anti - mullerian hormone ( amh ) , a polypeptide , secreted by the granulosa cells of the preantral and early developing antral follicles , has been shown to be a predictor of ovarian activity .
pcos patients show an increased number of antral follicles ; therefore they have higher circulating amh levels than the healthy women .
the interactions between amh and the hormonal profile characteristics in pcos require a better assessment .
the positive correlation between insulin resistance and serum amh levels suggests that insulin exerts an action on amh synthesis ; however this aspect is not yet fully understood .
polycystic ovary syndrome ( pcos ) is a common endocrine disorder , affecting 610% of women in reproductive age .
this syndrome is characterized by biochemical or clinical signs of hyperandrogenism , chronic anovulation , and polycystic ovaries .
evidence suggests that insulin resistance and its compensatory hyperinsulinemia play an important role in pcos pathogenesis [ 5 , 6 ] .
insulin is associated with hyperandrogenism ; it acts synergistically with luteinizing hormone to increase the androgen production of theca cells .
inositol ( hexahydroxycyclohexane ) belongs to the vitamin b complex group ; it is a 6-carbon ring compound , having a hydroxyl group linked to each carbon of the ring , with nine possible stereoisomeric forms depending on the epimerization of the six hydroxyl groups . among them , myoinositol ( myo - ins ) is the mostly represented isoform , with very relevant biological functions . increasing evidence
has demonstrated that myo - ins plays a key role in cell morphogenesis and cytogenesis , lipid synthesis , structure of cell membranes , and cell growth .
myo - ins administration improves hormonal profile , oocyte maturation , and insulin resistance ; furthermore , it promotes the meiotic progression of germinal vesicle oocytes [ 11 , 12 ] .
recent studies on pcos patients showed a decrease of androgen levels and an improvement in ovulation and metabolic parameters after treatment with myo - ins and d - chiro - inositol ( d - chiro - ins ) , which is another stereoisomeric form of inositol .
it was highlighted that very promising results were achieved administering myo - ins plus d - chiro - ins at their physiological range in plasma ( i.e. , 40  :  1 ) to ensure better clinical results in the pcos therapy .
it is important to investigate the possibility of using amh as a marker of some parameters : improved insulin resistance and decreased lh and androgen levels .
combined oral contraceptive ( coc ) pills , especially containing antiandrogen , are commonly used in the treatment of pcos patients to suppress ovulation . in this study
our primary outcome was to investigate the effect of myo - ins or coc on the clinical features , biochemical parameters , and amh levels in pcos patients .
the secondary outcome of the study was to compare differences in changes after treatment with myo - ins or coc .
this is a randomized prospective trial carried out at the department of obstetrics and gynecology ( dokuz eylul university , izmir , turkey ) between may 2013 and january 2014 .
this study started after the approval by the clinical research ethics committee of the faculty of medicine , dokuz eylul university , and the turkish health ministry drug and medical device foundation .
137 patients diagnosed with polycystic ovary syndrome and admitted to dokuz eylul university faculty of medicine , department of obstetrics and gynecology , division of reproductive endocrinology and infertility outpatient clinic , were included in the study .
the patients with odd numbers were allocated to coc group ( n = 60 ) , whereas the even ones to myo group ( n = 77 ) ( figure 1 ) .
the coc used was 2  mg cyproterone acetate and 0.035  mg ethinylestradiol ( diane 35 ; schering ag , istanbul , turkey ) daily .
the coc was given for 21 days and in the following 7 days no drugs were given .
the product used contained 1 gram myoinositol and 100    gram folic acid ( inofolic ; lo.li .
the drug was used twice a day continuously for 1216 weeks . in initial assessment , for patients who had regular menstrual cycle for both myo and coc group , the blood samples for hormonal assessment except progesterone were conducted on day 2 or 3 of the menstrual cycle .
the assessment after three months was done when the treatment finished and the patients had her first menstrual cycle after three months of treatment .
for both groups the patients were not under medication during the assessment of the hormone levels .
in oligo / amenorrhoeic patients , blood samples were collected after withdrawal bleeding induced by oral progestin ( 5  mg medroxyprogesterone acetate twice a day ; tarlusal ; deva holding a. . ,
the diagnosis of pcos was made according to the rotterdam criteria ; as prescribed , two out of three features were detected in the patients : oligomenorrhea ( fewer than six menstrual periods in the preceding year ) and/or anovulation ; clinical and/or biochemical signs of hyperandrogenism ; presence of 12 follicles in each ovary measuring 29  mm in diameter ; and/or increased ovarian volume ( > 10  ml ) .
smoking , hyperprolactinemia , hypogonadotropic hypogonadism , pregnancy , thyroid disease , congenital adrenal hyperplasia , androgen - secreting tumors , and cushing 's syndrome were ruled out during the screening phase .
none among the enrolled patients had taken , at least in the previous six months , oral contraceptives , antiandrogens , or any drug that could influence carbohydrate metabolism .
clinical evidence of hyperandrogenism was determined by the ferriman gallwey score 8 that reveals the presence of hirsutism and/or acne .
initial physical examination included weight , height , and waist and hip circumferences , to calculate waist / hip ratio ( whr ) and body mass index ( bmi ) .
the waist circumference was measured at the midpoint of lowest margin of 12th rib and the lateral iliac crest during the normal expiration .
resting systolic and diastolic pressures were measured ; after 2 minutes , the second measurement was performed and the mean values were determined .
serum levels of fasting plasma glucose and insulin , c - reactive protein ( crp ) , anti - mullerian hormone ( amh ) , apoprotein b ( apo b ) , apoprotein a1 ( apo a1 ) , high density lipoprotein ( hdl ) and low density lipoprotein ( ldl ) , total cholesterol , triglyceride , follicle stimulating hormone ( fsh ) , luteinizing hormone ( lh ) , total and free testosterone , dehydroepiandrosterone sulfate ( dheas ) , and sex hormone binding globulin ( shbg ) were measured .
normal insulin sensitivity was defined by fasting serum glucose and insulin levels with homeostatic model of insulin resistance ( homa - ir ) .
homa - ir was calculated by the formula : homa - ir = fasting blood glucose ( mg / dl )  fasting insulin ( iu / ml)/405 .
fasting venous blood samples were taken between 08:00 am and 10:00 am after a 12-hour overnight fast .
the blood samples were immediately centrifuged for 10 minutes and kept at 80c in eppendorf tubes until assayed .
serum anti - mullerian hormone ( amh ) levels were analyzed with commercial kit according to manufacturer instructions based on the principle of competitive enzyme linked immunosorbent assay ( elisa ) method ( catalog number : csb - e12756h , cusabio biotech co. , usa ) .
the microplate in the kit is precoated with goat - anti - rabbit antibody specific to amh .
standards and undiluted samples are loaded into the appropriate microtiter plate wells with horseradish peroxidase ( hrp ) conjugated amh and antibody specific to amh .
the competitive inhibition reaction was launched between hrp labeled amh and unlabeled amh with the antibody . following a wash to remove any unbound substances , substrate a and b solutions
the color development is stopped and the intensity of the color is measured spectrophotometrically at a wavelength of 450  nm .
a standard curve of known concentration ( 0 , 0.375 , 1.31 , 4.69 , 28.12 , and 150  ng / ml ) of amh was established and the concentration of analyte in the samples was calculated accordingly .
the elisa assays of amh had a sensitivity of 0.375  ng / ml ; a detection range of 0.375150  ng / ml ; intra - assay coefficient of variation < % 10 , interassay coefficient of variation < % 15 , respectively .
for transvaginal ultrasonography , the probes used were ev49/10ed center frequency 6.5  mhz and er49/10ed center frequency 6.5  mhz . for virgin patients transabdominal ultrasonography was used .
the presence of 12 follicles in each ovary measuring 29  mm in diameter was recorded .
longitudinal , transverse , and anteroposterior ovarian diameters were measured and multiplied by 0.5 to calculate the ovary volume .
data were analyzed by using statistical program for social sciences ( spss , version 16 ) .
the level of significance was accepted when p < 0.05 . in table 1 , the test used was independent t - test . in table 2 , the test used was independent t - test . in table 3 , the test used was paired samples test . in table 4 , the test used was paired samples test .
106 patients were analyzed , among them 54 patients were coc receivers ( group 1 ) , and 52 were myo - ins receivers ( group 2 ) .
baseline demographic , clinical characteristics , and ultrasound results of all patients are presented in table 1 .
the mean age was 22.79  4.13 years and 24.44  4.78 years for groups 1 and 2 , respectively . in group 1 , 41 ( 75.9% ) of 54 patients showed menstrual irregularity . at the end of the study period , patients in group 1 showed no menstrual irregularity , whereas the decrease of menstrual irregularity in group 2 was from 40 ( 76.9% ) to 8 ( 15.4% ) patients .
the baseline biochemical and hormonal values showed that patient groups were matched in these parameters except sex hormone binding globulin ( shbg ) level which was higher in group 1 ( table 2 ) .
