Prolactin and pain of endometriosis

As mentioned above, a substantial number of physiological processes from reproductive and endocrine functions, metabolism and angiogenesis to immune and stress responses are controlled by prolactin signaling. Numerous medical conditions are associated with either a deficiency or abnormally high serum prolactin levels (i.e., hypoprolactinemia or hyperprolactinemia, respectively). While some of these prolactin-related pathological conditions may indirectly result in pain, recent preclinical studies have identified a previously unrecognized direct role of prolactin signaling in female specific pain sensitization (reviewed in: Al-Karagholi, Kalatharan, Ghanizada, Dussor, & Ashina, 2023 ; Y. Chen, Navratilova, et al., 2020 ; M. J. Patil, Henry, & Akopian, 2014 ). Pain is a subjective experience composed of sensory, affective, and cognitive components. Pain in endometriosis can therefore result from processes involving activation of peripheral nociceptive pathways as well as dysfunction of the immune system, central sensitization, and alteration of pain processing in the brain. ( Lamvu et al., 2021 ; Masciullo et al., 2021 ; Morotti, Vincent, & Becker, 2017 ). Psychosocial, environmental, and cultural influences and pain education can also shape the perception of pain. We highlight the role of prolactin in sensitization of peripheral nociceptive pathways (i.e., peripheral sensitization) as an important factor underlying pain in endometriosis. Pain is typically initiated by activation of nociceptors, peripheral sensory neurons residing in the dorsal root (DRG) or trigeminal (TG) ganglia, that transmit noxious stimuli (i.e., high intensity stimuli that are capable of tissue damage) through the spinal cord to the brain. Tissue or nerve injury, inflammation or disease may sensitize nociceptors lowering their threshold for activation and amplifying the intensity of sensory inputs ( Hucho & Levine, 2007 ; Stein et al., 2009 ). This neuroplastic mechanism, called peripheral sensitization, leads to pain from normally innocuous stimulation (i.e., allodynia) and increased pain from painful stimulation (i.e., hyperalgesia). While peripheral sensitization is an evolutionarily beneficial mechanism that helps protect the injured part of the body and allows healing, long-term sensitization that remains unresolved even after recovery from injury, is maladaptive and may result in chronic pain. Many female-prevalent chronic pain conditions, including migraine ( Navratilova, Fillingim, & Porreca, 2021 ; Slatculescu & Chen, 2018 ), chronic pelvic pain and female-specific pain syndromes such as dysmenorrhea ( Brasil et al., 2020 ; Takeda, Tadakawa, Koga, Nagase, & Yaegashi, 2013 ) typically peak during reproductive age, are exacerbated during the menstrual cycle, and regress or disappear after menopause. These observations suggested involvement of female hormones in promoting pain, but direct and female selective sensitization of nociceptors was unexpected. The first study establishing sexually dimorphic role of prolactin in nociceptor sensitization in vivo used exogenous application of prolactin to rodent eyes and showed that while prolactin alone did not elicit eye-wiping pain behaviors, it potentiated eye-wiping responses to capsaicin, an agonist at the heat sensitive TRPV1 channel ( Diogenes et al., 2006 ). This study also showed that capsaicin-evoked excitability of cultured female sensory neurons was increased by pretreatment with prolactin. Subsequent studies demonstrated female selective sensitization to heat and mechanical stimulation following local injection of prolactin in the hindpaw ( M. Patil, Belugin, et al., 2019 ; Scotland et al., 2011 ). Similarly, prolactin injection onto the dura mater induced migrainelike behavior in naïve female mice, but much higher concentration was necessary to promote pain in males ( Avona et al., 2021 ; Y. Chen, Moutal, et al., 2020 ). The role of endogenous prolactin signaling in nociceptor sensitization was studied using D2 dopamine receptor agonists, cabergoline or bromocriptine, known to decrease serum prolactin levels. Alternative strategies manipulated prolactin signaling at the prolactin receptor using a selective PRLR antagonist (Δ1–9-G129R-hPRL), PRLR knock-out mice, or CRISPR/Cas-9 gene editing technology to reduce the expression of PRLR in specific regions of the nervous system. These studies provide evidence in rodents that prolactin system is recruited endogenously to sensitize female peripheral nociceptors in models of postoperative, inflammatory, and migraine-like pain as well as during opioid induced hyperalgesia ( Y. Chen, Moutal, et al., 2020 ; Ikegami et al., 2022 ; M. J. Patil, Green, et al., 2013 ; Scotland et al., 2011 ). Physiological or psychological stress is an important factor in many chronic pain conditions, including endometriosis and other pelvic pain syndromes, migraine, fibromyalgia, and others. Most of these conditions are more prevalent in women and stress is frequently cited as a triggering event associated with worsening of pain. Chronic or repeated stress represents a risk factor in promoting the transition to a chronic pathological pain state. Stress activates the hypothalamic pituitary adrenal (HPA) axis and is accompanied by a transitory increase in serum prolactin levels both in humans and in rodents. We and others have demonstrated that repeated exposure of mice to stress promotes long-term sensitization (priming) to normally subthreshold triggers that elicit pain only in primed but not naïve animals ( Mason, Kallianpur, Price, Akopian, & Dussor, 2022 ; Watanabe et al., 2022 ). This priming model is therefore relevant to increased pain vulnerability in patients with stress-related pain conditions, likely including endometriosis. Importantly, these findings demonstrate that repeated stress priming is mediated by dysregulation of prolactin receptor isoforms in peripheral nociceptors as described below. Previous work has shown different intracellular signaling of prolactin through different PRLR isoforms ( Belugin et al., 2013 ). Thus, in sensory neurons, signaling at the PRLR-L homodimer is associated with transcriptional changes while signaling at the PRLR-S homodimer is pronociceptive through mechanisms that include phosphorylation and sensitization of transducer channels including TRPV1, TRPA1 and TRPM8 ( M. J. Patil, Ruparel, et al., 2013 ). Importantly, when long and short isoforms join to form heterodimers, they silence each other's signaling ( Bentley & Wallis, 1987 ; Chiu, Koos, & Wise, 1992 ; Giss & Walker, 1985 ), suggesting that increased relative proportion of PRLR-S, either by upregulation of PRLR-S or by down-regulation of PRLR-L, could bias signaling towards the pronociceptive pathway ( Fig. 1 ). This concept was validated using the CRISPR/Cas-9 gene editing approach to decrease PRLR-L expression in mouse DRG neurons in vivo. Intrathecal injection of a CRISPR PRLR-L plasmid decreased PRLR-L expression in DRGs and resulted in mechanical hypersensitivity to normally innocuous hindpaw stimulation (allodynia) in naïve female but not male mice ( Y. Chen, Moutal, et al., 2020 ). Allodynia was then reversed either by decreasing serum prolactin levels with administration of cabergoline, or by CRISPR/Cas-9 deletion of the short PRLR isoform, confirming that pronociceptive effects were mediated by prolactin signaling at the PRLR-S. Subsequent studies demonstrated that down-regulation of PRLR-L and sensitization of peripheral DRG or TG neurons occurs physiologically following repeated stimulation of pituitary prolactin secretion by a variety of mechanisms including repeated stress, medications, or injury ( Y. Chen, Moutal, et al., 2020 ; Ikegami et al., 2022 ; Kopruszinski et al., 2022 ; Watanabe et al., 2022 ). Peripheral sensitization and dysregulation of PRLR isoforms persists for many days and appears to bestow enhanced vulnerability to pain in animal models of opioid induced hyperalgesia or in migraine priming models induced by repeated stress or repeated administration of migraine medications. Cumulatively, these preclinical studies demonstrate that high and repeated prolactin secretion can selectively sensitize female peripheral neurons in vivo by shifting the ratio of PRLR-L and PRLR-S expression in favor of signaling via the pronociceptive short isoform. Findings of co-occurrence of hyperprolactinemia in some women with endometriosis suggests that this mechanism could also promote pelvic pain and heightened vulnerability to comorbid pain conditions demonstrated in this disease. The prolactin connection to endometriosis would also suggest that targeting prolactin signaling at PRLR-S could prevent nociceptor sensitization and alleviate endometriosis related pain as well as reduce vulnerability to developing comorbid pain conditions.

Several findings in the literature support prolactin's involvement in the pathogenesis of endometriosis. For example, studies have shown that infertile women with endometriosis have elevated levels of prolactin or abnormal diurnal fluctuations of prolactin. Accordingly, prolactin-lowering drugs such as dopamine receptor D2 (D2R) agonists effectively reduced lesion burden in preclinical experiments in mice as well as in clinical studies in hyperprolactinemic women suffering from endometriosis. How common is the association of endometriosis with high prolactin levels, however, has not been comprehensively investigated. To determine this connection, we performed a comprehensive PubMed and EMBASE search of clinical studies reporting blood prolactin levels in patients with endometriosis. The search was conducted on February 9th 2023 using the following string of terms: (“prolactin“[All Fields] OR “PRL”[All Fields] OR “hyperprolactinemia”[All Fields]) AND “endometriosis”[All Fields]. We followed the reporting guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) ( Moher et al., 2009 ) ( Fig. 2 ). A total of 660 studies were identified in the two databases. After removal of 149 duplicates, 480 were found that fulfilled the search criteria. This set of articles was screened, and records were excluded if they had a nonexperimental methodology, involved only preclinical experiments, the study was not relevant to our question, or the full text of the article was not available. In the retrieved 56 articles the titles and abstracts were screened for eligibility as experimental clinical studies assessing the involvement of prolactin in endometriosis. The full texts of the 18 eligible articles were obtained (See Table 1 .). In all 18 selected clinical studies, serum or peritoneal prolactin levels were compared between endometriosis patients and non-endometriosis patients or healthy control groups. Ten studies were conducted in infertile women. Among them, four studies compared infertile endometriosis group versus infertile non-endometriosis group, three studies compared infertile endometriosis group versus fertile non-endometriosis group and three studies compared infertile endometriosis group, fertile endometriosis group and infertile non-endometriosis group. The remaining eight studies did not specify the fertility status. Three studies measured prolactin levels in peritoneal fluid. All reports except one found elevated serum prolactin level in the endometriosis group. Two of these studies showed positive correlation between the level of serum prolactin and the stage of endometriosis ( Kokot, Piwowar, Jedryka, Solkiewicz, & Kratz, 2021 ; Yarmolinskaya, Suslova, Tkachenko, Molotkov, & Kogan, 2020 ). Three studies reported a higher number of hyperprolactinemia patients in the endometriosis group compared to the control group ( Acien, Lloret, & Graells, 1989 ; Cunha-Filho et al., 2001 ; Muse, Wilson, & Jawad, 1982 ). Only one study showed no significant differences among infertile patients with endometriosis, infertile patients without endometriosis and fertile women ( Haney, Handwerger, & Weinberg, 1984 ). Overall, the literature review suggests that higher levels of serum prolactin might be associated with both the presence of endometriosis and its progression to a