Genital Development Research Articles

Testosterone undecanoate supplementation together with human chorionic gonadotropin does not impair spermatogenesis in males with isolated hypogonadotropic hypogonadism: a retrospective study.

Gonadotropin therapy is commonly used to induce virilization and spermatogenesis in male isolated hypogonadotropic hypogonadism (IHH) patients. In clinical practice, 5.6%–15.0% of male IHH patients show poor responses to gonadotropin treatment; therefore, testosterone (T) supplementation can serve as an alternative therapy to normalize serum T levels and promote virilization. However, treatment with exogenous T impairs spermatogenesis and suppresses intratesticular T levels. This retrospective study aimed to determine whether oral testosterone undecanoate (TU) supplementation together with human chorionic gonadotropin (hCG) would negatively affect spermatogenesis in IHH patients compared with hCG alone. One hundred and seven IHH patients were included in our study. Fifty-four patients received intramuscular hCG and oral TU, and 53 patients received intramuscular hCG alone. The median follow-up time was 29 (range: 12–72) months in both groups. Compared with the hCG group, the hCG/TU group required a shorter median time to normalize serum T levels (P < 0.001) and achieve Tanner stage (III and V) of pubic hair and genital development (P < 0.05). However, there were no significant differences in the rate of seminal spermatozoa appearance, sperm concentration, or median time to achieve different sperm concentration thresholds between the groups. In addition, there were no significant differences in side effects, such as acne and gynecomastia, observed in both groups. This study indicates that oral TU supplementation together with hCG does not impair spermatogenesis in treated IHH patients compared with hCG alone, and it shortens the time to normalize serum T levels and promote virilization.

AMA Acts on Treatment of Children at Border, Gun Violence, Pediatric Decision Making

AMA Acts on Treatment of Children at Border, Gun Violence, Pediatric Decision Making

Long-term effects of episiotomy on urinary incontinence and pelvic organ prolapse: a systematic review.

To focus attention on the long-term effects of episiotomy on urinary incontinence and pelvic organ prolapse. A systematic review was conducted including only studies with mean follow-up ≥ 5years. We searched using combinations of the following keywords and text words: "episiotomy", "perineal laceration", "perineal tear", "perineal damage" and "long term", "long term outcomes", "prolapse", "pelvic organ prolapse", "pelvic floor", "pelvic floor dysfunction", "urinary incontinence", "hysterocele", "cystocele" and "rectocele". The electronic database search provided a total of 6154 results. After exclusions, 24 studies were included yielding the following results: (1) episiotomy might be detrimental with respect to urinary incontinence symptoms; (2) the relationship between episiotomy and anti-incontinence surgery is not clear; (3) episiotomy does not seem to negatively influence genital prolapse development and might even be protective with respect to prolapse severity and prevalence; (4) episiotomy does not seem to affect genital prolapse surgery rate. We did not find evidence for a long-term beneficial effect of episiotomy in the prevention of urinary incontinence symptoms and anti-incontinence surgery. Episiotomy does not seem to negatively influence genital prolapse development and might even be protective with respect to prolapse severity and prevalence without affecting surgery rates.

Baseline Characteristics of Infants With Atypical Genital Development: Phenotypes, Diagnoses, and Sex of Rearing.

PurposeLittle is known about the phenotypes, diagnoses, and sex of rearing of infants with atypical genital development in the United States. As part of a multicenter study of these infants, we have provided a baseline report from US difference/disorder of sex development clinics describing the diagnoses, anatomic features, and sex of rearing. We also determined whether consensus guidelines are followed for sex designation in the United States.MethodsEligible participants had moderate-to-severe genital atypia, were aged <3 years, and had not undergone previous genitoplasty. Karyotype, genetic diagnosis, difference/disorder of sex development etiology, family history, and sex of rearing were collected. Standardized examinations were performed.ResultsOf 92 subjects, the karyotypes were 46,XX for 57%, 46,XY for 34%, and sex chromosome abnormality for 9%. The median age at the baseline evaluation was 8.8 months. Most 46,XX subjects (91%) had congenital adrenal hyperplasia (CAH) and most 46,XY subjects (65%) did not have a known diagnosis. Two individuals with CAH underwent a change in sex of rearing from male to female within 2 weeks of birth. The presence of a uterus and shorter phallic length were associated with female sex of rearing. The most common karyotype and diagnosis was 46,XX with CAH, followed by 46,XY with an unknown diagnosis. Phenotypically, atypical genitalia have been most commonly characterized by abnormal labioscrotal tissue, phallic length, and urethral meatus location.ConclusionsAn increased phallic length was positively associated with rearing male. Among the US centers studied, sex designation followed the Consensus Statement recommendations. Further study is needed to determine whether this results in patient satisfaction.

Association of phthalates, parabens and phenols found in personal care products with pubertal timing in girls and boys.

Are in-utero or peripubertal exposures to phthalates, parabens and other phenols found in personal care products associated with timing of pubertal onset in boys and girls? We found some associations of altered pubertal timing in girls, but little evidence in boys. Certain chemicals in personal care and consumer products, including low molecular weight phthalates, parabens and phenols, or their precursors, are associated with altered pubertal timing in animal studies. Data were from the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) longitudinal cohort study which followed 338 children in the Salinas Valley, California, from before birth to adolescence. Pregnant women were enrolled in 1999-2000. Mothers were mostly Latina, living below the federal poverty threshold and without a high school diploma. We measured concentrations of three phthalate metabolites (monoethyl phthalate [MEP], mono-n-butyl phthalate and mono-isobutyl phthalate), methyl and propyl paraben and four other phenols (triclosan, benzophenone-3 and 2,4- and 2,5-dichlorophenol) in urine collected from mothers during pregnancy and from children at age 9. Pubertal timing was assessed among 179 girls and 159 boys every 9 months between ages 9 and 13 using clinical Tanner staging. Accelerated failure time models were used to obtain mean shifts of pubertal timing associated with concentrations of prenatal and peripubertal biomarkers. In girls, we observed earlier onset of pubic hair development with prenatal urinary MEP concentrations and earlier menarche with prenatal triclosan and 2,4-dichlorophenol concentrations. Regarding peripubertal biomarkers, we observed: earlier breast development, pubic hair development and menarche with methyl paraben; earlier menarche with propyl paraben; and later pubic hair development with 2,5-dichlorophenol. In boys, we observed no associations with prenatal urinary biomarker concentrations and only one association with peripubertal concentrations: earlier genital development with propyl paraben. These chemicals are quickly metabolized and one to two urinary measurements per developmental point may not accurately reflect usual exposure. Associations of peripubertal measurements with parabens may reflect reverse causality: children going through puberty early may be more likely to use personal care products. The study population was limited to Latino children of low socioeconomic status living in a farmworker community and may not be widely generalizable. This study contributes to a growing literature that suggests that exposure to certain endocrine disrupting chemicals may impact timing of puberty in children. This study was funded by the National Institute of Environmental Health Sciences and the US Environmental Protection Agency. The authors declare no conflicts of interest. N/A.

Association of blood leukocyte DNA methylation at LINE-1 and growth-related candidate genes with pubertal onset and progression

ABSTRACTPuberty is a developmentally plastic phase. Variations in pubertal tempo have implications for the risk of later adult diseases. Influences on pubertal tempo have been widely discussed, but the underlying biological mechanisms remain unclear. Epigenetic modifications are known to regulate development processes; they could play an important role in affecting pubertal outcomes. We conducted a population-based analysis to investigate the association of peripubertal blood DNA methylation at LINE-1 and growth-related candidate genes with pubertal onset and progression in healthy adolescents. The analytic sample included 114 males and 129 females aged 10 to 18 years. DNA methylation at growth-related candidate loci IGF2, H19, HSD11B2, as well as LINE-1 repetitive elements were quantified. Cox survival and ordinal regression models were used to examine sex- and locus-specific associations of epigenetic markers with pubertal development using physician-assessed Tanner stages and self-reported menarche, adjusted for covariates. Among boys, DNA methylation at H19 was associated with later pubarche. HSD11B2 methylation was associated with earlier onset of pubic hair and genitalia development and slower pubertal progression. IGF2 was associated with later onset of genital development. Among girls, LINE-1 methylation was associated with later onset of breast development. For each percent increase of methylation at H19, there was 5% increased odds in the earlier onset of breast development. DNA methylation of IGF2 was associated with earlier onset of pubic hair. DNA methylation at genes known to influence early-life growth may also influence pubertal outcomes.

Maternal age at menarche and pubertal development in sons and daughters: a Nationwide Cohort Study.

Is maternal age at menarche associated with pubertal development in sons and daughters? Maternal age at menarche was associated with pubertal development in both sons and daughters. Studies have shown that age at menarche is greatly inherited from mother to daughter, but it remains largely unknown to what extent age at menarche in mothers is associated with timing of puberty in sons. In this population-based study we used data from the Puberty Cohort nested within the Danish National Birth Cohort. Live-born singletons aged 11 were followed from 2012 to 2016. In total, 15 822 children (7697 sons and 8125 daughters) gave half-yearly information on puberty from the age of 11 years until full sexual maturity or 18 years of age through self-administrated questionnaires (participation rate 71%). Information on maternal age at menarche was reported by the mothers during pregnancy. Maternal age at menarche was used both as a continuous and as a categorical variable (earlier, same time or later than peers). A multivariable regression model for interval-censored data was used. Maternal age at menarche was positively associated with timing of genital development, pubic hair development, first ejaculation of semen, voice break, axillary hair development and acne in sons, and with timing of breast development, pubic hair development, menarche, axillary hair development and acne in daughters. In sons, the associations were of similar strength for all pubertal markers, whereas in daughters, the associations were strongest for breast development and menarche. Age at menarche was recalled during pregnancy. However, studies indicate that age at menarche is recalled moderately in adulthood. Information on puberty was self-reported, but inaccuracy of data would probably cause non-differential misclassification. Early maternal age at menarche was associated with earlier pubertal development, and late maternal age at menarche was associated with later pubertal development in both sons and daughters. The largest effect-estimates were for the associations between maternal age at menarche and the daughters' age at menarche and age at breast development. The study was funded by the Danish Council for Independent Research (4183-00152). There are no competing interests. N/A.

Timing of puberty in boys and girls: A population-based study.

BackgroundA secular trend towards earlier puberty has been observed in girls, while a similar trend has been more uncertain in boys. We estimated current ages at pubertal development in both boys and girls.MethodsIn this population‐based cohort study, 14 759 of 22 439 invited boys and girls born from 2000 to 2003 in the Danish National Birth Cohort gave half‐yearly self‐reported information on puberty from the age of 11.5 years and throughout puberty. This late start of follow‐up limits the estimation of age at onset of puberty but not later pubertal milestones. We estimated mean age at attaining the following pubertal milestones in years with 95% confidence intervals (CI): age at menarche, voice break, first ejaculation of semen and Tanner stages for pubic hair development and breast development or genital development. Further, the difference in mean age at menarche between mothers and daughters was estimated.ResultsIn boys, voice break occurred at 13.1 (95% CI 13.0, 13.1) years, first ejaculation of semen occurred at 13.4 (95% CI 13.3, 13.4) years, and Tanner Genital Stage 5 occurred at 15.6 (95% CI 15.5, 15.6) years. In girls, age at menarche occurred at 13.0 (95% CI 13.0, 13.1) years and Tanner Breast Stage 5 occurred at 15.8 (95% CI 15.7, 15.9) years. Daughters had menarche 3.6 (95% CI 3.1, 4.2) months earlier than their mothers had.ConclusionThese data indicate that age at menarche has declined and to some extent support a decline in age at attaining other markers of pubertal development among boys.

Developmental asynchrony and antagonism of sex determination pathways in a lizard with temperature-induced sex reversal

Vertebrate sex differentiation follows a conserved suite of developmental events: the bipotential gonads differentiate and shortly thereafter sex specific traits become dimorphic. However, this may not apply to squamates, a diverse vertebrate lineage comprising of many species with thermosensitive sexual development. Of the three species with data on the relative timing of gonad differentiation and genital dimorphism, the females of two (Niveoscincus ocellatus and Barisia imbricata) exhibit a phase of temporary pseudohermaphroditism or TPH (gonads have differentiated well before genital dimorphism). We report a third example of TPH in Pogona vitticeps, an agamid with temperature-induced male to female sex reversal. These findings suggest that for female squamates, genital and gonad development may not be closely synchronised, so that TPH may be common. We further observed a high frequency of ovotestes, a usually rare gonadal phenotype characterised by a mix of male and female structures, exclusively associated with temperature-induced sex reversal. We propose that ovotestes are evidence of a period of antagonism between male and female sex-determining pathways during sex reversal. Female sexual development in squamates is considerably more complex than has been appreciated, providing numerous avenues for future exploration of the genetic and hormonal cues that govern sexual development.

Knockdown of rotund gene through larval RNA interference affects genital and elytral morphology in the ground beetle Carabus maiyasanus (Coleoptera: Carabidae)

AbstractInterspecific variation in genital morphology contributes to prezygotic reproductive isolation in many insect species. However, developmental genes affecting genital morphology are poorly understood. We undertook larval RNA interference (RNAi) experiments with rotund (rn) gene in the ground beetle Carabus (Ohomopterus) maiyasanus to examine the knockdown effects of this gene on genital development. We injected double‐stranded RNA (dsRNA) into third instar larvae of C. maiyasanus. The injection of rn dsRNA resulted in the absence or deformation of the copulatory piece in the male genitalia, which is a characteristic part playing a role in species‐specific genital coupling between the sexes. In addition, the rn dsRNA injection resulted in shortened elytra in both sexes, which is related to the known role of rn in forewing formation in Drosophila. Our study suggests that rn plays a previously underappreciated role in male genital development and potentially contributes to genital diversity in this beetle group.

Impact of In Utero and Adolescent Phthalate Exposure on Sexual Maturation Progression

Phthalates are endocrine disrupting compounds commonly found in consumer products, exposure to which may influence reproductive maturation in adolescence. Differential effects from exposure in utero versus later in life are understudied. We examined sex-stratified longitudinal associations between the geometric mean of 3 trimester-specific spot urine phthalate measurements and sexual maturation at two points in adolescence (8-14, 9-18 years), as well as cross-sectional associations at 9-18 years. Sexual maturation was assessed using Tanner stages and menarche onset for girls (N&amp;#61;246) and Tanner stages and testicular volume for boys (N&amp;#61;226). Generalized estimating equations for correlated ordinal multinomial responses were used to model relationships between phthalates and odds of transitioning to the next Tanner stage, while generalized additive (GA) mixed models were used to assess the odds of menarche. Cross-sectional analyses employed GA models (female N&amp;#61;281; male N&amp;#61;264). Among girls, MEP in utero exposure was associated with increased odds of being at a higher Tanner stage for breast development at 8-14 years, but with slower progression of breast development over the follow-up period (OR&amp;#61;0.74 per year; 95% CI: 0.57, 0.96) after adjustment for child age, BMI z-score and time between study visits. Similar results were found for breast development with in utero exposure to MEOHP, MBzP, and MCPP. There were no significant associations of in utero exposures with pubertal stage among boys, but concurrent MEP exposure was associated with lower odds of progression of genital development (OR&amp;#61;0.87; 95% CI: 0.76, 0.99). These results suggest in utero exposure to phthalates may disrupt estrogenic pathways to impact breast development during puberty, and that exposure results in earlier onset but slower progression of sexual development. In addition, MEP exposure during late adolescence may impede genital development among boys.

Maternal Smoking During Pregnancy and Timing of Puberty in Sons and Daughters: A Population-Based Cohort Study.

Because early puberty has been linked to diseases later in life, identification of modifiable causes of early puberty is of interest. We explored the possible associations between maternal smoking during pregnancy and pubertal development in sons and daughters. Between 2012 and 2017, 15,819 children from the Danish National Birth Cohort, born during 2000–2003, provided half-yearly information on puberty from the age of 11 years. We estimated adjusted age differences (in months) at attaining various pubertal milestones, including Tanner stages, per 10 daily cigarettes smoked in the first trimester of gestation. In sons, exposure to smoking in utero was associated with earlier genital development (Tanner 2, −1.3 months, 95% confidence interval (CI): −2.5, 0.0; Tanner 5, −3.7 months, 95% CI: −5.3, −2.0), pubic hair development (Tanner 2, −1.8 months, 95% CI: −2.9, −0.6; Tanner 5, −2.9 months, 95% CI: −4.2, −1.7), and voice break (−2.4 months, 95% CI: −3.6, −1.3). In daughters, maternal smoking was associated with earlier breast development (Tanner 2, −3.4 months, 95% CI: −5.3, −1.5; Tanner 5, −4.7 months, 95% CI: −6.5, −2.9), pubic hair development stages 3–5 (Tanner 5, −2.5 months, 95% CI: −4.1, −1.0), and menarche (−3.1 months, 95% CI: −4.0, −2.3). Fetal exposure to tobacco smoke might advance timing of puberty in boys and girls.

Prenatal Exposure to Persistent Organic Pollutants and Anogenital Distance in Children at 18 Months

Background: Anogenital distance (AGD) is a measure of in utero exposure to hormonally active agents. The aim of the present study was to evaluate the association between prenatal exposure to persistent organic pollutants (POPs) and AGD. Methods: POP levels were measured in pregnant women, and the AGD was recorded in 43 offspring at 18 months. We used linear regression models to analyze the association between maternal POP exposure and offspring AGD. We defined the anogenital index (AGI) as AGD divided by weight at 18 months (AGI = AGD / weight at 18 months [mm/kg]) and included this variable in the regression models. Results: AGI measure was 2.35 (0.61) and 1.38 (0.45) in males and females, respectively. AGI was inversely associated with lipid-adjusted concentrations of PBDE-99 (β = −0.28, 95% confidence interval [CI]: −0.51, −0.04) and PBDE-153 (β = −0.61, 95% CI: −1.11, −0.11) in males. We did not find any statistically significant relationship between any POPs and AGI in females. Conclusions: Environmental exposure to POPs may affect genital development and result in reproductive tract alterations with potentially relevant health consequences in maturity.

Anogenital distance and reproductive outcomes in 9- to 11-year-old boys: the INMA-Granada cohort study.

Studies examining the association of anogenital distance (AGD), a biomarker of prenatal androgen exposure, with sexual development in children are lacking. To assess the association between AGD measures and reproductive outcomes, including puberty onset, testicular volume, reproductive hormone levels, and urogenital malformations in boys aged 9-11years. A cross-sectional study was conducted among children belonging to the Spanish Environment and Childhood (INMA) Project, a population-based birth cohort study. The present sample included 279 boys for whom data were available on AGD, pubertal stage, testicular volume, and relevant covariates. Out of the boys with AGD data, 187 provided a blood sample for hormone analysis. AGD was measured from the center of the anus to the base of the scrotum. Pubertal development was assessed according to Tanner stage of genital development (G1-G5), and testicular volume was measured with an orchidometer. After adjusting for potential confounders, logistic regression analysis showed that AGD was positively associated with testicular volume but not with Tanner stage (>G1 vs. G1), serum hormone levels, or undescended testis. Regardless of their age, body mass index, and Tanner stage (G1 or >G1), boys with longer AGD showed increased odds of a testicular volume >3mL (OR=1.06, 95%CI=1.00-1.19 per 10% increment in AGD; and OR=3.14, 95%CI=0.99-9.94 for AGD >42 mm vs. <33mm). Longer AGD was associated with testicular growth, an indicator of gonadarche, but not with other reproductive outcomes. Although AGD was positively associated with testicular volume, it remains unclear whether AGD predicts testis size at puberty or is related to puberty onset.

Changes to sleep-wake behaviors are associated with trajectories of pubertal timing and tempo of secondary sex characteristics

We examined relations between sleep-wake behaviors and pubertal development from age 8.5 through 15.5 years in a US-based sample of 488 boys (75% White) and 478 girls (78% White). Applying conditional nonlinear growth models to 7-waves of longitudinal data, we examined how sleep-wake behaviors are related to individual differences in the developmental timing and tempo of secondary sex characteristics. For girls, results supported the hypothesis that circadian changes in bedtimes, wake times, sleep duration, and eveningness preference were uniquely related to development of discrete aspects of secondary sex characteristics. For boys, the hypothesis was generally not supported. Different endocrine systems related to discrete secondary sex characteristics may be responsible for more relations between sleep and pubic hair development than for breast or genital development. Further research into associations between adrenarche and sleep during puberty may help researchers understand more about the origins and timing of adolescent sleep changes.

Evaluation of testicular function in prepubertal children.

