This is the first report of the presence of ultrastructurally normal primordial and primary follicles, 13 months after autotransplantation of frozen–thawed human ovarian tissue. The stroma contained ...numerous viable and ultrastructurally normal blood vessels but showed poor cellular density.
What is the contamination rate by cancer cells and spermatogonia numbers in immature testicular tissue (ITT) harvested before the start of gonadotoxic therapy in boys with a hematological malignancy?
...Among our cohort of boys diagnosed with acute lymphoblastic leukemia (ALL) and lymphomas, 39% (n = 11/28) had cancer cells identified in their tissues at the time of diagnosis and all patients appeared to have reduced spermatogonia numbers compared to healthy reference cohorts.
Young boys affected by a hematological cancer are at risk of contamination of their testes by cancer cells but histological examination is unable to detect the presence of only a few cancer cells, which would preclude autotransplantation of cryobanked ITT for fertility restoration, and more sensitive detection techniques are thus required. Reduced numbers of spermatogonia in ITT in hematological cancer patients have been suggested based on results in a limited number of patients.
This retrospective cohort study included 54 pre- and peri-pubertal boys who were diagnosed with a hematological malignancy and who underwent a testicular biopsy for fertility preservation at the time of diagnosis before any gonadotoxic therapy between 2005 and 2021.
Among the 54 patients eligible in our database, formalin-fixed paraffin-embedded (FFPE) testicular tissue was available for 28 boys diagnosed either with ALL (n = 14) or lymphoma (n = 14) and was used to evaluate malignant cell contamination. Hematoxylin and eosin (H&E) staining was performed for each patient to search for cancer cells in the tissue. Markers specific to each patient's disease were identified at the time of diagnosis on the biopsy of the primary tumor or bone marrow aspiration and an immunohistochemistry (IHC) was performed on the FFPE ITT for each patient to evidence his disease markers. PCR analyses on the FFPE tissue were also conducted when a specific gene rearrangement was available.
The mean age at diagnosis and ITT biopsy of the 28 boys was 7.5 years (age range: 19 months-16 years old). Examination of ITT of the 28 boys on H&E stained sections did not detect malignant cells. Using IHC, we found contamination by cancerous cells using markers specific to the patient's disease in 10 of 28 boys, with a higher rate in patients diagnosed with ALL (57%, n = 8/14) compared with lymphoma (14%, n = 2/14) (P-value < 0.05). PCR showed contamination in three of 15 patients who had specific rearrangements identified on their bone marrow at the time of diagnosis; one of these patients had negative results from the IHC. Compared to age-related reference values of the number of spermatogonia per ST (seminiferous tubule) (Spg/ST) throughout prepuberty of healthy patients from a simulated control cohort, mean spermatogonial numbers appeared to be decreased in all age groups (0-4 years: 1.49 ± 0.54, 4-7 years: 1.08 ± 0.43, 7-11 years: 1.56 ± 0.65, 11-14 years: 3.37, 14-16 years: 5.44 ± 3.14). However, using a cohort independent method based on the Z-score, a decrease in spermatogonia numbers was not confirmed.
The results obtained from the biopsy fragments that were evaluated for contamination by cancer cells may not be representative of the entire cryostored ITT and tumor foci may still be present outside of the biopsy range.
ITT from boys diagnosed with a hematological malignancy could bear the risk for cancer cell reseeding in case of autotransplantation of the tissue. Such a high level of cancer cell contamination opens the debate of harvesting the tissue after one or two rounds of chemotherapy. However, as the safety of germ cells can be compromised by gonadotoxic treatments, this strategy warrants for the development of adapted fertility restoration protocols. Finally, the impact of the hematological cancer on spermatogonia numbers should be further explored.
The project was funded by a grant from the FNRS-Télévie (grant n°. 7.4533.20) and Fondation Contre le Cancer/Foundation Against Cancer (2020-121) for the research project on fertility restoration with testicular tissue from hemato-oncological boys. The authors declare that they have no conflict of interest.
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Abstract During the last decade, new technologies in reproductive medicine have emerged to preserve the fertility of women whose gonadal function is threatened by premature menopause or gonadotoxic ...treatments. To offer an individualized approach to these patients, different experimental procedures are under investigation, including oocyte cryopreservation and cryopreservation and transplantation of ovarian tissue in the form of cortical fragments, whole ovary or isolated follicles. This review shows that transmission electron microscopy (TEM), combined with other in-vivo and in-vitro analysis techniques, is a valuable tool in the establishment of new experimental protocols to preserve female fertility. Ultrastructural studies allow in-depth evaluation of the oocyte's unique morpho-functional characteristics, which explain its low cryotolerance, and provide essential information on follicular, stromal and endothelial cell integrity, as well as cellular interactions crucial for normal folliculogenesis. In order to be able to offer appropriate and efficient options in every clinical situation, oocyte in-vitro maturation and ovarian tissue transplantation need to be optimized. Further development of new approaches, such as follicular isolation and whole ovary transplantation, should be encouraged. Fine ultrastructural details highlighted by TEM studies will be useful for the further optimization of these emerging technologies.
