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BOR - Papers in Press, published online ahead of print March 23, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038018
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BIOLOGY OF REPRODUCTION 73, 139–148 (2005)
DOI: 10.1095/biolreprod.104.038018
© 2005 by the Society for the Study of Reproduction, Inc.

Perturbations in the Biochemical Composition of Fetal Fluids Are Apparent in Surviving Bovine Somatic Cell Nuclear Transfer Pregnancies in the First Half of Gestation1

Ning Li , David N. Wells , A. James Peterson , and Rita S.F. Lee 2 

Reproductive Technologies Group, AgResearch, Ruakura Research Centre, Hamilton 2001, New Zealand


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amniotic and allantoic fluid volumes and composition change dynamically throughout gestation. Cattle that are pregnant with somatic cell nuclear transfer (NT) fetuses show a high incidence of abnormal fluid accumulation (particularly hydrallantois) and fetal mortality from approximately midgestation. To investigate fetal fluid homeostasis in these pregnancies, Na, K, Cl, urea, creatinine, Ca, Mg, total PO4, glucose, fructose, lactate, total protein, and osmolalities were measured in amniotic and allantoic fluids collected at Days 50, 100, and 150 of gestation from NT pregnancies and those generated by the transfer of in vitro-produced embryos or by artificial insemination. Deviations in fetal fluid composition between NT and control pregnancies were apparent after placental and fetal organ development, even when no gross morphological abnormalities were observed. Individual NT fetuses were affected to varying degrees. Elevated allantoic Na was associated with lower K and increased allantoic fluid volume or edema of the fetal membranes. Total PO4 levels in NT allantoic and amniotic fluid were elevated at Days 100 and 150. This was not accompanied by hypophosphatemia at Day 150, suggesting that PO4 acquisition by NT fetuses was adequate but that its readsorption by the kidneys may be impaired. Excessive NT placental weight was associated with low allantoic glucose and fructose as well as high lactate levels. However, the fructogenic ability of the NT placenta appeared to be normal. The osmolality of the fetal fluids was maintained within a narrow range, suggesting that the regulation of fluid composition, but not osmolality, was impaired in NT pregnancies.

female reproductive tract, placenta, placental transport, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cloning of cattle by somatic cell nuclear transfer (NT) is characterized by a high rate of pregnancy complications and failure. The predominant cause of fetal mortality from our experience at AgResearch is the hydrops syndrome, which is an acute, excessive accumulation of allantoic fluid (hydrallantois) and, sometimes, amniotic fluid (hydramnios) from midgestation onward [1, 2]. Hydrops and its associated complications account for approximately 60% of pregnancy failures associated with NT from Day 120 onward. Although the incidence of hydrops may vary with the use of different cell lines as nuclear donors, the syndrome is more prevalent by far in NT pregnancies than in pregnancies conceived to natural mating, artificial insemination (AI), or after transfer of in vitro-produced (IVP) embryos. In a field study [3], the incidence of hydrops in cattle that were pregnant by natural mating or after AI was 1 in 1400 births (0.07%), compared with 1 in 55 births (1.8%) in pregnancies resulting after transfer of IVP embryos. Moreover, the method of in vitro embryo production appears to have bearing on the incidence of hydrops.

Fetal fluid homeostasis in cattle is still not fully understood. The ability of the fetus to maintain plasma volume is critical to its survival and development. This is achieved by balancing the fluid volume and composition of the amniotic and allantoic compartments through exchange between the maternal and fetal circulations. In cattle, amniotic fluid first accumulates after closure of the amnion at Day 22, and allantoic fluid first accumulates at Day 24, after the emergence of the allantoic sac from the hindgut. Initially, allantoic fluid accumulates at a faster rate than amniotic fluid, and between Days 27 and 33, the allantoic sac grows and fills to distend the gravid horn, eventually occupying both horns for the remainder of the pregnancy. Although the conceptus has become immobilized in the uterine lumen by Day 20 or 21 through the formation of trophoblastic papillae, which invade the endometrial glandular lumen [4], the hydrostatic pressure pushing the allantoic and trophoblastic membrane against the uterine wall probably facilitates chorioallantoic placental formation at the caruncles. During the first trimester, the allantoic fluid volume far exceeds the amniotic fluid volume, but as the fetus grows, amniotic fluid accumulates more rapidly so that by Day 100, the amniotic volume (~1000 ml) exceeds the allantoic volume, remaining in excess until approximately 150 days of gestation [5]. After that, the allantoic fluid volume increases rapidly, whereas the rate of amniotic fluid accumulation slows. At parturition, the allantoic fluid volume is approximately 15 L, and the amniotic fluid volume is approximately 5 L [5].

The biochemical constituents of bovine fetal fluid also change between Days 115 and 265 of gestation [6]. However, studies in sheep showed that the composition of fetal fluid in early pregnancy (Days 22–44) also changed [7]. This change in composition probably reflects changing metabolic and transport activity as well as alteration in the relative contribution of the fetal and placental tissues to the amniotic and allantoic compartments. Before the formation of the placentomes, transmembrane transport and the secretory activity of the extraembryonic membranes most likely are the major mechanisms for fluid accumulation. With further development, the predominant contributors to allantoic fluid are secretions from the mesonephros, then the metanephros, and finally the kidneys, so the allantoic fluid composition becomes more similar to fetal urine composition as the pregnancy progresses. However, allantoic fluid consistently differs in composition from that of fetal urine [6, 8], suggesting a modulating influence of placental, transmembranous, and intramembranous exchange [9]. In contrast, secretions from the respiratory tract, buccal cavity, gut, and fetal skin before its keratinization are the main contributors to the amniotic fluid [9], which has a composition that better resembles that of extracellular fluid. Toward the end of gestation, fetal urine is increasingly diverted from the allantoic sac into the amniotic fluid through the urethra as the urachus, through which urine previously passed, becomes progressively occluded.

We report here the composition of fetal fluids collected from a study comparing fetal and placental development in pregnancies resulting from the transfer of NT and IVP embryos and from AI. The fetal and placental morphometric data of surviving conceptuses at Days 50, 100, and 150 of gestation have been previously reported [10]. Amniotic and allantoic fluids collected from conceptuses described in that study were analyzed for Na, K, Cl, urea, creatinine, Mg, total Ca, total PO4, total protein, glucose, fructose, and lactate concentrations as well as for osmolality. Fetal serum from Day 150 also was analyzed for the first eight electrolytes and for osmolality. The data provide information regarding the dynamic changes in bovine fetal fluid composition in normal pregnancies during the first half of gestation, when the placentomes and fetal organs are being formed. In addition, the present study provides direct comparison of the fluid composition in viable NT fetuses with that in contemporary half-sib controls up to the period when hydrallantois is commonly encountered, thus providing information relevant to understanding the pathogenesis of hydrallantois.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All manipulations and treatments of animals involved in the present study were conducted in accordance with the regulations of the New Zealand Animal Welfare Act of 1999. The generation of embryos for transfer to recipients and the AI of heifers were carried out in two replicates of the same experiment conducted 2 mo apart with equivalent numbers of animals in each replicate. All fetuses were offspring of the same sire. Details of the experimental design and the generation of the conceptuses have been described previously [10]. The cell line used for NT is as described by Wells et al. [1]. At Days 50, 100, and 150, pregnant animals were fasted overnight and then slaughtered in the abattoir, and the uteri and contents were recovered. Morphometric measurements were made as described previously [10]. A portion of the allantoic and amniotic fluid was collected from each conceptus, placed on ice, and later centrifuged at 3000 x g for 10 min to remove cellular and particulate matter. Samples were frozen at –20°C until analysis. Fetal blood was collected from the umbilical vessels of Day 150 fetuses, and the serum was stored at –20°C. In some instances, either the allantoic or amniotic fluid from a particular fetus was not available for analysis because of loss caused by rupture of the uterus at slaughter, cross-contamination when one compartment leaked into another, or accidental contamination with fetal blood. Insufficient fetal serum was available for analysis in some cases.

Because the time between slaughter and fluid sampling could potentially affect the biochemistry of the fluids, particularly lactate levels, we subsequently collected, from three pregnant cows going through the abattoir, uterine tracts with fetuses of approximately 120, 160, and 250 days of gestation based on crown-rump lengths. At half-hourly intervals, from just after removal from the carcass to 3 h later, 2 ml of fluid were sampled with a syringe from the amniotic or the allantoic compartments of the intact uterus held at room temperature. The fluid samples were treated similarly as described above before analysis.

Fetal Fluid Biochemical Analysis

A panel of electrolytes (Na, K, Cl, Mg, total Ca, total PO4, urea, and creatinine) was measured by the former Animal Health Laboratories (Hamilton, New Zealand) with semiautomated equipment used for routine clinical chemical analyses with the appropriate quality controls. All samples from the above-described experiment were analyzed in one batch. Abnormally low or high results were repeated using another aliquot. Levels of Na, K, and Cl were determined using Roche reagents (Roche Diagnostics, Basel, Switzerland); levels of Ca, Mg, total PO4, urea, and creatinine were measured using the Hitachi 717 autoanalyzer.

Glucose and fructose were assayed simultaneously from the same sample using enzyme-linked biochemistry (former Boehringer-Mannheim Kit, catalog no. 139 106). Glucose was first converted to glucose-6-phosphate by hexokinase, then to gluconate-6-phosphate by glucose-6-phosphate dehydrogenase in an NADP-linked reaction. Fructose concentration was calculated from the difference in glucose concentration before and after conversion of fructose by hexokinase to fructose-6-phosphate, then to glucose-6-phosphate by phosphoglucose isomerase. The method was modified and adapted for use with 96-well microtiter plates with a total assay volume of 150 µl before addition of the enzymes. Before assay, samples were first heated to 65°C for 10 min to inactivate endogenous enzymatic activity, then centrifuged at 10 000 x g for 5 min to clear any precipitates. A standard curve for D-glucose and D-fructose was constructed. A glucose standard curve also was constructed in the presence of the highest fructose concentration in the fructose standard curve to determine if high fructose had any effect on glucose estimation. Lactate concentrations were measured using the enzymatic method described previously by Gardner and Leese [11] that coupled the conversion of lactate to pyruvate with the generation of NADH. The assay was adapted for use with 96-well microtiter plates using a total assay volume was 100 µl. L-Lactate dehydrogenase (rabbit muscle, L2500) and the L-lactic acid standard solution (826-10) were obtained from Sigma Chemical Co. (St. Louis, Mo); NAD was obtained from Roche (Basel, Switzerland). For both the fructose/glucose and the lactate assay, NADPH and NADH formation was measured as a difference in absorbance at 340 nm between the addition of the enzymes and the end point using a microtiter-plate reader (ELx800uv; Bio-Tek Instruments, Winooski, VT). A standard curve was constructed for each plate, and all standards and samples were analyzed in duplicate.

Osmolality was measured by freezing-point depression using the 3D3 osmometer (Advanced Instruments, Inc., Norwood, MA). Total protein was measured in duplicate using the method described by Bradford [12], with bovine serum albumin as standard.

Statistical Analyses

Factorial analyses of variance were done with and without log transformation on all data. In more than 50% of cases, the large difference in residual variance of the variables between stages of gestation, even after log transformation, made these analyses invalid. Hence, the treatments (AI, IVP, and NT) were compared for each variable at each stage of gestation using ANOVA, with and without log transformation. Comparisons between gestation means when variance heterogeneity was a problem were done on rank-transformed data or with individual comparisons using a t-test with or without log transformation, allowing for the unequal variance. Significance was accepted at P < 0.05. Relationships between relevant variables were examined using linear regression, with log transformation when appropriate. Some ratios between variables also were calculated and analyzed with ANOVA as described above. Comparisons between amniotic and allantoic levels of variables within animals were made using the sign test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Data (mean ± SEM) for each solute in amniotic and allantoic fluids and in fetal serum are presented in Tables 1–3. The sample numbers for amniotic and allantoic fluid for some treatment groups (Tables 1 and 2) were not the same in some instances because of accidental loss of one or the other, as outlined in Materials and Methods. Figures 1–5 show graphically the change in mean values with stage of gestation for each treatment group, with the average SEDs at each gestation shown as vertical bars. The NT means are joined by dotted lines. Only significant differences between treatment groups within a gestation stage are indicated by asterisks in the graphs. All conceptuses recovered at the three stages of gestation were viable at the time of slaughter.


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  		TABLE 1. Values (mean ± SEM) for the biochemical analysis of amniotic fluid from Days 50, 100, and 150


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  		TABLE 3. Values (mean ± SEM) for the biochemical analysis of fetal serum from Day 150


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  		TABLE 2. Values (mean ± SEM) for the biochemical analyses of allantoic fluid from Days 50, 100, and 150



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  		FIG. 1. Mean amniotic (AM) and allantoic (AL) fluid osmolalities at Days 50, 100, and 150 of gestation. Vertical bars represent the average SED for each gestation. The gestation (in days) is shown on the horizontal axes. Diamonds indicate AI, squares IVP, and triangles NT



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  		FIG. 5. Mean amniotic (AM) and allantoic (AL) fluid glucose, fructose, and lactate concentrations (in mM), with the average SED for each gestation shown as vertical bars. The gestation (in days) is shown on the horizontal axes. Diamonds indicate AI, squares IVP, and triangles NT. Significant differences among the treatment groups are indicated by asterisks. Glucose: Day 50 amniotic NT < IVP (P < 0.05) and AI (P = 0.06); Day 100 allantoic NT < IVP (P < 0.05). Fructose: Day 50 amniotic AI > IVP and NT (P < 0.05)

Osmolality of Fetal Fluids

No treatment-by-stage interaction was observed, but a significant effect of stage was, on either mean amniotic and allantoic fluid osmolality (P < 0.05 and P < 0.001, respectively; untransformed data). Mean amniotic and allantoic fluid osmolalities were similar between the treatment groups at each stage of gestation (Fig. 1). At Day 100, the amniotic fluid osmolality was lower than that on either Day 50 or Day 150. For individuals from which both fluid samples were obtained, allantoic fluid osmolality was lower than amniotic fluid osmolality (P < 0.001). No apparent relationship was observed between the osmolality and fluid volume for either amniotic or allantoic fluid at any stage (results not shown). Mean fetal serum at Day 150 generally was hyperosmotic compared with allantoic fluid, but the osmolality could be greater or lesser than that of amniotic fluid. Fetal serum osmolality (313 ± 4 mOsm/kg) was greater than maternal serum osmolality (279 ± 1 mOsm/ kg). At Day 150, the estimated allantoic osmolality derived from the sum of the analyzed solutes did not add up to the measured osmolality; between 20% and 40% of the osmolality was not accounted for. This was not the case in amniotic fluid. Despite the marked changes in the concentration of some of the constituents with increasing gestation and fluid volume, the osmolalities of the two fluids were maintained within a relatively narrow range in most cases, at least up to Day 150.

Na, K, and Cl

No treatment-by-stage interaction, but a significant effect of stage (P = 0.005, untransformed data), was observed on mean amniotic Na concentrations. Mean amniotic Na was similar among the treatment groups at all stages (Fig. 2). At Day 50, amniotic and allantoic fluid Na levels were closer together but diverged with increasing gestation because of a progressive decrease in allantoic Na concentration; however, amniotic fluid Na was higher than allantoic levels in most cases (P < 0.001). Mean fetal serum Na was higher than amniotic fluid levels at Day 150 (P < 0.001).



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  		FIG. 2. Mean amniotic (AM) and allantoic (AL) fluid Na, K, and Cl concentrations (in mM), with the average SED for each gestation shown as vertical bars. The gestation (in days) is shown on the horizontal axes. Diamonds indicate AI, squares IVP, and triangles NT

There was clear heterogeneity but no evidence of a treatment-by-stage interaction on mean allantoic Na concentrations. A significant effect of stage (P < 0.001, rank-transformed data) was, however, observed. Mean allantoic Na in all treatment groups decreased from Day 50 to Day 150. However, several NT individuals had elevated Na levels. One Day-100 fetus with an unusually high Na concentration of 120 mM had disproportionately smaller kidneys, a large heart, excessive placental growth, and edema of the fetal membranes. At Day 150, three of the eight NT samples had Na concentrations greater than 2 SD above the AI mean; two of these had excessive allantoic fluid. Thus, impaired Na handling was evident in some NT pregnancies by Day 100.

No treatment-by-stage interaction, but a significant effect of stage (P < 0.001, untransformed data), was observed on both mean amniotic and allantoic K concentrations. Mean amniotic K was similar for all treatment groups and increased between Days 50 and 100, then decreased so that by Day 150, the levels were similar to those seen at Day 50 (Fig. 2). In contrast, mean allantoic K increased from Day 50 to Day 150 in all treatment groups (Fig. 2). No significant difference was seen in the allantoic means among the treatment groups at any stage. Fetal serum K (Table 3) was almost twice as high as amniotic K (Table 1). Whether this was caused by postmortem elevation in blood with the passage of time after death is unknown. In the commercial preparations of fetal calf serum used for cell culture, the K concentration was 12.8 mM.

The Na concentrations in intra- and extracellular fluids tend to vary inversely with the concentration of K. This relationship was observed in bovine allantoic fluid, but it was not obvious in amniotic fluid. In allantoic fluid from all stages, a significant inverse correlation was seen between log K and the actual Na concentration in the AI (R = –0.94, P < 0.001) and NT (R = –0.84, P < 0.001) groups. Because of low sample numbers and variability within the IVP group, this relationship was not significantly correlated in this group.

No treatment-by-stage interaction (untransformed data), but a significant effect of stage (P < 0.05), was see on mean amniotic Cl concentrations. No difference was observed in mean amniotic Cl among the treatment groups at any stage (Fig. 2). A positive linear correlation was found between amniotic Na and Cl concentrations at all stages in the AI (R = 0.87, P < 0.001) and NT (R = 0.74, P < 0.0001) groups; the correlation was not significant in the IVP group (R = 0.468). The mean Na:Cl ratio decreased from 1.3 at Day 50 to 1.0 at Day 100 and remained unchanged after this time. In fetal serum, the mean Na:Cl ratio at Day 150 was 1.2.

Also, no treatment-by-stage interaction, but a significant effect of stage (P < 0.001, untransformed data), was found on mean allantoic Cl concentrations. Mean allantoic Cl for all three treatments dropped from 67 mM at Day 50 to 41 mM at Day 150; no significant difference was observed between the means at any stage. A significant positive linear correlation also was seen between allantoic Na and Cl concentrations at all stages (results not shown). In the AI group, a significant decrease (P < 0.01) was observed in the mean Na:Cl ratio between Days 50 and 150 (from 1.6 to 0.7, respectively); this decrease was not significant in either the IVP or NT groups.

Ca, Total PO4, and Mg

No significant treatment-by-stage interaction was observed, but a significant effect of stage (P < 0.05, log-transformed data) on mean amniotic Ca concentrations was. Mean amniotic fluid Ca levels increased slightly with stage of gestation (P = 0.015); however, no effect of treatment was found on the mean levels at any stage (Fig. 3). Clear heterogeneity but no evidence of a treatment-by-stage interaction was observed with mean allantoic Ca. However, a significant effect of stage (P < 0.05, rank-transformed data) was evident on mean allantoic Ca. Mean allantoic Ca levels increased between Days 50 and 100 in the AI and IVP groups (Fig. 3) but decreased so that by Day 150, levels were similar to those for Day 50. Although the mean NT allantoic Ca at Day 50 was significantly higher (P < 0.05) than the AI mean (but not the IVP mean), the mean NT allantoic Ca at Days 100 and 150 was not significantly different from the control group. In fetal serum, mean total Ca (Table 3) was higher than amniotic, but similar to allantoic, Ca.



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  		FIG. 3. Mean amniotic (AM) and allantoic (AL) fluid Ca, PO4, and Mg concentrations (in mM), with the average SED for each gestation shown as vertical bars. The gestation (in days) is shown on the horizontal axes. Diamonds indicate AI, squares IVP, and triangles NT. Significant differences among the treatment groups are indicated by asterisks. Ca: Day 50 allantoic NT > AI (P < 0.05). PO4: Day 150 amniotic NT > AI and IVP (P < 0.05); Day 50 allantoic NT < AI (P < 0.05); Day 100 and 150 allantoic NT > AI and IVP (P < 0.05). Mg: Day 150 amniotic NT > AI (P < 0.05)

Analysis suggested a treatment-by-stage interaction (P = 0.16, log-transformed data) and a significant effect of stage (P < 0.001) on mean amniotic total PO4. Mean amniotic PO4 was lower at Day 100 than at either Day 50 or Day 150 in all treatment groups (Fig. 3). In the NT group, mean amniotic PO4 was similar to that in the control groups at Days 50 and 100 but was significantly higher (P < 0.05) at Day 150. Allantoic fluid PO4 showed a different pattern of variation with stage of gestation compared to amniotic fluid (Fig. 3). We saw clear heterogeneity and a significant treatment-by-stage interaction (P < 0.001, log- or rank-transformed data) on mean allantoic PO4 levels. A significant effect of stage also was evident (P < 0.001). Mean allantoic PO4 was unchanged between Days 50 and 100 in the AI and IVP groups but increased ninefold in the NT group. At Day 50, the mean NT allantoic PO4 was significantly lower (P < 0.05) than the AI mean but was significantly higher than either the AI or IVP mean (P < 0.05) at Days 100 and 150. One Day-100 NT sample had a PO4 of 7.3 mM compared with the AI mean of 1.48 ± 0.44 mM. This conceptus was developmentally retarded, with the lowest body and fetal membrane weight and only 36 placentomes (compared with ~100 in normal pregnancies). A weak positive linear correlation was observed between PO4 and Na concentration in amniotic fluid, but a negative linear correlation in allantoic fluid, in all groups from Days 50 to 150. Mean total PO4 in fetal serum (Table 3) was 7- to 10-fold and 2- to 5-fold higher than amniotic and allantoic mean levels, respectively, and significantly higher (P < 0.05) in the NT compared with the AI, but not the IVP, group.

Mean Mg concentrations increased with gestation for both amniotic and allantoic fluid, although at different rates (Fig. 3). Clear heterogeneity with no treatment-by-stage interaction, but a significant effect of stage (P < 0.001, log- and rank-transformed data), was observed on mean amniotic and allantoic Mg. Mean amniotic Mg was not significantly different among the treatment groups at Day 50 or Day 100, but at Day 150, the NT amniotic mean was significantly higher (P < 0.05) than the AI mean. There was no effect of treatment on mean allantoic Mg at any stage. However, one Day-100 NT allantoic Mg was 14.3 mM, compared with the AI mean of 3.35 ± 1.59 mM. The same conceptus also had an amniotic Mg of 2.3 mM, compared with an AI mean of 0.49 ± 0.36 mM. The fetal and placental weights were normal, but only 39 placentomes were present. At Day 150, two of the NT conceptuses had allantoic levels of 28.0 and 29.2 mM, compared with the AI mean of 10.52 ± 3.70 mM. No gross abnormalities were noted with either of these Day-150 fetuses. The mean fetal serum Mg (Table 3) was significantly higher than amniotic fluid, but was significantly lower than allantoic fluid, concentrations (P < 0.001). Mean NT serum Mg was significantly higher (P < 0.05) than the AI, but not the IVP, mean.

Creatinine, Urea, and Total Protein

Clear heterogeneity but no evidence of a treatment-by-stage interaction was observed with both mean amniotic and allantoic creatinine. There was, however, a significant effect of stage (P < 0.001, rank-transformed data) on both. Although amniotic and allantoic creatinine levels were similar at Day 50 (Fig. 4), the levels increased at different rates (amniotic creatinine, 7-fold; allantoic creatinine, 55-fold) so that by Day 150, the mean allantoic creatinine was 20-fold higher than the amniotic mean levels. No effect of treatment was seen on mean amniotic creatinine levels at any stage. However, mean amniotic total creatinine was significantly higher in the IVP and NT groups compared with the AI group at Day 150 (P < 0.01). No effect of treatment was observed on allantoic concentration or total creatinine at any stage. A positive linear correlation between fetal weight at Day 150 (data not shown) and total creatinine was seen with amniotic (R = 0.75, P < 0.005), but not allantoic, fluid. Fetal serum creatinine (Table 3) was similar to amniotic fluid levels at Day 150.



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  		FIG. 4. Mean amniotic (AM) and allantoic (AL) fluid creatinine (in µM), urea (in mM), and total protein (in mg/ml) concentrations, with the average SED for each gestation shown as vertical bars. The gestation (in days) is shown on the horizontal axes. Diamonds indicate AI, squares IVP, and triangles NT. Total protein: Day 50 amniotic NT > IVP (P < 0.05); Day 100 allantoic NT > AI and IVP (P < 0.05), as indicated by an asterisk

Analysis suggested a treatment-by-stage interaction (P = 0.156, untransformed data) and a significant effect of stage (P < 0.005) on mean amniotic urea concentrations. Mean amniotic urea remained relatively unchanged between Days 50 and 150 in the AI and, to a lesser extent, the IVP group (Fig. 4), whereas the NT mean increased with gestation and was significantly higher at Day 150 compared with Day 50 (P < 0.005). No significant treatment-by-stage interaction or effect of stage or treatment was found on allantoic mean urea levels. Fetal serum (Table 3) as well as amniotic and allantoic urea levels were similar. Mean NT serum urea was significantly higher (P < 0.05) than the AI mean.

A significant treatment-by-stage interaction was observed on mean total protein concentrations in amniotic and allantoic fluid (P = 0.034 and P = 0.008, respectively; log-transformed data), and a significant effect of stage (P < 0.001) was found on mean amniotic, but not allantoic, protein concentration. Mean total amniotic protein concentrations in all treatments dropped between Days 50 and 100, then rose again at Day 150 for the AI and IVP groups, although not for the NT group (Fig. 4). In contrast, the change in allantoic total protein concentration with stage of gestation was different for each treatment group (Fig. 4). Mean NT allantoic protein was significantly higher (P < 0.05) at Day 100 but was not significantly different from either control group at Day 50 or Day 150. Three of the NT samples had protein concentrations of 0.74, 1.52, and 1.65 mg/ml, compared with 0.38 ± 0.19 and 0.26 ± 0.19 mg/ml for the AI and IVP means, respectively. In general, protein levels in allantoic fluid were higher than those in amniotic fluid (P < 0.001).

Glucose, Fructose, and Lactate

Mean glucose levels (Tables 1 and 2) generally were higher in amniotic than in allantoic fluid at every stage and in all treatment groups (P < 0.001). No correlation was apparent between amniotic and allantoic glucose concentrations. Analysis suggested a treatment-by-stage interaction (P = 0.16 and P = 0.072 for amniotic and allantoic, respectively; untransformed data) on mean glucose levels and a significant effect of stage on mean allantoic (P < 0.005), but not amniotic (P = 0.051), glucose levels. Mean amniotic glucose tended to be lower in the NT group at Day 50 (Fig. 5) compared with the AI (P = 0.06) and IVP (P < 0.05) groups but was not significantly different from the control values at Day 100 or Day 150. Mean NT allantoic glucose was significantly lower than the IVP (P < 0.05), but not the AI mean at Day 100; no difference was observed among the treatment groups at Day 50 or Day 150. Three of the Day-100 NT conceptuses had unusually low allantoic glucose, and of these, two had lower amniotic glucose as well. One of these was associated with exceptionally high caruncle and, hence, placentome weights, and another had only 36 placentomes and a developmentally retarded fetus, suggesting fetal growth restraint.

Fructose levels were higher than glucose in both fetal fluids in almost all cases (P < 0.001). A significant treatment-by-stage interaction (P < 0.05, log-transformed data) and a significant effect of stage (P < 0.001) were found on mean amniotic fructose. Mean amniotic fructose (Fig. 5) remained relatively unchanged from Day 50 to Day 150 in the AI group but increased significantly from Day 100 and Day 150 (P < 0.05) in the IVP and NT groups, respectively. At Day 50, the AI mean amniotic fructose was significantly higher (P < 0.05) than either the IVP or NT means; no significant difference was found among the groups at Days 100 and 150.

Clear heterogeneity was seen, and analysis suggested a treatment-by-gestation interaction (P = 0.145, rank-transformed data), but a significant effect of gestation (P < 0.001), on mean allantoic fructose. Between Days 50 and 100, mean allantoic fructose increased in all treatments (AI and IVP, P < 0.05; NT, P > 0.05), then decreased between Days 100 and 150 in the AI and IVP, respectively, but not in the NT group (Fig. 5). All Day-100 conceptuses with low allantoic glucose also had low allantoic fructose, suggesting that glucose probably is limiting for synthesis of fructose. The mean fructose:glucose ratio in amniotic fluid was 1.9, 1.7, and 3.8 at Days 50, 100, and 150, respectively. In allantoic fluid, the mean ratios were approximately 9, 10, and 15 for Days 50, 100, and 150, respectively. Thus, fructose is a greater contributor than glucose to fetal fluid osmolality.

Lactic acid is generated from the metabolism of glucose, fructose, and amino acids. No treatment-by-stage interaction (untransformed data), but a significant effect of stage (P < 0.001), was seen on both mean amniotic and allantoic lactate levels. Mean lactate levels in both amniotic and allantoic fluids decreased between Days 50 and 100 (Fig. 5) with the formation of the placentomes but was mainly unaltered between Days 100 and 150. No effect of treatment was found on the mean values at any stage in either fluid. However, some individuals had considerably elevated lactate levels, such as a Day-100 NT fetus with an allantoic lactate of 6.55 mM (compared with the AI mean of 1.28 ± 0.80 mM) and unusually low allantoic glucose and fructose. This fetus was of normal size but had the highest placental weight among the treatment groups at this stage. Similarly, another NT fetus at Day 150 with unusually high allantoic lactate (4.18 vs. the AI mean of 1.26 ± 0.40 mM) and low allantoic and amniotic glucose as well as fructose also had increased placental weight. The weight of this fetus was greater than 2 SD above the mean AI fetal weight (data not shown), and hydrops was observed.

We were concerned that the time between slaughter and fetal fluid sampling could affect the lactate levels, because lactate could still be produced after death. Generally, samples were recovered less than 3 h after slaughter. Lactate levels did increase with postmortem time but leveled off after 1.5–2 h. Allantoic lactate levels increased between slaughter and 3 h postmortem from 0.45 to 0.69, 0.49 to 0.61, and 0.58 to 0.79 mM in the three separate samples. Amniotic lactate increased from 1.15 to 1.31 and 2.70 to 2.89 mM in the first two samples and remained unchanged in the third sample. In these same fluid samples, Na, K, Cl, Mg, urea, and creatinine levels were unchanged (results not shown). Levels of PO4 increased 30%, and levels of Ca increased 60%, in the Day-120 allantoic fluid sample but not in the other two samples. Thus, any delay in the collection of samples was unlikely to be the cause for the very high lactate levels seen in some NT allantoic samples.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
From the present and previous studies, it is evident that amniotic fluid is maintained at a composition different from that of allantoic fluid despite the two membranes being in close apposition with each other and with the same overlying chorion for most of fetal development. The blood vessels emanating from the umbilical cord form one circulatory network that transports nutrients and metabolites between the fetal and maternal compartments and perfuse the placentomes that overly both the amniotic and allantoic sacs. That the fluid composition in the two compartments is different and changes differently with stage of gestation, despite intramembranous fluid and solute transport, suggests that the primary determinants of amniotic and allantoic fluid composition are the fetal components that directly contribute to the fluid. Differences in the permeability of the two membranes to various solutes also influence the fluid composition [13].

Both Na and K, because of their relative abundance, are the most important electrolytes regulating the movement and retention of body water, whereas Cl tends to track with Na transport. The ability to regulate intra- and extracellular Na, K, and Cl levels is an intrinsic requirement for fluid homeostasis. Although the transport of Na and K are coupled via the action of Na,K-ATPases, Na transport also is coupled to the transport of other molecules, such as glucose, PO4, amino acids, and hydrogen ions. The apparent lack of correlation between Na and K levels in amniotic fluid suggests that the cotransport of Na with these other molecules may play a significant role in influencing the Na: K ratio here. Before midgestation, all fetal urine goes into the allantoic sac [8] and so has negligible influence on amniotic fluid composition. The fall in allantoic Na and Cl and the increase in K with gestation are reflections of the increasing ability of the fetal kidney to reabsorb Na and Cl and the rising Na,K-ATPase activity in the renal tubules [14]. That the allantoic fluid Na is significantly less than fetal plasma or amniotic fluid levels suggests that fetal accumulation of Na from the mother must occur through the placental circulation and that little or no transport or exchange occurs by the intramembranous route [9] through the chorioallantoic membranes. Elevated allantoic Na in NT pregnancies may result from increased Na excretion because of renal hypoxia or decreased renal tubular Na reabsorption. The latter probably is linked to K transport, because in cases when Na levels were elevated, K levels were correspondingly lower. Increased Na secretion in these fetuses probably was accompanied by increased water accumulation to maintain the osmolality; hence, the increase in allantoic fluid volume. Such a link has been made with naturally occurring bovine and ovine hydrallantois [13, 15].

Both Ca and PO4 are actively transported from the mother to the fetus against a concentration gradient [16, 17]. In many species, fetal blood Ca is maintained at a higher level than in maternal circulation by the fetal-placental unit, which appears to act independently of maternal Ca regulation [16]. Almost all fetal Ca is associated with the skeleton, with the remaining Ca being involved in physiologically important processes, such as intracellular signaling, maintenance of cell membrane stability, and blood coagulation. Placental transport of Ca may be mediated by the membrane Ca-ATPase and by the cytosolic Ca-binding proteins, the calbindins. In cattle, calbindin9K-D has been localized by immunocytochemistry to most of the trophoblast cells of the intercotyledonary membranes but only to the binucleate cells of the cotyledonary membranes and to the uterine epithelial cells in the placentome [18]. The rise in Ca within amniotic and allantoic fluid between Days 50 and 100 may reflect an increase in fetal uptake with the formation of the placentomes, whereas the decrease between Days 100 and 150 in the allantoic fluid suggests increased fetal retention because of growth and ossification of the bones. Fetal crown-rump lengths increased approximately twofold, and fetal weight increased approximately nine-fold, between Days 100 and 150 [10].

Although PO4 is believed to be actively transported to the fetus, the regulation of its transport remains unknown [16]. Approximately 80% of PO4 is combined with Ca in bone. The Ca:PO4 ratio is an important determinant of bone ossification [19]. One of the most striking observations in NT fetal fluid composition is the apparent impairment in PO4 handling. In NT pregnancies, PO4 levels were elevated in both amniotic and allantoic fluids at Days 100 and 150. Elevated PO4 in amniotic fluid may indicate decreased intestinal absorption and/or fetal incorporation or increased loss or turnover, whereas elevated allantoic levels suggest impaired reabsorption and fetal retention as Na and PO4 are cotransported and readsorbed in the proximal tubules of the kidney [20]. That the Day-150 mean serum PO4 levels of NT fetuses were higher than the AI mean levels suggests that the transport of PO4 to the fetus in NT pregnancies was not impaired. However, the ability to incorporate this PO4 into fetal tissues, particularly the skeleton, may be diminished, or greater bone turnover may occur. Whether this eventually contributes to the spinal fractures reported in neonatal cloned and transgenic calves [21] is not presently known. The increase in both mean amniotic and allantoic PO4 between Days 100 and 150 in all treatment groups may indicate increased turnover from bone remodeling during growth.

Approximately 70% of Mg is combined with Ca in bone, with the rest being the principal divalent cation of soft tissues and body fluids. Exchange of Mg is reported to occur between the extracellular fluid compartment and bone [22]. The accumulation of Mg in the allantoic fluid with gestation is not easily explained given the exponential increase in tissue deposition with gestation. An impaired ability to handle Mg was evident in some Day-150 NT fetuses.

Urea is the secreted end product of nitrogen metabolism and is made in the liver from ammonia by an energy-requiring process. Urea concentrations in allantoic and amniotic fluid and in fetal and maternal plasma were essentially similar; thus, urea formed by the fetus is removed constantly and disposed of through the maternal circulation. Placental clearance and urea excretion rate in the fetal lamb were found to be higher than those in the adult [23]. Creatinine is formed mainly in the muscle by the irreversible, nonenzymatic removal of water from creatine phosphate. In both amniotic and allantoic fluids, creatinine levels increased dramatically with gestation. Thus, creatinine secreted by the fetus does not cross easily to the maternal compartment and, therefore, accumulates in the fetal fluids.

It is widely believed that the predominant contributor of NT pregnancy failure is abnormal placental development and function. One of the main functions of the placenta is to maintain an adequate supply of glucose to the fetus. Glucose transport is mediated, in part, by members of the facilitative glucose transporter family, as shown in horses [24] and, similarly, in sheep and cattle (F.B.P. Wooding, personal communication). In the absence of extreme maternal undernutrition, the supply of glucose to the fetus normally is maintained at the expense of maternal needs. Thus, the low glucose levels seen in some NT pregnancies are unlikely to result from maternal restriction in this group, because these animals were grazed alongside the control animals. The significantly lower amniotic glucose levels in Day-50 NT pregnancies may not necessarily be indicative of impaired transport but, rather, may be indicative of greater utilization. Placental tissues use 60–75% of the glucose leaving the uterine circulation [25]. Although NT fetal weight was not different from that in controls at Day 50, fetal membranes were significantly heavier, and the fetal cotyledons were better developed, than in AI or IVP controls [10]. The lower allantoic glucose in Day-100 NT pregnancies also was associated with increased fetal and placental mass or increased fetal membrane weight. Presumably, this results from greater utilization by these tissues rather than impaired placental glucose transport.

In ungulates, like in the ruminants, fructose is synthesized by the placenta and accumulates in fetal blood and fluids [26]. Glucose is converted first to sorbitol, then to fructose in the placenta [27]. In sheep, the fetal liver also can convert sorbitol to fructose [28]. Although fructose is accumulated, glucose is still preferentially used by the ovine fetus [29]. The lower allantoic fructose in our Day-100 NT pregnancies was associated with low glucose levels and increased placental and fetal mass, which implicates decreased placental fructose synthesis caused by glucose limitation. Neither decreased fructose nor glucose levels were seen in surviving NT pregnancies at Day 150. Generally, the ability of the NT placenta to synthesize fructose was not impaired.

The decrease in lactate levels between Days 50 and 100 is most likely caused by a switch to more aerobic metabolism with the formation of the placentomes. Placental tissues contribute to a significant proportion of lactate that enters the fetal circulation. Lactate itself is an important energy source for the fetus and placenta. High allantoic lactate in several NT samples was associated with low glucose and fructose and with excessive placental growth and edema of the fetal membranes in one Day-100 NT fetus. A Day-150 NT conceptus with excessive allantoic and amniotic fluid also had high allantoic, but normal amniotic, lactate. Previously, infusion of fetal sheep with sodium lactate led to severe polyhydramnios [30], whereas infusion of an equivalent amount of NaCl did not [31]. Whether increased lactate production results in increased fluid accumulation, such as that seen in the NT hydrops cases, is currently unknown, and it is not yet possible to attribute hydrops in NT to high lactate levels.

Fetal fluid homeostasis is regulated by several endocrine mediators. Pregnant ewes ovariectomized at 3 wk of pregnancy and supported thereafter by exogenous progesterone developed hydrallantois unless estrogens were administered as well [32, 33]. The mechanism of action remains unknown, and it was unclear whether the duration of exposure to abnormal estrogen:progesterone ratio was critical for subsequent development of hydrops. Short-term maternal infusion of glucocorticoids in sheep at 0.4 gestation resulted in increased allantoic fluid volume through increased fetal urine output [8]. Allantoic Na concentration was unchanged, however. Hormones regulating the transport of Na are atrial naturetic factor and cortisol, which both increase renal Na secretion [14]. Aldosterone promotes Na retention, whereas the transport of K and Mg is responsive the aldosterone and to acid-base balance [14]. Fetal uptake and handling of Ca is responsive to hormones elaborated by the parathyroid gland [16], but exactly when the bovine fetal gland becomes active is unknown. Fetal thyroparathyroidectomy in sheep during the last month of gestation resulted in hypocalcemia and reduced placental transport of Ca [34]. In the human, the fetal parathyroid glands are active from as early as 10 wk of gestation [22]. The kidney also contributes to the transplacental transport of Ca through the synthesis of vitamin D, which in turn regulates calcitonin gene expression [22]. In addition, the kidney may regulate Ca and PO4 levels by adjusting the excretion and readsorption through the renal tubules in response to factors such as the parathyroid hormones. These hormonal regulators, however, operate only after the fetal organs that synthesize and elaborate the hormones are developed and have acquired these functions. Before this, the acquisition of electrolytes critical to embryonic and fetal growth must be regulated by other mechanisms, such as maternal hormones or autocrine/paracrine factors acting on the absorptive and secretory epithelia of fetal membranes.

In summary, it is evident that the ability of surviving NT fetuses in the present study to handle some electrolytes was affected. To a smaller degree, electrolyte homeostasis also was sometimes perturbed in IVP pregnancies, suggesting an effect of in vitro culture on subsequent development. Despite the same nuclear genetics, not every NT individual was affected in the same way. Indeed, we previously reported greater variability in these NT fetuses compared with the controls derived by AI or IVP [10]. Most of the physiological abnormalities were not obvious until after development of the placentomes and the fetal organs, nor were they always associated with gross morphological abnormalities. The impairment of Na and PO4 handling suggests an underlying problem of kidney function. Thus, a combination of abnormal placental function and altered fetal endocrinology affecting fluid homeostasis may contribute to the high incidence of hydrops seen in NT pregnancies. Whether similar abnormalities are seen in NT fetuses generated from other cell lines is currently unknown.


   ACKNOWLEDGMENTS
 
We thank Neil Cox for his invaluable contribution in the statistical analysis. We also thank Anita Ledgard, Susan Ravelich, Martyn Donnison, and Martin Berg for help with sample collection and Debbie Berg and Sue Beaumont for advice on establishment of the lactate assay.


   FOOTNOTES
 
1 Supported by a grant from the Foundation for Research, Science, and Technology, New Zealand, grant C10 x 0018. Back

2 Correspondence: Rita S.F. Lee, Reproductive Technologies Group, AgResearch, Ruakura Research Centre, East St., Private Bag 3123, Hamilton 2001, New Zealand. FAX: 64 7 838 5628; rita.lee{at}agresearch.co.nz Back

Received: 14 November 2004.

First decision: 16 December 2004.

Accepted: 17 March 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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