A Retrospective Cohort Study Investigating the Effect of Intraovarian Platelet-Rich Plasma Therapy on the Oxidative State of Follicular Fluid in Women with Diminished Ovarian Reserve.

Infertility is a global issue, affecting approximately 10 to 15 percent of couples. 1 Treatments like in vitro fertilization (IVF) necessitate a sufficient number of high-quality oocytes. 2 One of the causes of female infertility is diminished ovarian reserve (DOR), with 6–35% of women visiting infertility clinics suffering from this condition. 3 Women with DOR often require IVF and typically have lower oocyte yields and pregnancy rates compared to those with normal ovarian reserves, necessitating special attention during the IVF process. Reactive oxygen species (ROS) play crucial roles in cell growth and death as primary or secondary messengers. Key transcription factors in cell signaling pathways related to proliferation, differentiation, apoptosis, and physiological processes in germ cells are influenced by redox status. Both positive and negative effects of oxidative stress on fetal growth have been documented. 4 Oxidative stress is a significant factor in pathological female infertility. Maintaining a balance between pro-oxidants and antioxidants is essential for the proper functioning of metabolic mechanisms, including the reproductive process. 5 An inverse correlation has been observed between the oxidative stress index (OSI) of follicular fluid and both the number of retrieved oocytes and the number of metaphase II (MII) oocytes. 6 Disruptions in membrane function, protein structure changes, and DNA damage due to redox homeostasis disruption negatively affect embryo growth and quality. 7 High ROS levels can cause genetic and epigenetic changes in the embryo, affecting the success rate of assisted reproduction and potentially leading to intergenerational effects. 8 Using antioxidant compounds to maintain redox balance can optimize pregnancy outcomes in both natural and assisted reproduction scenarios. 9 Platelet-rich plasma (PRP) is critical in the regeneration process due to its high content of bioactive, antioxidant, and anti-inflammatory factors. PRP is a blood product with a concentration approximately five times that of whole blood. 10 Over the past two decades, PRP (derived from autologous hematological components) has emerged as a key therapeutic tool for treating various medical conditions due to its role in enhancing repair processes. 11 PRP supports mitochondrial activity in mature oocytes during maturation, providing optimal conditions for IVF fertilization. 12 Autologous platelet concentrate promotes endometrial acceptance and improves pregnancy outcomes. 13 While some studies have found no positive association between PRP intrauterine injection and embryo implantation, pregnancy rate, or live birth rate, others have shown beneficial effects of PRP on endometrial cells. Additionally, positive results have been reported in patients with low ovarian reserve, premature ovarian failure, and menopausal women. 14 15 Given the importance of improving assisted reproductive treatments and the limited studies on PRP functionality, this study aims to investigate the effect of intra-ovarian PRP injection on the oxidative state of follicular fluid in women with DOR.

The average age of the women participating in this study was 34.68±0.719 years, and their body mass index (BMI) was 26.49±0.562 kg/m 2 . Most participants experienced primary infertility due to DOR, while approximately 40% had secondary infertility. The hormonal status of the serum and the outcomes of IVF procedures before and after PRP treatment are presented in Table 1 . Levels of FSH and LH did not significantly change before and after PRP therapy. However, the concentrations of E2 and AMH significantly increased post-PRP treatment ( Table 1 ). Additionally, although the number of oocytes and embryos obtained after PRP treatment increased, this rise was not statistically significant. While the number of embryos obtained post-intraovarian injection in patients with DOR increased, the improvement was not statistically significant; however, the quality of the embryos did improve. Of the 25 patients who completed the study, only 7 underwent embryo transfer before PRP therapy, none of whom had a positive β-hCG test result. After PRP therapy, 7 additional patients underwent embryo transfer. Among these women, the biochemical pregnancy success rate, based on positive β-hCG test results, was 42.9%. These findings suggest that PRP treatment increases the likelihood of biochemical pregnancy. Furthermore, the results indicated a significant increase in enzymatic antioxidants and TAC following PRP administration ( Fig. 1 ). Additionally, there was a notable decrease in TOS and MDA levels post-PRP treatment ( Fig. 2 ).

