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Induction of Ovulation in Rhesus Monkeys: A Comparative Study
Induction of Ovulation in Rhesus Monkeys:
A Comparative Study LIVIA S. WAN, M.D., and HOWARD BALIN, M.D., M.Sc.MED.
THE
of various ovulation-inducing agents in recent years has opened new avenues in management of certain types of female infertility. Although a great deal of investigation has been conducted at the clinical level with various agents, there appear to be many advantages to the performance of select in-depth studies in the subhuman primate such as Macaca mulatta. Use of the experimental monkey permits direct observation and photographic recording of the status of the ovaries while such medications are being administered, as well as biopsy of the ovary and/or endometrium for histologic examination. . The reproductive anatomy and physiology of the rhesus monkey have been well and thoroughly studied over the last 40 years.s, 12 The value of the rhesus monkey for investigation of problems in the field of human reproduction is that its menstrual cycle resembles that of the human and its reproductive tm~t is anatomically similar.14 Goss considers the rhesus monkey a highly evolved organism, a primate second only to man in psychic development. It responds to the stress of caphue and shipment by ceasing to menstruate. It also is said to have seasonal biologic rhythm, becoming anovulatory in the summer months,S, 7,12 but others have found that in captivity, especially in air-conditioned cages, seasonal anovulatory cycles are not evident. 28 However, the occurrence of anovulation under different stress conditions offers an ideal prototype anovulatory experimental animal for evaluation of the efficacy of compounds thought to have ovulation-inducing properties. In the past 4 years, comparative studies have been conducted in our research monkey colony. We have had the opportunity to compare by INTRODUCTION
From the Gynecic Research Section, Division of Obstetrics and Gynecology, Pennsylvania Hospital, Philadelphia, Pa. Presented at the 24th Annual Meeting of the American Fertility Society, San Francisco, Calif., Mar. 28-30, 1968.
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following the si\me protocols a human menotropin and two impeded estrogens for the purpose of induction of ovulation. RESEARCH PLAN AND MATERIALS Research Plan
Mature female Macaca rhesus monkeys observed in our research laboratory for at least 6 months were selected for the study. Other requisites for inclusion in our study were (1) history of 3-6 months of amenorrhea or oligomenorrhea and (2) evidence of anovulation by vaginal cytologic studies. The indices used for evaluation of ovarian response while in control and treatment cycles included (1) menstrual pattern, ( 2 ) vaginal smears, ( 3) laparotomy with ovarian biopsy at expected "postovulatory" phase, ( 4) endoscopic observation via ovarian chamber! or direct observation via a specially designed abdominal window, (5) photographic recording of laparotomy or. endoscopic findings, and (6) endometrial biopsy (abdominal approach). Materials and Methods
Human Menopausal Gonadotropin (HMG)jHuman Chorionic Gonadotropin (HCG). The human gonadotropic hormones-FSH and LH-are proteins containing carbohydrates. FSH preparation can be extracted either from pituitary glands (HPG) obtained at autopsy or from human menopausal urine (HMG). Pure pituitary LH principle has not yet been isolated; thus human chorionic gonadotropin (HCG) which exhibits luteotropic activity is used in its stead. The preparation of HMG (Pergonal*) used in our study exhibits primarily FSH activity with only a miniscule contaminating LH content. There have been numerous reports 15 , 20, 24, 27 about the use of HMG in combination with HCG for induction of ovulation in the human. The major drawback for large-scale clinic use has been the persistent, albeit infrcquent, occurrence of serious side effects due to overstimulation of the gonads, such as multiple pregnancies, large ovarian cyst with ascites, etc. This menotropic preparation has also been utilized for experimental reasons in the subhuman primates. 4 , 22, 23 Two dosage regimens used in our study included (1) 1 ampule of Pergonal (75 I.U.) intramuscularly for 5-8 consecutive days-depending upon estrogen response of the daily vaginal smear, followed by ~~ ampule (35.5 *Cutter Laboratories, Berkeley, Calif.
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J.U.) of Pergonal and 2000 J.U. HCG (APL*) for 3 days,23 and (2) 1 ampule of Pergonal for 6-7 consecutive days followed by a single large dose of HCG (5000 J.U.) intravenously (Fig. 2). A total of 23 monkeys were treated with these two regimens for a total of 30 cycles. OH •• OH
WY-5090 (dJ-l8- methyl-estriol)
CLOMIPHENE CITRATE
Fig. 1. Agents capable of inducing ovulation: (1) .gonadotropic honnones (glycoproteins)-FSH (HPC, HMC), LH (HCC); (2) nonsteroidal (clomiphene citrate); ( 3) steroidal (WY-5090) . SCHEDULE 1
o
HMG 1. U •
75
~ HCG-
I:'lJ.'
1
',000 2,000
nnnnnn rn [] [] SCHEDULE
75
f 4,000 2,000
2
~nnnDnnl
1 2 3 4 5 .6 7 8 9 Fig. 2. Experimental dosage schedules of HMC/HCC for induction of ovulation in rhesus monkeys. ~J
The therapy for the first treatment cycle was started arbitrarily on any day in animals with amenorrhea and on CyCle Day 5 in animals with anovulatory menstrual cycles. For subsequent cycles, the treatment was initiated on Cycle Day 5 if induction of ovulation was successful.
Clomiphene Citrate and Its Isomers. Clomiphene citrate (Clomidt) is a synthetic nonsteroidal impeded estrogen (Fig. 1). It is weakly estrogenic in the absence of endogenous estrogen as observed in experimental animals, but in the presence of estrogen .it exhibits strong anti-estrogenic activity.6. 19 Clomiphene was originally studied as a gonadotropin-inhibiting * Ayerst Laboratories, New York, N. Y. tWilliam S. Merrell Company, Cincinnati, Ohio.
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and antifecundity agent in rats. 13 In human studies, however, because of its observed strong anti-estrogenic property, it has been used with success as an ovulation-inducing agent. 10 • 11. 17. 26 Although its mode of action is still not understood completely, it appears to stimulate the release of gonadotropins either by direct action on the pituitary or by mediation through its anti-estrogenic activity via the hypothalamus. 1o . 1b Clomiphene citrate is a racemic mixture composed of two geometric isomers-Trans- (Isomer A) and Cis clomiphene (Isomer B) in an approximatel: 1 ratio. Recent studies have revealed that although each isomer is pharmacologically active, they appear to differ in their biologic potency.ll Of a total of 15 monkeys included in this study, 4 were treated with racemic mixture and 5 with each isomer (Table 1). All three were administered for 5 consecutive days. Later in the study, in pursuit of an optimal dosage, 0.2 mg.jkg.jday was also used for both isomers. The therapy was started arbitrarily on any day in animals with amenorrhea and on Cycle Day 1 in animals with menstrual cycles. The medication was administered orally in a vehicle which was incorporated into the monkey's food pellet. The pellets so medicated were offered to the animals as their first feeding in the early morning, when the monkey characteristically is eager for food. No other nourishment was allowed until the medicated pellets were completely ingested. This technic proved successful and avoided the necessity for forced feedings.
