Pathogenesis and epidemiology of precocious puberty. Effects of exogenous oestrogens

Precocious puberty is generally defined as the appearance of secondary sex characteristics before age 8 years in girls (or menarche before age 9 years) and before 9 years in boys. The overall incidence of sexual precocity is estimated to be 1:5,000 to 1:10,000 children. The female-to-male ratio is approximately 10:1. In addition to the psychosocial disturbances associated with precocious puberty, the premature pubertal growth spurt (with less time for prepubertal growth) and the accelerated bone maturation result in reduced adult height. Precocious puberty may be gonadotrophin-dependent [i.e. of central origin with premature activation of the gonadotrophin-releasing hormone (GnRH) pulse generator] or gonadotrophin-independent (i.e. peripheral where the GnRH pulse generator is suppressed). This can be determined by GnRH testing. The pathophysiology is the basis for different diagnostic and therapeutic strategies, i.e. in the first case a stimulated LH/FSH ratio >1 and suppressive treatment with GnRH agonists (e.g. in hypothalamic hamartoma), and in the second decreased gonadotrophins and removal or suppression of the endogenous or exogenous sex steroid source (e.g. congenital adrenal hyperplasia). While several cases of gonadotrophin-independent precocious puberty due to oestrogen exposure via the transdermal, oral, or inhalative route have been reported, no case is known with the development of subsequent secondary central precocious puberty. Food contamination with oestrogens is theoretically possible, but would most probably be sporadic and, thus, would not lead to precocious puberty. As steroid hormones in meat production are banned in the European Union, no data on the impact of environmental oestrogenic substances on human maturation are currently available. In conclusion, the risk for children to develop precocious puberty through exposure to oestrogens (or androgens) in the environment or in food is very low. Nevertheless, studies of the effects of defined environmental oestrogenic substances on the human reproductive system and on pubertal development are warranted.     

The role of proteins of the transforming growth factor-beta superfamily in the intraovarian regulation of follicular development

Ovarian follicular development occurs in a hierarchical manner with each follicle having a unique biochemical composition at any moment in time. It has long been understood that a precise coordination between the growth and maturation of the oocyte and adjacent follicular cells (i.e. somatic cells) is essential in order to produce an oocyte that is fully competent to undergo fertilization and embryo development. In addition to the critical endocrine signalling pathways between the hypothalamus, pituitary and ovary, it is now evident that the oocyte itself is important in influencing the microenvironment of the developing follicle by regulating, via paracrine and autocrine mechanisms, its own maturation as well as somatic cell proliferation, differentiation and ovulation rate. Several of the key oocyte-derived regulating factors are members of the transforming growth factor-beta (TGF-beta) superfamily and to date the best understood are growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and BMP6. Significant species differences appear to exist in the relative importance of these growth factors and much remains to be elucidated about their roles in the human ovary. More information on the roles of these factors during ovarian follicular development is likely to advance new therapeutic applications for management of fertility as well as our understanding of how better to assess oocyte quality.     

Effects of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its influence on oocyte developmental capacity

There has been an improvement in the blastocyst rates achieved following in-vitro embryo production that can largely be attributed to improved embryo culture conditions based on an increased knowledge of the in-vivo environment, as well as the metabolic needs of the embryo. Despite this, in-vitro oocyte maturation (IVM) conditions have remained largely unchanged. Within the antral follicle, numerous events affect oocyte maturation and the acquisition of developmental competency, including: interactions between somatic cells of the follicle (in particular cumulus cells) and the oocyte; the composition of follicular fluid; and the temperature and vascularity of the follicular environment. Many of these factors change with follicle size and oocyte growth. In contrast, culture conditions for IVM are based on somatic cells that often do not reflect the follicular environment, and/or have complex compositions or additives such as macromolecule supplements that are undefined in nature. Metabolites included in media such as glucose, pyruvate, oxygen and amino acids have been shown to have differential influences on oocyte maturation and competency. Manipulation of these factors and application of gained knowledge of the in-vivo environment may result in improved in-vitro oocyte maturation and overall in-vitro embryo production.     

