Hypogonadism and ecdysteroid production in Loa loa and Mansonella perstans filariasis

Acta Troptca, 46(1989)249-256 Elsevaer

249

ATP 00033

Hypogonadism and ecdysteroid production in Loa loa and Mansonella perstans filariasis J. Lansoud-Soukate 1, A. Dupont 1, M.L. De Reggl 2, G E. Roelants 1 and A. Capron 3 1International Centre for Medz~al Research ( CIRMF), Francevdle, Gabon, 2Irnmunotech, S A , Marsedle, France, and 3Centre d'lmmunologae et de Blologw Parasltatre, umtb mt',te 1NSERM, Instttut Pasteur, Ldle, France

Possable endocrlnologacal repercussions of mfecnon with Loa loa and Mansonella perstans filanae were studaed in Gabonese subjects Mlcrofilaremlc males were compared wath amacrofilaremlc controls In the infected group 13/105 subjects (12%) presented only abnormally low serum levels of testosterone ( < 4 ng/ml), 25/105 (24%) only abnormally high serum levels of gonadotrophms, FSH (> 15 mIU/ml) and LH ( > 20 mIU/ml), and 22/105 (21%) presented anomahes m both testosterone and gonadotrophm levels One out of 68 control subjects had 3 6 ng/ml serlc testosterone and all had normal levels of gonadotrophms Ecdysteroads were detected ( > 0 025 ng/ml) m the serum of 87/97 (90%) m~crofilaremlc subjects (GM 0 123 ng/ml) compared to 12/64 (19%) controls (GM 0 030 ng/ml) Ecdysteroads were detected m the urine of all subjects, infected (GM 8 468 ng/ml) as well as control (GM 1 245 ng/ml) The hormonal perturbations were correlated wath the levels of Loa loa macrofilaremla but not wath those of serum and urinary ecdysterolds These results demonstrate that macrofilaremlc subjects often show endocnnal signs of hypogonadasm and present apprecmble levels of ecdysteroads m serum and urine A darect role for parasitic ecdysteroads in hypogonadasm remains to be demonstrated Key words Fdarlasls, Loa loa, Mansonella peratans, Hypogonadasm, Ecdysteroads

Introduction

It is estimated that 300 mflhon people in the world are infected wtth filarlae (Faro, 1981a) Loa loa filarlasts affects 2 to 3 milhon subjects m Central Africa (Faro, 1981 b) and Mansonella perstans filanasts 40 to 80% of mdlvtduals hvmg m Central and Western Afraca (Hawking, 1977) The geographical dlstrtbution of loalasts is coincident with a zone of hypofecundlty with btrth rates of only 2 0 to 3 5%, whereas surroundmg regions have rates of about 6 0% (INSEE, 1960-61, Romantuk, 1968, Meheus et al, 1986) Certam filarlases such as those caused by Wuchererla bancroJtt (Ltchtenberg and Medma, 1957, Iturregut-Pagnan et al, 1976) and, to a lesser extent, Onchocerca volvulus (Strangway and Strangway, 1951, Nelson, 1958, Gratama, 1969), can reduce lesxons of the male gemtal tract That the same is true for L loa and M perstans infections is not clear (Gordon et al, 1950, Langutllat et al, 1978a, b) To our knowledge, the endocrine repercussions m the host of filarla infectxons have Correspondence address Dr J Lansoud-Soukate, Centre Internanonal de Recherches M6dlcales de Francevllle, B P 769, Francevflle, Republlque Gabonalse 0001-706X/89/$03 50 c~ 1989 Elsevier Science Pubhshers B V (Biomedical Division)

250 however not been investigated Thus we have looked for endocrine abnormahtles associated with L loa and M perstans mlcrofilaremla in naturally infected subjects Ecdysterolds are hormones controlling essential events of development and reproductIon m arthropodes and probably other protostomlan phylae and have been detected in helminths including filariae (Koolman, 1984, Rees and Men&s, 1984) In certain cases they have been shown in the biological fluids of parasitized vertebrates (Koolman, 1984, Nlrd6 et a l , 1984a,b) The possible correlation of parasite ecdysterolds and endocrine repercussions of L loa and M petstans' refections is investigated

Materials and Methods We tested 776 men aged 20 60 years from the province of Haut-Ogoou6, Southeastern G a b o n , for the presence of L loa and/or M perstans mlcrofilaremla O f 367 mlcrofilaremlc subjects, 262 were rejected for presenting other parasites in blood and/or stool, of 409 amlcrofilaremac subjects, 341 were rejected for the same reason Mlcrofilarlae detected, using the K n o t t ' s concentration method (Markell and Voge, 1981), were distributed as follows m the blood of the remaining 105 mlcrofilaremlc subjects 18 L loa only, 48 M perstans only and 39 with both N o n e of these mlcrofilaremlc subjects had obvious signs of hypogonadlsm on physical examination in the field Controls consisted of the remaining 68 amlcrofilaremxc subjects (hmlt of detection 1 organlsm/ml of blood) without clinical signs or history of filanasls N o other parasites were detected in the blood or stool of the 173 subjects selected for this study All samples were collected between 10 00 h and 15 00 h All subjects were of c o m p a r a b l e SOClO-economical status and no signs of malnutrition were detected Serum testosterone was measured for all subjects by the competitive radiolmmunoassay of A b r a h a m et al (1977) (using T R K 402 Amersham-France reagents) The results are expressed as ng/ml, the limit of detection was 0 1 ng/ml and normal values were considered to be 4 to 8 ng/ml Serum levels of folhcle-stlmulating hormone (FSH) and lutelnizlng-hormone (LH) were measured by radloimmunoassay using commercial kits ( C E A - S O R I N , OnsIndustries, St Quentin en Yvellnes, France) The results are expressed as mIU/ml, the limit of detection was 1 5 m I U / m l and normal values were considered to be 5 to 15 m I U / m l for FSH and 4 to 20 m I U / m l for L H Quantity of material available allowed ecdysterolds extraction from the serum and urine of only 97 mlcrofilaremlc and 64 control subjects Ecdysterolds were measured according to the method of De Reggl et al (1975) Results are expressed as ng 20hydroxy-ecdysone equivalent per ml, the limit of detection was 0 025 ng/ml Levels of ecdysteroids, testosterone, FSH and LH were log-normally distributed, with the exception of ecdysterolds in urine, and central tendencies were measured using the geometric mean The slgmficance of group differences was analysed by the Student's t-test, the Behrens-Fisher's test, the F-test and/or the ~2 test with Yate's correction (Armltage, 1971) The significance of group differences for ecdysterolds In urine, which were not normally distributed, was analysed by the Kruskal-Walhs' Htest (Siegel, 1956) Regression curves of correlations were calculated and eventually produced (Fig 2) with the Microsoft Chart R on a Macintosh R computer

251

Results

The presence of mlcrofilarlae in blood was independent of subject age in the present study L loa mlcrofilanae ranged from 1-86 200/ml (median 540/ml) and M perstans from 1-4 300/ml (median 100/ml) Hormonal levels in all groups described below were also independent of subject age In the mlcrofilaremlc group, 13/105 subjects (12%) presented only abnormally low levels of serum testosterone (< 4 ng/ml), 25/105 (24%) only abnormally high levels of serum gonadotrophms FSH (>15mIU/ml) and LH (>20mIU/ml), and 22/105 (21%) presented both low testosterone and high gonadotrophm levels Only one out of 68 control subjects had borderhne (3 6 ng/ml) serum testosterone and all had normal levels of gonadotrophms Means are presented in Fig 1 Mean hormonal m [U/m I ng/ml 14

