Prediction and detection of ovulation by the measurement of urinary pregnanetriol-3 alpha-glucuronide. A multicentre study.

The urine excretion pattern of pregnanetriol 3 alpha-glucuronide (PT-3G) throughout the menstrual cycle in 26 normal ovulating women was evaluated in a multicentre study. The concentration of PT-3G was measured by radioimmunoassay in daily samples of early morning urine (EMU) from 20 women for three consecutive cycles and from 6 women who conceived during the period of study. PT-3G was also measured in 24-h urine samples from 5 additional women. The peak of urine LH was used as a reference point for ovulation (Day 0). The EMU concentration of PT-3G in the follicular phase of 60 normal ovulatory cycles was 5.10 mumol/l +/- 0.11 (arithmetic mean +/- SE). The first PT-3G defined rise (CUSUM analysis) occurred during the late follicular phase (Days -3 to 0) with a PT-3G maximum excretion (9.69 mumol/1 +/- 0.55) on Day 0, whereas a PT-3G excretion peak occurred during mid-luteal phase (Days +5 to +9). The overall PT-3G excretion during the luteal phase (8.06 mumol/1 +/- 0.17) was significantly higher than that of the follicular phase (P less than 0.001). A further sustained increase in PT-3G excretion was noted after day +11 in the conceptional cycles. The 24-h excretion profile of PT-3G was similar to that obtained in EMU samples. No inter-centre significant variation was noticed in terms of PT-3G concentration values. The results were interpreted as demonstrating that the PT-3G excretion profile throughout the cycle exhibits a close resemblance to that of serum 17-OH progesterone. The data also indicates that although the immunoanalytical measurement of this urine steroid metabolite does not give an early sign for the occurrence of ovulation, it can be used for both the immediate prediction and the detection of ovulation.

The radioimmunoassay of pregnanetriol 3 alpha-glucuronide.

The hapten (5 beta-pregnane-17,20 alpha-diol-3 alpha-yl glucuronide) required to develop the radioimmunoassay was synthesized by an unambiguous chemical synthesis. An immunogenic complex was synthesized by coupling bovine serum albumin (BSA) to the glucuronide by mixed acid anhydride reaction. The immunogenic complex was used to induce the formation of specific antibodies in rabbits. In addition, the required radioligand [6,7-3H]5 beta-pregnanetriol 3 alpha-glucuronide was prepared with appropriate specific radioactivity. The characteristics of the antibody were determined with regard to specificity and sensitivity and the precision and accuracy of the assay method established. As applied to urine samples this method is superior to existing procedures in that they avoid the hydrolysis step and can be used directly on diluted urine. Examples have been given of useful application of this technique in clinical practice.

A possible method for the detection of ovulation and the determination of the duration of the fertile period.

PIP: In attempt to find a more reliable and earlier predictor of ovulation, radioimmunoassay methods were investigated which could measure the levels of steroid glucuronides (follicular estrogens), varying levels of which would warn of approaching ovulation well before (up to 96 hours) its occurrence so that abstinence can be practiced during the period of sperm viability before the egg is released. To this end, daily urine collections were made on 9 subjects and the steroid glucuronides were totalled. In addition, the excretion of 5 beta-pregnanediol-3 alpha-glucuronide was measured throughout the menstrual cycle. Measurement of this parameter seemed of more practical value since the ratio of estrone-3-glucuronide/pregnanediol-3 alpha-glucuronide rises during the periovulatory period and falls sharply after the formation of the corpus lutuem. This parameter, unlike measurement of total steroid glucuronides, is not dependent on the rate of excretion or on concentration. In all 9 subjects, a significant increase in this ratio (P .05) was detected 48-120 hours before the plasma lutropin maximum. For assay, this ratio value is easier to assess because the assay kit would be applicable to early morning urine samples and would not involve any measurement of urine volume or time. A short discussion follows the experimental presentation.^ieng

The direct radioimmunoassay of oestrogen glucuronides in human female urine.

Radioimmunoassays for five oestrogen metabolites in urine are described; they are oestrone 3-glucuronide, oestradiol 3-glucuronide, oestradiol 17 beta-glucuronide, oestriol 3-glucuronide and oestriol 16 alpha-glucuronide. These assays have proved accurate and reliable and can be performed rapidly; they have been carried out directly in diluted menstrual cycle urine and pregnancy urine. No sample pretreatment was required. Preliminary results suggest that clinically useful information can be obtained by performing these assays on random urine specimens.

The radioimmunoassay of steroid glucuronides. The oestrogen C-3 glucuronides as haptens.

Antisera were prepared against three related oestrogen ring-A glucuronides, oestrone 3-glucuronide, oestradiol 3-glucuronide and oestriol 3-glucuronide. The corresponding 6,7-3H-labelled conjugates were synthesized as radioligands and the cross-reactions of the antisera against ring-A oestrogen glucuronides and other steroid conjugates were examined. The specificity of the antiserum against oestriol 3-glucuronide was compared with that raised against oestriol 16alpha-glucuronide, and the measurement of the former conjugate in late-pregnancy urine is discussed.

The incidence of cestradiol receptor sites in breast tissues.

PIP: Portions of tumors from 41 patients with benign breast disease, 105 with breast cancer, and 6 of normal breast tissue were studied. Tissues were stored up to 1 month at -20 degrees C in air-tight containers prior to assay. Of the 41 benign tumors 3 had measurable receptor sites. 69 of the 105 cancers showed receptor sites, mostly of a higher order than the benign group. There was a statistically significant difference (p= .02) between the binding site concentration in the malignant tumors from pre- and postmenopausal patients. Concentrations were greater in the postmenopausal patients. The addition of increasing concentrations of dithioerythritol (DTT) in the buffers was found to enhance progressively the affinity for binding in 4 of tumors. In the 6 normal breast tissues, 2 sowed binding with DTT but none by the standard method. Using DTT to increase the sensitivity of the method may improve the prediction of later response to hormone manipulation.^ieng

Oestradiol receptors in carcinoma and benign disease of the breast: an in vitro assay.

An assay is described for measuring the concentration of specific, high-affinity oestradiol receptors in the cell supernatant fraction of breast tumour biopsies. The method has been applied to biopsies from 94 patients with malignant and benign diseases of the breast. Of the 53 biopsies classified as carcinomas, 37 contained high-affinity oestradiol receptors in concentrations ranging from 0·3-22·6 × 10(-15) moles/mg. tissue, 2 were borderline, and 14 did not contain any receptor. The proportion of positive results and the range of concentrations were found to be somewhat higher in postmenopausal than in premenopausal patients. Despite detailed examination, no histological feature was found which could explain the variation in receptor concentration; neither could it be accounted for by differences in the cellularity of the biopsies. Of the 41 benign breast biopsies examined only 3 contained any high-affinity oestradiol receptor and in these the concentration was very low, ranging from 0·3-0·6 × 10(-15) moles/mg. tissue. The receptor has not been detected in normal breast tissue. The relationship between the presence of oestrogen receptors and hormone responsiveness in tumours is discussed.