Innovative approach in assessing the role of neurogenesis, angiogenesis, and lymphangiogenesis in the pathogenesis of external genital endometriosis.

Abstract Endometriosis is a chronic, progressive, relapsing estrogen-dependent disorder characterized by the growth of tissue structure and function similar to the endometrium outside the normal mucosa of the uterine cavity localization. Endometriosis is found in 10–15% of women in reproductive age and it is one of the main causes of pelvic pain syndrome and infertility. Mechanisms of the development of endometriosis and related pathological pain impulses are still poorly understood and therapeutic approaches do not always have a sufficient effect; in this connection, the study of the pathogenesis of endometriosis and endometriosis-associated pain currently is perspective. Identification of significant factors of angiogenesis, lymphangiogenesis, and neurogenesis in the external genital endometriosis will promote the development of non-invasive early diagnosis and pathogenetic therapy.


Introduction
Endometriosis is a chronic disease manifested by the presence of foci of tissue, similar to the endometrium of the uterus outside its cavity. Endometriosis remains the subject of controversy: discussions are under way about its etiology, pathogenesis, and treatment. It can cause chronic pelvic pain, dyspareunia, diskhezii, and infertility in women.
Now there is no one doubt that links between infertility and endometriosis.
According to the current literature, from 25% to 50% of women with endometriosis have infertility, and 30-50% of women with endometriosis are infertile [1]. According to Guo [2], the incidence of endometriosis in women with chronic pelvic pain syndrome is more than 33%. Meuleman et al. in 2009 evaluated the occurrence of endometriosis in infertile women with ovulatory cycles and a healthy partner. The frequency of endometriosis in this category of women was 47%, while in 63% of the cases detected I and II stages of the pathology as classified by the American Fertility Society 1996 [1].
One of the most common clinical symptoms of endometriosis is pelvic pain. The mechanisms associated with endometriosis pain are complex as the pathogenesis of the disease. It is well known that there is no clear correlation between the degrees of prevalence of endometriosis is observed, such as laparoscopy and the severity of pain symptoms [3]. However, the degree of the localization of rectovaginal endometriosis pain intensity is associated with the depth of the spread of endometriosis [4].
On one hand, observations suggest that endometriosis is found in the one-third of the cases of women undergone laparoscopy for chronic pelvic pain [5]. On the other hand, according to some investigators, asymptomatic endometriosis was found in 14% of women who have undergone surgical sterilization [6].
A serious problem of endometriosis is fairly early start of menstrual pain (an average age of patients with endometriosis is 25-30 years) and a significant time delay (7-11 years) between the detection of the disease and the diagnosis [6]. About 70% of adults who complained of dysmenorrhea were eventually diagnosed endometriosis, which can be the basis for a more careful examination of young patients with corresponding symptoms [7]. On an average, women consulted seven specialists before being diagnosed and begin treatment [6].
Making a true estimate of the incidence of endometrial disease is difficult, as the only reliable way to diagnose is currently endometriosis visualization during diagnostic laparoscopy and subsequent histological confirmation of the diagnosis.
Thus, endometriosis is a disease that affects the majority of women of reproductive age, negatively affecting their physical, psychological, and social condition that significantly reduces the quality of life in this category of patients [1,2].
Despite extensive research, mechanisms for the development of endometriosis and related pathological pain impulses are still poorly understood, and there are several theories about the origin of endometriosis.
The most well-explained theory seems to be first proposed in 1921 by Sampson, the theory of retrograde implantation of endometrial fragments of menstrual blood thrown into the abdominal cavity and its growth, with the formation of endometrioid heterotopias and their dissemination in the lymphatic and circulatory vasculature.
The processes of angiogenesis, lymphangiogenesis, and neurogenesis, providing formation and growth of blood and lymphatic vessels and nerve fibers, underlie the pathogenetic mechanisms of development of endometriotic lesions [8].
Neurogenesis, angiogenesis, and lymphangiogenesis are crucial process in the endometrium and in particular, endometriosis, because of their role in the healing of injuries, growth, and spread of tumors and the generation of pain. The mother angiogenesis, lymphangiogenesis, and neurons are vital to repair, regeneration, growth, and differentiation of the endometrium which are controlled by estrogen and progesterone. In contrast to angiogenesis and lymphangiogenesis, little is known about the mother of endometrial neurogenesis; however, it is extremely important in the generation of the value of endometriosis-associated pain [9].
Development of pain includes a number of mechanisms and the interactions between the peripheral and the central nervous system [9]. Studies indicate the existence of a direct innervation of endometrioid heterotopias [10], as well as for changes in the peripheral and the central nervous system in women with endometriosis-associated pain [11].