more severe stage. Three studies measured the levels of prolactin in the blood as well as in peritoneal fluid ( Haney et al., 1984 ; Lima, Moura, & Rosa e Silva, A. A., 2006 ; Yarmolinskaya et al., 2020 ). One of them showed no differences in prolactin levels in both serum and peritoneal fluid in infertile patients with or without endometriosis ( Haney et al., 1984 ). The other two studies demonstrated elevated blood prolactin levels of patients with endometriosis, but only one of them showed elevated prolactin in peritoneal fluid ( Yarmolinskaya et al., 2020 ). These patients also reported chronic pain due to endometriosis. The other study was conducted in infertile patients with endometriosis and did not find a significant difference in peritoneal prolactin between the endometriosis and the control groups ( Lima et al., 2006 ). This study also examined an association between stress and endometriosis by measuring cortisol and prolactin levels in serum, peritoneal and follicular fluid ( Lima et al., 2006 ). The results show elevated serum levels of both cortisol and prolactin in endometriosis patients compared to control subjects while there were no differences in follicular and peritoneal fluids, demonstrating that while stress increases pituitary prolactin, it does not appear to directly modulate follicular and peritoneal prolactin levels. This study concluded that stress might contribute to the development of endometriosis. Overall, the findings of this literature search provide strong evidence demonstrating the association between high levels of prolactin and endometriosis. However, the causal relationship and the source of elevated prolactin, cannot be determined from these investigations. The selected studies also do not provide any information on the role of prolactin in promoting endometriosis-induced pain. Mechanistic studies in preclinical models, analyses of tissue explants from patients with endometriosis and evaluations of responses of primary cell cultures from endometriotic lesions are necessary to increase our understanding of the pathology of the disease and the factors that promote endometriosis pain.

Human prolactin is a large polypeptide hormone encoded by the prolactin gene located on chromosome 6 ( Abramicheva & Smirnova, 2019 ). Mature polypeptide is formed by 199 amino acids that can undergo posttranslational modifications resulting in a molecular mass of approximately 23 kDa. Prolactin is known to perform many physiological functions in the body, primarily those relevant to female reproductive processes during pregnancy, mammary gland development and lactation ( Karayazi Atici, Govindrajan, Lopetegui-Gonzalez, & Shemanko, 2021 ; Phillipps, Yip, & Grattan, 2020 ). However many additional metabolic, endocrinologic and immune functions are modulated by prolactin including effects on water and salt balance, growth, development, immunoregulation and angiogenesis ( Carre & Binart, 2014 ; Montgomery, 2001 ; Ramos-Martinez, Ramos-Martinez, Molina-Salinas, Zepeda-Ruiz, & Cerbon, 2021 ). More recently, preclinical studies have found a sexually dimorphic role of prolactin in promoting pain sensitivity selectively in females ( Al-Karagholi, Kalatharan, Ghanizada, Dussor, & Ashina, 2023 ; Y. Chen et al., 2020; Y. Chen, Navratilova, Dodick, & Porreca, 2020 ; M. Patil et al., 2019). The main source of prolactin in the blood is its release from pituitary lactotrophs ( Phillipps et al., 2020 ). Additionally, prolactin can also be synthesized in extra-pituitary tissues such as the mammary gland, uterus, ovaries, immune cells, endothelial cells, and adipocytes ( Marano & Ben-Jonathan, 2014 ). Prolactin binds to prolactin receptors that are expressed broadly in multiple organs including the reproductive organs, the immune system, and the peripheral and central nervous systems, reflecting the wide range of prolactin's physiological functions (Al-Karagholi et al., 2023; Binart, Bachelot, & Bouilly, 2010 ). The primarily source of serum prolactin are lactotroph cells located in the anterior pituitary ( Phillipps et al., 2020 ). Blood prolactin levels fluctuate during the day, with increases observed shortly after the onset of sleep. During the menstrual cycle, blood prolactin levels increase slightly in the luteal phase due to the stimulatory influence of estrogen. In pregnant women serum prolactin levels rise gradually throughout pregnancy, reaching a maximum during childbirth, and remain high throughout lactation, when further spikes of prolactin can be triggered by suckling ( Phillipps et al., 2020 ). Transient prolactin surges can also be triggered by exercise and emotional or physical stress in all individuals, including men, although to a lesser extent. Pituitary prolactin secretion is under tonic inhibitory control by dopamine released from hypothalamic dopaminergic neurons including especially the tuberoinfundibulary dopamine (TIDA) neurons located in the arcuate nucleus ( Grattan & Kokay, 2008 ). Dopamine is released from these neurons into the pituitary portal circulation, from where it can reach dopamine D2 receptors expressed on pituitary lactotrophs to inhibit prolactin secretion. Dopamine is therefore the main prolactin inhibitory factor (PIF). Dopamine D2 receptor agonists, bromocriptine, quinagolide and cabergoline, have been used clinically to suppress high prolactin levels observed in various cases of hyperprolactinemia for example due to prolactinomas or other pituitary gland tumors ( Ghoreshi et al., 2022 ; Shemanko, 2016 ). Other molecules, referred to as prolactin releasing factors (PRF), include for example thyrotropinreleasing hormone (TRH), estrogen and vasoactive intestinal peptide (VIP), all of which have been shown to stimulate prolactin secretion ( Phillipps et al., 2020 ). Prolactin is also synthesized at multiple non-pituitary sites, including the uterus ( Eyal, Jomain, Kessler, Goffin, & Handwerger, 2007 ; Tanaka et al., 1996 ). Extra-pituitary prolactin (ePRL) is encoded by the same gene and is biologically indistinguishable from pituitary prolactin, however, it is transcribed from an alternative distal promoter ( Ben-Jonathan, LaPensee, & LaPensee, 2008 ; Gellersen, Kempf, Telgmann, & DiMattia, 1994 ). Mechanisms regulating the synthesis of ePRL are therefore different from those regulating pituitary prolactin and are usually cell or tissue specific. To limit the scope of this review, we will focus on ePRL sources that are relevant to endometriosis, particularly the potential release from endometrial stromal cells, immune cells and adipocytes that are common constituents of endometrial lesions. In healthy endometrium, prolactin synthesis is stimulated by progesterone and therefore follows similar temporal changes during the menstrual cycle. Endometrial prolactin synthesis increases in the mid secretory phase and reaches maximum in the late luteal phase, coinciding with decidualization of endometrial stromal cells ( Auriemma et al., 2020 ). Endometrial prolactin synthesis and release are under a different control from pituitary prolactin and its concentration does not correlate with serum prolactin levels ( Arie et al., 2000 ). Little is known about prolactin secretion from endometriotic lesions in patients with endometriosis. However, cultured stromal cells derived from peritoneal, ovarian, and deeply infiltrating endometriotic lesions were shown to retain the ability to synthesize prolactin and release it into the culture medium, although the concentrations were lower than in cultured stromal cells obtained from healthy endometrium ( Klemmt, Carver, Kennedy, Koninckx, & Mardon, 2006 ). Thus, endometriotic stromal cells themselves may produce prolactin that can act locally in an autocrine or paracrine fashion impacting the growth and development of the lesion. In addition to endometrial stromal cells, immune cells are also known to infiltrate the lesions and influence cellular functions in the local micro-environment by releasing a variety of pro- and antiinflammatory mediators and other substances. Prolactin has been shown to be produced by virtually all subtypes of immune cells ( Montgomery, 2001 ). Immune cells also express prolactin receptors, suggesting they could respond to both pituitary and local prolactin through endocrine, paracrine, and autocrine mechanisms. Prolactin appears to promote both inflammatory and anti-inflammatory functions depending on its concentration, exposure time and inflammatory environment by stimulating release of a variety of cytokines and chemokines ( Ramos-Martinez et al., 2021 ). Many autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, autoimmune thyroid disease, and type 1 diabetes are often associated with high concentrations of prolactin, demonstrating physiological importance of prolactin signaling on immune functions ( Borba, Zandman-Goddard, & Shoenfeld, 2019 ). Although the source of prolactin in autoimmune diseases remains unclear, an extra-pituitary production from lymphocytes has been suggested. Immune cells within the endometriotic lesions influence the pathology and symptomatology of the disease, therefore local prolactin secretion from these cells could contribute to pain sensitization (see below). Histological evaluations of endometrial lesions reveal the presence of white adipose tissue (WAT) within the lesions. Release of prolactin from human breast adipose tissue was initially found serendipitously in Dr. Ben-Jonathans laboratory ( Brandebourg, Hugo, & Ben-Jonathan, 2007 ; Zinger, McFarland, & Ben-Jonathan, 2003 ). Later, de novo synthesis and release were also confirmed in human subcutaneous and visceral adipose explants and in cultured adipocytes at different stages of adipogenesis ( Hugo, Borcherding, Gersin, Loftus, & Ben-Jonathan, 2008 ). As with other extra-pituitary prolactin sources, the transcriptional regulation of adipose-derived prolactin is provided by the distal, rather than pituitary, prolactin promoter. However, unlike prolactin secretion from endometrial and immune cells, synthesis and release from adipocytes is inhibited by dopamine and by D2 dopamine receptor agonist, bromocriptine, through inactivation of PKA/CREB response elements within the distal prolactin promoter ( Borcherding et al., 2011 ). Therefore, systemic administration of D2 dopamine receptor agonists inhibits not only pituitary prolactin release but may also suppresses extra-pituitary secretion from adipose tissue, while secretion from endometrial and immune cells in unaffected. These pharmacological mechanisms need to be considered when assessing the influence of pituitary and extra-pituitary prolactin on the pathology of endometriotic lesions. The physiological effects of prolactin are mediated by its binding and activation of prolactin receptor (PRLR) ( Ben-Jonathan & Hugo, 2015 ; Ramos-Martinez et al., 2021 ). PRLRs are present in almost all organs and tissues, including the peripheral and central nervous system (M. Patil et al., 2019). PRLR is a single transmembrane receptor that belongs to the class I cytokine receptor superfamily. The PRLR does not have intrinsic tyrosine kinase activity but can be phosphorylated by associated intracellular kinases, in particular the Janus kinases 2 (JAK2). Although the receptor is encoded by a single gene, alternative splicing produces distinct isoforms, which differ in the lengths of their intracellular domains ( Ben-Jonathan & Hugo, 2015 ). The most studied isoforms are the long (PRLR-L) and the short isoforms (PRLR-S), although intermediate isoforms are also present in humans and rats. The cytoplasmic domain contains two regions (box 1 and box 2) that are crucial for activation of intracellular signaling. While box 1 is present in all transmembrane PRLR isoforms, box 2 is not found in the short isoforms. Binding of prolactin to its receptor induces receptor dimerization and activation of JAK2 kinases which cross-phosphorylate tyrosine residues within box 2 of the intracellular domains ( Goffin, Bernichtein, Touraine, & Kelly, 2005 ). Phosphorylation of these tyrosine residues allows binding of proteins with the Src-homologous domain (SH2) including the transcription factors of the STAT family (signal transducer and activator of transcription), in particular STAT5 ( DaSilva et al., 1996 ). Once phosphorylated, STAT proteins form homo- or heterodimers and translocate to the nucleus where they regulate gene transcription involved among others in proliferation, differentiation, and cell survival. In addition to the JAK2/STAT5 pathway, prolactin can activate the MAPK/ERK1/2 (mitogen activated protein kinase/extracellular signal-regulated kinase 1/2) and PI3K/AKT (phosphoinositide 3-kinase/protein kinase B) pathways ( Abramicheva & Smirnova, 2019 ). Because PRLR-S lacks box 2, SH2 domain-containing proteins, such as STAT5, are unable to bind to this isoform. Therefore, after heterodimerization with the long isoform, PRLR-S prevents signaling through the JAK2/STAT5 pathway, inhibiting all down-stream functions. The detailed roles of prolactin signaling via the PRLR-S homodimer are not well characterized, however, preclinical studies in rodents suggest that signaling at the PRLR-S homodimers promotes sexually dimorphic sensitization of peripheral nociceptors ( Y. Chen, Moutal, et al., 2020 ; M. J. Patil, Green, Henry, & Akopian, 2013 ; M. J. Patil, Ruparel, Henry, & Akopian, 2013 ), providing a rationale for higher prevalence of pain in women.

Our comprehensive literature search demonstrates that high levels of prolactin have been repeatedly reported and implicated in the pathophysiology of endometriosis. Preclinical investigations provide a mechanism by which high prolactin levels, or possibly repeated surges of prolactin, can sensitize peripheral nociceptors and promote pain in women. These findings lead us to suggest that in women with endometriosis, high levels of circulating prolactin in combination with local prolactin secretion from endometriotic tissue can sensitize nociceptors that innervate endometriotic lesions to promote pelvic pain ( Fig. 1 ). In addition, nociceptors throughout the body may undergo priming from increased systemic prolactin, which could explain why endometriosis is often associated with comorbid pain conditions including, for example migraine. Reducing prolactin levels and prolactin signaling is therefore a strategy to inhibit nociceptor sensitization and decrease the likelihood of pain outbreaks. Dopamine D2 receptor agonists (bromocriptine, cabergoline, quinagolide) are used clinically to reduce release of prolactin from the pituitary. These medications, however, do not influence local prolactin secretion at the lesion. Despite this lack of effect on local prolactin, these drugs are effective in treating endometriosis in many patients, reducing not only pain ( Amit Kyal, Mukhopadhyay, & Mukhopadhyay, 2018 ) but also the size of the lesions ( Gomez et al., 2011 ). In a prospective randomized study involving 80 women with endometriosis receiving either a standard hormone therapy with medroxyprogesterone or a trial treatment with cabergoline, both groups reported decreased pain over a three month treatment period that continued for additional three months after treatment termination ( Amit Kyal et al., 2018 ). After a four month treatment with quinagolide, the sizes of endometrial lesions were reported to show either a reduction or complete disappearance of the lesions in most patients following laparoscopic evaluation. Another study compared the efficacy of cabergoline and luteinizing hormone releasing hormone (LHRH) agonist in decreasing the size of endometrioma ( Hamid, Madkour, Moawad, Elzaher, & Roberts, 2014 ). Approximately 65% of patients had significant decrease in endometrioma size after three months of cabergoline treatment, which was a higher percentage than with the LHRH agonist. While these effects could result from decreased circulating prolactin, it should be noted that dopaminergic agonists can also inhibit angiogenesis and produce analgesic effects that contribute to the observed clinical efficacy. Angiogenesis plays an important role in the development and maintenance of endometriotic lesions. VEGF is the key mediator of angiogenesis in normal tissue, but it is significantly upregulated in different types of benign and cancer tumors. Dopamine and dopamine receptor agonists reduce angiogenesis through endocytosis of the VEGF receptor-2 (VEGFR-2) and through upregulation of the anti-angiogenic pathways. D2 receptors are expressed in endometrium and in ectopic lesions from women with endometriosis. It was suggested that dopamine agonists may reduce the size of endometrial lesions in part by inhibiting angiogenesis. Indeed, in patients with hyperprolactinemia dues to endometriosis, reduction of the size of endometriosis lesions following quinagolide treatment was accompanied by a decreased concentration of VEGF within the lesions ( Gomez et al., 2011 ). Similarly, cabergoline treatment of women with endometriosis significantly decreased the serum levels of angiogenic factors, decreased the size of the endometriotic lesions and reduced pelvic pain reported by the patients ( DiVasta et al., 2021 ; Hamid et al., 2014 ). Dopamine receptors are also expressed in dorsal root (DRG) and trigeminal (TG) ganglion neurons, in the spinal cord and the brain. Therefore, dopamine agonists may exert direct effects on peripheral and central nervous system. However, in rodent DRG neurons, selective D2 agonists had no significant effect on nociception ( Chakraborty, Rebecchi, Kaczocha, & Puopolo, 2016 ). In contrast, intrathecally administered D2 receptor agonists were shown to elicit antinociception likely by engaging the dopaminergic descending pain modulatory pathways that project to the spinal cord from higher neural centers ( Barasi et al., 1987 ; Barasi & Duggal, 1985 ; Gao, Zhang, & Wu, 2001 ; Jensen & Yaksh, 1984 ; Liu, Qiao, & Dafny, 1992 ; Millan, 2002 ). Additionally, activation of striatal D2 receptors has been demonstrated to inhibit hypersensitivity in rodent models of neuropathic and inflammatory pain ( Ansah, Leite-Almeida, Wei, & Pertovaara, 2007 ; D. Sato et al., 2022 ). The involvement of striatal dopamine D2 receptors in