Hum. Reprod. Advance Access originally published online on September 22, 2006
Human Reproduction 2007 22(2):527-535; doi:10.1093/humrep/del371
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Ovarian leukocyte distribution and cytokine/chemokine mRNA expression in follicular fluid cells in women with polycystic ovary syndrome
1 Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of China 2 Department of Obstetrics and Gynaecology, Hirosaki University School of Medicine, Hirosaki, Aomori, Japan and 3 Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia, Australia
4 To whom correspondence should be addressed at: Women’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, People’s Republic of China. E-mail: wurj{at}zju.edu.cn
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Abstract |
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BACKGROUND: Polycystic ovary syndrome (PCOS) affects 5–10% of reproductive-aged women and is commonly associated with anovulatory infertility. Leukocytes, together with granulosa cells, may contribute to the pathogenesis of PCOS via their ability to secrete an array of cytokines implicated in follicle growth. The aim of this study was to examine leukocyte subtypes in follicular phase ovaries and to quantify cytokine and chemokine mRNA expression in follicular fluid cells obtained at the time of oocyte retrieval before IVF in women with and without PCOS. METHODS: Ovaries were immunostained for various leukocyte markers [CD3, CD4, CD14, CD15, CD45, CD45RA, CD45RO, CD57 and major histocompatibility complex (MHC) class II]. In addition, follicular fluid cells were subjected to quantitative RT–PCR to evaluate colony-stimulating factor-1 (CSF-1), granulocyte-macrophage (GM)-CSF, interleukins (IL-1
, IL-6, IL-8 and IL-10), monocyte chemotactic protein (MCP-1) and tumour necrosis factor (TNF
) mRNA expression relative to -actin. RESULTS: CD45RO+ cells (activated/memory T lymphocytes) were reduced by 60% in the theca layer of follicles from PCOS women. The relative abundance of macrophages and neutrophils was unchanged. Cytokine and chemokine mRNA transcripts examined were not affected by PCOS status. There was an association between high BMI and high TNF and low IL-6 mRNA expression in follicular cells. IL-6 expression was higher in women who subsequently achieved pregnancy. CONCLUSIONS: T lymphocytes potentially play a role in the local pathological mechanisms of PCOS. Further studies are required to identify their contribution to the aetiology of this common condition.
Key words: chemokines/cytokines/follicular fluid/leukocyte/PCOS
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Introduction |
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Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in reproductive medicine (Knochenhauer et al., 1998
; Diamanti-Kandarakis et al., 1999) and is characterized by the elevation of plasma androgen levels, chronic oligomenorrhoea or amenorrhoea and the presence of a characteristic ovarian morphology (Zawadzki and Dunaif, 1992). This syndrome is also associated in some women with hyperinsulinism, hyperlipidaemia, diabetes mellitus, obesity, hirsutism, acne and an increased incidence of endometrial cancer (Norman and McVeigh, 1999; Norman, 2001).
Granulosa cells make up the primary component of intrafollicular cells, and dysfunction of these cells may contribute to the abnormal ovarian pathology observed in PCOS. Granulosa cells derived from follicles of women with anovulatory PCOS produce more estradiol (E2) in response to FSH than normal granulosa cells (Mason et al., 1994) and respond prematurely to LH. Coupled with the higher E2 levels, this may cause follicle growth arrest (Willis et al., 1998; Franks et al., 2000). Recently, it has been suggested that early follicle development is abnormal in polycystic ovaries, with an increased number of pre-antral follicles also present in these ovaries (Webber et al., 2003).
