ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY
The association of antim€ ullerian hormone levels with menstrual-cycle type and dysmenorrhea in young asymptomatic women Shoko Konishi, Ph.D.,a,b Yukiko Nishihama, M.S.,c Ayaka Iida, B.S.,c,d Jun Yoshinaga, Ph.D.,c and Hideki Imai, Ph.D.e a Department of Human Ecology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; b Department of Anthropology, University of Washington, Seattle, Washington; c Department of Environment Systems, Graduate School of Frontier Science, The University of Tokyo, Tokyo, Japan; d School of Allied Health Sciences, Kitasato University, Sagamihara, Japan; and e Faculty of Nursing, Tokyo Healthcare University, Tokyo, Japan
Objective: To examine the association between antim€ ullerian hormone (AMH) levels and menstrual-cycle and lifestyle characteristics among young Japanese women. Design: Cross-sectional study. Setting: A university. Patient(s): Female students aged 20–22 years (n ¼ 65) who had never used oral contraceptives. Intervention(s): Participants completed a questionnaire on reproductive and lifestyle characteristics, and kept a menstrual-cycle diary for 5 consecutive months. Serum AMH was measured once during the study period. Main Outcome Measure(s): Serum AMH concentration. Result(s): Compared with women with very mild menstrual pain, serum AMH concentration was 49.6% (95% CI 6.5%–72.8%) lower among women with severe menstrual pain. Higher AMH concentration was associated with irregular menstrual cycles. Even after adjusting for menstrual-cycle regularity and its interaction, more-severe menstrual pain was associated with significantly lower AMH concentration. Conclusion(s): Circulating AMH concentration was significantly lower among young Japanese women who had more-severe menstrual pain. Underlying physiological mechanisms need to be addressed in future studies. Use your smartphone (Fertil SterilÒ 2014;-:-–-. Ó2014 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Antim€ ullerian hormone, menstrual-cycle regularity, menstrual pain, young and connect to the women Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/konishis-amh-menstrual-cycle-dysmenorrhea-young-women/
A
ntim€ ullerian hormone (AMH) has been widely used in clinical settings, because it reflects the number of antral and pre-antral follicles present in the ovaries and can predict the ovarian response to hyperstimulation (1, 2). Circulating AMH levels peak in women's early 20's and decline
with age; they thus serve as a marker of reproductive aging (3, 4). On the other hand, the ability of AMH to predict fecundability (the probability of a woman conceiving within a given period of time) in a nonclinical population was suggested in 1 study (5), but not replicated in a following study (6).
Received May 8, 2014; revised and accepted July 31, 2014. S.K. has nothing to disclose. Y.N. has nothing to disclose. A.I. has nothing to disclose. J.Y. has nothing to disclose. H.I. has nothing to disclose. Reprint requests: Shoko Konishi, Ph.D., Department of Human Ecology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail: moe@humeco. m.u-tokyo.ac.jp). Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.07.1255 VOL. - NO. - / - 2014
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
The inability of AMH to predict fecundability may be related partly to large interindividual variation of circulating AMH even within the same age (7, 8), which is related to various lifestyle and reproductive characteristics. For example, lower AMH concentration has been found in women who are currently using oral contraceptives (3, 9), have mild/ minimal endometriosis (10), are obese (11), and who smoke (12). Higher AMH concentrations have been found in women with polycystic ovary syndrome (PCOS) (13). Among women who do not use oral contraceptives, a 1
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY regular (vs. irregular) and shorter menstrual cycle was associated with lower AMH levels (6, 9). Although there is no conventional biomarker of endometriosis that can be used in epidemiological studies, more-severe menstrual pain is associated with an increased risk for endometriosis (14). Therefore, in the present study, we examined the association between AMH levels and menstrual pain, in addition to the above-mentioned variables that are known to be associated with circulating AMH. Ethnicity is an independent predictor for AMH (15) and other reproductive outcomes across the life cycle (16), which suggests that association between AMH and its correlates, i.e., body mass index (BMI), smoking, and physical activity, may vary across ethnic groups. However, to our knowledge, no study has targeted Japanese women in a nonclinical setting to examine such associations. Given the fact that the AMH test is widely used in clinical settings in Japan as a marker of ovarian reserve, understanding the factors associated with individual variation of AMH levels among Japanese women is important. Therefore, the aim of the present study was to examine the association between circulating AMH levels, menstrual-cycle features, and lifestyle (e.g., smoking, physical activity) characteristics to find determinants of these hormone levels among young Japanese women.
