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1.
Reproduction ; 158(5): F45-F54, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31557725

RESUMO

Ovarian cryopreservation rapidly developed from basic science to clinical application and can now be used to preserve the fertility of girls and young women at high risk of sterility. Primordial follicles can be cryopreserved in ovarian cortex for long-term storage and subsequently autografted back at an orthotopic or heterotopic site to restore fertility. However, autografting carries a risk of re-introducing cancer cells in patients with blood-born leukaemias or cancers with a high risk of ovarian metastasis. For these women fertility restoration could only be safely achieved in the laboratory by the complete in vitro growth (IVG) and maturation (IVM) of cryopreserved primordial follicles to fertile metaphase II (MII) oocytes. Culture systems to support the development of human oocytes have provided greater insight into the process of human oocyte development as well as having potential applications within the field of fertility preservation. The technology required to culture human follicles is extremely challenging, but significant advances have been made using animal models and translation to human. This review will detail the progress that has been made in developing human in vitro growth systems and consider the steps required to progress this technology towards clinical application.


Assuntos
Preservação da Fertilidade , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/tendências , Oócitos/citologia , Animais , Células Cultivadas , Criopreservação/métodos , Feminino , Preservação da Fertilidade/métodos , Preservação da Fertilidade/tendências , Humanos , Oócitos/fisiologia , Oócitos/transplante , Folículo Ovariano/citologia , Folículo Ovariano/transplante , Ovário
2.
Reproduction ; 158(5): F35-F44, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31394506

RESUMO

Ovarian tissue cryopreservation and transplantation (OTCP-TP) has progressed over the past decade from a revolutionary experimental procedure to a well-accepted treatment in many centers for young patients with a high risk of ovarian failure after cancer treatment. The procedure is remarkably successful, with studies reporting return of ovarian function in up to 95% of graft recipients and pregnancy rates of between 30 and 50%. The most significant limitation of OTCP-TP is the massive loss of follicles that occurs following transplantation, which is primarily attributed to ischemic damage and follicle activation. We review the current approaches to reducing follicle loss and maximizing graft lifespan via pharmacological agents which reduce ischemic damage and follicle activation. We further discuss the value and disadvantage of inducing follicle activation in the graft as a means of generating mature follicles in the immediate short term.


Assuntos
Criopreservação/métodos , Preservação da Fertilidade/efeitos adversos , Folículo Ovariano/citologia , Folículo Ovariano/transplante , Reserva Ovariana/fisiologia , Ovário , Contagem de Células , Feminino , Preservação da Fertilidade/métodos , Humanos , Ovário/transplante , Gravidez , Taxa de Gravidez
3.
Reproduction ; 158(5): F27-F34, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31284266

RESUMO

Ovarian tissue cryopreservation (OTC) is mainly used for fertility preservation in girls and women facing a gonadotoxic treatment. If the woman subsequently becomes menopausal, the ovarian tissue may be transplanted to regain ovarian function, including fertility. The method was developed more than two decades ago and today thousands of women worldwide have undergone OTC. Fewer than 500 patients have had tissue transplanted and close to 100% of those regain ovarian function. Several technical aspects of OTC are now becoming more established, including high quantitative follicle survival, defining the size of the tissue resulting in optimal tissue revascularisation and follicle loss resulting from transport of ovarian tissue prior to freezing. We have used OTC to safeguard fertility in patients with genetic diseases, which for some diagnoses is purely experimental, as no transplantations is yet been performed. Usage of OTC beyond fertility is now also being considered; here, the endocrine function of follicles is the focus. It has been suggested that ovarian tissue stored in the reproductive years may be used to avoid premature ovarian insufficiency (POI) when there is a familial disposition or to postpone menopause in patients with an increased risk of osteoporosis or cardiovascular diseases. The benefit of OTC beyond fertility requires, however, actual clinical studies. The current review includes several recent technical aspects with contributions from Denmark building on some of the early work by Roger Gosden.


Assuntos
Criopreservação/métodos , Preservação da Fertilidade/métodos , Congelamento , Ovário , Dinamarca , Feminino , Fertilidade/fisiologia , Doenças Genéticas Inatas/terapia , Hormônios Esteroides Gonadais/metabolismo , Hormônios Esteroides Gonadais/fisiologia , Humanos , Infertilidade Feminina/etiologia , Infertilidade Feminina/prevenção & controle , Folículo Ovariano/fisiologia , Folículo Ovariano/transplante , Insuficiência Ovariana Primária/terapia
4.
J Ovarian Res ; 12(1): 46, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113493

