The effects of intraovarian injection of autologous menstrual blood-derived mesenchymal stromal cells on pregnancy outcomes in women with poor ovarian response.

BACKGROUND: Assisted reproduction faces a significant obstacle in the form of poor ovarian response (POR) to controlled ovarian stimulation. To address this challenge, mesenchymal stem cell therapy has been proposed as a potential treatment for female infertility and/or restoration of ovarian function in POR women. Our previous research has demonstrated that menstrual blood-derived-mesenchymal stromal cells (MenSCs) injected into the ovaries of women with POR can increase pregnancy rates. The objective of this study was to examine whether MenSC therapy could enhance ovarian reserve parameters and pregnancy outcomes in a larger population of individuals with POR. METHOD: This study consisted of 180 infertile individuals with POR who declined oocyte donation. Participants were divided into two groups: those who received bilateral MenSCs intraovarian injection and those who received no intervention. Our primary aim was to compare the rates of spontaneous pregnancy between the two groups, followed by an investigation of any alterations in the ovarian reserve parameters, such as serum FSH, AMH, and AFC levels, as well as the ICSI/IVF outcomes, in both groups of participants. RESULTS: The MenSC therapy exhibited a favourable tolerability profile and did not raise any safety concerns. Following the 2-month follow-up period, women who received MenSC treatment demonstrated a significantly higher rate of spontaneous pregnancy (P < 0.005) and an improvement in anti-Müllerian hormone (AMH) levels (P = 0.0007) and antral follicle count (AFC) (P < 0.001), whereas the control group demonstrated a considerable decline in these parameters (Both P < 0.001). The MenSC therapy led to a greater number of mature oocytes and embryos among women who underwent ICSI/IVF. Our age subgroup analysis demonstrated a significant difference in the number of spontaneous pregnancies and ICSI/IVF outcomes between the treatment and control groups only among individuals below 40 years of age. CONCLUSION: The results of our study indicate that MenSCs treatment may be a viable option for treating women experiencing POR. However, in order to be widely implemented in clinical practice, the clinical effectiveness of MenSCs therapy will need to be established through rigorous prospective randomized clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05703308. Registered 01/26/2023, retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05703308 . IRCT, IRCT20180619040147N4. Registered 08/01/2020.

Qualified sperm selection based on the rheotaxis and thigmotaxis in a microfluidic system.

Microfluidic systems with the ability to mimic the female reproductive tract (FRT) and sperm features have emerged as promising methods to separate sperm with higher quality for the assistant reproductive technology. Thereby, we designed and fabricated a microfluidic system based on FRT features with a focus on rheotaxis and thigmotaxis for passive sperm separation. In this regard, four various geometries (linear, square, zigzag, and sinusoidal) were designed, and the effect of rheotaxis and thigmotaxis were investigated. Although separated sperm in all microchannels were 100% motile, non-linear geometries were more effective than linear geometry in the term of separating the progressive sperm with high quality. In the presence of upstream flow, periodical changes in the slope of walls (in non-linear geometries) give rise to the periodical facing sperm with a high flow rate in the middle of microchannels, which was a reason for the high quality of separated sperm. However, because of sharp corners in the square and zigzag microchannels that create dead zones with a lack of upstream flow, which is noticeable via simulation results, these geometries have obstacles against sperm swimming toward the outlet, which was proved by image analysis. The sinusoidal geometry showed the highest enhancement level of the designed geometries compared to the linear geometry. Separated sperm exhibited 34.7% normal morphology, 100% motility, and 100% viability in the sinusoidal geometry. Therefore, the periodic change in the position of sperm from one wall to another wall can be a strategy for separating sperm with high quality. Graphical abstract: In the present study, we used a microfluidic system for studying the combined effects of thigmotaxis and rheotaxis for sperm separation process to achieve the successful Assisted reproductive technology (ART). The designed PDMS-based microfluidic system had four various geometries, including linear, square, zigzag, and sinusoidal. The functionality of separated sperm was evaluated by sperm tracking (ImageJ), motility assay (CASA software), and morphology assay (Papanicolaou ultrafast staining). Probing various geometries revealed 100% motility. In non-linear geometries, sperm's periodic detachment from the walls gave rise to the periodic interaction with the high flow velocity in the center of the channel, resulting in the separation of high-quality sperm with progressive motility. The collected data proved the influence of thigmotaxis on the quality of separated sperm. Morphologically improvement in separated sperm from the sinusoidal geometry was significant than others, which means the sinusoidal structure would be the best candidate for the sperm separation process. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-023-00294-8.

