Characterization of Vernix Caseosa: Water Content, Morphology, and Elemental Analysis

William L. Pickens,* Ronald R. Warner,² Ying L. Boissy,² Raymond E. Boissy*³ and Steven B. Hoath*³ *The Sciences Institute and Division of Neonatology, Children's Hospital Medical Center, Cincinnati, Ohio, U.S.A.; ²The Procter & Gamble Company, Cincinnati, Ohio, U.S.A.; ³Department of Dermatology, University of Cincinnati Medical Center, Cincinnati, Ohio, U.S.A.

Recent studies have prompted interest in the use of revealed ¯attened structures approximately 1±2 mmin epidermal barrier creams as protective bio®lms for thickness with distinct cellular envelopes indicative very low birthweight preterm infants. The key to of differentiated . Compared with mature understanding the role of epidermal barrier ®lms is corneocytes in adult , vernix cor- an elucidation of their interaction with water and a neocytes appeared swollen, the density of the keratin basic knowledge of their composition. In this study, ®laments was less, and there was a relative lack of we investigated the morphologic properties and ele- tono®lament orientation. Cryofractured specimens mental composition of the naturally occurring bio- were examined by cryoscanning electron microscopy ®lm, vernix caseosa. This bio®lm is typically lacking with subsequent elemental localization by X-ray in preterm infants and its production coincides in beam analysis. The ®ndings indicate the high water utero with terminal differentiation of the content of vernix is largely compartmentalized and formation of the stratum corneum. Signi®cantly, within fetal corneocytes. These results are consistent vernix (80.5 6 1.0% H2O) had a much higher water with the novel view of vernix as a ``¯uid phase'' stra- content than other barrier creams (Eucerin: 17.1 6 tum corneum consisting of a hydrophobic 0.6%, Aquaphor: 0.33 6 0.03%, Ilex: 0.19 6 0.02%, matrix with embedded fetal corneocytes possessing petrolatum: 0.03 6 0.01%; all p < 0.05). Phase con- unique biomechanical and water-binding properties. trast microscopy of vernix showed multiple cellular Key words: /epidermal barrier/fetal skin. J Invest elements with nucleic ``ghosts'' embedded in a puta- Dermatol 115:875±881, 2000 tive lipid matrix. Transmission electron microscopy

rior to birth, the fetal mammal exhibits an orderly preterm human infants (Nopper et al, 1996). These ®ndings support program of epithelial maturation. In order to cope with the hypothesis that preterm infant skin is missing a protective the exigencies of postnatal life, environmental interfaces mantle of barrier . In the term infant, cutaneous lipids with Psuch as the lung, gut, and skin must be functionally and barrier properties include intercorneocyte lamellar lipids synthe- structurally mature. Paramount among the changes that sized by keratinocytes and the naturally occurring bio®lm, vernix occur during the last trimester of human is the caseosa. In this report, we focus on characterization of the latter. development of a highly organized stratum corneum that is Vernix caseosa is a lipid-rich material consisting of wax and sterol required for postnatal epidermal barrier function. Many of the esters, squalene, , , and free sterols structural changes occurring in skin during human fetal develop- (Karkkainen et al, 1965) as well as cellular elements (Agoratos ment have been well documented (Holbrook and Odland, 1975, et al, 1988). To our knowledge, vernix is uniquely human, 1980). The presence of functional barrier competence by the time although other structures, such as the periderm in rodents may play of birth is evident by the ®nding that transepidermal water loss in a similar part in utero (Wickett et al, 1993; Okah et al, 1995b). Other the newborn term infant is lower than steady-state adult values animals, such as sheep, produce lanolin, which also contains wax (Cunico et al, 1977). and sterol esters (Harris et al, 2000); however, unlike vernix, this Preterm delivery results in a markedly immature epidermal sebaceous secretion has not been reported to contain desquamated barrier (Okah et al, 1995a; Rutter, 1996). Attempts have been made corneocytes. In humans, vernix progressively coats the infant in a to reduce transepidermal water loss in preterm infants by the use of cephalocaudal manner during the last trimester of gestation. The hydrophobic arti®cial polymer ®lms, such as polyethylene wraps high squalene and wax ester content in vernix strongly suggests that (Baumgart, 1984) and topical agents (Rutter and Hull, 1981). a signi®cant portion of the lipid content is of sebaceous origin Recently, it was reported that application of a topical barrier cream (Downing and Strauss, 1974). The onset of vernix production containing petrolatum resulted in improved epidermal barrier approximates the time of stratum corneum formation that begins function and a decrease in nosocomial infection in low birthweight anatomically in proximity to the pilosebaceous apparatus (Hashimoto, 1970; Hardman et al, 1999). Manuscript received February 9, 2000; revised June 27, 2000; accepted A review of the literature reveals a number of biochemical for publication August 2, 2000. studies characterizing the lipid content of vernix caseosa (Downing Reprint requests to: Dr. Steven B. Hoath, Skin Sciences Institute, 231 and Greene, 1968; Nicolaides et al, 1972; Wysocki et al, 1981; Bethesda Avenue, Cincinnati, OH 45267-0541. Email: [email protected] Stewart et al, 1982), but a paucity of morphologic characterization

