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The Transmission of Transfused Sickle-Trait Cells from Mother to Fetus
THE TRANSMISSION OF TRANSFUSED CELLS FROM MOTHER TO FETUS
SICKLE-TRAIT
NICHOLAS T. MACRIS, M.D., LOUIS M. HELLMAN, R. J. WATSON, M.D., BROOKLYN,N. Y. (From the Departments New York College
of
of Obstetrics and Gynecology of Medicine, Downstate Medical
and Pediatrics of the State Center, and the Kings County
M.D., AND
University Hospital)
I
N 1940 Landsteiner and Wiener1 made their first report on the Rh factor and This established that the antigen was located on human erythrocytes.’ was followed by the recognition of its role in erythroblastosis fetalis by Levine and his associates3 in 1941. In order to explain isoimmunization the old concept of the separate integrity of the fetal and maternal circulations was subjected to critical reappraisal. Several investigators approached this problem by devising experiments to show the transmission of maternal crythrocytes across the placental barrier. In 1946 Hedenstedt and Naeslund4 injected elliptocytic blood into 2 pregnant women. They demonstrated the elliptocytes in the blood of one of the infants and noted that the placenta in this case had many sites of bleeding and infarction. Naeslund and Nylinj injected erythrocytes tagged with radioactive phosphorus (P”‘) into 6 pregnant women prior to delivery. In one of the cases, they were able to demonstrate a significant degree of radioactivity in the infant’s erythrocytes. In 1951 NaeslundG transfused erythrocytes which had been tagged with radioactive iron (Fe5”) into 7 pregnant women. His findings in the blood of the infants of 4 of these women suggested that the radioactive red blood cells had crossed the placenta. In 1955 Mengert and his associates’ employed erythrocytes tagged with Fe59 and performed 29 experiments. Significant amounts of radioactivity were d:lrnonstrated in the fetal red blood cells in 25 of the experiments. The investigators considered the method with Fe5Qcumbersome and vulnerable to error. They did 2 additional experiments utilizing donor blood with the sickle-cell trait. This blood was injected into 2 pregnant women. Sickle cells were demonstrated in the blood of both infants, indicating that the sickle-trait cells had crossed the placenta.
Material
and Methods
The purpose of this investigation was to determine by yet another method whether intact maternal erythrocytes cross the placental barrier. Donor blood with the sickle-cell trait but without sickle-cell anemia was selected for this purpose. When erythrocytes with the sickle trait are exposed to a decreased oxygen tension, they assume a characteristic sickled configuration 1214
g;;,hzr~
1’1,ACENTAL
TRANSFER
OF
TRANSFUSED
SICKLE-TRAIT
CELLS
19lr - ,)
which is easily recognized. A group of pregnant women was therefore in.jccted wit.h sickle-trait blood and the blood of their infants was examined for sickle cells. Blood donated to the Kings County Hospital Blood Bank was examined for t,he sickle trait. A 1 per cent isotonic solution of sodium bisulfite in clistilled water was prepared and a large drop of the solution was mixed with a small drop of blood on a slide. A eoverslip glass was immediately applied with gentle pressure to prevent floating, and the excess solution-blood misture at the edges of the preparation was blott,ed. Care was taken to insure the freshness of the solution, since it deteriorates rapidly at, room temperature. With this method, sickling occurs promptly and is usually complete within ::O minutes to 1 hour. The Blood Bank donor requirements excluded individuals with sickle-cell anemia. Therefore, it was assumccl t,hat all donor bloods in which sickling was demonstrated came from individuals with the sicklcwll t,rait,. The subjects of this study were 25 Rh-posit,ive pregnant, women at or near terns. Specimens of their blood were examined for sickle cells and irl all except one none were found. When available, the husband’s blood was also examined for sickling. The selection of subjects in this study was governed largely by the blood group of the sickle-trait donor blood that was available. They ranged in age from l’i to 38 years. The distribution inrludeil 18 Kegroes, 4 Puerto Ricans, and 3 whites. Twenty-one of the subjects were transfused with 500 ml. each of sickletrait whole blood preserved in A.C.11. solution. In 3 inst.ances (Cases 1. 2, :?‘I, the supcrnatant plasma in 500 ml. of whole blood was removed, allowing t,he sickle-trait, cells to he given in a reduced volume. The elapsed time, between the end of the transfusion and delivery, ranged from 40 minutes to 153 hours and 2 minutes. Nineteen of the women were delivered vaginally and 6 II:,cesarenii section. Whole blood was collected both from the umhilica.1 cord at delivery an11 from the mother within 12 hours after delivery. A combination of drictl ammonium and potassium oxalates was used as the ant,icoagulant,. The blood from the umbilical cord was collected with the utmost pare to prevent cartaminat,ion with maternal blood. Sieklc-eel1 preparations of the mother’s and infant’s bloods were pr+ l)arcd by the method described above. In order to prevent drying, the preparations were covered with Petri dishes to which moist filt,er papers had been nttnvllcc1. They were examined for sickling at, int,ervals of 15 minutes, 30 minutes. and 1 hour. When sickle cells wcrc present. the sickling process occurred earlier and was more complete anlong the red 1~1ooclcells near t,hc edges of the preparation. A crude estimate of the number of sickle cells was made by rounting the number of sickle cells in 2,000 consecutive red blood cells near the edges of the coverslip glass, When sickle cells were found in the blood from an umbilical cord, the infant’s blood was, when possible. examined 8 weeks later in order to rule out the possibility of an inherited sickle trait.
Results The results of this study are listed in Table I. Sickle cells were found in the umbilical cord blood of the newborn infant in 3 of the 25 easesstudied. Case 2 was an M-year-old primigravida who was delivered vaginally with the aid of forceps. Case 6 was a 21-year-old multipara who had an uneventful vaginal delivery. Case 13, a 19-year-old primigravida, was deIivered 1~ ccsarean section because of a breech presentation and prolonged rupture 0-t:
AGE
Lt. w N
22
23 25 20 17 38 26
19
20
2
23
;
P.R.
36 33
:t
2”;
N
34
: N N
FR P:R:
19 35
fii
N
N
18
’
17 21 28 34 22 21
26
18
21
Yv
RACE AND NATIOh’ALITY
I I
I
11 :i
i 10
3 4 5
i
CASE NO.
I
--__~
-
TION
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Pos. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. ~__ Neg.
- 1
SICKLECELI,
i
b
0 0
ii
i x ii 0 ix vii 0 iv 0. 111
0
111
ii i. . .
0 0
ii 0 ii
PARITY
DATA
ii
/
I.
i iv i i ii i. . . 111
V
ii iv i ii xi. . . 111 i xii. . . “111 i
7.. 111
i. . . 111 i
GRAVIDITY .. . 111
TABLE
ON /
section
section
section section
section
section
STUDIED
DELIVERY
CASES
Vaginal Vaginal Vaginal Vasnal Vaginal Vaginal Vaginal Cesarean Vaginal Cesarean Vaginal Cesarean Cesarean Vaginal Vaginal Cesarean Vaginal Vaginal Vaginal Vaginal Vaginal Vaginal Vaginal Cesarean Vaginal
25 I
39 2 1
34
2” 35 5
2
153 32
-
1 79 0
13
k 26
7 21
1 10 0 7
HOURS
fi
55
29
50
10
56 54 02
30
30 30 47 20
13.3 12.7
12.2 13.5 11.3 13.0 12.3 14.3 12.1
12.1 10.8
9.6 11.9
10.6 12.1 13.4 0
11.9
40
12.1 13.7 13.0 13.5 12.4 10.3
IO
12
0
52
19
2i 40
11.5
SICKLE MOTHER
%
53
TO
/ MINUTES
DELIVERY
TIME FROM TRANSFUSION
: 0 0
0 0 0 0 0 0 n 0
0
2.3
0 0 0 0 0
0.2
0 0 0
2.6
0
1
INFANT
POST
BIRTH
I-
I
CELLS
-.I_
8
_-
-
-
-
-
-
0
0
WKS.
PARTUM
c;I:;;;;~TG(, PLACENTAL
TRANSFER
OF TRANSFUSED
SICKLE-TRAIT
mm3
1217
the membranes without labor. The placenta in each of the 3 cases was normal on gross inspection. The blood of the infants in Cases 2 and 6 was esamined for sickle cells when they were 8 weeks old. None were found, indicating that the sickle cells demonstrated in the umbilical cord blood ha{1 crossed the placenta and were not the result of an inherited sickle trait. ~1 similar follow-up study of the infant in Case 13 could not bc done. This infant had contracted a parenteral diarrhea and died at the age of 5?4 weeks. Sickle cells, when present, can be identified in histologic tissue sections. None wcrc found when the sections of the infant’s organs were examined. Case 15 presented an interesting natural experiment. This woman hatI the sickle-cell trait. She was included in the study because there was a possibility that her infant would not inherit the sickling trait. Two of the rnriables present in all of the other cases, the amount of sickle-trait bloocl injected anbe partum and the elapsed time from the end o-f the transfusion to tlelivery. were not factors in this case. All of her crythrocytes had the ability to sickle. Nevertheless, no sickle cells could bc dcmnnstrated in the infa.nt’s 1,lOOd.
Comment The placental passage of sickle-trait cells in 3 of 25 experimental subjects adds to the already existing evidence that in a small percentage of r)atients maternal red cells traverse the placental barrier and arr found in al)preciablc numbers in the fetal circulation. The mechanism of transfer is obscure. While breaks are known to occur in thr fetal circulation,8-“’ thrr pressure relationships are thought to be such that direcat passage must always occur from fetus to mother. The infant arterial pressure at birth is 60-70 mm. of mercury while the venous pressure in the umbilical cord is 30-40 mm. of mrrcury. This would make the capillary prcssurcx in the villi about 45 mm. of mercury.l’ The pressure of maternal blood in the intervillous space with the uterus at rest is about 10 mm. of mercury.12 While this pressure rises markedly with uterine contractions. a I)ropoTtionatc rise in fctnl pressure must also occur. Unless the known data on infants are not valid for the fetus in utcro, tlircct t,ransfcr of maternal cells against the pressure graclient is not feasible. Furthermore, although nothing is known about the pressure relationships of the human fetus in utero, the venous pressure cannot fall below the amniotic Auid pressure which for all intent,s and purposes is the same as the intervillous spnec pressare. The nnly remaining possibility is active transfer by the process of planocaytoGs which has been shown to occur for particulat,e matter.13 Making certain rough assumptions from the data in Table I, in at least one instance’ the fetus must have received at least 5 ml. of sickle-trait cells. Should the placenta be unable to distinguish between sickle-trait and normal maternal cells, the transfusion to accomplish this must have been a bit more than 40 ml., which is altogether unlikely. One is Ieft with the rather fantastic assumption that the microvilli distinguish between natural and unnatural maternal cells, engulfing only the latter. -___ *Case
2,
fetal
red-cell
mass
estimated
at
200
ml.
1218 The facts of transfer a baffling mystery.
can no longer
be denied.
The mechanism
remains
References A. 8.: 1. Landsteiner, K., and Wiener, Proc. Sot. Exper. Biol. & Med. 43: 223, 1940. 2. Landsteiner, Ii., and Wiener, A. S.: J. Exper. Med. 74: 309, 1941. 3. Levine, P., Burnham, L., Katzin, E. M., and Vogel, P.: AM. J. OBST. & GYNEC. 42: 925, 1941. 4. Hedenstedt, S., and Naeslund, J.: Acta med. scandinav., supp. 170, p. 126, 1946. 5. Naeslund, J.. and Nvlin. G.: Acta med. scandinav.. SUDD. 176. 1). 390. 1946. ’ ’ 6. Naeslund; J.I Aeta obst. et gynec. scandinav. 30: 231, f951. 7. Mengert, W. F., Rights, C. S., Bates, C. R., Jr., Reid, A. F., Wolf, 0. R., and Nabors, G. C.: AM. J. OBST. & GYNEC. 69: 678, 1955. 8. Javert. C.. ant1 Reiss. C.: Sure. Gvnec. & Obst. 94: 257. 1952. 9. Chown; Bruce: AM. J. OBST. &“GY~Ec. 70: 1298, 1955. ’ 10. O’Connor, W. J., Shields, George, Kohl, Schuyler G., and Sussman, Marvin: 9~. J. OBST. & GYNEC. 73: 768, 1957. 11. Prystowski, H.: Personal communication, 1958. 12. Hellman, L. M., Tricomi, V., and Gupta, 0.: Aan. J. OBST. & GYNEC. 74: 1018, 1957. 13. Uempscy, E. TV.: of the First ConTn Flexner, L. R., editor: Gestation : Transactions ference, New York, 1954, Josiah Macy, Jr., Foundation.
SICKLE-TRAIT
NICHOLAS T. MACRIS, M.D., LOUIS M. HELLMAN, R. J. WATSON, M.D., BROOKLYN,N. Y. (From the Departments New York College
of
of Obstetrics and Gynecology of Medicine, Downstate Medical
and Pediatrics of the State Center, and the Kings County
M.D., AND
University Hospital)
I
N 1940 Landsteiner and Wiener1 made their first report on the Rh factor and This established that the antigen was located on human erythrocytes.’ was followed by the recognition of its role in erythroblastosis fetalis by Levine and his associates3 in 1941. In order to explain isoimmunization the old concept of the separate integrity of the fetal and maternal circulations was subjected to critical reappraisal. Several investigators approached this problem by devising experiments to show the transmission of maternal crythrocytes across the placental barrier. In 1946 Hedenstedt and Naeslund4 injected elliptocytic blood into 2 pregnant women. They demonstrated the elliptocytes in the blood of one of the infants and noted that the placenta in this case had many sites of bleeding and infarction. Naeslund and Nylinj injected erythrocytes tagged with radioactive phosphorus (P”‘) into 6 pregnant women prior to delivery. In one of the cases, they were able to demonstrate a significant degree of radioactivity in the infant’s erythrocytes. In 1951 NaeslundG transfused erythrocytes which had been tagged with radioactive iron (Fe5”) into 7 pregnant women. His findings in the blood of the infants of 4 of these women suggested that the radioactive red blood cells had crossed the placenta. In 1955 Mengert and his associates’ employed erythrocytes tagged with Fe59 and performed 29 experiments. Significant amounts of radioactivity were d:lrnonstrated in the fetal red blood cells in 25 of the experiments. The investigators considered the method with Fe5Qcumbersome and vulnerable to error. They did 2 additional experiments utilizing donor blood with the sickle-cell trait. This blood was injected into 2 pregnant women. Sickle cells were demonstrated in the blood of both infants, indicating that the sickle-trait cells had crossed the placenta.
Material
and Methods
The purpose of this investigation was to determine by yet another method whether intact maternal erythrocytes cross the placental barrier. Donor blood with the sickle-cell trait but without sickle-cell anemia was selected for this purpose. When erythrocytes with the sickle trait are exposed to a decreased oxygen tension, they assume a characteristic sickled configuration 1214
g;;,hzr~
1’1,ACENTAL
TRANSFER
OF
TRANSFUSED
SICKLE-TRAIT
CELLS
19lr - ,)
which is easily recognized. A group of pregnant women was therefore in.jccted wit.h sickle-trait blood and the blood of their infants was examined for sickle cells. Blood donated to the Kings County Hospital Blood Bank was examined for t,he sickle trait. A 1 per cent isotonic solution of sodium bisulfite in clistilled water was prepared and a large drop of the solution was mixed with a small drop of blood on a slide. A eoverslip glass was immediately applied with gentle pressure to prevent floating, and the excess solution-blood misture at the edges of the preparation was blott,ed. Care was taken to insure the freshness of the solution, since it deteriorates rapidly at, room temperature. With this method, sickling occurs promptly and is usually complete within ::O minutes to 1 hour. The Blood Bank donor requirements excluded individuals with sickle-cell anemia. Therefore, it was assumccl t,hat all donor bloods in which sickling was demonstrated came from individuals with the sicklcwll t,rait,. The subjects of this study were 25 Rh-posit,ive pregnant, women at or near terns. Specimens of their blood were examined for sickle cells and irl all except one none were found. When available, the husband’s blood was also examined for sickling. The selection of subjects in this study was governed largely by the blood group of the sickle-trait donor blood that was available. They ranged in age from l’i to 38 years. The distribution inrludeil 18 Kegroes, 4 Puerto Ricans, and 3 whites. Twenty-one of the subjects were transfused with 500 ml. each of sickletrait whole blood preserved in A.C.11. solution. In 3 inst.ances (Cases 1. 2, :?‘I, the supcrnatant plasma in 500 ml. of whole blood was removed, allowing t,he sickle-trait, cells to he given in a reduced volume. The elapsed time, between the end of the transfusion and delivery, ranged from 40 minutes to 153 hours and 2 minutes. Nineteen of the women were delivered vaginally and 6 II:,cesarenii section. Whole blood was collected both from the umhilica.1 cord at delivery an11 from the mother within 12 hours after delivery. A combination of drictl ammonium and potassium oxalates was used as the ant,icoagulant,. The blood from the umbilical cord was collected with the utmost pare to prevent cartaminat,ion with maternal blood. Sieklc-eel1 preparations of the mother’s and infant’s bloods were pr+ l)arcd by the method described above. In order to prevent drying, the preparations were covered with Petri dishes to which moist filt,er papers had been nttnvllcc1. They were examined for sickling at, int,ervals of 15 minutes, 30 minutes. and 1 hour. When sickle cells wcrc present. the sickling process occurred earlier and was more complete anlong the red 1~1ooclcells near t,hc edges of the preparation. A crude estimate of the number of sickle cells was made by rounting the number of sickle cells in 2,000 consecutive red blood cells near the edges of the coverslip glass, When sickle cells were found in the blood from an umbilical cord, the infant’s blood was, when possible. examined 8 weeks later in order to rule out the possibility of an inherited sickle trait.
Results The results of this study are listed in Table I. Sickle cells were found in the umbilical cord blood of the newborn infant in 3 of the 25 easesstudied. Case 2 was an M-year-old primigravida who was delivered vaginally with the aid of forceps. Case 6 was a 21-year-old multipara who had an uneventful vaginal delivery. Case 13, a 19-year-old primigravida, was deIivered 1~ ccsarean section because of a breech presentation and prolonged rupture 0-t:
AGE
Lt. w N
22
23 25 20 17 38 26
19
20
2
23
;
P.R.
36 33
:t
2”;
N
34
: N N
FR P:R:
19 35
fii
N
N
18
’
17 21 28 34 22 21
26
18
21
Yv
RACE AND NATIOh’ALITY
I I
I
11 :i
i 10
3 4 5
i
CASE NO.
I
--__~
-
TION
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Pos. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. ~__ Neg.
- 1
SICKLECELI,
i
b
0 0
ii
i x ii 0 ix vii 0 iv 0. 111
0
111
ii i. . .
0 0
ii 0 ii
PARITY
DATA
ii
/
I.
i iv i i ii i. . . 111
V
ii iv i ii xi. . . 111 i xii. . . “111 i
7.. 111
i. . . 111 i
GRAVIDITY .. . 111
TABLE
ON /
section
section
section section
section
section
STUDIED
DELIVERY
CASES
Vaginal Vaginal Vaginal Vasnal Vaginal Vaginal Vaginal Cesarean Vaginal Cesarean Vaginal Cesarean Cesarean Vaginal Vaginal Cesarean Vaginal Vaginal Vaginal Vaginal Vaginal Vaginal Vaginal Cesarean Vaginal
25 I
39 2 1
34
2” 35 5
2
153 32
-
1 79 0
13
k 26
7 21
1 10 0 7
HOURS
fi
55
29
50
10
56 54 02
30
30 30 47 20
13.3 12.7
12.2 13.5 11.3 13.0 12.3 14.3 12.1
12.1 10.8
9.6 11.9
10.6 12.1 13.4 0
11.9
40
12.1 13.7 13.0 13.5 12.4 10.3
IO
12
0
52
19
2i 40
11.5
SICKLE MOTHER
%
53
TO
/ MINUTES
DELIVERY
TIME FROM TRANSFUSION
: 0 0
0 0 0 0 0 0 n 0
0
2.3
0 0 0 0 0
0.2
0 0 0
2.6
0
1
INFANT
POST
BIRTH
I-
I
CELLS
-.I_
8
_-
-
-
-
-
-
0
0
WKS.
PARTUM
c;I:;;;;~TG(, PLACENTAL
TRANSFER
OF TRANSFUSED
SICKLE-TRAIT
mm3
1217
the membranes without labor. The placenta in each of the 3 cases was normal on gross inspection. The blood of the infants in Cases 2 and 6 was esamined for sickle cells when they were 8 weeks old. None were found, indicating that the sickle cells demonstrated in the umbilical cord blood ha{1 crossed the placenta and were not the result of an inherited sickle trait. ~1 similar follow-up study of the infant in Case 13 could not bc done. This infant had contracted a parenteral diarrhea and died at the age of 5?4 weeks. Sickle cells, when present, can be identified in histologic tissue sections. None wcrc found when the sections of the infant’s organs were examined. Case 15 presented an interesting natural experiment. This woman hatI the sickle-cell trait. She was included in the study because there was a possibility that her infant would not inherit the sickling trait. Two of the rnriables present in all of the other cases, the amount of sickle-trait bloocl injected anbe partum and the elapsed time from the end o-f the transfusion to tlelivery. were not factors in this case. All of her crythrocytes had the ability to sickle. Nevertheless, no sickle cells could bc dcmnnstrated in the infa.nt’s 1,lOOd.
Comment The placental passage of sickle-trait cells in 3 of 25 experimental subjects adds to the already existing evidence that in a small percentage of r)atients maternal red cells traverse the placental barrier and arr found in al)preciablc numbers in the fetal circulation. The mechanism of transfer is obscure. While breaks are known to occur in thr fetal circulation,8-“’ thrr pressure relationships are thought to be such that direcat passage must always occur from fetus to mother. The infant arterial pressure at birth is 60-70 mm. of mercury while the venous pressure in the umbilical cord is 30-40 mm. of mrrcury. This would make the capillary prcssurcx in the villi about 45 mm. of mercury.l’ The pressure of maternal blood in the intervillous space with the uterus at rest is about 10 mm. of mercury.12 While this pressure rises markedly with uterine contractions. a I)ropoTtionatc rise in fctnl pressure must also occur. Unless the known data on infants are not valid for the fetus in utcro, tlircct t,ransfcr of maternal cells against the pressure graclient is not feasible. Furthermore, although nothing is known about the pressure relationships of the human fetus in utero, the venous pressure cannot fall below the amniotic Auid pressure which for all intent,s and purposes is the same as the intervillous spnec pressare. The nnly remaining possibility is active transfer by the process of planocaytoGs which has been shown to occur for particulat,e matter.13 Making certain rough assumptions from the data in Table I, in at least one instance’ the fetus must have received at least 5 ml. of sickle-trait cells. Should the placenta be unable to distinguish between sickle-trait and normal maternal cells, the transfusion to accomplish this must have been a bit more than 40 ml., which is altogether unlikely. One is Ieft with the rather fantastic assumption that the microvilli distinguish between natural and unnatural maternal cells, engulfing only the latter. -___ *Case
2,
fetal
red-cell
mass
estimated
at
200
ml.
1218 The facts of transfer a baffling mystery.
can no longer
be denied.
The mechanism
remains
References A. 8.: 1. Landsteiner, K., and Wiener, Proc. Sot. Exper. Biol. & Med. 43: 223, 1940. 2. Landsteiner, Ii., and Wiener, A. S.: J. Exper. Med. 74: 309, 1941. 3. Levine, P., Burnham, L., Katzin, E. M., and Vogel, P.: AM. J. OBST. & GYNEC. 42: 925, 1941. 4. Hedenstedt, S., and Naeslund, J.: Acta med. scandinav., supp. 170, p. 126, 1946. 5. Naeslund, J.. and Nvlin. G.: Acta med. scandinav.. SUDD. 176. 1). 390. 1946. ’ ’ 6. Naeslund; J.I Aeta obst. et gynec. scandinav. 30: 231, f951. 7. Mengert, W. F., Rights, C. S., Bates, C. R., Jr., Reid, A. F., Wolf, 0. R., and Nabors, G. C.: AM. J. OBST. & GYNEC. 69: 678, 1955. 8. Javert. C.. ant1 Reiss. C.: Sure. Gvnec. & Obst. 94: 257. 1952. 9. Chown; Bruce: AM. J. OBST. &“GY~Ec. 70: 1298, 1955. ’ 10. O’Connor, W. J., Shields, George, Kohl, Schuyler G., and Sussman, Marvin: 9~. J. OBST. & GYNEC. 73: 768, 1957. 11. Prystowski, H.: Personal communication, 1958. 12. Hellman, L. M., Tricomi, V., and Gupta, 0.: Aan. J. OBST. & GYNEC. 74: 1018, 1957. 13. Uempscy, E. TV.: of the First ConTn Flexner, L. R., editor: Gestation : Transactions ference, New York, 1954, Josiah Macy, Jr., Foundation.