Does adipose-tissue-derived stem cell conditioned medium (ASC-CM) supplementation enhance follicle and stromal cell outcomes in vitro?
Bovine ovaries (n = 8) were sectioned and cultured in vitro for ...8 days in two different groups: (i) standard culture (OT Ctrl D8); and (ii) culture with ASC-CM supplementation (OT + CM D8). Half of the culture medium was replaced every other day, and stored to measure the production of oestradiol. Follicle classification was established using haematoxylin and eosin staining. Follicle and stromal cell DNA fragmentation was assessed by TUNEL assays, while growth differentiation factor-9 (GDF-9) staining served as a marker of follicle quality. Additionally, three factors, namely vascular endothelial growth factor (VEGF), interleukin 6 (IL-6) and transforming growth factor beta 1 (TGF-β1), were evaluated in ASC-CM in order to appraise the potential underlying mechanisms of action of ASC.
The OT + CM D8 group showed a significantly higher proportion of secondary follicles (P = 0.02) compared with the OT Ctrl D8 group. The OT + CM D8 group also demonstrated significantly lower percentages of TUNEL-positive follicles (P = 0.014) and stromal cells (P = 0.001) compared with the OT Ctrl D8 group. Furthermore, follicles in the OT + CM D8 group exhibited a significant increase (P = 0.002) in expression of GDF-9 compared with those in the OT Ctrl D8 group, and oestradiol production was significantly higher (P = 0.04) in the OT + CM D8 group. All studied factors were found to be present in ASC-CM. VEGF and IL-6 were the most widely expressed factors, while TGF-β1 showed the lowest expression.
Addition of ASC-CM to culture medium enhances follicle survival, development and oestradiol production, and promotes the viability of stromal cells. VEGF, IL-6 and TGF-β1 could be paracrine mediators underlying the beneficial effects.
Rectal Tonsil as a Cause of Recurrent Rectal Prolapse Pire, Aurore; Pratte, Laurence; Camboni, Alessandra ...
Journal of pediatric gastroenterology and nutrition,
December 2020, 2020-December, 2020-12-00, 20201201, Letnik:
71, Številka:
6
Journal Article
How are markers of cell death, invasiveness and progesterone signalling expressed in endometrium and ectopic lesions from adenomyosis patients?
Formalin-fixed paraffin-embedded tissue was collected ...from 15 control and 15 adenomyosis participants . To assess cell survival capacity, caspase 3 and microtubule-associated proteins 1A/1B light chain 3B (LC3B) were immunolabelled as markers of apoptosis and autophagy respectively. Matrix metalloproteinase 9 (MMP9) expression served as a marker of extracellular matrix degradation and invasion activity. Progesterone receptors were immunostained to detect evidence of progesterone resistance.
Caspase 3 expression was significantly lower in the stromal (P = 0.0013) and epithelial (P = 0.0157) compartments of adenomyotic lesions than in healthy endometrial tissue. In the stroma, caspase 3 expression was significantly weaker in lesions than in corresponding eutopic endometrium (P = 0.0006). LC3B immunostaining was significantly decreased in adenomyotic stroma compared with corresponding eutopic endometrium (P = 0.0349). A significantly higher expression of MMP9 was detected in eutopic stroma from adenomyosis patients than in healthy tissue (P = 0.0295). Progesterone receptor immunostaining was found to be significantly weaker in the stroma of endometrium and ectopic lesions from adenomyosis patients than disease-free women (P = 0.0001; P = 0.0021).
Adenomyotic lesions show lower levels of apoptosis and autophagy, suggesting that aberrant cell survival may be involved in disease pathogenesis. MMP9 appears to contribute to endometrial invasiveness in adenomyosis, as its expression is more pronounced in endometrium from these women than women without the disease. Evidence of progesterone resistance can be found in endometrium and ectopic lesions from adenomyosis patients, and may drive disease development and account for the failure of certain patients to respond to progestogens.
To study the effect of freezing, in vitro culture (IVC) and grafting to chorioallantoic membrane (CAM) on follicle outcomes in human ovarian tissue.
An experimental study.
University-based research ...laboratory.
Fresh and cryopreserved ovarian tissue from 10 patients was donated to research with their consent and institutional review board approval.
Fresh and frozen-thawed ovarian cortical pieces were in vitro-cultured and compared (fresh-IVC vs FT-IVC). The FT-IVC fragments were then examined against fragments grafted to CAM (FT-CAM). After both IVC and CAM grafting, ovarian cortical pieces (4×2×1 mm3) were analyzed on days 0, 1, and 6.
Follicle analyses included histology (count and classification) and immunohistochemistry (Ki67 proliferation, caspase-3 apoptosis, 1A and 1B light chain 3B autophagy, p-Akt, FOXO1, and p-rpS6 PI3K activation). Droplet digital polymerase chain reaction further explored expression of PI3K pathway- and oocyte-related genes in tissue sections.
No major differences were detected between fresh-IVC and FT-IVC tissues in any conducted analyses. Although a significant drop was observed in primordial follicle (PF) proportions in the fresh-IVC and FT-IVC groups (d0 vs. d6, P<.002), they held steady in the FT-CAM group (d0 vs. d6, P>.05). The PF rates were also significantly higher in the FT-CAM group than the FT-IVC group on d6 (P=.02). Importantly, avian erythrocytes were already present in 30% of implants from d1. Apoptotic and autophagic follicle rates increased during IVC (P<.008), but remained significantly lower in the FT-CAM group (P<.01), confirming superior follicle preservation in CAM-grafted tissue. Upregulation of the PI3K/FOXO pathway was established in the IVC groups, demonstrating PF activation, whereas significant pathway downregulation was detected in the FT-CAM group (P<.03). The droplet digital polymerase chain reaction tests confirmed oocyte growth during IVC and follicle autophagy in all groups; however, the PI3K pathway appeared to be differentially modulated in tissues and follicles.
In vitro culture induces PF depletion with no additional impact of freezing. Grafting to CAM preserves the PF pool by curbing follicle activation, apoptosis, and autophagy, probably thanks to rapid graft revascularization and/or the circulating embryonic antimüllerian hormone. These findings highlight the importance of enhancing neoangiogenesis in ovarian grafts and investigating the potential benefits of administering antimüllerian hormone to prevent PF burnout.
Resultados de folículos en tejido ovárico humano: efecto del congelamiento, cultivo y trasplante.
Estudiar el efecto del congelamiento, cultivo in vitro (IVC) y trasplante en la membrana corioalantoidea (CAM) sobre los resultados de folículos en tejido ovárico humano.
Estudio experimental.
Laboratorio Universitario de investigación básica.
El tejido ovárico fresco y criopreservado de 10 pacientes fueron donados para investigación con su consentimiento y aprobación por el comité de revisión institucional.
Se compararon piezas de corteza ovárica cultivadas in vitro en fresco (Fresco-IVC) y cultivadas in vitro luego de congelamiento-descongelamiento (FT-IVC).
Los fragmentos FT-IVC fueron luego evaluados contra fragmentos trasplantados en CAM (FT-CAM). Los trasplantes de piezas corticales de ovario (421 mm) de IVC y CAM fueron analizados en los días 0, 1 y 6.
El análisis folicular incluyó la histología (conteo y clasificación) e inmunohistoquímica (Ki67 proliferación, caspasa 3 apoptosis, cadena ligera 3B de proteínas 1A y 1B autofagia, p-Akt, FOXO1 y p-rpS6 activación PI3K). Posteriormente se usó PCR digital para la explorar de la expresión de la vía PI3K- y genes relacionados a ovocitos de las secciones ováricas.
No se encontraron mayores diferencias entre tejidos fresco-IVC y FT-IVC en ninguno de los análisis realizados. Aunque se observó una disminución significativa de la proporción de folículos primordiales (PF) en los grupos fresco-IVC y FT-IVC (d0 vs. D6, P<.002), estos se mantuvieron estables en el grupo FT-CAM (d0 vs. D6, ns). Las tasas de PF fueron significativamente mayores en el grupo FT-CAM en comparación al grupo FT-IVC en d6 (P1/4.02).
De forma importante, eritrocitos aviares se encontraban presentes en un 30% de los trasplantes desde d1. Las tasas de apoptosis y autofagia incrementaron durante IVC (P<.008), pero se mantuvieron significativamente bajas en el grupo FT-CAM (P<.01), confirmando una mejor preservación en los trasplantes de tejidos CAM. Se estableció una regulación río arriba de la vía PI3K/FOXO en los grupos IVC, demostrando una activación PF, mientras que se detectó una regulación río abajo en el grupo FT-CAM (P<.03). La prueba de PCR confirmó el crecimiento ovocitario durante la IVC y la autofagia folicular en todos los grupos, sin embargo, la vía PI3K parecía estar modulada diferencialmente en tejidos y folículos.
El cultivo in vitro induce a la depleción de PF sin un impacto adicional del congelamiento. Injertar en CAM mantiene el conjunto de PF frenando la activación folicular, apoptosis, y autofagia, probablemente gracias a la rápida re-vascularización y/o a la hormona anti-mulleriana embrionaria circulante.
Estos hallazgos resaltan la importancia de mejorar la neo-angiogénesis en los trasplantes de ovario e investigar los potenciales beneficios de administrar la hormona anti-mulleriana para prevenir el agotamiento de PF.
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