Immunopotentiation reverses the embryotoxic effect of serum from women with pregnancy loss

Vol. 56, No.4, October 1991

FERTILITY AND STERILITY

Printed on ocid-free paper in U.S.A.

Copyright<> 1991 The American Fertility Society

lmmunopotentiation reverses the embryotoxic effect of serum from women with pregnancy loss*t

Mara Zigril, M.Sc.:j:§ Amos Fein, Ph.D.§ Howard Carp, M.B.B.S.§II Vladimir Toder, M.D., Ph.D.~ Sackler Sclwol of Medicine, Tel-Aviv University, Tel-Aviv, and Sheba Medical Center, Tel-Haslwmer, Israel

Objective: To assess the effect of sera of women with habitual abortions (AB) on attachment and spreading of mouse blastocysts in vitro. Design: Expansion, attachment, and spreading were the mouse blastocyst parameters utilized. Deoxyribonucleic acid (DNA) synthesis and cell markers expression were also assayed by autoradiography analysis and the indirect immunofluorescent technique. Setting: Sera were drawn from patients attending a habitual AB clinic in a tertiary care university hospital. Participants: Thiry-nine serum samples were drawn from habitually aborting women and the effect compared with 17 control AB sera. Intervention; Habitually aborting women were immunized with paternal leucocytes; 18 postimmunization sera were also assessed. Outcome and Results: After 48 hours, there was delayed attachment and spreading (4% of test blastocysts spread as compared with 50.5% of controls). This was more profound after 72 hours culture (7 .5% spread as compared with 72.8% of controls). Experimental sera were capable of reducing DNA synthesis, cytokeratin, fibronectin, or placental alkaline phosphata!)e expression by blastocyst cells. Leucocyte immunization of women with habitual ABs, clearly reversed the embryotoxic effect of the sera and enhanced cell markers expression. Conclusions: These data suggest that immunopotentiation may improve blastocyst survival in utero. Fertil Steril 56:653, 1991

Recently, strong evidence has accumulated that recurrent abortion (AB) may have an immunological or serologic basis via mechanisms reviewed by us

Received January 9, 1991; revised and accepted June 13, 1991.

* Supported by the Basic Research Foundation administered by Israel Academy of Sciences and Humanities, Jerusalem, Israel. t Presented at the 4th International Congress of Reproductive Immunology, Kiel, Germany, July 26 to 29, 1989. :j: Present address: IVF Unit, Department of Obstetrics and Gynecology, Rotschild Medical Center, Haifa, Israel. § Department of Embryology and Teratology, Sackler School of Medicine, Tel-Aviv University. II Department of Obstetrics and Gynecology, Sheba Medical Center. 1l Reprint requests: Vladimir Toder, M.D., Ph.D., Department of Embryology and Teratology, Sackler School of Medicine, TelAviv University, Tel-Aviv 69978, Israel. Vol. 56, No. 4, October 1991

elsewhere. 1 Serologic factors have been clearly shown to have an effect on early development. Although human pregnancy heat-treated serum supports the growth of mouse blastocysts in culture, heat-inactivated serum from habitually aborting women has been reported to inhibit mouse blastocyst development in vitro and cause anomalies. 2 Such serum is also capable of inhibiting the development of mouse embryos from the two-cell stage to the blastocyst. 3 Serum from women with other forms of reproductive failure was also found to reduce the number of preimplantation mouse embryos that can survive. 4 Serum from monkeys with pregnancy wastage can cause abnormalities to occur in rat embryos at the head-fold stage of development. 5 Based on the assumption that habitual abortion may be immunologically mediated, many workers (including Zigril et al.

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653

ourselves) have tried to potentiate the immune response to improve the outcome of the subsequent pregnancy. Most reports have claimed such immunization to be highly successful, 1•6- 8 but the mechanism remains obscure. Culture and reimplantation of mouse embryos may serve as a valuable model for identifying various embryotoxic factors in human serum. It could indicate the potential of the blastocyst to implant and develop. In this work, our purpose was multifold: (1) to determine the influence of serum from habitual aborters on the development of the blastocyst and the effect of immunization on embryo culture. Possible mechanisms of action of the sera were evaluated using deoxyribonucleic acid (DNA) synthesis and the expression of various cell markers by the blastocyst in culture, with the aim of assessing the blastocyst culture in vitro as a valid model for defining the population whose miscarriages are immunologically mediated and the potential effects of immunopotentiation of such patients. The following parameters were assessed: microscopic morphology, DNA synthesis, and the expression ofthe cell markers (cytokeratin, fibronectin, and placental alkaline phosphatase) shown in indirect immunofluorescent analysis. MATERIALS AND METHODS Collection of Sera

Serum was collected for testing from: (1) 25 women with three or more spontaneous abortions and no children (primary aborters); (2) 14 women with at least one child followed by three or more spontaneous abortions (secondary aborters); (3) 18 recurrently aborting women after leucocyte immunization; (4) 17 normal parous nonpregnant women (all these blood samples were taken in the nonpregnant state); and (5) normal pregnant women. Serum samples were centrifuged at 3,000 rpm for 5 minutes to separate debris, divided into aliquots of 150 ~tL, and stored at -20°C. Heat inactivation (56°C for 30 minutes) was performed on the day of experiment. All sera were collected under identical conditions. No woman was undergoing any drug therapy in the month before collecting serum. Immunization Procedure

Habitually aborting women were immunized by paternalleucocytes as previously described. 8 Before immunization, the presence of the following organ654

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Embryotoxicity and pregnancy loss

isms was excluded: hepatitis B virus, treponema pallidum, cytomegalovirus, and human immunodefficiency virus. Mononuclear cells were prepared for immunization from 100 cc of peripheral blood collected into heparinized tubes. After Ficoll hypaque density centrifugation, the ring at the Ficoll-serum interface was separated and washed. Approximately 70 to 80 X 106 cells were injected subcutaneously and intradermally to multiple injection sites in the forearm. Immunizations were repeated after 4 weeks. The cross-match for antipaternal complement-dependent antibody was repeated, and immunizations were boosted to four per week until antipaternal antibody was produced. Blastocyst Recovery and Embryo Culture

Blastocysts were obtained from 4-week-old ICR strain mice kept on a 14-hour light/10-hour dark cycle. Superovulation was induced by 5 IU Pregnant mare serum gonadotropin intraperitoneally (Organon, Oss, Holland), followed after 48 hours by 5 IU human chorionic gonadotropin (Sigma, St. Louis, MO). Females and males were mated; the presence of a vaginal plug the following morning was evidence of copulation and designated as day 1 of gestation. On day 4 of pregnancy, mice were killed by cervical, dislocation. The uterine horns were isolated and flushed with Biggers, Whitten, and Wittingham's (BWW) medium. Morphologically normal blastocysts were separated into fresh medium and incubated at 37°C in 5% C0 2 in air for 1 hour. Blastocysts were cultured in freshly prepared BWW medium supplemented with 20% voljvol human heat-inactivated serum for 72 hours. Five blastocysts were placed in drops of 10 ~tL medium and cultured on glass cover slips under oil. Morphological Observations

Various developmental stages of the blastocyst were assessed at 24-hour intervals through the binocular dissecting microscope, i.e., expansion or attachment after 24 hours in culture, attachment or spreading after 48 hours of culture, abnormal attachment or spreading after 72 hours in culture. Attachment and spreading were identified according to generally accepted criteria, i.e., attachment, when the blastocysts could not be separated from the Petri dish by gentle agitation; spreading was characterized by migration of trophectoderm when the cell populations could be identified, i.e., inner cell mass and trophoblast. Abnormal attachment was identified according to Chavez & Mclntyre's 2 criteria, i.e., agFertility and Sterility

gregation of the inner cell mass surrounded by only a few trophectodermal cells. Scores were recorded by at least two independent investigations as a proportion of the total number of blastocysts. The investigators were unaware of the clinical status of the patient.

Statistical Analysis

Statistical significance between the experimental and control groups was determined using the paired Student's t-test. The level for a statistically significant difference was set at· P < 0.05. Part of the results were evaluated by the X 2 test.

Deoxyribonucleic Acid Synthesis RESULTS

Deoxyribonucleic acid synthesis was measured by the incorporation of 3 H thymidine 72 hours after incubation. The coverslips were rinsed with BWW medium and incubated for 1 hour in BWW medium supplemented with 1.5 ~-tCi/mL 3 H-thymidine (specific activity 46 Ci/mmol; Nuclear Research Center, Beer-Sheva, Israel). After incorporation, the cultures were fixed for 10 minutes in Carnoy's solution and rinsed in 5% trichloroacetic acid for 5 minutes. The cultures were dried in room temperature and stored in a dark box at -4 °C. Dipping took place in a dark room using emulsion in jel-Ilford K-5 size A (1:2, 42°C; Ilford, Leeds, United Kingdom). Exposure time was 18 days. Development and fixation were performed in the dark room. A positive reaction was seen as black grains on the nuclei of the cells. Cell Markers

The development of cytoskeleton, membrane, and cytoplasmatic enzymatic markers was assessed by indirect immunofluorescence, using monoclonal and polyclonal antibodies. The cultures were fixed in cold acetone, incubated with the first antibody for 30 minutes at 4 oc in a humid camera, and washed three times in phosphate-buffered saline. Cultures were then incubated with fluorescein isothiocynate conjugate (FITC)-labeled secondary antibody for 30 minutes in the dark at 4 °C. The slides were washed covered by 90% glycerol in PBS, and examined un~ der a Zeiss fluorescent microscope. The cultured blastocysts were labeled for the following antigens: (1) cytokeratin: guinea-pig anticytokeratin immunoglobulin (Ig)G (1:20) (Bio Makor, Rehovot, Israel); (2) fibronectin: mouse antihuman fibronectin (1:200) (Clone FN-15; Bio Makor); (3) placental alkaline phosphatase (PLAP): rabbit antihuman PLAP (1:20) (Clone A268; Dakoppats, Glostrup, Denmark); (4) as secondary antibodies FITC-conjugated rabbit antiguinea pig IgG, (1:80); mouse antirabbit IgG (1:80); or rabbit antimouse IgG (1:80) (Bio Makor) were used; and (5) appropriate normal sera as the first antibody (1:2,500) (Bio Makor) were used for controls. Vol. 56, No.4, October 1991

Normal Pregnancy

The sera from pregnant women with normal obstetric histories were found to enhance normal mouse blastocyst attachment and outgrowth as compared with nonpregnant AB serum. Blastocysts, after 24 hours of culture, showed promoted development when supplemented with sera from pregnant women; 55.1% of 271 blastocysts reached the attachment stage, as compared with 24.8% of 333 blastocysts when the medium was supplemented by AB nonpregnant sera. This difference was not significant, however (Fig. 1). The enhancing effect of pregnant women's sera was also apparent after 48 hours of culture. Seventy-four percent ofblastocysts reached the spreading stage when cultured in medium supplemented with pregnant sera, as compared with 50.6% of blastocysts cultured in nonpregnant AB serum. This difference was significant (P < 0.05).

Survival In AS

a Pnlgnant Serum

100 % blastocysts developing

80

60

40

20

48 hours

24 hours

72 hours

Time in culture

D

AB pooled serum

-

Pregnant serum

Attachment assessed at 24 hours

Spreading assessed at 48 & 72 hours

Figure 1 The development of mice blastocysts in culture in the presence of pregnant and nonpregnant sera. Zigril et al.

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After 72 hours of culture, 85.5% of blastocysts reached the spreading stage when cultured in medium supplemented with pregnant sera, as compared with 72.8% ofblastocysts cultured in control serum. This difference was not significant.

(P < 0.001). After 72 hours of culture, 54.2% ofblastocysts cultured in the presence of secondary aborters' sera showed abnormal attachment, and only 6.2% reached the spreading stage, compared with 72.8% of blastocysts cultured in the presence of AB serum (P < 0.001).

Primary Aborters

Postimmunization Serum

Blastocyst development was inhibited in cultures supplemented with primary aborters' sera (Table 1). Inhibition of development was seen after 24 hours. No blastocyst reached the attachment stage. When cultured in the presence of AB sera, 24.8% of blastocysts attached. After 48 hours of culture, there was a significant difference at the spreading stage: 4.3% of 481 blastocysts in primary aborters' sera versus 50.6% (control sera). After 72 hours in culture, 57.8% of blastocysts cultured in the presence of primary aborters' sera were still at the attachment stage. There was a significant difference in the number of blastocysts that had reached the spreading stage: 9.0% of blastocysts cultured in primary aborters' sera and 72.8% of those cultured in the presence of control serum.

Blastocysts were cultured in the presence of sera drawn from primary aborters who had been immunized by paternal leucocytes and subsequently developed antipaternal complement-dependent antibody (Table 1). After 24 hours, 33% ofblastocysts cultured in the presence of these sera attached, compared with 0% blastocysts cultured in the presence of primary aborters sera (before immunization). After 48 hours, a larger number of blastocysts reached the spreading stage when supplemented with postimmunization sera to compare with preimmunization sera from primary aborters (35.8% and 4.3%, respectively) (P < 0.001). After 72 hours in culture, there was a significant difference in the proportion of blastocysts at the spreading stage. Significantly more blastocysts developed in the postimmunization sera than preimmunization sera (57.2% and 9.0%, respectively). Similarly, there was a significant decrease in the proportion ofblastocysts showing abnormal attachment (8.8% and 57.8%, respectively). When postimmunization sera was compared with preimmunization sera drawn from the same women (Table 2), there was a significant increase in the number of blastocysts spreading after 72 hours.

Secondary Aborters

Blastocysts incubated with secondary aborters' sera also showed clear inhibition of development (Table 1). After 24 hours of culture, no blastocyst developed to the attachment stage (24.8% of blastocysts incubated in AB control serum reached this stage). After 48 hours in culture, a significant difference was found in the number of blastocysts at the spreading stage: 1.0% of 266 blastocysts cultured in the presence of secondary aborters' sera compared with 50.6% of those supplemented with AB serum

Table 1

Deoxyribonucleic Acid Synthesis

Cultures ofblastocysts in the presence of AB sera were compared with cultures growing in the presence

Embryotoxicity of Sera From Habitual Aborters After Immunization With Paternal Leukocytes• 72h 24 h

Sera Control ABb (n = 17) Primary aborters (n = 25) Secondary aborters (n = 14) After immunization (n = 18)

48h

No. of blastocysts

Expanded

Attachment

Attachment

Spreading

333

65.0 ± 7.7

24.8 ± 8.0

36.1 ± 3.7

50.6 ± 6.8

481

73.7 ± 4.6

49.4±5.7

4.3 ± 2.6c

57.8 ± 7.9

9.0 ± 3.9c

266

85.8 ± 4.7

67.8 ± 6.4

1.0 ± 0.7c

54.2 ± 11.6

6.2 ± 4.3c

341

61.5 ± 8.0

49.0 ± 4.2

35.8 ± 3.7d

8.8 ± 3.3c

57.2 ± 4.5d

33.2 ± 8.0d

• Values are percentage means ± SE. b Control AB serum was obtained from parous nonpregnant women.

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Abnormal attachment

Spreading

72.8 ± 6.7

c Significantly different from control sera (P < 0.05). d Significantly different from primary aborters sera (P < 0.05).

Fertility and Sterility

Table 2

The Effect of Immunization on the Embryotoxic Effect of Sera From the Same Women With Pregnancy Loss

Sera

No. of blastocysts

Expanded

1. Preimmunization Postimmunization 2. Preimmunization Postimmunization 3. Preimmunization Postimmunization 4. Preimmunization Postimmunization 5. Preimmunization Postimmunization

20 19 10 20 23 20 10 20 20 18

95.0 100 40.0 40.0 95.6 70.0 30.0 20.0 30.0 44.4

a

Attachment

50.0 30.0 70.0 55.5

Attachment

Spreading

90.0 63.1 10.0 45.0 78.2 55.0 50.0 40.0 30.0 55.5

Abnormal attachment

Spreading

90.0 5.2

52.6

55.0 17.3 45.0

70.0 17.3 80.0

73.9 50.0

40.0

85.0

38.8

5.5

72.2

Values are percents.

of primary or secondary aborters' sera with respect to DNA synthesis in the presence of AB serum; 55% of the nuclei were labeled. In contrast, in the presence of primary or secondary aborters sera, labeling was only seen in 23.5% and 34.5%, respectively (Table 3). Cell Markers

