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Diagnosis of early pregnancy in the baboon
NT
DIAGNOSIS OF EARLY PREGN A. A. S h a i k h , C. A l l e n - R o w l a n d s , T . The South
est F o u n d a t i o n
for
Y IN
z i e r , D. C. K r a e
E BAB
E
I
N
e r , * a n d J . W. G o l d z i e h e r
sea~h and Education, San Antonio,
xas**
ABST M e t h o d s f o r t h e d i a g n o s i s o f e a r l y p r e g n a n c y i n t h e b a b o o n ...b y t h e d e t e c t i o n 0£ c l, or ion ic g o n a d o t r o p h i n ( C G ) ...i p! a s m a, ~u s i n g r a d i o i m o a s s a y ( R I A ) , o r in tl, e u r i n e b y h a e m a g g l u t i n a t i o n ...i n h i b i t i o n , a s well as b y easurement of pias~a estmgenSand progestins, ere c o p a r e d f o r tl~eir s e n s i t i v i t y , r e l i a b i i i t y ¢ a n d r a p i d i t y ; D e t e c t i o n : o f CG i n t h e u r i n e b y h a c m a g g l u t i n a t i o n i n h i b i t i o n w a s u n s a t i s f a c t o r y ..as . l a t e as d a y 2 5 a f t e r c o n c e p t i o n i O n d a y s 8 a n d 12, m e a s u r e e n t s o f e s t r o g e n ( E ) , ..p r o g e s t e r o n e (P), o r in p l a s m a c o u l d n o t b e
and P determinations ere obtained more quickly, and when evaluated according to a c o p u t e r-d e rived u 1a, w e r e 9 6 . 6 % su Cccs s fuI o n t a t d a y ....
INTRODUCTION In p r i m a t e s , e a r l y d e t e c t i o n o f p r e g n a n c y is o s t ..i p o r t a n t i f t h e y a be used e f f i c i e n t l y in fe~ ~;~ s t u d i e s , it p e r m i t s b e t t e r e x p e r i i n e n t a l d e s i g n a n d ~ d u c e s tlle ..c.. o s t involvedin ating ~ n u m b e r o f a n i m a l s w i t h u n c e r t a i n o u t c o e: A n a c c u r a t e a n d e a r l y d i a g n o s i s p e r l n its ) : treat n t o f a n i t a a l s in t!i e e a r l y s t a g e s o f regnaIlcy ~t . . o g i c s t u d ie ......s - P....~ ~~n a n,c jv de ,t e c ..... p o t e n t i a ! an t i-fe '~....a n. d. . .i s e s...... Sen t i al fo r t er. .a. t. .ol t io n c a n .....::~sation, ..... b e p c r f o r. . . . e d ,~ ......- e ,',~....... a gegl u t i n a t.i.o. .n. . . . e t h. .o. d. .S .......... f o r U r i n a ry; c h o r i o n:i C ..g...o...... nadot r o p I~in (1), b i o a ss "~'.......... . m o r c o n c e n t r a t e d u r i n e e x t ~ c t (22 5 ) , Or ~ d i o i m m u n o a s s a y o f c h o rio n ic go n a d o t r o p h i n o r i t s 13 u n it ( 6 )
W e l~ave evalua ted t|to re]iabi|ity an d rapidity o f ~cth ocls u tilizingRI A
r p|asm a C G
baboon,
*.. P
**
s....... e n t a d d r e 's"s ,
a s A & M U n i .v. e. .r.s. i. t y.,. . . . . . . . . T e x.... C o Iiege S t a t i o n , T e x as
o f : .V P h y...s i 0 1 og, ..... . e t e r i n a r y. .......
d r e s s r e p r i n t r e q u e s t s t o A. A . S h a i k l i , S o u t h w e s t F o u n d a t i o n E d u c a t i o n ; .O. B o x 2 8 1 4 7 , S a n A n t o n i o T e x a s 7 8 2 8 4
Accepted
OCTOBER
for
1976
publication
VOL.
14
July
NO.
4
26,
for Resea~h and ....
1976
391
13
TRACE
I .M .
Rl ALS A N D M
HODS
A total o f 60 m a t e d b a b o o n s i~ich sho e d t h e presence o f s p e a~ozoa in the:vagina! s e a r ere used i n this s t u d y .....In this species, seX skin deturgescence is ~ i d to Occur 3 days after ovulat ion (7). ougl~ studies in ou r i a b o r a t o r y indicate at ti0n in b a b o o n s actually occurs i d a y b e f o or on t h e first ofdeturgescence, e illbas~ dating for ....this . r e p o r t on the earlier published Tiius; the first:day o f de turge see n e e il I be called d a y 3 o f pregnancy, Blood samples Were collected in heparinized t u b e s on days i 2 , 16, 18, a n d 2 5 o f pregnancy. Blood p!es were obta d .~ .. 0 m n a d d i t i o n a l 19 I~aboons on ctay. 8 o f pregnancy. The plasma as st ed at -x0 C. Urine s a pies Were a l s o . o b t a i n e d On days 12, 16, ..18, a n d 25 o f p gnancy ..or, in several ....instances, d a i l y f r o m day 1 2 through 25 U r i n e was collected ithin 30-60 inutes o f voiding. o
.
.
.
.
.
.
Estroge ere e a s u r e d by RIA following the e t h o d o f Wu e t aL (8). N o prior c h r o a t o g r a p h y o f the .. pies as e ployed. T h e antiseru used in this study was kindly supplied by Dr. P. N. Rao; it cross-reacts 1:00% ith estradio|2iTtS; ......1 0 3 % w i t h estr0ne, and 6% estriol a n d i roxyestrone e values t us r e p r e s e n t " t o t a l eitrogens. T h e .... ........ , " aS t h e y were ........... ues for ..plasma progesterone also r e p r e s e n t " total progestins easu'r ed b y the CPB m e t h 0 d o f J o h a n s s o n ( 9 ) i t h o u t c h r o m
The hae agglutination inl~ibition test for urinary chorionic gonadotropllin e m p l o y e d the kit kindly supplied by the Hor one Distribution Officer, N1 DD, NIH. The tests ere carried out according to the pub|islied instructions (I). RIA for Plas
a Chorionic G o n a d o t r o
The antiseru used in this RIA as kindly supplied~by Dr. G. D. Hodgen and as developed in rabbits against the fl-subunit o f ovine LH (6). I o d i n a t e d (12s I) hu an chorionic g o n a d o t r o p h i n (HCG) as purchased fro Bm RIA, Montreal, Canada. Specific activities ranged from 112-131 ~Cilgg. Since no reference p r e p a r a t i o n for b a b o o ~ chorionic gonadotrophin s a liable, e prepared our o n reference standard from a seru pooI o b t a i n e d fro three b a b o o n s on day 25 of? pregnancy. ~his pooled seru as diluted 1:16 ith fetal calf seru and a dose response curve as c o n s t r u c t e d using volu es of 5-200 ~i (Fig. 1). ~ h e RIA procedure followed the e t h o d Of. Hodgen et al. (6), using 50 and 200 ~1 aliquots o f undiluted plas a. tal calf seru as added to both tl~e reference and u n k n o w n sa pies to ake tlle final seru c o n c e n t r a t i o n 20% by volu e. A plas a pool fro pregnant b a b o o n s (day 25 o f pregnancy) and a n o t h e r pool from non-pregnant animals at day 12 post-ovulation ere used as controls in each assay.
392
OCTOBER
1976
V O L . 14 N O . 4
C
ION
! gotive Contro!
0
600
40" c o
20"
......... t
5 in n o n o g r a
LN
:
~-
25
50
s;ot h er
samples
t ................ - .......................................... l
tOO
200
t
400
~n...... i c r o l i t e r s
Fig, I, Co parison o f d o s e r e s p o n s e curves using reference a n d c o n t r o I sa pies a n d a .. b a b o o n L H ..... (BLH) p r e p a r a t i o n , P e r c e n t c o u n t s p r e c i p i t a t e d are n o r alized to c o u n t s p r e c i p i t a t e d in t u b e s c o n t a i n i ~
A p o s i t i ~ diagnoMs o f p r e g n a n c y was ade w h e n the 200 ul sa pie p r e c i p i t a t e d b e t w e e n 0 . 5 and 9 0 % o f the c o u n t s , n o r alized to c o u n t s p r e c i p i t a t e d in the t u b e ,aining no antigen (Fig. 1)..A b a b o o n s c o n s i d e r e d n o t pregn It if the 2 0 0 ~I sa ple precipitated > 9 o f the- unts. T h e r e f o r e , t|lis R I A was used pri ily as a qualitative test to d e t e c t the presence or absence o f b ~ ~ o n c h o r i o n i c ~ n a d o t phin (B ). In a d d i t i o n , we have f o u n d that tl~e antiserum used in this s t u d y cross-reacts with b a b o o n LH (Fig. 1). Using 50-200 n g / t u b e o f b a b o o n , p i t u i t a w .....LH, a dose response cu si Liar to t h a t o b t a i n e d by using diluted p r e g n a n t b a b o o n seru wag o b t a i n e d . These. results a in disagreement ith published r e p o r t s o f ti~e~s p e c i f i c i t y Of the antiseru (1). Tlaerefore, a ~ I s e positive diagnosis o f p r e g n a n c y b e t w e e n ~ idmycle s u r e of~LH.~ RESU
I n s p e c t i o n o f . t h e plas sensitive in d i c a t o r 0 f
OCTOBER
~976
S
a steroid levels (Tables i,I1)~1o ed t h a t p r o g e s t i n s re a m o r e n cy t h a n estrogen~, b u t t h a t ~ ei t h e r d e t e r m i n a t i o n , , by itseI f,
VOL.
14 NO. 4
393
C O N ' I "R .
.
.
.
.
.
.
.
.
P~r l 0 N
2
~
~~
~
~
~;i¸¸''iI' ~
W , ~ ~~ 8 ~ ~i ~ ~!~ o ~~ :~~' ~,i ~ '~ ~~ ~W!I ~ ~ ~W ~ ~ ~~ ~ ,'~
W
W
Z
~
394
OCTOBER
1976
VOL.
i~,il~~
14 N O . 4
¢,rl
o
0
l..a
0
.,4
t-a
0
0
NEG POS NEG NEG NEG POS NEG NEG NEG NEG NEG
Urine 8CG (POS, NEG) E pgfml
11.1 ±1,5 15 16 (93.8}
6 16 (37.51
Note: Animal No. 18 was used in an ongoing experiment,therefore samplecollection was discontinued
Confirmation (%)
163.6±8,8
NEG POS POS POS POS
200.0 ± 13.0
POS POS POS POS POS NEG POS POS POS POS POS POS POS POS POS POS
Plasma BCG (PO5, NEG)
Mean ±$E
16.5 14.0 10.5 8,4 21.5 11,0 3.2 9.0 5.6 7.3 12.8 13~ 4,7 24.3 7,0 9.1
P ng/ml
210 225 238 135 197 !88 177 307 219 150 185 170
197 190 185 144 128 127 126 132 193 140 140 150 224 207 207 128
E pg/ml
D18
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 20 21 22 23 24 25 26 27 28 29 30
Animal No.
Table I (continued)
9,9 + 1.3
11.2 14.3 2.3 5.7 12.6 5~8 8.0 14.5 8,5 7.3 10.1 18.3
P ng/ml
D25
12 12 (1001
POS POS POS POS POS FOS POS POS POS POS POS POS
Plasma8CG (POS, NEG)
9 11 (81,8)
POS NEG POS POS NEG POS POS POS PO$ POS
POS
Urine BCG (POS, NEG)
O Z
C3
O Z
C3
~
~
~
~
~
~
o ~ o o o o ~ ~ 0
V
,~
,~8
VV
V
•••,•,
8
~
VV
~
Z
~~
~ e ~ o o o ~ o o o ~ ~ ~
VVVVVV
Z
~Mo
VVV
VVVV
V
~
"8
A
~
~w~ ~
8
~~
A
A
~
e S~~8~SS
~88s~~s$8
~ ~ssssss8
°
~ U..l~¸ ~
~
VV
VV
V
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V VVV~VV
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Z Z Z Z Z Z
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V
8
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C
.9 C
OCTOBER
~1 9 . 7 6
XzOL.
: : N O . .. 4
O
,,,1
w
4~
P
O
P~
O
¢3
E
0 8 (~1001
0 8 (100)
0.5 +0.0
< 0.5 <~ 03 ~ 0,5 < 0.5 < 0.5 <~ 0.5 '~ 0.5 0.7 < 0.5 < 0.5
P ng/ml
D25
0 10 (100)
NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG
Plasma BCG IPOS, NEG)
Note: Animal Nos, 1 and 21 to 30 started to recycleas determined by perinealsex skin turgescence,therefore no sampleswerecollected from theseanimals.
Confirmation (%)
0.6±0.1
113.8 +10,4
pg/ml
106.5±11.0
NEG
NEG NEG NEG NEG NEG NEG NEG
Urine BCG (POS, NEG)
Mean +SE
NEG NEG NEG NEG NEG NEG NEG
NEG
Plasma BCG (POS, NEG)
88 109 141 99 96 66 98 182 134 125
i.4
<~ 0.5 <~ 0.5 <~ 0.5 < 0.5 < 0.5 <~ 0.5 <~0.5
P ng/ml
139
149 92 78 67 81 131 115
E pg/ml
D18
10 11 12 13 14 15 16 17 18 19 2O
9
7 8
6
5
4
2 3
Animal No.
Table II (continued)
6 (100)
0
NEG NEG NEG NEG NEG NEG
Urine BCG (POS, NEG)
¢3
O Z
C
TRACE
ION
p o s s e s s e d s u f f i c i e n t d i s c r i m i n a t o r y p o e r in e a r l y p r e g n a n c y . W ! l e n b o t h h o r m o n e s ere e"| e ,V a t e~ d a b o v e t Ii e c o r ~ Sp o, 1d {n g va I u e s o b t a i n e d fr o, n k n o n n o n- p r e g n a n t a n i m a Is a t t 11a t t i m e Of t h e c y c l e , t l l e r e w a s a ~ r e~*' a t e r t l m n 9 5 % c e r t a i n t y o f p r e g n a n c y. ; . l i. k e. w. i s.e , . w . hen n e ti h e r, s ! o ed* e l e v a t i o n ,. t l .~ e r e. . a s a . g r e. a t e. r t.l l a n. 9 5 % c e r t a i n t y o f n o n - p r e g n a n c y . F o r those cases h e r e o n l y o n e v a l u e w a s e l e v a t e d o r w h e r e tllc rise in b o t h w a s b o r d e r i i n e , We a t tern p t e d t o d e re! o p a m a t h e a [ i c a l m o d e | w it ]~ o p t i m u m ....p r e d iC t ive p o e r. e o rigi n a| model was based d a t a f r o m t h e first 3 8 b a b o o n s , a n d tlle e q..u a t , o,~s e r e r e t e s t e d o n t h e entire set of data in 60 animals. E st r o g e n a n d p r o g c s t in v a l u c s f r o m p | a s m a o b t a i n c d o n d a y 1 2 w e r e a r r a i , g e d i n r a n k o r d e r , d i v i d e d i n t o q u a r.t.i.l.e. s ; a n d m e d i a n v a | u e s e x t r a c t e d By c o m p u t e r , ....e q u a t i o n s ere t t~e n d e r ive d t o fi t t i i e q u a r t i le u m b ers t o t 1!e e d i a n va i u e s for-e s t r o g e 11 ( E ) a n d p r o g e s t i n (P). F o r E, t h e equation~ t o o k t h e f o r m y = a + b forP t o o k tlle f o r t h ~ = a e b x , yielding for estrogen a quartile number = -23.32304 + 5.71769 (|n E value). For this e q u a t i o n, t ti e c o r r e la t io 1~ c o e ffi c i e n t (r) a s 0 . 9 8 . F o r p r o g e st i i i s. t i~e q u a r t i le n u m b e r = 0 . 8 6 2 2 3 e O" 1 5 8 4 ( P v a l u e ) itil r .... 0 . 9 9 . Th e t
o c q u a t i o n s w e r e t il e
c o i n b i n e d t o y i e 1d :
0 . 8 6 2 2 3 e x p ( 0 . 1 5 8 4 ....* P va I u e) = - 2 3 . 3 2 0 4 + 5 . 7 1 7 6 9 I n ( E va | u e ). S i n c e tile m e a n P v a l u e f o r p r e g n a n t a n i m a l s o n tllis d a y t h e a n i m a l is p g n a n t i f
as a b o u t 6 0 n g /
1,
e state that
6.0
0.1584 "!'hen ,, 0
e×p
![
.. -/ 0 . 8 6 2 2 3 e x ~ 0 . 1 5 8 4 x P v a Iu e_)_
+
23 32304 ........
E s t r o g e n (T)
5 . 7 1 7 69 For given l u e s o f P, E ( T ) p r o v i d e s a c u t o f f p o i n t : A c t u a l E v a l u e s les t l , a n tlle E ( T ) a s s o c i a t e d w i t h a p a r t i c u l a r v a l u e o f P s u g g e s t t l l a t t h e a n i m a l is n o t p r e g n a n t ; a c t u a l E values ~ e a t e r tllan the co p u t e d ~(T) for a given P value suggest p r e g n a n c y . When this ethod s a p p l i e d to t h e ra d a t a , it a p p e a r e d t h a t t h e p r o g e s t i n e q u a t i o n w a s n o t s e n s i t i v e e n o u g h t o d i s c r i i n a t e p r e g n a n c y u s i n g a v a l u e o f e x a c t l y 6 . 0 n g / ~ l . It w a s d e c i d e d to u s e a r a n g e o f 5 . 5 - 6 . 5 n g / m l as an e s t i m a t o r f o r p r e g n a n c y f o r d a y 12 d a t a . This procedure no p r o v i d e s t o esti a t e s f o r E ( T ) . I f t h e a c t u a l e s t r o g e n v a l u e falls b e l o w t h e lesser E ( T ) ( d e r i v e d f r o m t h e 5 . 5 n g / m l P v a l u e ) f o r a given s e t o f E a n d P v a l u e s , t h e a n i m a l is j u d g e d n o t p r e g n a n t . If t h e a c t u a l E v a l u e is g r e a t e r t h a n E ( T ) u s i n g 6 , 5 n g / m l , tile a n i m a l is j u d g e d p r e g n a n t , i f t h e a c t u a l E v a l u e lies b e t w e e n t h e t o v a l u e s f o r :E(T), t h e odel c a n n o t d e t e r ine the status. U s i n g tile e q u a t i o n s a n d v a l u e s g i v e n a b o v e , a c o p u t e r p r o g r a s written to generate a table containing values of P fro 0.1 to 8 ng/ml ith c o r r e s p o n d i n g ini u and axi u E ( T ) v a l u e s ( T a b l e 111).
398
OCTOBER
1976
VOL
14
~O.
4
h i e II1 Pregnancy Determination
ble
(Cont.)
Pregnancy Deter
ination Estr~en
stro~n Progestin
Ne~tive Maxi um
Positive Minimum
Progestin
177 174 171 168 165 162 160 157 155 153 150 148 146 144 142 140 138 136 134 133 131 129 128 126 125 123 122 120 119 118 116 115 114 I 13 112 111 109 108 107 106
216 211 207 3 199 195 192 188 185 182 178 175 172 169 167 164 161 159 156 154 152 149 147 145 143 141 139 137 135 134 132 130 129 127 126 124 123 121 120 I 18
4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5, I 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7~8 7.9 8.0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3, 7 3.8 3;9 4.0
ble
Ne~tive i a x i. . . . . . u
Positive Minimu
05 04
17 16
03 03
13
02 0i 00 99 98 97
97 96
95 94 94 93 92 92 91 90 9O 89 89 88 88 87 86
15 12 1! 0
09 08 07:
O6 .... 05 04 03
02 ol 00 00 99 98 97 96 96 5 94
8S 84
93 93 92 9I 91 90
84 83 83 83 82 82 81:
89 88 88 87 87 86
8O
s6 85
s6 85
9o ....
T o u s e .t h. i s. t.a b. l e , l o c.a. .t.e. .a.n. l al s P r o...g .e.s. t. l. n. v a l u........................ e , t h e n t o t h e r .i .g. h. .t. .~read t h e . . . .axz mt u ... . . . . . . . u. . . . . .a. .= ~ ................ e s t r o g e n v a l u e s : E x a m 16, if t h e p r o e s f i n V a l u e i s : 2 5) ~t h e n t h e e s t r o g e n ust be le~ than125 to conqrm .n.o. .n. - rpe gn a n c y. . .o. .r g r e a t .e.r. . .t.h. .a. n. . . .1. .4 3 t o. . . c. .o. .n. G . . r. . . . p r e g n. .a. .n.c y. . . .B. .e. t. w . e e n 1. 2. 5 . a.n d. . 1.4 ~. .r e.s u. l t. s . a .r e. m . c. o .n c. l u. s.l , . e
OCTOBER
1976
VOL. 14 NO. 4
399
suits o t ~ a d o u s
sts
T a b l e i su a r i z e s t h e results a f all tests p e r f o r ed in ani a!s hose p r e g n a n c y as c o n f i m l e d b y c a e s a r i a n s e c t i o n o n e i t h e r d a y 5 0 o r 70. ble iI gives tl!e C o r r e s p o n d i n g r e s u l t s in ....a n i m a l s hich p r o v e d n o t t o be p r e g n a n t , in ttie p r e g n a n t b a b o o n s , t h e t o t a l 5.2 pg/ Ito a day 25 e s t r o g e n vMues i n c r e a s e d gradually fro ...... .. a d a ) 8 mean value m e a n value o f 2 0 7 ± 1 2 . 0 pg/ i. By c o n t r a s t t e n o n - p r e g n a n t b a b o o n s y i e l d e d a d a y 8 can V a l u e o f 9 4 ± 7 . 4 hich ..re a i n e d essentially u n c h a n g e d . T h e c a n values o f 107 ± i 1.0 a n d i 14 +- 10.4 on d a y s ...18 a n d 2 5 , r e s p e c t i v e l y e r e n o t significant1,, ~:~iffercnt f r o m d a y 8 values. Tile p r o g e s t i n ues also i n c r e a s e d fro a day 8 ean v a l u e ~ o f 6 . I ± 1.1 n g / i t o 11.5 ± 1.8 o n d a y 25 H o w e v e r , t h e signific t c h a n g e s in p r o g e s t i n values o c c u r r e d b e t e e n d a y 8 a n d 16 a f t e r w h i c h t h e y stabilized, .... rr l m p r o g e s t i n values in n o n - p r e g n a n t ani als, b y c o n t st, sho ed a c o n t i n u o u s d e c l i n e , o n tile .a.. v erage, a n d b y d a y 18 w e r e b e i o the li its o f de te ct ab i I y i n M | b u t o n e a n i m a 1. Usiffg tile R I A e t h o d for d e t e c t i o n o f plas a BCG, t h e success rate in c o n f i r m i n g p r e g n a n c y o n d a y s 8 i 2 16 18 a n d 25 as 0, 6.5, 9 6 . 6 , 9 3 . 8 a n d i 0 0 % , r e s p e c t i v e l y . In c o n t r a s t , ...p r e g n a n c y c o n f i r m a t i o n by d e t e c t i o n o f BCG in u r i n e ith t h e b a e m a g g l u t i n a t i o n i n h i b i t i o n test as successful o n l y in 6 . 7 9 . I , 3 7 . 5 , a n d 8 2 % oil d a y s 12, 16, 18, a n d 25,
sho
A p p l y i n g t h e T a b l e IiI f o r u | a to t h e d a t a given in T a b l e s I a n d lI p r o v i d e d t h e r e s u l t s n in | a b | e IV. Success in ..c o n f i r ing p r e g n a n c y .. as 3 8 . 5 , 5 1 . 7 , 9 6 . 6 , a n d 1 0 0 % o n blc IV Co p a r i s o n o f B a b o o n Plas a C b o r i o n i c G o n a d o t r o p l l i n (BCG) D e t e c t i o n by R I A a n d the P r e g n a n c y D e t e r i n a t i o n ble for E s t r o g e n a n d Progestin V a l u e s in the Diagnosis o f Early ~P r e g n a n c y
Du t i o n of Pregnancy
Pregnant P r e d i c t i o n fro B Detection T a b l e 111 b y RI A
Non-P P ~ d i c t i o n fro ~ a b l e Ill
, nant B tection by R I A
y8 rrect Inco ct Unknown
0 13
3 2 1
6 0
15 8 6
2 29
22 6 2
30 0
28 0
28 1
28 1
~o 0
5 4 4
Day 12 at
Incorrect Unknown y16 rrcct l n c o e~t Unkno n
1
1
ylg rrect Inco ct Unknown
0
15
0 1
15 1
8
8
0 0
0
OCToBER:I
6
VOL. 14 NO. 4
CONTRACE
I
days 8, 12, 16, a n d 18, r e s p e c t i v e l y . T h e h a e a g g l u t i n a t i o n ? h i b i t i o n t e s t as n o t reliable even ...as l a t e as d a y 2 5 , a n d was n o t i n c l u d e d i n "tlie ble IV c o ~ p ~ i s o n . d a y 18, b o t h R I A - B C G a n d E + P m e t h o d s ere i 0 0 % c o ~ e c t ....It s h o u l d be n o t e d t h a t since the plas a B t e s t was a l m o s t 100% i n a c c u ~ t e in d e t e c t i n g p r e g n a n c y b y d a y in t h e .. p r e g n a n t g r o u p , ...it i s n o t surprising ...t h a t it w a s 1 0 0 % c o e c t in t h e e v a l u a t i o n o f t h e n o n , p r e g n a n t g r o u p s on d a y s 8 a n d 12.
DISCUSSION o ~ne d a t a o f bles I a n d II, it uld see easy co c o n f i r pregnancy by co paring tile E and P values o f d a y 8 with d a y 16 or d a y 12 ith d a y 16, b e c a u s e o f the significant increases o c c u r r i n g a t t h e s e fi es. H o w e v e r , c o parisons o f the individual ani als' d a t a for d a y 12 vs. d a y I 6 revealed six inst ces in 30 here the P lues:actually declined. ~ h e p r e g n a n t b a b o o n s s h o w e d no c h a n g e . O n l y 21 o f t h e 30 h a d arked increases. Co parison o f d a y 8 vs. d a y 16 values sho ed i n e r t es in all 13 instances, b u t so e increases fall ithin one s t a n d a r d error o f the t a n . ~ h u s , such co p a r i s o n s e not sufficiently reliable. H o w e v e r , o n e c a n get a very g o o d p su ptive i n d i c a t i o n since t h e is, by co p a r i s o n , a significant d r o p b e t t e n d a y 8 and 12 in n o n - p r e g n a n t a n i m a l s . Also, n o n e o f the p r e g n a n t b a b o o n s s h o e d P values Iess t h a n 2 ng/ 1 on d a y 16. T h e E values increased f r o m d a y 12 to d a y 16 in 25 b a b o o n s ; o n l y t h r e e did n o t sl~o a n y c h a n g e , It t h e r e f o r e a p p e ed tllat so e h e l p f u l c o r r e l a t i o n c o u l d be established b e t t e n tile rising t r e n d in tile values o f E and P. ~ h i s led us to t h e a t h e atical for u l a t i o n e b o d i e d in | a b l e III. Using this table for d i a g n o s i n g pre a n c y , o u r success rate as 9 6 . 6 % o n d a y 16 or 9 4 . 9 % i n c l u d i n g also the n o n p r e g n a n t cases. In t o instances t h e diagnosN as inconclusive, a n d it was i n c o r r e c t o n c e h e n tl~e ani al as not p r e g n a n t T h e earliest t h a t o n e c o u l d i d e n t i f y p r e g n a n c y ith s u f f i c i e n t relmbility (96. ) ing RIA for plas a B e G as d a y 16, b u t t h e result c o u l d n o t be o b t a i n e d until at least 18 h o u r s after the s ples e o b t a i n e d . Using rapid R I A and C P B p r o c e d u r e s (6 hr) for E and P, pregnanc-¢ c o u l d be c o n f i r ed o n day 16 itll the use o f T a b l e IIl and f u r t h e r s u p p o r t e d b y a co parison o f E a n d P values for d a y 8 or 12 w i t h t h o s e o f da-:, 16, if available. ~ h e r e f o r e , the d e t e c t i o n o f p r e g n a n c y by t h e easure ent o f t h e s e p}asrr~a steroids is ore r a p i d a n d convenient than the other m e t h o d s tested.
ACKNOwLEDGME
S
T h i s s t u d y .... as s u p p o r t e d b y N I C H D c o n t r a c t N O 1 - H D - 5 - 2 8 1 0 a n d b y a c o n t cc f r o m t h e E x p a n d e d ' Prograrn r i l e o R e s e ~ h in H u m rod ion, W H O : T h a n k s ~ e Mr. C. L: laya and G o eZ f o r t h e l r t e c h n i c a l assistance a n d t o M r , A. de la Penn f o r steroid ~ e a ~ r e m e nt s. FERE
ES
H o d g e n . G,D. a n d :Koss, G . T . P r e g n a n c y Diagnosis b y a He a g g l u t i n a t i o n Inhil0ition T e s t for Macaque G o n a d o c r o p i n ( M c G ) . J~ Clin .... Metab' 38 9 27-9 3 0 . 1 9 74
OCTOBER
1976
VOL. 14 NO. 4
1
CONTRACE
J:
ION
~ i¸' . . . . . .
......
,
i
¸
,t
t
~
',
~
Tullncr, W.W. and Hertz, R. C orlonm Gonadotropln Levels in the Rhesus Monkey During Early Pregnancy. Endocrinology 78:204-207, 1966,
2~
s!an, M. Meyer, R.K., and Wolf, R.C. Chorionic Gona otropin in the Blood and Urine o f Pregnant ........esus Monkeys ( M a c ulatta). Proc. Soc. Exp. Biol. Med. 125: 349-352, 1967.
1
Tullner, W.W. Urinary Chorionic Gonadotropin Excretion in the Monkey (Macao u ia t ta) ~ Ear !y Phase ....En do c ri n oi ogy 82 87 75, ..1968. .
4.
Hoagen, G.D., Dufau, M.L., C a t t , K.J., and Tullner, W.W. s t r o g e n s , Progesterone aud Chgrionic Gonadotropin in Pregnant Rhesus Monkeys .....EndoCrinology 91"896-900, 1972.
SO
Hodgen, G.D., Tullner, W.W., Vaitukaitis, J.L., Ward, D.N., and Ross, G.T. Specific .Radioi . unoassay o f Chorionic G o n a d o t r o p i n D u r i n g I ~ p l a n t a t i o n in R|lesus Monkeys. J. Clin Endocrinol. Metab 39:457-464, I974. Hendrickx, A.G. and Krae er, D.C. Obse ations on the Menstrual Cycle, Opti Mating Ti e, and Pre-I plantation ~ bryos of the Baboon, Anubis and , J. p rod. Fert., Suppl. 6:119-128, 1969.
4
Wu, C.-H.,. Lundy, L.E., and Lee, S.G[ A Rapid Estrogen. A Obstet. Gynccoi 115:169-180, 1973.
8.
dioi
a!
un0assay-for Plas a
Johansson, E.D.B. Progesterone Levels in Peripheral Plasma During tile Luteal Phase of the Nor al Hu an Menstrual Cycle Measured by a Rapid Co petitive Protein Binding Tec h n ique. Ac ta E nd oc rin oi. 6 i 5 9 2-6 0 6, .....19 6 9.
0
2
OCTOBER
1976
VOL. 14
4
DIAGNOSIS OF EARLY PREGN A. A. S h a i k h , C. A l l e n - R o w l a n d s , T . The South
est F o u n d a t i o n
for
Y IN
z i e r , D. C. K r a e
E BAB
E
I
N
e r , * a n d J . W. G o l d z i e h e r
sea~h and Education, San Antonio,
xas**
ABST M e t h o d s f o r t h e d i a g n o s i s o f e a r l y p r e g n a n c y i n t h e b a b o o n ...b y t h e d e t e c t i o n 0£ c l, or ion ic g o n a d o t r o p h i n ( C G ) ...i p! a s m a, ~u s i n g r a d i o i m o a s s a y ( R I A ) , o r in tl, e u r i n e b y h a e m a g g l u t i n a t i o n ...i n h i b i t i o n , a s well as b y easurement of pias~a estmgenSand progestins, ere c o p a r e d f o r tl~eir s e n s i t i v i t y , r e l i a b i i i t y ¢ a n d r a p i d i t y ; D e t e c t i o n : o f CG i n t h e u r i n e b y h a c m a g g l u t i n a t i o n i n h i b i t i o n w a s u n s a t i s f a c t o r y ..as . l a t e as d a y 2 5 a f t e r c o n c e p t i o n i O n d a y s 8 a n d 12, m e a s u r e e n t s o f e s t r o g e n ( E ) , ..p r o g e s t e r o n e (P), o r in p l a s m a c o u l d n o t b e
and P determinations ere obtained more quickly, and when evaluated according to a c o p u t e r-d e rived u 1a, w e r e 9 6 . 6 % su Cccs s fuI o n t a t d a y ....
INTRODUCTION In p r i m a t e s , e a r l y d e t e c t i o n o f p r e g n a n c y is o s t ..i p o r t a n t i f t h e y a be used e f f i c i e n t l y in fe~ ~;~ s t u d i e s , it p e r m i t s b e t t e r e x p e r i i n e n t a l d e s i g n a n d ~ d u c e s tlle ..c.. o s t involvedin ating ~ n u m b e r o f a n i m a l s w i t h u n c e r t a i n o u t c o e: A n a c c u r a t e a n d e a r l y d i a g n o s i s p e r l n its ) : treat n t o f a n i t a a l s in t!i e e a r l y s t a g e s o f regnaIlcy ~t . . o g i c s t u d ie ......s - P....~ ~~n a n,c jv de ,t e c ..... p o t e n t i a ! an t i-fe '~....a n. d. . .i s e s...... Sen t i al fo r t er. .a. t. .ol t io n c a n .....::~sation, ..... b e p c r f o r. . . . e d ,~ ......- e ,',~....... a gegl u t i n a t.i.o. .n. . . . e t h. .o. d. .S .......... f o r U r i n a ry; c h o r i o n:i C ..g...o...... nadot r o p I~in (1), b i o a ss "~'.......... . m o r c o n c e n t r a t e d u r i n e e x t ~ c t (22 5 ) , Or ~ d i o i m m u n o a s s a y o f c h o rio n ic go n a d o t r o p h i n o r i t s 13 u n it ( 6 )
W e l~ave evalua ted t|to re]iabi|ity an d rapidity o f ~cth ocls u tilizingRI A
r p|asm a C G
baboon,
*.. P
**
s....... e n t a d d r e 's"s ,
a s A & M U n i .v. e. .r.s. i. t y.,. . . . . . . . . T e x.... C o Iiege S t a t i o n , T e x as
o f : .V P h y...s i 0 1 og, ..... . e t e r i n a r y. .......
d r e s s r e p r i n t r e q u e s t s t o A. A . S h a i k l i , S o u t h w e s t F o u n d a t i o n E d u c a t i o n ; .O. B o x 2 8 1 4 7 , S a n A n t o n i o T e x a s 7 8 2 8 4
Accepted
OCTOBER
for
1976
publication
VOL.
14
July
NO.
4
26,
for Resea~h and ....
1976
391
13
TRACE
I .M .
Rl ALS A N D M
HODS
A total o f 60 m a t e d b a b o o n s i~ich sho e d t h e presence o f s p e a~ozoa in the:vagina! s e a r ere used i n this s t u d y .....In this species, seX skin deturgescence is ~ i d to Occur 3 days after ovulat ion (7). ougl~ studies in ou r i a b o r a t o r y indicate at ti0n in b a b o o n s actually occurs i d a y b e f o or on t h e first ofdeturgescence, e illbas~ dating for ....this . r e p o r t on the earlier published Tiius; the first:day o f de turge see n e e il I be called d a y 3 o f pregnancy, Blood samples Were collected in heparinized t u b e s on days i 2 , 16, 18, a n d 2 5 o f pregnancy. Blood p!es were obta d .~ .. 0 m n a d d i t i o n a l 19 I~aboons on ctay. 8 o f pregnancy. The plasma as st ed at -x0 C. Urine s a pies Were a l s o . o b t a i n e d On days 12, 16, ..18, a n d 25 o f p gnancy ..or, in several ....instances, d a i l y f r o m day 1 2 through 25 U r i n e was collected ithin 30-60 inutes o f voiding. o
.
.
.
.
.
.
Estroge ere e a s u r e d by RIA following the e t h o d o f Wu e t aL (8). N o prior c h r o a t o g r a p h y o f the .. pies as e ployed. T h e antiseru used in this study was kindly supplied by Dr. P. N. Rao; it cross-reacts 1:00% ith estradio|2iTtS; ......1 0 3 % w i t h estr0ne, and 6% estriol a n d i roxyestrone e values t us r e p r e s e n t " t o t a l eitrogens. T h e .... ........ , " aS t h e y were ........... ues for ..plasma progesterone also r e p r e s e n t " total progestins easu'r ed b y the CPB m e t h 0 d o f J o h a n s s o n ( 9 ) i t h o u t c h r o m
The hae agglutination inl~ibition test for urinary chorionic gonadotropllin e m p l o y e d the kit kindly supplied by the Hor one Distribution Officer, N1 DD, NIH. The tests ere carried out according to the pub|islied instructions (I). RIA for Plas
a Chorionic G o n a d o t r o
The antiseru used in this RIA as kindly supplied~by Dr. G. D. Hodgen and as developed in rabbits against the fl-subunit o f ovine LH (6). I o d i n a t e d (12s I) hu an chorionic g o n a d o t r o p h i n (HCG) as purchased fro Bm RIA, Montreal, Canada. Specific activities ranged from 112-131 ~Cilgg. Since no reference p r e p a r a t i o n for b a b o o ~ chorionic gonadotrophin s a liable, e prepared our o n reference standard from a seru pooI o b t a i n e d fro three b a b o o n s on day 25 of? pregnancy. ~his pooled seru as diluted 1:16 ith fetal calf seru and a dose response curve as c o n s t r u c t e d using volu es of 5-200 ~i (Fig. 1). ~ h e RIA procedure followed the e t h o d Of. Hodgen et al. (6), using 50 and 200 ~1 aliquots o f undiluted plas a. tal calf seru as added to both tl~e reference and u n k n o w n sa pies to ake tlle final seru c o n c e n t r a t i o n 20% by volu e. A plas a pool fro pregnant b a b o o n s (day 25 o f pregnancy) and a n o t h e r pool from non-pregnant animals at day 12 post-ovulation ere used as controls in each assay.
392
OCTOBER
1976
V O L . 14 N O . 4
C
ION
! gotive Contro!
