1. Chpter 9 Normal and Abnormal Sexual Development
CLINICAL GYNECOLOIC ENDOCRINOLOGY AND INFERTILITY
Chpter 9 Normal and Abnormal Sexual Development
OBGY R1 Lee Eun Suk

2. Normal Sex Differentiation
Gender identity the result of the following determinants
Genetic sex
Gonadal sex
Internal & external genitalia
The secondary sexual characteristics that appear at puberty
The role assigned by society
Four major steps which constitute normal sexual differentiation
Fertilization and determination of genetic sex
Formation of organs common to both sexes
Gonadal differentiation
Differentiation of the internal ducts and external genitalia

3. Fertilization and Determination of Genetic Sex
Step 1 in sex differentiation: Determination of genetic sex
Egg (23,X) + Sperm (23,X)=46,XX genetic female OR
Egg (23,X) + Sperm (23, Y)=46, XY genetic male karyotype

4. Formation of Organs Common to Both Sexes
The fertilized egg multiplies to form a large number of cells
Differentiation of the sex organs in this development
; At that stage, both 46,XX & 46,XY fetuses have similar sex
organs, specifically
Gonadal ridges
Internal ducts
External genitalia

5. Internal reproductive organs

7. Gonadal Differentiation
The important event in gonadal differentiation is of the gonadal ridge to become either an ovary or a testis
In males, the gonadal ridge develops into testes as a result of a product from a gene located on the Y chromosome
“Testis determining factor" (TDF)
: Gonadal medullary resion -> Sertoli cell
“Sex determining region of the Y chromosome" (SRY)
In females, the absence of SRY, due to the absence of a Y chromosome, permits the expression of other genes
which will trigger the gonadal ridge to develop into ovaries

8. Gonadal Differentiation
Pseudoautosomal region
The distal ends of the short arms of the X and Y chromosomes
During meiosis the homologous distal short arms of the X and Y chromosomes pairs, and interchange of genetic material occurs in autosomes
Gene deletions in this area of the X chromosome (Xp22.3) are associated with various conditions short stature , mental retardation, X- linked ichthyosis, Kallmann’s sydrome

9. Gonadal Differentiation
Subsequent sexual differentiation requires direction by various genes with TDF
SRY: The Y chromosome sex determinants region
SOX9: An autosomal testis-determining gene
DAX1: A potential testis-suppressing gene on X-chromosome
SF1: The link between SRY and the male development pathway
WT1: necessary for normal renal and gonadal development
WNT4: A potential ovary-determining gene on an autosome

10. Gonadal Differentiation
SRY
Sex determining region of the Y chromosome
Locate on the short arm of the Y chromosome
Transcription factor contains HMG (high-mobility group) box
- a DNA binding domain => conrol of gene transcrition
Investigations of the DNA-binding properties of the protein of SRY in the promoter P450 aromatase (conversion of testosterone to estradiol that is down-regulated in the embryo) & anti-mullerian hormone (responsible for regression of the mullerian ducts)
The expression of SRY in the tissue destined to become a gonad directs the cells of this gonadal primordium to differentiate as Sertoli cell

11. Gonadal Differentiation
SOX9 : An autosomal testis-determining gene
(SRY-like box) genes are similar in sequence to SRY
: an extra copy of SOX9 developes males, even if they have no
SRY gene – XX mice made transgenic for SOX9 develop testes
SF1 ; Steroidogenic factor, necessary to make the bipotential gonad
Collaboration with SOX9  elevate levels of AMH transcription
Wnt4 : Activate DAX1 expression
Lack the Wnt4 gene
 Ovary fail to form properly, express testis specific markers

12. Postulated cascades leading to the formation of the sexual phenotypes

13. Summary of key genetic events in early sex determination
Migration of primordial germ cells to the urogenital ridge
Differentiation of the bipotential gonadal tissue under the direction of WT-1 and SF-1
SRY activation of male-specific genes, especially SOX9, to produce the testes by cell proliferation, differentiation, migration and vascularization
Ovarian differentiation by suppression of SOX9 through the activity of DAX1 and Wnt4

14. Summary of the genetics of gonadal dysgenesis
Gonadal streaks without germ cells in XX or XY (female phenotype) :
Deficiencies in WT-1 or SF-1
Lack of testicular development in XY individuals
pure gonadal dysgenesis (female phenotype) :
Deficiencies in SRY or SOX9
Male phenotype in a 46,XX individual :
Presence of SRY
Mixed gonadal dysgenesis in mosaics (varying phenotype) :
Excess DAX1

15. Internal reproductive organs - Embryonic development
Urinary and genital tract
Closely related, anatomically and embryologically
Embryologic urinary system -> important inductive influence on developing genital system
Anomalies in one system are often mirrored by anomalies in another system
Urinary system, internal reproductive organs & external genitalia
Develop synchronously at an early embryologic age

16. Kidney, renal collecting system & ureters from nephrogenic cord

17. Mesonephric (Wolffian) duct
Singular importance for the following reasons
Grows caudally in developing embryo to open an excretory channel into the primitive cloaca and outside world
Serves as starting point for development of the metanephros which becomes definitive kidney
Differentiates into the sexual duct system in male
Although regressing in female fetuses, inductive role in development of the paramesonephric or mullerian duct

18. Mullerian duct Paramesonephric or mullerian ducts - development
Form lateral to mesonephric ducts
Grow caudally and then medially to fuse in midline
Contact urogenital sinus in region of the post. urethra at slight thickening known as sinusal tubercle

19. Duct System Differentiation - Male
TDF
Results in degeneration of gonadal cortex and differentiation of the medullary region of the gonad into Sertoli cells
Sertoli cells
Secrete glycoprotein known as anti-mullerian hormone(AMH)
Regression of paramesonephric duct system in male embryo
Signal for differentiation of Leydig cells from the surrounding mesenchyme

20. Duct System Differentiation - Female fetus
In the absence of TDF, medulla regresses and cortical sex cords break up into isolated cell clusters (-> primordial follicles)
In the absence of AMH & testosterone
Mesonephric duct system degenerates
Then, paramesonephric duct system develops
Inf. fused portion
Uterovaginal canal -> uterus and upper vagina
Cranial unfused portions
Open into celomic cavity (future peritoneal cavity)
Fallopian tubes

21. Anti-Müllerian Hormone
A member of the transforming growth factor-ß family
Regression of Müllerian duct system in male embryo
AMH has an inhibitory effect on oocyte meiosis
Plays a role in the descent of the testes
Inhibits surfactant accumulation in the lungs
Proteolytic cleavage of AMH produces fragments that have the ability to inhibit growth of various tumor ( a potential therapeutic application)

22. Duct System Differentiation
Leydig cells
Produce testosterone & dihydrotestosterone with 5a-reductase
Testosterone
Responsible for evolution of mesonephric duct system into vas deferens, epididymis, ejaculatory duct & seminal vesicle
At puberty, leads to spermatogenesis & changes in primary and secondary sex characteristics
DHT(dihydrotestosterone)
Results in development of the male external genitalia , prostate and bulbourethral glands

23. External Genitalia Differentiation
In the female, absence of androgens permits the external genitalia to remain feminine
The genital tubercle becomes the clitoris
The labioscrotal swellings → the labia majora
The urogenital folds → the labia minora
In the male, fetal androgens from the testes masculinize the external genitalia
The genital tubercle grows to become the penis
The labioscrotal swellings fuse to form the scrotum

24. External Genitalia

25. Abnormal Sexual Development

26. CLASSIFICATION OF INTERSEXUALITY
Disorders of fetal Endocrinology

27. CLASSIFICATION OF INTERSEXUALITY
Primary gonadal defect – Swyer syndrome

28. How many children are born with intersex conditions?
A conservative estimate is that 1 in 2000 children born will be affected by an intersex condition
98 % of affected babies are due to congenital adrenal hyperplasia

