ARTICLEOpen Access

Intraovarian Injection of Lyophilized Platelet-Rich Plasma in Patients with Poor Ovarian Response

Reproduction Clinic Tokyo, Tokyo, Japan

Corresponding author: Aya Karino, M.D., Reproduction Clinic Tokyo, Shiodome City Center 3F, 1-5-2 Higashi-Shimbashi, Minato-ku, Tokyo 105-7103, Japan.

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Hidehiko Matsubayashi

Reproduction Clinic Tokyo, Tokyo, Japan

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Saki Saito

Reproduction Clinic Tokyo, Tokyo, Japan

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Yurino Ishibashi

Reproduction Clinic Tokyo, Tokyo, Japan

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Kento Usui

Reproduction Clinic Tokyo, Tokyo, Japan

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Masakazu Doshida

Reproduction Clinic Tokyo, Tokyo, Japan

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Takumi Takeuchi

Reproduction Clinic Tokyo, Tokyo, Japan

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Yosuke Suzuki

Reproduction Clinic Osaka, Osaka, Japan

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Yasuhiro Ohara

Reproduction Clinic Osaka, Osaka, Japan

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, and

Tomomoto Ishikawa

Reproduction Clinic Tokyo, Tokyo, Japan

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https://doi.org/10.1142/S2661318224500087Cited by:0 (Source: Crossref)

Abstract

Objective: To demonstrate the effect of intraovarian injection of lyophilized platelet-rich plasma (PRP) in poor ovarian responders.

Methods: This retrospective cohort study evaluated data for 262 women who underwent intraovarian injection of lyophilized PRP at two private fertility clinics. Women treated from December 2021 to September 2022 and who met the Bologna criteria were included. Intraovarian injection of lyophilized PRP prepared from 50mL of autologous peripheral blood was followed by two centrifugations and activation with 2% CaCl₂, in which cells were removed with a filter and stored at room temperature. The differences in the follicle-stimulating hormone and antral follicle count (AFC), and the number of retrieved oocytes, mature oocytes, fertilized embryos, cleavage embryos, and blastocysts were measured before and 3 months after a single injection. A biological pregnancy with embryos generated after injection was also examined.

Results: Patients’ median age and anti-Mullerian hormone (AMH) levels were 44 years and 0.38ng/mL, respectively. The number of retrieved oocytes or mature oocytes and fertilized embryos or cleavage embryos was higher after injection. The number of cleavage embryos in women $\geq 40$ years significantly increased after injection. Of the 262 patients who underwent intraovarian injection of lyophilized PRP, 71 underwent embryo transfers using embryos obtained 3 months after injection, and 14 had biological pregnancies. Seven of these patients were in their 40s. Out of 14, one had a live birth, six are ongoing pregnancies, six had chemical abortions, and one had a miscarriage.

Conclusions: Intraovarian injection of lyophilized PRP improved egg retrieval results, especially in patients older than 40 years. Furthermore, this treatment may contribute to biological pregnancy in patient with poor ovarian response but needs to be further investigated in more cases in the future.

Keywords:

INTRODUCTION

Platelet-rich plasma (PRP) contains several cytokines and growth factors induced by platelet activation and is assumed to be involved in angiogenesis and wound healing (Alsousou et al., 2009). PRP injection has been used to treat mandibular reconstruction and tendonitis since the 1990s (Mishra and Pavelko, 2006; Tayapongsak et al., 1994). Therefore, PRP is derived from autologous blood and has been used for several medical indications due to its low invasiveness and safety. Currently, PRP is used in the fields of orthopedics, sports medicine, dentistry, otolaryngology, neurosurgery, ophthalmology, urology, wound healing and cosmetic, cardiothoracic, and maxillofacial surgery (Sampson et al., 2008). PRP therapy has also been clinically applied in reproductive medicine, with Chang et al. publishing the first report on PRP injection into the uterine cavity in 2015. In the study by Chang et al., five patients with recurrent implantation failure due to a thin endometrium underwent the procedure (Chang et al., 2015).

The first report on PRP injection into the ovary was presented at the European Society of Human Reproduction and Embryology meeting in 2016 by Pantos et al., who studied eight peri-menopausal women. Subsequently, several reports, including case reports (Pantos et al., 2019; Sfakianoudis et al., 2019; Sills et al., 2018) and cohort studies (Cakiroglu et al., 2020, 2022; Jackman et al., 2020; Melo et al., 2020; Sfakianoudis et al., 2020a; Sills et al., 2020; Stojkovska et al., 2019) have been published; however, no randomized study has been reported.

The specific factors present in PRP that contribute to folliculogenesis are not known. However, four factors that are thought to be important are transforming growth factor- $β$ , vascular endothelial growth factor, insulin-like growth factor, and platelet-derived growth factor (Hajipour et al., 2021).

Lyophilized PRP (freeze-dried PRP) has been introduced to replace fresh (conventional) PRP because it has long-term stability. Wolkers et al. were the first to report that platelets can be preserved by freeze-drying (FD) with trehalose, a carbohydrate that can withstand desiccation (Wolkers et al., 2001). The researchers demonstrated that the membrane and protein components of trehalose-loaded platelets before FD and after rehydration were remarkably similar to those of fresh platelets, allowing long-term storage of platelet-derived factor concentrate (PFC) at room temperature and retaining bioactivity (Andia et al., 2020). However, no report exists on the effect of injecting lyophilized PRP into the ovary.

This study was conducted to demonstrate the effect of intraovarian injection of lyophilized PRP (PFC-FD^TM, CellSource Co. Ltd., Japan) in patients with poor ovarian response.

MATERIALS AND METHODS

We performed a retrospective study of 339 patients who underwent intraovarian PFC-FD^TM injection between December 2021 and September 2022 at the Reproduction Clinic Tokyo and Reproduction Clinic Osaka. Patients were included if they had (1) experienced at least one oocyte retrieval in our clinics using their own oocytes and (2) poor ovarian response, as defined by the Bologna criteria (Ferraretti et al., 2011). In this study, we chose the Bologna criteria instead of the Poseidon criteria because about 80% of patients were 40 years or older. Of these 339 patients, 45 were excluded because they had not started their oocyte retrieval cycle after the PFC-FD^TM injection. The exclusion criteria were (1) at least one positive test for an infectious disease, (2) a platelet count of $\leq 80, 000 ∕ μ L$ , or (3) with no ovaries or ovaries in a position where puncture was impossible.

The study design was quasi-experimental, and all participants provided written informed consent prior to intraovarian PFC-FD^TM injection, including future analytical use of data related to the treatment. They were responsible for their all treatment costs. Institutional Review Board approval was obtained (Reproduction Clinic Institutional Review Board number RT20131882).

PFC-FD^TM was created at the Regenerative Medicine Center of CellSource Co., Ltd. (Tokyo, Japan), a facility licensed to manufacture specific cell products. Briefly, PFC-FD^TM is prepared according to the following steps: (1) 50mL of autologous peripheral venous blood undergoes primary centrifugation (280g, 10min) to separate the plasma from the blood cell layer; (2) secondary centrifugation (1,400g, 10min) takes place to separate the plasma components and produces 1mL of PRP in the precipitated portion; (3) 5mL of phosphate-buffered saline containing 2% CaCl₂ is added to the PRP and stirred to stimulate the platelets and release physiologically active substances rich in growth factors; (4) a filter is used to remove unnecessary cells in the PRP and render it acellular; and (5) the extracted components are dispensed into vials and freeze-dried for stable storage at room temperature. The product is restored to its original state by dissolving it in physiological saline. Storing at room temperature, and dissolving do not affect the growth factor components and their functions for at least 6 months (in-house document, CellSource Co., Ltd). From blood collection to the delivery of two vials of PFC-FD^TM to our institutes takes 3 weeks.

The intraovarian injection is ideally performed at the time of oocyte retrieval. However, in this study, for patients for whom oocyte retrieval was not achieved, intraovarian injection was performed at any time of the menstrual cycle if bilateral ovaries were confirmed by transvaginal ultrasonography. One vial of PFC-FD^TM was dissolved in 0.8mL of physiological saline immediately before injection. A long, thin needle (23G, 20cm; CN-23G200, Kitazato Corporation, Japan) and rocking syringe (1mL; 08040, Nipro Company, Japan) were used to inject 0.4mL of PFC-FD^TM into each ovary under transvaginal ultrasound guidance without anesthesia. The dosage of PFC-FD^TM was chosen according to the manufacturer’s recommendation. Previous studies reported that the effects of PRP appeared about 3 months after injection (Melo et al., 2020); therefore, we evaluated the differences in parameters before and 3 months after a single PFC-FD^TM injection. We evaluated follicle-stimulating hormone, AFC, and the number of retrieved oocytes, mature oocytes, fertilized embryos with intracytoplasmic sperm injection (ICSI) (two pronuclei), cleavage embryos, and blastocysts. The number of achieving biological pregnancy with embryos developed within 3 months after PFC-FD^TM injection was also examined.

Ovarian stimulation was started 3 months after the PFC-FD^TM injection. Ovarian stimulation was performed with clomiphene citrate only, antagonist protocol, agonist flare protocol or progestin-primed ovarian stimulation (PPOS) protocol, or none (natural cycle) were administered according to the patient’s anti-Mullerian hormone (AMH) levels and AFC on cycle day 3 of the pre-intervention. Similar ovarian stimulation methods (e.g., dosage of rFSH) were performed in the oocyte retrieval cycles before and after intraovarian PFC-FD^TM injection. Transvaginal ultrasound-guided oocyte retrieval was performed 36 hours after administering 5,000–10,000 IU of human chorionic gonadotropin.

Spermatozoa were prepared using two-layer Percoll density gradients. Collected semen samples were centrifuged using two Percoll solutions (90% and 45% SpermGrad; Vitrolife, V. Frölunda, Sweden) for 15 minutes at 500 g and then centrifuged in washing buffer for 5 minutes at 150g. After the sperm was pelleted, the swim-up method was performed. The procedure followed for ICSI is described elsewhere (Morimoto et al., 2023). Time-lapse imaging was used to monitor the culture system (Geri, Genea Biomedx, Sydney, Australia); incubation took place in one step medium (Sage; Origio, Måløv, Denmark) under a humidified gas mixture of 6.0% CO₂, 5.0% O₂, and 89.0% N₂ at 37^∘C.

All statistical analyses were performed with EZR version 1.61, a modified version of R Commander designed to add statistical functions frequently used in biostatistics (Kanda, 2013). The Wilcoxon signed-rank test was used to compare parameters before and after PFC-FD^TM injection. Logistic regression analysis was performed to detect the factors contributing to increasing the number of cleavage embryos by one or more after PFC-FD^TM injection (odds ratios and confidence intervals were calculated). Stratified analysis was evaluated using the Wilcoxon signed-rank test. Statistical significance was set at $P < 0.05$ .

RESULTS

This study included a total of 262 patients, excluding 32 patients who did not reach oocyte retrieval (Fig. 1). There were 109 women who successfully obtained cleavage embryos out of the 262 women who underwent oocyte retrieval, whereas the remaining 153 women did not. The demographic characteristics of the patients are shown in Table 1. Among the 262 patients, 1 was in her 20s, 36 were in their 30s, 217 were in their 40s, and 8 were in their 50s; the median age was 44 years (25–53). The median age of the male partners at the time of oocyte retrieval was 45 years (28–60). Ovarian stimulation methods included antagonist protocol in 150 (25–53 years old, median age 44), PPOS in 27 (30–49 years old, median age 44), clomiphene citrate only or natural cycle in 77 (34–52 years old, median age 44.5), and agonist flare protocol was used in 8 patients (37–47 years old, median age 42 years old).

**Table 1. Demographic characteristics of the participants, $n = 262$ .**
Characteristic	Value
Median female age, years	44 (25–53)
Median male age, years	45 (28–60)
Median AMH level, ng/mL	0.38 ( $< 0.01$ –2.01)
Median BMI, kg/m²	21.1 (16.4–34.2)
Number of pregnancies	121 (46%)
Number of childbirths	26 (9%)
Median number of previous cycles	12 (1–64)
Median duration of infertility, months	52 (5–280)
Ovarian stimulation protocol
Antagonist protocol	150
Progestin-primed ovarian stimulation	27
Clomiphene citrate only or natural cycle	77
Agonist flare protocol	8

AMH $=$ anti-Mullerian hormone; BMI $=$ body mass index.

Table 2 presents the pre- and post-treatment parameters for this study. Each number represents the mean values for the 262 women. The numbers of retrieved oocytes and mature oocytes were significantly increased after PFC-FD^TM injection compared with before injection, as were the numbers of fertilized embryos (two pronuclei) with ICSI and cleavage embryos. The number of cases in which cleavage embryos increased by one or more after PFC-FD^TM injection was 27.9% (73/262). A breakdown of patient ages in 73 cases showed 0/11 (0%) patients under 34 years, 3/26 (11.5%) aged 35–39 years, 23/104 (22.1%) aged 40–44 years, 44/113 (38.9%) aged 45–49 years, and 3/8 (37.5%) aged 50 years or older. Also, the breakdown of ovarian stimulation methods in 73 cases was as follows: antagonist protocol 49/150 (32.7%), PPOS 9/27 (33.3%), oral medications only or natural 13/77 (16.9%), and agonist flare protocol 2/8 (25.0%).

**Table 2. Comparison of evaluation parameters before and after PFC-FD^TM injection, $n = 262$ .**
Parameter	Before injection	After injection	Pvalue
FSH level, mIU/mL	21.08±1.02	18.70±0.82	0.077
Antral follicle count	1.76±0.10	1.93±0.12	0.259
Number of retrieved oocytes	1.37±0.11	1.74±0.14	$< 0.001$ *
Number of mature oocytes	1.04±0.08	1.39±0.10	$< 0.001$ *
Number of fertilized embryos^a	0.70±0.07	1.06±0.08	$< 0.001$ *
Number of cleavage embryos	0.33±0.04	0.61±0.06	$< 0.001$ *
Number of blastocysts	0.14±0.03	0.20±0.03	0.069

The Wilcoxon signed-rank test was used to compare factors before and after PFC-FD^TM injection.

Statistical significance was set at $P < 0.05$ .

^aFertilized embryos $=$ 2 pronuclei with intracytoplasmic sperm injection.

Logistic regression analysis revealed that the factors contributing to the number of women cleavage embryos increasing by one or more after PFC-FD^TM injection were female age and oral medication only or natural cycle, and the number of fertilized embryos before injection (Table 3). Univariate regression analysis also showed a significant correlation between age and the number of cleavage embryos ( $P < 0.001$ ).

**Table 3. Factors that contributed to increasing the number of cleavage embryos by one or more after PFC-FD^TM injection, $n = 73$ .**
Factor	Odds ratio	95% Confidence interval	Pvalue
Female age, years	1.12	1.03–1.22	0.012^∗
AMH level, ng/mL	1.75	0.87–3.53	0.117
BMI, kg/m²	1.02	0.92–1.13	0.719
Number of pregnancies	1.02	0.53–1.94	0.956
Number of childbirths	0.39	0.11–1.33	0.131
FSH level, mIU/mL	0.99	0.96–1.01	0.158
Antral follicle count	1.02	0.81–1.28	0.833
Antagonist protocol	1.85	0.98–3.50	0.056
Oral medication only or a natural cycle	0.25	0.11–0.58	0.002*
Number of retrieved oocytes	1.32	0.86–2.03	0.209
Number of mature oocytes	0.58	0.30–1.12	0.106
Number of fertilized embryos^a	0.59	0.36–0.98	0.041*

Logistic regression analysis was used to determine contributing factors.

Statistical significance was set at $P < 0.05$ .

^aFertilized embryos $=$ 2 pronuclei with intracytoplasmic sperm injection. AMH $=$ anti-Mullerian hormone; BMI $=$ body mass index.

Stratified analysis of age revealed that the number of cleavage embryos retrieved from patients 40 years or older was significantly higher after PFC-FD^TM injection compared with the number retrieved before injection ( $0.68 \pm 0.07$ vs. $0.33 \pm 0.04$ , $P < 0.001$ ); there was no significant difference in patients younger than 40 years ( $0.22 \pm 0.10$ vs. $0.35 \pm 0.10$ , $P = 0.246$ ). A stratified analysis of the number of blastocysts before injection was difficult because of the large case-to-case variation.

One hundred and nine women successfully developed one or more embryos from oocyte retrieval 3 months after PFC-FD^TM injection. Among the 109 women, 71 have undergone embryo transfers so far. The remaining 38 women continued their oocyte retrieval cycles without embryo transfer. In patients who underwent embryo transfers, only one patient had a fresh embryo transfer and 70 had frozen-thawed embryo transfers. Fourteen frozen-thawed embryo transfers (25–47 years old) resulted in a pregnancy. Half of these, seven cases were in her 40s. As for pregnancy outcomes, one patient had a live birth, six had ongoing pregnancies, six had chemical abortions, and one had a miscarriage (Table 4).

**Table 4. Pregnancy outcomes of embryo transfers with embryos that developed within 3 months after injection of PFC-FD^TM, $n = 71$ .**
	Total	Under 40 years	40 years or older
Number of cases	71	8	63
Median age	44 (25–49)
Mean number of embryos transferred after the PFC-FD^TM injection	1.7
Positive pregnancy test*	19.7% (14/71)	87.5% (7/8)	11.1% (7/63)
Chemical abortion	8.5% (6/71)	37.5% (3/8)	4.8% (3/63)
Miscarriage	1.4% (1/71)	None	1.6% (1/63)
Ongoing pregnancy**	8.5% (6/71)	37.5% (3/8)	4.8% (3/63)
Live birth	1.4% (1/71)	100.0% (1/1)	None

Positive pregnancy test was defined as $hCG > 20$ mIU/mL on 16th day of ovulation or progesterone supplementation (Suzuki et al., 2023)

Ongoing pregnancy was defined as over 16 weeks’ of gestation.

No adverse events, such as intraperitoneal hemorrhage, infection, fever, and damage to other organs, were observed during or after the intraovarian injection of PFC-FD^TM.

DISCUSSION

In this study, the numbers of retrieved oocytes, mature oocytes, fertilized embryos, and cleavage embryos significantly increased after intraovarian PFC-FD^TM injection compared to those were retrieved before injection. These changes were remarkable in patients 40 years or older. The lack of significant changes in the number of blastocysts may be partly due to the small number of cases that aimed to reach the blastocyst stage. Contributing factors to the number of cleavage embryos increased by one or more after PFC-FD^TM injection were female age $\geq 40$ years and ovarian stimulation methods, and the number of fertilized embryos. It is unclear why the number of fertilized embryos contributed to the increase in the number of cleaved embryos, the presence or absence of fertilization failure may have been an influential factor. Women with poor ovarian response who are unsuitable for conventional ovarian stimulation are often treated with oral medications only or natural cycles. In the present study, these methods were also performed on women with poor ovarian response (median age: 44.5 years). They resulted in a significant increase in cleavage embryos compared to other ovarian stimulation methods. The stratified analysis of ages and ovarian stimulation methods suggests that older women may be a good indication for intra-ovarian injection of lyophilized PRP.

Biological pregnancy was confirmed in 14 cases, 19.7% of the total number of embryos transferred (71 cases). Seven of these patients were in their 40s, three of whom continued their pregnancies. In the analysis of cumulative live birth rates in low-prognosis patients according to the POSEIDON criteria by Li et al., live birth rates in the first cycle of POSEIDON groups 3 and 4 were 14.73% and 6.58%, respectively (Li et al., 2019). Previous studies have reported that intraovarian injection of PRP in patients with poor ovarian response diagnosed by POSEIDON criteria improved ovarian function, IVF, and pregnancy outcomes (Cakiroglu et al., 2022). The pregnancy rates for patients included in these studies were reported to be 14.6% (Farimani et al., 2021) and 19.5% (Cakiroglu et al., 2022). The present study also showed an increase in the number of fertilized embryos and cleavage embryos in patients with ovarian dysfunction; pregnancy rates were similar to those reported in the past. Since the patients were women with poor ovarian response who also fall into POSEIDON groups 3 and 4, the pregnancy rate in this study was not low. Therefore, PRP (previous reports) and lyophilized PRP (current data) may be equally effective in poor ovarian response regarding pregnancy rate.

