Oestrone, oestradiol-17beta and oestriol levels in human foetal plasma during gestation and at term.

PIP: Marked rises in both unconjugated and sulphoconjugated estrone, estradiol-17-beta and estriol were observed in human fetal plasma between midgestation and term. Significant arterio-venous differences were found in the umbilical cord plasma. No consistent arterio-venous differences were found in the umbilical cord plasma. This indicates that all 3 estrogens are secreted from the placenta into the fetal circulation in the unconjugated form. Mean unconjugated estrogen (estrone + estradiol-17-beta + estriol) levels rose from 22.7 ng/ml at 17-20 weeks of gestation to 108.9 ng/ml at term in umbilical venous plasma and from 4.3 ng/ml to 23.3 ng/ml in umbilical arterial plasma. This represents a secretion rate of approximately 30 mg estrogen/day into the umbilical vein at term. Mean estrogen sulphate levels rose from 128 ng/ml to 313 ng/ml in the cord plasma during the same period. Of the 3 estrogens measured, estriol was quantitatively the major estrogen in fetal plasma. It consistently represented about 78% of the unconjugated fraction and 95% of the sulphate fraction at all stages of gestation. The method of delivery did not have a significant effect on the estrogen levels in uncomplicated pregnancies. Similar estrogen levels were found in fetal heart blood after either hysterotomy at spontaneous abortion at 16-20 weeks of gestation, and no significant differences were found for estrogen levels in cord plasma after elective Caesarean section at 38-39 weeks when compared with estrogen levels after normal delivery at term. A significant rise in fetal unconjugated estrogens at a time when fetal corticosteroids are increasing may be of physiological importance for fetal maturation in women.^ieng

Studies of human chorionic gonadotrophin, placental lactogen, and progesterone secretion in vitro.

The relative ability of explants of villous tissue from 8- to 10-week placenta to synthesize and release human chorionic gonadotrophin (HCG), human placental lactogen (HPL) and progesterone in culture was investigated. It was found that the synthesis and release of all three hormones was two to four times greater than the initial tissue content in the first 24 hours in culture. Between 24 and 48 hours of the secretion of HCG, HPL and progesterone was reduced to about 40 per cent, 20 per cent and 60 per cent respectively of that produced in the previous 24 hours. These results support previous findings that the production of HPL is more labile than HCG in culture, and suggest that progesterone secretion under the present conditions is least affected. When azastene (4, 4, 17 alpha-trimethylandrost-5-eno (2,3-d) isoxazol-17-ol), an inhibitor of gonadal and placental 3 beta-hydroxy steroid dehydrogenase conversion of pregnenolone to progesterone in rhesus monkeys, was added to the culture medium at a concentration of 0.3-150 mumol/l, no marked effect on the secretion of progesterone was observed. This indicates that the dehydrogenase in human placental tissue is either less accessible to azastene or that the inhibitory activity in the rhesus monkey was due to a metabolite of azastene.

The secretion of hormones during the culture of human preimplantation embryos with corona cells.

Following in vitro fertilization of human preovulatory oocytes from spontaneously ovulating women, determinations were made of the secretion into the culture medium of progesterone, estradiol, human chorionic gonadotropin (hCG), and the prostaglandins (PG) E2 and F2 alpha, over a 3- to 4-day period of embryo development. It was found that the corona cells associated with the egg could account, between days 2 and 3, for a mean daily secretion of 50 ng of progesterone and approximately 100 pg of estradiol, PGE2, and PGF2 alpha, respectively. Mechanical removal of the corona cells after about 48 hours for the examination of the egg for cleavage reduced the mean amount of progesterone produced on day 3 to 2 ng, and a concomitant decrease in estradiol. PGE2, and PGF2 alpha was observed. Steroid secretion could be restored on day 3 to more than 50% of that secreted on day 2 by returning some detached corona cells to the culture medium containing the embryo. hCG was not detected (less than 2 mIU/ml) in the culture medium at any stage of embryo culture over the 3- to 4-day period.

Effects of ovine corticotropin-releasing factor and vasopressin on plasma beta-endorphin, cortisol and behavior after minor surgery in sheep.

The evidence for an analgesic effect arising from increased peripheral concentrations of beta-endorphin (beta-EP) in various animal species is controversial, and has not been fully evaluated in the sheep. To stimulate beta-EP release, ovine corticotropin-releasing factor (oCRF) and arginine vasopressin (AVP) were injected intravenously (iv) into a group of 12 out of 24 sheep, 15 min prior to minor surgery on all sheep. This brought about significant increases (P less than 0.01) in plasma beta-endorphin (beta-EP) and cortisol concentrations, relative to the non-injected control sheep, 15-30 min after injection. Ultrafiltration indicated that less than 30% of the released beta-EP immunoreactivity was present as higher molecular weight forms (mol. wt greater than 10,000) and that the majority (about 75%) of the beta-EP was probably bound to plasma proteins. By 75 min after injection there was no significant difference in plasma beta-EP or cortisol concentrations between the two groups of sheep. Consistent with previous observations the sheep showed a characteristic aversive behavior to the human handler following surgery, lasting several days. This behavior appeared to be unaffected by a pre-operative increase in peripheral plasma beta-EP, and may indicate that this increase in beta-EP was not sufficiently analgesic to block the cognitive response to the operation, or long-lasting enough to prevent the perception of post-operative soreness.

