Received: June 28, 2011 Accepted: September 7, 2011

Transcription

1 Review An ti-mülle rian hor mo ne: A unique bioc he mi cal mar ker of go na dal de ve lop me nt and fer ti li ty in hu ma ns Ivana Zec 1*, Dubravka Tislaric-Medenjak 1, Zeljka Bu ko vec Meg la 1, Iva na Kucak 2 1 Laboratory of Endocrinology, Clinics of Oncology and Nuclear Medicine, Medical School University Hospital Sestre Milosrdnice, Zagreb, Croatia 2 Clinical Institute of Chemistry, Medical School University Hospital Sestre Milosrdnice, Zagreb, Croatia *Corresponding author: ivana.zec@kbcsm.hr Abstract An ti-mülle rian hor mo ne (AMH) is a glycop ro tein be lon gi ng to the tran sfor mi ng growth fac to rs (TGF-β). AMH plays a fun da men tal ro le in the regres sion of Mülle rian duc ts in ma le em bryo. In its ab sen ce, Mülle rian duc ts de ve lop in to fe ma le in ner rep ro duc ti ve or ga ns. In boys, it is sig ni fi can tly pro du ced in Ser to li cel ls of tes tes un til pu ber ty and then slowly dec rea ses to re si dual va lues for the re st of the me n s li fe. AMH ser ves as a bioc he mical mar ker of the pre sen ce of tes tes in cryptor chi dic ma les. In fe ma les, AMH is sec re ted by gra nu lo sa cel ls of sma ll fol lic les in the ova ry. Se rum va lues ar e alm os t undete ct able dur ing inf anc y an d t hen r ap idly inc r eas e w i t h t he o n s e t of p u b er t y, r e fl e c t i ng t he ini tial rec ruit me nt of pri mor dial fol lic les. AMH is pro du ced in growi ng fol lic les un til they rea ch a sta ge when do mi na nt fol lic le is de tac hed from a co ho rt of an tral fol lic les. The mea su re me nt of s e r um A MH levels dur ing woman s r epr oduct ive life rep re sen ts an ideal tool for the as ses sme nt of the ova rian follicular reserve. The advantage of AMH in relation to the ovarian steroid hormones is that serum levels do not fluctuate significantly during the men strual cycle. In ad di tion, cir cu la ting AMH strongly correlates with antral follicle count (AFC), visualized by ultrasound in the follicular phase of the cycle. As the num ber and qua li ty of the oo cytes di mi ni sh throug hout the wo ma n s rep ro duc tive life, serum concentrations of AMH gradually decrease and fall below detectable levels in m enop aus e. T his co uld b e of p art ic ular inter es t in subf ert ile an d in f er t i le wo m en un der g oi ng as sis te d rep ro duc ti ve tec hniques (ART) in ac hie vi ng preg nan cy. Key wor ds: ant i - Müller ian ho rm one; ovar ian r es erve; sp ermato g e ne sis; in f er t i li t y; s e x- di ff e r en t ia t io n Received: June 28, 2011 Accepted: September 7, 2011 In tro duc tion An ti-mülle rian hor mo ne (AMH) is a 140-kDa di meric glycop ro tein hor mo ne and be lon gs to the transforming growth factor-β (TGF-β) family. AMH is synthe si zed as a lar ge pre cur sor wi th a sho rt sig nal sequence followed by the pre-prohormone that forms homodimers. Prior to secretion, the mature hormone undergoes glycosylation and dimerization to pro du ce a 144-kDa di mer com po sed of identical disulphide-linked 72-kDa monomer subunits. Each monomer contains an N-terminal domain (al so cal led the pro re gio n ) and a C-ter minal do main (al so cal led the ma tu re re gio n ); it is believed that N-terminal domain accentuates the acti vi t y of the C-ter mi nal do main in whi ch re si des the bioac ti vi ty of the mo le cu le (1). Du ri ng cytop - las mic tran sit, be tween 5 and 20% of AMH is cleaved at a spe ci fic si te be tween the N-ter mi nal and the C-ter mi nal do main of the 72-kDa mo no mer, to fo rm two po lypep ti des of 58-kDa (pro re gion) and 12-kDa (ma tu re re gion). The se two par ts of the mo le cu le re main in non co va le nt at t ac hme nt. T he hu man ge ne co di ng for AMH is lo ca ted on the sho rt arm of chro mo so me 19; it has been sequenced and iso la ted (2). AMH signaling pathway Tar get or ga ns for AMH in ma les are Mülle rian ducts and, in bo th sexes, go na ds. The struc tu re of the 219

2 specific re cep tor for AMH has been iso la ted and cha rac te ri zed (3). As a mem ber of the TGFβ fa mi ly of growth fac to rs, it is thoug ht that AMH uses the sig nal tran sduc tion system that has been iden tified for the ot her fac to rs of the fa mi ly, no tab ly TGFβ itself, activin and the bone morphogenetic proteins (BMPs). These factors signal through a serine threonine ki na se re cep tor com plex con sis ti - ng of ligand -sp eci fic type II re cep to rs and mo re general type I receptors, also known as activin re cep tor- like protein k i na ses (AL K s). An ac ti va te d receptor complex phosphorylates and activates cytop las mic Smad pro tei ns that tran slo ca te to the nucleus and directly or indirectly affe ct ge ne expres sion (Figu re 1). For AMH one type II re cep tor has been iden ti fied (AMHRII) and shown to be specific and ne ces sa ry for AMH sig na li ng (4). The ge ne co di ng for AMH re cep tor is lo ca ted on the lo ng arm of chro mo so me 12 (5). The AMHRII ge ne is specifi cal ly expres sed in the go na ds and in the mesenchymal cells adjacent to the Müllerian ducts. Besides the exclusive AMHRII, three candidate AMH t y p eireceptor s ha ve b e en iden ti fied to be involved in AMH-induced Müllerian duct regression. However, the relative contribution of these three type I re cep to rs to AMH sig na li ng in the ovary remains to be determined (6). AMH-RI Smad proteins Gene expression AMH AMH-RII FI GU RE 1. AMH re cep tor bin di ng. The pri ma ry re cep tor, type II, bin ds its li ga nd and is co-expres sed wi th type I re cep tor which ser ve as a sig nal tran sdu cer. Af ter bei ng ac ti va ted, the type I re cep tor is phos pho ryla ted by AMH-RII. The ac ti va ted type I re cep tor, in tu rn, phos pho ryla tes downstream Sma ds pro tei ns. Ph o sp h or y l ate d r e ce pto r- sp e ci fi c Sma ds tran slo ca te to the nucleus whe re they re gu la te ge ne expres sion (60). AMH throug hout the hu man li fe Prenatal stage AMH plays fun da men tal ro le in fe tal sex differentiation. Before the 7 th week of ges ta tion, ma le and fe ma le fe tu ses ha ve in diffe re nt go na ds, bi po ten - tial exter nal ge ni ta lia, and two pai rs of uni po tential in ter nal duc ts (the Mülle rian duc ts and the Wolffian duc ts). In the XY fe tus, the expres sion of the SRY ge ne trig ge r s tes ti cu lar differentiation (7). From the 8 th gestational week, the somatic cells of the developing testes, the Leydig cells and Sertoli cells, produce three hormones that are essential for the cor re ct differentiation of the individual in the male direction. The steroid hormone testosterone, produced by the Leydig cells, stimulates development of the male sex characteristics, such as differentiation of the Wolffian duc ts in to epididymides, vasa deferentia and seminal vesicles (7). Insulin-li ke fac tor 3, a mem ber of the re laxin in su lin fami ly, is al so pro du ced by the Leydig cel ls, and is an es sen tial sig nal for the fir st pha se of tes tes des cent. AMH is pro du ced by the Ser to li cel ls of the testes and is res pon sib le for the reg res sion of the Mülle rian duc ts that in the fe ma le fe tus differentia te in to the ovi duc ts, the ute rus and the up per p or tion of the va gi na (8,9). Ab se nt du ri ng fe ma le sex differentiation, AMH is first expressed after 36 we e k s of in trau te ri ne li fe in the gra nu lo s a cel ls of ovaries (10). From bir th to pu ber ty In pre pu ber tal boys, Leydig cel ls pro du ce low amoun ts of tes tos te ro ne. Ser to li cel ls are sti ll immature and spermiogenesis is arrested in a premeiotic stage, while AMH remains secreted at high level till the onset of puberty (9). Therefore, serum AMH in ma le in fan ts reflec ts the fun ction of tes tes re liab ly and can al so be used in cryptor chi dic males as the ini tial la bo ra to ry te st to as se ss the presen ce of the tes tes (9). In newborn girls, ovaries contain primordial follicle pool con tai ni ng 1-2 mil lion of oo cytes ar res ted in the dip lo te ne sta ge of meio tic prop ha se I and surroun ded by flat te ne d pre - gra nu lo s a cel ls (11). T he - se pri mor dial fol lic les stay in their ar res ted sta te for yea rs ti ll the on set of pu ber ty. AMH is expres sed in 220

3 granulosa cells of growing follicles and continues to be present throughout the reproductive ages (4). In girls, AMH circulating values are almost undetectable at birth with a slight increase within the fir st yea rs of age, prior to pu ber ty. Pu ber ty and adul thood In boys, wi th the on set of pu ber ty the sec re tion of hy p othalamic go na dot ro pin re lea si ng hor mo ne (GnRH) and both go na dot ro pi ns, lu tei ni zi ng hormo ne (LH) and fol lic le -s ti mu la ti ng hor mo ne (FSH), increase. Leydig cells undergo further differentiation. Testosterone s y nthe sis in crea ses, whi ch al so invo kes maturation of Ser to li cel ls. G er mi nal cel ls undergo meiosis and sp er ma to ge ne sis b e gi ns. T he inhibitor y effe ct of tes tos te ro ne pre vai ls over FSH s timulation, re sul ti ng in down - re gu la tion of AMH e xpression and the cir cu la ti ng le ve ls ra pid ly dec rea se. The sec re tion of AMH reac hes va lues in adult males and is maintained almost constant until the re st of me n s li fe (9). In gir ls, the ri se of ova rian es tra diol synthe sis and acti va tion of the hy p ot ha la mus pi tui t a r y ova rian axis oc cu rs wi th the on set of pu ber ty. Oo cytes at the first meiotic prophase undergo atresia or resume meiosis after ac ti va tion by an ovu la tio n - in du - ci ng LH sur ge to fo rm the hap loid ga me te for fer tili za tion (11). AMH is pro du ced by ova ries and secre ted in to the blood stream. It is fir st expres sed in granulosa cells of the recruited primordial follicles and con ti nues to be expres sed in the growi ng follic les in the ova ries un til they ha ve reac hed the size of about 4-6 mm. At that differentiation state, usually one follicle is selected for dominance and its fur ther growth is con trol led by ac tion of pi tui tary FSH (12). Pos sib le ac tio ns of AMH in the ova ry are pre sen ted in figure 2. Physio lo gy and pat hop hysio lo gy of AMH in ma les An ti-mülle rian hor mo ne is the ear lie st Ser to li ce ll hormone secreted in males and, together with inhi bin B and FSH, is an im por ta nt in di ca tor of Ser toli cell function (13). The postnatal proliferation of Ser to li cells is essen tial for sper ma to ge ne sis, du ri - Posible actions of AMH in the ovary: Inhibition of follicular activation and growth Inhibition of FSH stimulated growth Inhibition of granulosa cell growth Inhibition of aromatase FSH primordial AMH AMH primary preantral antral FI GU RE 2. Model of AMH ac tio ns in the ova ry. AMH is expressed in pri ma ry, pre-an tral and sma ll an tral fol lic les. Ini tial recruit me nt of pri mor dial fol lic les ta kes pla ce as a con ti nuous proce ss, whe reas cyclic rec ruit me nt is dri ven by a ri se in FSH se rum le ve ls at the end of a pre vious men strual cycle. AMH in hi bi ts ( ) the ini tial rec ruit me nt of pri ma ry fol lic les from the res ti ng primor dial fol lic le pool and the sti mu la to ry effe ct of FSH on the growth of prean tral and sma ll an tral fol lic les (+) (4). ng whi ch the tran sie nt GnRH sur ge oc cu rs. In the fir st wee ks of newbo rn ma le, the in fan ti le GnRH sur ge in du ce a sig ni ficant increase of gonadotropi ns (LH and FSH), fol lowed by the in crea sed testosterone production in Leydig cells, as well as AMH and in hi bin B in Ser to li cel ls of tes tes (9). These unexpected high levels of androgens and AMH in the fir st mon ths af ter bir th are explai ned by the physio lo gi cal an dro gen in sen si ti vi t y of Ser to li cel ls in the se pe rio ds due to the la ck of an dro gen receptor expression in Sertoli cell nuclei (9). This sta ge, so cal led mi ni-pu ber ty, has been propo sed as the fir s t op p or tu ni t y to de te c t pa tien t s with hypogonadotropic hypogonadism (HH), since it can not be ob ser ved in boys wi th con ge ni tal HH (14). Thus, dec rea sed AMH se rum le vel, com pared to that in in fan ts of the cor res pon di ng age, is an early biochemical marker of congenital central hy p o go na di sm. AMH leve ls re main hi gh du ri ng the whole prepubertal phase and are downregulated in puberty by increased testosterone levels. Se rum AMH levels in males, ac cor di ng to age, are shown in tab le 1 (15)

4 TAB LE 1. Se rum AMH in nor mal ma les. Age group N Serum AMH (pmol/l) Mean ± SEM* 95% Cl** <15 days ± days 1 year ± yea rs ± yea rs ± yea rs ± >9 yea rs ***I ± >9 yea rs *** II ± >9 yea rs *** III 8 79 ± >9 yea rs *** IV V 8 48 ± Adul ts ± * SEM stan da rd er ror of the mean = SD/(square root of sam ple si ze) ** 95% con fi den ce in ter val *** Pu ber tal sta ges ac cor di ng to Mar sha ll and Tan ner (16). The as ses sme nt of AMH (a nd in hi bin B) can be useful in the differential diagnosis of constitutional de lay of growth and pu ber ty when AMH se rum levels remain at high prepubertal values, while low AMH (a nd in hi bin B) le ve ls at the age of physio logical puberty are indicative for the congenital hypogonadism (9). Klinefelter syndrome is the most common congenital cause of hypergonadotropic hypogonadism. The main clinical features include gynecomastia, sma ll testes, abse nt sp er ma to ge ne sis, nor mal or re du ced Leydig ce ll fun ction and ele va ted FSH levels. This disorder is caused by a supernumerary X chro mosome (k a r yo t y p e 47, X X Y or mo s ai ci sm 46,XY/47,XXY). At the ti me when nor mal pu ber ty is e xp e cte d, lab orato r y findings are characterized by the significantly increased FSH and LH levels, low testosterone, decreased AMH and undetectable inhibin B serum levels (17). Assessment of AMH (and inhibin B) represents a promissory approach in other causes of male hypogonadism. For example, acquired hypergonadotropic hypogonadism can be caused by autoimmune disea ses, che mot he ra py and /or ra dia - tion therapy for ma lig na nt ne op la sm that affe ct testicular tissue and function (18). T he mea su re me nt of cir cu la ti ng AMH can al so b e use f ul in in f an t s wi th bi la te ral no n - pal pab le go nad (cryptor chi di sm) as evi den ce that the tes tes can be present but not descended. If necessary, the hcg stimulation test is recommended to assess the presence of functioning testicular tissue and the detection of defects in testosterone biosynthesis by Leydig cel ls and its ac tion (18). AMH see ms to be a good can di da te mar ker for incom ple te or re du ce d sp er ma to ge ne sis sin ce it is specifi cal ly sec re ted by Ser to li cel ls in bo th se rum and seminal fluid in detectable concentrations. Serum AMH see ms to be sig ni fi can tly lower in no n- obstructive azoospermic men than in those with obstructive azoospermia and normal fertile man. H owe ver, the wi de over lap pi ng of va lues b e t we en sub jec ts pre ven ts this hor mo ne from bei ng of clinical utility (19). If Ser to li cel ls are unab le to pro du ce AMH, be cau se of mu ta tio ns in the AMH ge ne, or if tar get or ga ns are unresponsive to its action through mutations of the AMHRII ge ne, Mülle rian du c t de ri va ti ves wi ll be retained in genetic male, otherwise normally mas cu li ni zed. This con di tion is known as the persis te nt Mülle rian du ct syndro me (PMDS). In pa tients wi th mu ta tio ns of the AMH ge ne, le ve ls of cir cu - lati ng AMH are extremely low, even be fo re pu berty, when AMH le ve ls are nor mal ly hi gh. AMH concentrations are higher in patients with receptor type II (AMHRII) gene mutations (20). The ro le of AMH in ova rian re ser ve evaluation AMH is pro du ced by gra nu lo sa cel ls of growi ng follicles, from pre-antral to antral stage, until they rea ch the si ze of 4-6 mm (21). AMH expres sion disappears in follicle of increasing size and is almost lo st in fol lic les lar ger than 8 mm (22). The main physio lo gi cal ro le of AMH in the ova ry see ms to be targeted to the inhibition of primordial follicle recruitment, thus preventing too early depletion of the follicular reserve. Both in vi vo and in vit ro experi men ts ha ve in di ca ted that the tran si tion from pri - mordial into growing follicles becomes enhanced in ab sen ce of AMH (23). Fur ther mo re, the in hi bition of sen si ti vi ty to FSH by AMH is a de ci si ve step 222

5 in fol li cu lar se lec tion and it may oc cur due to the variable expression of AMH receptors among the recruited small pre-antral follicles. In such circumstances, out of the initially recruited follicle units, on ly tho se wi th lower AMH expres sion be co me sen si ti ve to FSH, of whi ch usual ly one is per mit ted for dominance. Therefore, AMH acts as an autocrine f actor that regu la tes do mi na nt fol lic le se le c tion. Cur re nt theo ries al so sug ge st a ro le for AMH as a co - re gulator of s te roi do ge ne sis in gra nu lo s a cel ls, as AMH le ve ls ap pear to be re la ted to es tra diol levels in follicular fluid from sma ll an tral fol lic les (24). This is con fir med in a stu dy whi ch showed that polymor phis ms in the ge ne for AMH or AMHRII seem to b e relate d to fol li cu lar pha se es tra diol le ve ls, sug ges ti ng a ro le for AMH in the FSH-in du ced steroidogenesis in the human ovary (25). The expres sion of AMH in pre-an tral and sma ll antral follicles mainly contributes to its serum levels du ri ng the reproduc ti ve li fe. An ac ti ve co ho r t of growing follicles represents the antral follicle count (AFC), whi ch al so ser ves as a mar ker of ova rian reserve and strongly correlates with serum AMH leve ls (26). Despite this cor re la tion, AFC poor ly refle ct the pre-an tral fol lic le pool and, mo reo ver, requires state-of-the-art ultrasound machine and e xp e rien ce d ul tra so nog rap hi st (27). Therefore, AMH may provide a more accurate assessment of the oo cyte/follicle pool. Con tra ry to the ot her ovarian re ser ve mar ke rs, AMH can be mea su red at any ti me du ri ng the men strual cycle due to in sig ni fi cant intra-cycle variability, which is a great advantage in clinical practice (28,29). In the longitudinal stu dy of se rum AMH le ve ls in whi te, bla ck and Hispanic women, authors found certain, but insignificant differences in measured values in relation to ra ce/ethnicity (30). AMH le ve ls are not chan ged during pregnancy, during which endogenous gonadotropin release is substantially diminished (31). In addition, AMH le ve ls re main s t ab le du ri ng oral contraceptive administration (32). These findings are consistent with the concept that AMH levels reflect the continuous FSH-independent noncyclic growth of sma ll fol lic les in the ova ry. Hen ce, AMH is a unique en doc ri ne pa ra me ter for the investigation of the ovarian reserve (33). Still, the Freeman s cross-sectional study in healthy, regularly menstruating women has shown a signifi ca nt in ver se as so cia tion of AMH se rum le ve ls and bo dy-ma ss in dex (BMI) (34). In obe se wo men, mean AMH le ve ls we re 65% lower than AMH le ve ls of no n - o b e se wo men. In ad di tion, a mul ti va riab le regression analysis of independent association of va riab les BMI, age, me no pau sal status and ra ce with AMH con firmed that BMI remained significantly associated with AMH levels. The authors suggested two pos sib le expla na tio ns for the finding: either obesity may be associated with decreased ovarian reserve, or obesity is associated with follicular dysfun ction. The weak ne ss of the stu dy is that blood sam ples we re se lec ted in a co ho rt of women in la te rep ro duc ti ve ages (35-47 yea rs) and the distribution of studied variables may not be representative for the entire range of reproductive life. AMH as in di ca tor of ova rian agi ng throughout reproductive life Tren ds in mo de rn so cie ties ha ve been ac com plished also with maternity postponement and the increasing demand for assisted reproduction techniques (ART) (35). Be cau se of a con si de rab le proportion of subfertile women, the assessment of ovarian reserve becomes of crucial interest in women presenting to fertility clinics. As a wo man ages the re is a gra dual dec rea se in b o th quan ti t y and qua li t y of the o o c y tes re si di ng within follicles, accompanied by a concomitant dec rea se in AMH se rum con cen tra tion. It has been de mon s tra te d that AMH le ve ls cor re la te s tron gly wi th AFC and are mo re re liab le in pre dic ti ng oocyte sup ply than wo man s age and ot her con ventional serum markers (FSH, estradiol) (36). Investigation of age-dependent reference values for AMH should provide a better insight in cutoff le vels throug hout the rep ro duc ti ve li fe of wo men. Re - cen tly, in two exten si ve stu dies, age-spe ci fic dis tribu tio ns of AMH in fe ma le po pu la tion we re exa mi - ned (37,38). In wo men, be tween 25 and 35 yea rs of age, the ave ra ge dec rea se in the me dian se rum AMH va lue is 1.4 pmol/l (0.2 ng/ml) per year, while after age of 35, the average yearly decrease is

6 pmol/l (0.1 ng/ml). The ra te of dec li ne in mean AMH va lues is 1.4 pmol/l (0.2 ng/ml) per year throu gh age 40 and di mi nis hes to 0.7 pmol/l (0.1 ng/ml) per year the reaf ter (37). During the years before the final menstrual period, o o c y tes undergo an ac ce le ra te d ra te of lo ss un til their pool is mos tly dep le ted. A hypot he sis stan ds that this pro ce ss is ini tia ted when the to tal of number of oocytes reaches approximately 25,000 and that woman, under physiological condition, reaches this thres ho ld at about yea rs of age (42). At me no pau se, the num ber of oo cytes has dec reased to so me hun dre ds (43). Perhaps, one of the outstanding challenges nowadays is the la ck of re fe ren ce va lues of AMH in healthy female p opula tion of rep ro duc ti ve age (39). This is mos tly due to the fa ct that the da ta ha ve been obtained from infertility clinics and/or center s for reproductive me di ci ne whe re the ma jo ri t y of women undergo la bo ra to ry tes ts for ART pro ce du - res. Shebl and coworkers presented basal serum AMH le ve ls in wo men who at ten ded an ART program be cau se of ma le fac tor (38). This pre su mab ly heal thy cohor t con sis te d of ovu la to r y wo men in reproductive ages, without issues related to abnor mal ova rian fun ction (e.g., tu bal fac tor, en dometriosis, polycystic ovaries). Evaluated median concentrations and confi den ce in ter va ls for ba s al se rum AMH ac cor di ng to age are shown in tab le 2 (supplementary data available online). Anot her two stu dies from Scot la nd ha ve been pub lishe d mos t re cen tly, pro p o si ng a p o pu la tion model for serum AMH levels (40,41). According to the observations, AMH values exhibit the greatest hete rogeneit y and spread in ab so lu te values in youn ger wo men, sug ges ti ng that they are pre senting with a wider range of development of AMHexpressing follicular co hor ts as com pa red wi th ol - der wo men. Al so, it may be that they exhi bit differen tial AMH expres sion for their age mat ched fol licu lar pool (40). The mo del of the as so cia tion of AMH concentrations with age confir med a non linear dec rea si ng ra te of AMH wi th age; the no mogram for wo men be tween 25 and 45 yea rs is be st des cri bed by a quad ra tic equa tion: log AMH = a + b x Age + c x Age 2. In this equa tion, log AMH is natural logarithm of AMH, a = (95% con fidence in ter val CI, to 1.973), b = ( to ) and c = ( to ) (40). In the subsequent study, Kelsey and coworkers generated the first validated model of serum AMH in healthy females from conception to menopause; the re sul ts ha ve been de ri ved from 3,260 da ta extracted from previously published control and/ or pros p e c ti ve s tu dies in heal thy fe ma le sub je c t s (41). They ha ve even in clu ded the da ta from co rd blood of pre te rm in fan ts up to tho se in fe ma les of 54.3 yea rs of age. As to the mo del of se rum AMH through the females life, concentrations fall shortly af ter bir th, in crea se slig htly at about two yea rs of age and then ri se sig ni ficantly with the onset of pu ber ty. In te res tin gly, the aut ho rs sug ge st that AMH se rum le ve ls rea ch a peak at about 24.5 yea rs of age and then gra dual ly dec rea se, in a non li near manner, through the period of women s reproductive life (41). For yea rs, se rum le ve ls of FSH and es tra diol ha ve been the de ci si ve bioc he mi cal mar ke rs in as sessment of diminishing ovarian function, but FSH ten ds to ri se over ba sal va lues in fol li cu lar pha se of the men strual cycle on ly when ova rian re ser ve is significantly exhausted and ovarian function already com pro mi se d (4 4). M o re f a vo rab le re sul t s ha ve been ob tai ned usi ng AMH as a mar ker, for it reflects the follicular cohort as a whole, including still inactive follicles and those being transited from pri mor dial to the ear ly an tral sta ge. It has been assu med that a thres ho ld of 2.8 pmol/l (0.39 ng/ml) for AMH has the optimal combined sensitivity and specifi ci ty for pre dic tion of the on set of me no pause in six years, with a positive predictive value of 0.90 (95% CI, ) and ne ga ti ve pre dic ti ve value of 0.76 (95% CI, ), res pec ti ve ly (45). AMH as a mar ker of pre ma tu re ova rian senescence It is con si de re d that fol li cu lar e xhaus tion b e co mes ob vious in the four th de ca de of wo man s li fe when chan ges in the go na dal en vi ron me nt oc cur, al though the ma jo ri ty of wo men of that age con ti nue wi th ap pa ren tly nor mal men strual cycles (46). The mo st re ce nt stu dy in a po pu la tion of pre su mab ly heal thy wo men has shown that in ap proxi ma te ly 5% of wo- 224