regulation of pain was also demonstrated in humans using positron emission tomography (PET) imaging ( Hagelberg et al., 2004 ). Therefore, in addition to inhibiting prolactin release from the pituitary gland, dopamine D2 agonists may have direct inhibitory effect on angiogenesis and development of endometriotic lesions, as well as direct antinociceptive effects by engaging D2 receptors in nociceptive circuits. In clinical studies investigating the efficacy of dopamine D2 receptor agonists in endometriosis, there is typically a subset of women that do not respond to these medications ( Gomez et al., 2011 ; Hamid et al., 2014 ). These findings imply that lowering systemic prolactin levels may not be sufficient to treat endometriosis in patients where high local concentrations are produced within the lesion itself. In this regard, extra-pituitary sources of excess prolactin were suspected in several documented cases of women with gynecological diseases where hyperprolactinemia persisted following treatment with dopamine D2 receptor agonists. These dopamine agonist resistant hyperprolactinemia patients achieved normalization of prolactin levels only after hysterectomy, providing evidence that in some patients, an extra-pituitary source of prolactin may contribute to increased plasma levels ( Sachdev, Reyes, & Snyder, 2020 ; H. Sato et al., 2018 ; Sendur, Aktoz, Usubutun, Tuncer, & Erbas, 2019 ). Therefore, sequestering of prolactin from extra-pituitary sources would be clinically desirable.

Many pain conditions show a high female prevalence. Additionally, healthy women are more sensitive to experimental nociceptive stimulation than men ( Bartley & Fillingim, 2013 ; Fillingim, King, Ribeiro-Dasilva, Rahim-Williams, & Riley 3rd., 2009 ). These observations suggest the likely contribution of hormonal influences to nociceptor sensitivity. Moreover, stress is an important risk factor for the development of chronic pain conditions and can contribute to exacerbation of pain in patients with existing pain disorders ( Martin, 2016 ). Hormonal and behavioral responses to stress also differ considerably between men and women ( Bangasser & Wicks, 2017 ), again pointing to a likely hormonal impact on pain in women. The role of pituitary and gonadal hormones, including estrogen and testosterone, have been extensively studied for relevance to sex differences in pain ( Gupta, McCarson, Welch, & Berman, 2011 ), however, the mechanisms are complex and so far largely unknown. Recently a neuroendocrine link between stress-related hypothalamic circuits and female-selective activation of nociceptors by circulating prolactin and PRLR isoforms have been uncovered in rodent pain models ( Y. Chen, Moutal, et al., 2020 ; Y. Chen, Navratilova, et al., 2020 ; Watanabe et al., 2022 ). These findings demonstrate that repeated stress elicits prolactin release through engagement of hypothalamic kappa opioid receptors on hypothalamic dopaminergic cells. The resulting increase in circulating prolactin can down-regulate the PRLR-L isoform and “tune up” the excitability of female nociceptors. Establishment of this sensitized state, referred to as “latent sensitization”, results in reduced thresholds to sensory stimuli, increasing the vulnerability to pain from normally innocuous stimuli. In this review, we suggest that prolactin mediated sensitization of peripheral nociceptors may occur in women with endometriosis due to either increased systemic levels of prolactin or local secretion of prolactin from endometriotic lesions or both ( Fig. 1 ). Sensitization of neurons innervating the lesion could explain the presence of pelvic pain commonly experienced by women with endometriosis. In addition, high levels of serum prolactin may lead to sensitization of nociceptors throughout the body, increasing the vulnerability for developing comorbid pain conditions. It should be noted that clinical assessment of serum prolactin levels is not able to determine the source of the hormone. Prolactinoma, hypothyroidism, chronic kidney disease, and other medical conditions, or certain drugs can lead to elevated serum prolactin. Physical and psychological stress activates the hypothalamic pituitary axis resulting in release of stress hormones, including prolactin. Therefore, chronic stress could also contribute to increased levels or repeated surges of prolactin. Endometriotic lesions are another local source of extra-pituitary prolactin, which could provide high concentrations within the microenvironment of the lesion, and in some cases could also contribute to increased prolactin blood levels. While our systematic literature search of clinical studies clearly demonstrates an association of hyperprolactinemia and endometriosis, establishment of a causal relationship between prolactin levels and endometriosis was not possible. It is noteworthy, that although the vast majority of women experience retrograde menstruation, believed to initiate the formation of endometriotic lesions, a much smaller proportion of women will end up developing the disease suggesting the importance of other mechanisms. High serum prolactin levels may predispose women to developing endometriosis by providing favorable conditions for ectopic endometrial cells to survive and grow. On the other hand, high prolactin levels may be the consequence of the disease through increased local or pituitary prolactin secretion. Our emerging point of view is that high local concentration of prolactin at sensory nerve endings results in sensitization of peripheral nociceptors selectively in females through a shift in prolactin signaling to the pronociceptive PRLR short isoform. This mechanism is especially relevant in women with endometriosis or other gynecological conditions, where prolactin is secreted by pathological tissues. Dopamine D2 agonists reduce the contribution of systemic prolactin, but secretion of prolactin by the lesion is mainly unaffected. Antibodies against prolactin or PRLR could represent novel effective therapies for treatment of endometriosis and other pain conditions in women.

