Objective
Knowledge of the stereology of human myometrium in pregnancy is limited. Uterine contractile performance may be altered in association with maternal obesity and advanced maternal age. The aim of this study was to investigate the stereology of human myometrium in pregnancy, and to evaluate a potential influence of maternal body mass index (BMI) and age.
Study Design
Biopsies of human myometrium were obtained from 57 women at cesarean section (n = 26, n = 13, n = 18 normal, overweight and obese BMI, respectively), and volume fractions of smooth muscle and extracellular matrix were assessed using stereologic techniques.
Results
The smooth muscle constituted 65.2% ± 8.9% (standard deviation) and the extracellular matrix 32.6% ± 7.7% (standard deviation) (n = 57). There was no correlation observed between maternal BMI, age, or parity with the fractional volumes of either smooth muscle or extracellular matrix.
Conclusion
These results outline the stereology of human myometrium in pregnancy. Putative functional differences in contractility, pertaining to obese or older mothers, are not related to smooth muscle content.
The myometrium, which is the major tissue component of the uterus, is composed predominantly of smooth muscle and extracellular matrix, and both of these tissue components are synchronously regulated to enable the uterus to vary its contractile state considerably, from relative quiescence before labor, to one of maximal contractility and force development during labor. Our knowledge of the number, or relative proportion, of smooth muscle fibers that exist in normal myometrium, is limited.
There is some evidence that the smooth muscle content of the uterus progressively diminishes caudally, ie, from fundus to cervix, where it may be as low as 10% in the latter location. It is also held that there is more smooth muscle in the inner layers of the myometrium, in comparison to the outer layers, and in the anterior and posterior walls of the uterus, in comparison to the lateral walls. However, apart from the belief that myometrial muscle fibers undergo hypertrophy during pregnancy and that there may be an increase in collagen and elastin during pregnancy there are no data, to our knowledge, evaluating the relative proportions of smooth muscle and extracellular matrix (ECM) in human myometrium obtained during pregnancy, in any clinical state.
Whether the proportions of smooth muscle and ECM that exist in myometrial tissue exert an influence on the efficacy of contractility, ie, function, is unclear, but it is reasonable to presume that they may be linked. For example, it has been demonstrated that women with slow progress of labor had higher concentrations, and lower extractability, of collagen in the uterine isthmus and cervix when compared with women with normal labor. Other clinical states that have been suggested as having an influence on functional uterine contractility include maternal obesity and advanced maternal age. Obesity, which is currently a major health problem in the developed world, is a condition that has been associated with poor uterine contractility for many clinical reasons. In addition, there are recent scientific reports demonstrating the potential inhibitory effects of obesity on uterine myocyte function, and contractility. However, this concept of poor uterine contractility in obese women remains controversial, and recent evidence suggests that the in vitro contractile properties of human myometrium are similar, irrespective of maternal body mass index (BMI). There is similar controversy about the clinical and physiologic effects of advancing maternal age on the efficacy of uterine contractility.
The primary aim of this study was to evaluate the smooth muscle cell and ECM content of human myometrium obtained from women in pregnancy. The secondary aim was to compare the findings observed in myometrium from women of normal BMI to those observed in women from overweight and obese BMI categories, and to investigate a possible correlation with maternal BMI. Finally, the effects of maternal age on the findings were examined.
Materials and Methods
The women included in the study were all attending Galway University Hospital, Ireland, for antenatal care during the period July 2009-June 2010, and had an elective prelabor cesarean section performed. The BMI was calculated at the time of the initial antenatal visit in the first trimester of pregnancy. The World Health Organization (WHO) international classification was used, whereby a BMI calculation of 18.50-24.99 was regarded as normal, 25.0-29.9 was overweight, and a value of 30.0 or above was regarded as obese. For recruitment, patients were provided with an information sheet, and written consent for tissue collection was obtained. Ethics Committee (institutional review board) approval for tissue collection was obtained from the Research Ethics Committee at Galway University Hospital. Biopsies of human myometrium were dissected, as previously described, from the upper lip of the uterine incision made at elective lower segment cesarean section operations, ie, from a location where the putative upper and lower segments of the uterus meet. Immediately on collection the biopsies were placed in physiologic salt solution (PSS). The PSS was of the following composition: 4.7 mmol/L potassium chloride, 118 mmol/L sodium chloride, 1.2 mmol/L magnesium chloride, 1.2 mmol/L calcium chloride, 1.2 mmol/L potassium phosphate, 25 mmol/L sodium bicarbonate, and 11 mmol/L glucose (Sigma-Aldrich, Dublin, Ireland). After collection, the biopsy specimens were dissected free of decidua and serosa, where appropriate, and myometrial strips were prepared, with a macroscopic longitudinal orientation, (ie, along the direction of the fibers), measuring approximately 2 × 2 × 10 mm. In cases where presumptive scar tissue was noted, this was also dissected away. This process has been outlined digramatically in Figure 1 , demonstrating representative tissue samples.