In the past decade, increasing recognition has been given to the contribution of the immune system to the regulation of ovarian function. Infiltrating leukocytes, as well as resident leukocytes, are thought to play important roles in the cyclic tissue remodelling events of the ovary due to their ability to secrete numerous inflammatory and immunomodulating molecules (Brannstrom and Norman, 1993; Norman and Brannstrom, 1996; Wu et al., 2004). Granulosa cells and leukocytes can secrete numerous cytokines and chemokines implicated in mediating intraovarian events, such as colony-stimulating factor (CSF)-1, granulocyte-macrophage (GM)-CSF, interleukin (IL)-1, -6, -8, monocyte chemotactic protein-1 (MCP-1) and tumour necrosis factor alpha (TNF) (Brannstrom and Norman, 1993; Jasper et al., 1996; Bukulmez and Arici, 2000; Carlberg et al., 2000; Kawano et al., 2001; Fujii et al., 2003). Both cell types and their secreted products mediate diverse functions in ovarian processes such as folliculogenesis, ovulation and corpus luteum formation and regression (Brannstrom and Norman, 1993; Vinatier et al., 1995; Terranova and Rice, 1997; Bukulmez and Arici, 2000). IL-1 and TNF have been detected in human pre-ovulatory follicular fluid (Wang et al., 1992; Wang and Norman, 1992), with mRNA concentrations of IL-1 increasing as follicle rupture approaches, suggesting that this molecule may be an important regulator of ovulation (Adashi, 1998).
Circulating cytokine levels are altered in women with PCOS, with increased serum TNF levels in these women (Gonzalez et al., 1999), and Turi et al. (1988) reported that some subsets of peripheral leukocytes are decreased in PCOS patients compared with normal controls. Recent studies have shown that follicular fluid from PCOS patients undergoing IVF has elevated IL-6, IL-13 and TNF and activated T-lymphocyte numbers and reduced IL-12 concentrations compared with non-PCOS patients (Gallinelli et al., 2003), with some cytokine levels in the follicular fluid being significantly higher than in serum, suggesting a local production or accumulation of these cytokines (Amato et al., 2003). In contrast to these studies, our group has previously shown in unstimulated cycles that follicular fluid TNF and IL-1 levels do not differ between PCOS and non-PCOS patients (Jasper and Norman, 1995). The difference between results may reflect the use of stimulatory drugs in IVF and different size of ovarian follicles investigated.
Macrophages and T lymphocytes are increased in the laser-drilled site of sheep ovaries after the acute inflammatory phase, demonstrating that the effectiveness of this surgery, when used on patients with PCOS, may be attributable to increased leukocyte numbers and their secreted products (Tozawa et al., 1995). The important role macrophages play in reproductive function has been demonstrated in CSF-1-deficient mice (Cohen et al., 1997) and in the rat following depletion of ovarian macrophages (Van der Hoek et al., 2000), each model displaying decreased ovulation rates and disturbed oestrous cycles.
Several studies suggest that immune regulation may be involved in the aetiology of PCOS. However, the number of investigations into the contribution of leukocytes to reproductive disorders such as PCOS is relatively small because of the limited availability of human tissue and the lack of animal models for these disorders. In the light of these previous studies examining the involvement of leukocytes, granulosa cells and their cytokine production in the aetiology of PCOS, we designed a study to investigate (i) the immunohistochemical distribution of various leukocytes in follicular phase ovaries and (ii) the mRNA expression of various cytokines and chemokines in follicular fluid cells collected at the time of oocyte retrieval for IVF from women with or without PCOS.
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Materials and methods |
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The study protocols and consent forms were approved by the Research Ethics Committees of The Queen Elizabeth Hospital, The University of Adelaide and The Royal Adelaide Hospital or The Women’s and Children’s Hospital. Written, informed consent was obtained from each patient before study participation. PCOS was diagnosed according to established National Institutes of Health (NIH) consensus criteria derived from the 1990 NIH/National Institute of Child Health and Human Development (NICHHD) conference (Zawadzki and Dunaif, 1992
). Briefly, the criteria include (i) menstrual dysfunction; (ii) clinical hyperandrogen (hirsutism, acne and androgenic alopecia); and (iii) exclusion of other related disorders, such as hyperprolactinaemia, non-classic adrenal hyperplasia or thyroid disease.
Immunohistochemical localization of leukocyte subtypes
Ovarian tissue
Whole ovaries were obtained from women of reproductive age (21–45 years old, median age 35 years) during the follicular phase of the menstrual cycle. All women had undergone total hysterectomy and bilateral salpingo-oophorectomy for either endometrial cancer following PCOS (PCOS, n = 5) or menstrual disorders in the presence of normal testosterone and in the absence of polycystic ovary histology (non-PCOS, n = 4). After surgical removal, ovaries were fixed in 10% buffered formalin and embedded in paraffin for routine histological studies following haematoxylin and eosin staining. The diagnosis of PCOS was confirmed histologically by the presence of multiple small antral follicles with a diameter of 2–8 mm, increased ovarian stroma and thickening of the tunica albuginea. No follicles >12 mm in size were observed in any ovaries. None of the women had been prescribed hormones or medications known to influence reproductive function, and none had any evidence of infection or inflammation.
Immunohistochemistry
A panel of mouse anti-human monoclonal antibodies (kindly provided as hybridoma supernatants by P. McCardle, Flinders Medical Centre, Adelaide, Australia) specific for various cell lineages were used for immunohistochemical localization of leukocytes (Table I). The specific reactivity of the monoclonal antibodies used in this study was validated using fluorescence-activated cell-scanning analysis and immunohistochemical staining of fresh frozen human peripheral blood mononuclear cell smears. The working concentration of each antibody was determined by immunohistochemical staining of formalin-fixed, paraffin-embedded sections of human spleen and tonsil using the protocol described below.
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Paraffin sections (5 µm) were mounted on poly-L-lysine-coated slides and deparaffinized in xylene. After hydration in descending concentrations of ethanol, the sections were immersed in 3% hydrogen peroxide in methanol for 20 min and washed twice in phosphate-buffered saline (PBS; GibcoBRL, Grand Island, NY, USA) for 3 min. Subsequently, the procedure for either heat-induced antigen retrieval or enzymatic digestion was performed as summarized in Table I. After pretreatment, the sections were blocked with 5% normal human serum (NHS) for 20 min at room temperature and then incubated in a humidified chamber overnight at 4°C, with primary antiserum diluted, as summarized in Table I, in PBS containing 1% bovine serum albumin (BSA, Fraction V, Sigma, St. Louis, MO, USA). After washing three times in PBS, biotinylated goat anti-mouse immunoglobulins (Vector Laboratories, Burlingame, CA, USA) were applied to the sections for 1 h at 4°C. After washing in PBS, slides were incubated with horse-radish peroxidase-conjugated streptavidin (LSAB2 system; Dako, Carpinteria, CA, USA) for 45 min at 4°C, according to the manufacturer’s instructions. Reaction products were developed during an 8 to 10 min incubation with 3,3'-diaminobenzidine (DAB) (Sigma). Slides were then counterstained with haematoxylin. Negative control sections were incubated without primary antibody and were uniformly negative.
Quantitative evaluation
The distribution of positive cells in the immunostained sections was evaluated in three separate tissue compartments: the theca layer surrounding all primary to antral follicles, the ovarian hilus and the stromal compartment. The area of positive stain in each specific region was evaluated by video image analysis (VIA) using Video Pro 32 software (Leading Edge, Adelaide, Australia), as previously described (Jasper et al., 2000). Results were expressed as percentage positivity, calculated as percentage area of brown (DAB) positive stain, normalized to total stained area (the area of haematoxylin counterstain plus DAB stain). For each specific region, four to eight randomly chosen fields were analysed, and the mean of these values was calculated. All slides were analysed by a scorer blinded to the origin of the samples.
Quantitative analysis of cytokine and chemokine mRNA expression by follicular fluid cells
Patient recruitment and follicular cell collection
Follicular fluid aspirates were obtained at the time of oocyte retrieval from 14 patients diagnosed as having PCOS and 17 normally ovulating women (non-PCOS) matched for age, weight and BMI undergoing IVF for other causes of infertility, including tubal disease (n = 5), male factor (n = 9) and unexplained infertility (n = 3). Age, weight, BMI, plasma testosterone and IVF outcome (positive pregnancy defined as the presence of a fetal heartbeat by ultrasonography 
7.5 weeks following a positive serum hCG test 14 days after oocyte retrieval) for each group are summarized in Table II. Patients underwent a standard IVF protocol; with recombinant FSH-stimulated follicles (N.V. Organon, the Netherlands) (15–25 mm in diameter) aspirated 32–34 h after hCG (Profasi, Serono, Rockland, MA, USA) administration. After removal of the cumulus–oocyte complexes, follicular fluid aspirates were pooled for each patient and immediately placed on ice. Before oocyte retrieval, a general clinical examination was performed and plasma hormones evaluated, including baseline levels of testosterone, LH, sex hormone-binding globulin (SHBG), FSH and mid-luteal phase E2 and progesterone. No steroid measurements were performed on the follicular fluid.
