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1.
Reprod Biomed Online ; 2020 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-33288476

RESUMO

In recent years, some countries and fertility preservation networks have started adopting 24 h transportation for ovarian tissue, a practice that has the potential to spread very quickly due to the high costs and bureaucracy involved in the establishment of ovarian tissue cryobanks. While pregnancies and live births have been reported after such long periods of transportation, this, however, remains an empirical procedure. This review aims to prompt reflection on ovarian tissue transport, highlighting the lack of knowledge in humans by providing a counterpoint looking into more than 40 studies published in different animal models. By discussing these studies in animals, the findings of various models can be deciphered, and light shed on the patterns identified. Like the development of different assisted reproductive technology procedures, this is an important step in creating guidelines for future studies on human ovarian tissue transportation.

2.
Fertil Steril ; 114(6): 1330-1338, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32828497

RESUMO

OBJECTIVE: To characterize ovarian tissue from pediatric patients by evaluating development and vascularization in follicle populations and comparing it with adult tissue after xenografting. DESIGN: Prospective experimental study. SETTING: Academic research center. PATIENT(S): Five children (median age 3 years) and seven women (median age 28 years). INTERVENTION(S): Hematoxylin and eosin staining, immunofluorescence, and transmission electron microscopy (TEM) evaluation before and after grafting. MAIN OUTCOME MEASURE(S): Follicle density, morphology, classification, and size, ovarian tissue vascularization, follicle ultrastructure. RESULT(S): Frozen-thawed ovarian tissue was divided into three fragments: nongrafted controls, TEM, and xenografting for 20 weeks. Follicle density was statistically significantly higher in pediatric than adult patients; even though it decreased in both groups after transplantation, it remained higher in pediatric patients. In the pediatric group, quiescent-stage follicles were the majority of the follicle pool before and after grafting, while growing follicles statistically significantly increased in both groups after grafting. Abnormal and atretic follicles were also observed in pediatric tissue and declined with age and after grafting. Pediatric ovarian tissue contained more and larger immature vessels, while mature vessels were larger in adults. The TEM analysis of abnormal pediatric follicles showed loss of shape and vacuolization of the cytoplasm without organelle damage. CONCLUSION(S): Statistically significant differences in follicle density were observed between pediatric and adult patients, but the follicle proportions were similar before and after grafting, with the exception of atretic and abnormal follicles. Pediatric tissue contains more and larger immature vessels than adult tissue, and the posttransplantation revascularization process is accelerated in this group.

3.
Exp Mol Pathol ; 113: 104374, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31917966

RESUMO

The endocrine disruptive effects caused by bisphenol A (BPA) are well known. Despite this, to date, evaluation of its long term effects is limited, meaning that there is still much to be unveiled in terms of alterations caused by perinatal exposure to BPA. Our aim was to determine if perinatal exposure to two different doses of BPA causes long term morphological and molecular alteration effects in the mammary gland (MG). We evaluated MG from Mongolian gerbil offspring exposed perinatally (during gestation and lactation) to 50 or 5000 µg/kg/day BPA. At 90 days of age the animals were subjected to a single dose of N-nitroso-N-methylurea in order to mimic a carcinogenic environment. At 6 months of age, animals in estrous were euthanized for morphological evaluation of the MGs. The MG architecture presented considerable changes in terms of detached epithelial cells, inflammation, glandular hyperplasia, and collagen fiber deposition. Furthermore, a higher index of epithelial cell proliferation was detected in comparison to the intact control group. In addition, we verified a higher molecular expression of EZH2 in the vehicle treated group, indicating that corn oil applied alone can alter the expression of this epigenetic biomarker. In conclusion, BPA perinatal exposure promotes significant changes in glandular cytoarchitecture and increases glandular epithelium proliferation rate, leading to the retention of stem-like properties. This event could compromise the fate and differentiation potential of mammary epithelium.


Assuntos
Envelhecimento/patologia , Compostos Benzidrílicos/toxicidade , Glândulas Mamárias Animais/patologia , Fenóis/toxicidade , Efeitos Tardios da Exposição Pré-Natal/patologia , Actinas/metabolismo , Animais , Proliferação de Células , Colágeno/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Gerbillinae , Histonas/metabolismo , Glândulas Mamárias Animais/efeitos dos fármacos , Gravidez
4.
J Assist Reprod Genet ; 37(1): 101-108, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31732846

RESUMO

PURPOSE: Our aim was to elucidate the mechanisms involved in follicle activation of the ovarian reserve after human ovarian tissue transplantation, with specific focus on the role of the effectors of the PI3K (mTOR and FOXO1) and Hippo (YAP) signaling pathways and whether they are somehow altered. METHODS: Frozen-thawed ovarian tissue was collected from six women (age 25-35 years) undergoing surgery for non-ovarian pathologies and divided into 4 fragments in each case: one for non-grafted controls and three for grafting to immunodeficient mice for 3, 7 and 21 days. The tissue was processed for hematoxylin and eosin staining, immunohistochemistry and immunofluorescence at different timepoints before and after grafting. Activation of the PI3K and Hippo signaling pathways was investigated by analysis of mTOR phosphorylation, FOXO1 cytoplasmic localization and YAP nuclear localization. RESULTS: No change in mTOR levels was observed in primordial follicles post-transplantation, but a significant upturn was recorded in growing follicles compared with primordial follicles, irrespective of grafting time. A higher percentage of primordial follicles was also found with FOXO1 in the cytoplasm after 3 days of transplantation than in non-grafted controls. Finally, a greater proportion of primordial follicles was detected with YAP in the nucleus at all timepoints after grafting. CONCLUSIONS: This study supports the hypothesis that follicle activation may occur as an early event after transplantation, with follicle growth and death both contributing to the burnout phenomenon. This is the first time that the effectors of the PI3K and Hippo pathways have been investigated in grafted human ovarian tissue and their role in burnout documented.


Assuntos
Proteína Forkhead Box O1/metabolismo , Folículo Ovariano/fisiologia , Reserva Ovariana , Ovário/transplante , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adulto , Animais , Feminino , Proteína Forkhead Box O1/genética , Humanos , Camundongos , Camundongos SCID , Folículo Ovariano/citologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética
5.
Ecotoxicol Environ Saf ; 188: 109918, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31753310

RESUMO

Hormonal regulation controls mammary gland (MG) development. Therefore some hormone-related factors can disrupt the early phases of MGs development, making the gland more susceptible to long term modifications in its response to circulating hormones. Endocrine disruptors, such as bisphenol A (BPA), are able to cause alterations in hormone receptor expression, leading to changes in the cell proliferation index, which may expose the tissue to neoplastic alterations. Thus, we evaluated the variations in hormone receptor expression in the MG of 6-month old Mongolian gerbils exposed to BPA and 17ß estradiol during the perinatal period. Receptors for estrogen alpha (ERα), beta (ERß), progesterone (PGR), prolactin (PRL-R), and co-localization of connexin 43 (Cx43) and ERα in gerbils were analyzed, and serum concentrations of estradiol and progesterone were assessed. No alterations in body, liver, and ovary-uterus complex weights were observed. However, there was an increase in epithelial ERα expression in the 17ß estradiol (E2) group and in PGR in the BPA group. Although immunohistochemistry did not show alterations in ERß expression, western blotting revealed a decrease in this protein in the BPA group. PRL-R was more present in epithelial cells in the vehicle control (VC), E2, and BPA groups in comparison to the intact control group. Cx43 was more frequent in E2 and BPA groups, suggesting a protective response from the gland against possible malignancy. Serum concentration of estradiol reduced in VC, E2, and BPA groups, confirming that alterations also impacts steroid levels. Consequently, perinatal exposure to BPA and the reference endogenous estrogen, 17ß estradiol, are able to increase the tendency of endocrine disruption in MG in a long term manner, since repercussions are observed even 6 months after exposure.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Estradiol/toxicidade , Glândulas Mamárias Animais/efeitos dos fármacos , Fenóis/toxicidade , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Receptores Estrogênicos/metabolismo , Receptores de Progesterona/metabolismo , Animais , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Gerbillinae , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente
6.
J Cell Physiol ; 234(7): 10148-10156, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30417361

RESUMO

Fertility preservation of prepubertal girls subjected to invasive cancer therapy necessitates defining protocols for activation of isolated primordial follicles. Granulosa (GCs) and cumulus cells (CCs) play pivotal role in oocyte development. Although GCs and CCs share some similarities, they differ in growth factors production. The current study was conducted to evaluate the effects of GCs, CCs and their conditioned media on mice primordial follicles activation. One-day-old mice ovaries were subjected to 6-day culture with base medium (BM), GC conditioned medium (GCCM), GC coculture (GCCC), CC conditioned medium (CCCM) or CC coculture (CCCC). Follicular growth and primordial to primary follicle transition was observed during 6-day culture, and follicular activation rate tended to be greater in GCCM than other groups (0.05


Assuntos
Meios de Cultivo Condicionados/farmacologia , Células do Cúmulo/metabolismo , Preservação da Fertilidade/métodos , Células da Granulosa/metabolismo , Folículo Ovariano/efeitos dos fármacos , Animais , Feminino , Camundongos , Folículo Ovariano/metabolismo , PTEN Fosfo-Hidrolase/metabolismo
7.
Fertil Steril ; 110(3): 534-544.e3, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29960702

RESUMO

OBJECTIVE: To characterize oxidative stress and metabolic activity in xenografted human ovarian tissue using microdialysis. DESIGN: Prospective experimental study. SETTING: Gynecology research unit at a university hospital. PATIENT(S): Cryopreserved ovarian cortex from five women 27-35 years of age. INTERVENTION(S): Frozen-thawed human ovarian tissue fragments were xenografted to the back muscle of ten nude mice. Before grafting, a microdialysis probe was placed inside each fragment. MAIN OUTCOME MEASURE(S): Daily reactive oxygen species (ROS), lactate, and glucose levels were collected by means of microdialysis. Follicle loss (hematoxylin and eosin), murine and human vascularization, and vessel stability (CD31, von Willebrand factor, and α-smooth muscle actin triple immunofluorescence) were analyzed on post-grafting days 10 and 21. RESULT(S): Lactate levels were significantly higher than glucose levels until day 10, after which time the lactate-glucose ratio stabilized at ∼1:1. Regarding ROS generation, there were two peaks on post-grafting days 10 and 17. Total vascularization increased significantly up to day 10 and remained similar up to day 21. However, murine vessel area and stabilization significantly increased up to day 21. Major follicle loss occurred in the first 10 days after transplantation. CONCLUSION(S): Our data validated microdialysis as a tool to characterize metabolic behavior and oxidative stress in grafted ovarian tissue. Three different post-grafting periods were identified according to the metabolic activity of grafted tissue, showing a long progression from anaerobic to aerobic metabolism and a protracted period of ROS generation. Oxidative stress was observed relatively late, after the most critical period of follicle loss, and lasted until the tissue vasculature stabilized.


Assuntos
Microdiálise/métodos , Ovário/metabolismo , Ovário/transplante , Estresse Oxidativo/fisiologia , Transplante Heterólogo/métodos , Adulto , Animais , Feminino , Humanos , Redes e Vias Metabólicas/fisiologia , Camundongos , Camundongos Nus , Ovário/citologia , Transplantes/citologia , Transplantes/metabolismo , Transplantes/transplante
8.
Biopreserv Biobank ; 16(2): 120-127, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29363997

RESUMO

In vitro culture of ovarian follicles is a new technique in reproductive technology, which helps in understanding the process of folliculogenesis. The in vitro culture of follicles could be carried out using three-dimensional (3D) natural scaffolds that mimic the ovarian tissue stroma. Selection of the right matrix and culture media in these scaffolds could increase the survival and maturation of the follicles. In this work, the applicability of matrigel-alginate (MA) and fibrin-alginate (FA) 3D scaffolds for folliculogenesis was assessed. The ovaries of 13-day-old Naval Medical Research Institute (NMRI) mice were isolated and distributed into control and vitrification groups. Preantral follicles (mean diameter: 120-140 µm) were mechanically isolated from control and vitrified-warmed ovaries, encapsulated in MA or FA scaffold and cultured for 12 days. Follicle survival, growth, maturation, and quantitative expression of oocyte maturation genes (Gdf9, Bmp15, Fgf8, KitL, Kit, and Amh) and proteins (GDF9 and BMP15) were assessed. Survival rate of culture preantral follicles in control groups was found to be significantly higher than vitrified follicles. Antrum formation was similar in all groups. Follicle diameters were significantly increased in all groups during culture period. A decreasing pattern of gene expression was seen for all genes in all groups. This trend was verified through evaluation of protein expression, during which there was strong staining in antral follicles from all groups in the last day of in vitro culture. The better survival and maturation rate of follicles in the MA compared to FA scaffold indicates that the MA matrix, being rich in extracellular matrix components, could mimic the ovarian condition better and presents a good environment for follicle development.


Assuntos
Alginatos/química , Fibrina/química , Folículo Ovariano , Técnicas de Cultura de Tecidos/métodos , Tecidos Suporte/química , Animais , Antígenos de Diferenciação/biossíntese , Feminino , Regulação da Expressão Gênica , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Masculino , Camundongos , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo
9.
J Assist Reprod Genet ; 35(1): 41-48, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29236205

RESUMO

PURPOSE: The aim of this study is to optimize fibrin matrix composition in order to mimic human ovarian tissue architecture for human ovarian follicle encapsulation and grafting. METHODS: Ultrastructure of fresh human ovarian cortex in age-related women (n = 3) and different fibrin formulations (F12.5/T1, F30/T50, F50/T50, F75/T75), rheology of fibrin matrices and histology of isolated and encapsulated human ovarian follicles in these matrices. RESULTS: Fresh human ovarian cortex showed a highly fibrous and structurally inhomogeneous architecture in three age-related patients, but the mean ± SD of fiber thickness (61.3 to 72.4 nm) was comparable between patients. When the fiber thickness of four different fibrin formulations was compared with human ovarian cortex, F50/T50 and F75/T75 showed similar fiber diameters to native tissue, while F12.5/T1 was significantly different (p value < 0.01). In addition, increased concentrations of fibrin exhibited enhanced storage modulus with F50/T50, resembling physiological ovarian rigidity. Excluding F12.5/T1 from further analysis, only three remaining fibrin matrices (F30/T50, F50/T50, F75/T75) were histologically investigated. For this, frozen-thawed fragments of human ovarian tissue collected from 22 patients were used to isolate ovarian follicles and encapsulate them in the three fibrin formulations. All three yielded similar follicle recovery and loss rates soon after encapsulation. Therefore, based on fiber thickness, porosity, and rigidity, we selected F50/T50 as the fibrin formulation that best mimics native tissue. CONCLUSIONS: Of all the different fibrin matrix concentrations tested, F50/T50 emerged as the combination of choice in terms of ultrastructure and rigidity, most closely resembling human ovarian cortex.


Assuntos
Órgãos Artificiais , Fibrina/química , Ovário , Materiais Biomiméticos/química , Composição de Medicamentos , Elasticidade , Feminino , Dureza , Humanos , Fenômenos Mecânicos , Folículo Ovariano/transplante , Folículo Ovariano/ultraestrutura , Ovário/química , Ovário/citologia , Ovário/ultraestrutura
10.
J Ovarian Res ; 10(1): 71, 2017 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-29061149

RESUMO

BACKGROUND: Ovarian tissue cryopreservation followed by transplantation after cancer remission is the most commonly applied fertility restoration approach in very young girls and women who require immediate cancer therapy. However, clinicians strongly advise against reimplantation of one's own ovarian tissue when there is a high risk of recurrence after grafting. For these patients, development of an alternative strategy, namely a transplantable artificial ovary, offers future hope of conceiving. The first essential requirement for an artificial ovary is the set-up of a safe and effective follicle isolation procedure. Despite encouraging results with different variants of this technique, none of them take into the account the physiology and great variability in follicular density inside individual tissue fragments and between different patients. The goal of this study was to improve our previously applied follicle isolation procedure in order to develop a tailored isolation procedure for human follicles according to individual tissue properties. To this end, enzymatic digestion was divided into three time intervals in order to initially recover the first follicles to be isolated, and then further dissociate undigested fragments of tissue containing entrapped follicles. RESULTS: After thawing frozen human ovarian tissue using a modified and tailored follicle isolation method, already 35% of follicles were fully isolated and recovered after 30 min of enzymatic digestion. Indeed, this protocol resulted in a higher follicle yield (p < 0.01) and greater numbers of primordial and primary follicles (p < 0.05) than the previous approach. However, no significant difference was found in caspase-3-positive and Ki67-positive staining between the two isolation protocols. In addition, greater follicle quality was demonstrated. When human follicles isolated using the modified protocol were encapsulated in a fibrin matrix with high concentrations of fibrinogen and thrombin and xenografted to a SCID mouse, more follicles were found to be healthy after one week of transplantation than in a previous our study. CONCLUSIONS: With the modified follicle isolation method, we were able to maximize the number and quality of isolated primordial and primary follicles, and develop a tailored follicle isolation procedure according to individual tissue properties. Moreover, improved follicle survival inside an artificial ovary prototype was detected after one week of xenografting.


