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Effect of length of gestation on maternal cellular immunity to human trophoblast antigens
Effect of length of gestation on maternal cellular immunity to human trophoblast antigens V. TAYLOR
PAMELA G.
GOWLAND
K.
W.
J.
HANCOCK
S. SCOT-I
Leeds, England
Maternal lymphocyte reactivity to human trophoblast antigens was studied in placentas of gestational ages 8 to 14 weeks and 32 to 34 week, respectively. Sign$cant trophoblast lysis beca.me apparent after 24 hours’ incubation in the latter case compared with a time lag of 72 hours in the terminated gestations. Maternal cellular immunity, therefore, was not detected during thejrst 3 ‘/z months of pregnancy, but was detectable by the time of parturition. The possible signl$icance is discussed with respect to the antigenic stimulus and survival of the fetal allograft.
WHATEVER the explanation for the survival of the fetal allograft may be,’ there is evidence that a maternal humoraP 3 and cellular4~ ’ immune response to fetal antigens has often occurred in normal pregnancy by the time of parturition. The nature of the antigenic stimulus and the time of onset and duration of the maternal response are undetermined. However, the macrophage migration inhibition technique has been used to show that maternal cell-mediated immunity to placental antigens could not be detected until the fourth to fifth month of pregnancy in primigravid women, with a subsequent increase in the degree of immunity during gestation.” In the present study using the placental monolayer cultures recently evaluated,’ maternal lymphocyte reactivity to trophoblast antigens in vitro was assessed in placentas of 8 to 14 weeks’ and 32 to 42 weeks’ gestational age.
Materials and methods The cultures were prepared by trypsinization chorionic villous tissue by methods described.? From the Departments of Obstetrics and Gynaecology, Immunolog?i, The University of Leeds, Received
for publication
Accepted June
April
8, 1975.
19, 1975.
Reprint reqwsts: Dr. P. V. Taylor, Department Obstetrics, University of Leeds, 17 Springfield Leeds 2, England.
of Mount,
of The and
culture medium consisted of 10 per cent calf serum 01 10 per cent maternal serum in medium 199 (Wellcome) and the cells were grown on glass coverslips in Leighton culture tubes at an initial concentration of 1 X 106cells per culture. A leukocyte-rich fraction was obtained from heparinized (200 U. per milliliter) maternal blood by sedimentation with one tenth its volume of 5 per cent dextran (molecular weight 110,000) for approximately 30 minutes at 4” C. The leukocytes were washed three times in medium 199 and 2 x lo6 cells added to each culture 24 hours after the cultures were set up, the time of addition being taken as day 0. Control cultures received fresh medium containing no maternal leukocytes. In some cases controls were set up with leukocytes from unrelated donors, i.e., male and nulliparous female volunteers. Viability of the trophoblast cultures was assessed on days 1, 3, 4, and 5, a minimum of two cultures being counted on each occasion. The viability test relied on the ability of living cells to adhere to the coverslip and was carried out as described.’ A reduction in the number of target cells in experimental compared with control groups has been attributed to killing and is considered to reflect a cytotoxic effect of the lymphocytes.g The cell counts in each culture have been expressed as a percentage of those in the appropriate controls. Statistical analyses of the data were performed by applying Student’s t test and Wilcoxon’s sum of ranks test.
Volume Number
125 4
Table
I. Viable
Gestation and cellular immunity to trophoblast antigens
trophoblast
cell counts of immature
placentas*
(yr.)
Duration of present pregnancy (wk.)
0
-
14
4
7
8
4
3%
0
-
8
0
-
8
61 73
2
8
14
90 96
79 76
2
4%
12
99 104 122 118
121 122 -
0
-
12
0
-
10
110.2 -c 13.7 66.1 ” 42.8
111.8 k 34.4 51.7 2 43.8
No. of
Day
previous pregnancies
1
3
4
5
I. (i) (ii)
100
111
-
113 5
129 0
(i) (ii) 3. (i) (ii) 4. (i) (ii) 5. (i) (ii) 6. (i) (ii) 7. (i) (ii) 8. (i) (ii) 9. (i) (ii)
100 130
121 151
125 12
167 0
100 95 100 100
144 118
100 48 130 85
114 39 -
100 87
114 63
113 30
-
100 86
85 88
100 97
100 80
111 126
100 102 100 96
92 92 113 112
2.
Mean S.D.
-+ (i)
(ii)
p value
100 98.6
2 14.2
111.4 107.1
+ 17.9 k 26.6
Not
Not
significant
significant
529
Time ehpsed since last
Pregnancr
12
*Immature placentas: normalized viable trophoblast cell counts on days 1, 3, 4, and 5 in cultures incubated with (i) no maternal lymphocytes or (ii) 2 x lo6 maternal lymphocytes.
Results The viable trophoblast cell counts of the nine immature placentas investigated are shown in Table I, together with some clinical details. Levels of significance are those calculated by Wilcoxon’s test. Student’s t test gave a lower level of significance than Wilcoxon’s test in only one case, and this is probably due to the relative independence of the latter of the form of distribution of the data. Significant killing of trophoblast cells by maternal lymphocytes became evident only after 72 hours’ incubation, the difference in the number of viable cells in test and control cultures being significant on days 4 and 5. Maternal lymphocytes showed a wide spectrum of reactivity to homologous trophoblast varying from complete destruction to no cytopathogenicity at all. The viable trophoblast cell counts of the ten mature placentas investigated are shown in Table II. Killing of trophoblast cells by maternal lymphocytes became evident after 24 hours, the difference in the number of viable cells in test and control cultures being significant on days 1, 3, 4, and 5. Again there was considerable variation in the extent of cytotoxicity, but in contrast to the immature situation all the term trophoblast cul-
tures were almost completely destroyed by day 5. The killing of trophoblast, whether immature or term, at between days 3 and 5 did not appear to be related to parity or time lapse between pregnancies. Lymphocytes from third-party individuals also exerted a cytotoxic effect on both early and term trophoblast, significant killing of the trophoblast cells occurring only after 72 hours’ incubation. In all the trophoblast cultures, whatever the gestational age of the placenta, the presence of maternal serum in the medium completely prevented trophoblast lysis.
Comment The lysis by both maternal and unrelated donor lymphocytes of cultured trophoblast cells has been described9 and attributed to a primary sensitization in vitro to trophoblast antigens.8 The nature of this antigenicity remains to be determined but histocompatibility factors may well be involved,’ which is possibly one explanation for the observed wide spectrum of maternal lymphocyte reactivity to trophoblast cells. Experiments in which mother and baby are tissue-typed are under way in order to elucidate this
530
June 15, 1976 Am. J. Obstet. Gynecol.
Taylor et al.
Table II. Viable trophoblast
cell counts of mature
placentas*
Day 1
.
3
4
5
No. of previouc prepunties
Tim elapsed since last pregnancy cvr.,
Duration CI/ present pregnan CT (wk.)
1. (i) (ii)
100 102
85 62
57 4
54 6
2
2%
41
2. (i) (ii)
100 108
100 97
105 11
100 15
0
-
42
3. (i) (ii)
100 91
105 98
88 11
70 7
0
-
39
4. (i) (ii)
100 73
65 19
61 4
64 3
0
36
5. (i) (ii)
100 72
91 75
X8 2
88 4
0
42
6. (i) (ii)
100 92
103 107
102 5
82 4
0
41
7. (i) (ii)
100 96
100 127
92 14
103 18
I
2
40
8. (i) (ii)
100 86
86 101
96 18
93 8
2
1%
40
9. (i) (ii)
100 95
95 75
88 43
78 32
0
-
32
10. (i) (ii)
100 64
75 60
72 27
62 27
3
Mean SD.
+ (i) (ii)
p value *Mature lymphocytes
100 88 + 14.12 -Co.05
> 0.01
90.5 2 12.94 82.1 lr 30.62
84.9 f 16.3 13.9 + 12.77
79.4 -t 16.7 12.4 2 10.29
0.05
placentas: normalized viable trophoblast or (ii) 2 x 10” maternal lymphocytes.
cell counts
problem. The crucial difference in this interaction between trophoblast and maternal cells when immature or mature placentas were studied was the time of appearance of significant trophoblast lysis, i.e., on day 4 in the former case and on day 1 in the latter, suggesting that maternal cellular immunity had developed by 32 weeks’ gestation-the shortest duration of pregnancy investigated in the mature group. This is emphasized by the fact that lymphocytes from unrelated donors had no significant cytotoxic effect during the first 72 hours’ incubation, which has also been reported by other workers.‘O No sensitization, therefore, was detected during the first 3% months of pregnancy, in agreement with the findings of Youtananukorn and associates,” although in both studies the in vitro techniques used may not have detected very low levels of cellular immunity. The apparent change in maternal lymphocyte reactivity could be attributed to maturation of trophoblast antigens rather than ontogeny of the maternal immune response. However, the response of maternal lymphocytes to cultured immature trophoblast suggests that
on days
1, 3, 4, and 5 in cultures
2
incubated
39
with
(i) no maternal
antigens are present on trophoblast cells at this early stage. If quantitative differences were involved, the response of third-party allogeneic lymphocytes to cultured trophoblast would probably alter according to whether early or late trophoblast was involved, and this was not found to be the case. The concept of a change in the maternal immune status would agree with the results of Youtananukorn and associates,6 who demonstrated an ontogony of the maternal immune response with lymphocytes from mothers at various stages of gestation with term placental antigens. It has been found” that reactivity to human placental antigens was maintained in the early postpartum period, i.e., on the fourth day after delivery. In the present study, some of the immature placentas were obtained from parous women (see Table I). The time elapsing between the last previous pregnancy and the present pregnancy ranged from 3% to 8 years, suggesting that detectable levels of cellular immunity had disappeared during this time interval. In the case of humoral immunity, the earliest disappearance of leukoagglutinins from the maternal circulation oc-
I
Volume Number
125 4
curred less than 2 months post partum, 22 per cent of women with such antibodies losing them by the third year. ‘r The apparent alteration as pregnancy proceeds in the immune status of the mother, which may have to occur de nova in each gestation, emphasizes the concept of a changing balance of mechanisms to ensure survival of the fetal allograft. The evidence of maternal cellular immunity to fetal antigens also implies that one
Gestation and cellular immunity to trophoblast antigens
531
function of these mechanisms must be to impede the efferent limb of the immune response, possibly by the production of enhancing antibodies or immune complexes. I2 This is supported by the finding in the present study that the presence of maternal serum in the cultures inhibits trophoblast lysis, a protective effect which probably has specific and nonspecific components.*
REFERENCES
1. Billingham, R. E.: The transplantation biology of mammalian gestation, AM. J. OBSTET. GYNECOL. 111: 469, 1971. 2. Hulka, J. F., Hsu, K. C., and Beiser, S. M.: Antibodies to trophoblast during the post partum period, Nature 191: 510, 1961. 3. Terasaki, P. I., Mickey, M. R., Yamazaki, J. N., and Vredevoe, D.: Maternal fetal incompatibility. I. Incidence of HL-A antibodies and possible association with congenital anomalies, Transplantation 9: 538, 1970. 4. Youtananukorn, V., and Matangkasombut, P.: Human maternal cell-mediated immune reaction to placental antigens, Clin. Exp. Immunol. 11: 549, 1972. K. E., Hellstrom, I., and Brawn, J.: Abroga5. Hellstrom, tion of cellular immunity to antigenically foreign mouse embryonic cells by a serum factor, Nature 224: 914, 1969. Matangkasombut, P., 6. Youtananukorn, V., and Osathanondh, V.: Onset of human maternal cellmediated immune reaction to placental antigens during the first pregnancy, Clin. Exp. Immunol. 16: 593, 1974. P. V., and Hancock, K. W.: Viability of human 7. Taylor,
a. 9.
10. 11.
12. 13.
trophoblast in vitro. J. Obstet. Gynaecol. Br. Commonw. 80: 834, 1973. Taylor, P. V., and Hancock, K. W.: Antigenicity of trophoblast and some possible antigen-masking effects during pregnancy, Immunology 28: 973, 1975. Hellstrom, I., and Hellstrom, K. E.: Colony inhibition and cytotoxicity assays, in Bloom, B. R., and Glade, P. R., editors: In Vitro Methods in Ceil-Mediated Immunity, New York and London, 1971, Academic Press, Inc., p. 409. Currie, G. A.: Immunological studies of trophoblast in vitro, J. Obstet. Gynaecol. Br. Commonw. 74841, 1967. Douthwaite, R. M., and Urbach, G. I.: In vitro antigenicity of trophoblast, AM. J. OBSTET. GYNECOL. 109: 1023 1971. Payne, R.: The development and persistence of leukoannlutinins in oarous women. Blood 19: 41 I, 1962. Youta$\ukorn, V.: and Mantagkasombut, P.: Specific plasma factors blocking human maternal cell-mediated immune reaction to placental antigens, Nature New Biol. 242: 110, 1973.
PAMELA G.
GOWLAND
K.
W.
J.
HANCOCK
S. SCOT-I
Leeds, England
Maternal lymphocyte reactivity to human trophoblast antigens was studied in placentas of gestational ages 8 to 14 weeks and 32 to 34 week, respectively. Sign$cant trophoblast lysis beca.me apparent after 24 hours’ incubation in the latter case compared with a time lag of 72 hours in the terminated gestations. Maternal cellular immunity, therefore, was not detected during thejrst 3 ‘/z months of pregnancy, but was detectable by the time of parturition. The possible signl$icance is discussed with respect to the antigenic stimulus and survival of the fetal allograft.
WHATEVER the explanation for the survival of the fetal allograft may be,’ there is evidence that a maternal humoraP 3 and cellular4~ ’ immune response to fetal antigens has often occurred in normal pregnancy by the time of parturition. The nature of the antigenic stimulus and the time of onset and duration of the maternal response are undetermined. However, the macrophage migration inhibition technique has been used to show that maternal cell-mediated immunity to placental antigens could not be detected until the fourth to fifth month of pregnancy in primigravid women, with a subsequent increase in the degree of immunity during gestation.” In the present study using the placental monolayer cultures recently evaluated,’ maternal lymphocyte reactivity to trophoblast antigens in vitro was assessed in placentas of 8 to 14 weeks’ and 32 to 42 weeks’ gestational age.
Materials and methods The cultures were prepared by trypsinization chorionic villous tissue by methods described.? From the Departments of Obstetrics and Gynaecology, Immunolog?i, The University of Leeds, Received
for publication
Accepted June
April
8, 1975.
19, 1975.
Reprint reqwsts: Dr. P. V. Taylor, Department Obstetrics, University of Leeds, 17 Springfield Leeds 2, England.
of Mount,
of The and
culture medium consisted of 10 per cent calf serum 01 10 per cent maternal serum in medium 199 (Wellcome) and the cells were grown on glass coverslips in Leighton culture tubes at an initial concentration of 1 X 106cells per culture. A leukocyte-rich fraction was obtained from heparinized (200 U. per milliliter) maternal blood by sedimentation with one tenth its volume of 5 per cent dextran (molecular weight 110,000) for approximately 30 minutes at 4” C. The leukocytes were washed three times in medium 199 and 2 x lo6 cells added to each culture 24 hours after the cultures were set up, the time of addition being taken as day 0. Control cultures received fresh medium containing no maternal leukocytes. In some cases controls were set up with leukocytes from unrelated donors, i.e., male and nulliparous female volunteers. Viability of the trophoblast cultures was assessed on days 1, 3, 4, and 5, a minimum of two cultures being counted on each occasion. The viability test relied on the ability of living cells to adhere to the coverslip and was carried out as described.’ A reduction in the number of target cells in experimental compared with control groups has been attributed to killing and is considered to reflect a cytotoxic effect of the lymphocytes.g The cell counts in each culture have been expressed as a percentage of those in the appropriate controls. Statistical analyses of the data were performed by applying Student’s t test and Wilcoxon’s sum of ranks test.
Volume Number
125 4
Table
I. Viable
Gestation and cellular immunity to trophoblast antigens
trophoblast
cell counts of immature
placentas*
(yr.)
Duration of present pregnancy (wk.)
0
-
14
4
7
8
4
3%
0
-
8
0
-
8
61 73
2
8
14
90 96
79 76
2
4%
12
99 104 122 118
121 122 -
0
-
12
0
-
10
110.2 -c 13.7 66.1 ” 42.8
111.8 k 34.4 51.7 2 43.8
No. of
Day
previous pregnancies
1
3
4
5
I. (i) (ii)
100
111
-
113 5
129 0
(i) (ii) 3. (i) (ii) 4. (i) (ii) 5. (i) (ii) 6. (i) (ii) 7. (i) (ii) 8. (i) (ii) 9. (i) (ii)
100 130
121 151
125 12
167 0
100 95 100 100
144 118
100 48 130 85
114 39 -
100 87
114 63
113 30
-
100 86
85 88
100 97
100 80
111 126
100 102 100 96
92 92 113 112
2.
Mean S.D.
-+ (i)
(ii)
p value
100 98.6
2 14.2
111.4 107.1
+ 17.9 k 26.6
Not
Not
significant
significant
529
Time ehpsed since last
Pregnancr
12
*Immature placentas: normalized viable trophoblast cell counts on days 1, 3, 4, and 5 in cultures incubated with (i) no maternal lymphocytes or (ii) 2 x lo6 maternal lymphocytes.
Results The viable trophoblast cell counts of the nine immature placentas investigated are shown in Table I, together with some clinical details. Levels of significance are those calculated by Wilcoxon’s test. Student’s t test gave a lower level of significance than Wilcoxon’s test in only one case, and this is probably due to the relative independence of the latter of the form of distribution of the data. Significant killing of trophoblast cells by maternal lymphocytes became evident only after 72 hours’ incubation, the difference in the number of viable cells in test and control cultures being significant on days 4 and 5. Maternal lymphocytes showed a wide spectrum of reactivity to homologous trophoblast varying from complete destruction to no cytopathogenicity at all. The viable trophoblast cell counts of the ten mature placentas investigated are shown in Table II. Killing of trophoblast cells by maternal lymphocytes became evident after 24 hours, the difference in the number of viable cells in test and control cultures being significant on days 1, 3, 4, and 5. Again there was considerable variation in the extent of cytotoxicity, but in contrast to the immature situation all the term trophoblast cul-
tures were almost completely destroyed by day 5. The killing of trophoblast, whether immature or term, at between days 3 and 5 did not appear to be related to parity or time lapse between pregnancies. Lymphocytes from third-party individuals also exerted a cytotoxic effect on both early and term trophoblast, significant killing of the trophoblast cells occurring only after 72 hours’ incubation. In all the trophoblast cultures, whatever the gestational age of the placenta, the presence of maternal serum in the medium completely prevented trophoblast lysis.
Comment The lysis by both maternal and unrelated donor lymphocytes of cultured trophoblast cells has been described9 and attributed to a primary sensitization in vitro to trophoblast antigens.8 The nature of this antigenicity remains to be determined but histocompatibility factors may well be involved,’ which is possibly one explanation for the observed wide spectrum of maternal lymphocyte reactivity to trophoblast cells. Experiments in which mother and baby are tissue-typed are under way in order to elucidate this
530
June 15, 1976 Am. J. Obstet. Gynecol.
Taylor et al.
Table II. Viable trophoblast
cell counts of mature
placentas*
Day 1
.
3
4
5
No. of previouc prepunties
Tim elapsed since last pregnancy cvr.,
Duration CI/ present pregnan CT (wk.)
1. (i) (ii)
100 102
85 62
57 4
54 6
2
2%
41
2. (i) (ii)
100 108
100 97
105 11
100 15
0
-
42
3. (i) (ii)
100 91
105 98
88 11
70 7
0
-
39
4. (i) (ii)
100 73
65 19
61 4
64 3
0
36
5. (i) (ii)
100 72
91 75
X8 2
88 4
0
42
6. (i) (ii)
100 92
103 107
102 5
82 4
0
41
7. (i) (ii)
100 96
100 127
92 14
103 18
I
2
40
8. (i) (ii)
100 86
86 101
96 18
93 8
2
1%
40
9. (i) (ii)
100 95
95 75
88 43
78 32
0
-
32
10. (i) (ii)
100 64
75 60
72 27
62 27
3
Mean SD.
+ (i) (ii)
p value *Mature lymphocytes
100 88 + 14.12 -Co.05
> 0.01
90.5 2 12.94 82.1 lr 30.62
84.9 f 16.3 13.9 + 12.77
79.4 -t 16.7 12.4 2 10.29
0.05
placentas: normalized viable trophoblast or (ii) 2 x 10” maternal lymphocytes.
cell counts
problem. The crucial difference in this interaction between trophoblast and maternal cells when immature or mature placentas were studied was the time of appearance of significant trophoblast lysis, i.e., on day 4 in the former case and on day 1 in the latter, suggesting that maternal cellular immunity had developed by 32 weeks’ gestation-the shortest duration of pregnancy investigated in the mature group. This is emphasized by the fact that lymphocytes from unrelated donors had no significant cytotoxic effect during the first 72 hours’ incubation, which has also been reported by other workers.‘O No sensitization, therefore, was detected during the first 3% months of pregnancy, in agreement with the findings of Youtananukorn and associates,” although in both studies the in vitro techniques used may not have detected very low levels of cellular immunity. The apparent change in maternal lymphocyte reactivity could be attributed to maturation of trophoblast antigens rather than ontogeny of the maternal immune response. However, the response of maternal lymphocytes to cultured immature trophoblast suggests that
on days
1, 3, 4, and 5 in cultures
2
incubated
39
with
(i) no maternal
antigens are present on trophoblast cells at this early stage. If quantitative differences were involved, the response of third-party allogeneic lymphocytes to cultured trophoblast would probably alter according to whether early or late trophoblast was involved, and this was not found to be the case. The concept of a change in the maternal immune status would agree with the results of Youtananukorn and associates,6 who demonstrated an ontogony of the maternal immune response with lymphocytes from mothers at various stages of gestation with term placental antigens. It has been found” that reactivity to human placental antigens was maintained in the early postpartum period, i.e., on the fourth day after delivery. In the present study, some of the immature placentas were obtained from parous women (see Table I). The time elapsing between the last previous pregnancy and the present pregnancy ranged from 3% to 8 years, suggesting that detectable levels of cellular immunity had disappeared during this time interval. In the case of humoral immunity, the earliest disappearance of leukoagglutinins from the maternal circulation oc-
I
Volume Number
125 4
curred less than 2 months post partum, 22 per cent of women with such antibodies losing them by the third year. ‘r The apparent alteration as pregnancy proceeds in the immune status of the mother, which may have to occur de nova in each gestation, emphasizes the concept of a changing balance of mechanisms to ensure survival of the fetal allograft. The evidence of maternal cellular immunity to fetal antigens also implies that one
Gestation and cellular immunity to trophoblast antigens
531
function of these mechanisms must be to impede the efferent limb of the immune response, possibly by the production of enhancing antibodies or immune complexes. I2 This is supported by the finding in the present study that the presence of maternal serum in the cultures inhibits trophoblast lysis, a protective effect which probably has specific and nonspecific components.*
REFERENCES
1. Billingham, R. E.: The transplantation biology of mammalian gestation, AM. J. OBSTET. GYNECOL. 111: 469, 1971. 2. Hulka, J. F., Hsu, K. C., and Beiser, S. M.: Antibodies to trophoblast during the post partum period, Nature 191: 510, 1961. 3. Terasaki, P. I., Mickey, M. R., Yamazaki, J. N., and Vredevoe, D.: Maternal fetal incompatibility. I. Incidence of HL-A antibodies and possible association with congenital anomalies, Transplantation 9: 538, 1970. 4. Youtananukorn, V., and Matangkasombut, P.: Human maternal cell-mediated immune reaction to placental antigens, Clin. Exp. Immunol. 11: 549, 1972. K. E., Hellstrom, I., and Brawn, J.: Abroga5. Hellstrom, tion of cellular immunity to antigenically foreign mouse embryonic cells by a serum factor, Nature 224: 914, 1969. Matangkasombut, P., 6. Youtananukorn, V., and Osathanondh, V.: Onset of human maternal cellmediated immune reaction to placental antigens during the first pregnancy, Clin. Exp. Immunol. 16: 593, 1974. P. V., and Hancock, K. W.: Viability of human 7. Taylor,
a. 9.
10. 11.
12. 13.
trophoblast in vitro. J. Obstet. Gynaecol. Br. Commonw. 80: 834, 1973. Taylor, P. V., and Hancock, K. W.: Antigenicity of trophoblast and some possible antigen-masking effects during pregnancy, Immunology 28: 973, 1975. Hellstrom, I., and Hellstrom, K. E.: Colony inhibition and cytotoxicity assays, in Bloom, B. R., and Glade, P. R., editors: In Vitro Methods in Ceil-Mediated Immunity, New York and London, 1971, Academic Press, Inc., p. 409. Currie, G. A.: Immunological studies of trophoblast in vitro, J. Obstet. Gynaecol. Br. Commonw. 74841, 1967. Douthwaite, R. M., and Urbach, G. I.: In vitro antigenicity of trophoblast, AM. J. OBSTET. GYNECOL. 109: 1023 1971. Payne, R.: The development and persistence of leukoannlutinins in oarous women. Blood 19: 41 I, 1962. Youta$\ukorn, V.: and Mantagkasombut, P.: Specific plasma factors blocking human maternal cell-mediated immune reaction to placental antigens, Nature New Biol. 242: 110, 1973.