Background:Endometriosis is a benign gynaecological disorder characterised by the presence of endometrial-like tissue outside the uterus. Proliferation of endometrial tissue and neoangiogenesis are essential factors in the development of endometriosis. Vascular endothelial growth factor (VEGF) and insulin-like growth factors (IGF1, IGF2) can be effective in neoangiogenesis and cell proliferation. Imprinted long non-coding RNA (lncRNA) H19 is involved in endometriosis pathogenesis through the regulation of cellular proliferation and differentiation. Curcumin has antiangiogenic, anti-proliferative, and anti-invasive properties for various diseases, and it is hypothesised that it may have therapeutic effects on endometriosis. This study aimed to evaluate the effects of curcumin on the expression of VEGF, IGF1, IGF2, and H19 lncRNA, as well as on cell migration and proliferation in endometrial stromal cells isolated from women with endometriosis. Materials and Methods:In this experimental study, human normal endometrial stromal cells (N-ESCs) from healthy endometrial tissue and human eutopic endometrial stromal cells (EU-ESCs) from endometriosis patients were cultured and treated with different concentrations of curcumin (0-100 mM) for 72 hours. Then, the effects of curcumin on cell viability, proliferation, and migration were assessed using methylthiazole tetrazolium (MTT) and the scratch test, respectively. Expressions of genes involved in angiogenesis (VEGF), proliferation (IGF1, IGF2), and the H19 lncRNA gene were evaluated using real-time polymerase chain reaction (PCR). Results:Curcumin decreased cell migration and proliferation of endometriotic stromal cells in a time- and concentration- dependent manner. It also reduced VEGF expression and increased the expressions of H19, IGF1, and IGF2. Conclusion:The anti-migration effect of curcumin on EU-ESCs and its impact on the expression of proliferative and angiogenic genes indicate that curcumin can be a promising treatment for endometriosis.

AIMS:Some studies have shown that mesenchymal stem cells (MSCs) and their derived extracellular vesicles (MSC-EVs) can restore ovarian function in premature ovarian failure (POF), however, concerns about their efficacy are attributed to the heterogeneity of the cell populations and EVs. Here, we assessed the therapeutic potential of a homogeneous population of clonal MSCs (cMSCs) and their EVs subpopulations in a mouse model of POF. MAIN METHODS:Granulosa cells were treated with cyclophosphamide (Cy) in the absence or presence of cMSCs, or cMSCs-derived EV subpopulations (EV20K and EV110K, isolated by high-speed centrifugation and differential ultracentrifugation, respectively). In addition, POF mice were treated with cMSCs, EV20K and/or EV110K. KEY FINDINGS:cMSC and both EV types protected granulosa cells from Cy-induced damage. Calcein-EVs were detected in the ovaries. Moreover, cMSC and both EV subpopulations significantly increased body weight, ovary weight, and the number of follicles, restored FSH, E2, and AMH levels, increased the granulosa cell numbers and restored the fertility of POF mice. cMSC, EV20K, and EV110K alleviated inflammatory-related genes expression (Tnf-α and IL8), and improved angiogenesis via upregulation expression of Vegf and Igf1 at the mRNA level and VEGF and αSMA at the protein level. They also inhibited apoptosis through the PI3K/AKT signaling pathway. SIGNIFICANCE:The administration of cMSCs and two cMSC-EVs subpopulations improved ovarian function and restored fertility in a POF model. EV20K is more cost-effective and feasible in terms of isolation, particularly in good manufacturing practice (GMP) facilities for treatment of POF patients in comparison with conventional EVs (EV110K).

