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Research Article

Anti-Mullerian hormone levels in female cancer patients of reproductive age in Indonesia: A cross-sectional study

[version 1; peer review: 1 approved with reservations]
PUBLISHED 06 Feb 2019
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This article is included in the Oncology gateway.

Abstract

Background: Efforts in reproductive preservation for cancer patients have become one of the important aspects of cancer management. In fact, decline in reproductive function is known to occur after exposure to anti-cancer treatments. Measuring anti-Müllerian hormone (AMH) levels is known to be the best parameter in predicting ovarian reserves, which indicates reproductive function. In total, 68% of cancer survivors of reproductive age who underwent anti-cancer treatments suffer from infertility. Meanwhile, ovarian reserves also decrease with increasing age. There is ongoing debate on whether the ovarian reserves of cancer patients could be reduced long before exposure to anti-cancer therapy. Therefore, it is important to know whether ovarian reserves in cancer patients decrease before or after anti-cancer therapy. This can help predict the reproductive function in such cases and the effectiveness of ovarian preservation efforts.
Methods: A cross-sectional study was conducted, comparing the AMH levels of 44 female cancer patients of reproductive age before cancer therapy, to 44 non-cancer patients of reproductive age (age matched). The biological ages from both groups were adjusted using the Indonesian Kalkulator of Oocytes.
Results: The median age in both groups was 28 years old. The AMH levels in the cancer group were found to be significantly lower in contrast to those in the non-cancer group (1.11 [0.08-4.65] ng/ml vs. 3.99 [1.19- 8.7]; p- value <0.001). Therefore, the biological age in the cancer group was 10 years older than that of the non-cancer group, indicating that ovarian aging occurs earlier in cancer patients.
Conclusions: AMH levels of cancer patients of reproductive age were already reduced before cancer therapy, given an older biological age, in contrast to that of the non-cancer patients. Proper counseling and implementation of fertility-preserving methods is highly recommended in this group of patients.

Keywords

Anti Mullerian Hormone (AMH), Ovarian Reserve, Cancer in Reproductive, Biological Age

Introduction

Reproductive preservation efforts for cancer patients have become an important aspect of cancer management. Of all female cancer cases worldwide, 10% of cases occur during reproductive age1. Most cancer patients do not have an opportunity to preserve reproductive function prior to cancer treatment, and decline in reproductive function is known to occur after exposure to cancer treatments, whether it be chemotherapy or radiotherapy. In total, 68% of cancer survivors of reproductive age who have undergone anti-cancer treatments experience infertility, amenorrhea, reduction of ovarian reserves, and premature ovarian failure. Ovarian reserves also decrease with increasing age2,3, therefore, the American Society of Clinical Oncology (ASCO), and the American Society for Reproductive Medicine (ASRM) have recommended that consultation on fertility preservation methods prior to cancer therapy should be provided4. However, there is an ongoing debate on whether the ovarian reserves of cancer patients could be reduced long before exposure to anti-cancer therapy.

The measurement of the anti-Müllerian hormone (AMH) levels is known to be the best parameter for predicting ovarian reserves, which represent reproductive function5. Low AMH levels reflect a low ovarian reserve6,7.

Several studies have shown that in breast cancer and lymphoma patients, ovarian reserves are already decreased prior to cancer therapy. However, other studies have shown that a decreased ovarian reserve is only associated with increasing age. It is, therefore, important to know whether the ovarian reserves in cancer patients decrease before or after therapy. This will help predict the reproductive function in such cases and the effectiveness of ovarian preservation efforts.

Methods

Study design and patient population

This is a cross-sectional study that compares the AMH levels of reproductive age cancer patients before cancer treatment with non-cancer patients. We enrolled 88 subjects; 44 cancer patients of reproductive age before cancer treatment and 44 non-cancer patients (similar age). For cancer group data, patients were recruited from three places; Obstetrics and Gynecology Polyclinics, Hematology Oncology Polyclinics of Cipto Mangunkusumo Hospital, Obstetrics and Gynecology Polyclinics and Inpatient Unit of Dharmais Cancer Hospital, from May 2015 to December 2017. For non-cancer group data, AMH levels were obtained as secondary data from our previous study8, from which we selected patients with a similar age as patients in the cancer group. Inclusion criteria for this study were (a) Cancer and non-cancer patients aged 17–40 years (b) Cancer patients who have never had a history of cancer therapy: chemotherapy/radiation, de-bulking tumors (specifically gynecology); and exclusion criteria were (a) respondents who not willing to be a participant in this study and (b) incomplete of filling informed consent. The sample size was derived from the formula n=z2pqd2 where n is the minimum sample size, Z is the standard normal deviation, as in majority of studies p values are considered significant below 0.05 hence 1.96 is used in formula, p is expected proportion in population based on previous studies (0.36), q is 1-p (0.64), d is the absolute error or precision (0.1 or 10%). Therefore, the total sample size for this study was calculated to be 88 sample subjects and the comparison of cancer groups before cancer therapy and non cancer group was 1:1 meaning the total sample size for each group was 44 subjects. Unpaired numerical analysis formulas were used to compare AMH levels between cancer patients in reproductive age before cancer therapy with non-cancer patients. The standard deviation for each group are calculated separately, and result shows S1 was standard deviation of the cancer group by: 1,088 ng/dl (mean 1.4 ng/dl) and S2 was standard deviation of non-cancer group by : 2.20 ng/dl (mean 4.37 ng/dl). For calculate the minimum number of samples that can be used in this study, and also know the effect size and power of the study so all collected data were inputted to G Power software version 3.0.10. As a result, minimum sample subject count for each group of at least 7 subjects, with actual P 0.83.

Data collection

Data collection was performed by consecutive sampling. In cancer group participants, they were given an explanation of the purpose and benefits of the study, and as asked to provide written consent to participate in the study. The written approval sheet was signed by the patient and the researcher. Researchers registered cases and filled out research status forms (Supplementary File 1 and Supplementary File 2). Blood serum (3 ml) from the cubital vein was collected from the cancer group, and stored in vacutainer without anticoagulants. This serum then sent to FKUI Integrated Laboratory for AMH measurement using ELISA techniques by AMH GEN II ELISA Beckman Coulter REF A79765 (with VMax machines). ELISA was performed to the manufacturer’s protocol. After obtaining the results of AMH levels, data analysis was performed. Full method for AMH hormone examination for cancer groups were taken 3 ml of venous blood from the cubital vein of the media and stored in a vacutainer without anticoagulants. After liquefaction and at room temperature, the AMH examination was carried out manually with a micro ELISA technique read with a VMAX type kinetic microplate reader with Softmax Pro Software version 5.4.1.

Statistical analysis

Statistical analyses were performed using IBM SPSS version 20. Data on ovarian reserves expressed in AMH levels from the cancer patients and the non-cancer patients are presented as medians (minimum-maximum) because the data were not normally distributed. Mann-Whitney tests were performed to compare the differences in ovarian reserves (AMH levels) between the cancer group and the non-cancer group.

Ethical approval

The Ethics Committee of the Faculty of Medicine from Universitas Indonesia approved this study on May 5, 2015 (reference number: 286/UN2.F1/ATIK/2015). All prospective subjects received an explanation from the main researcher and additional researchers regarding the procedures for conducting research. The decision to follow or refuse to follow the research was taken by informed consent. All data will be kept confidential and the subject had the right to know all the results of the examination carried out.

Results

The 88 subjects participated in this study, with similar characteristics between the two arms. The mean age for cancer and non-cancer groups was the same, 28 years. For the cancer patient menarche age was 11 years. Body mass index for cancer patients was 20.7 ± 3.89, and non-cancer patients 19.5 ± 2.561. Both groups have the same parity average of 0. The mean AMH level for cancer patients was 1.11 ng/ml (0.08–4.65 ng/ml), and for non-cancer patients 3.99 ng/ml (1.19–8.7 ng/ml). The biological age for cancer patients based on an AMH level of 1.1 ng/ml was 38 years old, and for non-cancer patients 3.99 ng/ml corresponded to 28 years old. Characteristics of research subjects can be seen in Table 1. Of all the cancer patients recruited in the study, 14 subjects were gynecologic cancer patients and 30 subjects were non-gynecologic cancer patients, the distribution of cancer cases can be seen in Table 2.

Table 1. Baseline characteristics.

CharacteristicCancer group
N: 44
Non-cancer group
N: 44
p-value
Age, years (min-max)28 (18–37)28.5(22–37)0.364
Menarche, age in years (min-max)11 (8–13)11 (8–13)0.364
BMI, kg (SD)20,7 ± 3.8919,5 ± 2.5610.043
Parity, n (min-max)0 (0–2)0 (0–3)0.449
AMH, ng/dl (min-max)1.11 (0.08–4.65)3.99 (1.19–8.7)<0.001
Biological age* (min-max)3828-

Notes: *Biological age based on AMH level is in accordance with the Indonesian Calculator of Oocyte. BMI: Body mass index, AMH: Anti-Mullerian hormone

Table 2. Cancer distribution.

