Regulatory T cells, inflammation, and endoplasmic reticulum stress in women with defective endometrial receptivity Mario Galgani, Ph.D., Luigi Insabato,

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Regulatory T cells, inflammation, and endoplasmic reticulum stress in women with defective endometrial receptivity Mario Galgani, Ph.D., Luigi Insabato, M.D., Gaetano Calì, Ph.D., Anna Nunzia Della Gatta, M.D., Paola Mirra, Ph.D., Federica Papaccio, B.Sc., Marianna Santopaolo, B.Sc., Carlo Alviggi, M.D., Antonio Mollo, M.D., Ida Strina, M.D., Giuseppe Matarese, M.D., Francesco Beguinot, M.D., Giuseppe De Placido, M.D., Luca Ulianich, Ph.D. Fertility and Sterility Volume 103, Issue 6, Pages 1579-1586.e1 (June 2015) DOI: 10.1016/j.fertnstert.2015.03.014 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Inflammatory circulating molecules levels in women with normal fertility (FS) and patients with recurrent miscarriage (RM) and repeated in vitro fertilization failure (RIF). Scatter plots indicate the levels of soluble tumor necrosis factor receptor (sTNF-R), soluble intercellular adhesion molecule 1 (sICAM-1), leptin, resistin, monocyte myeloperoxidase (MPO), resistin, and interleukin 22 (IL-22) measured in duplicate serum samples of FS, RM, and RIF women. Data are shown as the mean. ∗P<.05; ∗∗P<.005 by Mann-Whitney test. Fertility and Sterility 2015 103, 1579-1586.e1DOI: (10.1016/j.fertnstert.2015.03.014) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Endometrial T cell subpopulations in women with normal fertility (FS) and patients with recurrent miscarriage (RM) and repeated in vitro fertilization failure (RIF). Immunostaining for CD3+, CD4+, CD8+, and CD5+ in timed midsecretory endometrium of FS, RM, or RIF women. Representative images shown were captured at 200× magnification. Graphs represent the average number of T cells in five high power fields with ×40 objective magnification (approximately 1 mm2). ∗P<.05; ∗∗P<.01. Fertility and Sterility 2015 103, 1579-1586.e1DOI: (10.1016/j.fertnstert.2015.03.014) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Reduced regulatory FoxP3+ cells in women with infertility problems. Immunofluorescence analysis for Foxp3+ regulatory cells (green staining) in timed midsecretory endometrium of women with normal fertility (FS) and patients with recurrent miscarriage (RM) and repeated in vitro fertilization failure (RIF). Nuclei were stained by DRAQ5 (blue staining). Representative images shown were captured at ×200 magnification. The graph represents the average number of Foxp3+ regulatory cells counted in four fields of 1 mm2. ∗∗P<.01. Fertility and Sterility 2015 103, 1579-1586.e1DOI: (10.1016/j.fertnstert.2015.03.014) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Deregulated heat shock 70-kDa protein 5 (HSPA5) and soluble X box binding protein 1 (sXBP-1) mRNA expression in the endometrium of patients with recurrent miscarriage (RM) and repeated in vitro fertilization failure (RIF). (A) Timed midsecretory endometrial biopsies from women with normal fertility (FS) and from RM or RIF patients were analyzed for the expression of HSPA5 (left panel) and sXBP-1 (right panel) transcripts using real-time reverse-transcription polymerase chain reaction. The transcript levels were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). AU = arbitrary units. ∗∗P<.01. (B) Immunostaining for HSPA5 in timed midsecretory endometrium of FS, RM, or RIF women. Scale bar: 100 μm. We assigned the following scores: 0 = no positivity; I = weak positivity; II = medium positivity; III = strong positivity. Shown here are a representative experiment (left panel) and a summarizing graph (right panel). Fertility and Sterility 2015 103, 1579-1586.e1DOI: (10.1016/j.fertnstert.2015.03.014) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions