Invasive Diagnostic Testing in Obstetrics: Amniocentesis and Chorionic Villus Sampling

Z36.0 Antenatal screening for chromosomal anomalies

Amniocentesis, a diagnostic procedure involving the extraction of amniotic fluid from the amniotic cavity using a transabdominal needle approach, serves as a valuable method for prenatal genetic diagnosis and evaluation of fetal health through laboratory tests. Amniotic fluid, primarily comprising fetal substances such as urine, secretions, exfoliated cells, and transudate, provides essential information for fetal diagnosis. This procedure was first performed in 1970¹.

Indications

Amniocentesis is primarily performed for diagnostic and therapeutic indications. Prenatal genetic testing for chromosomal anomalies represents the most common diagnostic indication (Figures 1-3 and Video 1). Amniocentesis and chorionic villus sampling (CVS) (discussed later) remain, at present, the only definitive diagnostic tests for fetal aneuploidy and genetic disorders during pregnancy. Other indications for assessment of amniotic fluid include evaluation of potential fetal infection or chorioamnionitis, fetal blood or platelet typing, investigation of hemoglobinopathies, etc. Past indications have included indirect assessment of fetal hemolytic anemia by analysis of bilirubin in amniotic fluid, confirmation of elevated serum alpha fetal protein and assessment of fetal lung maturity, but these indications are mainly of historical interest. . Additionally, amniocentesis can serve as a therapeutic procedure to alleviate symptomatic polyhydramnios.

Technique

The optimal transabdominal needle insertion site is carefully chosen, with efforts made to avoid the placenta whenever feasible (especially in Rhesus-negative women). The rates of fetal loss were comparable between the transplacental and transmembrane approaches, but the transplacental passage was associated with higher rates of bloody tap.^2–5 In cases where a transplacental approach is necessary, the needle is directed through the thinnest part of the placenta. The use of color flow mapping can assist in identifying and avoiding the umbilical cord insertion site and large chorionic vessels.
While navigating the needle between the skin and the uterine wall, it is crucial to prevent inadvertent puncture of intestinal loops. This risk arises from potential incomplete visualization of the needle trajectory, particularly when there is insufficient insonation laterally to the ultrasound beam. If the maternal bladder obstructs the needle's path, it is advised that the patient empties their bladder prior to needle insertion.⁶

Under continuous ultrasound guidance, it is recommended to use a 20-22-G needle for transabdominal insertion during amniocentesis to prevent tenting of the amniotic membrane⁶ (slides 3-4). In a randomized trial, it was observed that using a larger bore needle decreased the risk of intrauterine bleeding if the needle passed through the placenta and allowed for faster fluid retrieval compared to a 22 gauge needle, although it caused more discomfort to the mother⁷. Additionally, a retrospective study (n=793) reported similar rates of fetal loss with 20-G (1.57%), 21-G (1.47%), and 22-G (1.61%) needles⁸.

Ultrasound guidance during amniocentesis does not decrease the rate of fetal losses, but continuous visualization of the needle through ultrasonographic monitoring is recommended to prevent direct fetal injury, minimize punctures, and reduce the incidence of bloody fluid⁹.

The standard length of a spinal needle is 8.9 cm (excluding the hub), although longer needles measuring 15 cm are also available. The selection of needle length should consider factors such as the thickness of the maternal panniculus, the location of the target fluid pocket, and the potential for intervening events, like uterine contractions, which may increase the distance between the skin and the target site.

To minimize the risk of maternal cell contamination, it is common practice to discard the initial drop of 2 ml amniotic fluid that may contain maternal cells.¹⁰  This can help prevent low-level mosaicism in cytogenetic studies. Fluid aspiration may be performed by the operator, by an assistant or by using a vacuum device¹¹.For testing purposes, approximately 20 to 30 mL of amniotic fluid is usually aspirated into sterile syringes.

Accurate identification requires the meticulous labeling of the syringes with comprehensive demographic information, coupled with the essential step of the patient's thorough verification. Postprocedural care involves assessing and recording the fetal heart rate. Patients should be advised that immediate and self-limited post-procedure effects such as uterine cramping, transient spotting, and minor vaginal loss of amniotic fluid may occur, but persistent vaginal fluid loss or bleeding, severe uterine cramping lasting hours, or fever should be reported. Limiting physical or sexual activity after the procedure is unnecessary. Non-alloimmunized RhD-negative patients should receive Rho(D) immune globulin. The American College of Obstetricians and Gynecologists recommends routine administration of 300 micrograms dose of Rho(D) immune globulin following mid-trimester amniocentesis.¹²

Timing

Amniocentesis, can be performed at various gestational ages, but it is most commonly done between 15^0/7 to 17^6/7 weeks of gestation. Performing amniocentesis before 15 weeks (referred to as early amniocentesis) compared to mid-trimester is associated with higher rates of fetal loss¹³ and potential for talipes equinovarus^14,15 , an increase in requirement for multiple needle insertions (4.7% versus 1.7%; RR 2.79, 95% CI 1.92–4.04),¹⁵ increased risk of failed culture (1.8% vs. 0.2%) and potential for false negative results (0.05% versus 0.0%; RR 3.00, 95% CI 0.12–73.67).¹⁶

The cellular composition of amniotic fluid primarily consists of epithelioid cells, along with the presence of fibroblastic-type cells and amniotic fluid-specific cells. Cells shed from the amnion and lower fetal urinary tract contribute significantly to the proliferating cell population, yet only a small fraction of these cells have the ability to adhere to a culture substrate and form colonies, with cloning efficiency declining before 15 weeks and at 24 to 32 weeks of gestation, which explains some of the culture efficiency of amniocytes.¹⁷

While later second-trimester procedures are safe, they may pose challenges if pregnancy options change based on abnormal results. In some cases, late second- and third-trimester procedures are conducted when fetal abnormalities are detected later in gestation, or suspected amniotic infection, providing valuable information for counseling, parental preparation, and determining the best delivery time, route, and location.

In cases where amniocentesis is prompted by a cytomegalovirus (CMV) infection indication, historically, it has been believed that the highest sensitivity for CMV detection occurs after 21 weeks of gestation. However, a retrospective study ¹⁸ examining 264 pregnancies conducted between 17^0/7 weeks and 22^6/7 weeks of gestation, with an interval of more than 8 weeks between seroconversion and amniocentesis, found that the diagnostic sensitivity before and after 21 weeks was comparable (87.2 percent and 92.1 percent, respectively). The negative predictive value was also similar (93.6 percent before 21 weeks and 96.8 percent after 21 weeks).

Complications

Amniocentesis carries several significant potential complications, including membrane rupture, direct and indirect fetal injury, infection, and the risk of fetal loss. Maternal complications, such as amnionitis, are rare and occur in less than 1 out of every 1000 procedures.^19–23

Pregnancy loss

The first and only randomized controlled trial (RCT) that offered the initial estimate of the extra risk of pregnancy loss following amniocentesis was published in 1986.²⁴ According to the trial's findings, there was a 1% increase in risk. However, it is important to note that the estimate was not very precise, as the confidence intervals were quite wide, encompassing both the possibility of no additional risk and a 2% risk. Considering the circumstances, it appears doubtful that a comparable RCT of adequate size and quality will be conducted again in the future.

Numerous observational studies, including large national registry-based ones, have been conducted since then, reporting much lower complication rates for both amniocentesis and CVS, with no significant increase in pregnancy losses compared to the background rate.^10,24–33 . Methodologically robust studies examining complication rates should ideally focus on estimating the procedure-related risk for fetuses that are structurally and genetically normal, using cohorts with genetic testing for all pregnancy losses, irrespective of whether an invasive test was previously performed. Additionally, they should include control groups matched for essential confounders, such as maternal age. However, it is important to consider that even if these criteria are fulfilled, the absence of randomization may still result in clinically relevant differences between women undergoing invasive testing and those who do not. Nonetheless, the decreased pregnancy loss rates reported in recent studies could be attributed to advancements in technology, techniques, and experience.

Women should be properly informed that the extra risk of experiencing pregnancy loss following an amniocentesis, conducted by a suitably trained operator, is expected to be less than 0.5%. However, it is essential to understand that the risk of pregnancy loss can vary based on specific factors. Procedures conducted by less skilled operators or in units with limited experience may carry a higher risk. Moreover, cases where the fetus has chromosomal, genetic, or structural abnormalities are also likely to increase the risk.

