SUMMARY IMMUNO-ENDOCRINE INTERACTIONS IN THE PATHOGENESIS OF ENDOMETRIOSIS

Transcription

1 SUMMARY IMMUNO-ENDOCRINE INTERACTIONS IN THE PATHOGENESIS OF ENDOMETRIOSIS Scientific coordinator Prof. Dr. Carmen VULPOI Prof. Dr. Mircea ONOFRIESCU PhD student Daniela-Roxana ALBU (MATASARIU) IAŞI, 2017

2 The doctoral thesis includes: A general part structured in 4 chapters, comprising 40 pages A personal part structured in 8 chapters, comprising 99 pages References part includes 344 bibliographic references Abbreviations There are 69 tables and 88 figures inserted 2 ISI articles as first author 1 ISI proceedings article as first author 1 BDI article as first author 3 oral presentations at conferences as first author Note: the abstract selectively reproduces references and iconography of the full text, respecting the numbering and the summary of the thesis. Keywords: endometriosis, progesterone, osteopontin, quality of life, immunohistochemistry. The present research was also possible thanks to the scholarship I owned in the project: Parteneriat strategic pentru creșterea calității cercetării științifice din universitățile medicale prin acordarea de burse doctorale și postdoctorale - DocMed.net_2.0 POSDRU/159/1.5/S/ This paper was published under the frame of European Social Found, Human Resources Development Operational Programme , project no. POSDRU/159/1.5/ It is with immense gratitude that I acknowledge the support and help of late Professor Carmen Vulpoi, without whom this thesis would not have been possible. Because I share the credit of my career path with her, I owe her my deepest gratitude, respect and consideration. 1

3 CONTENTS ABBREVIATIONS...3 INTRODUCTION...4 STATE OF KNOWLEDGE...6 CHAPTER GENERAL ASPECTS REGARDING ENDOMETRIOSIS Epidemiology Factors that influence the incidence of endometriosis...6 CHAPTER ETIOPATHOGENY OF ENDOMETRIOSIS Ethiology Pathogenesis...10 CHAPTER DIAGNOSIS OF ENDOMETRIOSIS Clinical diagnosis Paraclinical diagnosis...22 CHAPTER TREATMENT OF ENDOMETRIOSIS Medical treatment Surgical treatment...38 PERSONAL CONTRIBUTIONS...44 CHAPTER MOTIVATION AND OBJECTIVES OF THE STUDY...44 CHAPTER THE INCIDENCE OF ENDOMETRIOSIS - A PUBLIC HEALTH PROBLEM Purpose of the study Material and method Results Discussions...49 CHAPTER THE IMPACT OF ENDOMETRIOSIS OVER THE PHYSICAL, MENTAL AND SOCIAL STATE OF HEALTH Purpose of the study Material and method Results Discussions...90 CHAPTER OSTEOPONTIN - BIOMARKER IN ENDOMETRIOSIS Purpose of the study Material and method Results Discussions

4 CHAPTER HORMONAL THERAPY OF ENDOMETRIOSIS PRE- AND POSTOPERATIVE RESULTS Purpose of the study Material and method Results Discussions CHAPTER THE INFLUENCE OF PROGESTERONE OVER THE IMMUNOHYSTOCHEMICAL MARKERS IN ENDOMETRIOSIS Purpose of the study Material and method Results Discussions CHAPTER CONCLUSIONS CHAPTER ORIGINALITY ASPECTS AND PERSPECTIVES OPENED BY THE THESIS REFERENCES ANNEXES List of scientific works concerning the research theme published during doctoral studies

5 ABBREVIATIONS AINS- nonsteroidal anti-inflmmatories Bcl-2 - B cell lymphoma 2 BDI- Beck Depression Inventory CA125 Cancer antigen -125 COC combined oral contraceptives COX - ciyclooxygenase CST Cesarean section CT computerized tomography DAB - 3,3 1 diaminobenzidine EEV endovaginal ultrasonography EHP endometriosis health profile ELISA - Enzyme-Linked Immunosorbent Assay ER estrogen receptor FPI fertility problem inventory GnRH gonadotropin-releasing hormone HE - hematoxylin eosin stain IHC immunohistochemistry IL - interleukin IMC body mass index MMP - matrix metalloproteinase NETA - norethindrone acetate OPN - osteopontin PBS phosphate buffer saline PDGF platelet-derived growth factor PR progesterone receptor RMN magnetic resonance imaging Sdr. - syndrome TCDD - 2,3,7,8-tetracloro-p-dioxin TNF-α tumor necrosis factor-α VEGF vascular endothelial growth factor 4

6 INTRODUCTION Even in the times we are living, endometriosis continues to be one of the biggest medical enigmas, since its first literature description made by Daniel Shroen in 1690, who described the first endometriosis lesions and until our days, when numerous researches couldn t establish its exact etiology, associated risk factors and why its incidence is continuingly rising (Shroen, 1690, Signorile, Baldi, 2010). This disease seems to defy all the efforts made by the researchers to discover what causes it, its action mechanisms and thus, its treatment. Endometriomas are difficult to excise due to their rich vascularization, and the fact that they are highly adherent to the ovarian tissue makes it very hard to surgically remove them without damaging the normal ovarian tissue. The recurrence rates of the endometriomas, following surgery can be reduced by prescribing medication that inhibits menstruation, like hormonal contraceptives (Aznaurova, et al. 2014). The quality of life in correlation to the health state of the patient is a multidimensional concept that implies physical, psychological and social aspects associated with the disease itself and its treatment. The precocious diagnosis and the correct treatment of the endometriosis cysts lead to an upgrade of the quality of life, by improving the symptoms and higher chances for a future pregnancy. The progesterone treatments are efficient to combat the main symptoms of endometriosis and, also have less adverse effects, with a better cost-benefit ratio than the gonadotropin-releasing hormone agonists (GnRH) and Danazole, drugs that cannot be used on a long period of time. Also, the progesterone can reduce the inflammation induced by the endometriosis in the pelvis. Unfortunately, there is not a clear consensus in the literature regarding the optimal dosage and the duration of the treatment for the endometriosis lesions to regress. More than this, there are only few information regarding which type of progesterone is the most effective and the opinions of those who support the prescription of this drug are in fact arbitrary (Vercelini, et al., 2011, Streuli, et al., 2013). In the present thesis, I considered all these problems and I tried to solve them by a thorough study of the literature, with an up-to-date presentation of the results. 5

