In the context of assisted reproductive techniques (ART), poor ovarian response (POR) refers to a condition where the ovaries exhibit reduced follicular response during ovarian stimulation. This typically results in a lower number of retrieved oocytes compared to expected levels (1). While egg donation remains the most successful treatment for patients with POR, emerging methods are providing new hope for conceiving with one’s own eggs (2). Recent studies have shown that platelet-rich plasma (PRP) injections in the ovaries can significantly enhance ovarian function, offering renewed optimism for women with poor ovarian response (3).

PRP is derived from a patient’s own blood, concentrated to include a high number of platelets, which are rich in growth factors. These growth factors play pivotal roles in cellular repair and regeneration, making PRP a promising treatment for ovarian rejuvenation. This is particularly relevant for women experiencing diminished ovarian reserve (DOR) or primary ovarian insufficiency (POI) (4, 5). The diverse bioactive molecules in PRP play a pivotal role in enhancing ovarian function, influencing follicular development, oocyte quality, and overall reproductive outcomes (6–10) (Fig. 1).

Our study aims to address the ongoing debate surrounding the use of PRP injection in poor ovarian responders. While some studies report promising results and remarkable effects, others find no significant efficacy. We will investigate the factors contributing to this variability and discuss methods to enhance future research and therapeutic strategies involving PRP injection for poor ovarian responders.

We conducted a comprehensive literature search using four databases, including PubMed, Cochrane Library, Embase, and Google Scholar. Our search query included the following terms: “Poor ovarian response” OR “Poor Ovarian Responders” OR “Poor responder”, in combination with “Platelet rich plasma” OR “Platelet-rich plasma” OR “PRP”. The inclusion criteria comprised all relevant articles investigating the intraovarian injection of PRP in poor ovarian responders, including case series, case-control studies, retrospective/prospective cohort studies, and clinical trials. Duplicate articles, review articles, systematic reviews, meta-analyses, and studies published in languages other than English were excluded, as well as studies for which the full texts were not available (Fig. 2). Two reviewers independently screened the articles, with an expert overseeing the process to address any discrepancies. The desired data, including details about study characteristics, information about PRP preparation, intra-ovarian injection methods, and the study outcomes, were extracted.

18 articles were included in our review and extracted data categorized into 4 tables based on Study characteristics and design, PRP preparation procedure, Injection methods, and outcomes (11–28).

1. Study characteristics and design

The outcomes of studies investigating the effect of intraovarian platelet-rich plasma (PRP) injections can vary significantly due to differences in study design, sample size, participant selection, confounding factors, and other methodological variations (Table 1).

Common limitations acknowledged by the studies were the small sample size and the absence of a control group (29–39). While initial studies, often pilot studies, case series, or retrospective/prospective cohorts without control groups, showed a significant and promising effect of intraovarian PRP injections in poor ovarian responders, recent randomized controlled trials (RCTs) did not demonstrate significant improvement (28, 40).

To obtain more realistic and reliable results, studies should be conducted using RCT designs with control groups. An acceptable sample size is essential for statistical power. While a sample size of at least 40 patients per group is reasonable based on available studies, it should be tailored to the specific research question and desired level of statistical significance (41).

In comparing the outcomes of PRP injection between the treatment and control groups, researchers must account for interventions in the control group. Whether it involves administering a volume (placebo or another substance) or needling the ovaries, these actions can indeed elicit physiological responses that may impact the study outcomes, as well as ethical considerations, blinding, and randomization (42, 43).

