Original Study

Evaluation of the Pro-inflammatory Cytokine Tumor NecrosisFactor-a in Adolescents with Polycystic Ovary Syndrome

Melissa Pawelczak MD1, Jamie Rosenthal MD1, Sarah Milla MD2, Ying-Hua Liu MD, PhD 3,Bina Shah MD1,*1Division of Pediatric Endocrinology, Department of Pediatrics, New York University School of Medicine, New York, New York2Department of Radiology, New York University School of Medicine, New York, New York3Department of Pediatrics, New York University School of Medicine, New York, New York

a b s t r a c t

Background: Patients with polycystic ovary syndrome (PCOS) oft

en suffer from comorbidities associated with chronic inflammationcharacterized by elevations in pro-inflammatory cytokines. There is limited data on markers of chronic inflammation, in particular TumorNecrosis Factor-alpha (TNF-a), in adolescents with PCOS.Objectives: To compare serum levels of TNF-a in overweight or obese adolescents with PCOS and obese controls. In the PCOS group, tocorrelate serum TNF-a levels with body mass index (BMI) z-score, severity of hyperandrogenism, degree of insulin resistance, and ovarianultrasonographic characteristics.Methods: We performed a cross-sectional retrospective analysis of clinical and biochemical findings in 23 overweight or obese adolescentfemales with PCOS (mean BMI z-score 2, mean age 15.2 yrs) and 12 obese age- and sex-matched controls (mean BMI z-score 2, mean age14.1 y). All subjects were post-menarchal. Serum TNF-a levels were compared between groups. In the PCOS group, cytokine levels werecorrelated with BMI z-score, androgen levels, fasting insulin and glucose levels as well as ovarian ultrasonographic features.Results: Both groups were comparable in age, BMI z-score, fasting glucose, and fasting insulin. Mean free testosterone was 9.76 � 5.13 pg/mL in the PCOS group versus 5 � 2.02 pg/mL in the control group (P5 .0092). Serum TNF-awas 7.4 � 4 pg/mL in the PCOS group versus 4.8� 3.16 pg/mL in the control group (P 5 .0468). There was no significant correlation between serum TNF-a and BMI z-score, free testos-terone, fasting insulin, or fasting glucose. No correlation existed between serum TNF-a and ovarian follicle number, distribution, or volume.Conclusions: Serum TNF-a is elevated in overweight/obese adolescents with PCOS. Chronic inflammation in adolescents with PCOS renderthem at a potential increased risk for the development of atherosclerosis, type 2 diabetes, cancer, infertility, and other comorbidities. Everyeffort should be made to identify adolescents with PCOS early and initiate aggressive therapy to prevent future complications.Key Words: Polycystic ovary syndrome, Tumor necrosis factor-a (TNF-a), Adolescents

Introduction

Polycystic ovary syndrome (PCOS) is an endocrine disor-der that afflicts 5%-11% of reproductive aged women and isthought to be the most common endocrinopathy in thispopulation.1,2 Recent clinical guidelines suggest that PCOSshould be diagnosed in adolescents based on a thoroughclinical picture including oligomenorrhea, increasedandrogen levels, clinical signs and symptoms of androgenexcess, and exclusion of other causes of hyper-androgenemia.3 PCOS is known to be associated with a stateof low-grade systemic inflammation.4-9 Multiple studies inadults with PCOS have demonstrated elevated levels ofinflammatory indices such as IL-6, IL-18, tumor necrosisfactor-a (TNF-a), and C-reactive protein.10,11 Asmany of thesefactors are produced by adipocytes and PCOS is often asso-ciated with obesity, data onwhether the increased levels area result of obesity, PCOS, or both have been conflicting.4-10

A potential link between PCOS and elevations in theproinflammatory cytokine TNF-a is of particular interest, as

The authors indicate no conflicts of interest.Supported in part by grant 1UL1RR029893 from the National Center for Research

Resources, National Institutes of Health.* Address correspondence to: Bina Shah, MD, 530 1

stAve, Suite 3A, New York, NY

10016; Phone: (212) 562-3793; fax: (212) 562-3273E-mail address: bina.shah@nyumc.org (B. Shah).

