Spatiotemporal Dynamics of Human Ovarian Cortex Transcriptome Following Vitrification and Thawing: Insights into the FOS/AP-1 Pathway

Spatiotemporal Dynamics of Human Ovarian Cortex Transcriptome Following Vitrification and Thawing: Insights into the FOS/AP-1 Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Spatiotemporal Dynamics of Human Ovarian Cortex Transcriptome Following Vitrification and Thawing: Insights into the FOS/AP-1 Pathway Wen Li, Fanghao Guo, Di Sun, Haixia Ding, Yanquan Li, Mengxi Guo, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6312954/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ovary is a vital and dynamic reproductive organ. Ovarian tissue cryopreservation (OTC) plays vital role in female fertility preservation, especially for adolescent female cancer patients. Yet, the sensitive cell populations and cryoinjury molecular mechanisms underlying cryopreservation remain elusive. This study employs single-cell RNA sequencing and spatial transcriptomics to dissect the impacts of temperature stress and cryoprotectant toxicity on the human ovarian cortex. The spatiotemporal molecular characteristics of eight ovarian cell types following vitrification-thawing had been comprehensively characterized. Granulosa, perivascular and stromal cells are identified as most sensitive cell types to OTC procedure. Further analysis using Smart-seq2 on oocytes highlights alterations in“cell cycle” and “DNA methylation” pathways. Notably, the FOS/AP-1 pathway emerges as a crucial response factor to stress and toxicity during cryopreservation. Inhibition of this pathway with T-5224 mitigates vascular damage and reduces apoptosis in vitrification-thawed ovaries. These findings provide insight into the spatiotemporal dynamics during ovarian vitrification and thawing, aiding in prioritizing therapeutic strategies for pre- and post-cryopreservation interventions. Biological sciences/Physiology/Reproductive biology/Reproductive disorders Biological sciences/Computational biology and bioinformatics/Cellular signalling networks Health sciences/Biomarkers/Predictive markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Over the past two decades, advancements in cancer therapy have resulted in significant improvements in survival rates. 1 And increasing numbers of young girls and women < 45 years of age can now be cured of their disease. 2 However, treatments such as chemotherapy (especially alkylating agents), radiotherapy, and surgery can lead to primary ovarian insufficiency (POI), highlighting the urgent need to address quality of life post-cancer remission. 3 , 4 , 5 , 6 Therefore, fertility preservation should be considered prior to any gonadotoxic treatment, particularly for adolescents and women of reproductive age. 7 Embryo and oocyte cryopreservation are well-established fertility preservation techniques, whereas ovarian tissue cryopreservation (OTC) is an emerging and promising approach. 8 OTC for fertility purposes has been performed over 25 years. Since the first report of a live birth after thawing and auto-transplantation of ovarian cortex in 2004, 9 the procedure has gained ground and is now accepted and performed all over the world. 6 , 10 , 11 , 12 As of 2022, more than 10,000 women worldwide have undergone ovarian tissue cryopreservation, and more than 200 live births have been achieved. 13 , 14 More specifically, OTC and subsequent transplantation is the sole option that enables both restoration of fertility and resumption of ovarian endocrine function, avoiding the morbidity associated with POI. 4 , 15 , 16 , 17 Clinical meta-analysis shows that over 90% of women regain ovarian function after OTC-transplantation (OTT). 18 However, clinical outcomes of ovarian transplantation post-cryopreservation are significantly worse than those from fresh ovaries, indicating adverse effects associated with the cryopreservation process. 19 For in vitro fertilization (IVF) after tissue cryopreservation and transplantation, the live birth rate (LBR) is only 21%, which is 41% for post-embryo cryopreservation. 18 This discrepancy between high ovarian function recovery rates and lower live birth rates warrants further detailed investigation. Therefore, to improve follicle development quality post-transplantation, it is crucial to elucidate the mechanisms through which temperature stress and cryoprotectant toxicity damage ovarian cells. 20 , 21 The exploration of ovarian cryoinjury faces multiple challenges, mainly from the following aspects: Firstly, human ovary is a complex structure consisting of numerous heterogeneous cell types including stromal cells, granulosa cells, endothelial cells, immune cell, smooth muscle cells and oocytes. 22 , 23 , 24 , 25 , 26 And current studies indicate that the ovary, due to its diverse cellular composition, is highly responsive to external environmental factors, chemical drug stimuli, and aging processes. 27 , 28 Secondly, vitrification is emerging as an efficient strategy for ovarian cryopreservation at the current stage, 29 which had been widely used for oocytes, embryos preservation. 16 , 30 , 31 , 32 Vitrification-thawing inevitably leads to alterations in the physiological metabolism of various cell types within the ovary. Up to date, preliminary investigation has been carried out to explore the impact of vitrification on ovarian tissue via morphological staining and transmission electron microscopy. 33 , 34 High-throughput omics analyses have been conducted on oocytes and follicle complexes post-vitrification; however, multi-omics analyses have not yet to be performed on the ovarian cortex post vitrification-thawing. 35 , 36 The impact of vitrification-thawing treatment on ovarian cells, especially at the single-cell transcriptome and Spatiotemporal levels, remains elusive. With advances of single-cell RNA sequencing technique, characterizing cell changes in ovarian tissue under physiological or pathological conditions has been carried out currently 23 , 24 , 25 , 26 , 28 , 37 , 38 . Smart-seq2 based single-cell sequencing strategy enables detection of abundant genes per cell, especially for oocyte. 39 , 40 Spatial enhanced resolution omics sequencing (Stereo-seq) is a novel and skillful spatial RNA sequencing method with nanoscale sensitivity and a large field of view. 41 In this study, we aim to explore the cellular spatiotemporal changes and key regulatory genes involved in human ovarian cortex cryoinjury. We present a spatiotemporal atlas that systematically describes the spatial archetypes and cellular heterogeneity post human ovarian vitrification-thawing procedure. Detailly, granulosa, smooth muscle/perivascular cells and stromal cells had been identified as vitrification sensitive cell types. And the in-situ effects of vitrification-thawing on oocytes within ovary had also been explored via smart-seq2 strategy. Further, a potentially crucial role of FOS/AP-1 pathway in ovarian vitrification-thawed procedure had been tentatively explored. In all, our data provide valuable inspiration for the mechanism and potential therapeutic targets to vitrification-thawed stress and cryoprotectant toxicity induced ovarian cryoinjury. 2. Results 2.1 scRNA-seq profiling and spatial location of human ovarian cortex cells. The overview of the study design for the human ovarian cryoinjury spatiotemporal atlas is shown in Figure 1A. Each of the three ovarian cortexes from gender reassignment surgery patients (GRPs) was divided into both fresh and cryopreserved groups (cryo). The age information for the three groups is as follows: 29 years for Fresh & Cyro-1; 34 years for Fresh & Cyro-2; 35 years for Fresh & Cyro-3. Following by collagenase IV digestion, single-cell RNA sequencing (scRNA-seq) was applied to 27,185 fresh ovarian cells. Post standard ovary vitrification-thawing procedure, the cryopreserved group had been digested and conducted with scRNA-seq on 25,480 cells. Since large cells cannot be wrapped in microdroplets (dimeter >40um), Smart-seq2 strategy had been applied to oocyte in the ovarian cortex. Cells with mitochondrial gene expression exceeding 25% of the total unique molecular identifiers (UMIs) were excluded (Figure S1B). And the total cellular RNA content and number of expressed genes showed no significant difference among the three groups (Figure S1A and C). More detailed information can be found in the supplementary Major Resources Table 1. Using the Uniform Manifold Approximation and Projection (UMAP) algorithm for the nonlinear dimensionality reduction analysis, we identified eight distinct cell types based on specific markers (Figure 1B and Figure S1D). These include granulosa cells (GCs; AMH+), oocytes (DDX4+), stromal cells (SCs; STAR+), smooth muscle/perivascular cells (PCs; MACM+), blood endothelial cells (BECs; VWF+), lymphatic endothelial cells (LECs; PROX1+), macrophage (M; CD68+) and natural killer T cells (NKTs; CD3D+). The expression levels of markers for different cell types in the ovarian cortex were visualized using Featureplot(Figure 1C). Additionally, the biological function of each cell cluster were analyzed through Gene Ontology (GO) analysis of differentially expressed genes (DEGs) (Figure 1D), uncovering unique characteristics of these ovarian subclusters. For example, GO terms specific to oocytes, such as ‘‘meiotic cell cycle’’ and ‘‘gamete generation’’, were identified. GO terms including ‘‘regulation of follicle-stimulating hormone secretion’’ and ‘‘reproductive system development’’ were enriched in GCs. Meanwhile, ‘‘Cytoplasmic translation’’ and ‘‘extracellular matrix organization’’ were prominent in SCs, indicating their role in providing nutritional and structural support for ovary. Subsequently, stereo-seq based single-cell spatial transcriptome sequencing strategy (ST-seq) were conducted on the fresh and cryopreserved groups. Specifically, the sections were obtained separately from the Fresh-1 and Cryo-2 groups. The sequencing depth of ST-seq reached 1000 genes/dot (Fresh: 934 genes/pot, Vitrification-thawing: 841 genes/pot) when bin = 50, indicating high data quality (Figure S1E and F). Utilizing the scRNA-seq atlas, factor analysis was conducted to infer the probable single-cell composition of each spot, effectively mapping all scRNA-seq clusters. We identified eight celltypes, including GCs, two subtypes of SCs (SCs1 marked by COL1A2+ and SCs2 marked by GPR78+), PCs, smooth muscle cells (SMCs), ECs, theca-interstitial cells, oocytes and red blood cells (RBCs) (Figure 1E-G). SpatialFeaturePlot visualizes the in situ spatial expression patterns of subgroup marker gene in the ovarian cortex (Figure S1G). Additionally, a heatmap displayed the top cluster markers, revealing significant overlap between cell cluster markers identified by both scRNA-seq and Stereo-seq (Figure 1H). Overall, spatial cell mapping elucidated the distribution of clusters predicted by scRNA-seq within the human ovarian cortex. 2.2 Transcriptional Shifts in Ovarian Cortex: Comparing Slow Frozen, Vitrified, and Fresh Conditions at the Single-Cell Level Comparing the differences between slow freezing—a method clinically approved and tested over decades for ovarian tissue cryopreservation—and vitrification is crucial. Due to the difficulty in obtaining human ovarian samples and ethical constraints, we are currently unable to perform single-cell library sequencing analysis on slow-frozen human ovarian cortex samples. Fortunately, researchers have conducted single-cell sequencing on slow-frozen human ovarian cortex tissue (M Wagner et.al. Nat Commun , 2020) 24 , allowing for direct integration and comparison with our data. The UMAP analysis results indicate that the eight major subgroups are consistently present across all three sample groups without notable absence (Figure 2A). In-depth analysis of differentially expressed genes revealed distinct variations among the groups (Figure 2B). Specifically, comparisons between slow-freezing and vitrification highlighted significant differences in genes associated with focal adhesion, the p53 signaling pathway, and the MAPK pathway (Figure 2C). DNA damage and apoptosis score analyses indicated that slow freezing resulted in slightly less negative outcomes compared to vitrification (Figure 2D and E). Furthermore, cellular crosstalk analyses conducted via CellPhoneDB and CellChat revealed that slow freezing significantly reduced intercellular interactions compared to vitrification (Figure 2G and H). We further explored the changes in Wnt and Notch pathways in the ligand-receptor interactions between oocytes and other cells in slow frozen, vitrified, and fresh groups. Dotplot results indicate that the Wnt-FZD and JAG-NOTCH pathways are nearly absent in the slow_frozen group (Figure 2I). These pathways are closely related to cell proliferation, which may also explain the more severe apoptosis observed in the slow frozen group. Overall we may draw a conclusion as follows: 1) We identified genes differentially expressed between slow-frozen and vitrified-thawed ovarian cortex, notably in intercellular communication and the MAPK signaling pathway. 2) Comparative analysis indicates less oocyte damage in slow freezing compared to vitrification. 3) Cell-cell communication analysis showed greater disruption in slow freezing than in vitrification, particularly affecting oocyte interactions. 2.3 Cell-specific changes following ovarian vitrification-thawing’s transcriptional programs A comparative analysis of gene expression patterns across various ovarian cell types in the fresh and vitrification-thawed (cryo) groups had been conducted (Figure 3A). UMAP plot showed seven ovarian cell clusters in each sample (Figure 3B). Quantitative analysis revealed that the proportion of PCs increased significantly in all three replicate samples, with statistical significance, while SCs showed the opposite trend (Figure 3C and D). To evaluate the reliability of Figure 2C, the publicly available single-cell datasets of GSE255690 (GEO No.) 28 was integrated with our dataset. As expected, most ovarian subcluster from the same tissue sources were clustered together (Figure S2A and B). Integration and cell proportion analysis also showed a significant upregulation in stromal cell subclusters post vitrification-thawing (Figure S2C). Additionally, eight cell types were further validated in the integrated single-cell data using specific cell markers (Figure S2D). Differentially expressed genes (DEGs; top 50 with |avg_logFC| > 0.25 and P adj< 0.05) between the fresh and vitrification-thawed groups in at least one cell type were identified and shown in heatmap (Figure 3E). Pathway analysis demonstrated that the DEGs are mainly involved in “focal adhesion” and “apoptosis” (Figure 3F). CellPhoneDB is a bioinformatics toolkit designed to infer cell-cell communication by analyzing the combined expression of multi-subunit ligand-receptor complexes. 42 Post vitrification-thawing process, there was a significant reduction in ligand-receptor interactions between ovarian cells, notably between granulosa and other cell types (Figure S3A). Further analysis of individual subpopulations revealed that certain DEGs, such as complement 3 (C3), were unique to specific subpopulations, predominantly observed in stromal and granulosa cells (Figure S3B). Which indicates that different cell types exhibited heterogeneous responses to the vitrification-thawing process. Based on the above results, apoptosis and extracellular matrix organization may play an important role in human ovarian cryoinjury. Post vitrification-thawing procedure, the signaling pathway score for focal adhesion decreased while apoptosis increased in the majority of ovarian cells (Figure 3G). Pathway analysis of spatial gene expression data further confirmed heightened enrichment of apoptosis and a diminished activity of focal adhesion pathway in the vitrification-thawed ovarian cortex (Figure 3H). Masson’s trichrome staining revealed that fresh ovarian tissue had a higher collagen content compared with vitrification-thawing ones (Figure 3I and Figure S3G). And transmission electron microscopy analysis showed noticeable shrinkage in oocytes and surrounding cells, confirming a reduction in intercellular communication (Figure 3J). Additionally, TdT-mediated dUTP nick end labeling (TUNEL staining) further demonstrated a significant increase in the number of cells undergoing apoptosis following vitrification-thawing treatment (Figure 3K). In all, our findings suggest that mesenchymal disruption and increased apoptosis are predominant features of cryoinjury in human ovary cortex. However, specific cell types within the ovarian cortex may exhibit distinct characteristics associated with cryoinjury. 2.4 Vitrification and thawing altered the gene expression profiles of ovarian oocytes in situ . Dormant primordial follicles, situated within the approximately 1 mm thick ovarian cortex (Figure S1E), are the primary focus of vitrification in ovarian tissue. This prompts significant interest in investigating the impact of vitrification on the physiological metabolism of oocytes within the ovarian cortex. We sequenced 115 oocyte, and 5 oocyte sequencing data had been deleted due to abnormally high expression of mitochondrial genes (>30%). Finally, single-cell sequencing data from 110 oocytes at various stages had been analyzed (66 oocytes from Vitrification group, 44 from Fresh group). During the process of oocytes isolation from the dissociated ovarian tissue, we can’t identify what stage of these oocytes just under light filed with microscopy. Fortunately, the expanding availability of transcriptomic data enables the identification of genes specifically expressed in unique stage of the oocyte cycle. 