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Adenomyosis in endometriosis--prevalence and impact on fertility. Evidence from magnetic resonance imaging.

BACKGROUND: The hypothesis is tested that there is a strong association between endometriosis and adenomyosis and that adenomyosis plays a role in causing infertility in women with endometriosis. METHODS. Magnetic resonance imaging of the uteri was performed in 160 women with and 67 women without endometriosis. The findings were correlated with the stage of the disease, the age of the women and the sperm count parameters of the respective partners. RESULTS: The posterior junctional zone (PJZ) was significantly thicker in women with endometriosis than in those without the disease (P<0.001). There was a positive correlation of the diameter of the PJZ with the stage of the disease and the age of the patients. The PJZ was thicker in patients with endometriosis with fertile than in patients with subfertile partners. The prevalence of adenomyotic lesions in all 160 women with endometriosis was 79%. In women with endometriosis below an age of 36 years and fertile partners, the prevalence of adenomyosis was 90% (P<0.01) CONCLUSIONS: With a prevalence of up to 90%, uterine adenomyosis is significantly associated with pelvic endometriosis and constitutes an important factor of sterility in endometriosis presumably by impairing uterine sperm transport.

Adolescent↗

Vascular endothelial growth factor gene +405 C/G polymorphism is associated with susceptibility to advanced stage endometriosis.

BACKGROUND: Vascular endothelial growth factor (VEGF) is known to play a pivotal role in the development of endometriosis. This study was performed to investigate whether the VEGF gene 5'-untranslated region polymorphism is associated with susceptibility to advanced stage endometriosis. METHODS: This study comprised 215 women with advanced stage endometriosis, 219 control women without endometriosis, and 70 fertile women. Following extraction of genomic DNA, genotyping of the -460 C/T and +405 C/G polymorphisms of the VEGF gene were performed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. RESULTS: The distribution of genotypes and allele frequencies of the -460 C/T polymorphism in the endometriosis group did not differ from those in the control group and the fertile women group. However, genotype distribution of the +405 C/G polymorphism was significantly different between patients with and without endometriosis (P = 0.01) and between patients with endometriosis and the fertile women (P = 0.02). Patients with endometriosis showed a higher incidence of the +405 CC genotype compared with the controls and the fertile women (P = 0.007 and 0.016 respectively). CONCLUSIONS: These findings suggest that the VEGF +405 C/G polymorphism may be associated with the risk of advanced stage endometriosis in the Korean population.

Alleles↗

High density of small nerve fibres in the functional layer of the endometrium in women with endometriosis.

BACKGROUND: Endometriosis is a common gynaecological disease and is frequently associated with recurrent and serious pelvic pain such as dysmenorrhoea and dyspareunia, but the mechanisms by which these symptoms are generated are not well understood. METHODS: Histological sections of endometrial tissue were prepared from endometrial curettings and hysterectomies performed on women with endometriosis (n=25 and n=10, respectively) and without endometriosis (n=47 and n=35, respectively). These were stained immunohistochemically for the highly specific polyclonal rabbit anti-protein gene product 9.5 (PGP9.5) and monoclonal mouse anti-neurofilament protein (NF) to demonstrate both myelinated and unmyelinated nerve fibres. RESULTS: Small nerve fibres were identified throughout the basal and functional layers of the endometrium in all endometriosis patients, but were not seen in the functional layer of the endometrium in any of the women without endometriosis (P<0.001). NF-immunoreactive nerve fibres were present in the basal layer in all endometriosis patients but not in non-endometriosis patients, with one exception (P<0.001). CONCLUSIONS: Small nerve fibres detected in the functional layer in all women with endometriosis may have important implications for understanding the generation of pain in these patients. The presence of nerve fibres in an endometrial biopsy may be a novel surrogate marker of clinical endometriosis.

Adult↗

Tumour necrosis factor-alpha blockers: potential limitations in the management of advanced endometriosis? A case report.