Cancer type(n)
Gynecologic cancer14
Cervical cancer7
Ovarian cancer5
Endometrial cancer1
Vaginal cancer1
Non-gynecologic cancer30
Breast cancer9
Non-Hodgkin Lymphoma7
Acute myelogenous leukemia1
Chronic myelogenous leukemia4
Acute Lymphocytic Leukemia2
Rectal cancer
Tongue cancer1
Myelodysplastic syndromes1
Nasopharyngeal cancer1
Vesical cancer1
Adenocystic carcinoma1

Overall, the AMH levels were abnormally distributed (Kolmogorov-Smirnov test for normality, p-value <0.001) with a median level of AMH 2.06 ng/ml (0.08–8.78 ng/ml). AMH levels in the cancer group were lower than that of the non-cancer group (1.11 [0.08–4.65] ng/ml vs. 3.99 [1.19–8.7]; p-value <0.001). This study also showed that there were no statistically significant differences between the AMH levels of the gynecologic cancer patients 0.965 ng/ml (0.08–2.65 ng/ml) and the non-gynecological cancer patients before treatments 1.49 ng/ml (0.08–4.65 ng/ml); p-value 0.162.

Dataset 1.All raw data and demographic information obtained from subject during the present study.

Discussion

In line with advances in cancer therapy, the survival rate of cancer patients also increases. Unfortunately, this rate of improvement does not coincide with an increased quality of life, especially in regards to reproductive function. Currently, the best parameter to measure ovarian reserves is by measuring serum AMH levels; AMH levels can also help predict biological age, which is of more importance when assessing reproductive function.

There are several types of cancer known to be associated with decreased ovarian reserves prior to cancer therapy, such as Hodgkin and Non-Hodgkin’s lymphoma, and breast cancer. Lawrenz et al. demonstrated that the AMH levels of lymphoma patients were lower than that in non-cancer patients, with mean AMH levels of 2.06 ng/ml vs. 3.20 ng/dl (p- value <0.05)9. Su et al. conducted a similar study wherein the AMH levels of the breast cancer patients were lower than that of the non-cancer patients, 0.6 ng/ml vs. 1.1 ng/ml (p- value <0.001)10. Van et al. also conducted a broader study in 2014 comparing the AMH levels in young cancer patients (<18 years) before treatment with non-cancer patients (age-matched). The results showed that AMH levels in the cancer group were significantly lower than that of the non-cancer group, 1.4 mg/l vs. 3.0 mg/l (p- value <0.001)11. The results of these three studies were similar to that of our study; the AMH levels of the 44 reproductive age cancer patients prior to cancer treatment were lower than that of the non-cancer patients (1.11 [0.08–4.65] ng/ml vs. 3.99 [1.19–8.7] ng/ml; p- value <0.001).

Although some studies have shown that the ovarian reserves of reproductive-age cancer patients before treatment are lower than that of the non-cancer patients, the factors that directly affect AMH levels are still unknown. Several studies have suggested genetic mutations, such as BRCA gene mutations, may affect ovarian reserves in breast cancer patients1214; the effects of high cytokines in lymphoma patients indirectly affect the ovarian reserves as well15,16. In non-cancer populations, Jung et al. has proven that only the age of menarche affected the concentration of AMH (age of menarche <12 years vs. ≥14 years, 0.90 ng/mL vs. 1.12 ng/mL), while ethnicity, BMI, education level, smoking status, height, and menstrual cycle are not associated with AMH concentrations17. Su et al. found that age, BMI, parity, and smoking status are not associated with decreased levels of AMH in patients with breast cancer10.

