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Cytogenetic analysis of couples with recurrent abortions Charlton, William Robert David 1976

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CYTOGENETIC ANALYSIS OF COUPLES WITH RECURRENT ABORTIONS by WILLIAM ROBERT DAVID CHARLTON B.Sc. U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f M e d i c a l G e n e t i c s We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA-, 1975 In p re sent ing t h i s t he s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t ha t permiss ion fo r ex ten s i ve copying of t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r ep re sen ta t i ve s . It i s understood that copying or p u b l i c a t i o n o f t h i s t he s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n permi s s ion . Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date ABSTRACT Cytogenetic s t u d i e s of r e c u r r e n t a b o r t i o n have e s t a -b l i s h e d t h a t a s i g n i f i c a n t l y higher frequency of t r a n s l o c a -t i o n s e x i s t w i t h i n the p o p u l a t i o n of couples p r e s e n t i n g w i t h h i s t o r i e s of repeated a b o r t i o n than i n the general p o p u l a t i o n . However, few of these s t u d i e s have been able to e s t a b l i s h a d i r e c t c o r r e l a t i o n between balanced p a r e n t a l rearrange-ment and the r e c u r r e n t a b o r t i o n . The present study was a cy t o g e n e t i c a n a l y s i s of couples w i t h r e c u r r e n t a b o r t i o n , and u t i l i z e d ( i ) the newer chromosome banding techniques and ( i i ) c y t o g e n e t i c and morphological data on one or more abortus specimen per couple. During the course of the study 50 couples and two women were s t u d i e d . Chromosomes were obtained from leukocyte blood c u l t u r e s and giemsa-banded. C-banding and/or f l u o r e s c e n t banding was employed only i f the karyo-type was suspected to be abnormal. One t r a n s l o c a t i o n was found among 102 i n d i v i d u a l s : a t(14ql5q) i n a woman who had aborted a specimen w i t h growth d i s o r g a n i z a t i o n and a karyotype 45,XY,t(14ql5q). The i m p l i c a t i o n s of t h i s f i n d i n g are d i s c u s s e d . M o r p h o l o g i c a l s t u d i e s on the abortuses i n d i c a t e d t h a t specimens w i t h growth d i s o r g a n i z a t i o n are more frequent among women who are r e c u r r e n t aborters w i t h no term pregnancies and, were found to rec u r among t h i s sub-population. The frequency of t r a n s l o c a t i o n s among couples w i t h r e c u r r e n t a b o r t i o n i s considered i n terms of the e f f e c t s of t r a n s l o c a t i o n s on r e p r o d u c t i v i t y . The need f o r c y t o g e n e t i c c u l t u r e data on abortuses i n s e r i a l s t u d i e s on r e c u r r e n t a b o r t i o n i s d i s c u s s e d . i i i TABLE OF CONTENTS Page ABSTRACT - i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i I . INTRODUCTION 1 A) Spontaneous A b o r t i o n 1 1. C y t o g e n e t i c s o f spontaneous a b o r t i o n 1 2. A b o r t u s morphology 4 3. E t i o l o g y o f spontaneous a b o r t i o n 5 B) R e c u r r e n t Spontaneous A b o r t i o n 7 1. C l i n i c a l a s p e c t s 7 2. C y t o g e n e t i c s t u d i e s o f r e c u r r e n t a b o r t i o n 9 C) Purpose o f t h e P r e s e n t Study 13 I I . MATERIALS AND METHODS 14 A) P a t i e n t A s c e r t a i n m e n t 14 B) E x p e r i m e n t a l P r o c e d u r e s 16 1. Chromosome p r e p a r a t i o n s 16 2. T r y p s i n - g i e m s a b a n d i n g 17 3. C-banding 18 4. F l u o r e s c e n t b a n d i n g 19 5. A n a l y s i s and e s t a b l i s h m e n t o f k a r y o t y p e 19. i v P a g e I I I . RESULTS 21 A) C y t o g e n e t i c Data 21 1. Case R e p o r t 24 B) M o r p h o l o g i c a l and R e p r o d u c t i v e Data • 25 IV. DISCUSSION 33 A) M o r p h o l o g i c a l and R e p r o d u c t i v e Data 33 B) Chromosomal V a r i a n t s 34 C) T r a n s l o c a t i o n and A b o r t i o n 36 1. Freq u e n c y i n t h e p o p u l a t i o n o f women w i t h r e c u r r e n t a b o r t i o n s 36 2. S i g n i f i c a n c e o f t h e p r e s e n t t r a n s l o c a t i o n c a s e 37 3. T r a n s l o c a t i o n and r e p r o d u c t i v i t y 38 LITERATURE CITED • 41 LIST OF TABLES T a b l e Page I Chromosome S t u d i e s i n Repeated A b o r t i o n s 11 I I C y t o g e n e t i c and R e p r o d u c t i v e Data 22 I I I K a r y o t y p e s P r e s e n t i n Normal and Growth D i s o r g a n i z a t i o n C a t e g o r i e s 27 IV O c c u r r e n c e o f Abnormal Specimens i n N u l l i p a r o u s and Parous P a t i e n t s 2 7 v i LIST OF FIGURES F i g u r e s Page 1. P a r t i a l k a r y o t y p e - 9qh+ v a r i a n t 29 2. P a r t i a l k a r y o t y p e - 16qh+ v a r i a n t 29 3. P e d i g r e e o f c a s e h i s t o r y 30 4. Giemsa banded k a r y o t y p e o f a b o r t u s specimen: 4 5 , X Y , 1 4 - , 1 5 - , t ( 1 4 q l 5 q ) + 31 5. Composite p a r t i a l C, Q and Giemsa banded m a t e r n a l k a r y o t y p e : 45,XX,14-, 1 5 - , t ( 1 4 q l 5 q ) + 32 v i i ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my thanks t o t h e members o f my t h e s i s committee a t The U n i v e r s i t y o f B r i t i s h C o l u m b i a f o r t h e i r s u p p o r t and g u i d a n c e d u r i n g t h e c o u r s e o f t h i s p r o -j e c t : Dr. J . R. M i l l e r , Department o f M e d i c a l G e n e t i c s ( C h a i r m a n ) ; Dr. B. J . P o l a n d , Department o f M e d i c a l G e n e t i c s ( S u p e r v i s o r ) ; Dr. F. J . D i l l , Department o f M e d i c a l G e n e t i c s ; Dr. C. W. R o b e r t s , Department o f P o u l t r y S c i e n c e ; Dr. D. G. Holm, Department o f Z o o l o g y . S p e c i a l t h a n k s i s e x p r e s s e d t o Dr. F. J . D i l l f o r h i s a s s i s t a n c e and t e a c h i n g , and t o t h e s t a f f and f a c u l t y o f The Department o f M e d i c a l G e n e t i c s and The Human Embryology Study f o r p r o v i d i n g such a warm and f r i e n d l y atmosphere d u r i n g t h e c o u r s e o f t h e s t u d y . I acknowledge t h e s u p p o r t o f a M e d i c a l R e s e a r c h C o u n c i l S t u d e n t s h i p from A p r i l 1974 t o August 1975 t o Dr. B. J . P o l a n d . Thanks a r e e x p r e s s e d t o a l l t h e p h y s i c i a n s who a l l o w e d us t o s t u d y t h e i r p a t i e n t s and t o t h o s e p a t i e n t s who c o o p e r a t e d w i t h us and made t h i s s t u d y p o s s i b l e . F i n a l l y , I e x t e n d my warmest t h a n k s and r e g a r d s t o Dr. B. J . P o l a n d and Dr. J . R. M i l l e r f o r t h e i r g u i d a n c e and s u p p o r t . v i i i I . INTRODUCTION A) Spontaneous A b o r t i o n 1. C y t o g e n e t i c s o f spontaneous a b o r t i o n Spontaneous a b o r t i o n may be d e f i n e d as t h e n a t u r a l t e r m i n a t i o n o f a preg n a n c y b e f o r e v i a b i l i t y o f t h e f e t u s , t h e a c t u a l s e p a r a t i o n o f a b o r t i o n from s t i l l b i r t h d e p e n ding on t h e c r i t e r i a o f v i a b i l i t y . A 20-week g e s t a t i o n i s commonly used ( C a r r , 1 971a). I t i s e s t i m a t e d t h a t 15 t o 25% o f a l l p r e g n a n c i e s end i n r e c o g n i z a b l e a b o r t i o n ( I n h o r n , 1967; C a r r , 1972a) and t h a t a p p r o x i m a t e l y 80% o f a l l spontaneous a b o r t i o n s o c c u r i n t h e f i r s t t r i m e s t e r ( J a v e r t , 195 7; S t e v e n s o n e t a l , , 1959). The a c t u a l i n c i -dence o f p r e - c l i n i c a l a b o r t i o n i s unknown b u t i s t h o u g h t t o be h i g h . F o r i n s t a n c e , H e r t i g , Rock and Adams (195 6) d e s -c r i b e d 10 o f 34 e a r l y c o n c e p t u s e s from women o f p r o v e n f e r -t i l i t y and, on t h e b a s i s o f poor t r o p h o b l a s t development, c o n c l u d e d t h a t t h e p r e g n a n c i e s would have been l o s t b e f o r e b e i n g r e c o g n i z e d . The i n v o l v e m e n t o f a b n o r m a l i t i e s i n t h e development o f t h e human z y g o t e was w e l l e s t a b l i s h e d by t h e 1930's by M a l l and Meyer. I n 1943 H e r t i g and Sheldow r e p o r t e d 617 o b v i o u s a b n o r m a l i t i e s among 1,000 a b o r t u s e s . Most o f t h e abnor-m a l i t i e s were e i t h e r t o t a l absence o f an embryo o r a d i s -2 t i n c t i v e d i s o r d e r o f e m b r y o g e n e s i s . J a v e r t (195 7) examined 892 a b o r t u s e s and found 173 were d e v e l o p m e n t a l l y a r r e s t e d b e f o r e day 21 p o s t - c o n c e p t i o n , and o f t h e s e 99% were morpho-l o g i c a l l y a b n o r m a l . I n 1961, P enrose and D e l h a n t y r e p o r t e d t h e f i r s t o b s e r -v a t i o n o f a chromosome anomaly ( t r i p l o i d y i n a m a cerated f e t u s ) a s s o c i a t e d w i t h a b o r t i o n . The f i r s t s e r i a l c y t o -g e n e t i c s t u d i e s o f a b o r t i o n appeared i n t h e e a r l y 1960's ( C l e n d e n i n and B e n i r s c h k e , 1 9 6 3j C a r r , 1965) and s i n c e t h e n a l a r g e number o f s t u d i e s has appeared i n t h e l i t e r a t u r e . The number o f chromosome a n o m a l i e s r e p o r t e d among s p o n t a n -eous a b o r t u s e s t o t a l s o v e r 2,000 (Boue and Boue, 1974b) and i n v e s t i g a t o r s agree t h a t chromosome d i s o r d e r s a r e much more p r e v a l e n t among t h e s e specimens t h a n among t h e r a p e u t i c -a l l y i n d u c e d a b o r t u s e s (3%) ( S a s a k i e t a l . , 1967; C a r r , 1971b) and l i v e b o r n i n f a n t s (0.5%) (Court-Brown e t a l . , 1 9 6 5). The f r e q u e n c y o f c h r o m o s o m a l l y abnormal specimens v a r i e s c o n s i d e r a b l y from one s e r i e s t o a n o t h e r . Even among th e l a r g e r s t u d i e s , w i t h more t h a n 50 k a r y o t y p e d specimens, t h e i n c i d e n c e o f a b e r r a t i o n s r a n g e s from 8 t o 60% ( P a w l o -w i t z k i , 1 9 7 2 ) . The f a c t o r s t h a t can i n f l u e n c e t h e i n c i d e n c e o f chromosome a n o m a l i e s have been d i s c u s s e d i n d e t a i l e l s e -where ( C a r r , 1970; P a w l o w i t z k i , 1972) and a r e summarized a s : u n i d e n t i f i e d p h e n o t y p i c or g e s t a t i o n a l s e l e c t i o n o f a b o r t u s m a t e r i a l and v a r i a t i o n due t o l o c a l g e o g r a p h i c f a c t o r s . 