Current Insights in Prolactin Signaling and Ovulatory Function in Health and Disease

1. Introduction

Reports on the lactogenic (prolactogenic) activity of extracts from the anterior pituitary gland of cows first appeared in 1928 in the publication by P. Stricker and F. Grueter from the laboratory of Bouin at the University of Strasbourg, France [1,2]. Stimulation of lactation in rabbits via the injection of pituitary extracts was also confirmed in pigeons by O. Riddle, R.W. Bates and S.W. Dykshorn at the Cold Spring Harbor Laboratory on Long Island, New York, USA [3]. The latter team of authors made significant contributions to the identification, isolation and purification of a compound that acts as a polypeptide hormone and a tetrahelical cytokine, which was referred to as prolactin (PRL) because of its role in lactation [4,5].

After many years of intensive research, it is now known that PRL has a wide range of other diverse functions in the body [6,7,8,9] influencing not only the reproductive system [8,9,10,11,12,13,14] but also growth and development [15], metabolism [16,17,18,19], electrolyte transport [20], the integumentary system [21,22], behavior [23], the immune system [24,25,26] and carcinogenesis [27,28,29]. PRL exerts these versatile actions by acting on its receptor (PRLR), which is an archetype member of the class I cytokine receptor family [30,31,32].

Moreover, the development of precise bioassay methods has made it possible to discover that, under the control of the hypothalamus, anterior pituitary lactotrophs are not the only source of PRL. The multiple extrapituitary sites at which PRL is expressed and secreted in humans include the brain, endothelial cells, immune cells, decidua and ovarian tissue [9,33]. Pituitary PRL expression has been shown to be dependent on the Pit-1 transcription factor, which is a member of the Pit-Oct-Unc (POU) homeodomain protein family, whereas the extrapituitary PRL gene promoter that allows for PRL expression in several nonpituitary cells and tissues has dissimilar activity to the pituitary promoter, with Pit-1-independent activity and responsiveness to different regulators of gene expression [34].

The latter of the listed locations indicates that the human ovary not only serves as a target for endocrine PRL but also as a site of local PRL hormone production [9,35,36]. In ovarian tissue, PRL is expressed in both the antral follicle, theca and granulosa layers and is produced by granulosa cells [5,36,37,38]. Moreover, by acting locally as an autocrine or paracrine cytokine, this hormone is an important modulator of key processes such as follicular growth and development, angiogenesis, ovulation and steroidogenesis [13,39]. Despite the lack of consensus regarding their detailed comprehensive classification, ovulatory disorders are among the leading causes of infertility [40,41]. Therefore, abnormal PRL activity, both in the blood and at the level of ovarian tissue, can cause infertility due to the mechanism of anovulation or infrequent ovulation (oligo-ovulation) secondary to hormonal imbalances [42,43,44].

The aim of this review is to provide a comprehensive presentation of the current state of knowledge on the role of PRL in the process of ovulation and its disorders. Thus, a rationale for influencing PRL signaling pathways in therapeutic activities accompanying ovulation disorders is outlined.

2. Prolactin (PRL)

2.1. Structure

PRL is a globular protein hormone composed of a single-chain polypeptide with a molecular weight of 23 kDa that contains 199 amino acids in humans. There are three intramolecular disulfide bonds between the following six cysteine residues in the human PRL chain: Cys4-Cys11, Cys58-Cys174 and Cys191-Cys199 (Figure 1A) [5].

Analysis of the secondary structure of the PRL molecule showed that 50% of the length of the amino acid chain is arranged in α-helices, whereas the remaining part of the chain forms loops. According to the model of the tertiary structure of PRL, four long α-helices with four α-helical domains and two receptor binding sites are arranged in an antiparallel fashion (Figure 1B) [5,45].

Amino acid sequence homology with the human genome shows considerable species variation, ranging from 97% homology in primates to only 56% homology between primates and rodents [5,46]. In humans, PRL is structurally similar to growth hormone (GH) and human placental lactogen (hPL, also known as human chorionic somatotropin [hCS]). Together, these proteins form the "PRL/GH/hPL" family, which is characterized by a conserved helix bundle protein composition (Group I of the helix bundle protein hormones); consequently, PRL has similar biological activities to its representatives [47,48]. The genes encoding PRL, GH and hPL were generated as a result of the evolution of the common ancestor gene, which consisted of duplication of this gene. In humans, a single gene encoding PRL is located on chromosome 6 [49]. This gene is 10 kb in length and contains 5 exons and 4 introns. The PRL gene transcription process is regulated by two independent promoter regions: the proximal region that controls pituitary-specific expression and the more upstream 5,000-bp region that directs extrapituitary expression of PRL [50]. Preprolactin, which is a protein 2–3 kDa heavier than mature PRL, represents the initial product of PRL mRNA translation, from which PRL is produced after removal of the 28-amino acid signal peptide by proteolytic cleavage [5,7].

In humans, as in animals, many variants of the structure of the PRL molecule have also been described; these variants occur in small, often trace amounts as a result of alternative splicing of the primary transcript, as well as proteolytic cleavage and other posttranslational modifications (e.g., dimerization and polymerization, phosphorylation, glycosylation, sulfation and deamidation) of the amino acid chain [46,51]. These modifications indicate that, in addition to the monomeric form of PRL, in the human body, PRL also occurs as a dimer (molecular weight [MW] of 48-56 kDa) composed of glycosylated monomers and in the form of macro-PRL or big-big PRL (MW >150 kDa) composed of antigen-antibody complexes of monomeric PRL and immunoglobulin (most commonly, IgG) [52,53]. In a healthy person, monomeric PRL is the predominant form of circulating PRL and accounts for 85% of the total immunoreactive PRL, whereas dimeric PRL and macro-PRL each account for approximately 5-10% of the total PRL in the blood [53]. It should be noted that routine tests cannot distinguish between the three forms of PRL, which may be important in diagnosing disorders of PRL production [54,55,56].

