Preparation of Single-Somite Explants from Zebrafish Embryos.

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.

Ultrasonography and MRI features of the Mayer-Rokitansky-Küster-Hauser syndrome.

Although Mayer-Rokitansky-Küster-Hauser syndrome is a rare condition with a reported incidence of 1/4500 female live births, it represents the second most common cause of primary amenorrhea and has psychologically devastating consequences. The radiologist plays a pivotal role in both making the accurate initial diagnosis of this condition and assessing findings that may contribute to treatment planning. The purpose of this article is to provide an overview of the capabilities of ultrasound and magnetic resonance imaging (MRI) for the diagnosis and management of this syndrome with emphasis on the relevant clinical and surgical findings and to describe potential associated abnormalities and differential diagnosis.

Laparoscopic Davydov correction of a failed gracilis flap neovagina in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome with a pelvic kidney.

The laparoscopic Davydov procedure is a surgical technique for creation of a neovagina in patients with Mayer-Rokitansky-Kuster-Hauser syndrome (MRKH) or vaginal agenesis. Herein we report its use in creating a neovagina after failure of a previous surgical attempt in a patient with a pelvic kidney, which has not been described to date. The patient, a 28-year-old woman with MRKH in whom creation of a neovagina using bilateral gracilis flaps had been attempted, was unable to have intercourse because of a shortened and scarred vagina. We successfully used the laparoscopic Davydov procedure to create a functional neovagina despite the previous surgery and the presence of a pelvic kidney. The Davydov procedure is an option for use in patients with MRKH with history of unsuccessful neovaginal surgery and can be performed in the presence of a pelvic kidney.

Follow-up in women with vaginal agenesis after laparoscopic creation of neovagina.

STUDY OBJECTIVE: To compare 2 different methods to create a neovagina in patients with Rokitansky syndrome and to describe their functional results during the follow-up. DESIGN: Descriptive study on functional outcome in 32 patients with Rokitansky syndrome (Canadian Task Classification Force II-2). SETTING: University tertiary Hospital. DESIGN AND PATIENTS: 18 patients (group 1) underwent our original technique. 14 patients (group 2) were operated on with the modified technique. INTERVENTION: Comparison between 2 different techniques of laparoscopic approach. RESULTS: Both groups had excellent surgical outcomes. The modified technique (group 2) obtained an optimal vaginal depth in fewer days than in the first group (p < .001). Consequently, the hospital stay was shorter in the second group (p < .001). Follow-up was 85 months for group 1 and 17.2 months for group 2. Functional outcome was excellent during the follow-up in both groups. CONCLUSION: Laparoscopic creation of a neovagina is a safe, minimally invasive treatment with good functional and sexual results.

Uterine rudiments in patients with Mayer-Rokitansky-Küster-Hauser syndrome consist of typical uterine tissue types with predominantly basalis-like endometrium.

OBJECTIVE: To analyze the histologic and immunohistochemical structure of uterine rudiments focusing on the endometrium in a representative group of patients with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome compared with a control group. DESIGN: Prospective comparative study. SETTING: University hospital. PATIENT(S): Forty-two patients with MRKH syndrome and 13 control subjects. INTERVENTION(S): Representative biopsies or whole uterine rudiments were removed during surgery and processed by a standardized procedure including immunohistochemical staining and analysis. MAIN OUTCOME MEASURE(S): Histologic structure, tissue types, hormone receptor expression, endometrial proliferative capacity, and type in correlation with cycle phase. RESULT(S): Twenty-two of the uterine rudiments showed a duct-like structure or small cavity, 17 of which contained endometrial epithelium and CD10-positive stroma. All rudiments contained an intact myometrial layer. Tubal epithelium and stroma were found in three rudiment samples. No significant differences were observed with regard to estrogen receptor (ER) or progesterone receptor (PR) expression in endometrium or myometrium. Interestingly, patients showed predominantly basalis-like endometrium with specific lack of CD90 expression and significantly lower proliferation compared with controls. CONCLUSION(S): All typical uterine tissues can be found in uterine rudiments of patients with MRKH syndrome. Expression of hormonal receptors in the latter and controls did not differ significantly. Endometrium shows predominantly basalis-like features in uterine rudiments.

Vault prolapse of sigmoid neovagina 26 years after vaginoplasty in Mayer-Rokitansky-Küster-Hauser syndrome: a case report.

Various operative methods have been devised to create a neovagina for patients with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. Sigmoid vaginoplasty, a common modality for vaginal reconstruction, is believed to have satisfactory long-term anatomical and functional results. We herein report a patient with MRKH syndrome and vault prolapse of a sigmoid neovagina 26 years after vaginoplasty. Biopsies from the neovagina revealed colonic mucosa. Bilateral iliococcygeus fascia fixation of the neovaginal vault was performed vaginally. The patient had a low Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire-12 (PISQ-12) score preoperatively, which further decreased postoperatively. Therefore, the surgery failed to achieve a good functional result. No recurrence of the prolapse was observed 2 years postoperatively.

Vaginoplasty using acellular porcine small intestinal submucosa graft in two patients with Meyer-von-Rokitansky-Küster-Hauser syndrome: a prospective new technique for vaginal reconstruction.

The objective of this case study is to present our experience of a surgical approach for vaginal agenesis using an acellular porcine small intestinal submucosa (SIS) graft. The present report involved 2 patients diagnosed as having vaginal agenesis due to Meyer-von-Rokitansky-Küster-Hauser syndrome. The operation procedure involved the creation of a neovaginal tunnel and then a mold wrapped with the SIS graft was placed in the neovagina. The duration of surgery was less than 45 min with minimal blood loss and no operative and postoperative complications. Epithelialization of the neovagina was achieved within 2 months after surgery. The neovagina created with this procedure was the same as that of a normal adult vagina histologically and physiologically. In conclusion, the creation of a neovagina using a SIS graft resulted in a favorable outcome and this approach may be a potential alternative to the management of vaginal agenesis.

Renal and urological abnormalities occurring with Mullerian anomalies.

OBJECTIVE: To describe the spectrum and frequency of renal and urological abnormalities in a cohort of patients with uterine anomalies. MATERIAL AND METHODS: We conducted an ethics committee approved review of 164 women referred to the imaging department from a specialist gynaecological anomaly clinic. Imaging acquired over 8 years was assessed and this was sufficient for assessment of the morphology of the gynaecological and renal tracts in 113 patients. RESULTS: Absent unilateral kidney was the most common abnormality (31.8%), most frequent in Type 3 uterine anomalies. Absent kidneys occurred in other anomaly types at lower frequency. Pelvic ureteric remnants were found in 9 of 36 patients with absent kidneys; these inserted ectopically, most commonly into the vagina. Urological abnormalities were found in 11 patients with two kidneys, including ectopic ureters, scarred kidneys and dysplastic kidney. Obstructed hemivaginas were associated commonly but not invariably with an absent kidney. CONCLUSION: Previously unreported renal and urological abnormalities have been described in patients with congenital uterine anomalies. These have significant clinical impact. Ectopic ureters can cause incontinence, and potentially cause pain and become infected. Knowledge of the ureteric course, including ureteric remnants, is essential before complex laparoscopic and vaginal surgery.

[Evaluation of different techniques for vaginoplasty in the treatment of Mayer-Rokitansky-Kuster-Hauser syndrome]. / Evaluation des différentes techniques de vaginoplastie dans le cadre du traitment du syndrome de Mayer-Rokitansky-Küster-Hauser.

BACKGROUND: Rokitansky syndrome is an utero-vaginal aplasia with a frequency of 1 / 5000 female births. To correct this anomaly whose prognosis is mainly functional and psychological numerous surgical techniques have been described. AIMS: To report our experience about 13 patients with Rokitansky syndrome and having benefited from a vaginoplasty between 1993 and 2008 and to evaluate the results of the various anatomical techniques. METHODS: This is a retrospective study of 13 patients who underwent a vaginoplasty in the center of maternity and neonatal units, over a period from December 1993 to April 2008. During this period, two operative techniques were used: the technique of Mac-Indoe and the technique of Davydov. RESULTS: The mean age at the time of surgery was 22 years (15 years-32 years). In 6 patients operated using the technique of Davydov the average depth of neovagina was 6.83 cm, one treatment failure was noted. Among the 7 patients operated using the technique of Mac Indoe the average length of neovagina was 7 cm, 3 treatment failures were noted. It was noted that there was no failure of the anatomical results in patients who have had regular sex after surgery. It was noted that there was failure of the anatomical result in 4 of 9 patients who did not have sexual intercourse after the procedure regardless of the technique used, a failure rate of 44%. CONCLUSION: The choice of technique will be based on the experience of the teams as each technique has similar anatomical and functional results in the literature. Both anatomical and functional, the main factor that determines the success of treatment seems to be the motivation of the patient to have a married life and sexual relationship.