Endometriosis is a disease of the female reproductive system often associated with chronic pelvic pain and infertility ( Gruber & Mechsner, 2021 ). Endometriosis is estimated to affect approximately 10% of women of reproductive age ( Shafrir et al., 2018 ). However, the prevalence rises to 20% to 50% in infertile women ( Giudice & Kao, 2004 ) and to 70% in women with chronic pelvic pain ( Carter, 1994 ). It should be noted, that the overall prevalence, may be underestimated due to asymptomatic or misdiagnosed cases, because the symptoms of endometriosis are commonly attributed to other diseases with overlapping symptoms ( Horne, Daniels, Hummelshoj, Cox, & Cooper, 2019 ; Zondervan, Becker, & Missmer, 2020 ). The definitive diagnosis of endometriosis typically requires clinical and instrumental examinations performed by expert operators or laparoscopic visualization ( Becker, et al., 2022 ; Nnoaham, et al., 2011 ). Endometriosis can also be found in postmenopausal women, particularly in those on hormone replacement therapy ( Gemmell et al., 2017 ). Endometriosis is defined by the presence and growth of endometrium-like tissue outside of the uterus, found mainly in the pelvic area including the ovaries, ligaments, and peritoneal surfaces as well as the bowel and bladder. Endometriotic lesions are histologically similar to normal endometrium comprising primarily endometrial glands and endometrial stroma cells. Lesions are functionally capable of responding to hormonal stimuli and undergo cyclic changes like normal endometrium. Cyclic breakdown of ectopic endometrial tissue can promote a local inflammatory reaction and formation of adhesions and scar tissue. Fibrosis and hemosiderin laden macrophages are present in >70% of endometrial cases ( Clement, 2007 ). Endometriosis is classified into four stages (I-minimal, II-mild, III-moderate, and IV-severe) by the American Society for Reproductive Medicine (ASRM) according to surgical visualization of location, size, depth of the lesions and severity of adhesions. Minimal or mild endometriosis is characterized by superficial implants and mild adhesions. Moderate and severe endometriosis is characterized by deep implants with more severe adhesions. Clinical presentation of endometriosis varies among women. Many patients experience severe pelvic pain such as painful periods (dysmenorrhea), painful intercourse (dyspareunia), painful defecation (dyschezia) and painful urination (dysuria) ( Maddern, Grundy, Castro, & Brierley, 2020 ). Recent evidence suggests that endometriosis is more than a localized pelvic disease involving local inflammation and activation of peripheral nociceptive pathways. Endometriosis can lead to systemic inflammation as well as central sensitization, and alterations in the brain that cause increased pain aversiveness and mood and anxiety disorders, which are common manifestations observed in endometriosis patients ( L. C. Chen et al., 2016 ; Cuevas et al., 2012 ; Eriksen et al., 2008 ; Lamvu, Carrillo, Ouyang, & Rapkin, 2021 ; Low, Edelmann, & Sutton, 1993 ; Petrelluzzi, Garcia, Petta, Grassi-Kassisse, & Spadari-Bratfisch, 2008 ; Sepulcri Rde & do Amaral, 2009 ). The exact pathogenesis of endometriosis is not fully understood. However, there are several theories to explain the origin of endometriotic lesions, including retrograde menstruation, extrauterine-sourced stem cells, hematogenous or lymphatic spread, coelomic metaplasia, and Müllerian rest induction ( Bulun et al., 2019 ). The most widely accepted among these theories is retrograde menstruation, or reflux of menstrual debris containing viable endometrial cells through the fallopian tubes into the peritoneal cavity. This theory is supported by studies showing higher risk with early menarche, a short menstrual cycle and obstructed menstrual flow ( Shafrir et al., 2018 ; Smolarz, Szyllo, & Romanowicz, 2021 ). However, retrograde menstruation is a very common physiological process, occurring in >90% of menstruating women ( Halme, Hammond, Hulka, Raj, & Talbert, 1984 ). Hence other factors within the local environment likely determine the ability of endometrial cells to adhere to extrauterine tissues, proliferate, and develop into endometriotic lesions. These factors are thought to include hormonal influences, angiogenesis, neurogenesis, inflammation, presence of adipocytes and other factors ( Masciullo et al., 2021 ). Similar to the eutopic endometrium, the growth of ectopic tissue is thought to be regulated by ovarian steroid hormones such as estrogen and progesterone ( Marquardt, Kim, Shin, & Jeong, 2019 ). Estrogen promotes proliferation of epithelial cells in endometrium. Patients with endometriosis may have increased sensitivity to estrogen that fuels the growth of ectopic endometrium. Indeed, bioavailability of estradiol and the expression of estrogen receptor beta (ERβ) have been found to be elevated in ectopic endometriotic tissue. ERβ promotes the growth of endometriotic tissue by inhibiting apoptosis and enhancing cellular adhesion and proliferation. By contrast, progesterone inhibits estrogen-dependent epithelial