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Samples were centrifuged at 200 x g for 10 min at 4°C, and cells were aspirated and resuspended in Hanks’ balanced salt solution (HBSS, GibcoBRL) containing 0.06% gentamycin (DBL, Mulgrave, Australia), 0.01% sodium azide (Sigma) and 2 mM EDTA (Sigma) (HBSS/gent/EDTA/Az). The cells were then centrifuged over 1:1 (v : v) PBS : Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) at 400 x g for 20 min at 4°C to remove contaminating red blood cells. The layer of cells at the Percoll/PBS interface was removed and resuspended in HBSS/gent/EDTA/Az and centrifuged at 200 x g for 10 min. Finally, the cell pellet was resuspended in HBSS/gent/EDTA/Az, filtered through a 70-µm-diameter cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA) and washed with HBSS/gent/EDTA/Az containing 0.5% BSA. Total cells were counted and viability assessed by Trypan Blue dye exclusion (Sigma). The number of cells recovered from the follicular fluid of patients ranged from 5 x 106 to 30 x 106 cells, and cell viability as determined by Trypan Blue dye exclusion ranged from 50 to 85%.
RNA extraction and quantitative real-time RT–PCR
Total RNA was extracted from isolated follicular fluid cells by Tri-Reagent (Sigma) according to the manufacturer’s instructions. Contaminating residual DNA was eliminated by treatment with DNase I (Promega, Madison, WI, USA). Resulting RNA was dissolved in 20 µl ribonuclease-free water and concentration determined using a Ribogreen® RNA quantification kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. For each sample, 100 ng of RNA was diluted in PCR-grade water and reverse transcribed using random primers (Roche Diagnostics GmbH, Mannheim, Germany) and a Superscript II Reverse Transcription kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Negative controls omitting RNA or Superscript II enzyme were included.
Specific primers for the cytokines CSF-1, GM-CSF, IL-1, IL-6, IL-10, TNF and the chemokines IL-8 and MCP-1 were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA) and manufactured by Geneworks (Adelaide, Australia) (Table III). Primers for IL-1, IL-6, IL-8, IL-10 and MCP-1 all crossed intron/exon boundaries.
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Samples were assayed in triplicate on an ABI Geneamp 5700 sequence detection system (Applied Biosystems) and subjected to 40 cycles of amplification under varying conditions (Table III) using 3 µl of diluted cDNA (1:7), 10 µl of 2x SYBR green PCR master mix (Applied Biosystems) and 5–20 pmol of each primer. Negative controls omitting template cDNA were included in each run. The dissociation analysis performed following quantitative real-time RT–PCR showed that a single product had been amplified from each primer pair, as expected. Primer amplification efficiencies were also examined as previously described (Bonello et al., 2004
). Briefly, serially diluted cDNAs were assayed, and the resultant Ct values were plotted against log dilutions. The slopes of each graph were determined, and all primers were reported to have similar efficiencies. -Actin expression showed no significant difference between experimental groups when the Ct value for each sample was normalized to total RNA (µg) reverse transcribed (data not shown) and was therefore considered to be a suitable housekeeping gene for these samples. For all genes of interest, mRNA content was calculated for each sample relative to the housekeeping gene, -actin. This was performed using the equation 2–
Ct (ABI, Sequence Detector User Bulletin 2, Applied Biosystems), where Ct is the difference between the target gene and the housekeeping gene, and Ct is the change between the Ct for each sample and the control group (e.g. non-PCOS patients). For each gene of interest, results were described as the mean fold change from the non-PCOS group, where mean non-PCOS group was equivalent to 1.
Data analysis
Mann–Whitney U-tests (SigmaStat for Windows version 2.03, Jandel Corp, San Ramon, CA, USA) were used to examine statistical differences in percentage positivity following the immunohistochemical evaluation of leukocyte distribution in PCOS and non-PCOS ovaries. Owing to the small sample sizes, analysis of interactions between different parameters was not attempted. Mann–Whitney U-tests were also used to examine statistical differences in cytokine and chemokine mRNA expression in PCOS versus non-PCOS, high testosterone versus low testosterone, pregnant versus non-pregnant and high BMI versus low BMI groups. Statistical significance in differences between groups was concluded when P < 0.05.
