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Title: A prototype for ovulation detection: pros and cons.
Author: Lasley BL, Shideler SE, Munro CJ.
Journal: Am J Obstet Gynecol; 1991 Dec; 165(6 Pt 2):2003-7. PubMed ID: 1755459.
Abstract:
A noninstrumented enzyme immunoassay for urinary estrone conjugates was adapted from an instrumented microtiter plate enzyme immunoassay assay. The end point of the assay was a color change from green to clear, which was visible to the unaided eye. The visible color change was adjusted to allow 80 ng/ml estrone conjugates (on the basis of a sample size of 6.5 microliters urine) to be distinguished from an infinite dilution without instrumentation. The evaluation of human urine collected from ovulatory ovarian cycles demonstrated that early follicular phase concentrations (35.9 +/- 6.8 to 79.4 +/- 14.7 ng/ml, n = 10) produced a dark-green color, whereas late follicular phase concentrations (162.9 +/- 20.1 ng/ml, n = 10) produced no color. Daily urine samples throughout 10 ovulatory ovarian cycles produced parallel profiles when compared to measurements of estradiol in paired blood samples. Complete analysis of the data indicated that ovarian follicular dynamics can be accurately monitored through the noninstrumented analysis of daily estrone conjugates in urine samples. The purpose of this study of the timing of ovulation was to evaluate and compare the results of estrogen concentrations in serum using radioimmunoassay (RIA) with urine samples assessed by instrumented (EIA) and noninstrumented enzyme immunoassays (NEIA). This was done to determine the timing before ovulation of the 1st enzyme rise. The advantage of urinary analysis over serum analysis is that subjects can collect and freeze samples at home, toxic regulated substances are eliminated, time and cost is reduced, and the design allows for a larger population sample size. The study population was a group of women 23-40 years with normal menstrual cycles. Early morning urine samples and midmorning blood samples were obtained for 1 complete menstrual cycle. Reanalysis of 6 of the refrozen urine samples was made using NEIA. Diagnostic Product kits were used to establish ovulatory cycles, and Munro et al laboratory methods were used to analyze urinary estrone conjugated (EIC) and progesterone (PdG). The methods of Taussky was used to measure creatinine. The noninstrumented EIA was similar to the EIC EIA format of Czekala et al and the materials reported by Munro et al. However, there were 3 changes in the microtiter plate EIC EIA format: 1) high binding star tubes were substituted for the microtiter plate, 2) the EIC enzyme label was altered by eliminating the glucuronide moiety, and 3) small Whatman No. 1 filter paper pads (7.5 mm diameter) were used to measure and transfer urine samples (storage at 4 degrees Centigrade and dried completely). Reliability was comparable to micropipettors. The process is described. The results were that an increase of serum estradiol (E2) concentrations accurately and consistently predict the occurrence of future ovulation. It was also demonstrated that changes in urinary estrogen excretion can be used to predict ovulation, and can be detected with either EIA or NEIA. The rise in urinary EIC was detected by EIA (35.9 + or - 6.8 to 70.4 + or - 14.7 ng/ml and dark green color in the early follicular phase) and NEIA (70-80 ng/ml) between 6-2 days prior to the midcycle LH peak and comparable to estradiol rises in paired blood samples (r=0.88, p.01). E2 measurements are better predictors but eliminate self evaluation. The detection of the EIC rise (EIC values plus PdG and LH values) provide a complete and comprehensive monitor of all ovarian events of a complete menstrual cycle. The development of a noninstrumented test or urine osmolality will contribute to ending false positives or negatives.

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