increase in hdl and decrease in lh and lh / fsh ratio were determined statistically significant only in group 1 when compared with the baseline values ( for all p > 0.05 ) ( table 3 ) . in group 2 ,
the reduction of fasting glucose , ldl , dheas , total cholesterol , and prolactin levels was statistically significant ( for all p < 0.05 ) ( table 3 ) .
other parameters , such as total testosterone , dheas , and fasting glucose , were statistically improved in group 2 , while no particular improvement was present in group 1 ( table 3 ) .
after 3-month treatment , amh ( for group 1 p < 0.001 ; for group 2 p = 0.002 ) levels as well as ovarian volumes , ovarian antral follicle count , and total antral follicle counts showed a statistically significant decrease in both groups ( for all p < 0.001 ) ( table 3 ) .
when we evaluate progesterone levels at the end of the treatment , group 1 showed a statistically significant decrease whereas group 2 showed a statistically significant increase ( for group 1 p = 0.014 ; for group 2 p < 0.001 ) .
the lessening of ovarian volumes and amh levels was statistically significantly more in the myo group than coc group ( for amh ; p = 0.048 , for ovarian volumes p = 0.040 ) ( table 4 ) .
this prospective study observed the change in clinical , biochemical parameters and anti - mullerian hormone levels after treatment with two different drug supplementations , myoinositol and combined oral contraceptives .
study data demonstrated that myo - ins regimen in pcos patients positively affects metabolic parameters and modulates various hormonal factors deeply involved in the reproductive function and ovulation such as anti - mullerian hormone .
it is well known that pcos is characterized by hyperandrogenism and irregular menstrual cycles . concerning menstrual irregularity , using myo - ins papaleo et al .   and gerli et al
.   observed an improvement of 88% ( after six months ) and 70% ( after 14 weeks ) . in the current study ,
76.9% of patients in group 2 , treated with myo - ins , had menstrual irregularity initially .
use of myo - ins was associated with a significant improvement , and menstrual irregularity was strongly reduced in the study population .
this effect may be explained by the specific protein phosphorylation processes via protein kinase c , which modulates various cellular processes as a second messenger system .
anti - mullerian hormone is secreted primarily in the small antral follicles and amh measurements correspond to granulosa cell activity , total antral follicle count , and ovarian volume .
in addition , several studies showed that amh might correlate with the severity of this syndrome . in recent researches , positive correlations between amh levels , ovarian volume , and total antral follicle count
several studies in the literature support that coc treatment decreases ovarian volume and antral follicle [ 23 , 24 ] .
our analysis showed significant reduction in total antral follicle count and ovarian volume after treatment with coc and myo - ins ( for both groups p < 0.001 ) . in line with our study , the two researches of genazzani demonstrated reduction in ovarian volume after myo - ins administration .
however , they reported no changes in the total antral follicle count after myo - ins treatment [ 25 , 26 ] .
although there are no previously published data about changes in serum amh levels during myo - ins therapy , there are several studies that observed amh changes after treatment with coc [ 2729 ] .
examined 95 women in five different groups and they found no significant change in amh levels before and after treatment with combined oral contraceptives .
in contrast , decrease in amh levels after coc was shown in previous studies and it has been confirmed in our study [ 28 , 29 ] . the decline in amh levels may be related to the suppression of ovarian function with coc .
research has been mainly focused on coc 's activity and there are no clinical studies on myo - ins effect on amh levels .
reported that myo - ins supplementation , in pcos patients , affected metabolic parameters such as insulin sensitivity and modulated positively hormonal factors like lh , fsh , and testosterone .
there was one retrospective study which showed decreased amh levels after d - chiro - ins . to our knowledge , the present study is the first one in the scientific literature indicating statistically significant decrease in amh levels after 1216 weeks of myo - ins use .
we believe that suppression in amh levels may be explained by the reduction of total antral follicle count and ovarian volume .
however , the mechanism between decrease in total antral follicle count and myo - ins therapy is not clearly understood yet .
we hypothesize that whereas hyperinsulinemia may stimulate the development of antral follicles and recover the sensitivity of granulosa cells to fsh , therefore leading to increase in the number of follicles , ovarian volume , and amh levels , myo - ins may improve clinical and hormonal features of pcos patients by enhancing insulin sensitivity that decreases hyperinsulinemia . in our trial , we compared the ovarian volumes , total antral follicle count , and serum amh levels after treatment with coc and myo - ins .
patients in the study group showed significantly more relevant reduction in amh levels ( p = 0.048 ) and ovarian volume ( p = 0.040 ) than the coc group .
the decrease in total antral follicle count for myo - ins and coc group was similar ( p = 0.356 ) . based on our data
, we believe that myo - ins is more effective compared to coc in lowering amh levels and ovarian volumes .
