Research Article - Biomedical Research (2017) Volume 28, Issue 5
Anti-Mullerian hormone as an examination for ovulatory dysfunction of femaleLiu Honglian and Hong Li*
Department of Obstetrics and Gynecology, Renmin Hospital of Wuhan University, Wuhan, PR China
- *Corresponding Author:
- Hong Li
Department of Obstetrics and Gynecology
Renmin Hospital of Wuhan University
Wuhan, PR China
Accepted date: October 31, 2016
Objective: To investigate the relationship between anti-mullerian hormone (AMH) and ovulatory dysfunction.
Methods: The enzyme-linked immunosorbent assay (ELISA) was used to detect the serum level of AMH, the estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), the progestin (P), and the testosterone (T) in 106 participates with infertility (observation group)and carried on comparing with 102 women with pre-pregnancy physical examination (control group)Data were statistically analyzed.
Results: The serum hormone levels of AMH and LH in the observation group were higher than those in the control group (P<0.05). The FSH level of observation group was obviously lower than that in the control group (P<0.05). Pearson correlation analysis showed positive relationship between serum AMH and LH (P<0.05) as well as T (P<0.05). Serum AMH level in the observation group was negatively collated with FSH (P<0.05). No significant relationships were found between serum AMH and E2 level.
Conclusion: This study suggests that serum AMH level was associated with abnormal follicular development and would an effective measurement for ovulatory dysfunction of sterile female.
Infertility, Anti-mullerian hormone(AMH), Ovulatory dysfunction, Anovulation
As the higher incidence of infertility found in Chinese couples, there are more incidence of ovulatory dysfunction were found in women under reproductive age [1,2]. The diagnosis of clinical infertility should be examined by physicians in a range of medical specialties, including family medicine, paediatrics . Thus one reliable, validated diagnostic examination is desired for the worldwide.
Anti-Mullerian Hormone (AMH, also named Müllerian inhibiting substance, MIS), a glycoprotein produced in the granulosa cells of the ovary , regulates early follicular recruitment, secreted by preantral follicle and small antral follicle, is known as the most precise predictor of ovarian reserve in women . It is produced by the granulosa cells of small antral and preantral follicles and reflects the size of the pool of these follicles . AMH declines with age but remains stable during the menstrual cycle allowing its determination at random [4,6].
The purpose of this study was to obtain AMH levels in infertility females to categorize them as ovulation after treatment of ovulation stimulants . Through identification of treatment variables that increase the probability of ovulation, ovarian function in infertility females can be monitored more closely and if necessary prompt early referral, even in adolescence, to reproductive specialists. These patients might benefit from detailed discussions regarding reproductive potential and perhaps interventions for fertility preservation [8,9].
Materials and Methods
All subjects were obtained between December 2013 and December 2015 from the department of obstetrics and gynecology, Renmin Hospital of Wuhan University (Wuhan, China). The study obtained all informed written consent from all subjects and the study was approved by Ethics Committee of the Renmin Hospital of Wuhan University.
Infertile women with ovulatory dysfunction were recruited as the observation group, aged 22-40 years, inclusive with two patent fallopian tubes, two functional ovaries and a body mass index <37.0 kg/m2. The patient’s usual cycle length had to be either <21 or >35 days with six or more menses per year or >35 days with fewer than six menses per year and a positive response to a progestin challenge within the last 6 months. The subjects received 2 years’ follow-up. Women with normal ovulation who taking pre-pregnancy physical examination for pregnancy preparation as control group, aged 22-40 years, inclusive with two patent fallopian tubes, two functional ovaries and a body mass index <37.0 kg/m2. The patient’s usual cycle length between 21 and 35 days with six or more menses per year and the biphasic basal body temperature within the last 6 months. The subjects received long-term follow-up until pregnancy.
Blood samples for serum AMH and hormonal levels were obtained at the third day of the menstrual cycle. Transvaginal ultrasound was also performed on each subject at the same time. Transvaginal ultrasound was performed as routine and recorded the data of antral follicle count (AFC), peak systolic velocity (PSV), plasticity index (PI), and resistant index (RI) of ovary.
AMH and Hormone Assays Serum samples were collected in a 5 mL serum separator tube and processed within 4 h to avoid blood cell lysis. Samples not processed the same day were stored at 4°C. Samples were centrifuged for 10 minutes at 1500 rpm at room temperaturethen transferred into 1 mL Eppendorf tube and stored at 80°C. AMH levels were determined using the enzymatically amplified two-site AMH-Gen-II ELISA (Beckman Coulter, Immunotech, Webster, Texas, USA) according to the manufacturer’s specifications . Serum samples were dispensed into the wells which were coated with anti-AMH antibody, followed by the addition of the anti-AMH detection antibody labelled with biotin, 100 μl of the streptavidin-horseradish peroxidase (HRP) was added after washing, followed by the addition of 100 μl of substrate solution containing TMB for 8 to 12 min. Using an automatic ELISA reader (Bio- Rad, Hercules, CA) the degree of enzymatic turnover of the substrate was determined by dual wavelength absorbance measurement at 450 nm and between 600 and 630 nm. The absorbance measured was directly proportional to the AMH concentration in the samples which was calculated from the calibration curve. The results were expressed in ng/ml. Concentrations below 0.08 ng/ml were considered undetectable. Hormone levels were determined using ELISA , as done by routine.
Data are presented as mean ± SD, t-test was used to determine significant difference between two groups. Data were performed by one-way analysis-of-variance (ANOVA), followed by a post hoc Bonferroni’s multiple comparison test when appropriate. Pearson’s test was used to establish whether there was a linear relationship between two characteristics. P value of <0.05 was considered as significant difference. All data were analyzed by SPSS 22.
A total of 208 women enrolled in the study, of which 202 women completed it. Six women form the control group quitted the study. A third sterile female were found to be diagnosed with polycystic ovary syndrome (PCOS) which affect the development of dominant follicle and hormone secretion especially FSH, LH and T [12,13]. According to the results of trans-vaginal ultrasound, all women diagnosed with PCOS had polycystic ovaries . The AMH levels of women diagnosed with PCOS ranged from 5.3 to 12.6 ng/mL, while those of women who were not diagnostic with PCOS ranged from 4.6 to 8.2 ng/mL.
Compared the general condition of the observation group with control group in Table 1, there were significant difference on hormone levels except progestogen (p<0.05). It expressed lower AMH level, E2, LH and T, but less FSH level (p<0.05) . There was significant difference on AFC between the sterile female and the controls (p<0.05). While there was no difference on PSV, PI or RI of ovary (p>0.05).
|Observation group||Control group||P|
|Age||30.94 ± 6.27||29.24 ± 5.34||>0.05|
|AFC||5.5 ± 2.3||3.2 ± 1.3||<0.05|
|PSV (cm/s)||13.6 ± 11.41||13.2 ± 12.01||>0.05|
|PI||0.63 ± 0.31||0.64 ± 0.43||>0.05|
|RI||0.52 ± 0.03||0.51 ± 0.04||>0.05|
|AMH (ng/ml)||9.59 ± 4.41||4.45 ± 2.31||<0.05|
|E2 (pg/ml)||44.59 ± 17.41||33.45 ± 9.31||<0.05|
|P (ng/ml)||4.05 ± 3.28||3.45 ± 2.52||>0.05|
|FSH (mIU/ml)||8.45 ± 6.66||9.72 ± 3.45||<0.05|
|LH (mIU/ml)||7.32 ± 0.27||5.47 ± 2.63||<0.05|
|T (ng/ml)||0.84 ± 6.98||0.46 ± 5.38||<0.05|
Table 1. The general condition of subjects.
There are 64 women of the observation group were pregnancy within the follow-up time (60.37%), 87 women of the control group were pregnancy in the 2 years (90.62%), none spontaneous abortion occured. We further analyzed the correlation between possibilities of pregnancy in sterile female and the serum hormone level (Table 2). The women with success pregnancy were significantly younger than the women with non-pregnancy (p<0.05). It expressed lower AMH level in the women with non-success pregnancy (p<0.05). There was less AFC in the women with success pregnancy (p<0.05).
|Pregnancy women||Non-pregnancy women||P|
|Age||27.94 ± 4.57||30.12 ± 3.54||<0.05|
|AFC||3.1 ± 1.3||5.3 ± 2.3||<0.05|
|PSV(cm/s)||12.6 ± 10.91||13.4 ± 12.51||>0.05|
|PI||0.59 ± 0.34||0.62 ± 0.34||>0.05|
|RI||0.53 ± 0.02||0.50 ± 0.03||>0.05|
|AMH (ng/ml)||4.49 ± 3.31||3.35 ± 6.31||<0.05|
|E2 (pg/ml)||42.29 ± 10.41||39.45 ± 9.91||>0.05|
|P (ng/ml)||4.05 ± 3.28||3.45 ± 2.52||>0.05|
|FSH(mIU/ml)||8.45 ± 4.06||8.42 ± 3.45||>0.05|
|LH(mIU/ml)||5.38 ± 0.27||5.42 ± 0.33||>0.05|
|T(ng/ml)||0.54 ± 5.38||0.52 ± 4.98||>0.05|
Table 2. The correlation between probabilities of pregnancy and the AMH level in the sterile female.
In this report we describe the effectiveness of an AMH measurement as a diagnostic for ovarian reserve in sterile female. In this study it showed that there was a significant correlation between circulating AMH concentration and AFC. Many reports have mentioned that AMH is good correlated with ovarian follicle count [16,17]. Precious reports have mentioned that serum AMH would be an adjunct examination in the diagnosis of PCOS [18-20], we took a hypothesis that AMH would evaluated the function of ovarian and even regular ovulatory through detecting AMH [21,22]. However, little is known about the performance of AMH in predicting the number of retrieved oocytes and the follicular development even the success of live birth [23,24].
The probability of pregnancy is the most important issue for infertile couples and still remains a challenge for clinicians . One rapid and effective measurement of ovulatory dysfunction for women would be important for deciding the therapeutic regimen. In the study, it showed that AMH, LH and T could be a substantial cross-predictor of premature ovulatory dysfunction and specific ranges of AMH would be an independent predictor of successful pregnancy . The findings show patients with an AMH concentration below 4 ng/mL in the blood plasma are unlikely to achieve with successful pregnancy, whereas the blood plasma AMH concentration above 7 ng/mL is a substantial independent predictor of ovulation failure with PCOS as many studies have mentioned [26,27].
As more women are willing to delay the giving birth time, an increasing number of women seek the possibility of pregnancy when they appeared with ovulation failure . Woman age is a substantial independent predictor which would be the first factor taken into consideration by clinicians as the increasing risk of ovulation failure with age [29,30]. As previous reports have mentioned that AMH concentration were associated with age [31,32], in this study we limited the age range of all subjects and taking a general examination of the function of ovary to avoid confounding factors. While the results have showed that the women with success pregnancy were significantly younger than the women with non-pregnancy. Age may be an insurmountable factor for infertile couples [33,34].
In conclusion, in this study the results suggest that serum AMH testing could represent a useful and practical test to screen the general risk of ovulation failure for the masses and a potential independent predictor of dominant follicle ovulation and successful pregnancy. We will launch larger multicenter studies for better validate these preliminary results.
- Grynnerup AG, Lindhard A, Sørensen S. Recent progress in the utility of anti-Müllerian hormone in female infertility. Curr Opin Obstet Gynecol 2014; 26: 162-167.
- Takashima A, Takeshita N, Otaka K, Kinoshita T. Effects of bipolar electrocoagulation versus suture after laparoscopic excision of ovarian endometrioma on the ovarian reserve and outcome of in vitro fertilization. J Obstetrics Gynaecol Research 2013; 39: 1246-1252.
- Parahuleva N, Pehlivanov B, Orbecova M, Uchikova E, Ivancheva H. Anti-Mullerian hormone in the major phenotypes of polycystic ovary syndrome. Akush Ginekol (Sofiia) 2014; 53: 22-27.
- Claes A, Ball BA, Scoggin KE. The interrelationship between anti-Mullerian hormone, ovarian follicular populations and age in mares. Equine Veterinary J 2015; 47: 537-541.
- Grasa P, Ploutarchou P, Williams SA. Oocytes lacking O-glycans alter follicle development and increase fertility by increasing follicle FSH sensitivity, decreasing apoptosis, and modifying GDF9:BMP15 expression. FASEB J 2015; 29: 525-539.
- Fleming R, Seifer DB, Frattarelli JL, Ruman J. Assessing ovarian response: antral follicle count versus anti-Mullerian hormone. Reprod Biomed Online 2015; 31: 486-496.
- Ellakwa HE, Sanad ZF, Hamza HA, Emara MA, Elsayed MA. Predictors of patient responses to ovulation induction with clomiphene citrate in patients with polycystic ovary syndrome experiencing infertility. Int J Gynaecol Obstet 2016; 133: 59-63.
- Roosa KA, Zysling DA, Place NJ. An assessment of anti-Müllerian hormone in predicting mating outcomes in female hamsters that have undergone natural and chemically-accelerated reproductive aging. Gen Comp Endocrinol 2015; 214: 56-61.
- Iliodromiti S, Nelson SM. Ovarian response biomarkers: physiology and performance. Curr Opin Obstet Gynecol 2015; 27: 182-186.
- Themmen AP, Kalra B, Visser JA, Kumar A, Savjani G. The use of anti-Müllerian hormone as diagnostic for gonadectomy status in dogs. Theriogenology 2016; 86: 1467-1474.
- Mckinnon B, Mueller MD, Nirgianakis K, Bersinger NA. Comparison of ovarian cancer markers in endometriosis favours HE4 over CA125. Mol Med Rep 2015; 12: 5179-5184.
- De Conto E, Genro VK, da Silva DS. AMH as a Prognostic Factor for Blastocyst Development. JBRA Assist Reprod 2015; 19: 131-134.
- Zhou JY, Zhang XY, Yu ML, Lu SF, Chen X. Effect of Transcutaneuos Acupoint Electrostimulation on Serum Sex Hormone Levels and Expression of Ovarian Steroid Hormone Metabolic Enzymes in Polycystic Ovary Syndrome Rats. Zhen Ci Yan Jiu 2016; 41: 11-17.
- Tessaro I, Modina SC, Franciosi F, Sivelli G. Effect of oral administration of low-dose follicle stimulating hormone on hyperandrogenized mice as a model of polycystic ovary syndrome. J Ovarian Res 2015; 8: 64.
- Nardo LG, Yates AP, Roberts SA, Pemberton P, Laing I. The relationships between AMH, androgens, insulin resistance and basal ovarian follicular status in non-obese subfertile women with and without polycystic ovary syndrome. Hum Reprod 2009; 24: 2917-2923.
- Sonigo C, Simon C, Boubaya M. What threshold values of antral follicle count and serum AMH levels should be considered for oocyte cryopreservation after in vitro maturation? Hum Reprod 2016; 31: 1493-1500.
- Lukaszuk K, Liss J, Kunicki M. Anti-Mullerian hormone (AMH) is a strong predictor of live birth in women undergoing assisted reproductive technology. Reprod Biol 2014; 14: 176-181.
- Sahin Ersoy G, Altun Ensari T, Vatansever D, Emirdar V, Cevik O. Novel adipokines WISP1 and betatrophin in PCOS: relationship to AMH levels, atherogenic and metabolic profile. Gynecol Endocrinol 2016.
- Georgopoulos NA, Saltamavros AD, Decavalas G, Piouka A, Katsikis I. Serum AMH, FSH, and LH levels in PCOS. Fertil Steril 2010; 93: e13.
- Oncul M, Sahmay S, Tuten A, Acikgoz AS, Gurleyen HC. May AMH levels distinguish LOCAH from PCOS among hirsute women? Eur J Obstet Gynecol Reprod Biol 2014; 178: 183-187.
- Tolikas A, Tsakos E, Gerou S, Prapas Y, Loufopoulos A. Anti-Mullerian Hormone (AMH) levels in serum and follicular fluid as predictors of ovarian response in stimulated (IVF and ICSI) cycles. Hum Fertil (Camb) 2011; 14: 246-253.
- Martínez F, Clua E, Carreras O, Tur R, Rodríguez I. Is AMH useful to reduce low ovarian response to GnRH antagonist protocol in oocyte donors? Gynecol Endocrinol 2013; 29: 754-757.
- Gleicher N, Kushnir VA, Sen A. Definition by FSH, AMH and embryo numbers of good-, intermediate- and poor-prognosis patients suggests previously unknown IVF outcome-determining factor associated with AMH. J Transl Med 2016; 14: 172.
- Schram CA, Stephenson AL, Hannam TG, Tullis E. Cystic fibrosis (cf) and ovarian reserve: A cross-sectional study examining serum anti-mullerian hormone (amh) in young women. J Cyst Fibros 2015; 14: 398-402.
- Bakas P, Boutas I, Creatsa M. Can anti-Mullerian hormone (AMH) predict the outcome of intrauterine insemination with controlled ovarian stimulation? Gynecol Endocrinol 2015; 31: 765-768.
- Mumford SL, Legro RS, Diamond MP Baseline AMH Level Associated With Ovulation Following Ovulation Induction in Women With Polycystic Ovary Syndrome. J Clin Endocrinol Metab 2016; 101: 3288-3296.
- Vaiarelli A, Drakopoulos P, Blockeel C Limited ability of circulating anti-Mullerian hormone to predict dominant follicular recruitment in PCOS women treated with clomiphene citrate: a comparison of two different assays. Gynecol Endocrinol 2016; 32: 227-230.
- Tran ND, Cedars MI, Rosen MP. The role of anti-müllerian hormone (AMH) in assessing ovarian reserve. J Clin Endocrinol Metab 2011; 96: 3609-3614.
- Nikokavoura EA, Johnston KL, Broom J, Wrieden WL, Rolland C. Weight loss for women with and without polycystic ovary syndrome following a very low-calorie diet in a community-based setting with trained facilitators for 12 weeks. Diabetes Metab Syndr Obes 2015; 8: 495-503.
- Singh P, Srivastava RK, Krishna A. Effects of gonadotropin-releasing hormone agonist and antagonist on ovarian activity in a mouse model for polycystic ovary. J Steroid Biochem Mol Biol 2016; 163: 35-44.
- Shahrokh Tehraninezhad E, Mehrabi F, Taati R, Kalantar V, Aziminekoo E. Analysis of ovarian reserve markers (AMH, FSH, AFC) in different age strata in IVF/ICSI patients. Int J Reprod Biomed (Yazd) 2016; 14: 501-506.
- Meczekalski B, Czyzyk A, Kunicki M. Fertility in women of late reproductive age: the role of serum anti-Mullerian hormone (AMH) levels in its assessment. J Endocrinol Invest 2016.
- Bozkurt B, Erdem M, Mutlu MF. Comparison of age-related changes in anti-Mullerian hormone levels and other ovarian reserve tests between healthy fertile and infertile population. Hum Fertil (Camb) 2016; 19: 192-198.
- Hvidman HW, Bentzen JG, Thuesen LL. Infertile women below the age of 40 have similar anti-Mullerian hormone levels and antral follicle count compared with women of the same age with no history of infertility. Hum Reprod 2016; 31: 1034-1045.