http://informahealthcare.com/gye ISSN: 0951-3590 (print), 1473-0766 (electronic) Gynecol Endocrinol, Early Online: 1–5 ! 2014 Informa UK Ltd. DOI: 10.3109/09513590.2014.995618
ORIGINAL ARTICLE
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Oestrogen receptor alpha expression in neovaginal tissue of women following modified Abbe´-McIndoe technique and in premenopausal women Marcio Masashi Kajikawa1, Zsuzsanna Ilona Katalin Ja´rmy-Di Bella1, Juliane Dornelas1, Luciana Campanatti Crema1, Cla´udia Cristina Takano1, Gustavo Rubino de Azevedo Focchi2, Liliam Cristine Rolo3, Edward Araujo Ju´nior3, Rodrigo Aquino Castro1, Manoel Joa˜o Batista Castello Gira˜o1, and Marair Gracio Ferreira Sartori1 1
Department of Gynaecology at Paulista School of Medicine - Sa˜o Paulo Federal University (EPM-UNIFESP), Sa˜o Paulo-SP, Brazil, 2Department of Gynaecology at EPM-UNIFESP, Sa˜o Paulo-SP, Brazil, and 3Department of Obstetrics, Paulista School of Medicine – Federal University of Sa˜o Paulo (EPM-UNIFESP), Sa˜o Paulo-SP, Brazil Abstract The aim of the study was to compare the expression of oestrogen receptor alpha (ERa) in neovaginal tissue of patients with vaginal agenesis following neovaginoplasty using regenerated, oxidised cellulose in premenopausal women. A prospective, observational case– control study was performed on eight patients with vaginal agenesis following modified Abbe´McIndoe neovaginoplasty and 10 control premenopausal women following benign gynaecologic surgery. 6F11 monoclonal antibody was used to determine ERa expression in the vaginal mucosa. Quantitative and qualitative evaluations were performed, respectively, in vaginal epithelium and stroma. The thickness of the vaginal epithelium was determined as the vertical distance between the basal layer cells and the apical surface of the superficial layer. The percentage of ERa-expressing cells was higher in the control group, except in the superficial zone of the epithelium. In the stromal tissue, ERa was detected in only one patient from the neovagina group compared with nine women in the control group. The neovagina group had a statistically thinner epithelium. Our study suggests that women with vaginal agenesis following modified Abbe´-McIndoe neovaginoplasty using regenerated oxidised, cellulose experience relatively local hypo-oestrogenism in the first year after surgery, with repercussion in vaginal trophism.
Introduction Congenital malformations of the female reproductive tract represent a heterogeneous group of disorders and are a result of failure in the normal development of Mu¨llerian ducts during the embryologic phase. Abnormalities of Mu¨llerian ducts include aplasia/hypoplasia, abnormal fusion and canalisation of ducts and failure of septum resorption. Although the prevalence in the general population is unknown, reproductive tract malformations are common and some studies found these conditions in up to 7% of the female patient population [1]. The abnormal development of distal segments of Mu¨llerian ducts and urogenital sinus cause vaginal malformation, such as vaginal atresia, transverse vaginal septum, longitudinal vaginal septum and vaginal agenesis [2]. Vaginal agenesis is a congenital malformation characterised by the absence of the vagina. The most common cause of
Address for correspondence: Prof. Edward Araujo Ju´nior, Department of Obstetrics, Paulista School of Medicine – Federal University of Sa˜o Paulo (EPM-UNIFESP), Rua Carlos Weber, 956, apt. 113 Visage, Vila Leopoldina, Sa˜o Paulo-SP, CEP 05303-000, Brazil. Tel/Fax: +55 11 37965944. E-mail: [email protected]
Keywords Abbe´-McIndoe technique, neovagina, oestrogen receptor, premenopausal women History Received 22 August 2014 Revised 1 December 2014 Accepted 3 December 2014 Published online 29 December 2014
vaginal agenesis is Mayer–Rokitansky–Ku¨ster–Hauser Syndrome (MRKHS), characterized by variable degrees of aplasia of the Mu¨llerian ducts, resulting in the absence of the uterus and the upper portion of the vagina [2]. The incidence of MRKHS has been estimated as 1 in 4000–1 in 5000 female births. Although the majority of cases seem to be sporadic, cases of family have been reported, suggesting the involvement of a genetic component [3]. Clinically, women with MRKHS are presented by primary amenorrhea, normal development of secondary sexual characteristics, functional ovaries and normal 46, XX karyotypes. Other malformations associated with MRKHS include disruptions in the upper urinary tract, the skeleton, otologic sphere and cardiovascular anomalies [4]. The treatment of vaginal agenesis consists of the creation of a vaginal canal that permits satisfactory sexual activities. To achieve this objective, the canal should have anatomical and functional characteristics of a normal vagina [5]. Several nonoperative and surgical techniques have been developed for the treatment of vaginal agenesis. The ideal procedure should be able to be permanent, with minimal care for maintenance and be minimally invasive with low morbidity [6]. The American College of Obstetrics and Gynaecology suggests progressive dilatation as the first-line treatment for vaginal agenesis, and surgery is offered to those patients who failed the conservative techniques [7]. Surgical procedure options include use of bowel
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segments, peritoneum (Davydov’s technique), Vecchietti’s operation and Abbe´-McIndoe vaginoplasty [8–11]. The traditional Abbe´-McIndoe vaginoplasty consists of dissecting the vesicorectal space and covering the created canal with an autologous skin graft. Because this technique is minimally invasive, it is widely adopted for the treatment of vaginal agenesis [6]. However, disadvantages of this procedure sometimes limit its usage: local irritation caused by incorporation of hair-bearing skin, dryness, dyspareunia, graft shrinkage and formation of visible scars at the skin donor site [12,13]. Modifications of the original technique involving the use of different materials (amnion, autologous oral mucosa, in vitro cultured vaginal tissue and regenerated, oxidised cellulose) were developed to reduce the complications mentioned above [14–16]. Regenerated oxidised cellulose (InterceedÕ -Absorbable Adhesion Barrier; Johnson & Johnson, Cincinati, OH) is an absorbable synthetic mechanical barrier that has been used to reduce post-operative pelvic adhesions. It forms a gelatinous layer between the raw surfaces, preventing the formation of scar tissue and has local haemostatic efficacy and antibacterial activity. InterceedÕ was first used in the treatment of vaginal agenesis in the mid-1990s as a substitute for amnion to cover the stent placed in newly dissected vaginal canal [17]. The excellent results encouraged others to evaluate the efficacy of this material, concluding that vaginoplasty using regenerated oxidised cellulose is a safe and effective therapy for vaginal reconstruction. Patients who had this procedure reported satisfactory sexual intercourse and the neovagina acquired normal vaginal characteristics, with complete squamous epithelialisation within 6 months after the operation [18,19]. Vaginal trophism and epithelium proliferation are associated with sexual hormone status. It is well established that hypooestrogenism is associated with epithelium atrophy, dyspareunia and vaginal dryness, and oestrogen replacement in postmenopausal women is useful to relieve symptoms and to reverse atrophic anatomic changes [20]. Oestrogen effects are mediated by the interaction and activation of specific receptor proteins, named oestrogen receptor. Two types of oestrogen receptors (ER) were identified: ERa and ER beta (ERb). Although ERa is highly homologous to ERb, they are expressed in different types of tissue and mediate distinct responses. The presence of ERa in the vagina is well documented in humans and is considered to be the critical receptor for mediating specific vaginal responses to oestrogen [21]. The aim of this study was to compare the expression of ERa in neovaginal tissue of patients with MRKHS following vaginoplasty using regenerated oxidised cellulose and in the vagina of premenopausal women as control. The vaginal trophism was also evaluated by analysing the epithelium thickness in both groups.