RESUMO

PURPOSE: To study the quality of our human ovarian tissue cryopreservation technique as performed in the first official "International Fertility Protection Centre" in China in patients with certain cancer types using a mouse model, and to find the best site for tissue transplantation in the mouse. METHODS: Thirty-six BALB/C female nude mice were randomly divided into 3 groups, group 1: control group; group 2: ovariectomized group; group 3: ovarian tissue transplantation group. Seventy-two pieces obtained from six ovarian tissue samples from each of three cancer patients were transplanted into the ovarian bursa cavity (OBC), the subcutaneous thigh (TS) and the subcutaneous neck (NS) and removed after 1.5 and 2.5 months, respectively. Follicular growth rate (FGR), total follicle surviving rate (TFSR), tissue recovery rate (TRR), antral follicles (AF), follicle stimulating hormone (FSH), estradiol (E2) and anti-Mullerian hormone (AMH) levels were measured. RESULTS: No significant differences in FGR, OBC, NS (p > 0.05); TFSR was 100% in OBC, NS and TS. No significant differences in TRR (p > 0.05); AF were found only in OBC; TFSR was 100% after transplantation; significantly higher FGR in the 2.5 months compared to the 1.5 months-group (p < 0.05). AMH- and E2-level in group 1 and 3 were significantly higher than in group 2 (p < 0.05); in contrast, FSH was significantly lower. CONCLUSIONS: After transplantation in the mice, the thawed ovarian tissue survived and follicles developed. The ovarian fossa site was the best site for transplantation. Our animal experiments can verify that our human ovarian tissue cryopreservation technique can preserve the quality of ovarian tissue. This is the essential precondition for successful re-transplantation into the patients after performing chemo/radiotherapy to protect ovarian function and fertility.


Assuntos
Criopreservação , Xenoenxertos/fisiologia , Folículo Ovariano/fisiologia , Folículo Ovariano/transplante , Ovário , Adulto , Animais , Hormônio Antimülleriano/metabolismo , Estradiol/metabolismo , Feminino , Preservação da Fertilidade , Hormônio Foliculoestimulante/metabolismo , Sobrevivência de Enxerto , Xenoenxertos/crescimento & desenvolvimento , Xenoenxertos/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Ovário/cirurgia , Técnicas de Cultura de Tecidos , Transplante Heterólogo , Neoplasias do Colo do Útero/cirurgia
5.
Reproduction ; 158(5): F15-F25, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31075758

RESUMO

Increasing numbers of patients are now surviving previously fatal malignant diseases, so for women of childbearing age, fertility concerns are paramount once they are cured. However, the treatments themselves, namely chemo- and radiotherapy, can cause considerable damage to endocrine and reproductive functions, often leaving these women unable to conceive. When such gonadotoxic therapy cannot be postponed due to the severity of the disease or for prepubertal girls, the only way to preserve fertility is cryobanking their ovarian tissue for future use. Unfortunately, with some types of cancer, there is a risk of reimplanting malignant cells together with the frozen-thawed tissue, so it is not recommended. A safer approach involves grafting isolated preantral follicles back to their native environment inside a specially created transplantable artificial ovary for their protection. This bioengineered ovary must mimic the natural organ and therefore requires an appropriate scaffold to encapsulate not only isolated follicles, but also autologous ovarian cells, which are needed for follicles to survive and develop. Here we review the indications for use of this artificial ovary and advances in the field that are bringing us ever closer to clinical implementation.


Assuntos
Órgãos Artificiais , Preservação da Fertilidade/métodos , Folículo Ovariano/citologia , Folículo Ovariano/transplante , Ovário , Criopreservação/métodos , Feminino , Fertilidade/fisiologia , Humanos , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia
6.
Acta Obstet Gynecol Scand ; 98(5): 665-671, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30891730

RESUMO

There is now sufficient evidence to support the feasibility and efficacy of ovarian tissue (OT) cryopreservation and transplantation for both fertility preservation and restoration purposes. However, there are still issues to address regarding the grafting procedure itself, since transplanted tissue suffers massive follicle loss in the early post-grafting period. To improve follicle survival after transplantation, our group recently developed a two-step transplantation technique for OT transplantation in a xenografting model using adipose tissue-derived stem cells (ASCs). The aim of this narrative review is to describe and discuss the previously reported findings. ASCs were initially characterized by flow cytometry as positive for CD29, CD44, CD73, CD90, CD105 and CD166 (>95%) and negative for CD34, CD14, CD31, CD45 and Lin1. ASCs were used in a model of xenotransplantation, were they were embedded in a fibrin scaffold and transplanted to the peritoneum of immunodeficient mice. The goal of the first step was to increase levels of partial pressure of oxygen (pO2 ) and revascularization in the peritoneal transplantation site for further OT transplantation. As ASCs showed the ability to differentiate into endothelial-like cells and vessels in our model, OT transplantation was then performed with ASC grafts in a controlled experiment. At 7 days post-transplantation, the ASC group showed: (1) significantly higher pO2 levels; (2) significantly greater human and murine CD34-positive endothelial areas; (3) significantly higher primordial follicle survival rates; (4) and significantly lower numbers of apoptotic follicles compared with the control group. Our research model demonstrates that by adding ASCs to a fibrin scaffold before OT transplantation, faster and better graft reoxygenation and revascularization may be obtained, resulting in increased follicle survival and reduced follicle apoptosis.


Assuntos
Preservação da Fertilidade/métodos , Sobrevivência de Enxerto/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Folículo Ovariano , Transplante Heterólogo/métodos , Animais , Feminino , Camundongos , Modelos Animais , Folículo Ovariano/fisiologia , Folículo Ovariano/transplante
7.
Fertil Steril ; 111(6): 1217-1225.e3, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30922638

RESUMO

OBJECTIVE: To characterize ovarian follicles of girls and young women with Turner syndrome (TS) who underwent ovarian tissue cryopreservation (OTC). DESIGN: Retrospective case-control study. SETTING: University hospital. PATIENT(S): Fifteen girls and young women with TS aged 5-22 years at OTC were included, together with 42 control girls and young women aged 1-25 years who underwent OTC because of cancer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicle density (follicles/mm3), morphology, and health were assessed in ovarian cortex biopsies from TS patients and compared with controls. Hormone concentrations were measured in serum and follicle fluids. Immature cumulus oocyte complexes were obtained and matured in vitro. RESULT(S): Follicles were found in 60% of the biopsies (9 of 15) from TS ovaries. In 78% of the ovaries (7 of 9) with follicles, the follicle density was within the 95% confidence interval of the control group. There was a high rate of abnormal follicle morphology. Six follicle-specific proteins were expressed similarly in TS and control ovaries. However, apoptosis and zona pellucida protein expression were found to be abnormal in TS. Turner syndrome follicle fluid from small antral follicles had lower concentrations of estrogen and testosterone and higher concentrations of antimüllerian hormone than controls. Thirty-one cumulus oocyte complexes were collected from one patient and cultured for 48 hours in vitro, resulting in five metaphase II oocytes (maturation rate 16%, degeneration rate 19%). CONCLUSION(S): The benefits of OTC may be limited to a highly selected group of TS mosaic patients in whom a sizeable pool of normal follicles is present at OTC.


Assuntos
Criopreservação , Preservação da Fertilidade/métodos , Infertilidade Feminina/terapia , Folículo Ovariano/patologia , Ovário/patologia , Técnicas de Reprodução Assistida , Síndrome de Turner/patologia , Adolescente , Adulto , Biópsia , Criança , Pré-Escolar , Tomada de Decisão Clínica , Feminino , Fertilidade , Líquido Folicular/química , Predisposição Genética para Doença , Humanos , Técnicas de Maturação in Vitro de Oócitos , Lactente , Infertilidade Feminina/etiologia , Infertilidade Feminina/patologia , Infertilidade Feminina/fisiopatologia , Folículo Ovariano/química , Folículo Ovariano/transplante , Ovário/química , Ovário/transplante , Seleção de Pacientes , Fenótipo , Valor Preditivo dos Testes , Estudos Retrospectivos , Síndrome de Turner/complicações , Síndrome de Turner/genética , Adulto Jovem
8.
Methods Mol Biol ; 1920: 343-352, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30737702

RESUMO

In zebrafish and other externally developing animals, maternal effect gene products significantly contribute to embryonic and larval development. Several methods exist for both forward and reverse genetic manipulation of maternal gene products, yet it remains technically difficult to interfere with maternal gene products. Therefore, alternative methods to manipulate maternal factors in oocytes remain of interest. Here we describe a method which allows manipulation and subsequent transplantation of stage I-II follicles of zebrafish into recipient mothers where donor oocytes can develop into mature eggs and produce viable offspring. Additionally, we describe a simple microinjection protocol for injecting reporter constructs into follicles as a proxy for manipulating maternal effect genes.


Assuntos
Fertilização , Oócitos/metabolismo , Oócitos/transplante , Folículo Ovariano/transplante , Peixe-Zebra , Zigoto , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Desenvolvimento Embrionário , Feminino , Expressão Gênica , Genes Reporter , Microinjeções , Recuperação de Oócitos , Oogênese
9.
Hum Reprod ; 34(3): 506-518, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30597012

