Abnormal uterine bleeding: The well-known and the hidden face.

Abnormal uterine bleeding (AUB) is a bleeding from the uterine corpus that is abnormal in regularity, volume, frequency or duration. It encompasses heavy menstrual bleeding, irregular menstrual bleeding and intermenstrual bleeding, which are common symptoms among women of reproductive age, impacting their overall well-being. Menstruation involves interactions between endometrial epithelial and stromal cells, immune cell influx, and changes in endometrial vasculature. These events resemble an inflammatory response with increased vessel permeability, tissue breakdown, and the arrival of innate immune cells. However, the mechanisms of menstrual cessation are poorly understood. AUB can be related to structural causes (polyp, adenomyosis, leiomyoma, malignancy/hyperplasia) and nonstructural conditions (coagulopathy, ovulatory dysfunction, endometrial, iatrogenic). While transvaginal ultrasound is the primary method for the screening of intracavitary lesions, saline infusion sonohysterography is more accurate to detect endometrial polyps and submucous leiomyomas, while hysteroscopy with biopsy remains the reference method for a definitive diagnosis. The main goals in managing AUB are addressing and correcting the underlying primary cause, if possible, and establishing a regular bleeding pattern or amenorrhea, which can be done with antifibrinolytic agents, progestins, gonadotropin-releasing hormone agonists and antagonists, or surgical interventions, each one with specific indications and limitations. Further research is necessary to assess the effectiveness and the long-term effects of various medical and surgical treatments. Meanwhile, the availability of diagnostic methods such as transvaginal ultrasound and hysteroscopy and the universal distribution of medical treatments for AUB should be prioritized by policymakers to minimize the diagnostic and treatment delay and thus reduce the risk of AUB-related anemia and the need of hysterectomy.

Assessing the effect of adipose-tissue-derived stem cell conditioned medium on follicles and stromal cells in bovine ovarian tissue culture.

RESEARCH QUESTION: Does adipose-tissue-derived stem cell conditioned medium (ASC-CM) supplementation enhance follicle and stromal cell outcomes in vitro? DESIGN: Bovine ovaries (n = 8) were sectioned and cultured in vitro for 8 days in two different groups: (i) standard culture (OT Ctrl D8); and (ii) culture with ASC-CM supplementation (OT + CM D8). Half of the culture medium was replaced every other day, and stored to measure the production of oestradiol. Follicle classification was established using haematoxylin and eosin staining. Follicle and stromal cell DNA fragmentation was assessed by TUNEL assays, while growth differentiation factor-9 (GDF-9) staining served as a marker of follicle quality. Additionally, three factors, namely vascular endothelial growth factor (VEGF), interleukin 6 (IL-6) and transforming growth factor beta 1 (TGF-ß1), were evaluated in ASC-CM in order to appraise the potential underlying mechanisms of action of ASC. RESULTS: The OT + CM D8 group showed a significantly higher proportion of secondary follicles (P = 0.02) compared with the OT Ctrl D8 group. The OT + CM D8 group also demonstrated significantly lower percentages of TUNEL-positive follicles (P = 0.014) and stromal cells (P = 0.001) compared with the OT Ctrl D8 group. Furthermore, follicles in the OT + CM D8 group exhibited a significant increase (P = 0.002) in expression of GDF-9 compared with those in the OT Ctrl D8 group, and oestradiol production was significantly higher (P = 0.04) in the OT + CM D8 group. All studied factors were found to be present in ASC-CM. VEGF and IL-6 were the most widely expressed factors, while TGF-ß1 showed the lowest expression. CONCLUSIONS: Addition of ASC-CM to culture medium enhances follicle survival, development and oestradiol production, and promotes the viability of stromal cells. VEGF, IL-6 and TGF-ß1 could be paracrine mediators underlying the beneficial effects.

Solving the mysteries surrounding uterine fibroids: are we almost there?

A better understanding of uterine fibroid-related pathogenesis and symptoms like uterine bleeding and infertility is mandatory.

Linzagolix therapy versus a placebo in patients with endometriosis-associated pain: a prospective, randomized, double-blind, Phase 3 study (EDELWEISS 3).

