320 biologically URINARY EXCRETION OF ŒSTROGENS DURING THE MENSTRUAL CYCLE J. B. BROWN M.Sc. N.Z., Ph.D. Edin. From the Clinical Endocrinology Resea...

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URINARY EXCRETION OF ŒSTROGENS DURING THE MENSTRUAL CYCLE J. B. BROWN M.Sc. N.Z., Ph.D. Edin. From the Clinical Endocrinology Research Unit Research Council), University of Edinburgh










mixture of substances with different

activities, the results obtained have little quantitative significance. Smith et al. (1938) and Smith and Smith


cestrogens in the urine during different phases of the menstrual cycle. The most constant finding has been two peaks of excretion, one about the time of ovulation and the other during the luteal phase. However, most of the results have been obtained using bio-assay methods

(1952) obtained more reliable results by separating the oestrogens, before bio-assay, into cestriol, oestrone, and cestradiol fractions, and expressing their results in terms of the weights of pure crystalline oestrogens. Jayle and Crepy (1952) used the Kober colour reaction to measure total oestrogens and reported the results obtained


two menstrual


But these

figures, too, have limited quantitative meaning, since cestriol, cestrone, and oestradiol-17B all give different colour intensities in the Kober reaction. Engel et al. (1950), and Migeon (1953) CESTROGEN LEVELS




Engel (1950), attempted to



(Collected from



the urinary oestrogens excreted during the menstrual cycle by using counter-current distribution to separate and identify the individual oestrogens and fluorimetry to measure them. However, fluorescing contaminants interfered considerably with the measure. ment of cestrogens, and the main significance of the work was that Migeon obtained good evidence that oestnot, cestrone, and oestradiol are all present in pooled menstrual. cycle urine. More recently a convenient, accurate, and apparently specific chemical method for separately estimating cestriol, cestrone, and aestradiol-17B in human urine has been developed (Brown 1955a and b), and it is now possible measure

Fig. (-Subject ) ;; aged 36; para.-3. This figure and figs. 2-8 show the amounts of aestriol. oestrone, and oestradiot excreted per 24 hours, and also the variations in basal temperature. «<)«***’ = Menstrual period.

expressed in terms of animal units of total (see Gustavson et al. 1938, Pedersenactivity cestrogenic Bjergaard and Tonnesen 1948). Since at least three oastrogens (cestriol, oestrone, and oestradiol-17p) have been found in human urine, and it is erroneous to measure and have been



Fisher, M. (1954) Med. Offr, 92, 187. Foley, H., Parrot, L. (1954) See Bull. Hyg., Lond. 29, 602. Frew, H. (1954) Personal communication. Gaisford, W., Griffiths, M. (1951) Brit. med. J. ii, 702. Gedde-Dahl, T. (1952) Amer. J. Hyg. 56, 139. Gernez-Rieux, Ch. Breton, Houcke, Méreau, and Sevin (1947) Rev. Tuberc., Paris, 11, 727. Geser, A. (1954) Dis. Chest, 26, 62. Hertzberg, G. (1947) Tubercle, 28, 1. (1948) Achievements of B.C.G. Vaccination. Oslo. (1949) Acta tuberc. scand. 23, 338. International Tuberculosis Campaign (1952) Final report on work. July 1, 1948, to June 30, 1951. Copenhagen. Irvine, K. No. (1949) B.C.G. Vaccination in Theory and Practice. —


(1954) B.C.G. and Vole Vaccination. London. MacDougall, I. A., Mikhail, J. R., Tattersall, W. H. (1953) Brit. med. J. i, 64. McKinstry, R. N., Darling, A. S. (1953) Publ. Hlth, Lond. 66, 191. Meyer, J. (1954) Amer. Rev. Tuberc. 70, 402. Ministry of Health (1950) Memorandum 322/B.C.G./revised. O’Meara, R. A. Q., Price, D. (1953) Personal communication. Oppers, V. M. (1952) Rev. Tuberc., Paris, 6, 992. Palmer, C. E. (1952) Lancet, i, 935. Refvem, O. (1954) Acta tuberc. scand. 149, suppl. 294, p. 121. Research Committee of Tuberculosis, Japan (1954) Recent Investigations on B.C.G. Vaccine. Rosenthal. S. R. (1937) Amer. Rev. Tuberc. 35, 678. St. Ultan’s Hospital, Dublin (1953) Annual report, 1952. Sayé, L. (1953) Acta phthisiol. no. 9. Seibert, F., DuFour, E. H. (1954) Amer. Rev. Tuberc. 69, 585. Thrap-Meyer, H. (1954) Acta tuberculosea scandinavica, 29, 173-192. Ustvedt, H. J. (1950) Bull. World Hlth Org. 2, 501 ; Tubercle, 31, —


