Timing of spermatogonial stem cell transplantation affects the spermatogenic recovery outcome in mice.

Spermatogonial stem cell transplantation (SSCT) is a strategy that has demonstrated to be feasible to restore spermatogenesis in animal models when it is performed shortly after the gonadotoxic onset to destroy their endogenous germ cells. However, in the case of boys subjected to fertility preservation, future transplantations will be performed with a delay of many years. In order to study how timing of SSCT affects donor-derived spermatogenic recovery in mice, we compared the percentage of spermatogenic tubule cross-sections within testes of 59 C57BL/6NCrl mice distributed in 6 groups: group 1, untreated mice controls (n = 9); group 2, mice that received a single dose of busulfan 40 mg/kg (n = 10); group 3, mice that received two additional doses of busulfan 10 mg/kg every 5 weeks (n = 10); group 4 (SSCT-A), mice subjected to a standard SSCT performed 5 weeks after a single injection of busulfan 40 mg/kg (n = 10); group 5 (SSCT-B), mice subjected to a delayed SSCT performed 15 weeks after a single injection of busulfan 40 mg/kg (n = 10); and group 6 (SSCT-C), mice subjected to a delayed SSCT with two additional doses of busulfan 10 mg/kg every 5 weeks (n = 10). Spermatogenic recovery in standard SSCT-A and SSCT-C groups ranged between 22.29 and 22.65%, compared with a lower recovery rate of 11.54% showed in the SSCT-B group. However, donor contribution resulted higher in standard SSCT-A, representing a 69.71% of cross-sections, compared with the rest of conditions ranging from 34.69 to 35.42%. Overall, we concluded that a delay in the SSCT from the gonadotoxic onset decreases the efficiency of donor-derived spermatogenic recovery in mice.

Histologic Analysis of Testes from Prepubertal Patients Treated with Chemotherapy Associates Impaired Germ Cell Counts with Cumulative Doses of Cyclophosphamide, Ifosfamide, Cytarabine, and Asparaginase.

Cryopreservation of immature testicular tissue is an experimental strategy for the preservation of fertility in prepubertal boys that will be subjected to a gonadotoxic onset, as is the case of oncologic patients. Therefore, the objective of this study was to assess the impact of chemotherapeutic treatments on the testicular histologic phenotype in prepubertal patients. A total of 56 testicular tissue samples from pediatric patients between 0 and 16 years old (28 with at least one previous chemotherapeutic onset and 28 untreated controls) were histologically analyzed and age-matched compared. At least two 5-µm sections from testis per patient separated by a distance of 100 µm were immunostained for the germ cell marker VASA, the spermatogonial markers UTF1, PLZF, UCHL1, and SALL4, the marker for proliferative cells KI67, and the Sertoli cell marker SOX9. The percentage of tubule cross-sections positive for each marker and the number of positive cells per tubule cross-section were determined and association with the cumulative dose received of each chemotherapeutic drug was statistically assessed. Results indicated that alkylating agents, cyclophosphamide and ifosfamide, but also the antimetabolite cytarabine and asparaginase were associated with a decreased percentage of positive tubules and a lower number of positive cells per tubule for the analyzed markers. Our results provide new evidences of the potential of chemotherapeutic agents previously considered to have low gonadotoxic effects such as cytarabine and asparaginase to trigger a severe testicular phenotype, hampering the potential success of future fertility restoration in experimental programs of fertility preservation in prepubertal boys.

Criopreservación ovárica en niñas con cáncer: nuevos retos / Ovarian cryopreservation in girls with cancer: new challenges

Objetivos. La criopreservación de la corteza ovárica (CCO) para futuro autotrasplante permitirá hacer frente al fallo ovárico precoz y a las alteraciones de la capacidad reproductiva que afectan a algunas delas supervivientes de cáncer pediátrico. Material y métodos. En el Programa de Preservación de Fertilidad en Oncología Pediátrica se incluyen pacientes con alto riesgogonadotóxico: las que vayan a recibir radioterapia pélvica, trasplante de precursores hematopoyéticos, altas dosis de radioterapia craneal o agentes alquilantes, o aquéllas con patología ovárica bilateral. Antes del tratamiento oncológico y coincidiendo con otros procedimientos invasivos, se recoge el tejido ovárico por vía laparoscópica. Una vez descartada la malignidad en la muestra y confirmada la presencia de folículos primordiales, el equipo multidisciplinar de oncólogo, cirujano y especialista en fertilidad coordina la manipulación y envío de la corteza ovárica al Banco de Tejidos de la Comunidad Valenciana. Resultados. De julio de 2008 hasta mayo de 2010 se incluyeron en el programa a 8 pacientes, entre 8 y 18 años, con diagnóstico de: linfoma de Hodgkin (n= 2), leucemia aguda linfoide y mieloide (n= 2),sarcoma de Ewing pélvico, teratoma ovárico bilateral y meduloblastoma. Cinco pacientes recibieron quimioterapia no gonadotóxica previa a la CCO. De forma adicional, se practicaron 6 procedimientos en el mismo acto anestésico. Se realizó o oforectomía parcial en la mitad delos casos y total en el resto, asociando pexia ovárica en 1 ocasión. Todas las muestras fueron válidas. Conclusiones. La CCO de los casos seleccionados se realizó de forma segura, sin complicaciones ni demora del tratamiento oncológico. Podemos afi rmar que la primera experiencia nacional en este tipo de abordaje ha sido satisfactoria (AU)

Background. Ovarian cortex cryopreservation (OCC) for future autotransplant represents a treatment alternative for those paediatriccancer survivors affected of ovarian failure and fertility disorders. Methods. Patients with high gonadotoxic risk are included in the Oncology Paediatric Fertility Preservation Programme: those receiving pelvic radiotherapy, bone marrow transplantation, high doses of cranial radiotherapy or alquilating agents, or those with bilateral ovarian pathology. Prior to the oncological treatment, the ovarian tissue is harvested laparoscopically. At the same time, other invasive procedures are done. Once malignancy is ruled out of the specimen and the presence of primordial follicles is confirmed, the multidisciplinary team of oncologist, paediatric surgeon and fertility specialist coordinate the processing and delivery of the ovarian cortex to the Comunidad ValencianaTissue Bank. Results. From July 2008 to May 2010 eight patients have been included in the programme, aged between 8-18 years old and with diagnosis of: Hodgkin’s lymphoma (n= 2), Acute Myeloblastic and Lymphoblasticleukaemia (n= 2), pelvic Ewing’s sarcoma, bilateral ovarian Teratoma and Meduloblastoma. Five patients underwent non gonadotoxicchemo therapy before OCC. Six additional procedures were doneusing the same anaesthetic event. Partial oophorectomy was performed in half the cases, total oophorectomy in the rest of them, and an ovarianpexia was once associated. All taken samples were found to be valid. Conclusions. OCC of selected patients was performed safely, with neither postoperative complications nor delay of the oncological treatment. Therefore, the fi rst national experience in this procedure has been satisfactorily achieved (AU)

[Ovarian cryopreservation in girls with cancer: new challenges]. / Criopreservación ovárica en niñas con cáncer: nuevos retos.

BACKGROUND: Ovarian cortex cryopreservation (OCC) for future autotransplant represents a treatment alternative for those paediatric cancer survivors affected of ovarian failure and fertility disorders. METHODS: Patients with high gonadotoxic risk are included in the Oncology Paediatric Fertility Preservation Programme: those receiving pelvic radiotherapy, bone marrow transplantation, high doses of cranial radiotherapy or alquilating agents, or those with bilateral ovarian pathology. Prior to the oncological treatment, the ovarian tissue is harvested laparoscopically. At the same time, other invasive procedures are done. Once malignancy is ruled out of the specimen and the presence of primordial follicles is confirmed, the multidisciplinary team of oncologist, paediatric surgeon and fertility specialist coordinate the processing and delivery of the ovarian cortex to the Comunidad Valenciana Tissue Bank. RESULTS: From July 2008 to May 2010 eight patients have been included in the programme, aged between 8-18 years old and with diagnosis of: Hodgkin's lymphoma (n= 2), Acute Myeloblastic and Lymphoblastic leukaemia (n= 2), pelvic Ewing's sarcoma, bilateral ovarian Teratoma and Meduloblastoma. Five patients underwent non gonadotoxic chemotherapy before OCC. Six additional procedures were done using the same anaesthetic event. Partial oophorectomy was performed in half the cases, total oophorectomy in the rest of them, and an ovarian pexia was once associated. All taken samples were found to be valid. CONCLUSIONS: OCC of selected patients was performed safely, with neither postoperative complications nor delay of the oncological treatment. Therefore, the first national experience in this procedure has been satisfactorily achieved.

Long-term storage in liquid nitrogen does not affect cell viability in cardiac valve allografts.

Liquid nitrogen is the most common medium used by tissue banks for the storage of cryopreserved heart valves. This study evaluates the effect of the length of storage on human cryopreserved heart valves. Human tissues (14 aortic and 13 pulmonary) were frozen in a controlled-rate freezer (1 degrees C/min) and stored in the liquid phase of a nitrogen tank for 9.1+/-1.6 years. The preservative solution was medium M199 containing 5% human serum albumin and 10% Me(2)SO. After thawing in a water bath at 42 degrees C, the cryoprotectant was removed. Then, fragments from vascular wall and leaflet were dissected. Explant cultures and histological studies were performed in order to assess cell viability and structural integrity. CD90 and CD31 expression was analysed in cultured cells using flow cytometry. Light microscopy, immunofluorescence staining and laser scanning confocal microscopy were used to evaluate cell viability and extracellular matrix components. Electron microscopy was used for ultrastructural study. Cell cultures could be obtained from all the specimens assayed. Cells grew from explants showing a fibroblastic phenotype. CD90 expression was common in cultured cells but a low percentage of cells expressed CD31. Histological results showed a good preservation estructure in both leaflets and vascular walls. Morphological features of cellular irreversible damage were very rare. No differences which could be due to length of allograft storage period were observed. We concluded that allografts stored in liquid nitrogen up to 13 years did not significantly undergo loss of cell viability other than that due to disinfection, freezing and thawing protocols.