7 men of all ages, AMH se rum le ve ls are 7 pmol/l (1 ng/ml) (38). This finding has undoubtedly pointed to the fa ct that all age sub grou ps com pri se wo men wi th a re du ced ova rian re ser ve and that even young women are at risk of decreased reproductive potential. Premature ovarian failure is clinically defi ned as the com ple te ab sen ce of men strual cycles be fo re the age of 40, wi th FSH le ve ls excee di ng 40 IU/L and AMH levels below the menopausal threshold or even un de tec tab le in mo st of the ca ses (46). Pre mature ovarian ageing represents a milder degree of gonadal dysfunction which is characterized with ba se li ne FSH < 12 IU/L and/or ab nor mal ly low agespecific AMH (46,47). It has been ar gued that AMH offers better clinical specifi ci ty for the rea son that FSH is mos tly reflec ti ve of la te -sta ge, go na dot ro pi n- sensitive, follicles preceding ovulation (48). In another wor ds, AMH and FSH differently reflect ovarian reserve at different stages of life. The prestigious results in the recent Gleicher s studies postulate that the increasing triple CGG (Cytosine-Guanine-Guanine) repeats on the FMR1 (fra gile X) ge ne cor re la te wi th spe ci fic ovarian reserve parameters, and are associated with premature ovarian senescence and infertility in young women (49). The sig ni ficant associations between FMR1 genotypes and AMH levels could allow predictions about the functional ovarian reserve alrea dy in you ng ages (50). AMH as a pre dic tor of suc ce ss in as sis ted reproduction techniques (ART) T he reproducibilit y of AMH b e t we en con se cu ti ve cycles in the sa me wo man, in dis tin ction from FSH, estradiol and inhibin B, permits clinicians to have a reliable serum marker in prediction of ovarian response in assisted reproduction cycles. Much data show a s trong and p o si ti ve cor re la tion b e t we en ba sal AMH se rum le ve ls and the num ber of ret rieved oocytes in women undergoing ovarian stimula tion (35). Mo reo ver, ba sal se rum AMH va lue and an tral fol lic le cou nt (AFC) ha ve shown a si mi lar power in pre dic tion of the num ber of ret rie ved oocytes (35,51). In me ta-a na lysis of about 30 stu dies, receiver operating characteristic (ROC) curves for the prediction of ovarian response indicated no significant difference between the performances of AMH and AFC (52). The num ber of de ve lo ped fol lic les and the num ber of ret rie ved oo cytes are two of the mo st im por ta nt cri te ria for de fining poor response in women undergoing ovarian stimulation for in-vitro fertilization (IVF) (35). For instance, Barad and co-workers found that AMH cut-off va lue of 3.5 pmol/l (0.5 ng/ml) cou ld predict the retrieval of less than four oocytes with a spe ci fi ci ty of 84% and sen si ti vi ty of 87% (48). At this dis cri mi na to r y AMH va lue, the p o si ti ve and ne ga ti ve pre dic ti ve va lues we re 79.4% and 9 0 %, res pec ti ve ly. In con tra st, a va lue of FSH > 12 IU/L had sensitivity of 64.5% and specificity of 82.2%, wi th p o si ti ve and ne ga ti ve pre dic ti ve va lues of 71.4% and 77%, res pec ti ve ly (51). Ulti ma te ly im por ta nt is, howe ver, whet her as sessme nt of AMH can pre di ct chan ce for preg nan cy and li ve bir ths in IVF cycles. In the next stu dy of the mentioned authors, maximal ROC inflec tio ns, which differentiate between better and poorer live bir th chan ces in wo men wi th di mi nis hed ova - rian re ser ve (DOR), we re at AMH cu t-o ff < 7.5 pmol/l (1.05 ng/ml). Sti ll, it was de mon stra ted that cli ni cal preg nan cies cou ld be es tab lis hed at all AMH le vel, even in the ab sen ce of de tec tab le AMH (53). AMH as a mar ker of ova rian pat hop hysio lo gy Polycystic ovarian syndrome (PCOS) PCOS is the most frequent endocrine disorder of ova rian fun ction, wi th a pre va len ce of 6-7% among wo men of rep ro duc ti ve age (54). It has been known for yea rs that it rep re sen ts mo re com plex entity than solely an ovarian dysfunction. Increased androgen synthesis, disrupted folliculogenesis and insulin resistance are the manifestation of endocrine/reproductive and metabolic disturbances of the impaired molecular mechanisms known to underlie PCOS. According to the Rotterdam consensus, the diagnosis should meet at least two, of the three following criteria: chronic anovulation or oli go me nor r hea, cli ni cal or bioc he mi cal hype ran - drogenism and polycystic ovarian morphology (55). An ultrasound examination reveals increased 225

8 ova rian vo lu me and/or the pre sen ce of 12 fol licles in ea ch ova ry. It has been hypot he si zed that the increased intrafollicular AMH levels, found in PCOS, lower the follicle sensitivity to circulating FSH, thus preventing follicle selection and resulting in fol lic le ar re st at the sma ll an tral pha se (2-5 mm) (56). The ba lan ce be tween FSH and AMH might be cru cial for aro ma ta se ac ti vi ty at the ti me and after the selection of the dominant follicle. Decreased FSH action inhi bi ts aro ma ta se ac ti vi ty in synergy wi th AMH, hin de ri ng the con ver sion of an drogens to estrogens, thus contributing to ovarian androgen excess and reduction of follicular production of estradiol. The resulting low levels of estradiol may al so con tri bu te to the fai lu re of fol lic le se lection. In crea sed AMH le ve ls may al so act di rec tly on theca cells to stimulate androgen synthesis (54). Cir cu lating AMH le ve ls in wo men wi th PCOS are two to three ti mes hig her than in heal thy con tro ls, corresponding to the increase in the number