Cryopreservation of Human Ovarian Tissue: A Review Rivas Leonel, Ellen Cristina; Lucci, Carolina M.; Amorim, Christiani A.
Transfusion medicine and hemotherapy,
06/2019, Volume:
46, Issue:
3
Journal Article
Peer reviewed
Open access
Background: Cryopreservation of human ovarian tissue has been increasingly applied worldwide to safeguard fertility in cancer patients, notably in young girls and women who cannot delay the onset of ...their treatment. Moreover, it has been proposed to patients with benign pathologies with a risk of premature ovarian insufficiency. So far, more than 130 live births have been reported after transplantation of cryopreserved ovarian tissue, and almost all patients recovered their ovarian function after tissue reimplantation. Summary: This review aims to summarize the recent results described in the literature regarding human ovarian tissue cryopreservation in terms of methods and main results obtained so far. To cryopreserve human ovarian tissue, most studies describe a slow freezing/rapid thawing protocol, which is usually an adaptation of a protocol developed for sheep ovarian tissue. Since freezing has been shown to have a deleterious effect on ovarian stroma and granulosa cells, various research groups have been vitrifying ovarian tissue. Despite promising results, only 2 babies have been born after transplantation of vitrified/warmed ovarian tissue. Optimization of both cryopreservation strategies as well as thawing/warming protocols is therefore necessary to improve the survival of follicles in cryopreserved ovarian tissue. Key Messages: Human ovarian tissue cryopreservation has been successfully applied worldwide to preserve fertility in patients with malignant or nonmalignant pathologies that have a detrimental effect on fertility. Human ovarian tissue cryopreservation could also be applied as an alternative to postpone pregnancy or menopause in healthy women. Slow freezing and vitrification procedures have been applied to cryopreserve human ovarian tissue, but both alternatives require optimization.
Climatic variables can trigger physiological, biochemical, haematological and hormonal alterations that influence the maintenance of homeothermy and can affect production and productivity in sheep. ...Different mechanisms are responsible for tolerance to heat stress (HS) including coat and skin colour, body size, fat distribution, physiological reactions and not just coat type (hair/wool). This review looks at physical, physiological, molecular and genetic aspects of heat tolerance in sheep and how they affect hair and wool sheep. We propose that it is the adaptation to hot environments and not the type of coat (wool/hair) itself that determines the capacity of the resistance of the animal to HS, due to modifications in essential pathways such as energy metabolism, physiological responses and body size. When studied in similar environments, commercial wool breeds tend to show higher heat stress, but hair breeds tend not to differ from wool breeds that are adapted to hot environments.
•Within the same genetic group, white-coated animals are more tolerant.•Smaller animals are more tolerant than larger ones.•Wool animals from hot environments are more adapted than commercial wool animals.•Hair breeds that have undergone selection are less tolerant.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cryopreservation of ovarian tissue followed by transplantation represents a strategy to restore ovarian function and fertility. Stress from cryopreservation-thawing processes can lead to alterations ...and/or damage to mitochondrial structure and functionality. High resolution respirometry and histological analysis were used to evaluate the effect of cryopreservation and transplantation on ovarian tissue. Four different conditions were performed: Fresh non-transplanted tissue, Fresh transplanted tissue, Cryopreserved non-transplanted tissue and Cryopreserved transplanted tissue. All groups were able to respond to the substrates-uncoupler-inhibitor protocol. We found a dramatic decrease in general oxygen consumption in hemi-ovaries submitted to cryopreservation and/or transplantation. The effect of cryopreservation on mitochondrial metabolism was less intense than effect of transplantation, since the transplantation affected all of the mitochondrial states. A total of 2644 follicles were analyzed. Of these, 2198 were classified as morphologically normal. The percentage of morphologically normal follicles was significantly lower in the Cryopreserved transplanted group when compared to the Cryopreserved non-transplanted group and the Fresh transplanted group (p-value < 0.05). Despite decreased follicular viability and mitochondrial activity, the cryopreservation followed by transplantation of ovarian tissue proved feasible for attempts to restore ovarian function.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
STUDY QUESTION
Would a hydrogel with similar mechanical properties to the human ovarian cortex support preantral follicle development?
