Intra-ovarian injection of platelet-rich plasma in patients with poor ovarian response; a randomized controlled clinical trial.

This RCT was conducted at a tertiary referral institute (ROYAN Institute, Tehran, Iran). This study prospectively registered in http://www.Iranian Registry of Clinical Trials.ir (ID: IRCT20080831001141N40) at 01/17/2023. A total of 200 patients who presented to the outpatient clinic from February 2023 to July 2024 were included in this study. The participants were randomly assigned to the two groups with and without Intra-ovarian PRP injection according to a computer-generated, unconcealed randomization list. Inclusion criteria: Infertile women with a history of POR (the history of controlled ovarian stimulation (COS) cancelation due to inadequate response, no oocyte retrieval, no pronucleus (PN) formation, or retrieved oocytes < 5 in the previous COS); age < 43 years; body mass index (BMI) < 35 Kg/m 2 ; follicle-stimulating hormone (FSH) < 20 IU/mL; anti-mullerian hormone (AMH) < 1.2 ng/mL. Non-inclusion criteria: women with endocrine, hematologic, and autoimmune disorders; platelet count of less than 150🞨10 3 /µL; cancer history; chromosomal and genetic abnormalities; congenital uterine anomalies; fibromatous uterus, severe adenomyosis, endometriosis, history of ovarian surgery; hydrosalpinx, and severe male factor. Exclusion criteria: cervicitis; history of fever within one week before PRP infusion; use of steroid anti-inflammatories drugs at least two weeks before PRP infusion; use of non-steroid anti-inflammatories at least one week before PRP infusion; use of anticoagulants at least one week before PRP infusion; use of antibiotics at least three days before PRP infusion; use of garlic, onion and omega 3 and 6 supplements at least three days before PRP infusion [ 12 ]. The sample size for this study was calculated using Pass software (ve.11). We aimed to detect a clinically meaningful difference of 11% in the absolute change in clinical pregnancy between the experimental and control groups (based on findings from the study conducted by Melo et al. [ 13 ]). With these parameters, a sample size of 200 patients (100 per group) was determined to provide 80% power to detect the specified difference at a 5% significance level using a 2-tailed test, considering 10% loss to follow-up. A computer-generated randomization list was created by an independent methodologist blinded to the trial. To ensure balanced allocation, the block randomization method was used with an allocation ratio of 1:1. A block size of 4 was employed to accommodate the total sample size of 200 participants. The patients’ enrolment and assignment to different groups were carried out by a researcher in the clinic. Each patient participated in the study only once and if she had written consent. The researcher who followed the results of patients’ treatment and the methodologist who analyzed the data were uninformed regarding the type of protocol. Allocation concealment was maintained using sequentially numbered, opaque, sealed envelopes opened only after participant enrollment and consent. In order to preparing PRP using the ROOYAGEN kit (Arya Mabna Tashkhis, Iran) [ 12 ], 35 mL of peripheral venous blood and 5 mL of anticoagulant were centrifuged (Rotofix 32 A, Hettich, US) twice (1800 rpm for 10 min, and then, 3500 rpm for 6 min). Finally, 6 mL of PRP was obtained, which was 4–6 times more concentrated than the baseline platelet. To maximize consistency among PRP samples, all samples were prepared by a single technician. To ensure appropriate platelet concentration, the platelet count of the patient’s initial blood sample was measured prior to centrifugation, and the platelet count of the final PRP sample was measured again. This ensured that the platelet concentration of all samples was increased to the target range of 4–6 times the baseline platelet count. Then, a reproductive endocrinology and infertility physician injected 6 mL of non-activated PRP into the ovaries (3 mL in each ovary). All injections were performed by a single specialist physician. PRP injection was performed transvaginally under ultrasound guidance and under sedation anesthesia using a 32 cm 17 G single lumen needle (Reproline medical GmbH, D-53359 Rheinbach/ Germany). The injection was performed on days 8–12 of the menstrual cycle. When a dominant follicle was observed, the follicular fluid was first aspirated and then injected approximately 1 mL of PRP into the follicle. The remaining 2 mL was injected into 4 areas around the follicle. When no dominant follicle was detected in the ovaries, injections were administered to four regions of the ovarian stroma. The injection took place within one hour after PRP preparation. No intervention was done for the patients in the control group. All patients in the intervention group underwent physical examination and vaginal ultrasound by the physician 24 h after intra-ovarian PRP injection to confirm the absence of early complications, including hematoma formation or infection. If necessary, a follow-up visit was scheduled for one week later. Physician instructed patients to report symptoms such as fever, spotting or vaginal bleeding, and abdominal or pelvic pain. Patients in the intervention group avoided ovarian hormonal stimulation for three menstrual cycles following the injection, and physician advised them to attempt natural conception with their partners during this period. Control group participants received similar guidance to attempt natural pregnancy during three cycles before initiating ovarian stimulation. Following a three-month period designated for natural conception attempts, hormonal profiling was conducted for all participants in both groups. These assessments, performed on days 2–3 of the menstrual cycle, measured FSH, luteinizing hormone (LH), and AMH levels before ovarian stimulation commenced. Also, a radiologist performed a Doppler ultrasound to determine ovarian volume and antral follicle count (AFC). For all patients in the control group, as well as for patients in the intervention group who did not achieve spontaneous pregnancy within three months after intra-ovarian PRP injection, physicians performed at least one modified natural cycle. They monitored follicular growth in a natural cycle and administered human chorionic gonadotropin (HCG) to trigger ovulation when the follicle reached a diameter of 18–20 mm. If this cycle did not result in pregnancy, patients entered a COS for intracytoplasmic sperm injection (ICSI). The COS protocol included duo-stim with gonadotropin releasing hormone (GnRH) antagonist following E2 priming in the luteal phase of the preceding cycle [ 14 ]. If the specialist considered the patient unsuitable for a second ovarian stimulation cycle, a fresh embryo transfer (ET) was performed. If the patient qualified for a second cycle, embryos from both stimulation cycles were cryopreserved and later transferred during a frozen ET cycle. Endometrial preparation for frozen ET was implemented through a standard long GnRH-agonist protocol [ 12 ]. Based on embryo quantity and quality assessments, a reproductive endocrinology and infertility specialist transferred two to three embryos at either the cleavage or blastocyst stage into the uterine cavity using a ET catheter via a standard technique [ 15 ]. In fresh ET cycles, the luteal phase support was provided with the 50 mg per day progesterone intramuscular (IM) for two weeks. In frozen ET cycles, the luteal phase support was provided with the 50 mg per day progesterone IM and daily 6 mg of the estradiol valerate for two weeks. Then, the serum beta-HCG (β-HCG) was checked (14 days after the ET). After a β-HCG positive result, the same dose of progesterone in frozen or fresh ET and estradiol in FET was continued up 12 weeks of gestation [ 12 ]. All patients were followed for one year from the time of enrollment in the study, regardless of whether they received the intra-ovarian PRP intervention. Investigators recorded the spontaneous pregnancies, pregnancies from assisted reproductive technologies (ARTs), and their outcomes for all participants. The primary outcome was an increase in ovarian reserve, as indicated by higher serum AMH levels and AFC. The secondary outcome was the rate of clinical pregnancy. The clinical pregnancy rate was defined as the number of pregnancies confirmed by ultrasound showing a gestational sac. Statistical analysis was conducted using Statistical Package for Social Sciences (ver. 20.0; SPSS Inc., Chicago, USA). For categorical variables, Chi-square (χ2) test or Fisher’s exact test was conducted, where appropriate. For continuous variables, at first, the Kolmogorov–Smirnov test was used to determinate the normal distribution of variables and results were given as mean ± standard deviation (mean ± SD). The two-tailed t-test was used to compare means. The level of significance was set at p -value less than 0.05.

The flow diagram of the subjects according to the Consolidated Standards of Reporting Trials (CONSORT) guideline is shown in Fig.  1 . The demographic characteristics and medical history of patients are shown in Table  1 . Three months after PRP injection for patients in the intervention group and before patients entered the ovarian stimulation cycle, there were no significant differences in serum levels of FSH, LH and AMH, menstruation character, and ovaries volume between groups intervention and control. The AFC was significantly higher in intervention group ( p  = 0.001) (Table  2 ). Fig. 1 CONSORT flow diagram CONSORT flow diagram Table 1 Comparison of demographic characteristics of patients with/without intraovarian PRP injection Variable Intervention Group ( N  = 95) Control Group ( N  = 97) P -value Age (year) 36.23 ± 5.12 37.32 ± 4.61 0.148 BMI (kg/m 2 ) 26.03 ± 4.28 25.66 ± 4.24 0.593 Infertility 0.186  Primary 79/95 (83.2%) 87/97 (89.7%)  Secondary 16/95 (16.8%) 10/97 (10.3%)  Infertility duration 3.64 ± 1.83 3.74 ± 2.54 0.782 Previous ART outcome 0.284 Cancellation of ovarian stimulation due to inadequate response 34/95 (35.8%) 42/97 (43.3%)  No Oocyte 16/95 (16.8%) 12/97 (12.4%)  No PN 6/95 (6.3%) 9/97 (9.3%)  POR 1 39/95 (41.1%) 34/97 (35%) Values are presented as the mean ± SD and number (percent) BMI Body Mass Index, ART Assisted Reproductive Technology, PN Pronuclear, POR Poor Ovarian Response P -value obtained by independent t-test and chi square test. Statistically significant level < 0.05 1 Number of retrieved oocytes < 5 Comparison of demographic characteristics of patients with/without intraovarian PRP injection Values are presented as the mean ± SD and number (percent) BMI Body Mass Index, ART Assisted Reproductive Technology, PN Pronuclear, POR Poor Ovarian Response P -value obtained by independent t-test and chi square test. Statistically significant level < 0.05 1 Number of retrieved oocytes < 5 Table 2 Comparison of clinical characteristics of patients with/without intraovarian PRP injection Variable Intervention Group ( N  = 95) Control Group ( N  = 97) P -value Menstruation 0.345  Regular 88/95 (92.6%) 86/97 (88.7%)  Irregular 7/95 (7.4%) 11/97 (11.3%) Ovarian volume 1 (mm 3 ) 5.04 ± 1.97 4.69 ± 2.26 0.322 Antral follicle count 5.63 ± 2.34 3.93 ± 1.35 0.001* Hormonal profile  FSH (IU/L) 9.95 ± 4.65 8.89 ± 4.66 0.162  LH (IU/L) 5.11 ± 3.24 4.94 ± 4.48 0.787  AMH (ng/ml) 0.36 ± 0.25 0.32 ± 0.13 0.215 Values are presented as the mean ± SD and number (percent) FSH Follicle Stimulating Hormone, LH Luteinizing Hormone, AMH Anti Mullerian Hormone P -value obtained by independent t-test and chi square test. Statistically significant level < 0.05 ( * ) 1 Average volume of right and left ovary Comparison of clinical characteristics of patients with/without intraovarian PRP injection Values are presented as the mean ± SD and number (percent) FSH Follicle Stimulating Hormone, LH Luteinizing Hormone, AMH Anti Mullerian Hormone P -value obtained by independent t-test and chi square test. Statistically significant level < 0.05 ( * ) 1 Average volume of right and left ovary In total, 87 patients in the intervention group and 97 patients in the control group entered ovarian stimulation cycles. Seventeen patients in the intervention group and 15 patients in the control group did not undergo a second stimulation cycle, and fresh ET was performed for them. Consequently, the intervention group comprised 70 patients who completed two stimulation cycles and 17 who underwent only one, yielding a total of 157 stimulation cycles. Similarly, the control group included 82 patients who completed two stimulation cycles and 15 who underwent only one, producing a total of 179 stimulation cycles. The prescribed gonadotropin dose and the duration of COS did not differ significantly between the two groups. The intervention group showed lower rates of COS cancellations due to inadequate response and fewer cases of No PN compared with the control group; however, these differences did not reach statistical significance. The intervention group had significantly fewer cases of no oocyte retrieval ( p  = 0.040). The number of retrieved oocytes did not differ significantly between groups. However, the intervention group demonstrated significantly higher oocyte maturation rate ( p  = 0.005) and fertilization rate ( p  = 0.018). The total number of embryos obtained did not differ significantly, but the number of high-quality embryos was significantly greater in the intervention group ( p  = 0.042) (Table  3 ). Table 3 Comparison of ovarian stimulation data in patients with/without intraovarian PRP injection Variable Intervention Group ( N  = 87) (Number of cycles = 157) Control Group ( N  = 97) (Number of cycles = 179) P -value Gonadotropin dose 1 3221.59 ± 1285.11 3644.08 ± 1240.88 0.080 Duration of ovarian stimulation 1 10.41 ± 2.20 10.91 ± 2.61 0.282 Cycle cancellation due to  Inadequate response to ovarian stimulation 22/157 (14%) 36/179 (20.1%) 0.140  No oocyte 11/157 (7%) 25/179 (13.9%) 0.040*  No PN 15/157 (9.6%) 24/179 (13.4%) 0.271 Total oocytes retrieved 4.01 ± 3.04 4.12 ± 2.40 0.818 Oocyte maturation rate 2 (%) 84.87 ± 23.57 72.84 ± 27.22 0.005* Oocyte morphology  Morph 0.08 ± 0.36 0.02 ± 0.22 0.256  Slightly dysmorphic 2.24 ± 2.46 1.22 ± 1.50 0.002*  Dysmorphic 1.08 ± 2.11 1.72 ± 1.63 0.037*  Highly dysmorphic 0.60 ± 1.56 1.12 ± 2.02 0.077 Fertilization rate 3 (%) 83.64 ± 33.91 68.99 ± 38.34 0.018* Total embryos obtained 2.42 ± 2.08 1.95 ± 1.49 0.108 High quality embryo rate 4 (%) [ 16 ] 83.02 ± 29.77 71.03 ± 36.97 0.042* Blastulation rate 5 (%) 25.10 ± 5.29 17.74 ± 4.89 0.318 Number of embryos transferred 1.76 ± 0.64 1.86 ± 0.66 0.424 Implantation rate 6 (%) 71.88 ± 25.61 60.26 ± 23.11 0.216 Values are presented as the mean ± SD and number (percent) PN Pronuclear P -value obtained by independent t-test and chi square test. Statistically significant level < 0.05 (*) 1 Mean of Stim I & II 2 (MII oocytes/ Total oocytes retrieved) × 100 3 (Total 2PN/ Injected oocytes) × 100 4 (Excellent & good embryos/ Total embryos obtained) × 100 5 (The percentage of embryos that successfully develop into the blastocyst stage/ Total embryos obtained) × 100 6 (Number of gestational sacs/ Number of embryos transferred) × 100 Comparison of ovarian stimulation data in patients with/without intraovarian PRP injection Values are presented as the mean ± SD and number (percent) PN Pronuclear P -value obtained by independent t-test and chi square test. Statistically significant level < 0.05 (*) 1 Mean of Stim I & II 2 (MII oocytes/ Total oocytes retrieved) × 100 3 (Total 2PN/ Injected oocytes) × 100 4 (Excellent & good embryos/ Total embryos obtained) × 100 5 (The percentage of embryos that successfully develop into the blastocyst stage/ Total embryos obtained) × 100 6 (Number of gestational sacs/ Number of embryos transferred) × 100 In the intervention group, 10.5% of patients achieved spontaneous pregnancy within 3–8 months after intra-ovarian PRP injection. During the one-year follow-up period, no spontaneous pregnancies occurred in the control group. A total of 12.6% of patients in the intervention group and 34% of patients in the control group did not undergo ET after entering COS due to inadequate response, No oocyte, or No PN ( p  = 0.001). The number of fresh and frozen ET cycles did not differ significantly between the groups (Table  4 ). Table 4 Comparison of spontaneous pregnancy rate and ovarian stimulation outcome in patients with/without intraovarian PRP injection Variable Intervention Group ( N  = 95) Control Group ( N  = 97) P -value Spontaneous pregnancy 10/95 (10.5%) 0/97 0.001* Pregnancy after modified natural cycle 3/95 (3.2%) 2/97 (2.1%) 0.634 Entering the ovarian stimulation cycle 87/95 (91.6%) 97/97 (100%) 0.042* No ET 1 11/87 (12.6%) 33/97 (34%) 0.001* Fresh ET 17/87 (19.6%) 15/97 (15.5%) 0.699 Frozen ET 59/87 (67.8%) 49/97 (50.5%) 0.084 Values are presented as the number (percent) ET Embryo Transfer P -value obtained by independent t-test and chi-square test or Fisher’s exact test when more than 20% of cells with expected counts of less than 5 were observable. Statistically significant level < 0.05 ( * ) 1 Due to No oocyte, No PN, or cancellation of ovarian stimulation due to inadequate response Comparison of spontaneous pregnancy rate and ovarian stimulation outcome in patients with/without intraovarian PRP injection Values are presented as the number (percent) ET Embryo Transfer P -value obtained by independent t-test and chi-square test or Fisher’s exact test when more than 20% of cells with expected counts of less than 5 were observable. Statistically significant level < 0.05 ( * ) 1 Due to No oocyte, No PN, or cancellation of ovarian stimulation due to inadequate response Pregnancy outcomes are summarized in Table  5 . The clinical pregnancy rate following fresh ET was significantly higher in the intervention group ( p  = 0.024). Ectopic pregnancy (EP) occurred only in the intervention group, but the difference was not statistically significant. The overall clinical pregnancy rate (including spontaneous pregnancies and those achieved through ARTs was significantly higher in the intervention group ( p  = 0.011). The miscarriage rate did not differ significantly between the groups. The intervention group showed a higher live birth rate, but the difference was not statistically significant. Rates of twin pregnancy, preterm labor, and pregnancy complications were comparable between groups. No cases of fetal or neonatal anomalies were reported in either the intervention or control group. Patients’ pregnancy information is presented in Table  6 . Table 5 Comparison of pregnancy outcome in patients with/without intraovarian PRP injection Variable Intervention Group ( N  = 95) Control Group ( N  = 97) P -value Spontaneous pregnancy 10/95 (10.5%) 0 0.001*  Miscarriage 3/10 (14.28%) - -  Live births 7/10 (85.71%) - - Pregnancy from ovulation induction 3/95 (3.2%) 2/97 (2.1%) 0.634  Ectopic pregnancy 1/3 (33.3%) 0 -  Miscarriage 1/3 (33.3%) 2/2 (100%) -  Live births 1/3 (33.3%) 0 - Pregnancy from embryo transfer  Clinical pregnancy/ ET 17/76 (22.4%) 13/64 (20.3%) 0.811  Clinical pregnancy/ Fresh ET 7/17 (41.2%) 1/15 (6.7%) 0.024*  Clinical pregnancy/ Frozen ET 10/59 (16.9%) 12/49 (24.5%) 0.299  Ectopic pregnancy/ ET 1/76 (1.3%) 0 0.365  Ectopic pregnancy/ Pregnancy 1/17 (5.9%) 0 0.374  Miscarriage/ ET 6/76 (7.9%) 4/64 (6.25%) 0.745  Miscarriage/ Pregnancy 6/17 (35.3%) 4/13 (30.8%) 0.794  Live births/ ET 10/76 (13.2%) 9/64 (14.1%) 0.818  Live births/ Pregnancy 10/17 (58.8%) 9/13 (69.2%) 0.558 Total pregnancy  Clinical pregnancy/ patients 30/95 (31.6%) 15/97 (15.5%) 0.011*  Ectopic pregnancy/ patients 2/95 (2.1%) 0 0.155  Miscarriage/ patients 10/95 (10.5%) 6/97 (6.2%) 0.296  Live births/ patients 18/95 (18.9%) 9/97 (9.3%) 0.062 Birth weight (kg) 2793.75 ± 473.70 3153.33 ± 507.66 0.153 Preterm labor/ pregnancy 5/30 (16.7%) 2/15 (13.3%) 0.571 Twin pregnancy/ pregnancy 3/30 (3.3%) 1/15 (6.7%) 0.593 Pregnancy complications  Gestational diabetes mellitus/ pregnancy 1/30 (3.3%) 3/15 (20%) 0.064  Gestational hypertension/ pregnancy 2/30 (6.7%) 0 0.306 Values are presented as the mean ± SD and number (percent) ET Embryo Transfer P -value obtained by independent t-test and chi square test or Fisher’s exact test when more than 20% of cells with expected counts of less than 5 were observable. Statistically significant level < 0.05 (*) Comparison of pregnancy outcome in patients with/without intraovarian PRP injection Values are presented as the mean ± SD and number (percent) ET Embryo Transfer P -value obtained by independent t-test and chi square test or Fisher’s exact test when more than 20% of cells with expected counts of less than 5 were observable. Statistically significant level < 0.05 (*) Table 6 Pregnancy data of patients in the intervention and control groups case Group Age Pregnancy ET Outcome Twin pregnancy Labor Complication 1 Intervention 33 Spontaneous - Live birth - Preterm GH 2 Intervention 36 Spontaneous - Live birth - Term - 3 Intervention 39 Spontaneous - Live birth - Term - 4 Intervention 39 Spontaneous - Live birth - Term - 5 Intervention 39 Spontaneous - Live birth - Term - 6 Intervention 40 Spontaneous - Live birth - Term - 7 Intervention 41 Spontaneous - Live birth - Term - 8 Intervention 27 Spontaneous - Miscarriage - - - 9 Intervention 32 Spontaneous - Miscarriage - - - 10 Intervention 42 Spontaneous - Miscarriage - - - 11 Intervention 23 MNC - Live birth - Term - 12 Intervention 26 MNC - Miscarriage - - - 13 Intervention 27 MNC - EP - - - 14 Intervention 26 Fresh ET 1×C Live birth - Term - 15 Intervention 27 Fresh ET 1×C Live birth - Term - 16 Intervention 31 Fresh ET 2×B Live birth - Term - 17 Intervention 33 Fresh ET 2×C Live birth - Preterm - 18 Intervention 39 Fresh ET 1×C Live birth - Preterm - 19 Intervention 38 Fresh ET 2×B Miscarriage - - - 20 Intervention 38 Fresh ET 1×C Miscarriage - - - 21 Intervention 34 Frozen ET 2×B Live birth - Term GH 22 Intervention 36 Frozen ET 2×C Live birth - Term GDM 23 Intervention 40 Frozen ET 2×B Live birth Yes Term - 24 Intervention 35 Frozen ET 2×B Live birth Yes Preterm - 25 Intervention 37 Frozen ET 2×B Live birth Yes Preterm - 26 Intervention 39 Frozen ET 2×C Miscarriage - - - 27 Intervention 40 Frozen ET 2×C Miscarriage - - - 28 Intervention 41 Frozen ET 2×C Miscarriage - - - 29 Intervention 43 Frozen ET 2×C Miscarriage - - - 30 Intervention 38 Frozen ET 2×C EP - - - 31 Control 29 MNC - Miscarriage - - - 32 Control 34 MNC - Miscarriage - - - 33 Control 36 Fresh ET 2×C Live birth - Term - 34 Control 26 Frozen ET 2×C Live birth - Term GDM 35 Control 27 Frozen ET 2×B Live birth - Term - 36 Control 28 Frozen ET 2×B Live birth - Term - 37 Control 37 Frozen ET 2×B Live birth - Term - 38 Control 38 Frozen ET 2×B Live birth - Term - 39 Control 42 Frozen ET 2×B Live birth - Term - 40 Control 42 Frozen ET 2×B Live birth - Preterm GDM 41 Control 40 Frozen ET 2×C Live birth Yes Preterm GDM 42 Control 32 Frozen ET 3×C Miscarriage - - - 43 Control 39 Frozen ET 2×C Miscarriage - - - 44 Control 39 Frozen ET 1×C Miscarriage - - - 45 Control 42 Frozen ET 1×C Miscarriage - - - B Blastocyst, C Cleavage, MNC M odified Natural Cycle, EP Ectopic Pregnancy, ET Embryo Transfer, GDM Gestational Diabetes Mellitus, GH Gestational Hypertension Pregnancy data of patients in the intervention and control groups B Blastocyst, C Cleavage, MNC M odified Natural Cycle, EP Ectopic Pregnancy, ET Embryo Transfer, GDM Gestational Diabetes Mellitus, GH Gestational Hypertension No significant complications occurred after PRP injection, including allergic reactions, infection, intra-abdominal bleeding, intra-ovarian hematoma, bowel or bladder injury, or need for hospitalization. Mild pelvic pain within the first 24–48 h after injection was the most common and expected complaint among patients.

Diminished ovarian reserve (DOR), characterized by a reduction in both the number and quality of ovarian follicles, can significantly impair fertility. This condition compromises a woman’s ability to achieve natural pregnancy and may ultimately result in infertility [ 1 , 2 ]. In such cases, terms such as DOR or poor ovarian response (POR) are used, which are classified according to the Bologna criteria [ 3 ] or the POSEIDON criteria [ 4 ]. There is a clear need for new approaches to help women with low ovarian reserve, as there are few treatment options available to restore fertility in such women [ 5 ]. The effects of diminished vascular flow, restricted ovarian perfusion, and insufficient follicular oxygenation on reproductive pathology still require further investigation; however, interventions designed to enhance ovarian vascularity may prove advantageous. A new intervention recently proposed is the intraovarian injection of platelet-rich plasma (PRP) [ 6 ]. The role of platelet release in facilitating tissue regeneration is gaining more and more attention in contemporary medical research. Platelets are traditionally regarded as the primary responders in hemostasis and are acknowledged for their involvement in tissue repair, neovascularization, and immunomodulation [ 6 ]. Recent studies indicate that platelet secretory behavior encompasses not only granular deposits but also the synthesis and release of cytokines and other bioactive molecules. Bioactive proteins and molecules released by activated platelets exhibit numerous physiological effects, including the enhancement of angiogenesis, cell proliferation, cell differentiation, and apoptosis regulation [ 1 ]. The purpose of injecting PRP into ovarian tissue is to concentrate and deliver the aforementioned growth factors directly to the adult human ovary, which is the new target tissue. Platelet-derived mediators activate fibroblasts and recruit leukocytes, neutrophils, and macrophages, facilitating the removal of dead cells and cellular debris. Factors released by platelets also regulate the proliferation and migration of additional cells crucial for tissue repair. Another essential process for tissue repair is angiogenesis in injured tissue, which is controlled by platelets by releasing a variety of angiogenic mediators when they are activated [ 7 ]. Studies indicate that platelet-derived growth factors can initiate a process known as “ploidy rescue” in human embryos. The injection of autologous growth factors is hypothesized to deliver angiogenic inputs to suboptimal ovarian tissue. This process is expected to enhance perfusion, thereby increasing oxygen availability and transmitting genetic regulatory signals to ovarian stem cells or dormant, senescent oocytes. Each of these pathways is recognized for its utility in rectifying upstream abnormalities in oocyte-to-embryonic development, encompassing ovarian stem cell differentiation, mitochondrial dynamics, and mRNA storage [ 6 ]. Current evidence suggests that disruption of the ovarian microenvironment constitutes the primary mechanism underlying ovarian dysfunction. Among emerging therapeutic approaches, intraovarian PRP has demonstrated potential for improving the ovarian microenvironment, thereby restoring ovarian function and enhancing ovarian reserve [ 1 , 2 , 8 – 10 ]. We still have a rudimentary knowledge of the PRP platform and related elements of this innovative approach. The effects of PRP on the adult human ovary remain inadequately understood [ 7 ]. There is currently no definitive approach to validate the processes of “ovarian restoration” or “ovarian enhancement”. Although there are promising reports of PRP injections into human ovaries, we still have a limited understanding of the underlying mechanisms and safety of these injections. There is poor standardization in this regard among research and clinics groups [ 1 ]. It is important to comprehensively evaluate the safety and efficacy of newly introduced treatments before they are used in clinical settings. In the absence of robust and reliable data, intraovarian PRP injection must be regarded solely as an experimental procedure rather than a legitimate alternative for treating ovarian failure [ 11 ]. Based on available evidence, this randomized controlled clinical trial (RCT) aimed to determine the effect of intra-ovarian PRP injection in patients with a history of POR.

Studies on intra-ovarian PRP injection have been steadily increasing since 2016. Although these studies suggest potential benefits of intra-ovarian PRP in patients with DOR or POR, current evidence remains insufficient and inconclusive to confirm its effectiveness [ 17 ]. Meta-analyses emphasize the need for RCTs [ 18 – 21 ]. The present RCT was therefore conducted to evaluate the impact of intra-ovarian PRP injection in patients with POR. Our study found no significant differences between the intervention and control groups in terms of hormonal profile, including FSH, LH, and AMH. Similarly, ovarian volume did not differ significantly between groups. However, the AFC was considerably higher in the intervention group. Several studies have reported a significant reduction in serum FSH and LH levels, along with a notable increase in AMH and AFC, within 1–3 months after intra-ovarian PRP injection [ 17 , 19 , 21 , 22 ]. Other studies have reported these improvements as nonsignificant [ 18 , 20 ]. Because most of these investigations were prospective or retrospective before-and-after comparative studies, the overall quality of evidence remains low. A notable finding in our study was the lack of concordance between serum AMH and AFC. Although both serve as indicators of ovarian reserve, three months after PRP injection, we observed no significant difference in serum AMH, while AFC was significantly higher in the intervention group. In the study by Molinaro et al., only 13.5% of patients demonstrated concurrent improvement in both AFC and AMH; 22.9% showed improvement in AFC only, and 48.9% showed improvement in AMH only [ 23 ]. Herlihy et al., in their RCT using 4 ml PRP, reported no significant change in AFC or AMH [ 24 ]. Li et al. reported substantial improvement in AFC and nonsignificant improvement in AMH after a single PRP injection, but observed significant increases in both AFC and AMH after two PRP injections [ 