INTRODUCTION:

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy of reproductive system of women, it is a heterogeneous condition with several signs and symptoms and a controversial diagnosis that differs between countries depending on the used criteria, like National Institutes of Health (NIH) criteria, Androgen Excess Society (AES) criteria, and Rotterdam 2003 criteria^{1, 2}.

Nowadays, the proportion of women with the syndrome in the world is more than 10% in young women and increasing continuously,generally, the etiology and pathogenesis of PCOS not understood, yet³.Increased evidences confirming relationship of PCOS with obesity and insulin resistance (IR), which is an important cause of systemic complications like type 2 diabetes (DM2) and heart disease, and dermatological complications which associated with increased androgen level in plasma that lead to hirsutism, acne androgenic alopecia⁴, metabolic syndrome and autoimmune diseases⁵. Reproductive system complications lead to infertility in some cases^6,7, and all these complications lead to psychological complications like depression, anxiety and selflessness⁸.

Many studies explain the role of PCOS in development autoimmune disease in affected women like Graves' disease, Hashimoto's thyroiditis, myasthenia gravissystemic lupus erythematosus (SLE) and Celiac disease⁹.Other studies focus on whether the syndrome is an autoimmune disease by studying the level of some indicators that confirm the incidence of autoimmune disease, which are specific to the syndrome and do not appear in other autoimmune diseases¹⁰.

The current study was designed to find out relationship of new biochemical and immunological markers with PCOS in studied patients when compared with healthy control groupand study the possible relationship between PCOS and autoimmunity.

PATIENTS AND METHODS:

This study was conducted in Al-Nahrain University/ High Institute for Infertility Assisted Reproductive Technology- Baghdad, after taking approval by the scientific committee in the College of Pharmacy-Mustansiriyah University.

Ninety Iraqi women were participated in this study throughout a period from September to December /2018. Sixty women were already diagnosed with PCOS and the other thirty were healthy apparent.Proceeding to enrollment of participants, history information like age (ranged from 15 to 35 years), BMI (less than 30 i.e. not obese women), concomitant diseases, other treatment and hormonal profile including level of LH, FSH were taken after achieving diagnoses by a consultant gynecologistbased on abdominal ultrasound and depending on Rotterdam 2003 Criteria for PCOS, as shown in table 1 below.

Table1: Rotterdam criteria for PCOS diagnosis¹¹.

NO.	Criteria
1	Oligomenorrhea and/or anovulation.
2	Clinical and or biochemical signs of hyperandrogenesim.
3	Polycystic ovaries by ultrasound.

Exclusion criteria included all subjects with any medication known to interfere with the results of the present study, subjects with autoimmune diseases, DM1 or 2 and all subjects with any acute illness or infectious disease.

The body mass index was determined by dividing the individual weight (in kilograms) by the squared value of height (in meter) ¹².

Blood samples were collected from subjects, placed in gel tubes (contain clotting activator) and centrifuged within 10 minutes at speed (3000 r.p.m) for 3 min. Then sera of all blood samples were separated and storage at - 20 °C until the time of assay. The collected sera were used for detection of fetuin-A, sirtuin-1, auto-antibodies (AOA, AHA), and interleukins (IL-17, IL-23) by sandwich enzyme-linked immunosorbent assay (ELISA) technique,using readymade kits (mybioscource,USA).

STATISTICAL ANALYSIS:

Statistical Analysis System (SAS-2012) program was used to show the mean and standard error of mean (M± SE). Dependent t-testwas used to compare mean values. The probability considered significantly differ whenP<0.05, highly significant difference when P<0.01, and non-significant (NS) when P˃0.05.Correlation coefficient (r-value) was used to test the linear relationship between markers.The r-value or correlation coefficient was expressed as negative value when there is an inverse correlation, positive value when there is a direct correlation, and zero when there is no correlation¹³.

RESULTS:

Demographics and Baseline Characteristics:

The results recorded matching in mean ages and BMI between the two groups. The mean ages of patients group was 25.25 ± 0.63 years, there BMImean was 24.26 ± 0.39 kg/m².The mean ages of control group was 25.60 ± 0.96years, there BMI mean was 24.80 ± 0.51 kg/m², as illustrated in table2.

Table 2: Demographics and baseline characteristics.