Enhancing differentiation of menstrual blood-derived stem cells into female germ cells using a bilayer amniotic membrane and nano-fibrous fibroin scaffold.

Three-dimensional nanofiber scaffolds offer a promising method for simulating in vivo conditions within the laboratory. This study aims to investigate the influence of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold on the differentiation of human menstrual blood mesenchymal stromal/stem cells (MenSCs) into female germ cells. MenSCs were isolated and assigned to four culture groups: (i) MenSCs co-cultured with granulosa cells (GCs) using the scaffold (3D-T group), (ii) MenSCs using the scaffold alone (3D-C group), (iii) MenSCs co-cultured only with GCs (2D-T group), and (iv) MenSCs without co-culture or scaffold (2D-C group). Both MenSCs and GCs were independently cultured for two weeks before co-culturing was initiated. Flow cytometry was employed to characterize MenSCs based on positive markers (CD73, CD90, and CD105) and negative markers (CD45 and CD133). Additionally, flow cytometry and immunocytochemistry were used to characterize the GCs. Differentiated MenSCs were analyzed using real-time PCR and immunostaining. The real-time PCR results demonstrated significantly higher levels of VASA expression in the 3D-T group compared to the 3D-C, 2D-T, and 2D-C groups. Similarly, the SCP3 mRNA level in the 3D-T group was notably elevated compared to the 3D-C, 2D-T, and 2D-C groups. Moreover, the expression of GDF9 was significantly higher in the 3D-T group when compared to the 3D-C, 2D-T, and 2D-C groups. Immunostaining results revealed a lack of signal for VASA, SCP3, or GDF9 markers in the 2D-T group, while some cells in the 3D-T group exhibited positive staining for all these proteins. These findings suggest that the combination of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold with co-culturing GCs facilitates the differentiation of MenSCs into female germ cells.

Application of Menstrual Blood Derived Stromal (stem) Cells Exert Greater Regenerative Potency Than Fibroblasts/Keratinocytes in Chronic Wounds of Diabetic Mice.

Background: In this study we differentially showed the effects of cell-seeded bilayer scaffold wound dressing in accelerating healing process in diabetic ulcers that still remains as a major clinical challenge. The aim of the study was to compare immunomodulatory and angiogenic activity, and regenerative effect differences between Menstrual blood-derived Stem Cells (MenSCs) and foreskin-derived keratinocytes/fibroblasts. Methods: The streptozotocin-induced diabetic mice model was developed in male C57/BL6 mice. A bilayer scaffold was fabricated by electrospining silk fibroin nano-fibers on human Amniotic Membrane (AM). Dermal fibroblasts and keratinocyte isolated from neonatal foreskin and MenSCs were isolated from the menstrual blood of healthy women. The diabetic mice were randomly divided into three groups including no treatment group, fibroblast/keratinocyte-seeded bilayer scaffold group (bSC+FK), and MenSCs-seeded bilayer scaffold group. The healing of full-thickness excisional wounds evaluations in the diabetic mice model in each group were evaluated at 3, 7, and 14 days after treatment. Results: The gross and histological data sets significantly showed wound healing promotion via re-epithelialization and wound contraction along with enhanced regeneration in MenSCs-seeded bilayer scaffold group with the most similarity to adjacent intact tissue. Immunofluorescence staining of mouse skin depicted a descending trend of type III collagen along with the higher expression of involucrin as keratinocyte marker in the MenSCs-seeded bilayer nanofibrous scaffold group in comparison with other treatment groups from day 7 to day 14. Moreover, higher levels of CD31 and von Willebrand factor (VWF), and also a higher ratio of M2/M1 macrophages in association with higher levels of the neural marker were observed in the bSC+MenSCs group in comparison with bSC+FK and no treatment groups. Conclusion: Healing symptoms in wounds dressed with keratinocyte/fibroblast-seeded bilayer scaffold was significantly lower than MenSCs-seeded bilayer scaffold done on impaired diabetic wound chronicity.