29. FEMALE PSEUDOHERMAPHRODITISM
EXCESS FETAL ANDROGENS
Congenital adrenal hyperplasia
21 -hydrxylase deficiency
11-hydroxylase deficiency
3ß-hydroxysteroid
dehydrogenase deficiency
EXCESS MATERNAL ADROGEN
Maternal androgen secreting tumours (ovary, adrenal)
Maternal ingestion of androgenic drugs

30. 21-hydrxylase deficiency congenital adrenal hyperplasia
Cholesterol
Pituitary
Pregnenolone
Progesterone
ACTH
17-OH progesterone
Adrenal cortex
21-hydroxylase 
Androgens
Cortisol
Cortisol
Androgens

31. Masculinized females CONGENITAL ADRENAL HYPERPLASIA
There are several different forms of CAH, each related to one of the enzymes necessary to transform cholesterol to cortisol (hydrocortisone)
StAR / 20,22-hydroxylase, 3 -hydroxysteroid-dehydrogenase / hydroxylase / 21-hydroxylase and 11 -hydroxylase
When one of these enzymes is deficient, this leads to a hyperfunction and increased size (hyperplasia) of the adrenals
Among the various forms of CAH, the 21-hydroxylase deficiency is by far the most frequent, representing more than 95% of all cases
Defect in cortisol biosynthesis, with or without aldosterone def, androgen excess

32. Masculinized females CONGENITAL ADRENAL HYPERPLASIA - Biochemistry
Steroid 21 hyrdoxylase is a cytochrome p-450 enzyme located in ER
Catalyzed the conversion of 17-hydoxyprogesterone to 11-hydroxycortisol :precursor of cortisol
Conversion of progesterone to deoxycortisterone :precursor of aldosterone
This enzyme deficiency -> adrenal cortex is stimulated
& over production of cortisol precursor

33. Pathways of steroid biosynthesis in the adrenal cortex

34. Hypothalamic pituitary adrenal axis Renin-angiotensin-aldosterone axis

35. Clinical menifestation
Salt wasting type
: severe form with a concurrent defect in aldosterone synthesis
Simple virilizing type : normal aldosterone biosynthesis
Both are together termed classic 21-hydroxylase deficiency
There is also a mild,nonclassic form : may be asymptomatic
Classic 21-hydroxylase deficiency : 1 in 16,000 births

36. CONGENITAL ADRENAL HYPERPLASIA - salt wasting
75 percent of patients with classic 21-hydroxylase deficiency
 severely impaired 21-hydroxylation of progesterone
 cannot synthesis of aldosterone
Elevated of 21-hydroxylase precursor: aldosterone antagonist
Aldosterone deficiency
 hypovolemia and hypereninemia, and hyperkalemia (esp. in infant)
Cortisol deficiency
poor cardiac contractility, poor vascular resp. to catecholamine, and
GFR ↓ , antidiuretic H ↑

37. CONGENITAL ADRENAL HYPERPLASIA - salt wasting
So together : hyponatremic dehydration and shock
Adrenal medulla : depending on the glucocorticoid in part
 so salt wasting 21hyroxylase deficiency
 catecholamine deficiency
 exacerbating shock Identify : e, aldosterone, plasma renin
(hyperkalemia and low aldosterone and hyperreninemia)

38. Ambiguous genitalia Girls with classic 21-hydroxylase deficiency :
exposed to high level of adrenal androgen level (GA 7 wks)
Girls with ambiguous genitalia:
-a large clitoris
-rugated and partially fused labia majora
-uterus,fallopian tubes, and ovaries : normal
Boys :
-no overt signs of the disease
except variable and subtle hyperpigmentation
and penile en-largement

39. Postnatal virilization
Exposed to the high levels of sex steroids
Rapid growth (usually androgen effect)
Advanced bone age  premature epiphyseal closure (androgen’s extragonatal aromatization of estrogen)
Pubic and axillary hair: early develop
Girl : clitorial growth
Young Boys : penile growth
Long term stimulation : central precocious puberty

