Is anti-Müllerian hormone a marker of acute cyclophosphamide-induced ovarian follicular destruction in mice pretreated with cetrorelix?

Abstract

Objective: To define whether anti-Müllerian hormone (AMH) may be a marker of acute cyclophosphamide (CTX)-induced germ cell destruction in mice pretreated with the GnRH antagonist, cetrorelix.

Design: Controlled, experimental study.

Setting: Research laboratory in a federal research facility.

Animal(s): Balb/c female mice (6 weeks old).

Intervention(s): Mice were treated with GnRH antagonist (cetrorelix) or saline for 15 days followed by 75 mg/kg or 100 mg/kg of CTX or saline control on day 9.

Main outcome measure(s): Number of primordial follicles (PMF), DNA damage, AMH protein expression, and AMH serum levels.

Result(s): Ovaries in mice pretreated with cetrorelix had significantly more PMFs and reduced DNA damage compared with those exposed to CTX alone. Immunohistochemical staining for AMH expression and serum AMH levels did not differ significantly between treatment groups.

Conclusion(s): Cetrorelix protected PMFs and reduced DNA damage in follicles of mice treated with CTX, but AMH levels in tissue and serum did not correlate with germ cell destruction. Further research is needed to determine the mechanism responsible for the protective effects on PMF counts observed with cetrorelix.

Published by Elsevier Inc.

Figures

Figure 1

Effects of cetrorelix on primordial follicle counts in mice treated with cyclophosphamide. (A–F) H&E stains of representative sections from 56-day-old mice from the 6 treatment groups. Mice exposed to saline (Con), antagonist (Ant), cyclophosphamide 75 mg/kg (Cyc 75 mg/kg), cyclophosphamide 75 mg/kg with antagonist (Cyc 75 mg/kg + Ant), cyclophosphamide 100 mg/kg (Cyc 100 mg/kg), and cyclophosphamide 100 mg/kg with antagonist (Cyc 100 mg/kg + Ant). Scale bar = 100µm, magnification= 5×. (G) Primordial follicle counts (mean ± SEM) per 1000 micron2 ovarian area (y axis) were higher in the antagonist treated groups (x axis) (Cyc 75 vs Cyc 75 + Ant: 0.84 ± 0.30 vs 1.78 ± 0.33, p<0.0001; Cyc 100 vs Cyc 100 + Ant: 0.53 ± 0.33 vs 1.60 ± 0.33, p<0.0001). Each bar represents 5 mice. Error bars = ± SEM.

Figure 1

Effects of cetrorelix on primordial follicle counts in mice treated with cyclophosphamide. (A–F) H&E stains of representative sections from 56-day-old mice from the 6 treatment groups. Mice exposed to saline (Con), antagonist (Ant), cyclophosphamide 75 mg/kg (Cyc 75 mg/kg), cyclophosphamide 75 mg/kg with antagonist (Cyc 75 mg/kg + Ant), cyclophosphamide 100 mg/kg (Cyc 100 mg/kg), and cyclophosphamide 100 mg/kg with antagonist (Cyc 100 mg/kg + Ant). Scale bar = 100µm, magnification= 5×. (G) Primordial follicle counts (mean ± SEM) per 1000 micron2 ovarian area (y axis) were higher in the antagonist treated groups (x axis) (Cyc 75 vs Cyc 75 + Ant: 0.84 ± 0.30 vs 1.78 ± 0.33, p<0.0001; Cyc 100 vs Cyc 100 + Ant: 0.53 ± 0.33 vs 1.60 ± 0.33, p<0.0001). Each bar represents 5 mice. Error bars = ± SEM.

Figure 2

Cetrorelix reduced apoptosis in the granulosa cells of mice treated with cyclophosphamide. (A–F) Immunofluorescent detection of apoptotic cells (stained yellow) by TUNEL in ovaries exposed to saline (Con), antagonist alone (Ant), cyclophosphamide (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. Scale bar = 100µm, magnification= 20×. (I) Quantitative comparison of mean TUNEL scores of the ovaries receiving cyclophosphamide (100mg/kg) with and without antagonist. Percentage of apoptotic cells (mean ± SEM) in the cyclophosphamide-only (Cyc100) treated group vs the cyclophosphamide and antagonist group (Cyc100 + Ant), respectively (19% ± 6 vs 6% ± 6, p=0.0033, t-test). Each bar represents 3 mice.

Figure 2

Cetrorelix reduced apoptosis in the granulosa cells of mice treated with cyclophosphamide. (A–F) Immunofluorescent detection of apoptotic cells (stained yellow) by TUNEL in ovaries exposed to saline (Con), antagonist alone (Ant), cyclophosphamide (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. Scale bar = 100µm, magnification= 20×. (I) Quantitative comparison of mean TUNEL scores of the ovaries receiving cyclophosphamide (100mg/kg) with and without antagonist. Percentage of apoptotic cells (mean ± SEM) in the cyclophosphamide-only (Cyc100) treated group vs the cyclophosphamide and antagonist group (Cyc100 + Ant), respectively (19% ± 6 vs 6% ± 6, p=0.0033, t-test). Each bar represents 3 mice.

Figure 2

Cetrorelix reduced apoptosis in the granulosa cells of mice treated with cyclophosphamide. (A–F) Immunofluorescent detection of apoptotic cells (stained yellow) by TUNEL in ovaries exposed to saline (Con), antagonist alone (Ant), cyclophosphamide (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. Scale bar = 100µm, magnification= 20×. (I) Quantitative comparison of mean TUNEL scores of the ovaries receiving cyclophosphamide (100mg/kg) with and without antagonist. Percentage of apoptotic cells (mean ± SEM) in the cyclophosphamide-only (Cyc100) treated group vs the cyclophosphamide and antagonist group (Cyc100 + Ant), respectively (19% ± 6 vs 6% ± 6, p=0.0033, t-test). Each bar represents 3 mice.

Figure 3

Analysis of AMH expression in mouse ovarian tissue and serum. (A) Quantitation of AMH H score staining (y axis) between the 6 treatment groups (x axis). There was no difference in the mean H-scores (mean ± SEM) between the cyclophosphamide (Cyc) vs cyclophosphamide + antagonist (Cyc + Ant) treatment groups (75mg/kg: 3.1 ± 0.3 vs 3.1 ± 0.3, p= 0.6681 and 100 mg/kg: 3.2 ± 0.3 vs 2.9 ± 0.3, p=0.1128, t-test). Each bar represents a minimum of 6 mice. (B) Serum AMH levels on day 16 among 6 treatment groups. AMH levels (y axis, mean ± SEM) did not differ between the study groups (x axis). Control (Con) vs antagonist (Ant): 5.9 ± 2.4 vs 10.5 ± 2.4, p=0.1584; 75 mg/kg cyclophosphamide (Cyc75) vs 75mg/kg cyclophosphamide + antagonist (Cyc75 + Ant): 8.5 ± 2.4 vs 6.3 ± 2.4, p=1.0; 100 mg/kg cyclophosphamide (Cyc100) vs 100 mg/kg cyclophosphamide + antagonist (Cyc100 + Ant): 8.8 ± 2.4 vs 7.2 ± 2.4, p=1.0, t-test. Each bar represents a minimum of 5 mice.