British Journal of Obstetrics and Gynaecology April 1975. Vol. 82. pp. 262-264.
IMMUNOGLOBULIN M IN HUMAN AMNIOTIC FLUID AND ITS POSSIBLE ASSOCIATION WITH NEURAL-TUBE MALFORMATIONS BY
A. A. CANTUARIA Lecturer in Obstetrics and Gynaecology, University of Brasilia-DF., Visiting Research Fellow at the University of Newcastle upon Tyne AND
A. L. JONES Lecturer in Human Genetics, Department of Human Genetics, University of Newcastle upon Tyne Summary Immunoglobulin M (IgM) was found in the amniotic fluid from two fetuses with neural tube defects but not in that from normal fetuses. The possible associations of IgM with abnormalities of the central nervous system are discussed. THE quantitation of' a-fetoprotein (AFP) in amniotic fluid has enabled neural tube abnormalities to be detected antenatally with a high degree of specificity (Brock and Sutcliffe, 1972; Lorber et al., 1973; Nevin et al., 1973; Seller er al., 1973; Lindsey et al., 1973). However, the availability of additional indicators to couple with AFP estimation is highly desirable in view of the responsibility involved in terminating a pregnancy. The present preliminary report concerns the results of screening amniotic fluid for a number of other proteins in addition to AFP.
to have a lumbar myelomeningocoele; amniotic fluid was taken by aspiration from the intact amniotic sac. Transabdominal amniocentesis was used to obtain amniotic fluid from patient K at 16 weeks and on subsequent termination the fetus was observed to have a spina bifida. The remaining nine patients had uneventful pregnancies following amniocentesis. Quantitative estimations of albumin, immunoglobulin A @A), IgG, IgM, a-2-macroglobulin and AFP in the amniotic fluid were made by means of the single radial immunodiffusion technique (Mancini et al., 1965) using commercially available plates (Behringwerke AG).
AND METHODS MATERIALS AMNIOTIC fluid specimens were obtained from 11 patients and after centrifugation the supernatant was stored at -20 "C.All specimens except one (patient G) were sent to the Department of Human Genetics, University of Newcastle upon Tyne, for antenatal diagnosis of genetic disorders. Following termination by hysterectomy for multiple fibroids in patient G, the fetus proved
RESULTS The quantitative estimations of the proteins are summarized in Table I. Two specimens, G and K, had concentrations of AFP of 153 and 330 pg./ml. respectively. Albumin was detectable in all specimens with a range of 24 to 386 mg./ 100 ml. ; ct-2-macroglobulin was not detected in any specimen. Immunoglobulin A was undetectable in three patients and IgG in one. Immunoglobulin M was present in the specimens G and K at levels of 7.1 and 6 . 9 mg./100 ml. respectively, while in the remaining specimens it was not detectable.
A.A.C. was supported by a grant from CoordenaC5o do AperfeiGoamento de Pessoal de Nivel Superior (CAPES), Brazil. 262
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TABLE I Concentration of'Albumin, a2-Macroglobulin, IgA, IgC, IgM and AFP in amniotic fluid
A B C D E
F G H I J K
316 386 112 322 380 360 24 385 N.T. N.T. 3 50
N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.T. N.T. N.D.
2.0 0.8 N.D. 0.8 N.D. 1.3 N.D. 0.1 N.T. N.T. 0.6
32 26 6.5 39 32 35 N.D. 30 N.T. N.T. 32
N.D. N.D. N.D. N.D. N.D. N.D. 7.1 N.D. N.D. N.D. 6.9
25 25 17 19 24 19 153 29 22 35 3 30
N.D. = Not Detectable; N.T. = Not Tested Albumin, a2-macroglobulin and AFP measured on M Partigen plates; IgA and IgM measured on L. C.-Partigen plates; IgG measured on Tri-Partigen plates.
DISCUSSION Of the proteins studied other than AFP only IgM showed any increased level associated with neural tube defect. It is known that the human fetus can synthesize IgM in the liver from as early as 10: weeks (Gitlin and Biasucci, 1969), but that the levels of immunoglobulins, mainly IgM, produced by the fetus remain very low until after birth (van Furth et al., 1965). However, the fetus appears to be immunologically competent from about 20 weeks as intrauterine infections such as rubella lead to an increased IgM response after this time (Alford, 1965; Bellanti et al., 1965). IgM was not detectable in the amniotic fluid of the normal fetuses in the present series. This is in agreement with the work of Gitlin and Biasucci (1969) who were unable to detect IgM in amniotic fluid of 6k to 38 weeks gestation in spite of 20 to 40 times concentration. Similarly, Fischbacher and Quinlivan (1970) and Curl (1971) failed to detect IgM in amniotic fluid at term. The elevated IgM levels found in amniotic fluid from fetuses with neural tube defects were an unexpected finding for which there is no immediate explanation. It would appear unlikely that at 12 to 16 weeks the very low concentration of IgM in the serum of normal fetuses (van Furth et al., 1965) would be sufficient to account for the levels found in amniotic fluid in the cases
with open neural tube defects. This, however, might not be the case if IgM levels are raised in the serum of such fetuses. At present we have no information as to whether this is so or not. Maternal blood can be excluded as a source of IgM because a-Zmacroglobulin was not detected, and further, specimen G was obtained by aspiration from an intact amniotic sac after being carefully cleaned. There remains the possibility of local production of immunoglobulin within the central nervous system. Evidence for this comes from the observation that raised levels of IgM occur in the cerebrospinal fluid of patients with sleeping sickness (Greenwood and Whittle, 1973). Similarly, Lord et al. (1973) found a high level of IgM in the cerebrospinal fluid of a patient suffering from a combined immunodeficiency disease who had developed a chronic progressive encephalopathy. They concluded that the synthesis of IgM was restricted to lymphoid cells in the central nervous system because no IgM was detected in the serum. In view of these observations, it is reasonable to suppose that the lymphoid cells of the fetal central nervous system might produce IgM if exposed to antigenic stimulation, an occurrence which does not normally take place during early intrauterine life. Thus, if the amniotic fluid contains substances which are antigenic to the
264
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fetus, direct contact of this fluid with the central nervous system may give rise to a local production of IgM. In conclusion, these observations suggest that the presence of JgM in the amniotic fluid of two fetuses with open neura1 tube defects was probably the result of a direct stimulus to the lymphoid tissue of the central nervous system by foreign antigens in the amniotic fluid. Further work is in progress to confirm the validity of the present observations. REFERENCES Alford, C. A. (1965): American Journal of Diseases of Children, 110,455. Bellanti, J. A., Artenstein, M. S., Olsen, L., Buescher, E. L., Luhos, C. E., and Milstead, K. L. (1965): American Journal of Diseases of Children, 110,464. Brock, D. J. H., and Sutcliffe, R. G. (1972): Lancet, 2, 197. Curl, C. W. (1971): American Journal of Obstetrics and Gynecology, 109,408.
Fischbacher, P. H., and Quinlivan, W. L. G. (1970): American Journal of Obstetrics and Gynecology, 108, 1051. Gitlin, D., and Biasucci, A. (1969): Journal of Clinical Investigation, 48,1433. Greenwood, E. M., and Whittle, H. C. (1973): Lancet, 2, 525. Liudsey, D. A., Ferguson-Smith, M. A., Donald, I., Sweet, E. M., and Gibson, A. A. M. (1973): Lancet, 2, 522. Lorber, J., Stewart, C. R., m d Milford Ward, A. (1973): Lancet, 1,1187. Lord, R. A., Dupree, E., Goldblum, R. M., Storey, W. D., Forman, P. M., and Goldman, A. S. (1973): Lancet, 2, 528. Mancini, D. A., Carbonara, A. O., and Heremans, J. F. (1965): Zmmunochemistry,2,235. Nevin, N. C., Nesbitt, S., and Thompson, W. (1973): Lancet, 1, 1383. Seller, M. J., Campbell, S., Coltart, T. M., and Singer, J. D. (1973): Lancet, 2,73. Van Furth, R., Schuit, H. R. E., and Hijmans, W. (1965): Journal of Experimental Medicine, 122, 1173.
IMMUNOGLOBULIN M IN HUMAN AMNIOTIC FLUID AND ITS POSSIBLE ASSOCIATION WITH NEURAL-TUBE MALFORMATIONS BY
A. A. CANTUARIA Lecturer in Obstetrics and Gynaecology, University of Brasilia-DF., Visiting Research Fellow at the University of Newcastle upon Tyne AND
A. L. JONES Lecturer in Human Genetics, Department of Human Genetics, University of Newcastle upon Tyne Summary Immunoglobulin M (IgM) was found in the amniotic fluid from two fetuses with neural tube defects but not in that from normal fetuses. The possible associations of IgM with abnormalities of the central nervous system are discussed. THE quantitation of' a-fetoprotein (AFP) in amniotic fluid has enabled neural tube abnormalities to be detected antenatally with a high degree of specificity (Brock and Sutcliffe, 1972; Lorber et al., 1973; Nevin et al., 1973; Seller er al., 1973; Lindsey et al., 1973). However, the availability of additional indicators to couple with AFP estimation is highly desirable in view of the responsibility involved in terminating a pregnancy. The present preliminary report concerns the results of screening amniotic fluid for a number of other proteins in addition to AFP.
to have a lumbar myelomeningocoele; amniotic fluid was taken by aspiration from the intact amniotic sac. Transabdominal amniocentesis was used to obtain amniotic fluid from patient K at 16 weeks and on subsequent termination the fetus was observed to have a spina bifida. The remaining nine patients had uneventful pregnancies following amniocentesis. Quantitative estimations of albumin, immunoglobulin A @A), IgG, IgM, a-2-macroglobulin and AFP in the amniotic fluid were made by means of the single radial immunodiffusion technique (Mancini et al., 1965) using commercially available plates (Behringwerke AG).
AND METHODS MATERIALS AMNIOTIC fluid specimens were obtained from 11 patients and after centrifugation the supernatant was stored at -20 "C.All specimens except one (patient G) were sent to the Department of Human Genetics, University of Newcastle upon Tyne, for antenatal diagnosis of genetic disorders. Following termination by hysterectomy for multiple fibroids in patient G, the fetus proved
RESULTS The quantitative estimations of the proteins are summarized in Table I. Two specimens, G and K, had concentrations of AFP of 153 and 330 pg./ml. respectively. Albumin was detectable in all specimens with a range of 24 to 386 mg./ 100 ml. ; ct-2-macroglobulin was not detected in any specimen. Immunoglobulin A was undetectable in three patients and IgG in one. Immunoglobulin M was present in the specimens G and K at levels of 7.1 and 6 . 9 mg./100 ml. respectively, while in the remaining specimens it was not detectable.
A.A.C. was supported by a grant from CoordenaC5o do AperfeiGoamento de Pessoal de Nivel Superior (CAPES), Brazil. 262
NEURAL-TUBE DEFSCTS AND ISM
263
TABLE I Concentration of'Albumin, a2-Macroglobulin, IgA, IgC, IgM and AFP in amniotic fluid
A B C D E
F G H I J K
316 386 112 322 380 360 24 385 N.T. N.T. 3 50
N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.T. N.T. N.D.
2.0 0.8 N.D. 0.8 N.D. 1.3 N.D. 0.1 N.T. N.T. 0.6
32 26 6.5 39 32 35 N.D. 30 N.T. N.T. 32
N.D. N.D. N.D. N.D. N.D. N.D. 7.1 N.D. N.D. N.D. 6.9
25 25 17 19 24 19 153 29 22 35 3 30
N.D. = Not Detectable; N.T. = Not Tested Albumin, a2-macroglobulin and AFP measured on M Partigen plates; IgA and IgM measured on L. C.-Partigen plates; IgG measured on Tri-Partigen plates.
