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Medicina
Medicina (Kaunas) 2021;57:xxxxx www.mdpi.com/journal/medicina
Special Communication
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Appraisal of experimental menopause and infertility treatments:
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Intraovarian autologous platelet-rich plasma vs. condensed
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platelet-derived cytokines
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E. Scott Sills 1,2 *, Samuel H. Wood2,3
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1Regenerative Biology Group, FertiGen / CAG; San Clemente, California 92673 USA
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2Department of Obstetrics & Gynecology, Palomar Medical Center; Escondido, California 92029 USA
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3Gen 5 Fertility Center; San Diego, California 92121 USA
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*Plasma Research Section, FertiGen/CAG, P.O. Box 73910; San Clemente, California 92673 USA
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email: ess@prp.md Tel: +1 949-899-5686
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Abstract: The first published description of intraovarian platelet-rich plasma (PRP) appeared in mid-2016, when
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a new experimental technique was successfully used in adult human ovaries to correct the reduced fertility po-
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tential accompanying advanced maternal age. Considering the potential therapeutic scope of intraovarian acti-
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vated PRP and/or condensed platelet cytokines would likely cover both menopause treatment and infertility, the
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mainstream response has ranged from skeptical disbelief to welcome astonishment. Indeed, reports of restored
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menses in menopause (as an alternative to conventional hormone replacement therapy) and healthy term live-
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births for infertility patients (either with IVF or as unassisted conceptions) after intraovarian PRP injection con-
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tinue to draw notice. Yet any proper criticism of ovarian PRP applications will be difficult to rebut given the
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heterogenous patient screening, varied sample preparations, wide differences in platelet incubation and activa-
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tion protocols, surgical/anesthesia techniques, and delivery methods. Notwithstanding these features, no adverse
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events have been reported thus far and ovarian PRP appears well tolerated by patients. Here, early research guid-
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ing the transition of ‘ovarian rejuvenation’ from experimental to clinical is outlined. Likely mechanisms are pre-
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sented to explain results observed in both veterinary and human ovarian PRP research. Current and future chal-
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lenges for intraovarian cytokine treatment are also discussed.
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Keywords: platelets; cytokines; angiogenesis; embryo; menopause; fertility
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1. Introduction
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Platelet-rich plasma (PRP) represents a physiologic signaling aggregate comprising
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hundreds of platelet derived cytokines obtained from blood samples, collected by stand-
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ard venipuncture [1]. As a refinement of conventional PRP, growth factors of platelet
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origin may be further processed to enrich this releasate after activation [2]. Interest in PRP
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applications has grown over the past 15 years, and since 2016 it has attracted particular
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attention in experimental reproductive biology. Understandably, the claim to ‘rewind the
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biological clock’ [3] has been cautiously received. It is not known how many IVF clinics
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now offer ‘ovarian rejuvenation’ although it is a safe assumption the number was zero
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prior to 2016. In contrast, considerable experience with autologous PRP (fresh and frozen)
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has already been reported in cardiothoracic surgery [4], scalp hair regrowth [5], derma-
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tology [6], oral surgery [7], sports medicine [8] and other clinical fields [9]. While the ab-
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sence of randomized placebo-controlled clinical trials regarding intraovarian PRP must
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be acknowledged, this deficiency did not block IVF from entering mainstream fertility
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practice with no RCT support [10].
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Rejuvenation arrived on the gynecology stage with its (non-pharmacologic) promise
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to improve ovarian function [11]. Autologous PRP has also been used occasionally as an
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intrauterine lavage, aiming to boost endometrial receptivity and enhance embryo implan-
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tation [12]. In the follicular recruitment IVF space, intraovarian PRP joins a crowded cast
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of untested interventions such as human growth hormone, aspirin, heparin, DHEA,
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Citation: Sills & Wood
Medicina (Kaunas) 2021;57:xx.
https://doi.org/10.3390/xxx
Academic Editor: xxx
Received: xxx
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Medicina (Kaunas) 2021;57: FOR PEER REVIEW 2 of 9
antioxidants, and screening hysteroscopy [13]. But what is the basis of the proposed PRP
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pathway to ovarian rejuvenation?
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2. Therapeutic Rationale
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Platelets contain multiple granules which, upon activation, deliver numerous cargo
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proteins including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),
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vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming
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growth factor-beta 1 (TGF-β1), insulin-like growth factor (IGF), connecting tissue growth
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factor (CTGF), hepatocyte growth factor (HGF), and others [9,11]. The roster of releasate
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contents seems ever growing; these moieties orchestrate cellular growth and directs repair
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following tissue injury. For older or impaired ovarian tissue, small series and case report
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data suggest this signaling milieu can contribute to improved stromal perfusion, enable
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an enlarged follicle pool, recruit latent oocytes, and produce healthy term livebirths
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[14,15].
