THE AFFECT OF AGE ON SMOOTH MUSCLE MORPHOLOGY IN THE VAGINA
Megan A. Fawcett
Magee Womens Research Institute, PI: Dr. Steven Abramowitch
INTRODUCTION
Pelvic organ prolapse is a condition that occurs when the
vagina can no longer support the tissue and muscles of the
pelvic floor, such as the uterus, the bladder, and the rectum.
This results in a drop of the organs from their normal
orientation. This is estimated to affect half of women over age
50 [1]. Each year, an estimated 300,000 women undergo
surgery for this disorder, and 11% of these women will need a
second surgery [2]. Some risk factors for pelvic organ prolapse
include menopause, vaginal child birth, obesity, age, prior
surgery, and genetic predisposition [3].
Women who have prolapse must deal with functional,
social, and psychological distresses daily. It also becomes a
burden for women whose stage of prolapse requires surgery to
fix. There are five stages of prolapse, starting at stage 0 and
ending with stage 4. These stages are determined by a POP-Q
Exam and measure the displacement of the vagina with respect
to various pelvic organ landmarks [4]. Women typically require
surgery when they have stage 3 or 4 prolapse to correct their
normal waste expulsion daily activities and quality of life.
With a cost of 1 billion dollars a year [5], more research
needs to be completed to better understand this disorder. It has
been shown that the fraction of smooth muscle in a vagina is
almost double that of a vagina that has experienced prolapse
[5].
Currently, it is not known if decreased smooth muscle
content if a direct risk of pelvic organ prolapse [6]. Smooth
muscle is an important part of the vaginal wall. Smooth Muscle
is primarily located in the muscularis, one of the four layers of
the vagina. One factor that could influence smooth muscle
thickness and pelvic organ prolapse in women is her age.
The field of urogynocology is young discipline compared
to cardiovascular surgery or orthopedics [7]. The research being
done to in this lab is to learn more about pelvic organ prolapse,
risk factors and their associations, and what can be done to
decrease the risk that a woman would develop pelvic organ
prolapse.
OBJECTIVE
The objective of this project is to determine how age
impacts the thickness of the smooth muscle in the vagina and
therefore affects its function and support of the other pelvic
organs. Vaginal samples are from women ranging in age and
varying degrees of prolapse, as well as women who haven’t had
prolapse.
HYPOTHESIS/SUCCESS CRITERIA
The hypothesis of this study is that younger women and
control samples will have a higher percentage of large smooth
muscle bundles.
METHOD
Full thickness vaginal biopsies have previously been
collected prior to a women’s regularly scheduled surgery.
These samples are embedded in optimal cutting temperature
(OCT) compound, frozen with liquid nitrogen, and stored in a
-80 °C freezer to preserve the tissue quality.
A cryostat (Leica), a device used for histological
preparation of tissue samples, was used to slice the vaginal
samples to seven micrometers thick. Theses slices were placed
on microscope slides and would be used for a gross
morphological analysis.
Next, Mason’s Trichrom and Hematoxylin and Eosin
(H&E) staining techniques were used to stain the epithelial,
smooth muscle, and adventitia layers. A Nikon 90i microscope
and imaging program were used to take 20x magnification
images of the stained samples for conformation of the full
thickness vaginal tissues. If the tissues did not contain the
smooth muscle layer or if the tissues were degraded too much,
they were not used for any further experiments.
If the tissue samples do contain a smooth muscle layer,
they are then labeled for immunofluorescence analysis. A
primary antibody, mouse anti-smooth muscle actin, was applied
to one of two samples on a given microscope slide, and the
secondary antibodies, TexasRed (anti-mouse antibody), were
applied to both samples. DAPI a nucleus immunofluorescence
that is blue was also applied to all samples for verification of
cells.
Once all of the samples were labeled with primary and
secondary antibodies, they were imaged with the Nikon 90i and
software that was previously used. Nikon Elements, a software
package that provides a powerful interface for image
manipulation and data analysis, was used to measure different
areas of the smooth muscle in the vaginal samples. These five
different areas were then averaged to give an overall thickness.
These samples were categorized into four groups. Younger
women (< 50 years) without prolapse (YC), younger women
with prolapse (YP), older women (>= 50 years) without
prolapse (OC), and older women with prolapse (OP).
