Upsala J Med Sci 84: 21-35, 1979 Effects of Prolonged Luteinizing Hormone-releasing Hormone Therapy on Follicular Maturation, Ovulation and Corpus Luteum Function in Amenorrhoeic Women with Anorexia Nervosa Sven Johan Nillius and Leif Wide Depcirtmeni c?f Ohstetr-ics and Gynuecology u n d Department of Clinicul Chemistry, U 1 7 i ~ ~ e r s i t y H o s p i t u l , Uppsalu, Sii,eden ABSTRACT Nine a m e n o r r h o e i c women w i t h a n o r e x i a n e r v o s a (AN) w e r e g i v e n l o n g - t e r m t r e a t m e n t w i t h 500 ug of s y n t h e t i c l u t e i n i z i n g h o r m o n e - r e l e a s i n g hormone (LRH) e v e r y 8 h . A l l t h e women h a d i m p a i r e d l u t e i n i z i n g hormone (LH) s e c r e t i o n a n d no e v i d e n c e of endogenous o e s t r o g e n p r o d u c t i o n . T h r e e o f them a l s o h a d d e f i c i e n t f o l l i c l e - s t i m u l a t i n g hormone (FSH) s e c r e t i o n . The p i t u i t a r y r e s e r v e c a p a c i t y f o r g o n a d o t r o p h i n s e c r e t i o n w a s n o r m a l b u t t h e r e s p o n s e p a t t e r n t o LRH w a s s i m i l a r t o t h a t d e s c r i b e d i n p r e p u b e r t a l g i r l s . The c o n s t a n t a d m i n i s t r a t i o n o f LRH n o r m a l i z e d b a s a l LH and FSH s e c r e t i o n and i n d u c e d a c y c l i c a l g o n a d o t r o p h i n s e c r e t o r y p a t t e r n w i t h d i f f e r e n t i a l c h a n g e s o f t h e LH and FSH r e s p o n s e s t o LRH d u r i n g t h e t r e a t m e n t . LRH-induced g o n a d o t r o p h i n s e c r e t i o n p r o d u c e d f o l l i c u l a r growth and m a t u r a t i o n i n a l l t h e women. P r e s u m p t i v e o v u l a t i o n a l s o o c c u r r e d d u r i n g t h e 8 t r e a t m e n t c o u r s e s i n w h i c h o n l y LRH w a s a d m i n i s t e r e d . However, i n a d e q u a t e l u t e a l p h a s e s were o b s e r v e d d u r i n g 6 o f t h e s e 8 c y c l e s . Combined t h e r a p y w i t h LRH and human c h o r i o n i c g o n a d o t r o p h i n (HCG) d u r i n g 5 t r e a t m e n t c o u r s e s r e s u l t e d i n normal o v u l a t o r y c y c l e s w i t h a d e q u a t e c o r p u s l u t e u m f u n c - t i o n . I N T R O D U C T I O N A n o r e x i a n e r v o s a i s c h a r a c t e r i z e d e n d o c r i n o l o g i c a l l y by a n i m p a i r m e n t o f t h e g o n a d o t r o p h i n s e c r e t i o n from t h e a n t e r i o r p i t u i t a r y . The i m p a i r m e n t i s more m a r - ked f o r LH t h a n f o r FSH s e c r e t i o n . The p i t u i t a r y r e s p o n s i v e n e s s t o LRH i s re- duced i n a c u t e s t a g e s o f AN and r e s t o r e d t o normal a f t e r c l i n i c a l improvement w i t h r e g a i n o f body w e i g h t (20, 28, 3 2 , 3, 1 8 ) . R e p e a t e d s t i m u l a t i o n w i t h LRH can a l s o r e s t o r e t h e p i t u i t a r y r e s e r v e c a p a c i t y f o r g o n a d o t r o p h i n s e c r e t i o n t o normal ( 1 6 , 1 9 , 3 6 ) . By l o n g - t e r m t h e r a p y w i t h LRH it i s p o s s i b l e t o i n d u c e normal o v u l a t o r y m e n s t r u a l c y c l e s i n a m e n o r r h o e i c women w i t h AN ( 1 6 , 1 9 ) . How- e v e r , c y c l e s w i t h l u t e a l p h a s e d e f e c t s o f t e n o c c u r d u r i n g p r o l o n g e d t r e a t m e n t s w i t h Only LRH ( 1 6 , 1 9 ) . Here we a n a l y z e 13 m e n s t r u a l c y c l e s i n d u c e d by LRH i n 9 a m e n o r r h o e i c women w i t h AN i n a n a t t e m p t t o d i s c l o s e t h e mechanism(s) b e h i n d t h e l u t e a l p h a s e d e f e c t s . 21 MATERIAL and METHODS P a t i e n t s Nine women, aged 20-33 (mean 2 5 . 4 ) y e a r s , w i t h symptoms of AN v o l u n t e e r e d f o r t h e s t u d y . They a l l had s e c o n d a r y amenorrhoea of 8 months- t o 1 3 y e a r s - (median 20 months) d u r a t i o n . S i x women were j u d g e d by an e x p e r i e n c e d p s y c h i a - t r i s t t o f u l f i l l t h e d i a g n o s t i c c r i t e r i a o f t r u e AN ( 4 ) w h i l e t h e o t h e r t h r e e were l a b e l l e d a s a n o r e c t i c b e h a v i o u r , c o n s i d e r e d a s a m i t i g a t e d form of AN ( 7 ) . T h e i r mean body w e i g h t 4 2 . 