Human umbilical cord platelet-rich plasma to treat endometrial pathologies: methodology, composition and pre-clinical models

Abstract

Study question: Can human umbilical cord platelet-rich plasma (hUC-PRP) efficiently treat endometrial damage and restore fertility in a preclinical murine model?

Summary answer: Local application of hUC-PRP promotes tissue regeneration and fertility restoration in a murine model of Asherman syndrome and endometrial atrophy (AS/EA).

What is known already: AS/EA are well-described endometrial pathologies that cause infertility; however, there are currently no gold-standard treatments available. Recent reports have described the successful use of human platelet-rich plasma in reproductive medicine, and its regenerative potential is further enhanced using hUC-PRP, due to the ample growth factors and reduced pro-inflammatory cytokines in the latter.

Study design size duration: hUC-PRP (n = 3) was processed, characterized and delivered locally to endometrial damage in a murine model (n = 50). The hUC-PRP was either used alone or loaded into a decellularized porcine endometrium-derived extracellular matrix (EndoECM) hydrogel; endometrial regeneration, fertility outcomes and immunocompatibility were evaluated 2 weeks following treatment administration.

Participants/materials setting methods: Umbilical cord blood was obtained from women in childbirth. Endometrial damage (mimicking AS/EA) was induced using ethanol in 8-week-old C57BL/6 mice, and treated with the most concentrated hUC-PRP sample 4 days later. Characterization of hUC-PRP and immunotolerance was carried out with multiplex technology, while uterine samples were analyzed by immunohistochemistry and quantitative PCR. The number of embryos and their morphology was determined visually.

Main results and the role of chance: Platelet density was enhanced 3-fold in hUC-PRP compared to that in hUC blood (P < 0.05). hUC-PRP was enriched with growth factors related to tissue regeneration (i.e. hepatocyte growth factor, platelet-derived growth factor-BB and epidermal growth factor), which were released constantly (in vitro) when hUC-PRP was loaded into EndoECM. Both treatments (hUC-PRP alone and hUC-PRP with EndoECM) were immunotolerated and caused significantly regeneration of the damaged endometrium, evidenced by increased endometrial area, neoangiogenesis, cell proliferation and gland density and lower collagen deposition with respect to non-treated uterine horns (P < 0.05). Additionally, we detected augmented gene expression of Akt1, VEGF and Ang, which are involved in regenerative and proliferation pathways. Finally, hUC-PRP treatment restored pregnancy rates in the mouse model.

Large scale data: N/A.

Limitations reasons for caution: This proof-of-concept pilot study was based on a murine model of endometrial damage and the use of EndoECM requires further validation prior to clinical implementation for women affected by AS/EA.

Wider implications of the findings: The local administration of hUC-PRP has high impact and is immunotolerated in a murine model of AS/EA, as has been reported in other tissues, making it a promising candidate for heterologous treatment of these endometrial pathologies.

Study funding/competing interests: This study was supported by the Ministerio de Ciencia, Innovación y Universidades; Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital, Generalitat Valenciana; and Instituto de Salud Carlos III. The authors do not have any conflicts of interest to declare.

Keywords: endometrium; extracellular matrix hydrogel; human umbilical cord; platelet-rich plasma; regenerative medicine.

Figure 1.

Study design. (A) The hUC-PRP was generated by collecting hUC blood from women in childbirth, isolating the PRP fraction via commercial (closed) and manual (open) systems, and selecting the most concentrated sample. (B) In vitro hUC-PRP assays included the characterization of plasma components, the loading of the hUC-PRP into the EndoECM, and a comparison of the releasing kinetics of hUC-PRP with or without EndoECM. (C) To analyze the efficacy of hUC-PRP as a preclinical treatment, we studied the immunocompatibility of EndoECM and hUC-PRP, endometrial regeneration (n = 20) and fertility outcomes of a murine model (following natural mating; n = 30) of endometrial damage. α-sma, actin alpha 2, smooth muscle; Akt1, AKT serine/threonine kinase 1; Ang, angiogenin; EndoECM, decellularized porcine endometrium-derived extracellular-matrix hydrogel; hUC, human umbilical cord; hUC-PRP, platelet-rich plasma from the hUC; hUC-PPP, platelet-poor plasma from the hUC; VEGF, vascular endothelial growth factor.