Amniotic fluid leakage

In approximately 1-2% of all cases, minor complications from amniocentesis may occur, such as transient vaginal spotting or amniotic fluid leakage ³⁴. However, the perinatal outcomes in cases of amniotic fluid leakage after mid-trimester amniocentesis are remarkably better when compared to spontaneous rupture of membranes at a similar gestational age. The perinatal survival rate in such instances exceeds 90% ³⁵.

Needle injury

When amniocentesis is performed under continuous ultrasonographic guidance, needle injuries to the fetus have been reported but are rare. Additionally, amniotic fluid cell culture failure is observed in 0.1% of samples^36,37.

Chorioamnionitis

The likelihood of chorioamnionitis and uterine infection after genetic amniocentesis is  low, with a rate of less than 0.1%¹⁰ . In a very small number of cases, severe maternal complications  such as sepsis or even death, have been reported^38–42. These incidents are believed to be a result of inadvertent puncture of the bowel during the procedure. Furthermore, there is a risk of maternal infection due to the colonization of microorganisms in ultrasound gel and probes⁴³ .

Complications of third trimester amniocentesis

Third-trimester amniocentesis may be considered in cases involving newly identified fetal structural anomalies, suspected fetal infection, or fetal growth restriction. Although serious complications necessitating emergency birth are uncommon after third-trimester amniocentesis, two retrospective cohorts reported a preterm labor rate before 37 weeks of 4-8% and before 34 weeks of 3-4%^44,45.The procedure carries an increased risk of requiring more than one needle insertion (5% of procedures), and bloodstained samples have been reported in 5-10% of cases.⁴⁶ Additionally, there is a higher risk of culture failure following third-trimester amniocentesis, with an overall rate of 9.7% reported.⁴⁷ Another study analyzing amniocentesis procedures performed between 24^0/7 and 39^0/7 weeks from 2002 to 2014 found an increasing overall culture failure rate of 10.2%, escalating with gestational age from 2.1% between 24^0/7 and 27^0/7 weeks to 40.6% between 36^0/7 and 40^0/7 weeks.³⁵

Furthermore, it is important to note that culture failure primarily impacts karyotype analysis but generally does not affect chromosomal microarray or sequencing procedures, as DNA is extracted in the later cases.

Risk factors for complications

The presence of fetal structural anomalies itself elevates the baseline risk of miscarriage, which is further amplified following amniocentesis⁴⁸. When the amniotic fluid specimen appears bloody or discolored (brownish), indicating current intra-amniotic bleeding, it consistently signifies a higher risk of post-procedural fetal loss. This connection is attributed to the association of intra-amniotic bleeding with underlying placental disorders⁴⁹, ⁵⁰. It is worth highlighting that several of these variables can act as confounding factors in fetal loss. Women with conditions such as active bleeding, amniotic infections, or a history of miscarriage or late abortions have higher risk of fetal loss regardless of whether amniocentesis is performed.

A higher number of procedures performed annually, exceeding 100, has been associated with lower fetal loss rates⁴³. Conversely, an increased number of attempts, specifically three or more punctures, raises the risk of fetal loss. If more than two punctures are necessary, it has been recommended to delay the procedure by 24 hours^11,49. According to expert opinion, when the loss rates exceed 4 per 100 consecutive amniocenteses, the operator's competence should be reviewed^43,51.

Several risk factors have been suggested to increase the risk of fetal loss following amniocentesis, although their association has not been consistently proven. Plausible risk factors include uterine fibroids, Mullerian malformations, chorioamniotic separation, retrochorionic hematoma, previous or current maternal bleeding, maternal body mass index > 40 kg/m², multiparity (> 3 births), manifest vaginal infection, and a history of three or more miscarriages^52,53.

Chorionic villus sampling (CVS) involves the extraction of trophoblastic cells from the placenta. The chorionic villi consist of outer syncytiotrophoblast cells, a middle layer of cytotrophoblast cells, and an inner mesenchymal cell core. It was initially documented in China during the mid-1970s⁵⁴ and later integrated into medical practice in the early 1980s⁵⁵.

