Autologous Platelet-Rich Plasma Improves Pregnancy Outcomes of Patients with Thin Endometrium Regardless Endometrial Thickening: Multicenter Retrospective Study with Elimination of Embryonic Confounders

INTRODUCTION

Recurrent implantation failure (RIF) is generally defined as the failure to achieve clinical pregnancy in women under 40 years of age after embryo transfer (ET) of at least four high-quality embryos in at least three fresh or frozen-thawed embryo transfer (FET) cycles (Coughlan et al., 2014). RIF can be caused by chronic endometritis, imbalance of the bacterial flora, misexpression of genes related to the timing of implantation, and thinning of the endometrium. The latter is caused by primary factors as well as intrauterine surgeries, such as dilation and curettage (D&C), total curettage, and transcervical resection (TCR; Azumaguchi et al., 2017). Platelet-rich plasma (PRP) is a regenerative medicine that reportedly regenerates thin endometrium and improves fertility when administered in the uterine cavity. In 2015, it was reported for the first time that intrauterine infusion of PRP to patients with thin endometrium results in thickening of the endometrium and a significant increase in the rates of pregnancy and live birth (Chang et al., 2015). These findings were subsequently reconfirmed in several reports (Colombo et al., 2017; Zadehmodarres et al., 2017). In Japan, we introduced PRP as a fertility treatment in 2019 and initiated a clinical study on refractory infertility with thin endometrium. We also reconfirmed that PRP thickens thin endometrium and increases the pregnancy rate (Kusumi et al., 2020). As PRP treatment has become more widespread, attention has also focused on the non-endometrial thickening effects of PRP. Some have reported that PRP does not necessarily thicken the endometrium, but improves pregnancy outcomes (Enatsu et al., 2021). One possible reason for this is that PRP improves the endometrial environment for implantation, for example, by healing chronical endometritis (Sfakianoudis et al., 2019).

Therefore, in this study, we examined the relationship between endometrial thickening and pregnancy outcomes to determine the effect of PRP when the endometrium does not thicken after PRP administration. Furthermore, we considered the following: The actual success of implantation is largely dependent on embryonic factors such as whether the embryo has euploid or aneuploid chromosomes, in addition to endometrial factors. In particular, the average age for assisted reproductive technology in Japan in 2018 was 38.0 years (standard deviation $\pm 4.7$; Ishihara et al., 2021. In one study, the percentage of aneuploidy in blastocysts in 38-year-old women was 47.9% (Franasiak et al., 2014). Therefore, it is expected that the proportion of aneuploidy in the transferred embryos will be quite high in this study as well, and it will be difficult to accurately compare the effects of PRP in a population with a large embryonic bias. Therefore, we limited the transferred embryos to euploid embryos that underwent preimplantation genetic testing for aneuploidy (PGT-A) and analyzed the relationship between endometrial thickening and pregnancy outcomes after eliminating embryonic confounding factors. PGT-A is a test in which a portion of the trophectoderm (TE) of a blastocyst is biopsied and next-generation sequencing is used to determine chromosome ploidy. In addition to euploidy and aneuploidy, mosaicism is present. Euploid embryos are given first priority for transfer, but mosaic embryos are also considered based on chromosome number, type, and extent. The possibility of transfer is determined by expert opinion and genetic counseling (Cram et al., 2019). The transfer of euploid embryos with PGT-A can significantly increase pregnancy and live birth rates compared with the absence of PGT-A. Some reports indicate that the clinical pregnancy rate per transfer of euploid embryos is approximately 60%–70% and the live birth rate is approximately 50%–60% (Munné et al., 2019; Sato et al., 2019). In Japan, a clinical trial of PGT-A has been ongoing since January 2020 as approved by the Japan Society of Obstetrics and Gynecology (JSOG).

This study first examined the effectiveness of PRP by comparing pregnancy outcomes before and after PRP administration using the Japanese PRP registry database. We then examined whether there was a difference in pregnancy outcome when the endometrium was thickened or not thickened by PRP administration. To eliminate confounding factors in the embryos and to more accurately analyze the results, only euploid embryos obtained by PGT-A were selected for the same analysis.

