Introduction of Hair Collection Methods and Hair Progesterone Immuno-Assays Protocol

• In Event: Poster Session 14
  In Poster Session: PS 14 Topic - Methods, History, Theory

Abstract

Background. Saliva is a widely used noninvasive biofluid for measuring pubertal hormones, including progesterone. One pressing limitation is that salivary hormones fluctuate across moments, making it difficult to capture stable hormone levels. This limitation is especially problematic during developmental periods such as puberty and adolescence as these stages are marked by dramatically changing hormones across several time scales (e.g., momentary, diurnal, menstrual, saltatory). Hair assays are a promising biospecimen for basal hormones; hair provides a cumulative index that reflects the prior 1-5 months of hormone exposure (depending on the length of clippings). Emerging research has started integrating hair sex hormone assays (such as DHEA, testosterone and estradiol) into developmental studies. As this biospecimen gains traction, it is important to pay attention to method validation. To refine sample collection methodologies, we provide novel hair collection methodologies which are feasible for children and adolescents based on participants’ specific hair condition. Next, we validate an immunoassay protocol for progesterone measurement from hair given that progesterone is an important sex hormone for puberty/adolescent development.
Methods. For hair collection methods, participants with different hair conditions and types (long vs. short; straight vs. curly, African-American hair styles) were recruited as models for high-quality hair sample collection videos. Collection methods such as small bits, straight line, and tiny snips were demonstrated in a manner so that sufficient sample volume is generated for 4+ hormones (including progesterone) without undue participant burden or embarrassment. For hair progesterone protocol, 3-cm segments were washed twice in 5mL isopropanol for 3 min to eliminate impurities and then allowed to dry under forced air. Ten milligrams of hair were weighed and ground for 8min at 30 Hz. Powdered hair was extracted with 1.5mL methanol and incubated for 18 hours at room temperature with constant shaking. Samples were centrifuged at 5000 rpm for 5min to pellet the sample, and then 1 mL of supernatant was transferred to a new cryovial and dried down using a nitrogen evaporator. The steroid extract was reconstituted in assay diluent, put in 37oC water bath for 30min, vortexed for 1min, and assayed immediately using commercially available enzyme-immunoassays for progesterone.
Results. We assayed hair progesterone using 25mg, 15mg, 10mg, and 5mg of sample (N=6 samples, 4 females, 2 males). For each quantity, hormone was within assay range of sensitivity and there was a linear increase in progesterone of heavier samples (R2=.946). Samples were subsequently assayed with 10mg. Linearity was determined by assaying serial dilutions and spike-and-recovery assessment of 10mg samples and calculated as [observed-expected/expected]. For serial dilution, samples fell on average 5.43% above expected values. For spike-and-recovery, samples fell on average 4.62% above expected values.
Discussion. Hair collection videos are helpful for researchers to learn valid collection methods, opening the possibility for hair self-collection and with sufficient volume for multiple hormones. Results are encouraging that hair progesterone reflects the cumulative hormonal concentration and can be used as a stable hormonal index for both sexes. This opens the possibility for integrating hair progesterone into future psychobiological investigations during critical developmental periods.

Authors

• Wen Wang, Iowa State University
  Presenting Author

• Lotte van Dammen, Iowa State University
  Non-Presenting Author

• Shannin Nicole Moody, Iowa State University | Louisiana State Univeristy HSC
  Non-Presenting Author

• Allissa Van Steenis, Iowa State University
  Non-Presenting Author

• Elizabeth A. Shirtcliff, Iowa State University
  Non-Presenting Author