Several studies have shown that tumour necrosis factor (TNF)-alpha levels are increased in the peritoneal fluid of women with endometriosis, with correlation between TNF-alpha concentrations and the degree of disease. It is also likely that elevation of peritoneal fluids' TNF-alpha levels may play a role in the pathogenesis of infertility associated with endometriosis. Use of drugs such as etanercept, a TNF-alpha receptor immunoglobulin fusion protein which inhibits TNF-alpha activity, showed in an animal study to reduce the severity of the disease, and the size of endometriotic foci. TNF-alpha blockers were recommended as a possible new line of therapy for endometriosis. Our case involved a 35-year-old Para 0, with rheumatic arthritis and stage 4 endometriosis. After 6 years of constant use of etanercept, she showed no improvement of endometriosis as demonstrated at laparoscopy. However, she underwent a successful IVF after the first attempt. TNF-alpha-blocker medications might not be beneficial for patients with advanced endometriosis. However, we cannot exclude the possible effect of these medications on early-stage endometriosis, and further study is required. Some of the immunologic abnormalities in the pelvis of patients with endometriosis could be the consequence of the disease and not the cause, and possibly suppression of immune cells and their products may not have a major effect on endometriotic lesions at an advanced stage. This also could explain why suppression of TNF-alpha showed no effect on infertility. However, use of TNF-alpha-blockers before IVF might increase the success rate in advanced endometriosis.

Adult↗

Increased telomerase activity and human telomerase reverse transcriptase mRNA expression in the endometrium of patients with endometriosis.

BACKGROUND: Endometriosis is considered a frequent, benign disease with the ability to undergo neoplastic processes. The aim of this study was to evaluate the limitless replication potential of the endometrium in patients with endometriosis by examining human telomerase reverse transcriptase (hTERT) mRNA expression and telomerase activity. METHODS: Endometrium samples from 30 endometriosis patients and 30 patients without endometriosis were obtained via endometrial biopsy. The expression of hTERT mRNA was determined by real-time RT-PCR assay, and telomerase activity was measured by telomerase repeat amplification protocol (TRAP) assay. RESULTS: The mean normalized hTERT (N hTERT) mRNA level was significantly higher in the endometriosis than in the control group (P = 0.013). The mean hTERT mRNA levels during the proliferative phase and during the secretory phase were higher in the endometriosis group than in the control group, although the difference was only significant for the secretory phase (P = 0.036). We found a prominent difference in endometrial telomerase activity between moderate-to-severe endometriosis and the control group (P = 0.048). The levels of hTERT mRNA and telomerase activity increased as the disease became more severe (P = 0.038, P = 0.016). CONCLUSIONS: This study showed the overexpression of hTERT mRNA and telomerase activity in the endometrium of endometriosis patients. These finding suggest that replication potential of endometrial cells may have an important role in the pathogenesis of endometriosis.

Adult↗

CA125 and endometriosis.

This review covers the literature on CA125 and endometriosis; data on CA125 and oncology are not discussed. In normal women, plasma concentrations of CA125 are increased slightly at ovulation and significantly during menstruation. Marked increases are observed during pregnancy and following peritoneal irritation by infection or surgery. These data are consistent with the concept that CA125 in normal women is mainly derived from the endometrium and the irritated peritoneum. Plasma concentrations of CA125 are markedly elevated in women with cystic ovarian endometriosis and/or deeply infiltrating endometriosis, but not, or only slightly, in the luteal phase of women with minimal or mild endometriosis. This is consistent with the recent concept which considers minimal endometriosis as a normal condition occurring intermittently in many women, in contrast with deep endometriosis and cystic ovarian endometriosis which are called 'endometriotic disease'. Serum CA125 is not a good marker for endometriosis but it is a helpful additional parameter to diagnose endometriotic disease in patients with chronic pelvic pain. Following treatment of endometriosis, elevated plasma concentrations of CA125 could be used as an argument that treatment has been incomplete, or that the condition has recurred. Assaying CA125 in peritoneal fluid requires high sample dilutions or a modified immunoradiometric assay, and until now, its clinical value has been questionable.

Ascitic Fluid↗

Paracrine changes in the peritoneal environment of women with endometriosis.

