- Email: [email protected]
Chorionic Villus Sampling
principles and practice
-
Chorionic Villus Sampling JOAN S. HOCCE, MS, W. ALLEN HOGCE, MD, AND MITCHELL S. COLBUS, MD Chorionic villus sampling in the first trimester of pregnancy has the potential to become a major tool in the prenatal diagnosis and therapy of genetic disorders. Villus samples can be used for cytogenetic and biochemical studies as well as DNA analysis. However, little is known about the effects of chorionic villus sampling on a continuing pregnancy, or the long-term effects on the subsequently delivered infants. Despite these limitations, chorionic villus sampling appears to be a major breakthrough in prenatal diagnosis.
Chorionic villus sampling, a technique for obtaining fetal cells for prenatal diagnosis, is being investigated actively by both United States and European researchers as a possible replacement for amniocentesis.’ The idea behind the technique is not new, having been proposed as early as 1968.’ However, amniocentesis was being developed at the same time and quickly became the procedure of choice for prenatal diagnosis because of its relative safety and the ease of obtaining amniotic fluid in the second trimester. Because amniocentesis is a procedure of the second o r third trimester of pregnancy, researchers are now looking to chorionic villus sampling, done in the first trimester, as the future method of prenatal diagnosis. Detection of genetic defects by amniocentesis has contributed greatly to the field of medical genetics in the past decade and a half, but
Accepted: June 1984.
24
chorionic sampling may offer access to prenatal diagnosis to a much larger group of women who d o not find second trimester procedures acceptable. Approximately two weeks after implantation, the human trophectoderm proliferates and produces hundreds of villi that radiate in all directions (Figure 1). Villi on the implanting side of the chorion will form the placenta; villi away from the implantation site will disappear by approximately 14 weeks.3 Chorionic villi, fetal in origin, are a source of fetal genetic information because these cells reflect the chromosomes, enzymology, and DNA content of the fetus. Chorionic villus sampling is done at eight to 12 menstrual weeks because the villi begin to disappear after this period. A sampling of villi is usually obtained transcervically because the uterus is still in the pelvic cavity (Figure 2). Tissue obtained from the end of one or more villi at the site of implantation contains rapidly di-
viding fetal cells that can be analyzed rapidly for chromosomal and biochemical defects.’
HISTORY
In 1968, Mohr proposed fetal tissue sampling in the first trimester by use of a transvaginal hysteroscope.’ Tissue was obtained in seven out of eight attempts, and the only complication of the procedure was a tear in the membranes after biopsy in one case. In 1973, by direct vision with an endoscope, Kullander and Sandahl obtained villus biopsies in 39 patients requesting elective abortion between eight and 20 weeks.4 Specimens were successfully cultured for chromosomal analysis in 19 cases. These 19 patients were followed from seven to 43 days after the biopsy without any pregnancy complications. Kullander a n d Sandahl concluded that the ninth to tenth week of pregnancy offered the best opportunity for transcervical biopsy using the endoscope.
January/February 1986 JOG”
Spiral artery
Secondarv villi ,
j
intervillous space Intervillous
Fetus Amntotic cavity
Decidua capsularis fMfM& rnem-/
Figure 1. Diagram of the human embryo at approximately six weeks gestation. At the embryonic pole, the villi are numerous and well-formed; at the abembryonic pole, the villi are fewer in number and less well-developed.
During the 1970s, researchers continued to experiment with techniques for obtaining chorionic villi. Hahnemann, in 1974, described hysteroscopic biopsy in 95 pregnant women before elective termination of the pregnancy.' In 67 cases, the biopsy was taken immediately before abortion. The other 28 cases were observed for eight days after the biopsy; two of the 28 aborted following the procedure. Only 60% of the biopsies contained chorion or chorionic villi, but cell culture and karyotyp-
ing were successful in all 13 samples placed in culture. Two complications occurred; in one case, the amniotic membrane was punctured during biopsy, and bleeding obscured vision in another case. Rhine eta/.,in 1977, reported the use of the "antenatal cell extractor" for obtaining trophoblastic cells Cells from the endocervical for culture were obtained with a noninvasive procedure similar to that used for obtaining Pap smears. Fetal chromosomes were found in 25% of the samples. Goldberg eta/.
Amniotic cavity Zhorion frondosum
, k-\
Decidua capsularis
Catheter
Syringe/
Figure 2. Diagram of the uterus at 10 weeks gestation with biopsy catheter in position.
January/February 1986 JOGNN
attempted to confirm Rhine's findings but failed to do so, finding that the cultured cells were maternal.' In 1975, the Chinese reported using blind transcervical aspiration of chorionic villi to determine fetal sex.' Because tissue culture facilities were not available, chromosomal aberrations and biochemical abnormalities could not be determined. The sampling was performed at six to 14 weeks of gestation with a 94% accuracy rate in determining sex. The report included 100 pregnancies biopsied for sex determination. Thirty pregnancies were terminated; 29 because of a female prediction. Of the 70 continuing pregnancies, four cases ended in spontaneous abortion. The 1980s have brought more interest in the technique of chorionic villus sampling and its usefulness as a prenatal diagnostic tool. Kazy eta/.,in 1982, published a study of 165 cases in which sampling was performed at approximately six to 12 weeks gestation under real-time ultrasound guidance. They concluded that chorionic villus sampling was a valuable method of early prenatal diagnosis to detect accurately fetal sex and some enzyme defects."' lnterest in chorionic villus sampling was heightened in the United States and Europe when a group of researchers headed by Old and Weatherall used chorionic villi for restriction endonuclease analysis of fetal DNA and successfully diagnosed two fetuses at risk for hemoglobinopathies: one for betathalassemia and one for sickle cell anemia." Gosden et d.reported prenatal sex determination in three to four days by molecular analysis of DNA from chorionic villus specimens." The researchers suggested that rapid determination of sex would be valuable for t h e prenatal diagnosis of X-linked disorders. 25
mia.” This study added further evRecently, interest has increased idence for the safety of the in what chorionic villus sampling procedure. can offer for the future of prenatal diagnosis. Simoni and Brambati reported the diagnosis of trisomy CURRENT TECHNIQUES 21 in an obligate carrier of Duchenne muscular d y ~ t r o p h y . ’ ~ Some of the techniques currently Ward, Modell, and Petrou achieved used for prenatal diagnosis by an 89% success rate of obtaining chorionic villus sampling include specimens with transcervical asdirect vision biopsy with a hyspiration of chorionic villi under t e r o s ~ o p e , a ’ ~ transabdominal apreal-time ultrasound guidance.I4 In proach using aspiration with ultraaddition, Elles et af. showed that sound guidance,” and an aspirachorionic villi at nine to 10 weeks tion technique using a cannula gestation are a source of fetal DNA passed transcervically under realthat can be used for gene analysis time ultrasound guidance.’l Simoni with no detectable contamination eta/.reported four different methby maternal DNA.’’ These reports ods of sampling in an attempt to indicated that the procedure could find the safest and most efficient be done efficiently and that the tistechnique.” The four methods sue obtained yielded accurate reused were an endoscope system, sults. two different catheters for blind Chorionic villus sampling as a aspiration, and the Portex@ cathediagnostic tool became widely ter under ultrasound guidance. The publicized in the lay press in midresearchers concluded that villus 1983 following a report by Pergament et af. on the prenatal diag- aspiration with the Portex catheter under ultrasound guidance was the nosis of Tay-Sachs disease.I6 Tromost efficient technique, resulting phoblastic cells from the at-risk in a 96% success rate for obtaining pregnancy had normal levels of villus samples.” hexosaminidase A activity, which were confirmed in fetal fibroblasts ADVANTAGES obtained when the pregnancy was terminated because chromosome Chorionic villus sampling is a analysis revealed trisomy 16. A case reported by Tsvetkova et af. technique with wide clinical applidemonstrated that the prenatal di- cations. Samples of tissue obtained between eight and 12 weeks gesagnosis of metachromatic leukodystrophy was possible by deter- tation offer sufficient tissue for chromosome analysis, enzyme demining arylsulphatase A activity in terminations, and DNA analysis. villi obtained from a patient who For the diagnosis of most metahad two previous children with bolic disorders, the villi can be asmetachromatic leukodystrophy.” sayed directly for enzyme activity These reports confirmed the apbecause the amount of tissue is sufplicability of villus material for the ficient from the biopsy itself. detection of certain biochemical Couples at risk for a fetus with a disorders. Goossens et al. conchromosomal disorder are the firmed previous report^"*'^*'^ that largest group to which chorionic chorionic villus sampling allowed villus sampling is applicable. By the prenatal diagnosis of sickle cell analysis of the villus cells directly disease before the tenth gestaor after culture, an early diagnosis tional week by obtaining definitive can be made of the fetal karyotype. diagnoses by DNA analysis of choBecause chorionic villi contain rionic villus samples in five pregmany rapidly dividing cells, a nancies at risk for sickle cell ane26
chromosome spread can b e obtained in five to 24 DNA analysis can be carried out on the DNA obtained from chorionic villi for the prenatal diagnosis of hemoglobinopathies such as sickle cell anemia, alpha-thalassemia, and some beta-thalassemias. Because the DNA content of all fetal cells is’ identical, recombinant techniques can be used for first trimester diagnosis and would allow detection of disorders such as alpha-1-antitrypsin deficiency24 and phenylket~nuria.‘~ A Y-specific probe recently developed by Lau et af.allows fetal sexing in less than 24 hours.’6 As more restriction fragment length polymorphisms are found, linkages to genetic disorders may be found even before the biochemical basis of the disorder is under~tood.‘~ The possibilities offered by chorionic villus biopsy for fetal diagnosis in the first trimester may influence parents’ decisions about childbearing. Diagnosis before 12 weeks gestation greatly reduces parents’ emotional stress and the medical complications related to therapeutic abortion in the second trimester. Earlier diagnosis allows a couple more privacy in their pregnancy because it is not yet physically obvious t o others. Earlier diagnosis also may help families overcome some of the legal, social, and religious difficulties associated with amniocentesis and termination in the second trimester. Because the waiting period for obtaining results is significantly reduced (1-2 weeks for chorionic villus sampling as compared to 34 weeks for amniocentesis), families may experience less psychological stress.” The method of termination by suction evacuation may prove much more acceptable to gravidas and the community because of the relative safety of the procedure as compared to prostaglandin abortion in the second trimester.
January/February 1986 JOCNN
Earlier diagnosis holds the potential for improving fetal treatment by allowing earlier intervention. The advent for chorionic villus sampling should, therefore, stimulate more research in the area of fetal therapy, as well as in prenatal diagnosis.
DISADVANTAGES Currently, early fetal development is an unexplored area. Because so little is known about the procedure, the effects on the subsequently delivered infants must be determined. Chronologically, complications may be divided into those that are immediate, short-term, or longterm. One of the immediate complications is failure to obtain tissue. Investigators need to document the number of insertions and aspirations necessary to obtain a sample, and whether the timing of a repeat aspiration changes the loss rate related to the procedure. Another immediate complication related to the procedure is rupture of the membranes with leakage of amniotic fluid. Whether management of this complication should be conservative or immediate termination of the pregnancy should be recommended is unknown. Bleeding after the procedure is another complication, and any bleeding that does occur needs to be well documented as to the amount and duration, along with its relation to subsequent pregnancy loss or other complications. Spontaneous abortion is a shortterm complication. The spontaneous abortion rate could be as low as five to 6 percent2*or as high as 12%.*' However, the incidence of spontaneous abortion at this stage of gestation in control pregnancies demonstrating normal growth and cardiac activity by ultrasound is unknown. Control spontaneous abortion figures will have to be obJanuaryIFebruary1986 JOCNN
tained to determine the risk of the procedure. Infection is another potential problem. Should infection occur, no accepted form of management currently exists. The long-term effects of infection at this early stage of pregnancy, if successfully treated with antibiotics, is unknown. Maternal contamination of the specimen is a possible complication when sampling villi. Karyotyping should be done when assaying for biochemical disorders. For those patients at risk for biochemical disorder, follow-up amniocentesis as a confirmation may be needed until the true accuracy of the procedure is known. Long-term effects of chorionic villus sampling may include rhesus isoimmunization. To evaluate this possibility, a random, controlled study including Rhogam usage is needed. Secondly, careful evaluation of the neonate is necessary to determine any subtle anomalies that may result from chorionic villus sampling. In the case of still birth or neonatal death, an autopsy will be essential. Another important aspect of follow-up is confirmation at birth of the results, especially biochemical results. Other theoretical questions about chorionic villus sampling have been raised. Will an increased incidence of prematurity or placental abruption occur? What will be the long-term effects on the infant's growth? Will more growth retardation and fetal distress be seen as a result of removing villi early in pregnancy? Long-term follow-up needs to be done on these infants, with assessment of their growth, development, and intellectual function at one year of age or more to ascertain any subtle effects. Another important consideration is the possibility of monitoring the pregnancy with ultrasound for short-term or immediate complications. The optimal time for ultra-
sound follow-up is unknown. The expertise of the ultrasonographer will be important for the procedure and for the follow-up studies. Some cytogenetic considerations related to this technique that need to be addressed include 1) a method of efficient separation of chorionic villi from maternal cells, 2) the minimal amount of chorionic villi required for diagnostic testing, and 3) an improvement in the method for obtaining direct chromosome preparations. Currently, direct chromosome preparations do not yield high-quality banded chromosomes. Standardization of laboratory techniques needs t o be developed to ensure accurate and reliable results.