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Immunohistochemical localization of leukocyte subtypes in follicular phase ovaries
The results of the quantitative evaluation of positive immunohistochemical staining in various ovarian regions are summarized in Table IV. Positive staining was observed mainly in the theca and hilus, with relatively few leukocytes observed in the stromal compartment. The mean (± SEM) diameter of follicles studied was 3.1 ± 1.3 mm (range 1.0–7.0 mm). There was no positive cell staining in the vicinity of small pre-antral follicles observed within the stroma. Of the CD45-positive leukocytes, the most common leukocyte subpopulations were CD45RO-positive cells (activated/memory T lymphocytes) and CD14-positive cells (macrophages and monocytes), which were more common than CD15-positive neutrophils and CD3-positive T lymphocytes. There were fewer neutrophils than T lymphocytes. Very few natural killer (NK) cells were observed in either non-PCOS or polycystic ovaries.
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Of all the leukocyte subpopulations examined throughout the different ovarian compartments, only CD45RO-positive (activated/memory T lymphocytes) cells in the theca surrounding antral follicles were significantly reduced in PCOS compared with non-PCOS ovaries. Although not statistically significant, there were trends for reduced staining of other T lymphocyte markers CD3, CD4 and CD45RA in the theca of PCOS compared with non-PCOS ovaries, whereas macrophage and neutrophil numbers were comparable in all sites irrespective of PCOS status.
Cytokine and chemokine mRNA expression in follicular fluid cells
Age, weight, BMI, serum testosterone and resulting pregnancy outcome did not vary between PCOS and non-PCOS patients (Table II). The age of patients who became pregnant after their IVF-embryo transfer cycle was significantly lower than those who did not achieve pregnancy (P < 0.05, data not shown).
Messenger RNAs encoding CSF-1, IL-1, IL-6, IL-8, IL-10, MCP-1 and TNF were detectable by quantitative real-time RT–PCR in follicular fluid cells from all patients. GM-CSF mRNA was only detectable in four patients (2 of 14 PCOS and 2 of 17 non-PCOS; data not shown) and was therefore not included in further statistical analysis. One PCOS patient showed abnormally high mRNA expression of IL-6, IL-8, MCP-1 and TNF (as determined by >2 SD above the mean) and was therefore not included in the statistical analysis of these cytokines. Cytokine and chemokine mRNA profiles from follicular fluid cells of varying groups of patients including PCOS (n = 14) versus non-PCOS (n =17), high testosterone (
1.6 nmol—approximate median of all patients) (n = 14) versus low testosterone (n = 17), pregnant (n = 15) versus non-pregnant (n = 16) and high BMI (27 kg/m2—approximate median of all patients) (n = 12) versus low BMI (n = 19) were determined and revealed few differences between groups (Figure 1). No differences in cytokine or chemokine mRNA expression levels were observed between PCOS and non-PCOS patients. IL-6 mRNA was significantly higher in patients who became pregnant compared with the non-pregnant group. Although not statistically significant, there was a trend towards increased expression of IL-8 and IL-10 mRNA in follicular cells of pregnant versus non-pregnant women. TNF mRNA expression was significantly higher and IL-6 mRNA expression was significantly lower in the high BMI group compared with the low BMI group, with a trend towards increased expression of IL-10 mRNA in the high BMI group. No differences in cytokine or chemokine mRNA expression levels were observed between low versus high testosterone patients.
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Discussion |
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Leukocytes have been implicated in the paracrine regulation of ovarian processes due to their production of an array of biochemical mediators associated with ovarian events including the regulation of steroidogenesis, local vascular permeability, extracellularmatrix remodelling, angiogenesis and immunoregulation. Communication between leukocytes and resident ovarian cells (non-haemopoietic cells) through autocrine and paracrine cytokine networks is thought to be crucial for successful reproduction. PCOS follicle development is arrested, usually at <8 mm in diameter, and although this can be overcome by the administration of FSH, these follicles remain small without exogenous gonadotrophin stimulation. The roles of leukocytes, and locally synthesized cytokines and chemokines responsible for their recruitment into the ovary, in PCOS have been studied with equivocal results (Jasper and Norman, 1995
; Gonzalez et al., 1999; Amato et al., 2003; Gallinelli et al., 2003).