12

10

0

~ FSH

LH

Testosterone

Fig l Geometric m e a n + s t a n d a r d error of the mean of g o n a d o t r o p h m s FSH and LH (mlU/ml) and testosterone (ng/ml) m mlcrofilaremlc ( • ) and amlcrofilaremlc ([]) subjects

252 levels did not differ for the three types of mlcrofilaremla L loa alone, M perstans alone and both together However, the 18 subejcts presenting only L loa mlcrofilarlae showed a negative correlation between the levels of mlcrofilaremla and the serum testosterone (r = - 0 64, p < 0 005) and/or a positive correlation between the levels of mlcrofilaremla and FSH levels ( r - 0 71, p < 0 001) (Fig 2) These correlations were not found m subjects presenting pure M perstans mlcrofilaremla and there was no correlataon between L loa mlcrofilaremla and LH levels Table 1 shows the levels of ecdysterolds measured in serum and urine Serum ecdysterolds were detected m 87 of the 97 mlcrofilaremlc subjects for which sera were

TESTOSTERONE (ng/ml) 10

It 8

4

2

0 0

I

I

I

I

100

1000

10000

100000

Loa Ioa

rnJcrofilaremla

(/ml)

253

available and also m 12 of the 64 controls tested, albert m lower concentrations The difference between the means for the two groups was highly significant (p < 0 001) Urinary ecdysterolds were detected in all subjects although again m very significantly lower mean concentration in the control than m microfilaremic subjects (p < 0 001) There was no correlation between serum and urinary levels of ecdysteroids nor between the species and degrees of mlcrofilaremla and ecdysteroid levels m either serum or urine Host hormone levels were not correlated with ecdysterotd levels in etther blood or FSH

(mIU/ml)

80

B 70

60

50

40

30

20

10

o 10

I

I

I

I

100

1000

10000

100000

Loa Ioa

mmrofilaremta

(/rnl)

Fig 2 Regression curve of correlatmn between testosterone (A) or FSH (B) serum and Loa loa m~crofilaremm

254 TABLE 1 Ecdysterolds m subjects bearing microfilarlae and controls Ecdysterolds (ng/ml) Sample

Subjects

n

Geometric mean

Range

Serum

mlcrofilaremlcs controls

97 64

0 123a 0 030

< 0 025-3 605 < 0 025 0 485

Urine

mlcrofilaremlcs controls

97 64

8 468 b 1 245

0 275 87 215 0 255-44 045

aBehrens-Flsher's test d= 10 9, q=29 01, p <0 001 bKruskal-Walhs's test H - 4 6 5, p <0 001 urine Indeed, o f the 10 mlcrofilaremlc subjects w i t h o u t detectable e c d y s t e r o l d s in serum, 5 h a d h o r m o n a l a b n o r m a h t i e s