2.2. Prolactin receptor (PRLR)

PRLRs belong to the lactogen/hematopoietic cytokine receptor superfamily; additionally, as an archetype member of the class I cytokine receptor family that comprises systemic receptors with fundamental effects in the body, they play key roles in biology and as potential therapeutic targets. The same group of receptors as PRLRs, which consists of the simplest cytokine receptors, also includes the GH receptor, the interleukin-2 receptor (IL-2R), the erythropoietin receptor (EPOR) and the thrombopoietin receptor (TpoR, also known as MPL) [28,57]. Therefore, even with lower affinity, the PRLR can bind and be activated by hPL, GH, IL-2, EPOR and TpoR [31]. PRLR lacks an intrinsic kinase domain but possesses a Janus kinase 2 (JAK2)-associated region. Therefore, the receptor chain is dependent on the associated kinases to transduce phosphorylation-based signaling cascades. Upon PRL stimulation, the PRLR transduces signals through the activation of JAK2, thus leading to the phosphorylation of JAK2 [58].

The human PRLR gene is located at chromosomal region 5p14-p13. The genomic size of the human PRLR gene exceeds 200 kb, and the gene contains 11 exons, including six noncoding exons 1 that are alternatively spliced to a common noncoding exon 2, as well as exons 3-10 that encode the full-length activating long form of the receptor [59,60]. As a transmembrane receptor, the PRLR is composed of three main regions: the extracellular, transmembrane (a single transmembrane-spanning domain) and intracellular (cytoplasmic) regions. The extracellular region comprises two fibronectin type III domains known as D1 and D2. The WSXWS motif in D2, which is a generic cytokine class I receptor, acts as a molecular switch during ligand-bound activation of the PRLR [61]. Conversely, proline-rich sequence-mediated JAK2 association with the PRLR occurs in the intracellular domain. This JAK2/PRLR interaction is essential, although it alone cannot induce signal transduction through the Box-1/Box-2 subdomains. Further interaction of Box-1 with JAK2 and SRC family kinases (e.g., Fyn and c-Src) is required [62,63].

The long form of the PRLR may regulate signal transduction by exploiting the properties of the PRLR intracellular domain, which is intrinsically highly unstructured/disordered and binds to negatively charged lipids of the inner plasma membrane through conserved motifs resembling immune receptor tyrosine-based activation motifs [64]. Such lipid association of the PRLR intracellular domain is not accompanied by induced folding and is independent of specific tyrosine phosphorylation [64,65].

Intermediate and various short forms result from alternative splicing, variable promoter usage or other posttranscriptional (but unlikely) posttranslational modifications. Sequences from exon 11 are present only in the short forms of the receptor S1a and S1b and their respective variants [66,67]. Such proteome diversity in the PRLR results from the fact that, although many PRLR isoforms perform the same or similar biological roles, some of these isoforms have unique functions. Moreover, human PRLRs are located not only in the cell membrane but also in the cytosol (a soluble isoform) and, surprisingly, in cellular structures such as endosomes, the Golgi apparatus and lysosomes (here, they are presumably a degraded form of the receptor) [60,64,68]. All of the soluble forms of PRLR lack transmembrane and cytoplasmic regions [69,70]. A schematic illustration of the most common isoforms of human PRLR is included in Figure 2.

2.2.1. PRLR receptor signaling

PRLRs are expressed in a wide but varied range of human cells and tissues throughout an individual’s lifetime, starting from the preimplantation stage of the embryo and continuing further during intrauterine embryogenesis [6,71]. The binding of a PRL molecule produced in the pituitary or from extrapituitary sources to the extracellular ligand-binding domain of the PRLR initiates the signaling pathway within a systemic or local range, respectively. This also applies (to varying degrees and depending on the specific physiological or pathological conditions) to partial agonists of the PRLR (e.g., hPL, GH and IL-2) [31]. Studies have also shown that the binding affinity of human PRLR for nonhuman PRL is lower than that for human PRL [72]. Among the PRLR isoforms, the signaling pathways associated with the long form of the receptor are the most comprehensively characterized to date [68].

In canonical signaling, ligand binding to the PRLR results in stimulation of the tyrosine kinase activity of JAK2; moreover, with the exception of the short isoform of the receptor, multiple tyrosine residue phosphorylation within the PRLR occurs in the following ways. ❶ Subsequent activation of signal transducer and activator of transcription (STAT) proteins, particularly STAT5 (JAK2/STAT5 signaling), thus enforcing further downstream signaling [73,74]. Next, the tyrosine-phosphorylated STAT complex dissociates from the receptor, dimerizes and translocates into the nucleus, where it binds to the promoters of target genes. Although the JAK/STAT pathway is considered one of the major downstream pathways for cytokine receptor signaling, PRL also activates ❷ the Ras kinase/Raf kinase/mitogen-activated protein kinase/extracellular signal-regulated kinase ½ (Ras/Raf/MAPK/ERK1/2) pathway [75,76] and ❸ the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3-Kinase/AKT/mTOR) [76,77] downstream signaling pathway. In addition, in the absence of a physical association with STAT5a, ❹ signaling through the transcription factors RUSH-1α, RUSH-1β and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A, member 3 (RUSH/SMARCA3), has been identified (Figure 3) [78].

