AMH as an ovarian function predictor in premenopausal female breast cancer patients receiving chemotherapy is effective only for those over 35 years old

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This study is designed to evaluate the value of serum AMH clinically and theoretically. Methods We detected the serum estradiol, FSH and AMH in 144 pre-menopausal women with breast cancer receiving cyclophosphamide-based chemotherapy. The differences of hormones before and after chemotherapy were compared; the correlations among the hormones and amenorrhea, menstrual recovery were analyzed. In addition, serum AMH was detected randomly in 177 normal healthy women and 36 normal female C57BL/6J mice of different ages, meanwhile the status of ovary follicles was observed. Furthermore, 72 Balb/c nude mice with breast cancer were randomly assigned to three groups with different dosage of CTX (control, 100mg/kg, 200mg/kg), the alterations of serum AMH and ovary follicles were recorded and analyzed. Results Chemotherapy induced amenorrhea was associated with pre-chemo AMH level, E2 level and FSH level (P <0.0001). Recovery of menstruation was associated with pre-chemo AMH level (P 0.05). In breast cancer patients received chemotherapy, the serum AMH 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). In healthy women, the AMH level sharply declined after 35 years old ( P <0.0001) and remained relatively stable in early age. Similar results were obtained in normal mice experiments. The cancer-bearing mice exposed to 200mg/kg CTX endured significant decline of AMH levels and remarkable decrease of primordial and growing follicles (P <0.0001). Conclusion Our results indicate that AMH is effective for predicting post-chemo ovarian function only in premenopausal female breast cancer patients over 35 years old. Oncology Cancer Biology Anti-Müllerian hormone Ovarian function Cyclophosphamide Breast cancer Figures Figure 1 Figure 2 Figure 3 1. Background Anti-Müllerian hormone (AMH), a dimeric glycoprotein member of the transforming growth factor-β superfamily, is composed of two identical subunits and is also called as Müllerian inhibiting substance (MIS) [ 1 – 3 ]. In females, AMH is produced predominantly by granulosa cell and its expression begins in the primary follicles and peaks in the secondary and small antral follicles, but is not detected in primordial follicles and atretic follicles [ 4 , 5 ]. It has an inhibitory role in recruiting and developing the primordial follicles into growing follicles [ 6 ]. Previous investigations have validated serum AMH levels as a quantitative marker for ovarian reserve and ovarian dysfunction in in vitro fertilization (IVF) and polycystic ovary syndrome (PCOS) [ 7 , 8 ]. But the role of AMH on predicting ovarian function recovery in post-chemotherapy patients is still in controversy. Chemotherapeutic regimen including gonadotoxic molecules are usually used to treat those younger patients with advanced malignancies [ 9 , 10 ], while cyclophosphamide (CTX) is one of the most used cell-cycle non-specific cytotoxic drugs with frequent ovarian function damage [ 11 ]. Ovarian toxicity due to CTX usually manifests as amenorrhea or even premature ovarian failure (POF) [ 12 ]. Many serum markers, such as follicular stimulating hormone (FSH), luteinizing hormone (LH), inhibin B and AMH, have been employed to evaluate the ovarian reserve and the ability to conceive [ 13 – 17 ]. For AMH is relative stable for not been influenced by the menstrual cycle,5 many studies have adopted AMH as a prominent biomarker to assess ovarian function during the post-chemotherapy follow-up period [ 18 , 19 ]. However, the results of those studies were contradictory, and there is no consensus on the predictive value of AMH for ovarian function under chemotherapeutic damage till now [ 18 – 21 ]. Considering the controversies mentioned above, here we conduct this study to clarify whether AMH is an effective biomarker for predicting the ovarian function in premenopausal female breast cancer patients receiving chemotherapy, and to determine in which patients the serum AMH is the most valuable predictor of ovarian function. 2. Methods Study population During Jan 2016 to Dec 2017, we randomly recruited 144 pre-menopause women with breast cancer (27–49 years old, median 43y) who need post-operative chemotherapy in our breast center. The regimens used were TEC (docetaxol 75 mg/m 2 + epirubicin 60 mg/m 2 + CTX 500 mg/m 2 , D1) or FEC (5-fluorouracil 500 mg/m 2 + epirubicin 75 mg/m 2 + CTX 500 mg/m 2 , D1), every 3 weeks for 6 cycles. Patients with previous chemotherapy for malignancy or other disease, or with comorbidities related to fertility, and those breast cancer patients treated without chemotherapy were excluded. The serum estradiol (E2), FSH and AMH levels were detected twice in the breast cancer patients, once before chemotherapy and once six months after the last injection of chemotherapy. Amenorrhea occurred within six months after chemotherapy was recorded, and subsequent menstruation recovery was recorded one year after chemotherapy. At the same time 177 normal healthy women (17–75 years old, median 44y, without history of gynecological disease or any cancer) were recruited and serum AMH of each person was detected randomly. Written informed consent was obtained from all participants and ethical approval was granted by the Shanghai Jiao Tong University affiliated Shanghai Sixth People’s Hospital Medical Ethics Committee. Animals The project was approved by the Animal Ethics and Welfare Committee of Shanghai Jiao Tong University affiliated Shanghai Sixth People’s Hospital. Thirty-six young female wild-type C57BL/6J mice (obtained from Shanghai SLAC Laboratory Animal Co., Shanghai, China), aged 1, 4, 6, 8, 12, 16 months (6 mice per age group) were anesthetized and euthanasia by terminal cardiac puncture. Blood was collected from the heart and the ovaries were removed for further tests. In addition, 72 young female inbred Balb/c nude mice (obtained from Shanghai SLAC Laboratory Animal Co., Shanghai, China), aged 6 weeks and weighed 15–20 g, were randomly divided into three groups. 1*10 7 MCF7 cells were injected subcutaneously into every mice of each group. We observed and recorded the tumor growth every two days. At 8 weeks old, mice in group 1 (n = 24), group 2 (n = 24) and group 3 (n = 24) were treated with a single intraperitoneal dose of 0.1 ml saline, 100 mg/kg of CTX and 200 mg/kg of CTX, respectively. The dosages of CTX were based on a previous study that demonstrated a significant dose-dependent ovarian toxicity but not sterilization [ 15 , 22 ]. Six stochastic mice of each group underwent heart blood collection at the time of euthanasia (anesthetization and euthanasia by terminal cardiac puncture) and ovaries excision before chemotherapy. Then 1, 3, 5 weeks after chemotherapy, six stochastic mice of each group underwent blood collection before euthanasia and ovaries excision each time (Fig. 1 ). Ovaries were fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E), while the serums were isolated from blood samples for hormonal measurements. Serum E2, FSH assay Serum E2 and FSH in all 144 breast cancer patients were test by ADVIA Centaur XP Immunoassay System (SIEMENS) respectively. Serum AMH assay Human and mouse serum AMH levels were measured using the Human AMH ELISA kit (Cusabio Biotech, CATALOG #:11351) and Mouse AMH ELISA kit (XLPM0168) respectively. Assays were performed according to the manufacturer’s protocol and concentrations of AMH was determined from the standard curve. All serum samples were assayed in duplicate. Follicles counting We performed the follicles counting as previously described in reference[ 23 ]. Briefly, the total number of follicles in each ovary was estimated by counting the numbers of follicles in every five sections of H&E-stained whole ovaries and applying a fivefold correction factor. Five µm-thick sections were serially cut and every 5th section analyzed for follicles counting. There are five stages in all follicles: primordial, primary, secondary, antral, and corpus luteum follicles. Only follicles that had an oocyte nucleus were scored. Primary follicles, secondary follicles and antral follicles were collectively referred to growing follicles. Statistical analysis The data were expressed as the mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism 7 and SAS 8.02 software and differences among the chemotherapy groups and the control were determined using one-way analysis of variance followed by multiple comparison test. Correlations between menstruation status and hormones were performed using logistic regression analysis. P < 0.05 was considered to indicate a statistically significant difference. 3. Results 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. 2 A) 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. 2 B). 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. 2 C). While the number of growing follicles is increasing before 6 months old (r = 0.91, P < 0.0001, Fig. 2 D), 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. 2 F. 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. 2 E). 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. 3 D). 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. 4. Discussion In both species the decline in fertility is closely related to the reduction of the primordial follicle pool. Moreover, the relationships between ages, numbers of oocytes and fertility in virgin and multiparous mice have been confirmed years before. Furthermore, cytotoxic drugs, especially CTX, will cause various follicles destruction, which was more significant in primordial follicles [ 20 , 22 ]. The injury caused by CTX will eventually lead to ovarian dysfunction, although the specific mechanism is not very clear yet. It was reported that CTX injures the follicles in a direct way by DNA alkylation and subsequent disruption of normal cellular processes and depletes the primordial follicles by destruction of granulosa cells [ 11 , 12 , 22 , 24 , 25 ]. In the absence of AMH, ovarian follicles depletion was accelerated for more primordial follicles recruited into growing follicles [ 20 , 26 ]. Although it was ever reported that CTX-induced acute ovarian follicular destruction couldn’t be predicted by serum AMH levels [ 20 ]. According to our study, the happening of amenorrhea six months after chemotherapy was correlated with pre-chemo levels of AMH and E2 negatively, with pre-chemo FSH level positively. While the recovery of menstruation one year after chemotherapy was correlated with pre- and post-chemo AMH level positively, but not correlated with pre-chemo levels of E2 and FSH. That means patients with higher AMH levels are less likely to have amenorrhea and more likely to return to normal even after chemotherapy, whereas patients with lower AMH levels are more likely to have amenorrhea and less likely to recover after amenorrhea [ 27 ]. Although pre-chemo E2 and FSH levels were correlated with amenorrhea, we did not find a correlation between them and menstrual recovery after chemotherapy, which was in line with expectations: the levels of E2 and FSH fluctuated greatly with the physiological cycle, while the levels of AMH were relatively stable [ 5 ]. Further stratified analysis revealed interesting findings: in our clinical study there were no significant changes of AMH level in young patients accepted chemotherapy, while AMH levels declined dramatically in patients over 35 years old with chemotherapy [ 28 , 29 ]. It implied that in younger patients the sufficient primordial follicles reserve and strong compensation ability resulted in a stable AMH levels. Therefore, the serum AMH as a biomarker for ovarian function predicting will be more accurate in women receiving chemotherapy older than 35 years old, but not in women younger than 35 years old. There is no consensus on whether AMH can be used as a predictor of ovarian function in women undergoing chemotherapy, especially in younger patients [ 28 – 30 ]. Although studies have shown that AMH is of little value in predicting ovarian function in underage chemotherapy patients, no reasonable explanation has been found [ 20 , 21 , 28 , 30 ]. In our study on healthy women, the AMH level has a persistent trend of decline with ageing in women over 35 years old but remains relatively stable in women younger than 35 years old, which means AMH may not be an accurate and suitable biomarker of ovarian reserve for younger women [ 31 , 32 ]. We got a similar result in mice study, the AMH level decreased with ageing in mice after an increasing during the first 6 months. Following study revealed that the number of growing follicles has a close correlation with primordial follicles, as well as the serum level of AMH. And in mice toxicity experiment, 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. In mice older than 8 months, both the serum AMH level and the number of growing follicles underwent a steady decline, which is in accordance with the opinion of Scheffer et al [ 32 ]. It was suggested that in immature female mice and women, the level of AMH increases due to the increasing of growing follicles. After then, the number of growing follicles remained constant during the early reproductive period in female, accompanied with the constant levels of AMH. With the increasing of age, serum AMH level will gradually decrease accompanied with the decrease of growing follicles in female. It’s well understood that AMH regulates the rate of primordial follicles recruited for further growth, and the recruitment of primordial follicles is inhibited with the presence of AMH in mature ovaries [ 4 , 5 , 26 ]. In the juvenile, because the follicles were mostly primordial follicles, AMH could not accurately evaluate the ovarian functional impairment. However, in young adults, even if the growing follicles were largely killed by chemotherapy drugs, the remaining growing follicles and the timely replenishment of the primordial follicular pool could maintain the stability of AMH, so the serum AMH level could not accurately reflect the damage degree of ovarian function. On the contrary, AMH can accurately reflect the injury of ovarian function in middle-aged and elderly female due to the limited ovarian reserve [ 31 , 33 ]. 5. Conclusions According to our research, AMH is an effective biomarker for predicting the ovarian function in premenopausal female breast cancer patients receiving chemotherapy, but only in those older than 35 years old. AMH as a predictor of ovarian function and an ovarian protectant is worthy of further study [ 23 , 34 ]. Therefore, AMH may not only be used as a predictive and evaluation indicator for ovarian function in premenopausal female with multiple tumors, but also would be a potential therapeutic target. List of Abbreviations AMH: Anti-Müllerian hormone; MIS: Müllerian inhibiting substance; IVF: in vitro fertilization; PCOS: Polycystic Ovary Syndrome; CTX: Cyclophosphamide; POF: Premature ovarian failure; FSH: Follicular stimulating hormone; LH: Luteinizing hormone; E2: Estradiol; TNBC: Triple Negative Breast Cancer. Declarations Ethics approval and consent to participate The study was approved by the Institutional Research Committee, Animal Ethics and Welfare Committee of Shanghai Jiao Tong University affiliated Shanghai Sixth People’s Hospital. All patients had given consent before enrollment. Consent for publication Not applicable. Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This project was funded by the Science and Technology Commission of Shanghai Municipality medical program 15411966500, and by Zhejiang Medical Society cancer research program 2015ZYC-A07. Authors’ contributions XZ, XL, and SL participated in the study design. XL, YY, SL and RH conducted experiments. XL, YY, LM, and CX did the statistical analysis. XL and HW drew the figures using software. XL, RH, and XZ wrote the manuscript. All authors reviewed the report and gave final approval to submit for publication. Acknowledgements We would like to thank all clinical units and participants involved in the study. References Cate RL, Mattaliano RJ, Hession C, Tizard R, Farber NM, Cheung A, et al. Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells. Cell. 1986;45(5):685-698. Massagué J. The transforming growth factor-beta family. Annual review of cell biology. 1990;6(597-641. Behringer RR, Finegold MJ, Cate RL. Müllerian-inhibiting substance function during mammalian sexual development. Cell. 1994;79(3):415-425. Durlinger ALL, Gruijters MJG, Piet K, Bas K, Ingraham HA, Nachtigal MW, et al. Anti-Müllerian Hormone Inhibits Initiation of Primordial Follicle Growth in the Mouse Ovary. 2002;3):3. E. KM, F. MM, Piet K, N. vdL-BBM, H. dJF, P. GN, et al. Serum Anti-Müllerian Hormone Levels Reflect the Size of the Primordial Follicle Pool in Mice. 2006; Behringer RR, Cate RL, Froelick GJ, Palmiter RD, Brinster RL. Abnormal sexual development in transgenic mice chronically expressing müllerian inhibiting substance. Nature. 1990;345(6271):167-170. Visser JA, Jong FHD, Laven JSE, Themmen APNJR. Anti-M??llerian hormone: A new marker for ovarian function. 2006;131(1):1-9. Dumont A, Robin G, Catteau-Jonard S, Dewailly D. Role of Anti-Müllerian Hormone in pathophysiology, diagnosis and treatment of Polycystic Ovary Syndrome: a review. Reproductive biology and endocrinology : RB&E. 2015;13(137. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians. 2018;68(6):394-424. Fredholm H, Eaker S, Frisell J, Holmberg L, Fredriksson I, Lindman H. Breast cancer in young women: poor survival despite intensive treatment. PloS one. 2009;4(11):e7695. Torino F, Barnabei A, De Vecchis L, Sini V, Schittulli F, Marchetti P, et al. Chemotherapy-induced ovarian toxicity in patients affected by endocrine-responsive early breast cancer. Critical reviews in oncology/hematology. 2014;89(1):27-42. Bedoschi G, Navarro PA, Oktay K. Chemotherapy-induced damage to ovary: mechanisms and clinical impact. Future oncology (London, England). 2016;12(20):2333-2344. Broer SL, Broekmans FJM, Laven JSE, Fauser BCJM. Anti-Müllerian hormone: ovarian reserve testing and its potential clinical implications. Human reproduction update. 2014;20(5):688-701. Shuster LT, Rhodes DJ, Gostout BS, Grossardt BR, Rocca WA. Premature menopause or early menopause: long-term health consequences. Maturitas. 2010;65(2):161-166. Broer SL, van Disseldorp J, Broeze KA, Dolleman M, Opmeer BC, Bossuyt P, et al. Added value of ovarian reserve testing on patient characteristics in the prediction of ovarian response and ongoing pregnancy: an individual patient data approach. Human reproduction update. 2013;19(1):26-36. Brodin T, Hadziosmanovic N, Berglund L, Olovsson M, Holte J. Antimüllerian hormone levels are strongly associated with live-birth rates after assisted reproduction. The Journal of clinical endocrinology and metabolism. 2013;98(3):1107-1114. Broer SL, Mol BWJ, Hendriks D, Broekmans FJM. The role of antimullerian hormone in prediction of outcome after IVF: comparison with the antral follicle count. Fertility and sterility. 2009;91(3):705-714. Dezellus A, Barriere P, Campone M, Lemanski C, Vanlemmens L, Mignot L, et al. Prospective evaluation of serum anti-Müllerian hormone dynamics in 250 women of reproductive age treated with chemotherapy for breast cancer. European journal of cancer (Oxford, England : 1990). 2017;79(72-80. Fréour T, Barrière P, Masson D. Anti-müllerian hormone levels and evolution in women of reproductive age with breast cancer treated with chemotherapy. European journal of cancer (Oxford, England : 1990). 2017;74(1-8. Browne HN, Moon KS, Mumford SL, Schisterman EF, Decherney AH, Segars JH, et al. Is anti-Müllerian hormone a marker of acute cyclophosphamide-induced ovarian follicular destruction in mice pretreated with cetrorelix? Fertility and sterility. 2011;96(1): Detti L, Uhlmann RA, Lu M, Diamond MP, Saed GM, Fletcher NM, et al. Serum markers of ovarian reserve and ovarian histology in adult mice treated with cyclophosphamide in pre-pubertal age. Journal of assisted reproduction and genetics. 2013;30(11):1421-1429. Meirow D, Lewis H, Nugent D, Epstein M. Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide: clinical importance and proposed accurate investigative tool. Human reproduction (Oxford, England). 1999;14(7):1903-1907. Kano M, Sosulski AE, Zhang L, Saatcioglu HD, Wang D, Nagykery N, et al. AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy. Proceedings of the National Academy of Sciences of the United States of America. 2017;114(9):E1688-E1697. Tilly JL, Kolesnick RN. Sphingolipids, apoptosis, cancer treatments and the ovary: investigating a crime against female fertility. Biochimica et biophysica acta. 2002;1585(2-3):135-138. Yüce MA, Balkanli Kaplan P, Gücer F, Doğanay L, Altaner S, Canda T, et al. Prevention of cyclophosphamide-induced ovarian damage by concomitant administration of GnRHa in mice: a dose-dependent relationship? European journal of gynaecological oncology. 2004;25(5):628-631. Durlinger AL, Kramer P, Karels B, de Jong FH, Uilenbroek JT, Grootegoed JA, et al. Control of primordial follicle recruitment by anti-Müllerian hormone in the mouse ovary. Endocrinology. 1999;140(12):5789-5796. Kim H-A, Choi J, Park CS, Seong M-K, Hong S-E, Kim J-S, et al. Post-chemotherapy serum anti-Müllerian hormone level predicts ovarian function recovery. Endocrine connections. 2018;7(8):949-956. Decanter C, Morschhauser F, Pigny P, Lefebvre C, Gallo C, Dewailly D. Anti-Müllerian hormone follow-up in young women treated by chemotherapy for lymphoma: preliminary results. Reproductive biomedicine online. 2010;20(2):280-285. van Beek RD, van den Heuvel-Eibrink MM, Laven JSE, de Jong FH, Themmen APN, Hakvoort-Cammel FG, et al. Anti-Mullerian hormone is a sensitive serum marker for gonadal function in women treated for Hodgkin's lymphoma during childhood. The Journal of clinical endocrinology and metabolism. 2007;92(10):3869-3874. Lutchman Singh K, Muttukrishna S, Stein RC, McGarrigle HH, Patel A, Parikh B, et al. Predictors of ovarian reserve in young women with breast cancer. British journal of cancer. 2007;96(12):1808-1816. Wong QHY, Anderson RA. The role of antimullerian hormone in assessing ovarian damage from chemotherapy, radiotherapy and surgery. Current opinion in endocrinology, diabetes, and obesity. 2018;25(6):391-398. Scheffer GJ, Broekmans FJM, Looman CWN, Blankenstein M, Fauser BCJM, teJong FH, et al. The number of antral follicles in normal women with proven fertility is the best reflection of reproductive age. Human reproduction (Oxford, England). 2003;18(4):700-706. Bozza C, Puglisi F, Lambertini M, Osa E-O, Manno M, Del Mastro L. Anti-Mullerian hormone: determination of ovarian reserve in early breast cancer patients. Endocrine-related cancer. 2014;21(1):R51-R65. Steiner AZ, Pritchard D, Stanczyk FZ, Kesner JS, Meadows JW, Herring AH, et al. Association Between Biomarkers of Ovarian Reserve and Infertility Among Older Women of Reproductive Age. JAMA. 2017;318(14):1367-1376. Supplementary Files NC3RsARRIVEGuidelinesChecklistfillable.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-16366","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":389071,"identity":"14c9f1aa-adfc-4530-aa93-4c84f3c958d4","order_by":1,"name":"Xiaolin Li","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaolin","middleName":"","lastName":"Li","suffix":""},{"id":389072,"identity":"7f8553df-4404-497b-94e3-5c68a5027a20","order_by":2,"name":"Sixuan Liu","email":"","orcid":"","institution":"Shanghai Jiao Tong University Affiliated sixth people Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sixuan","middleName":"","lastName":"Liu","suffix":""},{"id":389073,"identity":"98249fe3-f0df-4c30-8d36-e613d58fef9c","order_by":3,"name":"Lisi Ma","email":"","orcid":"","institution":"Shanghai Sixth Peoples Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lisi","middleName":"","lastName":"Ma","suffix":""},{"id":389074,"identity":"0d71dec1-1ef8-4477-b89a-f12fd57e219e","order_by":4,"name":"Xuan Chen","email":"","orcid":"","institution":"Shanghai Sixth Peoples Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Chen","suffix":""},{"id":389075,"identity":"a29da905-7c20-4dea-a907-6fdde2f586ec","order_by":5,"name":"Huaiyu Weng","email":"","orcid":"","institution":"Shanghai Sixth Peoples Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huaiyu","middleName":"","lastName":"Weng","suffix":""},{"id":389076,"identity":"6f75fbde-fdd5-4ff9-8d97-eeaa08f2dfa5","order_by":6,"name":"Run Huang","email":"","orcid":"","institution":"Shanghai Sixth Peoples Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Run","middleName":"","lastName":"Huang","suffix":""},{"id":389077,"identity":"dd215319-4cc3-44fe-89d2-6fcde42f4703","order_by":7,"name":"Yang Yu","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Yu","suffix":""},{"id":389078,"identity":"c9252f43-2c48-4623-8ad8-d46b145d8044","order_by":8,"name":"XIANGYUN ZONG","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYDACZhiDvYFIHTxwLTwHiNUCZ0kkEKnFnp394uOKPzZ58pGvkz8XMNjJ6RJyIA8zT7HhGZ60YsPbudukZzAkG5sRciBQS5pkg8ThxI2zc7cx8zAcSNxGhJb0nw0GQC0zz27+TKQW9mOMDQmHE+dL8G6QJk7LYR5myYYDaYkbeIB+4TEgwi/s/ccffmz4Y5M4vx3ksAo7OYJagPYYgCkDsEoDgsrB9jwAU/INRKkeBaNgFIyCkQgAv709n7ym4+gAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8673-9003","institution":"Shanghai Sixth Peoples Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"XIANGYUN","middleName":"","lastName":"ZONG","suffix":""}],"badges":[],"createdAt":"2020-03-03 14:42:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-16366/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-16366/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":599829,"identity":"423ffcab-566e-49e6-9ce4-3c6567436ee9","added_by":"auto","created_at":"2020-03-05 15:41:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":682907,"visible":true,"origin":"","legend":"Flowchart of cancer bearing mice experiments\n72 Balb/c nude mice, aged 6 weeks and weighed 15–20 g, were randomly divided into three groups. 1*107 MCF7 cells were injected subcutaneously into every mice of each group. At 8 weeks old, mice in group 1 (n=24), group 2 (n=24) and group 3 (n=24) were treated with a single intraperitoneal dose of 0.1 ml saline, 100 mg/kg of CTX and 200 mg/kg of CTX, respectively. Six stochastic mice of each group underwent heart blood collection at the time of euthanasia and ovaries excision before chemotherapy. Then 9, 11, 13 weeks, six stochastic mice of each group underwent blood collection before euthanasia and ovaries excision each time.","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-16366/v1/Figure_1[1].png"},{"id":599830,"identity":"f28f5413-a1b4-42d1-8b5e-f592a8456537","added_by":"auto","created_at":"2020-03-05 15:41:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":594022,"visible":true,"origin":"","legend":"The trends of AMH level in healthy women and female mice and the correlation between follicle numbers and AMH level in mice.\nA, The AMH levels obviously declined in women over 35 years old. B, Serum AMH levels remained stable in 4-8 months old and declined after 8 months old. C, The number of primordial follicles decreased with increasing age. D, The number of growing follicles was significantly declined in mice older than 8 months. E, The correlation between AMH levels and growing follicles was strongly significant (r= 0.887, P \u003c0.0001, Pearson test). F. H\u0026E stained ovaries from mice of different age. (B-D, each bar represents a minimum of 6 mice, ANOVA test. #, no statistical significance; **, P \u003c0.001; ***, P \u003c0.0001.)","description":"","filename":"Figure21.png","url":"https://assets-eu.researchsquare.com/files/rs-16366/v1/Figure2[1].png"},{"id":599831,"identity":"b2addc9b-f8c6-4fe7-8ba6-cd4d5f25dadd","added_by":"auto","created_at":"2020-03-05 15:41:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2301159,"visible":true,"origin":"","legend":"Serum AMH levels and ovarian follicles of Balb/c nude mice.\nA, The levels of serum AMH remained stable in controlled group and declined significantly in CTX 200 mg/kg group (P \u003c0.0001). B, The number of primordial follicles was not changed obviously in controlled group (P \u003e0.05) and 100 mg/kg CTX group at early time, while the decrease in CTX 200 mg/kg group was apparent (P \u003c0.0001). C. The number of growing follicles remained stable in controlled group and 100 mg/kg CTX group (P \u003e0.05). However, it was decreased significantly in 200 mg/kg CTX group at 5 weeks after chemotherapy (P \u003c0.0001). D, H\u0026E stains of representative sections from all mice in different treatment groups. (A-C, each bar represents a minimum of 6 mice, ANOVA test)","description":"","filename":"Figure31.png","url":"https://assets-eu.researchsquare.com/files/rs-16366/v1/Figure3[1].png"},{"id":13492119,"identity":"c602e6c0-59a1-4dab-a9f0-77ac1c26e2d7","added_by":"auto","created_at":"2021-09-16 22:30:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1771058,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-16366/v1/76a78693-6a25-45fa-b2a7-2459eef14758.pdf"},{"id":599828,"identity":"30403cb6-079e-40c6-ae83-8d3c0ba0e044","added_by":"auto","created_at":"2020-03-05 15:41:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1155132,"visible":true,"origin":"","legend":"","description":"","filename":"NC3RsARRIVEGuidelinesChecklistfillable.pdf","url":"https://assets-eu.researchsquare.com/files/rs-16366/v1/NC3Rs ARRIVE Guidelines Checklist (fillable).pdf"}],"financialInterests":"","formattedTitle":"AMH as an ovarian function predictor in premenopausal female breast cancer patients receiving chemotherapy is effective only for those over 35 years old","fulltext":[{"header":"1. Background","content":" \u003cp\u003eAnti-M\u0026uuml;llerian hormone (AMH), a dimeric glycoprotein member of the transforming growth factor-β superfamily, is composed of two identical subunits and is also called as M\u0026uuml;llerian inhibiting substance (MIS) [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In females, AMH is produced predominantly by granulosa cell and its expression begins in the primary follicles and peaks in the secondary and small antral follicles, but is not detected in primordial follicles and atretic follicles [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It has an inhibitory role in recruiting and developing the primordial follicles into growing follicles [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Previous investigations have validated serum AMH levels as a quantitative marker for ovarian reserve and ovarian dysfunction in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e fertilization (IVF) and polycystic ovary syndrome (PCOS) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. But the role of AMH on predicting ovarian function recovery in post-chemotherapy patients is still in controversy.