Is anti-Müllerian hormone a marker of acute cyclophosphamide-induced ovarian follicular destruction in mice pretreated with cetrorelix?  Hyacinth N. Browne, M.D., Kimberly S. Moon, M.D., Sunni L. Mumford, Ph.D., Enrique F. Schisterman, Ph.D., Alan H. DeCherney, M.D., James H. Segars, M.D., Alicia Y. Armstrong, M.D., M.S.  Fertility and Sterility  Volume 96, Issue 1, Pages 180-186.e2 (July 2011) DOI: 10.1016/j.fertnstert.2011.04.008 Copyright © 2011 Terms and Conditions

Figure 1 Effects of cetrorelix on primordial follicle counts in mice treated with cyclophosphamide (CTX). (A–F) Hematoxylin and eosin (H & E) stains of representative sections from 56-day-old mice from the six treatment groups. Mice exposed to saline (Con), antagonist (Ant), CTX 75 mg/kg (CYC 75mg/kg), CTX 75 mg/kg with antagonist (CYC 75mg/kg + Ant), CTX 100 mg/kg (CYC 100mg/kg), and CTX 100 mg/kg with antagonist (CYC 100mg/kg + Ant). Bar = 100 μm; magnification, ×5. (G) Primordial follicle counts (mean ± SEM) per 1,000 micron2 ovarian area (y axis) were higher in the antagonist treated groups (x axis) (CYC 75mg/kg vs. CYC 75mg/kg + Ant: 0.84 ± 0.30 vs. 1.78 ± 0.33, P<.0001; CYC 100mg/kg vs. CYC 100mg/kg + Ant: 0.53 ± 0.33 vs. 1.60 ± 0.33, P<.0001). Each bar represents five mice. Error bars = ± SEM. Fertility and Sterility 2011 96, 180-186.e2DOI: (10.1016/j.fertnstert.2011.04.008) Copyright © 2011 Terms and Conditions

Figure 2 Cetrorelix reduced apoptosis in the granulosa cells (GC) of mice treated with cyclophosphamide (CTX). (A–F) Immunofluorescent detection of apoptotic cells (stained yellow) by TUNEL in ovaries exposed to saline (Con), antagonist alone (Ant), CTX (CYC) or pretreated with antagonist (CYC + Ant). (G) The positive control (Positive Con) was incubated with DNase 1, and (H) the negative control (Negative Con) was incubated with fluorescein-labeled dUTP. Bar = 100 μm; magnification, ×20. (I) Quantitative comparison of mean TUNEL scores of the ovaries receiving CTX (100 mg/kg) with and without antagonist. Percentage of apoptotic cells (mean ± SEM) in the CTX-only (Cyc100) treated group versus the CTX and antagonist group (Cyc100 + Ant), respectively (19% ± 6 vs. 6% ± 6, P=.0033, t-test). Each bar represents three mice. Fertility and Sterility 2011 96, 180-186.e2DOI: (10.1016/j.fertnstert.2011.04.008) Copyright © 2011 Terms and Conditions

Figure 3 Analysis of anti-Müllerian hormone (AMH) expression in mouse ovarian tissue and serum. (A) Quantitation of AMH H-score staining (y axis) between the six treatment groups (x axis). There was no difference in the mean H-scores (mean ± SEM) between the cyclophosphamide (CTX; Cyc) versus CTX + antagonist (Cyc + Ant) treatment groups (75 mg/kg: 3.1 ± 0.3 vs. 3.1 ± 0.3, P=.6681 and 100 mg/kg: 3.2 ± 0.3 vs. 2.9 ± 0.3, P=.1128, t-test). Each bar represents a minimum of six mice. (B) Serum AMH levels on day 16 among six treatment groups. The AMH levels (y axis, mean ± SEM) did not differ between the study groups (x axis). Control (Con) versus antagonist (Ant): 5.9 ± 2.4 versus 10.5 ± 2.4, P=.1584; 75 mg/kg CTX (Cyc75) versus 75 mg/kg CTX + antagonist (Cyc75 + Ant): 8.5 ± 2.4 versus 6.3 ± 2.4, P=1.0; 100 mg/kg CTX (Cyc100) versus 100 mg/kg CTX + antagonist (Cyc100 + Ant): 8.8 ± 2.4 versus 7.2 ± 2.4, P=1.0, t-test. Each bar represents a minimum of five mice. Fertility and Sterility 2011 96, 180-186.e2DOI: (10.1016/j.fertnstert.2011.04.008) Copyright © 2011 Terms and Conditions

Schematic of study design Schematic of study design. Groups 2, 4, and 6 were treated with GnRH antagonist (●) from days 1–15. On day 9, groups 1 (n = 10) and 2 (n = 10) were treated with a single intraperitoneal dose of 0.1 mL of saline (S), whereas groups 3 (n = 10) and 4 (n = 10) and groups 5 (n = 10) and 6 (n = 10) received a single intraperitoneal dose of 75 mg/kg or 100 mg/kg of cyclophosphamide (CTX; CYC), respectively. All mice were sacrificed on day 16 (Sac). Fertility and Sterility 2011 96, 180-186.e2DOI: (10.1016/j.fertnstert.2011.04.008) Copyright © 2011 Terms and Conditions

Analysis of anti-Müllerian hormone (AMH) expression in mouse ovarian tissue. (A–F) Ovarian sections were stained for AMH in mice treated with saline (No Ant), antagonist alone (Ant), cyclophosphamide (CTX; CYC), or pretreated with the antagonist and CTX (CYC + Ant). Brown staining represents AMH expression in granulosa cells (GC). (G) Negative control (Negative Con) did not receive primary antibody. Bar = 100 μm; magnification, ×5. Fertility and Sterility 2011 96, 180-186.e2DOI: (10.1016/j.fertnstert.2011.04.008) Copyright © 2011 Terms and Conditions