Studies relating to the ovarian monitor : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Abstract

Hormonal data contained on the Melbourne Women's Hospital Menstrual Cycle Database and data collected in the Palmerston North Centre of the World Health Organisation trial were analysed and compared. The analysis of the two sets of data showed that the utilisation of a threshold excretion rate for urinary Pregnanediol or Pregnanediol Glucuronide of 7 μmol 24 hr−¹ was an acceptable marker for the end of the fertile period. The data collected by the women participants in the World Health Organisation Trial also showed that the Ovarian Monitor, a home fertility test, provided the most simple, comprehensive and accurate marker of fertility status available. Lysozymes from several sources were examined as possible replacements for the hen egg white lysozyme in the Ovarian Monitor as a means of reducing the Estrone Glucuronide assay time. Unfortunately, although they were all found to possess a faster initial rate, the clearing curves were also more biphasic making them unsuitable for use in the current end-point assay. These differences were attributed to the presence of electrostatic fields on both the enzyme and the substrate. However, the human lysozyme obeyed second order kinetics for a significant percentage of the twenty minute clearing curve. Thus, the Estrone Glucuronide assay time could be significantly reduced by adapting the Ovarian Monitor to linearise the human lysoyzme clearing curve with an appropriate algorithm. Human lysozyme is very expensive thus, it was necessary to optimise conjugation condition for Estrone Glucuronide using the more economical hen egg white lysozyme. Also, chromatographic conditions for conjugate purification had to be established before the human lysozyme could be conjugated and the viability of the above proposal could be tested. Both the mixed anhydride and active ester conjugation methods were optimised. The most effective purification scheme involved pH 4.3 phosphate buffers using a Mono-S column followed by an Alkyl Superose column.