STUDIES ON OESTROGEN STIMULATED NEUROPHYSIN IN WOMEN WITH ANOVULATION

The effects of endogenous and exogenous oestrogen on serum concentrations of oestrogen stimulated neurophysin (ESN) were studied in women with regular menstrual cycles and patients with anovulation. In ten menstrual cycles serum ESN concentrations rose steadily from 305 ± 16 ng/l (mean ± SEM) in the early follicular phase to reach maximum concentrations at about the time of ovulation (601 ± 106 ng/l). There were significant correlations between ESN and oestradiol concentrations in nine of the ten cycles (P < 0·05). The concentration in the mid luteal phase (386 ± 43 ng/l) was also significantly greater than at the beginning of the cycle (P < 0·05). Stimulation of endogenous oestrogens by treatment with clomiphene was accompanied by a release of ESN. Oestrogen concentrations were significantly lower in 5 months in which ovulation did not occur than in ten treatments in which ovulation did occur (P < 0·05). The amount of ESN produced however, was similar in the two groups. The basal concentration of ESN was within the range found in the early follicular phase of the cycle in twenty of the thirty-seven patients with anovulation in whom it was measured. Concentrations tended to be low in patients with anorexia nervosa or hyperprolactinaemia and oestrogen concentrations were also low. Levels were high in oligomenorrhoea but oestradiol concentrations were normal. Serum ESN concentrations in oestrogen provocation and amplification tests reached maximum values in normal subjects 48 h after the administration of oestradiol benzoate: LH concentrations continued to rise until 72 h. In twenty-one of forty-one provocation tests on anovular patients the amount of ESN released was within the range for normal controls: twelve released less and eight released more. There was no correlation between the basal ESN values and the ESN released. There was intact positive LH feedback in sixteen of the forty-one tests performed. In all except two tests with positive LH feedback, ESN was also released, but in the twenty-five tests with no evidence of LH positive feedback there was normal release of ESN in twelve, reduced release in ten and excessive release in three. In the amplification tests LHRH was without effect on the ESN concentrations before or after oestrogen administration. It is concluded that either the release of LH stimulated by oestrogen is independent of the release of ESN, or it is stimulated at different threshold levels of oestrogen. ESN is more readily released than LH and the mechanism is probably less involved.     

HUMAN α-LACTALBUMIN AND HORMONAL FACTORS IN PREGNANCY AND LACTATION

Serum α-lactalbumin was monitored throughout pregnancy in twelve women and in a separate group of nineteen women during the first 3 months postpartum. During pregnancy α-lactalbumin rose significantly until the mid trimester (P < 0·001). From then until term, concentrations remained stable. Concentrations during labour were significantly higher (P < 0·01) than those seen at term, α-lactalbumin, 17β-oestradiol and progesterone concentrations behaved similarly during the first week of the puerperium in both lactating (n= 10) and non-lactating (n= 9) subjects. A large surge of α-lactalbumin closely followed the clearance of high circulating concentrations of sex steroids in both groups. Prolactin concentrations were significantly greater (P < 0·02) in lactating subjects by the third postpartum day. By the third postpartum week α-lactalbumin concentrations in lactating subjects had stabilized at labour levels in a milieu of high prolactin levels and depressed production on 17β-oestradiol and progesterone. Conversely, in non-lactating subjects α-lactalbumin concentrations fell, as did prolactin, coincidental with a rise in 17β-oestradiol, progesterone concentrations remaining barely detectable. The apparent control mechanisms for human α-lactalbumin secretion and thus, lactation, are discussed in the light of the data presented.     

THE DECREASED BASAL AND STIMULATED PROLACTIN LEVELS IN ISOLATED GONADOTROPHIN DEFICIENCY: A CONSEQUENCE OF THE LOW OESTROGEN STATE