Fig. 2-Subject 2 ;aged 34 ;nullipara.

321 reliable and detailed measurements of their excretion. I report here the results of applying this method to a study of the urinary excretion of oestrogens during a number of menstrual cycles of apparently normal women. This was a necessary preliminary to the study of abnormal conditions. A preliminary communication on some of these results has already been to make more

presented (Brown 1955a). Method

Complete 24-hour urine specimens were collected daily through the whole menstrual cycle of eight apparently normal women whose ages ranged from 17 to 40 years. In two cases (subjects 7 and 8) two whole menstrual cycles were


The amounts of costriol, cestrone, and

aestradiol-17&bgr; in each 24-hour urine specimen were determined by the chemical method described by Brown (1955b). Morning basal temperatures were taken orally throughout each cycle to correlate the oestrogen levels with times of expected ovulation. Results

The results are shown in figs. 1 to 8 and in the table. In both cases where two cycles were studied, the

Fig. 4-Subject 4aged 28; nuilipara.

of the cycle the changes in the oestrone one another, but the changes and cestradiol levels in the oestriol levels tend to lag about 24 hours behind the other two. This lag phenomenon is sometimes masked by the 24-hour interval over which the urine specimen is collected, but even so is clearly seen at the time of the ovulation peak and subsequent drop. After suddenly decreasing, the oestrogen levels tend to increase again to a second maximum (the " luteal maximum "), which occurs on about the 21st day, and then decrease before the onset of menstruation. During the luteal phase the cestriol lag is not apparent. The lowest levels are often reached several days after the onset of menstruation. While the pattern of cestrogen excretion is constant, the actual amounts found in the urine of different subjects vary over a considerable range. Those found at the onset of menstruation, at the ovulation peak, and at the luteal maximum are summarised in the table. No abnormal symptoms were recorded by any subject during the cycle studied. Furthermore, three subjects (4, 7, and 8), two of whom (7 and 8) were still collecting urine for assay, became pregnant shortly afterwards ; and a parous subject, whose oestrogen excretion was similar to that of subject 6, became pregnant during the cycle being studied. There is therefore no evidence of correlation between the relative or absolute amounts of oestrogens excreted and the subjective symptoms, parity, On the other hand it is or fertility of the woman. interesting to note that the youngest subject (5) excreted the lowest amounts of oestrogens.

During this phase

Fig. 3-Subject 3aged 40nullipara.

results obtained during the second cycle of each individual were almost identical with those obtained during the first cycle. Consequently only one cycle from each subject is included in the figures. A number of features common to all the cycles is apparent. The amounts of the three oestrogens excreted generally rise and fall together. The oestradiol levels are almost always lower than the corresponding oestrone levels, the ratio between the two being approximately constant at 1 : 2. There is no such constant ratio between the cestriol and oestrone levels, for the cestriol level may be equal to or greater than the corresponding oestrone level. The amounts excreted are’lowest during the first week of the cycle and begin to rise at about the 7th day to reach a well-defined maximum (the " ovulation peak ") at about the 13th day-or, more accurately, 14 days before the onset of the next menstrual period. This maximum occurs about the time of the temperature rise and is presumably related to ovulation. The peak is followed by a sudden decrease in oestrogen excretion.


Fig. 5-subject 5aged 17; nullipara.

