Cigarette smoke condensate exposure delays follicular development and function in a stage-dependent manner  Jean Clair Sadeu, M.D., Ph.D., Warren G. Foster, Ph.D.  Fertility and Sterility  Volume 95, Issue 7, Pages 2410-2417 (June 2011) DOI: 10.1016/j.fertnstert.2011.03.072 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Representative pictures of follicles during in vitro growth. Preantral follicles at days 1 and 4 in (A and B) Ctrl and (F and G) DMSO controls or cultured in (K and L) 30 μg/mL, (P and Q) 60 μg/mL, (U and V) 90 μg/mL, and (Z and AB) 130 μg/mL CSC. Preantral follicles defined as “follicular,” with the oocyte closely connected to its granulosa cells, surrounded by a basal membrane and adjacent thecal cells, or defined as (B, G, L, Q, W, and AC) “diffused,” with granulosa cells outgrowth through the surrounding basal membrane as the result of cell proliferation. Antral follicles at days 8 and 12 in (C and D) Ctrl and (H and I) DMSO controls or cultured in (M and N) 30 μg/mL, (R and S) 60 μg/mL, (X) 90 μg/mL, and (AD) 130 μg/mL CSC. In antral follicles granulosa cells differentiated into cumulus cells and mural granulosa cells, resulting in antral-like cavitiy (antrum) formation, characterized by appearance of translucent areas within the follicle. Immature oocyte with (AE) GV or (Y) GV breakdown, and mature oocytes with (E, J, O, and T) PB at day 13 of culture. GC = granulosa cells; O = oocyte; CC = cumuls cells; A = antrum, PB = polar body. Original magnification, ×400 (day 1, L, Q, V, AB, and day 13), ×200 (B and G), and ×100 (day 8 and day 13). Fertility and Sterility 2011 95, 2410-2417DOI: (10.1016/j.fertnstert.2011.03.072) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of CSC on follicular development and survival in vitro. Follicular development at days (A) 4, (B) 8, and (C) 12 of culture, and (D) follicular survival at day 12. Each experiment was repeated six times in duplicate plates per condition, n = 1,163 follicles (12–20 follicles per plate). Bars with different letters differ significantly (P<.05). Fertility and Sterility 2011 95, 2410-2417DOI: (10.1016/j.fertnstert.2011.03.072) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of CSC on steroid outputs during in vitro follicle growth. (A) Estradiol (ng/mL) at days 8 (n = 12) and 12 (n = 12) of culture, and (B) its relative [(day 12/day 8) × 100] increase between days 8 and 12. (C) Progesterone (ng/mL) at days 12 (n = 12) and 13 (n = 12) of culture, and (D) its relative [(day 13/day 12) × 100] increase between days 12 and 13. Bars with different letters differ significantly (P<.05). Fertility and Sterility 2011 95, 2410-2417DOI: (10.1016/j.fertnstert.2011.03.072) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of CSC on COC expansion in controls (Ctrl and DMSO) and CSC treatment groups (30, 60, 90, and 130 μg/mL). CC = cumuls cells; O = oocyte. Arrow indicates polar body oocyte; arrowhead indicates GV oocyte. Original magnification, ×200 (Ctrl and DMSO), ×400 (30, 60, 90, and 130 μg/mL). (A and B) Effect of CSC on oocyte growth and meiotic maturation. (A) Nuclear maturation and (B) oocyte growth throughout culture. Each experiment was repeated six times in duplicate plates per condition, n = 1,163 follicles (12–20 follicles per plates). Bars with different letters differ significantly (P<.05). Fertility and Sterility 2011 95, 2410-2417DOI: (10.1016/j.fertnstert.2011.03.072) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions