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- Tsiartas, Panos, et al.
(author)
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P–459 Ex vivo perfusion of whole ewe ovaries with follicular maturation for up to seven days: towards the development of an alternative fertility preservation method
- 2021
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In: Human Reproduction Vol 36 Issue Supplement 1. - : Oxford University Press (OUP). - 0268-1161.
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Conference paper (other academic/artistic)abstract
- Abstract Study question To develop an alternative fertility preservation method for young female cancer patients based on an ex vivo perfusion of whole ovaries serving as a platform for future ovarian stimulation studies. Summary answer It is possible to maintain viable follicles and to retrieve oocytes after ex vivo perfusion of ewe ovaries for up to 7 days. What is known already Some progress has been made in terms of follicular growth and the isolation of mature oocytes in vitro. However, full development, from early follicular stages to a viable offspring, has only been described in rodent models. The complex events controlling follicular expansion and the long time required for folliculogenesis and oocyte maturity in large mammalian species creating challenges and limitations for in vitro studies. Ex vivo perfusion of a whole ovary could potentially be a solution by exploiting the intact ovarian architecture to support folliculogenesis and oocyte maturation. Study design, size, duration Thirty-one ewe ovaries were divided into 4 groups and ex vivo perfused in a bioreactor. Group 1 (n=14) perfusion for 48hours with no hormone supplementation; Group 2 (n=4) perfusion 96–101hours with follicle stimulating hormone (FSH); Group 3 (n=3) perfusion 120–168hours with human menopausal gonadotropin (hMG); Group 4 (n=10) perfusion 72–144hours with hMG. Participants/materials, setting, methods Ewe ovaries from sexually mature ewes were ex vivo perfused in a bioreactor under normothermic conditions for up to 7 days (max total 168hours). Histomorphological, immunohistochemical, hormonal and biochemical analyses were performed to assess ovarian structure and viability after cold ischemia and after perfusion which was subsequently compared to control ovaries. Main results and the role of chance The perfused ovaries in group 2 and 3 showed no significant differences in follicular density, viability and oocyte quality after ischemia and perfusion compared to control ovaries. Estradiol and progesterone levels did not increase during the perfusion. The perfused ovaries in group 1 and 4 showed a significant decrease in the ovarian reserve and oocyte quality. In total, 16 GV-MI oocytes were retrieved from groups 3 and 4. Limitations, reasons for caution 1. Ovaries were retrieved from ewes of unknown cycle and reproductive history. 2. The perfusion medium was changed after 24hours from perfusion start to remove detrimental metabolites and this could affect the measured concentrations of hormones and metabolites in the perfusion medium. Wider implications of the findings: These results pave the way to propose ex vivo perfusion as a good platform for fertility preservation studies on whole mammalian and human ovaries to retrieve fully mature oocytes. Trial registration number Not applicable
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- Brännström, Mats, 1958, et al.
(author)
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Livebirth after uterus transplantation.
- 2015
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In: Lancet. - 1474-547X. ; 385:9968, s. 607-616
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Journal article (peer-reviewed)abstract
- Uterus transplantation is the first available treatment for absolute uterine infertility, which is caused by absence of the uterus or the presence of a non-functional uterus. Eleven human uterus transplantation attempts have been done worldwide but no livebirth has yet been reported.
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- Diaz-Garcia, César, et al.
(author)
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Ovarian cortex transplantation in the baboon: comparison of four different intra-abdominal transplantation sites.
