biosynthesis, role of glycosylation in folding pathways and in vitro and in vivo bioactivity of the hormone, as well as, identification of domains important for subunit association, receptor binding and subsequent signal transduction have been topics of active investigations. The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have unique hormone specific exodomain not present in other members of the GPCR family and characteristic seven transmembrane domains followed by a C terminal domain(5). Primary structure analysis of Glycoprotein hormone receptors family revealed sequence conservation, maximum homology being observed in the transmembrane domain (TMD)(6). Significant homologies could be observed in the hormone specific extracellular domains (ECD) also (7). Despite these homologies, the receptors exhibit exquisite specificity with very low cross reactivity with other members of the hormone family (8). Elucidation of the molecular details of the contacts between the hormone and the receptors has not been achieved so far. Various approaches have been employed to delineate the residues or domains of both hormone and receptors involved in interaction. These include testing of chimeras or mutants of hormones or receptors for changes in activity (9-12), chemical modifications(13) and competition with peptides from either hormones (14) or receptors (15). Polyclonal and monoclonal antibodies against glycoprotein hormones and various fragments of their receptors have been used to determine the role of different domains of both in binding and response (6, 16, 17). However, till date there is no consensus on the specific mechanisms by which the glycoprotein hormone docks onto its receptor. It was proposed that the initial contact between the hormone and the receptor occurs through high affinity binding of the hormone specific β subunit to the Leucine rich regions of the ECD that results in conformational changes in both hormone, as well as, the receptor and brings hormone/ECD complex closer to the TMD of the receptor. The secondary, relatively lower affinity interactions between the hormone and the receptor then take place through common α subunit and exoloops of TMD of the receptor resulting in signal generation (18, 19). Recently a different kind of model has been proposed which suggests that the hormone does not make any direct contacts with the TMD of the receptor. The signal is transduced by the change in contacts between ECD and TMD brought about by hormone’s interaction with ECD(8, 20). The present study was initiated with an overall objective of understanding the molecular details of the hormone receptor interactions of this family, particularly hCG- LH receptor interactions. Two different approaches were employed for this purpose; the first, direct approach being structure elucidation of the members of the glycoprotein hormone family while the second approach uses antibodies against hCG as tools to probe into hormone-receptor interactions. The results obtained using these two approaches have been consolidated in the present thesis and are organized as follows. Chapter 1 is an extensive review of the literature and it builds background for the present work while the exact aim and scope of the present work have been defined in Chapter 2. Chapter 3 describes cloning, expression and purification of recombinant glycoprotein hormones hLH, hCG and single chain derivative of hCG. The Chapter 4 gives details of the molecular aspects of hCG-LH receptor interaction dissected using hCG monoclonal antibodies (MAbs). Chapter 5 discusses implications of the observations made in the present study and states the future directions envisaged. There are a number of endocrinopathies associated with abnormal levels of glycoprotein hormones and treatments of such disorders often demand large quantities of either agonists or antagonists of the hormones. The structure-function relationship studies should help in identifying domains/residues important for subunit interaction, receptor binding, and signal transduction, which would also allow engineering of agonists and antagonists of hormone action. However, structure determination of the glycoprotein hormone family using X-ray crystallography has proved to be a difficult task and it is believed that the heterogeneity in glycosylation is the primary reason for this low success rate in the process of crystallization. The first crystal structure of hCG was that of completely deglycosylated hCG but such a molecule displays antagonistic behavior(2, 3). Use of NMR spectroscopy, the alternate method commonly used for structure determination is often limited by the availability of large quantities of biologically active hormones free of any contaminants. Large quantities of LH, hCG and FSH are also required for treatment of infertile patients suffering from gonadotropin deficiency. The first goal of the present study was thus to produce and purify biologically active recombinant hCG and hLH. Owing to the inherent features of glycoprotein hormones and their potential therapeutic applications, the recombinant expression of these hormones is an important goal from both basic research, as well as, commercial point of view. Considering the above mentioned features it is clear that the expression system used for the hyperexpression of these glycoprotein hormones should also serve as a model system for investigating structure–function relationships and folding of subunits during biosynthesis, in addition to providing sufficient quantities of the hormones for clinical applications. It has been demonstrated that N-linked glycosylation during biosynthesis facilitates protein folding and conformational maturation of glycoprotein hormone subunits into an assembly-competent, biologically active form (21). Therefore, the ideal recombinant expression system should also be able to glycosylate the protein during biosynthesis. The Pichia pastoris yeast expression system was chosen for hyperexpression of glycoprotein hormones as it blends the advantages of both bacterial and mammalian expression systems. Earlier, expression of biologically active hCG and the subunits of hCG and bovine FSH using Pichia pastoris expression system has been reported from the laboratory (22, 23). Chapter 3 (section 3.3.1) of the thesis describes hyperexpression of hLH. The expression of these heterologous proteins was scaled up using fermentation procedures to fulfill the requirements of large quantities of hormones for various applications. Purification of Pichia expressed hormones turned out to be a complex task as large quantity of the hormone was secreted out in the fermentation medium (10litre volume) that was of high ionic strength. Of several different strategies attempted for concentration and partial purification of recombinant hCG, hydrophobic interaction chromatography (HIC) using Phenyl Sepharose matrix emerged as the most efficient technique as a first step of purification. Subsequently, cation exchange chromatography using SP- Sepharose matrix yielded completely purified biologically active recombinant hCG (section 3.3.2). The preliminary data also suggested that Pichia cells express a biologically active form of hCG which appeared to be less glycosylated and of lower molecular weight. Using the same protocol purification of hLH, as well as, single chain derivative of hCG, phCGαβ was achieved (section 3.3.3). These recombinant proteins were characterized extensively using various biochemical, as well as, immunological criteria and were shown to be similar to their natural counterparts with respect to their ability to bind LH receptor and to transduce signal as judged by radioreceptorassays and in vitro bioassays respectively. The hydrophobic interaction chromatography proved an important starting point for purification of all the other members of the glycoprotein hormone family expressed using Pichia pastoris expression system. With the availability of purified, biologically active recombinant hCG in large quantities it was now possible to make attempts towards structure elucidation using NMR spectroscopy. The structure determination of such complex proteins by NMR spectroscopy is made relatively easier by labeling the proteins with magnetically more active, stable isotopes of carbon and nitrogen, 13C and 15N respectively however the cost is often prohibitively high. The Pichia pastoris expression system offers simple means of labeling the proteins as the cells can be grown on simple salts of carbon and nitrogen such as 13C labeled methanol, 15N labeled ammonium chloride or ammonium sulphate. The Chapter 3 also gives a brief account of the preliminary attempts made to label the recombinant hCG with 15N and the structural studies carried out with the carbohydrate moieties of the recombinant hCG using solution NMR spectroscopy. This work was carried out in collaboration with the laboratory of Prof. J.P Kamerling of the University of Utrecht, Netherlands and the efforts are currently underway to elucidate the complete structure of the Pichia expressed hCG. The common feature of receptors and antibodies against the ligand is that both display very specific, high affinity binding towards the ligand. Hence, it is logical to speculate that the antigen binding regions of the antibodies that inhibit hormone binding and/or response, exhibit homology with distinct domains of the receptor. By identifying the epitopes recognized by such antibodies, it should be possible to predict contact points between the hormone and the receptor. In the present study, this hypothesis has been tested using monoclonal antibodies (MAbs) against hCG recognizing different epitopes in the hormone molecule and having different effects on hormone binding and response (Chapter 4). These MAbs were classified as α subunit specific, β subunit specific or heterodimer specific depending on their abilities to bind either subunit in addition to the hormone itself. Interestingly, it was observed that the hCGβ subunit specific MAbs, as well as, heterodimer specific MAbs inhibited hCG receptor binding and hence the response generated by hCG, while the hCGα subunit specific MAbs inhibited only response to the hormone without interfering in binding (Section 4.3.1). To dissect out these interactions further the epitopes recognized by these antibodies on hCG molecule were determined (Section 4.3.2), single chain fragment variable (ScFv) were generated from each of these antibodies and it was shown that these ScFv retain the functionality of the original antibody (Section 4.3.3). Further, the amino acid sequence of each antibody was determined (Section 4.3.4) and finally shown that the antigen binding domains of antibodies show homology to the distinct regions of the LH receptor on sequence alignments between the two using three different programs (4.3.5). The hCGβ subunit specific MAb 52/28' displayed distinct homology with the ECD of LH receptor while the α subunit specific MAb C10 showed regions homologous to TMD of the receptor and the heterodimer specific MAb E12 was found to be similar to the hinge region of the receptor. This clearly indicates that the β subunit of hCG is in close contact with ECD of the receptor while the α subunit makes contacts with the TMD of receptor. The present study thus supports the existing model of hormone receptor interactions, which states that the hormone first binds to the exodomain of the receptor mainly through its β subunit while the integrity of the α subunit is critical for signaling. (24, 25). Also, the observations made in the present study exhibit an interesting possibility of antigen antibody complexes being used as surrogate models for gaining insights into hormone receptor complex. Further, it has been reported that hCG has immunocontraceptive potential(26). Active and passive immunization studies with hCG in primates and humans have demonstrated the possibility of controlling fertility by the antibodies capable of neutralizing hCG. This forms the basis for female contraceptive vaccine that has undergone Phase II clinical trials in India. The MAb E12 characterized in the present study displayed highly specific binding to heterodimeric hCG exclusively without showing any cross reactivity with hLH (Section 4.3.1). The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCGβ absent in hLHβ (Section 4.3.2). The MAb, IgG or its recombinant single chain fragment variable (ScFv), inhibited response to hCG, but not to hLH (4.3.3). Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception. The MAb E12 can also be used for passive immunization in case of emergency contraception. Another potential application of hCG specific antibodies is in homing and the treatment of tumors that secrete hCGβ subunit. The hCGβ subunit specific MAbs used in the present study 52/12 and 52/28' that inhibited hCG receptor binding as well as response generated by hCG can be used in treating such tumors. The functional ScFvs generated from these MAbs in the present study can be made use of on humanization. Thus, the present study has yielded some important molecules for therapeutic applications besides providing a new platform for structure-function relationship studies of the complex glycoprotein hormones.