Study of Free Testosterone in Polycystic Ovarian Syndrome

Reshma, A

doi:10.65188/nurexus.1057

Page 8

Journal of Medverse Research & Practice

ISSN: 3107-4278

Study of Free Testosterone in Polycystic Ovarian Syndrome

Dr. A Reshma

1

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

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

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

ORIGINAL ARTICLE

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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

Study Design

This hospital-based case-control study was conducted to evaluate serum free testosterone levels among

women with Polycystic Ovary Syndrome (PCOS) and compare them with healthy age-matched controls.

Study Setting and Duration

The study was carried out in the Department of Obstetrics and Gynecology, ACSR Government Medical

College, Nellore, over a period of 16 months from August 2022 to November 2023.

Study Population

The study population comprised women diagnosed with Polycystic Ovary Syndrome (PCOS) attending

the outpatient department and healthy age-matched women without PCOS who served as controls.

Sample Size

A total of 74 participants were included in the study, comprising 37 women with PCOS and 37 healthy

controls. The sample size was calculated based on the study by Hurjahan Banu et al. using the formula n

= 2(Zα + Z1-β)²σ² / d², considering a standard deviation of 17.39, an effect size of 11.26, 95% confidence

level, and 80% study power.

Inclusion Criteria

Women aged 18 years and above diagnosed with Polycystic Ovary Syndrome according to the Rotterdam

criteria who had not received metformin, clomiphene citrate, oral contraceptive pills, or other hormonal

medications for more than one month prior to enrolment were included in the case group. Healthy age-

matched women without clinical or ultrasonographic evidence of PCOS were included as controls.

Exclusion Criteria

Women with hyperprolactinemia, ovarian tumors, Cushing syndrome, current pregnancy, or those

unwilling to participate were excluded from the study.

Data Collection Tool

Following written informed consent, detailed demographic information, menstrual history, obstetric

history, medical history, and relevant clinical findings were recorded using a structured case record form.

General and systemic examinations were performed for all participants. Approximately 5 mL of venous

blood was collected under aseptic precautions, centrifuged to separate serum, and stored at –20°C until

biochemical analysis. Serum free testosterone levels were measured using a standardized Enzyme-Linked

Immunosorbent Assay (ELISA) technique following the manufacturer's instructions and quality control

procedures.

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Ethical Considerations

The study protocol was reviewed and approved by the Institutional Ethics Committee of ACSR

Government Medical College, Nellore, prior to commencement of the study. Written informed consent

was obtained from all participants before enrolment. Participant confidentiality was maintained by

assigning unique identification numbers, and all collected data were used exclusively for research

purposes.

Statistical Analysis

Data were entered into Microsoft Excel for coding and analyzed using the Statistical Package for the

Social Sciences (SPSS) software version 26.0. Continuous variables were expressed as mean ± standard

deviation, while categorical variables were presented as frequencies and percentages. Comparisons

between cases and controls were performed using the independent sample t-test for continuous variables

and the Chi-square test for categorical variables where appropriate. Pearson's correlation coefficient was

used to evaluate the relationship between serum free testosterone levels and clinical variables. A p-value

of less than 0.05 was considered statistically significant.

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

37

50

Control

37

50

Total

74

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)

18

0.000

Control

23.49 ± 3.849

(18–33)

15

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)

16

0.001

Control

10.70 ± 5.568

12.67 (2–27)

25

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).

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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)

Parameter

Mean difference

t statistic

Df

P value

Free testosterone in the case

9.66.

13.225

36

0.000

Free testosterone in control

10.698

11.686

36

0.000

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.

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

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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 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.

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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.

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