Continuing Education Activity
Amniocentesis is the aspiration of amniotic fluid from the amniotic cavity and is the most common invasive fetal testing procedure. It is usually performed for fetal aneuploidy testing. This activity reviews the indications of amniocentesis, highlights the procedural technique, and describes the role of the interprofessional team in performing this procedure.
Objectives:
- Identify the common indications and contraindications of performing amniocentesis.
- Describe the equipment required, the technique used, and the patient preparation needed for performing an amniocentesis.
- Outline and review the possible complications and clinical significance of amniocentesis.
- Review interprofessional team strategies for improving care coordination and communication to advance amniocentesis procedures and to improve clinical outcomes.
Introduction
Prenatal diagnosis enables the diagnosis of a wide spectrum of chromosomal abnormalities, gene disorders, X-linked conditions, neural tube defects and infections to be made before the birth of the fetus. The various invasive prenatal diagnostic tests are amniocentesis, chorionic villus sampling, and fetal blood sampling or cordocentesis.
Amniocentesis
Amniocentesis is an invasive technique. In this technique, amniotic fluid is withdrawn from the uterine cavity using a needle. This procedure is performed transabdominally and is performed under ultrasound guidance by a trained obstetrician. This was first performed for the diagnosis of genetic diseases (sex determination) of the fetus and by Fuchs and Riis in 1956 [1]. It is performed for diagnostic and therapeutic purposes. It is performed for diagnostic evaluation in form of chromosomal, biochemical, histopathological, and microbial assessment. When performed as a therapeutic procedure, this is done to reduce the volume of amniotic fluid in patients with polyhydramnios [2][3][4]. The amniotic fluid obtained consists of fetal exfoliated cells, transudates, urine, and other secretions. It can be performed between 15 - 20 weeks of gestation. Amniocentesis according to the weeks of gestation can be performed in early or later weeks of gestation [4][5]. Early amniocentesis is performed during 11-14 weeks of gestation. Due to the absence of fusion of membranes, there is an increased risk of procedure-related complications than other invasive procedures. There is an increased chance of performing the procedure in the later weeks of gestation as the cell culture failure rate is higher when performed early. There is an increased risk of amnionic fluid leakage, fetal demise, and talipes equinovarus. Counseling of the couple is done regarding the indications, risks, benefits, and limitations of the procedure [6]
Chorionic Villus Sampling
It is a prenatal invasive procedure. It is done under ultrasound guidance. In this procedure, ultrasonography is used to guide the catheter or needle into the chorion frondosum. It is done abdominally and is followed by aspiration of tissue (chorionic villi) for genetic or chromosomal analysis with a syringe containing tissue culture media. It is done in the first trimester for prenatal diagnosis between 10-13 weeks. Depending on the position of the uterus and bladder, gestational age of the patient, and placental localization it can be performed transabdominally or transcervically. Safer and early termination of pregnancy is possible as karyotype results are available within 7-10 days. It is indicated in chromosomal and genetic disorders. The samples collected are sent for DNA analysis. It is not performed in vaginal bleeding, in cases of cervical abnormalities, and severe infections. The major complications involved in this procedure are limb reduction defects, the presence of chromosomal abnormalities present in the extraembryonic tissue which are not found in the fetal tissue, intrauterine infections, membrane rupture, and fetal loss.
Fetal Blood Sampling or Cordocentesis
It is the technique in which under ultrasound guidance fetal blood sampling is performed through the maternal abdomen. It is usually performed after 18 weeks after visualization of cord insertion. As the lumen of the cord at earlier weeks of gestation is narrow hence it is considered safer to perform at 20-28 weeks of gestation. The blood sample obtained is sent to the laboratory for hematological, immunological, and biochemical analysis. The results are obtained within 24-72 hours. There is an increased risk of fetal loss and this risk is comparatively higher when compared with other invasive procedures.
Anatomy and Physiology
Amniotic fluid is a transparent fluid. It is light yellowish in colour. It is present in the amniotic sac. It creates a space for the fetus to grow and survive. It helps in performing fetal movements, which are necessary for the effective musculoskeletal development of the fetus. It helps in fetal swallowing and fetal breathing.
