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ORIGINAL ARTICLE  
Year : 2012  |  Volume : 55  |  Issue : 4  |  Page : 450-455
Biochemical evaluation of the supporting structure of pelvic organs in selected numbers of premenopausal and postmenopausal Malaysian women


Department of Obstetrics and Gynaecology and Pathology, International Medical University, Malaysia

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Date of Web Publication4-Mar-2013
 

   Abstract 

Context: Pelvic organ prolapse (POP) is associated with menopause and changes in the proteins of the pelvic supporting system, but there is scant data on the precise alterations in Malaysian women. Aim: The aim of this study is to determine the differences in the extracellular matrices (ECM) of uterosacral ligaments in premenopausal and postmenopausal Malaysian women with or without POP. Settings and Design: The observational study was conducted for 9 months in three general hospitals involving 30 women who underwent hysterectomies for various indications except for carcinoma of pelvic organs. Materials and Methods: Three groups were identified: Premenopausal women (Group 1), postmenopausal women without POP (Group 2), and postmenopausal women with POP (Group 3). Age, duration of menopause, body mass index (BMI), parity, and vaginal deliveries were documented. Only 21 samples of the uterosacral ligaments were stained immunohistochemically for collagen I and III, matrix metalloproteinases (MMPs) 1 and 2, elastin, and tenascin. Statistical Analysis Used: Image J software analysis was utilized for quantification, while non-parametric statistics (Kruskal-Wallis with post-hoc Dunns Multiple Comparison test) was used for result analysis. Results: The profile parameters were not significantly different except for mean age and duration of menopause in Group 3. Samples from Group 2 showed lower expression of almost all proteins except MMP1 and tenascin (higher) as compared to Group 1. The changes appeared to be exaggerated in Group 3, though statistically insignificant. Conclusion: A significant difference in the expression of ECM was apparent in postmenopausal subjects as compared to premenopausal ( P = 0.05), compromising the uterosacral ligament tensile strength. The findings are proven similar as those changes in women from other studies.

Keywords: Elastin, matrix metalloproteinase, pelvic organ prolapse, tenascin, uterosacral ligaments

How to cite this article:
Aznal SS, Meng FG, Nalliah S, Tay A, Chinniah K, Jamli M. Biochemical evaluation of the supporting structure of pelvic organs in selected numbers of premenopausal and postmenopausal Malaysian women. Indian J Pathol Microbiol 2012;55:450-5

How to cite this URL:
Aznal SS, Meng FG, Nalliah S, Tay A, Chinniah K, Jamli M. Biochemical evaluation of the supporting structure of pelvic organs in selected numbers of premenopausal and postmenopausal Malaysian women. Indian J Pathol Microbiol [serial online] 2012 [cited 2020 Sep 20];55:450-5. Available from: http://www.ijpmonline.org/text.asp?2012/55/4/450/107778



   Introduction Top


The incidence of pelvic organ prolapse (POP) among women is increasing due to prolonged life span after menopause. The disorder, which refers to the descent of pelvic organs into or out of the vaginal canal, is one of the commonest forms of female pelvic floor dysfunction. [1] There is a relatively high prevalence of POP in the aging population and is one of the most common indications for gynecological surgery in postmenopausal women. [2] While this disorder is related to defects in the pelvic organ supporting tissues, the exact pathophysiology is still unclear. [1],[3],[4],[5] POP results in various complications and it affects the quality of life significantly. Common risk factors are menopause, vaginal childbirth, and increased abdominal pressure, often due to obesity. [1] Though multiparity was demonstrated in the Oxford Planning Association Prolapsed Epidemiology Study as the most significant risk factor with an adjusted relative risk of 10.85, POP occurs in nulliparous women. In addition, women with positive family history of POP have a relative risk of 2.4-3.2. [6] Therefore, this indicates some form of genetic predisposition apart from connective tissue factors. [6],[7]

Changes in the extracellular matrix proteins of the supporting ligament have been implicated in POP. [8],[9] Moali et al.[10] alluded decrease in collagen I, III, and IV ratios in postmenopausal women who were not on hormonal therapy, compared to that of premenopausal women. A reduction in total collagen was not associated with elastin and smooth muscle content. Ewies [8] et al. found that some of the alterations in protein content were reversed in women with POP when treated with hormone therapy. Studies in mouse models as well as humans have shown some significant correlation between POP and the deficiency in either oestrogen receptors, matrix metalloproteinase-1, lysyl oxidase like-1 (LOXL-1) enzyme (which maintains the homeostasis of the elastic fiber), or the collagen (Type I, II, III, IV, V, VI) which form the matrix of pelvic supporting tissues. [11],[12]

Clinical trials have been conducted on the usage of non-surgical treatments such as tissue engineering for other pelvic floor dysfunction with or without incontinence. [13] As for POP, a better understanding of fibroblast reaction to mechanical stress, typing of collagen types, and matrix metalloproteinases production by fibroblasts would make it possible to develop newer and yet less invasive treatment to replace conventional surgical approaches. [12],[13],[14] Although research has thrown further light on the etiology and risk factors of POP, there is still scarcity of data on the extracellular matrix proteins of pelvic organ supporting tissue among Malaysian women. The current research would serve as a preliminary study to assess extracellular matrix proteins of the uterosacral ligaments in a selected group of premenopausal and postmenopausal Malaysian women.


   Materials and Methods Top


In the period between March and December 2010, 30 women who underwent standard procedure of hysterectomy transabdominally or vaginally for benign conditions including POP in three public hospitals from two states and who consented for this study were recruited. Both premenopausal and postmenopausal women (women who have been amenorrheic for more than a year) with or without POP were included. The exclusion criteria were those who were on hormonal therapy, had connective tissue disorders, severe pelvic adhesive disease, or previous pelvic surgery. Data on the age, duration of menopause, body mass index (BMI), parity, and number of vaginal births were obtained. Collected specimens were categorized into three groups: Group 1, premenopausal women without POP; Group 2, postmenopausal women without POP; and Group 3, postmenopausal women with POP.

During the surgery, a portion of uterosacral ligament 1 cm from its insertion into the cervix was identified and transected by the operating surgeon. The transected specimens were fixed in 10% buffered formaldehyde placed in specimen containers and transported to the research laboratory of the International Medical University, Malaysia, for embedding process in paraffin wax and blocks. The specimens were cut into seven 3 μm sections and placed on silanized slides. Immunohistochemistry (IHC) staining using the antigen retrieval method was then carried out on the processed slides according to the standard Dako's specification. [5] Primary antibodies, Abcam mouse monoclonal antibodies from "DakoDenmark A/S," were used for staining of collagen type I and III, elastin, and tenascin. The stock primary antibodies were diluted with "Dako" antibody diluents based on the value determined during trial run on test samples before actual staining. A series of dilution of antibodies were performed at different dilutional ratio and incubation time for each component of ECM with reference to the standard manufacturer dilution value [Table 1].
Table 1: Stock antibody to antibody diluents ratio (antibody diluents) and incubation time for each antibody during the optimization trial run

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Other IHC staining used were rabbit monoclonal antibodies for matrix metalloproteinase (MMP) 1 and rabbit polyclonal antibodies for MMP2. The IHC staining was further optimized with antigen retrieval methods with steps varied based on the type of antibody used for staining. [Table 2] shows the brief description.
Table 2: Antigen retrieval methods based on the type of antibodies applied during IHC staining

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Subsequently, image processing and analysis was carried out on individual slides using the microscope. At magnifications of 40×, 100×, 200×, and 400×, 10 randomly selected areas excluding areas containing smooth muscles, endothelial cells or glands, and blood vessels were analyzed to determine the percentage of area stained by the antibodies. [8],[15] The captured images were then sent to the Image J software to be processed at 100% size of each selected field where the characteristic brown color staining of 3,3' diamonibenzidine (DAB) substrate would be isolated by the Color Deconvolution plugin. Then, a standard color threshold value which was pre-determined from test samples was applied to select the brown color stain areas. [17] The selected areas will be calculated automatically by the software and the percentage area stained per field is given.

Non-parametric statistics such as Kruskal-Wallis with post-hoc Dunns Multiple Comparison tests were used for analysis of the results of all the groups [premenopausal (Group 1), postmenopausal (Group 2), and postmenopausal with POP (Group 3)]. Mann-Whitney test was performed when comparison was necessary between two groups. The levels of significance were set at P < 0.05, P < 0.01, and P < 0.001.