0
600
40" c o
20"
......... t
5 in n o n o g r a
LN
:
~-
25
50
s;ot h er
samples
t ................ - .......................................... l
tOO
200
t
400
~n...... i c r o l i t e r s
Fig, I, Co parison o f d o s e r e s p o n s e curves using reference a n d c o n t r o I sa pies a n d a .. b a b o o n L H ..... (BLH) p r e p a r a t i o n , P e r c e n t c o u n t s p r e c i p i t a t e d are n o r alized to c o u n t s p r e c i p i t a t e d in t u b e s c o n t a i n i ~
A p o s i t i ~ diagnoMs o f p r e g n a n c y was ade w h e n the 200 ul sa pie p r e c i p i t a t e d b e t w e e n 0 . 5 and 9 0 % o f the c o u n t s , n o r alized to c o u n t s p r e c i p i t a t e d in the t u b e ,aining no antigen (Fig. 1)..A b a b o o n s c o n s i d e r e d n o t pregn It if the 2 0 0 ~I sa ple precipitated > 9 o f the- unts. T h e r e f o r e , t|lis R I A was used pri ily as a qualitative test to d e t e c t the presence or absence o f b ~ ~ o n c h o r i o n i c ~ n a d o t phin (B ). In a d d i t i o n , we have f o u n d that tl~e antiserum used in this s t u d y cross-reacts with b a b o o n LH (Fig. 1). Using 50-200 n g / t u b e o f b a b o o n , p i t u i t a w .....LH, a dose response cu si Liar to t h a t o b t a i n e d by using diluted p r e g n a n t b a b o o n seru wag o b t a i n e d . These. results a in disagreement ith published r e p o r t s o f ti~e~s p e c i f i c i t y Of the antiseru (1). Tlaerefore, a ~ I s e positive diagnosis o f p r e g n a n c y b e t w e e n ~ idmycle s u r e of~LH.~ RESU
I n s p e c t i o n o f . t h e plas sensitive in d i c a t o r 0 f
OCTOBER
~976
S
a steroid levels (Tables i,I1)~1o ed t h a t p r o g e s t i n s re a m o r e n cy t h a n estrogen~, b u t t h a t ~ ei t h e r d e t e r m i n a t i o n , , by itseI f,
VOL.
14 NO. 4
393
C O N ' I "R .
.
.
.
.
.
.
.
.
P~r l 0 N
2
~
~~
~
~
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W , ~ ~~ 8 ~ ~i ~ ~!~ o ~~ :~~' ~,i ~ '~ ~~ ~W!I ~ ~ ~W ~ ~ ~~ ~ ,'~
W
W
Z
~
394
OCTOBER
1976
VOL.
i~,il~~
14 N O . 4
¢,rl
o
0
l..a
0
.,4
t-a
0
0
NEG POS NEG NEG NEG POS NEG NEG NEG NEG NEG
Urine 8CG (POS, NEG) E pgfml
11.1 ±1,5 15 16 (93.8}
6 16 (37.51
Note: Animal No. 18 was used in an ongoing experiment,therefore samplecollection was discontinued
Confirmation (%)
163.6±8,8
NEG POS POS POS POS
200.0 ± 13.0
POS POS POS POS POS NEG POS POS POS POS POS POS POS POS POS POS
Plasma BCG (PO5, NEG)
Mean ±$E
16.5 14.0 10.5 8,4 21.5 11,0 3.2 9.0 5.6 7.3 12.8 13~ 4,7 24.3 7,0 9.1
P ng/ml
210 225 238 135 197 !88 177 307 219 150 185 170
197 190 185 144 128 127 126 132 193 140 140 150 224 207 207 128
E pg/ml
D18
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 20 21 22 23 24 25 26 27 28 29 30
Animal No.
Table I (continued)
9,9 + 1.3
11.2 14.3 2.3 5.7 12.6 5~8 8.0 14.5 8,5 7.3 10.1 18.3
P ng/ml
D25
12 12 (1001
POS POS POS POS POS FOS POS POS POS POS POS POS
Plasma8CG (POS, NEG)
9 11 (81,8)
POS NEG POS POS NEG POS POS POS PO$ POS
POS
Urine BCG (POS, NEG)
O Z
C3
O Z
C3
~
~
~
~
~
~
o ~ o o o o ~ ~ 0
V
,~
,~8
VV
V
•••,•,
8
~
VV
~
Z
~~
~ e ~ o o o ~ o o o ~ ~ ~
VVVVVV
Z
~Mo
VVV
VVVV
V
~
"8
A
~
~w~ ~
8
~~
A
A
~
e S~~8~SS
~88s~~s$8
~ ~ssssss8
°
~ U..l~¸ ~
~
VV
VV
V
V
V VVV~VV
V
V ..
O
Z Z Z Z Z Z
.
.
.
.
.
.
.
.
.
+!
V
8
..
+~
C
.9 C
OCTOBER
~1 9 . 7 6
XzOL.
: : N O . .. 4
O
,,,1
w
4~
P
O
P~
O
¢3
E
0 8 (~1001
0 8 (100)
0.5 +0.0
< 0.5 <~ 03 ~ 0,5 < 0.5 < 0.5 <~ 0.5 '~ 0.5 0.7 < 0.5 < 0.5
P ng/ml
D25
0 10 (100)
NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG
Plasma BCG IPOS, NEG)
Note: Animal Nos, 1 and 21 to 30 started to recycleas determined by perinealsex skin turgescence,therefore no sampleswerecollected from theseanimals.
Confirmation (%)
0.6±0.1
113.8 +10,4
pg/ml
106.5±11.0
NEG
NEG NEG NEG NEG NEG NEG NEG
Urine BCG (POS, NEG)
Mean +SE
NEG NEG NEG NEG NEG NEG NEG
NEG
Plasma BCG (POS, NEG)
88 109 141 99 96 66 98 182 134 125
i.4
<~ 0.5 <~ 0.5 <~ 0.5 < 0.5 < 0.5 <~ 0.5 <~0.5
P ng/ml
139
149 92 78 67 81 131 115
E pg/ml
D18
10 11 12 13 14 15 16 17 18 19 2O
9
7 8
6
5
4
2 3
Animal No.
Table II (continued)
6 (100)
0
NEG NEG NEG NEG NEG NEG
Urine BCG (POS, NEG)
¢3
O Z
C
TRACE
ION
p o s s e s s e d s u f f i c i e n t d i s c r i m i n a t o r y p o e r in e a r l y p r e g n a n c y . W ! l e n b o t h h o r m o n e s ere e"| e ,V a t e~ d a b o v e t Ii e c o r ~ Sp o, 1d {n g va I u e s o b t a i n e d fr o, n k n o n n o n- p r e g n a n t a n i m a Is a t t 11a t t i m e Of t h e c y c l e , t l l e r e w a s a ~ r e~*' a t e r t l m n 9 5 % c e r t a i n t y o f p r e g n a n c y. ; . l i. k e. w. i s.e , . w . hen n e ti h e r, s ! o ed* e l e v a t i o n ,. t l .~ e r e. . a s a . g r e. a t e. r t.l l a n. 9 5 % c e r t a i n t y o f n o n - p r e g n a n c y . F o r those cases h e r e o n l y o n e v a l u e w a s e l e v a t e d o r w h e r e tllc rise in b o t h w a s b o r d e r i i n e , We a t tern p t e d t o d e re! o p a m a t h e a [ i c a l m o d e | w it ]~ o p t i m u m ....p r e d iC t ive p o e r. e o rigi n a| model was based d a t a f r o m t h e first 3 8 b a b o o n s , a n d tlle e q..u a t , o,~s e r e r e t e s t e d o n t h e entire set of data in 60 animals. E st r o g e n a n d p r o g c s t in v a l u c s f r o m p | a s m a o b t a i n c d o n d a y 1 2 w e r e a r r a i , g e d i n r a n k o r d e r , d i v i d e d i n t o q u a r.t.i.l.e. s ; a n d m e d i a n v a | u e s e x t r a c t e d By c o m p u t e r , ....e q u a t i o n s ere t t~e n d e r ive d t o fi t t i i e q u a r t i le u m b ers t o t 1!e e d i a n va i u e s for-e s t r o g e 11 ( E ) a n d p r o g e s t i n (P). F o r E, t h e equation~ t o o k t h e f o r m y = a + b forP t o o k tlle f o r t h ~ = a e b x , yielding for estrogen a quartile number = -23.32304 + 5.71769 (|n E value). For this e q u a t i o n, t ti e c o r r e la t io 1~ c o e ffi c i e n t (r) a s 0 . 9 8 . F o r p r o g e st i i i s. t i~e q u a r t i le n u m b e r = 0 . 8 6 2 2 3 e O" 1 5 8 4 ( P v a l u e ) itil r .... 0 . 9 9 . Th e t
o c q u a t i o n s w e r e t il e
c o i n b i n e d t o y i e 1d :
0 . 8 6 2 2 3 e x p ( 0 . 1 5 8 4 ....* P va I u e) = - 2 3 . 3 2 0 4 + 5 . 7 1 7 6 9 I n ( E va | u e ). S i n c e tile m e a n P v a l u e f o r p r e g n a n t a n i m a l s o n tllis d a y t h e a n i m a l is p g n a n t i f
as a b o u t 6 0 n g /
1,
e state that
6.0
0.1584 "!'hen ,, 0
e×p
![
.. -/ 0 . 8 6 2 2 3 e x ~ 0 . 1 5 8 4 x P v a Iu e_)_
+
23 32304 ........
E s t r o g e n (T)
5 . 7 1 7 69 For given l u e s o f P, E ( T ) p r o v i d e s a c u t o f f p o i n t : A c t u a l E v a l u e s les t l , a n tlle E ( T ) a s s o c i a t e d w i t h a p a r t i c u l a r v a l u e o f P s u g g e s t t l l a t t h e a n i m a l is n o t p r e g n a n t ; a c t u a l E values ~ e a t e r tllan the co p u t e d ~(T) for a given P value suggest p r e g n a n c y . When this ethod s a p p l i e d to t h e ra d a t a , it a p p e a r e d t h a t t h e p r o g e s t i n e q u a t i o n w a s n o t s e n s i t i v e e n o u g h t o d i s c r i i n a t e p r e g n a n c y u s i n g a v a l u e o f e x a c t l y 6 . 0 n g / ~ l . It w a s d e c i d e d to u s e a r a n g e o f 5 . 5 - 6 . 5 n g / m l as an e s t i m a t o r f o r p r e g n a n c y f o r d a y 12 d a t a . This procedure no p r o v i d e s t o esti a t e s f o r E ( T ) . I f t h e a c t u a l e s t r o g e n v a l u e falls b e l o w t h e lesser E ( T ) ( d e r i v e d f r o m t h e 5 . 5 n g / m l P v a l u e ) f o r a given s e t o f E a n d P v a l u e s , t h e a n i m a l is j u d g e d n o t p r e g n a n t . If t h e a c t u a l E v a l u e is g r e a t e r t h a n E ( T ) u s i n g 6 , 5 n g / m l , tile a n i m a l is j u d g e d p r e g n a n t , i f t h e a c t u a l E v a l u e lies b e t w e e n t h e t o v a l u e s f o r :E(T), t h e odel c a n n o t d e t e r ine the status. U s i n g tile e q u a t i o n s a n d v a l u e s g i v e n a b o v e , a c o p u t e r p r o g r a s written to generate a table containing values of P fro 0.1 to 8 ng/ml ith c o r r e s p o n d i n g ini u and axi u E ( T ) v a l u e s ( T a b l e 111).
398
OCTOBER
1976
VOL
14
~O.
4
h i e II1 Pregnancy Determination
ble
(Cont.)
Pregnancy Deter
ination Estr~en
stro~n Progestin
Ne~tive Maxi um
Positive Minimum
Progestin
177 174 171 168 165 162 160 157 155 153 150 148 146 144 142 140 138 136 134 133 131 129 128 126 125 123 122 120 119 118 116 115 114 I 13 112 111 109 108 107 106
216 211 207 3 199 195 192 188 185 182 178 175 172 169 167 164 161 159 156 154 152 149 147 145 143 141 139 137 135 134 132 130 129 127 126 124 123 121 120 I 18
4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5, I 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7~8 7.9 8.0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3, 7 3.8 3;9 4.0
ble
Ne~tive i a x i. . . . . . u
Positive Minimu
05 04
17 16
03 03
13
02 0i 00 99 98 97
97 96
95 94 94 93 92 92 91 90 9O 89 89 88 88 87 86
15 12 1! 0
09 08 07:
O6 .... 05 04 03
02 ol 00 00 99 98 97 96 96 5 94
8S 84
93 93 92 9I 91 90
84 83 83 83 82 82 81:
89 88 88 87 87 86
8O
s6 85
s6 85
9o ....
T o u s e .t h. i s. t.a b. l e , l o c.a. .t.e. .a.n. l al s P r o...g .e.s. t. l. n. v a l u........................ e , t h e n t o t h e r .i .g. h. .t. .~read t h e . . . .axz mt u ... . . . . . . . u. . . . . .a. .= ~ ................ e s t r o g e n v a l u e s : E x a m 16, if t h e p r o e s f i n V a l u e i s : 2 5) ~t h e n t h e e s t r o g e n ust be le~ than125 to conqrm .n.o. .n. - rpe gn a n c y. . .o. .r g r e a t .e.r. . .t.h. .a. n. . . .1. .4 3 t o. . . c. .o. .n. G . . r. . . . p r e g n. .a. .n.c y. . . .B. .e. t. w . e e n 1. 2. 5 . a.n d. . 1.4 ~. .r e.s u. l t. s . a .r e. m . c. o .n c. l u. s.l , . e
OCTOBER
1976
VOL. 14 NO. 4
399
suits o t ~ a d o u s
sts
T a b l e i su a r i z e s t h e results a f all tests p e r f o r ed in ani a!s hose p r e g n a n c y as c o n f i m l e d b y c a e s a r i a n s e c t i o n o n e i t h e r d a y 5 0 o r 70. ble iI gives tl!e C o r r e s p o n d i n g r e s u l t s in ....a n i m a l s hich p r o v e d n o t t o be p r e g n a n t , in ttie p r e g n a n t b a b o o n s , t h e t o t a l 5.2 pg/ Ito a day 25 e s t r o g e n vMues i n c r e a s e d gradually fro ...... .. a d a ) 8 mean value m e a n value o f 2 0 7 ± 1 2 . 0 pg/ i. By c o n t r a s t t e n o n - p r e g n a n t b a b o o n s y i e l d e d a d a y 8 can V a l u e o f 9 4 ± 7 . 4 hich ..re a i n e d essentially u n c h a n g e d . T h e c a n values o f 107 ± i 1.0 a n d i 14 +- 10.4 on d a y s ...18 a n d 2 5 , r e s p e c t i v e l y e r e n o t significant1,, ~:~iffercnt f r o m d a y 8 values. Tile p r o g e s t i n ues also i n c r e a s e d fro a day 8 ean v a l u e ~ o f 6 . I ± 1.1 n g / i t o 11.5 ± 1.8 o n d a y 25 H o w e v e r , t h e signific t c h a n g e s in p r o g e s t i n values o c c u r r e d b e t e e n d a y 8 a n d 16 a f t e r w h i c h t h e y stabilized, .... rr l m p r o g e s t i n values in n o n - p r e g n a n t ani als, b y c o n t st, sho ed a c o n t i n u o u s d e c l i n e , o n tile .a.. v erage, a n d b y d a y 18 w e r e b e i o the li its o f de te ct ab i I y i n M | b u t o n e a n i m a 1. Usiffg tile R I A e t h o d for d e t e c t i o n o f plas a BCG, t h e success rate in c o n f i r m i n g p r e g n a n c y o n d a y s 8 i 2 16 18 a n d 25 as 0, 6.5, 9 6 . 6 , 9 3 . 8 a n d i 0 0 % , r e s p e c t i v e l y . In c o n t r a s t , ...p r e g n a n c y c o n f i r m a t i o n by d e t e c t i o n o f BCG in u r i n e ith t h e b a e m a g g l u t i n a t i o n i n h i b i t i o n test as successful o n l y in 6 . 7 9 . I , 3 7 . 5 , a n d 8 2 % oil d a y s 12, 16, 18, a n d 25,
sho
A p p l y i n g t h e T a b l e IiI f o r u | a to t h e d a t a given in T a b l e s I a n d lI p r o v i d e d t h e r e s u l t s n in | a b | e IV. Success in ..c o n f i r ing p r e g n a n c y .. as 3 8 . 5 , 5 1 . 7 , 9 6 . 6 , a n d 1 0 0 % o n blc IV Co p a r i s o n o f B a b o o n Plas a C b o r i o n i c G o n a d o t r o p l l i n (BCG) D e t e c t i o n by R I A a n d the P r e g n a n c y D e t e r i n a t i o n ble for E s t r o g e n a n d Progestin V a l u e s in the Diagnosis o f Early ~P r e g n a n c y
Du t i o n of Pregnancy
Pregnant P r e d i c t i o n fro B Detection T a b l e 111 b y RI A
Non-P P ~ d i c t i o n fro ~ a b l e Ill
, nant B tection by R I A
y8 rrect Inco ct Unknown
0 13
3 2 1
6 0
15 8 6
2 29
22 6 2
30 0
28 0
28 1
28 1
~o 0
5 4 4
Day 12 at
Incorrect Unknown y16 rrcct l n c o e~t Unkno n
1
1
ylg rrect Inco ct Unknown
0
15
0 1
15 1
8
8
0 0
0
OCToBER:I
6
VOL. 14 NO. 4
CONTRACE
I
days 8, 12, 16, a n d 18, r e s p e c t i v e l y . T h e h a e a g g l u t i n a t i o n ? h i b i t i o n t e s t as n o t reliable even ...as l a t e as d a y 2 5 , a n d was n o t i n c l u d e d i n "tlie ble IV c o ~ p ~ i s o n . d a y 18, b o t h R I A - B C G a n d E + P m e t h o d s ere i 0 0 % c o ~ e c t ....It s h o u l d be n o t e d t h a t since the plas a B t e s t was a l m o s t 100% i n a c c u ~ t e in d e t e c t i n g p r e g n a n c y b y d a y in t h e .. p r e g n a n t g r o u p , ...it i s n o t surprising ...t h a t it w a s 1 0 0 % c o e c t in t h e e v a l u a t i o n o f t h e n o n , p r e g n a n t g r o u p s on d a y s 8 a n d 12.