During the past decade, macrophage-derived substances such as prostanoids, cytokines, growth factors and angiogenic factors have been detected in the peritoneal fluid of women with endometriosis. In particular, growth-promoting and angiogenic factors are considered to be substantially involved in the pathogenesis of endometriosis. In this study, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta) and intercellular adhesion molecule 1 (ICAM-1), substances recently detected in the peritoneal fluid of women with endometriosis, were assessed with regard to their concentrations in different stages of endometriosis and changes of the peritoneal paracrine activity after medical treatment with a gonadotrophin releasing hormone agonist (GnRHa). Peritoneal fluid was obtained from patients with endometriosis during laparoscopy before and after a 4-month treatment with a GnRHa. VEGF, TGF-beta and ICAM-1 could be detected in all women presenting with various stages of active endometriosis. After GnRHa therapy, all patients showed significant decreases in mean concentrations of VEGF (194+/-77 pg/ml), TGF-beta (902+/-273 pg/ml) and ICAM-1 (157+/-52 ng/ml). Patients with stage III and IV endometriosis (according to the rAFS score) had much higher concentrations of VEGF and TGF-beta before treatment compared with those patients with mild endometriosis (rAFS stages I and II). The most striking decrease in concentration was for TGF-beta, from 902 pg/ml before to 273 pg/ml after therapy. These results indicate an important role for paracrine activity in the establishment and maintenance of endometriosis. Indeed, treatment with a GnRHa may reduce paracrine activity in the peritoneal cavity via hypo-oestrogenism and provide proof of successful therapy.

Ascitic Fluid↗

The endometrium versus embryonic quality in endometriosis-related infertility.

In spite of a great deal of effort over many decades, the mechanisms that lead to infertility in women with endometriosis remain unknown. Moreover, controversial results in the literature add even more difficulties in the understanding of this issue. Since the introduction of IVF, we have had the opportunity to study the effects of endometriosis at specific stages of the reproductive process including folliculogenesis, fertilization, embryo development and implantation. Depending on the research group, there are conflicting data on the results of IVF in patients with endometriosis. Some researchers found impaired implantation in patients with endometriosis, but such a defect in implantation may be caused by either defective embryos or altered endometrium. The observation of a higher rate of arrested embryos in patients with endometriosis compared with disease-free women, and that women with this pathological situation undergoing ovum donation had the same chance of implantation as patients without endometriosis, initially suggested that impaired oocyte/embryo quality may be responsible for reduced implantation. Further investigations on follicular components have also found differences between women with endometriosis and those without the disease. In addition, recent advances on implantation research, mainly on markers of endometrial receptivity, also show features in the eutopic endometrium of women with endometriosis that are not found in endometrium of women without the disease, although there is no agreement on this point. In this review, we will focus on infertility-related endometriosis based on our own research and the available literature.

Embryo, Mammalian↗

Increased concentrations of soluble tumour necrosis factor receptor (sTNFR) I and II in peritoneal fluid from women with endometriosis.

Tumour necrosis factor alpha (TNFalpha), a proapoptotic cytokine, is known to be present in peritoneal fluid from women with endometriosis. An emerging view is that soluble TNF receptors (sTNFR) can modulate the effects of TNFalpha by acting as TNFalpha antagonists. To assess the relevance of sTNFRs in the pathophysiology of endometriosis, concentrations of sTNFR I, sTNFR II and TNFalpha in peritoneal fluid from women with endometriosis (n = 53) and without endometriosis (n = 40) were measured. Concentrations of both sTNFR I and sTNFR II in peritoneal fluid from women with endometriosis were significantly higher than in peritoneal fluid from women without endometriosis, both in the follicular and the luteal phases. TNFalpha concentrations did not differ in patients with and without endometriosis in both phases. When stratified by the stage of the disease, women with both stages I/II and stages III/IV exhibited significantly higher concentrations of sTNFR I and sTNFR II in peritoneal fluid, compared with women without endometriosis, whereas no appreciable difference in the concentrations was detected between stages I/II and stages III/IV. A significant correlation was found between the concentrations of sTNFR I and sTNFR II; while the correlations between TNFalpha and sTNFR I or sTNFR II, were either not significant or were very weak. Furthermore, mRNA for the membrane-associated TNF receptor type 1 and TNF receptor type 2, both of which convey the effects of TNFalpha, were shown to be expressed in endometriotic tissues as well as eutopic endometrium. Together, these findings suggest a possible involvement of sTNFRs in the pathophysiology of endometriosis.

Adult↗

Interleukin-10 gene promoter polymorphisms and their protein production in peritoneal fluid in patients with endometriosis.

