Male fertility restoration: In vivo and In vitro stem cell-based strategies using cryopreserved testis tissue - A scoping review.

IMPORTANCE: Advances in the treatment of childhood cancer have significantly improved survival rates, with more than 80% of survivors reaching adulthood. However, gonadotoxic cancer treatments endanger future fertility and prepubertal males have no option to preserve fertility by sperm cryopreservation. Also, boys with cryptorchidism are at risk of compromised fertility in adulthood. OBJECTIVE: This scoping review focuses on male fertility restoration, particularly relevant for prepubertal male cancer survivors and boys with cryptorchidism. The aim was to investigate current evidence for fertility restoration strategies, explore barriers to clinical implementation, and outline potential steps to overcome these barriers. EVIDENCE REVIEW: The review was conducted following the PRISMA-ScR criteria and previously published guidelines and examines studies using human testis tissue of prepubertal boys or healthy male adults. A literature search in PubMed was conducted and 72 relevant studies were identified, including in vivo and in vitro approaches. FINDINGS: In vivo strategies, such as testis tissue engraftment and spermatogonial stem cell (SSC) transplantation, hold promise for promoting cell survival and differentiation. Yet complete spermatogenesis has not been achieved. In vitro approaches focus on the generation of male germ cells from direct germ cell maturation in various culture systems, alongside human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). These approaches mark significant advancements in understanding and promoting spermatogenesis but achieving fully functional spermatozoa in vitro remains a challenge. Barriers to clinical implementation include the risk of reintroducing malignant cells and introduction of epigenetic changes. CONCLUSION: Male fertility restoration is an area in rapid development. Based on the reviewed studies the most promising and advanced strategy for restoring male fertility using cryopreserved testis tissue is direct testis tissue transplantation. RELEVANCE: This review identifies persistent barriers to the clinical implementation of male fertility restoration. However, direct transplantation of frozen-thawed testis tissue remains a promising strategy that is on the verge of clinical application.

Usefulness of alien sterilizing cytoplasms for the hybrid breeding of triticale (xTriticosecale Wittmack): preliminary results.

To be useful for cereal breeding, cytoplasmic male sterility (CMS) should express the complete sterility of maternal lines and the full restoration of the male fertility of F1 hybrids. The most reliable source of sterilizing cytoplasm for triticale is Triticum timopheevi; however, due to the low frequency of efficient non-restorer genotypes for this cytoplasm, new sources of CMS are needed. In this study, aside from T. timopheevi (T) cytoplasm, three alternative CMS sources were tested: Pampa (P) from Secale cereale L., Aegilops sharonensis (A), and Ae. ventricosa (V). The suitability of these cytoplasms for breeding was assessed based on the male fertility/sterility of F1 hybrids obtained through the manual pollination of CMS maternal lines with 36 triticale cultivars and breeding strains. About half of the hybrids with each type of cytoplasm were fully fertile and produced more than 30 grains per bagged spike. The highest percentage was found in hybrids with P cytoplasm (58.33%) and the lowest in hybrids with A cytoplasm (44.44%). Male sterility was observed in hybrids with P cytoplasm (16.67%) and A cytoplasm (16.67%) but not in hybrids with T or V cytoplasm. In terms of practical aspects, male sterility systems with P or A cytoplasm exhibit similarity in their ability to restore male fertility that differ from the T and V cytoplasms. Although all studied cytoplasms exhibited some disadvantages for breeding purposes, none should be definitively classified as unacceptable for future breeding programs regarding the development of triticale hybrid cultivars.

Evolution of biotechnological advances and regenerative therapies for endometrial disorders: a systematic review.