DISCUSSION Of the proteins studied other than AFP only IgM showed any increased level associated with neural tube defect. It is known that the human fetus can synthesize IgM in the liver from as early as 10: weeks (Gitlin and Biasucci, 1969), but that the levels of immunoglobulins, mainly IgM, produced by the fetus remain very low until after birth (van Furth et al., 1965). However, the fetus appears to be immunologically competent from about 20 weeks as intrauterine infections such as rubella lead to an increased IgM response after this time (Alford, 1965; Bellanti et al., 1965). IgM was not detectable in the amniotic fluid of the normal fetuses in the present series. This is in agreement with the work of Gitlin and Biasucci (1969) who were unable to detect IgM in amniotic fluid of 6k to 38 weeks gestation in spite of 20 to 40 times concentration. Similarly, Fischbacher and Quinlivan (1970) and Curl (1971) failed to detect IgM in amniotic fluid at term. The elevated IgM levels found in amniotic fluid from fetuses with neural tube defects were an unexpected finding for which there is no immediate explanation. It would appear unlikely that at 12 to 16 weeks the very low concentration of IgM in the serum of normal fetuses (van Furth et al., 1965) would be sufficient to account for the levels found in amniotic fluid in the cases
with open neural tube defects. This, however, might not be the case if IgM levels are raised in the serum of such fetuses. At present we have no information as to whether this is so or not. Maternal blood can be excluded as a source of IgM because a-Zmacroglobulin was not detected, and further, specimen G was obtained by aspiration from an intact amniotic sac after being carefully cleaned. There remains the possibility of local production of immunoglobulin within the central nervous system. Evidence for this comes from the observation that raised levels of IgM occur in the cerebrospinal fluid of patients with sleeping sickness (Greenwood and Whittle, 1973). Similarly, Lord et al. (1973) found a high level of IgM in the cerebrospinal fluid of a patient suffering from a combined immunodeficiency disease who had developed a chronic progressive encephalopathy. They concluded that the synthesis of IgM was restricted to lymphoid cells in the central nervous system because no IgM was detected in the serum. In view of these observations, it is reasonable to suppose that the lymphoid cells of the fetal central nervous system might produce IgM if exposed to antigenic stimulation, an occurrence which does not normally take place during early intrauterine life. Thus, if the amniotic fluid contains substances which are antigenic to the
264
A. A. CANTUARIA AND A. L. JONES
fetus, direct contact of this fluid with the central nervous system may give rise to a local production of IgM. In conclusion, these observations suggest that the presence of JgM in the amniotic fluid of two fetuses with open neura1 tube defects was probably the result of a direct stimulus to the lymphoid tissue of the central nervous system by foreign antigens in the amniotic fluid. Further work is in progress to confirm the validity of the present observations. REFERENCES Alford, C. A. (1965): American Journal of Diseases of Children, 110,455. Bellanti, J. A., Artenstein, M. S., Olsen, L., Buescher, E. L., Luhos, C. E., and Milstead, K. L. (1965): American Journal of Diseases of Children, 110,464. Brock, D. J. H., and Sutcliffe, R. G. (1972): Lancet, 2, 197. Curl, C. W. (1971): American Journal of Obstetrics and Gynecology, 109,408.
Fischbacher, P. H., and Quinlivan, W. L. G. (1970): American Journal of Obstetrics and Gynecology, 108, 1051. Gitlin, D., and Biasucci, A. (1969): Journal of Clinical Investigation, 48,1433. Greenwood, E. M., and Whittle, H. C. (1973): Lancet, 2, 525. Liudsey, D. A., Ferguson-Smith, M. A., Donald, I., Sweet, E. M., and Gibson, A. A. M. (1973): Lancet, 2, 522. Lorber, J., Stewart, C. R., m d Milford Ward, A. (1973): Lancet, 1,1187. Lord, R. A., Dupree, E., Goldblum, R. M., Storey, W. D., Forman, P. M., and Goldman, A. S. (1973): Lancet, 2, 528. Mancini, D. A., Carbonara, A. O., and Heremans, J. F. (1965): Zmmunochemistry,2,235. Nevin, N. C., Nesbitt, S., and Thompson, W. (1973): Lancet, 1, 1383. Seller, M. J., Campbell, S., Coltart, T. M., and Singer, J. D. (1973): Lancet, 2,73. Van Furth, R., Schuit, H. R. E., and Hijmans, W. (1965): Journal of Experimental Medicine, 122, 1173.
