Genetic Disorders and the Fetus. Группа авторов
3.6 Examples of toxicants reported in amniotic fluid
| Toxicant | References |
|---|---|
| Pesticides | 473, 490 |
| Dioxin | 513 |
| Organophosphates | 489 |
| Polychlorinated biphenyls | 513 |
| Herbicides | 473 |
| Chlorinated phenolic compounds | 473 |
| Perchlorate | 489 |
| Phthalates | 496 |
| Bisphenol A | 486, 487 |
| Phytoestrogens | 494 |
Not only have all the toxicants shown in Table 3.5 been found in AF, but organophosphates have been reported in postpartum meconium.518 Fetal exposure to certain phthalates may result in adult infertility, as shown in rats.519 We all have grave concerns about our toxic environment, but which government will act to safeguard fetal futures?
Amniotic fluid cell culture
Amniotic fluid cell (AFC) culture is a routine procedure in cytogenetics laboratories that has evolved considerably since its first application in the 1970s. What has changed over the years are improved speed to the final report, success rate of cell culture, and quality of the chromosome banding. Due to optimized cell culture media, the use of growth factor supplements, high‐quality plastic ware, sophisticated incubators, and robotic cell harvest equipment, culture failures occur in less than 0.2 percent of all specimens processed by experienced laboratories.
Alternatives to cell culture and metaphase karyotype analysis
The prediction that AFC culture would largely be replaced by CVS (see Chapter 9) analysis has not come true. Coelocentesis at 6–10 gestational weeks was proposed as an alternative to both CVS and amniocentesis but did not achieve wide acceptance.520 The use of early amniocentesis (9–14 gestational weeks) has been limited by increased fetal risk (see Chapter 9), failure to obtain fluid and operator inexperience rather than by failure to grow cells from such early specimens.521, 522 Although never widely adopted, rapid and quantitative PCR methods have been employed to identify the common autosomal and sex chromosome aneuploid states.523, 524 Interphase fluorescent in situ hybridization (FISH) technology was also predicted to replace cell culture and metaphase karyotype analysis. This prediction has not been realized except for occasional cases of malformations identified by ultrasound examination and then confirmed as aneuploid using FISH methods.525, 526 Most of these cases are still confirmed by cell culture and karyotype analysis, albeit after pregnancy termination. Interphase FISH analysis can identify 90–95 percent of the clinically relevant autosomal and sex chromosomal aneuploidy, although up to 25–30 percent of all cytogenetic changes (e.g. mosaics and balanced translocations) are detected only by G‐banded metaphase analysis.527–534 Prenatal chromosomal microarray is gaining rapid acceptance as an alternative to conventional chromosome analysis, and is now recommended as the first‐tier test when ultrasound examination identifies malformations.535–537
In cases of bladder outlet obstruction, it is possible to obtain fluid and cells for culture from the fetal bladder. In one study, karyotype analysis was successful in 95 percent of 75 samples, with six chromosome abnormalities identified.538 AF may not be accessible in cases of severe cystic hygroma, pleural effusion, renal agenesis, or bladder outlet obstruction. Cases presenting with severe cystic hygroma, ascites, or pleural effusion are at significant risk of having a chromosome abnormality, especially trisomy 21 or monosomy X.539 Fluid drawn from any of these sources will contain cells that can be cultured like amniocytes and usually also include lymphocytes that can be cultured with phytohemagglutinin (PHA).540–542
The increasing acceptance and sophistication of maternal serum screening tests and high‐resolution prenatal ultrasonography identified a greater number of at‐risk pregnancies, especially in women under age 35. Before the year 2000, this resulted in an increase in the number of amniocentesis procedures. However, since the early 2000s, the number of amniocenteses for karyotype studies has been falling. At first this was because women over age 35 used screening‐based risk figures to decide against prenatal diagnosis if their risks were deemed to be reduced based on triple, quadruple, or more complex screening tests that combine information from the first‐ and second‐trimester serum assays and ultrasound examinations (see Chapter 6). With each additional chemical component of maternal serum screening came a higher rate of detection and a lower false‐positive rate – hence fewer women were “screen positive” and many elected to forego amniocentesis.543, 544 As fetal DNA isolated and sequenced from maternal serum gains increased acceptance,545 the number of prenatal karyotype studies will continue to decline. False‐positive and false‐negative results will likely keep these methods as screening rather than diagnostic tests.546, 547
Amniotic fluid cell types
Cellular contents of native fluids
Few nucleated cells in second‐trimester amniocentesis fluids are capable of in vitro attachment and growth, even though many exclude trypan blue. These are cells with pale cytoplasm and small, densely staining nuclei.548, 549 Exfoliation of such cells from the fetal epidermis has been directly observed.550 It is not known why their number in a given fluid is so highly variable and whether this reflects the wellbeing of the fetus, for which other properties of the AF may be more predictive.551 Since the change of the fetal skin from a simple two‐layered structure to mature stratified epithelium takes place around the 16th week and occurs at different rates in different body zones, minor differences in gestational age might account for comparatively large differences in overall cornification and desquamation.552 Classic cytology and transmission or scanning electron microscopy have attempted a subdivision of cells in midtrimester fluids.550, 553–555
A variable number of cells attach to the culture substrate within 6–72 hours after incubation but the number of colony‐forming cells rarely exceeds 10 cells/mL fluid.556, 557 Cells that attach in less than 24 hours (rapidly adhering or RA cells), if present in clear fluids in large quantities, may indicate an NTD.551 Such cells often take on the characteristic elongated spindle‐like appearance of neural crest cells in monolayer culture. In AFC cultures from NTD pregnancies, the RA cells include monocytic cells that have phagocytic activity and cells of glial origin that lack phagocytic activity and stain positive for synaptophysin and neuron‐specific enolase.558, 559 Rapidly adhering, phagocytic, esterase‐ and Fc receptor‐positive cells are also found in AF from normal fetuses, albeit in more moderate quantities.560
In cases of abdominal wall defects, macrophage‐like cells and even lymphocyte‐like cells responding to PHA have been described.561 AF from distressed fetuses may likewise