Online Issues

ILAR Journal Vol 45(2)

Nonhuman Primates Contribute Unique Understanding to Anovulatory Infertility in Women

David H. Abbott, Shu C. Foong, Deborah K. Barnett, and Daniel A. Dumesic

David H. Abbott, Ph.D., is a Professor in the University of Wisconsin, Madison, Wisconsin (UW-Madison) Department of Obstetrics and Gynecology, and a Professor at the UW-Madison National Primate Research Center (NPRC). Shu C. Foong, M.D., is a Fellow in the Section of Reproductive Endocrinology and Infertility in the Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, Minnesota. Deborah K. Barnett, Ph.D., is an Assistant Researcher at the NPRC. Daniel A. Dumesic, M.D., is a Professor in the Departments of Obstetrics and Gynecology and Internal Medicine, Mayo Clinic, Rochester, Minnesota.

Abstract

Anovulatory infertility affects a large proportion of reproductive-aged women. Major improvements in successful clinical treatment of this prevalent disorder in women's health have been made possible because of biomedical research employing nonhuman primates. Experiments on female rhesus monkeys were the first to demonstrate that the key hypothalamic neurotransmitter, gonadotropin-releasing hormone, involved in stimulating pituitary gonadotropin synthesis, storage, and release was bioactive only when released in approximately hourly bursts. This breakthrough in understanding gonadotropin regulation enabled identification of hypogonadotropic, apparently normogonadotropic, and hypergonadotropic forms of anovulatory infertility, and development of appropriate stimulatory or inhibitory gonadotropin therapies. Treatments to overcome anovulatory infertility represent one of the major advances in clinical reproductive endocrinology during the last 25 yr. The future promise of nonhuman primate models for human ovulatory dysfunction, however, may be based on an increased understanding of molecular and physiological mechanisms responsible for fetal programming of adult metabolic and reproductive defects and for obesity-related, hyperinsulinemic impairment of oocyte development.

Key Words: anovulation; follicle-stimulating hormone; gonadotropin-releasing hormone; hypothalamic amenorrhea; luteinizing hormone; marmoset; polycystic ovary syndrome; rhesus monkey

Introduction

At the beginning of the 21st century, populations in many developed countries are aging, with increased longevity attributable to medical advances and improved standards of living (ESHRE Capri Workshop Group 2001). Consequently, naturally occurring human infertility is an increasing concern in terms of maintaining a sufficiently large and relatively youthful workforce. Naturally occurring infertility occurs in 13 to 21% of married couples of reproductive age, representing 5 million couples in the United States alone (Barbieri 1999; Hull 1987), with disorders of ovulation representing the largest single category (Table 1). For clinical purposes, anovulatory women can be classified into three groups: (1) hypogonadotropic (World Health Organization [WHO¹] Class I, e.g., hypothalamic amenorrhea), exhibiting low circulating luteinizing hormone (LH¹) and follicle-stimulating hormone (FSH¹) levels; (2) normogonadotropic (WHO Class II, e.g., hypothalamic dysfunction [patients who exhibit subnormal frequencies of gonadotropin-releasing hormone (GnRH¹) release, including luteal phase defects, and reflect a continuum of disorders that range between Class I and Class II] and polycystic ovary syndrome [PCOS¹]), exhibiting apparently normal circulating FSH levels (with or without LH hypersecretion); and (3) hypergonadotropic (WHO Class III, e.g., primary ovarian failure), exhibiting high circulating LH and FSH levels (Burgues and the Spanish Collaborative Group on Female Hypogonadotrophic Hypogonadism 2001; ESHRE Capri Workshop Group 1995; Laven et al. 2002). Success in the development of ovulation induction strategies for the first two of these three clinical scenarios has been based on an understanding of reproductive neuroendocrinology gained through an integration of human studies and nonhuman primate biomedical research. WHO Class III disorders are not addressed in this article because ovulation induction is not relevant for treating such infertility.

Insights into the mechanisms underlying anovulatory infertility in women have come from research studies employing primate and nonprimate mammals (Freeman 1994; Knobil and Hotchkiss 1994), mice with individual gene disruption (e.g., absence of estrogen receptors: Findlay et al. 2001; Korach et al. 1996), in vitro cell systems (e.g., fetal cell cultures of neurons and neuroglial cells: Richter et al. 2002; anterior pituitary explants: Vella et al. 2001), and immortalized cell lines (e.g., pituitary gonadotropes: Stanislaus et al. 1994; ovarian granulosa and theca cells: Husen et al. 2002; Wood et al. 2003). Unfortunately, nonprimate female mammals exhibit reproductive physiology that is functionally different from that in women (Barnett and Abbott 2003; Freeman 1994), and in vitro cell systems do not reflect the complex regulatory systems governing reproductive function in vivo (e.g., hypothalamic explants: Woller et al. 1998; immortalized gonadotrope cell lines: Maya-Nunez and Conn 2001). However, prospective clinical studies of anovulatory women with hypothalamic amenorrhea (Burgues and the Spanish Collaborative Group on Female Hypogonadotrophic Hypogonadism 2001) and PCOS (Filicori et al. 1994) demonstrate that much insight can be accomplished from the direct study of humans. Reproductive studies in women, nevertheless, have many limitations with respect to experimental design and ethical constraints.

