JBRA Assist. Reprod. 2023;27(4):682-688
ORIGINAL ARTICLE
doi: 10.5935/1518-0557.20230065
Abdominal Obesity May Play a Significant Role in inflammation Exacerbation in Polycystic Ovary Syndrome Patients
1Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
2Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
3Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
CONFLICT OF INTERESTS
The authors declare that they have no competing interests.
ABSTRACT
Objective: Polycystic ovary syndrome (PCOS) is an endocrine disorder that seems to be
pro-inflammatory at many levels, abdominal obesity (AO) is a prevalent
pro-inflammatory phenotype in PCOS patients, and it seems to contribute to
the initiation or worsening of inflammation in PCOS patients. In this study,
we investigated the role of the AO phenotype in the occurrence of other
obesity indicators (neck and arm) and augmentation of inflammation in the
follicular fluid (FF) of PCOS patients.
Methods: 40 patients under the age of 35 were divided into four groups: PCOS with AO,
PCOS without AO, non-PCOS with AO, and non-PCOS without AO. The FF samples
were collected from each patient. Clinical and anthropometric
characteristics of the participants, as well as tumor necrosis factor-α
(TNF-α) concentration in the FF samples, were quantitatively assessed using
enzyme-linked immunosorbent assays. The number of retrieved cumulus-oocyte
complexes (COC) and their quality were scored.
Results: The PCOS+AO+ group had significantly increased neck
circumference, compared to the other groups (p<0.001).
The concentration of TNF-α was significantly higher in the PCOS+AO+ group
than in the other groups (p<0.001). There were no
significant differences in the number of retrieved COC per patient and the
quality of oocytes between the groups (p>0.05).
Conclusions: Given the significant role of inflammation in the development of PCOS,
managing AO in PCOS patients may aid in reducing inflammation and could
potentially help in the design of customized treatment approaches.
Keywords: polycystic ovary syndrome, abdominal obesity, inflammation, tumor necrosis factor-α
INTRODUCTION
Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders and
reproductive abnormalities among women of reproductive age, with a prevalence
ranging from 5-18% (Costello et al.,
2019; Özer et al.,
2016; Zuo et al.,
2016). The Rotterdam criteria were created to confirm diagnosis of PCOS
in women who have at least two of the following symptoms; hyperandrogenism,
polycystic ovaries and oligo- and/or anovulation (Broekmans et al., 2006). PCOS patients have metabolic
disorders such as dyslipidemia, insulin resistance, pancreatic beta cell
dysfunction, endothelial dysfunction, early onset of type 2 diabetes, lipid profile
disorders, and visceral obesity, all of which affect health and fertility (Rocha et al., 2019). Other
factors such as obesity, lifestyle, and genetic and environmental factors can
contribute to the development of PCOS (Xie et al., 2019), associated with anovulation, which
stimulates excess androgen synthesis and chronic inflammation due to ovarian
dysfunction (Özer et al.,
2016). Pro-inflammatory mediators in PCOS patients can disrupt insulin
release and stimulate the production of androgens through ovarian theca cells,
playing an essential role in inducing hyperandrogenism (HA) (Al-Jefout et al., 2017; Bongrani et al., 2022). Low-grade chronic
inflammation has emerged as the main cause of long-term adverse outcomes in the
pathogenesis of polycystic ovary syndrome (Rojas et al., 2014).
Recent reports have it that obesity plays a functional role in the pathogenesis of
PCOS and has an increasing effect on developing metabolic disorders in PCOS
patients. Approximately 60-70% of PCOS patients have abdominal obesity (AO), even
normal-weight PCOS patients have excess visceral obesity and this adipose tissue may
contribute to inflammation in these patients (Al-Jefout et al., 2017; Nasiri et al., 2015). Of course, this information is
about abdominal obesity, and information about other indicators of obesity (neck and
arm) with increased inflammation in PCOS patients is not available. AO induces an
inflammatory response and reproductive disorders in PCOS patients via the secretion
of inflammatory cytokines and the activation of nuclear factors (NF- kB) (Kałużna et al., 2020). This
activation is related to the increase in the expression of pro-inflammatory
mediators such as tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6) (Nehir Aytan et al., 2016).
