Patent Application
20200371112
The invention relates to a method for determining the presence of a tissue injury and repair (TIAR) process in the uterus of a subject. The presence of TIAR is employed as a marker for the presence of an early or preliminary stage and/or increased risk of developing a medical disorder associated with abnormal formation of endometrial tissue. Such medical conditions are preferably benign, such as an archimetrosis, preferably endometriosis or adenomyosis. The method comprises determining a level of CXCL12 and/or CXCR4 in a sample from a subject.
Endometriosis is a non-malignant disease that affects many women predominantly during the reproductive period of life. With the cardinal symptoms, such as pelvic pain, bleeding disorders, and infertility, the disease has a tremendous impact on women's health.
Laparoscopy and the histological examination of the suspicious tissue constitute the standard procedure for the diagnosis of peritoneal endometriosis. It is often refrained from this invasive diagnostic method if not considered necessary, such as in a final sterility work-up and in situations of extreme complaints. In any case, the diagnosis is often delayed. Recent research utilizing imaging techniques, such as magnetic resonance imaging (MRI) and high resolution transvaginal sonography (TVS) has elucidated that pelvic endometriosis is significantly associated with uterine adenomyosis (Hricak et al., 1983; Reinhold et al. 1998; Leyendecker et al., 1998; Kunz et al., 2000; Leyendecker et al., 2000; Kunz et al., 2005; Leyendecker et al., 2009; Leyendecker et al., 2015; Van den Bosch et al., 2015) These imaging techniques could serve as non-invasive methods to identify uterine adenomyosis and thereby, indirectly, the probable presence of pelvic endometriosis in these women. However, as soon as the diagnosis of uterine adenomyosis can be based on these methods a considerable and irreversible destruction of the reproductive function of the utero-tubal system may have already been taken place.
Recent results from carefully taken histories of women with endometrioses revealed that the majority of them suffered from primary dysmenorrhea (Chapron et al., 2011; Leyendecker et al., 2015). There are, however, only scant data available with respect to the prevalence of the development of endometriosis and adenomyosis in a population of women with primary amenorrhea (Burnett et al., 2005). Thus, a simple, specific and non-invasive diagnostic test would help to identify these women at risk. Therefore, it is an urgent desideratum for research to establish a method that allows the diagnosis of the disease in a very early phase of development.
Uterine adenomyosis is caused by auto-traumatisation of the non-pregnant uterus by its genuine mechanical functions during the menstrual cycle (Leyendecker et al., 1998, 2009, 2015). It has also been described following iatrogenic trauma (Meyer, 1930; Kindermann, 1988). Moreover, some girls develop endometriosis and adenomyosis in the late puberty before menarche (Marsh and Laufer, 2005; Ebert et al., 2009; Janssen et al., 2013).
The term ‘Tissue Injury and Repair’ (TIAR) was coined to characterize the pathophysiological process that is operative in the processes of acute and chronic injury. It involves the local production and paracrine action of estradiol (Leyendecker et al., 2009) that, as shown in animal experiments, in turn results in an increased tissue expression of stromal cell-derived factor 1 (SDF 1), also known as the chemokine CXCL12 at the site of the lesion. By the binding to the corresponding receptor, CXCR4 that is expressed on mesenchymal stem cells (MSC), CXCL12 causes an increased attraction of MSC to the wound (Bouzaffour et al., 2009; Du and Taylor, 2997; Hufnagel et al., 2015).
The TIAR-process and the CXCL12/CXCR4 interaction are non-organ specific phenomena. They play essential roles in organogenesis and tissue regeneration, in inflammation and in wound healing as well as in benign and malignant proliferative processes (Dotan et al., 2010; Teicher and Fricker, 2010; Boudot et al., 2011; Lander et al., 2012).
Yet, as will be delineated below, these molecular processes can now be utilized as a novel tool for the early diagnosis of archimetrosis, such as uterine adenomyosis and peritoneal endometriosis in women that are at a high risk to acquire this disease entity.
Chemokines have been suggested as biomarkers for detecting endometriosis (Borrelli et al, “Can chemokines be used as biomarkers for endometriosis? A systematic review”, Human Reproduction, vol. 29, no. 2, 2013, 253-266), with a focus on CXCL8, CCL2 and CCL5. No mention is made of CXCL12 being used in this context. CXCR4 expression has been determined in endometriotic tissue using immunohistochemistry but is also expressed in surrounding healthy tissue (Van Den Berg et al: “Analysis of biomarker expression in severe endometriosis and determination of possibilities for targeted intraoperative imaging”, International Journal of Gynecology and Obstetrics, vol. 121, no. 1, 2013, pages 35-40).
CXCL12 has been suggested as a biomarker for endometriosis (WO 2016/011377, US 2016/017426), as has CXCR4 (WO 2016/094409), although no suggestion has been provided in the art to date that CXCL12/CXCR4 could be employed for determination of a TIAR process in the uterus, or in the prognosis or detection of the early stage of a medical disorder associated with abnormal formation of endometrial tissue, such as an archimetrosis. The present invention seeks to provide means for detecting the molecular and physiological determinants or markers prior to, or in an early stage of, an archimetrosis, thereby addressing a unique patient population, who are undergoing symptoms that require molecular clarification to support further diagnostic and therapeutic intervention.
Malgorzata Walentowicz-Sadlecka et al (“Stromal derived factor-1 (Sdf-1) and its receptors CXCR4 and CXCR7 in endometrial cancer patients”; PLOS ONE, vol. 9, no. 1, 2014, 84629) and WO 2013/017566 describe CXCR4 as a marker for endometrial cancer. No mention is made of this marker being used in determining an early stage of an archimetrosis.
As such, the field of reproductive health is in significant need of markers that may be employed as early as possible in order to identify pathogenic processes in the early stages of a developing archimetrosis before extensive endometriosis develops.
In light of the prior art the technical problem underlying the present invention is to provide alternative and/or improved means for determining an early stage of abnormal formation of endometrial tissue and/or for determining subjects at risk of developing a medical disorder associated with abnormal formation of endometrial tissue.
This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.
The invention therefore relates to a method for determining the presence of a tissue injury and repair (TIAR) process in the uterus of a subject, comprising:
a) providing a sample of said patient, and
b) determining a level of CXCL12 and/or CXCR4 in said sample,
c) wherein the level of CXCL12 and/or CXCR4 correlates with the presence of tissue injury and repair (TIAR) processes in the uterus of a subject.
The invention therefore relates further to a method for determining the presence of a tissue injury and repair (TIAR) process in the uterus of a subject as a marker for the presence of a preliminary stage or increased risk of developing a medical disorder associated with abnormal formation of endometrial tissue, comprising:
a) providing a sample of said patient, and
b) determining a level of CXCL12 and/or CXCR4 in said sample,
c) wherein the level of CXCL12 and/or CXCR4 correlates with the presence of tissue injury and repair (TIAR) processes in the uterus of a subject and/or the presence of a preliminary stage or increased risk of developing a medical disorder associated with abnormal formation of endometrial tissue.
The invention therefore relates further to a method for determining an early or preliminary stage of, and/or increased risk of developing, a benign proliferative disorder associated with abnormal formation of endometrial tissue, selected preferably from adenomyosis or endometriosis, comprising
a) providing a sample of said patient, and
b) determining a level of CXCL12 and/or CXCR4 in said sample,
c) wherein the level of CXCL12 and/or CXCR4 correlates with the presence of an early or preliminary stage of, and/or increased risk of developing, said disorder.
As described in more detail below, the tissue injury and repair (TIAR) process is a non-organ specific biological phenomenon that is employed as a marker for the early stage or increased risk of developing abnormal formation of endometrial tissue. It was a surprising aspect of the invention that determination of CXCL12 or its receptor CXCR4 could be used to determine the TIAR process in a subject at an early stage of or prior to abnormal formation of endometrial tissue.
Although CXCL12 has been suggested as a biomarker for endometriosis (WO 2016/011377), no suggestion has been provided in the art to date that CXCL12/CXCR4 could be employed for determination of a TIAR process in the prognosis or detection of the early stage of a medical disorder associated with abnormal formation of endometrial tissue. The present invention enables the analysis to be conducted at time points earlier than were previously thought possible, thereby representing an improvement over the prior art.
The present invention therefore represents a novel and surprising solution to the problem of providing molecular markers for a preliminary stage or an increased risk of developing a medical disorder associated with abnormal formation of endometrial tissue.
The present invention therefore relates to a method for the prognosis of a medical disorder associated with abnormal formation of endometrial tissue.
The present invention therefore relates to a method for the diagnosis of an early or preliminary stage of a medical disorder associated with abnormal formation of endometrial tissue.
In some embodiments, the medical disorder associated with abnormal formation of endometrial tissue is not cancer, and in particular is not endometrial cancer. In some embodiments, the medical disorder associated with abnormal formation of endometrial tissue is a benign (non-malignant) proliferative disease. According to the present invention, medical disorder associated with abnormal formation of endometrial tissue are those defined herein as archimetrosis, most preferably endometriosis or adenomyosis.
Endometriosis (archimetrosis) is a benign proliferative disease associated with an increased accumulation of archimetral (endometrial) stem cells (eg progenitor cells or mesenchymal stem cells, MSC) (a) in the subbasal stroma of the eutopic endometrium and (b) in the stroma of ectopic sites of endometriotic growth. The increased attachment of stem cells represents an imitation of archimetric embryology, but represents a misguided healing process after acute and/or chronic mechanical trauma. This injury initially occurs either as auto-traumatization by one's own mechanical activity of the non-gravid uterus or as iatrogenic trauma on the level of the eutopic endometrial stroma (endometrial-myometrial junction). This chronic mechanical irritation maintains a local traumatization which leads to infiltrating processes. Auto-traumatization of the uterus and the attachment of stem cells requires the functional effects of estradiol, which is mediated via the estradiol receptor alpha (ER-alpha) and the estradiol receptor beta (ER-beta; activation of CXCL12).
In the meaning of the present invention, an “early” or “preliminary stage” of a medical disorder associated with abnormal formation of endometrial tissue is characterized by a uterine auto-traumatization.
Such a traumatization can be determined via increased levels of bone marrow stem cells being recruited to the uterus in comparison to healthy controls.
In some embodiments, an “early” or “preliminary stage” of a medical disorder associated with abnormal formation of endometrial tissue may also be defined by the presence of endometrial tissue growth outside the uterus, but before cysts (endometriomas) occur, or while cysts are still small.
An auto-traumatization in an early stage of disease can also be determined by increased levels of estradiol, or the ER-alpha or ER-beta receptors, in the uterus of subjects compared to healthy controls.
The early or preliminary stage of such a disease can also be defined by increased levels of peristaltic activity in the uterus, without necessarily having pathologic endometriotic growth. The early or preliminary stage of such a disease can also be defined, preferably in young women, such as 18 or below, such as preferably of an age of 12, 13, 14, 15, 16 or 17, which exhibit a primary dysmenorrhea, and show an early stage of initial endometriotic growth.
The method may therefore be carried out on samples from subjects who exhibit early indications of an oncoming endometriosis, as may be defined, for example, by the presence of a local “micro-traumatization” of a part of the endometrium, wherein the damage or traumatization may in some embodiments be localized to a sub-region of the uterus, or to a sub-group of cells of the endometrium.
In some embodiments, such additional parameters may be measured with CXCL12 and/or CXCR4 in combination with the method of the present invention, or such measurements may be replaced by the method of the present invention. Such parameters for the early or preliminary phase may also be used as a definition of the patient group, without limitation to necessarily having tested the patients of the method for these parameters.
Conducting an assay for CXCL12 and/or CXCR4 levels at such an early stage is advantageous, as it enables subsequent treatment before the disease has been established.
According to preferred embodiments of the invention, the patient or patient group to be analyzed represents a novel aspect of the invention. For example, the analysis of patients prior to developing endometriosis or other diseases associated with abnormal formation of endometrial tissue represents a preferred embodiment of the invention, which has not previously been suggested in the prior art.
In one embodiment, the recruitment of bone marrow-derived stem cells, preferably mesenchymal stem cells, is evident in the uterus of said subject. MSCs can be detected according to the markers described in more detail below. The MSC recruitment is one characteristic of the TIAR process that indicates the likely onset of a medical disorder associated with abnormal formation of endometrial tissue.
In one embodiment of the invention, the subject exhibits symptoms of dysmenorrhea. Symptoms of dysmenorrhea include but are not limited to painful periods, menstrual cramps, or pain during menstruation. Dysmenorrhea, typically occurs around the time that menstruation begins and symptoms typically last less than three days. The pain is usually in the pelvis or lower abdomen. Other symptoms may include back pain, diarrhea, or nausea.
Carrying out the method of the present invention in women with symptoms of dysmenorrhea is a surprising and beneficial aspect of the invention. The present invention enables this patient group to be assessed for risk of an endometriosis or adenomyosis at a very early stage, before other symptoms occur, or before fully developed disease occurs.
In one embodiment, the medical disorder associated with abnormal formation of endometrial tissue is endometriosis.
In one embodiment, the medical disorder associated with abnormal formation of endometrial tissue is adenomyosis.
The above two medical conditions are defined primarily by the abnormal formation of benign endometrial tissue. Endometriosis is a condition in which the tissue that normally lines the uterus grows outside the uterus. Its primary symptoms include pain and infertility. Most often pain is associated with the ovaries, fallopian tubes, and tissue around the uterus and ovaries, but can occur elsewhere. Adenomyosis is a medical condition characterized by the abnormal presence of endometrial tissue (the inner lining of the uterus) within the myometrium (the thick, muscular layer of the uterus). In contrast, when endometrial tissue is present entirely outside the uterus, it represents a similar but distinct medical condition called endometriosis. The two conditions are found together but may also occur independently.
In one embodiment of the invention, the level of CXCL12 and/or CXCR4 positively correlates with the presence of a preliminary stage or increased risk of developing a medical disorder associated with abnormal formation of endometrial tissue. The positive correlation refers to an increasing risk or likelihood of having or developing said disease with increasing levels of said markers. In this context a cut-off or threshold level may be employed, or comparisons may be made to reference levels of healthy subjects.
In one embodiment of the invention, the sample comprises or consists of blood. In one embodiment of the invention, the sample comprises or consists of menstrual fluid. In one embodiment of the invention, the sample comprises a cervical test specimen or cervical test smear.
In a preferred embodiment, the sample is a menstrual fluid sample and said levels of CXCL12 and/or CXCR4 are elevated in said menstrual blood sample. In some embodiments, the levels of CXCL12 and/or CXCR4 are elevated in comparison to a peripheral blood sample, or to levels obtained from a sample of a healthy control subject.
In one embodiment of the invention, the determination of an increased risk of developing a medical disorder associated with abnormal formation of endometrial tissue is conducted prior to the occurrence of endometriosis and/or adenomyosis in said subject. This represents a particularly preferred embodiment of the invention, in which the prognosis or risk assessment can be carried out, and represents an entirely novel method compared to previous methods described in the art.
In further embodiments of the invention, the subject has been menstruating for a period of 3 years or less, preferably 2 years or less, and is preferably of an age of 12, 13, 14, 15, 16 or 17. The method may therefore be characterized by the patient group to be analyzed. It is particularly beneficial to conduct the method in subjects of a relatively young age who have not been menstruating for long periods of time.
The present method therefore also enables early treatment of the disease, thereby preventing establishment of the disease and its severe effects, such as infertility. Treatments may include but are not limited to progesterone or progestins (which counteract estrogen and inhibit the growth of the endometrium), avoiding products with xenoestrogens (which have a similar effect to naturally produced estrogen and can increase growth of the endometrium), hormone contraception therapy (such as in the form of oral contraceptives), treatment with danazol (danocrine) or gestrinone (which are suppressive steroids with some androgenic activity) or gonadotropin-releasing hormone (GnRH) agonists (which may decrease hormone levels) with or without estrogen add-back.
These treatment modalities were based on the concept of endometriosis constituting an estrogen dependent disease, however, without having taken the mechanical functions of the non-gravid uterus into consideration. The estradiol dependence certainly also holds true for the new concept of tissue injury and repair (TIAR) described herein, because the traumatizing functions, such as uterine peristalsis and neometral menstrual contractions, are essentially estradiol driven. The uncovering of the TIAR-concept and its relationship to CXCL12, now enables alternative and novel treatment modalities that are directed against the uterine hypercontractility.
As such, the present invention is associated with the additional advantage of enabling a new line of therapeutic approaches directly targeting uterine peristalsis and neometral menstrual contractions in the treatment and/or prevention of medical conditions associated with the abnormal formation of endometrial tissue.
Such treatments encompass medical interference with the hypothalamic-pituitary-ovarian axis, resulting for example in low-grade hypothalamic amenorrhea with lowered but still sufficient peripheral estradiol levels that still allow for normal tissue regeneration, such as on the level of the mucosa of the genital tract and the bone.
Another option relates to the interference with the oxytocin-oxytocin-receptor (OT/OTR) system that is directly controlling the uterine contractility. For example, approaches may be employed based on contraction suppression, for example such as Atosiban, which is an inhibitor of the hormones oxytocin and vasopressin. It is used as an intravenous medication as a labour repressant (tocolytic) to halt premature labor. Other Tocolytics (also called anti-contraction medications or labor suppressants), such as Terbutaline (Brethine), Ritodrine (Yutopar), Salbutamol (INN) or albuterol, Hexoprenaline (Gynipral) or Nifedipine (Procardia, Adalat), are alternative potential medications used to suppress contractions, that may directly target a pathological TIAR-related uterine peristalsis and neometral menstrual contractions. Painful contractions of the uterine muscle (similar to labor pains) are triggered by increased endometrial synthesis of prostaglandins, which appear in elevated amounts in the plasma and menstrual fluid of women with dysmenorrhea. Non-steroidal anti-inflammatory drugs, which have been used for years in arthritis, are effective prostaglandin inhibitors. Therefore, prostaglandin inhibitors may relieve dysmenorrhea in the majority of cases and lead to the treatment and/or prevention of medical conditions associated with the abnormal formation of endometrial tissue.
In some embodiments, the invention relates to a method for the treatment of medical disorder associated with abnormal formation of endometrial tissue comprising the method described herein in addition to subsequent administration of one or more therapeutic agents to a subject in need thereof.
Further preferred and non-limiting embodiments of the invention are provided in the detailed description of the invention below.
Historical Overview
With the beginning of the clinical and scientific interest in adenomyosis and endometriosis (Von Rokitansky, 1860) many theories have been presented to provide an understanding of the pathogenesis and pathophysiology of this enigmatic disease entity. The initial Wolffian duct theory put forward by von Recklinghausen had, already shortly after its formulation (Von Recklinghausen, 1896), to be replaced by the Müllerian duct theory that is considered valid until today (Cullen, 1896; Kossmann 1897). While many clinicians and scientists considered adenomyosis and endometriosis as a disease entity, particularly based on the Cullen's publications (Cullen, 1896; 1903; 1908; 1920), the focus of interest shifted to its peritoneal variety. In a contribution to a textbook Robert Meyer (1930) stated, in contrast to Cullen's findings (1920), that uterine adenomyosis is rarely associated with peritoneal endometriosis. In a large review the prevalence of endometriosis in adenomyosis ranged from about 10 to 80% (Emge, 1962). Most of the reported data were probably based upon casual findings during surgery and not upon data collected in a specific study focusing on that issue. In any case, peritoneal endometriosis was increasingly considered as a separate disease entity. This view was re-enforced by Robert Meyer's theory of coelomic metaplasia (Meyer 1919) that was later on extended by proposing a ‘secondary Müllerian system’. Robert Meyer did not defend his theory after a seemingly better one was proposed.
This was the theory of J. A. Sampson. Initially believing that peritoneal endometriosis would result from the rupture of ovarian endometrioma, (Sampson, 1922) he later proposed that peritoneal endometriosis is primarily due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. (Sampson, 1927). There is no doubt, as can be derived from his own arguments in support of his theory, that he in fact was convinced that during normal menstrual bleeding (“menstrual reaction”) vital endometrial cells would be disseminated into the peritoneal cavity. He observed this “menstrual reaction” in studying the bleeding from a recto-vaginal endometriotic lesion, of which he erroneously thought to be identical with that during normal menstrual desquamation. His theory was considered as plausible and, although its validity was doubted by some authorities in that field (Counseller, 1938; Philipp and Huber; 1939), his terminology was accepted. Without doubt, the theory of transtubal transplantation, meaning the transtubal route, is valid until today.
Sampson admitted that many of his patients with peritoneal endometriosis also presented with uterine adenomyoma. However, he did not consider a pathophysiological share between these lesions. He believed that uterine adenomyosis is caused by lymphatic transmission thus ignoring Cullen's work. Furthermore, his understanding of peritoneal endometriosis being composed of only endometrial epithelium and stroma, a view that is erroneously shared by many scientists up to today, was in contrast to the histological description of peritoneal adenomyoma (Cullen, 1920) irrespective whether they constitute superficial or deeply infiltrating lesions (Anaf et al., 2000; Leyendecker et al., 2002, Barcena de Arellano et al, 2011). Moreover, he did not take into account that the menstrual bleeding in women with endometriosis might differ from that of normal women. His theory of retrograde menstruation to cause peritoneal endometriosis is continued to be quoted in scientific work. It appears, however, that this refers more to the transtubal transplantation of endometrial tissue with the menstrual bleeding constituting merely the vehicle for this transport into the peritoneal cavity, rather than to a normal menstruation with transtubal reflux of the blood. In any way, definition and terminology often lack precision as already mentioned by Robert Meyer (1930). Moreover, premenarcheal peritoneal endometriosis (Marsh and Laufer, 2005; Ebert et al, 2009; Janssen et al., 2013) suggests that other factors than just bleeding might at least in addition also be responsible for the transtubal transport of endometrial fragments into the peritoneal cavity.
Sampson's theory had a strong impact on the understanding of endometriosis that was later-on re-enforced by laparoscopy (Batt, 2011). Peritoneal endometriosis and uterine adenomyosis were considered distinct disease entities without any pathophysiological shares (Parazzini et al., 1997). In the German literature, however, until today, uterine adenomyosis was still considered as a part of the disease process (Philipp and Huber, 1839; Kindermann, 1988). The basis of this understanding and the reconciliation of the divergent views was provided by Ridley's apodictic and, in fact, unproved view that ‘endometrium, where ever located, is inclined to invade subjacent tissue’ (Ridley, 1968). Thus, peritoneal endometriosis and uterine adenomyosis could co-exist in parallel. Ronald Batt (2016; personal communication) characterized the situation that he had realized during his medical work in the US: “With respect to endometriosis, in research, teaching and clinical management adenomyosis did not play any role”. Uterine adenomyosis that had initially addressed the interest to this disease entity fell into oblivion (Benagiano and Brosens, 2006).
During the last five decades, the clinical and scientific interest in endometriosis rose substantially. This is certainly owed to new methods available in the clinical management of the disease, such as laparoscopy and imaging techniques. The recognition of this disease, however, increased considerably in consequence of the dramatic change in the reproductive behavior of the society during this time. While in parous women the disease was often not diagnosed, with the postponing of child bearing endometriosis emerged as a disease of young non-parous women with an enormous impact on their lives.
Following the demonstration that transtubal ‘retrograde’ bleeding probably constitutes a normal phenomenon (Blumenkrantz et al., 1981; Halme et al., 1984) but only about 15% of young women acquire peritoneal endometriosis further theories have been proposed that were still based on the concept of retrograde menstruation, such as menstrual outflow obstruction, early menarche resulting in an increased number of menstruations and menstrual cycle irregularities (Mahmood and Templeton, 1990).
Newer concepts were increasingly based on laboratory data, such as those obtained in histological and immunological studies and in molecular biology. It was proposed that peritoneal endometriosis constitutes three different disease entities with ovarian endometriosis resulting from retrograde menstruation, superficial peritoneal endometriosis from metaplasia and deeply infiltrating recto-vaginal endometriosis constituting Müllerian remnants (Nisolle and Donnez, 1997). Brosens and Brosens (2000) distinguished superficial from deeply infiltrating endometriosis. Superficial endometriosis would arise from retrograde menstruation and the deeply infiltrating variety would constitute adenomyosis.
Cellular and molecular phenomena known from immunological diseases suggested that immunological defects on the level of the peritoneum were responsible for the growth of endometrial tissue on peritoneal surfaces (Halme et al, 1984; Bartosik et al., 1984; Leiva et al., 1994; Han et al, 2015). Peritoneal endometriotic lesions result from auto-transplantation of endometrial fragments and thus should, primarily, not cause immunological reactions. The immunological phenomena observed may, therefore, not be directed against the transplanted tissue but may rather constitute a reaction towards the cyclic changes of the implanted tissue, such as bleeding and cellular debris resulting from cyclic phenomena and cannot be externalized. It is therefore not justified to consider endometriosis primarily as an immunological disease.
In maintaining the view of retrograde menstruation, it was proposed, on the basis of results obtained in molecular biology, that peritoneal endometriosis resulted from the desquamation of intrinsically or epigenetically altered endometrium (Aghajanova et al., 2009; Bulun, 2009). These alterations in molecular biology observed, both, on the level of the endometrium and on the endometriotic lesions were of importance in the further understanding of the disease process. These views, however, did not take structural components in the disease process into consideration, such as the topography of the lesions within the uterine cavity as well as the zonal layers of the endometrium involved. This also holds true for recent publications, in that findings in biopsies taken from eutopic endometrium of affected women are considered as representative for the whole endometrium. Moreover, no causal relationship of uterine (auto)-traumatisation to the development of adenomyosis as the primary lesion is considered (Sakr et al., 2014; Wang et al., 2015; Plucino and Taylor, 2016; Moridi et al., 2017).
During the last two decades, the view had been advanced that peritoneal endometriosis developed primarily on the level of the uterus (Leyendecker et al., 1998, 2009). It can, without doubt, be considered as a paradigmatic change (Alabiso et al., 2016), first, to relate the development of the disease to basic processes of wound healing and, second, that the injury is caused by essentially physiological mechanical functions of the non-pregnant uterus (Leyendecker et al, 1998, 2009, 2015). For the understanding of such processes, however, the normal morphology of the non-pregnant uterus and its physiological mechanical functions had first to be elucidated and defined.
Uterine Morphology and the Mechanical Functions of the Non-Pregnant Uterus
The uterus was long considered to be a quiescent organ that becomes mechanically active only during the expulsion of the conceptus. With the advent of high resolution transvaginal ultrasound (TVS), hysterosalpingoscintigraphy (HSSG) and measurement of intrauterine pressure it became apparent that the uterus is a mechanically very active organ throughout the menstrual cycle. These functions consist of the archimyometrial peristaltic activity for directed sperm transport as well as of the neometral contractions for the discharge of the menstrual debris at the end of a non-conception cycle (Leyendecker et al., 2012; 2015).
The uterine corpus is composed of two organs, the neometra and the archimetra, that differ from each other with respect to embryology, structure and functions during the reproductive process (Werth and Grusdew, 1898; Leyendecker et al., 1998; Leyendecker et al. 199; Noe et al. 1999) (
The archimetra is phylogenetically and ontologically the oldest uterine structure. It is derived from the Müllerian ducts and it composed of the glandular endometrium and the glandular as well as the sub basal stroma, also termed the endometrial-myometrial junction, and the stratum subvasculare of the uterus as the muscular layer. This primordial uterus develops early during pregnancy. The archimetra constitutes the adult representation of the primordial uterus (Werth and Grusdew, 1898; Leyendecker et al., 1999; Noe et al., 1999).
The neometra is phylogenetically a younger uterine structure (Noe et al, 1999) and develops late during pregnancy, sometimes even after birth (Werth and Grusdew, 1898). The neometra is composed of two muscular layers, the stratum supravasculare with a longitudinal direction of muscular fibers and the stratum vasculare of the myometrium with a three-dimensional network of short muscular bundles. The neometra is of non-Müllerian origin. The two muscular layers develop from the connective tissue of the peritoneal serosa and the connective tissue of the vessels of the uterine stratum vasculare for example present in the rodents, respectively.
Because the stratum subvasculare is the ontological oldest muscular structure of the uterus, Werth and Grusdew (1898) coined the denomination archimyometrium. This prompted us to suggest the terms Archimetra and Neometra for the Müllerian and non-Müllerian parts of the human uterus, respectively (Leyendecker et a., 1998; Leyendecker et al., 1999, Noe at al., 1999) (
The archimetra is, more than the neometra, characterized by morphological and functional changes during the menstrual cycle. These changes pertain to all structures of the archimetra, such as the glandular epithelium and the stroma of the endometrium, the endometrial-myometrial junction as well as the archimyometrium.
Endometrium
A tripartite or quadripartite horizontal zonation exists in the human endometrium and in that of menstruating subhuman primates. The endometrial zones are microenvironments that differ by position, ultra structural differentiation and mitotic activity during the cycle (Kaiserman-Abramof and Padykula, 1987; Padykula et al., 1989). During the secretory phase of the rhesus menstrual cycle the functionalis and the spongiosa are characterised by progesterone induced mitotic inhibition, while the basalis not only escapes from that inhibition but rather exhibits increasing mitotic activity towards the end of the secretory phase (Padykula et al., 1989). It is reasonable to assume that these data are also pertinent to the human endometrium. Our data show that, in the human, proliferative phase ER and PR expression persist, though each at different levels, in the epithelium and/or the stroma of the basalis during the whole secretory phase even with an increase of the ER and PR expression during the late secretory phase. In the functionalis and in the spongiosa, however, the immunoreactive scores (IRS) of ER and PR expression decline progressively towards the end of the cycle. Thus, the basalis appears to constitute a highly vital endometrial compartment throughout the menstrual cycle, while most of the other layers are destined to cell death concomitant with the late luteal progesterone decline (Padykula et al., 1989; Leyendecker et al., 2002) (
The blood supply of the endometrium is pertinent to its cyclic changes and function (Okkels and Engle, 1938; Bartelmez, 1957; Ramsey, 1989; Rogers, 1996) (
It is of interest to note that only a single radial artery branching off from the arcuate artery provides, as a terminal vessel, the blood supply for a circumscribed segment that covers on the luminal surface an area of around 4-9 mm2. There is no horizontal communication of the vascular systems between such neighbouring segments (Rogers, 1996). Therefore, such a segment extending from the archimyometrium to the luminal surface and supplied by only one radial artery could be denominated the Hoxa-10-regulated archimetra micro-unit (AMU) (
In healthy women the level of desquamation is localized within the spongiosa and probably controlled by MMPs that are up-regulated following ischemia of the functionalis in consequence of the compression of the spiral artery by the process of endometrial shrinking (Rogers, 1996) (
Due to the special blood supply that is derived from the radial arteries the lower spongiosa and all of the basalis escape menstrual desquamation. In addition, the estradiol receptors are not fully down regulated by progesterone. In contrast, the immunoreactive scores (IRS) increase after a short postovulatory decline to nearly late proliferative phase levels at the end of the luteal phase and during menstruation. Thus, the basalis constitutes, during menstruation and the beginning of the secretory phase, a highly vital tissue (Leyendecker et al., 2002) (Figure. 4).
The regeneration (proliferation) of the functionalis after menstruation does not arise from bone marrow derived mesenchymal stem cells, although such cells are certainly involved in the normal and continuous process of endometrial tissue regeneration. In all of the menstrual basalis the epithelial cells are stained positive for estradiol receptor (ER.) (
Endometrial-Myometrial Junction
This thin layer of the archimetra is interposed between the endometrium and the archimyometrium and essentially constitutes the sub basal endometrial stroma. At the myometrial interface of the endometrial-myometrial junction cyclical metaplastic changes take place in that the stroma cells develop into fibro-muscular and muscular cells under the influence of progesterone and back into stromal cells during the proliferative phase of the cycle (Fujii et al., 1989).
The sub basal endometrial stroma and the stroma in the apical region of the endometrial glands are homing the MSC (Ibrahim et al., 2015). They are attracted via the vascular system to these sites because of the expression of CXCL12 in the glandular epithelium and they are the sources of the highly estrogen dependent continuous process of tissue regeneration (Ibrahim et al., 2015; Gargett et al., 2016).
Archimyometrium
The archimyometrium develops from the Müllerian mesenchyme early during ontology (Werth and Grusdew, 1898). Data from TVS and HSSG have shown that the archimyometrium serves directed sperm transport from the external cervical os into the isthmical part of the tube on the side of the dominant ovarian structure, the mature follicle bearing the egg, particularly during the late and immediate preovulatory phase of the menstrual cycle (Birnholz, 1984; Oike et al., 1988; Abramovicz and Archer, 1990; Devries et al., 1990; Lyons et al., 1991; Kunz et al., 1996). This function, the peristaltic activity of the archimyometrium, is under the control of the main ovarian steroids, estradiol and progesterone (Kunz et al., 1998a) (
In the sub human primate and in the human the two Müllerian ducts fuse early during embryonic development to form the unpaired uterus. The circular fibers of the archimyometrium separate on the mid-uterine level and continue on both sides as the circular muscular fibers of the uterine cornua and of the tubes. This separation forms, in the upper part of the uterus, a fundo-cornual-raphe that constitutes the gross morphological basis of directed sperm transport (Leyendecker et al., 1998; Leyendecker, 2000) (
Neometra
The muscular layers of the neometra, particularly the stratum vasculare, subserve the expulsion of the conceptus and in non-conception cycles, during menstruation, the discharge of the menstrual debris. During an ovulatory cycle the oxytocin receptors (OTR) accumulate in the stratum vasculare of the myometrium. Their formation is stimulated by follicular estrogen and by the subsequent increase of luteal progesterone (Maggi et al., 1992). Following the decline of progesterone in blood the OTRs are activated presumably by endometrial oxytocin (OT) (Zingg et al., 1995). A gradient of OTR concentration along the longitudinal uterine axis with highest density of the OTR in the fundal part of the uterus (Fuchs et al., 1998) ensures the orthograde discharge of menstrual debris and at the same time these contractions may occlude the intramural part of the tube, thus presumably minimizing the efflux of menstrual debris into the peritoneal cavity. The muscular mass of the neometra that is highest in the fundal region of the uterus (Wetzstein, 1965; Schwalm and Dubrauszky, 1966) and the increased concentration of OTR strongly suggests that the power of the neometral contractions during menstruation is highest in the fundal region of the uterus.
Evidence for a Uterine Role in the Disease Process
On the uterine level significant alterations are observed in women with endometriosis that pertain to the molecular biology of the endometrium and to morphological and functional alterations and dysfunctions of the muscular layers, respectively.
In the past two decades evidence was accumulating that the eutopic endometrium of women affected with endometriosis shares cellular and biochemical alterations with endometriotic lesions that are not or at least to a lesser extent found in the eutopic endometrium of healthy women. The endometrium of women with endometriosis is to a higher extent colonized with macrophages than that of women without the disease (Takebayashi et al., 2015). Furthermore, it was realized that, equipped with the COX2-enzyme and the P450 aromatase, the endometriotic lesions as well as the eutopic endometrium of affected women is, in contrast to healthy women, capable of producing estradiol from cholesterole (Fazleabas et al., 2003; Attar et al., 2009) In fact, it was shown that in women with endometriosis the concentration of estradiol in menstrual blood is higher than in peripheral blood. This difference does not exist in healthy women (Takahashi et a., 1989). Estradiol is produced by endometriotic and adenomyotic lesions and acts locally in a paracrine fashion. Recently, it was demonstrated that the chemotactic cytokine CRCI12 is significantly up-regulated in the epithelium of endometriotic lesions as well as in the eutopic endometrium of these women (Hufnagel et al., 2015).
There are several lines of evidence for the notion that dysfunctions of the uterus play a crucial role in the pathophysiology of endometriosis that may be summarized as follows:
1. Fragments of basal endometrium were found in the menstrual effluent with a higher prevalence in women with endometriosis than in controls. On the basis of these and other findings it was suggested that pelvic endometriosis results from the transtubal dislocation of fragments of basal endometrium (Leyendecker et al., 2002).
2. There is a significant association of pelvic endometriosis with uterine adenomyosis in women and in the baboon with life-long infertility (Barrier et al., 2004; Kunz et al 2005; Li and Guo, 2014; Leyendecker et al., 2015). In women, the reported prevalence, however, differs according to the study population chosen and to the criteria applied to the interpretation of MRI findings (
3. The uterine function of rapid and directed sperm transport into the ‘dominant tube’ is dysfunctional in women with endometriosis and is characterized by hyper- and dysperistalsis (Leyendecker et al, 1996) (
4. In comparison to normal controls and in contrast to peripheral blood estradiol levels are elevated in menstrual blood of women with endometriosis and adenomyosis (Takahashi et al., 1989).
5. The expression of the P450 aromatase is increased in adenomyotic tissue and in the ectopic and eutopic endometrium of women with endometriosis (Yamamato et al., 1993; Kitiwaki et al., 1997; Review: Leyendecker and Wildt, 2011).
3. Highly estrogen-dependent genes, such as VEGF and Cyr61, are up-regulated in eutopic endometrium of women with endometriosis and also in ectopic lesions as well as in experimental endometriosis (Absenger et al., 2005; Gashaw et al., 2006).
5. The peristaltic activity of the subendometrial myometrium can be dramatically increased by elevated peripheral levels of estradiol as they are observed during controlled ovarian hyperstimulation. The intensity of uterine peristaltic activity in women with endometriosis resembles that of women during controlled ovarian hyperstimulation although the peripheral estradiol levels are within the normal range (Leyendecker et al., 1998) (Figure. 16).
6. There is an increased intra uterine pressure in these women (Mäkäräinen, 1988; Bulletti et al., 2002). Histories taken of these women reveal a high prevalence of primary dysmenorrhea (Leyendecker et al., 2015). Primary dysmenorrhea is caused by an abnormally increased strength of the neometral contractions during menstruation (Dawood, 2006) (
Mechanism of Disease
Uterine Auto-Traumatisation
During the whole reproductive period of a woman's life, inevitably, the uterus is subjected to chronic mechanical strain due to its genuine mechanical functions, such as uterine peristalsis for directed sperm transport into the tube ipsilateral to the dominant follicle and due to rhythmic neometral contractions during menstruation for the externalization of endometrial debris. This results in chronic trauma. In autopsy, in about more than 60% of women uterine adenomyosis can be demonstrated. Following Sampson's theory, particularly under the influence of the American Society of Reproductive Medicine (ASRM) (American Fertility Society, 1985) and the European Society of Human Reproduction and Embryology (ESHRE) (Kennedy et al., 2005), it was long maintained that premenopausal uterine adenomyosis and peritoneal endometriosis constitute two different disease entities. A careful analysis of the available literature suggests that throughout the reproductive period of life uterine adenomyosis of perimenopausal and younger women is significantly associated with pelvic endometriosis (Moen and Muus, 1991; Muus, 1991; Kunz et al., 2005; Leyendecker et al., 2015)
The mechanical strain to the uterus is considerably increased in women who acquire disease early in their reproductive life. This is indicated by the fact that the intrauterine pressure during menstruation and the peristaltic activity are significantly enhanced over controls (Mäkäräinen, 1988; Salamanca and Beltran, 1995; Bulett et al., 2002). The intrauterine pressure exerted by neometral contractions during menstruation may exceed the blood pressure of arterioles not only during the contractions themselves but also between single contractions resulting in localized ischemia within archimetral tissue in addition to mechanical injury (
The peristaltic activity is increased with the doubling of the cervico-fundal peristaltic waves per minute. This pertains particularly to the early and mid-follicular phases of the menstrual cycle (
The archimyometrial peristaltic activities attain, like the neometral contractile activity, their strongest power physiologically in the more fundal part of the uterine corpus. In HSSG, it can be shown that the labeled macrospheres are injected deeply into the tubes and also into the peritoneal cavity in women affected from the disease (
Thus, in women with an exaggeration of both of these mechanical functions a massive mechanical strain is imposed on the more fundal part of the uterus chronically. This concurs with the finding that uterine adenomyosis primarily and predominantly develops in the more fundal part of the uterus (
The TIAR-mechanism results on the levels of the lesions in an increased tissue concentration of estradiol (Leyendecker et al., 2009). Because both mechanical functions of the uterus are controlled by ovarian estradiol, these exaggerated functions are further re-enforced by the paracrine action of the locally produced estradiol.
Because archimyometrial hyperperistalsis and neometral hypercontractility display various grades of severity it is justified to assume that, with respect to the exaggeration of the mechanical functions (and possibly also with respect to the susceptibility to mechanical strain), we are not dealing with a defined disease category completely separated from normal but rather with a ‘pathophysiological continuum’ ranging from mild to severe dysfunctions, such as in hypothalamic ovarian insufficiency (Leyendecker and Wildt, 1983). The development of premenopausal adenomyosis along a time axis in nearly all women supports this view. The disease development appears to be dependent on strength and chronicity of the mechanical injury. This renders the disclosure of a genetic background of adenomyosis in young women extremely difficult, in spite of clearly existing clinical and historical hints that suggest a hereditary component of endometriosis (Simpson et al., 1980; Malinak et al., 1980; Hadfield et al., 1997; Montgomery et al, 2008). Interestingly, dysmenorrhea and its severity are associated with increased contractility and over-expression of oxytocin receptors in women with symptomatic adenomyosis, which, however, could also be a sequel of local production and paracrine action of estradiol in adenomyosis (Guo et al., 2013).
Archimyometrial Hyperperistalsis Versus Neometral Hypercontractility and Compression of the Archimetra as the Primary Causative Factors
In young women affected by adenomyosis both mechanical functions of the non-pregnant uterus are intensified.
With a broadened ‘junctional zone’, In MRI early signs of the development of uterine adenomyosis can be identified close to the sagittal midline of the upper part of the uterus. This is the region of the lundo-cornual raphe, where the Müllerian ducts have fused and where, in directed sperm transport, hyperperistalsis may impose increased mechanical strain on stromal cells and myofibroblasts. This preponderance of the lesions near the uterine midline can still be observed in more advanced stages of uterine adenomyosis (
Further support for a role of hyperperistalsis in the development of adenomyosis comes from the finding of endometriosis in premenarcheal girls (Marsh and Laufer, 2005; Ebert et al., 2009; Janssen et al., 2013). With increasing ovarian function during puberty as demonstrated by rising estradiol levels in blood and the beginning growth of follicles up to the pre-ovulatory size (Peters, 1977) increased cervico-fundal peristalsis might abrade and transport cells and fragments of endometrium into the peritoneal cavity, where they might implant and cause endometriotic lesions
Adenomyosis does also develop under conditions without a fundo-cornual raphe, such as congenital malformations (Hansen et al., 2006; Su et al., 2005) suggesting that an additional mechanism inducing strain on the archimetra is operative (
Thus, the available data support the view that, both, archimyometrial hyperperistalsis and neometral compression are to a variable degree involved in the process of uterine auto-traumatisation.
The Microscopical Level (Microscopy)
The variable interaction of both hyper-activated mechanical functions may be demonstrated by the morphology of uterine adenomyotic lesions as they are frequently described in literature. Cullen (1903) pointed out that it was often difficult to demonstrate the Müllerian origin of the lesions in that they resulted from the proliferation of endometrial glands into the depth of the myometrium. Multiple microscopic sections had to be examined with scrutiny in order to demonstrate the glandular continuity between the lesions and the epithelial surface of the endometrium (Cullen, 1903; Otto. 1957) The lesions exhibited a cauliflower-like appearance with the stalk representing the primary proliferation from the apical zone of endometrial glands into the archimyometrium and the heads representing the bulk of the adenomyotic lesion extending in various directions within the myometrial wall. Cullen refrained from providing an explanation for this type of proliferation. On the basis of the knowledge accumulated now, however, a plausible explanation of the growth pattern of such a lesion may be attempted:
The primary archimetral injury may occur focally on the stromal level of several adjacent archimetral micro-units. The area affected by the trauma may be small and can be estimated by the size of the defect of the ‘junctional zone’ in TVS and MRI and, as can be seen under the microscope, by the amount of proliferating glandular systems each representing an archimetral micro-unit with apical glandular growth. Because there is no vascular communication between the units, the proliferative growth of the glands that depends on the increased attraction of MSC follows the blood supply provided by the radial artery of each archimetral micro-unit. With growing into the archimyometrium with hyperperistalsis of this muscular layer further mechanical strain, although this may also constitute the primary mechanical stress, is imposed on the proliferating lesions. With entering into the stratum vasculare of the neometra, in addition to further mechanical strain resulting from the neometral contractions during menstruation, a new vascular system is tapped allowing the proliferation of the lesion in various directions (Cullen, 1908; Goodall, 1944) (
Molecular Biology
Estradiol Receptor ßeta (ER-Beta); Chemokines and Mesenchymal Stem Cells (MSC)
ER-ßeta, chemokines and bone marrow derived mesenchymal stem cells are the main components in the disease process. Following identification of these genes and cellular elements many scientific efforts have been undertaken, to disclose their physiological roles and those in various disease processes.
With the identification of ER-ß after that of ER-alpha the dual existence of estradiol receptors, their distribution in various tissues, their physiological roles and their possible interaction are only incompletely understood (Hapangama et al., 2015). Both receptors have been shown to be expressed in many tissues of the body (Taylor and Al-Azzawi, 2000). With respect to the theme here for discussion it might be generalized that the preponderant role of ER-alpha constitutes for example in the correct functioning of the reproductive system, such as the hypothalamic-pituitary-ovarian axis and the utero-tubal complex (Franceschini et al. 2006), while ER-ßeta, concurring with the strong morphogenetic role of estradiol, is operative in embryonal morphogenesis, tissue regeneration and wound healing. In the basal endometrium, for example in patients with endometriosis and adenomyosis, both receptors are expressed as demonstrated by real-time PCR of menstrual effluent (Kissler et al., 2005, 2007; Bombail et al., 2008) (
Chemotactic cytokines (chemokines) are involved in cell trafficking during embryogenesis, in tissue regeneration and in wound healing (Wu et al., 2007; Stappenmbeck et al., 2009; Garbern et al., 2017). They are functionally closely related to the ER-beta. Three families of chemokines are identified with the CXC family being the preponderant one, such as CXCL12.
Bone marrow-derived MSC are a heterogeneous population of plastic adherent cells that represent only up to 0.01% of total marrow. They are defined to following criteria: (i) plastic adherent, (ii) positive for stromal cell surface markers, while negative for hematopoietic lineage markers and HLA-DR; and (iii) able to differentiate into bone, fat, and cartilage in vitro. These MSC express on their surface the receptor CXCR4 (Hocking, 2015).
These three components, ER-ß, CXCL12 and its receptor, CXCR4, on MSC surfaces can be termed the basic morphogenic complex that is acting within the archimetral micro-units and is playing a crucial role in the pathogenesis of endometriosis and adenomyosis.
Stromal Cell-Derived Factor 1 (CXCL12)
Stromal cell-derived factor 1 (SDF-1 or CXCL12) is a chemotactic cytokine (chemokine) that plays a predominant role in embryology, organogenesis, oncology, normal continuous tissue regeneration of virtually all tissues of the body such as the epidermis and intestinal mucosa, as well as in wound healing after injury and inflammation. It is highly expressed in tissues with a high cellular turnover, such as the intestinal mucosa and chronic skin disease such as psoriasis. CXCL12 sub-serves the attraction of bone marrow derived mesenchymal stem cells (MSC) that are equipped with the corresponding receptor CXCR4. The CXCL12/CXCR4-system regulates the need of various tissues for and the adequate supply with mesenchymal stem cells and it appears to be balanced in the healthy body with respect to normal tissue regeneration (Laird et al., 2011).
