Introduction and Context
Testosterone measurement is central to diagnosing and managing a range of endocrine disorders across the lifespan: polycystic ovary syndrome (PCOS), congenital adrenal hyperplasia (CAH), disorders of sex development, and suspected androgen deficiency. Yet, in women, adolescent girls, and children, circulating testosterone concentrations are substantially lower than in adult men and hover near the analytical limits of many assays. Two decades of efforts to standardize testosterone testing have improved accuracy at higher concentrations, but reference intervals (RIs) for females and pediatric age groups remain inconsistent across laboratories and platforms. This variability undermines diagnostic confidence, delays care, and complicates multicenter research.
On August 26, 2024, PCOS Challenge: The National Polycystic Ovary Syndrome Association partnered with the Centers for Disease Control and Prevention (CDC) Clinical Standardization Programs to convene a multidisciplinary stakeholder meeting. The goal: create a practical, evidence-based plan to generate harmonized, sex- and age-specific testosterone reference ranges for women, adolescent girls, and children using existing cohorts and CDC reference measurements. The meeting’s report was subsequently published in The Journal of Clinical Endocrinology & Metabolism (Ottey et al., 2026).
Why this consensus matters now
– Growing recognition of androgen-related morbidity in females (PCOS, metabolic and reproductive consequences) has increased demand for accurate biochemical evaluation.
– Advances in mass spectrometry and metrological traceability make harmonized RIs feasible, but standardized approaches for pooling cohort data are needed.
– Inconsistent RIs force clinicians to rely on clinical judgment or local lab cutoffs that may be inappropriate for specific ages or physiologic states (e.g., puberty, pregnancy).
New Guideline Highlights
The PCOS Challenge–CDC stakeholder meeting produced consensus recommendations across seven domains and a pragmatic four-phase work plan to produce standardized testosterone RIs for females and children. Key takeaways:
– Assay standardization is foundational: reanalysis of cohort specimens in a CDC-certified laboratory using reference methods (LC-MS/MS calibrated to CDC reference materials) is recommended to anchor harmonized RIs.
– Reference population characterization must be granular for age, Tanner stage, sex assigned at birth, menstrual/ovulatory status, medication use (e.g., hormonal contraception), and race/ethnicity.
– Study design and data-integration standards should be prespecified, using statistical normalization methods to combine cohorts while retaining clinically meaningful subgroup RIs.
– Education, global access, and inclusion of additional biomarkers (anti-Müllerian hormone [AMH], 11-oxygenated androgens) are priorities for future expansions.
Clinician-facing highlights
– Expect forthcoming peer-reviewed RIs anchored to CDC reference measurements; until published, recognize that adult male-derived cutoffs are inappropriate for females and children.
– When ordering testosterone tests in low-concentration contexts, prefer laboratories using validated LC-MS/MS methods with documented traceability and low limits of quantitation.
Updated Recommendations and Key Changes
The meeting did not issue clinical practice rules, but established consensus principles for RI creation and recommended operational steps that differ from prior informal practice:
– From local to centralized: Move from use of disparate lab-specific RIs to harmonized, population-specific RIs anchored to a central reference laboratory (CDC). Previous practice relied mainly on manufacturer-provided intervals or population samples from single centers.
– From adult-based to age- and sex-specific: Emphasize stratification by narrow age bands and Tanner stage for pediatric populations; acknowledge menstrual cycle and ovulatory status for adult women.
– From single-assay dependence to cross-platform harmonization: Recommend statistical normalization of existing cohort results to CDC reference values rather than wholesale replacement of assays across centers.
Table: Principal updates (summary)
– Assay anchoring: CDC reference measurements for reanalysis (new)
– Reference population granularity: narrow age bands, Tanner stage, menstrual/contraceptive status (expanded)
– Data integration: validated normalization/harmonization pipeline (new)
– Priority biomarkers for harmonization: testosterone now; plan to add AMH and 11-oxygenated androgens (new)
Evidence driving updates
The recommendations build on decades of analytical research showing immunoassays often overestimate or have poor precision at low female and pediatric testosterone concentrations, while LC-MS/MS provides better accuracy and specificity when properly standardized. The CDC’s ability to provide reference measurements enables retrospective harmonization of existing cohort data, increasing sample sizes for robust RIs without the cost and delay of new, prospective population sampling.
Topic-by-Topic Recommendations
1) Assay Standardization
– Use reference-method LC-MS/MS calibrated to CDC reference materials for reanalysis of archived and new specimens.
– Report lower limits of quantitation (LLOQ) and imprecision at female/pediatric concentration ranges.
– Participate in external quality assessment schemes and report method traceability in lab reports.
Recommendation grade: Consensus from stakeholder meeting; follows CDC HoSt principles and metrological best practices (evidence: analytical method comparison literature and CDC standardization program).
2) Reference Population Characterization
– Minimum required descriptors: age (in months for infants, narrow bands for children/adolescents), sex assigned at birth, Tanner stage (where relevant), race/ethnicity, BMI, pregnancy status, hormonal medication use, and presence/absence of known endocrine disorders.
– Exclude known androgen-affecting pathologies if the goal is a “healthy” reference interval; produce separate RIs for specific clinical states (e.g., post-oral contraceptive).
3) Study Design Parameters
– Use existing cohorts with well-annotated clinical metadata and stored serum/plasma specimens suitable for LC-MS/MS analysis.
– Aim for robust sample sizes within each stratum (target n per stratum defined by statistical power analyses); when a stratum is underpowered, present combined ranges with clear caveats.
– Predefine outlier-handling, statistical transformation, and percentile calculation methods.
4) Data Integration Standards
– Reanalyze cohort specimens at the CDC lab to provide a common anchor.
– Apply validated normalization algorithms to align original cohort measurements to CDC reference values when reanalysis of all specimens is not feasible.
– Provide transparency: publish normalization code, weighting strategies, and sensitivity analyses.
5) Educational Outreach
– Create clinician and laboratory guidance on interpretation of female and pediatric testosterone concentrations, including limitations of older immunoassays.
– Develop patient-facing materials explaining why testing approaches and reference ranges may change and what that means clinically.
6) Global Access and Equity
– Prioritize inclusion of diverse racial and ethnic groups, and cohorts from low- and middle-income regions where feasible.
– Share harmonization tools and protocols openly to enable local labs to align their RIs with CDC-anchored values.
7) Additional Biomarkers
– Recommend future standardization work for AMH and 11-oxygenated androgens, biomarkers increasingly recognized for reproductive and adrenal disorders.
Four-Phase Work Plan
The meeting proposed a pragmatic four-phase pipeline to create and publish harmonized RIs:
Phase 1 — Cohort Identification and Metadata Harmonization
– Identify candidate cohorts (the meeting identified nine women’s and adolescent health cohorts as starting points).
– Harmonize metadata fields.
Phase 2 — Specimen Reanalysis at CDC Laboratory
– Ship aliquots for reanalysis using CDC-traceable LC-MS/MS methods; establish measurement anchors.
Phase 3 — Statistical Analysis and Normalization
– Use predetermined statistical methods to calculate percentiles and RIs, including stratified analyses by age, Tanner stage, and clinical subgroups.
Phase 4 — Publication, Implementation, and Education
– Peer-reviewed publication of RIs and methods; dissemination to labs and professional societies; develop clinician and patient educational materials.
Expert Commentary and Insights
Committee perspectives
– Laboratory scientists emphasized that analytical accuracy at low concentrations is non-negotiable: small absolute differences can have large clinical impact in females and children.
– Clinicians highlighted the real-world harms from inconsistent RIs—missed diagnoses, overdiagnosis, and variable management decisions—and welcomed a harmonized standard.
– Patient advocates stressed the importance of transparency, inclusive cohort selection, and clear patient education when RIs change.
Controversies and unresolved issues
– How to handle specimens from patients on hormonal contraceptives or androgen-suppressing therapies: should separate RIs be constructed, or should clinicians interpret results using guidance on expected shifts? The meeting favored producing both “healthy unmedicated” RIs and context-specific reference ranges where data permit.
