The Ovarian Reserve of Primordial Follicles and the Dynamic Reserve of Antral Growing Follicles: What Is the Link? 1

BIOLOGY OF REPRODUCTION (2014) 90(4):85, 1–11Published online before print 5 March 2014.DOI 10.1095/biolreprod.113.117077

Minireview

The Ovarian Reserve of Primordial Follicles and the Dynamic Reserve of AntralGrowing Follicles: What Is the Link?1

Danielle Monniaux,2,3,4,5,6 Frederique Clement,7 Rozenn Dalbies-Tran,3,4,5,6 Anthony Estienne,3,4,5,6

Stephane Fabre,3,4,5,6 Camille Mansanet,3,4,5,6 and Philippe Monget3,4,5,6

3Institut National de la Recherche Agronomique (INRA), UMR85 Physiologie de la Reproduction et desComportements, Nouzilly, France4Centre National de la Recherche Scientifique (CNRS), UMR7247, Nouzilly, France5Universite Francois Rabelais de Tours, Tours, France6Institut Francais du Cheval et de l’Equitation (IFCE), Nouzilly, France7Institut National de Recherche en Informatique et en Automatique (INRIA), Paris-Rocquencourt Research Centre,Le Chesnay, France

ABSTRACT

The growing follicles develop from a reserve of primordialfollicles constituted early in life. From this pre-establishedreserve, a second ovarian reserve is formed, which consists ofgonadotropin-responsive small antral growing follicles and is adynamic reserve for ovulation. Its size, evaluated by direct antralfollicular count or endocrine markers, determines the success ofassisted reproductive technologies in humans and embryoproduction biotechnologies in animals. Strong evidence indi-cates that these two reserves are functionally related. The size ofboth reserves appears to be highly variable between individualsof similar age, but the equilibrium size of the dynamic reserve inadults seems to be specific to each individual. The dynamics ofboth follicular reserves appears to result from the fine tuning ofregulations involving two main pathways, the phosphatase andtensin homolog (PTEN)/phosphatidylinositol-3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDPK1)/v-aktmurine thymoma viral oncogene homolog 1 (AKT1) and thebone morphogenetic protein (BMP)/anti-Mullerian hormone(AMH)/SMAD signaling pathways. Mutations in genes encodingthe ligands, receptors, or signaling effectors of these pathwayscan accelerate or modulate the exhaustion rate of the ovarianreserves, causing premature ovarian insufficiency (POI) orincrease in reproductive longevity, respectively. With femaleaging, the decline in primordial follicle numbers parallels thedecrease in the size of the dynamic reserve of small antralfollicles and the deterioration of oocyte quality. Recent progress

in our knowledge of signaling pathways and their environmentaland hormonal control during adult and fetal life opens newperspectives to improve the management of the ovarianreserves.

aging, anti-Mullerian hormone, follicular development, ovary,premature ovarian failure

INTRODUCTION

Antral follicle count (AFC) has been the subject ofincreasing interest during the last decade in humans and largemammals. From the improvement of ovarian ultrasonographyand the validation of new endocrine markers, it is now possibleto evaluate the number of antral follicles larger than 1 mm indiameter present in the ovaries of women and large domestic orwild species [1, 2]. Presently, this evaluation is a routinepractice of primary importance to estimating the ovarianactivity of individual females for clinical or breedingapplications. AFC helps the clinicians to establish a diagnosisfor female infertility, and it participates in predicting thereproductive capacity of both human and animal species [3, 4].AFC can also predict the ovarian response of an individual togonadotropin-based stimulatory treatments, hence the successof assisted reproductive technologies in humans and embryoproduction biotechnologies in farm and wild animals [5, 6].

In all species, the antrum cavity is formed when the growingfollicle reaches a diameter between 200 and 300 lm. Theantrum grows by accumulating fluid derived from bloodflowing through the thecal capillaries and secretion products offollicular cells; some of them, such as hyaluronan andproteoglycans, generate an osmotic gradient that participatesin antrum growth [7]. In humans and large mammals, thefollicles grow slowly after antrum formation up to the stagewhen they become gonadotropin dependent and enter a phaseof rapid terminal development, occurring in a wave-like pattern[8–10]. Gonadotropin dependence is acquired at a givenfollicular diameter, characteristic of each species (e.g., 200 lmin rodents, 1 mm in pig, 2 mm in sheep, 3–5 mm in human andcow, and about 10 mm in horse [11]). Below this diameter, thesmall antral follicles constitute a pool of gonadotropin-responsive follicles, which is the reserve for ovulation andovarian biotechnologies. This is a dynamic reserve, since it is

1Supported by the INRA Prediction of Ovulation (PREDICTOV) project,by the Regulation of the Gonadotrope Axis (REGATE) INRIA/INRALarge Scale Initiative Action, by the Agence Nationale pour laRecherche (ANR-2010-BLAN-1608-01) and by French fellowships fromthe Region Centre and INRA to A.E. and C.M.2Correspondence: Danielle Monniaux, Physiologie de la Reproductionet des Comportements, Centre INRA Val-de-Loire, 37380 Nouzilly,France. E-mail: [email protected]

Received: 20 December 2013.First decision: 30 January 2014.Accepted: 19 February 2014.� 2014 by the Society for the Study of Reproduction, Inc.eISSN: 1529-7268 http://www.biolreprod.orgISSN: 0006-3363

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emptied by the cyclic follicle-stimulating hormone (FSH)-orchestrated entry of follicles in the follicular waves of terminaldevelopment, and renewed by the continuous growth of smallerfollicles [12].

When clinicians and zootechnicians talk about ovarianreserve, they usually refer to this dynamic reserve of smallantral follicles. However, the wording ‘‘ovarian reserve’’ canbe confusing, since the growing follicles themselves developfrom a first reserve of primordial follicles, which is constitutedearly in life. In this review, we consider these two differentovarian reserves: the former, the pre-established reserve ofprimordial follicles, and the latter, the dynamic reserve of smallantral follicles. The subject of this review concerns the generalfeatures of these reserves, the tools available for evaluating

them quantitatively and qualitatively, their interrelationships,and the regulation of the transitions between reservesthroughout life in physiological, pathological, and experimen-tal situations.

THE PRE-ESTABLISHED RESERVE OF PRIMORDIALFOLLICLES

The setting up of the reserve of primordial follicles occursduring fetal or neonatal life in all mammals, but the timing ofthe processes underlying the formation and the exhaustion ofthe reserve is clearly species specific [13] (Fig. 1). In allspecies, the formation of the germ cell reserve, calledoogenesis, begins with the migration of primordial germ cells

FIG. 1. Compared oogenesis and folliculogenesis: Temporal sequence of events in the life of different mammals. For each species, the developmentalphases corresponding to 1) meiosis initiation and formation of the reserve of primordial follicles, 2) folliculogenesis before puberty, and 3) folliculogenesisand occurrence of ovulation after puberty are mapped as blue, green, and orange bars, respectively. Ages are indicated under the bars. Before birth, agesare indicated as days postconception (dpc). After birth, ages are indicated as days, months, or years. The boundary between the blue and green barscorresponds to the stage at which most primordial follicles have been formed. Follicles can enter growth before the complete setting up of this reserve (asillustrated in Fig. 2). The boundary between the green and orange bars corresponds to the first occurrence of complete follicular development culminatingin ovulation. In most species, the reserve of primordial follicles is formed before birth or in the early postnatal period; however, meiosis is initiated onlyafter birth in rabbit. The complete exhaustion of the reserve occurs only in humans and some primates, likely associated with their long life span. Adaptedfrom Monniaux et al. 1997 [11] and Mauleon 1969 [13] with permission.

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into the gonadal ridges and their proliferation as oogonia withinovarian nests or cysts. Then oogonia enlarge and develop intoprimary oocytes, which initiate meiotic prophase. Thebreakdown of the cysts leads to the formation of 30-lm-diameter primordial follicles, each one consisting of a primaryoocyte arrested at the diplotene stage of prophase I of meiosis,surrounded by one layer of somatic cells, the pregranulosa, thecellular origin (ovarian or mesonephric surface epithelial cells)of which remains the subject of controversy [14, 15]. Abnormalfollicle formation is associated with a massive loss of oocytesat this stage. After forming, the primordial follicles may beginto grow either immediately or after a gap, depending on thespecies [16]. Alternatively, they become quiescent. In this lattercase, they will either degenerate or resume their growth severalmonths or years later. The molecular mechanisms underlying

oogenesis and follicle growth activation have been the subjectof recent reviews [17–20].

