Clinical outcome of intrauterine infusion of platelet‐rich plasma in patients with recurrent implantation failure

Abstract Purpose This study aimed to evaluate the effectiveness of intrauterine infusion of platelet‐rich plasma (PRP) before embryo transfer (ET) in recurrent implantation failure (RIF) cases. Methods The authors retrospectively analyzed 54 ET cycles involving frozen and thawed high‐quality blastocysts after intrauterine PRP infusion between September 2019 and November 2020. All patients had a history of at least two times of implantation failure on ET. A total of 54 patients were categorized into two groups: thin endometrium (39 patients) and unexplained implantation failure (15 patients). In the thin‐endometrium group, the endometrial thickness (EMT) was <8.0 mm at cycle days 12–14 in the prior ET cycle. Results Among the 54 ET cycles after PRP infusion, 31 (57.4%) were positive for human chorionic gonadotropin (hCG) and 27 (50%) achieved clinical pregnancy, which was significantly better than that in prior ET cycles without PRP infusion (27.2% and 9.6%, respectively). The EMT was not increased at ET date on the PRP cycle compared with that in the prior ET cycle in both patient groups. Moreover, EMT was not different between the hCG‐positive and hCG‐negative groups. Conclusion Although intrauterine PRP infusion had no superior effect on increasing the EMT than conventional therapeutic agents, it resulted in high pregnancy rates in patients experiencing RIF with or without thin endometrium.

miscarriage risk. 2,3 The reason is pathophysiologically explained by poor epithelial growth, reduced expression of vascular endothelial growth factor (VEGF), poor vascular development, and the high impedance of blood flow in the radial arteries of the uterine vasculature. 4 Although the endometrial thickness (EMT) reportedly improves through prolonged estrogen administration, aspirin, Vitamin E, and pentoxifylline, many women with thin endometrium remain nonresponsive with these treatments. 5,6 Platelet-rich plasma (PRP) is a new modality that has recently been suggested for thin-endometrium treatment. [7][8][9] PRP is prepared from fresh blood that is collected from a peripheral vein and processed to increase the concentration of platelets by separating various blood components. 10 Platelets contain a significant amount of growth factors, such as VEGF, epidermal growth factor, platelet-derived growth factor (PDGF), and transforming growth factor, which all stimulate proliferation and growth. 11 In the endometrium, angiogenesis is critical for endometrial growth after menstruation, and a vascularized receptive endometrium is essential for implantation. Therefore, growth factors and other cytokines found in PRP may promote endometrial thickening in patients with thin endometrium. 7,12 Until recently, intrauterine PRP infusion has been extensively reported to be effective in treating patients with a thin endometrium.
According to Kusumi et al., 8 EMT was increased by 1.27 mm after PRP administration compared with the previous ET cycle. Similarly, several researchers inferred that PRP infusion may effectively improve the endometrial growth and possibly, pregnancy outcomes in women with thin endometrium. 7,9,13 Nasari et al. 14 also reported that PRP infusion improved the implantation rate in patients with RIF.
However, reports on the effect of PRP infusion on women without thin endometrium are currently limited. Thus, we aimed to retrospectively investigate the effect of PRP infusion on patients with RIF and compare the outcomes between patients with RIF with thin endometrium and those without thin endometrium.

| MATERIAL S AND ME THODS
This retrospective study included 54 cycles of frozen and thawed

