Intra-ovarian injection of platelet-rich plasma into ovarian tissue promoted rejuvenation in the rat model of premature ovarian insufficiency and restored ovulation rate via angiogenesis modulation

Women’s reproduction disability, mentioned also as infertility, is defined as the failure achieving pregnancy in a duration of 1 year with unprotected sexual intercourses [1]. Both male and female patients almost equally suffer from infertility complications [2], which are caused by different factors, such as genetic background, obstructive disorders, and environmental elements. It has also been well documented that chemicals or anti-cancer gonadotoxic drugs are major inducers of sub-fertility in females before the physiological age, known as premature ovarian insufficiency (POI). In this regard, most POI patients derived to the fertility clinics have previously received chemotherapy [3, 4]. Moreover, several complications are associated with POI such as osteoporosis, depression, cardiovascular diseases, etc. [3, 5, 6]. The routine treatment strategies are in general gonadotropin-releasing hormone agonist (GnRHa), hormonal replacement therapy (HRT), and assisted reproductive technologies (ART). These procedures, however, are not providing satisfactory recovery to the patients [7, 8]. For example, HRT-based treatments have been linked to undesired secondary effects, like breast and ovarian cancers, venous thromboembolism [9, 10], and incapability of reforming uterine volume and/or endometrial thickness [3, 11]. In the case of ART, which considered as the primary strategy applied for patients dealing with POI [12], several unwanted effects were usually observed, such as inconsistent clinical outcomes, invasive surgical procedures, or prolonged ovulation induction protocols [7, 13]. In consequence, a new generation of therapeutics based on certain cell and cell products are being developed to palliate these deficiencies.

Platelet-rich plasma (PRP) is a remarkable example. It is composed by a high density platelet concentrate, and has been typically used for the treatment of various problems, such as surgeries or diabetic wounds, osteoarthritis, skin, and soft tissue damages [14‐18]. Platelets derived from megakaryocytes in the bone marrow are anucleated blood cells with a life span of 7 to 10 days in humans, and a bit shorter in mice [19]. They are easy to isolate and have an active role in wound healing and tissue repair due to their α-granules content [20, 21]. The α-granules contain more than 800 different proteins that have a paracrine effect on surrounding cells, especially on local mesenchymal stem cells (MSCs), promoting a rapid tissue regeneration [21].

Angiogenesis is a sophisticated biological phenomenon participating in physiological and pathological conditions, refers to the formation of de novo vascular units from the pre-existing vascular network. Importantly for POI reversal, the increase of vascularization in ovaries to provide an increase of blood nourishment, contributing to the acceleration of healing procedure and scavenging tissue debris [22]. The critical role of different ligands, notably angiopoietins (e.g. ANGPT1 or ANGPT2), and receptor tyrosine kinases (e.g. VEGFR-2 or KDR) has been well documented in relation to angiogenesis, and therefore we hypothesize that the measure of their expression after PRP administration in POI patients provides a good picture of the regeneration status [23].

The current investigation aimed to assess the effectiveness of PRP on ovarian rejuvenation in an experimentally 4-vinylcyclohexene dioxide (VCD) induced POI model in rats [24]. VCD, a side product of 4-Vinylcyclohexene (VCH), is usually employed as an adjuvant in epoxy resins; however it has been shown as well to selectively destruct both primordial and primary follicles [24‐26], similarly to what occurs in POI. Therefore, after POI induction in rats with VCD, PRP solution was injected at two different concentrations into ovarian tissue. Subsequently, fertility status, follicular developments, and angiogenesis-related transcript expressions of ANGPT2 and KDR were assessed using histological examination and transcriptomic assays. This study could help in addressing therapeutic alternatives in the restoration of ovarian tissue in POI patients.

It has been revealed that general clinical practices such as HRT, ART, and GnRHa, are not effective enough for POI conditions [3, 7‐11]. Nowadays, several alternative therapeutic approaches, such as cryopreservation and stem cell therapies, are at the new front line [29, 30], but to determine the most beneficial ones, several advancements have been made in the field of animal modeling.

