Objectives: Thin endometrium is a common problem encountered in the field of assisted reproductive technology. We explored the effects of platelet-rich fibrin in a thin endometrium rat model.
Materials and Methods: Twenty Sprague-Dawley rats were randomly divided into 2 groups. For the thin endometrium group, endometria of left uteri were injected with ethanol. For the experimental group, platelet-rich fibrin was sutured onto the left uteri of endometria injected with ethanol. Right uteri were kept as the normal (control) group. Two weeks after platelet-rich fibrin transplant, uteri were sampled for histology, immunohistochemistry, Western blot, and real-time reverse transcription-polymerase chain reaction.
Results: Endometrium thicknesses in normal, thin endometrium, and experimental groups were
632.2 ± 38.28, 434.80 ± 41.37, and 603.0 ± 40.93 μm, respectively. Endometrium thickness in the experimental group was significantly increased versus the thin endometrium group (P = .011). Immunohistochemical examination showed that expression levels of cytokeratin 18, vimentin, and leukemia inhibitory factor in the experimental group were significantly higher versus the thin endometrium group (P < .001, P < .006, and P = .001, respectively). In Western blot analysis, cytokeratin 18, integrin β3, leukemia inhibitory factor, and vimentin protein expressions were slightly higher in the experimental and normal groups versus the thin endometrium group. Real-time reverse transcription-polymerase chain reaction showed significantly higher octamer-binding transcription factor 4 mRNA levels in the experimental group versus the thin endometrium group (P < .001). Interleukin 6 and matrix metalloproteinase 9 mRNA levels were significantly upregulated in the experimental group versus the thin endometrium group (P= .004 and P < .001, respectively). Interleukin 1β mRNA expression was significantly lower in the experimental group versus the thin endometrium group (P < .007).
Conclusions: Application of platelet-rich fibrin has a therapeutic effect on thin endometrium in rats. Our results provide new insight on clinical treatment of thin endometrium.
Key words : Oct-4, c-Kit
In the human-assisted reproductive technology cycle, embryo implantation and clinical pregnancy rates are limited by endometrial receptivity, and a thin endometrium is considered an important clinical indicator affecting endometrial receptivity. Although no consensus has been reached on the threshold of thin endometrial thickness,1-3 most studies have used human chorionic gonadotropin day or corpus luteum characteristics to support vaginal ultrasonography examination on that day. If the endometrial thickness is less than 7 mm, then it is considered thin.4-6
Many clinical treatments have attempted to improve endometrial thickness, such as hormone replacement, growth hormone, aspirin, granulocyte colony-stimulating factor, and stem cells.1,4,7-17 Although these approaches have been somewhat effective, many problems have rendered the treatment unsatisfactory. For example, although stem cell therapy has an effect in clinical treatment, its safety is uncertain.18 Therefore, it is necessary to identify more effective and safe solutions.
Platelet-rich fibrin (PRF) is a new generation of autologous platelet-rich concentrate that replaces the first generation of platelet-rich plasma (PRP). It is simple to prepare and requires no biochemical treatment. Centrifugation during manufacture of PRF activates platelets, and the large particle degranulation enhances cytokine release. In other words, PRF can gradually release cytokines and promote fibrin matrix remodeling. Platelet-rich fibrin-rich growth factors can promote cell proliferation, migration, and angiogenesis and accelerate tissue healing and regeneration.19
Platelet-rich fibrin is used widely and effectively, such as for alveolar socket repair in the dental field, regeneration of periodontal defects in healing gingival lesions,20-23 facial defects and scars in the field of plastic surgery,24,25 and treatment of refractory wounds including diabetic foot and ulcers caused by venous thrombosis.26 Platelet-rich fibrin has gradually become accepted as a safe, effective, and natural artificial engineered growth factor. Its application can augment repair of musculoskeletal tissue, skin, and corneal ulcers and burns.27 However, few groups have investigated the application of PRF in the treatment of thin endometrium. The purpose of this study was to explore whether and how PRF may improve thin endometrium and enhance endometrial receptivity.
Materials and Methods
Eight-week-old female Sprague-Dawley rats were used in our study (Beijing Vital River Laboratory Animal Technology). All experimental rats had free access to food and water and were housed under a natural light and dark cycle (12 h light, 12 h darkness). Approximately 2 weeks later, vaginal cytology examinations were performed every morning to assess the estrous cycle. Thirty rats with a regular
4- to 5-day estrous cycle were selected for our study, and the estrous period was the time window for experiments. All rats were treated in accordance with the guidelines of the Animal Protection and Use Committee of Beijing Shijitan Hospital and followed the guidelines in the Animal Research: In Vivo Experiment Report. All rat procedures were approved by the Experimental Animal Ethics Committee of Beijing Shijitan Hospital. All researchers engaged in animal experiments hold animal experiment licenses issued by the Beijing Association on Laboratory Animal Care.
A rat model of thin endometrium was established with 30 rats with regular estrous cycle; animals were injected with absolute ethanol according to the method described by Zhao and colleagues.28 After all 30 rats were anesthetized, the left uterine horn was injected with ethanol and the right uterine horn was injected with normal saline (negative control). Efforts were made to protect the surrounding tissue when injecting absolute ethanol, which can cause severe intestinal adhesions, intestinal obstruction, and death in rats.
A preliminary experiment was performed to examine the rat endometrium before formal grouping (n = 10). Twenty rats were randomly divided into 2 groups. The experimental group was treated with ethanol in the left uterine horn, and PRF was given in the estrous period about 2 weeks after the procedure (n = 10). The thin endometrium group received injections of normal saline into the left uterine cavity about 2 weeks after the same procedure during the estrous period (n = 10). All rats underwent at least 2 procedures. After 3 additional estrous cycles (~2 weeks after PRF transplant), the uteri were removed under anesthesia, and all rats were euthanized. Uterine sections were stored in formalin and/or liquid nitrogen to await further study (Figure 1).
Platelet-rich fibrin preparation
We prepared the PRF according to the method of Dohan and colleagues.29 After anesthetization, ~5 mL of abdominal aortic blood was sampled from each rat, injected into a centrifuge tube without any anticoagulant, and centrifuged at 400 g for 10 minutes. Centrifugation ceased, and the sample remained undisturbed for 10 minutes, after which the PRF was visible as a translucent gel-like substance located between the red blood cell fragments at the bottom and the upper plasma. Next, the PRF was lightly squeezed into a thin sheet over clean gauze, which was cut into a long strip suitable for a rat’s uterine cavity and placed there before the incision was closed with sutures.
Surgical transplant of platelet-rich fibrin
After intraperitoneal injection of 10% chloral hydrate, a vertical midline incision was made in the lower abdomen of the rat to expose the uterus. In the experimental group, the left uterine horn was incised and received a suitable PRF, and then the wound was closed. The right uterine horn was used as a control without any treatment. In the thin endometrium group, the left uterine horn was injected with normal saline, whereas the right uterine horn was left untreated.
Scanning electron microscopy
The PRF was fixed with 3% glutaraldehyde at 4 °C overnight and dehydrated with increasing concentrations of acetone, and then the specimen was placed in tert-butanol. After the samples were dried and sublimated, these were sprayed with gold for scanning electron microscopy.
Transmission electron microscopy
The PRF was fixed with 3% glutaraldehyde at 4 °C overnight and dehydrated with increasing concentrations of acetone, and then the samples were embedded in resin for transmission electron microscopy.
Histology and immunohistochemical staining
Uterine specimens fixed in 4% paraformaldehyde and embedded in paraffin were cut into 5-μm sections for hematoxylin and eosin staining. The sections were immersed twice in xylene (10 minutes each time) and then hydrated in a decreasing ethanol series (100%, 100%, 95%, 85%, 75%, and 0%, each for 1 minute). The sections were stained in hematoxylin for 7 minutes, rinsed in tap water for 30 minutes, stained with eosin for 30 seconds, dehydrated with increasing concentrations of ethanol, cleared with xylene for 10 minutes, and then sealed with neutral resin. Endometrial thickness (ie, the vertical distance from the junction of the endometrial muscle layer to the uterine cavity) was measured at 4 image sites for each sample with ImageJ software (National Institutes of Health), and the average value was taken as the endometrial thickness of the slice. Endometrial thickness and morphology were evaluated and compared between groups.