dl-18-Methyl-Estriol (WY-5090).* This compound is the most recent addition to an ever-expanding formulary of experimental medications for inducing ovulation. It is a totally synthetic steroidal compound (Fig. 1) chemically related to estriol, and biologically characterized as a weakimpeded estrogen having one-tenth to one-fifth the potency of estriol. Although less potent than estriol and clomiphene, WY-5090 has heen found effective in studies with rodents, in blocking the secretion of pituitary gonadotropins." The rationale for testing this compound in the suhhuman primate lies in its similarity of biologic property to clomiphene. Human studies with this substances, however, have not as yet been conducted. A total of 9 monkeys were utilized in this study. The medication, ,I) mg. I day (approximately 1 mg. Ikg. Iday), was given orally for 5 consecutive days in the first treatment cycle. The dosage was then increased to 10 mg./dav in the second treatment cycle if ovulation had not heen induced at the .5-mg. level, and finally to 20 mg./day if the lO-mg. level failed to induce. The therapv was started arhitrarily on any day in animals with amenorrhea or on Cycle Day 5 in animals with menstrual cycles. *Wyeth Laboratories, Radnor, Pa.
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TABLE 1. Dosage-Time Regimens of Clomiphene Citrate for Induction of Ovulation in Rhesus Monkey Mixture
Dose (mg./kg./day)
Day
1.5
1-5 1-3
Clomiphene citrate (racemic mixture) Transclomiphene citrate (Isomer A) Cisclomiphene citrate (Isomer B)
0.61
1-5
o.2f o.9l 0.2f
1-5
RESULTS HMG/HCG
Twenty-three monkeys were treated with HMG jHeG regimen for a total of 30 cycles. Each monkey received one to three treatment courses. Ovulation was successfully induced in at least one treatment cycle in 17 (74%) of the 23 monkeys studied (Table 2). Of the 30 treatment cycles, 18 were proven to be ovulatory by 2 or more of the indexes used. A single site of ovulation was observed in 7 instances (54%) in which ovulation was confirmed by laparotomy (Table 3). Only in 6 instances ( 46%) was multiple ovulation noted (Fig. 3). This is in marked contrast to the findings of other investigators. 22 , 23, 29 Figure 4 is a photographic recording via a specifically designed abdominal window which illustrates TABLE 2. Comparison of Ovulation Induction with Different Substances ------
Monkeys Ovulatory* Nonovulatory Total Treated Cycles Ovulatory Nonovulatory Total
HMG/HCG
Clomiphene
WY-50.90
17 (74%) 6 (26%) 23
14 (93%) 1 ( 7%) 15
6 (67%) 3 (33%) 9
18 (60%) 12 (40%) 30
27 (59%) 19 (41%) 46
8 (32%) 18 (68%) 26
* Ovulated in at least 1 treatment cycle.
TABLE 3. Number of Ovulation Sites and/or Corpora Lutea Indentified After HMG/HCG Therapy in 13 Rhesus Monkeys -------
No. of monkeys Right Left
Single ovulation site
Multiple ovulation, sitp
7 (54%) 0110001 10X1110
6 (46%) 12.1231 lX/1111
X indicates ovary was previously removed.
.l .....
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Fig. 3 (top). Multiple ovulation induced by HMG/HCG therapy in rhesus monkey. Fig. 4 (bottom). Single site of ovulation induced by HMG/HCG therapy in rhesus monkey viewed through specially designed abdominal window.
the typical single ovulation site phenomenon which we observed when using HMGjHCG therapy in the amenorrheic or anovulatory monkeys. The use of a single large intravenous dose (5000 LV.) of HCG, as described in Schedule 2, proved to be slightly more effective than 3 divided smaller doses (2000 LV.) administered intramuscularly (Table 4). Furthermor~, with this time-dosage regimen, the time of ovulation can be predicted with more accuracy.
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TABLE 4. Ovulatory Response in Rhesus Monkeys with Different HMG/HCG Regimens Schedule
1 2 TOTAL
No. of cycles treated
Ovulation induced
No ovulation induced
23 7 30
13 (56.6%) 5 (71%) 18 (60%)
10 (43.3%)
2 (29%) 12 (40%)
The gross and histologic findings of the ovaries are summarized in Table 5. Multiple ovulation was designated in 3 instances in which only a single ovulation site was found on each ovary. In only one instance, three ovulation sites were identified on one ovary (Table 3). Grossly and microscopically, the majority of corpora lutea resultant from HMGjHCG therapy in either category was indistinguishable in all respects from those resulting from spontaneous ovulation, according to Corner's criteria. Figures 5 and 6 illustrate typical histologic findings of a corpus luteum and endometrium, respectively, resulting from HMG JHCG therapy in our anovulatory monkeys. In the majority of instances, serial vaginal cytologic studies demonstrated an estrogen peak typical of ovulation, followed by progestational reaction after ovulation occurred. Stimulated by the report regarding the changes found in zona pellucida TABLE 5. Ovarian Findings in Rhesus Monkeys after HMG/HCG Monkey No.
Histologic findings
Gross findings
SINGLE OVULATION GROUP
63-13 64-17 64-19 64-20 64-23 65-33 64-22
Normal corpus leteum, left Normal corpus luteum, left Normal corpus luteum, left Normal corpus luteum, right Normal corpus luteum, right, and large follicular cyst
Normal size Normal size Normal size Normal size Normal size Normal size Ovarian cyst
MULTIPLE OVULATION SITES
62-12 64-22 64-34 65-36 65-32 64-26
Normal corpora lutea, right and left 2 involuting corpora lutea, right Normal corpora lutea, right and left Normal corpora lutea, right and left Normal corpora lutea, right and left, and multiple follicles. Normal corpora lutea, right and left, and multiple luteinized follicles.
Normal size Normal size Normal size Normal size Cystic enlargement Cystic enlargement
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Fig. 5 (top). Portion of fresh corpus luteum resulting from HMG/HCG therapy in rhesus monkey. Fig. 6 (bottom). Secretory endometrium observed when ovulation was successfully induced by HMG/HCG therapy in rhesus monkey.
of baboon ovum after gonadotropin stimulation,16 we have examined the zona pellucida of 12 ovarian biopsy specimens from monkeys under RMG/ ReG therapy. These findings were compared with 8 ovarian biopsies obtained from monkeys without any medication. Intact zona pellucida were found in the majority of the specimens of both treated and unmedicated
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animals. Maruffo also reported that no alteration of zona pellucida was observed under light microscope in his recent study; however, fine vesiculation of the outer parts of the zona was noted under the electron microscope. 21 Cystic enlargement of the ovaries was observed in two animals with multiple ovulations. A large follicular cyst was noted in 1 animal in which a normal single ovulation site was found in adjacent normal ovarian tissue. No other ill effects were noted while monkeys were receiving therapy. Clomiphene Citrate and Its Isomers
A total of 15 monkeys was included in this study. They were treated with clomiphene (racemic mixture), Isomer A, or Isomer B for an aggregate of 46 cycles (Table 2). Ovulation was successfully induced in at least one treatment cycle in 14 of the 15 monkeys (94%). Each monkey received 1-4 courses of therapy. Of the total of 46 treatment cycles, 27 were proven to be ovulatory by laparotomy as well as other. peripheral indices. Single-site ovulation was observed uniformly in every instance where ovulation was successfully induced (Fig. 7). Isomers A and B were found to be equally effective in inducing ovulation in our anovulatory monkeys as their racemic compound (Table 6). Ovarian and endometrial biopsies performed during clomiphene-induced ovulatory cycles revealed findings identical to those expected in a spon-
Fig. 7. Single site of ovulation induced by clomiphene citrate in rhesus monkey.
