FURTHER OBSERVATIONS ON THE THERAPY OF ANOVULATORY INFERTILITY WITH SYNTHETIC LUTEINIZING HORMONE RELEASING HORMONE

FERTILITY.AND STERILITY Cpyright 1974 The American Fertility Sciety Va!. 25, N.1, January 1974 Printed in U.S.A. FURTHER OBSERVATIONS ON THE THERAPY OF ANOVULATORY INFERTILITY WITH SYNTHETIC LUTEINIZING HORMONE RELEASING HORMONE ARTURO ZARATE, ELIAS S. CANALES, JORGE SORIA, ARTURO GONZALEZ, ANDREW V. SCHALLY, AND ABBA J. KASTIN Department f Gyneclgic Endcrinlgy, Hspital Ginec Obstet N.1, lnstitut Mexican Segur Scial, Mexic, and Veterans Administratin Hspital and Tulane University, Schl f Medicine, New Orleans, Luisiana 70112 Luteinizing hrmne-releasing hrmne (LH-RH) is effective in inducing vulatin and pregnancy.1-4 Patients vary in sensitivity t LH-RH and it is therefre -(.. necessary t individualize the dsage fr each patient. Hwever, individual patients als vary in their respnses t the same dses in different curses f therapy. 3 The purpse f this cmmunicatin is t present ur experience with synthetic LH RH in the treatment f sterility in patients with an anvulatry syndrme. Fllicular maturatin was assessed by means f urinary estrgen determinatins in rder t individualize the therapy, the dse f LH-RH being adjusted accrding t the varian respnse. MATERIALS AND METHODS Patients. The study cncerned 42 infertile wmen in whm deficient hypthalamic-pituitary functin was the nly apparent cause f infertility. The patients' ages ranged frm 18 t 32 years (average, 24 years). Of the 42 patients, 24 had severe ligmenrrhea with cycles f 6 t 9 mnths' duratin, 12 had secndary amenrrhea f 8 t 18 mnths' duratin, and 6 had amenrrhea and galactrrhea withut enlargement f the sella -turcica. Preparatin f Synthetic LH-RH and Human Chrinic Gnadtrpin. Tw preparatins f synthetic LH-RH were Received May 15, 1973. 3 used. First, synthetic LH-RH was made by a slid-phase methd and purified by cunter-current distributin. 5 The LH-RH activity f this preparatin (AVS 77-65 N. 214-271) was 82% that f pure natural LH-RH. Ampules cntaining 500 p.g f LH-RH and diluted in 1 ml f 0.9% saline slutin were kept at 4 C until administratin. This preparatin will be refrerred t as "500 p.g LH-RH." Secnd, synthetic dept LH-RH (Hechst Pharmaceutical, Mexic City) was supplied in ampules, each f which cntained 0 p.g f LH-RH. This was diluted in saline slutin and t this was added an investigatinal carrier preparatin (Hechst). The preparatin will be referred t as "0 p.g LH-RH." The frm f human chrinic gnadtrpin (HCG) used was Pregnyl (Organn, Mexic City). Administratin f LH -RH. The 500 p.g LH-RH preparatin was given t 32 patients (Table 1). It was administered nce daily in the mrning as an intramuscular injectin t 15 patients and twice daily t ten thers. The remaining seven patients received three ampules f 500 p.g LH-RH daily, n days 5,, and 15 f a cycle induced by estrgen-prgestin medicatin. The varian respnse was evaluated by the daily estimatin f the 24-hur urinary excretin f ttal estrgens. Treatment was cntinued until the ttal urinary estrgen excretin apprximated 60 p.g/

4 ZARATE ET AL Vl. 25 Daily injectin frequency N. f patients TABLE 1. Results f Therapy With 500 /Lg LH-RH Ttal urinary Clinical diagnsisa Duratin f Ttal estrgens. IA GA POS thera py, days dse, mg JLg/24 hr Ovulatin 1>regnancy (range) (range) (range)' (n. f patients) (n. f patients) x1 15 12 0 3 5-20 2.5-.0 30-90 6 4 x2 6 3 1 5-5.0-.0 50-80 3 2 x3 7 3 3 3 (days 5. 45 16-50 0 0, and 15 nly) ala = idipathic anvulatin; GA = galactrrhea-amenrrhea syndrme; POS = plycystic vanan syndrme. bon last day f therapy. 24 hurs. Ovulatin was then attempted by single intramuscular llljectins f,000 IU f HeG n 2 cnsecutive days. The 24-hur urinary excretin f estrgens was determined each day until bleeding ccurred r 20 days had elapsed withut bleeding frm the last day f LH-RH administratin. The 0 p.g LH RH preparatin was given t the remaining ten wmen. Administratin was nce daily fr eight f the ten patients and twice daily fr tw patients (Table 2). Criteria fr Fllicular Respnse and Ovulatin. The varian respnse was assessed by urinary excretin f ttal estrgens. An increase in the estrgen excretin t at least 60 p.g/24 hurs was taken as evidence f fllicular maturatin. Presumptive vulatin was defined by a clear shift in the basal bdy temperature and an increase in urinary excretin f pregnanedil t at least 2.5 mg/24 hurs. Effect f LH-RH Administratin On Serum LH and FSH Cncentratins. Fur patients were studied after an vernight fast. An indwelling venus catheter was inserted and an infusin f a nrmal istnic saline slutin was started. Tw patients then received 500 p.g f LH-RH intramuscularly and anther tw received 0 p.g LH-RH intramuscularly. Venus bld samples were btained at -, 0, 15, 30, and 60 minutes and at 2, 3, and 4 hurs in all fur patients. Nine additinal patients were studied by daily serum determinatins f LH and FSH befre LH-RH administratin. Three mre patients had bld samples taken TABLE 2. Results f Therapy With 0 /Lg LH-RH Daily injectin Clinical Duratin f Ttal dse Ttal urinary Case n. frequency diagnsisa therapy, days flh-rh, mg estrgens, JLg/ 24 hr' 1 x1 la 8 0.8 40 2 x1 IA 1.0 32 3 x1 IA 8 0.8 48 4 x1 IA 16 1.6 60 5 x1 IA 1.0 62 14 1.4 70 6 x1 IA 9 0.9 63 7 x1 POS 12 1.2 63 8 x1 POS 9 0.9 67 9" x2 IA 5 1.0 94 x2 POS 5 1.0 60 ala = idipathic anvulatin; POS = plycystic varian syndrme. bon last day f therapy. COvulated and became pregnant....

~ " January 1974 LH-RH FOR ANOVULATORY INFERTILITY 5 TABLE 3. Results f Therapy With LH-RH in Anvulatry Sterility ~ ~ N. f Daily dse cases LH~RH preparatin (mg) 32 500 p.g vial (AVS 77-65 # 214-271) 0.5-1.5 0 p.g vial (Hechst) 0.1-0.2 Ttal 42 Ttal dse N. f N. f (mg) vulatins cnceptins 2.5-.0 9 6 0.8-1.6 7 '" befre and 30 minutes after administratin f LH -RH. Analytical Methds. Serum FSH and LH cncentratins were measured by a duble-antibdy radiimmunassay technique,6.7 in which results are expressed as ng/ml f serum and LER-907 is used as the reference standard. Highly purified FSH, LH, LER-907, and antisera were supplied by the Natinal Pituitary Agency and the Natinal Institutes f Arthritis and Metablic Diseases. Endgenus estrgen prductin was evaluated by means f the 24-hur urinary excretin f ttal estrgens measured accrding t the methd f Beling. 8 Urinary excretin f pregnanedil was determined by the technique f Gldzieher and Nakamura. 9 RESULTS The details f respnses LH -RH are given in Tables 1 and 2, and the results are summarized in Table 3. There were ten wmen wh vulated after LH-RH administratin and, f these, seven became pregnant. Injectins f at least 200 p.g LH-RH n cnsecutive days seemed t induce vulatin mst frequently (Tables 1, 2, and 3). Of the 42 wmen, 24 shwed an increase in the excretin f ttal urinary estrgens exceeding 60 p.g/24 hur but did nt vulate (Tables 1 and 2). Patients wh shwed a fllicular respnse required at least 1.0 mg LH-RH. Sme individual treatment cycles are illustrated in Figures 1 and 2. An increase in urinary estrgens abve 60 p.g ccurred in 11 ther patients; hwever,! LH RH 500!j~/OAY l'hr ' CERVICAL MUCUS SPINNBARKEIT (em) 15 14 13 * 0 12 II 80 TOTAL /ig/24hr 60 8 6 40 4 2 20 5 DAYS FIG. 1. Patient with secndary amenrrhea wh received LH-RH 500 p.g/day fr days. Ttal urinary estrgens reached 90 p.g/24 hur n the last day f therapy; then HCG was administered. Ovulatin and pregnancy ccurred in this case.

