Intrauterine administration of platelet‐rich plasma improves embryo implantation by increasing the endometrial thickness in women with repeated implantation failure: A single‐arm self‐controlled trial

Abstract Purpose The purpose of this study was to investigate the effectiveness of intrauterine administration of platelet‐rich plasma (PRP) in frozen embryo transfer (FET) cycle in Japanese patients with a thin endometrium. Method A prospective single‐arm self‐controlled trial was conducted in Japan. PRP administration was performed in 36 of the 39 eligible patients with a thin endometrium (≤7 mm). Hormone replacement therapy (HRT) with estrogen was performed for 2 menstrual cycles, and PRP was administrated on the 10th and 12th days of the second HRT cycle. The endometrial thickness was evaluated on transvaginal ultrasonography by two physicians at every visit, one an attending physician and the other a specialist physician blinded to the date and timing of the sonography. FET was performed during the second HRT cycle after PRP administration. Results After PRP administration, the mean (SD) endometrial thickness on the 14th day was significantly increased by 1.27 mm (P < .001) and 0.72 mm (P = .001) on the basis of the unblinded and blinded measurements, respectively. Of the 36 patients, 32 (88.9%) underwent FET. The clinical pregnancy rate was 15.6%. No adverse events occurred. Conclusions PRP therapy was safe and effective in increasing endometrial thickness improving possibly pregnancy rate.


| INTRODUC TI ON
Overall, the freeze-all/FET strategy in ART contributes to the improvement of pregnancy rates. This is because controlled ovarian stimulation increases the number of oocytes retrieved and prevents ovarian hyperstimulation syndrome, 2 enabling avoidance of the effect of a non-physiological endocrine environment due to controlled ovarian stimulation on implantation. 3 Moreover, for women aged ≥38 years, the predominance of FET is increasing especially with the improving pregnancy rates and decreasing abortion rates due to PGT-A. 4,5 Improvement in the implantation environment has become a major focus area of ART because of the prevalence of recurrent implantation failure (RIF). RIF represents cases of women aged ≤40 years who do not become pregnant even after at least 3 embryo transfers (≥4 embryos). 6  Administration of sildenafil for endometrial thinning may be an effective treatment but has not yet been verified in a randomized controlled trial (RCT). Therefore, new treatments are needed to improve endometrial thickness in patients with endometrial thinning.
Administration of platelet-rich plasma (PRP) may be used as one of the methods to increase the implantation rate of fertilized eggs.
PRP is easily prepared using plasma containing high concentrations of platelet-derived growth factor and cytokines obtained from centrifugation of blood samples. The components of PRP include platelet-derived growth factor, transforming growth factor beta, vascular endothelial growth factor, and epidermal growth factor. These components are considered to promote wound healing and tissue growth, and their use is expected to promote endometrial thickening in patients with a thin endometrium. 9,10 As stable implantation of fertilized eggs can be expected with thickening of the endometrium, PRP treatment is likely to contribute to increased implantation rates.
PRP has been applied in infertile patients with endometrial thinning. Chang et al 11 confirmed that in 5 patients whose endometrial thickness was <7 mm (range, 5.9-6.6 mm; mean, 6.3 mm), intrauterine injection of PRP in combination with hormone replacement therapy (HRT) thickened the endometrium to ≥7 mm (range, 7.0-8.0 mm; mean, 7.5 mm) between 48 and 72 hours after PRP injection, and all 5 patients became pregnant by subsequent embryo transfer. Zadehmodarres et al 12 administered HRT in combination with intrauterine PRP injection (twice on days 11-12 and days [13][14] to 10 patients in whom FET had been canceled because their endometrial thickness was ≤7 mm even after HRT. Thereafter, all the patients showed an endometrial thickness of ≥7 mm and underwent FET. The study showed that after FET, 5 patients became pregnant, including one whose pregnancy resulted in spontaneous abortion and 4 who maintained a normal pregnancy. Recent RCTs of PRP have shown that PRP improves endometrial thickness and increases implantation rates. 13 PRP has also been applied in patients with RIF 14 and may represent a potential therapy to optimize the preparation of the endometrium for successful implantation. We conducted a clinical study of PRP in patients with a thin endometrium (≤7 mm) who were scheduled to undergo FET with HRT to investigate changes in endometrial thickness in detail and pregnancy outcomes.

| Study design and patients
This study was a prospective single-arm self-controlled trial conducted in Japan. This study was conducted from February 2018 through January 2019 at Sanno Hospital, Center for Human Reproduction and Gynecologic Endoscopy. Recruitment of the study participants and follow-up survey was conducted at 7 fertility clinics in Japan. The objective of this study was to evaluate the efficacy and safety of PRP intrauterine infusion on endometrial thickness in women undergoing infertility treatments. In addition, we also performed a preliminary examination of the implantation rate. Women who had received ART and were going to receive FET with HRT were enrolled in this study. The inclusion criteria were age of 20-50 years, oocyte retrieval performed at age 42 years or younger, and endometrial thickness of ≤7 mm. The exclusion criteria were hepatic disorders, hemoglobin level of <11 g/dL, platelet count of <150 000/mm 3 , use of anticoagulants, and pregnancy. All PRP was prepared from autologous blood as follows: Peripheral blood was drawn from the forearm using vacuum blood collection tubes (Acti-PRP tube, Aeon International Inc, Taipei, Taiwan). Two tubes were used for collecting 20 mL (10 mL for each tube) of blood.
The blood was centrifuged at 2000 g for 6 minutes, and a total of 1 mL (0.5 mL for each tube) of PRP were obtained. The entire PRP volume (1 mL) was infused into the uterine cavity as soon as possible with ET catheter (Kitazato Medical Co., Ltd.) under transvaginal ultrasound guidance without clamp.

| Endpoints
The primary endpoint of the study was endometrial thickness. Three transvaginal ultrasonography images of the endometrium were taken for each patient, and the average of the three measurements from the three images, measured by two physicians, one an attending physician and the other a specialist physician blinded to the date and time of the transvaginal sonography, was recorded. The increments in thicknesses from the 10th to the 14th day of the second HRT cycle during with the PRP injections were compared with those of the first HRT cycle without PRP. The secondary endpoints were safety (adverse events) and patient pregnancy rate.

| Statistical analysis
Analyses of the safety of PRP were performed in all eligible patients, while the analyses of efficacy were performed in the patients who received PRP injections on the 10th and 12th days of the second HRT cycle. A descriptive analysis of the baseline patient characteristics was performed using the mean, SD, for continuous variables and percentage for categorical variables.
For the analysis of endometrial thickness, which was the primary endpoint, the data on both the unblinded measurements by the attending physicians and the blinded measurements by the specialist physician were used. To investigate the correlation between the unblinded and blinded measurements, the Pearson correlation coefficient and its 95% confidence interval (CI) were calculated.
Changes in endometrial thickness were calculated as "thickness on the 14th day-thickness on the 10th day of the second HRT cycle." Moreover, the following changes in endometrial thickness were cal-

| Patients
Forty patients were enrolled in the study, and 1 patient was judged to be ineligible before starting HRT; thus, 39 eligible patients were  (Table S1). The most common causes of infertility included uterine myoma, endometrial polyps, and Asherman's syndrome (Table S2)

| Efficacy and safety
Endometrial thickness increased during the second HRT cycle, and the mean (SD) increments in endometrial thickness between the 10th and 14th day of the second HRT cycle after PRP administration was 1.40 (0.84) mm on the basis of the unblinded measurements by the attending physicians and 0.50 (0.87) mm on the basis of the blinded measurements by the specialist physician ( Table 2). The correlation coefficient between the unblinded and blinded measurements was 0.885 (95% CI, 0.854-0.909) ( Figure S1).
Changes in the endometrium thickness after PRP administration were investigated using an analysis of variance for longitudinal data.

| D ISCUSS I ON
The endometrium plays a critical role in embryo implantation. The endometrium is a specific tissue that repeatedly falls and regenerates during each menstrual cycle, and appropriate endometrial thickness is required for implantation. 16 All study patients (n = 39) had experienced canceled embryo transfer cycles because their endometrium showed no response to hormone therapy due to various reasons.
In this study, intrauterine PRP injection (on the 10th and 12th days from menstruation) significantly increased endometrial thickness. However, in the previous RCT that investigated the effect of PRP on endometrial thickness, the control group also showed an increase in the endometrial thickness, 13   as compared with that in the other studies. In fact, in some patients, the endometrium was unresponsive to the PRP treatment ( Figure 1).
Although the sample size of this study was small, 3 patients with have been conducted to investigate the effect of PRP on the endometrium, PRP therapy has been suggested to contribute to endometrial regeneration, increase blood flow in the uterus, and alleviate endometritis, which is currently considered a major factor of RIF. Moreover, PRP therapy has attracted attention as an effective therapy for RIF. 14,20,21 Future research issues include investigation of the therapeutic effects of PRP on factors not limited to endometrial thickness, and the precise molecular mechanism of the effect of PRP must be elucidated.

ACK N OWLED G M ENTS
We thank the patients who participated in this study and their families. We are grateful to Drs Yasushi Odawara, Chikahiro Oka, Keiichi Kato, Toshihiro Kawamura, Yasushi Kuribayashi, Rikikazu Sugiyama, and Atsumi Yoshida for the patient recruitment and collaboration for this clinical research. Statcom Co., Ltd. provided statistical analysis and medical writing assistance.

Maki Kusumi, Tatsuji Ihana, Takako Kurosawa, Yasuo Ohashi, and
Osamu Tsutsumi declare that they have no conflict of interest.

H U M A N R I G HT S S TATE M E NT S A N D I N FO R M E D CO N S E NT
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients for being included in the study.

A PPROVA L BY E TH I C S CO M M IT TE E
The protocol has been approved by a suitably constituted Ethics Committee.
Clinical Trial Registry: The trial registration number is UMIN000030493.

DATA S H A R I N G A N D DATA ACCE SS I B I LIT Y
Not applicable.