A comparison of the contractile properties of human myometrium obtained from the upper and lower uterine segments
Correspondence: Dr M. J. M. Luckas, University of Liverpool, Department of Obstetrics and Gynaecology, Liverpool Women's Hospital, Crown Street, Liverpool L8 7SS, UK.
This study compared the contractile characteristics of myometrium taken from upper and lower uterine segments. Biopsies were obtained from women undergoing classical caesarean section. Myometrial strips were dissected and mounted in an organ bath, and the contractions were recorded. The cross sectional area of the contractile elements within the strips was measured enabling strips of differing dimensions to be compared. There were no significant differences in the contractile rate and force production produced by myometrium from the upper and lower segments. This study demonstrated that for contractile studies, the use of lower segment is appropriate. The results fail to demonstrate any functional regionality of the human uterus in terms of contractility.
The uterine body can be divided into upper (corpus) and lower segments (isthmus) on an anatomical basis. The proportion of the wall of the uterus in both regions during pregnancy that is made up by smooth muscle is approximately 50%. Nevertheless, the lower segment remains thinner and therefore more distensible than the upper segment, resulting in the lower segment being passively stretched during pregnancy and labour.
More recently, the idea of functional regionality of the uterus has been suggested. A number of physiological differences between the upper and lower uterine segments have been noticed. A fundus to cervix gradient has been described for prostaglandin receptors1, myometrial expression of cyclooxygenase 1 and 2, oxytocin receptors2 and progesterone receptors3. The myometrial gap junction protein connexin-43, strongly associated with increased uterine contractility and labour, also shows a fundus to cervix gradient in pregnant human myometrium4.
It is hypothesised that these regional differences make the upper segment more responsive to uterotonins and may underlie the propagation of uterine contractions from the fundus to the cervix4, the greater expression of relaxing prostaglandin EP2 and endothelinB receptors makes the lower segment relatively passive, thus enabling it to be thinned out and drawn up as labour progresses. Interestingly, Wedenberg et al.5 demonstrated a higher energy charge signified by the adenosine triphosphate:adenosine diphosphate ratio in the lower segment compared with the upper segment5, perhaps reflecting a lower use of energy by the relatively quiescent lower segment.
The most easily accessible source of human myometrium from pregnant women for research purposes is biopsy taken at the time of caesarean section. The vast majority of these operations are performed through the lower segment. A criticism of the use of this lower segment tissue is that perhaps it does not reflect what is going on in the uterus as a whole.
This study was designed to compare the contractile properties of myometrium obtained from upper and lower uterine segments.
Full thickness, paired uterine wall biopsies were obtained from 17 women undergoing classic caesarean section. The biopsies were obtained from the cephalic and caudal extremities of the caesarean section incision to obtain upper and lower segment tissue. The tissue was then placed in buffered physiological saline (Krebs) solution (composition: NaCl 154 mM, KCl 5.4 mM, MgSO4 1.2 mM, glucose 12 mM, CaCl2 2 mM, Hepes 10 mM, pH 7.40). Ethical approval was granted by the local ethics committee (Royal Liverpool University Hospital) and written informed consent was obtained from each participating woman. The indication for classical caesarean section was a poorly formed lower segment in 12 women: severe pre-eclampsia (n= 7); early onset intrauterine growth retardation (n= 4); and footling breech in premature labour (n= 1). In three women the indication was anticipated difficulty in delivering the fetus through a lower segment incision: transverse fetal lie (n= 2); and omphalocoele (n= 1). In two cases the indication was a previous classical caesarean section. Exclusion criteria were significant medical complications or medication likely to affect myometrial activity. The median gestation at tissue collection was 30 weeks (range 23 weeks 6 days to 41 weeks 4 days).
Under stereomicroscopic control, strips of longitudinal myometrial smooth muscle were dissected (3 × 10 mm). One strip from each paired biopsy was mounted in parallel on a standard organ bath (Linton Instruments, Norfolk, UK) and attached to a tension transducer (Grass FSG-01). The strips were stretched to 1.5-fold their resting length as this has been previously shown to enable maximum force generation6. Force production was recorded digitally and the tissue was perfused with oxygenated physiological saline at 37°C.
Once a stable pattern of contractile activity was established, spontaneous force production was recorded for two hours. After this, the strips were perfused with physiological saline containing 10 nm oxytocin (Sigma Chemical Company, Fancy Road, Poole, Dorset, UK). Again, force production was recorded for two hours. Lastly the strips were depolarised by perfusing with a high potassium (40 mM K+) physiological saline solution, made up by isosmotic replacement of sodium chloride with potassium chloride, and peak force production was recorded.
