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STEROID ASSAYS AND PREOVULATORY FOLLICULAR DEVELOPMENT IN HUMAN OVARIES PRIMED WITH GONADOTROPHINS
The Lancet Saturday
STEROID ASSAYS AND PREOVULATORY FOLLICULAR DEVELOPMENT IN HUMAN OVARIES PRIMED WITH GONADOTROPHINS
September
23
hyperfolliculation in infertile women. This paper presents analyses of the number and size of graafian follicles immediately before ovulation in patients given gonadotrophic hormones, and of the levels of oestradiol - 17 and progesterone in the follicular fluids.
R. G. EDWARDS
Physiological Laboratory, Cambridge University P. C. STEPTOE
G. E. ABRAHAM
Department of Obstetrics and Gynecology, Harbor General Hospital, Torrance, California, U.S.A. E. WALTERS
Agricultural
Group, Cambridge
University J. M. PURDY Physiological Laboratory, Cambridge University K. FOTHERBY
Department of Steroid Biochemistry, Royal Postgraduate Medical School, Hammersmith Hospital, London W.12 On average, 5-6 follicles were found in human ovaries primed with gonadotrophins. This number was only slightly correlated with the amount of urinary oestrogen and the amount of hormone administered. The levels of oestrogen in urine were determined by the number of large follicles (1·5 cm. in diameter or larger), each one contributing on average 15·37 µg. per day shortly before ovulation, and not by the total number of follicles. Some exceptional patients with few large follicles excreted large amounts of urinary oestrogen. The concentrations of œstradiol-17&bgr; and progesterone in follicular fluids were closely correlated, but there was considerable variation between follicles. Concentrations of œstradiol were between <12·5 and 1700 ng. per ml., and of progesterone between 65 and 18,000 ng. per ml. There was no correlation between the concentrations of steroids in follicular fluid and the size of the follicle, although concentrations were generally higher in patients given large amounts of H.M.G. A few follicles known to be immediately preovulatory also contained varying amounts of œstradiol and
Summary
progesterone. Introduction LITTLE is known about the growth and steroidal activity of the human ovarian follicle. Endogenous hormones or clomiphene can be given to induce 7778
Material and Methods Patients
Oldham General Hospital, Oldham
Research Council Statistics
1972
The patients were infertile because of occluded or missing oviducts. They were menstruating normally and their cycles were apparently ovulatory. They were attending an infertility clinic for examination of their ovarian response to exogenous hormones and for collection of preovulatory oocytes.1 Human gonadotrophins were used to induce the growth of follicles and their preovulatory changes. The patients were given a total of 150-900 l.u. of human menopausal gonadotrophin (H.M.G.,’Pergonal’), in three equal injections on days 2-3, 4-5, and 7-8 of their cycle. One injection of human chorionic gonadotrophin (H.C.G., Pregnyl’), almost always of 5000 i.u., was given between days 9 and 11. Laparoscopy was carried out 28-32 hours after the injection Of H.C.G., when ovulation is imminent 2,3 and the number of graafian follicles can be counted in I
most
patients.1
Follicles The contents of individual graafian follicles were aspirated separately. Clean samples were obtained from approximately a third of the follicles, and small amounts of heparin were added to other samples slightly con-
taminated with blood. Clean fluids were collected from up to 8 follicles in some patients. Graafian follicles were classified as large, medium, or small, according to their appearance under the laparoscope, the approximate diameters being 1.5 cm. or more, 0-75-1-0 cm., and 0-5 cm. or less. Follicles found deep in the ovary made classification difficult, so the volume of fluid taken from each follicle was estimated to confirm size. Fluids, granulosa cells, and oocytes were aspirated with a syringe or a small aspirator.1 Some graafian follicles could be classified as preovulatory or non-ovulatory from examination of the oocyte. Preovulatory follicles yielded an oocyte in diakinesis or a later stage of meiosis, or one that was fertilised within a few hours. Oocytes from non-ovulatory follicles If an oocyte was not recovered the were in dictyotene. follicle was " unclassified ". Hormone
Assays
The concentration of urinary oestrogens on the day after injection of H.C.G. was assayed by G. D. Searle & Co. Scientific Services Ltd., using the method described by Brown et awl.4 Concentrations of cestradiol-17pin follicular fluids were measured by radioimmunoassay.5 Proteinbinding assays of progesterone were carried out according to the method developed by johannson.6 Total progestins are measured bv this method and, in addition to nro-
612 gesterone, any 17 ex-hydroxyprogesterone in follicular fluid would also be measured. Towards the end of this series some samples of follicular fluid were assayed for progesterone by radioimmunoassay.’ Results
Correlation between Nufnber and Size of Follicles and Excretion of Urinary (Estrogen Observations were made on 68 patients, of whom 5 had only a single ovary. The total number of graafian follicles in a patient varied between 3 and 19, and the number of large follicles between 0 and 7 (fig. 1). The mean number of follicles per ovary increased slightly but non-significantly from 4-9 in patients excreting less than 20 g. urinary oestrogen a day to 6-2 in those with more than 100 jjLg. a day. In earlier workthe total number of follicles was independent of the amount of H.M.G. given over a range of 2251.
675
i.u.
multiple regression analysis carried out to investigate the relation between the levels of urinary oestrogen and the size of follicles gave the following regression equation: A
"1"’B1’’’’’’’
-1
I
Fig. 1-Number of large follicles in relation
"’......."".."-""
to amount of
urinarv cestroeen.
Steroids in Follicular Fluids The concentrations of oestradiol-17(3 and progesterone in follicular fluids were analysed in relation to follicular size, the levels of urinary oestrogens, the size of the follicle, and the type of follicle (preovulatory or
2. y=
-8-10+2-40 T+15-37 L
where y is the excretion of urinary oestrogen (.g. per day), T is the total number of follicles, and L is the number of large follicles. Thus the partial regression coefficients were 2-40+1’74 (p > 0.05) for total follicles, and 15-37+3-71 (p
following conclusions
were
drawn from the
(a) The total number of graafian follicles did a major influence on the amount of oestrogen in urine. This was established by the non-significance of the coefficient of T in the equation; this coefficient is a measure of the effect of T on y, the other variables, in this case L, being kept
non-ovulatory). i. Relative concentrations of aestradiol-17&bgr; and progesterone. were analysed, most of them from unclassified follicles. There was considerable variation in the concentrations of steroids in different follicles, values for oestradiol being between < 12-5 and 1700 ng. per ml.
-137 follicular fluids
analysis: not exert
.
FOLLICLES PRIMED WITH EXOGENOUS
GONADOTROPHINS. o
constant.
FOLLICLES ON DAY 13 OF A PATIENT WITH 28-DAY MENSTRUAL CYCLES.
(b) Large follicles exert a significant influence on the urinary levels of oestrogen, as measured by the coemcient of On average, each large L. follicle contributed an increment of 15-37 g. per day of urinary oestrogen. This value cannot be used predictively for individual patients, but merely
FOLLICLES ON DAY 12 OF A PATIENT WITH 35-DAY
MENSTRUAL CYCLES.
provides an average value for each large follicle. There were some exceptional observations -for example, one patient with 4 large follicles excreted low levels of oestrogen, and another with 1 large and 18 smaller follicles excreted large amounts of oestrogen. The relation between the number of large follicles and the amount of urinary oestrogen is plotted in 6s. 1.
-
.....
I
in
follicular fluids. Fig. 2-Relation between concentrations of cestradiol-17j3 and progesterone There was insufficient fluid from 2 follicles to complete the assays for progesterone.
( )
613 and for progesterone between 65 and 18,000 ng. per ml. (fig. 2). The data were submitted to a regression analysis designed to isolate the linear and quadratic components of the relationship between the two steroids. Data from all follicles were pooled, whether preovulatory, non-ovulatory, or unclassified. The equation was found to be:
E2= 37-44+0-178 P-(6’195 x 10-6) p2 E2 is the concentration of oestradiol, and P the concentration of progesterone. Both the linear and quadratic where
coefficients
were
found
to
be
significant (0-178±0-027
and
6-195 x 101-912 x 106, respectively). The
(a)
following
conclusions
were
drawn:
There was a significant linear increase in the levels of aestradiol-17 with increasing levels of progesterone. The linear relation is plotted in fig. 2. was also a progressive decline in increments of oestrogen with increasing levels of progesterone, as shown by the significant quadratic coefficient. Thus, as levels of progesterone rise, the corresponding increase in oestradiol becomes progressively smaller.
(b) There
(c) The
constant value of 37-44 ng. per ml. is the theoretical level of oestradiol in follicular fluid when progesterone levels are zero.
Some fluids were aspirated from graafian follicles of patients in their natural cycle, and 6 of these are indicated in fig. 2. The levels of oestradiol and progesterone in 2 of them taken from a patient on the 13th day of her cycle were similar to those found in primed ovaries. The others were taken from a patient on day 12 of a 35-day cycle. 1 of them contained much less progesterone, while the other 3 from this patient contained very low amounts of both steroids.
ii. Steroid concentrations in relation to the size of the follicles and dose of H.M.G: No correlation could be found between the concentrations of steroids and the size of the Concentrations varied greatly between follicles of the same size, the coefficients of variation being similar for large, medium, and small follicles. As shown below, some of these large follicles might have been
graafian follicles.
preovulatory. The mean values of oestradiol-170 and progesterone in follicular fluids for each dose of H.M.G. were compared. Significant correlations were detected between the concentrations of steroids and the amount of H.M.G. given, in CONCENTRATION OF
CESTRADIOL-17&bgr;
AND PROGESTERONE
(ng./ml.)
log CONCENTRATIONS OF
OESTRAD)OL]7(ngfmO
Fig. 3-Concentrations of
the sense that more steroids were present in the follicular fluids of patients given higher doses of H.M.G. than in those given lower doses, irrespective of the size of the follicle (F for both steroids 6, p < 0.01). =
iii. Steroid levels in follicular fluids in relation to the excretion of urinary cestrogen.-Variations in the steroid concentration of different follicles were compared in patients with high or low levels of urinary oestrogen. Data on 54 graafian follicles were available from 15 patients who were arbitrarily divided into " high " and " low " excretors at a dividing line of 30 µg. a day. In each group the concentrations of steroid in different follicles appeared to be equally variable (fig. 3). In the 9 patients where 4 or more fluids were analysed, 8 possessed follicles with high or low steroid concentrations. Follicles thus appeared to respond independently to the injection of exogenous gonadotrophins and not to respond as a group within a
patient. IN THE FLUIDS OF PREOVULATORY FOLLICLES AND IN ALL FOLLICLES
614 iv. Preovulatory and non-ovulatory follicles.—The samples of follicular fluid included a few from preovulatory follicles. Fluids from a further 9 preovulatory follicles, not included in the previous data, were assayed entirely by radioimmunoassay. The patients had been given between 675 and 900 i.u. of H.M.G. Concentrations of cestradiol-17&bgr;Q in these fluids were similar to those examined earlier, although concentrations of progesterone were generally higher. The two highest values-1900 ng. per ml. of oestradiol and 26,400 ng. per ml. of progesterone-were found in these 9 fluids. These data are included in the table. Concentrations of progesterone in all preovulatory follicles varied from 250 to 26,400 ng. per ml. and those of cestradiol-17&bgr;from 20 to 1900 ng. per ml. The data were too sparse for detailed analysis. Steroid concentrations appeared to be higher in larger follicles and in those from patients given high doses of H.M.G. (table). Concentrations of progesterone but not oestradiol were higher in preovulatory follicles than in all follicles combined (table). This comparison masks differences between the two groups, since values for all follicles also include those for preovulatory follicles. In a few non-ovulatory follicles, concentrations of œstradiol-17&bgr;were between 30 and 900 ng. per ml. and of progesterone between 210 and 5200 ng. per ml.
Discussion
This work has provided data on follicular activity in human ovaries when ovulation is imminent. Stimulation of animal ovaries with gonadotrophins induces many of the small follicles to develop into graafian follicles. In the cow ovary, for example, follicles 2-4 mm. in diameter respond to F.S.H. preparations. 8 2 follicles reach 1 mm. in diameter and form antra during the natural cycle of the rhesus monkey, but only 1 proceeds to ovulation.9 Information on human follicular growth and ovulation is scanty. In amenorrhoeic women given gonadotrophins, growth of a primordial follicle into a follicle capable of responding to H.C.G. requires 10 days, 10 and the interval between the injection of H.c.G. and ovulation is approximately 36 hours.1-3 In the present work, the mean number of graafian follicles found in each ovary was between 5 and 6, irrespective of the dose of H.M.G. given or the levels of urinary cestrogens. This estimate is perhaps the best that can be made of the number of follicles ready to respond to F.S.H. during any one cycle. Some available follicles might not have responded to the H.M.G. and others may have become atretic early in the treatment, as found in mice and sheep during their natural cycle. 11,12 Atresia late in development can probably be excluded because the follicles were apparently healthy, with good granulosa cells and clear follicular fluid. There was considerable variation between patients in the number of graafian follicles-a situation also well known in animals after injection of gonadotrophins. The levels of urinary oestrogen were correlated with the number of large follicles, each one contributing The effect of smaller on average 15-37 µg. a day. follicles was not graafian significant, although their contribution will become clearer as more information accumulates. They undoubtedly secrete steroids, for some patients with large amounts of urinary oestrogen possessed few large follicles and many small ones.
Patients with no large follicles excreted a daily average of approximately 15 µg. of urinary œstrogen—a value similar to that found in amenorrhoeic women with no evidence of follicular growth. 13 The concentrations of œstradiol-17&bgr; in blood would be expected to correlate closely with those in follicular fluid, as found in the sheep. 14 There was a great variation in the concentrations of œstradiol-17&bgr;and progesterone in fluids of different follicles, although the levels of the two steroids within each fluid were highly correlated. The concentrations depended partly on the amount of H.M.G. given to the patient, but not on the size of the follicle, which implies that larger follicles are no more active than smaller follicles, or that substrates diffuse more rapidly from The level of urinary oestrogens must be them. determined by the increased size of large follicles, and not because they are more active per unit of tissue. The sources of the two steroids in follicular fluids have not been fully defined. Increments of progesterone were higher than those of oestrogen, especially in preovulatory follicles, as would be expected since progesterone synthesis precedes oestradiol-17&bgr; in the ovarian metabolism of steroid horGranulosa cells taken from human follicles mones. by laparoscopy have 3&bgr;-hydroxysteroid dehydrogenase activity and can synthesise progesterone from pregnenolone 15; but they are less effective than thecal cells in converting progesterone to 17oc-hydroxyprogesterone, although these cell types in combination can synthesise steroids from acetate. 16, 17 Granulosa cells thus possess 3&bgr;-hydroxysteroid dehydrogenase activity but, in contrast to thecal cells, little aromatising activity, as also found in the mare. 18 The high concentration of progesterone in some of the human follicular fluids does not lead to excretion of unusually
large quantities of urinary pregnanediol (unpublished), although preovulatory progesterone can obviously be synthesised by ovarian follicles. Follicles stimulated by exogenous gonadotrophins may obviously differ from those developing during the natural cycle-for example, through the induced luteinisation of unripe follicles.10° Nevertheless, concentrations of steroids similar to those in primed follicles were found in some follicles sampled during the natural cycle, although others had much lower levels of steroids and especially of progesterone. How can the considerable variation in steroid concentrations of follicular fluids be explained ? This variation affected large preovulatory follicles just as much as medium and small follicles. Perhaps the H.M.G. caused differential stimulation of the follicles, but such an explanation hardly suffices for those follicles which enlarged and became preovulatory yet still contained low concentrations of steroid. A better explanation can be derived from recent work indicating that a regular and inherent pattern underlies follicle formation and utilisation, and the growth and secretions of graafian follicles and corpora lutea. The inherent oestrogenic activity of enlarging follicles seems to impose the regularity of the L.H. surge in midcycle," and the lifespan of the corpus luteum in women and rhesus monkeys appears to depend partly on inherent factors in the gland itself and partly on
615
secretion. 19,20 Some evidence indicates that follicles are formed and used in a definite sequence in mice.21 Two waves of follicular growth occur in human ovaries during the menstrual cycle-at the end of the follicular phase, and during the luteal phase Successive waves of follicular growth and enlargement are found during a single oestrous cycle in mice, sheep, and perhaps cattle, and the largest follicle can be replaced before ovulation by a more actively growing smaller follicle.11,12,23,24 The peaks of cestradiol secretion found in sheep during their oestrous cycle may be associated with follicular enlargement during each wave of follicular growth.25,26 Successive ovulations can be induced within a short time by post-ovulatory injections of gonadotrophins into mice, and both sets of oocytes can be fertilised and develop to full term. 27,28 L.H.