1083-3188/$ - see front matter � 2014 North American Society for Pediatric and Adolehttp://dx.doi.org/10.1016/j.jpag.2014.01.104

TNF-a has been shown to impact many aspects of ovarianfunction, including follicular growth, ovulation, and corpusluteum regression.12-14 Ovaries have been shown to bothproduceTNF-aandcontainTNF-a receptors.13,15 In rats, excesstestosterone as seen in PCOS has been shown to induce agreater release of TNF-a, thus leading to an increase in an-drostenedione and a decrease in estradiol production in therat ovary.16 It is therefore possible that altered TNF-a levels inPCOS contributes both to the short-term ovarian dysfunctionand hyperandrogenic state seen in the disease as well as tolong-term effects in the ovaries and in other organ systems.

There is a lack of data examining serum TNF-a in ado-lescents with PCOS. In this study we compared serum levelsof TNF-a in overweight or obese adolescents with PCOSand obese controls. In the PCOS group, we also correlatedTNF-a levels with body mass index (BMI) z-score, severityof hyperandrogenism, degree of insulin resistance, andovarian ultrasonographic (US) characteristics.

Subjects and Methods

Patient Population

The Institutional Review Board of New York UniversitySchool of Medicine and Bellevue Hospital approved thiscross-sectional retrospective pilot study. Data was reviewed

scent Gynecology. Published by Elsevier Inc.

M. Pawelczak et al. / J Pediatr Adolesc Gynecol 27 (2014) 356e359 357

from 2005-2010. All patients were followed by the PediatricEndocrinology service at Bellevue Hospital Center or NewYork University Medical Center, 2 large urban academictertiary care centers. We studied 23 females (age 12.3-17.7y) with PCOS and 12 age, BMI, and sex matched controls(age 11.4-16.5 y). Table 1 shows all subjects baseline de-mographic and clinical characteristics obtained from a chartreview. Exclusion criteria included pregnancy, ovarian orandrogen secreting tumor, thyroid dysfunction, hyper-prolactinemia, Cushing syndrome, and congenital adrenalhyperplasia. No subject was taking insulin sensitizers or anymedication known to influence the menstrual cycle. Thediagnosis of PCOS was based on the revised 2003 ESHRE/ASRM Rotterdam criteria, which require 2 of the 3 followingcharacteristics: (1) oligo/anovulation, (2) clinical/biochem-ical signs of hyperandrogenism, and/or (3) polycysticovaries on US (the presence of 12 or more folliclesmeasuring 2-9mm in diameter and/or ovarian volumeO 10ml). Hirsutism was defined as a Ferriman-Gallwey grade of8 or higher. Amenorrheawas defined as cessation of periodsfor more than 3 months. Oligomenorrhea was defined asless than 6 cycles per year. Only postmenarcheal subjectswere selected. Based on data from the National Health andNutrition Examination Survey, overweight was defined asa BMI between the 85th-95th percentile and obese as a BMIO 95th percentile for age and sex.

Hormonal Immunoassays

Blood samples from the PCOS group and controls wereobtained during routine Pediatric Endocrinology clinicvisits. Blood sample collection was not timed with respecttomenstrual cycle. Total testosteronewasmeasured bywellvalidated liquid chromatography/mass spectrometry withan analytical sensitivity of 1 ng/dL (Quest Diagnostics, NewYork, NY). The percent free testosterone was determined byequilibrium dialysis. The free testosterone level was calcu-lated based on total and percent free. Biochemical hyper-androgenismwas defined as a free testosterone of 7.5 pg/mLor greater. Fasting serum insulin was determined byradioimmunoassay with a lower limit of detectability of 2.5mU/mL (Quest Diagnostics). HOMA-IR was calculated by

