Chapter 24: CHORIONIC VILLUS SAMPLING

Sonographically guided chorionic villus sampling (CVS) has been available in the United States since the early 1980s and has offered couples at genetic risk an early and rapid prenatal diagnosis.¹ The procedure, which can be performed as early as 10 weeks of menstrual age, provides preliminary cytogenetic results within 48 hours and final culture results within 7 days. In contrast, genetic amniocentesis is not routinely performed until approximately 16 weeks of menstrual age with an additional 7 to 10 days required to culture the amniotic fluid cells. Fluorescent in situ hybridization (FISH) can be used for both of these techniques. Thus, the pregnancy is nearly half completed before a definitive diagnosis can be established with amniocentesis. If a significant fetal abnormality is identified, the prospective parents must make a difficult choice of whether to continue or terminate the pregnancy. Postponing this decision until the mid-trimester is extremely difficult because fetal movement has been perceived and significant bonding between the parent and fetus has occurred. In addition, the pregnancy is public knowledge, thereby precluding an element of privacy in decision making. If termination is chosen, maternal risks are greater than in the first trimester, with maternal mortality being up to 5 times higher.²

Despite the advantages of CVS, the procedure has struggled to become universally accepted. This has been due predominantly to a perception that the sampling and laboratory procedures are more complex than amniocentesis. In addition, there have been concerns that the procedure may induce fetal limb defects. Recently, however, enthusiasm for CVS has been renewed. First, contemporaneous studies have demonstrated the accuracy of the laboratory results, the reliability of the sampling, and the safety of the procedure if performed after 10 weeks of gestation by experienced operators. Second, studies have established the superior safety to CVS over first trimester amniocentesis.^{3,4, and 5} Additionally, over the last decade, the complication rates of CVS and mid-trimester amniocentesis are comparable due to the reduction of CVS problems.⁶ Third, the recent success of first trimester screening for fetal chromosomal abnormalities provides an impetus for a first trimester diagnostic procedure for fetal karyotype.⁷

For years, prenatal diagnosis has relied on the analysis of amniotic fluid fibroblasts as an indirect reflection of the fetal genetic makeup. Similarly, chorionic villi are fetal in origin, and as such are also an appropriate and useful source of tissue for the evaluation of fetal genetic disease. Their cytogenetic, molecular, and biochemical properties reflect those of the fetus. In addition, the villi are partly composed of cytotrophoblast cells, which are an actively dividing source of spontaneous mitoses that can be used to obtain a rapid chromosomal analysis. Finally, villi can be easily obtained without requiring puncture of the chorion or amnion membrane.

With the exception of α-fetoprotein analysis, the indications for CVS are essentially the same as those for amniocentesis. The major indications are listed in Table 24-1. Advanced maternal age (older than 35 years) is the most common indication, accounting for 90% of procedures.⁸ In addition, parents who have previously had a child with a chromosomal abnormality that may recur are likely to request early invasive testing, as are couples who are carriers of chromosome translocations or autosomal recessive biochemical or molecular diseases. First-trimester prenatal diagnosis is often requested by women who carry sex-linked diseases because of the 50% recurrence risk in male offspring. Recently, screening for trisomies 21 and 18 in the first trimester has become possible by using a combination of biochemical analysis (pregnancy-associated plasma protein A [PAPP-A] and human chorionic gonadotropin [hCG]) and measurement of the fetal nuchal translucency.⁷ If the preliminary work demonstrating almost 90% sensitivity is substantiated, a positive screen could become a major indication for CVS.

First-trimester prenatal diagnosis is not a new concept. The ability to sample and analyze villus tissue was demonstrated more than 25 years ago by the Chinese who, in an attempt to develop a technique for fetal sex determination, inserted a thin catheter into the uterus guided only by tactile sensation.⁹ When resistance from the gestational sac was felt, suction was applied and small pieces of villi aspirated. Although this approach seems relatively crude by today's standards of ultrasonically guided invasive procedures, the diagnostic accuracy and low miscarriage rate demonstrated the feasibility of first-trimester sampling.

In 1968, Hahnemann and Mohr attempted blind transcervical (TC) trophoblast biopsy in 12 patients using a 6-mm-diameter instrument.¹⁰ Although successful tissue culture was obtained, half of these subjects subsequently aborted. In 1973, Kullander and Sandahl used a 5-mm-diameter fiberoptic endocervoscope with biopsy forceps to perform TC CVS in patients requesting pregnancy termination.¹¹ Although tissue culture was successful in approximately half of the cases, 2 of the subjects subsequently became septic.

In 1974, Hahnemann described further experience with first-trimester prenatal diagnosis using a 2.5-mm hysteroscope and cylindrical biopsy knife.¹² Once again, significant complications, including inadvertent rupture of the amniotic sac, were encountered. By this time, the safety of midtrimester genetic amniocentesis had become well established, and further attempts at first-trimester prenatal diagnosis were temporarily abandoned in the Western hemisphere.

Two technological advances occurred in the early 1980s to allow reintroduction of CVS. The first of these was the development of real-time sonography, making continuous guidance possible. At the same time, sampling instruments were miniaturized and refined. In 1982, Kazy et al reported the first TC CVS performed with real-time sonographic guidance.¹³ That same year, Old reported the first-trimester diagnosis of β-thalassemia major using DNA from chorionic villi obtained by sonographically guided TC aspiration with a 1.5-mm-diameter polyethylene catheter.¹⁴ Using a similar sampling technique, Brambati and Simoni diagnosed trisomy 21 at 11 weeks of gestation.¹⁵

After these preliminary reports, several CVS programs were established in both Europe and the United States, with the outcomes informally reported to a World Health Organization (WHO)-sponsored registry maintained at Jefferson Medical College. This registry and single-center reports were used to estimate the safety of CVS until 1989, when 2 prospective multicentered studies, 1 from Canada¹⁶ and 1 from the United States,¹⁷ were published and confirmed the safety of the procedure.

Procedure-Related Anatomy

Between 9 and 12 weeks after the last menstrual period, the developing gestation does not yet fill the uterine cavity (Figure 24-1). The sac is surrounded by the thick leathery chorionic membranes within which are both the amniotic cavity and the extraembryonic coelom. The amniotic cavity contains the embryo and is enclosed by the thin, whispy, freely mobile amniotic membrane. The extraembryonic coelom is located between the amniotic and chorionic membranes, contains a tenacious mucoid-like substance, and disappears as the amniotic sac grows toward the chorion and the 2 membranes become juxtaposed.