Impact of endocrine disruptor chemicals in gynaecology

The potential hazardous effects that estrogen- and androgen-like chemicals may have both on wildlife and human health have attracted much attention from the scientific community. Endocrine disruptors (EDCs) are chemicals that have the capacity to interfere with normal signalling systems. EDCs may mimic, block or modulate the synthesis, release, transport, metabolism and binding or elimination of natural hormones. Even though potential EDCs may be present in the environment at only very low levels, they may still cause harmful effects, especially when several different compounds act on one target. EDCs include persistent pollutants, agrochemicals and widespread industrial compounds. Not all EDCs are man-made compounds; many plants produce substances (phytoestrogens) that can have different endocrine effects either adverse or beneficial in certain circumstances. Natural substances such as sex hormones from urban or farm wastes can become concentrated in industrial, agricultural and urban areas; thus, such wastes may be considered potential 'EDCs' for humans and/or wildlife. Much attention has focussed on changing trends in male reproductive parameters in relation to EDC exposure; however, studies on the female reproductive system have been less comprehensive. We have focussed this article on four major aspects of female reproductive health: fertility and fecundability, endometriosis, precocious puberty and breast and endometrial cancer.     

GPR54 and kisspeptin in reproduction

Kisspeptins, the peptide products of the KiSS-1 gene, were identified in 2001 as natural ligands of the previously orphan G protein-coupled receptor, GPR54. They include, among others, metastin and kisspeptin-10. The known biological functions of kisspeptins were initially restricted to their ability to suppress tumour metastasis, hence the name of metastin. However, in late 2003, two groups independently reported that loss-of-function mutations of the GPR54 gene are linked to absence of puberty onset and hypogonadotrophic hypogonadism in humans--a phenotype that was reproduced in GPR54-null mice. Those seminal observations revealed a totally unexpected, fundamental role of the KiSS-1/GPR54 system in control of puberty and reproductive function and boosted an extraordinary interest for the characterization of these novel facets of kisspeptin physiology. Indeed, in the last 2 years, metastin and kisspeptin-10 have been demonstrated as very potent stimulators of the gonadotrophic axis, in a number of species and through different routes of administration. In addition, the hypothalamic KiSS-1/GPR54 system has been proven as an essential gatekeeper of GnRH neurons, involved in their activation at puberty and their regulation by gonadal steroids and (probably) metabolic factors. This review comprehensively examines the experimental evidence obtained to date supporting a pivotal role of kisspeptins and GPR54 in the control of reproduction.     

Insulin-like growth factor binding protein-1 (IGFBP-1): a multifunctional role in the human female reproductive tract

Insulin-like growth factor-1 (IGFBP-1) is particularly important in human female reproductive physiology, where it is involved with other factors in a complex system which regulates menstrual cycles, puberty, ovulation, decidualization, implantation and fetal growth. This has implications for clinical obstetrics and gynaecology, where there is evidence for a pathophysiological role for IGFBP-1 in pre-eclampsia, intrauterine growth restriction, polycystic ovarian syndrome and trophoblast and endometrial neoplasms.     

The sensitivity of the child to sex steroids: possible impact of exogenous estrogens

The current trends of increasing incidences of testis, breast and prostate cancers are poorly understood, although it is assumed that sex hormones play a role. Disrupted sex hormone action is also believed to be involved in the increased occurrence of genital abnormalities among newborn boys and precocious puberty in girls. In this article, recent literature on sex steroid levels and their physiological roles during childhood is reviewed. It is concluded that (i) circulating levels of estradiol in prepubertal children are lower than originally claimed; (ii) children are extremely sensitive to estradiol and may respond with increased growth and/or breast development even at serum levels below the current detection limits; (iii) no threshold has been established, below which no hormonal effects can be seen in children exposed to exogenous steroids or endocrine disruptors; (iv) changes in hormone levels during fetal and prepubertal development may have severe effects in adult life and (v) the daily production rates of sex steroids in children estimated by the Food and Drug Administration in 1999 and still used in risk assessments are highly overestimated and should be revised. Because no lower threshold for estrogenic action has been established, caution should be taken to avoid unnecessary exposure of fetuses and children to exogenous sex steroids and endocrine disruptors, even at very low levels.     

Current perspective on primordial follicle cryopreservation and culture for reproductive medicine

Mature oocytes are rare and precious cells. A technology which generates larger numbers would be very welcome in clinical practice, animal production technology and research. Since de-novo formation of female germ cells has ceased by the time of birth, the most attractive strategy, in theory, is to harvest and culture primordial follicles, the most abundant stage in the ovary at all ages. So far, there has been more success with cryopreservation of primordial follicles than with culture, and frozen-thawed ovarian tissue grafts have restored fertility to a number of species after oophorectomy. However, in-vitro development of isolated follicles is not sustained beyond the primary follicle stage. To meet their requirements for growth, metabolism and differentiation, a multistage protocol will probably be required for the prolonged period of development to maturity. The mouse is the only model, to date, in which a live offspring has ever been produced after growing follicles completely in vitro. A triple-stage process was required, involving culture of ovarian explants followed by isolation of granulosa-oocyte complexes and, finally, suitable conditions for completing meiotic maturation. Achievement of this goal for the larger and more slowly developing follicles from human and farm animal ovaries is still a remote possibility.     

The importance of folate, zinc and antioxidants in the pathogenesis and prevention of subfertility

Current treatments of subfertile couples are usually empiric, as the true cause of subfertility often remains unknown. Therefore, we outline the role of nutritional and biochemical factors in reproduction and subfertility. A literature search was performed using MEDLINE, Science Direct and bibliographies of published work with both positive and negative results. The studies showed that folate has a role in spermatogenesis. In female reproduction, folate is also important for oocyte quality and maturation, implantation, placentation, fetal growth and organ development. Zinc has also been implicated in testicular development, sperm maturation and testosterone synthesis. In females, zinc plays a role in sexual development, ovulation and the menstrual cycle. Both folate and zinc have antioxidant properties that counteract reactive oxygen species (ROS). Thiols, such as glutathione, balance the levels of ROS produced by spermatozoa and influence DNA compaction and the stability and motility of spermatozoa. Oocyte maturation, ovulation, luteolysis and follicle atresia are also affected by ROS. After fertilization, glutathione is important for sperm nucleus decondensation and pronucleus formation. Folate, zinc, ROS and thiols affect apoptosis, which is important for sperm release, regulation of follicle atresia, degeneration of the corpus luteum and endometrial shedding. Therefore, the concentrations of these nutrients may have substantial effects on reproduction. In conclusion, nutritional and biochemical factors affect biological processes in male and female reproduction. Further research should identify pathways that may lead to improvements in care and treatment of subfertility.     

Mouse genetics provides insight into folliculogenesis,fertilization and early embryonic development

After colonization of the gonad, mouse female germ cells enter into the prophase of the first meiotic division as a mid-gestational hallmark of gender. Perinatally, oocytes interact with granulosa cells to form primordial follicles which, with cyclic periodicity, enter into a 3-week growth phase that culminates in meiotic maturation and ovulation. Successful fertilization in the oviduct results in the onset of embryogenesis. Genes expressed in oocytes encode maternal factors that control many of these developmental processes. The establishment of mouse models in which specific genes have been disrupted offers robust insights into molecular mechanisms that control oogenesis, folliculogenesis, fertilization and early embryogenesis. Although relatively few developmental circuits have been characterized in genetic detail, the ongoing revolution in mouse genetics holds great promise. These model systems provide novel information into the molecular basis of the pathways required for oocyte-specific processes as well as for interactions with the temporally changing environment of female germ cells. The similarities between the mouse and human genomes provide assurance that this knowledge will rapidly translate into a better understanding of human reproduction.     

Oocyte--granulosa cell interactions

In the past, different protocols of ovulation induction, aimed to overcome problems of anovulatory infertility in humans, have been developed during IVF programmes. However, administration of exogenous hormones may cause severe health problems, e.g. ovarian hyperstimulation syndrome. To overcome this problem an attractive alternative is to develop in-vitro systems that allow follicle and oocyte growth and maturation. This paper reviews the current status of research on oocyte-granulosa cell interactions and on the autocrine and paracrine factors involved in follicle development. The ovarian follicle is a morphological and functional unit in which the somatic and germ cell components are intimately associated and interdependent. The co-ordinate development of follicle and oocyte leads to a number of modifications in the growing oocyte necessary for the acquisition of competence to mature correctly and to undergo fertilization and embryo development. The search for the optimal culture conditions and the correct balance of hormones necessary to obtain a fertilizable oocyte in vitro is extremely important for clinical and agricultural applications.     