Methods Eight patients with MRKHS participated in the study to modify Abbe´-McIndoe neovaginoplasty using oxidised regenerated cellulose in the Department of Gynaecology at Sa˜o Paulo Federal University (UNIFESP) from November 2004 to December 2006. The women ranged in age from 18 to 27 years (a mean age of 21 years). Diagnostic criteria for MRKHS were primary amenorrhea, normal external genitalia and secondary sexual characteristics, and vaginal agenesis. All patients were submitted to pelvic nuclear magnetic resonance, investigation of urinary tract malformation and karyotype. This study was approved by ethical committee and all patients gave their informed consent before the surgical intervention. This study was approved by the local ethical committee and all patients signed the consent form.
Gynecol Endocrinol, Early Online: 1–5
Operative technique All patients underwent spinal anaesthesia and were placed in the lithotomy position. A transurethral catheter was inserted to empty the bladder and 20 ml of a solution containing 1:200 000 epinephrine was injected into the tissue between bladder and rectum. An H-shaped incision was made in the area of vaginal dimple and a tunnel was created bluntly until vaginal length of approximately 8–10 cm was achieved. Haemostasis was performed before the insertion of the stent covered with oxidised, regenerated cellulose. The labia were sutured together over the stent with polyglactin 2-0. The stent was removed 4–7 d after the surgery and the canal was irrigated with sterile saline solution. Before hospital discharge, the patients were instructed on the correct use of the stent to avoid closure of the neovagina. The subjects were followed up for physical examination and biopsy of vaginal mucosa. Control group Ten women were recruited as a control group at the Department of Gynaecology at Sao Paulo Federal University from April 2009 to June 2009. Inclusion criteria to the study were premenopausal women with regular menstrual cycles (25–35 d) in the last 3 months and indication for surgical treatment of benign gynaecologic diseases. Exclusion criteria were women using hormone therapy in the last 3 months, pregnancy, suspected malignant disease and use of any vaginal medications during the last 2 weeks. The women ranged in age from 24 to 35 years (a mean age of 28.8 years), and five were in the first half of the menstrual cycle and the other five were in the second half of the cycle at the time of sampling. Biopsy sampling In the neovagina group, the biopsies were performed after the evidence of complete vaginal epithelialisation (a mean time of 11 months after surgery). The biopsy specimens were obtained from the lateral vaginal walls with no need of local anaesthesia or topical haemostatic agents. In the control group, the vaginal tissue was obtained immediately after anaesthesia induction. All the biopsies were fixed in 10% formalin in phosphate buffer (pH 7.2) for 24 h at room temperature, and then dehydrated, oriented and embedded in paraffin. Determination of ERa expression by immunohistochemistry Sections of formalin-fixed and paraffin-embedded biopsies were cut at 5 mm thickness perpendicular to the epithelial surface and mounted on microscopic slides. Then, tissues were deparaffinised in xylene and rehydrated in ethanol. For antigen retrieval, we used citrate buffer (pH 6.0) in a pressure cooker, and hydrogen peroxide was applied to suppress endogenous peroxidase activity. 6F11 monoclonal antibody (mouse anti-ERa, NCL-ER-6F11, Novocastra, Newcastle Upon Tyne, UK) was used to determine the ERa expression in the vaginal mucosa. A biotin-conjugated secondary antibody (Novocastra, Newcastle Upon Tyne, UK) formulation was used to detect the primary antibody followed by streptavidin–peroxidase conjugate (Novocastra, Newcastle Upon Tyne, UK). 3,30 -Diaminobenzidine (Dako) was used as a chromogen, and the sections were counterstained with Harris haematoxylin (Merck, Darmstadt, Germany). ERa-staining evaluation Each slide was evaluated by two persons, and cells were considered positive if nuclear staining was present, and no
ER in neovagina
DOI: 10.3109/09513590.2014.995618
grading of staining intensity was performed. Eight consecutive microscopic fields of the epithelium and the stroma with 200 magnification were evaluated in each biopsy sample. The image of each microscopic field was obtained with a camera (Sony DXC-107A, Sony Electronics, Tokyo, Japan) and transferred to the computer screen. To manually determine the positively stained cells, we used the software Image Pro Pus 3.0.01 version (National Institutes of Health, Bethesda, MD). In the epithelium, the ERa expression was scored based on the percentage of positively stained cells. For a detail evaluation, the vaginal epithelium was divided into three zones: superficial zone, mid zone and deep zone. The basal cell layer was also evaluated separately in both groups. In the vaginal stroma, a qualitative evaluation of ERa was performed, focusing on fibroblasts and blood vessels.
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Epithelium thickness evaluation The thickness of the vaginal epithelium was determined as the vertical distance between the bottom surface of cells in the basal layer and the apical surface of cells in the superficial layer. The measurement was performed by two people using a transparent metric ruler coupled with the microscope. Statistical analysis All data were entered into Statistical Package for Social Sciences (SPSS) 16.0 (SPSS, Chicago, IL) for statistical analysis and graphic representation. The intraclass correlation coefficient (ICC) was used to assess the consistency between the results obtained by the two judges. The Shapiro–Wilk test was used to analyse whether the data were normally distributed or not. Then, the independent t-test and the Mann–Whitney test were used for comparison between neovagina and control group data with and without normal distribution, respectively. A significance level (p) of 0.05 was defined as statistical significance. To evaluate the correlation between ERa expression and vaginal epithelium thickness, Pearson’s correlation coefficient (r) and Spearman rank correlation test were used on data with and without normal distribution, respectively.
Results The neovaginoplasty was performed successfully in all subjects and no major intra-operative complication occurred. Six patients returned regularly until the observation of complete vaginal epithelialisation. The other two subjects were excluded from this study because they did not return for vaginal biopsy after 6 months of the operation. The vaginal samples were obtained between nine and 16 months after the surgery (a mean time of 11.6 months). The characteristics of the patients are shown in Table 1. ERa-expressing cells displayed brown nuclear staining, whereas nuclei not expressing ERa were identified by the colour blue on counter stain. ERa was detected in all samples of vaginal epithelium in both groups, and located predominantly
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in the basal cell layer, deep zone and mid zone. In the superficial zone, a minority of cells expressed ERa. In the stromal tissue, ERa positive fibroblasts or blood vessels were detected in only one patient in the neovagina group, and in nine women in the control group (Figure 1). The ICC values varied from 0.962 to 0.999 in both groups, revealing agreement between the measurements made by the different observers. For comparison between the neovagina and control groups, we used the mean value obtained by the two observers. The percentage of cells expressing ERa in the neovagina groups was 39.3% (20–70.5%) in the basal cell layer, 37.9% (21.3–53.7%) in the deep zone, 33.3% (18.67–68.07%) in the mid zone and 2.5% (0–5.09%) in the superficial zone. In the control group, the amount of cells expressing ERa was 70.7% (62.09– 86.65%) in the basal cell layer, 74.6% (66.37–83.03%) in the deep zone, 73.8% (64.37–79.17%) in the mid zone and 4.2% (0–8.82%) in the superficial zone. To compare these two groups, we used the Mann–Whitney test for deep and mid zone and independent t-test for basal cell layer and superficial zone. A significant difference was found in the frequency of ERa-expressing cells in the basal layer cell, deep and mid zone. There was no difference between the two groups in the superficial zone of the vaginal epithelium (Figure 2). In the neovagina group, the mean vaginal epithelium thickness was 2 mm (1–3 mm), and in the control group, it was 3.2 mm (2.5– 4.0 mm). To compare the two groups, we used the independent t-test, and a significant difference was found between them (p ¼ 0.002). There was a significant positive correlation between the percentage of ERa-expressing cells in the basal cell layer (p ¼ 0.004), deep zone (p ¼ 0.005) and mid zone (p ¼ 0.002) for the vaginal epithelium thickness. No correlation was found with the superficial zone (p ¼ 0.46).
Discussion The diagnosis of vaginal agenesis normally occurs during puberty, when the patient complains of the absence of menstrual bleeding or inability to have vaginal sexual intercourse. The knowledge of this condition causes emotional distress to the young women, since it is often associated with infertility and sexual disorders. The restoration of sexuality through the possibility of normal sex activity is the major objective of the treatment of vaginal agenesis, and this goal is achieved by different types of nonsurgical and operative techniques. Although all articles using oxidised, regenerated cellulose in the treatment of MRKHS are case series, the efficacy of this treatment is comparable with other materials used in the Abbe´-McIndoe neovaginoplasty [17–19]. Since this synthetic mesh is easily available with low complications related to the material, we considered this an ideal approach for vaginal reconstruction [22]. The quality of sexual life after neovaginoplasty in women with vaginal agenesis was assessed with different techniques using specific questionnaires associated with anatomic and functional evaluation. Overall, the patients showed satisfaction regarding
Table 1. Characteristics of patients who had neovaginoplasty.
Patient 1 2 3 4 5 6
Age at operation (years)
Vaginometry before operation (cm)
Karyotype
Abnormal intravenous urography
Other malformations
25 20 18 21 27 18
2 3 2 3 2 3
XX XX XX XX XX XX
Absent right kidney Normal Normal Normal Normal Absent right kidney
Right inguinal hernia, imperforate anus Normal Normal Normal Normal Normal
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Figure 1. ERa expression in human vaginal tissue (200 magnification). The vaginal epithelium was divided into three groups: superficial, mid and deep zone. In the deep zone, the basal cell layer (BCL) was also evaluated separately (A). In the stromal tissue (B), some fibroblasts and blood vessels cells expressed ERa (*). Figure 2. Percentage of ERa-expressing cells in vaginal epithelium. Significant differences in ERa expression were observed in the basal layer cell (p ¼ 0.009), deep zone (p ¼ 0.001) and mid zone (p ¼ 0.002). No differences were observed in the superficial zone (p ¼ 0.323).
their sexual life, with questionnaire scores comparable with healthy women, regardless of the technique used [10,14,22–24]. The main sexual complaints are dyspareunia and insufficient lubrication, and they are normally associated with anatomic and functional problems. Vaginal shortening, stricture formation, inadequate vaginal thickness and discharge are responsible for pain and discomfort during sexual intercourse. A thinner and drier vaginal mucosa is also observed in postmenopausal women, a consequence of systemic hypo-oestrogenism. This condition is improved with local or systemic hormone replacement [25]. Patients submitted to neovaginoplasty may experience transitory or permanent vaginal hypo-oestrogenism that leads to symptoms of genital atrophy. Since MRKHS is associated with normal ovarian function and adequate levels of oestrogens, lower expression of ER may be the reason of relative hormone deficiency in the neovagina mucosa. Motoyama et al. [18], for instance, observed the vaginal secretion in the neovagina was less compared with healthy women, although none of the patients complained of vaginal dryness. In the present study, we observed that ERa was expressed in the epithelium of all patients similarly as in the basal cell layer, deep and mid zones, and barely in the superficial zone. Only one patient had ERa-expressing fibroblasts and blood vessels in the vaginal stromal tissue. Similar results were obtained in a previous investigation that analysed healthy young women regardless of hormonal contraceptive usage [26]. In this study, ER was located
predominantly in the suprabasal part and along the basal membrane of the epithelium, and ER-positive cells were less abundant in the stromal tissue compared with the epithelium. Furthermore, the authors did not find differences between the follicular and the luteal phase biopsies concerning the percentage of ER expressing cells. Evaluation of women with stress urinary incontinence revealed that ERa was expressed in epithelial, stromal fibroblasts and smooth muscle cells of the vagina, but not in vaginal blood vessels. A significantly higher expression of ERa was observed in fertile and menopausal women with hormone therapy than menopausal women who were not on hormone replacement [27]. In our study, we compared the expression of ERa in neovaginal mucosa with vaginal tissue of healthy premenopausal women. Both epithelium and stroma of the control group had a higher expression of ERa. Only in the superficial zone of the epithelium was there no difference in the percentage of ERa positive cells. A previous investigation of ER expression in neovaginal epithelium after vaginoplasty using amnion graft also showed that the intensity of nuclear staining was higher in the deep and mid zone of the epithelium. However, in comparison with normal vaginal tissue, they did not find differences in the intensity of nuclear ER. Divergent results could be explained by the different types of material used in the Abbe´-McIndoe neovaginoplasty. Since amnion membrane has angiogenic properties, a precocious formation of a mature vaginal epithelium may
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DOI: 10.3109/09513590.2014.995618
be observed compared with regenerated, oxidised cellulose [28]. On the contrary, this last material is absorbed in 2 weeks and the epithelialisation of the vaginal mucosa from the dissected tissue may occur slowly. In our study, the vaginal samples of the neovagina group were obtained nine to 16 months after the surgery, and in this stage, the full properties of the epithelium could still not be achieved. New samples should be analysed to evaluate if the relative vaginal hypo-oestrogenism in the neovagina group is transient or permanent. The deficient stroma in patients after vaginoplasty shown to be the case of low expression of ERa and a normal stroma is required in order to have a normal epithelium. Kurita et al. [29] realized an experimental study in adult mice demonstrating that ERa was strongly detected in uterine and vaginal epithelia. Thus, proper expression of ERa is a prerequisite for normal functional differentiation of uterine and vaginal epithelial cells. To evaluate if the low expression of ERa in the neovagina had implications in the mucosa trophic condition, we compared the epithelium thickness in the neovagina and healthy young women. We observed that, 1 year after the operation, the neovagina epithelium is thinner than the control group. A positive correlation was found between the percentage of ERa positive cells in the basal cell layer, deep and mid zones for vaginal epithelium thickness. In this study, although no macroscopic differences are noted in neovagina and normal young women vagina, histochemical and microscopic studies revealed functional divergences in both epithelium and stroma. More studies are needed to conclude if this condition is permanent, since hypo-oestrogenism has substantial effects on the tissues of the urogenital system, leading to repercussion of some elements in the pelvic floor and lower urinary tract [30]. In this case, women submitted to neovaginoplasty could have a higher risk of developing urogenital disorders, such as urinary incontinence and genital prolapse.
Declaration of interest The authors report no declarations of interest.
References 1. Grimbizis GF, Campo M. Congenital malformations of the female genital tract: the need for a new classification system. Fertil Steril 2010;94:401–7. 2. Breech LL, Laufer MR. Mu¨llerian anomalies. Obstet Gynecol Clin North Am 2009;36:47–68. 3. Morcel K, Watrin T, Pasquier L, et al. Utero-vaginal aplasia (Mayer– Rokitansky–Kuster–Hauser syndrome) associated with deletions in known DiGeorge or DiGeorge-like loci. Orphanet J Rare Dis 2011; 6:9. 4. Morcel K, Camborieux L, Programme de Recherches sur les Aplasies Mu¨lle´riennes (PRAM), Guerrier D. Mayer-RokitanskyKu¨ster-Hauser (MRKH) syndrome. Orphanet J Rare Dis 2007;2:13. 5. Laufer MR. Congenital absence of the vagina: in search of the perfect solution. When, and by what technique, should a vagina be created? Curr Opin Obstet Gynecol 2002;14:441–4. 6. Panici PB, Ruscito I, Gasparri ML, et al. Vaginal reconstruction with the Abbe`-McIndoe technique: from dermal grafts to autologous in vitro cultured vaginal tissue transplant. Semin Reprod Med 2011; 29:45–54. 7. American College of Obstetrics and Gynecology; Committee on Adolescent Health Care. American College of Obstetrics and Gynecology. ACOG committee opinion. Nonsurgical diagnosis and management of vaginal agenesis. Int J Gynecol Obstet 2002; 79:167–70.
ER in neovagina
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8. El-Sayed HM, El-Lamie IK, Ibrahim AM, El-Lamie KI. Vaginal reconstruction with sigmoid colon in vaginal agenesis. Int Urogynecol J Pelvic Floor Dysfunct 2007;18:1043–7. 9. Marques HS, Santos FL, Costa PVL, et al. Creation of a neovagina in patients with Rokitansky syndrome using peritoneum from the pouch of Douglas: an analysis of 48 cases. Fertil Steril 2008;90: 827–32. 10. Fedele L, Bianchi S, Frontino G, et al. The laparoscopic Vecchietti’s modified technique in Rokitansky syndrome: anatomic, functional and sexual long-term results. Am J Obstet Gynecol 2008;198: 377e1–6. 11. McIndoe AH, Banniser JB. An operation for the cure of congenital absence of the vagina. J Obstet Gynaecol Br Emp 1938;45:490–4. 12. Davies MC, Creighton SM. Vaginoplasty. Curr Opin Urol 2007;17: 415–18. 13. Burgu B, Duffy PG, Cuckow P, et al. Long-term outcome of vaginal reconstruction: comparing techniques and timing. J Pediatr Urol 2007;3:316–20. 14. Fotopoulou C, Sehouli J, Gehrmann N, et al. Functional and anatomic results of amnion vaginoplasty in young women with Mayer–Rokitansky–Ku¨ster–Hauser syndrome. Fertil Steril 2010;94: 317–23. 15. Lin WC, Chang CYY, Shen YY, Tsai HD. Use of autologous buccal mucosa for vaginoplasty: a study of eight cases. Human Reprod 2003;18:604–7. 16. Panici PB, Bellati F, Boni T, et al. Vaginoplasty using autologous in vitro cultured vaginal tissue in a patient with Mayer von Rokitansky Kuster Hauser syndrome. Human Reprod 2007;22: 2025–8. 17. Jackson ND, Rosenblatt PL. Use of interceed absorbable adhesion barrier for vaginoplasty. Obstet Gynecol 1994;84:1048–50. 18. Motoyama S, Laoag-Fernandez JB, Mochizuki S, et al. Vaginoplasty with interceed absorbable adhesion barrier for complete squamous epithelialization in vaginal agenesis. Am J Obstet Gynecol 2003; 188:1260–4. 19. Sharma JB, Gupta N, Mittal S. Creation of neovagina using oxidized cellulose (surgicel) as a surgical treatment of vaginal agenesis. Arch Gynecol Obstet 2007;275:231–5. 20. Archer DF. Efficacy and tolerability of local estrogen therapy for urogenital atrophy. Menopause 2010;17:194–203. 21. Pessina MA, Hoyt Jr RF, Goldstein I, Traish AM. Differential regulation of the expression of estrogen, progesterone, and androgen receptors by sex steroid hormones in the vagina: immunohistochemical studies. J Sex Med 2006;3:804–14. 22. Dornelas J, Ja´rmy-Di Bella ZI, Heinke T, et al. Vaginoplasty with oxidized cellulose: anatomical, functional and histological evaluation. Eur J Obstet Gynecol Reprod Biol 2012;163:204–9. 23. Giannesi A, Marchiole P, Benchaib M, et al. Sexuality after laparoscopic Davydov in patients affected by congenital complete vaginal agenesis associated with uterine agenesis or hypoplasia. Human Reprod 2005;20:2954–7. 24. Brun JL, Belleanne´e G, Grafeille N, et al. Long-term results after neovagina creation in Mayer–Rokitansky–Ku¨ster–Hauser syndrome by Vecchietti’s operation. Eur J Obstet Gynecol Reprod Biol 2002; 103:168–72. 25. Mac Bride MB, Rhodes DJ, Shuster LT. Vulvovaginal atrophy. Mayo Clin Proc 2010;85:87–94. 26. Ildgruben A, Sjo¨berg I, Hammarstro¨m ML, Ba¨ckstro¨m T. Steroid receptor expression in vaginal epithelium of healthy fertile women and influences of hormonal contraceptive usage. Contraception 2005;72:383–92. 27. Fu X, Rezapour M, Wu X, et al. Expression of estrogen receptoralpha and -beta in anterior vaginal walls of genuine stress incontinent women. Int Urogynecol J Pelvic Floor Dysfunct 2003; 14:276–81. 28. Carvalho BR, Reis RM, Moura MD, et al. Neovaginoplasty using amniotic membrane in Mayer–Rokitansky–Ku¨ster–Hauser syndrome. Rev Bras Ginecol Obstet 2007;29:619–24. 29. Kurita T, Cooke PS, Cunha GR. Epithelial–stromal tissue interaction in paramesonephric (Mullerian) epithelial differentiation. Dev Biol 2001;240:194–211. 30. Sartori MG, Feldner PC, Jarmy-Di Bella ZI, et al. Sexual steroids in urogynecology. Climacteric 2011;14:5–14.
ORIGINAL ARTICLE
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Oestrogen receptor alpha expression in neovaginal tissue of women following modified Abbe´-McIndoe technique and in premenopausal women Marcio Masashi Kajikawa1, Zsuzsanna Ilona Katalin Ja´rmy-Di Bella1, Juliane Dornelas1, Luciana Campanatti Crema1, Cla´udia Cristina Takano1, Gustavo Rubino de Azevedo Focchi2, Liliam Cristine Rolo3, Edward Araujo Ju´nior3, Rodrigo Aquino Castro1, Manoel Joa˜o Batista Castello Gira˜o1, and Marair Gracio Ferreira Sartori1 1
Department of Gynaecology at Paulista School of Medicine - Sa˜o Paulo Federal University (EPM-UNIFESP), Sa˜o Paulo-SP, Brazil, 2Department of Gynaecology at EPM-UNIFESP, Sa˜o Paulo-SP, Brazil, and 3Department of Obstetrics, Paulista School of Medicine – Federal University of Sa˜o Paulo (EPM-UNIFESP), Sa˜o Paulo-SP, Brazil Abstract The aim of the study was to compare the expression of oestrogen receptor alpha (ERa) in neovaginal tissue of patients with vaginal agenesis following neovaginoplasty using regenerated, oxidised cellulose in premenopausal women. A prospective, observational case– control study was performed on eight patients with vaginal agenesis following modified Abbe´McIndoe neovaginoplasty and 10 control premenopausal women following benign gynaecologic surgery. 6F11 monoclonal antibody was used to determine ERa expression in the vaginal mucosa. Quantitative and qualitative evaluations were performed, respectively, in vaginal epithelium and stroma. The thickness of the vaginal epithelium was determined as the vertical distance between the basal layer cells and the apical surface of the superficial layer. The percentage of ERa-expressing cells was higher in the control group, except in the superficial zone of the epithelium. In the stromal tissue, ERa was detected in only one patient from the neovagina group compared with nine women in the control group. The neovagina group had a statistically thinner epithelium. Our study suggests that women with vaginal agenesis following modified Abbe´-McIndoe neovaginoplasty using regenerated oxidised, cellulose experience relatively local hypo-oestrogenism in the first year after surgery, with repercussion in vaginal trophism.
Introduction Congenital malformations of the female reproductive tract represent a heterogeneous group of disorders and are a result of failure in the normal development of Mu¨llerian ducts during the embryologic phase. Abnormalities of Mu¨llerian ducts include aplasia/hypoplasia, abnormal fusion and canalisation of ducts and failure of septum resorption. Although the prevalence in the general population is unknown, reproductive tract malformations are common and some studies found these conditions in up to 7% of the female patient population [1]. The abnormal development of distal segments of Mu¨llerian ducts and urogenital sinus cause vaginal malformation, such as vaginal atresia, transverse vaginal septum, longitudinal vaginal septum and vaginal agenesis [2]. Vaginal agenesis is a congenital malformation characterised by the absence of the vagina. The most common cause of
Address for correspondence: Prof. Edward Araujo Ju´nior, Department of Obstetrics, Paulista School of Medicine – Federal University of Sa˜o Paulo (EPM-UNIFESP), Rua Carlos Weber, 956, apt. 113 Visage, Vila Leopoldina, Sa˜o Paulo-SP, CEP 05303-000, Brazil. Tel/Fax: +55 11 37965944. E-mail: [email protected]
Keywords Abbe´-McIndoe technique, neovagina, oestrogen receptor, premenopausal women History Received 22 August 2014 Revised 1 December 2014 Accepted 3 December 2014 Published online 29 December 2014
vaginal agenesis is Mayer–Rokitansky–Ku¨ster–Hauser Syndrome (MRKHS), characterized by variable degrees of aplasia of the Mu¨llerian ducts, resulting in the absence of the uterus and the upper portion of the vagina [2]. The incidence of MRKHS has been estimated as 1 in 4000–1 in 5000 female births. Although the majority of cases seem to be sporadic, cases of family have been reported, suggesting the involvement of a genetic component [3]. Clinically, women with MRKHS are presented by primary amenorrhea, normal development of secondary sexual characteristics, functional ovaries and normal 46, XX karyotypes. Other malformations associated with MRKHS include disruptions in the upper urinary tract, the skeleton, otologic sphere and cardiovascular anomalies [4]. The treatment of vaginal agenesis consists of the creation of a vaginal canal that permits satisfactory sexual activities. To achieve this objective, the canal should have anatomical and functional characteristics of a normal vagina [5]. Several nonoperative and surgical techniques have been developed for the treatment of vaginal agenesis. The ideal procedure should be able to be permanent, with minimal care for maintenance and be minimally invasive with low morbidity [6]. The American College of Obstetrics and Gynaecology suggests progressive dilatation as the first-line treatment for vaginal agenesis, and surgery is offered to those patients who failed the conservative techniques [7]. Surgical procedure options include use of bowel
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segments, peritoneum (Davydov’s technique), Vecchietti’s operation and Abbe´-McIndoe vaginoplasty [8–11]. The traditional Abbe´-McIndoe vaginoplasty consists of dissecting the vesicorectal space and covering the created canal with an autologous skin graft. Because this technique is minimally invasive, it is widely adopted for the treatment of vaginal agenesis [6]. However, disadvantages of this procedure sometimes limit its usage: local irritation caused by incorporation of hair-bearing skin, dryness, dyspareunia, graft shrinkage and formation of visible scars at the skin donor site [12,13]. Modifications of the original technique involving the use of different materials (amnion, autologous oral mucosa, in vitro cultured vaginal tissue and regenerated, oxidised cellulose) were developed to reduce the complications mentioned above [14–16]. Regenerated oxidised cellulose (InterceedÕ -Absorbable Adhesion Barrier; Johnson & Johnson, Cincinati, OH) is an absorbable synthetic mechanical barrier that has been used to reduce post-operative pelvic adhesions. It forms a gelatinous layer between the raw surfaces, preventing the formation of scar tissue and has local haemostatic efficacy and antibacterial activity. InterceedÕ was first used in the treatment of vaginal agenesis in the mid-1990s as a substitute for amnion to cover the stent placed in newly dissected vaginal canal [17]. The excellent results encouraged others to evaluate the efficacy of this material, concluding that vaginoplasty using regenerated oxidised cellulose is a safe and effective therapy for vaginal reconstruction. Patients who had this procedure reported satisfactory sexual intercourse and the neovagina acquired normal vaginal characteristics, with complete squamous epithelialisation within 6 months after the operation [18,19]. Vaginal trophism and epithelium proliferation are associated with sexual hormone status. It is well established that hypooestrogenism is associated with epithelium atrophy, dyspareunia and vaginal dryness, and oestrogen replacement in postmenopausal women is useful to relieve symptoms and to reverse atrophic anatomic changes [20]. Oestrogen effects are mediated by the interaction and activation of specific receptor proteins, named oestrogen receptor. Two types of oestrogen receptors (ER) were identified: ERa and ER beta (ERb). Although ERa is highly homologous to ERb, they are expressed in different types of tissue and mediate distinct responses. The presence of ERa in the vagina is well documented in humans and is considered to be the critical receptor for mediating specific vaginal responses to oestrogen [21]. The aim of this study was to compare the expression of ERa in neovaginal tissue of patients with MRKHS following vaginoplasty using regenerated oxidised cellulose and in the vagina of premenopausal women as control. The vaginal trophism was also evaluated by analysing the epithelium thickness in both groups.
Methods Eight patients with MRKHS participated in the study to modify Abbe´-McIndoe neovaginoplasty using oxidised regenerated cellulose in the Department of Gynaecology at Sa˜o Paulo Federal University (UNIFESP) from November 2004 to December 2006. The women ranged in age from 18 to 27 years (a mean age of 21 years). Diagnostic criteria for MRKHS were primary amenorrhea, normal external genitalia and secondary sexual characteristics, and vaginal agenesis. All patients were submitted to pelvic nuclear magnetic resonance, investigation of urinary tract malformation and karyotype. This study was approved by ethical committee and all patients gave their informed consent before the surgical intervention. This study was approved by the local ethical committee and all patients signed the consent form.
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Operative technique All patients underwent spinal anaesthesia and were placed in the lithotomy position. A transurethral catheter was inserted to empty the bladder and 20 ml of a solution containing 1:200 000 epinephrine was injected into the tissue between bladder and rectum. An H-shaped incision was made in the area of vaginal dimple and a tunnel was created bluntly until vaginal length of approximately 8–10 cm was achieved. Haemostasis was performed before the insertion of the stent covered with oxidised, regenerated cellulose. The labia were sutured together over the stent with polyglactin 2-0. The stent was removed 4–7 d after the surgery and the canal was irrigated with sterile saline solution. Before hospital discharge, the patients were instructed on the correct use of the stent to avoid closure of the neovagina. The subjects were followed up for physical examination and biopsy of vaginal mucosa. Control group Ten women were recruited as a control group at the Department of Gynaecology at Sao Paulo Federal University from April 2009 to June 2009. Inclusion criteria to the study were premenopausal women with regular menstrual cycles (25–35 d) in the last 3 months and indication for surgical treatment of benign gynaecologic diseases. Exclusion criteria were women using hormone therapy in the last 3 months, pregnancy, suspected malignant disease and use of any vaginal medications during the last 2 weeks. The women ranged in age from 24 to 35 years (a mean age of 28.8 years), and five were in the first half of the menstrual cycle and the other five were in the second half of the cycle at the time of sampling. Biopsy sampling In the neovagina group, the biopsies were performed after the evidence of complete vaginal epithelialisation (a mean time of 11 months after surgery). The biopsy specimens were obtained from the lateral vaginal walls with no need of local anaesthesia or topical haemostatic agents. In the control group, the vaginal tissue was obtained immediately after anaesthesia induction. All the biopsies were fixed in 10% formalin in phosphate buffer (pH 7.2) for 24 h at room temperature, and then dehydrated, oriented and embedded in paraffin. Determination of ERa expression by immunohistochemistry Sections of formalin-fixed and paraffin-embedded biopsies were cut at 5 mm thickness perpendicular to the epithelial surface and mounted on microscopic slides. Then, tissues were deparaffinised in xylene and rehydrated in ethanol. For antigen retrieval, we used citrate buffer (pH 6.0) in a pressure cooker, and hydrogen peroxide was applied to suppress endogenous peroxidase activity. 6F11 monoclonal antibody (mouse anti-ERa, NCL-ER-6F11, Novocastra, Newcastle Upon Tyne, UK) was used to determine the ERa expression in the vaginal mucosa. A biotin-conjugated secondary antibody (Novocastra, Newcastle Upon Tyne, UK) formulation was used to detect the primary antibody followed by streptavidin–peroxidase conjugate (Novocastra, Newcastle Upon Tyne, UK). 3,30 -Diaminobenzidine (Dako) was used as a chromogen, and the sections were counterstained with Harris haematoxylin (Merck, Darmstadt, Germany). ERa-staining evaluation Each slide was evaluated by two persons, and cells were considered positive if nuclear staining was present, and no
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DOI: 10.3109/09513590.2014.995618
grading of staining intensity was performed. Eight consecutive microscopic fields of the epithelium and the stroma with 200 magnification were evaluated in each biopsy sample. The image of each microscopic field was obtained with a camera (Sony DXC-107A, Sony Electronics, Tokyo, Japan) and transferred to the computer screen. To manually determine the positively stained cells, we used the software Image Pro Pus 3.0.01 version (National Institutes of Health, Bethesda, MD). In the epithelium, the ERa expression was scored based on the percentage of positively stained cells. For a detail evaluation, the vaginal epithelium was divided into three zones: superficial zone, mid zone and deep zone. The basal cell layer was also evaluated separately in both groups. In the vaginal stroma, a qualitative evaluation of ERa was performed, focusing on fibroblasts and blood vessels.
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Epithelium thickness evaluation The thickness of the vaginal epithelium was determined as the vertical distance between the bottom surface of cells in the basal layer and the apical surface of cells in the superficial layer. The measurement was performed by two people using a transparent metric ruler coupled with the microscope. Statistical analysis All data were entered into Statistical Package for Social Sciences (SPSS) 16.0 (SPSS, Chicago, IL) for statistical analysis and graphic representation. The intraclass correlation coefficient (ICC) was used to assess the consistency between the results obtained by the two judges. The Shapiro–Wilk test was used to analyse whether the data were normally distributed or not. Then, the independent t-test and the Mann–Whitney test were used for comparison between neovagina and control group data with and without normal distribution, respectively. A significance level (p) of 0.05 was defined as statistical significance. To evaluate the correlation between ERa expression and vaginal epithelium thickness, Pearson’s correlation coefficient (r) and Spearman rank correlation test were used on data with and without normal distribution, respectively.
Results The neovaginoplasty was performed successfully in all subjects and no major intra-operative complication occurred. Six patients returned regularly until the observation of complete vaginal epithelialisation. The other two subjects were excluded from this study because they did not return for vaginal biopsy after 6 months of the operation. The vaginal samples were obtained between nine and 16 months after the surgery (a mean time of 11.6 months). The characteristics of the patients are shown in Table 1. ERa-expressing cells displayed brown nuclear staining, whereas nuclei not expressing ERa were identified by the colour blue on counter stain. ERa was detected in all samples of vaginal epithelium in both groups, and located predominantly
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in the basal cell layer, deep zone and mid zone. In the superficial zone, a minority of cells expressed ERa. In the stromal tissue, ERa positive fibroblasts or blood vessels were detected in only one patient in the neovagina group, and in nine women in the control group (Figure 1). The ICC values varied from 0.962 to 0.999 in both groups, revealing agreement between the measurements made by the different observers. For comparison between the neovagina and control groups, we used the mean value obtained by the two observers. The percentage of cells expressing ERa in the neovagina groups was 39.3% (20–70.5%) in the basal cell layer, 37.9% (21.3–53.7%) in the deep zone, 33.3% (18.67–68.07%) in the mid zone and 2.5% (0–5.09%) in the superficial zone. In the control group, the amount of cells expressing ERa was 70.7% (62.09– 86.65%) in the basal cell layer, 74.6% (66.37–83.03%) in the deep zone, 73.8% (64.37–79.17%) in the mid zone and 4.2% (0–8.82%) in the superficial zone. To compare these two groups, we used the Mann–Whitney test for deep and mid zone and independent t-test for basal cell layer and superficial zone. A significant difference was found in the frequency of ERa-expressing cells in the basal layer cell, deep and mid zone. There was no difference between the two groups in the superficial zone of the vaginal epithelium (Figure 2). In the neovagina group, the mean vaginal epithelium thickness was 2 mm (1–3 mm), and in the control group, it was 3.2 mm (2.5– 4.0 mm). To compare the two groups, we used the independent t-test, and a significant difference was found between them (p ¼ 0.002). There was a significant positive correlation between the percentage of ERa-expressing cells in the basal cell layer (p ¼ 0.004), deep zone (p ¼ 0.005) and mid zone (p ¼ 0.002) for the vaginal epithelium thickness. No correlation was found with the superficial zone (p ¼ 0.46).
Discussion The diagnosis of vaginal agenesis normally occurs during puberty, when the patient complains of the absence of menstrual bleeding or inability to have vaginal sexual intercourse. The knowledge of this condition causes emotional distress to the young women, since it is often associated with infertility and sexual disorders. The restoration of sexuality through the possibility of normal sex activity is the major objective of the treatment of vaginal agenesis, and this goal is achieved by different types of nonsurgical and operative techniques. Although all articles using oxidised, regenerated cellulose in the treatment of MRKHS are case series, the efficacy of this treatment is comparable with other materials used in the Abbe´-McIndoe neovaginoplasty [17–19]. Since this synthetic mesh is easily available with low complications related to the material, we considered this an ideal approach for vaginal reconstruction [22]. The quality of sexual life after neovaginoplasty in women with vaginal agenesis was assessed with different techniques using specific questionnaires associated with anatomic and functional evaluation. Overall, the patients showed satisfaction regarding
Table 1. Characteristics of patients who had neovaginoplasty.
Patient 1 2 3 4 5 6
Age at operation (years)
Vaginometry before operation (cm)
Karyotype
Abnormal intravenous urography
Other malformations
25 20 18 21 27 18
2 3 2 3 2 3
XX XX XX XX XX XX
Absent right kidney Normal Normal Normal Normal Absent right kidney
Right inguinal hernia, imperforate anus Normal Normal Normal Normal Normal
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Figure 1. ERa expression in human vaginal tissue (200 magnification). The vaginal epithelium was divided into three groups: superficial, mid and deep zone. In the deep zone, the basal cell layer (BCL) was also evaluated separately (A). In the stromal tissue (B), some fibroblasts and blood vessels cells expressed ERa (*). Figure 2. Percentage of ERa-expressing cells in vaginal epithelium. Significant differences in ERa expression were observed in the basal layer cell (p ¼ 0.009), deep zone (p ¼ 0.001) and mid zone (p ¼ 0.002). No differences were observed in the superficial zone (p ¼ 0.323).
their sexual life, with questionnaire scores comparable with healthy women, regardless of the technique used [10,14,22–24]. The main sexual complaints are dyspareunia and insufficient lubrication, and they are normally associated with anatomic and functional problems. Vaginal shortening, stricture formation, inadequate vaginal thickness and discharge are responsible for pain and discomfort during sexual intercourse. A thinner and drier vaginal mucosa is also observed in postmenopausal women, a consequence of systemic hypo-oestrogenism. This condition is improved with local or systemic hormone replacement [25]. Patients submitted to neovaginoplasty may experience transitory or permanent vaginal hypo-oestrogenism that leads to symptoms of genital atrophy. Since MRKHS is associated with normal ovarian function and adequate levels of oestrogens, lower expression of ER may be the reason of relative hormone deficiency in the neovagina mucosa. Motoyama et al. [18], for instance, observed the vaginal secretion in the neovagina was less compared with healthy women, although none of the patients complained of vaginal dryness. In the present study, we observed that ERa was expressed in the epithelium of all patients similarly as in the basal cell layer, deep and mid zones, and barely in the superficial zone. Only one patient had ERa-expressing fibroblasts and blood vessels in the vaginal stromal tissue. Similar results were obtained in a previous investigation that analysed healthy young women regardless of hormonal contraceptive usage [26]. In this study, ER was located
predominantly in the suprabasal part and along the basal membrane of the epithelium, and ER-positive cells were less abundant in the stromal tissue compared with the epithelium. Furthermore, the authors did not find differences between the follicular and the luteal phase biopsies concerning the percentage of ER expressing cells. Evaluation of women with stress urinary incontinence revealed that ERa was expressed in epithelial, stromal fibroblasts and smooth muscle cells of the vagina, but not in vaginal blood vessels. A significantly higher expression of ERa was observed in fertile and menopausal women with hormone therapy than menopausal women who were not on hormone replacement [27]. In our study, we compared the expression of ERa in neovaginal mucosa with vaginal tissue of healthy premenopausal women. Both epithelium and stroma of the control group had a higher expression of ERa. Only in the superficial zone of the epithelium was there no difference in the percentage of ERa positive cells. A previous investigation of ER expression in neovaginal epithelium after vaginoplasty using amnion graft also showed that the intensity of nuclear staining was higher in the deep and mid zone of the epithelium. However, in comparison with normal vaginal tissue, they did not find differences in the intensity of nuclear ER. Divergent results could be explained by the different types of material used in the Abbe´-McIndoe neovaginoplasty. Since amnion membrane has angiogenic properties, a precocious formation of a mature vaginal epithelium may
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DOI: 10.3109/09513590.2014.995618
be observed compared with regenerated, oxidised cellulose [28]. On the contrary, this last material is absorbed in 2 weeks and the epithelialisation of the vaginal mucosa from the dissected tissue may occur slowly. In our study, the vaginal samples of the neovagina group were obtained nine to 16 months after the surgery, and in this stage, the full properties of the epithelium could still not be achieved. New samples should be analysed to evaluate if the relative vaginal hypo-oestrogenism in the neovagina group is transient or permanent. The deficient stroma in patients after vaginoplasty shown to be the case of low expression of ERa and a normal stroma is required in order to have a normal epithelium. Kurita et al. [29] realized an experimental study in adult mice demonstrating that ERa was strongly detected in uterine and vaginal epithelia. Thus, proper expression of ERa is a prerequisite for normal functional differentiation of uterine and vaginal epithelial cells. To evaluate if the low expression of ERa in the neovagina had implications in the mucosa trophic condition, we compared the epithelium thickness in the neovagina and healthy young women. We observed that, 1 year after the operation, the neovagina epithelium is thinner than the control group. A positive correlation was found between the percentage of ERa positive cells in the basal cell layer, deep and mid zones for vaginal epithelium thickness. In this study, although no macroscopic differences are noted in neovagina and normal young women vagina, histochemical and microscopic studies revealed functional divergences in both epithelium and stroma. More studies are needed to conclude if this condition is permanent, since hypo-oestrogenism has substantial effects on the tissues of the urogenital system, leading to repercussion of some elements in the pelvic floor and lower urinary tract [30]. In this case, women submitted to neovaginoplasty could have a higher risk of developing urogenital disorders, such as urinary incontinence and genital prolapse.
Declaration of interest The authors report no declarations of interest.
References 1. Grimbizis GF, Campo M. Congenital malformations of the female genital tract: the need for a new classification system. Fertil Steril 2010;94:401–7. 2. Breech LL, Laufer MR. Mu¨llerian anomalies. Obstet Gynecol Clin North Am 2009;36:47–68. 3. Morcel K, Watrin T, Pasquier L, et al. Utero-vaginal aplasia (Mayer– Rokitansky–Kuster–Hauser syndrome) associated with deletions in known DiGeorge or DiGeorge-like loci. Orphanet J Rare Dis 2011; 6:9. 4. Morcel K, Camborieux L, Programme de Recherches sur les Aplasies Mu¨lle´riennes (PRAM), Guerrier D. Mayer-RokitanskyKu¨ster-Hauser (MRKH) syndrome. Orphanet J Rare Dis 2007;2:13. 5. Laufer MR. Congenital absence of the vagina: in search of the perfect solution. When, and by what technique, should a vagina be created? Curr Opin Obstet Gynecol 2002;14:441–4. 6. Panici PB, Ruscito I, Gasparri ML, et al. Vaginal reconstruction with the Abbe`-McIndoe technique: from dermal grafts to autologous in vitro cultured vaginal tissue transplant. Semin Reprod Med 2011; 29:45–54. 7. American College of Obstetrics and Gynecology; Committee on Adolescent Health Care. American College of Obstetrics and Gynecology. ACOG committee opinion. Nonsurgical diagnosis and management of vaginal agenesis. Int J Gynecol Obstet 2002; 79:167–70.
ER in neovagina
5
8. El-Sayed HM, El-Lamie IK, Ibrahim AM, El-Lamie KI. Vaginal reconstruction with sigmoid colon in vaginal agenesis. Int Urogynecol J Pelvic Floor Dysfunct 2007;18:1043–7. 9. Marques HS, Santos FL, Costa PVL, et al. Creation of a neovagina in patients with Rokitansky syndrome using peritoneum from the pouch of Douglas: an analysis of 48 cases. Fertil Steril 2008;90: 827–32. 10. Fedele L, Bianchi S, Frontino G, et al. The laparoscopic Vecchietti’s modified technique in Rokitansky syndrome: anatomic, functional and sexual long-term results. Am J Obstet Gynecol 2008;198: 377e1–6. 11. McIndoe AH, Banniser JB. An operation for the cure of congenital absence of the vagina. J Obstet Gynaecol Br Emp 1938;45:490–4. 12. Davies MC, Creighton SM. Vaginoplasty. Curr Opin Urol 2007;17: 415–18. 13. Burgu B, Duffy PG, Cuckow P, et al. Long-term outcome of vaginal reconstruction: comparing techniques and timing. J Pediatr Urol 2007;3:316–20. 14. Fotopoulou C, Sehouli J, Gehrmann N, et al. Functional and anatomic results of amnion vaginoplasty in young women with Mayer–Rokitansky–Ku¨ster–Hauser syndrome. Fertil Steril 2010;94: 317–23. 15. Lin WC, Chang CYY, Shen YY, Tsai HD. Use of autologous buccal mucosa for vaginoplasty: a study of eight cases. Human Reprod 2003;18:604–7. 16. Panici PB, Bellati F, Boni T, et al. Vaginoplasty using autologous in vitro cultured vaginal tissue in a patient with Mayer von Rokitansky Kuster Hauser syndrome. Human Reprod 2007;22: 2025–8. 17. Jackson ND, Rosenblatt PL. Use of interceed absorbable adhesion barrier for vaginoplasty. Obstet Gynecol 1994;84:1048–50. 18. Motoyama S, Laoag-Fernandez JB, Mochizuki S, et al. Vaginoplasty with interceed absorbable adhesion barrier for complete squamous epithelialization in vaginal agenesis. Am J Obstet Gynecol 2003; 188:1260–4. 19. Sharma JB, Gupta N, Mittal S. Creation of neovagina using oxidized cellulose (surgicel) as a surgical treatment of vaginal agenesis. Arch Gynecol Obstet 2007;275:231–5. 20. Archer DF. Efficacy and tolerability of local estrogen therapy for urogenital atrophy. Menopause 2010;17:194–203. 21. Pessina MA, Hoyt Jr RF, Goldstein I, Traish AM. Differential regulation of the expression of estrogen, progesterone, and androgen receptors by sex steroid hormones in the vagina: immunohistochemical studies. J Sex Med 2006;3:804–14. 22. Dornelas J, Ja´rmy-Di Bella ZI, Heinke T, et al. Vaginoplasty with oxidized cellulose: anatomical, functional and histological evaluation. Eur J Obstet Gynecol Reprod Biol 2012;163:204–9. 23. Giannesi A, Marchiole P, Benchaib M, et al. Sexuality after laparoscopic Davydov in patients affected by congenital complete vaginal agenesis associated with uterine agenesis or hypoplasia. Human Reprod 2005;20:2954–7. 24. Brun JL, Belleanne´e G, Grafeille N, et al. Long-term results after neovagina creation in Mayer–Rokitansky–Ku¨ster–Hauser syndrome by Vecchietti’s operation. Eur J Obstet Gynecol Reprod Biol 2002; 103:168–72. 25. Mac Bride MB, Rhodes DJ, Shuster LT. Vulvovaginal atrophy. Mayo Clin Proc 2010;85:87–94. 26. Ildgruben A, Sjo¨berg I, Hammarstro¨m ML, Ba¨ckstro¨m T. Steroid receptor expression in vaginal epithelium of healthy fertile women and influences of hormonal contraceptive usage. Contraception 2005;72:383–92. 27. Fu X, Rezapour M, Wu X, et al. Expression of estrogen receptoralpha and -beta in anterior vaginal walls of genuine stress incontinent women. Int Urogynecol J Pelvic Floor Dysfunct 2003; 14:276–81. 28. Carvalho BR, Reis RM, Moura MD, et al. Neovaginoplasty using amniotic membrane in Mayer–Rokitansky–Ku¨ster–Hauser syndrome. Rev Bras Ginecol Obstet 2007;29:619–24. 29. Kurita T, Cooke PS, Cunha GR. Epithelial–stromal tissue interaction in paramesonephric (Mullerian) epithelial differentiation. Dev Biol 2001;240:194–211. 30. Sartori MG, Feldner PC, Jarmy-Di Bella ZI, et al. Sexual steroids in urogynecology. Climacteric 2011;14:5–14.