Each stage of oogenesis overlaps in time with the precedingand the following ones. Oogonia, oocytes in meiotic prophase,primordial follicles, and growing follicles can coexist in theovarian cortex during oogenesis. Interestingly, they are locatedaccording to a centripetal gradient of development and growthactivation (as in the rat [21] and mouse [22, 23]). For instance,in the ovarian cortex of piglets, the first growing follicles(which were the first to be formed) are found in the deepest partof the cortex, near the medulla, whereas proliferating oogoniaare located close to the ovarian epithelium (Fig. 2A). Bycomparison, the ovarian cortex of young adults has clearlyenlarged and lost its previous spatial organization (Fig. 2B). Itcontains a lower density of quiescent primordial follicles, oftengrouped in nests [24], in the vicinity of growing ordegenerating preantral and antral follicles.

Oogenesis and folliculogenesis are directly responsible forthe changes in germ cell numbers observed in ovaries duringfetal and postnatal life (e.g., human [25] and cow [26]) (Fig. 3).After a huge peak of germ cells early in life, the reservedecreases sharply with time, falling to 300 000 at puberty andbeing nearly exhausted at menopause in humans and someprimates [27]. Germ cell renewal by neo-oogenesis has beenproposed as a possible mechanism to replenish the pool [28,29]; however, the importance of this process remains disputed[18, 20, 30]. Considering the low number of follicles that willactually undergo development until ovulation (approximately500 ovulations in a woman’s life), the follicular reserve atpuberty appears greatly oversized. Whether the exhaustion ofthe reserve is complete or incomplete depends on the loss rateand growing speed of the follicles on the one hand, and thelifetime of the species on the other.

The size of the reserve of primordial follicles seems to bequite variable between individuals of the same species. Fromthe few available histological analyses, more than 20-foldindividual variations in follicle numbers have been reported atbirth as well as at puberty (e.g., human [31] and cow [3]). Inhumans, without considering the pathological situations ofPOI, the large numerical differences existing between individ-uals at a young age may have important functional conse-quences on the length of the reproductive lifespan [31, 32].

FIG. 2. Histological appearance of pig ovarian cortex during oogenesisand after puberty. A) Formation of primordial follicles and follicle growthactivation in the ovarian cortex of a 21-day-old immature piglet. At thisstage, oogenesis and folliculogenesis coexist within the ovarian cortex.Proliferating oogonia, oocytes in meiotic prophase, primordial follicles,and early growing follicles are located according to a centripetal gradientof development and growth activation. B) Ovarian cortex of a 6-mo-old(young adult) gilt. At this stage, oogenesis has been completed, and theovarian cortex has enlarged and contains a lower density of primordialfollicles in the vicinity of growing follicles. a, antrum of a small antralfollicle; bv, blood vessels; g, granulosa; gf, growing follicles; mp, oocytesin meiotic prophase; o, oogonia; oe, ovarian epithelium; pf, primordialfollicles; t, theca. Bar ¼ 100 lm. (Estienne and Monniaux, unpublishedpictures.)

FIG. 3. Numerical changes in the ovarian reserve of germ cellsthroughout life in humans. Data were obtained by histological analyses.Months p.c., months postconception. Adapted from Baker 1963 [25] withpermission.

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Despite its indisputable interest, an accurate estimation of thesize of the reserve of primordial follicles of an individualremains difficult, due to the lack of molecular markers. Fromthe results of a recent study combining laser capturemicrodissection with transcriptome analysis in sheep ovary,oocyte-specific factors, such as DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 4 (DDX4) (previously known as VASA), which wasfound to be highly expressed in primordial follicles, might beproposed as possible molecular markers [33]; however, theirvalidation as accurate markers of the reserve in a fragment ofovarian cortex remains to be assessed.

THE DYNAMIC RESERVE OF SMALL ANTRAL FOLLICLES

The size of this reserve, estimated by AFC, appears to behighly variable between individuals of similar age (e.g., human[34] and cow [35–37]). As the small antral follicles are thedirect targets of gonadotropin treatments, the large between-individual variability in their numbers constitutes the mostlimiting factor in the success of assisted reproductivetechnologies in humans [38–40] and embryo productionbiotechnologies in other animals [36, 41–45]. Presently, AFCcan be performed by two different methods: ovarian ultraso-nography, which provides accurate estimations of folliclenumbers, and the measurement of endocrine markers, giving anindirect estimate of the reserve size. Concerning the latterapproach, the measurement of FSH and inhibin in serum canhelp to make a diagnosis, since the combination of high FSHand low inhibin concentrations are indicative of low numbersof growing follicles, yet they are not the most accurate markers.It is now well established that AMH is the best endocrinemarker of the population of small antral follicles in humans[46, 47] as well as in ruminants [37, 48, 49] (Fig. 4). The closerelationship existing between AFC and AMH is explained bythe facts that 1) AMH expression in female mammals is strictlyrestricted to granulosa cells of growing follicles and 2) thegranulosa cells of the largest preantral and the small antralhealthy growing follicles express the highest amounts of AMHin ovaries [50].

In the ovaries of young adults, both direct (AFC) andindirect (AMH concentrations) estimations of the size of thereserve of small antral follicles have highlighted littlenumerical change with time. For example, in cows, a highindividual repeatability of both AFC [35] and AMH plasmaconcentrations [37, 51, 52] has been observed over severalmonths and up to 1 yr. Moreover in the goat, a species that

presents a seasonal breeding activity, plasma AMH concentra-tions show little change with season [49]. Therefore, it can beassumed that the population of small antral follicles remains ina dynamic balance, with exits numerically compensated byentries, and that the equilibrium size of this dynamic reserve isspecific to each individual.

The mechanisms regulating the size of the dynamic reserveare not fully understood. Interestingly, in young adult heifers,animals with low numbers of primordial follicles also havehighly reproducible low AFC [53]. Moreover, when consider-ing populations of individuals with different ages, highlysignificant correlations have been found between the numbersof primordial follicles and AFC in mice [54] and humans [55].Altogether, these observations strongly indicate that the pre-established and dynamic reserves are functionally related. It isamazing that the size of the reserve of primordial folliclesformed early in life can be related to, and even seems todetermine, the ovarian activity of the adult. In fact, thisrelationship likely implies that finely tuned regulation of germand somatic cell survival, proliferation, and/or differentiationinvolves the same factors and the same molecular mechanismsfrom the formation of the primordial follicles up to the stagewhen the follicles become gonadotropin dependent and enterterminal development. From the available data, two mainsignaling pathways play major roles in all these processes. Thefirst one is the PTEN/PI3K/PDPK1 (previously known asPDK1)/AKT1 (previously known as AKT or PKB) signalingpathway, which regulates germ cell survival, as well asfollicular growth activation [56–59] and follicle growth [60].This pathway is activated by various hormones, growth factors,and cytokines. Among them, insulin, insulin-like growthfactors (IGF), and KIT ligand are known to be key regulatingfactors of the survival and differentiation of germ and somaticovarian cells [18, 61, 62]. The second important signalingpathway involves SMAD (vertebrate homolog of MothersAgainst Decapentaplegic [MAD]) transcription factors, whichare activated by factors of the transforming growth factor-b(TGFB) super family (i.e., BMP and AMH for the SMAD1/5/8pathway), and the TGFB and activins for the SMAD2/3pathway. It orchestrates the formation and development offollicles under the control of oocyte- (BMP15, GDF9) andsomatic cell (BMP2, BMP4, AMH, activins) -derived factors,which have been the subject of many recent investigations [63–67], including a multiscale modeling approach focused on thecoupling between somatic cell kinetics and oocyte growth [68].The main endocrine and paracrine mechanisms regulating the

FIG. 4. Relationship between the numbers of small antral follicles and plasma AMH concentrations in three ruminant species. A) In Holstein cows (n¼18), follicles were counted by ovarian ultrasonography. B) In Saanen goats (n¼ 15) and (C) in Merinos d’Arles ewes (n¼ 12), follicles were counted afterdissection from ovaries recovered from animals at slaughter. AMH concentrations were determined in plasma samples recovered at the time of follicularcounting. Each square represents data from one animal. The correlation coefficients in cows, goats, and sheep were 0.79 (P , 0.001), 0.89 (P , 0.001),and 0.62 (P , 0.05), respectively (Estienne and Fabre, unpublished results in C, and data in A and B adapted from Monniaux et al. 2013 [50] withpermission).

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dynamic reserve of small antral follicles are summarized inFigure 5.

For each individual, the size of the dynamic reserve couldresult from the fine tuning of these regulations. Even if a longerinterval from calving to conception has been reported in cowswith low AFC [69], it can be noted that female fertility isgenerally compatible with a large range of AFC values in eachspecies. Moreover, the number of ovulations (or naturalovulation rate) that is characteristic of each species, breed, orstrain does not depend on the size of the dynamic reserve.

Rather, it results from mechanisms of follicular selection that

have been described elsewhere [12, 70] and are outside the

scope of this review. The existence of a large between-

individual variability in the number of small antral follicles is

generally masked in normal conditions, but is revealed when

individuals are subjected to assisted reproductive technologies.

For instance, in ruminants, animals with low AFC are poor

embryo donors in the protocols of embryo production [42, 44,

49, 52, 71].

FIG. 5. Regulations of the transitions between the follicular reserves. A) Endocrine and paracrine regulations of the dynamic reserve of small antralfollicles: main current hypotheses. The dynamic reserve is filled by growing follicles originating from the pre-established reserve of primordial follicles,which is gradually emptied by both follicle growth activation and follicle degeneration. The transition between the reserves is regulated by various growthfactors, acting in a paracrine or/and endocrine way. Among them, activins and BMP are mitogenic and antiapoptotic factors for granulosa cells, whereasAMH produced by the granulosa cells of the large preantral and the small antral follicles restricts primordial follicle activation and modulates follicledevelopment. During the transition between the reserves, the development of each growing follicle also depends, importantly, on the molecular dialogestablished between the oocyte that produces the bone morphogenetic factors GDF9 and BMP15 and its surrounding granulosa cells that secrete theoocyte growth promoting factor Kit ligand (KITLG). Insulin and IGF, acting mostly in an endocrine way, can support the development of the growingfollicles by sensitizing their granulosa and theca cells to FSH and luteinizing hormone (LH), respectively. The dynamic reserve is emptied by the cyclicFSH-orchestrated entry of follicles in the follicular waves of terminal development. At this stage, a variety of intrafollicular regulation, involvingparticularly BMP, the inhibin/activin system, and the IGF system (IGFs and their binding proteins) can, importantly, modulate the gonadotropin-dependentdifferentiation of granulosa and theca cells, thus determining the fate of each antral follicle (i.e., atresia or development towards ovulation). B) Between-individual variations in the reserve sizes and transition rate between the reserves in a mono-ovulating species. One ovary per individual is schematized.Ovaries with a high reserve of primordial follicles display a high AFC. They waste their follicles as a result of the high recruitment and atresia rates of theprimordial and growing follicles, respectively. In contrast, both processes are markedly slowed down in ovaries with a small-sized reserve, whileintermediate situations with medium-sized reserves and moderate rates can also be encountered. The size of the dynamic reserve results from the finetuning of the regulations described in A. The number of ovulations (one in this example) is independent of the size of the reserve.

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OVARIAN RESERVE EXHAUSTION: GENETICDETERMINISM

Both ovarian reserves are exhausted at menopause inhumans and some primates, unlike in most mammals (Figs. 1and 3). In some pathological situations, a premature depletionof follicles is observed, which affects approximately 1%–2% ofwomen under the age of 40 yr and results in POI. Some geneticcomponent has been evidenced among the possible causes ofPOI [72].

Various natural or experimentally induced mutations ingenes encoding the ligands, receptors, or signaling effectors ofthe PTEN/PI3K/PDPK1/AKT1 or the SMAD signalingpathways can accelerate the exhaustion rate of the ovarianreserves and cause POI. The deletion of Pten, PDPK1,Ribosomal protein S6 (Rps6), or Forkhead box O3 (Foxo3,previously known as Foxo3A) in mouse oocytes induces adramatic activation and/or premature loss of the primordialfollicles, resulting in POI around the onset of sexual maturity[56–58]. In regard to the SMAD pathway, no data are availableon the exhaustion rate of the reserve of primordial follicles inSmad mutant mice [66]. However, mutations in some factors ofthe TGFB super family have been shown to affect the transitionof growing follicles between the two follicular reserves (Table1). In mice, the deletions of Amh [73, 74] or Inha [75–77], aswell as the overexpression of Bmp15 [78], accelerate thistransition, resulting in a premature depletion of the reserve ofprimordial follicles. In contrast, the deletion of Gdf9 blocks thegrowth of primary follicles, causing primary sterility [79], anda similar phenotype is observed in sheep carrying homozygousGDF9 mutations responsible for a loss of function of theprotein [80, 81]. Interestingly, the transition of folliclesbetween the reserves is not impaired in mice when Bmp15 isdeleted [82], whereas it is completely blocked in sheep carryinghomozygous loss-of-function mutations in BMP15 [83–86],indicating that the involvement of BMP15 in the control offollicular development is species specific. In humans, variousmutations in BMP15 and, to a lesser extent, in GDF9 andINHA, have been found to be associated with POI [72, 87, 88],and genetic variants of AMH and its receptor AMHR2 areassociated with different ages at menopause [89, 90],confirming the importance of these factors in the lifespan ofthe ovarian reserves.

As shown in the mouse genetic models described above, it isunusual to have beneficial effects of mutations on AFC withoutadverse side effects on the ovarian reserves in the long term(Table 1). However, in sheep carrying either heterozygous loss-of-function BMP15 mutations or the BMPR1BQ249R partial loss-of-function mutation, high AFC is not accompanied by thepremature depletion of the reserves [84, 91–93]. Rather, in thelatter genetic model, despite the presence of a low reserve ofprimordial follicles at birth, the ovarian reserves at 5 yr of ageare higher in ewes carrying the mutation, suggesting thatattenuation of the intraovarian signaling pathway of BMPs mayin fact be a successful means of limiting follicle consumption,hence preserving reproductive longevity and fertility [93].

Another promising way to increase the lifespan of theovarian reserves was proposed from the dramatic prolongationof the ovarian lifespan observed after Bax deletion in mice,suggesting that the mitochondrial factors of the Bcl2 family orthe death effector caspase cascade could modulate numericalchanges in the ovarian reserve through their pro- orantiapoptotic actions [94, 95]. However, up to now, nomutations in apoptosis regulator genes have been found to beassociated with the exhaustion rate of the ovarian reserves inhumans, so their importance in POI syndrome remainshypothetical.

DYNAMICS OF THE FOLLICULAR RESERVES DURINGLIFE

The reserve of primordial follicles is progressively depletedthroughout life, but the activation rate of primordial folliclesvaries as the ovary ages. In the young ovaries, the follicles thatwere formed first during oogenesis in the deepest part of thecortex massively activate first (Fig. 2), and are then relayed byfollicles located in outer parts of the cortex, and follicularactivation rate is progressively reduced [21]. Based on astatistical study of the human ovarian reserve size fromconception to menopause, the rate of follicular activation hasbeen estimated to increase from birth until approximately age14 yr, then to decrease toward menopause [31]. However, inanother study, an accelerated decay rate of the reserve wasfound before menopausal transition, resulting from enhancedfollicular activation rate in aging ovaries [96]. With aging, thedecline in primordial follicle numbers parallels the decrease in

TABLE 1. Experimental and natural mutations in genes encoding factors of the TGFB super family and their effects on the size of the two follicularreserves, the transition rate between them, and the occurrence of a premature depletion.

Animal species and genetic modelFirst

reserve* Transition rateSecondreserve$

Prematuredepletion� References

Mouse, Amh�/� Normal Accelerated rate andincreased atresia

High Yes [73, 74]

Mouse, Inha�/� Normal Accelerated High ND [76]Mouse, Bmp15�/� Normal Fairly normal Normal to low Reported in

some animals[82]

Mouse, Bmp15 overexpression Normal Accelerated rate andincreased atresia

Normal Yes [78]

Mouse, Gdf9�/� Normal No transition 0 / [79]Mouse, Gdf9þ/� Normal Normal Normal No [79]Mouse, Inha �/�Gdf9�/� Normal Accelerated Normal ND [75, 77]Mouse, Bmp15�/�Gdf9þ/� Normal Increased atresia Low Yes [82]Sheep, BMP15 homozygous loss-of-function mutations Normal No transition 0 / [83–85]Sheep, GDF9 homozygous loss-of-function mutations Normal No or few transition 0 to very low / [80, 81]Sheep, BMP15 heterozygous loss-of-function mutations Normal Unknown High No [91, 92]Sheep, BMPR1BQ249R partial loss-of-function mutation Low Slow High No [84, 92, 93]

* The pre-established reserve of primordial follicles.$ The dynamic reserve of small antral follicles.� ND, not determined, due to the premature death of animals by tumor development.

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size of the dynamic reserve of small antral follicles [97–99] andAMH blood concentrations [100, 101].

These numerical changes are accompanied by changes inthe quality of the follicles of the dynamic reserve. In ruminants,oocytes recovered from small antral follicles before pubertydisplay a suboptimal developmental competence in in vitroembryo production systems compared to oocytes from adults,and this poor oocyte quality is associated with differences inRNA stores [102]. Similarly, in humans, during childhood andadolescence there is a high proportion of morphologicallyabnormal follicles that show reduced capacity for in vitrogrowth [103]. In mice, the loss of follicles during theprepubertal period has been proposed to be an active processnecessary to eliminate the follicles that are in excess,containing poor quality oocytes [104]. Compromised oocytequality also occurs in aged ovaries [98, 105, 106], due tounfavorable microenvironment (vascularization defects, oxida-tive stress, influence of toxic compounds), leading to a highprevalence of aneuploidy [107]. During the premenopausal

period, the high FSH endocrine levels, resulting from thedecline in the number of inhibin-secreting follicles, may alsohave deleterious effects upon follicle and oocyte quality [108,109], while increasing multiple ovulations [110]. Thesequalitative and quantitative follicular changes contribute tofemale infertility and constitute an important limit to thesuccess of assisted reproductive technologies in premenopausalwomen [111]. The dynamics of the follicular reserves and theassociated endocrine changes throughout life are summarizedin Figure 6.

THE MANAGEMENT OF THE OVARIAN RESERVES:FUTURE DIRECTIONS

As described above, the existence of a large, between-individual variability in AFC constitutes the most limitingfactor in the success of assisted reproductive technologies [36,38–45]. Moreover, between-individual variability in thetransition rates between ovarian reserves and in the gonado-tropin-regulated development of the antral follicles toward

FIG. 6. Schematic representation of the ovarian reserves, including their exhaustion dynamics and associated endocrine changes in AMH, FSH, andinhibin throughout life. The reserves are represented by two tanks, the first containing the primordial follicles, which can either degenerate after activationand entry into growth or develop and enter the second tank, containing the small antral, gonadotropin-responsive follicles. The endocrine changes arerepresented by a balance between FSH and inhibin. The fulcrum of the balance is AMH, the endocrine levels of which are proportional to the number ofhealthy small antral follicles present in the second tank. Left panel: in early life, the first tank contains a high number of primordial follicles; most of theprimordial follicles, which were formed first during oogenesis (red balls), are also the first to be activated and to enter the second tank. At that point, thereis a burst of growing follicles, mirrored by high AMH endocrine levels and associated with high and low inhibin (INH) and FSH endocrine levels,respectively. These growing follicles enclose oocytes with suboptimal developmental competence after in vitro fertilization. In vivo, all growing follicleswill degenerate by atresia before or after entering the second tank. Middle panel: in the adult age period, the number of primordial follicles has decreasedand the first tank contains follicles formed later during oogenesis (green balls), which are able to give rise to growing follicles containing oocytes withoptimal developmental competence. The second tank has reached an equilibrium size (with exits broadly compensated by entries), which is specific toeach individual, correlated with the size of the first tank and mirrored by AMH endocrine levels. The antral follicles present in the second tank can enterterminal development, and some of them will be selected for ovulation. Right panel: in the presenescence period, the first tank contains a low number ofaged primordial follicles (black balls), which are able to give rise to growing follicles containing low-quality oocytes. The second tank has a reduced size,associated with low to undetectable AMH endocrine levels that can, therefore, enhance follicular growth activation. The high FSH endocrine levelspresent at this stage could be responsible for the increased occurrence of multiple ovulations in mono-ovulating species during aging (period ofpremenopause in women).