Development
On the 8 day, the trophoblast undergoes changes. Bilaminar germ disc formation occurs after the differentiation of embryoblast. On the dorsal aspect of the bilaminar germ disc, there is the presence of tall columnar cells. They are ectodermal in origin.
On the ventral aspect of the bilaminar germ disc, there is the presence of flattened polyhedral cells. They are endodermal in nature. Connecting stalk forms the umbilical cord later, it connects the bilaminar germ disc with the trophoblast.
There is the appearance of 2 cavities on either side of the germ disc:
- Amniotic cavity
- Yolk sac
Physiology of Amniotic Fluid
Maternal and fetal compartments are essential in the formation of amniotic fluid in the first trimester of pregnancy. Fetal skin is non keratinized in earlier weeks, hence allow free transfer of water and other small molecules and solutes through the amnion and chorion [7]. Amniotic fluid is similar to maternal and fetal extracellular fluid and it functions as a nonsterile aqueous electrolyte solution.
During the second trimester of pregnancy, the process of diffusion ceases as keratinization of fetal skin occurs, which makes the fetal skin impermeable to water and other solutes.
On transvaginal ultrasonography, urine is observed at 9 weeks in fetal bladder [8] and on transabdominal sonography, urine is seen at 11 weeks of gestation [9].During this period, the major component of the amniotic fluid is fetal urine. It is hypotonic (80–140 mOsm/litre) and as the fetal kidneys mature they contain increased concentrations of urea, uric acid and creatinine. At term, a fetus produces 500 to 700 ml of urine per day [10][11][12].
The average amniotic fluid volume at 12 weeks of gestation is 60 ml [9]. By 16 weeks, the mean volume is 175 ml [9][13]. From 20 weeks on, amniotic fluid volume varies. Amniotic fluid volume increases steadily throughout pregnancy to a maximum of 400 – 1200 ml at 34–38 weeks [13][14][15]. Near term, the net increase of amniotic fluid is only 5–10 ml/day in the third trimester. After 38 weeks, fluid volume declines by approximately 125 ml/week, to an average volume of 800 ml at 40 weeks [13][14][15].
Indications
Amniocentesis is a commonly performed procedure for several reasons. It is performed for :
- Diagnostic indications
- Therapeutic indications
Diagnostic Indications
1. It is used for chromosomal analysis: Karyotyping and DNA analysis (to diagnose Sex-linked disorders, inborn errors of metabolism, and neural tube defects).
2. It is done in cases of:
- Advanced maternal age (Age > 35years)
- Abnormal biochemical screening markers (maternal alpha-fetoprotein, human chorionic gonadotropin, unconjugated estriol) in 1st or 2nd trimester
- Ultrasound detection of an abnormality or soft tissue markers (Nuchal translucency, nasal bone hypoplasia, nuchal pad edema, etc.)
- Family or personal history of chromosomal abnormalities in previous pregnancies
- Abnormal parental karyotype
- Parental balanced translocation
3. Assessment of severity of Rh isoimmunisation:
5. To assess bilirubin levels in amniotic fluid and to assess the grade of severity of alloimmunization. Recently non-invasive tests, including the middle cerebral artery doppler has gained more importance over it.
6. Diagnosis of fetal infections [16]TORCH infections such as:
- CMV
- Parvovirus
- Toxoplasma Gondii
7. Fetal lung maturation (L/S ratio)
8. Diagnosis of chorioamnionitis
9. Biochemical analysis for alpha protein level and acetylcholinesterase level estimation.
Therapeutic Indications
1. In hydramnios, has a therapeutic role in relieving maternal discomfort and instillation of intraamniotic drugs.
2. Decompression amniocentesis in twin oligohydramnios-polyhydramnios sequence (TOPS): as it decreases the volume of amniotic fluid in the polyhydramnios sac, amniotic fluid pressures are decreased in both the sacs resulting in increased placental thickness thus improving uteroplacental circulation and improving fetal outcome [17].