Physiologically, pro-inflammatory cytokines are produced during follicular
development and are involved in ovulation induction, but long-term chronic
inflammation can impair follicle development and ultimately cause adverse
reproductive outcomes (Boots & Jungheim,
2015; Liu et al.,
2021). It appears that AO as a potential trigger of PCOS greatly
aggravates the pathogenic symptoms (Möhlig et al., 2004).
The follicular fluid (FF) and serum of PCOS patients have a high level of
inflammatory markers such as interleukin-1 beta (IL-1β), tumor necrosis factor- α
(TNF-α), and interleukin-6 (IL-6) (Szczuko et al., 2016). Serum and FF concentrations of TNF-α
are also elevated in PCOS patients (Gaafar et
al., 2014). TNF-α is a multifaceted cytokine secreted by
macrophages and causes the proliferation of granulosa cells and changes in ovarian
function (Gaafar et al.,
2014; Prins et al.,
2020). This cytokine is overexpressed in adipose tissue and causes
insulin sensitivity. The expression of this cytokine is increased in obesity and
plays an essential role in causing low-grade chronic inflammation and metabolic
syndrome disorders (Spritzer et
al., 2015).
Considering that AO is a pro-inflammatory phenotype, it seems that it can play a
significant role in exacerbating inflammation in polycystic ovary syndrome (PCOS),
which is a low-grade inflammatory disease, and can help to explain infertility in
PCOS patients. In the present study, we investigated the specific role of the
abdominal obesity (AO) phenotype in the occurrence of other obesity indicators (neck
and arm) and the exacerbation of FF inflammation among PCOS patients. For this
purpose, pro-inflammatory cytokine TNF-α concentrations in the FF samples of PCOS
patients were evaluated by an enzyme-linked immunosorbent assay technique.
MATERIALS AND METHODS
Patients
The medical ethical committee of the Royan Institute (Tehran, Iran) approved the study (Ethical code: NO.IR. ACECR.ROYAN.REC.1400.091) and written informed consent was obtained from the patients. Forty patients (25-35 years old) with in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) with symptoms at least three months have passed since their last ovulation stimulation, body mass index (BMI) >30, without systemic inflammatory diseases, diabetes, hypertension (Solak et al., 2016) and thyroid disorders (Sniezek & Francis, 2003), without continuous use of anti-inflammatory drugs (at least 6-8 weeks before the start of the study) were included in our study. Patients with ovarian hyperstimulation syndrome (OHSS) and poor response to ovarian stimulation were considered as exclusion criteria.
Study Design
In this observational cross-sectional study from October 2021 to June 2022, the
forty patients were referred to the Royan Institute (Tehran, Iran), at the start
of the IVF/ ICSI cycle and were divided into four groups based on the presence
of PCOS (diagnosed by the Rotterdam 2004 criteria) or absence of PCOS (normal
oogenesis women with a history of male factor, tubal factor, or egg donation),
and presence or absence of AO (waist/hip ratio ≥ 0.80):
Group 1: Women with PCOS; with abdominal obesity
(PCOS+AO+)
Group 2: Women with PCOS; without abdominal obesity (PCOS+AO)
Group 3: Non-PCOS women; with abdominal obesity
(PCOS-AO+)
Group 4: Non-PCOS women; without abdominal obesity (PCOS-AO).
Ovarian Stimulation
In accordance with the following antagonist protocol, all the study patients were
treated with standard controlled ovarian stimulation and oocyte retrieval.