8 kg ( r a n g e 3 6 - 4 7 ) which c o r r e s p o n d s t o 7 2 % ( r a n g e 63-88) o f mean i d e a l body w e i g h t ( 8 ) . No o r g a n i c c a u s e of t h e amenorrhoea and w e i g h t l o s s was found a t t h e c l i n i c a l i n v e s t i g a t i o n . Buccal smear c h r o m a t i n complement showed a normal f e m a l e p a t t e r n . X-ray e x a m i n a t i o n s of t h e s k u l l and p i t u i t a r y f o s s a were normal. A l l t h e p a t i e n t s were e u t h y r o i d , a s j u d g e d by c l i n i c a l e x a m i n a t i o n and t h y r o i d f u n c t i o n t e s t s . The 24-hour u r i n a r y e x c r e t i o n of 17-hydroxycorticosteroids and 1 7 - k e t o s t e r o i d s was normal i n a l l e x c e p t one p a t i e n t , who had low b a s a l l e v e l s which i n c r e a s e d n o r m a l l y a f t e r metyrapone. T e s t o s t e r o n e and p r o l a c t i n l e v e l s i n blood were w i t h i n t h e norm31 r a n g e s . A l l t h e p a t i e n t s had low o r a b s e n t endogenous o e s t r o g e n p r o d u c t i o n , a s j u d g e d b o t h by i n d i r e c t c l i n i c a l methods f o r e s t i m a t i n g o e s t r o g e n i c a c t i v i t y on t a r g e t o r g a n s (no w i t h d r a w a l b l e e d i n g a f t e r i n t r a m u s c u l a r p r o g e s t e r o n e e t c . ) and d i r e c t measurements o f o e s t r o g e n l e v e l s i n b l o o d . L R H t r e a t m e n t I n t r a v e n o u s LRH t e s t (100 ug, Hoechst) were performed b e f o r e , d u r i n g and a f t e r t h e t r e a t m e n t s . The FSH and LH r e s p o n s e t o LRH was d e f i n e d a s t h e d i f f e r - e n c e between t h e mean o f t h e two v a l u e s a t 30 and 45 m i n u t e s a f t e r t h e LRH i n j e c t i o n and t h e mean of t h e two c o n t r o l v a l u e s . F i v e hundred vg o f LRH ( H o e c h s t ) were a d m i n i s t e r e d s u b c u t a n e o u s l y o r i n t r a - m u s c u l a r l y e v e r y 8 h d u r i n g t h e t r e a t m e n t . Seven women were t r e a t e d w i t h o n l y LRH d u r i n g 8 t r e a t m e n t c y c l e s . During t r e a t m e n t f r e q u e n t d e t e r m i n a t i o n s of FSH, LH, o e s t r a d i o l (E ) and p r o g e s t e r o n e i n b l o o d were made. O e s t r o g e n m o n i t o r i n g w a s p e r f o r m e d by e s t i m a t i o n of t h e t o t a l u r i n a r y o e s t r o g e n (TE) e x c r e t i o n i n a l l b u t two o f t h e women. The t r e a t m e n t w i t h LRH a l o n e was c o n t i n u e d u n t i l m e n s t r u a t i o n o c c u r r e d . Four women were t r e a t e d w i t h LRH i n c o m b i n a t i o n w i t h HCG d u r i n g 5 t r e a t m e n t 2 c y c l e s m o n i t o r e d by d a i l y d e t e r m i n a t i o n s o f E 2 i n b l o o d o r TE i n u r i n e . When o e s t r o g e n s l e v e l s c o n s i s t e n t w i t h f o l l i c u l a r m a t u r a t i o n were r e a c h e d , t h e LRH i n j e c t i o n s were i n t e r r u p t e d and a s i n g l e i n t r a m u s c u l a r i n j e c t i o n o f 6-9000 IU o f HCG ( P r e g n y l , Organon) was a d m i n i s t e r e d . A f t e r t h a t , 1-4 i n j e c t i o n s of 1 5 0 0 - 6 0 0 0 I U of HCG were g i v e n a t i n t e r v a l s o f 3-7 d a y s . R 2 2 Hormone a s s a y methods Immunoreactive FSH and LH i n serum w e r e a s s a y e d by t h e radioimmunosorbent t e c h n i q u e w i t h i n d i r e c t l y c o u p l e d a n t i b o d i e s ( 3 3 ) . LH i n serum was measured by u t i l i z i n g human p i t u i t a r y LH ( 2 2 ) l a b e l l e d w i t h 1251 and r a b b i t antihuman p i t u i t a r y LH. The LH p r e p a r a t i o n had a b i o l o g i c a l a c t i v i t y of 9400 I U (2nd IRP- HMG) p e r mg. FSH i n serum was measured by u t i l i z i n g human p i t u i t a r y FSH ( 2 1 ) l a b e l l e d w i t h lz5I and g u i n e a - p i g anti-human p i t u i t a r y FSH. The FSH p r e p a r a t i o n had a b i o l o g i c a l a c t i v i t y o f 12000 IU (2nd IW-HMG) p e r mg. The r e s u l t s w e r e e x p r e s s e d i n ng o f t h e p u r i f r e d LH and FSH p r e p a r a t i o n s p e r m l o f serum. Com- p a r i s o n s between FSH and LH l e v e l s were made i n r e l a t i o n t o t h e g e o m e t r i c mean FSH/LH r a t i o ( a p p r o x i m a t e l y = 1) o f t h e normal m e n s t r u a l c y c l e . Immunoreactive E 2 w a s measured by a r a d i o i m m u n o l o g i c a l t e c h n i q u e u s i n g an a n t i s e r u m t o o e s t r a d i o l - 6 - o x i m e (11). P r o g e s t e r o n e was a s s a y e d by a s i m i l a r method ( 3 1 ) . One pg of E 2 / m l = 3.67 p m o l / l and l n g of p r o g e s t e r o n e / m l = 3.12 n m o l / l , RESULTS P r e t r e a t m e n t g o n a d o t r o p h i n l e v e l s i n serum b e f o r e and a f t e r i n t r a v e n o u s LRH a r e shown i n F i g . 1. The b a s a l LH l e v e l s were below t h e normal r a n g e i n a l l t h e p a t i e n t s w h i l e o n l y t h r e e of t h e p a t i e n t s had a b n o r m a l l y low b a s a l FSH l e v e l s . A l l b u t one o f t h e AN p a t i e n t s r e s p o n d e d t o LRH w i t h e v i d e n t r e l e a s e o f LH and FSH. The mean LH r e s p o n s e w a s s i m i l a r t o t h a t of t h e c o n t r o l group o f women i n t h e e a r l y f o l l i c u l a r p h a s e of t h e m e n s t r u a l c y c l e w h i l e t h e FSH r e s p o n s e was 4 t i m e s l a r g e r ( F i g . 1). The p r e t r e a t m e n t FSH/LH r a t i o of t h e g o n a d o t r o p h i n r e - s p o n s e s t o LRH w a s 1 . 0 4 i n comparison w i t h 0.22 f o r t h e c o n t r o l s u b j e c t s . T r e a t m e n t w i t h LRH a l o n e F i v e h u n d r e d pg of LRH was a d m i n i s t e r e d p a r e n t e r a l l y e v e r y 8 h o u r s t o 7 women u n t i l m e n s t r u a t i o n o c c u r r e d a f t e r 24-36 (mean 2 9 . 5 ) days of t r e a t m e n t . F o l l i c u l a r growth and m a t u r a t i o n , a s j u d g e d by i n c r e a s e d o e s t r o g e n l e v e l s i n u r i n e o r b l o o d , were i n d u c e d d u r i n g a l l t h e 8 t r e a t m e n t c y c l e s . O v u l a t i o n , as j u d g e d by p r o g e s t e r o n e v a l u e s o f more t h a n 3 ng/ml i n s i n g l e b l o o d samples t a k e n 3-10 days b e f o r e m e n s t r u a t i o n ( 9 ) , w a s a l s o i n d u c e d i n a l l t h e c y c l e s . B a s a l body t e m p e r a t u r e s were r e c o r d e d d u r i n g 5 t r e a t m e n t c y c l e s and a l l t h e c u r v e s were b i p h a s i c . The maximum p r o g e s t e r o n e c o n c e n t r a t i o n s o b s e r v e d w e r e l e s s t h a n . 1 0 ng/ml i n a l l b u t 2 c y c l e s (26.4 and 13.8 n g / m l ) . I n t h e f o u r c y c l e s w e r e b l o o d samples were o b t a i n e d a t l e a s t e v e r y 4 t h d a y , t h e maximum p r o g e s t e r o n e v a l u e was 7 . 7 ng/ml, s u g g e s t i n g i n s u f f i c i e n t c o r p u s l u t e u m func- t i o n . The l e n g t h o f t h e l u t e a l p h a s e was 12-16 d a y s . Hormone l e v e l s d u r i n g a p r e s u m p t i v e l y o v u l a t o r y m e n s t r u a l c y c l e i n d u c e d by LRH a l o n e are shcwn i n F i g . 2 . During t h e f i r s t t h r e e days of t r e a t m e n t t h e FSH r e s p o n s e s were l a r g e r t h a n t h e LH r e s p o n s e s . Then t h e FSH r e s p o n s e s 23 LH - 23 - 2 - 1 - 0, fM2S.E.M AN-Anorexia nervosa, n=9 C=Control, healthy women, early follicular phase, n=X) C AN [1 fiAf3 1 I I 0 C AN t 0 304560 C AN minutes F i g . 1. Mean LH and FSH l e v e l s b e f o r e and a f t e r i n t r a v e n o u s LRH ( l e f t ) and mean LH and FSH r e s p o n s e s t o LRH ( r i g h t ) b e f o r e LRH t r e a t m e n t o f 9 women A N . d e c r e a s e d w h i l e t h e LH r e s p o n s e s p r o g r e s s i v e l y i n c r e a s e d . T h e r e w a s s l o w i n - c r e a s e o f E 2 i n serum d u r i n g t h e f i r s t week o f t r e a t m e n t f o l l o w e d by a more r a p i d i n c r e a s e t o a peak l e v e l (350 pg/ml) c o n s i s t e n t w i t h f o l l i c u l a r matura- t i o n on t r e a t m e n t day 13. A t t h a t time LH i n serum r e a c h e d a maximum w i t h a l e v e l s i m i l a r t o t h a t s e e n d u r i n g t h e m i d c y c l e peak i n t h e normal m e n s t r u a l c y c l e . The LH peak was n o t accompanied by an e v i d e n t FSH peak. A f t e r t h e LH peak t h e E 2 l e v e l d e c r e a s e d somewhat and a t t h e same t i m e i n c r e a s e d p r o g e s t e - r o n e l e v e l s were o b s e r v e d , i n d i c a t i n g t h a t o v u l a t i o n presumably o c c u r r e d . Pro- g e s t e r o n e i n blood s l o w l y i n c r e a s e d to r e a c h a p l a t c a u of 1-14 ng/ml o n e w e e k l a t e r . I n c r e a s e d p r o g e s t e r o n e l e v e l s i n b l o o d were s e e n f o r 12-14 days b e f o r e m e n s t r u a t i o n . One week l a t e r , a new t r e a t m e n t c o u r s e w a s i n i t i a t e d ( F i g . 3 ) . The p r e - t r e a t m e n t LRH t e s t r e s u l t e d i n s m a l l g o n a d o t r o p h i n i n c r e a s e s from l o w b a s a l v a l u e s w i t h a g r e a t e r r e s p o n s e o f LH t h a n o f FSH. The FSH r e s p o n s e i n c r e a s e d s l i g h t l y d u r i n g t h e f i r s t days of t r e a t m e n t b u t t h e FSH l e v e l s a f t e r LRH 24 1 5 10 15 20 25 Dav of treatment Fig. 2. Basal LH and FSH levels i n serum as well as FSH and LH responses to LRH and serum levels of E 2 and progesterone before, during and after the first LRH treatment of a 22-year-old patient w i t h AN. I P E Fig. 3. LH, FSH, E and progesterone levels i n serum before, during and after the second LRH treatment of the AN patient KL from fig. 2. 2 25 1 5 x) 15 20 25 Day of treatment *_ J7 40 J 7 l P E - q 0 F i g . 4 . B a s a l LH and FSH l e v e l s i n serum as w e l l as LH and FSH r e s p o n s e s t o LRH and serum l e v e l s of E 2 and p r o g e s t e r o n e b e f o r e and d u r i n g t h e f i r s t t r e a t m e n t w i t h LRH i n c o m b i n a t i o n w i t h HCG of a 29-year-old woman w i t h AN. remained below t h e LH l e v e l s t h r o u g h o u t t h e t r e a t m e n t . T h e r e was a s m a l l and v e r y s l o w E 2 i n c r e a s e and t h e r e w a s no e v i d e n t m i d c y c l e E peak. On t r e a t m e n t day 1 9 , when t h e maximum LH r e s p o n s e t o LRH was o b s e r v e d , t h e E 2 l e v e l s was o n l y 1 2 0 pg/ml. A f t e r t h e LH peak, t h e r e w a s a s l o w i n c r e a s e of p r o g e s t e r o n e w i t h a maximum o f 8 . 2 ng/ml on day 29. 2 T r e a t m e n t w i t h LRH i n c o m b i n a t i o n w i t h H C G . F o l l i c u l a r m a t u r a t i o n and o v u l a t i o n were i n d u c e d by combined t h e r a p y w i t h LRH and HCG i n 5 t r e a t m e n t c y c l e s . The c o r p u s l u t e u m f u n c t i o n w a s a d e q u a t e , as j u d g e d by h i g h normal p r o g e s t e r o n e l e v e l s i n b l o o d . R e s u l t s from a combined LRH- HCG t r e a t m e n t o f an i n f e r t i l e a m e n o r r h o e i c p a t i e n t w i t h a c o m p l e t e l a c k o f p i t u i t a r y r e s p o n s i v e n e s s t o LRH b e f o r e t h e t r e a t m e n t , a r e shown i n F i g . 4 . D u r i n g t h e p r o l o n g e d LRH t r e a t m e n t b o t h LH and FSH r e s p o n s e s t o LRH a p p e a r e d . The FSH r e s p o n s e was more marked t h a n t h e LH r e s p o n s e d u r i n g t h e f i r s t 3 days and a p r e p u b e r t a l - l i k e FSH/LH r a t i o of t h e r e s p o n s e s were s e e n d u r i n g t h e f i r s t week o f t r e a t m e n t . Then t h e LH r e s p o n s e p r o g r e s s i v e l y i n c r e a s e d w h i l e t h e FSH 26 LH nglrnl . . e . I -. . .' F i g . 5. L H , FSH, E2 and p r o g e s t e r o n e l e v e l s i n serum as w e l l a s LH and FSH r e s p o n s e s t o LRH b e f o r e and d u r i n g t h e second LRH t r e a t m e n t o f t h e AN p a t i e n t BL from f i g . 4 . r e s p o n s e d e c r e a s e d . A t t h e same t i m e , t h e E s e c r e t i o n s t a r t e d t o r i s e . On t r e a t m e n t day 18, t h e LH l e v e l r e a c h e d a m i d c y c l e - l i k e p e a k , which w a s f o l l o w e d by i n c r e a s e d p r o g e s t e r o n e l e v e l s i n b l o o d . The c h a r a c t e r i s t i c f a l l o f t h e E l e v e l c o n c o m i t a n t w i t h t h e i n c r e a s e o f t h e p r o g e s t e r o n e l e v e l , found a t o v u l a - t i o n i n t h e normal m e n s t r u a l c y c l e , was s e e n d u r i n g t h e LRH i n d u c e d c y c l e o f t h i s p a t i e n t . The p r o g e s t e r o n e c o n c e n t r a t i o n was 1 0 . 7 ng/ml when HCG was admini- s t e r e d , i n d i c a t i n g t h a t o v u l a t i o n had a l r e a d y o c c u r r e d a t t h a t t i m e . Repeated i n j e c t i o n s o f H C G were t h e n g i v e n d u r i n g t h e l u t e a l p h a s e , which was s l i g h t l y p r o l o n g e d w i t h h i g h E2 and high-normal p r o g e s t e r o n e l e v e l s . 2 2 On m e n s t r u a l day 5 a new t r e a t m e n t w i t h o n l y LRH was i n s t i t u t e d ( F i g . 