Analysis of Glycosaminoglycans in the Parametrium and Vaginal Apex of Women with and without Uterine Prolapse

Introduction

Pelvic organ prolapse (POP) is a downward descent of pelvic organs that results in protrusions of the vagina, the uterus, or both. ¹ It is a disorder that can affect the anterior and posterior walls of the vagina or uterus or apex of the vagina, usually in some combination. ² In 1997, more than 225,000 surgical procedures for pelvic organ prolapse were undertaken in the United States (22.7 per 10,000 women), at an estimated cost of more than US$1 billion. ^3,4

Many risk factors for POP have been suggested. The cause of this disorder is likely to be multifactorial, attributable to a combination of risk factors varying from patient to patient. ⁵ Possible risk factors include genetic predisposition, parity (particularly vaginal birth), menopause, advancing age, prior pelvic surgery, connective tissue disorders, and factors associated with elevated intraabdominal pressure (eg, obesity or chronic constipation with excessive straining). ^{6 –9}

Anatomical support of pelvic viscera is mainly provided by the levator ani muscle complex and connective-tissue attachments of the pelvic organs. The endopelvic fascia is a connective-tissue network that envelops all organs of the pelvis and connects them loosely to supportive musculature and bones of the pelvis. Evidence suggests that abnormalities of connective tissue and connective-tissue repair might predispose some women to pelvic organ prolapse. ¹⁰ Individuals with prolapse might have altered collagen metabolism, including a decrease in type I collagen and an increase in type III collagen. ^{11 –13}

The integrity of the human body is largely dependent on the composition and arrangement of connective tissue. The connective tissue is composed of several key elements such as fibroblasts and an extracellular matrix (ECM) containing collagen, elastic fibrils, proteoglycans, and glycosaminoglycans (GAGs). They are organized similar to a hammock, which is an important determinant to their morphologic diversity and tissue functions.

In recent years, numerous studies have examined the relationship between ECM components, especially collagen, and different types of pelvic floor disorders, such as prolapse and urinary incontinence. ^{14 –18} However, the role of other ECM components has not yet been established.

GAGs are heteropolysaccharides commonly expressed in many tissues. The most common GAGs are chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate, hyaluronic acid, and heparin. In most cases, GAG chains are attached to core proteins, forming proteoglycans. Overall, GAGs contribute to the general architecture and permeability properties of connective tissue. In addition, they serve as anchors for cell-specific growth factors and enzymes in ECM and at the cell surface. ^19,20

To our knowledge there are still no data on the GAG composition of each part of the female body. The main objective of this paper is to evaluate and quantify the sulfated GAGs in the parametrium and vaginal apex of women with and without uterine prolapse.

Materials and Methods

Forty-two consecutive patients underwent surgery for uterine prolapse, for vaginal and abdominal hysterectomy, or for other gynecologic benign diseases. They were divided into two groups according to pelvic organ prolapse quantification (POP-Q): The stages 0–I group was composed of 25 patients, while there were 17 patients in the stages II–IV group.

Subjects were excluded if they had diabetes, gynecological cancer, vulvovaginal infections, a smoking habit, connective tissue disorders, alcoholism, chronic disabling diseases, or hypertension. Those who had used estrogen, progestogens, or androgens in the previous 12 months were not included.

All patients underwent a standard physical examination that included POP-Q according to the standardization of the terminology of female pelvic organ prolapse, ²¹ The examination included POP-Q in the supine position. The Valsalva maneuver or cough demonstrated the maximum descent of prolapse. The stage of prolapse in the superior compartment was determined by measurement of the uterine cervix (points C and D).

We used the methods, definitions, and units as determined by the International Continence Society, ² Clinical patterns were used to evaluate menopausal status. Women who reported current and regular periods were defined as premenopausal. Women who reported more than 12 months of amenorrhea were defined as postmenopausal. All patients gave their informed consent to participate in the study, and its protocol was approved by the local ethics committee.

During the surgical procedures, samples measuring roughly 10 mm in diameter were removed from the parametrium and/or the vaginal apex. Tissue was obtained with Metzenbaum scissors. There were no complications during these biopsies. Immediately after resection, each sample was rinsed in a cold phosphate-buffered saline (PBS) solution to remove blood excess. The specimens were placed into a purified glutaraldehyde primary fixative (2.5% glutaraldehyde in 0.1 mmol/L cacodylate buffered at pH 7.3) and frozen.

Extraction was performed separately for each site of biopsy. The frozen tissues were again washed in a cold PBS solution to remove glutaraldehyde excess. Afterward specimens were weighed and ground in 10 vol of acetone. After standing overnight at room temperature, tissue was collected by centrifugation and dried. The dry powder was incubated overnight with Maxatase, a serine endopeptidase, in order to purify the peptides (3 mg/ml in 0.06 M phosphate-cysteine buffer, pH 6.5, containing 20 mM EDTA) at 50°C. Trichloroacetic acid (90%) and sodium chloride were added to the supernatant up to 10% and 1 M final concentrations, respectively. The mixture was left to stand for 10 minutes at 4°C, and the precipitate formed was removed by centrifugation at 4,000 g for 20 minutes. GAGs were precipitated from the supernatant by the slow addition of 2 ml vols of ethanol with shaking. After 18 to 24 hours at −20°C, the precipitate was collected by centrifugation, vacuum-dried, resuspended—in 0.5 ml of a solution containing desoxyribonuclease I to denature contaminant DNA (1 mg/ml), a 0.05 M sodium acetate buffer, and 0.02 M magnesium chloride (pH 5.0)—and incubated at 30°C for 18 hours.

