Correlation between Anti-Mullerian Hormone Levels and Antral Follicle Numbers in Polycystic Ovary Syndrome

Introduction: Polycystic Ovary Syndrome (PCOS) is characterized by clinical and biochemical hyperandrogenism, polycystic ovarian morphology, altered gonadotropin secretion, insulin resistance and /or hyperinsulinemia associated with obesity. Objective: The objective of this study was to compare the correlations between levels of FSH, LH, estradiol, Anti-Mullerian Hormone (AMH), and antralfollicle numbers in women with polycystic ovary syndrome. Methods: PCOS was diagnosed when at least two out of the three parameters were present, according to the Rotterdam criteria. Patients were grouped according to the presence of oligomenorrhea and amenorrhea. Levels of AMH, FSH, LH and Estradiol were determined and a transvaginal ultrasound was performed to determine ovarian volume and the number of antral follicles. Results: Pearson’s correlation revealed a significant correlation (0.283, p =0.01) between AMH and antralfollicle number. It was appetent that the higher the number of antral follicles the concentrations of AMH is also higher. A linear correlation showed that the AMH concentration and the number of antral follicles correlated positively (r =0.303, p =0.002). While the levels of FSH correlated negatively with the number of antral follicles(r = -0.182, p = 0.05).The ovarian volume also correlated positively with the number of follicles (r =0.708, p = 0.000). Conclusions: A significant correlation of AMH with the number of antral follicleswas revealed.


INTRODUCTION
Several authors have described altered levels of gonadotropins, prolactin and sex steroids in PCOS 5 .Menstrual disturbances commonly observed in PCOS include oligomenorrhea and amenorrhea.About 70%-90% of women with oligomenorrhea have PCOS while 30%-40% of women with amenorrhea could have PCOS 6 . Women with PCOS often seek care for menstrual disturbances, clinical manifestations of hyperandrogenism, and infertility.PCOS is the most common cause of anovulatory infertility. 9Women with PCOS are likely to have not only a higher antral follicle count (AFC) but also a greater ovarian volume. 10C is essential to correlate biochemical and clinical markers to prognosticate infertility and ovarian reserve.Furthermore, women with higher serum levels of Anti-Mullerian Hormone (AMH) have longer menstrual cycles compared with those with lower levels.In females, .AMH is expressed in granulosa cells of growing follicles up to the antral stage, suggesting an important role in early ovarian folliculogenesis.AMH is able to inhibit the initiation of primordial follicle growth and may also decrease the sensitivity of antral follicles to follicle-stimulating hormone (FSH).In humans, however, the association between androgens and AMH remains uncertain, and its exact function in follicular recruitment and long-term effects is not well understood.Serum AMH levels could be used also to identify the girls at risk for PCOS in early adulthood, and allow early prevention by life style counselling.The aim of thisstudy was to compare the correlations between hormone levels of FSH, LH, estradiol, AMH, and antralfollicle numbers of women with polycystic ovary syndrome.

METHODS
This was a descriptive and analytical study.The selection of patients was randomly done by the Gynecology Service by two specialists.All patients underwent a complete medical history including reproductive background.In the participating Gynecology Service, a medical professional approached potential participants to ascertain interest in the study and obtain informed consent.Assurance was given that consent or refusal to participate would not affect the quality of care or eligibility for any future clinical studies and that all information provided would be confidential to the study team.Ethical approval was provided by the Institutional Ethics Committee of The University of Guanajuato with the Original PaPer www.slcog.lk/sljogapproval / ID CIDCSIC-1191304.
A total of 50 women were included, between 25-30 years of age, who were diagnosed with PCO according to the Rotterdam criteria. 11The PCOS was defined with at least two out of the three criteriawere present.The diagnosis was carried out by two experienced Gynecologists.The patients were separated in to two different groups; patients with oligomenorrhea were placed in group 1, while group 2 was set up by the patients with amenorrhea.In this study, amenorrhea is described as absence of at least 3 to 6 consecutive menstrual cycles or four or fewer menstrual period per year and oligomenorrhea is described as menstrual cycles fewer than 8 cycles per year, or the duration of the cycle exceeds 35 days.
The determinations of LH, FSH and estrogen levels were taken in the 1-3 daysof the menstrual cycle in patients with oligomenorrhea.The determinations levels of LH, FSH and estrogen were taken any day of the menstrual cycle in patients with amenorrhea.The determinations of levels of AMH were taken any day of the menstrual cycle in all women, during the second study visit.Patients who had a history of previous ovarian surgery and those who had been taking oral contraceptives were excluded from the present study.

Determination of hormone levels
Afterat least 12 hours of fasting 10ml of peripheral venous blood, on any dayof the menstrual cycle, was drawn for determining levels of AMH by an ultra sensitive ELISA(AMHGenII, Beckman CoulterInc.,Webster,TX,USA) with a sensitivity of 0.08ng/mL.To determine levels of LH, FSH and estrogen, 10 ml of peripheral venous blood were taken in the 1-3 days of the menstrual cycle of each patient and analyzed by means of radio immunoassay.

Transvaginal ultrasonography (USG)
Each patient had a USG performed by the same observer, a reproductive biologist with a 5 MHz transvaginal probe by Medison Sono Ace 6000C (Medison, Seoul, Korea).The measurements were performed in real time using the highest possible magnification to observe the ovaries.Transvaginal ultrasound was performed in all participants in the early follicular phase between day 2 and day 3 of the menstrual cycle.
Axes measured were length, thickness and width to calculate ovarian volume (OV). 12Ultrasonographic image was considered characteristic of PCOS in the presence of 12 or more follicles 2-9 mm in diameter located in the periphery of the ovaries by scanning each ovary from the inner to the outer margins in a longitudinal cross section, according to the Rotterdam criteria.The total ovarian volume (OV) was defined as the sum of the right and the left ovarian volumes, and the total antral follicle number was defined as the sum of the right and the left antral follicle count (AFC).

Statistical analysis
We used the statistical package SPSS version 21.0 for the statistical analysis.Descriptive statistics were used to represent the demographic characteristics of women.We performed Pearson's correlation between the number of antral follicles number and AMH levels, and a linear correlation between LH, FSH, estradiol, AMH, OV and antral follicle number.. In all cases the p value accepted as significant was <0.05.

RESULTS
The demographic and clinical characteristics of oligomenorrhea and amenorrhea patients with PCOS are given in Table 1.No significant differences in age, body mass index and education.The patients showed differences only in the presence of menstruation as expected.
When the women in the current study were classified according to their menstrual cycle into oligomenorrhea and amenorrhea (Table 2) it was found that FSH andLH concentrations were significantly higher in amenorrhea patients (p=0.024 and p=0.028, respectively).
Analysis of Pearson's revealed a significant correlation (0.283, p = 0.01) between AMH and the number of antral follicles, which showed that the higher the number of antral follicles more the concentrations of AMH.
The serum AMH level was not significantly related to the ovarian volume on ultrasonography.

DISCUSSION
In this study, we evaluated the relationship between levelsof FSH, LH, estradiol, AMH, and antral follicle numbers and ovarian volume in women with polycystic ovary syndrome.The results showed a significant correlation between serum AMH level and the number of antral follicles, which showed that the higher the number of antral follicles more the concentrations of AMH.
4] AMH is regarded as the most useful marker of the status of ovarian reserve. 15ellam et al.Other authors suggest that, serum AMH levels might be introduced as a marker to be utilized clinically in the differential diagnosis of hyperandrogenemic patient 16 .Our data confirm these findings, which showed that higher the number of antral follicles the more the concentration of AMH. 16tients who were diagnosed with PCOS-Amenorrhea showed elevated levels of FSH and AMH, ovarian volume and number of antral follicles.
In addition it has been reported that AMH levels between hyperandrogenic and non-hyperandrogenic PCOS patients are different. 17In the current study elevated FSH levels were reported in oligomenorrhea patient with PCOS.
Increased LH and normal to low FSH such as young PCOS women with hyperandrogenemia, serum AMH levels are increased, 18 this was seen in our study.
In contrast, we found not significant correlation between the serum AMH level and the ovarian volume, and this result is in disagreement with that of a previous study showing that the AMH levels positively correlate with the ovarian volume. 19One possible explanation is that the ovarian stromal volume is not significantly affected by the serum AMH level.
Hormone levels of FSH showed significant differences in the two groups.Stimulatory effect of FSH on AMH secretion in oligo/anovulatory PCOS women is known.Catteau-Jonard and Dewailly, suggest that the defective selection of a dominant follicle in anovulatory patients, results in a local inhibition of FSH action. 20rrent tests to determine the ovarian response, which include AMH are designed to predict how a woman can respond to controlled ovarian stimulation, so it is considered that assessing functional ovarian reserve through AMH is not enough.FSH levels increase with age, hindering the estimation of ovarian reserve and decrease possible fertility.The number of antral follicles, an indirect measure of ovarian reserve, seen through a transvaginal ultrasound is now reliable and allows us to have a more accurate estimation even when it comes to starting a treatment protocol for infertility.Antral follicle count with transvaginal sonography is a noninvasive and easy to perform method that provides predictive information about ovarian responsiveness before stimulation. 21Further research is needed to determine the relationship of AMH and PCOS.AMH may be a useful initial diagnostic test for PCOS subject to validation in prospective population. 22The study was restricted to women who sought treatment for infertility.Moreover, we believe that AMH can be used as a marker in conjunction with ultrasound values to assess ovarian reserve in the future.