Inaccuracies in laboratory testosterone testing have long hindered appropriate management in several patient groups, including women with polycystic ovary syndrome (PCOS) and men with suspected low testosterone, says Andrea Dunaif, MD, an expert in PCOS and androgenic disorders in women.
Dr. Dunaif, Chief of the Division of Endocrinology, Diabetes and Bone Disease, Icahn School of Medicine at Mount Sinai, has worked to address this issue for decades, including during her tenure as president of the Endocrine Society from 2005 to 2006, when she oversaw the creation of a task force to evaluate the accurate measurement of testosterone and make recommendations.
“The problem is that most commercially available testosterone assays are designed to measure testosterone in men, which is normally about tenfold higher than it is in women. In prepubertal children, it’s even lower. Therefore, it is not possible to accurately measure testosterone levels in women and children without specialized chromatographic assays. Levels determined in other types of assays can be falsely elevated or normal,” says Dr. Dunaif, the Lillian and Henry M. Stratton Professor of Molecular Medicine.
Free or non-sex hormone binding globulin (SHBG) bound testosterone (also known as bioavailable testosterone) levels are the most sensitive measurement for detecting androgen excess. The accurate assessment of these parameters also requires an accurate liquid chromatography tandem mass spectrometry (LC-MS/MS) measurement of total testosterone. The free and non-SHBG is then calculated using the concentrations of total testosterone, SHBG, and serum albumin. “However, many commercial labs use an assay that purports to directly measure free testosterone, the so-called direct free testosterone,” Dr. Dunaif says. “This assay has been found to be totally inaccurate and should never be used.”
“It is not possible to accurately measure testosterone levels in women and children without specialized chromatographic assays. Levels determined in other types of assays can be falsely elevated or normal.”
Andrea Dunaif, MD
In 2007, the Endocrine Society Task Force published a position statement recommending LC-MS/MS for total testosterone, and a calculation of free testosterone and non-SHBG bound from the total testosterone and the SHBG. The position statement also made a strong recommendation for overall standardization of testosterone testing, such that “normative values for total testosterone and free testosterone should be established taking into account all the appropriate variables, e.g., gender, age, race, stage of puberty, time of day, etc. We believe this goal can be accomplished. It has been done for cholesterol,” the society statement read.
In 2010, the Endocrine Society held another meeting on the topic, this time with the Centers for Disease Control and Prevention, the American Association of Clinical Endocrinologists, the American Thyroid Association, and the American Diabetes Association, along with other government and industry stakeholders. The result of the meeting was a consensus statement, endorsing the Endocrine Society’s prior guidance regarding testosterone assay standardization. The document made further recommendations, including a call for third-party payers and health care organizations to enable payment solely for such standardized assays, and for medical journals to require investigators to use those assays in research as a prerequisite for publication.
Unfortunately, many clinical laboratories have not implemented these recommendations and still utilize inaccurate methods for the measurement of total and free testosterone unless the clinician specifically orders the correct LC-MS/MS testosterone assay and calculated free or non-SHBG testosterone level. Further, it can be very difficult to determine the assay methodology when ordering these assays.
Ideally, the clinical laboratory should be using the correct methods for all testosterone assays, including those in men, where accurate measurement of non-SHBG bound testosterone is critical for the diagnosis of low testosterone. At the very least, clinical laboratories should default to the LC-MS/MS assay for women and children.
The LC-MS/MS assay takes longer and costs more, which partially explains why less reliable tests have persisted. “The bad assay is very quick. You can run it in an hour, so you get a result back that day,” Dr. Dunaif says. “But what's the point if the result is not informative? I’m sure if health care professionals were aware of these issues, they would insist on the correct assay.” Insurers will typically cover the correct test when it is ordered.
In collaboration with the PCOS patient group called PCOS Challenge: The National Polycystic Ovary Syndrome Association, the CDC held a meeting in August 2024 to discuss its efforts “to develop a plan for creating standardized reference intervals for testosterone in women and adolescent girls using existing data,” to support more accurate diagnosis and effective management of PCOS. Dr. Dunaif was a featured speaker at this CDC meeting. “We hope that the CDC initiative will finally lead to accurate testosterone measurements by clinical laboratories almost 20 years after the Endocrine Society position statement. It is exceptionally disappointing that major clinical laboratories have ignored the Endocrine Society and CDC recommendations. Health care provider education on the correct testosterone assay methodology will be critical for ensuring that clinical laboratories clearly indicate the correct assay methodology in their test ordering options,” says Dr. Dunaif.