5 ) . B e f o r e t h i s second LRH t r e a t m e n t , t h e r e was o n l y a s m a l l LH r e s p o n s e b u t no e v i d e n t FSH r e s p o n s e t o i n t r a v e n o u s LRH. During t h e f i r s t day of t r e a t m e n t , t h e FSH r e s p o n s e r e a p p e a r e d b u t t h e FSH l e v e l s a f t e r LRH n e v e r became h i g h e r t h a n t h e LH l e v e l s . A f t e r a b o u t 1 0 days of t r e a t m e n t t h e r e was a v e r y s m a l l and slow i n c r e a s e o f E2 i n b l o o d . When t h e maximal LH r e s p o n s e t o LRH was o b s e r v e d on t r e a t m e n t day 1 9 , t h e E 2 l e v e l was o n l y 1 1 9 pg/ml. P r o g e s t e r o n e s t a r t e d t o i n c r e a s e s l o w l y and t h e r e was a f u r t h e r E 2 i n c r e a s e t o a p l a t e a u w i t h l e v e l s between 150 and 200 pg/ml. I n c r e a s e d p r o g e s t e r o n e l e v e l s i n b l o o d were o b s e r v e d f o r 13 days b e f o r e m e n s t r u a t i o n w i t h maximum of 8.5 ng/ml on t r e a t m e n t days 29 and 31. Anorexia nerma Long-term LRH treatment n = 6 MZ5E.M A LH I Responses to LRH at the beginning of treatment E2= 28 pghl I] -*I- - I , - - , I - at follicular maturation E2= 345pg/ml F i g . 6 . Mean FSH and LH r e s p o n s e s t o LRH i n 6 women w i t h AN a t t h e b e g i n n i n g of t h e p r o l o n g e d LRH t r e a t m e n t and a t f o l l i c u l a r m a t u r a t i o n d u r i n g t h e t r e a t m e n t . Changes i n t h e p i t u i t a r y r e s p o n s i v e n e s s t o LRH d u r i n g p r o l o n g e d LRH t r e a t m e n t . The a l t e r a t i o n s i n t h e p i t u i t a r y g o n a d o t r o p h i n r e s p o n s e s t o LRH d u r i n g c h r o n i c a d m i n i s t r a t i o n of LRH a r e summarized i n F i g . 6 , which i s a c o m p o s i t e i l l u s t r a t i o n of FSH and LH l e v e l s a f t e r LRH a d m i n i s t r a t i o n a t t h e b e g i n n i n g o f and a f t e r 10-19 days of t r e a t m e n t w i t h 500 pg o f LRH e v e r y 8 h o u r s i n 6 o f t h e women w i t h AN. During t h e f i r s t days of t r e a t m e n t , a t a mean serum E 2 l e v e l of 28 pg/ml, t h e FSH l e v e l a f t e r LRH was t w i c e a s h i g h a s t h e LH l e v e l , an i n v e r t e d p r e p u b e r t a l - l i k e r e s p o n s e p a t t e r n . A f t e r 14 days o f t r e a t m e n t , on a v e r a g e , a t a mean E 2 l e v e l ( 3 4 5 pg/ml) c o n s i s t e n t w i t h f o l l i c u l a r m a t u r a t i o n , t h e s i t u a t i o n had changed d r a m a t i c a l l y . The mean LH c o n c e n t r a t i o n i n b l o o d a f t e r LRH had r i s e n t o a l e v e l s i m i l a r t o t h a t o b s e r v e d d u r i n g t h e m i d c y c l e peak i n t h e normal men- s t r u a l c y c l e . The LH l e v e l a f t e r LRH was 9 t i m e s h i g h e r t h a n t h e FSH l e v e l , which had d e c r e a s e d c o n s i d e r a b l y a t t h a t t i m e . The FSH and LH r e s p o n s e s t o LRH b e f o r e , d u r i n g and a f t e r two c o n s e c u t i v e LRH- HCG t r e a t m e n t s o f one o f t h e AN p a t i e n t s ( F i g . 7) i l l u s t r a t e t h e changes i n t h e p i t u i t a r y r e s p o n s i v e n e s s t o LRH which t a k e p l a c e d u r i n g p r o l o n g e d t r e a t m e n t s w i t h LRH. B e f o r e t h e f i r s t t r e a t m e n t c o u r s e , t h e b a s a l LH l e v e l s were v e r y low b u t t h e r e was a normal r e s p o n s e t o LRH. The b a s a l FSH l e v e l was normal and t h e FSH r e s p o n s e t o LRH w a s 10 t i m e s g r e a t e r t h a n t h e a v e r a g e FSH r e s p o n s e in h e a l t h y women i n t h e e a r l y f o l l i c u l a r p h a s e of t h e m e n s t r u a l c y c l e . During t h e t h e r a p y w i t h 500 ug o f LRH e v e r y 8 h o u r s , t h e r e w a s a p r o g r e s s i v e d e c r e a s e of t h e g r e a t FSH r e s p o n s e . A t f o l l i c u l a r m a t u r a t i o n on t r e a t m e n t day 10, t h e r e was a s l i g h t l y i n c r e a s e d LH r e s p o n s e b u t no l o n g e r any FSH r e s p o n s e t o LRH. HCG w a s 28 HCG 6000IU I M I - 4 - 2 O 0 0 . I ..