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Chorionic villus sampling in continuing pregnancies II. Cytogenetic reliability
Chorionic villus sampling in continuing pregnancies II. Cytogenetic reliability Alice 0. Martin, Ph.D., Joe Leigh Simpson, M.D., Barbara J. Rosinsky, B.S., and Sherman Elias, M.D. Chicago, Illinois Cytogenetic analysis was performed on 103 chorionic villus samples. Analysis of the 103 samples revealed six abnormalities. In three of the six the abnormalities were confirmed in fetal or neonatal tissue (47,XY, + 13; 46,XY, t(13q13q); 45,X). In three samples the abnormalities detected were not confirmed; in two of the three the abnormalities were detected only in long-term cultures, whereas in the other samples the abnormality was restricted to direct analysis of the villi after overnight incubation. Our initial experience leads us to conclude that certain abnormalities in chorionic villus sampling may not be indicative of fetal abnormalities; 45,X/46,XX or 45,X/46,XY mosaicism is such a complement. Discrepancies between cytogenetic analysis of intact villi processed soon after sampling and of cells grown in culture can be managed by adhering to several suggested guidelines and by liberal use of confirmatory amniocentesis. (AM J 0BSTET GYNECOL 1986; 154:1353-62.)
Key words: Chorionic villus sampling, cytogenetic reliability
Introduction of chorionic villus sampling as a routine diagnostic service not only requires confirmation of its safety but also its diagnostic accuracy. In our companion paper' we consider surgical techniques and safety of chorionic villus sampling. In this contribution we restrict our remarks to cytogenetic techniques and diagnostic accuracy in those 103 cases in which specimens were obtained. We propose guidelines for handling discrepancies between analysis of intact villi processed after overnight incubation and of cells derived from the villi and grown in tissue culture.
Material and methods General approach. Since initiation of our chorionic villus sampling program, laboratory methods have evolved and expanded to improve quality of cytogenetic preparations, to increase efficiency, and to maximize diagnostic accuracy. Our initial methods have been described previously. 2 We continue to use two types of preparations: ( 1) direct analysis of mitoses present in intact chorionic villi, these cells presumed to have originated from budding cytotrophoblasts, and (2) From the Section of Human Genetics, Prentice Women's Hospital and Maternity Center of Northwestern Memorial Hospital, Northwestern University. Supported by the National Institutes of Health (HD 02840, 19866, 52903) and by the March of Dimes. Presented at the Fifty-third Annual Meeting of The Central Association of Obstetricians and Gynecologists, New Orleans, Louisiana, October 10-12, 1985. Reprint requests: Alice 0. Martin, Ph.D., Suite 1176, Prentice Women's Hospital and Maternity Center, 333 East Superior St., Chicago, IL 60611.
analysis of cultured cells, the cultures having been initiated from mesenchyme cores of villi. We have performed cytogenetic analysis of cultured cells on all 103 subjects. "Direct" preparations were also successful in 79 cases. In some earlier patients analysis of intact villi was not always possible because ( 1) insufficient villi were obtained to attempt both types of preparations or (2) quality of the direct preparations was too poor to warrant analyses. However, only once among our last 59 specimens were both direct and culture methods not available. Our current protocol is to visualize the specimen immediately after it has been obtained, with use of a dissecting microscope transported to the operating suite. We seek to determine whether at least 5 mg of villi have been obtained. If not, the obstetrician may elect to perform up to two additional aspiration(s). The specimen(s) is then transported to the laboratory in a 100 mm Petri dish containing 15 ml nutrient media, usually Ham's F-12 supplemented with heparin, penicillin, and streptomycin. The specimen is further examined under a microscope ( 10 X) located in a horizontal flow hood (Edgeguard). If the specimen is extremely bloody, it is filtered through a 115 ml, 5 11 millipore filter unit (Nalgene Type A) before dissection. An estimate of the amount of material obtained is made by comparison with photographs of previously weighed villi (Fig. 1). Villi are then identified, dissected from nonvillus-appearing material, and washed in Hanks basic salt solution containing penicillin and streptomycin. If the total specimen is <5 mg, we may elect to proceed with a culture only, bypassing the direct method. 1353
1354 Martin et al.
June, 1986 Am J Obstet Gynecol
Fig. 1. Amount of dispersed chorionic villi in 5 to 40 mg aliquots.
Fig. 2. Portion of chorionic villi showing buds (short arrow) and capillaries (long arrow).
If both methods are to be attempted, villi are divided approximately equally for direct and culture preparations. The optimal-looking pieces (that is, villi with buds and capillaries) are allocated for the direct method. Preparation of intact villi for analysis (direct method). Villi with buds and capillaries (Fig. 2) are
necessary for the direct preparations. Such villi are placed in 2 ml of Chang's medium and incubated overnight at 37° C in an open system (5% carbon dioxide in balanced air). Specimens can also be processed immediately, but the mitotic index seems to increase when processed following overnight incubation (24 hours). Colcemid (final concentration, 0.15 IJ..g/ml) is added for at least 3 hours. The medium is then aspirated, and 2 ml of0.8% sodium citrate warmed to 37° C added. The specimen is left at room temperature for 20 minutes, following which the hypotonic solution is replaced with 2 ml of freshly prepared and chilled fixative (5 methanol/2 glacial acetic acid), and then chilled for 15 minutes at 4° C. This fixation is then repeated twice. The fixative is next aspirated, and the villi are allowed to air dry. They are then covered for 5 minutes with a few drops of 60% glacial acetic acid in distilled water. A small amount of the previously described 5: 2 fixative is then added, the villi are vigorously aspirated up and down the pipette, and the suspension transferred to microscope slides. Culture initiation. To initiate cultures, we first clean villi as already described. Photographs of villi are taken to document morphology of specimens used to initiate a given culture (A, B, C, etc.). This information will be used to determine (1) appearance of tissue in preparations in which maternal contamination is later demonstrated; (2) appearance of villi when a chromosomally abnormal complement is detected; (3) relationship between (a) villi appearance and quantity and (b) growth rate and quality of preparations. Two types of cultures are then prepared. In one type we use only prolonged trypsinization, whereas in the other we treat villi with trypsin ethylenediaminetetraacetate (EDTA) followed by collagenase. If only a few villi are available, the latter type is preferentiallly initiated because of the shorter time until analysis can proceed.
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Fig. 3. A growth center in a culture derived from chorionic villi mesenchyme.
Table I. Indications and results
Maternal age >35 years
Maternal age <35 years Previous trisomic offspring (two live-born, three amniocentesis, 1 chorionic villus sampling) Translocation heterozygosity45,XX,t(13q13q) Niemann-Pick disease
No.
46,XX
46,XY
Abnormalities
91
48
43
45,X 47,XY,+ 13 46,XY/47,XY,+ 15* 45,XY,t(4; 15)t 45,X/46,XX
4 6
4
4 2
46,XX,t(l3q 13q)
*Mosaicism was found in only direct cultures. Normal male fetus confirmed on amniocentesis. t45,XY,t(4; 15),(4qter ~4pl5 :: 5q ~ 15qter. Because of amniotic sac puncture, only one small piece of villus was obtained. These results are from one culture.
Trypsinization culture method. After dissection and cleaning, villi are placed in a 60 mm Petri dish containing 3 ml of 0.25% trypsin. The dish is placed on a agitator for 2 hours. If villi are large and multibranched, the trypsinized villi are then filtered through a wire mesh. If pieces are small or individually identified, they are merely "swished" in the trypsin to remove debris adhering to the mesenchyme core. In either case a further 30-minute exposure to trypsin is employed, again with the Petri dish placed on the agitator. Pieces of villi are then washed in several changes of Chang's medium (open or closed system types) and placed into flaskettes (Lab Tek). Trypsin-EDTA -collagenase culture method. After dissection and cleaning as previously described, villi are placed in 3 ml of trypsin-EDTA for 2 hours. Pieces of villi are then swished and washed to remove debris and placed in a 60 mm Petri dish with 5 ml of Chang's medium and I mg/ml of collagenase (Sigma Type V, 410 U/mg) overnight (I3 to I7 hours). The suspension is then centrifuged (1000 rpm for 10 minutes), washed
with supplemented medium (Chang's or minimal essential medium), and plated in flaskettes (Lab-Tek). Subsequent culture and harvest. Irrespective of whether the trypsinization method or the combined enzyme method is used, cells usually attach in I to 2 days. Growth centers (Fig. 3) are classified as C (chorionic villus type cells, which are morphologically similar to "AF" cells observed in amniotic fluid cultures); E2 (epithelial-type cells, but cells which are not similar to epithelial cells characteristically observed in amniotic fluid cultures); F (fibroblast-like cells)'; or other. Incidentally, chorionic villus growth centers appear different from those observed in amniotic fluid cultures. The former show well-defined, central aggregates of cells surrounded by peripherally growing cells. Cultures are monitored daily until the mitotic rate is sufficiently high to warrant harvest. The first cultures are ready for analysis at about 5 days and are those prepared by the trypsin-EDTA-collagenase method. Harvest is generally performed in situ on primary cultures, that is, enzymes are not used to disperse cells
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Table II. Chorionic villus sampling
Table IIIB. Incidence of aneuploidy in
abnormalities
chorionic villus sampling according to gestational age (all maternal ages)* Confirmation
UA UA + + UA
47,XY,+ 13 45,X/46,XX 46,XY,t( 13q; 13q) 45,X 45,XY,t(l4p; 15q) (unbalanced) 46,XY/47,XY,+ 15
+ +* + + +
Yes (abortus) No (abortus) Yes (abortus) Yes (abortus) No (amniocentesis) No (amniocentesis)
+
UA = Unavailable or unanalyzable. *Fifteen 45,X cells were obtained from two cultures said to be initiated with villi. Sixteen 46,XX cells were obtained from two cultures with less optimal specimens at initiation. Consequently it was assumed likely that the sixteen 46,XX cells were maternal in origin.
Table IliA. Incidence of aneuploidy in chorionic villus sampling according to maternal age (all gestational weeks) Abnormalities Sex chromosomes
Autosomes Week
n
35-39 >39
21/625 2!/271
I
%
n
3.4
8/625 4/271
7.6
I
% 1.3 1.5
*European Collaborative Data, 1984, from Mikkelsen.'
from their primary growth centers. However, cultures may be subcultured or treated with enzymes to disperse cells within the flaskette (l) if only a single culture is available, (2) if large numbers of metaphases are required for analysis, or (3) if especially high-quality karyotypes are needed. Culture harvest then proceeds. We use 0.7% of sodium citrate for 20 minutes as a hypotonic solution, followed by four changes of methanol/ glacial acetic acid (5: 2) fixative. Microscopic slides are prepared from the suspension-containing cells and are aged at least overnight before being treated with pancreatin and stained with Wright's Giemsa stain. Cytogenetic analysis. Ideally, 20 cells are analyzed from direct preparations and 20 from cultures. We especially prefer this number if a normal female chromosomal complement is encountered. If the complement is male, only 10 cells from each are counted. If mosaicism is detected, 50 to 100 cells are analyzed.
Results Cytogenetic analyses have been completed for I 03 patients with continuing pregnancies (Table I). Ninetyseven were diagnosed normal: 47 male (46,XY) and 50 female (46,XX). However, there was one instance
Abnormalities Week
n
%
6-7 8 9 10 11 12-13
2/19 15/145 29/516 17/465 6/185 6/169
10.5 10.3 5.6 3.7 3.2 8.7
*European Collaborative Data, 1984, from Mikkelsen.' of maternal cell contamination leading to a misdiagnosis of sex. There were six abnormal complements (Table II). Of the 97 normal complements, eight were confirmed by cytogenetic analysis of amniotic fluid cultures. (Amniocentesis is offered to all women whose chorionic villus sampling complement is 46,XX). An additional 35 diagnoses were confirmed at birth with respect to sex and/or normal phenotypes. One normal pregnancy was electively terminated, and 54 pregnancies are still in progress. The status of the six abnormal complements (Table II) is as follows: (1) The 47,XY,+ 13 case was confirmed on an abortus specimen, and thus was considered as validated. (2) The 45,X/46,XX mosaic was not confirmed on abortus material that was definitely fetal in origin; all 100 abortus cells were 46,XX. (3) The 45,XY,t(4;15)(4qter~4pl5::15ql5~15qter) unbalanced complement was confirmed at neither amniocentesis nor delivery (46,XY). A phenotypically normal male infant was delivered. This was not actually unexpected, since the initial abnormal complement was obtained from only one growth center in culture. This finding had thus been interpreted as a culture artifact, clonal in origin. The paucity of analyzable material occurred because only about 0.5 mg of villi were obtained, and these in very small fragments. Consequently, only one culture could be initiated, and only one center was analyzable. (4) The 46,XX,-13,+t(l3ql3q) complement was confirmed on abortus tissue. (5) The 46,XY/ 47,XY, + 15 mosaicism was not confirmed at amniocentesis. The pregnancy continued, and a normal male infant was delivered. However, blood for cytogenetic confirmation has not yet been obtained. Interestingly, this mosaicism was confined to direct preparations. (6) A 45,X complement was observed in both direct preparations and in cultured cells. The same complement was confirmed in abortus material. Overall then, we consider three abnormalities confirmed (Nos. 1, 4, and 6) and three not indicative of fetal anomalies (Nos. 2, 3, and 5).
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Comment The feasibility of routine cytogenetic evaluation of chorionic villi requires affirmative response to each of two general questions: (I) Do villi reflect fetal status, as opposed to being unrepresentative as the result of different embryonic origin? (2) Are villi obtained by aspiration of sufficient quality and quantity to render chromosomal analysis practical in a clinical setting? Early experiences of ourselves and others indicate that affirmative responses to both queries are likely. Notwithstanding our general optimism, however, several dilemmas must be resolved before chorionic villus sampling proves as reliable as amniocentesis for prenatal genetic diagnosis. Maternal cell contamination is one possible dilemma, albeit one that can be minimized with experience. Maternal decidual tissue can be identified and excluded from preparations. The problem appears greatest in cultured preparations. Maternally derived decidua appear to undergo mitoses much more slowly than fetally derived villi, thus erroneously including maternal cells is unlikely to yield spurious results in direct preparations. In cultured preparations, maternal cells may grow and must be excluded before initiation. The one diagnostic error resulting from maternal cell contamination occurred in our early experience. We believe repetition would be rare at present. That not all abnormal metaphases signify an abnormal infant is axiomatic in prenatal cytogenetic diagnosis. In chorionic villus sampling several special problems exist. First, relatively few cells may be available for analysis, and these cells may be derived from only a limited number of villi. Diagnostic errors caused by an abnormality restricted to a clone of cells are thus magnified. Second, clones of aneuploid or structurally abnormal cells may be especially troublesome in villi, which are not only rapidly dividing but not yet subject to the selective elimination that may occur later in pregnancy. We seek to resolve the inevitable diagnostic problems of chorionic villus sampling by initiating both direct as well as long-term (culture) preparations. Disagreements between results of direct preparations and results of cultures do arise. We thus developed and applied the following logic to determine our diagnosis: 1. Suppose one result is a male, and the other a female. The male complement is assumed to reflect fetal origin. 2. Suppose the complement is female, but results are only available from direct preparations and these are abnormal. This is considered likely to reflect fetal abnormalities. This type of preparation is less likely to show maternal contamination, especially if nonmosaic trisomy exists. 3. Suppose mosaicism occurs in both direct as well
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Table IV. Unbalanced translocations* Unbalanced segregant Parental abnormality
Robertsonian translocation Chorionic villus sampiing Amniocentesis Theoretical Reciprocal translocation Chorionic villus sampiing Amniocentesis Theoretical
n
%
5/25
20.8
13/245
5.3 33
7/19
38.9
34/259
13.1 50
*European Collaborative Data, 1984, from Mikkelsen.'
as cultured preparations. The mosaicism is then assumed truly to reflect fetal abnormalities; however, 45,X/46,XX or 45,X/46,XY mosaicism may still pose interpretive dilemmas even if there is "true" mosaicism by the operational definition. 4. Suppose mosaicism is detected only in direct preparations, but large numbers of cells (for example, 40 to 50) in cultured preparations show only a normal complement. Patients are informed of the disagreement, but the likelihood of a normal fetus is high. This approach is especially preferred if the chromosome involved is not one likely to be compatible with a live birth. Examples of "lethal" trisomies includes those for Nos. 2, 15, and 16. 5. Suppose mosaicism is found only in cultures. If possible, results are thus treated in similar fashion as that for interpretation of amniotic fluid cultures! That is, true mosaicism is defined as requiring the same abnormality to be found in more than one culture or clone. Of course, if only one culture is available for chorionic villus sampling, as is often the case, the operational definition of pseudomosaicism (that is, confirmed to one culture) cannot always not be applied. In such a situation, counseling will depend on the total number of analyzable cells from direct and culture preparations, the number of growth centers, and the chromosome involved (see 4). If the abnormal complement can be demonstrated in several growth centers of one culture, true mosaicism is more likely. Applying these and other conventional cytogenetic criteria led us to conclude that three of the six abnormalities we observed (Table II) were in fact truly reflective of fetal status. Three others probably reflected in vitro aberrations or clonal abnormalities restricted to certain villi. Many investigators attach less significance if mosaicism is confined to cultures, but we observed abnormalities apparently not reflective of fetal status in direct but not long-term cultures (case 6, Table II). Especially noteworthy was our failure to confirm
1358 Martin et al.
45,X/46,XX mosaicism that fulfilled criteria established for amniotic fluid analysis, namely, presence of the abnormality in more than one flask or clone. We now believe 45,X cells to be especially common in villi, and we liberally recommend confirmatory amniocentesis whenever 45,X/46,XX or 45,X/46,XY mosaicism is detected. This same reasoning holds when we detect a trisomy for an autosome lethal in triplicate (for example, Nos. 2, 5, and 16). Criteria for interpreting confirmatory amniocentesis are those published previously,5 irrespective of previous chorionic villus sampling results. Even after excluding chromosomal aberrations not reflective of fetal status, the proportion of abnormalities in chorionic villus sampling will remain relatively high. Our own data are limited, but Tables IliA and IIIB show outcome of chorionic villus sampling in a European Collaborative Series. The proportion of aneuploid fetuses in chorionic villus sampling is 4. 7% between maternal ages 35 to 39, and 9.2% over age 39. The proportion of abnormalities in amniocentesis is much lower, namely, only slightly ( 1.5 X) higher than the well-known frequencies observed in live-born infants.6 Aneuploidy in chorionic villus sampling is also greater at 8 weeks than at 10 or 11 weeks (Table IIIB). Table IV shows frequencies of unbalanced offspring conceived by parents with a balanced translocation; frequencies are much higher in chorionic villus sampling than amniocentesis, and these frequencies approach theoretical expectations. All the above emphasize that chorionic villus sampling is occurring before selective processes that are not completely finished by time of amniocentesis. None of the dilemmas considered above diminish our general enthusiasm for chorionic villus sampling; they merely point out that it remains investigational. Increasing experience will surely show trends that will continue to make interpretative dilemmas less common. Indeed, one must recall that pseudomosaicism occurs in 1% to 3% of amnniotic fluid analyses, but true mosaicism in only 0.1 to 0.2%. 6 Tetraploidy is now recognized as a normal phenomenon in amniotic fluid cultures, but this was once considered a dilemma. We suspect that the frequency of true mosaicism may prove more common in amniotic fluid cultures than in chorionic villus sampling, yet not so frequently as to pose insurmountable diagnostic problems. Liberal use of confirmatory amniocentesis is recommended while investigations continue to define the accuracy and reliability of chorionic villus sampling.
REFERENCES 1. Elias S, Simpson 1L, Martin AO, et al. Chorionic villus sampling in continuing pregnancies. I. Low fetal loss rates in initial 109 cases. AM1 0BSTET GYNECOL 1986;154: 1349. 2. Elias S, Simpson1L, Martin AO, Sabbagha RE, Gerbie AB,
June, 1986 Am J Obstet Gynecol
3. 4.
5. 6. 7.
Keith LG. Chorionic villus sampling for first trimester prenatal diagnosis (Northwestern University). AM 1 OBSTET GYNECOL 1985;152:204. Martin AO. Characteristics of amniotic fluid cells in vitro and attempts to improve culture techniques. Clin Obstet Gynecol1980;7:143. Simpson 1L, Martin AO, Verp MS, Elias S, Patel VA. Hypermodal cells in amniotic fluid cultures: frequency, interpretation, and clinical significance. AM 1 OBSTET GYNECOL 1982;143:250. Simpson 1L, Verp MS. Prenatal detection of genetic disorders. Clin Obstet Gynecol 1982;25:635. Benn P, Hsu LYF, Perlis T, Schanhaut A. Prenatal diagnosis of chromosome mosaicism. Prenatal Diagn 1984;4:1. Mikkelsen M. Cytogenetic findings in first trimester chorionic villi biopsies: a collaborative study. In: Fraccaro M, Simoni G, Brambati B, eds. First trimester fetal diagnosis. Berlin: Springer-Verlag, 1985: 109.
Editors' note: This manuscript was revised after these discussions were presented.
Discussion DR. FEDERICO G. MARIONA, Detroit, Michigan. This report by the Northwestern Memorial Hospital group reiterates their cautious and positive approach to the use of chorionic villus sampling in first-trimester genetic diagnosis. The fact that only 188 patients have been screened in over 1 year of clinical service demonstrates a seemingly nonaggressive counseling approach. Twenty-two percent of the patients were classified ineligible for chorionic villus sampling, and 26% of the remaining patients declined having the procedure performed. We can possibly contribute their technical success and low number of procedurally related losses to this stringent patient screening program. Their careful attention to patient selection can further by evidenced by the fact that the majority of patients were sampled between 9 and 10 weeks, with no patients being included after 11 weeks' gestation. This cautious attitude contributes to a laudable policy when we consider the self-imposed anxiety and pressure of some physician groups to accumulate cases. Generally speaking, their selection and exclusion criteria can be construed as being standard. We use a somewhat different catheter, which I no longer trim as I did during the experimental phase. The stylet is round ended and malleable and protrudes half a millimeter beyond the blunt end of a 21 or 26 em, 1.2 mm outer-diameter Teflon catheter. This catheter is under an investigational device exemption from the Food and Drug Administration. The catheter used by the authors of this paper is twice as thick. I am curious to know what prompted their decision to select that particular catheter. I noted that one of the diagnostic errors was related to the obtainment of an extremely small sample despite catheter size. Have you correlated the size of your samples with that of other groups using a smaller catheter or with those of your two obstetricians using differentsized catheters? My samples have gone from 0 (one case, 1.6%) to >50 mg, and one pass was sufficient in 73% of the cases. Our standard protocol for follow-up of all sampled
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patients includes a repeat ultrasound scan between 16 and 19 weeks' gestation, with concomitant obtainment of a sample of maternal serum a-fetoprotein. To date, we have not found a correlation between sample size, number of passes, and hypoechoic placental areas. Further, despite the significant increase in presample and postsample maternal serum a-fetoprotein, we still have not demonstrated a significant difference on the 16-and 19-week maternal serum a-fetoprotein reports between instrumented patients and noninstrumented patients. Have you monitored any of these parameters? This study reports one procedurally related loss in a patient for whom two passes were needed; an adequate sample size was obtained as well as a normal complement, but she bled vaginally. Postsampling vaginal spotting has occurred in 9.6% of our patients sampled to date. Have you looked at the frequency of this event in your population? The authors make a careful evaluation of the laboratory vagaries of their first 103 samples. I shall not attempt to verbally review their technical comments; however, I want to reference their efforts to establish formal recommendations for those involved in the cytogenetic analysis of complements from chorionic villi. Despite their stringent guidelines, they report one error in the sex determination, and six discrepancies, three of which were not confirmed. The diagnostic disparities deserve special consideration, since 50% nonconfirmatory results justify fully the statement that chorionic villus sampling remains an investigational technique. Three of the six disparities were taken to confirmatory amniocentesis; however, two chorionic villus sampling complements were not confirmed by amniocentesis. A 45,X complement was confirmed by amniocentesis, and I am wondering, What was the parental decision? With reference to the nonconfirmatory abortion tissue complement, was this abortion spontaneous or requested on the basis of chorionic villus sampling direct and long-term cultures? Based on the success rate of our short-term cultures, we stopped performing long-term cultures on a routine basis unless an abnormal complement was noted on the direct preparations. The latter has occurred in nine of our cases (14%). In considering the vagaries of the cytogenetic interpretations, the authors have developed a "response protocol" for abnormal findings. This deserves emphasis and appropriate consideration, since it is only through caution and experience that this process will not increase the existing level of parental anxiety. I am certainly interested in clinical details concerning the nine patients who opted for backup amniocentesis after a 46,XX complement was reported on chorionic villi. In our experience the perpetuation of the anxiety and emotional problems becomes a serious situation as the weeks go by. It is clear to this reviewer that the authors' aim is to call attention to the fact that chorionic villus sampling as a first-trimester fetal diagnostic technique remains in the investigational phase. Certainly the need exists
to continue assessing the risks and accuracy of chorionic villus sampling compared with that of the amniocentesis, which is the established technique.
Authors' note: Dr. Mariana has misunderstood our results. There were six abnormal complements detected. Four of these were considered reflective of the fetal complement: 45,X; 47,XY, + 13; 45,X/46,XX; and 46,XX,t( 13q 13q) [Table I]. Three of these four were confirmed; one was not (45,X/46,XX). The other two abnormalties were suspect; consequently amniocentesis was recommended. The 45, XY,t(4; 15) was in only one clone because of the extremely small amount of tissue received (0.05 mg). The other case was mosaic in direct preparations and normal in cultures, the mosaicism for a lethal chromosome, No. 15. Expectation of normal complements was confirmed at both amniocentesis and delivery. DR. JAMES E. NICKEL, Helena, Montana. Since the review of Rodeck' in 1983 in which he described the five biopsy techniques, one other technique has been added. Of these six techniques, the authors have chosen in their first paper to use the technique of guided catheter insertion with ultrasonic sector scanning. For a successful biopsy, three factors must be accomplished: yield of fetal tissue should be reliable, it should not be contaminated by maternal tissue, and there should be minimal risk to the mother and the fetus. The complication rate of this surgery is defined as the number of patients in whom the procedures failed or was thought to cause ill effects to the patient or the pregnancy, either immediately or in the initial followup. In a survey of the literature of the complication, rates have run from 0% to 67%, but in larger series and after a number of cases for operator experience, the complication rate seems to be approximately 2%. After an average number of cases, approximately 70, the complication rate drops from 6% to a 2%. In the large series of cases as reported by these authors and a series of other authors and from the international studies, it is now believed that the fetal mortality rate will not be much increased over that which is reported. One concern is the percentage of patients who bleed after this operation, which is believed to be 14%. This percentage was not reported by the authors, and we would like to ask what percentage of patients bleed following their procedures. It is thought by some authors that a number of complications occur with an increased number of repeated insertions of the catheter, these being related to bleeding, leaking of amniotic fluid, and abruptio placentae. Of major concern in this technnique is that of evaluation of sepsis. As reported in the Chorionic Villus Biopsy Newsletter, there are now a number of cases of sepsis with serious complications. I would ask the authors what the percentage of their infection rate is and whether they are currently doing cervical cultures. A further concern, which is currently being answered by the authors, is that if bacterial invasion does occur at the biopsy site, is the potential of weakening of the amniotic membranes a related problem in the ongoing
