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Serum oestradiol-17 beta in normal women.

Serum concentrations of oestradiol-17β were measured by radio-immunoassay during 40 menstrual cycles from 38 normal premenopausal women. Repeated samples were studied from 25 normal postmenopausal women. The method was highly specific and extremely sensitive. In premenopausal women the pattern of serum oestradiol-17β was remarkably constant but the concentrations varied with age. Women in the fourth decade of life had significantly higher concentrations of oestradiol-17β than either younger or older women. In postmenopausal women the concentration of oestradiol-17β was consistently very low.

IT seems clear from clinical (Schinzinger, 1889;Beatson, 1896), experimental (Loeb, 1919;Huggins, Briziarelli and Sutton, 1959) and biochemical evidence (Brown, 1958;Marmorston et al., 1965;Persson and Risholm, 1964;Nissen-Meyer and Sanner, 1963) that there is a relationship between ovarian function and cancer of the breast. Unfortunately it has not been possible to define this relationship in terms of hormonal activity because simple methods for the measurement of ovarian hormones in blood have not been available.
It is now possible by means of radioimmunoassay to measure simply and accurately the concentration of oestradiol-17,8 in serum (Abraham, 1969;Cameron and Jones, 1972) but the normal range has not yet been defined. Previous studies indicate that the concentrations are low during the follicular phase of the menstrual cycle, that there is a well defined peak at about the time of ovulation and a second rise to a fairly high plateau during the luteal phase. These variations, together with variability in the length of the menstrual cycle, have made the definition of a normal range extremely difficult. Studies of individual samples contribute little; it is necessary to examine daily samples taken throughout the menstrual cycle. In this study a group of normal premenopausal women was studied daily in an attempt to define the normal range and relate it to the age of the subjects. A further group of postmenopausal and menopausal women was studied repeatedly in a similar way. The object of the study was to provide a normal baseline with which to compare patients with benign and malignant breast disease.

MATERIALS AND METHODS
Samples of blood were taken daily or as often as possible during at least one menstrual cycle from 38 normal premenopausal women. Multiple samples were also obtained from 25 normal postmenopausal and 3 menopausal women (within one year of cessation of periods). None had a history of breast disease or gynaecological abnormality and none was taking any form of hormonal preparation. The ages of the 38 premenopausal subjects ranged from 21 to 53 years (mean 34 8). Of these, 14 were aged 20-29 years, 13 were 30-39 years, 10 were 40-49 years and one was 53 years of age. The ages of the normal postmenopausal subjects ranged from 47 to 64 years (mean 56). SERUM OESTRADIOL-17fl IN NORMAL WOMEN Diethyl ether (Pronalys peroxide-free) was obtained from May and Baker, and a freshly opened 500 ml bottle was used for each assay. Ethanol (analytical grade) was supplied by James Burroughs Ltd, London; benzene and methanol (scintillation grades) by Koch-Light and 1,4 dioxan (Analar) by BDH. Dextran T-70 was supplied by Pharmacia; gelatin by the Sterling Gelatine Co. Ltd, and Norit-A-charcoal by Sigma Ltd. Oestradiol-17f(E2) and scintillation grade 1,4-bis-2-(4-methyl-5-phenyloxazolyl) benzene (dimethyl POPOP) were purchased from Koch-Light and the 2-5 diphenyloxazole (PPO) from Fisons Scientific Apparatus Ltd, Loughborough. (2,4,6,7-3H4) oestradiol-17/3 (100 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, Bucks. The antiserum, anti-E2 17/3-6-BSA (Dean, Exley and Johnson, 1971) was a gift from Dr E. H. D. Cameron, Tenovus Institute, Cardiff. It was stored at -15°C and diluted to a concentration of 1 in 30,000 for use in the assay. The assay was performed in disposable glass tubes (12 x 75 mm). After use, other glassware was immersed overnight in a chromic acid bath, thoroughly rinsed with tap and deionized water and oven dried.
Collection of blood samples.-Peripberal venous blood (approximately 10 ml) was collected daily between 9 a.m. and 12 noon. The blood was allowed to clot, centrifuged and the serum removed and stored at -200C.
Radioimmunoassay of oestradiol-17f.-Oestradiol-17,B was extracted from samples of serum after addition of 15% v/v carbonate buffer (pH 9.4) by shaking with ether. The ether layer was removed with a Pasteur pipette and evaporated to dryness at 40°C under nitrogen. The assay was based on that described by Cameron and Jones (1972).
The high specificity of the anti-E217fl-6-BSA permitted precise determination of oestradiol without preliminary chromatographic separation. The cross-reaction with oestrone and oestriol was about 1% (Dean et al., 1971).
The antiserum, diluted to a concentration of 1 in 30,000 in 0-1% gelatin PBS, was then added and the mixture incubated overnight at 4°C. Free and bound fractions were separated by means of dextran-charcoal suspended in 0.1% gelatin PBS and the radioactivity of an aliquot of the supernatant fluid was counted by liquid scintillation spectrometry (Fig. 1).
When sera from premenopausal women were studied duplicate samples of 0-3 ml were extracted with ether and the extracts evaporated to dryness in the assay incubation tubes, where they were redissolved directly in gelatin PBS. Each cycle was assayed as a unit so that fluctuations would represent intra-rather than interassay variation.
When larger samples were required for postmenopausal sera (0.6-1-2 ml) 5 ml of ether were used for the extraction; the residues were redissolved in 800 ,ul ethanol and aliquots of 500 ,ll were transferred to the assay tubes and evaporated to dryness.
This procedure was also followed when internal tracer standard (2000 d/min tritiated oestradiol) was added to the plasma to test efficiency of extraction. Aliquots of 100 ,ul were transferred to vials and evaporated to dryness in the presence of a drop of solution of " cold " E217/3 in ethanol (10 dul/ml) for determination of the radioactivity in toluene scintillator. E217,p standards (5-150 pg) were prepared in ethanol solution. A constant volume (100 pl) of appropriate solution was delivered into each of the standard tubes, and 100 ,ul ethanol to all other tubes in the assay. Evaporation under nitrogen took place at 40°C. The appropriate volume of ether was added to all standard and control tubes and evaporated to dryness to compensate for any effect of the solvent on the standard curve.
Meaisurement of radioactivity.-An aliquot of 500 ,ul of the supernatant fluid from each sample was added to a vial which contained 10 ml of Bray's scintillator (Bray, 1960) and counted in a Nuclear Chicago Isocap/300 liquid scintillation system using Programme lB. Counting efficiency was approximately 25%. In order to check the efficiency of extraction, 100 ,ul of ethanolic extract plus added " cold " E217/3 were evaporated to dryness in a vial and the content of radioactivity determined after addition of 5 ml toluene scintillator which contained 033% PPO and 0.01% dimethyl POPOP. Effi- Criteria of the assay.-All assays were performed in duplicate and each assay included a standard curve.
Specificity: The only major cross-reacting steroid was 6-keto-17,B-oestradiol which does not occur in human biological fluids. Cross reaction to oestrone was 0.97% and to oestriol 1.2%.
Sensitivity: For each assay, 6 " water blanks " were taken through the whole procedure. The mean blank was 1-2 ± 0-2 (s.e. mean)pg for 68 assays; this indicates a sensitivity in terms of 95% confidence limits of a -reagent blank of 4-4 pg.
Accuracy: The accuracy of the method was estimated by recovery of added oestradiol. The mean recovery was 84-4 + 2.5% (s.e. mean) at the 20 pg/ml level and 83.3±2.2% at the 50 pg/ml level (n = 68). Precision: The interassay precision was clculated from 18 separate duplicate determinations of quality control samples prepared from pooled plasma. This gave a mean value of 61-0 + 3.3 (s.e. mean)pg/ml. (coefficient of variation 9-8%). Duplicate values from 4 complete menstrual cycles taken at random showed a standard error of reproducibility of 11 2 and a coefficient of variation of 8-9%.
Mea8urement of luteinizing hormone.-In 6 of the premenopausal women serum luteinizing hormone was also estimated by a standard double antibody procedure. The MRC HPLH Preparation 68/40 was used as the reference material.

