Retrospective study of the factors involved in the development of adenomyosis and the in vitro link between adenomyosis and breast cancer

Adenomyosis is a frequent benign uterine pathology in adult women, which is characterized by the presence of endometrial tissue (glands and stroma) in the myometrium. Women suffering from this condition may experience dysmenorrhea, abnormal uterine bleeding, pelvic pain or infertility, however, one third of cases are asymptomatic [ 1 ]. Until not long ago, the diagnosis of this disease could only be made retrospectively through the histological examination of the uterus, following hysterectomy. This pathology has become increasingly common in young women, and the new imaging techniques such as magnetic resonance imaging (MRI) and transvaginal pelvic ultrasound (TVUS) are taking steps towards diagnosing the disease, which is still little known [ 2 ]. The first information about the disorder appeared in 1860 when the German doctor Carl von Rokitansky noticed the presence of endometrial glands in the myometrium [ 3 , 4 ]. Later, in 1925, Frankl used the term “adenomyosis”, following that in 1972 the definition was reproduced by Bird et al. as “benign endometrial invasion into myometrium, diffusely enlarged uterus, ectopic, neoplastic endometrial glands, and stroma surrounded by hypertrophic and hyperplastic myometrium” [ 5 ]. The pathogenic mechanisms of adenomyosis are not fully elucidated, but in recent years it has been observed that inflammatory molecules, extracellular matrix enzymes, sex hormone receptors, as well as neuroangiogenic factors and growth factors play an important role [ 6 ]. Adenomyosis is considered to originate from the basal invagination of the endometrium into the myometrium, an invagination that occurs along the intramyometrial lymphatic system [ 7 ]. Another theory is that the condition may arise de novo from a metaplastic process starting from intramyometrial endometrial tissue [ 8 ]. Adenomyosis is a pathology characterized by hypertrophic or hyperplastic myometrium surrounding the stroma and glands, causing either diffuse or focal expansion of the uterus. Diffuse adenomyosis was defined as a widespread invasion of the basal glands in the myometrium, while focal adenomyosis was characterized by lesions in the inner, middle or outer myometrium, involving the posterior or anterior wall of the uterus [ 9 ]. The most diagnosed form of adenomyosis is the diffuse one, while the cystic variant, focal adenomyosis, is much rarer, found especially in women under 30 years of age [ 10 ]. In recent years, adenomyosis has transformed from a histopathological entity into a clinical condition, diagnosed by imaging techniques [ 11 ]. Transvaginal ultrasound has become the main analysis in the diagnosis of adenomyosis. Because adenomyosis is a gynecological condition that affects women of reproductive age, causing high rates of ovum implantation failure, pregnancy loss or premature birth [ 12 ], it has received special attention among medical specialists. Adenomyosis-related cancer is extremely rare; it occurs only in 1% of cases, especially in elderly people, but it can also be due to this disease [ 13 , 14 ]. Adenomyosis and cancers at this level present common manifestations, but the incriminating pathogenic mechanism of malignant changes remains unknown. A knowledge of the process by which the disease develops helps diagnose and institute the correct therapy [ 15 ]. Sex hormone imbalance and inflammation are the most known factors that promote the invasion of the myometrium with endometrial cells or the modification of Müllerian remnants in adenomyosis [ 16 ]. These factors are also involved in benign and malignant breast pathologies [ 17 ]. Breast cancer is one of the most common malignant pathologies worldwide [ 18 ]. Breast cancer is a heterogeneous pathology, with different factors that play a role in its development, such as age, personal history, and exposure to sex hormones [ 19 ]. The implication of sex hormones in the development of breast cancer lies in their ability to stimulate cell division [ 20 ]. Moreover, a study showed that women who underwent progestin treatment had a higher risk of developing breast cancer compared to women who administered estrogen-only medication [ 21 ]. Currently, the causes of the development of adenomyosis are not fully known, and this research aims to collect evidence in this regard. Our study focuses on the evaluation of the symptoms and more precisely the risk factors that influence the development of adenomyosis; information that will strengthen knowledge about this increasingly common disease. Further, following the data obtained in the retrospective study, we aimed to evaluate in vitro the relationship between levonorgestrel, which can influence the occurrence of adenomyosis, and breast cancer.