GAGs were then identified by agarose gel electrophoresis, quantified by densitometry at 525 nm, and compared with previously known and purified standard mixtures. The use of the agarose gel electrophoresis method has previously been described. ²²

The relative concentration of sulfated GAGs was determined by densitometry of toluidine-blue stained gel using a spectrophotometer with 525 nm of wavelength. The three peaks obtained were identified as CS, DS, and HS, using previously described procedures. ²³

Results

A total of 42 women underwent surgery and biopsy. The clinical data of patients are listed in Table 1. Patients were homogeneous for body mass index (BMI) and parity. However, we observed significant differences in age. Most patients without uterine prolapse were in premenopausal status. Most patients with uterine prolapse were in postmenopausal status.

In our study the parametrium and vaginal tissue showed three different types of sulfated GCAs, of which the predominant was DS, followed by CS and HS in all groups. The DS amounts varied from 1.53 to 2.37 mg/g of dry weight in the parametrium samples and from 2.25 to 5.59 mg/g in the vaginal apex tissue. HS varied from 0.24 to 0.40 mg/g of dry weight in the parametrium and from 0.16 to 0.30 mg/g in the vaginal apex. CS varied from 0.09 to 0.32 mg/g of dry weight in the parametrium and from 0.11 to 0.34 mg/g in the vaginal apex. In parametrium tissue the amounts of total GCAs—HS, DS, and CS—did not vary significantly. Similar patterns were observed for vaginal apex samples (Tables 2 and 3).

Discussion

In our study we did not observe significant differences in GAGs—HS, DS, or CS—in the parametrium and vaginal apex of patients with and without uterine prolapse. Studies involving sulfated GAGs in POP are still in the early stages. Tissue injury, age, and hormonal influences on GAGs have been studied in POP, but their relative roles have not been elucidated.

Our findings could be related to tissue injury or could be attributed to the fact that the groups were not homogeneous according to hormonal status. It is important to emphasize that when comparing pre- and postmenopausal patients there was obviously a significant difference regarding age. Therefore, we cannot draw conclusions in our study regarding uterine prolapse entirely without referring to tissue alterations inherent to the aging process.

De Deus et al. ²⁴ found that castration of adult rats reduced the GAG content in the bladder, and therapy with conjugated estrogens and medroxyprogesterone acetate (MPA) increased the GAG content to values similar to those of noncastrated rats. On the other hand, estrogen alone reverted the reduction promoted by castration, and MPA annulled the estrogen effect. In the urethra, there was a decrease in the DS/HS ratio of castrated rats with estrogen replacement. It was postulated that hypoestrogenism could decrease GAGs in the bladder of adult rats, and hormonal replacement could replace GAG quantity and quality.

Bezerra et al. ²⁵ found significant differences among GAGs in vaginal tissue. Total amounts of GAG, CS, DS, and HS in premenopausal women with prolapse stages I, II, and III were significantly higher than in postmenopausal patients with prolapse stages II and III. They also observed a significant decrease in total GAG, DS, and HS values in the postmenopausal group among women with prolapse stages I, II, and III, but no significant decrease in the amount of CS. It was postulated that decreases in GAGs in the vaginas of postmenopausal women could be a response to tissue injury of the ECM, which had undergone successive trauma because of childbirth, combined with the effects of hypoestrogenism.

However, data on changes in GAG metabolism are conflicting. The differences may partly be because of the analysis of different target tissue in patients with POP. Feldner et al. ²⁶ investigated sulphated GAGs from periurethral tissue in 35 women who underwent surgery for POP. The results showed an accumulation of GAG, DS, and CS in the tissues of these women.

The clinical relevance of these findings could be the result of a considerable impairment of the mechanical properties of the tissue. We cannot precisely determine the exact amounts of these substances in each part of the pelvic organs. But we can assume that the hypoestrogenism decreases the GAG content. On the other hand, it may be that there is more GAG content in the tissues of women with POP and stress urinary incontinence. It will require additional observation and research to understand better not only how all the components are connected but also the effect and importance of each on pelvic organ disorders.

Earlier studies of the ECM evaluated only the presence of collagen. In 1996, it was reported that patients with descent of the cervix to or beyond the introitus with an associated cystocele have a reduced collagen content, with a relatively high content of immature collagen cross-links. ²⁷ This newly formed collagen is degraded more easily than older glycated material, resulting in a decrease in collagen content, leaving behind glycated collagen and resulting in tissue with impaired mechanical strength. ²⁸ The researchers concluded that this deficiency, which is brittle and susceptible to rupture, is an important etiologic factor in uterine prolapse.

Since ours was a preliminary study, we acknowledge that it had some limitations. Although the site of the biopsy was standardized, the anatomy of pelvic support includes many different supporting tissues in extremely close proximity to each other. Tissue samples only millimeters apart will yield tissues of a vastly different nature. We also know that the conditions of the control group were not optimal. The sample size was small, and because of ethical limitations, we did not have an ideal group consisting of healthy women.

Conclusions

In summary, in this prospective study of women undergoing gynecologic surgery, we were unable to find differences in GAG content among those with and without POP. Therefore, prospective longitudinal studies are needed to clarify the role of the extracellular pathophysiological factors involved.