SUMMARY Chorionic villus sampling stands now as the possible future technique of prenatal diagnosis. The opportunity exists for good control studies to be done and for a n international registry of all cases to be established. Continuity of care and complete recording of data by those investigators doing chorionic villus sampling is needed to establish the relative safety of this procedure for both mother a n d fetus, and to document the reliability of the test results. Long-term, complete follow-up will be necessary to answer all of the questions now involved. Chorionic villus sampling has the potential t o become a major tool in the prenatal diagnosis and therapy of genetic diseases.
REFERENCES 1. Kolata G. First trimester prenatal diagnosis. Science 1983;221:1031. 2. Mohr J. Foetal genetic diagnosis: development of techniques for early sampling of foetal cells. Acta Pathol Microbiol Scand 1968;73:73. 3. Rhine SA, Milunsky A. Utilization
27
of trophoblast for early prenatal diagnosis. In: Milunsky A, ed. Genetic disorders and the fetus. New York: Plenum Press, 1979:527. 4. Kullander S, Sandahl B. Fetal chromosome analysis after transcervical placental biopsies during early pregnancy. Acta Obstet Gynecol Scand 1973;52:355. 5. Hahnemann N. Early prenatal diagnosis: a study of biopsy techniques and cell culturing from extraembryonic membranes. CIin Genet 1974;6:294. 6. Rhine SA, Palmer CG, Thompson TF. A simple alternative to amniocentesis for first-trimester prenatal diagnosis. Birth Defects 1977;13: 231. 7. Goldberg MF, Chen ATL, Ahn YW, Reidy JA. First-trimester fetal chromosomal diagnosis using endocervical lavage: a negative evaluation. Am J Obstet Gynecol 1980;138:436. 8. Department of Obstetrics and Gynecology, Tietung Hospital of Anshan Iron and Steel Co., Anshan, China. Fetal sex prediction by sex chromatin of chorionic villi cells during early pregnancy. Chin Med J 1975;1:117. 9. Williamson R, Eskdale J, Coleman DV, et al. Direct gene analysis of chorionic villi: a possible technique for first trimester antenatal diagnosis of haemoglobinopathies. Lancet 1981;Z:1 125. 10. Kazy Z, Ruzovsky IS, Bakharev VA. Chorion biopsy in early pregnancy: a method of early prenatal diagnosis for inherited disorders. Prenat Diagn 1982;2:39. 1 1 . Old JM, Ward RHT, Karagozlu F, et al. First-trimester fetal diagnosis for haemoglobinopathies: three cases. Lancet 1982;2:1414. 12. Gosden JR, Mitchell AR, Gosden CM, et al. Direct vision chorion bi-
28
opsy and chromosome-specific DNA probes for determination of fetal sex in first-trimester prenatal diagnosis. Lancet 1 9 8 2 3 14 16. 13. Brambati B, Simoni G. Fetal diagnosis of trisomy 21 in the first trimester of pregnancy. Lancet 19839 :586. 14. Ward RHT, Modell B, Petrou M. A method of chorionic villus sampling in the first trimester of pregnancy under real-time ultrasonic guidance. Br Med J 1983;286:1542. 15. Elles RG, Williamson R, Niazi M, et al. Absence of maternal contamination of chorionic villi used for fetal gene analysis. N Engl J Med 1983;308:1433. 16. Pergament E, Ginsberg N, Verlinsky Y, et al. Prenatal Tay-Sachs diagnosis by chorionic villi sampling. Lancet 1983;2:286. 17. Tsvetkova IV, Zolotukhina TV, Bakharev VA, et al. Prenatal exclusion of metachromatic leukodystrophy by estimation of arylsuphatase A activity in chorion and cultured amniotic fluid cells. Prenat Diagn 1983;3:233. 18. Goossens M, Dumez Y, Kaplan L, et al. Prenatal diagnosis of sickle cell anemia in the first trimester of pregnancy. N Engl J Med 1983;309: 831. 19. Gustavii B. First-trimester chromosomal analysis of chorionic villi obtained by direct vision technique. Lancet 1983;2:507. 20. Ward RHT. Personal communication, 1983. 21. Jackson L. Personal communication, 1983. 22. Simoni G, Brambati B. Danesino C, et al. Efficient direct chromosome analysis and enzyme determination from chorionic villi samples in the first trimester of pregnancy. Hum Genet 1983;63:349. 23. Blakemore KJ, Watson MS, Lieber
24.
25.
26.
27.
28.
WA, et al. Prenatal diagnosis in t h e first trimester using chorionic villi. Program and Abstracts, American Society of Human Genetics 1983378A. Kidd VJ, Golbus MS, Wallace BR, et al. Prenatal diagnosis of alpha1-antitrypsin deficiency by direct analysis of the mutation site in t h e gene. N Engl J Med 1984;310:639. Woo SLC, Lidsky A, Flemming G, et al. Cloned human phenylalanine hydroxylase gene allows prenatal diagnosis and carrier detection of classical phenylketonuria. Nature 1983;306:151. Lau Y-F, Huang JC, Dozy AM, Kan YW. Identification of Y-specific repeat sequences and their application for prenatal sex determination. Lancet 1984;1:4. Gusella JF, Wexler NS, Conneally MB. A polymorphic DNA marker genetically linked to Huntington’s disease. Nature 1983;306:234. Beeson D, Golbus MS. Anxiety engendered by amniocentesis. Birth Defects 1979;15:191.
Address for correspondence: W. A l l e n Hogge, MD, Assistant Professor, Dept. of Ob/Gyn, University of Virginia, P.O. Box 387, Charlottesville, VA 22908.
Joan S. Hogge is a genetic counselor in the Reproductive Genetics Unit at the University of California, San Francisco. W. Allen Hogge is assistant professor of Obstetrics, Gynecology, and Reproductive Sciences at the University of California, San Francisco.
Mitchell S. Golbus is professor of Obstetrics, Gynecology and Reproductive Sciences, and of Pediatrics at the University of California, San Francisco.
January/February 1986 JOCNN
-
Chorionic Villus Sampling JOAN S. HOCCE, MS, W. ALLEN HOGCE, MD, AND MITCHELL S. COLBUS, MD Chorionic villus sampling in the first trimester of pregnancy has the potential to become a major tool in the prenatal diagnosis and therapy of genetic disorders. Villus samples can be used for cytogenetic and biochemical studies as well as DNA analysis. However, little is known about the effects of chorionic villus sampling on a continuing pregnancy, or the long-term effects on the subsequently delivered infants. Despite these limitations, chorionic villus sampling appears to be a major breakthrough in prenatal diagnosis.
Chorionic villus sampling, a technique for obtaining fetal cells for prenatal diagnosis, is being investigated actively by both United States and European researchers as a possible replacement for amniocentesis.’ The idea behind the technique is not new, having been proposed as early as 1968.’ However, amniocentesis was being developed at the same time and quickly became the procedure of choice for prenatal diagnosis because of its relative safety and the ease of obtaining amniotic fluid in the second trimester. Because amniocentesis is a procedure of the second o r third trimester of pregnancy, researchers are now looking to chorionic villus sampling, done in the first trimester, as the future method of prenatal diagnosis. Detection of genetic defects by amniocentesis has contributed greatly to the field of medical genetics in the past decade and a half, but
Accepted: June 1984.
24
chorionic sampling may offer access to prenatal diagnosis to a much larger group of women who d o not find second trimester procedures acceptable. Approximately two weeks after implantation, the human trophectoderm proliferates and produces hundreds of villi that radiate in all directions (Figure 1). Villi on the implanting side of the chorion will form the placenta; villi away from the implantation site will disappear by approximately 14 weeks.3 Chorionic villi, fetal in origin, are a source of fetal genetic information because these cells reflect the chromosomes, enzymology, and DNA content of the fetus. Chorionic villus sampling is done at eight to 12 menstrual weeks because the villi begin to disappear after this period. A sampling of villi is usually obtained transcervically because the uterus is still in the pelvic cavity (Figure 2). Tissue obtained from the end of one or more villi at the site of implantation contains rapidly di-
viding fetal cells that can be analyzed rapidly for chromosomal and biochemical defects.’
HISTORY
In 1968, Mohr proposed fetal tissue sampling in the first trimester by use of a transvaginal hysteroscope.’ Tissue was obtained in seven out of eight attempts, and the only complication of the procedure was a tear in the membranes after biopsy in one case. In 1973, by direct vision with an endoscope, Kullander and Sandahl obtained villus biopsies in 39 patients requesting elective abortion between eight and 20 weeks.4 Specimens were successfully cultured for chromosomal analysis in 19 cases. These 19 patients were followed from seven to 43 days after the biopsy without any pregnancy complications. Kullander a n d Sandahl concluded that the ninth to tenth week of pregnancy offered the best opportunity for transcervical biopsy using the endoscope.
January/February 1986 JOG”
Spiral artery
Secondarv villi ,
j
intervillous space Intervillous
Fetus Amntotic cavity
Decidua capsularis fMfM& rnem-/
Figure 1. Diagram of the human embryo at approximately six weeks gestation. At the embryonic pole, the villi are numerous and well-formed; at the abembryonic pole, the villi are fewer in number and less well-developed.
During the 1970s, researchers continued to experiment with techniques for obtaining chorionic villi. Hahnemann, in 1974, described hysteroscopic biopsy in 95 pregnant women before elective termination of the pregnancy.' In 67 cases, the biopsy was taken immediately before abortion. The other 28 cases were observed for eight days after the biopsy; two of the 28 aborted following the procedure. Only 60% of the biopsies contained chorion or chorionic villi, but cell culture and karyotyp-
ing were successful in all 13 samples placed in culture. Two complications occurred; in one case, the amniotic membrane was punctured during biopsy, and bleeding obscured vision in another case. Rhine eta/.,in 1977, reported the use of the "antenatal cell extractor" for obtaining trophoblastic cells Cells from the endocervical for culture were obtained with a noninvasive procedure similar to that used for obtaining Pap smears. Fetal chromosomes were found in 25% of the samples. Goldberg eta/.
Amniotic cavity Zhorion frondosum
, k-\
Decidua capsularis
Catheter
Syringe/
Figure 2. Diagram of the uterus at 10 weeks gestation with biopsy catheter in position.
January/February 1986 JOGNN
attempted to confirm Rhine's findings but failed to do so, finding that the cultured cells were maternal.' In 1975, the Chinese reported using blind transcervical aspiration of chorionic villi to determine fetal sex.' Because tissue culture facilities were not available, chromosomal aberrations and biochemical abnormalities could not be determined. The sampling was performed at six to 14 weeks of gestation with a 94% accuracy rate in determining sex. The report included 100 pregnancies biopsied for sex determination. Thirty pregnancies were terminated; 29 because of a female prediction. Of the 70 continuing pregnancies, four cases ended in spontaneous abortion. The 1980s have brought more interest in the technique of chorionic villus sampling and its usefulness as a prenatal diagnostic tool. Kazy eta/.,in 1982, published a study of 165 cases in which sampling was performed at approximately six to 12 weeks gestation under real-time ultrasound guidance. They concluded that chorionic villus sampling was a valuable method of early prenatal diagnosis to detect accurately fetal sex and some enzyme defects."' lnterest in chorionic villus sampling was heightened in the United States and Europe when a group of researchers headed by Old and Weatherall used chorionic villi for restriction endonuclease analysis of fetal DNA and successfully diagnosed two fetuses at risk for hemoglobinopathies: one for betathalassemia and one for sickle cell anemia." Gosden et d.reported prenatal sex determination in three to four days by molecular analysis of DNA from chorionic villus specimens." The researchers suggested that rapid determination of sex would be valuable for t h e prenatal diagnosis of X-linked disorders. 25
