Stem cell therapy outcome for diabetic erectile dysfunction: A pilot analysis

Study design

This study is a randomized, double-blind, placebo-controlled trial conducted at Dr. Cipto Mangunkusumo Hospital in Indonesia from October 2020 to January 2023. The study received ethical approval from FKUI-RSCM Health Research Ethics Committee numbered “KET-936/UN2.F1.ETIK/PPM.00.02/2019”, approved on 12^th of August 2019. This study is a part of a larger clinical trial titled “Implantation Outcomes and Mechanisms of Action of Intracavernous Cord Mesenchymal Stem Cells in Patients with Erectile Dysfunction In Type 2 Diabetes Mellitus”. This study adheres with the Declaration of Helsinki. Participants provided written informed consent for their involvement in the study. The clinical study was registered at ClinicalTrials.gov (NCT04972890; 2021-07-06) (https://clinicaltrials.gov/study/NCT04972890?term=NCT04972890&rank=1), operated by the US National Institute of Health. When the trial first started, the authors of this study did not know about the policy of the International Committee of Medical Journal Editors (ICMJE), which requires all clinical trials to be registered prospectively. As soon as the authors became aware of this policy, this trial was immediately registered. The questionnaire and example consent form can be found as Extended data.^38,39 This study adheres to the CONSORT guidelines.^40,41

Participants and randomization

The study included sexually active men aged 18-65 who met the following criteria: they agreed to participate, had ED (as indicated by an IIEF-5 score of less than 22), had type 2 DM, and were not taking PDE-5 inhibitors. Patients with psychopathologies or mental retardation, a history of Peyronie’s disease, cerebrovascular disease, cancer, and moderate to severe risk of cardiovascular disease were excluded from the study. Subjects were randomly assigned to either the stem cells or placebo groups in a 1:1 ratio using simple random sampling with computer-generated random numbers. The investigators, treating clinicians, and subjects were all blinded.

MSC preparation

The MSC product was manufactured and distributed by the Stem Cells Medical Technology, Integrated Service Unit of the Faculty of Medicine at Universitas Indonesia – Dr. Cipto Mangunkusumo in Jakarta, Indonesia. The stem cell harvesting and culture stages were conducted following the methodology developed by Pawitan et al.,⁹ which utilizes xeno-free materials. Mesenchymal stem cells as much as 15 x 10⁶ MSC/2cc NaCl 0.9% were put into two sterile 1cc syringes. While a placebo was prepared using 2 cc of NaCl 0.9% solutions only. NaCl 0.9% solutions were supplied by Satoria Pharma, with an e-catalogue numbered “3526099002-OPA-034149472”.

Stem cell transplantations/intracavernosal injection

After completing mandatory screening, eligible participants received educational instruction from the research team regarding the procedure and the associated benefits, risks, and potential complications. Participants will be encouraged to inquire and articulate their comprehension of the research objectives and possible outcomes. At baseline, intracavernosal injections of either a placebo or stem cells were administered bilaterally in the penis. The patients were unaware of the intervention they received.

The patient underwent a one-hour post-procedure evaluation to determine the presence of complications before discharge. Side effect monitoring, including allergic reactions and pain, is conducted for 24 hours. Subjects were instructed to promptly notify the researcher of pain, swelling, bleeding, or any postoperative issues.

Follow-up

Every study participant received the same level of care. The participants received a standard treatment of PDE-5 inhibitors (specifically tadalafil) at a dosage of 1×2.5 mg for three months. A follow-up assessment was conducted on all experimental and control group participants at baseline, month 1, and month 3. The initial evaluation consisted of the IIEF-5 questionnaire, complete blood count, lipid profile, HbA1c, fasting blood glucose, total testosterone, and Doppler ultrasound. Testosterone and full blood count tests were conducted to rule out hypogonadism and other potential factors. The evaluation in the first and third months included administering the IIEF-5 questionnaire, lipid profile assessment, HbA1c and fasting blood glucose levels measurement, and Doppler ultrasound.

Outcomes

The study aimed to assess the efficacy and safety of MSC in treating ED in diabetic patients. The effectiveness of the intervention was assessed using the IIEF-5 questionnaire and PSV measurement throughout the follow-up period. Secondary objectives assessed lipid profile, HbA1c, and fasting blood glucose. Safety measures encompassed adverse effects, vital signs, and laboratory analysis.

Statistical analysis

The data were analysed using IBM SPSS Statistics version 25 software for Mac, where a copyright license has been obtained.¹⁰ Free alternative software include JASP, Jamovi and Sofa Statistics. Group comparisons were conducted using either the independent t-test or the Mann-Whitney test. Paired T-tests were used to compare individuals within the same group across different timelines. A repeated measures analysis of variance (ANOVA) was conducted to compare two groups at specific time intervals. A significance level of p<0.05 was used for all tests. The primary exploratory p-value was used to compare the means between the baseline and 3rd month of follow-up for most outcomes.

Recruitment

Between October 20, 2020, and January 20, 2023, 132 patients were screened for potential inclusion in the study. Ninety-six patients were excluded from the study due to their high risk of cardiovascular disease, history of cerebrovascular disease, cancer, or advanced age. The remaining ten patients opted out of participation due to concerns regarding genital injections. Therefore, the remaining 26 patients were randomly assigned to either the stem cells or the placebo group. Thirteen participants in the intervention group were administered stem cell therapy along with oral tadalafil, while another thirteen participants received a placebo along with oral tadalafil. One participant in the placebo group withdrew from the study due to an allergic reaction characterized by widespread skin rashes and itching, which was suspected to be caused by an allergy to tadalafil. Another patient was lost to follow-up. The provided diagram, labelled as Figure 1, is presented for reference. The patients were analysed according to their randomly assigned groups (intention-to-treat analysis). The IIEF-5 and laboratory comparison analysis included 24 patients, while the radiology analysis excluded two patients (one from each intervention group) due to incomplete data.

Figure 1. CONSORT flow diagram.

Baseline characteristics

Table 1³⁷ shows no statistically significant difference between groups in age, BMI, T2DM duration, ED duration, IIEF-5 value, fasting blood glucose (FBG), HbA1c, testosterone level, lipid profiles, and PSV dorsal artery.

Primary outcomes

Changes in erectile function

Erectile function was assessed subjectively using the IIEF-5 questionnaire and objectively measured through the PSV of the dorsal artery. There was a statistically significant improvement in IIEF-5 scores (p<0.001) at month 3 compared to baseline in both the placebo and stem cell groups. Both groups did not show a statistically significant difference (p>0.05). Table 2 presents the relevant data. Both the stem cells and placebo groups demonstrated a gradual improvement in the IIEF-5 parameter [Figure 2].

Figure 2. Mean values of erectile function assessed by IIEF-5 questionnaire.

Erectile function was objectively evaluated using a Doppler ultrasound examination to measure peak PSV in the dorsal artery. There was no statistically significant difference in PSV between the placebo and stem cell groups (p>0.05) [Figure 3]. Table 2 presents the relevant data.

Figure 3. Mean values of PSV across the periods.

Secondary outcomes

The analysis revealed no statistically significant variations in HbA1c, FBG, total cholesterol, HDL, LDL, and triglyceride levels between the placebo and the stem cell groups during the follow-up periods. Table 2 presents the relevant data. In both groups, inconsistent improvement trends were observed across the follow-up periods for the parameters mentioned [Figure 4, Figure 5, Figure 6, and Figure 7].

Figure 4. Mean of Fasting Blood Glucose Levels across the periods.

Figure 5. Mean of LDL Levels across the periods.

Figure 6. Mean of Total Cholesterol Levels across the periods.

Figure 7. Mean of HDL Levels across the periods.

Adverse events

Three subjects (11%) experienced a minor adverse event. No complications or significant adverse events were observed after the intervention in either treatment group. The minor adverse event observed was post-injection pain, with a Visual Analogue Scale (VAS) rating ranging from 3 to 6. The pain typically lasted for approximately 3 to 5 days following the injection. One patient experienced atypical penile pain for up to 5 days following the injection, with no subsequent adverse effects. No urination, bleeding, or inflammation issues were observed following the injection. No patients experienced aggravated erectile function during the follow-up period. We observed a patient who experienced an allergic reaction, characterized by a skin rash and itch symptoms, following a 5-day treatment of tadalafil 2.5 mg. The drug was discontinued on day 10, and the symptoms resolved three weeks after discontinuation. Therefore, the patient was excluded from the analysis due to incomplete follow-up.

Characteristics of subjects

The sample size in this study exceeded that of Bahk et al.’s 2010¹¹ study on umbilical cord mesenchymal stem cells, which included 10 subjects (7 in the experimental group and 3 in the control group). This study had a larger sample size than other clinical studies examining the efficacy of different stem cells in treating erectile dysfunction.^12–15

Bahk et al.¹¹ analyzed subjects with a mean age of 69.5 years (age range 57-87 years). The age of the subjects in that study differed from this study (51.08 ± 8.9 years in the stem cell group; 49.77 ± 11.18 years in the control group) because most of the subjects involved were elderly.

In this study, the duration of type 2 diabetes mellitus (DM) ranged from 12 to 52 years, with a mean duration of 29.4 years. However, in the stem cell group, the duration of type 2 DM was significantly shorter, with a mean duration of 8.27 ± 6.91 years. Similarly, in the placebo group, the mean duration of type 2 DM was only 6.08 ± 4.071 years. The study encompassed all patients with varying degrees of erectile dysfunction, regardless of their response to prior medical treatment. Meanwhile, Bahk et al.¹¹ recruited patients who had not responded to previous medical treatments (e.g., oral phosphodiesterase-5 inhibitors, prostaglandin E1 injections) for at least 6 months. All participants were in the process of receiving a penile prosthesis.

Changes in IIEF-5 questionnaire score

This study utilized a truncated version of the International Index of Erectile Function (IIEF-5) comprising five items to assess the occurrence and intensity of erectile dysfunction (ED). The five items were chosen based on their capacity to determine the presence of erectile dysfunction (ED), aligning with the definition of ED established by the National Institute of Health. The study examined the impact of various factors on erectile function and sexual satisfaction. The IIEF-5 was created and published by Rosen et al.¹⁶ This study utilized a truncated version of the International Index of Erectile Function (IIEF-5) comprising five items to assess the occurrence and intensity of erectile dysfunction (ED). The five items were chosen based on their capacity to determine the presence of erectile dysfunction (ED), aligning with the definition of ED established by the National Institute of Health. The study examined the impact of various factors on erectile function and sexual satisfaction. The IIEF-5 was created and published by Rosen et al.¹⁶ as a valid and straightforward diagnostic tool for clinical use. Laksita et al.¹⁷ has demonstrated the validity and reliability of the Indonesian IIEF-5 as a patient-reported outcome measure for assessing erectile dysfunction (ED) in illiterate middle-aged and adult men in Indonesia.

This study did not observe any statistically significant differences between the group receiving stem cells and the group receiving a placebo. Both groups showed an improvement in the IIEF-5 score during the follow-up phase. Adding stem cell injection did not yield significantly different outcomes in the treatment. This study presents contrasting findings compared to Bahk et al.¹¹ observed a beneficial impact of intracavernous umbilical cord mesenchymal stem cells on ED in patients with type 2 DM despite not achieving a level of sexual function considered normal. Most (85.7%) of the experimental group observed increased penile hardness. Nevertheless, this heightened level of hardness remains inadequate for achieving effective penetration. Two subjects were able to achieve penetration, maintenance, and orgasm during sexual intercourse with the use of a PDE-5 inhibitor (sildenafil citrate 100 mg). These abilities were maintained by the subjects even after five months of treatment. The occurrence of penile erections facilitated by PDE5 inhibitors involves a complex interplay of mechanisms, with the indirect involvement of nitric oxide (NO) being a crucial factor that should not be disregarded.¹¹

Erections are initiated by the release of NO from sinusoidal endothelial cells. Vasodilation induced by NO leads to venules being compressed between trabeculae and the tunica albuginea, resulting in blood retention within the blood vessels.¹⁸ Endothelial cells, cavernous smooth muscle cells, NO, and nerve cells play crucial roles in achieving an erection. However, DM has a detrimental effect on these components. Stem cells possess the capacity to differentiate into specific cell types, suggesting their potential for repairing damage and reinstating normal erectile function. Nevertheless, the precise mechanisms in this process still need to be understood and require additional investigation. This study employed unaltered human umbilical cord blood stem cells. Research has demonstrated that injecting mesenchymal stem cells modified with endothelial nitric oxide synthase (eNOS) into the corpus cavernosa of aged mice is more effective in improving ED.^19,20

Similar to Bahk et al., this study administered around 15 million stem cells per patient. The quantity of stem cells obtained from 1 unit of umbilical cord blood is significantly lower than that obtained from adipose tissue or bone marrow. Hence, the limited number of stem cells employed in the treatment could explain the absence of substantial improvement observed between the experimental and control groups. Ning et al.²¹ reported that adipose tissue-derived stem cells could localize on the sinusoidal endothelium and form tube-like structures. This localization and formation were believed to be facilitated by fibroblast growth factor-2 (FGF-2). Lin et al.²² found that the IGF/IGFR pathway is involved in neuronal differentiation, and the FGF-2 pathway is involved in endothelial differentiation. Hence, the improvement of erectile dysfunction (ED) in diabetic patients observed in this study may be attributed to the activation of endogenous stem cells in the cavernous tissue. This activation can be facilitated by humoral factors or factors secreted by the stem cells themselves.

Changes in radiology parameters

This study observed no significant difference in PSV between the stem cell treatment and control groups. No significant increase was observed in the analysis of PSV in groups when considering examination time. The color Doppler ultrasound examination of the penile vessels revealed that the average PSV of the dorsal artery in the intervention group was 16.64 ± 5.75, 21.3 ± 9.25, and 18.6 ± 8.46 at baseline, one month, and three months post-injection, respectively. This finding exhibits non-significant variations (p-value = 0.427). The mean PSV in the control group was 14.18±4.93, 18.93±11.22, and 18.6±8.46 at baseline, first month, and third month after injection, respectively. These values exhibited varying fluctuations but were not statistically significant (p = 0.355). The two groups had no significant difference in PSV (p = 0.990). The results of this study contrast with the findings of Levy et al.¹¹ observed a statistically significant rise in penile blood flow following the administration of chorionic placental stem cells. However, it should be noted that not all participants achieved satisfactory erections without the use of oral medications. Levy et al. conducted a study that examined the same treatment in individuals with Peyronie’s Disease.²³

Mirzaei et al.²⁴conducted a study with a comparable sample size of 20 subjects, utilizing oral mucosal mesenchymal stem cells. Their findings were consistent with the present study. Stem cell injection is thought to significantly affect the PSV index in penile Doppler ultrasound, which serves as a potential indicator of enhanced sexual function in patients. The potential enhancement of erectile function in diabetic patients may be attributed to the capacity of stem cells to differentiate into endothelial cells, leading to an increase in angiogenic tissue within the penis.²⁵ It is crucial to acknowledge that studies with a limited number of participants are likely to yield substantial improvements in PSV, leading to the need to consider the impact of small study effects on research.

Changes in other parameters

No significant changes in fasting blood sugar (GDP), HbA1C, and lipid profiles were observed in either group in this study. In contrast to the study conducted by Bahk et al.,¹¹ which utilized umbilical cord stem cells, the observed outcome of the present study was a reduction in GDP levels after a two-week treatment period. The experimental group patients maintained low blood glucose levels for 4 to 7 months compared to their levels before treatment. The experimental group demonstrated more consistent improvements in their DM conditions than the control group. Bahk et al.¹¹ hypothesized that infused stem cells may spread systemically through the bloodstream, supporting Min’s hypothesis.²⁶ They also predicted that these stem cells could migrate to other organs, such as the pancreas, positively affecting blood glucose levels. Ohta et al.²⁷ discovered that autologous adipose mesenchymal stem cells significantly impacted HDL, LDL, and remnant-like particle (RLP) cholesterol levels. The study concluded that these stem cells may help prevent the formation of macrophage foam cells.

Side effects

The study found no serious side effects during a three-month follow-up after stem cell injection. These findings align with the research results with various other types of stem cells. Bahk et al.,¹¹ Ichim et al.,²⁸ Garber et al.,²⁹ and Yiou et al.³⁰ Yiou et al.³⁰ noted a decrease in haemoglobin due to bone marrow stem cell aspiration, but blood transfusion was not required—the study by Haahr et al.¹³ reported mild effects at the injection site (redness, swelling, hematoma of the scrotum and penis) or at the liposuction site, all of which resolved on their own. Levy et al.¹² also reported mild irritation at the injection site that resolved within 48 hours. Al Demour et al.¹⁴ reported only minor side effects related to aspiration and injection site, with no side effects associated with the nervous, cardiovascular, respiratory, and gastrointestinal systems during two years of follow-up. Protogerou et al.³¹ also reported only minor side effects related to the injection site.

Factors affecting outcomes

Men with a diagnosis of type 2 DM often experience ED at an earlier stage, typically occurring 10 to 15 years earlier than in the general population.³² Additionally, individuals with this condition exhibit more severe symptoms of ED and experience a more significant decline in their overall quality of life when compared to the general population.³³ Moreover, ED resulting from DM tends to pose more significant challenges in terms of treatment with phosphodiesterase 5 (PDE-5) inhibitors.³⁴ Binmoammar et al. conducted a systematic review that examined five cross-sectional studies. Their findings indicate that patients with type 2 DM who have poor glycaemic control are more likely to experience ED.³⁵

Understanding the factors that influence response rates to PDE-5 inhibitors, which are common medications for the treatment of ED, is critical to maximizing treatment outcomes and exploring alternative therapies such as stem cell therapy. A cohort study evaluating individual response rates to PDE-5 inhibitors showed an overall response rate of 43%.³⁶ However, response rates may vary depending on the cause of ED, with veno-occlusive cases showing a higher response rate and neurogenic causes leading to a lower response rate. Several factors have been identified as potential predictors of response rate to PDE-5 inhibitors. These factors include age, smoking habits, DM, hypertension, hyperlipidaemia, IIEF-5) score before treatment, time interval to achieve erection, and duration of erection. Univariate analysis showed that these factors significantly influenced the response to PDE-5 inhibitors. However, in multivariate analysis, age and IIEF-5 score before treatment remained significant predictors.³⁶

Age has been identified as a significant factor in influencing response to PDE-5 inhibitors, with older individuals tending to experience decreased response due to physiological changes that occur with age. Smoking habits, which are known to have a negative impact on heart health, also emerged as a predictor. Smoking can cause damage to blood vessels, which may reduce the effectiveness of PDE-5 inhibitors in improving erectile function.³⁶ The pretreatment IIEF-5 score, which reflects erectile function before starting treatment, is also an important predictor. Individuals with lower baseline scores may experience a more significant increase in response to PDE-5 inhibitors. It should be noted that the predictor factors identified in this study likely also apply to response to stem cell therapy, which is a promising approach in the treatment of various conditions, including erectile dysfunction.³⁶ Factors such as age, smoking habits, and baseline erectile function, which affect the response to PDE-5 inhibitors, may also affect the effectiveness of stem cell therapy.

In this study, there were insignificant findings related to the outcomes observed. This may be due to the advanced age of the research subjects involved. In addition, relatively low pretreatment IIEF-5 scores may explain the nonsignificant findings.

Strength and weakness

This study is the first in Indonesia to assess the efficacy of stem cell therapy in treating ED in patients with type 2 DM. To mitigate research bias, this study utilized a clinical trial design with an intervention group and a control group (double arm). Nevertheless, this study has various limitations. Currently, the assessment of this condition relies on a limited set of parameters, including the IIEF-5 score for subjective evaluation, radiological parameters, and laboratory data such as blood sugar and lipid profiles. Molecular and histochemical parameters are challenging to analyse due to the lengthy specimen processing time. Furthermore, the current number of subjects falls below the minimum requirement due to challenges in recruiting eligible and willing participants for this study. Thus, the present analysis results need more research power.

Limitations

With time and funding limitations, the follow-up was short.

Further prospective studies should implement longer follow-ups.

The inclusion criteria could not guarantee that the participants got ED due to DM.