MATERIALS AND METHODS Subjects and Protocol The present data were obtained as part of a research project, targeting both men and women in Japan, on the effect of exposure to environmental chemicals on reproductive performance. Institutional review board (IRB) approval was obtained from the Tokyo Healthcare University and the Graduate School of Frontier Sciences, The University of Tokyo. All the junior students (n ¼ 97) at Tokyo Healthcare University were invited to participate in November 2012. A total of 97 students (88 women and 9 men) provided written informed consent. All the data analyzed in the present paper are based on a cross-sectional study consisting of one-time blood collection and a questionnaire survey, in addition to a menstrual-cycle diary that was kept prospectively and followed by a supplemental questionnaire. The female participants kept a diary to record menstrual bleeding for 5 consecutive months, which was used to categorize women by their menstrual-cycle regularity, as described below. They also filled out a questionnaire on lifestyle and menstrual-cycle characteristics at their convenience between December 2012 and May 2013. Specifically, the questionnaire included questions on age, height, weight, smoking, alcohol consumption, physical activity, age at menarche, and past oral contraceptive use. Body mass index was calculated using the self-reported height and weight reported in the questionnaire. Venous blood samples were collected in the early afternoon (1:00 PM to 3:00 PM), once from each woman in December 2012, irrespective of their menstrual-cycle phase, at the Tokyo Healthcare University. Upon completing the diary, the women answered a questionnaire on menstrual pain and oral contraceptive use during the diary-keeping period. Each woman was asked to characterize the most severe menstrual pain episode 2
during the 5-month diary-keeping period using the following 4 categories: very mild (‘‘almost none’’); mild (‘‘felt pain, but had no problem in daily activities’’); moderate (‘‘could go out, but felt difficulty in doing physical exercise’’); or severe (‘‘too painful to get up from a bed’’).
Hormonal Assays Serum samples were stored at 80 C until assay for AMH and testosterone (T) in July 2013. Serum concentration of T was measured, because women with PCOS show elevated concentrations of both AMH and T (13). The concentration of AMH and T in the serum samples was quantified using AMH Gen II ELISA and Roche electrochemiluminescence (ECLIA) testosterone II, respectively. Intra- (n ¼ 10) and inter-assay (n ¼ 5) coefficients of variation (CVs) for AMH at a concentration of approximately 5.0 ng/mL were 4.1% and 7.1%, respectively. Intra- (n ¼ 10) and inter-assay (n ¼ 5) CVs for T at a concentration of approximately 1.1 ng/mL were both 1.8%. All the assays were run in the laboratory of SRL Co. Ltd., Tokyo, Japan.
Statistical Analyses Menstrual bleeding was defined as R3 consecutive days of bleeding. Menstrual-cycle length was defined as the number of days between the first day of menstrual bleeding in one cycle and the first day of menstrual bleeding in the next cycle. Menstrual-cycle regularity was categorized according to established definitions (17), with some modifications. Because the 5-month recording period was set irrespective of menstrual-cycle phase, there were censored cycles in which either the starting or ending date of a menstrual cycle could not be obtained. Mean cycle length was calculated for each women using only noncensored cycles. We categorized each woman into 1 of 3 categories of menstrual-cycle regularity: regular cycles, with mean cycle length between 22 and 41 days; irregular cycles, with mean cycle length between 22 and 41 days and 1 cycle 41 days (both censored and noncensored); or oligomenorrhea, with mean cycle length >41 days. These criteria take into account both censored and noncensored cycles, reducing the possible bias due to overrepresentation of short cycles. Spearman's rank correlation coefficient was calculated to examine the correlation between the AMH and T concentrations. The concentration of AMH was log-transformed for further statistical analyses. Percentage differences in AMH concentration with each predictor variable were calculated using a univariate regression analysis to determine which factors are associated with various levels of AMH. Age was not adjusted in these models, because of the narrow age range of the participants. Menstrual-cycle regularity and menstrual pain were the only predictors that were significantly associated with AMH variation; thus, we used a 2-way analysis of variance (ANOVA) to examine the effects of the 2 factors and their interaction on AMH levels. For the ANOVA, very mild and mild menstrual-cycle pains were grouped into a ‘‘milder’’ category; moderate and severe pain were grouped VOL. - NO. - / - 2014
Fertility and Sterility® into a ‘‘more severe’’ category. Similarly, women with irregular cycles or oligomenorrhea were categorized as having ‘‘irregular,’’ as opposed to regular, cycles. Statistical analyses were performed with R (version 3.0.2; R Project for Statistical Computing, Vienna, Austria). Statistical significance was defined as P< .05.
RESULTS Among 88 women enrolled, 65 women gave a complete data set and had never used oral contraceptives. Age of the participants ranged from 20 to 22 years; mean (standard deviation) BMI was 20.4 (1.4) kg/m2 (Table 1). A statistically significant positive association was found between AMH and T concentrations after excluding one outlier (Fig. 1; rho ¼ 0.475, P< .001). Compared with women with regular menstrual cycles (n ¼ 42; median 4.03 ng/mL, interquartile range [IQR] 2.02– 6.54 ng/mL), those with irregular cycles (n ¼ 19; median 7.33 ng/mL, IQR 4.70–8.93 ng/mL) had 55.1% higher AMH levels (95% CI 4.7%–129.7%; Table 2). Compared with women with very mild menstrual pain (n ¼ 15; median 5.85 ng/mL, IQR 3.64–8.93 ng/mL), serum AMH concentration was 49.6% (95% CI 6.5%–72.8%) lower among women with severe menstrual pain (n ¼ 9; median 3.12 ng/mL, IQR 1.71–5.14 ng/ mL; Table 2). Even after adjusting for menstrual-cycle regularity and its interaction with menstrual pain, more-severe menstrual pain was associated with significantly lower AMH concentration (ANOVA; P¼ .036). The mean AMH concentration of women with irregular menstrual cycles and milder menstrual pain (median 7.3 ng/mL, IQR 4.1–9.2 ng/ mL) was more than 3 times higher than that of women with regular menstrual cycles and more-severe menstrual pain (median 2.2 ng/mL, IQR 1.7–5.0 ng/mL), whereas mean T concentration did not differ across the subgroups.
DISCUSSION Severe menstrual pain was associated with lower AMH among Japanese women aged 20–22 years. To our knowledge, no
FIGURE 1
A scatter plot depicting the positive association (rho ¼ 0.475, P
The association of antim€ ullerian hormone levels with menstrual-cycle type and dysmenorrhea in young asymptomatic women Shoko Konishi, Ph.D.,a,b Yukiko Nishihama, M.S.,c Ayaka Iida, B.S.,c,d Jun Yoshinaga, Ph.D.,c and Hideki Imai, Ph.D.e a Department of Human Ecology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; b Department of Anthropology, University of Washington, Seattle, Washington; c Department of Environment Systems, Graduate School of Frontier Science, The University of Tokyo, Tokyo, Japan; d School of Allied Health Sciences, Kitasato University, Sagamihara, Japan; and e Faculty of Nursing, Tokyo Healthcare University, Tokyo, Japan
Objective: To examine the association between antim€ ullerian hormone (AMH) levels and menstrual-cycle and lifestyle characteristics among young Japanese women. Design: Cross-sectional study. Setting: A university. Patient(s): Female students aged 20–22 years (n ¼ 65) who had never used oral contraceptives. Intervention(s): Participants completed a questionnaire on reproductive and lifestyle characteristics, and kept a menstrual-cycle diary for 5 consecutive months. Serum AMH was measured once during the study period. Main Outcome Measure(s): Serum AMH concentration. Result(s): Compared with women with very mild menstrual pain, serum AMH concentration was 49.6% (95% CI 6.5%–72.8%) lower among women with severe menstrual pain. Higher AMH concentration was associated with irregular menstrual cycles. Even after adjusting for menstrual-cycle regularity and its interaction, more-severe menstrual pain was associated with significantly lower AMH concentration. Conclusion(s): Circulating AMH concentration was significantly lower among young Japanese women who had more-severe menstrual pain. Underlying physiological mechanisms need to be addressed in future studies. Use your smartphone (Fertil SterilÒ 2014;-:-–-. Ó2014 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Antim€ ullerian hormone, menstrual-cycle regularity, menstrual pain, young and connect to the women Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/konishis-amh-menstrual-cycle-dysmenorrhea-young-women/
A
ntim€ ullerian hormone (AMH) has been widely used in clinical settings, because it reflects the number of antral and pre-antral follicles present in the ovaries and can predict the ovarian response to hyperstimulation (1, 2). Circulating AMH levels peak in women's early 20's and decline
with age; they thus serve as a marker of reproductive aging (3, 4). On the other hand, the ability of AMH to predict fecundability (the probability of a woman conceiving within a given period of time) in a nonclinical population was suggested in 1 study (5), but not replicated in a following study (6).
Received May 8, 2014; revised and accepted July 31, 2014. S.K. has nothing to disclose. Y.N. has nothing to disclose. A.I. has nothing to disclose. J.Y. has nothing to disclose. H.I. has nothing to disclose. Reprint requests: Shoko Konishi, Ph.D., Department of Human Ecology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail: moe@humeco. m.u-tokyo.ac.jp). Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.07.1255 VOL. - NO. - / - 2014
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
The inability of AMH to predict fecundability may be related partly to large interindividual variation of circulating AMH even within the same age (7, 8), which is related to various lifestyle and reproductive characteristics. For example, lower AMH concentration has been found in women who are currently using oral contraceptives (3, 9), have mild/ minimal endometriosis (10), are obese (11), and who smoke (12). Higher AMH concentrations have been found in women with polycystic ovary syndrome (PCOS) (13). Among women who do not use oral contraceptives, a 1
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY regular (vs. irregular) and shorter menstrual cycle was associated with lower AMH levels (6, 9). Although there is no conventional biomarker of endometriosis that can be used in epidemiological studies, more-severe menstrual pain is associated with an increased risk for endometriosis (14). Therefore, in the present study, we examined the association between AMH levels and menstrual pain, in addition to the above-mentioned variables that are known to be associated with circulating AMH. Ethnicity is an independent predictor for AMH (15) and other reproductive outcomes across the life cycle (16), which suggests that association between AMH and its correlates, i.e., body mass index (BMI), smoking, and physical activity, may vary across ethnic groups. However, to our knowledge, no study has targeted Japanese women in a nonclinical setting to examine such associations. Given the fact that the AMH test is widely used in clinical settings in Japan as a marker of ovarian reserve, understanding the factors associated with individual variation of AMH levels among Japanese women is important. Therefore, the aim of the present study was to examine the association between circulating AMH levels, menstrual-cycle features, and lifestyle (e.g., smoking, physical activity) characteristics to find determinants of these hormone levels among young Japanese women.
MATERIALS AND METHODS Subjects and Protocol The present data were obtained as part of a research project, targeting both men and women in Japan, on the effect of exposure to environmental chemicals on reproductive performance. Institutional review board (IRB) approval was obtained from the Tokyo Healthcare University and the Graduate School of Frontier Sciences, The University of Tokyo. All the junior students (n ¼ 97) at Tokyo Healthcare University were invited to participate in November 2012. A total of 97 students (88 women and 9 men) provided written informed consent. All the data analyzed in the present paper are based on a cross-sectional study consisting of one-time blood collection and a questionnaire survey, in addition to a menstrual-cycle diary that was kept prospectively and followed by a supplemental questionnaire. The female participants kept a diary to record menstrual bleeding for 5 consecutive months, which was used to categorize women by their menstrual-cycle regularity, as described below. They also filled out a questionnaire on lifestyle and menstrual-cycle characteristics at their convenience between December 2012 and May 2013. Specifically, the questionnaire included questions on age, height, weight, smoking, alcohol consumption, physical activity, age at menarche, and past oral contraceptive use. Body mass index was calculated using the self-reported height and weight reported in the questionnaire. Venous blood samples were collected in the early afternoon (1:00 PM to 3:00 PM), once from each woman in December 2012, irrespective of their menstrual-cycle phase, at the Tokyo Healthcare University. Upon completing the diary, the women answered a questionnaire on menstrual pain and oral contraceptive use during the diary-keeping period. Each woman was asked to characterize the most severe menstrual pain episode 2
during the 5-month diary-keeping period using the following 4 categories: very mild (‘‘almost none’’); mild (‘‘felt pain, but had no problem in daily activities’’); moderate (‘‘could go out, but felt difficulty in doing physical exercise’’); or severe (‘‘too painful to get up from a bed’’).
Hormonal Assays Serum samples were stored at 80 C until assay for AMH and testosterone (T) in July 2013. Serum concentration of T was measured, because women with PCOS show elevated concentrations of both AMH and T (13). The concentration of AMH and T in the serum samples was quantified using AMH Gen II ELISA and Roche electrochemiluminescence (ECLIA) testosterone II, respectively. Intra- (n ¼ 10) and inter-assay (n ¼ 5) coefficients of variation (CVs) for AMH at a concentration of approximately 5.0 ng/mL were 4.1% and 7.1%, respectively. Intra- (n ¼ 10) and inter-assay (n ¼ 5) CVs for T at a concentration of approximately 1.1 ng/mL were both 1.8%. All the assays were run in the laboratory of SRL Co. Ltd., Tokyo, Japan.
Statistical Analyses Menstrual bleeding was defined as R3 consecutive days of bleeding. Menstrual-cycle length was defined as the number of days between the first day of menstrual bleeding in one cycle and the first day of menstrual bleeding in the next cycle. Menstrual-cycle regularity was categorized according to established definitions (17), with some modifications. Because the 5-month recording period was set irrespective of menstrual-cycle phase, there were censored cycles in which either the starting or ending date of a menstrual cycle could not be obtained. Mean cycle length was calculated for each women using only noncensored cycles. We categorized each woman into 1 of 3 categories of menstrual-cycle regularity: regular cycles, with mean cycle length between 22 and 41 days; irregular cycles, with mean cycle length between 22 and 41 days and 1 cycle 41 days (both censored and noncensored); or oligomenorrhea, with mean cycle length >41 days. These criteria take into account both censored and noncensored cycles, reducing the possible bias due to overrepresentation of short cycles. Spearman's rank correlation coefficient was calculated to examine the correlation between the AMH and T concentrations. The concentration of AMH was log-transformed for further statistical analyses. Percentage differences in AMH concentration with each predictor variable were calculated using a univariate regression analysis to determine which factors are associated with various levels of AMH. Age was not adjusted in these models, because of the narrow age range of the participants. Menstrual-cycle regularity and menstrual pain were the only predictors that were significantly associated with AMH variation; thus, we used a 2-way analysis of variance (ANOVA) to examine the effects of the 2 factors and their interaction on AMH levels. For the ANOVA, very mild and mild menstrual-cycle pains were grouped into a ‘‘milder’’ category; moderate and severe pain were grouped VOL. - NO. - / - 2014
Fertility and Sterility® into a ‘‘more severe’’ category. Similarly, women with irregular cycles or oligomenorrhea were categorized as having ‘‘irregular,’’ as opposed to regular, cycles. Statistical analyses were performed with R (version 3.0.2; R Project for Statistical Computing, Vienna, Austria). Statistical significance was defined as P< .05.
RESULTS Among 88 women enrolled, 65 women gave a complete data set and had never used oral contraceptives. Age of the participants ranged from 20 to 22 years; mean (standard deviation) BMI was 20.4 (1.4) kg/m2 (Table 1). A statistically significant positive association was found between AMH and T concentrations after excluding one outlier (Fig. 1; rho ¼ 0.475, P< .001). Compared with women with regular menstrual cycles (n ¼ 42; median 4.03 ng/mL, interquartile range [IQR] 2.02– 6.54 ng/mL), those with irregular cycles (n ¼ 19; median 7.33 ng/mL, IQR 4.70–8.93 ng/mL) had 55.1% higher AMH levels (95% CI 4.7%–129.7%; Table 2). Compared with women with very mild menstrual pain (n ¼ 15; median 5.85 ng/mL, IQR 3.64–8.93 ng/mL), serum AMH concentration was 49.6% (95% CI 6.5%–72.8%) lower among women with severe menstrual pain (n ¼ 9; median 3.12 ng/mL, IQR 1.71–5.14 ng/ mL; Table 2). Even after adjusting for menstrual-cycle regularity and its interaction with menstrual pain, more-severe menstrual pain was associated with significantly lower AMH concentration (ANOVA; P¼ .036). The mean AMH concentration of women with irregular menstrual cycles and milder menstrual pain (median 7.3 ng/mL, IQR 4.1–9.2 ng/ mL) was more than 3 times higher than that of women with regular menstrual cycles and more-severe menstrual pain (median 2.2 ng/mL, IQR 1.7–5.0 ng/mL), whereas mean T concentration did not differ across the subgroups.
DISCUSSION Severe menstrual pain was associated with lower AMH among Japanese women aged 20–22 years. To our knowledge, no
FIGURE 1
A scatter plot depicting the positive association (rho ¼ 0.475, P
