Contraceptive efficacy of testosterone-estradiol implants in male rhesus monkeys

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CONTRACEPTIVE EFFI CACY 0F TESTOSTERONE ESTRADI OL I MPLANTS I N MALE RHESUS MONKEYS

T

t

J

~

LoblI

~

Ph D t

•

K. T. Kirton, Ph.D., A. D. Forbes,

L. L~ Ewing2 Ph.D, P. L....

, M . P . H . , and C. D e s j a r d i n s ,

Ph.D.3

F e r t i i i t y Research, The UpJohn Company, ~l amazoo

Mlchigan,

49001

ABSTRACT Rhesus monkeys I atta) were treated wi th testosterone (lO0~g/ s estrad oi (0.5 /kg/ ) via subcuta neous pol y di methyl s i I oxane (PDS Si i ast i c ) i mpl ants. Th i s t t e a t m e n t

c a u s e d a s t r ! k i ng r e v e r s i b 1 e s t e r i i i t y .

No....D gnancies

re observed in fe

les bred to the

s t e r o i d ....t r e a t e d .... 1 e s . I n c on t r a s t , t h e r e s no d i f f e f ence n pregnancy rate of fe les bred to control and steroid-treated monk s f o r 14 w e e k s , b e g i n n i ng 17 w e e k s

a f t e r removal of the s t e r o i d - f i i ! e d i

lants.

Submitted f o r publication December 7, 1982 Accepted f o r publ i cation january 12, ] 983

To wh 2

3

repr n

requests shou!d be ad r....O ssedt

D~vi sion of Reproducti ve Biology Depart nt of Population Dynamlcs The Johns Hopki ns School of Hygi ene and Pubiic Health 6~5 N. Wolfe St reet Baltimore, Maryiand 21205 Insti tute of Reproductive Biol ogy Depart nt of Zoo ogy The Uni versi ty of Texas aI; Austin Aust i n, Texas 78712 RIL 1983 VOL. 27 NO. 4

3 83

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of detectable side effects on s a t i c tlssues, the ionlc, chemlcal and f o r d ele nts of b!ood, or secondary sex structures including the ry gland (2). These r e s u l t s r a i s e d t h e q u e s t i o n o f t h e c o n t r a c e p t i v e e f f i c a c y , tn non-human p r i tes, of testosterone-estradiol subcutaneous PDS imp!ants pro gra d to e a s e t e s t o s t e r o n e and e s t diol at rates approxi ring those at whi ch tbe two stero i ds are produced endogenousI y. i n th s pa er we report that ie nkeys treated w~th }O0~g testosterone pl us O. 5ug estradio] per kg body w e i g h t p e r day a r e r e v e r s i b l y s t e r i l e . S t e r i l i t y i s a c c o m p a n i e d by a s t r i k n9 reduction n the nu~er of morphologically no I and v!gorous!y t i !;e s pe a t o z o a .... Re v e t s i b 1e s t e r i 1 i t y i s a c h i eve d wi thou t p e r t u rba t i n g the i o n i c , chemical and fo d ele nts of b!ood. ZRIALS AND METHODS GENEPAL Ten the sus nkeys were se! ected f r a group of f e r t i I e maI es in t h e Updohn f e r t i l i t y r e s e a r c h r h e s u s monkey c o l o n y . F~ve o f t h e s e les re assigned rand l y to and 5 to the tes iol treatn t g r o u p s .... C o n t r o l nkeys r e c e i v e d empty PDS ' i m p l a n t s . Treated nkeys received PDS i: lants designed to release lO0~g testosterone and 0 5ug estradiol per kg body weight as described e a r l i e r ( ! ) . EXPERIMENTAL DESIGN .... The study was di vi ded into three parts- pre-trea nt, trea nt, post-trea ent phases. The pre-trea nt phase began on O c t o b e r 6 , lgSO. The t r e a n t p h a s e began on November 17, i980~when t h e s t e r o i d - f f l e d ca ules were i lanted nto the ani is. The p o s t - t r e a t nt phase began when the stero~d-fi I led ~mplants re removed on August 27, 1981° The experi n t ended March 29, !g82. Breeding t r i a l s were carried out and b%ood and se n samples were co lected at various t ! s during the course of ....the study. Two breeding t r i a s re completed, one during the trea nt and another during.. the post-treat nt phase of the study .... The f l r s t breeding t r i a l lasted 23 weeks; i t began 17 eks a f t e r subcutaneous p! ace nt of s t e r o i d - f i l led capsules and continued u n t i l the end of the trea nt phase of the experi nt .... The second breeding t r l a l !asted !4 eks i t began 17 weeks a f t e r s t e r o i d - f i l l e d i lants re removed .. Se n sa es were collected thrice at bi0-weekly in rvals via electro e j a c u l a t i o n (1) d u r i n g t h e p r e - t r e a t n t p h a s e ; a t t h e 16tb and 4 0 t h week during the Crea nt phase; aC the 1 6 t h ek the nt phase. Biood samples re collected as described e a r l i e r (2)- at three h i - ekly intervals during the p r e - t r e a t n t phase; at 0 I , 4, 8, i2 16, 17, 20, 24, 28, 32, 36 and 40 ek~ during the trea nt phase; and at O, 4, 8, 12, !6, 20, 24, 28, and 31 eks during the post-trea nt phase of the study.