In this study, we examined leukocyte distribution in follicular phase ovaries and cytokine and chemokine mRNA expression in stimulated follicular cells in PCOS and non-PCOS patients. Our observations showed that in the unstimulated, natural cycling state, the distribution of most leukocytes was similar between PCOS and non-PCOS ovaries, apart from T lymphocytes and in particular the subset of CD45RO-positive cells. The density of these cells was significantly decreased in PCOS ovaries. CD45RO-positive cells are identified as expressing a specific isoform of CD45 and are generally recognized as a subset of activated or ‘memory’ T lymphocytes (Michie et al., 1992). Previous work describes reduced numbers of activated and T helper cells in follicular fluid of PCOS women and an inverse correlation between testosterone levels and activated T lymphocytes (Gallinelli et al., 2003), noting elevated testosterone to be characteristic of PCOS.
It is possible to speculate on functions in addition to steroidogenesis for T lymphocytes in ovarian events. Selection of a dominant follicle, paralleled with the demise of excess follicles, is an integral part of ovarian homeostasis. The determination of cell fate and requisite cell death for follicle reduction probably involves several pathways, potentially including either positive or negative selective roles for T cells. T cells might reasonably assist in follicle survival by the provision of trophic growth factors or by the suppression of adverse immune activity. Alternatively, T cells might facilitate follicle regression by delivering cytotoxic signals to induce cell death in oocytes or more probably their supporting granulosa cells (Kryczek et al., 2005). Such a function would necessitate adequate and appropriately distributed T-cell populations, which, if lacking in the PCOS ovary, might contribute to the failure of normal follicle selection and development processes.
The panel of cytokine and chemokine transcripts assessed in this study has been previously shown to be important in the ovary (Brannstrom and Norman, 1993; Terranova and Rice, 1997; Bukulmez and Arici, 2000). These cytokines can regulate follicular maturation and ovulation through paracrine and autocrine mechanisms. We have demonstrated that follicular fluid cells express mRNA for CSF-1, IL-1, IL-6, IL-8, IL-10, MCP-1 and TNF. It is likely that cytokine synthesis is contributed to by multiple cellular sources, as periovulatory follicular aspirates contain, in addition to granulosa cells, 5–15% tissue macrophages along with lymphocytes (Loukides et al., 1990; Machelon et al., 1994; Baranao et al., 1995).
We observed no differences in follicular fluid cell mRNA expression for the cytokines or chemokines examined from PCOS and non-PCOS ovaries. This is consistent with our earlier findings whereby immunoreactive IL-1 and TNF concentrations in the follicular fluid and culture media of unstimulated ovaries from PCOS were comparable with normal ovaries (Jasper and Norman, 1995). It has been previously reported that patients with PCOS had elevated serum and follicular fluid TNF and IL-6 concentrations than control women (Amato et al., 2003). The discrepancy in these results may be due to the different study populations and different analysis methods. In addition, translational regulation may be used as a mechanism to regulate protein production, which was not investigated in this study.
By contrast, we observed elevated TNF and reduced IL-6 mRNA expression in follicular fluid cells of patients with high BMI compared with patients with lower BMI. TNF has been shown to play roles in folliculogenesis, follicular maturation (Roby and Terranova, 1988; Adashi et al., 1990), androgen synthesis (Roby and Terranova, 1990), insulin resistance mediation (Paolisso et al., 1998) and inhibition differentiation of cultured granulosa cells (Darbon et al., 1989), and its abundance correlates with poor quality of oocytes (Carlberg et al., 2000; Lee et al., 2000). IL-6 has been shown to suppress TNF production and antagonizes its action (Gorospe et al., 1992; Tilg et al., 1994). A decrease in the TNF : IL-6 ratio in lower BMI women is also consistent with the known relationship between these cytokines and exercise (Petersen and Pedersen, 2005). During exercise, muscle fibre synthesis of IL-6 stimulates the appearance in the circulation of other anti-inflammatory cytokines and inhibits adipose tissue production of TNF. There is also evidence that TNF and IL-6 are involved in physiological sleep regulation, with links between TNF and degree of sleep disturbance and IL-6 with BMI (Vgontzas et al., 1997). These data raise the possibility that the mechanisms whereby TNF and IL-6 and BMI interact could have physiological links with the development of PCOS.