0022-202X/00/$15.00 ´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 875 876 PICKENS ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY and little information on vernix±water interactions. The present microscope. Alternatively, other vernix specimens were placed in 0.25% study reports direct measurement of the water content of vernix RuO4 in a 0.1 M cacodylate buffer for 1 h at 4°C, rinsed brie¯y in 0.1 M caseosa with a comparison with common epidermal barrier creams. cacodylate buffer and then dehydrated through a graded acetone series prior In order to identify the compartmentalization of water within to Epon embedding and overnight polymerization at 65°C. Thin sections were obtained using an ultramicrotome, counterstained with uranyl acetate freshly harvested human vernix, we also provide ultrastructural and lead citrate, and analyzed by transmission electron microscopy in a characterization of vernix by transmission and cryoscanning Philips CM12 at 100 KeV. electron microscopy accompanied by elemental X-ray analysis of carbon and oxygen. Cryoscanning electron microscopy and elemental analysis Freshly harvested vernix was transported from the delivery room to the laboratory MATERIALS ANDMETHODS under amniotic ¯uid. A small specimen was placed on a gold planchet, ¯ash-frozen in liquid nitrogen-cooled liquid ethane, then transferred to Vernix caseosa and standard barrier creams Within minutes of liquid nitrogen. On a cold stage under vacuum, the frozen specimen was delivery, vernix caseosa was gently scraped from the skin surface of fractured to expose a cross-section, coated with a 2 nm layer of gold/ newborn infants with a plastic spoon and stored in air-tight sterile culture palladium at ±110°C, and analyzed in an Hitachi S4500 scanning electron tubes at 4°C until analysis. Vernix collection was approved by the microscope ®tted with an Oxford cryostage thermocontrolled to ±110°C. Institutional Review Board of the University of Cincinnati Medical Image collection was performed with the electron beam acceleration Center. Aquaphor Healing Ointment and Eucerin Cream were voltage set at 2 KeV. X-ray spectra and elemental maps were obtained at a manufactured by Beiersdorf (Norwalk, CT). Ilex Skin Protectant Paste beam acceleration voltage of 4±6 KeV. The X-ray analysis was performed was manufactured by E.R. Squibb (Princeton, NJ). Petrolatum was with a Link Isis energy dispersive spectrophotometer with a thin window manufactured by CVS (Woonsocket, RI). silicon detector. Determination of the volatile content of vernix and standard RESULTS barrier creams Aliquots of freshly harvested vernix (n = 28) and standard barrier creams (n = 10) were thinly coated on to aluminum Determination of water content Specimens of freshly weigh pans and weighed using a Cahn model C-31 electrobalance. The harvested vernix caseosa and other topical barrier creams specimens were transferred to a vacuum chamber and maintained at 23°C routinely used in the newborn nursery were weighed then until constant weights were obtained. Dry weight to wet weight ratios and desiccated under vacuum until constant weights were obtained. the percentage of volatile component(s) were calculated. Dry weight to wet weight ratios and the percent of evaporative Quanti®cation of water content by Karl±Fischer titration Vernix weight loss were determined. Dry weight to wet weight ratios were specimens were obtained from three separate infants immediately after birth 0.19 6 0.01 and 0.83 6 0.02 (mean 6 SD) for vernix and Eucerin, and stored in air-tight containers at 4°C until analysis. Complete extraction of water was achieved by adding 200 mg of each vernix specimen to 25 ml of a solution containing 60% methanol and 40% formamide and incubating the mixture for 24 h at room temperature. A 3 ml aliquot of each extraction solution was removed for determination of water content by Karl±Fischer titration. The titration was performed at room temperature and pressure using a Mettler model DL-18 titrator. Karl±Fischer titration is a standard analytical technique used to determine water content. The analysis is based on the quantitative colorimetric reaction of water with an anhydrous solution of sulfur dioxide and iodine in the presence of a methanol-pyridine buffer (Houston and Poore, 1995).

Water release by vernix and standard barrier creams Aliquots of petrolatum, Aquaphor, Eucerin, and vernix (n = 6 for all groups) were thinly coated on to aluminum pans and weighed as described above. The specimens were inverted in normal saline and maintained at 37°C for 68 h. After careful blotting of excess saline, the specimens were reweighed to determine their capacity to hydrate. They were then maintained at room temperature (22±23°C) and humidity (43±46% RH) for the next 8 d. During this period, the specimens were weighed at regular intervals. The log percent of original weight was plotted over time to illustrate the rate of water release. Figure 1. Dry weight: wet weight ratios of vernix caseosa and standard topical creams used in the newborn nursery. The data Water uptake by vernix Following complete vacuum desiccation, indicate that vernix, obtained at birth from term infants, is approximately vernix specimens were rehydrated by submerging the specimens in tissue 80% volatile. Seventeen percent of Eucerin was volatile with negligible culture wells containing either deionized water or normal saline. volatility observed in the other preparations. All samples were dried to Rehydration was carried out in a humidi®ed standard tissue culture constant weight over a 1 wk period. The dry weight to wet weight ratios of environment thermoregulated to 37°C. Triplicate samples were removed at vernix and Eucerin were each statistically different from the other 24 h intervals for 5 d, excess liquid was blotted away, and the specimens preparations using one-way ANOVA and Bonferroni t test. Results are were weighed. The percentage of rehydration was determined by reported as mean 6 SD, *p < 0.05. comparison of these weights with the specimens' original weights, prior to desiccation. Table I. Determination of water content in freshly harvested Low magni®cation and phase contrast microscopy A whole mount specimen of native vernix was photographed at 4 3 magni®cation using a vernix Canon A1 35 mm camera with a macro lens. The same whole mount specimen was then gently pressed between two glass slides, examined and Karl±Fischer titration photographed by phase contrast microscopy using an Olympus IMT-2 phase contrast microscope. Gestational age of H2O content the infant (wk) (%) Transmission electron microscopy Specimens of vernix caseosa were ®xed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium 37 82.7 cacodylate buffer (pH 7.4), post®xed in 1% osmium tetroxide in 0.1 M 38 81.0 cacodylate buffer and embedded in Epon-Araldite. Sections were obtained 40 82.2 using a RMC-MT6000XL ultramicrotome and subsequently examined Mean 6 SD82.0 6 0.9 and photographed using a JEOL 100-CX transmission electron VOL. 115, NO. 5 NOVEMBER 2000 CHARACTERIZATION OF VERNIX CASEOSA 877 respectively, and uniformly high for the other preparations (Fig 1). Dehydration kinetics Freshly obtained vernix caseosa and These data indicate that approximately 80% of the original wet standard topical barrier creams were immersed in normal saline to weight of vernix is volatile. To ascertain the identity of the volatile assess their capacity to absorb water. The amount of hydration was component of vernix, Karl±Fischer titration was performed. Vernix determined by comparison of the weights before and after saline specimens from three infants were tested. Results showed that immersion. The change in weights following this hydration 82.0 6 0.9% (mean 6 SD) of the freshly harvested vernix is water procedure were ±2.6 6 1.4%, +1.0 6 0.5%, ±0.4 6 0.3%, and (Table I). These data are consistent with the weight ratios reported +0.0 6 0.7% for petrolatum, Aquaphor, Eucerin, and vernix, in Fig 1. respectively (mean 6 SD) (Fig 2a). Following hydration, the specimens were allowed to desiccate at room temperature and humidity. Data were plotted as log percent of original weight over time (Fig 2b). The resultant curvilinear plot indicates that dehydration follows ®rst-order kinetics suggesting that the release of water is a biphasic process. Rehydration of vernix caseosa Following complete desiccation, vernix was rehydrated in either deionized water or normal saline. The percent rehydration was determined by comparison with the original specimen weight prior to desiccation. Assuming linearity, lines of best ®t were determined over 5 d of rehydration. The slopes of the resultant regression lines indicate that vernix specimens rehydrated at a rate of 31% per day in deionized water and 13% per day in normal saline (Fig 3). After 3 d, vernix immersed in deionized water was resaturated to 92 6 14% (mean 6 SD) of its original weight, whereas vernix immersed in normal saline resaturated to only 42 6 4% (mean 6 SD) over the same period. Over the 5 d test period, vernix continued to rehydrate to 143 6 9% and 67 6 6% (mean 6 SD) of its original weight in deionized water and normal saline, respectively.

Figure 2. Dehydration kinetics of vernix caseosa and standard topical creams following hydration in normal saline. (a) Vernix and standard barrier creams were hydrated in normal saline over a 68 h period. Petrolatum and Aquaphor exhibited relatively small decreases in Figure 3. Rehydration kinetics of desiccated vernix caseosa in weight following hydration. Eucerin demonstrated a slight increase in either normal saline or deionized water. Following complete weight, whereas, on average, vernix caseosa weight did not change. (b) desiccation, vernix was rehydrated over a period of 5 d in either deionized Following hydration, the specimens were allowed to desiccate under water or normal saline. Rehydration rates and the percentage of ambient conditions. The rate of desiccation over 8 d is shown. All data are rehydration were determined by comparison with the original specimen reported as mean 6 SD. weight. Data are reported as mean 6 SD.

Figure 4. Low magni®cation and phase con- trast microscopy of vernix caseosa. These photomicrographs depict the dual nature of vernix caseosa: (a) A specimen of freshly harvested vernix caseosa seen under low magni®cation. To the naked eye, vernix appears to be a sticky white li- pid paste. (b) The same vernix viewed by phase contrast microscopy. A dense packing of fetal cor- neocytes can be seen surrounded by a presumptive lipid matrix. Of particular note is the observation that vernix caseosa is primarily a cellular moiety. 878 PICKENS ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Morphologic studies and elemental analysis Vernix was corneum, suggesting that they may be swollen with imbibed imaged under low magni®cation (Fig 4a). Because it is partially amniotic ¯uid. comprised of sebaceous-derived wax esters, vernix has the Transmission electron microscopy was used to reveal ultra- appearance and feel of a lipid material. When this same specimen structural features of both fetal corneocytes and intercellular lipids was ¯attened between two glass slides and viewed by phase contrast (Fig 5a±c). The fetal corneocytes are devoid of nuclei and other microscopy, it was apparent that in addition to the lipid organelles. They contain a sparse network of keratin ®laments and component, a large number of cells were present (Fig 4b). The exhibit little evidence of tono®lament orientation. The corneocytes phase contrast photomicrograph reveals a dense packing of fetal are approximately 1±2 mm in thickness. They lack desmosomes and corneocytes surrounded by a presumptive lipid matrix. The are surrounded by a thickened layer of amorphous lipid. In some diameter of these corneocytes is approximately 40 mm. Their size locations, the corneocytes appear malleable as evidenced by is greater than one sees from corneocytes from intact stratum bifurcation and curvature of the corni®ed cell envelope (Fig 5a). The intercellular lipid contains unidenti®ed inclusion bodies presumably of a proteinaceous nature (Fig 5b). Whereas most of the intercellular lipid is amorphous, occasionally, lamellar structures are observed between closely apposed corneocytes (Fig 5c). Cryofractured specimens of vernix caseosa were examined by cryoscanning electron microscopy. Here again, vernix caseosa is characterized by the numerous corneocytes embedded in lipid matrix as shown under relatively low magni®cation (Fig 6a). The corneocytes are covered with lipid that sometimes exhibits a layered orientation resulting in a lea¯et appearance (Fig 6b). Whereas much of this lipid covering appears smooth, intercorneo- cyte lipids sometimes contain granular inclusions (Fig 6c). Because cryopreparation preserves the original water content and distribu- tion, its localization within vernix can be ascertained. Water-®lled corneocytes were observed throughout vernix (Fig 7a). Elemental analysis was used to determine the distribution (Fig 7b) and the relative abundance (Fig 7c) of water in the specimens. X-ray maps of oxygen and carbon delineate the spatial relationship of water within vernix caseosa by identifying the oxygen (water) and carbon (lipid) compartments. Analysis of numerous sections revealed that all observed corneocytes were hydrated, although differential amounts of hydration were noted.

DISCUSSION The mechanisms whereby a competent epidermal barrier develops under conditions of aqueous immersion in utero are unclear. In human adult skin, extensive water exposure in vivo has been shown to disrupt both corneocytes and stratum corneum lipid lamellae (Warner et al, 1999). When in vitro human skin cultures are grown submerged in media, epidermal cells are relatively undifferentiated, whereas cultures lifted to an air±liquid interface exhibit epidermal strati®cation and corni®cation characteristic of normal human skin (Prunieras et al, 1983; Harriger and Hull, 1992). Likewise, lifting cultured fetal rat skin explants accelerates corni®cation and barrier maturation (Hanley et al, 1997). It has also been reported that control of the water gradient across the epidermis is a key factor in regulating lipid and DNA synthesis in barrier repair (Proksch et al, 1993). These ®ndings support the hypothesis that the fetal mammal possesses mechanisms regulating the transepidermal water gradient

Figure 5. Transmission electron microscopy of vernix caseosa. These photomicrographs illustrate ultrastructural characteristics of both the cellular and the intercellular lipid components of vernix. Panel (a) reveals features of the numerous fetal corneocytes (*) that comprise vernix. These corneocytes are devoid of nuclei and other organelles and contain a sparse network of keratin ®laments with little evidence of tono®lament orientation. In some locations, the corneocytes appear malleable as shown by bifurcation and curvature of the corni®ed cell envelope (arrowheads). No intercorneocyte desmosomal attachments are observed. Panels (b) and (c) are higher magni®cation photomicrographs that depict aspects of the intercellular lipids. Panel (b) shows amorphous intercellular lipids (#) that contain unidenti®ed inclusion bodies presumably of a proteinaceous nature (arrows). A corneocyte (*) with its sparse tono®lament content can be seen in the bottom portion of the photomicrograph. Whereas most of the intercellular lipid is amorphous, panel (c) reveals that occasional lamellar structures (arrow) are observed between closely apposed corneocytes (*). VOL. 115, NO. 5 NOVEMBER 2000 CHARACTERIZATION OF VERNIX CASEOSA 879

caseosa. To date, most of the literature pertaining to vernix caseosa has focused on its lipid components (Nicolaides et al, 1972; Wysocki et al, 1981; Stewart et al, 1982). A review of the literature revealed a single report that identi®ed and partially characterized the cellular component of vernix caseosa (Agoratos et al, 1988). In our study, we investigated the morphology of this naturally occurring bio®lm with particular emphasis placed on its cellular component and water content. Our initial ®nding that approximately 80% (wt/wt) of freshly harvested vernix was volatile suggested a high water content (Fig 1). Subsequent analysis by Karl±Fischer titration, an assay speci®c for water, con®rmed that the volatile component was, in fact, water (Table I). Paradoxically, vernix is a highly viscous material. For a substance to comprise 80% of its weight as water and retain high viscosity, its water must reside within a highly structured state. Hypothetically, this structure is conferred by the abundance of water-®lled fetal corneocytes found throughout vernix. Ultrastructural examination of the corneocytes in vernix caseosa revealed that these fetal cells can be easily distinguished from adult corneocytes in the stratum corneum by the lack of desmosomal attachments (Fig 5a). Subjectively, we observed apparent decreases in tono®lament orientation and keratin density and an increase in malleability of the fetal corneocytes compared with routine sections of adult stratum corneum. Additionally, these fetal corneocytes appear to be swollen. Normal adult corneocyte thickness is approximately 0.5 mm (Odland, 1991), whereas the thickness of vernix-embedded corneocytes was 1±2 mm. This increase in corneocyte volume is likely to be due to the high water content, an impression that is corroborated by both X-ray analysis and Karl± Fischer titration. Further studies need to be performed to quantify this initial impression. Both transmission and cryoscanning electron microscopy show granular inclusions in the matrix surrounding the corneocytes (Figs 5b,6c). These bodies may represent proteinac- eous material of keratinocyte origin or sebocyte debris. Whereas vernix caseosa initially loses water rapidly when exposed to air, complete desiccation at room temperature and humidity can take days to weeks. The curvilinear shape of our desiccation kinetics plot suggests that there are two separate compartments of water within vernix (Fig 2b). The rapidly released water may be associated with the granular inclusions described above or possibly with polar lipids in vernix, whereas the slowly released water is contained within fetal corneocytes. Our data also show that once vernix is completely dehydrated, rehydration to its original water content is a slow process (Fig 3). The slow rates of dehydration and rehydration are consistent with the histologic ®nding that vernix contains water saturable corneocytes surrounded by lipids that are resistant to aqueous diffusion. The associated ®nding that normal saline hydrates vernix caseosa more slowly and to a lesser degree than deionized water is hypothetically due to differences in osmolality. In previous work we reported that, following application to human skin, vernix caseosa had water handling capabilities and Figure 6. Cryoscanning electron microscopy of vernix caseosa. The hydration pro®les that distinguished it from other topical barrier orientation of fetal corneocytes (*) embedded within the lipid matrix of creams used on