Higher Improvement of Cardiac Function Following Myocardial Infarction using Menstrual Blood Stromal/Stem Cells (MenSCs) Suspended in Conditioned Medium versus Conditioned Medium Alone in Rat Model.

Background: To evaluate the efficiency of Menstrual blood Stromal/Stem Cells (MenSCs) administration in Myocardial Infarction (MI), the effects of MenSCs and their derived conditioned Medium (CM) on cardiac function in MI rat model was assessed. Methods: Animals were divided into four groups including sham group, MI group, MenSCs derived CM group (CM group), and MenSCs suspended in CM (MenSCs+CM) group. The injection of different groups was carried out 30 min after ligation of left anterior descending coronary artery into the infarct border zone. Results: The results showed a significant reduction in scar size after injection of MenSCs+CM compared to MI group. Ejection fraction and fractional shortening of MenSCs+CM group were higher than CM and MI group at day 28. Administration of MenSCs+CM led to much more survival of cardiomyocytes, and prevention of meta-plastic development. Moreover, human mitochondrial transfer from MenSCs to cardiomyocytes was seen in group treated by MenSCs+CM. Indeed, MenSCs+CM treatment evoked nuclear factor-κB (NF-κB) down-regulation more than other treatments. Conclusion: MenSCs+CM treatment could significantly ameliorate cardiac function by different mechanisms including inhibition of cartilaginous metaplasia, inhibition of NF-κB and mitochondrial transfer.

Systemic delivery of menstrual blood stem cells is more effective in preventing remote organ injuries following myocardial infarction in comparison with bone marrow stem cells in rat.

Objectives: Remote organ injury is a phenomenon that could happen following myocardial infarction (MI). We evaluated the potency of menstrual blood stromal (stem) cells (MenSCs) and bone marrow stem cells (BMSCs) to alleviate remote organ injuries following MI in rats. Materials and Methods: 2 × 106 MenSCs or BMSCs were administrated seven days after MI induction via the tail vein. Four weeks after cell therapy, activities of aspartate aminotransferase (AST), urea, creatinine, and Blood Urea Nitrogen (BUN) were evaluated. The level of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 were determined by ELISA assay. The expression of Nuclear Factor-κB (NF-κB) was evaluated by immunohistochemical staining. Apoptosis activity and tissue damage were also determined by TUNEL and H&E staining, respectively. Results: MenSCs and BMSCs administration caused a significant reduction in AST, urea, and BUN levels compared with the MI group. In addition, systemic injection of MenSCs significantly decreased the IL-1ß level compared with BMSCs and MI groups (P<0.05 and P<0.01 respectively). Apoptosis in injured kidneys was noticeably diminished in MenSCs-treated rats compared with BMSCs administrated and MI groups (P<0.05 and P<0.05, respectively). In hepatic tissue, limited numbers of TUNEL-positive cells were detected in all groups. Interestingly, MenSCs therapy evoked inhibition of NF-κB in the kidney strikingly. Although, no significant NF-κB expression was observed in hepatic tissue in any group (P>0.05). Conclusion: MenSCs are probably more protective than BMSCs on remote organ injuries following MI via decreasing cell death and immunoregulatory properties.

Intraovarian Administration of Autologous Menstrual Blood Derived-Mesenchymal Stromal Cells in Women with Premature Ovarian Failure.

BACKGROUND: Premature ovarian failure (POF) is a well-known cause of infertility, particularly in women under the age of 40. POF is also associated with elevated gonadotropin levels, amenorrhea and sex-hormone deficiency. AIM OF THE STUDY: In this study, the therapeutic potential of autologous mesenchymal stromal cells obtained from menstrual blood (Men-MSCs) for patients with POF was evaluated. METHODS: 15 POF patients were included in the study. The cultured Men-MSCs were confirmed by flow cytometry, karyotype, endotoxin and mycoplasma and were then injected into the patients' right ovary by vaginal ultrasound guidance and under general anesthesia and aseptic conditions. Changes in patients' anti-Müllerian hormone (AMH), antral follicle count (AFC), follicle-stimulating hormone (FSH), luteal hormone (LH), and estradiol (E2) levels, as well as general flushing and vaginal dryness were followed up to one year after treatment. RESULTS: All patients were satisfied with a decrease in general flushing and vaginal dryness. 4 patients (2.9%) showed a spontaneous return of menstruation without additional pharmacological treatment. There was a significant difference in AFC (0 vs. 1 ± 0.92 count, p value ≤0.001%), FSH (74 ± 22.9 vs. 54.8 ± 17.5 mIU/mL, p-value ≤0.05%), E2 (10.2 ± 6 vs. 21.8 ± 11.5 pg/mL p-value ≤0.01%), LH (74 ± 22.9 vs. 54.8 ± 17.5 IU/L,p-value ≤0.01%) during 3 months post-injection. However, there were no significant changes in AMH (p-value ≥0.05%). There were also no significant differences in assessed parameters between 3 and 6 months after cell injection. CONCLUSION: According to the findings of this study, administration of Men-MSCs improved ovarian function and menstrual restoration in some POF patients.

Greater angiogenic and immunoregulatory potency of bFGF and 5-aza-2'-deoxycytidine pre-treated menstrual blood stem cells in compare to bone marrow stem cells in rat model of myocardial infarction.

BACKGROUND: This study is designed to compare the menstrual blood stem cells (MenSCs) and bone marrow stem cells (BMSCs)-secreted factors with or without pre-treatment regimen using basic fibroblast growth factor (bFGF) and 5-aza-2'-deoxycytidine (5-aza) and also regenerative capacity of pre-treated MenSCs and/or BMSCs in a rat model of myocardial infarction (MI). METHODS: BMSCs and MenSCs were pre-treated with bFGF and 5-aza for 48 h and we compared the paracrine activity by western blotting. Furthermore, MI model was created and the animals were divided into sham, MI, pre-treated BMSCs, and pre-treated MenSCs groups. The stem cells were administrated via tail vain. 35 days post-MI, serum and tissue were harvested for further investigations. RESULTS: Following pre-treatment, vascular endothelium growth factor, hypoxia-inducible factor-1, stromal cell-derived factor-1, and hepatocyte growth factor were significantly increased in secretome of MenSCs in compared to BMSCs. Moreover, systemic administration of pre-treated MenSCs, leaded to improvement of cardiac function, preservation of myocardium from further subsequent injuries, promotion the angiogenesis, and reduction the level of NF-κB expression in compared to the pre-treated BMSCs. Also, pre-treated MenSCs administration significantly decreased the serum level of Interleukin 1 beta (IL-1ß) in compared to the pre-treated BMSCs and MI groups. CONCLUSIONS: bFGF and 5-aza pre-treated MenSCs offer superior cardioprotection compare to bFGF and 5-aza pre-treated BMSCs following MI.

Safety and efficacy study of allogeneic human menstrual blood stromal cells secretome to treat severe COVID-19 patients: clinical trial phase I & II.

BACKGROUND: Cell-free Mesenchymal stromal cells (MSCs) have been considered due to their capacity to modulate the immune system and suppress cytokine storms caused by SARS-CoV-2. This prospective randomized double-blind placebo-controlled clinical trial aimed to assess the safety and efficacy of secretome derived from allogeneic menstrual blood stromal cells (MenSCs) as a treatment in patients with severe COVID-19. METHODS: Patients with severe COVID-19 were randomized (1:1) to either MenSC-derived secretome treatment or the control group. Subjects received five intravenous infusions of 5 mL secretome or the same volume of placebo for five days and were monitored for safety and efficacy for 28 days after treatment. Adverse events, laboratory parameters, duration of hospitalization, clinical symptom improvement, dynamic of O2 saturation, lymphocyte number, and serial chest imaging were analyzed. RESULTS: All safety endpoints were observed without adverse events after 72 h of secretome injection. Within 28 days after enrollment, 7 patients (50%) were intubated in the treated group versus 12 patients (80%) in the control group. Overall, 64% of patients had improved oxygen levels within 5 days of starting treatment (P < 0.0001) and there was a survival rate of 57% in the treatment group compared to 28% in the control group was (P < 0.0001). Laboratory values revealed that significant acute phase reactants declined, with mean C-reactive protein, ferritin, and D-dimer reduction of 77% (P < 0.001), 43% (P < 0.001), and 42% (P < 0.05), respectively. Significant improvement in lymphopenia was associated with an increase in mean CD4+ and CD8+ lymphocyte counts of 20% (P = 0.06) and 15% (P < 0.05), respectively. Following treatment, percentage of pulmonary involvement showed a significant improvement in the secretome group (P < 0.0001). This improvement differed significantly between survivors and those who were dying (P < 0.005). CONCLUSIONS: For the first time, this study demonstrated that in hospitalized patients with severe COVID-19, therapy with MenSCs-derived secretome leads to reversal of hypoxia, immune reconstitution, and downregulation of cytokine storm, with no adverse effects attributable to the treatment. Given these outcomes, it may be possible to use this type of treatment for serious inflammatory lung disease with a mechanism similar to COVID-19 in the future. However, it is necessary to evaluate the safety and efficacy of MenSCs-derived secretome therapy in clinical trials on a larger population of patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05019287. Registered 24AGUEST 2021, retrospectively registered, https://clinicaltrials.gov/ct2/show/record/NCT05019287 . IRCT, IRCT20180619040147N6. Registered 04/01/2021.

Lower reactive oxygen species production and faster swimming speed of human sperm cells on nanodiamond spin-coated glass substrates.

The sperm selection stage is what assisted reproductive technologies have in common and is crucial as it affects the success of the treatment cycle. The employment of microfluidic platforms for sperm selection has emerged showing promising results. In microfluidic platforms, sperm cells encounter micro-confined environments meanwhile having contact with channel walls and surfaces. Modification of contact surfaces using nanoparticles leads to the alteration of surface characteristics which in turn affects sperm behavior especially motility which is an indicator for sperm health. In this article, we present the results of investigating the motility parameters of sperm cells in contact with surface-modified glass substrates using nanodiamond particles. The results show that the sperm swimming velocities are significantly improved within the range of 12%-52% compared to the control surface (untreated). Reactive oxygen species production is also decreased by 14% justifying the increase in swimming speed. Taken together, bonding these modified surfaces to sperm selection microfluidic devices could enhance their efficiency and further improve their outcomes offering new solutions to patients facing infertility.

The remarkable effect of menstrual blood stem cells seeded on bilayer scaffold composed of amniotic membrane and silk fibroin aiming to promote wound healing in diabetic mice.

INTRODUCTION: Impaired chronic wound healing frequently occurs in diabetic patients. We hypothesized that menstrual blood-derived mesenchymal stem cells (MenSCs) in combination with bilayer scaffold consisted of human amniotic membrane (AM) and electrospun silk fibroin nanofibers could potentially promote wound healing in diabetic mice. METHODS & METHODS: Two bilateral full-thickness wounds were created on dorsal skin of type-1 diabetic mice model and animals were equally divided in four groups including: no-treatment group (NT), amniotic membrane treated group (AM), bilayer scaffold treated group (bSC), and MenSCs-seeded bilayer scaffold treated group (bSC + MenSCs). Wound healing evaluations were performed at 3, 7, and 14 days after their treatment. The wound healing was analyzed by macroscopic and microscopic evaluations, and immunofluorescence staining of involucrin (IVL), type III collagen, CD31/ von Willebrand factor (vWF), and PGP9.5 were performed. Furthermore, number of neutrophils and macrophages and subpopulation of macrophages were assessed. In addition, the expression of Egr2, Mmp9, CXCL12, IDO1, Ptgs2 and VEGFA transcripts involved in wound repair were also analyzed. RESULTS: After 14 days, the best epidermal and dermal regeneration belonged to the cases received bSC + MenSCs as wound dressing. Moreover, the wound healing was typically faster in this group compared to other groups. Immunofluorescence evaluation represented higher levels of CD31 and VWF, higher ratio of M2/M1 macrophages, greater expression of IVL, and higher levels of the PGP9.5 in the bSC + MenSCs group in comparison with other groups. Expression analysis of assessed genes also supported assumption of more regeneration and healing in the bSC + MenSCs group versus other groups. CONCLUSION: These results indicate that enhanced immunomodulatory and reparative properties of MenSCs in conjunction with bilayer scaffold specified this cellular skin substitute for modulating wound chronicity and contribution to resolution of wound healing process in diabetic ulcer.