STUDY QUESTION: Does linzagolix administered orally once daily for up to 3 months at a dose of 75 mg alone or 200 mg in combination with add-back therapy (ABT) (1.0 mg estradiol; 0.5 mg norethindrone acetate, also known as norethisterone acetate [NETA]) demonstrate better efficacy than placebo in the management of endometriosis-related dysmenorrhea and non-menstrual pelvic pain? SUMMARY ANSWER: Combining 200 mg linzagolix with ABT was found to significantly reduce dysmenorrhea and non-menstrual pelvic pain at 3 months of therapy, while a daily dose of 75 mg linzagolix yielded a significant decrease only in dysmenorrhea at 3 months. WHAT IS KNOWN ALREADY?: A previously published Phase 2, dose-finding study reported that at a dose of 200 mg daily, linzagolix promotes full suppression of estradiol secretion to serum levels below 20 pg/ml and noted that the addition of ABT may be needed to manage hypoestrogenic side effects. At lower doses (75 mg and 100 mg/day), linzagolix maintains estradiol values within the target range of 20-60 pg/ml, which could be ideal to alleviate symptoms linked to endometriosis. STUDY DESIGN, SIZE, DURATION: EDELWEISS 3 was a multicenter, prospective, randomized, placebo-controlled, double-blind, double-dummy Phase 3 study to evaluate the safety and efficacy of linzagolix for the treatment of moderate-to-severe endometriosis-associated pain. Treatment was administered orally once daily for up to 6 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the EDELWEISS 3 trial, 486 subjects with moderate-to-severe endometriosis-associated pain were randomized at a 1:1:1 ratio to one of the three study groups: placebo, 75 mg linzagolix alone or 200 mg linzagolix in association with ABT. Pain was measured daily on a verbal rating scale and recorded in an electronic diary. MAIN RESULTS AND THE ROLE OF CHANCE: At 3 months, the daily 200 mg linzagolix dose with ABT met the primary efficacy objective, showing clinically meaningful and statistically significant reductions in dysmenorrhea and non-menstrual pelvic pain, with stable or decreased use of analgesics. The proportion of responders for dysmenorrhea in the 200 mg linzagolix with ABT group was 72.9% compared with 23.5% in the placebo group (P < 0.001), while the rates of responders for non-menstrual pelvic pain were 47.3% and 30.9% (P = 0.007), respectively. The 75 mg linzagolix daily dose demonstrated a clinically meaningful and statistically significant reduction in dysmenorrhea versus placebo at 3 months. The proportion of responders for dysmenorrhea in the 75 mg linzagolix group was 44.0% compared with 23.5% in the placebo group (P < 0.001). Although the 75 mg dose showed a trend toward reduction in non-menstrual pelvic pain at 3 months relative to the placebo, it was not statistically significant (P = 0.279). Significant improvements in dyschezia and overall pelvic pain were observed in both linzagolix groups when compared to placebo. Small improvements in dyspareunia scores were observed in both linzagolix groups but they were not significant. In both groups, hypoestrogenic effects were mild, with low rates of hot flushes and bone density loss of <1%. A daily dose of 200 mg linzagolix with ABT or 75 mg linzagolix alone was found to significantly reduce dysmenorrhea and non-menstrual pelvic pain also at 6 months of therapy. LIMITATIONS, REASONS FOR CAUTION: Efficacy was compared between linzagolix groups and placebo; however, it would be useful to have results from comparative studies with estro-progestogens or progestogens. It will be important to ascertain whether gonadotropin-releasing hormone antagonists have significant benefits over traditional first-line medications. WIDER IMPLICATIONS OF THE FINDINGS: Linzagolix administered orally once daily at a dose of 200 mg in combination with add-back therapy (ABT) demonstrated better efficacy and safety than placebo in the management of moderate-to-severe endometriosis-associated pain. The quality of life was improved and the risks of bone loss and vasomotor symptoms were minimized due to the ABT. The 75 mg dose alone could be suitable for chronic treatment of endometriosis-associated pain without the need for concomitant hormonal ABT, but further research is needed to confirm this. If confirmed, it would offer a viable option for women who do not want to wish to have ABT or for whom it is contraindicated. STUDY FUNDING/COMPETING INTEREST(S): Funding for the EDELWEISS 3 study was provided by ObsEva (Geneva, Switzerland). Analysis of data and manuscript writing were partially supported by ObsEva (Geneva, Switzerland), Theramex (London, UK) and Kissei (Japan) and grant 5/4/150/5 was awarded to M.-M.D. by FNRS. J.D. was a member of the scientific advisory board of ObsEva until August 2022, a member of the scientific advisory board of PregLem, and received personal fees from Gedeon Richter, ObsEva and Theramex. J.D. received consulting fees, speakers' fees, and travel support from Gedeon Richter, Obseva and Theramex, which was paid to their institution. C.B. has received fees from Theramex, Gedeon Richter, and Myovant, and travel support from Gedeon Richter-all funds went to the University of Oxford. He was a member of the data monitoring board supervising the current study, and served at an advisory board for endometriosis studies of Myovant. H.T. has received grants from Abbvie and was past president of ASRM. F.C.H. has received fees from Gedeon Richter and Theramex. O.D. received fees for lectures from Gedeon Richter and ObsEva and research grants for clinical studies from Preglem and ObsEva independent from the current study. A.H. has received grants from NIHR, UKRI, CSO, Wellbeing of Women, and Roche Diagnostics; he has received fees from Theramex. A.H.'s institution has received honoraria for consultancy from Roche Diagnostics, Gesynta, and Joii. M.P. has nothing to declare. F.P. has received fees from Theramex. S.P.R. has been a member of the scientific advisory board of Gedeon Richter and received fees from Gedeon Richter. A.P. and M.B. are employees of Theramex. E.B. was an employee of ObsEva, sponsor chair of the data monitoring board supervising the current study, and has been working as a consultant for Theramex since December 2022; she owns stock options in ObsEva. M.-M.D. has received fees and travel support from Gedeon Richter and Theramex. TRIAL REGISTRATION NUMBER: NCT03992846. TRIAL REGISTRATION DATE: 20 June 2019. DATE OF FIRST PATIENT'S ENROLLMENT: 13 June 2019.

Comprehensive Review of In Vitro Human Follicle Development for Fertility Restoration: Recent Achievements, Current Challenges, and Future Optimization Strategies.

Ovarian tissue cryopreservation (OTC) and subsequent transplantation (OTT) is a fertility preservation technique widely offered to prepubertal girls and young fertile women who need to undergo oncological treatment but are at a high risk of infertility. However, OTT is not considered safe in patients with certain diseases like leukemia, Burkitt's lymphoma, and ovarian cancer because of the associated risk of malignant cell reintroduction. In vitro follicle development has therefore emerged as a promising means of obtaining mature metaphase II (MII) oocytes from the primordial follicle (PMF) pool contained within cryopreserved ovarian tissue, without the need for transplantation. Despite its significant potential, this novel approach remains highly challenging, as it requires replication of the intricate process of intraovarian folliculogenesis. Recent advances in multi-step in vitro culture (IVC) systems, tailored to the specific needs of each follicle stage, have demonstrated the feasibility of generating mature oocytes (MII) from early-stage human follicles. While significant progress has been made, there is still room for improvement in terms of efficiency and productivity, and a long way to go before this IVC approach can be implemented in a clinical setting. This comprehensive review outlines the most significant improvements in recent years, current limitations, and future optimization strategies.

Uterine fibroid-related infertility: mechanisms and management.

Fibroids are a common pathology and increasingly observed in women seeking medical treatment for infertility. The longer reproductive horizon because of improvements in medical care and current trend for women to postpone childbearing are making fibroid-related infertility increasingly common. This review aimed to critically analyze the association between uterine fibroids and infertility, mechanisms by which uterine fibroids may impair fertility, and management of myoma-related infertility. The association of fibroids with infertility is a source of controversy. As the focus of this review is infertility, it is crucial to analyze the mechanisms by which fertility may be impaired by the presence of fibroids. Current management strategies involve mainly surgical interventions, including myomectomy by hysteroscopy, laparotomy, or laparoscopy, and nonsurgical approaches, such as uterine artery embolization and focused ultrasound performed under radiologic or echographic guidance. The risks and benefits of each option should be discussed with patients, and several factors need to be considered, including the skills of surgeons and availability of different resources in various centers. Concerning the efficacy of oral gonadotropin-releasing hormone antagonists (i.e., elagolix, relugolix, and linzagolix), they were shown to have a rapid impact on heavy menstrual bleeding (HMB) in >70% of women. When used without add-back therapy, these drugs cause a significant reduction in fibroid volume, namely, approximately 50% from baseline to week 24. Further studies are required to determine the best protocol and optimal dosage if a reduction in myoma volume is the main goal, as in case of myoma-related infertility.

Pathogenesis of uterine fibroids: current understanding and future directions.

Fibroids are benign uterine tumors characterized by the proliferation of uterine smooth muscle cells, embedded in an abundant extracellular matrix. Their prevalence is estimated to be >50% in women aged >45 years. Fibroids represent a considerable health burden. It is time to acquire a deeper mechanistic understanding of uterine fibroid-related etiology and pathogenesis, which may help pinpoint new strategies and an individualized approach. There is a need to gather prospective data and conduct studies to compare alternative approaches and assess long-term outcomes with respect to quality of life, recurrence of symptoms (bleeding and bulk symptoms), fertility, and even complications The goal of this review was to evaluate the widely accepted pathogenesis and identify risks factors and future directions for clinical and basic research into fibroids.

Endometriosis and adenomyosis: Similarities and differences.

Deep endometriosis and uterine adenomyosis are two frequently encountered conditions affecting approximately 200 million women worldwide. They are closely related, showing similar histological patterns and multiple common pathogenic features, and share the same symptoms. It is therefore not surprising that they are often thought to have a common developmental origin. Indeed, both deep endometriosis and adenomyosis appear to derive from estrogen-dependent overproliferation of endometrial tissue and its subsequent implantation in ectopic sites. Although the scientific community has shown increasing interest in these diseases over recent years, neither pathogenesis has yet been elucidated, so there are currently no efficient treatment options. Understanding the mechanisms underlying disease development, as well as discerning their relationship, are key to improving clinical management for millions of patients. The aims of this review are to summarize current knowledge on deep endometriosis and adenomyosis pathogeneses and discuss the possibility that these two entities are actually differential phenotypes of the same disease.

Impact of human ovarian tissue manipulation on follicles: evidence of a potential first wave of follicle activation during fertility preservation procedures.

PURPOSE: The aim of this study was to investigate the impact of processing human ovarian tissue on follicle activation dynamics. METHODS: Fresh ovarian tissue was retrieved from 9 women undergoing laparoscopic surgery for benign conditions. Biopsies from each patient were divided into 3 fragments, the first of which was immediately fixed in the operating room (T0) and the second and third just after processing at 25 (T25) and (T90) 90 min. To evaluate follicle activation, markers of the PI3K and Hippo signaling pathways were immunolabeled at each time point, targeting phospho-Akt (p-Akt) by immunohistochemistry and yes-associated protein (YAP) cellular localization in the granulosa cell layer by immunofluorescence. RESULTS: Four hundred forty primordial follicles were evaluated for p-Akt and 420 for YAP. Significantly stronger p-Akt expression was observed at T25 (23.01 ± 13.45%; p=0.04) and T90 (38.99 ± 25.21%; p<0.001) than at T0 (2.72 ± 3.35%). A significant nucleus-to-cytoplasm shift in YAP was detected at T25 (1.21 ± 0.25; p=0.015 compared to T0 (0.95 ± 0.09), while T90 (1.10 ± 0.16) values were similar to T25. CONCLUSION: Our data prove that ovarian tissue manipulation significantly impacts follicle dynamics by stimulating the PI3K and Hippo signaling pathways involved in primordial follicle activation. Further experimental evidence must nevertheless be gathered to understand and gain control of follicle activation mechanisms in non-physiological conditions (like ovarian tissue manipulation), in order to optimize fertility preservation and restoration strategies.

Are lower levels of apoptosis and autophagy behind adenomyotic lesion survival?

RESEARCH QUESTION: How are markers of cell death, invasiveness and progesterone signalling expressed in endometrium and ectopic lesions from adenomyosis patients? DESIGN: Formalin-fixed paraffin-embedded tissue was collected from 15 control and 15 adenomyosis participants . To assess cell survival capacity, caspase 3 and microtubule-associated proteins 1A/1B light chain 3B (LC3B) were immunolabelled as markers of apoptosis and autophagy respectively. Matrix metalloproteinase 9 (MMP9) expression served as a marker of extracellular matrix degradation and invasion activity. Progesterone receptors were immunostained to detect evidence of progesterone resistance. RESULTS: Caspase 3 expression was significantly lower in the stromal (P = 0.0013) and epithelial (P = 0.0157) compartments of adenomyotic lesions than in healthy endometrial tissue. In the stroma, caspase 3 expression was significantly weaker in lesions than in corresponding eutopic endometrium (P = 0.0006). LC3B immunostaining was significantly decreased in adenomyotic stroma compared with corresponding eutopic endometrium (P = 0.0349). A significantly higher expression of MMP9 was detected in eutopic stroma from adenomyosis patients than in healthy tissue (P = 0.0295). Progesterone receptor immunostaining was found to be significantly weaker in the stroma of endometrium and ectopic lesions from adenomyosis patients than disease-free women (P = 0.0001; P = 0.0021). CONCLUSIONS: Adenomyotic lesions show lower levels of apoptosis and autophagy, suggesting that aberrant cell survival may be involved in disease pathogenesis. MMP9 appears to contribute to endometrial invasiveness in adenomyosis, as its expression is more pronounced in endometrium from these women than women without the disease. Evidence of progesterone resistance can be found in endometrium and ectopic lesions from adenomyosis patients, and may drive disease development and account for the failure of certain patients to respond to progestogens.