The biopsy strips were fixed in 4% paraformaldehyde and processed for routine histology. Sections were stained using Masson’s Trichrome method. Simple point counting methods, as described by Howard and Reed, were then used to assess the volume fractions (Vv) of the smooth muscle cell (SMC) content and the ECM content. These were then converted to percentage of the total tissue. A uniform random sampling approach was used to capture images with a Leica brightfield microscope; an initial image was taken from a random area of the slide and subsequent images were taken at regular intervals from this starting point. A 36-point grid was superimposed on the image selected and each intersection of lines on this grid was considered a sampled point.
A representative tissue image is shown in Figure 2 , with the superimposed grid demonstrated, and examples of “points” highlighted. The spacing between the points was 36 μm. The numbers of points which coincided with SMC or ECM were counted. The volume fractions of SMC and ECM were then calculated by expressing the proportion of points hitting either tissue component as a fraction of the total number of points observed on any tissue. For each biopsy sample (representing an individual patient) counts were made from 10 different images resulting in a total point count, from which the final Vv was calculated. The optimum number of sampled points, or images counted, was determined using an average summation graph. This involves calculating a cumulative average Vv after each image that is counted. We determined the number of images at which this mean achieved a steady state wherein it did not vary by more than 5% after each subsequent image is added.
Data were tested for normality as well as equal variance. Data that were normally distributed, and of equal variance, were expressed as the mean ± standard deviation (SD), and were compared using the unpaired t test. When normality failed, data were compared using the Mann-Whitney U test. Multiple comparisons were made using analysis of variance (ANOVA) and Kruskal-Wallis tests and because no differences were found no post hoc tests were used. Comparisons of maternal age across the groups were made using an ANOVA for multiple testing, and because no differences were found no post hoc tests were used. Correlations were evaluated using GraphPad Prism. A P value of < .05 was considered significant. The sample size, n = 57, had 80% power to detect a correlation of 0.325 between the parameters measured.
Results
There were 57 women recruited for the study of whom 26 were in the normal BMI category, 13 in the overweight BMI category, and 18 in the obese BMI category. All the women had a cesarean section performed electively, ie, before labor onset. The median gestation at delivery was 39 weeks, (range, 38−40). The mean BMI was 22.90 (n = 26) in the normal group, 27.56 (n = 13) in the overweight group, and 33.21 (n = 18) in the obese group ( P < .001). The mean age of all women recruited was 33.8 years (range, 18−45, n = 57). There were no significant difference observed between the ages of the women in the 3 different BMI groups, which were as follows: normal (mean, 32.8; range, 18−42; n = 26); overweight (mean, 35.6; range, 27−42, n = 13); and obese (mean, 33.8; range, 22−45; n = 18) groups. The reasons for cesarean section were as follows: previous cesarean section n = 33, breech presentation n = 15, unstable fetal lie n = 1, fetal hydrocephalus n = 1, placenta previa n = 1, maternal request n = 1, previous myomectomy n = 2, maternal lung malignancy n = 1, multiple pregnancy n = 1, maternal hypertension n = 1. The parity details (median, range, n) before the index delivery in the 3 BMI groups were as follows: normal (1, 0−2, n = 26); overweight (1, 0−2, n = 13); obese (2, 0−4, n = 18).
In relation to smooth muscle content, the mean SMC for all women was 65.2 ± 8.9% (n = 57). The mean SMC values observed in the 3 groups were as follows: women of normal BMI it was 65.6 ± 8.2% (n = 26); women of overweight BMI group, it was 63.8 ± 6.6% (n = 13); women of obese BMI group was 65.6 ± 11.4% (n = 18). These data are diagrammatically represented in Figure 3 , A. There was no significant difference observed in the SMC across the 3 groups ( P = .818). No significant correlation was observed between SMC relative volume and maternal BMI ( P = .65), maternal age ( P = .17), or parity ( P = .42). These data are demonstrated in Figure 4 , A, C, and E, respectively.