At the end of the experiment, the strips were fixed in formal saline (4% formaldehyde in normal saline). At a later stage, they were embedded in paraffin and sliced on a standard microtome (5 μm slices). The tissues were then stained according to van Gieson's technique using standard histological techniques. The sections were then examined under light microscopy, the van Gieson's stain enabling the contractile and noncontractile elements to be differentiated. Using a standard haematological graticule, the cross sectional area of the contractile elements from each strip was measured. Five erythrocytes (also stained yellow by van Gieson's stain) were examined in ten different specimens. The mean diameters were calculated and compared using analysis of variance (Arcus Biomedical 1997, Ian Buchan, UK). The mean diameter of the erythrocytes did not significantly differ in different specimens indicating that fixation artefact was unlikely to result in a significant difference in the measured cross sectional area of individual specimens.
Analysis was performed using Microcal Origin 4.10 (Microcal Software Inc, Northampton, Massachusetts, USA). For the spontaneous and oxytocin augmented force production, five contractions were averaged and peak force measured in mN, the area under the curve was also recorded. Force was expressed as mN/mm2 of contractile tissue.
Contractile frequencies were expressed as median (range) and peak force production and area under the curve as mean (SD). Normally distributed data were compared using paired t tests and non-normally distributed data with Wilcoxon's signed rank test. Statistical significance was set at 5% (0.05). The methodology was validated by dissecting two strips of different thickness from five biopsies. Force production was recorded as described. The coefficient of variation for the methodology was 9.6%.
Of the 17 sets of pairs of myometrial strips obtained, two sets were discarded because one strip in each pair failed to contract, leaving 15 pairs of strips for analysis. Five of these 15 pairs were obtained from women at gestations > 37 weeks. There were no significant differences in contractile rates, peak force production and area under the curve for spontaneous contractile activity, oxytocin augmented contractions and lastly potassium depolarised contractions shown by tissue from the upper and lower uterine segments (Table 1). In addition, increasing gestation did not affect the contractile parameters outlined above or the amount of myometrium within the samples (data not shown).
Table 1. Contractile properties of paired muscle strips from the upper and lower uterine segment. Values are given as median [range] or mean (SD). AUC = area under the curve.
|Lower segment||0.17 [0.10–0.24]||44.8 (13.4)||1531.6 (262.8)||0.17 [0.12–0.22]||44.0 (10.6)||1632.8 (249.8)||50.4 (10.5)|
|Upper segment||0.17 [0.10–0.25]||47.2 (18.5)||1580.4 (325.3)||0.20 [0.11–0.23]||48.2 (17.6)||1597 (215.7)||53.3 (17.2)|
The mean (SD) proportion of the myometrial strips cross sectional area composed of contractile tissue was similar in the two regions; 65.4% (5.68) in the upper segment and 69.8% (12.32) in the lower segment.
This study found no differences in the in vitro contractile properties of myometrial strips obtained from the upper and lower segments of the uterus of pregnant women. Although this is in keeping with observations made by Wikland et al.1, where the authors made no attempt to correct for the physical differences in the myometrial strip dimensions. In a study primarily designed to investigate the relationship between myosin light chain phosphorylation and force generation, Word et al.6 also found no difference between upper and lower segment tissue from pregnant women in terms of contractile response to oxytocin and potassium chloride. The proportion of muscle in the myometrial strips examined in the study by Word et al.6 was similar (65% cross sectional area of the pregnant myometrium being composed of contractile elements) to that found in our study (65.4% in the upper segment and 69.8% in the lower segment being composed of contractile elements). However, to enable the comparison of the force generation of different strips, Word et al. calculated the cross sectional area of their strips using wet weight, stretched length and tissue density, and did not account for the noncontractile tissue within them. We believe our methodology was more robust.
Although we found no evidence of a difference in the contractile properties of tissue from the two regions of the uterus, our study is small. This is of necessity because of the rarity of obtaining upper segment tissue from pregnant women. Additionally, we can only comment on the in vitro contractile properties of the two areas. It must be remembered that the contractile properties of myometrium perfused in an organ bath may bear little resemblance to the in vivo state indicating the importance of the local environment. Nevertheless, we believe this work validates the use of lower segment tissue for in vitro research into human myometrial contractility.