Angiogenesis
The processes of angiogenesis are the most important mechanisms involved in the growth and remodeling of the endometrium. Endometrium is defined as vessels exposed to cyclic endometrial growth and regression during the menstrual cycle, under the influence of estrogen and progesterone [9]. Angiogenesis is regulated by a large number of different molecules: promoters and inhibitors [12].
Endometrioid heterotopias synthesized a number of factors that stimulate angiogenesis. Among these factors, the most important are VEGF-A and its receptor (VEGFR-2), angiopoietins -1 and -2. According to some studies, the expression of VEGF-A and its receptor in endometriotic lesions is significantly higher compared with the endometrium [13], according to other studieswith intact peritoneum [14].
According to the authors, different types of endometrioid heterotopias may exhibit varying degrees of expression of proangiogenic factors. For example, red endometriotic lesions are more pronounced processes of vascularization and cell proliferative activity [15]. Data on the increase of the concentration of soluble VEGF-A in peritoneal fluid in women with endometriosis are available in the literature [13,16].
In the presence of estradiol, a potent angiogenic factor, activated immune and neuroendocrine cells also promote angiogenesis, which is secreted by a variety of pro-angiogenic and pro-inflammatory cytokines (Il-1, Il-6, and Il-8), tumor necrosis factor -alpha (TNF-a) [18], endometrial cells secrete matrix metalloproteinases-1 and -2. Adhesion occurs by the activation of cell adhesion molecule sICAM-1, oxidative stress, and inflammatory processes which are supported by activated immune cells [19].

Lymphangiogenesis
Relatively little is known about lymphangiogenesis, but a number of studies have noted an increase in the expression of endometriotic lesion factors stimulating lymphangiogenesis, such as VEGF-C and D [14], and in a significant degree in the deep infiltrating endometriosis lesions compared with heterotopias, localized in the peritoneum and ovaries [20]. It was found to increase in endometriotic lesions [21,22], peritoneal fluid [23], and other growth factors such as IGF-1 and IGF-2 in patients with deep infiltrating endometriosis, at the same time a correlation between their concentration and proliferative activity of endometrial heterotopias was observed [24]. A significant increase in the density of lymphatic vessels in the stroma of endometrial lesions was found.

Neurogenesis
According to studies, the processes are involved in the neurogenesis growth and the development of endometriotic lesions. Violations of the local inflammatory response, hormonal and local angiogenesis contribute to the growth of nerve fibers. The presence of nerve fibers in the functional area of endometrial lesions plays a key role in the formation and perception of pain impulses; however, the precise mechanisms of endometriosisassociated pain are still unclear.
The nervous tissue in the endometriotic lesions presented sensitive, adrenergic, and cholinergic fibers. These fibers are involved in carrying the pain which may be associated with the formation of pain associated with endometriosis. An increase in the density of nerve fibers of the pelvic peritoneum causes hyperinnervation. These nerve fibers become a potential abnormally increased functionality. Thus, not only increasing the number of nerve fibers but also processes for their excitation can be broken as a result of abnormal neurogenesis [25].
Studies were performed in animal models reflecting the mutual cross-influencing each other endometrial foci and eutopic endometrium of endometriosis [26]. Neurotrophins and their receptors and other molecules expressed neuronal activity in endometriotic lesions. Neurotrophins -family of regulatory proteins of the nervous tissue -are synthesized by neurons and surrounding cells, and promote the proliferation, differentiation, and maintenance of viability and function of the peripheral and central neurons. Neurotrophins act locally at the point of release and especially intensively induces branching of the dendrites (arborisation) and neurite outgrowth (spruting) towards target cells.
To date results indicate the important role of neurogenesis in the processing of a number of molecules, including nerve growth factor (NGF), neurotrophins 3, -4/5, growth factor isolated from the brain (BDNF), neurotrophins 1B cell stimulation factor-3 (NNT-1/BSF-3), and glial cell neurotrophic factors (GDNF). NGF, neurotrophin-3, and their receptors TrkA and p75 are detected in the endometrial glands and stroma of peritoneal and ovarian endometriosis lesions and with deep infiltrative endometriosis [25]. According to various authors, ovarian endometriosis detects the expression of BDNF, NT-3, NT-4/5, slit ligand, and Robo receptors [25].

Endometriosis-associated pain
The formation of pain impulses in the development of endometriosis is caused by an inflammatory response involving macrophages, increased secretion of estrogen, cytokines, growth and remodeling tissue factors, and neurotrophins that stimulate the growth and activation of sensitive, sympathetic, and parasympathetic nerve fibers in the centers and the formation of persistent pain [27]. S76 T. Sheveleva et al.

