Investigation of the effect of intrauterine injection of platelet-rich plasma (PRP) on the results of in vitro fertilization (IVF): A cohort study

Farahnazsadat Ahmadi: Validation, Supervision, Project administration, Conceptualization. Fatemeh Nakhaei: Writing – original draft, Funding acquisition, Data curation. Mahboubeh Tajaldini: Writing – original draft, Investigation.

The mean number of previous failed embryo transfer cycles was 3.4 ± 0.7 in the control group and 3.3 ± 0.6 in the PRP group (P = 0.452), with all patients having experienced at least two blastocyst transfers or three cleavage-stage transfers without achieving clinical pregnancy. In this retrospective cohort study, a total of 100 women with RIF who were candidates for FET cycles were studied. The results of the study showed that intrauterine injection of PRP significantly increased pregnancy rates in women with RIF, especially in subgroups with primary infertility or pure IVF treatment. This effect was independent of endometrial thickness and hormonal parameters and was associated with minimal side effects. These findings provide a stronger basis for the clinical use of PRP in infertility centers. Table 1 summarizes the overall demographic and clinical characteristics of the 100 women enrolled in the study. The mean age of participants was 31.76 years (range 21–42), and the average BMI was 24.94. The duration of infertility ranged from 1 to 16 years, with a mean of 5.05 years. Thyroid function, assessed by TSH levels, showed a mean value of 2.33. These data indicate a relatively homogeneous study population with respect to age, BMI, infertility duration, and thyroid status ( Table 1 ). Table 1 Baseline characteristics and comparison between control and PRP groups. Variable Control (n = 49) PRP (n = 51) P-Value Age (years), mean ± SD 31.9 ± 5.6 31.6 ± 5.8 0.792* BMI (kg/m²), mean ± SD 25 ± 3.1 24.9 ± 3.1 0.871* Years of infertility, mean ± SD 5.1 ± 3.6 5.0 ± 3.5 0.889* TSH (mIU/L), mean ± SD 2.24 ± 1.3 2.41 ± 1.28 0.508* *Independent t -test. Baseline characteristics and comparison between control and PRP groups. *Independent t -test. The distribution of hysteroscopy history, hypothyroidism, levothyroxine use, type of infertility (primary vs. secondary), and menstrual cycle regularity was similar between groups. The results of the chi-square test showed that there was a significant difference between the two groups only in terms of the rate of hysteroscopy reported and the type of treatment (P-Value 0.05) ( Table 2 ). Table 2 Distribution of baseline characteristics of study participants and comparison between the two groups. Variable Total Groups Control (n = 49) PRP (n = 51) Number Percentage Number Percentage Number Percentage Hysteroscopy No 56 56.0 28 57.2 28 54.9 Yes 44 44.0 21 42.8 23 45.1 Hypothyroid No 68 68.0 35 71.4 33 64.7 Yes 32 32.0 14 28.6 18 35.3 Levothyroxine No 69 69.0 35 71.4 34 66.7 Yes 31 31.0 14 28.6 17 33.3 Type of infertility primary 59 59.0 37 75.5 30 58.8 secondary 41 41.0 12 24.5 21 41.2 Menstrual cycle type Regular 78 78.0 38 77.6 40 78.4 Irregular 22 22.0 11 22.4 11 21.6 Distribution of baseline characteristics of study participants and comparison between the two groups. Comparison between the two groups in terms of the distribution of other basic characteristics of the study participants: The results of the t -test showed that there was no statistically significant difference between the two groups in terms of average age, body mass index, number of years of infertility, and TSH hormone levels, indicating proper matching between the two groups (P-Value>0.05). Data are stratified by infertility type (primary vs. secondary) because these subgroups may differ in underlying etiology of RIF and baseline reproductive history (e.g., gravida, as shown in Table 3 ). Stratification allows assessment of whether the PRP treatment effect is consistent across these clinically distinct populations. Number of embryos transferred did not differ significantly between groups in either infertility type. All embryos were transferred at the blastocyst stage and were matched for quality and screened for other diseases, ensuring comparability. Gravida differed significantly in primary infertility (p = 0.002), reflecting expected baseline reproductive