= = = = = = = = = ANNALS
Of ANATOMY
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Vaginal reconstruction by skin grafts: A scanning electron microscopic evaluation F. Barberini, E. Vizza, M. Montanino, S. Marcoccia* and G. Montanino* Department of Anatomy and *2nd Department of Obstetrics and Gynecology, University of Rome "La Sapienza", Via Alfonso Borelli, 50, 1-00161 Rome, Italy
Summary. In order to evaluate the morphofunctional pattern
Key words: Neovagina - Skin autografts - Ultrastructure
pathogenesis has been and remains still controversial, since there are different opinions as to the embryological origin of the vagina. Two different organogenetic theories were advanced on this matter: a dualistic, and a unitary theory, each suggesting a different explanation for vaginal agenesis. According to the classic or dualistic embryogenetic theory (Koff 1933), only the lower part of the vagina is of ectodermal origin, deriving from the urogenital sinus, whereas the remaining part is of Mullerian (i.e. mesodermal) origin. With reference to the unitary theory suggested by Meyer (1934) and Bulmer (1957) and recently resumed by HoangNgoc Minh and others (1984), the vagina derives throughout from the vaginal plate (proliferation of the urogenital sinus) and, consequently, is of ectodermal origin. The Mullerian role in the development of the vagina would thus be indirect, with the interposition of a mesenchymal tissue inductor which would then stimulate a cranial proliferation of the vaginal plate. According to this theory, vaginal aplasia might depend upon a defect in the mesenchymal tissue inductor and may be associated with both functional and non-functional uteri. During this century, various surgical techniques of vaginal reconstruction have been proposed, some of which by using dermoepidermal autografts (McIndoe and Bamister 1938). Patients treated in this way refer, one year after operation, a satisfactory sexual activity, and the macroscopic features of the "neovagina" seem to confirm this. Therefore, a structural and ultrastructural investigation of the skin transplanted to create the neovaginae was undertaken, attempting a morphofunctional evaluation of the methods employed.
Introduction
Materials and methods
Vaginal agenesis, a congenital defect, occurs less frequently (1 : 4000) than the malformations of the upper parts of the female genital tract with which it is sometimes associated (Muller and Dellenbach 1967; Musset et al. 1973). Its
Three cases of vaginal agenesis have been examined. Two of these were associated with a rudimentary uterus (karyotype 46,xx "Rokitansky-Kuster-Hauser syndrome"), while the third case showed testicular feminization (karyotype 46,xy - "Morris syn
of transplanted skin structural and ultrastructural studies on biopsies taken from surgically created neovaginae in three cases of vaginal agenesis were performed one and two years postoperatively. Two of the cases were associated with a rudimentary uterus, while the remaining one involved testicular feminization. Neovaginae were constructed by directly prolonging a pre-existing perineal introitus, to form a vesicorectal pouch, which was lined inside with gluteal skin autografts. All three patients reported satisfactory psychosexual behavior one year after operation. Light microscopy of the bioptic fragments demonstrated, even two years after surgery, features generally similar to those of the normal dermis and epidermis, and a keratinization rate as a rule, that did not range significantly from the norm. Scanning electron microscopy also confirmed the maturation of the cornified cells, since these were often seen to be desquamating and exhibited structures usually found in normal skin, such as double edges on the luminal surface - usually considered to be the morphological expression of squamous cell overlapping - and microwrinkles on disrupted apical and basal cytoplasmic flaps, which are considered reciprocal cell-gearing devices. Our results seem to indicate, therefore, that the transplanted skin, maintaining, on the whole, its integrity and viability despite an environment differing somewhat from the external one with regard to moisture, pH and temperature, shows a good adaptability, at least until the above mentioned time lapses.
Ann. Anat. (1992) 174: 517-522 Gustav Fischer Verlag Jena
drome"). In all cases, a neovagina was created by directly prolonging a pre-existing, natural perineal introitus to form a vesicorectal pouch of about 10-12 cm in length. This pouch was then lined with autologous skin grafts taken from the gluteal regions and supported by means of a plastic stent. This stent was subsequently replaced with others, until complete adhesion of the grafts occurred. Biopsies of the surgically formed cavity wall (neovagina) were performed one and two years postoperatively, at 3 cm from the introitus, in each of the three patients (Fig. 1).
- ---lIeovoglno • Biopsy Fig. 1. This drawing illustrates the vesicorectal pouch (dotted line: neovagina) and the zone from which biopsies (black square, arrowhead) were taken. v = urinary bladder; r = rectum.
Fragments having a diameter of approximately 0.5 cm were fixed in 2.5 % 0.1 M glutaraldehyde solution buffered with sodium cacodylate to pH 7.35. After washing in running water for two hours, the fragments were dehydrated in ethanol and critical-point dried (Porter et al. 1972; Morta et al. 1977). The specimens were then shadowed with a gold and platinum coat in a high-vacuum sputter coater (Edwards) and examined in a Cambridge Stereoscan 150 scanning electron microscope (SEM) operating at 8 - 20 k V . For a better morphological evaluation of the keratinization rate, the samples already examined by SEM were embedded in Historesin and then cut in 3 !-tm-thick sections. These were stained with hematoxylin-eosin and Dane and Herman's tetrachromic method. The latter is specific for keratin and prekeratin identification in that these substances are dyed orange or reddish-orange, and the cell nuclei brown. Finally, the sections were examined and photographed with a Zeiss Photomicroscope II.
Results In overall views by SEM, biopsies taken one year after surgery exhibit a continuous carpet of flattened cells, often desquamating (Fig. 2). They have an irregularly rectangular or, more frequently, a polygonal shape, and are piled one on top of the other, so that the whole surface has a cobbled aspect. One may also detect the double cell edges specific to the squamous elements of the horny layer and due to edge overlapping, the outer edge corresponding to the real cell
edge and the inner one representing the pattern of the overlying exfoliated cell (Fig. 3). Our three-dimensional study at higher magnification stresses that the luminal side of the more superficial cell layer is characterized by a delicate pattern of microwrinkles, due to thin evaginations of the apical plasmalemma, rather similar on the whole to superficial epidermal microfolds. Similar formations were also noticed on the basal side of desquamating elements, to which correspond structures of identical significance on the opposite side of the underlying cells (Fig. 4). The presence of adnexal formations, such as hair and hairy follicles, was sometimes detected. In these locations, cells outlining the introitus may assume a typical vorticose pattern (Fig. 5). Samples taken two years after surgery show surface cells similar to those previously observed. In fact, epidermal cell layers consist of thin elements with edges tracing irregular polygons and detaching from underlying layers (Fig. 6). At higher resolution, cell boundaries are easily detected, as are double-edged images, typical of the overlapping elements described above (Fig. 7). In the interstitium between detaching cells and elements immediately subjacent to them, delicate microwrinkles of the apical and basal plasmalemma were also observed, as "gearing" microstructures between opposite cell surfaces (Fig. 8). Light microscopical (LM) observations performed two years postoperatively reveal an essentially normal aspect of the grafted skin, in both its epithelial (epidermis) and connective (dermis) components. The various layers of the epidermis still show substantially unaltered features, their morphology being easily recognizable throughout the epithelium. Likewise, the dermis does not show appreciable structural variations and, at the epidermal junction, rises into well developed papillae (Fig. 9a). The specific staining for keratin and prekeratin showed that even the epithelial cornified cytomorphosis remains, quantitatively, almost unchanged two years after surgery (Fig. 9b).
Discussion In cases of vaginal agenesis, the more or less invasive
surgical approaches to restore, even partially, the patient's anatomical integrity and sexual function have not always led to satisfactory results. Some of these involve the creation of a vesicorectal pouch and the introduction of a prosthesis (Wharton 1938) or an olive put under traction by abdominal way (Vecchietti 1965), or this pouch was subsequently lined inside with various tissues, such as amnion (Brindeau 1935; Burger 1947), bowel (Baldwin 1904; Schubert 1936; Runge 1951; Schmid 1956) or peritoneum (Bloch 1961). Other methods were bloodless, consisting in dilation of the urogenital sinus (Frank 1938; Speroff et al. 1986). We preferred to cover the surgically constructed vaginae with autologous skin grafts, according to the method of McIndoe and Barnister (1938), assuming that, since the cutaneous tissues closely resemble the ones lining the normal vaginal wall, these would be suitable.