7 PERSONAL CONTRIBUTIONS CHAPTER 5 MOTIVATION AND OBJECTIVES OF THE STUDY Endometriosis is a condition characterized by the implantation of stroma and endometrial glands outside the uterus, mainly on the ovaries and peritoneum (Nisolle, Donnez, 1997, Bulun, 2009, Giudice, 2010), by this inducing a chronic inflammatory reaction. The mechanism that triggers this inflammatory process is not fully known until now, endometriosis still being considered a disease of the theories, in spite of all the progress recorded in the research field. The state of health is directly correlated with the quality of life in a multidimensional concept that implies physical, psychological and social aspects of life, and the way they are influenced by the disease and its associated treatment. One of the best prevention methods for the avoidance of the suffering of the patients due to the disease is an early diagnosis of endometriosis, fact that could improve the management of the disease, thus the quality of life. Among the main objectives of the present research, I can count the following: evaluation of the incidence of endometriosis among the patients hospitalized in the Gynecology Department of the Clinical Hospital Cuza-Voda, Iasi; investigation of the psychological reactions associated with endometriosis and the level of depression in patients from the geographical region of Moldavia and comparison of these results with those cited in other international studies, keeping in mind the fact that the present study only included Caucasian patients; Correlation between a low quality of life with the infertility related stress and the depressive manifestations; Evaluation of the osteopontin (OPN) level in the patients` serum and correlation of these levels with the ones of the healthy patients and the surgical and medical treatment; Investigation upon the utility of the preoperatively hormonal treatment of the endometriosis cysts; Evaluation of the effectiveness of the postoperative hormonal treatment upon the prevention of the recurrence of endometriosis cysts. The considerations were largely debated in the text of this paper, being in fact the result of a detailed study over the patients, that included demographic aspects, the quantification of the impact of endometriosis over the quality of life, the evaluation of some possible future markers for an early diagnosis, and last but not least, future therapeutic options. 6

8 CHAPTER 6 THE INCIDENCE OF ENDOMETRIOSIS- A PUBLIC HEALTH PROBLEM 6.1 The purpose of the study Regarding the fact that the incidence of endometriosis varies between wide limits on a global scale, the main objective of the study was to identify the incidence and prevalence of this condition among the patients hospitalized and treated in the Clinical Hospital Cuza-Voda, Iasi. 6.2 Material and method The study included all the patients that were hospitalized and underwent surgery in the Department of Gynecology All the patients enrolled in the study signed a written consent, which was previously approved by the Ethical Medical Committee of the University of Medicine and Pharmacy Gr. T. Popa, Iasi. Following the selection criteria, from the total of patients hospitalized only 1100 were selected to take part to the study, as they had a clear diagnosis of endometriosis established by laparoscopy and histologic confirmation Results On years of study, the distribution of the cases in which surgery was performed for the diagnosis of ovarian endometriosis was shown to have a slightly increasing tendency, ranging from 114 surgical interventions performed in 2012, to 139 surgical interventions in The surgical interventions for peritoneal endometriosis were relatively constant between the years (72-75 cases). The number of surgical interventions for endometriosis located on the Cesarean scar, intestine, rectovaginal septum and Fallopian tubes, was low. I noted the fact that the patients with a higher level of education and with a good socio-economic status have a higher addressability, representing 94.5% of the patients with endometriosis from our Department. Analyzing the marital status of the patients, I found out that 847 patients were in a relationship. I also noticed that 953 of the patients with pelvic endometriosis were living in the city, only 147 of the patients having a stable residence in the country side Discussion The data from our casuistry show an incidence of 9.03%, being in total agreement with those found in the literature that describe an incidence up to 10% (Cramer, Missmer, 2002, Giudice, 2010). The yearly distribution over the 6 years of study identified an increasing tendency of the incidence of endometriosis in our Department. In 2012, a German study identified a prevalence of endometriosis of 0.8% (Abbas et al., 2012, Aznaurova et al., 2014), while another study from 2013 says that 62% of the patients that complain of chronic pelvic pain or dysmenorrhea actually suffer from endometriosis (Janssen et al., 2013, Aznaurova et al., 2014). In the followed period of time, between January 2010 and December 2015, the incidence of endometriosis showed a highhly increasing tendency, translated by the number of new cases diagnosed every year. This increase was objectified by the number of cases diagnosed with the condition, respectively 149 cases in 2010, with a prevalence of 6.69%, figures that are consistent with the data in the literature. Regarding the year 2015, the number 7