Radioimmunoassay of progesterone, 17-hydroxyprogesterone, estradiol-17beta and prostaglandin F in human corpus luteum.

Radioimmunoassay procedures have been adapted for the assay of progesterone, 17-hydroxyprogesterone, estradiol-17beta, and prostaglandin F in human corpus luteum. The method utilises a single homogenisation and extraction of the tissue followed by fractionation of the steroids on alumina, and separation of the prostaglandins of the F series from the E and A series on silica gel, prior to radioimmunoassay. An attempt has been made to validate the method for the progestins by comparison with results after fractionation of the progestins on Sephadex LH-20, for estradiol-17beta by comparison with values obtained with competitive protein-binding, and for prostaglandin F by comparison with values after additional purification. The results showed that peak concentrations of the three steroids in corpora lutea from women during the luteal phase of the menstrual cycle were comparable to those found in corpora lutea from women in early pregnancy. However, in six out of fourteen corpora lutea from non-pregnant women, prostaglandin F levels were higher than those found in corpora lutea from seven women in early pregnancy, i.e. 13-46 ng/g compared with 1-7 ng/g. Of the above six corpora lutea, four were on days 23-25 of the cycle, at a time when luteolysis would be commencing. The results in this paper support the conclusion that the corpus luteum is a major site of synthesis of the three steroids examined, although the site of synthesis of prostaglandin F is still equivocal.

Salivary and plasma cortisol as an index of stress in goats.

Total assayable cortisol in plasma was highly correlated (r = 0.97) with physiologically active free cortisol in plasma after routine management procedures in 1- to 3-weeks-old goats. Transport of adult goats caused significant increases (P less than 0.001) in free cortisol in saliva and in free and total cortisol in plasma. No difference (P greater than 0.05) between concentrations of free cortisol in saliva and in plasma was apparent before or after transport. The results demonstrated that the salivary cortisol method is a useful measure of stress in adult goats, and that the relationship between free and total cortisol in plasma, and the adrenocortical response to transport, appear to be similar in sheep and goats.

Stress in calves castrated surgically or by the application of rubber rings.

The effects of castration were studied in calves 4 to 11 weeks of age, using increases in salivary cortisol as an indicator of stress. Groups were castrated surgically or by rubber ring application or were non-castrated (control) females. The surgically castrated group showed more agitation during the operation, but both castrated groups resumed normal behaviour soon after the operation was completed. The short-term salivary cortisol response was significantly higher after surgical castration than after the application of rubber rings, where, in turn, it was significantly higher than in the control group. Salivary cortisol was elevated over a period from 15 min to 3 h following the castration, but at 4, 24 h and 6 days post-treatment there were no significant differences between treated groups and controls.

Development of a salivary cortisol method for detecting changes in plasma "free" cortisol arising from acute stress in sheep.

A simple device for collecting saliva (mainly parotid) from sheep is described. The collection of saliva, and the assay of "free" cortisol in saliva appears to offer certain advantages over the collection of blood, and the assay of serum cortisol, for the assessment of stress in sheep. With a little experience, it is easier to collect saliva than take blood samples when sheep are passing through a race. The "free" cortisol can be measured directly in saliva, whereas in serum, it is first necessary to separate "free" from protein-bound cortisol. Basal levels of "free" cortisol of less than 10 nmol/l were recorded in saliva and blood plasma or serum in unstressed sheep which had previous experience of being handled in a race, Significant increases in salivary cortisol and "free" and total ("free" plus protein-bound) cortisol in serum were found in sheep following adrenal stimulation with synacthen, or after 30 min of stressful transport. This indicates that the salivary cortisol technique is applicable to studies of stress in sheep, and should also be useful for other ruminants.

A comparison of stress in surgically and non-surgically mulesed sheep.

A comparison has been made in 9- to 10-month-old castrated male Merino sheep of the changes in plasma total cortisol concentration and behaviour after being treated by either the modified Mules operation or by topical application of a quaternary ammonium compound to achieve non-surgical mulesing. After surgical mulesing, plasma total cortisol concentration increased immediately and rapidly and reached a peak value in 15 minutes, whereas after non-surgical treatment an immediate rise did not occur, but a similar peak value was observed in blood samples collected 24 hours after treatment. The concentrations were lower in both groups at 48 hours. Likewise postural changes indicative of discomfort were immediately apparent in the surgically treated sheep, but not until 3 to 4 hours later in those treated non-surgically. Arena testing revealed that a lasting aversion to the person who restrained them during treatment developed in the surgically mulesed sheep, but not in those treated non-surgically. The non-surgical procedure did not create large open wounds, as did the surgical operation, but still achieved similar enlargement of the bare area on the breech, and healing was quicker in the non-surgically treated sheep.

