Ovarian tissue cryopreservation can help women who are undergoing cancer treatment, autoimmune diseases, or other treatments with radiation or/and aggressive chemotherapy, to preserve their ovarian tissue for function in future (1, 2). The key of this technology is a surgical extraction of ovarian tissue using laparoscopy, freezing and storage them in liquid nitrogen (3, 4). The main purpose of cryopreservation of ovarian tissue before anti-cancer treatment is preservation of primordial follicles, which after anti-cancer treatment and thawing can be used for auto-transplantation (5).

In fact, anti-cancer therapy may damage the ovarian tissue and lead to decreased or complete loss of fertility (6, 7). In the same time, cryopreservation of this tissue and auto-transplantation can increase the chances of patients to be pregnant (8). However, the success of the ovarian tissue auto-transplantation is depended from various factors: quantity and quality of follicles in this tissue, quality of tissue freezing and thawing as well as an age of patient (9, 10). In addition, this technology may also be limited by financial and ethical considerations for patients (11, 12).

In vitro culture of ovarian tissue is a technique of cultivating of this tissue under specific culture conditions (13). This technology can be used to study issues related to ovarian development, reproductive physiology, and reproductive toxicology. Cryopreservation includes the following stages: saturation by permeable cryoprotectants, freezing, thawing, removal of cryoprotectants, and in vitro or in situ culture. The last stage of cryopreservation (in vitro culture) is aimed to evaluate a quality of whole process of cryopreservation (14, 15). Once the ovarian tissues are obtained, it should be immediately placed in a nutrient solution to maintain the cells viability. Methods of handling of ovarian tissue for in vitro culture include removing of surrounding tissues (medulla) and blood vessels as well as dividing of cells into slices or pieces. The processed tissues should be placed in a cell culture medium. Special culture conditions, such as appropriate oxygen levels, hormone concentrations in the nutrient solution, and pH values, can be achieved by adjusting of gas and composition of the culture medium in incubators for cell culture (16).

Recent years can be characterized by increasing of attention to the molecular mechanisms of ovarian tissue in vitro culture. It was found that hormone concentrations at in vitro culture has a significant impact on the growth and development of ovarian tissue. Growth factors and cytokines can promote proliferation and differentiation of ovarian tissue. Extracellular matrix is a complex structure outside of cells, playing a crucial role in cell growth and differentiation (17).

It can be summarized that the molecular mechanisms of ovarian tissue in vitro culture are very complex, requiring a comprehensive consideration of various factors, including hormones, growth factors, extracellular matrix, and cellular metabolic processes such as autophagy and apoptosis (18).

The aim of our investigations was the evaluation of genetic risks and molecular alterations in human ovarian tissue during in vitro culture.

Except where otherwise stated, all chemicals were obtained from Sigma (Sigma Chemical Co., St. Louis, MO, USA). The primary experimental procedure of our experiments is shoved in Figure 1 .

Morphology of fresh fragments of ovarian tissue ( Figure 2A ) after seven days of in vitro culture, was changed ( Figures 2B-F ). It appears in a dense fibrotic capsule as well as in slightly decreased volume. The originally sharp edges of the ovarian cortical pieces have become rounded and are no longer angular.

Figures 2B, E demonstrate different degrees of fibrosis occurring in ovarian cortical slices after in vitro culture. Cortical slices have a higher degree of fibrosis, more follicular necrosis, compressed living space of the follicles and affecting development of mature follicles ( Figure 2E ). It was noted activated primordial follicles and cubic primary follicles with granulosa cells entering the growth and development trajectory, as well as some secondary follicles with multiple layers of granulosa cells and follicular membrane cells ( Figure 2C ). It was also detected necrotic degeneration of primordial follicles and the morphology and distribution of interstitial cells within fibroses tissue masses, characterized by the disordered arrangement of interstitial cells, necrosis of follicles, and flattened granulosa cells ( Figure 2F ).

It was detected the variable splicing classification and proportion for all samples, with each group of samples showing broadly similar data ( Figures 3A-E ). However, variable splicing occurred in all groups, and there was no significant difference in the types and proportions of variable splicing between two groups. After in vitro culture, the frequency of SE (skipped exon) decreased, while the frequency of RI (retained intron) increased ( Figures 3A-E ).

Differences in types and proportions of variable splicing may affect disease progression and prognosis. Different splicing events may lead to various gene expression. Exon skipping in genes such as TUBB6, FGFR1, cAMP-dependent protein kinase inhibitor gamma (PKIG), and METTL5 was observed, which could lead to the development of cancer or affect a normal function of nervous system ( Figures 3A-E ).

Alternative Splicing (AS) analysis in the Gene Ontology (GO) covers cellular and metabolic processes, biological regulation, and response to stimulus. In the Cellular Component category, it includes cell, cell part, organelle, organelle part, and membrane. In the Molecular Function category, it mainly covers: binding, catalytic activity, transcription regulator activity, molecular function regulation, and structural molecular activity ( Figure 4A ).

Functional Single Nucleotide Polymorphism (SNP)/Insertion-Deletion (InDel) mutation sites are primarily enriched in Human Papillomavirus (HPV) infection, Phosphoinositide 3-kinase (PI3K)-Akt signaling pathway, Mitogen-Activated Protein Kinase (MAPK) signaling pathway, Ras signaling pathway, Huntington disease, thermogenesis ( Figure 4B ).

Alternative Splicing is mainly enriched in the following Kyoto Encyclopedia of Genes and Genomes (KEGG) terms: metabolic pathways, lysosomes, endocytosis, ubiquitin mediated proteolysis as well as RNA transport ( Figure 4C ).

It is shown that the suspected deleterious events of SNVs are distributed on each chromosome after in vitro culture. Among them, there are 2075 mutations in the coding sequence and 572 suspected deleterious events in the exon region ( Figure 5 ). They are mainly distributed in the lipid metabolism pathway, including the activation and expression of the PPARA gene, and lipid particle organization. It is also detected that there are 1552 high-confidence deleterious sites and 502 low-confidence deleterious sites in the number of gene mutations after in vitro culture; there are 1309 high-confidence tolerated sites and 228 low-confidence tolerated sites ( Figure 5 ).

Fragments just after operation (“fresh”) were not presented in experiments because for transplantation were used only cryopreserved (frozen and thawed) ovarian tissue. In that way the effectiveness of in vitro culture of “fresh” cells is not actual. We need to do in vitro culture of cells after cryopreservation to answer two questions:

We can answer the first question without in vitro culture, when a piece of ovarian tissue is fixed and assessed immediately after surgery. In this case, we can see the morphology of this tissue, primarily the morphology of the follicles. But the fact is that for future transplantation we only have cryopreserved tissue that will be transplanted immediately after thawing, without in vitro culturing for one week.

We can confidently state that the optimal development of transplanted ovarian tissue will be observed in the patient’s “native” organism, i.e. after thawing and transplantation of such tissue. In fact, more optimal conditions for the further development of transplanted cells than in organism simply cannot exist. The aim of in vitro culture technology is to ensure that this technology maximally “copies” the parameters of the external environment after transplantation of these cells to the organism of patient.

We have used the only partial technological separation of medulla from cortex during preparation of ovarian fragments for cryopreservation. It is because the following processes occur after thawing and re-transplantation of ovarian tissue: 1) the cortex with follicles does not have blood vessels and the nutrition of these follicles occurs according to a diffuse principle; 2) for the integration of transplanted tissue, vessels are needed for formation of anastomoses; 3) blood vessels are contained in the medulla in the form of small vessels and stem cells partially differentiated into vessels; 4) complete removal of the medulla is a direct path to necrosis of the transplanted tissue.

For cryopreservation we used relatively large fragments of tissue. It is because cryobiology indicates that for slow (conventional, programmable) freezing, the shape and size of tissue fragments are not only insignificant, but do not play any role at all. Any fragment of ovarian tissue up to 4 mm thick (we use to 1.5 mm) will be completely saturated with permeable cryoprotectants (especially DMSO) within 5 min. from the beginning of equilibration, and this will happen regardless of the area of this fragment.

In our experiments, for thawing we have used 100°C water bath. For demonstration of this technological parameter the following descriptions can be used: 1) when thawing in boiling water, it is used a tampon that isolates the tissue fragment from the bottom of cryovial, which guarantees no overheating ( Figure 1 ); 2) the temperature of the tissue fragment after thawing does not rise above +14°C. For illustration: the cryo-vial after being removed from the boiling water after 1 sec. feels cold. This technique is used because a tenet of classical cryobiology states that all types of cells and tissues, regardless of cryopreservation method, must be thawed as quickly as possible.

In fact, fibroblasts, which are in the cortical layer of fragments, have more growth energy than follicular cells.

When a tissue fragment is attached to the bottom of a Petri dish, fibroblasts form a monolayer and their growth energy increases. This in turn leads to the suppression of other cells. Frequent replacement of liquid in a Petri dish is accompanied by frequent movement of the fragment along the bottom of the Petri dish and fibroblasts do not form a monolayer.

Upon microscopic examination of HE-stained sections, it was observed that the encapsulation of tissues cultured in vitro is dense, featuring a fibrous capsule that could influence the growth and development of follicles at the cortical cutting edges. Nonetheless, this phenomenon does not seem to hinder follicular development in the central inner parts. This prompts the question: which scenario yields better growth, tissues with dense fibrous encapsulation or those without encapsulation, especially at the edges of a gel scaffold? To address this inquiry, further tests and investigations are warranted in the future.

Recent studies have elucidated significant genetic and epigenetic alterations in gametes and embryos during in vitro culture, which hold importance for human assisted reproduction. El Hajj and Haaf highlighted profound epigenetic changes (25), while Kuijk et al. reported increased mutation rates in in vitro cultured stem cells, including pluripotent and adult types, primarily attributed to oxidative stress (26). These changes often result in genomic alterations in cells, raising concerns regarding their application in regenerative medicine. Consequently, further research is imperative to comprehend and mitigate these changes, with a specific focus on mutation sites and epigenetic modifications during embryonic development.