history differences rather than treatment effect. AMH, FSH, and TSH levels showed no significant differences between groups in either infertility category. Overall, Table 3 shows that ovarian reserve markers, hormonal profiles, and endometrial response were comparable between groups, with the exception of embryo count in secondary infertility ( Table 3 ). Table 3 Comparison of responses between the two groups participating in the study by type of infertility. Variable Type of infertility Groups t -test P-value Control PRP Average SD Average SD Endometrial thickness (mm) primary 9.91 1.56 9.98 1.45 -.217 .829 secondary 10.17 1.40 9.42 1.86 1.193 .243 Number of ova retrieved (n) primary 16.30 11.61 16.72 11.15 -.153 .879 secondary 20.67 18.31 11.95 7.00 1.579 .138 Number of embryos produced (n) primary 9.73 8.13 11.53 8.70 -.889 .377 secondary 15.75 12.86 7.11 5.18 2.217 .045 Number of embryos transferred (n) primary 3.51 .96 3.59 1.04 -.332 .741 secondary 3.83 1.11 3.11 1.20 1.693 .101 Gravida (number of previous pregnancies) primary .54 1.85 .03 .18 3.481 .002 secondary 1.42 .79 .47 .70 1.666 .104 Anti-Müllerian hormone – AMH (ng/mL) primary 5.66 4.14 5.43 4.81 0.20 0.834 secondary 3.49 3.30 2.72 2.79 .65 0.521 Follicle-stimulating hormone – FSH (mIU/mL) primary 7.09 3.21 6.41 3.96 0.78 0.43 secondary 5.65 1.38 6.73 2.64 −1.465 0.15 Thyroid-stimulating hormone – TSH (mIU/L) primary 2.24 1.27 2.33 1.10 -.289 .774 secondary 1.78 1.32 2.84 1.51 −2.005 .054 Comparison of responses between the two groups participating in the study by type of infertility. Given the significant difference in gravida between groups among women with primary infertility (P = 0.002, Table 3 ), we performed a logistic regression analysis to adjust for this baseline imbalance. After controlling for gravida, the association between PRP injection and clinical pregnancy remained statistically significant (adjusted OR = 4.12; 95% CI: 1.25–13.58; P = 0.019), confirming that the treatment effect was independent of gravida status. Table 4 summarizes pregnancy outcomes in the two groups. Table 4 Comparison of fertility outcomes between the two study groups. Groups Total P-Value Control PRP Pregnancy outcomes Did not conceive 33 28 61 0.089 Full term and cesarean section 7 11 18 Still pregnant 3 3 6 Full term and vaginal delivery 0 3 3 Twins/preterm and vaginal delivery 3 4 7 Miscarriage 2 1 3 PPROM and neonatal death 1 1 2 Total 49 51 100 Comparison of fertility outcomes between the two study groups. The proportion of women who did not conceive was slightly higher in the control group (33 vs. 28). The case group showed numerically higher rates of full‑term vaginal delivery, cesarean delivery, and twin/preterm vaginal delivery. Miscarriage and neonatal loss rates were low and similar between groups. The overall distribution of pregnancy outcomes did not reach statistical significance (p = 0.089). This indicates a trend toward improved pregnancy outcomes in the case group, although the difference was not statistically significant ( Table 4 ). Table 5 examines pregnancy success and failure separately for primary and secondary infertility. Table 5 Pregnancy by type of infertility in two study groups. Groups P-Value Control PRP Total failure of pregnancy Primary 20 18 38 0.045 Secondary 13 10 23 pregnancy Primary 10 15 25 0.650 Secondary 6 8 14 Pregnancy by type of infertility in two study groups. The difference in failure rates between groups was statistically significant (p = 0.045), indicating fewer failures in the case group. This table suggests that the intervention may have reduced pregnancy failure rates, particularly in primary infertility, although overall pregnancy rates were not significantly different. but no significant relationship was seen in the pregnant group (P-Value>0.05) (Table 6).

F.Ahmadi: Supervised the study throughout its development, guided the interpretation of findings, Contributed to the conception and design of the study, approved the final version of the manuscript, F. Nakhaei: Collected and organized clinical data, performed initial data analysis, drafted the manuscript, and approved the final version. M. Tajaldini : Assisted in statistical analysis, contributed to literature review and manuscript editing, approved the final version.