9 of cases reached 232, representing a 13.98% percent of the total number of patients, showing a significant increase of the prevalence of endometriosis in patients from the geographical region of Moldova. Thus, endometriosis is a disease whose incidence is in a continue raise, whose etiology is not fully known and understood and more than that, with a major impact upon the physical, mental and social health. Regarding the incidence of endometriosis, I managed to update the existing literature data and also bring a personal contribution over this aspect, taking into consideration the demographic considerations of the study group. CHAPTER 7 THE IMPACT OF ENDOMETRIOSIS OVER THE PHYSICAL, MENTAL AND SOCIAL STATE OF HEALTH 7.1. Purpose of the study The main objective of this study was to quantify the impact of endometriosis upon the quality of life Material and method This was an observational study that included 205 patients that were hospitalized in the 1 st Department of Gynecology of the Clinical Hospital Cuza-Voda, Iasi, between the years 2014 and All the patients enrolled in the study signed a written consent, which was previously approved by the Ethical Medical Committee of the University of Medicine and Pharmacy Gr. T. Popa, Iasi. For the development of this study, I used the following types of questionnaires: - Fertility Problem Inventory (FPI); - Endometriosis health profile (EHP); - Beck Depression Inventory (BDI). All the questionnaires were given to the patients only after they were translated into Romanian and validated Results Fertility Problem Inventory Synthesizing, the highest scores of infertility are found in the Need of parenthood domain (66.83%) and Social domain (50.53%), significantly higher compared to the Sexual domain (47.21%), Relationship domain (49.63%) and Rejection of a childfree lifestyle domain (48.48%). According to the epidemiological characteristics, the stress generated by infertility was significantly higher in patients who: don t have children (54.66% vs 52.59%; p=0.008); smokers (53.53% vs 47.46%; p=0.001); and overweight women (53.28% vs 51.29%; p=0.011). Endometriosis Health Profile (EHP) Part 1. The quality of life Synthesizing, the biggest scores regarding the quality of life were found in the following domains: Control (80.81%), Pain (77.03%) and Emotional (70.08%), significantly higher compared with the Social support (65.49%) or Self-care (33.33%). 8

10 The uniform score for EHP ranged from 53.33% to 86%, showing a lower quality of life in 70.49% of the questioned women. Part 2. Endometriosis-related stress Synthesizing, the highest scores for the endometriosis related stress are found in the following domains: Fertility (79.39%), Treatment (61.46%) and Work (57.07%), significantly higher compared with other domains like: Child relationship (34.29%) or Medical professions (34.76%). The uniform EHP score for the evaluation of the endometriosis caused stress ranged between 48.70% and 63.48%, showing a high level of stress in 57.09% of the questioned women. Beck Depression Inventory (BDI) The BDI score varied from 21 to 40, showing severe depression in 98.5% of the patients and mild depression in 1.5%. The average BDI score was significantly higher in the group of patients over 35 years old (35.87 vs 32.46; p=0.001) and in the group of unmarried patients (36 vs 33.86; p=0,007). The average BDI score was slightly higher in women without children (34.20 vs 33.74; p=0.514). Smoking (33.05 vs 34.24; p=0.180) or an overweight status (34.63 vs 34.02; p=0.383) do not induce a higher BDI average score. The average BDI score was slightly higher in patients that didn t suffer a surgical procedure (34.39 vs 33.93; p=0.395) Discussions The patients diagnosed with endometriosis are confronted daily with several unpleasant situations that have a damaging impact over the quality of life, increase the stress caused by infertility, the depression and anxiety, as this study has shown. For the quantification of the quality of life in women with endometriosis I used the EHP questionnaire. Analyzing the data in this study, I consider that the results obtained are relevant and in the same time very worrying. The patients with endometriosis are confronted on a daily basis with a series of symptoms such as chronic pelvic pain, dyspareunia, dysmenorrhea, emotional instability and a high level of stress, all of these having a negative impact upon the quality of life. I evaluated the effect of endometriosis over the work capability of women, and noted that in 87.8% of the cases, the women felt guilty for not being able to fulfill their tasks, due to their condition. The percentage is comparable with the one found in other studies that show a percent of 51% women that complain about endometriosis affecting their daily activity (De Graaf et al., 2013). Other studies (Xiaoli et al., 2016) showed that the incapacity to conceive generates a high level of stress in infertile women, fact that was mentioned in the present study, too. The failure of the fertility treatments has a major psychological impact upon the affected women. Fagervold et al. showed in 2009 that 7.7% of the women with endometriosis went through a breakup due to endometriosis, results comparable with the ones in this study. I estimated that approximately 22% of the women had the relationship with their partner altered if they lost a critical day for conceiving. The 9

11 same percent declared they don t always feel support from their partner (Fagervold et al., 2009, De Graaf et al., 2013). I must emphasize the importance of the sexual aspect and its impact upon the life of the patients. Dyspareunia, lack of desire for intercourse, especially due to the fear of failure in conceiving a baby, shows big alterations regarding the sexual aspect. Most of the time, the main concern of the doctor is to treat the somatic symptoms of endometriosis, such as pelvic pain, dyspareunia, spotting or infertility, without giving much importance to the psychological impairment of these patients. As it has been shown, the BDI score demonstrated the presence of a severe depressive mood in 98.5% of the patients who suffered of endometriosis associated with infertility, and moderate depression in 1.5% of the cases. These results are extremely alarming and suggest the fact that endometriosis in association with infertility is a trigger factor for depression, and more than this, it sustains a depressive state on a long term. Concluding, the descriptive analysis upon the infertility-caused stress suggests that the factors associated with a high level of stress are sadness, pessimism, the feeling of failure, self-blaming, irritability, lack of determination, an altered self-image, work difficulties and fatigue. CHAPTER 8 OSTEOPONTIN-BIOMARKER IN ENDOMETRIOSIS 8.1. Purpose of the study The main objective of this study was to evaluate the levels of OPN in the serum of patients with endometriosis, to determine the variations of these levels according to the treatment they followed, either it was surgical or with progesterone, and to compare the results with the levels of OPN from the serum of healthy patients Material and method The present reseatch is structured as a case-control, prospective study, that tried to identify a series of risk factors that in a cumulative manner form the very basis of endometriosis etiology and pathophysiology. Within the research, I analyzed the OPN serum level in 52 patients forming the study group that were divided as following: 40 patients with endometriosis and 12 healthy patients. Each patient that was enrolled in the study signed a written consent that was previously approved by the Ethical Medical Committee of the University of Medicine and Pharmacy Gr. T. Popa, Iasi. The patients enrolled in this study were previously diagnosed with endometriosis and were hospitalized in the 1 st Department of Gynecology from the Clinical Hospital Cuza-Voda, Iasi, for further investigations and laparoscopy, between the years The main characteristics of the study group: Ø The patients that were investigated in M1 (the moment of diagnosis) patients that didn t followed any kind of treatment (40 patients with endometriosis and 12 patients that form the study group); 10

12 Ø The patients investigated in M2 (6 months after a clear diagnosis, in the day of the surgical procedure) have been divided in two subgroups: 16 patients that followed treatment with 0.075mg desogestrel daily, for 6 months; and 24 patients that did not followed any type of treatment prior to surgery; Ø The patients investigated in M3 (6 months after the surgical procedure) have been divided in two subgroups: 25 patients followed treatment with 0.075mg desogestrel on a daily basis, for 6 months following surgery (16 continued the treatment with desogestrel, while 9 patients started the treatment only after surgery); and 15 patients who did not followed any treatment after the surgical procedure Results Both preoperatively and postoperatively, the levels of OPN were some kind homogenous, registering only a slight decrease of the average intraoperative level ( vs ; p=0.992). In patients that received treatment with Cerazette, the average preoperative level of OPN was significantly higher compared with the one obtained in the patients without treatment (465 vs ; p=0.05), and the average value of OPN was significantly raised in patients that received postoperative treatment with Cerazette (1410; p=0,001) Discussions On the studied casuistry, it is visible that the treatment with progesterone, given both before and after the surgical procedure to patients with endometriosis, has an increasing effect over the OPN serum levels. The patients that only underwent surgery, had a low level of OPN 6 months after surgery (M3), compared to those who followed treatment with Cerazette after the surgical procedure. These results are somehow curious, since it seems that the OPN serum level is more influenced by progesterone than by the disease itself. Moszynski et al. in 2013, in a study on OPN, stated that the average level of OPN was significantly lower in the serum of patients with endometriosis than the ones with other types of benign ovarian conditions, with a reference value gap between pg/ml. These results are consistent with the ones obtained from the studied casuistry, in contrast to those obtained by Cho et al. in 2009, that showed a high level of OPN in the serum of patients with endometriosis, compared to the ones who didn t suffer from this condition. Although in the literature the theme of this causal association is largely debated, the results are controversial. The study that I conducted managed to establish the lack of a direct association between endometriosis and OPN, the results being inconsistent with the existing literature data. The lack of OPN specificity for endometriosis, objectified through the present study, proves that OPN is only a marker for inflammation, not for endometriosis per se. The present study shows the fact that OPN failed to prove its utility as a biomarker for endometriosis, the results obtained stating the fact that its serum levels are not influenced by the disease, no matter the extent. I support this affirmation on two pieces of evidence: firstly, the level of OPN postoperatively, moment in which the patient is at least on a theoretical level considered cured, doesn t seem to suffer big 11

13 variations compared to the control group, and secondly, the serum level of OPN seems to be rather than otherwise influenced by the treatment with progesterone. CHAPTER 9 HORMONAL THERAPY OF ENDOMETRIOSIS- PRE- AND POSTOPERATIVE RESULTS 9.1. Purpose of the study The main objective of the study was to evaluate the differences between the results of the treatment with desogestrel, GnRH analogues and COCs, regarding the improvement in symptoms, ultrasonographic aspect and a series of surgical parameters, like intraoperative hemorrhage, adhesion formation and the easiness of cyst dissection Material and method I conducted a retrospective study that included 300 patients diagnosed with ovarian endometriosis cysts that were hospitalized and underwent surgery in the Clinical Hospital Cuza-Voda, Iasi, between the years , and followed medical treatment with various progestins for 6 months before surgery. Each patient that was enrolled in the study signed a written consent that was previously approved by the Ethical Medical Committee of the University of Medicine and Pharmacy Gr. T. Popa, Iasi. I evaluated the cysts dimensions, the aspect, shape, vascularization, the uterus and the contralateral ovary, the uniqueness or on the contrary, the bilaterality of the cysts, the presence of sludge and horizontal levels. As a part of the protocol of investigations, CA125 level was also dosed in the serum of the patients. Preoperatively, the patients were divided in 3 subgroups, as following: 100 patients followed treatment with desogestrel (Cerazette); 100 of the patients were treated with COCs (0,02 mg ethinyl estradiol/3mg drospirenone); 100 patients did not follow any kind of medical treatment, because they refused it or they presented contraindications to it, and underwent only the surgical procedure. Postoperatively, the patients were divided as following: 100 patients continued the treatment with mg desogestrel daily (Cerazette); 10 patients were treated with COCs (0.02 mg ethinyl estradiol/3mg drospirenone); 10 patients followed treatment with triptorelin 3.75mg intramuscular, monthly (Dipherelin); 10 patients followed treatment with gosereline 3.6 mg, subcutaneous, monthly (Zoladex); 5 patients followed treatment with dienogest 2 mg, daily (Visanne); 75 patients did not follow any medical treatment; 12

