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Induction of Ovulation with Human Urinary Gonadotrophins in the Monkey
Induction of' Ovulation with Human Urinary Gonadotrophins in the Monkey M. E. SIMPSON and G. VANWAGENEN
~HAVE
ALREADY reported the success attained in induction of ovulation in prepuberal and adult cyclic rhesus monkeys ( Macaca mulatta) with extracts of monkey pituitaries.8 - 10 A luteinizing agent, such as human chorionic gonadotrophin (HCG) was usually administered terminally, but in some instances ovulation occurred without such supplementation. It should be noted in this connection, however, that the monkey pituitary preparations injected were not purified, being 40% ethanol extracts which contained luteinizing hormone as well as the follicle-stimulating hormone. In the meantime similarly successful induction of ovulation has been induced in women following injection of human pituitary extracts (notably by Gemzell and co-workers4 • 5 and more recently by Buxton and Hermann1 • 2 ). It has now proved feasible to develop preovulatory follicles in the monkey by use of human postmenopausal urinary gonadotrophin ( HMG), and ovulation in monkeys so treated has followed administration of supplements of HCG. There appears to be a twofold justification for presentation of these results. Firstly, HMG is a more readily available source of a follicle stimulant than either human or monkey pituitaries, which are obviously limited and expensive as a source of hormone. Secondly, the use of an experimental
From the Department of Anatomy, University of California, San Francisco and Berkeley; and the Department of Obstetrics and Gynecology, Yale University School of Medicine. The study reported in this article was aided by Grants RG-4389 to University of California, and RG-5459 to Yale University, from the U.S. Public Health Service. We wish to thank Sven Johnsen (Statens Seruminstitut, Copenhagen) and P. Donini (Istituto Farmacologico Serono, Rome) for their generous assistance in supplying the preparations of human postmenopausal urinary gonadotrophins (J5 and Pergonal-23, respectively) used in this study. Presented at the Seventeenth Annual Meeting of the American Society for the Study of Sterili· ty, Apr. 21-23, 1961, Bal Harbour, Fla.
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primate is advantageous in that evidence of ovulation can be obtained by direct observation of the ovaries at laparotomy, and confirmation is possible by subsequent histological analysis of the ovaries. MATERIAL AND METHODS
The methods used, essentially the same as those in the previous study, are briefly as follows. Adult female macaque monkeys, known to be in good health and with regular menstrual cycles were injected with HMG from the fifth or sixth day of the cycle, twice daily, for 5 or 6 days. Two preparations of HMG were used, J5 and Pergonal-23. The former preparation when bioassayed in hypophysectomized female rats was found capable of initiating follicular growth within 3 days after a total dose of 75 p.g.; the minimal effective dose or rat unit of the other preparation was 150 p.g. Doses of 24 mg. of J5, or 75 mg. of Pergonal ( 320 and 500 RU, respectively) were adequate to cause preovulatory follicular development in monkeys. In the terminal phase of therapy, the last 3 or 4 days of the total 8- or 9-day injection period, continued support to follicles and their luteinization was accomplished by injection of half these doses of HMG together with HCG. This luteinizing agent was given in daily doses of 1750 to 3000 IU so that total doses of 5000-12000 IU HCG were administered in the 3- to 4-day period. A postinjection period of 6-9 days was allowed to elapse to permit development of the corpora lutea. Laparotomy was therefore performed on the nineteenth to twenty-second day of the cycle. The ovaries were examined in situ for the presence of fresh stigmata and newly formed corpora lutea, and one or both ovaries were then removed. The ovaries were fixed in Bonin solution, imbedded in paraffin, and cut in serial section at 6 or 8 /L· Incidence of recent ovulation was confirmed by study of the ovarian sections; and the degree of luteinization of corpora lutea was observed and the age of the corpora estimated. Changes in swelling of the sex skin and in its color were recorded during the course of injection and vaginal desquamation was followed by quantitative sedimentation6 as an aid in predicting the degree of follicular development and estrogen secretion. The uteri were examined histologically in those instances in which the animals were autopsied in order to determine whether they were under the influence of progesterone secreted by functional corpora lutea. RESULTS
Table 1 summarizes the results of injection of 5 monkeys and shows which dosages and time intervals proved successful.
TABLE I. Ovultion in Adult Macaca mulatta Following Follicular Stimulation by Human Menopausal Gonadotrophin (HMG) Injection (s.c.)
Monkey
Ovaries
Dosage/ dayt
HMG*
No.
Age (yr.)
Body wt. Day of cycle (kg.) Begin End
J5 (Johnsen) Mm 1032 Adult
5.3
5
Mm 1033 Adult
4.0
8
ca 9
6.3
5
ca 10
8.0
6
Mm 1047 Adult
4.4
5
Pergonal-23 Mm 892
Mm 908
IU
2 10 .5 1750 (787 RU) 2.5 12 2000 (67 RU) 15 13 8 2000 (713 RU) 15 14 3000 7.5 (700 RU) 15 13 7.5 3000 (700 RU) 13
Corpora lutea
HOG HMG (mg.)
Days No. after days inj.
Size gm.
em.
Follicles
No. of stigmata
Characteristics
2 4 3
7
L5.20 2.4 X 1.9 X 2.0 R 2.3 X 1.6 X 2.1
Many large L5 fall. & R6 cysts
2 3
2
L0.95 1.3 X 1.1 X 1.4 R 0.95 X .5x .7
5 4
6
L0.80 1.3 X .85 X 1.1 (absent) R
L9mm. LO No CL; cyst; luteinized R few small R 0 fall. fall. L large fall. L 3large Many CL, large 6+ small and small -
5 4
6
L2.47 1.9 X 1.4 X 1.6 R -
L large fall. L3 R2 -
Also CL with enclosed ova
5 4
9
L1.5 X 1.2 X 1.4 R 1.28 1.5 X 1.2 X 1.3
L4 R4
Also CL with enclosed ova
*MED (R U) for follicular development in hypophysectomized rats: .J5, 75 p.g.; Pergonal-23, 150 p.g. tTwo daily doses 6-8 hours apart.
Scanty luteinization
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Mm 1032 received a HMG ( J5) in a total preparatory dose of 44 mg. or 586 RU from the fifth to the tenth day of the cycle, followed by approximately half this dose mixed with 5250 IU HCG from the eleventh to the thirteenth day. Observation of the ovaries at laparotomy 7 days after termination of the injections showed six fresh stigmata present in the right ovary and, in the left ovary, five fresh stigmata as well as one fibrous stigma. Transillumination showed large follicles were also present. The ovaries were measured and were found to be enlarged to a comparable degree; the left, which was removed, weighed 5.2 gm., and was 10--20 times normal size. From histologic!ll examination the presence of fresh stigmata was unquestionable. The multiple corpora lutea of ovulation were less uniformly luteinized and appeared younger than anticipated from the postinjection interval. Many large follicles were present, and also follicles exceeding ovulation size, some of which had luteinized walls and enclosed eggs. It appeared that ovulation had taken place from the less grossly enlarged follicles. The corpora lutea were frequently crowded and deformed by the enlarged follicles (Fig. 1 and 2). As a result of cessation of injection of the follicular stimulant, also due to removal of one of the enlarged ovaries containing many follicles, the animal showed estrogen-deprival bleeding on the third day after laparotomy. Regular menstrual cycles were resumed 32 days later. The contralateral ovary (right), which during the experimental period had attained a size equal to the left ovary and contained multiple corpora lutea and large follicles, must then have quickly resumed normal function. Mm 1033 received what remained of the J5 preparation though the amount available was predicted to be entirely inadequate for preovulatory enlargement of follicles. The experience gained from injection of this small amount assisted in delimiting the amount of follicle-stimulating substance required. A total dose of 67 RU was injected during the eighth and ninth days of the cycle followed by a total of 6000 IU of HCG from the tenth to twelfth days. The left ovary, removed on the fourteenth day of the cycle, weighed 0.947 gm. and contained a number of moderately enlarged follicles besides one large, preovulatory one. Some luteinization of the walls of follicles had occurred and some hemorrhage into the antra, probably as a result of the effect of the HCG supplement. Bleeding occurred 4 days after removal of the ovary containing the large follicle (the 18th day of the cycle) and normal menstruation followed after an interval of 31 days. The three adult animals which received Pergonal-Mm 892, 908, and 1047 -were all treated in almost identical manner and with parallel results. Multiple ovulations occurred from both ovaries in all animals. Treatment of Mm 892 was begun on the fifth day of the cycle. A dose of 7.5 mg. HMG was given twice daily for 5 days to allow development of follicles. Following this,
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approximately half this dose, 4 mg., was mixed with 1000 IU HCG and administered twice daily for the next 4 days. On histological examination, the left ovary'* showed several fresh ovulation stigmata. At least three large corpora lutea of ovulation were identified; beside these corpora lutea of the more usual size there were several (six or more), small, well-luteinized structures which had ruptured to the surface. Hemorrhages in the luteinized structures were not uncommon. The corpora could have been 7-9 days old if ovulation had occurred soon after onset of HCG injection. ( HCG was given on the tenth to thirteenth day of the cycle and autopsy was performed 6 days later.) The corpora were, however, not as well developed as normal corpora lutea of this age (Fig. 3 and 4). Estrogen-deprival bleeding was observed beginning on the fifth day after injection and the uterus was bleeding on removal on the sixth day. The stromal cells were small, deeply stained, and closely packed and the uterine glands were straight and close together. The nuclei of the glands were basal, indicating ovulation had occurred; however, the epithelium was not a secreting epithelium so that bleeding was evidently due to withdrawal of estrogen. The indication was, therefore, that though ovulation had occurred corpus luteum function had not become effective. In Mm 908, injection of Pergonal was begun on the sixth day of the cycle. Doses of 7.5 mg. were given twice daily for 5 days after which half this dose, 3.75 mg., was mixed with 1500 IU of HCG and injected twice daily for the next 4 days. The last injection was given on the fourteenth day of the cycle and the ovaries were examined 6 days later (cycle day 20). The two ovaries were found to be of approximately the same dimension; the left weighed 2.47 gm. Three ovulation stigmata were observed in the left, and two in the right ovary. Beside the corpora lutea of ovulation, other· luteinized bodies were present containing enclosed eggs. Many follicles were also present, which distorted the corpora lutea (Plate I and Fig. 5 and 6). Mm 1047 was given a similar course of treatment, the only difference being the onset of injections on the fifth day of the cycle. The interval between the last injection and laparotomy was prolonged to 9 days. The two ovaries were enlarged to approximately the same degree, the right ovary weighing 1.278 mg. Four fresh stigmata were present in each ovary. The corpora lutea did not ::tppear to exceed 6 days in age. Some were hemorrhagic (Fig. 7 and 8). *Fetuses had been removed from the uterus of Mm 892 during four previous pregnancies in the period 1956-1959. The right ovary was removed in 1958. A live infant was successfully carried to term in 1959. Mm 908 had two uninterrupted pregnancies and two cesarian sections in the period 1956-1959.