Although associations of interleukin-10 (IL-10) gene promoter polymorphisms and their protein production with endometriosis risk have been reported, the correlations remain controversial. The objective of this study was to determine IL-10 gene promoter polymorphisms at -1082, -819 and -592 sites and their protein production in peritoneal fluid (PF) in patients with and without endometriosis. IL-10 gene promoter polymorphisms at -1082 site were detected by amplification refractory mutation system (ARMS)-PCR and that at -819 and -592 sites was genotyped by restriction fragment length polymorphism (RFLP)-PCR. Protein levels of IL-10 in PF were measured by enzyme-linked immunosorbent assay (ELISA). There were no significant differences in the genotype and allele frequencies of IL-10 gene promoter polymorphisms at position -1082 between the endometriosis and the control groups. However, the frequency of -819 or -592 C alleles was significantly increased in patients with endometriosis compared with controls. The protein levels of IL-10 in PF were statistically higher in the endometriosis group than in the control group. Moreover, the polymorphisms at -1082, -819 and -592 sites were associated with protein levels of IL-10 in PF in the endometriosis group while in the control group only the polymorphisms at position -1082 correlated with protein levels. Increased frequency of -819 or -592 C allele and increased protein production of IL-10 in PF in patients with endometriosis compared with controls and correlations of polymorphisms at -819 and -592 sites with protein levels of IL-10 in PF in patients with endometriosis may suggest that polymorphisms at -819 and -592 sites and their protein production are associated with endometriosis risk.

Ascitic Fluid↗

Effect of endometriosis on white blood cell subpopulations in peripheral blood and peritoneal fluid of baboons.

In women with endometriosis, changes in peripheral blood and peritoneal fluid white blood cell (WBC) populations have been reported, but it is known whether these alterations are casually related to or a consequence of endometriosis. The purpose of this study was to test the hypothesis that peripheral blood and peritoneal fluid WBC populations are altered in baboons with spontaneous and induced endometriosis compared to animals without disease. Peripheral blood and peritoneal fluid samples were obtained at laparoscopy from 60 baboons with a normal pelvis (n = 23), spontaneous endometriosis (n = 19) and induced disease (n = 12), luteal phase (n = 20), pregnancy or nursing (n = 11) and in non-cycling animals (n = 8). The WBC concentration was analysed with a Coulter counter and fluorescent antibody cell separation (FACS) analysis was used to measure cluster designation (CD)2, CD4, CD8, interleukin (IL)2R and leucine (Leu) M5 subsets. In peripheral blood, the percentage of CD4+ and IL2R+ cells was increased in baboons with stage II-IV spontaneous or induced endometriosis, suggesting that alterations in peripheral blood WBC populations may be an effect of endometriosis. In peritoneal fluid the WBC concentration and percentages of Leu M5+ macrophages and CD8+ lymphocytes were only increased in baboons with spontaneous endometriosis and not in animals with induced disease, suggesting that alterations in peritoneal fluid WBC populations may lead to the development of endometriosis. In summary, the results of this study suggest that peripheral blood and peritoneal fluid immune cell populations are affected in baboons with endometriosis.

Animals↗

Fluorescence of experimental endometriosis in rabbits, using tamoxifen-eosin association.

A major problem with endometriosis is the detection of microscopic and atypical lesions. An incomplete surgical eradication may lead to recurrence of the disease. This study aimed to investigate the diagnostic improvement of endometriosis by the use of tamoxifen-eosin induced fluorescence. The experimental study was performed on surgically induced endometriosis in the rabbit. Endometriosis was induced by grafting endometrium onto the broad ligament in 10 rabbits. After 5 weeks, the fluorescence of excised endometriosis was studied after systemic injection of tamoxifen and local application of eosin. Healthy peritoneal samples served as controls. The fluorescence of endometriotic foci was also compared with (n = 5) or without (n = 5) tamoxifen. Fluorescence excitation was carried out using a 150 W filtered lamp connected to an optical fibre. Fluorescence emission was measured using an optical fibre connected to a spectrofluorometer. Spectral analysis showed a specific fluorescence of endometriosis 72 h after systemic injection of tamoxifen and eosin application. This result is explained by binding to oestrogen receptors of tamoxifen which was protonized to form an ionic pair with eosin. Histological study of samples from the graft of endometrial tissue showed that experimental endometriosis had developed in eight out of the 10 rabbits. However, the fluorescence was not significantly different among the 10 rabbits. This observation was in accordance with previous studies in which endometriosis was confirmed by routine histological techniques or electron microscopy in 70-80% of cases. Consequently, the fluorescence of the two samples which did not present histological evidence of endometriosis indicates the presence of microscopic endometriotic foci.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Use of a new CA 125 assay in the diagnosis of endometriosis.