384

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BREEDING TRIALS - F e r t i l e female rhesus monkeys re se!ected f o r ting 15 of the ~r n s trua cyc I e Fema!es were ass i gned to m a l e s r a n d o m l y and p l a c e d

fe

in the male~ cage.

Males continued

les unti} each male had completed six (during trea

f i ve (du r i ng p o s t - t t e a tree n t p h a s e ) s u c ce s s f u I

to receive

nt phase) and

t i n g s a s e v i de rice d by

the presence of a seminal plug the cage. The bred fe les tanned u n t i l pregnancy s diagnosed by manuai palpation.

re ma~n

SEMEN ANALYSIS - E!ectroejaculated se n samples re. co! l e t t e d into glass coagulum was a l l o d to r e t r a c t f o r I hrs and the total ejacul ated vol u was asured An al iquot ( d i l uted {~i th 0 ....9% sai ine; w/v) s re~oved to qua ~~ta t i re] y eva I uate morpho] ogy an t i I ~ty by I ght microscopy as described el sewhere (3). One of Pronase sal ine so! ution (15 mg/ml) then was added to each ejaculate and the coagulum cut into pieces. A f t e r 4 to 5 hrs incubatlon at 37oC, the ejaculate was mixed thoroughly and a lO v a l i q u o t s transferred to i of phenoi solution (lOg and 40g NaHCO3Z~ . Nuclei of spe atozoa re counted on a he cy.... tO

ter.

BLOOD CHEMISTRY AND HE

OLOGY - A standard SMA-1260 p r o f l l e pl us electroleted on serum sa les .... Blood hematology s carried out on a Coult e r counter (Cou!t e r El ectronics, Inc., Hialeah, FL ) and d~fferen t i aI counts cutupi eted [ UpJohn Standard Opera i ng Procedure s f o r He tG~ogy (SOP 4.5) and Chemistries {SOPs 4.2 and 4.4) !980.....82]. STATISTICAL .ANALYSIS The resuits of the breeding t r i a l s owl ng equat i on.

re trans o~

d

X = 2 ARC SIN where P is p~..egnancy rate:, and N is the nu~er of observations. The average p r e g n a n c y r a t e f o r t h e g r o u p s w e r e c p a r e d u s i n g a t - t e s t on t h e t r a n s .... f o r ~ d values (4) The following assumptions were made in evaluating the significance of the results: 1) matings re c p l e t e l y independent of one another, 2) there is no t~ e f f e c t , and 3} the les represent a h ogeneous popuI at i on ... The se a 3 ....f a c t o r

n and blood chemistry and he r e p e a t e d .. a s u r e s de s i g n .

tology results were analyzed using

RESULTS FERTILITY - The results of the breeding t r i a l s carried out during the t r e a t ost

treat

nt phases

of this

study are

sh

n in Figure

I.

The

l e f t panel o f Figure I shows that the pregnancy rate of fe les bred to control les during the trea nt phase o f the study s 47~ (l fertile o f 30 s u c c e s s f u !

tings).

