PRENATAL DIAGNOSIS, VOL.
12,609-6 12 (1992)
FIRST-TRIMESTER AMNIOTIC FLUID AND EXTRAEMBRYONIC COELOMIC FLUID ACETYLCHOLINESTERASE ELECTROPHORESIS JAMES CAMPBELL, PETER CASS, NEVILLE WATHEN, ROSANA STONE* AND
NICHOLAS WALD* Combined Academic Departments of Obstetrics, Gynaecology and Reproductive Physiology, St Bartholomew's Hospital, London EClA 7BE, U.K.; *Department of Environmental and Preventive Medicine. Medical College of St Bartholomew's Hospital, Charterhouse Square, London ECI, U.K.
SUMMARY Acetylcholinesterase (AChE) gel electrophoresis was performed on samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasoundguided amniocentesis from 38 women between 8 and 12 weeks of pregnancy. AChE was positive in 33 per cent (12/36) of the amniotic fluid samples; the percentage of positive results decreased as gestation advanced. AChE was positive in 32 per cent (9/28) of the extraembryonic coelomic fluid samples. In 81 per cent (21/26) of matched samples, the AChE results were identical in the two fluids. Amniotic fluid and extraembryonic coelomic fluid AChE electrophoresis cannot be used to diagnose neural tube defects prior to 12 weeks of gestation. KEY WORDS
Acetylcholinesterase Early amniocentesis Amniotic fluid Extraembryonic coelom
INTRODUCTION Early genetic amniocentesis (10-12 weeks) is currently being evaluated in a number of centres (Nevin et d.,1990;Johnson and Godmilow, 1988).A potential advantage over chorionic villus sampling is the ability to screen for neural tube defects using alpha-fetoprotein and acetylcholinesterase. High resolution transvaginal ultrasonography has allowed detailed examination of early fetal development. In the first trimester, the two fluid compartments surrounding the fetus, namely the amniotic cavity and extraembryonic coelom, can be clearly identified and pure samples of amniotic fluid and coelomic fluid can be obtained using transvaginal ultrasoundguided needling techniques. Normal levels of alpha-fetoprotein in both amniotic and coelomic fluid have been established (Wathen el d., 1991). The aim of the present study was to determine whether acetylcholinesterase was present in both amniotic fluid and coelomic fluid in early pregnancy. SUBJECTS AND METHODS Transvaginal ultrasound-guided amniocentesis was performed on 38 women with apparently normal pregnancies prior to therapeutic termination of pregnancy between 8 and 12 weeks' gestation. Consent was obtained in every case and the study was approved by the District Ethical Committee. Women with uncertain menstrual Addressee for correspondence: Mr James Campbell, Department of Gynaecology, St Bartholomew's Hospital, West Smithfield, London ECl, U.K.
0197-385 1/92/070609-04$07.00 0 1992 by John Wiley & Sons, Ltd.
Received I0 September 1991 Accepted 6 December 1991
610
J. CAMPBELL ET AL.
Table 1. AChE electrophoresis results in samples of amniotic fluid and extraembryonic coelomic fluid at 8-12 weeks' gestation Gestation (weeks)
N
8
2
9
10
18 10
11 12
3 3
Amniotic fluid AChE+ AChE2 9 1
0 0
0 9 9 3 3
N
3 16
I 2 0
Coelomic fluid AChE+ AChE2 5 1 1
0
1 11 6 1
0
data or vaginal bleeding were excluded from the study. The lower genital tract was prepared with Betadine antiseptic solution. Transvaginal ultrasound was performed using an Aloka SSD 620 with a 5 mHz vaginal probe giving a 60" viewing angle. Gestational age was confirmed by measurement of the crown-rump length and fetal heart activity was identified in all cases. Ultrasound-guided needle aspiration of amniotic fluid and extraembryonic coelomic fluid was performed using an 18-gauge needle guide and a 22 cm 18-gauge needle with trocar (Casmed, U.K.). The needle was advanced through the vaginal vault and myometrium into either the amniotic fluid or the coelomic fluid, care being taken to avoid the placenta, cord, and fetus. After collection of the first sample, the trocar was reintroduced and the needle guided into the second fluid compartment. The first 2 ml aspirated was discarded to avoid contamination. Contaminated blood samples were also discarded. The coelomic fluid was in general noted to be straw-coloured, whilst the amniotic fluid appeared pale and clear. Matched samples of amniotic and coelomic fluid were obtained in 26 cases. Only amniotic fluid or coelomic fluid was obtained in 10 cases and 2 cases, respectively. Acetylcholinesterase (AChE) was determined by polyacrylamide gel electrophoresis (Smith et al., 1979). A positive AChE result was defined as one associated with the appearance of an unambiguous band in the gel at position 2, inhibited by the specific AChE inhibitor BW284C51. A faint AChE band at position 2 was regarded as negative.
