Regulation of Amniotic Fluid Volume

Placenta 28 (2007) 824e832

Regulation of Amniotic Fluid Volume M.H. Beall a,*, J.P.H.M. van den Wijngaard b, M.J.C. van Gemert b, M.G. Ross a b

a Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, 1000 W. Carson Street, Box 3, Torrance, CA 90502, USA Laser Center and Department of Obstetrics and Gynecology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Accepted 30 November 2006

Abstract Water arrives in the mammalian gestation from the maternal circulation across the placenta. It then circulates between the fetal water compartments, including the fetal body compartments, the placenta and the amniotic fluid. Amniotic fluid is created by the flow of fluid from the fetal lung and bladder. A major pathway for amniotic fluid resorption is fetal swallowing; however, in many cases the amounts of fluid produced and absorbed do not balance. A second resorption pathway, the intramembranous pathway (across the amnion to the fetal circulation), has been proposed to explain the maintenance of normal amniotic fluid volume. Amniotic fluid volume is thus a function both of the amount of water transferred to the gestation across the placental membrane, and the flux of water across the amnion. Water flux across biologic membranes may be driven by osmotic or hydrostatic forces; existing data suggest that intramembranous flow in humans is driven by the osmotic difference between the amniotic fluid and the fetal serum. The driving force for placental flow is more controversial, and both forces may be in effect. The mechanism(s) responsible for regulating water flow to and from the amniotic fluid is unknown. In other parts of the body, notably the kidney, water flux is regulated by the expression of aquaporin water channels on the cell membrane. We hypothesize that aquaporins have a role in regulating water flux across both the amnion and the placenta, and present evidence in support of this theory. Current knowledge of gestational water flow is sufficient to allow prediction of fetal outcome when water flow is abnormal, as in twinetwin transfusion syndrome. Further insight into these mechanisms may allow novel treatments for amniotic fluid volume abnormalities with resultant improvement in clinical outcome. Ó 2007 Published by Elsevier Ltd. Keywords: Amniotic fluid volume regulation; Aquaporin; Twinetwin transfusion syndrome

1. Introduction Amniotic fluid (AF) in the second half of human gestation is largely a product of fetal urine and lung fluid. Fluid is resorbed via fetal swallowing. An additional route of AF absorption is required to balance fluid output and absorption; this absorption is theorized to occur across the fetal amnion into the fetal vasculature, and has been named the intramembranous (IM) pathway. In addition, AF volume depends on fetal hydration. All water in the conception ultimately derives from the mother, therefore placental water flux is also a factor in determining AF volume.

* Corresponding author. Tel.: þ1 310 222 1970; fax: þ1 310 782 8148. E-mail address: [email protected] (M.H. Beall). 0143-4004/$ - see front matter Ó 2007 Published by Elsevier Ltd. doi:10.1016/j.placenta.2006.12.004

This accepted description of fetal fluid production and resorption provides little explanation as to how AF volume homeostasis is maintained throughout gestation, and does not account for gestational alterations in AF volume, or postterm or acute-onset oligohydramnios. As an example, the acute reduction in fetal swallowing and increase in urine output seen in response to hypoxia in the ovine model, would suggest an increase in AF volume, not the reduced AF volume noted in these fetuses [1]. To understand the mechanisms responsible for maintaining AF volume, recent research has addressed the regulation of water flow in the placenta and fetal membranes. We will discuss the possible mechanisms for the regulation of fetal water flow below, and follow with a description of our use of the biophysics of fetal water flow to develop a mathematical model of twinetwin transfusion syndrome.

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1.1. Mechanism of IM flow Studies in the ovine model suggest that flow through the IM pathway can be modulated to achieve AF volume homeostasis. Since fetal swallowing is a major route of AF fluid resorption, esophageal ligation would be expected to increase AF volume significantly. Although AF volume did increase significantly 3 days after ovine fetal esophageal occlusion [2], longer periods (i.e. 9 days) of esophageal ligation reduced AF volume in preterm sheep despite continued production of urine [3]. Similarly, esophageal ligation of fetal sheep over a period of one month did not increase AF volume [4]. In the absence of swallowing, with continued fetal urine production, normalized AF volume suggests an increase in IM flow. In addition, AF resorption increased markedly following infusion of exogenous fluid to the AF cavity [5] or after increased fetal urine output stimulated by a fetal intravenous volume infusion [6]. Collectively, these studies suggest that AF resorption pathways (likely IM flow) are under dynamic feedback regulation. That is, AF volume expansion increases IM resorption, ultimately resulting in a normalization of AF volume. Importantly, factors down-regulating IM flow are less well characterized; there is no evidence of reduced IM resorption as an adaptive response to oligohydramnios. Down-regulation of IM flow is possible, as prolactin reduces the up-regulation of IM flow due to osmotic challenge in the sheep model [7] and it may reduce diffusional permeability to water in human [8] and guinea pig [9] amnion. The specific mechanism and regulation of IM flow is key to AF homeostasis. A number of theories have been put forward regarding the forces driving IM flow. IM flow may be driven by the significant osmotic gradient between the hypotonic AF and isotonic fetal plasma [10] in the human and sheep, although in rats and mice the osmotic gradient does not favor AF to plasma flow [11e13]. One explanation may be that solute concentration at the membrane surface differs significantly from that in the plasma or AF as a whole, a phenomenon known as the ‘‘unstirred layer’’ effect [14]. Gross hydrostatic forces are unlikely to drive AF to fetal flow, as the pressure in the fetal vessels exceeds that in the amniotic cavity. Hydrostatic forces could be developed between the AF and interstitial space, with another force promoting water flow into the blood stream. Local changes in hydrostatic or osmotic pressure, have been proposed to drive IM flow, however, none of these has been demonstrated in vivo. A variety of mechanisms have been proposed for the regulation of IM flow. As esophageal ligation of fetal sheep resulted in up-regulation of fetal chorioamnion vascular endothelial growth factor (VEGF) gene expression [15], it was proposed that VEGF-induced neovascularization potentiates AF water resorption. These authors further speculated that fetal urine and/or lung fluid may contain factors that up-regulate VEGF, although their recent work demonstrates no effect of lung liquid on the rate of IM flow (J. Jellyman, personal communication). The association of increased VEGF, and presumably vessel growth and permeability [16], with increased IM flow, coupled with the difference noted between the asymmetrical flow in vivo and the

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symmetrical permeability of amnion in vitro have also led to the suggestion that the rate of IM flow is regulated by the fetal vessel endothelium, rather than the amnion [17]. At present, there is little other evidence for this hypothesis. In animals in which the fetal urine output had been increased by an intravenous volume load, an increase in AF resorption occurred, despite a constant membrane diffusional permeability to technetium [6]. In addition, artificial alteration of the osmolality and oncotic pressure of the AF revealed that IM flow was highly correlated with osmotic differences; however, there was a component of IM flow that was not osmotic-dependent. As this flow pathway was also permeable to protein with a reflection coefficient of near zero, this residual flow was felt to be similar to fluid flow in the lymph system [18]. These findings, in aggregate, have been interpreted to indicate active transport of bulk fluid (i.e. water and solutes) from the AF to the fetal circulation, either in the amnion or in the fetal vessel wall. Brace et al. [6] have proposed that this fluid transport occurs via membrane vesicles, and point out the high prevalence of intracellular vesicles seen on electron microscopy of the amnion [19]. This theory has not been widely accepted, as vesicle water flow has not been demonstrated in any other tissue and would be highly energydependent. Most authors believe that IM flow occurs through conventional para- and transcellular channels, driven by osmotic and hydrostatic forces, perhaps through an unstirred layer effect. Mathematical modeling indicates that relatively small IM sodium fluxes could be associated with significant changes in AF volume, suggesting that active transport of sodium may be a regulator of IM flow [20]. The observation that a portion of IM flow was independent of osmotic differences, however, suggests that other forces may also be significant [18]. Importantly, up-regulation of VEGF or sodium transfer alone cannot explain AF composition changes following fetal esophageal ligation, since AF electrolyte composition indicates that water flow increases disproportionately to solute (i.e. electrolyte) flow [21]. The passage of free water across a biological membrane is a characteristic of transcellular flow, a process mediated by cell membrane water channels (see below). Although water flow through these channels is passive, the expression and location of the channels can be modulated in order to regulate water flux. 1.2. Mechanism of placental flow In the placenta, the flow of water may be driven by either hydrostatic or osmotic/oncotic forces. Hydrostatic forces could be developed in the placenta by a difference between maternal and fetal circulatory pressure. Since the relative osmolalities of maternal and fetal plasma do not favor water flow to the fetus, osmotic water flow in the placenta would require the development of local osmotic gradients due to either active transport of solutes such as sodium or to the depletion of solvent from the local perimembrane environment. Either phenomenon may result in a local increase in osmolality due to the ‘‘unstirred layer’’ effect. In addition differences in the direction of blood flow may be important in establishing either

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osmotic or hydrostatic gradients within the placenta. The relative direction of maternal and fetal blood flows can be concurrent, countercurrent, crosscurrent, or a combination of these flows [22]. In rodent placentae, fetal and maternal blood circulate in opposite directions (i.e. countercurrent flow), potentially increasing the opportunity for exchange between circulations. Investigation has not revealed countercurrent blood flow in the ovine placenta [23]. The direction of maternal blood circulation in human placentae is from inside to outside of the placental lobule, and therefore at cross-current to the fetal blood flow [24]. It has not been possible to directly study pressure or osmotic differences at the level of the syncytium; therefore, theories regarding the driving forces for placental water flow are inferences from available data. Water transfer from mother to fetus may be driven osmotically by the active transport of solutes such as sodium [25]. Evidence suggests how this may occur in rodents. In the rat, inert solutes such as mannitol and inulin are transferred to the maternal circulation from the fetus more readily than from mother to fetus [26] and, conversely, sodium is actively transported to the fetus in excess of fetal needs. These data were taken to indicate that water was being driven to the fetal side by a local osmotic effect created by the sodium flux. Bulk fluid, composed of water with dissolved solutes, then returned from fetus to mother, probably by a paracellular route. These findings have been questioned [27], although most models suggest that transplacental water flow is driven by an osmotic mechanism. Perfusion of the guinea pig placenta with dextrancontaining solution demonstrated that the flow of water can also be influenced by colloid oncotic pressure [28]. The importance of osmotically driven water flow in the uninstrumented sheep is uncertain, as the maternal plasma was consistently hyperosmolar to the fetal although theoretical considerations have been used to argue that known electrolyte active transport, and a modest hydrostatic pressure gradient, could maintain maternal-to-fetal water flow against this osmotic gradient [29]. Others have argued that the driving force for water flux in the placenta is hydrostatic. In the perfused placenta of the guinea pig, reversal of the direction of the fetal blood flow reduced the rate of water transfer [30], suggesting that the arterial side of the fetal circulation was the site of fetal to maternal flow. Increasing the fetal-side perfusion pressure increased the fetal-to-maternal water flow both in the perfused guinea pig placenta [31] and in an intact sheep model [32]. Finally, pharmacologically increasing the blood pressure in the fetal vessels in a perfused human placenta was associated with increased fluid flow from the fetal to the maternal compartment [33]. These findings suggest that placental water transfer may be due to a hydrostatic mechanism. As a whole, the available data suggest that either osmotic or hydrostatic forces can promote placental water flow. The actual driving force in normal pregnancy is uncertain, and may vary with the species and/or the pregnancy stage. It also seems likely that the forces driving transplacental flow may be generated locally, as unstirred layers or as local hydrostatic differences; these forces may be nearly undetectable. Whatever

drives it, at least some part of placental water transfer involves the flow of solute-free water transcellularly, suggesting the involvement of membrane water channels in the process. We will review the characteristics of aquaporin water channels, and then comment on the evidence that aquaporins may be involved in regulating gestational water flow. 2. Aquaporins Aquaporins are cell membrane water channel proteins approximately 30 kD in size (26e34 kD). Although the majority of AQP structural studies have been performed on AQP1, similarities in sequence suggest that the three-dimensional structure of all AQPs is similar. AQPs are hydrophobic intramembranous proteins [34,35]. They organize in the cell membrane as tetramers, however, each monomer forms a hydrophilic pore in its center and functions independently as a water channel [35]. In addition to water, some AQPs also allow passage of glycerol, urea and larger molecules. These have also been called ‘‘aquaglyceroporins’’. Multiple AQPs have been identified (up to 13, depending on the species). Some are widely expressed throughout the body; others appear to be more tissue-specific. AQPs regulate water flux dependent upon cellular location. In the kidney, several AQPs are expressed in specific areas of the collecting duct. AQP3 and AQP4 are both present in the basolateral membrane of the collecting duct principal cells, while AQP2 is present in the apical portion of the membrane of these same cells [36]. The presence of these AQPs on different portions of the same cell is thought to regulate water transfer across the cell by differentially promoting water entry from the collecting duct lumen and from the interstitial fluid. AQP-regulated water flux is also dependent upon the cellular milieu. Regulation of AQP action may occur through the insertion or the removal of AQP into the membrane from the intracellular compartment. In the renal tubule, AQP2 is transferred from cytoplasm vesicles to the apical cell membrane in response to AVP [37] or forskolin [38]. AQP8 is similarly transferred from hepatocyte vesicles to the cell membrane in response to dibutyryl cAMP and glucagons [39]. In longer time frames, expression of various AQPs may be induced by external conditions. For example, AQP3 expression in cultured keratinocytes is increased in hypertonic medium [40]. In the intact organism, AQP3 expression in the kidney is up-regulated by dehydration [36], an effect mediated by AVP. Together with permeability data, these findings indicate that AQPs are important in the regulation of water flow across biological membranes, and that their expression and activity are regulated according to the needs of the organism. 2.1. Aquaporins in fetal membranes Studies have demonstrated AQP1, -3, -8 and -9 mRNA (gene) expression in fetal membrane and placenta in a variety of species (see below). These results suggest that AQPs 1, 3, 8 and 9 may all contribute to the regulation of conceptional water flow. A summary of these AQPs follows.

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2.1.1. Aquaporin 1 AQP1 is one of the most widely expressed water channels [41], present in red blood cells [42], apical and basolateral pulmonary membranes [43], renal proximal tubules and the descending thin limbs of Henle [44], corneal endothelium, and syncytiotrophoblasts in placenta [41], among other tissues. Transgenic mouse studies demonstrated that the AQP1 knockout (KO) is pathogenic, resulting in decreased proximal tubule water permeability and defective fluid absorption [45]. KO mice reproduce normally. AQP1 mRNA has been demonstrated in murine and ovine placentae, and it appears to be associated with the placental vessels [46,47]. In the sheep placenta, AQP1 is the earliest-expressed AQP, being found as early as 27 days of gestation [48]. Ovine placental AQP1 expression levels are highest early in pregnancy, with a decline thereafter, although there is a rise in expression near term [48]. In the mouse, AQP1 expression is found as early as e10 in both the placenta and fetal membranes, and the relative expression also decreases with increasing gestation [47]. AQP1 protein expression has been demonstrated in the fetal membrane at term in human pregnancies [49], though the cellular location of the protein has not been studied. 2.1.2. Aquaporin 3 AQP3 is an aquaglyceroporin, permeable to glycerol and urea, in addition to water [50,51]. AQP3 is a less effective water channel than AQP1, as rat AQP1 increased the water permeability of Xenopus oocytes up to twice as much as AQP3 [52,53]. AQP3 is expressed in the collecting duct, as well as other parts of the kidney in humans [54], rats [50,55] and mice [56]. It is also found in epithelia, particularly in the skin [57], gut [58], muscle [59], ear [60], and lung [61,62]. AQP3 KOs demonstrate a marked defect in urine concentrating ability with polyuria, although they reproduce successfully [63]. AQP3 deficiency is also associated with skin abnormalities, including defects in elasticity and hydration [57]. AQP3 message has been demonstrated in the placenta and fetal membranes of humans [49,64], mice [47] and sheep [46,48] and in rat placenta [65], while protein expression has been demonstrated in murine placenta and amnion and in ovine and human placenta. AQP3 protein is expressed on the apical membranes of human syncytiotrophoblast [64]. In the sheep placenta, AQP3 mRNA was detected by 60 days of gestation (term ¼ 150 days), reached a maximum at 100 days, and remained stable until term [46]. In the mouse placenta, AQP3 expression increased from e10 to e16 with no change from e16 to e19 (term 21 days) [47]. 2.1.3. Aquaporin 8 When expressed in Xenopus oocyte, the human, mouse, and rat AQP8 gene increased the water permeability of Xenopus oocyte by 10e20 fold [66e69], this increase is similar to that found with AQP1. Mouse, though not human or rat AQP8 is also permeable to urea [67e69]. In mouse, AQP8 mRNA can be detected in colon, liver and heart [69]. In humans,

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AQP8 gene expression is detected in pancreas, liver [68], and colon [70]. The function of AQP8 is uncertain, as AQP8 KO mice were not found to have any identifiable phenotypic defect [71]. AQP8 mRNA has been detected in the placenta and fetal membranes in the mouse [69] and human [72], and in ovine [48] placenta. In the mouse, AQP8 expression increased significantly in both tissues between e10 and e16, with no further change near term [47]. AQP8 protein expression has not been studied in these tissues. 2.1.4. Aquaporin 9 AQP9 is an aquaglyceroporin, permeable to the largest variety of small non-polar molecules of any AQP [73]. As a water channel, AQP9 is less effective than AQP1 and may be less effective than AQP3 [74]. AQP9 is expressed in the liver in rats [73] and humans [75], and in the rat testis, spleen and brain [76]; there is no measurable AQP9 in the kidney. AQP9 protein and mRNA have been demonstrated in human [64,77] and ovine [78] placenta, and fetal membranes. AQP9 message has also been demonstrated in murine placenta and fetal membranes, with AQP9 expression in the membranes increasing from e10 to e16 [47]. 2.2. AQPs and regulation of IM flow AQP expression in the amnion is subject to hormonal regulation. In work done in our lab, AQP1, -3, -8 and -9 expression is up-regulated in cultured human amnion cells following incubation with cAMP or forskolin, a cAMP-elevating agent [79e81], and AQP1 and -3 are up-regulated by AVP [82]. These effects may be significant, as AVP levels may be higher in the AF of fetuses with oligohydramnios [83] and recent data suggest that membrane AQP1 may regulate AF volume. Specifically, mice lacking the AQP1 gene have been reported to have significantly increased AF volume [84]. Furthermore, AQP1 expression was increased in human amnion derived from patients with increased AF volumes [85]; this up-regulation was postulated to be a compensatory response to polyhydramnios. AQP1 protein increased in ovine chorioallantoic membranes when the fetus was made hypoxic, suggesting a mechanism for the increased IM flow associated with ovine fetal hypoxia [86]. These data together support the hypothesis that AQP1 is a primary regulator of water flow out of the gestational sac across the amnion. 2.3. AQPs and regulation of placental water flow Indirect evidence suggests that AQPs may be involved in the regulation of placental water flow. The permeability of the placenta increases, and then stabilizes with advancing gestation in the human [87]. Interestingly, this pattern of increase is also seen in the placental expression of membrane AQP3 in both the sheep [48] and the mouse [88], suggesting that AQP3 may be involved in regulating placental permeability changes. These data together with the location of AQP3 on the apical membrane of the syncytiotrophoblasts make it a candidate

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regulator of placental water flow. In contrast, a recent report on AQP9 expression in the human placenta [89] suggests that AQP9 plays a significant role in transplacental urea flux; an effect on water flux was not seen. 3. Clinical correlation The principles of water flux outlined above can also be used to model conceptus water flow. We have chosen the example of twinetwin transfusion syndrome to illustrate how the principles of water flow described above can be applied to a clinical situation. 3.1. Modeling water transfer in the human conception in twinetwin transfusion TTTS is a serious clinical complication of monochorionic twin conception caused by a net fetofetal transfusion from one twin (the donor) to the other (the recipient) through placental vascular anastomoses. Asymmetries of fetal growth and AF volume develop, with the donor twin becoming both growth restricted and oligohydramniotic, while the recipient twin develops hydrops and polyhydramnios. We have modeled water flow in the human conception complicated by TTTS, and utilized this model to predict the outcome of various proposed treatments [90]. In our model, transplacental fluid flow from the maternal blood to the fetoplacental circulations was assumed to be the ultimate source of water for the increasing AF volume. Further, the transplacental flow was assumed to depend on a dynamic balance between the hydrostatic and colloid osmotic pressure gradients (starling equation, as above). We first modeled the fluid dynamics of normal pregnancy, estimating the net placental fluid flow for all gestational ages [91], this allows calculation of the transplacental filtration coefficient throughout gestation in the normal fetus. The assumption is made that the transplacental filtration coefficient as a function of gestation is constant, although clinically donor and recipient twins can develop discordant placental blood flows due to both altered placental vascular resistance and changes in other physiologic measures, such as blood viscosity. Our complete model describes fluid flow into and within the twin conception [92]. Briefly, the model consists of 10 differential equations for each twin, coupled by the net anastomotic fetofetal transfusion of blood and blood components, i.e. colloids, osmoles and renineangiotensinesystem (RAS) mediators. The 20 equations describe the development of fetal arterial and venous blood volumes, blood osmolality and colloid osmotic pressure, interstitial fluid volume and colloid osmotic pressure, intracellular fluid volume, AF volume and osmolality, and RAS mediator concentration in the fetal blood. Input parameters of the model are the following: (a) the diameter and length of all anastomoses throughout pregnancy, and (b) the placental sharing between the twins. Using the mathematical system to model a case of a net fetofetal transfusion of blood from donor to recipient twin, due to a single arteriovenous anastomosis, the fetofetal transfusion reduces the donor twin’s blood volume and total body fluid

volume, leading to hypotension, a reduced urine production rate, and reduced blood osmolality, and colloid osmotic pressure (Fig. 1). The decrease in fetal colloid osmotic pressure is greater than the decrease in the total capillary pressure of the donor, so transplacental fluid flow declines. The donor becomes growth retarded. The decrease in donor urine production is greater than the change in swallowing and intramembranous flow, causing the donor’s AF volume to increase more slowly than normal and even to decrease with a sufficiently large anastomotic flow. The donor twin finally becomes ‘‘stuck’’ to the uterine wall and immobilized. In the recipient twin, the converse applies. Here, the increased growth of the recipient blood volume leads to mild hypertension, increased urine production, increased COP, increased transplacental flow and finally polyhydramnios. Subsequently [92], the inability of the stuck donor twin to hydrate by swallowing AF aggravates its hypotension, leading to increased activation of its renineangiotensin system, which results in an increased transfusion of RAS mediators to the recipient. These excess RAS mediators now reduce the recipient’s polyuria, increasing the recipient’s venous and capillary blood pressures. As a consequence, fluid flow from the circulation to the interstitium increases, resulting in excess interstitial fluid volume (hydrops). Interestingly, because the excess fluid accumulating in the recipient fetus can either exit through the recipient bladder, contributing to polyhydramnios, or into the fetal interstitium contributing to hydrops, the development of hydrops may accompany reduction in AF. Because excess AF is known to compress the placenta, this likely affects the transplacental fluid flow. As the effect of the severity of polyhydramnios on transplacental flow was not included in the model, this relationship remains unexamined. 3.2. Modeling of treatment in twinetwin transfusion syndrome Therapeutic intervention in TTTS has long been controversial. Laser coagulation of anastamotic vessels has been shown to confer the best long-term fetal outcome in severe TTTS [93], but both amnioreduction and septostomy have been used, and have been described to have favorable short-term outcomes [94]. Many centers use the less-invasive techniques first, reserving laser treatment for resistant cases. The model described above can be used to evaluate likely outcomes of treatment, with known clinical outcomes used to evaluate the accuracy of the model. Amnioreduction, or removal of AF from the polyhydramniotic sac, has a long history as a treatment for TTTS. In the mathematical model, amnioreduction suddenly decreases AF pressure, acutely increasing transplacental fluid flow to both twins. Increased transplacental flow increases blood volume and blood pressure in the donor, which increases the donor’s urine production and AF volume. Ultimately, improvement in the donor blood pressure increases the fetofetal transfusion to the recipient. With time, the donor’s blood volume and arterial pressure decrease and its urine production again wanes [92]. Amnioreduction would therefore be expected to be of

