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16 pages, 1758 KiB

Open AccessArticle

Optimized Recovery of Immature Germ Cells after Prepubertal Testicular Tissue Digestion and Multi-Step Differential Plating: A Step towards Fertility Restoration with Cancer-Cell-Contaminated Tissue

by Sven De Windt, Dhoha Kourta, Marc Kanbar and Christine Wyns

Int. J. Mol. Sci. 2024, 25(1), 521; https://doi.org/10.3390/ijms25010521 - 30 Dec 2023

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Abstract

Undifferentiated germ cells, including the spermatogonial stem cell subpopulation required for fertility restoration using human immature testicular tissue (ITT), are difficult to recover as they do not easily adhere to plastics. Due to the scarcity of human ITT for research, we used neonatal [...] Read more.

Undifferentiated germ cells, including the spermatogonial stem cell subpopulation required for fertility restoration using human immature testicular tissue (ITT), are difficult to recover as they do not easily adhere to plastics. Due to the scarcity of human ITT for research, we used neonatal porcine ITT. Strategies for maximizing germ cell recovery, including a comparison of two enzymatic digestion protocols (P1 and P2) of ITT fragment sizes (4 mm³ and 8 mm³⁾ and multi-step differential plating were explored. Cellular viability and yield, as well as numbers and proportions of DDX4+ germ cells, were assessed before incubating the cell suspensions overnight on uncoated plastics. Adherent cells were processed for immunocytochemistry (ICC) and floating cells were further incubated for three days on Poly-D-Lysine-coated plastics. Germ cell yield and cell types using ICC for SOX9, DDX4, ACTA2 and CYP19A1 were assessed at each step of the multi-step differential plating. Directly after digestion, cell suspensions contained >92% viable cells and 4.51% DDX4+ germ cells. Pooled results for fragment sizes revealed that the majority of DDX4+ cells adhere to uncoated plastics (P1; 82.36% vs. P2; 58.24%). Further incubation on Poly-D-Lysine-coated plastics increased germ cell recovery (4.80 ± 11.32 vs. 1.90 ± 2.07 DDX4+ germ cells/mm², respectively for P1 and P2). The total proportion of DDX4+ germ cells after the complete multi-step differential plating was 3.12%. These results highlight a reduced proportion and number of germ cells lost when compared to data reported with other methods, suggesting that multi-step differential plating should be considered for optimization of immature germ cell recovery. While Poly-D-Lysine-coating increased the proportions of recovered germ cells by 16.18% (P1) and 28.98% (P2), future studies should now focus on less cell stress-inducing enzymatic digestion protocols to maximize the chances of fertility restoration with low amounts of cryo-banked human ITT. Full article

(This article belongs to the Special Issue Fertility Preservation and Restoration Options with Female and Male Gonadal Tissue or Cells)

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19 pages, 8907 KiB

Open AccessArticle

Overview of Gene Expression Dynamics during Human Oogenesis/Folliculogenesis

by Bastien Ducreux, Lucile Ferreux, Catherine Patrat and Patricia Fauque

Int. J. Mol. Sci. 2024, 25(1), 33; https://doi.org/10.3390/ijms25010033 - 19 Dec 2023

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Abstract

The oocyte transcriptome follows a tightly controlled dynamic that leads the oocyte to grow and mature. This succession of distinct transcriptional states determines embryonic development prior to embryonic genome activation. However, these oocyte maternal mRNA regulatory events have yet to be decoded in [...] Read more.

The oocyte transcriptome follows a tightly controlled dynamic that leads the oocyte to grow and mature. This succession of distinct transcriptional states determines embryonic development prior to embryonic genome activation. However, these oocyte maternal mRNA regulatory events have yet to be decoded in humans. We reanalyzed human single-oocyte RNA-seq datasets previously published in the literature to decrypt the transcriptomic reshuffles ensuring that the oocyte is fully competent. We applied trajectory analysis (pseudotime) and a meta-analysis and uncovered the fundamental transcriptomic requirements of the oocyte at any moment of oogenesis until reaching the metaphase II stage (MII). We identified a bunch of genes showing significant variation in expression from primordial-to-antral follicle oocyte development and characterized their temporal regulation and their biological relevance. We also revealed the selective regulation of specific transcripts during the germinal vesicle-to-MII transition. Transcripts associated with energy production and mitochondrial functions were extensively downregulated, while those associated with cytoplasmic translation, histone modification, meiotic processes, and RNA processes were conserved. From the genes identified in this study, some appeared as sensitive to environmental factors such as maternal age, polycystic ovary syndrome, cryoconservation, and in vitro maturation. In the future, the atlas of transcriptomic changes described in this study will enable more precise identification of the transcripts responsible for follicular growth and oocyte maturation failures. Full article

(This article belongs to the Special Issue Fertility Preservation and Restoration Options with Female and Male Gonadal Tissue or Cells)

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13 pages, 1411 KiB

Open AccessArticle

Reducing 3D Hydrogel Stiffness, Addition of Oestradiol in a Physiological Concentration and Increasing FSH Concentration Improve In Vitro Growth of Murine Preantral Follicles

by Mengxue Zheng, Jesús Cadenas, Susanne Elisabeth Pors, Tasnim Esa, Stine Gry Kristensen, Linn Salto Mamsen, Cristina Subiran Adrados and Claus Yding Andersen

Int. J. Mol. Sci. 2023, 24(15), 12499; https://doi.org/10.3390/ijms241512499 - 06 Aug 2023

Cited by 1 | Viewed by 952

Abstract

This study aimed to optimise culture conditions for murine preantral follicles to improve their growth and survival. Preantral follicles (diameter 100–130 µm) were isolated from prepubertal NMRI mice and individually cultured within alginate beads for 12 days. Three conditions were evaluated: (1) follicle [...] Read more.

This study aimed to optimise culture conditions for murine preantral follicles to improve their growth and survival. Preantral follicles (diameter 100–130 µm) were isolated from prepubertal NMRI mice and individually cultured within alginate beads for 12 days. Three conditions were evaluated: (1) follicle re-encapsulation on day 6 of culture-reducing alginate concentration (0.5% to 0.25% w/v), (2) the presence of oestradiol (E₂), and (3) increased follicle-stimulating hormone (FSH) concentration in the culture medium (from 10 to 100 mIU/mL FSH). Follicle morphology and growth, as well as anti-Müllerian hormone (AMH) production, were evaluated. From day 8, re-embedded follicles had a larger average diameter compared to follicles without alginate re-encapsulation (0.5% and 0.25% groups, p < 0.05). Oestradiol (1 µM) had a significantly positive effect on the mean follicular diameter and antrum formation (p < 0.001). Moreover, follicles cultured with 100 mIU/mL FSH showed faster growth (p < 0.05) and significantly higher antrum formation (p < 0.05) compared to the low FSH group. Nevertheless, AMH production was not affected by any of the culture conditions. In conclusion, the growth and survival of mouse preantral follicles during a 12-day period were improved by altering the alginate concentration midways during culture and adding E₂ and FSH to the culture medium. Full article

(This article belongs to the Special Issue Fertility Preservation and Restoration Options with Female and Male Gonadal Tissue or Cells)

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Review

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13 pages, 1844 KiB

Open AccessReview

In Vitro Growth of Human Follicles: Current and Future Perspectives

by Clara Malo, Sara Oliván, Ignacio Ochoa and Ariella Shikanov

Int. J. Mol. Sci. 2024, 25(3), 1510; https://doi.org/10.3390/ijms25031510 - 26 Jan 2024

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Abstract

Ovarian tissue cryopreservation is gaining importance as a successful method to restore fertility to girls and young women at high risk of sterility. However, there are concerns regarding the safety of transplantation after ovarian tissue cryopreservation due to the high risk of reintroducing [...] Read more.

Ovarian tissue cryopreservation is gaining importance as a successful method to restore fertility to girls and young women at high risk of sterility. However, there are concerns regarding the safety of transplantation after ovarian tissue cryopreservation due to the high risk of reintroducing cancer cells and causing disease recurrence. In these cases, the development of culture systems that support oocyte development from the primordial follicle stage is required. Notable achievements have been reached in human follicle in vitro growth in the past decade. Currently, systems for the in vitro culture of ovarian tissue are based on two-dimensional substrates that do not support the survival of follicles or recapitulate the mechanical heterogenicity in the mammalian ovary. Recognition of the importance of special arrangements between cells has spurred research in three-dimensional culture systems, and the provision of a precise culture system that maximizes the diffusion of nutrients and gases through the follicles has raised interest in advanced biomimetic models. The current review critically examines various culture systems employed for the in vitro development of follicles, with a particular focus on solutions utilizing Organ-on-a-Chip (OOC) technology. The emphasis on OOC technology underscores its role as a promising avenue in ensuring the successful cultivation and maintenance of follicular structures during the culture period. Full article

(This article belongs to the Special Issue Fertility Preservation and Restoration Options with Female and Male Gonadal Tissue or Cells)

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22 pages, 1999 KiB

Open AccessReview

The Role of microRNA in Spermatogenesis: Is There a Place for Fertility Preservation Innovation?

by Charlotte Klees, Chrysanthi Alexandri, Isabelle Demeestere and Pascale Lybaert

Int. J. Mol. Sci. 2024, 25(1), 460; https://doi.org/10.3390/ijms25010460 - 29 Dec 2023

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Abstract

Oncological treatments have dramatically improved over the last decade, and as a result, survival rates for cancer patients have also improved. Quality of life, including concerns about fertility, has become a major focus for both oncologists and patients. While oncologic treatments are often [...] Read more.

Oncological treatments have dramatically improved over the last decade, and as a result, survival rates for cancer patients have also improved. Quality of life, including concerns about fertility, has become a major focus for both oncologists and patients. While oncologic treatments are often highly effective at suppressing neoplastic growth, they are frequently associated with severe gonadotoxicity, leading to infertility. For male patients, the therapeutic option to preserve fertility is semen cryopreservation. In prepubertal patients, immature testicular tissue can be sampled and stored to allow post-cure transplantation of the tissue, immature germ cells, or in vitro spermatogenesis. However, experimental techniques have not yet been proven effective for restoring sperm production for these patients. MicroRNAs (miRNAs) have emerged as promising molecular markers and therapeutic tools in various diseases. These small regulatory RNAs possess the unique characteristic of having multiple gene targets. MiRNA-based therapeutics can, therefore, be used to modulate the expression of different genes involved in signaling pathways dysregulated by changes in the physiological environment (disease, temperature, ex vivo culture, pharmacological agents). This review discusses the possible role of miRNA as an innovative treatment option in male fertility preservation–restoration strategies and describes the diverse applications where these new therapeutic tools could serve as fertility protection agents. Full article

(This article belongs to the Special Issue Fertility Preservation and Restoration Options with Female and Male Gonadal Tissue or Cells)

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20 pages, 1281 KiB

Open AccessReview

BRCA Mutations and Fertility Preservation

by Joana Dias Nunes, Isabelle Demeestere and Melody Devos

Int. J. Mol. Sci. 2024, 25(1), 204; https://doi.org/10.3390/ijms25010204 - 22 Dec 2023

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Abstract

Hereditary cancers mostly affect the adolescent and young adult population (AYA) at reproductive age. Mutations in BReast CAncer (BRCA) genes are responsible for the majority of cases of hereditary breast and ovarian cancer. BRCA1 and BRCA2 act as tumor suppressor genes [...] Read more.

Hereditary cancers mostly affect the adolescent and young adult population (AYA) at reproductive age. Mutations in BReast CAncer (BRCA) genes are responsible for the majority of cases of hereditary breast and ovarian cancer. BRCA1 and BRCA2 act as tumor suppressor genes as they are key regulators of DNA repair through homologous recombination. Evidence of the accumulation of DNA double-strand break has been reported in aging oocytes, while BRCA expression decreases, leading to the hypothesis that BRCA mutation may impact fertility. Moreover, patients exposed to anticancer treatments are at higher risk of fertility-related issues, and BRCA mutations could exacerbate the treatment-induced depletion of the ovarian reserve. In this review, we summarized the functions of both genes and reported the current knowledge on the impact of BRCA mutations on ovarian ageing, premature ovarian insufficiency, female fertility preservation strategies and insights about male infertility. Altogether, this review provides relevant up-to-date information on the impact of BRCA1/2 mutations on fertility. Notably, BRCA-mutated patients should be adequately counselled for fertility preservation strategies, considering their higher sensitivity to chemotherapy gonadotoxic effects. Full article

(This article belongs to the Special Issue Fertility Preservation and Restoration Options with Female and Male Gonadal Tissue or Cells)

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21 pages, 2168 KiB

Open AccessReview

A Journey to Reach the Ovary Using Next-Generation Technologies

by Thuy Truong An Nguyen and Isabelle Demeestere

Int. J. Mol. Sci. 2023, 24(23), 16593; https://doi.org/10.3390/ijms242316593 - 22 Nov 2023

Viewed by 775

Abstract

Although effective in terms of the chances of future live birth, the current methods for fertility preservation, such as oocyte, embryo, or ovarian tissue cryopreservation, cannot be offered to all cancer patients in all clinical contexts. Expanding options for fertility preservation is crucial [...] Read more.

Although effective in terms of the chances of future live birth, the current methods for fertility preservation, such as oocyte, embryo, or ovarian tissue cryopreservation, cannot be offered to all cancer patients in all clinical contexts. Expanding options for fertility preservation is crucial to addressing the need to encompass all situations. One emerging strategy is pharmacoprotection, a non-invasive approach that has the potential to fill existing gaps in fertility preservation. In addition to the identification of the most effective therapeutic agents, the potential for off-target effects remains one of the main limitations of this strategy for clinical application, particularly when healthy ovarian tissue is targeted. This review focuses on the advances in pharmacoprotective approaches and the challenge of targeting the ovaries to deliver these agents. The unique properties of gold nanoparticles (AuNPs) make them an attractive candidate for this purpose. We discuss how AuNPs meet many of the requirements for an ideal drug delivery system, as well as the existing limitations that have hindered the progression of AuNP research into more clinical trials. Additionally, the review highlights microRNA (miRNA) therapy as a next-generation approach to address the issues of fertility preservation and discusses the obstacles that currently impede its clinical availability. Full article

(This article belongs to the Special Issue Fertility Preservation and Restoration Options with Female and Male Gonadal Tissue or Cells)

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18 pages, 4140 KiB

Open AccessReview

Overactivation or Apoptosis: Which Mechanisms Affect Chemotherapy-Induced Ovarian Reserve Depletion?

by Oren Kashi and Dror Meirow

Int. J. Mol. Sci. 2023, 24(22), 16291; https://doi.org/10.3390/ijms242216291 - 14 Nov 2023

Cited by 2 | Viewed by 1014

Abstract

Dormant primordial follicles (PMF), which constitute the ovarian reserve, are recruited continuously into the cohort of growing follicles in the ovary throughout female reproductive life. Gonadotoxic chemotherapy was shown to diminish the ovarian reserve pool, to destroy growing follicle population, and to cause [...] Read more.

Dormant primordial follicles (PMF), which constitute the ovarian reserve, are recruited continuously into the cohort of growing follicles in the ovary throughout female reproductive life. Gonadotoxic chemotherapy was shown to diminish the ovarian reserve pool, to destroy growing follicle population, and to cause premature ovarian insufficiency (POI). Three primary mechanisms have been proposed to account for this chemotherapy-induced PMF depletion: either indirectly via over-recruitment of PMF, by stromal damage, or through direct toxicity effects on PMF. Preventative pharmacological agents intervening in these ovotoxic mechanisms may be ideal candidates for fertility preservation (FP). This manuscript reviews the mechanisms that disrupt follicle dormancy causing depletion of the ovarian reserve. It describes the most widely studied experimental inhibitors that have been deployed in attempts to counteract these affects and prevent follicle depletion. Full article

(This article belongs to the Special Issue Fertility Preservation and Restoration Options with Female and Male Gonadal Tissue or Cells)

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