{"paper_id":"e9dc385f-b62c-464a-9448-8a212faaae40","body_text":"Female infertility is recognised by the World Health Organisation (WHO) as a global public health issue ( Macaluso  et al. , 2010 ), with more than one million cycles of IVF being performed globally each year since 2005 ( Zegers-Hochschild  et al. , 2014 ;  Adamson  et al. , 2016 ). Female infertility can arise from a range of conditions, including endocrine dysfunction, implantation failure, endometriosis and uterine fibroids, as well as pathologies related directly to the ovary, including polycystic ovary syndrome (PCOS), primary ovarian insufficiency (POI), environmental factors and inflammatory disease. However, ‘ovarian exhaustion’ is a natural part of the ageing process. In the past 50 years, the mean age at which women have their first child in the UK has increased from 23.8 to 30.7 years ( Office for National Statistics, 2020 ), suggesting that women are delaying childbearing. The impact of delayed childbearing means that women are moving closer to the period of climacteric for conception, and, in many cases, women are choosing not to reproduce until much later. One consequence of this has been a rise in fertility treatment and a rise in the age of women attending for medical investigation. Indeed, in the UK alone, the mean age of women attending for IVF treatment has hovered around 35 for the past 20 years ( Human Fertilisation and Embryology Authority, 2020 ). Since the advent of clinical IVF in 1978 ( Steptoe and Edwards, 1978 ) and associated Assisted Reproductive Techniques (ARTs), it has been possible to treat infertility in a number of cases. However, such approaches are reliant on a healthy oocyte for fertilisation and so have limited success in treating peri- or post-menopausal women without the use of donor eggs. Moreover, ARTs do little to tackle fundamental dysfunction within the ovary and in the oocytes that lead to female infertility and associated physiological adaptations.\nThe prospect of rejuvenating the exhausted ovary has been enticing ever since the description of oogonial stem cells (OSCs) in the ovarian cortex ( White  et al. , 2012 ), hinting at a possibility of therapeutic stimulation of post-natal folliculogenesis in subfertile women. In a study by  Niikura  et al.  (2009) , transplantation of ovarian stem cells from atrophic ovaries from aged mice into young, healthy counterparts resulted in their resumption of spontaneous oogenesis, suggesting that ovarian aging or insufficiency could be reversed if OSCs are provided a healthy environment. Further work has implicated a role for mitochondria in loss of oocyte quality associated with the aged ovary ( Cozzolino et al., 2019 ); indeed, methods to replenish mitochondria within aged oocytes are currently being explored as a means to rejuvenate them ( Labarta  et al. , 2019 ). However, as with IVF, efforts to improve egg quality do not address the wider aspects of age-related ovarian dysfunction.\nOne recently proposed option for ovarian rejuvenation is the intraovarian injection of platelet-rich plasma (PRP) which is being used increasingly in clinical settings for a number of soft tissues, including to support wound healing and ligament and muscle repair ( Suthar et al., 2017 ;  Hurley  et al. , 2019 ;  Verma  et al. , 2019 ;  Zhang  et al. , 2020 ). PRP was first described for ovarian rejuvenation by  Pantos  et al.  (2016) . Their work described how PRP, which is a component of blood, could, when in injected directly into the ovary, trigger the resumption of menstrual cycles in women exhibiting signs of the climacteric. In this review, we will briefly consider the concept of ovarian rejuvenation before describing what PRP is and how it is generated and finally reflecting on the current state of knowledge of ovarian rejuvenation with PRP.\n\nThe paradigm that the mammalian ovarian reserve is fixed at birth dates back to a nineteenth century hypothesis by Waldeyer in 1870, which was reaffirmed by Zuckerman in 1951 (reviewed in  Tilly  et al. , 2009 ). However, there is mounting evidence that this is only part of the story, and that it may be possible to replenish the ovarian follicle pool due to the presence of a population of oogonial stem cells (OSC) in adult ovaries ( Niikura  et al. , 2009 ). It is likely that both of these explanations are true in part; there is a fixed number of follicles at birth, which declines until exhaustion (typically 40+ years of age in the human), but that a population of OSC co-exist in the ovary and may be activated under specific circumstances ( Tilly and Telfer, 2009 ). However, spontaneous reactivation of OSCs is not yet believed to occur naturally  in vivo  in the adult human ovary. This is one principle that underpins the notion of ovarian rejuvenation.