Placental protein 14 production by human Fallopian tube epithelial cells in vitro.

We studied the in-vitro secretory function of non-polarized and polarized cultured Fallopian tube epithelial cells by measurement of the placental protein 14 (PP14) secretion in primary cultures and subcultures from Fallopian tubes obtained from eight premenopausal women in different phases of the ovarian cycle. Primary cultures were established in minimal essential medium in Earle's salts supplemented with fetal bovine serum and the cells were subcultured for six passages, in the polarized cell cultures, the cells being seeded on an extracellular matrix system. Cell freezing was carried out using 10% dimethyl sulphoxide. PP14 secretion into the culture media was measured by a radioimmunoassay using 125I-PP14 as label and rabbit anti-human PP14 serum. There was a large amount of PP14 secretion into the culture media in primary cultures, the secretion decreasing considerably after subculture 1. PP14 secretion after subculture 2 was not different from the control values. Polarized and non-polarized cells secreted similar amounts of PP14 and frozen-thawed cells did not appear to secrete PP14. Epithelial cells from Fallopian tubes obtained at different phases of the ovarian cycle did not appear to show any difference in PP14 secretion rates. Our data suggest that the in-vitro secretion of PP14 by human Fallopian tube epithelial cells is adversely affected by cell ageing and freezing.

Serum PAPP-A and free beta-hCG are first-trimester screening markers for Down syndrome.

Serum measurements of pregnancy-associated plasma protein A (PAPP-A) and the free beta-human chorionic gonadotrophin (hCG) subunit were made in 13 women with Down syndrome (DS) pregnancies and six other women with fetal aneuploidy ascertained at chorionic villus sampling (CVS), as well as 89 women with contemporaneous normal control pregnancies. Median serum PAPP-A measurements (0.31 MOM, 95 per cent confidence interval (CI) 0.22-0.65 vs. normal 1.06, 95 per cent CI 0.89-1.20) were lower and free beta-hCG subunit measurements (1.13 MOM, 95 per cent CI 0.93-2.63 vs. normal 0.91, 95 per cent CI 0.79-1.03) were higher at statistically significant levels. Receiver operator characteristics (ROC) curves showed that the highest sensitivity for detection, 71.2 per cent (95 per cent CI 54.7-87.6 per cent), was for depressed PAPP-A levels; the combination of low serum PAPP-A levels, maternal age, and elevated free beta-hCG levels yielded a detection rate of 78.9 per cent (95 per cent CI 64.9-92.8 per cent) of the affected pregnancies at 8-12 weeks' gestation.

Evidence for the in-vitro de-novo synthesis of immunoglobulin and a previously undescribed 17 kDa protein (TEP-2) by the mucosa of the fallopian tube.

De-novo synthesis and secretion of proteins by short-term explants of matched Fallopian tube mucosa and endometrium were studied using radiolabelled L-[35S]methionine and [3H]glucosamine. To compare directly each anatomical site of the Fallopian tube and endometrium from the same source, newly synthesized proteins were separated on one-dimensional polyacrylamide gel electrophoresis and examined by auto-radiography. De-novo synthesis of two protein bands provisionally designated as tubal epithelial protein 1 (TEP-1) and tubal epithelial protein 2 (TEP-2), was observed in explants of the Fallopian tube mucosa obtained from each anatomical site throughout the ovarian cycle (n = 20). TEP-2 was not apparent in tubal mucosa obtained from post-menopausal women (n = 5). De-novo synthesis of TEP-1 and TEP-2 was not apparent in autoradiographs of radiolabelled proteins from short-term explants of endometrium. From the autoradiographs the molecular mass of TEP-1 and TEP-2 was calculated to be 25 kDa and 17 kDa, respectively. Incorporation of glucosamine into newly synthesized protein occurred in TEP-2 but not TEP-1. TEP-1 was observed to be immunochemically identical to immunoglobulin kappa light chains.

In-vitro synthesis of total protein and placental protein PP14 by the fallopian tube mucosa: variation in relation to anatomical site, the ovarian cycle and the menopause.

De-novo synthesis and secretion of protein by short term explants of mucosa from each anatomical section of the Fallopian tube and endometrium of pre-menopausal (n = 25) and tubal mucosa of post-menopausal (n = 5) women were studied by demonstration of incorporation of radiolabelled L-[35S]methionine and one-dimensional SDS-polyacrylamide gel electrophoresis. A consistent finding in 25 pre-menopausal women was the presence of a 25 kDa protein band synthesized by tissue obtained throughout the ovarian cycle. Western blotting demonstrated that this protein band contained placental protein 14 (PP14)-like immunoreactivity in the proliferative (n = 2) and luteal phase (n = 2) of the ovarian cycle. To determine if there is quantitative variation in total protein and PP14 synthesis and secretion during the ovarian cycle, the total quantities of protein and PP14 synthesized were determined by Coomassie Brilliant Blue staining and radioimmunoassay respectively. Analysis of the results of total protein assay revealed statistically significant differences in relation to the anatomical origin of the study tissue (P < 0.01), the stage of the ovarian cycle (P < 0.04) and the manner in which each anatomical site varied during the ovarian cycle (P < 0.01), the endometrium being significantly different from the Fallopian tube. When the data for PP14 synthesized by the Fallopian tube mucosa were analysed, these effects were not seen. PP14 was not detected in the culture media of Fallopian tube mucosa obtained from post-menopausal women.

Low maternal serum levels of pregnancy associated plasma protein A (PAPP-A) in the first trimester in association with abnormal fetal karyotype.

OBJECTIVE: To assess the relation between maternal serum pregnancy associated plasma protein A (PAPP-A) in the first trimester and the outcome of pregnancy by karyotype. DESIGN: A retrospective study of PAPP-A levels in blood samples collected prior to chorionic villus sampling. SETTING: Milan, Italy. SUBJECTS: Five hundred twenty-two women aged 20 to 47, at 7 to 11 weeks gestation, prior to undergoing chorionic villus sampling. Four hundred forty-five women had a pregnancy with a normal karyotype; in 30 pregnancies the karyotype was abnormal (including 14 cases of Down's syndrome and 7 of trisomy 18). MAIN OUTCOME MEASURES: Normal or abnormal fetal karyotype. Serum PAPP-A at 6 to 11 weeks gestation measured by radioimmunoassay. RESULTS: The median value of PAPP-A in the abnormal group was 0.27 multiples of the normal median (MoM). This is significantly lower than the median value in the normal group (1.01 MoM) (95% CI for the difference 0.46-0.84 MoM; P < 0.00001 Mann-Whitney test). CONCLUSIONS: There is an association between low levels of PAPP-A in the first trimester with chromosome anomalies. Screening by measurement of PAPP-A might detect 60% of cases of Down's syndrome in the first trimester with a false positive rate of 5%.

Binding of steroid hormones and anabolic agents to bovine sex-hormone binding globulin.

Binding of some selected steroids and anabolic agents to bovine sex-hormone binding globulin (SHBG) was investigated. SHBG binding affinities, relative to the reference hormone 5 alpha-dihydrotestosterone, were estimated for the compounds. The results demonstrate that binding of steroid hormones to SHBG is facilitated by the 17 beta-hydroxyl group, possibly involving hydrogen binding, and by the methyl group at C-19 of the steroid moiety. Structural modifications at C-17 of a steroid molecule involving esterification, epimerization or reduction of the 17 beta-hydroxyl group, or introduction of a bulky 17 alpha group have the effect of decreasing the SHBG binding affinity of the steroid molecule.

Transfer of contraceptive steroids in milk of women using long-acting gestagens.

Levels of norethisterone and medroxyprogesterone acetate were measured in serum and milk of women receiving the injectable contraceptive formulations Norigest and DepoProvera, respectively, throughout complete injection intervals. In 5 of 10 women receiving Norigest, serum norethisterone levels were undetectable by 8 weeks after injection and only 2 women had detectable levels of norethisterone in milk at this time. In contrast, 8 of 10 women receiving DepoProvera had detectable levels of medroxyprogesterone acetate in both serum and milk 12 weeks after injection. The ratio of the milk:serum concentrations of norethisterone varied from 0.12 to 0.92 (mean 0.34) and for medroxyprogesterone acetate from 0.12 to 2.60 (mean 0.88). It is unlikely that these differences between the two formulations are due entirely to differences between the binding of norethisterone and medroxyprogesterone acetate to serum proteins. The area under the curve of serum steroid concentrations plotted against time was only 50% higher for women injected with DepoPovera than for those injected with Norigest but the area under the curve for milk values was 400 times higher. Assuming the infant ingests 600 ml daily, the daily intake of steroids in the first week after injection would be 0.5 to 2.4 micrograms for norethisterone and 1 to 13 micrograms for medroxyprogesterone acetate. By 8 weeks after injection, the amount of norethisterone ingested would be small but that of medroxyprogesterone acetate would still be significant.