Assuntos
Sobrevivência Celular , Criopreservação , Recuperação de Oócitos , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Animais , Contagem de Células , Feminino , Xenoenxertos , Humanos , Camundongos , Folículo Ovariano/transplante
11.
Fertil Steril ; 104(3): 672-80.e2, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26095134

RESUMO

OBJECTIVE: To evaluate the safety of our follicle isolation procedure in a model of ovarian tissue artificially contaminated with cancer cells, then to improve the procedure to effectively eliminate malignant cells from follicle suspensions without altering viability. DESIGN: Prospective experimental study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ten women undergoing laparoscopy for benign gynecologic disease. INTERVENTION(S): Follicle isolation from ovarian tissue artificially contaminated with marked fluorescent leukemic cells, either by the usual pickup technique without further treatment (group 1) or by washing three times after pickup (group 2). MAIN OUTCOME MEASURE(S): Evidence of leukemic cells in follicle suspensions using fluorescence microscopy and quantitative real-time polymerase chain reaction, and analysis of follicle viability. RESULT(S): In group 1, 196 leukemic cells were detected by fluorescence microscopy out of 499 follicles retrieved, while just one leukemic cell was found among 772 follicles after three washes. The BCR-ABL fusion transcript was detected when at least 19 cells were present in follicle suspensions; four samples were positive in group 1, and all were negative in group 2. Follicle viability was similar in both groups (95.6% vs. 96.4%). CONCLUSION(S): Cancer cells could inadvertently be picked up with isolated follicles in case of malignant contamination of ovarian tissue. A simple purging procedure consisting of three washes proved effective for eliminating leukemic cells while maintaining good follicle viability.


Assuntos
Separação Celular/métodos , Preservação da Fertilidade/métodos , Leucemia/patologia , Folículo Ovariano/patologia , Adulto , Biomarcadores Tumorais/genética , Biópsia , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia/genética , Microscopia de Fluorescência , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto Jovem
12.
Fertil Steril ; 101(4): 1149-56, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24462059

RESUMO

OBJECTIVE: To create an artificial ovary to provide an alternative way of restoring fertility in patients who cannot benefit from transplantation of cryopreserved ovarian tissue due to the threat of reintroducing malignant cells. DESIGN: In vivo experimental study. SETTING: Gynecology research unit in a university hospital. ANIMAL(S): Six-week-old female NMRI mice. INTERVENTION(S): Autografting of isolated preantral follicles and ovarian cells (OCs) encapsulated in two fibrin matrices containing low concentrations of fibrinogen (F; mg/mL) and thrombin (T; IU/mL): F12.5/T1 and F25/T4. MAIN OUTCOME MEASURE(S): Follicular density and development, OC survival and proliferation, inflammatory response, and vascularization. RESULT(S): After 1 week, the follicle recovery rate ranged from 30.8% (F25/T4) to 31.8% (F12.5/T1). With both fibrin formulations, all follicles were found to be alive or minimally damaged, as demonstrated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and at the growing stage (primary, secondary, and antral follicles), confirmed by Ki67 immunostaining. Isolated OCs also survived and proliferated after grafting, as evidenced by <1% apoptotic cells and a high proportion of Ki67-positive cells. Vessels were found in both fibrin formulations, and the global vascular surface area varied from 1.35% (F25/T4) to 1.88% (F12.5/T1). Numerous CD45-positive cells were also observed in both F25/T4 and F12.5/T1 combinations. CONCLUSION(S): The present study is the first to show survival and growth of isolated murine ovarian follicles 1 week after autotransplantation of isolated OCs in a fibrin scaffold. The results indicate that fibrin is a promising candidate as a matrix for the construction of an artificial ovary. Xenotransplantation of isolated human follicles and OCs is the necessary next step to validate these findings.


Assuntos
Órgãos Bioartificiais , Fibrina/química , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/transplante , Ovário/citologia , Ovário/crescimento & desenvolvimento , Tecidos Suporte , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Feminino , Camundongos , Folículo Ovariano/citologia
13.
Fertil Steril ; 100(5): 1350-7, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23953325

RESUMO

OBJECTIVE: To assess follicular development after long-term xenotransplantation and exogenous stimulation of cryopreserved prepubertal ovarian tissue. DESIGN: Pilot study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Cryopreserved ovarian fragments were obtained from five prepubertal patients aged 7.2-12.2 years. INTERVENTION(S): Xenografting of frozen-thawed prepubertal ovarian fragments to SCID mice for 5 months and exogenous stimulation. MAIN OUTCOME MEASURE(S): Follicular density, morphology, proliferation, development. RESULT(S): Follicular density varied between 1.05 and 47.89 follicles/mm(2) in frozen-thawed ovarian tissue and 0.48 and 32.74 follicles/mm(2) in grafted prepubertal ovarian tissue. Growing follicles at the last stage of follicular development were observed at significantly higher proportions after grafting. No statistical difference was evidenced in the number of primordial follicles, representing the largest proportion of the follicle pool (99.51% before grafting and 92.46% after grafting). Ki67 and antimüllerian hormone expression were observed in these growing follicles. CONCLUSION(S): This is the first description of transplantation of human cryopreserved prepubertal ovarian tissue to mice, demonstrating that a very high number of follicles survive after transplantation and a large pool of primordial follicles remains dormant. Growing follicles were observed, proving the responsiveness of prepubertal ovarian tissue to gonadotropins.


Assuntos
Criopreservação , Fármacos para a Fertilidade Feminina/farmacologia , Menotropinas/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/transplante , Animais , Hormônio Antimülleriano/metabolismo , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Criança , Feminino , Sobrevivência de Enxerto/efeitos dos fármacos , Hospitais Universitários , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Camundongos SCID , Folículo Ovariano/metabolismo , Projetos Piloto , Fatores de Tempo , Transplante Heterólogo
14.
Fertil Steril ; 98(5): 1291-8.e1-2, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22883570

RESUMO

OBJECTIVE: To assess the efficiency of two vitrification protocols to cryopreserve human preantral follicles with the use of a xenografting model. DESIGN: Pilot study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ovarian biopsies were obtained from seven women aged 30-41 years. INTERVENTION(S): Ovarian tissue fragments were subjected to one of three cryopreservation protocols (slow freezing, vitrification protocol 1, and vitrification protocol 2) and xenografted for 1 week to nude mice. MAIN OUTCOME MEASURE(S): The number of morphologically normal follicles after cryopreservation and grafting and fibrotic surface area were determined by histologic analysis. Apoptosis was assessed by the TUNEL method. Morphometric analysis of TUNEL-positive surface area also was performed. Follicle proliferation was evaluated by immunohistochemistry. RESULT(S): After xenografting, a difference was observed between the cryopreservation procedures applied. According to TUNEL analysis, both vitrification protocols showed better preservation of preantral follicles than the conventional freezing method. Moreover, histologic evaluation showed a significantly higher proportion of primordial follicles in vitrified (protocol 2)-warmed ovarian tissue than in frozen-thawed tissue. The proportion of growing follicles and fibrotic surface area was similar in all groups. CONCLUSION(S): Vitrification procedures appeared to preserve not only the morphology and survival of preantral follicles after 1 week of xenografting, but also their ability to resume folliculogenesis. In addition, vitrification protocol 2 had a positive impact on the quiescent state of primordial follicles after xenografting.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Folículo Ovariano/transplante , Ovário/transplante , Vitrificação , Adulto , Animais , Apoptose , Biópsia , Proliferação de Células , Sobrevivência Celular , Quebras de DNA , Feminino , Fibrose , Sobrevivência de Enxerto , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/patologia , Ovário/efeitos dos fármacos , Ovário/patologia , Projetos Piloto , Células Estromais/patologia , Células Estromais/transplante , Fatores de Tempo , Transplante Heterólogo
15.
Fertil Steril ; 95(4): 1229-34.e1, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20541745

RESUMO

OBJECTIVE: To compare cryopreservation of prepubertal testicular tissue after vitrification (V) and slow-freezing (SF). DESIGN: Prospective experimental study. SETTING: Academic research unit. ANIMAL(S): Six-day-old mice. INTERVENTION(S): After cryopreservation, viability tests (n = 10) and short-term culture (1 and 3 days) (n = 5) were performed. A comparison was made with fresh (FR) and noncultured controls (FR Ctrl). MAIN OUTCOMES MEASURE(S): Tissue viability was assessed by lactate dehydrogenase release assay. Apoptosis (caspase-3) and proliferation (Ki67) were evaluated by immunohistochemistry, and tubular diameter, integrity, and cell density by light microscopy. RESULT(S): Lactate dehydrogenase release was greater after SF than V (54.6% vs. 26.7%), whereas the mean number of apoptotic cells/tubule was higher after V than SF (2.13 vs. 0.07). On day 1, a decrease in cell density was noted in both cryopreserved groups, but this difference was not subsequently observed. On day 3, an increase in proliferation was seen in the SF and V groups versus FR tissue, and similar tubular diameter, integrity, and cell density were found in all cultured groups. CONCLUSION(S): This study shows that both SF and V protocols preserve survival, development, and integrity of prepubertal mouse testicular tissue in short-term organotypic culture. Additional investigation should now be conducted to assess tissue functionality.


