This depository contains all microscopy images presented in ‘Menstrual flow as a non-invasive source of endometrial organoids’ paper accepted at Communications Biology; Tereza Cindrova-Davies et al. present a method for deriving endometrial organoids using menstrual flow collected from women. The approach shows promise as a personalized and non-invasive way to investigate gynecological conditions such as endometriosis and failed implantation following IVF. Fig. 1b-d images present time line images detailing propagation of organoids from menstrual flow and scratch. Fig. 1b: The images represent the growth of menstrual organoids directly derived from menstrual blood. Fig. 1c-d: The images illustrate the growth of organoids from frozen menstrual (Fig. 1c) and scratch (Fig. 1d) digests of the same patient. Fig. 1e: Images depict organoids derived from an endometrial scratch and menstrual flow of the same patient; they are indistinguishable morphologically. Fig. 1f depicts single cell assay images to compare the growth of paired menstrual vs. scratch organoids derived from 4 patients. Images show organoids seeded from single cells (5,000 cells per 20 μl drop Matrigel) after 9d of growth. Fig. 1h shows representative images of immunostaining against the marker of proliferation, Ki67, in menstrual and scratch organoids of one patient. Fig. 3a images depict menstrual organoids seeded and grown for 4 d, followed by treatment with culture medium alone, or with hormones, which consisted of pre-treatment with β-estradiol (10 nM) for 2 d, followed by treatment with β-estradiol, progesterone (1 μM) and cAMP (1 μM) (EPC), EPC plus prolactin (20 ng/ml), or EPC plus prolactin (20 ng/ml), hPL (20 ng/ml) and hCG (100 ng/ml) for 4 d. At the end of each experiment, organoids were removed from Matrigel using Cell Recovery solution and processed for immunohistochemistry. The images show immunohistochemical upregulation of PAEP/GdA, acetylated tubulin, progesterone receptor (P4-R) and prolactin receptor (PRL-R) in response to hormones. Fig. 3e shows representative images of haematoxylin and eosin (H&E) staining of hormonally-treated menstrual and scratch organoids from one patient, showing morphological differences between untreated vs. hormonally treated organoids. Fig. 3f depics Bandeiraea simplicifolia -II (BSA-II) lectin staining of semi-thin resin sections of menstrual and scratch organoids treated with hormones. Suppl. Fig. 1: Derivation and characterisation of menstrual flow organoids. Time line images detailing propagation of organoids from menstrual flow and scratch. The top panel represents the growth of menstrual organoids directly derived from menstrual blood. The middle and bottom panels illustrate the growth of organoids from frozen menstrual and scratch digests of the same patient. Suppl. Fig. 2: Arachis hypogaea agglutinin (AHA) lectin staining of semi-thin resin sections of menstrual and scratch organoids treated with hormones. Menstrual and scratch organoids were grown for 4 d, and treated with culture medium alone or β-estradiol for 2 d followed by β-estradiol, progesterone and cAMP (EPC), EPC plus prolactin, or EPC plus prolactin, hPL and hCG for 4 d. Semi-thin resin sections were stained with AHA lectin. Suppl. Fig. 3: Periodic Acid Schiff (PAS) staining of menstrual and scratch organoids treated with hormones. Menstrual and scratch organoids were grown for 4 d, and treated with culture medium alone or β-estradiol for 2 d followed by β-estradiol, progesterone and cAMP (EPC), EPC plus prolactin, or EPC plus prolactin, hPL and hCG for 4 d. Paraffin-embedded sections were stained with PAS reagents to detect glycogen (purple). Arrows indicate purple glycogen staining, which is also secreted into the lumen of organoids treated with hormones. Suppl. Fig. 4: Haematoxylin and eosin (H&E) staining of hormonally-treated menstrual and scratch organoids from three patients. Menstrual and scratch organoids were grown for 4 d, and treated with culture medium alone or β-estradiol for 2 d followed by β-estradiol, progesterone and cAMP (EPC), EPC plus prolactin, or EPC plus prolactin, hPL and hCG for 4 d. Paraffin-embedded sections were stained with H&E reagents. The images show morphological differences between untreated vs. hormonally treated organoids from 3 patients. There is evidence of a columnar epithelial morphology with increased vacuole formation analogous to the hypersecretory phenotype of early pregnancy.