A hypothesis on the aetiology of adenomyosis: is there an association between cumulative number of menstruations and severity of adenomyosis on ultrasound?

Eva J.E. de Bock: Writing – review & editing, Writing – original draft, Visualization, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Cilla Verbeek: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Eva Klinkenberg: Methodology, Investigation, Formal analysis, Data curation. Jos W.R. Twisk: Methodology. Nicole B. Burger: Writing – review & editing, Supervision, Methodology, Conceptualization. Lynda J.M. Juffermans: Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization. Judith A.F. Huirne: Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization. Robert A. de Leeuw: Writing – review & editing, Supervision, Methodology, Conceptualization.

This exploratory retrospective cohort study was conducted at Amsterdam University Medical Centre, a tertiary referral centre for adenomyosis, as a sub-analysis of the prospective UteroVue cohort study. The study was approved by the institutional review board (NL83391.018.23). Participants for the UteroVue study were recruited via the benign gynaecology outpatient clinic and via flyering. Inclusion criteria were premenopausal participants aged ≥ 18 years with a uterus in situ. Exclusion criteria were current pregnancy and intrauterine device use. For this sub-analysis, additional exclusion criteria were a dominant uterine disorder other than adenomyosis, incomplete or invalid questionnaire data, and adenomyosis in the outer third of the myometrium presumed secondary to endometriosis. During the study consultation, clinical information was obtained, including current medication use, obstetric and surgical history, and current gynaecological symptoms. All participants subsequently underwent a transvaginal ultrasound examination using a HERA W10 machine with an EV2–10A transvaginal probe (Samsung Medison, Seoul, Republic of South Korea). After the study visit, two experienced sonographers (E.B. and N.B.) assessed the 2D and 3D B-mode and power Doppler scans by consensus, blinded to clinical data. The scans were evaluated on the presence of direct and indirect Morphological Uterus Sonographic Assessment (MUSA) features of adenomyosis [17] . Previously, the MUSA group proposed a classification of disease severity based on the estimated percentage of myometrium affected by MUSA features of adenomyosis, categorising adenomyosis as mild (50% affected myometrium) [18] , [19] . In the present study, the severity of adenomyosis was recorded as a continuous estimate of the percentage of affected myometrium. For this retrospective sub-analysis, participants were invited to complete a questionnaire about their menstrual history after their study visit. They reported age at menarche; cumulative weeks of pregnancy and breastfeeding; use and duration of hormonal menstruation suppression, including pill-free intervals; and a history of endometritis, pelvic inflammatory disease, chlamydia, and gonorrhoea, all retrospectively up to the time of the study visit. Data were verified after collection, and participants were contacted by telephone in case of discrepancies (e.g. reported medication use with a duration of 0 years). If participants could not be reached, a duration of 0.4 years was imputed, assuming that durations < 0.5 years may have been rounded down to zero. The cumulative number of menstruations was estimated accounting for pregnancy, lactation, and hormonal menstruation suppression use ( Fig. 1 ). Withdrawal bleeding during cyclical hormonal menstruation suppression (oral contraceptive pill, vaginal ring, contraceptive patch) was weighted at 0.7 of a natural menstruation to account for the approximately 30% less vaginal blood loss compared to menstruation [20] . Continuous hormonal menstruation suppression (hormonal intrauterine device, progestogen-only pill, contraceptive injection, contraceptive implant) was assumed to result in amenorrhoea. Fig. 1 Example of cumulative menstrual exposure estimation. Example of cumulative menstrual exposure estimation. As this was an exploratory study, no formal sample size calculation was performed. Statistical analyses were performed using IBM SPSS 28.0, with p -value < 0.05 considered statistically significant. If the outcome variable was skewed, a logarithmic transformation was applied; in such cases, a constant of 1 was added to all observations to accommodate zero values. The primary study endpoint was the association between cumulative menstruations and severity of adenomyosis. To assess this association, linear regression analyses were used. Based on the literature, potential confounding variables were identified and evaluated: vaginal births, prior uterine surgeries (caesarean sections, myomectomies, curettages, endometrial ablations), and age [7] , [15] , [21] , [22] . Confounders were retained if their inclusion resulted in a ≥ 10% change in the regression coefficient [23] . Potential multicollinearity was assessed using correlation analyses; variables showing collinearity ( r  > 0.7) were excluded. Subsequently, a multivariable linear regression analysis was performed, including cumulative menstruations and the selected confounders. Independent associations of each confounder with severity of adenomyosis were assessed within the same model.