Objective (s) One way to overcome the recurrence of cancer cells following ovarian tissue transplantation is to use decellularized tissues as a scaffold that does not have any cellular components. These cell-free scaffolds can be seeded with different type of stem cells for ovarian restoration. Materials and methods OSCs, PMSCs and BMSCs (oogonial, peritoneal and bone marrow mesenchymal stem cells, respectively) were seeded into human decellularized ovarian tissue as 4 groups: Scaffold + OSCs (SO), Scaffold + OSC + PMSCs (SOP), Scaffold + OSC + BMSCs (SOB) and Scaffold + OSC + PMSCs + BMSCs (SOPB). The produced grafts were transplanted into the sub-peritoneal space of ovariectomized NMRI mice as artificial ovary (AO). The expression of Vegf, CD34, Gdf9, Zp3, Ddx4, Amh and Lhr genes in AOs were measured by qRT-PCR. Also, histotechniques were considered to detect the anti GFP, PCNA, VEGF, GDF9, ZP3 and AMH proteins. Results H & E staining showed follicle-like structures in all groups; the number of these structures, in the SOP and SOB groups, were the highest. In SO group, differentiation ability to oocyte and granulosa cells was observed. Endothelial, oocyte, germ, and granulosa cell-like cells were specially seen in SOP and angiogenesis capability was more in SOB group. However, angiogenesis ability and differentiation to theca cell-like cells were more often in SOPB group. While none of the groups showed a significant difference in AMH level, estradiol levels were significantly higher in SOPB group. Conclusion Integration of OSCs + PMSCs and those OSCs + BMSCs were more conducive to oogenesis.

Microfluidic systems have been extensively studied in recent years as potential alternatives for problematic conventional methods of sperm selection. However, despite the widespread use of simple straight channels in these systems, the impact of channel geometry on selected sperm quality has not been thoroughly investigated. To explore this further, we designed and fabricated serpentine microchannels with different radii of curvature, inspired by the tortuous structure of the cervix. Our results showed that in the presence of gentle backflow, microfluidic channels with a 150 μm radius of curvature significantly enhanced the quality of selected sperms when compared to straight channels. Specifically, we observed significant improvements of 7% and 9% in total motility and progressive motility, respectively, as well as 13%, 18%, and 19% improvements in VCL, VAP, and VSL, respectively. Through careful observation of the process, we discovered a unique near-wall sperm migration pattern named boundary detachment-reattachment (BDR), that was observed exclusively in curved microchannels. This pattern, which is a direct consequence of the special serpentine geometry and sperm boundary-following characteristic, contributed to the superior selection performance when combined with a fluid backflow. After determining the best channel design, we fabricated a parallelized chip consisting of 85 microchannels capable of processing 0.5 ml of raw semen within 20 minutes. This chip outperformed conventional methods of swim-up and density gradient centrifugation (DGC) in terms of motility (9% and 25% improvements, respectively), reactive oxygen species (18% and 15% improvements, respectively), and DNA fragmentation index (14% improvement to DGC). Outstanding performance and advantages such as user-friendliness, rapid selection, and independence from centrifugation make our microfluidic system a prospective sperm selection tool in clinical applications.

ETHNOPHARMACOLOGICAL RELEVANCE:Ethnopharmacological studies for drug discovery from natural compounds play an important role for developing current therapeutical platforms. Plants are a group of natural sources which have been served as the basis in the treatment of many diseases for centuries. In this regard, Ceratonia siliqua (carob) is one of the herbal medicine which is traditionally used for male infertility treatments. But so far the main mechanisms for effects of carob are unknown. Here, we intend to investigate the ability of carob extract to induce spermatogenesis in an azoospermia mouse model and determine the mechanisms that underlie its function.AIM OF THE STUDY:This is a pre-clinical animal model study to evaluate the effect of carob extract in spermatogenesis recovery.METHODS:We established an infertile mouse model with the intent to examine the ability of carob extract as a potential herbal medicine for restoration of male fertility. Sperm parameters, as well as gene expression dynamics and levels of spermatogenesis hormones, were evaluated 35 days after carob administration.RESULTS:Significant enhanced sperm parameters (P < 0.05) showed that the carob extract could induce spermatogenesis in the infertile mouse model. Our data suggested an anti-apototic and inducer role in the expressions of cell cycle regulating genes. Carob extract improved the spermatogenesis niche by considerable affecting Sertoli and Leydig cells (P < 0.05). The carob-treated mice were fertile and contributed to healthy offspring that matured. Our data confirmed that this extract triggered the hormonal system, the spermatogenesis-related gene expression network, and signaling pathways to induce and promote sperm production with notable level (P < 0.05). We found that the aqueous extract consisted of a polar and mainly well water-soluble substance. Carob extract might upregulate spermatogenesis hormones via its amino acid components, which were detected in the extract by liquid chromatography-mass spectrometry (LC-MS).CONCLUSION:Our results strongly suggest that carob extract might be a promising future treatment option for male infertility. This finding could pave the way for clinical trials in infertile men. This is the first study that has provided reliable, strong pre-clinical evidence for carob extract as an effective candidate for fertility recovery in cancer-related azoospermia.

