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Asynchronous appearance of embryonic cardiac activity in multiple pregnancies following in-vitro fertilization
728
Letters to the Editor
A S Y N C H R O N O U S A P P E A R A N C E OF E M B R Y O N I C C A R D I A C A C T I V I T Y IN MULTIPLE PREGNANCIES FOLLOWING IN-vITRo FERTILIZATION
Sir,
INTRODUCTION
I m p l a n t a t i o n is a c o n t i n u i n g process that extends from five or six days up to about 12 days f o l l o w i n g ovulation. The e m b r y o becomes a b l a s t o c y s t on day 4 or 5 a f t e r fertilization. At this s t a g e it is capable after h a t c h i n g of a t t a c h m e n t to the e n d o m e t r i u m and of implantation. In single p r e g n a n c i e s f o l l o w i n g I n - V i t r o F e r t i l i z a t i o n (IVF) a d i f f e r e n c e up to 5 days in the a p p e a r a n c e of e m b r y o n i c heart a c t i v i t y has been d e m o n s t r a t e d (Schats et al. 1988). The factors d e t e r m i n i n g rapid or s l o w i m p l a n t a t i o n and s u b s e q u e n t d e v e l o p m e n t are disputed. M u l t i p l e p r e g n a n c i e s p r o v i d e the o p p o r t u n i t y to s t u d y d i f f e r e n t embryos in the same individual, t h e r e b y s t a n d a r d i z i n g for m a t e r n a l influences. The o b j e c t i v e of the p r e s e n t s t u d y was to e s t a b l i s h a d i f f e r e n c e in the d u r a t i o n of the i m p l a n t a t i o n stage in the h u m a n t h r o u g h d o c u m e n t a t i o n of the first a p p e a r a n c e of e m b r y o n i c c a r d i a c a c t i v i t y in m u l t i p l e pregnancies.
MATERIAL
and METHODS
B e t w e e n S e p t e m b e r 1987 and April 1988 a total of twelve p r e g n a n t s u b j e c t s c o n s e n t e d to p a r t i c i p a t e in the study. There were nine t w i n p r e g n a n c i e s and three triple pregnancies. All arose from IVF treatment, p r o v i d i n g exact k n o w l e d g e of the time of "ovulation" and t r a n s f e r of the embryos and thus of e m b r y o n i c age. O n l y p r e g n a n c i e s w i t h n o r m a l l y d e v e l o p e d fetuses d e t e c t e d by u l t r a s o u n d e x a m i n a t i o n Ii weeks after F o l l i c u l a r A s p i r a t i o n (FA) were included. All p a t i e n t s were s c a n n e d b e t w e e n I0. 00 and 12.00 a.m. using the D i a s o n i c s DRF 400 and the Toshiba SAL 77B t r a n s v a g i n a l probes. The f o r m e r was e q u i p p e d with a 5.0 and a 7.5 MHz m e c h a n i c a l s e c t o r s c a n t r a n s d u c e r and the l a t t e r w i t h a 5.0 MHz e l e c t r o n i c s e c t o r scan probe. F o l l o w i n g l o c a l i z a t i o n of the embryos, a s e a r c h for cardiac a c t i v i t y was made. If h e a r t a c t i v i t y was p r e s e n t the heart rate was d o c u m e n t e d by M-mode in c o n j u n c t i o n w i t h a real time scan. The heart rate was c a l c u l a t e d from the M - m o d e in Beats Per Minute (BPM). D a i l y e x a m i n a t i o n s for cardiac a c t i v i t y were s t a r t e d on day 25 a f t e r FA. This was b a s e d on a p i l o t s t u d y in 23 w o m e n in w h i c h cardiac a c t i v i t y c o u l d not be d e t e c t e d b e f o r e day 25 a f t e r FA. F o l l o w i n g d o c u m e n t a t i o n of first c a r d i a c a c t i v i t y in all c h o r i o n i c sacs w i t h i n the same pregnancy, f u r t h e r e x a m i n a t i o n s of the heart rate and C r o w n - R u m p L e n g t h (CRL) were c a r r i e d out in 2 - 3 day intervals until 42 days a f t e r FA.
Letters to the Editor
729
RESULTS C a r d i a c a c t i v i t y was first d o c u m e n t e d b e t w e e n day 26 and day 29 a f t e r FA (median day 27). The time d i f f e r e n c e in first a p p e a r a n c e of cardiac a c t i v i t y b e t w e e n d i f f e r e n t embryos in one and the same p r e g n a n c y v a r i e d b e t w e e n zero and two days (median I day) (Figure I). A s i g n i f i c a n t increase (p < 0.05 ; W i l c o x o n rank sum test) in CRL d i f f e r e n c e r e l a t i v e to the d i f f e r e n c e in a p p e a r a n c e of e m b r y o n i c cardiac a c t i v i t y was observed, w h e n all m u l t i p l e p r e g n a n c i e s were c o n s i d e r e d t o g e t h e r (Figure 2).
uN'!]
2.5] C ' R L L 21
E R
F
i
t.sq
0 F
R
i,l
F k T I E N T S
E
M 0
1 BFFERENCE IN APPEARANCEOF
!
ol
2
DIFFERENCEIN APPEARANCEOF EMBRYONICCARDIACACTIVITYIN DAYS
EMBRYONRCARDIACACTIV~Y IN DAY8 i g q ( e 1: D f f e r e n c e in a p p e a r a n c e l ~ ~_. are ac . a c t i v i t y in days in pregnancies.
This
is also d e m o n s t r a t e d
emb(yQnic mu/zLpte
in Figure
1TO
~ 150
R 14oi A T 1304 E 120~ I n 1104
t30
I 1211 n 110 100 90
for two embryos
R 150]
R 140
M
3, in w h i c h
to ~he cardiac
1707 H A 160~ E
~ 160
~ E
Figgre 2: pifference in C~L r e l a t i v e dtlTerenc¢ In ~ppea[aGce o l e~bryonlc activity tn m u l t i p l e /VP p r e g n a n c i e s .
pB 1004 i I : ~ i , I f i f , , , , , J , 25 26 27 28 29 30 31 32 33 34 35 38 37 38 39 40 41 42 DAY8 AFTER FOLLICLE ASPIRATION -X- EMBRYO1
90 I 0
~ , i i , , i i , i , , i J , , , t 2 3 4 6 6 7 8 9 10 1112 13 14 15 16 17 CROWN RUMPLENGTHkl MM
-~- EMBRYO2
: i s u r e 3a: Twin IVF pregnancy; to g e s t a t i o n a l age.
Heart
rate
- x - EMBRYO1 relative
ieure 3b: e~rt r a t e .
Twin
IVF
-~- EMBRYO2
pregnancy;
CRL
relative
to
from the same p r e g n a n c y the d i f f e r e n c e in heart rate d e t e r m i n e d by the s e q u e n t i a l onset of cardiac a c t i v i t y (Figure 3a) is m e r e l y r e d u c e d up to day 40 a f t e r FA f o l l o w i n g s t a n d a r d i z a t i o n for CRL (Figure 3b).
730
Letters to the Editor
DISCUSSION Until now it is i m p o s s i b l e to judge, on m o r p h o l o g i c a l grounds alone the q u a l i t y of oocytes after s t i m u l a t i o n and harvesting, and the q u a l i t y of the pre-embryos they produce after f e r t i l i z a t i o n (Acosta et al. 1988). Under i n - v i t r o conditions the d i f f e r e n c e in d e v e l o p m e n t a l potential of p r e - e m b r y o s has b e e n d o c u m e n t e d from the cleavage rate (Edwards et al. 1981, Lopata et al. 1982). This finding cannot be e x t r a p o l a t e d to the in-vivo s i t u a t i o n because of the d i f f e r e n t e n v i r o n m e n t in w h i c h the preembryos develop, in p a r t i c u l a r the q u a l i t y of the endometrium. I m p l a n t a t i o n is the means by which the early embryo establishes a fixed r e l a t i o n s h i p w i t h respect to the uterus. The nature of the i m p l a n t a t i o n process varies c o n s i d e r a b l y b e t w e e n species and may include s u c h events as the activation, s p a c i n g and o r i e n t a t i o n of the blastocyst, a d h e s i o n of the t r o p h o b l a s t to the luminal epithelium, i n v a s i o n of the endometrial stroma and the d e v e l o p m e n t of a decidual response. The successful c o m p l e t i o n of these events involves a complex series of s y n c h r o n i z e d changes in the b l a s t o c y s t and e n d o m e t r i u m under the control of o v a r i a n steroids. The very c o m p l e x i t y of i m p l a n t a t i o n makes this an e x t r e m e l y critical stage of early pregnancy. A m u l t i p l e IVF p r e g n a n c y offers the unique o p p o r t u n i t y to study the d e v e l o p m e n t of two or even three embryos in the same subject, thus s t a n d a r d i z i n g for i n t e r i n d i v i d u a l d i f f e r e n c e s such as m a t e r n a l age, parity, and other e n v i r o n m e n t a l variables. The d i f f e r e n c e in a p p e a r a n c e of cardiac a c t i v i t y b e t w e e n embryos from the same p r e g n a n c y may be d e t e r m i n e d by a s y n c h r o n y in onset of maternal n u t r i e n t supply, a l t h o u g h other factors such as microe n v i r o n m e n t a l d i f f e r e n c e s cannot be ruled out. However, after i m p l a n t a t i o n the growth potential for each of those embryos was v i r t u a l l y the same, as r e f l e c t e d by the increase of CRL and the heart rate. We th~erefore suggest that the d i f f e r e n c e in first a p p e a r a n c e of cardiac a c t i v i t y b e t w e e n embryos from the same p r e g n a n c y may be d e t e r m i n e d by the v a r i a b l e d u r a t i o n of the i m p l a n t a t i o n stage. Yours
Faithfully,
R. Schats, M.D., Dept. Obstetrics & Gynecology, A c a d e m i c Hospital of the Free University, Amsterdam, The Netherlands. C.A.M. Jansen, M.D., Ph.D., Dept. Obstetrics & Gynecology, D i a c o n e s s e n h u i s Voorburg, The Netherlands. J.W. Wladimiroff, M.D., Ph.D., Dept. Obstetrics & Gynecology, A c a d e m i c Hospital Dijkzigt, The Netherlands.
REFERENCES: Acosta, A.A.; Moon, S.Y.; Oehninger, S.; Muasher, S.J. ; Rosenwaks, Z; Matta, J.F. I m p l a n t a t i o n potential of each p r e - e m b r y o in m u l t i p l e pregnancies o b t a i n e d by in vitro f e r t i l i z a t i o n seems to be different. Fertil. Steril. 50: 906911; 1988. Edwards, R.G. ; Purdy, J.M.; Steptoe, P.C. ; Walters, D.E. The g r o w t h of human p r e i m p l a n t a t i o n embryos in vitro. Am. J. Obstet. Gynecol. 141: 408-415; 1981. Lopata, A. ; Martin, M.; Oliva, K.; Johnston, I. E m b r y o n i c d e v e l o p m e n t and b l a s t o c y s t i m p l a n t a t i o n f o l l o w i n g in vitro f e r t i l i z a t i o n and embryo transfer. Fertil. Steril. 38: 682690; 1982. Schats, R. ; Brandsma, G.; Cleveringa, L.M. ; Lankhorst, P.F.C. ; Vroegop, I.S. ; J a n s e n C.A.M. D e t e c t i o n and m o n i t o r i n g of e m b r y o n i c heart a c t i v i t y in early pregnancy. Hum. Reprod. 3: Suppl. 78 (Abstract); 1988.
Letters to the Editor
A S Y N C H R O N O U S A P P E A R A N C E OF E M B R Y O N I C C A R D I A C A C T I V I T Y IN MULTIPLE PREGNANCIES FOLLOWING IN-vITRo FERTILIZATION
Sir,
INTRODUCTION
I m p l a n t a t i o n is a c o n t i n u i n g process that extends from five or six days up to about 12 days f o l l o w i n g ovulation. The e m b r y o becomes a b l a s t o c y s t on day 4 or 5 a f t e r fertilization. At this s t a g e it is capable after h a t c h i n g of a t t a c h m e n t to the e n d o m e t r i u m and of implantation. In single p r e g n a n c i e s f o l l o w i n g I n - V i t r o F e r t i l i z a t i o n (IVF) a d i f f e r e n c e up to 5 days in the a p p e a r a n c e of e m b r y o n i c heart a c t i v i t y has been d e m o n s t r a t e d (Schats et al. 1988). The factors d e t e r m i n i n g rapid or s l o w i m p l a n t a t i o n and s u b s e q u e n t d e v e l o p m e n t are disputed. M u l t i p l e p r e g n a n c i e s p r o v i d e the o p p o r t u n i t y to s t u d y d i f f e r e n t embryos in the same individual, t h e r e b y s t a n d a r d i z i n g for m a t e r n a l influences. The o b j e c t i v e of the p r e s e n t s t u d y was to e s t a b l i s h a d i f f e r e n c e in the d u r a t i o n of the i m p l a n t a t i o n stage in the h u m a n t h r o u g h d o c u m e n t a t i o n of the first a p p e a r a n c e of e m b r y o n i c c a r d i a c a c t i v i t y in m u l t i p l e pregnancies.
MATERIAL
and METHODS
B e t w e e n S e p t e m b e r 1987 and April 1988 a total of twelve p r e g n a n t s u b j e c t s c o n s e n t e d to p a r t i c i p a t e in the study. There were nine t w i n p r e g n a n c i e s and three triple pregnancies. All arose from IVF treatment, p r o v i d i n g exact k n o w l e d g e of the time of "ovulation" and t r a n s f e r of the embryos and thus of e m b r y o n i c age. O n l y p r e g n a n c i e s w i t h n o r m a l l y d e v e l o p e d fetuses d e t e c t e d by u l t r a s o u n d e x a m i n a t i o n Ii weeks after F o l l i c u l a r A s p i r a t i o n (FA) were included. All p a t i e n t s were s c a n n e d b e t w e e n I0. 00 and 12.00 a.m. using the D i a s o n i c s DRF 400 and the Toshiba SAL 77B t r a n s v a g i n a l probes. The f o r m e r was e q u i p p e d with a 5.0 and a 7.5 MHz m e c h a n i c a l s e c t o r s c a n t r a n s d u c e r and the l a t t e r w i t h a 5.0 MHz e l e c t r o n i c s e c t o r scan probe. F o l l o w i n g l o c a l i z a t i o n of the embryos, a s e a r c h for cardiac a c t i v i t y was made. If h e a r t a c t i v i t y was p r e s e n t the heart rate was d o c u m e n t e d by M-mode in c o n j u n c t i o n w i t h a real time scan. The heart rate was c a l c u l a t e d from the M - m o d e in Beats Per Minute (BPM). D a i l y e x a m i n a t i o n s for cardiac a c t i v i t y were s t a r t e d on day 25 a f t e r FA. This was b a s e d on a p i l o t s t u d y in 23 w o m e n in w h i c h cardiac a c t i v i t y c o u l d not be d e t e c t e d b e f o r e day 25 a f t e r FA. F o l l o w i n g d o c u m e n t a t i o n of first c a r d i a c a c t i v i t y in all c h o r i o n i c sacs w i t h i n the same pregnancy, f u r t h e r e x a m i n a t i o n s of the heart rate and C r o w n - R u m p L e n g t h (CRL) were c a r r i e d out in 2 - 3 day intervals until 42 days a f t e r FA.
Letters to the Editor
729
RESULTS C a r d i a c a c t i v i t y was first d o c u m e n t e d b e t w e e n day 26 and day 29 a f t e r FA (median day 27). The time d i f f e r e n c e in first a p p e a r a n c e of cardiac a c t i v i t y b e t w e e n d i f f e r e n t embryos in one and the same p r e g n a n c y v a r i e d b e t w e e n zero and two days (median I day) (Figure I). A s i g n i f i c a n t increase (p < 0.05 ; W i l c o x o n rank sum test) in CRL d i f f e r e n c e r e l a t i v e to the d i f f e r e n c e in a p p e a r a n c e of e m b r y o n i c cardiac a c t i v i t y was observed, w h e n all m u l t i p l e p r e g n a n c i e s were c o n s i d e r e d t o g e t h e r (Figure 2).
uN'!]
2.5] C ' R L L 21
E R
F
i
t.sq
0 F
R
i,l
F k T I E N T S
E
M 0
1 BFFERENCE IN APPEARANCEOF
!
ol
2
DIFFERENCEIN APPEARANCEOF EMBRYONICCARDIACACTIVITYIN DAYS
EMBRYONRCARDIACACTIV~Y IN DAY8 i g q ( e 1: D f f e r e n c e in a p p e a r a n c e l ~ ~_. are ac . a c t i v i t y in days in pregnancies.
This
is also d e m o n s t r a t e d
emb(yQnic mu/zLpte
in Figure
1TO
~ 150
R 14oi A T 1304 E 120~ I n 1104
t30
I 1211 n 110 100 90
for two embryos
R 150]
R 140
M
3, in w h i c h
to ~he cardiac
1707 H A 160~ E
~ 160
~ E
Figgre 2: pifference in C~L r e l a t i v e dtlTerenc¢ In ~ppea[aGce o l e~bryonlc activity tn m u l t i p l e /VP p r e g n a n c i e s .
pB 1004 i I : ~ i , I f i f , , , , , J , 25 26 27 28 29 30 31 32 33 34 35 38 37 38 39 40 41 42 DAY8 AFTER FOLLICLE ASPIRATION -X- EMBRYO1
90 I 0
~ , i i , , i i , i , , i J , , , t 2 3 4 6 6 7 8 9 10 1112 13 14 15 16 17 CROWN RUMPLENGTHkl MM
-~- EMBRYO2
: i s u r e 3a: Twin IVF pregnancy; to g e s t a t i o n a l age.
Heart
rate
- x - EMBRYO1 relative
ieure 3b: e~rt r a t e .
Twin
IVF
-~- EMBRYO2
pregnancy;
CRL
relative
to
from the same p r e g n a n c y the d i f f e r e n c e in heart rate d e t e r m i n e d by the s e q u e n t i a l onset of cardiac a c t i v i t y (Figure 3a) is m e r e l y r e d u c e d up to day 40 a f t e r FA f o l l o w i n g s t a n d a r d i z a t i o n for CRL (Figure 3b).
