Beneficial effects of apigenin on ovarian histological changes and angiogenesis gene expression in rat model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common heterogeneous reproductive disorder and can affect approximately 10% of women of reproductive age. Abnormal vasculogenesis is a common event in polycystic ovary syndrome. This study planned to evaluate the antiangiogenic role of apigenin in ovarian histology, gene expression, and vascular density and stability in an experimental model of PCOS. Twenty-eight rats weighing 180-250 g were divided into 4 groups. Seven rats in the control group remain intact and without treatment. In 21 rats, an ovary polycystic model with a single injection of estradiol valerate was established. The PCOS rats were treated with vehicle, apigenin 10, or apigenin 20 mg/kg in three different PCOS groups for 14 days. At the end, a histological assessment of the ovaries was performed to determine collagen density and follicle counting. The endothelial or periendothelial cells were determined by immunohistochemical assay, and angiogenesis gene expression was determined using molecular assessments. Apigenin treatment partially restored follicular development, decreased the number of cysts, and increased corpora lutea in PCOS rats. Also, apigenin decreased the collagen density in the polycystic ovaries. However, apigenin administration mitigated ovarian angiogenesis by a reduction in endothelial and periendothelial cell numbers. A decrease in VEGF and VEGF R2 (kinase insert domain receptor, KDR) expressions was found after the treatment of rats with apigenin. Conclusively, our data revealed that apigenin improves ovarian histological alterations and follicular dynamics in polycystic ovary rats. The effect is partially mediated by suppression of the VEGF signaling system and reduction in endothelial and periendothelial cell proliferation.

Therapeutic potential of endometrial stem cells encapsulated in alginate/gelatin hydrogel to treat of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder in women, often leading to infertility due to anovulation. Recent advances suggest that endometrial stem cells (EnSCs) hold considerable promise for tissue regeneration, which could be pivotal in treating PCOS. To enhance the survival and stabilization of EnSCs within the ovary, the EnSCs were encapsulated in an injectable alginate/gelatin hydrogel (SC-H), which has excellent biocompatibility to support the survival of EnSCs. Polycystic ovary syndrome was induced in female Wistar rats using intraperitoneal injection of letrozole over 21 days. Then the rats were treated with SC, SC-H and clomiphene citrate for one-month post-PCOS induction. The effects of these treatments were evaluated based on changes in body and ovarian weights, inflammatory markers, endocrine profiles, and ovarian histology. The Induction of PCOS led to a significant increase in body and ovarian cyst weight, elevated serum levels of testosterone, luteinizing hormone (LH), and anti-Müllerian hormone (AMH), alongside reduced follicle-stimulating hormone (FSH) and progesterone levels. Histologically, there was a decrease in granulosa cells, immature follicles, and corpus luteum numbers. Treatment with SC and SC-H significantly mitigated these alterations, indicating improved PCOS conditions. Our findings demonstrate that SC and SC-H treatments can effectively ameliorate the symptoms of letrozole-induced PCOS in rats, primarily through their anti-inflammatory effects. This study lays the groundwork for potential clinical applications of EnSCs encapsulated in alginate/gelatin hydrogel as a novel therapeutic strategy for PCOS, highlighting the importance of biomaterials in stem cell-based therapies.

Testicular Cells Derived Conditioned Medium Supports Germ Cell Differentiation of Human Embryonic Stem Cells.

OBJECTIVE: There are ethical and technical challenges in studying human germ cell development. Therefore, the aim of the study is in vitro differentiation of human embryonic stem cells (hESCs), as pluripotent cells, to the germ cells which is a valuable tool for studying molecular and cellular aspects of gametogenesis and understanding causes of infertility. MATERIALS AND METHODS: In this experimental study, two different complete media [Dulbecco's Modified Eagle Medium (DMEM)+20% fetal bovine serum (FBS) and embryoid bodies (EBs) medium; KOSR/HES without basic fibroblast growth factor (bFGF)] were used in the both of test groups using testicular cells derived conditioned medium (TCCM) and control groups spontaneously differentiated (SD). Thereby, EBs from hESCs (Yazd2; 46XY) were cultured in different conditions EB medium; EB medium and conditioned EB medium; EB medium, DMEM, and FBS without conditioning; EB medium, conditioned DMEM, and FBS medium. EBs were collected after 4, 7, and 14 days and their gene expression profiles were assessed and compared to hESCs, as day 0, using IF and relative reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: An increase in the gametogenesis gene expression level in TCCM groups was showed in comparison with SD groups. Additionally, immunostaining of differentiated cells in all groups showed in vitro gametogenesis (IVG). CONCLUSION: Our findings showed that human TCCM could be used as a natural niche for in vitro male and female germ cell development. However, further studies are needed to define the factors and metabolites within the human TCCM.

Improving Fertility in Non-obstructive Azoospermia: Results from an Autologous Bone Mar-row-Derived Mesenchymal Stromal/Stem Cell Phase I Clinical Trial.

BACKGROUND: In this phase I clinical trial, our primary objective was to develop an innovative therapeutic approach utilizing autologous bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs) for the treatment of nonobstructive azoospermia (NOA). Additionally, we aimed to assess the feasibility and safety of this approach. MATERIALS AND METHODS: We recruited 80 participants in this non-randomized, open-label clinical trial, including patients undergoing NOA treatment using autologous BM-MSCs (n=40) and those receiving hormone therapy as a control group (n=40). Detailed participant characteristics, such as age, baseline hormonal profiles, etiology of NOA, and medical history, were thoroughly documented. Autotransplantation of BM-MSCs into the testicular network was achieved using microsurgical testicular sperm extraction (microTESE). Semen analysis and hormonal assessments were performed both before and six months after treatment. Additionally, we conducted an in-silico analysis to explore potential protein-protein interactions between exosomes secreted from BM-MSCs and receptors present in human seminiferous tubule cells. RESULTS: Our results revealed significant improvements following treatment, including increased testosterone and inhibin B levels, elevated sperm concentration, and reduced levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin. Notably, in nine patients (22.5%) previously diagnosed with secondary infertility and exhibiting azoospermia before treatment, the proposed approach yielded successful outcomes, as indicated by hormonal profile changes over six months. Importantly, these improvements were achieved without complications. Additionally, our in-silico analysis identified potential binding interactions between the protein content of BM-MSC-derived exosomes and receptors integral to spermatogenesis. CONCLUSION: Autotransplantation of BM-MSCs into the testicular network using microTESE in NOA patients led to the regeneration of seminiferous tubules and the regulation of hormonal profiles governing spermatogenesis. Our findings support the safety and effectiveness of autologous BM-MSCs as a promising treatment modality for NOA, with a particular focus on the achieved outcomes in patients with secondary infertility (registration number: IRCT20190519043634N1).

Sperm Selection Using Zona Pellucida-Binding Enhanced Embryo Morphokinetic and Clinical Outcomes in ICSI: A Sibling Oocytes Study.

The objective was to investigate the embryo morphokinitics using a time-lapse monitoring (TLM) system and assessment of clinical outcomes following intracytoplasmic sperm injection (ICSI) with zona pellucida (ZP)-bound sperm selection and conventional methods. A total of 371 metaphase II (MII) oocytes from 50 ICSI cycles were studied. Sibling oocytes were randomly divided into control (n = 199) and ZP-bound group (n = 172). All resulting zygotes were cultured and monitored in the TLM system up to Day 3 after ICSI. Fertilization rate, early embryo development, and clinical outcomes were evaluated. No significant differences were found in fertilization rate, time-lapse qualitative and quantitative measures, pronuclear fading time (PNF) t2, t3, t4, t5, t6, and t7 (times of cleavage to 2, 3, 4, 5, 6, and 7 cells), respectively. However, the t8 (time of cleavage to eight cells) and cc3 (duration of third cell cycle) revealed a significant difference between control and ZP-bound groups (p < .05). A significant difference between the two groups (p < .05) in the rates of Grade A embryos (according to Basile algorithm), chemical pregnancy, clinical pregnancy, and implantation was observed. Sperm selection using biological materials, such as ZP, improved both embryo quality and pregnancy outcomes, despite not affecting the early embryo development and morphokinetic parameters up to t8. This prospective randomized sibling oocyte trial was registered in October 2020 to January 2022 (IRCT20200705048021N1).

Xeno-free generation of new Yazd human embryonic stem cell lines (Yazd4-7) as a prior stage toward good manufacturing practice of clinical-grade raw materials from discarded embryos: A lab resources report.

Background: Xeno-free generation of human embryonic stem cells (hESCs) is important to prevent potential animal contaminations in culture for advanced cell-based therapeutic applications. Xeno-free production of hESCs is the first step for manufacturing clinical-grade hESC lines. Objective: To produce new hESC lines in xeno-free condition. Materials and Methods: This lab resources report was conducted at Stem Cell Biology Research Center, Yazd, Iran from 2019-2022. 4 new hESC lines from 11 (10 fresh and 1 frozen) donated surplus discarded human embryos were established. In this study, we report the xeno-free derivation of new Yazd hESC lines (Yazd4-7), without using immunosurgery, by culturing intact zona-free blastocysts obtained from discarded embryos onto the YhFF#8 cells as a feeder layer in a microdrop culture system. The pluripotency gene expression profile of the cell lines was assessed by reverse transcription polymerase chain reaction and the expression of specific surface markers was detected using immunofluorescent staining. In vitro differentiation was induced using embryoid body formation and gene expression profile of 3 germ layers and germ cells. Reverse transcriptase polymerase chain reaction was investigated to prove their pluripotent capacity. Results: In sum, we have been able to generate 4 new hESC lines (Yazd4-7) from 11 discarded embryos in xeno-free culture conditions using a micro drop culture system and YhFF#8 as a human source feeder layer. Conclusion: The outcome of this work can be the foundation for the future allogeneic cell-based therapeutic application using clinical grade good manufacturing practice-derived hESC derivatives.

The Effect of The Conditioned Medium from Human Embryonic Stem Cells on Mouse Oocytes In Vitro Maturation.

OBJECTIVE: Some reports have indicated that conditioned medium from growing mouse embryonic stem cells (ESCs) provides a supportive condition for small follicles growing, oocyte maturation, and following embryo growth. The aim of this study is assessing in vitro maturation (IVM) and consequent in vitro fertilization (IVF) outcome of immature mouse oocytes using human embryonic stem cells conditioned medium (HESCM). MATERIALS AND METHODS: In this experimental study, 240 germinal vesicle (GV) oocytes were took from NMRI female mice, aged 4-6 weeks, 48 hours before injection of 5 IU pregnant mare serum gonadotropin (PMSG). 120 GV oocytes without cumulus cells were cultured in each of the groups. 120 GV were cultured in HESCM as test groups and also 120 GV cultured in human embryonic stem cells medium (HESM) as control groups. After evaluating the metaphase II (MII) oocyte maturation rate at 8, 16 and 24 hours, the MII oocytes subsequently were fertilized in vitro and the two-cell embryo development rate was recorded at days 1, 2, and 3. Statistical analysis was performed by using the generalized estimating equations (GEE) method that calculated their rate ratio. RESULTS: Our data indicated there are significant differences between the maturation rates in HESCM and HESM (P=0.004), also the two-cell embryo development was significant between two culture media (P=0.00). CONCLUSION: Similar to some other studies, the secretome of the HESCM showed a significant impact on the IVM outcomes in mice.

Defined Physicochemical Cues Steering Direct Neuronal Reprogramming on Colloidal Self-Assembled Patterns (cSAPs).

Direct neuronal reprogramming of somatic cells into induced neurons (iNs) has been recently established as a promising approach to generating neuron cells. Previous studies have reported that the biophysical cues of the in vitro microenvironment are potent modulators in the cell fate decision; thus, the present study explores the effects of a customized pattern (named colloidal self-assembled patterns, cSAPs) on iN generation from human fibroblasts using small molecules. The result revealed that the cSAP, composed of binary particles in a hexagonal-close-packed (hcp) geometry, is capable of improving neuronal reprogramming efficiency and steering the ratio of the iN subtypes. Cells exhibited distinct cell morphology, upregulated cell adhesion markers (i.e., SDC1 and ITGAV), enriched signaling pathways (i.e., Hippo and Wnt), and chromatin remodeling on the cSAP compared to those on the control substrates. The result also showed that the iN subtype specification on cSAP was surface-dependent; therefore, the defined physicochemical cue from each cSAP is exclusive. Our findings show that direct cell reprogramming can be manipulated through specific biophysical cues on the artificial matrix, which is significant in cell transdifferentiation and lineage conversion.

Established Yazd human foreskin fibroblast lines (#8, #17, and #18) displaying similar characteristics to mesenchymal stromal cells: A lab resources report.

Background: Fibroblasts from different parts of the human body have been used in cell biology, drug discovery and cell therapy studies. One of the most available sources of human fibroblasts is neonatal foreskin. Not only do these cells have wound-healing applications, but they are also the most popular source for pluripotent stem cell biotechnology. Moreover, several studies have indicated that different sources of fibroblasts display similar features to mesenchymal stem cells. Objective: Generation and establishment of new human foreskin fibroblast cell lines called Yazd human foreskin fibroblasts (YhFFs). Materials and Methods: In this lab resources study, the production of 3 YhFF cell lines (YhFF#8, YhFF#17, and YhFF#18) is reported. Their biological features were characterized using immunofluorescence, polymerase chain reaction, and flow-cytometry for mesenchymal markers such as fibronectin, vimentin, CD44, CD73, CD90, CD105, and hematopoietic markers CD34 and CD45. Results: The YhFF cell lines were passaged more than 40 times and their normal karyotype was checked using G-binding. Similarly to previous reports, the flow cytometry analysis revealed that the YhFF cell lines displayed mesenchymal stromal cell characteristics. Conclusion: This study will contribute to the development of clinical-grade cell-based products such as micro-vesicles and exosomes for future therapeutic applications in regenerative medicine.

Mesenchymal stem cells-derived exosomes as a promising new approach for the treatment of infertility caused by polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a multifactorial metabolic and most common endocrine disorder that its prevalence, depending on different methods of evaluating PCOS traits, varies from 4% to 21%. Chronic low-grade inflammation and irregular apoptosis of granulosa cells play a crucial role in the pathogenesis of PCOS infertility. Mesenchymal stem cells (MSCs)-derived exosomes and extracellular vesicles (EVs) are lipid bilayer complexes that act as a means of intercellular transferring of proteins, lipids, DNA and different types of RNAs. It seems that this nanoparticles have therapeutic effects on the PCOS ovary such as regulating immunity response, anti-inflammatory (local and systemic) and suppress of granulosa cells (GCs) apoptosis. Although there are few studies demonstrating the effects of exosomes on PCOS and their exact mechanisms is still unknown, in the present study we reviewed the available studies of the functions of MSC-derived exosome, EVs and secretome on apoptosis of granulosa cells and inflammation in the ovary. Therefore, the novel cell-free therapeutic approaches for PCOS were suggested in this study.