SUMMARY ANSWER
Yes, our tailored PEGylated fibrin ...hydrogel was shown to significantly improve follicle growth in vitro.
WHAT IS KNOWN ALREADY
One of the main challenges in developing an engineered ovary is to provide a 3D matrix that supports the follicle architecture and the interaction between granulosa cells and the oocyte as they are essential for folliculogenesis. Thanks to its biocompatibility and bioactivity, fibrin has been employed to fabricate a 3D matrix to encapsulate ovarian follicles. However, follicles lose their physical support within a few days owing to rapid fibrin degradation. Therefore, different strategies, including physical and chemical modifications, have been developed to enhance the stability of fibrin.
STUDY DESIGN, SIZE, DURATION
By developing a matrix made of a synthetic (polyethylene glycol: PEG) and natural polymer (fibrin), we aimed to overcome fibrin degradation by the chemical reaction of PEGylation and tailor a PEGylated fibrin hydrogel formulation with mechanical strength similar to the ovarian cortex in women of reproductive age. To this end, response surface methodology was employed to obtain a tailored formulation of PEGylated fibrin. This hydrogel was then tested to encapsulate and support isolated human preantral follicles in vitro.
PARTICIPANTS/MATERIALS, SETTING, METHODS
A PEGylated fibrin formulation was tailored using mathematical modeling software to mimic the mechanical properties of human ovarian tissue at reproductive age. Human preantral follicles were isolated from 11 patients of reproductive age and encapsulated in the tailored hydrogels, which were cultured in vitro for 4 or 7 days. Follicle survival and diameter were assessed on Days 1 and 7. Furthermore, the follicles were subjected to confocal microscopy to evaluate their growth (Ki67 staining) on Day 7 and analyze cell–cell communication (connexin 43 and transzonal projection staining) on Day 4.
MAIN RESULTS AND THE ROLE OF CHANCE
In this study, mathematical modeling was applied to achieve the biomechanically tailored PEGylated fibrin formulation by targeting the specific goal of 3178 ± 245 Pascal, Young’s modulus of ovarian cortical tissue in reproductive-age women. Our results demonstrated that the PEGylated fibrin hydrogel consisting of 39.06 mg/ml of PEGylated fibrinogen and 50.36 IU/ml of thrombin was the optimum condition with the desirability of 97.5%. This tailored hydrogel yielded a high follicle survival rate (83%) after 7 days of in vitro culture and supported its development up to the secondary stage. Follicle growth was confirmed by the presence of Ki67-positive granulosa cells on Day 7. Additionally, connexin 43 and Phalloidin staining indicated the retention of connections between granulosa cells and the oocyte.
LARGE SCALE DATA
N/A.
LIMITATIONS, REASONS FOR CAUTION
In this study, our tailored hydrogel was only tested in vitro, which is not the same as the physiological environment. It is crucial to conduct a study assessing the follicles following their encapsulation in the tailored hydrogel and transplantation, which will be the next step of our investigation.
WIDER IMPLICATIONS OF THE FINDINGS
The findings from this study introduced a suitable biomaterial similar to the ovarian cortex in reproductive-age women in terms of biomechanical properties for encapsulating human preantral follicles. This biomaterial allowed the radial growth of follicles and preserved their viability. Furthermore, PEGylation improved the stability of fibrin and the physical support of follicles.
STUDY FUNDING/COMPETING INTEREST(S)
This study was supported by grants from the Fondation Louvain (PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer). The authors declare no competing interests.
Is the optimal timing for administering erythropoietin to minimize ischaemic injury in ovarian tissue transplantation before ovary removal for cryopreservation and subsequent transplantation or after ...transplantation?
