Automatic Evaluation for Bioengineering of Human Artificial Ovary: A Model for Fertility Preservation for Prepubertal Female Patients with a Malignant Tumor

Wang, Wanxue; Pei, Cheng; Isachenko, Evgenia; Zhou, Yang; Wang, Mengying; Rahimi, Gohar; Liu, Wensheng; Mallmann, Peter; Isachenko, V.

doi:10.3390/ijms232012419

Cited by 6 publications

(6 citation statements)

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“…The microtomography approach could offer a unique opportunity for resolving ovary structures or for evaluating the quality of ovarian fragments before and after cryopreservation, the latter in the context of autotransplantation protocols [ 32 ] of ovarian tissues for adolescent oncological patients. A better knowledge of the three-dimensional structure of human ovaries could also favor the setting of the artificial ovary, which is the most challenging and risky perspective in reproductive medicine [ 33 , 34 , 35 , 36 , 37 ].…”

Section: Introductionmentioning

confidence: 99%

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy

Gianoncelli

Sena

Kourousias

et al. 2023

IJMS

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An improved understanding of an ovary’s structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin’s solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues.

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Section: Introductionmentioning

confidence: 99%

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy

Gianoncelli

Sena

Kourousias

et al. 2023

IJMS

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“…Cryopreservation of ovarian tissue was performed according to our previously published protocol [42,43,44]. On the day of freezing, pieces of ovarian tissue were placed at room temperature in 20 mL freezing medium composed of basal medium supplemented with 6% dimethyl sulfoxide, 6% ethylene glycol, and 0.15 M sucrose.…”

Section: Methodsmentioning

confidence: 99%

“…In the laboratory, ovarian medulla was partially separated from cortex using forceps and a no. 22 scalpel [42,43,44].…”

Section: Methodsmentioning

confidence: 99%

“…On the day of freezing, pieces of ovarian tissue were placed at room temperature in 20 mL freezing medium composed of basal medium supplemented with 6% dimethyl sulfoxide, 6% ethylene glycol, and 0.15 M sucrose. Then, pieces were put into standard 5 mL cryo-vials (Thermo Fisher Scienti c, Rochester, NY, USA), which were previously lled by freezing medium and frozen in IceCube 14S freezer Thawing of tissue [42,43,44] was achieved by holding the vial for 30s at room temperature, followed by immersion in a 100°C (boiling) water for 60 s, and expelling the contents of the vial into the solution for removal of cryoprotectants. The exposure time in the boiling water was visually controlled by the presence of ice in the medium; as soon as the ice reached 2 to 1 mm apex, the vial was removed from the boiling water, at which point the nal temperature of the medium was between 4°C and 10°C.…”

Section: Methodsmentioning

confidence: 99%

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Assessment of molecular and morphological dynamics during long-time in vitro cultivation of cryopreserved human ovarian tissue: risk of genetic alterations

Wang,

Todorov,

Isachenko

et al. 2024

Preprint

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Cryopreservation of human ovarian tissue is a technology for protection of reproductive potential in patients undergoing aggressive anticancer treatments. This technology includes the following stages: saturation by permeable cryoprotectants, freezing, thawing, removal of cryoprotectants, and tissues in vitro or in situ culture. The aim of our investigations was the evaluation of genetic risks and molecular alterations in human ovarian tissue during in vitro culture. Ovarian tissue was frozen in 6% ethylene glycol and 6% dimethyl sulfoxide with speed of cooling 0.3°C/min and thawed at 100°C. After removal of cryoprotectants tissue fragments were in vitro cultured with the soluble extract of basement membrane protein (Matrigel) 3-D culture system for 7 days. Morphological and functional assessments were conducted using microscopic observation and RNA-Seq. Comparative analysis of tissue morphology before and after culture was performed with bioinformatics for gene expression and variant analysis, including functional annotation and study of protein-protein interaction. DNA and RNA analyses after cultivation indicated a rise in gene fusion and alternative splicing events, potentially affecting gene expression and cellular functions. It was concluded that long-time in vitro culture of human ovarian tissue results in substantial changes in its morphology and genetic alteration.

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“…The construction of artificial ovary was performed according to our previously published protocol, with fibrin encapsulation and follicle isolation using Tumor Dissociate Enzyme (TDE) [ 34 ].…”

Section: Methodsmentioning

confidence: 99%

RNA Transcripts in Human Ovarian Cells: Two-Time Cryopreservation Does Not Affect Developmental Potential

Zhou

Wang

Todorov

et al. 2023

IJMS

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Sometimes, for medical reasons, when a frozen tissue has already thawed, an operation by re-transplantation may be cancelled, and ovarian tissues should be re-frozen for transplantation next time. Research about the repeated cryopreservation of ovarian cells is rarely reported. It has been published that there is no difference in the follicle densities, proportions of proliferation of early preantral follicles, appearance of atretic follicles, or ultrastructural quality of frozen-thawed and re-frozen-rethawed tissue. However, the molecular mechanisms of a repeated cryopreservation effect on the developmental potential of ovarian cells are unknown. The aim of our experiments was to investigate the effect of re-freezing and re-thawing ovarian tissue on gene expression, gene function annotation, and protein–protein interactions. The morphological and biological activity of primordial, primary, and secondary follicles, aimed at using these follicles for the formation of artificial ovaries, was also detected. Second-generation mRNA sequencing technology with a high throughput and accuracy was adopted to determine the different transcriptome profiles in the cells of four groups: one-time cryopreserved (frozen and thawed) cells (Group 1), two-time cryopreserved (re-frozen and re-thawed after first cryopreservation) cells (Group 2), one-time cryopreserved (frozen and thawed) and in vitro cultured cells (Group 3), and two times cryopreserved (re-frozen and re-thawed after first cryopreservation) and in vitro cultured cells (Group 4). Some minor changes in the primordial, primary, and secondary follicles in terms of the morphology and biological activity were detected, and finally, the availability of these follicles for the formation of artificial ovaries was explored. It was established that during cryopreservation, the CEBPB/CYP19A1 pathway may be involved in regulating estrogen activity and CD44 is crucial for the development of ovarian cells. An analysis of gene expression in cryopreserved ovarian cells indicates that two-time (repeated) cryopreservation does not significantly affect the developmental potential of these cells. For medical reasons, when ovarian tissue is thawed but cannot be transplanted, it can be immediately re-frozen again.

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Automatic Evaluation for Bioengineering of Human Artificial Ovary: A Model for Fertility Preservation for Prepubertal Female Patients with a Malignant Tumor

Cited by 6 publications

References 58 publications

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy

Assessment of molecular and morphological dynamics during long-time in vitro cultivation of cryopreserved human ovarian tissue: risk of genetic alterations

RNA Transcripts in Human Ovarian Cells: Two-Time Cryopreservation Does Not Affect Developmental Potential

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