The possible role of the adipokine chemerin in the pathogenesis of polycystic ovarian syndrome (PCOS) in patients with obesity

I. Background: Polycystic ovarian syndrome (PCOS) can affect up to 10% of women of reproductive age and accounts for 75% of anovulatory infertility. PCOS is a heterogeneous syndrome. PCOS is more than just a disorder of reproduction and may be associated with significant metabolic alterations. These alterations can have important systemic sequelae that could contribute to long-term morbidity associated with diabetes and obesity. Obesity is a condition in which excess body fat has accumulated so that the Body Mass Index (BMI) is > 30. Obesity has been shown to negatively affect human health, including reproduction. Obesity is found in 50-70% of women with PCOS and is associated with insulin resistance. Women who are obese may have more severe hyperandrogenism and therapy-resistant anovulation than normal weight women with PCOS.. The pathogenesis of PCOS and the interrelationship between obesity and PCOS is complex and its etiology is not completely understood.

White adipose tissue (fat tissue) secretes a number of signalling peptides, collectively termed adipokines. Adipokines have important autocrine/paracrine roles in regulating adipocyte differentiation and metabolism, but also have endocrine/systemic actions in regulating lipid and glucose metabolism. The serum level of many adipokines is profoundly affected by the degree of adiposity. A recently identified adipokine chemerin is a secreted 18-kDa inactive pro-protein and converted to a 16-kDa active form by extracellular serine protease cleavage, which is present in plasma and serum (Goralski et al., 2007). The estimated concentration of active chemerin in human plasma and serum is about 3.0 nM and 4.4 nM, respectively (Zabel et al., 2006). In addition, plasma chemerin levels were significantly higher in obese subjects than those with normal weight (296.5 ± 61.2 vs. 222.7 ± 67.1 ng/ml) (Bozaoglu et al., 2007).

Previously studies have shown that visceral adipocytes contribute to the pathogenesis of metabolic disorders (e.g. impaired glucose intolerance, insulin resistance) by interrupting insulin intracellular signalling in these cells. Visceral adipocytes can also convert androstenedione to testosterone, its serum level is markedly higher (1.7 fold) in PCOS patients compared with its normal counterpart. Chemerin is an adipokine associated obesity and metabolic syndrome. It was recently reported that circulating chemerin levels increased in PCOS patients versus control (Tan et al., 2009). However, if and how chemerin contributes PCOS and the interrelationship between PCOS and obesity is unknown.

II. Objectives: The overall goal of this project is to investigate if and how adipokines (e.g. chemerin) regulate ovarian follicular development and to better understand the role of adipokines in the pathogenesis of PCOS and the interrelationship between PCOS and obesity.

III. Hypothesis: We hypothesized that the aberrant expression of chemerin in obese subjects contributes to the pathogenesis of PCOS. Elevated serum level of chemerin is associated with PCOS and metabolic disorders such as obesity.

IV. Experimental Approaches: My research program involves both biomedical and clinical research, which are co-supervised by a basic scientist (Dr. Benjamin Tsang) and clinical investigator (Dr. Arthur Leader), as described hereafter.

1). Biomedical Research
A). Objectives: The objectives for the basic study were first to establish an animal model that exhibits PCOS and obesity phenotypes, and to use this model to examine if chemerin plays a role in the pathogenesis of PCOS associated with obesity.
B). Methodology:
a) The establishment of rat PCOS/obesity model: Female rats age at 21 days were implanted subcutaneously a silicone capsule (3.4cm) containing dehydrotestosterone (DHT) (daily release of 83 µg) for 12 weeks to mimic the hyperandrogenic state in women with PCOS (as modified from Manneras et al., 2007). Rats receiving empty implant served as controls.
b) Characterization of the rat PCOS/obesity model: Animals were weighed weekly. As shown in the attached diagram. Insulin sensitivity test (IST) was performed at 10 wks of DHT delivery, by measuring plasma glucose levels at different time points (0, 5, 10, 20, 40, 80, 160 min.) after insulin challenge (0.2U/100g BW). The insulin sensitivity index KITT was calculated for each individuals. The oestrous cycle phases of rats were determined by microscopic analysis of vaginal smear obtained daily from 11-12 wks post-implantation of DHT capsule. Rats were euthanized at 15 wks of age (12 wks post-implantation). Serum samples were collected for analysis of chemerin levels (WB), sex steroid hormones (testosterone, androstenedione, 17β-estradiol, and progesterone (ELISA)). Omental and subcutaneous fat tissue extract, ovarian extract as well were obtained to determine chemerin production in these tissues. Ovaries were weighed before embedded or homogenized. Ovarian morphology (number of follicles, follicular cysts, follicular atresia, CL) was examined by microscope with ovarian sections stained with H&E.

c) Granulosa cells (GCs) culture: To examine if and how gonadotropin and/or chemerin are involved in dysregulation of ovarian steroidogenesis in PCOS animal, GCs isolated from PCOS and control rats were cultured for 48h with and without FSH (100 ng/ml) or rhChemerin (100ng/ml) in the absence or presence of testosterone (0.5 µM; substrate for estradiol (E2) production). Changes in E2 and progesterone (P4) levels in spent media were assessed by ELISA. Cell lysates were subjected to Western blot analysis to determine possible alterations in the contents of various steroidogenic enzymes (StAR, p450scc, 3-beta-HSD, 17beta-HSD, aromatase).

