Study of Free Testosterone in Polycystic Ovarian Syndrome

Reshma A et al | DOI: 10.65188/nurexus.1057

Page 7

Journal of Medverse Research & Practice

ISSN: 3107-4278

Study of Free Testosterone in Polycystic Ovarian Syndrome

Dr. A Reshma

Professor

Department of Obstetrics & Gynaecology, ACSR Government Medical College,

Nellore.

Email ID: reshmaa45@gmail.com,

Submission Date: 15.11.2025

Accepted Date: 16.12.2025

Published Date: 31.12.2025

DOI: 10.65188/nurexus.1057

article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits

unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Abstract

Background: Polycystic ovarian syndrome (PCOS) is a prevalent endocrine disorder in women of reproductive age,

characterized by hyperandrogenism, menstrual irregularities, and metabolic disturbances. Free testosterone is the

biologically active androgen, yet its levels may vary among different PCOS phenotypes.

Objective: To evaluate free testosterone levels in women with PCOS and compare them with those of age-matched

healthy controls.

Methods: A case-control study was conducted at the Department of Obstetrics and Gynecology, ACSR Government

Medical College, Nellore, from August 2022 to November 2023. A total of 74 women (37 diagnosed with PCOS and

37 healthy controls) were enrolled after informed consent. Serum-free testosterone was measured using ELISA.

Statistical analysis included descriptive statistics, independent t-tests, and Pearson correlation, with significance set

at p < 0.05.

Results: The mean age of participants was 24.24 ± 5.60 years in the PCOS group and 23.49 ± 3.85 years in

controls. Mean free testosterone levels were 9.66 ± 4.44 pg/mL in PCOS and 10.70 ± 5.57 pg/mL in controls, showing

a statistically significant difference (p < 0.05). Weak positive correlations between age and free testosterone were

observed in both groups (PCOS: r = 0.129; controls: r = 0.031).

Conclusion: Free testosterone levels in women with PCOS were slightly lower than those of controls, with minimal

correlation to age. These results underscore the heterogeneity of PCOS and suggest that assessing free testosterone

alone may not adequately reflect androgen excess. Comprehensive evaluation, including total testosterone, SHBG,

free androgen index, and phenotypic assessment, is recommended for accurate diagnosis and management.

Keywords: Polycystic ovarian syndrome; free testosterone; hyperandrogenism; case-control study; reproductive-age

women

Introduction

Polycystic ovarian syndrome (PCOS) is a multifaceted endocrine disorder affecting women of reproductive

age and is a leading cause of anovulatory infertility [1]. Its prevalence globally ranges from 5% to 20%,

depending on the diagnostic criteria applied, including Rotterdam, NIH, and AE-PCOS Society guidelines

[2]. In India, community-based studies report prevalence rates between 9% and 36%, highlighting an

increasing burden of reproductive and metabolic disorders among young women [3].

Clinically, PCOS manifests as menstrual irregularities, chronic anovulation, infertility, obesity, acne,

hirsutism, and metabolic abnormalities such as insulin resistance and dyslipidemia [4]. Hyperandrogenism

is a defining biochemical feature and may present clinically or be confirmed through

Reshma A et al | DOI: 10.65188/nurexus.1057

Page 8

elevated androgen levels [5]. Among androgens, free testosterone represents the biologically active fraction,

while total testosterone may be misleading due to variations in sex hormone-binding globulin (SHBG),

which is often reduced in PCOS secondary to hyperinsulinemia and obesity [6]. Therefore, free testosterone

measurement is considered a more reliable marker of androgen excess.

Accurate assessment of hyperandrogenemia remains challenging, as clinical practice frequently relies on

total testosterone, which may not reflect true androgen activity. Free testosterone correlates more closely

with clinical manifestations such as hirsutism, acne, and menstrual irregularities; however, measurement

accuracy depends on the assay employed [7]. Misestimation can lead to underdiagnosis or misclassification

of disease severity.

Moreover, free testosterone levels are associated with metabolic complications, including insulin resistance,

visceral adiposity, and increased cardiovascular risk [8]. Variability in assay methods further complicates

interpretation, as direct analog assays can be inaccurate while equilibrium dialysis, though more precise, is

expensive and not widely available [9]. Understanding the patterns of free testosterone in PCOS is therefore

essential for accurate diagnosis and optimal management.

Materials and Methods

This case-control study was conducted at the Department of Obstetrics and Gynecology, ACSR

Government Medical College, Nellore, from August 2022 to November 2023. Ethical approval was

obtained from the Institutional Ethics Committee, and all participants provided written informed consent.

Women aged over 18 years diagnosed with PCOS according to Rotterdam criteria and not using

medications such as metformin, clomiphene citrate, or oral contraceptives for more than one month were

included. Exclusion criteria included hyperprolactinemia, ovarian tumors, Cushing syndrome, current

pregnancy, or unwillingness to participate. Healthy age-matched women without PCOS served as

controls. Clinical history and physical examination were recorded. Five milliliters of venous blood were

collected from each participant, centrifuged, and the serum was stored at –20°C until analysis. Serum-free

testosterone was measured using ELISA. The sample size of 37 participants per group was calculated

based on a previous study by Hurjahan Banu, using the formula n = 2(Zα + Z1-β)²σ² / d², with a standard

deviation of 17.39 and an effect size of 11.26. Data were analysed using SPSS version 26.0. Continuous

variables were expressed as mean ± standard deviation, and categorical variables as percentages. Group

comparisons were performed using independent t-tests, and correlations were evaluated using Pearson

correlation coefficients. Statistical significance was defined as p < 0.05.

The present study received approval from the Institutional Ethics Committee of ACSR Government

Medical College (Ref No: IEC/ACSR-GMC/2024/55219). A detailed Participant Information Sheet was

provided to all participants, and written informed consent was obtained prior to their inclusion in the

study.

Results

This case-control study was conducted in the Department of OBG, Vanivilas Hospital, BMCRI, from

August 2022 to November 2023. Following Institutional Ethics Committee approval, 37 women with PCOS

and 37 controls meeting the inclusion criteria were enrolled after providing informed consent.

Table 1: Distribution of participants by group

Group

Frequency (n)

Percentage (%)

PCOS

Control

Reshma A et al | DOI: 10.65188/nurexus.1057

Page 9

Total

100

This table shows that the study included 74 participants, evenly divided between the PCOS group (37,

50%) and the control group (37, 50%).

Table 2: Age distribution between PCOS and Control groups

Group

Mean ± SD

Median (IQR)

Range

P value

PCOS

24.24 ± 5.595

23.00 (16-34)

0.000

Control

23.49 ± 3.849

(18–33)

0.001

The mean age of participants was slightly higher in the PCOS group (24.24 ± 5.60 years) compared to the

control group (23.49 ± 3.85 years). Median ages were 23 years (IQR 16–34) for PCOS and 23 years (IQR

18–33) for controls. The age difference between groups was statistically significant (P < 0.05).

Table 3: Free testosterone levels in PCOS vs Control groups

Group

Mean ± SD

Median (IQR)

Range

P value

PCOS

9.66 ± 4.443

10.85 (2–18)

0.001

Control

10.70 ± 5.568

12.67 (2–27)

0.000

The mean free testosterone level was slightly lower in the PCOS group (9.66 ± 4.44 pg/mL) compared to

controls (10.70 ± 5.57 pg/mL). Median levels were 10.85 pg/mL (IQR 2–18) for PCOS and 12.67 pg/mL

(IQR 2–27) for controls. The difference between groups was statistically significant (P < 0.05).

Figure 1: Free testosterone levels in the case PCOS groups vs the Control groups

Table 4: Comparison of free testosterone between groups (Independent t test)

The independent t-test comparing free testosterone levels between groups showed a mean of 9.66 pg/mL in

the PCOS group and 10.70 pg/mL in controls. Both comparisons were statistically significant (PCOS: t

= 13.23, df = 36, p < 0.001; Control: t = 11.69, df = 36, p < 0.001), indicating a significant difference

between the groups.

Parameter

Mean difference

t statistic

P value

Free testosterone in the case

9.66.

13.225

0.000

Free testosterone in control

10.698

11.686

0.000

Reshma A et al | DOI: 10.65188/nurexus.1057

Page 10

Table 5: Correlation between age and free testosterone

Parameter

Pearson correlation (r)

P value

Age vs Free Testosterone case

0.129

0.00

Age vs Free Testosterone control

0.031

0.00

There was a weak positive correlation between age and free testosterone in both groups (PCOS: r = 0.129;

Control: r = 0.031). Both correlations were statistically significant (P < 0.05), suggesting a minimal but

significant association between age and free testosterone levels.

Discussion

In the present case-control study involving 74 women (37 with polycystic ovary syndrome [PCOS] and 37

age-matched healthy controls), the mean serum free testosterone (FT) levels were 9.66 ± 4.44 pg/mL in the

PCOS group and 10.70 ± 5.57 pg/mL in the control group, with a statistically significant difference. Weak

positive correlations between age and free testosterone were observed in both groups. These findings

suggest that, in this cohort, biochemical hyperandrogenism as assessed by free testosterone was not

markedly elevated in women with PCOS, which differs from the classical expectation of consistently

increased androgen levels in this condition.

