Download Protein characterization of endometriosis lesion tissue.

QUANTIFICATION OF ENDOMETRIOSIS LESION TISSUE
Jacob Herman
McGowan Institute for Regenerative Medicine
Bioengineering Department, University of Pittsburgh
INTRODUCTION
Endometriosis is a women’s reproductive system disease in
which endometrial tissue that normally lines the inside of the
uterus, grows outside of the uterus [1]. Over five percent of
women throughout the world are affected by endometriosis
within their reproductive years [2]. Endometriosis is
characterized in stages from one through four, indicating the
increasing severity of inflammation and number of lesions
throughout the reproductive area. Many women do not
experience any symptoms until the disease becomes more
severe [3], where they experience chronic pelvic pain, long and
heavy periods, nausea, and increased rates of infertility during
the later stages of the disease.
The current gold standard for late stage endometriosis is
laparoscopic excision surgery [4]. This procedure is effective in
removing the diseased tissue, however leaves scarring at the
excision cite, providing no solution for restoring fertility levels
[5]. The shortcoming of available treatment options for
endometriosis creates a widespread need for a new treatment
that not only relieves pain, but also restores fertility.
The current research consensus cites the increase of proinflammatory
and
pro-angiogenic
cytokines
within
endometriosis lesion tissue [6,7]. Additionally, it has been
corroborated that the lesions are localized in the tissue areas
surrounding the uterus including: pelvic wall, cul de sac,
endometriomas, and others [6,7,8].
The research on the pathogenesis of endometriosis has no
consensus in the scientific community [3]. The controversy is
indicative of a lack of knowledge to screen for early stage
endometriosis, and stop the progression of the disease.
However, in order to diagnose endometriosis earlier, the key
differences between endometrial lesion tissue and healthy
uterine tissue must be well defined.
OBJECTIVE
The objective of this study was to collect core information
about the discrepancies between healthy uterine tissues and
endometriosis lesion tissues. This preliminary study aimed to
quantify the amount of soluble proteins within endometriosis
lesion tissues and within healthy tissues in the uterine area for
comparison and analysis.
SUCCESS CRITERIA
To accomplish this analysis, key differences between
endometriosis lesion tissue and control tissue protein
concentrations were examined for their significance, and
separated by excision location for further examination.
METHOD
For this preliminary study, 59 lesion tissues and 19 eutopic
control tissues excised from 23 women with endometriosis
were examined. The proteins were extracted, quantified, and
the resulting data was analyzed.
The endometriosis lesion tissue and the control tissue were
weighed to 100 mg before extraction. The tissue was diced with
a scalpel into 10 mg pieces. The tissue was transferred to 1 mL
scintillation vials. A salt buffer with detergent was then added
to the scintillation vial. The buffer lysed the cells and released
the intracellular proteins. A homogenizer (Fischer) was used to
break down the the extra cellular matrix. The homogenizer was
used for 1 min durations while the scintillation vile was
submerged in an ice water bath to avoid protein denaturation
from the solution heating. The homogenizer tip was cleaned
with ethanol and deionized water after each use to avoid cross
contamination. The homogenizer tip was also changed after
every 4 uses to further reduce contamination. The solution was
transferred to 1.5 mL microcentrifuge tubes and centrifuged for
10 min at 10,000 rpm. The soluble protein solution was aliquot
to 9 separate microcentrifuge tubes for storage in a -80°C.
The BioRad DC protein assay kit (Bio-Rad) relied upon
absorbency data to create a standard curve. The standards used
were 6 evenly spaced concentrations between 0 mg/mL and 2
mg/mL, predetermined by the protein assay kit instructions.
The standard curve allows for the determination of unknown
solution concentrations based upon measured absorbency data.
To ensure the highest accuracy, the soluble protein solutions
were diluted such that their diluted concentration were in
between the standard values of 0 mg/mL to 2 mg/mL. The
protein solutions were measured at different dilutions to
achieve the highest accuracy. The dilution factor was taken into
account when calculating final concentration. After dilution, the
absorbency was measured using a spectrophotometer. All of the
data was then compiled and analyzed for key discrepancies.