43, 44 Vlnplot analysis revealed high expression levels of LMOD3, RPS4X, and FIGLA in these oocytes, suggesting that they were predominantly at the primordial or primary stage (Figure S4A). AddModuleScore and Doheatmap analysis show genes specific to primary oocytes at the primordial stage are enriched in Cluster 1, while those marking later stages are upregulated in Cluster 2 (Figure S4B and C). This indicates that the majority of the oocytes used for smart-seq were derived from primordial follicles (~75 oocytes). Integrated analysis demonstrated substantial overlap in the expression profiles of oocytes from fresh and vitrified ovarian cortex, indicating generally identical (Figure 4A). Benefiting from the extensive sequencing depth of Smart-seq2 strategy, we identified a number of differentially expressed genes as shown in the heatmap analysis (Figure 4C). Notably, meiosis-related genes, namely, ZP family (including ZP2, ZP3 and ZP4), 45 OOEP and OOSP4B, were considerably downregulated (Figure 4B and C). In addition, cell cycle-related genes, including CKS1B and TUBB8, 46, 47 were significantly downregulated in vitrification ovarian oocytes, whereas CDCA7L and RAB18 were upregulated (Figure 4C). Intriguingly, gene related to heat response, namely, DNAJA2 (Hsp40), was also significantly upregulated in cryo-rewarming ovarian oocytes (Figure 4B). To validate the bioinformatic analysis claims, multiplex immunofluorescence staining had been employed. The results indicate an increased proportion of oocytes co-expressing SOHLH2 and DDX4 (Figure 4F and H), consistent with the analysis results of Featureplot (Figure 4G). And the upregulated gene SOHLH2 had been reported as primary ovarian insufficiency candidate genes. 48, 49 This prompts us to further explore the impact of changes in the expression levels of these genes on later embryonic development and physiological metabolism of offspring. GO analysis revealed that genes upregulated after vitrification-thawing were primarily associated with cell cycle-related pathways (Figure 4D and E). Intriguingly, our observations also indicate the involvement of epigenetic pathways, such as, “histone H2A acetylation” and “DNA methylation involved in gamete generation” (Figure 4D upper). These findings suggest that epigenetic regulatory mechanisms following ovarian vitrification-thawing merit further exploration. In addition, some pathway, related with apoptosis and embryonic development were observed in downregulated genes groups (Figure 4E upper). Furthermore, KEGG pathway analysis identified enrichment of several key pathways in vitrified ovarian oocytes, including “carbon metabolism”, “the citrate cycle”, “RNA degradation”, and “endocytosis” (Figure 4D and E bottom). Additionally, we compared the DNA damage and repair gene list and generated scores during ovarian vitrification and thawing. DNA damage scores decreased and DNA repair scores increased in vitrification-thawed ovarian oocytes (Figure S4D). Cell-cell communication analysis via CellPhoneDB revealed that, after vitrification-thawing, the oocyte in situ displayed a diminished interaction with granulosa, perivascular and stromal cells (Figure 4I). Detailly, the interaction between oocytes and peripheral cells via TGFβ (TGFB3, TGFB2) and its receptors is enhanced, indicating an activation of inflammation-related signaling pathways. While the Notching signaling pathway from Oocyte to peripheral cells (i.e., NOTCH1_DLK1, NOTCH1_DLL3&4 and NOTCH1_JAG1) decreased (Figure 4J and K). Intriguingly, the interaction between the AMH receptor (MIS receptor) and AMH from peripheral cells to oocytes was diminished, yet the reverse interaction remained unaffected (Figure 4J and K). The underlying molecular mechanism deserves further exploration. 2.5 PTGDS positive stromal cells (PTGDS + SCs) were sharply decreased post ovarian vitrification-thawing progress. Stromal cell subpopulations are now a focal point of investigation (Figure 5A). Quantitative analysis revealed a significant decrease in Subcluster 1 after the vitrification-thawing process (Figure 5B). A heatmap displayed the top genes predominantly expressed in Subcluster 1, including PTGDS, C3, SFRP4, and IGFBP6 (Figure 5C). GO analysis was conducted on the top differentially expressed genes in stromal cells following the vitrification-thawing procedure. Pathways related to “reactive oxygen responding”, stress-inflammatory response-related pathways such as “MAPK signaling pathway” are significantly activated (Figure 5D). Here, we defined subcluster 1 as PTGDS-positive subpopulations (PTGDS + SCs) (Figure 5E). Additionally, RNA from both fresh and vitrified-thawed ovarian cortex was enriched and analyzed via real-time PCR, with results confirming the bioinformatics findings (Figure 5F). ST data revealed decreased expression of C3, SFRP4, and PTGDS, particularly in the COL1A2+ stromal cell subtype located in the cortex (Figure 5G). To validate the bioinformatic analysis findings, RNAscope-based in situ hybridization (ISH) had been carried on human ovary tissues paraffin section. As expected, PTGDS positive cells are mainly distributed in the extracellular matrix. In the vitrification-thawing ovarian cortex section, the expression of PTGDS showed a downregulated profile compared with fresh control (Figure 5H) aligning with IHC results (Figure S5F). We replicated the ovarian cryopreservation strategy in murine ovaries. PTGDS also exhibited similar expression profile (including mRNA and protein expression level) changes as observed in human tissue samples (Figure 4I and Supplemental Figure S5G). This poses an intriguing question about the role of PTGDS-positive stromal cells and PTGDS genes in ovarian cryopreservation. The underlying pathways and metabolic processes involved warrant further exploration. The graphs showed that post vitrification-thawing, there was not only a decrease in PTGDS-positive cell subgroups but also an increase in subcluster 2 (Figure 5B), indicating a rapid and varied response of stromal cells to vitrification. Subsequent detailed analysis of cluster 2 revealed a subgroup with increased abnormalities post-vitrification (Figure S5A and B, Subgroup 4). Featureplot and temporal-based expression profiling both demonstrate that after vitrification and thawing, genes from the heat shock protein family, namely DNAJB1, HSP90A1, and HSP90B1, are notably enriched and expressed in the newly emerged subgroup 4 (Figure S5C and E). Additionally, Pseudo-temporal analysis showed that certain PTGDS-positive stromal cells displayed a tendency to transform into cells with higher expression of temperature-sensitive genes (Figure S5D and E). These findings collectively suggest the presence of heightened sensitivity and unique cryoinjury characteristics in SCs post ovarian vitrification-thawing. 2.6 The FOS/AP-1 signaling pathway is activated in smooth muscle/perivascular cells and granulosa cells following the vitrification-thawing procedure. Particular attention should be paid to the perivascular subtissue structures surrounding blood vessels, especially smooth muscle cells, as they participate in multicellular stratified systems. 50 As shown in Figure 1D, the smooth muscle/perivascular subcluster increased apparently after vitrification-thawing treatment. Reclustering analysis segmented the smooth muscle/perivascular cells into six clusters, with cluster 3 (FOS+) exhibiting a significant increase post vitrification-thawing (Figure 6A). The specifically highly expressed genes in cluster 3 predominantly include FOS, FOSB, JUN, JUNB, and JUND, as evidenced by the FeaturePlot and ViolinPlot (Figure 6G and Figure S6A). Meanwhile, heatmap results demonstrate consistent activation trends of AP-1 protein family members, including FOS, FOSB, and JUN, across three sets of replicate experiments (Figure 6C). Furthermore, pathway analysis indicated that DEGs following ovarian vitrification predominantly relate to “Amyotrophic lateral sclerosis”, “Huntington disease” et al., which are closely linked to muscle physiological dysfunction (Figure 6D). Granulosa cells play an important role in follicle development and oocyte maturation. 51, 52 Reclustering analysis revealed that granulosa cells had been divided into 3 subgroups. Moreover subcluster 3 (FOS/AP-1 active GCs) showed a significant increase in proportion post vitrification-thawing (Figure 6B). Intriguingly, the top expressing genes in cluster 3 including FOS, FOSB, JUN, JUNB and JUND as shown by Featureplot (Figure 6H). SpatialFeaturePlot indicates that the expression of FOS, FOSB, JUN, and JUNB significantly increased in situ following vitrification-thawing, corroborating the scRNA analysis findings (Figure S7A). Multi-immunofluorescence labeling had been conducted and revealed a significant increase in FOSB protein abundance in smooth muscle and perivascular cells following vitrification-thawing (Figure S7B). The above observation prompted a more detailed examination of the FOS/AP-1 pathway. Utilizing the gene scoring algorithm, both scRNA-seq and ST-seq data confirming FOS/AP-1 heightened activation in pericytes (PCs) compared to other subgroups (Figure 6E and F). Additionally, we had also focused on DNA damage repair, ROS-related genes, NF-κB pathway and AP-1 complex, but the effects of cryoinjury were not obvious (Figure S8). To validate the upregulation of FOS + perivascular cells, RNAscope-based ISH had been conducted. As expected, expression profile of FOS increased apparently in vitrification human ovarian cortex compared with fresh ones (Figure 6I), aligning with the FOSB’s immunochemical staining results (Figure 6J and Figure S6B). Furthermore, mRNA from both fresh and vitrified-thawed ovarian cortex was enriched and analyzed using real-time PCR, with results that corroborated the bioinformatics analysis (Figure 6K). The FOS family (including FOS, FOSB, FOSL1, and FOSL2) can dimerize with proteins of the JUN family, forming the transcription factor complex AP-1. 53, 54 FOS proteins are implicated as regulators of cell proliferation, differentiation, and transformation. Together, these results suggest that FOS/AP-1 pathway is activated immediately in response to temperature stress and cryoprotectant toxicity, providing insight into the mechanism underlying FOS’s role in preventing ovarian cellular cryoinjury. 2.7 FOS/AP-1 is rapidly activated in the early flow of vitrification process, independently of TGFβ mediation, and its inhibition maintains the viability of frozen-thawed ovaries in vitro . Previous studies have claimed that FOS/AP-1 pathway rapidly responds to external stimuli and stress, primarily facilitating the translation and accumulation of stress-related inflammation. 55, 56, 57 AP-1 activity is induced by numerous extracellular matrix and genotoxic agents, suggesting involvement in programmed cell death. 58 Cell-cell communication analysis showed a significant reduction in ligand-receptor pairs between FOS/AP-1-activated granulosa/perivascular cells and other cell types, especially oocytes (Figure 7A and Figure S9A). Furthermore, we focused on certain ligand-receptor sets involved in cell proliferation, differentiation and inflammation responses. As expected, FOS/AP-1-activated PCs and GCs exhibited a decrease in ligand-receptor interactions within NOTCH signaling which promotes proliferating (Figure 7B). Unexpectedly, although involved in the activation of FOS/AP-1, the TGFβ pathway showed a significant decrease in ligand-receptor pairs after vitrification-thawing (Figure 7B). This suggests that FOS/AP-1 activation is primarily driven by direct stress and chemical toxicity at this stage. The maintenance of cell identity involves the coordinated action of many regulators, among which transcription factors have been long recognized to play a central role. 59 SCENIC was used to identify transcription factors in granulosa cell to predict essential regulators involved in cryoinjury. Regulon specificity scores (RSSs) identified top 3 specific regulons including FOSB (Figure 7C). Here we further identified EGR1 as co-target to FOS and JUN (Figure 7D). Moreover, co-immunofluorescence and histochemical staining results indicated that EGR1 expression was significantly upregulated in smooth muscle/ perivascular cells following vitrification-thawing (Figure 7E and Figure S9B). And the key pathways involved in our research, as well as the molecules (FOS, FOSB, EGR1), are highly conserved in mice (Figure S9C and D). Additionally, we confirmed through Co-immunoprecipitation experiments that there is no direct interaction between FOS and EGR1 (Figure S10D). FOS is a transcriptional regulatory factor 60 , that we believe predominantly enters the cell nucleus after phosphorylation to regulate the upstream promoter of EGR1. These above intriguing results prompt us to systematically explore the specific flows and key points where and when the FOS/AP-1 pathway changes (Figure 7F). We refined the overall vitrification process into four key processes: obtaining fresh ovarian tissue cortex (FO), vitrification dehydration (VD), liquid nitrogen freezing (F) and thawing (T). Interestingly, western blotting revealed that FOS/AP-1 activation occurred at the onset of the vitrification dehydration phase and persisted after thawing (Figure 7G). Additionally, we conducted a preliminary exploration of the function of FOS/AP-1 in the ovary. FOS/AP-1 was activated by the agonist Phorbol 12-myristate 13-acetate (PMA) (Figure 10SA), which has been reported as an AP-1 agonist. Following this activation, the proliferation of human granulosa-like cell line (KGNs) had been inhibited (New Figure S10B). Activation of the FOS/AP-1 pathway increased apoptotic cell percentages in the KGN cell line. Concurrently, T5224 administration mitigated apoptosis induced by PMA treatment (New Figure S10C). It is important to know whether inhibiting FOS/AP-1 recovers the defect. T-5224 is a transcription factor FOS/AP-1 inhibitor with anti-inflammatory effects, which specifically inhibits the DNA binding activity of FOS/AP-1 without affecting other transcription factors. 61 Hence, we treated murine ovary with T-5224 adding in the cryoprotectants during vitrification-thawing (Figure 7H upper). Wholemount tissue immunofluorescence staining demonstrated that vascular network density in vitrification-thawed ovaries was well maintained following T-5224 adding in vitro (Figure 7H bottom). Additionally, TUNEL staining confirmed that T-5224 treatment reduced apoptosis in vitrified-thawed ovarian cells (Figure 7I & J). In summary, these findings suggest that inhibiting FOS/AP-1 via T-5224 improves ovarian function post vitrification-thawing. 3. Discussion This study conducted a comprehensive analysis of single-cell and spatial transcriptomic atlas of human ovaries throughout the cryopreservation procedure, illuminating spatial and temporal variations in gene expression during vitrification and thawing. The effects of vitrification-thawing on ovary had been investigated previously but very superficial and limited. 21 , 62 , 63 . The noteworthy contributions of our work are as follows: Firstly, we delineated gene expression signatures and spatial locations for eight types of human ovarian cells, pinpointing cell type-specific DEGs during ovarian cortex vitrification-thawing (Figs. 1 and 3 ). Both in vitrification-thawed and fresh human ovarian cortex, the stromal cells, granulosa cells, oocytes, endothelial cells, and smooth muscle/perivascular cells were classified according to their anatomical structures (Fig. 1 ). Generally, after vitrification-thawing, significant alterations were observed in pathways related to apoptosis, extracellular matrix synthesis, and fibrosis. Notably, genes such as FOS, C3, JUN, and JUNB were upregulated across all cell types (Fig. 3 ). Secondly, we focused on the differential effects of slow freezing and vitrification on ovarian cortex at the single-cell level, which is being reported for the first time. Interestingly, slow freezing caused more severe overall cell apoptosis but had less impact on oocytes. Additionally, intercellular communication was more significantly disrupted in the slow freezing group compared to vitrification (Fig. 2 ). Thirdly, via Smart-seq2 strategy, an exploration of cryoinjury-associated changes in gene expression highlighted the cell cycle response as a biological pathway involved in oocyte cryopreservation in situ (Fig. 4 ). Fourthly, subclustering analysis identified PTGDS-positive stromal cells as one of the primary cell types sensitive to vitrification-thawing, along with changes in transcriptomic features during cryopreservation (Fig. 5 ). Fifthly, integrating scRNA-seq data with ST-seq identified FOS/AP-1 as a potential key transcriptional factor for ovarian cellular cryoinjury, accelerating the transcription of EGR1 (Fig. 6 ). And inhibiting FOS via T-5224 improves maintenance of ovarian function in vitro post vitrification and thawing (Fig. 7 ). These findings provide new insights into human ovarian cortex cryopreservation and present potential targets for the treatment of ovarian cryoinjury. In vitrified-thawed ovaries, a notable observation is that most cell types exhibit characteristics associated with apoptosis. These changes include increased DNA repair scores, enhanced NF-κB and AP-1 pathway activity, and alterations in molecules related to the apoptosis pathway. Previous studies mainly focus on two aspects of vitrification-induced damage to the ovary: 1) direct cellular damage, such as DNA damage and apoptosis; 2) disruption of the intercellular microenvironment, including matrix collagen degradation. Significantly, there was a marked decrease in the stromal cell cluster, indicative of a reduced extracellular matrix phenotype. Concurrently, the rapid activation of the FOS/AP-1 pathway primarily in smooth muscle/perivascular cells (SM/PCs) and granulosa cells (GCs) suggests enhanced apoptosis and inflammation. The expression changes of oocyte within ovarian cortex after vitrification-thawing merit detailed examination. The oocyte-corona-cumulus complex (OCCC) development had been investigated systematically at single-cell level. 64 Recent study claimed that the effects of vitrification on the transcriptomes of mature human oocytes in vitro (metaphase II oocytes) are induced by the procedure itself rather than by the storage time 36 . While the effects of vitrification on oocytes in-situ are unclear. Here, cell cycle and meiosis-related physiological processes are identified as the primary pathways altered in oocytes within the ovary post vitrification-thawing. Moreover, some epigenetic related terms, such as histone H2A acetylation and DNA methylation, had also been observed in the DEGs GO analysis list. Additionally, the ligand-receptor interaction between oocyte and surrounding cells also are diminished, especially the NOTCH and AMH pathway related to developments. While our approach has certain limitations, it is important to note that due to variability in stages, we cannot entirely exclude the heterogeneity within the fresh or vitrification-thawed oocyte groups in vivo . Mesenchyme of ovarian stroma (herein called stromal cell), which is similar in morphology to fibroblasts, make up the connective tissue throughout the ovary and surround follicles. 65 The ovarian stroma comprises mostly incompletely characterized stromal cells (e.g., fibroblast-like, spindle-shaped, and stromal cells). 66 Recent studies have revisited the role of ovarian stromal cells, highlighting their significant contributions to folliculogenesis, particularly in the activation of primordial follicles and the differentiation of theca cells. 67 , 68 The plasticity of ovarian stromal cells is a key point of research, a recent study claimed that inhibition of ovarian fibrosis in part through regulating stromal cell differentiation via the TGF-β1/Smad3 signaling pathway can recover the function of ovarian function in POI rats. 69 Here, we identified a new vitrification sensitive stromal cell cluster, called PTGDS + stromal cells, and its biological function deserves further study. PTGDS (prostaglandin D2 synthase, PTGDS) is purified and firstly identified by Y Urade et al. 70 Previous study claimed that PTGDS catalyzes the conversion of PGH2 to PGD2, a prostaglandin involved in smooth muscle contraction/relaxation and a potent inhibitor of platelet aggregation. 71 This enzyme (PTGDS) is a dual-function protein; it acts as a PGD2-producing enzyme and also as a lipophilic ligand-binding protein. 72 , 73 Knocked-out(KO) mice of PTGDS are new model animals of aging, as they showing progressive age-related cartilage degradation 74 and adenomyosis. 75 Function of PTGDS had been studied in male reproductive system such as testis and epididymis. 76 , 77 , 78 , 79 Little is known about the function of PTGDS in human ovary. And we demonstrated that PTGDS + SCs is one of the most sensitive cell populations to cryopreservation. The up-downstream signaling pathways of PTGDS involved in ovarian function will be explored in further. The molecular network/key factors governing the cryoinjury of ovarian cells are not well understood. In this study, our analyses identified FOS/AP-1 as a crucial TF regulating cellular cryoinjury in the ovarian cortex. During vitrification-thawing, we noted an increasing in FOS expression in ovarian PCs and GCs cells. Stereo-seq analysis allows us to discern whether FOS/AP-1 pathway activation is due to single-cell suspension digestion or the cryopreservation procedure itself. As expected, the FOS/AP-1 was upregulated in situ during vitrification-thawing procedure. FOS is part of the AP-1 complex family, which includes FOS, FOSB, FOSL1, and FOSL2. These proteins dimerize with members of the JUN family to form the transcription factor complex AP-1. 80 , 81 Previous research reported that FOS/AP-1 regulates metabolic changes and cholesterol synthesis in human periovulatory granulosa cells. 82 Additionally, AP-1 can manifest oncogenic or anti-oncogenic effects by regulating genes involved in cell proliferation, differentiation, apoptosis, angiogenesis, and tumor invasion. 83 Furthermore, previous studies have claimed the ability of FOS/AP-1 to bind on FOXP3 gene locus and promote the expression of this master regulator of Treg identity, which leads to the accumulation of inflammation. 84 , 85 Consistently, we observed signification upregulation of inflammation- accumulation pathways, such as NF-κB signaling. Collectively, our data propose FOS/AP-1 as a potential target for recovering ovarian cryoinjury. And further investigation is needed to elucidate how the FOS/AP-1 pathway is activated, particularly determining whether cryoprotectant toxicity or temperature stress plays the dominant role. The novel benzophenone derivative T-5224 was rationally designed to serve as a potential drug to inhibit transcription regulated by AP-1. 86 , 87 In this study, we discovered its efficacy in improving ovarian function in vitro after vitrification and thawing. It is important to note that T-5224 is safe for oral administration to humans. 88 , 89 Therefore, the selective FOS/AP-1 inhibitor T-5224 appears suitable for development as a therapeutic agent for the recovery of ovarian degeneration in humans post cryopreservation and transplantation. In conclusion, we have mapped the spatiotemporal single-cell transcriptomic landscape of human ovarian cryoinjury and identified FOS/AP-1 as activated with vitrification-thawing and modulating ovarian recovery. The pharmacological silence of FOS, as inhibited by T-5224, emerges as a promising therapeutic strategy for mitigate ovarian damage post-vitrification-thawing. Our study deepens the understanding of human ovarian cryoinjury during vitrification-thawing, providing a valuable resource for investigating potential therapeutic interventions. Moving forward, we aim to explore FOS/AP-1 as a potential therapeutic target for restoring ovarian function after frozen-thawed transplantation. Materials and Methods Human samples and ethical statement Human ovaries were obtained from participants who underwent from gender reassignment surgery patients (GRPs). (Major Resources Table). The sample collection was approved by the local ethics committee (Approval No. B2022269P). And all participants provided informed consent and independent clinicians informed the patients about the study. Animal work All animal experiments were performed in accordance with the standard protocols, animal welfare regulations, and the institutional guidelines of International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University. We confirm that the study was carried out in compliance with the ARRIVE guidelines. Human ovarian vitrification and thawing The KITAZATO Ovarian Tissue Vitrification Kit (82212, 82222 and 81213 KITAZATO) was used for Human ovarian vitrification and thawing process. In brief, the ovarian cortex was cut into pieces (about 1cm X1cm) and washed with Ova Rinse. Then, tissue pieces were equilibrated in gradually increasing concentrations of cryoprotective agents (CPA) with a final concentration of 20% DMSO, 20% ethylene glycol (EG) and 0.4 M sucrose. 90 After equilibration, remove excess medium by placing the ovarian tissue on a piece of gauze. Lay the ovarian tissue on Ova Cryo Device with its superficial dimension spread out as much as possible. The excess solution was removed using blotting paper. Using fine tweezers, the Ova Cryo Device were plunged directly into liquid nitrogen and then gently put into cryovials under liquid nitrogen and the vial caps sealed. Finally, set the device to a cane and store in storage tank. After 2 weeks, the Ova Cryo Device with vitrified ovarian tissues were removed from the cryovials using tweezers while still under liquid nitrogen and then plunged directly into 37°C vitrification medium (Thaw1). After thawing for 30 s, tissues were transferred through a series of gradually decreasing concentrations of CPA for 5 min at each concentration to remove the CPA osmotically (20%, 10%, 5% and 0% for both DMSO and EG with 0.4 M sucrose, concluding with 0.2, 0.1 and 0 M sucrose). Then, tissues were washed in L-15 medium followed with digestion and fixation. Ovarian single cell suspension preparation For 10X Genomics strategy, the fresh and vitrification-thawing processed human ovarian cortex were cut into pieces with very sharp knives, followed by dissociating in collagenase I solution(2mg/ml) for 45 minutes at 37°C. Before FACS, the Blood cells were depleted by Red Blood cells lysis buffer (BD Pharmingen). Cells were then labeled with DAPI (4',6-diamidino-2-phenylindole, Cat number: D9452, Sigma, 1:1000), and DAPI − cells (live cells) were sorted through a MoFlo FACS machine (MoFlo Astrios EQ, Beckman Coulter) directly into PBS containing 0.04% BSA. For Smartseq2 strategy, fresh and vitrification-thawing processed human ovarian cortex were cut into pieces with very sharp knives, followed by dissociating in collagenase I solution(2mg/ml) for 45 minutes at 37°C. RPMI 1640 medium containing 10% FBS was used to stop digestion. The resulting cell suspension was then transferred into a 3.5 cm dish. Then, oocytes were manually picked under a dissection microscope and transferred to a PBS drop (containing 0.1% BSA) by mouth pipetting. Finally, the oocytes were placed separately into an individual PCR tube with lysis buffer and stored at -80°C for subsequent experiments. Single-cell RNA-seq library preparation and sequencing For 10X Genomics strategy based single-cell RNA sequencing, the single-cell RNA library was built via 10x Chromium platform, and FACS-enriched live ovarian cells were loaded per channel approximately. The libraries were built via the Chromium platform and Chromium Single Cell 3’ v2 chemistry. Finally, the libraries were loaded on an illumine NovaSeq 6000 System (Illumina, San Diego) with a PE150 sequencing strategy. For Smart-seq2 strategy based single-cell RNA sequencing, scRNA-seq libraries were constructed according to the published protocol with modifications 40 . Briefly, the constructed libraries of each oocyte will undergo preliminary screening through the marker genes (DDX4 for oocyte and NR5A2 for granulosa cells) of oocytes and granulosa cells via qPCR, aiming to exclude multi-cell samples. Then, the libraries were sequenced on an Illumina NovaSeq 6000 platform with a 150-bp paired-end read length by Novogene. Tissue processing for spatial transcriptomic experiment To collect tissues for Stereo-seq analysis, ovaries were dissected from Gender Reassignment Surgery patients (GRPs). The fresh tissues were rinsed in pre-chilled PBS twice to eliminate surface impurities and any remaining liquid or blood was gently wiped dry. Next, one piece of ovaries cortex had been treated with standard human ovarian vitrification and thawing procedure described above. The other one was washed with pre-chilled tissue freezing medium OCT (Leica, Germany) and embedded together in a new OCT. The ovaries were oriented with a blunt metal needle to ensure proper positioning. The entire OCT block was snap-frozen in liquid nitrogen that had been pre-chilled with isopentane and then transferred to a − 80°C freezer for storage before cryosection. Two weeks later, the treatment and OCT embedding methods adopted by the vitrification group were the same as those of the fresh group. To minimize RNA degradation, the fresh and cryo ones were embedded within 30 min and the entire dissection procedure was performed in a low-temperature environment. The OCT block was sliced transversely at a thickness of 10µm using Leica CM1950 cryostat (Leica). Total RNA was extracted from the sections using the RNeasy Mini Kit (Qiagen, USA) in accordance with the manufacturer’s protocol. A sample with an RNA integrity number (RIN) of 7–10, as measured by the 2100 Bioanalyzer (Agilent, USA), was used for the Stereo-seq. The targeted section was directly adhered to the surface of the Stereoseq chip (BGI, Qingdao, China), which had capture probes that contained a 25 bp coordinate identity (CID) barcode, a 10 bp molecular identifiers (MID), and a 22 bp polyT for in situ mRNA hybridization. The adjacent section was stained with H&E for tissue histology examination later. The section on the chip was incubated at 37°C for 3 min, fixed in pre-cooled methanol for 30 min at -20°C, and then stained with nucleic acid dye (Thermo Fisher Scientific) for ssDNA visualization. The Ti-7 Nikon Eclipse microscope (Nikon, Japan) was used for ssDNA and histological imaging. Library construction and sequencing were completed with the help of Oebiotech, China. Single-cell RNA-seq data analysis The Cell Ranger “count” pipeline (version 3.1.0) was applied with the FASTQ data produced to map the human reference genome (version hg19, GRCh38). The data matrixes in “outs” Files were then loaded in R (version 4.3.0) using the Seurat package (version 4.3.0.1). 91 After aggregation of the sequencing data from fresh and vitrification-thawed human ovarian cortex, R package Seurat 4.3.0 was used for cell filter, data normalization, variable gene selection, unsupervised clustering, and uniform manifold approximation and projection (UMAP) according to their recommended steps. 92 Briefly, Seurat objects were created from the aggregated library as matrix containing gene-by-cell expression data. Cells with less than 200 genes expression or a percentage of more than 0.05 mitochondrial genes were filtered out. Then data were log-normalized and scaled for subsequent analysis. Variable genes were found and used for principal component analysis (PCA), which was performed for dimension reduction. ElbowPlot function was used for the determination of the numbers of principal components, followed by unsupervised clustering and UMAP. FindAllMarkers function was used to identify the genes exclusively expressed in each cluster. Visualization of total profiles of each cluster was generated with Seurat function DimPlot. Visualization of gene expression with feature plot, dot plot, and heatmap was generated with Seurat function FeaturePlot, DotPlot and DoHeatmap, respectively. Differentially expressed genes (P < 0.01) between 2 identities were found with FindMarkers function. Gene ontology (GO) biological function analysis was performed with marker genes of each cluster found by FindMarkers function with average adjusted p < 0.05 on toppgene website ( https://toppgene.cchmc.org/ ) and then plotted with R package Goplot. Unsupervised clustering and bin clusters annotation of Stereo-seq data The downloaded GEM file was converted to the Seurat object and then processed with Seurat v4.4.4. The data were normalized, scaled, multiple samples were integrated, and unsupervised clustering was performed using bins (resolution = 0.4). Marker genes for each cell type were defined using the DEGs that were identified through the “FindAllMarkers” function. The cell identities of the clusters were then annotated based on the marker genes and histological morphology of the H&E images. To further confirm the relationship between cell types, the Pearson correlation was calculated across the matrix, and hierarchical clustering of the Stereo-seq clusters was performed using ComplexHeatmap package in R (v2.9.1). Integrated mapping of cell types in Stereo-seq spots with scRNA-seq data To combine scRNA-seq and Stereo-seq data, the “FindTransferAnchors” and “TransferData” functions of Seurat were employed to determine the possibility of anchors in single spots. The cell type associated with the highest probability among all cell types was subsequently identified as the cell type of the spot. The integrated mapping of cell types was then visualized using the “SpatialDimPlot” and “SpatialFeaturePlot” functions. Additionally, the gene and pathway score had been visualized by “AddModuleScore” and “ggplot2”. RNA in situ hybridization Human ovarian cortex sections from fresh and vitrification-thawed were fixed in 4% PFA overnight and embedded in paraffin wax. Eight µm thick sections were mounted on poly-L-lysine coated slides (ThermoScientific). For RNAscope® ISH analysis, Hs-PTGDS (431471), with the target region 15–808 in human PTGDS, probes (Advanced Cell Diagnostics) were used according to manufacturer’s instructions. Quantification of PTGDS positive signals had been performed by ImageJ via using equally sized region of interest (ROI) for the ovarian cortex zone and incorporating all cortex layers. Transmission electron microscopy For transmission electron ultrastructural analysis, fresh and vitrification-thawed tissues were fixed in PBS (Phosphate buffered saline), which is supplemented with 2% glutaraldehyde and 2% paraformaldehyde. After tissue processing, semithin (1 mm) and ultra-thin (700 nm) sections were cut; semi-thin sections were evaluated under a brightfield microscope (DMi8 Microscope; Leica), whereas ultra-thin sections were observed by transmission electron microscopy (TEM; LEO 906 E TEM 60 kV; Zeiss, Jena, Germany). Immunohistochemistry and H&E staining in human ovarian tissue For immunohistochemistry staining, tissues were fixed with 4% paraformaldehyde overnight, then washed by running water. Finally, tissues were embedded in paraffin according to the standard protocol. 5µm sections were pre-pared, deparaffinized by xylene for 10min twice. Subsequently, the sections were dehydrated by gradient alcohol, washed by PBS. Antigen retrievals were done using sodium citrate buffer (YEASEN, 36319ES60), then blocked with 5% bovine serum albumin (BSA), incubated by primary antibody at 4°C overnight, then done using an UltraSensitive™ SP (Mouse/Rabbit) IHC Kit (MXB, KIT-9710). Signal detection was applied by DAB systems (MXB, MAX- 001). Nuclear was stained by Mayer's hematoxylin (Beyotime). The sections were then dehydrated by gradient alcohol, transparentized with xylene, then mounted with neutral resin for microscopy. H&E staining was performed according to the kit (G1120, Solarbio). Briefly, washing the De-paraffinize slides to distilled water and staining slides with hematoxylin. Following a rinsing process, the sections are counterstained with eosin. Finally, shaking off excess dye and blot sections. Dehydrating in acetone, 20 dips. Then in Acetone-Xylene (1:1) solution, 20 dips. Clearing in xylene and mount in a synthetic mounting medium. Immunofluorescence staining The cryosections (7-µm thickness) were dried at room temperature for 1 hour, fixed by 4% PFA for 10 mins, and then rinsed with 1x PBS for three times. After being permeabilized with 0.1% Triton X-100 (T8787, Sigma) for 10 mins at room temperature, the cryosections were blocked in 1x PBS containing 5% bovine serum albumin (BSA) for 1 hour at room temperature and then incubated with primary antibodies at 4°C overnight in a humidified chamber. The primary antibodies utilized for these experiments were as follows: PTGDS (1:200, ab182141, Abcam), EGR1(10µg/ml; 55117-1-AP, Proteintech), FOSB (2µg/ml; 2251 Cell Signal Technology), SOHLH2 (1:100; bs-12279R Biossusa), αSMA (1:500; A5228 Sigma). DDX4 (2µg/ml; ab180642 abcam). Afterward, slides were rinsed with 1x PBSTx three times for 15 min, followed by the incubation with Alexa fluorescence-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Then the slides were rinsed with 1x PBSTx again three times and finally incubated with 4'6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) at room temperature for 15 min. Images were visualized using a fluorescence microscope (Leica, Germany). Ovarian Tissue Culture Ovaries at 7 dpp were subjected to organ culture as described below. The whole ovarian tissues were directly placed onto a twelve-well culture plate (LABSELECT, 12mm, 0.4µm). Each well contained 1.2 mL of Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture (Gibco, USA) supplemented with 5% Fetal Bovine Serum, 1% insulin–transferrin–selenium (ITS), and 100 UI/mL penicillin–streptomycin. We tested four different culture conditions: Fresh (The Control group), standards vitrification-thawing Treatment (The Cryo group), and with T-5224 post vitrification-thawing (The Cryo + T-5224 group, and during culture, T-5224 were added to the medium with the final concentration is 80uM per well). Ovarian tissues were cultured under each the four conditions for 2–4 days at 37°C, 5%CO2. Half of the culture media was replaced every other day. Chemicals T-5224 was provided by MedChemexpress (MCE) (New Jersey, USAs). T-5224 was dissolved in DMSO and diluted in cryoprotective agents (CPA) and ovarian culture medium to the target concentration for each experiment. Microscopy Specimens were mounted with fluorescence mounting medium (Dako, Glostrup, Denmark) or Mowiol mounting medium, and confocal images (e.g., cells and sections) were generally captured using a Leica TCS SP8 Confocal Microscope with 20X and 40X objectives. Images were processed with Car Zeiss software. Statistical Analysis For experimental studies, all quantitative data were evaluated whether they followed the normal distribution by the Shapiro-Wilk test and equal variance by F-test. For data passed both tests, data are expressed as means ± SEM. Student's t-test was used for the comparison between 2 groups, and for comparison among multiple groups, the data were analyzed by one-way ANOVA with Tukey's post hoc test. For the data that were not normally distributed, nonparametric test (Mann-Whitney U test) was performed and presented as median ± SD. All P values are 2-sided, and P < 0.05 was considered a statistically significant difference. Abbreviations OTC - Ovarian Tissue Cryopreservation scRNA-seq - single-cell RNA sequencing Stereo-seq - Spatial enhanced resolution omics sequencing PCs - perivascular cells SCs - Stromal Cells GCs - Granulosa Cells BECs - Blood vascular Endothelial Cells LECs - Lymphatic Endothelial Cells SMC - Smooth Muscle Cells GRPs -Gender Reassignment Surgery Patients DEGs - Differently Expressed Genes OCCC - oocyte-corona-cumulus complex Declarations Acknowledgments: We thank all members of the laboratory for critical discussion and comments on the article. We thank Prof. Shuo Xiao from Rutgers University for kindly supporting on murine ovarian in vitro culture. We express our gratitude to Prof. Yuxuan Zheng from the Human Phenome Institute, Fudan University, for his valuable advice on single-cell analysis techniques and methodologies. We thank Oebiotech (Inc. China) for its help in spatial transcriptome sequencing and analysis. This work was supported by the National Key Research and Development Project of China (2022YFC2703002), National Natural Science Foundation of China (82371726, 82071605, 82200541), The Joint Funds of the National Natural Science Foundation of China (U24A20658). Natural Science Foundation of Shanghai (No: 21ZR1428600), Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-ZDCX20212200), Shanghai Hospital Development Center Foundation (SHDC22022303) and Key project of Medical and Industrial intersection of Shanghai Jiao Tong University (YG2023ZD27). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions: F.G., D.S., H.D. and Y.L. contributed equally to this work. W.L., Q.Z. and M.Z. conceived the study. F.G., D.S., Y.L., and H.D. performed the experiments. F.G., D.S., Y.L., and M.G. analyzed and visualized the data. M.Z., Y.M., R.Q., S.L., and L.Z. provided the ideas and suggestions. M.G. and Q.Z. helped to collect clinical samples. F.G., and W.L. wrote and revised the manuscript. Conflict of Interest: Authors declare that they have no conflict of interest. Data and materials availability: All data are available in the main text or the supplementary materials. Data and code availability The RNA-sequencing data including have been deposited in Gene Expression Omnibus (GEO) under the accession number GSE267315 (reviewer token: ixctsiiqthybnsp). 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Stuart T , et al. Comprehensive Integration of Single-Cell Data. Cell 177 , 1888-+ (2019). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMaterial.docx Supplementary Material for Article File Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6312954","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":437225402,"identity":"fefd6b69-2875-458d-90d9-bc99f9272366","order_by":0,"name":"Wen 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13:46:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6312954/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6312954/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79911406,"identity":"4ae1ad57-01d2-432f-a3ec-ab7e9840c2e6","added_by":"auto","created_at":"2025-04-04 11:39:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1490256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003escRNA-seq profiling and spatial location of human ovarian cortex cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e The flow chart of spatio-temporally single-cell RNA sequencing strategy carried on human fresh and vitrification-thawed ovarian (including 10X genomics strategy, Smart-seq2 strategy and BGI’s Stereo-seq). Collagenase I was used to dissect ovarian cortex tissues. Fluorescence-activated cell sorting (FACS) was used to isolate live cells (DAPI\u003csup\u003e-\u003c/sup\u003e). Seurat 4.3 was used for single-cell analysis. Scale bar = 200um. scRNA-seq: single-cell RNA sequencing. \u003cstrong\u003eB)\u003c/strong\u003e Human ovarian tissues were classified into 8 populations. Colors denoted different populations. \u003cstrong\u003eC)\u003c/strong\u003e Featureplots characterized representative marker genes for different cell types and were used to define clusters of human ovarian. \u003cstrong\u003eD)\u003c/strong\u003e Left: heatmap showing expression signatures of top 50 specifically expressed genes in each cell type; the value for each gene is row-scaled Z score. Right: representative GO terms. \u003cstrong\u003eE)\u003c/strong\u003e The Spatial mapping of scRNA-seq cell clusters in representative slides of Fresh and Vitrification-Thawed human cortex tissue. \u003cstrong\u003eF)\u003c/strong\u003e Cell clusters identified by stereo-seq (Bin = 50, resolution = 0.4). \u003cstrong\u003eG)\u003c/strong\u003e The cell type mapping resulted from Panel E were shown separately. \u003cstrong\u003eH) \u003c/strong\u003eHeatmap showing marker genes expressed in the same cell types in both stereo-seq and scRNA-seq.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-6312954/v1/2516b72182d987494036cff7.png"},{"id":79910519,"identity":"a8ebcbdb-1f54-43fe-95c8-2d06c105c0b8","added_by":"auto","created_at":"2025-04-04 11:31:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1283901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe changes in transcriptional regulatory programs among Slow frozen, Vitrification and Fresh ovarian cortex at single-cell level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eUMAP showing the integration analysis of slow frozen vitrification-thawing and fresh human ovarian scRNA-seq data. \u003cstrong\u003eB) \u003c/strong\u003eHeatmap displaying top 30 differentially expressed genes between slow frozen, Vitrification and fresh samples. \u003cstrong\u003eC) \u003c/strong\u003eKEGG Pathway Analysis of total DEGs between slow frozen and vitrification-thawing treated cells within human ovarian cortex. \u003cstrong\u003eD) \u003c/strong\u003eKEGG Pathway Analysis of total DEGs between slow frozen and fresh treated cells within human ovarian cortex. \u003cstrong\u003eE) \u003c/strong\u003eGene set score analysis of Reactive Oxygen Species (ROS) metabolism pathway in all ovarian cells among slow frozen, vitrification-thawed and fresh group. \u003cstrong\u003eF) \u003c/strong\u003eGene set score analysis of Apoptosis pathway in all ovarian cells among slow frozen, vitrification-thawed and fresh group. \u003cstrong\u003eG) \u003c/strong\u003eTotal interaction strength in slow frozen, vitrification, and fresh groups. \u003cstrong\u003eH) \u003c/strong\u003eCircle plot illustrating cell-to-cell communication among 8 cell types in slow-frozen, vitrified, and fresh groups. The network diagram quantitatively displays nodes representing various cell types, with arrows showing signal interactions from ligand-expressing cells to receptor cells. Line thickness reflects the number of significant ligand-receptor pairs identified between different cell types. \u003cstrong\u003eI) \u003c/strong\u003eBubble plot of the communication probability of the significant ligand-receptor pairs that contributed to Wnt and Nocth signaling sent from Oocyte to each cell population in slow frozen, vitrification, and fresh groups. Notes: The dot color and size represent the communication probability and p-values, respectively. p-values were computed from one-sided permutation test.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-6312954/v1/7c116f28731dbf614c34520d.png"},{"id":79910520,"identity":"debf441b-78c4-4122-9f80-95774807ac8d","added_by":"auto","created_at":"2025-04-04 11:31:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1492342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell type-specific spatiotemporal changes in transcriptional regulatory programs throughout ovarian vitrification-thawing’s programs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e Integration analysis of fresh and vitrification-thawing human ovarian scRNA-seq data (only 10X Genomics strategy data). \u003cstrong\u003eB)\u003c/strong\u003e UMAP plots showing batch-dependent cell distribution. \u003cstrong\u003eC)\u003c/strong\u003e Quantitative analysis of the proportions of seven ovarian cell types changed during vitrification-thawing treatment. \u003cstrong\u003eD)\u003c/strong\u003e Statistical difference analysis of PCs and SCs after vitrification and thawing in three repeats. \u003cstrong\u003eE)\u003c/strong\u003e Heatmap displaying top 50 differentially expressed genes between fresh (left) and vitrified-thaw processed (right) samples with 3 individual repeats. \u003cstrong\u003eF)\u003c/strong\u003e KEGG Pathway Analysis of total DEGs between fresh and vitrification-thawing treated cells within human ovarian cortex. \u003cstrong\u003eG \u0026amp; H)\u003c/strong\u003e Gene set score analysis of Focal adhesin, Apoptosis and AP-1 pathways in various ovarian cell types of different groups. \u003cstrong\u003eJ)\u003c/strong\u003e Electron microscopy of fresh and vitrification-thawing human ovarian tissue. Abbreviations, GC: Granulosa Cells, O: Oocyte, M: mitochondria, N: nucleus, SC: Stromal Cells. Scale bar in upper = 10um. Scale bar in zoomed screen = 2um. \u003cstrong\u003eI)\u003c/strong\u003e Masson staining evaluates ovarian tissue fibrosis after vitrification-thawing in human ovary. \u003cstrong\u003eK)\u003c/strong\u003e TUNEL analysis of human ovarian cortex section after vitrification-thawing treatment.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-6312954/v1/1cb9c7d5f90e9238bc3daa97.png"},{"id":79910522,"identity":"0d9c9d2e-2d79-483f-9af9-e192927d3083","added_by":"auto","created_at":"2025-04-04 11:31:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1768081,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVitrification-thawing procedures altered the gene expression profiles of ovarian oocytes in situ.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003eThe UMAP plot showing two ovarian oocyte types. \u003cstrong\u003eB)\u003c/strong\u003e The Vlnplot showed the marker genes which is expressed predominantly in fresh and vitrification-thawing processed oocytes separately. \u003cstrong\u003eC),\u003c/strong\u003e The heatmap showed the top20 different expressed genes between fresh and vitrification-thawed treatment oocyte groups within ovary.\u003cstrong\u003e D and E)\u003c/strong\u003eEnriched GO terms and KEGG pathways of the up and downregulated genes in cryopreserved oocytes in situ. \u003cstrong\u003eF and H)\u003c/strong\u003e Dual Immunofluorescence staining of DDX4 and SOHLH2 in both fresh and vitrification-thawing treated murine ovarian section. scale bar=200μm. And quantification analysis of SOHLH2\u0026amp;DDX4 positive oocytes in panel \u003cstrong\u003eF\u003c/strong\u003e. N = 5.\u003cstrong\u003eG)\u003c/strong\u003eFeatureplot showing the expression profile changes of SOHLH2 in fresh and vitrification-thawed human ovarian cortex. \u003cstrong\u003eI)\u003c/strong\u003e Circle plot showing the intercellular communication between oocyte and major other cell types in Fresh and Vitrification-Thawing subjects, respectively.\u003cstrong\u003e J and K) \u003c/strong\u003eCellphoneDB analysis of Notch, TGFβ, Chemokines and Oocyte rich interactions between Oocyte and surrounding cell clusters in Fresh VS Vitrification-Thawing treated human ovarian cortex. Depicted are −log10 p-values (circle size) and log2 means (circle color) for the interacting pairs, for selected pairwise cluster combinations.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-6312954/v1/846894f397d39c6fe9d5bab3.png"},{"id":79910526,"identity":"e920a512-cfa4-4c26-b36f-14b66727f07d","added_by":"auto","created_at":"2025-04-04 11:31:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1319696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePTGDS positive stromal cells (PTGDS\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e SCs) were sharply decreased post ovarian vitrification-thawing progress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e 7 subclusters clustered from integrated stromal cells subset. \u003cstrong\u003eB)\u003c/strong\u003e The fraction of each subcluster calculated separately from fresh and cryo SCs. \u003cstrong\u003eC)\u003c/strong\u003e Heatmap showing expression signatures of top 50 specifically expressed genes in each subclusters. \u003cstrong\u003eD)\u003c/strong\u003e Pathway analysis of significant different expressed genes between fresh and vitrification-thawing processed stromal cells within ovary. \u003cstrong\u003eE)\u003c/strong\u003e Expression profiles of marker genes (PTGDS, C3, SFRP4 and IGFBP6) in subcluster 1 showed by FeaturePlot. \u003cstrong\u003eF)\u003c/strong\u003e Realtime PCR analysis of top genes’ mRNA expression profile changes post vitrification-thawing process in human ovarian cortex. N = 3. \u003cstrong\u003eG)\u003c/strong\u003e Gene set score analysis of C3, SFRP4, PTGDS and PTGDS+ cells’ top ten expressing genes in ST-seq data. \u003cstrong\u003eH)\u003c/strong\u003e In situ hybridization (ISH) was employed to detect the expression site of PTGDS in sections of human ovary tissue, both fresh and following vitrification-thawing treatment, using the RNAscope strategy. Scale bar = 200um. \u003cstrong\u003eI)\u003c/strong\u003e Western blot analysis was conducted to evaluate the changes in PTGDS protein expression levels in murine ovaries before and after vitrification.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-6312954/v1/da1ab3ab3ee65c1d00fe23ba.png"},{"id":79910541,"identity":"cc69224c-2d26-48c0-b562-f805bbf1c2c6","added_by":"auto","created_at":"2025-04-04 11:32:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1797595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe FOS/AP-1 signaling pathway is activated in perivascular and granulosa cells following the vitrification-thawing procedure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e Six susubclusters clustered from integrated smooth muscle/perivascular cells (SM/PCs) subset. \u003cstrong\u003eB)\u003c/strong\u003e Three clusters subclustered from integrated granulosa cells (GCs) subset. \u003cstrong\u003eC)\u003c/strong\u003e Heatmap displaying differentially expressed genes between perivascular and granulosa clusters in fresh (left) and vitrified-thaw processed (right) samples. \u003cstrong\u003eD) \u003c/strong\u003ePathway analysis of significate different expressed genes between fresh and vitrification-thawing processed perivascular cells within ovary. \u003cstrong\u003eE and F)\u003c/strong\u003e Gene set score analysis of stress-related expressing genes in scRNA-seq data and ST-seq data. \u003cstrong\u003eG)\u003c/strong\u003e Expression profiles of marker genes (FOS, FOSB, JUN, JUNB and JUND) in subcluster 3 (FOS\u003csup\u003e+\u003c/sup\u003e PCs) showed by FeaturePlot. \u003cstrong\u003eH)\u003c/strong\u003e Expression profiles of marker genes (FOS, FOSB, JUN, JUNB and JUND) in FOS/AP-1 active subsets showed by FeaturePlot. \u003cstrong\u003eI) \u003c/strong\u003eRNAscope strategy based in situ hybridization (ISH) was employed to detect the expression site of FOS in sections of human ovary tissue from fresh and following vitrification-thawing treatment. Scale bar = 200um.\u003cstrong\u003e J)\u003c/strong\u003e Immunofluorescence staining was employed to detect the expression site of FOSB in sections of human ovary tissue, both fresh and following vitrification-thawing treatment. Scale bar = 200um. \u003cstrong\u003eK)\u003c/strong\u003e Realtime PCR analysis of genes in AP-1 complex pathway expression profile changes post vitrification-thawing process in human ovarian tissues. N = 3.\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-6312954/v1/a4913d3e38397eac544446ce.png"},{"id":79911415,"identity":"dd47efcb-c109-4477-a1ba-f73e59aac154","added_by":"auto","created_at":"2025-04-04 11:40:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1813532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFOS/AP-1 is rapidly activated in the early flow of vitrification, independently of TGFβ mediation, and its inhibition maintains the viability of frozen-thawed ovaries \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e The circle plots demonstrated the crosstalk between PCs (left)/GCs (right) and other cell types in Fresh and Vitrification-Thawed subjects respectively. \u003cstrong\u003eB)\u003c/strong\u003e Dot plot of selected ligand/receptor (left) and receptor/ligand pair(right) interactions between FOS\u003csup\u003e+\u003c/sup\u003e PCs or FOS/AP-1 active GCs and other cell components, analyzed separately for fresh and vitrification-thawing groups. \u003cstrong\u003eC)\u003c/strong\u003e Regulon specificity scores (RSSs) identified top specific regulons in fresh and vitrification-thawing GCs. The top 6 regulons from each group are marked in red and annotated. \u003cstrong\u003eD)\u003c/strong\u003e Circle plot shows gene regulatory network of the TFs FOS and JUND and their target genes. \u003cstrong\u003eE)\u003c/strong\u003e Immunofluorescence staining was employed to detect the expression changes of EGR1 in human ovary tissue following vitrification-thawing treatment. Scale bar = 200um. \u003cstrong\u003eF) \u003c/strong\u003eSchematic diagram illustrating the vitrification and thawing process of mouse ovarian tissue, with corresponding protein collection points for Panel G. \u003cstrong\u003eG) \u003c/strong\u003eWestern Blotting of AP-1 complex family (FOS, FOSB and JUN) the upper stream protein (p-AKT and AKT) and down streams (EGR1) expression changes during the whole ovarian vitrification and thawing procedure. The protein collection time point: A: Fresh ovary (FO); B: Vitrification dehydration (VD); C: Liquid nitrogen freezing (F); D: Thawing (T). \u003cstrong\u003eH) \u003c/strong\u003eWhole-mount immunofluorescence was used to assess vascular (CD31) and extracellular matrix (αSMA) maintenance in in-vitro cultured mouse ovarian tissues across different treatment groups. \u003cstrong\u003eI) \u003c/strong\u003eTUNEL staining of ovaries from Fresh, Vitrification-thawed and T-5224 treated group. Data are presented as the mean ± s.e.m. n = 5 for each group (unpaired two-tailed t-test). \u003cstrong\u003eJ)\u003c/strong\u003e Schematic illustration showing that activation of FOS/AP-1 in PCs and GCs cells contributes to ovarian cryoinjury induced apoptosis.\u003c/p\u003e","description":"","filename":"Picture7.png","url":"https://assets-eu.researchsquare.com/files/rs-6312954/v1/ca34dd66fc8df306925820c5.png"},{"id":82137619,"identity":"5fd6f2fe-7895-476e-9e09-16d141bb6c58","added_by":"auto","created_at":"2025-05-07 06:18:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12791957,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6312954/v1/c4c44fbc-ac13-4f09-a7f3-abea403c8a03.pdf"},{"id":79911408,"identity":"9f3e3a07-fd43-43b2-9b95-b0ca06fe553c","added_by":"auto","created_at":"2025-04-04 11:39:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7999466,"visible":true,"origin":"","legend":"Supplementary Material for Article File","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6312954/v1/d0b06f08ce558e876e4faeb5.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Spatiotemporal Dynamics of Human Ovarian Cortex Transcriptome Following Vitrification and Thawing: Insights into the FOS/AP-1 Pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOver the past two decades, advancements in cancer therapy have resulted in significant improvements in survival rates.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e And increasing numbers of young girls and women\u0026thinsp;\u0026lt;\u0026thinsp;45 years of age can now be cured of their disease.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e However, treatments such as chemotherapy (especially alkylating agents), radiotherapy, and surgery can lead to primary ovarian insufficiency (POI), highlighting the urgent need to address quality of life post-cancer remission.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Therefore, fertility preservation should be considered prior to any gonadotoxic treatment, particularly for adolescents and women of reproductive age.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Embryo and oocyte cryopreservation are well-established fertility preservation techniques, whereas ovarian tissue cryopreservation (OTC) is an emerging and promising approach.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOTC for fertility purposes has been performed over 25 years. Since the first report of a live birth after thawing and auto-transplantation of ovarian cortex in 2004,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e the procedure has gained ground and is now accepted and performed all over the world.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e As of 2022, more than 10,000 women worldwide have undergone ovarian tissue cryopreservation, and more than 200 live births have been achieved.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e More specifically, OTC and subsequent transplantation is the sole option that enables both restoration of fertility and resumption of ovarian endocrine function, avoiding the morbidity associated with POI.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Clinical meta-analysis shows that over 90% of women regain ovarian function after OTC-transplantation (OTT).\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e However, clinical outcomes of ovarian transplantation post-cryopreservation are significantly worse than those from fresh ovaries, indicating adverse effects associated with the cryopreservation process.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e For in vitro fertilization (IVF) after tissue cryopreservation and transplantation, the live birth rate (LBR) is only 21%, which is 41% for post-embryo cryopreservation.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e This discrepancy between high ovarian function recovery rates and lower live birth rates warrants further detailed investigation. Therefore, to improve follicle development quality post-transplantation, it is crucial to elucidate the mechanisms through which temperature stress and cryoprotectant toxicity damage ovarian cells.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe exploration of ovarian cryoinjury faces multiple challenges, mainly from the following aspects: Firstly, human ovary is a complex structure consisting of numerous heterogeneous cell types including stromal cells, granulosa cells, endothelial cells, immune cell, smooth muscle cells and oocytes.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e And current studies indicate that the ovary, due to its diverse cellular composition, is highly responsive to external environmental factors, chemical drug stimuli, and aging processes.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Secondly, vitrification is emerging as an efficient strategy for ovarian cryopreservation at the current stage,\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e which had been widely used for oocytes, embryos preservation.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Vitrification-thawing inevitably leads to alterations in the physiological metabolism of various cell types within the ovary. Up to date, preliminary investigation has been carried out to explore the impact of vitrification on ovarian tissue via morphological staining and transmission electron microscopy.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e High-throughput omics analyses have been conducted on oocytes and follicle complexes post-vitrification; however, multi-omics analyses have not yet to be performed on the ovarian cortex post vitrification-thawing.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e The impact of vitrification-thawing treatment on ovarian cells, especially at the single-cell transcriptome and Spatiotemporal levels, remains elusive.\u003c/p\u003e \u003cp\u003eWith advances of single-cell RNA sequencing technique, characterizing cell changes in ovarian tissue under physiological or pathological conditions has been carried out currently\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Smart-seq2 based single-cell sequencing strategy enables detection of abundant genes per cell, especially for oocyte.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Spatial enhanced resolution omics sequencing (Stereo-seq) is a novel and skillful spatial RNA sequencing method with nanoscale sensitivity and a large field of view.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, we aim to explore the cellular spatiotemporal changes and key regulatory genes involved in human ovarian cortex cryoinjury. We present a spatiotemporal atlas that systematically describes the spatial archetypes and cellular heterogeneity post human ovarian vitrification-thawing procedure. Detailly, granulosa, smooth muscle/perivascular cells and stromal cells had been identified as vitrification sensitive cell types. And the in-situ effects of vitrification-thawing on oocytes within ovary had also been explored via smart-seq2 strategy. Further, a potentially crucial role of FOS/AP-1 pathway in ovarian vitrification-thawed procedure had been tentatively explored. In all, our data provide valuable inspiration for the mechanism and potential therapeutic targets to vitrification-thawed stress and cryoprotectant toxicity induced ovarian cryoinjury.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003e2.1\u003c/strong\u003e \u003cstrong\u003escRNA-seq profiling and spatial location of human ovarian cortex cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe overview of the study design for the human ovarian cryoinjury spatiotemporal atlas is shown in\u0026nbsp;Figure 1A. Each of the three ovarian cortexes from gender reassignment surgery patients\u0026nbsp;(GRPs)\u0026nbsp;was divided into both fresh and cryopreserved groups (cryo).\u0026nbsp;The age information for the three groups is as follows: 29 years for Fresh \u0026amp; Cyro-1; 34 years for Fresh \u0026amp; Cyro-2; 35 years for Fresh \u0026amp; Cyro-3.\u0026nbsp;Following by collagenase IV digestion, single-cell RNA sequencing (scRNA-seq) was applied to 27,185 fresh ovarian cells. Post standard ovary vitrification-thawing procedure, the cryopreserved group had been digested and\u0026nbsp;conducted with scRNA-seq on 25,480 cells. Since large cells cannot be wrapped in microdroplets (dimeter \u0026gt;40um), Smart-seq2 strategy had been applied to oocyte in the ovarian cortex.\u0026nbsp;Cells with mitochondrial gene expression exceeding 25% of the total unique molecular identifiers (UMIs) were excluded\u0026nbsp;(Figure S1B). And the total cellular RNA content and number of expressed genes showed no significant difference among the three groups (Figure S1A and C). More detailed information can be found in the supplementary\u0026nbsp;Major Resources Table 1.\u003c/p\u003e\n\u003cp\u003eUsing the Uniform Manifold Approximation and Projection (UMAP) algorithm for the nonlinear dimensionality reduction analysis,\u0026nbsp;we identified eight distinct cell types based on specific markers (Figure 1B and Figure S1D).\u0026nbsp;These include\u0026nbsp;granulosa cells (GCs; AMH+), oocytes (DDX4+), stromal cells (SCs; STAR+), smooth muscle/perivascular cells (PCs; MACM+), blood endothelial cells (BECs; VWF+), lymphatic endothelial cells (LECs; PROX1+), macrophage (M; CD68+) and natural killer T cells (NKTs; CD3D+).\u0026nbsp;The expression levels of markers for different cell types in the ovarian cortex were visualized using Featureplot(Figure 1C).\u0026nbsp;Additionally, the biological function of each cell cluster were analyzed through Gene Ontology (GO) analysis of differentially expressed genes (DEGs) (Figure 1D),\u0026nbsp;uncovering unique characteristics of these ovarian subclusters. For example, GO terms specific to oocytes,\u0026nbsp;such as ‘‘meiotic cell cycle’’ and ‘‘gamete generation’’, were identified.\u0026nbsp;GO terms including ‘‘regulation of follicle-stimulating hormone secretion’’ and ‘‘reproductive system development’’ were enriched in GCs.\u0026nbsp;Meanwhile,\u0026nbsp;‘‘Cytoplasmic translation’’ and ‘‘extracellular matrix organization’’ were\u0026nbsp;prominent in\u0026nbsp;SCs, indicating their role in providing nutritional and structural support for ovary.\u003c/p\u003e\n\u003cp\u003eSubsequently, stereo-seq based single-cell spatial transcriptome sequencing strategy (ST-seq) were conducted on the fresh and cryopreserved groups.\u0026nbsp;Specifically, the sections were obtained separately from the Fresh-1 and Cryo-2 groups.\u0026nbsp;The\u0026nbsp;sequencing depth of\u0026nbsp;ST-seq\u0026nbsp;reached 1000 genes/dot (Fresh: 934 genes/pot, Vitrification-thawing: 841 genes/pot) when bin = 50, indicating high data quality\u0026nbsp;(Figure S1E and F).\u0026nbsp;Utilizing the scRNA-seq atlas, factor analysis was conducted to infer the probable single-cell composition of each spot, effectively mapping all scRNA-seq clusters.\u0026nbsp;We identified eight celltypes, including GCs,\u0026nbsp;two subtypes of SCs (SCs1 marked by COL1A2+ and SCs2 marked by GPR78+),\u0026nbsp;PCs,\u0026nbsp;smooth muscle cells (SMCs), ECs, theca-interstitial\u0026nbsp;cells, oocytes and red blood cells (RBCs) (Figure 1E-G). SpatialFeaturePlot visualizes the in situ spatial expression patterns of subgroup marker gene in the ovarian cortex (Figure S1G).\u0026nbsp;Additionally, a heatmap displayed the top cluster markers, revealing significant overlap between cell cluster markers identified by both scRNA-seq and Stereo-seq\u0026nbsp;(Figure 1H).\u0026nbsp;Overall, spatial cell mapping elucidated the distribution of clusters predicted by scRNA-seq within the human ovarian cortex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTranscriptional Shifts in Ovarian Cortex: Comparing Slow Frozen, Vitrified, and Fresh Conditions at the Single-Cell Level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparing the differences between slow freezing—a method clinically approved and tested over decades for ovarian tissue cryopreservation—and vitrification is crucial. Due to the difficulty in obtaining human ovarian samples and ethical constraints, we are currently unable to perform single-cell library sequencing analysis on slow-frozen human ovarian cortex samples. Fortunately, researchers have conducted single-cell sequencing on slow-frozen human ovarian cortex tissue (M Wagner et.al. \u003cem\u003eNat Commun\u003c/em\u003e, 2020)\u003csup\u003e24\u003c/sup\u003e, allowing for direct integration and comparison with our data.\u0026nbsp;The UMAP analysis results indicate that the eight major subgroups are consistently present across all three sample groups without notable absence (Figure 2A). In-depth analysis of differentially expressed genes revealed distinct variations among the groups (Figure 2B). Specifically, comparisons between slow-freezing and vitrification highlighted significant differences in genes associated with focal adhesion, the p53 signaling pathway, and the MAPK pathway (Figure 2C). DNA damage and apoptosis score analyses indicated that slow freezing resulted in slightly less negative outcomes compared to vitrification (Figure 2D and E). Furthermore, cellular crosstalk analyses conducted via CellPhoneDB and CellChat revealed that slow freezing significantly reduced intercellular interactions compared to vitrification (Figure 2G and H).\u0026nbsp;We further explored the changes in Wnt and Notch pathways in the ligand-receptor interactions between oocytes and other cells in slow frozen, vitrified, and fresh groups. Dotplot results indicate that the Wnt-FZD and JAG-NOTCH pathways are nearly absent in the slow_frozen group (Figure 2I). These pathways are closely related to cell proliferation, which may also explain the more severe apoptosis observed in the slow frozen group.\u003c/p\u003e\n\u003cp\u003eOverall we may draw a conclusion as follows: 1) We identified genes differentially expressed between slow-frozen and vitrified-thawed ovarian cortex, notably in intercellular communication and the MAPK signaling pathway. 2) Comparative analysis indicates less oocyte damage in slow freezing compared to vitrification. 3) Cell-cell communication analysis showed greater disruption in slow freezing than in vitrification, particularly affecting oocyte interactions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Cell-specific changes following ovarian vitrification-thawing’s transcriptional programs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative analysis of gene expression patterns across various ovarian cell types in the fresh and vitrification-thawed (cryo) groups had been conducted (Figure 3A). UMAP plot showed seven ovarian cell clusters in each sample (Figure 3B). Quantitative analysis revealed that the proportion of PCs increased significantly in all three replicate samples, with statistical significance, while SCs showed the opposite trend (Figure 3C and D). To evaluate the reliability of Figure 2C, the publicly available single-cell datasets of\u0026nbsp;GSE255690 (GEO No.)\u003csup\u003e28\u003c/sup\u003e was integrated with our dataset. As expected, most ovarian subcluster from the same tissue sources were clustered together (Figure S2A and B).\u0026nbsp;Integration and cell proportion analysis also showed a significant upregulation in stromal cell subclusters post vitrification-thawing (Figure S2C). Additionally,\u0026nbsp;eight cell types were further validated in the integrated single-cell data using specific cell markers (Figure S2D).\u003c/p\u003e\n\u003cp\u003eDifferentially expressed genes (DEGs; top 50 with |avg_logFC| \u0026gt; 0.25 and P adj\u0026lt; 0.05) between the fresh and vitrification-thawed groups in at least one cell type were identified and shown in heatmap (Figure 3E). Pathway analysis demonstrated that the DEGs are mainly involved in “focal adhesion” and “apoptosis” (Figure 3F). CellPhoneDB is a bioinformatics toolkit designed to infer cell-cell communication by analyzing the combined expression of multi-subunit ligand-receptor complexes.\u003csup\u003e42\u003c/sup\u003ePost vitrification-thawing process, there was a significant reduction in ligand-receptor interactions between ovarian cells, notably between granulosa and other cell types\u0026nbsp;(Figure S3A).\u0026nbsp;Further analysis of individual subpopulations revealed that certain DEGs, such as complement 3 (C3), were unique to specific subpopulations, predominantly observed in stromal and granulosa cells (Figure S3B). Which indicates that different cell types exhibited heterogeneous responses to the vitrification-thawing process.\u003c/p\u003e\n\u003cp\u003eBased on the above results, apoptosis and extracellular matrix organization may play an important role in human ovarian cryoinjury. Post vitrification-thawing procedure, the signaling pathway score for focal adhesion decreased while apoptosis increased in the majority of ovarian cells (Figure 3G). Pathway analysis of spatial gene expression data further confirmed heightened enrichment of apoptosis and a\u0026nbsp;diminished activity\u0026nbsp;of focal adhesion pathway in the vitrification-thawed ovarian cortex (Figure 3H).\u0026nbsp;Masson’s trichrome staining revealed that fresh ovarian tissue had a higher collagen content compared with vitrification-thawing ones (Figure 3I and Figure S3G). And\u0026nbsp;transmission electron microscopy analysis showed noticeable shrinkage in\u0026nbsp;oocytes and surrounding cells, confirming a reduction in intercellular communication (Figure 3J).\u0026nbsp;Additionally, TdT-mediated dUTP nick end labeling (TUNEL staining) further demonstrated a significant increase in the number of cells undergoing apoptosis following vitrification-thawing treatment\u0026nbsp;(Figure 3K).\u0026nbsp;In all, our findings suggest that mesenchymal disruption and increased apoptosis are predominant features of cryoinjury in human ovary cortex. However, specific cell types within the ovarian cortex may exhibit distinct characteristics associated with cryoinjury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Vitrification and thawing altered the gene expression profiles of ovarian oocytes \u003cem\u003ein situ\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDormant primordial follicles,\u0026nbsp;situated within the approximately 1 mm thick ovarian cortex (Figure S1E),\u0026nbsp;are the primary focus of vitrification in ovarian tissue.\u0026nbsp;This prompts significant interest in investigating the impact of vitrification on the physiological metabolism of oocytes within the ovarian cortex.\u0026nbsp;We sequenced 115 oocyte, and 5 oocyte sequencing data had been deleted due to abnormally high expression of mitochondrial genes (\u0026gt;30%). Finally,\u0026nbsp;single-cell sequencing data from 110 oocytes at various stages had been analyzed\u0026nbsp;(66 oocytes from Vitrification group, 44 from Fresh group). During the process of oocytes isolation from the dissociated ovarian tissue, we can’t identify what stage of these oocytes just under light filed with microscopy. Fortunately, the expanding availability of transcriptomic data enables the identification of genes specifically expressed in unique stage of the oocyte cycle.\u003csup\u003e43, 44\u003c/sup\u003e Vlnplot analysis revealed high expression levels of LMOD3, RPS4X, and FIGLA in these oocytes, suggesting that they were predominantly at the primordial or primary stage\u0026nbsp;(Figure S4A). AddModuleScore and Doheatmap analysis show genes specific to primary oocytes at the primordial stage are enriched in Cluster 1, while those marking later stages are upregulated in Cluster 2\u0026nbsp;(Figure S4B and C). This indicates that the majority of the oocytes used for smart-seq were derived from primordial follicles (~75 oocytes).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIntegrated analysis demonstrated substantial overlap in the expression profiles of oocytes from fresh and vitrified ovarian cortex, indicating generally identical\u0026nbsp;(Figure 4A). Benefiting from the extensive sequencing depth of Smart-seq2 strategy, we identified a number of differentially expressed genes as shown in the heatmap analysis (Figure 4C).\u0026nbsp;Notably, meiosis-related genes, namely, ZP family (including ZP2, ZP3 and ZP4),\u003csup\u003e45\u003c/sup\u003e OOEP and OOSP4B, were considerably downregulated (Figure 4B and C).\u0026nbsp;In addition, cell cycle-related genes, including CKS1B and TUBB8,\u003csup\u003e46, 47\u003c/sup\u003e were significantly downregulated in vitrification ovarian oocytes, whereas CDCA7L and RAB18 were upregulated (Figure 4C). Intriguingly, gene related to heat response, namely, DNAJA2 (Hsp40), was also significantly upregulated in cryo-rewarming ovarian oocytes (Figure 4B). To validate the bioinformatic analysis claims, multiplex immunofluorescence staining had been employed. The results indicate an increased proportion of oocytes co-expressing SOHLH2 and DDX4 (Figure 4F and H), consistent with the analysis results of Featureplot (Figure 4G). And the upregulated gene SOHLH2 had been reported as primary ovarian insufficiency candidate genes.\u003csup\u003e48, 49\u003c/sup\u003e This prompts us to further explore the impact of changes in the expression levels of these genes on later embryonic development and physiological metabolism of offspring.\u003c/p\u003e\n\u003cp\u003eGO analysis\u0026nbsp;revealed that genes upregulated after vitrification-thawing were primarily associated with cell cycle-related pathways\u0026nbsp;(Figure 4D and E). Intriguingly,\u0026nbsp;our observations also indicate the involvement of epigenetic pathways,\u0026nbsp;such as, “histone H2A acetylation” and “DNA methylation involved in gamete generation” (Figure 4D upper). These findings suggest that epigenetic regulatory mechanisms following ovarian vitrification-thawing merit further exploration. In addition, some pathway, related with apoptosis and embryonic development were observed in downregulated genes groups (Figure 4E upper).\u0026nbsp;Furthermore, KEGG pathway analysis\u0026nbsp;identified enrichment of several key pathways in vitrified ovarian oocytes, including\u0026nbsp;“carbon metabolism”, “the citrate cycle”, “RNA degradation”, and “endocytosis” (Figure 4D and E bottom).\u0026nbsp;Additionally, we compared the DNA damage and repair gene list and generated scores during ovarian vitrification and thawing. DNA damage scores decreased and DNA repair scores increased in vitrification-thawed ovarian oocytes (Figure S4D).\u003c/p\u003e\n\u003cp\u003eCell-cell communication analysis via\u0026nbsp;CellPhoneDB revealed that, after vitrification-thawing, the oocyte in situ displayed a diminished interaction with granulosa, perivascular and stromal cells (Figure 4I). Detailly,\u0026nbsp;the interaction between oocytes and peripheral cells via TGFβ (TGFB3, TGFB2) and its receptors is enhanced, indicating an activation of inflammation-related signaling pathways. While the Notching signaling pathway from Oocyte to peripheral cells (i.e., NOTCH1_DLK1, NOTCH1_DLL3\u0026amp;4 and NOTCH1_JAG1) decreased (Figure 4J and K). Intriguingly,\u0026nbsp;the interaction between the AMH receptor (MIS receptor) and AMH from peripheral cells to oocytes was diminished, yet the reverse interaction remained unaffected\u0026nbsp;(Figure 4J and K). The underlying molecular mechanism deserves further exploration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 PTGDS positive stromal cells (PTGDS\u003csup\u003e+\u003c/sup\u003e SCs) were sharply decreased post ovarian vitrification-thawing progress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStromal cell subpopulations are now a focal point of investigation (Figure 5A).\u0026nbsp;Quantitative analysis revealed a significant decrease in Subcluster 1 after the vitrification-thawing process\u0026nbsp;(Figure 5B).\u0026nbsp;A heatmap displayed the top genes predominantly expressed in Subcluster 1, including PTGDS, C3, SFRP4, and IGFBP6\u0026nbsp;(Figure 5C).\u0026nbsp;GO analysis was conducted on the top differentially expressed genes in stromal cells following the vitrification-thawing procedure.\u0026nbsp;Pathways related to\u0026nbsp;“reactive oxygen responding”, stress-inflammatory response-related pathways such as “MAPK signaling pathway” are significantly activated (Figure 5D). Here, we defined subcluster 1 as PTGDS-positive subpopulations (PTGDS\u003csup\u003e+\u003c/sup\u003e SCs) (Figure 5E).\u0026nbsp;Additionally, RNA from both fresh and vitrified-thawed ovarian cortex was enriched and analyzed via real-time PCR, with results confirming the bioinformatics findings\u0026nbsp;(Figure 5F).\u0026nbsp;ST data revealed decreased expression of C3, SFRP4, and PTGDS, particularly in the COL1A2+ stromal cell subtype located in the cortex\u0026nbsp;(Figure 5G).\u003c/p\u003e\n\u003cp\u003eTo validate the bioinformatic analysis findings, RNAscope-based in situ hybridization (ISH) had been carried on human ovary tissues paraffin section. As expected, PTGDS positive cells are mainly distributed in the extracellular matrix. In the vitrification-thawing ovarian cortex section, the expression of PTGDS showed a downregulated profile compared with fresh control (Figure 5H) aligning with IHC results (Figure S5F).\u0026nbsp;We replicated the ovarian cryopreservation strategy in murine ovaries. PTGDS also exhibited similar expression profile (including mRNA and protein expression level) changes as observed in human tissue samples (Figure 4I and Supplemental Figure S5G). This poses an intriguing question about the role of PTGDS-positive stromal cells and PTGDS genes in ovarian cryopreservation. The underlying pathways and metabolic processes involved warrant further exploration.\u003c/p\u003e\n\u003cp\u003eThe graphs showed that post vitrification-thawing, there was not only a decrease in PTGDS-positive cell subgroups but also an increase in subcluster 2 (Figure 5B), indicating a rapid and varied response of stromal cells to vitrification. Subsequent detailed analysis of cluster 2 revealed a subgroup with increased abnormalities post-vitrification (Figure S5A and B, Subgroup 4). Featureplot and temporal-based expression profiling both demonstrate that after vitrification and thawing, genes from the heat shock protein family, namely DNAJB1, HSP90A1, and HSP90B1, are notably enriched and expressed in the newly emerged subgroup 4 (Figure S5C and E). Additionally, Pseudo-temporal analysis showed that certain PTGDS-positive stromal cells displayed a tendency to transform into cells with higher expression of temperature-sensitive genes (Figure S5D and E). These findings collectively suggest the presence of\u0026nbsp;heightened sensitivity\u0026nbsp;and unique cryoinjury characteristics\u0026nbsp;in\u0026nbsp;SCs post ovarian vitrification-thawing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 The FOS/AP-1 signaling pathway is activated in smooth muscle/perivascular cells and granulosa cells following the vitrification-thawing procedure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticular attention should be paid to the perivascular subtissue structures surrounding blood vessels, especially smooth muscle cells, as they participate in multicellular stratified systems.\u003csup\u003e50\u003c/sup\u003e As shown in Figure 1D, the\u0026nbsp;smooth muscle/perivascular\u0026nbsp;subcluster increased apparently after vitrification-thawing treatment.\u0026nbsp;Reclustering analysis segmented the smooth muscle/perivascular cells into six clusters, with cluster 3 (FOS+)\u0026nbsp;exhibiting\u0026nbsp;a significant increase post vitrification-thawing\u0026nbsp;(Figure 6A). The specifically highly expressed genes in cluster 3\u0026nbsp;predominantly include FOS, FOSB, JUN, JUNB, and JUND, as evidenced by the FeaturePlot and ViolinPlot\u0026nbsp;(Figure 6G and Figure S6A). Meanwhile, heatmap results demonstrate consistent activation trends of AP-1 protein family members, including FOS, FOSB, and JUN, across three sets of replicate experiments (Figure 6C).\u0026nbsp;Furthermore,\u0026nbsp;pathway analysis indicated that DEGs\u0026nbsp;following ovarian vitrification predominantly relate to\u0026nbsp;“Amyotrophic lateral sclerosis”, “Huntington disease” et al.,\u0026nbsp;which are closely linked to muscle physiological dysfunction\u0026nbsp;(Figure 6D).\u0026nbsp;Granulosa cells play an important role in follicle development and oocyte maturation.\u003csup\u003e51, 52\u003c/sup\u003e Reclustering analysis revealed that\u0026nbsp;granulosa cells had been divided into 3 subgroups. Moreover subcluster 3 (FOS/AP-1 active GCs) showed a significant increase in proportion post vitrification-thawing (Figure 6B). Intriguingly, the top expressing genes in cluster 3 including FOS, FOSB, JUN, JUNB and\u0026nbsp;JUND\u0026nbsp;as shown by Featureplot (Figure 6H).\u0026nbsp;SpatialFeaturePlot indicates that the expression of FOS, FOSB, JUN, and JUNB significantly increased in situ following vitrification-thawing, corroborating the scRNA analysis findings\u0026nbsp;(Figure S7A).\u0026nbsp;Multi-immunofluorescence labeling\u0026nbsp;had been conducted and revealed\u0026nbsp;a significant increase in FOSB protein abundance in smooth muscle and perivascular cells following\u0026nbsp;vitrification-thawing (Figure S7B).\u003c/p\u003e\n\u003cp\u003eThe above observation prompted a more detailed examination of the FOS/AP-1 pathway. Utilizing\u0026nbsp;the gene scoring algorithm,\u0026nbsp;both scRNA-seq and ST-seq data confirming FOS/AP-1 heightened activation in pericytes (PCs) compared to other subgroups (Figure 6E and F). Additionally, we had also focused on DNA damage repair, ROS-related genes, NF-κB pathway and AP-1 complex, but the effects of cryoinjury were not obvious (Figure S8).\u003c/p\u003e\n\u003cp\u003eTo validate the upregulation of FOS\u003csup\u003e+\u003c/sup\u003e perivascular cells,\u0026nbsp;RNAscope-based\u0026nbsp;ISH\u0026nbsp;had been conducted. As expected, expression profile of FOS increased apparently in vitrification human ovarian cortex compared with fresh ones (Figure 6I), aligning with the FOSB’s immunochemical staining results (Figure 6J and Figure S6B).\u0026nbsp;Furthermore, mRNA from both fresh and vitrified-thawed ovarian cortex was enriched and analyzed using real-time PCR, with results that corroborated the bioinformatics analysis (Figure 6K).\u0026nbsp;The FOS family (including FOS, FOSB, FOSL1, and FOSL2) can dimerize with proteins of the JUN family, forming the transcription factor complex AP-1.\u003csup\u003e53, 54\u003c/sup\u003e FOS proteins are implicated as regulators of cell proliferation, differentiation, and transformation. Together, these results suggest that\u0026nbsp;FOS/AP-1 pathway is activated immediately in response to temperature stress and cryoprotectant toxicity, providing insight into the mechanism underlying\u0026nbsp;FOS’s\u0026nbsp;role in preventing ovarian cellular cryoinjury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFOS/AP-1 is rapidly activated in the early flow of vitrification process, independently of TGFβ mediation, and its inhibition maintains the viability of frozen-thawed ovaries \u003cem\u003ein vitro\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have claimed that FOS/AP-1 pathway rapidly responds to external stimuli and stress, primarily facilitating the translation and accumulation of stress-related inflammation.\u003csup\u003e55, 56, 57\u003c/sup\u003e AP-1 activity is induced by numerous extracellular matrix and genotoxic agents, suggesting involvement in programmed cell death. \u003csup\u003e58\u003c/sup\u003e Cell-cell communication analysis showed a significant reduction in ligand-receptor pairs between FOS/AP-1-activated granulosa/perivascular cells and other cell types, especially oocytes\u0026nbsp;(Figure 7A and Figure S9A). Furthermore, we focused on certain ligand-receptor sets involved in cell proliferation, differentiation and inflammation responses. As expected, FOS/AP-1-activated PCs and GCs\u0026nbsp;exhibited a\u0026nbsp;decrease in ligand-receptor\u0026nbsp;interactions within\u0026nbsp;NOTCH signaling which promotes proliferating (Figure 7B). Unexpectedly, although involved in the activation of FOS/AP-1, the TGFβ pathway showed a significant decrease in ligand-receptor pairs after vitrification-thawing\u0026nbsp;(Figure 7B).\u0026nbsp;This suggests that FOS/AP-1 activation is primarily driven by direct stress and chemical toxicity at this stage.\u003c/p\u003e\n\u003cp\u003eThe maintenance of cell identity involves the coordinated action of many regulators, among which transcription factors have been long recognized to play a central role.\u003csup\u003e59\u003c/sup\u003e SCENIC was used to identify transcription factors in granulosa cell to predict essential regulators involved in cryoinjury. Regulon specificity scores (RSSs) identified top 3 specific regulons including FOSB (Figure 7C). Here we further identified EGR1 as co-target to FOS and JUN (Figure 7D). Moreover,\u0026nbsp;co-immunofluorescence and histochemical staining results indicated that EGR1 expression was significantly upregulated in smooth muscle/ perivascular cells following vitrification-thawing (Figure 7E and Figure S9B). And the key pathways involved in our research, as well as the molecules (FOS, FOSB, EGR1), are highly conserved\u0026nbsp;in mice (Figure S9C and D).\u0026nbsp;Additionally, we confirmed through Co-immunoprecipitation experiments that there is no direct interaction between FOS and EGR1 (Figure S10D). FOS is a transcriptional regulatory factor\u003csup\u003e60\u003c/sup\u003e, that we believe predominantly enters the cell nucleus after phosphorylation to regulate the upstream promoter of EGR1.\u003c/p\u003e\n\u003cp\u003eThese above intriguing results prompt us to\u0026nbsp;systematically explore the specific flows and key points where and when the FOS/AP-1 pathway changes (Figure 7F). We refined the overall vitrification process into four key processes: obtaining fresh ovarian tissue cortex (FO), vitrification dehydration (VD), liquid nitrogen freezing (F) and thawing (T). Interestingly,\u0026nbsp;western blotting revealed that FOS/AP-1 activation occurred at the onset of the vitrification dehydration phase and persisted after thawing\u0026nbsp;(Figure 7G). Additionally,\u0026nbsp;we conducted a preliminary exploration of the function of FOS/AP-1 in the ovary. FOS/AP-1 was activated by the agonist Phorbol 12-myristate 13-acetate (PMA) (Figure 10SA), which has been reported as an AP-1 agonist. Following this activation, the proliferation of human granulosa-like cell line (KGNs) had been inhibited (New Figure S10B).\u0026nbsp;Activation of the FOS/AP-1 pathway increased apoptotic cell percentages in the KGN cell line. Concurrently, T5224 administration mitigated apoptosis induced by PMA treatment\u0026nbsp;(New Figure S10C).\u0026nbsp;It is important to know whether inhibiting\u0026nbsp;FOS/AP-1\u0026nbsp;recovers the defect.\u0026nbsp;T-5224 is a transcription factor FOS/AP-1 inhibitor with anti-inflammatory effects, which specifically inhibits the DNA binding activity of FOS/AP-1 without affecting other transcription factors.\u003csup\u003e61\u003c/sup\u003e Hence, we treated murine ovary with T-5224 adding in the cryoprotectants during vitrification-thawing (Figure 7H\u0026nbsp;upper).\u0026nbsp;Wholemount tissue immunofluorescence staining demonstrated that vascular network density in vitrification-thawed ovaries was well maintained following T-5224 adding \u003cem\u003ein vitro\u003c/em\u003e (Figure 7H bottom). Additionally, TUNEL staining confirmed that T-5224 treatment reduced apoptosis in vitrified-thawed ovarian cells (Figure 7I \u0026amp; J). In summary, these findings suggest that inhibiting FOS/AP-1 via T-5224 improves ovarian function post vitrification-thawing.\u0026nbsp;\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThis study conducted a comprehensive analysis of single-cell and spatial transcriptomic atlas of human ovaries throughout the cryopreservation procedure, illuminating spatial and temporal variations in gene expression during vitrification and thawing. The effects of vitrification-thawing on ovary had been investigated previously but very superficial and limited.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The noteworthy contributions of our work are as follows: Firstly, we delineated gene expression signatures and spatial locations for eight types of human ovarian cells, pinpointing cell type-specific DEGs during ovarian cortex vitrification-thawing (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Both in vitrification-thawed and fresh human ovarian cortex, the stromal cells, granulosa cells, oocytes, endothelial cells, and smooth muscle/perivascular cells were classified according to their anatomical structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Generally, after vitrification-thawing, significant alterations were observed in pathways related to apoptosis, extracellular matrix synthesis, and fibrosis. Notably, genes such as FOS, C3, JUN, and JUNB were upregulated across all cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Secondly, we focused on the differential effects of slow freezing and vitrification on ovarian cortex at the single-cell level, which is being reported for the first time. Interestingly, slow freezing caused more severe overall cell apoptosis but had less impact on oocytes. Additionally, intercellular communication was more significantly disrupted in the slow freezing group compared to vitrification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thirdly, via Smart-seq2 strategy, an exploration of cryoinjury-associated changes in gene expression highlighted the cell cycle response as a biological pathway involved in oocyte cryopreservation \u003cem\u003ein situ\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Fourthly, subclustering analysis identified PTGDS-positive stromal cells as one of the primary cell types sensitive to vitrification-thawing, along with changes in transcriptomic features during cryopreservation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Fifthly, integrating scRNA-seq data with ST-seq identified FOS/AP-1 as a potential key transcriptional factor for ovarian cellular cryoinjury, accelerating the transcription of EGR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). And inhibiting FOS via T-5224 improves maintenance of ovarian function in vitro post vitrification and thawing (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings provide new insights into human ovarian cortex cryopreservation and present potential targets for the treatment of ovarian cryoinjury.\u003c/p\u003e \u003cp\u003eIn vitrified-thawed ovaries, a notable observation is that most cell types exhibit characteristics associated with apoptosis. These changes include increased DNA repair scores, enhanced NF-κB and AP-1 pathway activity, and alterations in molecules related to the apoptosis pathway. Previous studies mainly focus on two aspects of vitrification-induced damage to the ovary: 1) direct cellular damage, such as DNA damage and apoptosis; 2) disruption of the intercellular microenvironment, including matrix collagen degradation. Significantly, there was a marked decrease in the stromal cell cluster, indicative of a reduced extracellular matrix phenotype. Concurrently, the rapid activation of the FOS/AP-1 pathway primarily in smooth muscle/perivascular cells (SM/PCs) and granulosa cells (GCs) suggests enhanced apoptosis and inflammation.\u003c/p\u003e \u003cp\u003eThe expression changes of oocyte within ovarian cortex after vitrification-thawing merit detailed examination. The oocyte-corona-cumulus complex (OCCC) development had been investigated systematically at single-cell level.\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e Recent study claimed that the effects of vitrification on the transcriptomes of mature human oocytes in vitro (metaphase II oocytes) are induced by the procedure itself rather than by the storage time\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. While the effects of vitrification on oocytes in-situ are unclear. Here, cell cycle and meiosis-related physiological processes are identified as the primary pathways altered in oocytes within the ovary post vitrification-thawing. Moreover, some epigenetic related terms, such as histone H2A acetylation and DNA methylation, had also been observed in the DEGs GO analysis list. Additionally, the ligand-receptor interaction between oocyte and surrounding cells also are diminished, especially the NOTCH and AMH pathway related to developments. While our approach has certain limitations, it is important to note that due to variability in stages, we cannot entirely exclude the heterogeneity within the fresh or vitrification-thawed oocyte groups \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eMesenchyme of ovarian stroma (herein called stromal cell), which is similar in morphology to fibroblasts, make up the connective tissue throughout the ovary and surround follicles.\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e The ovarian stroma comprises mostly incompletely characterized stromal cells (e.g., fibroblast-like, spindle-shaped, and stromal cells).\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e Recent studies have revisited the role of ovarian stromal cells, highlighting their significant contributions to folliculogenesis, particularly in the activation of primordial follicles and the differentiation of theca cells.\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e The plasticity of ovarian stromal cells is a key point of research, a recent study claimed that inhibition of ovarian fibrosis in part through regulating stromal cell differentiation via the TGF-β1/Smad3 signaling pathway can recover the function of ovarian function in POI rats.\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e Here, we identified a new vitrification sensitive stromal cell cluster, called PTGDS + stromal cells, and its biological function deserves further study.\u003c/p\u003e \u003cp\u003ePTGDS (prostaglandin D2 synthase, PTGDS) is purified and firstly identified by Y Urade et al.\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e Previous study claimed that PTGDS catalyzes the conversion of PGH2 to PGD2, a prostaglandin involved in smooth muscle contraction/relaxation and a potent inhibitor of platelet aggregation.\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e This enzyme (PTGDS) is a dual-function protein; it acts as a PGD2-producing enzyme and also as a lipophilic ligand-binding protein.\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e Knocked-out(KO) mice of PTGDS are new model animals of aging, as they showing progressive age-related cartilage degradation\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e and adenomyosis.\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e Function of PTGDS had been studied in male reproductive system such as testis and epididymis.\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e Little is known about the function of PTGDS in human ovary. And we demonstrated that PTGDS\u003csup\u003e+\u003c/sup\u003e SCs is one of the most sensitive cell populations to cryopreservation. The up-downstream signaling pathways of PTGDS involved in ovarian function will be explored in further.\u003c/p\u003e \u003cp\u003eThe molecular network/key factors governing the cryoinjury of ovarian cells are not well understood. In this study, our analyses identified FOS/AP-1 as a crucial TF regulating cellular cryoinjury in the ovarian cortex. During vitrification-thawing, we noted an increasing in FOS expression in ovarian PCs and GCs cells. Stereo-seq analysis allows us to discern whether FOS/AP-1 pathway activation is due to single-cell suspension digestion or the cryopreservation procedure itself. As expected, the FOS/AP-1 was upregulated \u003cem\u003ein situ\u003c/em\u003e during vitrification-thawing procedure. FOS is part of the AP-1 complex family, which includes FOS, FOSB, FOSL1, and FOSL2. These proteins dimerize with members of the JUN family to form the transcription factor complex AP-1.\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e Previous research reported that FOS/AP-1 regulates metabolic changes and cholesterol synthesis in human periovulatory granulosa cells.\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e Additionally, AP-1 can manifest oncogenic or anti-oncogenic effects by regulating genes involved in cell proliferation, differentiation, apoptosis, angiogenesis, and tumor invasion.\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e Furthermore, previous studies have claimed the ability of FOS/AP-1 to bind on FOXP3 gene locus and promote the expression of this master regulator of Treg identity, which leads to the accumulation of inflammation.\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e Consistently, we observed signification upregulation of inflammation- accumulation pathways, such as NF-κB signaling. Collectively, our data propose FOS/AP-1 as a potential target for recovering ovarian cryoinjury. And further investigation is needed to elucidate how the FOS/AP-1 pathway is activated, particularly determining whether cryoprotectant toxicity or temperature stress plays the dominant role.\u003c/p\u003e \u003cp\u003eThe novel benzophenone derivative T-5224 was rationally designed to serve as a potential drug to inhibit transcription regulated by AP-1.\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e In this study, we discovered its efficacy in improving ovarian function in vitro after vitrification and thawing. It is important to note that T-5224 is safe for oral administration to humans. \u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e Therefore, the selective FOS/AP-1 inhibitor T-5224 appears suitable for development as a therapeutic agent for the recovery of ovarian degeneration in humans post cryopreservation and transplantation.\u003c/p\u003e \u003cp\u003eIn conclusion, we have mapped the spatiotemporal single-cell transcriptomic landscape of human ovarian cryoinjury and identified FOS/AP-1 as activated with vitrification-thawing and modulating ovarian recovery. The pharmacological silence of FOS, as inhibited by T-5224, emerges as a promising therapeutic strategy for mitigate ovarian damage post-vitrification-thawing. Our study deepens the understanding of human ovarian cryoinjury during vitrification-thawing, providing a valuable resource for investigating potential therapeutic interventions. Moving forward, we aim to explore FOS/AP-1 as a potential therapeutic target for restoring ovarian function after frozen-thawed transplantation.\u003c/p\u003e "},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eHuman samples and ethical statement\u003c/b\u003e \u003c/p\u003e\u003cp\u003eHuman ovaries were obtained from participants who underwent from gender reassignment surgery patients (GRPs). (Major Resources Table). The sample collection was approved by the local ethics committee (Approval No. B2022269P). And all participants provided informed consent and independent clinicians informed the patients about the study.\u003c/p\u003e\u003cp\u003e \u003cb\u003eAnimal work\u003c/b\u003e \u003c/p\u003e\u003cp\u003e All animal experiments were performed in accordance with the standard protocols, animal welfare regulations, and the institutional guidelines of International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University. We confirm that the study was carried out in compliance with the ARRIVE guidelines.\u003c/p\u003e\u003cp\u003e \u003cb\u003eHuman ovarian vitrification and thawing\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe KITAZATO Ovarian Tissue Vitrification Kit (82212, 82222 and 81213 KITAZATO) was used for Human ovarian vitrification and thawing process. In brief, the ovarian cortex was cut into pieces (about 1cm X1cm) and washed with Ova Rinse. Then, tissue pieces were equilibrated in gradually increasing concentrations of cryoprotective agents (CPA) with a final concentration of 20% DMSO, 20% ethylene glycol (EG) and 0.4 M sucrose.\u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e After equilibration, remove excess medium by placing the ovarian tissue on a piece of gauze. Lay the ovarian tissue on Ova Cryo Device with its superficial dimension spread out as much as possible. The excess solution was removed using blotting paper. Using fine tweezers, the Ova Cryo Device were plunged directly into liquid nitrogen and then gently put into cryovials under liquid nitrogen and the vial caps sealed. Finally, set the device to a cane and store in storage tank.\u003c/p\u003e\u003cp\u003eAfter 2 weeks, the Ova Cryo Device with vitrified ovarian tissues were removed from the cryovials using tweezers while still under liquid nitrogen and then plunged directly into 37°C vitrification medium (Thaw1). After thawing for 30 s, tissues were transferred through a series of gradually decreasing concentrations of CPA for 5 min at each concentration to remove the CPA osmotically (20%, 10%, 5% and 0% for both DMSO and EG with 0.4 M sucrose, concluding with 0.2, 0.1 and 0 M sucrose). Then, tissues were washed in L-15 medium followed with digestion and fixation.\u003c/p\u003e\u003cp\u003e \u003cb\u003eOvarian single cell suspension preparation\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFor 10X Genomics strategy, the fresh and vitrification-thawing processed human ovarian cortex were cut into pieces with very sharp knives, followed by dissociating in collagenase I solution(2mg/ml) for 45 minutes at 37°C. Before FACS, the Blood cells were depleted by Red Blood cells lysis buffer (BD Pharmingen). Cells were then labeled with DAPI (4',6-diamidino-2-phenylindole, Cat number: D9452, Sigma, 1:1000), and DAPI\u003csup\u003e−\u003c/sup\u003e cells (live cells) were sorted through a MoFlo FACS machine (MoFlo Astrios EQ, Beckman Coulter) directly into PBS containing 0.04% BSA.\u003c/p\u003e\u003cp\u003eFor Smartseq2 strategy, fresh and vitrification-thawing processed human ovarian cortex were cut into pieces with very sharp knives, followed by dissociating in collagenase I solution(2mg/ml) for 45 minutes at 37°C. RPMI 1640 medium containing 10% FBS was used to stop digestion. The resulting cell suspension was then transferred into a 3.5 cm dish. Then, oocytes were manually picked under a dissection microscope and transferred to a PBS drop (containing 0.1% BSA) by mouth pipetting. Finally, the oocytes were placed separately into an individual PCR tube with lysis buffer and stored at -80°C for subsequent experiments.\u003c/p\u003e\u003cp\u003e \u003cb\u003eSingle-cell RNA-seq library preparation and sequencing\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFor 10X Genomics strategy based single-cell RNA sequencing, the single-cell RNA library was built via 10x Chromium platform, and FACS-enriched live ovarian cells were loaded per channel approximately. The libraries were built via the Chromium platform and Chromium Single Cell 3’ v2 chemistry. Finally, the libraries were loaded on an illumine NovaSeq 6000 System (Illumina, San Diego) with a PE150 sequencing strategy.\u003c/p\u003e\u003cp\u003eFor Smart-seq2 strategy based single-cell RNA sequencing, scRNA-seq libraries were constructed according to the published protocol with modifications \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Briefly, the constructed libraries of each oocyte will undergo preliminary screening through the marker genes (DDX4 for oocyte and NR5A2 for granulosa cells) of oocytes and granulosa cells via qPCR, aiming to exclude multi-cell samples. Then, the libraries were sequenced on an Illumina NovaSeq 6000 platform with a 150-bp paired-end read length by Novogene.\u003c/p\u003e\u003cp\u003e \u003cb\u003eTissue processing for spatial transcriptomic experiment\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTo collect tissues for Stereo-seq analysis, ovaries were dissected from Gender Reassignment Surgery patients (GRPs). The fresh tissues were rinsed in pre-chilled PBS twice to eliminate surface impurities and any remaining liquid or blood was gently wiped dry. Next, one piece of ovaries cortex had been treated with standard human ovarian vitrification and thawing procedure described above. The other one was washed with pre-chilled tissue freezing medium OCT (Leica, Germany) and embedded together in a new OCT. The ovaries were oriented with a blunt metal needle to ensure proper positioning. The entire OCT block was snap-frozen in liquid nitrogen that had been pre-chilled with isopentane and then transferred to a − 80°C freezer for storage before cryosection. Two weeks later, the treatment and OCT embedding methods adopted by the vitrification group were the same as those of the fresh group.\u003c/p\u003e\u003cp\u003eTo minimize RNA degradation, the fresh and cryo ones were embedded within 30 min and the entire dissection procedure was performed in a low-temperature environment. The OCT block was sliced transversely at a thickness of 10µm using Leica CM1950 cryostat (Leica). Total RNA was extracted from the sections using the RNeasy Mini Kit (Qiagen, USA) in accordance with the manufacturer’s protocol. A sample with an RNA integrity number (RIN) of 7–10, as measured by the 2100 Bioanalyzer (Agilent, USA), was used for the Stereo-seq.\u0026nbsp;The targeted section was directly adhered to the surface of the Stereoseq chip (BGI, Qingdao, China), which had capture probes that contained a 25 bp coordinate identity (CID) barcode, a 10 bp molecular identifiers (MID), and a 22 bp polyT for in situ mRNA hybridization. The adjacent section was stained with H\u0026amp;E for tissue histology examination later. The section on the chip was incubated at 37°C for 3 min, fixed in pre-cooled methanol for 30 min at -20°C, and then stained with nucleic acid dye (Thermo Fisher Scientific) for ssDNA visualization. The Ti-7 Nikon Eclipse microscope (Nikon, Japan) was used for ssDNA and histological imaging. Library construction and sequencing were completed with the help of Oebiotech, China.\u003c/p\u003e\u003cp\u003e \u003cb\u003eSingle-cell RNA-seq data analysis\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe Cell Ranger “count” pipeline (version 3.1.0) was applied with the FASTQ data produced to map the human reference genome (version hg19, GRCh38). The data matrixes in “outs” Files were then loaded in R (version 4.3.0) using the Seurat package (version 4.3.0.1).\u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAfter aggregation of the sequencing data from fresh and vitrification-thawed human ovarian cortex, R package Seurat 4.3.0 was used for cell filter, data normalization, variable gene selection, unsupervised clustering, and uniform manifold approximation and projection (UMAP) according to their recommended steps.\u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e Briefly, Seurat objects were created from the aggregated library as matrix containing gene-by-cell expression data. Cells with less than 200 genes expression or a percentage of more than 0.05 mitochondrial genes were filtered out. Then data were log-normalized and scaled for subsequent analysis. Variable genes were found and used for principal component analysis (PCA), which was performed for dimension reduction. ElbowPlot function was used for the determination of the numbers of principal components, followed by unsupervised clustering and UMAP. FindAllMarkers function was used to identify the genes exclusively expressed in each cluster. Visualization of total profiles of each cluster was generated with Seurat function DimPlot. Visualization of gene expression with feature plot, dot plot, and heatmap was generated with Seurat function FeaturePlot, DotPlot and DoHeatmap, respectively. Differentially expressed genes (P \u0026lt; 0.01) between 2 identities were found with FindMarkers function. Gene ontology (GO) biological function analysis was performed with marker genes of each cluster found by FindMarkers function with average adjusted p \u0026lt; 0.05 on toppgene website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://toppgene.cchmc.org/\u003c/span\u003e\u003cspan address=\"https://toppgene.cchmc.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and then plotted with R package Goplot.\u003c/p\u003e\u003cp\u003e \u003cb\u003eUnsupervised clustering and bin clusters annotation of Stereo-seq data\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe downloaded GEM file was converted to the Seurat object and then processed with Seurat v4.4.4. The data were normalized, scaled, multiple samples were integrated, and unsupervised clustering was performed using bins (resolution = 0.4). Marker genes for each cell type were defined using the DEGs that were identified through the “FindAllMarkers” function. The cell identities of the clusters were then annotated based on the marker genes and histological morphology of the H\u0026amp;E images. To further confirm the relationship between cell types, the Pearson correlation was calculated across the matrix, and hierarchical clustering of the Stereo-seq clusters was performed using ComplexHeatmap package in R (v2.9.1).\u003c/p\u003e\u003cp\u003e \u003cb\u003eIntegrated mapping of cell types in Stereo-seq spots with scRNA-seq data\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTo combine scRNA-seq and Stereo-seq data, the “FindTransferAnchors” and “TransferData” functions of Seurat were employed to determine the possibility of anchors in single spots. The cell type associated with the highest probability among all cell types was subsequently identified as the cell type of the spot. The integrated mapping of cell types was then visualized using the “SpatialDimPlot” and “SpatialFeaturePlot” functions. Additionally, the gene and pathway score had been visualized by “AddModuleScore” and “ggplot2”.\u003c/p\u003e\u003cp\u003e \u003cb\u003eRNA in situ hybridization\u003c/b\u003e \u003c/p\u003e\u003cp\u003eHuman ovarian cortex sections from fresh and vitrification-thawed were fixed in 4% PFA overnight and embedded in paraffin wax. Eight µm thick sections were mounted on poly-L-lysine coated slides (ThermoScientific). For RNAscope® ISH analysis, Hs-PTGDS (431471), with the target region 15–808 in human PTGDS, probes (Advanced Cell Diagnostics) were used according to manufacturer’s instructions. Quantification of PTGDS positive signals had been performed by ImageJ via using equally sized region of interest (ROI) for the ovarian cortex zone and incorporating all cortex layers.\u003c/p\u003e\u003cp\u003e \u003cb\u003eTransmission electron microscopy\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFor transmission electron ultrastructural analysis, fresh and vitrification-thawed tissues were fixed in PBS (Phosphate buffered saline), which is supplemented with 2% glutaraldehyde and 2% paraformaldehyde. After tissue processing, semithin (1 mm) and ultra-thin (700 nm) sections were cut; semi-thin sections were evaluated under a brightfield microscope (DMi8 Microscope; Leica), whereas ultra-thin sections were observed by transmission electron microscopy (TEM; LEO 906 E TEM 60 kV; Zeiss, Jena, Germany).\u003c/p\u003e\u003cp\u003e \u003cb\u003eImmunohistochemistry and H\u0026amp;E staining in human ovarian tissue\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFor immunohistochemistry staining, tissues were fixed with 4% paraformaldehyde overnight, then washed by running water. Finally, tissues were embedded in paraffin according to the standard protocol. 5µm sections were pre-pared, deparaffinized by xylene for 10min twice. Subsequently, the sections were dehydrated by gradient alcohol, washed by PBS. Antigen retrievals were done using sodium citrate buffer (YEASEN, 36319ES60), then blocked with 5% bovine serum albumin (BSA), incubated by primary antibody at 4°C overnight, then done using an UltraSensitive™ SP (Mouse/Rabbit) IHC Kit (MXB, KIT-9710). Signal detection was applied by DAB systems (MXB, MAX- 001). Nuclear was stained by Mayer's hematoxylin (Beyotime). The sections were then dehydrated by gradient alcohol, transparentized with xylene, then mounted with neutral resin for microscopy.\u003c/p\u003e\u003cp\u003eH\u0026amp;E staining was performed according to the kit (G1120, Solarbio). Briefly, washing the De-paraffinize slides to distilled water and staining slides with hematoxylin. Following a rinsing process, the sections are counterstained with eosin. Finally, shaking off excess dye and blot sections. Dehydrating in acetone, 20 dips. Then in Acetone-Xylene (1:1) solution, 20 dips. Clearing in xylene and mount in a synthetic mounting medium.\u003c/p\u003e\u003cp\u003e \u003cb\u003eImmunofluorescence staining\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe cryosections (7-µm thickness) were dried at room temperature for 1 hour, fixed by 4% PFA for 10 mins, and then rinsed with 1x PBS for three times. After being permeabilized with 0.1% Triton X-100 (T8787, Sigma) for 10 mins at room temperature, the cryosections were blocked in 1x PBS containing 5% bovine serum albumin (BSA) for 1 hour at room temperature and then incubated with primary antibodies at 4°C overnight in a humidified chamber. The primary antibodies utilized for these experiments were as follows: PTGDS (1:200, ab182141, Abcam), EGR1(10µg/ml; 55117-1-AP, Proteintech), FOSB (2µg/ml; 2251 Cell Signal Technology), SOHLH2 (1:100; bs-12279R Biossusa), αSMA (1:500; A5228 Sigma). DDX4 (2µg/ml; ab180642 abcam). Afterward, slides were rinsed with 1x PBSTx three times for 15 min, followed by the incubation with Alexa fluorescence-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Then the slides were rinsed with 1x PBSTx again three times and finally incubated with 4'6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) at room temperature for 15 min. Images were visualized using a fluorescence microscope (Leica, Germany).\u003c/p\u003e\u003cp\u003e \u003cb\u003eOvarian Tissue Culture\u003c/b\u003e \u003c/p\u003e\u003cp\u003eOvaries at 7 dpp were subjected to organ culture as described below. The whole ovarian tissues were directly placed onto a twelve-well culture plate (LABSELECT, 12mm, 0.4µm). Each well contained 1.2 mL of Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture (Gibco, USA) supplemented with 5% Fetal Bovine Serum, 1% insulin–transferrin–selenium (ITS), and 100 UI/mL penicillin–streptomycin. We tested four different culture conditions: Fresh (The Control group), standards vitrification-thawing Treatment (The Cryo group), and with T-5224 post vitrification-thawing (The Cryo + T-5224 group, and during culture, T-5224 were added to the medium with the final concentration is 80uM per well). Ovarian tissues were cultured under each the four conditions for 2–4 days at 37°C, 5%CO2. Half of the culture media was replaced every other day.\u003c/p\u003e\u003cp\u003e \u003cb\u003eChemicals\u003c/b\u003e \u003c/p\u003e\u003cp\u003eT-5224 was provided by MedChemexpress (MCE) (New Jersey, USAs). T-5224 was dissolved in DMSO and diluted in cryoprotective agents (CPA) and ovarian culture medium to the target concentration for each experiment.\u003c/p\u003e\u003cp\u003e \u003cb\u003eMicroscopy\u003c/b\u003e \u003c/p\u003e\u003cp\u003eSpecimens were mounted with fluorescence mounting medium (Dako, Glostrup, Denmark) or Mowiol mounting medium, and confocal images (e.g., cells and sections) were generally captured using a Leica TCS SP8 Confocal Microscope with 20X and 40X objectives. Images were processed with Car Zeiss software.\u003c/p\u003e\u003cp\u003e \u003cb\u003eStatistical Analysis\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFor experimental studies, all quantitative data were evaluated whether they followed the normal distribution by the Shapiro-Wilk test and equal variance by F-test. For data passed both tests, data are expressed as means ± SEM. Student's t-test was used for the comparison between 2 groups, and for comparison among multiple groups, the data were analyzed by one-way ANOVA with Tukey's post hoc test. For the data that were not normally distributed, nonparametric test (Mann-Whitney U test) was performed and presented as median ± SD. All P values are 2-sided, and P \u0026lt; 0.05 was considered a statistically significant difference.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOTC - Ovarian Tissue Cryopreservation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003escRNA-seq - single-cell RNA sequencing\u003c/p\u003e\n\u003cp\u003eStereo-seq - Spatial enhanced resolution omics sequencing\u003c/p\u003e\n\u003cp\u003ePCs - perivascular cells\u003c/p\u003e\n\u003cp\u003eSCs - Stromal Cells\u003c/p\u003e\n\u003cp\u003eGCs - Granulosa Cells\u003c/p\u003e\n\u003cp\u003eBECs - Blood vascular Endothelial Cells\u003c/p\u003e\n\u003cp\u003eLECs - Lymphatic Endothelial Cells\u003c/p\u003e\n\u003cp\u003eSMC - Smooth Muscle Cells\u003c/p\u003e\n\u003cp\u003eGRPs -Gender Reassignment\u0026nbsp;Surgery\u0026nbsp;Patients\u003c/p\u003e\n\u003cp\u003eDEGs - Differently Expressed Genes\u003c/p\u003e\n\u003cp\u003eOCCC - oocyte-corona-cumulus complex\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all members of the laboratory for critical discussion and comments on the article. We thank Prof. Shuo Xiao from Rutgers University for kindly supporting on murine ovarian in vitro culture.\u0026nbsp;We express our gratitude to Prof.\u0026nbsp;Yuxuan Zheng from the Human Phenome Institute, Fudan University, for his valuable advice on single-cell analysis techniques and methodologies. We thank Oebiotech (Inc. China) for its help in spatial transcriptome sequencing and analysis. This work was supported by the National Key Research and Development Project of China (2022YFC2703002), National Natural Science Foundation of China (82371726, 82071605, 82200541), The Joint Funds of the National Natural Science Foundation of China (U24A20658). Natural Science Foundation of Shanghai (No: 21ZR1428600), Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-ZDCX20212200), Shanghai Hospital Development Center Foundation (SHDC22022303) and Key project of Medical and Industrial intersection of Shanghai Jiao Tong University (YG2023ZD27). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.G., D.S., H.D. and Y.L. contributed equally to this work. W.L., Q.Z. and M.Z. conceived the study. F.G., D.S., Y.L., and H.D. performed the experiments. F.G., D.S., Y.L., and M.G. analyzed and visualized the data. M.Z., Y.M., R.Q., S.L., and L.Z. provided the ideas and suggestions. M.G. and Q.Z. helped to collect clinical samples. F.G., and W.L. wrote and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the supplementary materials.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eData and code availability\u003c/h2\u003e \u003cp\u003eThe RNA-sequencing data including have been deposited in Gene Expression Omnibus (GEO) under the accession number GSE267315 (reviewer token: ixctsiiqthybnsp). The publicly available software applied in this study is listed and described in the Methods section. All codes are available from the corresponding authors on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMiller KD\u003cem\u003e, et al.\u003c/em\u003e Cancer treatment and survivorship statistics, 2022. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 409-436 (2022).\u003c/li\u003e\n\u003cli\u003eSiegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 17-48 (2023).\u003c/li\u003e\n\u003cli\u003eDonnez J, Dolmans MM. Fertility Preservation in Women. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e377\u003c/strong\u003e, 1657-1665 (2017).\u003c/li\u003e\n\u003cli\u003eDolmans MM. 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Ovarian tissue cryopreservation (OTC) plays vital role in female fertility preservation, especially for adolescent female cancer patients. Yet, the sensitive cell populations and cryoinjury molecular mechanisms underlying cryopreservation remain elusive. This study employs single-cell RNA sequencing and spatial transcriptomics to dissect the impacts of temperature stress and cryoprotectant toxicity on the human ovarian cortex. The spatiotemporal molecular characteristics of eight ovarian cell types following vitrification-thawing had been comprehensively characterized. Granulosa, perivascular and stromal cells are identified as most sensitive cell types to OTC procedure. Further analysis using Smart-seq2 on oocytes highlights alterations in“cell cycle” and “DNA methylation” pathways. Notably, the FOS/AP-1 pathway emerges as a crucial response factor to stress and toxicity during cryopreservation. Inhibition of this pathway with T-5224 mitigates vascular damage and reduces apoptosis in vitrification-thawed ovaries. These findings provide insight into the spatiotemporal dynamics during ovarian vitrification and thawing, aiding in prioritizing therapeutic strategies for pre- and post-cryopreservation interventions.\u003c/p\u003e","manuscriptTitle":"Spatiotemporal Dynamics of Human Ovarian Cortex Transcriptome Following Vitrification and Thawing: Insights into the FOS/AP-1 Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-04 11:31:54","doi":"10.21203/rs.3.rs-6312954/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c150e51f-ca94-4724-9737-ab4b6bce5969","owner":[],"postedDate":"April 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46554060,"name":"Biological sciences/Physiology/Reproductive biology/Reproductive disorders"},{"id":46554061,"name":"Biological sciences/Computational biology and bioinformatics/Cellular signalling networks"},{"id":46554062,"name":"Health sciences/Biomarkers/Predictive markers"}],"tags":[],"updatedAt":"2025-05-07T06:10:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-04 11:31:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6312954","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6312954","identity":"rs-6312954","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}