Gynecology-endocrinology

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1 Gyneclgy-endcrinlgy FERTILITY AND STERILITY Cpyright (<;') 1996 American Sciety fr Reprductive Medicine Vl. 65, N, 3, March 1996 Printed n acid-free paper in U. S. A. Circulating levels f fllistatin frm puberty t menpause*t L. Michael Kettel, M.D.:j: Luis V. DePal, Ph.D.II~ Arlene J. Mrales, M.D.:j: Dan Apter, M.D.:j:** Nichlas Ling, Ph.D.lltt Samuel S. C. Yen, M.D.:j::j::j: University f Califrnia-San Dieg, and the Whittier Institute fr Diabetes and Endcrinlgy, La Jlla, Califrnia Objective: T determine the changes in circulating levels f fllistatin, a binding prtein fr activin and inhibin, thrugh the reprductive life cycle in wmen. Design: An pen, prspective descriptive study. Setting: An academic endcrine research unit. Patients: Prepubertal (n = 1), midpubertal (n = 7), and pstpubertal (n = 25) (early adlescent) girls, nrmal cycling adult wmen (n = 8), pstmenpausal wmen (n = 17), and men (n = 13) were studied. Interventins: Nrmal cycling wmen were given Nal-Glu GnRH antagnist fr 3 days in the fllicular phase f the cycle. Main Outcme Measure: Serum cncentratins f fllistatin determined in a heterlgus RIA. Results: Mean fllistatin levels did nt change during puberty but were higher in adult and pstmenpausal wmen. Levels f immunreactive fllistatin in men were lwer than levels fund in nrmal cycling wmen and pstmenpausal wmen. Daily immunreactive fllistatin levels during the menstrual cycle remained cnstant and did nt change significantly after varian suppressin with GnRH antagnist. Cnclusin: Because dynamic changes f serum immunreactive fllistatin d nt ccur during varian activatin (puberty), suppressin, and age-related varian failure, the increase in immunreactive fllistatin levels in adult and pstmenpausal wmen may implicate surces ffllistatin ther than the vary. Fertil SteriI1996;65:472-6 Key Wrds: Fllistatin, activin, inhibin, menstrual cycle, puberty, menpause, Nal-Glu GnRH antagnist Inhibin and activin are nnsteridal peptides that have been lcalized thrughut the brain-pituitaryvarian axis and may be imprtant in the regulatin Received May 11, 1995; revised and accepted September 25, * Supprted by the Natinal Institute f Child Health and Human Develpment Center grant HD ; the University f Califrnia, San Dieg General Clinical Research Center, Natinal Institute f Health Divisin f Research Resurces grant MOl 827, Bethesda, Maryland; and in part by the Claytn Fundatin fr Research, Califrnia Divisin, La Jlla, Califrnia. t Presented in part at the 39th Annual Meeting f the Sciety fr Gyneclgic Investigatin, San Antni, Texas, March 18 t 21, :j: Department f Reprductive Medicine, University f Califrnia-San Dieg. Reprint requests: L. Michael Kettel, M.D., University f Califrnia-San Dieg Medical Center (8433), 2 West Arbr Street, San Dieg, Califrnia (FAX: ). 472 Kettel et al. Fllistatin levels in wmen f reprductive functin (1). Inhibin has specific FSH inhibiting actin and activin has FSH stimulating prperties (1). In additin, a nvel prtein with FSHinhibiting prperties was islated frm varian fllicular fluid f several species and was named fllistatin (2-4). Fllistatin is a single-chain glycsylated prtein with n structural similarity t the inhibin-activin family f peptides_ II Department f Mlecular Endcrinlgy, Whittier Institute fr Diabetes and Endcrinlgy. ~ Present address: Reprductive Sciences Branch, CPR, Natinal Institute f Child Health and Human Develpment (NICHD), Bethesda, Maryland. ** Present address: Department f Obstetrics and Gyneclgy, University f Helsinki, Helsinki, Finland. tt Present address: Neurcrine Bisciences, Inc., San Dieg, Califrnia. :j::j: S.s.C. Yen is a Claytn Fundatin Investigatr.

2 Recently, fllistatin has been shwn t functin as a fj subunit-specific binding prtein fr activin (fjfj) and inhibin (afj) (5,6). As such, fllistatin may participate in mdulating the wide range f physilgic functins f activin and inhibin (7-11). There is evidence that fllistatin, alng with activin and inhibin, may be imprtant in the regulatin f fllicular selectin and atresia (1-13). Cyclic changes in fllistatin and its messenger RNA (mrna) have been described during the rat estrus cycle with maximal expressin ffllistatin message in tertiary fllicles. On the ther hand, fllistatin prtein was detected nly in selected fllicles and nt in atretic fllicles (12). In human varies, we have described fllistatin gene expressin in granulsa and theca cells f bth small and large antral fllicles, suggesting an autcrine and/r paracrine rle fr fllistatin in flliculgenesis (14). Fllistatin is present in the peripheral circulatin in nrmally cycling wmen (11, 15). When measured in the serum f wmen underging varian hyperstimulatin fr IVF, fllistatin levels were fund t increase twfld t threefld in parallel with serum E2 (15). Circulating levels f immunreactive inhibin change in a dynamic, well-characterized pattern during the menstrual cycle (16, 17), whereas activin levels remain unchanged (18). In view f the imprtance f fllistatin as a ptential endcrine mdulatr fr inhibin and activin, we measured circulating fllistatin levels during puberty, thrughut the nrmal menstrual cycle, in pstmenpausal wmen, and in men. In additin, the effects f varian suppressin with Nal-Glu GnRH antagnist n fllistatin levels was assessed. MATERIALS AND METHODS Ten prepubertal girls (mean 9. ±.4 years), 7 midpubertal girls (mean 11.9 ±.6 years), 25 pstpubertal, early adlescent girls (mean 15.3 ±.3 years), 8 nrmal cycling wmen (mean 27.4 ±.5 years), 17 pstmenpausal wmen (mean 54.4 ± 1.9 years), and 13 men (mean 53.5 ± 2.4 years) were recruited fr study. The nrmal cycling wmen had dcumented cycle lengths f 26 t 32 days. Nne f the wmen had taken any hrmnal medicatins fr 3 mnths befre enrllment and, if apprpriate, used barrier cntraceptin thrughut the study. The prject was apprved by the Cmmittee n Investigatins Invlving Human Subjects f the University f Califrnia, San Dieg, and infrmed written cnsent was btained frm each subject befre enrllment. Cnsent was btained frm the parents f subjects < 18 years ld. All subjects, except the nrmal cycling wmen, had a single bld sample drawn fr determinatin Vl. 65, N.3, March 1996 f fllistatin cncentratin. The nrmal cycling wmen had daily bld samples drawn thrughut ne menstrual cycle and serum levels f LH, FSH, E2, P, inhibin, and fllistatin determined. In additin, five nrmal cycling wmen received 5 JLg/kg 1M Nal-Glu GnRH antagnist fr 3 cnsecutive days beginning n cycle day 7 during the next menstrual cycle and bld drawn n the last day f treatment. Serum fllistatin cncentratins were determined in duplicate by a sensitive hmlgus RIA using antisera raised against purified prcine fllistatin (15). The highly purified preparatin f prcine fllistatin used as tracer and reference standard cnsists fa mixture f32 (1%), 35 (7%), and 39 (2%) kd frms f the prtein. N crss-reactivity with FSH, LH, PRL, smatstatin, activin A, r inhibin A was seen. Displacement curves btained by increasing cncentratins f purified prcine fllistatin, human fllicular fluid, and serum were parallel (15), a finding in keeping with the fact that human fllistatin differs frm prcine fllistatin by nly fur amin acids. Hwever, activin-fllistatin cmplexes can displace radilabeled fllistatin frm antibdy-binding sites (unpublished bservatins) and, thus, displacement is referred t as fllistatinlike immunreactivity (immunreactive fllistatin). A serum sample f 1 JLL was used fr assay and the lwer limit f sensitivity fr the assay was apprximately 1 ng/ml. Serum LH, FSH, E2, P, and inhibin levels were als determined by RIA, as described previusly (17). The intra-assay and interassay cefficients f variatin fr the RIAs were, respectively, immunreactive fllistatin, 4.1% and 7.3%; LH, 2.8% and 8.7%; FSH, 1.4% and 6.%; E2, 2.7% and 9.3%; P, 2.7% and 9.1%; and inhibin, 4.% and 6.3%. Data were analyzed by tw-factr analysis fvariance, fllwed by Dunnett's test, paired r grup t tests as apprpriate. Results are presented as the mean ± SEM and P <.5 was cnsidered significant. RESULTS Mean ± SEM cncentratins f immunreactive fllistatin in pubertal girls, nrmal cycling wmen, pstmenpausal wmen, and men are seen in Figure 1. Levels in prepubertal, midpubertal, and pstpubertal girls were similar (4.9 ±.3 versus 4.9 ±.2 versus 5. ±.1 ng/ml, respectively). Levels fimmunreactive fllistatin were determined frm the daily samples btained thrughut the menstrual cycle in each nrmal cycling wmen and the results f the grup meaned. There was a significantly higher mean level f immunreactive fllistatin in the nrmal cycling wmen than the pubertal grup Kettel et al. Fllistatin levels in wmen 473

3 B DISCUSSION r-1 E " CJI 6 c Z... I- C I- III..... a ll. I PRE MID ADDL NC PM" MEN Figure 1 Mean ± SEM serum immunreactive fllistatin cncentratins in prepubertal, midpubertal, and pstpubertal girls, nrmal cycling wmen, pstmenpausal wmen, and men. PRE, prepubertal; MID, midpubertal; ADOL, pstpubertal; NC, nrmal cycling; PMW, pstmenpausal. (6.7 ±.2 t 5. ±.1 ng/ml, P <.1). Pstmenpausal wmen had levels f immunreactive fllistatin similar t nrmal cycling wmen (6.6 ±.3 versus 6.7 ±.2 ng/ml, respectively), but higher than pubertal girls (6.6 ±.3 t 5. ±.1 ng/ml, P <.1). In additin, nrmal cycling wmen and pstmenpausal wmen had higher levels f immunreactive fllistatin than men (6.7 ±.2 versus 6.6 ±.3 versus 5.4 ±.2 ng/ml, respectively, P <.1). Daily serum cncentratins f LH, FSH, E2, P, inhibin, and immunreactive fllistatin (mean ± SEM) during ne cmplete menstrual cycle are displayed in Figure 2. The expected dynamic secretry changes in LH, FSH, E2, and P ccurred in all eight nrmally cycling wmen studied. The daily levels f inhibin changed accrding t the pattern previusly described by McLachlan et al. (16). Levels f inhibin were lw during the early fllicular phase, with a gradual increase thrugh the midfllicular and late fllicular phases. After the midcycle LH surge, levels declined, and then rse again t reach a peak in the midluteal phase. Inhibin cncentratins then fell in parallel with E2 and P until menstrual bleeding began. Unlike the levels f inhibin, levels f immunreactive fllistatin remained cnstant thrughut the menstrual cycle. The slight decline in fllistatin levels in the late luteal phase was nt significant. When cmpared with levels during day 7 f the cntrl cycle, serum cncentratin f E2 and inhibin were reduced significantly by 3 days treatment with Nal-GIu GnRH antagnist (Fig. 3). Estradil levels declined 85.6% (P <.1) and inhibin levels fell 47.9% (P <.1). Hwever, levels fimmunreactive fllistatin declined insignificantly (6.1 ±.8 t 5.4 ±.4 ng/ml, P = NS) after Nal-GIu antagnist. In this study, we have demnstrated that circulating levels f immunreactive fllistatin d nt change during the nrmal menstrual cycle. In additin, serum immunreactive fllistatin levels did nt change significantly after 3 days f varian suppressin with a GnRH antagnist (Nal-GIu) despite significant declines in circulating LH and FSH. Treatment f variectmized rats with Nal-GIu als failed t change fllistatin mrna expressin in the anterir pituitary despite significant reductins in FSH (19). The failure f immunreactive fllistatin levels t change during the menstrual cycle r after GnRH antagnist treatment indicates a regulatry cntrl mechanism fr circulating levels f fllistatin that is nt dependent n FSH. The levels f fllistatin measured in this study were in the lwer range f the assay used and the ability t detect small differences in fllistatin levels is less than in the midprtin f the displacement curve. Hwever, using a hmlgus RIA, Khury et al. (2) have published their findings n circulating levels f fllistatin thrugh the menstrual cycle. They als reprt an 15 4 ~... 2 ~ 1 III J: C :I: 5...J "- r 12 2 I...J BOO "- III ~ 1 :l ~ 4 '" "- ;I J 11 ;:::;1 :l E 5 ;I Q 5 "- - N r UJ U DAYS FROM LH SURGE Figure 2 Mean ± SEM daily serum cncentratins f LH, FSH, inhibin, immunreactive fllistatin, E2 (cnversin factr t SI unit, 3.671), and P (cnversin factr t SI unit, 3.18) during ne cmplete menstrual cycle in eight wmen. Data are centered arund the LH surge..., ~..., 474 Kettel et ai. Fllistatin levels in wmen

4 4... E 2 c. => E CI c CONTROL E2 INH ** i-fs NAL -GLU Figure 3 Mean:+: 8EM serum cncentratins fe 2 (cnversin factr t 81 unit, 3.671), inhibin, and immunreactive fllistatin n cycle day 7 and after treatment with 5 Ilg/kg per day 1M Nal Glu GnRH antagnist fr 3 days in five wmen. absence f changes in fllistatin levels acrss the menstrual cycle. In a previus study by Sugawara et al. (15), serum levels f immunreactive fllistatin, determined using the same assay, increased after stimulatin f fllicular develpment with hmg. These wmen were enrlled in an IVF prgram and had received GnRH agnist (GnRH-a; 1 mg SC leuprlide acetate daily) t suppress varian functin befre receiving hmg. This study suggested the pssibility that gnadtrpin therapy had a direct effect n circulating levels ffllistatin. In this study, we were unable t detect a significant decline in immunreactive fllistatin levels after acute varian suppressin with a GnRH antagnist. The differences in fllistatin dynamics may be explained by several differences in study design. First, treatment with GnRH-a and hmg is begun in the luteal phase f the preceding cycle. Levels f immunreactive fllistatin reprted at baseline during GnRH-a and hmg treatment were much lwer (apprximately 1.5 ng/ml) than thse fund in either nrmal cycling r pstmenpausal wmen. The administratin f hmg resulted in a restratin f immunreactive fllistatin levels Vl. 65, N.3, March 1996 t cncentratins we have reprted thrughut the menstrual cycle. It is pssible that GnRH-a treatment had a different, maybe direct, effect n varian fllistatin prductin. Secnd, treatment with hmg results in cntrlled varian hyperstimulatin, which may stimulate a respnse in varian fllistatin prductin nt seen under cnditins f nrmal menstrual cyclicity. Third, the number f subjects given GnRH antagnist in this study was small (n = 5). We did detect a nnsignificant decline in immunreactive fllistatin levels after GnRH antagnist treatment. N changes in mean circulating levels fimmunreactive fllistatin were detected during pubertal varian activatin. Hwever, significantly higher serum levels f immunreactive fllistatin were bserved in wmen wh had established regular menstrual cyclicity. Levels f immunreactive fllistatin remained the same int menpause. Hwever, the grup f menpausal