Five key points to ensure reliable and accurate saliva testing for female hormones

Saliva-based assessments are a trustworthy and proven method for quantifying female hormone levels while being extremely precise and painless for the patient.1 This article discusses five key steps to keep in mind while setting up saliva-based testing for monitoring and measuring the imbalance of female hormones.

A hormone imbalance test for women can be based around saliva hormone testing. Image Credit: Tecan

Reassure yourself: Saliva-based testing works

While measuring female hormones to identify underlying reasons for female infertility, physicians should be able to measure the female sex hormones’ level throughout the menstrual cycle.

The same approach is followed while solving other hormonal problems, such as the hormone replacement therapy for menopause that requires personalized dosing.

This is because there are huge variations in the hormone concentration at each stage of the cycle, and the timing of menstrual cycle events, which differs in every woman. Differences in hormones are even present throughout multiple cycles for an individual, including occasional non-ovulatory cycles.2

For hormone analysis, recurrent serum sampling would be difficult, invasive and necessitates experts to draw the blood samples. Saliva, however, serves as an outstanding specimen for monitoring progesterone and estradiol levels during the menstrual cycle.

The specimens can even be self-collected on a daily basis at home. Also, the samples can be stored for at least a month in a freezer. With no extreme effects on assay results, the specimens can also be exposed to repeated freezing and thawing.3

Other works reveal that frozen samples can last at –20 °C for up to a year, and perhaps longer, with no remarkable variations in the steroid hormone concentration of samples.4

Image Credit: Tecan

Figure 1 reveals a classic profile acquired from monitoring estradiol and salivary progesterone in whole saliva samples, self-collected during the menstrual cycle, representing the peaks for estradiol and progesterone.

The graph clearly shows that skewed results are achieved even while restricting sampling to every other, proving the value of a straightforward, daily sampling method. Doing venous blood sampling with a daily sampling method to obtain a similar result would be unrealistic.

Figure 1. Representative patterns of salivary estradiol and progesterone levels across the normal menstrual cycle.1 Image Credit: Tecan

The saliva assessments are not only sensitive and accurate but are also outstanding in reliability for measuring free, bioavailable hormones levels.

As already discussed, around 95–99% of steroid hormones present in the blood circulation are bound to carrier proteins. Salivary concentrations provide a better depiction of the actual free hormones circulating levels, as saliva comprises unbound bioavailable hormones.

Collecting sample: The essentials for saliva testing

Initiated with a reliable, standardized sampling technique, saliva-based testing includes a receptacle for the sample. Hence, it can easily be stored in a freezer or fridge.

For maximum sensitivity, the testing lab should assure that the device used has been evaluated to reduce the communications between the salivary analyte being measured and the device surface.

It is crucial that the collection protocol utilized is compatible with the analyte being tested. Certain analytes can be only assessed with a passive drool sample. Employing a swab to quantify these analytes can lead to over-recovery or under-recovery of the measured analyte.

Using a swab that has been authenticated for measuring the analyte of interest is recommended. Swabs of some manufacturers are specific to cortisol and have only been validated for the cortisol measurement. It can lead to erroneous results if this swab is used to measure any other hormone, such as testosterone.

Since polyethylene collection tubes may adsorb steroids, they should be avoided.6 To minimize steroid adsorption issues, polypropylene tubes are recommended. However, cotton-based materials usage can also be challenging.

For instance, although the Salivette® (Sarstedt) sampling tubes’ usage with cotton swabs is agreed for the measurement of cortisol, results acquired for dehydroepiandrosterone (DHEA), progesterone, testosterone and estradiol with this method are highly erroneous.

This is believed to be due to the existence of plant sterols in the cotton, which cross-react in steroid immunoassays.7,8

Care should also be taken to ensure that blood contamination in the sample does not occur as it can skew the results. For instance, increased levels of testosterone are formed for at least 30 minutes post vigorous tooth-brushing.9

Test it out: Check how ELISA works

Compared with serum, steroid hormones exist in saliva in relatively low concentrations, even if the hormone is not bound and is predominantly free. Thus, quantitative assays for the assessment of salivary hormones need to be as sensitive as possible.

ELISA assays can offer the sensitivity needed, but they also need to be standardized within and between testing labs, ideally with an option to participate in inter-laboratory consistency testing as a contributor to the assay’s quality control. This way, a database of sample results can be made as numbers accumulate.

Later meta-analyses are performed, moving saliva-based testing to universal standardization.

Since mass spectroscopy (MS) is the established reference method for hormone quantification, it can be significant to consider whether a given saliva-based ELISA test has been measured against MS results.