RESUMO

STUDY QUESTION: Is it possible to eliminate metastasized cancer cells from ovarian cortex fragments prior to autotransplantation without compromising the ovarian tissue or follicles? SUMMARY ANSWER: Ex vivo pharmacological inhibition of YAP/TAZ by Verteporfin enabled us to efficiently eradicate experimentally induced small tumours, derived from leukaemia and rhabdomyosarcoma, from human ovarian tissue fragments. WHAT IS KNOWN ALREADY: Autotransplantation of ovarian tissue fragments that contain metastasized tumour cells may reintroduce the malignancy to the recipient. In order to enhance safety for the patient there is a strong need for protocols that effectively purges the ovarian tissue from malignant cells ex vivo prior to transplantation, without compromising ovarian tissue integrity. STUDY DESIGN, SIZE, DURATION: Tumour foci were experimentally induced in human ovarian cortex tissue fragments derived from at least three patients by micro-injection of cancer cell lines. Next, the tissue fragments were cultured to allow formation of metastasis-like structures followed by a 24 h ex vivo treatment with the YAP/TAZ inhibitor Verteporfin to eradicate the cancer cells. A control treatment was included in all experiments. The purged ovarian cortex fragments were cultured for an additional 6 days to allow any possibly surviving cancer cells to establish new metastatic foci. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human ovarian tissue was obtained after female-to-male sex reassignment surgery. Human rhabdomyosarcoma, leukeamia, breast cancer and Ewing's sarcoma cell lines were utilized for the induction of tumour foci. Tumour specific (immuno)histochemistry and RT-PCR were used for the detection of residual cancer cells after ex vivo treatment. Ovarian tissue and follicle integrity after exposure to Verteporfin was evaluated by histology, a follicular viability assay and a glucose uptake assay. MAIN RESULTS AND THE ROLE OF CHANCE: Metastasized rhabdomyosarcoma and leukaemia cells could be effectively purged from ovarian cortex tissue by a 24 h ex vivo treatment with Verteporfin, while breast cancer and Ewing's sarcoma did not respond to this treatment. Ovarian tissue integrity was not affected by purging, as no statistically significant difference (P > 0.05) was observed in the percentage of morphologically normal follicles, percentage of follicles with apoptotic cells, follicular viability or glucose uptake between the control treated ovarian cortex and Verteporfin treated ovarian cortex. LIMITATIONS, REASONS FOR CAUTION: Our tumour model is based on growth of human cancer cell lines. It is unclear whether these cells reflect the behaviour of malignant cells that have metastasized to the ovary during natural disease progression. Furthermore, the functionality of the ovarian tissue after ex vivo treatment requires further investigation in vivo. WIDER IMPLICATIONS OF THE FINDINGS: The results indicate that ex-vivo tumour cell purging of human ovarian cortex fragments intended for fertility preservation purposes is feasible by short-term pharmacological treatment. Effective purging of the ovarian cortex tissue enhances safety of ovarian cortex autotransplantation for the patient. This increases the likelihood that this form of fertility restoration may become an option for patients with malignancies for which ovarian cortex transplantation is currently considered unsafe. STUDY FUNDING/COMPETING INTEREST(S): Unconditional funding was received from Merck B.V. The Netherlands (Number 2016-FERT-1) and the foundation 'Radboud Oncologie Fonds' (Number KUN 00007682). The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: NA.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Leucemia/cirurgia , Neoplasias Ovarianas/cirurgia , Ovário/efeitos dos fármacos , Ovário/transplante , Rabdomiossarcoma/cirurgia , Transativadores/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Células K562 , Leucemia/metabolismo , Metástase Neoplásica , Países Baixos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/transplante , Neoplasias Ovarianas/tratamento farmacológico , Ovariectomia , Segurança do Paciente , Rabdomiossarcoma/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transplante Autólogo , Verteporfina/farmacologia , Adulto Jovem
10.
J Assist Reprod Genet ; 36(2): 349-359, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30390176

RESUMO

PURPOSE: To evaluate the efficiency of ovarian tissue treatment with Z-VAD-FMK, a broad-spectrum caspase inhibitor, to prevent follicle loss induced by ischemia/reperfusion injury after transplantation. METHODS: In vitro, granulosa cells were exposed to hypoxic conditions, reproducing early ischemia after ovarian tissue transplantation, and treated with Z-VAD-FMK (50 µM). In vivo, cryopreserved human ovarian fragments (n = 39) were embedded in a collagen matrix containing or not Z-VAD-FMK (50 µM) and xenotransplanted on SCID mice ovaries for 3 days or 3 weeks. RESULTS: In vitro, Z-VAD-FMK maintained the metabolic activity of granulosa cells, reduced HGL5 cell death, and decreased PARP cleavage. In vivo, no improvement of follicular pool and global tissue preservation was observed with Z-VAD-FMK in ovarian tissue recovered 3-days post-grafting. Conversely, after 3 weeks of transplantation, the primary follicular density was higher in fragments treated with Z-VAD-FMK. This improvement was associated with a decreased percentage of apoptosis in the tissue. CONCLUSIONS: In situ administration of Z-VAD-FMK slightly improves primary follicular preservation and reduces global apoptosis after 3 weeks of transplantation. Data presented herein will help to guide further researches towards a combined approach targeting multiple cell death pathways, angiogenesis stimulation, and follicular recruitment inhibition.


Assuntos
Clorometilcetonas de Aminoácidos/administração & dosagem , Apoptose/efeitos dos fármacos , Folículo Ovariano/transplante , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Inibidores de Caspase/administração & dosagem , Feminino , Células da Granulosa/efeitos dos fármacos , Humanos , Camundongos SCID , Folículo Ovariano/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Transplante Heterólogo/efeitos adversos
11.
Reprod Domest Anim ; 54(2): 216-224, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30203872

RESUMO

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.


Assuntos
Cabras/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/transplante , Transplante Heterólogo/veterinária , Vitrificação , Animais , Apoptose , Criopreservação/veterinária , Feminino , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Mensageiro/análise , Técnicas de Cultura de Tecidos/veterinária
12.
Fertil Steril ; 110(6): 1181-1183, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30396563

RESUMO

OBJECTIVE: To describe and demonstrate a simple and secure procedure for laparoscopic autotransplantation of fragmented ovarian cortical tissue in women with diminished ovarian reserve as part of in vitro activation (IVA) of ovarian follicles. DESIGN: Step-by-step video explanation of the surgical procedure with still pictures and surgical video clips to demonstrate the detailed technique. SETTING: Fertility clinic and obstetrics and gynecology department at a university hospital. PATIENT(S): Women with idiopathic diminished ovarian reserve and indication for in vitro fertilization (IVF), aged 25 to 39 years with an antral follicle count bilaterally of ≤5, antimüllerian hormone level of ≤5 pmol/L, and two ovaries. INTERVENTION(S): The laparoscopic autotransplantation consists of six steps: [1] obtaining ovarian cortical biopsy samples, [2] preparing the peritoneal pocket, [3] fragmenting the ovarian cortical tissue into pieces of approximately 1 mm3, [4] installing the tissue fragments into a catheter, [5] transplanting the tissue fragments into the peritoneal pocket, and [6] closing the peritoneal pocket with a surgical clip. After the procedure, the patients are evaluated with blood samples and ultrasound scans followed by controlled ovarian stimulation. Ethics committee approval was obtained. MAIN OUTCOME MEASURE(S): Feasibility of a six-step laparoscopic autotransplantation procedure using fragmented ovarian cortical tissue. RESULT(S): A simple, fast laparoscopic procedure for taking biopsy samples and autotransplanting cortical tissue fragments in an all-in-one procedure ensures the rapid handling and correct placement of the small tissue fragments. The procedure is performed in an outpatient setting with an operation time of 1 hour. We have performed this procedure on 20 patients with no complications. CONCLUSION(S): In vitro activation is a new, developing option for women in fertility treatment who have diminished ovarian reserve. Fragmentation of murine ovarian tissue has shown to suppress the Hippo pathway, thereby initiating proliferation and growth. This surgical procedure resembles that used when transplanting pieces of frozen-thawed ovarian tissue for fertility restoration, but the fragmented ovarian tissue is only 1 mm3, which makes it difficult to transplant. Until now no surgical procedures for transplanting small IVA fragments of cortical tissue has been published. With this video we report in detail a simple way of autotransplanting small fragments of IVA cortical tissue using what is already accessible in the operating theater. Among the many advantages of this procedure are its short duration (1 hour) and outpatient setting, which enable fast recovery and minimal postoperative pain. The procedure also allows fast handling and minimal manipulation of the tissue (limited to the fragmentation). The effect of autotransplantation of fragmented tissue in women with diminished ovarian reserve is currently being studied in ongoing trials. If the technique is combined with chemical IVA, a better outcome may be seen.


Assuntos
Laparoscopia/métodos , Folículo Ovariano/transplante , Reserva Ovariana/fisiologia , Feminino , Humanos , Transplante Autólogo/métodos
13.
J Assist Reprod Genet ; 35(12): 2205-2213, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30255455

RESUMO

PURPOSE: To report the results of cryopreserved ovary tissue transplantation for leukemia and other cancers, in a single US center. METHODS: One hundred eight females between age 6 and (median age 24) 35 were referred for possible ovary tissue cryopreservation over a 20-year period, with either slow freeze or vitrification. Thus far 13 patients returned up to 18 years later to have their tissue transplanted back. RESULTS: All 13 patients had return of ovarian function 5 months post transplant with regular menstrual cycling. AMH rose to very high levels as the FSH declined to normal. Four months later, the AMH again declined to very low levels. Nonetheless, the grafts remained functional for up to 5 years or longer. Ten of the 13 (77%) became spontaneously pregnant at least once, resulting in 13 healthy babies. A total of 24 healthy babies have been born 11 from fresh transplanted ovarian tissue and 13 from cryopreserved transplanted ovarian tissue. CONCLUSIONS: (1) Ovary tissue cryopreservation is a robust method for preserving a woman's fertility. (2) Cortical tissue pressure may be a key regulator of primordial follicle arrest, recruitment, and ovarian longevity. (3) This is the only such series yet reported in the USA.


Assuntos
Criopreservação , Preservação da Fertilidade/métodos , Folículo Ovariano/transplante , Ovário/transplante , Adulto , Feminino , Humanos , Neoplasias/patologia , Ovário/fisiologia , Gravidez , Insuficiência Ovariana Primária/patologia , Vitrificação
14.
J Ovarian Res ; 11(1): 76, 2018 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-30170634

RESUMO

BACKGROUND: The aim of this report was to describe a case of pregnancy after drug-free in vitro activation (IVA) of follicles and fresh tissue autotransplantation in primary ovarian insufficiency (POI) patient and to review the pertinent literature. METHODS: We present a case in wich a 32 - years old patient with POI became pregnant after IVA without tissue culture and with ovarian tissue transplantation. We also reviewed the literature using Pubmed database. CASE PRESENTATION: Pretreatment with estradiol/progesterone stopped the day before surgery. The removal of the ovarian cortex and autotransplantation were performed by laparoscopy in the same surgical act. Ovarian fragments were transplanted in contralateral ovary and peritoneal pocket near to the ovary. Immediately after surgery GnRH agonist together HMG injections started, leading the growth of 3 preovulatory follicles and the retrieval of two mature eggs. After IVF two embryos were transferred and singleton pregnancy was established and currently she is 25 weeks pregnant. RESULTS: A total of 51 patients with POI in whom an in vitro activation of ovarian tissue was performed, were collected from the revieew of the literature. In 29.4% of them, follicular development was obtained and in 4 of them a pregnancy. In all of them, a combined technique (fragmentation and activation) was performed in two laparoscopies. No case has been reported successfully after drug-free in vitro activation. CONCLUSIONS: This is the first report about a case with pregnancy after drug-free in vitro activation of follicles and fresh tissue autotransplantation in POI patient.