322 Discussion

The results reported here show two peaks of oestrogen excretion during the menstrual cycle, and in this respect similar to those

other workers. Quantisimilar to those reported by Smith et al. (1938) and Smith and Smith (1952). They present, however, a more detailed picture of some of the events which occur during the menstrual cycle. We see, for instance, that the rise to the ovulation peak is usually gradual; and the most satisfactory explanation for this is an increasing production (and secretion into the blood) of oestrogen from a rapidly enlarging follicle, rather than a sudden release of oestrogen-rich follicular fluid at ovulation. At the peak itself both factors may operate. The highly characteristic ovulation peak was a are

tatively, too, they

reported by are

Fig. 7-Subject 7aged 33; para-2.

oestrone and oestradiol levels should follow ovulation very closely and indicate its time more accurately than the temperature rise, which seems to depend on the more gradual and variable increase in progesterone production which also takes place at this time (Davis and Fugo 1948). The luteal maximum is probably due to oestrogens produced by a growing and then regressing corpus luteum. One possible explanation for the apparent absence of the cestriol lag at this time is that both cestriol and oestradiol-17[3 may be produced by the corpus luteum, and that the urinary oestriol may therefore be derived directly from the corpus luteum as well as from the normal metabolism of œstradiol-17&bgr;. Since, unless the circumstances are exceptional, the day-to-day variation in oestrogen excretion seems to be more important than the amounts excreted at any one time, single random estimates of urinary oestrogens in women during reproductive life will have little significance unless the specimen has been collected at a relevant and accurately known time in the cycle. Serial estimations

Fig. 6-Subject 6aged 32para-2.

constant finding in all the normal cycles studied. Since neither ovulation peak nor luteal rise have yet been observed in this laboratory in any woman having a uterus and suffering from ainenorrliceal the finding of such a peak may be good evidence of the occurrence and time of ovulation. The pattern of the three oestrogens about the time of ovulation-namely, oestradiol levels about half the oestrone levels, and the cestriol levels lagging a day behind-is essentially similar to that observed after the intramuscular injection of cestradiol17p (Brown 1955a). This furnishes further support for the generally accepted view that oestradiol-17(3 is the primary ovarian hormone at this time. The sudden decrease in oestrogen excretion after the ovulation peak could be due to a sudden change in follicular function caused by rupture at ovulation. If this is so, the drop in

Fig. 8—Subjest 8 ;aged 27, para-2.

323 umbilicus. The abdominal wall and peritoneum were locally infiltrated with 1% lignocaine hydrochloride (’Xylocaine’) and the injection needle was introduced through the anaesthetised area. It was possible to ascertain when the needle had been inserted into the uterine wall because the abdominal muscles moved relatively more than the uterus when the woman took a deep breath ; this caused the needle head to be deflected sharply downwards. When the needle was properly positioned, neither blood nor liquor amnii was withdrawn by suction on the

appear to be necessary before any accurate assessment of ovarian function can be made employing these methods.

Summary Results obtained using a new chemical method for estimating the urinary excretion of cestriol, cestrone, and œstradiol-17&bgr; during 10 normal menstrual cycles in women are recorded and discussed. I wish to thank Prof. G. F. Marrian, F.R.S., and Prof. R. J. Kellar, for their interest in this work. I am also grateful


to the female members of the staff and wives of colleagues who contributed the specimens, and to Mr. H. A. F. Blair and Miss

the nature and main features of the explained to the patient before -the investigation examination was started. Their most frequent reaction was one of interest and willingness to cooperate. The procedure caused them very little inconvenience. They felt only the introduction of the local anaesthetic through the skin and later a very transient, sharp, and deeply located prick when the uterine peritoneum was pierced. Many of them said they felt nothing. The injection of the radio-sodium was made as rapidly as possible. Care was taken to prevent leakage along the needle track by emptying the* syringe while the needle was fully inserted ; the skin was wiped free from blood and saline solution after the needle had been withdrawn. A screened end-window y-ray Greiger-Muller counter was immediately placed over the site of the injection and, to maintain its position relatively constant, fixed to the skin with adhesive tape. During the period of observation the counting-rates over the injection site were recorded at 1/2-min-ate intervals on a counting-rate meter. The results were expressed as the " time to half-value " after a correction had been made for the rising background caused by an increasing amount of 24Na appearing in the blood-stream after its removal from the injection pool. This corrected counting-rate (c - b) was calculated for each observed counting-rate (c) from the following formula :


Janet Mackie for their skilled technical assistance. REFERENCES —

hagen, 1, 38.

