Review Article methods of obtaining fetal tissues for karyotype, DISFA studies and enzyme assay(4). The procedure is done at 8-11 PRENATAL DIAGNOSIS OF weeks of gestation. The transcervical GENETIC DISORDERS sampling involves insertion of various types of catheters transcervically under ultrasound guidance for aspiration of chorionic villi. Transabdominal CVS is M.L. Kulkarni preferred as it can be done even in the Sajeev Vengalath second trimester of and is as- sociated with lesser risk of infection, bleeding and pregnancy loss(5). CVS Prenatal diagnosis of genetic disor- can be done in the second or third tri- ders has been possible because of great mester of pregnancy by direct aspiration advances in techniques of obtaining fetal of placental villi through transabdomi- tissues as well as development in cyto- nal route under ultrasound guidance. genetics, biochemical techniques and re- Coelocentesis is a new technique combinant DNA technology. The tech- that avoids puncture of the amnion. It niques currently in use or under investi- involves puncture of the chorion and as- gation for prenatal diagnosis are out- piration of the coelomic fluid from the lined in Tables I and II. extra-embryonic coelom. This proce- dure has not been used in clinical prac- tice(6). It is the withdrawal of amniotic fluid from the amniotic sac surrounding the Fetal Blood Sampling (l). If amniocentesis is done at 15- Initially this procedure was done by 16 weeks of gestation the process is passing a fetoscope transabdominally at called conventional(2), and if between 18-20 weeks, under ultrasound guid- 11-15 weeks early amniocentesis; the lat- ance. It never gained popularity because ter procedure is still experimental. Early of its difficulty and high rate of fetal loss amniocentesis is considered to be useful (approximately 4%). Fetal blood sam- for cytogenetic studies(3). pling has been replaced by Chorionic Villus Sampling (CVS) cordocentesis i.e., withdrawing blood from the umblical vein under ultra- This procedure is one of the earliest sound guidance(7). Cordocentesis has not only helped in the prenatal diagno- From the Department of Pediatrics, J.J.M. sis of genetic disorders, but also in un- Medical College, Davangere 577 004. derstanding of fetal physiology, devel- Reprint request: Dr. M.L. Kulkarni, Professor of opment and metabolism. The most im- Pediatrics, 2373, MCC A Block, Davangere portant indications for cordocentesis in- 577 004. clude: (i) rapid karyotyping for chromo- REVIEW ARTICLE

TABLE I-Techniques for Prenatal Diagnosis Fetal Tissues Technique Timing Studies Done Risk (weeks) on Tissues Amniotic fluid (a) Conventional 15-16 AFP I ACHE Abortion, needle amniocentesis hCG puncture injuries, (b) Early 11-14 placental abrupt- amniocentesis ion, chorio amni- nionitis, abruption, preterms labour. Amniocytes (a) Conventional 15-16 Cell culture for amniocentesis 11-14 karytopes, (b) Early enzyme assay, amniocentesis DNA studies, FISH Chorionic Villi (a) Transvaginal 8-11 Biochemical, 2% fetal loss, limb (b) Transabdominal 12-24 chromosmal, defects, mosaicism DNA and maternal bleedding Fetal blood (a) Fetoscopic 18-20 Coagulation 1 % fetal loss, aspiration factor Rhesus sensitiza- (b) Cordocentesis 16-20 Immunoglobulin tion, fetal antibodies infection, PROM estimation; DNA and enzyme study; karyotype, FISH. Fetal liver (a) Fetoscopic biopsy Enzyme assay (b) Percutaneous 18-20 as in OTC ? biopsy deficiency Fetal skin (a) Fetoscopic biopsy 18-20 Histopathology (b) Percutaneous biopsy ? Fetal muscle (a) Fetoscopic biopsy 18-20 Histopathology (b) Percutaneous biopsy ? Maternal serum Maternal blood 12-14 AFP/UE3/hCG Nil Fetal Cells in Flow cytometry, 1st FISH, maternal PCRI monoclonal trimester fetal sexing Nil circulation antibodies DNA tests Pre-implanation IVF 4-8 cell DNA, PCR, ? embryo biopsy Biopsy of stage enzyme assay blastocysts blastocyst AChE-Acetylcholine esterase; OTC-Ornithine transcarbamylase; PROM-Premature rupture of mem- branes.

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cutaneous albinism, and Sjogren- T ABLE II-Fetal Visualization Techniques Larsson syndrome(9). Techniques Route Timing Risk to fetus Fetal liver biopsy may rarely be nec- Embry- Trans- First tri- essary to diagnose inborn errors of me- oscopy vaginal mester ? tabolism limited to hepatic parenchymal Fetos- Trans- 18-20 enzyme abnormalities. Attempts have copy abdominal weeks 3% also been made to diagnose muscular Embryo- Trans- First tri- dystrophy by prenatal muscle biop- sy(10). Recent advances in DNA tech- abdominal mester ? nology have however allowed reliable Ultra- Trans- 15-20 diagnosis of muscle dystrophy and re- sound abdominal weeks nil duced the need for fetal muscle biopsies. T rans- First trim- vaginal ester nil Techniques for Fetal Imaging MRI All trim- Ultrasound : The role of ultrasound in esters ? prenatal medicine has expanded over Radio- Late the past two decades. As a result the graphy second possibilities for diagnosis and in utero and therapy and surgical interventions is third now possible. The timing for ultrasound trimester examination for various structural anomalies is shown in Table III (11).

Prenatal diagnosis of central nervous somal disorders and where either amni- system malformations especially that of otic fluid or CVS have shown chromo- somal mosaicism; (it) diagnosis and TABLE III-Optimal Timing for Ultrasound treatment of Rh isoimmunization; (iii) Examination immunoglobulin deficiency; (iv) clotting Indication Timing factor deficiencies; (v) hemoglobino- pathies; (vi) fetal platelet abnormalities; Anencephaly 10-12 weeks (vii) evaluation of nonimmune hydrops 16-18 weeks fetalis(8). Hydrocephaly/ 16 weeks onward Fetal Skin, Liver and Muscle Sampling microcephaly Fetal skin sampling was initially per- Cardiac abnormalities 18-22 weeks formed by fetoscopy, which has been re- Limb abnormalities 10 weeks onward placed by percutaneous insertion of bi- Renal disease 16 weeks onward opsy forceps under ultrasound guid- Face and mouth- 18-22 weeks ance. Conditions which can be detected by fetal skin sampling include anomlies anhidrotic and hypohidrotic ectodermal Anterior abdominal- 16-18 weeks dysplasia, epidermolysis bullosa, oculo- wall

1231 REVIEW ARTICLE neural tube defects is easy on ultra- sure, nonimmune hydrops and unex- sound imaging (Table IV). Many skeletal plained polyhydramnios(13). Trans- dysplasias and chromosomal anomalies vaginal and transabdominal ultrasono- including can be diag- graphy done at 11-14 weeks and 18-20 nosed on ultrasonography (Table V). The weeks respectively help to detect con- finding of a strawberry shaped head, genital heart disease(14). choroid plexus cysts, facial cleft, Esophageal atresia, pulmonary cys- micrognathia, exomphalocele and com- tic adenomatoid malformations, pulmo- plex hand and feet malformations sug- nary hypoplasia, diaphragmatic hernia, gest trisomy 18(12). hyper-echogenic bowel, obstructive The indication for fetal echocardio- lesiosn, ventral wall defects can be de- graphy include family history of con- tected during second and third trimes- genital heart diseases, diabetic mother, ter of pregnancy. intrauterine infection, teratogenic expo- Fetoscopy: A fine caliber endoscope is TABLE IV-Prenatal Diagnosis in Neural Tube inserted into the uterus. The direct visu- alization of the fetus is possible and fetal Defect blood sampling can be done. Because of MSAFP Increased the high risk of spontaneous abortion (2nd trimester) associated with fetoscopy, this tech- Ultrasound Transabdominal nique is rarely used. (2nd trimester) Etnbryoscopy: This is an experimental Transvaginal ultras- technique used in the first trimester of ound (1st trimester) pregnancy. A rigid endoscope inserted through the cervix into the space be- Anencephaly Absence of vault tween the amnion and chorion is used Spina bifida Spine anatomy- to visualize the embryo and for diagno- splaying of spine sis of structural malformations(15). Lemon sign" Banana sign.... Embryofetoscopy: Transabdominal Swelling, hydroc- embryofetoscopy is a new technique be- ephalus, lower ing evaluated for its utility in the first limb defects trimester diagnosis of structural abnor- Amniotic fluid Raised AFP, malities of the fetus. This technique has been possible because of availability of acetylcholinesterase new submillimetric fiberoptic endo- Chromosomal studies To exclude NTD, as scope(16). a part of chromoso- Magnetic Resonance Imaging: The mal disorder. clinical applications of this technique * Lemon sign: Forward scalloping of - the are limited due to fetal movements and frontal aspect of the fetal the cost involved in this mode of inves- head tigations(17). ** Banana sign: Banana shaped abnormal cerebellum. Radiography: The indications for con-

INDIAN PEDIATRICS VOLUME 32-NOVEMBER 1995

TABLE V-Prenatal Diagnosis in Down Syndrome Maternal serum markers AFP - decreased (14-16 weeks) UE3 - decreased (14 weeks) Triple test: hCG - increased (14 weeks) detection rate is 60% PAPP-A - increased 1st trimester Neutrophil alkaline phsophatase-increased Amniocentesis Early - Karyotype on cultured amniocytes Conventional - FISH on uncultured cells Cordocentesis (18 weeks) Fetal lymphocytes; Culture for karyotype; FISH on non-dividing fetal cells CVS (8-11 weeks) Direct preparation for karyotype, short term culture Ultrasound (11-20 weeks) Nuchal fold > 4 mm, double .bubble in abdo- men, short femur, renal pyelectasis, clinodac- tyly, macroglossia Fetal echocardiography Atrio-ventricular canal defects Fetal cells in maternal FISH circulation Figures in parenthesis indicate appropriate time for that test.

ventional radiographs are limited to di- rearrangements and fetal abnormalities agnosis of bone dysplasias in second or detected by ultrasound. The result of fe- third trimester(17).. tal karyotype are available within 72 hr with cord blood samples and CVS, and Prenatal Cytogenetics 2-3 weeks with amniotic fluid. All chromosomal and the majority of structural chromosomal The combination of molecular genet- abnormalities (deletion, translocation) ics and cytogenetics techniques (molec- can be detected prenataly. Cells used for ular cytogenetics) has given rise to the prenatal cytogenetic analysis includes powerful new technology of fluorescent amniocytes obtained by second trimister in situ hybridisation (FISH)(18). Briefly, amniocentesis, chorionic villi obtained a fluoroscently labelled DNA probe is by transcervical or transabdominal CVS hybridised to a standard chromosome or fetal blood sampling. Indications for preparation on a microscopic slide. This prenatal cytogenetic analysis include ad- method is useful in detecting chromo- vanced maternal age, previous child somal abnormalities, both numerical with , parental chromosome and microdeletion syndromes. For FISH

1233 REVIEW ARTICLE technique chromosome specific probes restriction enzyme: Restriction endo- (alpha satellite probe) which hybridise nucleases are bacterial enzymes that with pericentromeric region of the chro- recognise and cleave short specific base mosome are used. This technique is used sequences in double stranded DNA and for detecting chromosomal aneuploidy, are used to identify mutant genes, dele- that often involve X, Y, 13,18 and 21 tions within gene,, or characterize DNA chromosomes. polymorphism. A set of probes derived from whole (b) Polymerase Chain Reaction (PCR): single chromosome containing repetitive When the molecular basis of a disease is sequences that span the length of a known, the precise test can be designed. chromosome can be prepared and la- Most rely on the ability to amplify belled with fluorochromes (chromosome quickly and efficiently a specific region painting). A further use of FISH of DNA, using the PCR. This technique technique is the generation of region allows simple and rapid synthesis of mi- specific chromosome probes for the di- crogram quantities of DNA copies from agnosis of contiguous gene syn- template segments present in amounts dromes(19). Contiguous gene syn- as small as a single molecule. Various in dromes involves the loss of genes that herited diseases including alpha 1 may be functionally unrelated but are antitrypsin deficiency, Duchenne mus contiguous along the chromosome. cular dystrophy, phenylketonuria and These syndromes include Prader Willi fragile X syndrome can be diagnosed by syndrome, Miller-Dicker syndrome, PCR. Angelman syndrome and Di George (c) Allele Specific Oligonucleotide syndrome. FISH has also been used for Probes: This is an alternative way to de- the detection of Duchenne or Becker tect a known point mutation. The tech- muscular dystrophy carrier status in fe- nique uses two synthetic oligonucle- males whose affected male relatives otides, one specific for a normal gene have a known specific gene deletion. and the other specific for the known ge- netic mutation. In autosomal recessive DNA Based Prenatal Diagnosis disorders, in the affected individual This clinical application is based there will be two copies of mutated upon the fact that DNA complement is genes, while the normal individual will generally identical in every cell of the have two normal genes and the carrier body, and therefore a hereditary defect one mutated and one normal gene e.g., detectable at the DNA level, should be sickle cell disease, alpha 1 antitrypsin found in all nucleated cells from that in- deficiency. dividual(20,21). Any nucleated fetal (d) Expanding Trinucleotide Repeats: cells can be used to obtain DNA for di- This form of mutation is seen in 3 genet- agnosis. ic disorders, fragile X syndrome, A. Techniques for direct mutation myotonic dystrophy and Huntigton's disease. In these disorders normally analysis include: polymorphic trinucleotide repeat se- (a) Analysis of' the disorder through quences found at the disease locus ex-

1234 INDIAN PEDIATRICS VOLUME 32-NOVEMBER 1995 pand in affected individuals beyond the tiple family members from several gen- normal size range. In fragile X syn- erations. drome, CGG repeats extend to hundred The other types of polymorphisms or thousands of the triplets. Detection of becoming increasingly useful in diag- expanding trinucleotide repeats is rela- nostic linkage analysis are microsatel- tively simple. The simplest method is to lites or simple sequence repeats(24). amplify across the expanding re- gion(22,23). When the number of repeats Biochemical Screening is very large the expansion can be de- tected by DNA hybridization with a Alpha-Feto Protein (AFP) probe that is located close to the region AFP is a glycoprotein containing of expansion. about 4% carbohydrate which can be Other techniques for evaluation of detected in the human fetus by 29 days mutations include dot blot analysis and of life. It is produced initially both in the amplification refractory mutation sys- yolk sac and the liver, but the hepatic tems. contribution increases steadily. Its exact function is not known, but it acts as a B. Techniques for indirect mutation carrier protein and has an immunologi- analysis include: cal role. AFP is excreted into fetal serum (a) Linkage: In most genetic disorders and the main source of AFP in the amni- the mutation is unknown. By using cer- otic fluid is the fetal urine; fetal skin tain markers, that are close to mutant; may also allow passage of a small gene, one can indirectly infer about the amount of AFP. By 10 weeks of gesta- presence of mutant gene because of its tion AFP increases, peaks at 13 weeks proximity to the probe. and decreases thereafter by 12% every week. AFP is increased in neural tube (b) Restriction Fragment Length Poly- defects and decreased in chromosomal morphism (RFLP): This is a type of molec- aneuploidy like Down syndrome. ular marker produced when specific en- zymes are used to cut the DNA. Mea- During pregnancy the maternal se- surements of the fragment length to rum level of AFP (MSAFP) increases. which a DNA probe will hybridize can AFP is transferred to the maternal circu- be used to track the transmission of a lation by two routes, across the placenta variation in the DNA (polymorphism) directly from fetal serum and though through a family. Linkage of an RFLP to the fetal membranes from the amniotic the gene of interest can be used for ge- fluid. The median MSAFP levels mea- netic diagnosis. sures about 25 ng/ml at 16 weeks and About 80-85% of the thalassemic increases by about 15% per week until mutations can be detected by this meth- about 30 weeks, plateaus for the next 5 od. This can be used as indirect marker to 6 weeks and then falls rapidly. to detect single base substitution or de- Increased levels of MSAFP are seen letion in the beta globin gene. One major in with multiple gestation, disadvantage of these method is that neural tube defects, placental abnormal- linkage analysis requires a study of mul- ities, maternal disease like hepatocellu-

1235 REVIEW ARTICLE lar carcinoma, viral hepatitis and sys- agnosis would allow the selection and temic lupus erythematosus. The levels transfer of only healthy zygotes to the of MSAFP are decreased when the fetus uterus. has Down syndrome or other triso- Access to preimplantation embryo is mies(25). possible by either non-surgical uterine Unconjugated (UE 3) lavage or in vitro fertilization(28). The blastocyst with 100-200 cells obtained by UE 3 produced by the fetal adrenal the uterine lavage is subjected to gland is converted by the placenta and microbiopsy. A small slit is cut into the conjugated by the maternal liver. UE 3 zona pellucida of blastocyst with a mi- levels in maternal serum are approxi- cromanipulator. As the blastomere cells mately 25% lower in pregnancies with herniate through the incision they are Down syndrome. Measurement of UE3 excised from the embryo surface and levels in the maternal serum distinguish subjected for genetic diagnosis. After between Down syndrome and unaffect- the microbiopsy, the remaining blas- ed pregnancy as early as 9 to 11 weeks tomere may be cryopreserved in liquid of gestation. nitrogen. If the result of microanalytic Other proteins which can be detect- diagnostic technique on biopsy speci- ed in the maternal serum in Down syn- men proves to be normal, then the pre- drome include human chorionic gona- implantation embryo is replaced into dotropin(25), pregnancy associated pla- the uterine cavity. cental protein A(26) and neutrophil al- The cells obtained from the biopsy kaline phosphatase (Table V). specimen may be cultured to increase Fetal Cells in Maternal Blood the yield. The biopsied specimen of pre- implantation embryo is subjected to var- The nucleated fetal cells circulating in ious techniques for genetic diagnosis. the maternal blood can be utilized for Rapid advances in recombinant DNA, DNA extraction arid prenatal tests(27). monoclonal antibodies, flow cytometry Such tests can be done in the first tri- and cytogenetic techniques have made mester for population screening. They it possible to subject even a single cell carry no risk of maternal infection or fe- for genetic diagnosis. Micromanipula- tal miscarriage. The fetal cells isolated tion of the embryo, though no adverse include nucleated red blood cells, leuko- effects are reported, is potentially a ter- cytes and trophoblasts. These cells can atogenic procedure. be isolated from maternal blood by flow cytometry, monoclonal antibodies or DNA Based Diagnosis Available in PCR. India Prenatal Diagnosis Before Prenatal diagnosis using DNA tech- Implantation nology are being offered for beta- thalassemia and Duchenne muscular The diagnosis of genetic disorders in dystrophy at the Genetic Unit, Depart- preimplantation embryo is being ment of Pediatrics. All India Institute of evolved as a new specialty. Such a di- Medical Science, New Delhi 110 029.

1236 INDIAN PEDIATRICS VOLUME 32-NOVEMBER 1995

The cost for the DNA based study is ap- feet in the gene. It is ideally done before proximately Rs. 1500/- (Dr. I.C. Verma, mother is pregnant. This will tell wheth- New Delhi, personal communication). er prenatal diagnosis is possible or not. The Birth Defect Centre, A-ll, Elco During pregnancy at about 10-11 weeks Market Road, Bandra (W), Bombay 400 a CVS is obtained and the sex of the fe- 050 also provides similar services, tus determined. If the fetus is female, no though at comparatively higher rates. further tests are done. If fetus is male, the DNA extracted from CVS is tested Diagnosis of beta Thalassemia for abnormality of the gene. Prenatal diagnosis is done by DNA REFERENCES studies on the CVS obtained at 9-12 weeks, amniotic fluid cells obtained at 1. The NICHD National Registry for 14-15 weeks, or fetal blood obtained at Amniocentesis Study Group. Midtrimester amniocentesis for prena- 18 weeks. The desired method is CVS. tal diagnosis: safety and accuracy. Initially it has to be established that both JAMA 1976, 236: U71-U76/ parents are carriers. The birth of an af- 2. Elias S, Simpson JL. Amniocentesis. In: fected child makes them obligate carri- Essentials of Prenatal Diagnosis. Eds. ers. The blood level of Hb A2 are raised Simpson JL, Elias S. New York, in carriers. Blood is collected in a sterile Churchill Livingstone, 1993, pp 27-44. EDTA tube from both parents and the 3. Hanson FW, Tennant F, Hune S, affected child. The affected child should Brookhyser K. Early amniocentesis: not have received any blood transfusion outcome, risks, and technical prob- in the past 3-4 weeks. All the blood sam- lems at less than or equal to 12.8 ples should preferably be collected be- weeks. Am J Obstet Gynecol 1992,166: fore the CVS. A date is fixed with an ob- 1707-1711. stetrician for CVS. Laboratory diagnosis 4. Burtan BK, Scaulz CJ, Burd LI. Limb using DNA technology takes about 10- anomalies associated with chorionic 14 days. villus sampling. Obstet Gynecol 1992, 79: 726-730. Diagnosis of Duchenne Muscular 5. Jackson L, Wapner RJ. Chorionic vil- Dystrophy lous sampling. In: Essentials of Prena- tal Diagnosis. Eds. Simpson JL, Elias S. Definitive diagnosis in the affected New York, Churchill Livingstone, case is the first step in the diagnosis. 1993, pp 45-61. Next decide whether mother is a carrier 6. Roberts L, Rodeck CH. Prenatal diag- or not. This is done by pedigree analysis nosis. Medicine. International (Indian and high values of creatinine phosphok- edition) 1994, 3: 25-28. inase. However, it must be remembered 7. Bell JG, Weiner S: Cordocentesis. Curr that during pregnancy creatinine phos- Opinion Obstet Gynecol 1993, 5: 218- phokinase values are low. At least two 224. enzyme determinations must be made 8. Ryan G, Rodeck CH. Fetal blood sam- one week apart. pling. In: Essentials of Prenatal Diag- nosis. Eds. Simpson JL, Elias S. New The DNA of the affected child must Yok, Churchill Livingstone, 1993, pp be examined to determine the exact de- 63-75.

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9. Sheelman LP, Elias S. Percutaneous ploidies in uncultured aminocytes by umblical blood sampling, fetal skin using fluorescence in situ hybridization sampling and fetal liver biopsy. Semin (FISH). Am J Hum Genet 1992, 51: 55-65. Perinatol 1990,14: 456-464. 19. Emanuel B. Molecular cytogenetics. 10. Evans MI, Greb A, Kunkel LM, et al. In Towards dissection of the contiguous utero fetal muscle biopsy for the diag- gene syndromes. Am J Hum Genet nosis of Duchenne muscular dystropy. 1988, 43: 575-578. Am J Obstet Gynecol 1991, 165: 728- 20. Gilbert F, Marinduque B. DNA prena- 732. tal diagnosis. Curr Opinion Obstet 11. Gilmore DH, Aitken DA. Specific di- Gynecol 1990, 2: 226-235. agnostic techniques. In: Prenatal Diag- 21. Antenarakis SE. Diagnosis of genetic nosis in Obstetric Practice. Eds. Whit- disorder at the DNA level. N Engl J tle MJ, Conner JM. London, Blackwell Med 1989, 320:153-163. Scientific, 1989, pp 7-21. 22. Sutherland GR, Gedeon A, Kornman 12. Nybejg DA, Kramer D, Resta RG, et al. L, et al. Prenatal diagnosis of fragile X Prenatal sonograpic findings of tri- syndrome by direct detection of the somy 18: Review of 47 cases. J Ultra- unstable DNA sequence. N Engl J Med sound Med 1993, 2:103-113. 1991, 325: 1720-1722. 13. Haddow JE, Polomaki GE, Knight GJ, 23. Rousseau F, Heitz H, Biancalana V, et et al. Prenatal screening for Down syn- al. Direct diagnosis by DNA analysis drome with use of maternal serum of the fragile X syndrome of mental re- marker. N Engl J Med 1992, 327: 588- tardation. N Engl J Med 1991, 325: 593. 1673-1681. 14. Timor-Tretsch IE, Monteagudo A, 24. Hearne C, Ghosh S, Todd J. Warren WB. Transvaginal ultrasono- Microsatellites for linkage analysis of graphic definition of the central ner- genetic traits. Trends Genet 1992, 8: vous system in the first and early sec- 288-294. ond trimester. Am J Obstet Gynecol 25. Haddow JE, Palomaki GE. Prenatal 1991,164: 497-503. screening for Down syndrome. In: Es- 15. Cullen MT, Reece EA, Whetham J, sentials of Prenatal Diagnosis. Eds. Hobbins JG , Bmbryoscopy: descrip- Simplson JL, Elias S. New York, tion and utility of a new technique. Churchill Livingstone, 1993, pp 185- Am J Obstet Gynecol 1990,162: 82-86. 220. 16. Quinter RA, Abuhamad A, Hobbins 26. Evans MI, O'Brien JE, Dvorin E, JC, Mahoney MJ. Transabdominal Johnson MP. Biochemical screening. thin-gauge embryofetoscopy. A tech- Curr Opinion Obstet Gynecol 1994, 6: nique for early prenatal diagnosis and 453-458. its use in the diagnosis of a case of Meckel Gruber syndrome. Am J 27. Yeoh SC, Sargent I, Redman C. Fetal Obstet Gynecol 1993,168:1552-1557. cells in maternal blood and their use in 17. Committee on Genetics, American non invasive prenatal diagnosis. Academy of Pediatrics. Prenatal Ge- Progress. Obstet Gynecol 1993, 14: 51- netic Diagnosis for Pediatricians. Pedi 63. atrics 1994, 93:1010-1015. 28. McGowan D. Preirnplantation prena- 18. Klenger K, Landes G, Shook D, et al. tal diagnosis. Obstet Gynecol Clin Am Rapid detection of chromosome aneu- 1993, 3: 599-619.

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