BACKGROUND: The establishment and maintenance of pregnancy depend on endometrial competence. Asherman syndrome (AS) and intrauterine adhesions (IUA), or endometrial atrophy (EA) and thin endometrium (TE), can either originate autonomously or arise as a result from conditions (i.e. endometritis or congenital hypoplasia), or medical interventions (e.g. surgeries, hormonal therapies, uterine curettage or radiotherapy). Affected patients may present an altered or inadequate endometrial lining that hinders embryo implantation and increases the risk of poor pregnancy outcomes and miscarriage. In humans, AS/IUA and EA/TE are mainly treated with surgeries or pharmacotherapy, however the reported efficacy of these therapeutic approaches remains unclear. Thus, novel regenerative techniques utilizing stem cells, growth factors, or tissue engineering have emerged to improve reproductive outcomes. OBJECTIVE AND RATIONALE: This review comprehensively summarizes the methodologies and outcomes of emerging biotechnologies (cellular, acellular, and bioengineering approaches) to treat human endometrial pathologies. Regenerative therapies derived from human tissues or blood which were studied in preclinical models (in vitro and in vivo) and clinical trials are discussed. SEARCH METHODS: A systematic search of full-text articles available in PubMed and Embase was conducted to identify original peer-reviewed studies published in English between January 2000 and September 2023. The search terms included: human, uterus, endometrium, Asherman syndrome, intrauterine adhesions, endometrial atrophy, thin endometrium, endometritis, congenital hypoplasia, curettage, radiotherapy, regenerative therapy, bioengineering, stem cells, vesicles, platelet-rich plasma, biomaterials, microfluidic, bioprinting, organoids, hydrogel, scaffold, sheet, miRNA, sildenafil, nitroglycerine, aspirin, growth hormone, progesterone, and estrogen. Preclinical and clinical studies on cellular, acellular, and bioengineering strategies to repair or regenerate the human endometrium were included. Additional studies were identified through manual searches. OUTCOMES: From a total of 4366 records identified, 164 studies (3.8%) were included for systematic review. Due to heterogeneity in the study design and measured outcome parameters in both preclinical and clinical studies, the findings were evaluated qualitatively and quantitatively without meta-analysis. Groups using stem cell-based treatments for endometrial pathologies commonly employed mesenchymal stem cells (MSCs) derived from the human bone marrow or umbilical cord. Alternatively, acellular therapies based on platelet-rich plasma (PRP) or extracellular vesicles are gaining popularity. These are accompanied by the emergence of bioengineering strategies based on extracellular matrix (ECM)-derived hydrogels or synthetic biosimilars that sustain local delivery of cells and growth factors, reporting promising results. Combined therapies that target multiple aspects of tissue repair and regeneration remain in preclinical testing but have shown translational value. This review highlights the myriad of therapeutic material sources, administration methods, and carriers that have been tested. WIDER IMPLICATIONS: Therapies that promote endometrial proliferation, vascular development, and tissue repair may help restore endometrial function and, ultimately, fertility. Based on the existing evidence, cost, accessibility, and availability of the therapies, we propose the development of triple-hit regenerative strategies, potentially combining high-yield MSCs (e.g. from bone marrow or umbilical cord) with acellular treatments (PRP), possibly integrated in ECM hydrogels. Advances in biotechnologies together with insights from preclinical models will pave the way for developing personalized treatment regimens for patients with infertility-causing endometrial disorders such as AS/IUA, EA/TE, and endometritis. REGISTRATION NUMBER: https://osf.io/th8yf/.

Incorporation of Decidual Stromal Cells Derived Exosomes in Sodium Alginate Hydrogel as an Innovative Therapeutic Strategy for Advancing Endometrial Regeneration and Reinstating Fertility.

Intrauterine adhesion (IUA) stands as a prevalent medical condition characterized by endometrial fibrosis and scar tissue formation within the uterine cavity, resulting in infertility and, in severe cases, recurrent miscarriages. Cell therapy, especially with stem cells, offers an alternative to surgery, but concerns about uncontrolled differentiation and tumorigenicity limit its use. Exosomes, more stable and immunogenicity-reduced than parent cells, have emerged as a promising avenue for IUA treatment. In this study, a novel approach has been proposed wherein exosomes originating from decidual stromal cells (DSCs) are encapsulated within sodium alginate hydrogel (SAH) scaffolds to repair endometrial damage and restore fertility in a mouse IUA model. Current results demonstrate that in situ injection of DSC-derived exosomes (DSC-exos)/SAH into the uterine cavity has the capability to induce uterine angiogenesis, initiate mesenchymal-to-epithelial transformation (MET), facilitate collagen fiber remodeling and dissolution, promote endometrial regeneration, enhance endometrial receptivity, and contribute to the recovery of fertility. RNA sequencing and advanced bioinformatics analysis reveal miRNA enrichment in exosomes, potentially supporting endometrial repair. This finding elucidates how DSC-exos/SAH mechanistically fosters collagen ablation, endometrium regeneration, and fertility recovery, holding the potential to introduce a novel IUA treatment and offering invaluable insights into the realm of regenerative medicine.