Before each patient’s entered the cycle, basic information including antral
follicle count (AFC), anti-Müllerian hormone (AMH), luteinizing hormone (LH),
and follicle-stimulating hormone (FSH) was evaluated. Controlled ovarian
stimulation (COS), was started on day 2 or 3 of the cycle to ovulation
induction. During the first 6 days of the menstrual cycle, the patients received
regular, daily subcutaneous (SC) injections of 150 IU of the recombinant human
follitropin-b (rFSH) (Puregonw, MSD, Ballerup, Denmark). On day 6, the serial
vaginal ultrasonography was performed and based on the patient’s ovarian
response when two or three ovarian follicles reached a diameter of ≥13 mm,
patients received daily SC injections containing 0.25 mg of a GnRH antagonist
(Ganirelix-Orgalutranw; MSD, Ballerup, Denmark). Then ovarian stimulation
continued with rFSH along with the antagonist until the patients had at least
more than three follicles with an average diameter of 17-18 mm and E2 levels of
1000-3500 pg/mL and the number of follicles on both sides should be less than or
equal to 15 follicles and not had a risk of OHSS, received an SC injection of
6500IU or 13000 IU dosage of recombinant human chorionic gonadotrophin (rhCG)
(Ovitrellew; Merk Serono, Hellerup, Denmark) to induce final oocyte maturation.
And patients whose estradiol level was more than 3500 IU or high-risk patients
for OHSS with more than 16 follicles were excluded from the study and GnRH
agonists were used for all of them and all were frozen. A standard
ultrasonically guided follicular puncture was used to retrieve oocytes 36 to 38
hours after hCG injection. Subsequently, these patients underwent IVF / ICSI
process.
Collecting Follicular Fluid Sample
Follicular fluid was taken from the dominant follicles (18-24 mm) on the day of oocyte retrieval. The collected liquid was centrifuged for 15 minutes at a speed of 1200 rpm until the follicular cells settled and separated supernatants, and then were filtered (0.22 μm pore size). All the samples were heat-inactivated at 56°C for 30 minutes. Then the accumulated supernatants were kept at -80°C until biochemical analysis.
Oocyte maturity
Cumulus-oocyte complexes (COC) were retrieved 36 to 38 hours after the injection
of hCG. The evolution of oocyte quality was performed via morphological
assessment. The oocytes were graded into two groups: Metaphase II (MII) oocytes
with normal morphology (round with a smooth first polar body, dispersed
cytoplasmic granules, normal perivitelline space, homogeneous fine granularity,
and zona pellucida thickness (18 µm)) as good oocytes and those with
intracytoplasmic (vacuolization, accumulating saccules of smooth endoplasmic
reticulum, organelle clustering) and/ or extracytoplasmic anomalies (large
perivitelline space, abnormal and dark zona pellucida, granule in perivitelline
space) as fair oocytes.
Fertilization was assessed 17 hours after in vitro fertilization
(IVF) or intracytoplasmic sperm injection (ICSI). Embryos were transferred to
culture medium up to embryo transfer day.
TNF-α Concentration in Follicular Fluid Samples
According to the manufacturer’s protocol (CN: KPG-HT-NF-α48; pg/ml; LOT: HTNF0422004 Iran); TNF-α concentration was quantitatively investigated as an inflammatory marker in FF samples in all groups by enzyme-linked immunosorbent assays (ELISA).
Statistical Analysis
Data were reported as mean ± SD, and the graphs were plotted using the GraphPad Prism program (virgin 9). The data were statistically analyzed using analysis of the Kolmogorov–Smirnov test, and variances (Two-way ANOVA) followed by a post-Tukey test, and a p-value <0.05 was considered a significant difference.
RESULTS
Clinical and hormonal findings
A total of 40 follicular fluid samples from candidates with and without PCOS, who were undergoing oocyte retrieval for IVF/ICSI before ovarian stimulation and in the clinical laboratory of Royan Institute (Tehran, Iran) were evaluated. Table 1 shows the characteristics of the women in the four groups of the study. Clinical factors, including age, luteinizing hormone/follicle-stimulating hormone ratio, body mass index, thyroid stimulating hormone, and prolactin, did not significantly differ among the four groups (p>0.05), but free testosterone (as a biomarker of hyperandrogenism) and anti-müllerian hormone in PCOS+AO+ group were significantly higher compared with others (p<0.001).
Table 1. Clinical characteristics in studied groups classified according to
PCOS and AO.
Anthropometric findings
Table 2 shows the arm and neck circumference as upper body obesity index. Neck circumference was significantly increased in the PCOS+AO+ group compared with others (p<0.001).
Table 2. Anthropometric characteristics in studied groups classified according
to PCOS and AO.
