Postpartum infertility in common chimpanzees

American Journal of Primatology 24:245-255 (1991)

Post part u m I nf erti I ity in Common Chimpanzees CHARLES E. GRAHAM', ELAINE J. STRUTHERS', WILLIAM C . HOBSON', TRENT McDONALD', CHARLES FAIMAN', MAIRE T. BUCKMAN3, AND DELWOOD C. COLLINS4 'Primate Research Institute, New Mexico State University, Holloman Air Force Base; 'Faculty of Medicine, University of Manitoba, Winnepeg, Canada; "Department of Medicine, University of New Mexico School of Medicine, Albuquerque; 4Veteran Administration Medical Center, Atlanta, Georgia

The endocrine profile during normal postpartum amenorrhea in chimpanzees (Pan troglodytes) closely resembles that of women, and its duration is similarly extended by nursing. However, when infant chimpanzees in our colony were removed at birth, excessively prolonged postpartum amenorrhea (7-26 months duration) occurred in 24% of cases. Our endocrine studies indicate that such prolonged postpartum amenorrhea (PPAm) is a pathological condition associated with chronically elevated serum prolactin levels and galactorrhea.

In the absence of nursing, we sought an alternate behavioral basis for PPAm. Breast and genital auto- and partner-directed manipulation was compared in PPAm chimpanzees, normal 2-3 mo. postpartum chimpanzees (infants removed at birth), and regularly-menstruating chimpanzees. A statistically significant pattern of breast, but not genital, manipulation was observed in the PPAm group only, at levels comparable to normal suckling. In particular, a characteristic pattern of nipple auto-manipulation (spooling) occurred. Two partner-stimulated PPAm animals were also identified: when caged individually, they resumed cycling within a few days. In contrast, 2 self-stimulators did not resume cyclicity when isolated. These observations suggested that interruption of breast stimulation allowed cyclicity to resume. Treatment of PPAm chimpanzees with oral Bromocryptine Mesylate

(Sandoz, 2.5-5.0 mg b.i.d.1 was associated with depression of prolactin levels in most animals and resumption of cyclicity in 11/13 subjects within 4 months. Oral Pergolide (Eli Lilly, 200 kg once daily, 13-17 days), ap- peared much more effective, cyclicity resumed in 818 animals within 14 days of commencing treatment; both drugs significantly reduced postpartum amenorrhea duration.

Key words: ape, amenorrhea, prolactin, nursing

Received for publication August 7, 1989; revision accepted October 18, 1990.

Address reprint requests to Charles E. Graham, PhD., DSc., 301 Signers' Hall, University of Alaska Fairbanks, AK 99775.

0 1991 Wiley-Liss, Inc.

246 I Graham et al.

Fig. 1. Postpartum day of first menstrual cycle sexual swelling after 67 normal chimpanzee births. The y-axis shows the cumulative distribution of first postpartum day of sexual swelling associated with a menstrual cycle. Note the close fit of two exponential curves, with the intersection a t 140 days (vertical dashed line).

INTRODUCTION Our group has studied the physiology of postpartum amenorrhea and physio-

pathology of prolonged postpartum amenorrhea (PPAm) in great apes for several years.

We have shown that the profile of gonadotropins and steroid hormones in chimpanzees and organgutans closely resembles that of women, whether or not the infant is removed at birth [Nadler et al., 1981; Reyes et al., 19721. Estradiol, progesterone, and LH levels become stabilized at relatively low levels and FSH is relatively elevated until cyclicity resumes. Prolactin also appears to be elevated (see below). Prior to resumption of cyclicity, there appears to be a fall in circulating FSH levels and a rise in estradiol.

The interval to resumption of cyclicity in captive chimpanzees is related to the period of time the infant is allowed to nurse [Nadler et al., 19811, as it is in wild great apes and women. Our interest has focused on the interval to resumption of cyclicity in captive chimpanzees whose infants are removed a t birth because until the inception of the National Chimpanzee Breeding and Research Plan all our infants were nursery-raised to maximize production. We found that 24% of 67 postpartum periods associated with infant separation at birth were characterized by a period of amenorrhea in excess of 6 mo, a duration we considered excessive in view of the rapid return to cyclicity of the majority of animals [Graham & Stru- thers, 1988; Fig. 11. Obviously, prolonged postpartum amenorrhea decreases breeding productivity, hence our efforts to understand and correct this condition. PPAm chimpanzees were found to produce a milky breast secretion, indicative of a hyperprolactinemic etiology [Struthers, 19881, so suggesting the possibility of phar- macological reversal.