Mayer and colleagues in 2004 reported that daily IP injection of VCD (160 mg/kg) for 15 continuous days is an optimum dose to induce POI conditions, significantly diminishing the population of morphologically normal follicles within the ovaries [27]. In this study, POI physio-pathological features were achieved, like follicular depletion and atresia. From the therapeutic perspective, animal models have also been successfully employed, like in the work of Dehghani et al., in 2018. They reported that IP injection of PRP in POI rats increased the healthy primary and antral follicle counts, and they pointed out PRP as a protective agent against ovotoxic chemicals [31]. Along with the conduction of experiments involving intra-ovarian injection of PRP in animal models, some clinical trials have been started in human, opening hopes and therapeutic avenues in ovarian regeneration [32, 33].

Takehara and colleagues reported that administration of adipose derived mesenchymal stem cells (ADMSCs) could increase morphologically normal follicles counts in the treated rats [34]. In that line, our observations revealed that following intra-ovarian injection of PRP in both low and high concentrations, morphologically normal follicles were almost restored in all stages of follicular development, virtually reaching healthy control counts. As expected, no healthy primary follicles were observed in the POI rats. Evaluating the number of morphologically normal follicles (secondary and antral), there was a significant increase in the PRP-treated rats group versus the POI control. These observations illustrated the efficacy of PRP for the regeneration of ovarian tissue, possibly by shuttling growth factors into the site of injury [35]. Along with these changes, the count of atretic follicles was also obtained in a declining trend for PRP-treated as compared with the POI rats. In contrast to morphologically normal follicles, however, while atretic follicles were significantly reduced in PRP-a administered rats, a higher level of atresia was notified in the PRP-b group. This value could be due to the follicular dominance selection and negative feedback on other growing follicles leading the atresia [36]. Remarkably, the existence of different growth factors inside the platelet-derived granules could revert the cellular damage by increasing rejuvenation phenomenon [37]. Therefore, it is tempting to speculate that the increase of follicular reservoir in PRP-treated rats correlates with the inhibition of VCD-induced injuries.

In 2019, Ling and colleagues reported that human amnion-derived mesenchymal stem cells (hAD-MSCs) administration in POI rats caused overexpression of Bcl-2 and KDR transcript levels [38]. These findings probably were due to the decrease in apoptosis rates and the promotion of angiogenesis within the damaged organs, as well as regulation of follicular development and ovarian function [38]. In the abovementioned work of Takehara and colleagues, the transplantation of ADMSCs to POI rats also increased the gene expression levels of KDR and IGF-I compared to the non-treated groups, promoting angiogenesis rate and ovarian restoration [34]. In addition, Yao and co-workers reported that chemotherapy down-regulated the expression of VEGFR1 and KDR transcript levels, while human amnion epithelial cells (hAECs) transplantation into POI mice contributed to their up-regulation, likely by promoting pro-angiogenic signaling pathways [39]. In our study, VCD administration significantly elevated the transcript levels of ANGPT2 compared to healthy control rats, probably due to the inflammation caused by the chemical itself [40]. Higher expression levels of ANGPT2 and KDR were noticed in both PRP-a, and PRP-b treated groups compared to the POI rats, consistent with the increase of α-SMA⁺ small arteries in IHC imaging [37]. These results possibly indicate that the regeneration of POI ovarian tissue is done through the activation of pro-angiogenic pathways. Accordingly, some works have already highlighted the potent angiogenic activity of platelets via the secretion of, between others, SDF-1a, bFGF, VEGF and IL-8 [41‐43]. The highest expression levels in both treatment groups were 6 weeks after PRP administration, and in the subsequent weeks, a plateau pattern was reached. This could probably be due to the fact that the optimal concentration of PRP and a balanced growth factor delivery were reached [44‐46]. These findings illustrate that in the long term periods, the healing properties of PRP could diminish or, alternatively, that the stable conditions required for regeneration are possibly achieved. These data coincide with the recent report of Sfakianoudis and colleagues, in which six weeks after the autologous PRP treatment, a significant decline in serum FSH levels in the premature menopause patients were recorded [47].

