Hysteroscopic injections of autologous endometrial cells and platelet-rich plasma in patients with thin endometrium: a pilot randomized study

Abstract

The aim of this study was to evaluate the efficacy of hysteroscopically controlled injections of autologous platelet-rich plasma (PRP) and autologous endometrial cells as a treatment for infertile women with thin endometrium. The study enrolled 115 patients with thin endometrium (< 7 mm at implantation window) and infertility, who were divided into groups: Group 1 (the control) underwent conservative therapy; Group 2 received intraendometrial PRP injections instead of the conservative therapy; Group 3 received identical injections after conservative therapy; Group 4 received injections of the autologous endometrial cells suspended in PRP. A single injection dose of PRP contained 0.6-0.7 × 10¹¹ of platelets. The levels of PDGF-BB and VEGF in PRP were increased compared with ordinary plasma. The autologous endometrial cells, obtained from pipelle biopsies, constituted heterogeneous cell populations containing stromal and epithelial cells. Intraendometrial PRP injections had significant impact on endometrial thickness and local microcirculation in Group 2 and Group 3. In Group 4, injections of PRP reinforced with endometrial cells also facilitated a significant increase in endometrial thickness. This work describes a novel approach for infertility treatment in patients with refractory thin endometrium. PRP injections and injections of the endometrial cells suspended in PRP into endometrium enhanced cell proliferation and angiogenesis.

Figure 1

Endometrium parameters. Injection of autologous PRP – the image shows endoscopic needle inserted in the endometrium to the controlled depth of 2–3 mm (a). Endometrium thickness dynamics for the groups (b): * – p < 0,05; # – p < 0,05 for Group 4 vs Group 2. Histological image of endometrium with signs of fibrosis and atrophy (c): 1, fibrotic foci in the stroma adjacent to endometrial glands; 2, atrophic gland; hematoxylin and eosin, magnification × 100. Endometrium of an early stage of secretion in a patient who became pregnant ((d) – staining with hematoxylin and eosin, (f) – immunohistochemical expression of CD34); endometrium of an inferior secretion phase in a patient who did not become pregnant ((e) – staining with hematoxylin and eosin, (g) – immunohistochemical expression of CD34). Magnification × 200. Software: ImageScope RRID:SCR_014311.

Figure 2

Immunochemical characterization of PRP. (a) Images of western blotting membranes stained with specific antibodies to PDGF-BB and VEGF, representing ordinary plasma and PRP. (b) relative levels of PDGF-BB and VEGF in ordinary plasma and PRP, normalized to the total protein content; *p < 0.05. (c, d and e) representative flow cytometry dot plots of forward vs side scatter (FSC vs SCC) and SCC vs CD61 gating for CD63 vs P-selectin, quadrant axes set according to staining control. (f) percentages of positively stained cells. Software: (a) Image Lab Software RRID: SCR_014210, (b,f) GraphPad Prism RRID: SCR_002798, (c-e) BD CellQuest Pro RRID: SCR_014489.

Figure 3

Immunochemical characterization of the minimally manipulated cell products isolated from endometrial biopsies. (a) representative flow cytometry histograms with indicated percentages of CD146, CD90, vimentin, EPCAM and CD45 positive events; the pale-filled curves correspond to control staining. (b) a boxplot of cells positivity stained by markers. c, cell viability evaluation by anti-Ki-67 flow cytometry and methylene blue assay. (d) immunocytochemistry analysis with DAPI counterstaining for cell nuclei (blue): negative control (secondary antibodies only, left image) and staining with antibodies (right image) to vimentin (red) and EPCAM (green), magnification × 200. Software: (a,c) Flowing Software 2.5.1 RRID: SCR_015781, B, GraphPad Prism RRID: SCR_002798.