Human Reproduction, Vol. 14, No. 7, 1885-1889,
July 1999
© 1999 European Society of Human Reproduction and Embryology
Pregnancy is not associated with altered morphology of the femoral artery
1,3,42221,31 1Division of Cardiovascular Diseases, Department of Medicine, Guggenheim-7, Mayo Clinic and Foundation, Rochester, MN 55905, USA and 2 Department of Anatomy, Veterinary Faculty, Belgrade, Yugoslavia
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Abstract |
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While pregnancy is associated with adjustments in cardiovascular function, the morphology of the vascular system during pregnancy has been generally viewed as being very stable. However, recently we have demonstrated that pregnancy remodels the aorta and the carotid artery. In the present study, we assessed the morphological characteristics of the guinea-pig femoral artery during different stages of pregnancy using light and electron microscopy. There were no significant differences between external and internal diameters, wall thickness, total cross-sectional area and cross-sectional areas of lumen, intima, media, and adventitia of femoral arteries from non-pregnant and early-, mid- and late-pregnant guinea-pigs (n = 8–10). In previous studies, we have shown that the morphology of vascular smooth muscle and endothelial cells in the aorta and the carotid artery may be altered by pregnancy. Therefore, to test this possibility we measured diameters as well as cross-sectional areas of femoral arterial muscle and endothelial cells using electron microscopy. These parameters, at the electron microscopy level, were also not significantly changed by pregnancy (n = 8–10). It is concluded that the morphology of the guinea-pig femoral artery is not altered during pregnancy. In this regard, this study demonstrated that pregnancy-induced vascular remodelling varies between blood vessels that undergo the same functional alterations. Therefore, this may suggest that pregnancy-induced changes in blood flow through different vascular beds are not the most important factor involved in vascular remodelling observed during pregnancy. Rather, it is possible that haemodynamic-independent factors regulate pregnancy-mediated structural changes of the vascular wall.
Key words: endothelium/femoral artery/pregnancy/remodelling/stereology
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Introduction |
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It is known that cardiac output is increased during pregnancy, which is associated with increased rates of perfusion to both reproductive and non-reproductive organs (Rosenfeld, 1977
). This vascular adaptation of mothers to pregnancy is considered to be due to blunted vascular response to vasoconstrictors on the one hand (Weiner et al., 1991; Grbovi and Jovanovi, 1996, 1997; Jovanovi et al., 1998b), and increased response to vasodilators on the other (Weiner et al., 1989), although this concept has been repeatedly challenged (Jovanovi et al., 1994a, 1995a,b,c, 1997).
While pregnancy is associated with adjustments in cardiovascular function, the morphology of the vascular system during pregnancy has been generally viewed as being very stable (Rosenfeld, 1977; Peeters et al., 1980). However, recently we have demonstrated that pregnancy remodels the aorta and the carotid artery (Jovanovi and Jovanovi, 1997, 1998). Specifically, it has been shown that pregnancy induces hypotrophy of vascular endothelium in guinea pig aorta and carotid artery as well as hypotrophy of vascular smooth muscle in the guinea-pig carotid artery (Jovanovi and Jovanovi, 1997, 1998). Therefore, in the present study, we addressed further the effect of pregnancy on the morphological characteristics of the cardiovascular system, i.e. we assessed the morphological characteristics of the guinea-pig femoral artery during different stages of pregnancy.
Here, we report that pregnancy does not alter the morphology of the femoral artery.
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Materials and methods |
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Non-pregnant female guinea-pigs and early-pregnant (day 22 of pregnancy), mid-pregnant (day 44 of pregnancy) and late-pregnant (days 64–66 of pregnancy) guinea-pigs were used in this study (8–10 in each group), as previously described (Jovanovi
and Jovanovi, 1997, 1998). Briefly, guinea-pigs were anaesthetized with sodium pentobarbital (40 mg/100 g, i.p.), the femoral artery was removed, cut, fixed in 10% buffered neutral formaldehyde (48 h) (Fluka, Buchs, Switzerland), dehydrated through a graded series of ethanol solutions (70–100%) and embedded in paraffin wax. Resultant blocks were cut at 3 µm (Sorval JB-4 microtome, Newtown, CT, USA). Sections were stained with haematoxylin and eosin and each section was examined under x20 magnification (Olympus Vanox microscope, Tokyo, Japan). For electron microscopy femoral arteries were cut into rings (1 mm long), fixed in 3% glutaraldehyde (Fluka) for 24 h and after rinsing in 0.1 mol/l cacodylate buffer (pH 7.4) (Fluka) postfixed in 2% osmium tetroxide in cacodylate solution (Fluka) (1 h). The specimens were dehydrated in increasing concentrations (70–100%) of ethanol, and then passed through propylene oxide and embedded in Araldite (Jovanovi and Jovanovi, 1997, 1998; Jovanovi et al., 1998a). Ultrathin sections (50–70 nm) were cut with a diamond knife. The sections were stained with uranyl acetate (Fluka) and Reynold's lead citrate (Fluka) and then examined and photographed using a Philips EM 400-HMG electron microscope (Eindhoven, The Netherlands). The investigation conformed with the `Guide for the Care and Use of Laboratory Animals' (NIH publication 85–23, revised 1985).
Morphometric and stereological analysis
Light microscopy
Sections that included the entire circumference of each ring were cut from five different blocks of each animal and viewed with light microscopy. For each block five sections were examined. Maximal and minimal internal and external diameters were determined at magnification x20, and these values were used to determine the mean values of internal and external diameters. Wall thickness was calculated by subtraction of the two diameters and dividing by 2 (Jovanovi and Jovanovi, 1997, 1998; Jovanovi et al., 1998a). Cross-sectional areas were determined using the Weibel M42 standard point-counting system (Weibel, 1979; Jovanovi and Jovanovi, 1997, 1998). The cross-sectional areas were calculated using the following equation (Weibel, 1979; Lee et al., 1983): Am = (Pi/Pt)xAg; where Am = cross-sectional area, Pi = number of points falling on specific layer, Pt = total points of test grid and Ag = area of the whole test grid for the appropriate magnification (x20). Correction for eccentricity due to sectioning angle with reference to the long axis of the vessel was used for all calculations (Lee et al., 1983; Jovanovi and Jovanovi, 1997, 1998; Jovanovi et al., 1998a): correction = d1/d2 where d1 = minimal radius of vessel, d2 = maximal radius of vessel. The definite form of the equation used was Am = (Pi/Pt)xAgx(d1/d2).
Electron microscopy
Morphometric analyses were made from 20 random electron micrographs (five micrographs per block, four blocks per aorta) obtained at x2800–7900 magnification. The size of endothelial and smooth muscle cells, defined as cross-sectional area (Lee et al., 1983), were determined using the B 100 standard point-counting system (Weibel, 1979). To avoid errors in making average estimates of cell objects without having information about individual objects, the point counting was performed by eye, and the cross-sectional area of each individual cell profile was determined.
Cross-sectional area was calculated using the following equation A = Pixd' (Weibel, 1979); where A is the cell cross-sectional area; Pi is the number of points falling on cell cross-sectional area; d is the distance between the nearest points of the grid; d' is the distance between the nearest points of the grid corrected for magnification: d' = d/magnification (Weibel, 1979; Lee et al., 1983).
Statistical analysis
For all vessel parameters, separate data measurements of diameters, wall thickness and cross-sectional profiles of vascular components from each animal were respectively pooled and an average value recorded, so that in the analysis each animal contributed only one value for each parameter. All data were tested by the Kolmogorov Goodness of Fit test and were found to be normally distributed. The results are expressed as means ± SEM with range of values in parenthesis; n refers to the number of animals. One-way analysis of variance (ANOVA) was used when more than two groups were analysed and the individual pregnant groups were compared with the non-pregnant one, which served as a control. A value of P < 0.05 was considered to be statistically significant.
