1.    Introduction

Amniotic fluid surrounds the fetus during intrauterine development. This fluid is in a dynamic state throughout pregnancy and is essential to fetal well-being. This article will review the physiology of amniotic fluid exchange regulation, content, clinical significance, and abnormalities in both volume and content.

2.    Physiology of Amniotic fluid volume regulation (amniotic fluid dynamics)

2.1. Amniotic fluid dynamics in the first trimester of pregnancy

During the first trimester, the amniotic fluid composition is similar to that of fetal plasma. There is bi-directional diffusion between the fetus and the amniotic fluid across the skin that is not keratinized yet, and the surface of the amnion, placenta, and umbilical cord being freely permeable to water and solutes. The amniotic fluid serves as a physiologic buffer and an extension of the fetal extracellular compartment [1]. Neither fetal urination nor swallowing contributes significantly to the amniotic fluid volume until 14 weeks of pregnancy.

2.2. Amniotic fluid dynamics in the second and third trimester of pregnancy

Keratinization of fetal skin begins at 19 to 20 weeks of gestation and is usually complete at 25 weeks after conception. Numerous factors contribute to the formation and removal of amniotic fluid, following keratinization of the fetal skin [1,2]. Production of amniotic fluid is predominately accomplished by the fetal urine and lung fluid production. Fetal breathing movements contribute to the efflux of the lung secretion into the amniotic fluid. Other contributions consist of oral, nasal, and tracheal secretions [1,2,3,4]. Removal of the amniotic fluid is predominately accomplished by fetal swallowing. Additionally an intra-membranous pathway transfers fluid and solutes from the amniotic cavity to the fetal circulation across the amniotic membrane (across the network of blood vessels on the fetal surface of the placenta). Trans-membranous pathway involves direct exchange across fetal membranes between the fetus and maternal blood within the uterus and affects amniotic volume only minimally [2,5,6].

Amniotic fluid volume is dependent on gestational age, maintained within a fixed range, and appears to be highly regulated (although the precise regulation mechanism remains elusive). The volume peaks between the 36 and 38 weeks of gestation [2,7].

Amniotic fluid volume homeostasis is maintained by the delicate balancebetween inflow (fetal urine and to a lesser extend, lung secretion) and outflow(swallowing and intramembranous absorption) of fluid in theamniotic cavity [3,5,8,9]. Several studies demonstrate that the amount of fluid removed by fetal swallowing is significantly smaller than that produced by fetal urination [2,5,6]. Despite these considerably unequal parameters, amniotic fluid volume remains in relative equilibrium [3].Several studies suggest that neither lung fluid production nor urination serves as a regulatory role in the control of amniotic fluid volume. Whether fetal swallowing serves a regulatory role remains possible but inconclusive[10,11]. Studies suggest that the intramembranous pathway is responsible for the correction of imbalances and that it appears to play a significant role in establishing of the amniotic fluid volume [2,12].

Intramembranous absorption is known to be a significant pathwayfor fluid movement from the amniotic cavity to the fetal circulation across the amniotic membrane (across the network of blood vessels on the fetal surface of the placenta). Increase of the intramembranousabsorption would serve to limit the increase of the amniotic fluidvolume¹².Some studies suggest that the amnion is the structure limiting intramembranous water flow. Passive diffusion accounts for only part of the intramembranous fluid absorption. It is likely that much larger shifts of fluid and solutes occur by bulk transfer of amniotic fluid with all of its dissolved solutes into the fetal circulation perhaps via a trans-cellular vesicular transport mechanism [1,13]. Vascular endothelial growth factor (VEGF) in the ovine fetal membranes appears to be a mediator of this process. Other studies suggest that up regulation of VEGF gene expression in the amnion and chorion is associated with increased transfer of amniotic fluid into fetal blood. Vascular endothelial growth factor appears to involve regulation of the intramembranous blood vessel proliferation, influences the permeability of the microvessels and regulation of membrane transport via passive permeation as well as non-passive transcytotic vesicular movement of the fluid [14].

The demonstration of aquaporin proteins in fetal membranes suggests the possibility of water channels as another potential regulator of water flux across both the amnion and the placenta [4,15]. However, further studies are needed to identify the exact regulatory factors of amniotic fluid volume and the underlying mechanisms, which may allow better understanding and management of amniotic fluid abnormalities [4,9].