14 Among the intraoperative aspects with a role in the staging of the disease, I inspected the peritoneal cavity, identified the pelvi-peritoneal adhesions, the endometrioma, its uniqueness or bilaterality, its chocolate-like content and other possible endometriotic lesions located on the parietal peritoneum, uterus, bladder, intestine, utero-sacrate ligaments. As much as I could, I excised the endometriotic lesions or I coagulated them. After the surgical procedure, the patients were reevaluated every three months through clinical examinations and endovaginal ultrasound. As a part of the protocol of investigations, CA125 level was also dosed in the serum of the patients. I analyzed the results and included them in the present research, for observing the following: The correlation between the CA125 serum marker both with endometriosis and OPN; The impact of hormonal therapy over the serum marker CA125 and OPN; The postoperative correlation between the hormonal treatment and the ultrasonographic changes of the cyst The possible improvement of the surgical parameters with a hormonal therapy; The effects of a continuous hormonal treatment using progesterone (desogestrel/dienogest) vs COCs (0.02 mg ethinyl estradiol/3mg drospirenone) vs GnRH analogues (dipherelin/goserelin) in the prevention of postoperative recurrence of ovarian endometriotic cysts Results CA125 serum evolution Preoperatively, CA125 levels registered increased values in 57.5% of the patients with endometriosis (p=0.001), showing an estimated risk 1.71 times higher (RR=1.71; IC 95%: ). Both preoperatively and postoperatively, the values of the CA125 serum levels were homogenous. Preoperatively, the average level of CA125 exceeded the reference limit (53.34 U/mL), registering a significant intraoperative decrease (36.42 U/mL) and also postoperative decrease (25 U/mL) (p=0.002). OPN evolution The patients that followed postoperative treatment with Dipherelin or Zoladex registered a medium level of OPN significantly lower than the one found in patients that followed treatment with Cerazette (381; 583 vs 1410; p=0.001), but significantly higher than de average OPN level of the patients that did not follow any treatment. Correlation between OPN and CA125 The correlation between CA125 and OPN was indirect both pre- and intraoperative: if 59.2% of the preoperative patients (r=-0.592; p=0.011) and 32,9% intraoperative associated increased values of OPN with low values of CA125 (r=-0.329; p=0.041), postoperatively, 38.2% of the patients associated increased values of OPN with increased values of CA125 levels, results with statistical signification. The most obvious improvement regarding the diminishing in the size of the cyst and the formation of the sludge, objectified by ultrasonography, and was observed in the 13

15 patients that followed treatment with desogestrel. The diminishing of vascularization and the appearance of horizontal levels were similar in both study groups. I analyzed all 300 patients in means of the surgical aspects, observing the level of bleeding on the cleavage plan between the cyst wall and normal ovarian tissue, the presence of adhesions, conservative treatment and the duration of the surgical procedure. Intraoperatively, a lower level of bleeding was observed by the time of the dissection of the cysts, due to a better cleavage plan between the cyst wall and normal ovarian tissue. These effects were more visible in the group of patients that were treated with desogestrel. Thus, the duration of the procedure was lowered. Due to the better cleavage plan, the cystectomy was easily done, diminishing by this the using of coagulation as a method of hemostasis, which would imply a higher rate of ovarian damage. In the desogestrel group, 70% of the patients became pregnant in the first year after laparoscopy; in the group that followed treatment with COCs, 67% became pregnant, and in the group without treatment, 63% managed to obtain a pregnancy in the first year after surgery. The recurrence rate of the cysts was significantly lower, 6% in the group with desogestrel, 7% in the group with COCs Discussions In this study, the progestin treatment manages to optimize the intraoperative conditions and decreases the recurrence risk of endometriosis. Treatment with desogestrel, for 6 months preoperatively, leads to the diminishing of the cyst diameter, of the intraoperative bleeding, improving by these the dissection, with a highly better result. This study showed that progesterone treatment improves the surgical technique, especially by lowering the bleeding and increasing the tissue laxity between the cyst capsule and normal ovarian tissue. Both desogestrel and COCs, prescribed for a minimum of 6 months preoperatively have an decreasing effect over the endometriosis cyst, diminishes the intraoperative surgery and improves the dissection, desogestrel having way better results than COCs. Desogestrel could be considered a preoperative adjuvant therapy. The medical treatment should contribute to the fulfillment of two main objectives: the reduction of pain for long periods of time and the prevention of the evolution of the disease in the time interval between the conservative surgery and the moment of the pregnancy. The medical treatment of endometriosis can include progestins or a hypoestrogenic therapy, such as GnRH: triptorelin 3.75 mg (Dipherelin) or goserelin 3.6 mg (Zoladex), all of these having similar medical effects. However, each of them has a series of distinctive particularities. The continuous treatment with desogestrel can prevent the recurrence of endometriosis cysts and the clinical symptoms associated with endometriosis. Desogestrel offers a series of benefits, being a well-tolerated drug that allows to securely postpone a pregnancy, without having the risk of a relapse (Carr et al., 2014). Previous studies showed that desogestrel, as well as oral contraceptives that contain desogestrel are not only efficient but also secure to use as therapy for the endometriosis linked pain, as a postoperative treatment (Razzi et al., 2007). In 2017, Tanmahasamut et al. conducted a double-blind, randomized study that showed how a daily dose of 0.075m desogestrel can be an effective postoperative 14

