K.R. Laumas

In vitro binding of progesterone to receptors in the human endometrium and the myometrium.

High affinity, low capacity progesterone binding receptors have been identified in the cytosol fractions of the human endometrium and the myometrium. The endometrial and the myometrial progesterone binding proteins had sedimentation coefficients of 4.5 S and 4.1 S, respectively. Analysis of the bound steroids revealed that, along with progesterone, small amounts of its metabolites (20alpha-hydroxy-4-pregnene-3-one, 5alpha-pregnane-3,20-dione, 5alpha-pregnane-20alpha-ol-3-one) were also bound to the receptor proteins. Among the steroids studied for ligand specificity, 5alpha-pregnane-3,20-dione showed the highest competition for progesterone binding sites. Progestational steroids, like chlormadinone acetate and norgestrel, did not compete for the progesterone receptors. The endometrial and the myometrial progesterone binding receptors were thermolabile and protein in nature. The molecular weight of the endometrial progestrone binding protein was about 60,000-67,000 with a molecular (Stokes) radius of 33 A and the frictional ratio of 1.26. The myometrial progesterone binding protein had a molecular weight of 56,000-58,000 with a molecular (Stokes) radius of 31 A and a frictional ratio of 1.23. The binding of corticosterone to the myometrial cytosol was only 22-34%, whereas with progesterone it was 70-95%. A study of the immunoabsorption of the plasma proteins from the endometrial and the myometrial cytosol suggested the presence of specific progesterone binding receptors in the cytosol that were different to plasma proteins. The association constant of progesterone for the endometrial progesterone receptor was 1.9 x 10(9) M(-1) and for the myometrial progesterone receptor it was 1.4 x 10(9) M(-1), values that are higher than the association constant of progesterone for corticosteroid binding globulin, which is 7 x 10(8) M(-1). The evidence suggested that the human endometrial and the myometrial progesterone binding proteins are different to the corticosteroid-binding globulin.

Radioactivity in the breast milk of lactating women after oral administration of 3H-norethynodrel.

In an investigation of the excretion of oral contraceptives in the milk of lactating women, tritiated norethynodrel (the progestin component of Enovid) was administered after 2 days of Enovid to 4 patients with stillbirths. An average of 1.1% (range .45-1.52%) of the radioactive norethynodrel was excreted in the breast milk; on the basis of 5.0 mg of norethynodrel per Enovid tablet this amounts to an excretion of 55 mg of the progestin and its metabolites. Since such products could affect the infant, caution is urged in administration of oral contraceptives during lactation.

Long-term contraception by steroid-releasing implants. II A preliminary report on long-term contraception by a single silastic implant containing norethindrone acetate (ENTA) in women

A preliminary report on the long-term contraceptive effectiveness and acceptability of a single subdermal silastic implant containing norethindrone acetate (ENTA) is presented. The 4 types of implants used varied in length, wall thickness, and amount of ENTA; implant A contained 20-25 mg ENTA, implant B contained 30-35 mg ENTA, implant C contained 45-50 mg ENTA as was longer than all the other implants, and implant D contained 40 mg ENTA and had a greater wall thickness than all the other implants. 213 women volunteers received a single implant and were followed for a total of 909 cycles. 2 of 13 women receiving implant A, 2 of 39 women receiving implant B, 3 of 76 women receiving implant C, and none of the 85 women receiving implant D became pregnant. Implant D had the longest expected life-span (10 months). Menstrual irregularities were fewest with implants A (23%) and D (20%), and greatest with implant C (42%). there were no complaints of nausea, insomnia, tender breasts, or loss of libido. 4 patients with implant B and 6 patients with implant C had the capsules removed for medical reasons. More detailed studies of implant D are in progress.

Screening of anti-progestins using in vitro human uterine progesterone receptor assay system.

An in vitro receptor assay system was developed to screen new antiprogestin compounds and to estimate their relative binding affinity for uterine progesterone receptors. 62 steroidal and nonsteroidal compounds including the suspected antiprogestins were screened. Normal human uterine tissues were obtained at hysterectomy operations. Premenopausal women with normal menstrual cycles and without any hormonal treatment were selected for the study. Some synthetic progestin compounds displayed high relative binding affinity (RBA) while others (metabolites of progesterone and norethisterone) showed poor or no binding affinity for human uterine progesterone receptors. Removal of the axial 19-methyl group from progesterone as in 19-norprogesterone resulted in greater binding affinity while the presence of polar groups on the molecule decreased the binding affinity. Of the 30 suspected antiprogestins tested, R2323 showed maximum competition for progesterone binding sites. Biologically, R2323 prevents implantation in women and this is attributed to its high binding affinity for the progesterone binding sites; however, the compound is associated with bleeding irregularities in women. Other compounds with high RBA are STS557 and RMI14156. The study shows that a complexity of factors (e.g., hydrophobicity, hydrophilicity, spatial requirements, optimal contact, and steric hindrances) control the ligand interactions to human uterine cytosol receptors. Screening larger numbers of antiprogestins should be done. As some of the compounds have mixed antiprogestational, antiandrogenic, and antiestrogenic properties, a comparative study on the RBA of a particular compound for the respective receptors should be carried out to better understand the biological profile and molecular interactions of the compounds.