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ovulation can account for important differences betweenfemales in fertility and longevity of reproductive function.This variability likely has strong genetic components, but thecrucial question to answer now is: how to optimize thedynamics of the follicular reserves?

One strategy is to target the PTEN/PI3K/PDPK1/AKT1pathway, particularly by acting upon metabolism throughenvironmental and hormonal factors. This can apply to theimprovement of embryo production in animals, since increasesin insulin and IGF1 consecutive to a short-term feedingregimen with a high energy intake (flushing) have been shownto increase AFC and the follicular responses to gonadotropinsin ruminants [112, 113]. This could also be applied to humanclinics, particularly to restore the fertility of women withpolycystic ovary syndrome (PCOS), a common hyperandro-genic disorder often associated with metabolic alterations,chiefly insulin resistance and obesity. The ovaries of PCOSwomen are characterized by a slow transition betweenfollicular reserves, blocking of antral follicular development,and anovulation despite a high AFC [114, 115]; interestingly,improving insulin sensitivity by dietary therapy and exerciseregimens, or by administrating insulin-sensitizing drugs [116–118], can mobilize the growing follicles and restore ovulation.In a very interesting complementary perspective, new treat-ments for PCOS patients have been proposed recently toreinforce the activation of AKT1 signaling; they consist of thelocal disruption of the Hippo signaling pathway regulated byactin polymerization, as AKT1 and Hippo pathways can act inconcert to regulate follicular growth [119].

An alternative strategy is to target the BMP/AMH/SMADpathway. For example, in sheep, it has been recently shownthat the knockdown of AMH bioactivity by active immuniza-tion leads to a decline in the numbers of small growingfollicles, but an increase in AFC and ovulation numbers [120].In contrast, the administration of AMH would lower folliclegrowth activation and preserve more follicles for developmentlater in life, thereby causing a delay in the onset of menopause,as hypothesized in a lifelong model for the female reproductivecycle [121]. From these studies, AMH knockdown may haveshort-term therapeutic value in women who respond poorly toovarian stimulation, whereas AMH treatment might preservethe follicular reserves in the long term. From the sheep geneticmodels described above [84, 91–93], targeting the signalingpathway of BMPs by immunization or specific antagonists mayalso be a promising way to increase both AFC in the short termand reproductive longevity in the long term.

Moreover, the discovery of epigenetic regulation hashighlighted the importance of developmental programmingfor ovarian function. In sheep, testosterone exposure duringfetal life causes reproductive and metabolic disruptions,reproducing the human PCOS syndrome in adult animals[122]. This experimental situation might reflect, at least partly,some pathological cases, since endocrine disorders of PCOSoccur in female infants born to mothers with PCOS [115]. Asanother example, maternal nutrition and health during gestationin cows have been shown to influence AFC in prepubertal andadult offspring [123, 124]. All these observations indicate thatinteractions between genes and the maternal-fetal hormonalenvironment at the time of the formation of the reserve ofprimordial follicles may program postnatal ovarian function.

In conclusion, the pre-established reserve of primordialfollicles and the dynamic reserve of small antral follicles arefunctionally related and under both genetic and environmentalcontrol. From the recent improvements in the methodsavailable to estimate the reserve size, and our increasedknowledge of the cellular processes and signaling pathways

regulating their dynamics, new perspectives have been openedto improve the management of the ovarian reserves in the nearfuture.

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