3.    Role of Amniotic fluid

The amniotic fluid provides a number of important functions to the developing fetus. It contains nutrients and growth factors that facilitate fetal growth. It provides mechanical cushioning and antimicrobial effectors that protect the fetus.

Normal fluid is also important in the development of the gastrointestinal, pulmonary and musculoskeletal system [1], and a new source for stem cells [16].

3.1. The nutritive role of the amniotic fluid

Amniotic fluid contains carbohydrates, proteins and peptides, lipids, lactate, pyruvate, electrolytes, enzymes, and hormones [17,18].

Trophic effects of amniotic fluid have been demonstrated on cultured human fetal small intestinal cells [19]. This study suggests that growth factors found in the amniotic fluid, comparable to those in human milk, play a role in fetal growth and development. These trophic mediators include:

• Epidermal growth factor (EGF), which increases significantly during the second trimester, but is reduced in fetal growth restriction. The function of EGF in the human fetus is still largely unknown. In monkeys, in utero treatment with EGF improves lung maturity [20].

• Transforming growth factor beta-1 (TGF-b1) is found in amniotic fluid only during late stages of gestation. TGF-b1 is believed to induce terminal differentiation of intestinal epithelial cells and to accelerate the rate of healing of intestinal wounds by stimulating cell migration. TGF-b1 may also stimulate IgA production [19].

• Insulin-like growth factor I (IGF-I), and IGF-II receptors, as well as, insulin receptors, are found throughout the neonatal gut. Several studies in animals have shown that IGF-I in AF improves somatic growth, spleen weight, and bowel wall thickness [21].

• Granulocyte colony-stimulating factor (G-CSF) is found in amniotic fluid. Enteral administration of the G-CSF enhances intestinal growth of suckling mice [1].

• Erythropoietin is found in amniotic fluid, colostrum, and mature milk. In the neonatal rat, enteral erythropoietin is absorbed, stimulates erythropoiesis, and is a trophic factor for intestinal growth. The role of swallowed erythropoietin in the human fetus and neonate is not clear [1,22]. Its concentrations in human amniotic fluid have been correlated directly with increased umbilical cord blood erythropoietin concentrations, and so elevated amniotic fluid erythropoietin has been suggested as a marker for chronic fetal hypoxia[23,24].

Amniotic fluid plays an important protective role by providing a supportive cushion allowing fetal movement and growth. Amniotic fluid also has a significant defensive role as a part of the innate immune system.

3.1.1.   A part of innate immune system

The innate immune system is the first line of defense against pathogens and includes anatomic and physiologic barriers, enzymes and antimicrobial peptides, as well as phagocytosis and release of proinflammatory mediators by neutrophils and macrophages [1]. Many of the substances that comprise the innate immune system have been identified in amniotic fluid and vernix and have been shown to have significant antimicrobial properties; these include the human beta defensin that are a major family of vertebrate natural antimicrobials. Human beta defensins1-4 are expressed widely at mucosal surface [25,26]. Sarah J et al have shown in their study that in addition to the antimicrobial activity of human beta defensin, they have chemoattractant properties that suggest they interact between the innate and adaptive immune system. It is also considered an important chemokine that is involved in parturition [25]. Other antimicrobial agents include alpha defensin [HNP1-3] whose concentrations in the amniotic fluid increase with preterm labor, premature rupture of membranes, and chorioamnionitis probably due to release from neutrophils and, lactoferrin, lysozyme, bactericidal/permeability-increasing protein, calprotectin, secretory leukocyte protease inhibitor, psoriasin, and a cathelicidin [1,27,28]. These potent antimicrobial agents show broad-spectrum activity against bacteria, fungi, protozoa, and viruses [25,28]. However more work is required to demonstrate anti-infective properties of these antimicrobial peptides. A better understanding of such mechanisms may identify specific bioactive peptides as adjuncts to correct therapies for chorioamnionitis and neonatal infections. Such treatment modalities would target improved immunocompetency in the early gestational fetus or premature infant and potentially leads to better medical outcom [28].

The activity of the ‘‘cellular’’ innate immune system within amniotic fluid as a protective mechanism for the fetus is less well defined. The numbers of mononuclear phagocytes (i.e. monocytes, macrophages, histiocytes) in amniotic fluid are limited in normal pregnancies, while their numbers are increased in fetuses with neural tube defects. Whether these macrophages are present to prevent infection because of a disruption of the fetal skin or as scavenger cells to clean up neural debris is uncertain. Neutrophils are not normally identified in the amniotic fluid of healthy fetuses, but are useful as a marker of amniotic fluid infection [1]. However, further studies are needed for better understanding of the amniotic fluid protective role.

3.1.2.   Effect on fetal lung development

Fetal lung development is affected by numerous intrauterine factors including amniotic fluid adequacy, available thoracic spaces and neuromuscular functions [29]. Fetal lung growth, in part, appears to be stimulated by the distending force of lung fluid in the airways and to be inhibited by the absence of this fluid as occurs in oligohydramnios [29]. Lung development is regulated also by several transcription factors, such as thyroid transcription factor 1 family, hepatocyte nuclear family, and peptide growth factors. Growth factors are present in the amniotic fluid and they send signals, which are integrated with environmental influences, such as fluid volume and hyperoxia, to cause cellular proliferation and differentiation [30]. Fetal urine is an important component of amniotic fluid during late gestation and contributes to lung growth. During fetal development, the kidney is an example of major source of proline. Proline aids in the formation of collagen and mesenchyme in the lung, thus explaining the severe pulmonary hypoplasia in renal agenesis and dysplasias [30].

3.1.3.   Effect on fetal musculoskeletal system

As for fetal musculoskeletal system, amniotic fluid plays an important role in its development [31]. Frost proposed in his study that one of the primary factors in the development of bone strength is the load (force) placed on the bone. This load causes a strain on the bone, which is transmitted as an input signal to a sensor within the bone, Frost called the mechanostat. This mechanostat then directs an appropriate output to the effector cells, osteoblasts and osteoclasts [32]. Bone loading strongly influences bone modeling and during fetal life is determined primarily by fetal movement. Bone loading is far greater in the fetus in the intrauterine environment than in a newborn infant in the extrauterine environment because of the ability of the fetus to kick against the uterine wall in the buoyancy of the amniotic fluid. Fetal activity also promotes muscle growth, which contributes to bone loading [31,32]. Diminshed fetal movement and intrauterine confinement have been put forth as the underlying basis of temporary brittle bone disease [31,33],34].

3.1.4.   Effect on fetal gastrointestinal tract

Numerous studies have shown that the maturation of the fetal gastrointestinal tract is partially enhanced by swallowed amniotic fluid [35,36]. The micelles in the swallowed amniotic fluid might act as a promoter of fetal intestinal maturation [96].

Mulvihill, et al, have shown in their study that the esophageal ligation of fetal rabbit pups results in marked reductions in gastric and intestinal tissue weight and gastric acidity and these reductions were reversed by fetal intragastric infusion of amniotic fluid [37,38]. In similar studies on ovine fetuses, it has been noticed that the esophageal ligation induces some decrease of small intestine villous height as well as reduction in liver, pancreas, and intestinal weights [39,40]. Although ingestion of amniotic fluid nutrients may be necessary for optimal fetal growth, trophic growth factors as epidermal growth factor within the amniotic fluid also importantly contribute [41]. Other examples of trophic mediators in the amniotic fluid and their effect on the gastrointestinal tract development have been discussed above.

3.2. A Source of stem cells

Amniotic fluid contains multiple cell types derived from the developing fetus, including some that can give rise to differentiated adipose, muscle, bone, and neuronal cell lines [42]. De Coppi P et al, have identified in their study lines of broadly multipotent amniotic fluid-derived stem (AFS) cells that have the ability to differentiate into a wide range of lineages including those in all embryonic germ layer, thereby meeting the criterion for pluripotent stem cells [43]. Amniotic fluid stem cells have physical characteristics of both embryonic and adult stem cells, which suggest that amniotic fluid stem cells may exist at an intermediate stage between two stem cell types [42,44]. Stem cells isolated from amniotic fluid have several advantages over embryonic and adult stem cells: they are readily accessible, they replicate rapidly in culture (typically doubling every 36 hours), they do not require the support of other “feeder” cells that can cause contamination, and they do not form tumors in vivo [43].