In contrast to other types of non-gynecological cancers, reproductive preservation in gynecological cancers is not widely discussed. This is related to the fact that the genital organs themselves are involved in the cancer and the local treatments of such cancers are destructive to the organs of reproduction18. Therefore, in this study, we compared the AMH levels of gynecologic patients of reproductive age before receiving cancer treatment with the non-gynecological cancer patients, and we found no significant difference between AMH levels (0.965 ng/dl vs. 1.49 ng/dl; p- value 0.162). However, the unequal size of the gynecological and non-gynecological cancers subject groups in our study may have affected the results. With increasing age the capability of a woman to produce appropriate quality oocytes, and quantity of oocytes decreases; this process, called ovarian aging, is defined as a gradual decrease in both the quantity and quality of the oocytes. The decrease in the quality and quantity of these oocytes is related to chronological age and biological age19. Chronological age is determined by the passage of time from birth, while the biological age is determined by physiology20. Biological age affects the reproductive function more than chronological age, besides that ovarian reserve is a good marker for representing the biological age of the ovary. In this study, the biological age of the cancer group was 10 years higher than that of the non-cancer group, indicating that ovarian aging occurs earlier in cancer patients. This would indicate that reproductive preservation methods should be offered long before cancer patients undergo therapy.

There are many reproductive preservation techniques; however, one of technique for cancer patients is ovarian cryopreservation. This technique consists of two methods: the slow cooling method and vitrification ovary. Based on the research conducted by Wiweko et al, ovarian vitrification techniques are superior to the slow cooling method21. Ovarian vitrification does not alter the morphology of the granulosa cells, or the stromal and ovarian collagen components22. It has also been shown to not change the morphology of the pre-antral follicle nor does it increase the risk of cell apoptosis21,22. Other options that may still be offered to cancer patients of reproductive age are oocyte cryopreservation and embryo cryopreservation. In patients who already have a partner or are married, embryo cryopreservation can be offered, however, this may delay cancer therapy due to the ovarian stimulation process required for this technique. In addition to this, estrogen exposure given at the time of ovarian stimulation may have an adverse effect in estrogen sensitive tumors. Unlike embryo cryopreservation, cryopreservation of the oocytes can be used for single women who can undergo the stimulation cycle, but the effectiveness of this technique is very low. The pregnancy and delivery rates range from 1 to 5% per frozen oocyte23. The current weakness of this study is that the other gynecological abnormalities that may affect AMH levels in both groups were not included in the screening criteria for exclusion, such as genetic factors and the type/stage of cancer or other diseases which may affect the levels of AMH, were also not studied.

Conclusions

This study showed that the AMH levels in reproductive age patients before receiving therapy were lower in contrast to that in the non-cancer patients (1.11 ng/ml vs. 3.99 ng/ml; p- value <0.001). In addition to this, there was no significant difference in the AMH levels between the gynecologic and non-gynecologic cancer groups before treatment (0.965 ng/dl vs. 1.49 ng/dl; p- value 0.162).

Data availability

F1000Research: Dataset 1. All raw data and demographic information obtained from subject during the present study, https://doi.org/10.5256/f1000research.15728.d22918624.

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Harzif AK, Wiweko B, Addina P et al. Anti-Mullerian hormone levels in female cancer patients of reproductive age in Indonesia: A cross-sectional study [version 1; peer review: 1 approved with reservations]. F1000Research 2019, 8:159 (https://doi.org/10.12688/f1000research.15728.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
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Reviewer Report 10 Jun 2019
Nao Suzuki, Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kawasaki, Japan 
Approved with Reservations
VIEWS 28
This is a well-written manuscript about Anti-Mullerian hormone levels in female cancer patients of reproductive age in Indonesia. The aim of the study is to predict the reproductive function of cancer patients. The major limitation of the study is a ... Continue reading
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Suzuki N. Reviewer Report For: Anti-Mullerian hormone levels in female cancer patients of reproductive age in Indonesia: A cross-sectional study [version 1; peer review: 1 approved with reservations]. F1000Research 2019, 8:159 (https://doi.org/10.5256/f1000research.17166.r48874)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 12 Jul 2019
    Achmad Kemal, Division of Reproductive Endocrionolgy and Infertility, Department of Obstetrics and Gynecology Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, Indonesia
    12 Jul 2019
    Author Response
    We are grateful for the helpful feedback by the reviewer that helped us improve the quality of our manuscript. We carefully responded to all points and have modified the manuscript ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 12 Jul 2019
    Achmad Kemal, Division of Reproductive Endocrionolgy and Infertility, Department of Obstetrics and Gynecology Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, Indonesia
    12 Jul 2019
    Author Response
    We are grateful for the helpful feedback by the reviewer that helped us improve the quality of our manuscript. We carefully responded to all points and have modified the manuscript ... Continue reading

Comments on this article Comments (0)

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VERSION 3 PUBLISHED 06 Feb 2019
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Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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