invasive procedures  mainly chorionic villus sampling ( cvs ) and amniocentesis  are very common for prenatal diagnosis . nowadays , conventional prenatal chromosome analysis , fluorescence in situ hybridization ( fish ) , and array comparative genomic hybridization ( acgh ) analyses depend on samples acquired from invasive procedures .
chromosomal abnormalities that are compatible with life but cause considerable morbidity occur in 0.65% of newborns , and apparently balanced structural chromosomal rearrangements that will eventually affect reproduction occur in 0.2% of newborns .
the application of acgh or the single - nucleotide polymorphism ( snp ) chip has allowed the detection of submicroscopic abnormalities and genomic disorders ; the most commonly seen recurrent genomic disorders occur in approximately 0.18% of newborns . in 2009 ,
the american college of obstetricians and gynecologists ( acog ) recommended that conventional karyotyping should remain the principal cytogenetic tool in prenatal diagnosis , and that acgh should be an adjunct to prenatal care for women with abnormal ultrasound findings ( aus ) and a normal conventional karyotype .
however , many factors such as the risk that the fetus will have a chromosomal abnormality from direct fetal ultrasound imaging and indirect maternal serum markers , the risk of procedure - related miscarriage , the consequences of having an affected child , anxiety , ethnic background , and religion can affect a pregnant woman s decision to accept or reject an invasive procedure [ 57 ] . we expect that future application of cell - free fetal dna ( cff - dna ) screening for cytogenetic disorders will dramatically improve the diagnostic yield and reduce unnecessary invasive procedures ; therefore , it is time to summarize the clinical indications of invasive prenatal diagnosis .
in addition , cff - dna screening has many limitations   and many cytogenomic abnormalities , including chromosomal structural abnormalities and genomic abnormalities , can not be screened with it .
therefore , clinical indications accurately predicting the risk of cytogenomic abnormalities play an important role in prenatal genetic counseling and diagnosis .
current prenatal clinical indications include aus , abnormal maternal serum screening ( amss ) , advanced maternal age ( ama ) , family history ( fh ) of chromosomal abnormalities , and other events that could affect fetal health .
prenatal diagnosis is expected to move forward with more effective non - invasive prenatal indications and genome - wide analysis of cytogenomic abnormalities .
however , the efficacy of clinical indications in prenatal diagnosis could vary in different practice settings and different regions .
this study aimed to analyze the efficacies of indications of cvs and amniocentesis from a regional obstetric unit .
the results provide the diagnostic yields from single or combined clinical indications , and indicate which women need the invasive testing and future direction for better prenatal diagnosis of cytogenomic abnormalities .
this information will be useful for the obstetricians , clinical geneticists , and laboratory staff to further improve the quality of prenatal diagnosis .
yale cytogenetics laboratory is a regional reference laboratory for prenatal diagnosis in new haven county and surrounding areas . from 2000 to 2012 , the laboratory performed karyotype analysis on 3229 cvs and 4589 amniotic fluid ( af ) specimens ( excluding 17 culture failure samples ) .
fish using probes for the hira gene at 22q11.21 had been performed on 55 patients with aus of cardiac defects or fh of digeorge syndrome . since 2009 ,
acgh has been validated and offered to high - risk pregnancies after pre - testing counseling of technical specifications and limitations .
all test results and clinical indications of these 7818 cvs and amniocenteses were compiled from the laboratory s cytoaccess database .
the clinical indications for prenatal diagnosis included : ( 1 ) ama defined by age equal or greater than 35 years for singlet , 33 years for twin , and 31 years for triplet pregnancies ; ( 2 ) aus of increased nuchal translucency ( 3 mm ) , cystic hygroma and other kinds of suspected fetal anomalies ; ( 3 ) amss from first or second trimester maternal serum quad screen , including human chorionic gonadotropin ( hcg ) , -fetoprotein ( afp ) , unconjugated estriol , and inhibin a ; ( 4 ) fh of a chromosome abnormality in previous pregnancy , parents with abnormal karyotype , or history of abnormal offspring birth ; ( 5 ) multiple pregnancy ( mp ) , including twins and triplets ; and ( 6 ) other indications such as intrauterine fetal death or demise , radiation or medication exposure during pregnancy , anxiety , and consanguineous marriage .
of the 7818 cases , 68% , 30% , and 2% were referred by single indication , 2 indications , and 3 indications , respectively .
single and combined indications occurring in more than 1% of the samples ( except for mp ) were used to evaluate diagnostic yields .
conventional chromosome analysis was performed on g - band metaphases prepared from cultured af and cvs cells .
fish tests using aneuvision probes for rapid screening of aneuploidies of chromosomes x , y , 21 , 13 , and 18 and targeted probes for genomic disorders were performed following manufacturer s protocols ( vysis / abbott , abbott park , il ) .
genomic dna was extracted from cvs and cultured amniocytes using the gentra puregene kit ( qiagen , valencia , ca ) . the oligonucleotide acgh analysis using the agilent human genome cgh microarray 44k kit ( containing 44 913 60-mer oligonucleotides , using ncbi36/hg18 human genome assembly ) , and the 180k kit ( 177 873 60-mer oligonucleotides , using grch37/hg19 assembly ) ( agilent technologies , inc . , santa clara , ca ) was performed as previously described .
diagnostic yield was defined as percentage of the abnormal cases from cases referred by single , combined , or all indications .
time series regression analyses were performed for variance to evaluate the significance of trends and chi - square tests to compare rates , using spss for windows ( spss inc .
from 2000 to 2012 , the most commonly used indications were ama , amss , and aus .
for cvs cases , ama referrals declined from 55.8% to 51.1% ( p<0.05 ) , amss referrals increased from 0.5% to 11.2% ( p<0.05 ) , and aus referrals remained at the 10.63.3% level ( figure 1a ) . for af cases ,
ama referrals significantly declined from 55.9% to 35.2% ( p<0.001 ) , amss referrals remained at 26.82.4% , and aus referrals increased from 5% to 20% ( p<0.05 ) ( figure 1b ) .
the indications of fh , mp , and others remained in less than 12.33.1% and showed a relatively stable trend except for a decline in fh referrals for cvs .
the usage of multiple indications defined by the average number of indications per case showed an increasing trend during the 13 year period ( p<0.01 ) and cvs referrals required more indications than af ( p<0.01 ) ( figure 1c ) .
although the annual caseload of cvs and af showed a significant 57.2% decrease from 853 cases in 2000 to 365 cases in 2012 ( p<0.01 ) , there was no significance change in the number of abnormal findings ( figure 1d ) .
the diagnostic yield for chromosomal abnormalities showed a significant increase from 7.2% in 2000 to 13.4% in 2012 ( p<0.01 ) and the overall diagnostic yield was 9.6% .
clinical indications associated with detected chromosomal abnormalities were also counted and ordered as follows : ama 57.6% , aus 40.3% , amss 30.5% , fh 9.6% , mp 4.8% , and other indications 2.3% .
the efficacy of single or combined indications for detecting chromosomal abnormalities is summarized in table 1 .
the 3 highest diagnostic yields were noted in cases with ama / aus ( 51.4% for cvs , 24.2% for af ) , aus ( 34.7% for cvs , 14.5% for af ) , and ama / amss ( 17.8% for cvs , 9.9% for af ) . in this cohort ,
the most effective single indication was aus followed by amss or fh and then ama . mp was referred as indication of prenatal diagnosis due to our regional practice . however , there were no chromosomal abnormalities detected from mp referrals .
fish tests were used to detect 22q11.2 deletion on prenatal cases with aus of cardiac defects and fh of digeorge syndrome .
previous validation of acgh analysis on 11 cases with prenatally detected structural abnormalities successfully defined the genomic imbalances and gene content .
the acgh analysis performed on 237 prenatal cases with normal chromosome findings detected 6 pathogenic and likely pathogenic genomic abnormalities .
recurrent genomic disorders of 16p13.11 deletions inherited from a paternal carrier were detected in 2 unrelated families and a 22q11.2 duplication was seen in 1 case .
