CHORIONIC VILLUS SAMPLING

Chapter 24 

Definitions 

CHORIONIC VILLUS SAMPLING 

1. Chorionic villus sampling (CVS): an invasive procedure 

performed for first-trimester prenatal diagnosis. 

CVS is typically performed between 70 and 91 days 

after the LMP. In the procedure, tissue is withdrawn 

from the villi (vascular fingers) of the chorion, a part 

of the placenta, and examined. 

2. Chorion frondosum: the cellular, outermost extraembryonic 

membrane, composed of trophoblastic cells, 

and develops villi, and forms the fetal component of the 

placenta. 

3. Fluorescent in situ hybridization (FISH): rapid 

method of assessing targeted chromosomal abnormalities 

such as trisomy 21, 18, or 13. 

4. First-trimester screening: a method of screening for 

chromosomal abnormalities in the first trimester 

using two biochemical tests, PAPP-A and hCG as well 

as sonographic nuchal translucency measurement. 

INTRODUCTION 

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 

Ronald J. Wapner ● Eugene C. Toy 

Chapter 24 Chorionic Villus Sampling 

715 

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. 

3-5 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 

CONCEPTS AND INDICATIONS FOR 


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

716 Part 3 RISK ASSESSMENT AND THERAPY 

Table 24-1 

MAJOR INDICATIONS FOR 


Maternal age: 35 years or older at estimated date of delivery 

Previous child with nondisjunctional chromosome abnormality 

Parent is carrier of balanced translocation or other chromosome 

disorder 

Both parents are carriers of autosomal recessive disease 

Women who are carriers of a sex-linked disease 

Positive first-trimester screen for trisomy 21 or 18 

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. 

HISTORY OF CHORIONIC VILLUS SAMPLING 

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 6mm-diameter 

instrument. 10 Although successful tissue 

culture was obtained, half of these subjects subsequently 

aborted. In 1973, Kullander and Sandahl used a 5-mmdiameter 

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 

Canada 16 and 1 from the United States, 17 were published 

and confirmed the safety of the procedure. 

CHORIONIC VILLUS SAMPLING: THE 

PROCEDURE 

Procedure-Related Anatomy (Figure 24-1) 

Between 9 and 12 weeks after the last menstrual period, the 

developing gestation does not yet fill the uterine cavity. 

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

Extraembryonic 

border 

Amniotic 

membrane 

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. 

Figure 24-2. Sonogram at 10.8 weeks of gestation. The chorion frondosum 

(placenta) is located posteriorly and appears as a homogeneous 

hyperechoic area. 

Yolk sac 

Chorion laeve 

A 


Chorion frondosum 

20 cc syringe 

with 5 cc RPMI 

Tenaculum 

(optional) 

Deciduous border 

Figure 24-1. Diagram of first-trimester pregnancy illustrating relevant anatomic landmarks. 

3.5 mHz sector transducer 

with biopsy guide 

Transabdominal chorionic villus sampling 

717 

20 cc syringe 

with 5 cc RPMI 

20 G spinal 

needle 

int diam- .58 mm 

Portex 

catheter 

int. diam 

.89 mm 

Transcervical chorionic villus sampling 

B 

Figure 24-3. Diagram illustrating the technique of sonographically 

guided chorionic villus sampling: (A) transabdominal sampling and 

(B) transcervical sampling.


Table 24-2 

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). 

COMPARISON OF TRANSCERVICAL AND TRANSABDOMINAL CHORIONIC VILLUS 

SAMPLING PROCEDURES 

Transcervical Transabdominal 

Relative contraindications Cervical polyps, active cervical, or Interceding bowel 

vaginal herpes 

Ease of learning Somewhat more complex than Adaptation of amniocentesis technique but 

transabdominal approach learning curve still required 

Sample size Large sample; includes whole villi Smaller sample; includes many small pieces 

Patient discomfort Minimal to absent Moderate 

Placental location Better for posterior placenta Better for fundal placenta 

Figure 24-4. Cook catheter used for transcervical chorionic villus sampling. 

Note the general curvature of the distal end, which is aligned with 

the notch on the handle. This allows the operator to be aware of the direction 

of the curve. 

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 

Uterine contraction CVS catheter 

Figure 24-5. Transcervical chorionic villus sampling catheter forced 

anteriorly by posterior uterine contraction.

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. 

A 

B 

Figure 24-6. Sonogram illustrating sonographically guided transcervical 

chorionic villus sampling at 11.5 weeks of menstrual age. A: The tip of 

the catheter is visible at the internal os before farther advancement. 

B: The catheter is correctly placed within the corion frondosum parallel 

to the chorionic membrane. 


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 

719 

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-inchlong 

needle is sufficient for most patients, but a 5- or 6-inchlong 

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 

Figure 24-7. Sonogram illustrating transabdominal chorionic villus 

sampling. The needle is parallel to the chorionic plate.


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 

A B 

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. 

RISKS ASSOCIATED WITH CHORIONIC 

VILLUS 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 

Figure 24-8. A: Photograph of chorionic villus fragments in a Petri dish after collection by chorionic villus sampling. B: Magnified image of chorionic 

villus. Note the cytotrophoblastic bud. Within the center of the villus is the mesenchymal core and fetal blood vessels.

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. 28-30 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 lifethreatening 

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. 38-40 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 

721 

Multiple reports from individual centers have demonstrated 

the safety and low pregnancy loss rates after CVS. 8,44-51 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 procedureinduced 

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.

PREGNANCY LOSS: TRANSCERVICAL VERSUS 

TRANSABDOMINAL CHORIONIC VILLUS 

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 secondtrimester 

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. 

Table 24-3 


RISK OF FETAL ABNORMALITIES AFTER 


723 

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 al 63 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 oromandibularlimb 

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). 

STUDIES EVALUATING THE ASSOCIATION OF CHORIONIC VILLUS SAMPLING (CVS) AND LIMB 

REDUCTION DEFECT (LRD): PROCEDURES PERFORMED AFTER 63 DAYS 

No Association Association 

n Post-CVS n Post-CVS 

Reference Liveborns n LRDs Reference Liveborns n LRDs 

Jahoda et al. 120 3973 3 Burton et al. 131 394 4 

Halliday et al. 121 2071 3* Mastroiacovo et al. 132 2759 3 

Canadian group 13 905 0 Bissonnette et al. 129‡ 507 5 

Schloo et al. 122 3120 2 

Monni et al. 123 2752 2 

Blakemore et al. 124 3709 3 

Silver et al. 125 1048 1 ∗ 

Mahoney et al. 126 4588 8 ∗∗ 

Jackson et al. 127 12,863 5 

Smidt-Jensen et al. 128 2624 0 

Bissonnette et al. 129‡ 269 0 

Case Control Studies OR CI OR CI 

Dolk et al. 130 1.8 0.7–5 Mastroiacovo and Botto 133 19 9–37 

Williams et al. 73 Williams et al. 73 

Overall LRD 1.7 0.4–6 Terminal Digital LRD 6.4 1.1–38 

Transverse LRD 4.7 0.8–28 

∗ Uncertain association: There was no statistical increase in LRDs, but absolute incidence was higher than general risk. 

† Includes known syndromal defects. 

‡ Single report comparing two sampling sites. 

§ Less than 76 days. CI, confidence interval; OR, odds ratio.


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 

77-90 124-137 

reduction defects. 

PERINATAL RISKS AND IMPACT ON 

LONG-TERM DEVELOPMENT OF THE INFANT 

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 

LABORATORY ASPECTS OF CHORIONIC 

VILLUS SAMPLING 

CVS is now considered a reliable method of prenatal diagnosis, 

but early in its development incorrect results were 

reported. 93-95 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 

Syncytiotrophoblast 

Mesenchymal core 

Cytotrophoblastic 

cell column 

Cytotrophoblast 

A B 


725 

short incubation. 98-100 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. 

MATERNAL CELL CONTAMINATION 

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 determination8,102-104 and potentially to falsenegative 

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, 

Mesenchymal 

core 

Villus 

culture 

Villus tissue 

trypsin 

Cytotrophoblast 

suspension 

Direct 

preparation 

Figure 24-9. A: Diagram of normal villus architecture. B: Diagram outlining the laboratory technique for chorionic 

villus sampling direct chromosomal preparation and villus culture.


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. 

CONFINED PLACENTAL MOSAICISM 

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. 108,112-117 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. 118-125 

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 falsenegative 

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. 124-128 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. 

BIOCHEMICAL AND DNA PROCEDURES 

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 


727 

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 IN MULTIPLE 

GESTATIONS 

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 cases 120 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 

Table 24-4 

SAFETY OF CHORIONIC VILLUS 

SAMPLING (CVS) WITH TWINS 

COMPARED WITH AMNIOCENTESIS 

Success Pregnancy Loss 

n Rate Rate to 28 Weeks 

CVS 

Wapner et al. 115∗ 440 † 100% 2.8% 

Pergament et al. 116 128 99.2% 2.4% 

Brambati et al. 119 66 100% 1.6% 

Amniocentesis 

Wapner et al. 115∗ 73 100 2.9% 

∗ Contemporaneously collected comparison of CVS with amniocentesis in a 

single center. 

† Additional cases added since original publication. 

when selective termination is performed before 16 weeks 

of gestation. 135 

CHORIONIC VILLUS SAMPLING AND 

MULTIFETAL PREGNANCY REDUCTION 

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. 

SUMMARY 

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. 

KEY POINTS 

1. Chorionic villus sampling has a low complication rate 

comparable to mid-trimester amniocentesis in experienced 

hands. The learning curve is more difficult with 

CVS. 

2. CVS is the preferable method of fetal aneuploidy testing 

in the first trimester. 

3. CVS is generally performed after 9 completed weeks 

of gestation. In this setting, there does not seem to be 

an increased risk of limb reduction defects. 

4. CVS may be performed either transabdominally or 

transcervically. 

5. Caution should be exercised in the interpretation of a 

mosaic of chromosomal findings from CVS. 

6. The complications of CVS include miscarriage, bleeding, 

rupture of membranes, and infection. 

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CHORIONIC VILLUS SAMPLING

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