While advancing age is commonly linked to an increased risk of infertility due to DOR, this condition can also occur during reproductive years, well before old age. 19 21 Women with DOR often need specialized assisted reproductive techniques but typically show poor responses to standard methods. Exploring new interventional approaches and adjusting existing techniques can provide new hope for these women. Consistent with other studies, our results showed a significant rise in AMH levels after PRP treatment. Additionally, elevated E2 levels were observed in women with DOR. However, there was no significant change in MII oocyte counts post-PRP treatment, likely due to the small sample size. 19 The findings underscored the positive effect of PRP on antioxidant factors such as TAC, SOD, and catalase in the follicular fluid of women with DOR. Similarly, an animal model of polycystic ovary syndrome showed increased ovarian antioxidant levels after PRP administration, with a concurrent decrease in MDA levels. 22 Other studies have also reported increases in AMH and E2 levels following PRP treatment. 23 Given the crucial roles of AMH and E2, PRP's ability to boost these hormone levels suggests it could enhance fertility outcomes. Improvements in antioxidant status after PRP treatment have been noted in various clinical conditions. 24 For instance, a case report detailed PRP's beneficial role in a woman with DOR, where an increased number of follicles and improved oocyte quality led to a successful pregnancy in the second PRP treatment cycle. Although the study observed a higher number of oocytes and embryos post-PRP treatment, the difference was not statistically significant. Nevertheless, an increased pregnancy rate following PRP treatment was noted. 25 Discrepancies in the number of oocytes and embryos might be due to the reliance on visual evaluation techniques. High levels of ROS and oxidative stress in follicular fluid can adversely affect oocyte growth and quality, thereby impacting pregnancy outcomes. 6 26 One proposed mechanism for PRP's effectiveness is its ability to reduce oxidative stress in follicular fluid. In vitro studies have shown that PRP significantly impacts granulosa cells, promoting high proliferation rates and upregulating genes essential for reproduction. 27 Oxidative imbalance worsens numerous diseases, and recent studies suggest that PRP administration enhances antioxidant capacity, offering both protective and therapeutic benefits. 28 29 30 31 A meta-analysis indicated that PRP treatment significantly improves key fertility parameters in women with DOR, consistent with our findings. PRP injection led to increased AMH levels and biochemical pregnancy rates, along with notable improvements in the number of retrieved oocytes and embryos. 32 The detrimental effects of oxidative stress on male fertility have been well-documented. Intratesticular PRP injections have been shown to reduce MDA and IL-6 levels in mouse testis. 33 Additionally, PRP's protective role extends to human sperm freezing, improving sperm quality. 34 PRP incubation enhances sperm motility and reduces DNA fragmentation. 35 Although research in this area is still limited, PRP's rich growth factor content promotes tissue regeneration. Emerging evidence supports PRP's antioxidant effects, sustaining tissue healing by reducing protein and lipid oxidative damage and protecting tenocytes from oxidative stress-induced cell death. PRP also increases the capacity of some antioxidants. 24 Collectively, these findings suggest that PRP can serve as an effective adjunctive treatment in IVF. The main limitation of this study is the small sample size, as women with DOR often drop out of the treatment process for various reasons. The precise mechanisms and functions of PRP are not yet fully understood, underscoring the need to explore PRP's effects on factors that may enhance IVF success. Future research with larger sample sizes is essential to determine the extent to which PRP can benefit DOR patients and to address important questions regarding its efficacy in treating this condition. In conclusion, the findings of this study indicate that PRP probably improves IVF outcomes in women with DOR. Therefore, PRP as an adjuvant treatment, in conjunction with other therapeutic methods, may help reduce oxidative stress in follicular fluid and increase IVF success rates in patients with DOR.

This retrospective cohort study, conducted from 2022 to 2023, involved 25 women aged 30 to 38 years who were admitted to the Infertility Center of Fatemieh Hospital and Omid Infertility Center in Hamadan, Iran. The participants were selected for IVF based on the Bologna criteria for diagnosing DOR, 16 which is defined based on the presence of two of the following three critria: (1) maternal age over 40 years or any other risk factor for DOR, (2) fewer than three oocytes per cycle with a conventional stimulation, (3) abnormal ovarian reserve tests (i.e. anti-Müllerian hormone [AMH] levels of <0.5–1.1 ng/mL or an antral follicle count of <5–7), and no underlying conditions causing female infertility. Exclusion criteria included severe anemia preventing blood transfusion, sickle cell anemia, renal failure, respiratory infections, neutropenia, history or presence of malignancy or endometriosis, submucosal myoma, Asherman syndrome, untreated hypothyroidism, untreated hyperprolactinemia, tubal pathologies such as hydrosalpinx or tubal obstruction, underlying endocrine disorders, and any contraindications to pregnancy. Additionally, exclusion criteria for PRP preparation included a platelet count of <106/mL, hemoglobin levels of <10 mg/dL, and any contraindications for peripheral venous access. 