This literature review identified 16 original studies investigating cff nucleic acids in AF (Table ). This small number is in stark contrast to the numerous primary studies examining cff nucleic acids in maternal plasma, which have been the subject of several recent reviews (Maron and Bianchi, 2007
; Lo, 2009
; Wright and Burton, 2009
The reasons for this disparity are several. The most obvious is the risk of pregnancy loss associated with amniocentesis, which makes the collection of AF for only research indications unfeasible from an ethical viewpoint. Studies are therefore limited to using excess (discarded) fluid remaining from clinically indicated samples. This greatly reduces the numbers and gestational age range of the available samples for study.
The second major reason is that researchers have been justifiably focused on the non-invasive clinical applications of cff DNA for the diagnosis of fetal gender, rhesus genotyping and some single-gene disorders. These have transitioned into clinical care, resulting in reduced invasive testing rates of up to 45% in some populations (Chitty et al., 2007
). Eliminating the need for chorionic villus sampling or amniocentesis to accurately diagnose fetal aneuploidy—still the most common indication for an invasive procedure—would revolutionize the prenatal diagnostic sector. There is no doubt that pregnant women would embrace such an advance.
A third reason for the lack of attention paid to the cff nucleic acids in AF is the perceived lack of clinical utility of examining the cell-free portion of the sample when an invasive procedure is indicated. In the majority of cases, all of the clinically relevant diagnostic information can be obtained from the cellular portion of the AF using conventional techniques on cultured amniocytes. Occasionally, other analytes may be measured in the AF, but essentially the supernatant is usually discarded.
There are several conclusions that can be drawn from the results of the studies identified in this literature review. The first is that it is feasible to isolate AF cff nucleic acids from small volumes of AF in sufficient quality for further downstream applications such as microarray analysis. cff DNA in AF is present in a much higher concentration than in maternal plasma and is able to be extracted using commercial kits from fresh or stored clinical specimens (Bianchi et al., 2001
; Lapaire et al., 2008
). Successive technical refinements over the past decade have also made it possible to extract RNA of sufficient quality and quantity to perform functional genomic analysis (Larrabee et al., 2005a
; Slonim et al., 2009
Second, there is an overall paucity of information relating to the biology of cff nucleic acids in AF. Information regarding differences in fragmentation patterns according to karyotype, gestational age and sample storage time from two studies support the concept that fetal conditions have an impact on the size of cff DNA fragments in the AF, but other than this very little has been published regarding their tissue sources or kinetics (Lapaire et al., 2007a
; Peter et al., 2008
). What can be concluded from the studies in this review is that the fetus itself is the predominant source of the nucleic acids in the AF, with very little direct mRNA contribution from the placenta, as demonstrated by the microarray (Larrabee et al., 2005a
), and epigenetic studies (Lun et al., 2007
). Other studies confirm that the pool of cff nucleic acids in the AF is independent from the pool of placenta-derived plasma cff DNA and RNA (Ng et al., 2003
; Bischoff et al., 2005
; Tjoa et al., 2006
). Although it is possible to identify mRNA transcripts present in umbilical cord blood in the maternal circulation (Maron et al., 2007
), several studies have established that this trafficking is unidirectional from fetus to mother, making the prospect of contaminating maternal nucleic acids in the amniotic cavity extremely unlikely (Ng et al., 2003
; Sekizawa et al., 2003
; Maron et al., 2007
This review also highlights increasing total quantities of cff DNA and differences in mRNA expression as a function of gestational age (Lapaire et al., 2007a
). This suggests some similarities with plasma and serum biology. However, specific data on the kinetics, half-life and function of cff nucleic acids in AF are lacking. The issue of kinetics is relevant to AF mRNA, as transcription is a dynamic process that varies rapidly according to the physiological demands on the body, including diurnal rhythms. cff mRNA has a short half-life in maternal plasma of ~15 min (Chiu et al., 2006
), but mRNAs also decay at different rates according to their functional characteristics and sequence attributes (Yang et al., 2003
). How the results from other biological models relate to the steady-state population of cff nucleic acids in AF is unknown. This has significance for the interpretation of AF gene expression studies, as to whether they represent a real-time or a delayed snapshot of global fetal transcriptional activity.
Similarly, the relative contributions of various fetal tissues to the cff NA in AF has not been the subject of any research to date. Data from the proteomic studies of AF discussed in the introduction suggest that numerous fetal organs contribute to the protein content of AF, including those systems not in direct physical continuity with amniotic cavity, such as the fetal heart, brain and skeletal muscle. It seems reasonable to assume from these data that the tissue sources of cell-free nucleic acids in AF are similarly multiple, although whether they are in similar proportions to protein expression is uncertain, particularly given that the large number of placental proteins identified in proteomic studies is inconsistent with the low levels of placental mRNA transcripts found in AF. Furthermore, in the proteomic study of Cho et al.
, all keratin-related entries were removed as contaminants, so that fetal skin was not among the top 10 tissues with the most number of proteins in AF as one might expect (Cho et al., 2007
Related to both these unanswered questions is the unknown nature of its particle association. The filtration study by Larrabee et al.
) confirmed that filterable and non-filterable forms of cff mRNA exist in AF, supporting the existence of particle-associated forms like those in maternal plasma. However, no studies investigating the types of microparticles associated with NA in AF were identified by this review.
Amniotic fluid supernatant is a valuable biological sample
The final important conclusion from this review is that AF supernatant is a potentially valuable but under-utilized biological sample. Two studies have successfully performed gene expression analyses on cff mRNA in AF using a ‘discovery-driven’, rather than hypothesis-driven, approach (Larrabee et al., 2005a
; Slonim et al., 2009
). These reports establish that it is possible to identify differentially regulated genes in normal and abnormal fetuses, and employ bioinformatics tools to uncover the pathways and networks involved in the pathogenesis of specific diseases.