16 treatment for the prevention of the relapse of pain in patients with moderate-severe symptoms (Tanmahasamut et al., 2017). CHAPTER 10 THE INFLUENCE OF PROGESTERONE OVER THE IMMUNOHYSTOCHEMICAL MARKERS IN ENDOMETRIOSIS 10.1 Purpose of the study The main objective of the present research was to see the potential of progesterone treatment for patients with endometriosis, by observing the changes that occurred in the expression of the estrogen receptors (ER), the progesterone receptors (PR), B-cell lymphoma 2 (Bcl-2) and Ki-67 from the endometriosis tissue, in the stroma and endometrial glands Material and method This was a retrospective study that included 16 patients, who were given an ultrasound examination, with a diagnosis suspicion of endometriosis, that were further investigated by laparoscopy with biopsy, with a final diagnosis of endometriosis based on a histopathological examination, between the years All the patients enrolled in the study signed a written consent, which was previously approved by the Ethical Medical Committee of the University of Medicine and Pharmacy Gr. T. Popa, Iasi. The patients were diagnosed and hospitalized in the sight of the laparoscopic surgery in the 1 st Department Clinical Hospital Cuza-Voda, Iasi. The patients enrolled in the study were divided in two subgroups: the first group included 9 patients that followed progesterone treatment: 0.075mg desogestrel, daily (Cerazette), for 6 months before the surgical procedure, and the second group included 8 patients that did not followed any kind of treatment. Regarding the sampling and conservation processes of the human tissue fragments, ethical principles linked to the scientific research were met Results In tables no. 10.XVI and 10.XVII, I exposed the final results of the IHC reaction, in conformity with Allred and Suzuki semi-quantitative evaluation scores. Table 10.XVI. Concomitant summarization of the expression of ER and PR in the stroma and epithelium of the patients with and without treatment With treatment Without treatment No. ER PR ER PR E S E S E S E S 1 positive positive positive positive negative positive positive positive 2 positive positive negative positive positive positive positive positive 3 negative positive negative positive negative negative positive positive 4 positive positive positive positive positive positive positive positive 5 negative positive negative positive negative negative positive positive 15

17 6 negative positive negative positive negative positive negative positive 7 positive positive positive positive negative positive positive positive 8 positive positive positive positive / / / / 9 positive positive positive positive / / / / T * 6positive 3negative 9positive 5positive 4negative 9positive 2positive 5negative 5positive 2negative 6positive 1negative 7positive Table 10.XVII. Concomitant summarization of the expression of Ki-67 and Bcl-2 in the stroma and epithelium of the patients with and without treatment With treatment Without treatment No. Bcl-2 Ki-67 Bcl-2 Ki-67 E S E S E S E S 1 positive positive Negative positive negative positive positive positive 2 Positive positive positive negative positive negative positive positive 3 negative positive negative negative positive negative negative negative 4 positive positive negative negative positive positive positive positive 5 negative positive negative negative negative negative positive negative 6 negative positive negative negative negative positive negative positive 7 positive positive negative positive negative negative positive positive 8 positive positive positive negative / / / / 9 positive positive negative negative / / / / T 6positive 3negative 9positive 2positive 7negative 2positive 7negative 3positive 4negative 3positive 4negative 5positive 2negative 5positive 2negative The evaluation of the IHC reaction by determining the expression of the histological markers was made by noting the percentage of positive cells. In table no. 10.XVIII, I noted the average percentages obtained for each marker in the stroma and epithelium, in patients with and without treatment. Table 10.XVIII. The percentage assessment of the expression of the histological markers in women with and without treatment ER (%) PR (%) Bcl-2 (%) Ki-67 (%) Without epithelium 25,8 53,6 24,4 8 treatment stroma 45,8 71,4 27,3 20 With treatment epithelium 23, ,1 1,2 stroma 44, ,8 0,6 low high 16

