A tumor necrosis factor-α inhibitor reduces the embryotoxic effects of endometriotic peritoneal fluid  Yi-Jen Chen, M.D., Ph.D., Hua-Hsi Wu, M.D., Wan-Ting Liau, M.Sc., Chang-Youh Tsai, M.D., Ph.D., Hsiao-Wen Tsai, M.D., Kuan-Chong Chao, M.D., Yen-Jen Sung, M.D., Ph.D., Hsin-Yang Li, M.D., Ph.D.  Fertility and Sterility  Volume 100, Issue 5, Pages 1476-1485.e5 (November 2013) DOI: 10.1016/j.fertnstert.2013.07.1985 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Chocolate cyst peritoneal fluid (CH-PF) induces embryotoxicity and apoptosis. (A) The development of two-cell mouse embryos into blastocysts (3.5 days) when treated with nonendometriosis peritoneal fluid (N-PF) (negative control), 1% CH-PF, and 10% CH-PF. Bars = 100 μm. (B) Representative data from the evaluation of mouse embryos incubated with N-PF (negative control), 1% CH-PF, and 10% CH-PF by TUNEL assay. The positive controls for the TUNEL assay were incubated with 50 μL RQ1 RNase-free DNase. The negative controls were not exposed to the enzyme TdT. DAPI = 6-diamino-2-phenylindole. Bars = 100 μm. (C) The quantified results of the blastocyst developmental ratio after incubation with N-PF (negative control), 1% CH-PF, and 10% CH-PF. P<.01 across all incubations; P=.023 for 10% N-PF and 1% CH-PF; P<.01 for 1% CH-PF and 10% CH-PF. Fertility and Sterility 2013 100, 1476-1485.e5DOI: (10.1016/j.fertnstert.2013.07.1985) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The expression profiles of cytokines in CH-PF compared with N-PF. (A) A representative picture with the corresponding labeling. The significant difference in tumor necrosis factor-α expression is indicated by the red circles. (B) The quantification of tumor necrosis factor- α (TNF-α) expression in CH-PF and N-PF; *P<.001. The bars show the mean value μ SD. Fertility and Sterility 2013 100, 1476-1485.e5DOI: (10.1016/j.fertnstert.2013.07.1985) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Tumor necrosis factor-α (TNF-α) enhances embryotoxicity and apoptosis. Adalimumab abrogates the effects of TNF-α. (A) The growth of the two-cell mouse embryos to blastocysts (3.5 days) in the presence of M16, 0.01 ng/mL TNF-α, 0.1 ng/mL TNF-α, and TNF-α with 0.1 μg/mL adalimumab. Bars = 100 μm. (B) Representative cases of the TUNEL assay for mouse embryos treated with M16, 0.01 ng/mL TNF-α, 0.1 ng/mL TNF-α, and TNF-α with 0.1 μg/mL adalimumab. The positive control samples were incubated with 50 μL RQ1 RNase-free DNase. DAPI = 6-diamino-2-phenylindole. The negative control samples were not exposed to TdT. Bars = 100 μm. (C) The quantified results of the blastocyst developmental ratio after incubation with M16, TNF-α, and TNF-α plus adalimumab (Ada). P=.02 for 0.01 ng/mL TNF-α, 0.1 ng/mL TNF-α, and 0.01 ng/mL TNF-α with 0.1 μg/mL Ada; P=.01 for 0.1 ng/mL TNF-α, 0.01 ng/mL TNF-α with 0.1 μg/mL Ada, and 0.1 ng/mL TNF-α with 0.1 μg/mL Ada. Fertility and Sterility 2013 100, 1476-1485.e5DOI: (10.1016/j.fertnstert.2013.07.1985) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Adalimumab inhibits the embryotoxicity and apoptosis induced by chocolate cyst peritoneal fluid (CH-PF). (A) The growth of the two-cell mouse embryos to blastocysts (3.5 days) in the presence of M16, CH-PF, and CH-PF with adalimumab. PBS = phosphate-buffered saline. Bars = 100 μm. (B) The representative feature of apoptosis, as measured by the TUNEL assay, in the mouse embryos treated with M16, CH-PF, and CH-PF with adalimumab. The positive controls were incubated with 50 μL RQ1 RNase-free DNase, whereas the negative controls were not exposed to TdT. DAPI = 6-diamino-2-phenylindole; PBS = phosphate-buffered saline. Bars = 100 μm. (C) The quantified results of the blastocyst developmental ratio after incubation with M16, CH-PF, and CH-PF with adalimumab (Ada). P = .031 for CH-PF and CH-PF + Ada. Fertility and Sterility 2013 100, 1476-1485.e5DOI: (10.1016/j.fertnstert.2013.07.1985) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 The overexpression of cytokines in chocolate cyst peritoneal fluid (CH-PF), as demonstrated by cytokine arrays. (A) The cytokines expressed in CH-PF but not in nonendometriosis peritoneal fluid. (B) The representative features of the overexpressed cytokines in CH-PF. The arrow indicates a chocolate cyst/nonendometriosis (CH/N) ratio of one, which was used as a standard. Fertility and Sterility 2013 100, 1476-1485.e5DOI: (10.1016/j.fertnstert.2013.07.1985) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 The specificity and neutralization activity of adalimumab to human tumor necrosis factor-α (TNF-α). (A) The mouse embryos were incubated with different concentrations of adalimumab in the presence of TNF-α (0.1 ng/mL). The maximal blastocyst development ratio was noted in the 0.1 μg/mL adalimumab. Bars = 100 μm. (B) The mouse embryos were incubated with different concentrations of adalimumab in the presence of TNF-α (0.1 ng/mL) or 10% chocolate cyst peritoneal fluid (CH-PF). The maximal blastocyst development ratio was noted in the 0.1 μg/mL adalimumab. These experiments were using control antibody (Ab) treatment to prove the specificity of the effects. Bars = 100 μm. *P<.05. Fertility and Sterility 2013 100, 1476-1485.e5DOI: (10.1016/j.fertnstert.2013.07.1985) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions