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Comparison of 3-kit methods for determination of human chorionic somatomammotropin by radioimmunoassay
Clin. Biochem. 8, 229-233 (1975)
COMPARISON OF 3-KIT METHODS FOR DETERMINATION OF HUMAN CHORIONIC SOMATOMAMMOTROPIN B Y RADIOIMMUNOASSAY V. A. LAXDAL, J. A. TREW and T. B. MacLACHLAN*
Depar~nent of Pathology, Univevslty Hospital, Saskatoon, Saskatchewaet. (Accepted February 1, 1975)
CLBIA, 8, (4) 229-233 (1975)
Clin. Bioch6m. Laxdal, V. A., Trew, J. A. and MacLachlan, T. B.
Department of PafhoIogy, Univ6rsity Hospital, Saskatoon, Saskatch6wan. COMPARISON OF 3-KIT METHO.DS FOR DETERMINATION OF HUMAN CHORIONIC SOMATOMAM.MOTROPIN BY RADIOIMMUNOASSAY 1. Three commercially available kit procedures for human chorionic somatomammotropin (HCS) were investigated. 2. The comparison of the methods included procedure, accuracy, precision, sample requirement and time required for analyses. 3. While one kit gave significantly higher values and requires more time to obtain results, each of 3 procedures was concluded to provide clinically useful information.
IT HAS BEEN KNOWN FOR MANY YEARS that the placenta of several m a m m a l i a n species synthesizes and releases a hormone with lactogenic proper-
ties. Ito and Higashi 1 in 1961 isolated a protein from the human placenta which had prolactin activity. Josimovich and co-workers e,s and Kaplan and Grumbach~ verified the placental origin of the hormone and demonstrated its presence in placental extract, uteroplacental blood and peripheral serum from pregnant women. Sciarra and co-workers 5 using immunofluorescent techniques demonstrated that HCS was synthesized by the syncytiotrophoblast and was located in the cytoplasm of the syncytiotrophoblastic villus epithelium. Kaplan and Grumbach 6 showed that the hormone was secreted primarily into the maternal circulation and only small amounts were found in umbilical cord blood. This hormone has been known by a variety of names such as: Human Placental Lactogen (HPL), Purified Placental Protein, Chorionic Growth
*Department of Obstetrics & Gynecology, University Hospital, Saskatoon. Correspondence: Dr. V. A. Laxdal, Department of Pathology, University Hospital, Saskatoon, Saskatchewan.
230
LAXDAL et al
H o r m o n e P r o l a c t i n , as well as H u m a n (HCS:).
Chorionic" S o m a t o m a m m o t r o p i n
Spellacy a n d a s s o c i a t e s 7 s u g g e s t e d t h a t t h e level of H C S w o u l d b e a s e n s i t i v e a n d p r a c t i c a l i n d i c a t o r o f p l a c e n t a l f u n c t i o n a n d u s e f u l in t h e m a n a g e m e n t of C e r t a i n c o m p l i c a t e d p r e g n a n c i e s . L e t c h w o r t h a n d C h a r d s p r o p o s e d t h a t it w o u l d be f e a s i b l e to p e r f o r m t I C S a s s a y s as a s c r e e n i n g t e s t on all p r e g n a n t w o m e n a t e v e r y a n t e n a t a l visit. N i v e n a n d c o - w o r k e r s 9 s h o w e d t h a t t h e H C S t e s t in e a r l y p r e g n a n c y w a s a u s e f u l guide to t h e o u t c o m e of t h r e a t e n e d a b o r t i o n . Clinical s t u d i e s b y v a r i o u s i n v e s t i g a t o r s 19-1.5 h a v e d e m o n s t r a t e d a w i d e v a r i e t y o f n o r m a l values. T h i s p r o b l e m b e c a m e a p p a r e n t in o u r o w n clinical studies when using a kit procedure (the S c h w a r z / M a n n HCS kit). It w a s o b s e r v e d t h a t o u r values w e r e d e f i n i t e l y h i g h e r t h a n t h o s e r e p o r t e d b y others. A t t h e completion of o u r clinical studies, 2 o t h e r k i t s b e c a m e c o m m e r c i a l l y available f o r d e t e r m i n a t i o n o f H C ~ . T h e f o l l o w i n g is a r e p o r t o f t h e c o m p a r i s o n o f t h e 3 H C S k i t s w i t h r e s p e c t to reliability, p r e c i s i o n , a n d t i m e r e q u i r e d f o r a n a l y s i s u s i n g clinically a v a i l a b l e m a t e r i a l .
MATERIALS AND METHODS
Forty-three patients in the third trimester of pregnancy and classified as high-risk were studied. Four of the pregnancies resulted in intrauterine fetal deabh. Heparinized blood samples were ~btained from fasting subjects and the plasma stored at-2(~" until assayed. The following HCS kits were compared: 1. Human Placental Lactogen Radioimmunoassay Kit, Schwarz/Mann, Division of Beeten Dickinson and Co. The kit contains HPL-'nI, HPL antiserum, precipitating antiserum and HPL standard, supplied as lyophilized material. Analysis is performed on an aliquot of a 1:25 dilution of plasma in 0.04 M phosphate buffer with albumin and 0.02 M EDTA pH 7.4. The kit uses a precipitating antibody (anti-rabbit serum) to separate bound from free HCS. 2. HPL Immunoassay Kit, Amersham/Searle Corp., An activity of G. D. Searle & Co. The kit contains HP.L-'nI, rabbit anti-HPL serum, reference standards of HPL in serum (1,3,6,10 ~g/m|) and buffer supplied as lyophilized material. Separation of bound from free HPL is accomplished by alcohol precipitation. 3. Phadebas@ HCS Test Pharmaeia AB, Uppsala, Sweden. The kit contains Anti.HCS serum, HCS standard (16 ~g/ml), HCS-1nI, HCS free diluent and buffer substance, supplied as lyophilized material. Separation of bound from free HCS is accomplished using alcohol precipitation. Reagents and standards were reconstituted ~ccording to the manufacturers' instructions. Analyses were performed in duplicate and carried out as described by the manufacturer, except with the Schwarz/Mann kit where 20 ~1. of a 1 : 2 5 dilution was used instead of 10 ~1. of the above dilution.
HCS by RIA
231
TABLE 1 A COMPARISONOF THE ANALYTICALTECHNIQUES r
Test Kit
Anti-HCS Incubation
Sample (Volume)
Separation of Bound HCS
Fraction Counted
Test per Kit
Analytical (Delay) Time
Schwarz/ Mann
20 ~1. of 1:25 dilution
3 hr. @ 37 °
Antiserum 16-20 hr. @ 4°
Precipitate
200
20 hr.
Amersham/ Searle
50 td.
30 rain. @ room temp.
Alcohol Precipitation
Precipitate
50
1.5 hr.
Phadebas~)
i00 td.
60 min. @ roow temp.
Alcohol Precipitate Precipitation
50
2 hr.
TABLE 2 PRECISIONOF THE THREEMETHODSFOR HCS HC~ Concentration Mean . . . . . . . . . . . . . . . . . . . . . . S.E.M . . . . . . . . . . . . . . . . . . . . . . S.D. between Pairs . . . . . . . . .
Schwarz/Mann
Amersham/Searle
7.6 tLg/ml. 0.43 ~g/ml. 0.74 ~g/ml.
5.9 ttg/ml. 0.32 ~g/ml. 0.34 tLg/ml.
Phadebas® 5.2 tLg/ml. 0.25 ~g/ml. 0.51 t~g/ml.
Table 1 shows a comparison of these analytical techniques. Prior to comparison studies, practice runs were performed so that the analyst was completely familiar with the assay procedures. Analyses were conducted by the same analyst on the same day except where a second day was required to complete the test as with the Schwarz/Mann kit. 12ram x 75ram polypropylene tubes (Falcon Plastics #~053 DiD. of Becton, Dickinson Co.) were used for the assays. Radioactive counting of the bound HCS-125I was performed with a Nuclear Chicago Gamma Counting system Model. RESULTS T h e r e s u l t s o f t h e c o m p a r a t i v e s t u d i e s a r e s h o w n in T a b l e 2 a n d F i g . 1. The Schwarz/Mann procedure yielded values that were approximately 2 /~g/ml h i g h e r t h a n t h e o t h e r p r o c e d u r e s . T h e p a i r e d t - t e s t s h o w e d t h a t there was a significant difference between the 3 methods. The Amersham/Searle kit demonstrated the best precision as shown by the lower standard deviation between duplicate analyses. DISCUSSION T h e S c h w a r z / M a n n p r o c e d u r e u s e s a s e c o n d a n t i b o d y to s e p a r a t e b o u n d f r o m u n b o u n d H C S w h e r e a s t h e o t h e r m e t h o d s u s e alcohol p r e c i p i t a t i o n . T h i s a d d e d s p e c i f i c i t y c o u l d r e s u l t in h i g h e r levels, p a r t i c u l a r l y i f t h e
232
L A X D A L e t ~l
=L
12.0
U) U
Y= 0.84 + 1.145X
-l-
z
8.0
r= 0.87
4.0
V" 7.57
t !
N r,-
. ~.87
P < 0.001
PAIRED t - T E S T z
0
tn
I
I
I
I
4.0
8.0
12.0
16,0
AMERSHAM / SEARLE
HCSj # g / m l
'2.ol 1ira
..j"Y:
v) 8.0
".!
U Z
• . ~e
m 4.0 tU a < T O. 0
.
"
,,,÷ o e,, .
~e •
o.84
~,~Xl 5 . 8 7
"
•
5.21
I~IRED t - - T E S T I
4.0
I
8.0
,I
12.0
AMERSHAM / SEARLE
P < 0.001
I
16.0 HCS~ ~ g / m l
12.0 ¢D (/1
8.0
< m
4.0
; . ~ 1 . 2 0 + 0.53X • ."~."" r_- 0.88 7.
7
0
". • "
0
PAIRED f - - T E S T I
I
I
4.0
8.0
12.0
SCHWARZ / MANN
P < 0.001
I
16.0 HCS; # g / m l
F i g . 1. - - C o m p a r i s o = o f r e s u l t s o f H C S a s s a y s b y t h r e e m e t h o d s .
alcohol failed to completely precipitate all of the bound material. On the other hand, there is always the danger that some of the labelled HCS could be split from the HCS antisera during alcohol precipitation. This could cause false high values. There is no reference standard preparation of HCS which could result in antisera being produced with differing sensitivities and specificities. This may also explain some of the differences observed with different procedures.
HCS by RIA
233
When comparing the number of steps involved, as well as the total analytical time, the Amersham/Searle or the Phadebas procedures are to be preferred. These methods are sufficiently sensitive to measure HCS levels and to sense minute changes in concentration during the entire pregnancy. In spite of the higher values shown by the Schwarz/Mann procedure, this test was equally capable of predicting severe fetal distress or fetal death provided that normal ranges were well established for each week of gestation. ACKNOWLEDGEMENTS
Phadebas HCS Test was kindly supplied by Pharmacia Ltd., Montreal, Canada. HPL Immunoassay Kit was kindly supplied by Amersham/Searle Corp., Oakville, Ontario.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Ito, Y. and Higashi, K , (1961). Endocrinol. Jap., 8: 279. J0simovich, J. B., MacLaren, J. A., (1962). Endocrinol., 71: 209. Josimovich, J. B., Atwood, B.L., Goss, D. (1963). Endocrinol., 73: 410. Kaplan, S. L., Grumbach, M. M. (1964). J. Clin. End. Met., 25: 80. Sciarra J. J., Kaplan, S. L., Grumbach, M. M. (1963). Nature (London), 199: 1005. Kaplan, S. L., Grumbach, M. M. (1965). J. Clan. End. Met., 25: 1370. Spellacy, W., Cohen, B., Carlson, K. L. (1967). Am. J. Obstet. Gyn¢col., 97: 560. Letchworth, A. T., Chard, T. (1972). Th6 Lancet, pp. 704-706. Niven, P.A.R., Landon, J., Chard, T. (1972). Br. Med. J., 3: 799-801. Varma, K., Drisco]l, S., Emerson, K., Selenkow, H. (1971). Obs~e~. Crynecol., 38: 487. Spellacy, W., Teoh, E. S., Buhi, W., Berk, S., McCreary, S. (1971). Am. J. Obst6t. Gynecol., 109: 588. Genazzani, A. R., Casoli, M., Aubert, M., Fioretti, P. (1969). Lancet 2: 1385. Singer, W., Desjardin, P., Friesen, H. (1970). Obstet. Cryne¢ol., 36: 222. Saxena, B. N., Emerson, K., Selenkow, H. (1969). N. Eng. J. Med., 281: 225. Josimovieh, J. B., Kosor, B., Bocce-la, L., Mintz, D., Hutchinson, L. (1970). ObsteL Gyne¢ol., 36 : 244.
COMPARISON OF 3-KIT METHODS FOR DETERMINATION OF HUMAN CHORIONIC SOMATOMAMMOTROPIN B Y RADIOIMMUNOASSAY V. A. LAXDAL, J. A. TREW and T. B. MacLACHLAN*
Depar~nent of Pathology, Univevslty Hospital, Saskatoon, Saskatchewaet. (Accepted February 1, 1975)
CLBIA, 8, (4) 229-233 (1975)
Clin. Bioch6m. Laxdal, V. A., Trew, J. A. and MacLachlan, T. B.
Department of PafhoIogy, Univ6rsity Hospital, Saskatoon, Saskatch6wan. COMPARISON OF 3-KIT METHO.DS FOR DETERMINATION OF HUMAN CHORIONIC SOMATOMAM.MOTROPIN BY RADIOIMMUNOASSAY 1. Three commercially available kit procedures for human chorionic somatomammotropin (HCS) were investigated. 2. The comparison of the methods included procedure, accuracy, precision, sample requirement and time required for analyses. 3. While one kit gave significantly higher values and requires more time to obtain results, each of 3 procedures was concluded to provide clinically useful information.
IT HAS BEEN KNOWN FOR MANY YEARS that the placenta of several m a m m a l i a n species synthesizes and releases a hormone with lactogenic proper-
ties. Ito and Higashi 1 in 1961 isolated a protein from the human placenta which had prolactin activity. Josimovich and co-workers e,s and Kaplan and Grumbach~ verified the placental origin of the hormone and demonstrated its presence in placental extract, uteroplacental blood and peripheral serum from pregnant women. Sciarra and co-workers 5 using immunofluorescent techniques demonstrated that HCS was synthesized by the syncytiotrophoblast and was located in the cytoplasm of the syncytiotrophoblastic villus epithelium. Kaplan and Grumbach 6 showed that the hormone was secreted primarily into the maternal circulation and only small amounts were found in umbilical cord blood. This hormone has been known by a variety of names such as: Human Placental Lactogen (HPL), Purified Placental Protein, Chorionic Growth
*Department of Obstetrics & Gynecology, University Hospital, Saskatoon. Correspondence: Dr. V. A. Laxdal, Department of Pathology, University Hospital, Saskatoon, Saskatchewan.
230
LAXDAL et al
H o r m o n e P r o l a c t i n , as well as H u m a n (HCS:).
Chorionic" S o m a t o m a m m o t r o p i n
Spellacy a n d a s s o c i a t e s 7 s u g g e s t e d t h a t t h e level of H C S w o u l d b e a s e n s i t i v e a n d p r a c t i c a l i n d i c a t o r o f p l a c e n t a l f u n c t i o n a n d u s e f u l in t h e m a n a g e m e n t of C e r t a i n c o m p l i c a t e d p r e g n a n c i e s . L e t c h w o r t h a n d C h a r d s p r o p o s e d t h a t it w o u l d be f e a s i b l e to p e r f o r m t I C S a s s a y s as a s c r e e n i n g t e s t on all p r e g n a n t w o m e n a t e v e r y a n t e n a t a l visit. N i v e n a n d c o - w o r k e r s 9 s h o w e d t h a t t h e H C S t e s t in e a r l y p r e g n a n c y w a s a u s e f u l guide to t h e o u t c o m e of t h r e a t e n e d a b o r t i o n . Clinical s t u d i e s b y v a r i o u s i n v e s t i g a t o r s 19-1.5 h a v e d e m o n s t r a t e d a w i d e v a r i e t y o f n o r m a l values. T h i s p r o b l e m b e c a m e a p p a r e n t in o u r o w n clinical studies when using a kit procedure (the S c h w a r z / M a n n HCS kit). It w a s o b s e r v e d t h a t o u r values w e r e d e f i n i t e l y h i g h e r t h a n t h o s e r e p o r t e d b y others. A t t h e completion of o u r clinical studies, 2 o t h e r k i t s b e c a m e c o m m e r c i a l l y available f o r d e t e r m i n a t i o n o f H C ~ . T h e f o l l o w i n g is a r e p o r t o f t h e c o m p a r i s o n o f t h e 3 H C S k i t s w i t h r e s p e c t to reliability, p r e c i s i o n , a n d t i m e r e q u i r e d f o r a n a l y s i s u s i n g clinically a v a i l a b l e m a t e r i a l .