For immunohistochemical staining, sections were rinsed gently with phosphate-buffered saline 3 times for 3 minutes each time, fixed in a 3% hydrogen peroxide solution for 15 minutes to block endogenous peroxidase, and incubated with primary antibodies (anti-cytokeratin 18 [anti-CK18], ab668, 1:50; anti-vimentin, ab8978, 1:200; and anti-leukemia inhibitory factor [anti-LIF], ab113262, 1:50; all from Abcam) at 4 °C overnight. The next day, the wet box was removed and warmed to room temperature for 45 minutes, and then sections were rinsed with phosphate-buffered saline 3 times for 3 minutes each. The secondary antibody was added (50 μL/slide) and remained at room temperature for 30 minutes. Goat anti-rabbit immunoglobulin G antibody-horseradish peroxidase multimer was added dropwise to CK18 and LIF, and goat anti-mouse immunoglobulin G antibody-horseradish peroxidase multimer was added dropwise to vimentin. Slides were then rinsed 3 times in phosphate-buffered saline for 3 minutes each. After 3,3’-diaminobenzidine color development, slides were counterstained with hematoxylin and mounted to allow observation of the protein expression under the microscope. Immunohistochemical staining characteristics in the endometrium were observed in a high-power field of vision (×100). ImageJ software was used to calculate the average optical density values, and the average optical density method was used to semiquantitatively compare protein expression intensities, for which the average optical density was calculated as the integrated optical density divided by the area.
The tissue was homogenized in lysis buffer. The homogenate was centrifuged at 12 000 g for 10 minutes. The collected supernatant was the total protein, and the concentration was measured with the bicinchoninic acid method (BCA Protein Assay kit, Thermo Scientific). The same amount of protein was separated by 8% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked on a shaker with 5% skim milk at room temperature for 1 hour. Next, the membrane was incubated with the diluted primary antibody at 4 °C for 3 hours, followed by secondary antibody at a ratio of 1:3000, and then incubated at room temperature for 30 minutes in the dark. The protein bands were quantified with an Odyssey CLx infrared imaging system (LI-COR). Finally, the protein levels were quantified with ImageJ software.
Reverse transcription-polymerase chain reaction
Total RNA was extracted from uterine tissue with TRIzol RNA isolation reagent (Ambion, Life Technologies) according to the manufacturer’s instructions. We then measured RNA concentration and purity. Finally, reverse transcription was performed on a polymerase chain reaction instrument.
The cDNA amplification conditions were as follows: denaturation at 95 °C for 10 minutes for about 40 cycles, 15 seconds at 95 °C, and 60 seconds at 60 °C, with temperature during dissolution increased from 60 °C to 90 °C.
We used reverse transcription-polymerase chain reaction to explore the transcription of cytokines including vascular endothelial growth factor (VEGF), the stem cell factor receptor c-Kit, octamer-binding transcription factor 4 (Oct-4), interleukin 1β (IL-1β), IL-6, and matrix metalloproteinase 9 (MMP9). Glyceraldehyde-3-phosphate dehydrogenase was used as the internal reference. The polymerase chain reaction primers are listed in Table 1. This reaction was performed with the Applied Biosystems 7500 Real-Time PCR System. Gene expression levels were measured by the 2-ΔΔCT method.30
Data are presented as mean values ± SE and were analyzed with SPSS software (version 16.0) and GraphPad Prism software (version 6.0). Two-tailed t tests were used for statistical analyses to compare differences between the 2 groups, and P < .05 was considered significantly different.
Scanning electron microscopy and transmission electron microscopy
Scanning electron microscopy showed that a large amount of fibrin polymerized into a loose 3-dimensional network structure. In addition, transmission electron microscopy showed platelets aggregated in fibrin, intact alpha particles in platelets, and activation of some platelets (Figure 2).
Platelet-rich fibrin transplant increases endometrial thickness
To determine whether PRF transplant can improve endometrial thickness, uterine tissue was evaluated after hematoxylin and eosin staining. Endometrial thicknesses in the normal group, the thin endometrium group, and the experimental group were as follows: 632.2 ± 38.28, 434.80 ± 41.37, and 603.0 ± 40.93 μm, respectively. As shown in Figure 2, the thin endometrium and glands were significantly thinner from the effect of ethanol, and this difference was statistically significant compared with the normal group (P < .05). To verify that the procedure affected endometrial thickness, the values were compared between the saline-injected group and the normal group (all P > .05). There was a significant difference in endometrial thickness between the experimental group and the thin endometrium group (P < .05), which indicated that PRF did have a positive effect. For the experimental group, the endometrial epithelium and glandular epithelial cells appeared as a single layer of columnar cells, tightly and neatly arranged with no obvious edema in the stroma. For the thin endometrium group, the endometrium was thin, the epithelial cells of the endometrium were flat, and the stroma showed various degrees of edema. There was no significant difference in endometrial thickness between the experimental and normal groups(P > .05).