Before 9 weeks, chorionic villi cover the entire outer surface of the gestational sac. As growth continues, the developing sac begins to fill the uterine cavity, and most villi regress except at the implantation site, where they are associated with the decidua basalis (see Figure 24-1). Villi in this area rapidly proliferate to form the chorion frondosum, or fetal component of the placenta. Between 9 and 12 weeks of gestation, the villi float freely within the blood of the intervillus space and are only loosely anchored to the underlying decidua basalis.

Sampling Techniques

Sampling by CVS is generally performed between 70 and 91 days after the last menstrual period. This window is chosen to minimize the background spontaneous miscarriage rate that is higher in early pregnancy, yet still allows sufficient time for results to be available within the first trimester. The chorion frondosum is easily localized by ultrasound as a hyperechoic homogeneous area by this gestational age (Figure 24-2). In addition, fusion of the amnion and chorion has not yet occurred, thereby decreasing the risk of amnion rupture during the procedure. Sampling significantly earlier in gestation may be associated with an increased risk of fetal abnormalities and should not routinely be done.¹⁸ Transcervical sampling may be more difficult after 12 weeks of menstrual age due to the increasing distance between the cervix and placental site as uterine growth continues.

Chorionic villus sampling can be performed by either the TC or the transabdominal (TA) approach (Figure 24-3). The techniques are equally safe and efficacious, and the majority of patients can be sampled by either technique.¹⁹ In most cases, physician or patient preference will dictate which approach is used; however, in approximately 3% to 5% of patients, clinical circumstances will support one approach over the other (Table 24-2) requiring operators to be proficient in both.^19,20 Transcervical CVS is preferred when the placenta is located on the posterior uterine wall, whereas TA sampling is particularly useful when the placenta is implanted in a fundal or high anterior location. Transcervical sampling has the advantage of minimal patient discomfort but is somewhat more difficult to learn.²¹ Both approaches are best performed by using a 2-person technique, with one individual performing the sampling and the other guiding the ultrasound. Communication between the sonographer and sampler is imperative, and the best results have come from centers in which a limited number of samplers and sonographers perform CVS.

Transcervical Sampling

Transcervical CVS is performed by using a polyethylene catheter through which a stainless-steel malleable stylet has been inserted. The stylet fits snugly through the catheter and provides sufficient rigidity for adequate passage through the cervix and into the frondosum. The stylet has a rounded, blunt end that protrudes slightly beyond the end of the catheter to prevent sharp edges that may potentially perforate the membranes. The catheter has a luerlock end to accommodate a syringe. The Trophcan catheter (Portex Company, Concord, MA, USA) had been the one most frequently used in the United States. However, this catheter has recently been removed from the market by the manufacturer, leaving the catheter manufactured by the Cook Company (Spencer, IN, USA) as the only commercially available TC sampling device (Figure 24-4).

Before performing the CVS procedure, ultrasound scanning confirms fetal viability and establishes the area of the chorion frondosum. An approach is mentally mapped that allows catheter placement parallel to the chorionic membrane. Uterine contractions may be present and obstruct or alter the sampling path (Figure 24-5). They may also alter the appearance and location of the placenta by pulling it into unusual locations. When contractions significantly interfere with a proposed sampling path, delaying the procedure for 15 to 30 minutes until they abate is suggested. The presence of large placental lakes should also be noted so they can be avoided, because sampling through these lakes has been associated with increased postprocedure bleeding.²²

The maternal bladder should be sufficiently full to provide an acoustic window through which the vagina, cervix, and uterus can be visualized. Overfilling makes retrieval more difficult by increasing patient discomfort and displacing the uterus out of the pelvis, which extends and fixes the sampling path.

The procedure is performed in the lithotomy position on a standard examination table with foot stirrups. A speculum is inserted, and the vagina and cervix are cleansed with antiseptic solution. The catheter is prepared by slightly curving its distal 3- to 5-cm part with the guidewire in place to allow easy insertion through the cervix. In most cases, only a minimal amount of curvature is required. The cervical canal is then reimaged by ultrasound, and the catheter is introduced through the cervix until loss of resistance at the internal os is felt. Once the sonographer clearly identifies the catheter tip, it is guided by real-time sector scanning to the placental site (Figure 24-6A). The catheter is directed by gently maneuvering the curved periphery of the gestational sac. A greater amount of upward or downward movement of the tip can be accomplished by manipulating the speculum to redirect the angle of approach. Severe bending of the stylet is rarely, if ever, required, but occasionally use of a single-tooth tenaculum on the cervix is needed to alter uterine position.

Insertion of the catheter in the correct tissue plane between the inner uterine wall and gestational sac is critical to safe sampling. Although sonographic guidance is crucial, tactile sensation is equally important. The catheter can be easily advanced if it is in the proper tissue plane, whereas resistance is encountered if it is against the chorionic membrane or uterine wall. A gritty sensation is felt if the catheter is inserted too deeply into the decidua. Slight readjustment of the angle of direction corrects the problem. To ensure an adequate sample, the catheter should be advanced through the full length of the placenta. The guidewire is then removed, and a 20-cc syringe containing approximately 5 cc of a collection medium is attached. The sample is collected by aspiration using negative pressure as the catheter is slowly withdrawn. Slight distortion of the placental surface may be noted sonographically during this process, and larger villus fragments may be visualized as they pass through the catheter lumen.

Transabdominal Chorionic Villus Sampling

Two techniques for TA sampling are presently used. In the single-needle approach a 20-gauge spinal needle is used.²³ Alternatively, some operators perform a double-needle technique that uses an outer guide needle (18-gauge thin wall or a 16- to 17-gauge standard spinal needle) and a smaller sampling needle (20 gauge).²⁴ In general, a 3.5-inch-long needle is sufficient for most patients, but a 5- or 6-inch-long needle should be available for very obese women.

With the single-needle technique, a sampling path is chosen so that the tip of the needle passes within the chorion frondosum parallel to the chorionic membrane. Intervening bowel and bladder must be avoided. The needle tip is first inserted into the myometrium and then redirected parallel to the membrane (Figure 24-7). As with cervical sampling, the needle should be passed through as much villus tissue as possible and remain parallel to the chorionic membrane to avoid inadvertent puncture (Figure 24-6B). Once appropriately placed within the placenta, the stylet is removed and a syringe containing 5 cc of media is attached. Under continuous suction, 4 or 5 to-and-fro passes within the frondosum are made. The needle is then removed from the abdomen while suction is continued. This "vacuuming" technique is required to ensure retrieval of sufficient villus tissue because the diameter of the 20-gauge needle is slightly smaller than that of a TC catheter.

The 2-needle technique uses a slightly larger-gauge spinal needle as a trocar, which is inserted into the myometrium. A thinner (19- to 20-gauge) and longer sampling needle is passed through the trocar into the chorion frondosum. The stylet of the sampling needle is then replaced with a syringe, and sampling is performed as with a single needle.

Both TA sampling approaches appear to be equally safe. The 2-needle technique is theoretically less traumatic because the outer trocar remains still during sampling. It also has the advantage of allowing the operator to obtain additional villi by reinserting the sampling needle without requiring a second skin puncture. The single-needle approach is quicker, less uncomfortable, able to retrieve adequate tissue with minimal insertions, and appears to be the technique that has gained widest acceptance. Both techniques have a learning curve, and operator experience does seem to have a bearing on fetal loss rate.²⁵

Confirmation of Adequate Tissue Retrieval

The presence of adequate villus tissue can usually be confirmed by visual inspection of the syringe contents, but occasionally the sample may need to be evaluated under a dissecting microscope. Samples typically contain a mixture of predominantly villi with a small amount of maternally derived decidua. The chorionic villi appear as free-floating, white structures with fluffy, filiforme branches (Figure 24-8A). Contaminating decidua tissue has a more amorphous appearance and lacks distinct branches. Although these 2 tissues can usually be grossly distinguished by virtue of their respective morphology, confirmation under a dissecting microscope is required if there is uncertainty that adequate villi have been retrieved. Microsopically, the villi have a distinctive branched appearance. Their surface is punctuated by small buds consisting of an outer syncytiotrophoblast covering and a core of mitotically active cytotrophoblast cells (Figure 24-8B). Within the center of each villus is the mesenchymal core, through which capillaries carrying fetal blood cells course.

A minimum of 5 mg of villus tissue is required for most genetic analyses. If insufficient villi are present with the initial attempt, a second aspiration may be performed without additional risk.⁸ Pregnancy loss rates increase significantly when more than 2 insertions are required, and may be as high as 10% if 3 attempts are made.^17,26 Therefore, a third pass should only be attempted if successful retrieval seems certain. Before a third attempt, the anatomic relationships should be reevaluated, interfering contractions should have abated, and consideration should be given to sampling by the alternative route. In most experienced centers, more than 99% of patients can be successfully sampled with 2 or fewer insertions. In our center, we have not had a failed procedure in our last 15,000 patients.

Patients may resume normal physical activity after CVS, although strenuous exercise should be avoided for 24 hours. Sexual abstinence is recommended for a short period of time to minimize any risk of ascending infection. Patients may have some mild vaginal bleeding after CVS; therefore, they should be counseled about this possibility before sampling.

Bleeding

Vaginal bleeding is uncommon after TA CVS but is seen in 7% to 10% of patients sampled transcervically. Minimal spotting is a common occurrence and may occur in almost one-third of women sampled by the TC route.¹⁷ In most cases, the bleeding is self-limited and the pregnancy outcome is excellent. However, a subchorionic hematoma may be visualized immediately after sampling in up to 4% of TC samples.²⁷ The hematoma usually disappears before the 16th week of pregnancy and is only rarely associated with adverse outcome. Of the more than 15,000 CVS procedures performed in our center, we have never needed to terminate a pregnancy or admit a patient for excessive postprocedural bleeding.

Cases of heavy bleeding and resulting hematoma formation occur from accidental placement of the TC catheter into the vascular decidua basalis underlying the chorion frondosum. In extreme cases, the development of the hematoma can actually be seen on ultrasound. In most of these cases, a gritty feeling indicates penetration into the decidual layer. Careful attention to the feel of the catheter and avoidance of unnecessary manipulation can prevent most of these hemorrhagic episodes and minimize this complication.

Infection

Since the initial development of TC CVS, there has been concern that transvaginal passage of an instrument would introduce vaginal flora into the uterus. This possibility was confirmed by cultures that isolated bacteria from up to 30% of catheters used for CVS.^{28,29, and 30} However, in clinical practice, the incidence of post-CVS chorioamnionitis is low.^16,17,31,32 In a recently published US study of more than 2000 cases of TC CVS, infection was suspected as a possible etiology of pregnancy loss in only 0.3% of cases.¹⁷ Infection after TA CVS also occurs and has been demonstrated, at least in some cases, to be secondary to bowel flora introduced by inadvertent puncture by the sampling needle.

In our own series of more than 15,000 procedures in which prophylactic antibodies are not used, we have not observed any cases of chorioamnionitis requiring uterine evacuation. Our incidence of periabortion chorioamnionitis was 0.08% for both TC and TA sampling; this rate is about the same as that seen in series of spontaneous abortions that have not been sampled.^33,34 At present, because of the clinically low incidence of post-CVS chorioamnionitis, routine pre-CVS vaginal or cervical cultures for any organism other than gonococcus is not indicated.

Early in the development of TC CVS, 2 life-threatening pelvic infections were reported.^35,36 Each initially presented with a mild prodrome of maternal myalgias and low-grade fever without localized adnexal or uterine tenderness and subsequently led to maternal sepsis. Both occurred early in the respective center's experience, and in both the same catheter was used for repeat insertions. Since these reports, a practice of using a new sterile catheter for each insertion has been universally adopted, with only exceedingly rare reports of serious infectious complications.

Ruptured Membranes

Acute rupture of the membranes, documented by either obvious gross fluid leakage or a decrease in measurable amnionic fluid on ultrasound evaluation, is a very rare complication of CVS.^17,37 In our own experience, acute rupture of the membranes has not occurred. Experimental attempts to rupture membranes intentionally with a TC catheter have confirmed that the chorion can withstand significant punishment without perforation.

Gross rupture of the membranes days to weeks after the procedure is acknowledged as a possible post-CVS complication. Delayed rupture can result from either mechanical injury to the chorion at the time of sampling with rupture from exposure of the amnion, or chronic irritation or inflammation from a hematoma on low-grade infection, allowing exposure of the amnion to subsequent damage or infection. One group reported a 0.3% incidence of delayed rupture of the membranes after CVS,³² a rate confirmed by Brambati et al.²⁷

Unexplained mid-trimester oligohydramnios has been suggested as a rare complication of TC CVS and may occur from delayed chorioamnion rupture with slow leakage of amniotic fluid.³⁷ These cases are frequently associated with postprocedure bleeding and an elevated maternal serum α-fetoprotein (MSAFP). Operator experience will markedly reduce the risk of this complication, probably by decreasing hematoma formation with its potential to serve as either a nidus for a smoldering infection or a chemical irritant of the membranes.

Elevated MSAFP

An acute rise in MSAFP after CVS has been consistently reported, implying a detectable degree of fetal maternal bleeding.^{38,39, and 40} The elevation is transient, occurs more frequently after TA CVS, and appears to be dependent on the quantity of tissue aspirated.⁴⁰ Some studies have also demonstrated a correlation between the degree of elevation and the incidence of pregnancy loss.⁴¹ Levels will drop to normal ranges by 16 to 18 weeks, which allows neural tube defect (NTD) serum screening to proceed according to usual prenatal protocols.

Rh Isoimmunization

In Rh-negative women, the otherwise negligible fetal maternal bleeding that follows CVS accrues special importance because Rh-positive cells in volumes as low as 0.1 mL have been shown to cause Rh sensitization.⁴² Because all women with even a single pass of a catheter or needle show detectable rises in MSAFP, it seems prudent that all Rh-negative nonsensitized women undergoing CVS receive Rho (D) immunoglobulin subsequent to the procedure.

The potential for a CVS-induced maternal-to-fetal transfusion to worsen already existing Rh immunization has been described, suggesting that sampling sensitized patients represents a contraindication to the procedure.⁴³

Pregnancy Loss

Multiple reports from individual centers have demonstrated the safety and low pregnancy loss rates after CVS.^{8,44,45,46,47,48,49,50, and 51} In experienced centers, the rate of miscarriage from the time of CVS until 28 weeks of gestation is approximately 2% to 3%.¹⁹ However, to determine the incidence of procedure-induced pregnancy loss, adjustments for the relatively high background loss at this gestational age must be made.

First-trimester spontaneous abortion in women not undergoing CVS is a common event, occurring in 1 in every 6 clinically recognized pregnancies.⁵² However, miscarriage rates after ultrasound confirmation of a viable gestation are expected to be less. Simpson et al reported that, when ultrasound confirmation of fetal viability was noted at 8 weeks, 3.2% of 220 women with a mean age of 30 years aborted.⁵³ Christiaens and Stoutenbeek noted a 3.3% fetal loss rate in 274 women with proven fetal viability at 10 weeks.⁵⁴ Because the majority of women undergoing CVS are older than 35 years and the spontaneous miscarriage rate increases with advancing maternal age, this variable must also be considered. Wilson et al found a total fetal loss rate after proven viability by first-trimester ultrasonography of 1.4% in women younger than 30 years, 2.6% in those between 30 and 34 years old, and 4.3% in women older than 35 years.⁵⁵ It appears that the best estimate of the background spontaneous miscarriage rate in a population of women similar to those undergoing CVS is approximately 2% to 3%. Although this rate is similar to the postprocedure loss rate in other centers, a randomized clinical trial is necessary to quantify the procedure-induced risk precisely. Unfortunately, no randomized comparison of sampled with unsampled patients is likely; however, comparisons to amniocentesis have been performed.

Because the background loss rate is higher in the firsttrimester than in the second, any comparison of CVS to second-trimester amniocentesis must enroll all patients before the gestational age at which CVS is performed. The total loss rates can then be compared. All losses must be included, whether from a spontaneous miscarriage or an induced termination for abnormal results. This approach eliminates any bias that may occur when comparing procedures performed at significantly different gestational ages, and also takes into account cytogenetically abnormal embryos that miscarry before an amniocentesis, which would be electively terminated after CVS.

The largest demonstrations of data evaluating the relative safety of CVS and amniocentesis come from 3 recent collaborative reports. In 1989, the Canadian Collaborative CVS-Amniocentesis Clinical Trial Group reported its experience with a prospective, randomized trial comparing TC CVS with second-trimester amniocentesis.¹⁶ During the study period, patients across Canada were only able to undergo CVS in conjunction with the randomized protocol. There were 7.6% fetal losses (spontaneous abortions, induced abortions, and late losses) in the CVS group and 7.0% in the amniocentesis group. Thus, in desired pregnancies, an excess loss rate of 0.6% for CVS over amniocentesis was obtained; this difference was not statistically significant.

Two months after the publication of the Canadian experience, the first American collaborative report appeared.¹⁷ This study was a prospective, although nonrandomized, trial of more than 2200 women who chose either TC CVS or second-trimester amniocentesis. Patients in both groups were recruited in the firsttrimester of pregnancy. As in the Canadian study, advanced maternal age was the primary indication for prenatal testing. When the loss rates were adjusted for slight group differences in maternal and gestational ages at enrollment, an excess pregnancy loss rate of 0.8% referable to CVS over amniocentesis was calculated, which was not statistically significant.

Whereas both North American trials showed no statistical difference in pregnancy loss when CVS was compared with amniocentesis, a prospective, randomized collaborative comparison of more than 3200 pregnancies sponsored by the European MRC Working Party on the Evaluation of CVS demonstrated a 4.6% greater pregnancy loss rate after CVS (95% confidence interval [CI], 1.6% to 7.5%).³⁶ This difference reflected more spontaneous deaths before 28 weeks of gestation (2.9%), more terminations of pregnancy for chromosomal anomalies (1.0%), and more neonatal deaths (0.3%) in the CVS group.

The factors responsible for the discrepant results between the European and North American studies remain uncertain, but it is probable that inadequate operator experience with CVS accounted for a large part of this difference. Whereas the US trial consisted of 7 centers and the Canadian trial 11 centers, the European trial included 31 sampling sites. There were, on average, 325 cases per center in the US study, 106 in the Canadian study, and only 52 in the European trial. Although no significant change in pregnancy loss rate was demonstrated during the course of the European trial, it appears that the learning curve for both TC and TA CVS may exceed 400 or more cases.^56,57 Operators having performed fewer than 100 cases may have 2 or 3 times the postprocedure loss rate of operators who have performed more than 1000 procedures.

The consensus of the recent literature indicates that with experienced operators, the procedural complication rates with CVS and amniocentesis is comparable; however, CVS is more difficult to learn.⁵

There have been similar comparisons between CVS and early amniocentesis, defined as amniocentesis performed before 14 weeks of gestation. In these comparisons of 2 firsttrimester procedures, consideration of gestational age differences is not necessary. Nicolaides et al compared TA CVS with amniocentesis performed between 10 and 13 weeks and gestation.⁵⁸ In this prospective comparison, the spontaneous loss rate was significantly higher after early amniocentesis (5.3%) than after CVS (2.3%). Also, a significant increase in the incidence of talipes equinovarus was seen after early amniocentesis. In another recent comparison, Sundberg et al randomized patients to either amniocentesis between 11 and 13 weeks or TA CVS between 10 and 12 weeks.³ Although the initial end point of this trial was intended to be pregnancy loss, the trial was stopped early because of an increased risk of talipes equinovarus in the early amniocentesis group. Although the power of the trial to compare fetal loss rates was limited by the incomplete sample, no significant difference was demonstrated. The amniocentesis loss rate, however, was 0.6% higher. Leakage of amniotic fluid after sampling occurred significantly more frequently after amniocentesis. Overall, the higher loss rates, increased risk of fluid leakage, and subsequent club foot deformity with early amniocentesis suggest that CVS is the preferred technique for first-trimester sampling.

Randomized trials have compared the TC and TA approaches.^19,57,59'⁶¹ The US collaborative CVS project performed a randomized, prospective study and found no difference in the postprocedure pregnancy loss rates between the 2 approaches (TC, 2.5%; TA, 2.3%).¹⁹ Equally important was that the overall post-CVS loss rate in the study (2.5%) was 0.8% lower than that in the initial US study, which compared CVS with second-trimester amniocentesis. Because 0.8% was the quantitative difference in loss rates between amniocentesis and CVS in the original study, this finding suggests that, when centers become equivalently experienced, amniocentesis and CVS may have the same risk of pregnancy loss.

Smidt-Jensen et al, pioneers of TA CVS, added additional information to the comparative safety of the procedures.⁶¹ In a prospective, randomized study, they found no difference in pregnancy loss between TA CVS and second-trimester amniocentesis, but did demonstrate an increased risk for TC CVS, the procedure for which their center was least experienced. Chueh et al, in a retrospective review of more than 9000 CVS procedures, showed that in their center TC CVS had a slightly greater risk of pregnancy loss than TA sampling.⁶² It appears safe to speculate that fetal loss rates between TC and TA sampling will be similar in most centers once equivalent expertise is gained with either approach. Integration of both methods into the program of any single center will offer the most complete, practical, and safe approach to first-trimester diagnosis.

It has recently been suggested that CVS may be associated with the occurrence of specific fetal malformations. The first suggestion of this was reported by Firth et al⁶³ In a series of 539 CVS-exposed pregnancies, they identified 5 infants with severe limb abnormalities, all of which came from a cohort of 289 pregnancies sampled at 66 days of gestation or less. Four of these infants had the unusual and rare oromandibular-limb hypogenesis syndrome, and the fifth had a terminal transverse limb reduction defect. Oromandibular-limb hypogenesis syndrome occurs with a birth prevalence of 1 per 175,000 live births,⁶⁴ and limb reduction defects occur in 1 per 1690 births.⁶⁵ Therefore, the occurrence of these abnormalities in more than 1% of CVS-sampled cases raised strong suspicion of an association. In this initial report, all of the limb abnormalities followed TA sampling performed between 55 and 66 days of gestation.

Subsequent to this initial report, others added supporting cases to this list. Using the Italian multicenter birth defects registry, Mastroiacovo et al reported, in a case control study, an odds ratio of 11.3 (CI 5.6 to 2.13) for transverse limb abnormalities after first-trimester CVS.⁶⁶ When stratified by gestational age at sampling, pregnancies sampled before 70 days had a 19.7% increased risk of transverse limb reduction defects, whereas patients sampled later did not demonstrate a significantly increased risk. Other single-center and case control studies, however, have been inconclusive about an association of CVS with limb reduction defects, with the majority demonstrating no increased risk (Table 24-3).

There is support of the notion that CVS may increase the risk of limb defects when sampling is performed before 63 days of gestation. Most notably, Brambati et al, an extremely experienced group who have reported no increased risk of limb defects in patients sampled after 9 weeks, have reported a 1.6% incidence of severe limb reduction defects when patients were sampled at 6 and 7 weeks.⁶⁷ This rate decreased to 0.1% for sampling at 8 to 9 weeks. Hsieh et al, in a report of the Taiwan CVS experience, reported 29 cases of limb reduction defects after CVS from September 1990 until June 1992; 4 cases had oromandibular-limb hypogenesis syndrome.⁶⁸ There were 2 remarkable aspects of this report. First, although the gestational age at sampling was not known with certainty in all cases, the majority were performed at less than 63 days after the last menstrual period. Second, very inexperienced community-based operators performed the cases with limb reduction defects, whereas no defects were seen from the major centers. This experience suggests that very early sampling with excessive placental trauma may be etiologic in some reports of post-CVS limb reduction defects.

The question continues to be debated of whether CVS sampling after 70 days has the potential of causing more subtle defects, such as shortening of the distal phalanx or nail hypoplasia.⁶⁹ At present, there are few data to substantiate this concern. On the contrary, most experienced centers performing CVS after 10 weeks have not seen an increase in limb defects of any type. A recent review of more than 200,000 CVS procedures reported to the WHO registry was reported and demonstrated no increase in the overall incidence of limb reduction defects after CVS or in any specific type or pattern of defect.⁷⁰ In a similar review of more than 65,000 procedures performed in 10 of the most experienced centers in the world, no increase in limb reduction defects was identified.⁷¹

Mechanisms by which early CVS could potentially lead to fetal malformations continue to be disputed. Placental thrombosis with subsequent fetal embolization has been raised as a potential etiology, but is unlikely because fetal clotting factors appear to be insufficient at this early gestational age. Inadvertent entry into the extraembryonic coelom with resulting amnionic bands has also been raised as a potential mechanism, but appears unlikely as well, because actual bands have not been observed in the majority of the cases. In addition, many of the cases of oromandibular-limb hypogenesis syndrome had internal central nervous system anomalies that cannot be accounted for by fetal entanglement or compression.

Uterine vascular disruption appears to be the most plausible mechanism at present.⁶⁴ In this hypothesis, CVS causes placental injury or vasospasm that subsequently results in underperfusion of the fetal peripheral circulation. After the initial insult, there may be subsequent rupture of the thin-walled vessels of the damaged distal embryonic circulation, leading to further hypoxia, necrosis, and eventual resorption of preexisting limb structures. A similar mechanism leading to limb defects has been demonstrated in animal models after uterine vascular clamping, maternal cocaine exposure, or even simple uterine palpation.^71,72

In a recent report, Quintero et al added additional information about a possible etiology.^73,74 Using TA embryoscopic visualization of the first-trimester embryo, they demonstrated the occurrence of fetal facial, head, and thoracic ecchymotic lesions after traumatically induced detachment of the placenta with subchorionic hematoma formation. No changes in fetal heart rate were seen. Although these lesions consistently appeared after major physical trauma to the placental site, they were not able to be produced by the passage of a standard CVS catheter.

Any theory of CVS-induced limb defects must consider that there are different stages of fetal sensitivity and should demonstrate a correlation between the severity of the defects and the gestational age at sampling. Firth et al recently presented evidence that appears to illustrate that sampling before 9 weeks of gestation induces the most severe and proximally located fetal limb defects.⁷⁵ These severe defects are not seen after later CVS. Alternatively, Froster and Jackson reviewed the severity of the post-CVS limb defects reported to the WHO registry and found no such correlation.⁷⁰

At the present time, patients planning to have CVS can be counseled that there is no increased risk of severe limb defects if CVS is performed after 70 days of gestation.⁷⁶ They should be made aware of the present controversy concerning more subtle defects and reassured that this has not been seen in most experienced centers. If such a risk does exist, the magnitude based on case control studies can be estimated to be no higher than 1 in 3000.⁷⁶ Ideally, centers performing CVS should have aggressive follow-up systems in place and be capable of giving patients information about the rate of congenital abnormalities in their center. Sampling before 10 weeks of gestation should be limited to very exceptional cases, and these patients must be informed of a 1% or higher risk of limb reduction defects.^{77,78,79,80,81,82,83,84,85,86,87,88,89, and 90,124,125,126,127,128,129,130,131,132,133,134,135,136, and 137}

No increases in preterm labor, premature rupture of the membranes, small-for-gestational-age infants, maternal morbidity, or other obstetric complications have occurred in sampled patients.⁹¹ Although the Canadian collaborative study showed an increased perinatal mortality in CVS sampled patients, with the greatest imbalance being beyond 28 weeks, no obvious recurrent event was identified.¹⁶ To date, no other studies have seen a similar increase in perinatal loss.

Long-term infant follow-up has been performed by Chinese investigators, who evaluated 53 children from their initial placental biopsy experience of the 1970s. All were reported in good health, with normal development and school performance.⁹²

CVS is now considered a reliable method of prenatal diagnosis, but early in its development incorrect results were reported.^{93,94, and 95} The major sources of these errors included maternal cell contamination and misinterpretation of mosaicism confined to the placenta. Today, genetic evaluation of chorionic villi provides a high degree of success and accuracy, in particular with regard to the diagnosis of common trisomies.^96,97 In 1990, the US collaborative study reported a 99.7% rate of successful cytogenetic diagnosis, with 1.1% of the patients requiring a second diagnostic test, such as amniocentesis or fetal blood analysis to further interpret the results.⁹⁶ In most cases, the additional testing was required to delineate the clinical significance of mosaic or other ambiguous results (76%), and laboratory failure (21%) and maternal cell contamination (3%) also required follow-up testing. Continued experience has almost eliminated maternal cell contamination as a source of clinical errors. In addition, we now have a better understanding of the biology of the placenta so that confined placental mosaicism no longer leads to incorrect diagnosis, but provides us with information predictive of pregnancy outcome and can serve as a clue to the presence of uniparental disomy. Therefore, an understanding of villus morphology and CVS laboratory techniques is required to provide correct clinical interpretation.

Chorionic villi have 3 major components: (1) an outer layer of hormonally active and invasive syncytiotrophoblast, (2) a middle layer of cytotrophoblast from which syncytiotrophoblast is derived, and (3) an inner mesodermal core containing blood, capillaries for oxygen, and nutrient exchange (Figure 24-8B). After collection, the villi are cleaned of any adherent decidua and then exposed to trypsin to digest and separate the cytotrophoblast from the underlying mesodermal core. The cytotrophoblast has a high mitotic index, with many spontaneous mitoses available for immediate chromosomal analysis. The liquid suspension containing the cytotrophblast is either dropped immediately onto a slide for analysis or may undergo a short incubation.^{98,99, and 100} This "direct" chromosomal preparation can provide preliminary results within 2 to 3 hours. However, most laboratories now use overnight incubation to improve karyotype quality and thus report results within 2 to 4 days (Figure 24-9). The remaining villus core is placed in tissue culture and is typically ready for harvest and chromosome analysis within 1 week.¹⁰¹ The direct method has the advantage of providing a rapid result and minimizing the decidual contamination, whereas tissue culture is better for interpreting discrepancies between the cytotrophoblast and the actual fetal state. Ideally, both the direct and culture methods should be used because they each evaluate slightly different tissue sources. Abnormalities in either may have clinical implications. However, the direct preparation is labor intensive, adds additional cost, and is not routinely available in some laboratories.

Chorionic villus samples typically contain a mixture of placental villi and maternally derived decidua. Although specimens are thoroughly washed and inspected under a microscope after collection, some maternal cells may remain and grow in the culture. As a result, 2 cell lines, one fetal and the other maternal, may be identified. In other cases, the maternal cell line may completely overgrow the culture, thereby leading to diagnostic errors including incorrect sex determination^{8,102,103, and 104} and potentially to false-negative diagnoses, although there are no published reports of the latter. Direct preparations of chorionic villi are generally thought to prevent maternal cell contamination,^100,103 whereas long-term culture has a contamination rate ranging from 1.8% to 4%.¹⁰⁴ Because, in contrast to cytotrophoblast, maternal decidua has a low mitotic index, it is highly desirable for laboratories to offer a direct chromosomal preparation and a long-term culture on all samples of chorionic villus. Even in culture, the contaminating cells are easily identified as maternal and should not lead to clinical errors. Interestingly, for reasons still uncertain, maternal cell contamination occurs more frequently in specimens retrieved by the TC route.¹⁰⁴

Contamination of samples with significant amounts of maternal decidual tissue is almost always due to small sample size, making selection of appropriate tissue difficult. In experienced centers in which adequate quantities of villi are available, this problem has disappeared. Choosing only whole, clearly typical villus material and discarding any atypical fragments, small pieces, or fragments with adherent decidua will avoid confusion.¹⁰⁵ Therefore, if the initial aspiration is small, a second pass should be performed rather than risk inaccurate results. When proper care is taken and good cooperation and communication exists between the sampler and the laboratory, even small amounts of contaminating maternal tissue can be absent.

Fluorescent in situ hybridization (FISH) for common chromosomal abnormalities can be helpful in reaching a rapid diagnosis (within hours) without the concern for maternal contamination.

The second major source of potential diagnostic error associated with CVS is mosaicism confined to the placenta. Although the fetus and placenta have a common ancestry, chorionic villus tissue will not always reflect fetal genotype.^96,106 Although there was concern that this might invalidate CVS as a prenatal diagnostic tool, subsequent investigations have led to a clearer understanding of villus biology so that accurate clinical interpretation is now possible. This understanding has also revealed new information about the etiology of pregnancy loss, discovered a new cause of intrauterine growth retardation, and clarified the basic mechanism of uniparental disomy.

Discrepancies between the cytogenetics of the placenta and fetus occur because the cells contributing to the chorionic villi become separate and distinct from those forming the embryo in early development. Specifically, at approximately the 32- to 64-cell stage, only 3 to 4 become compartmentalized into the inner cell mass (ICM) to form the embryo, and the remainder become precursors of the extraembryonic tissues.¹⁰⁷ Mosaicism can then occur through 2 possible mechanisms.¹⁰⁸ An initial meiotic error in one of the gametes can lead to a trisomic conceptus that normally would spontaneously abort. However, if one of the early aneuploid precursor cells loses one of the chromosomes contributing to the trisomic set during subsequent mitotic divisions, the embryo can be "rescued" by reduction of a portion of its cells to disomy. This will result in a mosaic morula, with the percentage of normal cells dependent on the cell division at which rescue occurred. More abnormal cells will be present when correction is delayed to the second or a subsequent cell division. Because the majority of cells in the morula proceed to the trophoblast cell lineage (processed by the direct preparation), it is highly probable that that lineage will continue to contain a significant number of trisomic cells. Alternatively, because only a small number of cells are incorporated into the ICM, involvement of the fetus will depend on the chance distribution of the aneuploid progenitor cells. Involvement of the mesenchymal core of the villus, which also evolves from the ICM, is similarly dependent on this random cell distribution. Noninvolvement of the fetal cell lineage will produce confined placental mosaicism (CPM) in which the trophoblast and perhaps the extraembryonic mesoderm will have aneuploid cells, but the fetus will be euploid.

Alternatively, mitotic postzygotic errors can produce mosaicism, with the distribution and percentage of aneuploid cells in the morula or blastocyst dependent on the timing of nondisjunction. If mitotic errors occur early in the development of the morula, they may segregate to the ICM and have the same potential to produce an affected fetus as do meoitic errors. Mitotic errors occurring after primary cell differentiation and compartmentalization has been completed lead to cytogenetic abnormalities in only one lineage.

Meiotic rescue can lead to uniparental disomy (UPD). This occurs because the original trisomic cell contained 2 chromosomes from one parent and 1 from the other. After rescue, there is a theoretical 1 in 3 chance that the resulting pair of chromosomes came from the same parent, which is called uniparental disomy. UPD may have clinical consequences if the chromosomes involved carry imprinted genes in which expression is based on the parent of origin. For example, Prader-Willi syndrome may result from uniparental maternal disomy for chromosome 15. Therefore, a CVS diagnosis of confined placental mosaicism for trisomy 15 may be the initial clue that UPD could be present and lead to an affected child.^109,110 Because of this, all cases in which CVS reveals trisomy 15 (either complete or mosaic) should be evaluated for UPD by subsequent amniotic fluid analysis. In addition to chromosome 15, chromosomes 7, 11, 14, and 22 are felt to be imprinted and require follow-up.¹¹¹

Recently, there has been evidence that confined placental mosaicism (unassociated with UPD) can alter placental function and lead to fetal growth failure or perinatal death.^{108,112,113,114,115,116, and 117} The exact mechanism by which abnormal cells within the placenta alter fetal growth or lead to fetal death is unknown. However, the effect may be limited to specific chromosomes. For example, CPM for chromosome 16 leads to severe intrauterine growth restriction, prematurity, or perinatal death, with fewer than 30% of pregnancies resulting in normal full-term infants appropriate for gestational age.^{118,119,120,121,122,123,124, and 125}

CVS mosaic results require diligent follow-up by amniocentesis or fetal sampling to determine their clinical significance because, in most cases, if the mosaic results are confined to the placenta, fetal development will be normal. However, if the mosaic cell line also involves the fetus, there may be significant phenotypic consequences. Mosaicism occurs in about 1% of all CVS samples^{97,104,121,122} but is confirmed in the fetus in only 10% to 40% of these cases. The probability of fetal involvement appears to be related to the tissue source in which the aneuploid cells were detected and the specific chromosome involved.¹¹⁰ Mesenchymal core culture results are more likely than direct preparation to reflect a true fetal mosaicism.

In a recent review, Phillips et al demonstrated that autosomal mosaicism involving common trisomies (ie, 21, 18, and 13) was confirmed in the fetus in 19% of cases, whereas uncommon trisomies involved the fetus in only 3%.¹²³ When sex chromosome mosaicism was found in the placenta, the abnormal cell line was confirmed in the fetus in 16% of cases. When a nonfamilial marker chromosome was involved, it was confirmed in the fetus in more than one-fourth of cases, whereas mosaic polyploidy was confirmed in only 1 of 28 cases. Chromosomal structural abnormalities were confirmed in 8.6% of cases.

When placental mosaicism is discovered, amniocentesis is frequently performed to elucidate the extent of fetal involvement. When mosaicism is limited to the direct preparation only, amniocentesis appears to correlate perfectly with fetal genotype.¹²³ However, when a mosaicism is observed in tissue culture, both false-positive and false-negative amniocentesis results occur. In these cases, amniocentesis will predict the true fetal karyotype in approximately 94% of cases.¹²³ Most importantly, these discrepancies may involve the common autosomal trisomies. There have been 3 cases reported of mosaic trisomy 21 on villus culture and a normal amniotic fluid analysis, followed by a fetus or newborn with mosaic aneuploidy.⁹⁶

At present, the following clinical recommendations may be used to assist in the evaluation of CVS mosaicism. Analysis of CVS samples should, if possible, include both direct preparation and tissue culture. Although the direct preparation is less likely to be representative of the fetus, its use will minimize the likelihood of maternal cell contamination, and if culture fails, a nonmosaic normal direct preparation result can be considered conclusive, although rare cases of false-negative results for trisomies 21 and 18 have been reported.^{124,125,126,127, and 128} If mosaicism is found on either culture or direct preparation, follow-up amniocentesis should be offered. Under no circumstances should a decision to terminate a pregnancy be based entirely on a CVS mosaic result. For CVS mosaicism involving sex chromosome abnormalities, polyploidy, marker chromosomes, structural rearrangements, and uncommon trisomies, the patient can be reassured if amniocentesis results are euploid and detailed ultrasonographic examination is normal. However, no guarantees should be made and, as described above, in certain cases testing for UPD will be indicated. If common trisomies 21, 18, and 13 are involved, amniocentesis should be offered, but the patient must be advised of the possibilities of a false-negative result. Follow-up may include detailed ultrasonography, fetal blood sampling, or fetal skin biopsy. At present, the predictive accuracy of these additional tests is uncertain.

Most biochemical and molecular diagnoses that can be made from amnionic fluid or cultured amniocytes can also be made from chorionic villi. In many cases, the results will be available more rapidly and more efficiently by using villi, because sufficient enzyme or DNA is present in villus samples to allow direct analysis rather than wait for tissue culture. For example, the analysis of Tay-Sachs disease can be performed in less than 30 minutes using fresh villi.¹²⁹

A discussion of individual biochemical or molecular diagnoses is beyond the scope of this chapter and is impractical because techniques are changing so rapidly. A registry of diagnoses performed by CVS is kept and updated through the WHO by Dr Hans Galjaard in Rotterdam, The Netherlands, and a published summary of the early worldwide experience is available.¹³⁰

It cannot be assumed that biochemical or molecular results from villus tissue will always be a true reflection of the fetal state. Recently, misdiagnosis of the peroxisomal disorder, X-linked adrenoleukodystrophy, from cultured villus cells has been reported.¹³¹ In addition, tests requiring determination of DNA methylation status, such as that for fragile X,¹³² are also not always reliable in villus tissue. This does not, however, preclude CVS from making these prenatal diagnoses because other molecular approaches can be used. It does emphasize that all tests on villus tissue must be validated by testing sufficient numbers of affected and unaffected pregnancies before being used clinically. Because of the rarity and unique aspects of most biochemical and molecular disorders, specific diagnoses are usually performed by only a few laboratories. Before performing a CVS, the clinician should contact the center analyzing the tissue so that the details of testing can be discussed.

Chorionic villus sampling is a safe and effective approach to examining twins. Not only does it provide results early in pregnancy, but, if discordancy is discovered, the medical and psychological difficulties encountered with selective termination can be minimized. However, it can be technically more demanding because it requires an experienced operator and sonographer. The ideal time to perform a twin CVS is similar to that for singletons. Ultrasound initially identifies placental locations, determines chorionicity, and confirms fetal sizes and viability. Sampling of each sac is independently performed by either a TC or TA approach, with separate passes of a new sampling instrument for each attempt. Because no unique marker is available to ensure that the samples have been retrieved from distinct placentas, it is imperative that insertion of the instrument into each frondosum is certain. Longitudinal and transverse scanning planes should be used to ensure proper location. If any doubt exists, a repeat procedure is required, but with increased experience, the need for repeat procedure is rare.¹³³

Contamination of one sample with villi from the other sac is possible and occurs most commonly when retrieval is performed near the dividing membrane, or if a needle or catheter is dragged through one frondosum while sampling another. When the chorions appear fused, sampling near the cord insertion sites, with avoidance of the area of confluence of the 2 placentas, should prevent contamination and ensure sampling of each fetus. A combination of TC and TA sampling can minimize co-twin contamination by ensuring unique sampling paths. For example, if both chorion frondosa are situated along the anterior uterine wall, the lower one can be sampled transcervically and the upper transabdominally without contaminating either sample. Despite meticulous sampling techniques, co-twin contamination occurs in up to 4% of cases¹²⁰ but is rarely of clinical concern. If the cytogenetic laboratory is aware of the presence of a multiple gestation, the presence of both normal and aneuploid cells in the sample will be correctly interpreted. The possibility of contamination is of greater consequence if sampling is being performed for biochemical analysis, where a small amount of contaminating tissue could lead to an incorrect diagnosis. For this reason, instead of pooling a sample, we recommend analyzing individual villi when biochemical studies are to be performed. An experienced sampler and an aware and knowledgeable laboratory are extremely important in such cases.

The need to map the placental location clearly and accurately is mandatory, because the risk of discordant results is higher early in the pregnancy and selective termination may be required. The relative positions of the placentas will remain stable over the time frame that results are obtained, so that identification of the affected fetus can usually be determined even 2 or 3 weeks after the procedure. However, if there is uncertainty, a repeat CVS with direct villus analysis can confirm the position immediately before the termination.

Procedure-related loss rates after CVS sampling of twins are well studied. In experienced centers, no increased procedure-related loss risks are seen compared with second trimester amniocentesis.^133,134 Table 24-4 presents overall pregnancy loss rates to 28 weeks of 1.6% to 2.8% in sampled twins. When compared with a contemporaneously sampled group of patients choosing either CVS or second trimester amniocentesis, we found no difference in the overall risk of pregnancy loss (2.9% amniocentesis vs 3.2% CVS). There was, however, a slightly increased risk of losing one fetus in the group sampled by amniocentesis.¹³³

The choice of the appropriate technique for sampling twins depends on a number of factors including locally available skill and expertise. In centers in which amniocentesis and CVS are both available, CVS may be the preferred approach because it provides results 1 month sooner, thus providing earlier reassurance. When discordant results are encountered, complications and loss rates are decreased when selective termination is performed before 16 weeks of gestation.¹³⁵

Multifetal pregnancy reduction (MFPR) to improve perinatal outcome is an unfortunate but accepted part of reproductive medicine. As with selective termination, MFPR is most safely performed in the first-trimester.¹³⁶ Therefore, in high-order gestations at increased risk for a genetic abnormality, CVS before a reduction can avoid the potential need for a later selective termination. The CVS can be performed between 10 and 11.5 weeks of gestation, and a rapid karyotype can be available within 24 to 48 hours, after which the MFPR is performed. Villus mesenchymal core tissue culture is also performed, but results are usually not available for 7 to 10 days. Because of the small increased risk of confined placental mosaicism with the direct preparation, awaiting culture results when time allows is suggested. The positions of the sampled fetuses will be the same when the patient returns for the MFPR. In most cases, only the 2 fetuses most likely to remain after the MFPR are sampled. If an abnormality is identified, an additional fetus can be sampled at the time of the MFPR and the patient can return if this is also abnormal. Of 745 MFPR reductions performed at our center, 254 had an initial CVS. Abnormal chromosomal results were present in approximately 2.5% of pregnancies, and the abnormal fetus was terminated as part of the reduction procedure. The pregnancy loss rate to 24 weeks of gestation of those having a preceding CVS was 5.5% versus 5.6% in those having only MFPR. This encouraging outcome is similar to that described by Brambati et al¹³⁷ in which the cohort of patients undergoing CVS before MFPR demonstrated no increased risk of pregnancy loss, prematurity, or small-for-gestational-age infants.

Chorionic villus sampling is a safe technique for firsttrimester prenatal diagnosis of genetic disorders. Real-time sonography and technologic advances of sampling instruments have been critical in establishing a safe technique for retrieving villus tissue for genetic analysis. Clinical trials suggest that CVS carries a low risk of pregnancy loss, which is comparable to that of second-trimester amniocentesis. An understanding of the laboratory techniques and human embryology is essential in avoiding diagnostic errors related to confined placental mosaicism.

To maximize outcome, CVS should be performed by an experienced team of physicians, ultrasonographers, and genetic laboratory technicians. Before initiating a CVS program, operators should have considerable experience in the placement of the catheter, which can be achieved either in a formal training program with observation of 50 procedures followed by close hands-on supervision of another 100 cases,⁸ or by supervised practice on pregnancies undergoing subsequent abortion. In addition, it is advisable to limit the number of ultrasonographers assigned to assist in this procedure, because sampling success is equally dependent on skillful ultrasound guidance. Similar to the physician retrieving the villi, the guiding sonographer should be knowledgeable in the didactics of CVS sampling and should obtain adequate hands-on training before beginning work in this area. This can be achieved in a formal training program or by visiting centers performing this procedure.⁸ In this setting, CVS will continue to be an important, reliable, and safe contributor to prenatal genetic diagnosis.