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The correlation of serum asymmetric dimethylarginine and anti-Müllerian hormone in primary dysmenorrhea
Kaohsiung Journal of Medical Sciences (2016) 32, 414e419
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.kjms-online.com
ORIGINAL ARTICLE
The correlation of serum asymmetric ¨llerian dimethylarginine and anti-Mu hormone in primary dysmenorrhea ¨hha Bostancı a, Nermin Akdemir a,*, Fatma Behice Cinemre b, Mehmet Su ¨ nal a, Selc ¸uk Ozden a, Arif Serhan Cevrioglu a, Hakan Cinemre c, Orhan U d Zubeyde Kacal , Ramazan Akdemir e a
Department of Obstetrics and Gynecology, Sakarya University, Faculty of Medicine, Sakarya, Turkey b Department of Biochemistry, Sakarya University, Faculty of Medicine, Sakarya, Turkey c Department of Internal Medicine, Sakarya University, Faculty of Medicine, Sakarya, Turkey d Department of Medical Education and Ethics, Sakarya University, Faculty of Medicine, Sakarya, Turkey e Department of Cardiology, Sakarya, Sakarya University, Faculty of Medicine, Sakarya, Turkey Received 8 March 2016; accepted 4 July 2016
Available online 27 July 2016
KEYWORDS Anti-Mu ¨llerian hormone; Asymmetric dimethyl arginine; Ovarian reserve; Primary dysmenorrhea
Abstract In this study, we aimed to investigate the association of serum asymmetric dimethylarginine (ADMA) and anti-Mu ¨llerian hormone (AMH) levels in primary dysmenorrhea patients. The study employed a cross-sectional design. Eighty-nine female university students with primary dysmenorrhea were included in the study. All patients underwent complete clinical and laboratory investigations, including serum ADMA, AMH levels, pelvic ultrasonography, electrocardiography, and echocardiography. Pearson correlation and linear regression analysis were used to evaluate associations between continuous data. Categorical associations were evaluated using c2 test. Correlation analysis between serum ADMA and AMH levels in the study group showed a highly significant positive relationship (Pearson correlation Z 0.978, p Z 0.01). Our study has shown a significant positive correlation between serum ADMA and AMH levels in primary dysmenorrhea. Serum ADMA levels may have the potential to demonstrate ovarian reserve. Copyright ª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Conflicts of interest: All authors declare no conflicts of interests. * Corresponding author. Department of Obstetrics and Gynecology, Sakarya University, Faculty of Medicine, 54290, Sakarya, Turkey. E-mail address: [email protected] (N. Akdemir). http://dx.doi.org/10.1016/j.kjms.2016.07.001 1607-551X/Copyright ª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Serum ADMA and AMH levels in dysmenorrhea
Introduction Asymmetric dimethylarginine (ADMA) is an important marker in a variety of clinical settings, playing a role in endothelial dysfunction. It is associated with the pathogenesis of atherosclerosis, hypertension, and diabetic vascular complication, as well as primary dysmenorrhea (PD), polycystic ovary syndrome, and pregnancy-related disorders such as preeclampsia and hyperemesis gravidarum [1e3]. Changes in serum ADMA levels have also been investigated during normal menstrual periods. The menstrual cycle is divided into follicular and luteal phases and is regulated by hormones [4]. Estrogen and progesterone levels are lower in the early period of the follicular phase, but start to increase toward the end of the follicular phase, peaking during the surge of luteinizing hormone and follicle-stimulating hormone. In the early period of luteal phase and during the follicular phase, progesterone levels are very low, but start to increase in advancing of luteal phase [4]. Serum ADMA levels undergo small changes during the menstrual cycle. Levels are higher in the follicular phase of the cycle than in the luteal phase. These findings are well-correlated with flow-mediated dilatation, which indicates that endothelial dysfunction is highest in the follicular phase. Furthermore, oral contraceptive therapy reduces changes in serum ADMA levels in the normal menstrual cycle. Also, serum ADMA levels are significantly decreased by oral contraceptives in women with polycystic ovary syndrome. Orally administered hormone replacement therapy also has an effect on circulating ADMA levels [5]. Estrogen-containing pills decrease serum ADMA concentrations, whereas progestin-only pills do not, as compared with controls. Estrogen has a positive effect on endothelial functions, inducing arterial vasodilation by activating endothelial nitric oxide synthase. Estradiol may facilitate nitric oxide synthesis by reducing circulating levels of ADMA, which may then exert protective effects on the vasculature [6e8]. AMH is specifically produced by late preantral and small antral follicles. AMH controls folliculogenesis by restricting primordial follicle development, inhibiting the sensitivity of larger antral follicles to follicle-stimulating hormone and aromatase during ovulatory cycles. Serum AMH levels are generally accepted as a marker for prediction of the ovarian reserve. Women in the youngest age group, 18e30 years, show the highest levels of AMH, but then experience an agerelated decrease associated with ovarian reserve and levels of the ovarian follicular pool [9e14]. AMH is also a marker for prediction of in vitro fertilization success rates in terms of ovarian response to ovarian stimulation [15e17]. Factors such as ethnicity, body mass index (BMI), smoking, and physical activity affect serum AMH levels as well as serum ADMA levels [9]. The definite pathogenesis of PD remains unclear, but studies have shown that prostaglandins and vasoactive mediators are increased in the endometrium and menstrual specimens [18]. Recent studies have also shown that some hormonal or vascular endothelial functional changes, such as increased vasopressin and serum ADMA levels, cause vasoconstriction, uterine contractions, and eventually uterine ischemia related to the menstrual pain in PD. PD
415 also affects other systems and may lead to an increase in the risk of cardiac arrhythmia or mental status change during the menstrual period [4,18]. Serum AMH levels change during the menstrual cycle as well as serum ADMA levels, but peak concentrations of serum AMH occur in the late follicular phase w4e5 days before ovulation. In general, variations in serum AMH levels occur during the follicular and luteal phases. Serum AMH levels are used to detect the ovarian follicular pool and ovarian reserve in women. This is a useful technique for the prediction of quantitative and qualitative aspects and success estimation in assisted reproductive technologies [15]. Besides the common factors effecting ADMA/AMH and estrogen related common pathway mentioned above, our main hypothesis is the possible presence of a close relationship between serum AMH and ADMA levels in PD patients. A more potent ovarian reserve may lead to more prominent serum ADMA elevation. Thus, the objective of our study was to investigate the association of serum ADMA and AMH levels in PD patients.
Materials and methods Study group This is a substudy of a previous scientific project evaluating the pathophysiology and clinical aspects of primary dysmenorrhea in young women. The study cohort was selected among the faculty medical student population from January 2015 to October 2015. Eighty-nine female university students with primary dysmenorrhea were included in this study. The investigation conforms to the principles outlined in the Declaration of Helsinki and the Ethical Committee approved the study. Informed consent was obtained, and clinical and demographic data were collected from each participant.
Patients A detailed medical history was taken. Each participant underwent a complete gynecologic examination, including pelvic ultrasound; systemic physical examination, including the cardiac and cardiovascular systems; and laboratory tests such as biochemistry tests, electrocardiography, and echocardiography. Participants with any gynecological disease, rheumatic disorder, inflammatory bowel disease, fibromyalgia, known malignancy, known premature coronary artery disease, congenital cardiac disease, such as family history of premature coronary artery disease, diabetes mellitus, hypertension, any endocrine disease such as diabetes mellitus or thyroid disorders, endometriosis, ovarian cysts, and participants taking any oral contraceptive drugs were excluded. The participants’ main complaint was dysmenorrhea. Pain was mostly located in lower abdominal and pelvic area. Pain intensity measured by the Visual Analog Scale (VAS) was significantly correlated with scores measured by verbal and numeric scales. The VAS is a tool commonly used in research and clinical practice, and its reliability and validity in pain assessment have been clearly demonstrated [19]. The VAS score is derived from a 10-cm scale, with end points of 0 for “no pain” and 10 for “worst
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pain”. The use of the scale was clearly explained to all participants. Patients were asked to make a mark on the line representing their pain intensity. The distance from the “no pain” end to the patient’s mark was used to represent pain intensity level [19]. Grading from 1e4 was accepted as mild; 5e7 was accepted as moderate; and 8e10 was accepted as severe pain. The associations of ADMA and AMH levels in different pain groups were analyzed.
Laboratory analyses Blood samples were taken from each participant in the morning after 12 hours fasting on the 3rd day of menses. After clotting and centrifugation for 5 minutes at 3.00 g, serum for AMH and ADMA measurement was separated and stored at 80 C until the time of analysis. Blood glucose, serum creatinine, blood urea nitrogen, lactate dehydrogenase, alkaline phosphatase, aspartate and alanine aminotransferases, gamma glutamyl trans peptidase, and bilirubin were measured via an ARCHITECT c16000 auto analyzer (Abbott Laboratories, Abbott Park, Illinois, USA). ADMA and AMH levels were measured using commercial enzyme-linked immunosorbent assay kits (Shanghai Yehua Biological Technology, Shanghai, China) according to the manufacturer’s instructions. Intra-assay variability coefficients of ADMA and AMH ELISA assays were 9.2% and 10.6%, respectively. ADMA and AMH levels were expressed as micrograms per liter (mg/L) and nanogram per milliliter (ng/mL) in serum samples, respectively.
Statistics Data were analyzed via statistical software SPSS, version 10.0 [SPSS Inc, Chicago, IL, USA]. The distribution characteristics of continuous data were determined using
Table 1 Clinical, demographic, laboratory characteristics of patients with primary dysmenorrhea (n Z 89). Characteristics Age BMI (kg/m2) Smoking Alcohol Physical activity intensity
Age at menarche (y) Pain duration (day) Visual analogue scale (VAS) pain scores Asymmetric dimethylarginine (ADMA) Anti-Mu ¨llerian hormone (AMH) (VAS) Pain category VAS Cat 1e4 VAS Cat 5e7 VAS Cat 8e10
histogram examination and the one-sample KolmogoroveSmirnov test. Normally distributed data were presented as mean (standard deviation) and compared with one-way analysis of variance (ANOVA). Further analyses were performed using Tukey’s honestly significant difference test as needed. Pearson’s correlation and linear regression analysis were used to evaluate associations among continuous data. Categorical associations were evaluated via a c2 test. Data binning was applied in the case of embryo number. This process is a data preprocessing technique used to reduce the effects of minor observation errors. The statistical program placed the results into harmonic groups with optimal binning thresholds. Statistical significance was defined as p 0.05.