Timing

The first reports of possible connections between CVS and both oromandibular disruption and limb reduction defects surfaced in 1991, involving procedures carried out between 8^0/7 and 9^3/7 weeks' gestation⁵⁶. Since then, subsequent studies have arisen, refuting this association, though it is worth noting that most of these procedures were conducted after 10^0/7 weeks.⁵⁷  It is suggested that any potential risks may be dependent on the gestational age, which has led to guidelines discouraging CVS before 10^0/7 weeks⁵⁸. Additionally, performing CVS prior to 11^0/7 weeks' gestation can present greater technical challenges due to limited access and thinner, less developed placental tissue.

Technique

Access to the placenta can be achieved through either a transabdominal or transcervical approach. An RCT involving 3,873 pregnant people with singleton pregnancies (gestational age range: 7–12 weeks, mostly > 10 weeks) found similar fetal loss (2.3% vs. 2.5%) and successful sampling (95% vs. 94%) rates between the two methods.⁵⁹

Transabdominal CVS involves the insertion of a needle through the abdominal wall into the uterus and directing it into the placenta while maintaining strict aseptic conditions. Conversely, the transcervical approach entails the passage of an instrument, such as a cannula or forceps, through the cervical canal (Figures 5 and 6) and into the placenta under continuous ultrasound guidance. There are two main methods for achieving this: the free-hand technique and the biopsy adaptor method. Since there is no significant data comparing the safety or efficiency of these methods, the choice should be based on the operator's experience or preference. However, technical factors predominantly related to placental location favor one approach over the other in up to 5 percent of procedures^43,60 .

For the transabdominal approach, local anesthesia may be used . A single needle of 17–20 G or a two-needle set of outer 17/19 G and inner 19/20 G can be utilized ⁶¹. Once the needle reaches the target within the placenta, a series of back-and-forth needle  movements, ranging from one to ten, are performed while maintaining the vacuum for continuous aspiration^11,60,62. A retrospective cohort study published in 2017, looked at outcomes of 4862 CVS procedures. Out of these, 2833 procedures were performed transcervically, with 1787 using forceps and 1046 using cannula. The procedure-related pregnancy loss rate for transcervical CVS was 1.4%, while for transabdominal CVS, it was 1.0%. Interestingly, when the researchers analyzed the data further, they observed that the risk of pregnancy loss after transcervical CVS varied significantly depending on the instrument used. When forceps were used during transcervical CVS, the pregnancy loss rate dropped to only 0.27%, suggesting that forceps may provide a safety advantage in these cases. On the other hand, procedures conducted with cannula resulted in a higher pregnancy loss rate of 3.12%.⁴³

The amount of villi obtained in the sample must be visually checked. A minimum of 5 mg of villi in each sample is required to yield a valid result. ⁴³

Complications

Fetal loss

As previously mentioned, since the first RCTs, there have been no trials with appropriate control groups to determine the pregnancy loss rate at amniocentesis and CVS. Having an appropriate control group is of paramount importance. In a cohort study involving more than 22,000 patients undergoing first-trimester combined screening, the risk of miscarriage was approximately 1 percent higher in patients who underwent CVS (3613 patients) compared to those who did not undergo the procedure (2.1 versus 0.9 percent). However, this increased risk was influenced by distinct demographic and pregnancy characteristics, particularly aneuploidy ⁶³. When the aneuploidy risk was low, the risk of miscarriage following CVS rose (odds ratio [OR] 2.87, 95% CI 1.13-7.30), while paradoxically, the risk decreased when the aneuploidy risk was high (OR 0.47, 95% CI 0.28-0.76). This counterintuitive relationship may be attributed to the termination of pregnancies with major aneuploidies that would have otherwise resulted in spontaneous miscarriage. After accounting for these risk factors and restricting the analysis to low-risk pregnancies, CVS increased the risk of miscarriage approximately threefold above the patient's baseline risk (an increase from 0.1 to 0.3-0.5 percent)⁴³. While this represents a notable increase in relative terms, the absolute impact remains small. ⁶³

Failed cell culture

Failure of the cytotrophoblastic culture is reported to occur after  0.5-1% of procedures when at least 5 mg of chorionic villi are obtained ⁴³.

