First use of cord blood platelet-rich plasma in the treatment of vulvar lichen sclerosus: a preliminary study towards a randomized controlled trial

Vulvar lichen sclerosus (VLS) is a chronic inflammatory condition affecting the anogenital skin in both children and adult females 1 , 2 . Its prevalence among fertile women varies from 1 in 60 to 1 in 1,000 among adults and children in the United States. Mean age at time of diagnosis is 32 years, while symptoms appear at a mean age of 27 3 . Due to its nonspecific signs and symptoms, this condition is often underdiagnosed. Literature suggests VLS is not rare but rather a rarely diagnosed condition, as only 16% of patients are diagnosed at an early stage 4 . The main symptoms of VLS in adult females include vulvar and perianal pruritus , burning due to skin fissuring, dysuria, and sexual disfunction 5 , 6 . As what regards its pathogenesis, evidence suggests that dysregulated Th-1 and T-reg responses contribute to chronic inflammation, leading to tissue damage and activation of fibroblast pathways. The resulting inflammatory environment, characterized by the release of cytokines and reactive oxygen species, may increase the risk of vulvar malignancy by deactivating tumor suppressor genes and promoting cell proliferation 7 , 8 . Potent and ultra-potent topical corticosteroids (TCS) are crucial in the management of VLS 9 – 11 and are considered first-line treatment. Retrospective studies report that the rates of complete response to such treatment, defined as the total absence of symptoms and signs of the disease, may be as low as 30 to 50% 12 – 14 . Many patients experience only a partial response to TCS, due to residual sexual dysfunction and skin anelasticity and atrophy. In the recent years, there has been an increasing interest in exploring regenerative therapies as complementary treatments for VLS, including the use of platelet-rich plasma (PRP) and lipofilling 15 . In our recent study, we found that vulvar autologous PRP injections significantly improved patient satisfaction and symptoms in women with VLS who did not achieve complete remission of signs and symptoms following first line TCS treatment 16 . PRP, which is obtained via centrifugation of whole blood, consists in a concentrated platelet solution enriched with growth factors (GF2), inflammatory cells, and chemical mediators 17 , 18 . Its therapeutic efficacy relies on GFs such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), and nerve growth factor (NGF) 18 – 20 . The injection of PRP in injured sites increases the local concentration of GFs, cytokines, lysosomes, and adhesion proteins, initiating tissue repair processes 21 , 22 . These mechanisms, along with PRP’s immunomodulatory properties, support its use in the treatment of VLS 21 , 23 – 26 . However, allogeneic preparation from adult blood donations or cord blood units not available for hemopoietic transplant are possible alternatives 27 , 28 . In the present study, we decided to focus on allogeneic cord blood PRP (CB-PRP) rather than autologous PRP (PB-PRP) or adult donor PRP due to several advantages of this preparation. CB-PRP provides additional therapeutic benefits compared to PB-PRP due to its elevated levels of anti-inflammatory molecules 29 , 30 . It also addresses concerns regarding product quality related to patient age and associated comorbidities, which can affect PB-PRP. Additionally, CB-PRP offers lower production costs and is simpler to produce, as it is accessible from cord blood banks 30 . This pilot study is part of a randomized controlled trial aimed at comparing the effectiveness of CB-PRP with that of placebo (physiological saline solution) in the improvement of patient satisfaction in women with a partial response to standard treatment with TCS. The aim of the pilot study was to evaluate the safety and the tolerability of the treatment in this specific population. The secondary aim was to evaluate patient satisfaction, symptom improvement, sexual function, psychological well-being, quality of life, frequency of application of TCS as a maintenance treatment, vulvar trophism and architectural modifications following vulvar CB-PRP injections.

A total of 10 patients with a median age of 47 years [IQR: 31–53] were included in the pilot study. Diagnosis of VLS was histological in 9 cases ( Table I ). Nine/ten patients underwent three treatments with PRP, while one patient abandoned the study after the first vulvar injection of CB-PRP and was lost to follow-up. Therefore, results (apart from patient satisfaction) were analyzed for the remaining 9 patients who completed both treatment and follow-up. As what regards treatment tolerability, the mean pain score at time of needle insertion was 4,4 [median: 4; IQR 4–5] on an NRS scale, and the average NRS score for burning during PRP injection was 7.3 [median: 7; IQR: 6,75–8]. No moderate/severe or unexpected adverse reactions were observed in any of the participants. Three months after the last injection of PRP, five out ten (55.5%) patients were either satisfied or very satisfied with the treatment (55%, 95% CI: 24–76%), the remaining four patients (44.4%) declared themselves uncertain about their satisfaction with the treatment. None of the patients declared themselves unsatisfied. Patients’ global impression of change was improved in seven out of nine patients (78%). Patient symptoms at the time of enrollment and after treatment are reported in Table II . At time of enrollment, itching (89% of patients) and dyspareunia (88%) were the most prevalent symptoms, with median NRS scores of 6 [IQR: 2.5–7.5] and 6 [IQR: 2–10], respectively. Dyspareunia was the most severe symptom, while dysuria was not reported by any of the patients. Three months after treatment, median burning NRS scores were significantly decreased (p<0.05) ( Table II ). A significant improvement in sexual arousal and satisfaction (p<0.05) was observed in all women who reported having a sexual partner both before and after treatment. A positive trend was observed also for some of the remaining items of the FSFI questionnaire, i.e., lubrification and orgasm, although the difference didn’t reach significance ( Table II ). Regarding psychological wellbeing measures, the HADS questionnaire failed to show a statistically significant improvement in anxiety and depression post-treatment. Similarly, the SF-12 questionnaire did not yield a statistically significant improvement in physical health nor in mental wellbeing (MCS) following treatment ( Table II ). As what regards steroid treatment, at time of enrollment four out of nine patients (44.4%) were using ultra-potent TCS “on-demand”, two (22.2%) applied TCS twice a week and three (33.3%) applied the steroid cream more than twice a week. All patients also used vitamin E-based moisturizers daily. Following PRP treatment, patients were encouraged to continue using TCS “on demand”, i.e. when symptomatic. We observed a 55.5% absolute reduction (p=0.014) in the number of patients requiring maintenance treatment. Additionally, those who continued TCS use reduced its application frequency ( Table III ). No cases of vulvar intraepithelial neoplasia (VIN) or invasive vulvar cancer occurred during follow-up time. Vulvar skin elasticity and nourishment were improved in all patients and no significant differences in vulvar architecture were described ( Figure 3 ).

To our knowledge, this is the first human study evaluating treatment with CB-PRP in patients with VLS. In Italy, the Ministry of Health authorizes the use of cord blood components for non-transfusion purposes. Platelet-rich plasma is commonly used in its autologous form for various therapeutic applications 17 , 39 , 40 including VLS (level of evidence 2c according to the Italian National Blood Centre recommendations) 17 , 39 , 40 . Due to their regenerative and potentially anti-inflammatory properties, PB-PRP and CB-PRP could in fact represent valid therapeutic options in patients with VLS who do not achieve an optimal response with first-line TCS in terms of symptom resolution and functional restoration. The use of PB-PRP in clinical practice is limited by the variability in source materials and processing methods, which compromise the possibility of standardized final products. Moreover, in some cases, such as patients with hematological malignancies or elderly patients with coagulation disorders, it is contraindicated to obtain autologous PRP 41 . Such limitations may be overcome by the use of CB-PRP. CB-PRP has been studied less extensively than PB-PRP although, according to Rajesh Mani and co-workers, CB-PRP’s therapeutic effect is superior to that of PB-PRP, due to its increased concentration of growth factors, including PDGF-BB, FGF2, VEGF. Additionally, CB-PRP directs macrophage polarization from an M1 phenotype, which is pro-inflammatory, towards an M2 phenotype, which is associated with tissue regeneration 42 . Longo and colleagues compared adult blood platelet gel (ABPG) with cord blood-derived platelet gel (CBPG) and found that CBPG has a unique proteome composition linked to re-epithelialization and wound healing, while ABPG contains more pro-inflammatory components 43 . Moreover, CBPG’s production may be standardized and as such its composition is less variable than that of ABPG. The primary outcome of this pilot study was to assess the safety and tolerability of CB-PRP in treating VLS. The results indicate that CB-PRP is a safe treatment option, as no adverse effects such as allergic reactions, hematomas, or systemic side effects were observed. The treatment was generally well tolerated, with a mean scoring of 4 on the NRS scale for needle insertion pain scoring and an average scoring of 7 for burning sensation during injection. This is consistent with previous studies that reported minimal adverse effects following PRP treatments in other contexts. Patient satisfaction was high, with 55% of patients reporting being satisfied or very satisfied with the treatment, while the remaining 44% were uncertain but not dissatisfied. This improvement in satisfaction can be attributed to the significant reduction in median burning NRS scores (p<0.05) three months after treatment. These results are consistent with the literature, where PRP treatments have shown to alleviate symptoms effectively in various clinical studies 16 , 23 – 25 , 44 – 50 . A significant improvement in both sexual arousal and satisfaction, as measured by the FSFI questionnaire, was observed among patients who had a sexual partner at time of treatment. Other FSFI items such as lubrication and orgasm showed a positive trend following treatment, although they did not reach statistical significance. This improvement in sexual function is particularly important given that standard VLS treatments often is insufficient in improving sexual dysfunction. The HADS questionnaire failed to show a statistically significant improvement in anxiety and depression scores, and similarly, the SF-12 did not reveal any significant improvements in physical or mental wellbeing following treatment. These findings may be due both to the small sample size and to the short follow-up time. Previous studies have highlighted the psychological burden of VLS 25 , 51 suggesting that long-term studies with larger cohorts are necessary to fully understand the impact of PRP treatments on psychological health. A notable finding of our study was the reduction in the need for maintenance TCS treatment. The percentage of patients requiring ongoing TCS use dropped from 89% to 33%. This data supports CB-PRP’s potential to enhance tissue regeneration and reduce inflammation, as seen in studies on autologous PRP 43 , 52 . The results of this pilot study are promising and align well with existing literature on the use of PRP in dermatological and gynecological conditions. The absence of adverse effects and high patient satisfaction reinforce the safety and efficacy of CB-PRP. However, it being a pilot study, this study is limited by its small sample size and its relatively short follow-up time.