Comparison of the effects of Duphaston and Cetrotide on oocyte and embryo quality in women undergoing ICSI: A cross-sectional study.

BACKGROUND: Premature luteinizing hormone (LH) surge is one of the causes for assisted reproductive technology cycle cancellation, and it is needed to find novel approaches with improved efficacy and safety profile. OBJECTIVE: To compare the effects of Duphaston and Cetrotide on the prevention of premature LH surge and characteristics of retrieved follicles and embryos in women undergoing intracytoplasmic sperm injection. MATERIALS AND METHODS: In this retrospective cross-sectional study, 200 patients who were administrated recombinant follicle-stimulating hormone from the third day of menstruation cycle were included. When the follicular diameter reached above 13-14 mm, Cetrotide was prescribed in the control group, while in the case group, Duphaston was taken orally from the third day of cycle. The retrieved oocytes were fertilized in vitro by intracytoplasmic sperm. The level of hormones on the third day of menstruation and the characteristic of follicles, oocytes, and embryos were compared between the two groups. RESULTS: Duphaston successfully inhibits premature LH surge. There was no significant difference in the level of follicle-stimulating hormone, estradiol, and LH between the case and control groups (p > 0.05). However, results also showed that Duphaston causes more oocyte retrieval in comparison with Cetrotide (p = 0.04). Although, the number of follicles above 14 mm, mature oocyte, and the total number of viable embryos in the case group was slightly higher, it did not reach a significant difference compared with the control group (p > 0.05). CONCLUSION: Duphaston could be used as an appropriate medication instead of gonadotropin-releasing hormone antagonists in women undergoing controlled ovarian hyperstimulation. Duphaston prescription not only prevents premature LH surge but also improves the number of retrieved oocytes.

Safety and efficacy of engineered tissue composed of silk fibroin/collagen and autologous chondrocytes in two patients with cartilage defects: A pilot clinical trial study.

BACKGROUND: The objective of this pilot clinical trial study was to evaluate safety and effectiveness of the newly engineered tissue composed of autologous chondrocytes and collagen/fibroin scaffold in repair of osteochondral defects. METHODS: We implemented a pilot clinical study in two patients with knee osteochondral lesions using engineered tissue composed of scaffold and autologous chondrocytes. Patients were clinically evaluated using the International Repair Cartilage Society score and magnetic resonance imaging (MRI) for one year. RESULTS: Improved clinical outcomes and objective scores indicated a normal or nearly normal knee in both patients. International Knee Documentation Committee score was upgraded from 34.5 at baseline to 72.4 in the first patient, and 28.7 to 81.6 in the second patient. Visual analogue scale, showing the suffering pain score, was lowered from 8 to 0 in both patients, Western Ontario and McMaster Universities Osteoarthritis Index score representing the physical ability of the patients was changed from 68.1 to 87.1 in Patient 1 and 58.3 to 87.1 in Patient 2, the knee function score, related to the functional ability of the knee, was improved from 70 to 100 in the first patient and from 45 to 91 in the second patient. MRI showed great coverage and integration of the graft in patients, with no effusion, decreased edema and cartilage formation signals. CONCLUSIONS: The functional and clinical outcomes alongside MRI data showed promising results for regenerating osteochondral defects. A randomized clinical trial study is required to confirm feasibility of this novel engineered tissue in repair of osteochondral defects.

Quantification of human sperm concentration using machine learning-based spectrophotometry.

Spectrophotometry is an indirect non-invasive and quantitative method for specifying materials with unknown contents based on absorption behavior. This paper presents the first application of artificial neural network in spectrophotometry for quantification of human sperm concentration. A well-trained full spectrum neural network (FSNN) model is developed by examining the absorption response of sperm samples from 41 human subjects to different light spectra (wavelength from 390 to 1100 nm). It is shown that this FSNN accurately estimates sperm concentration based on the full absorption spectrum with over 93% prediction accuracy, and provides 100% agreement with clinical assessments in differentiating the samples of healthy donor from patient samples. We suggest the machine learning-based spectrophotometry approach with the trained FSNN model as a rapid, low-cost, and powerful technique to quantify sperm concentration. The performance of this technique is superior to available spectrophotometry methods currently used for semen analysis and will provide novel research and clinical opportunities for tackling male infertility.

