Stesura Seveso Archivio Italiano di Urologia e Andrologia 2023; 95(4):11669 1 ORIGINAL PAPER ronal degeneration, and fibrosis, all of which lead to the erectile dysfunction development (6). Although phosphodi- esterase type 5 inhibitors (PDE5i) are beneficial in treating erectile dysfunction in the majority of patients, their effec- tiveness is much reduced in diabetic people (7). This is likely due to the decreased generation of nitric oxide (NO) as a consequence of endothelial dysfunction (8). ED linked with diabetes is similarly very resistant to PDE5i therapy, with a 44% success rate opposed to an 85% suc- cess rate for hypogonadal ED patients (9). Patients with diabetes and erectile dysfunction had the greatest incidence of therapy termination with PDE5Is (28/36, or 78 percent) (10). Therefore, finding an effec- tive therapy for ED linked with diabetes is one of the most significant goals of modern ED research. Recent treatment techniques, like as gene therapy and stem cell therapy, are being investigated to treat diabetic ED more success- fully. Stem cell is a possible therapy for diabetic ED. Multiple stem cell types have been utilized to cure ED, including BMSC, ADSC, and USC (11-13). Stem cells may develop into several cell types, such as smooth muscle cells (SMC), neurons and vascular endothelial cells. In addition, they may emit paracrine substances that may boost angiogenesis and cell survival (14). A study was conducted in our Urology Department from March 2016 to September 2018. METHODS The study was conducted after ethical committee approval. For all men included in the study, explanation of the study procedures was done and informed consent was onbtained before enrollment. Inclusion criteria Diabetic adult men with HbA1c between 6.5% and 10% with diagnosis of diabetes mellitus dated more than 5 years. Having a consistent sexually active partner. Inadequate sexual activity in spite of taking the maximal dosage of oral PDE5I during the last eight weeks. Purpose: to assess safety and efficacy of autologous mesenchymal bone marrow stem cell injection in penile cavernosal tissue for erectile dysfunction therapy in diabetic men. Methods: The subjects of this study were diabetic men suffering erectile dysfunction, non-responding to maximum dose of oral PDE5I. Mesenchymal bone marrow stem cells were aspirated and injected after preparation in both corpora cavernosa at 3, 9 o’clock position. Erectile function was assessed by the International Index of Erectile Function and penile Doppler study, before and after 6 months after injection. Results: 4 patients out of 10 achieve hard erection adequate for satisfactory coitus, and 2 patients achieved penile hardness with addition of pharmacological therapy with sildenafil 100mg. Peak systolic velocity increased significantly in 4 patients (2 arteriogenic and 2 mixed erectile dysfunction), from 12∼22 cm/s to 32∼69 cm/s. Variations in end-diastolic velocity increased substantially in 2 patients with venogenic insufficien- cy alone at follow-up from 4∼5 cm /s to -4∼-3 cm/s. Conclusions: Despite promising stem cell treatment efficacy for patients with erectile dysfunction, more clinical studies and researches are still warranted. KEY WORDS: Erectile dysfunction; Diabetes; Stem cell. Submitted 21 August 2023; Accepted 7 September 2023 In 1995, the global erectile dysfunction (ED) prevalence was evaluated to be 152 million and is expected to reach 322 million by 2025 (2). ED may be organic, psychogenic or a combination. ED organic type is divided into vascu- logenic, neurogenic, cavernous, drug-induced, systemic disease-related and hormonal categories (2). Diabetic men tend to experience ED more frequently and 10-15 years earlier than non-diabetic men (3). Diabetes-related ED is a serious issue that has a significant influence on patients' life quality and family harmony (4). In addition, ED in diabetic people is more resistant and severe to treatment than in nondiabetic patients (5). Pathogenic impact of diabetic ED comprises endothelium malfunction, smooth muscle composition reduction, neu- Autologous mesenchymal stem cell therapy for diabetic men with erectile dysfunction. Is it promising? A pilot study Mohamed A. Alhefnawy 1, Emad Salah 2, Sayed Bakry 3, Taymour M. Khalifa 4, Alaa Rafaat 2, Refaat Hammad 4, Ali Sobhy 5, Ahmed Wahsh 2 1 Department of Urology, Benha University, Egypt; 2 Department of Urology, Al-Azhar University, Egypt; 3 Department of Embriology and Genetic engineering, Faculty of Science, Al-Azhar University, Egypt; 4 Department of Dermatology and Andrology, Al-Azhar University, Egypt; 5 Department of Clinical Pathology, Al-Azhar University, Egypt. DOI: 