\u003c/p\u003e \u003cp\u003eChemotherapeutic regimen including gonadotoxic molecules are usually used to treat those younger patients with advanced malignancies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while cyclophosphamide (CTX) is one of the most used cell-cycle non-specific cytotoxic drugs with frequent ovarian function damage [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Ovarian toxicity due to CTX usually manifests as amenorrhea or even premature ovarian failure (POF) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Many serum markers, such as follicular stimulating hormone (FSH), luteinizing hormone (LH), inhibin B and AMH, have been employed to evaluate the ovarian reserve and the ability to conceive [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For AMH is relative stable for not been influenced by the menstrual cycle,5 many studies have adopted AMH as a prominent biomarker to assess ovarian function during the post-chemotherapy follow-up period [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the results of those studies were contradictory, and there is no consensus on the predictive value of AMH for ovarian function under chemotherapeutic damage till now [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsidering the controversies mentioned above, here we conduct this study to clarify whether AMH is an effective biomarker for predicting the ovarian function in premenopausal female breast cancer patients receiving chemotherapy, and to determine in which patients the serum AMH is the most valuable predictor of ovarian function.\u003c/p\u003e "},{"header":"2. Methods","content":" \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eStudy population\u003c/span\u003e \u003c/p\u003e \u003cp\u003eDuring Jan 2016 to Dec 2017, we randomly recruited 144 pre-menopause women with breast cancer (27\u0026ndash;49\u0026nbsp;years old, median 43y) who need post-operative chemotherapy in our breast center. The regimens used were TEC (docetaxol 75\u0026nbsp;mg/m\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;epirubicin 60\u0026nbsp;mg/m\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;CTX 500\u0026nbsp;mg/m\u003csup\u003e2\u003c/sup\u003e, D1) or FEC (5-fluorouracil 500\u0026nbsp;mg/m\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;epirubicin 75\u0026nbsp;mg/m\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;CTX 500\u0026nbsp;mg/m\u003csup\u003e2\u003c/sup\u003e, D1), every 3 weeks for 6 cycles. Patients with previous chemotherapy for malignancy or other disease, or with comorbidities related to fertility, and those breast cancer patients treated without chemotherapy were excluded. The serum estradiol (E2), FSH and AMH levels were detected twice in the breast cancer patients, once before chemotherapy and once six months after the last injection of chemotherapy. Amenorrhea occurred within six months after chemotherapy was recorded, and subsequent menstruation recovery was recorded one year after chemotherapy.\u003c/p\u003e \u003cp\u003eAt the same time 177 normal healthy women (17\u0026ndash;75\u0026nbsp;years old, median 44y, without history of gynecological disease or any cancer) were recruited and serum AMH of each person was detected randomly. Written informed consent was obtained from all participants and ethical approval was granted by the Shanghai Jiao Tong University affiliated Shanghai Sixth People\u0026rsquo;s Hospital Medical Ethics Committee.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eAnimals\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe project was approved by the Animal Ethics and Welfare Committee of Shanghai Jiao Tong University affiliated Shanghai Sixth People\u0026rsquo;s Hospital. Thirty-six young female wild-type C57BL/6J mice (obtained from Shanghai SLAC Laboratory Animal Co., Shanghai, China), aged 1, 4, 6, 8, 12, 16 months (6 mice per age group) were\u003c/p\u003e \u003cp\u003eanesthetized and euthanasia by terminal cardiac puncture. Blood was collected from the heart and the ovaries were removed for further tests.\u003c/p\u003e \u003cp\u003eIn addition, 72 young female inbred Balb/c nude mice (obtained from Shanghai SLAC Laboratory Animal Co., Shanghai, China), aged 6 weeks and weighed 15\u0026ndash;20\u0026nbsp;g, were randomly divided into three groups. 1*10\u003csup\u003e7\u003c/sup\u003e MCF7 cells were injected subcutaneously into every mice of each group. We observed and recorded the tumor growth every two days. At 8 weeks old, mice in group 1 (n\u0026thinsp;=\u0026thinsp;24), group 2 (n\u0026thinsp;=\u0026thinsp;24) and group 3 (n\u0026thinsp;=\u0026thinsp;24) were treated with a single intraperitoneal dose of 0.1\u0026nbsp;ml saline, 100\u0026nbsp;mg/kg of CTX and 200\u0026nbsp;mg/kg of CTX, respectively. The dosages of CTX were based on a previous study that demonstrated a significant dose-dependent ovarian toxicity but not sterilization [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Six stochastic mice of each group underwent heart blood collection at the time of euthanasia (anesthetization and euthanasia by terminal cardiac puncture) and ovaries excision before chemotherapy. Then 1, 3, 5 weeks after chemotherapy, six stochastic mice of each group underwent blood collection before euthanasia and ovaries excision each time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Ovaries were fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (H\u0026amp;E), while the serums were isolated from blood samples for hormonal measurements.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eSerum E2, FSH assay\u003c/span\u003e \u003c/p\u003e \u003cp\u003eSerum E2 and FSH in all 144 breast cancer patients were test by ADVIA Centaur XP Immunoassay System (SIEMENS) respectively.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eSerum AMH assay\u003c/span\u003e \u003c/p\u003e \u003cp\u003eHuman and mouse serum AMH levels were measured using the Human AMH ELISA kit (Cusabio Biotech, CATALOG #:11351) and Mouse AMH ELISA kit (XLPM0168) respectively. Assays were performed according to the manufacturer\u0026rsquo;s protocol and concentrations of AMH was determined from the standard curve. All serum samples were assayed in duplicate.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eFollicles counting\u003c/span\u003e \u003c/p\u003e \u003cp\u003eWe performed the follicles counting as previously described in reference[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Briefly, the total number of follicles in each ovary was estimated by counting the numbers of follicles in every five sections of H\u0026amp;E-stained whole ovaries and applying a fivefold correction factor. Five \u0026micro;m-thick sections were serially cut and every 5th section analyzed for follicles counting. There are five stages in all follicles: primordial, primary, secondary, antral, and corpus luteum follicles. Only follicles that had an oocyte nucleus were scored. Primary follicles, secondary follicles and antral follicles were collectively referred to growing follicles.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eStatistical analysis\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. Statistical analysis was performed using GraphPad Prism 7 and SAS 8.02 software and differences among the chemotherapy groups and the control were determined using one-way analysis of variance followed by multiple comparison test. Correlations between menstruation status and hormones were performed using logistic regression analysis. \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e "},{"header":"3. Results","content":" \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003ePatient Characteristics\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe clinicopathological characteristics of the breast cancer patients were displayed on Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Most of the patients were diagnosed over 35\u0026nbsp;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\u0026rsquo; molecular subtype were HR+/HER2-, HR+/HER2+, HR-/HER2\u0026thinsp;+\u0026thinsp;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.