A New Cell Stem Concept for Pelvic Floor Disorders Prevention and Treatment – Endometrial Mesenchymal Stem Cells

Abstract

High rate complications and recurrences in reconstructive surgery using in situ synthetic/polypropylene (PP) meshes have driven to a new concept based on mesenchymal stem cells (MSCs) for homeostasis repair in pelvic floor disorders (PFD). Prevention and therapy with MSCs are up to date analyzed on small and large animal models, less in women trials. Cell based-vaginal/intraurethral, or systemically introduced, tissue engineering (TE) with new generation meshes/scaffolds MSCs seeded-bone marrow, adipose tissue and recently proposed the endometrial/menstrual MSCs (eMSCs/MenSCs) for PFDs, management. Easy collected, isolated with specific markers, cultured for number harvesting, without ethic and immune compatibility issues, with unique biologic properties eMSCs/MenSCs differentiate in many cellular types—smooth muscle, and fibroblast-like cells, preserving cell shape, and phenotype, without oncogenic risks, and collagen, elastin fibers; eMSCs/MenSCsare appropriate for PFDs management, respecting good protocols for human safety. The quick appeared regenerative effect-mediated by angiogenesis, apoptosis inhibition, cell proliferation, no chronic inflammation and low/no foreign body reactions, less thick collagen fibers, and fibrosis improve connective/neuromuscular tissues; less pelvic structures stiffness with more elasticity are advantages for new meshes/scaffolds generation in TE. Human eSMCs/MenSCs deliver bioactive factors by their exosomes/microvesicles/secretome for paracrine effects to injury site, facilitating in vivo tissue repair.

1. Introduction

Pelvic Floor Disorders (PFDs) are a continuous gynecologist’s challenge, reason for this paper to review their correction attempts, by better knowledge of pathophysiology, pelvic connective tissue structure and biology with the new concepts of regenerative medicine by mesenchymal stem cells (MSCs) and/or their bioactive mollecules, sometimes crucial. All these objectives are after the era of reconstructive surgery with native tissue or in situ synthetic/polypropylen (PP), non-absorbable meshes for fascia repair. The surgical procedures have immediate, and subsequent (within 5 years) complications (10–30% rate) [1], disorder’s recurrence or a new disorder, and host reactions to mesh—as a foreign body reaction (FBR), with fibrosis, mainly to transvaginal type, explaining abnormal functions after interventions. FDA (USA) had two public health notifications, warning since 2010 against vaginal mesh use in POP, because complications, recurrences, and litigations [2]; PP mesh use is prohibited for transvaginal POP surgery in Australia, New Zealand, USA, and UK [3]. Stem cells, as endometrial/menstrual mesenchymal stem cells (eMSCs/MenSCs) with their remarkable unique biological properties are considered a new potential tool for PFDs prevention and therapy, in a properly response to pathophysiology. MSCs are key regulatory components in the regenerating stem cell local microenvironment termed “stem cell niche” or for MSCs culture conditions, per se or by their secretome, facts that positively influence altered pelvic floor connective tissue, and contribute to tissue homeostasis restoration [4]. Allogeneic and autologus MSCS/MenSCs are proposed for these aims, being easy collected, isolated, purified with known perivascular surface markers for pericytes [5], and with the novel single perivascular marker Sushi Domain Containing 2 (SUSD2) for purifying eMSCs [6], and maintaining cells clonogenicity, reduced by flow cytometry used for their sorting [5, 6]. Different to other MSCs culture expandation imposing presence in culture medium of some constituents of their secretome [7, 8], for eMSCs and ERCs one adds transforming growth factor-β receptor inhibitor (TGF-β R inhibitor), that limits cells spontaneous differentiation to fibroblasts, and maintains the undifferentiated cells status in days following administration, to ensure efficacy [9]. eMSCs and ERCs have no ethic issue, or incompatibility risk, no need of immunosuppressive adjuvant drug, they intervene in repair and regeneration by new blood vessels formation, modulating host immune system, reducing chronic inflammation, FBRs, and fibrosis, with no scar as endometrium regenerates after each menstruation from the menarcha to menopause, under a normal estrogen/progesterone balance, and in postmenopause when on menopausal hormone therapy (MHT). One proposes cell based therapy-eMSCs/MenSCs direct placement in vaginal walls or intraurethral, or systemic administration [10, 11] to prevent postpartum POP or SUI, and tissue engineering (TE)/tissue grafts with eMSCc/MenSCs seeded in a new meshes/scaffold generation preferable biodegradable, obtained by a variety of technologies, as knitting (like old ones) from alternative synthetic and natural polymers, electrospinning, and three-dimensional printing [12], and a composite mesh of polyamide plus a gelatin layer [13], instead of PP meshes, with no ectopic tissue formation, or malignant tumor [14]. There are many small and large animal models, and few human clinical studies on MSCs for tissue restoration and repair when PFDs.

2. Contemporary burden of women’s pelvic floor disorders

PFDs named also Pelvic Floor Dysfunctions represent a women old pathology, with high incidence and prevalence in the last 70 years, associated to worldwide lifespan increase, independently to their different definitions and diagnosis criteria, last updated in 2010 by the International Urogynecological Association and International Continence Society [15], with over 250 definitions for clinical categories and subclassifications, and alphanumeric code for each definition. PFDs is an umbrella term for a group of clinical conditions caused by pelvic floor supportive tissue weakening, sometimes degenerative, occurring independently or simultaneously, connected to genetics, childbirth and aging, and it includes pelvic organs prolapse (POP) into vagina, as a hernia in the endopelvic fascia [16], and alterations in sensory or emptying abnormalities of the lower urinary and gastrointestinal tracts: urinary incontinence (UI) with clinical manifestations of stress urinary incontinence (SUI), overactive bladder syndrome (ORB), detrusor instability, urinary retention, and fecal incontinence, and sexual dysfunctions. PFDs are diagnosed in middle-aged women as stress urinary incontinence (SUI)—incidence peak around 46 years, and in elderly women when a second SUI peak between 70–71 years, being described in young women, also, around 18 years, with annual risk of 3.8 and 3.9 per 1000 women at that bimodal peaks; the risk for pelvic organ prolapse (POP) is increasing progressively from 50 to 60 years in Swedish population [17], to ages of 71 and 73 years, when annual risk is 4.3 per 1000 North American women evaluated for lifetime risks for SUI, or POP, or both surgery between 2007 and 2011 [18], and higher in institutionalized, and when dementia [19]. The recent Swedish national register-based cohort study [20] estimated a 12–19% lifetime risk for surgery for POP, similar to the earliear 19%-for Australian women [21]. PFDs have serious negative influences on women’s quality of life at any age [22]. SUI and POP are highly related to childbirth-associated pelvic floor injury [23], less influenced by route of delivery (vaginal, cesarean section), sometimes inherited, as a familial disorder, associated to other hereditary conditions—joint hypermobility and even Ehlers–Danlos syndrome, a mutation in the gene for collagen III [24, 25]. One recommends the extension of familial conditions lower than first degree generation relationship [26] for familial risk reduction and prolapse prevention, many cases being more frequently in postmenopause, without any pregnancy/delivery, when the association of ovarian aging hypoestrogenism, plus vasomotor syndrome, depressive mood, chronic constipation and cough, obesity, characteristics for this life period, are increasing PDFs medical and financial burden [27].

2.1 Synthetic mesh unfavorable results in surgery for pelvic floor disorders

In front of PFDs burden, one tried to correct the supportive tissue of cardinal, utero-sacral ligaments, levatorani muscle, and urethral sphincter damaged by gestation, parturition, and aging initially by non-surgical interventions: physical exercises (after delivery), pessaries—first choice for POP symptomatic women [28], or as a tool for decision of mid-urethral slim mesh, when one decides POP surgery, to avoid over and under-treatment if vaginal mesh is used for an nonexistent SUI or a urinary leakage when surgery for POP [29], pharmacological therapies, laser, nonablative monopolar radiofrequency [30], and surgery, when all these failure; either vaginal or abdominal route, or both with native tissue repair, through open surgery or laparoscopy, or reconstructive surgery with synthetic, monofilament PP mesh respecting the principles for different PFDs manifestations, classified in anterior and posterior compartment, and vaginal vault disorders. It was evident that the addition of mesh as reinforcement to vaginal walls provides better prolapse correction, compared with colporrhaphy using native tissue alone, by both objective and subjective criteria [31], as it was demonstrated for abdominal wall defects/hernia repair [32]. Unfortunately medical staff and patients wishes for pelvic floor normal functions restoration with PP meshes were not accomplished, or the results were not better than after native tissue repair, being reported high risks—immediate, subsequent (within first 5 years) readmissions for later postoperative complications, such as intractable pain, or mesh erosion or extrusion into the bladder, bowel or vagina [33], requiring surgical excision in ≥10% [28], or deterioration of vaginal biomechanical properties by high stiffness mesh implanted for prolapse [34], and further recurrences of previous disorder, or a de novo one, as incontinence surgery-incidence of 9.9% after surgery for POP without occult SUI (apical and non-apical) [35] or further prolapse surgery, sacro-spinous fixation with synthetic mesh being a risk factor for POP recurrence [36], andtransvaginal PP mesh having a higher risk than vaginal vault sacro–colpopexy [37, 38]. The population-based cohort study from Scotland [39] compared the primary outcome—immediate postoperative complications and subsequent (within 5 years) readmissions for later postoperative complications, further incontinence surgery, or further prolapse surgery in women older than 20 years, after first, single PP mesh (retropubic mesh, or single prolapse mesh procedure) to non-mesh procedures during 20 years. Immediate complications were lower after mesh procedures [adjusted relative risk (aRR) 0.44 (95%CI 0.36–055) and subsequent prolapse surgery [adjusted incidence rate ratio (air) 0.30 (0.24–0.39)], and a similar risk for further incontinence surgery [0.90 (0.73–1.11)], and later complications [1.12 (0.98–1.27)]. Anterior compartment prolapse PP mesh repair was associated to a similar risk of immediate complications as non-mesh surgery [aRR 0.93 (95%CI 0.49–1.79)], but with an increased risk of further incontinence [air 3.20 (2.06–4.96)], and prolapse surgery [1.69 (1.29–2.20)], and a substantially higher risk of later complications [3.15 (2.46–4.04)]. Posterior compartment mesh repair was associated to a similarly increased risk of repeat prolapse surgery and later complications as non-mesh surgery. Vaginal vault prolapse had similar outcome when vaginal and, separately, abdominal mesh repair were compared with vaginal non-mesh repair. Both vaginal and abdominal mesh procedures for vaginal vault prolapse repair are associated with similar effectiveness and complication rates to non-mesh repair.

The synthetic meshes used for pelvic floor reinforcement or reconstruction may provide the necessary mechanical support for damaged tissue, but implants’ biological actions interfere with host biology, inducing the growth of a fibrous tissue layer, as an additional physical support, but the scar may contract the mesh, and surrounding tissue up to 60% [40], Mesh parameters influence host tissues: microstructure-porosity (permeability for host’s cells, mainly immune cells, fibroblasts, macrophages, metabolites, oxygen at repair place, and also bacteria effects, by chronic inflammation), fiber filament type (monofilament: polyamide and polyetheretherketone monofilaments or multifilaments; synthetic/natural/composite), and diameter, mesh weight (light/ultralight/heavy), mechanical properties as stiffness, and elasticity, chemical properties, materials biodegradability, and integration in host organ by new blood vessels formation, facts that contributed to the new concept of stem cells in cells based therapy, and tissue engineering (TE) for PFDs prevention or repair [13, 41].

3. A new concept of stem cell use in prevention and therapy of women’s pelvic floor disorders

Tissue engineering is a new option in the field of pelvic floor repair when soft tissue reinforcement or reconstruction, or normal function are necessary. MSCs and/or their secretome are proposed after many studies regarding pelvic tissue biology, materials properties associated to stem cells designed to restore the anatomical functions, to provide a real pelvic floor mechanical support after damage, usually to be like a hammock, and to offer both the lost stiffness when under tension and the flexibility under bending [42].

3.1 Tissue homeostasis/remodeling behind repair in pelvic floor disorders

Pelvic floor—a complex resistance piece, keeps pelvic organs within the body, still allowing passage through urethra, vagina and anal canal, around which are designated striated muscles—levatorani, with its three fascicles, and superficialperinealmuscles or urogenital diaphragm [43], forming a functional neuromuscular unit, and fibrous connective tissue, generating endopelvic fascia, cardinal and utero-sacral suspensory ligaments, and vaginal dense fibromuscular-connective tissue. Pelvic connective tissue maintains the position of organs adjacent to vagina, and the close anatomical relationship among vagina, bladder, and rectum may contribute to the emergence of anatomical-functional failure of adjacent organs/systems, in PFDs [44], according to their normal different stiffness/elasticity. Animal models indicate that molecular changes in tissue composition, mainly protein content, coincide to altered biomechanical properties, in PFDs, mainly in POP, as one cites in Australian [45], European [46], North American analyses [47, 48]. Human and mouse pelvic floor provided similarities of pathological changes, centered on deep and superficial muscles, ligaments, and connective tissue, mainly on vaginal walls. Pelvic floor connective tissue contains stromal cells and a very complex extracellular matrix (ECM). The balance between ECM synthesis and degradation during women/females life is a key in pelvic floor properties, and the vaginal structures are strongly influenced during women, small and large animals life, ovine models demonstrating architectural and functionaldifferences according the reproductive status. The Australian ovine model [45] revealed the lowest total collagen content in virgin vaginal tissue, in contrast to parous tissue with highest total collagen and lowest elastin content with concomitant high maximum stress, in contrast to pregnant sheep with lowest collagen and highest elastin contents, and thickest smooth muscle layer and low maximum stress, and poor dimensional recovery following repetitive gestational loading. The vaginal tissue is anisotropic with some biomechanical properties—loading pressure, deformation rates, resistance to rupture, which were tested in ewes [49], and in vaginal specimens collected during surgery for POP [50] compared to specimens from cadavers without noticed PFDs (non-pelvic organ prolapse)—the first experimental study providing vaginal tissue mechanical behavior. The results highlight the non-linear relationship between stress (force per unit of surface) and strain, the vagina being hyperelastic and supporting very large deformation before rupture appearance, as in labor, and fetal expulsion. The vaginal walls tissue is stiffer in patients with POP than non-POP [51]. Comparison of biomechanical properties of the crucial organs of pelvic support [52], showed significant differences at large strain levels: vagina is more rigid, and less extendible than rectum, which, is more rigid than the bladder. The anterior and posterior vaginal walls have different stiffness, and the bladder tissue was anisotropic at large strain levels, facts very important for tissue repair: region with dysfunction/disorder, or procedure type.

ECM molecules are arranged in a matrix/scaffold, surrounding stromal cells (fibroblasts, myofibroblasts, smooth muscle) that synthesize collagen, as tropocollagen and elastin, as tropoelastin to form the fibers complex network, plus proteoglycans, and matricellular proteins, enzymes, according to their genes. All forming the complex network of pelvic floor support, recently updated [53]. EMC contains:

• tropocollagen, self-assembled into fibrils, aggregating to form a collagen type I (for tensile strength) and type III collagen fiber (for elastic properties, and increased collagen III reduces mechanical strength [54]; collagen form a cross-linked network intertwined with elastin—the elastic fibers core component, secreted by elastogenic cells as the monomer tropoelastin, and undergoes self aggregation, cross-linking and deposition on to microfibrils assemble into insoluble elastin polymers. A microfibril scaffold-primarily formed by the protein fibrilin-1, is required for elastic fibers formation [53]. Collagen and elastin fibers are surrounded by a viscous substance of proteoglycans-consisting of a core protein to which one or more glycosaminoglycan (GAG) chains—ashyaluronan or hyaluronic acid, heparan, dermatan sulfate and the small leucine-rich repeat proteoglycans (SLRPs)-decorin, lumican and fibromodulin [17] are covalently attached; SLRPs cover the surface of collagen fibers, contributing to fiber optimal formation [55]. Proteoglycans have key roles in controlling gradients and availability of potent growth factors, chemokines, cytokines, and morphogens, very important in tissue’s homeostasis, mechanical strength, development, and repair. One or more proteoglycans are cell surface or transmembrane receptors for adhesion molecules in all mammalian extracellular matrices [56], contributing to progenitor stem cells microenvironment/niche [57, 58].

• matricellular proteins or elastic fibers associated [59]: fibrilin-1, fibrilin-2, fibulin-3, -4, -5, are involved in elastic fibers synthesis and assembly. Fibulin-5 is a pivotal molecule with dual functions involving MMP-9 enzyme regulation, and in tethering polymerized monomeric form of elastin to surrounding cells in vaginal wall, and positively regulating coacervation, but negatively regulating maturation of coacervated elastin in vitro [60]. When pre pregnancy POP fibulin-5 knockout mice increases in severity after vaginal delivery [61, 62, 63].

• adhesion molecules: fibronectin; integrins—α, β or α-β integrins interaction with fibulin-5 is essential in vascular development, but dispensable for fibronectin fibrils assembly.

• enzymes:

  • matrix proteases as matrix metalloproteinase (MMP)-2, MMP-9 involved indisruption of collagen and elastin fibers, and particularly increased in POP, and in postmenopause comparative to premenopauseal asymptomatic cases; estrogens withdrawal or antiestrogenic therapy upregulates MMP-9; and TIMP-1, TIMP-2

  • lysyl oxidase-like–1 (LOXl-1)—predominantly catalyzes elastin cross-linking, its inhibition associated to increased MMP-9 led to subclinical POP [27, 64], because tropoelastin accumulation, according to the theory of antielastase-elastase inbalance in mice lacking LOXl-1, and the lack of deposit with normal elastic fibers in the uterine tract, and an abnormal postpartum heal of elastin, In LOXL1 knockout mice, smooth muscle cells stiffness and cells adhesion are altered, being proved the interplay between smooth muscle mechanics and ECM remodeling, mainly in postpartum [65, 66].

• water, very important for all body composition.

  The content, aspect and cross-link of collagen and elastic fibers, matricellular proteins, proteoglycans, and enzymes specially MMPs, LOXl-1 are negatively changed when POP; elastic and mechanical strength are decreased during gestation, and with age, beingconceivable that a loss of elastic fiber–associated proteins in pelvic floor connective tissues with aging, may disrupt the optimal balance between synthesis and degradation of vaginal elastic fibers, and lead to POP, fact associated to the critical negative proteases role in POP progression [48]. The fibers amount is less interested initially after vaginal delivery; their histomorphology is first changed, regarding length (shorter), and cross-linking in net-work [61], fibers density decreases later, by aging [67]. The quantification of collagen and elastic fibers shows a more important decrease of elastic fibers in superficial epithelial layers near vaginal epithelium, and less in the deepness of pelvic cavity, around muscles, and thin, irregular and disrupted collagen bundles, higher levels of collagen type III in the vaginal wall, and fragmentation of collagen fibers [68], being appreciated that epithelial-stromal interactions, and fibulin-5-integrin interactions—that suppress ROS generation, are critical in regulation of MMP-9 in mice vaginal wall [48], with an increased level of MMP-2, -9 in advanced prolapse [69, 70]. It is sure that such molecular changes are not corrected by surgical techniques with native tissue or with PP mesh, and the procedures of tissue engineering by different MSCs types, and/or their secretome may change pelvic floor future histomorphology and functionality with normal/near normal connective tissue appearance, that will be discussed in Section 2.5.

3.2 Genetics, gestational and postmenopausal influences on pelvic floor connective tissue disorders

Genes, sexual steroid hormones with their receptors, ligands and co-activators modulate pelvic floor structures, and volumes entire women’s life. One analyses Homeobox genes (HOXA-11involved in utero sacral ligaments fibroblasts proliferation and p53 regulation [71]), gene encoding LOXl1-generating a primarily failure of elastin postpartum healing in knockout mice, the decrease gene signal for production of three SLRPs-decorin, lumican and fibromodulin, which are collagen fiber assembly regulators with affected collagen fibrilogenesis and collagen fibrils shape, and impairment in elastic fiber assembly by down regulation of fibulin-5 in POP [17]. Genes encoding fibulin-5, fibulin-3, Upii-sv40t—involved in elastin fiber structure, are analyzed in PFDs associated to knockout mice aging [27].

Pregnancy induces adaptations in pelvic floor structures for vaginal delivery to withstand deformations with minimum damages, but vaginal delivery leads to floor disorders, damaging nerves, connective tissue, pelvic smooth and striated muscles. Pelvic connective tissue reduced stiffness and elasticity is essential, being demonstrated that the load carrying response (other than the functional response to the pelvic organs) of each fascia component, pelvic organ, smooth muscle, and ligament are assumed to be isotropic, hyperelastic, and incompressible [72]. There are parallel gestational changes in levatorani muscle: sarcomerogenesis, fiber elongation, and an increased ECM collagen content, with muscular stiffness [73], to avoid sarcomere hyperelongation resulted from mechanical strains imposed by vaginal delivery. Sometimes delivery related strains lead to acute sarcomere hyperelongation, and pregnancy pelvic floor muscles (PFMs) adaptation is exceeded [74]; with pelvic floor muscles (PFMs) avulsions discovered postpartum [73]. Human parturition needs PFMs elongation of 300% in resting muscle length to achieve fetal vaginal delivery, as computational models revealed [75]. An instrumental vaginal delivery with forceps may induce important damages of levator ani muscle visible at 3D/4D postpartum ultrasound [76], and one considers that the majority of vaginal deliveries are followed by subclinical damages. The postpartum pelvic floor repair is different from cervix uteri repair, with loss of pregestational EMC composition restoration. Each vaginal delivery, in special genes and familial heredity conditions, and also without these risk factors, may contribute to EMC damages, with changes of pelvic floor shape and in biochemical structure in pregnancy, and post delivery versus nullipara [77], gestation has the greatest impact on vaginal tissue composition and biomechanical properties proved in animal models (mice, sheep), and women. Important differences of pelvic floor EMC structures are described [45] between virgin, pregnant and parous females regarding total collagen, collagen III/I + III ratios, GAGs, and elastin, and in passive biomechanical properties-compliance and elasticity, and maximum stress and strain, with permanent strain following cyclic loading after each gestation. Vaginal tissue of virgin sheep had the lowest total collagen content and permanent strain, and parous tissue had the highest total collagen, and lowest elastin content with concomitant high maximum stress in contrast to pregnant sheep, that had the highest elastin and lowest collagen contents, and thickest smooth muscle layer, situation associated with low maximum stress, and poor dimensional recovery following repetitive loading. Vaginal biomechanical properties do not recover after pregnancy to those of virgins [44], and tensile strength appears to be linked to vaginal content: total collagen, elastin, and smooth muscles show a direct influence on tissue compliance—reduced after ovine consecutive pregnancies, different to rectum and bladder compliance which are stiffer than vaginal walls after many deliveries [78]. Vaginal distensibility pregnancy-induced and along vaginal delivery by tissue vaginal pressure is not recovered in late postpartum rats [79]. It was demonstrated in mice vaginal culture, the POP appearance and progression after each pregnancy and vaginal delivery, caused by a combination of inhibited elastin linking with tropoelastin accumulation [47], because inability to initiate damaged tissue necessary clear and replacement, with poor elastin properly self repair after each delivery, through abnormal enzymatic actions of MMP-2 (decrease)—TMP-4 (rise) [48], after an initial total elastin amount preservation.

Ageing associated hypoestrogenism is worsening pelvic floor condition. Sexual steroid hormones have receptors in all pelvic organs, not only in genitalia (Table 1) [80]. Uterine prolapse, but not SUI is diagnosed in nuliparous postmenopausal women.

	ERs	P4Rs	ARs
Vagina	+	+	+
Urethra	+
Urethral sphincter	+
Periurethal venous system	+
Bladder trigon	+	+	+
Pelvic floor; Pubo cervical muscle; Levator Ani muscle; Cardinal ligaments; Utero-sacral ligaments; Periurethral Fascia; Perivaginal tissue	+; ?(+/−); +; + (α, β); + (α, β); +	+; +; −; −

Table 1.

Sexual steroid hormones receptors distribution in pelvic floor structures.

Adapted from Rechberger and Skorupski [80] (creative common: License CC BY4.0 for adapt). ERs: estrogen receptors; P4Rs: progesterone receptors; ARs: androgens receptors.

Aging is associated to intrinsic aging stem cells-meaning self renewal reducing, through their genes aging [81] in the general dysfunctional frailty syndrome, where pro-inflammatory cytokines-TNF-α, IL-6 and C-reactive, are increased [82], and one may delay aging effects by menopausal hormone therapy (MHT), started from perimenopause for urogenital aging, with local estriol to reduce vaginal atrophy, and some symptoms of bladder aging [83, 84], and it is adjuvant in pre and long term postoperative care, associated to systemic MHT, around age of 50′ as new protocols advice, and different MSCs, types treatments, for frailty delay to safe health and function of organs/tissues, and one speaks about safe proper frailty treatment with MSCs, to increase health and function of organs/tissues [85, 86].

3.3 Mesenchymal stem/stromal cells for pelvic tissue repair and regeneration

High number surgical gynecological procedures proposed along 100 years showed limitations, low adequacy to PFDs pathophysiology, no restoring organs’ normal positions and functions, and better understanding by three dimensional digital models combining DeLancey JO’s theory [16] to Petros P integral theory of continence [interdependence between pelvic organ support systems, linking ligament fascia lesions, and clinical expression, with less critics after TVT (tension free vaginal tape) technique] [87], and tissue structure continuing to deteriorate by aging after correction, being far to be restored by surgery either by native tissue or by PP meshes. MSCs, and their secretome, are discussed since more than 10 years, specially after techniques for their potency enhancement by specific culture systems, including three-dimensional culture conditions, or their priming preconditioning with some molecules of their secretome [7, 65]. MSCs represent a pathophysiologic correction, and limitation of PDFs progression [88]. Tissue engineering is a new option to restore and maintain micturition normality via direct effects on damaged or dysfunctional tissues, or pelvic floor repair when soft tissue reinforcement is necessary [89, 90], to improve outcomes in POP management [91]. MSCs also referred as mesenchymal stromal cells belong to the pool of progenitor and adult stem cells (ASCs) family, from all postnatal organs and tissues, with specific properties for each one, ensuring the capacity of renewal after damage, and in aging [7]. Collected and isolated from various anatomical sites, more or less easy accessible, few ethics-related issues, MSCs are actually easily separated/purified [92], with specific markers, and cultured. Some consider multipotent MSCs to have a limited self-renewal capacity [93], in a specific microenvironment termed “stem cells niche”, first described by Schofield R [94] as “adult stem cell niche hypothesis”, which is reconsidered to be more dynamically than originally appreciated, with a bilateral influence from healthy or damaged tissue to MSCs, mainly by immunological and inflammatory signals in conjunction to MSCs’ paracrine effects. Others [95] consider that MSCs unique properties-high proliferative ability, self-renewal, differentiation to mesodermal lineages, appropriate to their location, are supporting tissue regeneration in physiologic and pathologic conditions, and are contributing to tissue homeostasis. MSCs are key regulatory components in the regenerating stem cell niche, by the increase of their own compound, or increasing physiologic cells turn-over [96] to support tissue regeneration after injury, or are activated in injured tissue, where they are inactive [97], or are attracting supporting cells to niche [4], or are activating tissue’s own cells to facilitate repair [98], capabilities that are different according to tissue type. These biological properties determined the change of “stem” cell nomination to “stromal” for a more appropriate connotation [7], and earlier Caplan AI [99] proposed the name of “Medicinal Signaling Cells” for a more accurate presentation: when systemically administered MSCs home in on sites of injury/disease, and exhibit a paracrine action, by secreting bioactive molecules as regulatory and growth factors, chemokines, cytokines, nucleic acids, packaged into extracellular vesicles or MSCs-derived exosomes, with trophic and immunomodulatory actions, reflecting that MSCs make therapeutic drugs in situ that are medicinal, important for tissue repair. MSCs fate in a tissue is influenced by local microenvironment or niche fixed compartment, where ASCs are in a dormant state (G₀) through signaling pathways inhibitory for growth and differentiation, often involving transforming growth factor-ß (TGF-ß), and bone morphogenetic protein (BMP) family members [57], being anchored to niche cells by adhesion molecules—cadherins, integrins [100] during stem cells’ periods of inactivity, and niche cells differentiation signals to resident stem cells [58]. The bioactive molecules produced through MSCs homing when systemic administrated, or when are added in cultures to potentiate MSCs action, as MSCs primers [65], are considered more important than cell engraftment and replacement. MSCs and their bioactive molecules have proangiogenic [101], antifibrotic, anti-inflammatory, and pro-inflammatory actions, which sustain proliferation [102], and stimulate effect of resident progenitor cells, in relationship to disease/organ/tissue type [103]. The damaged, ischemic tissue activates MSCs after injury, through different inflammatory signals (hypoxia, proinflammatory cytokines as IL-1β, IFN-γ, TNF-α, lipopolysaccharide) from host innate immune system and leads microenvironment to coordinate the production of immunomodulatory factors to sustain inflammation progression and rapid remove of allogenic MSCs, and production of growth factors to stimulate endothelial cells, fibroblasts, and tissue progenitor cells’ differentiation from MSCs niche, all contributing to tissue repair, in an orderly action by angiogenesis, EMC remodeling, and functional tissue restoration [7], MSCs have the ability to home to injured tissues to exert their paracrine actions when systemically administered, a very attractive feature for this chapter discussions (Figure 1) [104].

Figure 1.

MSCs role in damaged connective tissue repair. MSCs activation after tissue injury. The damaged tissue activates MSCs after injury through different inflammatory signals (hypoxia, cytokines asIL-1β, IFN-γ, TNF-α, LPS). MSCs activation leads microenvironment to coordinate the production of immunomodulatory factors, that sustain inflammation progression, and of growth factors to stimulate endothelial cells, fibroblasts, and tissue progenitor cells to differentiate, and all contribute to tissue repair, in an orderly action by angiogenesis, EMC remodeling, and functional tissue restoration. Adapted from Miceli et al. [7]. It is an open access article, distributed under the terms and conditions of the creative commons attribution (CCBY), licensee MDPI, Basel, Switzerland.

MSCs do not impose immunosuppresion, being immune-privileged due to their low expression of CD40, CD80, CD86, and major histocompatibility complex I (MHC I), and the lack of MHC II [92], or because they are immune evasive [105].

4. Endometrial and menstrual mesenchymal stem cells for prevention and therapy of pelvic floor disorders

MSCs from different tissues exhibit many common characteristics, their biological activity and some markers are different and depend on tissue origin: bone marrow (obtained by aspirate), adipose tissue (obtained by liposuction), placenta (for maternal MSCs), and umbilical cord, amniotic membranes and liquid (for fetal MSCs) collected at birth. All these MSCs have some limitations, as in vitro expansion [106] for their rarity in original tissue, invasive methods to harvest bone marrow aspirates, donor aging affects MSCs proliferative capacity [86], and the necessity to add their secretome’s active molecules in culture medium [7, 8, 107].

4.1 Unique characteristics of endometrial mesenchymal stromal/stem cells and menstrual mesenchymal stromal cells

In the last 15–20 years, a MSCs subpopulation of stem/progenitor/regenerative cells has been identified and characterized in human endometrium (eMSCs) and in menstrual blood (MenMSCs or ERCs), comparable to bone marrow and adipose tissue MSCs [95], but with unique biologic characteristics [1, 108], and knowledge plus new technique capabilities made them a very promising MSCs category in autologous and allogeneic cellular therapy, and TE. Uterine fragments of shedding endometrial tissue with their remarkable cells turn-over—like hematopoietic bone marrow, intestinal epithelium, epidermis, contribute to endometrial repair and renewal without scar, and gene profiling has demonstrated that the lysed stroma is enriched in genes involved in EMC dynamics, biosynthesis and degradation [109, 110], very promising in pelvic floor connective tissue repair/restoration, and other endometrial fragments from menstrual blood contain MenSCs. The concept of endometrial renewing after each menstruation (~400 cycles in woman’s life) by endometrial stem/progenitor cells located perivascular [6], in the basalis of endometrial glands near myometrium was first hypothetized by Prianishnikov VA, 1978 [111], reloaded by Padykula HA, 1989 [112], and it is continued in Australia at the Department of Obstetrics—Gynecology, Monash University and Centre for Women’s Health Research (Melbourrne, Victoria) by a team led by Gargett CE [113], who presented the first direct evidence that human endometrium contains rare populations of epithelial (0.22%), mesenchymal/stromal-eMSCs (1.25%), and endothelial progenitor cells, which exhibit the adult stem cells behavior in vitro, as clonogenicity, and later their differentiation potential (reviewed in 2016) (Figure 2) [115].

Figure 2.

Schematic perivascular localisation of human eMSCs. Co-expressing CD146 and PDGFRβ/CD140b and SUSD2⁺ eMSC in the endometrial basalis and functionalis layer, indicating eMSC will be shed into menstrual blood. Reprinted from Gargett and Masuda [114] with license permission: 535199193277 for Copyright Oxford University Press, 2022, July.

One may add a small side population (SP) cells which enriches endometrial stem cells fractions, according to their identity and differentiation potential [116]. eMSCs are responsible for cyclic regeneration of human, mice, and ovine endometrium [117, 118, 119, 120]. Other research group [121] demonstrated that the low number of human endometrial stromal/stem cells seems to belong to the family of MSCs, by possessing the minimal criteria of MSCs assessment [122] clonogenity, self-renewal, plastic adherence in culture, high proliferative potential and capacity and ability to differentiate into at least one type of mature functional progeny, but eMSCs have multilineage differentiation capacity [123]. eMSCs have proliferative capability to undergo 30 populations doubling before reaching senescence, generating 6.5 × 10¹¹ cells (Table 2) [124].

MSCs must be plastic-adherent when maintained in standard culture conditions. MSCs must express CD105, CD73 & CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 & HLA-DR surface molecules. MSCs must differentiate to osteoblasts, adipocytes and chondroblasts in vitro

Table 2.

Minimal criteria for defining multipotent MSCs.

International Society for Cellular Therapy position statement [122].

Taylor HS [125] at Yale University (USA) presented bone marrow (BM) as an exogenous source of eMSCs, which appear histologically as epithelial and stromal endometrial cells, expressing appropriate markers of endometrial cell differentiation, and cyclic mobilization of BM-derived stem cells is considered a normal physiologic process [126]. Menstrual blood, an usual waste tissue, but a “bio-waste” as recently reconsidered [127] (endometrial functionalis layer shed during menstruation) is an easy obtained source of MSCs, with no ethic issue, and isolated, cultured similarly to bone marrow aspirated. Menstrual stem cells (MenSCs), named also endometrial regenerative cells (ERCs) by the team from Bio-Communications Research Institute (Wichita, USA), who first isolated and cultured them [128], have the classic properties, and pattern of MSCs surface markers, are multipotent [129], retain a stable karyotype in culture [130], proved at more than 68 doublings without any karyotype or functional abnormalities [128, 131]. ECRs have similar capabilities for tissue repair and restoration as eMSCs, according to their secretome that ensure cells paracrine actions on endometrium (after menstruation, and Asherman syndrome) [132], ovaries [133], and on different organ [134, 135]. There are many controversies on eMSCs and ERCs makers [108], being demonstrated that endometrial/menstrual MSCs clones express MCSs markers [ITGB1 (CD29), CD44, NT5E (CD73), THY1 (CD90), ENG (CD105), PDGFRB (CD140B), MCAM (CD146)], but not endothelial or hemopoietic markers PECAM1 (CD31), CD34, PTPRC (CD45), and the pan-leukocyte marker CD45 [128]. The Australian team used these markers for eMSCs isolation and culture [124], plus two perivascular cell surface markers—CD146, and platelet-derived growth factor-receptor β (PDGF-Rβ) or CD140B, and have determined eMSCs location near blood vessels in human endometrium [114, 136, 137], blood vessel wall is considered the eMSCs niche, as it is for all MSCs [138, 139]. These markers were used in association to Sushi Domain Containing 2 (SUSD2) or W5C5, a special marker for eMSCs and MenSCs isolation, proposed as a novel single marker for purifying eMSCs, and to reconstitute endometrial stromal tissue in vivo from endometrial biopsies [6]. MenSCs need a selective marker enrichment to be consistent and efficacious as eMSCs obtained by endometrial biopsy. It is known that perivascular MSCs or pericytes are rare cells, difficult to harvest from adult tissues, and necessitate substantial ex-vivo culture expansion to achieve a sufficient number of potent cells, and prolonged culture of MSCs determine a spontaneous differentiation to fibroblasts, which limits culture expansion, and these significant limits challenged the special add in culture medium of a novel small molecule-transforming growth factor-β receptor inhibitor, namely A83-01, that limits the inconvenient and maintains eMSCs and other MSCs undifferentiated in the days following administration and ensure the therapeutic efficacy of a small proportion (2%) of cells which are estimated to remain in vivo in the days following administration [9, 91, 114, 140]. MenSCs secretome needs a special attention for its exceptional therapeutic effects, due to extracellular vesicles (EV) [135], including microvesicles, exosomes and apoptotic bodies transporting bioactive molecules. A total of 895 molecules are identified in exosomes [141], as micro RNA, lipids, growth factors (vascular endothelial growth factor, insulin-like growth factor-1, hepatocyte growth factor), chemokines, cytokines as regulators of immune response in different tissues [142], which can be isolated from menstrual blood as are MenSCs isolated [143]. The human MenSCs transcriptome and methylome profiles showed their most distinctive expression and epigenetic signature compared to human bone marrow and adipose MSCs [144]. MenSCs trandifferentiation capacity is extensively discussed, associated to their possibility to differentiate into mesodermal lineage (including chondrogenic, osteogenic, adipogenic, and cardiomyogenic fate), endodermal lineage (hepatocyte), and ectodermal lineage (neural and glial) [145], processes that varies considerably between each type, and it is different when one compares them to bone marrow and adipocyte MSCs [128]. Recently there were proved the beneficial effects of MenSCs and their secretome in pulmonary healing in severe acute adults lung cells injury (ARDS) from COVID, by increasing number of CD4 lymphocytes, reducing expression of inflammatory markers (C Reactive Proteine, ferritine, LDH), absorption of bilateral pleural exudates, better than other MSCs types (BM, adipose tissue) when systemic transplanted [146], having also the advantage of easy collection in emergency.