This diversity of signaling pathways corresponds to the numerous functions of PRL, which are associated with both physiological states and play important roles in the pathomechanism of diseases, especially endocrine disorders [6,7,9]. Moreover, these key PRL-mediated signaling pathways are integrated. The differences in the functions of PRLR according to the pleiotropic nature of PRL and other PRLR agonists are presented under selected physiological and pathological conditions in Figure 4. A detailed discussion of these activities, apart from those directly related to ovulation, is beyond the scope of this review.

3. Ovulation

In women, ovulation is a phase in the menstrual cycle when the rupture of the dominant follicle within one of the ovaries leads to the release of the egg (the secondary oocyte or an oocyte arrested in meiosis II at the stage of metaphase II), which enters the lumen of the fallopian tube and creates a chance for fertilization in the presence of fertile sperm [14,113]. This physiological process occurs around day 14 of a 28-day menstrual cycle (or generally at approximately 2 weeks before the onset of menstruation) in women of reproductive age, i.e., from menarche to menopause (roughly from ages 12 to 49 years). Typically, human ovaries produce a single dominant follicle that is approximately 25 mm in diameter and contains approximately 50 million granulosa cells and 7 ml of follicular fluid at the time of ovulation. For ovulation to occur during each menstrual cycle, the ovarian follicle (or a Graafian follicle), which becomes the dominant follicle as a result of a highly selective process, must undergo all stages of folliculogenesis (recruitment, selection and ovulation) within a strictly defined time frame (Figure 5) [114,115,116,117,118,119,120,121,122,123], including those related to gene expression (Table 1.).

The pool of ovarian follicles, numbering approx. 150,000 during a woman's puberty, is subject to recruitment and selection processes leading to the development of the dominant Graafian follicle, which undergoes ovulation. It is worth noting that while the cyclical, gonadotropin-dependent recruitment of the early antral follicles takes place over 14-day periods (follicular phase), reaching this stage through the earlier stages of folliculogenesis (gonadotropin-independent in the case of primordial follicles and gonadotropin-responsive in the case of secondary follicles) takes much longer. The initiation of the menstrual cycle during puberty and its subsequent course are determined by the variable, pulsatile secretion of gonadoliberin (GnRH - gonadotropin-releasing hormone) from the hypothalamus. GnRH stimulates the production and secretion of gonadotropins (FSH – follicle-stimulating hormone and LH – luteinizing hormone) in the anterior pituitary gland (adenohypophysis), which in turn stimulate the development of ovarian follicles and the production of ovarian steroids by thecal and granulosa cells (GC): estrogen (predominantly estradiol – E₂) and progesterone (P4). The negative feedback mechanism within the hypothalamic-pituitary-ovarian (HPO) axis plays a key role in regulating the levels of all hormones that control the menstrual cycle. However, there is an exception, when rising estradiol (E2) in the middle of the menstrual cycle paradoxically "switch" from being inhibitory on GnRH secretion ("negative feedback") to stimulating GnRH release ("positive feedback"), resulting in a surge in GnRH secretion and a downstream LH surge that triggers ovulation. The onset of LH surge usually precedes ovulation by 36 hours, whereas the peak serum level of LH occurs 10-12 hours before ovulation. Among the regulatory factors of the HPO axis negative feedback, the heterodimeric proteins produced by ovarian follicles inhibin A (αβA, InhA) and B (αβB, InhB) play a significant role. During the normal menstrual cycle, serum inhB levels are lowest in the early follicular phase, reach a mid-cycle peak coincident with decrease in FSH concentration and the preovulatory LH surge, and decrease during the luteal phase. Conversely, InhA concentrations are low during the selection and development of a dominant follicle, increase rapidly during ovulation, and are maximal during the mid-luteal phase. Changes in sex hormone concentrations are accompanied by cyclic changes in the endometrium (not shown in the figure), which enable implantation of the blastocyst after oocyte' fertilization or repetition of the cycle with the separation and expulsion of the functional layer of the uterine mucosa (onset of menstruation).

The mechanisms regulating the selection of antral ovarian follicles are poorly understood and are thought to rely on low estrogen levels, a decrease in follicle-stimulating hormone (FSH) levels and FSH receptor (FSHR) expression on the surface of granulosa cells. It has been assumed that a follicle capable of maximum expression of FSHR is able to maintain growth and becomes dominant in a given cycle [128,129]. However, such assumptions have not been confirmed under in vitro conditions, wherein apoptosis of human granulosa cells (hGLC) and transfected cell lines is induced by high doses of FSH or FSHR overexpression, whereas estrogens induce antiapoptotic signals via nuclear and a G protein-coupled estrogen receptor (GPER). Therefore, in vitro data suggest that antral follicle selection may be driven by underestimated, FSH-FSHR-dependent apoptotic signals due to transient maximization of FSHR expression and overload of cAMP signaling, thus prevailing on estrogen-dependent signals [130,131,132].

It is generally agreed in humans that a selected follicle becomes dominant at approximately one week before ovulation; specifically, this occurs as early as at 5 to 7 days of the cycle, at a time when the follicular diameter is approximately 10 mm. Only in the dominant follicle can FSH be detected at this point in time, which is also accompanied by a significant level of estradiol in the follicular fluid [121,122].