Alternative splicing, a process occurring during gene transcription, enables a single gene to undergo splicing, generating multiple distinct mRNA variants, each encoding a unique protein (27). Traditionally, splice isoforms have been categorized based on exon quantity and sequential arrangement (28). However, contemporary research has shifted toward alternative methodologies for splice isoform classification, incorporating criteria such as the nature of splicing events, functional dynamics of splicing factors, and metrics related to ribosomal stalling (29). Beyond splicing factors, the spliceosomal machinery is intricately regulated by an array of transcriptional regulatory elements, non-coding RNAs, and a diverse spectrum of molecular entities (30).

The pathogenesis of numerous diseases has been associated with the production of specific splice isoforms, with aberrant expression profiles of certain splicing factors implicated in various pathologies (31). For instance, SF3B1, a crucial constituent in the RNA splicing cascade, when mutated, disrupts normal splicing mechanisms, resulting in aberrant mRNA and protein synthesis, a phenomenon commonly observed in malignancies such as chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS) (32).

In the realm of therapeutics, small molecule agents like Pladienolide B and its analogs, which target SF3B1 and analogous splicing factors, are being rigorously investigated for their potential efficacy in treating malignancies characterized by SF3B1 mutations (33, 34). Additionally, in the context of non-small cell lung cancer (NSCLC), the use of small molecule inhibitors like H3B-8800, targeting splicing events associated with mutations in splicing factors such as SRSF2, SF3B1, and U2AF1, represents a novel strategy to disrupt the splicing apparatus and attenuate the proliferation of neoplastic cells (35, 36).

Gene fusion refers to the merging of two or more genes under certain conditions, resulting in a new protein-coding sequence. The mechanisms of gene fusion can be categorized into chromosomal structural variations and transcription/splicing abnormalities, primarily including three types: translocation, involving the transfer of chromosome fragments between chromosomes. Insertion, wherein a chromosome fragment is inserted into a new gap on the same or another chromosome and Inversion, characterized by the 180-degree rotation of a chromosome fragment. For example, EML4-ALK is generated by inversion, serving as one of the driver genes in non-small cell lung cancer (37, 38).

In this study, gene fusion events notably increased after in vitro culture, particularly intrachromosomal fusions. This augmentation could be attributed to chromosomal structural changes induced by cellular stress and mechanical instability resulting from fibrosis. However, due to the limited nature of this stress, it did not lead to a higher occurrence of gene fusion events between different chromosomes. Transcriptomic fusion can significantly impact gene expression levels and cellular function. Additionally, related studies have indicated that the rise in gene fusion events in ovarian tissue due to in vitro culture also occurs in gamete and zygote in vitro cultures (39).

Various in vitro culture techniques in assisted reproductive technology, including but not limited to IVM and blastocyst culture, extend the duration of human reproductive cell growth and development outside the body, thereby increasing the frequency of gene fusion events and subsequently elevating the lifetime cancer risk for post-birth offspring (40). Transcript fusion may alter the expression levels of the fused genes and/or the structure and function of the encoded proteins, thereby affecting cell functions. A deeper understanding of the regulatory mechanisms of fused genes can unveil more profound insights into gene expression and regulation. Transcript fusion is a significant outcome in transcriptome sequencing, crucial for enhancing our understanding of gene regulatory mechanisms, diagnosing and treating diseases, and refining transcriptome annotations (41).

InDels (Insertions-Deletions) and SNPs (Single Nucleotide Polymorphisms) are common forms of genetic variation. An InDel refers to an insertion or deletion of one or more bases in the genetic sequence, while a SNP involves the substitution of a single base with another. InDels are more likely to occur than SNPs due to the absence of a point mutation requirement, which is necessary for SNPs. Moreover, InDels generally have a more pronounced impact than SNPs as they can cause relatively larger frame shifts, altering the gene’s reading frame and subsequently changing the protein sequence and structure. SNPs are more suitable for broad distribution analysis across the genome and population genetic studies due to their involvement in single nucleotide variations. InDel and SNP variations are often investigated in tissues with a higher genetic predisposition to diseases such as cancer, neurological disorders, and cardiovascular diseases (42, 43). In the results, the distribution of InDels and SNPs in the in vitro cultured group significantly differed from that in the control group, exhibiting a higher frequency of occurrence. This suggests an increased likelihood of congenital defects (44, 45).

This study acknowledges inherent limitations stemming from the scarcity of human gamete samples (46). Given their invaluable nature, the limited quantity of available samples for research remains a persistent bottleneck in this field. Additionally, the academic composition of the research team presented a missed opportunity in integrating extensive bioinformatics, impeding the potential development of comprehensive machine learning algorithms and the establishment of relevant databases (47). However, with the anticipated convergence of multidisciplinary fields and the evolution of interdisciplinary sciences, these opportunities are expected to materialize in the future.

In conclusion, long-time in vitro culture of human ovarian tissue results in substantial changes in its morphology and genetic alteration.