RESULTS
The overall difference between the lesion tissue and the
control tissue protein concentration was compared in Figure 1.
This figure clearly shows that the protein concentration of the
lesion tissue protein is higher than the control tissue protein.
The lesion tissue has an average concentration of 2.22 ± 0.85
mg/mL whereas the control tissue had an average of 0.72 ±
0.22 mg/mL.
Additionally, the data was analyzed based on the location
of the endometriosis lesion tissue, shown in Figure 2. The
sample sizes for each location varied between 4 and 8 tissues.
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surrounding the uterus have varying protein concentrations.
Endometriomas were calculated to have higher protein
concentration. This is indicative of a potential connection
between endometriosis and endometriomas. Further studies are
needed to draw conclusions about a concrete connection.
Conversely, the cul de sac region behind the uterus has very
low protein concentration levels. While this could be due to a
small sample size, this area could be less prone to
endometriosis than other areas surrounding the uterus. Further
research could look for the mechanism behind the cul de sac
resistance to endometriosis lesions.
Figure 1. Comparison of average protein concentration of the
endometriosis lesions tissue versus the control tissue. Lesion
tissue was found to be higher than control tissue protein
concentration.
The black horizontal line is representative of the overall
endometriosis lesion tissue protein concentration of 2.22
mg/mL. As seen in Figure 2, the pelvic side-wall and
endometrioma had average protein concentrations higher than
the overall endometriosis lesion tissue protein concentration.
However, only the endometrioma protein concentration
standard error bars were above the endometriosis lesion tissue
average protein concentration. The endometrioma average
protein concentration was 3.90 ± 0.97 mg/mL.
Figure 2. Analysis of protein concentration of endometriosis
lesion tissue based on area of excision. The black horizontal
line represents the overall average endometriosis lesion tissue
protein concentration. Endometriomas were found to have
substantially higher soluble protein concentrations.
DISCUSSION
From Figure 1, it is clear that the lesion tissue has
substantially higher protein concentrations than the control
tissue. This result is consistent with the previous research that
indicates the presence of pro-inflammatory and pro-angiogenic
cytokines. More research is needed to confirm the presence and
amount of these pro-inflammatory and pro-angiogenic factors.
Furthermore, analyzing the data from Figure 2, the protein
concentrations within lesions excised from different areas
CONCLUSIONS
There was clear discrepancy found between the overall
protein concentration of endometriosis lesion tissue and control
tissue. In addition to the higher lesion tissue protein
concentration, endometriomas were found to have to potential
connection to endometriosis because of its high protein
concentration levels. Conversely, the cul de sac could have
potential resistance to endometriosis lesions based on its low
protein concentration. Future studies will characterize the
endometriosis lesion tissues to identify the cytokine profile
within the tissue. This foundation knowledge can help to create
a hydrogel seeded with endometriosis resistant factors to be
injected post laparoscopic surgery as a means to facilitate the
regrowth of healthy tissue and restoration of fertility.
ACKNOWLEDGMENTS
Thank you to Dr. Bryan Brown for mentoring me in the
first undergraduate laboratory experience. Thank you to Alexis
Nolfi, my graduate student mentor, and the entire staff of the
Brown Lab for their support and encouragement.
REFERENCES
[1] Rose GL. What is Endometriosis? Women’s Health
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[3] Schweppe KW, et al. Endometriosis – Pathogenesis,
Diagnosis, and Therapeutic Options for Clinical and
Ambulatory Care. J Reproductive Med and Endocrinology
2013; 10-1:102-119.
[4] Johnson NP, et al. Consensus on current management of
endometriosis. Human Reproduction 2013; 28-6:1552-1568.
[5] Jacobson TZ, et al. Laparoscopic Surgery for Pelvic Pain
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[6] Malutan AM, et al. Pro-inflammatory cytokines for
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[7] Ahn SH, et al. Pathophysiology and Immune Dysfunction in
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[8] Bedaiwy MA, et al. Abundance and Localization of
Progesterone Receptor Isoforms in Endometrium in Women
With and Without Endometriosis and in Peritoneal and Ovarian
Endometriotic Implants. Reproductive Sciences 2015; 22-9:
1153-11.
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