The permissive role of estrogen in normal tissue regeneration
Estrogens play a predominant role in tissue regeneration. The expression of CXCL12 is stimulated by estrogen via the estrogen-receptor ß (ER-ß) (Ruiz et al., 2010; Zhou et al-. 2ß15; Wang et al., 2015). In hypoestrogenic states such as the late postmenopause or following ovariectomy and in states of severe hypothalamic ovarian failure the expression of CXCL12 is reduced impairing the normal tissue regeneration that is critically dependent upon a normal incorporation of MSC into the tissue. Also the stem cells themselves appear to be of reduced quality. (Review: Gargett et al., 2016)
The Role of Estradiol in Tissue Injury and Repair (TIAR)
The local production of estrogen, both, on the level of the uterus in adenomyosis and of the ectopic peritoneal lesions is, undoubtedly, central to the understanding of the pathophysiology of the disease (Matsusaki et al., 2001). Recent studies have increasingly shown that estradiol is of utmost importance in the process of wound healing (Gilliver et al., 2007; Mowa et al., 2008). This action of estrogens appears to be mainly mediated by the estrogen receptor-beta (ER2). Animal experiments with chemotoxic and mechanic stress to astroglia (Garcia-Segura, 2008; Sierra et al., 2003; Lavaque et al., 2006) and urinary bladder tissue as well as studies with isolated connective tissue such as fibroblasts and cartilage (Yang et al., 2005; Jeffrey et al., 2007; Shioyama et al., 2008) have revealed that tissue injury and inflammation with subsequent healing is associated with a specific physiological process that involves the local production of estrogen from its precursors. Interleukin-1 induced activation of the cyclooxygenase-2 enzyme (COX-2) results in the production of prostaglandin E2 (PGE2), which in turn activates STAR (steroidogenic acute regulatory protein) and the P450 aromatase. Thus, with the increased transport of cholesterole to the inner mitochondrial membrane testosterone can be formed and aromatized into estradiol that exerts its proliferative and healing effects via the ER2. In studies with fibroblast it was surprising that the first steps of this cascade could be activated by seemingly minor biophysical strain) (Yang et al., 2005). Following termination of unphysiological strain and healing this process is down-regulated and the local production of estrogen or up-regulation of estrogen dependent genes ceases (Yang et al., 2005; Hadjiargyrou et al., 2000). This cascade can even be activated in tissue that normally does not express the P450aromatase indicating the basic physiological significance of the local production of estrogen in tissue injury and repair (TIAR) (Garcia-Segura et al., 1999). The similarity of the molecular biology of TIAR in various tissues with that described in endometriosis (Hudelist et al., 2007; Aghajanova et al., 2009; Bulun, 2009; Gurates and Bulun, 2003; Kissler et al., 2005, 2007; Attar et al., 2009) strongly suggests that this represents the common underlying mechanisms of both processes (
TIAR as an Emergency System for the Attraction of MSC to the Site of Injury
The TIAR mechanism provides ‘estrogen-rich niches’ at the site of tissue injury (Lander et al., 2012). In these niches MSC are accumulated to induce and accelerate the healing process. The molecular biology is similar to that of normal tissue regeneration. While the latter is dependent on the permissive action of systemic estradiol and constitutes an endocrine effect of estradiol, in the TIAR system the strong morphogenic effect of estradiol is utilized in that it is produced at the site of the lesion and acting in a paracrine way to result in an increased expression of CXCL12 that in turn attracts CXCR4 marked MSC. In the urodele it could be demonstrated that the healing of experimental heart injuries could be accelerated by local injections of estradiol into the lesion and that this effect was mediated by the increased expression of CXCL12. At the site of the lesion the MSC differentiate into the tissue specific progenitor cells. This mechanism seems also to be operative in limb regeneration of the axolotl. After amputation of a limb a blastema of stem cells is formed that covers the wound. By cell-to-cell interaction the stem cells are programmed to form the various tissues of a limb. This mechanism constitutes a re-activation of embryonic morphogenesis (Garbern et al., 2013).
In the normal endometrium CXCL12 is expressed in the glandular epithelium. Bone marrow derived stem cells marked by the CXCR4 are attracted by the chemokine and homed in the stroma of the endometrial-myometrial junction to substitute continuously for the loss of apoptotic endometrial cells. Isolated and transplanted endometrial stem cells (ESC; better: archimetral stem cells; ASC) have been shown to differentiate in all Müllerian tissue elements, such as endometrial glandular epithelium, endometrial stroma and metaplastic myometrium (Review: Gargett et al., 2016). In endometriosis, in consequence of the local production and the paracrine effect of estradiol the expression of CXCL12 is and the attraction of MSC are dramatically increased. Locally they differentiate into archimetral stem cells and form hoxa-10 regulated archimetral micro-units, such as “uteri en miniature” in adenomyotic (Cullen, 1903) and “mini-primordial uteri” in endometriotic lesions (Leyendecker et al., 2002).
The attraction MSC to the lesions in uterine adenomyosis does not result in healing. Because the causal trauma and, therefore, the attraction of MSC continue, the proliferative process is resulting with the adenomyotic lesion in aberrant Müllerian morphological structures of variable size, within the uterine wall.
Peritoneal Endometriosis and Peripheral Adenomyosis
There is ample evidence that basal endometrial fragments or only single viable cells of the basal endometrium or even only endometrial or archimetral stem cells (ESC; ASC) may be transported into the periphery of the body by the vascular system as indicated by vital Müllerian tissue in lymphnodes (Mechsner et al., 2008).
The transtubal transmission, however, certainly constitutes the preponderant route of dissemination (Sampson, 1927; Leyendecker 2000). Highly vital basal endometrial tissue elements are transported into the abdominal cavity, where they implant on peritoneal surfaces. Accounting his work Cullen (1920) describes the sites, where endometriotic/adenomyotic lesions persist. These are sites of enduring mechanical strain (
Impairment of Fertility
Already Freund (in: Recklinghausen, 1897) mentioned that sterility was one of the main symptoms of patients suffering from uterine adenomyosis. With the advent of laparoscopy it was realized that not only severe pelvic endometriosis with an overt impairment of ovarian and utero-tubal function but also minimal and mild forms of the disease with no involvement of the tubo-ovarian complex were associated with infertility. Surgical and medical eradication of these lesions did not result in a significant improvement of the conception rates in these patients (Hull et al., 1987; Adamson and Pasta, 1994; Marcoux et a., 1997).
Because no overt cause of the sterility could be identified in these patients the term “unexplained infertility” was introduced (Tummon et al., 1988; Cahil et al., 1995). At that time, the possible presence of uterine adenomyosis was not yet taken into consideration in these studies. That uterine adenomyosis could be a factor of sterility independent of peritoneal lesion was shown in couples with uterine adenomyosis constituting the sole identified factor of sterility, in MRI, the “junctional zone” was significantly broader than in sterile couples with an additional male factor of sterility (Kunz et al., 2005). It was assumed that uterine hyper- and dysperistalsis of the female with an impairment of directed sperm transport were the causative factors.
Already in small adenomyotic lesions hyper- and dysperistalsis can be observed. These lesions destroy the cervico-fundal propagation of the peristaltic waves. The correct propagation of these waves to ensure directed sperm transport into the dominant tube presumably requires the undisturbed interaction of the archimetral micro-units. Furthermore, the local production and paracrine action of estradiol in the TIAR process interferes with the endocrine regulation of directed sperm transport. As seen in real-time TVS, in women with endometriosis, the long cervico-fundal waves as seen in normal controls are replaced by a more convulsive action of the adenomyotic uterus (Kunz et al., 2000).
The level of HOXA 10 and CXCL12 have been described to be lowered in endometrium of women with endometriosis in comparison to controls. It was therefore suggested that the reduced implantation rates in endometriosis may be caused by the decrease of these levels (Moridi et al., 2017). This, however, does no concur with the clinical experience. In women with endometriosis and adenomyosis, following artificial reproductive technology (ART), such as IVF and ICSI, the implantation rate is at least as high as in normal women. The miscarriage rate, however, is increased (
Thus, taken together, there is strong direct and indirect evidence that the destruction of the functional morphology of the archimetra by mechanical strain constitutes a major cause of the development of infertility in young women affected by endometriosis.
Nosological Categorization of Adenomyosis and Endometriosis
Injury Due to Mechanical Strain Followed by Wound Healing with the Resumption of Embryonal Morphogenesis
There is ample evidence provided that the disease is caused by auto-traumatisation of the non-pregnant uterus by its genuine mechanical functions during the normal menstrual cycle and/or by iatrogenic trauma. Chronic mechanical strain is the causative factor in many diseases, such as atherosclerosis and arthrosis. With the TIAR-system resulting in the local production of estradiol and the chemo-attraction of stem cells by CXCL12 the disease utilizes the basic, evolutionarily highly conserved molecular biology of cell trafficking in embryology (Wierman et al., 2011), continuous tissue regeneration, such as that of the intestinal mucosa (Konstatzinopoulos et al., 2003; Wada-Hareike et al., 2006), chronic proliferative diseases, such as psoriasis (Zraggen et al., 2004) and wound healing following various kinds of injury (Hocking, 2015) such as inflammation i. e. of the pulpa of the teeth (Zhang et al., 2015) and other tissues as well as mechanical trauma, of virtually all organs and tissues of the body (Wu et al., 2007). Thus, the pathophysiology of adenomyosis is essential that of tissue injury and repair (TIAR) (Leyendecker et al., 2009). The proliferative process in endometriosis and adenomyosis consists in the attempt to reconstruct, following injury, archimetral micro-units in order to re-build the archimetra as a whole organ. It is reasonable to assume that there is a physiological background of this phenomenon.
With implantation, decidualization and hemochorial placentation the structure of the archimetra changes dramatically. The trophoblast invades the decidua deeply into the layer that had constituted the archimyometrium before pregnancy. One to two weeks after delivery the junctional zone myometrium (archimyometrium) can be, in MRI, identified again. The wound is quickly re-epithelialized and following termination of the lochia the archimetra presents with non proliferated endometrium as observed in hypoestrogenic states. In cases of breastfeeding this hypoestrogenic status persists for a longer period of time. In a recent publication it was estimated that endometrial stem cells would constitute about 30% of the cellular mass of the decidua. Endometrial stem cells are programmed to form all tissue elements of the archimetra, such as endometrial epithelium, endometrial stroma and metaplastic muscular fibers. Thus, decidual ESC would reconstruct the archimetra, guided by the system of archimetral micro-units that persists during pregnancy and ensures the correct positioning of these morphological and functional elements (Guo et al., 2010; Huang et al., 2012).
In the axolotl it was shown that experimental interference with the limb reconstruction resulted in disordered growth (Zhu et al, 2012). Clinically, in not properly performed postpartum and miscarriage-curettages a similar event might happen. Such curettages are the cause of most of the cases of iatrogenic uterine adenomyosis (Kindermann, 1988).
In the post-partum period the reconstruction of the archimetra is controlled by the genetic program (Noxa-10) that is also operative in the morphogenetic program of this organ during ontology. Chronic traumatisation and the permanent attraction of stem cells in consequence of the enduring paracrine action of locally elevated estradiol (TIAR), however, results in a proliferative process that destroys the functional morphology of the archimetra. Thus, it is the aim of the test to identify this destructive process as early as possible.
Principle and Mechanism of the Test
At the site of the archimetral lesion within the uterine cavity MMPs are up-regulated and the level of desquamation is horizontally moved from the spongiosa layer into the basalis layer. Thus, in contrast to healthy women, fragments of basalis are also desquamated and shed with the menstrual blood. With immunohistochemistry basal endometrial fragments can be identified in menstrual blood because the glandular epithelium of the basalis layer is stained estrogen receptor positive (Leyendecker et al., 2002). Moreover, these cells are vital in contrast to the cells of the shed functionalis layer. Some of the basal endometrial fragments may be disseminated via the tubes within the peritoneal cavity, where they have their further own fate of development (
Trauma results in an attraction of macrophages to the site of the lesion. Interleukin 1R released by the macrophages initiates the TIAR process in the stromal cells of the basal endometrium and of the endometrial myometrial junction. The locally produced estradiol, by binding to the ERR, dramatically increases the expression of CXCR12 in the basal endometrial epithelium, which in turn attracts MSC marked with CXCR4. They differentiate by cell-to-cell contact in archimetral (endometrial) stem cells that initiate the adenomyotic proliferative process (
The test identifies this process by the measurement of CXCL12 and/or CXCR4, preferably in an aliquot of menstrual blood. In women without the onset of the disease process this test will be negative, because no basal endometrium is desquamated during menstruation. The magnitude of the measured CXCL12 and CXCR4 levels in the menstrual blood aliquot will depend upon the extent of the archimetral lesion, the amount of basal endometrial fragments shed and collected with the test procedure. ER-alpha and ER-beta are highly expressed in the epithelium of the basal endometrium. Their parallel estimation in the menstrual blood aliquot would provide an estimation of the amount of basal endometrial fragments subjected to the test procedure (
The test utilizes non-organ specific processes of molecular biology. However, in that the test is performed in young women with the risk for the development of adenomyosis and the material is taken from the menstrual blood of these women high degrees of disease- and organ-specificity of the test are ensured.
The concept of uterine auto-traumatisation of the non-pregnant uterus during the reproductive period of life (Tissue Injury and Repair, TIAR) created a completely new basis for the understanding of the pathophysiology of adenomyosis and endometriosis and provides the perspective of a new rational approach to the prevention of the disease. The new insights are based upon a new understanding of the morphology of the non-pregnant uterus, its various functions in the early process of reproduction and their endocrine and paracrine regulation. They are furthermore based on new imaging techniques, such as hysterosalpingoscintigraphy (HSS, magnetic resonance imaging (MRI) and transvaginal sonography (TVS), as well as on new data obtained in molecular biology, not only with respect to the diseases under study but also with respect to more general principles of molecular biology regarding embryology, tissue injury and organ repair. Of utmost importance was, in the understanding of the pathophysiology, to relate the findings of the individual patient to her history. While uterine peristalsis for directed sperm transport had initially been considered the principal mechanical function of the non-pregnant uterus to result in lesions at the upper midline of the archimetra (fundo-cornual raphe), new data suggest that ‘neometral compression of the archimetra’ for orthograde menstrual discharge should be considered as another important cause of uterine auto-traumatisation. While about 60-80% of all women develop perimenopausal adenomyosis/endometriosis, about 10-15% being affected by an early onset of the disease. Uterine auto-traumatisation pertains to both forms. Genetic variations, such as an increased local production and paracrine action of estradiol following trauma (COX-2; P450arom) and/or hyperactivity of the oxytocin (OT)/OT-receptor system controlling the mechanical functions of the non-pregnant uterus, may be responsible for the increased uterine auto-traumatisation in young women affected by the disease.
The pathophysiology is characterized by three intertwined processes:
1. The organ-specific auto-traumatisation by uterine hyperperistalsis for directed sperm transport and by the neometral compression of the archimetra during menstruation. Both contractile mechanisms of the uterus are controlled by ovarian endocrine function.
2. The non-organ specific TIAR process that results in the production of estrogen at the site of traumatisation. The attraction of mesenchymal stem cells (MSC) to the site of the lesion is utilizing the estrogen-enhanced CXCL12/CXCR4 system.
3. The differentiation of the MSC at the site of the lesion into endometrial (ESC) or archimetral stem cells (ASC) with the genetic program that results in the proliferation of Müllerian tissue being composed of all layers of the archimetra such as endometrium, sub basal stroma and metaplastic muscular tissue (“uteri en miniature”, Cullen, 1903).
Iatrogenic adenomyosis develops principally in the same way.
Focal and diffuse proliferations of Müllerian tissue destroy the functional architecture of the ‘junctional zone myometrium’ (archimyometrium) resulting in dysperistalsis and impaired fertility. In MRI and TVS these proliferations can be visualized as permanent irregularities and expansions of the junctional zone (JZ) and “halo”, respectively.
There is a high association between adenomyosis and pelvic endometriosis. Indirect evidence suggests that in women with adenomyosis fragments of basal endometrium and stroma are disseminated within the abdominal cavity where they implant on peritoneal surfaces. They persist at sites of chronic mechanical strain. Such lesions are composed of all archimetral elements (“mini primordial uteri”, Leyendecker et al., 2002) and can therefore be regarded as external adenomyosis.
The test of the present invention aims at identifying these processes on the level of the uterus in an early stage of development in young women with primary dysmenorrhea that are at special risk to acquire the disease. With the test procedure, the appearances and the levels of CXCL12 and CXCR4 are determined preferably in menstrual blood. CXCL12 and CXCR4 are expressed in the endometrial epithelium and adjacent stroma of the basal endometrium, respectively. Because estradiol receptor (ER) is always expressed in the epithelium of the basal endometrium, the parallel measurement of estradiol receptor (ER) allows estimating whether or not basal endometrium is desquamated at all during menstruation. The levels of both, CXCL12 and CXCR4, preferably relative to the levels of ER, indicate initiation and extent of the attraction of MSC and thus of the proliferative process. An early diagnosis of the disease process allows taking adequate measures against a further progression of the proliferation in order to prevent the detrimental sequels of the disease such as causing infertility.
Methods for Detecting CXCL12 and/or CXCR4
The detection reagents for determining the level of CXCL12 and/or CXCR4 or fragment(s) thereof, are preferably selected from those necessary to perform the method, for example antibodies directed to CXCL12 and/or CXCR4, suitable labels, or alternative diagnostic reagents for molecular determination and preferably quantification of CXCL12 and/or CXCR4.
In one embodiment of the method described herein the level of CXCL12 and/or CXCR4 or fragment(s) thereof is determined using a method selected from the group consisting of an immunoassay (IA), nucleic acid amplication reaction, or mass spectrometry (MS). Non-limiting examples relate to a luminescence immunoassay (LIA), radioimmunoassay (RIA), chemiluminescence- and fluorescence-immunoassays, enzyme immunoassay (EIA), Enzyme-linked immunoassays (ELISA), luminescence-based bead arrays, magnetic beads based arrays, protein microarray assays, rapid test formats such as for instance immunochromatographic strip tests and automated systems/analyzers.
Antibodies against CXCL12 or CXCR4 are known to a skilled person, for example those obtained from ThermoFischer or Abcam.
The method according to the present invention can furthermore be embodied as a homogeneous immunoassay method, wherein the sandwich complexes formed by the antibody/antibodies and the marker, e.g., the CXCL12 and/or CXCR4 or a fragment thereof, which is to be detected, remains suspended in the liquid phase. In this case it is preferred, that when two antibodies are used, both antibodies are labelled with parts of a detection system, which leads to generation of a signal or triggering of a signal if both antibodies are integrated into a single sandwich.
In further embodiments of the method described herein, the method additionally comprises a molecular analysis of a sample from said patient. The sample used for the molecular analysis for detecting an infection preferably is a blood sample, more preferably menstrual blood or menstrual fluid sample.
According to some embodiments, CXCL12 and/or CXCR4 are detected by one or more methods for analysis of biomolecules selected from the group comprising nucleic acid amplification methods such as PCR, qPCR, RT-PCR, qRT-PCR or isothermal amplification, mass spectrometry, detection of enzymatic activity and immunoassay based detection methods.
Suitable primers and/or probes can be designed by a skilled person based on the nucleic acid sequences of CXCL12 and/or CXCR4, as described below. Primers may be designed to amplify nucleic acids corresponding to mRNA transcripts of CXCL12 and/or CXCR4, as is commonly employed in molecular diagnostic approaches. Further methods of molecular analysis are known to the person skilled in the art and are comprised by the method of the present invention.
The term “nucleic acid amplification” refers to any method comprising an enzymatic reaction, which allows the amplification of nucleic acids. One preferred embodiment of the invention relates to a polymerase chain reaction (PCR). Another preferred embodiment relates to real time PCR (RT-PCR) or quantitative RT-PCR (qRT-PCR), as it allows the quantification of the amplified target in real-time. The term “real-time PCR” is intended to mean any amplification technique which makes it possible to monitor the progress of an ongoing amplification reaction as it occurs (i.e. in real time). Data is therefore collected during the exponential phase of the PCR reaction, rather than at the end point as in conventional PCR. Measuring the kinetics of the reaction the early phases of PCR provides distinct advantages over traditional PCR detection. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. Traditional PCR methods may also be applied, and use separation methods, such as agarose gels, for detection of PCR amplification at the final phase of or end-point of the PCR reaction. For qRT-PCR no post-PCR processing of the unknown DNA sample is necessary as the quantification occurs in real-time during the reaction. Furthermore, an increase in reporter fluorescent signal is directly proportional to the number of amplicons generated.
Real-time PCR technique can be classified by the chemistry used to detect the PCR product, specific or non-specific fluorochromes. A non-specific DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity measured at each cycle.
Specific detection of PCR production can be achieved by using fluorescent reporter probes, which detect only the DNA containing the sequence complementary to the probe. Therefore, use of the reporter probe significantly increases specificity, and enables performing the technique even in the presence of other dsDNA. Using different-colored labels, fluorescent probes can be used in multiplex assays for monitoring several target sequences in the same tube. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers, which are undesirable potential by-products in PCR. The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe through hydrolysis by the 5′ to 3′ exonuclease activity of the polymerase used for the amplification reaction breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter. Due to the fact that this kind of assay is based on the 5′-exonuclease activity of the polymerase it is also called “5′-exonuclease assay”.
In a preferred embodiment, the invention relates to the detection of CXCL12 and/or CXCR4 mRNA transcripts obtained from menstrual blood samples via molecular diagnostics techniques.
In particular, quantitative or semi-quantitative PCR-based methods, such as those described above, may be applied using primers that hybridize with CXCL12 and/or CXCR4-encoding nucleic acid molecules.
In one embodiment of the method described herein, the method additionally comprises comparing the determined level of CXCL12 and/or CXCR4 thereof to a reference level, threshold value and/or a population average corresponding to CXCL12 and/or CXCR4 in patients who have been diagnosed as healthy, wherein said comparing may be carried out in a computer processor using computer executable code.
The methods of the present invention may in part be computer-implemented. For example, the step of comparing the detected level of a marker, e.g. the CXCL12 and/or CXCR4, with a reference level can be performed in a computer system.
The computer-system can be directly at the point-of-care (e.g. primary care, ICU or ED) or it can be at a remote location connected via a computer network (e.g. via the internet, or specialized medical cloud-systems, optionally combinable with other IT-systems or platforms such as hospital information systems (HIS)). Typically, the computer-system will store the values (e.g. marker level or parameters such as age, blood pressure, weight, sex, etc.) on a computer-readable medium and conduct a comparison in values based-on pre-defined and/or pre-stored reference levels or reference values, leading to a score. The resulting score will be displayed and/or printed for the user (typically a health professional such as a physician). Alternatively or in addition, the associated prognosis, diagnosis, assessment, treatment guidance, patient management guidance or stratification will be displayed and/or printed for the user (typically a health professional such as a physician).
Cut-off values and other reference levels of CXCL12 and/or CXCR4 thereof in patients who have been diagnosed as being healthy may be determined by previously described methods.
As used herein, “diagnosis” in the context of the present invention relates to the recognition and (early) detection of a clinical condition of a subject. Also, the assessment of the severity of the disease may be encompassed by the term “diagnosis”.
“Prognosis” relates to the prediction of an outcome or a specific risk for a subject based on a disease. This may also include an estimation of the chance of recovery or the chance of an adverse outcome for said subject.
The invention may also relate to risk assessment and/or risk stratification. In the present invention, the terms “risk assessment” and “risk stratification” relate to the grouping of subjects into different risk groups according to their further prognosis. Risk assessment also relates to stratification for applying preventive and/or therapeutic measures.
It is understood that in the context of the present invention “CXCL12 and/or CXCR4” or the like refers to determining any biological representation of CXCL12 and/or CXCR4. Included are measurement on the protein and/or nucleic acid level. Fragments of such molecules may also be employed in the analysis.
The CXCL12 gene encodes a stromal cell-derived alpha chemokine member of the intercrine family. The encoded protein functions as the ligand for the G-protein coupled receptor, chemokine (C-X-C motif) receptor 4, and plays a role in many diverse cellular functions, including embryogenesis, immune surveillance, inflammation response, tissue homeostasis, and tumor growth and metastasis. Mutations in this gene are associated with resistance to human immunodeficiency virus type 1 infections.
Multiple protein sequences have been detected for CXCL12, for example those recorded as stromal cell-derived factor 1 isoform 5 precursor [Homo sapiens], 103 aa protein, Accession: NP_001264919.1, GI: 489406390, stromal cell-derived factor 1 isoform delta precursor [Homo sapiens], 140 aa protein, Accession: NP_001171605.1, GI: 296011023, stromal cell-derived factor 1 isoform gamma precursor [Homo sapiens], 119 aa protein, Accession: NP_001029058.1, GI: 76563933, stromal cell-derived factor 1 isoform alpha precursor [Homo sapiens], 89 aa protein, Accession: NP_954637.1, GI: 40316924, stromal cell-derived factor 1 isoform beta precursor [Homo sapiens], 93 aa protein, Accession: NP_000600.1, GI: 10834988.