Platelet-rich plasma transplant promotes endometrial cell regeneration
Next, we explored the effect of PRF transplant on protein expression levels in the thin endometrium model. The expression levels of CK18 and LIF in the endometrial epithelium and expression level of vimentin in the endometrial stroma were assessed by immunohistochemistry. Positive expression was indicated by brown staining. The comparison showed that levels of CK18, LIF, and vimentin in the normal and experimental groups were significantly higher than in the thin endometrium group (all P < .05).
There was no significant difference between the experimental and normal groups (P > .05, Figure 3). Western blot also showed that the protein levels of CK18, integrin β3, LIF, and vimentin were higher in the experimental and normal groups compared with the thin endometrium group. There was no significant difference between the experimental and normal groups (P > .05, Figure 3).
Platelet-rich plasma transplant regulates mRNA levels of vascular endothelial growth factor, c-Kit, octamer-binding transcription factor 4, interleukin 1β, interleukin 6, and matrix metalloproteinase 9
To clarify the relationship between PRF transplant and changes in mRNA levels of VEGF, c-Kit, Oct-4, IL-1β, IL-6, and MMP9, we performed real-time reverse transcription-polymerase chain reaction for each group.
Levels of Oct-4 mRNA were significantly higher in the experimental group compared with the thin endometrium group (P < .05). Interleukin 1β mRNA in the experimental and normal groups was significantly downregulated compared with the thin endometrium group (P < .05).
As shown in Figure 4, IL-6 and MMP9 mRNA levels in the experimental group were significantly upregulated compared with the thin endometrium group, and IL-6 and MMP9 mRNA levels were significantly lower in the thin endometrium group compared with the normal group (all P < .05).
There was no significant difference in c-Kit mRNA levels between the experimental and thin endometrium groups, (P > .05; Figure 4), but these levels were slightly higher in the experimental group. Compared with the thin endometrium group, VEGF mRNA expression was slightly higher in the experimental group, although not significantly so (P > .05).
The present study provides the first evidence of the effectiveness of PRF for treatment of thin endometrium in rats. Placement of PRF in the rat uterine cavity after alcohol injection caused an increase in the endometrial thickness compared with the thin endometrium group. Notably, there was no significant difference between the endometrial and glandular epithelia in the experimental and normal groups, and the endometrial epithelium was relatively full in the experimental group (single layer of tall columnar cells) compared with the thin endometrium group (flat cells); also, the endometrial stroma in the thin endometrium group showed edema compared with the experimental group. Studies have reported that growth factors interact with endometrial epithelial and stromal cells through autocrine and/or paracrine pathways to participate in endometrial regeneration.31,32
Importantly, we also confirmed that placement of PRF in the rat uterine cavity can significantly increase expression of the hallmark protein CK18 in endometrial epithelial cells compared with the thin endometrium group; the endometrial stromal protein vimentin also increased significantly compared with the thin endometrium group. In other words, intrauterine PRF transplant can promote endometrial proliferation in rats. This coincides with the report by Jang and colleagues33that intrauterine injection with autologous PRP can promote and accelerate the regeneration of ethanol-damaged endometrium. Matsumoto and colleagues found that different subtypes of platelet-derived factors promote the proliferation and migration of human endometrial stromal cells.34
Leukemia inhibitory factor is an important regulator of endometrial function, and it is a marker of endometrial receptivity and plays an important role in embryo implantation.35 We found significantly higher LIF expression in the experimental group compared with the thin endometrium group but not compared with the normal group. This suggests that PRF implantation can enhance endometrial receptivity. Our results show that placement of PRF in the uterine cavity can promote endometrial regeneration and improve receptivity.