The Valencia Programme for Fertility Preservation.

INTRODUCTION: Fertility preservation needs have increased dramatically in recent years due to a rising cancer incidence and also a significant increase in survival rates. MATERIALS AND METHODS: We report on 200 women participating in the Valencia Programme for Fertility Preservation, of whom 55% were breast cancer patients, 25% Hodgkin's disease patients, and the remaining 20% suffered from other nonmalignant and malignant diseases. Mean age was 28.2 years (11-39). Before patients were submitted to oncological treatment, the right ovarian cortex was extracted by laparoscopy and cryopreserved. Once the patient was free of disease, the right ovarian cortex was thawed and implanted onto the left ovarian medulla (after extraction at the same surgical time of left ovarian cortex). RESULTS: In 95% of cases, a piece approximately 2 x 3 cm was obtained. The procedure did not cause any change in the cancer therapy schedule. Four implants had been performed (all of them in women who underwent chemotherapy prior to ovarian cortex extraction), from which two of the case achieved ovarian function resumption, in one case a month after the implant and in the other 5.5 months after. The remaining two implanted cases were performed 5 and 6 months prior to the writing of this paper, respectively. CONCLUSIONS: Ovarian tissue cryopreservation is a feasible option to preserve ovarian function and possibly fertility in adolescents and young women at risk of developing premature ovarian failure (POF) due to chemotherapy and/or radiotherapy.

The Valencia Programme for Fertility Preservation

INTRODUCTION: Fertility preservation needs have increased dramatically in recent years due to a rising cancer incidence and also a significant increase in survival rates. MATERIALS AND METHODS: We report on 200 women participating in the Valencia Programme for Fertility Preservation, of whom 55% were breast cancer patients, 25% Hodgkin's disease patients, and the remaining 20% suffered from other nonmalignant and malignant diseases. Mean age was 28.2 years (11-39). Before patients were submitted to oncological treatment, the right ovarian cortex was extracted by laparoscopy and cryopreserved. Once the patient was free of disease, the right ovarian cortex was thawed and implanted onto the left ovarian medulla (after extraction at the same surgical time of left ovarian cortex). RESULTS: In 95% of cases, a piece approximately 2 x 3 cm was obtained. The procedure did not cause any change in the cancer therapy schedule. Four implants had been performed (all of them in women who underwent chemotherapy prior to ovarian cortex extraction), from which two of the case achieved ovarian function resumption, in one case a month after the implant and in the other 5.5 months after. The remaining two implanted cases were performed 5 and 6 months prior to the writing of this paper, respectively. CONCLUSIONS: Ovarian tissue cryopreservation is a feasible option to preserve ovarian function and possibly fertility in adolescents and young women at risk of developing premature ovarian failure (POF) due to chemotherapy and/or radiotherapy (AU)

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Assessment of dietary adequacy for an elderly population based on a Mediterranean model.

This study analysed whether at different energy levels, a varied diet based on Mediterranean Diet patterns would meet the RDIs for specific nutrients in a population > 65 y. Based on RDIs for elderly persons > 65 y for PRO, FAT CHO, phosphorus, calcium, magnesium, iron, zinc, vitamin C, B6, folate and fibre, menu models based on Mediterranean diet food patterns were calculated for the following calorie levels: 1400, 1500, 1600, 1700 and 1800 kcals. 15 menu variations for each calorie level were then created based on the previously calculated models. Utilising the Program for Alimentation and Nutrition (PAN) database, nutritional analysis was carried out for all menus and the mean nutrient values for a 2 week period were calculated for each calorie level. Intakes at all calorie levels provided adequate amounts of folate, phosphorus, iron and Vitamin B6. Intakes were low in all groups for Calcium, Zinc, Magnesium and Vitamin E, with the exception of the 1800 kcal level for Vitamin E. Results show that at low energy levels, meeting nutrient needs was difficult and that even at higher calorie intakes, contrary to what was expected, certain nutrients were found to be inadequate.

Amperometric flow-injection determination of sucrose with a mediated tri-enzyme electrode based on sucrose phosphorylase and electrocatalytic oxidation of NADH.

A new sucrose electrode is described for the determination of sucrose without interference from glucose or fructose. The sucrose electrode is based on the tri-enzymatic system of sucrose phosphorylase, phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase, where NAD(P)H is produced from the last enzymatic reaction and recycled into NAD(P)+ through its electrocatalytic oxidation by Os(4,4'-dimethyl-2,2-bypyridine)2(1,10-phenanthroline-5,6-dione). The electrodes were optimised with respect to the various construction parameters and carrier composition in a FIA system and their response as a function of the pH and flow-rate was examined. The electrodes were suitable for operation in a FIA system and the analysis of real samples showed good agreement with the reference method. Typical optimised electrodes showed detection limits of 1 mM sucrose, response time of 5 min, sensitivity 1.010 nA mM(-1), and current density of 8.38 microA cm(-2), using 200 mM PIPES pH 7.25 with 10 mM phosphate and 5 mM MgCl2 as carrier.