10.4081/aiua.2023.11669 Summary Archivio Italiano di Urologia e Andrologia 2023; 95(4):11669 M.A. Alhefnawy, E. Salah, S. Bakry, et al. 2 Exclusion criteria History of bone marrow disorders, neurogenic ED, gen- tamycin hypersensitivity. History of severe cardiovascular disease (angina, arrhythmias, cardiac failure, and stroke), renal failure, and respiratory failure as life-threatening conditions. Positive HIV, HBV, HCV, and syphilis tests. Cancer history during the last five years. HbA1c levels more than 10 percent. Uncontrolled hypertension or hypotension (systolic blood pressure > 170 or 90 mm Hg, diastolic blood pressure > 100 or 50 mm Hg). Anticoagulant therapy. Severe infectious disease. Testosterone concentration less than 200ng/dL Having a penile implant or be open to getting one, Patients with alterations in penis morphology. Subjects participating in additional clinical studies in the previous 30 days. Subjects unable to comply with procedure. All partici- pants were subjected to detailed medical and sexual histo- ry including IIEF questionnaire, physical examination including (general and genital examination) and laborato- ry work up including testosterone level, HBA1C, pro- lactin, LH and FSH. Evaluation of general condition by cell blood count (CBC), liver function, renal function, and thy- roid function tests, lipid and coagulation profile was done for all patients. Penile Doppler Ultrasound was done for all patients. Normal peak systolic velocity was defined as a value ≥ 35 ml/second and normal end diastolic velocity was defined as a value ≤ 3 ml/second. IIEF Patient Questionnaire The 6-question IIEF Questionnaire (Table 1) is a validat- ed, multidimensional, self-administered study that has shown beneficial in clinical studies for assessing erectile dysfunction and treatment effects. This questionnaire in addition to the penile Doppler were performed before and 6 months after injection. Patients with poor IEEF scores (17 out of 30) in D (Erectile Function) were eligible for mesenchymal stem cell treatment. Procedures Under sterile conditions with local anesthetics, ten ml mesenchymal stem cell (MSCs) were aspirated from the bone marrow of the iliac crest of the candidate. After lab processing, the sample was brought to the operative the- ater. In the operation room, the patient was positioned in the supine position. Genitalia were sterilized and penile block was performed. Then genitalia were sterilized again, MSCs were injected in both corpora cavernosa at 3 and 9 o’clock position. Outcome measures Six months following injection, IIEF and Peak systolic velocity (PSV) and End diastolic velocity (EDV) were tested for changes from baseline (preoperative). Statistical analysis: Using statistical program for social sci- ences (SPSS) version 23.0 for Windows, the acquired data were edited, structured, tabulated, and statistically ana- lyzed. Data are displayed as mean, SD, frequency, and percentage. Student's t test was used to compare contin- uous variables (two-tailed). We compared categorical variables using the chi-square (2) and Fisher's exact tests (if needed). The level of significance was accepted if the P value < 0.05. RESULTS This study included 10 diabetic patients complaining of ED, mean age 52 years and HBA1c range from 6.5 -9.5. Hyperlipidaemia was detected in 6 (60%) patients. Table 2 demonstrates the demographic characteristics of the studied patients. Erectile function was assessed by IIEF- score and penile Doppler study. Two patients have pure arterial insuffi- ciency, 4 (40%) patients have pure veno-occlusive disor- Table 2. Demographic characteristics of the studied patients. Parameters All cases n = 10 Age (years) Mean + SD 52.3 ± 6.4 Range (Min-Max) 25 (40-65) Residence N (%) Rural 3 (30.0%) Urban 7 (70.0%) Education N (%) Illiterate 5 (50.0%) Secondary 3 (30.0%) High 2 (20.0%) Socio-economic level N (%) Low 4 (40.0%) Middle 3 (30.0%) High 3 (30.0%) Medical diseases N (%) Diabetes 10 (100.0%) Hyperlipidemia 6 (60.0%) Table 1. 