17 PRP was prepared following established protocols. 18 19 Ovarian stimulation was conducted using the Shanghai protocol. 20 Immediately following the initial follicular puncture, a 2 mL intra-ovarian PRP injection was administered under ultrasound guidance, followed by a second puncture for further stimulation. After the first IVF procedure and follicular puncture, the oocytes were separated. Next, follicular fluids were collected from all participants and transferred to sterile falcon tubes. Then, tubes were centrifuged at 1,000×g for 4 min at 21℃, and supernatants (follicular fluids) were separated. Finally, the follicular fluid samples were stored at −80℃ until the experiments. Two months after the first follicular puncture and intra-ovarian PRP injection, follicular fluid samples were collected similar to the first stage. Estradiol (E2), AMH, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels were measured at baseline and after two menstrual cycles to evaluate the treatment effects. In both phases, hormone levels were measured on the second and third days after the start of the menstrual cycle. This study received approval from the Ethics Committee of Hamadan University of Medical Sciences, Hamadan, Iran, with the registration code IR.UMSHA.REC.1401. 268. All procedures adhered to the ethical guidelines of the Declaration of Helsinki (1967; version 2013) and the ethical standards of the National Iranian Research Committee. Serum levels of E2, AMH, LH, and FSH were quantified using electrochemiluminescence assays on the Roche Cobas e 411 Immunoassay Analyzer (Roche Diagnostics GmbH, Germany). The detection limits for E2 ranged from 5 to 3,000 pg/mL. For AMH, the measurement range was 0.01 to 23 ng/mL, and for LH and FSH, the ranges were 0.1 to 200 mIU/mL. TAC in follicular fluid was measured using the Naxifer™ Kit (Navand Salamat Co., Iran). This assay utilizes the Ferric Reducing Antioxidant Power (FRAP) method, which assesses the sample's antioxidant potential by its ability to reduce ferric iron (Fe 3+ ) to ferrous iron (Fe 2+ ) The resulting color change was measured at 593 nm with a microplate reader. TAC levels were quantified against a standard curve (Fe 2+ solution) and expressed as mmol Fe 2+ /L. TOS in follicular fluid was evaluated using the Natos™ Kit (Navand Salamat Co., Iran). This assay is based on the oxidation reaction of amplifying molecules, where the color intensity is directly proportional to the quantity of oxidant molecules. The results were calibrated using hydrogen peroxide (H 2 O 2 ) and expressed as equivalent liquid peroxide per liter (µmol H 2 O 2 Eq/L). According to the manufacturer, the standard range for TOS levels is 0.156–10 µmol Eq/L, with a sensitivity of 0.023 µmol Eq/L. The oxidative stress index (OSI) was calculated as the ratio of TOS to TAC and expressed as µmol Eq H 2 O 2 /mmol Fe 2+ . Lipid peroxidation levels in follicular fluid were determined by measuring malondialdehyde (MDA) using the Nalondi TM Kit (Navand Salamat Co., Iran). MDA, a reactive aldehyde produced by lipid peroxidation, reacts with thiobarbituric acid (TBA) at high temperatures to form a pink MDA-TBA adduct. This adduct was quantified colorimetrically at 550 nm against a standard curve, with results expressed as nmol/mL. CAT activity in follicular fluid was measured using the Nactaz™ Catalase Activity Assay Kit (Navand Salamat Co., Iran). Approximately 10 6 granulosa cells were homogenized in 1 mL of lysing buffer and centrifuged at 8,000×g for 10 minutes. The supernatants were separated for enzyme activity assessment. The detection method relies on the peroxidative activity of catalase, which, in the presence of hydrogen peroxide (H 2 O 2 ) and methanol, produces formaldehyde. This formaldehyde reacts with a dye to form a chromogen that is detectable at 550 nm. Enzyme activity was reported as nmol/min/mg protein. SOD activity in granulosa cells was measured using the Nasdox™-Superoxide Dismutase Assay Kit (Navand Salamat Co., Iran). Briefly, 10 6 cells were centrifuged at 800×g for 2 minutes, and the supernatants were discarded. The cell pellets were then washed twice with PBS, mixed with 0.5 mL of lysing buffer, vortexed for 10 minutes, and centrifuged at 12,000×g for 5 minutes. The supernatants were collected for the SOD assay. SOD activity was measured through pyrogallol autoxidation, which is highly dependent on superoxide, serving as a substrate for SOD. In the presence of SOD, pyrogallol autoxidation was inhibited, and SOD activity was indirectly measured at 420 nm. A calibration curve was generated using purified SOD as a standard, and results were expressed as units per milligram of protein. Data analysis was conducted using SPSS software version 22.0 (IBM, USA) and GraphPad Prism software version 8.0 (GraphPad Software Inc., USA). Data were described using mean, standard deviation, minimum and maximum values, frequency, and percentage indicators. For pairwise comparisons of quantitative variables before and after treatment, a paired t-test was used if the data distribution was normal; otherwise, the non-parametric Wilcoxon test was applied. Qualitative data were analyzed before and after treatment using McNemar's test. Data were expressed as mean±SEM, and a p-value of less than 0.05 was considered statistically significant.