- 0 1 , , , l1 &&&,",: 1 3 5 7 9 11 13 bleeding - 2 - 0 t o -Fig. 7 . B a s a l LH and FSH l e v e l s i n serum a s w e l l a s LH and FSH r e s p o n s e s t o LRH b e f o r e , d u r i n g and a f t e r two c o n s e c u t i v e LRH t z e a t m e n t s of a 28-year-old woman w i t h A N . t h e n g i v e n t o i n d u c e o v u l a t i o n . A s e c o n d HCG i n j e c t i o n was g i v e n one week l a t e r t o s u p p o r t c o r p u s l u t e u m f u n c t i o n . M e n s t r u a t i o n o c c u r r e d 15 days a f t e r t h e f i r s t HCG i n j e c t i o n . A f t e r t h e f i r s t t r e a t m e n t , on m e n s t r u a l day 3, t h e b a s a l FSH l e v e l s were below t h e d e t e c t i o n l i m i t o f t h e a s s a y and t h e r e was no FSH r e s p o n s e t o LRH. Four days l a t e r , t h e r e was a v e r y s m a l l b u t s i g n i f i c a n t FSH r e l e a s e a f t e r LRH from unmeasurable b a s a l l e v e l s . During t h e f i r s t days of LRH t h e r a p y , t h e FSH r e s p o n s e t o LRH i n c r e a s e d markedly and r e a c h e d a h i g h maximum on t r e a t m e n t day 4 , when t h e E 2 l e v e l was s t i l l low. The FSH r e s p o n s e t h e n p r o g r e s s i v e l y de- c r e a s e d . On t r e a t m e n t day 10 t h e r e w a s no l o n g e r any FSH r e s p o n s e t o LRH. The LH l e v e l s 90 min a f t e r t h e 500 ug d o s e o f LRH i n c r e a s e d d u r i n g t h e t r e a t m e n t and were maximal a f t e r 1 2 - 1 4 d a y s . However, t h e s e LH l e v e l s were a p p a r e n t l y n o t h i g h enough t o i n d u c e o v u l a t i o n a s no p r o g e s t e r o n e i n c r e a s e was o b s e r v e d a t t h a t t i m e . HCG was t h e r e f o r e g i v e n t o i n d u c e o v u l a t i o n and pregnancy o c c u r r e d i n t h i s i n - f e r t i l e p a t i e n t w i t h 13 y e a r s ' o f amenorrhoea. One h e a l t h y c h i l d was d e l i v e r e d a t t e r m . Only two ( p a t i e n t KL and BL) o f t h e o t h e r 8 women were i n v o l u n t a r i l y s t e r i l e . They were t r e a t e d by LRH a l o n e o r i n c o m b i n a t i o n w i t h HCG f o u r t i m e s ( F i g s . 2-5) b u t d i d n o t c o n c e i v e d u r i n g t h e t r e a t m e n t s . 29 DISCUSSION This study shows that the impaired gonadotrophin secretion in women with 'AN can be restored to normal by long-term treatment with LRII. Constant administra- tion of the single gonadotrophin-releasing hormone not only normalized basal FSH and LH secretion but also induced a cyclical gonadotrophin secretory pattern with differential changes of the LH and FSH responses to LRH during the treat- ment. LRH-induced gonadotrophin secretion initiated follicular growth and a normal ovarian cycle with follicular maturation and ovulation could be produced in amenorrhoeic women who were devoid of ovarian activity before the treatment. The results suggest that the impaired gonadotrophin secretion in amenorrhoeic women with AN is due to a supra-pituitary disturbance with deficient secretion of endogenous gonadotrophin-releasing hormone from the hypothalamus. The LH secretion was reduced in all the 9 patients with AN but the pituitary reserve capacity for LH secretion was normal in all but one woman. The pitui- tary FSH secretion was unimpaired in most patients and the pituitary capacity to release FSH in response to LRH was, on average, four times greater than in healthy women in the early follicular phase of the menstrual cycle. The pre- treatment FSH/LH ratio, both in the basal state and after stimulation with LRH, was therefore much greater than in healthy women of fertile age and similar to that described in prepubertal children (10, 23, 5). One of the AN patients had neither any LH nor FSH response to LRH before the treatment but her pituitary responsiveness was restored to normal after repeated stimulation with LRH. Thus, unresponsiveness in a diagnostic LRH test does not necessarily indicate primary pituitary failure but may be due to dysfunction at the hypothalamic level with insufficient hypothalamic stimulation of the pituitary gonadotrophs by endo- genous gonadotrophin-releasing hormone. Nor does a lack of gonadotrophin re- sponse to acute LRH stimulation preclude successful results of long-term LRH therapy as shown by the induction of both follicular maturation and ovulation by LRH in the patient with no pretreatment gonadotrophin responses to LRH (Fig. 4 ) . Striking changes in the pituitary responsiveness to LRH were seen during the LRH treatments. The maximal FSH responses were obtained during the first days of treatment and at that time FSH levels which were greater than or equal to the LH levels after LRH were observed in most patients. However, the FSH re- sponses rapidly decreased during the treatment and this decrease became more marked when the oestrogen secretion from the ovaries started to rise. The