Increased concentrations of the fibronectin glycoprotein can cause ectopic tissue growth patients with endometriosis and the formation of various cancerous tumors. Furthermore, fibronectin binding to its receptors from the EDA (Extra Domain A) region contributes to promote tumorigenesis, metastasis and vasculogenesis. Thus, the EDA region can be considered a unique target for therapeutic intervention. Therefore, the present study used computational methods to identify the best fibronectin inhibitor(s) among FDA-approved drugs. First, docking-based virtual screening was performed using PyRx 0.8. Next, FDA-approved drugs that obtained favorable results in the docking phase were selected for further studies and analysis using molecular dynamics (MD) simulation. The preliminary findings of the virtual screening showed that 17 FDA-approved drugs (from 2471) had more favorable energy with their binding energy less than -9 kcal/mol. The MD simulation results of these 17 drugs showed that Avapritinib had a lower RMSD value and higher binding energy and hydrogen bonding than the other complexes in the EDA domain. Also, analyses related to the second structure changes displayed that Avapritinib in the EDA domain led to more changes in the second structure. According to the results, the anticancer drug Avapritinib forms a more stable complex with fibronectin than other FDA-approved drugs. Furthermore, this drug leads to more changes in the second EDA structure, which may have more serious potential for inhibiting EDA fibronectin.Communicated by Ramaswamy H. Sarma.

Background: The accurate identification and isolation of ovarian stem cells from mammalian ovaries remain a major challenge because of the lack of specific surface markers and suitable in vitro culture systems. Optimized culture conditions for in vitro expansion of ovarian stem cells would allow for identifying requirements of these stem cells for proliferation and differentiation that would pave the way to uncover role of ovarian stem cells in ovarian pathophysiology. Here, we used three-dimensional (3D) aggregate culture system for enrichment of ovarian stem cells and named them aggregate-derived stem cells (ASCs). We hypothesized that mimicking the ovarian microenvironment in vitro by using an aggregate model of the ovary would provide a suitable niche for the isolation of ovarian stem cells from adult mouse and human ovaries and wanted to find out the main cellular pathway governing the proliferation of these stem cells.Results: We showed that ovarian aggregates take an example from ovary microenvironment in terms of expression of ovarian markers, hormone secretion and supporting the viability of the cells. We found that aggregates-derived stem cells proliferate in vitro as long-term while remained expression of germline markers. These ovarian stem cells differentiated to oocyte like cells in vitro spontaneously. Transplantation of these stem cells in to chemotherapy mouse ovary could restore ovarian structure. RNA-sequencing analysis revealed that interleukin6 is upregulated pathway in ovarian aggregate-derived stem cells. Our data showed that JAK/Stat3 signaling pathway which is activated downstream of IL6 is critical for ovarian stem cells proliferation.Conclusions: We developed a platform that is highly reproducible for in vitro propagation of ovarian stem cells. Our study provides a primary insight into cellular pathway governing the proliferation of ovarian stem cells.

Abstract Background Asherman syndrome (AS), or intrauterine adhesions, is a main cause of infertility in reproductive age women after endometrial injury. Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) are promising candidates for therapies that repair damaged endometria. However, concerns about their efficacy are attributed to heterogeneity of the cell populations and EVs. A homogenous population of MSCs and effective EV subpopulation are needed to develop potentially promising therapeutic options in regenerative medicine. Methods AS model was induced by mechanical injury in adult rat uteri. Then, the animals were treated immediately with homogeneous population of human bone marrow-derived clonal MSCs (cMSCs), heterogenous parental MSCs (hMSCs), or cMSCs-derived EV subpopulations (EV20K and EV110K). The animals were sacrificed two weeks post-treatment and uterine horns were collected. The sections were taken, and hematoxylin–eosin was used to examine the repair of endometrial structure. Fibrosis was measured by Masson’s trichrome staining and α-SMA and cell proliferation by Ki67 immunostaining. The function of the uteri was explored by the result of mating trial test. Expression changes of TNFα, IL-10, VEGF, and LIF were assayed by ELISA. Results Histological analysis indicated fewer glands, thinner endometria, increased fibrotic areas, and decreased proliferation of epithelial and stroma of the uteri in the treated compared with intact and sham-operated animals. However, these parameters improved after transplantation of both types of cMSCs and hMSCs and/or both cryopreserved EVs subpopulations. The cMSCs demonstrated more successful implantation of the embryos in comparison with hMSCs. The tracing of the transplanted cMSCs and EVs showed that they migrated and localized in the uteri. Protein expression analysis results demonstrated downregulation of proinflammatory factor TNFα and upregulation of anti-inflammatory cytokine IL-10, and endometrial receptivity cytokines VEGF and LIF in cMSC- and EV20K-treated animals. Conclusion Transplantation of MSCs and EVs contributed to endometrial repair and restoration of reproductive function, likely by inhibition of excessive fibrosis and inflammation, enhancement of endometrial cell proliferation, and regulation of molecular markers related to endometrial receptivity. Compared to classical hMSCs, cMSCs were more efficient than hMSCs in restoration of reproductive function. Moreover, EV20K is more cost-effective and feasible for prevention of AS in comparison with conventional EVs (EV110K).

Spermatogonial stem cell (SSC) counterparts known as female germline stem cells (fGSCs) were found in the mammalian ovary in 2004. Although the existence of fGSCs in the mammalian postnatal ovary is still under controversy, fGSC discovery encourages investigators to better understand the various aspects of these cells. However, their existence is not accepted by all scientists in the field because isolation of fGSCs by fluorescent activated cell sorting (FACS) has not been reproducible. In this study, we used differential adhesion to isolate and enrich fGSCs from mouse and human ovaries and subsequently cultured them in vitro. fGSCs were able to proliferate in vitro and expressed germ cell-specific markers Vasa, Dazl, Blimp1, Fragilis, Stella, and Oct4, at the protein level. Moreover, mouse and human fGSCs were, respectively, cultured for more than four months and one month in culture. Both mouse and human fGSCs maintained the expression of germ cell-specific markers over these times. In vitro cultured fGSCs spontaneously produced oocyte-like cells (OLCs) which expressed oocyte-relevant markers. Our results demonstrated that differential adhesion allows reproducible isolation of fGSCs that are able to proliferate in vitro over time. This source of fGSCs can serve as a suitable material for studying mechanisms underlying female germ cell development and function.

Germ cell production from stem cells allows for studying the mechanisms involved in gamete development with the aim of helping infertile couples with the generation of healthy gametes. In this context, improving the protocols for in-vitro germ cell induction from stem cells is very important. Recently, SB4 small molecule has been introduced as a potent agonist for bone morphogenic protein 4 (BMP4). Herein, we investigated whether BMP4, is replaceable by SB4 for having affordable protocol for in vitro germ cell differentiation. We demonstrated that SB4 can induce Blimp1 (as the first gene induced germ line differentiation) expression significantly but at a lower level compared to BMP4. However, Tfap2c (a putative downstream target of Blimp1 during germ cell differentiation) expression level in SB4-induced aggregates was significantly higher than in BMP4-induced aggregates. Moreover, co-presence of both BMP4 and SB4 could increase the expression level of Prdm14, Nnose3 and Stella (Dppa3), and thereby improve establishment of the germ cell fate during in-vitro differentiation of embryonic stem cells. In summary, our data suggest that SB4 could improve germ line gene expression pattern induced by BMP4 during embryonic stem cells in-vitro differentiation.

The human endometrium is a dynamic tissue that undergoes cyclic changes in response to sex steroid hormones to provide a receptive status for embryo implantation. Disruptions in this behavior may lead to implantation failure and infertility; therefore, it is essential to develop an appropriate in vitro model to study endometrial changes in response to sex hormones. In this regard, the first choice would be human endometrial cells isolated from biopsies that could be used as monolayer cell sheets or to generate endometrial organoids. However, the need for fresh samples and short-time viability of harvested endometrial biopsy limits these approaches. In order to overcome these limitations, we sought to develop an efficient, simple, robust and reproducible method to cryopreserve human endometrial biopsies that could be stored and/or shipped frozen and later thawed to generate endometrial organoids and endometrial stromal cells (EnSCs). These cryopreserved biopsies could be thawed and used to generate simple endometrial organoids or organoids for co-culture with matched stromal cells that are functionally responsive to sex hormones as similar as the organoids generated from fresh biopsy. An optimal endometrial tissue cryopreservation method would allow the possibility for endometrial tissue biobanking to enable future organoid generation from both healthy tissues and pathological conditions, and open new venues for generate endometrial assembloids, consisting of epithelial organoids and primary stromal cells.

Endometriosis is a sex hormone-dependent, painful disease that affects 10-15% of women worldwide with no definitive cure, and current treatments are not always effective. This limitation is mainly due to gaps in our knowledge about the mechanisms involved in the pathogenesis of endometriosis at the cellular and molecular levels. Hormonal dysregulation appears to be responsible for inflammation, angiogenesis, endometrial non-receptivity, embryo implantation failure and infertility in women with endometriosis. Although correlative evidence about possible causes of hormonal dysregulations exists, the functional mechanisms remain unknown. Reliable research models of endometriosis are needed to investigate the exact mechanisms that underlie hormone disruptions. This Commentary discusses the available in-vivo and in-vitro systems for studying endometriosis. The authors emphasize the recently developed human endometriosis organoids as cutting-edge and innovative research models for endometriosis investigations, discuss their advantages and describe challenges that must be addressed to yield a reliable in-vitro model of human endometriosis. Moreover, it discusses microfluidic technology to address the present challenges for producing advanced endometriosis organoids and how to benefit from CRISPR technology to improve our knowledge about disturbed hormonal function in patients with endometriosis.

Because spermatogonia transmit genetic information across generations, their DNA must be protected from environmental damages, including exposure to zinc oxide nanoparticles (ZnO NPs), which are frequently used in modern technology. Here, we used an in vitro system enriched for spermatogonia and exposed them to 10 and 20 μg/ml ZnO NPs for one/seven days. We did not detect any significant cell death, chromosomal instability, or DNA fragmentation in the spermatogonia treated with the ZnO NPs following one-day treatment with 10 or 20 μg/ml ZnO NPs. However, ZnO NPs (both 10 and 20 μg/ml) induced chromosomal instability in the spermatogonia after seven days of treatment. Moreover, one-day exposure to these NPs induced reactive oxygen species (ROS) generation and upregulation of apoptotic pathway-related genes p53, Caspase3 and Il6, as an inflammatory factor. Taken together, our study provides preliminary evidence for possible damages induced by low concentrations of ZnO NPs in spermatogonia. We should pay increased attention when using these NPs because of the silent damages in spermatogonia that can be transmitted to the next generation and cause severe effects. However, more data and validation of these results are required to determine the extent of this concern.

RESEARCH QUESTION:Do human endometriosis organoids recapitulate aberrant progesterone signalling in the disease to serve as advanced experimental models for uncovering epigenetic mechanisms involved in attenuated progesterone response in endometriosis? DESIGN:Initially, the organoids were established from acquired biopsies (women with and without endometriosis) and characterized by morphological, histological and immunostaining analyses. RESULTS:A panel of endometriosis-related genes showed a pattern of expressions in cytochrome c oxidase subunit II (COX2), matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor of metalloproteinase-3 (TIMP3), transforming growth factor beta 1 (TGF-β1), and zinc finger E-box binding homeobox 1 (ZEB1), and a contradictory expression pattern for cadherin (CDH1), POU class 5 homeobox 1 (POU5F1; also known as OCT4), and Nanog homeobox (NANOG) in the endometriosis organoids that is concordant with published research. These endometriosis organoids failed to upregulate 17β-Hydroxysteroid dehydrogenase 2 (17HSDβ2), progestogen associated endometrial protein (PAEP), secreted phosphoprotein 1 (SPP1), and leukaemia inhibitory factor (LIF) in response to progesterone at the level observed in control endometrium organoids. Progesterone receptor B (PRB) gene expression significantly decreased in both eutopic and ectopic organoids compared with control endometrium organoids. DNA hypermethylation, as an epigenetic mechanism for suppression of transcription, was detected at the PRB promoter in the eutopic, but not ectopic, organoids. Therefore, other epigenetic mechanisms, such as histone modifications and microRNAs, may be responsible for PRB downregulation in ectopic organoids. CONCLUSIONS:Endometriosis organoids are powerful preclinical models that can be used to investigate the molecular mechanisms involved in endometriosis-associated progesterone resistance.