Table 1Demographic and Clinical Characteristics of PCOS Subjects and Obese Controls

PCOS Controls P

n 23 12Age (y) 15.2 � 1.84 14.08 � 1.7 .08Ethnicity .74Hispanic 18 (78%) 9 (75%)Asian 3 (13%) 1 (8.3%)Other* 2 (9%) 2 (16.7%)

BMI (kg/m2) Z Score 2.07 � 0.48 2.04 � 0.25 .84Age at Menarche (y) 11.63 � 1.31 11.35 � 1.45 .6Menstrual history 0.0001Regular 2 (9%) 11 (91.7%)Oligomenorrheic 14 (6.9%) 1 (8.3%)Amenorrheic 7 (30.4%) 0

Acne present 15 (65.2%) 8 (66.7%) .18Hirsute 17 (73.9%) 0 0.0001Acanthosis nigricans present 18 (78.3%) 10 (83.3%) .8

Data are expressed as n (%) and mean � SD.* Other 5 African American, Native American, or mixed ethnicity.

dividing the product of insulin (mU/mL) and glucose (mg/dL)by 405.17 The HOMA-IR cutoff point for diagnosis of insulinresistance was$3.16.18 Following collection and processing,serum samples for TNF-a were stored at �20�C until hor-mone analyses were performed. Serum TNF-a wasmeasured in duplicate using human multiplexing beadimmunoassays (Biosource, Camarillo, CA) utilizing Lumi-nexR in accordance with the manufacturer's instructions.All serum TNF-a measurements were performed in thesame lab using the same assay. The lower limit of detect-ability for this assay is 0.1 pg/mL.

Ultrasonographic Data

All PCOS subjects underwent transabdominal pelvic USas part of their PCOS diagnostic workup. The images wereobtained using a Philips HDI 5000 US machine (Andover,MA) with a curved 5 mHz transducer. No US datawas available for the control group. Ultrasonographic im-ages were re-reviewed with a pediatric radiologist toconfirm the reported measurements and to determineovarian morphology. Ovarian volume was calculated as0.5233 � length � width � thickness, derived from theformula for a prolate ellipsoid. An ovarian volume of 10 mlor more was considered enlarged. As transabdominal USlimits follicle visualization, the number of follicles wasdescribed as “few” when fewer than 5, “moderate” whenbetween 5 and 10, and “multiple” when greater than 10.Follicle distribution was described as central, peripheral, ormixed. The presence of cysts greater than 10mmwas noted.

Statistical Methods

A P value of !.05 was considered significant. Compari-sons of 2 groups on continuous variables were made usingthe Student t test; for comparisons of more than 2 groups,ANOVA tests were used. The c2 tests were performed toassess the correlations between 2 categorical variables. Thecorrelation between continuous variables was assessedusing Pearson's correlation coefficient. All analyses were2-tailed and performed using SAS. Results are reported asmean � SD.

Results

Clinical and Demographic Characteristics (Table 1)

There was no significant difference in mean age betweenthe PCOS group (15.2� 1.8 y) and controls (14.1�1.7 y) (P5.08). BMI Z-score was similar in the PCOS group (2.07 � .48)and controls (2.04� 0.25) (P5 .84). Themajority of subjectsin both groups were Hispanic (78% of the PCOS group, n 5

18, 75% of the control group, n 5 9, P 5 .74). Of theremaining PCOS subjects, 3 were Asian and 2 were of mixedethnicity. Of the remaining controls, 1 was Asian, 1 wasAfrican American, and 1 was Native American. The averageage at menarche was similar between the PCOS group (11.6� 1.3 y) and controls (11.4 � 1.5 y) (P 5 .6). Most PCOSsubjects were oligo- or amenorrheic (91%) while themajority of control subjects had regular menses (92%)

Table 2Hormonal and Biochemical Profiles of PCOS Subjects and Controls

n PCOS n Controls P value

TNF-a (pg/mL) 23 7.4 � 4.08 12 4.8 � 3.16 .05Free testosterone (pg/mL) 23 9.76 � 5.14 6 5 � 2.84 .01Fasting glucose (mg/dL) 19 88.63 � 12.82 10 89.2 � 8.61 .89Fasting insulin (mU/L) 19 21.12 � 10.5 7 18.96 � 7.61 .58HOMA-IR 19 4.79 � 2.45 7 4.55 � 1.43 .53

Data expressed as mean � SD.

Table 4Correlation between Serum TNF-a and PCOS Subjects' Pelvic US Indices, Degree ofObesity, and Hormonal Data

r P

Ultrasound indicesOvarian volumeLeft ovary �0.25 .25Right ovary 0.01 .95Mean ovarian volume �0.15 .49

Peripheral follicle distributionLeft Ovary .47Right Ovary .16

Follicle NumberLeft Ovary .69Right Ovary .62

Degree of obesityBMI Z Score 0.18 .41

Hormonal dataFree testosterone (pg/mL) �0.03 .91Fasting insulin (mU/mL) �0.07 .79

M. Pawelczak et al. / J Pediatr Adolesc Gynecol 27 (2014) 356e359358

(P ! .0001). One or more physical signs of hyperandro-genism (acne, alopecia, or male pattern baldness) waspresent in 87% of PCOS subjects compared with 41.67% ofcontrols (P5 .01). There was no significant difference in thepresence of acanthosis nigricans between the PCOS group(78.26%) and control group (88%) (P 5 .8).

Hormonal and Biochemical Profiles (Table 2)

Serum TNF-a was significantly higher in the PCOS group(7.4 � 4.08 pg/mL) compared with controls (4.8 � 3.16 pg/mL) (P 5 .0468). The PCOS subjects had significantly higherfree Testosterone levels (9.76 � 5.13 pg/mL) compared tocontrols (5 � 2.02 pg/mL) (P 5 .0092). Data on fasting in-sulin and glucose levels were available in 19 out of 23 PCOSsubjects and 7 out of 12 control subjects. Of these, therewasno significant difference in insulin resistance defined asa HOMA-IR $ 3.16 (P 5 .53).

Ovarian US Characteristics (Tables 3 and 4)

Transabdominal pelvic US data was available for all PCOSsubjects.

Ovarian VolumeMean left ovarian volume was 9.75 � 5.78 mL. Mean

right ovarian volume was 9.55 � 4.07 mL. Overall meanovarian volume was 9.65 � 4.58 mL. Ovarian volume wasgreater than 10 mL in 35% of images. Serum TNF-a did notsignificantly correlate with ovarian volume (left ovary r 5

�0.25, P 5 .25, right ovary r 5 0.01, P 5 .95).

Ovarian MorphologyPeripheral follicular distribution was noted in 76% of US

images (n5 35). In the remainder, the follicular distributionwas mixed (n 5 11). No significant correlation existed be-tween serum TNF-a and peripheral follicle distribution (leftovary P 5 .47, right ovary P 5 .16) In 38/46 US images, more

Table 3Pelvic US Characteristics of PCOS Group

Ovarian Volume (mL)Right ovarian volume 9.55 � 5.78Left ovarian volume 9.75 � 4.07Mean ovarian volume 9.65 � 4.58

Follicle Distribution (%)Peripheral 76%Mixed 24%

Follicle NumberFew 0Moderate 17Multiple 83

than 10 follicles were noted. In the remainder, between5 and 10 follicles were noted (83% of images demonstratedgreater than 10 follicles). There was no significant correla-tion between serum TNF-aand follicle number (P 5 .69 onleft ovarian images, P 5 .62 on right ovarian images).

Discussion

This pilot study demonstrates significantly increasedserum TNF-a levels in overweight and obese adolescentfemales with PCOS compared with BMI- and age-matchedcontrols. Elevated serum TNF- a reflects a state of chronicinflammation with potential negative implications for thefuture development of cardiovascular disease, metabolicdisease, and cancer in this young study population.