Amniotic fluid stem cells can serve as precursors to a broad range of differentiated cell types that potentially have therapeutic applications [43]. Recently, it has been suggested that amniotic fluid stem cells might be capable of repairing damaged tissues resulting from conditions such as spinal cord injuries, cartilage damage, diabetes, Alzheimer disease, and stroke [43,44,45]. The results of study by Tsai et al, suggest that besides being an easily accessible and expandable source of fetal stem cells, amniotic fluid will provide a promising source of neural progenitor cells that may be used in future cellular therapies for neurodegenerative diseases and nervous system injuries [46,47].

De Coppi J et al, stated that, banking of cells that would otherwise be discarded could provide a convenient source not only for autologous treatment later in life, but for matching of histocompatible donor cells with prospective recipients [43].

3.3. Diagnostic amniocentesis and amniotic fluid uses

Amniotic fluid contains amniocytes in addition to fetal cells from the skin, genitourinary system, and gut, along with biochemical products that may be removed for analysis [48]. Amniocentesis is the most widely performed invasive prenatal diagnostic procedure, most commonly used for diagnosing genetic and chromosomal abnormalities prenatally [49].

Most amniocenteses are performed to obtain amniotic fluid for karyotyping and the majority is undertaken from 15 completed weeks onwards [50]. Indications for fetal karyotyping include an abnormal screening test result as for trisomy 21, advanced maternal age, a sonographically detected structural abnormality, previous aneuploidy, and a known chromosomal translocation in either partner [48]. Amniocentesis to diagnose inborn errors of metabolism and cystic fibrosis by measuring the activity of fetal enzymes and their byproducts has been largely replaced by molecular DNA analysis [48]. Likewise, amniocentesis to measure alpha feto protein and acetylcholinestrase to diagnose a neural tube defect is rarely necessary because of the reliability of ultrasonography [48, 51]. Evaluation of amniotic fluid bilirubin level based on optical density has been used to predict the severity of fetal hemolysis in alloimmunized pregnancies. Currently, the combination of amniocentesis to assess optical density, Doppler flow studies of the intra-hepatic umbilical vein and the middle cerebral artery and fetal blood sampling by cordocentesis are recommended to closely monitor the anemic fetus [52,53,54]. Oepkes D et al, have shown in their study that middle cerebral artery Doppler peak velocity measurement shows better test characteristics in the prediction of fetal anemia than the traditional amniotic fluid spectrophotometry in Rh alloimmunized pregnancies [55]. Allele-specific polymerase chain reaction of amniotic fluid fetal cells can also be used to identify fetuses at risk for hemolytic disease of the newborn due to minor blood group incompatibilities [56,57].

The rate of miscarriage associated with amniocentesis is approximately 1%. More recent large uncontrolled series suggest that procedure-related loss rates around 0.5% can be achieved [58,59]. Fetal loss due to amniocentesis seems to occur within the first 2 to 3 weeks following the procedure [60,61]_.

Amniocentesis performed before 14 completed weeks of gestation is referred to as early. Early amniocentesis is not a safe alternative to second-trimester amniocentesis or CVS [50]. The CEMAT group (The Canadian Early and Midtrimester Amniocentesis Trial group), have reported in their randomized trial a significantly greater fetal loss and a higher incidence of talipes in the early amniocentesis cases compared with the ‘late’ ones (7.6% versus 5.9%) [62]. A randomized study by Nicolaides et al compared transabdominal chorionic villus sampling to early amniocentesis and suggested that loss rates might be higher in the latter [63]. Several studies showed that early amniocentesis had a significantly higher rate of amniotic fluid leakage than TA-CVS and mid-trimester amniocentesis[64,65,66].

Amniotic fluid assessment has been studied in patients with preterm labor and/or preterm premature rupture of membranes (PPROM) to investigate possible intra-amniotic infection (IAI). Amniotic fluid indicators suggestive of infection include elevated levels of matrix metalloproteinase (e.g., MMP-9) [67,