40. Linear growth A meta-analysis of data from 18 centers showed
: 1.4 SD below the population mean
Both undertreatment and overtreatment : risk for short stature
Undertreatment : causing premature epiphyseal closure induced
by high levels of sex steroids
Overtreatment : glucocorticoid-induced inhibition of the growth

41. Reproductive function
Girls : problem at the reproductive system
-oligomenorrhea, amenorrhea
-prenatal androgen exposure: effect to sex-role behavior
Boys : fewer problems

42. CONGENITAL ADRENAL HYPERPLASIA - Diagnosis

43. CONGENITAL ADRENAL HYPERPLASIA
Normal infant :100ng/dl (17 hydroxyprogesterone)
Affected infants:10000ng/dl ↑
New born -> screening test
10% (severe affected infant) : 17 hydroxyprogesterone level ↓
Preterm or sick infants -> 17 hydroxyprogesterone level ↑↑
The severity of hormonal abnormalities: depends on the type of 21-hydroxylase def
Salt wasting : 17-hydroxyprogesterone: ng/dl

44. CONGENITAL ADRENAL HYPERPLASIA
Random 17-hydroxyprogesterone(17-Ohpro) >80 ug/L or 242 nmol/L (nl : <2.95 ug/L or 9 nmol/L)
Salt losing > nonsalt loser
-> corticotropin stimulation test : unneeded
Genetic analysis -> prenatal testing
Nonclassic CAH : random 17-OHpro : nl
-> Screening
: early morning basal 17-OHpro level >1.5 ug/L (4.5 nmol/L) in children
& >2.0 ug/L(6nmol/L) in women during follicular phase of menstrual cycle
-> Corticotropin ST(250ug of tetracosactide(cosyntropin) )
: any time during the day => positive : 17-OHpro >15.0 ug/L(45nmol/L)

45. CONGENITAL ADRENAL HYPERPLASIA
Heterozygote carrier
: mild elevation, 17-OHpro <10 ug/L(30nmol/L) following CST
Carrier : symptoms or signs of the disease
Premature adrenarche 1/3 : heterozygote carriers of 21-OH def (recent)
17-OHpro :10-15 ug/L
-> DDx of heterozygote carrier or homozygote affected Pt
-> Genetic analysis

46. CONGENITAL ADRENAL HYPERPLASIA – Management
According to the clinical course & hormonal level
Purpose
: normal growth, B.Wt, pubertal development, optimal adult height
Growth velocity, body Wt velocity, bone age maturation F/U
Classic 21-OH def
-> glucocorticoid : adrenal androgen secretion ↓
-> mineralocorticoid : electrolytes & plasma renin activity normalization

47. Treatment Problems
Hypercortisolism (iatrogenic Cushing’s syndrome) - Sn & Sx
: obesity, growth failure, adult short stature, striae,
osteoporosis, hyperlipidemia
Symptoms of hyperandrogenism
: virilism, infertility of female, precocious virilisation of male,
early puberty, adult short stature

48. Glucocorticoid and mineralocorticoid therapy
Hydrocortisone of physiologic dose
-> corticotropin & androgen production suppression (Х): 6 mg/m2/day
Hydrocortisone 12-15mg/m2/day -> sufficient androgen suppression
20 mg/m2/day (neonatal period)
25 mg/m2/day -> do not use
Dose variability factor
Indivudual variation in the metabolism and sensitivity
Previous degree of hypothalamic-pituitary-adrenal axis suppression
High dose glucocorticoid short course therapy

49. Glucocorticoid and mineralocorticoid therapy
Mineralocorticoid (fludrocortisone)
: plasma renin activity ->mid normal range
Dose : ug/day
Nonsalt losing :elevated plasam renin activity
Infant with salt losing : NaCl supply ->1-2 g/day
17mEq Na/NaCl 1g