Awareness that follicular ently stable and that several
growth could be inherof follicles develop menstrual cycle makes it waves
within a single oestrous or easier to interpret the results of the present work and to design modified treatments for patients. The nonovulatory graafian follicles could be a heterogeneous population arising partly through the successive injections of H.M.G., some follicles being in early growth and others in later stages, so resulting in the observed variation in steroid levels. Many of these follicles might have responded to further injections of H.C.G., for preovulatory swelling requires less than 24 hours in many species. The preovulatory follicles might also have been in various stages of their development, even though their oocytes were in diakinesis or a closely similar stage of development. In the rabbit, levels of oestrogen and progestin in the fluids of preovulatory follicles rise soon after L.H. release but then decline rapidly to almost zero before ovulation, 29 and levels of plasma-steroids decline in sheep and monkeys while the preovulatory follicle is still intact.19,30,311 The human preovulatory follicles with low amounts of steroid were probably fully mature and awaiting ovulation, while those with higher levels may have been less mature and hastened through their
preovulatory changes by the H.C.G. Improvements in the treatment are possible by modifying the dosage schedule, or using gonadotrophic preparations with a different F.S.H./L.H. ratio for stimulation of follicular growth. Consistent rises in plasma-F.s.H. during follicular growth are achieved by seven or more daily injections of H.M.G. into amenorrhocic patients, although L.H. levels are lower than in the natural cycle.32,33 Treatment with H.M.G. or human pituitary gonadotrophin, when shortened to 1-3 days, is still sufficient to stimulate follicular growth for 10 days in anovulatory patients 10,34;such a brief regimen could be preferable to our present schedule for patients with a normal menstrual cycle, since their endogenous gonadotrophins could support the growing follicles, and the development of large numbers of small graafian follicles might be avoided. Problems could arise through the limited half-life of F.S.H. and L.H. 35,36and if levels of endogenous gonadotrophin were lowered by the feedback of oestrogen from the developing follicles. A wise precaution may therefore be administration of small supplementary
injections of H.M.G. H.C.G. induces a satisfactory L.H. peak, 32,33 the single injection probably sufficing because of its long half life. 37 There appears no need to use preparations with differing F.S.H./L.H. ratios; since urinary and pituitary preparations with a ratio of 1/1 (i.e., that present in pergonal) induced a better response in anovulatory patients than those containing more
F.S.H. 34
We thank the Ford Foundation, the Manchester Regional and District Hospital Management Committee for financial support; Prof. C. R. Austin for his encouragement; Searle Scientific Services for hormone assays; and Dr. A. Brand for permission to use unpublished data.
Hospital Board, and the Oldham
Requests for reprints should be addressed to R. G. E., Physiological Laboratory, Cambridge CB2 3EG, U.K. REFERENCES 1. Steptoe, P. C., Edwards, R. G. Lancet, 1970, i, 683. 2. Edwards, R. G. ibid. 1965, ii, 926. 3. Jagiello, G., Karnicki, J., Ryan, K. J. ibid. 1968, i, 178. 4. Brown, J. B., McLeod, S. C., McNaughton, C., Smith, M. A., Smyth, B. J. Endocr. 1968, 42, 5. 5. Abraham, G.E.J. clin. Endocr. Metab. 1969, 28, 866. 6. Johansson, E. D. B. Acta endocr., Copenh. 1969, 61, 591. 7. Abraham, G. E., Swerdloff, R., Tulchinsky, D., Odell, W. D. J. clin. Endocr. Metab. 1971, 32, 619. 8. Laster, D. B. J. Reprod. Fert. 1972, 28, 285. 9. Baker, T. G. W.H.O. Symposium on the Use of Non-human Primates in Research on Human Reproduction, Sukhumi, 1971 (edited by E. Diczfalusy and C. C. Standley). Karolinska Institute, Stockholm, 1972. 10. Gemzell, C., Johansson, E. D. B. in Control of Human Fertility (edited by E. Diczfalusy and U. Borell). Almqvist & Niksell, Stockholm, 1971. 11. Peters, H., Levy, E. J. Reprod. Fert. 1966, 11, 227. 12. Smeaton, T. C., Robertson, H. A. ibid. 1971, 25, 243. 13. Brown, J. B., Matthews, G. D. Rec. Progr. Hormone Res. 1962, 18, 337. 14. Brand, A. PH.D. thesis, State University of Utrecht, 1970. 15. Younglai, E. V., Edwards, R. G., Steptoe, P. C. Can. J. Biochem. 1972, 50, 233. 16. Ryan, K. J., Petro, Z. J. clin. Endocr. Metab. 1966, 26, 46. 17. Ryan, K. J., Petro, Z., Kaiser, J. ibid. 1968, 28, 355. 18. Younglai, E. V., Short, R. V. J. Endocr. 1970, 47, 321. 19. Knobil, E. W.H.O. Symposium on the Use of Non-human Primates in Research on Human Reproduction, Sukhumi, 1971 (edited by E. Diczfalusy and C. C. Standley). Karolinska Institute, Stockholm, 1972. 20. Vande Wiele, R. L., Bogumil, J., Dryenfirth, I., Ferin, M., Jewelewicz, R., Warren, M., Rizkallàh, T., Mikhail, G. Rec. Progr. Hormone Res. 1970, 26, 63. 21. Henderson, S. A., Edwards, R. G. Nature, 1968, 218, 22. 22. Block, E. Acta endocr., Copenh. 1951, 8, 33. 23. Rajakoski, E. ibid. 1960, suppl. 52, p. 1. 24. Pedersen, T. J. Reprod. Fert. 1970, 21, 81. 25. Scaramuzzi, R. J., Caldwell, B. V., Moor, R. M. Biol. Reprod. 1970, 3, 110. 26. Cox, R. I., Mattner, P. E., Thorburn, G. D. J. Endocr. 1971, 49, 345. 27. Edwards, R. G., Fowler, R. E. J. Endocr. 1958, 17, 223; 1960, 21, 147. 28. Kaufmann, M. H., Whittingham, D. G. J. Reprod. Fert. 1972, 28, 465. 29. Younglai, E. V. ibid. 1972, 30, 157. 30. Moore, N. W., Barrett, S., Brown, J. B., Schindler, I., Smith, M. A., Smyth, B. J. Endocr. 1969, 44, 55. 31. Bjersing, L., Hay, M. F., Kann, G., Moor, R. M., Naftolin, F., Scaramuzzi, R. J., Short, R. V., Younglai, E. V. J. Endocr. 1972, 52, 465. 32. Fogel, M., Rubin, B. L., Ossowski, R. Am. J. obstet. Gynec. 1972, 12, 629. 33. Czygan, P. J., Lehmann, F., Breckwoldt, M., Bettendorf, G. Acta endocr., Copenh. 1972, 70, 417. 34. Bertrand, P. V., Colemann, J. R., Crooke, A. C., MacNaughton, M. C., Mills, I. H. J. Endocr. 1972, 53, 231. 35. Coble, Y. D., Kohler, P. O., Cargille, C. M., Ross, G. T. J. clin. Invest. 1969, 48, 359. 36. Yen, S. S. C., Llerena, L. A., Pearson, O. H., Little, A. S. J. clin. Endocr. Metab. 1970, 30, 325. 37. Midgley, A. R., Jaffe, R. B. ibid. 1968, 28, 1712.
STEROID ASSAYS AND PREOVULATORY FOLLICULAR DEVELOPMENT IN HUMAN OVARIES PRIMED WITH GONADOTROPHINS
September
23
hyperfolliculation in infertile women. This paper presents analyses of the number and size of graafian follicles immediately before ovulation in patients given gonadotrophic hormones, and of the levels of oestradiol - 17 and progesterone in the follicular fluids.
R. G. EDWARDS
Physiological Laboratory, Cambridge University P. C. STEPTOE
G. E. ABRAHAM
Department of Obstetrics and Gynecology, Harbor General Hospital, Torrance, California, U.S.A. E. WALTERS
Agricultural
Group, Cambridge
University J. M. PURDY Physiological Laboratory, Cambridge University K. FOTHERBY
Department of Steroid Biochemistry, Royal Postgraduate Medical School, Hammersmith Hospital, London W.12 On average, 5-6 follicles were found in human ovaries primed with gonadotrophins. This number was only slightly correlated with the amount of urinary oestrogen and the amount of hormone administered. The levels of oestrogen in urine were determined by the number of large follicles (1·5 cm. in diameter or larger), each one contributing on average 15·37 µg. per day shortly before ovulation, and not by the total number of follicles. Some exceptional patients with few large follicles excreted large amounts of urinary oestrogen. The concentrations of œstradiol-17&bgr; and progesterone in follicular fluids were closely correlated, but there was considerable variation between follicles. Concentrations of œstradiol were between <12·5 and 1700 ng. per ml., and of progesterone between 65 and 18,000 ng. per ml. There was no correlation between the concentrations of steroids in follicular fluid and the size of the follicle, although concentrations were generally higher in patients given large amounts of H.M.G. A few follicles known to be immediately preovulatory also contained varying amounts of œstradiol and
Summary
progesterone. Introduction LITTLE is known about the growth and steroidal activity of the human ovarian follicle. Endogenous hormones or clomiphene can be given to induce 7778
Material and Methods Patients
Oldham General Hospital, Oldham
Research Council Statistics
1972
The patients were infertile because of occluded or missing oviducts. They were menstruating normally and their cycles were apparently ovulatory. They were attending an infertility clinic for examination of their ovarian response to exogenous hormones and for collection of preovulatory oocytes.1 Human gonadotrophins were used to induce the growth of follicles and their preovulatory changes. The patients were given a total of 150-900 l.u. of human menopausal gonadotrophin (H.M.G.,’Pergonal’), in three equal injections on days 2-3, 4-5, and 7-8 of their cycle. One injection of human chorionic gonadotrophin (H.C.G., Pregnyl’), almost always of 5000 i.u., was given between days 9 and 11. Laparoscopy was carried out 28-32 hours after the injection Of H.C.G., when ovulation is imminent 2,3 and the number of graafian follicles can be counted in I
most
patients.1
Follicles The contents of individual graafian follicles were aspirated separately. Clean samples were obtained from approximately a third of the follicles, and small amounts of heparin were added to other samples slightly con-
taminated with blood. Clean fluids were collected from up to 8 follicles in some patients. Graafian follicles were classified as large, medium, or small, according to their appearance under the laparoscope, the approximate diameters being 1.5 cm. or more, 0-75-1-0 cm., and 0-5 cm. or less. Follicles found deep in the ovary made classification difficult, so the volume of fluid taken from each follicle was estimated to confirm size. Fluids, granulosa cells, and oocytes were aspirated with a syringe or a small aspirator.1 Some graafian follicles could be classified as preovulatory or non-ovulatory from examination of the oocyte. Preovulatory follicles yielded an oocyte in diakinesis or a later stage of meiosis, or one that was fertilised within a few hours. Oocytes from non-ovulatory follicles If an oocyte was not recovered the were in dictyotene. follicle was " unclassified ". Hormone