Assuntos
Criopreservação/métodos , Testículo , Sobrevivência de Tecidos , Vitrificação , Fatores Etários , Animais , Animais Recém-Nascidos , Apoptose/fisiologia , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Técnicas de Cultura de Órgãos , Estudos Prospectivos , Sobrevivência de Tecidos/fisiologia
16.
Fertil Steril ; 95(4): 1241-6, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20638058

RESUMO

OBJECTIVE: To compare early follicular growth after fresh and frozen-thawed human ovarian tissue xenografting and to investigate whether expression of c-kit, kit ligand (KL), and growth differentiation factor-9 (GDF-9) is maintained after freezing and xenografting of human ovarian tissue. DESIGN: Prospective experimental study. SETTING: Gynecology research unit in a university hospital. ANIMAL(S): Ten nude (Swiss nu/nu) 6-week-old female mice. INTERVENTION(S): The ovarian biopsy samples were obtained from six women, aged 20 to 30 years. Fresh and frozen-thawed ovarian fragments intraperitoneally grafted into nude mice for 3 weeks. MAIN OUTCOME MEASURE(S): Histologic analysis and immunohistochemical evaluation of c-kit, KL, and GDF-9 expression before and after grafting. RESULT(S): The integrity and proportion of growing follicles increased similarly in both fresh (64%) and frozen-thawed (59%) xenografts compared with non-grafted tissue (fresh: 40%; frozen-thawed: 21%). Both C-kit and KL staining were detected in the oocytes and granulosa cells of preantral follicles, and GDF-9 expression was observed in the oocytes of preantral follicles in all groups. CONCLUSION(S): Freezing does not appear to have a major impact on early follicular growth after transplantation. This study shows, for the first time, expression of c-kit, KL, and GDF-9 in human preantral follicles after ovarian tissue cryopreservation and xenotransplantation.


Assuntos
Criopreservação/métodos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Folículo Ovariano/química , Folículo Ovariano/metabolismo , Transplante Heterólogo/métodos , Adulto , Animais , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Folículo Ovariano/citologia , Estudos Prospectivos , Fatores de Tempo , Adulto Jovem
17.
Fertil Steril ; 95(3): 1094-7, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21168134

RESUMO

OBJECTIVE: To test the effect of different vitrification solutions and procedures on the morphology of human preantral follicles. DESIGN: Pilot study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ovarian biopsies were obtained from nine women aged 22-35 years. INTERVENTION(S): Ovarian tissue fragments were subjected to [1] different vitrification solutions to test their toxicity or [2] different vitrification methods using plastic straws, medium droplets, or solid-surface vitrification before in vitro culture. MAIN OUTCOME MEASURE(S): Number of morphologically normal follicles after toxicity testing or vitrification with the different treatments determined by histologic analysis. RESULT(S): In the toxicity tests, only VS3 showed similar results to fresh tissue before and after in vitro culture (fresh controls 1 and 2). In addition, this was the only solution able to completely vitrify. In all vitrification procedures, the percentage of normal follicles was lower than in controls. However, of the three protocols, the droplet method yielded a significantly higher proportion of normal follicles. CONCLUSION(S): Our experiments showed VS3 to have no deleterious effect on follicular morphology and to be able to completely vitrify, although vitrification procedures were found to affect human follicles. Nevertheless, the droplet method resulted in a higher percentage of morphologically normal follicles.


Assuntos
Criopreservação/métodos , Crioprotetores/toxicidade , Folículo Ovariano/citologia , Técnicas de Cultura de Tecidos/métodos , Vitrificação , Adulto , Biópsia , Meios de Cultura/toxicidade , Dimetil Sulfóxido/toxicidade , Etilenoglicol/toxicidade , Feminino , Humanos , Folículo Ovariano/efeitos dos fármacos , Propilenoglicol/toxicidade , Soluções/toxicidade , Sacarose/toxicidade , Adulto Jovem
18.
Reprod Biomed Online ; 17(1): 136-50, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18616902

RESUMO

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.