3 A f t e r c o r r e c t i n g f o r b i a s due t o g e s t a t i o n a l and m a t e r n a l age, P a w l o w i t z k i (1972) e s t i m a t e s a f r e q u e n c y o f e x p e c t e d a n o m a l i e s i n spontaneous a b o r t u s e s o f 44%. A c o m p a r i s o n o f t h e p r i n c i p a l s u r v e y s shows a s i m i l a r i t y i n t h e f r e q u e n c y o f t h e v a r i o u s t y p e s o f a n o m a l i e s found i n spontaneous a b o r t u s e s (Boue and Boue, 1974b). The m a j o r i t y f a l l i n t o t h r e e major c a t e g o r i e s : a u t o s o m a l t r i s o m y , 45X monosomy, and t r i p l o i d y . T r i s o m i e s r e p r e s e n t a p p r o x i m a t e l y 40% o f t h e a n o m a l i e s r e p o r t e d i n t h e u n s e l e c t e d s e r i e s ( C a r r , 1972a) and o v e r 50% i n t h e l a r g e s t s e l e c t e d s e r i e s (Boue and Boue, 1 9 7 3 c ) . The f r e q u e n c y o f t h e d i f f e r e n t t r i s o m i e s among a b o r t u s e s i s not random and t h i s has been c l e a r l y documented by t h e use o f more p r e c i s e c y t o l o g i c a l t e c h n i q u e s . T r i s o m y 16 i s t h e most f r e q u e n t t r i s o m y among a b o r t u s e s and a c c o u n t s f o r 15 t o 20% o f a l l chromosomal a n o m a l i e s o b s e r v e d (Boue and Boue, 1974b; T h e r k e l s o n , 1973). Of a l l t h e p o s s i b l e 22'autosomal t r i s o m i e s , o n l y 4 have n o t as y e t been d e s c r i b e d ( K a j i i , 1973; M c C o n n e l l and C a r r , 1975). I n t h e o r y , monosomy s h o u l d be j u s t as common as t r i -somy, b u t i n a b o r t e d m a t e r i a l t h o s e f o u n d a r e a l m o s t e x c l u -s i v e l y 45X monosomy and a c c o u n t f o r 15 t o 25% o f a l l ano-m a l i e s ( C a r r , 1972a; Boue and Boue, 1974b). I f t h e hypo-t h e s i s t h a t m e i o t i c n o n d i s j u n c t i o n s o c c u r randomly f o r each chromosome p a i r i s a c c e p t e d , t h e n t h e t o t a l number o f mono-somies and t r i s o m i e s i n a b o r t e d m a t e r i a l c a n be e s t i m a t e d 4 from t h e numbers o f monosomy X and t r i s o m y 16 o b s e r v e d among a b o r t u s e s . I t has been c a l c u l a t e d t h a t monosomic and t r i -somic z y g o t e s r e p r e s e n t about 40% o f a l l z y g o t e s c o n c e i v e d (Boue and Boue, 19 73b). The t h i r d c l a s s o f chromosome anomaly i s p o l y p l o i d y . P o l y p l o i d c e l l s have chromosome numbers t h a t a r e m u l t i p l e s o f t h e germ c e l l number. T r i p l o i d y (3n) a c c o u n t s f o r about 20% o f a b o r t u s a n o m a l i e s and i s a l m o s t f o u r t i m e s as common as t e t r a p l o i d y (4n) (Boue and Boue, 1974b). The f r e q u e n c y o f s t r u c t u r a l chromosomal a n o m a l i e s found among spontaneous a b o r t u s e s i s v e r y low. Of 921 t o t a l ano-m a l i e s r e p o r t e d by Boue and Boue ( 1 9 7 4 b ) , o n l y 35 (3.8%) were f o u n d . These were 18 R o b e r t s o n i a n t r a n s l o c a t i o n s and 17 o t h e r a n o m a l i e s i n c l u d i n g d e l e t i o n and p a r t i a l t r i s o m y . M o s a i c i s m was even l e s s common. 2. A b o r t u s morphology Chromosome a n o m a l i e s appear t o be most common i n e a r -l i e r d e v e l o p m e n t a l p e r i o d s . Boue and Boue (1974b) found 66% i n a b o r t u s e s a r r e s t e d i n t h e i r development b e f o r e 8 weeks. T h i s f r e q u e n c y f e l l t o 23% among a b o r t u s e s a t 8-12 weeks o f development. Szulman (1965) and Mikamo (1970) r e p o r t e d s i m i l a r r e s u l t s . T h i e d e and M e t c a l f e (1966) f o u n d t h a t 88% o f z y g o t e s o f l e s s t h a n 4 weeks o f development were chr o m o s o m a l l y a b n o r m a l . There i s a good c o r r e l a t i o n between t h e f r e q u e n c y o f chromosomal anomalies and the frequency of morphological ano-malies i n abortuses. M i l l e r and Poland (1970) r e p o r t e d mor-p h o l o g i c a l a b n o r m a l i t i e s i n 88% of abortuses with a develop-ment l e s s than 5 weeks, i n 58% of the 5-6 week group and i n 32% of the 7-12 week group. On the other hand, 16% of s t r u c t u r a l l y normal embryos have a chromosomal anomaly (Singh and Carr, 1967). In the e a r l i e s t and most s e v e r e l y r e t a r d e d morphological d i s o r g a n i z a t i o n , i n c l u d i n g those pre-v i o u s l y d e s c r i b e d as b l i g h t e d ova ( i . e . empty s a c ) , there appears to'be an excess of t r i s o m i e s ( C a r r , 1972b; Poland and M i l l e r , 1973). To date, very l i t t l e m orphological des-c r i p t i o n has accompanied c y t o g e n e t i c r e p o r t s , so c o r r e l a t i o n between anatomical and c y t o g e n e t i c f i n d i n g s i n abnormal abortuses has been very slow to emerge. 3. E t i o l o g y of spontaneous a b o r t i o n The p o s s i b l e causes of spontaneous a b o r t i o n have, i n the past, been grouped i n three c a t e g o r i e s : ( i ) develop-mental events w i t h i n the embryo or f e t u s which are incom-p a t i b l e w i t h i n t r a u t e r i n e e x i s t e n c e ; ( i i ) maternal f a c t o r s and ( i i i ) complex i n t e r a c t i o n s between mother and embryo or f e t u s . The extent of the c o n t r i b u t i o n of the f i r s t category has been d i s c u s s e d and can be estimated from the many pub l i s h e d s e r i a l c y t o g e n e t i c s t u d i e s as at l e a s t 35%. This i s thought to be an u n d e r e s t i m a t i o n and w i l l f l u c t u a t e depending on the g e s t a t i o n a l age o f t h e m a t e r i a l s t u d i e s (Boue and Boue, 1 9 7 3 c ) . As w e l l , i t i s m e r e l y s p e c u l a t i o n as t o t h e p r o p o r -t i o n o f t h i s c l a s s c o n t r i b u t e d by e a r l y l e t h a l m e t a b o l i c e r r o r s i n m o r p h o l o g i c a l l y normal a b o r t u s e s . Incompetence o f t h e c e r v i c a l os and a n o m a l i e s o f t h e u t e r u s ( i . e . b i c o r n u a t e u t e r u s ) have been i m p l i c a t e d i n t h e second c l a s s o f e t i o l o g i c a l f a c t o r s . As w e l l , d e f e c t i v e c o r p u s l u t e u m r e g u l a t i o n has been s u g g e s t e d as a c a u s i t i v e f a c t o r i n c l i n i c a l a b o r t i o n , b u t t h i s r e m a i n s a d e b a t a b l e i s s u e . Some w o r k e r s b e l i e v e t h a t a b o r t i o n i s commonly a t t r i b u t e d , p erhaps w i t h o u t adequate r e a s o n , t o d e f i c i e n t s e c r e t i o n o f p r o g e s t e r o n e by t h e c o r p u s l u t e u m ( H e l l m a n and P r i t c h a r d , 1 9 71), w h i l e o t h e r s m a i n t a i n t h a t c o r p u s luteum i n a d e q u a c y i s commonly a s s o c i a t e d w i t h b o t h i n f e r t i l i t y and r e p e a t e d spontaneous a b o r t i o n (Jones e t a l . , 1 9 7 4). The r e l a t i o n o f m a t e r n a l age t o t h e d i f f e r e n t t y p e s o f a n o m a l i e s v a r i e s ( C a r r , 1971a; Boue and Boue, 1974a). No m a t e r n a l age i n f l u e n c e c a n be d e m o n s t r a t e d f o r monosomy X o r t r i p l o i d y , b u t t h e i n f l u e n c e o f i n c r e a s e d m a t e r n a l age i s marked i n t r i s o m y , p a r t i c u l a r l y t r i s o m i e s o f t h e a c r o c e n t r i c chromosomes. So f a r , no i n f l u e n c e o f p a t e r n a l age has been d e m o n s t r a t e d . The t h i r d c a t e g o r y i s d i f f i c u l t t o a n a l y z e . One pos-s i b l e f a c t o r i s ABO i n c o m p a t i b i l i t y . Matsunaga and I t o h ( 1 9 5 7 ) , f o u n d t h a t 15.3% o f a l l p r e g n a n c i e s t e r m i n a t e d i n a b o r t i o n s among i n c o m p a t i b l e m a t i n g s i n c o n t r a s t t o 10.3% i n 7 the compatible group, a f i n d i n g t h a t was s i g n i f i c a n t at the 0.001 p r o b a b i l i t y l e v e l . In a more recent study, among 78 f e t u s e s examined f o r ABO i n c o m p a t i b i l i t y , 44.7% were found to be inc o m p a t i b l e w i t h the mother (Takano and M i l l e r , 1972). B) Recurrent Spontaneous A b o r t i o n l'i C l i n i c a l aspects H a b i t u a l a b o r t i o n has been de f i n e d as the occurrence of three or more consecutive spontaneous a b o r t i o n s (Eastman, 1956; Reid, 1972). Some people d i s p u t e whether a c l i n i c a l c o n d i t i o n of h a b i t u a l a b o r t i o n a c t u a l l y e x i s t s , or whether i t merely r e f l e c t s the laws of chance, r e c a l l i n g t h a t 15 to 25% of a l l pregnancies terminate as r e c o g n i z a b l e a b o r t i o n s (Inhorn, 1967). There i s general agreement i n the l i t e r a -t u r e t h a t a pregnant woman i s more l i k e l y to abort i f she has already had an a b o r t i o n (Warburton and F r a s e r , 1964). For i n s t a n c e , p r o s p e c t i v e s t u d i e s on the outcome of preg-nancies f o l l o w i n g a spontaneous a b o r t i o n by Boue and Boue. (1974a) showed t h a t women w i t h no previous h i s t o r y of abor-t i o n (except the a b o r t i o n studied) had a subsequent a b o r t i o n frequency of 13.9%, whereas women wit h a h i s t o r y of previous a b o r t i o n had a frequency of 24.3%. These authors suggest the p o s s i b i l i t y of the e x i s t e n c e of p r e - d i s p o s i n g f a c t o r s f o r at l e a s t some of the cases of repeated a b o r t i o n . Cer-t a i n maternal f a c t o r s , f o r example e n d o c r i n o l o g i c a l and ana-t o m i c a l c o n d i t i o n s h a v e b e e n i m p l i c a t e d as p r e - d i s p o s i n g f a c t o r s . R e p e a t e d a n o m a l o u s e m b r y o n i c d e v e l o p m e n t h a s a l s o b e e n p o s t u l a t e d as a p o s s i b l e f a c t o r i n r e c u r r e n t a b o r t i o n . H o w e v e r , i n a s t u d y o f 73 s p e c i m e n s f r o m women w i t h r e c u r r e n t a b o r t i o n s t h e r e was no s i g n i f i c a n t i n c r e a s e i n f e t u s e s w i t h a b n o r m a l d e v e l o p m e n t ( P o l a n d , 1971). I n t h i s s t u d y , h o w e v e r , a n o m a l o u s g r o w t h d e v e l o p m e n t ( m o s t l y b l i g h t e d o v a ) o c c u r r e d more f r e q u e n t l y among t h e a b o r t u s e s f r o m n u l l i p a r o u s ( no v i a b l e b i r t h s ) women w i t h r e c u r r e n t a b o r t i o n s t h a n among a b o r t u s e s f r o m women w i t h r e c u r r e n t a b o r t i o n s who h a d h a d a v i a b l e c h i l d o r c h i l d r e n . T h e r e i s v e r y l i t t l e e p i d e m i o l o g i c a l i n f o r m a t i o n on r e -c u r r e n t a b o r t i o n a n d t h e c h r o m o s o m a l c o n s t i t u t i o n o f s u c c e s -s i v e a b o r t u s e s . A s t u d y b y Boue a nd Boue (19 73a) o n 30 women e a c h o f whom h a d two c o n s e c u t i v e a b o r t i o n s k a r y o t y p e d r e v e a l e d t h a t ( i ) when t h e a b o r t u s h a s a n o r m a l k a r y o t y p e t h e f i r s t t i m e , t h e k a r y o t y p e was a l s o n o r m a l t h e s e c o n d t i m e i n 8 c a s e s , w h e r e a s c h r o m o s o m a l a n o m a l i e s w e r e p r e s e n t i n o n l y 3 c a s e s , ( i i ) f o r 17 women whose f i r s t a b o r t i o n s h a d a c h r o m o -s o m a l a n o m a l y , t h e s e c o n d a b o r t i o n was a g a i n a b n o r m a l i n 14 c a s e s and n o r m a l i n 3 c a s e s , a n d ( i i i ) two women w e r e DqDq t r a n s l o c a t i o n c a r r i e r s . I n t h e l a t t e r i n s t a n c e , a l l f o u r a b o r -t u s e s e x a m i n e d i n v o l v e d t h e DqDq t r a n s l o c a t i o n and t h r e e o f t h e k a r y o t y p e s w e r e ' t r a n s l o c a t i o n t r i s o m i e s . A l b e r m a n e t a l . (1975) h a s r e p o r t e d t h a t m o t h e r s who h a d d e l i v e r e d a c h r o m o s o m a l l y n o r -m a l a b o r t u s more o f t e n h a v e a h i s t o r y o f r e p e a t e d a b o r t i o n s 9 w h i l e t h o s e w i t h c h r o m o s o m a l l y abnormal a b o r t u s e s tended t o have fewer r e p e a t e d a b o r t i o n s b u t an e x c e s s o f p r e v i o u s v i a b l e c h i l d r e n with'Down syndrome. Such s t u d i e s i n v o l v e s m a l l sample s i z e s and much more e p i d e m i o l o g i c a l i n f o r -m a t i o n must be g a t h e r e d b e f o r e any c o r r e l a t i o n s c a n be e s t a b l i s h e d r e g a r d i n g r e c u r r e n t spontaneous a b o r t i o n . 2. C y t o g e n e t i c s t u d i e s o f r e c u r r e n t a b o r t i o n I n t h e p a s t decade, t h e q u e s t i o n has been r a i s e d as t o whether f a m i l i a l c y t o g e n e t i c a b e r r a t i o n s may be r e s p o n s i b l e f o r some o f t h e c a s e s o f h a b i t u a l a b o r t i o n . Chromosomal r e a r r a n g e m e n t s a r e known t o o c c u r i n an a p p a r e n t l y h e a l t h y p e r s o n i n a b a l a n c e d f o r m . I n such c a s e s , d u r i n g gameto-g e n e s i s a p r o p o r t i o n o f u n b a l a n c e d germ c e l l s c a n be ex-p e c t e d t o be formed and upon f e r t i l i z a t i o n t o g i v e r i s e t o e i t h e r embryos w i t h g r o s s maldevelopment o r e a r l y a b o r t i o n . Schmid (1962) was t h e f i r s t t o r e p o r t t h e r e s u l t s o f chromosome a n a l y s i s i n . p a t i e n t s w i t h a h i s t o r y o f two or more spontaneous a b o r t i o n s . Of t h e t e n c o u p l e s he s t u d i e d , one p a t i e n t had an e x t e n s i o n o f t h e s h o r t arm o f a G c h r o -mosome. S i n c e Schmid's f i n d i n g , numerous a u t h o r s have r e p o r t e d chromosome s t u d i e s among p a t i e n t s w i t h r e c u r r e n t a b o r t i o n s . R e c i p r o c a l t r a n s l o c a t i o n , c e n t r i c f u s i o n ( R o b e r t s o n i a n t r a n s l o c a t i o n ) and m o s a i c i s m have been d e s -c r i b e d . Schmid' ( 1 9 6 2 ) , Wingate (1965) and C a r r (1965) i n d e -10 p e n d e n t l y n o t e d l o n g s h o r t arms o f group G chromosomes i n s u b j e c t s w i t h h i s t o r i e s o f r e p e a t e d spontaneous a b o r t i o n . S i n c e t h a t t i m e , t h i s and s i m i l a r f i n d i n g s have been n o t e d i n s e v e r a l normal i n d i v i d u a l s and t h e a s s o c i a t i o n w i t h r e -c u r r e n t a b o r t i o n i s now c o n s i d e r e d f o r t u i t o u s . Such c h r o -mosomal a b n o r m a l i t i e s a r e c o n s i d e r e d t o be normal v a r i a n t s and w i l l be d i s c u s s e d i n more d e t a i l l a t e r . R e p o r t s on t h e r e s u l t s o f chromosome i n v e s t i g a t i o n s o f t h e p a r e n t s w i t h c a s e s o f r e p e a t e d a b o r t i o n a r e l i s t e d i n T a b l e I . Chromosome a b e r r a t i o n s o r v a r i a t i o n s a s s o c i a t e d w i t h m u l t i p l e a b o r t i o n s were found i n 93 (7.2%) o f 1346 i n -d i v i d u a l s (626 males and 720 f e m a l e s ) . The i n c i d e n c e o f t r a n s l o c a t i o n s (2.2%) f o u n d i n t h i s p o p u l a t i o n i s more t h a n t e n t i m e s h i g h e r t h a n t h e 0.17% f o u n d i n t h e g e n e r a l p o p u l a t i o n (Hamerton e t a l . i n p r e s s ) . There a r e two main t y p e s o f t r a n s l o c a t i o n t o c o n s i d e r : t h o s e i n v o l v i n g two a c r o c e n t r i c chromosomes ( R o b e r t s o n i a n t r a n s l o c a t i o n s ) and t h o s e i n v o l v i n g r e c i p r o c a l i n t e r c h a n g e s . The t h e o r e t i c a l c o n s i d e r a t i o n s o f t h e b e h a v i o u r o f R o b e r t -s o n i a n t r a n s l o c a t i o n s a t m e i o s i s have been d i s c u s s e d by Ham-e r t o n (1968) w h i l e t h o s e f o r r e c i p r o c a l t r a n s l o c a t i o n a r e summarized by F o r d and C l e g g ( 1 9 6 9 ) . P e r i c e n t r i c i n v e r s i o n has been r e p o r t e d once i n c o n j u n c t i o n w i t h r e p e a t e d a b o r t i o n ( B h a s i n e t a l . 1 9 7 3). A l l o b s e r v a t i o n s i n i s o l a t e d c a s e s o f p e r i c e n t r i c i n v e r s i o n s , however, s u g g e s t n o n - i m p a i r e d f e r t i l i t y o f t h e i n v e r s i o n h e t e r o z y g o t e and no a s s o c i a t e d TABLE I . Chromosome S t u d i e s i n Repeated A b o r t i o n s 11 No. A u t h o r s No. s t u d i e d T o t a l T r a n s l o c a t i o n s Chromosome f e - a b n o r m a l -male male i t i e s 1. Schmid ('62) 10 10 1 2. Rowley e t a l . ( 1 6 3 ) 22 8 1 3. Wingate ('65) 22 22 5 4. S i n g h e t a l . ( ' 6 6 ) 22 19 0 5. McKay e t a l . ( ' 6 7 ) 42 42 2 (2 6. Shaw e t a l . ( ' 6 7 ) 30 30 1 (1 7. Bishum & M o r t o n ('68) 27 — 1 8. Perqament e t a l . ( ' 6 8 ) 43 39 6 (1 9. S t e n c h e v e r e t a l . ( ' 6 8 ) 41 36 1 (1 10. K a d o t a n i e t a l . ( ' 6 9 ) 71 71 8 (1 (1 (1 11. W i l s o n ('69) 50 50 10 (1 (1 (1 (1 12. Golob & Kunze-muhl ('71) 22 14 2 (1 13. S t e n c h e v e r & J a r v i s ('71) 28 28 3 (1 14. Lucas e t a l . ('72) 42 42 5 (1 (1 (1 15 . R o t t e t a l . ('72) 26 17 6 (1 16. Warter & F o u s s e r e a u ('72) 32 22 5 (2 (1 (1 17. De L a C h a p e l l e e t a l . ('73) 51 50 10 (1 18. Kaosaar & M i k e l s a a r ('73) 28 28 8 19. B h a s i n e t a l . ('73) 27 16 2 (1 20. Papp e t a l . ('74) 14 14 1 (1 21. S o u b i r o n s ('75) 32 32 10 (1 (1 (1 22. B r o u s t e t e t a l . ( ' 7 5 ) 30 30 — (1 (1 45, XX,D-,D-,t(DqDq)+ 46, XX 1 8 q i 45,XX,13-,15-,t(13q!5q) 45, X X , 1 4 - , 1 5 - , t ( 1 4 q l 5 q ) 46, XX,t(Bp+;Cq-) 45, XY,D-,G-,t(DqGq) 46, X Y , t ( C p t ; D q - ) 46,XX,t(Gp+;lp-) 46,XX,t(Gp+;18-) 46,XY,t(Gp+;Cq-) 45,XX,D-,D-,t(DqDq)+ 45,XX,D-,D-,t(DqDq)+ 4 5 , X X , 1 3 - , 1 5 - , t ( 1 3 q l 5 q ) 45, X X , 1 5 - , 1 5 - , t ( 1 5 q l 5 q ) 46, XX,t(5p+;10q-) 46 , X X , t ( 1 3 p + ; l q - ) 45, XX,D-D-,t(DqDq)+ 46, XX,t(Dq+;Bq-) 46,XY,t(Dp+;Fp-)' 46,XY,t(Cq+;Dq-) 46,XX,t(7g+;14p-) 45,XX,13-,15-,t(13q!5q) 45,XY,D-,G-,t(DqGq)+ 45,XY,D-,G-,t(DqGq)+ 45, XX,D-,D-,t(DqDq)+ 46, XY,t(Bp+;Cq-) 46,XX,t(5q+;6q-) 46,XX,t(10q+;5q-) T o t a l 720''-626 93 30 12 i n c r e a s e i n f e t a l w astage. U n b a l a n c e d gametes a r e an excep-t i o n and p r o b a b l y a r i s e from c r o s s i n g - o v e r w i t h i n t h e i n -v e r s i o n h e t e r o z y g o t e l o o p . These u n b a l a n c e d k a r y o t y p e s have been r e p o r t e d i n a s s o c i a t i o n w i t h mental, r e t a r d a t i o n , b u t n o t w i t h a b o r t i o n ( W i l s o n , 1971; T a y s i e t a l . , 1 9 7 3 ) . D i f f e r e n t w o r k e r s have used v a r i o u s d e f i n i t i o n s o f h a b i t u a l a b o r t i o n . Many o b s t e t r i c i a n s agree w i t h Eastman (1956) i n r e s e r v i n g t h e term f o r c a s e s i n w h i c h t h e f e t u s i s l o s t i n t h r e e o r more c o n s e c u t i v e p r e g n a n c i e s . C y t o g e n e t i -c i s t s , however, have u t i l i z e d a v a r i e t y o f c r i t e r i a i n t h e i r s t u d i e s , r a n g i n g f rom Eastman's o b s t e t r i c a l d e f i n i t i o n t o s i t u a t i o n s i n v o l v i n g two o r more n o n - c o n s e c u t i v e a b o r t i o n s and i n c l u d i n g l i v e b o r n s . The r a t i o n a l e , a c c o r d i n g t o W i l s o n (1969) i s t h a t chromosome causes o f a b o r t i o n would n o t be e x p e c t e d t o b r i n g about a b o r t i o n i n e v e r y p r e g n a n c y , t h u s s e r i e s u t i l i z i n g Eastman's c r i t e r i a would be b i a s e d a g a i n s t such a b n o r m a l i t i e s b u t would t e n d t o i n c l u d e an e x c e s s o f m a t e r n a l c a u s e s o f a b o r t i o n ( i . e . u t e r i n e d e f e c t s ) . However, i n many c a s e s t h e q u e s t i o n r e m a i n s whether t h e d e m o n s t r a t e d p a r e n t a l chromosomal a b e r r a t i o n i s t h e cause o f the r e c u r r e n t a b o r t i o n . I n v e r y few c a s e s i n t h e p u b l i s h e d s e r i a l i n v e s t i g a t i o n s were t i s s u e c u l t u r e r e s u l t s from t h e a b o r t u s a v a i l a b l e and o n l y i n one c a s e was t h e r e a c l e a r l y d e m o n s t r a t e d c o r r e l a t i o n : t h e t i s s u e c u l t u r e r e s u l t s r e -v e a l e d a 4 6 X Y , t ( 1 5 q l 5 q ) , 1 5 + a b o r t u s , l o s t t o a woman of chromosomal c o n s t i t u t i o n 4 5 X X , t ( 1 5 q l 5 q ) (Lucas e t a l . , 1 9 7 2). 13 C) Purpose o f t h e p r e s e n t s t u d y The p u rpose o f t h e p r e s e n t i n v e s t i g a t i o n was p r i m a r i l y t o add i n f o r m a t i o n f o r t h e d e t e r m i n a t i o n o f t h e i n c i d e n c e and t y p e s o f chromosomal a b e r r a t i o n s found i n c o u p l e s expe-r i e n c i n g r e p e a t e d spontaneous a b o r t i o n s . I n a d d i t i o n , t h e c l i n i c a l f i n d i n g s i n t h e m e d i c a l and o b s t e t r i c a l h i s t o r y o f such c o u p l e s was e v a l u a t e d i n terms o f whether o r n o t c h r o -mosome s t u d i e s s h o u l d be u n d e r t a k e n i n f u t u r e c a s e s . The d e s i g n o f t h e s t u d y was s i m i l a r t o t h e p r e v i o u s l y p u b l i s h e d , s t u d i e s , b u t f o r two a s p e c t s : ( i ) t h e i n v e s t i g a t i o n em-p l o y e d t h e newer chromosome b a n d i n g t e c h n i q u e s , s p e c i f i c a l l y t h e Giemsa b a n d i n g , w h i c h a l l o w more p r e c i s e r e s o l u t i o n and i d e n t i f i c a t i o n o f i n d i v i d u a l chromosomes and hence f o r a more c r i t i c a l a n a l y s i s o f s u s p e c t e d chromosomal a n o m a l i e s ; and ( i i ) a t l e a s t one a b o r t e d specimen per c o u p l e was a v a i -l a b l e f o r e x a m i n a t i o n . I n many c a s e s b o t h m o r p h o l o g i c a l and c y t o g e n e t i c i n f o r m a t i o n was a v a i l a b l e on an a b o r t u s . I n p r e v i o u s s t u d i e s i t was q u e s t i o n a b l e whether t h e d e m o n s t r a t e d p a r e n t a l a b e r r a t i o n was t h e e t i o l o g i c a l f a c t o r i n t h e r e c u r -r e n t a b o r t i o n s . By d e m o n s t r a t i n g c y t o g e n e t i c a b n o r m a l i t y i n t h e c o n c e p t u s , a c o r r e l a t i o n w i t h t h e b a l a n c e d p a r e n t a l a b n o r m a l i t y i s much more l i k e l y . 14 I I . MATERIALS AND METHODS A) P a t i e n t A s c e r t a i n m e n t A l l c o u p l e s were a s c e r t a i n e d t h r o u g h t h e Embryology Study, U n i v e r s i t y o f B r i t i s h C o l u m b i a , on t h e b a s i s o f t h e i r r e p r o d u c t i v e h i s t o r y , and i n each c a s e a t l e a s t one a b o r t i o n specimen was a v a i l a b l e f o r e x a m i n a t i o n . The embryos and f e t u s e s were o b t a i n e d t h r o u g h t h e Department o f P a t h o l o g y , Vancouver G e n e r a l H o s p i t a l . A specimen was a d m i t t e d i n t o t h e Embryology Study i f i t was s u f f i c i e n t l y i n t a c t t o o b t a i n a r e a s o n a b l e e s t i m a t e o f d e v e l o p m e n t a l age. Any d e v e l o p -m e n t a l a b n o r m a l i t y as e v i d e n c e d by i n t e r n a l o r e x t e r n a l e x a m i n a t i o n was r e c o r d e d ( M i l l e r and P o l a n d , 1973). A l l c o u p l e s s t u d i e d had a h i s t o r y o f a t l e a s t two c o n -s e c u t i v e spontaneous a b o r t i o n s . An i n i t i a l s u r v e y r e v e a l e d 51 c o u p l e s t h a t q u a l i f i e d as r e c u r r e n t spontaneous a b o r t e r s by t h e o b s t e t r i c a l d e f i n i t i o n o f h a v i n g had 3 o r more co n -s e c u t i v e spontaneous a b o r t i o n s . The s u r v e y a l s o r e v e a l e d an a d d i t i o n a l 65 c o u p l e s w i t h 2 c o n s e c u t i v e spontaneous a b o r t i o n s . The s t u d y commenced i n May, 1974 on c o u p l e s w i t h 3 o r more spontaneous a b o r t i o n s and f a i l i n g an adequate r e s -ponse from t h i s group was broadened i n December, 1974 t o i n -c l u d e t h e 2 o r more a b o r t i o n group. As w e l l as c o u p l e s a s c e r -t a i n e d by t h e i n i t i a l s u r v e y , c o u p l e s who s u b s e q u e n t l y 15 became known t o t h e Embryology Study by t h e a b o r t i o n o f a second o r t h i r d c o n s e c u t i v e specimen a l s o q u a l i f i e d f o r c y t o g e n e t i c e v a l u a t i o n . The t e r m i n a t i o n d a t e o f t h e p r e s e n t s t u d y had been d e f i n e d a t t h e o u t s e t as t h e d a t e on w h i c h 50 c o u p l e s had been a s c e r t a i n e d and a n a l y z e d c y t o g e n e t i c a l l y . Throughout t h e c o u r s e o f t h e s t u d y , c o u p l e s were co n -t a c t e d i n one o f two ways: ( i ) P a t i e n t s who a b o r t e d d u r i n g t h e s t u d y p e r i o d were i n i t i a l l y c o n t a c t e d i n t h e Vancouver G e n e r a l H o s p i t a l f o l l o w i n g t h e i r a b o r t i o n . A comprehensive h i s t o r y , i n c l u d i n g m e d i c a l , o b s t e t r i c a l and f a m i l y i n f o r m a -t i o n , was o b t a i n e d ' b e f o r e d i s c h a r g e from t h e h o s p i t a l . The p a t i e n t ' s p h y s i c i a n or s p e c i a l i s t was t h e n c o n t a c t e d by form l e t t e r as t o t h e e l i g i b i l i t y o f t h e p a r t i c u l a r p a t i e n t f o r e n t r y i n t o t h e s t u d y . F o l l o w i n g r e s p o n s e from t h e d o c t o r , an a p p o i n t m e n t f o r r e p r o d u c t i v e c o u n s e l l i n g o f t h e c o u p l e was a r r a n g e d by m a i l or t e l e p h o n e t h r o u g h t h e d o c t o r ' s o f f i c e . ( i i ) P a t i e n t s whose l a s t r e c o r d e d spontaneous a b o r t i o n p r e c e d e d t h e i n i t i a l s u r v e y were c o n t a c t e d , as above, t h r o u g h t h e i r p h y s i c i a n and/or s p e c i a l i s t . I n t h e s e c a s e s a more co m p r e h e n s i v e h i s t o r y was p r o c u r e d d u r i n g t h e c o u n s e l l i n g s e s s i o n . A s t a n d a r d p r o c e d u r e was f o l l o w e d t h r o u g h o u t t h e s t u d y w i t h r e g a r d t o r e p r o d u c t i v e c o u n s e l l i n g o f each c o u p l e . Both m a t e r n a l and p a t e r n a l m e d i c a l and f a m i l y h i s t o r y were t a k e n , i n t h e form o f f a m i l y p e d i g r e e w i t h emphasis on 16 s t e r i l i t y , a b o r t i o n , i n f e r t i l i t y , g e s t a t i o n a l c o m p l i c a t i o n s , n e o n a t a l d e a t h s and g e n e t i c d i s e a s e . The p o s s i b l e causes o f r e c u r r e n t a b o r t i o n were d i s c u s s e d and e m p i r i c r e c u r r e n c e r i s k s were p r e s e n t e d w i t h r e g a r d t o outcome o f f u t u r e p r e g -n a n c i e s . F i n a l l y , 3-5 cc o f venous b l o o d was t a k e n by s y r i n g e from each member o f t h e c o u p l e and a t l e a s t two b l o o d c u l t u r e s were i n i t i a t e d p e r i n d i v i d u a l . B) E x p e r i m e n t a l P r o c e d u r e s 1. Chromosome p r e p a r a t i o n s Chromosome p r e p a r a t i o n s o f c u l t u r e d human l e u k o c y t e s were p r e p a r e d by a m o d i f i c a t i o n o f t h e c u l t u r e method o f Moorhead e t a l . ( 1 9 6 0 ) . L e u k o c y t e c u l t u r e s were i n i t i a t e d by a d d i n g 0.25 c c (4-5 d r o p s ) o f whole b l o o d t o 5 m l . o f chromosome medium. Two d i f f e r e n t mediums were u t i l i z e d d u r i n g t h e c o u r s e o f t h e s t u d y : G i b c o chromosome medium 1A and D u l b e c c o ' s M o d i f i e d E a g l e medium, t h e l a t t e r t h e n b e i n g supplemented w i t h 11 m l . f e t a l c a l f serum, 1 m l . p e n i c i l l i n - s t r e p t o m y c i n and 2 m l . h e p a r i n p e r 100 m l . o f medium b e f o r e u s e . J u s t p r i o r t o i n c u b a t i o n o f c u l t u r e s c o n t a i n i n g D u l b e c c o medium, 0.1 c c o f p h y t o h e m a g g l u t i n (M form) was added per 5 m l . c u l t u r e t u b e . Each c u l t u r e was i n c u b a t e d f o r 72 h o u r s a t 3 7°C. C o l c e m i d a t a f i n a l c o n -c e n t r a t i o n o f 0.02 ug/ml was added 1 hour p r i o r t o t h e h a r -v e s t o f t h e c e l l s . A t t h e c o m p l e t i o n o f t h e i n c u b a t i o n p e r -i o d , c u l t u r e t u b e s were t r a n s f e r r e d t o a c l i n i c a l c e n t r i f u g e 17 and spun a t 800 rpm f o r 8 m i n u t e s . The s u p e r n a t a n t was r e -moved and t h e c e l l s suspended i n 5 m l . o f h y p o t o n i c s o l u t i o n (0.0 75 KC1) and c e n t r i f u g e d a t 800 rpm f o r 8 m i n u t e s . Upon remov a l o f t h e s u p e r n a t a n t , t h e c e l l s were g e n t l y r e s u s p e n d e d and f i x e d i n 3:1 a b s o l u t e m e t h y l a l c o h o l : g l a c i a l a c e t i c a c i d . The t u b e s were t h e n a l l o w e d t o s t a n d a t room tempera-t u r e f o r 30 m i n u t e s and t h e n t h e c e l l s were c e n t r i f u g e d and r e s u s p e n d e d s e v e r a l t i m e s i n f r e s h f i x a t i v e . F i n a l l y , r e -suspended c e l l s were dropped from a p i p e t t e onto p r e - c l e a n e d , c o l d , wet s l i d e s . The s l i d e s were f l a m e d , a i r - d r i e d and s t o r e d a t room t e m p e r a t u r e u n t i l u t i l i z e d . 2. T r y p s i n - g i e m s a b a n d i n g The t r y p s i n - g i e m s a b a n d i n g t e c h n i q u e u t i l i z e d was a m o d i f i c a t i o n o f S e a b r i g h t ' s t e c h n i q u e ( 1 9 7 1 ) . The f l a m e d r i e d s l i d e s were t r e a t e d 7 days a f t e r t h e i r p r e p a r a t i o n , by t a k i n g them t h r o u g h a s e r i e s o f s o l u t i o n s . The s l i d e s were f i r s t immersed i n t r y p s i n s o l u t i o n (1 m l . o f 5% t r y p s i n made up i n Hank's Ca and Mg f r e e s a l i n e , d i l u t e d t o 40 m l . i n 0.9% s a l i n e ) f o r 5-20 seconds, and t h e n immersed i n one wash o f 1% C a C l ^ and two washes o f d i s t i l l e d w a t e r . S l i d e s were t h e n s t a i n e d i n Giemsa s t a i n (2 m l . o f Giemsa s t a i n ( G u r r B1160), 20 m l . phosphate b u f f e r (pH 6.5) and 30 m l . d i s t i l l e d w a t e r ) f o r a p p r o x i m a t e l y 4 m i n u t e s , and t h e n r i n s e d i n d i s t i l l e d w a t e r , b l o t t e d d r y i n b i b u l o u s p a p e r , d i p p e d i n x y l o l and mounted i n P r o t e x mounting medium. 18 3. C-banding Two C-banding t e c h n i q u e s were used d u r i n g t h e c o u r s e o f th e s t u d y . One was a method o f K a j i i ' s ( K a j i i e t a l . 1974) w h i c h u t i l i z e d p r e v i o u s l y t r y p s i n - g i e m s a banded s l i d e s , and t h e o t h e r a method o f A r r i g h i and Hsu (1971) w h i c h u t i l i z e d f r e s h l y p r e p a r e d a i r - d r i e d s l i d e s . F o l l o w i n g t h e K a j i i t e c h n i q u e t r y p s i n - g i e m s a banded s l i d e s were d e s t a i n e d by s e q u e n t i a l i m m e r s i o n i n x y l o l ( t o remove t h e c o v e r s l i p ) , 70% e t h a n o l , 1:1 g l a c i a l a c e t i c a c i d : 5 0 % e t h a n o l , and d i s t i l l e d w a t e r . The C-banding was p e r -formed 12 weeks a f t e r i n i t i a l p r e p a r a t i o n o f t h e s l i d e s . F o l l o w i n g d e s t a i n i n g , s l i d e s were immersed i n d o u b l e s t r e n g t h t r y p s i n s o l u t i o n (2 m l . o f 5% t r y p s i n , made up i n Hank's Ca and Mg f r e e s a l i n e , d i l u t e d t o 40 m l . i n 0.9% s a l i n e ) f o r 3 m i n u t e s , r i n s e d i n d i s t i l l e d w a ter and s t a i n e d w i t h a v e r y s t r o n g Giemsa s t a i n (5 m l . Giemsa s t a i n , 20 m l . phosphate b u f f e r (pH 7.0) and 30 m l . d i s t i l l e d w a t e r ) f o r 10-60 m i n u t e s . S l i d e s were t h e n r i n s e d i n d i s -t i l l e d w a t e r and mounted i n P r o t e x . U s i n g t h e b a s i c method•of A r r i g h i and Hsu t h e p r o c e d u r e was as f o l l o w s : 1. 0.2 N HC1 a t room t e m p e r a t u r e f o r 30 m i n u t e s . 2. Three r i n s e s i n d i s t i l l e d w a t e r . A i r d r y . 3. P a n c r e a t i c RNase (100 ug/ml. i n 2xSSC, pH 7.25) a t 3 7°C i n a m o i s t chamber f o r 45 m i n u t e s . 4. Two r i n s e s i n each o f 2xSSC (pH 7.0), 70% e t h a n o l , 19 and 95% e t h a n o l . A i r d r y . 5. 0.07 N NaOH a t room t e m p e r a t u r e f o r 50 s e c o n d s . 6. Two r i n s e s each i n 70% e t h a n o l and 95% e t h a n o l . A i r d r y . 7. 2xSSC (pH 7.0) a t 65°C f o r 16 h o u r s . 8. Two r i n s e s each i n 70% e t h a n o l and 95% e t h a n o l . A i r d r y . 9. S t a i n f o r 10-15 m i n u t e s i n a Giemsa s o l u t i o n , p r e -p a r e d as f o l l o w s : 50 m l . d i s t i l l e d w a t e r , 1.5 m l . 0.1M c i t r i c a c i d , a d j u s t pH w i t h 0. 2M Na 2HPC< 4 t o pH=7.0, 1.5 m l . 100% m e t h a n o l , 5 m l . Giemsa s t a i n . 10. Two r i n s e s i n d i s t i l l e d w a t e r . A i r d r y . Mount i n P r o t e x m o unting medium. 4. F l u o r e s c e n t b a n d i n g The f l u o r e s c e n t t e c h n i q u e used was a m o d i f i c a t i o n o f C a s p e r s s o n ' s t e c h n i q u e (1969 a , b ) . A i r - d r i e d s l i d e s were immersed i n a 0.5 grams/100 m l . s o l u t i o n o f q u i n a c r i n e d i h y d r o c h l o r i d e f o r 20-30 m i n u t e s . S l i d e s were t h e n r i n s e d and mounted i n d i s t i l l e d w a t e r and t h e c o v e r s l i p s s e a l e d w i t h p a r a f f i n wax. 5. A n a l y s i s and e s t a b l i s h m e n t o f k a r y o t y p e A t l e a s t 25 t r y p s i n - g i e m s a banded metaphases were ana-l y z e d f o r chromosome number t o r u l e o u t t h e p o s s i b i l i t y o f m o s a i c i s m . Of t h e s e 10-15 w i t h t h e b e s t r e s o l u t i o n were 20 . phot o g r a p h e d under o i l i m m e r s i o n (X 100) u s i n g Kodak h i g h c o n t r a s t copy f i l m 5069. A l l o b s e r v a t i o n s were made w i t h a Z e i s s P h o t o m i c r o s c o p e under phase c o n t r a s t . P r i n t e d p h o t o -graphs were p r o d u c e d and k a r y o t y p e s were c o m p l e t e d on t h e b a s i s o f The P a r i s C o n f e r e n c e (1971) n o m e n c l a t u r e . A t l e a s t two k a r y o t y p e s were e s t a b l i s h e d p e r p a t i e n t and more i f a chromosomal a b n o r m a l i t y was s u s p e c t e d . C-banding and l e s s o f t e n f l u o r e s c e n c e was t h e n employed f o r more p r e c i s e c h a r a c t e r i z a t i o n o f t h e s u s p e c t e d v a r i a n t chromosome. C-band p h o t o g r a p h i c p r o c e d u r e was t h e same as f o r t h e t r y p s i n - g i e m s a banded p r e p a r a t i o n s . O b s e r v a t i o n s o f f l u o r e s c e n t p r e p a r a t i o n s were made w i t h a Z e i s s P h o t o m i c r o s c o p e w i t h an HB 200 mercury lamp u t i l i z i n g a BG 12 e x c i t e r f i l t e r and a '47' b a r r i e r f i l t e r . P h o tographs were t a k e n u s i n g I l l f o r d Pan F f i l m under o i l i m m e r s i o n w i t h an ex p o s u r e t i m e o f 20-40 s e c o n d s . 21 I I I . RESULTS Between May 1, 1974 and August 1, 1975, 50 c o u p l e s and two a d d i t i o n a l f e m a l e s w i t h h i s t o r i e s o f r e p e a t e d spontaneous a b o r t i o n s were e v a l u a t e d and c o u n s e l l e d t h r o u g h t h e Embryo-l o g y S t u d y, U n i v e r s i t y o f B r i t i s h C o l u m b i a . The c y t o g e n e t i c f i n d i n g s and r e p r o d u c t i v e h i s t o r i e s w i t h a v a i l a b l e i n f o r m a -t i o n r e g a r d i n g a b o r t u s morphology and k a r y o t y p e a r e p r e -s e n t e d i n T a b l e I I . A) C y t o g e n e t i c Data There was no e v i d e n c e o f chromosome m o s a i c i s m i n any o f t h e a d u l t s i n t h i s s e r i e s . S t r u c t u r a l changes c o n f i n e d t o one chromosome were i n t e r p r e t e d as normal m o r p h o l o g i c a l v a r i a n t s o f t h e human chromosome complement. E i g h t i n d i v i -d u a l s were f o u n d t o have such v a r i a n t s : ( i ) 3 males were f o u n d t o be h e t e r o z y g o u s f o r a chromosome 9 w i t h an e n l a r g e d s e c o n d a r y c o n s t r i c t i o n , as r e v e a l e d by C-banding; ( i i ) one woman was f o u n d t o be h e t e r o z y g o u s f o r a chromosome 16 w i t h an e n l a r g e d band o f h e t e r o c h r o m a t i n , as r e v e a l e d by C-banding; ( i i i ) one male was f o u n d t o have an u n u s u a l l y l o n g Y chromo-some as measured by c o n v e n t i o n a l Y/F i n d e x (Y/F = 1.00); ( i v ) 2 f e m a l e s were h e t e r o z y g o u s f o r a G-group chromosome 22 TABLE I I . C y t o g e n e t i c and R e p r o d u c t i v e Data Case # P a r i t y G r a v i d i t y Specimen Specimen P a r e n t a l M orphology K a r y o t y p e K a r y o t y p e s 1 1 5 s y s t e m i c d e f e c t s f i x e d 46XY ; 46XX 2 0 3 w. n. 1. * - 46XY9qh+; 46XX 3 1 5 i n c o m p l e t e 46XY 46XY9qh+; 46XX 4 2 6 i n c o m p l e t e 46XX 46XY ; 46XX 5 0 3 i ) i i ) GD 1 GD 2 f a i l e d f a i l e d 46XY , 46XX22S+ 6 1 4 w.n.1. — — 46XX 7 0 5 GD 1 46XX 46XY 46XX 8 0 2 i ) i i ) i n c o m p l e t e i n c o m p l e t e f a i l e d , f a i l e d 46XY 46XX 9 1 5 i ) GD 2 f a i l e d 46XY9qh+; 46XX i i ) GD 2 f a i l e d i i i ) w.n.1. 46XX 10 1 4 w.n.1. f a i l e d — 46XX 11 0 3 i ) i i ) i i i ) GD 2 GD 1 GD 2 47XXY 47XY,16+ f a i l e d 46XY 46XX 12 0 4 i n c o m p l e t e f a i l e d 46XY 46XX16h+ 13 1 4 i n c o m p l e t e f a i l e d 46XY 46XX . 14 0 4 i n c o m p l e t e 46XYq+ 46XYq+; 46XX 15 0 3 i ) GD 1 46XX, 22ph+ 46XY 46XX22ph+ i i ) GD 1 4 7XX,14+ 16 0 4 i ) i i ) i i i ) GD 1 GD 2', GD 2 f a i l e d f a i l e d f a i l e d 46XY 46XX 17 0 3 GD 2 f i x e d 46XY 46XX 18 0 4 s y s t e m i c d e f e c t s 46XY 46XY 46XX 19 0 4 GD 3 46XX 46XY 46XX 20 0 2 i n c o m p l e t e f a i l e d 46XY 46XX 21 0 4 i n c o m p l e t e f a i l e d 46XY 46XX 22 0 3 i n c o m p l e t e — 46XY 46XX 23 0 2 i ) i i ) GD 1 i n c o m p l e t e 4 7XX,2 2+ f a i l e d 46XY 46XX 24 0 2 GD 1 46XX 46XY,14ph+; 46XX 25 1 3 i n c o m p l e t e - 46XY 46XX 26 0 2 GD 2 46XX 46XY 46XX . w.n.1.* : w i t h i n normal l i m i t s TABLE I I . C y t o g e n e t i c and R e p r o d u c t i v e Data ( c o n t i n u e d ) Case # P a r i t y G r a v i d i t y Specimen Specimen P a r e n t a l Morphology K a r y o t y p e K a r y o t y p e s 27 0 3 w . n . l . * f i x e d 46XY; 46XX 28 1 5 i ) GD 2 i i ) GD 2 4 7XX,22+ f a i l e d 46XY; 46XX 29 0 3 i n c o m p l e t e f a i l e d 46XY; 46XX 30 0 4 GD 3 — 46XY; 46XX 31 0 2 s y s t e m i c d e f e c t s 69XX? 46XY; 46XX 32 0 3 GD 2 f a i l e d 46XY; 46XX 33 0 3 GD 1 f a i l e d 46XY; 46XX 34 0 2 i n c o m p l e t e - 46XY; 46XX 35 1 4 i n c o m p l e t e - 46XY; 46XX 36 0 2 i ) GD 1 i i ) GD 2 47XX,9+ f a i l e d 46XY; 46XX 37 0 3 GD 1 f i x e d 46XY; 46XX 38 0 2 i ) GD 2 45XY,t ( 1 4 q l 5 q ) 46XY; 4 5 X X , t ( 1 4 q l 5 i i ) GD 3 s y s t e m i c d e f e c t s 4 5 X Y , t ( 1 4 q l 5 q ) 39 0 2 GD 2 46XX 46XY; 46XX 40 1 4 i ) w . n . l . i i ) abnormal f a i l e d 46XY; 46XX 41 0 4 i ) i n c o m p l e t e i i ) i n c o m p l e t e i i i ) GD 3 f a i l e d f a i l e d 46XX 46XY; 46XX 42 1 4 s y s t e m i c d e f e c t s f a i l e d 46XY; 46XX 43 1 4 i ) w . n . l . i i ) w . n . l . 46XX 46XY; 46XX 44 0 2 i ) GD 1 i i ) i n c o m p l e t e f a i l e d f a i l e d 46XY; 46XX 45 1 5 GD 3 46XX 46XY; 46XX 46 • 0 2 i ) GD 1 i i ) GD 3 f a i l e d f a i l e d 46XY; 46XX 47 0 2 GD 1 f i x e d 46XY; 46XX 48 0 3 w . n . l . f a i l e d 46XY; 46XX 49 1 3 i ) GD 1 i i ) GD 3 f a i l e d 47XY,22+ 46XY; 46XX 50 1 5 w . n . l . f a i l e d 46XY; 46XX 51 2 8 i ) w.n.1. i i i ) i n c o m p l e t e i i i ) i n c o m p l e t e i v ) i n c o m p l e t e v ) i n c o m p l e t e 46XX 46XY f a i l e d 46XX 46XY 46XY; 46XX 52 0 6 w . n . l . f i x e d 46XY; 46XX 24 w i t h an e l o n g a t e d s a t e l l i t e and one male was f o u n d t o be h e t e r o z y g o u s f o r a chromosome 14 w i t h an e l o n g a t e d s a t e l l i t e . These v a r i a n t s w i l l be d i s c u s s e d l a t e r . P a r t i a l k a r y o t y p e s o f t h e two f o r m e r c a t e g o r i e s o f v a r i a n t s a r e p r e s e n t e d i n f i g u r e s 1 and 2 (page 2 9 ) . Giemsa b a n d i n g r e v e a l e d o n l y one i n d i v i d u a l w i t h a s t r u c t u r a l r e a r r a n g e m e n t and t h a t was a R o b e r t s o n i a n t r a n s l o c a t i o n i n v o l v i n g chromosomes 14 and 15. 1. Case r e p o r t T h i s husband and w i f e , aged 25 and 23 r e s p e c t i v e l y , were C a u c a s i a n and u n r e l a t e d . Her f i r s t spontaneous a b o r -t i o n o c c u r r e d a f t e r 12 weeks o f pregnancy i n J u l y , 1973. The p r o d u c t s o f c o n c e p t i o n were a n a l y z e d and f o u n d t o c o n -s i s t o f f r a g m e n t s o f d e c i d u a and p l a c e n t a o n l y . No embryo o r f e t u s was i d e n t i f i e d . F o l l o w i n g t h i s a b o r t i o n , o r a l c o n t r a c e p t i o n was i n i t i a t e d i n A u g u s t , 1973 and t e r m i n a t e d i n September, 1974. She r e p o r t e d one normal m e n s t r u a l p e r i o d i n November b e f o r e becoming p r e g n a n t . T h i s pregnancy p r o -ceeded n o r m a l l y u n t i l m i d - F e b r u a r y , 1975. A t t h i s t i m e b l e e d i n g o c c u r r e d , c o n t i n u e d f o r 10 d a y s , and i n A p r i l a d i a g n o s i s o f m i s s e d a b o r t i o n was made. The u t e r u s was e mptied by d i l a t a t i o n and c u r e t t a g e and t h e p r o d u c t s o f c o n c e p t i o n examined i n d e t a i l . The specimen c o n s i s t e d o f an i n t a c t c h o r i o n i c sac c o n t a i n i n g two a m n i o t i c s a c s . These s a c s were c o n s i d e r e d t o be m o n o z y g o t i c t w i n s . Twin "A" a m n i o t i c sac c o n t a i n e d an embryo 15 mm. crown-rump l e n g t h , w h i c h showed marked s y s t e m i c d i s o r g a n i z a t i o n i n c l u d i n g m i c r o c e p h a l y , f a c i a l a p l a s i a and r e t a r d e d l i m b buds. No l o w e r l i m b buds c o u l d be i d e n t i f i e d . Twin "B" c o n s i s t e d o f an a m n i o t i c sac 13 mm. i n d i a m e t e r c o n t a i n i n g a s m a l l p i e c e o f amor-phous embryonic t i s s u e 1.5 mm. i n d i a m e t e r w i t h no r e c o g -n i z a b l e e x t e r n a l f e a t u r e s . No p r e v i o u s f a m i l y h i s t o r y o f r e p e a t e d spontaneous a b o r t i o n was n o t e d on e i t h e r s i d e o f t h e f a m i l y ( f i g . 3 ) . T i s s u e c u l t u r e s f o r chromosomal a n a l y s i s were e s t a b l i s h e d from p i e c e s o f amnion from b o t h s a c s by t h e method o f K a j i i e t a l . ( 1 9 7 4 ) . Giemsa-banded k a r y o t y p e s from b o t h s a c s were 45, X Y , 1 4 - , 1 5 - , t ( 1 4 q l 5 q ) + ( f i g . 4 ) . L y m p h o c y t i c c u l t u r e s were e s t a b l i s h e d on t h e p a r e n t s , and t h e mother p r o v e d t o be t h e c a r r i e r f o r t h e same t r a n s l o c a t i o n ( f i g . 5 ) . Both Q-banding and C-banding were u t i l i z e d i n an a t t e m p t t o d e t e r m i n e t h e b r e a k p o i n t s o f t h e two chromosomes i n v o l v e d and t o d e t e r m i n e w h i c h c e n t r o m e r e was i n v o l v e d i n t h e t r a n s l o c a t i o n . I t appears t h a t a l l o f t h e c e n t r o m e r i c h e t e r o c h r o m a t i n o f b o t h chromosomes 14 and 15 i s p r e s e n t i n t h e t r a n s l o c a t i o n chromo-some, w h i c h s u g g e s t s t h a t b o t h e n t i r e l o n g arms a r e p r e s e n t . B) M o r p h o l o g i c a l and R e p r o d u c t i v e Data M o r p h o l o g i c a l and c y t o g e n e t i c i n f o r m a t i o n on t h e a b o r t e d m a t e r i a l i s p r e s e n t e d i n T a b l e I I I , 35% (9/26) o f a l l t h e s u c c e s s f u l l y c u l t u r e d specimens were ch r o m o s o m a l l y abnormal. A l l 9 o f t h e specimens w i t h abnormal k a r y o t y p e s had growth d i s o r g a n i z a t i o n (G.D.). 56% (9/16) o f a l l G.D. specimens w h i c h were s u c c e s s f u l l y c u l t u r e d were k a r y o t y p i c a l l y abnor-ma l . There appeared t o be a tendency towards an e x c e s s o f t r i s o m i e s i n t h e GD 1 group and an o v e r a l l l a c k o f t r i p l o i d and 45X spe c i m e n s . The r e l a t i v e l y s m a l l sample s i z e does n ot a l l o w f o r an adequate c o m p a r i s o n o f t h e f r e q u e n c y o f G.D. i n th e 3 major chromosomal c a t e g o r i e s f o u n d most f r e q u e n t l y i n spontaneous a b o r t i o n m a t e r i a l , as r e p o r t e d by P o l a n d and M i l l e r ( 1 9 7 3 ) . The c a t e g o r y p r e v i o u s l y d e s c r i b e d as GD 4 ( m u l t i p l e a n o m a l i e s and growth d i s o r g a n i z a t i o n ) was n o t e v i d e n t i n t h i s group o f a b o r t u s e s . F i v e specimens w i t h s y s t e m i c d e f e c t s ( i . e . s p i n a b i f i d a , c l e f t l i p , e t c . ) i n w h i c h a s p e c i f i c system i s i n v o l v e d were n o t e d : 1 w i t h c y s t i c tumours; 2 w i t h m u l t i p l e s y s t e m i c d e f e c t s (1 t r i p l o i d and 1 t r a n s l o c a t i o n h e t e r o z y g o t e ) ; 1 w i t h s e v e r e f a c i a l a p l a s i a ; and 1 w i t h l i m b d e f o r m i t i e s . The f r e q u e n c y o f s y s t e m i c and growth d i s o r g a n i z a t i o n specimens i n n u l l i p a r o u s and parous p a t i e n t s . i s p r e s e n t e d i n T a b l e IV. I t i s e v i d e n t t h a t ( i ) t h e s y s t e m i c d e f e c t s a r e e v e n l y d i v i d e d between t h e n u l l i p a r o u s and parous c o u p l e s w i t h r e c u r r e n t a b o r t i o n s and t h a t ( i i ) t h e o c c u r r e n c e o f G.D. appears t o be more f r e q u e n t among t h e spontaneous a b o r t i o n s o f n u l l i p a r o u s c o u p l e s w i t h r e c u r r e n t a b o r t i o n (57%) t h a n among t h e spontaneous a b o r t i o n s o f t h e parous c o u p l e s w i t h r e c u r r e n t a b o r t i o n ( 2 2 % ) . Among t h e 5 2 c o u p l e s p r e s e n t i n g w i t h r e c u r r e n t a b o r t i o n s , 27 TABLE I I I . K a r y o t y p e s P r e s e n t i n Normal and  Growth D i s o r g a n i z a t i o n C a t e g o r i e s C a t e g o r y T r i s o m y T r a n s -l o c a t i o n T r i p l o i d Normal F i x e d o r F a i l e d T o t a l GD 1 4 - - 2 9 (60%) 15 GD 2 2 1 - 2 10 (67%) 15-GD 3 1 - 1 3 1 6 Normal - - - 4 8 12 I n c o m p l e t e - - - 6 12 18 TOTAL 7 1 1 17 40 (65%) 66 TABLE IV. O c c u r r e n c e o f Abnormal Sp ecimens i n N u l l i p a r o u s and Parous P a t i e n t s Study T o t a l T o t a l Specimens S y s t e m i c GD s Specimens %GD s PARA 0; GRAV 2 13 17 2 10 69 PARA 0; GRAV 3+ 22 30 - 17 57 35 47 2 27 57 PARA 1; GRAV 3 2 3 — 2 66 PARA 1; GRAV 4+ 15 24 3 4 16 17 27 3 6 22 28 16 had c o n t r i b u t e d more t h a n one specimen t o t h e Embryology S t u d y . G.D. was fo u n d t o p e r s i s t e n t l y r e c u r amongst t h e a b o r t u s e s o f 9 o f t h e 16 p a t i e n t s . Ten o f t h e 16 r e c u r r e n t a b o r t e r s were n u l l i p a r o u s , and o f t h e s e 7 had a h i s t o r y o f r e c u r r e n t l y a b o r t i n g G.D. specimens. These 16 women c o n -t r i b u t e d 39 a b o r t u s e s c o l l e c t i v e l y , o f w h i c h 22 (56%) were G.D. sp e c i m e n s . I n f o r m a t i o n g a t h e r e d r e g a r d i n g m e d i c a l and f a m i l y h i s -t o r i e s was f o r t h e most p a r t u n r e m a r k a b l e . V e r y few r e l a t e d any abnormal e v e n t s t h a t had o c c u r r e d d u r i n g t h e c o u r s e o f th e p r e g n a n c i e s . However, o v e r 75% o f t h e women e x p e r i e n c e d v a g i n a l b l e e d i n g p r i o r t o t h e a c t u a l a b o r t i o n e v e n t ( s ) . R u b e l l a was n o t d e t e c t e d . I n f e r t i l i t y was r e p o r t e d i n 40% as h a v i n g o c c u r r e d a t some t i m e d u r i n g t h e c o u r s e o f t h e r e p r o d -u c t i v e h i s t o r y . Throughout t h e c o u r s e o f t h e s t u d y , s e v e r a l women were f o l l o w e d up r e g a r d i n g a p o t e n t i a l d i a g n o s i s o f l a t e l u t e a l phase p r o g e s t e r o n e i n a d e q u a c y . Based on b a s a l body tempera-t u r e d a t a and e n d o m e t r i a l b i o p s i e s , f i v e o f t h e s e women were t r e a t e d w i t h . o r a l p r o g e s t e r o n e d u r i n g t h e i r subsequent p r e g -n a n c i e s and i n f o u r c a s e s normal term d e l i v e r i e s o c c u r r e d . However, i n t h e s e c a s e s t h e r a p y had been i n i t i a t e d l a t e i n pregnancy and t h e r e f o r e any c a u s a l e f f e c t o f t r e a t m e n t r e g a r d -i n g outcome o f pregnancy i s s p e c u l a t i v e . I n t h e most r e c e n t t r e a t e d c a s e t h e t r e a t m e n t was i n i t i a t e d e a r l y ( t h i r d p o s t -o v u l a t o r y day) and t h e pregnancy i s c u r r e n t l y i n p r o g r e s s . II i i j m f 6 a a n 10 9qh+/ 9 Figure 1. P a r t i a l karyotype - 9qh+ variant ft 16qh+/16 Figure 2. P a r t i a l karyotype - 16qh+ variant o • o • I 0 4> (£) ® 4 6 6 0 = • o = o -t ( 14 :15 ) t r a n s l o c a t i o n c a r r i e r n o r m a l c h r o m o s o m e s un te s ted spontaneous a b o r t i o n O 6 vl <> u HI 11 i . i 14 14/15 15 16 17 19 2 0 21 22 Figure 4. Giemsa banded karyotype of abortus specimen: 45,XY,14-,15-,t(14ql5q)+ II •I If m 13 14 14/15 15 Figure 5. Composite partial C, Q and Giemsa banded maternal karyotype: 45,XX, 14-,15-,t(14ql5q)+ 33 IV. DISCUSSION A) Morphological and Reproductive Data Although the sample s i z e of the present study was l i m i t e d and the f a i l u r e r a t e of t i s s u e c u l t u r e of the aborted mate-r i a l was d i s a p p o i n t i n g l y h i g h , some p o i n t s m e r i t d i s c u s s i o n . F i r s t , as evidenced by Table I I I , 56% (9/16) of the abortuses w i t h growth d i s o r g a n i z a t i o n (G.D.) were k a r y o t y p i -c a l l y normal. 35% (9/26) of a l l the s u c c e s s f u l l y c u l t u r e d specimens were chromosomally abnormal. Both these f i n d i n g s are i n agreement w i t h those r e p o r t e d i n the l i t e r a t u r e (Singh and Carr, 1967). Second, 13% (5/38) of the developmentally abnormal specimens had systemic d e f e c t s (Table I V ) . This i s low when compared to p u b l i s h e d r e p o r t s which noted t h a t among spon-taneous abortuses, growth d i s o r g a n i z a t i o n and systemic ano-malies occurred w i t h approximately the same frequency, w h i l e among me d i c a l l y - i n d u c e d abortuses, 85% of the abnormal spec-imens were of the systemic type ( M i l l e r and Poland, 1973). This would tend to support the b e l i e f t h a t women w i t h r e c u r r e n t abo r t i o n s tend to abort more G.D. specimens than do the gen-e r a l p o p u l a t i o n of spontaneous aborters (Poland, M i l l e r and Jones, i n p r e s s ) . G.D. was found to be more frequent among the spon-taneous a b o r t u s e s o f n u l l i p a r o u s women w i t h r e c u r r e n t a b o r -t i o n s (57%) t h a n among t h e parous women w i t h r e c u r r e n t a b o r -t i o n s ( 2 2 % ) . Among t h o s e p a t i e n t s on whom t h e r e was m o r p h o l -o g i c a l i n f o r m a t i o n on more t h a n one a b o r t u s , specimens w i t h G.D. were f o u n d t o r e c u r among t h e p r e g n a n c i e s o f 7 o f 10 n u l l i p a r o u s women w i t h r e c u r r e n t a b o r t i o n s . T h i s s u p p o r t s o t h e r s t u d i e s i n s u g g e s t i n g t h a t growth d i s o r g a n i z a t i o n s t e n d t o r e c u r among n u l l i p a r o u s women w i t h r e c u r r e n t a b o r -t i o n s ( W a l l and H e r t i g , 1948; P o l a n d , 1971; P o l a n d , M i l l e r and J o n e s , i n p r e s s ) . Recent f o l l o w - u p s t u d i e s s u g g e s t t h a t n u l l i p a r o u s r e c u r r e n t a b o r t e r s have t h e h i g h e s t o v e r a l l r i s k (54%) f o r subsequent spontaneous a b o r t i o n ( P o l a n d , M i l l e r and J o n e s , i n p r e s s ) . A c a r e f u l e v a l u a t i o n o f t h e l u t e a l phase o f t h e c y c l e s h o u l d accompany t h e s t u d y o f women w i t h r e c u r r e n t s p o n t a -neous a b o r t i o n s , and s u p p o r t i v e hormone t h e r a p y i n s t i t u t e d where n e c e s s a r y . The e n d o c r i n o l o g i c a l r o l e i n t h e e t i o l o g y o f r e c u r r e n t a b o r t i o n r e m a i n s t o be i n v e s t i g a t e d . B) Chromosomal V a r i a n t s S i n c e one o f t h e pur p o s e s o f t h e p r e s e n t s t u d y was t o d i s c e r n , i n t h e s t u d y p o p u l a t i o n , chromosomal v a r i a n t s from s t r u c t u r a l r e a r r a n g e m e n t s , some d i s c u s s i o n r e g a r d i n g v a r i a n t s i s w a r r a n t e d . These v a r i a n t s , found i n 1.8 - 3.0% o f t h e gen-e r a l p o p u l a t i o n ( Court-Brown e t a l . , 1965; Hamerton e t a l . , i n p r e s s ) , were f r e q u e n t l y c o n f u s e d as t r a n s l o c a t i o n chromo-35 somes b e f o r e t h e newer b a n d i n g t e c h n i q u e s were d e v e l o p e d . They have been d e f i n e d as i n c l u d i n g e n l a r g e d s e c o n d a r y c o n s t r i c t i o n s , e n l a r g e d s h o r t arms and/or s a t e l l i t e s o f a c r o c e n t r i c chromosomes, and l a r g e Y chromosomes (Hamerton e t a l . , 1972 ). A l l t h r e e t y p e s o f v a r i a n t s were e n c o u n t e r e d i n t h e p r e s e n t s t u d y p o p u l a t i o n . The l o n g Y chromosome and s a t e l l i t e v a r i a n t s a r e t h e most common (Orye, 19 74) and have been o b s e r v e d i n a s s o c i a -t i o n w i t h m e n t a l r e t a r d a t i o n and a v a r i e t y o f c o n g e n i t a l d i s -o r d e r s ( K a d o t a n i e t a l . , 1969; Zeuthen and N i e l s e n , 1973). Chromosomes 1, 9 and 16 a r e e s p e c i a l l y i n c l i n e d t o v a r i a t i o n s o f e n l a r g e m e n t o f h e t e r o c h r o m a t i c s e c o n d a r y c o n s t r i c t i o n s , p r e s u m a b l y due t o i n s i t u d u p l i c a t i o n o r u n e q u a l c r o s s i n g o v er ( N i e l s e n e t a l . , 1974b). A l t h o u g h most i n v e s t i g a t o r s c o n s i d e r t h a t t h e s e v a r i a n t s e x e r t no d e t e c t a b l e i n f l u e n c e on phenotype (Court-Brown e t a l . , 1 9 65), t h e t r u e r e l a t i o n w i l l o n l y be u n c o v e r e d when t h e r e s u l t s o f s e v e r a l l a r g e p o p u l a t i o n s u r -v e y s , i n v o l v i n g b a n d i n g t e c h n i q u e s , a r e a v a i l a b l e . Some s t u d i e s have e x p l o r e d t h e p o s s i b i l i t y o f an i n c r e a s e d r i s k o f n o n d i s j u n c t i o n i n progeny o f v a r i a n t h e t e r o z y g o t e s (Ohno e t a l . , 1961; Hamerton e t a l . , 1965). N i e l s e n e t a l . (1974a) r e p o r t s a s i g n i f i c a n t i n c r e a s e o f t h e v a r i a n t 9qh+ chromo-some i n p a r e n t s o f c h i l d r e n w i t h major chromosome a b n o r m a l i -t i e s . There a r e no r e p o r t s o f t h e a s s o c i a t i o n o f such v a r i a n t s i n c o n j u n c t i o n w i t h spontaneous a b o r t i o n , and i t i s g e n e r a l l y b e l i e v e d t h a t any such a s s o c i a t i o n i s f o r t u i t o u s . 36 C) T r a n s l o c a t i o n and A b o r t i o n 1. F r e q u e n c y i n t h e p o p u l a t i o n o f c o u p l e s w i t h r e c u r r e n t a b o r t i o n s The f r e q u e n c y o f t r a n s l o c a t i o n s was l o w e r i n t h i s s e r i e s (1 i n 102 i n d i v i d u a l s * ) t h a n t h e 2.2% i n t h e combined s e r i e s p r e s e n t e d i n T a b l e I , b u t h i g h e r t h a n t h e 0.'17% r e p o r t e d f o r t h e g e n e r a l p o p u l a t i o n (Hamerton e t a l . , i n p r e s s ) . Marked d i f f e r e n c e s i n f r e q u e n c y o c c u r between t h e s e r i e s q u o t e d i n T a b l e I , r a n g i n g from 0 - 7.5 (Warter and F o u s s e r e a u , 1972) and the f a c t o r s w h i c h cause t h i s v a r i a t i o n must be c o n s i d e r e d . S m a l l sample s i z e i s one f a c t o r . I f t h e f i g u r e s quoted above f o r t h e f r e q u e n c y o f t r a n s l o c a t i o n i n t h e g e n e r a l pop-u l a t i o n and f o r t h e p o p u l a t i o n o f c o u p l e s p r e s e n t i n g w i t h r e -c u r r e n t a b o r t i o n a r e a c c e p t e d as t r u e e s t i m a t e s , t h e n a sam-p l e s i z e o f 2 78 would be r e q u i r e d t o be 90% s u r e o f d e t e c t i n g a s i g n i f i c a n t d i f f e r e n c e a t t h e 5% l e v e l . A sample s i z e o f 102 would be r e q u i r e d t o be 50% s u r e o f d e t e c t i n g t h e same d i f f e r e n c e . T h e r e f o r e , i t i s o n l y when t h e s e l i m i t e d s t u d i e s a r e p o o l e d t h a t a s i g n i f i c a n t d i f f e r e n c e i s d e m o n s t r a t e d . D i f f e r e n t d e f i n i t i o n s o f r e p e a t e d a b o r t i o n may a c c o u n t f o r some of t h e d i f f e r e n c e s . Thus the 2.2% i n c i d e n c e i n t h e combined s e r i e s i s f o r a nonhomogenous p o p u l a t i o n . Most a u t h o r s b e l i e v e t h a t t h e more s t r i n g e n t t h e c r i t e r i a , t h e more f r e q u e n t l y t r a n s l o c a t i o n s w i l l be f o u n d (De La C h a p e l l e e t a l . , 1 9 7 3). S i n e t e t a l . (1973) and Pernod e t a l . (1975) quote t h e r i s k o f c a r r y i n g a t r a n s l o c a t i o n as 0.5% f o r a sub-* A f r e q u e n c y o f 0.98% w i t h 95% c o n f i d e n c e l i m i t s o f 0.02 t o 5.45%. 37 j e c t who has had two spontaneous a b o r t i o n s ; 1.0% f o r t h r e e spontaneous a b o r t i o n s and up t o 10% f o r i n d i v i d u a l s who have had f i v e o r more a b o r t i o n s . I t has a l s o been i m p l i e d t h a n an i n c r e a s e i n chromosomal a b n o r m a l i t i e s would be e x p e c t e d i n f a m i l i e s h a v i n g a h i s t o r y o f a t l e a s t one abnormal o f f -s p r i n g i n a d d i t i o n t o r e p e a t e d a b o r t i o n s (Hsu e t a l . , 1970; C a r r , 1971b). 2. S i g n i f i c a n c e o f t h e p r e s e n t t r a n s l o c a t i o n c a s e There have been t h r e e p r e v i o u s l y r e p o r t e d c a s e s o f ( 1 4 q l 5 q ) R o b e r t s o n i a n f u s i o n ( S t e n c h e v e r e t a l . , 1968; Hecht and K i m b e r l i n g , 1971; and R a a i j m a k e r s - E n g e l e n , 1973). Two o f t h e s e c a s e s were a s c e r t a i n e d t h r o u g h p a t i e n t s w i t h e i t h e r m e n t a l r e t a r d a t i o n or c o n g e n i t a l a n o m a l i e s , t h e t h i r d , a n u l l i p a r o u s woman w i t h a h i s t o r y o f t h r e e a b o r t i o n s , t h r o u g h a s e r i a l s t u d y o f c o u p l e s w i t h r e c u r r e n t a b o r t i o n s ( S t e n c h e v e r e t a l . , 1968). The main c o n s i d e r a t i o n i s whether o r n o t t h e r e i s a c a u s a l r e l a t i o n between the t r a n s l o c a t i o n i n t h e p a t i e n t and h e r p regnancy l o s s e s and, more s p e c i f i c a l l y , whether t h e a b n o r m a l i t i e s f o u n d i n t h e t w i n embryos a r e r e l a t e d t o t h e t r a n s l o c a t i o n . The e x t e n s i v e growth d i s o r g a n i z a t i o n (G.D. 2 and G.D. 3) f o u n d i n t h e a b o r t e d specimens i s s i m i l a r t o t h a t w h i c h i s commonly a s s o c i a t e d w i t h t r i s o m i e s ( P o l a n d and M i l l e r , 1 973). However, n o t a l l i n s t a n c e s o f G.D. a r e a s s o c i a t e d w i t h abnormal chromosome complements. 38 S i n c e t h e mother i s p h e n o t y p i c a l l y n o r m a l , as i s t h e case i n t h e m a j o r i t y o f DqDq c a r r i e r s (Cohen, 1971) and t h e a b o r t e d specimens a r e b a l a n c e d f o r t h e same t r a n s l o c a t i o n chromosome, i t would seem t h a t t h e r e l a t i o n between mat e r -n a l k a r y o t y p e and/or i t s morphology i s most l i k e l y f o r t u i t o u s . 3. T r a n s l o c a t i o n and r e p r o d u c t i v i t y There i s no s i g n i f i c a n t e v i d e n c e t h a t t h e r e i s an i n c r e a s e i n a b o r t i o n r a t e i n f a m i l i e s c a r r y i n g a R o b e r t s o n i a n t r a n s l o -c a t i o n (Hamerton, 1968; J a c o b s e t a l . , 1970; F r i e d r i c h , 1 9 7 4 ). An i n c r e a s e d i n c i d e n c e o f a b o r t i o n among such t r a n s l o c a t i o n h e t e r o z y g o t e s was r e p o r t e d by Court-Brown (1967) b u t has s i n c e been a t t r i b u t e d t o as s e l e c t i o n a r t i f a c t i n a l i m i t e d s e r i e s by some a u t h o r s (Hamerton, 1968). The f a c t t h a t 50% (14/28) o f t h e t r a n s l o c a t i o n s t h u s f a r r e p o r t e d i n s e r i a l s t u d i e s o f r e c u r r e n t a b o r t i o n a r e R o b e r t s o n i a n , and t h a t i t s f r e q u e n c y i n t h i s p o p u l a t i o n i s more t h a n t e n t i m e s h i g h e r t h a n t h a t f o u n d i n t h e g e n e r a l p o p u l a t i o n , would t e n d t o argue i n f a v o u r o f a r o l e i n a b o r t i o n . Many s p o r a d i c c a s e r e p o r t s f u r t h e r t h i s b e l i e f ( f o r a r e v i e w see Khudr, 1974). S u r v e y s o f f a m i l i e s w i t h a utosomal r e c i p r o c a l t r a n s -l o c a t i o n s a s c e r t a i n e d t h r o u g h s e l e c t e d probands show a s i g n -i f i c a n t i n c r e a s e ( a t t h e 5% l e v e l ) i n t h e p r o p o r t i o n o f p r e g -n a n c i e s r e s u l t i n g i n s t i l l b i r t h s or a b o r t i o n s among t r a n s -l o c a t i o n h e t e r o z y g o t e s ( F o r d and C l e g g , 1969; Ja c o b s e t a l . , 1970; L e j e u n e and D u t r i l l a u x , 1 9 7 0 ) . The o v e r a l l f r e q u e n c y o f 39 a b o r t i o n among t r a n s l o c a t i o n h e t e r o z y g o t e s v a r i e s w i t h t h e f a m i l y and t h e chromosomes i n v o l v e d ( F o r d and C l e g g , 1969). I n f e r t i l i t y has been f r e q u e n t l y r e c o r d e d i n b a l a n c e d t(DqDq) h e t e r o z y g o t e s ( Y u n i s e t a l . , 1964; K j e s s l e r , 1964; W i l s o n , 1971). P o s t - z y g o t i c i n f e r t i l i t y c a u s e d by v e r y e a r l y a b o r t i o n o f i m b a l a n c e d z y g o t e s has been s u g g e s t e d as a p o s s i b i l i t y i n t h e s e i n s t a n c e s ( W i l s o n , 1971; Chandley e t a l . , 1 9 7 2 ). I n such c a s e s , t o t a l i n f e r t i l i t y would n o t be e x p e c t e d assuming t h a t s e g r e g a t i o n a t m e i o s i s i s random. T h i s has been s u p p o r t e d by t h e e x p e r i m e n t a l work o f Gropp (1974) and F o r d and Evans ( 1 9 7 4 ) . U t i l i z i n g s t r u c t u r a l l y h e t e r o z y g o u s m i c e , i n v o l v i n g f u s i o n m e t a c e n t r i c t r a n s l o c a -t i o n s , a n a l o g o u s t o R o b e r t s o n i a n t r a n s l o c a t i o n s , t h e y demon-s t r a t e d t h a t monosomic z y g o t e s were o b s e r v e d o n l y b e f o r e i m p l a n t a t i o n and were a l m o s t c o m p l e t e l y e l i m i n a t e d i n t h e e a r l y p o s t - i m p l a n t a t i o . n p e r i o d , whereas ''trisomy.. s e g r e g a t i o n p r o d u c t s l e d t o d i f f e r e n t t i m e s o f f e t a l d e a t h . There have been r e l a t i v e l y few r e p o r t s o f a b o r t u s e s w i t h u n b a l a n c e d p r o d u c t s o f t r a n s l o c a t i o n s . U n b a l a n c e d R o b e r t -s o n i a n t r a n s l o c a t i o n s i n h e r i t e d from a c a r r i e r p a r e n t have been d e s c r i b e d ( J a c o b s e n e t a l . , 1963; C a r r , 1971b; Boue and Boue, 1973a). K a j i i (1974) r e p o r t e d on t h r e e s u c c e s s i v e a b o r t u s e s , each w i t h an u n b a l a n c e d form o f a s e g r e g a t i n g r e c i p r o c a l t r a n s l o c a t i o n . Boue and Boue (1974b) f o u n d t h r e e a b o r t u s e s w i t h s e g r e g a t i o n p r o d u c t s o f a r e c i p r o c a l t r a n s -l o c a t i o n ; two were due t o a d j a c e n t s e g r e g a t i o n and one was 40 due t o 3:1 s e g r e g a t i o n . O t h e r s have been r e p o r t e d and a r e r e v i e w e d e l s e w h e r e (Lindenbaum and Bobrow, 1975). Thus, v e r y few cause and e f f e c t r e l a t i o n s have been documented between t r a n s l o c a t i o n and a b o r t i o n . I n p r e v i o u s s e r i a l s t u d i e s , o f t e n t h e f e t u s e s a r e assumed t o be chromo-s o m a l l y u n b a l a n c e d and t h e pregnancy l o s s e s c a u s a l l y r e -l a t e d t o t h e p a r e n t a l b a l a n c e d t r a n s l o c a t i o n . O f t e n o t h e r f a m i l y members a r e a l s o i d e n t i f i e d as t r a n s l o c a t i o n h e t e r o -z y g o t e s b u t w i t h no a p p a r e n t l y a s s o c i a t e d r e p r o d u c t i v e p r o -b l e ms. These d i f f e r e n c e s i n f e r t i l i t y may be c o m p l e t e l y f o r t u i t o u s , b u t i t has a l s o been s u g g e s t e d t h a t perhaps o t h e r g e n e t i c f a c t o r s may m o d i f y p o t e n t i a l l y d e l e t e r i o u s e f f e c t s o f t h e h e t e r o z y g o u s s t a t e ( W i l s o n , 1 9 7 1). The r e s u l t s o f t h e p r e s e n t s t u d y and a r e v i e w o f t h e l i t e r a t u r e would s u g g e s t t h e need f o r a more s y s t e m a t i c and c o m p r e h e n s i v e approach t o t h e c y t o g e n e t i c s t u d y o f r e c u r r e n t a b o r t i o n . An e s t a b l i s h e d e l e v a t e d f r e q u e n c y o f t r a n s l o c a t i o n i n t h i s p o p u l a t i o n i s m e a n i n g l e s s u n l e s s d i r e c t c o r r e l a t i o n between t r a n s l o c a t i o n and e f f e c t on r e p r o d u c t i v i t y i s e s t a b l i s h e d by a b o r t u s t i s s u e c u l t u r e d a t a . W i t h b e t t e r q u a l i t y i n f o r m a t i o n b ased on more e x t e n -s i v e e p i d e m i o l o g i c a l d a t a , i t w i l l be p o s s i b l e t o i d e n t i f y subgroups w i t h i n t h i s p o p u l a t i o n a t h i g h r i s k f o r b o t h c y t o g e n e t i c and n o n - c y t o g e n e t i c e t i o l o g i e s o f r e c u r r e n t a b o r t i o n . 41 LITERATURE CITED Alberman, E., E l l i o t t , M., C r e a s y , M., and D h a d i a l , R. 1975. P r e v i o u s r e p r o d u c t i v e h i s t o r y i n mothers p r e s e n t i n g w i t h spontaneous a b o r t i o n s . B r i t . J . o f Ob. and Gyn. May 82:366-373. A r r i g h i , F. E., and Hsu, T. C. 1971. L o c a l i z a t i o n o f h e t e r o c h r o m a t i n i n human chromosomes. C y t o g e n e t i c s 10:81-86. Bh a s i n y M.K., F o e r s t e r , W., and Fuhrmann, W. 1973. A c y t o g e n e t i c s t u d y o f r e c u r r e n t a b o r t i o n . Human-g e n e t i k 18:139-152. Bishum, N. P., and M o r t o n , W. R. M. 1968. Chromosomal s t u d i e s on women w i t h two o r more u n s u c c e s s f u l p r e g n a n c i e s . J . O b s t e t . Gynec. B r . Commonw. 75:66-70 Boue, J . G. and Boue, A. 1973a. Chromosomal a n a l y s i s o f two c o n s e c u t i v e a b o r t u s e s i n each o f 43 women. Humangenetik 19:2 75-280. Boue, J . G. and Boue, A. 1973b. Outcome o f p r e g n a n c i e s f o l l o w i n g a spontaneous a b o r t i o n w i t h chromosomal a n o m a l i e s . Am. J . O b s t e t . Gynec. 116:806-812. Boue, J . G. and Boue, A. 1973c. 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