Advanced strategies for single embryo selection in assisted human reproduction: A review of clinical practice and research methods.

Among the primary objectives of contemporary assisted reproductive technology research are achieving the births of healthy singletons and improving overall fertility outcomes. Substantial advances have been made in refining the selection of single embryos for transfer, with the aim of maximizing the likelihood of successful implantation. The principal criterion for this selection is embryo morphology. Morphological evaluation systems are based on traditional parameters, including cell count and fragmentation, pronuclear morphology, cleavage rate, blastocyst formation, and various sequential embryonic assessments. To reduce the incidence of multiple pregnancies and to identify the single embryo with the highest potential for growth, invasive techniques such as preimplantation genetic screening are employed in in vitro fertilization clinics. However, new approaches have been suggested for clinical application that do not harm the embryo and that provide consistent, accurate results. Noninvasive technologies, such as time-lapse imaging and omics, leverage morphokinetic parameters and the byproducts of embryo metabolism, respectively, to identify noninvasive prognostic markers for competent single embryo selection. While these technologies have garnered considerable interest in the research community, they are not incorporated into routine clinical practice and still have substantial room for improvement. Currently, the most promising strategies involve integrating multiple methodologies, which together are anticipated to increase the likelihood of successful pregnancy.

Therapeutic potential of exosomes in spermatogenesis regulation and male infertility.

BACKGROUND: Spermatogenesis is a fundamental process crucial for male reproductive health and fertility. Exosomes, small membranous vesicles released by various cell types, have recently garnered attention for their role in intercellular communication. OBJECTIVE: This review aims to comprehensively explore the role of exosomes in regulating spermatogenesis, focusing on their involvement in testicular development and cell-to-cell communication. METHODS: A systematic examination of literature was conducted to gather relevant studies elucidating the biogenesis, composition, and functions of exosomes in the context of spermatogenesis. RESULTS: Exosomes play a pivotal role in orchestrating the complex signaling networks required for proper spermatogenesis. They facilitate the transfer of key regulatory molecules between different cell populations within the testes, including Sertoli cells, Leydig cells, and germ cells. CONCLUSION: The emerging understanding of exosome-mediated communication sheds light on novel mechanisms underlying spermatogenesis regulation. Further research in this area holds promise for insights into male reproductive health and potential therapeutic interventions.

Testicular niche repair after gonadotoxic treatments: Current knowledge and future directions.

The testicular niche, which includes the germ cells, somatic cells, and extracellular matrix, plays a crucial role in maintaining the proper functions of the testis. Gonadotoxic treatments, such as chemotherapy and radiation therapy, have significantly improved the survival rates of cancer patients but have also been shown to have adverse effects on the testicular microenvironment. Therefore, repairing the testicular niche after gonadotoxic treatments is essential to restore its function. In recent years, several approaches, such as stem cell transplantation, gene therapy, growth factor therapy, and pharmacological interventions have been proposed as potential therapeutic strategies to repair the testicular niche. This comprehensive review aims to provide an overview of the current understanding of testis damage and repair mechanisms. We will cover a range of topics, including the mechanism of gonadotoxic action, repair mechanisms, and treatment approaches. Overall, this review highlights the importance of repairing the testicular niche after gonadotoxic treatments and identifies potential avenues for future research to improve the outcomes for cancer survivors.

In vivo and in vitro sperm production: an overview of the challenges and advances in male fertility restoration.

Male infertility can be caused by genetic anomalies, endocrine disorders, inflammation, and exposure to toxic chemicals or gonadotoxic treatments. Therefore, several recent studies have concentrated on the preservation and restoration of fertility to enhance the quality of life for affected individuals. It is currently recommended to biobank the tissue extracted from testicular biopsies to provide a later source of spermatogonial stem cells (SSCs). Another successful approach has been the in vitro production of haploid male germ cells. The capacity of SSCs to transform into sperm, as in testicular tissue transplantation, SSC therapy, and in vitro or ex vivo spermatogenesis, makes them ideal candidates for in vivo fertility restoration. The transplantation of SSCs or testicular tissue to regenerate spermatogenesis and create embryos has been achieved in nonhuman mammal species. Although the outcomes of human trials have yet to be released, this method may soon be approved for clinical use in humans. Furthermore, regenerative medicine techniques that develop tissue or cells on organic or synthetic scaffolds enriched with bioactive molecules have also gained traction. All of these methods are now in different stages of experimentation and clinical trials. However, thanks to rigorous studies on the safety and effectiveness of SSC-based reproductive treatments, some of these techniques may be clinically available in upcoming decades.