wmen studied was fairly yung (mean 54.4 ± 1.9 years) and changes with advancing age remain t be defined. Men had significantly lwer circulating levels f immunreactive fllistatin than either nrmal cycling wmen r pstmenpausal wmen. Taken tgether, the lack f change in serum immunreactive fllistatin levels after acute varian suppressin and the increase seen in immunreactive fllistatin after puberty may implicate surces f fllistatin ther than the vary. Ptential tissue cntributin t the circulatin are numerus because fllistatin mrna has been identified in cerebral crtex, pituitary, adrenal, thymus, pancreas, gut, kidney, heart, lung, uterus, and skeletal muscle (21). Within the rat vary, the cyclic changes in fllistatin mrna seen during the estrus cycle were nt unifrmly accmpanied by crrespnding changes in expressin ffllistatin prtein (12); althugh fllistatin mrna was present in granulsa cells f every develping fllicle, the prtein was present nly in selected dminant fllicles. This disparity may implicate that translatin f the message is a key regulatry step in fllistatin bilgic activity. These data suggest an imprtant lcal autcrine-paracrine rle f fllistatin in reprductin. Certainly fllistatin mrna has been lcalized in all imprtant reprductive structures, including the anterir pituitary, testis, vary, and uterus (21). Within the circulating cmpartment, a cnstant level f fllistatin may allw dynamic changes in circulating inhibinactivin t be expressed at the target tissue. Fllistatin is nt the nly binding prtein fr activin and inhibin. Recently, ll2-macrglbulin was shwn t bind bth inhibin and activin in human plasma (22). Mre imprtantly, ll2-macrglbulin may be the primary binding prtein fr inhibin and activin in Kettel et al. Fllistatin levels in wmen 475

5 human serum, whereas fllistatin is the majr binding prtein in fllicular fluid (23). In ther bilgic systems, such as erythrpiesis, fllistatin has been shwn t negate the stimulatry effects f activin (24). Thus, circulating fllistatin may functin as a carrier prtein t deliver inhibin-activin t the target site and, in ther systems, as a mdulatr f bilgic actin. In summary, circulating levels f immunreactive fllistatin increase between puberty and the establishment f regular menstrual cycles and then remain stable int menpause. N changes in circulating immunreactive fllistatin levels were detected after acute varian suppressin with GnRH antagnist. Because there was n significant change in immunreactive fllistatin during the activatin f varian cyclicity during puberty r after acute varian suppressin, the changes in peripheral immunreactive fllistatin levels may implicate surces f fllistatin ther than the vary. Assuming the binding sites f fllistatin are saturated, the cnstant level f immunreactive fllistatin in the peripheral circulatin may permit the dynamic changes in circulating inhibin-activin t exert their target cell actins. REFERENCES 1. Ling N, DePal LV, Bicsak TA, Shimasaki S. Nvel varian regulatry peptides: inhibin, activin, and fllistatin. Clin Obstet Gynecl 199;33: Uen N, Ling N, Ying S_Y, Esch F, Shimasaki S, Guillemin R. Islatin and partial characterizatin ffllistatin: a nvel Mr 35, mnmeric prtein that inhibits the release ffllicle stimulating hrmne. Prc Nat! Acad Sci USA 1987;84: Ying SY, Becker A, Swansn G, Tan P, Ling N, Esch F, et al. Fllistatin specifically inhibits pituitary fllicle stimulating hrmne release in vitr. Bichem Biphys Res Cmmun 1987; 149: DePal LV, Shimnaka M, Schwall RH, Ling N. In viv cmparisn f the fllicle-stimulating hrmne-suppressing activity ffllistatin and inhibin in