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eClinicopathological characteristics of the breast cancer patients\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eCharacteristics\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003eNumber of Patients (N\u0026thinsp;=\u0026thinsp;144)\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eAge (years)\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;35\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026ge;\u0026thinsp;35\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e20(13.9%)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e124(86.1%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eT stage\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e57(39.6%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e64(44.4%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e19(13.2%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e4(2.8%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eAxillary lymph node\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNegative\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e94(65.3%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePositive\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e50(34.7%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eHistology\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eInvasive ductal carinoma\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e129(89.6%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eInvasive lobular carinoma\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e7(4.9%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOthers\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e8(5.5%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eGrade\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eⅠ\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003eⅡ\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003eⅢ\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003eundifferentiated\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e35(24.3%)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e54(37.5%)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e40(27.8%)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e15(10.4%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eMolecular subtype\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eHR+/HER2-\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003eHR+/HER2+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e67(46.5%)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e41(28.5%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eHR-/HER2+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e12(8.3%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eTNBC\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e24(16.7%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eOperation\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eTotal mastectomy\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e12(8.3%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eModified radical mastectomy\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e92(63.9%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eBreast conserving surgery\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e40(27.8%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e displayed the clinicopathological characteristics (age, axillary lymph node, histology, and so on) of the breast cancer patients in this research.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eThe menstruation status and productive hormone levels in breast cancer patients with chemotherapy\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe relationship between menstruation status and hormones was analyzed by Logistic regression. As shown on Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, 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.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eMenstruation status in breast cancer patients with chemotherapy\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eTotal\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;35y\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026ge;\u0026thinsp;35\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eAmenorrhea\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e112/144(77.8%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e8/20(40.0%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e104/124(83.9%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eMenstruation recovery\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e28/112(25.0%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e6/8(75.0%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e22/104(21.2%)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eThe relationship between menstruation and reproductive hormone (Logistic regression analysis)\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAmenorrhea\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003eMenstruation recovery\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eOR (95%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e value\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003eOR (95%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e value\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eAMH\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePre-chemo\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003ePost-chemo\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.31(0.157\u0026ndash;0.612)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e0.319(0.167\u0026ndash;0.611)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.224(0.079\u0026ndash;0.631)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e0.161(0.053\u0026ndash;0.484)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.005\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eOestradiol\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003ePre-chemo\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003ePost-chemo\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.983(0.873\u0026ndash;0.994)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e0.980(0.969\u0026ndash;0.991)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.002\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e0.001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.003(0.991\u0026ndash;1.015)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e0.985(0.973\u0026ndash;0.988)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.637\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e0.023\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eFSH\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003ePre-chemo\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003ePost-chemo\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.022(1.004\u0026ndash;1.041)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e1.002(0.963\u0026ndash;1.943)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.016\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e0.913\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.027(0.959\u0026ndash;1.101)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e1.041(1.016\u0026ndash;1.068)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.444\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e0.002\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMoreover, 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\u0026nbsp;years old (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while dramatic decline was detected in patients over 35\u0026nbsp;years old (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It suggests that the AMH level will be more effective on assessing the chemo-induced ovarian injury in premenopausal women older than 35\u0026nbsp;years old.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eReproductive hormone levels in breast cancer patients before and after chemotherapy (Wilcoxon Signed Ranks Test)\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAll\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;35\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026ge;\u0026thinsp;35\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eAMH\u003c/span\u003e (ng/ml)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.13 (0.01, 5.32)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e0.97 (0.01, 4.02)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePre-chemo\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003ePost-chemo\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.22 (0.01, 8.68)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e1.08 (0.01, 8.52)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e3.29 (0.81, 8.68)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e3.24 (0.06, 8.52)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eP-value\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.897\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eOestradiol\u003c/span\u003e (pg/ml)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003ePre-chemo\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003ePost-chemo\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e122.43 (18, 335)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e63.88 (5, 287)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e173.2 (18, 335)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e134.7 (15, 287)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e119.44 (27, 242)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e64.56 (5, 234)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eP-value\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.361\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eFSH\u003c/span\u003e (mIU/ml)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003ePre-chemo\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003ePost-chemo\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e10.51 (1.32, 83.22)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e51.22 (3.45, 121.06)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e8.30 (1.55, 38.82)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e30.82 (3.56, 119.83)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e10.54 (1.32, 83.22)\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e51.82 (3.45, 121.06)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eP-value\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.014\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e*The data was described by mean, minimum, maximum.