SIMPSON
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A
B
Plate 1. Ovaries of macaque monkeys after treatment with human menopausal gonadotrophin (HMG) followed by human chorionic gonadotrophin. A. Mm 908 Right ovary, removed at laparotomy, showing two ovulation stigmata. There was a third stigma on the reverse side of the ovary. B. Mm 1047 Right ovary at laparotomy, before removal, showing ovulation sites of three young corpora lutea.
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1
2
3
4
Fig. 1. (Mm 1032, left ovary) Ovulation induced by HMG (J5) followed by HCG. (H&E, X40) Fig.2. (Mm1032,leftovary) Secondovulationpointinsame (Fig.1) ovary. (MAz, X 44) Fig. 3. (Mm 892, left ovary) Ovulation induced by HMG (Pergonal) followed by HCG. (MAz, X 63) Fig. 4. (Mm 892, left ovary) Stigma of another of the ovulations from this (Fig. 3) ovary. (H&E, X 125)
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6
6
7
8 Fig. 5. (Mm 908, left ovary) Corpus luteum with stigma. (MAz, X 19.5) Fig. 6. (Mm 908, left ovary) Another corpus luteum and stigma from the same (Fig. 5) ovary. (MAz, X 32) Fig. 7. (Mm 1047, right ovary) Section through whole ovary showing multiple corpora lutea and two recent stigmata. (H&E, X 63) Fig. 8. (Mm 1047, right ovary) Higher magnification of wall of typical corpus luteum. (H&E, X 125)
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DISCUSSION
The dosage of HMG which was used in the successful experiments may have been too large and the timing of injection, though feasible, may not have been optimal. As stated in the instance of Mm 1032, ovulation may have occurred only from the less grossly enlarged follicles, which suggests that the dose of HMG was too high and that the cystic structures formed had exceeded their capacity to shed eggs and become transformed into corpora lutea. The doses of chorionic gonadotrophin may appear to be very large in relation to the size of animals injected; however, the luteinization was scanty in Mm 1032, the animal which received a dose of HCG lower than that given the other animals. It should be noted, though not recorded here, that HCG injected aloneintramuscularly, intravenously, or subcutaneously-did not induce ovulation. Doses from 10,000 to 30,000 IU were administered to 4 adult female monkeys' at different times of the cycle (days 6-13, 9-16, 7-10, and 3-6) without accelerating follicular development or causing follicular luteinization or corpus luteum formation. Both incorrect dosage and timing may be contributory to the undesired ·feature of multiple ovulations instead of a single ovulation. It is to be noted, however, that multiple ovulations have been found characteristic in all species in which ovulation has been induced by administration of exogenous gonadotrophins, and the cause has not yet been ascertained. This report covers a purely preliminary study and makes no pretense of supplying the fundamental knowledge which we eventually must have regarding the specific hormones needed for ovulation. Assuming that FSH and ICSH are the specific pituitary hormones required for ovulation, this study does not give accurate information regarding the dosage or proportion in which these individual hormones are required. The monkeys used in the study possessed intact pituitaries. Their pituitaries, furthermore, were probably in different physiological states, even though as many experimental factors as possible were controlled, such a~ time of the cycle. The pituitaries of the experimental animals therefore could have contributed variable amounts of hormones to the reaction. This criticism can be made of all studies which have been conducted in normal animals in efforts to determine the factors necessary for ovulation. This statement naturally applies to studies on induction of ovulation in normal or amenorrheic women. Many of the fundamental questions regarding the control of ovulation can only be answered by use of animals deprived of their pituitaries, a condition
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impossible in the human and difficult in the monkey. 7 Even in studies in which intact individuals have been used the numbers of individuals studied has been far from adequate to answer the questions involved here. In studies of ovulation in the rat, in which it is feasible to study the animal after hypophysectomy and, furthermore, to employ the purest preparations available, many problems are still unresolved. Though several years of effort have been devoted already to this project and 5000 or more of such hypophysectomized rats have already been studied, the exact conditions for ovulation in this species are still incompletely determined. It appears from the studies reported here, using the macaque, that there is no danger of permanent maladjustment of the primate ovary after administration of doses which enlarg~ the ovary by many times. As noted, resumption of normal ovarian function and menstrual periods recurred at the regular interval. In