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While intraovarian PRP is usually regarded as a precursor to IVF, the ‘reset’ estab-
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lished after platelet cytokine treatment may confer benefits even if not followed by such
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complex treatments [15-17]. This could occur due to temporary resolution of tissue dys-
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function associated with hypoperfusion characteristic of the senescent ovary [11].
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3. Ovarian PRP: Veterinary and human research
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What might PRP accomplish in the setting of impaired or even obliterated reserve as
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with menopause? This question was explored in an animal model where intraperitoneal
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4-vinylcyclohexene dioxide administration was used as a gonadotoxin for total ovarian
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collapse. Next, rat intraovarian PRP injections were followed by documentation of cellular
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changes as well as expression of angiogenic-related transcripts ANGPT2 and KDR via
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real-time qPCR. While histopathological review confirmed an ovarian insufficiency (POI)
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state after initial conditions, injection of PRP substantially reduced the extent of follicular
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atresia and inflammatory response [18]. An uptick was also measured in ANGPT2 and
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KDR transcript expression in POI rats secondary to enhanced inflammation but reduced
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after PRP administration vs. controls. Perhaps most crucially, improvement in litter
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counts was documented among animals receiving PRP compared to the non-treated POI
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group [18]. This study also found intraovarian PRP also protected morphologically nor-
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mal follicles from degeneration. This parallels earlier observations among rats with exper-
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imentally induced PCOS, where improved ovarian antioxidant potential and enhanced
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follicular development after PRP mitigated deleterious oocyte effects in PCOS [19].
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Research from Milan [20] described bovine response to administration of autologous
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intraovarian PRP before programmed superovulation. A significant improvement was
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noted in mean follicle number between control vs. PRP injected ovaries, and significantly
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more high-grade blastocysts were generated following PRP use [20]. Additional animal
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experiments have found a beneficial (rescue) effect of PRP on ovarian function in female
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rats with ovarian damage induced by cyclophosphamide, concluding this approach could
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lead to improved primordial, primary, secondary, and antral follicle counts [21].
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Medicina (Kaunas) 2021;57: FOR PEER REVIEW 3 of 9
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Figure 1. At least two methods of PRP sample preparation are currently in use, including conven-
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tional activation (A) and condensed cytokines isolated after in vitro platelet (PLT) incubation/pro-
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cessing (B). Note that depleted platelets (DEP) are removed (in B) following concentration of platelet
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releasate. Although bioactivity for both is a function of multiple signaling moieties, the concentra-
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tion of such growth factors should be markedly increased along path B. Relevant platelet-derived
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cytokines include Vascular endothelial growth factor (VEGF), a signal protein promoting angiogen-
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esis; Ligand of CD40 (CD-40L), an inflammatory signal for endothelium, platelets, and leukocytes;
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Interleukin-1β (IL-1β), an inflammatory marker involved in cell growth, differentiation; Interleukin-
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8 (IL-8) which initiates angiogenesis, perfusion, and movement to injury/infection sites; PLT derived
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growth factor (PDGF), essential for vascular development, proliferation of fibroblasts, osteoblasts,
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tenocytes, vascular SMCs and mesenchymal stem cells; and PLT factor 4 (PF4), central in organizing
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platelet aggregation.
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4. Patient & protocol differences
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Both platelet concentration and derivative cytokine releasate show considerable in-
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dividual variation [22], and this aspect of PRP treatment is important to review with po-
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tential patients before ovarian rejuvenation is attempted. At least two techniques exist to
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process PRP (see Figure 1), although ‘best practice guidelines’ are not yet available to help
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screen patients or suggest a specific therapy. While a minimum platelet level necessary to
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elicit a regenerative response probably depends on the intended target tissue, animal re-
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search [23] found a threshold approaching 1M platelets/mL (i.e., a 3-8 fold enrichment
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over baseline) as sufficient for bone healing. Of note, these experts did acknowledge a
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‘more is not always better’ paradigm [23] to highlight the need for additional research.
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Prospective intraovarian PRP human data (n=182) identified significant differences
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in baseline platelet count among responders vs. non-responders [24], validating a relation
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between platelet count and subsequent ovarian reserve post-treatment. Even for women
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with platelet levels on the lower margin of normal, it might be possible to collect two
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samples within a few hours to pool autologous blood for aggregate processing and same
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day (fresh) injection. By facilitating ovarian PRP treatment with an augmented platelet
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concentration, this may overcome marginally low platelet counts which would otherwise
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be disqualifying. For cases with absolute thrombocytopenia (PLT <100K), formal hema-
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tology consult is appropriate as these patients would be high risk for IVF and pregnancy.
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The impact of handling technique on platelet lysate has also been studied [25], find-
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ing method of sample preparation can significantly influence the growth factor profile.
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Specifically, platelet-derived cytokine levels were markedly increased in non-calcium
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Medicina (Kaunas) 2021;57: FOR PEER REVIEW 4 of 9
activated PRP with a freeze-thaw-freeze incubation; a major disadvantage was also re-
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ported if room temperature incubation was used [25].
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Lower PRP concentrations might still block nasoseptal cells from losing their chon-
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drogenic potential due to in vitro expansion, thereby promoting recommitment [26]. Re-
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duced concentration of platelet releasate was able to augment mesenchymal stromal cells
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as noted by upregulated gene expression, sulfated glycosaminoglycan production, and
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compressive modulus after in vitro culture. Some markers of regenerative action were
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again impaired at higher concentration of platelet releasate (10%), emphasizing the need
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to define an optimal sample preparation method [26].
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Platelet-derived cytokines have been quantified following activation either with cal-
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cium alone or calcium/thrombin [27]. High concentrations of platelet-derived growth fac-
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tor, endothelial growth factor, and transforming growth factor (TGF) were secreted with
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interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-
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α. Unsurprisingly, no cytokines were secreted without platelet activation. TGF-β3 and
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IFNγ were absent in all studied fractions. Clots obtained after platelet coagulation re-
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tained a high cytokine concentration, including platelet-derived growth factor and TGF
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[27].
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Research from colleagues in Ukraine reported on 38 women with low ovarian reserve
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and at least two failed IVF cycles (age 31-45 yrs) who underwent ovarian PRP [28]. In their
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experience, route of PRP administration was either via laparoscopy or transvaginal ultra-
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sound and patients were monitored over one year. Significant improvement in ovarian
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function was noted after treatment, including 10 pregnancies and delivery of six healthy
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babies [28]. The largest single-center experience with intraovarian PRP probably remains
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at Genesis Athens (Greece) [1] where this team actively publishes results on specific pa-
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tient groups. For example, among menopausal women receiving ovarian PRP, 24 of 30
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attained restored menstruation and improved hormonal levels/ovarian antral follicle
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count – sometimes as soon as one month after injection [1]. This finding extends results
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from an earlier questionnaire study (n=80) on quality of life/non-reproductive outcomes
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where ovarian PRP was administered as an alternative to standard HRT [29]. Due to loss
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of follow-up after ‘menopause reversal’, no longitudinal data were available on this group
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to determine duration of treatment effects. Also in 2019, ovarian PRP data were published
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from a prospective matched cohort study where selected reproductive outcomes were
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tracked in 20 IVF patients receiving this treatment vs. 20 control IVF patients without
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ovarian PRP. In this pilot trial, a trend towards improved embryo implantation and live-
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birth rate was measured among IVF patients who received ovarian PRP [30]. One tech-
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nique (Segova) mentioned by experts in Serbia reports on a PRP processing method using
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‘special systems and machines’ to increase growth factor levels up to 18 times the initial
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concentration [31].
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5. Considerations & contraindications
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Before enrolling patients for ovarian PRP or intraovarian injection of condensed
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plasma cytokines, the same baseline considerations for IVF or HRT should apply. Ovarian
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PRP patient entry criteria followed during the NIH Clinical Trial [NCT03178695] included
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patients with at least one ovary, infertility of >1yr duration, at least one prior failed (or
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canceled) IVF cycle, or amenorrhea for at least three months. However, patients who are
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otherwise healthy but have undetectable reserve (serum AMH <0.03ng/mL) and consid-
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ered so unsuited for fertility treatment that they were never allowed to try IVF with native
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oocytes elsewhere, should not be excluded [32]. At exam, it is important to confirm safe
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ovarian access via transvaginal ultrasound prior to ovarian PRP. For those where back-
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ground medical conditions are uncertain, medical clearance is required. Patients with on-
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going pregnancy, current or previous IgA deficiency, ovarian insufficiency secondary to
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sex chromosome etiology, prior major lower abdominal surgery resulting in pelvic adhe-
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sions, anticoagulant use for which plasma infusion is contraindicated, psychiatric disor-
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der precluding study participation (including active substance abuse or dependence),
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Medicina (Kaunas) 2021;57: FOR PEER REVIEW 5 of 9
obesity, current smokers, ongoing malignancy, or chronic pelvic pain should be excluded
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[24].
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During pre-treatment screening it is important to query aspirin/NSAID use, as agents
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in this class will attenuate platelet function. Specifically, irreversible inhibition of platelet
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COX-1 by aspirin suppresses precursors required for downstream cytokine signaling [33].
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Recent research has clarified the mechanisms involved in aspirin’s brake effect on platelet
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activation [34] and since platelet activation is essential for ovarian PRP to achieve any
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therapeutic gain, requiring a NSAID-free window of at least 10d before planned intraovar-
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ian injection seems reasonable. Likewise, for patients taking pentoxifylline, this medica-
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tion also merits caution in advance of ovarian PRP as it impairs transforming growth fac-
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tor-beta and platelet-derived growth factor production [35]. Pentoxifylline also can block
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platelet-associated cytokine release in some settings [36] and should be avoided to opti-
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mize overall platelet functional potential.
