Chorionic villus sampling for first-trimester Prenatal diagnosis: Northwestern University Program

Magyar et al.

tient, and I would agree with him very much. We are very careful to inform our patients prior to the operation that these side effects may occur. Then if the effects are experienced in the postoperative period, the patient is thus forewarned and generally under these circumstances does very well, both physically and emotionally. Dr. Mattox referred to shoulder pain. This is something that, of course, we have all seen in instances of

May 15, 1985 Am J Obstet Gynecol

hemoperitoneum. We certainly see it after laparoscopy, but it has been our experience that we have not had patients complain of shoulder pain following the use of the intraperitoneal dextran. REFERENCE 1. Krinsky AH, Haseltine FP, Decherney A. Peritoneal fluid accumulation with dextran 70 instilled at time of laparoscopy. Fertil Sterill984;4l:647.

Chorionic villus sampling for first-trimester prenatal diagnosis: Northwestern University Program Sherman Elias, M.D., Joe Leigh Simpson, M.D., Alice O. Martin, Ph.D., Rudy E. Sabbagha, M.D., Albert B. Gerbie, M.D., and Louis G. Keith, M.D. Chicago, Illinois We present our initial experience in developing a chorionic villus sampling program at Northwestern University. In phase 1, we performed chorionic villus sampling in 58 patients prior to elective first-trimester abortion, assessing the reliability and reproducibility of obtaining adequate villus samples and performing cytogenetic analysis by means of both the direct and culture methods. Specimens were categorized according to quality: class I, multiple identifiable villi (n = 20); class II, few villi or villi mixed with decidua (n = 15); class III, no villi (n = 23). There was a positive trend between operator experience, amount of villi obtained, and quality of cytogenetic preparations. In March, 1984, we received Institutional Review Board approval to perform chorionic villus sampling in continuing pregnancies (phase 2). Among the first 20 cases we found two abnormalities (47,XY, + 13; 45,X). The remaining 18 pregnancies were continuing. Recommendations are made for developing a chorionic villus sampling program. (AM J OasTET GVNECOL 1985;152:204-13.)

Key words: Chorionic villus sampling, genetics, prenatal diagnosis Amniocentesis is currently the technique most commonly employed for prenatal diagnosis of genetic disorders. Although widely practiced, amniocentesis unfortunately cannot be safely performed prior to 15 to 16 weeks' gestation. Thus fetal diagnosis of chromosomal or biochemical abnormalities can rarely be established prior to 18 weeks' gestation. 1 Couples awaiting results frequently undergo considerable psychological stress. Moreover, if a genetic disorder is detected and the couple elects to terminate the pregnancy, an abortion must be performed well into the second trimester. These circumstances impose greater risks and expense

From the Section of Human Genetics and the Diagnostic Ultrasound Center, Prentice Women's Hospital and M atemily Center ofN orthwestern Memorial Hospital, Northwestern University. Presented at the Fifty-second Annual Meeting of The Central Association of Obstetricians and Gynecologists, Detroit, Michigan, October 11-13, 1984. Reprint requests: Sherman Elias, M.D., Suite 1176, Prentice Women's Hospital and Maternity Center, 333 East Superior St., Chicago, 1L 60611.

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to the woman compared with an outpatient first-trimester abortion. Chorionic villus sampling for first-trimester prenatal diagnosis has the potential of becoming an attractive alternative to genetic amniocentesis. Prior to becoming a standard, however, considerable caution and monitoring is necessary to establish the accuracy and safety of the procedure. We present here our initial experience in developing technical expertise in chorionic villus sampling and in assessing various laboratory methods (phase I). Thereafter we shall describe our own early experience in introducing chorionic villus sampling for prenatal diagnosis in continuing pregnancies (phase 2). We shall also provide recommendations for those contemplating initiation of a chorionic villus sampling program. Material and methods Phase 1. The objectives of this phase of the study were to (1) assess the quality of cytogenetic preparations as a function of chorionic villus specimen quality and

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CHORIONIC VILLUS SAMPLING

Uterus

Chorion Fetus

Fig. 1. Chorionic villus sampling procedure.

of operator experience, (2) develop laboratory methods for obtaining cytogenetic preparations, and (3) correlate the cytogenetic findings in chorionic villus samples and abortuses. In August, 1983, our experimental protocol was approved by the Institutional Review Board of Northwestern University, and informed consent of each study subject was obtained. Chorionic villus sampling procedures were performed principally by a single investigator (S. E.) in a series of 58 patients undergoing elective first-trimester (6 to 12 weeks' gestation) abortion. The mean age of these patients was 27.2 years, with a range of 14 to 41 years. The distribution of gestational ages, as determined by the onset of the last menses, is shown in Table I. One of two instruments was used: (1) a 2.4 mm outer diameter, 18 cm long modified pediatric plastic nasogastric feeding tube with two side openings and a blunt tip which has a removable aluminum stylet or (2) a 1.5 mm outer diameter, 21 cm long Portex plastic catheter (Portex, Inc., Wilmington, Massachusetts) which has a removable blunt-ended aluminum stylet that protrudes sufficiently to obliterate the open end. The sampling procedure was performed in a fashion similar to that developed by investigators from London 2 • 3 and Milan.' To describe briefly, with use of a real-time mechanical sector transducer (3.5 MHz) or a phased-array sector transducer (3.5 MHz), an ultrasound examination was first performed to confirm the gestational age (by measuring the crown-rump length) and to confirm fetal viability (by visualization of heart movements). Patients whose pregnancies were determined to be beyond 13 weeks' gestation or in whom fetal viability was not confirmed were excluded from the study. Under sterile conditions the chorionic villus sampling catheter was then guided transcervically into the substance of the chorion frondosum under

Fig. 2. Ultrasonographic scan (sector transducer, 3.5 MHz) showing chorionic villus sampling catheter approaching chorion frondosum .