Prolactin secretion has been evaluated in seven male and six female patients with isolated gonadotrophin deficiency (IGD). The subjects were challenged with the dopaminergic antagonist, metoclopramide (10 mg) and TRH (200 μg) before, during and after cessation of hormonal treatment. Five females received three consecutive 21-day courses of ethinyl oestradiol (0·1 mg daily) at monthly intervals and the remaining subject conjugated oestrogens (Premarin 0·625 mg daily) according to a similar protocol. Treatment of the males with hCG (pregnyl) 5000 iu twice weekly led to a rise in oestradiol and testosterone levels. Two males were receiving pergonal (human menopausal gonadotrophin) in addition. In the untreated state in both males and females, basal oestradiol and PRL levels were decreased as were the PRL responses to metoclopramide and TRH as compared with normal controls. During treatment in both groups, there was an increase in basal PRL levels as well as PRL response to the two stimuli, which became indistinguishable from the controls. Cessation of treatment was associated with a rapid decrease in basal PRL levels and PRL elevation following metoclopramide and TRH. In contrast to the effect of hCG, the administration of two non-aromatizable androgens (mesterolone and fluoxymesterone) had no effect on basal and TRH-induced PRL secretion. The administration of clomiphene citrate during hCG treatment in one male IGD patient produced a decrease in the basal and stimulated PRL response.
It is concluded that the low basal PRL levels and impaired PRL responses to stimulation are not an inherent component of the syndrome of IGD, but a consequence of the abnormal steroid milieu.     

INDIVIDUAL NEUROPHYSIN CONCENTRATIONS IN THE PITUITARY AND CIRCULATION OF HUMANS

Specific, homologous human neurophysin I and II radioimmunoassays were established and used to measure the individual, immunoreactive neurophysin concentrations in human plasma. Circulating levels of human neurophysin I in normal individuals were less than 1 ng/ml and neurophysin II levels were 1-2 ng/ml. During dehydration, there was a significant rise in plasma neurophysin I, together with an increase in neurophysin II. Haemorrhage also was associated with a rise in plasma neurophysin I and II, but the percent increase was greater for I than II. In two subjects in whom nicotine inhalation caused a rise in plasma neurophysin I, there was no detectable increase in plasma neurophysin II. These stimuli which have been reported to release vasopressin from the posterior pituitary also are associated with the differential release of neurophysin I. Plasma neurophysin II levels could more clearly be shown to rise independently of plasma neurophysin I during events thought to be related to oxytocin release. Plasma neurophysin II levels were significantly elevated in women taking oral contraceptives. Similarly during pregnancy there was a progressive rise in plasma neurophysin II concentration which was proportional to the period of gestation. Plasma neurophysin II concentrations in seven of fifteen nursing women rose significantly during suckling. There was no detectable change in plasma neurophysin I concentrations during any of these events. Plasma neurophysin I and II levels were both significantly elevated in fourteen patients with chronic renal failure and rose over haemodialysis, suggesting that the kidney may be the major route of clearance of the neurophysins. In humans the independent release of neurophysin II was associated with stimuli thought to release oxytocin, but neurophysin I showed only a differential release compared to neurophysin II in vasopressin stimulated events.     

THE RELATIONSHIP BETWEEN OESTROGEN STATUS AND BONE LOSS IN POST-MENOPAUSAL WOMEN

The relationships between the rate of change of metacarpal cortical width, urinary hydroxyproline/creatinine ratio, net absorption of calcium and maturation value of the vaginal smear have been examined in normal post-menopausal women and crush fracture cases. Rapid bone loss, high bone resorption and low calcium absorption are shown to be associated with poor oestrogen status, with crush fracture cases presenting an exaggerated view of the normal post-menopausal situation.     

HYPERCORTISOLAEMIA AND LACK OF SKELETAL RESPONSE TO OESTROGEN IN POSTMENOPAUSAL WOMEN

Measurements of plasma ‘cortisol’ and metacarpal mineral content were made in seventy-two postmenopausal women of whom one half had been taking 20-40 μg mestranol daily for 1-3 years. Urinary free ‘cortisol’ (UFC) was also measured in just over one half of these women. Significant increases in plasma ‘cortisol’ and metacarpal mineral content were found in the mestranol treated women. The greatest bone mineral response was found in those women with plasma ‘cortisol’ concentrations in the range 36-45 μg/100 ml. A significant inverse relationship was found between UFC and metacarpal mineral change. These findings imply that failure of the skeleton to respond to oestrogen therapy might result from a relative increase in adrenocorticoid activity. It is suggested that the measurement of plasma ‘cortisol’ and UFC may be of value in monitoring the treatment of patients on long-term oestrogen therapy.     