myoinositol can not replace coc for contraception and may be preferred for patients with infertility who desire to conceive pregnancy .
also , myoinositol can be an option in patients with hirsutism , oligoanovulation , and symptoms of hyperandrogenism who have a contraindication to use coc .
myoinositol is not a primary treatment ; it is an adjunct therapy to improve insulin resistance . in the present study
, we also observed the effect of coc and myo - ins on fasting insulin , fasting glucose , and homa - ir .
the results showed no significant change in insulin or homa - ir in both groups .
only pcos patients , who received myo - ins , presented a significant reduction in glucose levels .
however , we know that our study had a too short duration to be capable of detecting myo - ins effect on insulin and homa - ir .
in contrast with our data , zacch et al . demonstrated that fasting insulin and homa - ir values were decreased after myo - ins treatment .
reported that coc did not affect fasting insulin or fasting glucose levels or homa - ir . in minozzi et al .
's study , patients were divided into two groups as coc + myo - ins receivers and only coc receivers .
the results showed a significant reduction in glucose levels only in coc + myo - ins group , whereas no significant change was observed in coc group .
the results of minozzi et al . 's prospective study showed that the combination of coc and myo - ins was a more effective treatment for clinical symptoms of pcos and in controlling endocrine disorders and insulin resistance .
many studies have been carried out in pcos patients with high serum androgen levels to determine the clinical implications .
the results of this study showed that myoinositol treatment was able to improve some key parameters such as the total testosterone level .
the reduction of testosterone is a key outcome in the management of pcos women , because of the typical symptoms related to the hyperandrogenic status , affecting the pcos pathway
. these results could be due to the insulin sensitizing action of myoinositol and the sequent downregulation of the androgens production at ovarian level .
the distinct effect of myoinositol on testosterone , amh , and ovarian volume is surprising and this could be explained by a reduction in hyperinsulinism . in our study
, we did not observe significant reduction in insulin and homa - ir , but still there is a decrease in homa - ir blood levels . to observe the significant change
demonstrated a decrease in free testosterone and total testosterone after myo - ins treatment , but no significant change in androstenedione levels .
when we evaluate the changes in androgen levels in our study , we have seen that myo - ins treatment reduced dhea - s and total testosterone levels , but no significant differences in shbg and androstenedione were found . in the present study , whereas coc group showed a significant increase in shbg levels , statistical significance was not observed in the increase of dheas , free testosterone , and androstenedione and in the reduction in total testosterone levels .
literature data indicate that if the treatment duration was extended , changes in androgen levels might become significant .
therefore , it is more appropriate planning a long term therapy in the management of androgen excess , anovulation , and insulin resistance .
this study shows that the combination of myoinositol plus folic acid should be considered in the treatment of pcos patients . considering previous studies , myo - ins
studies in the literature indicate that myoinositol exerts positive effects on insulin resistance , but further researchers are still required to clarify the mechanism .
the present study is the first one reporting that myoinositol is superior to coc in terms of lowering androgens levels . also ,
 recently , myoinositol ( myo - ins ) and folic acid combination has gained an important role for treating polycystic ovary syndrome ( pcos ) , in addition to combined oral contraceptives ( coc ) . 
 we aimed to examine myo - ins effects on anti - mullerian hormone ( amh ) levels and compare them with those ones obtained administering coc . in this prospective study , 
 137 pcos patients , diagnosed according to rotterdam criteria and admitted to the reproductive endocrinology and infertility outpatient clinic at dokuz eylul university ( izmir , turkey ) , were included . 
 after randomization to coc ( n = 60 ) and myo - ins ( n = 77 ) arms , anthropometric measurements , blood pressure , modified ferriman gallwey scores were calculated . 
 biochemical and hormonal analysis were performed , and lh / fsh and apo b / a1 ratios were calculated . 
 data analysis was carried out in demographically and clinically matched 106 patients ( coc = 54 ; myo - ins = 52 ) . 
 after 3-month treatment , increase in hdl and decreases in lh and lh / fsh ratio were statistically more significant only in coc group when compared with baseline ( in both cases p > 0.05 ) . in myo - ins group , 
 fasting glucose , ldl , dheas , total cholesterol , and prolactin levels decreased significantly ( for all p < 0.05 ) . 
 progesterone and amh levels , ovarian volume , ovarian antral follicle , and total antral follicle counts lessened significantly in both groups ( for all p < 0.05 ) . in pcos 
 treatment , myo is observed more effective in reductions of total ovarian volume and amh levels . 
1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion