Prenatal Screening
Serum Biochemical Screening
Basic Principles
Serum biochemical screening involves detecting the concentrations of various biochemical markers in maternal serum using biochemical methods. By combining this data with maternal factors such as age, weight, and gestational week, the risk of fetal chromosomal abnormalities, including trisomy 21, trisomy 18, trisomy 13, and neural tube defects, can be predicted. Depending on the gestational period, serum biochemical screening is divided into first-trimester and second-trimester screening.
First-trimester markers include pregnancy-associated plasma protein A (PAPP-A) and free β-human chorionic gonadotropin (β-hCG). Normally, free β-hCG levels in maternal serum decrease, and PAPP-A levels increase after the 10th week of pregnancy. In cases of trisomy 21, free β-hCG levels are higher, while PAPP-A levels are lower compared to normal pregnancies. Greater deviations in these markers correspond to a higher risk of trisomy 21.
Second-trimester markers typically include alpha-fetoprotein (AFP), β-hCG, unconjugated estriol (uE3), and inhibin A. Compared to normal pregnancies, mothers carrying a fetus with Down syndrome show elevated levels of β-hCG and inhibin A, and reduced levels of AFP and uE3. By setting specific cutoff values and incorporating factors such as gestational week, maternal weight, diabetes status, the number of fetuses, and ethnicity, the risk of trisomy 21 can be calculated.
Screening for neural tube defects (NTDs) is based on the principle that NTD fetuses typically exhibit abnormally elevated AFP levels in maternal serum and amniotic fluid. By analyzing AFP levels, the risk of NTDs can be assessed.
Technical Features
Serum biochemical screening is noninvasive and does not increase the risk of fetal loss. It is relatively cost-effective and can screen for NTDs.
Timing of Screening
First-trimester screening is conducted between 11 weeks and 13 weeks + 6 days of gestation. Second-trimester screening is performed between 15 and 20 weeks of gestation.
Important Considerations
Serum biochemical screening is a prenatal screening method, not a diagnostic tool. Conventional methods for prenatal diagnosis cannot be replaced, and clinical decisions about pregnancy termination should not be made based solely on screening results.
Second-trimester biochemical screening is not recommended as a standalone method for screening Down syndrome in twin pregnancies.
AFP is useful for screening neural tube defects, especially open NTDs. Factors influencing maternal serum AFP levels include gestational age, maternal weight, ethnicity, diabetes, stillbirths, multiple pregnancies, fetal anomalies, and placental conditions. When positive results are encountered, these factors need to be comprehensively considered.
Noninvasive Prenatal Testing (NIPT)
Basic Principles
NIPT, also referred to as noninvasive prenatal DNA testing, is based on the detection of fetal cell-free DNA (cffDNA) present in maternal plasma. By collecting maternal peripheral blood, high-throughput sequencing is used to sequence cell-free DNA fragments (including fetal DNA) in the maternal plasma. Bioinformatic analysis then calculates the risk of fetal chromosomal abnormalities. NIPT is currently used to screen for trisomy 21, trisomy 18, trisomy 13, and sex chromosome aneuploidies. With advancements in sequencing technologies and algorithm optimization, NIPT is expected to expand to screening for chromosomal microdeletion/microduplication syndromes and single-gene disorders.
Technical Features
NIPT is noninvasive and does not increase the risk of fetal loss. Its sensitivity and specificity are higher than those of serum biochemical screening. For detecting trisomy 21, trisomy 18, and trisomy 13 in both singleton and twin pregnancies, NIPT is currently the most sensitive and specific screening method.
Timing of Screening
NIPT can be performed starting at 10 weeks of gestation, with the optimal time being between 12 and 22 weeks.
Important Considerations
NIPT is a screening tool, not a diagnostic method. Conventional diagnostic methods cannot be replaced, and high-risk results require genetic counseling and invasive diagnostic procedures for confirmation. Clinical decisions regarding pregnancy termination should not be based solely on NIPT results.
The fetal cffDNA detected in maternal blood originates from the placenta rather than the fetus itself, which may result in false positives. Causes include placental mosaicism, vanishing twin syndrome, and maternal tumors.
In dizygotic twin pregnancies, NIPT can only assess the overall risk without specifying which fetus carries greater risk, requiring invasive diagnostic procedures for clarification.
NIPT might not be suitable for the following populations:
- Individuals with a history of delivering chromosomally abnormal fetuses or with confirmed chromosomal abnormalities in one parent.
- Pregnant individuals who have undergone procedures in the past year that might interfere with high-throughput sequencing results, including blood transfusions, transplantation surgeries, cell therapy, or immune therapy.
- Cases where fetal imaging suggests a possibility of chromosomal microdeletion/microduplication syndromes or other chromosomal abnormalities.
- High-risk groups for specific genetic disorders.
Prenatal Ultrasound Screening
Due to its safety for the fetus, prenatal ultrasound examination serves as the primary method for screening fetal structural abnormalities. Prenatal ultrasound is divided into first-trimester and second- to third-trimester examinations. First-trimester ultrasound is further categorized into routine first-trimester ultrasound and nuchal translucency (NT) ultrasound conducted between 11 and 13+6 weeks of gestation. Conventionally, prenatal ultrasound screening for fetal structural abnormalities would be performed at 20–24 weeks of gestation. However, with advancements in equipment and techniques, experienced prenatal diagnostic ultrasound specialists are now able to conduct earlier screenings during the first trimester, particularly during the NT ultrasound period. Transvaginal ultrasound, in particular, significantly enhances image resolution of fetal structures and aids in identifying abnormalities during early pregnancy. Such abnormalities may include anencephaly, severe encephalocele, major open spina bifida, severe thoracoabdominal wall defects accompanied by visceral herniation, single atrium, megacystis, and omphalocele. These findings provide pregnant individuals with early options and reduce the risks associated with midterm labor induction.
For second- to third-trimester examinations, a hierarchical approach is used, dividing prenatal ultrasound into four levels (I, II, III, IV). Levels I to III are classified as screening, while level IV is considered diagnostic.
- Level I prenatal ultrasound is a basic examination focusing primarily on fetal growth parameters, without assessing fetal anatomical structures.
- Level II prenatal ultrasound is a routine examination aimed at screening for severe fetal structural anomalies, such as anencephaly, alobar holoprosencephaly, major encephalocele, open spina bifida with myelomeningocele, single ventricle, common arterial trunk, renal agenesis, severe thoracoabdominal wall defects with visceral herniation, and lethal skeletal dysplasias marked by significantly shortened limbs.
- Level III prenatal ultrasound involves systemic examination, providing detailed anatomical assessments to improve the detection rate of fetal abnormalities. Routine prenatal exams may select different levels of ultrasound screening based on the facility's capacity, the physician’s expertise, and the gestational week.
- Level IV prenatal ultrasound, or targeted ultrasound, falls under the category of diagnostic examination.
It is important to note that prenatal ultrasound screening has limitations. Ultrasound may not detect all fetal structural abnormalities, nor can it assess fetal intellectual development or physiological function. Detection rates differ depending on the type of fetal structural abnormality. Additionally, certain fetal anomalies develop dynamically and only become apparent as pregnancy progresses, resulting in a degree of missed diagnoses.
Prenatal Diagnosis
Currently, prenatal diagnosis of fetal diseases focuses on two main areas: structural abnormalities and genetic disorders. Structural abnormalities are primarily diagnosed using imaging techniques, including ultrasound and magnetic resonance imaging (MRI). Prenatal diagnosis of genetic conditions involves fetal tissue sampling and laboratory diagnostic techniques.
Examination of Fetal Structural Abnormalities
Ultrasound Examination
Prenatal ultrasound diagnostic examinations are conducted to investigate abnormalities discovered during screening, providing systematic, targeted evaluations and imaging diagnoses. Level IV prenatal ultrasounds fall under the scope of diagnostic examinations. These include specialized assessments of fetal cardiac, cranial, urogenital, and skeletal systems. Prenatal diagnostic ultrasound requires advanced technical and diagnostic skills from physicians, as well as collaboration with fetal medicine specialists, geneticists, and pediatric experts in related fields. This multidisciplinary approach ensures comprehensive and accurate evaluations of fetal abnormalities, including prognosis and further clinical management steps. However, ultrasound diagnosis has its limitations and may have varying misdiagnosis rates depending on the condition. Ultrasound findings should not be equated with clinical diagnoses and are not a substitute for pathological examination.
Magnetic Resonance Imaging (MRI)
MRI provides high contrast and resolution for soft tissue differentiation, offers multi-planar imaging capabilities, and covers a broad imaging field. It has become an effective supplementary method for diagnosing fetal structural abnormalities. Currently, MRI is not a routine screening method and is primarily utilized when ultrasound reveals fetal abnormalities but cannot confirm a diagnosis. Factors such as oligohydramnios, excessive maternal intestinal gas, or obesity may impair ultrasound resolution of fetal anatomical structures, making MRI a preferred alternative in such cases.
MRI, which does not involve ionizing radiation, is considered highly safe, and no reports have identified harmful effects of magnetic fields on the fetus. However, to ensure additional safety, MRI is generally avoided for fetuses under 3 months of gestation.
Examination of Fetal Genetic Disorders
Prenatal diagnostic techniques for genetic disorders play a crucial role in preventing the birth of individuals affected by hereditary diseases. These techniques primarily include fetal tissue sampling methods and laboratory technologies.
Sampling Techniques
Sampling techniques can be categorized into invasive and noninvasive approaches. Invasive techniques include amniocentesis, chorionic villus sampling (CVS), and cordocentesis (percutaneous umbilical blood sampling). Noninvasive techniques involve obtaining fetal DNA, RNA, or fetal cells through maternal peripheral blood for prenatal diagnosis or preimplantation genetic testing.
Amniocentesis
Amniocentesis, performed under ultrasound guidance, is currently the most widely used invasive prenatal diagnostic technique and is relatively safe.
Indications
These include cases requiring the extraction of amniotic fluid to obtain fetal cells or DNA for genetic testing.
Contraindications
These include:
- Presence of symptoms indicating miscarriage.
- Signs of infection.
- Abnormal coagulation function.
Timing of the Procedure
Amniocentesis is generally conducted after 15 weeks of gestation. Performing the procedure prior to 15 weeks increases the risk of miscarriage, amniotic fluid leakage, and fetal malformations.
Preoperative Preparation
This involves:
- Confirmation of indications for the procedure and informing the pregnant individual and their family about the purpose and risks of the procedure; obtaining informed consent.
- Completion of preoperative evaluations, including assessment of maternal vital signs, routine blood tests, coagulation profile, and fetal heart rate monitoring.
Procedure
After the maternal bladder is emptied, the individual lies in the supine position. The abdominal skin is sterilized and draped using standard protocols. Real-time ultrasound is used to assess fetal positioning within the uterine cavity and the location of the placenta, followed by the determination of the puncture path. Under continuous ultrasound guidance, a puncture needle with a stylet is inserted percutaneously into the amniotic sac, avoiding the fetus, placenta, and umbilical cord. The initial 2 mL of amniotic fluid is withdrawn and discarded to prevent contamination by maternal cells. A new syringe is attached to collect the required amount of amniotic fluid for laboratory testing. Postoperatively, fetal heart rate is monitored, and symptoms such as abdominal pain or vaginal bleeding are observed.
Complications
Potential complications include fetal loss, fetal injury, bleeding, chorioamnionitis, and amniotic fluid leakage. The overall complication rate of amniocentesis is relatively low. The fetal loss rate is approximately 0.5%, while rates of vaginal bleeding or amniotic fluid leakage range between 1% and 2%. The incidence of chorioamnionitis is less than 0.1%.
Key Considerations
These include:
- Strict aseptic technique is required to prevent infection.
- Punctures should not be performed during uterine contractions, and vigilance is necessary to monitor for amniotic fluid embolism by assessing changes in maternal vital signs or symptoms such as coughing, respiratory distress, or cyanosis.
- Efforts should be made to complete the procedure in a single attempt and repeat maneuvers should not exceed three attempts.
- Careful measures should be taken to avoid injury to the intestines and bladder.
- For Rh-negative pregnant individuals, Rh immunoglobulin administration is required following amniocentesis.
Chorionic Villus Sampling (CVS)
CVS, performed under ultrasound guidance, is the primary sampling method for early prenatal diagnosis. Its advantage lies in enabling genetic diagnosis during the first trimester, aiding decisions regarding pregnancy termination, and reducing the risks associated with second-trimester labor induction.
Indications
These include cases requiring the extraction of chorionic villus tissue for genetic testing.
Contraindications
These include:
- Presence of symptoms indicating miscarriage.
- Signs of infection.
- Abnormal coagulation function.
Timing of the Procedure
CVS is typically performed after 10 weeks of gestation. Conducting the procedure before 10 weeks increases the risk of miscarriage and fetal malformations.
Preoperative Preparation
This involves:
- Confirmation of indications for the procedure and informing the pregnant individual and their family about the purpose and risks of the procedure; obtaining informed consent.
- Completion of preoperative evaluations, including assessment of maternal vital signs, routine blood tests, coagulation profile, and fetal heart rate monitoring.
Procedure
CVS can be performed via either a transabdominal or transcervical approach, with the specific choice depending on the location of the placenta and the operator's expertise. For transabdominal CVS, the maternal bladder is emptied, and the individual lies in the supine position. The abdominal skin is sterilized and draped using standard protocols. Real-time ultrasound is used to assess fetal positioning within the uterine cavity and locate the placenta, followed by the determination of the puncture path. Local anesthesia is applied to the puncture site. Under continuous ultrasound guidance, a puncture needle with a stylet is inserted percutaneously into the placenta, and the stylet is withdrawn. Using a syringe with negative pressure, an adequate amount of villus tissue is aspirated for laboratory testing. Postoperatively, fetal heart rate is monitored, and symptoms such as abdominal pain or vaginal bleeding are observed.
Complications
Potential complications include fetal loss, bleeding, and chorioamnionitis. These complications are rare when CVS is performed by an experienced specialist. The fetal loss rate associated with transabdominal CVS is approximately equivalent to that of second-trimester amniocentesis.
Key Considerations
These include:
- Strict aseptic technique is required to prevent infection.
- Efforts should be made to avoid injury to the intestines and bladder.
- The procedure should ideally be completed in a single attempt, with repeat maneuvers not exceeding three attempts.
- About 1% of CVS cases may involve confined placental mosaicism (CPM), leading to inconclusive genetic testing results, which may require further analysis through amniotic fluid sampling.
- For Rh-negative pregnant individuals, Rh immunoglobulin administration is required following CVS.
Percutaneous Umbilical Cord Blood Sampling (PUBS)
Percutaneous umbilical cord blood sampling, also known as cordocentesis, is one of the prenatal diagnostic sampling techniques performed under ultrasound guidance. Compared with amniocentesis and chorionic villus sampling (CVS), cordocentesis carries relatively higher risks. Careful evaluation of the potential risks and benefits is necessary before deciding on its use.
Indications
These include cases that require fetal blood from the umbilical vein for genetic testing.
Contraindications
These include:
- Presence of symptoms indicating miscarriage.
- Signs of infection.
- Abnormal coagulation function.
Timing of the Procedure
PUBS is generally performed after 18 weeks of gestation. Performing the procedure before this gestational age increases the risk of fetal demise.
Preoperative Preparation
This involves:
- Confirmation of the procedural indications and communication with the pregnant individual and their family regarding the purpose and risks of the procedure, followed by obtaining informed consent.
- Completion of preoperative evaluations, including assessment of maternal vital signs, routine blood tests, coagulation profile, and fetal heart rate monitoring.
Procedure
The individual lies in the supine position after the bladder is emptied. The abdominal skin is sterilized and draped using standard protocols. Real-time ultrasound is used to assess fetal positioning within the uterine cavity, in addition to the location of the placenta and umbilical cord, in order to identify the puncture pathway. Local anesthesia is administered to the skin at the puncture site. Under continuous ultrasound guidance, a puncture needle with a stylet is inserted percutaneously into the umbilical vein. Once the needle is in place, the stylet is removed, and a syringe is used to aspirate an adequate amount of umbilical venous blood for laboratory testing. Postoperatively, fetal heart rate is monitored, and symptoms such as abdominal pain or vaginal bleeding are observed.
Complications
Complications associated with PUBS include fetal loss, fetal bradycardia, bleeding at the puncture site, umbilical cord hematoma, and chorioamnionitis. The risk of fetal loss is approximately 1%–2%. This risk is higher in cases involving fetal anomalies, fetal growth restriction, or fetal hydrops.
Key Considerations
These include:
- Strict aseptic technique is necessary to prevent infection.
- Punctures must not be performed during uterine contractions, and attention must be given to maternal vital signs and symptoms such as coughing, respiratory distress, or cyanosis that could indicate amniotic fluid embolism.
- Efforts must aim to complete the procedure in a single attempt, with repeat maneuvers not exceeding three attempts.
- Fetal heart rate changes, such as bradycardia, warrant immediate cessation of the procedure and, if necessary, intrauterine resuscitation.
- For Rh-negative pregnant individuals, Rh immunoglobulin administration is needed following PUBS.
Fetal Tissue Biopsy
Fetoscopy allows direct visualization of the fetal surface, including facial features, and facilitates fetal skin biopsy. However, this technique has high technical demands and carries a higher risk of complications. With the development of noninvasive ultrasound technologies and advancements in molecular genetics, fetoscopy is no longer performed solely for diagnostic purposes. Instead, it is used for the intrauterine treatment of certain fetal conditions, such as twin-to-twin transfusion syndrome or amniotic band syndrome.
Preimplantation Genetic Testing (PGT)
In cases where parents are affected by certain hereditary diseases, in vitro fertilization may be used to obtain embryos. These embryos can undergo genetic testing before implantation to reduce the risk of spontaneous abortion and prevent the transmission of hereditary diseases to offspring. As PGT typically involves examining only one or a subset of cells from the early embryo, the results may not fully represent the genetic traits of the entire embryo. Therefore, CVS or amniocentesis is recommended for individuals conceived through PGT to confirm the diagnosis.
Laboratory Techniques
Laboratory techniques refer to genetic testing conducted on fetal tissues from various sources. These techniques include cytogenetic methods, biochemical genetic methods, and molecular genetic methods, such as conventional chromosome karyotype analysis, chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH), quantitative fluorescence PCR (QF-PCR), multiplex ligation-dependent probe amplification (MLPA), copy number variation sequencing (CNV-seq), and DNA sequencing technologies.
Conventional Chromosome Karyotype Analysis
Chromosome karyotype analysis is considered the "gold standard" for diagnosing chromosomal abnormalities. By culturing fetal cells and analyzing their karyotypes, it is possible to detect numerical chromosomal abnormalities as well as structural abnormalities larger than 5–10 Mb. The main limitations of this technique include the lengthy cell culture process, a high demand for human resources, and a turnaround time of two weeks to one month. Additionally, karyotype analysis has a low resolution, making it challenging to detect or confirm subtle structural chromosomal anomalies, such as microdeletion and microduplication syndromes or marker chromosomes.
Chromosomal Microarray Analysis (CMA)
CMA, also known as molecular karyotyping, enables genome-wide scanning and detection of chromosomal imbalances in copy number variants (CNVs). Its primary advantage lies in its ability to identify microdeletions and microduplications. However, CMA cannot distinguish balanced translocations from normal karyotypes and is also unable to detect low-level mosaicism. A significant challenge in the application of this technology in prenatal diagnosis involves the interpretation of variants of uncertain significance (VUS) and the lack of established guidelines and standards.
Fluorescence In Situ Hybridization (FISH) and Quantitative Fluorescence PCR (QF-PCR)
These techniques are primarily utilized for detecting numerical abnormalities of chromosomes 13, 18, 21, X, and Y. Common chromosomal aneuploidies can be diagnosed within 1–2 days using these methods. FISH uses chromosome-specific DNA probes, while QF-PCR relies on chromosome-specific short tandem repeat markers. Collectively referred to as rapid aneuploidy detection methods, they complement karyotype analysis by facilitating the rapid identification of specific chromosomal abnormalities, though they do not provide comprehensive karyotype results.
Multiplex Ligation-Dependent Probe Amplification (MLPA)
MLPA is a technique used for qualitative and semi-quantitative analysis of target DNA sequences. It is primarily employed to detect copy number variants in specific regions, methylation levels, or hotspot mutations. This method combines DNA probe hybridization with PCR technology, offering a rapid and highly specific approach for analysis. However, it is not suitable for detecting unknown variants or balanced chromosomal translocations.
Copy Number Variation Sequencing (CNV-seq)
CNV-seq involves low-coverage whole-genome sequencing of DNA samples using high-throughput sequencing technologies. Sequencing data are aligned to the human reference genome, and bioinformatic analyses are used to identify copy number variations in the sample. CNV-seq has advantages such as high throughput, wide detection range, and the ability to detect low-level mosaicism. However, it is unable to identify triploidy, polyploidy, balanced chromosomal translocations, inversions, or loss of heterozygosity (LOH).
DNA Sequencing Technologies
DNA sequencing is widely employed in the diagnosis of single-gene disorders. Over the past half-century, significant advancements have been achieved in sequencing technologies, progressing through first-generation sequencing (Sanger sequencing), second-generation sequencing (high-throughput sequencing), and third-generation sequencing.
The first-generation sequencing method remains the "gold standard" for genetic testing. It is highly accurate and capable of generating long sequencing reads but is limited by high costs and low throughput, which restricts its large-scale application. It is mainly used for confirming known pathogenic mutation sites and validating results from second-generation sequencing.
Second-generation sequencing methods include targeted sequencing, whole-exome sequencing (WES), and whole-genome sequencing (WGS). The major advantages of second-generation sequencing are its high throughput, shorter turnaround time, and lower cost, making it widely applicable in research and clinical settings. However, its shorter read lengths pose limitations in diagnosing certain genetic conditions, particularly those involving tandem repeat sequences or complex structural rearrangements.
Third-generation sequencing, also termed long-read sequencing (LRS), is exemplified by single-molecule real-time (SMRT) sequencing and nanopore sequencing. Compared to second-generation sequencing, third-generation sequencing provides greatly extended read lengths, addressing limitations such as tandem repeat analysis, structural variants, and haplotype phasing. Despite these advantages, its clinical application remains limited due to lower sequencing accuracy compared to the first and second generations, as well as high costs.