PTEN and FOXO3 expression in the prenatal and postnatal human ovary.

PURPOSE: The objective of this study was to analyse the expression and cellular localization of FOXO3, pFOXO3 and PTEN throughout human ovary development both before and after birth. METHODS: Foetal, pubertal and adult paraffin-embedded ovarian samples were analysed by immunohistochemistry for cellular localization of FOXO3, pFOXO3 and PTEN proteins. Protein and mRNA expression were analysed by western blot and real time PCR, respectively, from fresh biopsies. RESULTS: PTEN was not detected by immunohistochemistry in germ cells and follicles of foetal, pubertal and adult ovaries. Occasional PTEN immunoreactive granulosa cells were found in atretic antral follicles in the adult ovary. Western blot analysis showed low levels of PTEN protein. Nuclear FOXO3-expressing primordial follicles represented a variable proportion of the ovarian reserve. The presence of FOXO3-expressing primordial follicles was very low in foetal ovary; although always represented in a low proportion, prevalence increased during pubertal and adult life. CONCLUSION: Our results seem to indicate that two subpopulations of primordial follicles, i.e. nuclear FOXO3-expressing and no FOXO3-expressing primordial follicles are found in the postnatal human ovary. This scenario suggests that FOXO3 could be acting as in the mouse model, preventing primordial follicle activation. However, the strategy would not be an "all or nothing" system as in mouse ovary but rather a selected subpopulation of primordial follicles preserved to ensure long-term fertility.

The infant and pubertal human ovary: Balbiani's body-associated VASA expression, immunohistochemical detection of apoptosis-related BCL2 and BAX proteins, and DNA fragmentation.

STUDY QUESTION: How do apoptosis-related BCL2 and BAX genes, known to regulate death or survival of germ cells in fetal and adult life, and germ-cell-specific VASA protein behave from birth to puberty in the human ovary? SUMMARY ANSWER: In resting primordial follicles in both infant and pubertal ovaries, BCL2 family members and germ-cell-specific VASA behave as in fetal life. After birth, once follicles leave the resting reserve to enter the growing follicular pool, detection of apoptosis-related genes moves from the germ cell to granulosa cells and VASA expression is lost. WHAT IS KNOWN ALREADY: In the human ovary, around 85% of the 7 × 106 potential oocytes produced at mid-gestation are lost before birth, an extra 10% before puberty, and loss continues throughout reproductive life until germinal exhaustion of the ovary. Oocyte loss is mainly driven through a balanced expression of BCL2 gene family members. Apoptosis-inducing BAX gene shows a sustained expression throughout fetal and adult life, whereas apoptosis-inhibiting BCL2 is detectable during the proliferative stage of primordial germ cells and oogonia in the fetal ovary and proliferation of granulosa cells in growing follicles in the adult ovary. The germ-cell marker VASA is detectable in the fetal ovary from early oogenesis and is conspicuously expressed in primordial follicles, where in late pregnancy it is associated with the Balbiani's vitelline space. VASA expression is not detectable in the adult ovary. STUDY DESIGN, SIZE, DURATION: This is a qualitative analysis involving infant/pubertal paraffin-embedded human ovaries screened for apoptosis-related proteins, DNA fragmentation and germ-cell identity. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovaries from 13 patients ranging in age from 4 to 16 years, undergoing gynaecological surgical procedures due to benign pathology, were studied. Tissues were fixed in 10% formalin, paraffin-embedded and processed for immunohistochemistry to screen the temporal and cellular localization of germ-cell-specific VASA protein and BCL2 and BAX apoptosis-related proteins. In addition, a terminal deoxynucleotidyl transferase-mediated deoxiuridinetriphosphate nick-end labelling (TUNEL) assay was performed to detect DNA fragmentation. General histology and tissue integrity were assessed by haematoxylin-eosin staining. MAIN RESULTS AND THE ROLE OF CHANCE: VASA showed a differential pattern of expression; in the resting primordial follicle reserve in infant and pubertal ovaries, it was associated with the Balbiani's body space in the germ cell. VASA remained detectable in primary follicles leaving the resting reserve, but once follicles entered the growing pool it became undetectable. This pattern of VASA expression is the same as in the fetal ovary. BAX was expressed both in the resting primordial reserve and in the pool of growing follicles, whereas BCL2 was detected only in granulosa cells in antral follicles in the growing pool. Apoptosis-related protein expression moved from the germ cell to the somatic stratum when primordial follicles left the resting reserve to enter the pool of growing follicles, irrespective of female age. Most TUNEL-positive cells were detected in the granulosa cells of antral follicles. No TUNEL-positive cells were found in resting primordial follicles. LIMITATIONS, REASONS FOR CAUTION: The study was limited by the qualitative nature of the immuno-histochemical analysis and the TUNEL assay. The results neither quantify the levels of germ-cell death nor exclude other concurrent cell death mechanisms that could act in the regulation of female germ-cell number. WIDER IMPLICATIONS OF THE FINDING: This study provides missing knowledge about apoptosis and germ-cell-specific VASA expression in the human ovary between birth and puberty and the participation of BCL2 and BAX genes in the balance between death and survival throughout female germ-line development. Intracellular localization of VASA in resting follicles emerges as a possible marker with prognostic value that needs further investigation, especially in infant patients entering ovarian cryo-preservation programmes. This knowledge will be valuable in optimizing the rescue and clinical use of germ cells to restore fertility in women. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for this study. The authors have no conflicts of interest to declare.

The ovarian germinal reserve and apoptosis-related proteins in the infant and adolescent human ovary.

BACKGROUND: Normal pubertal ovary displays all stages of follicular development and a biased BAX/BCL2 protein ratio in favor of pro-apoptotic BAX protein comparable to the adult ovary. However, adolescents suffering malignant extra-gonadal disease show a limited follicle development after cytotoxic drug treatment and a reduced capacity of in vitro follicle growth. We evaluated the expression of pro- and anti-apoptotic members of the BCL2 gene family, the FAS/FAS-L proteins from the extrinsic apoptosis pathway, the germ-cell-specific marker VASA, the pluripotency marker OCT3/4, and markers of early and late apoptosis in the ovary of pubertal patients with malignant extra-gonadal disease, which received or not pre-surgery chemotherapy, entering a cryopreservation program. RESULTS: Ovarian biopsies from 12 adolescent girls were screened for follicle count and expression of VASA, OCT3/4, BAX, BCL2, MCL1L and S, cleaved-BID, FAS/FAS-L and CASPASE 3 through immunohistochemistry, western blot and RT-PCR. All stages of folliculogenesis, from primordial to antral follicle, were present in all 12 patients analyzed. VASA and most of the screened apoptosis-related genes showed a pattern of immune-expression comparable to that previously reported. OCT3/4 showed a cytoplasmic localization in the great majority of the primordial follicles; however, in some cases the localization was nuclear. In addition, OCT3/4B showed a significant reduction compared to OCT3/4A. Unexpectedly, BCL2 was detected at all stages of folliculogenesis, associated to the Balbiani's body in the primordial follicles, regardless of whether patients had or had not received chemotherapy, ruling out the possibility that its expression is a protective response to chemotherapy. CONCLUSIONS: These findings reveal new information on the morphological status of the follicular reserve and the expression of apoptosis-related genes in histologically normal adolescent ovary from patients undergoing extragonadal cancer. The unexpected expression of apoptosis-inhibiting BCL2 protein, both in patients that had or had not received chemotherapy, opens a new avenue for thorough investigations. Moreover, the nuclear localization of OCT3/4 protein in primordial follicle-enclosed oocytes suggests a possible increased activity of ovarian stem cells in response to chemotherapy and/or extragonadal cancer. This new information can be essential for a better managing of in vitro culture of follicles that can be removed by filtration from preserved ovarian tissue, especially in girls that entered a cryopreservation program.

[Meconium periorchitis: A case report]. / Periorquitis meconial: A propósito de un caso.