- 2011
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In: Human reproduction. - : Oxford University Press (OUP). - 1460-2350 .- 0268-1161. ; 26:12, s. 3303-3311
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Journal article (peer-reviewed)abstract
- BACKGROUNDSeveral sites have been used for ovarian cortex transplantation (OCT) in humans. The present study was designed to evaluate different intra-abdominal transplantation sites in the baboon to gain further knowledge about alternative transplantation sites in a human setting.METHODSAutologous fresh OCTs were performed in 12 baboons (Papio anubis). Four different sites were tested: the free portion of the omentum (OMF), the portion of the omentum adjacent to the spleen (OMS), the pouch of Douglas (D) and the pelvic wall on the psoas muscle (PW). Cortex survival, follicle density, cyclicity and hormonal levels were compared between the different sites, 3 and 6 months after transplantation.RESULTSMacroscopically, antral follicles were only found in the OMS and OMF locations, which also showed a higher proportion of follicle-containing cortex at light microscopy (OMF 71.4%, OMS 83.3% versus PW 58.8% and D 40%, P< 0.05). Higher densities of primordial [OMF: 3.54 (0-13.18) follicles/grid, OMS: 3.85 (0-8.53), PW: 0 (0-13.25), D 0 (0-1.33), P< 0.05] and primary follicles [OMF: 3.54 (0-18.52), OMS: 3.85 (0-1), PW: 0 (0-4.58), D 0 (0-0.25), P< 0.05] was also found in the omental locations.CONCLUSIONSOmental locations provide a better site, in terms of follicle survival, for intra-abdominal OCT in the baboon compared with the pelvic wall and the D.
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- Milenkovic, Milan, 1966
(author)
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Experimental studies on ovarian cryopreservation and transplantation
- 2011
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Doctoral thesis (other academic/artistic)abstract
- Around 8% of all cancer victims are below 40 years of age and the survival after cancer treatment during childhood and reproductive years has increased considerably to be around 80% today. The clinical field of fertility preservation has emerged to enable cancer patients that are treated with potentially gonadotoxic chemotherapy-radiotherapy during childhood or reproductive ages, to preserve their fertility. In prepubertal girls and women of reproductive age, where immediate IVF is not an option, ovarian cryopreservation and later re-transplantation is today the only fertility option. Today 13 live births have been reported worldwide after ovarian cortex cryopreservation and avascular re-transplantation some years after the woman has been declared disease-free. However, the effectiveness of the method of ovarian cryopreservation is low. This thesis investigates several models to be used in improvement of ovarian cryopreservation protocols, including whole ovary cryopreservation, and in addition studies different transplantation sites for avascular cortex transplantation in a non human primate species. The ovine ovarian ovary was used to examine a slow freezing method with the cryoprotectant dimethylsulphoxide (DMSO). Sheep ovaries were cryopreserved in liquid nitrogen and after thawing several viability tests were used. It was shown that the presence of DMSO was advantages for steroid and cyclic AMP output during in vitro perfusion and in cultured ovarian cells. Light microscopy showed well preserved tissue in the DMSO group after perfusion and a higher density of small follicles as compared to ovaries cryopreserved without of CPA. This study shows that the sheep ovary is a suitable method for further studies on whole ovary cryopreservation, including comparisons of different cryopreservation protocols. The human postmenopausal ovary was evaluated as a tool for further cryopreservation research in the human. Naturally cryopreservation of human ovaries is aiming at preserving premenopausal ovarian ovaries or ovarian tissue. However, this study on post menopausal ovary shows that the aged ovary can be used as a valuable tool for the research, with special emphasises on the function of the stroma and the vascularity. The study showed that human post menopausal ovaries could be effectively cryopreserved in DMSO and that the stroma secreted androgens during in vitro perfusion. Electron microscopy showed a well-preserved morphology in these human ovaries. The rodents are commonly used in reproductive physiology research and there is a large knowledge about the ovarian function and folliculogenesis in these species. The present study developed a technique for cannulation of the vasculature to the rat ovary and cryopreservation of the rat ovary by either vitrifiction or slow freezing. The cryoprotectant used was DMSO in high and low concentration. The result of the study indicated that a whole rat ovary can successfully be cryopreserved and that the DMSO concentration of 1.5 M is optimal when evaluating a secretion of steroids and viability of primordial follicles after cryopreservation. Cryopreserved ovarian cortex tissue can either be transplanted back to an orthotopic or a heterotopic site. The live births reported in the human have all been from the orthotopic site but there are no comparative studies of different transplantation site in primate species. This study used the baboon as a model to compare different heterotopic intraabdominal transplantation sites. It was found that transplantation of the omentum was of advantage compared to transplantation to the pelvic wall or the pouch of Douglas. After a lag phase of 2-3 months the freshly transplanted ovarian tissue showed signs of growth of a large follicle and cyclicity of the animals. In summary, the study presents several usable models for viability tests after whole ovary cryopreservation. These models can be explored in further research in the area. In a primate species, the omentum has been found a suitable heterotopic ovarian site. This finding can be used also in a human situation where orthotopic ovarian cortex transplantation is impossible because of anatomical or pathophysiological reasons.
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| 8. |
- Milenkovic, Milan, 1966, et al.
(author)
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The human postmenopausal ovary as a tool for evaluation of cryopreservation protocols towards whole ovary cryopreservation.
- 2011
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In: Journal of assisted reproduction and genetics. - : Springer Science and Business Media LLC. - 1573-7330 .- 1058-0468.
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Journal article (peer-reviewed)abstract
- PURPOSE: Cryopreservation of a complete ovary may be a future method for fertility preservation in cancer patients. Difficulties exist in cryopreservation of the relatively large ovarian tissue mass. This study evaluates whether a human postmenopausal ovary can be used, as a complement to animal models, in studies of this research field. METHODS: Postmenopausal human ovaries (n=10) were isolated and flushed through ovarian arteries with either the cryoprotectant dimethylsulphoxide or Ringer-Acetate, followed by slow freezing. After thawing, production of androgens during in vitro perfusion and morphology (light/electron microscopy) were assessed. RESULTS: The dimethylsulphoxide-cryopreserved ovaries showed larger secretion of androgens during perfusion than Ringer Acetate-cryopreserved ovaries. Light microscopy showed well preserved morphology in both groups. Electron microscopy revealed normal appearance of stroma and vessels in the dimethylsulphoxide group. CONCLUSIONS: The study demonstrates the potential to use the postmenopausal human ovary for further studies aiming at optimizing cryopreservation protocols, with special reference to ovarian vascularity and stroma.
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| 9. |
- Milenkovic, Milan, 1966, et al.
(author)
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Viability and function of the cryopreserved whole rat ovary: comparison between slow-freezing and vitrification.
- 2012
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In: Fertility and sterility. - : Elsevier BV. - 1556-5653 .- 0015-0282. ; 97:5, s. 1176-1182
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Journal article (peer-reviewed)abstract
- OBJECTIVE: To investigate four different protocols for cryopreservation of the whole rat ovary with intact vasculature to evaluate whether differences exist in post-thawing viability of the ovary after either vitrification or slow freezing. DESIGN: Experimental study. SETTING: Obstetrics and gynecology department. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Ovaries were isolated with the vascular tree intact up to the bifurcation of the abdominal aorta and were subsequently cannulated. The ovaries were flushed with increasing concentrations of the cryoprotectant dimethyl sulfoxide (DMSO) to either 1.5 or 7 M. The ovaries underwent cryopreservation by vitrification or passive slow freezing. MAIN OUTCOME MEASURE(S): After thawing, the ovaries were subjected to neutral red viability staining to assess the density of viable small follicles and for long-term (48 hours) incubation evaluation of steroid secretion, histology, and apoptosis assay. RESULT(S): The follicular viability was decreased in both vitrification groups and in the slow-freezing group with the high concentration of DMSO, as compared with fresh controls. Estradiol levels in the incubation medium followed the same pattern. Light microscopy revealed well-preserved morphology in all groups after 48 hours' incubation. Apoptosis was increased in both vitrified and cryopreserved ovaries. CONCLUSION(S): We have developed a new method that can be used in basic studies to improve cryopreservation protocols. Our initial findings suggest that a moderate concentration of the cryoprotectant DMSO is superior to a high DMSO concentration for both vitrification and slow freezing.
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