Endometrial and Menstrual Blood Mesenchymal Stem/Stromal Cells: Biological Properties and Clinical Application.

A highly proliferative mesenchymal stem/stromal cell (MSC) population was recently discovered in the dynamic, cyclically regenerating human endometrium as clonogenic stromal cells that fulfilled the International Society for Cellular Therapy (ISCT) criteria. Specific surface markers enriching for clonogenic endometrial MSC (eMSC), CD140b and CD146 co-expression, and the single marker SUSD2, showed their perivascular identity in the endometrium, including the layer which sheds during menstruation. Indeed, cells with MSC properties have been identified in menstrual fluid and commonly termed menstrual blood stem/stromal cells (MenSC). MenSC are generally retrieved from menstrual fluid as plastic adherent cells, similar to bone marrow MSC (bmMSC). While eMSC and MenSC share several biological features with bmMSC, they also show some differences in immunophenotype, proliferation and differentiation capacities. Here we review the phenotype and functions of eMSC and MenSC, with a focus on recent studies. Similar to other MSC, eMSC and MenSC exert immunomodulatory and anti-inflammatory impacts on key cells of the innate and adaptive immune system. These include macrophages, T cells and NK cells, both in vitro and in small and large animal models. These properties suggest eMSC and MenSC as additional sources of MSC for cell therapies in regenerative medicine as well as immune-mediated disorders and inflammatory diseases. Their easy acquisition via an office-based biopsy or collected from menstrual effluent makes eMSC and MenSC attractive sources of MSC for clinical applications. In preparation for clinical translation, a serum-free culture protocol was established for eMSC which includes a small molecule TGFß receptor inhibitor that prevents spontaneous differentiation, apoptosis, senescence, maintains the clonogenic SUSD2+ population and enhances their potency, suggesting potential for cell-therapies and regenerative medicine. However, standardization of MenSC isolation protocols and culture conditions are major issues requiring further research to maximize their potential for clinical application. Future research will also address crucial safety aspects of eMSC and MenSC to ensure these protocols produce cell products free from tumorigenicity and toxicity. Although a wealth of data on the biological properties of eMSC and MenSC has recently been published, it will be important to address their mechanism of action in preclinical models of human disease.

Current evidence on immunological and regenerative effects of menstrual blood stem cells seeded on scaffold consisting of amniotic membrane and silk fibroin in chronic wound.

Wound healing process is a complex of overlapping and coordinated events progresses beyond the inflammatory phase toward wound resolution, whereas chronic wounds fail to terminate inflammatory phase and could not develop toward regenerative state. The immunopathology of chronic wounds has been attributed to the prolonged inflammation and dysregulation of microenvironments responsible for imbalance between pro-inflammatory and anti-inflammatory states, as well as cellular and tissue senescence. We here discuss that menstrual blood-derived mesenchymal stem cells (MenSCs) with their authentic functions especially immunosuppressive, angiogenic and migratory properties in combination with a bilayer amniotic membrane/nano-fibrous fibroin scaffold could bring about effective regenerative effects in healing of chronic wound. To debate, following evidences have been cumulated : 1) Persistent pro-inflammatory state in chronic wound bed could inhibit wound resolution; 2) MenSCs exhibit noticeable regenerative, immunosuppressive effects and immunomodulatory activity, 3) The migratory characteristics of MenSCs may not be sufficient for their homing to chronic wounds site, and 4) Bilayer scaffold composed of amniotic membrane and silk fibroin induces MenSCs differentiation into keratinocyte-like cells and stimulates skin regeneration.

Improvement of Pregnancy Rate and Live Birth Rate in Poor Ovarian Responders by Intraovarian Administration of Autologous Menstrual Blood Derived- Mesenchymal Stromal Cells: Phase I/II Clinical Trial.

