Buhimschi, Catalin S. MD1; Buhimschi, Irina A. MD1; Zhao, Guomao1; Funai, Edmund MD1; Peltecu, Gheorghe MD2; Saade, George R. MD3; Weiner, Carl P. MD4
Cesarean delivery is the most frequently performed major surgical procedure in the United States.1 Since the original 1926 description by Kerr, the transverse lower uterine segment cesarean delivery has gained wide acceptance because of a lower mean blood loss and lower rate of uterine rupture in subsequent pregnancies.2 Unlike the evolution in the types of abdominal wall incisions,3–5 the surgical technique for the transverse incision on the lower uterine segment has changed little over the ensuing years. For instance, creation of a bladder flap is a typical part of a cesarean delivery although there is evidence that dissecting the urinary bladder from the lower uterine segment is not necessarily clinically beneficial.6
The formation of the uterus and upper vagina begins with the fusion of the two paramesonephric ducts.7 The fusion begins caudally and proceeds cranially to form a tube with a single lumen. This tube, called the uterovaginal canal, becomes the uterus, the cervix, and the upper portions of the vagina, whereas the unfused cranial portion of the paramesonephric ducts become the fallopian tubes. The mucous membrane that lines the cervix may also originate from the endodermal epithelium of the urogenital sinus. Interest in the isthmus, the portion of the uterus between the internal os of the cervix and the endometrial cavity, has grown over the years. Although it continues to be debated,8 the embryological origin of the isthmus could have a profound effect on the structure and function of the lower uterine segment during gestation.9
The anatomical relationship between the human cervix, uterine body, and lower uterine segment is the subject of conflicting theories.10 Some believe the cervix is pulled up during human parturition to become part of the lower uterine segment,11 whereas others suggest the lower uterine body extends downward toward the cervix to cover the inferior pole of the fetus.12 As a result of cervical dilation during active labor, the topographical arrangement of the lower uterine segment relative to the reflection of the visceral peritoneum may shift. Thus, the hysterotomy may occur at anatomically different sites, depending on whether or not the visceral peritoneal bladder flap is created.
We hypothesize that the arrangement and composition of the uterine wall matrix varies significantly with the site of the uterine incision (above or below the bladder flap reflection) and that such differences translate to divergent biomechanical properties of the lower uterine segment. We tested this hypothesis by examining the relationships between structural composition and tensile strength properties of the lower uterine segment above and below the reflection of the urinary bladder flap in women undergoing a clinically indicated cesarean delivery at term for dystocia.
MATERIALS AND METHODS
Lower uterine segment biopsies were obtained from 40 nulliparous laboring women undergoing a clinically indicated primary low-transverse cesarean delivery at term for dystocia. The study was approved by the Human Investigational Committee of Yale and Wayne State Universities. Subjects were recruited consecutively (March 2002 to April 2004) based on the availability of one of the investigators (C.S.B.), who was present for all surgical procedures. Each subject provided written informed consent and met the criteria for dystocia (failure to dilate or descend) despite adequate uterine contractility (assessed by intrauterine pressure catheter),13 and all subjects had requested epidural (or combined spinal-epidural) analgesia. The patient’s primary care obstetrician, who was not a member of the research team, made the decision for cesarean delivery. Inclusion criteria required that the women be in active labor (regular contractions and more than 4 cm cervical dilatation) with a singleton fetus in a vertex presentation. Exclusion criteria included gestational age less than 37 weeks, placental abnormalities (low-lying or complete placenta previa, placental abruption), uterine structural abnormalities or previous uterine scar, and fetal heart rate abnormalities at enrollment.
