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. 2022 Oct 17;23(20):12419.
doi: 10.3390/ijms232012419.

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

Wanxue Wang  1 , Cheng Pei  1 , Evgenia Isachenko  1 , Yang Zhou  1 , Mengying Wang  1 , Gohar Rahimi  1 , Wensheng Liu  1   2 , Peter Mallmann  1 , Vladimir Isachenko  1
Affiliations

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

Wanxue Wang et al. Int J Mol Sci. .

Abstract

Introduction: The in vitro culture of primordial follicles is the only available option for preserving fertility in prepubertal girls with malignant tumors. The cultivation of primordial follicles in scaffolds as artificial ovaries is a promising approach for this.

Methods: Dissociated follicles were placed into an artificial ovarian scaffold composed of fibrinogen and thrombin. The follicles were cultured in a dish dedicated to live cell imaging and observed for growth using immunofluorescence and development via optical microscopy. The morphology of the follicles in the scaffold was three-dimensionally reconstructed using the Imaris software. Growth and development were also quantified.

Results: The morphology of artificial ovaries began to degrade over time. Within approximately 7 days, primordial follicles were activated and grew into secondary follicles. A comparison of optical and confocal microscopy results revealed the superior detection of live cells using confocal microscopy. The three-dimensional reconstruction of the confocal microscopy data enabled the automatic enumeration and evaluation of the overall morphology of many follicles.

Conclusions: The novel artificial ovary-enabled primordial follicles to enter the growth cycle after activation and grow into secondary follicles. The use of a fibrin scaffold as a carrier preserves the developmental potential of primordial germ cells and is a potentially effective method for preserving fertility in prepubertal girls.

Keywords: artificial ovary; fertility preservation; imaris reconstruction; prepubertal cancer; primordial follicles.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Vitality and morphology evaluations of isolated follicles. (A) Freshly isolated follicles in an enzyme solution before manipulated enrichment using a capillary. The follicles were stained with neutral red. (B) Follicles manipulated and enriched using a 125 µm capillary in MOPS solution. (C) Optical microscopy image of isolated primordial follicles stained with neutral red. (D) Hoechst-stained follicles observed via fluorescence microscopy. (E) Calcein-AM-stained follicles after isolation observed via fluorescence microscopy. (F) Merged image of the images shown in panels (D,E). (G) Hoechst staining of an isolated secondary follicle. (H) Merged confocal photography of Hoechst and Calcein-AM-stained secondary follicle. (I) Spherical screenshot of the isolated secondary follicle.
Figure 2
Figure 2
Photography of follicles in fibrin scaffolds. (A) Fibrinogen–thrombin complex including follicles in the unsolidified state. (B,C) General view of an artificial ovary on day 0 (B) and day 7 with a dissociated scaffold (C). (D) Optimal microscopy image of artificial ovary on day 0. (E,F) Viable primordial follicle in fibrin on day 0 (E) and day 3 (F). (G) Dead primary follicle on day 7. (H) Viable primordial follicle on day 7. (I) Viable secondary and primordial follicles in fibrin on day 3. (J) Dying secondary follicle in fibrin on day 7. (K) Viable secondary follicle in fibrin on day 3. (L) Viable secondary follicle in fibrin on day 7.
Figure 3
Figure 3
Automatic calculation and analysis system for the human artificial ovary. (A) Three-dimensional reconstruction of artificial ovaries imaged using Imaris 9.0 software with RedDot staining. (B) Distribution of follicles for analytical counting after image denoising. (C) Interface and general parameter settings of automatic counting and analysis system. (D) After automatic counting, the system generated a data file in txt format.
Figure 4
Figure 4
Construction of the artificial ovary from tissue to scaffold.
See this image and copyright information in PMC

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