Vitrified-warmed Ovarian Tissue Research Articles

Vitrified-Warmed Ovarian Tissues From Poultry Can Re-Organize After Transfer Into A Recipient

Vitrified-Warmed Ovarian Tissues From Poultry Can Re-Organize After Transfer Into A Recipient

In vitro Activation Prior to Transplantation of Human Ovarian Tissue: Is It Truly Effective?

Research Question: What are the true benefits, if any, of disrupting the Hippo signaling pathway and stimulating the Akt pathway in xenotransplanted human ovarian tissue using an in vitro activation (IVA) approach?Design: Human ovarian tissue was retrieved from 18 young patients by laparoscopy and grafted to 54 severe combined immunodeficient mice. The experiment was conducted using fresh ovarian tissue (group I; n = 6 women), slow-frozen-thawed ovarian tissue (group II; n = 6 women), and vitrified-warmed ovarian tissue (group III; n = 6 women). Slow-freezing and vitrification procedures were performed according to Gosden's and Kawamura's protocols, respectively. The tissue (fresh, slow-frozen, and vitrified) was fragmented into small cubes (1 × 1 × 1 mm) to disrupt the Hippo signaling pathway and cultured or not in IVA medium for 48 h with Akt stimulators (PI3K stimulator and PTEN inhibitor), before being transplanted to the mice. All the grafts were maintained for 28 days.Results: (1) Follicular density: Follicular density decreased in all groups after transplantation, most significantly in the vitrification group. Culture with IVA had no impact. (2) Follicle activation: Addition of PI3K stimulator and PTEN inhibitor for 48 h prior to grafting did not significantly change the proportion of primordial follicles in any of the groups (fresh, slow-frozen, or vitrified tissue) compared to 48 h of control culture without these molecules. Particularly, vitrification and culture in IVA medium yielded no benefits in terms of growing follicle percentages or follicle proliferation rates. The large proportion of growing follicles in the vitrified tissue group after grafting may have been responsible for the higher rate of atresia.Conclusion: We were unable to demonstrate any significant benefits of cutting ovarian tissue into small cubes and applying IVA with Akt stimulators. The association of vitrification and transplantation was actually found to be the most deleterious combination with respect to the follicle reserve, and even worse when culture with Akt stimulators was performed.

Long-term follow-up of vitrified and autografted baboon (Papio anubis) ovarian tissue.

Can vitrified baboon ovarian tissue survive >5 months after autotransplantation and subsequently restore fertility? Our results show that ovarian tissue grafts can survive at least 18 months, but fertility restoration could not be confirmed due to lack of pregnancy. Ovarian function in baboons can be re-established after autografting of vitrified-warmed ovarian tissue fragments. Ovaries from five adult female baboons were used for this study. Unilateral ovariectomy was performed in each animal, followed by vitrification of ovarian fragments. Orthotopic autotransplantation of the vitrified-warmed ovarian fragments was carried out 1 day after ovariectomy. One month later, the other ovary was removed from each animal and vitrified. The next day, the ovarian samples were warmed and orthotopically autografted. Biopsies of grafted tissues were taken after 12 and 18 months for stromal tissue and follicle evaluation. Control samples were collected before vitrification. After grafting, follicle survival, growth and function and also the quality of stromal tissue were assessed histologically and by immunohistochemistry. Estrogen levels were measured, and cyclicity was monitored. All five animals were mated several times. Male baboons used in the mating experiments were not of proven fertility. After vitrification, warming and long-term grafting, follicles were able to grow. However, their function may have been negatively affected by vitrification and/or transplantation, as expression of kit ligand and c-kit differed from fresh ungrafted tissue (P < 0.05). Corpora lutea and/or ovulation stigma were observed in grafts, indicating successful ovulation in all the baboons, with estrogen levels comparable to those in adult female baboons. Not applicable. Despite our promising findings on ovarian function restoration, the vitrification procedure could not be validated. Moreover, male baboons used for mating were not of proven fertility. Our protocol of ovarian tissue vitrification successfully re-established ovarian function in a baboon model of autotransplantation. While more studies are required to determine whether this approach can indeed restore fertility, it may prove an easy way of cryopreserving ovarian tissue with a view to recovering ovarian function. This study was supported by grants from the Fonds National de la Recherche Scientifique (FNRS) (C.A.A. is an FRS-FNRS Research Associate; grant 5/4/150/5 awarded to M.M.D.), Fonds Spéciaux de Recherche, Fondation Saint Luc, Stichting tegen Kanker (Fondation contre le Cancer), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil; grant # 013/14 CAPES/WBI awarded to C.M.L.), Wallonie-Bruxelles International (awarded to C.A.A.), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant awarded to Sarah R. Scalercio) and donations from the Ferrero family. None of the authors have any competing interests to declare.