Interestingly, we also found that IL-6 mRNA levels in follicular cells were increased in patients who subsequently achieved pregnancy following IVF-embryo transfer. The elevated levels of IL-6 could contribute to regulating the oocyte microenvironment, as IL-6 is a potent angiogenic substance that facilitates vascularization and increased delivery of FSH to the growing follicle (Mori, 1990). IL-6 is also thought to play a role in the regulation of ovarian steroid production (Van der Hoek et al., 1998). This finding contrasts with other studies, showing no correlation between follicular fluid IL-6 and pregnancy rates (Hammadeh et al., 2002a,b).
A caveat in this study was that access to human ovarian tissue at defined stages of the menstrual cycle was extremely difficult to obtain, therefore necessitating a less than ideal study size. Cells and tissues from unstimulated menstrual cycles are equally rare, such that it was necessary to collect samples from different populations of women and extrapolate across data sets. It is unlikely that use of tissue from PCOS patients with endometrial cancer would be a source of confounding factors related to the neoplasm, although not impossible. Peripheral changes have been noted in women with endometrial cancer, including elevated IL-6 and activin A and reduced cytotoxic activity of circulating NK cells and reduced superoxide production in granulocytes (Garzetti et al., 1994; Petraglia et al., 1998; Bellone et al., 2005; Lampe et al., 2006). By and large, these altered immune and inflammatory parameters return to baseline levels after surgery. Furthermore, there is little likelihood that endometrial cancer results in changes specific to the ovary of greater extent or intensity than those reflected systemically, because the two organs are spatially discrete, and there are no reports describing ovarian pathologies of women who have endometrial cancer. Notwithstanding these limitations, this study provides insight into parameters that would usefully be targeted in larger future studies with more rigorously defined study populations.
In normal cycles, PCOS follicles tend to be <8–10 mm in diameter when their development is arrested. Therefore, a further consideration in the interpretation of the current data is that measurement of cytokine profiles might well be influenced by follicle exposure to FSH and hCG. It seems unlikely that these artificially supported follicles remain representative of the 10-mm follicles from which they were derived. It therefore remains possible that despite the lack of difference in cytokine expression in large follicles, an underlying problem in cytokine expression in the small follicle might be instrumental in developmental follicle arrest that ensues without gonadotrophin support. Furthermore, although NIH criteria were used to assess PCOS status in this study, there is the potential for subsets of patients of differing aetiologies to comprise the PCOS group. Discrepancies in this study and the scientific literature so far may relate to the criteria used to assess PCOS. This highlights the importance of consistent use of the NIH criteria across clinics and laboratories in order that studies might be sensibly compared.
In summary, we have found that T lymphocytes may be dysregulated in the processes leading to PCOS. This finding suggests that the immune system contributes to follicle maturation through the agency of T lymphocytes as well as through the previously well-characterized roles of cytokines and chemokines. The mechanisms underpinning how T lymphocytes contribute to local follicle selection and survival are unclear. It will be of interest to investigate the activities of T cells in follicle development, with a view to identifying possible suppressive and/or cytotoxic functions as well as synthesis of signature T-cell cytokines not investigated in this study.
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Acknowledgements |
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The authors are grateful to Dr Priya Duggal, Mr Clyde Milner and Mr Fred Amato for their technical assistance. R.Wu and S.Fujii were supported by overseas research scholarship from the Chinese Scholarship Council, State Education Ministry and the Japanese Ministry of Education, respectively. This study was funded by project and programme grants from the National Health and Medical Research Council of Australia.
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Submitted on July 25, 2005; resubmitted on February 13, 2006; resubmitted on April 26, 2006; resubmitted on June 2, 2006; accepted on June 22, 2006.
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