Amniotic fluid, with its nutritive and protective functions, is essential for fetal well-being and development ¹ . It is contained by the amniotic cavity enclosed by the amnion and chorion ² . Amniotic fluid originally comes from maternal plasma and passes through the fetal membranes, being fueled by secretions from the fetal respiratory and gastrointestinal tract, from the oral cavity, and from fetal skin before keratinization occurs ^{1, 3– 5} . Furthermore, cells from the amnion layers secrete proteins into the amniotic fluid ² . At the beginning of pregnancy, the biochemical composition of human amniotic fluid resembles fetal extracellular fluid, probably due to passage across the still unkeratinized fetal skin ⁶ . Given the interplay between amniotic fluid and fetal physiology, amniotic fluid has been used as an important source of potential biomarkers for fetal pathologies ⁷ .

Fetal endogenous steroids, including cortisol, cortisone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), estradiol, estriol, and testosterone are involved in broad and essential physiological functions, including glucose- and energy-metabolism, development, timing of pregnancy, adaptation to the extra uterine environment, and immune processes [e.g. 8, 9]. Dysfunction or dysregulation of underlying hormone systems (e.g. the hypothalamic-pituitary-adrenal axis) is seen in major endocrine diseases (e.g. Cushing’s syndrome, Addison’s disease) ^{10, 11} and non-endocrine disorders (including mental disorders and stress-related conditions) ^{12– 16} . Further, information on prenatal steroid hormone concentrations is of relevance for elucidating fetal origins of health, disease, and variation in mental processes ^{17– 24} . Hence, the assessment of these hormones is of clinical and scientific relevance for a range of different disciplines, including endocrinology, psychiatry, psychobiology, psychology, neonatology, pediatrics, gynecology, and internal medicine [e.g. 22].

Therefore, there is a strong need for powerful and easily applicable analytical procedures for research as well as diagnostic purposes, to assess fetal steroids in amniotic fluid, overcoming previous limitations of sensitivity and specificity. However, as yet, respective methods are scarce. Steroid hormones can be quantified by traditional immunoassays but with several limitations as a lack of sensitivity making the quantification of small amounts of steroids difficult or a lack of specificity due to antibody cross-reactivity resulting in results higher than true concentrations ²⁵ . The presence of interfering substances such as autoantibodies, exogenous substances, hemolysis or lipaemia in patient samples can alter the measurable concentration of the analyte ²⁶ . The matrix effect problems also affect immunoassays. Furthermore, a relatively large sample volume is required as immunoanalysis provides only single-analyte assays and this is particularly a problem for precious samples such as amniotic fluid. Mass-spectrometric techniques are an alternative to address immunoassays issues. It allows for highly sensitive and specific simultaneous assessment of multiple biomarkers, for example in the context of metabolic and proteomic profiling in amniotic fluid ^{7, 25} . Recently, first studies reported on the successful application of different mass-spectrometric approaches, including gas-chromatography mass spectrometry (GC-MS) and liquid-chromatography tandem mass spectrometry (LC-MS/MS), for concomitant assessment of multiple selected steroids in amniotic fluid ^{26– 28} .

The main objective of this study was to develop highly sensitive and specific analytical ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods for determination of cortisol, cortisone, DHEA, DHEAS, estradiol, estriol, and testosterone concentrations in human amniotic fluid, and provide proof-of-principle for the method’s applicability in amniotic fluid samples of pregnant women, collected within obstetric care.

We developed an analytical method to simultaneously measure cortisol, cortisone, DHEAS, and testosterone, and an analytical method to simultaneously measure estradiol and estriol in human amniotic fluid, using an LC-MS/MS assay, with good linearity, sensitivity, specificity and accuracy without interferences between the molecules and a low LOQ. Five out of the seven hormones were quantifiable in all amniotic fluid samples, while DHEA was quantifiable only in a few samples and testosterone was primarily quantifiable in samples from women carrying a male fetus.

The results add to previous findings that indicated that different mass-spectrometric approaches, including gas-chromatography mass spectrometry (GC-MS) and LC-MS/MS, can be applied for assessment of steroids in amniotic fluid ^{27, 28} .

Comparisons of our results on steroid concentrations in human amniotic fluid with data from previous studies are hampered by substantial heterogeneity across previous reports. For example, even when restricting comparisons to findings also based on LC-MS/MS, cortisol concentrations in human amniotic fluid have previously been reported as higher ²⁸ as well as substantially lower ²⁹ than the here reported values. Reasons for such heterogeneity may include – besides the analytical procedures themselves – differences across studies with regard to i) indication for amniotic fluid collection, ii) gestational age at collection, iii) other sociodemographic or obstetric characteristics related to the pregnancy, iv) procedures applied for amniotic fluid collection and storage, and v) the interval between collection and biochemical analyses of amniotic fluid [cf. 1, 29– 31]. Overall, the here reported concentrations are compatible in magnitude with previous findings on cortisol, cortisone, DHEA, DHEA-S, and testosterone concentrations in human amniotic fluid, assessed via mass spectrometric approaches ^{27, 28} , and with previous findings on estradiol and estriol concentrations in human amniotic fluid, assessed via immunoassay-based approaches ^{32, 33} (we are not aware of any respective information from previous studies using mass spectrometric approaches).

Our study has several strengths. We developed analytical methods to quantify a range of steroid hormones in human amniotic fluid with low minimal amount of sample and a total analytical run time of 11 minutes, allowing simultaneous assessment of several hormones. Using state-of-the-art approaches, we confirmed high sensitivity and specificity, good accuracy and reproducibility of the analyses. Notably, the UPLC-MS/MS methods show no interference in contrast to many immunoassays. Last but not lest, by providing proof-of-principle for using the methods to quantify hormone concentrations in human amniotic fluid samples, we open the way for their further development for a wide range of potential applications in scientific and clinical settings.

Our study has several limitations. First, for one out of the seven analytes, we were unable to quantify the steroids in all samples and for another one out of the seven analytes, we were unable only in samples from females to quantify the steroids, most likely because concentrations in the respective samples were below our LOQ or even LOD. For DHEA, the analytical method was not sufficiently sensitive to detect a concentration under 0.75 ng/ml. Although recoveries were greater than 80%, detection limits of the UPLC-MS/MS system have been reached for this molecule. The analytical study of steroids in amniotic fluid of midgestation made by Fahlbusch et al. showed DHEA concentrations of 0.64 ± 0.48 ng/mL for male group of fetuses and 0.56 ± 0.36 ng/mL for female group of fetuses ²⁸ . According to these results, LOQ should be at least 0.1 ng/mL to detect DHEA in 275 samples analyzed. Similarly, the lack of analytical sensitivity also affects testosterone results. According to Fahlbusch et al., testosterone concentrations in amniotic fluid were 0.30 ± 0.15 ng/mL for male group of fetuses and 0.02 ± 0.02 ng/mL for female fetuses ³⁰ . Kushnir et al. determined serum pediatric references ranges below 0.37 ng/mL for males aged 6 to 24 months and below 0.09 ng/mL for females of similar age ³⁴ . In the same, Soldin et al. showed testosterone concentrations in serum range from 0.04 to 0.31 ng/mL for males (0–6 years) and range from 0.02 to 0.1 ng/mL for females (0–5 years) ³⁵ . In view of these studies, the LOQ of our method was not sensitive enough to detect a low concentration of testosterone in amniotic fluid particularly for female fetuses. However, identifying a concentration as being below LOQ may also be of diagnostic value. Second, timing of amniotic fluid collection was not evenly distributed across pregnancy, so we cannot draw conclusions regarding changes in amniotic fluid steroid concentrations throughout gestation. Third, amniotic fluid samples were not immediately assessed after collection, so we cannot exclude degradation of analytes between sample collection and analysis, even though storage conditions would have made substantial degradation rather unlikely. Finally, as amniotic fluid is not routinely collected during every pregnancy, we cannot assume that our sample of study participants is representative with regard to the population of pregnant women. However, we drew our participants from women, undergoing amniotic fluid collection during obstetric care just for the reason that the pregnant women were above 30 years of age. Hence we expect our sample of study participants being largely comparable to a typical sample of women undergoing amniotic fluid testing.

Future studies should further address the role of storing conditions and time interval between collection and analyses. Moreover, future studies should aim at further increasing the sensitivity of the methods. Finally, further studies should test the functional significance of the methods and provide data on their validity and applicability in clinical and research settings.

The methods may provide new opportunities for future applications in a range of fields, such as endocrinology and beyond, providing a tool to determine steroid concentrations in pathological conditions characterized by disturbances of steroid hormones and related enzymatic activities.

We here demonstrated that cortisol, cortisone, DHEAS, estradiol, estriol, and testosterone concentrations are easily detectable and quantifiable in human amniotic fluid, using state-of-the-art and reproducible LC-MS/MS techniques with good linearity, sensitivity, specificity, and accuracy without interferences for the molecules and with a low LOQ. The methods may provide an appropriate approach for a wide range of clinical and research applications, including the field of endocrinology and others.

Individual (non-aggregated) data cannot be made publicly available, due to ethical restrictions. In order to access this data, data must be requested from the corresponding author. Data requestors will have to provide: i) written description and legally binding confirmation that their data use is within the scope of the study, as outlined in the ethical board request and written informed consent (respective information will be provided by the corresponding author on request); ii) detailed written description and legally binding confirmation of their actions to be taken to protect the data (e.g., with regard to transfer, storage, back-up, destruction, misuse, and use by other parties), as legally required and to current national and international standards (data protection concept); and iii) legally binding and written confirmation and description that their use of these data is in line with all applicable national and international laws (e.g., the General Data Protection Regulation of the EU). In case of requests of hormone data only, less restrictive regulations may apply.