Oocyte retrieval after heterotopic transplantation of ovarian tissue cryopreserved by closed vitrification protocol.

A device for closed vitrification was designed to reduce the risk of contamination and investigated on its efficacy for ovarian function recovery after cryopreservation and heterotopic transplantation. Ovarian tissues from green fluorescence protein transgenic mice (10 GFP mice) were vitrified using the device, and warmed ovarian tissues were transplanted into the ovarian bursa region in wild-type female mice (6 mice). Fresh ovarian tissues were similarly transplanted as a control. After recovery of the estrous cycle, mice were mated with male mice. Ovarian tissues from six cynomolgus monkeys were vitrified and warmed with the device for autologous, heterotopic transplantation. Fresh tissue transplantation was not performed for the control. Ovarian function was examined by recovery of the hormonal cycle. Histological examination was conducted. The number of live pups per recipient mouse was not significantly different after transplantation of fresh or vitrified-warmed ovarian tissue, although the pregnancy rate was reduced with vitrified tissues. The hormonal cycle was restored in 5/6 monkeys after heterotopic transplantation of vitrified-warmed ovarian tissue. Follicles were harvested at eight sites in the omentum and 13 sites in the mesosalpinx. In vitro maturation (IVM)/IVF produced embryo but did not develop. Resumption of the hormonal cycles, follicle development, and oocyte retrieval from vitrified-warmed ovarian tissue transplants may indicate that the use of vitrification for ovarian tissue in a closed system has a potential of clinical application without the risk of contaminations. More detailed analyses of the effects of vitrification on ovarian tissue, such as gene expression patterns in oocytes and granulosa cells, may be needed for establishing a standard procedure for cryopreservation of ovarian tissues in human.

Three-dimensional culture of fresh and vitrified ovarian tissue in a novel tyramine substituted hyaluronan gel

Follicle culture in matrices that allow 3-D growth may better mimic in vivo conditions by preserving follicle architecture and critical granulosa-oocyte interactions. This study explores methodology for culturing fresh and vitrified ovarian tissue. We further describe the application of a relatively new biomaterial for in vitro follicle maturation and the production of functional metaphase II oocytes. Mouse pre-antral follicle model for IVM and testing of a hyaluronan matrix. Ovaries from 12 to 14 day B6D2F1 pups were removed. Ovaries were vitrified using an ethylene glycol (EG)/DMSO protocol. Ovaries were equilibrated in chilled vitrification solutions (VS) containing 5, 10, 15 and finally 20% EG/DMSO in L-15 with 20% SSS. Ovaries were loaded on a nylon mesh and plunged in to a specially vented cryovial filled with LN2. Pre-antral follicles (FL) and ovarian follicle clusters were collected from fresh and vitrified-warmed ovaries either by mechanical isolation using needles or by enzymatic digestion in collagenase. The HA hydrogel (3 mg/ml) cross-linking was initiated by mixing with 0.03% hydrogen peroxide in a 25:1 ratio. HA gel was then quickly pipetted into the culture dish. Isolated preantral follicles or intact clusters with 4-6 follicles were rapidly seeded into the HA before gelling was completed. Follicle culture was performed in α-MEM supplemented with 5% FBS, 100 mIU/ml FSH, and ITS. Dishes were incubated at 37°C with 6% CO2. Final maturation was triggered with hCG (1.5 IU/ml) and EGF (5 ng/ml) after 12 days of growth. Outcome parameters monitored were follicle morphology, survival in culture, germinal vesicle breakdown, oocyte maturation and meiotic spindle retardance using Oosight Imaging system. Vitrified-warmed ovarian tissue was easily dissected into clusters with 4-5 intact follicles without exposure to enzyme. In contrast, enzymatic follicle isolation was more efficient and preferable for fresh tissue. With fresh ovaries, maturation rate in FL-clusters was similar to that observed with isolated follicles. Meiotic spindle retardance in ovulated oocytes from both culture types did not differ. The overall percent oocyte maturation to metaphase II was lower with vitrified tissue. HA is a natural matrix and supported follicle growth and oocyte maturation. The 3-D culture of FL-clusters provided a method to further mimic in vivo growth by preserving interfollicle connections and architecture. Functional competence of oocytes needs further assessment. Enzymatic treatment of vitrified-warmed ovaries for follicle isolation may not be the best approach due to tissue fragility.Table 1IVM summary data for fresh and vitrified tissueFrozenaFreshbFollicle - ClusterFollicle - IsolatedFollicle - ClusterFollicles observed during IVC69130154Antrum formationna51% (66/130)53% (81/154)Ovulated complexes after hCG93% (64/69)71% (92/130)66% (101/154)GVBD (MI)52% (33/64)30% (28/92)28% (28/101)Maturation to MII34% (22/64)59% (54/92)55% (56/101)Spindle Retardance (Range)na1.34- 3.641.62- 3.51aMechanically isolated follicle cluster. bEnzymatic isolation. Open table in a new tab

Effect of treatment with angiopoietin-2 and vascular endothelial growth factor on the quality of xenografted bovine ovarian tissue in mice.

Cryopreservation and transplantation of ovarian tissue (OT) represents a method for fertility preservation. However, as the transplantation is performed without vessel anastomosis, unavoidable ischemic damage occurs. To reduce this ischemic damage and improve outcomes after transplantation, we used two kind of angiogenic factors, angiopoietin-2 (ang-2) and vascular endothelial growth factor (VEGF). Fresh or vitrified-warmed bovine OTs were prepared for xenotransplantation (XT). Fresh OTs were immediately xenografted into nude mice (XT-Fresh). Vitrified-warmed OTs were xenografted into four subgroups of mice, which were injected intraperitoneally before XT with saline (XT-Vitri), Ang-2 (XT-Ang-2), VEGF (XT-VEGF), and a combination of Ang-2 and VEGF (XT-Combined). Seven or 28 days post-grafting, grafted OTs and blood samples were collected for evaluation. Follicle normality was higher in the angiogenic factor-treated groups than in the XT-Vitri group. The XT-VEGF and the XT-Combined showed higher (P<0.05) follicular density than the XT-Vitri group. The highest apoptotic follicle ratio was observed in the XT-Vitri group on day 7; this was decreased (P<0.05) in the XT-Combined group. Microvessel densities were higher in the angiogenic factor-treated groups than in the XT-Vitri group. The largest fibrotic area was showed in the XT-Vitri group on day 28, and it was decreased (P<0.05) in the XT-combined group. Based on these results, administration of Ang-2 and VEGF to recipients prior to XT appeared to alleviate ischemic damage by enhancing angiogenesis, which resulted in the maintenance of follicle integrity and density, and reduced follicle apoptosis and OT fibrosis.

