La crioconservación de los embriones por congelación o vitrificación reduce la supervivencia de los embriones de conejo (Oryctolagus cuniculus) entre un 20 y 50% dependiendo de la estirpe genética y el procedimiento utilizado. En embriones vitrificados de conejo se ha observado un elevada mortalidad tras la implantación que sugeriría que el proceso tiene efectos tardíos y negativos sobre el desarrollo fetal. El objetivo de la tesis fue estudiar el transcriptoma embrionario previo a la implantación (6 días) y el desarrollo embrionario y fetal de embriones de conejo crioconservados mediante dos procedimientos habituales en esta especie. Específicamente, el objetivo del capítulo I fue la evaluación de los efectos del procedimiento de congelación sobre el desarrollo y expresión génica de embriones de conejo. Para ello, evaluamos primero la distribución de pérdidas durante la gestación analizando las tasas de desarrollo a blastocisto tardío, implantación y nacimiento. Después comparamos el perfil transcriptómico de embriones de 6 días, previamente congelados y transferidos en el estadio de mórula, con embriones desarrollados in vivo (6 días post-inseminación). Para ello, llevamos a cabo un análisis microarray de dos colores y usamos una plataforma genérica específica de conejo. Las tasas de desarrollo a blastocisto tardío, implantación y nacimiento fueron más bajas para los embriones congelados. Así mismo, también se observaron diferencias a nivel de expresión, y los embriones viables de 6 días del grupo congelado presentaron 70 genes diferencialmente expresados. Esta fue la primera aproximación que nos proporcionó una lista de genes candidatos que podrían provocar fallos en el diálogo materno-embrionario, implantación y formación de la placenta. El capítulo II evaluó la distribución de pérdidas durante la gestación debidas al proceso de vitrificación. Sin embargo, en este caso, para evaluar los efectos de la vitrificación sobre la expresión génica de blastocistos tardíos usamos la técnica de PCR cuantitativa (qPCR) con 10 genes candidatos, elegidos por estar diferencialmente expresado en los embriones congelados del anterior trabajo (SCGB1A1, EMP1, C1QTNF1, ANXA3, EGFLAM y TNFAIP6 ), o por su rol en el desarrollo embrionario e implantación (OCT4, VEGF, HBA and LAMA 4 ). Además, introdujimos también datos ecográficos sobre el tamaño del saco embrionario, feto, la placenta fetal y materna desde el día 10 hasta el 14 de gestación. Los resultados mostraron dos picos principales de pérdidas durante la gestación: Uno situado antes de la implantación y el otro después. Asimismo, también detectamos una reducción en el tamaño del feto y de la placenta materna entre los días 10 y 14. Finalmente, pudimos observar que, comparado con los embriones de 6 días desarrollados en condiciones in vivo, la expresión relativa de los transcritos SCGB1A1, EMP1, C1QTNF1, ANXA3, EGFLAM y TNFAIP6 estaba significativamente alterada en los embriones vitrificados, siguiendo además el patrón previamente observado en los embriones congelados. En el capítulo III se compararon directamente los transcriptomas de los embriones de 6 días obtenidos de embriones congelados y vitrificados de 3 días. Siguiendo los mismos procedimientos que en los trabajos anteriores, evaluamos la distribución de pérdidas a lo largo de la gestación analizando las tasas de desarrollo a blastocisto tardío, implantación y nacimiento. Asimismo, empleando la misma plataforma genérica microarray del primer experimento realizamos un análisis dos colores microarray en el que comparamos directamente el perfil transcriptómico a día 6 de desarrollo de embriones congelados y vitrificados. Los resultados reportaron que la