In conclusion, CB-PRP appears to be a safe and potentially effective treatment for VLS, offering improvements in symptoms and sexual function while reducing the need for maintenance TCS therapy. These promising results justify further investigation in a larger randomized controlled trial to validate the efficacy and safety of CB-PRP in the treatment of VLS.

This prospective single-arm interventional pilot study is the first stage of a double-blind randomized placebo-controlled trial. The primary outcome of this pilot study was the safety and tolerability of CB-PRP in women with VLS. The entire trial (including this pilot phase) was approved by the local Ethics Committee (Milan 21/06/2022, Comitato Etico Milano Area 2) (protocol ID 2994, study number 6245). The entire clinical trial is registered at ClinicalTrials.gov with Unique Protocol ID: NCT06661382 . Patients were recruited between December 2022 and January 2023 at our Center of Vulvar Diseases, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy. Written informed consent was obtained from each participant/patient for study participation, image acquisition and data publication. Eligibility criteria included women aged 18 to 80 with a clinical or histological diagnosis of VLS who had partially responded to standard first-line three-month treatment with TCS. Exclusion criteria comprised presumed or confirmed pregnancy, coagulopathies, platelet disorders, cardiovascular and respiratory diseases, major infections, and prior vulvar surgery involving fat grafting or injection of PRP. Ten women were included in this pilot study (the randomized trial will be performed on a sample of 100 women, 50 of whom will undergo vulvar injections with umbilical cord PRP and 50 of whom will receive placebo-vulvar injections of normal saline; for a total sample of 110 patients). At time of enrollment, all patients were receiving maintenance treatment, consisting of TCS as needed. The sample size of 10 patients in this pilot phase was chosen to refine the procedure for the entire trial and to assess safety, as this is the first study using CB-PRP in VLS. Although small, this sample size was deemed sufficient to detect early safety signals and offer preliminary efficacy insights. No formal stopping rules were established due to the exploratory nature of the study, but the study would have been halted in case of serious adverse effects. Adverse events were classified as mild (self-limiting discomfort), moderate (requiring conservative treatment), or severe (requiring medical intervention or hospitalization). All patients underwent a pre-treatment clinical assessment by means of a vulvoscopy, a method which provides a magnified vision of the vulvar area. Vulvoscopy was aimed at ruling out pre-cancerous (VIN) or invasive lesions, evaluating vulvar tissue elasticity, and classifying vulvar architecture using the CIV classification 31 . Patients were also asked to complete the following self-administered questionnaires: a 0–10 numerical rating scale (NRS) was used to assess the intensity of pruritus , burning, dyspareunia, and dysuria (0 indicated absence of symptoms, while 10 denoted the most severe intensity imaginable) 32 ; the Female Sexual Function Index (FSFI) questionnaire was utilized to evaluate six dimensions of sexual function (desire, arousal, lubrication, orgasm, pain, satisfaction), each scored on a scale of 0–5 or 1–5. A total score of 26.55 or lower indicates sexual dysfunction 33 ; the Hospital Anxiety and Depression Scale (HADS) for the assessment of psychological well-being. It consists of two 7-item scales: one for anxiety (HADS-A) and one for depression (HADS-D). Each scale is scored from 0 to 21 with higher scores indicating poorer psychological conditions 34 ; the Short Form-12 item questionnaire (SF-12) for the assessment of the impact of health on patients’ well-being. It consists of a physical component summary score (PCS) and a mental component summary score (MCS). Higher scores indicate better well-being 35 . Additional information regarding menopausal status and frequency of TCS use was collected. CB-PRP was manufactured at the Transfusion Center of the IRCCS Ca’ Granda Foundation, Ospedale Maggiore Policlinico in Milan. Cord blood was obtained from donors recruited at the Mangiagalli Center’s delivery room during childbirth and stored in the Transfusion Center’s Cord Blood Bank. Eligible women for cord blood donation were healthy individuals without parenteral, sexual, or behavioral risk factors for transfusion-transmissible infections. They tested negative for markers of HIV, HCV, HBV, and syphilis and had successfully completed a full-term pregnancy without complications. Cord blood collection was performed by trained midwives immediately after childbirth using a sterile closed system with specialized collection bags. The process involved venipuncture of the umbilical cord into a sterile bag, allowing the blood to flow naturally ( Figure 1 ). On average, approximately 75 mL of Cord Blood (CB) was collected from each umbilical cord. Like peripheral blood, CB consists of leukocytes, erythrocytes, thrombocytes, and plasma. CB also contains various types of stem cells, including hematopoietic stem cells, which can be used for hematologic transplants. To ensure proper preservation, CB units were transported from the maternity ward to the Cord Blood Bank at a controlled temperature of 4°C and were processed within 48 hours. CB-PRP was prepared by specialized biologists in the Department of Transfusion Medicine and Hematology at our institution. The preparation was performed under sterile conditions, specifically in a Class A laminar flow setup. This ensures the utmost cleanliness and integrity of PRP samples, minimizing the risk of contamination and maintaining the highest standards of quality throughout the preparation process. Whole CB underwent a double centrifugation process, the former at a low speed and the latter at a high speed. During the first centrifugation, plasma and platelets were separated from red blood cells and leukocytes. During the second centrifugation, platelets were further concentrated to obtain PRP, and a portion of Platelet-Poor Plasma 36 ( Figure 2a ). The platelet pellet was then suspended in plasma ( Figure 2b ), in order to obtain a CB-PRP concentration of approximately 1,000×10 9 /L (optimal range between 800–1,200×10 9 /L). This process ensures the preparation of a highly concentrated PRP product for therapeutic use ( Figure 2c ). The median volume of CB-PRP was 3.65 mL (IQR, 1 mL) per preparation, and the median platelet count in the CB-PRP was 1,082×10 9 /L (IQR, 293.5×10 9 /L). CB-PRP was then securely stored in a designated freezer until the serological, nucleic acid and sterility tests conducted at the conclusion of the preparation process were concluded. Once all the test results were confirmed to be negative, the prepared unit was considered validated and approved for clinical use. This rigorous testing and validation process ensures the safety and quality of the CB-PRP unit before it is used for therapeutic purposes. Adherence to strict quality and safety requirements is of utmost importance in the preparation of blood components, including PRP. Improper processing procedures may in fact increase the risk of bacterial contamination and subsequent infections. The patients received one unit of CB-PRP per injection, with a total of 3 units of CB-PRP per patient. The injections were carried out by a gynecologist specialized in vulvar diseases. Before the procedure, a topical cream containing 2.5% lidocaine and 2.5% prilocaine was applied to the vulvar area to minimize pain. PRP was injected across the entire vulvar region using a 30-gauge needle in a clockwise pattern, targeting specific areas such as the clitoral hood, the labia minora, the interlabial sulci, the vestibule, the posterior fourchette, and the perianal area. The number of injections varied depending on PRP availability. The median number of injections per patient was 8, with a maximum of 10 injections. There was no observed correlation between the number of injections and either treatment efficacy or adverse effects. The treatment’s tolerability, measured by needle-insertion pain and burning sensation during PRP injection, was assessed using a 0–10 Numerical Rating Scale (NRS) 32 . Each patient was planned to undergo three treatments, spaced 4–6 weeks apart. Patients underwent a vulvoscopic examination 15 days after each PRP injection to detect potential adverse reactions such as infections and bruising. The final assessment occurred three months after the third injection. At this time, patients filled out the same set of questionnaires they had completed prior to treatment as well as the following: a patient satisfaction questionnaire, based on a 5-point Likert scale (from “1: very satisfied with the PRP procedure” to “5: very unsatisfied”); the Global Impression of Change questionnaire that provides patients’ subjective overall impression of change on a scale ranging from “very much worse” to “very much improved” 37 , 38 . Vulvoscopy was also performed to rule out the presence of pre-cancerous (VIN) or invasive lesions, evaluate the elasticity of vulvar tissues, and describe vulvar architecture using the CIV classification 31 . Data was prospectively collected in an Excel spreadsheet specifically created for this study. Statistical analyses were performed using SPSS 25.0 Statistics for Mac, IBM Corp. (Armonk, NY, USA). Continuous data, such as numerical measurements, is reported using means and standard deviations (SD) or medians and interquartile ranges (IQR), depending on the distribution of the data. Categorical data is presented as absolute values and percentages. Wilcoxon’s test (a non-parametric test for related samples) was used to compare self-reported intensity of symptoms, sexual function, psychological wellbeing, and quality of life at baseline and three months after treatment. A p value ≤0.05 was used to determine significance.