Platelet-rich plasma promotes the development of isolated human primordial and primary follicles to the preantral stage

This study aimed to assess the effects of platelet-rich plasma (PRP) on growth and survival of isolated early human follicles in a three-dimensional culture system. After fresh and vitrified-warmed ovarian tissue was digested, isolated early preantral follicles and ovarian cells were separately encapsulated in 1% alginate (w/v). The encapsulated follicles and ovarian cells were cultured together in a medium supplemented with foetal bovine serum (FBS), PRP, PRP + FBS, or human serum albumin (HSA) for 10 days. Growth and survival of the follicles were assessed by measurement of diameter and staining with trypan blue. Follicular integrity was assessed by histological analysis. After culturing, all follicles increased in size, but growth rate was greater in follicles isolated from fresh samples than those from vitrified-warmed ones (P < 0.001). Similarly, follicular viability of fresh samples after culturing was higher than that of vitrified-warmed ones. The growth and survival rates of follicles from both fresh and vitrified groups cultured in PRP supplemented media were significantly higher than those of other groups (growth P < 0.001 and survival P < 0.05, in both groups). In conclusion, media supplementation with PRP can better support viability and growth of isolated human early preantral follicles in vitro.

A combination of simvastatin and methylprednisolone improves the quality of vitrified-warmed ovarian tissue after auto-transplantation.

Does the preoperative administration of simvastatin and methylprednisolone enhance mouse ovarian quality after auto-transplantation of vitrified-warmed ovarian tissue (OT)? Treatment with combined simvastatin and methylprednisolone enhances the quality of transplanted mouse OTs. The prevention of ischemic injury after transplantation of OT is critical for preserving the ovarian follicles. Preoperative administration of simvastatin (a cholesterol-lowering drug) has beneficial effects on various organ transplantations. Moreover, donor treatment with simvastatin and methylprednisolone (main effects are on immune response) prevents ischemia-reperfusion injury and has a beneficial effect on allograft survival in rat cardiac allografts. A total of 232 6-week-old B6D2F1 mice were randomly distributed into fresh control, vitrified-warmed control and experimental groups (n = 10-17 per group). The experimental groups were as follows: sham control, simvastatin, methylprednisolone and co-treatment groups. In the experimental groups, the mice were administered simvastatin (5 mg/kg, orally), methylprednisolone (15 mg/kg, i.v.) or a combination of simvastatin and methylprednisolone 2 h before ovariectomy, whereas the sham control mice received normal saline. Whole ovaries were removed from the mice and vitrified by two-step vitrification procedures. The vitrified ovaries were warmed 1 week later and auto-transplanted under the bilateral kidney capsules. The ovaries and blood samples were collected 2, 7 and 21 days (D) after transplantation for histological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, immunohistochemistry for CD31 and serum anti-Mullerian hormone (AMH) level estimation. Embryonic development was evaluated after IVF of oocytes obtained from the transplanted ovary. The group that received simvastatin and methylprednisolone showed a significantly improved intact (Grade 1) follicle ratio (D2: P < 0.001, D7: P < 0.05 and D21: P < 0.001), apoptotic follicle ratio (D21: P < 0.05), CD31-positive area (D7: P < 0.05 and D21: P < 0.05) and serum AMH level (D7: P < 0.001) after transplantation when compared with the sham control. However, no difference was noted in the fertilization and blastocyst formation rates, number of total and apoptotic blastomeres per blastocyst and inner cell mass/trophectoderm ratio among the four transplantation groups. Although we evaluated the beneficial effects of simvastatin and methylprednisolone in the present study, we did not unravel the corresponding protective mechanisms. Our results suggest that a combination of simvastatin and methylprednisolone has beneficial effects on the quality and functioning of transplanted OT. This combined treatment can potentially be applied clinically to humans and domestic animals subject to further studies.

Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified-warmed ovarian tissue survival

What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified-warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of α-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified-warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4-58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6-92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

Extended uterine receptivity for blastocyst implantation and full-term fetal development in mice with vitrified-warmed ovarian tissue autotransplantation.