congelación y la vitrificación tienen los mismos efectos dañinos sobre el desarrollo a día 6, pero la distribución de pérdidas difiere durante la implantación y el desarrollo, siendo las tasas de implantación y nacimiento mayores para el grupo vitrificado. Las similitudes a día 6 de desarrollo también se reflejaron en el patrón de expresión génica, porque no se detectaron diferencias a nivel transcriptómico entre los dos tipos de embriones. En el capítulo IV se analizó el transcriptoma de los embriones vitrificados de 6 días y placentas fetales de 14 días frente al transcriptoma de embriones y placentas control no vitrificados pero que fueron recuperados y transferidos, aislando así el efecto del proceso de vitrificación. Al igual que observamos en el capítulo II, los embriones vitrificados que eran capaces de llegar al estadio de blastocisto tardío eran también capaces de implantar, pero no todos los embriones implantados tenían la capacidad de finalizar la gestación. Teniendo en cuenta los resultados de las ecografías del trabajo anterior, tomamos datos del peso del feto, la placenta fetal y materna a día 14 de desarrollo y del peso al nacimiento. En los resultados detectamos un descenso en los pesos del feto y la placenta materna, así como un incremento en el peso al nacimiento de los embriones vitrificados. En este experimento, para la evaluación de la expresión génica introdujimos unas cuantas modificaciones en el diseño experimental. Así, empleamos una plataforma microarray específica para embrión de conejo y realizamos un análisis microarray de un color. En el caso de los embriones de 6 días no encontramos diferencias significativas en la expresión génica, pero en el caso de las placentas identificamos 60 genes sobreexpresados. Llegados a este punto, realizamos un análisis 2D-DIGE en as placentas fetales para encontrar diferencias a nivel proteómico. Así, detectamos 89 proteínas diferencialmente expresadas en las placentas de 14 días de desarrollo derivadas de embriones vitrificados. Debido a los anteriores resultados de alteraciones a nivel transcriptómico y proteómico en las placentas pocos días después de la implantación, el capítulo V lo centramos en el estudio de las alteraciones proteómicas en placentas fetales de 24 días de desarrollo. En este trabajo pretendimos comparar los perfiles proteicos de placentas fetales de embriones vitrificados y controles en la última parte de la gestación, pocos días antes del nacimiento. Tras realizar un análisis 2D-DIGE encontramos que había 32 proteínas diferencialmente expresadas entre los grupos experimentales. Estos resultados demostraron que la vitrificación induce cambios sustanciales en la expresión de proteínas placentarias al final de la gestación, y que aparte de las consecuencias a corto plazo, la crioconservación embrionaria puede provocar consecuencias a largo plazo en esos fetos que al final nacen. Los resultados de esta tesis nos permiten suponer que la crioconservación embrionaria, bien sea por congelación o vitrificación, no debe ser neutral. Además, por primera vez se han observado modificaciones en los embriones vitrificados ya implantados. Basándonos en estos resultados, nuestro trabajo deja abierta la cuestión de si los efectos causados por la vitrificación durante el desarrollo fetal pueden conllevar algún tipo de alteraciones metabólicas o fisiológicas en la vida adulta. / Saenz De Juano Ribes, MDLD. (2014). Estudio del efecto de la crioconservación sobre la expresión génica de embriones de conejo [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/36957 / TESIS / Premios Extraordinarios de tesis doctorales

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No description available.