The assessment of testicular function is not currently performed in childhood. The aim of this review was to address the usefulness of serum Anti-Müllerian Hormone (AMH), inhibin B, and testicular volume (TV) evaluation in children. Serum AMH and inhibin B levels reflect Sertoli cells (SCs) health and number, SCs maturation degree and their exposure to FSH and to intra-tubular testosterone (T). These hormones might be helpful in discriminating between congenital central hypogonadism (cHH) and constitutional delay in growth and puberty (CDGP) and in case of clinical suspicious of precocious puberty. Furthermore, low AMH and/or inhibin B levels have been observed in children with primary testicular disorders, suggesting the existence of SC dysfunction. TV also provides useful information on testicular health. Recently, a medical calculator, requiring testis length and the stage of genital development, has been developed to easily derive TV. The evidence supports the usefulness of AMH, inhibin B and TV evaluation for the early diagnosis of puberty disorders and primary testicular damage. We suggest the measurement of TV by using the medical calculator in all children and to reserve AMH and inhibin B measurements to those cases of no testicular growth, clinical suspicious of puberty disorders or in children at risk for spermatogenesis damage. This work-up might allow the early detection of testicular tubular damage which, in turn, may be useful to prevent the oncoming male infertility in adulthood.

In utero exposure to both high- and low-dose diethylstilbestrol disrupts mouse genital tubercle development.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) during in utero development has been linked to the increasing incidence of disorders of sexual development. Hypospadias, the ectopic placement of the urethra on the ventral aspect of the penis, is one of the most common DSDs affecting men, and can also affect women by resulting in the misplacement of the urethra. This study aimed to comprehensively assess the resulting hypospadias phenotypes in male and female mice exposed in utero from embryonic day 9.5 to 19.5 to the potent estrogenic endocrine disruptor, diethylstilbestrol, at a high, clinically relevant dose, and a low, previously untested dose, administered via water. The anogenital distance of male pups was significantly reduced and hypospadias was observed in males at a high frequency. Females exhibited hypospadias and urethral-vaginal fistula. These results demonstrate the ability of an estrogen receptor agonist to disrupt sexual development in both male and female mice, even at a low dose, administered via drinking water.

Sensitive periods in development of endometriosis

The hypothesis of three sensitive periods (SP) in development of common gynecological disease external endometriosis (EM) is suggested: I prenatal female genital system development from the stem cells (SC) of Mullerian ducts; II uterine endometrial cells transition into mesenchymal meSC; III endometriotic lesions implantation and growth within pelvic lining. Genetic and epigenetic drivers operative at each SP of EM are outlined and their implication for understanding EM origin and development are briefly discussed.

Atypical Genital Development and Hypospadias: a Pediatric Urology perspective from Etiology to Surgery

Atypical Genital Development and Hypospadias: a Pediatric Urology perspective from Etiology to Surgery

Development of urogenital system in the Spix cavy: A model for studies on sexual differentiation

This study documented, for the first time, the morphological patterns of differentiation of male and female genital organs of Spix cavy (Galea spixii) using histological and ultrastructural analyses, with immuno-localization of steroidogenic enzymes, cytochromes P450 aromatase (P450arom) and 17α-hydroxylase/17, 20-lyase (P450c17), involved in the synthesis of estrogens and androgens respectively throughout fetal sexual development. Undifferentiated gonads of Spix cavy develop into ovaries in females after 25 days of gestation (DG), exhibiting P450arom immunoreactivity. After 25 DG, paramesonephric ducts develop and form oviducts, uterine horns and cranial portion of the vagina. The caudal portion of the vagina originates from the urogenital sinus, and a vaginal closure membrane is present at the end of gestation. Partial channeling of the urethra into the clitoris occurs after 40 DG, but complete channeling never occurs. A preputial meatus emerges near the tip of organ. In males, undifferentiated gonads develop into testes at 25 DG and develop immunoreactivity for P450c17, which is required for androgens synthesis and likely maintenance of mesonephric ducts. Mesonephric ducts develop subsequently, forming the epididymis and ductus deferens. The pelvic urethra develops after 25 DG with channeling into the penis occurring around 30 DG. This is the first morphological study describing the process of sexual differentiation during gestation in a hystricomorph rodent and one of the most comprehensive analyses conducted in any mammal. Male genital organ development follows the general pattern described in other domestic mammals, but does not include formation of the baculum as occurs in mice and rats. In females, clitoral development includes partial canalization by the urethra and development of a preputial meatus. Further studies are required to clarify the mechanisms involved in the differentiative processes described.