The remaining follicles that are recruited in a given cycle undergo atresia, the main mechanism of which in mammals involves programmed cell death (apoptosis). Therefore, follicular atresia is a physiological phenomenon that allows for the regulation of the number of follicles growing within a given pool by the crosstalk between cell death and cell survival signals. Accordingly, the fate of the individual follicle (growth to ovulation or inhibited growth with subsequent atresia) is dependent on a precise balance in the expression of specific receptors (mainly for endocrine hormones and gonadotropins) and the action of intraovarian factors (such as gonadal steroids, cytokines and growth factors) regulating follicular cell proliferation, growth and differentiation, as well as those promoting apoptosis. It is estimated that in each ovulatory cycle, the average ovary loses 1,000 follicles during the process of selecting a dominant follicle that will be released. Therefore, the total pool of follicles must be sufficiently large [133,134].

Indeed, the fetal ovary at the twentieth week of gestation contains 6-7 million oocytes, which results from the mitosis of approximately 500 to 1,300 primordial germ cells; this theoretically represents the entire cohort of oocytes capable of participating in reproduction during a woman’s lifetime [135]. These germ cells subsequently begin meiosis and arrest at meiotic prophase 1, wherein they form germ cell cysts and, ultimately, primordial follicles. Consisting of an oocyte surrounded by a single layer of cuboidal granulosa cells that initiates follicle development, primordial follicles are the first class of follicles that develop in mammalian ovaries. The process of follicular recruitment begins soon after this time point and proceeds continuously in both gonadotropin-independent (before puberty) and gonadotropin-dependent (after puberty) manners. Consequently, the number of follicles in the ovaries decreases to approximately 500,000 at birth, 150,000 at puberty and 1,000 at menopause [136]. Thus, out of the entire pool of ovarian follicles that are present on the day of the woman’s birth, ovulation will increase in only 0.1% of patients, whereas ovulation will increase in 99.9% of patients throughout the process of follicular atresia [137,138]. The continuity of folliculogenesis means that an active ovary always contains follicles in various stages of development [139].

The luteal phase occurs in the second half of the menstrual cycle, wherein it starts immediately after ovulation. This phase is usually 14 days long in most women. After the oocyte with cumulus cells is released from the ovulating follicle, the remaining granulosa cells continue to enlarge, become vacuolated, and begin to accumulate a yellow pigment known as lutein. Luteinized granulosa cells with newly formed theca-lutein cells and surrounding stroma give rise to a new structure known as the corpus luteum. The primary hormone produced by this transient endocrine organ is progesterone; however, it also produces inhibin A and estradiol. The primary function of the corpus luteum is to prepare the estrogen-primed endometrium for implantation of the fertilized ovum. This transformation of the endometrium is known as decidualization. In contrast to most mammals, decidualization of the human endometrium does not require embryo implantation. Instead, this process is driven by the postovulatory rise in progesterone levels and increasing local cAMP production [140]. Eight or nine days after ovulation, approximately around the time of expected implantation, peak vascularization is observed within the corpus luteum. This time period also corresponds to peak serum levels of progesterone and estradiol [117]. The lifespan of the corpus luteum depends upon continued LH support. Corpus luteum function declines by the end of the luteal phase, unless human chorionic gonadotropin (hCG) is produced during a pregnancy. If pregnancy does not occur, the corpus luteum undergoes luteolysis under the influence of estradiol and prostaglandins, during which a scar tissue known as the corpus albicans is formed [117]. In response to decreasing progesterone levels, spontaneous decidualization causes menstrual shedding and cyclic regeneration of the endometrium.

3.1. Hormonal regulation of ovulation

In addition to menstrual bleeding, ovulation is the most crucial element of fluctuations in hormonal balance; this process is repeated cyclically every 28 days (with a range of 21-40 days) and is referred to as the menstrual cycle. Occurring during the course of the menstrual cycle, the morphological changes in the ovaries leading to ovulation and the formation of the corpus luteum, as well as functional changes in the endometrium that enable blastocyst implantation in the event of fertilization of the ovum, are primarily regulated by hormones [141,142,143]. The pulsatile secretion of gonadotrophin-releasing hormone (GnRH) from the hypothalamus (under the control of the kisspeptin-neurokinin B-dynorphin [KNDγ] pathway) stimulates the anterior pituitary to secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which correspondingly stimulate the development of ovarian follicles and the production of the ovarian steroids estrogen (predominantly estradiol) and progesterone (P4) [143,144,145,146]. A negative feedback mechanism within the hypothalamic-pituitary-ovarian (HPO) axis is crucial for the control and regulation of the menstrual cycle, particularly during the following three phases: follicular (which begins with menstrual bleeding), ovulatory, and luteal phases. However, there is an exception in females, wherein rising estradiol (E2) during the middle of the menstrual (or estrous) cycle paradoxically "switches" from being inhibitory on GnRH secretion ("negative feedback") to stimulating GnRH release ("positive feedback"), thus resulting in a surge in GnRH secretion and a downstream LH surge that triggers ovulation (Figure 6B) [147,148]. Additionally, the granulosa cells of the growing follicle secrete a variety of peptides that may play an autocrine/paracrine role in stimulating follicles (activins), thus inhibiting the development of adjacent follicles (e.g., inhibins and follistatin) or suppressing/unsuppressing the biosynthesis of FSH (inhibins vs. activins) [149,150,151,152]. The luteal phase of the cycle is relatively constant in all women, with a duration of 14 days. The variability of cycle length is usually derived from varying lengths of the follicular phase of the cycle, which can range from 10 to 16 days [117]. The general characteristics of the main endocrine hormones involved in the menstrual cycle and their importance during the process of ovulation are summarized in Table 2.