forming judgments regarding the induction of corpus luteum formation it is important to give these bodies sufficient time to mature so that there is no possibility of confusing them with follicles artificially ruptured at laparotomy. In the present study it will be noted that a period of 6-9 days was allowed after injections ceased before examination of the ovaries. In previous studies it had been found that ovulation usually followed within a day or two after beginning administration of HCG, provided the ovaries had been properly prepared by a follicle stimulant, the time of ovulation being judged by comparison with normally ageing corpora as described by Corner.3 In this series the evidence is not as clear for ovulation following immediately after administration of the luteinizing agent. The rupture of follicles smaller than normal preovulatory follicles, and formation of very small corpora lutea, is a result not previously encountered. The distortion of the large corpora lutea by coexisting large follicles which did not rupture was also conspicuous in this series of experiments. The policy of injection early in the cycle (onset of injections being on day 5 or 6) has been maintained here, as this has been the time of onset in previously successful experiments. Efforts to induce ovulation by injection of monkey pituitary preparations or sheep gonadotrophins during the last half of the menstrual period were not successful. It is important to know that the HMG can be used in the primate for stimulation of follicles and that pituitary preparations are not essential, since the former is cheaper and more available, and so more feasible for therapeutic use. However, confusion should not be introduced by the assumption that when injecting HMG one is administering pituitary hormone. The pituitary may reasonably be assumed to be the ultimate source of the hormone in the blood stream and urine of postmenopausal women; however, the substance
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excreted is known to have biological properties different from pituitary extracts. The postmenopausal product most closely resembles partially purified pituitary FSH, but at higher doses it has the capacity to repair deficient interstitial cells and to cause luteinization. The proportion of follicle-stimulating and interstitial-cell-stimulating properties of the two preparations of HMG used in this study can be seen in Table 2. It is also possible by reference to this table to compare the biological properties in postmenopausal hormone with those of primate pituitary preparations. TABLE 2. Biological Differences Between Human Menopausal Gonadotrophin and Pituitary Follicle-Stimulating Fractions MEDin ii !i? rats (p.g.)
sa
Interstitial cell repair IP
Ratio
98 20-25 320 250 187 50-60 1.7 75 150
32 15 320 250 187 250 120 750t 300
3:1 1.5:1 1:1 1:1 1:1 1:5 1:70 1:10 1:2
Follicle stimulation Source
Fraction
Pituitary human monkey
30% Et0H 0 WDIV-50A Saline 40% EtOH 40% EtOH NIH-FSH WDIV-19B J5 Pergonal-23
sheep
HMG
*Merck L581-034. t Higher doses required for luteinization.
CONCLUSIONS
Multiple ovulations were induced in adult Macaca mulatta following follicle development by injection of human postmenopausal gonadotrophin and subsequent luteinization by administration of human chorionic gonadotrophin. The 4 adult females which received adequate unitages of HMG shed 3-6 ova from each of their ovaries. REFERENCES 1. BuxTON, C. L., and HERRMANN, W. Induction of ovulation in the human with human gonadotropins. Yale f. Biol. & Med. 33:145, 1960. 2. BUXTON, C. L., and HERRMANN, W. Induction of ovulation in the human with human gonadotropins. Am. f. Obst. & Gynec. 81:584, 1961. 3. CoRNER, G. W. Development, organization and breakdown of the corpus luteum in the Rhesus monkey. Contrib. Embryol. 31:111, 1945; 4. GEMZELL, C. A., DICZFALUSY, E., and TILLINGER, G. Clinical effect of human pituitary follicle-stimulating hormone (FSH). f. Clin. Endocrinol. 18:1333, 1958.
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5. GEMZELL, C. A., DICZFALUSY, E., and TILLINGER, K. G. Human pituitary folliclestimulating hormone. I. Clinical effect of a partially purified preparation. Ciba Foundation Coli. on Endoc., Human Pituitary Hormones 18:191, 1960. 6. HARTMAN, C. G. Studies in the reproduction of the monkey Macacus (Pithecus) rhesus, with special reference to menstruation and pregnancy. Contrib. Embryol. 28:1,
1932. 7. KNoBIL, E., KosTYo, J. L., and CREEP, R. 0. Production of ovulation in the hypophysectomized rhesus monkey. Endocrinology 65:487, 1959. 8. SIMPSON, M. E., and VAN WAGENEN, G. Experimental induction of ovulation in the macaque monkey. Fertil. & Steril. 9:386, 1958. 9. VANWAGENEN, G., and SIMPSON, M. E. Induction of multiple ovulation in the Rhesus monkey (Macaca mulatta). Endocrinology 61:316, 1957. 10. VAN WAGENEN, G., and SIMPSON, M. E. Experimentally induced ovulation in the Rhesus monkey (Macaca mulatta). Rev. Suisse de Zool. 64:807, 1957.
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~HAVE
ALREADY reported the success attained in induction of ovulation in prepuberal and adult cyclic rhesus monkeys ( Macaca mulatta) with extracts of monkey pituitaries.8 - 10 A luteinizing agent, such as human chorionic gonadotrophin (HCG) was usually administered terminally, but in some instances ovulation occurred without such supplementation. It should be noted in this connection, however, that the monkey pituitary preparations injected were not purified, being 40% ethanol extracts which contained luteinizing hormone as well as the follicle-stimulating hormone. In the meantime similarly successful induction of ovulation has been induced in women following injection of human pituitary extracts (notably by Gemzell and co-workers4 • 5 and more recently by Buxton and Hermann1 • 2 ). It has now proved feasible to develop preovulatory follicles in the monkey by use of human postmenopausal urinary gonadotrophin ( HMG), and ovulation in monkeys so treated has followed administration of supplements of HCG. There appears to be a twofold justification for presentation of these results. Firstly, HMG is a more readily available source of a follicle stimulant than either human or monkey pituitaries, which are obviously limited and expensive as a source of hormone. Secondly, the use of an experimental
From the Department of Anatomy, University of California, San Francisco and Berkeley; and the Department of Obstetrics and Gynecology, Yale University School of Medicine. The study reported in this article was aided by Grants RG-4389 to University of California, and RG-5459 to Yale University, from the U.S. Public Health Service. We wish to thank Sven Johnsen (Statens Seruminstitut, Copenhagen) and P. Donini (Istituto Farmacologico Serono, Rome) for their generous assistance in supplying the preparations of human postmenopausal urinary gonadotrophins (J5 and Pergonal-23, respectively) used in this study. Presented at the Seventeenth Annual Meeting of the American Society for the Study of Sterili· ty, Apr. 21-23, 1961, Bal Harbour, Fla.
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primate is advantageous in that evidence of ovulation can be obtained by direct observation of the ovaries at laparotomy, and confirmation is possible by subsequent histological analysis of the ovaries. MATERIAL AND METHODS
The methods used, essentially the same as those in the previous study, are briefly as follows. Adult female macaque monkeys, known to be in good health and with regular menstrual cycles were injected with HMG from the fifth or sixth day of the cycle, twice daily, for 5 or 6 days. Two preparations of HMG were used, J5 and Pergonal-23. The former preparation when bioassayed in hypophysectomized female rats was found capable of initiating follicular growth within 3 days after a total dose of 75 p.g.; the minimal effective dose or rat unit of the other preparation was 150 p.g. Doses of 24 mg. of J5, or 75 mg. of Pergonal ( 320 and 500 RU, respectively) were adequate to cause preovulatory follicular development in monkeys. In the terminal phase of therapy, the last 3 or 4 days of the total 8- or 9-day injection period, continued support to follicles and their luteinization was accomplished by injection of half these doses of HMG together with HCG. This luteinizing agent was given in daily doses of 1750 to 3000 IU so that total doses of 5000-12000 IU HCG were administered in the 3- to 4-day period. A postinjection period of 6-9 days was allowed to elapse to permit development of the corpora lutea. Laparotomy was therefore performed on the nineteenth to twenty-second day of the cycle. The ovaries were examined in situ for the presence of fresh stigmata and newly formed corpora lutea, and one or both ovaries were then removed. The ovaries were fixed in Bonin solution, imbedded in paraffin, and cut in serial section at 6 or 8 /L· Incidence of recent ovulation was confirmed by study of the ovarian sections; and the degree of luteinization of corpora lutea was observed and the age of the corpora estimated. Changes in swelling of the sex skin and in its color were recorded during the course of injection and vaginal desquamation was followed by quantitative sedimentation6 as an aid in predicting the degree of follicular development and estrogen secretion. The uteri were examined histologically in those instances in which the animals were autopsied in order to determine whether they were under the influence of progesterone secreted by functional corpora lutea. RESULTS
Table 1 summarizes the results of injection of 5 monkeys and shows which dosages and time intervals proved successful.
TABLE I. Ovultion in Adult Macaca mulatta Following Follicular Stimulation by Human Menopausal Gonadotrophin (HMG) Injection (s.c.)
Monkey
Ovaries
Dosage/ dayt
HMG*
No.
Age (yr.)
Body wt. Day of cycle (kg.) Begin End
J5 (Johnsen) Mm 1032 Adult
5.3
5
Mm 1033 Adult
4.0
8
ca 9
6.3
5
ca 10
8.0
6
Mm 1047 Adult
4.4
5
Pergonal-23 Mm 892
Mm 908
IU
2 10 .5 1750 (787 RU) 2.5 12 2000 (67 RU) 15 13 8 2000 (713 RU) 15 14 3000 7.5 (700 RU) 15 13 7.5 3000 (700 RU) 13
Corpora lutea
HOG HMG (mg.)
Days No. after days inj.
Size gm.
em.
Follicles
No. of stigmata
Characteristics
2 4 3
7
L5.20 2.4 X 1.9 X 2.0 R 2.3 X 1.6 X 2.1
Many large L5 fall. & R6 cysts
2 3
2
L0.95 1.3 X 1.1 X 1.4 R 0.95 X .5x .7
5 4
6
L0.80 1.3 X .85 X 1.1 (absent) R
L9mm. LO No CL; cyst; luteinized R few small R 0 fall. fall. L large fall. L 3large Many CL, large 6+ small and small -
5 4
6
L2.47 1.9 X 1.4 X 1.6 R -
L large fall. L3 R2 -
Also CL with enclosed ova
5 4
9
L1.5 X 1.2 X 1.4 R 1.28 1.5 X 1.2 X 1.3
L4 R4
Also CL with enclosed ova
*MED (R U) for follicular development in hypophysectomized rats: .J5, 75 p.g.; Pergonal-23, 150 p.g. tTwo daily doses 6-8 hours apart.
Scanty luteinization
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Mm 1032 received a HMG ( J5) in a total preparatory dose of 44 mg. or 586 RU from the fifth to the tenth day of the cycle, followed by approximately half this dose mixed with 5250 IU HCG from the eleventh to the thirteenth day. Observation of the ovaries at laparotomy 7 days after termination of the injections showed six fresh stigmata present in the right ovary and, in the left ovary, five fresh stigmata as well as one fibrous stigma. Transillumination showed large follicles were also present. The ovaries were measured and were found to be enlarged to a comparable degree; the left, which was removed, weighed 5.2 gm., and was 10--20 times normal size. From histologic!ll examination the presence of fresh stigmata was unquestionable. The multiple corpora lutea of ovulation were less uniformly luteinized and appeared younger than anticipated from the postinjection interval. Many large follicles were present, and also follicles exceeding ovulation size, some of which had luteinized walls and enclosed eggs. It appeared that ovulation had taken place from the less grossly enlarged follicles. The corpora lutea were frequently crowded and deformed by the enlarged follicles (Fig. 1 and 2). As a result of cessation of injection of the follicular stimulant, also due to removal of one of the enlarged ovaries containing many follicles, the animal showed estrogen-deprival bleeding on the third day after laparotomy. Regular menstrual cycles were resumed 32 days later. The contralateral ovary (right), which during the experimental period had attained a size equal to the left ovary and contained multiple corpora lutea and large follicles, must then have quickly resumed normal function. Mm 1033 received what remained of the J5 preparation though the amount available was predicted to be entirely inadequate for preovulatory enlargement of follicles. The experience gained from injection of this small amount assisted in delimiting the amount of follicle-stimulating substance required. A total dose of 67 RU was injected during the eighth and ninth days of the cycle followed by a total of 6000 IU of HCG from the tenth to twelfth days. The left ovary, removed on the fourteenth day of the cycle, weighed 0.947 gm. and contained a number of moderately enlarged follicles besides one large, preovulatory one. Some luteinization of the walls of follicles had occurred and some hemorrhage into the antra, probably as a result of the effect of the HCG supplement. Bleeding occurred 4 days after removal of the ovary containing the large follicle (the 18th day of the cycle) and normal menstruation followed after an interval of 31 days. The three adult animals which received Pergonal-Mm 892, 908, and 1047 -were all treated in almost identical manner and with parallel results. Multiple ovulations occurred from both ovaries in all animals. Treatment of Mm 892 was begun on the fifth day of the cycle. A dose of 7.5 mg. HMG was given twice daily for 5 days to allow development of follicles. Following this,
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approximately half this dose, 4 mg., was mixed with 1000 IU HCG and administered twice daily for the next 4 days. On histological examination, the left ovary'* showed several fresh ovulation stigmata. At least three large corpora lutea of ovulation were identified; beside these corpora lutea of the more usual size there were several (six or more), small, well-luteinized structures which had ruptured to the surface. Hemorrhages in the luteinized structures were not uncommon. The corpora could have been 7-9 days old if ovulation had occurred soon after onset of HCG injection. ( HCG was given on the tenth to thirteenth day of the cycle and autopsy was performed 6 days later.) The corpora were, however, not as well developed as normal corpora lutea of this age (Fig. 3 and 4). Estrogen-deprival bleeding was observed beginning on the fifth day after injection and the uterus was bleeding on removal on the sixth day. The stromal cells were small, deeply stained, and closely packed and the uterine glands were straight and close together. The nuclei of the glands were basal, indicating ovulation had occurred; however, the epithelium was not a secreting epithelium so that bleeding was evidently due to withdrawal of estrogen. The indication was, therefore, that though ovulation had occurred corpus luteum function had not become effective. In Mm 908, injection of Pergonal was begun on the sixth day of the cycle. Doses of 7.5 mg. were given twice daily for 5 days after which half this dose, 3.75 mg., was mixed with 1500 IU of HCG and injected twice daily for the next 4 days. The last injection was given on the fourteenth day of the cycle and the ovaries were examined 6 days later (cycle day 20). The two ovaries were found to be of approximately the same dimension; the left weighed 2.47 gm. Three ovulation stigmata were observed in the left, and two in the right ovary. Beside the corpora lutea of ovulation, other· luteinized bodies were present containing enclosed eggs. Many follicles were also present, which distorted the corpora lutea (Plate I and Fig. 5 and 6). Mm 1047 was given a similar course of treatment, the only difference being the onset of injections on the fifth day of the cycle. The interval between the last injection and laparotomy was prolonged to 9 days. The two ovaries were enlarged to approximately the same degree, the right ovary weighing 1.278 mg. Four fresh stigmata were present in each ovary. The corpora lutea did not ::tppear to exceed 6 days in age. Some were hemorrhagic (Fig. 7 and 8). *Fetuses had been removed from the uterus of Mm 892 during four previous pregnancies in the period 1956-1959. The right ovary was removed in 1958. A live infant was successfully carried to term in 1959. Mm 908 had two uninterrupted pregnancies and two cesarian sections in the period 1956-1959.
SIMPSON
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A
B
Plate 1. Ovaries of macaque monkeys after treatment with human menopausal gonadotrophin (HMG) followed by human chorionic gonadotrophin. A. Mm 908 Right ovary, removed at laparotomy, showing two ovulation stigmata. There was a third stigma on the reverse side of the ovary. B. Mm 1047 Right ovary at laparotomy, before removal, showing ovulation sites of three young corpora lutea.
•
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1
2
3
4
Fig. 1. (Mm 1032, left ovary) Ovulation induced by HMG (J5) followed by HCG. (H&E, X40) Fig.2. (Mm1032,leftovary) Secondovulationpointinsame (Fig.1) ovary. (MAz, X 44) Fig. 3. (Mm 892, left ovary) Ovulation induced by HMG (Pergonal) followed by HCG. (MAz, X 63) Fig. 4. (Mm 892, left ovary) Stigma of another of the ovulations from this (Fig. 3) ovary. (H&E, X 125)
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6
6
7
8 Fig. 5. (Mm 908, left ovary) Corpus luteum with stigma. (MAz, X 19.5) Fig. 6. (Mm 908, left ovary) Another corpus luteum and stigma from the same (Fig. 5) ovary. (MAz, X 32) Fig. 7. (Mm 1047, right ovary) Section through whole ovary showing multiple corpora lutea and two recent stigmata. (H&E, X 63) Fig. 8. (Mm 1047, right ovary) Higher magnification of wall of typical corpus luteum. (H&E, X 125)
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DISCUSSION
The dosage of HMG which was used in the successful experiments may have been too large and the timing of injection, though feasible, may not have been optimal. As stated in the instance of Mm 1032, ovulation may have occurred only from the less grossly enlarged follicles, which suggests that the dose of HMG was too high and that the cystic structures formed had exceeded their capacity to shed eggs and become transformed into corpora lutea. The doses of chorionic gonadotrophin may appear to be very large in relation to the size of animals injected; however, the luteinization was scanty in Mm 1032, the animal which received a dose of HCG lower than that given the other animals. It should be noted, though not recorded here, that HCG injected aloneintramuscularly, intravenously, or subcutaneously-did not induce ovulation. Doses from 10,000 to 30,000 IU were administered to 4 adult female monkeys' at different times of the cycle (days 6-13, 9-16, 7-10, and 3-6) without accelerating follicular development or causing follicular luteinization or corpus luteum formation. Both incorrect dosage and timing may be contributory to the undesired ·feature of multiple ovulations instead of a single ovulation. It is to be noted, however, that multiple ovulations have been found characteristic in all species in which ovulation has been induced by administration of exogenous gonadotrophins, and the cause has not yet been ascertained. This report covers a purely preliminary study and makes no pretense of supplying the fundamental knowledge which we eventually must have regarding the specific hormones needed for ovulation. Assuming that FSH and ICSH are the specific pituitary hormones required for ovulation, this study does not give accurate information regarding the dosage or proportion in which these individual hormones are required. The monkeys used in the study possessed intact pituitaries. Their pituitaries, furthermore, were probably in different physiological states, even though as many experimental factors as possible were controlled, such a~ time of the cycle. The pituitaries of the experimental animals therefore could have contributed variable amounts of hormones to the reaction. This criticism can be made of all studies which have been conducted in normal animals in efforts to determine the factors necessary for ovulation. This statement naturally applies to studies on induction of ovulation in normal or amenorrheic women. Many of the fundamental questions regarding the control of ovulation can only be answered by use of animals deprived of their pituitaries, a condition
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impossible in the human and difficult in the monkey. 7 Even in studies in which intact individuals have been used the numbers of individuals studied has been far from adequate to answer the questions involved here. In studies of ovulation in the rat, in which it is feasible to study the animal after hypophysectomy and, furthermore, to employ the purest preparations available, many problems are still unresolved. Though several years of effort have been devoted already to this project and 5000 or more of such hypophysectomized rats have already been studied, the exact conditions for ovulation in this species are still incompletely determined. It appears from the studies reported here, using the macaque, that there is no danger of permanent maladjustment of the primate ovary after administration of doses which enlarg~ the ovary by many times. As noted, resumption of normal ovarian function and menstrual periods recurred at the regular interval. In forming judgments regarding the induction of corpus luteum formation it is important to give these bodies sufficient time to mature so that there is no possibility of confusing them with follicles artificially ruptured at laparotomy. In the present study it will be noted that a period of 6-9 days was allowed after injections ceased before examination of the ovaries. In previous studies it had been found that ovulation usually followed within a day or two after beginning administration of HCG, provided the ovaries had been properly prepared by a follicle stimulant, the time of ovulation being judged by comparison with normally ageing corpora as described by Corner.3 In this series the evidence is not as clear for ovulation following immediately after administration of the luteinizing agent. The rupture of follicles smaller than normal preovulatory follicles, and formation of very small corpora lutea, is a result not previously encountered. The distortion of the large corpora lutea by coexisting large follicles which did not rupture was also conspicuous in this series of experiments. The policy of injection early in the cycle (onset of injections being on day 5 or 6) has been maintained here, as this has been the time of onset in previously successful experiments. Efforts to induce ovulation by injection of monkey pituitary preparations or sheep gonadotrophins during the last half of the menstrual period were not successful. It is important to know that the HMG can be used in the primate for stimulation of follicles and that pituitary preparations are not essential, since the former is cheaper and more available, and so more feasible for therapeutic use. However, confusion should not be introduced by the assumption that when injecting HMG one is administering pituitary hormone. The pituitary may reasonably be assumed to be the ultimate source of the hormone in the blood stream and urine of postmenopausal women; however, the substance
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excreted is known to have biological properties different from pituitary extracts. The postmenopausal product most closely resembles partially purified pituitary FSH, but at higher doses it has the capacity to repair deficient interstitial cells and to cause luteinization. The proportion of follicle-stimulating and interstitial-cell-stimulating properties of the two preparations of HMG used in this study can be seen in Table 2. It is also possible by reference to this table to compare the biological properties in postmenopausal hormone with those of primate pituitary preparations. TABLE 2. Biological Differences Between Human Menopausal Gonadotrophin and Pituitary Follicle-Stimulating Fractions MEDin ii !i? rats (p.g.)
sa
Interstitial cell repair IP
Ratio
98 20-25 320 250 187 50-60 1.7 75 150
32 15 320 250 187 250 120 750t 300
3:1 1.5:1 1:1 1:1 1:1 1:5 1:70 1:10 1:2
Follicle stimulation Source
Fraction
Pituitary human monkey
30% Et0H 0 WDIV-50A Saline 40% EtOH 40% EtOH NIH-FSH WDIV-19B J5 Pergonal-23
sheep
HMG
*Merck L581-034. t Higher doses required for luteinization.
CONCLUSIONS
Multiple ovulations were induced in adult Macaca mulatta following follicle development by injection of human postmenopausal gonadotrophin and subsequent luteinization by administration of human chorionic gonadotrophin. The 4 adult females which received adequate unitages of HMG shed 3-6 ova from each of their ovaries. REFERENCES 1. BuxTON, C. L., and HERRMANN, W. Induction of ovulation in the human with human gonadotropins. Yale f. Biol. & Med. 33:145, 1960. 2. BUXTON, C. L., and HERRMANN, W. Induction of ovulation in the human with human gonadotropins. Am. f. Obst. & Gynec. 81:584, 1961. 3. CoRNER, G. W. Development, organization and breakdown of the corpus luteum in the Rhesus monkey. Contrib. Embryol. 31:111, 1945; 4. GEMZELL, C. A., DICZFALUSY, E., and TILLINGER, G. Clinical effect of human pituitary follicle-stimulating hormone (FSH). f. Clin. Endocrinol. 18:1333, 1958.
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5. GEMZELL, C. A., DICZFALUSY, E., and TILLINGER, K. G. Human pituitary folliclestimulating hormone. I. Clinical effect of a partially purified preparation. Ciba Foundation Coli. on Endoc., Human Pituitary Hormones 18:191, 1960. 6. HARTMAN, C. G. Studies in the reproduction of the monkey Macacus (Pithecus) rhesus, with special reference to menstruation and pregnancy. Contrib. Embryol. 28:1,
1932. 7. KNoBIL, E., KosTYo, J. L., and CREEP, R. 0. Production of ovulation in the hypophysectomized rhesus monkey. Endocrinology 65:487, 1959. 8. SIMPSON, M. E., and VAN WAGENEN, G. Experimental induction of ovulation in the macaque monkey. Fertil. & Steril. 9:386, 1958. 9. VANWAGENEN, G., and SIMPSON, M. E. Induction of multiple ovulation in the Rhesus monkey (Macaca mulatta). Endocrinology 61:316, 1957. 10. VAN WAGENEN, G., and SIMPSON, M. E. Experimentally induced ovulation in the Rhesus monkey (Macaca mulatta). Rev. Suisse de Zool. 64:807, 1957.
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