Endometriosis is a benign gynecological disease that is manifested by the presence and growth of endometrial cells and glands outside the uterine. Active angiogenesis, migration, and invasion of endometrial tissue outside the uterine are critical for the development of endometriosis and lead to the survival and growth of endometriotic lesions. Metformin, as an anti-diabetic agent, represents anti-angiogenic property. Here, we performed a study using human normal endometrial stromal cells (N-ESCs) from healthy endometrial tissue and human eutopic endometrial stromal cells (EU-ESCs) and ectopic endometrial stromal cells (ECT-ESCs) from endometriosis patients. ESCs were cultured and treated with different concentrations of Metformin (0-20 mmol/l) for 72 h to evaluate Metformin effect on cell viability, proliferation, migration was measured by methyl thiazolyl tetrazolium (MTT) assay and scratch test respectively as well as expression of angiogenesis and migration markers. The Metformin reduced cell migration, and proliferation of endometriotic stromal cells in a time and concentration dependently manner. Furthermore, Metformin attenuated the expression of angiogenic and inflammatory genes in human endometriotic stromal cells. The direct anti-proliferative effect on ECT-ESCs combined with the effects of Metformin on inflammatory and angiogenesis-related genes expression supports its therapeutic potential for endometriosis. Metformin could be used as an effective adjuvant in endometriosis treatment.

Ethnopharmacological relevance: Ethnopharmacological studies for drug discovery from natural compounds play an important role for developing current therapeutical platforms. Plants are a group of natural sources which have been served as the basis in the treatment of many diseases for centuries. In this regard, Ceratonia siliqua (carob) is one of the herbal medicine which is traditionally used for male infertility treatments. But so far the main mechanisms for effects of carob are unknown. Here, we intend to investigate the ability of carob extract to induce spermatogenesis in an azoospermia mouse model and determine the mechanisms that underlie its function.Aim of the study: This is a pre-clinical animal model study to evaluate the effect of carob extract in spermatogenesis recovery.Methods: We established an infertile mouse model with the intent to examine the ability of carob extract as a potential herbal medicine for restoration of male fertility. Sperm parameters, as well as gene expression dynamics and levels of spermatogenesis hormones, were evaluated 35 days after carob administration.Results: Enhanced sperm parameters showed that the carob extract could induce spermatogenesis in the infertile mouse model. Our data suggested an anti-apototic and inducer role in the expressions of cell cycle regulating genes. Carob extract improved the spermatogenesis niche by affecting Sertoli and Leydig cells. The carob-treated mice were fertile and contributed to healthy offspring that matured. Our data confirmed that this extract triggered the hormonal system, the spermatogenesis-related gene expression network, and signaling pathways to induce and promote sperm production. We found that the aqueous extract consisted of a polar and mainly well water-soluble substance. Carob extract might upregulate spermatogenesis hormones via its amino acid components, which were detected in the extract by liquid chromatography-mass spectrometry (LC-MS).Conclusion: Our results strongly suggest that carob extract might be a promising future treatment option for male infertility. This finding could pave the way for clinical trials in infertile men. This is the first study that has provided reliable, strong pre-clinical evidence for carob extract as an effective candidate for fertility recovery in cancer-related azoospermia.

Demystifying the mechanisms that underlie germline development and gamete production is critical for expanding advanced therapies for infertile couples who cannot benefit from current infertility treatments. However, the low number of germ cells, particularly in the early stages of development, represents a serious challenge in obtaining sufficient materials required for research purposes. In this regard, pluripotent stem cells (PSCs) have provided an opportunity for producing an unlimited source of germ cells in vitro. Achieving this ambition is highly dependent on accurate stem cell niche reconstitution which is achievable through applying advanced cell engineering approaches. Recently, hydrogel microparticles (HMPs), as either microcarriers or microcapsules, have shown promising potential in providing an excellent 3-dimensional (3D) biomimetic microenvironment alongside the systematic bioactive agent delivery. In this review, recent studies of utilizing various HMP-based cell engineering strategies for appropriate niche reconstitution and efficient in vitro differentiation are highlighted with a special focus on the capabilities of droplet-based microfluidic (DBM) technology. We believe that a deep understanding of the current limitations and potentials of the DBM systems in integration with stem cell biology provides a bright future for germ cell research.