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Pre-intake considerations notwithstanding, a published opinion accurately identified
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weaknesses in ovarian PRP reports currently available [37]. Particularly noted were the
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paucity of delivery data after ovarian PRP, the heterogeneity of commercial systems used
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in plasma processing, and absence of pre- vs. post-PRP antral follicle count [37]. Regard-
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ing the first critique, the nascent phase of autologous plasma factor research explains why
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delivery data remain confined to small series and case reports. Highlighting the underde-
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veloped state of ovarian PRP is proper and the call for delivery rate information is a nec-
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essary message. This is a familiar deficiency, and warrants acceptance against a larger
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unresolved debate about how best to report ‘success’ for IVF in general [38,39]. The second
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point on differences in PRP sample handling, processing, and administration is also com-
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pelling, and represents a serious hurdle to be cleared if usable comparisons are to be de-
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livered. Few published PRP protocols use the exact same kit for specimen collection and
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processing, centrifugation ‘spin’ parameters, or which reagent is used for platelet activa-
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tion. Such differences can impact substrate platelet concentration, its cytokine profile, and
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efficiency of growth factor release. Normal temporal and biological factors can influence
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platelet availability make assessments across multiple PRP platforms difficult to compare
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[9,40]. This variation with ovarian PRP methods presents problems for meaningful cross-
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center comparisons, yet with description of sample preparation, surgical approach, and
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full reporting of findings it could actually accelerate better understanding of which PRP
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technique works best for patients. Concerning antral follicle count data to score response
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to intraovarian PRP, collecting this information would probably add little to ovarian reju-
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venation given its limited reproducibility and low measurement consistency secondary to
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operator and/or ultrasound equipment variation. Serum AMH, in contrast, is much easier
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to standardize and thus represents a preferred marker for follicular potential/ovarian
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function [41].
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Reliable measurement of selected constituent molecular signals derived from plate-
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lets as a function of activation reagent and in vitro incubation method can offer descriptive
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information for reproductive biology. Because multiple ways exist to perform ‘ovarian
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rejuvenation’, it will be useful to document differences in cytokine concentrations by tech-
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nique. Case report and small series reports, while interesting, are most beneficial for the
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possible rather than the probable. It has been acknowledged that many IVF techniques
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now accepted as routine clinical practice once were experimental with humble or obscure
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origins. As noted here, the safety and efficacy of such treatments should be supported by
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data ideally from multiple randomized clinical trials [42]. We agree with Kamath et al [13]
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as caution is appropriate where use of early, investigational techniques are proposed.
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6. Conclusions
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The central question of whether or not the adult human ovary retains the capacity to
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produce de novo oocytes remains open. In postnatal CNS tissues also once thought irre-
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placeable, a similar issue is found regarding functional recovery and cellular regeneration
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[43,44]. So under certain conditions, the key objective of cellular regeneration here like-
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wise may need reconsideration. Working with a murine eye model, upregulation of
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Medicina (Kaunas) 2021;57: FOR PEER REVIEW 6 of 9
specific genes has restored a ‘youthful’ DNA methylation pattern as well as axonal re-
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growth following tissue damage—an approach enabling vision to return after blindness
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injury in mice [45]. Relevant genes implicated in this transcriptome expression include
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Sox2, Oct4, and Klf4 [45]. Of note, human platelet lysate has been shown to promote
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mRNA expression of such ‘mitotic bookmarking factors’ [46]. Building on such studies,
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reproductive biology can gain much to determine if these (or other) signals are operant in
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postnatal ovarian function. Small series and case data now exist to describe reproductive
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outcomes after intraovarian PRP or platelet-derived cytokines. While experience with se-
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rum AMH (as an estimate of ovarian reserve) following autologous ovarian PRP requires
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multicenter validation, additional research to characterize specific PRP cytokine compo-
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nents will be even more useful.
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Observations discussed here from clinical work in ovarian rejuvenation favor a hy-
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pothesis for derivative neovascularity to modulate oocyte competence, by augmenting
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cellular oxygenation and/or reducing levels of intraovarian reactive oxygen species [11].
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Of note, subsequent experimental animal research found ovarian function and follicular
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development were indeed promoted after VEGF-mediated vascular remodeling [47].
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The need for rigorous RCT data on intraovarian insertion of platelet-derived cyto-
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kines before this innovation enters accepted IVF practice should be viewed as familiar
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terrain for reproductive medicine [10,48]. Declining ovarian reserve and ineffective fertil-
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ity responses have become more formidable with advanced maternal age and cannot be
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defeated by gonadotropins alone. Similarly, perimenopause marks a symptomatic decay
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in female sex hormone output which at present is usually managed by exogenous hor-
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mone replacement therapy [49,50]. For both patient populations, the prospect of effective
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‘ovarian rejuvenation’ would hold considerable appeal. Could autologous platelet cyto-
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kines help meet this need? While a postnatal folliculogenesis model might explain im-
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proved ovarian function among older women, important challenges remain [51,52]. A
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population of adult ovarian germline stem cells might be latent—but available—for dif-
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ferentiation, the exact process through which cytokines induce such development requires
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additional study. What has been suggested from early reports on platelet cytokines sug-
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gests these moieties can initiate morphogenetic processes normally seen during evolution-
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ary development [53]. Of note, recent observations in two species of sacoglossan snail
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(gastropoda) have demonstrated extreme regeneration wherein a severed head was able
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to regrow an entire new body within approximately 20 days [54]. Unless a parallel process
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can be discovered to achieve limited postnatal replenishment of the human oocyte pool,
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reliance on IVF with donor oocytes will continue. Meanwhile, characterization of the ac-
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tivated PRP substrate, its derivative growth factor profile, receptor targets, optimal sam-
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ple delivery, and ideally RCT data to support this treatment are still needed.
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Authors’ Contributions
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Both authors contributed equally to the work and approved the final version of the
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manuscript.
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Acknowledgment
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Ann-Marie C. Sills (Fundación Santiago Apóstol, Villanueva de la Cañada; Madrid SPAIN)
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is thanked for editorial assistance with this manuscript.
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Declaration of Conflicting Interests
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The authors have been awarded a provisional U.S. Patent for process and treatment using
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autologous platelet-derived cytokines for ovarian therapy.
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Funding
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Medicina (Kaunas) 2021;57: FOR PEER REVIEW 7 of 9
This research received no specific grant from any funding agency in public, commercial,
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or not-for-profit sectors.
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References
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1. Sfakianoudis K, Simopoulou M, Grigoriadis S, Pantou A, Tsioulou P, Maziotis E et al. Reactivating ovarian function
282
through autologous platelet-rich plasma intraovarian infusion: Pilot data on premature ovarian insufficiency, perimeno-
283
pausal, menopausal, and poor responder women. J Clin Med 2020;9:1809. DOI: http://dx.doi.org/10.3390/jcm9061809
284
2. Sills ES, Rickers NS, Svid CS, Rickers JM, Wood SH. Normalized ploidy following 20 consecutive blastocysts with chromo-
285
somal error: Healthy 46,XY pregnancy with IVF after intraovarian injection of autologous enriched platelet-derived growth
286
factors. Int J Mol Cell Med 2019;8:84-90. DOI: http://dx.doi.org/10.22088/IJMCM.BUMS.8.1.84
287
3. Pantos K, Nitsos N, Kokkali G, Vaxevanoglou T, Markomichali C, Pantou A et al. Ovarian rejuvenation and folliculogenesis
288
reactivation in peri-menopausal women after autologous platelet rich plasma treatment [abstract]. ESHRE 32nd annual
289
meeting; 2016 Jul 3-6; Helsinki, Finland. Hum Reprod 2016;Suppl 1:i301. DOI: https://sa1s3.patientpop.com/as-
290
sets/docs/111052.pdf
291
4. Yao D, Feng G, Zhao F, Hao D. Effects of platelet-rich plasma on the healing of sternal wounds: A meta-analysis. Wound
292
Repair Regen 2020 Oct 31. DOI: http://dx.doi.org/10.1111/wrr.12874
293
5. Dervishi G, Liu H, Peternel S, Labeit A, Peinemann F. Autologous platelet-rich plasma therapy for pattern hair loss: A
294
systematic review. J Cosmet Dermatol 2020;19:827-35. DOI: http://dx.doi.org/10.1111/jocd.13113
295
6. Dunn A, Long T, Kleinfelder RE, Zarraga MB. The adjunct use of platelet-rich plasma in split-thickness skin grafts: A sys-
296
tematic review. Adv Skin Wound Care 2020 Dec 4. DOI: http://dx.doi.org/10.1097/01.ASW.0000722764
297
7. Sethi Ahuja U, Puri N, More CB, Gupta R, Gupta D. Comparative evaluation of effectiveness of autologous platelet rich
298
plasma and intralesional corticosteroids in the management of erosive oral Lichen planus- a clinical study. J Oral Biol Cra-
299
niofac Res 2020;10:714-8. DOI: http://dx.doi/org/10.1016/j.jobcr.2020.09.008
300
8. Everts P, Onishi K, Jayaram P, Lana JF, Mautner K. Platelet-rich plasma: New performance understandings and therapeutic
301
considerations in 2020. Int J Mol Sci 2020;21:7794. DOI: http://dx.doi.org/10.3390/ijms21207794
302
9. Gentile P, Garcovich S. Systematic review-The potential implications of different platelet-rich plasma (PRP) concentrations
303
in regenerative medicine for tissue repair. Int J Mol Sci 2020;21:5702. DOI: http://dx.doi.org/10.3390/ijms21165702
304
10. Evers JLHH. Do we need an RCT for everything? Hum Reprod 2017;32(3):483-484. DOI: http://dx.doi.org/10.1093/hum-
305
rep/dex003
306
11. Wood SH, Sills ES. Intraovarian vascular enhancement via stromal injection of platelet-derived growth factors: Exploring
307
subsequent oocyte chromosomal status and in vitro fertilization outcomes. Clin Exp Reprod Med 2020;47:94-100. DOI:
308
http://dx.doi.org/10.5653/cerm.2019.03405
309
12. Bos-Mikich A, Ferreira MO, de Oliveira R, Frantz N. Platelet-rich plasma or blood-derived products to improve endome-
310
trial receptivity? J Assist Reprod Genet 2019;36:613-20. DOI: http://dx.doi.org/10.1007/s10815-018-1386-z
311
13. Kamath MS, Mascarenhas M, Franik S, Liu E, Sunkara SK. Clinical adjuncts in in vitro fertilization: a growing list. Fertil
312
Steril 2019;112:978-86. DOI: http://dx.doi.org/10.1016/j.fertnstert.2019.09.019
313
14. Sfakianoudis K, Simopoulou M, Nitsos N, Rapani A, Pantou A, Vaxevanoglou T et al. A case series on platelet-rich plasma
314
revolutionary management of poor responder patients. Gynecol Obstet Invest 2019;84:99-106. DOI:
315
http://dx.doi.org/10.1159/000491697
316
15. Farimani M, Heshmati S, Poorolajal J, Bahmanzadeh M. A report on three live births in women with poor ovarian response
317
following intra-ovarian injection of platelet-rich plasma (PRP). Mol Biol Rep 2019;46:1611-6. DOI:
318
http://dx.doi.org/10.1007/s11033-019-04609-w
319
16. Sills ES, Rickers NS, Wood SH. Intraovarian insertion of autologous platelet growth factors as cell-free concentrate: Fertility
320
recovery and first unassisted conception with term delivery at age over 40. Int J Reprod Biomed 2020;18:1081-6. DOI:
321
http://dx.doi.org/10.18502/ijrm.v18i12.8030
322
17. Sfakianoudis K, Rapani A, Grigoriadis S, Retsina D, Maziotis E, Tsioulou P et al. Novel approaches in addressing ovarian
323
insufficiency in 2019: Are we there yet? Cell Transplant 2020;29:963689720926154. DOI:
324
http://dx.doi.org/10.1177/0963689720926154
325
18. Ahmadian S, Sheshpari S, Pazhang M, Bedate AM, Beheshti R, Abbasi MM et al. Intra-ovarian injection of platelet-rich
326
plasma into ovarian tissue promoted rejuvenation in the rat model of premature ovarian insufficiency and restored ovula-
327
tion rate via angiogenesis modulation. Reprod Biol Endocrinol 2020;18:78. DOI: http://dx.doi.org/10.1186/s12958-020-00638-4
328
19. Anvari SS, Dehgan GH, Razi M. Preliminary findings of platelet-rich plasma induced ameliorative effect on polycystic
329
ovarian syndrome. Cell J 2019;21:243-52. DOI: http://dx.doi.org/10.22074/cellj.2019.5952
330
20. Cremonesi F, Bonfanti S, Idda A, Anna LC. Improvement of embryo recovery in Holstein cows treated by intraovarian
331
platelet rich plasma before superovulation. Vet Sci 2020;7:16. DOI: http://dx.doi.org/10.3390/vetsci7010016
332
21. Ozcan P, Takmaz T, Tok OE, Islek S, Yigit EN, Ficicioglu C. The protective effect of platelet-rich plasma administrated on
333
ovarian function in female rats with Cy-induced ovarian damage. J Assist Reprod Genet 2020;37:865-73. DOI:
334
http://dx.doi.org/10.1007/s10815-020-01689-7
335
22. Sundman EA, Cole BJ, Fortier LA. Growth factor and catabolic cytokine concentrations are influenced by the cellular com-
336
position of platelet-rich plasma. Am J Sports Med 2011;39:2135–40. DOI: http://dx.doi.org/10.1177/0363546511417792
337
Medicina (Kaunas) 2021;57: FOR PEER REVIEW 8 of 9
23. Weibrich G, Hansen T, Kleis W, Buch R, Hitzler WE. Effect of platelet concentration in platelet-rich plasma on peri-implant
338
bone regeneration. Bone 2004;34:665–71. DOI: http://dx.doi.org/10.1016/j.bone.2003.12.010
339
24. Sills ES, Rickers NS, Petersen JL, Li X, Wood SH. Regenerative effect of intraovarian injection of activated autologous plate-
340
let-rich plasma: Serum anti-Mullerian hormone levels measured among poor-prognosis in vitro fertilization patients
341
[NCT03178695]. Int J Regenerative Med 2020;3:1-5. DOI: https://dx.doi.org/10.31487/j.RGM.2020.01.02
342
25. Steller D, Herbst N, Pries R, Juhl D, Hakim SG. Impact of incubation method on the release of growth factors in non-Ca2+-
343
activated PRP, Ca2+-activated PRP, PRF and A-PRF. J Craniomaxillofac Surg 2019;47:365-72. DOI:
344
http://dx.doi.org/10.1016/j.jcms.2018.10.017
345
26. do Amaral RJ, Matsiko A, Tomazette MR, Rocha WK, Cordeiro-Spinetti E, Levingstone TJ et al. Platelet-rich plasma re-
346
leasate differently stimulates cellular commitment toward the chondrogenic lineage according to concentration. J Tissue
347
Eng 2015;6:2041731415594127. DOI: http://dx.doi.org/10.1177/2041731415594127
348
27. Machado ES, Leite R, Dos Santos CC, Artuso GL, Gluszczak F, de Jesus LG et al. Turn down - turn up: a simple and low-
349
cost protocol for preparing platelet-rich plasma. Clinics (Sao Paulo) 2019;74:e1132. DOI: http://dx.doi.org/10.6061/clin-
350
ics/2019/e1132
351
28. Amable PR, Carias RB, Teixeira MV, da Cruz Pacheco I, Corrêa do Amaral RJ, Granjeiro JM et al. Platelet-rich plasma
352
preparation for regenerative medicine: optimization and quantification of cytokines and growth factors. Stem Cell Res Ther
353
2013;4:67. DOI: http://dx.doi.org/10.1186/scrt218
354
29. Petryk N, Petryk M. Ovarian rejuvenation through platelet-rich autologous plasma (PRP)-a chance to have a baby without
355
donor eggs, improving the life quality of women suffering from early menopause without synthetic hormonal treatment.
356
Reprod Sci 2020;27:1975-82. DOI: http://dx.doi.org/10.1007/s43032-020-00266-8
357
30. Sills ES, Li X, Rickers NS, Wood SH, Palermo GD. Metabolic and neurobehavioral response following intraovarian admin-
358
istration of autologous activated platelet rich plasma: First qualitative data. Neuroendocrinol Lett 2019;39:427-33. PMID:
359
30796792.
360
31. Stojkovska S, Dimitrov G, Stamenkovska N, Hadzi-Lega M, Petanovski Z. Livebirth rates in poor responders' group after
361
previous treatment with autologous platelet-rich plasma and low dose ovarian stimulation compared with poor responders
362
used only low dose ovarian stimulation before in vitro fertilization. Open Access Maced J Med Sci 2019;7:3184-8. DOI:
363
http://dx.doi.org/10.3889/oamjms.2019.825
364
32. Tinjic S, Abazovic D, Ljubic D, Vujovic S, Vojvodic D, Božanovic T et al. Ovarian rejuvenation. Donald Sch J Ultrasound
365
Obstet Gynecol 2019;13:64-8. DOI: http://dx.doi.org/10.5005/jp-journals-10009-1587
366
33. U.S. National Library of Medicine. Autologous platelet-rich plasma (PRP) infusions and biomarkers of ovarian rejuvenation
367
and ageing mitigation [NCT03178695]; March 7, 2017. https://clinicaltrials.gov/ct2/show/NCT03178695
368
34. Schrör K. Aspirin and platelets: the antiplatelet action of aspirin and its role in thrombosis treatment and prophylaxis.
369
Semin Thromb Hemost 1997;23:349-56. DOI: http://dx.doi.org/10.1055/s-2007-996108
370
35. Qi Z, Hu L, Zhang J, Yang W, Liu X, Jia D et al. PCSK9 enhances platelet activation, thrombosis, and myocardial infarct
371
expansion by binding to Platelet CD36. Circulation 2020 Sep 29. DOI: http://dx.doi.org/10.1161/CIRCULA-
372
TIONAHA.120.046290
373
36. Wu J, Zern MA. Hepatic stellate cells: a target for the treatment of liver fibrosis. J Gastroenterol 2000;35(9):665-72. DOI:
374
http://dx.doi.org/10.1007/s005350070045
375
37. Tong Z, Dai H, Chen B, Abdoh Z, Guzman J, Costabel U. Inhibition of cytokine release from alveolar macrophages in
376
pulmonary sarcoidosis by pentoxifylline: comparison with dexamethasone. Chest 2003;124:1526-32. DOI:
377
http://dx.doi.org/10.1378/chest.124.4.1526
378
38. Urman B, Boza A. Reply: Every established treatment had been experimental at the beginning. Hum Reprod 2020;35:1720-1.