variectmized rats. Endcrinlgy 1991; 128: Nakamura T, Taki K, Et Y, Shibai H, Titani K, Sugin H. Activin-binding prtein frm rat vary is fllistatin. Science 199; 247 : Shimnaka M, Inuye S, Shimasaki S, Ling N. Fllistatin binds t bth activin and inhibin thrugh the cmmn betasubunit. Endcrinlgy 1991; 128: Stuffer RL, Wdruff TK, Dahl KD, Hess DL, Mather JP, Mlskness TA. Human recmbinant activin-a alters pituitary luteinizing hrmne and fllicle-stimulating hrmne secretin, fllicular develpment, and steridgenesis, during the menstrual cycle in rhesus mnkeys. J Clin Endcrinl Metab 1993; 77: Et Y, Tsuji T, Takegawa M, Takan S, Ykgawa Y, Shibai H. Purificatin and characterizatin f erythrid differentiatin factr (EDF) islated frm human leukemia cell line THP-1. Bichem Biphys Res Cmmun 1987;142: Ttsuka Y, Tabuchi M, Kjima I, Shibai H, Ogata E. A nvel actin f activin A: stimulatin f insulin secretin in rat pancreatic islets. Bichem Biphys Res Cmmun 1988; 156: Wdruff TK, Lyn RJ, Hansen SE, Rice GC, Mather JP. Inhibin and activin lcally regulate rat varian flliculgenesis. Endcrinlgy 199; 127: Schneyer AL, O'Neil DA, Crwley WF. Activin-binding prteins in human serum and fllicular fluid. J Clin Endcrinl Metab 1992; 127: Nakatani A, Shimasaki S, DePal LV, Ericksn GF, Ling N. Cyclic changes in fllistatin messenger ribnucleic acid and its prtein in the rat vary during the estrus cycle. Endcrinlgy 1991; 129: DePal LV, Bicsak TA, Ericksn GF, Shimasaki S, Ling N. Fllistatin and activin: a ptential intrinsic regulatry system within diverse tissues. Prc Sc Exp Bii Med 1991; 198: Rberts VJ, Barth S, EI-Reiy A, Yen SSC. Expressin f inhibinlactivin subunits and fllistatin mrnas and prteins in varian fllicles and the crpus luteum during the human menstrual cycle. J Clin Endcrinl Metab 1993;77: Sugawara M, DePal L, Nakatani A, DiMarz SJ, Ling N. Radiimmunassay f fllistatin: applicatin fr in vitr fertilizatin prcedures. J Clin Endcrinl Metab 199; 71: McLachlan RI, Rbertsn DM, Healy DL, Burger HG, de Kretser DM. Circulating immunreactive inhibin levels during the nrmal human menstrual cycle. J Clin Endcrinl Metab 1987;65: Rseff SJ, Bangah ML, Kettel LM, Vale W, Rivier J, Burger HG, et al. Dynamic changes in circulating inhibin levels during the luteal-fllicular transitin f the human menstrual cycle. J Clin Endcrinl Metab 1989;69: Demura R, Suzuki T, Tajima S, Mitsuhashi S, Odagiri E, Demura H, et al. Human plasma free activin and inhibin levels during the menstrual cycle. J Clin Endcrinl Metab 1993; 76: DePaul LV, Mercad M, Gu Y, Ling N. Increased fllistatin (activin-binding prtein) gene expressin in rat anterir pituitary tissue after variectmy may be mediated by pituitary activin. Endcrinlgy 1993; 132: Khury RH, Wang QF, Crwley WF, Hall JE, Schneyer AL, Tth T, et al. Serum fllistatin levels in wmen: evidence against an endcrine functin f varian fllistatin. J Clin Endcrinl Metab 1995;8: Michel U, Albistn A, Findlay JK. Rat fllistatin: gnadal and extragnadal expressin and evidence fr alternative splicing. Bichem Biphys Res Cmmun 199; 173: Vaughn JM, Vale W. Cl'2-Macrglbulin is a binding prtein f inhibin and activin. Endcrinlgy 1993; 132: Krummen LA, WdruffTK, DeGuzman G, Cx ET, Baly DL, Mann E, et al. Identificatin and characterizatin f binding prteins fr inhibin and activin in human serum and fllicular fluid. Endcrinlgy 1993; 132: Shizaki M, Sakai R, Tabuchi M, Nakamura T, Sugin K, Sugin H, et al. Evidence fr the participatin f endgenus activin Alerythrid differentiatin factr in the regulatin f erythrpiesis. Prc Nat! Acad Sci USA 1992;89: Kettel et al. Fllistatin levels in wmen