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TABLE 6. Comparison of Ovulation Induction with Clomiphene Citrate and Its Isomers Monkeys Ovulatory* Nonovulatory Total Cycles Ovulatory Nonovulatory Total
Racemic
Trans (A)
Cis (B)
Total
4 1 5
5 0 5
5 0 5
14 (94%) 1 ( 6%) 15
6 5 11
11 6 17
10 8 18
27 (59%) 19 (41%) 46
* Ovulated in at least one treatment cycle.
taneous ovulatory cycle (Fig. 8 and 9). Vaginal smears did not reveal the antiestrogenic changes found when clomiphene was used in lower animals; instead, a typical ovulatory cytologic pattern was noted when ovulation was successfully induced. Endometrial findings were compatible with the status of ovarian activity. No ovarian cyst formation was observed in our experimental group of animals during the study period. dl-18-Methyl-Estriol (WY-5090)
Nine monkeys were treated with this compound for a total of 26 cycles. Ovulation was successfully induced in at least one treatment cycle in 6 (67%) of the 9 monkeys (Table 2). When the dosage of WY-5090 was increased, the successful rate of induction of ovulation was not enhanced. Each monkey was treated for 2-4 cycles. In the total of 26 treatment cycles, single surface ovulation sites were found in 5 instances (Fig. 10). In another 3 instances, a fresh site of ovulation was not identified on the surface of the ovary; however, a biopsy revealed a covert corpus luteum which on histologic section was characterized by an aggregation of luteinized cells under a layer of normal ovarian cortical tissues. Although these findings were highly suggestive of ovulation they might merely represent luteinization of follicles without discharge of ova. Vaginal cytologic findings and endometrial biopsies were generally compatible with the ovarian findings at laparotomy or ovarian biopsies. DISCUSSION
Multiple ovulation occurred only in 46% of cycles following HMGjHCG therapy. In these instances, the greatest number of ovulation sites noted
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Fig. 8 (top). Portion of fresh corpus luteum resulting from clomiphene;citrate therapy in rhesus monkey. Fig. 9 (bottom). Early secretory endometrium obser:ved when ovulation was successfully induced by clomiphene citrate therapy in rhesus monkey.
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Fig. 10. Single site of ovulation induced by WY-5090 in rhesus monkey.
was 3. Generally, however, only 1 ovulation site was noted on each ovary. This is in marked contrast with the work of Simpson and van \Vagenen,2:;' 21> who reported 5-6 ovulation sites per ovary. As these investigators used adult rhesus monkeys with regular ovulatory menstrual cycles, their results can be best characterized as due to "superovulation." The feature of multiple ovulation noted in the great majority of their animals treated with HMG might be dosage-related, due to the additive role of HMG plus already-existing levels of endogenous gonadotropins. In contradistinction, in our experiments we attempted to "induce" ovulation in anovulatory monkeys rather than "superovulate" normal ovulating animals. Although our protocol did not include serial FSH and LH titrations, our findings in subhuman primates appeared to be more in harmony with the observations of Taymor and Sturgis on HMG use in the anovulatory human. Taymor has recently reported that in his experimental series, those amenor· rheic and anovulatory patients with a normal pretreatment level of endogenous gonadotropin were particularly prone to overstimulation ( superovulation phenomenon?).25 Human pituitary gonadotropin (HPG*) is also currently under study in our laboratory. It would be interesting to observe if the findings in anovulatory animals are similar to those in animals treated with HMGfHeG. The result of this study will be reported at a later date. Of the monkeys treated with clomiphene or its isomers, 93% ovulated in at least one of their treatment cycles. However, the overall rate of ovula*HPG was supplied through the courtesy of Dr. S. L. Steelman, Merck Institute for Therapeutic Research, Rahway, N. J.
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tion in the total of 46 treatment cycles was no higher than that of the HMGjHCG series (59% vs. 60%). Unlike our experience with human menotropins, however, we did not find ovarian overstimulation a problem with clomiphene or its isomers in the amenorrheic or anovulatory monkeys under study. In no instance in which ovulation was induced successfully did we note more than a single ovulation site. The exact mechanism of clomiphene in induction of ovulation is not as yet completely known. However, many studies l l , 18 indicated that it stimulates the release of gonadotropins through an intact hypothalamic-pituitary axis, and then the gonadotropins in turn trigger the growth of follicles and discharge of an ovum. Therefore, the gonadotropic levels play a role equally important in clomiphene-treated animals as in those treated with HMG JHCG. It appeared that clomiphene-induced gonadotropin secretion in primates is better balanced by an intact hypothalamic-pituitary-ovarian axis than by direct replacement of gonadotropins-HMGjHCG, with which there are difficulties in regulating dosages. Our finding of single ovulation resulting from clomiphene therapy is also in contrast with clinical experience in humans, where multiple ovulations as well as ovarian enlargement has been noted as a side effect.8, 11, 17 Is this difference merely due to a species variation (subhuman versus human primate), or does the existing endogenous gonadotropic level also playa role in this aspect? Without further study, this question cannot be answered. The utilization of serum radio-immunoassay technic for the titration of FSH and LH, when extended to monkeys, may offer an answer in future studies. In the search to find newer, safer, and more effective agents for the purpo.se of induction of ovulation, dl-18-methyl-estriol (WY-5090) has shown great promise in lower animals. 5 Its ability to induce ovulation in subhuman primates, however, was not as effective as that of the other two agents here reported. Further study with various dosage-time regimens will be necessary to establish an optimal dosage for induction of ovulation before any conclusion regarding its efficacy can be drawn. An interesting finding was noted in the WY-5090 study. In 3 animals in which ovulation was implied histologically, no gross sign of ovulation could be identified on the surface of the ovary; on section, however, a distended follicle with a slightly yellowish tint was noted. Although these areas were histologically reported as subcortical corpora lute a, they may merely represent luteinization of follicles without actual discharge of ova. In these animals every peripheral index was indicative of ovulation. This finding is not considered to be coincidental since it was not noted with other agents in our comparative studies.