18 10.4. Discussion Brandenberger et al. in 1999 and then Brătilă et al. in 2015 were concluding that ER and PR expressions were significantly modified in the ectopic endometrial tissue, compared to the normal endometrium. Through the results of this study, regarding the patients that followed treatment with desogestrel, I demonstrated that the PR expression was significantly increased in the stroma, in comparison with those that did not followed a medical treatment (p=0.025). This fact is highly suggestive for a rising of the progesterone sensibility in the stroma. In the study conducted by Brătilă et al. in 2015, the ER expression was lower than the one of the PR, results consistent with the ones presented in this study. The present research stated that oral progesterone has a limited effect over the ER both in the stroma and epithelium, and the expression of these receptors is not influenced by the treatment. Thus, treatment with desogestrel seems to be ineffective over the ER, on the used dosage. In 2017, Tanmahasamut et al. conducted a double-blind, randomized study that showed that a daily 0.075mg dose of desogestrel is an efficient preoperative treatment in the prevention of the endometriosis-linked pain relapse, in patients with moderate to severe symptoms (Tanmahasamut et al., 2017). The study conducted by Goumenou et al. in 2004 that compared the apoptosis level with the expression of Bcl-2 in the stromal and epithelial cells in women with ovarian endometriosis, concluded that the stromal cells have a high apoptosis level, but a low Bcl-2 expression, compared to the glandular cells, showing that there is no difference between apoptosis and the Bcl-2 expression in women with endometriosis (Goumenou et al., 2004, Braun et al. 2007). Nguyen et al. in 2016 showed in his study that the percentage of Ki-67 positive cells was significantly lower both in the epithelial and stromal cells of the endometriosis cysts, in women that followed treatment with dienogest (Nguyen et al., 2016). The present study shows that an oral dose of 0.075mg desogestrel, given daily for 6 months, dramatically decreases the Ki-67 expression both in the endometrial glands and especially in the stroma of the endometriosis cyst (from an average of 20% of positive cells, to 0.66% positive cells in the stroma). This fact proves that exogenous progesterone has a strong inhibitory action over the cellular proliferation. This inhibitory action over the stroma in the wall of the endometrioma, that comes in direct contact with normal ovarian stroma, is clinically seen as a stagnation or even decrease in the dimensions of the cyst and also as an improvement of the intraoperative environment, especially as a better dissection of the cyst s wall, and an increasing in the laxity of the tissues that lay between the cyst s wall and normal ovarian tissue. The results of this study are new evidences over the utility of progesterone treatment over endometriosis, and are an impulse to continue the research in this field, for a more precise identification of the underlying mechanisms that characterize this condition. The oral treatment with 0.075mg desogestrel proved its benefits over the endometrioma, by acting on a molecular level, increasing the expression of PR and Bcl-2 and decreasing Ki-67, effects that are seen in the clinical practice as an improvement of the symptoms, a decrease in the dimensions of the cyst and an improvement of the operative environment. 17

19 CHAPTER 11 CONCLUSIONS 1. The incidence of endometriosis was approximated at 9.03%, with an increasing rate in the past 6 years, most probably due to the improvement in the diagnosis techniques and also due to the increasing preoccupation of the patients for the infertility topic. 2. Demographically speaking, the patient with pelvic endometriosis is an urban living, married, with higher education, with a high socio-economical level woman, that complains of chronic pelvic pain, dysmenorrhea, dyspareunia and/or infertility. 3. The patients with endometriosis are fighting daily with a series of symptoms like chronic pelvic pain, dyspareunia, dysmenorrhea, emotional instability and a high level of stress, all of these having a negative impact over the quality of life. 4. The BDI score showed the presence of a severe depressive condition in 98.5% of the patients with endometriosis associated with infertility and moderate depression in 1.5% of cases. 5. The descriptive analysis over the infertility associated stress suggests that the associated factors with a high level of stress are sadness, pessimism, the feeling of failure, self-blaming, irritability, lack of determination, an altered self-image, the work difficulties and fatigue. 6. On the studied casuistry, it is shown that in the patients with endometriosis, the progesterone treatment before or after the surgical procedure increases the OPN serum level. 7. The patients that were treated only by surgical means, have a low level of OPN, 6 months postoperatively, compared to the ones that followed treatment with Cerazette after surgery, the average level of OPN in the last subgroup being times higher than in the subgroup without treatment. 8. The present research shows the fact that OPN did not proved its utility as a biomarker in the investigation protocol of endometriosis, the results suggesting that its serum levels are not correlated with the expression of the disease in the body. 9. Treatment with desogestrel for 6 months preoperatively determined a decrease in the cyst diameter and in the intraoperative hemorrhage level, improving by these the dissection environment, with better results. 10. Treatment with mg oral desogestrel managed to prove its benefits over the endometrioma, acting on a molecular level, by increasing the PR expression, the Bcl-2 and decreasing Ki-67, effects that are clinically seen as an improvement of the symptoms, with a decrease of the cyst diameter and an improvement of the intraoperative environment. 18

20 CHAPTER 12 ORIGINALITY ASPECTS AND PERSPECTIVES OPENED BY THE THESIS The originality aspects of the present thesis are seen both in its novelty character and in the approaches that I used in the attempt to explore the study s topic. Along the review of the literature, I performed an empirical research, approaching a topical theme, endometriosis being one of the medical enigmas, a debatable theme that continues to surprise through its novelty elements that come to surface, often succeeding to mislead even the ones that are closely investigating it. Also, the impact of endometriosis over the patients was detailed in the present thesis, with a broad presentation of the results that have a major statistical signification, especially regarding the causality link between endometriosis and depression. It is important to mention as an originality mark of this thesis the fact that the materials used for the accomplishment of this study-the EHP, FPI and BDI questionnaires, although highly known on an international level as quantification tools for the quality of life, were for the first time translated, validated and applied in Romania, as a part of this study. Both the theme of the incidence of endometriosis and the one of the impact of the disease over women were studied on big groups of patients. However, they were limited by the geographical distribution of the patients, all of them living in the Moldova region. That is why as future perspectives opened by the thesis I can mention the necessity of a multicenter study that would integrate all the Romania s regions, for a better view of these parameters on a national level and for a comparison between the regional centers and why not, a comparison with the international studies. Also, a multicenter study has the advantage to identify some possible new risk factors for endometriosis. Another highly original study that was incorporated in this thesis was the one of a possible association between endometriosis and OPN, with a possible use of OPN as a serum marker for the detection of the disease. Although this possible association is largely debated in the literature, the results are contradictory. The study I conducted established the lack of association between endometriosis and OPN, results that are somehow opposed to those in the literature. The lack of OPN specificity for endometriosis is objectified in this study, proving that OPN is just an inflammatory marker. Another worth mentioning aspect as an originality mark is the impact of progesterone treatment over the clinical presentation and the intraoperative environment, with an improvement of the last one, and the quantification of the imagistic aspects of endometriosis cysts, with its improvement after the therapeutic cure with progesterone. The only published Romanian study that investigates a series of histologic markers and their roles in endometriosis, is the one conducted by Brătilă et al. in 2015, mentioning the fact that none of the patients included in the study followed medical treatment. The results I obtained following the IHC analysis were concordant with the study from Compared to it, the present research comes with an additional exposure regarding the evolution of these histological markers successive to progesterone treatment. Thus, the increasing of the PR expression, the decreasing of the Ki-67 proliferation factor and the increasing of the anti-apoptotic marker Bcl-2 as 19