Assays
The concentration of urinary oestrogens on the day after injection of H.C.G. was assayed by G. D. Searle & Co. Scientific Services Ltd., using the method described by Brown et awl.4 Concentrations of cestradiol-17pin follicular fluids were measured by radioimmunoassay.5 Proteinbinding assays of progesterone were carried out according to the method developed by johannson.6 Total progestins are measured bv this method and, in addition to nro-
612 gesterone, any 17 ex-hydroxyprogesterone in follicular fluid would also be measured. Towards the end of this series some samples of follicular fluid were assayed for progesterone by radioimmunoassay.’ Results
Correlation between Nufnber and Size of Follicles and Excretion of Urinary (Estrogen Observations were made on 68 patients, of whom 5 had only a single ovary. The total number of graafian follicles in a patient varied between 3 and 19, and the number of large follicles between 0 and 7 (fig. 1). The mean number of follicles per ovary increased slightly but non-significantly from 4-9 in patients excreting less than 20 g. urinary oestrogen a day to 6-2 in those with more than 100 jjLg. a day. In earlier workthe total number of follicles was independent of the amount of H.M.G. given over a range of 2251.
675
i.u.
multiple regression analysis carried out to investigate the relation between the levels of urinary oestrogen and the size of follicles gave the following regression equation: A
"1"’B1’’’’’’’
-1
I
Fig. 1-Number of large follicles in relation
"’......."".."-""
to amount of
urinarv cestroeen.
Steroids in Follicular Fluids The concentrations of oestradiol-17(3 and progesterone in follicular fluids were analysed in relation to follicular size, the levels of urinary oestrogens, the size of the follicle, and the type of follicle (preovulatory or
2. y=
-8-10+2-40 T+15-37 L
where y is the excretion of urinary oestrogen (.g. per day), T is the total number of follicles, and L is the number of large follicles. Thus the partial regression coefficients were 2-40+1’74 (p > 0.05) for total follicles, and 15-37+3-71 (p
following conclusions
were
drawn from the
(a) The total number of graafian follicles did a major influence on the amount of oestrogen in urine. This was established by the non-significance of the coefficient of T in the equation; this coefficient is a measure of the effect of T on y, the other variables, in this case L, being kept
non-ovulatory). i. Relative concentrations of aestradiol-17&bgr; and progesterone. were analysed, most of them from unclassified follicles. There was considerable variation in the concentrations of steroids in different follicles, values for oestradiol being between < 12-5 and 1700 ng. per ml.
-137 follicular fluids
analysis: not exert
.
FOLLICLES PRIMED WITH EXOGENOUS
GONADOTROPHINS. o
constant.
FOLLICLES ON DAY 13 OF A PATIENT WITH 28-DAY MENSTRUAL CYCLES.
(b) Large follicles exert a significant influence on the urinary levels of oestrogen, as measured by the coemcient of On average, each large L. follicle contributed an increment of 15-37 g. per day of urinary oestrogen. This value cannot be used predictively for individual patients, but merely
FOLLICLES ON DAY 12 OF A PATIENT WITH 35-DAY
MENSTRUAL CYCLES.
provides an average value for each large follicle. There were some exceptional observations -for example, one patient with 4 large follicles excreted low levels of oestrogen, and another with 1 large and 18 smaller follicles excreted large amounts of oestrogen. The relation between the number of large follicles and the amount of urinary oestrogen is plotted in 6s. 1.
-
.....
I
in
follicular fluids. Fig. 2-Relation between concentrations of cestradiol-17j3 and progesterone There was insufficient fluid from 2 follicles to complete the assays for progesterone.
( )
613 and for progesterone between 65 and 18,000 ng. per ml. (fig. 2). The data were submitted to a regression analysis designed to isolate the linear and quadratic components of the relationship between the two steroids. Data from all follicles were pooled, whether preovulatory, non-ovulatory, or unclassified. The equation was found to be:
E2= 37-44+0-178 P-(6’195 x 10-6) p2 E2 is the concentration of oestradiol, and P the concentration of progesterone. Both the linear and quadratic where
coefficients
were
found
to
be
significant (0-178±0-027
and
6-195 x 101-912 x 106, respectively). The
(a)
following
conclusions
were
drawn:
There was a significant linear increase in the levels of aestradiol-17 with increasing levels of progesterone. The linear relation is plotted in fig. 2. was also a progressive decline in increments of oestrogen with increasing levels of progesterone, as shown by the significant quadratic coefficient. Thus, as levels of progesterone rise, the corresponding increase in oestradiol becomes progressively smaller.
(b) There
(c) The
constant value of 37-44 ng. per ml. is the theoretical level of oestradiol in follicular fluid when progesterone levels are zero.