Changes in the endometrium of endometriosis
There is growing evidence that the endometrium in women with endometriosis is fundamentally different from the endometrium of healthy women, although routine histological study did not show significant differences. The combination of reducing apoptosis [28], depression immune surveillance [29], as well as the ability to increase the adhesion of the proteolytic activity [30], angiogenic potential proliferation [31], and estrogen production [32] in violation of the innervation determines the ability of this tissue to enhance the implantation and growth on the surface of the peritoneum and, ultimately, the formation, and the occurrence of endometriosis with corresponding symptoms.

Angiogenesis in the endometrium
A study comparing the endometrium of healthy women and women affected by endometriosis showed significant differences in the process of angiogenesis, manifested in the increase of expression of proangiogenic growth factors including vascular endothelial growth factor-A (VEGF-A)angiopoietins -1 and -2 as well as receptors for these molecules [13][14][15]24]. Thus, there was a reduction in the expression of factors that inhibit angiogenesis, such as thrombospondin and prokinetitsin [33].
These data suggest a higher angiogenic potential of endometrial tissue of women with endometriosis and expected to increase the ability to ectopic endometrial invasion and survival in the abdominal cavity [34].

Lymphangiogenesis in the endometrium
There exists a considerable overlap between the mechanisms that control angiogenesis and lymphangiogenesis, involving molecules promoters and inhibitors, such as angiopoietins, integrins, fibroblast growth factor, hepatocyte growth factor, vascular growth factors C and D (VEGF-C, VEGF-D), insulin-like growth factors [35].
Relatively little is studied about the natural inhibitors of lymphangiogenesis. It is proved that the inhibitory effect was exerted on him by vazogibin-1, interferon-alpha, beta transforming growth factor, semaphorin 3F, and neostatin. In addition to the increase of the angiogenesis activity, there is an evidence of changes in the local lymphangiogenesis in women with endometriosis: reduced expression of vascular growth factor C; increased expression of vascular growth factor receptor 2, semaphorin E, and matrix metalloproteinase 7 [36]. There are observations which describe the spread of ectopic endometrial tissue in the lymph vessels and lymph nodes outside the endometrial cavity of the uterus [37].

Neurogenesis in the endometrium
According to various authors, in women with endometriosis expression of neurotrophins and their receptors and other neuronal active in eutopic endometrium of the uterus was increased as compared with healthy women. Thus, the expression of NGF and its receptor TrkA and p75 [38], as well as the expression BNDF and NT-4 were significantly increased [39].
Other irregularities in the eutopic endometrium of endometriosis associated with neurogenesis included increase of neuroendocrine number and immune cells producing neurotrophins including T cells, B cells, macrophages, NK-cells, dendritic cells, mast cells [40].
In addition to the detection of increasing of the neurogenesis promoters, expression in eutopic endometrium of women with endometriosis were found in the functional layer small unmyelinated nerve fibers (sensory C fibers) that are not detected in healthy endometrium [25].

Treatment
Treatment for endometriosis has three main objectives: (a) the decrease in the intensity of pain; (b) preventing recurrence of the disease; (c) increase of the likelihood of pregnancy [24,40].
The treatment of endometriosis must use individual approachthe development of an optimum diagnostic and therapeutic individual program [40]. The goal of the treatment should be determined by the patient's main problems (elimination of pain symptoms and/or bleeding, restore fertility, or just to improve the quality of life).
The first stage to diagnose and eliminate the anatomical substrate of endometriosis is often carried out with medicaldiagnostic laparoscopy, which is the ''gold'' standard [41]. However, during the surgery, only visible endometriosis was removed and, in future, patients continue to suffer from pelvic pain. Laparoscopy should be performed only by a physician, who can, if necessary, carry out an adequate surgical treatment in full [42].