Stress responses in lambs docked and castrated surgically or by the application of rubber rings.

A comparative study has been made in lambs 3 to 6 weeks of age of the behavioural responses and changes in plasma immunoreactive beta-endorphin (ir beta-endorphin) and cortisol after docking or docking plus castration by the application of rubber rings or by surgery. The use of rubber rings on lambs at this age was characterised by very agitated behaviour indicative of considerable distress for a period of up to 1 h. In contrast, surgery was accompanied by some post-operative shock exhibited by reduced exploratory and social behaviour, at least in the lambs exposed to docking plus castration. In the latter group there were highly significant increases in both plasma ir beta-endorphin and cortisol concentrations that may be consistent with the induction of stress-induced analgesia. We conclude that surgery caused less distress than the rubber rings, and is therefore preferable for docking and castration of lambs 3 to 6 weeks of age.

Comparison of total and free cortisol in bovine serum and milk or colostrum.

Total (free plus protein-bound) and free cortisol concentrations in paired samples of serum and skim milk or colostrum were determined by specific radioimmunoassay after extraction or ultrafiltration. For 11 cows in established lactation mean total cortisol concentrations +/- SE in serum and milk were 4.5 +/- .7 ng/ml and .35 +/- .06 ng/ml, respectively. Concentrations of free cortisol in serum and milk were about .3 ng/ml, which is at the lower limit of the present assay. Following 200 IU Synacthen injection to 5 cows, total cortisol in serum and milk rose to 67 +/- 6.0 ng/ml and 11.4 +/- 1.5 ng/ml by 8 h postinjection, while free concentrations increased to 4.3 +/- .7 ng/ml and 6.5 +/- .8 ng/ml in serum and milk, respectively. Eight additional cows were sampled at 1 to 2 d after parturition, and total cortisol in serum and colostrum was 16.6 +/- 1.6 ng/ml and 4.4 +/- 1.3 ng/ml, and free cortisol was 2.4 +/- .4 ng/ml and 1.8 +/- .5 ng/ml, respectively. Free cortisol concentrations in colostrum or in milk from cows in established lactation, before or after adrenal stimulation, are directly related to free cortisol concentrations in the blood, while the bound cortisol in milk is more related to these two stages of lactation rather than the bound hormone in the blood. Measurement of free cortisol in milk should allow the monitoring of changes in plasma free cortisol in studies of stress in dairy cows.

Changes in estrogen levels in maternal venous plasma after intra-amniotic infusion of prostaglandin F2 for therapeutic abortion.

PIP: Peripheral plasma levels of estrone, estradiol-17beta and estriol were measured by the method of Shutt and Cox in 10 women following intra-amniotic infusion of prostaglandin F2alpha (PGF2a) for therapeutic abortion. Initial dose was 30 mg, followed if necessary, by doses of 15 mg at 24 hours and 42 hours. Gestational age of pregnancies ranged from 14 to 19 weeks, with a mean of 16 weeks. All 10 women completely aborted. Mean induction-abortion interval was 24 + or - 12 hours. The mean estrone, estradiol 17beta and estriol levels declined to about half of the pre-infusion levels after 80% of the induction-abortion interval had elapsed. The main decline in estrogen levels occurred in individual women either during the 1st quarter or during the last quarter of the induction-abortion interval. There were no significant relationships between changes in estrogen levels and the interval from 1st administration of PGF2a to subsequent abortion.^ieng

Two cases of placental sulphatase deficiency with unusual endocrine features.

Two cases of placental sulphatase deficiency are described both of which showed typical clinical features, associated with very low urinary and serum oestrogens. In the one case that reached term there was a spontaneous rise in serum oestradiol levels (from 16 to 44 nmol/l) after 38 weeks, although no oestradiol rise occurred after DHEAS loading. She later achieved a vaginal delivery. Although no steroid sulphatase activity could be detected in the placenta of case 2, there was a small transient rise in serum oestradiol (from 15.4 to 32.7 nmol/l) after maternal DHEAS loading. However, the absolute levels reached were very low compared to the response seen in normal pregnancies. The spontaneous rise in basal oestradiol levels between 38 and 41 weeks and the transient rise following DHEAS loading have not been previously reported for patients with placental sulphatase deficiency. Umbilical cord blood levels of several steroids in case 2 showed some similarities to previous case reports. Levels of DHEA, 16 alpha OH DHEA, 17OH pregnenolone and cortisol were significantly lower than control values. DHEAS, 16 alpha OH DHEAS, 17OH progesterone and androstenedione were within the normal range.