Our clinical study was carried out over a period of 9 months (January–September 2023) in the Obstetrics-Gynecology Clinic of the “Pius Brinzeu” County Emergency Clinical Hospital in collaboration with the “Victor Babes” University of Medicine and Pharmacy. The protocol was approved by the Scientific Research Ethics Committee of the “Victor Babes” University of Medicine and Pharmacy. The study method consisted in analyzing the following parameters: age, demographic data, symptoms, obstetric and gynecological antecedents - childbirths/abortions, associated pathologies and treatment with progestogens. The analyzed group included patients hospitalized between January and September 2023 in which the diagnosis of adenomyosis was confirmed in all cases by ultrasound. We therefore retrospectively analyzed all cases in which a hysterectomy was performed and adenomyosis was confirmed by ultrasound examination. Patients included in the study were selected based on strict inclusion and exclusion criteria. patients admitted to the Obstetrics-Gynecology Clinic of the “Pius Brinzeu” County Emergency Clinical Hospital patients ≥ 18 years old confirmed diagnosis of adenomyosis patients with incomplete available data Levonorgestrel, trypsin-EDTA solution, and dimethyl sulfoxide (DMSO) were acquired from Sigma Aldrich, Merck KgaA (Darmstadt, Germany). The trypsin-EDTA solution, fetal bovine serum (FBS), penicillin/streptomycin combination, and the specific cell medium, Dulbecco’s Modified Eagle’s medium (DMEM) were procured from Pan-Biotech GmbH (Aidenbach, Germany). The MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) kit was purchased from Roche Holding AG (Basel, Switzerland), and the lactate dehydrogenase (LDH) kit was bought from ThermoFisher Scientific (Waltham, MA, USA). All reagents utilized were of high analytical standard purity. The MDA-MB-231 (ATCC HTB-26™) breast cancer line was purchased from ATCC (American Type Cell Collection, Lomianki, Poland). The in vitro analyses were carried out on the MDA-MB-231 breast adenocarcinoma cell line grown in Dulbecco’s Modified Eagle’s medium supplemented with 10% FBS, and 1% penicillin/streptomycin (100 U/mL/100 μg/mL). The cells were cultured and kept under standard conditions (5% CO 2 and 37 °C) in a humidified atmosphere. Cell viability was analyzed by the MTT assay. MDA-MB-231 cancer cells were seeded in 96-well plates (1 × 10 4 cells/100 μL/well) and were stimulated with two concentrations of levonorgestrel (LG) solution (0.5 μM and 15 μM) in DMSO for 24 and 48 h. After the stimulation period, 100 μL of fresh media and 10 μL of tetrazolium salt solution (10 μL) were added to each well and incubated for 3 h. Lastly, 100 μL of solubilization buffer was added for 30 min, at room temperature, without light. The absorbance values for reduced MTT were read at 570 nm using Cytation 5 from BioTek Instruments Inc. (Winooski, VT, USA). To complement the results of the MTT test, a possible cytotoxic effect of LG on MDA-MB-231 cells was investigated using the LDH method. Therefore, the cells were grown under the same conditions as for the MTT method (1 × 10 4 cells/100 μL/well) and then the cells were stimulated with the two concentration of LG solution for 24 h and 48 h, respectively. After the stimulation period, a volume of 50 μl from each well was dispensed into a new plate (96 wells), on top of which 50 μL of the reaction mixture was placed for 30 min at room temperature. Finally, 50 μL of stop/well solution was added. Absorbances were measured at 490 and 680 nm, respectively, using Cytation 5 from BioTek Instruments Inc. In vitro results are expressed as means ± standard deviation (SD). The software GraphPad Prism version 9.4.0 for Windows (GraphPad Software, San Diego, CA, USA) was used for data interpretation. One-way ANOVA test and Dunett’s multiple comparisons post-test were conducted to compare the results. The statistically significant differences between results were marked with * (** p < 0.01, *** p < 0.001, and **** p < 0.0001).