Assuntos
Fertilidade , Infertilidade/etiologia , Microscopia Eletrônica de Transmissão/métodos , Neoplasias/complicações , Ovário/transplante , Técnicas de Reprodução Assistida , Núcleo Celular/metabolismo , Criopreservação/métodos , Feminino , Humanos , Meiose , Mitocôndrias/metabolismo , Oócitos/metabolismo , Ovário/ultraestrutura , Fuso Acromático/metabolismo , Zona Pelúcida/metabolismo
19.
Fertil Steril ; 85 Suppl 1: 1077-81, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16616077

RESUMO

OBJECTIVE: To determine the behavior of isolated primordial follicles that were exposed to different concentrations of dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PROH), and glycerol (GLY). DESIGN: Isolated primordial follicles were exposed to the cryoprotectant (CPA) solution and photographed to calculate their volume at different periods of exposure. SETTING: Laboratorio Renzo Giuliani, University of Florence, Italy. ANIMAL(S): Lambs, 30-40 days old. INTERVENTION(S): Isolation of primordial follicles and subsequent exposure to CPA. MAIN OUTCOME MEASURE(S): Follicular volume. RESULT(S): At 2 minutes of CPA exposure, all follicles appeared to be shrunken. At approximately 5 minutes, shrinkage ceased, and follicles started to swell, absorbing the CPA and water to maintain osmotic equilibrium. When DMSO was tested, follicular dehydration in all concentrations did not exceed 17%; with PROH and EG, it reached 33% and 27%, respectively. The highest degree of dehydration (48%) was seen with GLY. In almost all tested concentrations, follicular shrinkage occurred up to 5 minutes. CONCLUSION(S): Volume changes in isolated primordial follicles can fluctuate according to the CPA used and its concentration.


Assuntos
Permeabilidade da Membrana Celular , Crioprotetores/farmacocinética , Soluções para Preservação de Órgãos/farmacocinética , Folículo Ovariano/fisiologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Crioprotetores/administração & dosagem , Citoproteção/fisiologia , Relação Dose-Resposta a Droga , Feminino , Soluções para Preservação de Órgãos/administração & dosagem , Permeabilidade/efeitos dos fármacos , Ovinos
20.
Fertil Steril ; 81 Suppl 1: 735-40, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15019803

RESUMO

OBJECTIVE: To verify the viability of isolated primordial follicles to different propylene glycol (PROH) and glycerol (GLY) concentrations before and after cryopreservation. DESIGN: Isolated primordial follicles were stained with trypan blue to evaluate the effect of different PROH and GLY concentrations before and after cryopreservation. SETTING: Laboratorio Renzo Giuliani, University of Florence, Italy. PATIENT(S): Thirty- to forty-day-old lambs. INTERVENTION(S): : Isolation of primordial follicles with subsequent exposure to cryoprotectant and freezing. MAIN OUTCOME MEASURE(S): Histologic structure and follicular mortality. RESULT(S): After the isolation procedure (control), the mean number of live primordial follicles/mL was 2,688 and 4,452 in the GLY and PROH groups, respectively. When GLY was used, the number of live follicles before cryopreservation was 820, 756, 640, 524, 564, and 460 follicles/mL with concentrations of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mol/L, respectively. After cryopreservation, this number decreased to 0, 12, 36, 100, 84, and 68 follicles/mL, respectively, with the same concentrations. When PROH was used, the number of live follicles before cryopreservation was 4,216, 3,880, 3,560, 1,812, 704, and 568 follicles/mL with concentrations of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mol/L, respectively. After cryopreservation, this number decreased to 0, 116, 336, 472, 360, and 244 follicles/mL, respectively, with the same concentrations. CONCLUSION(S): Both cryoprotectants were shown to preserve isolated primordial follicles after cryopreservation.


Assuntos
Criopreservação , Crioprotetores/administração & dosagem , Glicerol/administração & dosagem , Folículo Ovariano/efeitos dos fármacos , Propilenoglicol/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Feminino , Folículo Ovariano/anatomia & histologia , Folículo Ovariano/fisiologia , Ovinos , Sobrevivência de Tecidos/efeitos dos fármacos , Testes de Toxicidade
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