In c o n t r a s t ,

females bred to steroid ~rea ted Thus,

s teroid~reated

no p r e g n a n c i e s

were observed

i es (0 f e r t i ! e of 30 successfu!

males were sterile

for

at

17 eks a f t e r imp!antati on of testosterone p r o b a b i ! i t y t;hat s t e r i l l t y occurred by chance is

least

23

eks;

in the

tings ).

beginning

PDS capsules. The ess than 2 In i000.

The r i g h t panel of Figure i docu nts the pregnancy rate during the post t r e a t n t phase of the study; n t r o l and treated les i regnated 48% (12

RIL

1983 VOL

27 NO. 4

.385

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,ION - T h e l e f t panel perce nt pregnant f o r contro (cross-hatched bar) and trea ted monkeys during the treatment phase of .....the study. The r i g h t panel shows the percent pregnant f o r cont ro I .( c ro ss hatched

. "

~\~

\ 30 ¸

U E W

~-

"

,.

~ -~

bar) and treated (black bar) monk ~ s dur ~ng the post -

\~

The I n s e r t ~n each bar sh

tO

IO

treatment phase of the study. "

NO~[ CON

TRE

TR

0

;ONtnOL

MENT

T

T

¢0

~IOD

s

the number of attained per the number of successfui t i rigs.

f e r t i l e of 25 successful t i n g s ) and 56% (I f e r t i l e of :25 successful tlngs) of the fe l e s , spectively. Thus, the f e r t i ! I t y o f the ste id-treated males returned to cont levels at least 17 weeks a f t e r testosterone....estradlol PDS impi ants red SEMER CHARACTERISTICS .... As anticipated, semen characteri s t i cs of control .and s varied dramatical ! y during the trea ent phase of

t h i s study {Table i ) . spe

Spe

tozoa, and percent no

per e j a c u l a t e ,

rate, percent motile

I : rphology. . . . re s i g n i f i c a n t l y ( P < O )

lower

- The Effect of Testosterone-gstra iol Trea nt on Se n Vo~u Spe tozoa Numbers, Motil i t y and Morphology During the T a CONTROL

Se n V o l u (ml) Sper to.zoalEjaculate x ~06 Moti I i R a t e (0-4) Mot i I e Spe z o a (%) No ! Morphology (~)

TREATED

1.8 ± O.2Z 1046 ± iOZ

2.0 0.4 74 ± 28**

2.4 ± 0.3 8Z ~ 3

0.2 ± O . l * * 18 , 6**

75 ± 4

I l ± 4**

IObservations were de on se n m p l e s col lected from both control and treated monkeys 16 eks (n=5) and 40 eks (n=5) a f t e r ~ ~ I a n ~ t i on of cont ce)tive steroids 2The values represent t h e vations **

3

an ÷_ standard error of the : an of lO obser....

(P<0.01).

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in contracepted than in control nk~s ..... In contrast, the contraceptive steroid formulat on did not i n f uence semlnal vo ume (P>O. 5). The semen cha c t e r i s t i c s of control nkeys during the t r e a t nt phase of this study (Table I ) re identlcal to those obtalned durlng-the p - t r e a t nt and trea nt phases of the study which were . 2 0 . 1 (X • S E ) I 1 7 8 ± 98, 2.i ~ O . , 79 ± 2 6, and 76) ± 3~ respective!y, f o r se n vo u (ml), s~er tozoa per ejaculate (X 0 ), i t y rate (0....4), motl e spe tozoa (~) and normal rphology (% . . . . . Semlnal characterlstics of control and steroid-treated nkeys were Identical (P>O.15) during the pre ....and post-trea nt periods (resul ts not sho ) w i t h a single exception. The percent of sper tozoa with nor l morphoi "ogy was st i l I si gni f i cant i y (P<0.05) Iower i n contra cepte d than control monkeys during the post.....treat nt phase of the study; 52 lO versus 81 +_ 6%, respectively. BLOOD CHEMIS?RY AND HE

OLOGY-There was no effect of the cont ceptive t i tuents of bl odd: ca ! ¢i urn, sodlum, potas si urn, chl orl de, inorganic phosphate, g)ucose, BUN, cholesterol, total protein, al bumin, total b i I i rub i n al kal i ne phosphata se, g utamic oxal acetl c tra nsaminase, white blood c e l l , red blood c e i l , hemoglobin, eosinophil, basophll numbers, hematocrit, an corpuscular volu , ~ a n corpuscular an corpuscular he globln concentra on, l phocyte, nocyte and p l a t e l e t numbers (results not ). ..... DISCUSSION FERTIL ITY - The s t r i k i ng vers i bI e st e r i i i i nduced tes tos rone-est r a i ~ i a n t s in rhesus nk~s is similar ....to that observed in le rats (5) and to that observed in hu n es taking o I comblnations of thy! testosterone and ethynyioestradio! (6). I n f e r t i l i t y in the contrace)~d n s is not due to a defect in sexual behavior because semlnal p ugs a present in the cage with the sa f quency In contro)and t r e a t e d les. Again, this is conslstent wlth the behavior of le rats (5) and bu ns (6) trea ted wi th androgen-e st roge n contrace pt ive fo rmu! at i ons. i nteres t i ngI y, the c ~l ete and reve r s i bl e ste r i I i ty i n contracep ted nk s s achleved desplte the fact that a l l the nkeys.were not azoospe ic (Table i ) . Spe tozoa per ejaculate dropped below I x 106 in only I of 5 contracepted nkeys, in one of these65 s t e r i l e monk s the lo s t spe tozoa count per ejaculate was 162 x lO~. Obviously, a hlgh contraceptive e l f cacy can be obta in oiigosRermic rhesus n k e y s . Thls co fete sterl I i ty presu b y stems from the s t r i ki ng reducti on i n the perce,ntage and rate of motile sper tozoa in ejacula of contracepted monkeys (Table I ). These l a t t e r results suggest that sperm I I t y ....f e r t i l l t y tu tion function of the ep~dl ~s ~ y be impai d by the testosterone-est dlol trea n t .. I t is becoming increasingly clear that the epidi i s has a profound androgen dependency ( ) and that fete ~ s t i s f l u i d contains unknown factors required to Intain certain aspects of caput epidi l function (7). Thus, the suppresslon of LH re!ease t e s t i c u i a r t e s sterone productlon, and, ! ~ subsequently, spe togenesis by the test te r one~ estradiol contraceptive fo ulatlon (1) also ~ y Interfere with the spe tu tion and/or storage functl on....of the epi i dymi s...