Our previous study demonstrated that vitrified-warmed ovarian tissue autotransplantation (VOAT) into estrus cycle-ceased ovariectomized mice restored fertility to achieve full-term fetal development for transferred embryos, while less steroidogenesis in the corpus luteum was observed in VOAT mice. It has been reported that the window of uterine receptivity for blastocyst implantation is extended at lower estrogen levels. Therefore, we hypothesized that duration of the window in VOAT mice could be extended. Blastocysts were transferred into VOAT mice on day 5 of pseudopregnancy. Immunohistochemical analysis was performed to examine the potential in VOAT ovarian tissues. The rate of live birth pups from embryos transferred on day 5 of pseudopregnant VOAT mice was not different from that of embryos transferred on day 4 of pseudopregnancy in VOAT mice, while embryo transfer on day 5 into intact mice showed no pregnancy. Immunohistochemical analysis of the corpus luteum of day 8 pseudopregnant VOAT mice with uteri having decidualization induced on day 5 showed less steroidogenesis and blood vessel formation as compared to intact mice. Uterine receptivity was extended in VOAT mice. Less steroidogenesis and blood vessel formation in the transferred ovarian tissues may be associated with the extended uterine receptivity.

Vitrified-warmed ovarian tissue autotransplantation into ovariectomized mice restores sufficient ovarian function to support full-term pregnancy.

Our previous study demonstrated that heterotopic autotransplantation of fresh ovarian tissue followed by transfer of blastocysts supported full-term pregnancy in the mouse. In the present study, to address whether vitrified-warmed ovarian tissue has the potential to support uterine preparation for implantation and subsequent pregnancy to full term, we examined vitrified-warmed ovarian tissue autotransplantation (VOAT) in mice. VOAT into kidney capsules was performed for sexual cycle-ceased mice after 7 days of ovariectomy. Uterine potential of decidualization was examined by oil infusion on day 4 of pseudopregnancy. Immunohistochemical analysis was performed to examine the potential in VOAT ovarian tissues. Blastocysts were transferred into uteri on day 4 of pseudopregnancy. In VOAT mice, uterine decidualization on day 8 of pseudopregnancy was the same as that in intact mice. Blastocyst transfer into the pseudopregnant VOAT mice showed the same rates of pregnancy and live birth pups as intact mice, while less steroidogenesis in the corpus luteum was detected in VOAT mice. The autotransplantation of vitrified-warmed ovarian tissues after 7 days of ovariectomy restored their sexual cycle and then supported their pregnancy and production of offspring.

Vitrified-Warmed Ovarian Tissue Autotransplantation into Ovariectomized Mice Restores Sufficient Ovarian Function to Support Term Pregnancy.

Vitrified-Warmed Ovarian Tissue Autotransplantation into Ovariectomized Mice Restores Sufficient Ovarian Function to Support Term Pregnancy.

Effective method for in-vitro culture of cryopreserved human ovarian tissue

It is widely accepted that it is possible to successfully cryopreserve human ovarian tissue by direct plunging into liquid nitrogen using permeable cryoprotectants only, without disaccharides. This study aimed to search for and test a new method for in-vitro culture of vitrified tissue. Ovarian biopsies were obtained during operative laparoscopy. Pieces of ovarian tissue were vitrified and warmed. After warming, tissue pieces were randomly distributed into three groups for further culture: in 2 ml of culture medium which was regularly renewed (group 1), in 30 ml of culture medium without agitation (group 2) and in 30 ml of culture medium with agitation (group 3). During the 2-week and 6-week culture, the growth of follicles within the vitrified-warmed ovarian tissue pieces was investigated. After 2 weeks of culture, mean numbers of non-degenerated follicles per mm 2 of tissue were 1.5, 1.7 and 4.5 for groups 1, 2 and 3 respectively (groups 1 and 2 versus group 3, P < 0.05). Agitation during culture of ovarian tissue is beneficial, and can be used as a prognostic tool for future warming and autotransplantation of ovarian tissue.