Obstetrics & Gynecology:
Serum and Follicular Fluid Cytokines in Polycystic Ovary Syndrome During Stimulated Cycles
Amato, Giovanni MD; Conte, Marisa MD; Mazziotti, Gherardo MD; Lalli, Eleonora MD; Vitolo, Gabriella MD; Tucker, Arthur T. PhD; Bellastella, Antonio MD; Carella, Carlo MD; Izzo, Alfredo MD
Institutes of Endocrinology and Obstetrics & Gynecology, 2nd University of Naples, Naples, Italy; and The Ernest Cooke Microvascular Unit, St. Bartholomew's Hospital, London, United Kingdom.
Address reprint requests to: Giovanni Amato, MD, Via Orsi 33, 80128 Napoli, Italy.; E-mail: firstname.lastname@example.org.
Received July 15, 2002. Received in revised form November 30, 2002. Accepted December 4, 2002.
OBJECTIVE: To investigate the serum and intrafollicular tumor necrosis factor–α and interleukin-6 concentrations in infertile women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF).
METHODS: Thirty-one patients with PCOS undergoing IVF were studied. Thirty-nine normally ovulating women matched for age and body mass index and undergoing IVF for male infertility were the control group. Serum tumor necrosis factor–α, interleukin-6, and estradiol levels were assayed before recombinant follicle-stimulating hormone stimulation under gonadotropin-releasing hormone analogue suppression and 34–36 hours after human chorionic gonadotropin (hCG) administration at the time of the oocyte retrieval. Cytokine and estradiol concentrations were also evaluated in the follicular fluids obtained at the time of oocyte retrieval.
RESULTS: The patients with PCOS had higher serum and follicular fluid tumor necrosis factor–α and interleukin-6 concentrations (P < .001) and lower follicular fluid estradiol levels (P < .05) than control women. In both groups, the serum tumor necrosis factor–α, interleukin-6, and estradiol values increased significantly after hCG stimulation. In both groups, the follicular fluid cytokine concentrations were higher than those found in the serum. In the PCOS women the follicular fluid tumor necrosis factor–α values were significantly and inversely correlated to the follicular fluid estradiol values (ρ = −0.79; P < .001); this correlation was not found in the control subjects.
CONCLUSION: In infertile women with PCOS, 1) serum and follicular fluid interleukin-6 and tumor necrosis factor–α values were higher than those found in control women, 2) the cytokine concentrations were higher in the follicular fluid than in the serum, and 3) the intrafollicular tumor necrosis factor–α concentrations were significantly and inversely correlated to the estradiol levels. These results suggest an involvement of the immune system in PCOS.
Polycystic ovary syndrome (PCOS) is the most frequently occurring ovarian disorder associated with impaired ovulation. 1 Immune mechanisms may play an important role in determining PCOS, as suggested by the high concentrations of white blood cells in polycystic ovaries, 2 the high prevalence of antiovarian antibodies, 3 and the altered lymphocyte subset in peripheral blood of women with the disease. 4
Cytokines, soluble polypeptides of immunological origin, are local regulators of ovarian function. 5 They are involved in regulation of gonadal steroid secretion and corpus luteum function as well as in the modulation of the ovulatory process. 6–8 Tumor necrosis factor–α and interleukin-6 have been shown to exert a pleiotropic effect on ovary function. 7,9–14 Both cytokines have been shown to be in the follicular fluid, 15–17 suggesting their production by the granulosa cells. 10,18 Indeed, the gonadotropins can modulate cytokine production, 10 causing marked fluctuations in relation to the different stages of folliculogenesis. 19 Recently, the role of these cytokines in PCOS has suggested that they are involved in ovary hyperstimulation 20 as well as in hyperandrogenism of PCOS. 21 However, the data reported are few, and the results are controversial. 17,20–23
In this controlled study, our purpose was to evaluate the serum and follicular fluid levels of tumor necrosis factor–α and interleukin-6 in infertile women with PCOS undergoing in vitro fertilization (IVF). The evaluation of women during a standardized ovarian stimulation protocol allowed us to obtain follicular fluids from follicles of the same developmental phase and serum specimens at specific time points. By this model, we investigated the cytokine behavior in a temporal relationship with the gonadotropin-mediated stimulation and different stages of folliculogenesis.
