Abnormal anti-Müllerian hormone level may be a trigger for breast cancer in young women: A case-control study

Abstract Background Anti-Müllerian hormone (AMH) is a known sensitive biomarker for fertility and ovarian reserve. The results of in vivo and human studies showed inconsistency with respect to the relation between AMH and breast cancer. Objective To compare the AMH level of young Iranian women with early breast cancer who have not received any treatment compared to that of healthy women. Materials and Methods In this case-control study, 58 breast cancer cases were recruited from the breast oncology clinic of two university hospitals. They were diagnosed with an in situ or invasive breast cancer before any anticancer treatment between August 2018 and April 2019. Healthy controls (n = 58) were selected from women referred to a gynecologic outpatient clinic without any symptoms of cancer or infertility. AMH was measured by the AMH enzyme-linked immunosorbent assay kits in one laboratory. Results Final analysis showed that the AMH means of case and control were not statistically significant (3.36 ± 2.95 vs 3.13 ± 1.79). However, the lower and higher AMH level categories are more prevalent in breast cancer compared to the control. Pearson's correlation test showed that the AMH level was negatively correlated with age (r = -0.44, p< 0.001). The results of logistic regression analysis considering confounding factors showed the positive association between breast cancer and lower (Odds Ratio [OR] = 5.98, p = 0.02) and higher quartile of AMH level (OR = 4.95, p = 0.01). Conclusion Our results suggest that abnormal AMH level is more frequent in young breast cancer patients. Further investigation considering AMH determinants is required.


Introduction
Breast cancer is the most commonly diagnosed cancer in most regions of the world (1).
Approximately 9.5% of new cases of breast cancer and 6.7% of breast cancer occur among women aged < 45 yr (2). A recent study has shown that the incidence of breast cancer in young women is increasing (3); the mean age of the women with breast cancer in Iran is < 50 yr (4).
Scientists are looking for different biomarkers to prevent or treat cancers including breast cancer.

One of the researchers' favorite biomarkers is
Müllerian-inhibiting substance, also known as anti-Müllerian hormone (AMH). Besides the use of AMH as a sensitive biomarker for fertility and ovarian reserve, it serves as a biomarker in several other conditions (5). For instance, AMH concentration is used as a reliable marker if at any time there are minor fluctuations in serum concentrations during a normal menstrual cycle (6).
AMH is a member of the transforming growth factor-β superfamily of the growth and differentiation response modifiers. Because most gynecologic tumors originate from Müllerian duct-derived tissues, and since Müllerianinhibiting substance/AMH causes regression of the Müllerian duct in male embryos, it is expected to have an inhibiting effect on the growth of gynecologic tumors (7).
Based on laboratory evidence in cell-line and mouse models, an inverse association between AMH level and breast tumor development has been illustrated (8)(9)(10)(11), and it seems that AMH has a protective effect against breast cancer and can suppress the growth of mammary tumors.
These findings have led to the suggestion that AMH could be used in the treatment of breast and other gynecologic cancers (7). Although the exact role of AMH in mammary glands is still unclear, they are possible target tissue of AMH.
However, different results have been reported in epidemiological human studies. Some of them assert that a lower AMH is associated with a higher rate of breast cancers (12,13), while others have shown a positive relationship between AMH level and breast cancer (14)(15)(16)(17). Since AMH plays a role in regulating folliculogenesis and the interaction between AMH and estradiol is not fully understood (18)

AMH measurement
Hospital nurses collected 5 ml of blood samples from the cubital vein. All samples were stored at room temperature and transferred to the same laboratory within 2 hr. The laboratory doctor and personnel were blinded about case and control groupings. AMH was measured by the AMH enzymelinked immunosorbent assay kits (Beckman Coulter, AMH gene II assay, Brea, CA, USA).
The limit of detection (LOD) was 0.08 ng/ml intra-assay and inter-assay variations were 7.7%.

Sample size calculation
Based on the results of a study (14) and considering the OR of higher quartile compared to the reference quartile, we calculated that at least 44 samples would be required in each group to detect an association with a power of 80% and α = 0.05 by using Epi Info site (http: www.cdc.gov/epiinfo). In each group, 60 samples were recruited for possible losses and further analysis. Appropriate written informed consent was obtained from all participants prior to blood sample collection.

International Journal of Reproductive BioMedicine
Jalaeefar et al.

Results
The final analysis was performed on the data collected from 58 women in each group. Table I presents the Characteristics of cases and controls.
Overall, breast cancer patients had lower single status women compared to the control group, and women in the control group were more educated than those in the case group with borderline significance (p = 0.07). With respect to the medical history of the participants, thyroid diseases were common and other diseases were very rare.
Therefore, self-history of thyroid diseases is mentioned in Table I

Discussion
The serum levels of AMH in young women with early breast cancer prior to any treatment was compared to healthy women in the present study considering confounding factors. We found a positive association between lower and higher AMH levels and breast cancer before anticancer treatment.
Our results were not consistent with preclinical studies in cell-line and animal models. Those studies reported that AMH inhibits tumor cell growth and migration through nuclear factor kappa-light-chain-enhancer of activated B cellsmediated pathways, and that after AMH injection, the ratio of apoptotic cells in murine mammary tissue increases eightfold (8)(9)(10)(11). Therefore, an inverse association between AMH and breast tumor development was demonstrated. However, those models have evaluated the basal-like breast cancer subtypes, which account for the minority of breast cancers. As mentioned in Table III, the AMH level in triple-negative patients is this study was 2.34 ± 1.42. It seems that AMH assessment in other subtypes of breast cancer is necessary.
The present study result shows that the low level of AMH in young patients is associated with breast cancer, which has also been confirmed by other studies (12,13 It is well-known that high breast density is associated with an increased risk of breast cancer (21). A recent study examined the relation of AMH with mammographic breast density.
They found that women with higher AMH levels had a significantly lower fatty breast (22). This study supported the hypothesis of the relationship between AMH levels and breast cancer. Meanwhile, this study could support the previous findings which explained the possible effect of AMH on breast tissue.
The first prospective study in premenopausal and Her2-negative tumors (p = 0.03) (Table III).
Although our results require further investigation due to the small sample size and lack of access to IHC of all patients, Ge and co-authors study also confirmed the increasing trend of AMH for tumors positive for both estrogen and progesterone receptors (17). It seems that further evaluation of AMH level in subtypes of breast cancer is needed for appropriate fertility preservation counseling.
A recent study showed that breast cancer patients with BRCA mutation have significantly lower serum AMH levels and recommended that fertility preservation should be considered more aggressively in these patients (28). Therefore, for the accurate evaluation of the association between AMH level and breast cancer, BRCA mutation assessment is necessary.
The present study has some advantages.
Based on our knowledge, this is the first evaluation of the AMH level in young Iranian breast cancer patients. In this study, we measured AMH levels for 2 hours after sampling and used the AMH Gene II assay kit for measurement, which is highly specific and reproducible (29).

Limitation
The first limitation of the current study is that since the AMH level was evaluated after a breast cancer diagnosis, it might not reflect the AMH level before cancer development. Another limitation might the small sample size of the study compared with large cohorts.

Conclusion
In conclusion, we found the lower and higher AMH level categories are more prevalent in young breast cancer patients who were in premenopausal status and didn't receive any cytotoxic treatment. Further studies are necessary with a larger sample size, considering age, menopausal status, oral contraceptive use, and previous history of other diseases such as PCOS as well as genetic assessment for BRCA mutation.
As young women are more likely to benefit from preventive drug intervention, individual risk assessment is particularly valuable in young women and can encourage them to pursue screening for early detection of breast cancer, or offer them new biomarker-based treatments.