Cryopreservation of oocytes from pre-antral follicles

Cryobiology is a very important tool in reproductive biology. Research in this area focuses on the possibility of restoring fertility in women with reproductive problems or after cancer treatments. Another goal is to establish a genetic resource bank for endangered or commercially important animal species. Cryopreservation of oocytes from pre-antral follicles has been studied during the past decade. Procedures can be divided between the cryopreservation of either ovarian tissue or isolated follicles. Most studies describe a slow freezing/rapid thawing protocol to cryopreserve ovarian fragments. Histology shows that the follicles maintain their morphological integrity, and transplantation of ovarian tissue demonstrates that the follicles can restart their growth and eventually ovulate. Some research groups have obtained offspring using this procedure in mice and sheep. With regard to the cryopreservation of isolated follicles, the few studies reported in this area used the same freezing protocol, and some of them described follicular growth using in-vitro culture. The best result was obtained in mice, with animal birth after follicular cryopreservation and culture. However, additional studies are necessary for a better understanding of the events during follicular cryopreservation and to establish a standard protocol for ovarian transplantation or follicle culture.     

The use of LH activity to drive folliculogenesis: exploring uncharted territories in ovulation induction

LH plays critical roles in the control of folliculogenesis and ovarian function in humans. LH activity administration during gonadotrophin ovulation induction can enhance ovarian response and optimise treatment. More specifically, LH activity (both LH and low-dose hCG) can support the growth and stimulate the maturation of larger ovarian follicles as a result of specific granulosa cell receptors that develop after a few days of FSH priming. This action of LH is independent of FSH, and it has been shown recently that the last stages of follicular development can be supported by sole administration of LH activity in the form of low-dose hCG, without causing premature luteinization. Reproductively competent oocytes and pregnancy can be obtained with this regimen. Furthermore, LH activity is capable of reducing the development of small ovarian follicles (<10 mm) that may predispose patients to developing complications such as the ovarian hyperstimulation syndrome. Thus, better understanding of the dynamics and mechanisms that control human folliculogenesis and a more rational and selective use of LH activity administration may allow a reduction in cost and increased safety, while maintaining a high efficacy of the ovulation induction regimens used in assisted reproduction.     

Clinical management of low ovarian response to stimulation for IVF: a systematic review

Poor response is not a rare occurrence in ovarian stimulation. Although not fully accepted, the most dominant criteria for poor ovarian response are small numbers of follicles developed or oocytes retrieved, and low estradiol (E2) levels after the use of a standard stimulation protocol. There is no ideal predictive test as the poor responder is revealed only during ovulation induction; however, increased levels of day 3 FSH and E2 as well as decreased levels of inhibin B can be used to assess ovarian reserve. Several protocols have been proposed for clinical management of low ovarian response in IVF. Although high doses of gonadotrophins have been used by the vast majority of authors, results have been controversial and prospective randomized studies have shown little or no benefit. The few available relevant studies do not indicate that recombinant FSH improves outcome. Flare-up GnRH agonist protocols (including all dosage varieties) produce better results than standard long luteal protocols. Luteal initiation GnRH agonist 'stop' protocols were shown to improve ovarian response according to prospective studies with historical controls, but this was not confirmed by well-designed prospective, randomized, controlled studies. The few available data obtained with GnRH antagonists have not shown any benefits. Adjuvant therapy with growth hormone (GH) or GH-releasing factors results in no significant improvement. The use of corticosteroids reduces the incidence of poor ovarian response in women undergoing IVF treatment. The limited data obtained with nitric oxide donors are encouraging. Pretreatment with combined oral contraceptives prior to stimulation may help ovarian response. No benefit was observed with standard use of ICSI or assisted hatching of zona pellucida. Finally, natural cycle IVF has produced results which are comparable with those obtained with stimulated cycles in true poor responders. Well-designed, large-scale, randomized, controlled trials are needed to assess the efficacy of these different management strategies.     

Neonatal exposure to potent and environmental oestrogens and abnormalities of the male reproductive system in the rat: evidence for importance of the androgen-oestrogen balance and assessment of the relevance to man

The effects on reproductive tract development in male rats, of neonatal exposure to potent (reference) oestrogens, diethylstilboestrol (DES) and ethinyl oestradiol (EE), with those of two environmental oestrogens, octylphenol and hisphenol A were systematically compared. Other treatments, such as administration of a gonadotrophin-releasing hormone antagonist (GnRHa) or the anti-oestrogen tamoxifen or the anti-androgen flutamide, were used to aid interpretation of the pathways involved. All treatments were administered in the neonatal period before onset of puberty. The cellular sites of expression of androgen receptors (AR) and of oestrogen receptor-alpha (ERalpha) and ERbeta were also established throughout development of the reproductive system. The main findings were as follows: (i) all cell types that express AR also express one or both ERs at all stages of development; (ii) Sertoli cell expression of ERbeta occurs considerably earlier in development than does expression of AR; (iii) most germ cells, including fetal gonocytes, express ERbeta but not AR; (iv) treatment with high, but not low, doses of potent oestrogens such as DES and EE, induces widespread structural and cellular abnormalities of the testis and reproductive tract before puberty; (v) the latter changes are associated with loss of immunoexpression of AR in all affected tissues and a reduction in Leydig cell volume per testis; (vi) none of the effects in (iv) and (v) can be duplicated by treating with high-dose octylphenol or bisphenol A; (vi) none of the reproductive tract changes in (iv) and (v) can be induced by simply suppressing androgen production (GnRHa treatment) or action (flutamide treatment); and (vii) the adverse changes induced by high-dose DES (iv and v) can be largely prevented by co-administration of testosterone. Thus, it is suggested that many of the adverse changes to the testis and reproductive tract induced by exposure to oestrogens result from a combination of high oestrogen and low androgen action. High oestrogen action or low androgen action on their own are unable to induce the same changes.     

Genetic studies to identify genes underlying menopausal age

Menopausal age is important as a retrospective marker for ovarian senescence, an early menopausal age is associated with an increased risk of cardiovascular diseases and osteoporosis, whereas a later menopausal age has been associated with an increased risk of breast cancer. The worldwide average for age at natural menopause is approximately 51 years and is more or less normally distributed with a range roughly between 40 and 60 years. Environmental factors explain only a small part of the variance and it has been proposed that genetic factors are the main source of variation. Menopausal age may be considered a continuous complex trait. Complex traits are defined as traits that are influenced by both multiple genetic and environmental factors. A category of complex traits comprises those that are measured on a continuous scale. The genomic loci that make up the genetic component are called 'quantitative trait loci' or QTLs. The first linkage study on menopausal age suggests that the involvement of the X-chromosome may not be limited to premature ovarian failure (POF), but may influence the broader spectrum of menopausal age. A potentially new locus for variation in menopausal age was allocated to chromosome 9. Further studies need to identify new candidate genes to help unravel the pathophysiology of menopausal age. It is becoming increasingly clear that, in any speciality, it should be acknowledged that genetic factors are involved in many traits and that uncovering these factors may provide insight into pathogenesis and ultimately advance prevention and treatment of disease. In this review we discuss methods and basic principles of gene finding for such traits, exemplified by menopausal age as phenotype. Furthermore, we give an overview of the state of the art of candidate gene studies and linkage studies.     

Perinatal outcome and developmental studies on children born after IVF

Since the first birth after IVF, many scientific papers have been published on the technical aspects of the IVF procedure, but few studies have addressed the issue of the perinatal outcome of IVF pregnancies and of the children's development and well-being. A high rate of adverse outcome has been demonstrated in a large group of IVF pregnancies. Prematurity, low birth weight and perinatal mortality are higher than in the general population. The majority of these complications are related to multiple births, but they are also found in singleton pregnancies. An analysis of the multiple risk factors involved in these complications is needed. The infertile status of IVF patients clearly plays a role in the risk of adverse outcome. Age and parity may be important factors. The role of IVF itself has not been demonstrated convincingly. The effect of ovarian stimulation deserves further study. Most of the studies published on the follow-up of IVF children are reassuring, but it is clear that these studies are not sufficient to eliminate without doubt any adverse effects on the well-being of IVF children. All IVF pregnancies should be followed with great care, not because they are more precious than spontaneous pregnancies, but because they are exposed to an increased risk of complications. The main problem of IVF remains the high rate of multiple pregnancies, including twins.     

Regulation of primordial follicle assembly and development

The assembly of the primordial follicles early in ovarian development and the subsequent development and transition of the primordial follicle to the primary follicle are critical processes in ovarian biology. These processes directly affect the number of oocytes available to a female throughout her reproductive life. Once the pool of primordial follicles is depleted a series of physiological changes known as menopause occur. The inappropriate coordination of these processes contributes to ovarian pathologies such as premature ovarian failure (POF) and infertility. Primordial follicle assembly and development are coordinated by locally produced paracrine and autocrine growth factors. Endocrine factors such as progesterone have also been identified that influence follicular assembly. Locally produced factors that promote the primordial to primary follicle transition include growth factors such as kit ligand (KL), leukaemia inhibitory factor (LIF), bone morphogenic proteins (BMP's), keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF). Factors mediating both precursor theca-granulosa cell interactions and granulosa-oocyte interactions have been identified. A factor produced by preantral and antral follicles, Müllerian inhibitory substance, can act to inhibit the primordial to primary follicle transition. Observations suggest that a complex network of cell-cell interactions is required to control the primordial to primary follicle transition. Elucidation of the molecular and cellular control of primordial follicle assembly and the primordial to primary follicle transition provides therapeutic targets to regulate ovarian function and treat ovarian disease.     

Varicocele treatment in the light of evidence-based andrology

Treatment of varicoceles became the most common treatment for male infertility merely on an empirical basis. However, in the age of evidence-based medicine it is surprising that only a few, and mainly recent, randomized controlled clinical trials with relevant outcome parameters have been published to allow adequate judgement of treatment effectiveness. Moreover, difficulties in study design could also be detected in most of these high-quality studies. Despite these difficulties and in contrast to the majority of uncontrolled studies on varicocelectomy, meta-analysis of these randomized controlled clinical studies involving 385 patients showed no significant treatment benefit and questions the common practice of varicocelectomy. Even the high-quality studies show conflicting results and therefore the topic of varicocele treatment will remain controversial and further randomized clinical trials should readdress this issue. For the time being, intervention by surgical or angiographic occlusion of the spermatic vein cannot be recommended.     

The effects of oestrogens on linear bone growth

Regulation of linear bone growth in children and adolescents comprises a complex interaction of hormones and growth factors. Growth hormone (GH) is considered to be the key hormone regulator of linear growth in childhood. The pubertal increase in growth velocity associated with GH has traditionally been attributed to testicular androgen secretion in boys, and to oestrogens or adrenal androgen secretion in girls. Research data indicating that oestrogen may be the principal hormone stimulating the pubertal growth spurt in boys as well as girls is reviewed. Such an action is mediated by oestrogen receptors (ER-alpha and ER-beta) in the human growth plate, and polymorphisms in the ER gene may influence adult height in healthy subjects. Prepubertal oestradiol concentrations are significantly higher in girls than in boys, explaining sex-related differences in pubertal onset. Men with a disruptive mutation in the ER gene (oestrogen resistance) or in the CYP19 gene (aromatase deficiency) who have no pubertal growth spurt and continue to grow into adulthood due to lack of epiphyseal fusion supports this notion. Furthermore, phenotypic females with complete androgen insensitivity syndrome have a normal female growth spurt despite lack of androgen action. Oestrogens may also influence linear bone growth indirectly via modulation of the GH-insulin-like growth factor-I (IGF-I) axis. Thus, ER blockade diminishes endogenous GH secretion, androgen receptor (AR) blockade increases GH secretion in peripubertal boys, and non-aromatizable androgens [oxandrolone or dihydrotestosterone (DHT)] have no effect on GH secretion. Treatment with aromatase inhibitors reduces circulating IGF-I concentrations in healthy males, and reduces growth in boys with testotoxicosis. Taken together, these findings suggest that oestrogens may, in addition to their direct effects, stimulate GH secretion and thereby increase circulating IGF-I, which in turn may stimulate growth. Thus, oestrogens have important biphasic actions on longitudinal growth in boys as well as in girls. Very low levels of oestrogens may stimulate bone growth without affecting sexual maturation directly at the growth plate as well as through stimulation of the GH-IGF axis, which in turn may stimulate growth. Conversely, higher levels of oestrogens stimulate secondary sexual characteristics and epiphyseal fusion.