<p>Preterm birth is the leading cause of newborn mortality, with 15 million babies born premature worldwide every year. Children that do survive early delivery are more likely to develop cognitive abnormalities, motor deficits, heart disease, cerebral palsy, and more. While little is known about the pathophysiology of preterm birth, several pregnancy-related complications are related to preterm birth, namely cervical insufficiency and preeclampsia. In the former, premature cervical remodeling and softening can result in the shortening of the cervix, increasing a woman’s risk of preterm birth; this condition is called cervical insufficiency (CI), which is the inability of the cervix to remain closed as a result of weakened tissues. CI is currently measured by a one-dimensional sonographic cervical length, where < 25 mm indicates shortening. Preeclampsia is a disorder that can be explained through the Page kidney phenomenon: compression of the left renal vein (LRV) causes renal venous outflow obstruction, leading to elevated intrarenal pressure and hypertension. The supine pressor test (SPT) is a diagnostic tool for preeclampsia where a positive test is defined by an increase of 20 mmHg in diastolic blood pressure (BP) when shifting from the left lateral recumbent to the supine position. Due to the intense risk of morbidity and mortality for both the mother and the fetus, the need to monitor BP changes is critical. Currently, there is an unmet clinical need to characterize the hemodynamic and geometric properties of the female reproductive organs throughout gestation. Utilizing ultrasound imaging can increase our knowledge about the 3D anatomy and systemic changes during pregnancy, unravel risk factors, establish preventative methods, and standardize treatment plans. In this thesis research, we developed a murine model to 1) examine the pathophysiology of renal vein stenosis, and 2) investigate the effects of stenosis on various cervical dimensions. Renal vein stenosis was found to greatly impact blood flow velocities, as well as cervical width (<i>p<0.05</i>). LRV and cervical area and height also trend towards significance, and there is negative damage to the left kidney and placentae within the stenosed cohort. We also conducted a human study that showed reduced change in postural BP in patients with higher body mass index (BMI). Systolic and diastolic BP in the supine position was significantly greater than in the lateral position for all BMIs with a baseline increase in BP of approximately 9-14 mmHg. These findings suggest that therapeutic positioning and close monitoring of BP could mitigate the risk of developing related disorders in pregnancy.</p>

The thesis examines the relationship of birthweight to risk factors and markers, such as proteinuria and glomerular filtration rate, for chronic disease in postnatal life. It made use of the Australian Diabetes, Obesity and Lifestyle Study (AusDiab). The AusDiab study is a cross sectional study where baseline data on 11,247 participants were collected in 1999-2000. Participants were recruited from a stratified sample of Australians aged ≥ 25 years, residing in 42 randomly selected urban and non-urban areas (Census Collector Districts) of the six states of Australia and the Northern Territory. The AusDiab study collected an enormous amount of clinical and laboratory data. During the 2004-05 follow-up AusDiab survey, questions about birthweight were included. Participants were asked to state their birthweight, the likely accuracy of the stated birthweight and the source of their stated birthweight. Four hundred and twelve chronic kidney disease (CKD) patients were approached, and 339 agreed to participate in the study. The patients completed the same questionnaire. Medical records were reviewed to check the diagnoses, causes of kidney trouble and SCr levels. Two control subjects, matched for gender and age, were selected for each CKD patient from participants in the AusDiab study who reported their birthweight. Among 7,157 AusDiab participants who responded to the questionnaire, 4,502 reported their birthweights, with a mean (standard deviation) of 3.4 (0.7) kg. The benefit and disadvantages of these data are discussed in chapter three. The data were analysed for the relationship between birthweight and adult body size and composition, disorders of glucose regulation, blood pressure, lipid abnormalities, cardiovascular diseases and glomerular filtration rate. Low birthweight was associated with smaller body build and lower lean mass and total body water in both