To begin with, our study aimed to identify as much objective data as possible, to understand the predisposing factors in the development of adenomyosis, which can be helpful in future cases. It should be remembered that the treatment in the case of adenomyosis is surgical, and the dominant symptoms of this condition are often common to other pathologies that are easier to diagnose and which, in most cases, have other therapeutic methods. The group of patients analyzed, during the 9 months of the study, includes 99 patients who underwent hysterectomy interventions. Out of a total of 99 hysterectomies performed during this period, the diagnosis of adenomyosis was confirmed by ultrasound in 28.28% of cases ( Table I ). The other hysterectomies were performed for different pathologies, mainly for uterine fibroids, uterine prolapse and atypical endometrial hyperplasia. Figure 1 presents the analysis of the incidence of hysterectomies during the study period and highlights the prevalence of the diagnosis of adenomyosis from these interventions. Following the graphic representation, we can observe a Gaussian distribution of the diagnosis of adenomyosis, with most cases being observed in April. The clinical observations, the enlarged uterus with dysmenorrhoea and menorrhagia, suggest adenomyosis, but a clear diagnosis is needed, and an initial step is done through the ultrasound examination. Ultrasound is the most used technique in the diagnosis of gynecological pathologies because it is easily available in the outpatient clinic, it is relatively less expensive than other imaging techniques. In our study, we evidence the changes observed in a 40-year-old patient diagnosed with diffuse adenomyosis. More precisely, there were differences in the thickness of the anterior wall compared to the posterior one, a mixed sandy appearance, and possibly also cystic adenomyosis in the thickness of the anterior wall, as can be seen in figure 2 . In addition, the patient had severe menstrual pain and prolonged menstruation. The next two images ( Figure 3a . and Figure 3b .) are from another patient, diagnosed with focal adenomyosis, in which the difference in thickness between the anterior wall and the posterior uterine wall, the hypervascularization of the uterine walls, the mixed sandy ultrasound appearance and the change in the trajectory of the endometrium can again be observed. The linear appearance of the uterine cavity changes into an S-shaped appearance due to the infiltration of the endometrial glands at the level of the myometrium. In addition to the clear appearance of adenomyosis, the presence of two intramural fibromatous nodules can be noted, which may have a component of cystic adenomyosis. In image 3a, the difference in thickness between the anterior and posterior uterine wall can be seen, and this is also confirmed in image 3b with vascular Doppler. Furthermore, the ovaries cannot be visualized, a proof that the pathology has not extended to the ovaries. In this study, once adenomyosis was confirmed, we wanted to identify the risk factors that could lead to the development of this pathology. Although it is considered that older age can have a direct impact on the appearance of the disease, among our patients we can see that most cases developed in women between 40 and 45 years old, more precisely 53.57% cases out of the 28 diagnosed ( Figure 4 ). A first analysis was performed regarding the patients’ environment of origin, it was compared whether the rural or urban environment can have a direct influence on the occurrence of adenomyosis. From our data, 15 cases of adenomyosis were observed from rural areas and 13 from urban areas. The area of origin could be considered a factor in the development of this pathology, but the results are insignificant ( Figure 5 ). A series of data were extracted from the patient’s file, from symptomatology to personal history and comorbidities; based on which conclusions were drawn regarding the impact on the development of adenomyosis ( Table II ). In the present case, the patients complained of painful symptoms, such as dysmenorrhea, deep dyspareunia, and pelvic pain. One of the 28 women diagnosed with adenomyosis was asymptomatic, while the others presented in most cases all the other 3 symptoms (71.42%). Age, various uterine operations, and previous pregnancies can influence the appearance of adenomyosis. Thus, it was observed that the development of adenomyosis was associated with the number of births and was more frequent in the case of abortions. Multiple pregnancies can influence the development of this uterine pathology. In our study, out of the 28 patients diagnosed with adenomyosis, 32.14% of these are multiparous women, 60.71% are primiparous women and 7.14% are women without pregnancy up to that point. We notice that most women had only one pregnancy but were still diagnosed with adenomyosis. This aspect may be due to the fact that of the 17 primiparous women, 61.58% gave birth by cesarean section, and 3 of the patients had twin pregnancies, thus having a history of uterine operations. Therefore, it is not clear whether previous uterine surgical interventions act directly on the development of adenomyosis, but there is enough data to show that it can influence the appearance of this pathology. Moreover, among our patients, it became evident that the age between 40–45 years is the most prone to the occurrence of adenomyosis because half of them also went through an abortion, be it surgical, medicinal, or spontaneous. As can be seen in the current study, most people diagnosed with adenomyosis also had other associated pathologies, the largest proportion being represented by urogenital disorders. Noting that most of the patients had at least three associated pathologies, we can say that coexisting pathologies can influence the development of adenomyosis. Since it is known that sex hormones can influence the development of breast cancer and observing that a large number of patients were receiving treatment with progestogens, we chose the most used molecule and tested it further in vitro on the triple-negative mammary cancer cell line. Initially, the MTT test was performed to evaluate the impact of LG on cell viability on the MDA-MB-231 cancer line after 24 and 48 hours of treatment. The obtained data show us that at the two concentrations tested, LG stimulates the proliferation of breast cancer cells depending on the concentration and exposure periods. Thus, after 24 hours, an increase in cell viability of up to 126% for the highest concentration was highlighted, and after 48 hours up to 120% for the concentration of 0.5 μM and 139% for 15 μM, as can be seen in Figure 6 . We can state from the obtained results that LG can stimulate the proliferation of MDA-MB-231 breast cancer cells. To strengthen the results previously obtained with the help of the MTT method, we performed the LDH cytotoxicity test. According to the data, we observe that the tested concentrations of LG do not have a cytotoxic effect on MDA-MB-231 cells neither at 24 h nor at 48 h of stimulation. After a 48-hour stimulation, at the highest concentration a value of approximately −15% was obtained, which indicates that LG does not have a cytotoxic effect on the MDA-MB-231 tumor line ( Figure 7 ).