Group	Mean ± SE
Group	Age (year)	BMI (kg/m²)
Control	25.60 ± 0.96	24.80 ± 0.51
Patients	25.25 ± 0.63	24.26 ± 0.39
T-test	2.237 ^NS	1.328 ^NS
P-value	0.756	0.427

NS=Non-Significant difference (P˃0.05).

Serum levels of luteinizing hormone and follicular stimulating hormone:

The results demonstrated that the mean level of LH has been elevated significantly in sera of patients group (11.58 ± 0.94IU/ml) in comparison with control group (5.79 ± 0.58 IU/ml), (P<0.01), as clarified in table 3 and figure 1a.

Regarding FSH hormone, the results reported that there is no significant difference in the mean level of this hormone between patients (8.70 ± 1.28 IU/ml) and control group (6.24± 0.51 IU/ml) (P>0.05), as shown in table 3 and figure1b.

Table 3: Mean serum level of luteinizing hormone and follicular stimulating hormone in patients and control group.

Groups	Mean ± SE
Groups	LH (IU/ml )	FSH (IU/ml )
Control	5.79 ± 0.58	6.24 ± 0.51
Patients	11.58 ± 0.94	8.70 ± 1.28
T-test	2.872 **	3.688 ^NS
P-value	0.0001	0.1868

LH= Luteinizing hormone, FSH= Follicular stimulating hormone.

NS= Non-Significant difference (P˃0.05).

**=highly significant difference (P<0.01).

Figure 1a: Mean serum level of luteinizing hormone in patients and control group.

LH= Luteinizing hormone.

**=highly significant difference (P<0.01).

Figure 1b: Mean serum level of follicular stimulating hormone in patients and control group.

FSH = Follicular stimulating hormone.

NS= Non-Significant difference (P˃0.05).

Serum levels of fetuin-A:

The result clarified that there was a highly significant difference (P<0.01) in mean levels of serum fetuin-A between the control and patients group. Figure 2 and table 4shown that the mean of fetuin-Aserum levels in patients group (56254.25 ± 34030.36ng/ml) was significantly higher when compared with control group (966.89 ± 283.52 ng/ml).

Table 4: Mean serum level of fetuin-A in patients and control group.

Groups	fetuin-A Mean ± SE (ng/ml )
Control	966.89 ± 283.52
Patients	56254.25 ± 34030.36
T-test	959.13 **
P-value	0.00273

**= highly significant difference (P<0.01).

Figure 2: Mean serum level of fetuin-A in patients and control group.

**= highly significant difference (P<0.01).

Serum levels of sirtuin-1:

The results of current study showed that there was highly significant difference in the mean level of serum situin-1 between patients (56.99 ± 2.06ng/ml) and control group (4.67 ± 0.27 ng/ml) (P<0.01) as clarified in table5 and figure 3.

Table 5: Mean serum level of sirtuin-1 in patients and control group.

Groups	Sirtuin-1 Mean ± SE (ng/ml)
Control	4.67 ± 0.27
Patient	56.99 ± 2.06
T-test	5.831 **
P-value	0.0001

**= highly significant difference (P<0.01).

Figure 3: Mean serum level of sirtuin-1 in patients and control group.

**= highly significant difference (P<0.01).

Serum levels of auto-antibodies (anti-histone and anti-ovarian antibody)

Current results demonstrated a highly significant difference (P<0.01) between both groups, regarding AHA and AOA. The mean serum levels of AHA were (1.598 ± 0.11, 3.11 ± 0.06U/ml) respectively, for control and patients group. On the other hand, the mean serum levels of AOA were (6.03 ± 3.43, 11.02 ± 1.27 U/ml) respectively, for control and patients group, as illustrated in table 6 and figures 4a, 4b.

Table 6: Mean serum level of auto-antibodies (anti-histone antibody and anti-ovarian antibody) in patients and control group.

Groups	Mean ± SE
Groups	AHA (U/ml )	AOA (U/ml )
Control	1.598 ± 0.11	6.03 ± 3.43
Patients	3.11 ± 0.06	11.02 ± 1.27
T-test	0.250 **	3.584 **
P-value	0.0001	0.0001

AHA= anti-histone antibody, AOA= anti-ovarian antibody.

**= highly significant difference (P<0.01).

Figure 4a: Mean serum level of anti-histone antibody in patients and control group.