Endometrial mesenchymal stromal/stem cells improve regeneration of injured endometrium in mice.

BACKGROUND: The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. METHODS: Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. RESULTS: Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. CONCLUSIONS: Our findings suggest eMSC improves regeneration of injured endometrium in mice.

Editing of ORF138 restores fertility of Ogura cytoplasmic male sterile broccoli via mitoTALENs.

Cytoplasmic male sterility (CMS), encoded by the mitochondrial open reading frames (ORFs), has long been used to economically produce crop hybrids. However, the utilization of CMS also hinders the exploitation of sterility and fertility variation in the absence of a restorer line, which in turn narrows the genetic background and reduces biodiversity. Here, we used a mitochondrial targeted transcription activator-like effector nuclease (mitoTALENs) to knock out ORF138 from the Ogura CMS broccoli hybrid. The knockout was confirmed by the amplification and re-sequencing read mapping to the mitochondrial genome. As a result, knockout of ORF138 restored the fertility of the CMS hybrid, and simultaneously manifested a cold-sensitive male sterility. ORF138 depletion is stably inherited to the next generation, allowing for direct use in the breeding process. In addition, we proposed a highly reliable and cost-effective toolkit to accelerate the life cycle of fertile lines from CMS-derived broccoli hybrids. By applying the k-mean clustering and interaction network analysis, we identified the central gene networks involved in the fertility restoration and cold-sensitive male sterility. Our study enables mitochondrial genome editing via mitoTALENs in Brassicaceae vegetable crops and provides evidence that the sex production machinery and its temperature-responsive ability are regulated by the mitochondria.

Identification and characterization of circular RNAs involved in the fertility stability of cotton CMS-D2 restorer line under heat stress.

BACKGROUND: As a vital type of noncoding RNAs, circular RNAs (circRNAs) play important roles in plant growth and development and stress response. However, little is known about the biological roles of circRNAs in regulating the stability of male fertility restoration for cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) cotton under high-temperature (HT) stress. RESULTS: In this study, RNA-sequencing and bioinformatics analysis were performed on pollen grains of isonuclear alloplasmic near-isogenic restorer lines NH [N(Rf1rf1)] and SH [S(Rf1rf1)] with obvious differences in fertility stability under HT stress at two environments. A total of 967 circRNAs were identified, with 250 differentially expressed under HT stress. We confirmed the back-splicing sites of eight selected circRNAs using divergent primers and Sanger sequencing. Tissue-specific expression patterns of five differentially expressed circRNAs (DECs) were also verified by RT-PCR and qRT-PCR. Functional enrichment and metabolic pathway analysis revealed that the parental genes of DECs were significantly enriched in fertility-related biological processes such as pollen tube guidance and cell wall organization, as well as the Pentose and glucuronate interconversions, Steroid biosynthesis, and N-Glycan biosynthesis pathways. Moreover, we also constructed a putative circRNA-mediated competing endogenous RNA (ceRNA) network consisting of 21 DECs, eight predicted circRNA-binding miRNAs, and their corresponding 22 mRNA targets, especially the two ceRNA modules circRNA346-miR159a-MYB33 and circRNA484-miR319e-MYB33, which might play important biological roles in regulating pollen fertility stability of cotton CMS-D2 restorer line under HT stress. CONCLUSIONS: Through systematic analysis of the abundance, characteristics and expression patterns of circRNAs, as well as the potential functions of their parent genes, our findings suggested that circRNAs and their mediated ceRNA networks acted vital biological roles in cotton pollen development, and might be also essential regulators for fertility stability of CMS-D2 restorer line under heat stress. This study will open a new door for further unlocking complex regulatory mechanisms underpinning the fertility restoration stability for CMS-D2 in cotton.

Results from the first autologous grafting of adult human testis tissue: a case report.

Fertility restoration using autologous testicular tissue transplantation is relevant for infertile men surviving from childhood cancer and, possibly, in men with absent or incomplete spermatogenesis resulting in the lack of spermatozoa in the ejaculate (non-obstructive azoospermia, NOA). Currently, testicular tissue from pre-pubertal boys extracted before treatment with gonadotoxic cancer therapy can be cryopreserved with good survival of spermatogonial stem cells. However, strategies for fertility restoration, after successful cancer treatment, are still experimental and no clinical methods have yet been developed. Similarly, no clinically available treatments can help men with NOA to become biological fathers after failed attempts of testicular surgical sperm retrieval. We present a case of a 31-year-old man with NOA who had three pieces of testis tissue (each ∼2 × 4 × 2 mm3) extracted and cryopreserved in relation to performing microdissection testicular sperm extraction (mTESE). Approximately 2 years after mTESE, the thawed tissue pieces were engrafted in surgically created pockets bilaterally under the scrotal skin. Follow-up was performed after 2, 4, and 6 months with assessment of reproductive hormones and ultrasound of the scrotum. After 6 months, all engrafted tissue was extracted and microscopically analyzed for the presence of spermatozoa. Furthermore, parts of the extracted tissue were analyzed histologically and by immunohistochemical analysis. Active blood flow in the engrafted tissue was demonstrated by doppler ultrasound after 6 months. No spermatozoa were found in the extracted tissue. Histological and immunohistochemical analysis demonstrated graft survival with intact clear tubules and normal cell organization. Sertoli cells and spermatocytes with normal morphology were located near the basement membrane. MAGE-A and VASA positive spermatogonia/spermatocytes were detected together with SOX9 positive Sertoli cells. Spermatocytes and/or Sertoli cells positive for γH2AX was also detected. In summary, following autologous grafting of frozen-thawed testis tissue under the scrotal skin in a man with NOA, we demonstrated graft survival after 6 months. No mature spermatozoa were detected; however, this is likely due to the pre-existing spermatogenic failure.

Endometrial mesenchymal stromal/stem cells improve regeneration of injured endometrium in mice

BACKGROUND: The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. METHODS: Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. RESULTS: Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. CONCLUSIONS: Our findings suggest eMSC improves regeneration of injured endometrium in mice.

Nano-biomaterial Fibrinogen/P(LLA-CL) for prevention of intrauterine adhesion and restoration of fertility.

Endometrial damage resulting from surgical procedures is a significant cause of intrauterine adhesion, thin endometrium, and subsequent miscarriage and infertility. Unfortunately, there is currently no effective clinical solution to promote endometrial regeneration after severe injury. In this study, we combined fibrinogen (Fg) and P(LLA-CL) by electrostatic spinning to form a stable nano-biomaterial Fg/P(LLA-CL), which can promote endometrial regeneration. After inducing physical injury to rat endometrium, we found that Fg/P(LLA-CL) membranes placed in the uterine cavities increased endometrial thickness and the number of glands after injury, while reducing the area of endometrial fibrosis. In addition, Fg/P(LLA-CL) increased neovascularization and decreased COL1A1 deposition. The expression of TGF-ß1, a cytokine that promotes fibrosis, was down-regulated in the early stage of injury. Finally, fertility assays confirmed that Fg/P(LLA-CL) improved the pregnancy rate in rats with endometrial injury, and its safety was verified by blood tests and pathological examination of heart, liver, spleen, lung, and kidney. Therefore, Fg/P(LLA-CL) shows great potential as a safe and nontoxic biomaterial for endometrial regeneration, ultimately improving pregnancy outcomes in patients with intrauterine adhesion.

Use of mesenchymal stem cells to enhance or restore fertility potential: a systematic review of available experimental strategies.

STUDY QUESTION: To what extent does regenerative medicine with stem cell therapy help to address infertility issues for future clinical application? SUMMARY ANSWER: Regenerative medicine using different stem cell sources is yielding promising results in terms of protecting the ovarian reserve from damage and senescence, and improving fertility potential in various preclinical settings. WHAT IS KNOWN ALREADY: Regenerative medicine using stem cell therapy is emerging as a potential strategy to address a number of issues in the field of human reproduction. Indeed, different types of adult and fetal mesenchymal stem cells (MSCs) have been tested with promising results, owing to their ability to differentiate into different tissue lineages, move toward specific injured sites (homing), and generate a secretome with wound-healing, proangiogenic, and antioxidant capacities. STUDY DESIGN SIZE DURATION: Guided by the checklist for preferred reporting items for systematic reviews and meta-analyses, we retrieved relevant studies from PubMed, Medline, and Embase databases until June 2023 using the following keywords: 'mesenchymal stem cells' AND 'ovarian follicles' OR 'ovarian tissue culture' OR 'ovarian follicle culture' OR 'cumulus oocyte complex'. Only peer-reviewed published articles written in English were included. PARTICIPANTS/MATERIALS SETTING METHODS: The primary outcome for the experimental strategies was evaluation of the ovarian reserve, with a focus on follicle survival, number, and growth. Secondary outcomes involved analyses of other parameters associated with the follicle pool, such as hormones and growth factors, ovarian tissue viability markers including oxidative stress levels, oocyte growth and maturation rates, and of course pregnancy outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: Preclinical studies exploring MSCs from different animal origins and tissue sources in specific conditions were selected (n = 112), including: in vitro culture of granulosa cells, ovarian tissue and isolated ovarian follicles; ovarian tissue transplantation; and systemic or intraovarian injection after gonadotoxic or age-related follicle pool decline. Protecting the ovarian reserve from aging and gonadotoxic damage has been widely tested in vitro and in vivo using murine models and is now yielding initial data in the first ever case series of patients with premature ovarian insufficiency. Use of MSCs as feeder cells in ovarian tissue culture was found to improve follicle outcomes and oocyte competence, bringing us one step closer to future clinical application. MSCs also have proved effective at boosting revascularization in the transplantation site when grafting ovarian tissue in experimental animal models. LIMITATIONS REASONS FOR CAUTION: While preclinical results look promising in terms of protecting the ovarian reserve in different experimental models (especially those in vitro using various mammal experimental models and in vivo using murine models), there is still a lot of work to do before this approach can be considered safe and successfully implemented in a clinical setting. WIDER IMPLICATIONS OF THE FINDINGS: All gathered data on the one hand show that regenerative medicine techniques are quickly gaining ground among innovative techniques being developed for future clinical application in the field of reproductive medicine. After proving MSC effectiveness in preclinical settings, there is still a lot of work to do before MSCs can be safely and effectively used in different clinical applications. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR T.0077.14, FNRS-CDR J.0063.20, and grant 5/4/150/5 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, and the Fondation St Luc. None of the authors have any competing interest to disclose. REGISTRATION NUMBER: N/A.

Genetic Analysis of Novel Fertility Restoration Genes (qRf3 and qRf6) in Dongxiang Wild Rice Using GradedPool-Seq Mapping and QTL-Seq Correlation Analysis.

The improvement of grain yield, quality, and resistance can be achieved through the utilization of heterosis. The combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) gene(s) greatly facilitates the commercial development of three-line hybrid rice based on heterosis. The basis for investigating the relationship between CMS and Rf genes lies in the rapid localization of wild rice fertility restoration genes. A set of the BC4F5 population derived from interspecific crosses between Xieqingzao B (XB) and the BC1F9 XB//Dongxiang wild rice (DWR)/XB line L5339 was used to detect quantitative trait loci (QTL) for fertility restoration. The population was then crossed with two male sterile lines, Zhong9A (Z9A) and DongB11A (DB11A), in order to generate a testcrossing population for investigating spikelet fertility. Based on the linkage mapping, seven QTLs were detected on chromosomes 1, 3, 5, 6, 8, and 10, explaining 2.76 to 12.46% of the phenotypic variation. Of them, two novel fertility restoration QTLs, qRf3 and qRf6, can restore fertility of the CMS-DWR line DB11A by 16.56% and 15.12%, respectively. By employing joint QTL-seq and GradedPool-Seq methods, two novel Rf QTLs for DB11A, qRf3 and qRf6, were identified at the physical locations of 10,900,001-11,700,000 bp and 28,016,785-31,247,556 bp, respectively. These findings are useful for exploring the natural variations of Rf genes in rice. Therefore, rice's new genetic resources for the selection and breeding of rice restorer lines provide promising candidates for QTL fine localization and clarification.

Dose effects of restorer gene modulate pollen fertility in cotton CMS-D2 restorer lines via auxin signaling and flavonoid biosynthesis.

KEY MESSAGE: Dose effects of Rf1 gene regulated retrieval mechanism of pollen fertility for CMS-D2 cotton. Cytoplasmic male sterility conditioned by Gossypium harknessii cytoplasm (CMS-D2) is an economical pollination control system for producing hybrid cotton seeds compared to artificial and chemical emasculation methods. However, the unstable restoring ability of restorer lines is a main barrier in the large-scale application of "three-line" hybrid cotton in China. Our phenotypic investigation determined that the homozygous Rf1Rf1 allelic genotype had a stronger ability to generate fertile pollen than the heterozygous Rf1rf1 allelic genotype. To decipher the genetic mechanisms that control the differential levels of pollen fertility, an integrated metabolomic and transcriptomic analysis was performed at two environments using pollen grains of four cotton genotypes differing in Rf1 alleles or cytoplasm. Totally 5,391 differential metabolite features were detected, and 369 specific differential metabolites (DMs) were identified between homozygous and heterozygous Rf1 allelic genotypes with CMS-D2 cytoplasm. In addition, transcriptome analysis identified 2,490 differentially expressed genes (DEGs) and 96 unique hub DEGs with dynamic regulation in this comparative combination. Further integrated analyses revealed that several key DEGs and DMs involved in indole biosynthesis, flavonoid biosynthesis, and sugar metabolism had strong network linkage with fertility restoration. In vitro application of auxin analogue NAA and inhibitor Auxinole confirmed that over-activated auxin signaling might inhibit pollen development, whereas suppressing auxin signaling partially promoted pollen development in CMS-D2 cotton. Our results provide new insight into how the dosage effects of the Rf1 gene regulate pollen fertility of CMS-D2 cotton.

Minimal Infiltrative Disease Identification in Cryopreserved Ovarian Tissue of Girls with Cancer for Future Use: A Systematic Review.

BACKGROUND: Ovarian tissue cryopreservation and transplantation are the only available fertility techniques for prepubertal girls with cancer. Though autotransplantation carries a risk of reintroducing malignant cells, it can be avoided by identifying minimal infiltrative disease (MID) within ovarian tissue. METHODS: A broad search for peer-reviewed articles in the PubMed database was conducted in accordance with PRISMA guidelines up to March 2023. Search terms included 'minimal residual disease', 'cryopreservation', 'ovarian', 'cancer' and synonyms. RESULTS: Out of 542 identified records, 17 were included. Ovarian tissues of at least 115 girls were evaluated and categorized as: hematological malignancies (n = 56; 48.7%), solid tumors (n = 42; 36.5%) and tumors of the central nervous system (n = 17; 14.8%). In ovarian tissue of 25 patients (21.7%), MID was detected using RT-qPCR, FISH or multicolor flow cytometry: 16 of them (64%) being ALL (IgH rearrangements with/without TRG, BCL-ABL1, EA2-PBX1, TEL-AML1 fusion transcripts), 3 (12%) Ewing sarcoma (EWS-FLI1 fusion transcript, EWSR1 rearrangements), 3 (12%) CML (BCR-ABL1 fusion transcript, FLT3) and 3 (12%) AML (leukemia-associated immunophenotypes, BCR-ABL1 fusion transcript) patients. CONCLUSION: While the majority of malignancies were found to have a low risk of containing malignant cells in ovarian tissue, further studies are needed to ensure safe implementation of future fertility restoration in clinical practice.

Fertility restoration in mice with chemotherapy induced ovarian failure using differentiated iPSCs.

BACKGROUND: Treatment options for premature ovarian insufficiency (POI) are limited to hormone replacement and donor oocytes. A novel induced pluripotent stem cell (iPSC) transplant paradigm in a mouse model has potential translational applications for management of POI. METHODS: Mouse ovarian granulosa cell derived-iPSCS were labelled with green fluorescent protein (GFP) reporter and differentiated in vitro into oocytes. Differentiated cells were assayed for estradiol and progesterone secretion by enzyme-linked immunosorbent assays. After Fluorescence-Activated Cell Sorting (FACS) for the cell surface marker anti-Mullerian hormone receptor (AMHR2), enriched populations of differentiated cells were surgically transplanted into ovaries of mice that had POI secondary to gonadotoxic pre-treatment with alkylating agents. A total of 100 mice were used in these studies in five separate experiments with 56 animals receiving orthotopic ovarian injections of either FACS sorted or unsorted differentiated iPSCSs and the remaining animals receiving sham injections of PBS diluent. Following transplantation surgery, mice were stimulated with gonadotropins inducing oocyte development and underwent oocyte retrieval. Nine transplanted mice were cross bred with wild-type mice to assess fertility. Lineage tracing of resultant oocytes, F1 (30 pups), and F2 (42 pups) litters was interrogated by GFP expression and validation by short tandem repeat (STR) lineage tracing. FINDINGS: [1] iPSCs differentiate into functional oocytes and steroidogenic ovarian cells which [2] express an ovarian (GJA1) and germ cell (ZP1) markers. [3] Endocrine function and fertility were restored in mice pretreated with gonadotoxic alkylating agents via orthotopic transplantation of differentiated iPSCS, thus generating viable, fertile mouse pups. INTERPRETATION: iPSC-derived ovarian tissue can reverse endocrine and reproductive sequelae of POI. FUNDING: Center for Infertility and Reproductive Surgery Research Award, Siezen Foundation award (RMA). Reproductive Scientist Development Program, Marriott Foundation, Saltonstall Foundation, Brigham Ovarian Cancer Research Fund (K.E).

New Solutions for Old Problems: How Reproductive Tissue Engineering Has Been Revolutionizing Reproductive Medicine.

Acquired disorders and congenital defects of the male and female reproductive systems can have profound impacts on patients, causing sexual and endocrine dysfunction and infertility, as well as psychosocial consequences that affect their self-esteem, identity, sexuality, and relationships. Reproductive tissue engineering (REPROTEN) is a promising approach to restore fertility and improve the quality of life of patients with reproductive disorders by developing, replacing, or regenerating cells, tissues, and organs from the reproductive and urinary systems. In this review, we explore the latest advancements in REPROTEN techniques and their applications for addressing degenerative conditions in male and female reproductive organs. We discuss current research and clinical outcomes and highlight the potential of 3D constructs utilizing biomaterials such as scaffolds, cells, and biologically active molecules. Our review offers a comprehensive guide for researchers and clinicians, providing insights into how to reestablish reproductive tissue structure and function using innovative surgical approaches and biomaterials. We highlight the benefits of REPROTEN for patients, including preservation of fertility and hormonal production, reconstruction of uterine and cervical structures, and restoration of sexual and urinary functions. Despite significant progress, REPROTEN still faces ethical and technical challenges that need to be addressed. Our review underscores the importance of continued research in this field to advance the development of effective and safe REPROTEN approaches for patients with reproductive disorders.

Refinement of Rf1-gene localization and development of the new molecular markers for fertility restoration in sunflower.

BACKGROUND: Ability to restore male fertility is important trait for sunflower breeding. The most commonly used fertility restoration gene in the production of sunflower hybrids is Rf1. The localization of Rf1 on the linkage group 13 has been previously shown, however, its exact position, its sequence and molecular mechanism for fertility restoration remain unknown. Therefore, several markers linked to Rf1 gene, commonly used for MAS, don't always allow to identify the genotype of plants. For this reason, the search for new markers and precise localization of the Rf1 gene is an urgent task. METHODS AND RESULTS: Based on previously identified single nucleotide polymorphisms (SNPs) at LG13, significantly associated with the ability to restore male fertility, two markers have been developed that have performed well after careful evaluation. These markers, together with other Rf1 markers, were applied for genotyping 72 diversity panel accessions and 291 individuals of F2 segregating population, obtained from crossing the cytoplasmic male sterility (CMS) AHO33 and restorer RT085HO lines. The analysis revealed no recombinants between Rf1 gene and SRF833 marker, the distance between Rf1 and SRF122 marker was 1.0 cM. CONCLUSIONS: Data obtained made it possible to specify the localization of the Rf1 gene and reduce the list of candidate genes to the 3 closely linked PPR-genes spanning a total of 59 Kb. However, it cannot be ruled out that analysis of the candidate region in the genome of fertility restorer lines can reveal new candidate genes in this locus that are absent in the cytoplasmic male sterility maintainer reference sequence.

Amniotic fluid-derived exosomes attenuated fibrotic changes in POI rats through modulation of the TGF-ß/Smads signaling pathway.

In the current study, we investigated the regenerative effects of amniotic fluid exosomes (AF-Exos) in a rat model for premature ovarian insufficiency (POI). POI is a condition characterized by a decrease in ovarian function that can lead to infertility. We induced POI by administering cyclophosphamide (CTX) for 15 consecutive days, and then transplanted AF-Exos directly into both ovarian tissues. Four weeks later, we measured the serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2), and performed histopathological evaluations using H & E and Masson's trichrome staining. We also monitored the expression of genes related to the TGF-ß signaling pathway using real-time PCR and examined the fertility rate of POI rats after AF-Exos therapy. Histological analysis showed an increase in atretic follicles and a decrease in healthy follicle count after POI induction. Four weeks post-AF-Exos intervention, the healthy follicle count increased (p < 0.01) while the atretic follicle count decreased (p < 0.001). In parallel, the deposition of collagen fibers also decreased following AF-Exos transplantation. The concentrations of FSH and LH hormones in sera remained unchanged after injection of AF-Exos, while E2 levels increased (p < 0.05). The expression of Smad-4 (p < 0.01) and Smad-6 (p < 0.05) was upregulated in POI rats that received AF-Exos, while Smad-2, TGF-ß1, TNF-α, and IL-10 remained statistically unchanged. Our records showed a notable increase in litter number after AF-Exos compared to the non-treated POI rats. These results suggest that AF-Exos transplantation has the potential to restore ovarian function through the TGF-ß/Smads signaling pathway in POI rats.

Genomic survey of high-throughput RNA-Seq data implicates involvement of long intergenic non-coding RNAs (lincRNAs) in cytoplasmic male-sterility and fertility restoration in pigeon pea.

BACKGROUND: Long-intergenic non-coding RNAs (lincRNAs) originate from intergenic regions and have no coding potential. LincRNAs have emerged as key players in the regulation of various biological processes in plant development. Cytoplasmic male-sterility (CMS) in association with restorer-of-fertility (Rf) systems makes it a highly reliable tool for exploring heterosis for producing commercial hybrid seeds. To date, there have been no reports of lincRNAs during pollen development in CMS and fertility restorer lines in pigeon pea. OBJECTIVE: Identification of lincRNAs in the floral buds of cytoplasmic male-sterile (AKCMS11) and fertility restorer (AKPR303) pigeon pea lines. METHODS: We employed a computational approach to identify lincRNAs in the floral buds of cytoplasmic male-sterile (AKCMS11) and fertility restorer (AKPR303) pigeon pea lines using RNA-Seq data. RESULTS: We predicted a total of 2145 potential lincRNAs of which 966 were observed to be differentially expressed between the sterile and fertile pollen. We identified, 927 cis-regulated and 383 trans-regulated target genes of the lincRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the target genes revealed that these genes were specifically enriched in pathways like pollen and pollen tube development, oxidative phosphorylation, etc. We detected 23 lincRNAs that were co-expressed with 17 pollen-related genes with known functions. Fifty-nine lincRNAs were predicted to be endogenous target mimics (eTMs) for 25 miRNAs, and found to be associated with pollen development. The, lincRNA regulatory networks revealed that different lincRNA-miRNA-mRNA networks might be associated with CMS and fertility restoration. CONCLUSION: Thus, this study provides valuable information by highlighting the functions of lincRNAs as regulators during pollen development in pigeon pea and utilization in hybrid seed production.

Turning Garlic into a Modern Crop: State of the Art and Perspectives.

Garlic is cultivated worldwide for the value of its bulbs, but its cultivation is challenged by the infertility of commercial cultivars and the accumulation of pathogens over time, which occurs as a consequence of vegetative (clonal) propagation. In this review, we summarize the state of the art of garlic genetics and genomics, highlighting recent developments that will lead to its development as a modern crop, including the restoration of sexual reproduction in some garlic strains. The set of tools available to the breeder currently includes a chromosome-scale assembly of the garlic genome and multiple transcriptome assemblies that are furthering our understanding of the molecular processes underlying important traits like the infertility, the induction of flowering and bulbing, the organoleptic properties and resistance to various pathogens.