Earlier studies have documented higher free testosterone concentrations in women with PCOS. Penttilä

reported significantly elevated serum free testosterone levels in women with PCOS compared to healthy

controls, supporting the role of biochemical hyperandrogenism as a diagnostic feature of the syndrome [12].

Similarly, Legro et al. demonstrated that androgen excess, including elevated testosterone levels, was a

prominent feature in several PCOS phenotypes and influenced both diagnosis and treatment approaches

[13]. Grassi et al., using liquid chromatography tandem mass spectrometry (LC-MS/MS), further confirmed

significantly higher testosterone and androstenedione levels in women with PCOS, highlighting the

sensitivity of advanced analytical techniques in detecting androgen excess [14].

The apparent discrepancy between our findings and previous reports may be explained by the well-

recognized heterogeneity of PCOS. Legro et al. described substantial phenotypic variation within PCOS,

noting that certain phenotypes, particularly those without overt hyperandrogenism, may exhibit androgen

levels comparable to healthy controls [13]. Grassi et al. also emphasized that androgen profiles differ

significantly depending on the PCOS phenotype and the analytical method used, suggesting that cohorts

enriched with less androgenic phenotypes may demonstrate lower free testosterone levels [14].

Methodological differences in androgen assessment may also contribute to variability across studies.

Lerchbaum and Obermayer-Pietsch highlighted that free testosterone levels are strongly influenced by assay

methodology, timing of sample collection, and circulating sex hormone-binding globulin concentrations,

which can lead to under- or overestimation of biochemical hyperandrogenism [15]. Patil et al. similarly

emphasized that reliance on a single marker, such as free testosterone, may be insufficient, advocating for

the use of the free androgen index and complementary androgen measurements for accurate evaluation

[16].

Recent evidence suggests that androgens beyond testosterone play a significant role in the hyperandrogenic

milieu of PCOS. O’Reilly et al. demonstrated that 11-oxygenated androgens, particularly 11-

ketotestosterone, are markedly elevated in women with PCOS and may better reflect androgen excess than

conventional testosterone measurements [17]. Wang et al. reinforced this concept, identifying androgen

excess as a hallmark of PCOS and highlighting the contribution of non-classical androgens to its

pathophysiology [18]. Studies focusing on specific populations, including Indian women,have further

shown that LC-MS/MS–measured androgen profiles can reveal patterns not captured by routine

Reshma A et al | DOI: 10.65188/nurexus.1057

Page 11

immunoassays, underscoring the importance of population-specific assessment [19,20].

With respect to age, the weak correlations observed in the present study are consistent with recent findings

indicating minimal influence of age on free testosterone levels within reproductive-age women with PCOS.

Kugelman et al. reported that basal total and free testosterone levels did not vary significantly with age and

did not impact reproductive outcomes in women with PCOS undergoing assisted reproduction [22].

Similarly, Zhang et al. found that androgen levels were not strong age-dependent predictors in reproductive

outcome analyses among women with PCOS [23].

From a clinical perspective, these findings highlight that biochemical hyperandrogenism, as assessed solely

by free testosterone, may not be universally present in all women with PCOS. Exclusive reliance on free

testosterone may therefore lead to underdiagnosis or misclassification of androgen excess. A

comprehensive diagnostic approach incorporating total testosterone, sex hormone-binding globulin, free

androgen index, androstenedione, 11-oxygenated androgens, and detailed phenotypic assessment is

essential for accurate diagnosis, appropriate risk stratification, and individualized management of women

with PCOS.

Limitations

The study had several limitations. The sample size was modest, which may limit the generalizability and

statistical power of the findings. We did not measure SHBG, total testosterone, or other androgen

metabolites, limiting the ability to fully characterize androgen status. Blood samples were not controlled

for menstrual cycle phase, which could influence hormone levels. Furthermore, the cross-sectional design

precludes evaluation of longitudinal changes or causal relationships between androgen levels and clinical

outcomes. Finally, the study cohort was drawn from a single tertiary care center, which may limit

applicability to broader populations.

Conclusion

In this case-control study, women with PCOS exhibited slightly lower free testosterone levels compared to

healthy controls, and only weak positive correlations were observed between age and free testosterone.

These findings emphasize the heterogeneity of PCOS and suggest that not all patients exhibit biochemical

hyperandrogenism. Reliance on free testosterone alone may underestimate androgen excess, potentially

leading to an incomplete diagnosis. A comprehensive approach incorporating total testosterone, SHBG,

free androgen index, other androgen metabolites, and phenotypic evaluation is recommended for accurate

diagnosis and optimal management of PCOS.

Conflict of interest: Nil

Source Of Fund: Nil

Acknowledgement: I sincerely thank my department for their invaluable support throughout the study,

and I am grateful to the management of the institution for providing the resources and environment

necessary for the successful completion of this work.

Reference

1. Teede H, Misso M, Costello M, et al. Recommendations from the international evidence-based guideline

for the assessment and management of polycystic ovary syndrome. Clin Endocrinol (Oxf). 2018;89(3):251-

268.Azziz R, Woods KS, Reyna R, et al. The prevalence and features of the polycystic ovary syndrome in

an unselected population. J Clin Endocrinol Metab. 2004;89(6):2745-2749.

2. Dunaif A. Insulin resistance and the polycystic ovary syndrome. Endocr Rev. 1997;18(6):774-800.

Reshma A et al | DOI: 10.65188/nurexus.1057

Page 12

3. Rosner W, Auchus RJ, Azziz R, et al. Utility, limitations, and pitfalls in measuring testosterone. J Clin

Endocrinol Metab. 2007;92(2):405-413.

4. Escobar Morreale HF. Polycystic ovary syndrome. Nat Rev Endocrinol. 2018;14(5):270-284.

5. Carmina E, Azziz R, Bergfeld W, et al. Female pattern hair loss and androgen excess. J Clin Endocrinol

Metab. 2019;104(7):2875-2891.

6. Goldman AL, Bhasin S, Wu FC, et al. A reappraisal of testosterone binding in circulation. Endocr Rev.

2017;38(4):302-324.

7. Sam S. Obesity and polycystic ovary syndrome. Obes Manag. 2007;3(2):69-73.

8. Lim SS, Davies MJ, Norman RJ, Moran LJ. Overweight, obesity and central obesity in women with

polycystic ovary syndrome. Hum Reprod Update. 2012;18(6):618-637.

9. Hussein S, et al. Reference intervals and biologic variation of testosterone in women: comparison between

PCOS and controls. Fertil Steril. 2014;101(6):1695 1701.

10. Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the

polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab. 2004;89(6):2745 2749.

11. Penttilä TA. Serum free testosterone in polycystic ovary syndrome. Fertil Steril. 1996;65(3):693 698.

12. Legro RS, Kunselman AR, Dodson WC, Dunaif A. Phenotypic variation in polycystic ovary syndrome:

impact on diagnosis and treatment. Fertil Steril. 1998;69(6):1051 1058.

13. Grassi G, Polledri E, Fustinoni S, Chiodini I, Ceriotti F, D’Agostino S, et al. Hyperandrogenism by Liquid

Chromatography Tandem Mass Spectrometry in PCOS: Focus on Testosterone and Androstenedione. J Clin

Med. 2021;10(1):119.

14. Lerchbaum E, Obermayer-Pietsch B. Hyperandrogenemia in Polycystic Ovary Syndrome. PLoS One.

2014;9(5):e97530.

15. Patil P, et al. Free-androgen index in women with polycystic ovarian syndrome. J Hum Reprod Sci.

2015;8(4):222 228.

16. O’Reilly MW, Kempegowda P, Jenkinson C, et al. 11-oxygenated androgens in PCOS: implications for

diagnosis and therapy. J Clin Endocrinol Metab. 2017;102(9):3330 3339.

17. Wang K, et al. Androgen excess: a hallmark of polycystic ovary syndrome. Front Endocrinol (Lausanne).

2023;14:1273542.

18. Subramaniam K, et al. Insights from liquid chromatography-mass spectrometry-measured androgens in

Indian women with PCOS. Gynecol Endocrinol. 2024;40(5):375 382.

19. Chen F, Chen M, Zhang W, Yin H, Chen G, Huang Q, Yang X, Chen L, Lin C, Yin G. Comparison of the

efficacy of different androgens measured by LC MS/MS in representing hyperandrogenemia and an

evaluation of adrenal origin androgens with a dexamethasone suppression test in patients with polycystic

ovary syndrome. J Ovarian Res. 2021;14:32.

20. Mansour A, Noori M, Hakemi MS, Haghgooyan Z, Mohajeri Tehrani MR, Mirahmad M, Sajjadi Jazi SM.

Hyperandrogenism and anthropometric parameters in women with polycystic ovary syndrome. BMC Endocr

Disord. 2024;24:201.

21. Kugelman N, Digby A, Rotshenker Olshinka K, Bellemare V, Pooni A, Son W Y, Dahan MH. Basal serum

luteinizing hormone, total testosterone, and free testosterone levels do not impact in vitro fertilization

outcomes in patients with polycystic ovary syndrome. J Turk Ger Gynecol Assoc. 2024;25(4):192–199.

22. Zhang Y, Liu X, Li Z, et al. The discriminatory capability of anthropometric measures in predicting

reproductive outcomes in Chinese women with polycystic ovary syndrome. J Ovarian Res. 2024;17:186.