RESEARCH PROPOSAL

PUTI Q2

Study of Macrophage Response to Oxidized LDL (Low Density Lipid) in Low Grade
Chronic Inflammation in Obesity and Non-Obesity Polycystic Ovary Syndrome

Prof. Dr. dr. Andon Hestiantoro, SpOG(K), MPH

NIP. 196011271985121001

Faculty of Medicine
Universitas Indonesia
2023
 Abstract

Polycystic ovary syndrome (PCOS) is a multifactorial disease, resulting from the interaction
of various genetic predispositions and environmental factors, especially low-grade chronic
inflammatory conditions. The exact pathophysiology and pathomechanism of the causal
relationship between PCOS and chronic low-grade inflammation is still not fully understood.
Obesity and increased levels of fat are thought to be one of the main triggers for this
condition. The increased amount of Ox-LDL circulating in the body can then be captured by
macrophage cells via the CD-36 receptor. CD-36 is a protein molecule that is expressed on
the surface of macrophage cells. Binding of oxidized LDL by macrophage cell receptors
causes polarization of M1 macrophages to become more numerous which causes
accumulation of secreted inflammatory markers. Therefore, this study was conducted to
provide an overview of the mechanism of low-grade chronic inflammation in PCOS cases
differentiated based on body mass index, namely normal and obesity, in terms of CD-36
expression in macrophages exposed to oxidized LDL and the inflammatory markers they
produce. This study is expected to provide an overview of the pathomechanism of the chronic
inflammatory response in women with PCOS. Knowledge of the causes and initial
mechanisms is expected to provide further understanding in the diagnosis of polycystic ovary
syndrome
 Background

Polycystic Ovary Syndrome (PCOS) is a multifactorial fertility disorder in women of

reproductive age. Several studies have shown a link between PCOS and low-grade chronic
inflammation. The study found that there is not only a correlation, but also a causal
relationship between chronic inflammation and obesity and hyperandrogenism, which are
clinical manifestations of Polycystic Ovary Syndrome. Several inflammatory mediators, such
as C Reactive Protein (CRP), Interleukin 1B (IL-1B), Interleukin 6 (IL-6), and Tumour
Necrosis Factor alpha (TNF-a) were found to be higher in women with PCOS symptoms.
However, chronic inflammation that occurs often only releases pro-inflammatory cytokines
that are significant and permanent, but in such low amounts that they are difficult to detect in
the blood circulation system.
The exact pathophysiology and pathomechanism of the causal relationship between
PCOS and chronic low-grade inflammation is still not fully understood. Obesity and
increased levels of fat are thought to be one of the main triggers for this condition. Increased
fat content will make adipocyte cells in adipose tissue increase in size to accommodate and
compensate for the increased fat content. The event of increasing the size of adipocytes is
called hypertrophy. When experiencing hypertrophy, adipocytes will experience hypoxia and
release Nuclear Factor kappa B which causes adipocytes to become more susceptible to
inflammation, apoptosis, fibrosis, and release of fatty acids. The release of a transcription
factor will also cause an increase in MCP1. Increased MCP1 expression in PCOS cases led to
more monocyte recruitment in adipose tissue and polarization of monocytes to become more
M1 macrophages compared to M2 macrophages.
M1 and M2 macrophages are two different groups of monocyte subsets that have
opposing activities. 9 Classically activated macrophages or commonly called M1 are
macrophages that play a role in secreting pro-inflammatory cytokines and chemokines and
inhibiting cell division. In contrast, M2 macrophages activated via alternative pathways play
a greater role in the secretion of anti-inflammatory cytokines and chemokines and stimulate
cell proliferation. The imbalance of polarization of M1 and M2 macrophages, with more M1
polarization, in PCOS cases is thought to cause a systemic chronic inflammatory status due to
the release of more pro-inflammatory cytokines. Meanwhile, the lack of M2 macrophage
activation causes a decrease in the number of anti-inflammatory cytokines, one of which is
Interleukin-10 (IL-10). This pro- and anti-inflammatory marker and cytokine imbalance
causes chronic inflammatory conditions in PCOS cases.
Apart from being driven by increased MCP1 expression, other studies have shown
that chronic inflammation in Polycystic Ovary Syndrome is also triggered by an increase in
fatty acids. 14 Women who experience PCOS, especially those with an obese body mass
index and above, tend to experience increased fatty acids which then undergo changes fat
profile into oxidized Low-Density Lipid (ox-LDL). The increased amount of Ox-LDL
circulating in the body can then be captured by macrophage cells via the CD-36 receptor.
CD-36 is a protein molecule that is expressed on the surface of macrophage cells. Binding of
oxidized LDL by macrophage cell receptors causes polarization of M1 macrophages to
become more and more which causes accumulation of secreted inflammatory markers.
However, not much has been studied regarding the expression of CD-36 receptors in
macrophages of PCOS cases.
The study of the imbalance between pro- and anti-inflammatory cytokines and
macrophage response can assist in understanding and managing chronic low-grade
inflammatory conditions such as Polycystic Ovary Syndrome. This study is expected to
provide an overview of the pathomechanism of the chronic inflammatory response in women
with PCOS. Knowledge of the causes and initial mechanisms is expected to provide further
understanding in the diagnosis of polycystic ovary syndrome.
 Objective

General objectives:
This study was conducted to examine the expression levels of CD-36 and inflammatory
markers (TNF-a, IL-1B, and IL-10) in Women with Polycystic Ovary Syndrome with normal
body mass index (BMI) and obesity.

Special objectives:
1. Knowing the difference in CD-36 expression in macrophages of PCOS women compared
to normal women
2. Knowing the relationship between increased expression of CD-36 and pro- and anti-
inflammatory mediators (TNF-α, IL-1B, and IL-10) in PCOS women
3. Knowing the differences in CD-36 expression in macrophages of Obese and non-Obesity
PCOS women
4. Knowing the differences in the response of macrophages to oxidized LDL in Obese and
non-Obesity PCOS women analyzed based on pro and anti-inflammatory mediators (TNF-a,
IL-1B, and IL-10) in in vitro culture
 Methods

All research subjects will be briefed on the research protocol and must provide written
informed consent if they are willing to participate. Peripheral blood samples will be taken
from all samples. Demographic data collected includes Anthropometric data and the
Ferriman-Gallwey Score.
From the peripheral blood samples obtained, monocyte cells will be isolated which
will then be cultured and activated to become macrophages. The macrophage cells obtained
will then be treated in the form of ox-LDL exposure. After exposure, the remaining culture
medium will be analyzed to measure TNF-a, IL-1B, and IL-10. Meanwhile, macrophage cells
will be analyzed for CD-36 expression using Flow Cytometry.
Statistical analysis will be performed using SPSS software version 25. All tests are
two-tailed and a p value <0.05 will be considered significant. Continuous variables are
summarized as mean ± SD or median (minimum – maximum), according to the normality of
the data distribution. Bivariate correlations were assessed using Pearson's or Spearman's
coefficients.
 Sample Collection

Demographic data collection Blood collection

Isolation of Mononuclear Cells

Macrophage activation

Ox-LDL Non -x-LDL

exposure exposure

Mcrophage Culture Macroph Culture

media age media

Flow ELISA Flow ELISA

cytometri cytometri

Data Analysis
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