In this paper we shall review our previous research, present new data, and synthesize our findings in order to draw important new conclusions for breeding management of the great apes.

METHODS Animals in this study were housed in indoor-outdoor cages, usually in female-

female pairs, exposed to the natural photoperiod, except during infrequent inclem- ent winter weather, when they were confined indoors in a 12/12 hours photoperiod. For blood samples, animals were anesthetized with i.m. ketamine hydrochloride

Postpartum Infertility in Chimpanzees I 247

TABLE I. Ethogram for Evaluation of Stimulatory Behavior in Chimpanzees

ACTOR MODE OBJECT

Auto (A) Oral (0) Breast (B) Partner (P) Manual (M) Nipple (N)

Genital (G) Other (mainly grooming)

(10 mg/kg) and serum was subjected to radioimmunoassay for prolactin, using a double antibody method [Reyes et al., 19751. For steroids and gonadotropins, methods cited in Graham et al. [19721 were used; for each assay the interassay and intra-assay variation were less than 8% and 12%, respectively. Note that gonadotropin values are not expressed as International Units, as equivalency of chimpanzee gonadotropins to human gonadotropins has not been established, although serial dilutions do react in a parallel manner in human assay systems [Graham et al., 19721.

Breast secretions were obtained under ketamine anesthesia by compressing the breast and collecting milk into a clean vessel. Lactose was measured by the method of Marier and Boulet [1959]. Milk from normally nursing mothers was obtained 2-4 months after birth, and secretion was obtained at various intervals >6m after birth from PPAm subjects. Interval after suckling was not recorded.

For behavioral evaluation, 6 (unless otherwise stated) 2-hour focal sessions on each subject were conducted from the outside run on each subject a t varying times during the day, always by the same observer (E.J.S.); 2 normal postpartum animals were examined for only 2 and 4 sessions, respectively. The duration of specific behavior patterns was timed, employing an ethogram we developed [Table I; Gra- ham & Struthers, 19881. During the continuous observation, the frequency, duration and episodic pattern of the behaviors were recorded.

Bromocryptine (Sandoz) was dissolved in fruit juice and offered at a dose of 2.5-5.0 mg at approximately 8.00h and 15.00h; controls received vehicle alone and were switched to the treatment group after approximately 5 months, unless cyclicity occurred first. Treatment and controls were matched according to duration of amenorrhea as far as possible. Pergolide mesylate (Eli Lilly) was offered once per day at 200 pghubject on a fruit slice. Drug-treated animals were all mature fe- males, but varied greatly in age and parity; the animals were not characterized by atypical menstrual cycle patterns. Their behavioral characteristics were not evaluated. Drug treatment began 7-32 months after parturition.

Standard analyses of variance were used in this study, except as mentioned. To determine the significance of Bromocryptine and Pergolide treatment, historical data shown in Figure 1 [Graham & Struthers, 19881 was used to compute the probability of each chimpanzee spontaneously returning to cyclicity during the treatment interval. In considering that treatment started at differing intervals after birth, we found that the remaining interval to cyclicity is independent on the time elapsed from parturition; this property is characteristic of exponential distributions, which we found by an independent method best fits our data. This means that the probability of any chimpanzee returning to cyclicity within the period of drug treatment is equal no matter when after parturition treatment began (as- suming no treatment effect). Thus we tested the null hypothesis that treatment for 5 months in the case of Bromocryptine, and 2 weeks in the case of Pergolide did not significantly alter the interval to return of cyclicity.

248 I Graham et al.

This study was conducted in 4 main phases. Initially longitudinal endocrine data were studied. Later, the effects of anesthesia and Bromocryptine treatment were examined in a separate group of animals. Pergolide was studied in a third group. Finally, behavioral and breast secretion data were studied in a fourth group. There was no overlap in the types of data studied between groups, except as mentioned in the text. Animals for each phase were all those available at the time that met the criteria for PPAm; hence there was no recognizable bias in selection of subjects.

RESULTS Postpartum Duration

Our initial definition of prolonged postpartum amenorrhea (> 6 months) was arbitrary. We re-analyzed the distribution of postpartum durations in chimpanzees whose infants were removed at birth by piecewise linear regression on log-trans- formed data. We found that the data are in fact best described by 2 distributions (r2 = 0.99), with a break point at 140 days postpartum as shown in Figure 1. This suggests that excessively long postpartum periods represent a separate population that can be more completely characterized.

Lactation We have conducted a preliminary study of lactose content in milky breast

secretion from prolonged postpartum chimpanzees, as an indicator of lactogenesis [Struthers, 1988; Struthers et al., 19891. The mean lactose concentration (mean 2 SE: 29.8 t 3.4) in breast secretion from four prolonged postpartum amenorrhea chimpanzees was about half that of nursing chimpanzees (64 rt 2.1; P < 0.0001). No fluid could be expressed from the breast of normally cycling animals for comparison. These figures correspond closely with human figures for nursing and galactorrhea milk. [Vorheer, 19781. Consequently, we conclude postpartum amenorrhea of greater than 6 months duration in chimpanzees is a hyperprolactinemic amenorrhealgalactorrhea syndrome.

Endocrinology We have previously published mean gonadotropin and ovarian steroid levels in

chimpanzees and orangutans that nursed their infants and which had infants removed at birth, including serial data from 1 chimpanzee that had its infant removed at birth and resumed cyclicity within 170 days. [Nadler et al., 19811. We here present serial data from 2 chimpanzees with prolonged amenorrhea, one of whose infants was removed at birth, the other a t 42 weeks postpartum (Figs. 2,3) . It is interesting that the gonadotropin and steroid profiles are similar among all the animals and to women [Reyes et al., 19721, namely depressed ovarian steroids and LH, elevated FSH, until shortly before cycle resumption, when in most cases there was a dramatic reversal of the gonadotropin ratios.

We observed a significant effect of ketamine anesthesia on serum prolactin in menstruating animals (elevation) when compared with voluntary samples obtained before anesthesia; but in prolonged postpartum subjects there was an op- posite, but non-significant trend (Table 11).

Prolactin levels in normal, alert menstruating animals were usually less than half of those for alert prolonged postpartum subjects (P < 0.051, although there was some overlap in range (Table 11); however, the mean values obtained under ketamine anesthesia approximately 15 minutes later from the 2 groups were almost identical. It should be noted that the levels in the animals bled alert are also rather high, compared with values previously reported from our group both for chimpan-

Postpartum Infertility in Chimpanzees / 249

POS 1 PART UM END 0 C R IN E C H A N G E S IN CHIMPANZEE; INFANT REMOVED AT BIRTH

1

0 4 8 12 16 2 0 24 28 32 36 40 44

WEEKS POSTPARTUM

Fig. 2. Postpartum endocrine changes in chimpanzee, infant removed a t birth. D indicates first sexual swelling detumescence, approximately co-incident with first postpartum ovulation. Note relative elevation of FSH and suppression of other hormones until shortly before spontaneous cycle resumption in this case of prolonged postpartum amenorrhea.

zees and humans [Reyes et al., 1972, 19751: this observation suggests that the animals bled alert may nevertheless be subject to some stress; we plan to address this question in the future.

Behavior A preliminary series of 1-3 2-hour focal observation sessions, on all the 5

PPAm subjects available at the time was undertaken because of anecdotal reports that breast stimulation was occurring. We found that 2 subjects were manually stimulating their own nipples, and 2 were frequently receiving stimulation from their female cage partner. The fifth animal showed no evidence of such stimulation. Prior to undertaking a formal analysis of behavioral patterns associated with prolonged amenorrhea, we placed these subjects in individual metabolism cages to collect urine samples. To our surprise, 2 of these animals resumed cyclicity within 6 days of confinement in individual cages: these animals turned out to be the partner-stimulated animals. In contrast, the 2 animals that had been characterized as self-stimulators did not resume cyclicity within 5 months. This result is exactly what one would predict if breast stimulation is a factor maintaining prolonged amenorrhea: animals separated from partners that provided breast stimu-