SERUM FREE 1,25-DIHYDROXYVITAMIN D AND THE FREE 1,25-DIHYDROXYVITAMIN D INDEX DURING A LONGITUDINAL STUDY OF HUMAN PREGNANCY AND LACTATION

The changes in three different indices of 1,25-dihydroxyvitamin D (1,25(OH)2D) biological activity were studied longitudinally in 35 women during late pregnancy and lactation and in 26 control women. Measurements were made of maternal serum total 1,25(OH)2D and free 1,25(OH)2D concentration (by centrifugal ultrafiltration) and the free 1,25(OH)2D index (the molar ratio of total 1,25(OH)2D and vitamin D binding protein (DBP]. During late pregnancy total 1,25(OH)2D concentrations were significantly elevated when compared to controls, as were free 1,25(OH)2D and DBP concentrations and the free 1,25(OH)2D index. Serum total 1,25(OH)2D, free 1,25(OH)2D and DBP concentrations all fell dramatically during the first 2 weeks of lactation with total 1,25(OH)2D and free 1,25(OH)2D concentrations falling to levels below those of controls. During the course of lactation both total 1,25(OH)2D and free 1,25(OH)2D levels rose significantly although they were not different from controls at 18 weeks of lactation. In contrast, the free 1,25(OH)2D index fell during the first 2 weeks of lactation, but remained at this level, significantly lower than controls. Neither urinary calcium excretion nor dietary calcium intake correlated with total or free 1,25(OH)2D, DBP, or the free 1,25(OH)2D index. The disagreement in the results of free 1,25(OH)2D concentration and free 1,25(OH)2D index demonstrates that these two approaches to measuring biologically active 1,25(OH)2D are not equivalent. In attempting to account for the increased calcium requirements of human reproduction we conclude that in pregnancy any of the 1,25(OH)2D measurements may be appropriate. In lactation, however, either 1,25(OH)2D is not a major factor or 1,25(OH)2D biological activity is inadequately represented by any of the currently available methods.     

CORTISOL BINDING CAPACITY AND OESTROGEN CONCENTRATIONS IN MATERNAL AND CORD PLASMA IN PREGNANCIES WITH NORMAL AND ANENCEPHALIC FETUSES

The cortisol binding capacity of maternal and cord plasma samples obtained at delivery from fifteen women and their normal infants and from seven women and their anencephalic infants was measured at 4°C by a gel filtration technique. The concentration of oestrogen in these samples was measured by radioimmunoassay. There was no significant difference (t test) between the cortisol binding capacity of peripheral plasma from women with normal infants (1.5±0.24 μmol/l, mean ± SD) and from those who delivered anencephalic infants (1.35 ± 0.30μmol/l), nor between the cortisol binding capacity of cord plasma from anencephalic infants (0.47 ± 0.04 μmol/l) and that of normal infants (0.37 ± 0.10 μmol/l). However, mean oestrogen concentrations in maternal and cord plasma from the pregnancies with an anencephalic fetus were significantly lower (P<0.01) than in the corresponding samples from normal pregnancy. It is concluded that oestrogen concentrations in maternal and cord plasma in normal pregnancy at delivery are much greater than those required to account for the increase in plasma cortisol binding capacity. Since plasma cortisol binding capacity in pregnancy with an anencephalic fetus is not diminished, the reduced excretion of corticosteroids relative to normal pregnancy in this condition is unlikely to be due to alterations in cortisol metabolism associated with a lower plasma cortisol binding capacity.     

OESTROGEN AND PROGESTERONE RECEPTORS IN MEN WITH BILATERAL OR UNILATERAL PUBERTAL MACROMASTIA

Abnormalities at the tissue receptor level may be important in the pathophysiology of pubertal macromastia, which may be unilateral or bilateral. We studied breast tissue removed from seven boys of age 16-17 years, five with bilateral and two with unilateral gynaecomastia. We confirmed that their physical features, karyotype, and plasma concentrations of testosterone, oestradiol, LH, FSH, and prolactin were all normal for adolescent males. Oestrogen and progesterone receptors were measured with a steroid binding (dextran coated charcoal) assay which was used for breast cancer receptor studies. Oestrogen receptors were not detectable in any of the 12 breasts studied. Progesterone receptors were detectable at a low level in two patients with bilateral gynaecomastia, one breast from each patient. We conclude that although the development of bilateral or unilateral male macromastia in puberty may yet be mediated by a local tissue receptor abnormality, this disorder is probably not mediated by an abnormal increase in oestrogen receptor number.     

