Researchers have devised a cheap, non-invasive and specific means to monitor progesterone production in women. The new technique uses saliva and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The research was published in the Annals of Clinical Biochemistry.
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The scientists circumvented the need for limited-specificity immunoassays by employing LC-MS/MS which provides the added advantage of being high-throughput. This novel assay enables profiling of progesterone, an indicator of ovulation, throughout menstruation; a contributor to successful means of both assisted and non-assisted reproductive channels.
Progesterone levels are a strong indicator of peak fertility and are essential for pregnancy. As such, monitoring of progesterone concentrations is a crucial part of reproductive therapies and a means to reduce the incidence of preterm births.
Current techniques rely on multiple repeated blood sample collection from women under the surveillance of clinicians. Thus, the use of saliva abrogates the need for clinical personnel, which presents both an effective, cost-cutting and non-invasive means of self-administered, multi-sample acquisition.
Immunoassays versus LC-MS/MS
Traditional, commercially available quantification methods of progesterone rely on immunoassays which suffer from limited specificity. LC-MS/MS represents an attractive means of preventing this, yet its application has been stymied by the lengthy sample preparation and processing times required. Thus, the need for a high-throughput and reliable assay, as developed by researchers from the University of Birmingham, present an improved means of future progesterone profiling.
Prior research has indicated that saliva, in comparison to blood, provides a more accurate measurement of the bioactive form of progesterone, accurately reflecting the free concentration of the hormone which is strongly correlated with total concentration during pregnancy.
Previous salivary assays have been used to diagnose birth defects and have therefore been suggested as a biomarker for preterm birth.
The team, led by Professor Schiffner and colleagues have developed a single preparatory step, followed by a high-speed LC-MS/MS assay for the quantification of progesterone in whole saliva samples. Validation of the analytical performance demonstrated the method’s suitability for routine use, measuring progesterone throughout the menstruation period.
Salivary samples are an ‘advantageous’ alternative
The method was developed and tested on nine saliva samples from healthy women with regular menstrual cycles, taking no oral contraception. Participants were asked to avoid brushing teeth prior to sample collection to avoid blood contamination (the latter is known to falsely elevate salivary steroid levels). Samples were subject to a single step requiring only liquid-extraction for the isolation of progesterone which was reconstituted in 40% ethanol.
The researchers subsequently performed ultra-high-pressure liquid chromatography (UPLC) which was subsequently coupled with tandem MS (MS/MS). The potential for progesterone concentration falling below the limit of detection and the absence of standardization of samples was overcome by the LC-MS/MS assay which provides the specificity and selectivity necessary for routine application.
Stringent validation of the analytical performance of the devised LC-MS/MS assay was carried out to ensure accuracy and precision.
No interference from steroid-related compounds
The assay demonstrated high selectivity and was free of impaired detector response (known as ion suppression) which subsequently indicated intra- and inter-assay precision. Moreover, samples remained stable following reconstitution.
The one-step sample preparation of the whole saliva enabled rapid processing, with a run time of 5.5 minutes. The success of the LC-MS/MS assay was reflected by the observed increase in the salivary concentration of progesterone in seven of the nine samples during the luteal phase, concurrent with peak ovulation mid-menstrual cycle.
Maximal progesterone concentrations fell within the limits imposed by the standard concentration. The assay successfully demonstrated its application in high-throughput monitoring of physiological progesterone concentrations at several points during the menstrual cycle in a non-invasive manner. Crucially, unlike immunoassays, the LC-MS/MS assay demonstrates no interference by steroid-related compounds.
In addition, some samples reached a progesterone concentration below the lower limit of quantification (LLOQ) (20 pmol/L) at certain time points of the menstrual cycle. The researchers indicate, however, that the small sample size (n=9) subsequently precludes the definition of a healthy progesterone range and natural variations.
The development of the high-throughput LC-MS/MS salivary progesterone assay by Professor Schiffer and colleagues lays the groundwork for the development of robust reference ranges for progesterone concentrations throughout the menstrual cycle from large population-wide studies.
It is hoped that the technique will be extended to include application in disease therapy – specifically in congenital adrenal hyperplasia, where progesterone is a biomarker for inadequate steroid replacement therapy.
A novel high-throughput assay for the measurement of salivary progesterone by liquid chromatography tandem mass spectrometry. Annals of clinical biochemistry. 2018. DOI: 10.1177/0004563218780904
This study had full ethical approval from the Science, Technology, Engineering and Mathematics Ethical Review Committee of the University of Birmingham.