This study was approved by the Ethics and Research Committee of the International Medical University, Malaysia, and has received ethics approval from the National Medical Research Registry Malaysia (NMRR-10-608-6141) and it is of the vested interest of the authors and institution.


   Results Top


Clinical Profile

Out of 30 uterosacral ligament samples obtained, 20 were from premenopausal women, of which only 11 were stainable and 10 samples from postmenopausal group represented women with POP (n = 6) and without POP (n = 4). The clinical profiles of the subjects are shown in [Table 3].
Table 3: Clinical profiles of premenopausal and postmenopausal women with and without pelvic organ prolapse

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The mean menopausal age group ranged from 60.5 to 68.3 years. There was no significant difference between Group 1 and Group 2. The duration of menopause ranged from 12 to 17.8 years, with a longer duration seen in postmenopausal women with POP (17.8 ± 1.0 vs. 12.0 ± 2.9 years). Difference was not significant. No significant differences were apparent in parity, BMI, and vaginal births.

Biochemical Evaluation of The Extracellular Proteins of Samples of Uterosacral Ligaments

The mean percentage areas of the individual slides, i.e. collagen type I and III, elastin, and tenascin, MMP1 and 2, were analyzed after the IHC staining process at 400× magnifications using the Image J software [Figure 1]a-f and [Table 4].
Figure 1: (a-f) IHC photomicrographs of collagen I and III, MMP1 and MMP2, elastin, and tenascin of the uterosacral ligaments of Groups 1-3 at 400x magnification

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Table 4: Mean percentage area of ECM proteins after IHC staining in the three groups

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Samples from the uterosacral ligament of Group 2 had characteristically lower expression of collagen type I and elastin [Figure 1]a and c, but they had higher expression of collagen III, MMP1, and tenascin [Figure 1]b, e, and d when compared to Group 1. There was no difference in the expression of MMP2 for Group 1 and Group 2 [Figure 1]f. The changes in expression of all types of protein appeared to be exaggerated in Group 3 [Figure 1]a-f. The differences between Group 2 and Group 3 were, however, not statistically significant.


   Discussion Top


The prevalence of POP has been quoted to be as high as 20-75% among women though severe degree of prolapse is uncommon. [18],[19] Racial differences, aging, the menopausal state, and child bearing, especially vaginal deliveries, have a significant impact on POP due to alteration in protein matrix and damage of the pelvic floor supporting structures. The current study is a preliminary evaluation of extracellular matrix (ECM) in a selected group of Malaysian women. POP in all the four samples occurred in postmenopausal women who had been in a longer duration of menopausal state, compared to those with no prolapse (12 ± 2.9 vs. 17.8 ± 1). The multivariate analysis did not show significant difference in other factors, viz. BMI, parity, and number of vaginal deliveries, in the three groups. Though duration of menopause was longer in women who had POP, it was not statistically different from postmenopausal women without POP.

The strength of the pelvic organ supporting structures is dependent on the integrity of its ECM protein which is made up of different types of collagen (types I-IV). Many biochemical studies focus on collagen content, MMPs, elastin, and tenascin. [2],[19],[20],[21] The supporting structure's elasticity and extensibility are determined by formation of fibrils or degradation by collagenases. In the vagina and endopelvic fascia including the supportive ligaments, collagen I and III fibers are the main structural components which determine the tensile strength. Studies have postulated that the quality of ligament strength can be determined using the ratio of collagen I:III, indicating greater tensile strength if there was a high ratio and vice versa. [20],[21],[22],[23]

Our study shows a decrease in collagen type I in the postmenopausal group with lowest value in those with POP. This finding concurs with the findings of others. Loss of collagen type I expression in postmenopausal women is possibly attributed to estrogen depletion and tissue decompensation due to the aging process. [23],[24] The ratio of collagen I: III was shown to be the lowest in postmenopausal group with POP. Despite this finding, it is uncertain if the reduced ratio is associated with the occurrence of POP as there was no statistically significant difference between the postmenopausal women with and without POP. A large sample size would be needed for statistical evaluation. The decrease is consistent with findings of Moalli et al. [10] who showed a decreased ratio, however, in the arcus tendineus fasciae pelvis, another important pelvic supportive tissue in postmenopausal women. A plausible theory is such biochemical changes set the motion for POP, especially in the presence of other risk factors mentioned. It would not be incorrect to surmise that these biochemical changes set into motion a series of processes which lead to POP in the presence of risk factors like vaginal birth and obesity. The proteinases MMP1 and MMP2 have been demonstrated to be uniformly present in all the uterosacral ligament samples. We have demonstrated a significant increase of MMP1 in postmenopausal group, especially with POP (P < 0.001) which may be an important indicator of increased collagen degradation by the interstitial collagenase in the supporting structure. This is comparable to the findings of others who have reported similar increase of the proteinases in the pelvic supporting ligaments of women with POP. [12],[24] MMP2 expressions, on the other hand, showed no discernible difference in the premenopausal and postmenopausal groups. This probably alludes to a lesser role when aging and estrogen depletion are factored in. This is in line with the findings in our study where an increase of MMP1 is apparent, but not of MMP2, in women with menopause associated with estrogen deficiency and aging.

Elastin and tenascin are two other distinct proteins analyzed in the samples. Elastin seemed to decrease in expression as compared to tenascin in the postmenopausal group, especially the group with POP. This is consistent with many other studies. It is shown that the presence of estrogen is capable of stimulating enzymatic activity that can accelerate the maturation of collagen type I and elastin in ECM. Even though elastin is synthesized in response to cyclic stretching, injury, UV radiation, and in pathological conditions such as emphysema, it has not been possible to induce formation of elastic fibers in adult tissue. [25],[26] This would probably explain the decrease of elastin in postmenopausal women, suggesting that re-synthesis does not occur with the estrogen-deficient state. Tenascin, on the other hand, appears to play a promotive role as it is largely involved in morphogenic movements, tissue patterning, and repair. [27] This is particularly seen in higher quantities in the postmenopausal uterosacral ligaments. Adult tissues undergoing inflammation or active remodeling are associated with increasing amount of tenascin. Although not demonstrated in our study, this concept is able to explain its relationship to previous childbirth, estrogen deficiency, and aging, all capable of stressing the supporting ligaments of the pelvis paving the way to POP.


   Conclusion Top


This preliminary study confirms the pattern of ECM in the uterosacral ligaments in a selected group of Malaysian women to be similar to the findings of others. [22],[23],[24] A significant difference in the expression of ECM proteins is apparent when premenopausal subjects are compared to postmenopausal subjects. Clearly, there is a compromise of uterosacral ligament caliber and biochemical composition as demonstrated by the differences of the ECM proteins, though not significant. The duration of menopause impacted on the increased possibility of the occurrence of POP. Other variables like BMI and parity might have also affected the constitution of the pelvic organ ligaments. Although exaggerated difference in the quality of ECM proteins was demonstrated among women with and without POP, the limited sample size did not enable demonstrating statistical significance. This study may form the basis for a larger study to compare the variables mentioned to reach statistical significance.

Limitations and Further Studies

The small sample size due to short duration of study period and limited funding has substantially implicated the statistical values of the findings. There were many unsuitable samples for staining due to improper collection of ligaments and slicing. As the sampling is also opportunistic (patients who have diseased pelvic organs undergoing removal of the uterus), there may be other confounding factors affecting the elements in the ECM of the supporting ligaments.

Further research should be conducted with a larger sample size to study the possible distinction of profiles like racial group or hormonal status in relation to the difference of ECM in pelvic supporting ligament. Other components like fibroblast activities, endothelin T-I, estrogen, and progesterone receptors could be investigated, especially in their relation to possible genetic factors, i.e., micro RNA or allele that controls the activity of enzymes or proteinases. Stem cell therapy or genetic manipulation as an alternative treatment is thus possible. A similar diagnostic kit test to the changes indermal collagen could be developed on changes in the pelvic supportive ligaments should the ECM of the structures studied is comparable to tissues like dermal collagen.


   Acknowledgments Top


This research was made possible by the funding provided by the International Medical University Malaysia [Research Grant No.: BMSc 1-01/2010 (09)] and assistance and approval from the Ministry of Health Malaysia. The help of staffs of Hospital Tuanku Jaafar and the Head of Department of Obstetrics and Gynaecology is acknowledged.

 
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Correspondence Address:
Sharifah Sulaiha Syed Aznal
Department of Obstetrics and Gynaecology, International Medical University, Clinical School, Seremban, Negeri Sembilan
Malaysia
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Source of Support: Research Grant from International Medical University, Conflict of Interest: None


DOI: 10.4103/0377-4929.107778

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