The study was aimed to evaluate the safety and efficacy of cell therapy using autologous menstrual blood derived- mesenchymal stromal cells (Men-MSCs) in fertility potential of poor ovarian responders (PORs). POR women were divided into mesenchymal stroma cell (MSC) therapy (n = 15) and routine ICSI (n = 16) groups. The cultured Men-MSCs were autologously injected into left ovary of MSC group after approval by flow cytometry, karyotyping, endotoxin, sterility and mycoplasma tests. Changes in anti-Mullerian hormone (AMH), antral follicles count (AFC), oocytes and embryos number, clinical pregnancy rate and live birth rate were followed in both groups up to one year after treatment. 4 of 15 participants in MSC group got naturally pregnant during 3 months after cell administration, in contrast to no natural conception in control group (P = 0.04). The mean AMH level did not significantly differ with that of previous cycle or control group. Although mean AFC and oocytes number in MSC group did not indicate considerable difference with those of control group, raise of these parameters in comparison with previous cycle was significant (both P = 0.01). Nonetheless, oocyte fertilization rate and embryo number in MSC group were higher than control group (P = 0.04 and P = 0.008, respectively). Altogether, 7 of 15 women in MSC group and 2 of 16 women in routine ICSI group had clinical pregnancy that resulted in 5 live births in main group and one birth in control group. In conclusion, cell therapy using Men-MSCs could be considered as a potential treatment to restore fertility capability of POR women.The trial registration number (TRN): IRCT20180619040147N2.Date of registration: 2018-08-21.

Differentiation of Menstrual Blood Stem Cells into Keratinocyte-Like Cells on Bilayer Nanofibrous Scaffold.

Skin tissue engineering is a high-throughput technology to heal the wounds. Already, considerable advances have been achieved using stem cells for wound healing applications. Menstrual blood stem cell (MenSC) is an available and accessible source of stem cells that have differentiation potential into a wide range of lineages like keratinocytes. Extracellular matrix like substratum plays an impressive role in skin regeneration as an attachment site for stem cells by transmitting the bioactive signals and provoking stem cells to differentiate into keratinocyte lineage. The biomimetic nanofibrous scaffold especially in bilayer format has been extensively utilized to develop skin equivalents. This chapter explains detailed protocols of keratinocyte differentiation of MenSCs on bilayer scaffold comprising amniotic membrane and fibroin nanofibers. The isolated MenSCs are seeded on the nanofibers and subsequently differentiated into keratinocyte lineage in co-culture with foreskin-derived keratinocytes. Immunofluorescence staining is used to evaluate the development of seeded MenSCs in bilayer scaffold into keratinocyte-like cells.

Efficient Wound Healing Using a Synthetic Nanofibrous Bilayer Skin Substitute in Murine Model.

Treatment of full-thickness skin wounds with minimal scarring and complete restoration of native tissue properties still exists as a clinical challenge. A bilayer skin substitute was fabricated by coating human amniotic membrane (AM) with electrospun silk fibroin nanofibers, and its in vivo biological behavior was studied using murine full-thickness skin wound model. Donut-shaped silicon splints were utilized to prevent wound contraction in mouse skin and simulate re-epithelialization, which is the normal path of human wound healing. Skin regeneration using the bilayer scaffold was compared with AM and untreated defect after 30 d. Tissue samples were taken from healed wound areas and investigated through histopathological and immunohistochemical staining to visualize involucrin (IVL), P63, collagen I, CD31, and vascular endothelial growth factor. In addition, mRNA expression of IVL, P63, interleukin-6, and cyclooxygenase-2 was studied. The application of bilayer scaffold resulted in the best epidermal and dermal regeneration, demonstrated by histopathological examination and molecular analysis. In regenerated wounds of the bilayer scaffold group, the mRNA expression levels of inflammatory markers (interleukin-6 and cyclooxygenase-2) were downregulated, and the expression pattern of keratinocyte markers (IVL and P63) at both mRNA and protein levels was more similar to native tissue in comparison with AM and no-treatment groups. There was no significant difference in the expression level of collagen I, CD31, and vascular endothelial growth factor among different groups. Conclusively, these promising results serve as a supporting evidence for proceeding to clinical phase to examine the capacity of this bilayer scaffold for human skin regeneration.