At our institute, many patients who require artificial reproductive technology treatment are older than 40 years, reflecting the situation throughout Japan. The Japan Society of Obstetrics and Gynecology reported that 449,900 cycles of artificial reproductive technology were performed in 2020. According to the ART data book 2020 published by the Japan Society of Obstetrics and Gynecology, in approximately 40% of the cycles, the patients were 40 years or older. Fertility decreases in women over the age of 35 years (Balasch, 2010; Tatone, 2008); the decrease in fertility with age is related to both a decrease in the number of available oocytes and an increase in the aneuploidy of oocytes (Pellestor et al., 2003). Consequently, oocyte donation is favored as maternal age increases and ovarian reserve decreases (Cabry et al., 2014; Serour et al., 2010). However, oocyte donation is not practically available in Japan, as no established laws or guidelines exist for oocyte donation. Therefore, autologous oocytes must be used. The underlying ovarian dysfunction that causes infertility is often difficult to address with artificial reproductive technology, resulting in a growing interest in treatments that may rejuvenate dysfunctional ovaries.

Stem cell therapy, autologous ovarian transplantation, artificial ovaries, artificial gametes, mitochondrial transplantation, and PRP are candidates for improving ovarian function (Sfakianoudis et al., 2020b). PRP injection, however, is the most realistic method considering cost, level of invasiveness, adverse effects, and ethical issues (Sfakianoudis et al., 2020b).

To date, including the most recent report by (Patel et al., 2023), 13 case reports and 18 cohort studies have been published regarding intraovarian injection of PRP. The study participants had primary ovarian insufficiency, premature ovarian failure, or poor ovarian response, or were perimenopausal or menopausal. The methods detailed in these studies were inconsistent in terms of volumes of blood drawn, infusion volumes, and parameters for evaluation (Atkinson et al., 2021; Hajipour et al., 2021; Pacu et al., 2021; Panda et al., 2020; Seckin et al., 2022; Sfakianoudis et al., 2020a). Improvements in several parameters (i.e., AMH and follicle stimulating hormone levels, AFC, follicle count, retrieved oocytes, resumption of the menstrual cycle, clinical pregnancy rate, and live birth rate) were also inconsistent among the studies. Since no randomized controlled studies have been reported, the true efficacy of intraovarian injection with PRP is unclear.

Of the 18 cohort studies on intraovarian PRP injection, only two used control groups (Melo et al., 2020; Stojkovska et al., 2019); sham injection was not performed in the control groups. Mechanical stretching and/or mild injury to the ovary following the puncture injection procedure may elicit several inflammatory responses sufficient to temporarily restore ovarian function (Atkinson et al., 2021). Laparoscopic ovarian drilling is a treatment option for clomiphene-resistant polycystic ovarian syndrome (Lebbi et al., 2015), and the ovarian puncture PRP injection procedure may provide similar stimulation and contribute to the success of the injection. However, there is evidence for the effect of PRP injection alone. A report examined ovarian rejuvenation by intraovarian PRP injection in a rat model of premature ovarian dysfunction induced by gonadotoxic chemicals. This report compared intraovarian injections of PRP with saline solution in a rat model of premature ovarian dysfunction. In the PRP-injected group, protection of morphologically normal follicles from degeneration and atresia caused by gonadotoxic chemicals and stimulation of angiogenesis was observed.

Moreover, the PRP-injected group showed tissue rejuvenation, inflammation regulation, minimizing histopathological damages, and promoting litter counts. No such changes were observed in the saline infusion group. Therefore, it was demonstrated that PRP could partially restore the function of the ovaries in a state of premature ovarian dysfunction, not due to mechanical stimulation by the puncture injection procedure (Ahmadian et al., 2020).