Age (old age), is a risk factor for adenomyosis, demonstrated in various epidemiological studies, thus more than 70% of women undergoing hysterectomy after adenomyosis are in their fifth decade of life and multiparous [ 22 – 24 ]. However, adenomyosis can be found in women at the beginning of childbearing age [ 25 ]. Following a study conducted on women between 18 and 30 years old, it was found through ultrasound diagnosis that more than 30% of them show signs of adenomyosis, and in another study on women under 40 years old, it was observed through MRI, the presence of pathology in over 55% of cases [ 26 , 27 ]. The reports suggest that this pathology causes dysmenorrhea and pelvic pain in teenage girls [ 28 ], so we can conclude that the clinical age at the diagnosis of adenomyosis can be much earlier than previously estimated, as was also observed in the present study. Our results can be correlated with the data from the literature, thus observing that the symptomatology known until now for adenomyosis is similar to that observed in the case of our patients. Several studies claim that pelvic pain is common to adenomyosis, which occurs in approximately 50% of diagnosed women, and the intensity of the pain can be associated with the depth of myometrial invasion of ectopic endometrial areas [ 29 , 30 ]. Pelvic pains from adenomyosis can be due either to a weakened immune system that secretes neurogenic and algogenic substances [ 31 ] or due to increased contractility of the myometrium following the response to immune and hormonal changes [ 32 ]. Abundant menstruation and intermenstrual bleeding are common in patients with adenomyosis. It was found that the frequency of these bleedings increases the more the myometrium is affected by ectopic entities. Abnormal bleeding can be due to tissue damage, damage that occurs as a result of cyclic bleeding of the ectopic endometrium, which leads to inflammation and the activation and disruption of the vascular system at the local level [ 33 ]. Human studies indicate that adenomyosis could be associated with fertility impairment, but it is insufficiently described and the mechanisms involved are unclear. According to a meta-analysis, the prevalence of adenomyosis in infertile women undergoing fertilization varied from approximately 7% to 35% [ 34 ]. In animals, a closer link between adenomyosis and infertility has been observed. Thus, in a study conducted on 37 baboons with histologically confirmed adenomyosis, the prevalence of infertility was greatly increased in the presence of adenomyosis [ 35 ]. Infertility can be due to immunological changes in the ectopic endometrium, local estrogenic growth, alteration of cell proliferation, and cell adhesion to the endometrium, which lead to implantation abnormalities [ 16 ]. Studies in the literature report that a large percentage of women with adenomyosis are multiparous [ 22 , 23 ]. The possible explanation lies in the fact that pregnancy can facilitate the formation of the disease, by including the adenomyotic foci in the myometrium due to the invasive character of the trophoblast on the prolongation of the myometrial fibers [ 24 ]. Moreover, the adenomyotic tissue may have a greater number of estrogen receptors and the high hormonal titer may favor the development of the ectopic endometrium; knowing how many changes can occur in a pregnant woman [ 36 ]. There are studies in which women diagnosed with adenomyosis have not previously undergone any surgical intervention, there being no direct association between them [ 22 , 37 ]. However, in the study conducted by Whitted et al. an increased prevalence of adenomyosis was observed in patients who had previously undergone a cesarean section, data was also obtained in our study [ 38 ]. Moreover, other study exposed the fact that women who suffered from termination of pregnancy through different methods, the most common being curettage, presented higher rates of adenomyosis compared to women who did not go through such interventions [ 39 ]. The disease may present as one or a combination of thickening of the internal myometrium, areas of focal or diffuse disease, and involvement of the external myometrium. One idea is that trauma occurs at the endometrial myometrial interface encouraged by increased peristalsis. This process results in local trauma and a repair mechanism that leads to increased local estradiol levels that further accentuate hyperperistalsis and increased local damage that allows endometrial