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Fig. 1. Amniotic fluid volume, with inflows and outflows, in twinetwin transfusion syndrome. Numbers calculated using a mathematical model for a single arteriovenous (AV) anastomosis in an equally shared placenta (see text). A: amniotic fluid volume across gestational age for the donor twin and recipient twin as compared to normal. B: urine output. C: swallowed volumes. Note that the swallowed volume of the donor twin is solely lung fluid after 22 weeks’ gestation, and that lung fluid production is independent of transfusion status in this model. D: intramembranous flow rates.

transient, but not long-term benefit. In clinical practice, amnioreduction is used serially, with transient improvement after each episode. Serial removal of large amounts of AF may, however, be associated with an increased risk of pregnancy loss, and the long-term benefit of amnioreduction in severely affected TTTS pregnancies is doubtful [95,96]. Septostomy, or conversion of a diamniotic to a monoamniotic pregnancy has been proposed as a treatment for TTTS. Mathematical modeling of monoamniotic twins reveals no change in fetal outcome with the change in amnionicity [97], suggesting that septostomy may be of limited longterm benefit. Small clinical studies have reported results as good, or better than, those obtained with amnioreduction [98] and a larger, randomized trial, showed no difference in twin survival between septostomy and amnioreduction [99]. As there was no untreated group in this trial, the effect of either treatment could be negligible. Fetoscopic laser therapy, with coagulation of the anastomotic vessels, has been used successfully in the treatment of TTTS. Mathematical modeling of laser therapy [100] predicts an ongoing benefit to both donor and recipient fetuses. Clinically, laser therapy was superior to amnioreduction in a randomized, controlled trial [93], and was of especial benefit in fetuses with more severe disease [95]. All of these findings suggest that the proposed mathematical model is a reliable reflection of clinical reality.

4. Conclusion The circulation of water between mother and fetus, and within the fetal compartment, is complex, and the mechanisms regulating water flow remain poorly understood. Water flow across the placenta must increase with increasing fetal water needs, and must be relatively insensitive to transient changes in maternal status. Water circulation within the conception must sustain fetal growth and plasma volume, while also allowing for appropriate amounts of AF for fetal growth and development. The flow of water into and among the fetal water compartments appears to be regulated largely by the water permeability of biological membranes. Water permeability in the placenta appears to be regulated at the level of the syncytiotrophoblast, and to vary with gestational age. Placental water flux may be altered both by changes in maternal or fetal osmolality or oncotic pressure, or by changes in relative blood pressure or placental blood flow. Once in the gestational sac, water is exchanged between the fetus, placenta and AF. AF volume is influenced by intramembranous flow across the amnion into the fetal vessels. Intramembranous flow can be altered to maintain AF volume, although the mechanisms responsible for this regulation remain controversial. We have proposed that intramembranous water flow is dependent on aquaporin water channels; abnormalities in aquaporin 1 have been

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described in association with disordered AF volume. Further work is necessary to determine whether abnormalities of water transfer are responsible for idiopathic oligohydramnios and polyhydramnios, and whether manipulation of water transfer can influence the course of these conditions. Using the physical description of the water flow into and within the conception, we have developed a mathematical model of water flow in the twinetwin transfusion syndrome, and we have used our model to assess the various treatment modalities proposed for this condition. Our predictions of treatment effectiveness are in good general agreement with experimental data, suggesting that the model of fetal water dynamics presented here is generally reliable. This model may assist in developing therapies for abnormal AF volume based on the biophysics of membrane water flux.

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