Assuntos
Preservação da Fertilidade/métodos , Folículo Ovariano/transplante , Insuficiência Ovariana Primária/complicações , Transplante Autólogo/métodos , Adulto , Feminino , Humanos , Gravidez
15.
Stem Cell Res Ther ; 9(1): 252, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30257706

RESUMO

BACKGROUND: The increasing number of patients with ovarian insufficiency due to autoimmune disorders, genetic predisposition, or iatrogenic effects of treatment such as cancer therapies necessitates an urgent measure to find a safe and transplantable alternative ovary. A bioengineered ovary is one of the strategies on which the researchers have recently been working. An engineered ovary should be able to mimic the natural ovary aspects. Recent studies suggest that the decellularized organ-specific extracellular matrix-based scaffolds can serve as a native niche to bioengineering artificial organs. Therefore, we established a human decellularized ovarian scaffold based on a sodium lauryl ester sulfate (SLES)-treated process, as an optimized protocol. METHODS: The human ovary samples were decellularized with 1% SLES for 48 h followed by DNase I in PBS for 24 h, and then thoroughly rinsed in PBS to remove the cell remnants and chemical reagents. Efficient cell removal was confirmed by DNA content analysis, hematoxylin and eosin, and Hoechst staining. Preservation assessment of the extracellular matrix structures was performed by immunohistochemistry, histological staining, and scanning electron microscopy. An MTT test was done to assess the in vitro scaffold's cytocompatibility, and finally in vivo studies were performed to evaluate the biocompatibility, bioactivity, and secretion functions of the ovarian grafts made of primary ovarian cells (POCs) on the decellularized scaffolds. RESULTS: Evidence provided by SEM, histochemical, and immunohistochemical analyses showed that the ovarian extracellular matrix was preserved after decellularization. Moreover, MTT test indicated the suitable cytocompatibility of the scaffolds. The in vivo assessment showed that the POCs kept their viability and bioactivity, and reconstructed the primordial or primary follicle-like structures within the scaffolds after transplantation. Immunostaining characterized somatic cells that were capable of expressing steroid hormone receptors; also, as a marker of granulosa cell, inhibin-α immunostaining demonstrated these cells within the grafts. Additionally, hormone assessment showed that serum estradiol and progesterone levels were significantly higher in ovariectomized rats with ovarian cells-seeded grafts than those with or without decellularized scaffold grafts. CONCLUSIONS: A human ovary-specific scaffold based on a SLES-decellularized protocol as a biomimicry of the natural ovarian niche can be an ideal scaffold used to reconstruct the ovary.


Assuntos
Folículo Ovariano/citologia , Ovário/citologia , Insuficiência Ovariana Primária/terapia , Tecidos Suporte , Animais , Células Cultivadas/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Feminino , Humanos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/transplante , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Ovário/transplante , Insuficiência Ovariana Primária/patologia , Ratos , Dodecilsulfato de Sódio/farmacologia , Engenharia Tecidual/métodos
16.
J Assist Reprod Genet ; 35(10): 1831-1841, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30043336

RESUMO

OBJECTIVE: To determine whether recombinant AMH (rAMH) could prevent post-transplant follicular depletion by acting on the stemness markers Oct-4, Sox2, and NANOG. MATERIALS AND METHODS: This was an experimental study where 12 ovariectomized nude mice were xenotransplanted with vitrified/warmed ovarian cortex obtained from a pre-pubertal girl and Alzet pumps delivering rAMH, or placebo (control), were inserted intra-abdominally. Previously vitrified/warmed ovarian cortex fragments were transplanted after 7 days and then harvested after 14 days from pump placement. We performed real-time RT-PCR analyses, ELISA for AMH, FSH, and estradiol, histologic measurement of ovarian follicles, and immunohistochemistry for Ki67 and TUNEL. The main outcome measures were serum levels and tissue expression of the parameters under investigation and follicle count. RESULTS: Serum AMH, FSH, and estradiol reflected post-ovariectomy profiles and were mildly influenced by rAMH administration. Ovarian cortex expression of AMH, AMH-R2, VEGF, GDF9, Oct-4, and Sox2 was lower in rAMH mice than in controls, while NANOG was upregulated. There was a non-significant decrease in primordial follicles after vitrification-warming, and xenotransplantation further decreased this number. There were lower cell replication and depressed apoptosis in the rAMH group. CONCLUSIONS: Administration of recombinant AMH in the peri-transplant period did not protect the initial follicular depletion but decreased apoptosis and cellular activation and regulated stem cell markers' tissue expression. These results aid our understanding of the inhibitory effects of AMH on follicular development and show the benefit of administering exogenous AMH at the time of pre-pubertal ovarian cortex transplant to protect the follicles from pre-activation and premature depletion.