Because of these circumstances, nonhuman primates play key roles in addressing many experimental shortcomings while, at the same time, providing a high degree of homology to reproductive function in women. Hypothalamic regulation of the preovulatory LH surge in primates does not require a concomitant change in the frequency or amplitude of GnRH (Knobil and Hotchkiss 1994), whereas dramatic increases in these GnRH release dynamics are required in female nonprimates (Karsch et al. 1997). The LH-chorionic gonadotropin (CG¹) endocrine system is unique to higher primates because CG is absent in lower primates and nonprimates (Gromoll et al. 2003). While Old World primates, such as female rhesus monkeys (Macaca mulatta), exhibit hypothalamic GnRH regulation of pituitary LH release (Woller et al. 1992) that may be similar to that of women, New World primates, such as common marmosets (Callithrix jacchus), exhibit hypothalamic GnRH regulation (Abbott et al. 1998) of pituitary CG release (Gromoll et al. 2000, 2003). In New World primates, exon 10 is absent from the LH receptor, which abolishes LH but not CG action, and pituitary gonadotropes express CG rather than LH (Gromoll et al. 2003).

Nonhuman Primates as Biomedical Models of Choice for Studies of Ovulatory Dysfunction in Women

Primates also exhibit ovarian cycle characteristics that delineate them from nonprimates. Many primate species, particularly Old World primates, have a more prolonged follicular phase compared with nonprimate mammals, and the postovulatory luteal phase ends with menstrual bleeding (Barnett and Abbott 2003). Corpus luteum secretion of inhibin and estradiol suppresses pituitary FSH secretion (Stouffer et al. 1994), which contributes to follicular phase prolongation. Such FSH suppression constrains primate ovarian follicular development during the luteal phase (Gilchrist et al. 2001; Gougeon 1998), whereas little or no luteal constraint on FSH secretion and follicle development is found in nonprimates (Ginther et al. 1996, 2001). Consequently, primates exhibit relatively small, less well-developed follicles at the onset of the follicular phase compared with nonprimates (Barnett and Abbott 2003). Prolongation of the follicular phase also extends estrogen-stimulated proliferation and growth of the uterine endometrium, compared with nonprimates, probably to accommodate a rapidly invading placenta during conceptive cycles. Progressive development of these reproductive traits across the primate order may have been necessary to accommodate timely differentiation and growth of an increasingly large-brained fetus (Barnett and Abbott 2003; Ellison 2001; Martin 1974).

Given such clear reproductive differences between female primate and nonprimate reproductive function, it is not surprising that primates are increasingly used in reproductive research. Between the 1960s and 1990s, the number of papers published on female primate reproduction increased approximately 10-fold (from ~32 to ~330 papers/year, respectively; PrimateLit Bibliographic database website maintained by the Wisconsin Primate Research Center). Depending on the infertility research question asked, appropriate selection of a primate species can provide close emulation of human physiological function, with similarly complex paracrine and neuroendocrine regulatory systems (Tables 2 and 3). For example, female rhesus monkeys can be used to manipulate and quantify hypothalamic neurotransmitter release directly into the hypophyseal portal circulation and to identify neurotransmitters critical for initiating puberty (Mitsushima et al. 1994) and menopause (Woller et al. 2002). Novel or unconventional approaches also can be used when the selected primate species exhibits specific differences from humans in paracrine or neuroendocrine regulation. Specifically, social dominance suppression of ovulatory function in common marmosets has been used to investigate hypothalamic neurotransmitters involved in repeatable and reversible inhibition and reactivation of pituitary gonadotropin release (Abbott et al. 1997b, 1998). Nonhuman primate research, however, is not without its own constraints from logistical problems of limited animal numbers (e.g., naturally low breeding rates), expensive animal maintenance (e.g., prolonged longevity), risk of asymptomatic zoonotic infection (e.g., herpes B virus; Huff and Barry 2003), and limitations on the size of biological samples obtained from small-bodied primates (e.g., ~0.3- to 0.4-kg common marmosets).

The choice of appropriate nonhuman primate species for studies of ovulatory dysfunction, nevertheless, is usually determined by the biological characteristics of a particular species rather than by logistical constraints. In Table 2, the major traits of Old and New World primates are described relevant to studies of ovulation, and Table 3 includes examples of species used for studies of ovulatory dysfunction. As can be seen from these tables, there are a number of species appropriate for reproductive studies, and some can be more appropriate for certain research studies than others. For instance, mechanistic studies of LH-related ovulatory dysfunction would be better performed on Old World species such as rhesus, cynomolgus (Macaca fascicularis), or vervet (Chlorocebus aethiops) monkeys instead of New World monkeys because of (1) diminished functional capacity of the LH receptor in New World monkeys, and (2) CG, rather than LH, secretion from the anterior pituitary (Gromoll et al. 2003). However, studies requiring strict control over ovulation or onset of the follicular phase would be better performed on New World species, such as common marmosets, because reliable luteolysis is readily achieved by prostaglandin F2 alpha analogue administration resulting in controlled timing of the next ovulation (Summers et al. 1985). Such ovulatory control is not possible in Old World species. The possible merits and demerits of each species are therefore many, with both Old and New World monkeys providing a wide degree of choice depending on the research question (Tables 2 and 3).

Hypothalamic Focus

The key breakthrough in our understanding of the physiology of hypothalamic-pituitary regulation of ovulation in women stems from nonhuman primate studies conducted in the mid- to late 1970s (Knobil and Hotchkiss 1994). These nonhuman primate experiments now serve as the foundation for our understanding of the different pathologies underlying major forms of anovulatory infertility in reproductive-aged women. They include studies that identify pulsatile hypothalamic GnRH release as a functional necessity for pituitary gonadotropin release, as well as hypogonadotropic and normo- to hypergonadotropic mechanisms of anovulation. Such studies continue to drive the development of clinical treatments for similar ovulatory disorders in women and have critically changed our understanding of ovulatory function in women. In doing so, nonhuman primate research has contributed directly to improvements in women's health and clinical care.

Biomedical Studies of Nonhuman Primates Establish Pulsatile Hypothalamic GnRH Release as a Necessity for Effective Pituitary Gonadotropin Release

Experiments performed by Knobil and colleagues (Knobil and Hotchkiss 1994) mark the first demonstration of an hourly or "circhoral" pulsatile pattern of LH secretion in ovariectomized rhesus monkeys. Presumption of a central nervous system signal generator ("GnRH pulse generator") controlling rhythmic LH release (Dierschke et al. 1970) is supported by the localization of a key component of the GnRH pulse generator in rhesus monkeys in the medial basal hypothalamus. Ablation of the arcuate nucleus within the medial basal hypothalamus abolishes pituitary gonadotropin secretion (Plant et al. 1978), whereas deafferentation of the medial basal hypothalamus does not (Krey et al. 1975). The integrity of the arcuate nucleus within the medial basal hypothalamus appears to be critical for the maintenance of pulsatile gonadotropin secretion from the anterior pituitary, regardless of whether neuronal connections with the rest of the brain are maintained. In arcuate nucleus-lesioned rhesus monkeys, administration of exogenous GnRH, in hourly pulses of 6-min duration each, successfully restores pulsatile LH secretion, whereas continuous GnRH infusion severely inhibits LH release (Belchetz et al. 1978; Nakai et al. 1978). Intermittent or pulsatile release of GnRH is apparently essential for normal (pulsatile) release of gonadotropin from anterior pituitary gonadotrope cells. Endogenous, pulsatile release of GnRH was subsequently confirmed by electrophysiological recording of increased multiunit activity from the medial basal hypothalamus in association with pituitary LH release (Wilson et al. 1984) as well as by in vivo measurement of pulsatile GnRH release from the median eminence using push-pull perfusion techniques (Gearing and Terasawa 1988; Levine et al. 1985). These primate studies, along with in vitro perfusion studies of human medial basal hypothalami exhibiting pulsatile GnRH release, all confirm the importance of pulsatile GnRH release for physiological gonadotropin secretion (Rasmussen et al. 1989).

The true nature and location of the GnRH pulse generator, however, was not known until neuronal cell culture systems derived from fetal rhesus monkeys were established. These in vitro cultures have illustrated the unique, intrinsic, pulsatile nature of GnRH neurons (Terasawa et al. 1993) and have suggested that the entire population of ~2000 GnRH neurons scattered across the preoptic area and hypothalamus (Silverman 1988) acts as a single, integrated pacemaker (Teresawa 1998, 2002). Approximately every 40 to 60 min, about 80% of these GnRH neurons simultaneously release GnRH (Terasawa 2002; Terasawa et al. 1999). Such reliable, cohesive, and organized release of GnRH is clearly evident in these neurons well before their migration is complete from the embryonic olfactory placode to the preoptic area and anterior hypothalamus (Terasawa 2001). The synchronization of GnRH release from widely dispersed GnRH neurons is made possible by the active participation of neuroglial cells in rapidly transmitting an ATP-synchronized calcium "wave" that activates the widely dispersed neurons (Keen et al. 2002; Terasawa 2002). These recent in vitro findings may help reconcile how the original electrophysiological recordings of increased multiunit activity in the medial basal hypothalamus (Wilson et al. 1984), well removed from the anatomical locations of most GnRH neuron cell bodies (Silverman et al. 1986), were coincident with pituitary LH release. The electrophysiological recordings may have reflected intracellular changes in calcium concentrations in non-neuronal glial cells involved in the calcium wave and not action potentials from GnRH neurons (Terasawa 2001).

Necessity of Normal, Pulsatile GnRH and Gonadotropin Release for Maintaining Ovulatory Function

The additional observation that hourly pulses of GnRH infusion initiate normal ovulatory menstrual cycles in prepubertal female rhesus monkeys establishes a critical link between activation of the hypothalamo-pituitary unit and ovarian follicle development (Wildt et al. 1980). In these prepubertal females receiving fixed GnRH pulses, ovarian estrogen production capable of exceeding a circulating estradiol level of 150 pg/mL for at least 36 hr elicits a midcycle LH surge, with subsequent ovulation (Knobil 1980). Ovulatory menstrual cycles also are reproduced in arcuate-lesioned adult rhesus monkeys after pulsatile GnRH administration (Knobil 1980). The ability of parenteral estradiol to elicit a serum LH surge in arcuate-lesioned, ovariectomized adult rhesus monkeys receiving hourly GnRH pulses further emphasizes the importance of estrogen positive feedback on pituitary LH release. This phenomenon is substantiated by in vitro studies of rhesus and human fetal pituitary cells showing estradiol sensitization to GnRH (Dumesic et al. 1987; Frawley and Neill 1984a; Jaffe and Keye 1974). Despite such clearly demonstrable pituitary integration of estradiol-induced LH pulses with unvarying GnRH pulse frequency, estradiol-induced increases in GnRH pulse frequency also are demonstrated at the time of the LH surge in female rhesus monkeys (Terasawa 1998). In primates, and probably in humans, therefore, redundant mechanisms appear to exist for generating the midcycle ovulatory LH surge.