\nAs an illustration of this concept, mice rendered sterile from chemotherapeutic drugs can have fertility restored and can produce viable offspring through natural mating after undergoing an OSC transplant from neonatal or adult mouse ovaries ( Zou  et al. , 2009 ). It was further demonstrated that when ovarian tissue containing premeiotic germ cells from aged mice was transplanted into young host mice, the germ cells produced NOBOX-expressing oocytes and formed follicles ( Niikura  et al. , 2009 ). Combined, these studies show OSC transplantation may restore fertility and that it may be possible to produce oocytes from OSC from aged mammalian ovaries in the correct milieux.\nAlthough data from animal models support the notion of OSCs, the presence of equivalent stem cell populations in humans remains disputed. For example,  Virant-Klun  et al.  (2008)  confirmed that ovarian stem-like cells were present on the surface epithelium of post-menopausal women and women with premature ovarian failure (POF), which aligns with the reported location of OSC in the ovaries of juvenile and young-adult mice ( Tilly and Telfer, 2009 ). By contrast, when analysing the cell populations in the human ovarian cortex,  Wagner  et al.  (2020)  were unable to identify a population of germline stem cells. Of course, it must be acknowledged that studies on normal ovarian function in humans is rather constrained since substantial ovarian tissue from healthy, reproductive-aged women is rarely available. Furthermore, tissue from dysfunctional ovaries may not exhibit the full range of physiological function, and biopsies may not be reflective of the whole ovary as stem cells may not be uniformly spread ( Horan and Williams, 2017 ). These factors make it challenging to determine definitively if a population of stem cells is present within the adult ovary.\nIf present, ovarian OSC may offer the potential for women experiencing ovarian failure as a result of menopause or POF to be treated for their infertility beyond the only current option of IVF using a donor egg. This has provided an underpinning of attempts to initiate ovarian rejuvenation in clinical settings, including investigating the utility of PRP in four pilot studies of different reproductive pathologies: POI, poor ovarian responders (POR), perimenopause and menopause ( Sfakianoudis  et al. , 2020b ).\n\nThe blood platelet is a tiny, anucleate cell responsible for the initiation of formation of a thrombus ( Fig. 1 ). Platelets are formed from a fragment of megakaryocyte membrane that is pre-packaged with a myriad of molecules and complexes necessary for its primary function, which is to sense signs of trauma within the vasculature and aggregate together to stem the loss of blood. One of the primary steps in thrombus formation is platelet activation, which is driven by ‘outside-in’ signalling, initiated through a vast repertoire of G-protein coupled receptors, integrins and glycoprotein channels on the surface of the platelet ( Li  et al. , 2010 ). The activation of platelets can occur through numerous mechanisms by a seemingly endless number of agonists, including but not limited to, thrombin, collagen, adenosine diphosphate (ADP), thromboxanes, serotonin, oxidised LDL and extracellular divalent cations ( Lopez-Vilchez  et al. , 2009 ;  Li  et al. , 2010 ;  Wraith  et al. , 2013 ;  Shen  et al. , 2017 ).\nGranule release in activated platelets.  Platelets express numerous glycoprotein, integrin and G- protein-coupled receptors that bind to a myriad of soluble and matrix proteins and molecules, resulting in tightly orchestrated intracellular signalling. This intracellular signalling significantly increases cytoplasmic calcium levels and causes drastic changes in the platelet cytoskeleton, resulting in ashape change in the platelet to an ‘echinocytic’ formation. During this process, granular storage compartments migrate inwards to the centre of the platelet and fuse with the plasma membrane and release their contents into the extracellular milleiu. PAR1/4, protease-activated receptors 1/4; GPVI, glycoprotein VI; TXA2, thromboxane A2; TP, thromboxane protstanoid receptor; 5-HT, 5-hydroxytryptomine; P2Y, purinergic receptor 2Y; vWF, von Willebrand Factor; IL-8, interleukin-8; CCL5, chemokine ligand 5; SDF-1a, stromal cell-derived factor 1 alpha; FGF, fibroblast growth factor; EGF, endothelial growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; TGFb1, transforming growth factor beta 1.