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Early in our studies of the basic characteristics of the menstrual ovarian cycle of rhesus monkeys, we used the HMGJHCe regimen to overcome our local newly established colony's problem of a propensity of anovulatory animals. This work, which involved continuous temperature telemetry, has been previously reported. 2 Prior to a decision to use HMGjHCe to correct the anovulatory status of monkeys to induce ovulation, we were apprehensive about the possibility of a high incidence of overstimulation, as had been reported by others.22.23 It appeared to us that multiple ovulations or ovarian cyst formation could distort our telemetry experiments since the resultant alteration in estrogen and progesterone might greatly alter the physiologic events (tubal, uterine, and cervical) that characterize a spontaneous ovulatory cycle. Our apprehension was relieved by the surprisingly high incidence of single ovulation resulting from HMG jHee therapy. Furthermore, with this technic, the time of ovulation can be predicted with considerable precision. Later in our studies, we were delighted to find that clomiphene citrate and its isomers were even more successful in not only inducing but also in closely simulating what appeared to be normal ovulatory physiology. It is our suggestion that in many experiments in reproductive physiology and its alteration (for contraceptive purposes), controlled induction of ovulation, as des'cribed above, offers an alternative method to superovulation for investigators who desire to study the physical, chemical, or biologic events occurring at the time of ovulation or ovum collection as a pertinent part of their studies. The technic offers a method of timing ovulation with more accuracy than spontaneous ovulation and without the resultant disadvantage of superovulation with its accompanying abberation of estrogen and progesterone secretion and distortion of physiologic events of ovarian cycles. It also offers investigators a technic by which to study comparatively anovulatory and ovulatory physiology in the same animal (i.e., allows the animal to be used as its own control). SUMMARY
1. Three agents capable of inducing ovulation-human gonadotropins (HMGjHCG), clomiphene citrate, and dl-18-methyl-estriol (WY-5090)were studied comparatively in the anovulatory subhuman primate-the rhesus monkey. The work reported upon is in contrast to previous experiments by others where superovulating properties were studied. 2. A total of 23 amenorrheic or anovulatory monkeys was treated with HMGjHCG for a total of 30 treatment cycles. Ovulation was successfully induced in 60% of the treated cycles. Clomiphene citrate and its isomers
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were used in 15 monkeys for a total of 46 treatment cycles with a resultant ovulation of 59%. dl-18-Methyl-estriol-a new steroidal impeded estrogen was also evaluated on 9 monkeys for 26 cycles. Ovulation was observed n 32% of the treated cycles; however, in some instances the observance of subcortical corpora lutea left doubt as to the true nature of ovulatory events. 3. The incidence of multiple ovulation in HMG/HCG-treated animals was observed in only 46% and the number of ovulation sites on each ovary was considerably less than that reported by others. Single ovulation was observed uniformly when clomiphene and WY-5090 were employed. 4. Ovarian enlargement was noted as a result of multiple ovulation in 2 monkeys after HMG/HCG therapy. A large follicular cyst was also found in 1 monkey when only a single ovulation was induced. No side effect was observed in monkeys treated with clomiphene and WY-5090. 5. It is suggested that actual induction of ovulation be used by investigators if they require a timetable, rather than superovulation with its accompanying physiologic distortions.
Pennsylvania Hospital 8th and Spruce St. Philadelphia, Pa. 19108
REFERENCES 1. BALIN, H., HALPERN, B. D., and ISRAEL, S. L. ExperImental techniques for continuous direct observation of cyclic ovarian function. Fertil Steril 14:654, 1963. 2. BALIN, H., BUSSER, J. H., FROMM, E., WAN, L. S., and ISRAEL, S. L. Biotelemetry as an adjunct to the study of ovarian physiology. Fertil Steril16:1, 1965. 3. CORNER, G. W. Ovulation and menstruation in Macacus rhesus. Contrib Em-
bryol 75:75, 1923.
4. DEDE, J. A., and PLENTL, A. A. Induced ovulation and artificial insemination in a rhesus colony. Fertil Steril17:757, 1966. 5. EDGREN, R. A. Personal communication. 6. EM MENS, C. W. Estrogenic, antiestrogenic and antifertility activities of various compounds. ] Reprod Fertil 9:277, 1965. 7. ERICKSON, L. E. Light-dark periodicity and the rhesus-monkey menstrual cycle.
Fertil Steril15:352, 1964. 8. GOLDFARB, A. F., MORALES, A., and RAKOFF, A. E. Critical review of 160 clomi· phene-related pregnancies. Obstet Gyneo 31:342, 1968. 9. Goss, L. J. Care and diseases of the research monkey. Ann NY Acad Sci 85. 990, 1960. 10. GREENBLATT, R. B., Roy, S., MAHESH, V. B., BORFIELD, W. E., and JUNGEK, E. C. Induction of ovulation with clomiphene citrate Amer ] Obstet Gynec 84:900, 1962. 11. GREENBLATT, R. B., MAHESH, V. B., and PICO, I. R. "Stimulation of Ovulation i~ the Human with Clomiphene Citrate." In Proceedings of the Fifth World Congress of Gynecology and Obstetrics. 1967, p. 301. 12. HARTMAN, C. G. Studies in the reproduction of the monkey Macacus (Pitheus) rhesus with special reference to menstruation and pregnancy. Contrib Embryol 23:1,1932.
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13. HOLTKAMP, D. E., GRESLIN, J. G., ROOT, C. A., and LERNER, L. J. Gonadotropin inhibiting and anti-fecundity effects of clomiphene. Proc Soc Exper Biol M.ed 105: 197, 1960. 14. JACOBSON, H. M., and WINDLE, W. F. Observations on mating, gestation, birth, and postnatal development of Macaca rnulatta. Biol Neonat 2: 105, 1960. 15. KAsE, N., MROUCH, A., and BUXTON, C. L. Pergonal therapy of anovulatory infertility. ArnerJ Obstet Gynec 100:176, 1968. 16. KATZBERG, A. A. Gonadotropin-induced anomalies of the zona pellucida of the baboon ovum. Science 151: 1225, 1966. 17. KISTNER, R. 'V. Induction of ovulation with clomiphene citrate. Ohstet Gynec Surv 20:873, 1965. 18. KOBAYASHI, T., KATO, J., and VILLEE, C. A. "Effect of Clomiphene on the Uptake of Estradiol by the Anterior Hypothalamus and Hypophysis." In Proceedings of the Fifth World Congress of Gynecology and Obstetrics. 1967, p. 35l. 19. LERNER, L. J., HOLTHAUS, F. G., and THOMPSON, C. R. A nonsteroidal estrogen antagonist. Endocrinol 63:295, 1958. 20. LUNENFELD, B. Treatment of anovulation by human gonadotropins. ] Int Feel Gynaec Obstet 1:153, 1963. 21. MARUFFO, C. A. Zona pellucida of rhesus monkey ovum after gonadotropin stimulation. Science 157: 1313, 1967. 22. MASTROIANNI, L., JR., SUZUKI, S., MANABE, Y., and WATSON, F. Further observations on the influence of the intrauterine device on ovum. Amer ] Ohstet Gynec 99:649, 1967. 23. SIMPSON, M. E., and VAN \VAGENEN, G. Induction of ovulation with human urinary gonadotropins in the monkey. Fertil Steril 13: 140, 1962. 24. TAYMOR, M. L., STURGIS, S. H., LIEBERMAN, B. L., and GOLDSTEIN, D. P. Induction of ovulation with human postmenopausal gonadotropin. I. Case selection and results of therapy. Fertil Steril17:731, 1966. 25. TAYMOR, M. L., and STURGIS, S. H. Induction of ovulation with human postmenopausal gonadotropin. II. Probable causes of overstimulation. Feriil Stcril 17:736, 1966. 26. TOWNSEND, S. L., BROWN, J. B., ADEY, A. D., JOHNSTONE, J. W., EVANS, J. H., and TAFT, H. P. Induction of ovulation. J Obstet Gynaec Brit COlHm 73:.529, 1966. 27. VANDE WIELE, R. L., and TURKSOY, N. Treatment of amenorrhea and anovulation with human menopausal and chorionic gonadotropins. J Clin Endocrinol 23: 369,1965. 28. VERMANDE-VAN ECK, G. J. Effect of low dosage X-irradiation UPOIl pituitary gland and ovaries of rhesus monkeys. Fertil Steril2:294, 1956. 29. VAN WAGENEN, G. Induction of ovulation in ·Macaca mulalta. Fertil Steril 19: 15, 1968.
A Comparative Study LIVIA S. WAN, M.D., and HOWARD BALIN, M.D., M.Sc.MED.
THE
of various ovulation-inducing agents in recent years has opened new avenues in management of certain types of female infertility. Although a great deal of investigation has been conducted at the clinical level with various agents, there appear to be many advantages to the performance of select in-depth studies in the subhuman primate such as Macaca mulatta. Use of the experimental monkey permits direct observation and photographic recording of the status of the ovaries while such medications are being administered, as well as biopsy of the ovary and/or endometrium for histologic examination. . The reproductive anatomy and physiology of the rhesus monkey have been well and thoroughly studied over the last 40 years.s, 12 The value of the rhesus monkey for investigation of problems in the field of human reproduction is that its menstrual cycle resembles that of the human and its reproductive tm~t is anatomically similar.14 Goss considers the rhesus monkey a highly evolved organism, a primate second only to man in psychic development. It responds to the stress of caphue and shipment by ceasing to menstruate. It also is said to have seasonal biologic rhythm, becoming anovulatory in the summer months,S, 7,12 but others have found that in captivity, especially in air-conditioned cages, seasonal anovulatory cycles are not evident. 28 However, the occurrence of anovulation under different stress conditions offers an ideal prototype anovulatory experimental animal for evaluation of the efficacy of compounds thought to have ovulation-inducing properties. In the past 4 years, comparative studies have been conducted in our research monkey colony. We have had the opportunity to compare by INTRODUCTION
From the Gynecic Research Section, Division of Obstetrics and Gynecology, Pennsylvania Hospital, Philadelphia, Pa. Presented at the 24th Annual Meeting of the American Fertility Society, San Francisco, Calif., Mar. 28-30, 1968.
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following the si\me protocols a human menotropin and two impeded estrogens for the purpose of induction of ovulation. RESEARCH PLAN AND MATERIALS Research Plan
Mature female Macaca rhesus monkeys observed in our research laboratory for at least 6 months were selected for the study. Other requisites for inclusion in our study were (1) history of 3-6 months of amenorrhea or oligomenorrhea and (2) evidence of anovulation by vaginal cytologic studies. The indices used for evaluation of ovarian response while in control and treatment cycles included (1) menstrual pattern, ( 2 ) vaginal smears, ( 3) laparotomy with ovarian biopsy at expected "postovulatory" phase, ( 4) endoscopic observation via ovarian chamber! or direct observation via a specially designed abdominal window, (5) photographic recording of laparotomy or. endoscopic findings, and (6) endometrial biopsy (abdominal approach). Materials and Methods
Human Menopausal Gonadotropin (HMG)jHuman Chorionic Gonadotropin (HCG). The human gonadotropic hormones-FSH and LH-are proteins containing carbohydrates. FSH preparation can be extracted either from pituitary glands (HPG) obtained at autopsy or from human menopausal urine (HMG). Pure pituitary LH principle has not yet been isolated; thus human chorionic gonadotropin (HCG) which exhibits luteotropic activity is used in its stead. The preparation of HMG (Pergonal*) used in our study exhibits primarily FSH activity with only a miniscule contaminating LH content. There have been numerous reports 15 , 20, 24, 27 about the use of HMG in combination with HCG for induction of ovulation in the human. The major drawback for large-scale clinic use has been the persistent, albeit infrcquent, occurrence of serious side effects due to overstimulation of the gonads, such as multiple pregnancies, large ovarian cyst with ascites, etc. This menotropic preparation has also been utilized for experimental reasons in the subhuman primates. 4 , 22, 23 Two dosage regimens used in our study included (1) 1 ampule of Pergonal (75 I.U.) intramuscularly for 5-8 consecutive days-depending upon estrogen response of the daily vaginal smear, followed by ~~ ampule (35.5 *Cutter Laboratories, Berkeley, Calif.
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J.U.) of Pergonal and 2000 J.U. HCG (APL*) for 3 days,23 and (2) 1 ampule of Pergonal for 6-7 consecutive days followed by a single large dose of HCG (5000 J.U.) intravenously (Fig. 2). A total of 23 monkeys were treated with these two regimens for a total of 30 cycles. OH •• OH
WY-5090 (dJ-l8- methyl-estriol)
CLOMIPHENE CITRATE
Fig. 1. Agents capable of inducing ovulation: (1) .gonadotropic honnones (glycoproteins)-FSH (HPC, HMC), LH (HCC); (2) nonsteroidal (clomiphene citrate); ( 3) steroidal (WY-5090) . SCHEDULE 1
o
HMG 1. U •
75
~ HCG-
I:'lJ.'
1
',000 2,000
nnnnnn rn [] [] SCHEDULE
75
f 4,000 2,000
2
~nnnDnnl
1 2 3 4 5 .6 7 8 9 Fig. 2. Experimental dosage schedules of HMC/HCC for induction of ovulation in rhesus monkeys. ~J
The therapy for the first treatment cycle was started arbitrarily on any day in animals with amenorrhea and on CyCle Day 5 in animals with anovulatory menstrual cycles. For subsequent cycles, the treatment was initiated on Cycle Day 5 if induction of ovulation was successful.
Clomiphene Citrate and Its Isomers. Clomiphene citrate (Clomidt) is a synthetic nonsteroidal impeded estrogen (Fig. 1). It is weakly estrogenic in the absence of endogenous estrogen as observed in experimental animals, but in the presence of estrogen .it exhibits strong anti-estrogenic activity.6. 19 Clomiphene was originally studied as a gonadotropin-inhibiting * Ayerst Laboratories, New York, N. Y. tWilliam S. Merrell Company, Cincinnati, Ohio.
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and antifecundity agent in rats. 13 In human studies, however, because of its observed strong anti-estrogenic property, it has been used with success as an ovulation-inducing agent. 10 • 11. 17. 26 Although its mode of action is still not understood completely, it appears to stimulate the release of gonadotropins either by direct action on the pituitary or by mediation through its anti-estrogenic activity via the hypothalamus. 1o . 1b Clomiphene citrate is a racemic mixture composed of two geometric isomers-Trans- (Isomer A) and Cis clomiphene (Isomer B) in an approximatel: 1 ratio. Recent studies have revealed that although each isomer is pharmacologically active, they appear to differ in their biologic potency.ll Of a total of 15 monkeys included in this study, 4 were treated with racemic mixture and 5 with each isomer (Table 1). All three were administered for 5 consecutive days. Later in the study, in pursuit of an optimal dosage, 0.2 mg.jkg.jday was also used for both isomers. The therapy was started arbitrarily on any day in animals with amenorrhea and on Cycle Day 1 in animals with menstrual cycles. The medication was administered orally in a vehicle which was incorporated into the monkey's food pellet. The pellets so medicated were offered to the animals as their first feeding in the early morning, when the monkey characteristically is eager for food. No other nourishment was allowed until the medicated pellets were completely ingested. This technic proved successful and avoided the necessity for forced feedings.
dl-18-Methyl-Estriol (WY-5090).* This compound is the most recent addition to an ever-expanding formulary of experimental medications for inducing ovulation. It is a totally synthetic steroidal compound (Fig. 1) chemically related to estriol, and biologically characterized as a weakimpeded estrogen having one-tenth to one-fifth the potency of estriol. Although less potent than estriol and clomiphene, WY-5090 has heen found effective in studies with rodents, in blocking the secretion of pituitary gonadotropins." The rationale for testing this compound in the suhhuman primate lies in its similarity of biologic property to clomiphene. Human studies with this substances, however, have not as yet been conducted. A total of 9 monkeys were utilized in this study. The medication, ,I) mg. I day (approximately 1 mg. Ikg. Iday), was given orally for 5 consecutive days in the first treatment cycle. The dosage was then increased to 10 mg./dav in the second treatment cycle if ovulation had not heen induced at the .5-mg. level, and finally to 20 mg./day if the lO-mg. level failed to induce. The therapv was started arhitrarily on any day in animals with amenorrhea or on Cycle Day 5 in animals with menstrual cycles. *Wyeth Laboratories, Radnor, Pa.
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TABLE 1. Dosage-Time Regimens of Clomiphene Citrate for Induction of Ovulation in Rhesus Monkey Mixture
Dose (mg./kg./day)
Day
1.5
1-5 1-3
Clomiphene citrate (racemic mixture) Transclomiphene citrate (Isomer A) Cisclomiphene citrate (Isomer B)
0.61
1-5
o.2f o.9l 0.2f
1-5
RESULTS HMG/HCG
Twenty-three monkeys were treated with HMG jHeG regimen for a total of 30 cycles. Each monkey received one to three treatment courses. Ovulation was successfully induced in at least one treatment cycle in 17 (74%) of the 23 monkeys studied (Table 2). Of the 30 treatment cycles, 18 were proven to be ovulatory by 2 or more of the indexes used. A single site of ovulation was observed in 7 instances (54%) in which ovulation was confirmed by laparotomy (Table 3). Only in 6 instances ( 46%) was multiple ovulation noted (Fig. 3). This is in marked contrast to the findings of other investigators. 22 , 23, 29 Figure 4 is a photographic recording via a specifically designed abdominal window which illustrates TABLE 2. Comparison of Ovulation Induction with Different Substances ------
Monkeys Ovulatory* Nonovulatory Total Treated Cycles Ovulatory Nonovulatory Total
HMG/HCG
Clomiphene
WY-50.90
17 (74%) 6 (26%) 23
14 (93%) 1 ( 7%) 15
6 (67%) 3 (33%) 9
18 (60%) 12 (40%) 30
27 (59%) 19 (41%) 46
8 (32%) 18 (68%) 26
* Ovulated in at least 1 treatment cycle.
TABLE 3. Number of Ovulation Sites and/or Corpora Lutea Indentified After HMG/HCG Therapy in 13 Rhesus Monkeys -------
No. of monkeys Right Left
Single ovulation site
Multiple ovulation, sitp
7 (54%) 0110001 10X1110
6 (46%) 12.1231 lX/1111
X indicates ovary was previously removed.
.l .....
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Fig. 3 (top). Multiple ovulation induced by HMG/HCG therapy in rhesus monkey. Fig. 4 (bottom). Single site of ovulation induced by HMG/HCG therapy in rhesus monkey viewed through specially designed abdominal window.
the typical single ovulation site phenomenon which we observed when using HMGjHCG therapy in the amenorrheic or anovulatory monkeys. The use of a single large intravenous dose (5000 LV.) of HCG, as described in Schedule 2, proved to be slightly more effective than 3 divided smaller doses (2000 LV.) administered intramuscularly (Table 4). Furthermor~, with this time-dosage regimen, the time of ovulation can be predicted with more accuracy.
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TABLE 4. Ovulatory Response in Rhesus Monkeys with Different HMG/HCG Regimens Schedule
1 2 TOTAL
No. of cycles treated
Ovulation induced
No ovulation induced
23 7 30
13 (56.6%) 5 (71%) 18 (60%)
10 (43.3%)
2 (29%) 12 (40%)
The gross and histologic findings of the ovaries are summarized in Table 5. Multiple ovulation was designated in 3 instances in which only a single ovulation site was found on each ovary. In only one instance, three ovulation sites were identified on one ovary (Table 3). Grossly and microscopically, the majority of corpora lutea resultant from HMGjHCG therapy in either category was indistinguishable in all respects from those resulting from spontaneous ovulation, according to Corner's criteria. Figures 5 and 6 illustrate typical histologic findings of a corpus luteum and endometrium, respectively, resulting from HMG JHCG therapy in our anovulatory monkeys. In the majority of instances, serial vaginal cytologic studies demonstrated an estrogen peak typical of ovulation, followed by progestational reaction after ovulation occurred. Stimulated by the report regarding the changes found in zona pellucida TABLE 5. Ovarian Findings in Rhesus Monkeys after HMG/HCG Monkey No.
Histologic findings
Gross findings
SINGLE OVULATION GROUP
63-13 64-17 64-19 64-20 64-23 65-33 64-22
Normal corpus leteum, left Normal corpus luteum, left Normal corpus luteum, left Normal corpus luteum, right Normal corpus luteum, right, and large follicular cyst
Normal size Normal size Normal size Normal size Normal size Normal size Ovarian cyst
MULTIPLE OVULATION SITES
62-12 64-22 64-34 65-36 65-32 64-26
Normal corpora lutea, right and left 2 involuting corpora lutea, right Normal corpora lutea, right and left Normal corpora lutea, right and left Normal corpora lutea, right and left, and multiple follicles. Normal corpora lutea, right and left, and multiple luteinized follicles.