MATERIALS AND METHODS

We organized the Gynecologic PRP Study Group and a case-registration system. This study was a retrospective analysis of FET cycles using data from April 2019 to October 2021 in the registration database. Of the 674 cases registered in the system by 14 fertility clinics in Japan that administered PRP for endometrial thinning or RIF, a total of 208 patients who underwent single blastocyst transfer (SBT) after PRP due to thin endometrium ($<8$mm) in their previous cycle were included in the study. The clinical pregnancy rate, live birth rate, and miscarriage rate between the PRP cycles and the past cycles before PRP administration were compared. The transferred embryos in historical control were also basically SBT. Next, the group of patients whose endometrium became thicker than the maximum endometrial thickness of the previous cycle after PRP administration was defined as the “thickened endometrium” group and the group of patients whose endometrium did not become thicker was defined as the “unthickened endometrium” group. The clinical pregnancy rate, live birth rate, and miscarriage rate in the two groups were compared. In addition, 28 patients who underwent single euploid blastocyst transfer (SEBT) with PGT-A were also divided into two groups using the same method, and their pregnancy outcomes were compared.

Ethical statement

All patients provided written informed consent prior to PRP administration. This study was conducted in accordance with the principles of the Declaration of Helsinki and the Act on the Safety of Regenerative Medicine. The study was approved by the Certified Committee for Regenerative Medicine (clinical No. PB3170046) and the Ethics Review Committee of International University of Health and Welfare (approval No. 21-S-7).

The following subsections detail the protocols performed at the Center for Human Reproduction and Gynecologic Endoscopy, Sanno Hospital. The same or similar protocols were used at 13 other fertility centers.

Preparation of PRP

To prepare PRP from autologous blood, peripheral blood was collected from the forearm using a vacuum blood collection tube (Acti-PRP tube, Aeon Biotherapeutics Corp, Taipei, Taiwan); 20mL of blood was collected using two tubes (10mL per tube). Each volume of blood was centrifuged at 2,000g for 6 minutes. A total of 1mL of PRP was obtained (0.5mL per tube); 1mL of total PRP was injected into the uterine cavity as soon as possible without clamping using an ET catheter (Kitasato Medical, Inc., Tokyo, Japan) under transvaginal ultrasound guidance.

TE biopsy and PGT-A analysis

Embryos were cultured to the blastocyst stage on day 5 or 6 after oocyte retrieval and fertilization. TE biopsies were then performed: a portion of the zona pellucida was opened using an OCTAX laser (Vitrolife, Västra Frölunda, Sweden). Five to ten TE cells were biopsied from the opening. The harvested cells were washed thoroughly, placed in PCR tubes, and frozen at −20^∘C. Samples were then transported to an analysis company (OVUS Corporation, Aichi, Japan) and processed according to their protocol. Whole genome amplification was performed using the SurePlex WGA Kit (Illumina, San Diego, CA) according to the manufacturer’s protocol. Nextera libraries were prepared from the amplified DNA and subsequently sequenced using the VeriSeq PGS assay system by MiSeq (Illumina). Sequencing data were analyzed using BlueFuse Multi analysis software v4.5.

Blastocyst classification with PGT-A

Blastocysts undergoing PGT-A were classified into four groups by the JSOG system: A, euploids; B, euploids with suspicious mosaicism; C, aneuploids; D, undiagnosable. Only groups A and B were used for ET. The priority of transfer was higher in group A than in group B. Group B was divided into low- and high-level mosaicism according to whether the extent of mosaicism was$<$or $>50\%$, and low-level mosaics were considered for transfer after genetic counseling. The embryos actually transferred in SEBT were mostly euploids ($>90\%$), with a few low-level mosaics.

Intrauterine infusion of PRP and FET

As a rule, frozen-thawed SBT was performed during the hormone replacement cycle. Transdermal or oral estrogen preparations were started on day 3 of the menstrual cycle, PRP was injected into the uterine cavity on cycle days 10 and 12, progesterone was started transvaginally around cycle day 14, and FET was performed at the appropriate time. FET was cancelled when patients had uterine bleeding on the day of the ET or when the endometrium was extremely thin, such as less than 4 mm. Endometrial thickness was measured at each time point by transvaginal ultrasonography. Basically, the same attending physician performed ultrasound measurements of endometrial thickness on the same patient to eliminate interobserver variability.