3. Amnioinfusion in oligohydramnios to prevent fetal lung hypoplasia, and umbilical cord compression during labor.
4. Fetal blood transfusion in fetuses having severe hemolysis.
Contraindications
1. There are no absolute contraindications for amniocentesis.
2. Relative contraindications for amniocentesis are:
- Hepatitis B and HIV infections can be transmitted from maternal circulation to the fetus during the procedure.
- Decreased amniotic fluid (oligohydramnios) [18]
- Oral anticoagulation therapy must be stopped 48-72 hours before the procedure and patients may be shifted to low molecular weight heparin.
Equipment
The procedure is done under continuous ultrasound guidance. The following equipments are required:
- Ultrasonography machine
- Sterile swabs and drapes
- Syringe 2 ml, 10 ml
- Needle 20 gauge to 22 gauge
- Containers for collection and sample transport
- 5% Povidine iodine solution
Personnel
1. Couples should undergo genetic counselling.
2. Written consent should be taken.
3. Risks, benefits, indications, procedures and complications related to mother and fetus must be explained in detail to the couple. The fetal and maternal risks associated with the procedure should also be discussed in detail. The time required to obtain results, failure to culture cells, and the type of cytogenetic test being performed on the sample obtained should be discussed.
4. In Rh-negative women, anti-D must be administered.
5. Proper documentation of the procedure should be done.
6. Ultrasonography prior to the procedure is done to document the number of fetuses, viability of the fetus, placental location, gestational age, site of cord insertion and any obvious fetal malformation.
7. Both the operator and assistant must scrub with antiseptic and use sterile gloves. The exposed abdominal surface area must be cleaned with povidine iodine with sterile gauze and antiseptic solutions.
8. The ultrasound probe must be covered with a sterile plastic cover. The gel must be kept at the inner surface, as it helps in better transmission of ultrasound waves. Ideally, sterile gel should be used, to decrease the risk and spread of contamination.
Preparation
1. Aseptic precautions must be established at the beginning of the procedure by preparing the skin and ultrasound probe.
2. A local anaesthetic is not required.
3. Prophylactic antibiotics are not required.
Technique
After proper genetic counselling and informed written consent, a detailed ultrasound is performed in order to assess the gestational age, placental localization, gross congenital anomalies, maximum vertical pocket (MVP), fetal position, fetal movements and amniotic fluid volume. Skin is prepared with povidine iodine and sterile ultrasonography gel is applied. After confirming the prerequisites and once the preparation is complete, amniocentesis is performed using the aseptic technique. The technique is performed under ultrasound guidance. A 20 gauge to 22 gauge spinal needle is used to enter the amniotic cavity under continuous ultrasound guidance. The needle is directed under proper visualization in the clear region of amniotic fluid. It must be ensured that fetal parts, umbilical cord or placenta are not present in the region of needle insertion. Transplacental puncture is usually not performed in cases of alloimmunization or infections to the mother like human immunodeficiency virus (HIV), hepatitis B virus (HBV) or hepatitis C virus (HCV) [19][20]. A firm entry into the amniotic cavity is recommended to prevent the tenting of the amniotic membrane[21] . Once entry into the cavity is confirmed, amniotic fluid is slowly aspirated. The initial 1 ml to 2 ml of amniotic fluid is discarded because it has the highest chance of maternal cell contamination[21]. Approximately 18 ml to 20 ml of amniotic fluid is required for karyotype testing, and 2 ml to 5 ml is required to test for enzyme deficiency testing. The needle is removed after adequate amniotic fluid has been obtained.
Complications
Both maternal and fetal complications can occur with amniocentesis.
Maternal Complications
1. An estimated 2.6% risk of fetomaternal hemorrhage.
2. Maternal isoimmunization in Rh-negative cases.
3. There is minimal chance of the introduction of skin bacteria into the amniotic cavity. The risk of chorioamnionitis and uterine infections is less than 0.1%.