Thirty Swiss mice (nu/nu) were divided into three groups: treatment control group (n = 10); erythropoietin before harvesting group (EPO-BH) (n = 10) and erythropoietin after transplantation group (EPO-AT) (n = 10). Animals underwent bilateral ovariohysterectomy and their hemiovaries were cryopreserved by slow freezing. At the same time, previously cryopreserved hemiovaries were transplanted subcutaneously in the dorsal region. Erythropoietin (250 IU/kg) and sterile 0.9% saline solution were administered every 12/12 h over 5 consecutive days in the EPO-AT and EPO-BH groups, respectively.
Administration of erythropoietin in the EPO-AT group improved the viability of ovarian follicles, reducing degeneration and increasing the number of morphologically normal growing follicles at 14 days after transplantation compared with the EPO-BH group (P = 0.002). This group also showed higher percentages of proliferative follicles at 7 days after transplantation (P ≤ 0.03), increased blood vessel count (P ≤ 0.03) and greater tissue area occupied by blood vessels at days 7 and 14 after transplantation (P ≤ 0.03), compared with hormone administration before cryopreservation (EPO-BH group) and the treatment control group. Additionally, treatment with erythropoietin before or after transplantation reduced fibrotic areas at 7 days after transplantation (P = 0.004).
Erythropoietin treatment after transplantation reduced ischaemic damage in transplanted ovarian tissue, increased angiogenesis, maintenance of ovarian follicle proliferation and reduced fibrosis areas in the grafted tissue.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Even though sheep embryo cryopreservation is a commonly used procedure the survival and pregnancy outcomes can vary greatly. This study investigated whether cryopreservation was causing subtle ...changes in ultrastructure, mitochondrial activity or cytoskeletal integrity. Sheep embryos were either slow cooled in 1.5M EG (n=22), or vitrified in 20% EG+20% DMSO with 0.5M sucrose in Open Pulled Straws (OPS) (n=24). One hour after warming the cryopreserved embryos differed from control embryos in that they had no mitochondrial activity combined with cytoskeletal disorganization and large vesicles. Vitrified embryos also showed many points of cytoskeleton disruption. Ultrastructural alterations resulting from actin filaments disorganization were observed in both cryopreserved groups. This includes areas presenting no cytoplasmic organelles, Golgi complex located far from the nucleus and a decrease of specialized intercellular junctions. Additionally, large vesicles were observed in vitrified morulae and early blastocysts. The alterations after cryopreservation were proportional to embryo quality as assessed using the stereomicroscope. Even in the absence of mitochondrial activity, grade I and II cryopreserved embryos contained mitochondria with normal ultrastructure. Embryos classified as grade I or II in the stereomicroscope revealed mild ultrastructural alterations, meaning that this tool is efficient to evaluate embryos after cryopreservation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Could a modification in the ovarian tissue freezing protocol improve follicle survival after cryopreservation and xenotransplantation?
Ovarian tissue was used from 13 adult patients, frozen either ...with our original protocol, or a modified version involving a higher concentration of dimethyl sulphoxide (DMSO), larger volume of cryopreservation solution and lower seeding temperature. After thawing, the ovarian fragments were xenotransplanted to six mice with severe combined immunodeficiency (SCID) for 3 weeks.
The proportion of primordial follicles decreased, and the proportion of growing follicles increased significantly (all P < 0.01) after cryopreservation and xenografting compared with fresh controls for both protocols. Follicle density, development, ultrastructure and function were similar between treatments.