\nA core platelet response to activation is the release of the contents of intracytoplasmic granules. Platelets contain two main granule stores, the alpha and dense granules, both of which replete with factors critical for an effective platelet response to vascular damage ( Fig. 1 ). Where alpha and dense granules are lacking, the conditions grey platelet syndrome and delta storage pool deficiency can arise. Both of these conditions are associated with an increased bleeding tendency ( Bolton-Maggs  et al. , 2006 ). It is also of note that more recently, platelet secretory behaviour has been shown to extend beyond the realm of granular stores and also involves activation-dependent synthesis and release of cytokines and other bioactive molecules ( Heijnen and van der Sluijs, 2015 ). It is, therefore, clear that the contents of platelet intracytoplasmic granules and  de   novo  synthesis of agents are essential for the haemostatic response, and the descriptions on the functions of platelet releasate have historically focussed on its role in haemostasis ( Rendu and Brohard-Bohn, 2001 ). However, the catalogue of bioactive proteins and molecules released by activated platelets can have multiple physiological effects which include increased angiogenesis, cell proliferation, cell differentiation and regulation or attenuation of apoptosis ( Bir  et al. , 2011 ;  Au  et al. , 2014 ;  Golebiewska and Poole, 2015 ). The therapeutic role of the platelet releasate in driving tissue regeneration is of growing interest throughout modern medicine.\nPRP is a term used to describe a fraction of the blood after processing. It is typically isolated from autologous whole blood retrieved by phlebotomy into a citrate-based anticoagulant. This is then subjected to differential centrifugation, resulting in the removal of red blood and immune cells, leaving behind a high concentration of platelets within plasma. Commercial sources of PRP are available, which can provide a predetermined concentration of platelets. However, in many cases, PRP is derived ‘in-house’, produced according to many subtle protocol variations. It is not uncommon for resulting PRP to retain varying concentrations of RBCs and WBCs; such contamination and absence of standardisation may result in conflicting findings regarding the effects of PRP in different applications.\nIn recent years, there has been significant interest in exploiting PRP in regenerative medicine. Particular attention has been paid to musculoskeletal ( Scully  et al. , 2019 ,  2020 ), oral-maxillofacial ( Xu  et al. , 2020 ) and osteoarthritis ( Evans  et al. , 2020 ) applications to name but a few. For a more comprehensive account, the reader is referred to a review ( Scully  et al. , 2018 ).\n\nOver the past decade, there have been a growing number of studies that have reported that injection of PRP directly into the ovary can increase folliculogenesis and egg harvest. One of the earliest studies reporting this approach was from  Callejo  et al.  (2013) , who implanted cryopreserved ovarian tissue within the peritoneum. PRP was used as a pro-angiogenic and proliferative agent, and the approach supported a successful live birth. The proangiogenic effect of PRP was further highlighted in a study by  Bakacak  et al.  (2015) , who used a rat model of ovarian ischaemia induced by torsion. In that study, PRP treatment in all conditions significantly increased peritoneal vascular endothelial growth factor (VEGF) and provided protection from ROS-induced oxidative damage during reperfusion.\nMore recently, direct injection of PRP into ovaries has been reported. In 2016, a short communication at the ESHRE Annual Meeting indicated that infusion of PRP into the ovary of perimenopausal women led to resumption of menstrual cycles ( Pantos  et al. , 2016 ). The study included only eight women but was the first reported use of PRP for rejuvenation of the perimenopausal ovary. Since then, there have been several limited investigations into the utility of PRP injection into the ovaries of perimenopausal women which are summarised in  Table I .  Sills  et al.  (2018)  reported that for healthy women with a history of infertility, ovarian PRP infusion produced several MII oocytes for cryopreservation, with one individual proceeding to successful embryo transfer at time of publication. Other studies have reported similar cases; commonly ovarian PRP therapy has caused AMH to increase and FSH levels to fall in previous non-responders, leading to folliculogenesis, significant levels of oocyte retrieval, and in a handful of cases, spontaneous pregnancy ( Sfakianoudis  et al. , 2018 ;  Farimani  et al. , 2019 ;  Pantos  et al. , 2019 ;  Hsu  et al. , 2020 ).\nSummary of reports on the effect of PRP infusion in ovarian rejuvenation .\nContinued .\nVCD administration successfully reduced the presence of morphologically normal follicles to none and increased the atretic follicle count, also mildly increasing FSH levels although not significantly\nPRP intraovarian injection reduced follicular atresia in POI-induced rat ovaries and saw an increase in litter counts, as well as higher expression of  ANGPT2  and  KDR  when compared to the other groups. After PRP intervention, FSH levels declined, although not statistically significant, with the greatest decline observed in the higher platelet concentration of PRP\nIn the only preclinical study on the effect of PRP injection into human ovaries,  Hosseini  et al.  (2017)  obtained healthy donated ovaries from deceased donors. PRP injection led to an increase in follicle size and their viability at 10 days compared to treatment with foetal calf serum (FCS) alone. Surprisingly, a combination of FCS and PRP did induce follicular growth, which is an interesting observation worthy of further investigation.\nWhile these case studies appear encouraging, it is important to reflect on the experimental designs. A common feature of the first studies of the effect of PRP infusion is the absence of a sham injection group. It is conceivable that the mechanical stretching and/or mild injury to the ovary resulting from the procedure is sufficient to elicit an inflammatory response leading to temporary resumption of ovarian function. For example, laparoscopic ovarian ‘drilling’ is a therapeutic option for the treatment of clomiphene-resistant PCOS ( Lebbi  et al. , 2015 ) and, thus, a comparable ovarian needle stick injury may be a causative factor in the success of PRP therapy. Importantly, the recent study of  Ahmadian et al. (2020)  used a sham injection group, which showed no morphologically normal follicles, and the same result was observed in the ‘no injection’ group. This demonstrated that injection with saline is not sufficient to reverse the effects of premature ovarian insufficiency in this animal model, nor can it elicit a comparable response to the two groups with different concentrations of PRP, which show reduced follicular atresia and increased follicular quality. It is vital that future studies control for this component of the intervention.\nAn important study was published by  Melo et al. (2020)  who reported findings from a non-randomised interventional study involving 83 subfertile women, 46 of whom opted for several infusions of 200 µl of autologous PRP into each ovary, and 37 who opted for no treatment. These two arms were further subdivided into groups who opted for IVF, and those who continued with unassisted conception. Overall, significantly higher antral follicle counts were observed in women who received PRP infusion compared to those women who received no treatment. In addition, embryo quality was scored higher from those obtained through PRP therapy, although there was no difference in the fertilisation rate of oocytes from either group. The authors concluded that ovarian injection of PRP did lead to increased egg yield in subfertile women and prompted changes within the oocyte which may lead to increased ‘quality’ of subsequent embryos. In both the IVF and spontaneous conception groups, those receiving PRP therapy developed 13 clinical pregnancies, compared to 2 in the control group although there were insufficient data on live births to draw any definitive conclusions. Although these data are encouraging, the absence of randomisation may have led to a socioeconomic selection bias, since PRP intervention was adopted only by couples able to pay for the treatment. Examples such as this illustrate the necessity that case studies are scrutinised in detail. Ideally, a properly controlled randomised clinical trial will be necessary to confirm the efficacy of ovarian PRP therapy.\n\nGiven the complexity of platelet signalling and activation, the precise details of how platelets initiate their full range of physiological effects remain unclear. However, it is well established that platelets release a range of cytokines in response to activation ( Roh  et al. , 2016 ). Cytokine signalling is increasingly being shown to be involved in the interrelationship among the oocyte, granulosa and thecal cells, with dysfunction in this ecosystem resulting in deficiencies in follicle maturation, ovulation and luteinisation ( Orisaka  et al. , 2006 ;  Field  et al. , 2014 ). A number of the cytokines that regulate follicle development are released by platelets through secretion of their alpha and dense granule contents during platelet activation ( Table II ). Therefore, a working hypothesis is that PRP may provide a readily accessible, individualised, cost-effective blend of proangiogenic, proliferative and proinflammatory factors which may stimulate de-novo oogenesis and/or follicle maturation.\nFactors released by platelets with known effects in the ovary .\nHussein  et al.  (2005)\nKalén  et al.  (2008)\nDemiray  et al.  (2017)\nLedee  et al.  (2008)\nMachlus  et al.  (2016)\nBen-Ezra  et al.  (1990)\nReizel  et al.  (2010)\nArici (1996)\nHuang  et al.  (2017)\nHart  et al.  (1990)\nNilsson  et al.  (2006)\nValeri  et al.  (2006)\nPinkas  et al.  (2008)\nPascuali  et al.  (2015)\nYeh  et al.  (2016)\nDeuel  et al.  (1981)\nPervushina  et al.  (2004)\nHuang  et al.  (2016)\nGeng  et al.  (1997)\nMerten and Thiagarajan (2000)\nKryczek  et al.  (2005)\nHolt  et al.  (2006)\nMassberg  et al.  (2006)\nNishigaki  et al.  (2011)\nAmireault and Dubé (2005)\nBrenner  et al.  (2007)\nHenriksen  et al.  (2012)\nCloutier  et al.  (2018)\nAssoian  et al.  (1983)\nDragovic  et al.  (2007)\nMeyer  et al.  (2012)\nJaffe  et al.  (1982)\nDisdier  et al.  (1989)\nKõks  et al.  (2009)\nBender  et al.  (2019)\nRival  et al.  (2019)\nZaslavsky  et al.  (2010)\nShweiki  et al.  (1993)\nWynendaele  et al.  (1999)\nDuncan  et al.  (2008)\nItaliano  et al.  (2008)\nRadomski  et al.  (2002)\nBu  et al.  (2006)\nRaidem  et al.  (2003)\nLee  et al.  (2008)\nPeralta  et al.  (2013)\nNilsson  et al.  (2001)\nPintucci  et al.  (2002)\nBen-Haroush  et al.  (2005)\nOno  et al.  (2013)\nCheng  et al.  (2015)\nUrtz  et al.  (2015)\nPors  et al.  (2020)\nBMP, bone morphogenic protein; CCL5/RANTES, chemokine (C-C motif) ligand 5; EGF, endothelial growth factor; IL-8, interleukin-8; PDGF, platelet-derived growth factor; PF4, platelet factor 4; CXCL4, chemokine (C-X-C motif) ligand 4; SDF-1a, stromal-cell derived factor 1 alpha; CXCL12, chemokine (C-X-C motif) ligand 12; TGF-b1, transforming growth factor beta 1; VEGF, vascular endothelial growth factor; TIMP-4, tissue inhibitor of matrix metalloprotease; TSP-1, thrombospondin-1; GM-CSF, granulocyte-monocyte colony stimulating factor; FGF, fibroblast growth factor; S1P, sphingosine-1-phosphate;  CCN2 , connective tissue growth factor.\nOne possible explanation of the observed effects of PRP on the ovary might be that it acts in a proangiogenic manner ( Kakudo  et al. , 2014 ) via action of platelet-released cytokines ( Table II ), including, for example VEGF. Primordial follicles typically rely on stromal blood vessels, but become progressively encapsulated in a thecal capillary network during maturation, a process which is mirrored by increased VEGF expression that persists through to corpus luteum formation ( Gordon  et al. , 1996 ;  Barboni  et al. , 2000 ;  Danforth  et al. , 2003 ;  Pauli  et al. , 2005 ). Heterozygous knockdown of the hypoxia-response element within the VEGFA promoter or VEGFR antagonism in mouse ovaries leads to vascular malformation, resulting in a poor ovarian response to stimulation ( Feng  et al. , 2017 ), indicative of a role for VEGF in follicle development and the overall importance of correctly regulated vascularisation in follicle development. Another major constituent of platelet releasate, platelet-derived growth factor (PDGF), has also been implicated in regulating vessel formation and maturity. This was demonstrated via intraovarian injection with an anti-PDGF antibody in rats by  Pascuali  et al.  (2015) , who consequently observed a reduction in follicle maturation paired with an increase in follicle atresia. This direct evidence for the importance of proangiogenic factors in follicle development supports the idea that PRP and/or platelet releasate can increase blood supply to the immature follicle pool and/or OSCs and encourage their maturation.\nAn additional potential explanation for the positive effects of PRP on the ovary is via sphingosine-1-phosphate (S1P) ( Ono  et al. , 2013 ;  Urtz  et al. , 2015 ). S1P has been isolated from follicular fluid at high nanomolar concentrations ( Von Otte  et al. , 2006 ) and there is evidence to suggest that it can promote follicle maturation, likely through increased expression of CCN2, a connective tissue growth factor shown to drive follicle maturation ( Cheng  et al. , 2015 ). Platelet alpha granules contain abundant stores of S1P, which is released upon activation and have been measured at over 300 nM per 1 × 10 7  platelets. If a linear relationship between S1P concentrations and platelet count exists, this would estimate that in studies that have infused activated PRP into the ovary, the amount of S1P delivered is approximately 9 µM, close to the range reported to be beneficial by  Cheng  et al.  (2015) . However, a recent study involving both murine and human follicles and human-to-murine xenotransplantation reported that although  CCN2  expression was elevated in response to supraphysiological S1P doses, there was no increase in the number of follicles. By contrast, ovaries receiving S1P treatment suffered a significant reduction in follicle number compared to control counterparts ( Pors  et al. , 2020 ). These findings again highlight the uncertainty of the effect of factors released by activated platelets on oocyte and follicle development and clinicians must be careful when considering such approaches.\nAlthough it is theorised that PRP supports the development of follicles from OSCs, alternative explanations must be considered. In a study by  Hosseini  et al.  (2017) , PRP was found to improve the growth and viability  in vitro  of preantral follicles isolated from human ovaries post-mortem, supporting the notion that PRP may aid ovarian rejuvenation through supporting development of existing primordial follicles. However, this application relies on the patient having a supply of oocyte-containing follicles, thus, rendering the approach unsuitable for women who have experienced ovarian exhaustion.  Panda  et al.  (2020)  expressed the need for better-controlled studies to confirm the conclusions drawn by  Cakiroglu  et al.  (2020) , which found that the number of remaining follicles within the ovaries of women with POI determines their response to PRP infusion, and that women without any antral follicles are unlikely to respond to PRP.\nThe prospective pilot study by  Sfakianoudis  et al.  (2020b ) determined that perimenopausal women and women deemed to be POR benefitted the most from the treatment, more so than POI and menopausal patients. In an article by  Sfakianoudis  et al.  (2020a ), they describe how novel techniques (such as PRP, ovarian stem cells transplant and ovarian tissue transplant) may effectively treat ovarian insufficiency by reactivating follicular growth through restoring the microenvironment of the ovary. Therefore, it should be acknowledged that PRP infusion may only be an appropriate treatment for select ovarian disorders.\n\nA primary consideration of the effect of PRP in any aspect of regenerative medicine is the ‘activation status’ of the platelet ( Fig. 1 ). As previously discussed, platelets have the capacity to respond to agonists and release a range of molecules, creating a ‘releasate’ ( Piersma  et al. , 2009 ;  Parsons  et al. , 2018 ). Indeed, PRP from resting platelets differs markedly to that containing activated platelets, and the mode of activation will influence the composition of the releasate. Despite this, there is considerable variation in the activation status of platelets used in studies of ovarian rejuvenation; some studies describe using calcium ( Sills  et al. , 2018 ;  Hsu  et al. , 2020 ;  Melo et al., 2020 ) or thrombin ( Hosseini  et al. , 2017 ), while others inject quiescent platelets or simply do not state their activation status ( Callejo  et al. , 2013 ;  Farimani  et al. , 2019 ;  Pantos  et al. , 2019 ). The importance of reporting the activation status and the methods therein, paired with the use of appropriate controls, is critical, given reported effects of thrombin or calcium alone in the regulation of ovarian function. For example, thrombin has been shown to regulate progesterone synthesis in the preovulatory ovary homogenates, with multiple cell types within the ovary readily expressing PAR1 and PAR4 receptors ( Cheng  et al. , 2012 ) through which thrombin elicits biological function directly. In addition, there is good evidence of an interaction between calcium signalling and ovarian steroidogenesis (reviewed in  Kouba  et al. , 2019 ).\nTo date, it appears that efforts to investigate the role of platelet activation in the context of ovarian rejuvenation remain limited. For example, platelets possess CD40 and αIIbβ 3  on their surface in a resting state ( Inwald  et al. , 2003 ;  Li  et al. , 2010 ). Thus, it is conceivable that these adhesive receptors and ligands are sufficient to elicit folliculogenesis or to recruit immune cells to the ovary without the need for platelets within the PRP to have become activated prior to injection. By contrast, activation and subsequent degranulation may be the critical function required for PRP to elicit an effect and quiescent PRP may become activated through exposure to platelet-activating matrices within the ovarian stroma. Differentiating the effects of stimulated versus unstimulated PRP should be a focus of future investigations and may help isolate the most effective agents that cause the reported regenerative effect in the ovary, paving the way for more defined interventions.