Meconium periorchitis is uncommon. In the unborn child the peritoneum vaginal canal is open and, secondary to intestinal perforation due to any cause (intestinal atresia, volvulus, and others), meconium peritonitis occurs. The intestinal content reaches the scrotal vaginal cavity. Meconium peritonitis can heal spontaneously and without consequences. Calcified remnants of this event may remain in the peritoneal cavity and/or scrotum. In the newborn, a hydrocele and scrotal mass can be observed; the ultrasound will show a heterogeneous image with calcifications. Meconium periorchitis or meconium vaginalitis resolves spontaneously. The lack of awareness of this disease could lead to unnecessary surgery in the newborn. We present a 33 days old patient with a scrotal mass in whom surgery was performed with the pathological diagnosis of meconium periorchitis.

Periorquitis meconial: A propósito de un caso / Meconium periorchitis: A case report

La periorquitis meconial es infrecuente. En el feto, está abierto el conducto peritoneo vaginal y, ante una perforación intestinal por cualquier causa (atresia intestinal, vólvulo, entre otras), se produce una peritonitis meconial. El contenido intestinal puede pasar hacia la cavidad vaginal escrotal. La peritonitis meconial puede resolverse espontáneamente y sin consecuencias. Restos calcificados de este evento pueden quedar en la cavidad peritoneal y/o en el escroto. En el recién nacido, se observará un hidrocele y una masa palpable intraescrotal, ecográficamente heterogénea y con calcificaciones. La periorquitis meconial o vaginalitis meconial se resuelve espontáneamente. Su desconocimiento podría llevar a cirugías innecesarias en el neonato. Se presenta el caso de un paciente de 33 días de vida con una masa escrotal, en quien se realizó una cirugía exploratoria. El diagnóstico anatomopatológico fue de periorquitis meconial.

Meconium periorchitis is uncommon. In the unborn child the peritoneum vaginal canal is open and, secondary to intestinal perforation due to any cause (intestinal atresia, volvulus, and others), meconium peritonitis occurs. The intestinal content reaches the scrotal vaginal cavity. Meconium peritonitis can heal spontaneously and without consequences. Calcified remnants of this event may remain in the peritoneal cavity and/or scrotum. In the newborn, a hydrocele and scrotal mass can be observed; the ultrasound will show a heterogeneous image with calcifications. Meconium periorchitis or meconium vaginalitis resolves spontaneously. The lack of awareness of this disease could lead to unnecessary surgery in the newborn. We present a 33 days old patient with a scrotal mass in whom surgery was performed with the pathological diagnosis of meconium periorchitis.

Endometriosis en la etapa infanto-juvenil: presentación de nueve casos en un hospital pediátrico / Endometriosis in the infant-juvenile period: presentation of nine cases from a pediatric hospital

RESUMENEN. La bibliografía médica se registra un claro incrementoen los últimos años de casos de endometriosisen la etapa infanto-juvenil, algunos, en niñaspremenárquicas. Se presentan 9 pacientes con endometriosisestudiadas y tratadas en el HospitalNiños “Dr. Ricardo Gutiérrez” en el período 2000-2004. En la mayoría de los casos, el motivoconsulta fue dismenorrea. Las pacientes se evaluaroncon examen físico, ecografía pelviana y examenlaparoscópico para confirmar el diagnóstico y efectuarel tratamiento. Se destaca la necesidad de realizarestos estudios en pacientes con dismenorrearesistente a los tratamientos comunes, para contribuiral diagnóstico y tratamiento precoz de la afección,de modo de preservar la fertilidad futura.Palabras clave: endometriosis infanto-juvenil, dismenorrea,laparoscopia.

Síndrome de Rokitansky (agenesia úterovaginal): aspectos clínicos, diagnósticos y terapéuticos / Rokitansky syndrome(uterine vaginal agenesis): clinical, diagnostic and therapeutic aspects