Three cell markers, cytokeratin, fibronectin, and PLAP, were examined in the blastocyst cultures using indirect immunofluorescent analysis (Table 4). Monoclonal or polyclonal antibodies to these three antigens were used. As a negative control, appropriate normal sera were used as the first antibody. Cytokeratin

Clear differences were found between blastocyst cultures growing in the presence of AB sera and sera from primary or secondary aborters. Blastocysts cultured in the presence of AB control sera showed strong staining of the trophoblast cells. Staining appeared in the keratin type filamentous organization (not shown). Blastocysts cultured in the presence of sera from habitual aborters showed much weaker staining. Blastocysts cultured in medium supple-

Table 3 The Effect of Sera From Primary or Secondary Aborters on DNA Synthesis in Blastocyst Cultures Sera

Labeled

AB control Primary aborters Secondary aborters

71/129 (55.0) a 16/68 (23.25) 28/81 (34.5)

a

72 h

48h

24h

Values in parentheses are percents.

Vol. 56, No.4, October 1991

Significance

x2

=

x2 =

17.93 P < 0.0001 8.37 P < o.oos

mented with sera from habitual aborters after immunization showed stronger staining of the trophoblast cells. More filaments could be seen than in cultures containing preimmunization sera (Table 4). Fibronectin

The various cultures showed different staining patterns. Trophoblast cells of blastocysts cultured in the presence of AB sera showed clear and strong membranal staining as did inner cell mass cells and the cells between the inner cell mass and trophoblast (not shown). In blastocysts cultured in the presence of habitual aborters sera, there was weak staining of inner cell mass cells and very faint staining of trophoblast cells. Blastocysts cultured in the presence of sera from habitual aborters after immunization showed stronger and clearer staining of inner cell mass and trophoblast cells (Table 4). Placental Alkaline Phosphatase

Cultures supplemented with control sera showed granular staining of the cytoplasm of trophoblast cells and of the cells between the inner cell mass and trophoblast (not shown). The inner cell mass also showed positive but weaker staining for PLAP. Cultures of blastocysts supplemented with sera from

Table 4 Cell Markers' Expression on Murine Blastocyst in Vitro Treatment

Cytokeratin

Fibronectin

PLAP

Control medium Habitual aborters' sera Postimmunization sera

++++

++ ++

++

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++

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primary aborters before the immunization showed much weaker staining of the trophoblast and the border between the cells. Blastocysts cultured in the presence of sera after immunization showed less granular staining than the controls but stronger staining of the trophoblast (Table 4). DISCUSSION

This work assessed the influence of sera from women with successful pregnancy, habitual abortion, and women after paternal leucocyte immunization on the in vitro growth of mouse preimplantation blastocysts. Blastocysts developed normally when cultured in medium supplemented with AB control sera. Supplementing the culture medium with sera from pregnant women enhanced blastocyst development, at least in the attachment and spreading stages. One possible explanation for the enhanced blastocyst development could be that in pregnancy various growth factors are present, such as pregnancy-associated growth factor, which is a T-dependent polyclonal activator of B cells. 9 Human placenta has been reported to be an excellent source of colony-stimulating factors and is capable of stimulating the proliferation of decidua and trophoblast cells in a dose-dependent fashion. 10 Cultures supplemented with sera from primary and secondary habitual aborters clearly inhibited blastocyst development. This first became apparent at the attachment stage after 24 hours in culture. After 48 hours in culture, an abnormal attachment stage was seen, characterized by damaged trophoblast cells but with no visible damage to the inner cell mass. The spreading stage was also significantly inhibited. These results concur with those of Chavez and Mclntyre 2 as far as primary aborters are concerned. However, in this series, a similar picture was seen with secondary aborters, whereas Chavez and Mclntyre 2 found this effect in primary aborters alone. In the present study, the inhibition of blastocyst development could be seen at the level of DNA synthesis and cell markers. Blastocyst cultures supplemented by sera from primary and secondary habitual aborters showed a decrease of 3 H -thymidine-labeled nuclei in trophoblast cells. There was inhibition of the expression of the cytokeratin-cytoskeleton marker, associated with cell differentiation.U Because this cytoskeleton is the first type of intermediate filament expressed during early mouse development, 11 the cytoskeleton of the trophoblast cells could be one of the targets for the sera from 658

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habitual aborters to attack. On this basis, it can be assumed that trophoblast damaged in its cytoskeleton could fail to implant normally. There was inhibition in the expression of fibronectin-membrane marker. Fibronectin, either present in the serum used in the growth medium or synthesized by the cells promotes adhesion and spreading of cells on the growth substratum. 12•13 In cultured mouse blastocysts, fibronectin was found in the trophoblast cells, particularly at the sites of cell spreading and in the inner cell mass. Consequently, it has been suggested that the adhesiveness of the fibronectin helps the blastocyst to attach to the endometrium. The character of cell migration may also assist the trophoblast to grow and penetrate. 12 This is supported by the observation that in the early human chorionic villi, fibronectin is localized to the trophoblastic basement membranes. 13 Placental akaline phosphatase activity was also inhibited. In the preimplantation mouse, PLAP activity has been reported between the 8 and 16-cell stages and continues until the late morula stage. 14 Placental alkaline phosphatase activity is greatly reduced during blastocyst formation but reappears in relatively high levels in trophoblast derivatives of early postimplantation embryos; it then becomes one of the predominant glycoproteins located at the membrane surface of placental microvilli. 15 Placental alkaline phosphatase has been observed in tumors of trophoblastic and nontrophoblastic origin. 16 It has been reported that transformation of normal to malignant trophoblast is associated with expression of a distinct placental type alkaline phosphatase gene product. 17 Therefore, the inhibition of blastocyst development by serum from habitually aborting women could be because of moderation of the production or function of vital enzymes such as PLAP, which are important in implantation. Therefore sera from habitual aborters may influence various levels of trophoblast development. The success of the fetal allograft must depend on preventing a potentially harmful maternal immune response. Women with habitual abortions may have an impaired immune response toward the fetus. Paternal leucocyte immunization has been reported to modulate this situation. In this study, a significant improvement was seen in blastocyst development when the medium was supplemented with sera from women immunized with paternal leucocytes. This was expressed as a significant increase in the number ofblastocysts attaching and spreading, as compared with those before immunization. This improvement was accompanied by an enhanced expression of troFertility and Sterility

phoblast cell markers. Thus, increased cytokeratin filaments, fibronectin, and PLAP expression clearly indicated the improvement of the cytoskeleton structure, cell attachment, and enzymatic functioning of cultured blastocysts. The mechanism of habitual abortion is still unclear. Numerous mechanisms that may be immunologically mediated have been described. 1S-21 Immunization of habitual aborters may enhance the production of growth factors that promote the development of the trophoblast and enhance fetal protection.1o,22,2s At present, no laboratory test is available to diagnose the population who may have immunologically mediated abortions. Immunization at present is entirely empirical. Similarly, after immunization no investigation is available to show that the required change has occurred. Various nonspecific vague indicators of immune function have been used such as changes in mixed lymphocyte reactivity, 24 blocking antibody, 25 and development of antipaternal antibody .1·6•8 The mouse blastocyst culture model could be more specific for the embryotoxic effect of habitual aborters' sera and may be a good indicator of a change after immunization. A clinical trial is currently under way to clarify the correlation between the results obtained in this model and the clinical outcome of pregnancy.

REFERENCES 1. Carp HJA, Toder V, Gazit E, Orgad S, Mashiach S, Serr D, Nebel L: Paternal leucocyte immunization and habitual abortion. Contemp Rev Obstet Gynaecol 1:49, 1988 2. Chavez DJ, Mcintyre JA: Sera from women with histories of repeated pregnancy losses cause abnormalities in mouse perimplantation blastocysts. J Reprod Immunol 6:273, 1984 3. Oksenberg JR, Brautbar C: In vitro suppression of murine blastocysts growth by sera from women with reproductive disorders. Am J Reprod Immunol Microbiol 11:118, 1986 4. Porter AJ, Singh SM, Jung JH: Evaluation of serum associated embryotoxicity/mutagenicity in females with reproductive disfunctions using preimplantation mouse embryos in vitro. Mutagenesis 3:403, 1988 5. Klein NW, Plenefish JD, Carey SW, Federickson WT, Saclett GB, Burlacher TM, Parker RM: Serum from monkeys with histories of fetal wastage causes abnormalities in cultured rat embryos. Science 215:66, 1982 6. Mowbray JF, Underwood JL, Michel M, Forbes PB, Beard RW: Immunization with paternal lymphocytes in women with recurrent miscarriage. Lancet 2:679, 1987 7. Beer ARE, Semprini AE, Zhu Z, Quebbman JF: Pregnancy outcome in human couples with recurrent spontaneous abortions: HLA antigen profiles; HLA antigen sharing; female serum MLR blocking factors; and paternal leukocyte immunization. Exp Clin Immunogenetics 2:137, 1985

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8. Carp HJA, Toder V, Gazit E, Orgad S, Mashiach S, Serr DM, Nebel L: Immunization by paternalleucocytes for prevention of primary habitual abortion: results of a matched controlled trial. Gynecol Obstet Invest 29:16, 1990 9. Carlino JA, Morse JH: Pregnancy associated growth factor. II. AT-dependent polyclonal activator of human adult peripheral blood lymphocytes (PBL). J Immunol134:1702, 1985 10. Toder V, Altaratz H, Shepshelovitch J, Fein A, Nebel L: Immunoregulation of cytokines at the placenta. In Reproductive Immunology, Materno-fetal Interface Relationship, Vol. 154, Edited by G Chaouat. Paris, Colloque INSERM, 1987, p 131 11. Jackson BW, Grund C, Schmidt E, Burki K, Franke WW, Illmansee K: Formation of cytoskeletal elements during mouse embryogenesis, intermediate filaments of the cytokeratin type and desmosomes in preimplantation embryos. Differentiation 17:161, 1980 12. Yokaichiya T, Hoshiai H, Yajima A: Fibronectin localization in mouse embryos from blastocyst stage to the egg cylinder stage in in vitro. Tohoku J Exp Med 154:261, 1988 13. Yamaguchi Y, Ijemura M, Yoshizava Z, Kurosawa K, Yoshinagaci K, Sato A, Suzaki M: Changes in the distribution of fibronectin in the placenta during normal human pregnancy. Am J Obstet Gynecol 152:715, 1985 14. Johnson LV, Galarco PB, Siiebect NL: Alkaline phosphatase activity in the preimplantation mouse embryo. J Embryo! Exp Morpho! 40:83, 1977 15. Carlson RW, Wada HG, Sussman NH: The plasma membrane of human membrane of human placenta. J Bioi Chern 251:4139, 1976 16. Fishman NH: Perspectives on alkaline phosphatase isoenzymes. Am J Med 56:617, 1979 17. Ovitt CE, Strauss AW, Alpers DH, Chou JV, Bairn I: Expression of different sized placental alkaline phosphatase in placenta and choriocarcinoma cells. Proc Nat! Acad Sci USA 83:3781, 1986 18. Mowbray JF, Gibbings CR, Sidgewick AS, Ruzkiewicz M, Beard RF: Effect of blood transfusion in women with recurrent abortion. Transplant Proc 10:896, 1983 19. Underwood JL, Ruskiewicz M, Barnden KL, Beard RW, Mowbray JF, Sanderson AR: Does antigenic modulation cause the absence of major histocompatility complex antigens on the syncytiotrophoblast. Transplant Proc 17:921, 1985 20. Singal DP, Fagnili L, JosephS: Blood transfusion induces antiidiotypic antibodies in renal transplant patients. Transplant Proc 15:1005, 1983 21. Clark DA, Mowbray J, Underwood L, Liddel H: Histopathologic alterations in the decidua in human spontaneous abortion: loss of cells with large cytoplasmic granules. Am J Reprod Immunol Microbiol 13:19, 1987 22. Wegmann TG: Fetal protection against abortion. Is it immunosuppression or immunostimulation? Res Immunol 135D:309, 1984 23. Clark DA, Chaput A, Walker C, Rosenthal K: Active suppression of host versus graft reaction in pregnant mice. Soluble inhibitor of the response to interleukin and obtained from decidua associated non T cells. J Immunol 134:1659, 1985 24. Beer AE: Immunologic aspects of normal pregnancy and recurrent spontaneous abortion. Semin Reprod Endocrinol 6: 163, 1988 25. Unander AM, Lindholm A: Transfusions of leucocyte rich erythrocyte "concentrates": a successful treatment in selected cases of habitual abortion. Am J Obstet Gynecol 154:516, 1986

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