Additional fragments are:
Processed forms SDF-1-beta(3-72) and SDF-1-alpha(3-67) are produced after secretion by proteolytic cleavage of isoforms Beta and Alpha, respectively. The N-terminal processing is probably achieved by DPP4. Isoform Alpha is first cleaved at the C-terminus to yield a SDF-1-alpha(1-67) intermediate before being processed at the N-terminus. The C-terminal processing of isoform Alpha is reduced by binding to heparin and, probably, cell surface proteoglycans.
The corresponding nucleic acid sequences, of the encoding CXCL12 gene and mRNA transcripts for molecular determination, may be obtained from Gene ID: 6387 (NCBI).
CXCR4 is the receptor for the C-X-C chemokine CXCL12/SDF-1 that transduces a signal by increasing intracellular calcium ion levels and enhancing MAPK1/MAPK3 activation. Acts as a receptor for extracellular ubiquitin; leading to enhanced intracellular calcium ions and reduced cellular cAMP levels. Involved in hematopoiesis and in cardiac ventricular septum formation. Also plays an essential role in vascularization of the gastrointestinal tract, probably by regulating vascular branching and/or remodeling processes in endothelial cells. Involved in cerebellar development. In the CNS, could mediate hippocampal-neuron survival.
Additional isoforms of this protein are also known, such as:
Multiple transcript variants encoding different isoforms of CXCL12 have been found for this gene. Reference: Gene ID: 6387, NCBI.
The corresponding nucleic acid sequences for CXCR4, of the encoding gene and mRNA transcripts for molecular determination, may be obtained from Gene ID: 7852 (NCBI).
In some embodiments, the method comprises a polymerase chain reaction (PCR) to determine a level of CXCL12 and/or CXCR4 in said sample, wherein said PCR comprises primers that hybridize with CXCL12 and/or CXCR4-encoding nucleic acid molecules according to one or more of SEQ ID NO 8-17.
In some embodiments, the invention relates to the method described herein, characterized in that primer sequences are used in the PCR reaction to determine a level of CXCL12 and/or CXCR4 in said sample, wherein the primer sequences comprise or consist of nucleotide sequences according to one or more of SEQ ID NO 22, 23, 24 and/or 25.
A further aspect of the invention relates to the method as described herein, employing primers in the PCR method as follows:
In some embodiments, the method comprises an immunoassay using one or more antibodies that bind CXCL12 and/or CXCR4 according to one or more of SEQ ID NO 1-7 to determine a level of CXCL12 and/or CXCR4 in said sample.
As used herein, the term “sample” is a biological sample that is obtained or isolated from the patient or subject. “Sample” as used herein may, e.g., refer to a sample of bodily fluid or tissue obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest, such as a patient. Preferably herein, the sample is a sample of a bodily fluid, such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusions, cells, a cellular extract, a tissue sample, a tissue biopsy, a stool sample and the like. Particularly, the sample is a sample of menstrual fluid, also known as menstrual blood.
In certain aspects of the invention, the method of determining CXCL12 and/or CXCR4 is one or more immunoassays comprising the steps of:
a) contacting the sample with
b) detecting the binding of the two antibodies or antigen-binding fragments or derivates thereof to said CXCL12 or CXCR4.
Preferably, one of the antibodies can be labeled and the other antibody can be bound to a solid phase or can be bound selectively to a solid phase. In a particularly preferred aspect of the assay, one of the antibodies is labeled while the other is either bound to a solid phase or can be bound selectively to a solid phase.
The level of the marker of the present invention, e.g. CXCL12 or CXCR4, can also be determined by a mass spectrometric (MS) based methods. Such a method may comprise detecting the presence, amount or concentration of one or more modified or unmodified fragment peptides of e.g. CXCL12 or CXCR4 in said biological sample or a protein digest (e.g. tryptic digest) from said sample, and optionally separating the sample with chromatographic methods, and subjecting the prepared and optionally separated sample to MS analysis. For example, selected reaction monitoring (SRM), multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) mass spectrometry may be used in the MS analysis, particularly to determine the amounts of ADM or fragments thereof. Herein, the term “mass spectrometry” or “MS” refers to an analytical technique to identify compounds by their mass. The skilled person is aware how quantify the level of a marker in the sample by mass spectrometric methods. For example, relative quantification “rSRM” or absolute quantification can be employed as described above.
The invention further relates to kits, the use of the kits and methods wherein such kits are used. The invention relates to kits for carrying out the herein above and below provided methods. The herein provided definitions, e.g. provided in relation to the methods, also apply to the kits of the invention.
In particular, the invention relates to kits for determining the presence of a tissue injury and repair (TIAR) process in the uterus of a subject as a marker for the presence of a preliminary stage or increased risk of developing a medical disorder associated with abnormal formation of endometrial tissue, wherein said kit comprises
The kit may additionally comprise items useful for obtaining a sample, such as a blood or menstrual fluid sample, for example the kit may comprise a container, wherein said container comprises a device for attachment of said container to a canula or syringe, is a syringe suitable for blood isolation, exhibits an internal pressure less than atmospheric pressure, such as is suitable for drawing a pre-determined volume of sample into said container, and/or comprises additionally detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, such as guanidinium isothiocyanate, guanidinium hydrochloride, sodium dodecylsulfate, polyoxyethylene sorbitan monolaurate, RNAse inhibitor proteins, and mixtures thereof, and/or A filter system containing nitro-cellulose, silica matrix, ferromagnetic spheres, a cup retrieve spill over, trehalose, fructose, lactose, mannose, poly-ethylen-glycol, glycerol, EDTA, TRIS, limonene, xylene, benzoyl, phenol, mineral oil, anilin, pyrol, citrate, and mixtures thereof.
The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.
The present invention is further described by reference to the following non-limiting figures and examples.
The invention is further described by the following figures. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.
b) a peritoneal endometriotic lesion (×400) as an ectopic “microarchimetra”. With endometrial glands. endometrial stroma and peristromal muscular tissue the lesion is composed of all elements of the archimetra.
c.) The primordial uterus of the 23rd week of pregnancy (×50) is composed of the elements of the archimetra, such as endometrium and archimyometrium (specific actin staining) (top right). The archimetra is essentially the adult representation of the primordial uterus.
d.) The “halo” in transvaginal sonography represents the archimyometrium as does e.) the “junctional zone” in MR imaging. Transvaginal sonography (TVS) and magnetic resonance imaging (MRI) of the uterus of a 29 years old woman unaffected with endometriosis and adenomyosis. Sagittal scans of the uterine midline are shown. The myometrial-endometrial lining is sharp and smooth; the “halo” in TVS and the “junctional zone” in MRI are unaltered; there is symmetry with respect to the anterior and posterior myometrial walls and the texture of the myometrium in TVS appears to be homogenous. Modified from Leyendecker et al. (2004, Annals of the New York Academy of Sciences 1034:338-355).
A=archimyometrium (a). Due to the high intrauterine pressure in consequence of the contraction of the neometra the archimyometrium ruptures in the cornual angles and fragments of basal endometrium are dislocated into myometrial wall, where they develop into endometriotic cysts (a and c). At the same time basal stromal cell at the fundo-cornual raphe are chronically overstretched resulting in the initiation of the TIAR mechanism and the development of an adenomyotic lesion.
(1) Uterine adenomyosis; (2) obvarian endometriosis; (3) intestinal endopmetriosis; (4) intestinal-uterine adhesion due to an endometriotic lesion; (5) lesion in sacro-uterine ligament; (6) retrocervical-vaginal endometriosis and in the cul de sac; (7) lesion atserosa of the urinary bladder and the anterior wall of the uterus; (8) umbilical endometriosis; (9) endometriosis in the abdominal wall; (10) inguinal endometriosis. These sites are characterized by chronic mechanical strain. Modified from Cullen 1920
The invention is further described by the following examples. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.
In order to illustrate a practical embodiment of the invention, the following is carried out:
ELISA Assay for CXCL12 Detection
A sandwich ELISA assay is carried out using a menstrual blood samples obtained from female subjects of ages 12 to 20.
(1) A standard plastic ELISA plate is coated with a capture antibody directed against CXCL12; the surface is prepared with a known quantity of capture antibody. Any nonspecific binding sites on the surface are blocked. The plate is washed.
(2) The menstrual blood sample is added, and any CXCL12 present in the sample binds to the capture antibody immobilized on the solid surface; The plate is washed to remove unbound components.
(3) A secondary detecting antibody is added, which also binds to CXCL12, and which is labelled with a horseradish peroxidase enzyme; This enzyme-linked secondary antibody is applied as a detection antibody and binds to the already immobilized CXCL12 from the sample. The plate is washed to remove the unbound antibody-enzyme conjugates.
(4) A substrate is added, and is converted by the enzyme of the secondary antibody to a detectable form. Essentially, a chemical is added to be converted by the enzyme into a color or fluorescent or electrochemical signal. The absorbance or fluorescence or electrochemical signal of the plate wells is measured in a standardized plate reader to determine the presence and quantity of CXCL12.
The above analysis is conducted using samples from multiple subjects who are diagnosed as healthy subjects after internal physical examination, or show symptoms of dysmenorrhea but prior to pathological indications of endometriosis. All subjects are monitored for further disease symptoms and are eventually categorized into patient groups with or without abnormal formation of endometrial tissue. Comparisons between the CXCL12 levels determined in the two subject groups indicate an elevated level of CXCL12 in subjects who went on to develop a medical condition of abnormal formation of endometrial tissue. Similar analysis on the basis of CXCR4 reveals a similar correlation.
Pilot Study: RT-PCR Analysis of CXCL12 in Menstrual Blood Samples in Patients with Archimetrosis:
Introduction
The test is based on the inventive concept of the pathogenesis and pathophysiology of uterine adenomyosis and endometriosis (archimetrosis). Adenomyosis and endometriosis (archimetrosis) are caused by auto-traumatisation or iatrogenic traumatization of the uterus (Leyendecker et al. 1998; Leyendecker et al. 2015). The auto-traumatisation results from genuine biomechanical functions of the non-pregnant uterus during the menstrual cycle. These functions consist of the uterine peristalsis of the archimyometrium (subvascular layer of the myometrium) for directed sperm transport into the tube ipsilateral to the dominant follicle and high fundal implantation of the blastocyst as well as of rhythmic contractions of the stratum vasculare of the myometrium for the externalization of the menstrual debris at the end of menstrual cycle.
The adult uterus is composed of two phylogenetically and ontogenetically different structures: The archimetra and the neometra. The archimetra is the adult representation of the primordial uterus and derived from the Müllerian duct. It develops early in phylogeny and ontogeny. The neometra, composed of the the supravascular and the vascular layers of the myometrium, respectively, is of non-Müllerian origin develops late during phylogeny and in the human late in pregnancy and even after birth (Werth and Grusdew, 1898; Leyendecker et al. 1998; Leyendecker et al. 1999; Noe et al. 1999).
During the first ten postmenarcheal years following the establishment of regular ovulatory cycles (13-23 years of age) 5 to 6 million peristaltic contraction waves occur. This estimation is based on the observation that, in healthy women, about 2 contraction waves per minute occur during the early follicular phase of the cycle (Leyendecker et al. 1996).
In addition, during this period of reproductive life at least 110 to 140 thousand neometral contractions occur (Leyendecker et al. 2015). This estimation is based on the observation that, during the menstrual period, these contractions occur with a frequency of about 24-36 contractions per hour for a duration of about 36 hours.
These contractile activities of the non-pregnant uterus during and at the end of the menstrual cycle are dramatically increased in women that are suffering from endometriosis/adenomyosis. The increased peristaltic activity (hyperperistalsis) in women suffering from endometriosis could be demonstrated by hystero-salpingo-scintigraphy (HSSG) and video-sonography of uterine peristalsis (VSUP) (Kunz et al. 1996; Leyendecker et al. 1996). In women with endometriosis, the frequency of the peristaltic waves is doubled during the early, mid-follicular and mid-luteal phases of the cycle as compared to controls.
Primary dysmenorrhea constitutes the clinical symptom of neometral hypercontractility. The intrauterine pressure in these women during menstruation might exceed the blood pressure in arterioles not only on the height of the contractions but also between the contractions (Leyendecker et al. 2015). Thus, the menstrual pain may be caused by both the power of the contractions and a concomitant contractions-induced ischemia.
In this study the symptoms primary and secondary dysmenorrhea are defined and used in a temporal and not in a causal context.
Accordingly, for the purpose of this study, primary dysmenorrhea is defined as menstrual pain that develops early in the period of reproductive life, shortly after menarche with the onset of regular ovulatory cycles.
Accordingly, for the purpose of this study, secondary dysmenorrhea is defined as the menstrual pain that develops after a somewhat longer period of regular menstruations without pelvic pain.
Intensity of Primary Dysmenorrhea
There is circumstantial evidence that the auto-traumatization and the development of archimetrosis depends upon on strength and duration of the myometrial contractile activity. The prevalence of archimetrosis in 60-80% of premenopausal women (Emge, 1962) and the increasing prevalence of archimetrosis in symptom-free women attempting tubal sterilization with increasing time lapse after the last pregnancy support this view (Moen and Muus 1991; Muus 1991). It is further supported by the observation that the morphological destruction of the uterus due to adenomyosis was more pronounced in women with extreme primary dysmenorrhea than in women with less serious grades of dysmenorrhea (Leyendecker et al. 2015). This strength-duration characteristic allows the conclusion that the likelihood of developing archimetrosis at a young age increases with the severity of the uterine hypercontractility and, clinically and historically, with the severity of primary dysmenorrhea.
It is, therefore, necessary to apply in this study a grading system of the intensity of primary dysmenorrhea when taking the history of the patient. Instead of using a pain score based on subjective sensation historical data that easily can be remembered, such as the use of analgesics, are preferred (Leyendecker et al. 2015):
Mild Primary Dysmenorrhea:
The menstrual pain could be tolerated without the use of analgesics.
Moderate Primary Dysmenorrhea:
To control menstrual pain analgesics were used more or less occasionally.
Severe Primary Dysmenorrhea:
The menstrual pain could only be tolerated each month by the use of analgesics
Extreme Primary Dysmenorrhea:
Absenteism from school and work during menstruation
Change of Character of Dysmenorrhea (Quality and Intensity)
During many years of the early reproductive period of life the character of pelvic menstrual pain may not change. Some women, however, report that, despite of continuing regular cycles, the menstrual pain decreased about two to four years after menarche. This might be due to the continuous loss of follicles and the continuing slight decrease in estradiol levels with increasing age. Also, during a phase of anovulatory cycles, the pain might be reduced or might have even disappeared.
Alternatively, the pain might change in character in that it is more localized and may be also intensified. This could indicate that the disease is growing and spreading, in that larger adenomyoma and/or pelvic endometriosis is developing. In such circumstances the patient might be convinced that the genuine disease has begun with the onset of this particular pain and considers the menstrual pain and discomfort before as “normal”. This is probably the reason why “secondary dysmenorrhea” was regarded as the principal symptom of endometriosis
This indicates that the menstrual history has to be taken with scrutiny. Special questions might help the patients to remember the history of their menstrual pain, such as, whether the use of oral contraceptives was started solely in order to obtain relief from pain or solely for, primarily for or together with attempting contraception.
The prevalence of primary dysmenorrhea is about 50% (Burnett 2005). In a recent study of women affected by archimetrosis (uterine adenomyosis and/or peritoneal endometriosis) of 116 women 109 reported and 7 denied dysmenorrhea. Of the 109 women with dysmenorrhea 17 had secondary and 92 primary dysmenorrhea. With more than 70% the occasional and constant use of analgesics prevailed in women with primary dysmenorrhea. Conversely, such women are at increased risk to acquire uterine adenomyosis and peritoneal endometriosis (archimetrosis).
Examination of the Menstrual Effluent in Women with and without Archimetrosis
Tissue Culture:
In a large review Philipp and Huber (1939) reported that in healthy women the tissue culture of menstrual debris was not possible while cyclic endometrium easily grew in culture. Commenting on Sampson theory (1927) that peritoneal endometriosis would result from retrograde menstruation they argued that the endometrial tissue, disseminated within the peritoneal cavity and growing on peritoneal surfaces must be derived from tissue of deaper layers of the endometrium shed during menstruation.
Immunhistochemistry of the Menstrual Effluent:
Histological examination of menstrual effluent showed cellular death of the endometrial fragments shed in women without endometriosis, while in women with endometriosis the shed tissue fragments showed the characteristics of vital cells. Moreover, immunohistochemistry of the menstrual effluent showed the expression of estradiol and progesterone receptors in women with endometriosis and not in women without the disease. Because estradiol and progesterone receptors are, at the end of the cycle and during the early menstrual cycle only expressed in the basal and not in the functional layer of the endometrium, it was concluded that in women with endometriosis fragments of basal endometrium are shed, which is not the case in disease free controls. Therefore, it was suggested that peritoneal endometriosis would result from the menstrual desquamation and transtubal dissemination of fragments of basal endometrium (Leyendecker et al. 2002).
Molecular Biology of the Menstrual Effluent: Estradiol:
In women with archimetrosis the levels of estradiol in menstrual blood are higher than in peripheral blood taken at the same time. In healthy control the respective estradiol levels do not differ from each other.
TIAR:
This is the acronym of Tissue Injury And Repair. It describes the capacity of mesenchymal tissue to express of enzymes that catalize the synthesis of estradiol from cholesterol. In various tissues tissue cultures and experiments with isolated stromal cells it had been demonstrated that this synthetic pathway and parts of it are stimulated by strain that may be mechanical (overstretching of cells, mechanical tissue injury) or inflammatory in character. This results, at the site of traumatization, in the local production of estradiol. Several investigations have shown that this cascade of synthesis of estradiol is also activated in endometriotic tissue and in the endometrium of affected women and not in controls.
Circumstantial evidence indicates that, on the level of the non-pregnant uterus, this mechanical strain and traumatization result from the genuine mechanical functions of the uterus, such as the peristaltic activity of the archimyometrium for directed sperm transport and the menstrual neometral contractions for the externalization of menstrual debris.
There is also clinical and experimental evidence for a strain-duration characteristics of these contractile activities to cause trauma because nearly all women develop, with time, uterine adenomyosis (and endometriosis) and women with hypercontractility, such as hyper- and dysperistalsis and increased neometral contractions as indicated by primary dysmenorrhea develop this disease (archimetrosis) early in their reproductive life.
In 2009, for the first time, the theory was developed and published that the local production of estradiol within uterine tissue serves healing of the uterine wounds induced by auto-traumatization and also iatrogenic trauma (Leyendecker et al. 2009) and subsequently further elaborated (Leyendecker et al. 2015)
Following the finding that estrogen-receptor positive endometrial tissue is detached during menstruation and found in the menstrual effluent of women with endometriosis, which is not the case in disease free women (Leyendecker et al. 2002), real time PCR of ER-alpha, ER-beta, PR and COX2 was performed in the menstrual effluent of women with endometriosis and controls. Particularly the concentration of ER-beta in the menstrual effluent of women with endometriosis was dramatically and significantly increased in comparison to controls. In this study, however, the PCR of the P450 arom was invalid because of the application of a wrong primer (Kissler et al. 2002; 2007). Nevertheless, available experimental data suggest that the P450arom may be also significantly increased in the menstrual effluent of women with adenomyosis/endometriosis because it catalizes, within the TIAR process the final step, the aromatization of testosterone to estradiol.
CXCL12, CXCR4:
In various tissues wound healing is attained by the attraction of mesenchymal stem cells (MSC) into the site of trauma. This is achieved by the dramatically increased expression of the chemokine CXCL12 by estradiol and mediated by the ER-beta. This results in the local proliferation of endometrial or archimetral stem cells (ESC or ASC), in stroma-epithelial transformation and in stromal metaplasia giving in turn rise to the formation of uterine adenomyoma. From these local sites of proliferation vascular dissemination, and most importanly, transtubal dissemination of vital endometrial cells (basal epithelium and stroma; eventually only ASC) may occur resulting in peripheral and peritoneal endometriosis, respectively.
Archimetrosis Constitutes a Potential Sequel of Auto-Traumatisation Due to Myometrial Hypercontractility
The expression of the basal morphogentic complex (ER-Beta, CXCL12, CXCR4) is not organ and disease specific. The relative but nevertheless high specificity of the test results from the test to be performed in the menstrual effluent of a defined group of women in their early or middle reproductive period of life that display a characteristic symptomatology such as a history of primary dysmenorrhea, pelvic pain, unexplained infertility and eventually bleeding disorders (pre- and postmenstrual spotting).
Adenocarcinoma of the endometrium constitutes a malign Müllerian proliferation. Abnormal uterine bleeding constitutes the characteristic symptom of this disease. However, the prevalence of this malign process is in the climacteric period of life.
Expected Results of the Test
Previous studies performed with the menstrual effluent using real-time PCR showed a significant increase of ER-ßeta expression in the menstrual effluent of women with endometriosis as compared to healthy controls. ER-ßeta is a constituent of what is defined as the “basal morphogenetic complex” constisting of ER-ßeta, CXCL 12 and CXCR4. This basal morphogenetic complex requires estradiol for stimulation which is provided, in a prakrine fashion, by the TIAR process that is induced by mechanical strain and enables the normal endometrial stromal cells to convert into cells that are capable to synthezise estradiol from cholesterol. Both ER-ßeta and the chemokine CXCL12 are expressed in the same tissue compartment of the endometrium, of which fragments are shed with the menstrual blood in women with endometriosis.
In consequence, it can be expected that the MSCs expressing CXCL4 on their surface and that are, via the endometrial capillary system (terminal vessels), attracted by CXCL12 to this endometrial compartment are significantly increased in the menstrual effluent as well. The detection of increased levels of CXCL12 and CXCR4, therefore, is indicative of a wound healing process and, in a defined population of young women, indicative of the beginning or the presence of archimetrosis.
Subjects and Methods
Subjects: The tests will be performed in women (17 to 35 yrs of age) with and without archimetrosis (uterine adenomyosis and pelvic endometriosis). The presence or absence of archimetrosis has to be demonstrated by laparoscopy. and/or high resolution vaginal sonography.
A careful menstrual history (menarche; regularity of cycles), history of pelvic pain (primary and secondary dysmenorrhea; dyspareunia; dysuria; dyschezia, first onset and aggravation of pelvic pain) and bleeding disorders has to be taken. The presence or absence of cyclic pelvic pain prior to menarche and peri-menstrual pain at distal (extragenital) parts of the body have to be documented.
A careful gynecological examination has to be performed with special emphasis being laid upon finding special sites of pain, such as the sacro-uterine ligaments, the recto-vaginal septum, bowel and ovarian adhesions to the uterus.
Exclusion Criteria:
Previous pregnancy; previous intrauterine surgery (hysteroscopy; curettage); irregular cycles; anovulatory cycles; hormonal therapy such as oral contraception, if not terminated 6 month ago; use of intrauterine device; use of COX2 inhibitors during the test phase; fibroids of the uterus.
High Resolution Transvaginal Sonography of the Uterus (TVS):
All relevant sonographic data of the uterus have to be documented (size; position; anterior, posterior and fundal walls, junctional zone myometrium (“halo”; archimyometrium). The presence or absence of signs of uterine adenomyosis has to be documented. TVS is performed during the mid-luteal phase of the cycle (day 20-24 of the cycle) prior to sample collection in order to demonstrate a luteal phase appearance of the endometrium.
Sample Collection:
Venous blood is drawn in the mid-luteal phase for measurement of serum progesterone levels in order to document a normal luteal phase.
Menstrual blood is collected on the morning after onset of menstruation (Leyendecker et al. 2002). This could be the 1st or 2nd day of menstruation. During the previous night, just a sanitary pad is used.
Processing of the Menstrual Blood Specimen:
The menstrual blood sample is centrifuged (600×g for 5 minutes) and the supernatant is decanted. RNAlater RNA Stabilization Reagent (Quiagen, Order No. 76104) is added to the pellet. RNA stays stable for 7 days at room temperature (15-25° C.).
On the day of sample collection or one day later the specimen is transferred to the “Institut für experimentelle Chirurgie der Universität des Saarlandes”, Homburg/Saar, Germany for cryopreservation and for the later implementation of the RT-PCR.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17204073.5 | Nov 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/082879 | 11/28/2018 | WO | 00 |
