Urinary pregnandiol-3glucuronide and estrone conjugates to creatinine ratios in early pregnancies complicated by vaginal bleeding.

There is no simple and rapid test available to predict the outcome of an early pregnancy complicated by vaginal bleeding. In this prospective study, 15 women with normal pregnancies collected a weekly urine sample between 6 and 13 weeks’ gestation. A single random urine sample was obtained from 15 women with bleeding who continued to carry their child and 50 women who proceeded to have a spontaneous abortion (SAB). Pregnandiol-3-glucuronide (PDG) was determined with the use of enzyme-multiplied immunoassay technique (EMIT) and estrone conjugates (E1C) were measured by radioimmunoassay (RIA).
The ratios of these metabolites to creatinine (C) were calculated. PDG/C ratios in normal women rose gradually from 6 weeks on. All women with bleeding during a normal pregnancy had ratios in the normal range, but 94% of women with a SAB had ratios below the normal range. The E1C/C ratio remained unchanged from 6 to 11 weeks and then rose rapidly. Until 11 weeks, there was no clear separation between the E1C/C ratios of the women with a SAB and the women with bleeding who continued their pregnancies. The prognosis of threatened abortion can be made by a urinary PDG/C ratio but not by an E1C/C ratio. EMIT is simple and quick and uses technology present in many laboratories.
Journal: Fertility and Sterility

October/15/1987
A simple, rapid, and sensitive solid-phase immunoassay procedure for the determination of estrone-3-glucuronide (E1-3-G), which uses chemiluminescence as the end point in unextracted morning urine, is described. Thirty-one patients undergoing induction of ovulation in an in vitro fertilization (IVF) unit participated in the study. From day 3 of the menstrual cycle until the day of hCG administration, morning blood samples and morning urine specimens were collected for the determination of serum 17 beta-estradiol (E2) and urine E1-3-G, respectively. A good correlation was noted between E2 measured by radioimmunoassay (RIA) and the E1-3-G measured by chemiluminescence immunoassay (CIA), from day 5 up to the day of hCG administration (0.6 less than r less than 0.85, P less than 0.001). It is evident from this study that the CIA measurement of E1-3-G in morning urine is an accurate and rapid (2.5 hours) method and is convenient for monitoring induction of ovulation with human menopausal gonadotropins.

The development of over-the-counter (OTC) assays for pregnanediol-<em>3</em>-<em>glucuronide</em> and <em>estrone</em>-B,D-<em>glucuronide</em>.

The development of simple tests for estrone-B,D-glucuronide (E1G) and pregnanediol-3-glucuronide (P3G) in urine is described. The haptens P3G and E1G, coupled to bovine serum albumin (BSA), were used as immunogens against which specific monoclonal antibodies were made by fusion of variants of P3.X63.Ag8.653 with spleen lymphocytes from immunized mice. When covalently bonded to gelatin or BSA and passively adsorbed to a microtiter plate, the hapten provides the solid phase for an ELISA. Peroxidase-labelled monoclonal antibody is premixed with a urine specimen and the mixture is immediately added to the plate.
After a brief incubation and washing, a mixture of tetramethylbenzidine chromogen and hydrogen peroxide is added to serve as substrate and generate color. The ELISA can be used to monitor levels of E1G and P3G during menstrual cycles and provides a simple, noninvasive method which can be used in a laboratory. A similar competitive assay can be performed using colloidal gold as the label instead of peroxidase. The replacement of peroxidase by colloidal gold further simplifies the procedure and could be used as the basis for an OTC test.

Correlation between plasma estradiol and <em>estrone</em>-<em>3</em>-<em>glucuronide</em> in urine during the monitoring of ovarian induction therapy.

An improved radioimmunoassay was developed to determine estrone-3-glucuronide in daily urine. Resulting levels were compared with those of estradiol in plasma of 10 healthy women and 14 undergoing ovulation induction therapy with human menopausal gonadotropin and human chorionic gonadotropin. A highly significant correlation between plasma estradiol and urinary estrone-3-glucuronide in normal (r = .9209; P less than .01) and stimulated (r = .9229; P less than .01) women was demonstrated.
These results proved that the pattern of excretion of estrone-3-glucuronide perfectly reflected the changes in plasmatic estradiol levels when monitoring ovarian induction and that estrone-3-glucuronide determinations can provide clinically useful information in human induction therapy.

Measurement of <em>estrone</em>-<em>3</em>-<em>glucuronide</em> and pregnanediol-<em>3</em> alpha-<em>glucuronide</em> in early morning urine samples to monitor ovarian function.

The determination of the concentration of estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide has been performed by a chemiluminescent immunoassay in early morning urine samples of 14 normal menstruating women and 11 women affected by luteal phase defect. The early morning urine samples were daily collected for an entire menstrual cycle. We have employed a timed and measured volume collection procedure as correction factor. The integrated values of the hormonal data in definite time intervals were used to create a nomogram. By means of this method, it was possible to completely separate normal from luteal insufficiency subjects and to distinguish two different types of luteal phase defects.
Moreover, the same approach was applied to the study of the role and the frequency of luteal phase defect in 15 patients affected by habitual abortion and in 17 premenopausal women who had undergone quadrantectomy for T1a No Mo breast cancer. A luteal phase defect was detected in nine of the aborting patients (60%) and in eight women affected by breast cancer (47%). Finally estrone-3-glucuronide was measured in early morning urine samples of 96 prepubertal and pubertal girls in different pubertal stages and in one patient affected by precocious puberty, before and during an agonist GnRH treatment. The urinary test of ovarian function seems to be suitable for diagnostic purposes and for clinical studies.

Use of a monoclonal antibody to <em>estrone</em>-<em>3</em>-<em>glucuronide</em> in an enzyme-linked immunosorbent assay (ELISA).

A direct urinary ELISA for estrone-3-glucuronide has been produced following cloning and characterisation of a monoclonal antibody to the above estrogen metabolite. The ELISA follows our established pattern of absorbing a thyroglobulin conjugate, to which estrone-3-glucuronide has been coupled, to the wells of a microtitre plate using guanidine hydrochloride. A competition reaction between either standards/samples and the adsorbed hormone compete for antibody combining sites.
The assay is completed by addition of an anti-mouse Ig-peroxidase complex and read at 492 nm following additions of O-phenylenediamine substrate in under 4 h. The correlation between urinary “total estradiol” and “total estrone and estradiol” is very good and, in conjunction with our ELISA for pregnanediol glucuronide, has allowed for the improved clinical management of infertile and subfertile women.

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Determination of the time of ovulation in chimpanzees by measurement of LH, <em>estrone</em> sulfate, and pregnanediol <em>3</em> alpha-<em>glucuronide</em> in urine: comparison with serum hormone patterns.

The concentrations of LH, total estrogens, and pregnanediol 3 alpha-glucuronide (PdG) were determined by specific radioimmunoassays on daily overnight urine samples obtained in 13 menstrual cycles of six adult female chimpanzees during the periods of increasing, maximal, and decreasing tumescence of the perineal sex skin.
The peaks of estrogens and LH and the rise in PdG in urine accurately reflected the peaks of estradiol-17 beta and LH and the subsequent rise in progesterone in the serum of the same animals during the same menstrual cycles, and can be used to predict and verify the occurrence of ovulation, thus avoiding the repeated tranquilizations necessary to obtain daily blood samples.

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