RESULTS The results are shown in Table 1. AChE was positive in 12/36 (33 per cent) amniotic fluid samples; the percentage of positive results decreased as gestation advanced. Acetylcholinesterase was positive in extraembryonic coelomic fluid in 9/28 samples (32 per cent). In 21/26 (81 per cent) matched samples, the AChE result was the same in both amniotic and coelomic fluid. Three cases at 9 weeks' gestation had a positive result in amniotic fluid but a negative coelomic fluid AChE result. One case at 9 weeks had a negative amniotic fluid AChE band but a positive coelomic fluid result, as did one case at 11 weeks' gestation. DISCUSSION We have shown that AChE is present in both amniotic fluid and extraembryonic coelomic fluid in early pregnancy. In most cases, the results were similar in the two
AMNIOTIC AND EXTRAEMBRYONIC COELOMIC FLUIDS
61 1
fluids. The source of AChE in coelomic fluid in the first trimester is unknown. AChE in amniotic fluid is believed to be fetal in origin (Chubb et al., 1979). It is released from both nerve and muscle tissue; secretion from both these sources may diminish as the neural axis matures (Chubb et al., 1979). Muller et al. (1989) have suggested that the neural tube is the main source of amniotic fluid AChE and that following its closure at 6 weeks’ gestation, enzyme levels gradually decrease. It might therefore be expected that AChE levels would be higher in amniotic fluid than in extraembryonic coelomic fluid; the amniotic fluid surrounds the source of AChE (fetus) while it is effectively surrounded by the extraembryonic coelom and separated from it by the amnion. AChE in coelomic fluid may be derived from amniotic fluid or may be transferred from fetal serum via the placenta, yolk sac, and umbilical cord. In our study, most positive AChE samples occurred within 3 weeks of neural tube closure. Cytochemical studies have also shown that AChE may be released in higher quantities in early pregnancy by immature nerve terminals (Chubb et al., 1979) and/or the molecular form of AChE may alter as the fetus matures, making it less soluble (Muller et al., 1985). Samples of amniotic fluid contaminated with fetal blood may have increased AChE levels (Report of the Collaborative Acetylcholinesterase Study, 1981). In this study, we were careful to avoid traumatizing the placenta, cord, and fetus whilst collecting the fluids, and contamination is unlikely as a possible reason for the positive AChE results. Our results are similar to those of Muller et al. (1989), who performed AChE estimation on amniotic fluid samples in early pregnancy (4-1 5 weeks). All samples at 8 weeks or less were positive and all samples after 12 weeks’ gestation were negative. Between 9 and 1 1 weeks, the number of positive specimens progressively decreased. Drugan et al. (1988) tested 18 amniotic fluid samples from pregnancies of less than 13 weeks’ gestation; 13 were negative and the remainder were inconclusive or equivocal. Many centres are currently performing early amniocentesis for karyotyping but relatively few are performed before 12 weeks’ gestation (Lilford, 1991). Although the numbers in our study are small, the results show that amniotic fluid and coelomic fluid AChE electrophoresis cannot be used to diagnose neural tube defects prior to 12 weeks of gestation. REFERENCES Chubb, J.W., Pilowski, P.M.,Springell, H.J. (1979). Acetylcholinesterase in human amniotic fluid; an index of fetal neural development?, Lancet, i, 688490. Drugan, A,, Syner, F.N., Greb, A., Evans, M. (1988). Amniotic fluid alpha-fetoprotein and acetylcholinesterase in early genetic amniocentesis, Obstet. Gynecol., 72,35-38. Johnson, A., Godmilow, L. (1988). Genetic amniocentesis at 14 weeks or less, Clin. Obstet. Gynaecol., 31,345-352. Lilford, R.J.(1991). Invasive diagnostic procedures in the first trimester. In: Drife, J.O., Donnai, D. (Eds). Antenatal Diagnosis of Fetal Abnormalities, London: Springer-Verlag, 79-95. Muller, F., Dumez, Y., Massoulie, J. (1985). Molecular forms and solubility of acetylcholinesterase during the embryonic development of rat and human brain, Brain Res., 33, 295-302. Muller, F., Oury, J. F., Boue, A. (1989). First-trimester amniotic fluid acetylcholinesterase electrophoresis, Prenat. Diagn., 9, 173-1 75.