A total of 264 participants provided informed consent between 2024 and 2025. After excluding participants due to dominant uterine fibroids (n = 91), malignancy (n = 4), PEComa (n = 1), uterine necrosis (n = 1), incomplete questionnaires (n = 45), implausible data resulting in a negative number of estimated menstruations (n = 2), adenomyosis presumed secondary to endometriosis (n = 1), the analysis included 119 participants. Among the included participants, 13% had endometriosis (n = 16). Of the participants with endometriosis, 38% had mild adenomyosis, 38% moderate adenomyosis, and 25% severe adenomyosis. Their general characteristics and hormonal menstruation suppression medication use are shown in Table 1 . Table 1 General characteristics and hormonal menstruation suppression medication use of the participants. Characteristic Value, N = 119 Age a (years) 27.0 (24.0 – 34.0) Body Mass Index a (kg/m 2 ) 23.5 (21.1 – 26.7) Ancestral origin b   Africa 5 (4%)   Asia 3 (3%)   Europe 98 (82%)   South America 4 (3%)   Mixed 9 (8%) Age at menarche c (years) 12.7 ± 1.5 Gravidity b   0 86 (72%)   1 10 (8%)   ≥2 23 (19%) Parity b   0 94 (79%)   1 9 (8%)   ≥2 16 (13%) Prior gynaecological inflammatory conditions b   Chlamydia 17 (14%)   Pelvic inflammatory disease 1 (1%)   Endometritis 15 (13%) Endometriosis 16 (13%) Prior uterine surgery b 17 (14%)   Caesarean section 4 (3%)   Hysteroscopic myomectomy 1 (1%)   Curettage 10 (8%)   Endometrial ablation 1 (1%) Menstrual cycle phase at time of study visit b   Pre-ovulatory 36 (30%)   Post-ovulatory 25 (21%)   Suppressed menstrual cycle 58 (49%) Gynaecological symptoms b   No symptoms 95 (80%)   Heavy menstrual bleeding 7 (6%)   Dysmenorrhea 10 (8%)   Chronic pelvic pain 13 (11%)   Deep dyspareunia 9 (8%)   Subfertility 1 (1%) Cyclical hormonal menstruation suppression   Oral contraceptive pill b 103 (87%)    Duration in years a 7.0 (3.0-11.0)   Vaginal ring b 9 (8%)    Duration in years a 1.0 (0.7-1.0)   Contraceptive patch b 2 (2%)    Duration in years a 1.3 (1.0-1.3) Continuous hormonal menstruation suppression   Hormonal intrauterine device b 38 (32%)    Duration in years a 3.0 (1.0-5.0)   Progestogen-only pill b 24 (20%)    Duration in years a 1.0 (0.1-1.0)   Contraceptive injection b 12 (10%)    Duration in years a 1.0 (0.5-1.8)   Contraceptive implant b 10 (8%)    Duration in years a 1.0 (0.8-3.6) Estimated myometrium affected by adenomyosis   No adenomyosis (0%) 10 (8%)   Mild adenomyosis (1-24%) 74 (62%)   Moderate adenomyosis (25-50%) 20 (17%)   Severe adenomyosis (>50%) 15 (13%) Direct MUSA features of adenomyosis   Subendometrial lines and/or buds 102 (86%)   Myometrial cysts 36 (30%)   Hyper-echoic islands 76 (64%) Indirect MUSA features of adenomyosis   Fan-shaped shadowing 54 (45%)   Globular uterus 10 (8%)   Asymmetrical thickening 21 (18%)   Irregular junctional zone 98 (82%)   Interrupted junctional zone 58 (49%) a Median (25 th percentile - 75 th percentile). b Count of participants (percentage). c Mean ± standard deviation. MUSA = Morphological Uterus Sonographic Assessment. General characteristics and hormonal menstruation suppression medication use of the participants. Median (25 th percentile - 75 th percentile). Count of participants (percentage). Mean ± standard deviation. MUSA = Morphological Uterus Sonographic Assessment. In the unadjusted analyses, a statistically significant association was observed between the cumulative number of menstruations and severity of adenomyosis (log-transformed β = 0.005 on the ln( percentage affected myometrium +1) scale, Exp( β ) = 1.005, 95% confidence interval (CI)= 1.002–1.007, p  < 0.01). Age showed a moderate correlation with the cumulative number of menstruations ( r  = 0.72), indicating collinearity; therefore, age was not included in adjusted models. After adjustment for number of vaginal births and uterine surgeries, included as confounders due to ≥ 10% change in the regression coefficient for cumulative menstrual exposure, the association between cumulative number of menstruations and severity of adenomyosis remained statistically significant (log-transformed β =0.003 on the ln( percentage affected myometrium +1) scale , Exp( β )= 1.003, 95% CI= 1.001–1.006, p  < 0.05). In addition, the confounders included in the model, vaginal births and uterine surgeries, were also independently associated with severity of adenomyosis (both p  < 0.05).

Our findings suggest that cumulative menstrual exposure is associated with severity of adenomyosis, supporting the TIAR hypothesis. However, causality cannot be inferred and the role of age remains uncertain. Further prospective studies are needed to clarify the relationship between menstrual exposure, age, and severity of adenomyosis.