Assuntos
Hormônio Antimülleriano/genética , Xenoenxertos/metabolismo , Folículo Ovariano/transplante , Ovário/transplante , Animais , Hormônio Antimülleriano/administração & dosagem , Hormônio Antimülleriano/sangue , Apoptose/genética , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Regulação da Expressão Gênica no Desenvolvimento , Xenoenxertos/efeitos dos fármacos , Xenoenxertos/crescimento & desenvolvimento , Humanos , Camundongos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Ovariectomia , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Fatores de Transcrição SOXB1/genética , Transplante Heterólogo , Vitrificação
17.
J Ovarian Res ; 11(1): 35, 2018 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-29716634

RESUMO

BACKGROUND: Fertility preservation by whole ovary cryopreservation and transplantation (WOCP&TP) with vascular anastomosis requires successful cryopreservation. In this study, we investigated the possibility of restoring ovarian function and natural fertility after WOCP&TP in a premature ovarian insufficiency (POI) rat model. The influence of cryopreservation on the offspring of rats following WOCP&TP was also explored. METHOD: Rats aged 8-10 weeks were used as donors and recipients for allotransplantation. Fifteen rat whole ovaries were divided into three groups: the optimized group, the conventional group, and the fresh group. Different perfusion modes were used before cryopreservation and after thawing. Whole ovaries were observed by morphologic analysis, immunohistochemical staining, and transferase-mediated deoxyuridine triphosphate nick end-labeling assay. Ovarian function and fertility after WOCP&TP were then observed in 25 cyclophosphamide-induced POI rats for 8 months. Ovarian function was assessed by vaginal smears and blood hormone levels. Fertility restoration was quantified as the live birth rate after mating. The filial generation of rats was mated at 8-10 weeks of age. Offspring were observed for birth defect. RESULTS: Histological evaluation demonstrated intact morphology of follicles in all groups, with 77.6% of the total number of follicles identified as intact in the optimized group. The apoptotic rates of ovarian cells in the optimized group were significantly lower than that in the conventional group. Of the 20 live POI rats, 14 (70%) began to recover ovarian function after 2 weeks of transplantation, with normal hormone levels achieved 4 weeks after transplantation. Four of 14 rats were pregnant and delivered live offspring. One rat had a second pregnancy and delivered a second litter of live offspring. When the offspring matured, they were mated, and second and third generations of rats were born. All offspring had no abnormalities in appearance. CONCLUSIONS: High rates of restoration of ovarian function and natural fertility with multiple generations of offspring were obtained following WOCP&TP in a cyclophosphamide-induced POI rat model by utilizing optimized perfusion. Cryopreservation did not affect the viability of successive generations.


Assuntos
Fertilidade/fisiologia , Folículo Ovariano/transplante , Ovário/transplante , Insuficiência Ovariana Primária/terapia , Animais , Criopreservação , Ciclofosfamida/toxicidade , Modelos Animais de Doenças , Feminino , Preservação da Fertilidade/métodos , Humanos , Menopausa Precoce , Folículo Ovariano/fisiopatologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Gravidez , Insuficiência Ovariana Primária/induzido quimicamente , Insuficiência Ovariana Primária/fisiopatologia , Ratos , Reprodução/fisiologia , Vitrificação
18.
Hum Reprod ; 33(6): 1107-1116, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29635371

RESUMO

STUDY QUESTION: Do adipose tissue-derived stem cells (ASCs) enhance vascularization and follicle survival in xenografted ovarian tissue using a two-step transplantation approach? SUMMARY ANSWER: Higher rates of oxygenation and vascularization of ovarian tissue, as well as increased follicle survival rates, were detected in the early post-grafting period. WHAT IS KNOWN ALREADY: ASCs have multilineage differentiation potential, proangiogenic properties and enhance vascularization in a peritoneal grafting site. Some studies suggest that using ASCs may improve ovarian tissue quality by enhancing graft angiogenesis. STUDY DESIGN, SIZE, DURATION: A total of 15 severe combined immunodeficient (SCID) mice were intraperitoneally grafted with frozen-thawed human ovarian tissue (OT) from five different patients. A peritoneal transplantation site had been previously prepared in a first step using either empty fibrin (Fi+OT group [n = 5]) or ASC-loaded fibrin (Fi/ASCs+OT group [n = 5]) for 14 days prior to grafting. Five mice underwent the standard one-step transplantation procedure and served as controls (OT group). Lithium phthalocyanine (LiPc) crystals were inserted into all grafted human ovarian tissue before transplantation. Levels of partial pressure of oxygen (pO2) in grafts were monitored in vivo by electron paramagnetic resonance (EPR) oximetry on Days 3 and 7. Samples for histology and immunohistochemistry (IHC) were collected after euthanizing the mice on Day 7 following EPR. One piece of ovarian tissue per patient was fixed for analysis to serve as non-grafted controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), histological (haematoxylin and eosin staining), immunohistochemistry (anti-mouse and human double CD34 and anti-human Ki-67) and TUNEL analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant increase in pO2 was observed in all groups between Days 3 and 7 (P < 0.001). A significantly higher pO2 level was observed in the Fi/ASCs+OT group compared to the OT group on Day 7 (P = 0.028). Total CD34-positive vessel area on Day 7 was greater in the Fi/ASCs+OT group than in any other group (vs non-grafted group: P = 0.0014; vs OT group: P = 0.013; vs Fi+OT group: P = 0.018). Primordial follicle survival rates after grafting were higher in the Fi/ASCs+OT group than in the OT (P = 0.0059) or Fi+OT groups (P = 0.0307). TUNEL-positive follicle percentages after grafting were significantly lower in the Fi/ASCs+OT group than in any other grafted tissue (vs OT group: P = 0.045; vs Fi+OT group: P = 0.0268). Percentages of Ki-67-positive primordial follicles were significantly higher in all grafted groups compared to non-grafted tissue controls (P < 0.01). LIMITATIONS REASONS FOR CAUTION: As demonstrated by our results, the proposed two-step ovarian tissue transplantation procedure using ASCs enhances vascularization in the early post-grafting period, leading to increased follicle survival rates and decreased apoptosis. However, mechanisms involved in the proangiogenic behavior of ASCs remain to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that the proposed transplantation procedure with ASCs is a promising step towards potentially solving the problem of massive follicle loss after ovarian tissue grafting. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, grant Télévie No. 7.6515.16 F to DDM and grant 5/4/150/5 awarded to MMD and CAA is research associate, FRS-FNRS), Fonds Spéciaux de Recherche, Fondation St Luc, and Foundation Against Cancer, and donations from the Ferrero family.