379
DOI: http://dx.doi.org/10.1093/humrep/deaa122
380
39. Kushnir VA, Barad DH, Gleicher N. Defining assisted reproductive technology success. Fertil Steril 2013;100:e30. DOI:
381
http://dx.doi.org/10.1016/j.fertnstert.2013.08.036
382
40. Gunderson S, Jungheim ES, Kallen CB, Omurtag K. Public reporting of IVF outcomes influences medical decision-making
383
and physician training. Fertil Res Pract 2020;6:1. DOI: http://dx.doi.org/10.1186/s40738-020-00070-7
384
41. Dhurat R, Sukesh MS. Principles and methods of preparation of platelet-rich plasma: A review and author's perspective. J
385
Cutan Aesthet Surg 2014;7:189-97. DOI: http://dx.doi.org/10.4103/0974-2077.150734
386
42. Liu L, Zhou C. Anti-Mullerian hormone and antral follicle count differ in their ability to predict cumulative treatment
387
outcomes of the first complete ovarian stimulation cycle in patients from POSEIDON groups 3 and 4. J Obstet Gynaecol Res
388
2020;46:1801-8. DOI: http://dx.doi.org/10.1111/jog.14269
389
43. Goldberg JL, Klassen MP, Hua Y, Barres BA. Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion
390
cells. Science 2002;296:1860–1864. DOI: http://dx.doi.org/10.1126/science.1068428
391
44. Yun MH. Changes in regenerative capacity through lifespan. Int J Mol Sci 2015;16(10):25392-432. DOI:
392
http://dx.doi.org/10.3390/ijms161025392
393
45. Lu Y, Brommer B, Tian X, Krishnan A, Meer M, Wang C et al. Reprogramming to recover youthful epigenetic information
394
and restore vision. Nature 2020;588(7836):124-129. DOI: http://dx.doi.org/10.1038/s41586-020-2975-4
395
46. Laner-Plamberger S, Oeller M, Mrazek C, Hartl A, Sonderegger A, Rohde E et al. Upregulation of mitotic bookmarking
396
factors during enhanced proliferation of human stromal cells in human platelet lysate. J Transl Med 2019;17(1):432. DOI:
397
http://dx.doi.org/10.1186/s12967-019-02183-0
398
Medicina (Kaunas) 2021;57: FOR PEER REVIEW 9 of 9
47. Cho J, Kim TH, Seok J, Jun JH, Park H, Kweon M et al. Vascular remodeling by placenta-derived mesenchymal stem cells
399
restores ovarian function in ovariectomized rat model via the VEGF pathway. Lab Invest 2021;101:304-317. DOI:
400
http://dx.doi.org/10.1038/s41374-020-00513-1
401
48. Harper J, Jackson E, Sermon K, Aitken RJ, Harbottle S, Mocanu E et al. Adjuncts in the IVF laboratory: where is the evidence
402
for 'add-on' interventions? Hum Reprod 2017;32(3):485-491. DOI: http://dx.doi.org/10.1093/humrep/dex004
403
49. Urman B, Boza A, Balaban B. Platelet-rich plasma another add-on treatment getting out of hand? How can clinicians pre-
404
serve the best interest of their patients? Hum Reprod 2019;34:2099-103. DOI: http://dx.doi.org/10.1093/humrep/dez190
405
50. Sills ES. The scientific and cultural journey to ovarian rejuvenation: Background, barriers, and beyond the biological clock.
406
Medicines (Basel) 2021;8(6):29. DOI: http://dx.doi.org/10.3390/medicines8060029
407
51. Hanna CB, Hennebold JD. Ovarian germline stem cells: an unlimited source of oocytes? Fertil Steril 2014;101;20-30. DOI:
408
http://dx.doi.org/10.1016/j.fertnstert.2013.11.009
409
52. Dunlop CE, Telfer EE, Anderson RA. Ovarian stem cells—potential roles in infertility treatment and fertility preservation.
410
Maturitas 2013;76:279-83. DOI: http://dx.doi.org/10.1016/j.maturitas.2013.04.017
411
53. Yuan J, Zhang D, Wang L, Liu M, Mao J, Yin Y et al. No evidence for neo-oogenesis may link to ovarian senescence in adult
412
monkey. Stem Cells 2013;31:2538-50. DOI: http://dx.doi.org/10.1002/stem.1480
413
54. Mitoh S, Yusa Y. Extreme autotomy and whole-body regeneration in photosynthetic sea slugs. Curr Biol 2021;31:R233-R234.
414
DOI: http://dx.doi.org/10.1016/j.cub.2021.01.014
415
