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During the third trimester of human gestation, there is a progressive increase in the turbidity of the amniotic fluid surrounding the fetus (1). This turbidity has been assayed as an index of fetal lung maturity by various methods ranging from spectrophotometry to visual examination (2, 3). The precise etiology of amniotic fluid turbidity is unclear and potentially mutifactorial. Amniotic fluid turbidity, for example, has been related to an increase in lung-derived phospholipids and, more recently, to the presence of lamellar bodies derived from type II pneumocytes (4, 5). Alternatively, turbidity may result primarily from detachment of vernix caseosa from the fetal skin surface (1, 6). To our knowledge, there is no information on the mechanism of vernix detachment from the skin surface into the amniotic fluid during late gestation.

Structurally, vernix caseosa is a biologic film, containing lipids and proteins, with both hydrophobic and hydrophilic domains (7). This biofilm is the putative result of a surge in sebaceous gland activity coupled with the desquamation of fetal corneocytes during the last trimester of pregnancy (8). A gradual build up of this proteolipid film coats the fetal skin surface during the critical period of adaptation before birth. With advancing gestational age, vernix on the skin surface detaches into the amniotic fluid (1). Following detachment, the fetus presumably swallows the vernix, exposing the developing gut to this material. The physiologic significance of this exposure has not been investigated.

In this study, we tested the hypothesis that increasing phospholipid concentrations of pulmonary origin would induce a “roll up” phenomenon (9) leading to detachment of vernix from the skin surface. Hypothetically, it is the detachment of vernix caseosa that is the primary factor leading to increased amniotic fluid turbidity toward term. Herein, we report the development of an in vitro assay for studying the mechanism of vernix detachment. The rheological (flow) properties of vernix were also assessed as a function of physiologic temperature. The results indicate a potential mechanism for inducing amniotic fluid turbidity and suggest a novel physiologic interaction between the skin and lung during late fetal development.

METHODS

Vernix collection.

Vernix was harvested from full-term infants without antenatal complications born at University Hospital in Cincinnati and stored in sterile vials at the time of delivery. Samples were kept under refrigeration at 4°C until the time of experiment. Exclusion criteria included prenatal steroid therapy, chorioamnionitis, and blood or meconium contamination of vernix.

Turbidity assay.

A typical assay was performed as follows: 10 mg of vernix pooled from three to four term infants, was applied to the interior wall of 1.5-mL polypropylene microfuge tubes and spread with a glass pestle to form an even coating. One milliliter of normal saline with 0, 25, 50, 100, or 200 μg phospholipid was added to the tubes. Microfuge tubes containing comparable phospholipid concentrations without vernix were used as controls. In the first experiment, phospholipid was derived from Survanta® (Ross Laboratories, Columbus, OH, U.S.A.), a clinically used pulmonary surfactant suspension isolated from bovine lung lavage. The capped tubes were gently agitated by slow rotation on a rotisserie/shaker containing 22 slots for the assay tubes (Labquake®-Barnstead/Thermolyne, Dubugue, IA, U.S.A.). Two such rotisseries were used in each experiment and placed within an Air Shields incubator for thermal equilibration at 37°C overnight. The liquid was then decanted and spectophotometrically analyzed at 650 nm. Increased OD correlated with increased solution turbidity secondary to detachment of vernix from the walls of the microfuge tubes. Absorbance values from control samples were subtracted from the vernix-coated tubes to determine the final OD.

The assay was also performed with a synthetic mixture of phospholipids that closely approximated pulmonary surfactant lipid content but was devoid of surfactant-associated proteins. This synthetic phospholipid mixture (PLM) was prepared in a mixture of chloroform:methanol (3:1) using dipalmitoylphosphatidylcholine, 65.0%; phosphatidylcholine, 20.0%; dipalmatoylphosphatidylglycerol, 10.0%; phosphatidylinositol, 2.5%; and cholesterol, 2.5%. All lipids were purchased from the Sigma Chemical Co. (St. Louis, MO, U.S.A.). The solvent was evaporated under a nitrogen stream and the resultant residue was suspended in an appropriate volume of Tris-buffered saline, pH 7.4 (Sigma Chemical Co.), to yield the desired concentration. This mixture was sonicated for 2 min on ice and stored at –20°C until use. Experiments were repeated multiple times. Representative results are discussed and shown in the figures.

Rheology.

Flow curves were obtained on a Haake RS 150 controlled stress rheometer (Haake, Paramus, NJ, U.S.A.), with circulating water bath temperature control. The sensor system was attached to a 20-mm diameter parallel plate with measurement gap set to 1.00 mm. The measurement program consisted of a stress ramp from 1.0 to 10,000 pascal over 3 min, following a 5-min equilibrium period. Vernix specimens were tested at two different physiologically relevant temperatures;i.e. 23°C (room temperature) and 37°C (body temperature).

Statistical analyses.

All experiments were performed using pooled vernix samples derived from three to four term infants. From experiment to experiment, different pooled samples were used. Consequently, within a given experiment, the vernix itself was not a source of variability. This was an important factor to control given the focus of this study on pulmonary surfactant-vernix interactions. Methodological constraints included the fact that each point required its own control because increasing phospholipid automatically increased the turbidity of the sample. Given these constraints, the experimental system had small intra-assay variability, which allowed statistical comparisons to be made between the experimental conditions. The t test was used for statistical comparisons with a p value <0.05 being considered significant.

RESULTS

Effect of Survanta® on solution turbidity.

Vernix-coated microfuge tubes were incubated overnight with a range of concentrations of phospholipids derived from Survanta as described in the “Methods” section. Figure 1 shows a dose-dependent increase in turbidity, indicating increasing detachment of vernix from the walls of the microfuge tubes.

Figure 1
figure 1

Effect of Survanta® to promote detachment of vernix caseosa. Vernix-coated microfuge tubes were incubated overnight with increasing concentrations of phospholipids derived from bovine pulmonary surfactant (Survanta) as described in the “Methods” section. A dose-dependent increase in turbidity (OD = 650 nm) was observed. All data are expressed as mean ± SEM. N = 4 for each data point.

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Effect of synthetic pulmonary phospholipid mixture on solutionturbidity.

The assay was repeated with Survanta as described above, and contrasted with an assay that used an equimolar synthetic PLM. This synthetic mixture approximates natural pulmonary phospholipid but is devoid of surfactant-associated proteins. As shown in Figure 2, there was no increase in turbidity observed using the PLM, whereas phospholipids derived from Survanta showed a dose-dependent response as in the previous experiment. This lack of response observed using PLM supports the hypothesis that surfactant-associated proteins facilitate vernix detachment.

Figure 2
figure 2

Effect of synthetic PLM on vernix detachment. The turbidity assay was repeated with a synthetic PLM that closely mimics natural pulmonary phospholipid, but is devoid of surfactant-associated proteins. As shown in this representative experiment, a lack of response was observed when using the PLM. All data are expressed as mean ± SEM. N = 4 for each data point.

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Effect of temperature on solution turbidity.

To assess the effect of physiologic temperature on the development of turbidity, 200 μg/mL of phospholipid derived from Survanta was incubated overnight in vernix containing tubes at either body temperature (37°C) or room temperature (23°C). As shown in Figure 3, vernix detachment was significantly higher at body temperature than at room temperature. The assayed turbidity was 2.2 times greater at the higher temperature. This temperature-dependent increase in turbidity suggests that in utero spreading and detachment of vernix is facilitated by the near-constant core body temperature.

Figure 3
figure 3

Effect of temperature on vernix detachment. Two hundred μg/mL of phospholipid (Survanta®) was incubated overnight in the presence of 10 mg of vernix at either body temperature (37°C) or room temperature (23°C). The solution turbidity was significantly higher at body temperature than at room temperature, indicating a greater amount of detached vernix. All data are expressed as mean ± SEM. N = 4 for each data point.

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Effect of temperature on the rheological properties of vernix.

Rheological measurements were made at 23°C and 37°C, as described in the “Methods” section. As shown in Figure 4, vernix behaves as a relatively fluid material at body temperature compared with room temperature. The minimum shear stress required to initiate flow, i.e. the yield value, was 4000 Pa versus 10,000 Pa at 37°C and 23°C, respectively. These data corroborate the results depicted in Figure 3.

Figure 4
figure 4

Effect of temperature on the rheological properties of vernix . Rheolological measurements were performed using a Haake model RS150 controlled-stress rheometer. Flow measurements were made at 23°C and 37°C. At physiologic temperature, vernix exhibits a yield value of approximately 4000 Pa. When the sample was tested at room temperature, the yield value increased to 10,000 Pa.

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DISCUSSION

One of the most important advances in the perinatal management of high-risk pregnancies is the ability to reliably ascertain the degree of pulmonary maturity. In general, amniotic fluid is either clear or turbid at the time of amniocentesis, and this turbidity has been correlated with lung maturity by many authors over the last two decades (10–13). Based on a visual comparison between mature and immature unspun amniotic fluid specimens, a correct distinction (confirmed by biochemical markers) was recorded 87% of the time (10). Strong et al. (11) reported a similar association based on the ability to read newsprint through a test tube containing amniotic fluid. The inability to read print through amniotic fluid correlated with mature lungs. Turner and Read (12) as well as other investigators (13) have shown an association between amniotic fluid turbidity assayed at an OD of 650 nm and fetal lung maturity (13). Consequently, we chose OD measurement at 650 nm to quantify turbidity.

In this study, we hypothesized that the increase in amniotic fluid turbidity with advancing gestational age was secondary to an interaction between pulmonary surfactant and vernix caseosa. Late in the second trimester, and particularly in the third trimester, the sebaceous gland becomes a large, complex, hyperplastic structure (8, 14). Sebum, the end product of sebaceous gland secretion, forms a primary constituent of vernix caseosa (14). Vernix also contains desquamated fetal corneocytes indicating a contribution from terminal differentiation of the epidermis (15). The lung develops and matures in parallel over the same time period. At approximately 20–24 wk of human gestation, pulmonary type II cells become identifiable by the presence of lamellar inclusion bodies. These bodies contain concentric whorls of phospholipids that are slowly released into the amniotic fluid (16). Similarities between the epidermis and the lung are presented in Table 1.

Table 1 Similarities between the skin and the lung
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In this study, we used an in vitro assay to demonstrate that a progressive increase in phospholipid concentration results in an increase in vernix detachment from the surface of microfuge tubes (Fig. 1). The phospholipid concentrations used are within the physiologic range expected in the amniotic fluid toward the end of a term gestation. Total lipids in amniotic fluid are reported to increase from approximately 40 μg/mL at 20 wk gestation to 386 μg/mL at term (17). Approximately 65% of total lipids are lecithin. Lecithin concentrations increase from 43 μg/mL at 34–35 wk gestation to 147 μg/mL at term before labor. Lecithin levels are known to further increase during term labor to approximately 232 μg/mL (17).

The results of our in vitro assay lead us to speculate that amniotic fluid turbidity in vivo is secondary to “roll up” and detachment of skin surface vernix induced by increasing physiologic concentrations of pulmonary surfactant. Pulmonary surfactant is rich in phosphatidylcholine and other lipids, with proteins constituting less than 10% of its mass (18). Surfactant proteins, particularly SP-B, are known to alter phospholipid membrane organization, enhancing the surface reducing properties of phospholipids (19). In our study, we demonstrate a lack of response in the turbidity assay with a phospholipid mixture that closely mimics pulmonary surfactant but is devoid of surfactant proteins (Fig. 2). The lack of response observed when using the synthetic PLM may indicate that surfactant-associated proteins are necessary to elicit vernix detachment.

At present, there is little consensus regarding the potential role of vernix during the last trimester of pregnancy. A diffuse coating of vernix is hypothesized to provide a water-impermeable barrier to the developing fetus, which is immersed in amniotic fluid. In our study, we show that vernix detachment was significantly higher at body temperature compared with room temperature (Fig. 3). Vernix is also significantly less viscous at core body temperature than at room temperature (Fig. 4). These findings suggest that the coating, spreading, and detachment of vernix is facilitated by the thermal environment in utero. The detached vernix is subsequently swallowed by the fetus with unknown effects on the fetal foregut. Of interest, in this regard, is a recent report that vernix is rich in nitrogen-containing amino acids such as glutamine and asparagine (20). Glutamine has recently received increased attention as a possible trophic factor for the preterm infant and has been implicated in gut maturation (21, 22).

This is the first report suggesting a unique, time-dependent, and potentially relevant physiologic interaction between vernix caseosa and pulmonary surfactant during the third trimester of human gestation. These findings support a surfactant-mediated induction of vernix detachment from the fetal skin surface leading to an increase in amniotic fluid turbidity. Prenatally, the existence of a lung-skin interaction juxtaposes two major epithelial systems involved in postnatal environmental coupling. Clearly, more work must be performed to determine the nature of the interaction between pulmonary surfactant and vernix caseosa. The assay, while possessing the virtue of simplicity, contains considerable interassay variability. This may be attributable, among other factors, to differences in vernix samples, handling, or assay conditions. The lack of response of the PLM containing samples is intriguing (Fig. 2). Further work is required to assess the role, if any, of surfactant-associated proteins in altering the biophysical properties of vernix.