We examined obese and overweight adolescent femaleswith and without PCOS ages 11-17 years and found anelevation in serum TNF- a in those with PCOS without cor-relation to BMI. Although it is difficult to delineate whetherraised serum TNF- a contributes to the pathogenesis of PCOSor is an effect of PCOS, these results suggest that a factorintrinsic to PCOS may contribute to the elevated levels.Although TNF-a impacts follicular growth, ovulation, andcorpus luteum regression, we did not find a significant cor-relation between serum TNF-a and ovarian US characteristicscommon in PCOS, such as enlarged ovarian volume,increased follicular number, or peripheral follicle dis-tribution.12e14 This may also be related to recent suggestionthat multifollicular ovaries are a feature of normal pubertythat may be difficult to distinguish from PCOS morphology.3

There are potential implications of elevated serum TNF-aonmultiple organ systems. TNF-a promotes the synthesis ofIL-6, which regulates the synthesis of C-reactive protein, anacute phase reactant.9 Both IL-6 and C-reactive protein areconsidered strong risk markers for cardiovascular events.9

TNF-a has been implicated in plaque extension in athero-sclerosis as well as autoimmune diseases, such as systemiclupus erythematous.19,20 TNF-a may also help induce oste-oporosis by helping to activate osteoclasts.19 The release ofTNF-amay promote viral infections, such as HIV.21 Elevatedserum TNF-a may play a role in ovarian carcinogenesis andmetastasis.22 Studies have shown increased expression of

M. Pawelczak et al. / J Pediatr Adolesc Gynecol 27 (2014) 356e359 359

TNF-a in ovarian carcinoma tissue compared to normalovarian epithelial cells and elevated serum TNF-a levels inpatients with ovarian cancer compared to controls.12,23,24 Apositive correlation has been identified between TNF-a andendometrial cancer, the development of which may bemore common in women with PCOS.25,26

Our study demonstrates increased serum TNF-a inoverweight/obese adolescents with PCOS. The data re-ported here represent a pilot study and, therefore, did notbenefit from the advantages inherent to a larger samplesize with greater power. As this was a retrospective study,it was not possible to follow subjects over time to examineoutcome measures such as the possible development ofdisease states with inflammatory pathogenesis. In addi-tion, although in this cross-sectional retrospective study,US characteristics did not correlate with levels of TNF-a, itwould have been ideal to have longitudinal US data aswell as control group data for comparison. It was anadvantage of the study to have BMI-matched subjects andcontrols. However, serum TNF-a levels in normal weightadolescents with PCOS would have allowed for furthercontrol of the potentially confounding factor of increasedvisceral fat.

Serum TNF-a levels may be elevated in adolescent pa-tients with PCOS. These high levels may contribute to thepotential development of infertility, atherosclerosis, type 2diabetes, and certain cancers over an extended period oftime. Future studies are needed to clarify the effects of highserum TNF-a in PCOS patients and better delineate the longterm implications.

References

1. Norman RJ, Dewailly D, Legro RS, et al: Polycystic ovary syndrome. Lancet 2007;370:685

2. Franks S: Polycystic ovary syndrome. N Engl J Med 1995; 333:8533. Legro RS, Arslanian SA, Ehrmann DA, et al: Diagnosis and treatment of

polycystic ovary syndrome: an endocrine society clinical practice guideline. JClin Endocrinol Metab 2013; 98:4565

4. Deligeoroglou E, Vrachnis N, Athanasopoulos N, et al: Mediators of chronicinflammation in polycystic ovarian syndrome. Gynecol Endocrinol 2012; 28:974

5. Sun L, Hu W, Liu Q, et al: Metabonomics reveals plasma metabolic changes andinflammatory marker in polycystic ovary syndrome patients. J Proteome Res2012; 11:2937

6. Xiong YL, Liang XY, Yang X, et al: Low-grade chronic inflammation in theperipheral blood and ovaries of women with polycystic ovarian syndrome.Eur J Obstet Gynecol Reprod Biol 2011; 159:148