A preliminary pharmacokinetic and pharmacodynamic evaluation of depot-medroxyprogesterone acetate and norethisterone oenanthate.

PIP: 2 populations attending WHO centers, one in Sweden and one in India, participated in a comparative, pilot trial of 2 increasingly popular injectable progestin-only female contraceptives, Depo-Provera and Norigest. The purpose of the study was to assess the pharmacokinetic and pharmacodynamic properties of the 2 formulations (depot medroxyprogesterone acetate and norethisterone enanthate). Differences were found between Swedish women and Indian women in their reactions to the 2 drugs: 1) Norigest was detectable in blood samples a significantly shorter time after injection of the agent in Indian women than in Swedish women; this difference was not apparent with Depo-Provera. 2) Although there was no difference at the 2 centers in the time of ovulation return for subjects receiving Norigest, 0 of 4 Swedish women ovulated more than 156 days after Depo-Provera injection, whereas all 4 Indian women ovulated within 73 days of Depo-Provera injection; in the Swedish women, the levels of medroxyprogesterone were undetectable at time of return to ovulation, whereas Indian women had levels of .6 ng/ml when ovulation resumed. 3) In both cultures, Depo-Provera users had significantly more episodes of bleeding and spotting than Norigest users. This preliminary report emphasizes the variety of responses possible to injection of different contraceptive progestins among various populations and points to the need for further culturally comparative studies.^ieng

Bioavailability of Lynestrenol.

The bioavailability of lynestrenol and ethynyloestradiol from a capsule formulation was superior to a normal tablet formulation. Bioavailability, as measured by the area under the plasma concentration curves, showed, particularly in the case of the capsule formulation, that all of the administered lynestrenol was converted to norethisterone.

PIP: An in vivo research study demonstrated that the bioavailability of lynestrenol and ethynyloestradiol administrered in capsule form was equal to or slightly greater than when it was administered in normal tablet form. Lynestrenol was metabolized to norethisterone both in vivo and in vitro in previous investigations. 11 normal males were administer either 1 capsule containing 2.5 mg lynestrenol and 0.05 mg ethynyloestradiol or tablets containing the same dose of steroids. Blood samples were taken just before and 1,2,4,8, and 24 hours after the drugs were administered. 8 days later the subjects were given the opposite formulations and blood samples were again obtained at the same time intervals. Radioimmunoassay was used to assess levels of free and conjugated ethynyloestradiol and norethisterone in the serum samples. Biovailability assessments were made by calculating the areas under the time-concentration curves. Results demonstrated that the conversion of lynestrenol to norethisterone in vivo was complete. Unconjugated ethynyloestradiol mean values, 2 and 4 hours after administration of the drugs, were higher for capsules than for tablets. For blood samples taken at other time periods the mean values of unconjugated ethynyloestradiol were similar for both capsules and tablets. For conjugated ethynyloestradiol the mean values and the areas under the curve, for blood samples obtained at each time interval, showed no significant difference for tablets and capsules.

Blood levels of medroxyprogesterone acetate after multiple injections of depoprovera or cycloprovera.

Mean levels of MPA in blood taken 90 days after injection were not significantly different between women who had received a single injection of DepoPrevera and those who had received eight injections at 90-day intervals. There was a wide variation in plasma MPA concentration in the two groups of women. In women who had received 31 to 45 injections of CycloProvera, plasma levels of MPA 28 days after injection were significantly higher than those of women who had received a single injection. The levels were also higher than those found in women 90 days after injection of DepoProvera. The results suggest that the dose of MPA in CycloProvera could be reduced.

Plasma levels of norethisterone measured by radioimmunoassay and gas chromatography-mass fragmentography.

PIP: Assays designed to quantitate the plasma levels of norethisterone (NET) are compared. Results from radioimmunoassay were compared with those from gas chromatography-mass fragmentography which were established in 5 subjects after a 1-mg oral dose of the steroid. In general, the levels of steroid measured by radioimmunoassay were usually higher than those measured by gas chromatography-mass fragmentography; there was however a correlation coefficient of .96 (P .001). For concentrations of the steroid above 500 pg/ml, the mean overestimate by radioimmunoassay was less than 30%.^ieng

Rate of metabolism of norethisterone in women from different populations.

The rate of metabolism of orally administered norethisterone was compared in fourteen centres by measuring plasma levels of the steroid by radioimmunoassay at varying times after oral administration of a 1 mg dose. The inter-centre differences were of the same order as the intra-centre differences. Variations in metabolism appeared not to be due to variations in body size.

Occurrence of ovulation in women receiving the injectable contraceptive norethisterone oenanthate.

Ovarian function was studied in ten women receiving a single intramuscular injection of 200 mg norethisterone oenanthate. Two of the ten women appeared to ovulate within 60 days of injection and a further four within 90 days. Follicular activity probably not followed by ovulation occurred in five subjects within 60 days of the injection and in a further subject within 90 days. Only one subject showed no evidence of follicular or luteal activity during the course of this study. There was wide variation in the rate at which the administered gestagen was metabolised and no correlation between the rate of metabolism of norethisterone and the return of ovarian function or the ponderal index, height or weight of the subjects.

Plasma levels of norethisterone after single and multiple injections of norethisterone oenanthate.

PIP: To study the likelihood of accumulation of norethisterone enanthate (NE) with repeated administrations, 15 women were given 200 mg NE in a single intragluteal injection (Group 1) and 15 others weere given from 5 to 11 such injections at from 12- to 10-week intervals (Group 2). Blood samples were taken and serum NE concentrations were determined. NE levels fell on a straight regression line when plotted semilogarithmically against number of days postinjection in both groups, but the slopes differed significantly (p .01) with plasma NE decreasing more slowly over time in Group 2. Extrapolated estimations of the time required for NE levels to become undetectable were 107 days for Group 1 and 152 days for Group 2. It is suggested that the steroid metabolic rate may have decrease in Group 2. These findings also offer some rationale for the suggestion that NE be administered at 60-day intervals for the 1st 3 injections and at 90-day intervals thereafter.^ieng

A radioimmunoassay for serum medroxyprogesterone acetate.

PIP: When injected intramuscularly in a dose of 150 mg, Depo Provera, a microcrystalline suspension of medroxyprogesterone acetate (MPA), will provide a contraceptive effect for at least 3 months. This paper describes a sensitive radioimmunoassay for MPA which has been used in the author's laboratory for the past 2 years. MPA was converted to MPA-3-CMO and the oxime was conjugated with bovine serum albumin (BSA) by the mixed anahydride method. 4 rabbits were immunized with the antiserum. A high titre of MPA antibodies was detected 6 months after immunization. Serum from the rabbit with the highest titre of antibodies to MPA was subjected to radioimmunoassay. 7 days after the intramuscular injection of 150 mg Depo-Provera, serum levels of MPA were found in the range of 1750 to 9000 pg/ml. By 75 days, the levels had decreased to 680-2600 pg/ml. The method was found to have adequate accuracy, precision and sensitivity.^ieng

Levels of contraceptive seroids in breast milk and plasma of lactating women.

PIP: Levels of contraceptive steroids in breast milk and plasma of lactating women were investigated. 7 lactating women received 150 mg medroxyprogesterone acetate (MPA) 1 week after delivery. 5 women (lactating) took 350 mcg norethisterone (NET) and 2 took 150 mcg d-norgestrel (d-ng) + 30 mcg ethinyl estradiol. Milk and blood samples were drawn and radioimmunoassay of the contraceptive drug levels were performed. In all 7 women taking MPA, drug was detected in the breast milk and plasma in a ratio of 1:1 for a period of 87 days. NET was also present in milk and plasma but the concentration in milk was about 1/10 of the amount in plasma. d-Ng levels were also less than 1/10 that in plasma.^ieng