Fertility and Sterility

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Fertility and Sterility Local application of danazol-loaded hyaluronic acid hydrogel to endometriosis in a rat model Kazuhito Nomura, M.D., Koichi Murakami, M.D., Ph.D., Makio Shozu, M.D., Ph.D., Tsuyoshi Nakama, Ph.D., Nobuhiko Yui, Ph.D., Masaki Inoue, M.D., Ph.D. Fertility and Sterility Volume 85, Pages 1157-1167 (April 2006) DOI: 10.1016/j.fertnstert.2005.08.057 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 A [3H]-thymidine incorporation assay of cultured epithelial cells from rat endometrium. Rat endometrial epithelial cells were cultured after enzymatic digestion. Culture medium was changed to Dulbecco’s Modified Eagle’s Medium (DMEM)–F12 (1:1) containing 0.2% bovine serum albumin (BSA) at approximately 50% confluence. After 24 hours of incubation, culture medium was replaced with DMEM–F12 (1:1, phenol red free) supplemented with 5% dextran charcoal–treated fetal bovine serum (FBS) with or without sodium hyaluronate (1 mg/mL). Examined reagents were added to the medium, and incubation was continued for 24 hours. Incorporated [3H]-thymidine was counted 3 hours after addition of 1 μCi [3H]-thymidine. Each bar represents mean ± SE of three different experiments, each performed with cells in triplicate wells. (A) Dose-dependent effects of Danazol (DZ) on [3H]-thymidine incorporation. (B) Effects of E2 on [3H]-thymidine incorporation in DZ-treated epithelial cells. *P<.05 compared with control (Kruskal-Wallis test). **P<.01 compared with control (Kruskal-Wallis test). Nomura. A DDS for endometriosis. Fertil Steril 2006. Fertility and Sterility 2006 85, 1157-1167DOI: (10.1016/j.fertnstert.2005.08.057) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Microscopic appearance of endometrioma at 4 weeks after injection (HE stain, ×200; immunostaining for macrophage, ×200). (A) Control group: endometrial epithelial cells are cuboidal and contain normal volumes of cytoplasm. (B) Immunohistochemical staining of macrophages in the control group: many macrophages are found in the endometriotic stromal lesion beneath the epithelial cells. (C) Hyaluronic acid (HA) gel group: epithelial cells resemble those in the control group. (D) High-dose Danazol-loaded hyaluronic acid hydrogel (DZ-HA) gel group: endometrial epithelial cells show atrophic changes (decreased cytoplasm, perinuclear halo, and condensed nuclei). (E) Oophorectomized group: epithelial cells resemble those in DZ-HA gel groups. Nomura. A DDS for endometriosis. Fertil Steril 2006. Fertility and Sterility 2006 85, 1157-1167DOI: (10.1016/j.fertnstert.2005.08.057) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Microscopic appearance of endometrioma at 4 weeks after injection (HE stain, ×200; immunostaining for macrophage, ×200). (A) Control group: endometrial epithelial cells are cuboidal and contain normal volumes of cytoplasm. (B) Immunohistochemical staining of macrophages in the control group: many macrophages are found in the endometriotic stromal lesion beneath the epithelial cells. (C) Hyaluronic acid (HA) gel group: epithelial cells resemble those in the control group. (D) High-dose Danazol-loaded hyaluronic acid hydrogel (DZ-HA) gel group: endometrial epithelial cells show atrophic changes (decreased cytoplasm, perinuclear halo, and condensed nuclei). (E) Oophorectomized group: epithelial cells resemble those in DZ-HA gel groups. Nomura. A DDS for endometriosis. Fertil Steril 2006. Fertility and Sterility 2006 85, 1157-1167DOI: (10.1016/j.fertnstert.2005.08.057) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Immunohistochemical findings of endometrioma at 4 weeks after treatment (counterstained using hematoxylin, ×200). (A) Control group: scattered apoptotic cells contain ssDNA-positive condensed nuclei and perinuclear halos. (B) High-dose Danazol-loaded hyaluronic acid hydrogel (DZ-HA) gel group: endometrial epithelium with increased number of apoptotic cells, containing ssDNA-positive condensed nuclei and perinuclear halos. (C) Oophorectomized group: epithelial cells display atrophic changes but few apoptotic cells are apparent. Nomura. A DDS for endometriosis. Fertil Steril 2006. Fertility and Sterility 2006 85, 1157-1167DOI: (10.1016/j.fertnstert.2005.08.057) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Changes of endometrioma volume after Danazol-loaded hyaluronic acid hydrogel (DZ-HA) gel treatment. A 0.1-mL aliquot of HA gel or DZ-HA gel was injected after evacuation of cyst contents at week 0. Size of the endometrioma was measured weekly using micrometer calipers. Size at gel injection (week 0) was considered 100%. Each point represents mean ± SE of cyst size in each group. Control: untreated; OVX: oophorectomy conducted at week 0. *P<.05 compared with control (Scheffé). **P<.01 compared with control (Scheffé). Nomura. A DDS for endometriosis. Fertil Steril 2006. Fertility and Sterility 2006 85, 1157-1167DOI: (10.1016/j.fertnstert.2005.08.057) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions