Patient Characteristics
The clinicopathological characteristics of the breast cancer patients were displayed on Table 1. Most of the patients were diagnosed over 35 years old (86.1%) and primarily diagnosed with a T1 (39.6%) or T2 (44.4%) tumor. Histological diagnoses comprised invasive duct carcinomas (89.6%), lobular carcinomas (4.9%), and others (5.5%); In this cohort, 67 (46.5%), 41 (28.5%), 12 (8.3%), and 24(16.7%) patients’ molecular subtype were HR+/HER2-, HR+/HER2+, HR-/HER2 + and TNBC, respectively. A total of 12(8.3%), 92(63.9%), and 40(27.8%) women received total mastectomy, modified radical mastectomy, breast conserving surgery, respectively.
Table 1
Clinicopathological characteristics of the breast cancer patients
Characteristics | Number of Patients (N = 144) |
Age (years) < 35 ≥ 35 | 20(13.9%) 124(86.1%) |
T stage | | |
1 | | 57(39.6%) |
2 | | 64(44.4%) |
3 | | 19(13.2%) |
4 | | 4(2.8%) |
Axillary lymph node | | |
Negative | | 94(65.3%) |
Positive | | 50(34.7%) |
Histology | | |
Invasive ductal carinoma | | 129(89.6%) |
Invasive lobular carinoma | | 7(4.9%) |
Others | | 8(5.5%) |
Grade | | |
Ⅰ Ⅱ Ⅲ undifferentiated | | 35(24.3%) 54(37.5%) 40(27.8%) 15(10.4%) |
Molecular subtype | | |
HR+/HER2- HR+/HER2+ | | 67(46.5%) 41(28.5%) |
HR-/HER2+ | | 12(8.3%) |
TNBC | | 24(16.7%) |
Operation | | |
Total mastectomy | | 12(8.3%) |
Modified radical mastectomy | | 92(63.9%) |
Breast conserving surgery | | 40(27.8%) |
Table 1 displayed the clinicopathological characteristics (age, axillary lymph node, histology, and so on) of the breast cancer patients in this research.
The menstruation status and productive hormone levels in breast cancer patients with chemotherapy
The relationship between menstruation status and hormones was analyzed by Logistic regression. As shown on Tables 2 & 3, Chemotherapy induced amenorrhea was significantly correlated with pre-chemo levels of AMH, E2, FSH and post-chemo levels of AMH, E2. Recovery of menstruation was significantly correlated with pre-chemo AMH level but not E2 and FSH levels. This indicates that amenorrhea may be predicted through the pre-chemo levels of AMH, E2 and FSH, but menstruation recovery can only be predicted through pre-chemo AMH level.
Table 2
Menstruation status in breast cancer patients with chemotherapy
| Total | < 35y | ≥ 35 |
Amenorrhea | 112/144(77.8%) | 8/20(40.0%) | 104/124(83.9%) |
Menstruation recovery | 28/112(25.0%) | 6/8(75.0%) | 22/104(21.2%) |
Table 3
The relationship between menstruation and reproductive hormone (Logistic regression analysis)
| Amenorrhea | Menstruation recovery |
| OR (95%) | P value | OR (95%) | P value |
AMH | | | | |
Pre-chemo Post-chemo | 0.31(0.157–0.612) 0.319(0.167–0.611) | < 0.001 < 0.001 | 0.224(0.079–0.631) 0.161(0.053–0.484) | < 0.005 < 0.001 |
Oestradiol Pre-chemo Post-chemo | 0.983(0.873–0.994) 0.980(0.969–0.991) | 0.002 0.001 | 1.003(0.991–1.015) 0.985(0.973–0.988) | 0.637 0.023 |
FSH Pre-chemo Post-chemo | 1.022(1.004–1.041) 1.002(0.963–1.943) | 0.016 0.913 | 1.027(0.959–1.101) 1.041(1.016–1.068) | 0.444 0.002 |
Moreover, in breast cancer patients treated with chemotherapy, the serum AMH and E2 did not differ significantly between pre- and post- chemotherapy in patients younger than 35 years old (P > 0.05), while dramatic decline was detected in patients over 35 years old (P < 0.0001) (Table 4). It suggests that the AMH level will be more effective on assessing the chemo-induced ovarian injury in premenopausal women older than 35 years old.
Table 4
Reproductive hormone levels in breast cancer patients before and after chemotherapy (Wilcoxon Signed Ranks Test)
| All | < 35 | ≥ 35 |
AMH (ng/ml) | | | 1.13 (0.01, 5.32) 0.97 (0.01, 4.02) |
Pre-chemo Post-chemo | 1.22 (0.01, 8.68) 1.08 (0.01, 8.52) | 3.29 (0.81, 8.68) 3.24 (0.06, 8.52) |
P-value | < 0.001 | 0.897 | < 0.0001 |
Oestradiol (pg/ml) Pre-chemo Post-chemo | 122.43 (18, 335) 63.88 (5, 287) | 173.2 (18, 335) 134.7 (15, 287) | 119.44 (27, 242) 64.56 (5, 234) |
P-value | < 0.0001 | 0.361 | < 0.0001 |
FSH (mIU/ml) Pre-chemo Post-chemo | 10.51 (1.32, 83.22) 51.22 (3.45, 121.06) | 8.30 (1.55, 38.82) 30.82 (3.56, 119.83) | 10.54 (1.32, 83.22) 51.82 (3.45, 121.06) |
P-value | < 0.0001 | 0.014 | < 0.0001 |
*The data was described by mean, minimum, maximum. |
The trends of AMH level in healthy women and female mice and the correlation between follicle numbers and AMH level in mice.