25 ]. Tickoo et al. reported simultaneous improvement in AFC and AMH after three consecutive cycles of 2 ml activated PRP injection [ 26 ]. These findings suggest that further studies are needed to elucidate the mechanism of intra-ovarian PRP and its impact on ovarian reserve. It is also possible that the discrepancy between serum AMH levels and AFC is influenced by the timing of measurements of these variables, and that improvements in serum AMH levels may become apparent over a longer time frame. Accordingly, serial monthly assessment of serum AMH levels for at least six months following PRP injection may help clarify this issue. In our study, the total dose of gonadotropin and the duration of COS did not differ significantly between groups. Similarly, the RCT by Barrenetxea et al., conducted after 4 ml of activated PRP injection, reported no significant differences in gonadotropin dose or stimulation duration [ 27 ]. Retrospective studies also did not show significant reductions in gonadotropin dose or stimulation duration [ 23 , 25 ]. The cycle cancellations due to inadequate response and No PN were lower in the intervention group, although the difference was not statistically significant. In contrast, cases of no oocyte retrieval were significantly lower. Although most before–and–after comparative studies reported a substantial reduction in cycle cancellation rates [ 20 , 22 ], the RCT by Barrenetxea et al. did not observe such a difference [ 27 ]. This finding may be explained by the use of two consecutive stimulation cycles immediately after PRP injection, without allowing sufficient time for ovarian recovery. Although we observed no significant difference in the total number of retrieved oocytes, the proportion of metaphase two (MII) oocytes relative to the total number of oocytes was significantly higher in the intervention group. Patients in the intervention group also showed oocytes with better morphology. Barrenetxea et al. similarly reported a considerably higher number of MII oocytes in the intervention group compared with controls [ 27 ], whereas Herlihy et al. found no significant difference [ 24 ]. Despite both being RCTs, their limited sample sizes reduce the strength of their evidence. Most retrospective and prospective non-RCTs have reported a significant increase in the number of retrieved oocytes and MII oocytes [ 17 , 19 – 22 , 28 ], while a few have shown nonsignificant improvements [ 18 ]. In our study, the fertilization rate and the rate of high-quality embryos were significantly higher in the intervention group, whereas the total number of embryos obtained and the blastulation rate were higher but not statistically significant. In both existing RCTs, similar to our study, the blastulation rate was not significant [ 24 , 27 ]. Some studies have reported a considerable improvement in fertilization rates [ 23 , 29 ], while others have found no significant difference [ 13 , 30 ]. The small sample sizes and methodological differences across studies make direct comparison difficult. Most reports describe an increase, whether substantial or nonsignificant, in the number of embryos obtained after PRP injection [ 17 , 19 – 22 , 28 ]. Only a limited number of studies addressed embryo quality. These studies, in line with ours, reported better embryo quality following PRP injection. However, since we no utilized the time-lapse embryo imaging technique for assessment, we cannot comment on this with certainty. 10.5% of our patients in the intervention group achieved spontaneous pregnancy within 3–8 months after intra-ovarian PRP injection, whereas no spontaneous pregnancies occurred in the control group during one year of follow-up. According to two meta-analyses, spontaneous pregnancy rates after intra-ovarian PRP injection in patients with POR or DOR range between 5% and 7% [ 17 , 28 ]. Based on the spontaneous pregnancy rates, we consider the present study a success. Some may consider the effects of the needle entering the ovarian tissue and the resulting inflammation to be effective in the regeneration of ovarian tissue and the occurrence of spontaneous pregnancy [ 1 ]. Nevertheless, needle entry into the ovarian tissue alone appears insufficient to explain these effects. In the control group, 57% of patients underwent FP within three months before enrollment, and 76% underwent FP after study entry. Therefore, if ovarian puncture alone induced spontaneous pregnancy, we would have observed cases in the control group as well. In our study, the clinical pregnancy rate per fresh ET was significantly higher in the intervention group compared with the control group (41.2% vs. 6.7%). Similarly, Simalvi et al. reported a clinical pregnancy rate of 32.6% in a retrospective study after fresh ET [ 31 ]. Based on these findings, we recommend intra-ovarian PRP followed by fresh ET in patients with POR or DOR, rather than pursuing consecutive ovarian stimulation cycles with embryo accumulation and frozen transfer. In our study, the overall clinical pregnancy rate (including spontaneous and ART-derived pregnancies) was also significantly higher in the intervention group compared with the controls (31.6% vs. 15.5%). The live birth rate was higher in the intervention group (18.9% vs. 9.3%), although this difference was not statistically significant. The two existing RCTs reported no significant difference in clinical pregnancy rates [ 24 , 27 ], most likely due to their limited sample sizes. Two meta-analyses reported clinical pregnancy rates of 18–25% and live birth rates of 11–16% [ 17 , 21 ]. Therefore, we consider the performance achieved in our study acceptable. Most existing studies have focused on the impact of intra-ovarian PRP injection on ovarian reserve parameters and ART outcomes, while pregnancy outcomes have received less attention. In our research, EP occurred only in the intervention group, but the difference between groups was not statistically significant. Similarly, no significant differences were observed between the intervention and control groups in rates of miscarriage, preterm labor, twin pregnancy, gestational diabetes mellitus (GDM), or gestational hypertension. No cases of fetal or neonatal anomalies were reported in either group. Although these findings are encouraging, confirming the efficacy and safety of intra-ovarian PRP requires