TNF α levels
The concentration of TNF-α as pro-inflammatory cytokine was considerably higher in the PCOS+AO+ group compared to the others (p<0.001) (Figure 1).
Figure 1. The concentration of TNF-α in follicular fluid in four groups was
measured by ELISA Data are mean ± SEM; n=10 per group;
*p<0.05, **p<0.01,
****p<0.0001 were considered statistically
significant.
Quality of oocytes
The number of retrieved COC per patient and quality of oocytes did not significantly differ between groups (p>0.05) (Table 3).
Table 3. Anthropometric characteristics in studied groups classified according
to PCOS and AO.
DISCUSSION
Metabolic abnormalities such as hyperinsulinemia, insulin resistance, dyslipidemia,
and obesity are often present in PCOS women (Liu et al., 2022). Recent studies have used the
measurement of the waist-to-hip ratio of PCOS patients as a simple, cheap,
non-invasive, available method to establish the amount of visceral obesity.
Measuring the neck and arm circumference in PCOS patients as an anthropometric
parameter representing the upper body’s subcutaneous adipose tissue can also be an
innovative tool for screening abdominal obesity distribution, which reflects
abdominal obesity and metabolic disorders (Liu et al., 2022; Yang et al., 2021). The results of the present study also
show that patients with excessive adiposity (AO) have more upper-body obesity
(around the neck and arms), which worsens hyperandrogenism. Of course, arm obesity
was not significant, probably due to the small sample size in our study, but studies
with larger sample sizes need to verify this.
The results of the study show that the TNF-α in the FF of PCOS patients with AO is
significantly higher than in the other groups. This result is consistent with
several other studies that reported high levels of pro-inflammatory cytokines in
PCOS patients (Adams et al., 2016; Amato et al., 2003; Liu et al., 2021; Zhang et al., 2017). Most researchers consider inflammation as a key characteristic in
PCOS patients and PCOS as a chronic inflammatory disease (Ghowsi et al., 2018). Although the exact
mechanism is not fully understood yet, studies have shown that inflammation in these
patients is caused by an increase in androgen synthesis by the ovary, which by
stimulating androgen secretion, causes ovarian and adrenal hyperandrogenism (Repaci et al., 2011). The
increase in androgens hinders the synthesis of sex hormone-binding globulin (SHBG),
raises blood glucose levels, and leads to the accumulation of fat in the abdominal
area. Consequently, it disrupts the normal menstrual cycle, interferes with
follicular maturation, and contributes to the development of complications
associated with PCOS (Nehir Aytan et
al., 2016; Rudnicka et al., 2021).
Inflammation resulting from reduced expression of the glucose transporter gene type 4
(GLUT4) and excessive production of TNF-α in adipose tissue leads to insulin
resistance (Samy et al.,
2009). The phosphorylation of insulin receptor substrate- 1 (IRS-1) by
intracellular serine kinases leads to the disruption of signaling events and
decreased insulin sensitivity (Ghowsi et
al., 2018). In PCOS patients, the balance of
pro-inflammatory cytokines such as TNF-α, IL-6, and IL-18 and anti-inflammatory
cytokines such as IL-27, IL-35, and IL-37 is disturbed, and the levels of
pro-inflammatory cytokines increases dramatically. As a consequence, the ovulation
process is disrupted due to the increase in unregulated inflammation, leading to
infertility (Ghowsi et al.,
2018).
The buildup of adipose tissue exacerbates the inflammation seen in these patients
(Boots & Jungheim, 2015). Adipose
tissue plays a crucial role in regulating glucose and lipid metabolism, which can
affect energy consumption, inflammation, and cardiovascular and reproductive
functions. Adipose tissue releases various cytokines, acute phase proteins, and
other inflammatory mediators, which can have autocrine, paracrine, or systemic
effects that impact glucose metabolism, energy balance, and proin-flammatory or
anti-inflammatory activities (Coelho et
al., 2013). Abdominal obesity (AO) is prevalent in 38-88% of
PCOS patients, leading to insulin resistance by inhibiting insulin receptor tyrosine
kinase in fat muscles (Oróstica et
al., 2016). Obese women with PCOS experience high levels of
free testosterone, androgens, insulin resistance, and a relative increase in blood
sugar compared to women with normal weight, which leads to infertility, frequent
miscarriages, menstrual and ovulation abnormalities, type 2 diabetes, high blood
pressure, and implantation problems include decreasing the implantation rate (Velez et al., 2021).
Hyperandrogenism as an inflammation trigger can be independent of obesity or
associated with excessive AO (Nehir Aytan et
al., 2016). Studies have shown that hyper-androgenism exists
in both obese and lean PCOS patients; but obesity, especially AO can increase
hyperandrogenism (Mohammadi et al.,
2017; Velez et al.,
2021). Excessive androgens can cause hypertrophy of fat cells, leading to
hypoxia, production of reactive oxygen species, and fat cell necrosis. In addition,
fat tissue can activate the nuclear factor NF-κB (an inflammatory factor) by
inducing oxidative stress and reducing antioxidant capacity, resulting in an
increase in inflammatory cytokines such as TNF-α (Velez et al., 2021). There is thus a close correlation
between inflammation, obesity, hyperinsulinemia, hyperandrogenism, and PCOS; they
are interconnected and reinforce each other through several signaling pathways
(Thathapudi et al.,
2014).
In conclusion, the buildup of visceral adipose tissue is a crucial factor that
contributes to the metabolic syndrome features associated with PCOS and chronic
low-grade inflammation (Mohammadi et
al., 2017). inflammatory cytokines play an important role in
the proliferation of follicular theca cells, in the development of chronic low-grade
inflammation, cancer, and the regulation of ovarian activity during the menstrual
cycle. These cytokines are considered to be the primary candidates in molecular
events, activation, and regulation of pro-inflammatory cascade in PCOS patients
(Mohammadi et al.,
2017). Furthermore, excessive production of TNF-α by fatty tissue can disrupt
insulin function in various cells, including endothelial, epithelial, fibroblast,
and endometrial tissue, especially in PCOS patients, and impairs their reproductive
function (Rostamtabar et al.,
2021).
In addition to the mild pro-inflammatory environment generated in the endometrium of
these women, obesity also creates higher levels of inflammation in the endometrium
of obese patients with PCOS. The current study showed that the level of TNF-α
expression was highest in PCOS patients with AO compared with other groups,
consistent with the results of previous studies. Consequently, the expressions of
inflammatory factors in the serum and FF of PCOS patients are greatly increased,
disrupting the ovulatory process and successful fertility in these patients (Oróstica et al., 2016).
Inflammation plays a key physiological role in folliculogenesis and ovulation, and an
unperturbed inflammatory response is essential for proper folliculogenesis. Mounting
evidence suggests that abnormal inflammation can disrupt normal ovarian follicle
dynamics and result in impaired oocyte quality. Failure to ovulate and decreased
implantation can result in infertility (Boots &
Jungheim, 2015).
The findings of the current study show that the pro-inflammatory state of PCOS plays
an essential role in causing the complications of this disease. In addition, AO
associated with PCOS significantly increases inflammatory status and
hyperandrogenism (Liu et al.,
2021). The results of our study were consistent with Niu’s finding that
increased inflammation in the group of PCOS with AO was associated with a decrease
in the number of good-quality oocytes (Niu et al., 2017) (Figure
2).
Figure 2. The schematic illustration of the association between AO and
inflammation in PCOS patients in decreasing fertility potential.
The combination of visceral obesity and chronic inflammation in PCOS patients likely leads to a disruption of oocyte quality and a significant reduction in fertility in obese PCOS women. inflammatory changes in women with PCOS may have a crucial role in drug approaches, treatment response, and metabolic and reproductive impairments in women with this syndrome.
CONCLUSION
Considering the role of AO in aggravating hyperandrogenism and subsequently increasing pro-inflammatory cytokine levels and inducing inflammation, more patients should be included in the study, and if the same results are repeated, it is recommended that a new therapeutic protocol based on lifestyle improvement (including diet therapy, exercise, and weight loss) and pharmacotherapy should be selected for these patients before entering the treatment cycle.