The current retrospective cohort study was carried out at the Nahal Infertility Center, Sayyad Shirazi Hospital, Golestan University of Medical Sciences, Gorgan, Iran during the period of January-December 2023 (Bahman-Azar 1402 Persian calendar). The Institutional Review Board (IRB) of Golestan University of Medical Sciences gave approval to the study protocol (ethical code: IR.GOUMS.REC.1403.220) and carried out the study following the Declaration of Helsinki. Informed consent was not obtained written because the analysis was retrospective, and all patients had already given general consent to the use of de-identified information in research on admission. Medical data of all infertile women with frozen-thawed embryo transfer (FET) cycles were filtered. Inclusion criteria were: (1) age 21–42 years; (2) primary or secondary infertility over 12 months; (3) recurrent implantation failure (RIF), defined as failure to achieve clinical pregnancy after at least three transfers of high-quality cleavage-stage embryos or after two transfers of blastocysts (day 5/6 grade A or B) in at least two separate IVF/FET cycles, in accordance with the European Society of Human Reproduction and Embryology (ESHRE) working group recommendations; (4) normal hysterosalpingography or diagnostic hysteroscopy (less than 12 months prior to index cycle); (5) endometrial thickness ≥ 7 mm on day of progesterone introduction; (6) body-mass index (BMI) < 30 kg/m². The exclusion criteria included: endometriosis, untreated hydrosalpinx, congenital anomalies of the uterus, submucous myomas greater than 2 cm, intraoperative adhesions, autoimmune diseases, or incomplete electronic medical records. A minimum sample of 94 subjects was calculated with the use of the OpenEpi software (v. 3.01) and was based on the difference in clinical pregnancy rate observed in a pilot local dataset (27 vs. 8) to give 80 % power with an alpha of 0.05 (two-sided). A total of 100 eligible cycles (51 PRP, 49 control) were eventually analyzed to compensate 5% possible loss to follow-up. Each subject was given a standardized hormone-replacement therapy (HRT) protocol of endometrial priming: oral estradiol valerate (Aburaihan Co., Iran) 6 mg daily from cycle day 2, and raised to 8 mg after 7 days in case the endometrial thickness was less than 7 mm. Endometrial monitoring was done through trans-vaginal ultrasound every 3–4 days starting on the 10 th day. In case of the endometrial thickness of ≥ 7 mm and with at least one leading follicle of not more than 10 mm, intramuscular progesterone (50 mg day −1 ; Caspian Tamin, Iran) was administered and embryo transfer was planned on the 6th day of the progesterone administration. Vitrification of all embryos had been carried out on day 5/6 (blastocyst stage). In case the pregnancy was confirmed, luteal-phase support by progesterone was kept up until 12 gestation weeks. The preparation of autologous PRP was done in the morning of progesterone initiation (day −6 of FET). A 17 mL portion of peripheral blood was poured into two 10 mL tubes with 1 mL of acid-citrate-dextrose solution A (ACD-A, 3.2 months). Erythrocytes separation was done using the centrifugation technique at 1200 × g and 12 min at room temperature. The topmost layer of the plasma was pipetted into sterile tubes and centrifuged again at 1000x g /10 min. Sterile collection of the lower 34 mL of platelet-rich layer (35x platelet baseline concentration) was taken. In 30 min of preparation, 0.5–0.7 mL PRP were infused gradually into the uterine cavity with the help of an IUI catheter (Laboratoire CCD, France) with the aid of ultrasound. The patients were kept on a supine position of 15 min and discharged. To ensure that the embryology team was blinded, the control group was subject to empty catheteric transfer. Primary outcome: A clinical pregnancy which is the existence of no less than one intrauterine gestational sac containing a heartbeat at 6–7 weeks of gestation. Secondary outcomes: Biochemical pregnancy (= 25 IU L −14 days after transfer), continuing pregnancy (= 12 weeks), implantation (= number of sacs per number of embryos transferred), miscarriage, live-birth, and neonatal outcomes. The endometrial thickness was noted at progesterone intake day. Two blinded researchers extracted all the data out of the electronic medical records. The data collected from this study were analyzed using SPSS 26 statistical software. Descriptive statistics and necessary statistical graphs were presented, and the t -test or its nonparametric equivalent was used to compare pregnancy outcomes between groups for quantitative variables, and the Chi-Square test was used for qualitative variables. Proportions were compared using the Chi-square test. P < 0.05 was considered statistically significant.

According to the findings the present study and related studies, it can be concluded that PRP provides better conditions of the implantation of embryos due to the regulation of the local immune response, the promotion of angiogenic processes, cell adhesion, and the modification of growth factor and cytokine expression in the endometrium. The current research, was the first to examine the impact of PRP in a group of RIF patients with a homogenous FET program and based on actual clinical data bridged the scientific gap of the research in the field. Moreover, since the thickness of the endometrium has not been significantly altered, and on the contrary, the pregnancy rate is also increasing; this study suggests the paradigm of thickness as an assessment of endometrial readiness to be challenged and the new paradigm of assessing the endometrium to be based on qualitative measurements using modern biomarkers. Since this method is relatively inexpensive, minimally invasive, and does not require the use of complex equipment, it can be offered as an effective adjunct procedure alongside standard IVF therapies, particularly among those individuals with a history of frequent implantation failure.