Confined placental mosaicism

Confined placental mosaicism (CPM) is a condition characterized by the presence of chromosomally abnormal cells exclusively within the placenta, while the chromosomes of the developing fetus remain normal⁶⁴. CPM can originate from post-zygotic errors, such as non-disjunction in a diploid conception. Alternatively, it may occur through a trisomic rescue mechanism, where a viable trisomic conceptus loses one chromosome during anaphase lagging, resulting in the development of a diploid cell line⁶⁵. CVS has a higher risk of CPM than amniocentesis (2.3 versus 0.4 percent, RR 5.7, 95% CI 1.9-16.2)⁵⁸. CPM is observed in approximately 1-2% of procedures⁴³ .

Limb-reduction defects and oromandibular hypogenesis

The generally accepted lower limit for CVS procedures is ten weeks of gestation, as an increased rate of transverse limb abnormalities has ⁶⁶ been reported when CVS is performed before 9 weeks of gestation ^57,67–69.

Fetomaternal hemorrhage

Fetomaternal hemorrhage (FMH) has been documented based on an increase in maternal serum alpha-fetoprotein (MSAFP) following CVS. It has been proposed as one cause of fetal loss after CVS, particularly when MSAFP is very high or continues to rise after CVS  ⁷⁰. Release of fetal blood into the maternal circulation can also cause isoimmunization; therefore, RhD-negative patients should receive Rho(D) immune globulin following the procedure. CVS-related FMH may augment the maternal immune response in patients already sensitized and lead to early, severe erythroblastosis fetalis⁷¹. MSAFP levels should drop to baseline levels by 16 to 18 weeks of gestation ⁷². The incidence of culture failure, amniotic fluid leakage, or infection after CVS is less than 0.5%⁶⁶.

Antibiotic prophylaxis

The available evidence remains inconclusive and of limited quality to make a definitive assessment of the efficacy of antibiotic prophylaxis before invasive procedures.⁷³ Based on the available evidence, the standard of care for amniocentesis does not include the use of Prophylactic antibiotics⁷⁴.

In cases where the amniotic fluid appears cloudy or purulent upon inspection, or if the woman exhibits clinical signs indicative of intra-amniotic infection, the operator should strongly consider sending a small sample of the amniotic fluid for microbiological analysis and consider antibiotic treatment⁴³.

Pre-procedure ultrasound

Initially, an obstetric ultrasound examination is conducted to assess number of fetuses, fetal viability, position, biometry, placenta location, and gestational age⁷⁵.

Aseptic environment

Maintaining asepsis is crucial during invasive procedures to minimize the risk of infection for both the fetus and the pregnant person. To ensure aseptic conditions, it is recommended to use a tray containing sterile gloves, gauze pads, forceps, and needles.⁷⁵ Prior to transabdominal CVS or amniocentesis, the abdominal skin should be cleansed using an antiseptic solution such as chlorexidine or iodine, followed by the application of a sterile drape. It is common practice to enclose the probe in a sterile bag, although disinfection of the probe itself is also acceptable. To prevent bacterial contamination, it is strongly advised to use separate sterile gel. For transcervical CVS, a sterile speculum is inserted, and both the cervix and vaginal walls are cleansed using an antiseptic solution.^6,75

Local anesthesia

Local anesthesia is usually unnecessary for amniocentesis as most patients experience mild discomfort, and no intervention has been proven to effectively reduce this discomfort^76–78 . However, for transabdominal CVS, the use of local anesthesia can be considered to alleviate patient discomfort resulting from the use of a larger needle.⁷⁹ A randomized control study that checked the impact of using N₂O compared to local anesthesia found that N₂O was as efficient and even superior to local anesthesia for both pain and anxiety reduction during CVS⁸⁰. There is currently no available data regarding the use of local anesthesia before transcervical CVS.

Uterine relaxants

The available data on procedure-related complications of amniocentesis, obtained from studies incorporating ultrasound guidance for needle placement visualization, do not provide evidence supporting the association of specific techniques (such as uterine relaxants) with a reduced risk of complications⁷³.

Maternal viral infection (HIV, Hepatitis B and Hepatitis C)

Recent studies have shown a very low risk of mother-to-child transmission (MTCT) of HIV in pregnant people on highly active antiretroviral therapy (HAART) with undetectable viral loads. Several retrospective cohorts spanning from 1985 to 2015, totaling 317 procedures, found no cases of MTCT in pregnant people on HAART ^81–84. Another multicenter retrospective case-control study involving 166 amniocentesis procedures found a non-significant increase in MTCT for pregnant people without antiretroviral therapy (25.0% versus 16.2%) and those treated with certain antiretroviral regimens (6.1% versus 3.3%), while no MTCT cases were found in pregnant people on HAART ⁸³ . It is important for operators to inform pregnant people that most evidence is based on amniocentesis, and data on CVS are limited. When screening results for blood-borne viruses are unknown, testing should be delayed until HIV status is determined. In cases where screening is declined, informed consent should include a discussion of the risk of MTCT, particularly for pregnant people with higher viral load and not on antiretroviral treatment ^81,83.

Regarding Hepatitis B, the overall risk of MTCT is low, but increased viral load poses a transmission risk. A small retrospective case-control study of infants born to pregnant people with positive HBsAg, of whom 63 out of 642 had amniocentesis, showed a significant increase in MTCT when the HBV DNA was over 500 copies/ml (4.2% versus 17.4%; OR 4.76 [95% CI 1.17–19.33]) ⁸⁵. As for Hepatitis C, there is an absence of high-quality data on the risk of MTCT following amniocentesis, but currently, there is no evidence of higher risk ⁸⁶.

Multiple gestation

Counseling patients regarding the risk of aneuploidy and diagnostic testing in multiple gestations is more complex than for singleton pregnancies due to the presence of more than one fetus and limited data on multiple gestations. Information from a large European population registry indicates that the adjusted relative risk of Down syndrome per fetus from multiple gestations is approximately only half that of singletons⁸⁷.

When counseling for multiple gestations, a discussion of pregnancy management options if only one fetus has aneuploidy should be included. These options encompass continuing the pregnancy, terminating the entire pregnancy, and selective second-trimester termination of the affected fetus.

Expertise in ultrasound scanning is crucial for operators performing amniocentesis or CVS in multiple pregnancy ⁸⁸. Careful fetal mapping, chorionicity and mapping of the placental site and dividing membrane are essential before any invasive procedure to ensure proper identification and appropriate sampling of each fetus, with samples correctly labeled for analysis.

For amniocentesis in dichorionic twins, most operators commonly choose double uterine entry during amniocentesis or CVS. However, the limited data available suggest that there is no significant difference in the risk between amniocentesis performed with single or double uterine entry ⁸⁹. For CVS, there appears to be no significant differences in pregnancy loss with transabdominal versus transcervical CVS, or with the use of a single versus double needle technique, or single versus double uterine entry⁸⁹. The risk of cross-contamination during CVS is approximately 1%, and using a double uterine entry technique may help mitigate this risk ⁸⁹. Based on the currently available evidence, operators are advised to utilize the technique with which they feel most comfortable.

In monochorionic diamniotic twin pregnancies, it is advisable to conduct sampling of a single sac when chorionicity has been clearly established via ultrasound before 14 weeks, and when there is concordance in fetal growth and anatomy. However, if these criteria are not met, the consideration of double sampling is warranted ⁹⁰. The two-sampling approach may also be appropriate after in-vitro fertilization (IVF) or in situations involving discordant anomaly/growth, with a small risk of heterokaryotypia. If clinical indications necessitate the sampling of two sacs, the recommended technique is the two-puncture method to prevent iatrogenic monoamnionicity ⁹⁰.

Recent studies in the last years suggest that the actual risk of amniocentesis-related pregnancy loss in twins might be less than 1%, as these studies have consistently reported no significant increase in pregnancy losses above the background risk.⁹¹ The available data on procedure-related pregnancy losses following CVS are more limited. Agarwal et al. conducted a meta-analysis of four observational studies involving CVS in twin pregnancies (632 procedures) and reported an overall pregnancy loss rate of 3.8% (95% CI 2.5–5.5) following CVS⁸⁹. Due to data heterogeneity, it was not feasible to estimate the risk of pregnancy loss before 24 weeks with CVS ⁸⁹. Furthermore, for both amniocentesis and CVS, the majority of studies have not categorized their outcomes by chorionicity, making it challenging to accurately determine procedure-related losses separately for monochorionic diamniotic and dichorionic diamniotic twins.

In late second or early third-trimester procedures aimed at evaluating infection in preterm pre-labor rupture of membranes or preterm labor, amniocentesis only requires sampling the lower-most (presenting) sac. This approach is sufficient since infection typically follows an ascending route from the vagina.

While amniotic fluid culture serves as the gold standard for detecting intraamniotic infection, its time-consuming nature necessitates the use of alternative indicators. The presence of bacteria and leukocytes (at least six per high-power field) is indicative of infection in gram stain, considering amniotic fluid is typically sterile in uncomplicated pregnancies with intact membranes.⁹².

Additional swift indicators encompass assessing glucose concentration (≤14 mg/dL suggests infection), determining WBC concentration (abnormal result ≥30 cells/mm3), and evaluating leukocyte esterase activity. Recognizing an amniotic fluid IL-6 level exceeding 2.6 ng/mL is now widely accepted as diagnostic for intra-amniotic inflammation. The rapid bedside detection of MMP-8 (>20 ng/mL) in amniotic fluid is associated with a notably higher rate of intra-amniotic inflammation. A composite profile, including positive Gram stain, leukocyte esterase, low glucose, and elevated WBC, provides 90% sensitivity and 80% specificity in predicting positive amniotic fluid culture results.⁹³

Karyotype, FISH, QFPCR

The choice of test depends on the indication, with chromosomal analysis (karyotype) taking 7 to 14 days, while interphase fluorescence in situ hybridization (FISH) and Quantitative Fluorescent Polymerase Chain Reaction (qfPCR) offer a limited karyotype within 24 to 48 hours, mainly detecting aneuploidy in chromosomes 13, 18, 21, X, and Y⁹⁴.  Abnormalities detected by FISH/qfPCR and reliably detectable by chromosome analysis should be confirmed by conventional cytogenetic analysis (Figures 7 and 8).

Moreover, FISH can be utilized to detect other chromosome abnormalities if appropriate probes are available. Autosomal trisomies for 21, 18, and 13; sex chromosome aneuploidy; and triploidy account for 80 percent of clinically significant chromosomal conditions diagnosed prenatally.⁹⁴

The concordance rates between FISH and karyotype analysis of uncultured amniocytes are exceedingly high, surpassing 99 percent⁹⁵. Nonetheless, confirmation through karyotyping remains necessary to ascertain the presence of a translocation. It should be noted that normal FISH results necessitate additional testing, either through karyotype analysis or chromosomal microarray (CMA). Relying solely on FISH would overlook alterations in chromosome structure and less common aneuploidies.

Chromosomal microarray

Karyotyping has long been the standard approach for prenatal diagnosis, but the use of CMA is on the rise. CMA offers several advantages, including its ability to detect small gains and losses of genetic material, known as copy number variants (CNVs), typically in the range of 50 to 100 kilobases. These CNVs may not be identifiable through traditional karyotyping methods, yet they have the potential to lead to significant phenotypic abnormalities. In contrast, karyotyping typically achieves a resolution of only 5 to 10 megabases (Figures 9 and 10).

A systematic review of prenatal CMA revealed that a clinically significant CNV was detected in 5.6% of euploid fetuses exhibiting ultrasound anomalies restricted to a single anatomical system. In euploid fetuses with multiple anomalies, the detection rate rose to 9.1% ⁹⁶. Similar estimates from other reviews indicate that significant CNVs in euploid fetuses with ultrasound anomalies range from 5.1 to 10.0 percent ^97,98. Furthermore, the use of CMA has unveiled the identification of a pathogenic or likely pathogenic CNV in approximately 1.7% of individuals with normal ultrasound findings and a normal karyotype.¹³

One notable advantage of CMA is its ability to provide results without the need for cell culture, thereby reducing the turnaround time. However, the increased depth of molecular analysis with CMA raises the likelihood of incidental findings, which adds complexity to prenatal counseling. For instance, CMA may identify variants of unknown significance, previously unrecognized genetic variants in one or both parents, or fetal genes associated with adult-onset diseases. It is important to note that while CMA offers improved resolution compared to karyotyping, it is unable to detect certain clinically significant genetic findings, such as gene sequence alterations that could impact gene function, balanced structural rearrangements, and unbalanced translocations.