612
J. CAMPBELL ETAL.
Nevin, J., Nevin, N.C., Dornan, J.C., Sim, D., Armstrong, J.C. (1990). Early amniocentesis: experience of 222 consecutive patients, 1987-1988, Prenat. Diugn., 10,7983. Report of the Collaborative Acetylcholinesterase Study (1 98 1). Amniotic fluid acetylcholinesterase electrophoresis as a secondary test in the diagnosis of anencephaly and open spina bifida in early pregnancy, Lancet, ii, 321-324. Smith, A.D., Wald, N.J., Cuckle, H.S., Stirrat, G.M., Bobrow, M., Lagercrantz, H. (1979). Amniotic fluid acetylcholinesterase as a possible diagnostic test for neural tube defects in early pregnancy, Lancet, i, 685488. Wathen, N.C., Cass, P., Kitau, M.J., Chard, T. (1991). Human chorionic gonadotrophin and alpha-fetoprotein levels in matched samples of amniotic fluid, extraembryonic coeiomic fluid and maternal serum in the first trimester of pregnancy, Prenat. Diagn., 11, 145-151.
12,609-6 12 (1992)
FIRST-TRIMESTER AMNIOTIC FLUID AND EXTRAEMBRYONIC COELOMIC FLUID ACETYLCHOLINESTERASE ELECTROPHORESIS JAMES CAMPBELL, PETER CASS, NEVILLE WATHEN, ROSANA STONE* AND
NICHOLAS WALD* Combined Academic Departments of Obstetrics, Gynaecology and Reproductive Physiology, St Bartholomew's Hospital, London EClA 7BE, U.K.; *Department of Environmental and Preventive Medicine. Medical College of St Bartholomew's Hospital, Charterhouse Square, London ECI, U.K.
SUMMARY Acetylcholinesterase (AChE) gel electrophoresis was performed on samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasoundguided amniocentesis from 38 women between 8 and 12 weeks of pregnancy. AChE was positive in 33 per cent (12/36) of the amniotic fluid samples; the percentage of positive results decreased as gestation advanced. AChE was positive in 32 per cent (9/28) of the extraembryonic coelomic fluid samples. In 81 per cent (21/26) of matched samples, the AChE results were identical in the two fluids. Amniotic fluid and extraembryonic coelomic fluid AChE electrophoresis cannot be used to diagnose neural tube defects prior to 12 weeks of gestation. KEY WORDS
Acetylcholinesterase Early amniocentesis Amniotic fluid Extraembryonic coelom
INTRODUCTION Early genetic amniocentesis (10-12 weeks) is currently being evaluated in a number of centres (Nevin et d.,1990;Johnson and Godmilow, 1988).A potential advantage over chorionic villus sampling is the ability to screen for neural tube defects using alpha-fetoprotein and acetylcholinesterase. High resolution transvaginal ultrasonography has allowed detailed examination of early fetal development. In the first trimester, the two fluid compartments surrounding the fetus, namely the amniotic cavity and extraembryonic coelom, can be clearly identified and pure samples of amniotic fluid and coelomic fluid can be obtained using transvaginal ultrasoundguided needling techniques. Normal levels of alpha-fetoprotein in both amniotic and coelomic fluid have been established (Wathen el d., 1991). The aim of the present study was to determine whether acetylcholinesterase was present in both amniotic fluid and coelomic fluid in early pregnancy. SUBJECTS AND METHODS Transvaginal ultrasound-guided amniocentesis was performed on 38 women with apparently normal pregnancies prior to therapeutic termination of pregnancy between 8 and 12 weeks' gestation. Consent was obtained in every case and the study was approved by the District Ethical Committee. Women with uncertain menstrual Addressee for correspondence: Mr James Campbell, Department of Gynaecology, St Bartholomew's Hospital, West Smithfield, London ECl, U.K.