Table 5 depicts the age distribution of all healthy women and the median AMH levels by age group. The AMH level sharply declined in women over 35 years old (P < 0.0001, Fig. 2A) and remained relatively stable in early age.
Table 5
Serum AMH level in normal woman (n = 177)
| AMH level(ng/ml) |
Age group (yr) | N | Mean | Max | Min |
< 26 | 6 | 3.61 | 8.04 | 1.56 |
26–34 | 20 | 3.29 | 7.24 | 1.19 |
35–39 | 26 | 1.58 | 5.32 | 0.31 |
40–44 | 44 | 0.89 | 1.66 | 0.01 |
45–49 | 45 | 0.36 | 1.18 | 0.01 |
50–54 | 15 | 0.05 | 0.47 | 0.01 |
55+ | 21 | 0.01 | 0.01 | 0.01 |
Table 5 depicts the age distribution of all healthy women and the median AMH levels by age group.
Serum AMH levels were determined in C57BL/6J wild-type female mice of various ages. It was increasing at the early time and declined significantly with increasing age (Fig. 2B). We found three phases in the changes of AMH level. At the first phase the serum AMH levels were increasing obviously before 4 months of age (r = 0.97, P < 0.0001). At the second phase mice of 4–8 months of age had relatively stable AMH levels (r = 0.97, 4 months vs. 6 months P = 0.28, 4 months vs. 8 months P = 0.98). A significant decline of the AMH level was observed in mice elder than 8 months (r = 0.97, P < 0.0001), which represents mice being with an irregular cycle and getting into anestrus.
Analysis of the follicles counting revealed that the number of primordial follicles declined with increasing age (r = 0.99, P < 0.0001, Fig. 2C). While the number of growing follicles is increasing before 6 months old (r = 0.91, P < 0.0001, Fig. 2D), remaining stable in 6–8 months old (r = 0.91, 6 months vs. 8 months, P = 0.21), and declined significantly after 8 months old (r = 0.91, P < 0.0001). Histological appearance was reveled in the Fig. 2F. It’s correlated strongly between the serum AMH levels and the number of growing follicles (r = 0.887, P < 0.0001, Table 6 and Fig. 2E).
Table 6
The correlation between AMH levels and growing follicles by age group
Age in months | 1 | 4 | 6 | 8 | 12 | 16 |
AMH level (ng/ml) | 107.8 ± 11.6 | 186.7 ± 7.7 | 195.4 ± 7.1 | 184.4 ± 10.8 | 129.9 ± 6.1 | 71.2 ± 5.4 |
Growing follicles | 207.8 ± 16.3 | 341.0 ± 14.7 | 380.5 ± 24.7 | 354.5 ± 18.8 | 280.5 ± 23.5 | 217.5 ± 26.6 |
Pearson’s correlation | r = 0.887, P < 0.0001 |
Table 6 showed the strongly correlation between the serum AMH levels and the number of growing follicles (r = 0.887, P < 0.0001).
The effect of cyclophosphamide on AMH level and follicles in Balb/c nude mice.
There was no significant difference of the tumors size among the three groups before chemotherapy (Control vs. CTX100 vs. CTX200, P > 0.05). The growth of tumors in CTX 100 mg/kg group was similar to that in controlled group, while the tumor growth in CTX 200 mg/kg was slowered down more effectively (Control vs. CTX200, P < 0.0001). The trends of serum AMH, primordial follicles and growing follicles counting at 8 weeks old (pre-chemotherapy), 9weeks old (1week after CTX or saline), 11weeks old (3weeks after CTX or saline), 13weeks old (5weeks after CTX or saline) in Balb/c nude mice with breast cancer were shown in Fig. 3(A-C). The relation between AMH levels and follicle numbers in control and CTX-treated group was shown on Table 7. No significant difference of serum AMH levels was observed in control group among mice aged from 8 to 13 weeks, as well as among mice at 8 weeks old (pre-chemo) in each group. In 200 mg/kg CTX group,the AMH levels were going down significantly over time (P < 0.0001). Interestingly, in 100 mg/kg CTX group, the AMH levels were rising slightly at 9weeks, declining sharply at 11weeks, and then increasing at 13weeks eventually. What’s more, the primordial follicles consistently declined by time lapse in each group (P < 0.0001), while the trend was dramatic in 200 mg/kg CTX group. The number of growing follicles was always in accordance with the changes of serum AMH levels. Changes of the number of growing follicles were revealed by H&E sections (Fig. 3D).
Table 7
The comparison of AMH levels and follicle numbers in control and CTX-treated groups (ANOVA test)
Comparison Time point | P value |
G1 vs. G2 | G1 vs. G3 |
AMH | PDF | GF | AMH | PDF | GF |
Pre-CTX | ns | 0.03 | ns | ns | ns | ns |
1 week after CTX | 0.037 | ns | < 0.001 | < 0.0001 | 0.001 | ns |
3 week after CTX | < 0.001 | ns | ns | < 0.0001 | < 0.0001 | < 0.0001 |
5 week after CTX | ns | 0.028 | ns | < 0.0001 | < 0.0001 | < 0.0001 |
Note: G1, Control group; G2, CTX100 group; G3, CTX200 group; PDF, primordial follicles; GF, growing follicles; ns, not significant. |