Table I. Distribution of the cases in phase 1 of the study according to gestational age as calculated from the beginning of the last menses Week of gestation

No. of cases

6 7 8 9 10

6 8

II

12

23

8 8 3 2

continuous ultrasound (sector scan) monitoring (Figs. I and 2). After removing the stylet villus material was aspirated into a 20 ml syringe containing 5 ml of Ham's Fl2 media supplemented with penicillin (l00 IU/ml), streptomycin sulfate (50 tJ..g/ml), and heparin (10 lUi ml). No more than four insertions of the catheter were performed in any single patient in attempting to obtain a sample. Specimens were transported immediately to the laboratory, where they were transferred to Petri dishes and examined under an inverted microscope (x 100). To assess the quality of the specimens, we categorized them into one of three groups: class I, identifiable villi with at least one arborizing cluster; class II, small amounts of villi or mixed tissue, including some villi; class III, unidentifiable tissue, questionable villi, or no villi. Chorionic villus specimens were processed for cytogenetic analysis in one of two ways: (I) the so-called "direct method" initially described by Simoni et aI.' and later modified by Gregson and Seabright,S or (2) a culture method. In the direct method chromosome preparations are obtained from spontaneous mitoses freed from the cytotrophoblast, which permits cytogenetic

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Table II. Proportion of each chorionic villus sampling specimen class as a function of operator experience Chronologie order of cases 1-20 Class of specimen

n

I II III

6 9 5

I

21-40

%

n

30

5 5 10

45

25

I

41-58

%

n

25 25

9

50

I

8

I

% 50 5.6 44.4

analysis within 24 to 48 hours. Culturing villi provides a greater number of meta phases and permits cytogenetic analysis in 5 to 7 days. For the direct method, villi are rinsed in fresh media and minced as necessary. Arborizing clusters of villi are transferred to 35 mm plastic Petri dishes containing 1 ml Chang media (Hana Biologics, Berkeley, California) and maintained at 37° C in an open system (5% carbon dioxide/95% balanced air). Either immediately or after an overnight incubation, cultures are exposed to 1 x 10- 6 mol/L concentration of colchicine for 1 to 3 hours. Media are then replaced with a hypotonic solution of 2 ml of 1% sodium citrate for 10 to 15 minutes, rinsed briefly in a 1 : 1 absolute methanol/glacial acetic acid fixative, and stored in a 5: 2 fixative overnight at - 20° C. Prior to slide preparation the villi are rinsed several times with 5: 2 fixative and then treated with 60% to 70% acetic acid for 5 to 10 minutes. Very small drops of the cell suspension are distributed by means of a micropipette onto dry prewarmed slides. The slides are then dried at least 2 hours in an oven at 40° to 45° C. G-banding is achieved by pretreatment with pancreatin and staining with a Wright-Giemsa solution. In the culture method the villi are washed in Hanks balanced salt solution and transferred to 35 mm Petri dishes containing 3 ml of calcium- and magnesium-free 0.25% trypsin solution; they are then agitated on a girator for 2 hours. The villi and cell suspension are then filtered through a fine wire mesh with use of syringe suction. Villi are removed from the filter, transferred to a 35 mm Petri dish containing 2 ml of calcium- and magnesium-free 0.25% trypsin, and gently agitated for 30 to 60 minutes. Villi are rinsed several times in Chang media and then distributed into 16 ml Flaskettes (Lab-Tek, Miles Scientific, Naperville, Illinois). If necessary, an additional amount of Chang media is added to moisten the growth surface of the Flaskette. Cultures are incubated in an open system (5 % carbon dioxide/95% balanced air) at 37° C and monitored daily for growth. When the cultures show adequate mitotic activity (usually 5 to 7 days), they are sequentially exposed to a 2 x 10 -7 mol/L concentration of colchicine for I to 2 hours, a 0.7% sodium citrate

solution for 20 minutes, and several changes of 5: 2 absolute methanol/glacial acetic acid fixative. The bottom of the Flaskette (which is a microscope slide) is removed and G-banding is achieved as described for the direct method. In 10 cases, cytogenetic analyses were performed on the abortuses (methods were a modification of those previously described by Elias et a1. 5 ) as well as the chorionic villus sampling samples. Phase 2. Our initial experience (described later) was sufficiently encouraging that in March , 1984, we sought and obtained approval from the Institutional Review Board of Northwestern University to offer chorionic villus sampling in continuing pregnancies. The following were considered to be indications for offering chorionic villus sampling: (I) advanced parental age (usually maternal age of ;,,35 years), (2) prior child with a chromosomal abnormality, and (3) fetus at high risk for a detectable mendelian disorder (for example, TaySachs disease, sickle cell disease). Chorionic villus sampling was performed only in patients whose pregnancies were between 7 and II weeks gestation as determined by the onset of the last menses, and confirmed by ultrasonographic measurement of the crown-rump length (that is, between 9 mm and 41 mm) . Patients were excluded from the study if (1) fetal viability was not confirmed (by ultrasonographic visualization of fetal heart motion); (2) multiple gestation was diagnosed by ultrasonographic scanning; (3) pathological conditions were present which conferred increased risk in performing chorionic villus sampling (for example, active genital herpes simplex infection, severe cervicitis, active uterine bleeding); (4) large submucous uterine leiomyomas were uitrasonographically visualized, and reasonable access to the chorionic villi was considered impo5Sible. The techniques for chorionic villus sampling and the processing of specimens were similar to that described in phase I except that a 2.1 mm outer diameter radiopaque Teflon catheter (E-Z Cath, Desert Pharmaceutical Co. , Inc., Sandy, Utah) cut to 16 em was used for sampling. Also, the chorionic villus sampling specimens were examined in the operating room under an inverted microscope (x 50) to assess the quality of the specimen and the necessity of performing additional samplings. The chorionic villus sampling procedures were performed in an ambulatory surgical suite. Following chorionic villus sampling, fetal heart movements were verified by ultrasonographic visualization. Because of the uncertain risk of maternal cell contamination in the chorionic villus sampling specimen which could lead to an erroneous fetal diagnosis, the investigative protocol provided the option for each patient whose results showed a 46,XX complement to have a subsequent genetic amniocentesis at no add i-

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Table V. Summary of chorionic villus sampling procedures in the first 20 diagnostic cases

Table III. Success of culture growth as a function of specimen quality Culture growth

Week of Case gestation

Growth Specimen quality

n

I II III

13 I II

87 14 69

2 6 5

Total

25

66

13

I

%

Total

No growth

IS 7 16 38

Table IV. Success of culture growth from class I specimens as a function of laboratory experience Successful growth

No. of cultures initiated

Specimens from Cases 1-30 Specimens from Cases 31-58 Total

6 9 IS

207

r---.--n

4 9 13

I

%

67 100 87

tional cost. All patients were scheduled for a follow-up level II ultrasound evaluation at 18 weeks' gestation to survey for gross fetal malformations, particularly neural tube defects, and to assess fetal growth.

Results Phase 1. In 25 cases the catheter was transcervically passed into the uterus once in an attempt at obtaining chorionic villi, in 25 cases the catheter was passed twice, in seven cases the catheter was passed three times, and in one case the catheter was passed four times. Table II shows that there was a positive trend between the chronologie order of the chorionic villus sampling (that is, increasing operator experience) and an ability to obtain the best quality specimen (class I). Among 38 specimens in which cultures were initiated from chorionic villi, growth occurred in 66% (Table III). Not surprisingly, the best results (87%) were derived from the 15 cultures initiated from class I specimens. Growth occurred in 67% of cultures initiated from class I specimens obtained during the first 30 cases of the series, but in all class I specimens initiated from the last 28 cases (Table IV). Metaphases showing at least 600 bands were consistently obtained from cultured chorionic villus sampling specimens. Among 39 chorionic villus sampling specimens in which the direct methods were used, we obtained metaphases in 11 (28%); however, the banding quality was not as optimal as that achieved by the culture method. Among 10 cases in which chromosomal analysis was available from both the chorionic villus sampling spec-

I 2 3 4 5 6 7 8 9 10 11 12 13 14 IS 16 17 18 19 20

11 10.5 9 10 9 10.5 10 10 8 10 10 9.5 8.5 10 7 9 10.5 10 9.5 9.5

Number of passes of Cytogenetic analysis from chorionic villus chorionic villus sampling specimen sampling catheter

2

2 3 5 4 3 3 3 5 3 2 2 I

2

4 1 2 2 I 3

46,xX 46,XY 46,XX* 47,XY,+ 13 46,XX 46,XX 46,XX 46,XX 45,X 46,XX 46,XX 46,XY 46,XX 46,XY 46,XX 46,XY 46,XX 46,XX 46,XY 46,XX

* Amniocentesis at 16 weeks' gestation revealed a 46,XY complement, presumably indicating maternal cell contamination in the chorionic villus sampling specimen.

imens and the abortus, there was concordance for sex in 9 of 10 cases. In one class III chorionic villus Sampling specimen (poorest quality) the chromosomal complement was 46,XX, but the abortus was 46,XY, indicating maternal cell contamination. Phase 2. Among our first 30 women who received formal genetic counseling and were offered chorionic villus sampling, 22 women elected to undergo the procedure. In all except two cases the indications were advanced maternal age (35 years or older). One patient was a 30-year-old woman who had a previous child with trisomy 21, and the other was a 30-year-old woman who had a previous child with trisomy 13. In all cases the fetal heart movement was ultrasonographically visualized, indicating fetal viability. One woman was determined to have a large posterior leiomyoma, and it was believed that access to the chorionic villi would be difficult; chorionic villus sampling was therefore not attempted and the patient was scheduled for an amniocentesis at 16 weeks' gestation. In another woman a twin gestation was ultrasonographically diagnosed, and she was also scheduled for an amniocentesis at 16 weeks' gestation. The remaining 20 patients underwent the chorionic villus sampling procedure. Table V summarizes the pertinent data. The total amount of chorionic villus sampling specimen per patient ranged from 1 mg to 30 mg as estimated by comparison to a predetermined reference standard in which varying amounts of villi were weighed. In all 20 cases cytogenetic analysis was

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made by means of the culture method; in 12 of the 20 cases the direct method was also performed. In seven of the 12 direct analysis was believed reliable and indeed the results correlated with that from the culture method. In four of these 12 cases the metaphases were insufficiently spread to permit cytogenetic analysis, whereas no meta phases were observed from one of the cases in which direct preparations were attempted. Case 4 revealed a 47,XY, + 13 complement and case 9 a 45,X complement. Both patients electively terminated their pregnancies. In case 3 a class III chorionic villus sampling specimen was determined to be 46,XX. We therefore recommended an amniocentesis at 16 weeks' gestation. This revealed a 46,XY complement, presumably indicating maternal cell contamination in the chorionic villus sampling specimen. Except for the two cases in which patients underwent elective abortion because of a chromosomal abnormality, all pregnancies are currently continuing, (that is, as of October 10, 1984, there had been no spontaneous abortions in our series thus far, with patients having been followed for between 1 and 28 weeks after the chorionic villus sampling procedure). Four patients have experienced spotting per vagina for up to 6 days following chorionic villus sampling. Otherwise, all pregnancies have been uncomplicated.

Comment Historical aspects. Sampling fetal tissues in the first trimester for prenatal diagnosis was first attempted over a decade ago. In 1973 Kullander and Sandahl' studied a series of 39 women undergoing elective pregnancy termination between 8 and 20 weeks of gestation, 25 being between 8 and 12 weeks of gestation. Subjects underwent transcervical chorionic biopsy with use of a 5 mm endocervicoscope and biopsy forceps. Cytogenetic analyses were completed in 20 cases, all of which were normal and concordant with respect to the sex of the abortus. In 19 cases, pregnancy termination was delayed between 7 and 43 days; the only complications consisted of two patients who developed infection. In 1975 the faculty at Tietung Hospital of Anshan Iron and Steel Company, Anshan, China," reported 100 cases in which pregnancy was allowed to continue after a 3 mm diameter cannula was "blindly" introduced transcervically to aspirate chorionic villi. Fetal sex was determined by X-chromatin analysis. Four pregnancies aborted spontaneously after the procedure, 30 were terminated on the basis of sex prediction, and the remaining 66 pregnancies were allowed to continue. Among these 66 the accuracy rate (that is, correct fetal sex prediction) was 94%. A different approach was attempted in 1977 by Rhine et al. 9 These workers devised an "antenatal cell

May 15, 1985 Am J Obstet Gynecol

extractor" to obtain exfoliated trophoblastic cells by lavage of the lower uterine segment. Unfortunately, our group and others also tried this technique and concluded that first-trimester prenatal diagnosis was not feasible with use of cells obtained by this method. Improved success for chorionic villus sampling became possible with the development of ultrasound technology that allowed precise placement of sampling devices. In 1982 Kazy et al. 10 in Moscow studied 165 cases between 6 and 12 weeks' gestation. Transcervical chorionic villus sampling was performed in one of two ways: (1) following real-time or gray-scale ultrasonographic evaluation by means of a 1.7 mm diameter fetoscope with a direct-vision biopsy forceps (55 cases) or (2) with use of a 2 mm diameter flexible biopsy forceps under simultaneous real-time ultrasonographic guidance (110 cases). In 26 cases chorionic villus sampling was performed in pregnancies allowed to continue. The indications included hemophilia A or B (14 cases); Duchenne muscular dystrophy (six cases); Xlinked hypocephalus (two cases); prior Turner syndrome (four cases). Eleven women elected to terminate their pregnancies because a male fetus was diagnosed; two pregnancies were terminated "at the mother's request." The remaining 13 patients continued their pregnancies. Of these, 11 were delivered of normal infants and two pregnancies were continuing at the time of the report. In 1982 Old et al. II reported 63 patients in whom chorionic villus sampling was performed prior to elective first-trimester abortion (7 to 13 weeks' gestation) via transcervical introduction of a 1.5 mm diameter plastic catheter with a malleable aluminum obturator (Portex Ltd., Hythe, Kent) directed under simultaneous real-time ultrasound guidance. In none of these cases was there any untoward effects following chorionic villus sampling. These investigators then applied similar techniques in three continuing pregnancies. One of two fetuses at risk for l3-thalassemia was affected, and the patient elected to terminate the pregnancy. In the other case the fetus was unaffected, and the pregnancy was continued. One case at risk for sickle cell anemia later showed a missed abortion. In March, 1983, Brambati and Simoni l2 described ultrasonographically directed chorionic villus sampling (Portex catheter) in a 37-year-old woman heterozygous for Duchenne muscular dystrophy. Cytogenetic analysis revealed a 47,XX, + 21 fetus, and the patient elected to terminate her pregnancy. By late spring of 1983 investigators from around the world reported additional case reports and small series describing chorionic villus sampling for first-trimester prenatal diagnosis for a variety of genetic disorders. The largest reported experience from a single center

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is from Simoni and colleagues 4 in Milan, Italy. In 1983 they reported 372 cases in which chorionic villus sampling was performed prior to pregnancy termination. In 62 cases the procedure was performed by transcervical fetoscopic sampling, in 159 by blind aspiration of villi by means of a plastic tube, in 48 by blind passage of the Portex catheter, and in 103 by ultrasonographically guided passage of a Portex catheter. In the last series (Portex catheter plus ultrasound studies) the success rate for obtaining villi was 96%. This group utilized a "direct method" to obtain meta phases within hours after chorionic villus sampling. They also assayed eight enzymes directly from villi, thus showing that villi were suitable for rapid diagnosis of metabolic diseases. Subsequently, Simoni et al." reported their first 100 diagnostic chorionic villus sampling procedures in continuing pregnancies, including two sets of twins. In two cases they failed to obtain adequate specimens for analysis. Fetal cytogenetic diagnosis was successful in each of the 96 pregnancies in which fetal material was obtained; in two additional cases villi were not obtained. The abortion rate following chorionic villus sampling was stated to be 8%, and only a few of the pregnancies had been delivered at the time of the publication. Safety and accuracy. Although numerous groups around the world have now initiated chorionic villus sampling programs, few data are currently available regarding either diagnostic accuracy or safety. There are several potential problems. First, aspirated cells could be of maternal origin (decidua), clearly an issue if the chorionic villus sampling specimen proves to be 46,XX. Fortunately, decidua can usually be distinguished from villi with use of a dissecting microscope; however, potential problems still exist, particularly in inexperienced hands. Second, trophoblastic cells may not necessarily reflect fetal status. Trisomic lines are known to exist in trophoblasts but not in embryonic tissue. Third, in vitro aberrations may arise, as they do in amniotic fluid cells. Overall, substantially more data must become available before assuming that chorionic villus sampling has the same diagnostic accuracy as amniotic fluid culture. Fourth, and perhaps more troublesome, is the fact that the complication rate as a result of chorionic villus sam piing has yet to be established. Of course, any assessment of procedure-related losses must take into account the absolute fetal loss rate as compared to the natural loss rate. Surprisingly, the natural loss rate is unknown. Abortion rates by week of gestation are available, but times of losses in these studies were based on the clinical recognition of fetal death (for example, passage of tissue). However, a fetus could have died weeks prior to the clinical manifestation of spontaneous abortion. Thus the appropriate comparison for chorionic

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209

villus sampling studies would be losses occurring in women who show an ultrasonographically viable fetus (that is, a well-defined gestational sac, fetal heart motion, and appropriate crown-rump length) appropriately corresponding to the gestational age as determined by the onset of the last menses. Several sources of data indicate that loss rate after a normal 8 to 10 week ultrasound study is only 2% to 4%, depending upon age and other variables. These sources of data include (1) one prospective collaborative study by our group and others in which pregnancies are diagnosed by [3-human chorionic gonadotropin radioimmunoassay within 21 days of conception,14 and (2) studies in which ultrasonography is routinely performed at the time of initial obstetrical registration. IS More accurate figures should be available soon. It is apparent, however, that ultragraphically normal pregnancies are thus lost much less often than previously believed. Moreover, losses becoming clinically evident after 8 weeks usually reflect fetal deaths occurring weeks earlier (socalled missed abortions). Recently, Jackson and Wapner l6 summarized pooled data of diagnostic cases from multiple centers, including our own. Among 1232 registered cases there were 1084 continuing pregnancies (the 148 cases terminated for diagnostic reasons were excluded). None of the 140 infants delivered at the time of the report showed congenital anomalies. In the 1084 pregnancies, 55 (5.1%) of the fetuses were lost for all reasons. In their own experience Jackson and Wapner l6 had only two fetal losses among 170 diagnostic cases. These workers concluded that available data " ... demonstrate that chorionic villus sampling is safe enough to permit a carefully controlled clinical assessment of its safety and accuracy." Indeed, a prospective and controlled study has been initiated through National Institute of Child Health and Human Development funding. Seven centers in the United States (Baylor Medical School, Jefferson Medical College, Michael Reese Hospital, Mt. Sinai Medical School, Northwestern University, University of California at San Francisco, Yale University) have agreed upon a common protocol in order to assess safety and accuracy. However, it will be several years before definitive data will be available. Recommendations for introducing chorionic villus sampling as a clinical service. Our initial experience in developing a chorionic villus sampling program at Northwestern University has provided us the basis for making the following recommendations. First, all aspects of chorionic villus sampling performed either before elective pregnancy terminations or for prenatal diagnosis in continuing pregnancies must be under the approval of an Institutional Review Board with informed consent from each subject. Subjects who are

210

Elias et al.

candidates for prenatal diagnosis must specifically understand that chorionic villus sampling is investigational and not yet an accepted alternative to genetic amniocentesis. Second, our data in termination cases (phase 1) not surprisingly indicates that the success in obtaining adequate chorionic villi samples and in obtaining accurate cytogenetic analysis is directly correlated with operator and laboratory experience. Accordingly, we concur with the recommendations of Ward et al. 3 in suggesting that the surgical and laboratory methods for chorionic villus sampling should be developed with the help of volunteers undergoing elective first-trimester abortions. Only thereafter should chorionic villus sampling be offered for prenatal diagnosis in continuing pregnancies in which a research protocol is used. Although the number of cases performed on patients prior to elective abortions will vary, we found about 50 cases to be sufficient. Third, in our experience culture methods routinely provide numerous high-quality meta phases (600 bands or greater). However, the direct method has not been so successful in consistently providing high quality metaphases. Modifications of the direct method alone may yet prove sufficient for accurate cytogenetic diagnosis. However, we currently utilize the culture method on all chorionic villus sampling specimens, while also performing the direct method if sufficient material is available. Fourth, we address the potential problem of maternal cell contamination by offering either a repeat chorionic villus sampling or a genetic amniocentesis if the chorionic villus sampling specimen is of poor quality or if only analyses from the culture method are available showing a 46,XX complement. Contamination could yield cytogenetic, biochemical, or DNA restriction endonuclease analyses that are not reflective of the fetal status. If an adequate sample of chorionic villi (;;.5 mg) is obtained, the accuracy of prenatal diagnosis is optimized. We wish to acknowledge the following individuals for their cooperation in this investigation: Drs. H. M. Arof, J. B. Asher, L. A. Carrow, M. Fredriksen, M. V. Gerbie, R. E. Lane, R. A. McDermott, L. S. Myers, R. J. Peirce, M. A. Rosner, D. Sawyer, R. Siupik, R. F. Valle. REFERENCES I. NICHD National Registry for Amniocentesis Study Group. Midtrimester amniocentesis for prenatal diagnosis: safety and accuracy. JAMA 1976;236:1471. 2. Old JM, Ward RHT, Karagozlu F, Petrou M, Modell B. First-trimester fetal diagnosis for haemoglobinopathies: three cases. Lancet 1982;2:1413. 3. Ward RHT, Modell B, Petrou M, Karagozlu F, Douratosos E. Method of sampling chorionic villi in first trimester of pregnancy under guidance of real time ultrasound. Br MedJ 1983;286:1542. 4. Simoni G, Brambati B, Danesino C, et al. Efficient direct

May 15, 1985 Am J Obstet Gynecol

5. 6. 7. 8.

9. 10.

II. 12. 13. 14.

15. 16.

chromosome analyses and enzyme determinations from chorionic villi samples in the first trimester of pregnancy. Hum Genet 1983;63:349. Gregson NM, Seabright M. Handling chorionic villi for direct chromosome studies. Lancet 1983;2: 1491. Elias S, LeBeau M, SimpsonJL, Martin AO. Chromosome analysis of ectopic human conceptuses. AM 1 OBSTET GyNECOL 1981; 141:698. Kullander S, Sandahl B. Fetal chromosome analysis after transcervical placental biopsies during early pregnancy. Acta Obstet Gynecol Scand 1973;52:355. Department of Obstetrics and Gynecology, Tietung Hospital of Anshan Iran and Steel Company, Anshan. Fetal sex prediction by sex chromatin of chorionic villi cells during early pregnancy. Clin Med J 1975; 1: 117. Rhine SA, Palmer CG, Thompson F. A simple alternative to amniocentesis for first-trimester prenatal diagnosis. Birth Defects 1977;13(3D):231. Kazy Z, Rozoovsky IS, Bakharev VA. Chorion biopsy in early pregnancy: a method of early diagnosis for inherited disorders. Prenat Diagn 1982;2:39. Old JM, Ward RHT, Karagozlu F, Petrou M, Modell B. First trimester fetal diagnosis for haemoglobinopathies: three cases. Lancet 1982;2:1413. Brambati B, Simoni G. Fetal diagnosis of trisomy 21 in the first trimester of pregnancy. Lancet 1983; 1:586. Simoni G, Brambati B, Danesino C, et al. Diagnostic application of first trimester trophoblast sampling in 100 pregnancies. Hum Genet 1984;66:252. Simpson JL. Low fetal loss rate after normal ultrasound at eight weeks gestation: implications for chorionic villus sampling (CVS)-the NICHD Diabetes in Early Pregnancy Project. Am1 Hum Genet 1984;36:197S. Wilson RD, Kendrick V, Wittman BK, McGilivary BC. Risk of spontaneous abortion in ultrasonographically normal pregnancies. Lancet 1984;2:920. Jackson L, Wapner RJ. Chorionic biopsy. N Engl J Med 1984;311:539.

Discussion DR. FEDERICO G. MARIONA, Detroit, Michigan. The authors have presented a narrative of their tribulations in organizing a clinical program for first-trimester prenatal diagnosis through chorion villi sampling. They have concomitantly supplied a series of comments that contain a review of the work of other groups worldwide and supported this with 16 references, thus indicating the interest in this technique for the last 11 years. Any team that has seriously initiated a project of this magnitude must recognize the importance of their recommendations and I fully concur and support those comments. Our own experience involved the performance of over 100 cases of transcervical chorion aspirations on volunteers undergoing elective first-trimester abortion (8 to 12 weeks); the study was initiated in May, 1983, and protocolized in November, 1983. Like that of the authors, our phase 1 was designed to assess the safety of chorionic villi aspiration along with the consistency and reliability of ultrasound examination for placental location and catheter guidance and the adequate performance of the cytogenetics laboratory for sample examination, microdissection, and culture techniques. We consider this phase critical in

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terms of evaluating sample size and cleanliness of the fronds as it related to accurate laboratory determination. Twenty-six such determinations were performed, and except for one culture contaminated with yeast, all villi karyotypes matched the karyotypes obtained from abortion material. We noticed small differences in the laboratory techniques and realize that they are probably guided by the local experience. Obviously, the purity of the sample, its size, and attention to details increase accuracy and metaphase yield and decrease maternal cell contamination.' Our laboratory utilizes both the direct and the culture technique. The decision as to which technique will be used is made at the time of the sampling, since the tissue is observed and evaluated immediately. A very adequate sample (10 mg) will lend itself for direct and culture procedures. A smaller sample is primarily processed for culture, avoiding the waste of valuable material. We extended our study and provided cultured fibroblasts to the biochemical laboratory for enzyme determination. 2 I would like to ask the authors what their experience has been in this regard . The authors opted for a 2.1 mm diameter catheter for their clinical samples as opposed to continuing with the 1.5 mm catheter used for the cases in phase 1. The catheter was also shorter (16 cm) as opposed to 21 cm in phase 1. In our experience a thicker, shorter catheter that is stiffer introduces easily and may not necessitate a stylet; it obtains a larger but not so clean sample. The lack of standardization of catheters is apparent, since several types are utilized around the world. Our protocol includes presampling and postsampling maternal serum a-fetoprotein determination. Did the authors perform this test? Have they seen any differences? I would like to hear their comments on the value of endocervical cultures obtained prior to sampling, especially in the clinical cases. I am surprised that of their class III specimens (those called unidentifiable tissue, questionable villi, or no villi) 69% grew. Exactly what is it that grew? Usually we attempted no more than three catheter insertions, and the maximum in clinical cases has been two. Therefore, I am somewhat concerned that their group attempted five insertions, even though I am aware of the pressure one is under to obtain an adequate sample. Moreover, four of their patients exhibited vaginal spotting for one week; did they correlate this to the number of insertions? I noticed that in their clinical cases they leaned toward the tenth week (53%) versus 13% in phase 1. Was there any particular reason for this? I definitely agree with their second selection. Since they established a qualitative classification for their samples, for what reason did they switch to the standard reference we all use (sample weight) for their clinical samples? The authors discuss the natural embryo loss rate. We have found an unexpected rate of blighted ova and missed abortion at the time of our presample sono-