In the study comparing fetuses affected by polyhydramnios with normal fetuses, Larrabee et al.
) identified a water transporter gene transcript, aquaporin-1, that is statistically significantly increased by up to 18-fold in recipient twins affected by TTTS. Similarly, Slonim et al.
) provided new insights into the pathogenesis of Down syndrome by identifying a role for oxidative stress, ion transport and immune and stress responses in the abnormal development of the trisomy 21 fetus.
Implications for future research
Functional genomics in obstetrics and fetal medicine
Gene expression studies on live human fetuses have been restricted by access to appropriate tissue such as fetal blood. Post-partum gene expression studies using stillborn, neonatal or placental samples are inherently limited in their ability to reflect prenatal events. Until recently, the main applications of RNA-based functional genomics in obstetrics have been in the study of normal parturition (Aguan et al., 2000
; Havelock et al., 2005
; Hassan et al., 2009
; Montenegro et al., 2009
) and spontaneous preterm birth (Romero et al., 2006
; Enquobahrie et al., 2009
), using myometrial, cervical or amniotic membrane specimens. More recently, researchers have applied a global gene expression approach to pre-eclampsia examining various tissues such as maternal blood (Donker et al., 2005
; Sun et al., 2009
), post-partum placentas (Zhou et al., 2006
; Centlow et al., 2008
; Enquobahrie et al., 2008
; Toft et al., 2008
; Sitras et al., 2009
; Zhu et al., 2009
), umbilical vein epithelial cells (Hoegh et al., 2006b
), endometrial epithelial cells (Hoegh et al., 2006a
) and first-trimester chorionic villus samples (Founds et al., 2009
). Furthermore, mRNA-based microarray analysis has also recently been used to describe the ‘transcriptome of fetal inflammation’ derived from umbilical cord blood (Madsen-Bouterse et al., 2010
Despite this rapidly growing interest in utilizing functional genomics in obstetrics, very few global gene expression studies have been performed on live ongoing pregnancies for specific abnormalities of fetal development. First-trimester chorionic villus samples have been investigated with microarray analysis to study the mechanisms involved with enlarged nuchal translucencies in euploid fetuses (Farina et al., 2006
) and for the prediction of pre-eclampsia (Founds et al., 2009
), but these represent placental rather than fetal transcription profiles. An alternative approach in which global gene expression is compared between antenatal, post-natal and neonatal samples to identify significantly up-regulated fetal transcripts in maternal blood has been successfully employed to produce new insights into fetal development in normal term babies (Maron et al., 2007
) and to develop potential fetal biomarkers for congenital heart disease (Arcelli et al., 2010
). However, beyond the two studies identified in this review (Larrabee et al., 2005a
; Slonim et al., 2009
), no other reports specifically used AF cff mRNA to advance knowledge regarding fetal development.
AF microparticles and cff mRNA
Another potential direction for future research would be to integrate developments in the biology of microparticles in plasma with those in cff nucleic acids in AF to determine whether cff mRNA has a functional role as a novel form of cell-to-cell genetic signalling within the amniotic cavity. Until recently, cff nucleic acids have been conceived of as by-products of cell turnover and it is unknown if they possess any biological activity in the amniotic cavity.
Exosomes are small vesicles <100 nm diameter of endosomal origin that are released by a wide variety of cells into the extracellular space (Simpson et al., 2009
). They contain proteins and RNA derived from the parent cell and are thus able to be characterized according to cell-specific markers. They are relevant to cell-free nucleic acid biology because they contain mRNA and miRNA sequences that are transferrable to other cells, thus leading to the translation of unique proteins in in vitro
and in vivo
models (Valadi et al., 2007
). This suggests that cell-free RNA can play an active role in cell-to-cell genetic exchange, as so-called exosomal shuttle RNA.
Exosomes are secreted by many different cell types and are present in adult and neonatal urine (Pisitkun et al., 2004
; Keller et al., 2007
). Using the CD24 membrane protein as a marker of renal cell origin, Keller et al.
) showed that AF from second-trimester pregnancies contains exosomes that are secreted by the fetal kidney. Their function in the AF is not known, but it is interesting to speculate on whether they have role in fetal and AF homeostasis, particularly as they were shown to contain aquaporin-2. If functional mRNA could be demonstrated in kidney-derived AF exosomes, then this may provide a novel mechanism for the fetal kidneys to directly influence the intra-membranous route of fluid transport in amniotic membranes.
This proposed method of fetal-maternal communication via AF exosomes could extend to other systems than fluid balance. In the only other report on exosomes in AF, Asea et al.
) suggest a possible role for exosomes in immune regulation. They detected the presence of heat shock protein-containing exosomes in second-trimester AF specimens. Heat shock protein is known to play a role in microbial-induced and sterile forms of inflammation (Quintana and Cohen, 2005
). AF contains decidual natural killer cells. AF exosomes may therefore be a mechanism by which the fetus and the maternal immune system communicate within the uterine cavity.
Neither of these two AF exosome studies has specifically sought to confirm the presence of fetal mRNA in these microparticles. Although the body of literature on exosomes in other body fluids supports the hypothesis that AF exosomes should also contain functional mRNA, it would be an important research priority to confirm this. Delineating the functional significance of these exosomal forms of cff nucleic acids in AF would then open up an entirely new field of research, one in which various fetal organ systems could communicate on a genetic basis with each other, the fetal membranes, placenta and mother.