MATERIALS AND METHODS
Forty-three patients in the third trimester of pregnancy and classified as high-risk were studied. Four of the pregnancies resulted in intrauterine fetal deabh. Heparinized blood samples were ~btained from fasting subjects and the plasma stored at-2(~" until assayed. The following HCS kits were compared: 1. Human Placental Lactogen Radioimmunoassay Kit, Schwarz/Mann, Division of Beeten Dickinson and Co. The kit contains HPL-'nI, HPL antiserum, precipitating antiserum and HPL standard, supplied as lyophilized material. Analysis is performed on an aliquot of a 1:25 dilution of plasma in 0.04 M phosphate buffer with albumin and 0.02 M EDTA pH 7.4. The kit uses a precipitating antibody (anti-rabbit serum) to separate bound from free HCS. 2. HPL Immunoassay Kit, Amersham/Searle Corp., An activity of G. D. Searle & Co. The kit contains HP.L-'nI, rabbit anti-HPL serum, reference standards of HPL in serum (1,3,6,10 ~g/m|) and buffer supplied as lyophilized material. Separation of bound from free HPL is accomplished by alcohol precipitation. 3. Phadebas@ HCS Test Pharmaeia AB, Uppsala, Sweden. The kit contains Anti.HCS serum, HCS standard (16 ~g/ml), HCS-1nI, HCS free diluent and buffer substance, supplied as lyophilized material. Separation of bound from free HCS is accomplished using alcohol precipitation. Reagents and standards were reconstituted ~ccording to the manufacturers' instructions. Analyses were performed in duplicate and carried out as described by the manufacturer, except with the Schwarz/Mann kit where 20 ~1. of a 1 : 2 5 dilution was used instead of 10 ~1. of the above dilution.
HCS by RIA
231
TABLE 1 A COMPARISONOF THE ANALYTICALTECHNIQUES r
Test Kit
Anti-HCS Incubation
Sample (Volume)
Separation of Bound HCS
Fraction Counted
Test per Kit
Analytical (Delay) Time
Schwarz/ Mann
20 ~1. of 1:25 dilution
3 hr. @ 37 °
Antiserum 16-20 hr. @ 4°
Precipitate
200
20 hr.
Amersham/ Searle
50 td.
30 rain. @ room temp.
Alcohol Precipitation
Precipitate
50
1.5 hr.
Phadebas~)
i00 td.
60 min. @ roow temp.
Alcohol Precipitate Precipitation
50
2 hr.
TABLE 2 PRECISIONOF THE THREEMETHODSFOR HCS HC~ Concentration Mean . . . . . . . . . . . . . . . . . . . . . . S.E.M . . . . . . . . . . . . . . . . . . . . . . S.D. between Pairs . . . . . . . . .
Schwarz/Mann
Amersham/Searle
7.6 tLg/ml. 0.43 ~g/ml. 0.74 ~g/ml.
5.9 ttg/ml. 0.32 ~g/ml. 0.34 tLg/ml.
Phadebas® 5.2 tLg/ml. 0.25 ~g/ml. 0.51 t~g/ml.
Table 1 shows a comparison of these analytical techniques. Prior to comparison studies, practice runs were performed so that the analyst was completely familiar with the assay procedures. Analyses were conducted by the same analyst on the same day except where a second day was required to complete the test as with the Schwarz/Mann kit. 12ram x 75ram polypropylene tubes (Falcon Plastics #~053 DiD. of Becton, Dickinson Co.) were used for the assays. Radioactive counting of the bound HCS-125I was performed with a Nuclear Chicago Gamma Counting system Model. RESULTS T h e r e s u l t s o f t h e c o m p a r a t i v e s t u d i e s a r e s h o w n in T a b l e 2 a n d F i g . 1. The Schwarz/Mann procedure yielded values that were approximately 2 /~g/ml h i g h e r t h a n t h e o t h e r p r o c e d u r e s . T h e p a i r e d t - t e s t s h o w e d t h a t there was a significant difference between the 3 methods. The Amersham/Searle kit demonstrated the best precision as shown by the lower standard deviation between duplicate analyses. DISCUSSION T h e S c h w a r z / M a n n p r o c e d u r e u s e s a s e c o n d a n t i b o d y to s e p a r a t e b o u n d f r o m u n b o u n d H C S w h e r e a s t h e o t h e r m e t h o d s u s e alcohol p r e c i p i t a t i o n . T h i s a d d e d s p e c i f i c i t y c o u l d r e s u l t in h i g h e r levels, p a r t i c u l a r l y i f t h e
232
L A X D A L e t ~l
=L
12.0
U) U
Y= 0.84 + 1.145X
-l-
z
8.0
r= 0.87
4.0
V" 7.57
t !
N r,-
. ~.87
P < 0.001
PAIRED t - T E S T z
0
tn
I
I
I
I
4.0
8.0
12.0
16,0
AMERSHAM / SEARLE
HCSj # g / m l
'2.ol 1ira
..j"Y:
v) 8.0
".!
U Z
• . ~e
m 4.0 tU a < T O. 0
.
"
,,,÷ o e,, .
~e •
o.84
~,~Xl 5 . 8 7
"
•
5.21
I~IRED t - - T E S T I
4.0
I
8.0
,I
12.0
AMERSHAM / SEARLE
P < 0.001
I
16.0 HCS~ ~ g / m l
12.0 ¢D (/1
8.0
< m
4.0
; . ~ 1 . 2 0 + 0.53X • ."~."" r_- 0.88 7.
7
0
". • "
0
PAIRED f - - T E S T I
I
I
4.0
8.0
12.0
SCHWARZ / MANN
P < 0.001
I
16.0 HCS; # g / m l
F i g . 1. - - C o m p a r i s o = o f r e s u l t s o f H C S a s s a y s b y t h r e e m e t h o d s .
alcohol failed to completely precipitate all of the bound material. On the other hand, there is always the danger that some of the labelled HCS could be split from the HCS antisera during alcohol precipitation. This could cause false high values. There is no reference standard preparation of HCS which could result in antisera being produced with differing sensitivities and specificities. This may also explain some of the differences observed with different procedures.
HCS by RIA
233
When comparing the number of steps involved, as well as the total analytical time, the Amersham/Searle or the Phadebas procedures are to be preferred. These methods are sufficiently sensitive to measure HCS levels and to sense minute changes in concentration during the entire pregnancy. In spite of the higher values shown by the Schwarz/Mann procedure, this test was equally capable of predicting severe fetal distress or fetal death provided that normal ranges were well established for each week of gestation. ACKNOWLEDGEMENTS
Phadebas HCS Test was kindly supplied by Pharmacia Ltd., Montreal, Canada. HPL Immunoassay Kit was kindly supplied by Amersham/Searle Corp., Oakville, Ontario.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Ito, Y. and Higashi, K , (1961). Endocrinol. Jap., 8: 279. J0simovich, J. B., MacLaren, J. A., (1962). Endocrinol., 71: 209. Josimovich, J. B., Atwood, B.L., Goss, D. (1963). Endocrinol., 73: 410. Kaplan, S. L., Grumbach, M. M. (1964). J. Clin. End. Met., 25: 80. Sciarra J. J., Kaplan, S. L., Grumbach, M. M. (1963). Nature (London), 199: 1005. Kaplan, S. L., Grumbach, M. M. (1965). J. Clan. End. Met., 25: 1370. Spellacy, W., Cohen, B., Carlson, K. L. (1967). Am. J. Obstet. Gyn¢col., 97: 560. Letchworth, A. T., Chard, T. (1972). Th6 Lancet, pp. 704-706. Niven, P.A.R., Landon, J., Chard, T. (1972). Br. Med. J., 3: 799-801. Varma, K., Drisco]l, S., Emerson, K., Selenkow, H. (1971). Obs~e~. Crynecol., 38: 487. Spellacy, W., Teoh, E. S., Buhi, W., Berk, S., McCreary, S. (1971). Am. J. Obst6t. Gynecol., 109: 588. Genazzani, A. R., Casoli, M., Aubert, M., Fioretti, P. (1969). Lancet 2: 1385. Singer, W., Desjardin, P., Friesen, H. (1970). Obstet. Cryne¢ol., 36: 222. Saxena, B. N., Emerson, K., Selenkow, H. (1969). N. Eng. J. Med., 281: 225. Josimovieh, J. B., Kosor, B., Bocce-la, L., Mintz, D., Hutchinson, L. (1970). ObsteL Gyne¢ol., 36 : 244.