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-It was reported In an earl l e r paper { 2 ) t h a t the testosteronei r a cept i ve t re atreent a I tore d bi ood serum coricentra t i ons of cho.... l e s t e r o i , alkai ine phosphatase and glucose, and seminal volu . In the present study there was no e f f e c t of the contraceptive steroid treatment on these c r i te!'la. r) Seminal volume of control and treated monk.s is shown in Table I. The concent t i on of thole ste rol a! kaI i ne phosphata se and gl ucose i n per i pheral b l o o d re 159 +_ 42 C)T ~ SE), i28 ± 40, and 99 ± 21 f o r control )monkeys and 151 +_ 18, llO ) 39, and 87 ) !8 f o r sterold treate onkeys, respectlvely. Test~s s ze, ho vet, was d!mlni:shed by the sterold t r e a t nt. Testis wi dt;h (l at i tud i nal ax i s )of the I e f t tes t i s a sured through the scrotum w! th calipers s 29 and n~ f o r control and treated n k ~ s , respectively, a t the end of the trea nt phase of the study .... However, t e s t i s width recovered by the end of the post-treatment phase of the study; 28 versus 27 f o r con t r o 1 a n d t t e a t e d mon key s , r e s pe c t i ve 1y . A widely discussed issue not add :ssed in t h i s study ~s whether abno l sper tozoa in the ejaculate of a contracepted oligospe ic human male....can f e r t i l i z e an o o c y t e and r e s u i t i n an i n c r e a s e d i n c i d e n c e o f c o n g e n i t a l ab ndr~l ities. Al though t h i s issue can only be resolved by f u r t h e r experi ntation, several points are worthy of discussion, r s t none of the ollgospermic rhesus los ~n study re f e r t i l e suggesting that the ak3y motile and/or abno f l y shaped sper tozoa were incapable of cre~ atlng a pal pabl e embryonic-pl acental ss. S~cond, there was no evi dence of pro1 onged e s t r o u s c y c l e s i n r h e s u s f i es i~regnated by s t e r i ! e , ol i g o sperm c rhesus maies, suggesti ng that there ~(as no i ncrea sod i nc dence of early f e t a l reso tlon ....(results not sho ). Th rd) se n from rt~le n capable of conceiving no I offspring contain a high percentage of abhor 1 f o ~ s (8). In f a c t , a human se n sample is considered nor by so authors (8) even i f i t contains 40% abno l fo s. F i n a l l y , the incidence of early f e t a l ioss in the hu n fe l e is surprlsing!y high suggesting that under no al circumstances ma ryos f a l to develop. B ggers (9) reported that "b .... t h e t i ) pregnan i s r e c o g n i z a b l e , h a l f t h e e m b r y o s have been l o s t " . ' Bi~gers . (9) . . f u. r t h e r noted that, dur ng the nder pregnancy, anot her quar te r of the emb~ o s per i sh or are absor bed ". Thus, abnorma! emb~os y be screened out by the hlgh emb~onic loss in h u ns. H applicable is thl s contraceptive strategy to the human le? Briggs and Briggs (6) already have contracepted hu n ~ i e s with an oral fo l a~ tlon of ~ t h y l testosterone and eth~nyloestradiol. Further, consideration of th s quest i on must beg~n by evaI ua t i ng the op C! ona i t es tos t erone/es trad i o1 requ i red f o r contracepti on i n the human I e. These cha c t e r i sties might be v e ~ d i f f e r e n t in n than in nk s a s e v i d e n c e d the fact that the r a t i o o f te terone/estradiol needed to i n h i b i t sper togenesi s varies fr 200 300, I000 in nkeys, rats and dogs, re e c t i v e l y . Second) the lonq-te effects of the testosterone-est dlol cont ceptive f o r lat~on on c a n i n e so t i c t i s s u e s a n d p r o s t a t i c s t r u c t u r e and f u n c t i o n s h o u i d be d e te ined because the aged dog, i ike : n, has a' s i g n i f i c a n t incidence of ben t gn p r o s t a t i c h y p e r p 1 a s i a . F i na 11 y , h i gh ....d o s e t o x t c o l ogy s t u d i e s on s t i c tissues, breast, accessory sex organ structure and function, must be c ieted in the non-hu n pr~ te befo the f u l l l i c a t i o n of this con.... t c e p t i v e s t r a t e g y can be c o n t e f a t e d f o r t h e hu n m a l e . The co f e t e and reversible s t e r i l i t y caused by the contraceptive fo I a t i on c o u p I e d w t t h o u r f a i 1 u r e t o d e t e c t s e c o n d a r y compl t ca t i ons a t t r i b u -

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table to the steroid t r e a t nt offers j ust l f I ca t i on f o r f u r t h e r eva!uat i on o f a contraceptive strategy in the hu n based on administering n a t u r a l l y occurring steroids a t rates approxi t l n g those which are produced endog..... enously. AC~OWLEDGEMENT

The technlcal ass!stance of d. Moran In asurlng serum chemistrles Is g r a t e f u l l y acknowledged. This research s supported in part the Updohn Company and by The National i n s t i t u t e of Child Health and Hu n Develop n t through Research Contract 3-2745, Research Grant 07204 and Population Center Grant 06268o

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Ewing, L. L., Huber, A. C., Strandberg, J° D., Adams, R J . , Cochran, R. C. a d De ard ns C. S t i c t~ssue responses of le rhesus nk s trea ted with a contraceptl ve stero~ d f o r I at ! on .... Contracep i on 2 7 : 3 6 3 ....3 8 1 ,

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Valerlo, D. A., Leverage, W. E...., and Munster, J. H. macaques. L a b . Anl. Care 1970.

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Robalre, B., Scheer, H. and Hachey, C. Regulation of epi d tabol i z i ng en es ~ Bi oregui at o t s o f Repro t i on, ( j a g i e l l o and H....J. Vogel.°~ ~ademlc Press, NeW York, 1981.

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