4. The procedure increases the risk of preterm, preterm premature rupture of membrane, and oligohydramnios.
5. There is a 2% to 3% risk of vaginal bleeding.
6. Post-procedure pain and maternal discomfort: Mean pain intensity described is 1.6+/-1.3 when noted on a scale of 0-7 [22].
7. Amniotic fluid embolism.
8. Hematoma over maternal skin, intestinal or internal organ injures.
Fetal Complications
1. The fetal loss rate associated with amniocentesis on average is 0.11%. The loss is 0.56% within 28 days, 0.09% within 42 days [21].
2. Amniotic fluid leak: 1% to 2%, and usually associated with spontaneous sealing of membranes [21]. It may also result in:
- Fetal lung hypoplasia
- Respiratory distress
3. Fetal injuries like bleeding from cord, ocular injuries, postural deformities like talipes equinovarus (clubfoot) might occur.
The risk of complications is high when more than or equal to 3 pricks are used to obtain amniotic fluid. In ideal conditions, if an adequate fluid sample is not obtained in 2 pricks, the procedure should be abandoned for 24 hours, where after it can be re-attempted. In experienced hands, people performing more than 300 procedures/year; the risk is less. The risk of fetal loss is higher in women who are otherwise at a higher risk of miscarriage, such as women carrying fetuses with structural malformations, fibroids, retroplacental hematoma, obese women, women with vaginal infection at the time of the procedure. Amniocentesis in up to 86.0% of the patients was safe and free from any complications [23].
Clinical Significance
After the amniocentesis procedure, the sample of amniotic fluid will be taken to a laboratory for testing. Once the amniotic fluid is obtained, it is sent for a conventional cell culture report, which is obtained in 14 days. There are rapid chromosomal preparations available that give results in 1 to 2 days, including fluorescent in-situ hybridization (FISH) and quantitative fluorescence polymerase chain reaction (QF-PCR). The procedure is relatively safe, with fewer complications amongst experienced hands. The location of the placenta is an important factor for amniocentesis. While performing the procedure, one should try to avoid penetration of the placenta. The anterior and fundal placenta is associated with a higher number of complications, including multiple pricks, blood-stained liquor; however, it is not associated with an increase in the number of fetal loss rates [24]. Passing the needle through the placenta is slightly associated with an increase in rates of preterm birth [25].
There are two different types of tests:
1. A rapid test
2. A full karyotype
1. Rapid test: A rapid test looks for abnormalities on specific chromosomes. A rapid test can identify a number of chromosomal conditions that cause physical and mental abnormalities. This test is almost 100% accurate. These are:
- Down's syndrome: caused by extra chromosome 21
- Edward's syndrome: caused by extra chromosome 18
- Patau's syndrome: caused by an extra chromosome 13
2. Full karyotype:
A. The cells in the sample of amniotic fluid are grown for upto 10 days in a laboratory before being examined under a microscope to check for:
- Number of chromosomes
- Appearance of chromosomes
B. Results from a full karyotype will usually be available in two or three weeks. After amniocentesis, the sample of amniotic fluid is sent to a lab for analysis. Results usually take 10 days to 3 weeks depending upon the laboratory. In the laboratory, genetic and chemical tests are done. For genetic tests, certain chromosomes and genes are analyzed.
For chemical tests, proteins, minerals, and other compounds in the amniotic fluid are analyzed. Amniocentesis results will either be positive or negative.
Negative test: It means fetus doesn't have the abnormality for which they have been tested for.
Positive test: It means the baby has the disorder for which baby was tested for.
For the majority of the chromosomal conditions there is no cure, so regarding the continuation of pregnancy couple needs to be counseled properly.
Enhancing Healthcare Team Outcomes
The decision to perform amniocentesis and convey the results to the couple requires communication between geneticists and fetal medicine experts. The patient has to be counselled by geneticists for them to know the true possibility of the fetus being affected by some genetic disease. Following the counseling session, the patient is referred for the procedure. While performing the procedure, coordination amongst the team is required. Once the needle is inside the amniotic cavity, the assistant should carefully withdraw the amniotic fluid so as the procedure is performed in the minimal time possible and with minimal risk of needle displacement.