Serum CA 125 concentrations are elevated in some women with endometriosis; however, the low sensitivity and specificity of the assay have made it impractical to use it as a diagnostic test for endometriosis. Recently a second-generation CA 125 assay has been developed. The purpose of this study was to compare serum CA 125 concentrations in women with and without endometriosis using both the older assay and the new CA 125 assay and to determine if the new assay concentration improves the clinical utility of CA 125 in the diagnosis of endometriosis. A total of 123 patients with endometriosis, pelvic pain or infertility were enrolled. Blood for CA 125 was drawn in the cycle preceding laparoscopy or laparotomy, and concentrations found by the older and newer assays were correlated with the patients' endometriosis stage using the revised American Fertility Society classification of endometriosis. The CA 125 concentrations determined by the new assay were highly correlated with concentrations determined by the older assay in patients with and without endometriosis (r = 0.96). The sensitivity and specificity were slightly improved using the new CA 125 assay; however, this assay did not dramatically improve detection of endometriosis.

Biomarkers↗

Effect of peritoneal fluid from infertile women with endometriosis on ionophore-stimulated acrosome loss.

The effect of peritoneal fluid (PF) from endometriosis patients was studied in spontaneous and stimulus-induced (Ca-ionophore; A23187) acrosome reactions. PF samples were obtained from 21 infertile women with endometriosis and five normal women (controls). Sperm acrosomes were examined by staining with Pisum sativum agglutinin labelled with fluorescein isothiocyanate. The incidence of spontaneous acrosome reaction after 1 and 6 h of incubation (6.7 +/- 1.6 and 6.9 +/- 1.4 respectively) was significantly (P < 0.001) lower when the incubation was performed with PF from endometriosis patients in comparison with spermatozoa incubated in PF from the control group (12.8 +/- 1.1 and 12.8 +/- 0.8). Similarly, the incidence of A23187-induced acrosome reaction after 1 and 6 h of incubation (19.8 +/- 2.7 and 20.0 +/- 2.4) was significantly (P < 0.001) lower when spermatozoa were incubated with PF from endometriosis patients in comparison with spermatozoa incubated with PF from the control group (34.6 +/- 9.8 and 34.4 +/- 1.1). The incidence of A23187-inducible acrosome reaction was also significantly (P < 0.001) lower when the incubation was performed with PF from endometriosis patients (13.1 +/- 2.8 and 13.1 +/- 2.4) when compared with that from the control group (21.8 +/- 2.6 and 21.6 +/- 1.5). No relationship was found between the stage of endometriosis and the incidence of acrosome loss. In conclusion, the PF from endometriosis patients decreased both spontaneous and stimulus-induced acrosome reaction. This may represent a mechanism for the detrimental effect of the PF from endometriosis patients on the spermatozoa-oocyte interaction and partially explain the aetiology of infertility in patients with endometriosis.

Acrosome↗

Is a long period without childbirth a risk factor for developing endometriosis?

Endometriosis was found in 42 (20%) of 206 symptom-free, parous Norwegian women with a mean age of 37 years at the time of sterilization. Case-control analysis between the 42 women with endometriosis and the 164 without endometriosis revealed an increased risk of endometriosis with increasing number of years since the last childbirth. After 10 years without a birth, the odds ratio for endometriosis was 4.5 compared with the first 5 years after the last delivery. An increased risk of endometriosis with increasing age of the women could not be detected. If these findings from women with voluntary infertility are transferred to women with endometriosis associated with involuntary infertility, endometriosis could be a consequence of a long period without childbirth. This might explain why endometriosis is often diagnosed in infertile women.

Adult↗

Endometriosis and pain.