AHA= anti-histone antibody.

**= highly significant difference (P<0.01).

Figure4b: Mean serum level of anti-ovarian antibody in patients and control group.

AOA= Anti-ovarian antibody.

**= highly significant difference (P<0.01).

Serum levels of interleukins (17 and 23).

The mean serum levels of interleukins (17 and 23) for control group were (33.28 ± 3.30, 174.23 ± 19.14pg/ml) respectively, while in patients group were (225.88 ± 9.71, 537.20 ± 19.03 pg/ml) respectively.

The results of the current study shown that there were a highly significant difference between patients and control group (P<0.01) in serum levels of interleukins (17 and 23), as in table 7 and figures 5a, 5b.

Table 7: Mean serum level of interleukins (17 and 23) in patients and control group.

Groups	Mean ± SE
Groups	IL-17 (pg/ml )	IL-23 (pg/ml )
Control	33.28 ± 3.30	174.23 ± 19.14
Patients	225.88 ± 9.71	537.20 ± 19.03
T-test	27.778 **	59.932 **
P-value	0.0001	0.0001

IL = interleukins.

**= highly significant difference (P<0.01).

Figurer 5a: Mean serum level of interleukins-17 (pg/ml) in patients and control group.

IL = interleukins.

**= highly significant difference (P<0.01).

Figurer 5b: Mean serum level of interleukin-23 (pg/ml) in patients and control group.

IL = interleukins.

**= highly significant difference (P<0.01).

Correlation coefficient between Ages, BMI, and studied markers in patients group

The result shown that there was a direct correlation coefficient betweenage of patients and LH, fetuin-A, AHA and AOA, but statistically wasn’t significant (P>0.05). Meanwhile, an inverse correlation with a non significant difference (P>0.05) was recorded between age of patients and FSH, sirt-1, IL-17 and 23. On the other hand, all markers have inverse correlation with BMI of patients group, at a non-significant level (P>0.05), as in table 8.

Table 8: Correlation coefficients between Ages, BMI and studied markers in patients group.

Markers	Correlation coefficient (r)
Markers	Age	Level of significant	BMI	Level of significant
LH	0.01	NS	-0.02	NS
FSH	-0.10	NS	-0.19	NS
Fetuin-A	0.04	NS	-0.06	NS
Sirt-1	-0.04	NS	-0.10	NS
AHA	0.04	NS	-0.04	NS
AOA	0.11	NS	-0.11	NS
IL-17	-0.03	NS	-0.05	NS
IL-23	-0.02	NS	-0.14	NS

LH= luteinizing hormone, FSH= follicular stimulating hormone, Sirt-1= sirtuin-1, AHA= anti-histone antibody, AOA= anti-ovarian antibody IL= interleukins.

NS= Non-Significant difference (P˃0.05).

Correlation coefficient between different studied markers.

The results of the current study clarified that LH have a non- significant direct correlation (P>0.05) with FSH, significant direct correlation (P<0.05) with ILs, and highly significant correlation (P<0.01) with sirtuin-1 and auto-antibodies, but have non-significant inverse correlation with fetuin-A (P>0.05).

Regarding FSH, there was a direct correlation coefficient with all markers at a non-significant level (P>0.05) except fetuin-A, which exhibit inverse correlation coefficient at non-significant level (P>0.05).

Meanwhile, ILs have a direct correlation coefficient between each other and with all studied markers at highly significant level (P<0.01), except fetuin -A at non-significant level (P>0.05), as show in table 9.

Table 9: Correlation coefficient between different studied markers.

Markers	Correlation coefficient (r)	Level of sig.
LH & FSH	0.08	NS
LH & IL-17	0.22	*
LH & IL-23	0.23	*
LH &sirtuin-1	0.34	**
LH & fetuin-A	-0.08	NS
LH & AOA	0.33	**
LH & AHA	0.36	**
FSH & IL-17	0.09	NS
FSH & IL-23	0.08	NS
FSH & sirtuin-1	0.11	NS
FSH & fetuin-A	-0.04	NS
FSH & AOA	0.09	NS
FSH & AHA	0.08	NS
IL-17 & IL-23	0.79	**
IL-17&sirtuin-1	0.69	**
IL-17 & fetuin-A	0.09	NS
IL-17 & AOA	0.40	**
IL-17 & AHA	0.64	**
IL-23 & sirtuin-1	0.72	**
IL-23 & fetuin-A	0.07	NS
IL-23 & AOA	0.45	**
IL-23 & AHA	0.66	**
sirtuin-1& fetuin-A	0.09	NS
sirtuin-1 & AOA	0.58	**
sirtuin-1 & AHA	0.69	**
fetuin-A & AOA	0.04	NS
fetuin-A & AHA	0.07	NS
AOA & AHA	0.51	**