Notwithstanding the findings described above, serum LH pulses, and presumably GnRH release, in normal cycling female rhesus monkeys and women vary throughout the menstrual cycle. Luteinizing hormone pulses occur in greater frequency, but smaller amplitude, during the follicular phase versus the luteal phase, and they increase in frequency before the midcycle ovulatory LH surge (Filicori et al. 1986; Hall et al. 1992; Midgley and Jaffe 1971; Terasawa et al. 1987; Yen et al. 1972). Abnormal neuroregulation of rhythmic GnRH release during this time can interfere with ovulation, thereby decreasing fertility in women from prolonged folliculogenesis or abnormal corpus luteum function.

Hypogonadotropic Anovulation (Relevant to WHO Class I, but Including a Continuum of Disorders from WHO Class I to Class II)

Subnormal Hypothalamic Function

Hypothalamic amenorrhea in nonhuman and human primates refers to chronic anovulation and acyclic gonadotropin secretion from reduced endogenous GnRH release (Leyendecker et al. 1980; Miller et al. 1983). Primate menstrual cycles that lack sufficient GnRH secretion (pulse frequency or amplitude) do not support normal follicular maturation or subsequent corpus luteum function (Knobil and Hotchkiss 1994). In female rhesus monkeys that have intact ovaries, but bear hypothalamic lesions that abolish endogenous GnRH release, pulsatile GnRH administration at hourly intervals induces ovulation. In contrast, administration of GnRH pulses at more prolonged intervals of 3 hr causes a progressive decline in follicular development leading to anovulation. This effect of diminished GnRH pulse frequency on ovulatory function is accompanied by significant reductions in serum estradiol and LH levels, without accompanying changes in serum FSH levels (Pohl et al. 1983). Such nonhuman primate experiments replicate the decreased pulsatile GnRH secretion observed in women with abnormal follicular maturation (Marshall et al. 2001; Reame et al. 1985) and link hypothalamic dysfunction, in which serum FSH and estradiol levels are normal (Davajan and Kletzky 1991), with several abnormalities of ovulation in women experiencing chronic illness, weight loss, eating disorders, strenuous exercise, hyperprolactinemia, and physical or emotional stress (Perkins et al. 1999, 2001; Yen 1999). The degree to which pulsatile hypothalamic GnRH release slows results in a continuum of ovulatory disorders (Yen 1999) that range from subtle abnormalities of luteal progesterone secretion (Suh and Betz 1993; Wuttke et al. 2001) to hypothalamic amenorrhea, in which acyclic gonadotropin secretion from absent endogenous GnRH release causes chronic anovulation with estrogen deficiency (Leyendecker et al. 1980; Miller et al. 1983).

Weight Loss and Strenuous Exercise

Experiments involving nonhuman primates have been particularly useful in determining a more mechanistic understanding of hypothalamic amenorrhea from weight loss (including anorexia and eating disorders; Laughlin et al. 1998) or excessive exercise (Loucks and Thuma 2003). Weight loss, achieved through controlled dietary restriction, may (Lujan et al. 2003a) or may not (Colman et al. 1999; Mattison et al. 2003) induce hypogonadotropic anovulation in female rhesus monkeys. The absence of anovulation in the latter studies may reflect a less severe calorie restriction (30% reduction) than that imposed by the former (40% reduction). The less severe calorie restriction may have permitted reduction of the resting metabolic rate (Blanc et al. 2003) to match or exceed energy intake, thus avoiding a physiological state of negative energy balance associated with anovulation (Wade and Schneider 1992). Anovulation from chronic metabolic energy deficiencies is considered an adaptive response to environmental conditions that are unfavorable for successful production and rearing of offspring (Schneider and Wade 2000). Hypogonadotropic anovulation from strenuous exercise may reflect another aspect of this adaptive response to chronic deficiency in metabolic energy. During strenuous exercise training, female cynomolgus macaques abruptly exhibit anovulation at varying intervals from the onset of exercise, as determined by decreased circulating levels of gonadotropins and estradiol, increased follicular phase length, decreased luteal phase progesterone secretion, and amenorrhea (Williams et al. 2001a). Such hypogonadotropic anovulation is readily reversed by feeding the monkeys supplementary calories while continuing the exercise regimen (Williams et al. 2001b). Exercise-induced anovulation is thus considered a physiological response to metabolic energy limitations and is no longer viewed as a stress response to intense physical activity in nonhuman primates (Williams et al. 2001b) and humans (Loucks 2003).

Role for Leptin?

There is also emerging evidence for a role of leptin in the metabolic control of ovulatory function. Leptin, which is secreted in a circadian rhythm by adipocytes and other tissues, can accelerate GnRH pulsatility in rodents (Lebrethon et al. 2000). Subcutaneous infusion of leptin into prepubertal female rhesus monkeys prematurely increases circulating LH and estradiol levels, increases skeletal size, and causes early menarche (Wilson et al. 2003). This pubertal precocity is reminiscent of increased skeletal size and early menarche induced by testosterone propionate treatment of juvenile female rhesus monkeys (Van Wagenen 1949). The similarity in effects induced by leptin and testosterone may reflect anabolic signaling to the hypothalamus of excess metabolic energy that permits accelerated maturation of the neuroendocrine mechanisms regulating puberty and initiation of ovulation. Detailed understanding of such regulatory neuroendocrine mechanisms, however, remains elusive.

In humans, 1 yr of leptin administration to a prepubertal girl with congenital leptin deficiency activated pulsatile gonadotropin secretion and the normal progression of puberty (Farooqi et al. 2003). This result suggests that long-term leptin therapy can induce pubertal activation of the hypothalamic-pituitary unit in individuals previously unexposed to endogenous leptin. Such an effect of leptin on primate reproduction, however, remains controversial because leptin administration to male rhesus monkeys fails to induce premature puberty (Barker-Gibb et al. 2002; in contrast to findings in females), and it increases circulating LH levels inconsistently in short-term fasted, adult animals (Finn et al. 1998; Lado-Abeal et al. 2000). Moreover, continuous infusion of leptin into the lateral ventricle of adult female rhesus monkeys fails to reverse weight loss-induced hypogonadotropic anovulation (Lujan et al. 2003a). Low serum leptin levels and absence of the diurnal leptin secretion, nevertheless, are typical features of hypothalamic amenorrhea in women (Andrico et al. 2002; Laughlin and Yen 1997), along with inhibitory effects of leptin, in obesity-related diseases (Moschos et al. 2002), on ovarian steroidogenesis (Ghizzoni et al. 2001; Spicer and Francisco 1997).

Stress Responses

It is well established that chronic or prolonged activation of physiological stress responses induces hypogonadotropic anovulation in many mammalian species (Sapolsky 2002), including nonprimates (Cameron 1997) and humans (Yen 1999). The neuroendocrine and endocrine mechanisms mediating stress responses are by no means straightforward and can involve the release of catecholamines from the sympathetic nervous system and adrenal medulla, activation of the hypothalamic-pituitary-adrenal (HPA¹) axis, hyperprolactinemia, and a variety of other endocrine responses, including suppression of hormones related to anabolism and growth (Orth and Kovacs 1998; Sapolsky et al. 2000). Much attention has been paid to the activation of the HPA axis and the role of glucocorticoids in suppressing ovulation. Hypothalamic release of corticotropin-releasing hormone (CRH¹) can inhibit pulsatile gonadotropin release (Feng et al. 1991; Olster and Ferin 1987; Xiao and Ferin 1997). In nonhuman primates, experimental activation of the HPA axis that increases CRH release suppresses GnRH pulsatility (Xiao et al. 1999; see Xiao and Ferin 1997 for discussion). Exogenous administration of ACTH also induces anovulation (Moberg et al. 1982). When stress responses are experimentally induced, in nonhuman primates, however, the neuroendocrine mechanism inhibiting LH secretion is far from clear. Insulin-induced hypoglycemia, restraint, and receipt of same sex aggression reliably activate the HPA axis and inhibit LH secretion (Table 4). Unexpectedly, CRH is not consistently implicated in the inhibition of LH secretion during hypoglycemia or restraint-induced stress. Vasopressin (co-released with CRH from hypothalamic CRH neurons) is also not convincingly implicated in LH inhibition induced by hypoglycemia (Table 4). Endogenous opioid peptides, however, are implicated in LH suppression induced by restraint with or without prior receipt of same sex aggression, but are not implicated in LH suppression induced by hypoglycemia (Table 4). The inhibitory effects of these neurotransmitters on LH secretion may reflect inhibitory action on GnRH release from the hypothalamus rather than on diminished pituitary gonadotrope responsiveness to endogenous GnRH (Lujan et al. 2003b). Not surprisingly, moderate short-duration stress may not induce amenorrhea, although it may reduce the reproductive potential of the given cycle, along with the quality of subsequent cycles (Xiao et al. 1999).

Social subordination is a stressor in some nonhuman primate societies (Abbott et al. 2003b) and can be associated with the activation of the HPA axis and hypogonadotropic anovulation (e.g., cynomolgus monkeys) (Adams et al. 1985). However, the relation between subordinate status, activated HPA axis, and hypogonadotropic anovulation has not been identified consistently in studies involving, for example, the common marmoset (Saltzman et al. 1994) and the cotton top tamarin (Ziegler et al. 1995), probably because activation of the HPA axis in female subordinates occurs only in species in which subordinate status attracts high rates of physical or psychosocial stressors from dominants (Abbott et al. 2003b). Specialized, but undefined, neuroendocrine mechanisms appear to induce hypogonadotropic anovulation in female subordinates in the absence of obvious stress responses (Abbott et al. 1997b).

Although the mechanism responsible for the slowing of GnRH secretion in hypothalamic amenorrhea is unclear, excessive levels of endogenous hypothalamic opioid activity (Khoury et al. 1987), particularly β-endorphins, inhibit the pulsatile release of GnRH and LH (Jaffe et al. 1994). Morphine administration to ovariectomized monkeys or to isolated human medial basal hypothalamus perifused in vitro abolish pulsatile GnRH activity, and this effect is reversed by concomitant administration of naloxone, an opiate cell receptor antagonist (Grosser et al. 1993; Rasmussen et al. 1989). Furthermore, naloxone can block the effect of CRH on the GnRH pulse generator in monkeys (Gindoff and Ferin 1987; see Xiao and Ferin 1997), possibly explaining the beneficial effect of naloxone in some patients with hypothalamic amenorrhea (Wildt and Leyendecker 1987). Endogenous opioids, however, may best modulate pituitary LH secretion during conditions of relatively high gonadal steroid production because increased LH pulse frequency occurs only with naloxone infusion during the late follicular to mid-luteal phase of the menstrual cycle (Rossmanith et al. 1988).

