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

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

On the distribution of urine output in normally cycling women

It has been suggested that it is possible to monitor the menstrual cycle by measuring the concentration of urinary reproductive steroids. This neglects the variation in void volume and in urine production rate. In neither case has any systematic analysis been reported previously. Overnight urine samples were collected each day for one complete cycle by 24 women and the void volumes and intervoid times were recorded. The void volume and urine production rate were approximately lognormally distributed and the intervoid time was approximately normally distributed. Using these distributions we consider the implications of the variation in void volume and urine production rate for the comparison of the concentrations of a urinary analyte in two samples

Expressing the quantity of urinary analytes: a discussion of some issues arising from the monitoring of the menstrual cycle

Practical domestic monitoring of the menstrual cycle requires measurements of urinary metabolites of reproductive hormones: oestrone glucuronide (E1G) and pregnanediol glucuronide (PdG). Data reported in the literature are expressed as (i) concentration, without or with either creatinine- or specific gravity correction, or (ii) excretion rates. This variation in such a fundamental issue prompts consideration of the relationships between the four measures. Because the menstrual cycle kinetics of E1G and PdG are complex, we consider measurements of urinary creatinine, urea, galactose, xylose and inulin which tend to be more stable. We show that uncorrected concentration measurements of these urinary analytes can be positively correlated, negatively correlated or uncorrelated with the serum concentration. Based on measurements of urinary creatinine concentrations, urinary specific gravity and creatinine excretion rates, we conclude that urinary analyte concentration are likely to be more reliable when creatinine-corrected rather than corrected using specific gravity, but that both are less reliable than measurements of the excretion rate. This has implications for the quantitation of any urinary analyte, but especially for the monitoring of the menstrual cycle in which changes in E1G and PdG from one day to the next can be physiologically significant for a woman monitoring her fertility

Does the use of ovulation monitors really increase pregnancy rates? Some things women should know

Ovulation monitors are widely used by women wishing to achieve pregnancy. However, there are few data substantiating claims that these devices enhance the probability of becoming pregnant. In one report it is concluded from the cumulative pregnancy rate that the use of the Clearblue Easy Fertility Monitor increased the pregnancy rate. In a second report, it is argued that the use of the Clearblue Digital Ovulation Test reduces the time taken to conceive. We reconsider these previously published data by analysing each cycle and show that use of such devices might have a small effect (ϕ ≈ 0.12, odds ratio = 2.1-2.2, relative risk = 1.9) in the first month of use, but has no significant effect (ϕ ≈ 0.01, odds ratio = 1.2, relative risk = 1.1-1.2) in the second month. However, the subjects recruited for these two trials had single cycle pregnancy rates (7-11%) that were more similar to those of women avoiding pregnancy (about 6%) than women trying to conceive (about 25%). Given this, there is a reason to suspect that the data that are available might not be representative of all women. Further work is required to test whether even this small transient effect can be replicated in women with higher pregnancy rates. Women should be aware of the limitations of these ovulation monitors

The spurious correlation between concentration and creatinine-corrected concentration in urine

The use of urinary analytes to monitor physiological processes relies on making the correct measurement. Three alternatives are commonly contemplated: concentration, creatinine-corrected concentration and excretion rate. Of these, the latter is the most reliable, but is perceived by some to be difficult to measure. This has led to the more frequent reliance on concentration and one of the justifications for this is the reported linear relationship between the concentration and the creatinine-corrected concentration. We show that this correlation is spurious in that the magnitude of the correlation coefficient depends on the ratio of the standard deviations of the creatinine and analyte concentrations. As an example urinary analyte we use pregnanediol (Pd) which is an important tool for women wishing to monitor their own fertility. Urinary Pd concentration is not a reliable substitute for creatinine-corrected Pd concentration or the Pd excretion rate

Does the Use of Ovulation Monitors Really Increase Pregnancy Rates? Some Things Women Should Know

Ovulation monitors are widely used by women wishing to achieve pregnancy. However, there are few data substantiating claims that these devices enhance the probability of becoming pregnant. In one report it is concluded from the cumulative pregnancy rate that the use of the Clearblue Easy Fertility Monitor increased the pregnancy rate. In a second report, it is argued that the use of the Clearblue Digital Ovulation Test reduces the time taken to conceive. We reconsider these previously published data by analysing each cycle and show that use of such devices might have a small effect (ϕ ≈ 0.12, odds ratio = 2.1-2.2, relative risk = 1.9) in the first month of use, but has no significant effect (ϕ ≈ 0.01, odds ratio = 1.2, relative risk = 1.1-1.2) in the second month. However, the subjects recruited for these two trials had single cycle pregnancy rates (7-11%) that were more similar to those of women avoiding pregnancy (about 6%) than women trying to conceive (about 25%). Given this, there is a reason to suspect that the data that are available might not be representative of all women. Further work is required to test whether even this small transient effect can be replicated in women with higher pregnancy rates. Women should be aware of the limitations of these ovulation monitors

Homogeneous and heterogeneous enzymeimmunoassays for the home detection of fertility : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Biochemistry at Massey University

The physiology of the menstrual cycle has been reviewed and the suitability of the ovarian steroid urinary metabolites estrone glucuronide and pregnanediol glucuronide as markers of fertility and as utilised by the home Ovarian Monitor fertility assay was discussed. The biomaterials for the homogeneous enzymeimmunoassay which forms the basis of the Ovarian Monitor home fertility assay were prepared. One of the major difficulties in preparing a signal generator for use in home tests is the separation of the unconjugated enzyme material from the desired signal generators. In this thesis a new procedure was developed for the purification and isolation of the complex range of signal generators formed during acylation of hen egg white lysozyme with estrone glucuronide. A cation exchange column in the presence of 7 M urea allowed the separation to be carried out in the absence of the hydrophobic effects which complicate other schemes. Even under these conditions complex behaviour was seen which could be rationalised in terms of the tertiary structures of the conjugates and their electrostatic fields. A second step involving hydrophobic interaction chromatography gave two pure mono conjugated estrone glucuronide lysozyme conjugates in good yield the activities of which were highly inhibited (>90%) by anti-estrone glucuronide antibodies. The availability of the pure conjugates allowed the effect of tertiary structure on the immunoassays to be evaluated. The results showed that both mono acylated hen egg white lysozymes could be used to give good standard curves for use in monitoring menstrual cycles for the naturally occurring periods of fertility and infertility. Since the specific activity of human lysozyme is three times that of hen egg white lysozyme, in an attempt to provide a more rapid test for fertility human lysozyme estrone glucuronide conjugates were synthesised with estrone glucuronide for the first time. However, despite the fact that these two enzymes had very similar tertiary structures they behaved completely differently in the protein chemistry and immunological experiments reported in this thesis. The human enzyme was more easily acylated to give pure mono acylated conjugates in high yield and the conjugates were more easily purified to give highly inhibitable conjugates (>95%). The differences in behaviour could be accounted for in terms of the sequence differences between the two lysozymes and the relative exposure of the lysine residues. A fast assay (1-2 minutes) was developed for urinary estrone glucuronide using the three new signal generators. However, the sensitivity of the assays was less than half that of the hen egg white conjugates making them unsuitable for use in home assays for fertility. The assays could be useful for women using fertility drugs such as clomiphene. The lack of sensitivity of the assays and other binding behaviour indicated a much tighter binding to the antibody than with the hen egg white conjugates. This important difference was accountable on the basis of the extra extension of the lysine residues in the human enzyme. A new method for producing estrone glucuronide conjugates of the active enzyme horse radish peroxidase was evaluated. The mono substituted hemin conjugates reconstituted with the apo protein to give active peroxidases with good specific activities (~50%) and good stability. The procedure is such that any small molecule can be attached to the enzyme using the same procedures and a large range of signal generators can be formed for immunoassays. However, both assay formats examined failed to produce an assay in this thesis. The reconstituted enzymes, although binding to the immobilised anti- estrone glucuronide antibodies as required did not produce the necessary colours. The reasons for this were discussed

Self-monitoring of fertility hormones: a new era for natural family planning?

Natural family planning (NFP) methods have served many generations well, and in particular, the symptothermal or symptohormonal methods. The comparison of daily mucus and temperature records for individual cycles with daily hormone measurements, which is now possible, shows that some of the assumptions underlying NFP may not be completely accurate. The various methods are inadvertently depending on an element of chance, which, of course, cannot be known by the NFP user. However, it is statistically inevitable that such errors will result eventually in an unexpected pregnancy, and these discrepancies are the likely reason for the method failures. Further research and integration of home hormone measurements with NFP symptoms are needed

Monitoring the menstrual cycle using urinary oestrone glucuronide: the relationship between excretion rate and concentration

Oestrone glucuronide (E1G) is monitored during the menstrual cycle because it reflects the follicular production of oestradiol (E2), the rate of which increases as the follicles grow. Despite several recent assertions to the contrary, the urinary E1G concentration is an inadequate indicator of E2 because it is confounded by variations in the rate of urine production. We show that (a) the serum E2 concentration is proportional to the E1G excretion rate, which is the product of the urinary concentration of E1G and the urine production rate, (b) that it is almost as important to measure the urine production rate as it is to measure the urinary E1G concentration itself, and (c) that the distribution of urine production rate is positively skewed, from which it is inferred that extreme values are more likely than would be expected were the distribution normal. If urinary E1G is to be used to monitor the menstrual cycle it is essential that the concentration is corrected for fluctuations in urine production rate: the E1G excretion rate is reliable, urinary E1G concentration is not

Identifying ovulatory cycles and the day of ovulation by the mis-use of pregnanediol glucuronide excretion rate thresholds

[Extract] Sir In a recent paper in this journal, Rosetta et al. [1] compared methods of identifying (i) ovulatory cycles and (ii) the day of ovulation based on intermittent urinary hormone metabolite measurements using expert assessment of the data as a 'gold' standard. Some of their work is based on our pregnanediol-3-glucuronide (PdG) excretion rate threshold(s) which were developed to (i) identify the end of the fertile window of the cycle and (ii) distinguish between different categories of the menstrual cycle [2]. Several issues arise from their report, but we wish to consider only those concerning (a) the implicit assumption that different measures of PdG are equivalent, (b) the criteria used in the expert assessment and (c) the history of intermittent sampling