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graphic evaluation. Have the authors any comments? I would like to add that the 0.9% spontaneous loss rate in the National Institute of Child Health and Human Development diabetes study as opposed to the variable rate reported worldwide for chorion villi sampling (0% to 2.8% and 6.5%) requires careful evaluation and clear discussion with the prospective parents. In summary, by late August, 1984, 2021 women had been sampled worldwide, 1952 of them successfully, and 348 had been delivered. The adjusted loss rate remains at 3.2%. Chorion villi sampling may demonstrate that its safety and accuracy is equal to or better than that of amniocentesis at 16 weeks, once a randomized, prospective, and controlled study such as the one presently being conducted in Canada, has been completed in the United States (I realize that is easier said than done). Once these limitations, contraindications, and risks are definitely and properly established and the patients and physicians clearly understand them, chorion villi sampling may offer a distinct advantage in decreasing parental anxiety and maternal morbidity related to advanced second-trimester abortion. Until then, a scientific de minimis approach is strongly recommended. REFERENCES I. Elies RG, Williamson R, Niazi M, Coleman D, Horwell D.

Absence of maternal contamination of chorionic villi used for fetal-gene analysis. N Engl J Med 1983;308: 1433. 2. Bhatia R, Mariona F, Koppitch, Fleisher L. First trimester prenatal diagnosis by chorionic villi sampling. Presented at the annual Great Lakes perinatal research conference, September, 1984. 3. Niazi M, Coleman D, Leomer F. Trophoblast sampling in early pregnancy, culture of rapidly dividing cells from immature placental villi . Br J Obstet GynaecoI1981;88:1081. 4. Fleisher L, Mitchell D, Koppitch F, Mariona F, Evans M, Goodman S, Nadler H. Chorionic villous samples (CVS) for the prenatal diagnosis of amino acidopathies. J Hum Genet (in press).

DR. ROBERT J. CARPENTER, Houston, Texas. Within the field of obstetrics, no specific area has changed more rapidly than prenatal diagnosis of fetuses at risk for genetic disease. Currently, chorionic villus sampling is being utilized in multiple centers around the world to obtain first-trimester results in women at risk for genetic disease. The paper under discussion today by Dr. Elias and his colleagues demonstrates in a correct fashion the phases of evaluation which must be utilized by all individuals attempting to do this procedure. One of the most important pieces of information presented by these investigators is that operator efficiency is directly related to extensive preclinical application of this technique. There is a high rate of failed acquisition of adequate specimens for all operators during their initial phase-one procedure, and as they have demonstrated and as seen in our own experience, high-resolution sector scanning is required for defining the location of cord insertion and utilizing it as a marker for insertion of the catheter. A second factor that is extremely critical in the se-

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Elias et al.

lection of patients for the procedure and is discussed briefly in their manuscript is the time of gestation. Although the volume of cases is insufficient in continuing pregnancies to demonstrate this, acquisition of specimen is most appropriately reserved for those patients who are at 9 to II or 12 weeks' gestation when placental mass and localization can be adequately described by ultrasound scanning to allow passage of the catheter. The two major United States centers with more than 300 cases each have adequately demonstrated this. One center samples only patients of >9 weeks' gestation and has a catheter pass rate of 1.1 insertion per patient, whereas at a second center where 7 and 8 weeks' gestations are routinely sampled, a pass rate of 2.2 per patient is seen in their statistics. Although some may attribute this difference to operator proficiency, both in Dr. Elias's brief experience and in our own, pregnancies at ~9 weeks' gestation are much easier to sample, with a larger volume of sample recovered and with a higher percentage of class I villi. In their discussion concerning phase 2 study, multiple issues are raised which require accurate assessment. When these data are available, a reassessment of patients in whom chorionic sampling is appropriate may be required. The major concern for most individuals performing prenatal diagnosis is the overall loss rate intrinsic to chorionic villus sampling, which is above and beyond that of the loss rate at that same gestational age. Only a prospective randomized study stratified for gestational age will provide these data. However, from preliminary attempts by the seven National Institutes of Health centers to initiate this, it appears that patients will often self-select themselves for sampling, which will not result in routine randomization. If this were not the major issue of chorionic villus sampling, one would not be concerned with this patient selection process. However, amniocentesis has a known loss rate of 0.5% (one in 200 patients) in the first 2 weeks following the procedure and can be defined as a safe and accurate procedure. If the loss rate with chorionic villus sampling as seen in the major United States centers with good statistical data is 3%, then women having chorionic villus sampling are at increased risk over those having amniocentesis. Therefore the potential concern as noted by the authors regarding the indications for chorionic villus sampling procedure should be considered carefully. Should the patient at age 35 with a I % to 2% risk of a chromosomal abnormality be allowed to have chorionic villus sampling in the first trimester when the risk of loss of pregnancy may be at least twice that of her risk for a defective child? Should advanced maternal age for chorionic villus sampling be raised to age 38, 39, or 40? Or should the sampling be reserved for women with high-risk disease such as balanced translocation carriers or metabolic or X-linked disease in which the risk is 25% to 50% and would certainly warrant a 3% loss rate, which is currently the risk for fetoscopy and fetal blood acquisition for hemophilia testing. This is one of several major issues raised by the whole area of chorionic villus

May 15, 1985 Am J Obstet Gynecol

sampling. The authors' data with continuing pregnancies will not allow any conclusions to be made. However, their data as well as those of others in the National Institutes of Health collaborative effort and those of our colleagues across the world should be sufficient for this assessment to be made. Other questions that must be answered are (1) What is the risk of prematurity associated with the procedure? (2) What is the risk of intrauterine growth retardation compared to the general population? (3) Will the developmental outcome of the infants in these samplings be any different from th~t of those for whom sampling was by amniocentesis? DR. ELIAS (Closing). With respect to the first question, our series was limited to cytogenetic analysis. We have not yet performed biochemical analysis on chorionic villus sampling specimens. We are currently initiating such studies with collaborators at Children's Memorial Hospital. We did not perform u-fetoprotein testing before and after chorionic sampling. This has been considered by other groups. There is some question as to whether the sensitivity for u-fetoprotein testing performed so early in gestation is sufficiently precise to detect any significant changes. With respect to the question of cervical cultures, we did perform cervical cultures for Neisseria gonorrhoeae in many of our patients. However, other groups around the world have recently indicated that the yield from such a screening program is very small. The issue of whether class III specimens provide diagnostically useful information is indeed a concern. If one gets a 46,XX complement, one would have to seriously consider the possibility of maternal cell contamination (that is, the source of tissue is possibly decidua). On the other hand, we feel that initiation of the culture is very impo~tant even if one has a class III specimen because if the results show a 46,XY or a chromosomal abnormality, it would presumably reflect fetal status. Dr. Mariona expressed concern about our willingness to pass the catheter up to five times. That was only in phase I of our study. In continuing pregnancies we limit our number of passages to three. Another question raised was in regard to the differential between the gestational ages in our phase 1 and phase 2 patients, that is, the phase I patients were generally at an earlier gestation compared to patients undergoing diagnostic chorionic villus sampling. The reason was that patients who were in phase 1 were having voluntarY interruptions of pregnancy and presented earlier for their interruptions compared to patients undergoing antenatal diagnosis. As Dr. Carpenter had mentioned, our current opinion is that testing between 8 and 10 weeks is optimal. Dr. Carpenter inquired about the indications for chorionic villus sampling and suggested we consider limiting the procedure to high-risk groups (that is, those at risk for mendelian disorders). In our genetics unit we recommend flexibility in offering patients antenatal diagnosis. We do not adhere to rigid criteria. Each cou-

Chorionic villus sampling

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pIe must weigh the risks!benefits ratio for themselves in deciding upon chorionic villus sampling or any diagnostic procedure. Finally, current data would suggest that although amniocentesis may ultimately be of less risk with respect to a pregnancy loss as compared to chorionic villus sampling, the surprisingly low spon-

taneous abortion figures that we have encountered thus far are very encouraging and would, in my opinion, make antenatal diagnosis through chorionic villus sampling a potentially attractive option for indications like advanced maternal age.

Noninvasive prediction of hyaline membrane disease: An optimized classification of sonographic placental maturation George M. Kazzi, M.D., Thomas L. Gross, M.D., RobertJ. Sokol, M.D., and s. NadyaJ. Kazzi, M.D. Detroit, Michigan, and Cleveland, Ohio Accurate prediction of fetal pulmonary maturity by means of a less invasive procedure than amniocentesis would be desirable. Sonographic diagnosis of a Grade III placenta has been reported to be an excellent predictor of fetal lung maturity. The standard classification of placental grading assigns grade according to the most advanced portion of the placenta. Using this classification, we studied 230 patients. In 80 pregnancies with Grade III placenta, three of the neonates developed respiratory distress syndrome. With reclassification of the placentas as immature, (no Grade III areas), intermediate, (only a portion of the placenta being Grade III), or mature, (Grade III placenta throughout), it was found that no neonatal hyaline membrane disease occurred in the 41 pregnancies with mature placentas, whereas 12% of the neonates in the immature group and 8% in the intermediate group developed hyaline membrane disease. These findings suggest that when sonographic examination of the placenta shows both Grade'" and non-Grade '" sections, there is still a risk for an immature amniotic fluid lecithin/sphingomyelin ratio and neonatal hyaline membrane disease. The placentas should be considered mature only when Grade III changes are present in all sections examined by ultrasound. (AM J OBSTET GVNECOL 1985;152:213-9.)

Key words: Hyaline membrane disease, sonographic placental maturation Prematurity remains a major cause of perinatal morbidity and mortality. Amniocentesis to assess fetal pulmonary maturity is a widely accepted procedure to predict neonatal outcome. However, it is an invasive procedure associated with morbidity and even fetal mortality.' Previous reports have suggested that less invasive methods for predicting hyaline membrane disease might be useful. For example, in a previous study by the authors· a sonographically determined fetal biparietal diameter of 90 mm was found to be associated with 100% absence of hyaline membrane disease. Recent advances in ultrasonography have provided the From the Departments of Obstetrics and Gynecology and Pediatrics, Wayne State University/Hutzel Hospital, and The Perinatal Clinical Research Center, Cleveland Metropolitan General Hospital! Case Western Reserve University. Presented at the Fifty-second Annual Meeting of The Central Association of Obstetricians and Gynecologists, Detroit, Michigan, October 11-13, 1984. Reprint requests: George M. Kazzi, M .D., Department of Obstetrics and Gynecology, Hutzel Hospital, 4707 St. Antoine, Detroit, M1 48201.

clinicians with the ability to describe the maturational changes in the placenta as pregnancy advances. Grannum et al. 3 assessed sonographically diagnosed placental changes and assigned Grades 0, I, II, and III to the progressively occurring appearance. A Grade III placenta had a perfect correlation with fetal pulmonic maturity. However, this finding was not confirmed by Quinlan et al.,' who reported that Grade III placenta could be associated with both an immature lecithin! sphingomyelin ratio and neonatal hyaline membrane disease. The standard classification of placental grading assigns grade according to the most advanced portion of the placenta3 ; therefore, if Grade III changes are seen in one area of a placenta, the placenta is classified as Grade III. We have recently used this grading system in a study of 230 patients for whom ultrasound studies of the placenta and amniotic fluid phospholipids were evaluated.5 Among the group of 80 patients who were delivered following a Grade III placenta, three neonates developed hyaline membrane disease. The pur213