Hyperprolactinemia

Hypothalamic amenorrhea also can result from hyperprolactinemia, which can reduce pulsatile GnRH and gonadotropin secretion (Bohnet et al. 1976). Prolactin, synthesized by pituitary lactotrophs, may directly affect GnRH neurons (Milenkovic et al. 1994), or indirectly affect GnRH secretion through hypothalamic dopaminergic and opioidergic systems (Moult et al. 1982; Quigley et al. 1979). Experimental manipulation of prolactin secretion in nonhuman primates has contributed to our understanding of the causes and consequences of hyperprolactinemia. To this end, hyperprolactinemia can be induced in nonhuman primates with dopamine antagonists (Aso et al. 1982; Horie et al. 1986; Martensz and Herbert 1982; Moro et al. 1999), ovarian hyperstimulation (Collins et al. 1984a,b; Simon et al. 1988), estrogen/progesterone therapy (Groff et al. 1990; Olive et al. 1989; Williams et al. 1981, 1985), social subordination/stress (Bowman et al. 1978), and pituitary allografting (Bethea et al. 1991).

Hypothalamic dopamine has been established as the major neuroendocrine inhibitor of pituitary prolactin secretion through studies of hypophysial-stalk transected rhesus macaques, in which dopamine delivery into the portal circulation was interrupted, leading to hyperprolactinemia (MacLeod and Lehmeyer 1974; MacLeod et al. 1970). Dopamine antagonists such as sulpride and domperidone also induce hyperprolactinemia and anovulation in marmosets (Moro et al. 1999), baboons (Aso et al. 1982), and rhesus macaques (Martensz and Herbert 1982). Induced hyperprolactinemia can be reversed by infusion of dopamine (Frawley and Neill 1984b; Neill et al. 1981; Vaughan et al. 1980) or the use of dopamine receptor agonists, bromocriptine, or cabergoline (Collins et al. 1984b; Moro et al. 1999). Ovulation can be restored in anovulatory marmosets with the use of cabergoline to suppress sulpiride-induced hyperprolactinemia (Moro et al. 1999). Bromocriptine therapy during the luteal phase also prevents hyperprolactinemia in cynomolgus macaques undergoing ovarian hyperstimulation for in vitro fertilization (IVF¹; Collins et al. 1984b) and counteracts hyperprolactinemia-induced inhibition of the estradiol-induced LH surge in socially subordinate female talapoin monkeys (Bowman et al. 1978). These nonhuman primate studies lend support for the use of both bromocriptine and cabergoline in the clinical treatment of hyperprolactinemia (Besser and Thorner 1976; Ferrari et al. 1986), in which low serum LH levels in hyperprolactinemic anovulatory women can be increased by bromocriptine therapy (Moult et al. 1982; Quigley et al. 1979).

Oligo-ovulation and Luteal Phase Defects

Reduced pulsatility of GnRH also may diminish fertility through luteal phase deficiency, a clinical abnormality characterized by impaired corpus luteum progesterone secretion and delayed endometrial development (Suh and Betz 1993; Wuttke et al. 2001). Luteal phases of shortened duration and/or impaired function occur in human and nonhuman primates (McNeely and Soules 1988; Stouffer 1990; Wilks et al. 1976). In clinical treatment of luteal phase deficiency, such patients can be classified as WHO Class I or WHO Class II, as these women appear to represent a continuum of disorders between the two classifications. Nonhuman primate models of luteal dysfunction, utilizing naturally occurring and experimentally induced luteal phase defects in rhesus, bonnet, and cynomolgus macaques (DiZerega and Hodgen 1981a,b; Ravindranath and Moudgal 1990; Stouffer 1990; Stouffer and Hodgen 1980; Wilks et al. 1976), link problems during follicular development (e.g., lower serum FSH and a low serum FSH:LH ratio) with subsequent corpus luteum defects (DiZerega and Hodgen 1981b; Wilks et al. 1976). Spontaneous luteal phase defects occur in rhesus monkeys during the summer months, when these monkeys can exhibit an increased incidence of oligomenorrhea and amenorrhea, even in a controlled, nonseasonal environment (Dailey and Neill 1981; Nusser et al. 2001; Riesen et al. 1971). Rhesus monkeys housed outdoors, however, exhibit complete cessation of ovulatory cycles during the summer months (Herndon et al. 1985; Walker et al. 1984). Seasonally occurring luteal phase defects accompanying oligomenorrhea have suggested that functional deficiencies of the hypothalamo-pituitary unit during follicular development may have functional consequences on corpus luteum function (Walker et al. 1983, 1984; Wilks et al. 1976, 1977). The annual periods of reduced ovarian function make rhesus macaques a unique model for studying normal and abnormal luteal development within the same individual. However, such environmental regulation of ovulatory function may be less desirable in neuroendocrine studies, in which a nonseasonal (e.g., cynomolgus monkey) versus a seasonal breeder (e.g., rhesus monkey, Table 3) might be a more appropriate biomedical model.

Luteal defects are induced in nonhuman primates by transient reduction in FSH levels during the follicular phase (DiZerega and Hodgen 1981a; Ravindranath and Moudgal 1990; Stouffer and Hodgen 1980). Suppression of serum FSH, but not LH, with charcoal-extracted porcine follicular fluid (pFF¹) reduces preovulatory rises in E₂ and induces luteal defects in rhesus macaques (DiZerega and Hodgen 1981a; Stouffer and Hodgen 1980). Progesterone secretion in pFF-treated animals mimics spontaneously occurring luteal phase defects of unmanipulated animals (Stouffer and Hodgen 1980). Similarly, treatment of female bonnet monkeys with antiserum to FSH on days 5, 6, or 7 of the cycle reduces luteal phase length and prevents pregnancy in treated females during that cycle (Ravindranath and Moudgal 1990). Stress in rhesus macaques also produces reduced serum LH and progesterone levels with inadequate luteal function (Xiao et al. 2002). When early cycle FSH therapy is combined with pFF treatment, serum progesterone levels at the beginning of the luteal phase are improved, strengthening the link between early cycle FSH levels and luteal phase defects (DiZerega and Hodgen 1981b). In addition to abnormal progesterone secretion, pFF treatment impairs the responsiveness of the corpus luteum to human chorionic gonadotropin (hCG¹). Exogenous hCG is ineffective at rescuing corpora lutea in pFF-treated macaques, and the luteal cells from pFF-treated animals are unresponsive to hCG in vitro, suggesting that the deficient corpus luteum may be unable to establish a pregnancy (Stouffer and Hodgen 1980).