ET cycles performed after intrauterine PRP infusion at Hanabusa
Women's Clinic between September 2019 and November 2020.
The inclusion criteria of this study were patients with RIF, defined as having a history of at least two consecutive cycles of implantation failure and 25-45 years of age during ET. To exclude the influence of embryo quality, we only analyzed ETs with high-quality blastocysts.
Blastocysts were graded according to Gardner's classification, 15 and a blastocyst grade of 3BB or better was considered as a high quality.
The exclusion criteria were hepatic disorder, hemoglobin level <11 g/ dl, platelet count <150 million/mm 3 , anticoagulant administration, and congenital uterine anomalies. This study was approved by the Frozen and thawed ET was performed with a hormone replacement cycle, which was provided after ovarian suppression using a similar method reported previously. 16 Figure 1  Platelet-rich plasma was prepared from autologous peripheral blood using a similar method reported previously. 8 Using two vacuum blood collection tubes (Acti-PRP tube, Aeon International Inc.), we collected 20 ml of peripheral blood from the forearm. Then, the blood was centrifuged at 2000 g for 6 min and divided into three layers: bottom layer (containing red and white blood cells), supernatant layer (containing cellular plasma), and a buffy coat layer (located between the two layers). We obtained 1 ml (0.5 ml per tube) of PRP from the bottom of the supernatant layer. Immediately after collecting the PRP, we infused the entire PRP volume (1 ml) into the uterine cavity using a soft catheter (Kitazato Medical Co., Ltd.). Correct fluid infusion was confirmed by transvaginal ultrasonography.
Transvaginal and oral progesterone was administered on CD 15; the date was adjusted as CD 15 even when the progesterone start date was changed. ET was performed basically on CD 20 (5 days after progesterone administration), but the date was changed in cases which the previous ERA test recommended another preferable date.
In this study, the primary outcome was clinical pregnancy. At CD 30 (10 days after ET), serum human chorionic gonadotropin (hCG) was evaluated; positive hCG was considered when the hCG level was >5 mIU/ml. Further, we defined clinical pregnancy as the presence of a gestational sac with heart beats in the uterus via transvaginal ultrasonography 3 weeks after the ET. The secondary outcome was the increase in EMT; the increments in EMT from CD 10 (first PRP infusion) to CD 14 (second PRP infusion) and CD 20 (ET date) were compared with those of the prior ET cycles without PRP infusion.
All statistical data were calculated using Student's t-test or chisquare test and analyzed using Excel (Office 365, Microsoft USA) and EZR (Saitama Medical Center), which is a graphical user interface for R (The R Foundation for Statistical Computing). Moreover, p-values <0.01 were considered significant. Table 1 summarizes the characteristics of 54 included patients, of which 15 constituted the unexplained implantation failure group and 39 belonged to the thin-endometrium group. Age, number of prior ART cycles, number of prior clinical pregnancy, and number of spontaneous abortion were not significantly different between two groups. These factors were also not different between the hCGpositive group and hCG-negative group. Table 2 represents the changes in EMT between the PRP cycles and the prior ET cycles. Naturally, the EMT at CDs 12-14 was thinner in the thin-endometrium group than in the unexplained implantation failure group; the EMT was almost the same in PRP infusion cycles and prior ET cycles in both groups. Regardless of the EMT at CDs 12-14, the endometrium did not thicken at the ET date on the PRP infusion cycles. When comparing the EMT between the hCG-positive and hCG-negative groups, no difference was found in both the unexplained implantation failure group and the thinendometrium group.

TA B L E 3 Pregnancy outcomes in
patients who underwent frozen and thawed embryo transfer after PRP instillation in comparison with previous cycles. The analyses contain only highquality blastocyst transfer in hormone replacement cycles both in PRP cycles and prior ET cycles transmissible diseases, such as HIV and hepatitis. Based on the pioneering and long-term clinical experience on the application of PRP in the oral-maxillary field and thousands of patients having received this therapy so far, the use of PRP is considered safe. 18 In this study, no adverse events, such as injury and infection, were observed.
Nazari et al. 14  The main limitation of this study is its retrospective study design with a relatively small sample size. In addition, patient's recruitment depended on physician's and patient's preference; therefore, selection bias may occur. Moreover, the underlying mechanism and biological pathways are still unclear, especially for patients with unexplained implantation failure.

H U M A N R I G HT S S TATE M E NT A N D I N FO R M E D CO N S E NT
All patients were well informed and written informed consent was obtained prior to the treatment period.

A N I M A L R I G HTS
This article does not contain any studies with animal subjects performed by the any of the authors.