Assuntos
Tecido Adiposo/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Neovascularização Fisiológica/fisiologia , Folículo Ovariano/transplante , Ovário/irrigação sanguínea , Ovário/transplante , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos SCID , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Ovário/citologia , Estudos Prospectivos
19.
J Assist Reprod Genet ; 35(4): 615-626, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29497951

RESUMO

PURPOSE: Even with 86 live births reported globally so far, the mechanism of primordial follicle loss following autotransplantation of the frozen-thawed ovarian tissue needs further evaluation. Pten, Tsc1, p27, and Amh are the inhibitor proteins that play crucial roles in suppressing the transition from the primordial follicle to primary state, maintaining the primordial follicle reserve. In this study, we aimed to evaluate whether the expression patterns of these proteins change and it may be related to the global primordial follicle loss after autotransplantation of the frozen-thawed ovarian tissue. METHODS: Four groups were established in rats: fresh-control, frozen/thawed, fresh-transplanted, and frozen/thawed and transplanted. After slow freezing and thawing process, two ovarian pieces were transplanted into the back muscle of the same rat. After 2 weeks, grafts were harvested, fixed, and embedded into the paraffin block. Normal and atretic primordial/growing follicle count was performed in all groups. Ovarian tissues were evaluated for the dynamic expressions of the Pten, Tsc1, p27, and Amh proteins using immunohistochemistry, and H-score analyses were done. RESULTS: Ovarian tissue cryopreservation does not change the expression patterns of inhibitory proteins that control ovarian reserve. Both in fresh and frozen/thawed autotransplanted groups, the expression of inhibitory proteins and Amh decreased significantly in primordial follicles and in growing follicles, respectively. In control group and in frozen/thawed group, primordial follicle counts were similar but decreased by almost half in both fresh-transplanted and frozen/thawed and transplanted groups. CONCLUSIONS: One of the causes of primordial follicle loss after transplantation of ovarian graft may be decreased expression of the inhibitory proteins that guard the ovarian reserve and transplantation itself seems to be the major cause for disruption of inhibitory molecular signaling. Our findings highlight important molecular aspects for future clinical applications for fertility preservation in humans.


Assuntos
Criopreservação/veterinária , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Folículo Ovariano/metabolismo , Reserva Ovariana/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Feminino , Preservação da Fertilidade , Folículo Ovariano/citologia , Folículo Ovariano/transplante , Ratos , Ratos Wistar , Transplante Autólogo , Proteína 1 do Complexo Esclerose Tuberosa
20.
Cryobiology ; 80: 101-113, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29154909

RESUMO

The aim of this study was to assess the follicular development and the patterns of EphrinB1 and PDGFA immunostaining in vitrified mouse ovarian tissue (OT) with and without transplantation. Histological evaluation was performed on fresh and vitrified OTs, whether transplanted or not. RT-PCR was performed on fresh and vitrified ovarian samples (OSs) and vitrified OS graft. Vitrification alone did not significantly reduce the normal primordial, primary, and secondary follicles except antral ones (p > 0.05). However, transplantation decreased all the follicle types. The EphrinB1 immunoexpression showed high intensity in all follicular types in vitrified OT and the significant increased was detected in secondary and antral follicles (p < 0.05). PDGFA protein immunoexpression of primordial and primary follicles was decreased in vitrified OT (p < 0.05). However, the lowest immunoexpression of EphrinB1 and PDGFA was detected after transplantation (p < 0.05). The levels of ephrinb1 and pdgfa mRNA expressions in vitrified OS and vitrified OS grafts were found as comparable to the fresh OS. In conclusion, vitrification has no detrimental effect on the follicles at the different developmental stages, majority of ovarian follicular loss takes place after transplantation rather than vitrification. Overall, vitrification and grafting do not change the ephrinb1 and pdgfa gene expressions. In addition, EphrinB1 and PDGFA are expressed during different stages of folliculogenesis in a different manner in fresh, vitrified, or grafted OTs. Vitrification and/or grafting appear to affect the follicular expression of EphrinB1 and PDGFA. These findings suggest that these proteins could have several functions related to the development of follicles and angiogenesis after transplantation.


Assuntos
Criopreservação/métodos , Efrina-B1/biossíntese , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/transplante , Fator de Crescimento Derivado de Plaquetas/biossíntese , Vitrificação , Animais , Feminino , Camundongos
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