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Results |
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Light microscopy
The values of external and internal diameters and wall thickness of femoral artery from guinea-pigs in different stages of pregnancy are shown in Table I
. There were no statistically significant differences between those values (n = 8–10). To examine more precisely the influence of pregnancy stage on the morphology of the femoral artery, we calculated the cross-sectional areas of different arterial layers in non-pregnant, early-, mid- and late-pregnant animals (Figure 1). The values of cross-sectional areas were not significantly different between animals in different stages of pregnancy (Figure 1; n = 8–10).
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Electron microscopy
Obtained values of maximal and minimal diameters of endothelial and smooth muscle cells are presented in Tables II and III
respectively. Photomicrographs of endothelial and muscle cells from non-pregnant and late-pregnant guinea-pig femoral arteries are presented in Figure 2, while values for cross-sectional areas of endothelial and muscle cells during all stages of pregnancy are presented in Figure 3. Statistical analysis revealed that diameters and cross-sectional areas of individual endothelial and smooth muscle cells were not significantly changed by pregnancy in both cases (n = 8–10).
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Discussion |
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In different animal species, including humans, it has been reported that pregnancy is associated with changes of blood flow throughout the body (Peeters et al., 1980
; Easterling et al., 1991; Magness et al., 1991). It has been postulated that this cardiovascular adaptation to pregnancy may be due to altered vascular reactivity toward neurotransmitters and humoral factors, leading to general maternal vasodilatation (Conrad et al., 1991; Grbovi and Jovanovi, 1996; Jovanovi et al., 1998b).
As opposed to the aorta and the mesenteric and uterine arteries, the effect of pregnancy on the function and morphology of the femoral artery has been less investigated. In fact, there are no reports that addressed the outcome of haemodynamic changes on the femoral artery morphology. So far, it has been demonstrated that the reactivity of the femoral artery to vasodilator substances is increased during pregnancy (Ahokas et al., 1991). Previously, Awal et al. (1995) have shown that, as observed in the uterine artery (Osol and Cipolla, 1993; Cipolla and Osol, 1994), pregnancy increases the diameter of the femoral artery. In contrast, here we demonstrate that the diameter of the guinea-pig femoral artery is not altered by pregnancy. This disagreement may be due to the differences in species examined in these two studies. Specifically, Awal et al. (1995) performed their study on rat blood vessels, while the current study used guinea-pigs. Guinea-pigs were specifically used in the present study, having in mind that functional changes in the cardiovascular system during guinea-pig pregnancy are apparently similar to that in humans (Jovanovi et al., 1994b,c, 1995b,c,d, 1998c).
To test definitively the possibility that changes in the morphology of the femoral artery during pregnancy do occur, we applied morphometry and stereology on electron micrographs, as previously described (Jovanovi and Jovanovi, 1997, 1998; Jovanovi et al., 1998a, 1999). This analysis confirmed our findings obtained with light microscopy, and provided more evidence for the notion that the morphology of the intima and media of the guinea-pig femoral artery is not altered by pregnancy. Therefore, the use of precise morphometric and stereological methods, at the level of both light and electron microscopy, allows us to conclude that pregnancy is without effects on the morphology of the guinea-pig femoral artery. Previously, we have reported that pregnancy remodels guinea-pig aorta and carotid artery (Jovanovi and Jovanovi, 1997, 1998) in a similar way. However, during pregnancy blood flow is increased through the aorta, but decreased through the carotid artery (Rosenfeld, 1977). Here, we demonstrated that the femoral artery, which is characterized by pregnancy-associated functional alterations, is not morphologically changed by pregnancy. Taken together, these studies, as well as the present findings, may suggest that pregnancy-induced changes in blood flow through different vascular beds are not the most important factor involved in the vascular remodelling observed during pregnancy. Rather, it is possible that haemodynamic-independent factors regulate pregnancy-mediated structural changes of the vascular wall.
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Acknowledgments |
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This work was supported by the Merck Company Foundation and the American Heart Association.
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Notes |
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3 Present address: Department of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 4HN, Scotland, UK
4 To whom correspondence should be addressed: Department of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 4HN, Scotland, UK
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References |
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Submitted on December 29, 1998; accepted on March 22, 1999.
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