Entrapment of a highly specific antiprogesterone antiserum using polysiloxane copolymers.

Antiprogesterone antiserum was entrapped within a polysiloxane copolymer prepared from a 3:1 mixture of tetraethoxysilane and 3-aminopropyltriethoxysilane monomers. 400 microliters of this monomer mixture entrapped 70 mg of the 140 mg of immunoglobulins which were added, and the protein could not be washed from the highly stable copolymer which formed. Approximately half of the entrapped antiprogesterone antiserum was found to retain progesterone binding capacity with an apparent Ka equal to that of free antiserum in solution and was insensitive to effects of pH between 3 and 7. These preliminary observations and the unique chemistry of polysiloxane polymer formation suggest that such polymers may be useful in the entrapment of proteins for a variety of applications.

Reanalysis of antisera specificities and binding characteristics of rat pituitary hormone assays.

Rat pituitary hormone radioimmunoassays (RIAs) are widely used in reproductive research, yet data on specificity and binding characteristics of many of the antisera are not widely available. This report characterizes one set of rat antisera supplied by the National Institutes of Health (USA). Rat follicle-stimulating hormone (FSH) and thyrotropin-stimulating hormone (TSH) antisera appear specific, but TSH exhibited significant competition in the rat luteinizing hormone (LH) assay. In addition, statistically significant nonparallelism was demonstrable in all three assay systems. This creates further problems in characterizing antisera cross-reactivity and may make potency estimates for pituitary standards inaccurate.

Alterations in progesterone metabolism and luteal function in infertile women with endometriosis.

The concentrations of pregnanediol-3-glucuronide (PGD) and pregnanolone (PN) were measured in daily morning urine specimens from 66 infertile women (40 with varying degrees of endometriosis and 26 control subjects) and correlated with daily changes in basal body temperature (BBT) and with midluteal levels of serum progesterone (P). PN and BBT rose at midcycle in women with endometriosis, as expected, indicating secretion of some P at that time. However, PGD, the major endpoint of P metabolism, was delayed in its excretion. Endometrial biopsies were similarly delayed (out of phase) in women with endometriosis, and a significantly higher incidence of follicular luteinization was seen. It appears that while P secretion begins at midcycle, the bulk of P secretion is delayed, perhaps because of the process of follicular luteinization, and that a shortened functional luteal phase thus exists in women with endometriosis.

Relationship of luteinizing hormone, pregnanediol-3-glucuronide, and estriol-16-glucuronide in urine of infertile women with endometriosis.

The concentration of luteinizing hormone (LH), pregnanediol-3-glucuronide (PGD), and estriol-16-Glucuronide (E3G) were measured in daily morning urine specimens from 53 infertile women. In 26 of 29 women with various degrees of proven endometriosis, two distinct midcycle peaks of LH were found 2 or 3 days apart. Patients with LH peaks separated by 3 days had significantly more severe endometriosis than those with a single peak. Maximum concentrations of E3G were found to be delayed until after the first LH peak in these patients, and PGD concentrations did not rise until the time of the second LH peak, making actual luteal function of shorter duration than normal. From the data on LH, it appears that an inappropriate hormonal feedback mechanism is operative in endometriosis.

Ability of implants of polymer-entrapped antiprogesterone antiserum to absorb and deplete progesterone from serum of the pregnant rat.

A highly specific antiprogesterone antiserum (APA) produced by immunization of sheep with an 11 alpha-hydroxyprogesterone hemisuccinate-thyroglobulin conjugate was purified, and the IgG fraction was entrapped within a polysiloxane matrix. The matrix immobilized APA but allowed penetration and binding of progesterone (P) to the APA. In this entrapped form APA implanted intraperitoneally in rats on the tenth day of pregnancy resulted in a decline in serum P from 50 to 12 ng/ml within 12 hours and to less than 2 ng/ml within 36 hours. Free serum P measured by equilibrium dialysis fell to less than 0.2 ng/ml at 36 hours. Concomitant with the decline in serum P was a rise in both serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and eventual fetal resorption.

Identification of a 17-hydroxyprogesterone-binding immunoglobulin in the serum of a woman with periodic rashes.

A 30-year-old patient with primary infertility had a history of recurrent oral and perineal rashes that appeared just prior to the midcycle rise in basal body temperature and subsided with the onset of menses. The rashes did not appear during periods of treatment with oral contraceptives or danazol, and were suppressed by administration of cortisol. Sera obtained during the follicular and luteal phases of her cycle were found to contain a progestin-binding component with high affinity for 17-hydroxyprogesterone and a somewhat lower affinity for progesterone. Analysis of these sera showed the progestin-binding component to have properties of IgG, with an apparent binding affinity for 17-hydroxyprogesterone of 2 X 10(10) M-1. This report is the first characterization of an antibody to an endogenous steroid in human serum.

Effects of antiprogesterone antiserum on serum and ovarian progesterone, gonadotropin secretion, and pregnancy in the rat.

A high affinity antiprogesterone antibody (APA) was tested in both pseudopregnant and pregnant rats for its ability to alter serum and ovarian concentrations of total and free progesterone and serum gonadotropins and for contragestational effects. APA injected ip on the sixth day of pseudopregnancy resulted in rapidly increased levels of total progesterone in serum, but no detectable change in free progesterone as measured by equilibrium dialysis. Serum gonadotropins similarly increased after APA injection. Progesterone concentrations in various body tissues indicated an initial increase in progesterone production followed by a marked decrease apparently in response to induce luteolysis. Similar levels of hormones were obtained in pregnant rats after ip administration of APA on day 10 of gestation. Fetal resorption followed in all animals and was complete within 96 h. The effect of APA on fetal loss appears to be a combination of a direct reduction of uteroovarian progesterone concentration and a negative feedback of progesterone on the pituitary release of gonadotropins, thus resulting in luteolysis and a further reduction in available progesterone.

Radioimmunoassay of pregnanediol concentrations in early morning urine specimens for assessment of luteal function in women.

A radioimmunoassay has been developed that employs a readily available radioligand 3H-20alpha-hydroxy-4-pregnen-3-one for the determination of pregnanediol glucuronide in urine. The unextracted steroid is assayed directly after dilution of urine with the use of an antiserum produced to a thyroglobulin conjugate of pregnanediol glucuronide. The assay has been validated, and the ability to assess luteal function by measurement of the concentration of pregnanediol glucuronide in early morning urine specimens has been demonstrated.