RENIN-ANGIOTENSIN SYSTEM IN NORMAL AND IN HYPERTENSIVE DISEASE OF PREGNANCY

Plasma-renin activity, plasma-renin substrate, and plasma-angiotensinase activity have been determined in seven normotensive and six hypertensive pregnant women. Plasma-renin activity and plasma-renin substrate were significantly elevated in both groups; but there were no significant differences between the normotensive and the hypertensive pregnant women. Plasma-angiotensinase activity was increased in the normotensive pregnant group; by contrast, the hypertensive group did not show this increase, and plasma-angiotensinase activity was significantly lower in the post-partum period in hypertensive pregnant women. This study favours the hypothesis that decreased inactivation of angiotensin II may have a role in the pathogenesis of hypertensive disease of pregnancy.     

OESTRONE SULPHATE IN PLASMA FROM POSTMENOPAUSAL WOMEN AND THE EFFECTS OF OESTROGEN AND ANDROGEN THERAPY

The concentration of oestrone sulphate in peripheral plasma from postmenopausal women was investigated using a method which involved extraction of the conjugate, which was then hydrolysed with acid, and determination (by radioimmunoassay) of the purified oestrone fraction obtained. The concentration of unconjugated oestrone in the same plasma samples was also measured. Postmenopausal women had concentrations of oestrone sulphate in plasma (1.1 +/- 0.36 nmol/l, mean +/- SD, n = 39) similar to those found in women in the follicular phase of the menstrual cycle and less than those found in males (3.2 +/- 0.61 nmol/l, n = 21). The mean ratio of the concentration of oestrone sulphate to that of oestrone in plasma from postmenopausal women (7.9 +/- 3.3) was significantly lower (P less than 0.001, t test) than the mean ratio in men (19.8 +/- 3.8). Treatment with conjugated oestrogens, oestradiol in a cream, oestradiol valerate or ethinyl oestradiol, increased the concentration of oestrone sulphate in the peripheral circulation. In contrast, chronic corticosteroid therapy reduced the level of oestrone sulphate (0.5 +/- 0.11 nmol/l, n = 10) but this was partly restored (to 0.7 +/- 0.13 nmol/l) by concomitant oral dehydroepiandrosterone. Ingestion of piperazine oestrone sulphate (Harmogen, 1.5 mg) by three fasting postmenopausal women was followed 4 h later by oestrone sulphate concentrations five to ten times those found at midcycle.     

THE EFFECT OF ENDOGENOUS OESTROGEN ON PLASMA AND URINARY CALCIUM AND PHOSPHATE IN OOPHORECTOMIZED WOMEN

Using a specific radioimmunoassay, significant levels of plasma oestradiol can be detected in the blood of oophorectomized women. In these women the plasma concentration of oestradiol correlates positively with the body fat content. Low circulating concentrations of oestradiol are associated with increased values for serum phosphate and alkaline phosphatase, but no significant change in serum calcium. The fasting urinary calcium creatinine ratio is inversely related to circulating plasma oestradiol concentration which also correlates, in a more complex way, with the renal threshold for phosphate (TmPO₄/GFR). It is suggested that oestrogen production may be an important factor in determining bone loss in post-menopausal women.     

NURSING BEHAVIOUR, PROLACTIN AND POSTPARTUM AMENORRHOEA DURING PROLONGED LACTATION IN AMERICAN AND KUNG MOTHERS

In order to determine the effects of protracted nursing in American women, blood was collected hourly for 24 h and nursing periods recorded in 20 mothers, 10 amenorrhoeic, 3 3/4 to 17 1/4 months postpartum (PP), and 10 menstruating, 5 1/4 to 46 months PP. These data were compared to the daytime nursing behaviour and 1000-1100 h PRL of women among !Kung hunter-gatherers of Botswana, a non-contraceptive using population with a birth space interval of greater than 3 years. Intense nursing behaviour maintained amenorrhoea and hyperprolactinaemia for 1 to nearly 2 years PP in both American and !Kung mothers. Among Americans, 80 min of nursing per day, in conjunction with a minimum of six nursing episodes, was highly predictive of remaining amenorrhoeic up to 18 months PP. Amenorrhoea was always accompanied by hyperprolactinaemia, but delay in the onset of menses was related more to nursing behaviour than to a particular 24 h PRL level. The 1000-1100 h sample is equivalent to and about half of the 24 h mean in high and low intensity nursers, respectively. The !Kung women were similar to the high intensity nursing American women in 1000-1100 h PRL, percent amenorrhoeic, and the number of minutes of daytime nursing.