Results There were 89 women with primary dysmenorrhea in the study. Mean (standard deviation; SD) age was 21 (2.2). Mean (SD) BMI was 22.4 (1.9). Other demographic and clinical data, as well as serum ADMA and AMH levels of the study group, are presented in Table 1. After patients were grouped according to VAS categories, serum ADMA and AMH levels were compared between groups. ANOVA univariate analysis revealed there was significant difference in ADMA levels between groups (p Z 0.008). Further analysis was
Table 2 ADMA and AMH levels according to VAS categories. VAS category
n
Serum ADMA, mgyL mean (SD)
Serum AMH, ngymL mean (SD)
1e4 (Mild) 5e7 (Moderate) 8e10 (Severe)
24 39 26
35.2 (24) 54.0 (28.7) 56.5 (32.8)
2.9 (1.2) 6.2 (0.8) 8.4 (0.7)
ADMA Z asymmetric dimethylarginine; AMH Z anti-Mu ¨llerian hormone; VAS Z visual analogue scale.
Mean (SD), % 21.08 2.21 22.4 1.9 8 (8.9) 5 (5.6) Lowemoderate: 65 (73), Intense: 26 (27) 13.2 1.3 1 0.76 5.9101 2.18 49.6594 29.63 9.8762 5.50 Frequency 24 (26.8) 39 (43.8) 26 (29.2)
(%)
BMI Z body mass index; Cat Z category; SD Z standard deviation.
Figure 1. ADMA levels according to VAS groups. ADMA Z asymmetric dimethylarginine; VAS Z visual analogue scale.
Serum ADMA and AMH levels in dysmenorrhea performed using Tukey’s test, showing that the VAS 5e7 and VAS 8e10 groups had significantly higher serum ADMA levels compared with VAS 1e4 group (p Z 0.018 and 0.012, respectively), whereas there were no significant differences in mean serum ADMA levels between VAS 5e7 and VAS 8e10 groups (p Z 0.845). Similarly, there was a significant difference in serum AMH between VAS groups (p Z 0.010). Further analysis via Tukey’s test revealed that serum AMH levels were significantly higher in VAS 5e7 and VAS 8e10 groups compared with VAS 1e4 group (p Z 0.015 and 0.024, respectively), whereas there were no significant
417 differences in mean serum ADMA levels between the VAS 5e7 and VAS 8e10 groups (p Z 0.984) (Table 2, Figures 1 and 2). Correlation analysis between serum ADMA and AMH levels in the whole study group showed a significant positive relationship (Pearson correlation Z 0.978, p Z 0.01) (Figure 3). Thus, we proceeded with linear regression analysis. The regression model included only serum ADMA and AMH. It revealed that ADMA was a significant independent predictor of serum AMH (Table 3). Assumptions of linear regression were met and adjusted R2 was 95.6%.
Discussion
Figure 2. AMH levels in different VAS groups. AMH Z antiMu ¨llerian hormone; VAS Z visual analogue scale.
This study has demonstrated a highly significant positive correlation between serum ADMA and AMH levels in the PD patient group. We graded dysmenorrhea pain as mild, moderate, or severe and evaluated the association of serum ADMA and AMH levels. In comparisons among pain groups, serum ADMA and AMH levels significantly differed between the severe and mild pain intensity groups. However, there was no significant difference between moderate to severe pain groups in serum ADMA and AMH levels. Thus, we also found that more severe pain was associated with higher serum ADMA and AMH levels. This study has demonstrated the association of serum ADMA and AMH levels for the first time in the literature. In a normal menstrual period, changes in serum ADMA and AMH levels, although minimal, are similar due to menstrual physiology. This may explain the highly significant positive correlation between serum ADMA and AMH levels in our study [10e16].
Figure 3. Scatterplot depicting correlation between serum ADMA and AMH. ADMA Z asymmetric dimethylarginine; AMH Z antiMu ¨llerian hormone.
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Table 3 Linear regression analysis of AMH where ADMA is the independent predictor. Model Constant ADMA
B
Standard Error
Beta
t
p
0.858 0.182
0.255 0.004
d 0.978
3.37 41.181
0.001 <0.001
ADMA Z asymmetric dimethylarginine; AMH Z anti-Mu ¨llerian hormone; B Z unstandardized coefficient; t Z statistic.
biochemical research are needed to clarify the association of serum ADMA and AMH levels in an unselected population. There is a significant positive correlation between serum ADMA and AMH levels in PD. These results may be explained by two factors: (1) both share similar changes during the cycle; and (2) they share similar genetic signaling pathways. Serum ADMA levels have the potential to be used to evaluate ovarian reserve.
Acknowledgments In a mouse model, increased serum ADMA levels were found to be positively correlated with bone morphogenic protein receptor-2 gene mutation. This was also seen in the pathogenesis of a primary pulmonary hypertension study, which concluded that changes in bone morphogenetic protein signaling and enzymes involved in arginine methylation may contribute to explaining the pathophysiological significance of elevated ADMA levels [20]. Three AMH receptors have been described in studies into the genetic relations of AMH. These Type I receptors, ALK2, ALK3, and ALK 6, are shared with bone morphogenetic proteins (BMPs). Subsequently, similar to BMPs, AMH signaling is mediated through the downstream signaling molecules Smad1, Smad5, and Smad8. However, the relative contributions of these three Type I receptors to AMH signaling in the ovary remain undetermined [20e23]. As we discussed above, both ADMA and AMH use similar signal pathways and the same signaling system. Therefore, increased serum ADMA levels may trigger the excess synthesis of AMH or vice versa. Serum ADMA levels may be said to show ovarian reserve as well as serum AMH levels, whereas serum AMH levels may be used to evaluate vascular endothelial function [20e22]. Gene expressions in endothelial cells are triggered by very small changes in the concentration of ADMA. The identification of pathways regulated by ADMA may aid our understanding of how ADMA contributes to a wide range of pathologies. Two pathways of specific interest have been identified: BMP signaling and enzymes involved in arginine methylation. The effects on BMP signaling may be particularly important in renal disease, as well as in the link between raised ADMA and pulmonary hypertension. Serum AMH levels may also be increased in those diseases, as in our study of the PD patient group [20]. Finally, it should be noted that there are conflicting data in the literature regarding chronic pelvic pain and serum AMH levels. For example, contrary to our study, severe menstrual pain was associated with lower AMH among Japanese women aged 20 to 22 years. This may be explained by inclusion of a high percentage of patients with endometriosis in that study, whereas endometriosis causes chronic pelvic pain similar to PD, AMH levels have been shown to be lower [10].
Limitations Those results may not represent the population at large, as our study group was chosen from among PD patients. The absence of a control group, absence of follow-up data, single blood sampling, and small sample size are limitations of this study. Further studies incorporating genetic and
This study was supported by Sakarya University (2012-1100-002) with Ethics approval number 21. Date: February 12, 2014.
References [1] Akdemir N, Cinemre H, Bilir C, Akin O, Akdemir R. Increased serum asymmetric dimethylarginine levels in primary dysmenorrhea. Gynecol Obstet Invest 2010;69:153e6. [2] Jensen MP, Karoly P, Braver S. The measurement of clinical pain intensity: a comparison of six methods. Pain 1986;27: 17e26. [3] Jones KR, Fink R, Hutt E, Vojir C, Pepper GA, ScottCawiezell J, et al. Measuring pain intensity in nursing home residents. J Pain Symptom Manage 2005;30:519e27. [4] Valtonen P, Punnonen K, Saarelainen H, Heiskanen N, Raitakari OT, Juonala M, et al. ADMA concentration changes across the menstrual cycle and during oral contraceptive use: the cardiovascular risk in Young Finns Study. Eur J Endocrinol 2010;162:259e65. [5] Charitidou C, Farmakiotis D, Zournatzi V, Pidonia I, Pegiou T, Karamanis N, et al. The administration of estrogens, combined with antiandrogens, has beneficial effects on the hormonal features and asymmetric dimethyl-arginine levels, in women with the polycystic ovary syndrome. Atherosclerosis 2008;196: 958e65. [6] Holden DP, Fickling SA, Whitley GS, Nussey SS. Plasma concentrations of asymmetric dimethylarginine, a natural inhibitor of nitric oxide synthase, in normal pregnancy and preeclampsia. Am J Obstet Gynecol 1998;178:551e6. [7] Verhoeven MO, Hemelaar M, van der Mooren MJ, Kenemans P, Teerlink T. Oral, more than transdermal, oestrogen therapy lowers asymmetric dimethylarginine in healthy ostmenopausal women: a randomized, placebo-controlled study. J Intern Med 2006;259:199e208. [8] Cevik D, Unay O, Durmusoglu F, Yurdun T, Bilsel AS. Plasma markers of NO synthase activity in women after ovarian hyperstimulation: influence of estradiol on ADMA. Vasc Med 2006;11:7e12. [9] Hampl R, Snajderova ´ M, Mardesic T. Antimu ¨llerian hormone (AMH) not only a marker for prediction of ovarian reserve. Physiol Res 2011;60:217e23. [10] Konishi S, Nishihama Y, Iida A, Yoshinaga J, Imai H. Association of antimu ¨llerian hormone levels with menstrual-cycle type and dysmenorrhea in young asymptomatic women. Fertil Steril 2014;102:1439e43. [11] Lambert-Messerlian G, Plante B, Eklund EE, Raker C, Moore RG. Levels of antimu ¨llerian hormone in serum during the normal menstrual cycle. Fertil Steril 2016;105:208e13. [12] Kissell KA, Danaher MR, Schisterman EF, Wactawski-Wende J, Ahrens KA, Schliep K, et al. Biological variability in serum antiMu ¨llerian hormone throughout the menstrual cycle in ovulatory and sporadic anovulatory cycles in eumenorrheic women. Hum Reprod 2014;29:1764e72.
Serum ADMA and AMH levels in dysmenorrhea [13] Souza AH, Carvalho PD, Rozner AE, Vieira LM, Hackbart KS, Bender RW, et al. Relationship between circulating antiMu ¨llerian hormone (AMH) and superovulatory response of high-producing dairy cows. J Dairy Sci 2015;98:169e78. [14] Merhi Z, Zapantis A, Berger DS, Jindal SK. Determining an antiMullerian hormone cutoff level to predict clinical pregnancy following in vitro fertilization in women with severely diminished ovarian reserve. J Assist Reprod Genet 2013;30:1361e5. [15] La Marca A, Sighinolfi G, Radi D, Argento C, Baraldi E, Artenisio AC, et al. Anti-Mullerian hormone (AMH) as a predictive marker in assisted reproductive technology (ART). Hum Reprod Update 2010;16:113e30. [16] Gnoth C, Roos J, Broomhead D, Schiffner J, Godehardt E, Freundl G, et al. Antimu ¨llerian hormone levels and numbers and sizes of antral follicles in regularly menstruating women of reproductive age referenced to true ovulation day. Fertil Steril 2015;104:1535e43. ¨, C [17] Keskin U, Go ¸ akir E, Ceyhan ST, Ercan CM, Baser I. ¨ktolga U Asymmetrical dimethylarginine levels on the implantation success of in vitro fertilization and embryo transfer. Gynecol Endocrinol 2012;28:805e8.
419 [18] Iacovides S, Avidon I, Baker FC. What we know about primary dysmenorrhea today: a critical review. Hum Reprod Update 2015;21:762e78. [19] Paice J, Cohen F. Validity of a verbally administered numeric rating scale to measure cancer pain intensity. Cancer Nurs 1997;20:88e93. [20] Jeppesen JV, Anderson RA, Kelsey TW, Christiansen SL, Kristensen SG, Jayaprakasan K, et al. Which follicles make the most anti-Mullerian hormone in humans? Evidence for an abrupt decline in AMH production at the time of follicle selection. Mol Hum Reprod 2013;19:519e27. [21] Smith CL, Anthony S, Hubank M, Leiper JM, Vallance P. Effects of ADMA upon gene expression: an insight into the pathophysiological significance of raised plasma ADMA. PLoS Med 2005;2:e264. [22] Visser JA. AMH signaling: from receptor to target gene. Mol Cell Endocrinol 2003;211:65e73. [23] Gruijters MJ, Visser JA, Durlinger AL, Themmen AP. AntiMu ¨llerian hormone and its role in ovarian function. Mol Cell Endocrinol 2003;211:85e90.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.kjms-online.com
ORIGINAL ARTICLE
The correlation of serum asymmetric ¨llerian dimethylarginine and anti-Mu hormone in primary dysmenorrhea ¨hha Bostancı a, Nermin Akdemir a,*, Fatma Behice Cinemre b, Mehmet Su ¨ nal a, Selc ¸uk Ozden a, Arif Serhan Cevrioglu a, Hakan Cinemre c, Orhan U d Zubeyde Kacal , Ramazan Akdemir e a
Department of Obstetrics and Gynecology, Sakarya University, Faculty of Medicine, Sakarya, Turkey b Department of Biochemistry, Sakarya University, Faculty of Medicine, Sakarya, Turkey c Department of Internal Medicine, Sakarya University, Faculty of Medicine, Sakarya, Turkey d Department of Medical Education and Ethics, Sakarya University, Faculty of Medicine, Sakarya, Turkey e Department of Cardiology, Sakarya, Sakarya University, Faculty of Medicine, Sakarya, Turkey Received 8 March 2016; accepted 4 July 2016
Available online 27 July 2016
KEYWORDS Anti-Mu ¨llerian hormone; Asymmetric dimethyl arginine; Ovarian reserve; Primary dysmenorrhea
Abstract In this study, we aimed to investigate the association of serum asymmetric dimethylarginine (ADMA) and anti-Mu ¨llerian hormone (AMH) levels in primary dysmenorrhea patients. The study employed a cross-sectional design. Eighty-nine female university students with primary dysmenorrhea were included in the study. All patients underwent complete clinical and laboratory investigations, including serum ADMA, AMH levels, pelvic ultrasonography, electrocardiography, and echocardiography. Pearson correlation and linear regression analysis were used to evaluate associations between continuous data. Categorical associations were evaluated using c2 test. Correlation analysis between serum ADMA and AMH levels in the study group showed a highly significant positive relationship (Pearson correlation Z 0.978, p Z 0.01). Our study has shown a significant positive correlation between serum ADMA and AMH levels in primary dysmenorrhea. Serum ADMA levels may have the potential to demonstrate ovarian reserve. Copyright ª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Conflicts of interest: All authors declare no conflicts of interests. * Corresponding author. Department of Obstetrics and Gynecology, Sakarya University, Faculty of Medicine, 54290, Sakarya, Turkey. E-mail address: [email protected] (N. Akdemir). http://dx.doi.org/10.1016/j.kjms.2016.07.001 1607-551X/Copyright ª 2016, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Serum ADMA and AMH levels in dysmenorrhea
Introduction Asymmetric dimethylarginine (ADMA) is an important marker in a variety of clinical settings, playing a role in endothelial dysfunction. It is associated with the pathogenesis of atherosclerosis, hypertension, and diabetic vascular complication, as well as primary dysmenorrhea (PD), polycystic ovary syndrome, and pregnancy-related disorders such as preeclampsia and hyperemesis gravidarum [1e3]. Changes in serum ADMA levels have also been investigated during normal menstrual periods. The menstrual cycle is divided into follicular and luteal phases and is regulated by hormones [4]. Estrogen and progesterone levels are lower in the early period of the follicular phase, but start to increase toward the end of the follicular phase, peaking during the surge of luteinizing hormone and follicle-stimulating hormone. In the early period of luteal phase and during the follicular phase, progesterone levels are very low, but start to increase in advancing of luteal phase [4]. Serum ADMA levels undergo small changes during the menstrual cycle. Levels are higher in the follicular phase of the cycle than in the luteal phase. These findings are well-correlated with flow-mediated dilatation, which indicates that endothelial dysfunction is highest in the follicular phase. Furthermore, oral contraceptive therapy reduces changes in serum ADMA levels in the normal menstrual cycle. Also, serum ADMA levels are significantly decreased by oral contraceptives in women with polycystic ovary syndrome. Orally administered hormone replacement therapy also has an effect on circulating ADMA levels [5]. Estrogen-containing pills decrease serum ADMA concentrations, whereas progestin-only pills do not, as compared with controls. Estrogen has a positive effect on endothelial functions, inducing arterial vasodilation by activating endothelial nitric oxide synthase. Estradiol may facilitate nitric oxide synthesis by reducing circulating levels of ADMA, which may then exert protective effects on the vasculature [6e8]. AMH is specifically produced by late preantral and small antral follicles. AMH controls folliculogenesis by restricting primordial follicle development, inhibiting the sensitivity of larger antral follicles to follicle-stimulating hormone and aromatase during ovulatory cycles. Serum AMH levels are generally accepted as a marker for prediction of the ovarian reserve. Women in the youngest age group, 18e30 years, show the highest levels of AMH, but then experience an agerelated decrease associated with ovarian reserve and levels of the ovarian follicular pool [9e14]. AMH is also a marker for prediction of in vitro fertilization success rates in terms of ovarian response to ovarian stimulation [15e17]. Factors such as ethnicity, body mass index (BMI), smoking, and physical activity affect serum AMH levels as well as serum ADMA levels [9]. The definite pathogenesis of PD remains unclear, but studies have shown that prostaglandins and vasoactive mediators are increased in the endometrium and menstrual specimens [18]. Recent studies have also shown that some hormonal or vascular endothelial functional changes, such as increased vasopressin and serum ADMA levels, cause vasoconstriction, uterine contractions, and eventually uterine ischemia related to the menstrual pain in PD. PD
415 also affects other systems and may lead to an increase in the risk of cardiac arrhythmia or mental status change during the menstrual period [4,18]. Serum AMH levels change during the menstrual cycle as well as serum ADMA levels, but peak concentrations of serum AMH occur in the late follicular phase w4e5 days before ovulation. In general, variations in serum AMH levels occur during the follicular and luteal phases. Serum AMH levels are used to detect the ovarian follicular pool and ovarian reserve in women. This is a useful technique for the prediction of quantitative and qualitative aspects and success estimation in assisted reproductive technologies [15]. Besides the common factors effecting ADMA/AMH and estrogen related common pathway mentioned above, our main hypothesis is the possible presence of a close relationship between serum AMH and ADMA levels in PD patients. A more potent ovarian reserve may lead to more prominent serum ADMA elevation. Thus, the objective of our study was to investigate the association of serum ADMA and AMH levels in PD patients.
Materials and methods Study group This is a substudy of a previous scientific project evaluating the pathophysiology and clinical aspects of primary dysmenorrhea in young women. The study cohort was selected among the faculty medical student population from January 2015 to October 2015. Eighty-nine female university students with primary dysmenorrhea were included in this study. The investigation conforms to the principles outlined in the Declaration of Helsinki and the Ethical Committee approved the study. Informed consent was obtained, and clinical and demographic data were collected from each participant.
Patients A detailed medical history was taken. Each participant underwent a complete gynecologic examination, including pelvic ultrasound; systemic physical examination, including the cardiac and cardiovascular systems; and laboratory tests such as biochemistry tests, electrocardiography, and echocardiography. Participants with any gynecological disease, rheumatic disorder, inflammatory bowel disease, fibromyalgia, known malignancy, known premature coronary artery disease, congenital cardiac disease, such as family history of premature coronary artery disease, diabetes mellitus, hypertension, any endocrine disease such as diabetes mellitus or thyroid disorders, endometriosis, ovarian cysts, and participants taking any oral contraceptive drugs were excluded. The participants’ main complaint was dysmenorrhea. Pain was mostly located in lower abdominal and pelvic area. Pain intensity measured by the Visual Analog Scale (VAS) was significantly correlated with scores measured by verbal and numeric scales. The VAS is a tool commonly used in research and clinical practice, and its reliability and validity in pain assessment have been clearly demonstrated [19]. The VAS score is derived from a 10-cm scale, with end points of 0 for “no pain” and 10 for “worst
416
N. Akdemir et al.
pain”. The use of the scale was clearly explained to all participants. Patients were asked to make a mark on the line representing their pain intensity. The distance from the “no pain” end to the patient’s mark was used to represent pain intensity level [19]. Grading from 1e4 was accepted as mild; 5e7 was accepted as moderate; and 8e10 was accepted as severe pain. The associations of ADMA and AMH levels in different pain groups were analyzed.
Laboratory analyses Blood samples were taken from each participant in the morning after 12 hours fasting on the 3rd day of menses. After clotting and centrifugation for 5 minutes at 3.00 g, serum for AMH and ADMA measurement was separated and stored at 80 C until the time of analysis. Blood glucose, serum creatinine, blood urea nitrogen, lactate dehydrogenase, alkaline phosphatase, aspartate and alanine aminotransferases, gamma glutamyl trans peptidase, and bilirubin were measured via an ARCHITECT c16000 auto analyzer (Abbott Laboratories, Abbott Park, Illinois, USA). ADMA and AMH levels were measured using commercial enzyme-linked immunosorbent assay kits (Shanghai Yehua Biological Technology, Shanghai, China) according to the manufacturer’s instructions. Intra-assay variability coefficients of ADMA and AMH ELISA assays were 9.2% and 10.6%, respectively. ADMA and AMH levels were expressed as micrograms per liter (mg/L) and nanogram per milliliter (ng/mL) in serum samples, respectively.
Statistics Data were analyzed via statistical software SPSS, version 10.0 [SPSS Inc, Chicago, IL, USA]. The distribution characteristics of continuous data were determined using
Table 1 Clinical, demographic, laboratory characteristics of patients with primary dysmenorrhea (n Z 89). Characteristics Age BMI (kg/m2) Smoking Alcohol Physical activity intensity
Age at menarche (y) Pain duration (day) Visual analogue scale (VAS) pain scores Asymmetric dimethylarginine (ADMA) Anti-Mu ¨llerian hormone (AMH) (VAS) Pain category VAS Cat 1e4 VAS Cat 5e7 VAS Cat 8e10
histogram examination and the one-sample KolmogoroveSmirnov test. Normally distributed data were presented as mean (standard deviation) and compared with one-way analysis of variance (ANOVA). Further analyses were performed using Tukey’s honestly significant difference test as needed. Pearson’s correlation and linear regression analysis were used to evaluate associations among continuous data. Categorical associations were evaluated via a c2 test. Data binning was applied in the case of embryo number. This process is a data preprocessing technique used to reduce the effects of minor observation errors. The statistical program placed the results into harmonic groups with optimal binning thresholds. Statistical significance was defined as p 0.05.
Results There were 89 women with primary dysmenorrhea in the study. Mean (standard deviation; SD) age was 21 (2.2). Mean (SD) BMI was 22.4 (1.9). Other demographic and clinical data, as well as serum ADMA and AMH levels of the study group, are presented in Table 1. After patients were grouped according to VAS categories, serum ADMA and AMH levels were compared between groups. ANOVA univariate analysis revealed there was significant difference in ADMA levels between groups (p Z 0.008). Further analysis was
Table 2 ADMA and AMH levels according to VAS categories. VAS category
n
Serum ADMA, mgyL mean (SD)
Serum AMH, ngymL mean (SD)
1e4 (Mild) 5e7 (Moderate) 8e10 (Severe)
24 39 26
35.2 (24) 54.0 (28.7) 56.5 (32.8)
2.9 (1.2) 6.2 (0.8) 8.4 (0.7)
ADMA Z asymmetric dimethylarginine; AMH Z anti-Mu ¨llerian hormone; VAS Z visual analogue scale.
Mean (SD), % 21.08 2.21 22.4 1.9 8 (8.9) 5 (5.6) Lowemoderate: 65 (73), Intense: 26 (27) 13.2 1.3 1 0.76 5.9101 2.18 49.6594 29.63 9.8762 5.50 Frequency 24 (26.8) 39 (43.8) 26 (29.2)
(%)
BMI Z body mass index; Cat Z category; SD Z standard deviation.
Figure 1. ADMA levels according to VAS groups. ADMA Z asymmetric dimethylarginine; VAS Z visual analogue scale.
Serum ADMA and AMH levels in dysmenorrhea performed using Tukey’s test, showing that the VAS 5e7 and VAS 8e10 groups had significantly higher serum ADMA levels compared with VAS 1e4 group (p Z 0.018 and 0.012, respectively), whereas there were no significant differences in mean serum ADMA levels between VAS 5e7 and VAS 8e10 groups (p Z 0.845). Similarly, there was a significant difference in serum AMH between VAS groups (p Z 0.010). Further analysis via Tukey’s test revealed that serum AMH levels were significantly higher in VAS 5e7 and VAS 8e10 groups compared with VAS 1e4 group (p Z 0.015 and 0.024, respectively), whereas there were no significant
417 differences in mean serum ADMA levels between the VAS 5e7 and VAS 8e10 groups (p Z 0.984) (Table 2, Figures 1 and 2). Correlation analysis between serum ADMA and AMH levels in the whole study group showed a significant positive relationship (Pearson correlation Z 0.978, p Z 0.01) (Figure 3). Thus, we proceeded with linear regression analysis. The regression model included only serum ADMA and AMH. It revealed that ADMA was a significant independent predictor of serum AMH (Table 3). Assumptions of linear regression were met and adjusted R2 was 95.6%.
Discussion
Figure 2. AMH levels in different VAS groups. AMH Z antiMu ¨llerian hormone; VAS Z visual analogue scale.
This study has demonstrated a highly significant positive correlation between serum ADMA and AMH levels in the PD patient group. We graded dysmenorrhea pain as mild, moderate, or severe and evaluated the association of serum ADMA and AMH levels. In comparisons among pain groups, serum ADMA and AMH levels significantly differed between the severe and mild pain intensity groups. However, there was no significant difference between moderate to severe pain groups in serum ADMA and AMH levels. Thus, we also found that more severe pain was associated with higher serum ADMA and AMH levels. This study has demonstrated the association of serum ADMA and AMH levels for the first time in the literature. In a normal menstrual period, changes in serum ADMA and AMH levels, although minimal, are similar due to menstrual physiology. This may explain the highly significant positive correlation between serum ADMA and AMH levels in our study [10e16].
Figure 3. Scatterplot depicting correlation between serum ADMA and AMH. ADMA Z asymmetric dimethylarginine; AMH Z antiMu ¨llerian hormone.
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Table 3 Linear regression analysis of AMH where ADMA is the independent predictor. Model Constant ADMA
B
Standard Error
Beta
t
p
0.858 0.182
0.255 0.004
d 0.978
3.37 41.181
0.001 <0.001
ADMA Z asymmetric dimethylarginine; AMH Z anti-Mu ¨llerian hormone; B Z unstandardized coefficient; t Z statistic.
biochemical research are needed to clarify the association of serum ADMA and AMH levels in an unselected population. There is a significant positive correlation between serum ADMA and AMH levels in PD. These results may be explained by two factors: (1) both share similar changes during the cycle; and (2) they share similar genetic signaling pathways. Serum ADMA levels have the potential to be used to evaluate ovarian reserve.
Acknowledgments In a mouse model, increased serum ADMA levels were found to be positively correlated with bone morphogenic protein receptor-2 gene mutation. This was also seen in the pathogenesis of a primary pulmonary hypertension study, which concluded that changes in bone morphogenetic protein signaling and enzymes involved in arginine methylation may contribute to explaining the pathophysiological significance of elevated ADMA levels [20]. Three AMH receptors have been described in studies into the genetic relations of AMH. These Type I receptors, ALK2, ALK3, and ALK 6, are shared with bone morphogenetic proteins (BMPs). Subsequently, similar to BMPs, AMH signaling is mediated through the downstream signaling molecules Smad1, Smad5, and Smad8. However, the relative contributions of these three Type I receptors to AMH signaling in the ovary remain undetermined [20e23]. As we discussed above, both ADMA and AMH use similar signal pathways and the same signaling system. Therefore, increased serum ADMA levels may trigger the excess synthesis of AMH or vice versa. Serum ADMA levels may be said to show ovarian reserve as well as serum AMH levels, whereas serum AMH levels may be used to evaluate vascular endothelial function [20e22]. Gene expressions in endothelial cells are triggered by very small changes in the concentration of ADMA. The identification of pathways regulated by ADMA may aid our understanding of how ADMA contributes to a wide range of pathologies. Two pathways of specific interest have been identified: BMP signaling and enzymes involved in arginine methylation. The effects on BMP signaling may be particularly important in renal disease, as well as in the link between raised ADMA and pulmonary hypertension. Serum AMH levels may also be increased in those diseases, as in our study of the PD patient group [20]. Finally, it should be noted that there are conflicting data in the literature regarding chronic pelvic pain and serum AMH levels. For example, contrary to our study, severe menstrual pain was associated with lower AMH among Japanese women aged 20 to 22 years. This may be explained by inclusion of a high percentage of patients with endometriosis in that study, whereas endometriosis causes chronic pelvic pain similar to PD, AMH levels have been shown to be lower [10].
Limitations Those results may not represent the population at large, as our study group was chosen from among PD patients. The absence of a control group, absence of follow-up data, single blood sampling, and small sample size are limitations of this study. Further studies incorporating genetic and
This study was supported by Sakarya University (2012-1100-002) with Ethics approval number 21. Date: February 12, 2014.
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