Preterm birth and the immature cardiovascular system

Preterm birth (defined as birth prior to 37 completed weeks of gestation) affects 8-10% of all live births worldwide. Preterm birth introduces the cardiovascular system of the baby to the ex utero environment before cardiovascular development and maturation is complete. Being born necessarily causes a profound haemodynamic change within the cardiovascular system, including a shift from right ventricular dominance to left ventricular dominance, closing of the ductus arteriosus and foramen ovale, and an increase in heart rate and arterial blood pressure. The broad aim of this thesis was to understand how preterm birth affects the development of the immature cardiovascular system. To achieve this aim I have used an ovine model of preterm birth, and autopsy material from human infants. Due to the imposition of ex utero life on an immature and therefore ill-prepared cardiovascular system, it was hypothesised that preterm birth would lead to an early termination of cardiomyocyte proliferation, abnormal cardiomyocyte maturation, increased interstitial collagen deposition, and thicker aortic blood vessels with a reduced lumen diameter. Prior to the commencement of the studies described in this thesis, relatively little was known about how preterm birth affected the development and maturation of the cardiovascular system. The aim of the first experimental chapter in this thesis (Chapter 2) was to examine the effect of preterm birth on the development and maturation of the cardiomyocytes (heart muscle cells). Preterm lambs were born at a time-point approximating moderately preterm birth (ie. 133 days of the 147 day gestation in sheep (0.9 of term); this is developmentally equivalent to ~32 weeks of human gestation. Lambs born at term were used as controls. The hearts of the lambs were examined at 9 weeks post-term equivalent age. To determine whether or not preterm birth had an effect on the development and maturation of cardiomyocytes, I utilised stereology to examine the number of cells within the heart, confocal microscopy to measure the size, nuclearity and ploidy (the number of genome copies per nucleus) of the cardiomyocytes, and picrosirius red staining to examine the amount and distribution of collagen within the heart. This study demonstrated that at 9 weeks post-term equivalent age, lambs born preterm, compared with controls, had hearts with enlarged cardiomyocytes in both the left and right ventricles, abnormal maturation of the cardiomyocytes (remaining mononucleated but became polyploid) and increased interstitial collagen deposition. The publication arising from this study was the first published study to examine the effect of preterm birth on the development and maturation of cardiomyocytes. The aim of the second experimental chapter (Chapter 3) was to examine how preterm birth affects the morphometry and composition of the great vessels (the aorta and pulmonary artery), using the same ovine model of preterm birth as in the previous chapter. This is important as the aorta and pulmonary artery change their roles dramatically at the time of birth. The aorta in utero is exposed to relatively low systolic/diastolic pressures, but high systolic/diastolic pressure after birth. The pulmonary artery in utero supplies a large proportion of the blood to the whole body (via the ductus arteriosus) and experiences systemic arterial pressure, whereas ex utero it supplies only the pulmonary circulation and its pressure is considerably lower than systemic arterial pressure. The results of this study showed that preterm birth led to thickening of the aortic wall coupled with a narrower lumen, but without any changes in pulmonary artery morphometry. The composition of vessel walls was also altered, in that the aortae from the preterm lambs had increased elastin and reduced smooth muscle; the pulmonary artery only had increased elastin deposition. The most concerning finding were areas of wall injury in the aortae from preterm lambs, which was not observed in lambs born at term. Chorioamnionitis is an inflammatory process, usually resulting from a microbial infection, affecting the chorion, amnion, umbilical cord and amniotic fluid; it is the most common antecedent of human preterm birth. The aim of the third experimental chapter (Chapter 4) was to determine how chorioamnionitis affects the development and maturation of cardiomyocytes, using an ovine model of acute chorioamionitis. On day 120 of the 147 day gestational period (0.82 of term), LPS (lipopolysaccharide, a protein from the outer coat of gram negative bacteria) was injected under ultrasound guidance into the amniotic sac. Seven days later (127 days, 0.86 of term), the fetus was humanely killed and the heart removed for examination. I used confocal microscopy to examine the number, size, maturation and ploidy of the cardiomyocytes, immunohistochemistry to measure cell proliferation and picrosirius red staining to measure the amount and distribution of interstitial collagen within the heart. Lambs exposed to LPS, compared to controls, had smaller mononucleated (immature) but larger binucleated (mature) cardiomyocytes, a reduction in estimated cardiomyocyte number and increased capillary density. The reduction in estimated cardiomyocyte number was offset by an 8-fold rise in cardiomyocyte proliferation in LPS exposed lambs. The aim of the fourth experimental chapter (Chapter 5) was to assess the effects of preterm birth on development of the human heart. To achieve this aim I obtained archival heart tissue from the Women’s and Children’s Hospital in Adelaide, South Australia; the fixed infant cardiac tissue was obtained at perinatal autopsies following written consent from the parents. I analysed heart tissue from 13 infants born preterm, but who subsequently died having lived for at least one day. As controls I analysed heart tissue from 17 stillborn infants who died in utero from acute causes. I used confocal microscopy to examine cardiomyocyte volume, maturation, and ploidy. Cell proliferation was measured using immunohistochemistry and interstitial collagen deposition was measured using picrosirius red staining and image analysis. The results of this study demonstrated that there was no effect of preterm birth on heart weight (absolute or relative to bodyweight) or on cardiomyocyte volume, maturation and ploidy. Cardiomyocyte proliferation was negligible in all but one of the preterm born infants, as early as 24 hours of birth. There was a small, but statistically significant increase in collagen deposition in the hearts of preterm born infants. In order to perform the cardiac analyses necessary for these studies, I developed an improved method for morphometrically analysing cardiomyocytes in thick paraffin sections (Chapter 6). Ordinarily, it is almost impossible to identify the boundaries of cardiomyocytes in paraffin sections stained with haematoxylin and eosin. Wheat germ agglutinin (a lectin) bound to a fluorescent dye has previously been used to delineate cell boundaries in many tissues. I used wheat germ agglutinin-Alexa Fluor 488 to stain the cardiomyocyte cell membranes and a nuclear dye (e.g. DAPI, YOYO-3) to identify the nuclei. Utilising confocal microscopy and image analysis I was able to quantitatively analyse cardiomyocytes in thick (≥40µm) paraffin sections including measurement of cell volume, nuclearity (number of nuclei per cell) and ploidy (number of genome copies per nucleus). This improved technique enabled the studies described in this thesis to proceed, and will be helpful to others in better characterising heart tissue embedded in paraffin, such as autopsy specimens. In conclusion, the results of my studies demonstrate that preterm birth, and one of the major risk factors for preterm birth (intrauterine inflammation), has potentially serious adverse effects on the development and maturation of the heart and major arteries. My findings suggest that people born preterm are likely to be at increased risk of developing cardiovascular disease in later life; hence it is important in future studies to closely examine the postnatal growth and structure and function of hearts and arteries from adults who were born moderately, very and extremely preterm. The monitoring of cardiovascular health throughout life in individuals born preterm would enable early intervention and possibly prevention of overt cardiovascular disease