To further confirm the above results, we performed another Western blot experiment. The results showed that protein levels of CK18, integrin β3, LIF, and vimentin were slightly higher in the experimental group compared with the thin endometrium group. There was an increasing trend, but there was no statistical difference, possibly a result of the small number of animals. Additional studies with larger sample sizes are needed to confirm our findings.
At the mRNA level, IL-1β levels in the experimental and normal groups were significantly lower than that in the thin endometrium group. Compared with the thin endometrium group, IL-6 and MMP9 mRNA levels were significantly upregulated in the experimental group, whereas these were significantly downregulated in the thin endometrium group compared with the normal group. This indicates that PRF has anti-inflammatory and antifibrotic effects, which is consistent with previous studies.36,37 Some groups have suggested that factors such as IL-6 can stimulate and mobilize bone marrow mesenchymal stem cells.38,39 mRNA expression of Oct-4 in the experimental group was significantly higher than that in the thin endometrium group. mRNA expression of c-Kit in the experimental group was not significantly different than in the thin endometrium group. This is inconsistent with the findings of Jang and colleagues,33 and additional work is needed to understand this discrepancy.
Vascular endothelial growth factor is known to play a central role in angiogenesis and its regulation.40 However, there was no significant change in VEGF mRNA level in the experimental group, but it tended to be higher compared with the thin endometrium group. This difference might be significant if a larger number of animals were included.
There are several reasons why we used PRF to treat thin endometrium in rats. Platelet-rich fibrin is a replacement platelet concentrate of PRP, which is a gel formed by the slow polymerization of its own coagulation factors.18 Previously reported results were consistent with what we observed, ie, its 3-dimensional network is rich in platelets and growth factors. Compared with PRP, growth factors are slowly released for up to 10 days.41 There are 3 major advantages of PRF. First, the preparation is simpler than that of PRP, as described by Dohan and colleagues.29 Second, PRF has retention and slow-release properties. During PRF production, fibrin slowly circulates growth factors and platelet networks in the blood during the slow polymerization process. Specifically, fibrin dissolution activates platelets and releases growth factors.42 Third, it is relatively safe because it is autologous.
One limitation of our study is that it is not convenient to place PRF in rats, so it cannot be transplanted multiple times. However, there are no restrictions on numbers of placements in human patients. Transplant can be performed multiple times according to the cycle, which may achieve better effects. Of course, clinical use is subject to the formal approval of ethics committees and the patient’s informed consent.
Because traditional treatments have not solved the difficult problem of improving thin endometrium, it may be appropriate to use PRF to treat thin endometrium clinically, which is presently under investigation.
Application of PRF in the rat model of thin endometrium has a therapeutic effect, but the mechanism requires further study. It is hoped that our work will facilitate additional basic research and clinical application of treatments for thin endometrium.
DOI : 10.6002/ect.2020.0199
From the 1Department of Obstetrics and Gynecology, Beijing Shijitan Hospital, Capital Medical University, Beijing; the 2Department of Obstetrics and Gynecology, Chaoyang Central Hospital, Chaoyang City; and the 3Department of Plastic Surgery, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China
Acknowledgements: We thank Mr. Jin Xu of Peking University First Hospital’s Electron Microscopy Laboratory for assistance with scanning electron microscopy. This study was supported by Beijing Natural Science Foundation of China (7202075), Beijing Hospitals Authority Ascent Plan, Code:DFL20190701. Other than described here, the authors have not received any funding or grants in support of the presented research or for the preparation of this work and have no additional declarations of potential conflicts of interest.
Corresponding author: Wenpei Bai, Department of Obstetrics and Gynecology, Beijing Shijitan Hospital, Capital Medical University, Tieyilu 10, Yangfangdian, Haidian District, Beijing, 100038, People's Republic of China
Figure 1. Schematic of the Experimental Procedure
Table 1. Primers for Real-Time Polymerase Chain Reaction
Figure 2. Plasma-Rich Fibrin Under Electron Microscope and Morphological Observations
Figure 3. Expression Levels of Cytokeratin 18, Leukemia Inhibitory Factor, and Vimentin by Immunohistochemistry
Figure 4. mRNA Expression of Vascular Endothelial Growth Factor, c-Kit, Octamer-Binding Transcription Factor 4, Interleukin 1?, Interleukin 6, and Matrix Metalloproteinase 9