6 ZARATE ET AL Vl. 25 CERVICAL MUCUS SPINNBARKEIT (em) +15 12 8 6 4 2 1 LH-RH500~g/DAY l'hr ' I I + + + + FERNING + &++ * +++ 120 0 80 TOTAL 60,.;/24 hr 40 20 5 15 DAYS FIG. 2. LH-RH therapy fr days fllwed by HCG shwed n fllicular grwth as evaluated by urinary estrgen determinatin. Changes in cervical mucus were misleading. Patient presented neither withdrawal bleeding nr vulatin after LH-RH. this nly ccurred 5 days after HeG administratin, at which time withdrawal bleeding ccurred. The respnse t LH-RH therapy accrding t the clinical diagnsis is given in Table 4. Of the ten patients wh TABLE 4. Results f LH-RH Therapy Accrding t Clinical Diagnsis N. f N. f N. f Clinical diagnsis cases vulatins cnceptins Stein-Leventhal syndrme 24 6 5 Idipathic anvulatin 12 4 2 Amenrrheagalactrrhea syndrme 6 0 0 Ttal 42 7 vulated six had the Stein-Leventhal syndrme, and five f these six became pregnant. The remaining vulatins and pregnancies ccurred in cases f idipathic anvulatin. Effect f LH-RH n Serum LH and FSH Values. The Serum LH values after intramuscular injectin f the 500 p.g f LH-RH and the 0 p.g f LH-RH preparatins are illustrated in Figure 3. Serum LH values increased sharply 15 t 30 minutes after injectin and peaked at 30 t 60 minutes after injectin. Serum LH values were maintained at this value fr the fllwing 60 minutes and then decreased; the values returned t the preinjectin values 3 hurs after injectin. LH ",/ml 2000 1!500 00.00.00 400.00 FSH nglml '00 - LH-RH SOOM9 x---x LH-RH 0 M9(DEPOT) '00. e e e--. 200 e ~y,... x / ~x I" t=t, ---... ----X.J < -$ 4 ~-lc--)(h' 0 ~......--"1C----~ X-X-- - 0 15 30 ~NUTES c:j.0 120 HOURS FIG. 3. Effects f intramuscular LH-RH administratin n serum LH and FSH levels. Tw patients received LH-RH 500 p.g and tw mre received 0 p.g LH-RH. Increase in bth LH and FSH was essentially similar with the tw dses f LH-RH. -4 <II

January 1974 LH-RH FOR ANOVULATORY INFERTILITY 7....... Serum FSH values after injectin f LH-RH are als shwn in Figure 3. These peaked in 30 t 45 minutes and then declined. At 3 hurs after injectin, serum FSH values tended t remam higher than the basal values. In these few patients there was n appreciable difference between the LH and FSH respnses t the tw dses f LH-RH. Daily bld tests befre injectin f LH HH shwed nly mild variatins in LH and FSH serum levels in three representative patients (Fig. 4, 5, and 6). Hwever, three patients whse prgress was fllwed by daily determinatins f gnadtrpins befre and 30 minutes after LH-RH administratin shwed a peak in LH values after each injectin and, t a lesser extent, a peak in FSH values (Fig. 7 and 8). LH nq/ml FSH n9/ml :1 TOTAL 40 1(9/24 hr 30 20 CERVICAL MUCUS SPINNBARKEIT em 12 HCC24y. POLYCYSTIC OVARIES I LH-RHO.l m. DAY r l A 15 20 DAYS Of THE CYCLE 25 I MENSES I FIG. 4. 0 fj-g f LH-RH fr days induced abrupt increase in urinary excretin f estrgens as well as changes in cervical mucus; hwever. vulatin did nt ccur and patient menstruated days after discntinuatin f LH-RH and HeG. Nte basal variatins f bth LH and FSH serum levels during the days f study. L H nljl/ml GSS 24y IDIOPATHIC ANOVULATION l LH-RHOim.,m/DAY ::1~ Iii FSH 2001 n/mi ':&~ TOTAL /{/24hr 30 I 60 l -r MENSES 20 21 DAYS OF TREATMENT FIG. 5. 0 p,g f LH-RH fr 5 days induced fllicular grwth as estimated by urinary excretin f estrgens; hwever, 3 days later ttal estrgens declined despite cntinuatin f therapy. Daily serum determinatins f basal FSH and LH shwed slw decline in cncentratins, particularly FSH. DISCUSSION The results cnfirmed that LH-RH has a therapeutic effect in sme cases f sterility. Hwever, studies in a larger grup f patients have shwn that it is difficult t assess the crrect dse f LH-RH s that the dsage fr each patient has t be individualized. Even s, as the present study demnstrates, the varied respnse t LH-RH treatment makes it difficult t establish a definitive therapeutic regimen. It des seem, thugh, that vulatin ccurred primarily in thse patients wh received at least 200 p,g LH-RH n each f 5 cnsecutive days (Tables 1, 2, and 3). Administratin f daily dses f nly 0 p,g LH-RH r larger dses n 3 nncnsecutive days was less successful. Mnitring fllicular grwth by determinatins f the urinary estrgens didnt permit the assessment f the length f LH-RH therapy; that is, patients with a high estrgen prductin did nt necessarily vulate. This finding remains t

8 ZARATE ET AL Vl. 25 2001 ngl~i. 0 300] ":~=, "'J TOTAL 60] ~gl24hr 30 GAZ 26 Y.O. POLYCYSTIC OVARIES r,-------,--------r-------, r,---------r--------,---------, 5 15 DAYS OF TREATMENT FIG. 6. LH-RH fr 15 days in 26-year-ld with plycystic varian syndrme. Fllicular grwth assumed t ccur because urinary estrgens exceeded 50 I'g/24< hur. Ovulatin did nt ensue and withdrawal bleeding was induced by chlrmadinne 20 days after cmpletin f LH-RH cycle. Baseline LH and FSH values were determined during the 15 days f therapy; irregular FSH values were incnsistent. l G. G. L. 24 y. D 30min. auer lh-rh SECONDARY AMENORRHEA. II BASAL 6001 1 400 ng/ml LH 0 j 200 22'~ FSH 0 ng/ml LH-RH 0.5 mg.i.m. ~ I + + + + + + + + + +..--.1 URINARY ::~~ ~ 20 "j l.i1g 124hi) I I 5, DAYS OF THERAPY + I I 50 FIG. 7. Daily administratin f LH-RH demnstrated that pituitary maintains ability t respnd with LH secretin despite repeated stimulatin; but FSH secretin varied during LH-RH therapy. Urinary excretin f estrgens shwed clser crrelatin with degree f LH respnse t LH-RH. 01 03 05 07 09 6 6 400 ngl/~i'008 ~ ~~9S/~1 0----0 300 200-0-,0...0. 300..,(7- -....0- '0- -(1--0---00- -0- ' -- J>-O--_.-..._.. -0.'00 0--_-0--- 0 200 4006 t::j btj b 500 z ~-c>--- _......0.-0--.0--_-0.. 300 0,I>-r>-- -0."_-0-.0.'0-.0-0-.O"..._--- -~... 2.00 12341234123412341234 HOURS HOURS HOURS HOURS HOURS ~ T M E FIG. 8. Tw patients with secndary amenrrhea received 500 I'g f LH-RH daily fr days. Serum LH and FSH values were determined at hurly intervals (1 t 4< hurs) n days 1, 3, 5, 7, and 9 f therapy. Effect f LH-RH administratin (arrws) shws that pituitary maintained ability t secrete LH, but FSH secretin was relatively variable.