females and males. In addition low birthweight was associated with central obesity and higher body fat percentage in females, even after taking into account current physical activity and socioeconomic status. Fasting plasma glucose, post load glucose and glycosylated haemoglobin were strongly and inversely correlated with birthweight. In those with low birthweight (< 2.5 kg), the risks for having impaired fasting glucose, impaired glucose tolerance, diabetes and all abnormalities combined were increased by 1.75, 2.22, 2.76 and 2.28 for females and by 1.40, 1.32, 1.98 and 1.49 for males compared to those with normal birthweight (≥ 2.5 kg), respectively. Low birthweight individuals were at higher risk for having high blood pressure ≥ 140/90 mmHg and ≥ 130/85 mmHg compared to those with normal birthweight. People with low birthweight showed a trend towards increased risk for high cholesterol (≥ 5.5 mmol/l) compared to those of normal birthweight. Females with low birthweight had increased risk for high low density lipoprotein cholesterol (≥ 3.5 mmol/l) and triglyceride levels (≥ 1.7 mmol/l) when compared to those with normal birthweight. Males with low birthweight exhibited increased risk for low levels of high density lipoprotein cholesterol (<0.9 mmol/l) than those with normal birthweight. Females with low birthweight were at least 1.39, 1.40, 2.30 and 1.47 times more likely to have angina, coronary artery disease, stroke and overall cardiovascular diseases respectively, compared to those ≥ 2.5 kg. Similarly, males with low birthweight were 1.76, 1.48, 3.34 and 1.70 times more likely to have angina, coronary artery disease, stroke and overall cardiovascular diseases compared to those ≥ 2.5 kg, respectively. The estimated glomerular filtration rate was strongly and positively associated with birthweight, with a predicted increase of 2.6 ml/min (CI 2.1, 3.2) and 3.8 (3.0, 4.5) for each kg of birthweight for females and males, respectively. The odd ratio (95% confidence interval) for low glomerular filtration rate (<61.0 ml/min for female and < 87.4 male) in people of low birthweight compared with those of normal birthweight was 2.04 (1.45, 2.88) for female and 3.4 (2.11, 5.36) for male. One hundred and eighty-nineCKD patients reported their birthweight; 106 were male. Their age was 60.3(15) years. Their birthweight was 3.27 (0.62) kg, vs 3.46 (0.6) kg for their AusDiab controls, p<0.001 and the proportions with birthweight<2.5 kg were 12.17% and 4.44%, p<0.001. Among CKD patients, 22.8%, 21.7%, 18% and 37.6% were in CKD stages 2, 3, 4 and 5 respectively. Birthweights by CKD stage and their AusDiab controls were as follows: 3.38 (0.52) vs 3.49 (0.52), p=0.251 for CKD2; 3.28 (0.54) vs 3.44 (0.54), p=0.121 for CKD3; 3.19 (0.72) vs 3.43 (0.56), p= 0.112 for CKD4 and 3.09 (0.65) vs 3.47 (0.67), p<0.001 for CKD5. The results demonstrate that in an affluent Western country with a good adult health profile, low birthweight people were predisposed to higher rates of glycaemic dysregulation, high blood pressure, dyslipidaemia, cardiovascular diseases and lower glomerular filtration rate in adult life. In all instances it would be prudent to adopt policies of intensified whole of life surveillance of lower birthweight people, anticipating this risk. The general public awareness of the effect of low birthweight on development of chronic diseases in later life is of vital importance. The general public, in addition to the awareness of people in medical practice of the role of low birthweight, will lead to a better management of this group of our population that is increasingly surviving into adulthood.

The thesis examines the relationship of birthweight to risk factors and markers, such as proteinuria and glomerular filtration rate, for chronic disease in postnatal life. It made use of the Australian Diabetes, Obesity and Lifestyle Study (AusDiab). The AusDiab study is a cross sectional study where baseline data on 11,247 participants were collected in 1999-2000. Participants were recruited from a stratified sample of Australians aged ≥ 25 years, residing in 42 randomly selected urban and non-urban areas (Census Collector Districts) of the six states of Australia and the Northern Territory. The AusDiab study collected an enormous amount of clinical and laboratory data. During the 2004-05 follow-up AusDiab survey, questions about birthweight were included. Participants were asked to state their birthweight, the likely accuracy of the stated birthweight and the source of their stated birthweight. Four hundred and twelve chronic kidney disease (CKD) patients were approached, and 339 agreed to participate in the study. The patients completed the same questionnaire. Medical records were reviewed to check the diagnoses, causes of kidney trouble and SCr levels. Two control subjects, matched for gender and age, were selected for each CKD patient from participants in the AusDiab study who reported their birthweight. Among 7,157 AusDiab participants who responded to the questionnaire, 4,502 reported their birthweights, with a mean (standard deviation) of 3.4 (0.7) kg. The benefit and disadvantages of these data are discussed in chapter three. The data were analysed for the relationship between birthweight and adult body size and composition, disorders of glucose regulation, blood pressure, lipid abnormalities, cardiovascular diseases and glomerular filtration rate. Low birthweight was associated with smaller body build and lower lean mass and total body water in both females and males. In addition low birthweight was associated with central obesity and higher body fat percentage in females, even after taking into account current physical activity and socioeconomic status. Fasting plasma glucose, post load glucose and glycosylated haemoglobin were strongly and inversely correlated with birthweight. In those with low birthweight (< 2.5 kg), the risks for having impaired fasting glucose, impaired glucose tolerance, diabetes and all abnormalities combined were increased by 1.75, 2.22, 2.76 and 2.28 for females and by 1.40, 1.32, 1.98 and 1.49 for males compared to those with normal birthweight (≥ 2.5 kg), respectively. Low birthweight individuals were at higher risk for having high blood pressure ≥ 140/90 mmHg and ≥ 130/85 mmHg compared to those with normal birthweight. People with low birthweight showed a trend towards increased risk for high cholesterol (≥ 5.5 mmol/l) compared to those of normal birthweight. Females with low birthweight had increased risk for high low density lipoprotein cholesterol (≥ 3.5 mmol/l) and triglyceride levels (≥ 1.7 mmol/l) when compared to those with normal birthweight. Males with low birthweight exhibited increased risk for low levels of high density lipoprotein cholesterol (<0.9 mmol/l) than those with normal birthweight. Females with low birthweight were at least 1.39, 1.40, 2.30 and 1.47 times more likely to have angina, coronary artery disease, stroke and overall cardiovascular diseases respectively, compared to those ≥ 2.5 kg. Similarly, males with low birthweight were 1.76, 1.48, 3.34 and 1.70 times more likely to have angina, coronary artery disease, stroke and overall cardiovascular diseases compared to those ≥ 2.5 kg, respectively. The estimated glomerular filtration rate was strongly and positively associated with birthweight, with a predicted increase of 2.6 ml/min (CI 2.1, 3.2) and 3.8 (3.0, 4.5) for each kg of birthweight for females and males, respectively. The odd ratio (95% confidence interval) for low glomerular filtration rate (<61.0 ml/min for female and < 87.4 male) in people of low birthweight compared with those of normal birthweight was 2.04 (1.45, 2.88) for female and 3.4 (2.11, 5.36) for male. One hundred and eighty-nineCKD patients reported their birthweight; 106 were male. Their age was 60.3(15) years. Their birthweight was 3.27 (0.62) kg, vs 3.46 (0.6) kg for their AusDiab controls, p<0.001 and the proportions with birthweight<2.5 kg were 12.17% and 4.44%, p<0.001. Among CKD patients, 22.8%, 21.7%, 18% and 37.6% were in CKD stages 2, 3, 4 and 5 respectively. Birthweights by CKD stage and their AusDiab controls were as follows: 3.38 (0.52) vs 3.49 (0.52), p=0.251 for CKD2; 3.28 (0.54) vs 3.44 (0.54), p=0.121 for CKD3; 3.19 (0.72) vs 3.43 (0.56), p= 0.112 for CKD4 and 3.09 (0.65) vs 3.47 (0.67), p<0.001 for CKD5. The results demonstrate that in an affluent Western country with a good adult health profile, low birthweight people were predisposed to higher rates of glycaemic dysregulation, high blood pressure, dyslipidaemia, cardiovascular diseases and lower glomerular filtration rate in adult life. In all instances it would be prudent to adopt policies of intensified whole of life surveillance of lower birthweight people, anticipating this risk. The general public awareness of the effect of low birthweight on development of chronic diseases in later life is of vital importance. The general public, in addition to the awareness of people in medical practice of the role of low birthweight, will lead to a better management of this group of our population that is increasingly surviving into adulthood.

The thesis examines the relationship of birthweight to risk factors and markers, such as proteinuria and glomerular filtration rate, for chronic disease in postnatal life. It made use of the Australian Diabetes, Obesity and Lifestyle Study (AusDiab). The AusDiab study is a cross sectional study where baseline data on 11,247 participants were collected in 1999-2000. Participants were recruited from a stratified sample of Australians aged ≥ 25 years, residing in 42 randomly selected urban and non-urban areas (Census Collector Districts) of the six states of Australia and the Northern Territory. The AusDiab study collected an enormous amount of clinical and laboratory data. During the 2004-05 follow-up AusDiab survey, questions about birthweight were included. Participants were asked to state their birthweight, the likely accuracy of the stated birthweight and the source of their stated birthweight. Four hundred and twelve chronic kidney disease (CKD) patients were approached, and 339 agreed to participate in the study. The patients completed the same questionnaire. Medical records were reviewed to check the diagnoses, causes of kidney trouble and SCr levels. Two control subjects, matched for gender and age, were selected for each CKD patient from participants in the AusDiab study who reported their birthweight. Among 7,157 AusDiab participants who responded to the questionnaire, 4,502 reported their birthweights, with a mean (standard deviation) of 3.4 (0.7) kg. The benefit and disadvantages of these data are discussed in chapter three. The data were analysed for the relationship between birthweight and adult body size and composition, disorders of glucose regulation, blood pressure, lipid abnormalities, cardiovascular diseases and glomerular filtration rate. Low birthweight was associated with smaller body build and lower lean mass and total body water in both females and males. In addition low birthweight was associated with central obesity and higher body fat percentage in females, even after taking into account current physical activity and socioeconomic status. Fasting plasma glucose, post load glucose and glycosylated haemoglobin were strongly and inversely correlated with birthweight. In those with low birthweight (< 2.5 kg), the risks for having impaired fasting glucose, impaired glucose tolerance, diabetes and all abnormalities combined were increased by 1.75, 2.22, 2.76 and 2.28 for females and by 1.40, 1.32, 1.98 and 1.49 for males compared to those with normal birthweight (≥ 2.5 kg), respectively. Low birthweight individuals were at higher risk for having high blood pressure ≥ 140/90 mmHg and ≥ 130/85 mmHg compared to those with normal birthweight. People with low birthweight showed a trend towards increased risk for high cholesterol (≥ 5.5 mmol/l) compared to those of normal birthweight. Females with low birthweight had increased risk for high low density lipoprotein cholesterol (≥ 3.5 mmol/l) and triglyceride levels (≥ 1.7 mmol/l) when compared to those with normal birthweight. Males with low birthweight exhibited increased risk for low levels of high density lipoprotein cholesterol (<0.9 mmol/l) than those with normal birthweight. Females with low birthweight were at least 1.39, 1.40, 2.30 and 1.47 times more likely to have angina, coronary artery disease, stroke and overall cardiovascular diseases respectively, compared to those ≥ 2.5 kg. Similarly, males with low birthweight were 1.76, 1.48, 3.34 and 1.70 times more likely to have angina, coronary artery disease, stroke and overall cardiovascular diseases compared to those ≥ 2.5 kg, respectively. The estimated glomerular filtration rate was strongly and positively associated with birthweight, with a predicted increase of 2.6 ml/min (CI 2.1, 3.2) and 3.8 (3.0, 4.5) for each kg of birthweight for females and males, respectively. The odd ratio (95% confidence interval) for low glomerular filtration rate (<61.0 ml/min for female and < 87.4 male) in people of low birthweight compared with those of normal birthweight was 2.04 (1.45, 2.88) for female and 3.4 (2.11, 5.36) for male. One hundred and eighty-nineCKD patients reported their birthweight; 106 were male. Their age was 60.3(15) years. Their birthweight was 3.27 (0.62) kg, vs 3.46 (0.6) kg for their AusDiab controls, p<0.001 and the proportions with birthweight<2.5 kg were 12.17% and 4.44%, p<0.001. Among CKD patients, 22.8%, 21.7%, 18% and 37.6% were in CKD stages 2, 3, 4 and 5 respectively. Birthweights by CKD stage and their AusDiab controls were as follows: 3.38 (0.52) vs 3.49 (0.52), p=0.251 for CKD2; 3.28 (0.54) vs 3.44 (0.54), p=0.121 for CKD3; 3.19 (0.72) vs 3.43 (0.56), p= 0.112 for CKD4 and 3.09 (0.65) vs 3.47 (0.67), p<0.001 for CKD5. The results demonstrate that in an affluent Western country with a good adult health profile, low birthweight people were predisposed to higher rates of glycaemic dysregulation, high blood pressure, dyslipidaemia, cardiovascular diseases and lower glomerular filtration rate in adult life. In all instances it would be prudent to adopt policies of intensified whole of life surveillance of lower birthweight people, anticipating this risk. The general public awareness of the effect of low birthweight on development of chronic diseases in later life is of vital importance. The general public, in addition to the awareness of people in medical practice of the role of low birthweight, will lead to a better management of this group of our population that is increasingly surviving into adulthood.