21 a support form for the progesterone action over the first two markers is only a small part of the data that are present in this study. Also, the lack of change in the ER expression opens up the prospect of future treatments that can supplement and potentiate the progesterone treatment, without having bad interactions with it. Finally, I consider that I evoked all of these aspects in a highly academic manner, with the fulfillment of desiderates that I submitted in the beginning of this thesis. SELECTIVE BIBLIOGRAPHY 1. Abbas S, Ihle P, Koster I, Schubert I. Prevalence and incidence of diagnosed endometriosis and risk of endometriosis in patients with endometriosisrelated symptoms: findings from a statutory health insurance-based cohort in Germany. Eur J Obstet Gynecol Reprod Biol. 2012; 160: Aznaurova YB, Zhumataev MB, Roberts TK, et al. Molecular aspects of development and regulation of endometriosis. Reprod Biol Endocrin. 2014; 12: Brandenberger AW, Lebovic DI, Tee MK et al. Oestrogen receptor (ER)- alpha and ER-beta isoforms in normal endometrial and endometriosis-derived stromal cells. Mol Hum Reprod. 1999; 5(7): Braun DP, Ding J, Shaheen F et al. Quantitative expression of apoptosisregulating genes in endometrium from women with and without endometriosis. Fertil Steril. 2007; 87(2): Brătilă E, Brătilă CP, Comandașu DE et al. The assessment of immunohistochemical profile of endometriosis implants, a practical method to appreciate the aggressiveness and recurrence risk of endometriosis. Rom J Morphol Embryol. 2015; 56(4): Bulun SE. Endometriosis. N. Engl. J. Med. 2009; 360: Carr B, Dmowski WP, O Brien C et al. Elagolix, an oral GnRH antagonist, versus subcutaneous depot medroxyprogesterone acetate for the treatment of endometriosis: effects on bone mineral density. Reprod Sci. 2014; 21(11): Cho S, Ahn YS, Choi YS et al. Endometrial osteopontin mrna expression and plasma osteopontin levels are increased in patients with endometriosis. Am J Reprod Immunol. 2009; 61: Cramer DW, Missmer SA. The epidemiology of endometriosis. Ann N Y Acad Sci. 2002; 955: Discussion 34-16, De Graaff AA, D Hooghe TM, Dunselman GA, et al. The significant effect of endometriosis on physical, mental and social wellbeing: results from an international cross-sectional survey. 2013; 28(10): Fagervold B, Jenssen M, Hummelshoj L, Moen MH. Life after a diagnosis with endometriosis a 15 years follow-up study. Acta Obstet Gynecol Scand. 2009; 88: Giudice LC. Clinical practice. Endometriosis. N. Engl. J. Med. 2010; 362(25):

22 13. Goumenou AG, Matalliotakis IM, Tzardi M et al. Apoptosis and differential expression of apoptosis- related proteins in endometriotic glandular and stromal cells. J Soc Gynecol Invest. 2004;11: Janssen EB, Rijkers AC, Hoppenbrouwers K et al. Prevalence of endometriosis diagnosed by laparoscopy in adolescents with dysmenorrhea or chronic pelvic pain: a systematic review. Hum Reprod Update. 2013; 19(5): Moszynski R, Szubert S, Szpurek D et al. Role of osteopontin in differential diagnosis of ovarian tumors. Obstet Gynaecol Res. 2013; 39(11): Nisolle M, Donnez J. Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the recto-vaginal septum are three different entities. Fertil Steril. 1997; 68: Nguyen TT, Hachisuga T, Urabe R et al. Immunohistochemical analysis of the effect of dienogest on ovarian endometriotic cysts. J Uoeh. 2016; 38(4): Razzi S, Luisi S, Ferretti C, et al. Use of a progestogen only preparation containing desogestrel in the treatment of recurrent pelvic pain after conservative surgery for endometriosis. Eur J Obstet Gynecol Reprod Biol. 2007; 135: Shroen D. Disputatio inauguralis medica de ulceribus uteri. Jena:Krebs. 1690; Signorile PG, Baldi A. Endometriosis: new concepts in pathogenesis. Int J Biochem Cell Biol. 2010; 42: Streuli I, Ziegler D, Santulli P, et al. An update on the pharmacological management of endometriosis. Expert Opin Pharmacother. 2013; 14: Tanmahasamut P, Saejong R, Rattanachaiyanont M et al. Postoperative desogestrel for pelvic endometriosis-related pain: a randomized controlled trial. Gynecol Endocrinol. 2017; doi: / Vercellini P, Crosignani CG, Somigliana G et al. Waiting for Godot : a commonsense approach to the medical treatment of endometriosis. Hum Reprod. 2011; 26(1): Xiaoli S, Mei L, Junjun B et al. Assessing the quality of life of infertile Chinese women: a cross-sectional study. Taiwan J Obstet Gynecol. 2016; 55(2):