Abstract Study question Are there correlations between HE4 levels and the presence of ultrasonographic findings of adenomyosis in infertile women, and how does this relationship effect pregnancy outcomes? Summary answer The presence of subendometrial linear lines as marker of endo-myometrial invasion, is correlated with serum HE4 levels and negatively impacts pregnancy outcomes. What is known already Adenomyosis observed in women of reproductive age can present with infertility, particularly as implantation is adversely affected in junctional zone abnormalities. The endo-myometrial invasion in the pathogenesis of adenomyosis has been variously defined based on ultrasound criteria outlined in the MUSA (Morphological Uterus Sonographic Assessment) consensus. In patients with adenomyosis, while implantation rates may remain unchanged in some studies, an increased rate of first-trimester miscarriages has been reported in others. This study was conducted based on the hypothesis that HE4 (Human Epididymis Protein 4) marker may play a role in this potential invasiveness in adenomyosis and negatively affect embryo implantation. Study design, size, duration 82 infertile patients diagnosed with adenomyosis based on the MUSA consensus ultrasound criteria were included in our 10-month prospective study. HE4 testing was performed on serum samples collected from the patients. Our primary outcome focused on the association between ultrasonographic findings and the serum HE4 levels. The secondary outcomes comprised assessing the relationship between HE4 levels and the outcomes of implantation and pregnancy in the patients who underwent embryo transfer. Participants/materials, setting, methods In this study, infertile patients aged 20-40 diagnosed with adenomyosis via ultrasonography were included. Patients with a history of myoma or myomectomy, high-dose biotin intake, chronic organ failure, and a history of malignancy were excluded from the study. Ultrasonographic features were documented, and 39 patients underwent frozen embryo transfer. Pregnancy outcomes were categorized as clinical pregnancy or miscarriage for analysis. Main results and the role of chance As a primary outcome of the study, a statistically significant correlation between serum HE4 levels and subendometrial linear lines, as indicated in the MUSA consensus ultrasound criterias is found (p = 0.015). Logistic regression analysis showed that HE4 independently increased subendometrial linear striations [OR (95% CI) = 1.05 (1.01-1.10); model significance p = 0.020]. ROC analysis suggested that serum HE4 levels above the 37.4 pmol/L cut-off value may be associated with the presence of subendometrial linear striations. Although no relationship was found between HE4 levels and implantation rates, statistically significant higher levels of HE4 were observed in cases of miscarriage compared to ongoing pregnancies (p = 0.038). Limitations, reasons for caution Study limitations include reliance on 2D-TVUSG without 3D and Doppler. Enhanced evaluation using 3D-TVUSG and Doppler for JZ and subendometrial areas is recommended. Larger sample sizes could yield more impactful findings. Wider implications of the findings HE4 correlates with observed subendometrial linear lines, suggesting a link to endo-myometrial invasion. While it doesn’t impact transfer cycle implantation rates, it may be linked to higher early pregnancy losses. Trial registration number not applicable