730
Letters to the Editor
DISCUSSION Until now it is i m p o s s i b l e to judge, on m o r p h o l o g i c a l grounds alone the q u a l i t y of oocytes after s t i m u l a t i o n and harvesting, and the q u a l i t y of the pre-embryos they produce after f e r t i l i z a t i o n (Acosta et al. 1988). Under i n - v i t r o conditions the d i f f e r e n c e in d e v e l o p m e n t a l potential of p r e - e m b r y o s has b e e n d o c u m e n t e d from the cleavage rate (Edwards et al. 1981, Lopata et al. 1982). This finding cannot be e x t r a p o l a t e d to the in-vivo s i t u a t i o n because of the d i f f e r e n t e n v i r o n m e n t in w h i c h the preembryos develop, in p a r t i c u l a r the q u a l i t y of the endometrium. I m p l a n t a t i o n is the means by which the early embryo establishes a fixed r e l a t i o n s h i p w i t h respect to the uterus. The nature of the i m p l a n t a t i o n process varies c o n s i d e r a b l y b e t w e e n species and may include s u c h events as the activation, s p a c i n g and o r i e n t a t i o n of the blastocyst, a d h e s i o n of the t r o p h o b l a s t to the luminal epithelium, i n v a s i o n of the endometrial stroma and the d e v e l o p m e n t of a decidual response. The successful c o m p l e t i o n of these events involves a complex series of s y n c h r o n i z e d changes in the b l a s t o c y s t and e n d o m e t r i u m under the control of o v a r i a n steroids. The very c o m p l e x i t y of i m p l a n t a t i o n makes this an e x t r e m e l y critical stage of early pregnancy. A m u l t i p l e IVF p r e g n a n c y offers the unique o p p o r t u n i t y to study the d e v e l o p m e n t of two or even three embryos in the same subject, thus s t a n d a r d i z i n g for i n t e r i n d i v i d u a l d i f f e r e n c e s such as m a t e r n a l age, parity, and other e n v i r o n m e n t a l variables. The d i f f e r e n c e in a p p e a r a n c e of cardiac a c t i v i t y b e t w e e n embryos from the same p r e g n a n c y may be d e t e r m i n e d by a s y n c h r o n y in onset of maternal n u t r i e n t supply, a l t h o u g h other factors such as microe n v i r o n m e n t a l d i f f e r e n c e s cannot be ruled out. However, after i m p l a n t a t i o n the growth potential for each of those embryos was v i r t u a l l y the same, as r e f l e c t e d by the increase of CRL and the heart rate. We th~erefore suggest that the d i f f e r e n c e in first a p p e a r a n c e of cardiac a c t i v i t y b e t w e e n embryos from the same p r e g n a n c y may be d e t e r m i n e d by the v a r i a b l e d u r a t i o n of the i m p l a n t a t i o n stage. Yours
Faithfully,
R. Schats, M.D., Dept. Obstetrics & Gynecology, A c a d e m i c Hospital of the Free University, Amsterdam, The Netherlands. C.A.M. Jansen, M.D., Ph.D., Dept. Obstetrics & Gynecology, D i a c o n e s s e n h u i s Voorburg, The Netherlands. J.W. Wladimiroff, M.D., Ph.D., Dept. Obstetrics & Gynecology, A c a d e m i c Hospital Dijkzigt, The Netherlands.
REFERENCES: Acosta, A.A.; Moon, S.Y.; Oehninger, S.; Muasher, S.J. ; Rosenwaks, Z; Matta, J.F. I m p l a n t a t i o n potential of each p r e - e m b r y o in m u l t i p l e pregnancies o b t a i n e d by in vitro f e r t i l i z a t i o n seems to be different. Fertil. Steril. 50: 906911; 1988. Edwards, R.G. ; Purdy, J.M.; Steptoe, P.C. ; Walters, D.E. The g r o w t h of human p r e i m p l a n t a t i o n embryos in vitro. Am. J. Obstet. Gynecol. 141: 408-415; 1981. Lopata, A. ; Martin, M.; Oliva, K.; Johnston, I. E m b r y o n i c d e v e l o p m e n t and b l a s t o c y s t i m p l a n t a t i o n f o l l o w i n g in vitro f e r t i l i z a t i o n and embryo transfer. Fertil. Steril. 38: 682690; 1982. Schats, R. ; Brandsma, G.; Cleveringa, L.M. ; Lankhorst, P.F.C. ; Vroegop, I.S. ; J a n s e n C.A.M. D e t e c t i o n and m o n i t o r i n g of e m b r y o n i c heart a c t i v i t y in early pregnancy. Hum. Reprod. 3: Suppl. 78 (Abstract); 1988.