MATERIALS AND METHODS
Our study group consisted of 31 infertile patients with PCOS who underwent IVF treatment. Polycystic ovary syndrome was diagnosed based upon the elevation of one or more plasma androgen levels (free testosterone greater than 0.8 ng/mL, total testosterone greater than 70 ng/dL, dehydroepiandrosterone sulfate greater than 300 ng/dL), chronic oligo- (six or fewer menses per year) or amenorrhea, and the presence of ten or more follicles (2–10 mm in diameter) in one or both ovaries with an enlarged stroma at ultrasound evaluation. 24,25 21-hydroxylase deficiency, hyperprolactinemia, and androgen-secreting tumors were excluded in these patients by appropriate diagnostic tests. The indications of IVF in PCOS were failure of five to six cycles of increasing doses of clomiphene citrate and failure to conceive after ovulation induced by gonadotropins. Thirty-nine normally ovulating women diagnosed with male factor infertility of greater than 3 years and undergoing IVF, with age and body mass index (BMI) comparable to the study groups, were selected as the control group (Table 1). Regular menses and levels of progesterone above 10 ng/mL in the luteal phase of the preceding menstrual cycles demonstrated normal ovulation. At study entry, the exclusion criteria were 1) BMI above 30 kg/m2, to avoid the effects of obesity on serum tumor necrosis factor–α levels 26,27; 2) diabetes or any other systemic disease; 3) thyroid diseases; and 4) treatment with insulin-sensitizing agents in the last year.
The study was approved by the local ethical committee. All patients gave written informed consent before participation in the study, according to the Declaration of Helsinki of 1975, revised 1983.
As previously described, 28 oocyte retrieval was performed 34–36 hours after human chorionic gonadotropin (hCG) administration with the use of transvaginal, sonographically guided puncture. Samples of follicular fluids were obtained from 176 follicles (range 18–24 mm) at oocyte collection after recombinant follicle-stimulating hormone (rFSH) and hCG administration in all patients participating in IVF. For each patient, at least two follicles were aspirated to provide a more accurate evaluation of the intrafollicular cytokine concentrations. Each follicle was aspirated separately, and fluids were collected in a 15-mL conical tube. Subsequently, for each individual, a pooled follicular fluid sample was obtained by combining equal aliquots from two or more fluid collection devices. Part of each sample was centrifuged (2000g) to separate serum.
Tumor necrosis factor–α, interleukin-6, and estradiol (E2) levels were assayed in the follicular fluids obtained at oocyte collection; they were also evaluated in sera obtained from all IVF patients before rFSH stimulation under gonadotropin-releasing hormone analogue suppression (suppression phase) and 34–36 hours after hCG administration at the time of the oocyte retrieval (pickup phase).
Follicular fluid and sera were processed rapidly, and all samples were stored at −20C. The assays were performed at the end of the enrollment of all 70 women.
Interleukin-6 and tumor necrosis factor–α were measured in serum and follicular fluid by a solid-phase enzyme-linked immunosorbent assay (Medgenix Diagnostics, Fleurus, Belgium). Intrassay coefficients of variation in our laboratory were 4.2% for interleukin-6 and 3.3% for tumor necrosis factor–α, respectively. Estradiol was measured in serum and follicular fluid by a radioimmunoassay commercial kit (Sorin, Saluggia, Italy). Intra-and interassay coefficients obtained were between 5.1% and 11.3% for E2.
The Kolmogorov–Smirnov test was applied to both groups. The results were significant, suggesting an asymmetric distribution of the data for each variable. Median and range were used to present the data, unless otherwise indicated. Paired and unpaired data were compared using Wilcoxon signed-rank and Mann–Whitney tests. The Spearman rank correlation was used to demonstrate correlation between variables. Hypothesis tests were two sided, with probability values of less than 5% taken to represent statistical significance.
Table 2 shows the results of tests performed on sera from the two groups of women. In the suppression phase, PCOS patients showed serum tumor necrosis factor–α and interleukin-6 values significantly higher than those found in the control group; however, E2 values did not statistically differ between the two groups (P = .053). No correlation was found between serum tumor necrosis factor–α and E2 (PCOS group: ρ = −0.26, P = .15; control group: ρ = 0.05, P = .74) or between serum interleukin-6 and E2 (PCOS group: ρ = −0.25, P = .17; control group: ρ = 0.17, P = .29). After gonadotropin stimulation (pickup phase), the serum values of all three parameters increased significantly both in women with PCOS and in control subjects. At the pickup phase, the patients with PCOS showed serum tumor necrosis factor–α and interleukin-6 values significantly higher than those of the control group, whereas the difference in E2 values was not significant (Table 2). The median percentage of variation of tumor necrosis factor–α and interleukin-6 in the two groups did not significantly differ (data not shown).
Table 3 shows the result of tests performed on follicular fluids from the two groups of women. Polycystic ovary syndrome women showed higher follicular fluid tumor necrosis factor–α and interleukin-6 levels and lower follicular fluid E2 levels than those found in the control group. In both groups, the follicular fluid interleukin-6 and tumor necrosis factor–α levels were higher than those found in the serum (P < .001 for both comparisons). In patients with PCOS, follicular fluid tumor necrosis factor–α levels were significantly correlated to follicular fluid interleukin-6 (ρ = 0.36, P < .05) and follicular fluid E2 levels (ρ = −0.79, P = .001) (Figure 1). No significant correlation was found between follicular fluid interleukin-6 and follicular fluid E2 values in patients with PCOS (ρ = −0.23, P = .21). In the control group, no significant correlation was found between follicular fluid interleukin-6 and tumor necrosis factor–α (ρ = 0.08, P = .61), follicular fluid interleukin-6 and E2 (ρ = −0.06, P = .69), and follicular fluid tumor necrosis factor–α and E2 values (ρ = 0.21, P = .18).