This study showed that, although the higher DMSO concentration did not improve survival of preantral follicles, it did not seem to induce any major toxicity in the follicle population either.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
This work presents a long-term follow-up (300 days) of rats after a single intravenous injection of DMSA-coated magnetite nanoparticles (DMSA-MNP). The animals were systematically evaluated by ...hematological, biochemical, and ultrasound examinations, monitoring the same animal over time. In addition, oxidative stress evaluation, DMSA-MNP biodistribution, computerized tomography for ex vivo organs, and histopathology analysis were performed at the end of the experiment period. Overall, DMSA-MNP administration did not cause serious damage to the rats’ health over the course of 300 days post-administration. All animals presented hematological parameters within the normal limits, and no alterations on serum creatinine, urea, ALT, and AST were related to DMSA-MNP administration. Liver and spleen showed no important alterations in any of the examinations. The kidneys of treated animals displayed intermittent pelvis dilation at ultrasound analysis, but without damage to the organ parenchyma after 300 days. The lungs of treated animals presented a light interalveolar septal thickening, but the animals did not present any clinical respiratory symptom. Nanoparticles were not detected in the vital organs of treated animals 300 days after administration. This work represents the first assessment of the long-term effects of DMSA-MNP and goes a step further on the safety of its use for biomedical applications.
Many feline species are currently threatened with extinction. Therefore, germplasm bank establishment has become imperative. However, cryoinjury and ischemia-reperfusion injury pose significant ...obstacles to both cryopreservation and xenotransplantation. In this regard, erythropoietin (Epo) represents a potential alternative strategy due to its properties. This study aimed to assess the incubation of domestic cat ovarian tissue in Epo, both before and after cryopreservation, and investigate its effectiveness in promoting revascularization following xenotransplantation. Sixteen ovaries from 8 healthy cats were sliced following elective bilateral ovariohysterectomy (OHE). Subsequently, 8 fragments measuring 3 mm³ each were obtained from the cortical region of each ovary. The fragments were allocated into 3 treatment groups: Cryo group, fragments were cryopreserved, thawed and immediately transplanted; Cryo + Epo group, fragments were first cryopreserved in nitrogen, thawed, incubated in Epo (100 IU) for 2h and transplanted; and the Epo + Cryo group, in which fragments were first incubated in Epo (100 IU) for 2h, cryopreserved, thawed and immediately transplanted. The fragments were then xenotransplanted into the dorsal subcutaneous region of ovariectomized female nude mice and retrieved at 7, 14, 21, and 28 days post-transplantation. The results indicated that Epo effectively enhanced follicular survival, preservation of viability, and tissue revascularization. The Epo + Cryo group displayed better revascularization rates on D14 and D21 post-transplantation and an increase in primordial and growing follicles on D28, the Cryo + Epo group exhibited significantly more follicles on D14 and D21, with fewer degenerated follicles.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Alterations to DNA methylation have been attributed to in vitro culture and may affect normal embryo development. We chose to analyze DNA methylation reprogramming in the rabbit which, of the species ...with delayed transcriptional activation of the embryonic genome, allows easy comparisons between in vivo-developed (IVD) and in vitro-cultured (IVC) embryos. In this species, variations in DNA methylation had not previously been quantified, even in IVD embryos. IVD and IVC embryos were recovered at the 2, 4, 8 and 16-cell, morula and blastocyst stages. Immunostaining for 5-methyl-cytidine and normalization of the quantity of methylated DNA vs. the total DNA content were then performed. Our quantitative results evidenced DNA demethylation during pre-implantation development in both IVD and IVC embryos, but with different kinetics. Demethylation occurred earlier in vitro than in vivo between the 2 and 8-cell stages in IVC embryos, reaching its lowest level, while it only started at the 4-cell stage and ended at the 16-cell stage in IVD embryos. We also showed that an absence of serum from the culture medium significantly altered the degree of DNA demethylation. Finally, at the blastocyst stage, ICM was more methylated than the trophectoderm in all cases. Despite a morphological delay observed in in vitro cultured blastocysts, the difference in DNA methylation between ICM and trophectoderm cells appeared at the same time post-fertilization in IVD and IVC embryos, which may reflect another difference in the dynamics of DNA methylation during blastocyst formation. Our data thus clearly establish an effect of embryonic environment on DNA methylation reprogramming during pre-implantation development in a non-rodent species.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
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