Pregnancy assessment

The primary endpoints were clinical pregnancy rate, live birth rate, and miscarriage rate in the “thickened endometrium” and “unthickened endometrium” groups. Clinical pregnancy was determined when a gestational sac was identified by transvaginal ultrasonography. Miscarriage was diagnosed when, after confirming the gestational sac, the pregnancy had stopped progressing or the fetal heartbeat had stopped by 22 weeks’ gestation.

Statistical analysis

Statistical data were analyzed using Paired-samples t-test and Chi-square test in Excel (Office 365; $\mathrm{\text{Microsoft}}<\mathrm{\text{Redmond}}$, WA). The Mann–Whitney U-test was used to analyze the data for the SEBT with PGT-A group. Statistical significance was set at $P<0.05$.

RESULTS

The enrollment and assignment of eligible patients is shown in Fig. 1. From the 676 patients who received PRP before FET from April 2019 to October 2021, 327 patients with thin endometrium with a maximum endometrial thickness of less than 8.0mm in the previous cycle were selected. From the 327 patients, we excluded 103 patients who underwent cleavage ET, 12 patients who underwent double ET, and 4 patients whose ET was cancelled; 208 patients who underwent SBT were included.

Table 1 shows the basic characteristics of the 208 patients. Overall, mean endometrial thickness significantly increased from $6.3\pm 1.0$mm in the past to $7.1\pm 1.5$mm after PRP administration ($P<0.01$); 118 (56.9%) had a history of intrauterine surgery such as D&C or TCR. The average number of previous oocyte retrievals and ETs was greater than 4. Figure 2 shows a comparison of clinical pregnancy rate, live birth rate, and miscarriage rate in 208 patients with and without PRP. All outcomes were significantly improved with PRP administration (34.1 vs 20.0, $P<0.01$; 22.6 vs 3.9, $P<0.01$; 33.8 vs 73.4, $P<0.01$). No major complications were observed after PRP administration.

The 208 patients were divided into two groups after PRP administration, the “thickened endometrium” group or “unthickened endometrium” group, and the basic characteristics of each group are shown in Table 2. Of the 208 patients, 136 (65.4%) were assigned to the “thickened endometrium” group and 72 (34.6%) to the “unthickened endometrium” group. In the “thickened endometrium” group, the endometrial thickness significantly increased from $6.2\pm 1.1$mm in the previous cycle to $7.7\pm 1.5$mm after PRP administration ($P<0.01$). There were no significant differences in the number of previous intrauterine surgeries, previous oocyte retrievals, or previous ETs. Figure 3 shows a comparison of pregnancy outcomes in the two groups. The clinical pregnancy rate, live birth rate, and miscarriage rate were all not significantly different between the “thickened endometrium” group and the “unthickened endometrium” group (36.0 vs 30.5%, $P=0.43$; 25.7 vs 16.7%, $P=0.11$; 28.5 vs 45.4%, $P=0.16$, respectively).

Table 3 shows the basic characteristics of the 28 patients in the SEBT group with PGT-A. Of the 28 patients, 22 (78.6%) were assigned to the “thickened endometrium” group and 6 (21.4%) to the “unthickened endometrium” group. In the “thickened endometrium” group, endometrial thickness significantly increased from $6.4\pm 1.0$mm in the previous cycle to $8.8\pm 1.7$mm after PRP administration ($P<0.01$). There was no difference in background. Figure 4 shows a comparison of pregnancy outcomes between the two groups. There were no statistically significant differences in the clinical pregnancy and live birth rates, and the miscarriage rate was 0% in both groups (36.4 vs 50%, $P=0.30$; 36.4 vs 50%, $P=0.30$; 0 vs 0%, respectively).

DISCUSSION

In this study, intrauterine infusion of PRP was found to improve the pregnancy outcomes in patients with thin endometrium, although it should be noted that historical controls tend to overestimate the treatment effect. Furthermore, the use of PGT-A more accurately demonstrated that pregnancy outcome improves even without endometrial thickening after PRP administration. This study suggests that PRP may have mechanisms other than endometrial thickening that improve the implantation environment, and that PRP may be effective in patients with refractory thin endometrium who are unable to achieve endometrial thickening.