LH= luteinizing hormone, FSH= follicular stimulating hormone, Sirt-1= sirtuin-1, AHA= anti-histone antibody, AOA= anti-ovarian antibody IL= interleukins.

Non-Significant difference (P˃0.05).

*= significant difference (P<0.05).

**= highly significant difference (P<0.01).

DISCUSSION:

Polycystic ovary syndrome (PCOS) consider the most common cause of chronic hyperandrogenism, anovulation and infertility in young women ¹⁴. Considerable evidence suggests that PCOS has diverse causes, arising as a complex trait with contributions from both heritable and environmental factors that affect ovarian steroidogenesis. Insulin resistant leading to hyperinsulinuma, in part related to coexistent obesity, is the most common non-steroidogenic factor in polycystic ovaries, where this complex interaction generally mimics an autosomal dominant trait¹⁵.

In the current study, the role of some hormones has been studied, in addition to the role of biochemical and immunological markers of PCOS, where serum LH levels were elevated with a highly significant difference in patients group when compared with control, as in table 3 and figure 1a, this result agree with other study which explained the role of LH in PCOS and its direct correlation with anti-müllerian hormone (AMH) ¹⁶.

In the current study, FSH was studied and a non-significant difference in serum FSH level was observed in patients group when compared with control, as shown in table 3 and figure 1b, this agree with study which explain maintaining serum FSH level within normal range in PCOS patients who didn't have IR status¹⁷.

In the present study, the results of serum fetuin-A levels indicated a highly significant difference between patients and control group, as shown in table 4 and figure 2. This result agree with other studies which showed the relation of fetuin-A with IR, oxidative stress and androgen hormones in PCOS, these studies proved that serum levels of fetuin-A be elevated in the case of IR in different diseases, like DM2 or PCOS¹⁸. Certain studies confirm the relationship of fetuin-A with IR which is a major cause leading to development of metabolic disease and it can lead to elevated serum fetuin-A level in more advance stages of disease or when not responding to treatment¹⁹.

Regarding serum sirtuin-1 level, the results recorded a highly significant elevation in patients group when compared with control, as shown in table 5 and figure 3. These findings agreed withother study which explain the role of sirtuin-1 in immune response like inflammation, insulin metabolism and metabolic syndrome, like PCOS, and recorded a highly significant elevation in serum sirtuin-1 level in patients group when compared with control ²⁰. The highly elevation in serum sirtuin-1 levelsmay causediseases like atherosclerosis, cancer, and fatty liver, considering sirtuin-1 as an important hepatic metabolic derangements, while the slight elevation in serum sirtuin-1 levels providing a protection from aging and Alzheimer's disease ²¹. This elevation in serum sirtuin-1 levels probably caused by a high fat nutrition of patients, especially bad fats from fried foods, processed foods, and non-sport lifestyle ²².

In the current study, the results of serum AHA level recorded a highly significant differences between patients and control group, as shown in the table 6 and figure 4a, this agree withotherstudy which revealed a high AHA serum level in PCOS patients group with unexplained infertility and there is a significant difference when compared with control group ²³, and consisting with other study which found an increase in serum AHA level in PCOS patients when using this marker as one of the indicators of autoimmunity for this syndrome²⁴.

In the present study, serum AOA levels in patients group were elevated with a highly significant difference when compared with control group, as shown in table 6 and figure 4b. This result corresponds with many studies emphasizes the presence of different types of auto-antibodies in PCOS, including AOA ²⁵.

On the other hand, in the current study the serum IL-17 levels in patients group were elevated with a highly significant difference when compared with control, this agree with other study which measured serum IL-17 levels in PCOS patients to explain the role of this marker in complications of PCOS ²⁶. Other findings of the current study showed a highly significant difference in serum IL-23 levels between patients and control group, that agree with result ofother study which examined the role of IL-23 in inflammatory diseases (like PCOS) and stimulating the immune system ²⁷.

The present study recorded a direct correlation between serum LH and serum sirtuin-1 levels at a highly significant level, scanning of literatures revealed no previous comparative studies to prove their relationship, this highly elevation in serum sirtuin-1 level associated with LH probably occurred because of PCOS and lifestyle of patients. The results of the current study have shown a highly significant direct correlation of LH with serum auto-antibodies (AHA, AOA) levels that correspond with other study which recorded that patients suffering from PCOS have low number of anti-inflammatory regulatory T cells and elevated serum autoantibodies levels, like antihistone and anti-double stranded DNA antibodies, and in agreement with other studies which confirm the present of auto-antibody likeanti-dsDNA antibody with increase serum LH level in PCOS ²¹.Results of the current study was found a direct correlation between LH and IL-17 at significant level, and direct correlation with IL-23 at significant level which was parallel with many studies that confirm the results of the current study. The elevation in serum IL-17 and 23 level in PCOS patients considered the syndrome as a low- grade chronic inflammation and these ILs members of the pro-inflammatory cytokines act as significant components of the immune system during certain inflammatory diseases due to stimulation of immunological processes²⁸.

Other findings explain a direct correlation between sirtuin-1 and other markers in addition to its correlation with LH that has been mentioned, and the results recorded a direct correlation between sirtuin-1 and auto-antibodies (AHA, AOA) at a highly significant level respectively, but there were no previous studies regarding this correlation in PCOS, while there are many studies dealing with the correlation of AHA with autoimmune diseases like SLE²⁹ , this contributes to clarify the role of AHA in autoimmunity, and the results of the current study showed that the correlation between sirtuin-1 and auto-antibodies (AHA, AOA) may be belong to the over stimulation of immune system caused by elevation of serum sirtuin-1 levels in patients with PCOS ³⁰.

The present study noticed that sirtuin-1 has a direct correlation with both ILs (17 and 23) at highly significant levels, also there were no previous literature on the correlation between sirtuin-1 and IL-17. Interleukin-17 stimulate chronic inflammation and play a crucial role in development of autoimmune disorders by stimulating of T-helper cell due to release other types of ILs which can cause autoimmunity with the presence of sirtuin-1 and auto-antibodies, other study confirmed this result and explain the role of sirtuin-1 in regulation of IL-23 and its balanced role in immune system³¹, which underscores its importance in PCOS as a low-grade inflammatory disease which can develop into autoimmune disease ³².

On the other hand, the search in literatures revealed no previous comparative studies regarding the correlation of auto-antibodies (AHA, AOA) with IL-17 and IL-23 which have a direct correlation at high significant levels in the PCOS current study patients, but these findings confirm the role of ILs and auto-antibodies in PCOS which may developed as autoimmune disease³³.

In the current study, the auto-antibodies (AHA, AOA) have a direct correlation with each other at high significant levels. There were no previous studies regarding this correlation in PCOS, this finding was helped in trying to diagnose PCOS as an autoimmune disease through the presence of different types of auto-antibodies ³⁴. Other results of the current study showed a direct correlation at high significant level between IL-17 and IL-23, this agree with result of other study which explain the level of IL-17 and IL-23 in follicular fluid of PCOS patients and find a direct correlation between the two types of ILs ³⁵.

The hypothesis which have been assumed previously in the objective of this study to assess the possible relationship between autoimmunity and PCOS may have an importance value in preventing, diagnosis and therapeutic intervention in PCOS women. The findings of the current study, especially the presence of high levels of auto antibodies, suggested that PCOS is associated with immune reaction. This immunological reaction may leads to inflammatory reaction, and can stimulate abnormal production of cytokines, like IL-17 and 23, which elevated also in the current study, leading to overt biochemical changes in PCOS. Serum sirtuin-1 levels was elevated also due to over stimulation of immune system. These entire factors confirm the possibility that PCOS is an autoimmune disease.

ACKNOWLEDGMENTS:

The authors would like to thank Mustansiryiah University/ College of pharmacy (www.uomustansiriyah.edu.iq), Baghdad-Iraq, for its support in the present work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 20.06.2019 Modified on 19.08.2019

Research J. Pharm. and Tech 2020; 13(7): 3171-3178.

DOI: 10.5958/0974-360X.2020.00561.2