Understanding the aetiology of adenomyosis is crucial for advancing treatment and prevention approaches. In this study, we investigated the TIAR hypothesis by examining the relationship between menstrual exposure and the severity of adenomyosis on ultrasound. We found that the cumulative number of menstruations was significantly associated with disease severity. Specifically, each menstruation corresponded to a 0.3% increase in affected myometrium, indicating a clinically relevant impact over a lifetime, considering an average of approximately 450 menstruations [24] , [25] . These findings support the hypothesis that repeated microtrauma to the endo-myometrial interface during menstruation enables endometrial invasion, driving the development of adenomyosis. This is the first empirical study on the relationship between menstrual exposure and adenomyosis. Thereby, it provides an important contribution to the largely theoretical literature on TIAR [26] . Furthermore, our findings support the invagination theory on which the TIAR model builds, as uterine surgeries and vaginal birth were both associated with adenomyosis. This is in line with previous studies [15] , [27] , [28] , [29] . Several limitations should be considered. First, menstrual history data were collected retrospectively, which may have introduced recall bias. Second, selection bias might have occurred because only participants who completed the menstrual history questionnaire were included. This might have resulted in underrepresentation of participants with complex medication histories, who might have found it difficult to report their extensive medication history. Third, age was moderately correlated with cumulative menstrual exposure, hindering our ability to assess their independent effects. These variables could not be separated, as the limited sample size prevented subgroup analyses of similarly aged participants with varying menstrual exposures. Fourth, the relatively young age of the study population may limit generalisability. Fifth, the weighting factor applied to withdrawal bleeding contributed to imprecision in menstrual exposure estimation. Given these limitations, our findings should be interpreted with caution. Nonetheless, such limitations are expected in an initial exploration of the understudied TIAR hypothesis. The potential impact of menstrual exposure on progression of adenomyosis is increasingly relevant, as lifetime menstrual exposure has risen substantially over time. In industrialised countries today, the lifetime number of ovulatory menstruations is roughly three to ten times higher than in the 18th century, due to fewer pregnancies, shorter durations of lactational amenorrhea, earlier menarche, and later menopause [24] , [25] . If the TIAR hypothesis is correct, this would imply that the lifetime risk of severe adenomyosis in these countries is higher nowadays as a consequence of increased menstrual exposure. Under this assumption, it may be worthwhile to explore whether hormonal menstruation suppression could prevent progression of adenomyosis. To disentangle the effects of age and menstrual exposure, further research with larger populations with broad variation in cumulative menstrual exposure within age groups is required to enable age-based subgroup analyses. Furthermore, prospective studies with serial ultrasound exams are necessary to observe disease progression over time. Lastly, assessment of disease severity may benefit from incorporating additional tissue characteristics into the ultrasound exam, such as microcirculation and tissue stiffness, through the use of contrast-enhanced ultrasound and elastography [30] , [31] .

Few gynaecological conditions remain as mysterious as adenomyosis. This uterine disorder is characterised by endometrial glands and stroma within the myometrium and is associated with dysmenorrhea, heavy menstrual bleeding, and subfertility [1] . These symptoms can be profoundly disruptive to daily life [2] . Beyond this knowledge, much about the condition remains unknown. This is reflected in the wide variation in reported prevalence, ranging from 7% to 67% depending on study population and diagnostic modality (ultrasound, magnetic resonance imaging, or histopathology) [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] . The lack of effective treatments further illustrates these knowledge gaps: hysterectomy is unsuitable for those desiring pregnancy, and hormonal therapies often fail to provide adequate symptom relief [12] . To develop effective treatment and prevention strategies, it is essential to uncover the underlying aetiology. Two main hypotheses have been proposed: the “metaplasia” and “invagination” theories [13] . The latter postulates that disruption of the endo-myometrial interface enables endometrial invasion into the myometrium. Initially, the invagination theory focused on macrotrauma, such as vaginal birth and surgeries. However, the “tissue injury and repair” (TIAR) hypothesis posits that, in addition to macrotrauma, repeated microtrauma from physiological uterine menstrual contractions damages the endo-myometrial interface [14] . This microtrauma triggers a chronic injury-repair cycle, characterised by inflammation, local oestrogen production and enhanced uterine contractility. The resulting vicious cycle weakens the endo-myometrial interface and induces cellular changes in basal endometrial cells, increasing their proliferation, migratory capacity, and resistance to apoptosis. This facilitates endometrial invasion [14] . Based on the TIAR hypothesis, a higher cumulative number of menstruations may cause more microtrauma, possibly resulting in more extensive adenomyosis. Supporting this, studies have shown that early menarche and short menstrual cycles are associated with adenomyosis [15] , [16] . Nevertheless, the TIAR hypothesis remains debated. Therefore, we explored whether the cumulative number of menstruations is associated with the severity of adenomyosis on ultrasound.

The authors have nothing to declare.