The mechanism by which PRP regenerates and proliferates the endometrium is thought to involve various growth factors and cytokines that include vascular endothelial growth factor, transforming growth factor beta (TGF-$\beta$), platelet-derived growth factor, insulin-like growth factor 1, CXC motif chemokine ligand 12, and CC chemokine ligand 5 released from platelet granules that induce activation of endometrial stromal fibroblasts and proliferation and migration of mesenchymal stem cells. One in vitro study showed that activated PRP increased cell migration and proliferation in a variety of human endometrial cells involved in tissue regeneration and promoted matrix protein expression (Aghajanova et al., 2018).

Conversely, patients in the group whose endometrium did not grow may have damaged or depleted stromal cells or stem cells, or may have insufficient or dysfunctional growth factors or cytokines in the PRP. However, even in such cases, we suspect that PRP may have other mechanisms to improve the implantation environment through other processes. An example of a reported effect of PRP other than endometrial growth is the healing of chronical endometritis. In animal studies, PRP administration to mares with chronic degenerative endometritis was effective in reducing the inflammatory response of the exacerbated uterus to semen (Reghini et al., 2015). Another bovine in vitro study reported that PRP-cultured endometrial cells were effective in decreasing the gene expression of inflammatory factors such as interleukin (IL)-1$\beta$, IL-8, prostaglandin endoperoxide synthase 2, and inducible nitric oxide synthase (Marini et al., 2016), suggesting the possibility of treating endometritis, which is considered a factor in RIF. Cytokines, such as tumor necrosis factor alpha (TNF-$\alpha$) and TGF-$\beta$, which are present in PRP are also thought to improve the crosstalk between the embryo and endometrium (Fujiwara, 2006) and promote the adhesion of trophoblast cells to the extracellular matrix (Irving and Lala, 1995).

In addition, this study also reconfirmed the low miscarriage rate in the case of SEBT. Most miscarriages are due to aneuploidy of the embryo, and most aneuploid embryos, even if implanted, are spontaneously eliminated over the course of the pregnancy (Azmanov et al., 2007; Nagaoka et al., 2012; Ogasawara et al., 2000). The miscarriage rate after euploid ET with PGT-A is estimated to be about 10% regardless of maternal age (Munné et al., 2019; Sato et al., 2019), which is significantly lower than the miscarriage rate after ET without PGT-A. The reason why there was not a single miscarriage in the SEBT group in this study is thought to be due to the use of euploid embryos with PGT-A, as well as chance due to the small sample size.

The results of this study suggest that PRP may be a treatment option not only for endometrial growth in patients with thin endometrium, as initially expected, but also for improving the endometrial environment for implantation. It may also be worthwhile to test the hypothesis that PRP is effective for unexplained RIF.

Limitations of this study are its retrospective design and small sample size. Power to detect a difference was low due to the small sample size. It may just be that the samples were not large enough to detect differences. Prospective studies with larger sample sizes are needed to reconfirm the results of this study.

Finally, regarding the safety of PRP, none of the patients experienced any major complications after PRP administration, suggesting that the treatment is generally safe for the patients. Although there are no clear reports on the safety of PRP for the embryo itself, the half-life of the growth factors and cytokines contained in PRP is short, for example, 18.2 minutes for TNF-$\alpha$ (Oliver et al., 1993), and it is unlikely that they will remain in the uterine lumen by the time of ET after PRP administration. Therefore, PRP is not expected to have a direct negative effect on the embryo itself either.

DECLARATION

Human rights statements and informed consent: All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients for inclusion in the study.

Approval by the ethics committee: The protocol was approved by a suitably constituted ethics committee.

Data sharing and data accessibility: Not applicable.

CONFLICT OF INTEREST

Satoshi Suzuki, Maki Kusumi, Tomoko Maeda, Kiyotaka Kawai, Toshihiro Kawamura, Eri Okamoto, Toushun Jo, Ryo Tsutsumi, Satoru Takamizawa, Takahiro Nakayama, Motowo Nabeta, Yukio Nishiyama, Mari Nomiyama, Kenji Furui, Yoshiharu Morimoto and Osamu Tsutsumi declare that they have no conflict of interest.

Funding: This research was supported by AMED under Grant Number JP22lk0310083.

ACKNOWLEDGMENTS

We thank the patients who participated in this study and their families.