Prevention of relapse (for surgery)
According to the recommendations of the American Association of Reproductive Medicine (ASRM), endometriosis should be treated as a chronic disease that requires a long-term development plan for conducting patient with the maximum use of drug therapy in order to avoid repeated surgical interventions [43].
Most international recommendations for a first-line drug therapy for a long time attributed combine oral contraceptives (COCs) and progestins. Only in the case of unsuccessful outcome, their application is for 3 months. Recommended a second-line therapy including agonist of gonadotropin-releasing hormone (GnRHa) to ''return'' (''add-back'') treatment/therapy cover or left norgestrel-releasing system (Mirena), although not having formally registered indications for the treatment of endometriosis [41]. Treatment by other means, or a palliative (analgesics, nonsteroidal anti-inflammatory drugs -NSAIDs), causes a large number of side effects (danazol and gestrinone) or experimental (aromatase inhibitors, selective estrogen receptor modulators, (SERMs), agonists of estrogen receptor-b (ER b), antiangiogenic agents, etc.) [42].
The hormonal treatment of endometriosis should be guided by the basic principle that no drug can eliminate the morphological substrate of endometriosis, but only has an indirect effect on it, which explains the symptomatic and clinical benefit [42].
COCs are widely used to treat the symptoms of endometriosis, but judging according to research, the effect is negligible. A metaanalysis suggests that even though COCs suppress ovulation, which reduces the risk of endometriosis, estrogen component in their structure may actually stimulate the development of the disease [44].
Danazol is an androgenic steroid, which is sufficiently effective in the treatment of endometriosis. According to studies, more than 80% of the patients noted the disappearance or significant pain relief [45], but its use is limited by side effects.
GnRH agonists are considered the gold standard for the treatment of endometriosis because of their high efficiency for the relief of pain, but their use is accompanied by estrogen-deficiency symptoms: hot flashes, vaginal dryness, decreased libido. Unfortunately, their hopes for the agonist of gonadotropinreleasing hormone and antigonadotropiny fully justified [45].
A special attention is focused on medication dienogest, which is recommended as monotherapy for endometriosis in Europe, Japan, and others. It is a synthetic progestin oral progestogen with severe and moderate antigonadotropnym effects, but without androgen, glucocorticoid, and mineralocorticoid activity. The experiments on animals also showed that dienogest induces apoptosis of ovarian granulosa cells [46].
The use of aromatase inhibitors is a new promising method for the treatment of endometriosis. These drugs reduce the local synthesis of estrogen in the endometrial heterotopias and also inhibit the formation of estrogen in the ovaries, brain, and adipose tissue [47]. The systematic review of these studies has shown that aromatase inhibitors significantly pronounced a decrease in pain as compared with GnRH agonists [48]. It is important to know that aromatase inhibitors cause significant loss of bone density with prolonged use and cannot be appointed as a monotherapy in premenopausal women.
Currently, a large number of studies in animal models are aimed at understanding the mechanisms that suppress pathological angiogenesis, and neurogenesis limfangio-, as well as the development of drugs is aimed at key pathogenetic links of endometriosis. Drugs are divided into groups of exclusive preparations, the only known function is to inhibit angiogenesis, and inclusive preparations of angiogenic activity which is related to other functions. The first group includes funds from direct (endogenous and synthetic angiogenesis inhibitors: endostatin, Short synthetic endostatin peptides, Caplostatin, Lodamin) and indirect sources (Pro-angiogenic signaling blockers: Anti-VEGF-A antibodies, Soluble truncated VEGF-R1, VEGF-targeted gene therapy, Bevacizumab, 2-Methoxyestradio, SU5416, SU6668, Sorafenib). The second group can be formed based on hormones and functionally related substances: progesterone, dydrogesterone, dihydrodydrogesterone, dienogest, danazol, leuprolide acetate; dopamine agonists; antiinflammatory drugs; immunomodulators; statins; phytochemical agents and other compounds; PPAR ligands [49].
At the same time, the researchers have three main issues: the effectiveness of the treatment, the risk of drug resistance, and the possibility of side effects. The desired effect can be achieved by simultaneous inhibiting effects on several pro-angiogenic signaling pathway [50].
According to various authors, however, there is a risk of cumulative adverse effects by combining several drugs that inhibit angiogenesis [48][49][50]. Nevertheless, the concept of a combination of various inhibitors opens up new perspectives in the therapy of endometriosis.
Thus, treatment of endometriosis -medical and surgicalshould be seen as complementary therapies. Personalized selection of the optimal medical and surgical component improves the efficiency of treatment and improves the prognosis.

Conclusions
Endometriosis is a disease that causes a severe pain in women of reproductive age. It increases neurogenesis in eutopic endometrium and in endometriotic heterotopia, as shown by the expression of a number of neurotrophins, their receptors, and increase of the nerve fibers density as compared with control tissues. Increase in the nerve fibers density and increased expression of various neurotrophins play a significant role in the formation of endometriosis-associated pain.
It requires further study of the pathogenetic basis of angiogenesis, lymphangiogenesis, and neurogenesis, which will develop new and effective methods of diagnosis, prevention, and treatment of disease.
Thus, many researchers believe that the study of angiogenesis, lymphangiogenesis, and neurogenesis allow us to find an innovative approach to the diagnosis of endometriosis. Therapy for endometriosis that is found on the principle of the translational medicine will effectively fight disease, improve quality of life of patients, and restore the endocrine and reproductive functions.

Declaration of interest
The authors report that they have no conflicts of interest.