7. Repaci A, Gambineri A, Pasquali R: The role of low-grade inflammation in thepolycystic ovary syndrome. Mol Cell Endocrinol 2011; 335:30

8. Sathyapalan T, Atkin SL: Mediators of inflammation in polycystic ovarysyndrome in relation to adiposity. Mediators Inflamm 2010; 2010:758656

9. Diamanti-Kandarakis E, Paterakis T, Kandarakis HA: Indices of low-gradeinflammation in polycystic ovary syndrome. Ann N Y Acad Sci 2006; 1092:175

10. Duleba AJ, Dokras A: Is PCOS an inflammatory process? Fertil Steril 2012; 97:711. Fulghesu AM, Sanna F, Uda S, et al: IL-6 serum levels and production is related

to an altered immune response in polycystic ovary syndrome girls with insulinresistance. Mediators Inflamm 2011; 2011:389317

12. Norman RJ, Br€annstr€om M: Cytokines in the ovary: pathophysiology andpotential for pharmacological intervention. Pharmacol Ther 1996; 69:219

13. Br€annstr€om M, Norman RJ: Involvement of leukocytes and cytokines in theovulatory process and corpus luteum function. Hum Reprod 1993; 8:1762

14. Terranova PF, Rice VM: Review: cytokine involvement in ovarian processes. AmJ Reprod Immunol 1997; 37:50

15. Balchak SK, Marcinkiewicz JL: Evidence for the presence of tumor necrosisfactor alpha receptors during ovarian development in the rat. Biol Reprod1999; 61:1506

16. Figueroa F, Davicino R, Micalizzi B, et al: Macrophage secretions modulate thesteroidogenesis of polycystic ovary in rats: effect of testosterone onmacrophage pro-inflammatory cytokines. Life Sci 2012; 90:733

17. Quon MJ: Limitations of the fasting glucose to insulin ratio as an index of insulinsensitivity. J Clin Endocrinol Metab 2001; 86:4615

18. Keskin M, Kurtoglu S, Kendirci M, et al: Homeostasis model assessment is morereliable than the fasting glucose/insulin ratio and quantitative insulinsensitivity check index for assessing insulin resistance among obese childrenand adolescents. Pediatrics 2005; 115:e500

19. Locksley R, Killeen N, Lenardo M: The TNF and TNF receptor superfamilies:integrating mammalian biology. Cell 2001; 104:487

20. Jacob CO, Lewis GD, McDevitt HO: MHC Class II-associated variation in theproduction of tumor necrosis factor in mice and humans: relevance to thepathogenesis of autoimmune diseases. Immunol Res 1991; 10:156

21. Fauci AS, Schnittman SM, Poli G, et al: Mechanisms in human immunodeficiencyvirus (HIV) Infection. Ann Intern Med 1991; 114:678

22. Muthukumaran N, Miletti-Gonz�alez KE, Ravindranath AK, et al: Tumor necrosisfactor-alpha differentially modulates CD44 expression in ovarian cancer cells.Mol Cancer Res 2006; 4:511

23. Szlosarek PW, Grimshaw MJ, Kulbe H, et al: Expression and regulation of tumornecrosis factor alpha in normal and malignant ovarian epithelium. Mol CancerTher 2006; 5:382

24. Hassan MI, Kassim SK, Laban M, et al: Ovarian cancer-induced immunosup-pression: relationship to tumor necrosis factor-alpha (TNF-alpha) release fromovarian tissue. Anticancer Res 1990; 19:5657

25. Dossus L, Becker S, Rinaldi S, et al: Tumor necrosis factor (TNF)- a, soluble TNFreceptors and endometrial cancer risk: the EPIC study. Int J Cancer 2011; 129:2032

26. Chittenden BG, Fullerton G, Maheshwari A, et al: Polycystic ovary syndromeand the risk of gynaecological cancer: a systematic review. Reprod BiomedOnline 2009; 19:398