LH responses to LRH, on the other hand, progressivley increased during the treat- ment and, in most patients,reached maximal midcycle-peak levels at high oestro- gen levels consistent with follicular maturation. After that increased progest- erone levels began to appear in the blood, suggesting that ovulation occurred. During the luteal phase of the cycle, the LH responses decreased somewhat but . ... - 30 remained much g r e a t e r t h a n t h e FSH r e s p o n s e s t h r o u g h o u t t h e r e m a i n d e r of t h e c y c l e . Thus, t h e i n v e r t e d p r e t r e a t m e n t g o n a d o t r o p h i n r e s p o n s e s t o LRH changed d u r i n g t h e p r o l o n g e d LRH t r e a t m e n t and became s i m i l a r t o t h o s e s e e n i n h e a l t h y a d u l t women. The changes i n t h e p i t u i t a r y r e s p o n s i v e n e s s t o LRH may b e due t o t h e f a c t t h a t LRH s t i m u l a t e s s y n t h e s i s and r e l e a s e of p r e d o m i n a n t l y LH and t h a t modulatory f e e d b a c k e f f e c t s of t h e o v a r i a n hormones, p a r t i c u l a r l y E 2, t h e n a c t a t t h e p i t u i t a r y l e v e l t o g e t h e r w i t h s e l f - p r i m i n g e f f e c t s of LRH t o c a u s e a f u r t h e r marked i n c r e a s e of t h e LH r e s p o n s i v e n e s s t o LRH ( 2 5 , 1, 35). These a l t e r a t i o n s are v e r y s i m i l a r t o t h o s e s e e n i n t h e normal f e m a l e p a s s i n g from t h e p r e p u b e r t a l t o t h e p o s t p u b e r t a l s t a g e ( 1 0 , 5 ) . S i m i l a r e n d o c r i n e and p h y s i c a l changes of p u b e r t y h a v e b e e n s e e n d u r i n g p r o l o n g e d LRH t h e r a p y i n hypogonado- t r o p i c men ( 1 5 ) . It c o u l d b e c o n s i d e r e d t o b e a n a l o g o u s t o t a k i n g t h e s e p a t i e n t s through p u b e r t y by t h e h i g h d o s e LRH t r e a t m e n t . F o l l i c u l a r growth and m a t u r a t i o n were i n d u c e d d u r i n g t h e LRH t r e a t m e n t and p r e s u m p t i v e e v i d e n c e of o v u l a t i o n , i . e . i n c r e a s e d p r o g e s t e r o n e s e c r e t i o n , w a s a l s o o b t a i n e d . However, t h e p r o g e s t e r o n e v a l u e s d u r i n g t h e p r e m e n s t r u a l p e r i o d were r a t h e r low i n 6 of t h e 8 c y c l e s where o n l y LRH w a s a d m i n i s t e r e d , s u g g e s t i n g i n s u f f i c i e n t c o r p u s l u t e u m f u n c t i o n . The l u t e a l p h a s e s w e r e of normal l e n g t h and t h e l u t e a l p h a s e d e f e c t s were t h e r e f o r e more s i m i l a r t o t h a t d e s c r i b e d a s t h e i n a d e q u a t e l u t e a l p h a s e by Sherman & Korenman ( 2 7 ) t h a n t o t h e s h o r t l u t e a l p h a s e d e f e c t d e s c r i b e d by S t r o t t e t a l . ( 2 9 ) . Subnormal p r e o v u l a t o r y FSH l e v e l s h a v e b e e n found d u r i n g m e n s t r u a l c y c l e s w i t h l u t e a l p h a s e d e f e c t s ( 2 9 , 2 6 , 2 7 ) . S t r o t t and co-workers p o s t u l a t e d t h a t a r e l a t i v e FSH d e f i c i e n c y d u r i n g t h e f o l l i c u l a r p h a s e r e s u l t s i n abnormal f o l l i c u l a r development and s u b s e q u e n t i n - a d e q u a t e c o r p u s l u t e u m f o r m a t i o n o r f u n c t i o n ( 2 9 ) . P a t i e n t s of t h e p r e s e n t s t u d y , who had i n v e r t e d p r e t r e a t m e n t r e s p o n s e p a t t e r n s w i t h g r e a t FSH releases a f t e r LRH, responded promptly t o t h e LRH t r e a t m e n t w i t h marked E 2 i n c r e a s e s c o n s i s t e n t w i t h f u l l f o l l i c u l a r m a t u r a t i o n and t h e y h a d normal c o r p u s l u t e u m f u n c t i o n , as j u d g e d by t h e p r o g e s t e r o n e c o n c e n t r a t i o n i n b l o o d ( e . g . p a t i e n t KL, t r e a t m e n t I , F i g . 2 ) . P a t i e n t s w i t h low FSH r e s p o n s e s i n c o m b i n a t i o n w i t h h i g h e r LH r e s p o n s e s b e f o r e t h e t r e a t m e n t r e s p o n d e d s l o w l y w i t h much l o w e r p r e o v u l a t o r y o e s t r o g e n i n c r e a s e s f o l l o w e d by i n a d e q u a t e l u t e a l p h a s e s ( e . g . p a t i e n t B L , t r e a t - ment 11, F i g . 