RESULTS
Nineteen samples in total were used for analysis. Three
samples were in the YC, YP, and OC groups each. The OP
group had ten samples. As shown in Figure 1, the pink areas of
the Trichrome stain were identified as the smooth muscle layer,
the muscularis. For the immunofluorescence labeling, the red
areas of tissue were identified as the smooth muscle bundles
and analyzed as such.
When analyzed, the vaginal smooth muscle bundles were
categorized into three categories: small, medium, and large.
The small category included bundles with a diameter less than
20 microns, the medium included bundles with diameters of 2050 microns, and the large included bundles with diameters
1
greater than 50 microns. These percentages were found based
off of total smooth muscle content found in each sample, and
then averaged per category. These results are shown in Figure
1.
Figure 1: Smooth muscle bundles categorized by patient status
and smooth muscle bundle size
In Figure 2, the expected results were shown with the OP
women having a higher percentage of small smooth muscles as
opposed to the YP. The higher percentage of small smooth
muscles indicates weakness and could explain why there was
prolapse. With weaker bundles, it makes it harder for the
vagina to support the other organs in the pelvic floor.
Using a Student’s t-test with a p value of < 0.05, the only
statistically significant results were found between the young,
control women and the older, prolapse women for all three
smooth muscle bundle categories. For the small, medium, and
large categories, there were values from the t-test of 0.044,
0.045, and 0.05, respectively.
Another significant result was found in the medium bundle
size between the young prolapse group and young control
group with a value of 0.034.
Figure 2: Comparison of young and old samples for prolapse.
As shown in Figure 2, the YP women had a lower
percentage of small smooth muscle bundles as compared to the
OP. However, for the control, YC women had a higher
percentage of small bundles, shown in Figure 3.
DISCUSSION
As shown in Figures 1-3, there is a much higher percentage
of smooth muscle bundles that fall into the small category as
opposed to the medium and large categories.
In Figure 2, the expected results were shown with the OP
women having a higher percentage of small smooth muscles as
opposed to the YP women. The higher percentage of small
smooth muscles indicates weakness and could explain why
there was prolapse. With weaker bundles, it makes it harder for
the vagina to support the other organs in the pelvic floor.
In Figure 3, the unexpected results were shown with the
control groups and the OC women having a lower percentage
of small smooth muscle bundles than the YC women. It was
expected to see the YC women having more evenly dispersed
smooth muscle bundles across the small, medium, and large
categories. The YC women were not expected to have a high
percentage of small smooth muscle bundles because this
indicates weakness within the pelvic floor. It was expected that
the medium and large smooth muscle bundle categories would
have higher percentages, because this indicates a strong,
supported vagina, which would help support the other pelvic
organs.
One limitation of this study was having such a small
sample size for each category (excluding OP). More research
needs to be completed to better understand how age really
impacts the smooth muscle content of the vagina. Pelvic organ
prolapse affects about 10% of the total people in the United
States each year. With further research, better therapies,
surgeries, and preventative measures can be taken to help
women correct this disease.
ACKNOWLEDGMENTS
I would like to thank Dr. Steven Abramowitch, Dr. Rui
Liang, Dr. Pamela Moalli, Stacy Palcsey, and Alexis Nolfi for
their support through my research process. Additionally, I
would like to thank Dr. Savio Woo and Jonquil Flowers for
their valuable critiques of my work throughout this semester.
REFERENCES
[1] Drutz HP, A.M., Pelvic organ prolapse: demographics and
future growth prospects.
[2] Boyles, S.H., A.M. Weber, and L. Meyn, Procedures for
pelvic organ prolapse in the United States
[3] Lukacz, E.S., et al., Parity, mode of delivery, and pelvic
floor disorders.
[4] POP-Q Exam Reference Guide.
[5] Shanshan Mei, et al. The Role of Smooth Muscle Cells in
the Pathophysiology of Pelvic Organ Prolapse.
[6] Boreham, Muriel, et al. Morphometric Analysis of Smooth
Muscle in the Anterior Vaginal Wall of Women with Pelvic
Organ Prolapse
[7] Bump, R.C., et al., The standardization of terminology of
female pelvic organ prolapse and pelvic floor dysfunction
Figure 3: Comparison of young and old samples for control.
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