RESULTS
cycles varied greatly (17-39 days) so that Premenopaasal women a reference point other than the first (a) The normal range.-A constant day of the cycle was needed for compattern was found in 36 of the 40 cycles parative purposes. Others have used the studied (Fig. 2). The lengths of the time of the mid-cycle peak of luteinizing hormone as a reference point. In the 6 subjects in whom luteinizing hormone was measured, this peak was consistently 24 hours after the ovulatory oestradiol peak (Fig. 3). Consequently the latter was chosen as the reference point and referred to as Day 0. Preceding days were referred to as negative numbers and the days following as positive numbers. When orientated in this way, the mean concentrations of oestradiol-17,/ in 30 completely studied cycles were as follows: (1) follicular phase (Days 11 to 4) 35.3 ± 4.39 (s.e. mean)pg/ml; (2) ovulatory peak (Day 0) 192-9 AE 12-7 (s.e. mean)pg/ml; (3) luteal phase (Days +4 to + 12) 67-3 ± 1-47 (s.e. mean)pg/ml. (Fig. 4). Six incomplete cycles in which Day 0 could not be exactly defined were omitted from the statistical analyses. (b) Effect of age. During the luteal phase there were marked differences in concentration of oestradiol between women in the third, fourth and fifth decades of life (Fig. 5).
When examined by the method of paired statistical comparisons the concentrations in women in the fourth decade were significantly higher than those of women in the third (P < 0.01). These in turn were significantly higher than those of women in the fifth decade (P <-0.001). The magnitude of the ovulatory peak was greater during the fourth decade than in the other two decades (Table). When the concentrations in the fourth and third decades were compared the difference was significant (P < 0 01). (c) Abnormal cycles.-Four subjects had a pattern which differed from the others. In 2 there were high peaks (240 and 220 pg/ml) on the fourth and fifth days respectively; in a further cycle from one of these the abnormality was no longer apparent. The other 2 subjects, aged 47 and 53 years, had no ovulatory peak but plateaux were apparent later in the cycle.

Postmenopausal women
In postmenopausal women the concentrations were consistently low in each of the 25 subjects (mean 5.7 i 0 33) (s.e. mean)pg/ml.

Menopausal women
Bizarre results were found in each of the 3 women studied at the time of the menopause (Fig. 6).

DISCUSSION
The advantages of the method used in this study were its specificity and the high degree of sensitivity. Only small quantities of serum were required from both premenopausal (0 3 ml) and postmenopausal women (0 6-1*2 ml).
The considerable variation in the length of the menstrual cycle (17-39 days) and in particular the variation in timing of the ovulatory peak (8th-18th day) demanded that, for comparative purposes, cycles be synchronized around a reference point other than the first day of menstruation. Most authors (Abraham et al., 1972;Mishell et al., 1971) have related their data to the sharply defined mid-cycle peak of luteinizing hormone. Our results and those of other workers (Mishell et al., 1971;Korenman and Sherman, 1973) indicate that the peak of luteinizing hormone occurs consistently 24-36 hours after the ovulatory peak of oestradiol-17/3. The luteinizing hormone peak is transient and may be easily missed without comprehensive serial assays (Shaaban and Klopper, 1973), so it seemed simpler to use the ovulatory peak of oestradiol itself as the reference point. The results for the 30 complete cycles studied in this way were remarkably consistent and the standard error throughout the range was small.
It is difficult to explain why women in the fourth decade of life had higher concentrations of oestradiol than younger and older women. Women in their late twenties and thirties are known to have a more stabilized menstrual pattern, whereas younger and older women have a tendency to be more erratic, especially with regard to the length of their cycles (Treloar et al., 1967).
In 2 women a peak of oestradiol occurred very early in the cycle while menstruation was still taking place and in one at least it was related to a peak of luteinizing hormone. It is suggested that the peak of oestradiol normally acts as the trigger for ovulation (Vande Weile et al., 1970) but it is difficult to believe that ovulation was taking place at this very early stage of the cycle. Estimations were repeated in one of these subjects throughout a further cycle, which followed a normal pattern. The pattern in a further 2 subjects also differed from the normal. The ovulatory peak of oestradiol did not occur but a plateau in the latter part of the cycle was present. Both subjects were approaching the age of the menopause and it may be that their cycles were anovulatory.
As expected, the concentrations of oestradiol-17,8 in postmenopausal women were extremely low. The source of these small amounts of oestrogen may be from endogenous conversion of androgens and not from ovarian secretion (Barlow, Emerson and Saxena, 1969;Longcope, 1971).
The results from 2 of the 3 women whose periods had recently ceased were high, and this suggests that there may be considerable ovarian function for at least one year after cessation of menstruation. In one of these women the concentration had fallen dramatically when she was studied a second time one year after her periods had ceased.
With these few exceptions, the pattern throughout the study was remarkably consistent and the data seemed to provide a normal baseline with which to compare patients with endocrine abnormalities and in particular, for our purposes, those with diseases of the breast. A study in which the serum concentrations of oestradiol-17,8 in premenopausal women with fibroadenosis and cystic disease of the breast and in women with cancer of the breast were compared with the normal pattern, has just been completed. The results will be reported.