Discussion T h e levels o f m i c r o f i l a r e m l a f o u n d in the subjects studied here covered the range r e p o r t e d m the l i t e r a t u r e ( F a i n , 1981, H a w k i n g , 1977) Such h o r m o n a l d i s t u r b a n c e s as those detected in 60/105 ( 5 7 % ) microfilaremlc subjects in o u r study c o u l d p l a y a role in h y p o f e c u n d i t y in C e n t r a l Africa. Parallel decreases o f t e s t o s t e r o n e levels a n d increases o f g o n a d o t r o p h l n levels m i g h t indicate a p r i m a r y testlcular defect ( H u a n g et a l , 1984, I b r a h l m et a l , 1983) D e c r e a s e in t e s t o s t e r o n e level w i t h o u t elevation o f g o n a d o t r o p h l n s , o b s e r v e d in 13/105 ( 1 2 % ) o f mlcrofilaremlc individuals, could also p o i n t o u t to a supra-test~cular m e c h a n i s m T h a t testosterone c o u l d be efficiently m e t a b o h z e d by p a r a s i t e s has yet to be excluded E c d y s t e r o I d s were f o u n d in serum a n d urine o f m~crofilaremic i n d i v i d u a l s as has been the case in o t h e r paraslUzed v e r t e b r a t e s ( K o o l m a n et a l , 1984, Nlrd6 et a l , 1984a,b) E c d y s t e r o l d s were also f o u n d m low c o n c e n t r a t i o n s in 19% a m i c r o f i l a r e m i c c o n t r o l s This could be due to s o m e subjects c a r r y i n g a d u l t w o r m s w i t h o u t mlcrofilarla, as has been d e m o n s t r a t e d for 22 o u t o f 33 subjects p r e s e n t i n g with a L loa eye passage ( D u p o n t et a l , 1988) M o r e o v e r , s o m e o t h e r h e l m l n t h p a r a s i t e infections c o u l d have escaped detection F u r t h e r m o r e , h u m a n f o o d c o n t a i n s p h y t o e c d y s o n e s which are r a t h e r w i d e s p r e a d an p l a n t s Be t h a t as it m a y , e c d y s t e r o l d levels were significantly higher m the mJcrofilarem~c t h a n in the a m i c r o f i l a r e m i c g r o u p H o r m o n a l p e r t u r b a t i o n seems h o w e v e r c o r r e l a t e d with the degree o f mlcrofilaremxa a n d n o t with e c d y s t e r o i d levels W h i l e e c d y s t e r o l d s c o u l d p l a y a role m h y p o g o n a d l s m , e g t h r o u g h their a c t i o n on cyclic A M P m e t a b o l i s m ( C a t t et a l , 1979, 1980, C a t a l a n et a l , 1979), a role for these parasitic h o r m o n e s in this p h e n o m e n o n r e m a i n s to be d e m o n s t r a t e d

Acknowledgments C I R M F is 70% financed b y the State o f G a b o n a n d 30% by funds p r o v i d e d by ElfG a b o n W e t h a n k the e n d o c r i n o l o g y l a b o r a t o r y o f C I R M F for m e a s u r i n g testos-

255

terone, FSH and LH, Mrs L Trotteln and Mr E Ruelle for excellent assistance in computer analysis, Drs C Chlzzollm, R W Cooper, T E Egwang, D Mavoungou, and M Plnder for rewewlng the manuscript and Mrs M A Molto for expert secretarial assistance

