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Slush nitrogen vitrification of human ovarian tissue does not alter gene expression and improves follicle health and progression in long-term in vitro culture

      Objective

      To study whether slush nitrogen (SN) vs. liquid nitrogen (LN) vitrification affects human ovarian tissue gene expression and preserves follicle health during extended in vitro culture.

      Design

      Randomized experimental study.

      Setting

      University research laboratory.

      Patient(s)

      Ovarian biopsies collected by laparoscopic surgery from patients with benign gynaecologic conditions.

      Intervention(s)

      None.

      Main Outcome Measure(s)

      Ovarian strips were vitrified with LN or SN, warmed, and analyzed before or after culture for 9 days (d9) in gas-permeable dishes. Expression of genes involved in stress and toxicity pathways was analyzed in fresh and warmed strips by polymerase chain reaction (PCR) array and quantitative real-time–PCR. Fresh and vitrified/warmed strips were analyzed for follicle quality, progression, and viability before or after culture.

      Result(s)

      The SN vitrification preserved follicle quality better than LN (% grade 1 follicles: fresh control, 54.2; LN, 29.3; SN, 48.8). Quantitative reverse transcription–PCR demonstrated a noticeable up-regulation of 13 genes in LN samples (range, 10–35) and a markedly lower up-regulation of only 5 genes (range, 3.6–7.8) in SN samples. Long-term in vitro culture evidenced worse follicle quality and viability in LN samples than in both fresh and SN samples (% grade 1 follicle: fresh d0, 51.5; fresh d9, 41; LN d9, 16.4; SN d9, 55) and a highly significant reduction of primordial follicles and a concomitant increase of primary and secondary follicles in all samples. Follicle growth to the secondary stage was significantly higher in vitrified tissue than in fresh tissue, being better in SN than in LN vitrified tissue.

      Conclusion(s)

      Follicle quality, gene expression, viability, and progression are better preserved after SN vitrification.
      La vitrificación de tejido ovárico humano mediante nieve carbónica no altera la expresión génica y mejora la calidad y la progresión de los folículos en cultivo in vitro a largo plazo

      Objetivo

      Estudiar si la vitrificación mediante nieve carbónica (NC) vs. nitrógeno líquido (NL) afecta la expresión génica del tejido ovárico humano y preserva la calidad de los folículos durante el cultivo in vitro prolongado.

      Diseño

      Estudio experimental aleatorizado.

      Lugar

      Laboratorio de investigación universitaria.

      Paciente(s)

      Biopsias de ovario obtenidas mediante cirugía laparoscópica de pacientes con afecciones ginecológicas benignas.

      Intervenciones

      Ninguna.

      Medidas de los resultados principales

      Las tiras de ovario fueron vitrificadas con NL o NC, descongeladas y analizadas antes o después del cultivo durante 9 días (D9) en placas permeables a gases. Se analizó la expresión de los genes implicados en las rutas de estrés y toxicidad mediante Q3 Array reacción en cadena de la polimerasa (PCR) y PCR cuantitativa a tiempo real en las tiras de ovario frescas y descongeladas. Se analizó la calidad de los folículos, así como la progresión y viabilidad de las tiras frescas y vitrificadas/descongeladas antes o después del cultivo.

      Resultado(s)

      La vitrificación mediante NC conservó la calidad de los folículos mejor que la vitrificación mediante NL (% de folículos grado 1: fresco control 54,2; NL 29,3; NC 48,8). PCR cuantitativa mostró una notable regulación al alza de 13 genes en las muestras NL (rango: 10-35) y una regulación al alza notablemente menor de solo 5 genes (rango: 3,6-7,8) en las muestras NC. El cultivo in vitro a largo plazo mostró una peor calidad y viabilidad de los folículos en las muestras vitrificadas mediante NL que en las muestras frescas y vitrificadas mediante NC (% de folículos grado 1: fresco D0 51,5; fresco D9 41; NL D9 16,4; NL D9 55) y una significativa reducción de los folículos primordiales, así como un aumento concomitante de los folículos primarios y secundarios en todas las muestras. El crecimiento del folículo en estadio secundario fue significativamente mayor en el tejido vitrificado que en el tejido fresco, siendo mejor en NC que en tejido vitrificado mediante NL.

      Conclusión(es)

      La calidad, expresión génica, viabilidad y progresión de los folículos, se conserva mejor después de la vitrificación mediante NC.

      Palabras clave

      Folículos, expresión génica, histología, criopreservación ovárica, viabilidad

      Key Words

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