0197-385 1/92/070609-04$07.00 0 1992 by John Wiley & Sons, Ltd.
Received I0 September 1991 Accepted 6 December 1991
610
J. CAMPBELL ET AL.
Table 1. AChE electrophoresis results in samples of amniotic fluid and extraembryonic coelomic fluid at 8-12 weeks' gestation Gestation (weeks)
N
8
2
9
10
18 10
11 12
3 3
Amniotic fluid AChE+ AChE2 9 1
0 0
0 9 9 3 3
N
3 16
I 2 0
Coelomic fluid AChE+ AChE2 5 1 1
0
1 11 6 1
0
data or vaginal bleeding were excluded from the study. The lower genital tract was prepared with Betadine antiseptic solution. Transvaginal ultrasound was performed using an Aloka SSD 620 with a 5 mHz vaginal probe giving a 60" viewing angle. Gestational age was confirmed by measurement of the crown-rump length and fetal heart activity was identified in all cases. Ultrasound-guided needle aspiration of amniotic fluid and extraembryonic coelomic fluid was performed using an 18-gauge needle guide and a 22 cm 18-gauge needle with trocar (Casmed, U.K.). The needle was advanced through the vaginal vault and myometrium into either the amniotic fluid or the coelomic fluid, care being taken to avoid the placenta, cord, and fetus. After collection of the first sample, the trocar was reintroduced and the needle guided into the second fluid compartment. The first 2 ml aspirated was discarded to avoid contamination. Contaminated blood samples were also discarded. The coelomic fluid was in general noted to be straw-coloured, whilst the amniotic fluid appeared pale and clear. Matched samples of amniotic and coelomic fluid were obtained in 26 cases. Only amniotic fluid or coelomic fluid was obtained in 10 cases and 2 cases, respectively. Acetylcholinesterase (AChE) was determined by polyacrylamide gel electrophoresis (Smith et al., 1979). A positive AChE result was defined as one associated with the appearance of an unambiguous band in the gel at position 2, inhibited by the specific AChE inhibitor BW284C51. A faint AChE band at position 2 was regarded as negative.
RESULTS The results are shown in Table 1. AChE was positive in 12/36 (33 per cent) amniotic fluid samples; the percentage of positive results decreased as gestation advanced. Acetylcholinesterase was positive in extraembryonic coelomic fluid in 9/28 samples (32 per cent). In 21/26 (81 per cent) matched samples, the AChE result was the same in both amniotic and coelomic fluid. Three cases at 9 weeks' gestation had a positive result in amniotic fluid but a negative coelomic fluid AChE result. One case at 9 weeks had a negative amniotic fluid AChE band but a positive coelomic fluid result, as did one case at 11 weeks' gestation. DISCUSSION We have shown that AChE is present in both amniotic fluid and extraembryonic coelomic fluid in early pregnancy. In most cases, the results were similar in the two
AMNIOTIC AND EXTRAEMBRYONIC COELOMIC FLUIDS
61 1
fluids. The source of AChE in coelomic fluid in the first trimester is unknown. AChE in amniotic fluid is believed to be fetal in origin (Chubb et al., 1979). It is released from both nerve and muscle tissue; secretion from both these sources may diminish as the neural axis matures (Chubb et al., 1979). Muller et al. (1989) have suggested that the neural tube is the main source of amniotic fluid AChE and that following its closure at 6 weeks’ gestation, enzyme levels gradually decrease. It might therefore be expected that AChE levels would be higher in amniotic fluid than in extraembryonic coelomic fluid; the amniotic fluid surrounds the source of AChE (fetus) while it is effectively surrounded by the extraembryonic coelom and separated