infants, such as petrolatum-based ointments and vernix caseosa is depicted at (a) low and (b) high magni®cation. Panel (a) water-in-oil emulsions (Bautista et al, 2000). In this study, we Shows a stacking arrangement of the fetal corneocytes, whereas (b) attempted arti®cially to hydrate vernix caseosa and selected topical illustrates that the lipid covering the corneocytes is arranged in a smooth barrier creams in normal saline (Fig 2a). Paradoxically, petrolatum lea¯et-like orientation (arrowheads). A corneocyte (*) is depicted at the left- and Aquaphor each lost a small amount of weight. These weight hand side of the photomicrograph. Because this portion of the cell is devoid losses can be explained by the partial dissolution of the materials as of lipid covering and partially fractured, the interior tono®laments are evidenced by the presence of ®lms on the top of the hydrating exposed and present an irregular surface. Panel (c) reveals granular inclusions in the intercorneocyte lipid (arrows). Presumably, these inclusions saline solutions. In contrast, Eucerin absorbed approximately 1% of are the same type of structures that were noted in Fig 5(b). The asterisks its initial weight. It is possible that this absorption of water is due to denote lipid-covered corneocytes. the hygroscopicity of the glycerin within the formulation. Vernix, however, neither absorbed nor lost water as observed by the lack of any appreciable weight change. The osmolality of amniotic ¯uid at with facilitation of epidermal barrier development in an aqueous term has been reported to be 252 6 16 mmol per kg (Benzie et al, intrauterine environment. 1974), which approximates that of normal saline. The hydration of One putative mechanism for the modulation of the transepi- vernix in utero, however, may not be solely a function of amniotic dermal water gradient is the production of the fetal bio®lm, vernix ¯uid osmolality. Other factors such as the presence or absence of 880 PICKENS ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY and its associated proteins may play a role. levels of cholesterol and wax esters in the newborn are These components of amniotic ¯uid have been shown to interact indistinguishable from the levels seen at puberty, another period with vernix, affecting its rheologic properties (Narendran et al, when there is a marked increase in sebaceous activity (Ramasastry 2000). et al, 1970). Whereas much of the lipid content of vernix is likely to The ®ndings of several investigators indicate that sebaceous be of sebaceous origin, lamellar lipids were occasionally observed secretions are abundant during late gestation. As early as 1936, it (Fig 5c). These lamellar lipids may represent intercorneocyte lipids was documented that skin surface lipid levels in the term neonate from detached sheets of fetal stratum corneum associated with are higher than those found in adults (Emanuel, 1936). It has been covalently bound lipid envelopes. Our working hypothesis is that reported that the last trimester of pregnancy is marked by a surge in antenatal sebaceous secretions act synergistically with the develop- fetal activity (Ponchi, 1982) and that skin surface ing epidermis to promote terminal differentiation and establishment of a rudimentary stratum corneum. Consistent with this hypothesis are reports that corni®cation begins in the vicinity of the pilosebaceous units (Hashimoto, 1970; Hardman et al, 1999). It is likely that prior to corni®cation, sebaceous lipids cover the skin surface and establish a hydrophobic barrier between it and the aqueous uterine compartment. Amniotic ¯uid would therefore, be excluded from the surface of the precorni®ed cells resulting in a potential ``xeric stress'' that would be the functional equivalent of air-lifting cultured skin. As the stratum corneum matures in utero, fetal corneocytes detach and become embedded in the overlying lipid. Therefore, rather than the traditional view of vernix caseosa as a thick lipid paste, vernix would be more accurately described as a combination of fetal corneocytes surrounded by a matrix of sebaceous and epidermal lipids. We surmise that an important function of vernix caseosa is to modulate water activity at the skin surface and thereby promote corni®cation and protect the developing epidermal barrier from the deleterious effects of extended and excessive water exposure. In this context, the corneocytes in vernix would act as ``sinks'' to interdict water moving across the fetal skin, whereas the sebaceous lipids in vernix would provide a hydrophobic barrier. Stratum corneum lipids, hypothetically, also comprise a component of the lipid mixture within vernix, although the typical lamellar structure is only rarely observed. In any case, the control of transepidermal water movement by synthesis of a super®cial lipid ®lm that is progressively infused with a large number of cellular ``sponges'' may be an important mechanism for facilitating and maintaining corni®cation in utero under aqueous conditions. Finally, it should be noted that vernix provides a very different type of barrier to stratum corneum desiccation compared with commercially available topical preparations. The high water content, the rich contingent of embedded corneocytes, the surrounding matrix of physiologic lipids and the human fetal origin of this material are unique for a barrier cream. These results are consistent with the novel view of vernix as a ``mobile'' or ``¯uid phase'' stratum corneum lacking rigid desmosomal connections. Experimentally, this viewpoint may prove useful in studying the role of sebaceous secretions and their interaction with corneocytes in the formation of the human skin surface lipid ®lm (Sheu et al, 1999).