Normal size Normal size Normal size Normal size Cystic enlargement Cystic enlargement
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Fig. 5 (top). Portion of fresh corpus luteum resulting from HMG/HCG therapy in rhesus monkey. Fig. 6 (bottom). Secretory endometrium observed when ovulation was successfully induced by HMG/HCG therapy in rhesus monkey.
of baboon ovum after gonadotropin stimulation,16 we have examined the zona pellucida of 12 ovarian biopsy specimens from monkeys under RMG/ ReG therapy. These findings were compared with 8 ovarian biopsies obtained from monkeys without any medication. Intact zona pellucida were found in the majority of the specimens of both treated and unmedicated
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animals. Maruffo also reported that no alteration of zona pellucida was observed under light microscope in his recent study; however, fine vesiculation of the outer parts of the zona was noted under the electron microscope. 21 Cystic enlargement of the ovaries was observed in two animals with multiple ovulations. A large follicular cyst was noted in 1 animal in which a normal single ovulation site was found in adjacent normal ovarian tissue. No other ill effects were noted while monkeys were receiving therapy. Clomiphene Citrate and Its Isomers
A total of 15 monkeys was included in this study. They were treated with clomiphene (racemic mixture), Isomer A, or Isomer B for an aggregate of 46 cycles (Table 2). Ovulation was successfully induced in at least one treatment cycle in 14 of the 15 monkeys (94%). Each monkey received 1-4 courses of therapy. Of the total of 46 treatment cycles, 27 were proven to be ovulatory by laparotomy as well as other. peripheral indices. Single-site ovulation was observed uniformly in every instance where ovulation was successfully induced (Fig. 7). Isomers A and B were found to be equally effective in inducing ovulation in our anovulatory monkeys as their racemic compound (Table 6). Ovarian and endometrial biopsies performed during clomiphene-induced ovulatory cycles revealed findings identical to those expected in a spon-
Fig. 7. Single site of ovulation induced by clomiphene citrate in rhesus monkey.
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TABLE 6. Comparison of Ovulation Induction with Clomiphene Citrate and Its Isomers Monkeys Ovulatory* Nonovulatory Total Cycles Ovulatory Nonovulatory Total
Racemic
Trans (A)
Cis (B)
Total
4 1 5
5 0 5
5 0 5
14 (94%) 1 ( 6%) 15
6 5 11
11 6 17
10 8 18
27 (59%) 19 (41%) 46
* Ovulated in at least one treatment cycle.
taneous ovulatory cycle (Fig. 8 and 9). Vaginal smears did not reveal the antiestrogenic changes found when clomiphene was used in lower animals; instead, a typical ovulatory cytologic pattern was noted when ovulation was successfully induced. Endometrial findings were compatible with the status of ovarian activity. No ovarian cyst formation was observed in our experimental group of animals during the study period. dl-18-Methyl-Estriol (WY-5090)
Nine monkeys were treated with this compound for a total of 26 cycles. Ovulation was successfully induced in at least one treatment cycle in 6 (67%) of the 9 monkeys (Table 2). When the dosage of WY-5090 was increased, the successful rate of induction of ovulation was not enhanced. Each monkey was treated for 2-4 cycles. In the total of 26 treatment cycles, single surface ovulation sites were found in 5 instances (Fig. 10). In another 3 instances, a fresh site of ovulation was not identified on the surface of the ovary; however, a biopsy revealed a covert corpus luteum which on histologic section was characterized by an aggregation of luteinized cells under a layer of normal ovarian cortical tissues. Although these findings were highly suggestive of ovulation they might merely represent luteinization of follicles without discharge of ova. Vaginal cytologic findings and endometrial biopsies were generally compatible with the ovarian findings at laparotomy or ovarian biopsies. DISCUSSION
Multiple ovulation occurred only in 46% of cycles following HMGjHCG therapy. In these instances, the greatest number of ovulation sites noted
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Fig. 8 (top). Portion of fresh corpus luteum resulting from clomiphene;citrate therapy in rhesus monkey. Fig. 9 (bottom). Early secretory endometrium obser:ved when ovulation was successfully induced by clomiphene citrate therapy in rhesus monkey.
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Fig. 10. Single site of ovulation induced by WY-5090 in rhesus monkey.
was 3. Generally, however, only 1 ovulation site was noted on each ovary. This is in marked contrast with the work of Simpson and van \Vagenen,2:;' 21> who reported 5-6 ovulation sites per ovary. As these investigators used adult rhesus monkeys with regular ovulatory menstrual cycles, their results can be best characterized as due to "superovulation." The feature of multiple ovulation noted in the great majority of their animals treated with HMG might be dosage-related, due to the additive role of HMG plus already-existing levels of endogenous gonadotropins. In contradistinction, in our experiments we attempted to "induce" ovulation in anovulatory monkeys rather than "superovulate" normal ovulating animals. Although our protocol did not include serial FSH and LH titrations, our findings in subhuman primates appeared to be more in harmony with the observations of Taymor and Sturgis on HMG use in the anovulatory human. Taymor has recently reported that in his experimental series, those amenor· rheic and anovulatory patients with a normal pretreatment level of endogenous gonadotropin were particularly prone to overstimulation ( superovulation phenomenon?).25 Human pituitary gonadotropin (HPG*) is also currently under study in our laboratory. It would be interesting to observe if the findings in anovulatory animals are similar to those in animals treated with HMGfHeG. The result of this study will be reported at a later date. Of the monkeys treated with clomiphene or its isomers, 93% ovulated in at least one of their treatment cycles. However, the overall rate of ovula*HPG was supplied through the courtesy of Dr. S. L. Steelman, Merck Institute for Therapeutic Research, Rahway, N. J.
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tion in the total of 46 treatment cycles was no higher than that of the HMGjHCG series (59% vs. 60%). Unlike our experience with human menotropins, however, we did not find ovarian overstimulation a problem with clomiphene or its isomers in the amenorrheic or anovulatory monkeys under study. In no instance in which ovulation was induced successfully did we note more than a single ovulation site. The exact mechanism of clomiphene in induction of ovulation is not as yet completely known. However, many studies l l , 18 indicated that it stimulates the release of gonadotropins through an intact hypothalamic-pituitary axis, and then the gonadotropins in turn trigger the growth of follicles and discharge of an ovum. Therefore, the gonadotropic levels play a role equally important in clomiphene-treated animals as in those treated with HMG JHCG. It appeared that clomiphene-induced gonadotropin secretion in primates is better balanced by an intact hypothalamic-pituitary-ovarian axis than by direct replacement of gonadotropins-HMGjHCG, with which there are difficulties in regulating dosages. Our finding of single ovulation resulting from clomiphene therapy is also in contrast with clinical experience in humans, where multiple ovulations as well as ovarian enlargement has been noted as a side effect.8, 11, 17 Is this difference merely due to a species variation (subhuman versus human primate), or does the existing endogenous gonadotropic level also playa role in this aspect? Without further study, this question cannot be answered. The utilization of serum radio-immunoassay technic for the titration of FSH and LH, when extended to monkeys, may offer an answer in future studies. In the search to find newer, safer, and more effective agents for the purpo.se of induction of ovulation, dl-18-methyl-estriol (WY-5090) has shown great promise in lower animals. 5 Its ability to induce ovulation in subhuman primates, however, was not as effective as that of the other two agents here reported. Further study with various dosage-time regimens will be necessary to establish an optimal dosage for induction of ovulation before any conclusion regarding its efficacy can be drawn. An interesting finding was noted in the WY-5090 study. In 3 animals in which ovulation was implied histologically, no gross sign of ovulation could be identified on the surface of the ovary; on section, however, a distended follicle with a slightly yellowish tint was noted. Although these areas were histologically reported as subcortical corpora lute a, they may merely represent luteinization of follicles without actual discharge of ova. In these animals every peripheral index was indicative of ovulation. This finding is not considered to be coincidental since it was not noted with other agents in our comparative studies.
