1. Lessons Learned/Challenges Corticotropin Releasing Hormone (CRH)

2. Why Measure CRH? 41 amino acid neuropeptide, plays a major role in the physiological response to stress The principal hypothalamic regulator of the pituitary-adrenal axis Higher levels of CRH associated with adverse pregnancy outcomes

3. What Medium and Collection Method to Use? Medium – Plasma Collection Method – Collection tube EDTA Protease inhibitor – Time Timing of sample collection – no circadian rhythm Effects on plasma samples-process within 12 hrs Timing during pregnancy – Temperature Continuously chilled at 4°C

4. Considerations when Processing Sample Processing – Refrigerated Centrifugation 3,000-4,500 rpm for 15 minutes Aliquots siphoned and analyzed or stored – Storage -80°C for < 2 years – Plasma extraction Removes CRH from plasma Methanol vs C18 Sep-Column Immediately prior to assay

5. Factors in Selection of Assay Factors to consider – Cost – Ease – Reliability/sensitivity of tests ELISA vs RIA – Elisa (easier, rapid turnover time) – RIA (well documented in literature, greater sensitivity)

6. Lessons Learned Challenges Designing research study – Timing of collection in relation to EGA – Stratifying for ethnicity/race Identify published protocol, consult with author/experts Consider role of lab manager Evaluate each plate result with consultant- assess standardized curve Which method of assay?

7. References Glynn, L.M., Dunekl Schetter, C., Chicz-DeMet, A., Hobel, C.J., & Sandman, C.A. (2006). Ethnic differences in adrenocorticotroic hormone, cortisol and corticotropin- releasing hormone during pregnancy. Peptides, 28, 1155- 1161. Latendress, G. & Ruiz, J. (2008). Bioassay research methodology: measuring CRH in pregnancy. Biological Research for Nursing, 10, 54-62. Strong, E.F., Kleinman, K.P., Gillman, M.W. et al. (2006). Measuring corticotropin-releasting hormone in pregnant women-comparing blood collection protocols for epidemiological studies. Paediatric and Perinatal Epidemiology, 20, 67-71.