[Mayer-Rokitansky-Küster-Hauser syndrome. A report of two cases]. / Síndrome de Mayer-Rokitansky-Küster-Hauser: reporte de dos casos y revisión de la bibliografía.

The Mayer-Rokitansky-Kuster-Hauser is a rare congenital anomaly characterized by lack of vaginal and uterine development variable and normal ovaries. It results from agenesis or hypoplasia Müller duct system. Cervicovaginal agenesis as part of the complex syndrome, is even rarer. We report two cases: adolescent patient with primary amenorrhea, cervicovaginal agenesis and chronic pelvic pain, and a 28-year-old patient with primary amenorrhea, congenital absence of uterus and vagina.

Congenital developmental defects of derivates of müllerian ducts.

Congenital developmental defects of Müllerian derivates, understandable with the knowledge of embryological development of Wolffian and Müllerian ducts, are defects of canalisation (= gynatresias), defects in fusing, combined defects and uterovaginal agenesis. Gynatresias should be suspected in the newborn, but distinguished in puberty, on the basis of menstrual blood retention, as hymeneal atresia (haematocolpos), aplasia partis distalis vaginae (haematocolpos partialis), transversal vagina septum and aplasia of vagina and uterine cervix (isolated haematometra). Particular operations are described. Defects in the fusing of Müllerian ducts from the point of view of surgery could be single-coated or double-coated. Incomplete reduplication with unilateral renal aplasia syndrome could present as hemihaematocolpos, hemihaematometra and haematometra in rudimental horn. For diagnosis of these disorders menstrual blood retention is necessary. Surgical treatment in the first two types includes resection of the common wall and haematometra in rudimental horn needs metroplasty or hemihysterectomy. Congenital absence of uterus and vagina (Rokitanski Küster) appears in genetically, endocrinologically and psychosexually normal females. Diagnosis is based on clinical examination and ultrasonography. Initial examination should be nonsurgical (dilatation method). All surgical corrections create a place for the future vagina and reach its epithelization. Different procedures are criticised. The author recommends and describes Vecchietti's laparoscopic surgery.

Vaginal malformations: a proposed classification based on embryological, anatomical and clinical criteria and their surgical management (an analysis of 167 cases).

BACKGROUND: Although it is virtually impossible to formulate a scheme that can satisfactorily collect all different types of vaginal malformations, a simple classification would be of a considerable value and would permit logical operative decisions. Many classifications of anomalies of uterus and vagina have been proposed: we find them unsatisfactory and confusing, being either too simple or too complex. We propose a new classification, focused only on vagina and based on embryological, anatomical, clinical and surgical criteria. METHODS: In over 30 years, 167 females with vaginal malformations have been observed in our department. Intersex cases were excluded. The encountered anomalies have been divided into six types and ten subtypes: type I vaginal agenesis (IA associated with uterine agenesis/17 Pts, IB isolated/1 Pt); type II vaginal atresia (IIA proximal/1 Pt, IIB distal/4 Pts); type III vaginal atresia with urethrovaginal fistula-urogenital sinus (IIIA proximal fistula-high sinus/42 Pts, IIIB distal fistula-low sinus/55 Pts); type IV vaginal atresia with transverse septum (IVA transverse septum/6 Pts, IVB imperforate hymen/17 Pts); type V disorders of müllerian ducts fusion (VA vaginal duplication/4 Pts, VB longitudinal septum/4 Pts); type VI cloaca/16 Pts. Of each type and subgroup of malformation the appropriate surgical correction is reported. Types of malformation and surgical treatment are analysed: IA vaginal reconstruction using a sigmoid conduit, IB vaginal reconstruction using a sigmoid conduit, atresic cervical resection and uterus-new vagina anastomosis according to Schmid; II perineal vaginal pull-through; IIIA anterior sagittal transanorectal vaginal pull-through, IIIB perineal flap vaginoplasty; IVA excision with abdominovaginal approach, IVB hymen incision; VA tubularization, VB septectomy via perineal approach; VI posterior sagittal anorectal-vaginal-urethroplasty. RESULTS: Most of the patients had good aesthetic and functional results. Type III showed relatively more complications: four redo operations (IIIA), four revisions of the vaginoplasty (IIIB). One patient is still waiting for definitive surgical correction. CONCLUSIONS: An early diagnosis is desirable to correct adequately vaginal malformations, which becomes mostly evident around puberty. The better timing for surgery is early age, to obtain better results and to avoid many of the psychological problems that arise at a later age.

Vaginoplasty with oxidized cellulose: anatomical, functional and histological evaluation.

OBJECTIVE: To present and evaluate the histological, anatomical and functional results of the McIndoe procedure, as modified by the application of oxidized cellulose (Surgicel™) in women with vaginal agenesis. STUDY DESIGN: Eleven patients with vaginal agenesis underwent vaginoplasty using a mould that had been wrapped with oxidized cellulose. The surgeries were performed between January 2009 and January 2010. Eight of the patients had been diagnosed with Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, and the remaining three had been diagnosed with cervicovaginal agenesis (CVA). The mean follow-up time was 14 months (range, 6-24 months), and it included clinical examinations and evaluation of the Female Sexual Function Index (FSFI). Neovaginal biopsies were taken at the time of surgery and 1-12 months after surgery. The histology of the samples was evaluated to determine squamous epithelialization of the neovaginal tissue over time, and the total collagen content of the neovaginas were compared with normal control subjects. For statistical analysis we employed the ANOVA test and the t-test. RESULTS: At 6 months, anatomical success was achieved in 100% of the MRKH syndrome patients (neovaginal length ≥ 6 cm), and functional success was achieved in 100% of the patients who started their sexual life (FSFI score ≥ 30). Biopsy results showed complete epithelialization of the neovagina after 5 months in all samples, and the collagen content was comparable to that of a normal vagina. One major postoperative complication occurred in a patient with CVA, which culminated in death. The uterovaginal canalization procedure was unsuccessful at creating an outflow tract for regular menses in all cases. CONCLUSIONS: The procedure described here offers patients a functional vagina by means of a simple and low-cost procedure that elicits squamous epithelialization of the neovaginal vault, with total collagen content similar to that of normal vaginal tissue. It is a potential alternative therapeutic approach for MRKH syndrome but not applicable to cases of CVA.

Management of vaginal agenesis.

Rokitansky syndrome and complete androgen insensitivity syndrome are the most common causes of vaginal agenesis. Treatment should be deferred until adolescence to allow informed consent and compliance. The best treatment for vaginal agenesis remains controversial although vaginal dilation therapy is still widely considered the first line treatment because success rates are high and associated risks are low. A variety of surgical options are also available, each with enthusiastic proponents. Long-term outcome studies on most surgical techniques, however, are still lacking and until recently most studies have reported on success rate in terms of anatomical success only, without including sexual function. Moreover, the medical literature lacks prospective comparative outcome studies, meaning that current choice of surgical procedure relies greatly on the surgeon's preference and experience.

Avian pelvis originates from lateral plate mesoderm and its development requires signals from both ectoderm and paraxial mesoderm.

The pelvic girdle is composed of three skeletal elements: ilium, pubis, and ischium. In comparison with other parts of the postcranial skeleton, its development is not well known to date. To elucidate the embryonic origin of the avian pelvic girdle and the signaling centers that control its development, we have performed extirpation and quail-to-chick grafting experiments. The results reveal that the entire pelvic girdle originates from the somatopleure at somite levels 26 to 35. No somitic cell contribution to skeletal elements of the pelvis has been detected. Removal of the surface ectoderm covering the lateral plate mesoderm has revealed that ectodermal signals control the development of the pelvic girdle, especially the formation of the pubis and ischium. The impaired development of the ischium and pubis correlates with the downregulation of Pax1 and Alx4, two transcription factors that control the normal development of the ischium and pubis. Although of somatopleural origin, the development of the ilium depends on somitic signals. Insertion of a barrier between somites and somatopleure disrupts the expression of Emx2 and prevents normal development of the ilium but does not affect the expression of Pax1 or Alx4 and the development of the pubis and ischium. Thus, the development of the ilium, but not of the pubis and ischium, depends on somitic and ectodermal signals.