Figure 2.

hUC-PRP characterization, composition and in vitro releasing kinetics. (A) Comparison of platelet enrichment using commercial and manual methods for PRP extraction. Platelet enrichment was defined as the number of platelets in the hUC-PRP, divided by the number of platelets in whole hUC blood. The most enriched sample (indicated with an arrow) was selected for subsequent analyses. (B) Comparison of platelet density in hUC-PRP, hUC-PPP and whole hUC blood. (C) Predominant results from the multiplex protein assay for cytokines, chemokines and growth factors in hUC-PRP. (D) Comparative heat-map of protein differences found among hUC-PRP and hUC-PPP extracted with a commercialized system, PRP from adult peripheral blood and hUC-PRP extracted with double centrifugation. (E–G) Releasing kinetics of (E) HGF, (F) PDGF-BB and (G) EGF in EndoECM + hUC-PRP, EndoECM and hUC-PRP conditions. The cumulative release was defined as the cumulative concentration released at each point, with respect to the total concentration released on day 14. Data in A–D are presented as a mean of three replicates ± SD. *P < 0.05. EGF, epidermal growth factor; EndoECM, decellularized porcine endometrium-derived extracellular-matrix hydrogel; HGF, hepatic growth factor; hUC, human umbilical cord; hUC-PRP, platelet-rich plasma from hUC; hUC-PPP, platelet-poor plasma from hUC; IP10, C-X-C motif chemokine 10; MIP-1β, C-C motif chemokine 4; PDGF-BB, platelet-derived growth factor-BB; SCF, stem cell factor; SDF1α, stromal cell-derived factor 1 A; VEGF A, vascular endothelial growth factor.

Figure 3.

hUC-PRP immunocompatibility. Concentration of (A) IL-5, (B) IL-6, (C) TNFa and (D) IFNg in murine plasma collected 14 days following administration of saline, hUC-PRP or EndoECM + hUC-PRP, or a sham intervention, n = 5 per group. Data are presented as a mean of three replicates ± SD. P < 0.05. EndoECM, decellularized porcine endometrium-derived extracellular-matrix hydrogel; hUC-PRP, platelet-rich plasma from hUC; IFNg, interferon gamma; IL-5, interleukin 5; IL-6, interleukin 6; TNFa, tumor necrosis factor alpha.

Figure 4.

Histological analysis of the damaged murine endometrium 14 days post-treatment. Representative cross-sections of murine uterine horns that were either damaged with ethanol and treated with saline, hUC-PRP or EndoECM + hUC-PRP, or manipulated without causing any damage (sham). Tissue regeneration was evaluated using Masson’s Trichrome staining for (A) endometrial area, (B) gland density and collagen deposition, (C) Ki-67 immunostaining and (D) GSL I (green) and α-sma (red) double immunostaining of new (green) and mature (red and green) blood vessels in the endometrium. Scale bars are set to 500 µm (A) or 100 µm (B to D). Histological quantification of (E) endometrial area, (F) gland density, (G) fibrosis, (H) proliferation and (I) neoangiogenesis. *, P < 0.05; **, P < 0.01; EndoECM, decellularized porcine endometrium-derived extracellular-matrix hydrogel; hUC-PRP, platelet-rich plasma from human umbilical cord blood.

Figure 5.

Molecular analysis of the damaged murine endometrium 14 days post-treatment. Murine uterine horns were damaged with ethanol and treated with saline, hUC-PRP or EndoECM + hUC-PRP, and mRNA was extracted. Relative gene expression of aSMA (A), Akt1 (B), VEGF (C) and Ang (D). Data were normalized with respect to the gene expression from the sham group and presented as a mean ± SD. For all the experiments, n = 5 per group. P < 0.05 was considered statistically significant. *, P < 0.05. Akt1, thymoma viral proto-oncogene 1; Ang, angiogenin; aSMA, actin alpha 2, smooth muscle, aorta; EndoECM, decellularized porcine endometrium-derived extracellular-matrix hydrogel; hUC-PRP, platelet-rich plasma from human umbilical cord blood; VEGF, vascular endothelial growth factor.

Figure 6.

Achievement of pregnancy 14 days following unilateral treatment to the uterine horns of mice with ethanol-damaged endometrium. Fertility restoration was assessed by (A) pregnancy rate (number of embryos per horn by total number of embryos), (B) number of embryos per horn, (C) embryo weight and (D) representative images of gestational uterine horns and embryo morphology, 14 days after confirmed mating by vaginal plug (stage E14.5 of embryo development). Right uterine horns were treated with saline, hUC-PRP or EndoECM + hUC-PRP, while contralateral left horns only underwent sham surgery. (A–C) Data are presented as mean ± SD, n = 10 per group. A statistical significance of P < 0.05 is indicated by the asterisk. hUC-PRP, platelet-rich plasma from human umbilical cord; EndoECM, decellularized porcine endometrium-derived extracellular-matrix hydrogel.