In women with luteal phase deficiency, there is a relatively high incidence of abnormal serum LH surges with lower peak serum LH levels or prolonged duration, causing impaired follicular growth (Ayabe et al. 1994). A variety of clinical conditions such as hyperprolactinemia, hyperandrogenic states, weight loss, and stress may cause luteal phase deficiency (Ginsburg 1992). In nonhuman primates, a short luteal phase is the first sign of hyperprolactinemia after bromocriptine treatment ends (Seppala et al. 1976; see Bethea et al. 1991). In addition to its previously described inhibitory effect on gonadotropin secretion (Bohnet et al. 1976), hyperprolactinemia also may predispose to luteal phase deficiency and pregnancy loss by directly disrupting corpus luteum function (Seppala et al. 1976), suppressing granulosa cell steroidogenesis (McNatty and Sawers 1975; McNatty et al. 1974), and impairing follicle development (Kauppila et al. 1984) and endometrial development (Corenblum et al. 1976; Jones et al. 1998; Martinez et al. 2002; Tseng and Mazella 1999).

GnRH and Gonadotropin Replacement

Administration of exogenous pulsatile GnRH to women with hypothalamic amenorrhea, as predicted by the earlier rhesus studies of Knobil and colleagues (Knobil 1980), establishes ovulatory menstrual cycles in most of these patients, implicating reduced endogenous GnRH pulsatility (Leyendecker et al. 1980; Marshall et al. 2001; Miller et al. 1983). As a direct result of these nonhuman primate findings, pulsatile GnRH pumps are available to women with hypothalamic amenorrhea for ovulation induction and are associated with a lower risk of multiple birth than daily parenteral gonadotropin administration (Hurley et al. 1984; Leyendecker et al. 1980; Martin et al. 1993). This treatment, however, has been complicated and expensive, making it difficult for women to wear a portable GnRH pump for prolonged time intervals (ESHRE Capri Workshop Group 1995). Consequently, daily injections of gonadotropins are a commonly used alternative to pulsatile GnRH administration for ovulation induction in women with hypogonadotropic anovulation (Burgues and the Spanish Collaborative Group on Female Hypogonadotrophic Hypogonadism 2001).

Knowledge that pulsatile GnRH administration to arcuate nucleus-lesioned rhesus monkeys successfully restored pulsatile LH secretion, whereas continuous GnRH infusion suppresses LH release, has led to the concept of pituitary desensitization to GnRH (Belchetz et al. 1978; Nakai et al. 1978). The subsequent development of long-acting GnRH agonists to induce pituitary desensitization to GnRH in women rapidly has resulted in the clinical use of GnRH agonists for several hormone-dependent reproductive pathologies, including sexual precocity, uterine leiomyomata, endometriosis, and ovarian hyperandrogenism (Andreyko et al. 1987; Azziz et al. 1995). The use of GnRH analogues to induce pituitary desensitization to GnRH also has significantly improved assisted reproductive techniques (Porter et al. 1984) by preventing estradiol-induced LH surges during gonadotropin therapy and thereby reducing IVF cycle cancellation while improving pregnancy rates (Hughes et al. 1992).

Normogonadotropic Anovulation (Relevant to WHO Class II)

PCOS is an infertility disorder of reproductive-aged women characterized by exaggerated GnRH pulsatility, LH hypersecretion, anovulation, ovarian hyperandrogenism, and insulin resistance (Dunaif 1997; Erhmann et al. 1995; Franks 1995; Zawadski and Dunaif 1992). Normal to low circulating FSH levels, however, classify PCOS as a "normogonadotropic" anovulatory condition (WHO Class II; Laven et al. 2002). This anovulatory syndrome is the most prevalent form of anovulatory infertility, which accounts for 73 to 75% of women with secondary amenorrhea (Dunaif 1997; Hull 1987).

In adulthood, female rhesus monkeys exposed to androgen excess during fetal life present with signs and symptoms (Abbott et al. 1998) that mimic a diagnosis of PCOS in women (Zawadzki and Dunaif 1992). These nonhuman primates provide the first insight into the developmental origins of this highly prevalent syndrome in women (Abbott et al. 2002a). Androgen excess is induced in fetal female rhesus monkeys by daily subcutaneous injection of their pregnant mothers with 10 mg of testosterone propionate dissolved in oil during discrete periods of gestation. Such treatment of the mother overcomes the capacity of the rhesus monkey fetal-placental unit to metabolize testosterone and is successful in elevating circulating levels of testosterone in the female fetus to those found in the fetal male (Resko et al. 1987). The postnatal outcomes of masculinized genitalia and behavior are typical for female mammals, including humans, exposed to androgen excess during hormonally sensitive periods of somatic and neural differentiation (Baum et al. 1990; Mortola 1997). In addition, prenatally androgenized females evidence a significant degree of anovulation. Prenatally androgenized females have periods of anovulation at an approximately 12-fold greater incidence than found in age- and weight-similar controls (Abbott et al. 1998). Anovulation in prenatally androgenized females is significantly associated with hyperinsulinemia and increased adiposity (Abbott et al. 1998), a metabolic link that is not found in controls. Also similar to reproductive dysfunction in PCOS women, prenatally androgenized female rhesus monkeys exhibit (1) enlarged ovaries with polyfollicular ovarian morphology (Abbott et al. 2002b), and (2) circulating testosterone and LH levels that are approximately 52% and 25% higher, respectively, than in controls (Abbott et al. 1997a).