Long-term contraception by steroid releasing implants VI serum concentrations of norethindrone in women bearing a single silastic implant releasing norethindrone acetate

A single silastic implant D containing 40 mg norethindrone acetate was inserted subdermally into 6 normal cycling women of reproductive age. Serum norethindrone concentrations were measured by radioimmunoassay involving an antiserum raised against norethindrone 11alph-hemisuccinate-bovine serum albumin. There was an initial high level of circulating steroid up to 2 months when it declined to 80 ng/100 ml serum in 5 subjects. The other subject had an unusually high serum norethindrone level throughout. The serum levels of norethindrone were elevated again when the 1st implant was replaced with the 2nd. A serum concentration of about 80 ng/100 ml of norethindrone is effective in providing contraception. The initial high release of steroid may contribute to menstrual disturbances experienced by some women.

Characterization of progesterone receptors and metabolism of progesterone in the normal and cancerous human mammary gland

The molecular interaction of progesterone with receptor proteins was investigated in 18 cancerous and 12 normal human mammary gland tissues. The time course analysis of progesterone localization showed that it was initially concentrated in the cytosol and subsequently transferred to the nucleus. In the cancerous tissue the nuclear fraction had significantly higher amounts of the steroid than in the cytosol fraction while in the normal mammary gland tissue localization of progesterone in the nuclear fraction was only slightly higher compared with the cytosolic fraction. In both the normal and cancerous mammary tissue progesterone was bound to a 4.3S receptor but a characteristic 6.7S binding component was noted in the normal mammary tissue. The number of specific binding sites in the normal and cancerous cytosol was of the order of 2.1 and 1.5 pmol/mg protein and the dissociation constant was 0.42 x > 10 -9 10 -9 < M -1 respectively. The binding component was a thermolabile protein but different than plasma proteins. Ligand specificity studies revealed that along with progesterone other progestins compete for the binding sites. In the mammary gland progesterone was metabolized mainly to 20-hydroxy-4-pregnen-3-one 5x-pregnane-320-dione and polar compounds. The breast carcinoma tissue showed higher metabolism than the normal breast tissue. In the breast carcinoma 20beta-hydroxy-4-pregnen-3-one was the major metabolite whereas in the normal mammary gland 20x-hydroxy-4-pregnen-3-one was the major metabolite. (Authors modified)

Progestin and antiprogestin interactions with progesterone receptors in human myomas

Progesterone receptors (PRs) in human fibromyomata (myomas) and normal myometrium were characterized by gel filtration, sucrose gradient sedimentation analysis, isoelectrofocusing and ligand specificity. The PR population in myoma tissues, 1242 ± 505 fmol/mg protein, was 3‐fold higher than normal myometrial tissues under similar hormonal milieu. The increased PR concentration in myomas appears to be of potential importance in physiopathology of fibromyoma.

Evaluation of 15 (S)15 methyl-PGF2α methyl ester suppositories with a two dose schedule for termination of early pregnancy

Abstract The abortifacient effectiveness of 15-methyl-PGF 2α vaginal suppositories in two dose schedules, was tested in forty women for termination of 30–56-day pregnancies. The dose schedule consisted of 1.0 mg or 1.5 mg vaginal suppositories administered 3 hourly for the maximum of 4 suppositories. Success rates were 70 per cent and 65 per cent, respectively. Incidence of vomiting and diarrhea was comparatively higher with the 1.5 mg schedule. Estimation of serum progesterone and HCG was done before the administration of suppositories and two weeks later. A significant decline in the serum progesterone and HCG levels, two weeks post-treatment, occurred in successful cases and further confirmed the effectiveness of the therapy.

Local tissue response to silastic implant containing norethindrone acetate in women

Abstract The effect of a silastic implant made of polydimethyl siloxane tubing and containing 40 mg of norethindrone acetate has been studied in relation to its effect on the subcutaneous tissue of fifty women. Clinically, induration over the implant was detected in only one of them. The histopathology revealed connective tissue response to the silastic implant. The connective tissue showed progressive condensation and maturation of collagen and was associated with chronic inflammatory cell reaction,mainly mononuclear cells and foreign body giant cells. New capillary formation was also seen. However, no anaplastic changes of the fibroblasts were observed.

In vitro biosynthesis of estrogens in placentas from normal and toxemic pregnancies

Abstract In vitro biosynthesis of estrogens in microsomal fractions obtained from placentas from 7 normal and 7 toxemic cases has been investigated under identical experimental conditions. 19-Hydroxyandrost-4-ene-3, 17-dione, androst-4-ene-3, 17-dione, and testosterone were used as substrates and studied for their conversion into estrogens. The conversion of these substrates was found to be low in the toxemic placenta compared to the normal placenta. Kinetic studies with 19-hydroxyandrost-4-ene-3, 17-dione which has been considered to be an obligatory intermediate in the biosynthesis of estrogens showed that in both normal and toxemic placentas the maximum conversion was attained within one hour, and this remained unchanged when incubations were carried out even up to 4 hours. In kinetic studies also the toxemic placenta showed a lower conversion compared to the conversion found in normal cases. Human chorionic gonadotropin was found to have no effect on the conversion of 19-hydroxyandrost-4-ene-3, 17-dione to estrogens in either normal or toxemic placentas. The present finding of low estrogen biosynthesis in toxemic cases is discussed.