mia.” This study added further evRecently, interest has increased idence for the safety of the in what chorionic villus sampling procedure. can offer for the future of prenatal diagnosis. Simoni and Brambati reported the diagnosis of trisomy CURRENT TECHNIQUES 21 in an obligate carrier of Duchenne muscular d y ~ t r o p h y . ’ ~ Some of the techniques currently Ward, Modell, and Petrou achieved used for prenatal diagnosis by an 89% success rate of obtaining chorionic villus sampling include specimens with transcervical asdirect vision biopsy with a hyspiration of chorionic villi under t e r o s ~ o p e , a ’ ~ transabdominal apreal-time ultrasound guidance.I4 In proach using aspiration with ultraaddition, Elles et af. showed that sound guidance,” and an aspirachorionic villi at nine to 10 weeks tion technique using a cannula gestation are a source of fetal DNA passed transcervically under realthat can be used for gene analysis time ultrasound guidance.’l Simoni with no detectable contamination eta/.reported four different methby maternal DNA.’’ These reports ods of sampling in an attempt to indicated that the procedure could find the safest and most efficient be done efficiently and that the tistechnique.” The four methods sue obtained yielded accurate reused were an endoscope system, sults. two different catheters for blind Chorionic villus sampling as a aspiration, and the Portex@ cathediagnostic tool became widely ter under ultrasound guidance. The publicized in the lay press in midresearchers concluded that villus 1983 following a report by Pergament et af. on the prenatal diag- aspiration with the Portex catheter under ultrasound guidance was the nosis of Tay-Sachs disease.I6 Tromost efficient technique, resulting phoblastic cells from the at-risk in a 96% success rate for obtaining pregnancy had normal levels of villus samples.” hexosaminidase A activity, which were confirmed in fetal fibroblasts ADVANTAGES obtained when the pregnancy was terminated because chromosome Chorionic villus sampling is a analysis revealed trisomy 16. A case reported by Tsvetkova et af. technique with wide clinical applidemonstrated that the prenatal di- cations. Samples of tissue obtained between eight and 12 weeks gesagnosis of metachromatic leukodystrophy was possible by deter- tation offer sufficient tissue for chromosome analysis, enzyme demining arylsulphatase A activity in terminations, and DNA analysis. villi obtained from a patient who For the diagnosis of most metahad two previous children with bolic disorders, the villi can be asmetachromatic leukodystrophy.” sayed directly for enzyme activity These reports confirmed the apbecause the amount of tissue is sufplicability of villus material for the ficient from the biopsy itself. detection of certain biochemical Couples at risk for a fetus with a disorders. Goossens et al. conchromosomal disorder are the firmed previous report^"*'^*'^ that largest group to which chorionic chorionic villus sampling allowed villus sampling is applicable. By the prenatal diagnosis of sickle cell analysis of the villus cells directly disease before the tenth gestaor after culture, an early diagnosis tional week by obtaining definitive can be made of the fetal karyotype. diagnoses by DNA analysis of choBecause chorionic villi contain rionic villus samples in five pregmany rapidly dividing cells, a nancies at risk for sickle cell ane26
chromosome spread can b e obtained in five to 24 DNA analysis can be carried out on the DNA obtained from chorionic villi for the prenatal diagnosis of hemoglobinopathies such as sickle cell anemia, alpha-thalassemia, and some beta-thalassemias. Because the DNA content of all fetal cells is’ identical, recombinant techniques can be used for first trimester diagnosis and would allow detection of disorders such as alpha-1-antitrypsin deficiency24 and phenylket~nuria.‘~ A Y-specific probe recently developed by Lau et af.allows fetal sexing in less than 24 hours.’6 As more restriction fragment length polymorphisms are found, linkages to genetic disorders may be found even before the biochemical basis of the disorder is under~tood.‘~ The possibilities offered by chorionic villus biopsy for fetal diagnosis in the first trimester may influence parents’ decisions about childbearing. Diagnosis before 12 weeks gestation greatly reduces parents’ emotional stress and the medical complications related to therapeutic abortion in the second trimester. Earlier diagnosis allows a couple more privacy in their pregnancy because it is not yet physically obvious t o others. Earlier diagnosis also may help families overcome some of the legal, social, and religious difficulties associated with amniocentesis and termination in the second trimester. Because the waiting period for obtaining results is significantly reduced (1-2 weeks for chorionic villus sampling as compared to 34 weeks for amniocentesis), families may experience less psychological stress.” The method of termination by suction evacuation may prove much more acceptable to gravidas and the community because of the relative safety of the procedure as compared to prostaglandin abortion in the second trimester.
January/February 1986 JOCNN
Earlier diagnosis holds the potential for improving fetal treatment by allowing earlier intervention. The advent for chorionic villus sampling should, therefore, stimulate more research in the area of fetal therapy, as well as in prenatal diagnosis.
DISADVANTAGES Currently, early fetal development is an unexplored area. Because so little is known about the procedure, the effects on the subsequently delivered infants must be determined. Chronologically, complications may be divided into those that are immediate, short-term, or longterm. One of the immediate complications is failure to obtain tissue. Investigators need to document the number of insertions and aspirations necessary to obtain a sample, and whether the timing of a repeat aspiration changes the loss rate related to the procedure. Another immediate complication related to the procedure is rupture of the membranes with leakage of amniotic fluid. Whether management of this complication should be conservative or immediate termination of the pregnancy should be recommended is unknown. Bleeding after the procedure is another complication, and any bleeding that does occur needs to be well documented as to the amount and duration, along with its relation to subsequent pregnancy loss or other complications. Spontaneous abortion is a shortterm complication. The spontaneous abortion rate could be as low as five to 6 percent2*or as high as 12%.*' However, the incidence of spontaneous abortion at this stage of gestation in control pregnancies demonstrating normal growth and cardiac activity by ultrasound is unknown. Control spontaneous abortion figures will have to be obJanuaryIFebruary1986 JOCNN
tained to determine the risk of the procedure. Infection is another potential problem. Should infection occur, no accepted form of management currently exists. The long-term effects of infection at this early stage of pregnancy, if successfully treated with antibiotics, is unknown. Maternal contamination of the specimen is a possible complication when sampling villi. Karyotyping should be done when assaying for biochemical disorders. For those patients at risk for biochemical disorder, follow-up amniocentesis as a confirmation may be needed until the true accuracy of the procedure is known. Long-term effects of chorionic villus sampling may include rhesus isoimmunization. To evaluate this possibility, a random, controlled study including Rhogam usage is needed. Secondly, careful evaluation of the neonate is necessary to determine any subtle anomalies that may result from chorionic villus sampling. In the case of still birth or neonatal death, an autopsy will be essential. Another important aspect of follow-up is confirmation at birth of the results, especially biochemical results. Other theoretical questions about chorionic villus sampling have been raised. Will an increased incidence of prematurity or placental abruption occur? What will be the long-term effects on the infant's growth? Will more growth retardation and fetal distress be seen as a result of removing villi early in pregnancy? Long-term follow-up needs to be done on these infants, with assessment of their growth, development, and intellectual function at one year of age or more to ascertain any subtle effects. Another important consideration is the possibility of monitoring the pregnancy with ultrasound for short-term or immediate complications. The optimal time for ultra-
sound follow-up is unknown. The expertise of the ultrasonographer will be important for the procedure and for the follow-up studies. Some cytogenetic considerations related to this technique that need to be addressed include 1) a method of efficient separation of chorionic villi from maternal cells, 2) the minimal amount of chorionic villi required for diagnostic testing, and 3) an improvement in the method for obtaining direct chromosome preparations. Currently, direct chromosome preparations do not yield high-quality banded chromosomes. Standardization of laboratory techniques needs t o be developed to ensure accurate and reliable results.
SUMMARY Chorionic villus sampling stands now as the possible future technique of prenatal diagnosis. The opportunity exists for good control studies to be done and for a n international registry of all cases to be established. Continuity of care and complete recording of data by those investigators doing chorionic villus sampling is needed to establish the relative safety of this procedure for both mother a n d fetus, and to document the reliability of the test results. Long-term, complete follow-up will be necessary to answer all of the questions now involved. Chorionic villus sampling has the potential t o become a major tool in the prenatal diagnosis and therapy of genetic diseases.
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Address for correspondence: W. A l l e n Hogge, MD, Assistant Professor, Dept. of Ob/Gyn, University of Virginia, P.O. Box 387, Charlottesville, VA 22908.
Joan S. Hogge is a genetic counselor in the Reproductive Genetics Unit at the University of California, San Francisco. W. Allen Hogge is assistant professor of Obstetrics, Gynecology, and Reproductive Sciences at the University of California, San Francisco.
Mitchell S. Golbus is professor of Obstetrics, Gynecology and Reproductive Sciences, and of Pediatrics at the University of California, San Francisco.
January/February 1986 JOCNN