RESUMEN. Introducción. El síndrome de Rokitansky es unaanomalía grave del tracto reproductivo femenino,con ausencia de menarca e imposibilidad deembarazo.Objetivo. Analizar las características clínicas, diagnósticas,terapéuticas y evolutivas de un grupo deadolescentes afectadas.Material y métodos. Nueve adolescentes (15,2 ± 1,5años) que consultaron por ausencia de menarca odolor pelviano se evaluaron mediante examen físico,ecografía ginecológica, resonancia nuclear magnéticaabdominopelviana, cariotipo, dosajes hormonalesy laparoscopia. El diagnóstico final fue desíndrome de RokitanskyResultados. Todas las pacientes tuvieron caracteressexuales secundarios normales. Tres presentaronanomalías renales y tres anomalías esqueléticas.La ecografía mostró útero ausente, rudimentarioo asimétrico, este último con hematómetra evidenciadopor resonancia nuclear magnética. Lalaparoscopia detectó anomalías tubarias u ováricasen seis casos. Mediante estos estudios, cinco denuestras pacientes se clasificaron como pertenecientesa la forma B (atípica) del síndrome. Dospacientes tuvieron cariotipos anormales: 46, X, del(X) (q22q24) y 46,XX, t (3;22) (q13.3, q 12.1), ambassin insuficiencia ovárica. Seis pacientes tuvieronconcentraciones hormonales normales, una presentóhiperplasia suprarrenal congénita de formano clásica, confirmada por estudio molecular y otramostró alteraciones compatibles con síndrome depoliquistosis ovárica. En seis pacientes se efectuóplástica de vagina con resultado satisfactorio

Estruma ovárico benigno: diagnóstico y tratamiento en la adolescencia / Benign struma ovari: diagnosis and treatment in adolescence

Los teratomas de ovario son los blastomas ováricos más frecuentes en la etapa infanto-juvenil en la mujer: derivados de las células germinales totipotenciales pueden contener tejidos del ectodermo,mesodermo y endodermo. El estruma ovárico es un teratoma con predominio de tejido tiroideo. Son excepcionales en la etapa infanto-juvenil. Existen estrumas benignos y malignos y se presentan con una frecuencia de 2 por ciento y 0,3 por ciento respectivamente dentro de la frecuencia total de los teratomas. Asintomáticos en su mayoría, pueden manifestarse como tumor abdominopélvico y producir ascitis e hidrotórax.Se presenta un caso de estruma ovárico benigno en una adolescente de 15 años, que consultó por tumor abdominal de crecimiento lento, lo que motivó rechazo social en su medio escolar por presunción de embarazo.

Importancia de la gonadectomía temprana en pacientes con síndrome de Turner portadoras de cromosoma Y / Importance of early gonadectomy in patients with Turner syndrome with presence of Y chromosome material

Introducción: la presencia de material del cromosoma Y en mujeres con Síndrome de Turner se asocia con el desarrollo de tumores gonadales, teniendo un incremento del riesgo relacionado con la edad. Objetivo: investigar la correlación entre la presencia de cromosoma Y ó secuencias específicas del Y y los hallazgos histopatológicos de las gónadas en pacientes con Síndrome de Turner. Material y métodos: se estudiaron cinco niñas con síndrome de Turner, con edades comprendidas entre los 4.4 y 13.4 años de edad. En todas las pacientes se realizó el análisis cromosómico con bandeo G y técnicas de alta resolución, tales como análisis molecular por PCR amplificando las secuencias específicas del cromosoma Y (SRT, DYZ1, DYZ3 y AMGL). Tres niñas tenían un cariotipo conteniendo el cromosoma Y, mientras que en las otras dos niñas restantes fueron detectadas secuencias del cromosoma Y por análisis molecular. En todas la niñas fue realizada la gonadectomía bilateral con técnica laparoscópica. Resultados: el procedimiento laparoscópico fue adecuado y bien tolerado por las niñas. Tres de las pacientes (de 5.4, 8 y 13.4 años de edad) que tenían cromosoma Y presentaron tumores gonadales: gonadoblastoma uni o bilateral, y en una de ellas se encontró un disgerminoma. En las dos niñas restantes se encontraron las típicas gónadas tipo "streak" del Síndrome de Turner, sin tejido neoplásico. Conclusión: el hallazgo de tumores gonadales en 3 de las 5 niñas con Síndrome de Turner refuerza la importancia de la indicación de la gonadectomía a edades tempranas cuando las pacientes son portadoras de material del Y en sus cromosomas. La técnica laparoscópica es una elección adecuada para el tratamiento