from it by the amnion. AChE in coelomic fluid may be derived from amniotic fluid or may be transferred from fetal serum via the placenta, yolk sac, and umbilical cord. In our study, most positive AChE samples occurred within 3 weeks of neural tube closure. Cytochemical studies have also shown that AChE may be released in higher quantities in early pregnancy by immature nerve terminals (Chubb et al., 1979) and/or the molecular form of AChE may alter as the fetus matures, making it less soluble (Muller et al., 1985). Samples of amniotic fluid contaminated with fetal blood may have increased AChE levels (Report of the Collaborative Acetylcholinesterase Study, 1981). In this study, we were careful to avoid traumatizing the placenta, cord, and fetus whilst collecting the fluids, and contamination is unlikely as a possible reason for the positive AChE results. Our results are similar to those of Muller et al. (1989), who performed AChE estimation on amniotic fluid samples in early pregnancy (4-1 5 weeks). All samples at 8 weeks or less were positive and all samples after 12 weeks’ gestation were negative. Between 9 and 1 1 weeks, the number of positive specimens progressively decreased. Drugan et al. (1988) tested 18 amniotic fluid samples from pregnancies of less than 13 weeks’ gestation; 13 were negative and the remainder were inconclusive or equivocal. Many centres are currently performing early amniocentesis for karyotyping but relatively few are performed before 12 weeks’ gestation (Lilford, 1991). Although the numbers in our study are small, the results show that amniotic fluid and coelomic fluid AChE electrophoresis cannot be used to diagnose neural tube defects prior to 12 weeks of gestation. REFERENCES Chubb, J.W., Pilowski, P.M.,Springell, H.J. (1979). Acetylcholinesterase in human amniotic fluid; an index of fetal neural development?, Lancet, i, 688490. Drugan, A,, Syner, F.N., Greb, A., Evans, M. (1988). Amniotic fluid alpha-fetoprotein and acetylcholinesterase in early genetic amniocentesis, Obstet. Gynecol., 72,35-38. Johnson, A., Godmilow, L. (1988). Genetic amniocentesis at 14 weeks or less, Clin. Obstet. Gynaecol., 31,345-352. Lilford, R.J.(1991). Invasive diagnostic procedures in the first trimester. In: Drife, J.O., Donnai, D. (Eds). Antenatal Diagnosis of Fetal Abnormalities, London: Springer-Verlag, 79-95. Muller, F., Dumez, Y., Massoulie, J. (1985). Molecular forms and solubility of acetylcholinesterase during the embryonic development of rat and human brain, Brain Res., 33, 295-302. Muller, F., Oury, J. F., Boue, A. (1989). First-trimester amniotic fluid acetylcholinesterase electrophoresis, Prenat. Diagn., 9, 173-1 75.
612
J. CAMPBELL ETAL.
Nevin, J., Nevin, N.C., Dornan, J.C., Sim, D., Armstrong, J.C. (1990). Early amniocentesis: experience of 222 consecutive patients, 1987-1988, Prenat. Diugn., 10,7983. Report of the Collaborative Acetylcholinesterase Study (1 98 1). Amniotic fluid acetylcholinesterase electrophoresis as a secondary test in the diagnosis of anencephaly and open spina bifida in early pregnancy, Lancet, ii, 321-324. Smith, A.D., Wald, N.J., Cuckle, H.S., Stirrat, G.M., Bobrow, M., Lagercrantz, H. (1979). Amniotic fluid acetylcholinesterase as a possible diagnostic test for neural tube defects in early pregnancy, Lancet, i, 685488. Wathen, N.C., Cass, P., Kitau, M.J., Chard, T. (1991). Human chorionic gonadotrophin and alpha-fetoprotein levels in matched samples of amniotic fluid, extraembryonic coeiomic fluid and maternal serum in the first trimester of pregnancy, Prenat. Diagn., 11, 145-151.