Endometriosis generally causes pain that is cyclic and generally responds to medication and/or surgery. When endometriosis is found coincidentally, it may need no treatment because many women have endometriosis as a self-limited disease. In other women, the biologic behavior is much more unpredictable. Severe dysmenorrhea, focal pelvic tenderness, and deep dyspareunia are suggestive of endometriosis. Diagnosis at laparoscopy includes concerns about subtle appearance, endometriosis hidden within adhesions, retroperitoneal disease, and intra-ovarian lesions. Negative laparoscopy results do not mean that patients have no endometriosis. In contrast, a response to GnRH agonists can occur in patients with no endometriosis because conditions other than endometriosis are estrogen sensitive. Coexistent disease can confuse the picture at the time of surgery. Some coexistent diseases also can cause pain that is similar to that of endometriosis. Distinguishing those patients who need no treatment from those who need intermediate or extensive treatment can be very difficult. Care is needed to ensure that patients are neither overtreated or undertreated. An integrated approach involving a multidisciplinary team is needed in some. Other patients respond to primary care techniques.

Acupuncture Therapy↗

Immunohistochemical analysis of alphavbeta5 and alphavbeta6 integrins in the endometrium and endometriosis.

We performed a case-controlled study to study the immunoreactivity patterns and staining intensity of alphavbeta5 and alphavbeta6 integrins in ectopic and eutopic endometrium from women with endometriosis and compared them with those in normal control endometrium. Forty eutopic and ectopic samples from patients with endometriosis were compared with 12 control endometrial samples. Staining was evaluated using a computerized image analysis system by a blinded, independent observer. The immunoreactivity patterns for both alphavbeta5 and alphavbeta6 were very similar. In eutopic endometrium from women with or without endometriosis, the immunostaining intensity score for both integrins was stronger during the secretory than the proliferative phase. Immunoreactivity in endometriosis was greater than in eutopic endometrium. There were no significant menstrual cycle-related staining differences in endometriosis. The percentage of blood vessels immunolabeled was greater in endometriosis than eutopic endometrium from endometriosis patients and greater in eutopic endometrium from these patients compared with control endometrium. Ectopic tissue from patients with stages I and II endometriosis showed significantly higher staining intensity than ectopic tissue from patients with stages III and IV disease. Determination of the specific functional consequences of the differences observed in alphavbeta5 and alphavbeta6 integrin immunostaining may provide an increased understanding regarding the pathogenesis of endometriosis.

Antigens, Neoplasm↗

Genetic polymorphism in the fibrinolytic system and endometriosis.

OBJECTIVE: Although most women experience retrograde menses during their reproductive life, endometriosis develops only in a small percentage. We hypothesized that persistence of a fibrin matrix in peritoneal pockets, as a result of hypofibrinolysis, could allow menstrually deposited endometrial fragments to initiate endometriosis. Fibrinolysis is modulated by several factors, and polymorphisms in the plasminogen activator inhibitor-1 (PAI-1) gene are considered to be one of the important determinants. The objective of this study was to evaluate PAI-1 genotypes in a group of women with or without endometriosis. METHODS: In 118 women (75 with laparoscopically confirmed endometriosis and 43 controls), genomic DNA was extracted from blood and the PAI-1 promoter genotype was determined by polymerase chain reaction amplification of DNA using specific primers for the 4G or 5G allele followed by gel electrophoresis. A portion of the polymerase chain reaction product was purified and sequenced to confirm the gel electrophoresis results. RESULTS: Endometriosis was more likely in patients with 4G/5G (odds ratio 38; 95% confidence interval [CI] 6-229) or 4G/4G (odds ratio 441; 95% CI 53-3,694) compared with 5G/5G PAI-1 genotype. Fifty-two of 75 women with endometriosis (69 %, 95% CI 58-79%) had the 4G/4G genotype compared with only 5 of 43 (12%; 95% CI 4-25%) controls. In contrast, the 5G/5G genotype associated with normal fibrinolysis was found in 2 of 75 (3%; 95% CI 0-9%) women with endometriosis compared with 24 of 43 (56%; 95% CI 40-71%) controls. CONCLUSION: Hypofibrinolysis, associated with the 4G allele of the PAI-1 gene, was found significantly more often in women with endometriosis compared with controls. Persistence of fibrin matrix could support the initiation of endometriotic lesions in the peritoneal cavity, explaining why some women with retrograde menstruation develop endometriosis while others do not. LEVEL OF EVIDENCE: II-2.

Adult↗