– Race/ethnicity stratification: the group agreed to report RIs by race/ethnicity only when evidence supports clinically meaningful differences and recommended against using race as a biological proxy without careful context.
– Optimal age/Tanner bins for adolescents: balancing statistical power with clinical relevance remains a methodological challenge; the meeting proposed narrow bands where data permit and combined bands with clear caveats otherwise.
Future research needs
– Larger, prospectively collected cohorts with standardized specimen handling to validate age- and sex-specific RIs.
– Standardization and clinical validation of 11-oxygenated androgens and AMH across platforms.
Practical Implications for Clinicians and Laboratories
For clinicians
– When interpreting testosterone in women and children, ask the laboratory about assay methodology, LLOQ, and whether the reported RI is aligned to reference-method standards.
– Avoid relying solely on a single numerical cutoff; integrate biochemical results with clinical signs, menstrual history, growth and pubertal staging, and other hormones.
– Anticipate updates to published reference ranges and be prepared to re-interpret prior results in light of new, harmonized RIs.
For laboratories
– Document assay traceability and performance metrics at low concentrations; participate in external quality assurance programs.
– If LC-MS/MS is not available locally, establish pathways to send specimens for reference testing or adopt validated normalization approaches.
A brief clinical vignette
Maya, a 16-year-old high school athlete, presents with irregular menses and new-onset hirsutism. Her local lab issued a testosterone level that falls within the lab’s “normal” adult female RI, but the clinician suspects androgen excess. On questioning, the clinician learns the lab uses an older immunoassay with limited precision at low female concentrations. The clinician orders a confirmatory LC-MS/MS test from a CDC-traceable lab. The result, interpreted using emerging harmonized adolescent RIs, confirms mild testosterone elevation. This anchors further evaluation for PCOS and individualized management. The vignette illustrates how assay choice and appropriate RIs directly affect diagnostic accuracy.
References
– Ottey S, Pokuah F, Cree MG, Sherif K, Vesper HW, Lyle AN, Azziz R. Testosterone reference ranges in women, adolescent girls, and children: the 2024 PCOS Challenge-CDC stakeholder meeting. J Clin Endocrinol Metab. 2026 Sep 16;111(10):e2290-e2299. PMID: 42444555. https://pubmed.ncbi.nlm.nih.gov/42444555/
– Teede HJ, Misso ML, Costello MF, et al. Recommendations from the International Evidence-Based Guideline for the Assessment and Management of Polycystic Ovary Syndrome. Hum Reprod. 2018;33(9):1602–1618. doi:10.1093/humrep/dey256
– Legro RS, Arslanian SA, Ehrmann DA, et al. Diagnosis and Treatment of Polycystic Ovary Syndrome: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2013;98(12):4565–4592. doi:10.1210/jc.2013-2350
– Centers for Disease Control and Prevention. Hormone Standardization (HoSt) Program. CDC Laboratory Quality Assurance and Standardization Programs. https://www.cdc.gov/labstandards/hs/index.html (accessed 2026).
– Stanczyk FZ, Clarke NJ. Advantages and Challenges of Mass Spectrometry Assays for Steroid Hormone Measurements. J Steroid Biochem Mol Biol. 2010;121(3-5):491–495. doi:10.1016/j.jsbmb.2010.04.016
Practical next steps for readers
– Clinicians: During diagnostic workups for androgen disorders in females and children, document assay method and request LC-MS/MS testing when results are near clinical decision points.
– Laboratory directors: engage with CDC standardization programs and professional societies to align local practice with the planned harmonized RIs.
– Researchers: consider contributing well-annotated cohort data and stored specimens to collaborative RI projects.
The PCOS Challenge–CDC stakeholder meeting represents a pragmatic, multidisciplinary effort to overcome the long-standing problem of variable testosterone reference ranges in females and children. By anchoring harmonization efforts to CDC reference measurements and combining existing cohorts with robust metadata and transparent statistical methods, the field can expect clinically useful, evidence-based RIs that will improve diagnosis, management, and research of androgen-related conditions.