further high-quality studies. Overall, evaluating the effectiveness of intra-ovarian PRP remains challenging due to heterogeneity in study populations, methodological differences, small sample sizes, and variations in PRP preparation protocols. For instance, different procedures for the preparation of PRP can result in different qualities, purities, and quantities. This may include variations in the speed and duration of centrifugation, which can result in a higher platelet concentration at higher speeds or compromised platelet integrity [ 32 ]. The volume of PRP injected per ovary has ranged from 0.2 to 5 ml across studies, with 4 ml being the most frequently reported. Most studies did not specify the extent to which platelet concentration in PRP exceeded baseline serum platelet levels, making it difficult to assess outcomes relative to PRP dose. In some reports, patients received more than one injection; in these cases, 0.2–2.5 ml PRP was administered during 2–3 consecutive cycles [ 13 , 25 , 26 , 30 , 33 ]. Because of their small sample sizes, these studies cannot provide conclusive evidence regarding the efficacy of repeated injections. The interval between intra-ovarian PRP injection and initiation of ovarian stimulation has varied from 1 to 3 months. Most studies have limited follow-up to a maximum of three months, making it difficult to assess long-term efficacy. It is worth noting that none of the existing studies have specified the depth of injection within the ovarian tissue, and this aspect requires further evaluation. Collectively, current evidence indicates that no standardized protocol exists for intra-ovarian PRP injection. Weak standardization reduces the likelihood of consensus on its effectiveness. Researchers are therefore encouraged to report methodological details thoroughly in clinical studies. Concerns also remain regarding the safety of intra-ovarian PRP [ 22 ]. PRP has found extensive application across various regenerative medical approaches, with studies confirming its capacity to induce differentiation of diverse cell types. Given that PRP may create favorable conditions for ovarian stem cell differentiation, the potential risk of tumorigenic or malignant transformation warrants careful consideration [ 1 , 7 , 8 ]. While no serious complications were observed during the one-year follow-up in this study, long-term safety evaluation of this method requires follow-up extending beyond one year. Given the limited number of RCTs, large-scale randomized controlled studies are required to identify suitable candidates for intra-ovarian PRP, define age cutoffs, determine optimal PRP preparation protocols, establish the timing and frequency of injections, and evaluate both safety and long-term efficacy. Very few RCTs have investigated intra-ovarian PRP, while most reports consist of retrospective studies or uncontrolled prospective before–and–after studies. Conducting the present study as a RCT with a relatively large sample size is therefore its main strength. Another strength is that, unlike most previous studies, we examined pregnancy outcomes in detail. A limitation of our study was the lack of blinding. Intraovarian PRP injection was performed under anesthesia; therefore, administering a placebo intraovarian injection would have also required anesthesia, and for ethical reasons, we decided to forgo it. Although we acknowledge that implementing blinding would have increased the statistical power of the study. Most available studies have evaluated the effect of intra-ovarian PRP in DOR patients defined by the Bologna criteria or in POR patients classified as POSEIDON groups 3 and 4. Future studies should investigate POR patients classified as POSEIDON groups 1 and 2. These patients, despite acceptable ovarian reserve markers such as AMH and AFC, experience poor ART and fertility outcomes. Many of them, despite retrieving a sufficient number of oocytes after ovarian stimulation, lack MII oocytes or fail to achieve embryos. Others experience adverse pregnancy outcomes such as second-trimester miscarriage, intrauterine fetal demise (IUFD), or unexplained preterm labor before 30 weeks. Considering the regenerative potential of PRP, its use in this patient population may also prove beneficial. Also, the intricate mechanism of action of intraovarian injection of autologous PRP is not completely elucidated. Some studies indicate that PRP may activate dormant follicles, promoting their growth and development. Furthermore, PRP may promote the differentiation of ovarian stem cells into new oocytes, which could result in the generation of high-quality oocytes [ 21 ]. Studies have shown that the diversity of platelet cargo proteins suggests the target response of intraovarian PRP is probably not restricted to oocytes or follicles. For example, PRP influences signaling networks that enhance perfusion, modulate HOX gene expression, regulate ion channels, stabilize telomeres, and restore ribosomal and mitochondrial function in older oocytes [ 34 ]. In the present study, despite the absence of a statistically significant difference in serum AMH levels and the number of retrieved oocytes, the intervention group demonstrated superiority in terms of oocyte and embryo quality. Therefore, the improved pregnancy outcomes observed in the intervention group should be interpreted in light of a clearer understanding of the precise mechanisms underlying intraovarian PRP injection. Nevertheless, the results of the present study support the notion that the effect of intraovarian PRP injection is qualitative rather than quantitative. There for, further research should also evaluate the effect of intra-ovarian PRP on follicular fluid parameters to understand its mechanisms of action better.

Our study demonstrated that the injection of 3 mL PRP with a fivefold platelet concentration compared to baseline serum resulted in increased AFC, reduced ART cycle cancellations, and improved quantitative and qualitative parameters of oocytes and embryos. In addition, intra-ovarian PRP was associated with a considerable rate of spontaneous pregnancy and pregnancy after fresh ET, without significantly increasing rates of pregnancy complications such as GDM, gestational hypertension, preterm labor, or fetal and neonatal anomalies.

Supplementary Material 1. Supplementary Material 1.