4.2 Potential application of endometrial and menstrual mesenchymal stem cells in pelvic floor disorders prevention and therapy

eMSCs with autologous and allogeneic origins are easily procured from endometrial biopsies—during reproductive years, under contraceptives, in postmenopause [115], without anesthesia or from menstrual blood (source that can be repeatedly used at every menstrual cycle, much easier obtained vs. other sources of ASC). with remarkable differentiation capabilities, plus paracrine actions from their secretome, proved by preclinical and some phase III trials—much discussed and criticized in USA [147], because industry commercial enthusiasm [148]. FDA (USA) approved in 2011 clinical trials with ERCs, which can be used for PFDs prevention and therapy. Actually eMSCs are not accessible for human trials all over the world, or there are no legally approved banks for eMSCs/MenMSCs, these cells being used in some countries only for academic/scientific health centers or not-for profit public institutions. European Medicine Agency has a “hospital exemption” clause, with existence of many unknowns/controversies on eMSCs/MenMSCs-based therapies in human (allogeneic or autologous), as they are used in animal models. Actually one must consider that amalgamation of highly specialized disciplines such as tissue engineering, stem cell therapy and personalized medicine provide important approaches and tools to respond to these challenges in PFDs prevention and therapy, as there were found regulatory approval and deployment for disorders with unmet medical needs [149].

4.2.1 Endometrial/menstrual mesenchymal stem cells based for pelvic floor disorders prevention and therapy

Cell therapy is an emerging field in clinical practice, bone marrow, adipose tissue being subjects for trials in chronic and degenerative disease. Basic evidences provide the preventive role of MSCs autologous cell based therapy in rats SUI by local-urethral administration [150], which is a minimally invasive procedure, and intravenous (i.v.) route [10]. MSCs are homing in damaged pelvic organs, where they are attracted by cytokines [151] and chemokines [152]. After i.v. administration, post vaginal distension (VD)—a model for childbirth injury, GFP-labeled cells were depicted at 4 to 10 days in urethra, vagina, rectum, and levator ani muscle of sacrificed animals, with significantly more MSCs homing at 4 days versus after sham VD, and reduction of GFP intensity at 10 days after VD [153]. It is discussed PFDs prevention with MSCs in high risk women by genetic predisposition, with postpartum SUI and/or POP, and the possibility to induce homing a long time after injury or to increase homing after an acute injury via stem cells genetic modification to express a greater number of homing ligands [154] or an electrical stimulation to the paravaginal region, which induces neural stem cells migration [155], or by MSCs paracrine effects in damaged tissues [growth factors as Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), angiopoietin-2, and Platelet-Derived Growth Factor (PDGF-BB)], which were proved to be in a rate of about 10–100,000 times more for MenMSCs than the control mesenchymal cell lines derived from umbilical cord blood, and by stimulating metalloproteinases involved in elastin postpartum [128]. Post infusion febrile reaction is the sole adverse event associated to bone marrow MSCs i.v. [156]. One discusses the i.v. dose differentiation between small (rodents), large (ovine) animals, and human: 50 million MSCs/kg for rodents, and 1–2 million cells/kg and never more than 12 million cells/kg for human. MSCs pulmonary entrapment, with their quick phagocytisation, by lung tissue macrophages [157], and the genetic instability and tumorigenicity [156], not valuable for eMSCs/MenMSCs [14]. ERCs presenting more than 68 doublings without any karyotype or functional abnormalities [128, 131].

Australia has programs based on techniques to purify eMSCs by magnetic beads and special markers, for production of large number of cells under Good Manufacturing Practice (GMP) conditions to offer women’s own eMSCs when they need for PFDs, for TE. Actually there are only animal studies, no human trial on eMSCs, /MenSCs for PFDs, being discussed other MSCs types. The Cochrane Database Syst Rev. (2017) [158] found in Cochrane Incontinence Group Specialized Trials Register only one small RCT for SUI, on injection of autologous MSCs with fat origin vs placebo, terminated early because of safety concerns, and afterwards there are mentioned some clinical trials for SUI treated with autologous muscle derived MSCs compared to placebo [159], or transurethral and periurethral intrasphincteric injections of cellular suspension for SUI, with limited accuracy of results [160], or a preliminary randomized study of stem cells from adipose tissue implanted for fecal incontinence [161].

4.3 Tissue engineering with endometrial and menstrual mesenchymal stromal cells for pelvic floor disorders

Tissue engineering (TE) for pelvic floor disorders treatment combines principles of eMSCs/MenSCs biology with materials science (scaffolds, meshes for pelvic implantation), and biomedical engineering [162]. The most discussed PFDs beneficiary of eMSCs therapy are POP and SUI—de novo, persistent or recurrent after failure of surgery with native tissue or reconstruction. There are necessary multidisciplinary trained teams. and special protocols, as are presenting Ichim T (2008) CEO at Medistem Laboratory (San Diego, USA), and Gargett CE (2013)—head of the Australian Stem Cell Centre.

The key to safe and efficacious TE in PFDs was the generation of some tissue substitute by materials with nano-architecture/nanofiber technology [163] or 3D printing, mimicking EMC pelvic floor topography, mainly vaginal EMC, or to induce favorable tissue mechanical responses, or to add some EMC constituents (as tropoelastin-the elastin core in EMC network, besides collagen, lost in POP) [164], for production of mesh/scaffold new generation, which allows entrapment, and persistence of seeded eMSCs/MenSCs up to 14 days after implant [41]. eMSCs are in vitro optimized in serum free conditions—fibronectine is the optimal substrate for human eMSCs attachment [91], and eMSCs transcriptome reveals improved potential for cell based therapy after adding TGF-β receptor inhibitor in culture medium (to prolong their undifferentiated status after implantation, by eMSCs apoptosis, and senescence prevention, and maintaining the percentage of SUSD2⁺ cells to more than 90% for all samples) [165]. Another proposal for eMSCs tissue repair efficacy augmentation is the add of a protective delivery system, as a compatible bio-hydrogel carrier that encapsulates eMSCs in mesh/scaffold and improves cells retention at site from host immune system actions to rapid their remove, due to loss of vascular niches [107], and by their encapsulation in hydrogel MSCs can promote endogenous cellular repair [140]. There are many composite meshes produced from different materials: nondegradable polyamide meshes, as those of polyamide/gelatin seeded with 100,000 human or ovine MenSCs/cm², which stimulate angiogenesis, host synthesis type 1 and type III collagen, lower leukocytes infiltrate at 90 days postimplantation (when tested on rats) [166], andthe new biomimetic tissue generation of degradable nano/microstructured meshes [167], meshes obtained through new technologies of electrospinning and 3 D bioprinted endometrial stem cells on an aloe vera–alginate (AV-ALG) injectable hydrogel or on melt electrospun poly epsilon-caprolactone mesh, with the largest open pore diameter and the lowest thickness that promotes eMSCs encapsulated in the hydrogel attachment, which reduces FBRs associated to eMSCs same action [168, 169]. The meshes designed with nanoscale fibers using electrospinning techniques promote cell–cell and cell-biomaterial interactions, being appreciated in Australia [91, 167, 170], and Nederland studies [171] that biometric properties of this nanostructured mesh can improve the integration, overcome erosion, and offer good outcomes in POP reconstructive surgery. The mesh type added to eMSCs have different persistence time after implantation, the natural ones have the shortest “life” duration after implant; the non-degradable polyamide/gelatin mesh plus autologuse MSC persisted 3 days in immunocompetent mice, 1–2 weeks in immunocompromised rodent model when xenogenic human eMSCs, and 90 days in ovine; the longest duration is at 3 D printed mesh [168]. The Chinese study [12] shows that a composite mesh based on synthetic and natural polymers seems to provide the best combination for an ideal pelvic floor mesh material, because natural polymers can provide ligands for cell adhesion and growth factors that promote tissue remodeling, while synthetic polymers provide mechanical strength. New scaffolds proposed for POP provide a three-dimensional environment, and are mimicking EMC network, specially by their micro/nanoscale architecture [172], offering a larger area for EMC constituent proteins, and more binding sites for cell membrane receptors, and adhesion molecules, growth factors, genes, immunomodulatory agents, and external stimuli (electrical, or magnetic pulses), which are delivered simultaneously to target sites after scaffold implantation for promotion of new healthy tissue [173]. No ideal mesh/scaffold for PFDs exists to day, and one may choose the mesh/scaffold according to patient history, and implants’ properties, such as material type: natural (as purified human/animal collagen, chitosan, gelatin, elastin, fibrin, silk, and fibronectin for human eSMCs [174, 175], or synthetic, or by the old criteria used for PP meshes, as pore sizes, mesh’s weight, and the host tissue response to mesh/scaffold material or to eMSCS/ERCs, understanding that reaction to implant materials are crucial for balance between their own elasticity and stiffness, vaginal tissue capacity of strain, which are absent when PP meshes are used. Novel blends of electrospun synthetic and natural polymers combined with eMSC in new generation of implants show that this approach promotes host cell infiltration and slows biomaterial degradation that has potential to strengthen the vaginal wall during healing [164, 165], actioning like intrinsic ECM, but with some limitation regarding small pore size of electrospun nanofiber meshes and toxic organic solvents used for their production [12]. /Human eMSCs/MenSCs modulate host tissue response to implanted materials, by stimulating tissue proper stem cells proliferation, their own high proliferation rate [128], and scaffolds’ eMSCs infiltration, and constituents of their secretome as growth factors, enzymes as MMPs—important for elastin postpartum recovery [128], influence mesh mechanical behavior after implantation [166], by fibrosis and FBRs reducing, when nondegradable polyamide mesh implant, through influencing macrophage polarization switching from an M1 to M2 phenotype, as in rodent and ovine models [166, 176, 177]. eMSCs seeded in degradable nano/microstructured meshes improve mesh tissue integration, eMSCs are entraped over 6 weeks in vivo, by cells with immunomodulatory effects, and by increasing local angiogenesis reduce FBRs to mesh implanted in mice with POP [166, 167, 169], and induce an up-regulation of M2 markers—as CD206 and Arg1, Mrc1, and Il10 genes in host tissue macrophages, parallel to reduction of cellular infiltration and secretion of inflammatory cytokines Il-1β and Tnf-α [175].

The Australian researchers [140] appreciate that tissue engineered mesh inserted transvaginally in large animal models will aid the validation of these constructs prior to clinical translation by assessing their integration with host tissue, and FBRs through histological analysis, immunoassays and gene profiling. Research has commenced with the completion of multiple xenogenic small and large animals studies assessing eMSC/PAG constructs, as mentioned above [166]. These animal models will be crucial in further assessing the efficacy of locally delivered eMSC and further determination of their action mechanisms. Based on these findings, multiple heterologous small and large animal studies are underway to assess the efficacy of other biomimetic degradable materials such as PLCL and 3D-PCL meshes seeded with eMSC/MenSCs.

5. Future proposals for stem cell prevention and therapy in pelvic floor disorders

The Australian research teams recognize some unknown data about eMSCs/MenSCs, mediation on cellular migration and recruitment by their paracrine effects, how eMSCs mediate M2 immunomodulatory responses during the FBR after implantation of bioengineered constructs. One discusses eMSCs secretome constituents as future associated to new generation implants for tissue engineering in PFDs. The Canadian and North American and Chinese researchers recognize the high financial burden for the studies and introduction in human clinical practice according to legislation issues which are incomplete resolved in Europe and North America, in comparison to Australia and Japan.

Conflict of interest

None.