518
Fig. 2. Surface layer of dermo-epidermal autograft, showing flattened, desquamating epithelial cells. One year after surgery; SEM, x455. Fig. 3. The squamous cells appear irregularly polygonal and assume, on the whole, a stonelike aspect. Note the presence of double cell edges (---"), due to overlapping of piled-up elements. One year after surgery; SEM, xl ,820. Fig. 4. An exfoliating cell showing upset of a cytoplasmic flap. The apical (white arrow) and basal (black arrow) surfaces are characterized by slight rnicrowrinkles (mw) of fhe plasmalemma, similar to the ones found in the erpidermis. One year after surgery; SEM, x9,100. Fig. 5. A hair arising from its follicle projects toward fhe inside of the neovaginal lumen. The cells surrounding fhe introitus of fhe follicular cavity (*) assume a vorticose arrangement. One year after surgery; SEM, x540. 6. Cells of fhe superficial layer of fhe dermo-epidermal autograft, basically similar to fhose seen in Fig. 1. ec = exfoliated cells. Two years after surgery; SEM, x450. Fig. 7. The squamous elements exhibit a clearly polygonal shape and well defined boundaries, which are often double (---"). Two years after surgery; SEM, 1,820.
519
Fig. 8. During epithelial sloughing, slight microwrinkles (mw) are seen in the opposite cell surfaces (apical: white arrow; basal: black arrow), and are meant for reciprocal gearing. (Two years after surgery; SEM, x2,300.)
Fig. 9. Dermo-epidermal autograft sections: a) All epidermal cell layers are well recognizable and do not denote appreciable histologic alterations. The dermis, too, seems to be substantially unaffected; b) The cutaneous keratinization rate does not present significant quantitative variations as compared to the rule . hc == cornified cells; be = basal cells; d-e j = dermoepidermal junction; d = dermis; p = dermal papilla. (Two years after surgery; LM, X 112; a) hematoxylin-eosin; b) Dane and Herman's tetrachrome method for keratin and prekeratin.)
520
Morphological studies of skin grafted in order to form neovaginae are very scarce (Holmstrom et al. 1976; Lathrop et al. 1985). The results we obtained by LM and SEM demonstrate that dermo-epidermal autografts have, one or two years after surgery, morphological features generally similar to those of the pre-grafted tissue. In fact, the grafted epidermis shows structural and ultrastructural aspects closely resembling those occurring during the normal process of corneal cytomorphosis. In particular, all cell layers of the graft are well differentiated: from the basal layer, made up of prismatic elements, to the superficial one, consisting of flattened cornified cells, piled up in columns . In addition, no significant reduction in thickness of the cornified layer was noted . This is in contrast to the opinion that an environment different from the skin surface because of high humidity, pH and bacterial flora, may induce a loss of the most superficial cell layers (Holmstrom et al. 1976) . The results of our LM studies using specific stains for keratin clearly show that the normal keratinization rate is preserved. Further, our SEM observations reveal the presence of low, steplike depressions, double cell contours and structures of reciprocal "gearing", such as superficial and basal microwrinkles, being all morphological features found in normal epidermal squamous elements (Menton and Eisen 1971 ; Hashimoto and Kanzaki 1975). The regular arrangement ofthe cornified layer and the preservation of its specific structural features, such as the piled-up aspect seen in normal cells, as well as the desquamation of the surface elements, are a sign of a valid cell turnover and consequently of morphofunctional integrity. Furthermore, considering the regular columnar arrangement of the cornified layer, it is logical to assume that the mitotic activity of the normal basal layer is maintained, this being an essential premise for a systematic layout of overlying layers (Menton and Eisen 1971). As far as the dermis is concerned, no significant morphological changes were observed in comparison to the structure of the dermis in normal skin. However, two years after surgery, we have not been able to confirm the presence of adnexal formations such as hair and hairy follicles, which, on the contrary, were seen before. Neverthless, this is not conclusive enough evidence for justifying a modification in the graft viability, after a given lapse of time. In fact, we believe that such formations cannot occur in all bioptic fragments , because of their small size. Our structural and ultrastructural observations lead us to the conclusion that grafted skin, while maintaining its morphological integrity and even showing a clear viability, has good adaptive capacities in spite of altered conditions, at least during the first two years after surgery. In fact, the major properties of skin, such as imperviousness and resistance to exogenous thermal, chemical and mechanical stresses, are mainly based upon the keratinization and integrity of the stratum corneum. Such properties, absent in the normal vaginal mucosa , are a limit, of course, to the result of surgery, sometimes making intercourse difficult and causing dyspareunia, because of scarce superficial lubrication and minor elastic relaxation of the walls. Owing to these first results, the present study of the
grafted skin might be extended over longer time intervals, so as to evaluate possible time-dependent morpho functional modifications , and to determine the overall long-term fate of the autologous neovagina. Acknowledgments. The authors would like to thank Prof. Pietro M. Motta, for critically reading the paper and Mr. David W. Finn in drawing up the manuscript. This investigation was supported by grants from M. P. I. (60%), Italy , 1987/1988.
References Baldwin JF (1904) Formation of an artificial vagina by intestinal transplantation. Am Surg 40: 398-409 Bloch P (1961) Un nouveau traitment chirurgical de I'absence du vagin. Ber III. WeltkongreB fUr Gynlikologie , Bd II, Wien Brindeau A (1935) Kiinstliche Scheide mit Hilfe einer reifen Eihaut. Zbl Gynlik 59 : 1196-1197 Bulmer D (1957) The development of the human vagina. J Anat 91: 490-509 Burger K (1947) Weitere Erfahrungen iiber die kiinstliche Scheidebildung mit Eihliuten. Zbl Gynlik 69: 1153-1158 Frank RT (1938) Formation of an artificial vagina without operation. Am J Obstet Gynecol 35 : 1053-1055 Hashimoto K, Kanzaki T (1975) Surface ultrastructure of human skin . Acta Dermatovener 55: 413-430 Hoang-Ngoc Minh, Smadja A, Belaisch J, Chayo M (1984) Les malformations congenitales du vagin : interpretations embryogenetiques . Gynecologie 3: 195-204 Hol~strom H, Heyden G, Johanson B (1976) Congenital absence of the vagina . A clinical , histological and histochemical study on 17 patients with graft-constructed vaginas. Scand J Plast Reconstr Surg 10: 231-236 Koff AK (1933) Development of the vagina in the human fetus. Contributions to embryology. Carnegie Institution of Washington 140: 59-90 Lathrop JC, Jung Ree H, McDuff HC (1985) Intraepithelial neoplasia of the neovagina. Obstet Gynecol 65: 91S-94S McIndoe AH , Barnister DB (1938) An operation for the cure of congenital absence of the vagina. J Obstet Gynecol Brit Emp 45: 490-494 Menton DN, Eisen AZ (1971) Structure and organization of mammalian stratum corneum. J Ultrastr Res 35: 247-264 Meyer R (1934) Zur Frage der Entwicklung der menschlichen Vagina. Teill. Von der Bildung des distalen Endes der Miillerschen Vagina bis zum beginnenden Ersatz des Miillerschen Epithels durch Sinusepithel. Arch Gynecol 158: 639-738 Motta PM, Andrews PM, Porter KR (1977) Microanatomy of cell and tissue surfaces. An atlas of scanning electron microscopy. Lea & Febiger, Philadelphia Miiller P, Dellenbach P (1967) Malformations congenitales des trompes et de l'uterus . Encyc10pedie Medico-Chirurgicale 123 Musset R, Poitout P, Paniel BJ (1973) Malformations congenitales du vagin, de l'isthme et du col uterin . Encyclopedie MedicoChirurgicale 120 Porter KR , Kelley D, Andrews PM (1972) The preparation of cultured cells and soft tissues for scanning electron microscopy.
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In: Proceedings of the 5th Annual Stereoscan Colloquium. Kent Cambridge Scient, pp 1-19 Runge H (1951) Zur Technik der Vaginalplastik nach Burger bei Atresia vaginae. Zbl Gyniik 79: 559-567 Schmid HH (1956) Scheidenbildung aus dem S-fOrmigen Dickdarm. Fischer, Jena Schubert G (1936) Die kiinstliche Scheidenbildung aus dem Mastdarm nach Schubert. Enke, Stuttgart
Speroff L, Glass RH, Kase NG (1986) Endocrinologia ginecologica clinica e infertilita'. Ed. Ermes, Milano, pp 323- 348 Vecchietti G (1965) Neovagina nella sindrome di RokitanskyKuster-Hauser. Attual Ost Ginec II: 131-143 Wharton LR (1938) A simple method of constructing a vagina. Ann Surg 107: 842-849 Accepted July II, 1989
522
Of ANATOMY
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Vaginal reconstruction by skin grafts: A scanning electron microscopic evaluation F. Barberini, E. Vizza, M. Montanino, S. Marcoccia* and G. Montanino* Department of Anatomy and *2nd Department of Obstetrics and Gynecology, University of Rome "La Sapienza", Via Alfonso Borelli, 50, 1-00161 Rome, Italy
Summary. In order to evaluate the morphofunctional pattern
Key words: Neovagina - Skin autografts - Ultrastructure
pathogenesis has been and remains still controversial, since there are different opinions as to the embryological origin of the vagina. Two different organogenetic theories were advanced on this matter: a dualistic, and a unitary theory, each suggesting a different explanation for vaginal agenesis. According to the classic or dualistic embryogenetic theory (Koff 1933), only the lower part of the vagina is of ectodermal origin, deriving from the urogenital sinus, whereas the remaining part is of Mullerian (i.e. mesodermal) origin. With reference to the unitary theory suggested by Meyer (1934) and Bulmer (1957) and recently resumed by HoangNgoc Minh and others (1984), the vagina derives throughout from the vaginal plate (proliferation of the urogenital sinus) and, consequently, is of ectodermal origin. The Mullerian role in the development of the vagina would thus be indirect, with the interposition of a mesenchymal tissue inductor which would then stimulate a cranial proliferation of the vaginal plate. According to this theory, vaginal aplasia might depend upon a defect in the mesenchymal tissue inductor and may be associated with both functional and non-functional uteri. During this century, various surgical techniques of vaginal reconstruction have been proposed, some of which by using dermoepidermal autografts (McIndoe and Bamister 1938). Patients treated in this way refer, one year after operation, a satisfactory sexual activity, and the macroscopic features of the "neovagina" seem to confirm this. Therefore, a structural and ultrastructural investigation of the skin transplanted to create the neovaginae was undertaken, attempting a morphofunctional evaluation of the methods employed.
Introduction
Materials and methods
Vaginal agenesis, a congenital defect, occurs less frequently (1 : 4000) than the malformations of the upper parts of the female genital tract with which it is sometimes associated (Muller and Dellenbach 1967; Musset et al. 1973). Its
Three cases of vaginal agenesis have been examined. Two of these were associated with a rudimentary uterus (karyotype 46,xx "Rokitansky-Kuster-Hauser syndrome"), while the third case showed testicular feminization (karyotype 46,xy - "Morris syn
of transplanted skin structural and ultrastructural studies on biopsies taken from surgically created neovaginae in three cases of vaginal agenesis were performed one and two years postoperatively. Two of the cases were associated with a rudimentary uterus, while the remaining one involved testicular feminization. Neovaginae were constructed by directly prolonging a pre-existing perineal introitus, to form a vesicorectal pouch, which was lined inside with gluteal skin autografts. All three patients reported satisfactory psychosexual behavior one year after operation. Light microscopy of the bioptic fragments demonstrated, even two years after surgery, features generally similar to those of the normal dermis and epidermis, and a keratinization rate as a rule, that did not range significantly from the norm. Scanning electron microscopy also confirmed the maturation of the cornified cells, since these were often seen to be desquamating and exhibited structures usually found in normal skin, such as double edges on the luminal surface - usually considered to be the morphological expression of squamous cell overlapping - and microwrinkles on disrupted apical and basal cytoplasmic flaps, which are considered reciprocal cell-gearing devices. Our results seem to indicate, therefore, that the transplanted skin, maintaining, on the whole, its integrity and viability despite an environment differing somewhat from the external one with regard to moisture, pH and temperature, shows a good adaptability, at least until the above mentioned time lapses.
Ann. Anat. (1992) 174: 517-522 Gustav Fischer Verlag Jena
drome"). In all cases, a neovagina was created by directly prolonging a pre-existing, natural perineal introitus to form a vesicorectal pouch of about 10-12 cm in length. This pouch was then lined with autologous skin grafts taken from the gluteal regions and supported by means of a plastic stent. This stent was subsequently replaced with others, until complete adhesion of the grafts occurred. Biopsies of the surgically formed cavity wall (neovagina) were performed one and two years postoperatively, at 3 cm from the introitus, in each of the three patients (Fig. 1).
- ---lIeovoglno • Biopsy Fig. 1. This drawing illustrates the vesicorectal pouch (dotted line: neovagina) and the zone from which biopsies (black square, arrowhead) were taken. v = urinary bladder; r = rectum.
Fragments having a diameter of approximately 0.5 cm were fixed in 2.5 % 0.1 M glutaraldehyde solution buffered with sodium cacodylate to pH 7.35. After washing in running water for two hours, the fragments were dehydrated in ethanol and critical-point dried (Porter et al. 1972; Morta et al. 1977). The specimens were then shadowed with a gold and platinum coat in a high-vacuum sputter coater (Edwards) and examined in a Cambridge Stereoscan 150 scanning electron microscope (SEM) operating at 8 - 20 k V . For a better morphological evaluation of the keratinization rate, the samples already examined by SEM were embedded in Historesin and then cut in 3 !-tm-thick sections. These were stained with hematoxylin-eosin and Dane and Herman's tetrachromic method. The latter is specific for keratin and prekeratin identification in that these substances are dyed orange or reddish-orange, and the cell nuclei brown. Finally, the sections were examined and photographed with a Zeiss Photomicroscope II.
Results In overall views by SEM, biopsies taken one year after surgery exhibit a continuous carpet of flattened cells, often desquamating (Fig. 2). They have an irregularly rectangular or, more frequently, a polygonal shape, and are piled one on top of the other, so that the whole surface has a cobbled aspect. One may also detect the double cell edges specific to the squamous elements of the horny layer and due to edge overlapping, the outer edge corresponding to the real cell
edge and the inner one representing the pattern of the overlying exfoliated cell (Fig. 3). Our three-dimensional study at higher magnification stresses that the luminal side of the more superficial cell layer is characterized by a delicate pattern of microwrinkles, due to thin evaginations of the apical plasmalemma, rather similar on the whole to superficial epidermal microfolds. Similar formations were also noticed on the basal side of desquamating elements, to which correspond structures of identical significance on the opposite side of the underlying cells (Fig. 4). The presence of adnexal formations, such as hair and hairy follicles, was sometimes detected. In these locations, cells outlining the introitus may assume a typical vorticose pattern (Fig. 5). Samples taken two years after surgery show surface cells similar to those previously observed. In fact, epidermal cell layers consist of thin elements with edges tracing irregular polygons and detaching from underlying layers (Fig. 6). At higher resolution, cell boundaries are easily detected, as are double-edged images, typical of the overlapping elements described above (Fig. 7). In the interstitium between detaching cells and elements immediately subjacent to them, delicate microwrinkles of the apical and basal plasmalemma were also observed, as "gearing" microstructures between opposite cell surfaces (Fig. 8). Light microscopical (LM) observations performed two years postoperatively reveal an essentially normal aspect of the grafted skin, in both its epithelial (epidermis) and connective (dermis) components. The various layers of the epidermis still show substantially unaltered features, their morphology being easily recognizable throughout the epithelium. Likewise, the dermis does not show appreciable structural variations and, at the epidermal junction, rises into well developed papillae (Fig. 9a). The specific staining for keratin and prekeratin showed that even the epithelial cornified cytomorphosis remains, quantitatively, almost unchanged two years after surgery (Fig. 9b).
Discussion In cases of vaginal agenesis, the more or less invasive
surgical approaches to restore, even partially, the patient's anatomical integrity and sexual function have not always led to satisfactory results. Some of these involve the creation of a vesicorectal pouch and the introduction of a prosthesis (Wharton 1938) or an olive put under traction by abdominal way (Vecchietti 1965), or this pouch was subsequently lined inside with various tissues, such as amnion (Brindeau 1935; Burger 1947), bowel (Baldwin 1904; Schubert 1936; Runge 1951; Schmid 1956) or peritoneum (Bloch 1961). Other methods were bloodless, consisting in dilation of the urogenital sinus (Frank 1938; Speroff et al. 1986). We preferred to cover the surgically constructed vaginae with autologous skin grafts, according to the method of McIndoe and Barnister (1938), assuming that, since the cutaneous tissues closely resemble the ones lining the normal vaginal wall, these would be suitable.
518
Fig. 2. Surface layer of dermo-epidermal autograft, showing flattened, desquamating epithelial cells. One year after surgery; SEM, x455. Fig. 3. The squamous cells appear irregularly polygonal and assume, on the whole, a stonelike aspect. Note the presence of double cell edges (---"), due to overlapping of piled-up elements. One year after surgery; SEM, xl ,820. Fig. 4. An exfoliating cell showing upset of a cytoplasmic flap. The apical (white arrow) and basal (black arrow) surfaces are characterized by slight rnicrowrinkles (mw) of fhe plasmalemma, similar to the ones found in the erpidermis. One year after surgery; SEM, x9,100. Fig. 5. A hair arising from its follicle projects toward fhe inside of the neovaginal lumen. The cells surrounding fhe introitus of fhe follicular cavity (*) assume a vorticose arrangement. One year after surgery; SEM, x540. 6. Cells of fhe superficial layer of fhe dermo-epidermal autograft, basically similar to fhose seen in Fig. 1. ec = exfoliated cells. Two years after surgery; SEM, x450. Fig. 7. The squamous elements exhibit a clearly polygonal shape and well defined boundaries, which are often double (---"). Two years after surgery; SEM, 1,820.
519
Fig. 8. During epithelial sloughing, slight microwrinkles (mw) are seen in the opposite cell surfaces (apical: white arrow; basal: black arrow), and are meant for reciprocal gearing. (Two years after surgery; SEM, x2,300.)
Fig. 9. Dermo-epidermal autograft sections: a) All epidermal cell layers are well recognizable and do not denote appreciable histologic alterations. The dermis, too, seems to be substantially unaffected; b) The cutaneous keratinization rate does not present significant quantitative variations as compared to the rule . hc == cornified cells; be = basal cells; d-e j = dermoepidermal junction; d = dermis; p = dermal papilla. (Two years after surgery; LM, X 112; a) hematoxylin-eosin; b) Dane and Herman's tetrachrome method for keratin and prekeratin.)
520
Morphological studies of skin grafted in order to form neovaginae are very scarce (Holmstrom et al. 1976; Lathrop et al. 1985). The results we obtained by LM and SEM demonstrate that dermo-epidermal autografts have, one or two years after surgery, morphological features generally similar to those of the pre-grafted tissue. In fact, the grafted epidermis shows structural and ultrastructural aspects closely resembling those occurring during the normal process of corneal cytomorphosis. In particular, all cell layers of the graft are well differentiated: from the basal layer, made up of prismatic elements, to the superficial one, consisting of flattened cornified cells, piled up in columns . In addition, no significant reduction in thickness of the cornified layer was noted . This is in contrast to the opinion that an environment different from the skin surface because of high humidity, pH and bacterial flora, may induce a loss of the most superficial cell layers (Holmstrom et al. 1976) . The results of our LM studies using specific stains for keratin clearly show that the normal keratinization rate is preserved. Further, our SEM observations reveal the presence of low, steplike depressions, double cell contours and structures of reciprocal "gearing", such as superficial and basal microwrinkles, being all morphological features found in normal epidermal squamous elements (Menton and Eisen 1971 ; Hashimoto and Kanzaki 1975). The regular arrangement ofthe cornified layer and the preservation of its specific structural features, such as the piled-up aspect seen in normal cells, as well as the desquamation of the surface elements, are a sign of a valid cell turnover and consequently of morphofunctional integrity. Furthermore, considering the regular columnar arrangement of the cornified layer, it is logical to assume that the mitotic activity of the normal basal layer is maintained, this being an essential premise for a systematic layout of overlying layers (Menton and Eisen 1971). As far as the dermis is concerned, no significant morphological changes were observed in comparison to the structure of the dermis in normal skin. However, two years after surgery, we have not been able to confirm the presence of adnexal formations such as hair and hairy follicles, which, on the contrary, were seen before. Neverthless, this is not conclusive enough evidence for justifying a modification in the graft viability, after a given lapse of time. In fact, we believe that such formations cannot occur in all bioptic fragments , because of their small size. Our structural and ultrastructural observations lead us to the conclusion that grafted skin, while maintaining its morphological integrity and even showing a clear viability, has good adaptive capacities in spite of altered conditions, at least during the first two years after surgery. In fact, the major properties of skin, such as imperviousness and resistance to exogenous thermal, chemical and mechanical stresses, are mainly based upon the keratinization and integrity of the stratum corneum. Such properties, absent in the normal vaginal mucosa , are a limit, of course, to the result of surgery, sometimes making intercourse difficult and causing dyspareunia, because of scarce superficial lubrication and minor elastic relaxation of the walls. Owing to these first results, the present study of the
grafted skin might be extended over longer time intervals, so as to evaluate possible time-dependent morpho functional modifications , and to determine the overall long-term fate of the autologous neovagina. Acknowledgments. The authors would like to thank Prof. Pietro M. Motta, for critically reading the paper and Mr. David W. Finn in drawing up the manuscript. This investigation was supported by grants from M. P. I. (60%), Italy , 1987/1988.
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