\nThe contents of platelet granules may not all be beneficial for re-establishing female fertility among all disease settings. As a theoretical example, thrombospondin-1 has been implicated in follicle development ( Kõks  et al. , 2010 ;  Bender  et al. , 2019 ), yet it inhibits the proangiogenic action of VEGF ( Greenaway  et al. , 2007 ) which may be undesirable where perfusion of the ovaries is limited. In addition, increased intraovarian VEGF and blood flow is thought to play a role in the pathogenesis of PCOS ( Chan  et al. , 2003 ;  Carmina  et al. , 2005 ;  Peitsidis and Agrawal, 2010 ). Conversely,  Anvari  et al.  (2019)  recently reported that PRP therapy partially re-established hormonal balance in a rat model of PCOS. Here, PRP treatment increased the expression of oestrogen receptors α and β and of superoxide dismutase and glutathione peroxidase in ovarian homogenates. PRP-treated ovaries had significantly more pre-antral and antral follicles up to 30 days after treatment, suggesting that PRP may be a viable option for driving folliculogenesis in females with PCOS. In addition, platelets also release significant quantities of IL-15 when activated ( de Miguel-Gómez et al., 2020 ). Increased IL-15 concentrations in follicular fluid have been negatively correlated with pregnancy outcomes via IVF, indicating that this cytokine may be detrimental to follicle maturation ( Spanou  et al. , 2018 ). Interestingly, it is highly expressed in immature follicles, and falls during their maturation, which raises the potential importance of IL-15 in the activation of germline stem cells, as IL-15 is a potent regulator of other stem cell types ( Huntington  et al. , 2009 ;  Gómez-Nicola  et al. , 2011 ). This interplay and opposing effects of PRP constituents in different contexts serve to illustrate the importance of detailed studies of the mechanisms of how PRP might act on the ovary, and much additional work is required before any conclusions can safely be drawn.\n\nEven though there are other biological derivatives, such as human umbilical cord plasma, which may provide additional benefits in the sphere of ART ( de Miguel-Gómez et al., 2020 ), the appeal of PRP lies in its balance among therapeutic effect, cost effectiveness, ease of isolation and autologous nature. However, with the increased interest of ovarian PRP injection inconclusive efficacy and lack of understanding of mechanism of action, fundamental research into the effect of this therapy on the cellular level is required. Dysregulation of early processes in oocyte maturation and subsequent embryo development can lead to drastic changes in the growing foetus, possibly leading to increased risk of disease in early years and onwards. Indeed, the long-term safety of new treatments in ART must be robustly assessed ( Harper  et al. , 2012 ), especially given the context that there is still concern that ART itself may increase the risk of birth defects ( Luke  et al. , 2020 ). Perhaps most of most relevance in the context of PRP, the precise physiological causes of premature ovarian failure or primary ovarian insufficiency outside the natural ageing process remain poorly understood, and further work to understand the role of putative OSCs is required.\nWith ovarian PRP therapy in its infancy, understandably, there is poor standardisation among research groups and clinics, however, this must soon be addressed to form a consensus as to the efficacy of this treatment. To assist this, we would propose that authors carrying out research in this area commit to reporting of key basic information regarding PRP. At a minimum, we suggest that such studies should include platelet count, activation status, activation agent (if any), platelet function testing, origin of PRP, volume infused, anticoagulant used, clinical account of menstrual status based on AMH level, and a detailed reporting of the participant’s fertility history.\nAdditionally, while there are encouraging data supporting the notion that PRP treatment might have some future use in the ART setting, it is paramount that we undertake robust and detailed basic studies to understand mechanism of action and to try to identify unintended outcomes, before moving into whole animal studies. These precursors would be an important bedrock on which to carry out well-designed clinical studies, allowing us to investigate this new technology with all rigour currently available. There are, at the time of writing, 13 registered clinical trials investigating the effect of PRP on ovarian rejuvenation either recruiting or underway. Strikingly, few of these trials describe the inclusion of appropriate PRP controls. It is only from well-controlled trials, built on detailed mechanistic understanding that we can clarify the platelet-mediated effects of PRP therapy in ovarian rejuvenation and folliculogenesis.\n\nNo new data were generated or analysed in support of this research.","source_license":"CC-BY-4.0","license_restricted":false}