50. CONGENITAL ADRENAL HYPERPLASIA – Monitoring therapy
Commonly, serum 17-hydroxyprogesterone & androstenedione level
Testosterone level in female and prepubertal male
Test time : early morning(8:00 AM)
Target level of 17-OHpro : 4-12 ug/L (12-36 nmol/L)

51. CONGENITAL ADRENAL HYPERPLASIA – Prenatal therapy
Classic CAH fetus – pregnant women
: Dexamethasone-> fetal pituitary adrenal axis inhibition
& affected female’s genital ambiguity↓
Risk : mother & father - carrier
-> classic CAH female’s 1/8
Masculinisation of external genitalia
: 6weeks gestation -> start Tx
Chorionic villi sampling or amniocentesis
-> male or unaffected female -> Tx stop
Affected female ->70% normal birth or sl virilization

53. Incompletely Masculized Males

54. What is AIS?
A genetic condition where affected people have male chromosomes & male gonads with complete or partial feminization of the external genitals
An inherited X-linked recessive disease with a mutation in the Androgen Receptor (AR) gene resulting in:
Functioning Y sex chromosome
Abnormality on X sex chromosome
Types
CAIS (completely insensitive to AR gene) -External female genitalia -Lacking female internal organs
PAIS (partially sensitive-varying degrees) -External genitalia appearance on a spectrum (male to female)
MAIS (mildly sensitive, rare) -Impaired sperm development and/or impaired masculinization
Also called Testicular Feminization

55. Androgen Receptor Gene
AIS results from mutations in the androgen receptor gene, located on the long arm of the X chromosome (Xq11-q12)
The AR gene provides instructions to make the protein called androgen receptor, which allows cells to respond to androgens, such as testosterone, and directs male sexual development
Androgens also regulate hair growth and sex drive
Mutations include complete or partial gene deletions, point mutations and small insertions or deletions

56. The Process of Sexual Development
In AIS the chromosome sex and gonad sex do not agree with the phenotypic sex
Phenotypic sex results from secretions of hormones from the testicles
The two main hormones secreted from the testicles are testosterone and mullerian duct inhibitor
Testosterone is converted into dyhydrotestosterone
Mullerian duct inhibitor suppresses the mullerian ducts and prevents the development of internal female sex organs in males
Wolffian ducts help develop the rest of the internal male reproductive system and suppress the Mullerian ducts
Defective androgen receptors cause the wolffian ducts & genitals to be unable to respond to the androgens testosterone and dihydrotestosterone

57. AIS Fetus Development
Each fetus has non-specific genitalia for the first 8 weeks after conception
When a Y-bearing sperm fertilizes an egg an XY embryo is produced and the male reproductive system begins to develop
Normally the testes will develop first and the Mullerian ducts will be suppressed and testosterone will be produced
Due to the inefficient AR gene cells do not respond to testosterone and female genitalia begin to form
The amount of external feminization depends on the severity of the androgen receptor defect
CAIS: complete female external genitalia
PAIS: partial female external genitalia
MAIS: Mild female external genitalia, essentially male

58. 5-alpha reductase deficiency
Normal internal genitalia
: testes secrete T, MIH causes Mullerian ducts to degenerate
Lack of DHT leads to inadequate masculinization of external genitalia at birth
Testes in labia or inguinal canal
Urogenital sinus: urethra & blind vagina
Prostate gland: small or absent
At puberty, lots of T
 testes descend, scrotum darkens, phallus enlarges, muscular &
deep voice

59. Testing for AIS Tests During Pregnancy
Chorionic Villus Sampling (9-12 weeks)
Ultrasound and Amniocentesis (after 16 weeks)
After Birth
Presence of XY Chromosomes
Buccal Mouth Smear
Blood Test
Pelvic Ultrasound
Histological Examination of Testes

60. Treatments Surgery Orchidectomy or gonadectomy Removal of the testes
Vaginal lengthening
Genital plastic surgery
Reconstructive surgery on the female genitalia if masculinization occurs
Phalloplasty
Vaginoplasty
Pressure dilation
Clitorectomy