This study was the first situation in Iran where the impact of PRP injection on the fertility outcomes of women with RIF undergoing FET cycles were studied. Our findings revealed that PRP injection had a significant potential of enhancing clinical pregnancy, without influencing endometrial thickness and hormones levels such as AMH and FSH. These findings suggest that the beneficial PRP action is likely to be through regulating the endometrial immune environment, increasing angiogenesis, and improving embryo-endometrial interaction, but not necessarily by raising endometrial thickness. Given that endometrial thickness was matched between groups and all transferred embryos were blastocysts of comparable quality, the observed improvement in pregnancy outcomes is likely attributable to functional rather than structural endometrial changes. According to a study by Chang et al. (2019), PRP injection had a significant effect on the increase of endometrial thickness and pregnancy rate in thin-endometrium patients [10] . Whereas, in our study, the endometrial thickness did not significantly differ between the two groups, still the pregnancy rate was higher in the intervention group. This could be explained by the fact that patient inclusion criteria were different; in the study by Chang, patients were predominantly thin (<7 mm) endometrium whereas in our study, patients were selected based on a history of RIF, not just endometrial thickness. A recent meta-analysis published in 2023 by Maged et al., which included 20-study meta-analysis that identified more than 3308 participants, indicated that PRP injection did not only significantly increase implantation rates (OR = 2.65), clinical pregnancy (OR = 1.79), and live birth (OR = 1.83) but also did so significantly [11] . These findings are consistent with our study, although a significant reduction in the rates of miscarriages was also reported in the aforementioned meta-analysis, whereas no significant difference in miscarriage rates was observed in our study. This contradiction can be ascribed to a variation in sample size or varying time of PRP injection. In a study by Zamaniyan et al., very close to our results, PRP injection in patients with a history of RIF led to higher clinical pregnancy rate (46% vs. 11% in the control group) in a study carried out in Iran [12] , which is very similar to our results (27.45% vs. 8.16%). These similarities in findings reinforce the possible validity of PRP application among the Iranian population. However, some studies have also reported different results. Using the case of Ershadi et al., in a study in Zahedan, they found that there was no significant difference in the pregnancy or implantation rates in the PRP group and the control group [13] . In this study, the thickness of the endometrium among the patients was more than 8 mm and it is perhaps that the impact of PRP on fertility would be less evident among patients with normal endometrium. This hypothesis is also partially confirmed in our study, since the mean endometrial thickness of both groups exceeded more than 9 mm, however, a positive effect of PRP on pregnancy was not excluded. a recent 2025 study by Nguyen and colleagues, utilizing RNA sequencing of primary endometrial epithelial and stromal cells from RIF patients treated with platelet lysate (a derivative of PRP), demonstrated significant upregulation of genes involved in extracellular matrix organization and degradation, including MMP1, MMP9, and ADAMTS18, alongside activation of cell cycle pathways (BUB1, CDK1, MKI67). Critically, this study also showed that treatment increased trophoblast spheroid attachment by 26–29% (P < 0.01) in RIF patient-derived endometrial epithelial cells, providing direct evidence that PRP enhances embryo-endometrial adhesion independent of thickness changes [14] . A study by Chen et al. (2025) investigating PRP in patients with persistent chronic endometritis found that intrauterine PRP infusion significantly modulated the endometrial immune microenvironment. Following PRP treatment, the proportions of endometrial CD8⁺ T cells, CD56⁺ natural killer (NK) cells, Foxp3⁺ regulatory T cells, and T-bet⁺ Th1 cells were significantly decreased. Transcriptomic analysis revealed upregulation of genes implicated in endometrial receptivity and antimicrobial defense, while genes involved in immune response processes were downregulated. Functional enrichment analysis specifically highlighted the critical role of leukocyte chemotaxis-related genes in reshaping the uterine immune environment [15] . These findings suggest that PRP improves pregnancy outcomes not by increasing endometrial mass, but by reconstructing the local immune microenvironment toward a tolerogenic state favorable for implantation. Mao et al. (2023) demonstrated that PRP promotes endometrial regeneration through upregulation of the retinol metabolism pathway and extracellular matrix receptor interaction pathway, with melanotransferrin (MELTF) identified as a key upregulated gene. Additionally, PRP was shown to inhibit endometrial epithelial cell death by downregulating ferroptosis, autophagy, and pyroptosis—forms of regulated cell death that can compromise endometrial integrity and receptivity [16] . Pregnancy rates in our study also increased although there was no significant difference in the endometrial thickness implying that PRP influenced the quality and not the quantity of the endometrium. This attribute could encompass a modification in the endometrial transcriptome, local immune control, or a hyperirritability of estrogen and progesterone receptors, which requires future molecular studies. Although the exact