Figure 7. Cryoscanning electron microscopy and elemental analysis of vernix caseosa with emphasis on the water content and orientation. Specimens of vernix caseosa were cryofractured and imaged by cryoscanning electron microscopy. These photomicrographs illustrate the orientation of water within vernix. (a) Depicts a single corneocyte (*) that is partially fractured to reveal intermediate ®laments within the cell. Using elemental analysis, the distribution of water in vernix is illustrated in (b). The image in the upper left panel of (b) depicts the normal appearance of a single corneocyte by cryoscanning electron microscopy. This corneocyte is indicated throughout the four panels in (b) by asterisks. Corresponding X-ray maps for oxygen (lower left panel) and carbon (upper right panel) show the distribution of water and lipid, respectively. The X- ray map in the lower right panel re¯ects the distribution of sulfur and is used as a background element to check for X-ray absorption artifacts due to surface topography effects. These X-ray maps reveal compartmentalization of water and lipid but not sulfur. The spectrum of these data, shown in (c), illustrates the high level of oxygen (water) relative to carbon (lipid) in this specimen. VOL. 115, NO. 5 NOVEMBER 2000 CHARACTERIZATION OF VERNIX CASEOSA 881

The authors would like to thank the nurses and residents in Labor and Delivery at Houston TE, Poore MW: The application of the Karl Fischer oven for the determination of water in consumer products. J Air Waste Mgt Assoc 46:990± University Hospital. This work was funded in part by grant RO1NR03699 from 992, 1995 the National Institute of Nursing Research and by the National Occupational Karkkainen J, Nikkari T, Ruponen S, Haahti E: Lipids of vernix caseosa. J Invest Research Agenda (NORA) of the Institute of Occupational Safety and Health. Dermatol 44:333±338, 1965 Narendran V, Pickens WL, Wickett RR, Hoath SB: Interaction between pulmonary surfactant and vernix: a potential mechanism for induction of amniotic ¯uid turbidity. Pediatr Res 48:120±124, 2000 REFERENCES Nicolaides N, Hwei C, Anari MNA, Rice GR: The fatty acids of wax esters and Agoratos T, Hollweg G, Grussendorf EI, Paploucas A: Features of vernix caseosa sterol esters from vernix caseosa and from human skin surface lipid. Lipids cells. Am J Perinatol 5:253±259, 1988 7:506±517, 1972 Baumgart S: Reduction of oxygen consumption, insensible water loss, and radiant Nopper AJ, Horii KA, Sookdeo-Drost S, Wang TH, Mancini AJ, Lane AT: Topical heat demand with use of a plastic blanket for low-birth-weight infants under ointment therapy bene®ts premature infants. J Pediatr 128(Part 1):660±669, radiant warmers. Pediatrics 74:1022±1028, 1984 1996 Bautista MI, Wickett RR, Visscher MO, Pickens WL, Hoath SB: Characterization of Odland GF. Structure of the skin. In: Goldsmith LA (eds). Physiology, Biochemistry, vernix caseosa as a natural bio®lm: comparison to standard oil-based ointments. and Molecular Biology of the Skin. New York: Oxford University Press, 1991, p. Pediatr Dermatol 17:253±260, 2000 10 Benzie RJ, Doran TA, Harkins JL, Jones Owen VM, Porter CJ: Composition of the Okah FA, Wickett RR, Pickens WL, Hoath SB: Surface electrical capacitance as a amniotic ¯uid and maternal serum in pregnancy. Am J Obstet Gynecol 119:798± noninvasive bedside measure of epidermal barrier maturation in the newborn 810, 1974 infant. Pediatrics 96:668±692, 1995a Cunico RL, Maibach HI, Kahn H, Bloom E: Skin barrier properties in the newborn. Okah FA, Pickens WL, Hoath SB: Effect of prenatal steroids on skin surface Transepidermal water loss and carbon dioxide emission rates. Biol Neonate hydrophobicity in the premature rat. Pediatr Res 37:402±408, 1995b 32:117±182, 1977 Ponchi P. The sebaceous gland: In: Maibach HI, Boisits EK (eds). Neonatal Skin Downing DT, Greene RS: Double bond positions in the unsaturated fatty acids of Structure and Function. New York: Marcel Dekker, 1982, pp 67±80 vernix caseosa. J Invest Dermatol 50:380±386, 1968 Proksch E, Holleran WM, Menon GK, Elias PM, Feingold KR: Barrier function Downing DT, Strauss JS: Synthesis and composition of surface lipids of human skin. J regulates epidermal lipid and DNA synthesis. Br J Dermatol 128:473±482, 1993 Invest Dermatol 62:228±244, 1974 Prunieras M, Regnier M, Woodley D: Methods for cultivation of keratinocytes with Emanuel SV: Quantitative determinations of the sebaceous gland's function, with an air±liquid interface. J Invest Dermatol 81(Suppl. 1):28s±33s, 1983 particular mention of the method employed. Acta Derm Venereol 17:444, 1936 Ramasastry P, Downing DT, Pochi PE, Strauss JS: Chemical composition of human Hanley K, Jiang Y, Elias PM, Feingold KR, Williams ML: Acceleration of barrier skin surface lipids from birth to puberty. J Invest Dermatol 54:139±144, 1970 ontogenesis in vitro through air exposure. Pediatr Res 41:293±299, 1997 Rutter M, Hull D: Reduction of skin water loss in the newborn: Effect of applying Hardman MJ, Moore L, Ferguson MW, Byrne C: Barrier formation in the human topical agents. Arch Dis Child 56:669±672, 1981 fetus is patterned. J Invest Dermatol 113:1106±1113, 1999 Rutter N: The immature skin. Eur J Pediatr 155:S18±S20, 1996 Harriger MD, Hull BE: Corni®cation and basement membrane formation in a Sheu HM, Chao SC, Wong TW, Yu-Yun Lee J, Tsai JC: Human skin surface lipid bilayered human skin equivalent maintained at an air±liquid interface. J Burn ®lm: an ultrastructural study and interaction with corneocytes and intercellular Care Rehabil 13:187±193, 1992 lipid lamellae of the stratum corneum. Br J Dermatol 140:385±391, 1999 Harris I, Hoppe U: Lanolins. In: Loden M, Maibach HI (eds). Dry Skin and Moisturizers: Chemistry and Function Dermatology. Clinical and Basic Science Stewart ME, Quinn MA, Downing DT: Variability in the composition of Series. New York: CRC Press, 2000, pp 259±267 wax esters from vernix caseosa and it possible relation to sebaceous gland Hashimoto K: The ultrastructure of the skin of human embryos IX. Formation of the activity. J Invest Dermatol 78:291±295, 1982 hair cone and intraepidermal hair canal. Arch Klin Exp Dermatol 238:333±345, Warner RR, Boissy YL, Lilly NA, Spears MJ, McKillop K, Marshall JL, Stone KJ: 1970 Water disrupts stratum corneum lipid lamellae: Damage is similar to surfactants. Holbrook KA, Odland GF: The ®ne structure of the developing human epidermis: J Invest Dermatol 113:960±966, 1999 Light, scanning and transmission electron microscopy of the periderm. J Invest Wickett RR, Mutschelknaus JL, Hoath SB: Ontogeny of water sorption-desorption Dermatol 65:16±38, 1975 in the perinatal rat. J Invest Dermatol 100:407±411, 1993 Holbrook KA, Odland GF: Regional development of the human epidermis in the Wysocki SJ, Grauaug A, O'Neill G, HaÈhnel R: Lipids in forehead vernix from ®rst trimester embryo and the second trimester fetus (ages related to the timing newborn infants. Biol Neonate 39:300±304, 1981 of amniocentesis and fetal biopsy). J Invest Dermatol 74:161±168, 1980