invasion into the myometrium [ 8 , 40 ]. Several ultrasonographic criteria have been used to diagnose adenomyosis, including the enlargement of the uterus, the asymmetry of the thickness of the uterine wall, the presence of heterogeneous myometrial areas, and the discovery of myometrial cysts [ 41 ]. In particular, the performed ultrasounds describe adenomyosis by highlighting myometrial cysts, by the presence of poorly defined myometrial heterogeneity, and by foci of abnormal myometrial echotexture. Hysteroscopy is a major diagnostic and therapeutic tool for uterine conditions. The standard treatment for adenomyosis is hysterectomy, which was also used in our patients. However, this method cannot always be considered, especially when the woman wants to maintain her fertility or cannot undergo such surgery due to complications. Adenomyosis is a challenge in the field of gynecology and often hysterectomy is used to treat premenopausal and perimenopausal women. Hormonal suppressive treatments, such as oral contraceptives, high doses of progestogens, and hormone-releasing intrauterine devices, are known to temporarily stop the regression of the pathology and can be used in this sense [ 42 ], but may increase the risk of breast cancer [ 43 ], as highlighted in our preliminary in vitro study. In our study, hysterectomy was resorted to with the approval of the patients, the great majority already having a child, being only two cases of nulliparous women. The present in vitro study was conducted to investigate the impact of the synthetic sex hormone levonorgestrel on the behavior of MDA-MB-231 breast cancer cells, to identify a link in the development of breast cancer. The data reported that LG stimulates the proliferation of breast tumor cells depending on concentration and time; the highest values were observed when stimulated with the highest concentration of synthetic hormone after a 48-hour application. Sex hormones play an important role in the etiopathogenesis of several types of cancer, including breast cancer [ 44 ]. Moreover, oral contraceptives with synthetic estrogens and progestogens are currently considered promoters of breast cancer progression [ 45 ]. In a study by Hasan et al., a concentration-dependent stimulatory effect of cell proliferation (1 pM-10 μM) of both progesterone and estrogen was evident on the MCF-7 breast cancer cell line after a 24-hour exposure h, but on the MDA-MB-231 cell line, the effect was not dependent on the dose [ 46 ]. In another study, a high percentage of cell proliferation was observed in two sex hormone receptor-positive breast cancer cell lines (HCC1500 and ZR75-1) following treatment with estradiol and ethinylestradiol, but also a negative influence on the expression of apoptotic markers and proliferative [ 47 ]. On the other hand, Zhou and co-workers obtained a concentration-dependent inhibition of the proliferation of MDA-MB-231 cells following the application of progesterone, but at lower concentrations (20–80 ng/mL) [ 48 ]. Moreover, the results obtained by Kadioğlu et al. showed that patients with adenomyosis present a high risk of high mammographic breast density [ 49 ]. This increase in breast density is closely related to the risk of breast cancer [ 50 ].

Clinically, adenomyosis is a heterogeneous disease, which differs from patient to patient. The specialized literature and our study reported the fact that clinical symptoms along with risk factors and more so personal antecedents can influence myometrial lesions, leading to diffuse or focal adenomyosis. Therefore, these characteristics may vary depending on the phenotype of the pathology. Regarding the results of the in vitro evaluation of the synthetic sex hormone, levonorgestrel, we can state that it can stimulate the proliferation of MDA-MB-231 breast cancer cells at higher concentrations and with prolonged exposure. Moreover, the preliminary in vitro study is the first step in making a connection between the impact of adenomyosis in the development of breast cancer. However, this aspect requires a thorough evaluation in the future to be able to say with certainty if a factor that influences the appearance of adenomyosis has a direct impact on the development of breast cancer. Finally, some limitations should be noted. The limitations of the present study refer to the small number of patients included and the heterogeneity of the risk factors highlighted in the group. Therefore, multicentric prospective studies are warranted.