250 I Graham et al.

m Z POSTPARTUM ENDOCRINE CHANGES I N CHIMPANZEE; n INFANT REMOVED WEEK 4 2 -

9

4 7 = 6

4 4

m\ 2

2 i 8

8 5

s7 a = 1 2 E 0

2

2

w 15.8

15.6

(3 0.6 m w

4 I 0.4 s- 0.2 m E a\ 2 y 0.0

0 4 8 12 16 20 24 28 32 36 4 0 44 48 52

WEEKS POSTPARTUM

- l o o h x 7

rn -0 n

Fig. 3. Postpartum endocrine changes in chimpanzee, infant removed at week 42 (I). The first postpartum cycle may have been anovulatory, based on absence of a detectable progesterone elevation. Note the similarity with the endocrine profile in Figure 2 , except that a clear gonadotropin ratio reversal did not precede return to cyclicity, perhaps because gonadotropin determinations were terminated before the ovulatory cycle occurred.

TABLE 11. Serum Prolactin Concentration in Menstruating and Prolonged Postpartum Chimpanzees, Before and After Anesthesia

Mean ? SE concentration Range

Number (ng/ml) (ngiml)

Menstruating Alert 8 40.2 -t 14.2",b 11-111 Anesthetized 8 69.1 2 15.9"3d 18-150

Amenorrheic Alert 5 87.8 ? 5.1b,c 57-129 Anesthetized 5 68.4 ? 12.6",d 56-86

Paired comparisons: P < 0.01; = P < 0.05; c,d = P > 0.05.

lation would resume cyclicity, whereas autostimulators would not [Graham et al., 1987; 19881.

Based on these results we subsequently undertook a more formal analysis of stirnulatory behavior in 6 2-hour focal periods in each of 6 subjects (all of the PPAm subjects available at the time), using the ethogram described above. Because genital stimulation has been suspected to induce prolactin secretion, we sought evidence of both breast and genital stimulation.


TABLE 111. Duration (D; Minute/Hour) and Incidence (I;/Hour) of Self-Directed Breast and Nipple Contact

N D-Oral I-Oral D-Manual I-Manual Status N

Prolonged PPAm 6 36 0.35" 0.50" 1.71 2.26b (exclude Betty) 5 30 0.11 0.15 Normal PPAm 6 30 0.07 0.08 0.07 0.18 Menstruating' 6 36 < 0.01 0.01 0.01 0.10

"Mainly attributed to 1 subject (Betty; 1.3 timesihour; 1.8 minuteshour). bMost subjects, most sessions (highest rate, 18410 observed time). 'No effect of presence or absence of male partner.

In this analysis, the 6 PPAm animals were compared with 6 normal 2-3 month postpartum animals with no prior history of PPAm whose infants were separated at birth, and with 6 normal menstruating animals (Table 111). Five PPAm subjects exhibited a consistent pattern of autostimulation: they manually manipulated their nipples and breasts in a characteristic way, typically crossing their arms and rotating the contralateral nipples in a manner reminiscent of winding a bobbin (hence our name for this pattern; spooling [Graham & Struthers, 1987, 19881. A sixth female vigorously sucked her own nipples frequently (the intensity of this behavior was evidenced by her greatly enlarged nipples). No additional partner- stimulated animals were observed.

The incidence and duration of nipple stimulation in the PPAm group was significantly higher than in the normal postpartum and menstruating groups described above (P < 0.05). Genital stimulation occurred at a very low frequency in all groups, with no significant differences.

Individual serum prolactin levels in the 3 groups (6 subjects each) were highly variable; mean levels (ng/ml * SE) were: prolonged postpartum animals: 83.8 2 22.8; normal nursing animals: 21.5 +- 9.6; cycling animals: 34.8 * 12.4. The normal postpartum prolactin levels were significantly lower than those of the PPAm animals (P < 0.05), a difference for which we have no explanation, although in view of data presented earlier it may represent an anesthesia effect. However, these data support the hypothesis that the prolonged postpartum animals which exhibited a high incidence of self-stimulation are hyperprolactinemic.