2) Clinical Research
A) Objectives: To compliment our basic science studies and further test our hypothesis that the aberrant expression of chemerin in obese subjects contributes to the pathogenesis of PCOS.
B) Methodology: We have first proposed a pilot clinical study that involves four groups of patients (ten subjects per group): a) PCOS with obesity; b) PCOS without obesity; c) Control subjects with obesity; d) Control subjects without obesity. The pilot study has been approved by the Ottawa Health Research Ethics Board (OHREB) at the Ottawa Hospital Research Institute (OHRI) and the Ottawa Fertility Centre (OFC), and patients undergoing assessment for both infertility and PCOS at the Ottawa Fertility Center are being recruited. After informed consent, an additional blood sample will be collected for chimerin analysis. This pilot study will address the following questions:

a) Are serum chemerin levels higher in PCOS with obesity group than in the other three groups?
b) Are circulating chemerin levels associated with dysregulation of steroidogensis in PCOS subjects?
c) Are circulating chemerin levels associated with metabolic disorders (such as insulin resistance, hyperinsulemia, dyslipidemia) in PCOS subjects?

Demographic information of patients was collected from the clinical record (age, BMI, waist-hip ratio (WHR) etc.). Serum chemerin levels will be assessed (WB). Metabolic factors that are associated with obesity and insulin resistance will be assessed by measuring fasting serum cholesterol, triglyceride (TG), insulin and glucose. Regular clinical protocols and assessments will be used for clinical management, including FSH, LH, testosterone, DHEAS, TSH levels will be determined by ELISA as described in basic study.

V. Results:
A rat PCOS model that show both ovarian feature (follicles arrest at small antral stage) and metabolic feature (obesity, insulin resistance etc.) has been successfully established.
Our data have shown that PCOS rat exhibits:
1) Irregularity in oestrous cycle. Oestrous cycle is disrupted in PCOS rats in comparison to control rats (94% vs. 22%, N = 17).
2) Ovary weight reduced in size. Ovary weight in PCOS rats is lower than their control counterpart (54.96 ± 6.85 vs. 99.74 ± 2.88 g, N = 15, P < 0.001).
3) Body overweight. Weight gain is significantly higher than control animal over the experimental period (340.32 ± 20.66 vs. 256.49 ± 21.31g, N = 18, P < 0.001) beginning at 5 weeks after implant.
4) Insulin resistance. Insulin sensitivity in PCOS rats is reduced compared to control (KITT = 1.91 ± 0.15 vs. 2.34 ± 0.22, N = 17, P = 0.063).
5) Aberrant chemerin expression. Serum chemerin levels, chemerin content in omental and subcutaneous fat tissues are significantly increased in PCOS rats compared to controls (3.67 ± 0.46 vs. 2.21 ± 0.34, P < 0.05; 1.84 ± 0.19 vs. 1.27 ± 0.08, P < 0.01; 1.57 ± 0.29 vs. 0.96 ± 0.12, P < 0.05, respectively).
6) Dysregulation of steroid hormone production in cultured GC from PCOS rats. The stimulatory effect of FSH on E2 and P4 secretion by cultured granulosa cells was lower in PCOS rats than control rats. Whereas chemerin had no effects on E2 and P4 secretion in cultured GC from control rats in vitro, it stimulated E2 but inhibited P4 secretion by GC from PCOS rats

The clinical study is on-going and results are expected to be forthcoming soon.

VI. Conclusion:
We have developed a rat model for PCOS which recapitulates many of the reproductive and metabolic phenotypes of human PCOS. These findings suggest that the DHT-treated rat may be useful model for investigating of molecular and cellular basis of PCOS associated with reproductive and metabolic disorders. Elevated serum chemerin levels in these PCOS/obese rats reflect the changes in fat tissues, indicating chemerin may be associated with PCOS.

Clinical implications: The pathophysiology of PCOS is complex and is multi-factorial. The possibility of aberrant expression of chemerin contributing to the pathogenesis of PCOS associated with obesity offers a novel and exciting avenue for the investigation on the metabolic component of this syndrome. The identification of serum chemerin as an early diagnostic biomarker and therapeutic target for PCOS with obesity is worth exploring.
Challenges and Unanswered Questions: However, some challenges remain to be addressed. For instance, although our findings demonstrated an association of serum chemerin levels in obese rats with PCOS phenotypes, whether elevated chemerin is a direct cause of PCOS is still unknown. In addition, while chemerin has a modulatory role on steroidogenesis in granulosa cells, if the theca cells and the oocytes are indeed cellular target for this adipokine, particularly in the context of androgen production and oocyte maturation, remains to be determined. Mechanistic studies are also urgently required. The following are additional challenges and unanswered questions:

1. Though the heterogeneity of PCOS, the experimental rodent PCOS model is obviously induced by DHT. However, our hypothesis is that aberrant (elevated) chemerin produced by adipocytes contribute to PCOS. So, a controversial concept exists between obesity and PCOS. Which is the chicken, which is the egg?
2. The discrepancy between the experimental rodent PCOS model and human PCOS has been shown in the present study (increases in ovary size in human PCOS, decrease in ovary size in the DHT-induced rat PCOS). What contributes to the differences in the changes in ovarian weigh between human and rat? Does DHT have an inhibitory effect on follicular fluid accumulation (in rat) that is not shared by androstenedione (in human)?
3. Hyperandrogenism is a hallmark in PCOS, both from the point of view of symptoms and etiology. There are different forms of androgen (testosterone, androstenedione, dehydroepiandrosterone sulfate (DHEA-S)) in different mammals. Which one is better indicator in PCOS than others? Are they different between human and rat?
4. Metabolic phenotypes of PCOS such as obesity, insulin resistance account for more than 50% human PCOS populations. If and how adipokines (e.g. chemerin in this case) regulate insulin signalling within the ovary and insulin sensitivity in other organs that express insulin receptor?