Follicle Transition Research Articles

Effective combination therapy for telogen hair loss

Background. Telogen hair loss is one of the most common forms of alopecia, associated with a disruption in the phases of the hair growth cycle – specifically, the premature transition of the hair follicle into the telogen phase. The primary causes are the influence of triggering factors such as increased stress levels and previous illnesses. The main goal of therapy is to prolong the anagen phase and stimulate hair growth through the use of topical peptides and a multivitamin complex.Objective. To study the effectiveness of a hair growth restoration method in the treatment of telogen alopecia using cosmetic hair growth products and dietary supplements.Methods. The study involved 20 patients diagnosed with telogen alopecia who received treatment with the topical cosmetic products «Time to Grow» and the dietary supplement «Thick and Strong Hair» for 9 weeks. The effectiveness of the treatment was assessed at 3, 6, and 9 weeks after the start of therapy.Results. The analysis of the study showed statistically significant changes in trichoscopy indicators in patients with telogen alopecia, demonstrating the effectiveness of using hair growth cosmetics (p<0.05). At the final assessment point, a reduction in hair loss was observed in 100 % of the subjects, along with improvements in hair quality and volume. A survey evaluating the participants’ subjective experiences demonstrated satisfaction with the treatment and adherence to the therapy.Conclusion. The use of hair growth products and the dietary supplement «Time to Grow» has proven effectiveness in the treatment of telogen hair loss.

Transcriptomic analysis of regulatory mechanisms in the telogen-anagen transition of ovine hair follicles

BackgroundDorper sheep are celebrated for their fast maturation and superior meat quality, with some shedding their wool each spring. Wool shedding occurs naturally due to the hair follicle (HF) cycle, but its regulatory mechanisms remain unclear and need further investigation.ResultsIn this study, shedding and non-shedding sheep were selected from the same Dorper flock. Skin samples were collected in September of the first year and January and March of the following years. RNA sequencing was performed on these samples. Principal component analysis (PCA) was used to assess the results. A total of 2536 differentially expressed genes (DEGs) were identified. Using a clustering heatmap and fuzzy clustering analysis three distinct gene expression patterns were identified: A pattern (high expression in anagen), T1 pattern, and T2 pattern (high expression in telogen). For each pattern, differentially expressed genes (DEGs) were analyzed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Combining this with pathway expression analysis, six A-pattern and fourteen T-pattern pathways linked to telogen-anagen transition in the HF cycle were identified. Networks of key pathways were then constructed. Additionally, key genes were identified in the telogen-anagen transition, including one A-pattern gene and seven T-pattern (T1, 1; T2, 6) genes, using the Maximal Clique Centrality (MCC) tool in Cytoscape. Predicted transcription factors (TFs) involved in key pathways, such as LEF and STAT5B, were identified. Finally, RNA-seq results were confirmed by RT-qPCR.ConclusionThis study highlights critical genes and pathways in the telogen-anagen transition, and transcriptome sequencing along with bioinformatics analysis provides new insights into the regulatory mechanisms of the HF cycle and development.

Facile fabrication of cedrol nanoemulsions with deep transdermal delivery ability and high safety for effective androgenic alopecia treatment

The successful treatment of androgenic alopecia (AGA) hinges on an effective transdermal drug delivery strategy, a challenge that requires urgent attention. In this study, we introduce a novel approach utilizing cedrol nanoemulsions (CD-NEs) for AGA treatment. Our findings demonstrate that cedrol (CD) effectively inhibits the expression of 5α-reductase 2 (5αR2), akin to the FDA-approved drug finasteride, while also surpassing it in terms of promoting cell proliferation. Through formulation optimization, we have developed stable CD-NEs suitable for large-scale production. Experimental and molecular dynamics simulations further reveal that CD-NEs enhance transdermal penetration by altering lipid organization, facilitating CD delivery to deeper skin layers via a trans-epidermal pathway. In vivo studies conducted on AGA model C57BL/6 mice demonstrate that CD-NEs significantly promote hair follicle regeneration, accelerating the transition of hair follicles from the telogen phase to the anagen phase. Moreover, CD-NEs treatment leads to a significant reduction in dihydrotestosterone (DHT) levels in the skin while maintaining stasis levels in serum, underscoring its efficacy in targeting androgenetic pathways with high safety. This comprehensive analysis sheds light on the efficacy and mechanism of action of CD-NEs in hair regeneration, offering valuable insights for future clinical applications in AGA treatment.

Terminalia bellirica (Gaertn.) Roxb. Extracts reshape the perifollicular microenvironment and regulate the MAPK pathway for androgenetic alopecia treatment

Ethnopharmacological relevanceTerminalia bellirica (Gaertn.) Roxb. (TBR), a popular herbal remedy in India and Southeast Asia, has been demonstrated to possess multiple pharmacological activities. However, systematic studies on the medicinal effects and mechanism of TBR for the androgenetic alopecia (AGA) treatment are deficient. Materials and methodsHuman Umbilical Vein Endothelial Cells (HUVECs) and testosterone-induced AGA mice were used to evaluate the hair regrowth activity of TBR extracts. Chemical constituents and potential active components of TBR extracts were analyed by UPLC-Q-TOF-MS in vitro/vivo. The hair regrowth mechanisms of TBR were elucidated through network pharmacology and experimental validation. ResultsTotally 28 chemical constituents in TBR were identified, of which 15 were predicted as potential active components for AGA therapy. TBR could significantly scavenge ROS, promote VEGF level/cell migration of HUVECs, and inhibiting type II 5α-reductase activity (the inhibit rate: 82.35 ± 1.02 %). Pharmacodynamic evaluation suggested that TBR effectively led to hair regrowth in C57BL6 mice compared to minoxidil. TBR promoted the hair follicle (HF) transition from the telogen phase to anagen phase by decreasing MDA levels, increasing VEFG expression and up-regulating phosphorylated P38/ERK protein levels in the MAPK signalling pathway. ConclusionsTBR reversed AGA via inhibiting SRD5A2 activity and stimulating the MAPK pathway. Meantime, TBR could remodel the follicle microenvironment by reducing oxidative stress and increasing angiogenesis.

Bioengineered 3D ovarian model for long-term multiple development of preantral follicle: bridging the gap for poly(ε-caprolactone) (PCL)-based scaffold reproductive applications

BackgroundAssisted Reproductive Technologies (ARTs) have been validated in human and animal to solve reproductive problems such as infertility, aging, genetic selection/amplification and diseases. The persistent gap in ART biomedical applications lies in recapitulating the early stage of ovarian folliculogenesis, thus providing protocols to drive the large reserve of immature follicles towards the gonadotropin-dependent phase. Tissue engineering is becoming a concrete solution to potentially recapitulate ovarian structure, mostly relying on the use of autologous early follicles on natural or synthetic scaffolds. Based on these premises, the present study has been designed to validate the use of the ovarian bioinspired patterned electrospun fibrous scaffolds fabricated with poly(ε-caprolactone) (PCL) for multiple preantral (PA) follicle development.MethodsPA follicles isolated from lamb ovaries were cultured on PCL scaffold adopting a validated single-follicle protocol (Ctrl) or simulating a multiple-follicle condition by reproducing an artificial ovary engrafted with 5 or 10 PA (AO5PA and AO10PA). The incubations were protracted for 14 and 18 days before assessing scaffold-based microenvironment suitability to assist in vitro folliculogenesis (ivF) and oogenesis at morphological and functional level.ResultsThe ivF outcomes demonstrated that PCL-scaffolds generate an appropriate biomimetic ovarian microenvironment supporting the transition of multiple PA follicles towards early antral (EA) stage by supporting follicle growth and steroidogenic activation. PCL-multiple bioengineering ivF (AO10PA) performed in long term generated, in addition, the greatest percentage of highly specialized gametes by enhancing meiotic competence, large chromatin remodeling and parthenogenetic developmental competence.ConclusionsThe study showcased the proof of concept for a next-generation ART use of PCL-patterned scaffold aimed to generate transplantable artificial ovary engrafted with autologous early-stage follicles or to advance ivF technologies holding a 3D bioinspired matrix promoting a physiological long-term multiple PA follicle protocol.

P02 Accelerated ageing of hair follicles in androgenetic alopecia

Abstract Introduction and aims Androgenetic alopecia (AGA) is defined by hair loss from the frontal, parietal and vertex regions of the scalp; however, occipital follicles are spared. Hair follicles in AGA undergo miniaturization, which is the transition of terminal follicles into vellus follicles. Similarly with age, the diameter of the hair shaft decreases with loss of pigmentation, which is also seen in miniaturized follicles. To investigate whether frontal follicles have accelerated ageing compared with occipital follicles, we looked at methylation levels of genes associated with ageing. As the thickness and pigmentation of hair fibre are influenced by the dermal papilla (DP), we conducted microarray analysis of frontal and occipital DP to establish whether accelerated ageing of follicles is driven by DP. Methods The biological age of frontal and occipital follicles was calculated using methylation status of ELOVL2, ASPA, EDARADD and PDE4C (n = 3). For microarray analysis, DP was microdissected from the frontal (FDP) and occipital (ODP) follicles from patients undergoing hair transplantation surgeries to treat AGA (n = 4). Results Frontal follicles were found to have significantly higher biological age compared with patient-matched occipital follicles suggesting accelerated ageing of follicles in AGA. Transcriptomic analysis revealed significant increase in expression of several genes in FDP and notably EGR1, HSPB1, DNAJB1 and APOD, which have been shown to be upregulated in the DP of aged mice compared with young mice. We found downregulation of TRPS1 in FDP, which represses EGR1. Mutations in TRPS1 underlie trichorhinophalangeal syndrome which is associated with vellus-type hair. Conclusions Our transcriptional and molecular analysis revealed that FDP and frontal follicles have an advanced biological age relative to ODP. This increased age is also reflected in the transcriptional signature of FDP. We hypothesize that these changes are in part due to regulation of signalling by the transcriptional repressor TRPS1, whose expression is decreased in FDP.

Proteo-transcriptomic profiles reveal key regulatory pathways and functions of LDHA in the ovulation of domestic chickens (Gallus gallus)

BackgroundIn poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying follicular ovulation in avians is a complex biological process that remains unclear.ResultsCritical biochemical events involved in ovulation in domestic chickens (Gallus gallus) were evaluated by transcriptomics, proteomics, and in vitro assays. Comparative transcriptome analyses of the largest preovulatory follicle (F1) and postovulatory follicle (POF1) in continuous laying (CL) and intermittent laying (IL) chickens indicated the greatest difference between CL_F1 and IL_F1, with 950 differentially expressed genes (DEGs), and the smallest difference between CL_POF1 and IL_POF1, with 14 DEGs. Additionally, data-independent acquisition proteomics revealed 252 differentially abundant proteins between CL_F1 and IL_F1. Perivitelline membrane synthesis, steroid biosynthesis, lysosomes, and oxidative phosphorylation were identified as pivotal pathways contributing to ovulation regulation. In particular, the regulation of zona pellucida sperm-binding protein 3, plasminogen activator, cathepsin A, and lactate dehydrogenase A (LDHA) was shown to be essential for ovulation. Furthermore, the inhibition of LDHA decreased cell viability and promoted apoptosis of ovarian follicles in vitro.ConclusionsThis study reveals several important biochemical events involved in the process of ovulation, as well as crucial role of LDHA. These findings improve our understanding of ovulation and its regulatory mechanisms in avian species.

Efficacy and Pain Tolerance of Alexandrite Laser Hair Removal at Different Stages of the Menstrual Cycle.

Several different parameters play a role in the transition of hair follicles to the anagen phase, with the role of androgens, progesterone, and estrogen hormones and receptors being significant. The effectiveness of laser hair removal (LHR) and pain tolerance during procedure were investigated during 3 different phases of the menstrual cycle. Forty-eight axillae were randomly divided into 3 groups: menstruation, ovulation, and luteal. Three laser sessions were performed on each axilla at a 1-month interval. Blood hormone levels were measured in the patients. An alexandrite laser was applied during LHR sessions. Before each LHR session and 1 month after the third session, hair follicles in 4-cm2 areas in the center of the axillae were counted. Patients self-assessed the pain they felt during the laser application in each session with a visual pain scale. The average values for hair counting in the groups were as follows (M, menstruation; O, ovulation; L, luteinization): M0 = 47.6, M1 = 27.4, M2 = 16.1, M3 = 9.9; O0 = 41.8, O1 = 21.1, O2 = 13.8, O3 = 8.6; and L0 = 49.4, L1 = 27.1, L2 = 15.1, L3 = 9.8. The average values on the visual analog scale scores in the groups were: M1 = 3.94, M2 = 3.06, M3 = 1.94; O1 = 3.50, O2 = 3.06, O3 = 1.69; and L1 = 3.63, L2 = 2.50, L3 = 1.56. Statistical analysis was conducted with Tukey post hoc analysis after analysis of variance. The results of LHR are not affected by changes in hormone levels during the menstrual cycle in females. Although not statistically significant, it has been observed that pain tolerance during laser application is lower during the menstruation cycle.

Cardamonin-loaded liposomal formulation for improving percutaneous penetration and follicular delivery for androgenetic alopecia.

Androgenic alopecia (AGA) has a considerable impact on the physical and mental health of patients. Nano preparations have apparent advantages and high feasibility in the treatment of AGA. Cardamonin (CAR) has a wide range of pharmacological activities, but it has the problems of poor solubility in water and low bioavailability. There are few, if any, researches on the use of nano-loaded CAR to improve topical skin delivery of AGA. In this study, a CAR-loaded liposomal formulation (CAR@Lip and CAR@Lip Gel) was developed and characterized. The prepared CAR@Lip exhibited a uniform and rounded vesicle in size. CAR@Lip and CAR@Lip Gel can significantly improve the cumulative release of CAR. Additionally, CAR@Lip can obviously promote the proliferation and migration of human dermal papilla cells (hDPCs). Cell uptake revealed that the uptake of CAR@Lip significantly increased compared with the free drug. Furthermore, both CAR@Lip and CAR@Lip Gel groups could markedly improve the transdermal performance of CAR, and increase the topical content of the drug in the hair follicle compared with CAR. The ratchet effect of hair follicles could improve the skin penetration depth of nanoformulations. Notably, Anti-AGA tests in the mice showed that CAR@Lip and CAR@Lip Gel groups could promote hair growth, and accelerate the transition of hair follicles to the growth stage. The anti-androgen effect was revealed by regulating the expression of IGF-1, VEGF, KGF, and TGF-β, participating in SHH/Gli and Wnt/β-catenin pathways. Importantly, the nanoformulations had no obvious skin irritation. Thus, our study showed that CAR-loaded liposomal formulation has potential application in the treatment of AGA.

Hair Growth-Promoting Effects of Rosehip (Rosa canina L.) Seed Oil in C57BL/6 Mice.

Rosehip (Rosa canina L.) seeds, a by-product of the food processing industry, contain various bioactive compounds that have potential cosmetic and pharmacological applications. Rosehip seed oil (RHSO) has been shown to exert therapeutic effects in skin disorders, but its role in promoting hair growth remains unknown. In this study, we aimed to elucidate the hair growth-promoting activity of RHSO and the related mechanisms of action. The depleted dorsal skin of telogenic C57BL/6 mice was topically treated with RHSO for 21 days, and the extent of hair regrowth was assessed. The results indicated that RHSO stimulated hair growth by inducing the early transition of hair follicles from telogen to anagen phase. Histological analysis revealed significant increases in hair follicle density, hair bulb size, and skin thickness. RHSO treatment also upregulated the expression of hair growth-associated genes, including β-catenin, phospho-glycogen synthase kinase-3 beta, Sonic hedgehog, smoothened, cyclin D1, cyclin E, and insulin like growth factor 1. These findings suggest that RHSO stimulates hair growth and may show promise as a preventive and/or therapeutic agent for hair loss.

Carbon Dioxide Fractional Laser Treatment Induces Lgr5+ Stem Cell Activation and Hair Regrowth Through the Canonical Wnt/β-Catenin Pathway.

Different types of alopecia have negative impacts on patients. Recently, some kinds of laser or light therapies have been reported to effectively alleviate hair loss. Carbon dioxide fractional laser (CO2FL) treatment is one of the most effective laser treatments, but its beneficial effects and exact mechanism in hair regrowth have not been reported in detail. The purpose of this study was to investigate the effect and molecular mechanism further. C57 and Lgr5-Cre: Rosa-mTmG mouse models of hair regrowth were established by CO2FL treatment, and the parameters that induced the best effect were determined. Tissues were harvested on the day prior to the treatment day and on days 3, 5, 7, 10 and 14 after CO2FL. H&E and immunofluorescence staining, RNA sequencing (RNA-seq), quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and related inhibitor were used to determine the molecular mechanism underlying the effect of CO2FL treatment on the hair cycle and hair regrowth. In clinical trial, five participants were treated three sessions at 1-month intervals to obverse the effects. Hair regrew and covered the treatment area on the tenth day after CO2FL treatment with the best parameters, while the control group showed signs of hair growth on the 14th day. H&E and immunofluorescence staining showed that the transition of hair follicles (HFs) from telogen to anagen was accelerated, and the rapid activation and proliferation of Lgr5+ hair follicle stem cells (HFSCs) were observed in the treatment group. The RNA-seq, qPCR and WB results indicated that the Wnt pathway was significantly activated after CO2FL treatment. Improvement achieved with CO2FL treatment in clinical trial. The results of this study suggest that CO2FL treatment can promote hair regrowth by activating Lgr5+ HFSCs and upregulating the Wnt/β-catenin pathway. Clinical trial results demonstrated that CO2FL treatment will be a promising therapeutic regimen for alopecia. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment.

Do ovarian stromal cells (OSCs) influence the viability and growth of human preantral follicles in vitro? A feeder layer of OSCs promotes the growth and transition of low developmental stage follicles to the primary/secondary stage while maintaining a high proportion of viable follicles. In the ovary, follicles rely on the support of ovarian cells, which secrete essential factors for their survival and development. This phenomenon has also been demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of OSCs. This co-culture notably enhances follicle survival and growth. Pre-antral follicles were isolated from human frozen-thawed ovarian tissue biopsies and then encapsulated in 1% alginate scaffolds. These embedded preantral follicles were either placed directly on the OSCs feeder layer or at the bottom of a culture dish for a 7-day in vitro culture (control). The study compared follicle viability, growth, and hormone production between the different groups. Primordial/intermediate and primary follicles were isolated from frozen-thawed ovarian tissue of cancer patients (n = 6). OSCs were then isolated from ovarian tissue of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 using an inverted microscope to assess their development based on the increase in diameter. Viability was evaluated by staining a subset of follicles (n = 87) with calcein AM and ethidium homodimer-I, followed by classification into healthy/minimally damaged and damaged/dead follicles using confocal fluorescence microscopy. Additionally, estradiol levels were measured using ELISA. A total of 382 human preantral follicles (370 primordial/intermediate and 12 primary) with a mean diameter of 40.8 ± 9.9 µm (mean ± SD) were isolated, embedded in 1% alginate hydrogel, and placed either on a monolayer of OSCs or directly on the plastic. By Day 7, the preantral follicles showed a significant size increase under both culture conditions (P < 0.0001 for D0 vs D7). The mean diameter of follicles (quiescent and growing) cultured on the feeder layer was 80.6 ± 11.0 μm compared to 67.3 ± 7.2 μm without it (P = 0.07). During the 7-day in vitro culture, the viability of the follicles significantly decreased only in the group without an OSCs monolayer compared to the D0 viability (P < 0.05). Additionally, more follicles transitioned to a higher developmental stage in the presence of OSCs (D0 primordial/intermediate: 184, primary: 7 vs D7 primordial/intermediate: 51, primary/secondary: 93) compared to those cultured without OSCs (D0 primordial/intermediate: 186, primary: 5 vs D7 primordial/intermediate: 84, primary/secondary: 65; P < 0.001). Specifically, 66 and 44 follicles reached the secondary stage (75< x <200 μm) in the presence and absence of OSCs, respectively. Moreover, the estradiol level was significantly higher (P = 0.006) in the alginate beads containing primordial and growing follicles cultured on the OSCs (54.1 ± 14.2 pg/ml) compared to those cultured without OSCs (29.9 ± 4.0 pg/ml). N/A. This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further in vitro studies are required to investigate follicular developmental capacity, physiology, and steroidogenesis in alginate scaffolds with human OSCs. Activating and growing human primordial/intermediate follicles to a secondary stage in in vitro short-term culture has posed a longstanding challenge. However, co-culturing with human OSCs has shown the potential to overcome this limitation. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., PhD scholarship awarded to H.V.), Fondation Louvain (awarded to C.A.A.; PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer), Foundation Against Cancer (grant 2018-042 awarded to A.C.), and the European Community Structural Funds and Lithuanian Research Council (Agreement registration No. D-19-0874). The authors have no conflicts of interest to declare.

Co-delivery of minoxidil and tocopherol acetate ethosomes to reshape the hair Follicular Microenvironment and promote hair regeneration in androgenetic alopecia

The most prevalent kind of hair loss is androgenic alopecia (AGA), which is characterized by hair follicle miniaturization and microenvironment dysfunction. Although topical Minoxidil (MXD) was considered to be a safe and effective treatment for AGA, excess reactive oxygen species (ROS) and lower sulfotransferase activity in the hair follicular microenvironment led to an unsatisfactory treatment of AGA. Here, we developed the ethosome (MTE) load of minoxidil and tocopherol acetate to improve the therapeutic effect of MXD on androgenic alopecia. It could regulate the microenvironment around hair follicles, promote the telogen-to-anagen transition of hair follicles, and boost hair regeneration, thus achieving a synergistic effect of 1 + 1 > 2. The results proved that MTE showed excellent stability, biosafety, and good dermal and follicular permeability in vitro. The hair regeneration ability of AGA model mice showed that the co-delivery ethosome might regulate the microenvironment around the hair follicles and improve hair regeneration in comparison to the commercial minoxidil tincture alone. As a result, the strategy provided a promising new strategy for the treatment of AGA.

P-410 First wave of primordial follicle activation during human ovarian tissue manipulation for fertility preservation

Abstract Study question Does ovarian tissue biopsy, transportation and processing for fertility preservation and restoration trigger primordial follicle activation? Summary answer Early manipulation of ovarian tissue is sufficient to trigger follicle activation by stimulating PI3K/Akt and disrupting the Hippo pathway. What is known already Primordial follicle recruitment occurs continuously in physiological conditions by modulation of autocrine and paracrine factors, like the PI3K/Akt and Hippo pathways, to ensure follicle growth over time. During fertility preservation and restoration procedures, follicle activation may be disrupted and follow nonphysiological patterns. The ability to control activation dynamics by up- or downregulation of these pathways may enhance fertility restoration outcomes in a number of ways. Indeed, downregulation of follicle activation shortly after transplantation may protect the ovarian reserve from early depletion. Conversely, ovarian tissue in vitro culture may benefit from upregulation of primordial follicle activation to boost further growth. Study design, size, duration Fresh ovarian tissue was retrieved from nine women undergoing laparoscopic surgery for benign conditions. Three timepoints were investigated. One-third of collected tissue per patient was immediately fixed in the operating room, without any manipulation (time zero, T0). The remaining tissue was transferred to the laboratory and dissected to remove any surplus medulla. It was then cut into small cortical fragments, half of which were fixed after 25 minutes(T25) and the other half after 90 minutes(T90). Participants/materials, setting, methods All cortical fragments were fixed in 4%formaldehyde and embedded in paraffin for histology. In order to explore follicle activation,markers of the PI3K/Akt and Hippo signaling pathways were immunolabeled at each timepoint, targeting: (i) phospho-Akt (p-Akt) in primordial follicles by immunohistochemistry as a marker of early PI3K/Akt pathway activation; and (ii) Yes-associated protein (YAP) cellular localizationin the granulosa cell layer of primordial follicles by immunofluorescence as a marker of Hippo disruption. Main results and the role of chance An upturn in p-Akt expression was observed at T25 (22,34 ± 0.13%; p = 0.0233) and T90 (39,01 ± 0.22%, p = &amp;lt; 0.0001) compared to T0 (2,87 ± 0,03%). In terms of YAP cellular localization, a significant nucleus-to-cytoplasm shift was detected at T25 (1.11 ± 0.09; p = 0.0428) compared to T0 (0.97 ± 0.10), while T90 (1.07± 0.14) values were similar to T25. Our data prove that ovarian tissue manipulation triggers primordial follicle activation very early, involving both the PI3K/Akt and Hippo signaling pathways, which appear to cooperate in primordial-to-primary follicle transition. Our results indicate that the first stages of any fertility preservation or restoration procedure involving ovarian tissue manipulation contribute to dysregulation of the very mechanisms responsible for the ovarian reserve maintenance and follicle growth. Additional strategies are required to gain the control of follicle activation mechanisms in nonphysiological conditions (ex vivo ovarian tissue manipulation), in order to exploit ovarian reserve dynamics to serve the need of patients. Limitations, reasons for caution Analyses in the study were limited to histology and immunolabeling to acquire a descriptive picture of pathway activation kinetics over time. Further investigations using dynamic experimental models are essential to advance our understanding of signaling pathway synergy in vivo. Wider implications of the findings Since dysregulation of follicle activation in nonphysiological conditions appears to be associated with poor oocyte quality, enhancing our ability to control the relevant signaling pathways is crucial to optimizing fertility preservation procedures. Trial registration number not applicable

P-407 Artificial ovary construction: short-term in vitro culture of human primordial and primary follicles in a 3D environment on a stromal cell feeder layer

Abstract Study question Do ovarian stromal cells (OSCs) influence viability and growth of human pre-antral follicles in vitro? Summary answer Feeder layer of OSCs advanced growth and transition of primordial follicles to primary/secondary stage while keeping a high proportion of viable follicles. What is known already In the ovary, follicles require the support of ovarian cells through the secretion of essential factors for their survival and development. This was also demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of ovarian stromal cells. This co-culture significantly increased follicle survival and growth. Study design, size, duration Pre-antral follicles were isolated from human frozen-thawed ovarian tissue (OT) biopsies and encapsulated in 1% alginate scaffolds. Embedded pre-antral follicles were placed directly on the OSCs feeder layer or bottom of a culture dish for a 7-day in vitro culture (control). Follicle viability and growth and hormone production were compared between groups. Participants/materials, setting, methods Primordial and primary follicles were isolated from frozen-thawed OT of cancer patients (n = 6). OSCs were isolated from OT of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 under an inverted microscope. Viability was assessed by staining a proportion of follicles (n = 87) with calcein AM and ethidium homodimer-I. They were classified (V1/V2: healthy/minimally damaged; V3/V4: damaged/dead follicles) using confocal fluorescence microscopy. Estradiol levels were measured by ELISA. Main results and the role of chance A total of 379 human pre-antral follicles (primordial=360; primary=19) were isolated and embedded in 1% alginate hydrogels and classified according to their viability. Most follicles (96%) were viable after isolation with a diameter of 40.8±9.9 µm (mean±SD). At Day 7, pre-antral follicles have grown significantly in size in both culture conditions: on the OSCs feeder layer and without it (p &amp;lt; 0.0001 for D0 vs. D7). However, no significant difference (p = 0.07) between culture conditions was observed. The mean diameter of follicles cultured on the OSCs layer was 80.6±11.0 μm and without it, 67.3±7.2 μm. Nonetheless, the distribution pattern of different quality grade follicles was significantly different on OSCs (V1: 62%, V2: 25%, V3: 2%, V4: 11%) and follicles without OSCs (V1: 35%, V2: 38%, V3: 0%, V4: 23%; p = 0.03). Additionally, more follicles have been activated and reached a higher developmental stage on OSCs (D0 primordial: 184, primary: 7 vs. D7 primordial: 53, primary/secondary: 93) than without stromal cells (D0 primordial: 186, primary: 4 vs. D7 primordial: 84, primary/secondary: 64; p &amp;lt; 0.001) with 66 and 43 follicles reaching a secondary stage (75&amp;lt;x&amp;lt;200 μm), respectively. Estradiol level was significantly (p = 0.006) higher in media of follicles cultured on the OSCs, 54.1±14.2 pg/ml vs. 29.9±4.0 pg/ml. Limitations, reasons for caution This study was performed only in a short-term culture and no primordial/primary follicles have reached the antral stage. Further in vitro studies on follicular developmental capacity, physiology and steroidogenesis in alginate scaffold with human ovarian stromal cells are needed. Wider implications of the findings Activation and growth of human primordial follicles during in vitro short-term culture to a secondary stage has been a long-lasting challenge. Co-culture with human ovarian stromal cells can help to overcome this limitation. Trial registration number not applicable

3D bioprinting of calcium molybdate nanoparticles-containing immunomodulatory bioinks for hair regrowth

The regeneration of hair follicles in skin defect area remains a huge challenge all over the world. However, there is no effective strategy to solve this problem due to the difficulty of harvesting hair follicles in anagen period. Herein, we design a bioink based on calcium molybdate (CM) nanoparticles, laden with dermal papilla cells (DPCs) and macrophages. Interestingly, an anti-inflammatory microenvironment is established by the release of molybdenum (Mo) ions, thereby achieving favorable immunomodulation of bioactive ions-mediated hair regrowth. It is demonstrated that in the CM nanoparticles-containing multicellular bioinks, Mo polarizes macrophages into M2 phenotype to secret anti-inflammatory cytokines. Furthermore, it is shown that the anti-inflammatory immune microenvironment of the scaffolds induces the secretion of hair follicle anagen-related cytokines in DPCs, promoting hair regrowth in vivo. Finally, transcriptomic analysis reveals the underlying mechanism that Mo activates the mammalian target of rapamycin (mTOR) signaling pathway in macrophages and up-regulates the expression of growth factors (HGF and IGF-1), which accelerate the transition of hair follicles to anagen period. This study offers a new insight and strategy for the reconstruction of complex skin appendages through bioprinting immunomodulatory multicellular scaffolds with inorganic bioinks.

The transcriptome reveals the molecular regulatory network of primordial follicle depletion in obese mice

To explore the dynamic transcriptional regulatory network of primordial follicle fate in obese mice to elucidate the potential mechanism of primordial follicle depletion. Experimental study and transcriptomic analysis. Healthy (n=15) and obese (n=15) female mice. Six-week-old CD-1 mice were divided into healthy and high-fat diet groups and fed continuously for 12 weeks. The diet of healthy mice contained 10% fat. The diet of high-fat mice contained 60% fat. Primordial to primary follicle transition rate, gene expression changes, enriched Kyoto Encyclopedia of Genes and Genomes pathways, and ferroptosis. Primordial follicle depletion was increased in the ovaries of obese mice. We found that deposited fat around primordial and primary follicles of obese mice was higher than that for healthy mice. The proliferation of granulosa cells around primary follicles was increased in obese mice. In addition, we uncovered specific gene signatures associated with the primordial to primary follicle transition (PPT) in obese mice using laser capture microdissection RNA sequencing analysis. Gene set enrichment analysis indicated that ferroptosis, cell oxidation, vascular endothelial growth factor, and mammalian target of rapamycin signaling were increased significantly in the primordial follicles of obese mice. Notably, the ferritin, acyl CoA synthetase long-chain family member 4, and solid carrier family 7 member 11 associated proteins of the ferroptosis signaling pathway were significantly increased in the PPT phase of obese mice. Our work suggests that ferroptosis is a key pathway activated within immature ovarian follicles in the context of obesity and that the process may be involved in the physiological regulation of the PPT as well.

The mechanisms that control the preantral to early antral follicle transition and the strategies to have efficient culture systems to promote their growth in vitro.

Preantral to early antral follicles transition is a complex process regulated by endocrine and paracrine factors, as well as by a precise interaction among oocyte, granulosa cells and theca cells. Understanding the mechanisms that regulate this step of folliculogenesis is important to improve in vitro culture systems, and opens new perspectives to use oocytes from preantral follicles for assisted reproductive technologies. Therefore, this review aims to discuss the endocrine and paracrine mechanisms that control granulosa cell proliferation and differentiation, formation of the antral cavity, estradiol production, atresia, and follicular fluid production during the transition from preantral to early antral follicles. The strategies that promote in vitro growth of preantral follicles are also discussed.

Isoxazole 9 (ISX9), a small molecule targeting Axin, activates Wnt/β-catenin signalling and promotes hair regrowth.

Isoxazole 9 (ISX9) is a neurogenesis-promoting small molecule compound that can up-regulate the expression of NeuroD1 and induce differentiation of neuronal, cardiac and islet endocrine progenitors. So far, the molecular mechanisms underlying the action of ISX9 still remain elusive. To identify a novel agonist of the Wnt/β-catenin, a cell-based SuperTOPFlash reporter system was used to screen known-compound libraries. An activation effect of ISX9 on the Wnt/β-catenin pathway was analysed with the SuperTOPFlash or SuperFOPFlash reporter system. Effects of ISX9 on Axin1/LRP6 interaction were examined using a mammalian two-hybrid system, co-immunoprecipitation, microscale thermophoresis, emission spectra and mass spectrometry assays. The expression of Wnt target and stemmness marker genes were evaluated with real-time PCR and immunoblotting. In vivo hair regeneration abilities of ISX9 were analysed by immunohistochemical staining, real-time PCR and immunoblotting in hair regrowth model using C57BL/6J mice. In this study, ISX9 was identified as a novel agonist of the Wnt/β-catenin pathway. ISX9 targeted Axin1 by covalently binding to its N-terminal region and potentiated the LRP6-Axin1 interaction, thereby resulting in the stabilization of β-catenin and up-regulation of Wnt target genes and stemmness marker genes. Moreover, the topical application of ISX9 markedly promoted hair regrowth in C57BL/6J mice and induced hair follicle transition from telogen to anagen via enhancing Wnt/β-catenin pathway. Taken together, our study unravelled that ISX9 could activate Wnt/β-catenin signalling by potentiating the association between LRP6 and Axin1, and may be a promising therapeutic agent for alopecia treatment.

SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells.

The primordial to primary follicle transition (PPT) in the ovary is critical to maintain sustainable reproductive resources in female mammals. However, it is unclear how granulosa cells (GCs) of the primary follicle participate in regulating PPT. This study focused on exploring the role of transcription factor Sp1 (SP1)in regulating PPT based on the fact that SP1 is pivotal for pregranulosa cell proliferation before primordial follicle formation. The results showed that mice fertility was prolonged when Sp1 was specifically depleted from GCs (GC- Sp1 -/- ). Besides, the PPT in GC- Sp1 -/- mice was reduced, resulting in more primordial follicles being preserved. Single-cell RNA-seq also indicated that the level of cholesterol metabolism was downregulated in GC- Sp1 -/- mice. Additionally, the PPT was promoted by either overexpression of ferredoxin-1 (FDX1), one of the key genes in mediating cholesterol metabolism or supplementing cholesterol for cultured fetal ovaries. Collectively, SP1 in GCs participates in the metabolism of cholesterol partially by regulating the transcription of Fdx1 during the PPT.