This measurement for a given test can give extra assurance, especially when a lab is initiating saliva-based ELISA testing and requires empirical evidence that it is functioning efficiently.10

Saliva-based ELISA tests need to be standardized and reproducible in experimental terms: inter-assay, intra-assay, inter-lab, intra-lab and batch-to-batch, with CVs that are within defined tolerance limits.

Preferably, an ELISA inter-assay CV, whether it is between people, batches or labs, needs to be less than 15%, and the intra-assay CV over triplicates needs to be less than 10%.

Automation can be taken into account if sufficient assessments are performed, for instance, in the 1000s or even more per week, with respect to ELISA plates.

The results given by Automated ELISA tests are usually equivalent to a skilled manual worker while eliminating huge possibilities for human error.

Check the math: Saliva testing is economically viable

Saliva collection is 48% less costly than blood collection when comparing prices that are specific to each method.11 Also, saliva can be home-collected, and the samples are effortlessly stored without any damage, reducing the requirement to repeat sample collections.

This practicality in sample collection is essential for measuring female hormone imbalance, as daily samples are needed, and the measurement’s cyclical nature implies that any missing samples could delay a possible diagnosis and therapy for a further month.

After completing the sample collection, additional costs will be based on the technology platform used (for example, manual versus automated ELISA and ELISA versus MS), although so far the increased cost will be incurred from the clinical consultations and follow-up treatments, and not from the tests themselves.

Although saliva-based testing might be a part of a broader scheme to examine defined conditions, like infertility, the female hormone imbalance testing is not regularly covered by public healthcare or insurance.

In certain cases, where women self-refer, for example, while looking to relieve menopausal symptoms, it can be noted that there is a number of tests available online.

Some of these tests are saliva-based, and other tests are blood-based. Generally, they have a one-off, non-refundable price, that comprises a basic online consultation fee.

Diversify: Use saliva testing for other applications too

Saliva testing is not only practical to set up in the lab, but it is also good to know that the setup is not limited to female hormone levels testing alone.

Saliva can be used to quantify many other biomarkers, which implies the user can capitalize on the investment that is put into infrastructure development, adapting the workflow to include male hormone levels and other steroid types.12

Saliva-based tests have already been developed in several other areas, which include — but are not limited to — psychology, sports medicine and occupational medicine. For example, high levels of cortisol are linked to stress and burnout.

Cortisol was also one of the first markers to be assessed in saliva samples, making it a true target for quantifying stress levels in the workplace.13

It is also noted that many labs that test female hormone imbalance also check for adrenal status as part of an overall hormone health check, and hence, measure DHEA levels and cortisol to provide a complete understanding of a woman’s hormone status.

As a result, there are several overlaps in the various hormonal pathways. However, according to the individual test instructions, the exact clinical application for which a particular test is relevant should be considered.


  1. Gandara, B. K., Leresche, L., & Mancl, L. (2007). Patterns of salivary estradiol and progesterone across the menstrual cycle. Annals of the New York Academy of Sciences 1098, 446-450. PubMed ID: DOI:
  2. Becker, J. B., Arnold, A. P., Berkley, K. J., Blaustein, J. D., Eckel, L. A., Hampson, E., Herman, J. P., Marts, S., Sadee, W., Steiner, M., Taylor, J., & Young, E. (2005). Strategies and methods for research on sex differences in brain and behavior. Endocrinology, 146(4), 1650–1673. PubMed ID: DOI:
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  4. Schultheiss, O. C., & Stanton, S. J. (2009). Assessment of salivary hormones. In E. Harmon-Jones & J. S. Beer (Eds.), Methods in social neuroscience (p. 17–44). Guilford Press.
  5. Bellagambi, F.G., Lomonaco, T., Salvo, P., Vivaldi F., Hangouet, M., Ghimenti, S., Biagini, D., Di Francesco, F. & Fuoco, R. & Errachid, A. (2020). Saliva sampling: Methods and devices. An overview. Trends in Analytical Chemistry. Elsevier. Volume 124, March 2020, 115781 Elsevier ID: DOI:
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  10. Stanczyk, F. Z., & Clarke, N. J. (2010). Advantages and challenges of mass spectrometry assays for steroid hormones. The Journal of steroid biochemistry and molecular biology, 121(3-5), 491–495. PubMed ID: DOI:
  11. Accessed 21 July 2020.
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  13. van den Heuvel, L. L., Wright, S., Suliman, S., Stalder, T., Kirschbaum, C., & Seedat, S. (2019). Cortisol levels in different tissue samples in posttraumatic stress disorder patients versus controls: a systematic review and meta-analysis protocol. Systematic reviews, 8(1), 7. PubMed ID: DOI:

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Last updated: May 6, 2024 at 9:17 AM


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