Introduction
Patients who undergo more than three unsuccessful embryo transfer (ET) attempts despite having good-quality embryos (GQEs), whether in fresh or frozen-thawed cycles, are typically diagnosed with recurrent implantation failure (RIF). RIF is a multifactorial condition influenced by maternal age, lifestyle behaviors (e.g., smoking), body mass index, stress, cytokine levels, immunological factors, ovarian and uterine factors, sperm characteristics, genetic factors, and endometrial receptivity [1,2]. In recent years, growth factors such as granulocyte–macrophage colony-stimulating factor have been incorporated into strategies for cultivating human embryos from patients with RIF [3,4], and co-culture systems using human-derived cells such as endometrial cells [5,6] have been designed to improve embryo quality and increase the likelihood of a successful pregnancy.
Platelet-rich plasma (PRP) is a blood plasma fraction with high concentrations of growth factors, such as platelet-derived growth factor, vascular endothelial growth factor (VEGF), transforming growth factor (TGF), and epidermal growth factor (EGF), as well as cytokines. These growth factors are stored in platelet alpha granules, promote cell proliferation and differentiation, and contribute to various processes, including anticancer effects and angiogenesis [7-9]. The concentrations of these factors in PRP are typically three to five times higher than in regular plasma. Due to its capacity to support natural healing and tissue regeneration, PRP is currently used in clinical applications for pain reduction, inflammation control, autologous cartilage regeneration, and joint tissue repair [10]. PRP has also been proposed as an approach to increase pregnancy rates among individuals undergoing in vitro fertilization (IVF).
Recent studies have shown that PRP injections significantly increase endometrial thickness in women with thin endometrium (<7 mm), leading to more pregnancies in IVF treatment cycles [9] and frozen-thawed ET cycles [11]. Furthermore, research indicates that the administration of autologous PRP into the ovaries of patients with poor response not only has a positive effect on the growth and differentiation of various cell types but also improves pregnancy outcomes [12].
Studies of immature caprine [13] and bovine [14,15] oocytes have supported PRP as an effective substitute for serum during in vitro oocyte maturation that helps preserve mitochondrial activity in mature oocytes. Moreover, the use of PRP during human sperm incubation reduces DNA fragmentation and oxidative stress while improving sperm motility and viability [16]. These studies suggest that the various cytokines and growth factors present in PRP hold promise as novel supplements to culture media for human reproduction. However, no reports are yet available on the effect of incorporating PRP into embryo culture media on the development of human embryos in IVF treatment cycles.
Therefore, in this study, we investigated the effects of introducing autologous PRP into human embryo culture medium by evaluating its impact on embryo development and pregnancy rates among individuals with RIF.
Methods
1. Study population
This observational pilot study included patients with RIF who underwent IVF cycles at Pyeongchon Maria IVF Center in the Republic of Korea between June 2022 and May 2023. Using G*Power 3.1.9.7 (University of Düsseldorf), the significance level for regression analysis was set at 0.05, the medium effect size at 0.5, and the power at 0.80, in accordance with Cohen’s law. Assuming one tested predictor, the minimum required sample size was 128 [17]. Individuals with RIF were considered eligible if they had undergone at least three consecutive unsuccessful IVF cycles, intracytoplasmic sperm injection (ICSI) treatments, or frozen-thawed ETs despite having four or more high-quality embryos [18-20]. Accordingly, patients who had undergone ET for more than three cycles through previous in vitro procedures but did not conceive were selected first. A total of 244 patients with RIF underwent IVF during the study period. Among these patients, 32 were excluded due to clear uterine structural factors (e.g., congenital uterine anomalies), a history of viral diseases or sexually transmitted diseases, or a requirement for cryopreservation of all embryos. In total, 77 patients who consented to the PRP protocol were included in the PRP group, while 124 who did not consent or did not respond were included in the control group. To analyze the effect of PRP on clinical outcomes, patients were divided into groups based on common female infertility factors (tubal issues, endometriosis, poor ovarian response, and polycystic ovary syndrome [PCOS]), male factors, multiple factors, and unexplained factors.
Eligible patients were informed about the use of PRP, and written informed consent was obtained from all participating patients (Figure 1). This study was conducted in compliance with internationally recognized standards for research practices and reporting and with the 1964 Helsinki Declaration. Ethical approval for this study was obtained from the Maria Fertility Hospital Institutional Review Board (Maria IRB No. 2022-004). All ongoing and related trials in the study were registered (Clinical Research Information Service: KCT0008597; http://cris.nih.go.kr/cris/search/search_result_st01.jsp?seq=24911).
2. Stimulation protocols
In this study, ovarian stimulation was performed using either a gonadotropin-releasing hormone (GnRH) agonist or antagonist protocol. Recombinant follicle-stimulating hormone (Gonal-F, Merck Serono; or Follitrope, LG Life Sciences) was administered daily at doses of 150 to 450 IU. The dose was determined based on factors such as patient age, anti-Müllerian hormone level, and response to follicular stimulation. In the agonist group, a GnRH agonist was administered subcutaneously 10 days before the start of the menstrual cycle, followed by gonadotropin beginning 14 days after agonist injection.
Conversely, in the antagonist group, gonadotropin administration commenced on day 3 of the menstrual cycle, and a GnRH antagonist such as Cetrotide (Merck Serono) or ganirelix (Ganilever; LG Life Sciences) was injected subcutaneously once the follicles reached 13 mm, continuing daily until follicle maturation was induced. After 5 days of gonadotropin stimulation, the first ultrasound assessment was performed. If ultrasound examination showed that one or more follicles had reached 18 mm or that three or more follicles had reached 16 mm, recombinant human chorionic gonadotropin (Ovidrel; Merck Serono) was administered to trigger ovulation. Luteal phase support was provided with vaginal and oral progesterone starting the day after oocyte retrieval.
3. Preparation of PRP
PRP was obtained from blood samples collected from patients who consented to PRP culture on the day of oocyte retrieval. To separate PRP from blood, a 13-cc blood sample was obtained using a 20-cc syringe coated with 2 cc of anticoagulant. This sample was then transferred to a PRP centrifugal separation kit (Ycellbio Medical Co. Ltd.) and centrifuged at 1,200 ×g for 3 to 5 minutes. Centrifugation yielded a buffy coat forming a monolayer between the plasma and blood cells, which was designated as PRP. The final concentration was adjusted to 1×109 platelets/mL using a hemocytometer [21] and activated by freezing at −196 °C and thawing at 4 °C three times [22,23]. The platelet bodies were then separated via centrifugation at 3,000 ×g for 30 minutes, and the product was stored at 4 °C until use. To prepare the autologous PRP-supplemented culture medium, 50 µL of the recovered PRP supernatant was thoroughly mixed with 5 mL of culture medium (GEMS one-step media; Merck Serono). This mixture was filtered through a 0.2-µm membrane filter to remove aggregates and suspended matter, yielding the final autologous PRP-enhanced culture medium. The medium included 5% PRP [13].
4. Fertilization, embryo culture, and ET
Fertilization was carried out according to patient-specific indications using conventional insemination or ICSI. Fertilization was assessed 16 hours after insemination by the presence of two distinct pronuclei. Zygotes were cultured in groups of four or fewer in a 30-µL droplet of GEMS one-step media under paraffin oil. After ET, surplus embryos were cultured to the blastocyst stage.
PRP was not added to the media used for washing, fertilization, or transfer; it was added only to the culture medium. Embryos in both groups were cultured under identical conditions (37 °C, 5% O2, 6% CO2). ET was performed on day 3 under abdominal ultrasound guidance following the assessment of embryo quality. The number of embryos transferred (with a maximum of three) was determined based on embryo condition and patient age.
5. Morphological assessment and clinical outcomes
Embryo quality was evaluated based on the blastomere number and uniformity, degree of fragmentation, presence or absence of multinucleation, and cytoplasmic morphology [24]. To minimize intra- and interobserver variation, three highly experienced embryologists performed the assessments. Embryos with six to eight blastomeres of uniform size, no multinucleation, and <10% fragmentation were classified as grade A; embryos with <25% fragmentation together with two to three nonuniform blastomeres or one to two multinucleated blastomeres were classified as grade B. All other embryos were classified as low-quality (grade C or below). In this study, GQEs were defined as those with at least six cells on the day of ET (grades A and B). Top-quality embryos were defined as eight-cell embryos of good quality (grade A). Chemical pregnancy was defined as a positive serum human chorionic gonadotropin test on day 14 after oocyte collection, while clinical pregnancy was considered to be indicated by the presence of a distinct gestational sac on ultrasound on day 28. Implantation rate was calculated as the total number of gestational sacs divided by the total number of embryos transferred. Ongoing pregnancies, multiple pregnancies, miscarriages, and ectopic pregnancies were also recorded for the final analysis.
6. Statistical analysis
Data were analyzed with IBM SPSS Statistics ver. 28 (IBM Corp.). Between-group comparisons were performed using the chi-square test or Fisher exact test. The Student t-test was used to compare continuous variables between groups, and p-values of less than 0.05 were considered to indicate statistical significance. Data are also presented as odds ratios with 95% confidence intervals.
Results
A total of 201 patients with RIF who underwent a fresh IVF‒ET cycle with day-3 transfer were included: 77 in the PRP group and 124 in the control group. The PRP and control groups were compared regarding female age, infertility duration, number of IVF cycles, proportion of ICSI, stimulation period or protocol type, and ovarian response to stimulation. No significant differences in these baseline characteristics were observed between groups (Table 1). Additionally, when causes of infertility were compared, no between-group differences were found for female factors (57/77 [74.0%] vs. 90/124 [72.6%]), male factors (4/77 [5.2%] vs. 6/124 [4.8%]), multiple factors (7/77 [9.1%] vs. 7/124 [5.7%]), or unexplained factors (9/77 [11.7%] vs. 21/124 [16.9%]). Among female factors, the rates of tubal issues, PCOS, poor response, and endometriosis did not differ significantly between groups (Table 1).
The embryological findings are summarized in Table 2. The numbers of oocytes retrieved and the fertilization and cleavage rates exhibited no significant differences between groups. However, the percentage of GQEs with at least six cells on day 3 was higher in the PRP group (154/346 [44.5%]) than among the control patients (205/547 [37.5%]) (p=0.041). In the PRP group, the mean number of top-quality transferred embryos was significantly greater than in the control group (0.5±0.7 vs. 0.4±0.6, p=0.039), whereas the mean number of blastomeres per transferred embryo did not differ significantly between groups.
In a comparison of clinical outcomes between PRP and control groups, the clinical pregnancy rate (26/77 [33.8%] vs. 23/124 [18.5%], p=0.015), implantation rate (28/188 [14.9%] vs. 26/300 [8.7%], p=0.033), and ongoing pregnancy rate (19/77 [24.7%] vs. 14/124 [11.3%], p=0.013) were all significantly higher in the PRP group than in the control group (Table 3). When outcomes were analyzed by infertility etiology, clinical pregnancy rates were significantly higher in the PRP group for tubal factor infertility (5/11 [45.5%] vs. 1/20 [5.0%], p=0.006) and for unexplained infertility (6/9 [66.7%] vs. 3/21 [14.3%], p=0.004). Miscarriage rates were similar between groups, indicating no significant effect of PRP-supplemented culture on miscarriage. We also assessed births to date and confirmed normal births in 16 of the 19 ongoing pregnancies in the PRP group and in 10 of the 12 ongoing pregnancies in the control group. Among the five cases that could not be confirmed, most patients did not consent to release information about the final birth outcome (Table 4).
Discussion
In vivo, normally fertilized embryos achieve stable implantation and successful pregnancy through interactions with a variety of substances, including cytokines, growth factors, and transcription factors, as well as with fallopian tube and uterine cells [25]. However, in vitro conditions have certain limitations, which can reduce pregnancy success. To address these limitations, the use of PRP has been explored in many treatments and studies related to infertility [26-29]. To date, the direct relationships between the various constituents of PRP and pregnancy outcomes remain unclear [26]. Furthermore, few studies have directly used PRP in embryo culture.
This study focused on human embryo culture. We found that the use of PRP improved embryo quality, pregnancy rates, and implantation rates, and we confirmed successful birth outcomes without birth defects. Notably, the positive effect of PRP was particularly pronounced in patients with tubal factor infertility and unexplained infertility. Further research is needed to confirm that PRP supplementation has a beneficial effect in these groups. Although not included in the data presented, one birth occurred after a freeze-thaw cycle. These findings suggest that PRP can be applied not only in fresh cycles but also in freeze-thaw cycles.
A recent study by Hassaneen et al. [30] evaluated the use of allogeneic PRP in bovine oocyte IVF and in vitro culture by supplementing in vitro maturation and development media with allogeneic PRP (5%), fetal calf serum (FCS; 5%), mixed PRP+FCS (2.5%+2.5%), or platelet-poor plasma (PPP; 5%) and assessing embryo development in high- and low-quality oocytes. For good-quality oocytes, the PRP group had significantly higher cleavage (p<0.05) and blastocyst (p<0.01) rates. Among low-quality oocytes, the PRP-alone and PRP+FCS groups had higher cleavage rates than the PPP group (p<0.05). The blastocyst rate for good-quality oocytes was also higher in the PRP group than in the FCS and PPP groups, indicating that allogeneic PRP is effective in stimulating bovine oocyte maturation and embryo development in vitro. These findings align with those of the present study and support our conclusion regarding the usefulness of PRP supplementation in both animals and humans.
Molecular-level studies examining how growth factors in PRP may affect embryo development and clinical outcomes remain to be conducted. However, prior research allows us to speculate that growth factors present in PRP—such as EGF, TGF, fibroblast growth factor (FGF), and insulin-like growth factor (IGF)—positively influence embryonic development and clinical outcomes. Cleavage-stage blastomeres, the inner cell mass, and the trophectoderm (TE) of the human embryo express receptors for EGF, TGF, FGF, and IGF [31,32]. These receptors‚ which are located on the cell membrane, bind signaling molecules, triggering intracellular events that influence cell behavior and ultimately shape embryonic development. This development is a complex process that requires precise spatial and temporal regulation of cellular processes, including cell migration, differentiation, and proliferation, and depends on coordinated and rapid cellular communication [33]. In vitro research has demonstrated that the addition of IGF-1 to culture medium activates the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway and increases the rate of blastocyst development in humans [34]. Similar results have been reported with IGF-2 in humans and cattle [35]. These results indicate that the IGF system is related to placental, fetal, and postnatal growth in mammals and is also involved in cellular differentiation in early embryos.
In bovine embryos, supplementing in vitro culture with EGF increases the embryonic development rate and TE capacity for invasion [36]. EGF alone does not improve blastocyst development in cattle, with similar findings reported in sheep. Some studies have demonstrated expression of EGF and its receptors in the early embryos of mice, rabbits, sheep, and pigs, but this expression has not been noted in other mammals, such as goats [37]. In vitro, the addition of TGF increases the rate of blastocyst development in sheep, goats, and cattle [26,38]. In early bovine embryos (two- to eight-cell stages), expression of TGF has been shown to increase the relative abundance of Nanog (a transcription factor important in embryonic stem cells), suggesting an early role of TGF [39]. The expression of the FGF-2 receptor is evident in mouse blastocysts but not in human blastocysts [40]. These data suggest that TE expansion in early development depends on FGF in mice, whereas FGF-dependent expansion in humans occurs later. FGF family members also play key roles in the early development of pig embryos. FGF-4 regulates TE formation and elongation, and the level of FGF-2 increases in endometrial tissues between days 15 and 20 of pregnancy, supporting its relevance in embryo elongation and implantation [41]. In vitro studies have shown that VEGF supplementation in bovine and pig embryo cultures increases cytoplasmic maturation and blastocyst development rates [42]. Overall, studies on the functions of the growth factors described above could provide direct or indirect evidence that the addition of PRP to the culture medium positively impacts human embryo development and clinical outcomes. In addition, the autocrine and paracrine effectors present in PRP warrant further investigation regarding their effects on embryo development.
This study evaluated various growth factors through the addition of autologous PRP; thus, each embryo with potential deficiencies could utilize the growth factors it needed. Although concerns exist about exposing embryos to the multiple growth factors present in PRP, we observed a higher ongoing pregnancy rate in the PRP group than in the group without PRP. In addition, the likelihood of adverse outcomes appeared low, as no fetal abnormalities were observed among patients whose embryos were cultured with PRP. However, given the limited clinical application in this study, long-term observation with broader clinical use is necessary.
The incorporation of cytokines or other growth factors into cell culture necessitates a precise approach to identify the most effective and suitable concentration. This precision is essential because the optimal concentration can vary by cell type, sometimes even within the picomolar or nanomolar range. In this study, we set the culture concentration based on previous research [13]. However, the amount of blood collected from patients was limited, and the PRP yield after final processing varied widely among patients. In some cases, very little PRP was added to the culture; therefore, subsequent studies should establish an acceptable concentration range for PRP supplementation. Notably, embryo culture carries inherent risks and constraints, necessitating careful consideration of the ethical dimensions associated with embryo research. In the context of this study, the use of autologous PRP—a substance derived from each patient’s own body—helped mitigate potential pathological concerns during embryo culture.
In conclusion, this study supports that the addition of PRP to conventional human embryo culture systems can be effective and useful. Our findings suggest an alternative avenue for addressing infertility, offering a new opportunity for pregnancy in a specific group of patients with RIF. Further follow-up studies with larger cohorts and individual tracking of embryo development should be conducted to provide more statistically and practically significant conclusions. In addition to RIF cycles, we propose applying PRP in various specific protocols, such as those for patients with a poor response or immature IVF cycles.
PDF Links
PubReader
ePub Link
XML Download
Full text via DOI
Full text via PMC
Download Citation