the aus findings of iugr , cystic hygroma , echogenic bowel , and tetralogy of fallot ( tof ) were noted in cases with recurrent genomic disorders .
the clinical indications and the genomic abnormalities from fish and acgh analyses are shown in table 2 .
variants of unknown significance ( vous ) were detected in 5 cases ; 2 of them pursued parental study and were confirmed as familial variants of maternal origin ( data not shown ) .
the estimated diagnostic yield from the fish and acgh analyses for genomic abnormalities on tested prenatal cases was 4% ( 12/303 ) .
our cytogenetics laboratory has provided prenatal chromosome , fish , and microarray analyses for yale affiliated hospitals with stable referrals from new haven county and surrounding areas .
consistent with previous studies , our data demonstrated a continuous trend of reduced invasive procedures .
the caseload reduction and procedure shifting were largely attributed to better knowledge and more informative counseling from clinical indications .
the revised 2007 acog screening and invasive testing guidelines recommended that genetic screening and invasive diagnostic testing for aneuploidy should be available to all pregnant women , regardless of maternal age .
ama , a known factor for increased risk of down syndrome , is still the leading single indication in many reports . in our practice
, ama is the most common indication , followed by amss in second place and aus in third place .
however , due to the development of sensitive ultrasonic technology and maternal serum markers , the indication of amss for cvs and aus for af increased from 2000 to 2012 , with a significant decrease in ama .
analysis of diagnostic yields from single and combined clinical indications can provide useful information for prenatal genetic counseling and diagnosis .
for single indications for cvs and af , ama , amss , aus , and fh accounted for 39% , 13% , 11% , and 4% of referrals , but 19% , 10% , 23% , and 4% of total abnormal findings , respectively .
for combined indications , ama / aus , ama / amss , and ama / fh accounted for 3% , 13% , and 5% of referrals , but 13% , 17% , and 4% of total abnormal findings , respectively ( table 1 ) .
it was obvious that ama / aus , aus , and ama / amss are the most effective indications and together they accounted for 26% of total referrals and 52% of total abnormal findings .
however , the remaining 48% of chromosomal abnormalities from other single and combined indications are indispensable in prenatal practice . in the present cohort ,
only 40% of cases detected with chromosome abnormalities had aus findings , suggesting the absence of ultrasound visible fetal structural anomalies in about 60% of prenatal chromosomal abnormalities .
further advancement of prenatal ultrasonography with better imaging and the inclusion of soft markers will improve the diagnostic yield . however , some fetuses with chromosomal abnormalities could lack ultrasound - detectable fetal anomalies . with a population of 0.86 million in new haven county and 2.17 million in the surrounding counties and a 1.3% birth rate by the 2012 united states census
, it is estimated that each year there are 11 180 newborns in new haven county and 27 430 newborns in the surrounding counties . in this prenatal cohort during a 13-year period , chromosome analysis detected 292 fetuses ( average 22 cases per year ) with trisomy 21 for the diagnosis of down syndrome ; fish and acgh detected 7 fetuses with deletions and duplication in the digeorge syndrome region ( 0.5 case per year ) . in a general population ,
the occurrence of down syndrome ( omim#190685 ) is about 1/650 to 1/1000 live births and occurrence of digeorge syndrome ( omim#188400 ) is 1 in 4000 , indicating a 6:1 to 4:1 ratio of down vs. digeorge . using a 1/800 occurrence for down syndrome , prenatal diagnosis of 22 fetuses inferred an annual basis of 17 600 pregnant women , which could be translated to a population of 1.35 million , consisting of the 0.86 million in the new haven county and 23% of the 2.17 million from the surrounding counties . from the same population basis ,
postnatal cytogenomic analysis was performed on 1354 pediatric patients from 2006 to 2009 and detected 7 cases with trisomy 21 ( 1.75 cases per year ) , 13 cases with the 22q11.21 deletion , and 3 cases with the reciprocal duplication ( 4 cases per year ) .
combined the prenatal and postnatal data , the annual detection of 24 down syndrome and 4~5 digeorge region deletions and duplications matches the occurrence and down vs. digeorge ratio from the estimated 17 600 pregnancies .
but only 10% ( 0.5/4~5 ) of digeorge deletions and duplications were detected prenatally in the current setting .
the lower diagnostic yield of genomic disorders in prenatal practice is likely due to the lack of sensitive and specific clinical indications . since 20082010 we found that the number of invasive procedures began to change significantly , perhaps because non - invasive prenatal testing became popular from that time .
recently , a non - invasive prenatal screening method using massive parallel sequencing of maternal plasma cff - dna has been validated and rapidly integrated into prenatal genetic evaluation .
a rapid acmg statement for this newly introduced cff - dna - based screening has been issued .
currently , this newly integrated procedure has demonstrated high sensitivity and specificity in detecting common aneuploidies .
this progress has also re - energized efforts to develop non - invasive prenatal diagnosis using maternal circulating fetal cells [ 8,1922 ] .
further improvement of cff - dna based screening for genomic imbalances is technically feasible and is expected to become a direct indication of genomic abnormalities in prenatal counseling for recommending further invasive procedures .
from 2000 to 2012 , with the development of sensitive ultrasonic technology and maternal serum markers , the indication of amss and aus were increasing while ama were significantly decreasing , although ama is the most common indication . largely due to this change
of13 years , prenatal evaluation using ama / aus was the most effective in detecting chromosomal abnormalities , but better indications for genomic abnormalities are needed .
the occurrence of chromosomal morbidity , reproductive defects , and common genomic disorders in 0.65% , 0.2% , and 0.18% , respectively , of newborns indicated that the baseline of prenatal cytogenomic diagnosis should be set on at least 1% but most likely up to 3% of pregnant women if including other genomic abnormalities and gene mutations .
diagnostic prenatal cytogenomic tests should be performed on 176 to 528 fetuses from 17 600 pregnancies ; the 2012 caseload of 365 in this laboratory is within the baseline level .
the goal of invasive procedures for prenatal diagnosis is to achieve much higher diagnostic yield for cytogenomic abnormalities . toward that end ,
prenatal genetic evaluation using multiple screening methods of ama , aus , amss , and cff - dna and combined diagnostic chromosome and acgh tests will become highly effective in detecting cytogenomic abnormalities .
 backgroundbecause the future application of cell - free fetal dna screening is expected to dramatically improve the diagnostic yield and reduce unnecessary invasive procedures , it is time to summarize the indications of invasive prenatal diagnosis . 
 this retrospective study was performed to evaluate the changes and efficacies of indications of invasive procedures for detecting cytogenomic abnormalities from 2000 to 2012.material/methodsfrom our regional obstetric unit , 7818 invasive procedures were referred by indications of advance maternal age ( ama ) , abnormal ultrasound findings ( aus ) , abnormal maternal serum screening ( amss ) , and family history ( fh ) . 
 chromosome , fluorescence in situ hybridization ( fish ) , and array comparative genomic hybridization ( acgh ) analyses were performed on chorionic villus sampling ( cvs ) and amniotic fluid ( af ) specimens at the yale cytogenetics laboratory . 
 the abnormal findings from single or combined indications were compared to evaluate the diagnostic yield.resultsthe annual caseload declined by 57.2% but the diagnostic yield increased from 7.2% to 13.4% . 
 chromosomal and genomic abnormalities were detected in 752 cases ( 9.6% , 752/7818 ) and 12 cases ( 4% , 12/303 ) , respectively . 
 significantly decreased ama referrals and increased aus and amss referrals were noted . 
 the top 3 indications by diagnostic yield were ama / aus ( 51.4% for cvs , 24.2% for af ) , aus ( 34.7% for cvs , 14.5% for af ) , and ama / amss ( 17.8% for cvs , 9.9% for af).conclusionsover a period of 13 years , the indication of amss and aus were increasing while ama was decreasing for prenatal diagnosis of cytogenomic abnormalities , and there was a continuous trend of reduced invasive procedures . prenatal evaluation using ama / aus was the most effective in detecting chromosomal abnormalities , but better indications for genomic abnormalities are needed . 