DISCUSSION o ~ne d a t a o f bles I a n d II, it uld see easy co c o n f i r pregnancy by co paring tile E and P values o f d a y 8 with d a y 16 or d a y 12 ith d a y 16, b e c a u s e o f the significant increases o c c u r r i n g a t t h e s e fi es. H o w e v e r , c o parisons o f the individual ani als' d a t a for d a y 12 vs. d a y I 6 revealed six inst ces in 30 here the P lues:actually declined. ~ h e p r e g n a n t b a b o o n s s h o w e d no c h a n g e . O n l y 21 o f t h e 30 h a d arked increases. Co parison o f d a y 8 vs. d a y 16 values sho ed i n e r t es in all 13 instances, b u t so e increases fall ithin one s t a n d a r d error o f the t a n . ~ h u s , such co p a r i s o n s e not sufficiently reliable. H o w e v e r , o n e c a n get a very g o o d p su ptive i n d i c a t i o n since t h e is, by co p a r i s o n , a significant d r o p b e t t e n d a y 8 and 12 in n o n - p r e g n a n t a n i m a l s . Also, n o n e o f the p r e g n a n t b a b o o n s s h o e d P values Iess t h a n 2 ng/ 1 on d a y 16. T h e E values increased f r o m d a y 12 to d a y 16 in 25 b a b o o n s ; o n l y t h r e e did n o t sl~o a n y c h a n g e , It t h e r e f o r e a p p e ed tllat so e h e l p f u l c o r r e l a t i o n c o u l d be established b e t t e n tile rising t r e n d in tile values o f E and P. ~ h i s led us to t h e a t h e atical for u l a t i o n e b o d i e d in | a b l e III. Using this table for d i a g n o s i n g pre a n c y , o u r success rate as 9 6 . 6 % o n d a y 16 or 9 4 . 9 % i n c l u d i n g also the n o n p r e g n a n t cases. In t o instances t h e diagnosN as inconclusive, a n d it was i n c o r r e c t o n c e h e n tl~e ani al as not p r e g n a n t T h e earliest t h a t o n e c o u l d i d e n t i f y p r e g n a n c y ith s u f f i c i e n t relmbility (96. ) ing RIA for plas a B e G as d a y 16, b u t t h e result c o u l d n o t be o b t a i n e d until at least 18 h o u r s after the s ples e o b t a i n e d . Using rapid R I A and C P B p r o c e d u r e s (6 hr) for E and P, pregnanc-¢ c o u l d be c o n f i r ed o n day 16 itll the use o f T a b l e IIl and f u r t h e r s u p p o r t e d b y a co parison o f E a n d P values for d a y 8 or 12 w i t h t h o s e o f da-:, 16, if available. ~ h e r e f o r e , the d e t e c t i o n o f p r e g n a n c y by t h e easure ent o f t h e s e p}asrr~a steroids is ore r a p i d a n d convenient than the other m e t h o d s tested.
ACKNOwLEDGME
S
T h i s s t u d y .... as s u p p o r t e d b y N I C H D c o n t r a c t N O 1 - H D - 5 - 2 8 1 0 a n d b y a c o n t cc f r o m t h e E x p a n d e d ' Prograrn r i l e o R e s e ~ h in H u m rod ion, W H O : T h a n k s ~ e Mr. C. L: laya and G o eZ f o r t h e l r t e c h n i c a l assistance a n d t o M r , A. de la Penn f o r steroid ~ e a ~ r e m e nt s. FERE
ES
H o d g e n . G,D. a n d :Koss, G . T . P r e g n a n c y Diagnosis b y a He a g g l u t i n a t i o n Inhil0ition T e s t for Macaque G o n a d o c r o p i n ( M c G ) . J~ Clin .... Metab' 38 9 27-9 3 0 . 1 9 74
OCTOBER
1976
VOL. 14 NO. 4
1
CONTRACE
J:
ION
~ i¸' . . . . . .
......
,
i
¸
,t
t
~
',
~
Tullncr, W.W. and Hertz, R. C orlonm Gonadotropln Levels in the Rhesus Monkey During Early Pregnancy. Endocrinology 78:204-207, 1966,
2~
s!an, M. Meyer, R.K., and Wolf, R.C. Chorionic Gona otropin in the Blood and Urine o f Pregnant ........esus Monkeys ( M a c ulatta). Proc. Soc. Exp. Biol. Med. 125: 349-352, 1967.
1
Tullner, W.W. Urinary Chorionic Gonadotropin Excretion in the Monkey (Macao u ia t ta) ~ Ear !y Phase ....En do c ri n oi ogy 82 87 75, ..1968. .
4.
Hoagen, G.D., Dufau, M.L., C a t t , K.J., and Tullner, W.W. s t r o g e n s , Progesterone aud Chgrionic Gonadotropin in Pregnant Rhesus Monkeys .....EndoCrinology 91"896-900, 1972.
SO
Hodgen, G.D., Tullner, W.W., Vaitukaitis, J.L., Ward, D.N., and Ross, G.T. Specific .Radioi . unoassay o f Chorionic G o n a d o t r o p i n D u r i n g I ~ p l a n t a t i o n in R|lesus Monkeys. J. Clin Endocrinol. Metab 39:457-464, I974. Hendrickx, A.G. and Krae er, D.C. Obse ations on the Menstrual Cycle, Opti Mating Ti e, and Pre-I plantation ~ bryos of the Baboon, Anubis and , J. p rod. Fert., Suppl. 6:119-128, 1969.
4
Wu, C.-H.,. Lundy, L.E., and Lee, S.G[ A Rapid Estrogen. A Obstet. Gynccoi 115:169-180, 1973.
8.
dioi
a!
un0assay-for Plas a
Johansson, E.D.B. Progesterone Levels in Peripheral Plasma During tile Luteal Phase of the Nor al Hu an Menstrual Cycle Measured by a Rapid Co petitive Protein Binding Tec h n ique. Ac ta E nd oc rin oi. 6 i 5 9 2-6 0 6, .....19 6 9.
0
2
OCTOBER
1976
VOL. 14
4