References Abraham, G E , Manhmos, F S and Garza, R (1977) Radlolmmunoassay of Steroids In B M Jaffre and H R Behrman (eds), Handbook of Radlolmmunoassay, Academic Press, New York, NY, p 590 Armltage, P (1971) Statistical Methods in Medical Research, Blackwell, Oxford Catalan, R E , Aragones, M D and Martlnez, A M (1979) Effect of ecdysterone on cyclic AMP and cyclic GMP in mouse plasma Blochem Blophys Res Commun 87, 1018 1023 Catt K J , Hardwood, J P , Agullera G and Dufour, M L (1979) Hormonal regulations of peptlde receptors and target cell responses Nature 280 109-116 Catt, K J , Hardwood, J P , Clayton, R N , Davies, T F Chan, V , Katlklneml M , Nozu, K and Dufour, M C (1980) Regulation of peptlde hormone receptors and gonadal steroldogenesls Rec Prog Horm Res 36, 557-622 De Reggl, M L , Him, M H and Delaage, M A (1975) Radlolmmunoassay ofecdysone An application to drosophila larvae and pupae Biochem Blophys Res Commun 66, 1307 1315 Dupont, A , Zue Ndong, J and Plnder, M (1988) Common occurrence of amlcrofilaremlc Loa loa filarlasls within the endemic region Trans R Soc Trop Med Hyg 82, in press Falm A (1981a) Pourquol faut-ll combattre les filarloses Ann Soc Beige Med Trop 61, 589-599 Fain, A (1981b) Epldemlologle et pathologle de la loase Ann Soc Beige M6d Trop 61 277 285 Gordon, R M , Kershaw, E , Crewe, W and Oldro)d, H (1950) The problem of lolasls in West Africa with special reference to recent investigations at Kumba in the British Cameroons and at Sapele in Southern Nigeria Trans R Soc Trop Med Hyg 44, 11 47 Gratama, S (1969) The pathogenesls of hydrocele in filarlal infections Trop Geogr Med 21 254-258 Hawking, F (1977) The distribution of human filarlasls throughout the world Part IlI Africa Trop Dis Bull 74, 649 679 Huang, H F S, Zaldl, P and Nleschlag, E (1984) Pituitary testlcular interrelationships during germinal lnvoluhon in the vitamin A deficient rat J Endocrlnol 100, 33-41 Ibrahlm, I I , Barakat, R M R , Bassloun~¢, H K , Hanna, L S, Aboul-Atta, A M , Bayard, M A , Ghaly, 1 M , Abdalla, M I and Hafy, E S E (1983) Effect of urinary bllharzlal infection on male pubertal development and endocrine functmns Arch Androl 11 59 64 lnstltUt National de la Statlstlque et des Etudes Economlques (INSEE), Mlnlstere de la Cooperation, France Recensement et enqu&e demographlque 1960 1961, ensemble du Gabon resultats definltlfs Iturregul-Pagnan, J R Fortuno, R F and Noy M A (1976) Genital manifestations of filarlasls Urology 8, 207-209 Koolman, J , Walter, J and Zahner, H (1984) Ecdysterolds in helmlnths In J Hoffman and M Porchet (eds), Blos.',nthesls, Metabolism and Mode of Action of Invertebrate Hormones, Springer, Berlin, Heidelberg, pp 323 330 Langulllat, G , Albert, M Tursz, A and Blot, P (1978a) Enqu&e sur Fettologle de 1 hypofecondlte au Gabon oriental I Protocole et premiers resultats Rev Epldemlol Sante Pub 3, 237 257 Langulllat G , Garln, Y , Tursz, A , Beau~als, B and Larl~lere, M (1978b) Enqu&e sur l'&lologle de l'hypof&ondlte au Gabon Oriental lit Endemle hlarlenne (Loa loa, D per~tans) frequence des mlcrofilmres dans les hydroceles Rev Epldemlol Sante Publ 3, 273 282 Llchtenberg, F and Medina, R (1957) Bancroftan filarlasls in the etiology of fumculo-epldldymltlS, perlorchltlS and hydrocele in Puerto-Rico Am J Trop Med Hyg 6, 739 751 Markell, E K and Voge, M (1981) Medical Parasitology, 5th edn, Saunders, Philadelphia PA, p 337 Meheus, A , Renlers J and Collet, M (1986) Determinants oflnfertlhty in Africa Afr J Sex Tran Dis 2,31 35 Nelson, G S (1958) "Hanging groin" and hernia complication ofonchocerclasls Trans R Soc Trop Hyg 52 222 225 Nlrde, P , Torpler, G Capron, A , Delaage, M and De Reggl, M L (1984a) Ecdysterolds In schlstosomes and host-parasite relationship Trans R Soc Trop Hyg 52, 331-337

256 Nlrd6, P , De Reggl, M L , Tsanpras, G , Torpler, G , Fessancourt, P and Capron, A (1984b) Excretion of ecdysterolds by schlstosomes as a marker of parasite refection FEBS Lett 168, 235 240 Rees, H H and Mendls, A H W (1984) The occurrence and possible physiological slgntficance of ecdysterolds during nematode and cestode development FEBS Lett 168, 338 345 Romanmk, A (1968) L'lnfecondlt~ en Afrlque tropmale In La Population de l'Afnque, Population Council Edlhons, New York, NY pp 286-300 Siegel, S (1956) Non parametric statistics for the beha~loural sciences, McGraw-Hill, Tokyo Strangway W E and Strangway, A K (1951) Omhocerca voh'ulus m Angola Can Med Assoc J 64, 427-429