5 ) . The r e s u l t s s u g g e s t t h a t a p o s s i b l e e x p l a n a t i o n f o r t h e l u t e a l p h a s e d e f e c t s might be an a b s o l u t e o r r e l a t i v e FSH d e f i c i e n c y which l e a d s t o d e f e c t i v e f o l l i c u l a r m a t u r a t i o n and s u b s e q u e n t i n s u f f i c i e n t c o r p u s l u t e u m func- t i o n . An a l t e r n a t i v e e x p l a n a t i o n f o r abnormal f o l l i c u l a r development might b e t h e r e l a t i v e l y h i g h LH l e v e l s i n d u c e d by LRH d u r i n g t h e f o l l i c u l a r p h a s e of t h e t r e a t m e n t c y c l e s . The LH/FSH r a t i o w a s s i m i l a r t o t h a t d e s c r i b e d i n women w i t h t h e p o l y c y s t i c o v a r y syndrome ( 1 4 , 3 4 ) where f o l l i c u l a r m a t u r a t i o n i s i m p a r i e d . The r a i s e d LH l e v e l s may s t i m u l a t e t h e o v a r i e s t o an i n c r e a s e d androgen s e c r e t i o n . 31 which inhibits follicular development. Ross and co-workers reported that small doses of HCG or LH to oestrogen-treated hypophysectomized immature female rats resulted in decreased granulosa cell proliferation and increased follicular atresia and showed that this inhibitory effect was mediated by local intra- ovarian effects of androgens, secreted by the ovary in response to HCG and LH (12, 13). In regularly menstruating women, elevation of LH activity in blood by HCG administration during the early follicular phase has been shown to cause luteinization of the theca interna with degeneration of tertiary follicles and delay or suppression of ovulation (30, 6). It can not be excluded that the LRH- induced LH elevations during LRH stimulation of follicular growth and maturation may have had deleterious effects on the follicular development and subsequent corpus luteum function. Thirdly, it might be that during treatment with only LRH the LH peak levels at follicular maturation were not high enough for sufficiently long periods for normal ovulation and corpus luteum formation to occur. To secure an adequate preovulatory LH surge, HCG was therefore administered during five additional treatment cycles after induction of follicular maturation by LRH. A l l these LRH- HCG treatment cycles were ovulatory with normal luteal phases, as judged by the progesterone values. In two of the cycles (e.g. Fig. 4), the E2 and progesterone patterns in blood suggested that ovulation had already occurred when HCG was given. During the postovulatory phase additional HCG injections were given to support corpus luteum function. In the cycles where only LRH was given, the luteal phase LH levels after LRH were presumably high enough for further support of the corpus luteum. However, it seems necessary to continue the LRH treatment throughout the luteal phase as we observed short luteal phases during treatment cycles where the LRH injections were interrupted during the postovulatory phase of the cycle (17). One may question whether ovulation really occurred during the treatment cycles with signs of luteal phase insufficiency. The increased progesterone levels in blood are only indirect indices of ovulation and may be caused by luteinization of granulosa cells of the follicles without ovulation. In summing up results of treatment with LRH, Schally and co-workers concluded that although ovulation can be induced with LRH in sterile women the percentage of ovulations and pregnan- cies is relatively low (24). This might possibly be explained by a high percent- age of cycles with luteal phase insufficiency. Only three of the nine women in the present study were involuntarily sterile. One of them became pregnant during her second LRH-HCG treatment (Fig. 7 ) and by that she proved that normal folli- cular maturation can be induced by treatment with LRH alone in women with im- paired gonadotrophin secretion and absent pretreatment ovarian activity. For treatment of anovulatory infertility, it may be necessary to combine LRH with HCG or LH to secure normal ovulation and adequate corpus luteum function. 32 ACKNOWLEDGEMENTS T h i s work w a s s u p p o r t e d by t h e S w e d i s h M e d i c a l R e s e a r c h C o u n c i l ( g r a n t No. 13X-3145). We a r e i n d e b t e d t o D r s . F. Enzmann a n d M . v a n d e r Ohe, F a r b w e r k e H o e c h s t AG, F r a n k f u r t / M a i n , FRG, f o r g e n e r o u s s u p p l y o f s y n t h e t i c LRH and t o Mrs. Anna-Lena Barmark, Mrs. B i r g i t t a Bohman, M r . 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