proliferation, and acts as an anti-inflammatory agent. Dysregulation of the progesterone receptors or alteration of progesterone signaling pathways causes progesterone resistance in endometriotic lesions ( Vercellini, Vigano, Somigliana, & Fedele, 2014 ). This aberrant progesterone response facilitates an estrogen dominant, proinflammatory environment, that can result in a systemic inflammatory disease. Endometriotic lesions require an adequate blood supply to guarantee oxygen and essential nutrients for survival in their ectopic sites. Endometriotic lesions are typically characterized by a dense vascularization ( Chung & Han, 2022 ). Numerous blood vessels are observed around the active endometriotic lesions at laparoscopy and the implant itself is strongly vascularized upon histological examination ( Shafrir et al., 2018 ). Moreover, increased angiogenic activity has been demonstrated in peritoneal fluid from women with endometriosis and strong expression of vascular endothelial growth factor (VEGF) has been shown in active lesions ( Donnez, Smoes, Gillerot, Casanas-Roux, & Nisolle, 1998 ; McLaren, Prentice, Charnock-Jones, & Smith, 1996 ; Rein et al., 2010 ). These findings show that angiogenesis likely plays a significant role in endometrial growth. Accumulating evidence indicates that neurogenic processes are involved in endometriotic lesion development and maintenance ( Velho, Taube, Sehouli, & Mechsner, 2021 ). Sensory, sympathetic, and parasympathetic nerves have been confirmed in peritoneal lesions, and with significantly higher expression than in normal peritoneum ( Tokushige, Markham, Russell, & Fraser, 2006 ; Wang et al., 2009 ). In addition, endometriotic lesions have the ability to attract their own nerve supply when invading surrounding tissues by secreting neurotrophic factors (NTFs) that promote intralesional neural sprouting ( Mechsner et al., 2007 ). This enriched innervation may play a key role in hypersensitivity to touch or other stimuli as well as ongoing pain through lowering the sensory thresholds of nociceptors ( Mechsner et al., 2007 ). Clinical studies have shown that patients with higher density of nerve fibers within the endometriotic lesions report to have more severe pain ( McKinnon, Bersinger, Wotzkow, & Mueller, 2012 ; Mechsner et al., 2009 ). It is presumed that these nerve fibers play a role in the generation of chronic pelvic pain, but the exact mechanisms have yet to be delineated. Inflammation and altered immunity are essential features for endometriotic lesion survival. Ectopic tissue elicits a localized immune and inflammatory response with the production of cytokines, leukocytes, chemokines, and prostaglandins ( Abramiuk et al., 2022 ; Galandrini et al., 2008 ; Machairiotis, Vasilakaki, & Thomakos, 2021 ). This proinflammatory microenvironment in endometriotic lesions promotes their proliferation, vascularization, and activation of nociceptors ( Laux-Biehlmann, d'Hooghe, & Zollner, 2015 ). The population of macrophages is significantly elevated in peritoneal lesions of patients with endometriosis and dysfunction of macrophages plays an important role in endometriotic lesion growth and inflammation. Macrophages in peritoneal lesions show decreased phagocytic capacity, and increased activation of proinflammatory cytokines and angiogenetic factors ( Chuang, Wu, Shoji, & Tsai, 2009 ; Lousse et al., 2008 ). Moreover, local natural-killer-cell cytotoxic activity is impaired in women with endometriosis, which may contribute to implantation of endometrial cells in an ectopic area and enhanced progression and survival of the lesions ( Sciezynska, Komorowski, Soszynska, & Malejczyk, 2019 ). Adipose tissue, body fat, is also commonly found in endometriotic lesions ( Kubo et al., 2021 ; Kyuragi et al., 2017 ; Malysheva, Kopteva, & Krylova, 2020 ). Adipose tissue is composed of adipocytes which store body fat to produce energy and generate heat. It also plays an active role in pathological conditions such as inflammatory diseases and cancer. Inflammation of adipose tissue is a pathophysiological feature of many gynecological diseases. Indeed, fatty acid-binding protein 4 (FABP4), primarily expressed in adipocytes and macrophages, acts at the interface of metabolic and inflammatory pathways, and was found to be significantly higher in patients with endometriosis compared to control subjects ( Kubo et al., 2021 ). Inflammation of adipose tissue is related to hyperprolactinemia. Adipose tissue expresses prolactin receptors, and also secrets prolactin ( Ben-Jonathan & Hugo, 2015 ). This extra-pituitary prolactin is modulated by autocrine/paracrine mechanisms which do not depend on the same control factors involved in the regulation of prolactin secretion from the pituitary gland. During adipose inflammation, release of prolactin is increased ( Bouckenooghe et al., 2014 ). Recently, evidence has begun to accumulate suggesting pathogenic role of prolactin in gynecological diseases ( Auriemma et al., 2020 ). This evidence also implies possible involvement of prolactin release from adipose tissue in the development of endometriotic lesion.