Endometriosis is major gynecological disease that affects over 10% of women worldwide and 30%-50% of these women have pelvic pain, abnormal uterine bleeding and infertility. The cause of endometriosis is unknown and there is no definite cure mainly because of our limited knowledge about its pathophysiology at the cellular and molecular levels. Therefore, demystifying the molecular mechanisms that underlie endometriosis is essential to develop advanced therapies for this disease. In this regard, HOX genes are remarkable because of their critical role in endometrial development and receptivity during implantation, which is attributed to their ability to mediate some of the sex steroid functions during the reproductive period. Access to the expression profiles of these genes would provide the necessary information to uncover new genes for endometriosis and assist with disease diagnosis and treatment. In this study we demonstrate an altered expression pattern for the HOX clusters (A-D) and their cofactors in both eutopic and ectopic conditions compared to control tissue biopsies. Remarkably, most of the intensive changes occurred in eutopic samples from endometriosis patients compared to control tissue biopsies. Pathway analysis revealed the involvement of differentially expressed genes in cancer that correlate with an association between endometriosis and cancer. Our results suggest critical roles for the HOX cluster and their cofactors in endometriosis pathophysiology.

Recent developments in organoid technology are revolutionizing our knowledge about the biology, physiology, and function of various organs. Female reproductive biology and medicine also benefit from this technology. Organoids recapitulate features of different reproductive organs including the uterus, fallopian tubes, and ovaries, as well as trophoblasts. The genetic stability of organoids and long-lasting commitment to their tissue of origin during long-term culture makes them attractive substitutes for animal and in vitro models. Despite current limitations, organoids offer a promising platform to address fundamental questions regarding the reproductive system’s physiology and pathology. They provide a human source to harness stem cells for regenerative medicine, heal damaged epithelia in specific diseases, and study biological processes in healthy and pathological conditions. The combination of male and female reproductive organoids with other technologies, such as microfluidics technology, would enable scientists to create a multi-organoid-on-a-chip platform for the next step to human-on-a-chip platforms for clinical applications, drug discovery, and toxicology studies. The present review discusses recent advances in producing organoid models of reproductive organs and highlights their applications, as well as technical challenges and future directions.

BACKGROUND Premature ovarian failure（POF） affects many adult women less than 40 years of age and leads to infertility. According to previous reports, various tissue-specific stem cells can restore ovarian function and folliculogenesis in mice with chemotherapy-induced POF. Human embryonic stem cells（ES） provide an alternative source for mesenchymal stem cells（MSCs） because of their similarities in phenotype and immunomodulatory and anti-inflammatory characteristics. Embryonic stem cell-derived mesenchymal stem cells（ES-MSCs） are attractive candidates for regenerative medicine because of their high proliferation and lack of barriers for harvesting tissue-specific MSCs. However, possible therapeutic effects and underlying mechanisms of transplanted ES-MSCs on cyclophosphamide and busulfan-induced mouse ovarian damage have not been evaluated.AIM To evaluate ES-MSCs vs bone marrow-derived mesenchymal stem cells（BMMSCs） in restoring ovarian function in a mouse model of chemotherapy-induced premature ovarian failure.METHODS Female mice received intraperitoneal injections of different doses of cyclophosphamide and busulfan to induce POF. Either human ES-MSCs or BMMSCs were transplanted into these mice. Ten days after the mice were injected with cyclophosphamide and busulfan and 4 wk after transplantation of the ESMSCs and/or BM-MSCs, we evaluated body weight, estrous cyclicity, folliclestimulating hormone and estradiol hormone concentrations and follicle count were used to evaluate the POF model and cell transplantation. Moreover, terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling, real-time PCR, Western blot analysis and immunohistochemistry and mating was used to evaluate cell transplantation. Enzyme-linked immunosorbent assay was used to analyze vascular endothelial growth factor, insulin-like growth factor 2 and hepatocyte growth factor levels in ES-MSC condition medium in order to investigate the mechanisms that underlie their function.RESULTS The human ES-MSCs significantly restored hormone secretion, survival rate and reproductive function in POF mice, which was similar to the results obtained with BM-MSCs. Gene expression analysis and the terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling assay results indicated that the ES-MSCs and/or BM-MSCs reduced apoptosis in the follicles. Notably, the transplanted mice generated new offspring. The results of different analyses showed increases in antiapoptotic and trophic proteins and genes.CONCLUSION These results suggested that transplantation of human ES-MSCs were similar to BM-MSCs in that they could restore the structure of the injured ovarian tissue and its function in chemotherapy-induced damaged POF mice and rescue fertility. The possible mechanisms of human ES-MSC were related to promotion of follicular development, ovarian secretion, fertility via a paracrine effect and ovarian cell survival.