The incidence of nipple self-stimulation is similar to stimulation produced by nursing infants, in studies by Nadler et al. [1981] and Dienske and van Vreeswijk [1987]. Since the endocrine profile in nursing women and prolonged postpartum amenorrhea chimpanzees is similar, and it is well established that suckling in humans and other species prolongs lactation and amenorrhea [Konner & Worth- man, 19801, it seems probable that self- or partner-stimulation of the nipple in chimpanzees has a similar effect.

Treatment We have evaluated the efficacy of dopamine receptor agonists (prolactin an-

tagonists) in restoring cyclicity in PPAm chimpanzees, since they are efficacious in reducing hyperprolactinemia-associated amenorrhea in women. We have previously reported preliminary data on this subject [Graham & Faiman, 19831, and now can add additional information.

Bromocryptine treatment (2.5-5.0 mg/day) of 12 PPAm chimpanzees was associated with a reduction in circulating prolactin levels (determined under ketamine anesthesia): mean (2 SE; ng/ml) serum prolactin fell from a pretreatment

252 I Graham et al.

TABLE IV. Days Postpartum Pergolide (200 pgld) Initiated, and Treatment Day of Sex Swelling Resumption

Chimp Days Days to Pre-treatment ID# postpartum swelling Prl levels

143 477 621 627 683 879 257 896 t

916 264 795 300 426 253 199 210 420.4

11 11 14 6 9 6 4 4

8.125

55 27 77 70 59

160 129 85 83

level of 106.5 t 30.7 to 44.3 ? 13.4 after 2 months, and to 26.8 +- 10.9 after 4 months. By comparison, 4 controls with an initial value of 86.3 ? 10.3 had mean prolactin levels of 109.8 * 11.6 after 2 months (4-month values not determined). We found ketamine anesthesia did not affect prolactin levels during Bromocryp- tine treatment [Graham et al., 19891.

Bromocryptine treatment was associated with restored cyclicity in 11/13 animals within 4 months (7 within 1 month, and 4 more within 2-3 months). Al- though the probability of any one of the animals returning to cyclicity within 150 days is 0.57, the probability that 11 or more animals doing so is calculated using the Binomial distribution to be only 0.038, significant a t the P < 0.05 level. Four vehicle controls did not resume cyclicity during a 5-month period: a Fisher’s Exact Test on a 2 x 2 table yields P < 0.01, indicating a treatment effect.

We suspected that the variable, and often delayed, responses to Bromocryptine were due to inability to maintain adequate circulating levels in the animals for logistical reasons (especially inability to administer 3 doses a t the recommended 8 hour intervals). Consequently we turned to Pergolide, a more potent analog with longer duration of action. This compound is administered as a single daily dose of 200 pg to women. With this treatment, 8/8 animals with typical postpartum amenorrhea and mean pretreatment serum prolactin levels of 82.8 ? 15.1 ng/ml re- turned to cyclicity within 14 days (Table IV); because of the unexpected rapid response, prolactin levels were not obtained during Pergolide administration. Sta- tistical comparison with historical data yielded a probability of P = 0.08 for individual animals returning to cyclicity within 2 weeks of commencing treatment, and a probability of P < 0.0001 of all 8 animals resuming cyclicity within this interval, strongly indicating a treatment effect. However, for logistical reasons, there was no vehicle control in the Pergolide experiment, so we cannot prove that this effect is specifically due to the drug, as opposed to a placebo effect.

Clearly for both treatments, there is a remarkable acceleration of return to cyclicity, and i t seems highly unlikely that this is due to a placebo effect.

DISCUSSION The results reported here clearly indicate that prolonged postpartum amenor-

rhea in chimpanzees is associated with hyperprolactinemia and galactorrhea. The identification of a behavioral component which prolongs postpartum infertility in an estimated 27% of pregnancies by mimicking suckling, and the demonstration that prolactin antagonists effectively reverse the amenorrhea represent important

Postpartum Infertility in Chimpanzees I 253

advances in the management of postpartum infertility when infants are removed at birth (or even later, since Alford [19861 has reported frequent prolonged amenorrhea when infants were separated up to a year after birth). In a future study we plan to examine the association between prolonged amenorrhea, auto- or allo- stimulation, galactorrhea, elevated prolactin levels, and response to antidopamin- ergic drugs in the same individual animals, in order to further strengthen our conclusions concerning the relationship between these phenomena.