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eThe trends of AMH level in healthy women and female mice and the correlation between follicle numbers and AMH level in mice\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e 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\u0026nbsp;years old (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and remained relatively stable in early age.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eSerum AMH level in normal woman (n\u0026thinsp;=\u0026thinsp;177)\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003eAMH level(ng/ml)\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAge group (yr)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eN\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eMean\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eMax\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eMin\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;26\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e3.61\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e8.04\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.56\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e26\u0026ndash;34\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e20\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e3.29\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e7.24\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.19\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e35\u0026ndash;39\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e26\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.58\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.32\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.31\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e40\u0026ndash;44\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e44\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.89\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.66\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.01\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e45\u0026ndash;49\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e45\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.36\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.18\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.01\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e50\u0026ndash;54\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e15\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.05\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.47\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.01\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e55+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e21\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.01\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.01\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.01\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e depicts the age distribution of all healthy women and the median AMH levels by age group.\u003c/p\u003e \u003cp\u003eSerum 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). 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\u0026thinsp;=\u0026thinsp;0.97, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). At the second phase mice of 4\u0026ndash;8 months of age had relatively stable AMH levels (r\u0026thinsp;=\u0026thinsp;0.97, 4 months \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003evs\u003c/span\u003e. 6 months \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.28, 4 months \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003evs\u003c/span\u003e. 8 months \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.98). A significant decline of the AMH level was observed in mice elder than 8 months (r\u0026thinsp;=\u0026thinsp;0.97, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), which represents mice being with an irregular cycle and getting into anestrus.\u003c/p\u003e \u003cp\u003eAnalysis of the follicles counting revealed that the number of primordial follicles declined with increasing age (r\u0026thinsp;=\u0026thinsp;0.99, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). While the number of growing follicles is increasing before 6 months old (r\u0026thinsp;=\u0026thinsp;0.91, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), remaining stable in 6\u0026ndash;8 months old (r\u0026thinsp;=\u0026thinsp;0.91, 6 months \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003evs\u003c/span\u003e. 8 months, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.21), and declined significantly after 8 months old (r\u0026thinsp;=\u0026thinsp;0.91, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Histological appearance was reveled in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF. It\u0026rsquo;s correlated strongly between the serum AMH levels and the number of growing follicles (r\u0026thinsp;=\u0026thinsp;0.887, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eThe correlation between AMH levels and growing follicles by age group\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAge in months\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e4\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e6\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e8\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e12\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cdiv class=\"SimplePara\"\u003e16\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAMH level (ng/ml)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e107.8\u0026thinsp;\u0026plusmn;\u0026thinsp;11.6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e186.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e195.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e184.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cdiv class=\"SimplePara\"\u003e129.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cdiv class=\"SimplePara\"\u003e71.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eGrowing follicles\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e207.8\u0026thinsp;\u0026plusmn;\u0026thinsp;16.3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e341.0\u0026thinsp;\u0026plusmn;\u0026thinsp;14.7\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e380.5\u0026thinsp;\u0026plusmn;\u0026thinsp;24.7\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e354.5\u0026thinsp;\u0026plusmn;\u0026thinsp;18.8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e280.5\u0026thinsp;\u0026plusmn;\u0026thinsp;23.5\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cdiv class=\"SimplePara\"\u003e217.5\u0026thinsp;\u0026plusmn;\u0026thinsp;26.6\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePearson\u0026rsquo;s correlation\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c9\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003er\u0026thinsp;=\u0026thinsp;0.887, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e showed the strongly correlation between the serum AMH levels and the number of growing follicles (r\u0026thinsp;=\u0026thinsp;0.887, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eThe effect of cyclophosphamide on AMH level and follicles in Balb/c nude mice\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThere was no significant difference of the tumors size among the three groups before chemotherapy (Control \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003evs\u003c/span\u003e. CTX100 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003evs\u003c/span\u003e. CTX200, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The growth of tumors in CTX 100\u0026nbsp;mg/kg group was similar to that in controlled group, while the tumor growth in CTX 200\u0026nbsp;mg/kg was slowered down more effectively (Control \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003evs\u003c/span\u003e. CTX200, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(A-C). The relation between AMH levels and follicle numbers in control and CTX-treated group was shown on Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. 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\u0026nbsp;mg/kg CTX group,the AMH levels were going down significantly over time (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Interestingly, in 100\u0026nbsp;mg/kg CTX group, the AMH levels were rising slightly at 9weeks, declining sharply at 11weeks, and then increasing at 13weeks eventually. What\u0026rsquo;s more, the primordial follicles consistently declined by time lapse in each group (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while the trend was dramatic in 200\u0026nbsp;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\u0026amp;E sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eThe comparison of AMH levels and follicle numbers in control and CTX-treated groups (ANOVA test)\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cdiv class=\"SimplePara\"\u003eComparison\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003eTime point\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e value\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eG1\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003evs\u003c/span\u003e. \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eG2\u003c/span\u003e\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eG1\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003evs\u003c/span\u003e. \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eG3\u003c/span\u003e\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eAMH\u003c/span\u003e\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003ePDF\u003c/span\u003e\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eGF\u003c/span\u003e\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eAMH\u003c/span\u003e\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003ePDF\u003c/span\u003e\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eGF\u003c/span\u003e\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePre-CTX\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.03\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e1 week after CTX\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.037\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e3 week after CTX\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e5 week after CTX\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.028\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003ens\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.0001\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: G1, Control group; G2, CTX100 group; G3, CTX200 group; PDF, primordial follicles; GF, growing follicles; ns, not significant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e "},{"header":"4. Discussion","content":" \u003cp\u003eIn both species the decline in fertility is closely related to the reduction of the primordial follicle pool. Moreover, the relationships between ages, numbers of oocytes and fertility in virgin and multiparous mice have been confirmed years before. Furthermore, cytotoxic drugs, especially CTX, will cause various follicles destruction, which was more significant in primordial follicles [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The injury caused by CTX will eventually lead to ovarian dysfunction, although the specific mechanism is not very clear yet. It was reported that CTX injures the follicles in a direct way by DNA alkylation and subsequent disruption of normal cellular processes and depletes the primordial follicles by destruction of granulosa cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the absence of AMH, ovarian follicles depletion was accelerated for more primordial follicles recruited into growing follicles [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough it was ever reported that CTX-induced acute ovarian follicular destruction couldn\u0026rsquo;t be predicted by serum AMH levels [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. According to our study, the happening of amenorrhea six months after chemotherapy was correlated with pre-chemo levels of AMH and E2 negatively, with pre-chemo FSH level positively. While the recovery of menstruation one year after chemotherapy was correlated with pre- and post-chemo AMH level positively, but not correlated with pre-chemo levels of E2 and FSH. That means patients with higher AMH levels are less likely to have amenorrhea and more likely to return to normal even after chemotherapy, whereas patients with lower AMH levels are more likely to have amenorrhea and less likely to recover after amenorrhea [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Although pre-chemo E2 and FSH levels were correlated with amenorrhea, we did not find a correlation between them and menstrual recovery after chemotherapy, which was in line with expectations: the levels of E2 and FSH fluctuated greatly with the physiological cycle, while the levels of AMH were relatively stable [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Further stratified analysis revealed interesting findings: in our clinical study there were no significant changes of AMH level in young patients accepted chemotherapy, while AMH levels declined dramatically in patients over 35\u0026nbsp;years old with chemotherapy [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It implied that in younger patients the sufficient primordial follicles reserve and strong compensation ability resulted in a stable AMH levels. Therefore, the serum AMH as a biomarker for ovarian function predicting will be more accurate in women receiving chemotherapy older than 35\u0026nbsp;years old, but not in women younger than 35\u0026nbsp;years old.\u003c/p\u003e \u003cp\u003eThere is no consensus on whether AMH can be used as a predictor of ovarian function in women undergoing chemotherapy, especially in younger patients [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although studies have shown that AMH is of little value in predicting ovarian function in underage chemotherapy patients, no reasonable explanation has been found [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In our study on healthy women, the AMH level has a persistent trend of decline with ageing in women over 35\u0026nbsp;years old but remains relatively stable in women younger than 35\u0026nbsp;years old, which means AMH may not be an accurate and suitable biomarker of ovarian reserve for younger women [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We got a similar result in mice study, the AMH level decreased with ageing in mice after an increasing during the first 6\u0026nbsp;months. Following study revealed that the number of growing follicles has a close correlation with primordial follicles, as well as the serum level of AMH. And in mice toxicity experiment, the primordial follicles consistently declined by time lapse in each group (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while the trend was dramatic in 200\u0026nbsp;mg/kg CTX group. The number of growing follicles was always in accordance with the changes of serum AMH levels. In mice older than 8 months, both the serum AMH level and the number of growing follicles underwent a steady decline, which is in accordance with the opinion of Scheffer et al [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. It was suggested that in immature female mice and women, the level of AMH increases due to the increasing of growing follicles. After then, the number of growing follicles remained constant during the early reproductive period in female, accompanied with the constant levels of AMH. With the increasing of age, serum AMH level will gradually decrease accompanied with the decrease of growing follicles in female.\u003c/p\u003e \u003cp\u003eIt\u0026rsquo;s well understood that AMH regulates the rate of primordial follicles recruited for further growth, and the recruitment of primordial follicles is inhibited with the presence of AMH in mature ovaries [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the juvenile, because the follicles were mostly primordial follicles, AMH could not accurately evaluate the ovarian functional impairment. However, in young adults, even if the growing follicles were largely killed by chemotherapy drugs, the remaining growing follicles and the timely replenishment of the primordial follicular pool could maintain the stability of AMH, so the serum AMH level could not accurately reflect the damage degree of ovarian function. On the contrary, AMH can accurately reflect the injury of ovarian function in middle-aged and elderly female due to the limited ovarian reserve [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e "},{"header":"5. Conclusions","content":" \u003cp\u003eAccording to our research, AMH is an effective biomarker for predicting the ovarian function in premenopausal female breast cancer patients receiving chemotherapy, but only in those older than 35\u0026nbsp;years old. AMH as a predictor of ovarian function and an ovarian protectant is worthy of further study [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, AMH may not only be used as a predictive and evaluation indicator for ovarian function in premenopausal female with multiple tumors, but also would be a potential therapeutic target.\u003c/p\u003e "},{"header":"List of Abbreviations","content":"\u003cp\u003eAMH: Anti-Müllerian hormone; MIS: Müllerian inhibiting substance; IVF: in vitro fertilization; PCOS: Polycystic Ovary Syndrome; CTX: Cyclophosphamide; POF: Premature ovarian failure; FSH: Follicular stimulating hormone; LH: Luteinizing hormone; E2: Estradiol; TNBC: Triple Negative Breast Cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Institutional Research Committee, Animal Ethics and Welfare Committee of Shanghai Jiao Tong University affiliated Shanghai Sixth People\u0026rsquo;s Hospital. All patients had given consent before enrollment.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis project was funded by the Science and Technology Commission of Shanghai Municipality medical program 15411966500, and by Zhejiang Medical Society cancer research program 2015ZYC-A07.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; contributions\u003c/p\u003e\n\u003cp\u003eXZ, XL, and SL participated in the study design. XL, YY, SL and RH conducted experiments. XL, YY, LM, and CX did the statistical analysis. XL and HW drew the figures using software. XL, RH, and XZ wrote the manuscript. All authors reviewed the report and gave final approval to submit for publication.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe would like to thank all clinical units and participants involved in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCate RL, Mattaliano RJ, Hession C, Tizard R, Farber NM, Cheung A, et al. Isolation of the bovine and human genes for M\u0026uuml;llerian inhibiting substance and expression of the human gene in animal cells. Cell. 1986;45(5):685-698.\u003c/li\u003e\n\u003cli\u003eMassagu\u0026eacute; J. The transforming growth factor-beta family. Annual review of cell biology. 1990;6(597-641.\u003c/li\u003e\n\u003cli\u003eBehringer RR, Finegold MJ, Cate RL. M\u0026uuml;llerian-inhibiting substance function during mammalian sexual development. Cell. 1994;79(3):415-425.\u003c/li\u003e\n\u003cli\u003eDurlinger ALL, Gruijters MJG, Piet K, Bas K, Ingraham HA, Nachtigal MW, et al. Anti-Müllerian Hormone Inhibits Initiation of Primordial Follicle Growth in the Mouse Ovary. 2002;3):3.\u003c/li\u003e\n\u003cli\u003eE. KM, F. MM, Piet K, N. vdL-BBM, H. dJF, P. GN, et al. Serum Anti-Müllerian Hormone Levels Reflect the Size of the Primordial Follicle Pool in Mice. 2006;\u003c/li\u003e\n\u003cli\u003eBehringer RR, Cate RL, Froelick GJ, Palmiter RD, Brinster RL. Abnormal sexual development in transgenic mice chronically expressing m\u0026uuml;llerian inhibiting substance. Nature. 1990;345(6271):167-170.\u003c/li\u003e\n\u003cli\u003eVisser JA, Jong FHD, Laven JSE, Themmen APNJR. Anti-M??llerian hormone: A new marker for ovarian function. 