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Early in our studies of the basic characteristics of the menstrual ovarian cycle of rhesus monkeys, we used the HMGJHCe regimen to overcome our local newly established colony's problem of a propensity of anovulatory animals. This work, which involved continuous temperature telemetry, has been previously reported. 2 Prior to a decision to use HMGjHCe to correct the anovulatory status of monkeys to induce ovulation, we were apprehensive about the possibility of a high incidence of overstimulation, as had been reported by others.22.23 It appeared to us that multiple ovulations or ovarian cyst formation could distort our telemetry experiments since the resultant alteration in estrogen and progesterone might greatly alter the physiologic events (tubal, uterine, and cervical) that characterize a spontaneous ovulatory cycle. Our apprehension was relieved by the surprisingly high incidence of single ovulation resulting from HMG jHee therapy. Furthermore, with this technic, the time of ovulation can be predicted with considerable precision. Later in our studies, we were delighted to find that clomiphene citrate and its isomers were even more successful in not only inducing but also in closely simulating what appeared to be normal ovulatory physiology. It is our suggestion that in many experiments in reproductive physiology and its alteration (for contraceptive purposes), controlled induction of ovulation, as des'cribed above, offers an alternative method to superovulation for investigators who desire to study the physical, chemical, or biologic events occurring at the time of ovulation or ovum collection as a pertinent part of their studies. The technic offers a method of timing ovulation with more accuracy than spontaneous ovulation and without the resultant disadvantage of superovulation with its accompanying abberation of estrogen and progesterone secretion and distortion of physiologic events of ovarian cycles. It also offers investigators a technic by which to study comparatively anovulatory and ovulatory physiology in the same animal (i.e., allows the animal to be used as its own control). SUMMARY
1. Three agents capable of inducing ovulation-human gonadotropins (HMGjHCG), clomiphene citrate, and dl-18-methyl-estriol (WY-5090)were studied comparatively in the anovulatory subhuman primate-the rhesus monkey. The work reported upon is in contrast to previous experiments by others where superovulating properties were studied. 2. A total of 23 amenorrheic or anovulatory monkeys was treated with HMGjHCG for a total of 30 treatment cycles. Ovulation was successfully induced in 60% of the treated cycles. Clomiphene citrate and its isomers
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were used in 15 monkeys for a total of 46 treatment cycles with a resultant ovulation of 59%. dl-18-Methyl-estriol-a new steroidal impeded estrogen was also evaluated on 9 monkeys for 26 cycles. Ovulation was observed n 32% of the treated cycles; however, in some instances the observance of subcortical corpora lutea left doubt as to the true nature of ovulatory events. 3. The incidence of multiple ovulation in HMG/HCG-treated animals was observed in only 46% and the number of ovulation sites on each ovary was considerably less than that reported by others. Single ovulation was observed uniformly when clomiphene and WY-5090 were employed. 4. Ovarian enlargement was noted as a result of multiple ovulation in 2 monkeys after HMG/HCG therapy. A large follicular cyst was also found in 1 monkey when only a single ovulation was induced. No side effect was observed in monkeys treated with clomiphene and WY-5090. 5. It is suggested that actual induction of ovulation be used by investigators if they require a timetable, rather than superovulation with its accompanying physiologic distortions.
Pennsylvania Hospital 8th and Spruce St. Philadelphia, Pa. 19108
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bryol 75:75, 1923.
4. DEDE, J. A., and PLENTL, A. A. Induced ovulation and artificial insemination in a rhesus colony. Fertil Steril17:757, 1966. 5. EDGREN, R. A. Personal communication. 6. EM MENS, C. W. Estrogenic, antiestrogenic and antifertility activities of various compounds. ] Reprod Fertil 9:277, 1965. 7. ERICKSON, L. E. Light-dark periodicity and the rhesus-monkey menstrual cycle.
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13. HOLTKAMP, D. E., GRESLIN, J. G., ROOT, C. A., and LERNER, L. J. Gonadotropin inhibiting and anti-fecundity effects of clomiphene. Proc Soc Exper Biol M.ed 105: 197, 1960. 14. JACOBSON, H. M., and WINDLE, W. F. Observations on mating, gestation, birth, and postnatal development of Macaca rnulatta. Biol Neonat 2: 105, 1960. 15. KAsE, N., MROUCH, A., and BUXTON, C. L. Pergonal therapy of anovulatory infertility. ArnerJ Obstet Gynec 100:176, 1968. 16. KATZBERG, A. A. Gonadotropin-induced anomalies of the zona pellucida of the baboon ovum. Science 151: 1225, 1966. 17. KISTNER, R. 'V. Induction of ovulation with clomiphene citrate. Ohstet Gynec Surv 20:873, 1965. 18. KOBAYASHI, T., KATO, J., and VILLEE, C. A. "Effect of Clomiphene on the Uptake of Estradiol by the Anterior Hypothalamus and Hypophysis." In Proceedings of the Fifth World Congress of Gynecology and Obstetrics. 1967, p. 35l. 19. LERNER, L. J., HOLTHAUS, F. G., and THOMPSON, C. R. A nonsteroidal estrogen antagonist. Endocrinol 63:295, 1958. 20. LUNENFELD, B. Treatment of anovulation by human gonadotropins. ] Int Feel Gynaec Obstet 1:153, 1963. 21. MARUFFO, C. A. Zona pellucida of rhesus monkey ovum after gonadotropin stimulation. Science 157: 1313, 1967. 22. MASTROIANNI, L., JR., SUZUKI, S., MANABE, Y., and WATSON, F. Further observations on the influence of the intrauterine device on ovum. Amer ] Ohstet Gynec 99:649, 1967. 23. SIMPSON, M. E., and VAN \VAGENEN, G. Induction of ovulation with human urinary gonadotropins in the monkey. Fertil Steril 13: 140, 1962. 24. TAYMOR, M. L., STURGIS, S. H., LIEBERMAN, B. L., and GOLDSTEIN, D. P. Induction of ovulation with human postmenopausal gonadotropin. I. Case selection and results of therapy. Fertil Steril17:731, 1966. 25. TAYMOR, M. L., and STURGIS, S. H. Induction of ovulation with human postmenopausal gonadotropin. II. Probable causes of overstimulation. Feriil Stcril 17:736, 1966. 26. TOWNSEND, S. L., BROWN, J. B., ADEY, A. D., JOHNSTONE, J. W., EVANS, J. H., and TAFT, H. P. Induction of ovulation. J Obstet Gynaec Brit COlHm 73:.529, 1966. 27. VANDE WIELE, R. L., and TURKSOY, N. Treatment of amenorrhea and anovulation with human menopausal and chorionic gonadotropins. J Clin Endocrinol 23: 369,1965. 28. VERMANDE-VAN ECK, G. J. Effect of low dosage X-irradiation UPOIl pituitary gland and ovaries of rhesus monkeys. Fertil Steril2:294, 1956. 29. VAN WAGENEN, G. Induction of ovulation in ·Macaca mulalta. Fertil Steril 19: 15, 1968.