January 1974 LH-RH FOR ANOVULATORY INFERTILITY 9 be clarified, because assessment f fllicular grwth by estrgen determinatin has prved t be a reliable index when human menpausal gnadtrpin therapy is used.' The results f the administratin f LH-RH by the intramuscular rute cnfirmed its efficacy in releasing LH and, t a lesser extent, FSH. Mrever, the data fr patients wh received LH-RH daily fr t 16 days shwed that the pituitary is capable f respnding t repeated stimulatin with LH secretin; the release f FSH, hwever, tended t be mre variable. Our results indicate that a higher daily and ttal dse f LH-RH results in a higher percentage f vulatin (Table 3). Nevertheless, it is pssible that different treatment mdalities, and perhaps higher dses, wuld yield mre psitive effects. Fr example, the use f lng-acting preparatins f LH-RH might maintain LH and FSH at cnstantly elevated values and s avid the temprary surge f LH and FSH that ccurs after administratin f the shrt-acting LH-RH. In view f the imprtance f finding better treatment fr female sterility, different regimens f LH-RH administratins shuld be tried. Our results als indicate that wmen with the Stein-Leventhal syndrme were mre readily respnsive t LH-RH. Patients wh vulated and became pregnant have prved previusly t respnd t clmiphene citrate with gnadtrpin secretin. These bservatins permit ne t cnclude that the patients wh are mre likely t respnd t LH-RH therapy are thse with an apparently relatively nrmal tnic FSH and LH hypthalamic functin. Studies f LH-RH administratin in patients either with lng-standing amenrrhea r primary amenrrhea are in prgress. SUMMARY Synthetic LH-RH was used in an attempt t induce vulatin and pregnancy in 42 wmen with suspected hypthalamic anvulatin. Of the 42 wmen, ten vulated and seven f them cnceived; 24 f the 42 patients shwed signs f fllicular grwth but did nt vulate. Mnitring fllicular develpment by urinary estrgen excretin did nt permit the assessment f the apprpriate length f LH-RH therapy. Injectin f at least 200 /Lg LH RH n cnsecutive days induced vulatin mst ften. The intramuscular administratin f LH-RH cnfirmed its efficacy in releasing LH and t a lesser extent, FSH. Patients wh received LH-RH daily fr several days shwed that the pituitary was capable f respnding t repeated stimulus with LH secretin, and yet the release f FSH tended t be mre variable. The results f the present study demnstrate that LH-RH given intramuscularly induces vulatin, but it remains difficult t assess the crrect dse f LH-RH due t the individual varied respnse t treatment. REFERENCES 1. Zarate A, Canales ES, Ayala L, et al: Successful inductin f vulatin with synthetic luteinizing hrmne-releasing hrmne (LH RH) in hypthalamic infertility. Sern Fundatin cnference. Acapulc, Mexic, June, 1972 2. Kastin AJ, Zarate A, Midgley AR, et al: Ovulatin cnfirmed by pregnancy after infusin f Prcine LH-RH. J Clin Endcrinl Metab 33:980, 1971 3. Zarate A, Canales ES, Schally AV, et al: Successful inductin f vulatin with synthetic luteinizing hrmne-releasing hrmne in anvulatry infertility. Fertil Steril 23: 672, 1972 4. Keller P1: Inductin f vulatin by synthetic luteinizing hrmne-releasing factr in infertile wmen. Lancet 2:570, 1972 5. Matsu H, Arimura A, Nair RMG, et al: Synthesis f the prcine LH and FSH releasing hrmne by the slid-phase methd. Bichem Biphys Res Cmmun 45:822, 1971

.. ZARATE ET AL Vl. 25 6. Midgley AR Jr: Radiimmunassay: a methd fr human chrinic gnadtrpin and human luteinizing hrmne. Endcrinlgy 79:, 1966 7. Midgley AR Jr: Radiimmunassay fr human fllicle stimulating hrmne. J Clin Endcrinl Metab 27:295, 1967 8. Beling CG: Gel filtratin f cnjugated urinary estrgens and its applicatin in clinical assays. Acta Endcrinl (Kbh) 43: Suppl 79:9, 1963 9. Gldzieher JW, Nakamura Y: A clinical methd fr the determinatin f urinary pregnanedil and pregnanetril. Acta Endcrinl (Kbh) 4:371, 1972. Canales ES, Zarate A, Gnzalez A, et al: Therapy f anvulatry sterility with menpausal and chrinic gnadtrpins. Assessment f fllicular maturatin by urinary estrgen determinain. Int J Fertil 18:26, 1973