Some fluids were aspirated from graafian follicles of patients in their natural cycle, and 6 of these are indicated in fig. 2. The levels of oestradiol and progesterone in 2 of them taken from a patient on the 13th day of her cycle were similar to those found in primed ovaries. The others were taken from a patient on day 12 of a 35-day cycle. 1 of them contained much less progesterone, while the other 3 from this patient contained very low amounts of both steroids.
ii. Steroid concentrations in relation to the size of the follicles and dose of H.M.G: No correlation could be found between the concentrations of steroids and the size of the Concentrations varied greatly between follicles of the same size, the coefficients of variation being similar for large, medium, and small follicles. As shown below, some of these large follicles might have been
graafian follicles.
preovulatory. The mean values of oestradiol-170 and progesterone in follicular fluids for each dose of H.M.G. were compared. Significant correlations were detected between the concentrations of steroids and the amount of H.M.G. given, in CONCENTRATION OF
CESTRADIOL-17&bgr;
AND PROGESTERONE
(ng./ml.)
log CONCENTRATIONS OF
OESTRAD)OL]7(ngfmO
Fig. 3-Concentrations of
the sense that more steroids were present in the follicular fluids of patients given higher doses of H.M.G. than in those given lower doses, irrespective of the size of the follicle (F for both steroids 6, p < 0.01). =
iii. Steroid levels in follicular fluids in relation to the excretion of urinary cestrogen.-Variations in the steroid concentration of different follicles were compared in patients with high or low levels of urinary oestrogen. Data on 54 graafian follicles were available from 15 patients who were arbitrarily divided into " high " and " low " excretors at a dividing line of 30 µg. a day. In each group the concentrations of steroid in different follicles appeared to be equally variable (fig. 3). In the 9 patients where 4 or more fluids were analysed, 8 possessed follicles with high or low steroid concentrations. Follicles thus appeared to respond independently to the injection of exogenous gonadotrophins and not to respond as a group within a
patient. IN THE FLUIDS OF PREOVULATORY FOLLICLES AND IN ALL FOLLICLES
614 iv. Preovulatory and non-ovulatory follicles.—The samples of follicular fluid included a few from preovulatory follicles. Fluids from a further 9 preovulatory follicles, not included in the previous data, were assayed entirely by radioimmunoassay. The patients had been given between 675 and 900 i.u. of H.M.G. Concentrations of cestradiol-17&bgr;Q in these fluids were similar to those examined earlier, although concentrations of progesterone were generally higher. The two highest values-1900 ng. per ml. of oestradiol and 26,400 ng. per ml. of progesterone-were found in these 9 fluids. These data are included in the table. Concentrations of progesterone in all preovulatory follicles varied from 250 to 26,400 ng. per ml. and those of cestradiol-17&bgr;from 20 to 1900 ng. per ml. The data were too sparse for detailed analysis. Steroid concentrations appeared to be higher in larger follicles and in those from patients given high doses of H.M.G. (table). Concentrations of progesterone but not oestradiol were higher in preovulatory follicles than in all follicles combined (table). This comparison masks differences between the two groups, since values for all follicles also include those for preovulatory follicles. In a few non-ovulatory follicles, concentrations of œstradiol-17&bgr;were between 30 and 900 ng. per ml. and of progesterone between 210 and 5200 ng. per ml.
Discussion
This work has provided data on follicular activity in human ovaries when ovulation is imminent. Stimulation of animal ovaries with gonadotrophins induces many of the small follicles to develop into graafian follicles. In the cow ovary, for example, follicles 2-4 mm. in diameter respond to F.S.H. preparations. 8 2 follicles reach 1 mm. in diameter and form antra during the natural cycle of the rhesus monkey, but only 1 proceeds to ovulation.9 Information on human follicular growth and ovulation is scanty. In amenorrhoeic women given gonadotrophins, growth of a primordial follicle into a follicle capable of responding to H.C.G. requires 10 days, 10 and the interval between the injection of H.c.G. and ovulation is approximately 36 hours.1-3 In the present work, the mean number of graafian follicles found in each ovary was between 5 and 6, irrespective of the dose of H.M.G. given or the levels of urinary cestrogens. This estimate is perhaps the best that can be made of the number of follicles ready to respond to F.S.H. during any one cycle. Some available follicles might not have responded to the H.M.G. and others may have become atretic early in the treatment, as found in mice and sheep during their natural cycle. 11,12 Atresia late in development can probably be excluded because the follicles were apparently healthy, with good granulosa cells and clear follicular fluid. There was considerable variation between patients in the number of graafian follicles-a situation also well known in animals after injection of gonadotrophins. The levels of urinary oestrogen were correlated with the number of large follicles, each one contributing The effect of smaller on average 15-37 µg. a day. follicles was not graafian significant, although their contribution will become clearer as more information accumulates. They undoubtedly secrete steroids, for some patients with large amounts of urinary oestrogen possessed few large follicles and many small ones.