Smith, G. V. S., Smith, O. W., Pincus, G. (1938) Amer. J. Physiol. 121, 98. Smith. O. W., Smith, G. V. S. (1952) Recent Progr. Hormone Res.

7, 209.




S. B. OSBORN B.Sc. Lond., A.Inst.P.




H. PAYLING WRIGHT B.Sc., Ph.D. Lond., L.M.S.S.A.

ground =


THE thesis that pre-eclampsia might be associated with placental ischaemia led Browne and Veall (1953) to attempt the measurement of placental blood-flow. Using an isotope technique they were able to locate the placenta in women during the last trimester of pregnancy. In patients in whom the placenta was placed anteriorly they injected isotonic saline solution containing 24N aCI through the uterine wall into the placenta. Browne and Veall showed that the rate at which the injected material was removed from the placental site varied considerably ; in normotensive women the removal was extremely rapid, whereas in patients with raised blood-pressure, either from pre-eclampsia or from essential hypertension, it was

notably delayed. might result from inade-

quate development of, or from pathological changes within, the

placenta itself or from a reduction of the bloodsupply from the uterine wall. To elucidate this question it seemed of value to attempt comparative measurements of uterine (as opposed to placental) blood-flow in normall and in pre-eclamptic pregnancies. Method

In the present series of observations 0.3-0-4 ml. ofE isotonic saline solution containing 24NaCl with an activity of 5-10 µC was injected directly into the uterine wall. As a standard procedure the area chosen was the lowerr left quadrant, about 4 em. below and to the side of the3 .



initial count per min., b = corrected backcount per min., bi initial background count per min., b2 = final background count per min., and observed counting-rate to be corrected for backc ground. The final background (b2) was obtained by observing the counting-rate over the contralateral area of the uterus at the end of the experiment. For this purpose it was assumed that this count represented the radioactivity in a cone of tissue corresponding to that containing the injection site but free from radiations arising from the residual pool of the radioactive material. This technique was essentially similar to that described by Kety (1948, 1949) to measure the clearance-rate of locally injected radioactive sodium from the site of injection. Though such clearance depends on many interacting factors (McGirr 1952) it is essentially based upon the postulate that the rate of removal of a diffusable substance is proportional to, and offers a measurement of, local tissue circulation. It must, however, be emphasised that the technique measures the effectiveness of local tissue circulation rather than the absolute blood-flow in the area. In the present study certain factors which influence the clearance-rate of the injected sodium may be considered to be uniform. Thus, the temperature of the uterine wall may be assumed to be independent of environmental conditions ; since the patient is confined to bed, moreover, the restricted skeletal muscle activity which is possible can have little influence on the uterine blood-flow. Finally, the uniformity of texture of the unstriped musculature of this organ renders it improbable that spurious results will arise from leakage- of the tracer substance from the site of injection along fascial where I



This diminished clearance-rate



Brown, J. B. (1955a) Mem. Soc. Endocrin. 3 (in the press). (1955b) Biochem. J. (in the press). Davis, E. D., Fugo, N. W. (1948) J. clin. Endocrin. 8, 550. Engel, L. L. (1950) Recent Progr. Hormone Res. 5, 335. Slaunwhite, W. R., Carter, P., Nathanson, I. T. (1950) J. biol. Chem. 185, 255. Gnstavson, R. G., Mason, L. W., Hays, E. E., Wood, T. R., D’Amour, F. E. (1938) Amer. J. Obstet. Gynec. 35, 115. Jayle, M. F., Crèpy, O. (1952) Ann. Biol. Clinique, 10, 44. Migeon, C. J. (1953) J. clin. Endocrin. & Metab. 6, 674. Pedersen-Bjergaard, K., Tønnesen, M. (1948) Acta endocr., Copen-


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