The mechanism by which PRP improves ovarian function is considered to have two possible courses: (1) dormant follicles start to develop, and (2) new oocytes (follicles) are created from stem cells in the ovaries (Seckin et al., 2022). The first possibility is based on the finding that menopausal women have about 1,000 dormant follicles remaining in their ovaries (Nelson, 2009), and the second on the finding that the ovaries contain stem cells (Atkinson et al., 2021; White et al., 2012).

Lyophilized PRP has been shown to be stable at room temperature through quantification of platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor, and endothelial growth factor (da Silva et al., 2018; Shiga et al., 2017), which stimulated the proliferation of human umbilical endothelial cells or fibroblasts (da Silva et al., 2018). Using porcine PRP, the levels of transforming growth factor- $β$ 1 and vascular endothelial growth factor increased significantly in lyophilized PRP after Ca-activation compared to fresh PRP, Ca-activated PRP, or Ca-activated PRP after lyophilization (Pan et al., 2016). Lyophilized PRP has been utilized for tissue regenerative therapies, wound healing, and injection into the uterine cavity (Gangaraju et al., 2023; Ngah et al., 2021; Saputro et al., 2022), but no reported studies on intraovarian injection of lyophilized PRP exist.

The differences between conventional and lyophilized PRP are summarized in Table 5. The presence or absence of cell components can determine differences in terms of legal regulations. Lyophilized PRP is a humoral therapy and is therefore not subject to the Ensuring Safety of Regenerative Medicine in Japan Act. This means that lyophilized PRP is not legally regulated in Japan—which eliminates the need for complicated paperwork and procedures—and can easily be adopted by many facilities in Japan.

**Table 5. Differences between conventional and lyophilized platelet-rich plasma.**
Factor	Conventional platelet-rich plasma $^{a}$	Lyophilized platelet-rich plasma $^{b}$
Cell components	Yes	No
Required time for production	The day of drawing blood	3 weeks
Expiration date for use	The day of drawing blood	6 months at room temperature
Surgeon in charge	Application is necessary for each doctor	Not restricted
Administration site	Designation is necessary beforehand	Not restricted
Legal regulations	Yes	No

^aConventional platelet-rich plasma $=$ fresh platelet-rich plasma.

^bLyophilized platelet-rich plasma $=$ platelet-derived factor concentrates freeze-drying (PFC-FD^TM).

This is the first study to examine the effect of intraovarian injection of lyophilized PRP in patients with poor ovarian responses. However, this study has some limitations. This is a retrospective study without any control group. The final treatment goals— pregnancy and childbirth—were studied in a small number of cases because many of the cases used in this study had not yet reached the stage of embryo transfer.

Since this is a new treatment that is available, it is necessary to establish an optimal procedure for PFC-FD^TM injection, such as site, volume, and intervals between injection administration. Future clinical research using randomized controlled trials with a sham injection group is necessary to demonstrate the effectiveness of PRP and PFC-FD^TM injection.

CONCLUSIONS

We retrospectively showed the effect of intraovarian PFC-FD^TM injection in women with poor ovarian response. The numbers of retrieved oocytes, mature oocytes, fertilized embryos, and cleavage embryos were significantly increased after intraovarian PFC-FD^TM injection compared to before injection. These changes were remarkable in patients 40 years and older. Therefore, women of advanced age might be a good indication for intraovarian injection of lyophilized PRP. However, this add-on should not be recommended outside strict research protocols for infertile women until more data on efficacy and safety, especially from randomized settings, are available.

DISCLOSURES

Conflict of Interest Statement

All authors declare no conflict of interests for this article.

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 with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients for inclusion in the study.

Animal Studies

None.

Approval by Ethics Committee

Institutional Review Board approval was obtained (Reproduction Clinic Internal Review Board number RT20131882).

Clinical Trial Registry: Not applicable

ACKNOWLEDGEMENTS

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

ORCID

Aya Karino https://orcid.org/0000-0002-3876-8352

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Vol. 06, No. 02

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Received 10 June 2023

Accepted 3 March 2024

Published: 3 June 2024

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This is an Open Access article published by World Scientific Publishing Company. It is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 (CC BY-NC-ND) License which permits use, distribution and reproduction, provided that the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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