Patients with no large follicles excreted a daily average of approximately 15 µg. of urinary œstrogen—a value similar to that found in amenorrhoeic women with no evidence of follicular growth. 13 The concentrations of œstradiol-17&bgr; in blood would be expected to correlate closely with those in follicular fluid, as found in the sheep. 14 There was a great variation in the concentrations of œstradiol-17&bgr;and progesterone in fluids of different follicles, although the levels of the two steroids within each fluid were highly correlated. The concentrations depended partly on the amount of H.M.G. given to the patient, but not on the size of the follicle, which implies that larger follicles are no more active than smaller follicles, or that substrates diffuse more rapidly from The level of urinary oestrogens must be them. determined by the increased size of large follicles, and not because they are more active per unit of tissue. The sources of the two steroids in follicular fluids have not been fully defined. Increments of progesterone were higher than those of oestrogen, especially in preovulatory follicles, as would be expected since progesterone synthesis precedes oestradiol-17&bgr; in the ovarian metabolism of steroid horGranulosa cells taken from human follicles mones. by laparoscopy have 3&bgr;-hydroxysteroid dehydrogenase activity and can synthesise progesterone from pregnenolone 15; but they are less effective than thecal cells in converting progesterone to 17oc-hydroxyprogesterone, although these cell types in combination can synthesise steroids from acetate. 16, 17 Granulosa cells thus possess 3&bgr;-hydroxysteroid dehydrogenase activity but, in contrast to thecal cells, little aromatising activity, as also found in the mare. 18 The high concentration of progesterone in some of the human follicular fluids does not lead to excretion of unusually
large quantities of urinary pregnanediol (unpublished), although preovulatory progesterone can obviously be synthesised by ovarian follicles. Follicles stimulated by exogenous gonadotrophins may obviously differ from those developing during the natural cycle-for example, through the induced luteinisation of unripe follicles.10° Nevertheless, concentrations of steroids similar to those in primed follicles were found in some follicles sampled during the natural cycle, although others had much lower levels of steroids and especially of progesterone. How can the considerable variation in steroid concentrations of follicular fluids be explained ? This variation affected large preovulatory follicles just as much as medium and small follicles. Perhaps the H.M.G. caused differential stimulation of the follicles, but such an explanation hardly suffices for those follicles which enlarged and became preovulatory yet still contained low concentrations of steroid. A better explanation can be derived from recent work indicating that a regular and inherent pattern underlies follicle formation and utilisation, and the growth and secretions of graafian follicles and corpora lutea. The inherent oestrogenic activity of enlarging follicles seems to impose the regularity of the L.H. surge in midcycle," and the lifespan of the corpus luteum in women and rhesus monkeys appears to depend partly on inherent factors in the gland itself and partly on
615
secretion. 19,20 Some evidence indicates that follicles are formed and used in a definite sequence in mice.21 Two waves of follicular growth occur in human ovaries during the menstrual cycle-at the end of the follicular phase, and during the luteal phase Successive waves of follicular growth and enlargement are found during a single oestrous cycle in mice, sheep, and perhaps cattle, and the largest follicle can be replaced before ovulation by a more actively growing smaller follicle.11,12,23,24 The peaks of cestradiol secretion found in sheep during their oestrous cycle may be associated with follicular enlargement during each wave of follicular growth.25,26 Successive ovulations can be induced within a short time by post-ovulatory injections of gonadotrophins into mice, and both sets of oocytes can be fertilised and develop to full term. 27,28 L.H.
Awareness that follicular ently stable and that several
growth could be inherof follicles develop menstrual cycle makes it waves
within a single oestrous or easier to interpret the results of the present work and to design modified treatments for patients. The nonovulatory graafian follicles could be a heterogeneous population arising partly through the successive injections of H.M.G., some follicles being in early growth and others in later stages, so resulting in the observed variation in steroid levels. Many of these follicles might have responded to further injections of H.C.G., for preovulatory swelling requires less than 24 hours in many species. The preovulatory follicles might also have been in various stages of their development, even though their oocytes were in diakinesis or a closely similar stage of development. In the rabbit, levels of oestrogen and progestin in the fluids of preovulatory follicles rise soon after L.H. release but then decline rapidly to almost zero before ovulation, 29 and levels of plasma-steroids decline in sheep and monkeys while the preovulatory follicle is still intact.19,30,311 The human preovulatory follicles with low amounts of steroid were probably fully mature and awaiting ovulation, while those with higher levels may have been less mature and hastened through their
preovulatory changes by the H.C.G. Improvements in the treatment are possible by modifying the dosage schedule, or using gonadotrophic preparations with a different F.S.H./L.H. ratio for stimulation of follicular growth. Consistent rises in plasma-F.s.H. during follicular growth are achieved by seven or more daily injections of H.M.G. into amenorrhocic patients, although L.H. levels are lower than in the natural cycle.32,33 Treatment with H.M.G. or human pituitary gonadotrophin, when shortened to 1-3 days, is still sufficient to stimulate follicular growth for 10 days in anovulatory patients 10,34;such a brief regimen could be preferable to our present schedule for patients with a normal menstrual cycle, since their endogenous gonadotrophins could support the growing follicles, and the development of large numbers of small graafian follicles might be avoided. Problems could arise through the limited half-life of F.S.H. and L.H. 35,36and if levels of endogenous gonadotrophin were lowered by the feedback of oestrogen from the developing follicles. A wise precaution may therefore be administration of small supplementary
injections of H.M.G. H.C.G. induces a satisfactory L.H. peak, 32,33 the single injection probably sufficing because of its long half life. 37 There appears no need to use preparations with differing F.S.H./L.H. ratios; since urinary and pituitary preparations with a ratio of 1/1 (i.e., that present in pergonal) induced a better response in anovulatory patients than those containing more
F.S.H. 34
We thank the Ford Foundation, the Manchester Regional and District Hospital Management Committee for financial support; Prof. C. R. Austin for his encouragement; Searle Scientific Services for hormone assays; and Dr. A. Brand for permission to use unpublished data.
Hospital Board, and the Oldham
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