Establishment Of Reference Interval Of Anti-mullerian Hormone Of Fertile Women In Zhejiang Province

Background:Fertility and reproduction are important parts of women’lives, the evaluations of the reproductive capability of a woman are of significant importance. Ovarian reserve (OR), which represents the quantity and quality of the follicles in the ovary, reflects female reproductive capability and is one of the most valuable markers of women’functional potential of the ovary. Traditionally, OR can be measured through some markers, including age, follicle stimulating hormone (FSH), inhibin B (INHB) and antral follicle count (AFC). All these markers have some unavoidable limitations. Take FSH and INH B for example, samples can only be taken at the third day of menstrual cycle. The results of AFC are affected by subjective factors, such as the ability of the examiners and the conditions of the patients. In recent years, a new marker, named anti-Mullerian hormone (AMH), has been shown to be a better marker that reflects declining OR. Serum AMH level has been widely used in fertility therapy with individualized stimulation protocols to predict response to stimulation with exogenous gonadotropin in women undergoing in vitro fertilization (IVF). In addition, AMH has been shown useful in identifying a good prognosis group for IVF and gonadotropin stimulation in women of advanced reproductive age. Furthermore, in adult women, serum AMH levels decline steadily with age and are highly predictive for age at menopause. The decline of serum AMH levels appears to occur before changes in other age-related variables such as FSH, which rises late in female reproductive lifespan. AMH has also been employed as a tool to identify women with premature ovarian insufficiency or polycystic ovary syndrome. By using AMH as a marker, extensive efforts have been put into evaluation of the effect of a variety of diseases on ovarian reserve, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), human immunodeficiency virus (HIV) infection associated diseases and cancers, and the extent of OR damage following various treatments, including surgeries involving endometriomas, radiotherapy and chemotherapy of female cancers. For example, it has been well established that chemotherapy of breast cancer results in loss of fertility and premature ovarian failure, and thus examination of serum AMH levels will offer these cancer patients opportunity of informed decision made for cryopreservation of ovarian cortical tissue prior to cancer treatment.Despite extensive clinical application of AMH as a marker, the only documented reference values for healthy fertile women were derived from a study on French fertile women, and no AMH reference data have been reported for Chinese fertile women. Thus, establishing appropriate reference values (RIs) for AMH in Chinese fertile women according to their defining characteristics will provide valuable information for clinical application of AMH as a marker in Chinese women. So the objectives of this study were to study AMH changes of Chinese Fertile Women and evaluate the influence of some clinical character factors on serum concentration of AMH and to establish RIs for serum AMH levels among Zhejiang Province fertile women.Methods:The study was carried out at the Women’s Hospital, School of Medicine, Zhejiang University in China. Chinese fertile women, who were admitted to the hospital, were included in this prospective cross-sectional study. All the included subjects were in their first trimester of pregnancy, and had no history of serious diseases. Previous studies had shown no significant changes in AMH levels during the first trimester of pregnancy when compared with the AMH levels before conception. Therefore, women in their first trimester of pregnancy, indicating their fertility, were chosen as the ideal population for obtaining reference values of AMH in this study. The trained physician conducted face-to-face interviews with the participants. Details of the research were explained to all the patients, who, at the same time, were handed a written informed consent and answered the questions in the questionnaires, if they agreed to participate. The serum samples were collected from the residue serum samples of the usual blood tests carried out in the first trimester of pregnancy. Serum AMH levels were measured using a second generation AMH enzyme-linked immunodsorbent assay. Data processing and analyses were carried out using Microsoft Excel2007and the Statistical Package for the Social Science (SPSS version20.0). According to the requirements of International Federation of Clinical Chemistry (IFCC) for reference intervals, a nonparametric statistical method with500bootstrap iterations and a90%confidence interval was employed to calculate reference values of AMH levels.Results:1. This study comprised358women,100%participants have signed their consents, approximately96.37%of the participants completed the entire questionnaire. The number were119、82、59、52and46for the age ranges of17-24、25-29、30-34、35-39and40-48years, respectively.2. In total, the median of age was28yr and the25-75percentiles were23and35.3yr (range17-48yr). The median of height was160cm and the25-75percentiles were158and164cm (range146-178cm). The median of weight was52.5Kg and the25-75percentiles were45.0and57.0Kg (range37-92Kg). The median of BMI was20.2Kg/m2and the25-75percentiles were18.8and22.0Kg/m2(range14.4-33.9 Kg/m2). The median of gestational weeks was7.1weeks and the25-75percentiles were6.3and8.6weeks (range4.4-12Kg). The number of smokers is19, account for5.2%. The number of nulliparous women is187, account for51.7%. The number of priminarous women is151, account for41.7%. The number of multiparous women is24, account for6.6%.3. The difference of height between five age groups doesn’t have significant meanings (P=0.969). The difference of weight between five age groups has significant meanings (P=0.000). The difference of BMI between five age groups has significant meanings (P=0.000). The difference of gestational weeks between five age groups has significant meanings (P=0.013). The difference of nulliparous women between five age groups has significant meanings (P=0.000). The difference of primiparous women between five age groups has significant meanings (P=0.000). The difference of multiparous women between five age groups has significant meanings (P=0.000).4. Correlation tests showed a strong correlation between AMH concentration and age, while no correlations were found between serum AMH levels and other factors such as height, weight and BMI after AMH levels being adjusted for age through parity correlation tests. In addition, no correlations were detected between gestational age and serum AMH levels in different age groups. Regression analysis results showed that the relationship between AMH concentration and age was best represented by a linear function:Log AMH=-0.378+0.088*Age-0.0018*Age2(R2=0.322, P=0.000)5. AMH levels tend to diminish as age advances and this trend could be properly found from the data below:the median and the25-75percentiles for the AMH levels were5.36(3.58-7.00).4.65(3.12-6.35),3.90(3.02-5.60),2.89(1.85-4.82) and1.29(0.76-2.45) ng/ml for the age ranges of17-24,25-29,30-34,,35-39and40-48years, respectively. The reference intervals for serum AMH concentration were established in the five age groups. Conclusions:We established RIs of serum AMH levels in a group of fertile women in their first trimester of pregnancy in Zhejiang province. In this study we found that the range of AMH concentration for women aged from17-48year are20.22-0.16ng/ml. A progressive decline of serum AMH levels was observed with the increase of subject ages in this study. Regression analysis results showed that the relationship between AMH concentration and age was best represented by a linear function:Log AMH=-0.378+0.088*Age-0.0018*Age2(R2=0.322, P=0.00). The results will provide useful information for clinical practice involving ovarian reserve among women of child-bearing age.