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation.

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle loss occurring shortly after reimplantation due to abnormal follicle activation and death. Rodents are benchmark models for investigating follicle activation, but the cost, time, and ethical considerations are becoming increasingly prohibitive, thus driving the development of alternatives. The chick chorioallantoic membrane (CAM) model is particularly attractive, being inexpensive and maintaining natural immunodeficiency up to day 17 postfertilization, making it ideal to study short-term xenografting of human ovarian tissue. The CAM is also highly vascularized and has been widely used as a model to explore angiogenesis. This gives it a remarkable advantage over in vitro models and allows the investigation of mechanisms affecting the early post-grafting follicle loss process. The protocol outlined herein aims to describe the development of a CAM xenografting model for human ovarian tissue, with specific insights into the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

Hysteroscopic management of molar pregnancy: A series of 36 cases.

Background: Hydatidiform Mole (HM) is the most common form of gestational trophoblastic disease. Dilatation and curettage is the classical treatment of this affection. Hysteroscopic resection (HsR) is an alternative for the treatment of intra-uterine pathology. Objective: To describe the feasibility of HsR for the management of HM. Result: Case series of patients who had a complete or partial HM confirmed by histological examination of the trophoblastic tissue resected by operative hysteroscopy between 2007 and 2019. After approval of our ethics committee, we evaluated 36 patients who underwent hysteroscopic resection for molar pregnancy. Histological analysis showed partial HM in 28 patients (77.8%) and complete HM in 8 (22.2%). Main surgical complications were uterine perforation in one patient and glycine resorption in 10 patients with two cases of hyponatremia corrected by standard treatment. We performed an ultrasound control 1 month after the intervention in 19 patients (52.8%) as they had slow decrease of HCG or bleeding complaints and found retained product of conception (RPOC) in six patients (16.7%). Conclusion: This first report on a small number of patients demonstrate that hysteroscopic resection is a feasible procedure for the management of molar pregnancy. Direct visualization of the procedure helps the surgeon to control the resection. Further studies are mandatory to compare this technique with D&C in term of RPOC and fertility outcomes as it remains the standard treatment.

Assessment of folliculogenesis in ovarian tissue from young patients with Turner syndrome using a murine xenograft model.

OBJECTIVE: To study the impact of aneuploid granulosa and stromal cells on folliculogenesis of small ovarian follicles from patients with mosaic Turner syndrome (TS) using a murine xenograft model. DESIGN: Laboratory study. SETTING: University hospital. PATIENT(S): Ovarian cortical tissue was obtained by laparoscopic surgery from 18 patients with mosaic TS (aged 5-19 years) and 13 controls (aged 5-18 years). INTERVENTION(S): Part of each tissue fragment was used to karyotype ovarian cells in nongrafted tissue by fluorescence in situ hybridization. The remaining tissue was xenografted to severe combined immunodeficient mice for 5 months. Grafted tissue was analyzed for aneuploidy, and follicle density and morphology were determined. Expressions of proliferating cell nuclear antigen and anti-Müllerian hormone were investigated by immunohistochemistry. MAIN OUTCOME MEASURE(S): The impact of aneuploid granulosa and stromal cells on folliculogenesis. Fluorescence in situ hybridization of ovarian tissue before grafting was performed to determine the level of aneuploidy in stromal cells and oocytes and granulosa of small follicles. After xenografting, the level of aneuploidy of the newly formed layers of granulosa cells was again determined in secondary and antral follicles. RESULT(S): Follicle density in ovarian tissue from patients with TS was significantly lower than in controls before grafting. Fluorescence in situ hybridization analysis confirmed that 101 of 104 oocytes from nongrafted tissue of patients with TS showed normal X chromosome content, whereas granulosa and stromal cells were mainly 45,X. Fragments from 12 patients with TS contained follicles at all stages after xenografting, including secondary and antral follicles. Follicle density in patients with TS and controls decreased significantly after grafting. Moreover, a shift from high to low proportions of 45,X granulosa cells was observed during folliculogenesis. Expression of proliferating cell nuclear antigen in follicles from patients with TS increased significantly during grafting. Secretion of anti-Müllerian hormone was impaired before grafting in peripubertal/postpubertal girls with TS, but recovered after grafting. CONCLUSION(S): Our study showed that small follicles from patients with mosaic TS undergoes folliculogenesis, despite the presence of aneuploid granulosa and stromal cells. Ovarian tissue cryopreservation could therefore be a valid option to preserve fertility in young patients with mosaic TS if sufficient numbers of follicles are present, thus preferably before the age of 12.

