Radioimmunoassay of 17-hydroxyprogesterone

A method is described for the determination of 17-hydroxyprogesterone in peripheral venous plasma (0.1–1.0 ml) from men and women using an antiserum to 17-hydroxyprogesterone-3-carboxymethyl oxime bovine serum albumin (BSA).

The coefficients of variation on replicate analyses ranged from 7–16%. The louest level of 17-hydroxy-progesterone uhich may be determined is 5 ng/100 ml plasma. The concentration (mean ± S.D.; ng/100 ml plasma) in a group of healthy men (aged 20–40 yrs) uas 123 ± 65. From women during days 1–10 of the menstrual cycle the value uas 40 ± 15, during days 18–32 of the cycle 134 ± 57 and during pregnancy (12th week to term) 622 ± 262. Progesterone was determined in the same samples using an antiserum to 11-hydroxyprogesterone-11-hemisuccinate-BSA.     

Amniotic fluid and decidual prolactin during pregnancy in rhesus macaques

In rhesus macaques, the concentration of immunoreactive prolactin in the amniotic fluid remains low during most of the first trimester of pregnancy and then increases abruptly at 60-80 days of gestation. During the second half of pregnancy, large amounts of prolactin accumulate in the amniotic fluid. Much of this amniotic fluid prolactin may originate from the superficial endometrium (decidua). This hypothesis is supported by the increasing amounts of decidual prolactin (dPRL) measured in endometrium obtained at early (50 days), mid-(80 days), and late (greater than or equal to 150 days) gestation. In culture, late pregnancy endometrium released more dPRL than did early pregnancy endometrium. When tissues were cultured in medium without progesterone, the amounts of dPRL measured in the medium declined steadily over 6 days, regardless of the gestational age of the endometrium. dPRL was consistently measured in medium harvested from cultures that received either progesterone or medroxyprogesterone; however, progesterone did not induce an increase in the amounts of dPRL released by cultures prepared from early pregnancy endometrium. This suggests that factors in addition to progesterone may stimulate the increase in dPRL that occurs at midgestation in rhesus macaques.     

Amniotic fluid zinc in risk pregnancies

The zinc concentration of amniotic fluid (AF) of 129 pregnant women was analyzed by the flame atomic absorption spectrometry. This prospective study was performed in order to find out whether the determination of the AF zinc concentration can be used to monitor the growth and development of the fetus. There were two groups of patients: early stage (15th–19th gestation wk) in which the amniocentesis was performed as a prenatal genetic examination, and late stage (26th–40th wk) in which the amniocentesis was performed due to obstetric reasons. The average AF zinc concentrations were 1.2 and 1.0 μmol/L in the early and late gestation group, respectively. The AF zinc concentration did not correlate with the weight, height, or Apgar scores of the newborn nor with the maternal diseases, age, or parity. The AF zinc concentration in the late gestation group was significantly lower if the fetus was male than if it was female. If the AF was greenish the zinc concentration was elevated. One malformation, congenital nephrosis, with an exceptionally high zinc concentration (9.0 μmol/L), was found.     

Production of 17-hydroxypregnenolone and 17-hydroxyprogesterone by the immature rat ovary

The concentration of 17-hydroxyprogesterone (17-OH-prog), but not 17-hydroxypregnenolone (17-OH-preg), was detectable in the sera of 5 day old female rats. The level of 17-OH-preg increased dramatically between day 5 and 10, remained high for 5 days and then decreased to low levels by day 25; a second increase was found on day 35. 17-OH-prog began to increase in the serum after day 10, reached a peak by day 20 and then decreased by day 25 and remained the same through day 35. Stimulation of the ovaries of intact females with 20 IU of pregnant mare's serum gonadotropin resulted in a prompt increase in both progestins, but a much larger increase in 17-OH-preg than in 17-OH-prog. Increases were similar, but quantitatively less, in hypophysectomized females. The results demonstrate that the ovaries of immature rats contain an active 17-hydroxylase system.     

