A Systematic Review Evaluating the Efficacy of Intra-Ovarian Infusion of Autologous Platelet-Rich Plasma in Patients With Poor Ovarian Reserve or Ovarian Insufficiency

The emergence of autologous platelet-rich plasma (PRP) therapy reflects a break-through for infertile patients with premature ovarian failure. To study the efficacy of intra-ovarian infusion of autologous PRP on the improvement of ovarian reserve parameters and the subsequent artificial reproductive technique (ART) cycle outcomes in infertile women with poor ovarian reserve or premature ovarian insufficiency, a systematic search in electronic databases like Medline (through PubMed), Embase, Scopus, Web of Science, and Cochrane was done using relevant search terms. Except for case series, case reports, and review articles, all other types of studies, those evaluated for the effects of intra-ovarian infusion of PRP in subfertile women for decreased ovarian reserve (DOR) or premature ovarian insufficiency (POI) were included in our systematic review. The data were extracted from each eligible study and cross-checked by two authors. Intra-ovarian PRP infusion appears to be effective in ovarian rejuvenation, and the results of the subsequent intracytoplasmic sperm injection (ICSI) cycle are encouraging. PRP intervention was found to be beneficial in terms of an improvement in ovarian reserve parameters (increase in serum anti-mullerian hormone or antral follicle count or decrease in serum follicular stimulating hormone). ICSI cycle performance in terms of the total number of oocytes retrieved, number of two-pronuclei embryos, fertilization rate, number of cleavage stage embryos, number of good quality embryos, and cycle cancellation rate were found to be improved after intra-ovarian PRP infusion as compared to their previous cycle without PRP infusion.


Introduction And Background
A steady decline in the quantity and quality of the oocyte reserves associated with ovarian ageing acts as the principal limiting factor for success in both spontaneous conception and assisted reproductive technology (ART) [1][2]. These aged oocytes are also more prone to errors in deoxyribonucleic acid (DNA) synthesis and cell division, resulting in increased rates of aneuploidy and congenital defects in the resulting conceptions [3]. In the absence of any effective treatment to prevent, delay, or reverse ovarian senescence, various therapeutic strategies like antioxidant dietary supplements containing vitamins C and E, melatonin, dehydroepiandrosterone (DHEA), and coenzyme Q10 have been used to address this issue [4][5][6][7][8]. However, evidence in their support are inconclusive, and their overall effectiveness remains sparse [9][10]. At present, treatment strategies to tackle infertility associated with ovarian insufficiency are commonly in vitro fertilization (IVF) treatment in conjunction with an oocyte donation program or adoption [11][12][13][14][15][16]. Of late, ovarian function restoration approaches are being investigated rigorously, which can result in healthy, genetically linked offspring in these patients. The successful use of platelet-rich plasma (PRP) in regenerative medicine has led investigators to study its effect in the treatment of conditions like decreased ovarian reserve, premature ovarian failure, etc. [17]. The emergence of autologous PRP therapy reflects a breakthrough approach, showcasing promising results. However, at present, there are very few studies addressing this issue.
This systematic review was conducted to study the effects of intra-ovarian instillation of autologous PRP on ovarian rejuvenation. It was designed to study the efficacy of intra-ovarian infusion of autologous plateletrich plasma (PRP) on the improvement of ovarian reserve parameters and the subsequent artificial reproductive technique (ART) cycle outcomes in infertile women with poor ovarian reserve or premature ovarian insufficiency. This systematic review was done to assess the effectiveness of intra-ovarian infusion of PRP in sub-fertile women in terms of improvement in ovarian reserve parameters and outcomes after assisted reproduction. We followed the recommendations of the Cochrane Handbook for Systematic Reviews of Interventions and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines ( Figure 1).

PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses
A systematic search in electronic databases like Medline (through PubMed), Embase, Scopus, Web of Science, and Cochrane was performed from January 2000 to November 2020. We used search terms like: ("In Vitro Fertilization" OR "IVF" OR "Intracytoplasmic sperm injection" OR "ICSI" OR "Embryo transfer" AND "Platelet-rich plasma" OR "PRP" OR "Autologous platelet-rich plasma" OR "Platelet-rich plasma" and "premature ovarian failure" OR "decreased ovarian reserve" OR "premature menopause" etc). Literature published only in the English language was included in our study.

Inclusion and exclusion criteria
All studies that evaluated the effects of intra-ovarian infusion of PRP in subfertile women for decreased ovarian reserve (DOR) or premature ovarian insufficiency (POI) were included in our systematic review. This review included all types of studies, including case-control studies, cohort studies, or clinical trials. Data regarding ovarian reserve parameters and the intracytoplasmic sperm injection (ICSI)/in vitro fertilization (IVF) cycle characteristics were analyzed in this systematic review. Case series, case reports, and review articles were excluded from our systematic review.

Meta-analysis
A meta-analysis was planned; however, due to the paucity of available studies and the heterogeneity of the included outcomes, it was deemed inapplicable.

Summary of the Literature Search
The initial electronic literature search yielded 908 publications. After excluding duplicates and irrelevant publications, we found 16 potentially eligible studies. After reading the full text of those 16 articles, 12 articles (three case series, two case reports, one article with insufficient data, one study involving in-vitro experimental design, and five review articles) were excluded. Finally, four studies were included in our systematic review [19][20][21][22]. The flow diagram of the literature search and selection of studies is shown in Figure 1.

Study Characteristics
In this systematic review, we have included four studies that evaluated the effectiveness of PRP in infertile women diagnosed with decreased ovarian reserve or premature ovarian failure or menopause. Table 1 outlines the characteristics of all included studies [19][20][21][22].  Out of four included studies, one was a non-randomized clinical trial and three were quasi-experimental studies (uncontrolled before and after studies). All studies compared the ovarian reserve parameters (AMH, FSH, and antral follicle count (AFC)) before and after PRP infusion. The analysis of the outcome of the ICSI cycle was done in three out of four studies [19][20][21]. The method of preparation of PRP and time schedule of injection are described in Table 2

Ovarian reserve parameters
Serum Anti-Mullerian Hormone (AMH) As shown in Table 3, the study conducted by Sfakianoudis et al. [21] showed an increase in serum AMH level after treatment with PRP for the cohorts of poor ovarian response, premature ovarian insufficiency, perimenopause, and menopause, respectively. The difference was found to be statistically significant with pvalues of less than <0.0001 in all groups.  Similarly, Melo et al. found an increase in the median value of the post-treatment level of serum AMH (in ng/ml) from the respective pretreatment level [20]. The median difference was 0.5 (0.43 to 0.57), and the increase in serum AMH with PRP infusion was found to be statistically significant (p-value <0.001). This has been depicted in Table 3.
At the same time in the study by Cakiroglu et al. [19] and Sills et al. [22], the level of serum AMH was also found to be increased ( Table 3) after intra-ovarian PRP infusion with a statistically significant difference (pvalues of <0.01 and 0.0016 for the respective studies). Sills et al. [22] found platelet count as a parameter likely to predict AMH response to intra-ovarian PRP injection.

Serum Follicular-Stimulating Hormone (FSH)
In the study by Sfakianoudis et al., the serum FSH levels were decreased post-treatment with PRP for all the studied cohorts [21]. Except for the cohort of women with poor ovarian reserve (p-value=0.1342), all other cohorts were having a statistically significant decrease in serum FSH level (p-values of <0.0001, 0.0024, and <0.0001 for cohorts of premature ovarian insufficiency, perimenopause, and menopause respectively).
Melo et al. found a decrease in the median of post-treatment values of serum FSH with a median difference of − 5.5 (− 6.3 to − 4.9) and the difference was found to be statistically significant ( p-value -0.001) [20].
In the study by Cakiroglu et al., the average of pretreatment and post-treatment values of serum FSH (all values in mIU/ml) were found to be 41.9 ± 24.7 and 41.6 ± 24.7 [19]. But this decrease in serum FSH levels was not statistically significant (p-value=0.87).
Moreover, Sills et al. found an increase in the post-treatment serum FSH value of 64.68 ± 5.5 from its pretreatment value of 52.67 ± 4.64 with a statistically significant difference (p-value<0.0001) [22].
These findings are tabulated in Table 3.

Antral Follicle Count (AFC)
As far as antral follicle count is concerned, the studies by Sfakianoudis et al. [21] (in all of their studied cohorts) and Melo et al. [20] found an increase in AFC after treatment with PRP, and this increase was statistically extremely significant with p-values of <0.0001 in each of the studies. This is shown in Table 3.
Again in the study by Cakiroglu et al. [19], the average of pretreatment and post-treatment values of antral follicle count were found to be 0.5 ± 0.5 and 1.7 ± 1.4 indicating a statistically significant increase (p-value= <0.01).

Outcomes of the ICSI Cycle
Sfakianoudis et al. compared the post-PRP ICSI cycle with the prior ICSI cycle as control [21]. They found the average of total number of retrieved oocytes ( Table 4) [20].   In the study by Cakiroglu et al., ART was attempted in 201 women who had at least one antral follicle after PRP out of which oocyte retrieval was performed in 130 (64.7% were stimulated) women [19]. Out of this, in 82 women (40.8% of stimulated cycles), at least one cleavage-stage embryo was obtained and embryo cryopreservation or fresh embryo transfer was performed. These embryos were grade 1/2 morphologically. The mean number of oocytes per retrieval was 1.81 ± 1.30. The mean numbers of two-pronuclei (2 PN) and cleavage-stage embryos obtained in women who developed embryos were 1.24 ± 0.49 and 1.18 ± 0.39, respectively. Among the 82 women who developed embryos, 25 preferred to store cryopreserved embryos for transfer at a later stage and 57 underwent embryo transfer. Of those who underwent embryo transfer, 28/57 (49.1%) were fresh embryo transfers and 29/57 (50.9%) were frozen-thawed embryo transfers; 7/28 (25.0%) of fresh embryo transfers and 6/29 (20.7%) of frozen-thawed embryo transfers resulted in a pregnancy ( Table  4).

Discussion
This systematic review involved the data analysis of 663 subfertile women who were intervened with an intra-ovarian infusion of PRP from four studies. PRP intervention was found to be beneficial in terms of improvement in ovarian reserve parameters (increase in serum AMH or antral follicle count or decrease in serum FSH). The outcome of the ICSI cycle was studied in three out of four included studies. The outcome of the ICSI cycle in terms of the total number of oocytes retrieved, number of two-pronuclei embryos, fertilization rate, number of cleavage stage embryos, number of good quality embryos, and cycle cancellation rate were found to be improved after the intra-ovarian PRP infusion as compared to their previous cycle without PRP infusion.
A study by Cakiroglu et al. found that women who did not have an antral follicle at the time of PRP injection were less likely to respond to treatment as compared to those who had one or two antral follicles [19]. Similarly, women with the lowest quartile for serum AMH and the highest for serum FSH were less likely to respond. The final conclusion for the same study was that PRP helps activate existing preantral and/or early antral follicles and that the number of remaining follicles in the ovaries of women with POI likely determines the extent of their response [19]. However, at this point in time, we cannot generalize this finding and there is an obvious need for future well-controlled studies to identify the subpopulation that can get the maximum benefit from PRP infusion.
Many studies have documented that the use of PRP can reduce the features of inflammation, postoperative blood loss, infection, and narcotic requirements. Also, PRP has a role in the acceleration of osteogenesis and wound and soft tissue healing [23][24]. The granules in platelets contain certain important growth factors such as transforming growth factor-β, insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor, and hepatocyte growth factor (HGF), which forms the basis for the tissue regenerative properties of PRP [17,[25][26].
These are the same growth factors that are considered to be vital for cell migration and differentiation, as well as for proliferation, activation of angiogenesis, and tissue regeneration [27][28]. Of note, studies have found an inverse relationship between aging and concentrations of growth hormone and IGF-1 [29].
Specifically, the application of PRP in ovarian rejuvenation has not been studied in detail. Till now, only a few studies have addressed this issue [30]. In their study, Bakacak et al. found that PRP can have a significant effect on preventing ischemia and reperfusion damage in rats following bilateral adnexal torsion and surgical detorsion [31]. They concluded that this action was mainly through an increase in VEGF. Few studies in the form of case series got encouraging results after evaluating the application of PRP for managing a thin endometrium, recurrent implantation failure, and poor response to controlled ovarian stimulation [32][33][34].
A case was reported documenting a biochemical pregnancy and subsequent miscarriage following autologous PRP intra-ovarian infusion in an infertile woman with premature ovarian failure [35]. Similarly, cases documenting live birth in poor responders following PRP infusion have also been reported [32]. Additionally, other studies support a contribution of PRP treatment to follicular growth and maturation [36].
Similar to our study, recently, a few case series also highlighted the efficacy of intra-ovarian PRP injection. In the case series described by Pantos et al. [37], Sills et al. [38], and Sfakianoudis et al. [32], the pretreatment serum AMH was increased after the intra-ovarian infusion of PRP. However, this increase in serum AMH levels was statistically significant only in the study by Pantos et al. [37] (p-value 0.0395), and in the studies by Sfakianoudis et al. [32] and Sills et al. [38], the difference was statistically not significant (p-values 0.4546 and 0.17, respectively).
Similarly, in these case series, the pretreatment serum FSH values were decreased after the intra-ovarian infusion of PRP [32,[37][38]. This decrease was found to be statistically significant in all of the above studies with p-values of 0.0310, 0.01, and 0.0053, respectively.
In their case series, Pantos et al. found a post-treatment increase in the mean values of AFC (0 ±0 and 2 ± 1.41, respectively) [37]. However, this was not found to be statistically significant with a p-value of 0.0699.

Strengths and limitations
This is a systematic review evaluating the effect of intra-ovarian infusion of PRP on ovarian reserve parameters and ICSI cycle performance in women with decreased ovarian reserve or premature ovarian failure. However, some limitations should be considered in the interpretation of this systematic review. The less number of studies (n = 4) and lack of homogeneity among the included studies is the foremost limitation. This is the reason that we could not perform a meta-analysis. In most of the studies, pregnancy characteristics, such as clinical pregnancy rate, miscarriage rate, chemical pregnancy rate, and live birth rate have not been evaluated. Only a few studies have evaluated the ICSI cycle performance. Lastly, most of the included studies are quasi-experimental studies and not a single RCT is included. However, as the intraovarian infusion of PRP is a rare and the newest form of therapy in the field of infertility, such limitations are expected while conducting a systematic review. Moreover, the encouraging result of our study has paved the way for conducting future, well-organized, randomized controlled trials.

Conclusions
Our systematic review showed that intra-ovarian autologous PRP infusion increases the ovarian reserve parameters resulting in increased mature oocyte yield, fertilization rate, as well as the formation of goodquality embryos. Thus, this sensational novel therapy is particularly a great finding in the field of reproductive medicine, as this has the potential to put a full stop to our long search for the question of poor ovarian reserve and getting a genetically linked baby. Definitely, there is a great need for future, high-quality randomized controlled trials to estimate its efficacy in terms of clinical pregnancy and live birth rate. Also, there is a need to identify an optimum level of serum AMH or another marker of ovarian reserve for the success of intra-ovarian PRP infusion and identify the subpopulation that would get the most benefit from PRP.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.