The increased LH pulse frequency in PCOS women may result from the reduced ability of steroids to suppress the hypothalamic GnRH pulse generator, combined with increased LH responsiveness to GnRH from enhanced pituitary sensitivity to GnRH (Marshall and Eagleson 1999; Pastor et al. 1998; Rebar et al. 1976). Hyperfunction of the hypothalamic-pituitary unit in PCOS women leads to an increase in both the amplitude and frequency of LH release compared with normal measurements (Burger et al. 1985; Kazer et al. 1987; Waldstreicher et al. 1988). Despite increased LH levels seen in PCOS, FSH levels remain in the relatively low to normal range. Data from ovariectomized rhesus monkeys demonstrate that increased frequencies of GnRH pulsation preferentially increase LH secretion over FSH (Wildt et al. 1981). This effect may represent differential regulation of gonadotropin subunit genes by GnRH, whereby GnRH pulses induce a preferential increase in LH-β versus FSH-β mRNA, resulting in higher secretion of LH than FSH (Kaiser et al. 1995). The high serum LH levels, in turn, stimulate theca cells to increase ovarian androgen production. The ovary is certainly one source of hyperandrogenism in prenatally androgenized females, as demonstrated by increased circulating testosterone levels following an intramuscular injection of hCG (Eisner et al. 2002) that emulate the ovarian hyperandrogenism evident in women with PCOS (Ehrmann et al. 1995).

The prenatally androgenized female rhesus monkey model of PCOS has demonstrated that androgen excess during early gestation induces multiple metabolic and reproductive defects that closely resemble those of PCOS women. Preferential accumulation of abdominal fat in prenatally androgenized females (Eisner et al. 2003) appears to be a key contributing factor for insulin defects, which in turn are associated with impaired reproductive function (Abbott et al. 2002a). Elevations in circulating levels of both LH and insulin during IVF cycles in prenatally androgenized female rhesus monkeys are implicated in the impaired developmental competence of oocytes retrieved (Dumesic et al. 2002), suggesting potential endocrine mechanisms for the high rates of miscarriage found in PCOS women undergoing IVF cycles (Barbieri 1999).

Future Promise of Nonhuman Primate Research for Anovulatory Infertility in Women

Although future major breakthroughs in our understanding of anovulatory infertility may still emerge from further examination of hypothalamic function (e.g., Terasawa et al. 2002), a new research potential is emerging: fetal androgen excess programming of insulin resistance, type 2 diabetes mellitus, anovulation, and infertility in adult females (Abbott et al. 1998, 2003a; Birch et al. 2003; Sharma et al. 2002). A nonhuman primate model now exists to investigate asynchronous follicle differentiation and oocyte maturation in PCOS women, in whom abnormal follicle differentiation is associated with LH hypersecretion, hyperinsulinemia, and impaired oocyte and embryo development (Dumesic et al. 2002, 2003). Because any attempts to investigate the mechanisms governing oocyte development in PCOS women are limited by ethical and experimental constraints on the use of human embryos for biomedical research, this nonhuman primate model is the quintessence of translational research, merging our understanding of pathophysiology in nonhuman primates with that of clinical disease in PCOS women. It allows investigators to examine whether abnormal follicle luteinization and impaired oocyte development in nonhuman primates are caused by adverse effects of hyperinsulinemia on follicle maturation, and, if so, whether such abnormalities are reversed by improved insulin sensitivity. It also provides direct evidence for translation of nonhuman primate findings into clinical research, which can then corroborate whether a similar phenomenon exists in PCOS women. Using similar, but not identical, techniques in both PCOS women and nonhuman primates may be the only means by which the causes for impaired oocyte development and the origins of reduced fecundity in PCOS women are understood. It is very likely that careful integration of studies in human and nonhuman primates will yield new clinical strategies that improve pregnancy outcome and minimize pregnancy loss in women with disorders of insulin action, including PCOS, obesity, and type 2 diabetes mellitus.

Acknowledgments

This manuscript was supported by National Institutes of Health (NIH) grants RR13635, HD44405, HD44650, and RR00167. Preparation of the manuscript was facilitated by the staff and resources of the Wisconsin Primate Research Center (WPRC) Library, partly supported by NIH grant RR15311. Nonhuman primates at the WPRC were maintained in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals (NRC 1996), and experimental protocols were reviewed and approved by the Graduate School Animal Care and Use Committee of the University of Wisconsin (UW)- Madison. The WPRC is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) as part of the UW-Madison Graduate School. This manuscript is publication number 43-004 of the WPRC.

¹Abbreviations used in this article: CG, chorionic gonadotropin; CRH, corticotropin-releasing hormone; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; hCG, human chorionic gonadotropin; HPA, hypothalamic-pituitary-adrenal; IVF, in vitro fertilization; LH, luteinizing hormone; PCOS, polycystic ovary syndrome; pFF, porcine follicular fluid; WHO, World Health Organization.

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