In this study, we observed that infertile women with PCOS undergoing IVF showed higher serum and follicular fluid tumor necrosis factor–α and interleukin-6 levels than the control subjects. Moreover, we demonstrated that follicular fluid concentrations of tumor necrosis factor–α were significantly and inversely correlated to follicular fluid E2 levels.
The role of the cytokines in PCOS has been investigated, with controversial results in relation to the different analysis procedures and the different study populations. 17,20–23 We choose to evaluate the tumor necrosis factor–α and interleukin-6 values in infertile PCOS women undergoing IVF. This study model was suggested by the need to recognize the different phases of the folliculogenesis that have been shown to markedly influence cytokine production. Previous studies reported high serum tumor necrosis factor–α concentrations in nonstimulated PCOS women. 21,22 In the present study we confirmed this finding, and we found a strict correlation between tumor necrosis factor–α and interleukin-6 values. These findings disagree with previous observations that do not demonstrate any significant increase of the latter cytokine in PCOS patients in the course of spontaneous cycles. 17,20 Furthermore, we demonstrated that such difference was evident both in the early phase of the folliculogenesis and at the time of ovulation. The gonadotropin stimulation, however, induced a significant and comparable increase of interleukin-6 and tumor necrosis factor–α either in the patients with PCOS or in the normal group. This finding is suggestive for a physiologic modulation of cytokine secretion by these pituitary hormones, although in our study protocol the gonadotropin stimulation was performed with pharmacological doses. Indeed, previous studies demonstrated that FSH was able to induce interleukin-6 expression from granulosa cells, 10 and a possible regulation of interleukin-6 expression by luteinizing hormone (LH) was also suggested. 29 The rise of interleukin-6 levels could be in relation to the angiogenesis occurring during the ovarian follicular development. 12
In our PCOS patients, the intrafollicular interleukin-6 and tumor necrosis factor–α concentrations were higher than those found in the serum, suggesting a local production for these cytokines. Indeed, even if both cytokines are mainly produced by white blood cells, they can be also elaborated by granulosa cells. 30 Besides the source of production, however, the high intrafollicular levels of tumor necrosis factor–α and interleukin-6, as well as the inverse correlation between the tumor necrosis factor–α and E2 values, could be suggestive of an involvement of these cytokines in the pathophysiology of PCOS. This conclusion follows the hypothesis of the existence of a relationship between the endocrine and immune systems in such disease, 4 as demonstrated in other pathologic diseases of the reproductive system. 31,32 Because the present study was performed in a homogeneous group of PCOS patients undergoing stimulation, we cannot demonstrate either the pathophysiologic mechanisms or the eventual involvement of the above cytokines in all pathologic aspects of the syndrome. Actually, both interleukin-6 and tumor necrosis factor–α seem to exert many effects at the reproductive axis. In particular, interleukin-6 has a stimulatory effect on LH secretion, 29,33 which pulse secretion is impaired in the patients with PCOS. 34 Also, tumor necrosis factor–α has been shown to influence folliculogenesis, follicular maturation, 7,9,10 and gonadotropin-induced steroidogenesis by stimulating the proliferation of theca-luteinized and interstitial cells, 35 androgen synthesis, 36 and inhibition of differentiation of cultured granulosa cells. 37 Moreover, high tumor necrosis factor–α concentrations in follicular fluid were linked to a poor quality of oocytes, 31,32,38 and some evidence suggests that tumor necrosis factor–α could mediate insulin resistance, 39 which is a common finding in patients with PCOS. In addition to these effects, the immunological properties of tumor necrosis factor–α should be considered in this physiopathologic context. Tumor necrosis factor–α is a proinflammatory cytokine associated with the Th1 immune response. 40 As a Th1-inducing factor, tumor necrosis factor–α seems to play a critical role in modulating the apoptotic process in experimental models of autoimmune diseases. 41 Abnormality in apoptotic process has been described in PCOS, 42 and the modulatory role of type 1 cytokines has been investigated. 43 The results of our study suggest an involvement of the immune system in the pathogenesis of disease. 3 The future characterization of these physiopathologic aspects might favor new diagnostic and therapeutic perspectives in the management of patients with PCOS.
Our study demonstrated that in infertile women with PCOS in the course of IVF, 1) serum interleukin-6 and tumor necrosis factor–α levels were higher than those found in control women, 2) the cytokine concentrations were higher in the follicular fluid than in the serum, and 3) the intrafollicular tumor necrosis factor–α concentrations were significantly and inversely correlated to the E2 levels. Further studies are needed to clarify both the specific role of these cytokines in the disease and the eventual application of cytokine measurement, as well as the anticytokine treatment, in the management of patients with PCOS.
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