Could IVF replace reproductive surgery? No, reproductive surgery is still very much alive.

Could IVF replace reproductive surgery? The answer is no. Reproductive surgery still has a place, at least in some indications that will be explored in this contribution. While IVF can offer infertile couples the chance to have a healthy baby, it should be acknowledged that reproductive surgery can heal or harm the organs where reproduction takes place. This paper reviews different diseases and conditions with an impact on fertility, which may benefit from the technological innovations of recent decades, novel applications and the skill of reproductive surgeons. Reproductive surgery is certainly not dead. It lives on with the promise of restoring the functional anatomy to enhance the chances of pregnancy. It is our responsibility to train young residents adequately in this field to provide the right treatment at the right time.

Summary of the ISFP congress, Brussels, 10-12 November, 2022.

The 7th International Congress of the ISFP was held in Brussels in November 2022. Hundreds of attendees from all over the world had the rare opportunity to hear the most distinguished leaders discuss and debate the latest advances in the field. Participants were also able to attend workshops under the guidance of skilled practitioners. Numerous topics were considered, including a recap on fertility preservation approaches in cancer and benign pathologies and a section on male factor infertility. Other aspects covered were in vitro maturation and poor responders, the impact of chemotherapy on the ovary, and future perspectives. Participants had the chance to listen to a symposium on fertility preservation techniques, and finally, a keynote lecture on fertility preservation in gynecological cancers brought this prominent and highly influential event to a close.

M2 macrophages enhance endometrial cell invasiveness by promoting collective cell migration in uterine adenomyosis.

RESEARCH QUESTION: Are M2 macrophages implicated in endometrial invasiveness in adenomyosis? DESIGN: Seventeen formalin-fixed paraffin-embedded uterine samples and 16 fresh endometrial biopsies were collected from women with or without adenomyosis. Double immunofluorescence was performed to determine the predominant macrophage population in adenomyosis between M1 and M2 phenotypes. The invasion capacity of endometrial cells was assessed by invasion assays and quantitative polymerase chain reaction for genes involved in cell motility and epithelial-mesenchymal transition (EMT). Specific mechanisms of invasion were investigated by immunohistochemistry for E-cadherin, N-cadherin and matrix metalloproteinase 9 (MMP9). RESULTS: Only M2 macrophages were found to accumulate in adenomyosis, in higher numbers in both eutopic endometrium (P = 0.0109) and lesions (P = 0.0267) than healthy tissue. Co-culture with M2 macrophages significantly boosted invasion capacity in endometrial epithelial (P = 0.0002; P = 0.002) and stromal cells (P = 0.0469; P = 0.0047) from both adenomyosis patients and healthy controls. No gene expression differences indicating EMT were noted, either between co-cultured and control cells, or between healthy and adenomyotic cells. E- and N-cadherin protein expression did not differ significantly between endometrium from adenomyosis subjects and healthy tissue but MMP9 expression was increased in eutopic stroma from adenomyosis patients (P = 0.0492). In adenomyosis, both E-cadherin (P = 0.0379) and N-cadherin (P = 0.0196) were more extensively expressed in basal glands than functional glands. CONCLUSIONS: M2 macrophages accumulate in adenomyosis and enhance invasion capacity of adenomyotic and even healthy endometrial cells, implying that macrophage infiltration alone may be sufficient to promote the disease. This study failed to detect any changes pointing to EMT, suggesting an alternative mode of invasion. Strong E- and N-cadherin-positive intercellular junctions in basal (invasive) glands suggest the involvement of collective cell migration in the invasion process of endometrium.