of small follicles in ovaries (54). As a diag nos tic mar ker of PCOS, AMH has been fou nd to offer a re la ti ve ly hi gh spe ci ficity and sensitivity (92% and 67%, respectively) (56). Moreover, eva lua tion of ba sal AMH le ve ls in wo men wi th PCOS plays an im por ta nt ro le in pre dic ti ng the ri sk for ovarian hyper-response and informing a clinician to direc t the ap pli ca tion of mi ld s ti mu la tion protocols in order to avoid ovarian hyperstimulation syndro me (OHSS) (35,36). Po lymor phis ms of AMH and AMHRII ge nes as causes of infertility Two ge nes are res pon sib le for the AMH sig nal transduction pathway (AMH ge ne and its re cep tor AM- HRII ge ne), con firming their association with follicu lar pha se es tra diol le vel (25). AMH is pro du ced by granulosa cells in early developing follicles, which indicates its role during the initiation of primordial fol lic le growth and in re gu la tion of FSH sen si tivi ty. As shown in a re ce nt stu dy, ge ne tic va rian ts of AMH and AMHRII ge nes seem to be as so cia ted with idiopathic infertility in normo-estrogenic and normo - ovulator y wo men (57). T he re sul t s sug ge s t that AMH polymorphisms could modify hormone bio lo gical activities, playi ng a ro le in con trol li ng follicle recruitment and growth. Although in the sma ll sam ple si ze, the ge no typi ng has shown that al le le frequen cies of 482 A>G, IVS 5 6 C>T, IVS A>G, 146T>G polymorphisms are statistically significantly increased in the infertile women compared with the controls (57). Although it seems plausible that polymorphisms of AMH and AM- HRII mig ht re du ce the hor mo ne bio lo gic ac ti vi t y, ma ki ng fol lic les mo re sen si ti ve to FSH, fur ther investigations are expected to clarify their association with unexplained infertility in women. Müllerian aplasia or Mayer-Rokitansky-Küster- Hauser syndrome Müllerian agenesis, also named the Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS), is the second most common cause of primary amenorrhea (58). Al thou gh the etio lo gy of the syndro me remains unclear, evidences suggest that MRKHS is tran smit te d in an au to so mal do mi na nt man ner causing defects in prenatal development in the female genital tract. It is characterized by the congenital absence of the Müllerian structures including the Fal lo pian tu bes, the ute rus, and the in ter nal por tion of the va gi na in an ot he rwi se chro mo somally normal 46,XX subject. Females with MRKHS have functioning ovaries and normal external genitalia. Still, molecular analyses of AMH and AMHRII genes have not detected any deleterious mutations res pon sib le for the syndro me (59). This finding refle c t s nor mal ly re gu la te d AMH gene promoters, tran scrip tion and AMH pro tein pro duc tion in adu lt MRKH pa tien ts. As blood AMH le ve ls dec rea se over time and are associated with the number of remaining antral follicles, AMH therefore represents a specific mar ker for ova rian age and cou ld be helpful for predicting successful IVF or surrogacy pregnancies in such cases (58). AMH as a mar ker of gra nu lo sa ce ll tu mo rs The sex co rd stro mal neop las ms are de ri ved from me sen chymal stem cel ls in the ova rian cor tex; they include granulosa theca cell tumors and granulosa ce ll tu mo rs (GCTs), whi ch con sti tu te 1-3% of all ova rian tu mo rs (60,61). Two dis tin ct types of GCTs ha ve been des cri bed on the ba sis of cli ni cal presentation and histological characteristics: the juveni le and the adu lt fo rm (60). GCTs are hor mo nese c re ti ng ne op las ms; they may pro du ce es tra diol 226

9 and progesterone and they secrete peptide hormo nes, su ch as in hi bin and AMH (61). The re fo re, se rum AMH ser ves as bioc he mi cal mar ker in diagno sis and monito ri ng of the di sea se. Howe ver, se - rum AMH is ele va ted on ly in sex co rd stro mal tumo rs, whi le in hi bin may be ele va ted in different types of can cer (60). Glo bal ly, AMH se rum le ve ls seem to be ele va ted in 76-93% of GCT pa tien ts (61). Al thou gh AMH is con si de red a power ful in hibitor in granulos a ce ll pro li fe ra tion, s tu dies ha ve shown that the inhibitory signal is functional only in the ini tial sta ge of tu mor de ve lop me nt, but not when the tumor becomes progressive and reach a lar ge di men sion (60). One of the pos sib le theo ries is that, in progressive tumors, the ability of binding of AMHRII re cep tor by its li ga nd is lo st and that the inhibitory signal of AMH becomes ineffi cie nt (61). Un for tu na te ly, the agen ts used to treat the ma lignan cies are ab le to des troy a grea ter or les ser number of ova rian pri mor dial fol lic les in can cer sur vivors (62). Radiothe ra py and che mot he ra py, es pe - cial ly alkylating agen ts, cyclop hos pha mi de, bi sul - phan, melphalan, cytosine, procarbazine and cispla tin are extre me ly har mful to the ova rian re ser ve and fun ction (63). In su ch ca ses, the re is a de finite and unquantifiab le b e ne fit of mo ni to ri ng se rum AMH levels, because girls and young women are at increased risk of premature ovarian failure as we ll as of reduce d rep ro duc ti ve abi li t y. Ba th and coworker s have shown that, amo ng pa tien t s wi th regular menstrual cycles, cancer surviving women have signifi can tly lower ba sal AMH va lues than con tro ls (13.0 ± 3.0 vs ± 3.4 pmol/l; P < 0.05) and higher early follicular phase FSH levels (7.5 ± 1.4 vs. 4.2 ± 0.3 IU/L; P = 0.02) (63). In anot her stu dy, Par tri ge and cowor ke r s in ves ti ga te d pa ra me te r s of ova rian reserve in wo men trea te d wi th t a moxi fen and radiotherapy for early stage breast cancer (64). In uni va ria te lo gis tic ana lyses, AFC, FSH, AMH and inhibin B levels were all significantly different between survivors and controls, though in a multiple logistic regression analysis, AMH levels distinguished sur vi vo rs from con tro ls wi th the grea te st statistical difference (odds ratio 7.63) (64). Although the both studied groups, patients and controls, we re ade quately mat che d, the li mi t a tion of the study is that the parameters of ovarian reserve had not been measured before chemotherapy started. Development of immunoassays for AMH measurement The first reported AMH immunoassay used a pair of monoclonal antibodies raised against human re com bi na nt AMH, b o th di re c te d to epi to p es in the pro re gion (65). Howe ver, the use of this as say showed re sul ts wi th va ria bi li ty in AMH con cen tra - tions due to storage and freeze-thaw instability. The se co nd type of as say used a pair of mo noc lonal an ti bo dies, one di rec ted to the pro re gion and the ot her to the ma tu re re gion of the AMH mo lecule (61). A third assay, developed more recently, uses a monoclonal antibody pair directed to epitopes in the pro re gion (66). The new AMH Gen II assay uses a pair of monoclonal antibodies directed to epi to pes in the ma tu re re gion of AMH (67). The ad van ta ge of this as say is that mea su red va lues are not affec ted by pro teo lysis of AMH in the sam ple as the ma tu re re gion is mo re stab le again st proteo lysis com pa red to the pro re gion, in pa rt due to its multiple cystine residues (67). In addition, the Gen II as say mea su res AMH in hu man, mon key, bovine, other mammalian species with improved per for man ce. The lowe st amou nt of AMH in a sample that can be de tec ted wi th 95% pro ba bi li ty is 0.57 pmol/l (0.08 ng/ml). The mo noc lo nal an ti bodies are de ve lo ped again st the epi to pes of the matu re re gion of AMH whi ch pre ven ts any of cro ss reac tio ns wi th in hi bin A, LH and FSH at 2 ti mes their physiological concentration. In addition, potential interfering substances from serum matrix (he mo g lo bin, trig lyce ri des and bi li ru bin) do not de te rio ra te ac cep t ab le li mi t s of ± 10 % difference to the va lue of con trol spe ci men (68). AMH Gen II as say, de ve lo ped by Bec kman Coul ter Company (Beckman Coulter Inc., California, USA), uses an ti b o dies de ve lo p e d by Diag nos tic Sys te ms La bo ra to ries (DSL Inc., Texas, USA) and has been stan dar di zed to the Im mu no te ch ca lib ra tion (Im - munotech a.s., Check Republic) (67). The lowest amou nt of AMH in a sam ple that can be de tec ted with 95% pro ba bi li ty is 0.56 pmol/l (0.08 ng/ml). Passi ng-bab lok reg res sion ana lysis has shown a good cor re la tion wi th the Im mu no te ch AMH ELISA (r = 0.99; P < 0.001; y = 1.0x) (67). Yet, it has to be ta ken in to ac cou nt that la bo ra to ries im ple men ti ng Bec k- 227

10 man Coul ter AMH Gen II as say shou ld expe ct an increase in AMH values of approximately 40% (69). As it is a re la ti ve ly new as say, a need exis ts for in te r- labo rator y compa ri son in the fo rm of an ex ter nal qua li ty assurance sche me (70). Even mo re, la bo ra to - ries that have implemented AMH immunoassay in a rou ti ne practice shou ld en ro ll in the exter nal pro ficiency testing program. The latest has recently been established under UK NEQAS scheme design (71). Conclusion E x ten sive investiga tion of cli ni cal sig ni ficance of AMH serum values undoubtedly confir ms that it is not ju st a ha nd of bio lo gic wat ch. One may thi nk that the scien tis ts ha ve re sear ched all the con di tions when AMH ser ves as a bioc he mi cal mar ker of male and female physiology and patophysiology. Cer tain ly, we shou ld be awa re of an out stan di ng ro le of AMH in different stages of human life. Perha ps mo re pros pec ti ve lo ng te rm stu dies in healthy population are needed to evaluate the reliable re fe ren ce va lues ac cor di ng to ages, es p e cial ly in women within the reproductive life span. This could fa ci li ta te a cor re ct eva lua tion of ova rian re ser ve in in fer ti le sub jec ts, al low a mo re ac cu ra te pre diction of reproductive potential and improve counse li ng in par ti cu lar ca ses. Po ten tial con fli c t o f in te r e st None declared. References 1. Wil son C, Cle men te N, Eh ren fe ls C, Pe pin sky R, Jos so N, Vi gier B, et al. Mullerian inhibiting substance requires its N-terminal domain for maintenance of biological activity, a novel finding within the transforming growth-factor-beta superfamily. Mol En doc ri nol 1993;7: Pi ca rd JY, Be na rou st R, Guer rier D, Jos so N, Khan A. Clo ni ng and expres sion of cdna for an ti-mul le rian hor mo ne. 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