Amniotic fluid and blood lactate concentrations in mares and foals in the early postpartum period

Amniotic fluid (AF) lactate concentration and time-dependent changes in blood lactate concentration in mares after parturition have never been evaluated. In this study, the venous blood lactate concentration of mares and foals during the first 72 h of the postpartum period was assessed, and the concentration of lactate in the AF collected during delivery and the utility of its measurement for evaluating the foal's health were investigated. This prospective observational study was carried out on mares attended at delivery. They were divided into mares delivering healthy (Group 1) and sick (Group 2) foals. The following samples were collected: AF and umbilical blood at delivery, mare's and foal's jugular blood every 12 hours from parturition until 72 h postpartum (T0-T72). Sixty-two mares were enrolled in Group 1 and 19 in Group 2. In Group 2, the survival rate was 68.4%. The median blood lactate of the foals at T0 was 3.60 mmol/L in Group 1 and 5.05 mmol/L in Group 2. The monitoring of the blood lactate concentration showed a significant time-dependent decrease from T24 in the foals (P < 0.01) and from T12 in the mares (P < 0.01). Lactate concentration over time was significantly different between healthy and sick foals (P < 0.01) but not between mares with normal and dystocic delivery (P = 0.08). A significant difference (P = 0.04) was detected as regards AF lactate concentration between Group 1 (median 14.99 mmol/L) and Group 2 (median 12.61 mmol/L). For the first time, AF lactate concentration was evaluated during parturition, and significantly higher levels were found in mares delivering healthy foals. This was an unexpected and very interesting result which warrants further investigation involving a larger number of mares. Additional studies are needed before either mare's blood or AF lactate concentration can be used in a clinical setting.     

Metabolism of 17-hydroxyprogesterone by a Bacillus species.

Fermentation of 17-hydroxyprogesterone with a Bacillus species (IICB-301) in a modified nutrient medium under aerobic conditions yielded androst-4-ene-3,17-dione and 15 alpha,17-dihydroxypregn-4-ene-3,20-dione in addition to a new pregnane analogue, 6 beta,17,20 alpha-trihydroxypregn-4-ene-3-one. Each microbial metabolite was characterized by the application of various spectroscopic techniques. The availability of the new metabolite, 6 beta,17,20 alpha-trihydroxypregn-4-ene-3-one, enabled complete elucidation of its 13C-n.m.r. spectrum.     

The production and characterisation of antisera to 17-hydroxyprogesterone

Sheep were immunised with 11-deoxycortisol-21-hemisuccinate-bovine serum albumin (11-deoxycortisol-21-HS-BSA) or with 17-hydroxyprogesterone-7 alpha-carboxyethyl thioether-keyhole limpet haemocyanin (17-OHP-7 alpha-CETE-KLH) or with 17-OHP-3-(O-carboxymethyl)oxime-KLH (17-OHP-3-CMO-KLH). The resultant antisera were assessed using [3H]17-OHP and dextran-coated charcoal to separate the antibody bound and free fractions. All sheep produced antisera with an apparent affinity constant of from 1.4 to 6.6 X 10(9) 1/mol. Those raised against 11-deoxycortisol-21-HS-BSA had titres ranging from 1:12,000 to 1:78,000 but showed significant cross-reactivity with many of the steroids tested. Sheep immunised with 17-OHP-7 alpha-CETE-KLH had antisera titres of from 1:102,000 to 1:180,000 and only 17-hydroxypregnenolone cross-reacted significantly (10-20%). The best antisera were raised in sheep immunised with 17-OHP-3-CMO-KLH. Titres ranged from 1:168,000 to 1:390,000 and there were about 8 g/l of specific antibodies which cross-reacted 5.7% or less with 17-hydroxypregnenolone, and less than 0.5% with progesterone, 11-deoxycortisol and the other steroids studied. The antisera to 17-OHP-3-CMO-KLH were further assessed using [125I]17-OHP; titres ranged from 1:5,700,000 to 1:18,000,000 with affinity constants of from 1.67 to 2.5 X 10(10) 1/mol. They showed minimal or no cross-reactivity with the steroids studied. Reimmunisation after an 8-month interval produced antisera with a higher affinity constant and even lower cross-reactivity with other steroids.     

Amniotic fluid cell morphology in early antenatal prediction of abortion and low birth weight.

The morphology of rapidly adherent (RA) amniotic fluid cells was examined in 201 pregnant women referred for amniocentesis because of two sequential high serum alpha-fetoprotein (AFP) concentrations. Out of 43 amniotic fluid samples containing increased amounts of AFP, 42 had neural or peritoneal cells predominating among the RA cells, the outcome being an infant with a neural-tube defect or exomphalos. In the other case with a raised amniotic fluid AFP concentration but only anterior placental cells the infant was normal. In 25 amniotic fluid samples containing normal amounts of AFP distinctive new patterns of RA cells were observed, termed fetal distress cells. These pregnancies resulted in five spontaneous abortions and 20 infants with birth weights under 2500 g. Fetal distress cells were not detected in any of the remaining 133 samples. One pregnancy was terminated because of a chromosomal abnormality, and there were seven twin pairs not recognised on ultrasonography before amniocentesis. The remaining 125 pregnancies went to term, resulting in infants with birth weights exceeding 2500 g. The results suggest that RA-cell morphology will prove to be of value in the early antenatal prediction of spontaneous abortion and low birth weight.     

Isolation ofAcholeplasma oculi from human amniotic fluid in early pregnancy

Acholeplasmas have been isolated from a variety of animals, insects, and plants, but onlyAcholeplasma laidlawii has previously been found in humans. We have isolatedAcholeplasma oculi in pure culture from the amniotic fluid of a woman at 19 weeks of gestation. The organism was positively identified by growth inhibition, epi-immunofluorescence, and arbutin hydrolysis. Demonstration of organisms directly in amniotic fluid by DNA fluorochrome and immunofluorescence staining provided additional evidence that the isolate was genuine and not a medium contaminant. The remainder of the pregnancy was unremarkable, and a full-term male infant was delivered without complications. Even though there is some evidence possibly associatingA. oculi with various diseases in livestock, the prevalence and significance ofA. oculi in humans has not been determined.     

Adequacy of saliva 17-hydroxyprogesterone determination using various collection methods

Steroids determination in saliva offers several advantages. The collection of saliva is a noninvasive, less stressful technique than blood withdrawal and reflects the circulating unbound fractions. The suitability of saliva for 17-hydroxyprogesterone and cortisol determinations has been documented in healthy subjects as well as in diseases like Congenital Adrenal Hyperplasia and Cushing syndrome. The aim of the study was to compare the influence of different collection methods on the results of 17-hydroxyprogesterone measurement in saliva collected by different ways, using commercially available RIAs developed for plasma. 17-hydroxyprogesterone was determined in 64 healthy adult volunteers (30 males, 34 females) in serum (Group SE) and in saliva collected before meals at 8-10 p.m. by directly spitting into a plastic tube (Group SP), using a cotton swab (Group SA) and using a polyester swab Salivette (Group SB). We used a commercially available direct radioimmunoassay without separation technique. The 17-hydroxyprogesterone mean values (ng/ml) were 1.16+/-1.3 (Group SE), 0.056+/-0.046 (Group SP), 0.089+/-0.048 (Group SA) and 0.058+/-0.049 (Group SB). The detection limit was 0.010 ng/ml. The correlations between the values in serum (Group SE) and in saliva were: r=0.77, p<0.05 (Group SP); r=0.62, p<0.05 (Group SA); r=0.70, p<0.05 (Group SB). The saliva values corresponding to the serum cut-off point of 3 ng/ml upper limit of normal values were in ng/ml 0.13 (Group SP), 0.16 (Group SA) and 0.11 (Group SB). In conclusion, 17-hydroxyprogesterone determinations in saliva using commercially available RIAs primarily developed for serum, is a reliable and easy to perform procedure. The three different methods of saliva collection showed 17-hydroxyprogesterone concentrations to have good agreement.     

Inhibition of 17,20(17-hydroxyprogesterone)-lyase by progesterone

¹⁴C-17-Hydroxyprogesterone was incubated with 7000 × g × 20 min supernatants of rat testis homogenates in the presence of various concentrations of ³H-progesterone, both under conditions where metabolism would take place and where it would be prevented. When metabolism was prevented, the ratio of progesterone to 17-hydroxyprogesterone in the microsomal fraction was 3 times that which was added to the incubation medium.Progesterone competitively inhibited 17,20-lyase action on added 17-hydroxyprogesterone but not on 17-hydroxyprogesterone formed from the added progesterone. The rate of formation of 17-hydroxyprogesterone from progesterone, however, was inhibited by added 17-hydroxyprogesterone. The results indicate that there is no free exchange of an intermediate between progesterone and androstenedione with the soluble fraction, either inside or outside the microsomal vesicle. The limited exchange with 17-hydroxyprogesterone in solution probably represents exchange with an enzyme-bound intermediate.     

Amniotic fluid arginase in women at term]

Study on 34 cases. A considerable arginasic activity is observed in amniotic fluid in women at the end of pregnancy. This activity is weak, but increased by Mn2+ (about 5 to 8 times). The total amniotic fluid is more active than the surpernatant, the deposit of which has been eliminated. An important part of arginasic activity results from the elements of the deposit (amniotic cells, foetal cells and eventualy erythrocytes in hemorragic amniotic fluid).     

Amniotic fluid 5-hydroxyeicosatetraenoic acid in preterm labor

5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonate lipoxygenase product capable of stimulating human uterine contractility in a dose-dependent manner in vitro. The purpose of this study was to determine if preterm labor is associated with changes in the concentration of this metabolite in amniotic fluid. Amniotic fluid was obtained by transabdominal amniocentesis from three groups of women with preterm labor: group 1 - women without intraamniotic infection who responded to tocolysis (n = 32); group 2 - women without intraamniotic infection who failed to respond to tocolysis (n = 22); and group 3 - women with intraamniotic infection (n = 14). 5-HETE was determined by radioimmunoassay. The median amniotic fluid concentration of 5-HETE in women who responded to tocolysis (median = 1412 pg/ml; range: 111-3547) was significantly lower than in women who delivered despite tocolysis (median = 2052 pg/ml; range: 136-7774) and women with intraamniotic infection (median = 1876 pg/ml; range: 543-7033) [p less than 0.05]). No difference in amniotic fluid concentrations' of 5-HETE were found between women in groups 2 and 3 (p greater than 0.05).     

Amniotic fluid 5-hydroxyeicosatetraenoic acid in preterm labor

5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonate lipoxygenase product capable of stimulating human uterine contractility in a dose-dependent manner in vitro. The purpose of this study was to determine if preterm labor is associated with changes in the concentration of this metabolite in amniotic acid. Amniotic fluid was obtained by transabdominal amniocentesis from three groups of women with preterm labor: group 1 — women without intraamniotic infection who responded to tocolysis (n = 32); group 2 — women without intraamniotic infection who failed to respond to tocolysis (n = 22); and group 3 — women with intraamniotic infection (n = 14). 5-HETE was determined by radioimmunoassay. The median amniotic fluid concentration of 5-HETE in women who responded to tocolysis (median = 1412 pg/ml; range: 111–3547) was significantly lower than in women who delivered despite tocolysis (median = 2052 pg/ml; range 136–7774) and women with intraamniotic infection (median = 1876 pg/ml; range: 543–7033) [p < 0.05]). No difference in amniotic fluid concentrations' of 5-HETE were found between women in groups 2 and 3 (p > 0.05).     

Amniotic fluid prostaglandin E2 in preterm labor

These studies were designed to determine amniotic fluid concentrations of prostaglandin E2 (PGE) in women with preterm labor. Amniotic fluid was retrieved by transabdominal amniocentesis from 68 women with preterm labor (less than 37 weeks). Patients were divided into three groups according to the response to tocolysis and the presence or absence of an intraamniotic infection. Amniotic fluid concentrations of PGE2 were significantly greater in women with preterm labor and intraamniotic infection than in women without infection. Patients unresponsive to tocolysis without intraamniotic infection had a significantly greater concentration of PGE2 in amniotic fluid than those responsive to tocolysis.     

Amniotic fluid lipoxygenase metabolites during spontaneous labor and after RU486 treatment during late pregnancy in rhesus macaques

To determine changes in amniotic fluid (AF) lipoxygenase metabolites prior to spontaneous labor and after RU486 administration, we implanted AF and vascular catheters and myometrial electromyographic (EMG) electrodes in 8 rhesus macaques at 120-130 days of pregnancy (term = 167 days). Four animals had AF samples taken serially until they delivered their infants normally at term. The other four animals received RU486 (20 mg/kg/day) for 3 days. AF samples were collected every 2-3 days and at 12 hour intervals for 72 hours before and after treatment with RU486. Uterine activity was monitored continuously. LTB4, 5-HETE and 15-HETE were measured by radioimmunoassay. In untreated animals, LTB4 and 5-HETE concentrations in AF increased significantly (P less than 0.05) 4 days before delivery with no change in 15-HETE. After RU486, mean levels of LTB4 and 5-HETE were increased although the difference was not statistically significant. No change in 15-HETE levels was observed. In conclusion, LTB4 and 5-HETE increase in AF before the onset of spontaneous labor. Progesterone receptor blockade by RU486 does not reproduce the changes in AF lipoxygenase metabolites observed during normal parturition.