Regarding the evaluation of fertility status after the PRP injection, the mating trial retrieved a significantly higher number of healthy litters in both PRP-treated groups compared to the non-treated ones. In agreement with our findings, and having stem cell infusion as the more recent gold standard [34], Mohamed and colleagues transplanted umbilical cord blood mesenchymal stem cells (UCMSCs) to POI mice, getting a higher number of born litters than in the untreated POI group [48]. Similar results were obtained as well from Lai and co-workers in 2014 and Su and colleagues in 2016, in where skin derived mesenchymal stem cells (SMSCs) and ADMSCs combined with collagen scaffold were used, respectively [8, 49]. Several other reports confirmed as well the effectiveness of cell therapy on fertility restoration and litter counts [7, 34, 49].

Analyzing the FSH hormonal status after intervention, it revealed a minor decline in treated versus non-treated rats, which was not statistically significant. The possible reason of these little differences could be the selective blocking effect of VCD on the c-Kit receptor (a tyrosine kinase receptor) in oocytes [24]. In fact, the main ligand of c-Kit (KITLG), which is a type of growth factor produced by granulosa cells, has an essential role in follicular survival [24]. In a recently published article, Carolino and colleagues studied the endocrine profile of POI rats induced by VCD. They reported that the FSH levels in POI rats did not significantly change for at least 90 days after the VCD administration [50]. In another study, VCD did not influence the FSH levels until 240 days from the start of the experiment [24], and Mayer and colleagues reported that VCD injection to mice do not significantly increase the FSH levels until 37 days [27]. Taking everything together, it could be concluded that an eight-week follow up might be not a long enough period to alter FSH levels in an animal model of POI induced by VCD.

Despite the superior effect of PRP at higher concentration compared to the low concentration in terms of follicle count in almost all time points, litter counts following mating, FSH serum levels and atretic follicle numbers, were not statistically different between the groups. In terms of gene expression analysis, although not statistically significant, PRP-a treated rats exhibited better results, probably due to the negative feedback from a high concentration of platelets in the PRP-b treated subjects. These findings highlight the critical role of PRP concentration in the modulation of angiogenesis signaling pathway within the injured ovaries.

Nowadays, several clinical centers have initiated PRP therapies for various reproductive-related complications, including POI and poor responding patients. As a clear advantage, the PRP-therapy can easily be used as co-adjuvant in patients undergoing chemotherapy, which are already being treated with other fertility procedures, such as ovarian tissue preservation. Additionally, PRP therapy possesses some interesting improvements that surpasses the benefits of other current cell sources for transplants. For instance, derived from its autologous origin, PRP administration does not induce auto-immune response after transplant, a highly prevalent and detrimental secondary effect of the actual therapies. Another example could be that, due to the existence of numerous granules, the introduction of PRP provides a high concentration of growth factors and regenerative modulators directly into the injured sites [18]. Consequently, it is noteworthy to highlight this cell source as a powerful healing product, which can potentially be used in a wide range of pathological processes like, between others, the POI.

Talking about suitable clinical settings to administrate PRP, transvaginal ultrasound- guided injection is a good candidate, since it is a nonsurgical technique that allows the clinicians to inject biological products like cells/PRP directly into the ovarian tissue. This technique is performed under sedation and is basically the same method used for an egg aspiration/retrieval during IVF/ICSI operation [32, 51]. As described above, stem cell transplantation is another putative therapeutic option for POI patients and potentially more powerful in combination with PRP; however, stem cell therapy is sometimes associated with tumorigenesis, making this approach less safe than others [52]. Therefore, PRP treatment could be listed as one of the least invasive procedures, even though, all the possible complications should be evaluated carefully in the future works.

We have demonstrated that PRP can partially restore the function of POI ovaries. It can protect morphologically normal follicles from degeneration and atresia caused by ovotoxic chemicals and stimulate angiogenesis. Moreover, PRP showed a good capacity for tissue rejuvenation, inflammation regulation, minimizing the histopathological damages, and promoting the litter counts. Importantly, the PRP concentration has an important effect on the therapeutic outcome of PRP administration. If excessive, PRP will reduce its efficacy due to possibly a negative feedback generated by its components. It is therefore needed to find an optimized dose for PRP in any specific application to gain the best outcomes. Besides, PRP therapy is relatively cheap, safe, and does not require complicated procedures for its administration thus, overall, PRP could be considered as a putative alternative strategy for a variety of complications linked with female infertility, including POI.