Prolactin in Human Reproduction

Introduction

Prolactin (PRL) is a single chain (23 KDa) polypeptide hormone, which is secreted by anterior pituitary lactotroph cells. Several lines of evidence indicate that PRL has an essential role in reproduction and lactation. In addition, animal data have supported a role for PRL in a variety of metabolic processes. However, such PRL actions have not been unequivocally confirmed in humans. The present chapter reviews PRL physiology, followed by a discussion of the role of PRL in pathologic states, including hyperprolactinemia and PRL deficiency. Data on the epidemiology, pathology, clinical evaluation, and management of PRL-secreting pituitary adenomas (prolactinomas) are then reviewed, including data relevant to prolactinomas in the setting of preconception and pregnancy.

Lactotroph Development

Lactotroph cells develop under carefully orchestrated control by several transcription factors.
Lactotroph hyperplasia is physiologic during pregnancy and is reversible postpartum.

Pituitary lactotrophs are relatively abundant in the human anterior pituitary gland, accounting for up to 25% of cells in individuals of both genders. During embryogenesis, the pituitary gland develops from ectodermal primordial cells destined to form the anterior and intermediate lobe and neuroectodermal tissue arising from the floor of the diencephalon, which ultimately forms the posterior lobe. During development, inductive interactions and a host of transcription factors have a critical role in the formation of the pituitary and differentiation into mature functioning cells. Transcription factors and signaling molecules that have been implicated in pituitary ontogenesis include those involved in the initiation of pituitary formation (SIX1, SIX6, HESX1, OTX2, PITX1, PITX2, PITX3, ISL1, LHX3, LHX4, SOX2, beta catenin, NOTCH1, NOTCH2), those involved in the migration and proliferation of cells forming the Rathke’s pouch (BMP2, BMP4, FGF8, FGF10, FGF18, SHH) and those involved in lactotroph differentiation (PROP1, POU1F1, GATA2, LHX3).

Lactotroph and somatotroph cells generally develop from common progenitor cells (mammosomatotrophs) under the influence of several transcription factors, though it is possible that some lactotrophs may develop through other precursor cell lines. ^, In particular, the transcription factor POU1F1 (also known as PIT1), a member of the POU homeodomain transcription factor family, is critical in the differentiation and proliferation of lactotrophs, somatotrophs and thyrotrophs and the expression of genes encoding PRL, growth hormone (GH) and the beta subunit of thyrotropin (TSH beta). ^, Patients with inactivating mutations in the POU1F1 gene lack lactotrophs, somatotrophs, and thyrotrophs, resulting in PRL, GH, and thyrotropin deficiency, respectively. In addition, patients with the anti-PIT1 antibody syndrome develop PRL, GH, and thyrotropin deficiency as a consequence of autoimmune damage to the respective pituitary cell populations. Another homeodomain transcription factor, known as prophet of PIT1 (PROP1), has a critical role in the expression of POU1F1. ^, Patients with inactivating mutations in the PROP1 gene may have PRL, GH, thyrotropin deficiency as well as gonadotropin (follicle stimulating hormone [FSH] and luteinizing hormone [LH]) deficiency. Patients with inactivating mutations in genes encoding other transcription factors, including HESX1, LHX3, and LHX4, generally have multiple pituitary hormone deficiencies as well as other midline cranial defects. ^,

Prolactin Gene

A single gene encodes prolactin in humans.
Prolactin gene transcription is increased by estradiol and inhibited by thyroid hormone.
Dopamine, the major factor regulating prolactin secretion in humans, acts by inhibiting adenyl cyclase–dependent signaling pathways.

In humans, the gene encoding PRL consists of 5 coding exons, one noncoding exon, and four introns. It spans approximately 10 Kb in length and is located on chromosome 6. ^, There are several regulatory elements located in the 5’ region of the PRL gene, including areas responsible for stimulation of PRL gene transcription in response to POU1F1 or estradiol, as well as those responsible for suppression of PRL gene transcription by thyroid hormone. In addition to its direct effects, estradiol also modulates dopamine (DA)-induced inhibition on PRL gene transcription. The stimulatory effects of POU1F1 on PRL gene transcription are also subject to modulation by a variety of factors, including cyclic adenosine monophosphate (cAMP), glucocorticoids, estradiol, thyrotropin-releasing hormone (TRH) and epidermal growth factor (EGF). ^,

Dopamine is the major inhibitory factor regulating PRL secretion and acts through the D2 dopamine receptor to inhibit adenyl cyclase and the cAMP-dependent protein kinase A (PKA) pathway. In contrast, TRH stimulates PRL secretion via the phosphoinositide pathway, leading to the activation of membrane calcium channels and the release of calcium ions from the endoplasmic reticulum into the cytoplasm, which in turn activate protein kinase C (PKC), causing the downstream phosphorylation of other proteins, and also bind to calmodulin or topoisomerase II, which have a variety of downstream actions. Of note, dopamine inhibits PRL secretion caused by intracellular calcium ion release. This dopamine action is antagonized by several calcium channel antagonists in vitro, including verapamil, diltiazem, and nimodipine. Interestingly, verapamil causes the opposite effect from what would be predicted by in vitro data; that is, it leads to an increase in PRL levels in humans. ^, This observation was associated with decreased tuberoinfundibular dopamine release and may involve an effect of verapamil on N-type calcium channels present in neurons. Other types of calcium channel antagonists, including dihydropyridines and benzothiazepines, do not affect PRL levels in vivo.

Vasoactive intestinal peptide (VIP) stimulates adenyl cyclase, leading to PRL secretion. Several factors that stimulate PRL secretion, including TRH, neurotensin, and angiotensin II, may also act via the stimulation of phospholipase A2, leading to the release of arachidonic acid, which causes an increase in calcium influx. This effect can be antagonized by dopamine and phospholipase A2 inhibitors. ^,

Prolactin Synthesis in Pituitary Lactotrophs

Several posttranslational prolactin modifications occur and influence prolactin bioactivity.
Macroprolactin species are large prolactin aggregates that have decreased bioactivity.
A 16 kDa prolactin fragment has been implicated in the pathogenesis of peripartum cardiomyopathy and preeclampsia.

The PRL gene is transcribed to mRNA, which undergoes processing in the nucleus to yield a mature, 1 Kb mRNA species, encoding a 227 amino acid PRL precursor. This contains a 28 amino acid signal peptide sequence, which is cleaved posttranslationally to yield a 199 amino acid PRL protein. ^, Additional posttranslational modifications of the PRL molecule include glycosylation, phosphorylation, cleavage, and polymerization.

Of note, the large majority (80%–90%) of the circulating PRL is monomeric, whereas about 10% of circulating PRL is dimeric (“big PRL,” molecular mass ∼50 KDa) and approximately 5% of circulating PRL is multimeric (“big big PRL”). Such high molecular mass PRL species are collectively called “macroprolactin” and may additionally contain bound immunoglobulin. Macroprolactin has been reported to exhibit decreased binding to PRL receptors and has lower receptor binding affinity and decreased bioactivity in most, but not all, assays. ^, Patients with macroprolactinemia, who have elevated total serum PRL as a consequence of elevated multimeric PRL, appear to have normal pituitary-gonadal function, likely as a consequence of decreased bioactivity of multimeric PRL species. ^,

Cleavage of the 23 KDa PRL species by metalloproteases or cathepsin D may occur in peripheral tissues, leading to the generation of an N-terminal 16 KDa PRL variant. This PRL species has antiangiogenic, proapoptotic, and proinflammatory properties and has been implicated in the pathogenesis of peripartum cardiomyopathy and preeclampsia, based on animal and human data. Bromocriptine therapy decreases serum PRL and improves cardiac function in women with peripartum cardiomyopathy.

Prolactin Synthesis in the Decidua and Other Tissues

Extrapituitary prolactin secretion occurs in the decidua and other tissues.
Prolactin of decidual origin appears to promote immunological tolerance of the fetus in utero.
The physiologic role of extrapituitary prolactin in other tissues remains incompletely understood in humans.

Several lines of evidence suggest that PRL is synthesized in the decidua. ^, Very high PRL levels (10–100 times those in maternal serum) have been found in amniotic fluid. ^, In culture, decidual and chorion cells secrete PRL. This PRL species is identical in sequence and activity to pituitary PRL and is expressed under control by an alternative promoter, which is located upstream from the transcription initiation site used in pituitary lactotrophs. ^, Of note, PRL secretion in the decidua is stimulated by progesterone (either alone or together with estrogen), relaxin, insulin, and insulin-like growth factor I (IGF-I) but is not influenced by dopamine agonists or antagonists (in contrast to pituitary PRL). Decidual PRL appears to have an important role in maintaining pregnancy by downregulating interleukin 6 and 20 alpha hydroxysteroid dehydrogenase. Of note, decidual PRL synthesis was reduced in decidual tissue from women who had suffered a miscarriage, wherein proinflammatory cytokines were elevated. In aggregate, these data suggest that decidual PRL supports pregnancy by promoting immunological tolerance of the fetus in utero. ^,

Extrapituitary PRL expression has been documented in the mammary gland, ovaries, testes, prostate, endothelial cells, brain, skin, adipose tissue, lymphocytes, and cochlea in a variety of animal paradigms. The physiologic role of extrapituitary PRL synthesis is under investigation. It has been proposed that extrapituitary PRL may promote tumorigenesis (including tumor initiation and/or propagation) in the breast and prostate. This is an area of ongoing debate and uncertainty since some studies have found little PRL expression in human tumors. Gain-of-function PRL receptor variants do not appear to be associated with a higher risk of breast cancer or fibroadenomas. Nevertheless, available data have prompted the development of a variety of inhibitors of PRL receptors or PRL signaling, and their study as potential therapies in human prostate or breast cancer.

Prolactin Assays

Prolactin is commonly measured using two-site antibody immunoassays.
Several artifacts and conditions may influence the results of these assays, including the hook effect, the presence of macroprolactin, heterophilic antibodies, and biotin.

Serum PRL is currently measured using two-site (“sandwich”) immunometric assays, including immunoradiometric (IRMA), chemiluminescent (ICMA), and electrochemiluminescent (ECLIA) platforms, which use a two-antibody system. A “capture” antibody is used to collect the PRL molecules present in the specimen to a solid substrate (such as microparticle beads or coated tubes). Subsequently, a second (“reporter”) antibody attaches to the PRL–capture antibody complex and is used to generate a detection signal through its radiolabeled or chemiluminescent tag. Measured PRL levels can vary considerably between different immunoassays. Assay interference may occur and should be suspected in the presence of nonlinearity in serially diluted specimens or discrepancy between measured hormone levels and the clinical presentation. Several factors and mechanisms, including the “hook effect,” the presence of macroprolactin, heterophilic antibodies, or exogenous biotin, may lead to assay interference.

Hook Effect

The hook effect may occur when serum specimens being assayed contain very high PRL levels, as can be the case in patients with large PRL-secreting pituitary macroadenomas. ^, In these patients, PRL is present in vast stoichiometric excess to the two assay antibodies in the test solution, thus preventing the formation of the heterotrimeric complex (capture antibody–PRL–reporter antibody). The hook effect can cause substantial under-reporting of PRL levels. This artifact is clinically significant since patients with adenomas that are thought to be PRL-secreting will be generally offered a trial of medical therapy. In contrast, failure to diagnose a prolactinoma based on a normal or minimally elevated PRL level in a patient with a large pituitary adenoma may likely lead to a recommendation for surgery rather than medical therapy. In one study, the hook effect was reported in 5.6% of 69 patients with macroadenomas. The frequency of hook effect using current assay techniques is uncertain. To detect this possible artifact, PRL measurements should be obtained both in undiluted and diluted (1:100) serum specimens in patients with large macroadenomas. ^,

Macroprolactin

Monomeric PRL normally accounts for approximately 90% of circulating PRL, the rest consisting of PRL multimers of varying molecular mass, sometimes associated with immunoglobulin. Multimeric PRL species are collectively termed “macroprolactin” (as already mentioned). Macroprolactin can be resolved and separated from monomeric PRL by gel filtration. In routine practice, polyethylene glycol (PEG) precipitation has been used to separate macroprolactin from PRL monomers (only the latter is left in the supernatant after centrifugation). ^, Of note, hyperprolactinemia can be solely attributed to the presence of macroprolactin only if the monomeric PRL level present in the serum is within the reference range.

Macroprolactin appears to have decreased bioactivity in some, but not all, bioassays. ^, Women with macroprolactinemia who were treated with dopamine agonists were reported to experience resolution of galactorrhea (if originally present) but had no consistent improvement in menstrual cyclicity. ^, Macroprolactinemia should be considered in patients with hyperprolactinemia who lack typical symptoms attributable to PRL excess. ^, However, the presence of macroprolactinemia does not obviate the need for pituitary imaging, since some patients with pituitary adenomas may exhibit macroprolactinemia. Therefore, the clinical utility of assessing macroprolactin in symptomatic patients is limited.

Heterophilic Antibodies

Heterophilic antibodies, including human anti-mouse or other anti-animal immunoglobulins and the rheumatoid factor autoantibody, may cause assay interference by bridging the capture and reporter antibodies in the absence of analyte (i.e., PRL), thus giving rise to artifactually high PRL levels. ^, Heterophilic antibodies may also confound many other assays, in addition to PRL immunoassays. Using blocking animal sera or heterophilic antibody blocking tubes can help prevent this type of interference.

Exogenous Biotin

Several immunoassay platforms use biotinylated capture antibodies that collect the analyte being assayed onto streptavidin-coated microbeads. Measurement of hormone levels in patients taking biotin supplements in large doses is subject to possible interference if the immunoassay platform in use employs the biotin-streptavidin reaction. In these cases, ingested biotin may prevent the formation of the streptavidin-biotin complex in the assay solution, leading to under-reporting of the analyte being measured by sandwich immunoassays (including PRL) or overreporting of hormones measured using competition assays. ^, If it is clinically permissible, patients taking biotin supplements should discontinue them for 3 days before serum specimens are obtained for immunoassays. ^, Alternatively, immunoassay formats that do not use biotinylated antibodies can be used. ^,

Prolactin Secretion

Prolactin secretion is pulsatile.
Serum prolactin levels rise in many physiologic states, including pregnancy and lactation.

Prolactin secretion is pulsatile, involving approximately 14 secretory peaks per 24 hours, each peak lasting 67 to 76 minutes. ^, The PRL pulse amplitude increases during slow-wave sleep, with pulses beginning about 60 minutes after sleep onset. In addition, PRL levels increase by 50% to 100% about 30 minutes after meals as a result of stimulation of PRL secretion by amino acids being absorbed postprandially (particularly tyrosine, phenylalanine, and glutamate). ^,

Changes in Prolactin Levels With Age

Immediately following delivery, PRL levels in neonates are elevated about 10 times above baseline, presumably as a consequence of high levels of placental estrogen, and decrease toward baseline over several months. Subsequently, PRL levels rise somewhat during adolescence. In women, PRL levels decrease by approximately 50% within about 2 years after menopause. In older men, PRL levels also decline by about 50% in comparison with young adults.

Prolactin Levels During Physiologic Stress

Acutely, exercise leads to an increase in serum PRL levels, which is not sustained long-term in long-distance runners. ^, Other forms of physiologic stress, such as acute illness or injury, also lead to a two-to-three-fold increase in PRL levels, lasting about 1 hour, which is not sustained in patients with prolonged illness. ^, Women have a more robust prolactin response than men.

Prolactin Levels During the Menstrual Cycle, Pregnancy, and the Postpartum State

Prolactin levels are lower in the follicular phase and rise during the luteal phase in some women. In addition, PRL and LH secretion are synchronous in the luteal phase, possibly in response to gonadotropin-releasing hormone (GnRH) stimulation of PRL secretion during that period.

During pregnancy, PRL levels increase continuously and may reach more than tenfold or higher levels in nonpregnant women. This is a consequence of estrogen secretion by the placenta, which leads to lactotroph hyperplasia and stimulates PRL secretion, preparing the mammary gland for lactation postpartum. ^, Lactotroph hyperplasia is physiologic during pregnancy and is reversible within several months after delivery but is delayed by nursing. ^,

After delivery, basal PRL levels remain elevated in women who are nursing. ^, In addition, PRL secretory peaks occur rapidly at the time of each suckling event as a result of neurogenic stimulation of PRL secretion. ^, Within several months, there is a gradual decline in basal PRL levels toward normal as well as a decrease in the amplitude of secretory spikes in response to suckling. ^, These events occur as a consequence of a gradual decrease in the intensity of breastfeeding, while formula is being introduced into the infant’s diet. ^, Menses resume as postpartum hyperprolactinemia abates. Nipple stimulation, either acute or chronic (by nipple rings), may cause hyperprolactinemia in some healthy women who are not nursing. ^,

Regulation of Systemic Prolactin Levels

Prolactin secretion is primarily under inhibitory control by hypothalamic dopamine.
Thyrotropin-releasing hormone, vasoactive intestinal peptide, serotonin, and other factors may have some role in stimulating prolactin secretion.

Hypothalamic regulation of PRL secretion is primarily mediated via inhibitory factors (predominantly dopamine), as evidenced by an increase in PRL secretion and systemic PRL levels in patients who suffered pituitary stalk damage. ^, In addition, several releasing factors may have a role in modulating PRL secretion ( Fig. 3.1 ).

Fig. 3.1, Factors and pathways involved in the regulation of prolactin secretion.

Prolactin Inhibitory Factors

Several lines of evidence indicate that hypothalamic dopamine is the most important PRL inhibitory factor under physiologic conditions. Dopamine is present in hypothalamic portal vessels at levels that are sufficient to suppress PRL secretion. ^, Stimuli that elicit PRL secretion also lead to a decrease in dopamine levels in the hypophyseal portal circulation. ^, Mice with targeted disruption of D2 receptors, whose absence prevents dopamine action in pituitary lactotrophs, develop hyperprolactinemia, lactotroph hyperplasia, and multiple prolactinomas. Infusion of dopamine in low doses leads to suppression of PRL secretion in humans. ^, Estradiol partially reverses the effects of dopamine infusion on PRL secretion. Pharmacologic agents that inhibit dopamine receptors lead to hyperprolactinemia in humans.

Dopamine is synthesized in neurons whose perikarya are located in the dorsal arcuate nucleus and the ventromedial nucleus of the hypothalamus. This neuronal pathway is termed the tuberoinfundibular dopamine pathway. Axon terminals originating from these neurons terminate in the median eminence, where dopamine is released, enters the hypophyseal portal system, and traverses the pituitary stalk to reach pituitary lactotrophs in the pars distalis, where dopamine activates D2 receptors to suppress PRL secretion. Mice with disrupted PRL genes do not synthesize any PRL and have substantially decreased dopamine in the tuberoinfundibular dopamine pathway, consistent with the existence of a positive effect of secreted PRL on hypothalamic dopamine release, generating a short loop feedback mechanism of secreted PRL that negatively regulates its own secretion.

Whether other physiologically relevant PRL inhibitory factors exist is a matter of controversy. Some data suggest that gonadotropin-releasing hormone (GnRH)-associated protein (GAP), neuromedin U as well as gamma-aminobutyric acid (GABA) can suppress PRL in experimental paradigms, but their relevance in humans is unclear. Animal data have also suggested an autocrine and paracrine role for PRL in regulating its own secretion.

Prolactin-Releasing Factors

Several factors have been proposed as being stimulatory of PRL secretion (as detailed below). However, their physiologic relevance is uncertain in many cases. Overall, their role appears relatively minor in comparison with the inhibitory role of dopamine on PRL secretion.

Thyrotropin-Releasing Hormone

Thyrotropin-releasing hormone (TRH) induces PRL secretion in vitro and in vivo in humans when administered in doses that also cause TSH secretion. ^, On the other hand, targeted disruption of the TRH gene in mice leads to central hypothyroidism but does not influence PRL levels. In addition, nipple stimulation during suckling leads to a PRL secretory spike but does not influence TSH levels. Thus, it appears that TRH does not have a major role as a releasing factor under normal conditions. It should be noted, however, that patients with primary hypothyroidism have elevated TSH and PRL levels, which are normalized by levothyroxine replacement, suggesting that endogenous TRH may be relevant in PRL regulation in these patients but does not exclude the possibility that changes in dopaminergic tone in the hypothyroid state may also contribute to hyperprolactinemia. Patients with hyperthyroidism have normal PRL levels but do exhibit an abnormally low PRL response to TRH administration, which is restored after euthyroidism is achieved.

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