The patients were randomized into one of two types of lower uterine segment incisions: one in which a bladder flap was developed (“yes” group) and another where the incision was performed without dissection of a bladder flap (“no” group). Subjects enrolled in the “no bladder flap group” had their hysterotomy incision performed approximately 2 cm above the reflection of the visceral peritoneum. In the women assigned to the “yes bladder flap” group, the peritoneum above the reflection of the upper margin of the bladder was grasped in the midline with a forceps and incised laterally with scissors. The lower flap of the peritoneum was elevated, and the urinary bladder was gently separated from the lower uterine segment by blunt dissection. The hysterotomy incision was performed approximately 2 cm below the upper margin of the peritoneal dissection. Randomization was based on a random number generator and the assigned incision type was sealed in consecutively numbered envelopes.
Before surgery and opening of the randomization envelope, an abdominal ultrasound survey (Acuson Sequoia 512, Acuson Corporation, Mountain View, CA [Wayne State] and Voluson 730 Expert, General Electric, Zipf, Austria [Yale]) was performed by using 5.0 or 7.5 MHz transabdominal probes. One investigator (C.S.B.) performed all ultrasound examinations and measurements without knowledge of the group assignment. The lower uterine segment myometrium was identified sonographically as the echo homogeneous layer between the serosa and the decidua. Myometrial thickness was measured at two different sites: 2 cm above (“above” measurement) and 2 cm below (“below” measurement) the reflection of the partially full urinary bladder over the lower uterine segment, as previously described (Fig. 1).14 The intraobserver coefficient of variation for the measurement of myometrial thickness varied from 5% to 8%.
Full thickness uterine wall biopsies were obtained from the upper and lower edges of the hysterotomy incision immediately after delivery of the fetus and placenta using Metzenbaum scissors. There were no complications related to the biopsy. The myometrial wall samples from the upper and lower edges of the hysterotomy incision were divided into three sections. The first strip was frozen immediately in liquid nitrogen and stored at −80°C for biochemical assessment of total collagen, measurement of collagen pyridinoline-deoxypyridinoline (measure of collagen cross-link), and sulfated glycosaminoglycans content. The second strip was fixed in 10% buffered formaldehyde for 24 hours and then wax-embedded in paraffin for histological examination. The third strip was transported in phosphate buffer solution to the laboratory where the biomechanical properties were tested within 1 hour of collection.
Hydroxyproline (Hyp) was measured in tissue hydrolysates by using a modification of a method originated by Reedy and Enwemeka.15 Standard aliquots of L-Hyp (2–50 μg/mL) were prepared in water from a stock solution of 100 mg/mL. Citrate-acetate buffer was prepared by adding 30 g citric acid, 15 g sodium hydroxide, and 90 g sodium acetate trihydrate to 500 mL water. Isopropanol (290 mL) was added slowly while stirring rapidly; the pH was adjusted to 6.0. One hundred microliters of tissue hydrolysate in 6N hydrogen chloride (HCl) was neutralized with 100 μL 6N sodium hydroxide (NaOH). The samples and standards were oxidized for 20 minutes at room temperature by the addition of 100 μL chloramine T reagent (0.7 g chloramine T in 5 mL water and 45 mL citrate-acetate buffer). Color development was achieved over a 20-minute incubation at 65°C with 100 μL fresh Ehrlich’s reagent. The Ehrlich’s reagent was prepared by suspending 1 g of 4-dimethylaminobenzaldehyde in 4 mL propanol and 1.8 mL 60% perchloric acid. One hundred microliters of reacted sample or standard was added in triplicate to a 96-well microtiter plate and the absorbance read at 550 nm using a VERSAmax microplate reader with Softmax Pro 3.1.1 software (Molecular Devices, Sunnyvale, CA). Hydroxyproline induces a change in the color of the Ehrlich’s reagent from bright yellow to magenta. All measurements were done in one assay to eliminate interassay variability. The intra-assay variability was 2%. Protein concentrations in neutralized hydrolysates were measured using a bicinchoninic acid/cupric sulfate reagent (BCA kit; Pierce, Rockford, IL). The total collagen concentration in the hydrolysate was based on the assumption that collagen contains approximately 14% Hyp.16
Quantification of collagen cross-links pyridinoline and deoxypyridinoline was performed by reversed phase high pressure liquid chromatography (HPLC) as previously published.17,18 Briefly, 0.1 g of lower uterine segment tissue was minced and hydrolyzed for 16 hours at 150°C in 6N HCl. Cross-links were partially purified by subjecting the clarified hydrolysates to cellulose chromatography (Sigma Chemical, St. Louis, MO). The aqueous eluant was lyophilized to dryness, resuspended in 1% heptafluorobutyric acid, and applied to a Waters Nova-Pak C18 column (Waters, Milford, MA) for reversed-phase high-performance liquid chromatography (HPLC) in 0.18% heptafluorobutyric acid/14.65% acetonitrile using a Waters 625 HPLC system. Pyridinoline and deoxypyridinoline cross-links were detected by autofluorescence at 297 nm excitation and 395 nm emissions using a Waters model 474 HPLC fluorescence detector. They were identified and quantitated based on a co-elution and comparison with pyridinoline and deoxypyridinoline calibration standards (Metra; Quidel, San Diego, CA). The values were initially expressed in picomoles per milliliter of extract and then normalized to the derived collagen concentration based on the hydroxyproline measurement. Final values are presented as nanomoles per milligram of collagen. The ratio of pyridinoline to deoxypyridinoline was calculated as a measure of collagen turnover.
The lower uterine segment tissue level of sulfated glycosaminoglycans was determined using a quantitative dye-binding assay based on the ability of the chains of sulfated glycosaminoglycans to bind Alcian Blue dye.19 The assay system was optimized to exclude interference from proteins, nucleic acids and hyaluronan (a nonsulfated glycosaminoglycan). Briefly, myometrium was pulverized in liquid nitrogen to assure homogeneity of the sample, and 50 mg of the frozen material digested in 1 mL phosphate buffer (pH=7) containing 50 mmol/L sodium ethylenediaminetetraacetic acid (EDTA), 50 mmol/L cysteine hydrochloride, and 1 mg crude papain. After a 3-hour incubation in a shaking water-bath at 65°C, the concentration of sulfated glycosaminoglycans released into the supernatant was measured using a standardized assay system (sulfated glycosaminoglycans K-assay; Kamiya Biomedical, Seattle, WA) against a six-point standard curve of chondroitin sulfate ranging from 12.5 to 400 μg/mL. The results are expressed as micrograms per gram of tissue.
Collagen birefringence was assessed under polarized light after staining the tissue sections with picosirius red. The polyazo dye, Sirius red F3B (Aldrich Chemical, Milwaukee, WI) ionically binds with the positively charged collagen. Briefly, duplicate sections were deparaffinized, hydrated, and stained with Weigert’s hematoxylin for 10 minutes. After washing, the sections were stained for 1 hour in picosirius red (1 g/L Sirius red in saturated picric acid) then washed with two changes of acidified water (0.5% acetic acid), dehydrated, cleared in xylene, and mounted in resinous medium. Large collagen fibers appear brilliant yellow-orange on a black background when epipolarization microscopy is used secondary to the double refraction of light. According to Junqueira et al,20 the birefringence is highly specific for collagen and represents an index of the organization of collagen fibers. For each subject, five fields (100× magnification) were analyzed at random, and digital images were acquired with an Olympus U-STP polarizing light microscope equipped with an Olympus OLY-200 digital camera (Olympus, Melville, NY). Fixed light settings were maintained for all images during digital acquisition. The images were analyzed using Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA); mean luminosity (light intensity of the pixels) in the image was considered proportional to the amount of birefringent collagen per field. The results are expressed as units of luminosity. Luminosity was quantified from 14 (background luminosity) to 255 (bright white). This methodology was used previously to quantify the relative abundance and orientation of collagen fibers in the human myometrium.21 By averaging the luminosity values from the five fields, a semiquantitative estimation of the amount of collagen is obtained for each tissue biopsy.
We sought to determine whether structural differences at the incision site translated into different biomechanical properties. Samples of myometrium were placed on a marked plastic plate for dissection and cut into uniform 2 cm×0.5 cm strips, as previously described.21 The strips were then mounted in a tissue bath containing 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/phosphate buffer solution (pH=7.4), with one pole secured to the platform of a Shimadzu EZ-test Instrument (Shimadzu North America, Columbia, MD) and the other anchored to a movable hook on the instrument. We used a stretching protocol to emulate characteristics of labor,22 modified from the original design by Downing and Sherwood.23 The device stretches the tissue by 0.42 mm every minute, with sampling rate: 20 Hz; duration of stretching: 0.7 seconds; duration or equilibration: 59.3 seconds. Online stress-strain curves were computer generated.24
Parameters such as slope (a measurement of stiffness/elasticity), yield point (the moment when tissue changes its proprieties from elastic to plastic), and break point (a measure of tissue strength) were recorded and interpreted in relation to the biochemical and histological constituents of the tissue at the biopsy location (Fig. 2). Once intermittent stretching was initiated, it was continued past the yield point until the break point was reached. The tissue deformed irreversibly beyond the yield point, but before the break point. After each stretch, the stress on the tissue rose abruptly and then decreased to a plateau higher than the one achieved during the prior stretch. This accommodation created a force-displacement curve with a saw-tooth appearance, particularly during the initial phase of stretch.21,22 Straight lines were fitted to the linear portion of the force-displacement curve, the slope of the regression line calculated and used to characterize the sample’s resistance to stretch. A larger slope indicated a higher resistance to stretch, and therefore, increased stiffness. All parameters were normalized to the dry weight of the strip. Because all the strips were cut at a consistent length and width, the dry weight of the sample was used as a surrogate for the cross-section.21,22
The normality of the data distribution was tested using the Kolmogorov-Smirnov test. Analysis was performed with the aid of SigmaSTAT 2.03 (Jandel, San Rafael, CA). The data are presented as mean and standard error of the mean, if normally distributed, or as median and range if not. Comparisons between groups included Student t tests, Mann Whitney U test, or one-way or two-way analysis of variance (ANOVA), as appropriate. Although the arithmetic means or medians are reported in the text, statistical testing using two-way ANOVA was performed following logarithmic transformation of the data. Differences in proportions were examined with Fisher exact test. Pearson product moment correlation was used to estimate association between variables. Nineteen to 20 subjects per group confers sufficient power to identify 50% differences in slope between the two groups (power=0.8, α=0.05). P<.05 was considered to indicate statistical significance.
Ninety percent of women solicited for enrollment consented to the study, and all enrolled patients were included in the final analysis. There were no significant differences in maternal, fetal, and labor characteristics of the study population (Table 1). There were no differences in cervical dilatation, station, or uterine contractility at the time of cesarean delivery between the two groups. The ultrasonographic thickness of the lower uterine segment myometrium before cesarean delivery did not differ significantly between the two sites (2 cm above and 2 cm below the reflection of the urinary bladder) within each group or among the two study groups at each site (yes bladder flap: above 0.51±0.04 versus below 0.52±0.02 cm; no bladder flap: above 0.56±0.04 versus below 0.55±0.05 cm; two-way ANOVA, P=.96 for site and P=.30 for bladder flap).
The myometrium from the lower edge of the hysterotomy had a higher collagen content than that from the upper, whether the hysterotomy incision was performed above or below the reflection of the bladder flap peritoneum (yes bladder flap: upper edge 63.8±8.2 versus lower edge 222.8±38.3 μg/mg protein; no bladder flap: upper edge 66.1±7.2 versus lower edge 263.2±64.7 μg/mg protein (two-way ANOVA P<.001 for edge examined and P=.92 for bladder flap) (Fig. 3A). In contrast, the lower edge of the hysterotomy contained decreased collagen cross-linking independent of whether or not a bladder flap was created (yes bladder flap: upper edge 33.5±11.3 versus lower edge 8.1±1.2 nmol pyridinoline per milligram collagen; no bladder flap: upper edge 28.4±2.9 versus lower edge 9.2±1.7 nmol pyridinoline per milligram collagen (P<.001 for edge examined and P=.97 for bladder flap) (Fig. 3B). The pyridinoline:deoxypyridinoline ratio did not vary between biopsy sites (P=3.0) or between groups (P=.63).
Representative collagen birefringence images observed under polarized light microscopy after picosirius red staining are displayed in Figure 4. There was no significant difference in collagen birefringence of the upper versus the lower edge of the myometrium, whether or not a bladder flap was created (yes bladder flap: upper edge 37.2±1.9 versus lower edge 37.9±2.1 units; no bladder flap: upper edge 36.2±2.2 versus lower edge 39.7±2.5 units; two-way ANOVA, P=.75 for edge position; P=.68 for bladder flap) (Fig. 3C).
The quantity of sulfated glycosaminoglycans in the myometrium collected from lower uterine segment of laboring women was similar at each of the biopsy sites (yes bladder flap: upper edge 427.5±39.7 versus lower edge 444.8±39.8 μg/g tissue; no bladder flap: upper edge 441.5±40.5 versus lower edge 474.2±32.3 μg/g tissue; two-way ANOVA, P=.81 for edge position; P=.86 for bladder flap) (Fig. 3D).
Myometrial stiffness was similar between groups and did not vary by site of biopsy (slope: yes bladder flap: upper edge 7.6±1.3 versus lower edge 9.1±1.7 N/g; no bladder flap: upper edge 8.1±1.2 versus lower edge 5.7±0.7 N/g; two-way ANOVA, P=.49 for edge position; P=.78 for bladder flap). There was no statistically significant interaction between the flap and the level of uterine biopsy (P=.16). Likewise, there were no differences in the force required to reach the yield point between groups: (yes bladder flap: upper edge 117.9±22.6 versus lower edge 114.1±20.9 N/g; no bladder flap: upper edge 110.2±16.6 versus lower edge 101.7±13.4 N/g;) or for break point (yes bladder flap: upper edge 1134.2±161.5 versus lower edge 1250.1±192.3 N/g; no bladder flap: upper edge 1067.7±158.2 versus lower edge 1142.3±116.2 N/g; P=.66 for edge position; P=.41 for bladder flap). There was no correlation between the total glycosaminoglycans measured and either the myometrial slope, yield point, or break point for the upper or lower edge of the uterine incision, whether or not a bladder flap was surgically created. Similarly, neither the total tissue collagen nor the degree of collagen cross-linking was related to either the slope, break point, or the yield point for any of the study groups.
The biomechanical properties of connective tissues play fundamental physiological roles in nearly every organ, including the uterus.24 Living tissues display visco-elasticity (combination of solid-like characteristics such as elasticity, strength, and stability, but also liquid-like properties such as flow), which indirectly reflects mechanical properties of each of the constituents combined with their structural arrangements in the tissue matrix.25 Although elastin and glycosaminoglycans are important determinants of tissue mechanical properties, collagen remains perhaps the most critical component responsible for maintenance of tissue structural integrity.26 Therefore, one might reasonably expect the amount of collagen to be the primary determinant of a tissue’s mechanical properties. The results of the present study reveal significant differences in collagen and collagen cross-linking by location in the lower uterine segment. Yet, these differences do not translate into differences in the biomechanical properties of the lower uterine segment. Thus, our working hypothesis that structural differences in the lower uterine segment influence the biomechanical properties of the myometrium cannot be supported.
One prior investigation concluded that different connective tissues with differing collagen content can exhibit similar mechanical behavior to meet the needs of an organ.25 We find that this conclusion is applicable to the lower uterine segment. Perhaps it should not be surprising considering that the visco-elasticity of a biological tissue is not just the sum of the biomechanical properties of its individual constituents (collagen, glycosaminoglycans, elastin). Rather, it is the result of a complex interaction between cells and extracellular matrix products (glycosaminoglycans, elastin) that maintain normal tissue integrity.27 Glycosaminoglycans seem to have little if any role in determining the biomechanical behavior of the lower uterine segment because their tissue levels did not differ between lower uterine segment sites and there was no correlation between the total glycosaminoglycans measured and tensile visco-elastic properties of the tissue.
The triple helix arrangement allows collagen to cross-link into complex three-dimensional networks of fibrils, fibers, and bundles that dictate both firmness and mechanical strength during parturition.28–30 A growing body of evidence suggests that the orientation and thickness of collagen fibers correlates with mechanical strength.30 Collagen has a natural birefringence due its three-dimensional arrangement of fibers, and this property is enhanced by Sirius red dye.20,31 The fibers’ thickness and orientation determine polarization colors. The similarity in collagen birefringence among sites, despite significant differences in collagen content and the degree of cross-linking, confirms that orientation of collagen fibers, rather than amount, plays a more important role in maintenance of the strength and, thus, the integrity of the lower uterine segment.
The results of the tensile visco-elastic properties of the myometrial tissue are intriguing from the perspective of the four sites tested. One might intuitively expect that the structural and mechanical properties of the tissue vary gradually from the lowest (lower edge in the bladder flap group) to the highest (upper edge in the no bladder flap group). Yet, there is no difference in either biochemical or mechanical results. The upper edge in the “yes flap” looks like the upper edge in the “no flap” group, whereas the same applies for the lower edge. One explanation may be related to the mechanisms that govern myometrial contractility and tissue retraction in the immediate puerperium. Our prior ultrasonographic studies reveal that there is significant myometrial thickening of the anterior uterine wall with loss of the lower uterine segment after delivery of the fetus.14 Certainly, one limitation of the current study rests with the inability to mark precisely the edges of the uterine incision. Therefore, although the hysterotomy incision is easily placed above or below the reflection of the urinary bladder before delivery of the fetus, the retraction of the lower uterine segment wall toward the uterine body or cervix, respectively, at the time of delivery may significantly change the anatomical relationships and, hence, explain the similarities between the groups and the sites of the biopsy.
The findings of the current investigation have important clinical and research implications. Previous randomized studies intended to evaluate the effects of not forming a bladder flap at the time of cesarean delivery.6 Omission of the bladder flap provided short-term advantages, such as a reduction in the incision-to-delivery interval, total surgical time, reduced blood loss, and a lower need for analgesics. Yet, whether the site of the uterine incision may impact long-term effects such as wound scarring remained unexplored. Although cesarean delivery remains the most common major surgical intervention worldwide,32 our understanding of myometrial repair and regeneration after surgery is extremely limited.33 We previously proposed that the process of myometrial wound healing determines the future morphology, functional behavior, and risk of uterine rupture.34 The finding that structural differences are related to the edge but not the level of the lower uterine segment incision suggests that healing of the uterine scar will probably not be influenced by the hysterotomy site as long as the incision is 2 cm above or below the reflection of the urinary bladder.
Lastly, functional genomic approaches have identified intrinsic processes related to human parturition.35,36 These studies note that parturition is characterized by a massive down- or up-regulation of a large panel of inflammatory, contractile, and apoptosis-related genes. Most of these studies were conducted using myometrial samples collected from the lower uterine segment of laboring or nonlaboring women at the time of cesarean delivery. Similarly, conclusions concerning the contractile properties of human myometrium were also frequently based on myometrial samples collected from the lower uterine segment. Because the lower uterine segment elongates to allow passage of the fetus, it was proposed by one investigator that findings in myometrial samples obtained from the lower uterine segment mostly reflect temporal and functional regulation of remodeled cervical, rather than uterine, body tissue.37 Thus, the current findings may seem to suggest indirectly that spatial and temporal variability in gene expression in the myometrium may not only be detected in samples from the uterine fundus versus the lower uterine segment, but also from the upper versus the lower edge of the lower uterine segment hysterotomy incision.37 Further studies are needed to elucidate whether the upper and lower edge myometria display significant differences in the functionality of their muscular contractile components.
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