1360 Martin et al.
series? Have they, since the initial gathering of these data, noticed any increased rate of premature rupture of the membranes? It is noted in the paper that the risk of teratogenesis is low, since organogenesis is nearly completed by 8 weeks and their biopsies are apparenntly being done after 8 weeks. The risk of fetal malformation in this series is very low, and I wonder whether the authors have noticed, in other series in which the insertions were accomplished before 8 weeks, an increased rate of teratogenesis. The paper of Warren, 2 in Lancet revealed that in approximately 49% of all cases having chorionic villus biopsy, maternal hemorrhage occurs. Do the authors use or plan on using tests such as the Kleihauer-Betke test to determine whether fetal/maternal hemorrhage has occurred? The study by Perry' in 1985 reported experience similar to that of the authors, with a success rate of93% and the only immediate complication occurring in approximately 5% of their cases. They were the first to report on patient attitudes toward this technique. It was found in their paper that an equal number of patients preferred chorionic villus biopsies as compared to amniocentesis. However, if it were known that a less than 5% complication rate occurred, then over 70% of patients were found to prefer chorionic villus biopsy. In the summary of their paper their studies revealed that chorionic villus biopsy was not universally accepted because of the 5% complication rate. I would ask the authors whether in their presurgical counseling they suggest a <5% complication rate and whether this was a major factor in the decision of 38 patients who elected not to have chorionic villus biopsy, as noted in the authors' summary table. It was found in review of the literature at the end of 1984 that 43 centers had reported an excess of 3000 cases with a 97% success rate. The total chorionic villus biopsy fetal loss rate was 4.1 %, and it was found that centers with the most experience had a fetal loss rate of 3.4%. It is suggested that the advantage of the chorionic villus biopsy is a possible reduction in the emotional and physical distress of reproduction for couples at genetic risk because of the ease of performing it in the first trimester. However, current concern is ( 1) that chorionic villus biopsy is more costly than amniocentesis, (2) that it takes longer to accomplish this technique, and (3) that it requires a higher level of ultrasound input. The preparation of the villi for cultures takes longer, and the direct preparations are labor intensive, requiring a much higher level of skill in the team. Have the authors currently evaluated the costeffectiveness of this study and do the authors have suggestions as to what legal precautions we may take in freeing ourselves of the potential liability of this technique? In their second paper the authors have explained the use of the technique of dissecting microscope and direct preparation, as well as cultured villi. This represents a shortened version of the report of Holzgreve4 of the
June, 1986 Am J Obstet Gynecol
University of California in San Francisco in which three different methods of chromosome analysis are used. The authors have shown a marked improvement in their technique because even with use of this technique Martinville et a!." had reported 9% failures of cultured tissue. As can be seen from the paper the results of this technnique rely entirely upon the purity of villus tissue. In a report of possible sources of error Ridler6 indicated that the prenatal diagnosis by means of chorionic villus sampling has not yet fulfilled its early promise. This was because of the variable success in attaining villi pure enough for culture, and because in direct preparation for karyotyping with the chorionic biopsy, the likelihood of contamination with maternal cells is much greater. It was thought by these authors that artificial mosaicism is much more common in trophoblastic tissue rather than fetal tissue, and herein may lie the possibility of the error. This point has been clearly brought up and discussed by the authors. In the excellent summary by Blakemore7 in 1984, with use of a modification of the Niazi culturing techniques for obtaining metaphase cells and the use of quinacrine-banded karyotypes, the results could be obtained in 5 hours from uncultured villus. The authors have not reported to us regarding the time interval for initial diagnosis. It was in the summary in Blakemore's paper that they indicated that their technique did show a high degree of reliability; however, it was felt by these authors that at the current time they were spending one and a half to two times the effort and resources used in chorionic villus biopsy compared to that for chromosomal diagnosis. I would ask the authors whether they have done a cost-analysis comparison between this technique and routine amniocentesis techniques. Do the authors feel they would have a higher failure rate currently with their techniques of villus samples that were <5 mg? Of concern to all people using this technique is the significance of mosaicism and polyploidy, which needs to be established in direct preparations and harvest of cells. It is well known that the amniocytes demonstrate a high frequency of tetraploidy, which is of no recognized significance itself, and this has been reported on numerous occasions in animal studies. I would like to ask the authors whether they are planning an ongoing comparison study with early prenatal karyotyping done simultaneously from blood samples to establish the reliability of this technique concurrently with their technique. I would also like to ask the authors about the number of technicians required in their approach, the time required in preparations, and the length of time between the biopsy and the laboratory diagnosis. The authors have presented a very comprehensive plan to evaluate the samples if they get questionable results on direct preparation and should be complimented on this approach. If the authors do not get enough tissue, do they eliminate the direct preparation and go directly to use of the cultures? I would like to thank the authors for supplying me
Volume 154 Number 6
with a summary outline of their papers before the time of the meeting and would like to compliment them for their ongoing work in what is considered at this point an alternate technique to amniocentesis. Also, I would like to ask the authors how they currently handle the liability situation, which must arise with use of this experimental technique. REFERENCES I. Rodeck CH. Development of an improved technique for
2. 3. 4.
5. 6. 7. 8.
first-trimester microsampling of chorion. Br J Obstet Gynaecol1983;90:1113. Warren RC. Does chorionic villus sampling cause fetomaternal haemorrhage? Lancet 1985;691. Perry TB. Chorionic villi sampling: clinical experience, immediate complications, and patient attitudes. AM J OBSTET GYNECOL 1985; 151:161. Holzgreve W, Hogge WA, Golbus MS. Chorion villi sampling (CVS) for prenatal diagnosis of genetic disorders: first results and future research. Eur J Obstet Gynecol Reprod Biol1984;17:121. de Martinville B. DNA analysis of first-trimester chorionic villous biopsies: test for maternal contamination. Am J Hum Genet 1984;36:1357. Ridler MA, Grewol MS. Possible source of error in prenatal diagnosis via chorionic villus biopsy [letter). Lancet 1984; I: 1081. Blakemore KJ. A method of processing first-trimester chorionic villous biopsies for cytogenetic analysis. Am J Hum Genet 1984; 1386. Gregson NM. Handling chorionic villi for direct chromosome studies. Lancet 1983; 1491.
DR. ROBERT CARPENTER, Houston, Texas. One point raised by Dr. Mariana that Dr. Simpson might discuss really needs a lot of emphasis, and that is the aspect of counseling. The question was raised by him as to the number of patients who subsequently elect not to have chorionic villus sampling done. We have had 100 procedures done in our center, and at least 50% to 60% of the patients who requested the procedure did not have it done. The reasons for this are very diverse, but I think the major one is that aspect raised by Dr. Nickel, that is, what is the loss rate associated with the procedure. Couples fear that they may experience a loss due to chorionic villus sampling even when the data for an individual center show a loss rate of 2%. In our particular center the loss rate associated with the procedure that is quoted to patients is the loss rate experienced in 9000 worldwide series of about 4%. We have had five losses in our 100 procedures. To date, those five losses have occurred from 3 days to 7 weeks after the procedure. So far we have not had preterm labor in any of the patients at more than 21 weeks' gestation. Therefore the critical questions of loss rate and infection are also addressed and are extensively handled in our counseling sessions. The risk of infection is the one risk we cannot quantitate, and there have been relatively few cases in the 9000 procedures that have been done. Those two particular issues, when raised, will scare many patients, and I think it is very appropriate in any
Chorionic villus sampling. II
1361
counseling center dealing with chorionic villus sampling that those two issues be emphasized and emphasized again. We are probably overly counseling, and in effect, instead of being nondirective, we are being biased to a more directive conservative view. All of the other things discussed by Dr. Elias as far as procedural techniques are concerned should be done to minimize the risk of infection. One other question was raised concerning cervical cultures. We are doing cultures in all cases at the time of chorionic villus sampling. We currently have a continuous series of 60. We are finding a significant culture-positive rate for both mycoplasma and for Ureaplasma, and also we are establishing cultures from the catheter on its removal from the uterine cavity. We are also finding similar organisms as well as other organisms, including E. coli and group B streptococci, which did not show up on the initial culture. The meaning of that information will be seen in the future as more prospective data are collected. DR. C. BRANDON CHENAULT San Antonio, Texas. Has consideration been given to Rh sensitization? What do you do about it as far as counseling and treatment are concerned? DR. ELIAS (Closing). Let me begin by addressing the question of vaginal bleeding. We have followed up all of our patients very carefully and have found that about half of them had some spotting within 48 hours after the procedure. On follow-up the vast majority reflected some old blood in the vaginal vault from the site of the tenaculum grasping. With respect to infection, we have not had any in our series to date. We routinely perform cultures for gonorrhea as a screening process, in accordance with the National Institutes of Health protocol, and we have not found any to be positive yet. We do not perform cultures for other organisms because at this time we are not sure about the significance of the inevitable cadre of organisms that one could find. We recognize that there is a very significant potential for sepsis in these patients and we have alerted all of our physicians about this risk. We have sent out a special letter to this effect. In addition, we give our patients a handout to take home with them, which specifically discusses the warning signs and gives them telephone numbers to call in case of suspected problems. We follow up all of our patients 3 to 4 days after the procedure to find out if there have been any untoward effects. With respect to the issue of premature rupture of the membranes, we did have one case that I discussed in the presentation. This patient had premature rupture of the membranes at 27 weeks' gestation and was delivered at 29 weeks' gestation with the infant dying of (3-hemolytic streptococcal pneumonia. This is one of the major focuses of the National Institutes of Health collaborative study, and the answer to the problem of premature rupture of the membranes will hopefully be forthcoming. From personal communications with some of the other centers, we have some concerns about second-trimester rupture of the membranes associated
1362 Martin et al.
with oligohydramnios, and we will have to keep a very close watch on this possible association. With respect to Rh sensitization, at the American Society of Human Genetics meeting in Salt Lake City, Utah, Dr. Karen Blakemore and her colleagues from Yale reported a study in which maternal serum a-fetoprotein levels were assessed both before and after chorionic villus sampling in 51 cases, and in 32 cases they found increases of ~5 ng/ml. That means that in twothirds of the cases there was some degree of fetomaternal bleeding. There was also a positive correlation with the amount of villi that were aspirated and the level of fetoprotein. We routinely administer Rh immune globulin after chorionic villus sampling to all appropriate Rh-negative candidates. The acceptance of chorionic villus sampling is, of course, a very complex issue. We and others are currently collaborating on psychological profile studies trying to assess the various anxieties that patients undergo in making decisions between no prenatal diagnosis, chorionic villus sampling or amniocentesis. Obviously proper counseling in a neutral fashion is critical. A question was asked concerning the cost of the procedure. We can say it is essentially the same as for a genetic amniocentesis at our institution. We do, however, add about $200 to the procedure for the ambulatory surgery suite as well as for the postoperative follow-up, which is about 45 minutes. Legal precautions in chorionic villus sampling are germane in the context of developing a new surgical technique. We can only say that we feel it is very important to have institutional review board approval of this new procedure because, as we have tried to indicate, both the safety and the accuracy have not yet been fully delineated. We believe that before proceeding to use chorionic villus sampling in continuing pregnancies it is required to have Food and Drug Administration approval for the catheter to be used. One does not want to be caught in a situation of having a complication when the Food and Drug Administration has specifically said that only approved centers should be doing the procedure. The most important "legal precaution" is that one ought to fully adhere to the spirit as well as the letter of informed consent, and I believe that good medicine will result in good legal outcomes.
June, 1986 Am J Obstet Gynecol
The time for results generally takes about 48 hours with direct analysis and 5 to 7 days thereafter for the cultures. Initially we found our laboratory processes to be very labor intensive, but more recently we have actually found that the time per technician is reduced because of the advantage of not having to follow cultures for prolonged periods of time. Currently we feel that a cytogenetics technician can process approximately 150 samples per year. The question was asked whether we were doing any work with peripheral maternal blood or trophoblastic cytogenetic analysis. We have initiated a collaborative effort with Dr. Harold Miller at Michigan State University, and of course we all have hopes of developing noninvasive methods for prenatal monitoring. If a small amount of tissue is obtained at chorionic villus sampling, as Dr. Simpson indicated, we would generally process the specimen by the culture technique because this offers a greater number of metaphases. However, each case must be individualized. With respect to Dr. Carpenter's comment about counseling, this is obviously crucial. We would much rather have a patient decline chorionic villus sampling than have a patient agree to it who is not fully committed to the process. Our genetic counselor spends approximately 45 minutes with each couple, and we follow this up by an additional 10 to 15 minutes of discussion before the procedure. We have also found, as others have, that the optimal time of sampling is between 9 and 11 weeks from the onset of the last menses. At less than 8 weeks one begins to have trouble documenting fetal compromise. More important, there is a very dramatic drop-off rate in the frequency of cytogenetic abnormalities after this time. In other words, if you were to sample at 6 to 7 weeks you would be ascertaining a greater number of chromosomally abnormal fetuses that would have otherwise spontaneously aborted. Finally, in evaluating various catheters in our initial trials, we determined that samples can be more reliably obtained with the larger-sized catheter (that is, 16 gauge outer diameter).
Key words: Chorionic villus sampling, cytogenetic reliability
Introduction of chorionic villus sampling as a routine diagnostic service not only requires confirmation of its safety but also its diagnostic accuracy. In our companion paper' we consider surgical techniques and safety of chorionic villus sampling. In this contribution we restrict our remarks to cytogenetic techniques and diagnostic accuracy in those 103 cases in which specimens were obtained. We propose guidelines for handling discrepancies between analysis of intact villi processed after overnight incubation and of cells derived from the villi and grown in tissue culture.
Material and methods General approach. Since initiation of our chorionic villus sampling program, laboratory methods have evolved and expanded to improve quality of cytogenetic preparations, to increase efficiency, and to maximize diagnostic accuracy. Our initial methods have been described previously. 2 We continue to use two types of preparations: ( 1) direct analysis of mitoses present in intact chorionic villi, these cells presumed to have originated from budding cytotrophoblasts, and (2) From the Section of Human Genetics, Prentice Women's Hospital and Maternity Center of Northwestern Memorial Hospital, Northwestern University. Supported by the National Institutes of Health (HD 02840, 19866, 52903) and by the March of Dimes. Presented at the Fifty-third Annual Meeting of The Central Association of Obstetricians and Gynecologists, New Orleans, Louisiana, October 10-12, 1985. Reprint requests: Alice 0. Martin, Ph.D., Suite 1176, Prentice Women's Hospital and Maternity Center, 333 East Superior St., Chicago, IL 60611.
analysis of cultured cells, the cultures having been initiated from mesenchyme cores of villi. We have performed cytogenetic analysis of cultured cells on all 103 subjects. "Direct" preparations were also successful in 79 cases. In some earlier patients analysis of intact villi was not always possible because ( 1) insufficient villi were obtained to attempt both types of preparations or (2) quality of the direct preparations was too poor to warrant analyses. However, only once among our last 59 specimens were both direct and culture methods not available. Our current protocol is to visualize the specimen immediately after it has been obtained, with use of a dissecting microscope transported to the operating suite. We seek to determine whether at least 5 mg of villi have been obtained. If not, the obstetrician may elect to perform up to two additional aspiration(s). The specimen(s) is then transported to the laboratory in a 100 mm Petri dish containing 15 ml nutrient media, usually Ham's F-12 supplemented with heparin, penicillin, and streptomycin. The specimen is further examined under a microscope ( 10 X) located in a horizontal flow hood (Edgeguard). If the specimen is extremely bloody, it is filtered through a 115 ml, 5 11 millipore filter unit (Nalgene Type A) before dissection. An estimate of the amount of material obtained is made by comparison with photographs of previously weighed villi (Fig. 1). Villi are then identified, dissected from nonvillus-appearing material, and washed in Hanks basic salt solution containing penicillin and streptomycin. If the total specimen is <5 mg, we may elect to proceed with a culture only, bypassing the direct method. 1353
1354 Martin et al.
June, 1986 Am J Obstet Gynecol
Fig. 1. Amount of dispersed chorionic villi in 5 to 40 mg aliquots.
Fig. 2. Portion of chorionic villi showing buds (short arrow) and capillaries (long arrow).
If both methods are to be attempted, villi are divided approximately equally for direct and culture preparations. The optimal-looking pieces (that is, villi with buds and capillaries) are allocated for the direct method. Preparation of intact villi for analysis (direct method). Villi with buds and capillaries (Fig. 2) are
necessary for the direct preparations. Such villi are placed in 2 ml of Chang's medium and incubated overnight at 37° C in an open system (5% carbon dioxide in balanced air). Specimens can also be processed immediately, but the mitotic index seems to increase when processed following overnight incubation (24 hours). Colcemid (final concentration, 0.15 IJ..g/ml) is added for at least 3 hours. The medium is then aspirated, and 2 ml of0.8% sodium citrate warmed to 37° C added. The specimen is left at room temperature for 20 minutes, following which the hypotonic solution is replaced with 2 ml of freshly prepared and chilled fixative (5 methanol/2 glacial acetic acid), and then chilled for 15 minutes at 4° C. This fixation is then repeated twice. The fixative is next aspirated, and the villi are allowed to air dry. They are then covered for 5 minutes with a few drops of 60% glacial acetic acid in distilled water. A small amount of the previously described 5: 2 fixative is then added, the villi are vigorously aspirated up and down the pipette, and the suspension transferred to microscope slides. Culture initiation. To initiate cultures, we first clean villi as already described. Photographs of villi are taken to document morphology of specimens used to initiate a given culture (A, B, C, etc.). This information will be used to determine (1) appearance of tissue in preparations in which maternal contamination is later demonstrated; (2) appearance of villi when a chromosomally abnormal complement is detected; (3) relationship between (a) villi appearance and quantity and (b) growth rate and quality of preparations. Two types of cultures are then prepared. In one type we use only prolonged trypsinization, whereas in the other we treat villi with trypsin ethylenediaminetetraacetate (EDTA) followed by collagenase. If only a few villi are available, the latter type is preferentiallly initiated because of the shorter time until analysis can proceed.
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1355
Fig. 3. A growth center in a culture derived from chorionic villi mesenchyme.
Table I. Indications and results
Maternal age >35 years
Maternal age <35 years Previous trisomic offspring (two live-born, three amniocentesis, 1 chorionic villus sampling) Translocation heterozygosity45,XX,t(13q13q) Niemann-Pick disease
No.
46,XX
46,XY
Abnormalities
91
48
43
45,X 47,XY,+ 13 46,XY/47,XY,+ 15* 45,XY,t(4; 15)t 45,X/46,XX
4 6
4
4 2
46,XX,t(l3q 13q)
*Mosaicism was found in only direct cultures. Normal male fetus confirmed on amniocentesis. t45,XY,t(4; 15),(4qter ~4pl5 :: 5q ~ 15qter. Because of amniotic sac puncture, only one small piece of villus was obtained. These results are from one culture.
Trypsinization culture method. After dissection and cleaning, villi are placed in a 60 mm Petri dish containing 3 ml of 0.25% trypsin. The dish is placed on a agitator for 2 hours. If villi are large and multibranched, the trypsinized villi are then filtered through a wire mesh. If pieces are small or individually identified, they are merely "swished" in the trypsin to remove debris adhering to the mesenchyme core. In either case a further 30-minute exposure to trypsin is employed, again with the Petri dish placed on the agitator. Pieces of villi are then washed in several changes of Chang's medium (open or closed system types) and placed into flaskettes (Lab Tek). Trypsin-EDTA -collagenase culture method. After dissection and cleaning as previously described, villi are placed in 3 ml of trypsin-EDTA for 2 hours. Pieces of villi are then swished and washed to remove debris and placed in a 60 mm Petri dish with 5 ml of Chang's medium and I mg/ml of collagenase (Sigma Type V, 410 U/mg) overnight (I3 to I7 hours). The suspension is then centrifuged (1000 rpm for 10 minutes), washed
with supplemented medium (Chang's or minimal essential medium), and plated in flaskettes (Lab-Tek). Subsequent culture and harvest. Irrespective of whether the trypsinization method or the combined enzyme method is used, cells usually attach in I to 2 days. Growth centers (Fig. 3) are classified as C (chorionic villus type cells, which are morphologically similar to "AF" cells observed in amniotic fluid cultures); E2 (epithelial-type cells, but cells which are not similar to epithelial cells characteristically observed in amniotic fluid cultures); F (fibroblast-like cells)'; or other. Incidentally, chorionic villus growth centers appear different from those observed in amniotic fluid cultures. The former show well-defined, central aggregates of cells surrounded by peripherally growing cells. Cultures are monitored daily until the mitotic rate is sufficiently high to warrant harvest. The first cultures are ready for analysis at about 5 days and are those prepared by the trypsin-EDTA-collagenase method. Harvest is generally performed in situ on primary cultures, that is, enzymes are not used to disperse cells
1356 Martin et al.
June, 1986 Am J Obstet Gynecol
Table II. Chorionic villus sampling
Table IIIB. Incidence of aneuploidy in
abnormalities
chorionic villus sampling according to gestational age (all maternal ages)* Confirmation
UA UA + + UA
47,XY,+ 13 45,X/46,XX 46,XY,t( 13q; 13q) 45,X 45,XY,t(l4p; 15q) (unbalanced) 46,XY/47,XY,+ 15
+ +* + + +
Yes (abortus) No (abortus) Yes (abortus) Yes (abortus) No (amniocentesis) No (amniocentesis)
+
UA = Unavailable or unanalyzable. *Fifteen 45,X cells were obtained from two cultures said to be initiated with villi. Sixteen 46,XX cells were obtained from two cultures with less optimal specimens at initiation. Consequently it was assumed likely that the sixteen 46,XX cells were maternal in origin.
Table IliA. Incidence of aneuploidy in chorionic villus sampling according to maternal age (all gestational weeks) Abnormalities Sex chromosomes
Autosomes Week
n
35-39 >39
21/625 2!/271
I
%
n
3.4
8/625 4/271
7.6
I
% 1.3 1.5
*European Collaborative Data, 1984, from Mikkelsen.'
from their primary growth centers. However, cultures may be subcultured or treated with enzymes to disperse cells within the flaskette (l) if only a single culture is available, (2) if large numbers of metaphases are required for analysis, or (3) if especially high-quality karyotypes are needed. Culture harvest then proceeds. We use 0.7% of sodium citrate for 20 minutes as a hypotonic solution, followed by four changes of methanol/ glacial acetic acid (5: 2) fixative. Microscopic slides are prepared from the suspension-containing cells and are aged at least overnight before being treated with pancreatin and stained with Wright's Giemsa stain. Cytogenetic analysis. Ideally, 20 cells are analyzed from direct preparations and 20 from cultures. We especially prefer this number if a normal female chromosomal complement is encountered. If the complement is male, only 10 cells from each are counted. If mosaicism is detected, 50 to 100 cells are analyzed.
Results Cytogenetic analyses have been completed for I 03 patients with continuing pregnancies (Table I). Ninetyseven were diagnosed normal: 47 male (46,XY) and 50 female (46,XX). However, there was one instance
Abnormalities Week
n
%
6-7 8 9 10 11 12-13
2/19 15/145 29/516 17/465 6/185 6/169
10.5 10.3 5.6 3.7 3.2 8.7
*European Collaborative Data, 1984, from Mikkelsen.' of maternal cell contamination leading to a misdiagnosis of sex. There were six abnormal complements (Table II). Of the 97 normal complements, eight were confirmed by cytogenetic analysis of amniotic fluid cultures. (Amniocentesis is offered to all women whose chorionic villus sampling complement is 46,XX). An additional 35 diagnoses were confirmed at birth with respect to sex and/or normal phenotypes. One normal pregnancy was electively terminated, and 54 pregnancies are still in progress. The status of the six abnormal complements (Table II) is as follows: (1) The 47,XY,+ 13 case was confirmed on an abortus specimen, and thus was considered as validated. (2) The 45,X/46,XX mosaic was not confirmed on abortus material that was definitely fetal in origin; all 100 abortus cells were 46,XX. (3) The 45,XY,t(4;15)(4qter~4pl5::15ql5~15qter) unbalanced complement was confirmed at neither amniocentesis nor delivery (46,XY). A phenotypically normal male infant was delivered. This was not actually unexpected, since the initial abnormal complement was obtained from only one growth center in culture. This finding had thus been interpreted as a culture artifact, clonal in origin. The paucity of analyzable material occurred because only about 0.5 mg of villi were obtained, and these in very small fragments. Consequently, only one culture could be initiated, and only one center was analyzable. (4) The 46,XX,-13,+t(l3ql3q) complement was confirmed on abortus tissue. (5) The 46,XY/ 47,XY, + 15 mosaicism was not confirmed at amniocentesis. The pregnancy continued, and a normal male infant was delivered. However, blood for cytogenetic confirmation has not yet been obtained. Interestingly, this mosaicism was confined to direct preparations. (6) A 45,X complement was observed in both direct preparations and in cultured cells. The same complement was confirmed in abortus material. Overall then, we consider three abnormalities confirmed (Nos. 1, 4, and 6) and three not indicative of fetal anomalies (Nos. 2, 3, and 5).
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Comment The feasibility of routine cytogenetic evaluation of chorionic villi requires affirmative response to each of two general questions: (I) Do villi reflect fetal status, as opposed to being unrepresentative as the result of different embryonic origin? (2) Are villi obtained by aspiration of sufficient quality and quantity to render chromosomal analysis practical in a clinical setting? Early experiences of ourselves and others indicate that affirmative responses to both queries are likely. Notwithstanding our general optimism, however, several dilemmas must be resolved before chorionic villus sampling proves as reliable as amniocentesis for prenatal genetic diagnosis. Maternal cell contamination is one possible dilemma, albeit one that can be minimized with experience. Maternal decidual tissue can be identified and excluded from preparations. The problem appears greatest in cultured preparations. Maternally derived decidua appear to undergo mitoses much more slowly than fetally derived villi, thus erroneously including maternal cells is unlikely to yield spurious results in direct preparations. In cultured preparations, maternal cells may grow and must be excluded before initiation. The one diagnostic error resulting from maternal cell contamination occurred in our early experience. We believe repetition would be rare at present. That not all abnormal metaphases signify an abnormal infant is axiomatic in prenatal cytogenetic diagnosis. In chorionic villus sampling several special problems exist. First, relatively few cells may be available for analysis, and these cells may be derived from only a limited number of villi. Diagnostic errors caused by an abnormality restricted to a clone of cells are thus magnified. Second, clones of aneuploid or structurally abnormal cells may be especially troublesome in villi, which are not only rapidly dividing but not yet subject to the selective elimination that may occur later in pregnancy. We seek to resolve the inevitable diagnostic problems of chorionic villus sampling by initiating both direct as well as long-term (culture) preparations. Disagreements between results of direct preparations and results of cultures do arise. We thus developed and applied the following logic to determine our diagnosis: 1. Suppose one result is a male, and the other a female. The male complement is assumed to reflect fetal origin. 2. Suppose the complement is female, but results are only available from direct preparations and these are abnormal. This is considered likely to reflect fetal abnormalities. This type of preparation is less likely to show maternal contamination, especially if nonmosaic trisomy exists. 3. Suppose mosaicism occurs in both direct as well
1357
Table IV. Unbalanced translocations* Unbalanced segregant Parental abnormality
Robertsonian translocation Chorionic villus sampiing Amniocentesis Theoretical Reciprocal translocation Chorionic villus sampiing Amniocentesis Theoretical
n
%
5/25
20.8
13/245
5.3 33
7/19
38.9
34/259
13.1 50
*European Collaborative Data, 1984, from Mikkelsen.'
as cultured preparations. The mosaicism is then assumed truly to reflect fetal abnormalities; however, 45,X/46,XX or 45,X/46,XY mosaicism may still pose interpretive dilemmas even if there is "true" mosaicism by the operational definition. 4. Suppose mosaicism is detected only in direct preparations, but large numbers of cells (for example, 40 to 50) in cultured preparations show only a normal complement. Patients are informed of the disagreement, but the likelihood of a normal fetus is high. This approach is especially preferred if the chromosome involved is not one likely to be compatible with a live birth. Examples of "lethal" trisomies includes those for Nos. 2, 15, and 16. 5. Suppose mosaicism is found only in cultures. If possible, results are thus treated in similar fashion as that for interpretation of amniotic fluid cultures! That is, true mosaicism is defined as requiring the same abnormality to be found in more than one culture or clone. Of course, if only one culture is available for chorionic villus sampling, as is often the case, the operational definition of pseudomosaicism (that is, confirmed to one culture) cannot always not be applied. In such a situation, counseling will depend on the total number of analyzable cells from direct and culture preparations, the number of growth centers, and the chromosome involved (see 4). If the abnormal complement can be demonstrated in several growth centers of one culture, true mosaicism is more likely. Applying these and other conventional cytogenetic criteria led us to conclude that three of the six abnormalities we observed (Table II) were in fact truly reflective of fetal status. Three others probably reflected in vitro aberrations or clonal abnormalities restricted to certain villi. Many investigators attach less significance if mosaicism is confined to cultures, but we observed abnormalities apparently not reflective of fetal status in direct but not long-term cultures (case 6, Table II). Especially noteworthy was our failure to confirm
1358 Martin et al.
45,X/46,XX mosaicism that fulfilled criteria established for amniotic fluid analysis, namely, presence of the abnormality in more than one flask or clone. We now believe 45,X cells to be especially common in villi, and we liberally recommend confirmatory amniocentesis whenever 45,X/46,XX or 45,X/46,XY mosaicism is detected. This same reasoning holds when we detect a trisomy for an autosome lethal in triplicate (for example, Nos. 2, 5, and 16). Criteria for interpreting confirmatory amniocentesis are those published previously,5 irrespective of previous chorionic villus sampling results. Even after excluding chromosomal aberrations not reflective of fetal status, the proportion of abnormalities in chorionic villus sampling will remain relatively high. Our own data are limited, but Tables IliA and IIIB show outcome of chorionic villus sampling in a European Collaborative Series. The proportion of aneuploid fetuses in chorionic villus sampling is 4. 7% between maternal ages 35 to 39, and 9.2% over age 39. The proportion of abnormalities in amniocentesis is much lower, namely, only slightly ( 1.5 X) higher than the well-known frequencies observed in live-born infants.6 Aneuploidy in chorionic villus sampling is also greater at 8 weeks than at 10 or 11 weeks (Table IIIB). Table IV shows frequencies of unbalanced offspring conceived by parents with a balanced translocation; frequencies are much higher in chorionic villus sampling than amniocentesis, and these frequencies approach theoretical expectations. All the above emphasize that chorionic villus sampling is occurring before selective processes that are not completely finished by time of amniocentesis. None of the dilemmas considered above diminish our general enthusiasm for chorionic villus sampling; they merely point out that it remains investigational. Increasing experience will surely show trends that will continue to make interpretative dilemmas less common. Indeed, one must recall that pseudomosaicism occurs in 1% to 3% of amnniotic fluid analyses, but true mosaicism in only 0.1 to 0.2%. 6 Tetraploidy is now recognized as a normal phenomenon in amniotic fluid cultures, but this was once considered a dilemma. We suspect that the frequency of true mosaicism may prove more common in amniotic fluid cultures than in chorionic villus sampling, yet not so frequently as to pose insurmountable diagnostic problems. Liberal use of confirmatory amniocentesis is recommended while investigations continue to define the accuracy and reliability of chorionic villus sampling.
REFERENCES 1. Elias S, Simpson 1L, Martin AO, et al. Chorionic villus sampling in continuing pregnancies. I. Low fetal loss rates in initial 109 cases. AM1 0BSTET GYNECOL 1986;154: 1349. 2. Elias S, Simpson1L, Martin AO, Sabbagha RE, Gerbie AB,
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3. 4.
5. 6. 7.
Keith LG. Chorionic villus sampling for first trimester prenatal diagnosis (Northwestern University). AM 1 OBSTET GYNECOL 1985;152:204. Martin AO. Characteristics of amniotic fluid cells in vitro and attempts to improve culture techniques. Clin Obstet Gynecol1980;7:143. Simpson 1L, Martin AO, Verp MS, Elias S, Patel VA. Hypermodal cells in amniotic fluid cultures: frequency, interpretation, and clinical significance. AM 1 OBSTET GYNECOL 1982;143:250. Simpson 1L, Verp MS. Prenatal detection of genetic disorders. Clin Obstet Gynecol 1982;25:635. Benn P, Hsu LYF, Perlis T, Schanhaut A. Prenatal diagnosis of chromosome mosaicism. Prenatal Diagn 1984;4:1. Mikkelsen M. Cytogenetic findings in first trimester chorionic villi biopsies: a collaborative study. In: Fraccaro M, Simoni G, Brambati B, eds. First trimester fetal diagnosis. Berlin: Springer-Verlag, 1985: 109.
Editors' note: This manuscript was revised after these discussions were presented.
Discussion DR. FEDERICO G. MARIONA, Detroit, Michigan. This report by the Northwestern Memorial Hospital group reiterates their cautious and positive approach to the use of chorionic villus sampling in first-trimester genetic diagnosis. The fact that only 188 patients have been screened in over 1 year of clinical service demonstrates a seemingly nonaggressive counseling approach. Twenty-two percent of the patients were classified ineligible for chorionic villus sampling, and 26% of the remaining patients declined having the procedure performed. We can possibly contribute their technical success and low number of procedurally related losses to this stringent patient screening program. Their careful attention to patient selection can further by evidenced by the fact that the majority of patients were sampled between 9 and 10 weeks, with no patients being included after 11 weeks' gestation. This cautious attitude contributes to a laudable policy when we consider the self-imposed anxiety and pressure of some physician groups to accumulate cases. Generally speaking, their selection and exclusion criteria can be construed as being standard. We use a somewhat different catheter, which I no longer trim as I did during the experimental phase. The stylet is round ended and malleable and protrudes half a millimeter beyond the blunt end of a 21 or 26 em, 1.2 mm outer-diameter Teflon catheter. This catheter is under an investigational device exemption from the Food and Drug Administration. The catheter used by the authors of this paper is twice as thick. I am curious to know what prompted their decision to select that particular catheter. I noted that one of the diagnostic errors was related to the obtainment of an extremely small sample despite catheter size. Have you correlated the size of your samples with that of other groups using a smaller catheter or with those of your two obstetricians using differentsized catheters? My samples have gone from 0 (one case, 1.6%) to >50 mg, and one pass was sufficient in 73% of the cases. Our standard protocol for follow-up of all sampled
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patients includes a repeat ultrasound scan between 16 and 19 weeks' gestation, with concomitant obtainment of a sample of maternal serum a-fetoprotein. To date, we have not found a correlation between sample size, number of passes, and hypoechoic placental areas. Further, despite the significant increase in presample and postsample maternal serum a-fetoprotein, we still have not demonstrated a significant difference on the 16-and 19-week maternal serum a-fetoprotein reports between instrumented patients and noninstrumented patients. Have you monitored any of these parameters? This study reports one procedurally related loss in a patient for whom two passes were needed; an adequate sample size was obtained as well as a normal complement, but she bled vaginally. Postsampling vaginal spotting has occurred in 9.6% of our patients sampled to date. Have you looked at the frequency of this event in your population? The authors make a careful evaluation of the laboratory vagaries of their first 103 samples. I shall not attempt to verbally review their technical comments; however, I want to reference their efforts to establish formal recommendations for those involved in the cytogenetic analysis of complements from chorionic villi. Despite their stringent guidelines, they report one error in the sex determination, and six discrepancies, three of which were not confirmed. The diagnostic disparities deserve special consideration, since 50% nonconfirmatory results justify fully the statement that chorionic villus sampling remains an investigational technique. Three of the six disparities were taken to confirmatory amniocentesis; however, two chorionic villus sampling complements were not confirmed by amniocentesis. A 45,X complement was confirmed by amniocentesis, and I am wondering, What was the parental decision? With reference to the nonconfirmatory abortion tissue complement, was this abortion spontaneous or requested on the basis of chorionic villus sampling direct and long-term cultures? Based on the success rate of our short-term cultures, we stopped performing long-term cultures on a routine basis unless an abnormal complement was noted on the direct preparations. The latter has occurred in nine of our cases (14%). In considering the vagaries of the cytogenetic interpretations, the authors have developed a "response protocol" for abnormal findings. This deserves emphasis and appropriate consideration, since it is only through caution and experience that this process will not increase the existing level of parental anxiety. I am certainly interested in clinical details concerning the nine patients who opted for backup amniocentesis after a 46,XX complement was reported on chorionic villi. In our experience the perpetuation of the anxiety and emotional problems becomes a serious situation as the weeks go by. It is clear to this reviewer that the authors' aim is to call attention to the fact that chorionic villus sampling as a first-trimester fetal diagnostic technique remains in the investigational phase. Certainly the need exists
to continue assessing the risks and accuracy of chorionic villus sampling compared with that of the amniocentesis, which is the established technique.
Authors' note: Dr. Mariana has misunderstood our results. There were six abnormal complements detected. Four of these were considered reflective of the fetal complement: 45,X; 47,XY, + 13; 45,X/46,XX; and 46,XX,t( 13q 13q) [Table I]. Three of these four were confirmed; one was not (45,X/46,XX). The other two abnormalties were suspect; consequently amniocentesis was recommended. The 45, XY,t(4; 15) was in only one clone because of the extremely small amount of tissue received (0.05 mg). The other case was mosaic in direct preparations and normal in cultures, the mosaicism for a lethal chromosome, No. 15. Expectation of normal complements was confirmed at both amniocentesis and delivery. DR. JAMES E. NICKEL, Helena, Montana. Since the review of Rodeck' in 1983 in which he described the five biopsy techniques, one other technique has been added. Of these six techniques, the authors have chosen in their first paper to use the technique of guided catheter insertion with ultrasonic sector scanning. For a successful biopsy, three factors must be accomplished: yield of fetal tissue should be reliable, it should not be contaminated by maternal tissue, and there should be minimal risk to the mother and the fetus. The complication rate of this surgery is defined as the number of patients in whom the procedures failed or was thought to cause ill effects to the patient or the pregnancy, either immediately or in the initial followup. In a survey of the literature of the complication, rates have run from 0% to 67%, but in larger series and after a number of cases for operator experience, the complication rate seems to be approximately 2%. After an average number of cases, approximately 70, the complication rate drops from 6% to a 2%. In the large series of cases as reported by these authors and a series of other authors and from the international studies, it is now believed that the fetal mortality rate will not be much increased over that which is reported. One concern is the percentage of patients who bleed after this operation, which is believed to be 14%. This percentage was not reported by the authors, and we would like to ask what percentage of patients bleed following their procedures. It is thought by some authors that a number of complications occur with an increased number of repeated insertions of the catheter, these being related to bleeding, leaking of amniotic fluid, and abruptio placentae. Of major concern in this technnique is that of evaluation of sepsis. As reported in the Chorionic Villus Biopsy Newsletter, there are now a number of cases of sepsis with serious complications. I would ask the authors what the percentage of their infection rate is and whether they are currently doing cervical cultures. A further concern, which is currently being answered by the authors, is that if bacterial invasion does occur at the biopsy site, is the potential of weakening of the amniotic membranes a related problem in the ongoing
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series? Have they, since the initial gathering of these data, noticed any increased rate of premature rupture of the membranes? It is noted in the paper that the risk of teratogenesis is low, since organogenesis is nearly completed by 8 weeks and their biopsies are apparenntly being done after 8 weeks. The risk of fetal malformation in this series is very low, and I wonder whether the authors have noticed, in other series in which the insertions were accomplished before 8 weeks, an increased rate of teratogenesis. The paper of Warren, 2 in Lancet revealed that in approximately 49% of all cases having chorionic villus biopsy, maternal hemorrhage occurs. Do the authors use or plan on using tests such as the Kleihauer-Betke test to determine whether fetal/maternal hemorrhage has occurred? The study by Perry' in 1985 reported experience similar to that of the authors, with a success rate of93% and the only immediate complication occurring in approximately 5% of their cases. They were the first to report on patient attitudes toward this technique. It was found in their paper that an equal number of patients preferred chorionic villus biopsies as compared to amniocentesis. However, if it were known that a less than 5% complication rate occurred, then over 70% of patients were found to prefer chorionic villus biopsy. In the summary of their paper their studies revealed that chorionic villus biopsy was not universally accepted because of the 5% complication rate. I would ask the authors whether in their presurgical counseling they suggest a <5% complication rate and whether this was a major factor in the decision of 38 patients who elected not to have chorionic villus biopsy, as noted in the authors' summary table. It was found in review of the literature at the end of 1984 that 43 centers had reported an excess of 3000 cases with a 97% success rate. The total chorionic villus biopsy fetal loss rate was 4.1 %, and it was found that centers with the most experience had a fetal loss rate of 3.4%. It is suggested that the advantage of the chorionic villus biopsy is a possible reduction in the emotional and physical distress of reproduction for couples at genetic risk because of the ease of performing it in the first trimester. However, current concern is ( 1) that chorionic villus biopsy is more costly than amniocentesis, (2) that it takes longer to accomplish this technique, and (3) that it requires a higher level of ultrasound input. The preparation of the villi for cultures takes longer, and the direct preparations are labor intensive, requiring a much higher level of skill in the team. Have the authors currently evaluated the costeffectiveness of this study and do the authors have suggestions as to what legal precautions we may take in freeing ourselves of the potential liability of this technique? In their second paper the authors have explained the use of the technique of dissecting microscope and direct preparation, as well as cultured villi. This represents a shortened version of the report of Holzgreve4 of the
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University of California in San Francisco in which three different methods of chromosome analysis are used. The authors have shown a marked improvement in their technique because even with use of this technique Martinville et a!." had reported 9% failures of cultured tissue. As can be seen from the paper the results of this technnique rely entirely upon the purity of villus tissue. In a report of possible sources of error Ridler6 indicated that the prenatal diagnosis by means of chorionic villus sampling has not yet fulfilled its early promise. This was because of the variable success in attaining villi pure enough for culture, and because in direct preparation for karyotyping with the chorionic biopsy, the likelihood of contamination with maternal cells is much greater. It was thought by these authors that artificial mosaicism is much more common in trophoblastic tissue rather than fetal tissue, and herein may lie the possibility of the error. This point has been clearly brought up and discussed by the authors. In the excellent summary by Blakemore7 in 1984, with use of a modification of the Niazi culturing techniques for obtaining metaphase cells and the use of quinacrine-banded karyotypes, the results could be obtained in 5 hours from uncultured villus. The authors have not reported to us regarding the time interval for initial diagnosis. It was in the summary in Blakemore's paper that they indicated that their technique did show a high degree of reliability; however, it was felt by these authors that at the current time they were spending one and a half to two times the effort and resources used in chorionic villus biopsy compared to that for chromosomal diagnosis. I would ask the authors whether they have done a cost-analysis comparison between this technique and routine amniocentesis techniques. Do the authors feel they would have a higher failure rate currently with their techniques of villus samples that were <5 mg? Of concern to all people using this technique is the significance of mosaicism and polyploidy, which needs to be established in direct preparations and harvest of cells. It is well known that the amniocytes demonstrate a high frequency of tetraploidy, which is of no recognized significance itself, and this has been reported on numerous occasions in animal studies. I would like to ask the authors whether they are planning an ongoing comparison study with early prenatal karyotyping done simultaneously from blood samples to establish the reliability of this technique concurrently with their technique. I would also like to ask the authors about the number of technicians required in their approach, the time required in preparations, and the length of time between the biopsy and the laboratory diagnosis. The authors have presented a very comprehensive plan to evaluate the samples if they get questionable results on direct preparation and should be complimented on this approach. If the authors do not get enough tissue, do they eliminate the direct preparation and go directly to use of the cultures? I would like to thank the authors for supplying me
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with a summary outline of their papers before the time of the meeting and would like to compliment them for their ongoing work in what is considered at this point an alternate technique to amniocentesis. Also, I would like to ask the authors how they currently handle the liability situation, which must arise with use of this experimental technique. REFERENCES I. Rodeck CH. Development of an improved technique for
2. 3. 4.
5. 6. 7. 8.
first-trimester microsampling of chorion. Br J Obstet Gynaecol1983;90:1113. Warren RC. Does chorionic villus sampling cause fetomaternal haemorrhage? Lancet 1985;691. Perry TB. Chorionic villi sampling: clinical experience, immediate complications, and patient attitudes. AM J OBSTET GYNECOL 1985; 151:161. Holzgreve W, Hogge WA, Golbus MS. Chorion villi sampling (CVS) for prenatal diagnosis of genetic disorders: first results and future research. Eur J Obstet Gynecol Reprod Biol1984;17:121. de Martinville B. DNA analysis of first-trimester chorionic villous biopsies: test for maternal contamination. Am J Hum Genet 1984;36:1357. Ridler MA, Grewol MS. Possible source of error in prenatal diagnosis via chorionic villus biopsy [letter). Lancet 1984; I: 1081. Blakemore KJ. A method of processing first-trimester chorionic villous biopsies for cytogenetic analysis. Am J Hum Genet 1984; 1386. Gregson NM. Handling chorionic villi for direct chromosome studies. Lancet 1983; 1491.
DR. ROBERT CARPENTER, Houston, Texas. One point raised by Dr. Mariana that Dr. Simpson might discuss really needs a lot of emphasis, and that is the aspect of counseling. The question was raised by him as to the number of patients who subsequently elect not to have chorionic villus sampling done. We have had 100 procedures done in our center, and at least 50% to 60% of the patients who requested the procedure did not have it done. The reasons for this are very diverse, but I think the major one is that aspect raised by Dr. Nickel, that is, what is the loss rate associated with the procedure. Couples fear that they may experience a loss due to chorionic villus sampling even when the data for an individual center show a loss rate of 2%. In our particular center the loss rate associated with the procedure that is quoted to patients is the loss rate experienced in 9000 worldwide series of about 4%. We have had five losses in our 100 procedures. To date, those five losses have occurred from 3 days to 7 weeks after the procedure. So far we have not had preterm labor in any of the patients at more than 21 weeks' gestation. Therefore the critical questions of loss rate and infection are also addressed and are extensively handled in our counseling sessions. The risk of infection is the one risk we cannot quantitate, and there have been relatively few cases in the 9000 procedures that have been done. Those two particular issues, when raised, will scare many patients, and I think it is very appropriate in any
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counseling center dealing with chorionic villus sampling that those two issues be emphasized and emphasized again. We are probably overly counseling, and in effect, instead of being nondirective, we are being biased to a more directive conservative view. All of the other things discussed by Dr. Elias as far as procedural techniques are concerned should be done to minimize the risk of infection. One other question was raised concerning cervical cultures. We are doing cultures in all cases at the time of chorionic villus sampling. We currently have a continuous series of 60. We are finding a significant culture-positive rate for both mycoplasma and for Ureaplasma, and also we are establishing cultures from the catheter on its removal from the uterine cavity. We are also finding similar organisms as well as other organisms, including E. coli and group B streptococci, which did not show up on the initial culture. The meaning of that information will be seen in the future as more prospective data are collected. DR. C. BRANDON CHENAULT San Antonio, Texas. Has consideration been given to Rh sensitization? What do you do about it as far as counseling and treatment are concerned? DR. ELIAS (Closing). Let me begin by addressing the question of vaginal bleeding. We have followed up all of our patients very carefully and have found that about half of them had some spotting within 48 hours after the procedure. On follow-up the vast majority reflected some old blood in the vaginal vault from the site of the tenaculum grasping. With respect to infection, we have not had any in our series to date. We routinely perform cultures for gonorrhea as a screening process, in accordance with the National Institutes of Health protocol, and we have not found any to be positive yet. We do not perform cultures for other organisms because at this time we are not sure about the significance of the inevitable cadre of organisms that one could find. We recognize that there is a very significant potential for sepsis in these patients and we have alerted all of our physicians about this risk. We have sent out a special letter to this effect. In addition, we give our patients a handout to take home with them, which specifically discusses the warning signs and gives them telephone numbers to call in case of suspected problems. We follow up all of our patients 3 to 4 days after the procedure to find out if there have been any untoward effects. With respect to the issue of premature rupture of the membranes, we did have one case that I discussed in the presentation. This patient had premature rupture of the membranes at 27 weeks' gestation and was delivered at 29 weeks' gestation with the infant dying of (3-hemolytic streptococcal pneumonia. This is one of the major focuses of the National Institutes of Health collaborative study, and the answer to the problem of premature rupture of the membranes will hopefully be forthcoming. From personal communications with some of the other centers, we have some concerns about second-trimester rupture of the membranes associated
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with oligohydramnios, and we will have to keep a very close watch on this possible association. With respect to Rh sensitization, at the American Society of Human Genetics meeting in Salt Lake City, Utah, Dr. Karen Blakemore and her colleagues from Yale reported a study in which maternal serum a-fetoprotein levels were assessed both before and after chorionic villus sampling in 51 cases, and in 32 cases they found increases of ~5 ng/ml. That means that in twothirds of the cases there was some degree of fetomaternal bleeding. There was also a positive correlation with the amount of villi that were aspirated and the level of fetoprotein. We routinely administer Rh immune globulin after chorionic villus sampling to all appropriate Rh-negative candidates. The acceptance of chorionic villus sampling is, of course, a very complex issue. We and others are currently collaborating on psychological profile studies trying to assess the various anxieties that patients undergo in making decisions between no prenatal diagnosis, chorionic villus sampling or amniocentesis. Obviously proper counseling in a neutral fashion is critical. A question was asked concerning the cost of the procedure. We can say it is essentially the same as for a genetic amniocentesis at our institution. We do, however, add about $200 to the procedure for the ambulatory surgery suite as well as for the postoperative follow-up, which is about 45 minutes. Legal precautions in chorionic villus sampling are germane in the context of developing a new surgical technique. We can only say that we feel it is very important to have institutional review board approval of this new procedure because, as we have tried to indicate, both the safety and the accuracy have not yet been fully delineated. We believe that before proceeding to use chorionic villus sampling in continuing pregnancies it is required to have Food and Drug Administration approval for the catheter to be used. One does not want to be caught in a situation of having a complication when the Food and Drug Administration has specifically said that only approved centers should be doing the procedure. The most important "legal precaution" is that one ought to fully adhere to the spirit as well as the letter of informed consent, and I believe that good medicine will result in good legal outcomes.
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The time for results generally takes about 48 hours with direct analysis and 5 to 7 days thereafter for the cultures. Initially we found our laboratory processes to be very labor intensive, but more recently we have actually found that the time per technician is reduced because of the advantage of not having to follow cultures for prolonged periods of time. Currently we feel that a cytogenetics technician can process approximately 150 samples per year. The question was asked whether we were doing any work with peripheral maternal blood or trophoblastic cytogenetic analysis. We have initiated a collaborative effort with Dr. Harold Miller at Michigan State University, and of course we all have hopes of developing noninvasive methods for prenatal monitoring. If a small amount of tissue is obtained at chorionic villus sampling, as Dr. Simpson indicated, we would generally process the specimen by the culture technique because this offers a greater number of metaphases. However, each case must be individualized. With respect to Dr. Carpenter's comment about counseling, this is obviously crucial. We would much rather have a patient decline chorionic villus sampling than have a patient agree to it who is not fully committed to the process. Our genetic counselor spends approximately 45 minutes with each couple, and we follow this up by an additional 10 to 15 minutes of discussion before the procedure. We have also found, as others have, that the optimal time of sampling is between 9 and 11 weeks from the onset of the last menses. At less than 8 weeks one begins to have trouble documenting fetal compromise. More important, there is a very dramatic drop-off rate in the frequency of cytogenetic abnormalities after this time. In other words, if you were to sample at 6 to 7 weeks you would be ascertaining a greater number of chromosomally abnormal fetuses that would have otherwise spontaneously aborted. Finally, in evaluating various catheters in our initial trials, we determined that samples can be more reliably obtained with the larger-sized catheter (that is, 16 gauge outer diameter).