6-question IIEF Questionnaire. Archivio Italiano di Urologia e Andrologia 2023; 95(4):11669 3 Autologous mesenchymal stem cell therapy for diabetic men with erectile dysfunction der and 4 (40%) patients mixed arteriogenic and vascu- logenic insufficiency as declared by penile Doppler study, Table 3 shows the ED type among the studied patients. The effect of single intracavernosal injection of BMSc on erectile function were assessed using IIEF-6 questions as shown in Table 4 and penile Doppler study at 6 months, as in Table 5 and 6, comparing the PSV and EDV before and after injection at 6 months. DISCUSSION DM is a systematic disease that affects every part of the body. In the penis, it is associated with reduced contents of all three key components for erectile function, namely, cavernous nerve (CN), cavernous endothelial cells (CEC) and smooth muscle cells (CSMC) (15-19). The reduction of CEC content is likely due to DM-induced apoptosis in CEC as demonstrated by immune-histochemical analysis of corpora cavernosa (CC) samples between diabetic and nondiabetic patients (20).Thus, how to prevent and/or reverse these pathological processes is critically important for the effective treatment of DM-associated ED. In this regard, stem cells (SC) therapy has been considered prom- ising, due to SC’s well-known regenerative capacity (18). MSCs, initially isolated from bone marrow, have later been isolated from many adult tissues such as adipose tissue, skeletal muscle, brain and skin. As their name suggests, MSCs are defined by their ability of self- renewal and dif- ferentiation into various phenotypes (multipotency). The therapeutic effect of MSCs has consistently been demonstrated and these benefits are mostly attributed to their ability to produce an array of bioactive molecules. This is known as paracrine action of MSCs and it involves stimulation of angiogenesis and revascularization, modu- lation of immune and inflammatory responses, inhibition of apoptosis and trophic effects such as stimulation of mitosis, proliferation and differentiation of intrinsic stem progenitor cells (21). Different routes have been suggested for the delivery of stem cells, and research continues to assess the most effective route of instillation. Some studies have involved the direct injection of cells into the organ of concern (22- 24). Other studies have investigated intraperitoneal or intravenous injections of stem cells (25). Studies have shown that less than 1% of stem cells infused via the intravenous route reach the target tissue, and those that do reach the target tissue dissipate after a few days (26). In preclinical studies, the intravenous injection of ADSCs has been shown to lead to improvements in erectile func- tion (27). The intracorporal injection of stem cells for ED treatment has been commonly evaluated in preclinical studies, as it is both straightforward and logical (28). Periprostatic injection, with or without a concurrent intracorporal injection, has also been attempted (29, 30). Many preclinical trials have been performed to investigate the safety, efficacy, and mechanisms of stem-cell therapy for ED in animal models. Soebadi et al. in 2016 summa- rized these studies (31). As stated by those authors, these preclinical trials have provided ample data on the utiliza- tion of both bone marrow stem cells and ADSCs for ED. Almost all of the studies reported improved erectile func- tion in various animal models of CN injury, vascular insuf- ficiency, diabetes mellitus, hyperlipidemia, and aging. Human data on stem-cell therapy for ED are finally emerging approximately 10 years after the first reports on animal models. We have 4 very important published human clinical trials which employed stem cells in patients with ED, as summarized below. Bahk et al. (24) injected 1.5×107 umbilical MSC into the corpora of 7 ED patients with DM and noted improve- ment when coupled with oral PDE5i. The International Index of Erectile Function (IIEF)-5, global assessment ques- tionnaire, erection diary, blood glucose diary, and med- ication dosage were monitored for 9 months. Three par- ticipants regained morning erections in 1 month, 2 partic- ipants achieved erection successful for penetration in con- junction with PDE5i for 6 months. Yiou et al. (22) admin- istered BMSC in men with ED after radical prostatectomy. Four equal groups of patients were given escalating doses of BMSC (2 ×107, 2 × 108, 1 × 109, and 2 × 109, respec- tively). IIEF-15, erection hardness scale, penile duplex and penile NO release tests were all used to assess erectile function. Significant improvement was noted in 9 of 12 patients treated in combination with an oral PDE5i. Haahr et al. (23) injected ASC into 17 men with a history of prostatectomy to determine safety and efficacy. Five patients had minor adverse events related to liposuction, Table 3. Classification of erectile dysfunction in the studied patients. All cases n = 10 Type N (%) Single 6 (60.0%) Mixed 4 (40.0%) Table 4. Comparison of International Index of Erectile Dysfunction before and after injection (Total n = 10). Before treatment After treatment P-value Mean ± SD Mean ± SD All cases n = 10 12.7 ± 2.16 19.2 ± 5.75 0.026 S Table 5. Comparison of peak systolic velocity (PSV) before and after injection at 6 months (Total n = 10). Before injection After injection P-value Mean ± SD Mean ± SD All cases n = 10 25 ± 12.5 40.9 ± 18.9 0.016 S Mixed cases n = 4 13.75 ± 6.13 25.5 ± 13.2 0.16 NS Arteriogenic cases n = 6 16.33 ± 6.21 38.8 ± 22.8 0.036 S Table 6. Comparison of End Diastolic velocity EDV before and after injection at 6 months (Total n = 10). Before injection After injection P-value Mean ± SD Mean ± SD All cases n = 10 3.9 ± 5.4 0.9 ± 4.09 0.026 S Mixed cases n = 4 5.07 ± 2.08 3.33 ± 1.15 0.37 NS Arteriogenic cases n = 6 6.12 ± 2.7 2.37 ± 3.8 0.022 S Archivio Italiano di Urologia e Andrologia 2023; 95(4):11669 M.A. Alhefnawy, E. Salah, S. Bakry, et al. 4 2 men has redness or swelling at the injection site, and 1 patient developed a scrotal and penile hematoma. They used IIEF-5 to evaluate erectile function and found 8 of 17 men able to achieve an erection for successful intercourse with no mention of use of oral medications. Levy et al. (32) injected adult placental-matrix-derived stem cells (unknown cell number) in a study of 8 men with ED, and assessed peak systolic velocity, end-diastolic velocity, stretched penile length, penile width. Five patients at 3 months achieved erections for successful intercourse with use of PDE5i. The only measure significantly improved was peak systolic velocity. We summarized this previously mentioned 4 published clinical trials on stem-cell therapy for ED in Table 7. The present study includes 10 diabetic patients type 2 aged 40-65 complaining of erectile dysfunction. After clinical examination and evaluation, the cause of erectile dysfunction in these patients has been cleared: four patients had mixed arteriogenic and vasculogenic insuffi- ciency, four patients had pure venogenic insufficiency and two patients had pure arteriogenic cause. These patients cannot satisfy sexual activity with proper sexual stimulation in spite of taking maximum dose of oral PDE5I within last 8 weeks. In this study we injected patients with MSC-derived stem cells and followed them for 6 months with Doppler parameters and the IIEF questionnaire. All patients agreed that bone marrow stem-cell therapy had some effect on ED, although it was insufficient in some patients. The effects of treatment on erectile func- tion and penile vascular parameters were assessed using the IIEF-15 and by color duplex Doppler ultrasound. The peak systolic velocity was found to have improved to a statistically significant extent in 4 patients (2 arterio- genic and 2 mixed ED), from 12~ 22 cm/s to 32~69 cm/s. Changes in end-diastolic velocity were found to have improved to statistically significant extent in 2 patients with venogenic insufficiency at follow-up from 4~5 cm/s to -4~-3 cm/s. At follow-up, in two patients (mixed ED), changes in PSV were not statistically significant for both PSV and EDV. Two patients with venogenic ED had no improvement in EDV measurement after injection of MSCs. Changes in end-diastolic velocity were found to be not statistically significant in four patients (two withe venogenic insufficiency and two with mixed ED). Four patients achieved hard erection adequate for satisfac- tory coitus, and two achieved penile hardness with addi- tion of pharmacological therapy with sildenafil 100 mg. As regard to our study all patients with arteriogenic ED have significant improvement in duplex penile U/S and penile hardness, and improvement in PSV was observed in two patients with mixed ED; in four patients with venogenic ED, two patients had significant improvement in duplex U/S and penile hardness and two patients no sig- nificant improvement; two patients with mixed ED had no significant increase in both peak systolic velocity and end diastolic velocity and no improvement in penile hardness. CONCLUSIONS Autologous stem-cell therapy is considered a viable treat- ment option for diabetic ED patients. 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Correspondence Mohamed Abdelrahman Alhefnawy, MD (Corresponding Author) dr.mohamedalhefnawy@gmail.com Assistant Professor of Urology, Benha University Fareed Nada Street 13518, Banha, Egypt Emad Salah, MD emadeldeen_salah@hotmail.com Alaa Rafat, MD dralaarafaat@gmail.com Ahmed Wahsh, MD hudaahmed320@gmail.com Professor of Urology Al-Azhar University, Egypt Sayed Bakry, MD sbakry@azhar.edu.eg Professor of Genetic Engineering, Al-Azhar University, Egypt Taymour Khalifa, MD taymour.khalifa@gmail.com Professor of Dermatology, Venereology and Andrology, Al-Azhar University, Egypt Refaat Hammad, MD refaat-ragab@yahoo.com Assistant Professor of Dermatology and Andrology, Al-Azhar University, Egypt Ali Sobhy, MD dralisobhy@azhar.edu.eg Assistant Professor of Clinical Pathology, Egypt Conflict of interest: The authors declare no potential conflict of interest.