2006;131(1):1-9.\u003c/li\u003e\n\u003cli\u003eDumont A, Robin G, Catteau-Jonard S, Dewailly D. Role of Anti-M\u0026uuml;llerian Hormone in pathophysiology, diagnosis and treatment of Polycystic Ovary Syndrome: a review. Reproductive biology and endocrinology : RB\u0026amp;E. 2015;13(137.\u003c/li\u003e\n\u003cli\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians. 2018;68(6):394-424.\u003c/li\u003e\n\u003cli\u003eFredholm H, Eaker S, Frisell J, Holmberg L, Fredriksson I, Lindman H. Breast cancer in young women: poor survival despite intensive treatment. PloS one. 2009;4(11):e7695.\u003c/li\u003e\n\u003cli\u003eTorino F, Barnabei A, De Vecchis L, Sini V, Schittulli F, Marchetti P, et al. Chemotherapy-induced ovarian toxicity in patients affected by endocrine-responsive early breast cancer. Critical reviews in oncology/hematology. 2014;89(1):27-42.\u003c/li\u003e\n\u003cli\u003eBedoschi G, Navarro PA, Oktay K. Chemotherapy-induced damage to ovary: mechanisms and clinical impact. Future oncology (London, England). 2016;12(20):2333-2344.\u003c/li\u003e\n\u003cli\u003eBroer SL, Broekmans FJM, Laven JSE, Fauser BCJM. Anti-M\u0026uuml;llerian hormone: ovarian reserve testing and its potential clinical implications. Human reproduction update. 2014;20(5):688-701.\u003c/li\u003e\n\u003cli\u003eShuster LT, Rhodes DJ, Gostout BS, Grossardt BR, Rocca WA. Premature menopause or early menopause: long-term health consequences. Maturitas. 2010;65(2):161-166.\u003c/li\u003e\n\u003cli\u003eBroer SL, van Disseldorp J, Broeze KA, Dolleman M, Opmeer BC, Bossuyt P, et al. Added value of ovarian reserve testing on patient characteristics in the prediction of ovarian response and ongoing pregnancy: an individual patient data approach. Human reproduction update. 2013;19(1):26-36.\u003c/li\u003e\n\u003cli\u003eBrodin T, Hadziosmanovic N, Berglund L, Olovsson M, Holte J. Antim\u0026uuml;llerian hormone levels are strongly associated with live-birth rates after assisted reproduction. The Journal of clinical endocrinology and metabolism. 2013;98(3):1107-1114.\u003c/li\u003e\n\u003cli\u003eBroer SL, Mol BWJ, Hendriks D, Broekmans FJM. The role of antimullerian hormone in prediction of outcome after IVF: comparison with the antral follicle count. Fertility and sterility. 2009;91(3):705-714.\u003c/li\u003e\n\u003cli\u003eDezellus A, Barriere P, Campone M, Lemanski C, Vanlemmens L, Mignot L, et al. Prospective evaluation of serum anti-M\u0026uuml;llerian hormone dynamics in 250 women of reproductive age treated with chemotherapy for breast cancer. European journal of cancer (Oxford, England : 1990). 2017;79(72-80.\u003c/li\u003e\n\u003cli\u003eFr\u0026eacute;our T, Barri\u0026egrave;re P, Masson D. Anti-m\u0026uuml;llerian hormone levels and evolution in women of reproductive age with breast cancer treated with chemotherapy. European journal of cancer (Oxford, England : 1990). 2017;74(1-8.\u003c/li\u003e\n\u003cli\u003eBrowne HN, Moon KS, Mumford SL, Schisterman EF, Decherney AH, Segars JH, et al. Is anti-M\u0026uuml;llerian hormone a marker of acute cyclophosphamide-induced ovarian follicular destruction in mice pretreated with cetrorelix? Fertility and sterility. 2011;96(1):\u003c/li\u003e\n\u003cli\u003eDetti L, Uhlmann RA, Lu M, Diamond MP, Saed GM, Fletcher NM, et al. Serum markers of ovarian reserve and ovarian histology in adult mice treated with cyclophosphamide in pre-pubertal age. Journal of assisted reproduction and genetics. 2013;30(11):1421-1429.\u003c/li\u003e\n\u003cli\u003eMeirow D, Lewis H, Nugent D, Epstein M. Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide: clinical importance and proposed accurate investigative tool. Human reproduction (Oxford, England). 1999;14(7):1903-1907.\u003c/li\u003e\n\u003cli\u003eKano M, Sosulski AE, Zhang L, Saatcioglu HD, Wang D, Nagykery N, et al. AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy. Proceedings of the National Academy of Sciences of the United States of America. 2017;114(9):E1688-E1697.\u003c/li\u003e\n\u003cli\u003eTilly JL, Kolesnick RN. Sphingolipids, apoptosis, cancer treatments and the ovary: investigating a crime against female fertility. Biochimica et biophysica acta. 2002;1585(2-3):135-138.\u003c/li\u003e\n\u003cli\u003eY\u0026uuml;ce MA, Balkanli Kaplan P, G\u0026uuml;cer F, Doğanay L, Altaner S, Canda T, et al. Prevention of cyclophosphamide-induced ovarian damage by concomitant administration of GnRHa in mice: a dose-dependent relationship? European journal of gynaecological oncology. 2004;25(5):628-631.\u003c/li\u003e\n\u003cli\u003eDurlinger AL, Kramer P, Karels B, de Jong FH, Uilenbroek JT, Grootegoed JA, et al. Control of primordial follicle recruitment by anti-M\u0026uuml;llerian hormone in the mouse ovary. Endocrinology. 1999;140(12):5789-5796.\u003c/li\u003e\n\u003cli\u003eKim H-A, Choi J, Park CS, Seong M-K, Hong S-E, Kim J-S, et al. Post-chemotherapy serum anti-M\u0026uuml;llerian hormone level predicts ovarian function recovery. Endocrine connections. 2018;7(8):949-956.\u003c/li\u003e\n\u003cli\u003eDecanter C, Morschhauser F, Pigny P, Lefebvre C, Gallo C, Dewailly D. Anti-M\u0026uuml;llerian hormone follow-up in young women treated by chemotherapy for lymphoma: preliminary results. Reproductive biomedicine online. 2010;20(2):280-285.\u003c/li\u003e\n\u003cli\u003evan Beek RD, van den Heuvel-Eibrink MM, Laven JSE, de Jong FH, Themmen APN, Hakvoort-Cammel FG, et al. Anti-Mullerian hormone is a sensitive serum marker for gonadal function in women treated for Hodgkin's lymphoma during childhood. The Journal of clinical endocrinology and metabolism. 2007;92(10):3869-3874.\u003c/li\u003e\n\u003cli\u003eLutchman Singh K, Muttukrishna S, Stein RC, McGarrigle HH, Patel A, Parikh B, et al. Predictors of ovarian reserve in young women with breast cancer. British journal of cancer. 2007;96(12):1808-1816.\u003c/li\u003e\n\u003cli\u003eWong QHY, Anderson RA. The role of antimullerian hormone in assessing ovarian damage from chemotherapy, radiotherapy and surgery. Current opinion in endocrinology, diabetes, and obesity. 2018;25(6):391-398.\u003c/li\u003e\n\u003cli\u003eScheffer GJ, Broekmans FJM, Looman CWN, Blankenstein M, Fauser BCJM, teJong FH, et al. The number of antral follicles in normal women with proven fertility is the best reflection of reproductive age. Human reproduction (Oxford, England). 2003;18(4):700-706.\u003c/li\u003e\n\u003cli\u003eBozza C, Puglisi F, Lambertini M, Osa E-O, Manno M, Del Mastro L. Anti-Mullerian hormone: determination of ovarian reserve in early breast cancer patients. Endocrine-related cancer. 2014;21(1):R51-R65.\u003c/li\u003e\n\u003cli\u003eSteiner AZ, Pritchard D, Stanczyk FZ, Kesner JS, Meadows JW, Herring AH, et al. Association Between Biomarkers of Ovarian Reserve and Infertility Among Older Women of Reproductive Age. JAMA. 2017;318(14):1367-1376.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anti-Müllerian hormone, Ovarian function, Cyclophosphamide, Breast cancer","lastPublishedDoi":"10.21203/rs.3.rs-16366/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-16366/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground The predicting value of AMH for ovarian dysfunction after chemotherapy is controversial. This study is designed to evaluate the value of serum AMH clinically and theoretically. Methods We detected the serum estradiol, FSH and AMH in 144 pre-menopausal women with breast cancer receiving cyclophosphamide-based chemotherapy. The differences of hormones before and after chemotherapy were compared; the correlations among the hormones and amenorrhea, menstrual recovery were analyzed. In addition, serum AMH was detected randomly in 177 normal healthy women and 36 normal female C57BL/6J mice of different ages, meanwhile the status of ovary follicles was observed. Furthermore, 72 Balb/c nude mice with breast cancer were randomly assigned to three groups with different dosage of CTX (control, 100mg/kg, 200mg/kg), the alterations of serum AMH and ovary follicles were recorded and analyzed. Results Chemotherapy induced amenorrhea was associated with pre-chemo AMH level, E2 level and FSH level (P \u0026lt;0.0001). Recovery of menstruation was associated with pre-chemo AMH level (P \u0026lt;0.0001) but not E2 and FSH level (P \u0026gt;0.05). In breast cancer patients received\u0026nbsp;chemotherapy, the serum AMH did not differ significantly between pre- and post- chemotherapy in patients younger than 35 years old (P\u0026gt;0.05),\u0026nbsp;while dramatic decline was detected in patients over 35 years old (P\u0026lt;0.0001).\u0026nbsp;In healthy women, the AMH level sharply declined after 35 years old ( P \u0026lt;0.0001) and remained relatively stable in early age. Similar results were obtained in normal mice experiments. The cancer-bearing mice exposed to 200mg/kg CTX endured significant decline of AMH levels and remarkable decrease of primordial and growing follicles (P \u0026lt;0.0001).\u0026nbsp;Conclusion Our results indicate that AMH is effective for predicting post-chemo ovarian function only in premenopausal female breast cancer patients over 35 years old.\u003c/p\u003e","manuscriptTitle":"AMH as an ovarian function predictor in premenopausal female breast cancer patients receiving chemotherapy is effective only for those over 35 years old","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-03-05 15:41:09","doi":"10.21203/rs.3.rs-16366/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f90affa-563e-4698-ba20-95043658dbeb","owner":[],"postedDate":"March 5th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":66395,"name":"Oncology"},{"id":66396,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2020-05-01T15:41:00+00:00","versionOfRecord":[],"versionCreatedAt":"2020-03-05 15:41:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-16366","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-16366","identity":"rs-16366","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}