mechanism of PRP's effect on the endometrium is not yet fully understood, experimental studies have shown that PRP can release growth factors such as VEGF, PDGF, and TGF-β, which lead to the proliferation of endometrial mesenchymal stem cells (EnMSCs), increased angiogenesis, and reduced local inflammation [17] . Recent studies have also indicated that PRP has the capacity of enhancing the expression of adhesion molecules including α 3 and L -selectin in the endometrium, which play an important role in implantation [18] , [19] . These mechanisms require molecular and immunohistochemical investigations in future studies to accurately identify the pathways involved in the effectiveness of PRP. Several limitations should be acknowledged. First, the retrospective cohort design inherently carries a risk of selection bias, as treatment allocation was not randomized. Although baseline characteristics were largely comparable between groups ( Table 2 ), and logistic regression adjusted for identified imbalances (gravida), the possibility of unmeasured confounding cannot be excluded. Second, the sample size (n = 100), while adequately powered to detect the primary outcome difference, limits the robustness of subgroup analyses (e.g., by infertility type or age strata). Third, we did not perform molecular or immunohistochemical analyses on endometrial samples, which would be necessary to elucidate the mechanisms underlying PRP's qualitative effects. Fourth, long-term follow-up of offspring was not conducted, and perinatal outcomes beyond the neonatal period are unknown. Therefore, while our findings are promising, they require confirmation in larger, multicenter, prospective randomized controlled trials with extended follow-up. Such studies should also be adequately powered for subgroup analyses to identify which RIF patients derive the greatest benefit from PRP therapy.

Fertility is one of the significant reproductive health issues of the current century, most of which is characterized by the inability to conceive after one year of consistent intercourse in an absence of contraceptives. Although assisted reproduction has made tremendous progress, recurrent implantation failure (RIF) is among the primary challenges to the success of in vitro fertilization (IVF) cycles [1] , [2] . RIF is typically determined following three to more high-quality embryo transfers without pregnancy and its occurrence in the group of infertility centers has been identified to range between 10% and 15% [1] , [3] . A number of factors are implicated in the RIF of which the most significant of them cover uterine abnormalities, genetic disorders, maternal immunodeficiency, low-quality endometria, and ineffective embryo-endometrial interaction. Nevertheless, in many cases, the cause of RIF remains unknown and hence its management is difficult. in the recent years, the attention of researchers was paid to new approaches to make endometrial receptivity more active and enhance the conditions of implantation [4] . The application of platelet-rich plasma (PRP) is one of the new techniques that have demonstrated promise in preliminary research to enhance fertility success. The growth factors present in PRP include PDGF, VEGF, TGF- 3 and EGF among others that are of significant importance in repairing the tissues, angiogenesis, cell proliferation, and immune response regulation [5] , [6] . PRP was intrauterinely injected first in 2015 in patients with thin endometrium and demonstrated encouraging results of endometrial growth and pregnancy rates [7] , [8] . Although initial research has shown positive results, further, controlled research as to the efficacy of PRP on IVF outcomes is still lacking, particularly on patients with RIF. Most of the available research are observational, with limited sample size, and brief follow up with the vice versa some inconsistencies observed in PRP preparation regimens, injection schedule, and injection volume administered, have rendered comparison of the outcomes challenging [9] . Additionally, the mechanism of action of PRP on the endometrium and implantation remains undetermined, and additional research on the subject is required in the format of interventional studies with a proper design and the appropriate sample size. In this regard, the current research was aimed at determining the efficacy of intrauterine PRP injection on the fertility outcome of women undergoing fertility medical care through using IVF therapy with a history of implantation failure. This study attempts to bridge the gap in the scientific literature by applying a retrospective cohort study design and applying actual clinical data and give a clearer response to the underlying research question that of whether PRP may be used as an effective and safe adjunct in the management of RIF.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Despite the promising results obtained in this study, there are limitations which include retrospective study design, small sample size, not investigating the molecular processes, lack of long-term follow-up of the infants which make it worrying to generalize the results of the study. Therefore, it is recommended that future randomized and double-blind studies with bigger sample sizes investigating the immune and molecular biomarkers (i.e., cytokine levels, miRNA expression, and growth factors) in the endometrium be conducted to further clarify the precise mechanism of the effect of PRP on implantation. In addition, exploring the role of PRP in a particular subgroup of patients with thin endometrium, with history of recurring miscarriage, or with abnormalities in the uterus could provide valuable information.