3D Printing of Alginate/Chitosan-Based Scaffold Empowered by Tyrosol-Loaded Niosome for Wound Healing Applications: In Vitro and In Vivo Performances.

This study introduces a tyrosol-loaded niosome integrated into a chitosan-alginate scaffold (Nio-Tyro@CS-AL), employing advanced electrospinning and 3D printing techniques for wound healing applications. The niosomes, measuring 185.40 ± 6.40 nm with a polydispersity index of 0.168 ± 0.012, encapsulated tyrosol with an efficiency of 77.54 ± 1.25%. The scaffold's microsized porous structure (600-900 µm) enhances water absorption, promoting cell adhesion, migration, and proliferation. Mechanical property assessments revealed the scaffold's enhanced resilience, with niosomes increasing the compressive strength, modulus, and strain to failure, indicative of its suitability for wound healing. Controlled tyrosol release was demonstrated in vitro, essential for therapeutic efficacy. The scaffold exhibited significant antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus, with substantial biofilm inhibition and downregulation of bacterial genes (ndvb and icab). A wound healing assay highlighted a notable increase in MMP-2 and MMP-9 mRNA expression and the wound closure area (69.35 ± 2.21%) in HFF cells treated with Nio-Tyro@CS-AL. In vivo studies in mice confirmed the scaffold's biocompatibility, showing no significant inflammatory response, hypertrophic scarring, or foreign body reaction. Histological evaluations revealed increased fibroblast and macrophage activity, enhanced re-epithelialization, and angiogenesis in wounds treated with Nio-Tyro@CS-AL, indicating effective tissue integration and repair. Overall, the Nio-Tyro@CS-AL scaffold presents a significant advancement in wound-healing materials, combining antibacterial properties with enhanced tissue regeneration, and holds promising potential for clinical applications in wound management.

Polycaprolactone/Testicular Extracellular Matrix/Graphene Oxide-Based Electrospun Tubular Scaffolds for Reproductive Medicine: Biomimetic Architecture of Seminiferous Tubules.

Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively replicating the spatial characteristics of native tissue, poses a challenge in maintaining the requisite cellular arrangement essential for spermatogenesis. In order to mimic the structural properties of seminiferous tubules, the objective is to fabricate a biocompatible tubular scaffold. Following the decellularization process of the testicular tissue, validation of cellular remnants' elimination from the specimens is conducted using 4',6-diamidino-2-phenylindole staining, hematoxylin and eosin staining, and DNA content analysis. The presence of extracellular matrix (ECM) components is confirmed through Alcian blue, Orcein, and Masson's trichrome staining techniques. The electrospinning technique is employed to synthesize the scaffolds using polycaprolactone (PCL), extracted ECM, and varying concentrations of graphene oxide (GO) (0.5%, 1%, and 2%). Subsequently, comprehensive evaluations are performed to assess the properties of the synthetic scaffolds. These evaluations encompass Fourier-transform infrared spectroscopy, scanning electron microscopy imaging, scaffold degradation testing, mechanical behavior analysis, methylthiazolyldiphenyl-tetrazolium bromide assay, and in vivo biocompatibility assessment. The PCL/decellularized extracellular matrix with 0.5% GO formulation exhibits superior fiber morphology and enhanced mechanical properties, and outperforms other groups in terms of in vitro biocompatibility. Consequently, these scaffolds present a viable option for implementation in "in vitro spermatogenesis" procedures, holding promise for future sperm production from spermatogonial cells.

Ultrastructural study: in vitro and in vivo differentiation of mice spermatogonial stem cells.

Mouse testicular tissue is composed of seminiferous tubules and interstitial tissue. Mammalian spermatogenesis is divided into three stages: spermatocytogenesis (mitotic divisions) in which spermatogonial stem cells (SSCs) turn into spermatocytes, followed by two consecutive meiotic divisions in which spermatocytes form spermatids. Spermatids differentiate into spermatozoa during spermiogenesis. Various factors affect the process of spermatogenesis and the organization of cells in the testis. Any disorder in different stages of spermatogenesis will have negative effects on male fertility. The aim of the current study was to compare the in vitro and in vivo spermatogenesis processes before and after transplantation to azoospermic mice using ultrastructural techniques. In this study, mice were irradiated with single doses of 14 Gy 60Co radiation. SSCs isolated from neonatal mice were cultured in vitro for 1 week and were injected into the seminiferous tubule recipient's mice. Testicular cells of neonatal mice were cultured in the four groups on extracellular matrix-based 3D printing scaffolds. The transplanted testes (8 weeks after transplantation) and cultured testicular cells in vitro (after 3 weeks) were then processed for transmission electron microscopy studies. Our study's findings revealed that the morphology and ultrastructure of testicular cells after transplantation and in vitro culture are similar to those of in vivo spermatogenesis, indicating that spermatogenic cell nature is unaltered in vitro.

Melatonin Promotes Differentiation of Human Spermatogonial Stem Cells Cultured on Three-Dimensional Decellularized Human Testis Matrix.

PURPOSE: The use of 3D (3-Dimensional) culture systems supported cell-to-cell and cell-to-extracellular matrix (ECM) interactions, proliferation, and differentiation of SSCs (Spermatogonial stem cells). The potential advantages of ECM-based scaffolds for in vitro spermatogenesis have been indicated in human and animal experiments. Furthermore, the strong antioxidant and anti-inflammatory activities of melatonin have improved in vitro manipulation of human SSCs in culture conditions. MATERIALS AND METHODS: SSCs were isolated from the testis of three dead-brain patients and then propagated for four weeks. The characterization of SSC colonies was done using real-time PCR (Polymerase chain reaction), ICC (Immunocytochemistry), and xenotransplantation to mice model. Decellularization of the human testis was performed using 0.3% sodium dodecyl sulfate (SDS) solution and 1% Triton X-100. Also, various characterizations of DTM (Decellularized testicular matrix ) were carried out using histological staining and DNA content analysis. The optimum dose of melatonin was selected by MTT (Methyl thiazol tetrazolium). SSCs were cultured in 4 groups: control, melatonin, ECM, and ECM-melatonin in a differentiation medium for four weeks. The expression of differentiation genes was evaluated by real-time polymerase chain reaction. In addition, the viability of cultured cells was assessed by MTT assay. RESULTS: The results of ICC and real-time PCR showed the expression of undifferentiated SSC markers (PLZF and GRFA1) in SSC colonies following the 2D culture of isolated SSCs. The presence of testicular ECM components after different staining methods; and the reduction of DNA content confirmed the proper decellularization process. Germ cell apoptosis significantly decreased in melatonin and ECM groups, and the higher viability of SSCs was seen in the ECM-melatonin group. The relative expression of GFRA1 and PRM2 decreased and increased in ECM and ECM-melatonin groups, respectively. CONCLUSION: Our study showed that the addition of melatonin to the human naturally-derived ECM scaffold could provide a suitable platform for inducing the differentiation and preserving the viability of SSCs.

Doxorubicin-loaded Niosomes functionalized with gelatine and alginate as pH-responsive drug delivery system: A 3D printing approach.

Despite many efforts, breast cancer remains one of the deadliest cancers and its treatment faces challenges related to cancer drug side effects and metastasis. Combining 3D printing and nanocarriers has created new opportunities in cancer treatment. In this work, 3D-printed gelatin-alginate nanocomposites containing doxorubicin-loaded niosomes (Nio-DOX@GT-AL) were recruited as an advanced potential pH-sensitive drug delivery system. Morphology, degradation, drug release, flow cytometry, cell cytotoxicity, cell migration, caspase activity, and gene expression of nanocomposites and controls (Nio-DOX and Free-DOX) were evaluated. Results show that the obtained niosome has a spherical shape and size of 60-80 nm. Sustained drug release and biodegradability were presented by Nio-DOX@GT-AL and Nio-DOX. Cytotoxicity analysis revealed that the engineered Nio-DOX@GT-AL scaffold had 90 % cytotoxicity against breast cancer cells (MCF-7), whereas exhibited <5 % cytotoxicity against the non-tumor breast cell line (MCF-10A), which was significantly more than the antitumor effect of the control samples. Scratch-assay as an indicator cell migration demonstrated a reduction of almost 60 % of the covered surface. Gene expression could provide an explanation for the antitumor effect of engineered nanocarriers, which significantly reduced metastasis-promoting genes (Bcl2, MMP-2, and MMP-9), and significantly enhanced the expression and activity of genes that promote apoptosis (CASP-3, CASP-8, and CASP-9). Also, considerable inhibition of metastasis-associated genes (Bax and p53) was observed. Moreover, flow-cytometry data demonstrated that Nio-DOX@GT-AL decreased necrosis and enhanced apoptosis drastically. The findings of this research can confirm that employing 3D-printing and niosomal formulation can be an effective strategy in designing novel nanocarriers for efficient drug delivery applications.

In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility.

Men's reproductive health exclusively depends on the appropriate maturation of certain germ cells known as sperm. Certain illnesses, such as Klinefelter syndrome, cryptorchidism, and syndrome of androgen insensitivity or absence of testis maturation in men, resulting in the loss of germ cells and the removal of essential genes on the Y chromosome, can cause non-obstructive azoospermia. According to laboratory research, preserving, proliferating, differentiating, and transplanting spermatogonial stem cells or testicular tissue could be future methods for preserving the fertility of children with cancer and men with azoospermia. Therefore, new advances in stem cell research may lead to promising therapies for treating male infertility. The rate of progression and breakthrough in the area of in vitro spermatogenesis is lower than that of SSC transplantation, but newer methods are also being developed. In this regard, tissue and cell culture, supplements, and 3D scaffolds have opened new horizons in the differentiation of stem cells in vitro, which could improve the outcomes of male infertility. Various 3D methods have been developed to produce cellular aggregates and mimic the organization and function of the testis. The production of an artificial reproductive organ that supports SSCs differentiation will certainly be a main step in male infertility treatment.

3D-printed placental-derived bioinks for skin tissue regeneration with improved angiogenesis and wound healing properties.

Extracellular matrix (ECM)-based bioinks has attracted much attention in recent years for 3D printing of native-like tissue constructs. Due to organ unavailability, human placental ECM can be an alternative source for the construction of 3D print composite scaffolds for the treatment of deep wounds. In this study, we use different concentrations (1.5%, 3% and 5%w/v) of ECM derived from the placenta, sodium-alginate and gelatin to prepare a printable bioink biomimicking natural skin. The printed hydrogels' morphology, physical structure, mechanical behavior, biocompatibility, and angiogenic property are investigated. The optimized ECM (5%w/v) 3D printed scaffold is applied on full-thickness wounds created in a mouse model. Due to their unique native-like structure, the ECM-based scaffolds provide a non-cytotoxic microenvironment for cell adhesion, infiltration, angiogenesis, and proliferation. In contrast, they do not show any sign of immune response to the host. Notably, the biodegradation, swelling rate, mechanical property, cell adhesion and angiogenesis properties increase with the increase of ECM concentrations in the construct. The ECM 3D printed scaffold implanted into deep wounds increases granulation tissue formation, angiogenesis, and re-epithelialization due to the presence of ECM components in the construct, when compared with printed scaffold with no ECM and no treatment wound. Overall, our findings demonstrate that the 5% ECM 3D scaffold supports the best deep wound regeneration in vivo, produces a skin replacement with a cellular structure comparable to native skin.

Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds.

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.

The protective effects of astaxanthin on pre-antral follicle degeneration in ovine vitrified/warmed ovarian tissue.

This study assesses the protective effects of astaxanthin (AST) against vitrification/warming-induced cryoinjuries of ovarian tissue slices in sheep. Cortical slices of slaughterhouse acquired-ovine ovaries were randomly distributed in different groups: fresh, toxicity, and five vitrification groups including vitrification in presence of 0 (control group), 1, 10 and 100 µM astaxanthin or 100 µM vitamin E. After vitrification/warming and 24 h culturing, the samples were subjected to histological studies, antioxidant evaluation by TAC and TBAR assays, and assessment of relative expression of BMP4, BMP15, GDF9 and KITLG genes related to folliculogenesis and follicular growth regulation. According to the results, vitrification reduced the percentage of morphologically intact follicles compared to the fresh and toxicity groups (p < 0.05). In vitrification groups, vitamin E and all three concentrations of AST increased the percentage of intact pre-antral follicles and antioxidant activity relative to the vitrified control (p < 0.05). This enhancement significantly occurred in 10 µM AST group more than vitamin E (p < 0.05). Also, 10 µM concentration of AST enhanced the expression of all the examined genes compared to the control (p < 0.05), while the expression of BMP4, BMP15 and KITLG was higher in the AST than vitamin E (p < 0.05). The latter could increase only the expression of GDF9 compared to the control group (p = 0.011). In conclusion, AST is a highly effective antioxidant for maintaining the survival of pre-antral follicles, retaining cell density, increasing total antioxidant capacity, and increasing the expression of some genes related to follicular development after short-term culture of vitrified/warmed ovarian tissue slices.

Protective effects of human amniotic membrane derived mesenchymal stem cells (hAMSCs) secreted factors on mouse spermatogenesis and sperm chromatin condensation following unilateral testicular torsion.

Testicular torsion is considered a urological disorder that requires immediate detorsion surgery. Ischemia/reperfusion (I/R) injury after testicular torsion detorsion causes of drastic impairment of spermatogenesis and infertility. Cell-free-based approaches seem to be a promising strategy to prevent I/R injury, they have more stable biological properties, and they contain paracrine factors of mesenchymal stem cells. The purpose of this study was to evaluate the protective effects of human amniotic membrane derived mesenchymal stem cells (hAMSCs) secreted factors on mouse sperm chromatin condensation and spermatogenesis improvement after I/R injury. hAMSCs were isolated and characterized by RT- PCR and flow cytometry, preparation of hAMSCs secreted factors was performed. Forty male mice were randomly divided into 4 groups: sham-operated, torsion detorsion, torsion detorsion+ intratesticular injection of DMEM/F-12, and torsion detorsion+ intratesticular injection of hAMSCs secreted factors. After one cycle of spermatogenesis, the mean number of germ cells, Sertoli, Leydig, myoid as well as tubular parameters, Johnson score, and spermatogenesis indexes were evaluated by H& E and PAS stainings. Sperm chromatin condensation and relative expression of c-kit and prm 1 genes were assessed by aniline blue staining and real-time PCR, respectively. The mean number of spermatogenic cells, Leydig, myoid, Sertoli, spermatogenesis parameters, Johnson score, as well as germinal epithelial height and diameters of seminiferous tubules decreased significantly after I/R injury. The thickness of basement membrane and percentage of sperm with excessive histone significantly increased, while the relative expression of c-kit and prm 1 significantly decreased in torsion detorsion group (p 0.001). hAMSCs secreted factors remarkably restored normal sperm chromatin condensation, spermatogenesis parameters and histomorphometric organization of seminiferous tubules via intratesticular injection (p 0.001). Thus, hAMSCs secreted factors may potentially salvage torsion-detorsion-induced infertility.

In vitro production of mouse morphological sperm in artificial testis bioengineered by 3D printing of extracellular matrix.

Since autologous stem cell transplantation is prone to cancer recurrence, in vitro sperm production is regarded a safer approach to fertility preservation. In this study, the spermatogenesis process on testicular tissue extracellular matrix (T-ECM)-derived printing structure was evaluated. Ram testicular tissue was decellularized using a hypertonic solution containing triton and the extracted ECM was used as a bio-ink to print an artificial testis. Following cell adhesion and viability examination, pre-meiotic and post-meiotic cells in the study groups (as testicular suspension and co-culture with Sertoli cells) were confirmed by real-time PCR, flow-cytometry and immunocytochemistry methods. Morphology of differentiated cells was evaluated using transmission electron microscopy (TEM), toluidine blue, Giemsa, and hematoxylin and eosin (H&E) staining. The functionality of Leydig and Sertoli cells was determined by their ability for hormone secretion. The decellularization of testicular tissue fragments was successful and had efficiently removed the cellular debris and preserved the ECM compounds. High cell viability, colonization, and increased expression of pre-meiotic markers in cultured testicular cells on T-ECM-enriched scaffolds confirmed their proliferation. Furthermore, the inoculation of neonatal mouse testicular cells onto T-ECM-enriched scaffolds resulted in the generation of sperm. Morphology evaluation showed that the structure of these cells was quite similar to mature sperm with a specialized tail structure. The hormonal analysis also confirmed production and secretion of testosterone and inhibin B by Leydig and Sertoli cells. T-ECM printed artificial testis is a future milestone that promises for enhancing germ cell maintenance and differentiation, toxicology studies, and fertility restoration to pave the way for new human infertility treatments in the future.

The role of myo-inositol supplement in assisted reproductive techniques.

Assisted reproductive techniques can help many infertile couples conceive. Therefore, there is a need for an effective method to overcome the widespread problems of infertile men and women. Oocyte and sperm quality can increase the chances of successful in vitro fertilisation. The maturation environment in which gametes are present can affect their competency for fertilisation. It is well established that myo-inositol (MI) plays a pivotal role in reproductive physiology. It participates in cell membrane formation, lipid synthesis, cell proliferation, cardiac regulation, metabolic alterations, and fertility. This molecule also acts as a direct messenger of insulin and improves glucose uptake in various reproductive tissues. Evidence suggests that MI regulates events such as gamete maturation, fertilisation, and embryo growth through intracellular Ca2 + release and various signalling pathways. In addition to the in-vivo production of MI from glucose in the reproductive organs, its synthesis by in vitro-cultured sperm and follicles has also been reported. Therefore, MI is suggested as a therapeutic approach to maintain sperm and oocyte health in men and women with reproductive disorders and individuals of reproductive age.

Proliferation of human spermatogonial stem cells on optimized PCL/Gelatin nanofibrous scaffolds.

Improvement of culture system and increasing the proliferation of spermatogonia stem cells under in vitro condition are the essential treatment options for infertility before autologous transplantation. Therefore, the present study aimed to evaluate the proliferation of human spermatogonia stem cells on the electrospun polycaprolactone/gelatin nanocomposite. Therefore, for this purpose, nanofiber porous scaffolds were prepared using the electrospinning method and their structures were then confirmed by SEM. After performing swelling, biodegradability and cell adhesion tests, human spermatogonia stem cells were cultured on scaffolds. In addition, both cell viability and proliferation were assessed using immunocytochemistry, flow cytometry and real-time PCR techniques in culturing during a 3-week period. SEM images indicated the presence of fibres with suitable diameters and arrangement as well as a sufficient porosity in nanocomposite scaffolds, showing good biocompatibility and biodegradability. The results show a significant increase in the number of spermatogonia stem cells in the cultured group on scaffold compared with the control group (p ≤ 0.05). As well, the results show that the expressions of integrin ɑ6 and ß1 and Plzf genes estimated using real-time PCR in nanofiber scaffolds were significantly higher than those of the control group (p ≤ 0.05). However, the expression of c-Kit gene in the 3D group showed a significant decrease compared with the 2D group. Flow cytometry analysis also showed that the number of Plzf-positive cells was significantly higher in nanofiber porous scaffolds compared with the control group (p ≤ 0.05). Additionally, immunocytochemistry findings confirmed the presence of human spermatogonia stem cell colonies. In general, it seems that the designed nanocomposite scaffold could provide a suitable capacity for self-renewal of human spermatogonia stem cells, which can have a good application potential in research and reconstructive medicine related to the field of male infertility.

The therapeutic potential of adipose tissue-derived mesenchymal stromal cells in the treatment of busulfan-induced azoospermic mice.

PURPOSE: The generation of germ cells from mesenchymal stromal cells (MSCs) provides a valuable in vitro platform for infertility modeling. The establishment of these cells is a new approach for assisted reproductive technology (ART) to help infertile patients who lack functional gametes. METHODS: Human adipose-derived MSCs were isolated and then characterized for multipotency by flow cytometry, differentiation capacity, and cytogenetic assays. These cells were used in a male germ cell differentiation study. The expression of male germ cell markers was evaluated at day 21 of differentiation using an immunofluorescence assay, flow cytometry, and RT-qPCR. Undifferentiated MSCs were used for transplantation in busulfan-induced azoospermic mice. RESULTS: In this study, MSCs were successfully isolated from human adipose tissues which were positive for cell markers such as CD90, CD105, CD73, and CD29 but negative for CD34 and CD45. The results of flow cytometry, immunocytochemistry, and RT-qPCR analysis at day 21 of differentiation showed that the undifferentiated adipose-derived MSCs are able to differentiate into male germ cells. Additionally, transplantation of undifferentiated MSCs in busulfan-induced azoospermic mice caused spermatogenesis recovery in the majority of seminiferous tubules. CONCLUSION: In this study, we showed that differentiation of human adipose-derived MSCs into male germ cells is a useful tool for in vitro study of human germ cell development. Our results demonstrated that cell therapy with adipose-derived MSCs could help the repair of pathological changes in testicular seminiferous tubules. Therefore, it may have a clinical application for the treatment of azoospermia in infertile patients.

Artificial testis: a testicular tissue extracellular matrix as a potential bio-ink for 3D printing.

Testicular scaffolds may be an option for fertility preservation. The aim was to develop various procedures for the decellularization of testicular tissue and to design a bio-ink to construct a bioartificial testis. Ram testicular tissue fragments were decellularized using NaCl buffer, NaCl buffer-Triton, SDS and SDS-Triton. The removal of the cells from the tissues was confirmed by DAPI and H & E staining, as well as the evaluation of the DNA content. Alcian blue, Orcein and Masson's trichrome staining methods were also used to confirm that T-ECM was preserved intact. Then, the optimal decellularization protocol was selected to determine the parameters of the bio-ink and printing of the scaffold. The extracted T-ECM was used to print the hydrogel scaffold in combination with alginate-gelatin. The printability, morphological, mechanical and biological properties of the printed hydrogels were characterized. Decellularization of testicular tissue fragments using the NaCl buffer-Triton protocol was significantly more efficient than other decellularization methods in removing the cellular debris and preserving the T-ECM compounds. The 3D printed scaffold with 5% T-ECM showed a uniform surface morphology with high cell attachment and cyto-biocompatibility properties for spermatogonia stem cells in vitro and in vivo compared to other groups. It is concluded that T-ECM can be used as a biomimetic material to make an artificial testis with possible in vitro sperm production.

Male Factors: the Role of Sperm in Preimplantation Embryo Quality.

Few studies have been conducted on the paternal effects on embryogenesis as compared with studies on maternal effects. The fertility potential of sperm decreases with genomic material abnormalities. Damaged DNA of sperm has been correlated with poor fertilization, reduced implantation and pregnancy rates, and increased production of aneuploid embryos. Evidence suggests that the role of sperm in embryogenesis goes beyond genomic material transfer, and centrosomes, sperm-derived cytoplasmic factors, paternal mRNA, and small RNAs are essential for early embryonic development. Epigenetic factors like histone modification and DNA methylation participate in the regulation of gene expression in sperm. The etiology of sperm chromatin abnormalities is important in male fertility and may affect reproductive outcomes. Success in implantation depends on the quality of the fertilized sperm and oocyte as well as the type of assisted reproductive techniques. Therefore, male factors affecting development of embryo can play a role in the failure of assisted reproductive techniques. Further studies are needed to evaluate clinical aspects and the risks of transmitting genetic or epigenetic disorders to provide safe therapies for infertility.