Several societies, including the American College of Obstetricians and Gynecologists (ACOG) and the Society for Maternal-Fetal Medicine (SMFM), have recommended the use of CMA for prenatal diagnosis. ACOG's committee opinion states that CMA should be employed instead of karyotyping when one or more major structural abnormalities are identified through ultrasound⁹⁹. Additionally, ACOG advocates for making CMA available to any patient opting for invasive diagnostic testing.

Next generation sequencing

When standard chromosomal analysis, including CMA, fails to provide a diagnosis, molecular genomic techniques are employed for specific genetic conditions. Advanced sequencing methods like next-generation sequencing (NGS) offer various options, such as examining individual genes, selected gene panels, whole  exome (constituting 1-2% of the genome), or the entire genome. These approaches result in higher diagnostic rates ¹⁰⁰. Targeted gene sequencing reduces the likelihood of uncertain genomic variants or incidental findings and offers quicker turnaround times compared to exome sequencing (ES) or whole genome sequencing (WGS). However, focusing on specific gene panels may limit the examination of other significant conditions ¹⁰⁰.

Targeted gene sequencing involves testing specific genes known to be associated with certain genetic conditions, often relying on a positive family history and previously identified pathogenic variants. For instance, sequencing the FGFR3 gene can confirm a diagnosis of achondroplasia ¹⁰¹.

Exome sequencing (ES) analyzes the exome through NGS, which contains a substantial proportion of disease-causing variants. However, ES does not sequence the noncoding regions of the DNA, limiting the detection of certain abnormalities ¹⁰² (Figure 11). A meta-analysis showed a 31% incremental yield of ES for prenatal diagnosis of fetal structural anomalies when karyotype or CMA results were normal. The yield varied based on factors like the expertise of the multidisciplinary team and the specific anomaly type ¹⁰³. Nevertheless, the routine clinical application of ES may present challenges, including result interpretation, limited phenotypic data, the need for standardized prenatal curation, and dealing with variants of unknown significance.^104–107

Whole genome sequencing (WGS) provides comprehensive analysis of sequence variation, detecting small copy number variants, structural variants, and more. The turnaround time for WGS is often faster than ES ¹⁰². However, routine use of prenatal sequencing for indications other than fetal anomalies is not supported due to limited validation data and insufficient knowledge of its benefits and limitations ¹⁰⁸.

Methylation studies are valuable when abnormal methylation or imprinting is suspected in genetic disorders, such as Beckwith-Wiedemann syndrome, which can be detected through methylation studies ¹⁰². Decisions regarding prenatal sequencing should be made with genetic specialists considering result interpretation, timeliness, and comprehensive patient and family counseling ¹⁰⁸.

In the context of clinical genetic diagnostics, the primary goal is to determine whether the patient has a pathogenic or potentially pathogenic variant, as this information significantly impacts patient care and the management of their family members. To achieve this, the first step involves a comprehensive examination of all available evidence to assess the potential pathogenicity of variants.

According to the American College of Medical Genetics and Genomics guidelines, the results of CMA and NGS are classified into five categories:¹⁰⁹

• Pathogenic variants: These variants are considered to be the cause of the patient's disease.
• Likely pathogenic variants: The identified variant is highly likely to be the cause of the patient's disease. However, some uncertainty remains, and caution should be exercised when using this information for clinical decision-making.
• Variants of unknown significance (VUS): These variants exhibit characteristics of an independent disease-causing mutation, but there is insufficient or conflicting evidence to establish their significance definitively.
• Likely benign variants: The variant is unlikely to be the cause of the tested disease.
• Benign variants: These variants are not considered to be the cause of the tested disease.

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