Importantly, all ovarian hormones that regulate the course of the menstrual cycle are synthesized under precisely defined conditions and time frames and act in an autocrine and paracrine manner. A comprehensive understanding of the mechanisms of influence at this level in physiological states and disorders (e.g., ovulation) requires further research [130,192,193,194].

3.2. Mechanism of follicle rupture during ovulation

The initial hypothesis that the rupture of the wall of the ovulating follicle occurs as a result of an increase in intrafollicular pressure has not been confirmed by different studies. Despite a significant and rapid increase in the volume of follicular fluid before ovulation, which is mainly due to the inflow of fluid from the extravascular space, no significant changes in intrafollicular pressure have been detected at that time [195].

Therefore, it is important that the preovulatory surge in gonadotropins (primarily LH) triggers a series of changes in the wall of the dominant follicle, the enzymatic degradation of which leads to rupture and determines the extrusion of the oocyte. Therefore, the assessment of the molecular mechanisms of follicle wall rupture during ovulation has focused on creating a model of interconnected signaling networks that initiate a proteolytic enzyme cascade that has a local effect. Many mediators of these LH-induced signaling cascades are associated with inflammation, including the production of mediators of the inflammatory response by granulosa and theca cells, such as steroids, prostaglandins, chemokines, and proteolytic enzymes (e.g., collagenase and plasmin) [173]. It has been demonstrated that granulosa cells in vivo produce increasing amounts of plasminogen activator as ovulation approaches and that the enzyme is solely produced by cells obtained from follicles destined to ovulate [196]. The production of plasminogen activator is regulated by gonadotropins [196,197].

The concentrations of prostaglandins E and F and hydroxyeicosatetraenoic acid (HETE) reach a peak in the follicular fluid just prior to ovulation [198,199].

Prostaglandins may stimulate both proteolytic enzymes and oocyte release by inducing smooth muscle fiber contractions within the ovary, whereas HETE may stimulate angiogenesis and hyperemia [117,199]. The point at which the dominant follicle is closest to the ovarian surface where digestion and subsequent rupture of the wall occur is known as the stigma.

In the context of the obvious similarities between ovulation and the inflammatory response, it is worth noting that at the level of ovarian follicles, PRL (which is also locally produced in the ovarian tissue) acts both as a hormone (endocrine and paracrine) and as a proinflammatory cytokine [5,200]. Thus, under certain conditions, the expression of PRLRs plays both pro- and anti-gonadal roles in the regulation of ovarian functions, including ovulation. For example, PRL may inhibit ovulation by stimulating granulosa cell apoptosis or promote ovulation by triggering a cascade of proteolytic processes in the wall of the ovarian follicle. However, neither the pattern of expression of PRLRs nor its regulatory mechanisms during follicle development have been clearly defined [201,202].

3.3. Ovulatory disorders

The term "ovulatory disorders" describes a set of abnormalities that occur continuously or intermittently, thus resulting in the absence of ovulation (anovulation) or its infrequent, irregular occurrence (oligoovulation) [203]. During oligoovulation, the menstrual cycle is typically longer than normal (21 to 35 days), thus resulting in eight or fewer menstrual periods per year. In addition to abnormal uterine bleeding, ovulatory disorders may disappear in some cases. Ovulatory disorders, which typically result from episodic or chronic dysfunction of the hypothalamic–pituitary–ovarian (HPO) axis, represent a major cause of infertility, as the lack of an optimal quality oocyte that is regularly released every month significantly reduces the chances of fertilization. Anovulation and oligoovulation are estimated to constitute 25% of the known causes of female infertility [204,205].

Although there are numerous known causes and contributors to ovulatory disorders, the entire spectrum of mechanisms of pathogenesis remains to be fully elucidated. Ovulatory disorders are often observed due to coexisting endocrinopathy, neoplasms, or emotional states requiring psychological/psychiatric assistance and as a consequence of the use of medications. Therefore, optimally effective research, teaching, and clinical management of ovulatory disorders must be based on a generally accepted and possibly simple classification. Intensive activities that have been recently conducted by international bodies (International Federation of Gynecology and Obstetrics, or FIGO) have resulted in a consensus and the presentation of a proposal for a new classification; according to its authors, this proposal may replace the existing ones, especially the widely used World Health Organization (WHO) classification of ovulatory dysfunction [205,206,207]. Both classifications are summarized in Figure 6.

The WHO classification clearly takes into account the states of hyperprolactinemia in groups V and VI (highlighted on yellow background).

In the FIGO classification, the effect of PRL on ovulation is not directly separated at various levels (hypothalamic, pituitary, or ovarian) of physiological and pathological (PCOS) regulation; in contrast, the WHO classification clearly considers the states of hyperprolactinemia in Groups V and VI.

According to various estimates, polycystic ovary syndrome (PCOS), which is the most common cause of oligoovulation and anovulation (anovulatory infertility), may affect 5 to even 20% of women of reproductive age [208,209,210,211]. In PCOS, abnormal hormone levels prevent ovarian follicles from growing and maturing to ovulation. As a result, immature follicles accumulate in the ovaries, thus resulting in a typical picture of polycystic morphology via ultrasound examination [212]. The pathophysiology of PCOS is heterogeneous and associated with interactions between reproductive function disorders and altered metabolism [213]. Hyperandrogenism and insulin resistance are part of a vicious disease cycle that exacerbates the course of PCOS and is also affected by dysfunction of the hypothalamus-pituitary-ovarian (HPO) axis [214]. Notably, in 30% of PCOS patients, an increase in the serum PRL concentration can be detected in both the follicular and luteal phases of the menstrual cycle [215,216]. It is well documented that hyperprolactinemia may limit the number of growing ovarian follicles and inhibit ovulation [217,218].

3.3.1. PRL and ovulatory disorders

The average basal level of PRL in women is 13 ng/ml (277 mIU/L), whereas the upper normal limit of serum PRL in most laboratories is 15 to 20 ng/ml (320 to 425 mIU/L) [219]. Elevated PRL (hyperprolactinemia) is the most common endocrine disorder of the hypothalamic-pituitary axis. Clinical data clearly indicate an association between hyperprolactinemia and infertility, the main cause of which is ovulatory disorders due to suppression of the HPO axis via inhibition of pulsatile gonadotropin releasing hormone [44].

The vast majority of causes of hyperprolactinemia can be classified as physiological, pathological, or drug-induced (iatrogenic) [42,219,220]. However, despite continuous progress in pathophysiology and diagnostic methods, cases of idiopathic hyperprolactinemia of unknown or no clear cause, as well as hyperprolactinemia, are misdiagnosed due to the use of inadequate analytical techniques [221,222]. Table 3 categorizes the most important causes of hyperprolactinemia and discusses the known or probable underlying mechanisms.

Hypothalamic dopaminergic cells, which are known as tuberoinfundibular dopamine (TIDA) neurons, are located in the arcuate nucleus and send axonal projections to the median eminence. Dopamine is tonically (i.e., continuously) secreted by these dopaminergic neurons to ensure an appropriate level of inhibition of PRL release from pituitary lactotrophs [7]. Therefore, increased PRL secretion under both physiological and pathological conditions mostly results from the removal of dopamine-inhibiting pathways. Hence, most prolactin-relasing factors (PRFs) act indirectly through the disactivation of the TIDA system, although direct effects on lactotrophs and other mechanisms of action (which have not been completely clarified) are certainly present [261,262,263]. Although PRL is well known to stimulate hypothalamic dopamine secretion, thereby exerting negative feedback regulation on its own release, autocrine or paracrine actions of PRL on lactotroph cells have also been suggested [264].

The most common cause of hyperprolactinemia is prolactinoma, which involves a benign (noncancerous) prolactin-releasing tumor, the main prevalence of which is estimated to be approximately 30 per 100,000 people, with a peak incidence occurring at ages 25 to 34 years [265]. Moreover, even in approximately 40% of nonfunctioning pituitary adenomas (NFPAs), benign adenohypophyseal tumors are not associated with clinical evidence of hormonal hypersecretion, and hyperprolactinemia occurs as a result of pituitary lactotroph disinhibition (the stalk effect) [266]. Interestingly, most patients with increased intrasellar pressure caused by large pituitary tumors do not exhibit hyperprolactinemia. Lactotroph insufficiency accompanying pituitary failure may be responsible for 87% of cases of normoprolactinemia among patients with pituitary tumor sizes > 20 mm [267]. Similarly, cases of hypoprolactinemia in the context of ovulatory disorders should be considered accompanying damage to the entire glandular part of the pituitary gland (e.g., in Sheehan syndrome); therefore, it can be combined with gonadotropin deficiencies [268].

As shown in Table 3, a large group of medications can cause an increase in PRL levels, thus impairing dopaminergic activity in the central nervous system (CNS) in various ways, including by acting as a D2 receptor antagonist, a false neurotransmitter, an H2 receptor antagonist, a Ca2+ channel blocker, a selective 5-HT reuptake inhibitor (SSRI) or an inhibitor of the vesicular monoamine transporter type 2 (VMAT2) [269,270]. Latrogenic causes of hyperprolactinemia may be a clinical problem in which the incidence is underestimated, especially in patients who are not taking antipsychotics or antidepressants [103,249,271]. For example, hyperprolactinemia caused by treatment of pregnancy-induced hypertension (PIH) with methyldopa may be associated with an increased incidence of postpartum depression and maternity blues (also known as baby blues) [272].

3.3.1.1. PRL and the release of gonadotropins.

A sufficiently high PRL concentration suppresses hypothalamic gonadotropin-releasing hormone (GnRH, also known as gonadoliberin), which is part of a family of peptides that play pivotal roles in reproduction by stimulating the synthesis and secretion of LH and FSH from the adenohypophysis [81].

When considering the physiological activities of PRL, other than those related to lactation, it can be assumed that a certain level of PRL circulating in the blood, as well as what is produced locally in reproductive tissues, may be necessary for optimal oocyte development, formation and maintenance of the corpus luteum, as well as blastocyst implantation, steroidogenesis in early pregnancy and modulation of the immune system [26,44,273,274,275,276].

However, persistently high levels of PRL can lead to (secondary to GnRH inhibition) hypogonadotropic hypogonadism related to the suppression of both LH and FSH, as well as fertility problems that, in women, are most often a result of ovulation disorders, including the complete disappearance of ovulation [43]. Perhaps surprisingly, only a very small percentage of GnRH neurons express PRLRs or exhibit STAT5 phosphorylation (pSTAT5) in response to an acute PRL stimulus. Moreover, the interaction of PRL with the cell membrane of GnRH neurons is not accompanied by significant modulation of its excitability [277,278]. In addition to changes in PRLR expression under physiological and pathological conditions, the abovementioned findings indicate that PRL does not act directly on GnRH neurons; therefore, the effects of PRL on gonadotropin secretion are mediated by another population of hypothalamic neurons, including those related to Kisspeptin secretion [12,279,280].

3.3.1.2 PRL-kisspeptin interaction

Kisspeptins are a set of peptide fragments encoded in humans by the KISS1 gene [281]. All of these polypeptides have very similar affinities for the kisspeptin receptor KISS-1R (also known as GPR54), which is a G protein-coupled receptor and is considered the canonical receptor for neuropeptides that are products of the KISS1 gene [282,283].

In the mammalian hypothalamus, kisspeptin neurons are mainly located in two areas: the rostral region, which is associated with the preoptic area (POA), and the caudal region, which is associated with the arcuate nucleus (ARC) [282,284]. It has been proposed that neurons in the ARC that coexpress kisspeptin, neurokinin B and dynorphin (KNDy cells) play key roles in GnRH pulse generation, with kisspeptin driving GnRH release and neurokinin B (NKB) and dynorphin acting as start and stop signals, respectively [285,286]. KNDy neurons in the ARC also appear to mediate the negative feedback effects of E2 and are thought to be the main regulators of pulsatile LH secretion. Moreover, KNDy neurons may also be involved in the positive feedback of E2 to induce the LH surge [287]. However, the role of kisspeptin neurons in the POA has not been determined. KNDy neurons within the POA are hypothesized to be involved in the modulatory effects of E2 on thermoregulation [288].

KISS-1R is expressed by GnRH neurons and is directly activated by kisspeptin to stimulate GnRH release [289]. GnRH-secreting neurons form a relatively small population of cells (e.g., approximately 800 neurons in mice and 1,000-2,000 in humans) scattered between the POA and ARC in the shape of an “inverted Y” [290].

Kisspeptin is able to induce GnRH secretion both by stimulating KISS-1R in the neuronal stroma and in GnRH nerve terminals located in the mediobasal hypothalamus (MBH) region [290,291]. By acting directly on GnRH release and directly and/or indirectly on LH and FSH secretion, the kisspeptin/KISS-1R system has been widely reported to be a key factor in the regulation of the hypothalamic-pituitary-gonadal (HPG) axis [146,292]. Loss-of-function mutations in the kisspeptin/KISS-1R system disrupt puberty and infertility in both humans and animal models, whereas mutations that activate this system lead to precocious puberty in humans [293,294,295,296]. Consistently, it has been hypothesized that abnormal kisspeptin signaling may be responsible for stress-induced fertility disorders, including ovulation disorders [145,146,281,297].

Many studies have shown that the significant actions of PRL in modulating the HPG axis are the result of interactions with neurons expressing the KISS1 gene [12,298,299,300]. This is because most KISS1-expressing neurons coexpress functional PRLRs (those receptors through which PRL induces pSTAT5) [301,302]. After acute intraperitoneal (i.p.) administration of PRL to female mice in diestrus, pSTAT induction was observed in approximately 80% of KISS1-expressing neurons in the ARC of the hypothalamus [12,300]. Further confirmation that the PRL/PRL-kisspeptin interaction plays an important role in the regulation of the HPG axis was obtained by demonstrating that systemic or intracerebroventricular (icv) infusion of PRL inhibited KISS1 expression in the hypothalamus, with a subsequent reduction in plasma LH levels [299,303]. Therefore, secondary to the inhibition of GnRH secretion, hypogonadotropic hypogonadism due to hyperprolactinemia is the result of the downregulation of the kisspeptin/KISS-1R signaling system [148,304].

Interestingly, it has been suggested that kisspeptin may have a direct effect on PRL production in lactotrophs of the adenohypophysis under the influence of E2, which may regulate KISS-1R expression and function [305]. KISS-1R appears to be essential for mediating the effect of kisspeptin on PRL secretion, although TIDA neurons do not express KISS-1R and are electrically unresponsive to kisspeptin. It follows that kisspeptin can directly stimulate PRL secretion via KISS-1R in nondopaminergic neurons, whereas the effect on expression of tyrosine hydroxylase TIDA neurons causing an increase in PRL secretion is indirect, which results from inhibition of dopamine release with subsequent pituitary lactotrophs disinhibition. The latter effect doubles the well-documented inhibitory effect of gamma-aminobutyric acid (GABA) within TIDA neurons on dopamine secretion [306]. The kisspeptin-dependent inhibition of dopamine release from TIDA neurons is most likely mediated by the neuropeptide FF receptor 1 (NPFFR1), which is differentially expressed on dopaminergic neurons in the hypothalamus [307]. The occurrence of increased PRL secretion after kisspeptin treatment of TIDA neurons is also dependent on E2 and the expression of estrogen receptor-α (ER-α) on dopaminergic neurons [308].

Therefore, the variability of the functional balance within the PRL-kisspeptin interaction may determine the dominant influence of GnRH or PRL on a given stage of the sexual cycle, including ovulation [12,309,310,311].

The interplay between PRL and kisspeptin in influencing the sexual cycle and ovulation via the HPG axis is shown in Figure 7.

4. Concluding remarks

PRL is a pleiotropic neuroendocrine hormone that is synthesized and secreted mainly by lactotroph cells in the anterior pituitary gland [5,6]. The principal role of PRL in mammals is to regulate lactation [7]. Although the pool of PRL circulating in the blood is almost exclusively of pituitary origin, one should not forget about the extrapituitary sites of PRL synthesis, which may play a role in local regulation. One such location is the ovarian tissue, where depending on the variable expression of PRLRs during the menstrual cycle, PRL (which acts both in an endocrine and paracrine manner) can significantly influence ovulation and the function of the corpus luteum [33,35,36]. However, the nature of these processes corresponding to the physiological state and ovulatory disorders in vivo has still been insufficiently explained, and the results are often contradictory. For example, few studies have reported of a negative relationship between follicular fluid PRL levels and oocyte competence [312,313], whereas other authors have shown that higher follicular PRL levels characterize preovulatory follicles [314] and are also favorable predictors of oocyte maturation and fertilization ability [44,315,316,317]. Notably, the only PRL isoform that has been detected in follicular fluid is the most potent small PRL [318].

When considering the abovementioned effects, it can be assumed that the influence of PRL-dependent granulosa cell apoptosis may constitute a future therapeutic target in the treatment of anovulation and/or corpus luteum dysfunction [202,319]. Moreover, PRL binding activates PRLR, which is a type-1 family cytokine receptor, which then stimulates a signaling cascade through the activation of STAT5. Thus, in patients with hyperprolactinemia, a shift in the immune system balance between proinflammatory and anti-inflammatory factors may manifest itself at the ovarian level as an ovulation disorder (e.g., resulting from abnormal rupture of the wall of the Graafian follicle). In this situation, attempts to influence PRLR expression/signaling via PRLRs in ovarian follicle tissue may prove promising [201,320]. Additionally, regardless of ovulation, the PRL/PRLR axis may play an oncomodulatory role and promote tumor aggressiveness in patients with ovarian cancer [321]. Thus, increased PRLR expression may provide a basis for the development of PRLR antagonists or PRLR-drug conjugates to improve the selective uptake of anticancer drugs [28,321].

Unlike intraovarian mechanisms, the regulatory effect of PRL on the HPG axis in females at the level of the CNS is much better understood.

As previously mentioned, ovulation disorders caused by hypoprolactinemia rarely occur in an isolated form because they are most often the result of damage to the entire glandular part of the pituitary gland, e.g., as a result of puerperal ischemia and necrosis (Sheehan's syndrome). Deficiencies in gonadotropins, such as FSH and LH (but not PRL or GnRH), are responsible for the clinical picture of secondary ovarian failure in these patients [268].

Ovulation disorders involving more or less severe symptoms of hypogonadotropic hypogonadism accompanied by hyperprolactinemia absolutely require diagnostic methods to exclude/confirm prolactinoma and side effects of the utilized drugs (iatrogenic causes) and, subsequently, less common causes of elevated PRL [246,322,323]. Prolactinomas are treated with surgery or DA agonists (e.g., bromocriptine, lisuride, quinagolide, and cabergoline) depending on the adenoma size, clinical factors and patient preference. In microadenomas, patient preference for observation or hormonal replacement therapy (HRT) can also be considered depending on menopausal and gonadal status [324]. It is estimated that treating hyperprolactinemia with DA agonists leads to the normalization of PRL levels and the return of ovulatory cycles in approximately 80% of patients. In treatment-resistant patients, the DA agonist is recommended to be changed [325]. In hyperprolactinemia induced by drugs that cannot be discontinued due to the underlying disease, DA administration may be pointless or even dangerous. Once a pituitary adenoma has been ruled out, the use of sex steroids (HRTs) is recommended in this situation (if necessary) to prevent osteoporosis [325,326].

Kisspeptin also helps to reverse the suppressive effects of hyperprolactinemia on GnRH neurons. It has been demonstrated that the administration of kisspeptin in hyperprolactinemia induces hypothalamic pulsatile GnRH secretion with subsequent pulsatile LH secretion from the adenohypophysis [327]. Clinical algorithms for kisspeptin treatment in ovulation disorders are constantly being developed due to the beneficial effect of this neuropeptide on follicle maturation, as assessed by maturity-related gene expression [328].

Finally, an important consideration in the clinical approach to treating hyperprolactinemia is macroprolactinemia. Macroprolactinemia, which is defined as the quantitative predominance of an isoform of a greater molecular weight than PRL known as macroprolactin (big-big PRL), is a common cause of hyperprolactinemia. According to various data, among patients with hyperprolactinemia, 10-46% were diagnosed with macroprolactinemia [55,56,329]. However, due to the fact that it is usually composed of a PRL monomer and an IgG molecule that has a prolonged clearance rate similar to that of immunoglobulins, macroprolactin is characterized by limited bioavailability (as confined to the vascular system) and much lower bioactivity than PRL. Therefore, the predominance of macroprolactin, which is the main molecular form of PRL in the serum of patients with a normal concentration of monomeric PRL, is associated with no symptoms or a mild course of hyperprolactinemia [52,55,222]. Due to the fact that macroprolactin interferes with many immunological assays that are commonly used for the detection of PRL, macroprolactinemia is a frequent cause of misdiagnosed hyperprolactinemia in clinical practice for the treatment of ovulation disorders [52,330]. Patients with undiagnosed macroprolactinemia may then be unnecessarily exposed to pituitary imaging and to futile treatment with anti-PRL drugs (DA agonists), to which they are resistant. Given the high prevalence of macroprolactinemia among women with elevated PRL levels and the difference in the management of patients with macroprolactinemia compared to true monomeric hyperprolactinemia, all patients with persistently elevated PRL levels, especially asymptomatic patients, should be screened with tests appropriate for the diagnosis of macroprolactinemia [52,331,332]. Due to the fact that the main cause of macroprolactinemia involves anti-PRL antibodies, the diagnosis of a patient with infertility and ovulation disorders should include accompanying autoimmune diseases [333].