The definition of POR varies across studies. While the Bologna criteria provide a framework for defining POR, there is ongoing debate about the optimal thresholds for antral follicle count (AFC), Anti-Mullerian hormone (AMH), and age (44). The POSEIDON system, on the other hand, considers female age, ovarian reserve markers, ovarian sensitivity to gonadotropins, and the number of retrieved oocytes. It stratifies patients into four groups based on expected or unexpected impaired ovarian response to stimulation (45). Despite the establishment of the Bologna and POSEIDON criteria, discrepancies in defining POR persist, creating challenges for consistent interpretation of trial outcomes (46). Clinical trials often enroll poor ovarian responders based on either the POSEIDON or Bologna criteria. However, it’s important to recognize that POR exists along a spectrum rather than as a fixed diagnosis. This variability among participants can impact trial results and their subsequent interpretations.

In several meta-analyses, we’ve noticed the inclusion of studies based on both the POSEIDON and Bologna criteria. However, this dual inclusion can complicate comparisons and interpretation of results, potentially impacting overall conclusions (5, 47–49). To enhance reliability and comparability, systematic reviews and meta-analyses investigating the efficacy of PRP injection in the ovaries would benefit from using specific inclusion criteria, thereby minimizing heterogeneity among the included cases. In a study comparing the impact of PRP injection across the four POSEIDON groups, the most significant change in total oocyte count was observed in Group 4 (50). However, another study comparing Groups 3 and 4 found no significant difference (28). These findings indicate diverse outcomes in different subgroups, even when using unified criteria.

The POSEIDON criteria include age as one of the subgroup classifications. Given that age significantly influences ovarian reserves and function, utilizing the POSEIDON criteria enables researchers to create more homogeneous study groups (45). This enhances the reliability of findings related to PRP treatment in patients with POR. Additionally, it’s essential for researchers to rigorously compare candidates’ outcomes based on subgroups to gain meaningful insights and tailor treatments effectively.

Our remarkable blood system allows our bodies to tolerate a wide range of platelet counts without symptoms. Even though the typical range for platelets in blood is 150,000 to 450,000 per microliter, individuals with platelet counts exceeding 50,000 (thrombocytopenia) or those with thrombocytosis (more than 450,000 platelets per mcL) may remain asymptomatic (51, 52).

This variation in platelet counts, even without causing noticeable signs or symptoms, can significantly influence the outcomes of PRP injections in poor ovarian responders. Notably, one patient may receive up to ten times more platelets compared to another patient (53). Despite some studies employing a platelet count cut-off or excluding patients with thrombocytopenia, it is advisable to assess platelet concentration after preparing PRP and before injection, to achieve more consistent outcomes and find the best therapeutic dosage. (Table 2,3)

2. Platelet-rich plasma Preparation procedure

The variability in PRP preparation methods across studies contributes to diverse results. Different approaches in PRP processing, including factors such as the initial blood volume, centrifugation speed, recentrifugation, anticoagulants, and activation protocols, can lead to significantly different outcomes. A literature review on the optimal PRP preparation protocol revealed the complexity of implementing a standardized approach (54). Various studies employ different models for PRP preparation procedures (Table 2). When preparing PRP, the concentration of platelets depends on the volume of blood processed. Smaller blood volumes result in more concentrated PRP, with a higher platelet count per milliliter. Conversely, larger blood volumes yield more diluted PRP due to the relative proportion of platelets across the total volume. This concentration variation occurs because platelets are not evenly distributed throughout the blood; they tend to be more concentrated in the initial sample (55–57).

For optimal results, it is better to consider recentrifugation when dealing with larger blood samples for PRP preparation. This step ensures consistent platelet concentration and maximizes the therapeutic potential. Researchers and physicians should prioritize this practice to achieve reliable outcomes. During centrifugation to concentrate platelets, consider their integrity and activation while safeguarding against aggregation and fragmentation. Lower speeds tend to result in less effective separation and more aggregation (58, 59), while higher speeds lead to better separation and less contamination of other cells. Additionally, increased centrifuge speed tends to enhance platelet activation without aggregation or fragmentation (60). Although platelets are primarily considered to reside in the buffy coat (61), a recent study found that the optimal platelet count was achieved by aspirating 0.5 mL below the buffy coat and extending 0.5 mL above it, rather than starting directly at the buffy coat and aspirating only plasma (62).

While no direct comparison exists between activated and non-activated PRP for ovarian function, a recent meta-analysis demonstrated that exogenously activated PRP is more effective in improving pain and function than non-activated PRP in patients with knee osteoarthritis (63). The activation of PRP leads to platelet degranulation, releasing nearly 100% of growth factors within 1 hour (64). This enhanced release of growth factors, including platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and Transforming Growth Factor beta (TGF-β), may contribute to the observed effectiveness. Also, Platelet function is influenced by storage, freezing, and thawing. Fresher platelets tend to have higher levels of growth factors. (65, 66) Selecting the most effective activator and determining the optimal dosage during PRP preparation play crucial roles in promoting tissue healing and regeneration while minimizing excessive clot formation (67). A 10% calcium chloride solution, with a ratio of 1-part CaCl₂ to 5 parts PRP by volume (approximately 50 microL CaCl₂ per mL of PRP), effectively activates platelets and releases their growth factors. Once activated, platelets should be used within five minutes to minimize clot formation (67–69).

Normal ovarian volume in adult women is approximately 6–7 mL, as reported in several studies (70–72). However, this volume is even smaller in poor ovarian responders due to diminished ovarian reserve and a reduced number of follicles (73). Injecting an excessive volume can lead to damage, rupture, or complications. Additionally, physicians must consider that injections may result in leakage, potentially causing a portion of the PRP to be missed (29, 74). While no specific rule governs the selection of injection doses, it appears that a minimum of 2 mL is necessary for a therapeutic effect in the resulting reaction (75). Thus, a Mid-volume (2 mL), highly concentrated (achieved through double centrifugation), and activated PRP may be more favorable for improving ovarian function in poor ovarian responders.

3. Injection techniques

The outcomes of PRP injections can vary based on the specific method used. The Injection volume, injection method, whether unilateral or bilateral, laparoscopic or guided by transvaginal ultrasound, the specific injection spot (intra-ovarian, intra-follicular, or systemic intravenous), and whether the injection is multifocal or uniformly distributed significantly impact treatment outcomes (Table 3).

In the context of bilateral or unilateral PRP injections, studies suggest more promising results with bilateral injection. (75) Bilateral injections are generally preferred when both ovaries are accessible and healthy. However, it’s essential to consider that unilateral injection is sometimes used due to anatomical issues (such as blocked access to one ovary), previous surgery, or specific patient conditions (such as unilateral ovarian pathology). (35) These factors may contribute to lower pregnancy outcomes in cases of unilateral treatment.

Timing is crucial in ART. Administering PRP at the time of oocyte collection might not be ideal (76). Luteinization could have already started due to the stimulation, and concurrently, the basement membrane breaks down, leading to vascularization of the granulosa cells (77). This increased blood flow within the ovaries during stimulation can cause PRP to be reabsorbed into the vascular system, potentially limiting its full effectiveness (76). Additionally, the heightened vascularity may increase the risk of ovarian abscess and subsequent bacteremia (78).

While TVS is a powerful tool for diagnosing and monitoring ovaries, it does present challenges for intra-ovarian injections that can impact accuracy (79). These challenges include anatomical variability due to factors such as body habitus, bladder filling, and uterine position—all of which affect ovarian visibility (80). Additionally, small ovarian size, particularly in poor ovarian responders, and senescent changes in older patients may further complicate the procedure. (73) On the other hand, laparoscopic intra-ovarian injection is more invasive but offers the advantage of precise localization (81). In situations where TVS fails to visualize the ovaries due to factors such as abnormal localization, large fibroids, or interference from intestines or blood vessels, laparoscopic access is employed to ensure safety and accurate assessment (82).

In the context of intraovarian injections, utilizing a 17-gauge needle with a length of approximately 30–35 cm ensures effective delivery of substances to the targeted areas, consistent with findings from studies reported in Table 3. Multifocal spots injection targets multiple specific areas within the ovary, stimulating various regions simultaneously. This approach may enhance overall ovarian function by treating different follicles and tissues. In contrast, a single-site uniform distribution spreads PRP evenly throughout all layers of the ovary. It ensures that PRP reaches both the cortical (outer) and medullary (inner) compartments, supporting all follicles, regardless of their location within the ovary (83). While single-site uniform distribution is more beneficial for comprehensive ovarian support, the multifocal spots technique is particularly useful when treating a specific targeted area. Considering that the problem with poor ovarian responders primarily lies in their follicles and given that primordial and preantral follicles are usually located in the cortical compartment of the ovaries (84), targeting the cortex should be a key consideration when using the multifocal spot technique (Table 3). However, further studies are necessary to compare the efficacy of both methods and determine which one is more proficient overall (83).

4. Studies Outcomes

Different Assisted Reproductive Technology methods

To date, no studies have investigated which stimulation protocol or ART technique is superior after intra-ovarian administration of PRP. While Various studies have employed different types of stimulation protocols, these diverse approaches can lead to varying outcomes, independent of the impact of PRP (Table 4).

In a study, the GnRH-a long protocol resulted in a higher number of retrieved oocytes and mature oocytes compared to the GnRH-a miniflare protocol. (85) In a review study, the GnRH Agonist Long Protocol demonstrated improved folliculogenesis and higher pregnancy rates. (86) Notably, in comparing DuoStim (Shanghai protocol) and minimal ovarian stimulation, two recent studies reached very different outcomes. Jingzhe Li et al. found that the number of frozen embryos in the DuoStim group was significantly lower than in the two consecutive mild stimulations group in the elderly POR group (ages ≥ 35 years). In contrast, Saharkhiz et al. found that the DuoStim protocol can lead to significantly more metaphase II (MII) oocytes and embryos compared to minimal stimulation (87, 88). However, despite some minor discrepancies, studies have shown that in cases using in vitro fertilization (IVF) or Intracytoplasmic Sperm Injection, the live birth rates and embryo quality are similar (89, 90).

The most significant effects of PRP in the ovaries are short-term, starting from 2 weeks to 3 months after injection (91, 92). Most studies measure outcomes 2–3 months post-injection (47). It is also suggested to wait about 2 to 3 months after the PRP injection before beginning ovarian stimulation (93).

As indicated in Table 4, the studies reported their findings using various parameters, including changes in hormonal levels (such as AMH, LH, and FSH), the number of oocytes, oocyte quality, embryo quality, and pregnancy and fertilization rates.

While PRP treatment aims at ovarian rejuvenation in patients with POI or early menopause, the focus shifts toward optimizing oocyte quality and quantity for poor ovarian responders and those with DOR (4, 5).

When addressing poor ovarian responders seeking fertility treatment, including PRP injection, it’s essential to focus on improving their chances of pregnancy and successful IVF cycles (94). While assessing factors like anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels is important, the goal is to enhance live birth rates. (95)

Despite the growing interest in intra-ovarian PRP therapy for poor ovarian responders, studies are not unanimous on its effectiveness. The discrepancies arise from variations in study protocols and characteristics, differences in PRP preparation methods, diverse techniques and strategies for intra-ovarian injection, and the measurement of different parameters and outcomes. Randomized controlled trials must be conducted with a standardized sample size, ensuring interventions are applied to both groups. Candidates should be selected more homogeneously, adhering to specific inclusion criteria. Baseline and concentrated platelet levels must be considered, and PRP preparation methods should be standardized. Intra-ovarian injection parameters, including PRP volume, use of TVS or laparoscopy, injection site, timing, and administration frequency, should be tailored to patient characteristics. Ultimately, employing the optimal stimulation protocol, determining the optimal interval after PRP injection, and prioritizing outcomes such as live birth rates should be the focus of future studies and therapeutic efforts.