As many as 27% of postpartum periods may be abnormally prolonged, based on our statistically-based redefinition of prolonged amenorrhea as > 140 days. (The provisional definition of PPAm, > 6 months, used throughout this paper proved to be reasonable and conservative).

This report may also contribute to our understanding of normal regulation of postpartum amenorrhea duration. The exact mechanism by which the prolactin antagonists inhibit postpartum amenorrhea and the relation of this effect to depression of prolactin levels is not clear. Our observations in the chimpanzee suggest that the postpartum period of amenorrhea is closely associated with raised levels of prolactin, as is the case in the human [Duchen & McNeilly, 1980; Konner & Worthman, 19801. However it is unlikely that prolonged postpartum amenorrhea is directly related to elevated prolactin levels. In women, the prolactin in- crease induced by suckling is not related to an inhibition of basal or pulsatile secretion of LH [Dawood et al., 1981; Glasier et al., 19841, and cyclicity can return in the face of continuing prolactin elevation in women [Cardenas, personal com- munication]. Thus, the relationship between prolactin and the resumption of ovarian activity may be indirect, i.e., both the decline in prolactin levels and the resumption of normal LH secretion are dependent on a decrease in suckling, which otherwise maintains the amenorrheic state [Duchen and McNeilly, 19801.

The role of dopamine in mediating the incoming neural signal to the hypotha- lamicipituitary axis is most puzzling, because in chimpanzees dopamine agonists bring about both a fall in prolactin and resumption of cyclicity. Dopamine is the major factor inhibiting release of prolactin [Ben-Jonathan, 19851. Exogenous dopamine receptor agonists such as Pergolide effectively block prolactin secretion because dopamine is a natural prolactin inhibiting factor. It seems likely that dopamine agonists block the influence of nipple stimulation on the GnRH pulse generator by an unknown means; apparently the effect on prolactin is not directly in this pathway. The site of action might be on the hypothalamus, or a t a lower level in the hypothalamicipituitaryiovarian axis. The self-stimulating chimpanzee model may provide an opportunity to investigate these questions, since it should be possible to determine the sequence of behavioral and neuro-endocrine events fol- lowing administration of a prolactin antagonist and subsequent cycle resumption. However, progress may depend on development of a urinary prolactin assay, so that variations due to stress and episodic secretion can be avoided.

The identification of a behavioral phenomenon of self-stimulated lactational amenorrhea in the chimpanzee, taken with reports of a rare psychotic amenorrhea/ galactorrhea syndrome in women that is associated with nipple self-stimulation [Vorheer, 19781 clearly indicates the importance of understanding the hypotha- lamic mechanisms that link the suckling stimulus to gonadotropin secretion.

It is clear from these experiments that postpartum infertility and its control can have a major impact on the breeding efficiency of captive chimpanzees. In addition we have described a naturally-occurring model system that offers opportunity for improved understanding of the regulation of postpartum fertility in higher primates, including man.

254 / Graham et al.

CONCLUSIONS 1. Auto- or partner-breast stimulation is a primary etiological cause of PPAm

in captive chimpanzees. 2. Hyperprolactinemia and galactorrhea is closely associated with PPAm in

chimpanzees. 3. PPAm is the most common recognized form of infertility in female chim-

panzees; the ability to limit its duration pharmacologically should significantly improve breeding productivity in these valuable and scarce species. Our data strongly suggest this approach is feasible.

ACKNOWLEDGMENTS Partially supported by NIH grant RR 03582. We gratefully acknowledge gifts

of Bromocryptine Mesylate and Pergolide from Sandoz, Inc., and Eli Lilly, Inc., respectively. Table I11 is reproduced from Graham and Struthers [1988] with per- mission of the publisher. We thank J.D. Neil1 for conducting gonadotropin assays.

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Postpartum infertility in common chimpanzees