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OG BASIC SCIENCE
S.No Topic Page No
1. Barr Body Test 2
2. Applied Anatomy Of Internal Iliaic Artery 4
3. Fragile X Syndrome 7
4. Sex Differrentiation And Sex Determination 9
5. Disorders Of Sex Development (Dsd) 12
6. Congenital Adrenal Hyperplasia 19
7. Premalignant Lesions Of Vulva 23
8. Mosaicism 28
9. Congenital Hyperbilirubinemia: 31
10. Endocrinology Of Puberty: 35
11. Iodine Metabolism: 38
12. Anatomy Of Anal Sphincter: 40
13. Micronutrients In Pregnancy: 42
14. Pain Pathway And Labour Analgesia: 44
15. Wound Healing: 48
16. Karyotyping In Gynecology: 52
17. Pulmmonary Surfactanat: 55
18. Prevention Of Parent To Child Transmission (Pptct) 58
19. Laboratory Diagnosis Of Hiv Infection: 62
20. Haart Therapy: 64
21. Pcr And Its Clinical Implications: 65
22. Antimetabolites: 69
23. Screening Methods For Osteoporosis 72
24. Energy Sources In Endoscopic Surgery: 75
25. Antimalarials In Pregnancy: 79
26. Kreb’s Citric Acid Cycle: 81
27. Stem Cell Banking And Stemcell Therapy: 84
28. Obstetric Outcome In Maternal Pyelonephritis: 86
29. Group B Streptococci Colonization In Pregnancy: 87
30. Disinfection Of Operation Theater: 90
31. Nonimmune Hydrops Fetalis: 93
32. Lymphatic Drainage Of Vulva: 97
33. Physiology Of Micturition: 99
34. Role Of Cytokines In Preterm Labour: 102
35. Anatomical And Physiological Changes Of Respiratory System In 104
Pregnancy:
36. Fluid And Electrolyte Balance In Labour: 106
37. Fluid Management In Obstetric Shock: 108
38. Double Bhor Effect: 115
39. Double Bhor Effect: 116
40. Torch Infection: 118


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OG BASIC SCIENCES
BARR BODY STUDY:
1. Sex chromatin is an approximately 1 micron clump of chromatin seen usually at the periphery of
female nuclei in certain tissues like corneal epithelium, buccal mucosa, oral and vaginal mucosa,
fibroblasts etc. and called “Barr body” and as a drumstick in polymorphonuclear neutrophils nuclei in
the blood smears. Sex chromatin was found in all tissues except in liver and pancreatic acinar cells
2. It refers to the inactive X chromosome found inside the nuclei of somatic cells of female in the
interphase of cell cycle. It could be maternal or paternal in origin and inactivation occurs very early in
embryogenesis and is permanent.
3. Mary Lyon put forth a hypothesis that one of the X-chromosomes of females is randomly inactivated
and this chromosome could be of maternal or paternal origin. The inactivation is stable and occurs at
embryogenesis. It was hypothesized that this was to compensate for the extra gene products
produced in females who have two X-chromosomes and is called dosage compensation.
4. The condensed X chromosome lags in replication of DNA (it is out of sync with the rest of
chromosomes). There is X inactivation gene on the long arm of X chromosome.
5. The inactivated X is observed as a darkly stained body in the nucleus attached to the nuclear
membrane. It is either triangular, oval or dumbbell shaped and is always one per each inactivated X
chromosome.
6. It is usually found in cells of buccal mucosa (adjacent to nuclear membrane), neurons (attached to
nucleolus) and neutrophils (drum-stick from nucleus).
7. This condensed clump of chromatin is intensely basophilic. It is named Barr body after its discoverer
Murray L Barr.
Significance of Barr Bodies
1. The number of Barr bodies is one less than the number of X chromosomes. Therefore, by
counting the number of Barr bodies or body (by simple test of buccal smear) one can understand
the numerical anomalies of sex chromosomes.
2. Normal male XY is Barr body negative while normal female XX is positive. Turner syndrome (XO) is
Barr body negative female. Kleinfelter (XXY) is bar body positive male.
3. This test can be offered as a provisional diagnostic test in ambiguous genitalia.

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Sex identification using Barr body
1. The fact that the number of X chromosomes is directly related to the number of Barr bodies in the
nucleus is commonly used to confirm the sex of women athletes.
Condition Sex Chromosomes Number of Barr Bodies
Normal Female XX 1
Normal Male XY None
Trisomy X XXX 2
Turner's Syndrome X None
Kleinfelter's Syndrome XXY 1
Clinical Indications for Sex Chromatin Studies
1. Ambiguous genitalia
2. Somatic growth disturbance
3. Mental retardation with sex anomalies
4. The Klinefelter and Turner syndromes
5. Primary amenorrhea and oligomenorrhea
6. Delayed puberty
7. Hypogonadism
8. Abnormal development at puberty.
Buccal smear:
1. A buccal smear is a test where cells are taken from the tongue. Cells are collected by scraping the
tongue with a spatula. The cells are then placed on a slide and the sample is taken to the
laboratory for evaluation. The cells are evaluated for the presence of Barr bodies (a mass seen in
a normal female sex chromosome). The buccal smear test can confirm whether the patient is a
male or female.
2. The cells which line the inside of your cheeks are classified as a stratified squamous
epitheliumtissue and are the surface of a mucous membrane. These flat, scale-like buccal cells are
shed constantly as the tissue is renewed. By gently scraping the inside of cheek, these cells can be
harvested, and when smeared and stained, may be used to illustrate a number of important
biological phenomena including cell and tissue structure, oral bacterial flora and morphology, etc.
This tissue is non-keratinized and therefore the surface cells are living and still possess their
nuclei, in contrast with shed epidermal cells.
3. The patient should rinse the mouth with mouthwash and then with water. A sterilized disposable
swab strip is drawn along the buccal surface of the cheek. The cellular material is quickly smeared
on the two clean slides and two smears are made. A thin film of cells on the slides are done and
kept them for air-drying. Air-dried smear was kept in 95% Ethyl alcohol fixation for 30-35 minutes.
4. The working solution of Giemsa and methylene Blue stains are prepared by dilution methods with
distilled water from stock solution. On one slide, Giemsa stain is poured and allowed to stand for
20 – 25 minutes. After staining, the slide is washed with distilled water to remove the excess
stain. Another slide, Methylene blue is also poured and allowed to stand as similar as above
procedure. Finally, the slides with two different stains were kept for air-drying and then observed
under the microscope.
5. Violet Barr bodies are observed inside a pink nucleus over the Giemsa stained slide and light blue
Barr bodies are observed inside blue coloured nucleus with Methylene Blue stained slide.
6. A total of 10 cells per slide are counted. To determine the presence of Barr bodies, there are
modified guidelines as follow:
a. Only cells with clearly visualized without folding and easily distinguishable nuclear border.
b. Located near the nuclear membrane as a bar, semi-disc, or triangle.
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c. Length of chromatin body had to exceed 1µm in size.
d. Doubtful cells were considered negative.
e. Count of morphologically acceptable Barr bodies was expressed as a %.
7. Subjects with chromatin count ≤ 2 were recorded as male and those with > 2 were recorded as
female PAP stain is the most commonly used staining technique which very recently has been
replaced by acridine orange for its specificity
Drumsticks
1. Drumsticks are present in peripheral blood of individuals carrying more than one X-chromosome
in at least part of their granulocytes. The average incidence of drumsticks is about 1 per 38
neutrophils in the blood films from females, and they are absent in males
PCR
Now the polymerase chain reaction is carried out to detect the presence of the SRY gene, which is
supposed to be the true determinant for maleness.

APPLIED ANATOMY OF INTERNAL ILIAIC ARTERY
The internal iliac artery is the major artery of the pelvis. It originates at the bifurcation of the common
iliac artery into its internal and external branches, and approximately occurs at vertebral level L5-S1.
The artery descends inferiorly, crossing the pelvic inlet to enter the lesser pelvis. During its descent, it is
situated medially to the external iliac vein and obturator nerve. At the superior border of the greater
sciatic foramen, it divides into anterior and posterior trunks.

The anterior trunk
It gives rise to numerous branches that supply the pelvic organs, the perineum, and the gluteal and
adductor regions of the lower limb.
1. Obturator artery – Travels through the obturator canal, accompanied by the obturator nerve and
vein. It supplies the muscles of the thigh’s adductor region.
2. Umbilical artery – Gives rise to the superior vesical artery, which supplies the superior aspect of
the urinary bladder. In utero, the umbilical artery transports deoxygenated blood from the fetus
to the placenta.
3. Inferior vesical artery – Supplies the lower aspect of the bladder. In males, it also supplies the
prostate gland and seminal vesicles.
4. Vaginal artery (female) – Descends to the vagina, supplying additional branches to the inferior
bladder and rectum.
5. Uterine artery (female) – Travels within the cardinal ligament to reach the cervix, where it
ascends along the lateral aspect of the uterus. At origin of the fallopian tubes, it anastomoses
with the ovarian artery. During its course, it crosses the ureters superiorly.
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6. Middle rectal artery – Travels medially to supply the distal part of the rectum. It also forms
anastomoses with the superior rectal artery (derived from the inferior mesenteric) and the
inferior rectal artery (derived from the internal pudendal)
7. Internal pudendal artery – Moves inferiorly to exit the pelvis via the greater sciatic foramen.
Accompanied by the pudendal nerve, it then enters the perineum via the lesser sciatic foramen. It
is the main artery responsible for the blood supply to the perineum.
8. Inferior gluteal artery – The terminal branch of the anterior trunk. It leaves the pelvic cavity via
the greater sciatic foramen, emerging inferiorly to the piriformis muscle in the gluteal region. It
contributes to the blood supply of the gluteal muscles and hip joint.
Posterior Trunk
The posterior trunk gives rise to arteries that supply the lower posterior abdominal wall, posterior pelvic
wall and the gluteal region. There are typically three branches:
1. Iliolumbar artery – Ascends to exit the lesser pelvis, dividing into a lumbar and iliac branch. The
lumbar branch supplies psoas major, quadratus lumborum and the posterior abdominal wall. The
iliac branch supplies the muscles and bone around the iliac fossa.
2. Lateral sacral arteries (superior and inferior) – Travel infero-medially along the posterior pelvic
wall to supply structures in the sacral canal, and the skin and muscle posterior to the sacrum.
3. Superior gluteal artery – The terminal branch of the posterior trunk. It exits the pelvic cavity via
the greater sciatic foramen, entering the gluteal region superiorly to the piriformis muscle. It is
the major blood supply to the muscles and skin of the gluteal region.
Surgical anatomy
1. Branching pattern of the internal iliac artery:
a. Knowing the variability of the internal iliac branching pattern is extremely important for
endovascular radiologists, surgeons, obstetricians – gynecologists, orthopedic surgeons,
and urologists.
b. The organization of the branching pattern can be studied with pelvic embolization. This
can avoid unnecessary embolization, embolectomy or ligation, and iatrogenic injury to
the internal iliac artery can be prevented if the branching pattern is known.
2. Internal iliac ligation:
a. It is required for hemostasis in cases of severe pelvic hemorrhage e.g. severe postpartum
hemorrhage or open-book pelvic fractures. As there are extensive anastomoses, collateral
circulation develops soon after ligation.
b. Clinical anatomy The level of bifurcation of the common iliac artery is quite constant, and
there are two easily identifiable guides. These are the sacral promontory and a line drawn
between both anterior superior iliac spines. The bifurcation of the common iliac artery is
found at the level of both of these landmarks in the majority of patients.
c. The important anatomical relations of the internal iliac (hypogastric) artery can be
summarized as follows:
i. Anterior medial – covered by peritoneum (the internal iliac artery is entirely
retroperitoneal);
ii. Anterior – the ureter (retroperitoneal and attached to the peritoneum);
iii. Posterolateral – the external iliac vein and the obturator nerve;
iv. Posteromedial – the internal iliac vein;
v. Lateral – the psoas major and minor muscles. Physiology of internal iliac artery
ligation Because of the excellent collateral circulation in the pelvis, vascular
compromise does not occur when one or both internal iliac arteries are ligated.
3. Variations of Internal Iliac Artery:
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a. For a successful ligation of the IIA, the operating surgeon should be cognizant of the level
of origin, the level of division of the artery; unilateral or bilateral ligation of the IIA can be
lifesaving in patients with massive postpartum hemorrhage, after vaginal and abdominal
hysterectomy; in massive broad ligament hematoma, cervical carcinoma, and
retroperitoneal bleeding after pelvic fractures, bilateral IIA ligation is done. In a study, in
40 % specimens, variant branches were observed
b. Adachi proposed that umbilical artery was a continuation of the main stem of the internal
iliac artery and the superior gluteal, the inferior gluteal and the internal pudendal arteries
were principal branches of the umbilical artery from an embryological point of view. His
scheme is as follows:
i. TYPE I: The superior gluteal artery arises separately from internal iliac artery, and
the inferior gluteal and internal pudendal vessel are given off by a common trunk
.If the latter divides within the pelvis it is considered to be TYPE IA, where as if the
bifurcation occurs below the pelvic floor it is considered as TYPE IB.
ii. TYPE II: The superior and inferior gluteal arteries arise by a common trunk and the
internal pudendal vessel separately. In this category TYPE II A includes those
specimens in which the trunk common to the two gluteal arteries divides within
the pelvis, and TYPE II B in which the division occurs outside the pelvis.
iii. TYPE III: Three branches arise separately from the internal iliac artery.
iv. TYPE IV: The three branches arise by a common trunk. TYPE IV a: The trunk first
gives rise to the superior gluteal artery before bifurcating into the other two
branches. TYPE IV b: First vessel is emerging from common trunk, which then
divides into superior and inferior gluteal arteries.
v. TYPE V: The internal pudendal and the superior gluteal arteries arise from
common trunk, and the inferior gluteal has a separate origin.

Schematic representation of Adachi's classification of branching pattern of internal iliac artery.
IIA: Internal Iliac Artery; IGA: Lower Gluteal Artery; IPA: Internal Pudendal Artery;
SGA: Superior Gluteal Artery; UA: Umbilical Artery.
4. Aneurysms of the internal iliac artery: These are rare but are not easy to diagnose. The aneurysm
causes obstructive symptoms by compressing the ureters or rectum leading to constipation or
difficulty passing urine. Rupture of the aneurysm can be fatal due to severe hemorrhagic shock.
Treatment is endovascular stenting in asymptomatic aneurysms or surgical ligation in
symptomatic aneurysms.
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FRAGILE X SYNDROME
Introduction:
1. Fragile X syndrome is an X-linked dominant disorder. Fragile X syndrome is the name given to this
condition because some affected individuals have an X chromosome that looked as if it had
“broken” or was “fragile” and was held together by the slightest of ties. This technique is no
longer used in the diagnosis of this syndrome because it is both less accurate and costlier than are
molecular techniques.
2. Fragile X syndrome is caused by an expansion mutation in the fragile X mental retardation 1
(FMR1) gene located on the X chromosome. It characteristically leads to some degree of mental
retardation.
3. Normally, this DNA segment is repeated from 5 to about 40 times. In people with fragile X
syndrome, however, the CGG segment is repeated more than 200 times. The abnormally
expanded CGG segment turns off (silences) the FMR1 gene, which prevents the gene from
producing FMRP. Loss or a shortage (deficiency) of this protein disrupts nervous system functions
and leads to the signs and symptoms of fragile X syndrome.
4. Males and females with 55 to 200 repeats of the CGG segment are said to have an FMR1 gene
premutation. Most people with this premutation are intellectually normal.
5. Recently recognized manifestations in premutation carriers include premature ovarian failure and
tremor/ataxia. Premature ovarian failure occurs in up to 20 percent of women who are
premutation carriers of the FMR1 gene.
6. Fragile X–associated tremor/ ataxia syndrome (FXTAS) affects 30 percent of premutation carrier
men between the ages of 50 and 60 years, and its prevalence increases with age.

Epidemiology
1. Fragile X syndrome, the phenotype associated with full mutation, occurs in approximately one in
4,000 men and one in 6,000 to 8,000 women.
2. The premutation in the FMR1 gene occurs in approximately one in 800 men and up to one in 100
to 200 women.
Clinical Presentation
1. Although fragile X syndrome occurs in males and females, females generally present with milder
symptoms. The first clinical clue in children often is delayed attainment of one or more
developmental milestones.
2. With few exceptions, affected males have mental retardation, generally of moderate degree.
About one third of affected females have mild to severe mental retardation. There is a specific
pattern of deficits in abstract reasoning, sequential processing, and mathematics.
3. Clinical findings during early childhood may include macrocephaly and frontal bossing (unusually
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prominent forehead). After puberty, macroorchidism is present in affected men. Additional
findings may include strabismus and mild connective tissue dysplasia, such as mitral valve
prolapse, hyperextensible joints, and pes planus.
4. Behavior is characterized by attention deficits, hand flapping, hand biting, and gaze aversion.
However, the average age of diagnosis currently is eight years, reflecting the subtlety of features
in young children.
5. FXTAS is a neurodegenerative disorder with progressive intention tremor and cerebellar ataxia.
Affected persons present with Parkinsonism, peripheral neuropathies, and dementia after age 50
years.
Premutation:
1. Premature ovarian failure may occur as an isolated clinical finding in women with premutations.
Follicle-stimulating hormone (FSH) levels are elevated in these women even before the onset of
premature ovarian failure.
2. Women who are infertile and have prematurely elevated FSH levels should be considered for
carrier status testing of the FMR1 premutation.

Diagnosis
1. The diagnosis of fragile X syndrome is confirmed by molecular genetic testing of the FMR1 gene.
Prenatal testing is available.
2. FMR1 is characterized by a repetitive CGG trinucleotide sequence, which is repeated six to 50
times in unaffected persons. A full mutation consists of more than 200 CGG repeats in the FMR1
gene, plus hypermethylation, which leads to an inability to produce the FMR1 protein. Almost all
males and more than one half of females with full mutations have fragile X syndrome.
3. Premutation carriers, who have between 50 and 200 CGG repeats, are not cognitively affected
but may have physical or psychiatric findings. In addition, they are susceptible to developing
premature ovarian failure and FXTAS.
4. Rarely, the fragile X phenotype occurs in a premutation carrier if hypermethylation is present.
Conversely, the phenotype may be absent in a person with a full mutation without
hypermethylation, confirming that fragile X syndrome results from the absence of FMR1 protein.
Genetic Counseling and Inheritance
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1. Fragile X syndrome is an X-linked inherited disorder. It is important to diagnose affected patients
as early as possible to provide early intervention and supportive care (i.e., specific developmental
therapy and an individualized education plan) and to inform parents for further family planning.
2. One half of families in a 2002 survey reported having an additional child with fragile X syndrome
before the older affected child was diagnosed.
3. Family history collection should include questions about other family members, with particular
attention to developmental delay, mental retardation, and psychiatric disorders. In addition, a
family history of women with premature ovarian failure and men with FXTAS should be
ascertained.
4. A positive family history in a proband with developmental delay should prompt consideration of
genetic testing of the FMR1 gene.
5. The American College of Medical Genetics recommends testing, regardless of family history, for
all males and females with mental retardation of unknown etiology.
Management
1. Currently, treatment is symptomatic. Individuals diagnosed with FXS should be provided
with special educational services to meet their specific cognitive disabilities, PT/OT and
consultation with a speech/language pathologist. Seizures should be controlled with
carbamazepine or valproic acid. FXS patients may also require referral to a child psychiatrist for
treatment of ADHD, either with methylphenidate or clonidine and guanfacine. SSRIs can be used
to treat anxiety, risperidone can be used for agression and low dose aripiprazole for behavioral
problems.
2. As more has been learned about the biochemical pathways FRMP is involved in, new therapeutic
targets have been identified. Pilot studies and small RCTs have shown promising results in
mGluR5 (metabotrophic glutamate receptor 5) antagonists, Lithium (involved in mGluR5
signaling) and GABA agonists, but more trials are needed.
3. Additionally, women with a child or a family history of Fragile X should be evaluated for carrier
status and may undergo pre-implantation or prenatal genetic testing.

SEX DIFFERRENTIATION AND SEX DETERMINATION
Components of reproductive system:
1. Sex chromosomes
2. Gonads
3. Internal genitalia
4. External genitalia
sex Chromosomes Gonad Internal genitalia External genitalia
Male XY Testis Wolffian (Epididymis, Vas Penis, scrotum
deferens)
Female XX Ovary Mullerian (Uterus, Fallopian Clitoris, vulva
tube)

Sex development can be divided into two sequential processes:
1. Sex determination – refers to the undifferentiated gonads that commit to developing into either
testes or ovaries
2. Sex differentiation – i.e. gonadal hormones act on the internal and external genitalia resulting in
differentiation into male and female
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3. All fetuses develop with a female blueprint. Conversion female into male fetus is done by Y
chromosome. In the absence of Y chromosome, the bipotential gonad will develop into an ovary
aided by the expression of other ovary-specific genes
4. The TESTES are essential for male sexual differentiation, but the ovaries are NOT necessary for
female differentiation
5. The genetic sex of the embryo is established at with the inheritance of an X or Y chromosome
from the father
6. The Y chromosome, through the testis-determining gene Sry, acts dominantly to trigger
differentiation of testes from the indifferent gonads that would otherwise develop as ovaries
7. If Sry gene is translocated to X chromosome in the father an XX offspring can develop into male
phenotype. Similarly if Sry gene undergoes deletion in the father, an XY offspring can develop into
female phenotype.
Gonads:
1. There are 3 different cell lineages of bipotential fate present in the gonads
2. The supporting cell lineage will give rise to Sertoli cells in the testis and follicle cells in the ovary.
3. The steroidogenic cell lineage produces the sexual hormones that will contribute to the
development of the secondary sexual characteristics of the embryo. In the male these are the
Leydig cells in the female the Theca cells.
4. The connective cell lineage will contribute to the formation of the organ as a whole
Male differentiation:
1. Sertoli cells secrete anti-Mullerian hormone (AMH) &Leyding cells secrete testosterone.
2. Testosterone secretion is stimulated by placental human chorionic gonadotropin (hCG), which
peaks at 8-12 wk. In the latter half of pregnancy, lower levels of testosterone are maintained by
luteinizing hormone (LH) secreted by the fetal pituitary.
3. AMH causes the regression of the Mullerian structures that would otherwise develop into the
Fallopian tubes, uterus, cervix and the upper third of the vagina in the female
4. Testosterone stimulates the Wolffian ducts will develop into male internal genitalia: – Epididymis
– vas deferens – vesicula seminalis
5. The development of external male genitalia is dependent on the conversion of testosterone to
dihydrotestosterone (DHT) by the enzyme 5a-reductase
6. Under the influence of DHT, the genital tubercle will form the penis, the urethral folds will form
the urethra & the genital folds form the scrotum
7. DHT is also produced via an alternative biosynthetic pathway from androstanediol, and this
pathway must be intact for normal and complete prenatal virilization to occur. A functional
androgen receptor, produced by an X-linked gene, is required for testosterone and DHT to induce
these androgen effects.
Female differentiation:
1. A chromosome complement of 46,XX will lead to the development of ovaries. Both the long and
short arms of the X chromosome contain genes for normal ovarian development.
2. DAX1, SOX3 and Wnt4 are the three candidate genes currently known to suppress testicular
differentiation. DAX1 and SOX3 inhibit SF1 and SOX9, preventing Sertoli cell differentiation,
favoring granulosa cell development.
3. In female fetuses, Wolffian structures regress in the absence of testosterone and Mullerian ducts
persist in the absence of AMH.
4. Wnt4 arrests the Leydig cell precursors and induces development of Mullerian ducts into fallopian
tubes, uterus, cervix and upper third of vagina.
5. FOXL2 helps in follicular development in the ovary and in suppression of somatic testis formation
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in genetic females.
6. Female sexual development does not depend on the presence of ovaries or hormones. The lack of
testosterone will lead to a clitoris, urethra, the rest of the vagina and the labia for the female.

SEXUAL MATURITY RATING (SMR)
1. Generally, adolescence begins at age 11–12 years and ends between ages 18 and 21. Most
teenagers complete puberty by age 16–18 years;
2. Most girls enter puberty at age 9 or 10 years, reach menarche on average at 12 1/2, and achieve
full fertility by age 15 or 16. Boys begin pubertal development somewhat later than girls, at age 11
or 12 years, produce sperm (spermarche) and have their first ejaculation on average at age 13 or
14, and achieve full fertility at approximately age 16.
3. Puberty consists of a series of predictable events. The staging system is published by Marshall and
Tanner and the sequence of changes, commonly referred to as "Tanner stages".
4. Sexual maturity rating (SMR) includes stage of pubic hair growth, penis and testis development in
boys, and breast maturation in girls.
Tanner staging in Boys:
SMR STAGE PUBIC HAIR PENIS TESTES
1 None Preadolescent Preadolescent
2 Scanty, long, slightly pigmented Minimal change/ Enlarged scrotum,
Enlargement pink, texture altered
3 Darker, starting to curl, small amount Lengthens Larger
4 Resembles adult type, but less Larger; glans and Larger, scrotum
quantity; coarse, curly Breadth increase in size Dark
5 Adult distribution, spread to medial Adult size Adult size
surface of thighs
Tanner staging in Girls:
STAGE PUBIC HAIR BREASTS
1 Preadolescent Preadolescent
2 Sparse, lightly pigmented, straight, medial Breast and papilla elevated as small
border of labia mound; diameter of areola increased
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3 Darker, beginning to curl, increased amount Breast and areola enlarged,
No contour separation
4 Coarse, curly, abundant, but less than in adult Areola and papilla form secondary
mound
5 Adult feminine triangle, spread to medial Mature, nipple projects, areola part
surface of thighs of general breast contour

DISORDERS OF SEX DEVELOPMENT (DSD)
1. DSDs are defined as conditions involving the following elements:
a. Congenital development of ambiguous genitalia (e.g., 46,XX virilizing congenital adrenal
hyperplasia; clitoromegaly; micropenis)
b. Congenital disjunction of internal and external sex anatomy (e.g., Complete Androgen
Insensitivity Syndrome; 5-alpha reductase deficiency)
c. Incomplete development of sex anatomy (e.g., vaginal agenesis; gonadal agenesis)
d. Sex chromosome anomalies (e.g., Turner Syndrome; Klinefelter Syndrome; sex chromosome
mosaicism)
e. Disorders of gonadal development (e.g., ovotestes)
2. DSD conditions usually present with:
a. Atypical genitalia in the newborn period or as
b. Delayed puberty in an adolescent.
3. DSDs include anomalies of
a. The sex chromosomes,
b. The gonads,
c. The reproductive ducts (internal genitalia)
d. The external genitalia.
4. The current system for the classification of DSD was introduced in the Chicago Consensus in 2005.
There are three broad groups:
a. 46,XX DSD
b. 46,XY DSD
c. Sex chromosome DSD
CLASSIFICATION:
1. 46,XX DSD: (formerly known as female pseudohermaphroditism)
a. Androgen Exposure
i. Fetal/Fetoplacental Source
1. 21-Hydroxylase (P450c21 or CYP21) deficiency
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2. 11β-Hydroxylase (P450c11 or CYP11B1) deficiency
3. 3β-Hydroxysteroid dehydrogenase II (3β-HSD II) deficiency
4. Cytochrome P450 oxidoreductase (POR)
5. Aromatase (P450arom or CYP19) deficiency
6. Glucocorticoid receptor gene mutation
ii. Maternal Source
1. Virilizing ovarian tumor
2. Virilizing adrenal tumor
3. Androgenic drugs
b. Disorder of Ovarian Development
i. XX gonadal dysgenesis
ii. Testicular DSD (SRY+, SOX9 duplication)
c. Undetermined Origin
i. Associated with genitourinary and gastrointestinal tract defects
2. 46,XY DSD (formerly known as male pseudohermaphroditism)
a. Defects in Testicular Development
a. Denys-Drash syndrome (mutation in WT1 gene)
b. WAGR syndrome (Wilms tumor, aniridia, genitourinary malformation, retardation)
c. Deletion of 11p13
d. Campomelic syndrome (autosomal gene at 17q24.3-q25.1) and SOX9 mutation
e. XY pure gonadal dysgenesis (Swyer syndrome)
f. Mutation in SRY gene
g. XY gonadal agenesis
h. Unknown cause
b. Deficiency of Testicular Hormones
a. Leydig cell aplasia
b. Mutation in LH receptor
c. Lipoid adrenal hyperplasia (P450scc or CYP11A1) deficiency; mutation in StAR
(steroidogenic acute regulatory protein)
d. 3β-HSD II deficiency
e. 17-Hydroxylase/17,20-lyase (P450c17 or CYP17) deficiency
f. Persistent müllerian duct syndrome because of antimüllerian hormone gene
mutations or receptor defects for antimüllerian hormone
c. Defect in Androgen Action
a. Dihydrotestosterone deficiency because of 5α-reductase II mutations or
AKR1C2/AKR1C4 mutations
b. Androgen receptor defects:
i. Complete androgen insensitivity syndrome
ii. Partial androgen insensitivity syndrome (Reifenstein and other syndromes)
c. Smith-Lemli-Opitz syndrome (defect in conversion of 7-dehydrocholesterol to
cholesterol, DHCR7)
3. Ovotesticular DSD
a. XX
b. XY
c. XX/XY chimeras
4. Sex Chromosome DSD
a. 45,X (Turner syndrome and variants)
 14
b. 47,XXY (Klinefelter syndrome and variants)
c. 45,X/46,XY (mixed gonadal dysgenesis, sometimes a cause of ovotesticular DSD)
d. 46,XX/46,XY (chimeric, sometimes a cause of ovotesticular DSD)
Evaluation of Ambiguous genitalia and determination of sex
A. Definition. Ambiguous genitalia are present when the sex of an infant is not readily apparent after
examination of the external genitalia.
B. Incidence.
a. Congenital adrenal hyperplasia is often considered the most common cause with anincidence
quoted from 1 in 14,000 to 1 in 28,000, followed by androgen insensitivity and mixed gonadal
dysgenesis.
b. Hypospadias has a frequency of about 1 in 300 births, but only a minority of these patients
has a disorder of sex development
C. Embryology.
a. The early fetus, regardless of the genetic sex (XX or XY), is bipotential and can undergo either
male or female differentiation. The innate tendency of the embryo is to differentiate along
female lines.
b. Development of the gonads.
i. Gonadal development occurs during the embryonic period (the third through the
seventh to eighth weeks of gestation).
ii. Testicular differentiation. Gonadal differentiation is determined by the absence or
presence of the Y chromosome. If the Y chromosome (more specifically, the sex-
determining region of the Y or SRY gene) is present, the gonads differentiate as
testes. The testes then produce and release testosterone, which is converted to
dihydrotestosterone (DHT) in the target organ cells by 5α-reductase. DHT induces
male differentiation of the external genitalia. The testes descend behind the
peritoneum and normally reach the scrotum by the eighth or ninth month.
iii. Ovarian differentiation. In the female fetus, where the Y chromosome/SRY gene is
absent, the gonads form ovaries (even in 45,X Turner syndrome, histologically
normal ovaries are present at birth). As ovaries do not produce testosterone,
female differentiation proceeds. Two X chromosomes are needed for
differentiation of the primordial follicle. If part or all of the second X chromosome is
missing, ovarian development fails, resulting in atrophic, whitish, streaky gonads by
1–2 years of age.
c. Development of external genitalia.
i. This part of sexual differentiation occurs in the fetal period, beginning in the
seventh week of gestation, and proceeds up to the 14th week
ii. Normal male. At ~9 weeks postconceptional age, in the presence of systemic
androgens (especially DHT), masculinization begins with lengthening of the
anogenital distance. The urogenital and labioscrotal folds fuse in the midline
(beginning caudally and progressing anteriorly), leading to the formation of the
scrotum and the penis.
iii. Normal female. In the female fetus, the anogenital distance does not increase. The
urogenital and labioscrotal folds do not fuse and instead differentiate into the labia
majora and minora. The urogenital sinus divides into the urethra and the vagina.
D. Pathophysiology
a. Virilization of female infants (female pseudohermaphroditism).
i. Many belong to this group. They have a 46,XX karyotype, are SRY negative, and
 15
have exclusively ovarian tissue.
ii. The degree of masculinization of the female infant depends on the potency of the
androgenic exposure.
iii. The most common cause of excess fetal androgens congenital adrenal hyperplasia
(CAH) and excessive production of adrenal androgens (dehydroepiandrosterone
and androstenedione) and testosterone.
iv. Most common is 21-hydroxylase deficiency, which causes adrenal hyperplasia, and
excessive production of adrenal androgens (dehydroepiandrosterone and
androstenedione) and testosterone. Two forms of CAH:
1. Classic:
a. a simple virilizing form – cortisol deficiency
b. a salt-losing form – aldosterone deficiency
2. Non classic: mild and delayed onset
v. 11-Hydroxylase enzyme deficiency is less common and associated with salt
retention, volume expansion, and hypertension.
vi. Other, less common causes.
1. Virilizing maternal or fetal tumors
2. Maternal androgen ingestion or topical use.
b. Inadequate virilization of male infants (male pseudohermaphroditism).
i. This condition is caused by:
1. Inadequate androgen production or
2. Incomplete end-organ response to androgen.
ii. These patients have a 46,XY karyotype and exclusively testicular tissue.
iii. Decreased androgen production:
1. 3β-HSD II deficiency
2. 17-Hydroxylase/17,20-lyase (P450c17 or CYP17) deficiency
3. Deficiency of Müllerian-inhibiting substance
4. Testicular unresponsiveness to human chorionic gonadotropin (hCG) and
luteinizing hormone (LH); and
5. Anorchia (absent testes caused by loss of vascular supply to the testis during
fetal life).
iv. Decreased end-organ response to androgen:
1. Defect in the androgen receptor
2. Unknown defect with normal receptors.
v. 5α-Reductase deficiency. Results in failure of the external genitalia to undergo male
differentiation because of the lack of DHT
c. Disorders of gonadal differentiation
i. True hermaphroditism. The presence of both a testis and an ovary (or ovotestes) in
the same individual is a rare cause of ambiguous genitalia. Most individuals with
true hermaphroditism have a 46,XX karyotype, but mosaics of
46,XX/45,X/46,XY/multiple X/multiple Y have all been reported. The appearance of
the genitalia is variable; fertility is poor.
ii. Gonadal dysgenesis
1. Pure gonadal dysgenesis. Characterized by the presence of a streak gonad
bilaterally (complete gonadal dysgenesis) or unilaterally (partial gonadal
dysgenesis).
2. Mixed gonadal dysgenesis. Characterized by the presence of a unilateral
 16
functioning testis and a contralateral streak gonad. All patients have a Y
chromosome and some degree of virilization of the external genitalia. Mixed
gonadal dysgenesis is associated with a high incidence of gonadal malignancy
in mid to late childhood.
d. Chromosome abnormalities, syndromes, and associations.
i. In general, chromosomal abnormalities do not usually lead to genital abnormalities.
However, disruption of normal sex development has been reported occasionally in
trisomies 13 and 18, triploidy, and a number of other chromosomal anomalies.
ii. Single-gene disorders and syndromes such as Smith-Lemli-Opitz syndrome, Rieger
syndrome, CHARGE syndrome (coloboma, heart defects, choanal atresia, retarded
growth and development, genital abnormalities, and ear anomalies),
iii. Camptomelic dysplasia, and others can also be associated with external sexual
ambiguity
iv. VATER/VACTERL (vertebral, anal, tracheal, esophageal, renal dysplasia/vertebral
defects, anal atresia, cardiac malformations, tracheoesophageal fistula, renal
dysplasia, and limb abnormalities) association can include abnormally developed
genitalia.
VI. Clinical presentation.
a. History.
a. Family history of early neonatal deaths (a death in early infancy accompanied by
vomiting and dehydration may be secondary to CAH),
b. Consanguinity of the parents (increased risk for autosomal recessive disorders), and
female relatives with amenorrhea and infertility (male pseudohermaphroditism or
chromosomal anomalies)
c. Ingestion or topical use of drugs during pregnancy (particularly androgens or
progestational agents).
b. Physical examination
a. General examination.
i. Dysmorphic features (syndromes and chromosomal abnormalities),
ii. Hypertension or hypotension, areolar hyperpigmentation, and signs of
dehydration (as signs of CAH).
b. Genitalia. Gonads: The number, size, and symmetry of gonads should be evaluated.
Palpable gonads below the inguinal canal are usually testes. Ovaries are not found in
scrotal folds or in the inguinal region. However, the testes may be intra-abdominal.
c. Phallus length: Measured from the pubic ramus to the tip of the glands, a stretched
penile length in a full-term infant should be ≥2.0 cm.
d. Urethral meatus: Look for hypospadias (usually accompanied by chordee).
e. Labioscrotal folds: Findings can range from unfused labia majora, variable degrees of
posterior fusion, and bifid scrotum to fully fused, normal-appearing scrotum. The
presence of a vaginal opening or urogenital sinus should be determined. A rectal
examination, to determine presence of a uterus, may be considered.
VII. Diagnosis
A. Laboratory studies
1. Initial evaluation. An important test in the initial evaluation is the chromosome analysis. Most
cytogenetic laboratories can now provide preliminary results of a karyotype in a few days.
Fluorescent in situ hybridization techniques (FISH) allow even faster determination of the sex
chromosome status; X- and Y-specific probes are available.
 17
2. Buccal smears are unreliable and therefore obsolete. The remainder of the diagnostic
evaluation depends on the sex chromosome status.
3. Blood for basic biochemical studies can be obtained at the same time as the karyotype,
including 17-hydroxyprogesterone (17-OHP), testosterone, dihydrotestosterone, sodium, and
potassium levels.
4. Normal 46, XX karyotype. This finding implies virilization of a genetic female and is caused by
excessive maternal or fetal androgen. If the mother is not virilized, the infant almost always
has virilizing adrenal hyperplasia. To confirm the diagnosis, measure the following:
a. 17-hydroxyprogesterone (17-OHP). This is the precursor to the enzyme defect in 21-
hydroxylase and 11-hydroxylase enzyme deficiency. In infants with either defect, the
serum or plasma level of 17-OHP will be 100–1000 times the normal infant level.
b. Daily serum measurements of sodium and potassium. Infants with 21-hydroxylase
enzyme deficiency usually have relative or absolute aldosterone deficiency and begin
to demonstrate hyperkalemia at days 3–5 and hyponatremia 1–2 days later. If
hyperkalemia becomes clinically significant before the 17-OHP result is available,
empirical treatment with intravenous saline, cortisol, and fludrocortisone may be
needed.
c. Serum testosterone. About 3% of infants with ambiguous genitalia are true
hermaphrodites, and most have a 46,XX karyotype. If the 17-OHP is not elevated and
there is no maternal virilization, a high testosterone level suggests hermaphroditism
or fetal testosterone-producing tumor.
5. Normal 46,XY karyotype.
a. Testosterone (T) and dihydrotestosterone (DHT). These hormone levels should be
measurable and are higher in newborns than later in childhood. In the male
pseudohermaphrodite, testosterone is low in any defect in testosterone production.
The T-to-DHT ratio should be between 5:1 and 20:1 when expressed in similar units. A
high T-to-DHT ratio suggests 5α-reductase deficiency.
b. Androstenedione levels are measured to diagnose 17-ketosteroid reductase
deficiency.
c. LH and follicle-stimulating hormone (FSH). These hormones are also higher in infancy
than they are in childhood. A diagnosis of gonadotropin deficiency is suspected if
these values are low in a reliable assay but can be confirmed in infancy only if there
are other pituitary hormone deficits. Note that growth hormone and ACTH deficiency
are manifested in the newborn period as hypoglycemia. In primary gonadal defects
and some androgen-resistant states, LH and FSH are elevated.
d. hCG stimulation test. hCG is administered to stimulate gonadal steroid production
when testosterone values are low (as in gonadotropin deficiency or a defect in
testosterone synthesis). A rise in the testosterone level confirms the presence of
Leydig cells and, by implication, testicular tissue. In patients with 5α-reductase
deficiency, the basal T-to-DHT ratio may be normal but elevated after hCG
stimulation.
e. Assessment of pituitary function. If gonadotropin deficiency due to impairment of
pituitary function is suspected (eg, microphallus/micropenis combined with
hypoglycemia), thyroid function tests, growth hormone levels, ACTH stimulation test,
and imaging studies of the pituitary gland may be indicated.
6. Abnormal karyotype.
a. Mixed gonadal dysgenesis with a dysplastic gonad may be present in infants with
 18
abnormal karyotype and abnormal genitalia. Hormone studies are unlikely to be
revealing in this scenario.
b. These techniques may allow detection of SRY gene material in 46,XX phenotypic
males and be useful in determining whether Y material is present in a 45,X individual,
placing the patient at risk for gonadoblastoma.
B. Radiographic studies
1. Ultrasonography to evaluate adrenal and pelvic structures. Although a uterus is sometimes
palpable on rectal examination shortly after birth (because of enlargement in response to
maternal estrogen), ultrasonography seems less invasive. The presence and localization of
gonads may also be clarified by ultrasonography. Adrenal ultrasonography is sufficiently
sensitive to determine adrenal abnormalities in the majority of patients with untreated
adrenal hyperplasia.
2. Contrast studies to outline the internal anatomy (sinography, urethrography,
vesicocystoureterography, and intravenous urography) may be indicated in complex cases
and before reconstructive surgery.
3. Magnetic resonance imaging (MRI) has been used to evaluate patients with disorders of sex
development but, at least in the neonatal period, sensitivity may only be marginally improved
over ultrasound.
VIII. Management
1. General considerations. It is very important to protect the privacy of child and parents while
diagnostic studies are in progress. A multidisciplinary team should assist the patient and
family throughout the diagnostic process and beyond.
2. Once a diagnosis has been established, gender should be assigned and a team of specialists
should supervise medical treatment (steroid replacement, gonadal removal, reconstructive
surgery, etc.) and treatment of psychosocial aspects.
3. Circumcision should be delayed in any infant with a DSD until completion of multidisciplinary
evaluation and gender assignment.
4. Medical management in the neonatal period and early infancy
a. Congenital adrenal hyperplasia.
i. Glucocorticoid therapy. Should be initiated as soon as possible.
Maintenance cortisol replacement therapy is often given orally.
Hydrocortisone is the oral preparation of choice. (See Chapter 148.) Initial
doses usually range from 10 to 20 mg/m2/d given as 3 divided doses and
often require adjustments for growth and during periods of stress.
Alternatively, intramuscular cortisone acetate is sometimes used in children
<6 months of age out of concern that oral hydrocortisone may be absorbed
erratically in these infants.
ii. Mineralocorticoid therapy. Fludrocortisone acetate at a dose of 0.05–0.1
mg daily (given orally) is often used. Unlike hydrocortisone, the dose of
fludrocortisone does not change with increase of body size or during stress.
Some endocrinologists also recommend sodium supplementation (1–5
mEq/kg/d).
5. Incompletely virilized genetic male. Treatment with depo-testosterone might be considered
by the team of specialists depending on the results of the diagnostic evaluation.
IX. Prognosis, gender assignment, long-term care.
It is now believed that prenatal and early exposure of the brain to androgens, if present,
influences gender-specific behavioral patterns and sexual identity in addition to the external
 19
appearance of the genitalia or their future function.

CONGENITAL ADRENAL HYPERPLASIA
1. Synonyms: Adrenogenital syndrome; CAH
2. A group of autosomal recessive disorders resulting from the deficiency of one of the enzymes
required for cortisol synthesis in the adrenal cortex.
a. Cortisol deficiency increases secretion of corticotrophin (ACTH), which, in turn, leads to
adrenocortical hyperplasia and overproduction of intermediate metabolites.
b. In most cases, adrenal hyperplasia also involves a deficiency in aldosterone, which results in
mild to severe loss of body sodium.
c. In some, it also involves overproduction of adrenal androgens, which, in affected females,
results in prenatal virilization with an ambiguous /male external genitalia at birth.
3. Types:
a. 21-hydroxylase deficiency accounts for the vast majority of cases (90%),
b. 11-hydroxylase (5%),
c. cholesterol desmolase,
d. 17-hydroxylase
e. 3-hydroxysteroid dehydrogenase

21- OH DEFICIENCY (commomnest type)
1. Classic 21-hydroxylase deficiency occurs in approximately 1 in 15,000 - 20,000 births
1. More than 90% of CAH cases are caused by 21-hydroxylase deficiency. This P450 enzyme
hydroxylates as follows:
a. progesterone à11-deoxycorticosteroneà aldosterone
b. 17-hydroxyprogesterone à11-deoxycortisolà cortisol
2. More than 90% of mutations causing 21-hydroxylase deficiencies are recombination between
CYP21 and CYP21P.
3. Accumulation of 17-hydroxyprogesterone leads to high levels of androstenedione that are
converted outside the adrenal gland to testosterone. This problem begins in 8-10 wk of gestation
and leads to abnormal genital development in females
 20
4. 21-hydroxylase deficiencies can be divided into classic deficiency with and without salt wasting
(cortisol and aldosterone deficiencies) and non-classic type.
a. Classic
i. Virilizing syndrome- affected females are virilized in utero and present at birth with
cliteromegaly, labial fusion, and normal internal organs. This is the most common
cause of ambiguous genitalia in females.
ii. Salt losing > 75% of 21-OH deficiency has salt losing because there is inadequate
production of aldosterone. These neonates will fail to gain weight, vomit, have
hyponatremia and hyperkalemia, hypoglycemia and be acidotic. They may appear
septic.
b. Non-Classic- will often present later on in childhood with signs and symptoms of androgen
excess.
Affected female infant:
1. Enlargement of the clitoris and by partial or complete labial fusion.
2. The vagina usually has a common opening with the urethra (urogenital sinus).
3. The clitoris may be so enlarged that it resembles a penis; because the urethra opens below this
organ, some affected females may be mistakenly presumed to be males with hypospadias and
cryptorchidism.
4. The severity of virilization is usually greatest in females with the salt-losing form of 21-
hydroxylase deficiencies.
5. The internal genital organs are normal, because affected females have normal ovaries and not
testes and thus do not secrete antimüllerian hormone.
Behavior problems:
1. Prenatal exposure of the brain to high levels of androgens may influence subsequent sexually
dimorphic behaviors in affected females.
2. Girls may demonstrate aggressive play behavior, tend to be interested in masculine toys such as
cars and trucks, and often show decreased interest in playing with dolls.
3. Women may have decreased interest in maternal roles. There is an increased frequency of
homosexuality in affected females.
4. Nonetheless, most function heterosexually and do not have gender identity confusion or
dysphoria. It is unusual for affected females to assign themselves a male role except in some with
the severest degree of virilization.
Untreated female children:
a. The clitoris may become further enlarged in affected females.
b. Although the internal genital structures are female, breast development and menstruation
may not occur unless the excessive production of androgens is suppressed by adequate
treatment.
Male infant:
1. Male infants appear normal at birth. Thus, the diagnosis may not be made in boys until signs
of adrenal insufficiency develop.
2. Because patients with this condition can deteriorate quickly, infant boys are more likely to die
than infant girls. For this reason, all 50 American states and many countries have instituted
newborn screening for this condition.
Untreated male children
1. Child may have rapid somatic growth and accelerated skeletal maturation. Thus, affected patients
are tall in childhood but premature closure of the epiphyses causes growth to stop relatively
early, and adult stature is stunted.
 21
2. Muscular development may be excessive. Pubic and axillary hair may appear, and acne and a
deep voice may develop.
3. The penis, scrotum, and prostate may become enlarged in affected boys; however, the testes are
usually prepubertal in size so that they appear relatively small in contrast to the enlarged penis.
4. Occasionally, ectopic adrenocortical cells in the testes of patients become hyperplastic similarly to
the adrenal glands, producing testicular adrenal rest tumors.
Nonclassic forms:
1. Similar but usually milder signs of androgen excess may occur in non classic 21-hydroxylase
deficiency. In this attenuated form, cortisol and aldosterone levels are normal and affected
females have normal genitals at birth.
2. Males and females may present with precocious pubarche and early development of pubic and
axillary hair.
3. Hirsutism, acne, menstrual disorders, and infertility may develop later in life, but many females
and males are completely asymptomatic.
Adrenomedullary Dysfunction
1. Development of the adrenal medulla requires exposure to the extremely high cortisol levels
normally present within the adrenal gland. Thus patients with classic CAH have abnormal
adrenomedullary function.
2. They may have blunted epinephrine responses, decreased blood glucose, and lower heart rates
with exercise.
3. Ability to exercise is unimpaired and the clinical significance of these findings is uncertain.
LABORATORY FINDINGS
1. Salt-losing disease: hyponatremia, hyperkalemia, metabolic acidosis, and, often, hypoglycemia;
abnormalities can take 10-14 days or longer to develop after birth.
2. Blood levels of 17-hydroxyprogesterone are markedly elevated. However, levels of this hormone
are high during the 1st 2-3 days of life even in unaffected infants and especially if they are sick or
premature.
3. Blood levels of cortisol are low in patients with the salt-losing type of disease. They are often
normal in patients with simple virilizing disease but inappropriately low in relation to the ACTH
and 17-hydroxyprogesterone levels.
4. In addition to 17-hydroxyprogesterone, levels of androstenedione and testosterone are elevated
in affected females;
5. Testosterone is not elevated in affected males, because normal infant males have high
testosterone levels compared with those seen later in childhood.
6. Levels of urinary 17-ketosteroids and pregnanetriol are elevated but are now rarely used clinically
because blood samples are easier to obtain than 24 hr urine collections.
7. ACTH levels are elevated but have no diagnostic utility over 17-hydroxyprogesterone levels.
Plasma levels of renin are elevated, and serum aldosterone is inappropriately low for the renin
level.
8. Diagnosis of 21-hydroxylase deficiency is most reliably established by measuring 17-
hydroxyprogesterone before and 30 or 60 min after an intravenous bolus of 0.125-0.25 mg of
cosyntropin (ACTH 1-24). Nomograms exist that readily distinguish normals and patients with
nonclassic and classic 21-hydroxylase deficiency.
9. Genotyping is clinically available and may help confirm the diagnosis, but it is expensive.
10. Prenatal diagnosis of 21-hydroxylase is possible late in the 1st trimester by analysis of DNA
obtained by chorionic villus sampling or during the 2nd trimester by amniocentesis.
NEWBORN SCREENING
 22
1. Because 21-hydroxylase deficiency is often undiagnosed in affected males until they have severe
adrenal insufficiency, newborn screening is mandatory.
2. These programs analyze 17-hydroxyprogesterone levels in dried blood obtained by heelstick and
absorbed on filter paper cards; the same cards are screened in parallel for other congenital
conditions, such as hypothyroidism and phenylketonuria.
3. The nonclassic form of the disease is not reliably detected by newborn screening.
4. The main difficulty with current newborn screening programs is that to reliably detect all affected
infants, the cutoff 17-hydroxyprogesterone levels for recalls are set so low that there is a very
high frequency of false-positive results
TREATMENT
Glucocorticoid Replacement
1. Cortisol deficiency is treated with glucocorticoids. 15-20 mg/m2/24 hr of hydrocortisone daily
administered orally in 3 divided doses.
2. Double or triple doses are indicated during periods of stress, such as infection or surgery.
Glucocorticoid treatment must be continued indefinitely in all patients with classic 21-hydroxylase
deficiency but may not be necessary in patients with nonclassic disease unless signs of androgen
excess are present. Therapy must be individualized.
Monitoring:
1. Growth: crossing to higher height percentiles may suggest under treatment, whereas loss of
height percentiles often indicates overtreatment with glucocorticoids. Overtreatment is also
suggested by excessive weight gain.
2. Pubertal development should be monitored by periodic examination
3. Skeletal maturation is evaluated by serial radiographs of the hand and wrist for bone age.
4. 17-hydroxyprogesterone and androstenedione, should be measured early in the morning
5. Menarche may be delayed in girls with suboptimal control.
6. Children with simple virilizing disease true precocious puberty may be treated with a
gonadotropin hormone–releasing hormone analog such as leuprolide. Males with 21-hydroxylase
deficiency who have had inadequate corticosteroid therapy may develop testicular adrenal rest
tumors, which usually regress with increased steroid dosage. Testicular MRI, ultrasonography, and
color flow Doppler examination help define the character and extent of disease.
Mineralocorticoid Replacement
1. Patients with salt-wasting disease require mineralocorticoid replacement with fludrocortisone.
Infants: 0.1-0.3 mg daily in 2 divided doses with sodium supplementation (sodium chloride, 8
mmol /kg) in addition to the mineralocorticoid. Older infants and children: 0.05-0.1 mg daily of
fludrocortisone
2. Therapy is evaluated by monitoring of vital signs; tachycardia and hypertension are signs of
overtreatment with mineralocorticoids.
3. Serum electrolytes should be measured frequently in early infancy as therapy is adjusted. Plasma
renin activity is a useful way to determine adequacy of therapy; it should be maintained in or near
the normal range but not suppressed.
Surgical Management of Ambiguous Genitals
1. Virilized females usually undergo surgery between 2-6 mo of age. The clitoris is reduced in size,
with partial excision of the corporal bodies and preservation of the neurovascular bundle;
however, moderate clitoromegaly may become much less noticeable even without surgery as the
patient grows.
2. Vaginoplasty and correction of the urogenital sinus usually are performed at the time of clitoral
surgery; revision in adolescence is often necessary.
 23
3. Female sexual dysfunction increases in frequency and severity in those with the most significant
degrees of genital virilization and with the degree of enzymatic impairment (prenatal androgen
exposure) caused by each patient’s mutations.
Sex assignment:
1. Confused gender identity is not common with CAH; it occurs mostly in females with the salt-
wasting form of the disease and the greatest degree of virilization.
2. Educate the patient, family, and others about how to deal with the intersex condition.
3. They propose that surgery should be delayed until the patient decides on what, if any, correction
should be performed.
4. Severely virilized genotypic (XX) females raised as males have generally functioned well in the
male gender as adults.
5. Adrenalectomy (with hormone replacement) may be an alternative to standard medical hormone
replacement therapy, but patients treated in this way may be more susceptible to acute adrenal
insufficiency.
Prenatal Treatment
1. Mothers with pregnancies at risk are given dexamethasone, a steroid that readily crosses the
placenta, in an amount of 20 μg/kg prepregnancy maternal weight daily in 2 or 3 divided doses.
This suppresses secretion of steroids by the fetal adrenal, including secretion of adrenal
androgens.
2. If started by 6 wk of gestation, it ameliorates virilization of the external to determine the sex and
genotype of the fetus; therapy is continued only if the fetus is an affected female.
3. Chorionic villus biopsy is then performed to determine the sex and genotype of the fetus; therapy
is continued only if the fetus is an affected female.
4. Maternal side effects of prenatal treatment have included edema, excessive weight gain,
hypertension, glucose intolerance, cushingoid facial features, and severe striae.

PREMALIGNANT LESIONS OF VULVA
Terminology and classification:
1. The term “carcinoma in situ” (CIS) was introduced by Broders in 1932 to describe precancerous
lesions of the squamous epithelium.
2. The term “dysplasia” goes back to Reagan, who first used it in 1953 to describe all atypical and
abnormal differentiations of the squamous epithelium
3. The Cervical intraepithelial neoplasia (CIN) terminology, which divides the lesions into 3 main
groups and is still widely used today, was introduced by Richart in 1968. He used it to describe a
continuous progression from mild (CIN 1) to moderate dysplasia (CIN 2) and finally to severe
dysplasia or carcinoma in situ (CIN 3).
4. The classification schemes and terminologies for intraepithelial neoplasia have evolved
significantly over the past several decades. For many years, the vulvar intraepithelial neoplasia
(VIN) nomenclature, introduced in the 1980s by the International Society for the Study of
Vulvovaginal Disease (ISSVD), in which lesions were stratified into three tiers of severity was the
dominant classification scheme.
5. As the role of HPV in VIN lesions was elucidated, subsequent revision by the ISSVD has designated
HPV-associated VIN, which comprises the majority of cases, as VIN of the usual type (uVIN) and
the less common HPV-independent lesions as VIN, differentiated type (dVIN).
6. The most recent revision to the terminology has been introduction of the use of a unified
terminology for all HPV-related intraepithelial squamous lesions throughout the lower anogenital
 24
tract, which replaces the previously used "intraepithelial neoplasia (CIN)" term with the term
"squamous intraepithelial lesion (SIL)".
Premalignant vulval lesions include:
1. Vulval intraepithelial neoplasia (VIN).
2. Paget’s disease.
3. Lichen sclerosus.
4. Squamous cell hyperplasia.
5. Condyloma accuminata
Vulval intraepithelial neoplasia (VIN)
The different degrees of VIN are:
1. VIN–I: Corresponds to mild cellular atypia. The lesion is limited to the deeper one-third of the
epithelium.
2. VIN–II: Corresponds to moderate cellular atypia. The lesion is limited up to middle-third of the
epithelium.
3. VIN–III: Corresponds to severe cellular atypia and carcinoma-in-situ. The abnormal cells involve
whole thickness of the epithelium. There is no stromal invasion.
The following facts are related to VIN:
1. It is more frequent in patients in the age group 20–40 years, i.e. at a younger age group compared
to vulval carcinoma. The average age is as low as 33 years.
2. It is often related with STD such as condyloma accuminata, herpes simplex virus II, gonorrhea,
syphilis or Gardnerella vaginalis.
3. HPV associated VIN is seen more in young women. HPV 6 and 11 are associated with vulval
condylomas. HPV 16, 18, 31, 35 are associated with VIN lesions.
4. There is increased prevalence of associated CIN (10–80%).
5. Regression frequently occurs in young woman, during pregnancy or when it is caused by viral
infection.
6. Rarely progresses to invasive carcinoma (< 10%). It takes 20–30 years (for CIN 10–15 years) to
progress to invasive cancer.
DIAGNOSIS
1. The chief complaints may be pruritus vulvae, lump or bleeding from a vulval ulcer. It may be even
symptomless.
2. Local examination reveals a lesion in the vulva with white, grey, pink or dull red color. Lesions
look rough, raised from the surface and often multifocal.
3. Application of 5 percent acetic acid turns VIN lesions white with punctuation and mosaic patterns.
These changes are best seen with a colposcope.
4. Cytologic screening of the vulva is not useful and unreliable.
5. Confirmation of diagnosis is done by biopsy. Usually 3–5 mm diameter dermal punch is taken
under local anesthetic. The small amount of bleeding is controlled using Monsel’s solution
(ferrous sulphate).
6. Larger biopsy when required may be taken using a scalpel. Multiple site biopsies are useful.
7. A complete pelvic examination is to be done. To exclude vaginal or cervical neoplasia, cytologic
evaluation has to be performed.
Histology
1. The cells exhibit features of malignancy. There is complete loss of polarity and stratification.
Cellular immaturity, nuclear abnormalities and mitotic activity vary depending upon the grade of
VIN.
2. There is hyperkeratosis, acanthosis (hyperplasia of epidermis) and chronic inflammatory cell
 25
infiltration. Koilocytes may be present. The rete ridges are large and elongated. Basement
membrane remains intact. There is no evidence of involvement of the dermis.
Treatment of VIN
1. The fact remains that VIN has got low malignant potential. The risk of progression to invasive
disease is less than 10 percent. Hence conservative management is generally accepted.
Medical
1. Medical treatment is done for young woman to control the symptoms. Topical fluorinated steroid
ointment can be applied twice daily for a period of about 6 months. Commonly used
preparations are 0.1 percent triamcinolone acetonide, or 0.1 percent betamethasone valerate.
Local application of chemotherapy in the form of 5 percent 5-fluorouracil (5-FU) cream may be
used in recurrent lesions. The result is however unsatisfactory.
2. Whatever therapy is employed, it is mandatory for regular follow up. Biopsies to be taken freely
whenever an abnormal area is detected.
Surgery:
The following are the types of surgery:
1. Local excision: Wide local excision with 1 cm margin is reserved in young patient with localized
lesion.
2. Laser therapy: CO2 laser vaporization is gaining increasing popularity in the treatment of vulval
epithelial atypia. It gives better cosmetic results with lower recurrence rate.
3. In multifocal lesion, simple excision of the individual lesion(s) is done. Skinning vulvectomy is less
commonly done. Cryosurgery and loop electrosurgical excision procedure (LEEP) has been used.
4. Simple vulvectomy: It is employed in diffuse type especially in postmenopausal women. Long-
term follow-up is needed as the risk of recurrence is high (40–70%).
PAGET’S DISEASE
1. This is a special type of VIN. The lesion grows horizontally within the epidermis. The rete ridges
tend to push into the dermis without actually penetrating it. The characteristic histologic picture
is presence of Paget cells in the epidermis. The cells are large - round or oval in shape with
abundant pale cytoplasm.
2. There may be presence of mucopolysaccharides in most of the cells. Nuclear mitotic figures are
rare. Associated adenocarcinoma of apocrine gland (adenocarcinoma in situ) is present in about
10 percent of the cases. There is high incidence (30%) of associated carcinomas of other organs
(breast, cervix, ovary, GI tract and bladder).
3. Symptoms are mainly pruritus, vulvar soreness, pain or bleeding.
4. Local examination reveals—labia majora appear red, scaling, with elevated lesion. There may be
associated white lesions. The skin is usually indurated.
Treatment
1. Simple vulvectomy is done. Multiple biopsies are to be taken to exclude associated
adenocarcinoma of the apocrine glands.
2. If it is found positive, bilateral lymph node dissection should be done at a second stage.
LICHEN SCLEROSUS:
1. Lichen sclerosus et atrophicus (LSA) is an infrequent, chronic atrophic disease most commonly
affecting the vulva of postmenopausal white women,2,3 although it may appear in any age, in all
races and both sexes4
2. The epithelium is metabolically active. It usually occurs following menopause; may occur even in
childhood but in that case, it resolves after menarche.
3. Distribution of lesion: The entire vulva is involved. Lesion encircles the vestibule. It involves
clitoris, labia minora, inner aspects of labia major and the skin around the anus. It is usually
 26
bilateral and symmetrical in a figure of eight distribution. It does not involve the vestibule or
extend into the vagina or anal canal. It may even extend to the perineum and beyond the
labiocrural folds to the thighs. It may also affect other parts of the body (trunk and limbs—18%).
The distribution of lesion is thus wider.
Histology:
1. The epithelium is thin with epidermal atrophy. At times, there is hyperkeratosis, parakerotosis,
acanthosis and elongation of rete ridges with evidence of collagen hyalinization. Fibroblasts are
absent.
2. There is presence of inflammatory cells including lymphocytes and plasma cells
Clinical features:
1. Pruritus is more than soreness. There is dyspareunia (excluding childhood), sleeplessness and
dysuria. Dyspareunia is due to stenosis of the vulval outlet. The skin is thin and looks white.
Inflammatory adhesions of the labia minora and fusion may cause difficulty with micturition and
even retention of urine. There may be narrowing of vaginal introitus. Pruritus is related to active
inflammation with erythema. Scratching results in subepithelial hemorrhages (ecchymosis).
Diagnosis:
1. The diagnosis should be confirmed by biopsy having characteristic changes. Biopsy can be taken
with a punch biopsy forceps (keyes) under local anesthetia as an outpatient procedure.
2. Monsel’s solution (ferric subsulfate) is applied to control bleeding.
Management:
1. The risk of malignancy is about 1-4 percent.
2. Ultrapotent topical steroids clobetasol is very effective. Local application has to be continued
nightly daily for 1 month followed by alternate night for the second month and thereafter twice
weekly. Usually, there is improvement following topical use of clobetasol for a period of 3
months.
3. Lesions resistant to corticosteroids need treatment with tacrolimus and pimicrolimus. There are
immunosuppressants and calcineurin inhibitors. Surgery and CO2 laser vaporization may be
needed to release adhesion for the treatment of urinary retention or narrow vaginal introitus.
SQUAMOUS CELL HYPERPLASIA:
(Hyperplastic Dystrophy, Previously Known as Leukoplakia)
1. Chronic irritation or chronic vulvovaginal infection often leads to benign epithelial thickening and
hyperkeratosis. Some of these women suffer from autoimmune diseases such as diabetes,
thyroiditis, achlorhydria.
2. During the acute phase, the lesions may appear red and moist due to secondary infection. As
epithelial thickening develops, the environment of the vulva causes maceration and a raised
white lesion which may be circumscribed or diffuse; it looks rubbery. It may involve any part of
the vulva, perianal area, perineum or skin of the adjacent thighs. These lesions have also been
designated lichen simplex chronicus or neurodermatitis.
3. Patients suffer from pruritis, soreness, discharge and dyspareunia. The woman is often
premenopausal. The lesion begins as white polygonal papules which coalesce to form plaques
giving the appearance of being ‘splashed with white wash’—fissures may develop due to
scratching.
4. Microscopic examination reveals irregular down growth of the rete pegs deep into the dermis.
The cells of the basal layers show active mitosis, the prickle cell layer is increased in thickness, and
there is a heavy accumulation of keratin on the surface. The dermis reveals infiltration with
inflammatory cells.
Management:
 27
1. Ten to thirty per cent of these cases develop malignant change. Initial treatment with oestrogens
is worthwhile. Oral administration of 0.625 mg of conjugated equine oestrogen (Premarin) helps
to control vulval pruritus. Bland local medicaments like Calamine lotion, crotamine or zinc oxide
paste are soothing. In case of suspected superadded inflammation, steroid ointment containing
1% hydrocortisone, betamethasone, fluocinolone with or without antimicrobial agents like
neomycin, Soframycin (antibiotic), miconazole or chiniofon (antifungal) are very useful.
2. A prescription for a mild sedative at bedtime to ensure adequate rest helps recovery and prevents
patients from scratching. Two per cent lignocaine ointment also relieves pain. Clobetasol 0.05%
cream is also used.
3. In case malignancy is suspected, multiple-site biopsies are mandatory. Lesser degrees of dysplasia
require extended observation, but in more advanced lesions surgical excision is indicated to
relieve pruritus as well as to remove the potential site of malignancy. Colposcopic inspection
using acetic acid and toluidine blue is desirable. One per cent aqueous toluidine blue is applied
and washed off after 1 min with 1% acetic acid. Blue areas are biopsied.
CONDYLOMATA ACUMINATA:
1. Also called venereal warts, these are caused by the HPV, which is a small DNA double-ended virus.
These warts spread diffusely over the whole of the vulval area. The verrucous growths may
appear discrete or coalesce to form large cauliflower-like growths. They affect the skin of the
labia majora, perineum, perianal region and vagina.
2. The growths are seen in women of the childbearing age and are mainly sexually transmitted.
Vaginal discharge, oral contraceptives and pregnancy favour their growth. There are several
varieties of the HPV of which HPV 6, 11, 16 and 18 as well as 31, 32 and 33 are of significance to
the gynaecologist. HPV 6, 16 and 18 are implicated in the development of condyloma
acuminatum and cancers of the cervix and vulva.
3. The presence of koilocytes constitutes the histological marker for the virus. Apart from koilocytes,
other histological features are perinuclear halo, multinucleation, organophilic cytoplasm
acanthosis and chronic inflammatory infiltrate.
4. Dysplasia may be seen in warts in elderly women. The typing of virus is based on DNA, DNA
hybridization and polymerase chain reaction (PCR). A small DNA virus, 55 nm in diameter, is
epitheliotropic and contributes to 15% of all cancers. In young women, the infection is transient in
90% and disappears without any alteration in DNA. In older women, it often persists and
progresses to carcinoma in situ in 30% cases in 1–3 years and cancer of the cervix, both adeno-
carcinoma and squamous cell cancer.
5. Condyloma is associated with vulval, vaginal and cervical cancers in 20% cases. Liver and cervical
cancers account for about 80% virus-related cancers.
Diagnosis
1. Colposcopic study of this lesion aided by acetic acid application is important in the diagnosis of
lesions on the cervix and 1% toluidine blue staining for the vulval lesions. The abnormal vulval skin
with the abnormal nuclei retains the blue dye, whereas the normal skin allows the dye to be
washed off. Acetic acid can cause burning in the vulva and it should be diluted to 50% before use.
Vulval skin is first smeared with water-soluble K-Y jelly and treated with dilute acetic acid. The
vascular pattern is studied. The abnormal areas stained with toluidine blue are biopsied.
2. Cytology. Koilocytes, with perinuclear halo, multinucleation and orangeophilic cytoplasm.
3. Histology. Acanthosis, chronic inflammatory infiltration, and sometimes dysplasia cells.
4. Viral tests. DNA test, hybridization, PCR staining. CD4 count shows immune functioning.
5. Colposcopic Findings
Meisels described colposcopic appearance of condylomas as patches of raised projection of
 28
acetowhite epithelium with speckled appearance. Immunochemical technique can demonstrate
viral antigen in the tissue sections.
Treatment
1. Young women with flat condyloma may be observed for 6 months, especially when it develops
during pregnancy, because the lesions often disappear spontaneously. Local application of
podophyllin 25% in alcohol or podophyllin 20% in tincture benzoin for 6 h daily or 25%
trichloracetic acid plus 5% fluorouracil causes sloughing off of small warts in 3–4 days in 70–80%
cases. The treatment may need to be repeated weekly as the warts recur at 3–6 weeks’ interval.
Local podophylline cream (podofilox) is also available.
2. This treatment is, however, contraindicated in the first trimester of pregnancy because the drug is
absorbed into the circulation and is cytotoxic causing abortion and peripheral neuropathy. This
treatment is also contraindicated in vaginal and cervical lesions because of severe inflammatory
reaction provoked at these sites.
3. The larger lesions are best removed by diathermy loop or laser ablation. The surgical excision of a
localized growth is another alternative. Associated syphilis and malignancy need to be excluded.
The husband should be treated simultaneously or protected from infection by advising the use of
condoms. Vulval and vaginal warts during pregnancy mandate caesarean section to avoid
papilloma laryngitis in the neonate.
4. Lately, Ikic et al advocated interferon local ointment or cream or intralesional injection. The
cream is applied four to five times daily 1 g each time (1 g contains 2 x 106 IU), with total daily
dose of 6 g for 8 weeks. Ninety per cent lesions regress by then.
5. Intramuscular injection of 2 x 106 IU of interferon daily for 10 days yields 90% success. Side
effects are fever, myalgia and headache. Cream is preferred to injection as the latter is painful.
Interferon inhibits the viral and cellular growth.
6. Apart from surgery, the warts can be removed by cryosurgery, diathermy or laser. Needless to say
that biopsy is mandatory to rule out malignancy.
7. Pap smear of the cervix is also required to rule out cervical malignancy.
8. Other measures include the following:
a. Improve body immunity with antioxidants such as vitamin C and folic acid.
b. No smoking.
9. Inosiplex is immunomodulator used as adjunct to conventional therapy. Orally, it is given 5 mg/kg
daily for 12 weeks. About 20% complete response and 40% partial response is reported.
10. Imiquimod cream applied three times a week for 4 months cures 75% cases, but recurrence
occurs in 15% cases. Some develop local erythematous reaction to the cream
Vaccines
a. Vaccine at 0, 1 and 6 months before exposure to sexual activity in adolescent girls and boys are
available, though expensive. Bivalent vaccine against HPV 16 and 18 is known as Cervarix.
b. Quadrivalent vaccine against HPV 6, 11, 16 and 18 is known as Gardasil or Silgard. The high cost
of vaccine precludes the prophylactic use in general population as of today.
c. Cervarix is given at 0, 1 and 6 months. Gardasil at 0, 2 and 6 months.

MOSAICISM
Introduction:
1. Chromosomal mosaicism is defined as the presence of two or more chromosomally distinct cell
lines within an individual. At its core, chromosomal mosaicism is the failure of chromosomes to
properly segregate during mitosis, leading to the gain or loss of whole chromosomes, a
phenomenon known as aneuploidy.
 29
2. Chromosomal mosaicism has been implicated in genetic diseases, miscarriages and
preimplantation embryo wastage. Moreover, mosaicism has been shown in cancer and in an
increased incidence of trisomy 21 conceptions, and has been associated with aging.
General mosaicism
General mosaicism is the presence of a two or more cell lines throughout the entire organism. In
order for the mosaic cell lineage to be present within the entire organism, the aneuploidy
(abnormal no of chromosome) in question must derive from a mitotic event during the first days
of embryonic development, prior to any cellular differentiation. At this stage, mosaicism has been
found to range between 65 and 70%.
Confined mosaicism
Confined mosaicism refers to chromosomal mosaicism that is only present in a particular area.
For example, confined chromosomal mosaicism has been reported in the brain, placenta and
gonads, amongst other places.
MECHANISMS
1. There are three main mechanisms by which chromosomal mosaicism can occur leading to a gain
and/or loss of chromosomes: nondisjunction, anaphase lagging and chromosome gain referred to
as endoreplication.
2. Endoreplication and anaphase lagging account for the majority of chromosomal errors during
embryonic development, while nondisjunction occurs to a lesser extent.
Non-disjunction
1. Non-disjunction is the failure of sister chromatids to separate during mitosis. Instead of
separating, the entire chromosome (two chromatids) is pulled to one cell, creating a cell with a
monosomy and another cell with a trisomy.
2. If non-disjunction occurs prior to cell differentiation, for example in a preimplantation embryo, a
general mosaic is created. Non-disjunction can also lead to a confined mosaicism. For example, if
the non-disjunction event occurs in the trophoblast, after differentiation, then only the placenta
will contain the mosaic cell lines, while the embryo proper could be euploid.
Anaphase lagging
1. Anaphase lagging is the failure of a single chromatid to be incorporated into the nucleus resulting
in a monosomy of that chromosome in one cell and a disomy in the corresponding chromosome
in the other cell. Anaphase lagging occurs when the chromatid fails to attach to the spindle or
when the chromatid attaches to the spindle but then fails to be incorporated in the nucleus.
2. If this mechanism occurs prior to differentiation, then the organism will contain two distinct cell
lines, thereby creating a general mosaic. If this event occurs after differentiation in the
trophoblast, then the placenta will contain a normal and monosomic cell line.
Endoreplication
Endoreplication is the replication of a chromosome without division. This results in a trisomic
chromosome in one cell and a disomic chromosome in the other.
Uniparental disomy
1. As UPD implies, there are two chromosomes present; however, instead of one maternal and
paternal chromosome, there are two copies of either a maternal or paternal chromosomes. This
may be the result of a trisomic rescue event after an error during meiosis
2. The most frequent chromosome that UPD occurs in is chromosome 15, where two paternal
copies is referred to as Angelman syndrome or two maternal copies is known as Prader-Willi
syndrome. Other chromosomes that can present with UPD are chromosomes 7, 11 and 16
ORIGINS
Paternal origin
 30
1. The centrosome is inherited from the sperm and is responsible for the first mitotic divisions
within the human embryo. The disruption of the sperm centrosome can produce mosaicism in the
preimplantation embryo.
2. Studies have shown that chromosomal aneuploidies are more prevalent in patients with severe
male factor infertility.
Maternal origin
1. As previously stated, the centrosome is paternally inherited but the mitochondria and mRNA
stores necessary for proper chromosome division originate from the oocyte. Research has
indicated that mitochondrial function is affected by maternal age, possibly influencing
chromosome segregation.
2. Abnormalities in spindles are also more prevalent in older women. For example, meiotic spindles
from older women can have an abnormal shape and produce more chromosome misalignment
compared with spindles of younger women, which appear to have normal configurations.
External influences
1. External factors also contribute to mosaicism. For example, the production of oocytes for in vitro
fertilization (IVF) requires controlled ovarian hyperstimulation of ovaries by exogenous follicle-
stimulating hormone. Hyperstimulation has been implicated in increased rates of cleavage stage
aneuploidy.
3. Improper culture conditions can compromise embryo quality. Furthermore, 5% oxygen versus
atmospheric oxygen levels has been found to improve embryo quality and decrease sex
chromosome mosaicism when compared with culture in atmospheric oxygen levels.
Incidence in preimplantation embryos
Cleavage embryos
1. Mosaicism occurs in 15–90% of all cleavage stage human embryos. It is believed that if a majority
of the cells are diploid, then these embryos could be deemed viable, even though they are
general mosaics.
2. It seems that mosaicism and aneuploidies are routine during the first cleavage divisions in human
preimplantation development.
Blastocysts
1. The blastocyst is composed of two distinct parts, the trophectoderm, which will become the
placenta and the inner cell mass, which will become the fetus. Thus, the blastocyst represents the
first stage of cellular differentiation in human embryonic development whereby totipotent cells
become pluripotent cells.
2. Compared with cleavage-stage embryos, similar rates of mosaicism appear to exist in the human
blastocyst. Regardless of the variability in different studies, mosaicism is still prevalent at the
blastocyst stage.
Incidence postimplantation
1. Confined placental mosaicism (CPM) is detected via Chorionic villus sampling (CVS) at 10–12
weeks and is conducted on patients who are at an increased risk of genetic abnormalities;
therefore, the true incidence of CPM in a general population is unknown.
2. Research indicates that 1–2% of viable pregnancies present with a chromosomally abnormal
placenta. A majority of these abnormalities within CPM are autosomal trisomies

 31

Clinical consequences
1. Mosaicism during the preimplantation stage has a greater consequence than postimplantation
mosaicism. It is believed that the actual fetus only derives from three cells of the inner cell mass.
Any cells not destined to become the fetus may not be a true representation of the fetus itself but
rather of extraembryonic tissue (i.e. EEM, cytotrophoblasts, placenta, chorion, etc.).
2. The clinical significance of mosaicism is directly associated with the ratio of abnormal to normal
cells
3. Mosaic cell lines can arise from either mitotic or meiotic circumstances. If a meiotic error has
occurred and the error is corrected at the cleavage stage, mosaicism will result. The meiotic
errors are typically more devastating due to the initial onset of the abnormality, which allows the
mosaic cell line to dominate.
4. However, mitotic errors may be as severe as meiotic errors depending on when they occur. If a
mitotic error occurs in the first one or two divisions, then the mosaic cell line could be present
through all embryonic tissues. However, if the mitotic error occurs further along in development,
the mosaic cell line could be confined type.
5. The effects of the mosaicism depend greatly on the location of the mosaic cell line along with
what chromosome is involved.

CONGENITAL HYPERBILIRUBINEMIA:
1. Neonatal hyperbilirubinemia is defined as serum bilirubin > 5 mg/dl
2. Classification of Hyperbilirubinemia
1) Physiological: appear after 24 hour and always indirect hyperbilirubinemia
 32
2) Pathological: appear witn in 24 hours indirect or direct hyperbilirubinemia
1. Indirect:
I. Hemolytic
1. ABO
2. Rh
3. Enzyme defect- G6PD
4. Membrane defect: Spherocytosis
5. Hemoglobin defect: Thalasemia
II. Non Hemolytic
1. Breast milk jaundice
2. Criggler najar I&II
3. Gilbert
4. Infection
2. Direct:
1. Familial:
a. Dubin Jhonson
b. Rotar
2. Infection
a. Neonatal hepatitis (TORCHS)
a. Neonatal sepsis
b. Giant cell hepatitis
3. Obstructive:
a. Biliary Obstruction
b. Choledochal cyst
4. Metabolic:
a. Cystic fibrosis
b. Galactosemia
c. Alpha1-antitrypsin deficiency
d. Tyrosinemia

Congenital hyperbilirubinemia refers to:
1. Disorders of hepatic uptake: Gilbert Syndrome
2. Disorders of conjugation: Crigler-Najjar Syndrome Type I; Crigler-Najjar Syndrome Type II; Lucey-
Driscoll Syndrome
3. Dosorders of excretion: Dubin Jhonson and Rotar

CRIGLER-NAJJAR SYNDROME
Crigler-najjar syndrome type I (glucuronyl transferase deficiency)
1. This is inherited as an autosomal recessive trait. Enzyme bilirubin uridine diphospho glucuronate
glucuronosyltransferase is completely absent (UGT1A1). CN type II is due to decreased UGT1A1
activity.
2. CN type I is inherited as an autosomal recessive trait.
3. Clinical Manifestations
a. Severe unconjugated hyperbilirubinemia develops in homozygous affected infants in the 1st 3
days of life, and without treatment, serum unconjugated bilirubin concentrations of 25-35
mg/dL are reached in the 1st month.
 33
b. Kernicterus, an almost universal complication of this disorder, is usually 1st noted in the early
neonatal period; some treated infants have survived childhood without clinical sequelae.
c. Stools are pale yellow.
d. Persistence of unconjugated hyperbilirubinemia at levels >20 mg/dL after the 1st wk of life in
the absence of hemolysis should suggest the syndrome.
2. Diagnosis
a. The diagnosis of CN type I is based on the early age of onset and the extreme level of bilirubin
elevation in the absence of hemolysis.
b. In the bile, bilirubin concentration is <10 mg/dL compared with normal concentrations of 50-
100 mg/dL; there is no bilirubin glucuronide.
c. Definitive diagnosis is established by measuring hepatic glucuronyl transferase activity in a
liver specimen obtained by a closed biopsy; open biopsy should be avoided because surgery
and anesthesia can precipitate kernicterus.
d. DNA diagnosis is also available and may be preferable. Identification of the heterozygous
state in parents also strongly suggests the diagnosis.
3. Treatment
a. The serum unconjugated bilirubin concentration should be kept to <20 mg/dL for at least the
1st 2-4 wk of life; in low birthweight infants, the levels should be kept lower by repeated
exchange transfusions and phototherapy.
b. Phenobarbital therapy should be considered to determine responsiveness and differentiation
between type I and II.
c. The risk of kernicterus persists into adult life (serum bilirubin usually >35 mg/dL).
d. Adjuvant therapy using agents that bind photobilirubin products such as calcium phosphate,
cholestyramine, or agar can be used to interfere with the enterohepatic recirculation of
bilirubin.
e. Prompt treatment of intercurrent infections, febrile episodes help prevent the later
development of kernicterus
f. Orthotopic liver transplantation cures the disease and has been successful in a small number
of patients; isolated hepatocyte transplantation has been reported in fewer than 10 patients,
but all patients eventually required orthotopic transplantation.
g. Other therapeutic modalities have included plasmapheresis and limitation of bilirubin
production. The latter option, inhibiting bilirubin generation, is possible via inhibition of heme
oxygenase using metalloporphyrin therapy.

CRIGLER-NAJJAR SYNDROME TYPE II
(PARTIAL GLUCURONYL TRANSFERASE DEFICIENCY)
1. CN type II is an autosomal recessive disease; it is caused by homozygous missense mutations in
UGT1A1, resulting in reduced (partial) enzymatic activity
2. Type II disease can be distinguished from type I by the marked decline in serum bilirubin level that
occurs in type II disease after treatment with phenobarbital secondary to an inducible phenobarbital
response element on the UGT1A1 promoter.
Clinical Manifestations
1. When this disorder appears in the neonatal period, unconjugated hyperbilirubinemia usually
occurs in the 1st 3 days of life; serum bilirubin concentrations can be in a range compatible with
physiologic jaundice or can be at pathologic levels. The concentrations characteristically remain
elevated into and after the 3rd wk of life, persisting in a range of 1.5-22 mg/dL; concentrations in
 34
the lower part of this range can create uncertainty about whether chronic hyperbilirubinemia is
present.
2. Development of kernicterus is unusual.
3. Stool color is normal, and the infants are without clinical signs or symptoms of disease. There is
no evidence of hemolysis.
Diagnosis
1. Concentration of bilirubin in bile is nearly normal in CN type II.
2. Jaundiced infants and young children with type II syndrome respond readily to 5 mg/kg/24 hr of
oral phenobarbital, with a decrease in serum bilirubin concentration to 2-3 mg/dL in 7-10 days.
3. Treatment
4. Long-term reduction in serum bilirubin levels can be achieved with continued administration of
phenobarbital at 5 mg/kg/24 hr.
5. Orlistat, an irreversible inhibitor of intestinal lipase, induces a mild decrease in plasma bilirubin
levels (~10%) in patients with CN I and II.

GILBERT SYNDROME
1. Gilbert's syndrome is usually an autosomal recessive disorder and is a common cause of unconjugated
hyperbilirubinaemia. There have been some reports of heterozygous cases, mainly within Asian
populations.
2. Gilbert's syndrome affects 5-10% of people in Western Europe. The worldwide prevalence of Gilbert's
syndrome varies considerably depending on which diagnostic criteria are used. Men are more
commonly affected than women.
3. Gilbert syndrome is caused by mutation in the promoter region of UGT1A1 that leads to decreased
glucuronyl transferase activity to < 30%.
4. Gilbert's syndrome is characterised by unconjugated hyperbilirubinemia, no evidence of haemolysis,
normal liver enzyme levels and no evidence of liver disease.
5. It is usually diagnosed around puberty, and aggravated by intercurrent illness, stress, fasting or after
administration of certain drugs.
6. The increase in serum concentrations of unconjugated bilirubin can lead to intermittent episodes of
non-pruritic jaundice, which can be precipitated by fasting, infections, dehydration, surgery, physical
exertion and lack of sleep. Symptoms, including tiredness, that occur during episodes of jaundice are
caused by the precipitating factor and do not result directly from Gilbert's syndrome.
7. It can remain unnoticed for many years, but usually presents in adolescence with:
a. Intermittent jaundice noticed after fasting, lack of sleep, vigorous exercise or during an
intercurrent illness.
b. Exposure to certain medications, which may precipitate jaundice - eg, chemotherapy. Adverse
effects of anticancer agents have been observed in Gilbert's syndrome patients due to reduced
drug or bilirubin glucuronidation.

LUCY - DRISCOL SYNDROME
1. Lucey Driscoll syndrome is characterized by severe jaundice in the first 4 days of life.
2. It is a transient condition and is due to presence of an inhibitor substance in the mother's blood
that prevents the action of an enzyme in the baby's liver that conjugates the bilirubin (the
pigment that accumulates in access in jaundice).
3. As a result of high bilirubin, the child has jaundice and sometimes even convulsions. Since, this
inhibitor is present only for a limited time, the condition is transient.
 35
4. The treatment is light in the form of photo therapy, drugs such as phenobarbitone and even blood
transfusion in the form of exchange transfusion.

DUBIN-JHONSON SYNDROME
1. Dubin-Johnson syndrome is an autosomal recessive inherited defect with variable penetrance in
hepatocyte secretion of bilirubin glucuronide.
2. The defect in hepatic excretory function is not limited to conjugated bilirubin excretion but also
involves several organic anions normally excreted from the liver cell into bile.
3. Bile acid excretion and serum bile acid levels are normal.
4. Total urinary coproporphyrin excretion is normal in quantity but coproporphrin I excretion increases
to ∼80% with a concomitant decrease in coproporphyrin III excretion. Normally, coproporphyrin III is
>75% of the total.
5. Cholangiography fails to visualize the biliary tract and x-ray of the gallbladder is also abnormal.
6. Liver histology demonstrates normal architecture, but hepatocytes contain black pigment similar to
melanin.
ROTOR SYNDROME
1. Patients with Rotor syndrome have an additional deficiency in organic anion uptake; however, the
genetic defect has not yet been elucidated.
2. Unlike Dubin-Johnson syndrome, total urinary coproporphyrin excretion is elevated, with a relative
increase in the amount of the coproporphyrin I isomer.
3. The gallbladder is normal by roentgenography, and liver cells contain no black pigment.
4. In Dubin-Johnson and Rotor syndromes, sulfobromophthalein excretion is often abnormal.

ENDOCRINOLOGY OF PUBERTY:
Definitions:
1. Puberty is the developmental stage during which a child becomes a young adult, characterized by
the maturation of gametogenesis, secretion of gonadal hormones, and development of secondary
sexual characteristics and reproductive functions.
2. Thelarche denotes the onset of breast development, an estrogen effect.
3. Pubarche denotes the onset of sexual hair growth, an androgen effect.
4. Menarche indicates the onset of menses and spermarche the appearance of spermatozoa in
seminal fluid.
5. Gonadarche refers to the onset of pubertal function of the gonads, which produce most of the
sex hormones that underlie the pubertal changes in secondary sex characteristics.
6. Adrenarche refers to the onset of the adrenal androgen production that contributes to pubarche.
The Hypothalamic-Pituitary-Gonadal Axis:
1. Normal puberty results from sustained, mature activity of the HPG axis. In response to a single
gonadotropin releasing hormone (GnRH), the pituitary gland releases two gonadotropins:
luteinizing hormone (LH) and follicle-stimulating hormone (FSH). GnRH is secreted by specialized
neurons of the hypothalamus in a pulsatile fashion.
2. Pituitary LH and FSH secretion consequently is pulsatile and can be sustained only in response to
pulsatile GnRH signals. LH acts primarily on the specialized interstitial cells of the gonads to
stimulate formation of androgens, and FSH acts primarily on the follicular/tubular compartment
to stimulate formation of estrogen from androgen precursors, inhibin, and gametes.
3. The HPG axis is active during three phases of development: fetal, neonatal, and adult, with
puberty being the period of transition to mature function.
 36
4. The HPG axis is established during the first trimester. Its activity in the second trimester
contributes to the establishment of normal penile size and the inguinalscrotal phase of testicular
descent. In the latter half of pregnancy, activity is suppressed by the high estrogens elaborated by
the fetoplacental unit.
5. The HPG axis promptly functions at a pubertal level in the newborn after withdrawal from
maternal estrogens. This “minipuberty of the newborn” is subclinical, except for contributing to
genital growth, acne, and transient thelarche in the neonate.
6. HPG function subsequently comes under gradual central nervous system restraint at the end of
the neonatal period. The HPG axis becomes increasingly active again in the late prepubertal
period, as central nervous system restraint recedes, followed by an increasing tempo throughout
puberty. The gonads account for the most important circulating estrogen (estradiol) and
androgen (testosterone).
7. Gonadal function accounts for more than 90% of estradiol production in the female (50% in the
male) and more than 90% of testosterone production in the male (50% in the female)

Adrenarche, the “Puberty” of the Adrenal Gland
1. Adrenarche is actually a re-onset of adrenal androgen production. The fetal zone of the adrenal
cortex elaborates large amounts of dehydroepiandrosterone sulfate (DHEAS), which is important
as the major substrate for placental estrogen formation during pregnancy. This zone then
regresses over the first several postnatal months.
2. The adrenal gland secretes more than 90% of DHEAS in children and women and more than 70%
in adult men, while 50% of testosterone in the female and less than 10% of testosterone in the
male is produced by the adrenal. Adrenal androgen stimulates apocrine odor and mild acne and
pubic hair growth after about 10 years of age.
Interactions Between Pubertal Hormones and the Growth Hormone/Insulin-like Growth Factor-I Axis
1. Pituitary GH secretion increases during puberty in response to sex steroids. This rise in GH causes
a rise in insulin-like growth factor-I concentrations to peaks in late puberty that is above those of
adults, sometimes in the adult acromegalic range. Half of the characteristic pubertal growth spurt
is due to the direct effect of sex steroids on epiphyseal growth and half to GH stimulation.
2. GH is necessary for optimal gonadotropin effects on gonadal growth and sex steroid effects on
secondary sex characteristics. For example, selective GH resistance is characterized by small
testes and micropenis, poor breast and sexual hair development, and absence of a pubertal
 37
growth spurt.
Regulation of the Onset and Progression of Puberty
1. There is no single “trigger” for puberty; rather, puberty results from a gradual increase in GnRH
pulsatility that arises from maturation of central nervous system. Puberty is associated with
changing sensitivity of the neuroendocrine system to negative feedback by gonadal hormones.
2. Increasing central activation during puberty permits sex steroids to rise to adult concentrations
before exerting negative feedback effects. The major GnRH-inhibitory systems are GABAergic and
opioidergic; the major excitatory systems involve glutamate and kisspeptin, with glial cells
facilitating GnRH secretion.
3. Kisspeptin is a hypothalamic neuropeptide discovered in the search for the molecular basis of
hypogonadotropic hypogonadism; it acts as an important signal for pubertal GnRH release via
GPR54, a G-protein coupled receptor located on GnRH neurons.
4. It has been estimated that at least half of the variation in the timing of puberty is genetically
determined, and ethnicity is one such factor. Sex hormones, hormonally active environmental
chemicals, diverse somatic stimuli (including nutrition, the growth hormone/insulin-like growth
factor system, thyroid hormones), and general health all affect the pubertal process.
5. Pubertal and skeletal maturation appear to have common somatic determinants. Children
generally enter puberty when they achieve a pubertal bone age. Pubertal stage normally
correlates better with the bone age than with chronologic age. Thus, for example, the onset of
breast development normally occurs at a bone age of about 10 years and menarche occurs at a
bone age of about 12.5 years, whether the child is 9 or 14 years of age.
6. Optimal nutrition is necessary for initiation and maintenance of normal reproductive function.
Early to mid-childhood may be a critical period for weight to influence the onset of puberty.
Suboptimal nutrition related to socioeconomic conditions is an important factor in the late onset
of puberty in underdeveloped countries. Conversely, obesity is an important factor in advancing
the onset of puberty.
7. Leptin, a hormone secreted by fat cells, appears to be an important link between nutrition and
the attainment and maintenance of reproductive competence. Leptin acts on the hypothalamus
to reduce appetite and stimulate gonadotropin secretion. Leptin deficiency causes obesity and
gonadotropin deficiency. Blood leptin concentrations rise throughout childhood and puberty to
reach higher values in girls than boys. Attainment of a critical threshold appears to signal that
nutritional stores are sufficient for mature function of the GnRH pulse generator and, thus,
permits puberty.
Hormonal Changes of Normal Puberty
1. The first hormonal change of puberty is a sleep-related increase in the pulsatile release of LH by
the pituitary gonadotropes. At the beginning of puberty, a unique diurnal variation of pubertal
hormones occurs, with little LH secretion during the day and a significant increase inpulsatile
secretion during sleep. After menarche, this diurnal variation no longer exists.
2. LH stimulates the interstitial cells of the ovaries (theca cells) to form androgenic precursors of
estradiol and those of the testes (Leydig cells) to secrete testosterone itself.
3. FSH acts on the granulosa cells of the ovary and Sertoli cells of testes to stimulate gametogenesis
and gonadal growth. In granulosa cells, FSH strongly stimulates aromatase to form estradiol from
thecal androgens. As the gonads become increasingly sensitized to gonadotropin stimulation,
they grow and secrete sex hormones at steadily increased rates.
4. The hormonal increases culminate in positive feedback in girls, secreting a mid-cycle surge of LH
and the ovary is prepared for ovulation by sustained level of estrogen secretion.
5. Estrogen stimulates the target tissues: the female genital tract (eg, endometrial growth, cervical
 38
mucus secretion) and breasts. Androgen stimulates the classic male target tissues (eg, sexual hair
and sebaceous gland). Both stimulate sexual drive and function. Both sex steroids account for the
pubertal growth spurt, directly and indirectly via growth hormone. Both directly stimulate
epiphyseal growth and epiphyseal maturation, which is indexed by bone age radiographs and
peak bone mass accrual.
6. During puberty, estrogen promotes lipogenesis and lower body fat distribution. In contrast,
androgens generally are lipolytic, although they favor the development of visceral fat stores, and
promote muscular development. The similar increase of body mass index during puberty in girls
and boys, thus, is due to differences in body composition, with a higher percent being body fat in
girls and lean body mass in boys.
7. The menstrual cycle arises from cyclic maturation of ovarian follicles that result in cyclic changes
in sex hormones, particularly estradiol and progesterone, which entrain cyclic changes in
gonadotropin concentrations. The biologic goal of this monthly variation is to select and nurture
one dominant follicle to the point of ovulation for potential fertilization. A normal average 28-day
cycle consists of two phases: the follicular phase (variable in duration, averaging 14 days at
maturity) and the luteal phase (14_1 SD days), with the latter occurring only in ovulatory cycles.
The follicular phase begins with the onset of menses and culminates in the mid-cycle LH surge,
which induces ovulation from the follicle.
8. The empty follicle forms the corpus luteum, initiating the luteal phase. Progesterone increases
steadily to be sustained at very high levels for several days, along with lesser but substantial
increases in estradiol. Progesterone and estradiol secretion from the corpus luteum maintain the
endometrial layer of the uterus in preparation for potential pregnancy. If pregnancy does not
occur, with its resultant increase in human chorionic gonadotropin, the corpus luteum life span is
exhausted, which results in withdrawal of female sex steroids, followed by endometrial sloughing
and menstrual flow.
9. Daytime pubertal hormone concentrations may not indicate the early stages of puberty
accurately because of diurnal and cyclic variations. For this reason, GnRH-stimulated values may
be necessary to diagnose pubertal disorders. A peak LH value greater than approximately 4.0 U/L
in response to GnRH or GnRH agonist testing has been suggested as indicative of the onset of
puberty.

IODINE METABOLISM:
Introduction
1. Iodine is a micronutrient of crucial importance for the health and well-being of all individuals. It is
a trace element, just 5 gm of which are sufficient to meet the life-time needs of an individual with
a life-span of 70 years.
2. Iodine is mostly concentrated in thyroid gland. A healthy adult body contains 15-20 mg of iodine,
70-80% of which is stored in the thyroid gland. Daily intake of iodine by an individual amounts to
500 micrograms; daily physiological requirement during adult life is 150 micrograms; during
pregnancy and lactation period is 200 micrograms; and during neonatal period is 40 micrograms.
Normally about 120 micrograms of iodide are taken up by the thyroid gland for the synthesis of
thyroid hormones.
Source:
1. Oceans are the world's main repositories of iodine and very little of earths iodine is actually found
in the soil. The deposition of iodine in the soil occurs due to volatilization from ocean water, a
process aided by ultraviolet radiation.
2. The coastal regions of the world are much richer in iodine content than the soils further inland;
 39
3. Iodine is mostly obtained from food sources particularly vegetables grown on iodine-rich soil; the
remaining requirement is met from drinking water.
4. Iodine is found in nature in various forms: inorganic sodium and potassium salts (iodides and
iodates); inorganic diatomic iodine (molecular iodine or I), and organic monoatomic iodine.
Iodine metabolism
1. Thyroid gland plays a central role in the metabolism of iodine. Iodine trapping is the first step in
the metabolism of iodine. The process commences with the uptake of iodide from the capillary
into the follicular cell of the gland by an active transport system.
2. Synthesis and secretion of thyroglobulin is the second step. It occurs within the follicular cell.
3. Thyroid hormone has two constituents, tyrosine and iodine. These two are assembled on the
glycoprotein thyroglobulin (Tg) and synthesis occurs in the central colloid. Iodide enters the
cell across the basolateral membrane via a sodium symporter. This process is driven
by secondary active transport mediated by a Na+/K+-ATPase pump.
4. At the luminal membrane, iodide is activated to iodine by thyroperoxidase (TPO) before
entering the colloid via the ion exchanger Pendrin. Thyroglobulin, containing tyrosine, is
produced by Golgi complexes and the endoplasmic reticulum in the follicular cells.
The thyroglobulin/tyrosine complex is exocytosed across the luminal membrane into the
colloid.
5. Within the colloid, one iodide may attach to a tyrosine (again attached to a thyroglobulin) to
form monoiodotyrosine (MIT). This reaction is catalysed by TPO. A second iodide may attach to
MIT to form di-iodotyrosine (DIT).
6. Coupling of MIT and DIT yield the thyroid hormones. One MIT and one DIT
gives triiodothyronine (T3) while two DITs give thyroxine (T4).
7. When stimulated (by TSH) the surrounding follicular cells phagocytose a portion of the
colloid. Once within the follicular cell, this vesicle is subject to lysosomes, which act to release
the T3/T4 from Tg.
8. The thyroid hormones T3/T4 may then diffuse into the blood stream. Any remaining MIT and
DIT is deiodinated (a reaction catalysed by iodinase) and recycled.
9. The stored hormones can meet the body requirements for up to 3 months.
10. In the blood stream, T4 and T3 may circulate in the bound or free form; whereas 99 percent of T4
and T3 circulate in the bound form, less than 1 percent circulates in an unbound form. The
binding proteins include thyroxine binding globulin (TBG), thyroxine binding prealbumin (TBPA)
and thyroxine binding albumin (TBA). Binding of hormones apart from serving as a reservoir also
helps to prevent urinary loss of hormones. The unbound hormones are biologically active. About
80 percent of circulating T3, the most active thyroid hormone is derived from peripheral
deiodination of T4 hormone.
11. Thyroid secretion is regulated by pituitary gland through TSH which operates on a feed-back
mechanism tuned to T4 level in blood. A fall in T4 level stimulates the pituitary to increase its TSH
secretion which in turn stimulates the thyroid gland to release T4 in circulation to maintain
normal level of the hormone in blood.
12. While major portion of iodine is concentrated in the thyroid gland, the non hormonal iodine is
found in a variety of body tissues including mammary glands, eye, gastric mucosa, cervix and
salivary glands. With the exception of mammary tissue the function of iodine in these tissues is
still not clear. Accumulation of iodine in the breast plays an important role during breast feeding
in fetal and neonatal development; however such iodine has also proven to have antioxidant
function.
 40
13. In the presence of hydrogen peroxide and peroxidase, iodide acts as an electron donor, thereby
decreasing damage by free oxygen radicals.
Iodine deficiency disorders
1. Iodine Deficiency Disorder (IDD) is the most common endocrinopathy in the world and also the
most preventable cause of mental retardation.
2. IDD is a term that collectively reflects the clinical and sub-clinical manifestations of iodine
deficiency. Iodine being an indispensable component of T3 and T4 hormones, its deficiency
interferes seriously with the synthesis of these hormones.
3. In a situation of severe iodine deficiency, while the level of T4 remains low, the level of TSH
remains high. Under continuing TSH stimulation in endemic areas, thyroid gland undergoes
hypertrophy and hyperplasia of follicular cells and in the process, enlarges in size and appears as a
goiter, which may in certain cases attain an enormous size.
4. The deficiency not only leads to goiter formation but also to severe retardation of growth,
development and maturation of nearly all the tissues of the body, especially those that are fast
developing. The critical period in endemic areas extend from second trimester to second year of
life. Deficient intake of iodine during this period can lead to devastating consequences resulting
from permanent damage to brain. Administration of iodine during second trimester of pregnancy
reverses the damage caused by iodine deficiency. However, the damage sustained after the end
of the second trimester of pregnancy is permanent. Owing to maternal IDD, it is estimated that
about one-fifth of pregnant women in India will give birth to children who will not reach their
optimum physical and mental potential.
5. A growing fetus in the womb of an iodine deficient mother is at high risk. The pregnancy may end
in abortion, still birth, congenital anomalies or low birth weight outcome. Infants born to iodine
deficient mothers, who survive the critical postnatal phase, may develop endemic cretinism.
Iodine supplementation strategies
1. Since iodine is released from the body through urine, the best way to determine iodine deficiency
across a large population is to measure the amounts of iodine in urine samples. The WHO defines
iodine deficiency as a median urinary iodine concentration less than 50 μg/L in a population.
2. Iodine supplementation in areas deprived of iodine rich food is viewed as the most cost effective
solution to address the problem of IDD. Iodine deficiency can be corrected by adding iodine to
dietary media like salt, oil, water, sauces etc. The methods of proven value for mass use are
iodized salt and iodized oil.

ANATOMY OF ANAL SPHINCTER:
There are two sphincters, (internal and external), around the anal canal, which provide powerful
sphincteric mechanism at the distal end of the gastrointestinal tract.
INTERNAL SPHINCTER
1. It is formed by thickened circular muscle coat of the anal canal and surrounds the upper two-third
of the canal.
2. Above, it is continuous with the circular muscle coat of the rectum and below reaches up to the
level of the white line of Hilton.
3. It is surrounded by the deep and superficial parts of the external anal sphincter.
EXTERNAL SPHINCTER
The external anal sphincter as the name implies is outside the internal anal sphincter. It surrounds
the whole length of the anal canal and consists of three parts: deep, superficial, and
subcutaneous.
Deep Part
 41
The deep part surrounds the upper part of the internal sphincter. It has no bony attachments. The
puborectalis blends with the deep part of external sphincter behind and forms a sling around the
anorectal junction, which is attached anteriorly to the back of the pubis. In the resting state, the
anorectal tube is angled forward at this level and contraction of puborectalis sling will increase
this angle, an important factor in the continence mechanism.
Superficial Part
The superficial part is elliptical in shape. It arises from the tip of coccyx and anococcygeal raphe
behind, surrounds the lower part of internal sphincter and then gets inserted into the perineal
body in front.
Subcutaneous Part
The subcutaneous part lies below the internal sphincter in the perianal space and surrounds the
lower part of the anal canal. It has the form of flat band, about 15 mm broad. It has no bony
attachment. It is traversed by fibroblastic septa derived from the conjoint longitudinal coat.
Nerve Supply of the Sphincter
1. The internal sphincter is made up of smooth muscle and supplied by the autonomic nerve fibres
(sympathetic and parasympathetic), hence it is involuntary.
2. The external anal sphincter is made up of striated muscle and hence, supplied by the somatic
nerve—inferior rectal nerve and perineal branch of 4th sacral nerve. It is therefore under
voluntary control.
Anorectal ring:
While doing per rectal examination, the muscular ring on which the flexed finger rests just over an
inch above the anal margin is the “anorectal ring”. It is formed by the fusion of the deep part of
the external sphincter, the internal sphincter, and puborectalis, and demarcates the junction
between the anal canal and the rectum. Surgical excision of this ring results in incontinence.
Conjoint longitudinal coat of the anal canal:
It is formed at the anorectal junction by the fusion of puborectalis and longitudinal muscle coat of
the rectal wall. It runs vertically downward between the internal and external anal sphincters, and
at the level of white line it breaks up into a number of fibroelastic septa which fan out, traverse
through the subcutaneous part of the anal canal to be attached to the skin around the anal
orifice. The most lateral of these septa forms perianal fascia which passes laterally to be attached
to obturator fascia enclosing the pudendal canal. The space between the perianal fascia and the
skin is called perineal space. The most medial septum of the conjoint longitudinal coat forms the
anal intermuscular septum, which passes medially and gets attached to Hilton’s line.

 42

MICRONUTRIENTS IN PREGNANCY:
Introduction:
1. Micronutrients and trace elements have an important influence on the health of both mother and
fetus. Deficiency of micronutrients during pregnancy may give rise to complications such as
anaemia and hypertension, as well as impairing fetal function, development and growth.
2. A recent meta-analysis evaluating the effects of antenatal multimicronutrient supplementation on
pregnancy outcomes has revealed a significant reduced risk of low birth weight and improved
birth weight in comparison to iron/folic acid supplementation only.
3. Factors leading to deficiency of these micronutrients include poor diet, gene polymorphisms,
malabsorption syndromes, and in certain populations, diseases such as malaria or intestinal
parasites.
Iron
1. Iron is one of the major trace elements required during pregnancy. It is suggested that a lack of
iron in pregnancy can result in preterm delivery and maternal anaemia. Adequate iron is required
from conception, throughout the pregnancy and during lactation. Iron deficiency during lactation
may be associated with mental retardation. The recommended daily iron intake for pregnant
women is 27 mg/day.
2. Good dietary sources of iron (ie. more than 2 mg/serve) include liver, beef, fortified cereals,
cashew nuts and baked beans. Although it is believed that iron excess can hinder the absorption
of other vitamins, this hypothesis has yet to be fully substantiated.
Calcium
1. Calcium is required for the maintenance of skeletal, neuromuscular, and cardiac function. Studies
have shown the maternal skeleton is not the source for fetal calcium needs, thus adequate
calcium supplementation during pregnancy and lactation (~1000 mg/day) is essential for fetal
bone mineralisation.
2. In pregnant women who are vegetarians, calcium absorption can be poor from foods rich in oxalic
acid (beans and spinach) or phytic acid (nuts and grains). In comparison to dairy milk, calcium
absorption from soymilk may be as efficient, while calcium absorption from dried beans is about
50% and from spinach only about 10%.
Zinc
1. Zinc is an important constituent of various enzymes that help maintain structural integrity of
proteins and regulate gene expression. It is recommended that pregnant women take 11 mg/day,
and 12 mg/day during lactation.
 43
2. Nevertheless, it is important to be aware that dietary intake of iron at levels found in some
supplements may impair zinc absorption.
Phosphorus
1. Phosphorus, in the form of phosphate, is thought to regulate acid base balance in the
bloodstream and activate catalytic proteins. Deficiency in this mineral, although rare, may result
in symptoms such as anaemia, muscle weakness, bone pain, rickets, parenthesis, anorexia, ataxia,
confusion, and possibly death.
2. Phosphorus is available in a wide range of foods and the recommended intake in pregnancy is
similar to that of nonpregnant women.
Iodine
1. Iodine is essential for fetal and neonatal growth and development. Severe iodine deficiency may
result in abortion or stillbirth, congenital anomalies, neurological cretinism, or mental deficiency
with deafness, spastic diplegia, squint and myxoedematous cretinism.
2. It is recommended that pregnant women consume no less than 220 µg/day.
Selenium
1. Selenium is thought to have an antioxidant property as well as a role in cellular function, muscle
maintenance, fertility and cancer prevention. Deficiency in selenium can result in osteoarticular
disorder, cardiac enlargement, heart failure, arrhythmia and premature death.
2. In conjunction with iodine deficiency, selenium deficiency has also been reported to increase the
risk of cretinism. It is recommended that pregnant women increase their daily intake of selenium
to 65 µg/day and to 70 µg/day during lactation.
Vitamin A
1. Deficiency in vitamin A has been associated with intrauterine growth retardation, preterm birth,
low birth weight, placental abruption, and increased mortality of the mother. Research has also
ascertained that vitamin A supplementation can increase haemoglobin concentrations by about 4
g/L in marginally deficient maternal populations.
2. Daily intake of 800 µg is recommended in pregnant women. Breast milk is a good source of
vitamin A. However, adequate maternal dietary vitamin A is essential to maintain adequate levels
in breast milk.
Vitamin B group
1. The group of B vitamins, which consists of thiamine (B1), riboflavin (B2), niacin or nicotinic acid
(B3), B6, folate (B9) and B12, is essential for enhancing the immune system as well as reducing
the plasma concentration of homocysteine.
2. Elevated maternal plasma homocysteine levels as a result of vitamin B deficiency may lead to pre-
eclampsia, premature birth and low birth weight. Most cases of B12 deficiency in infants are
related to maternal veganism or malabsorption, such as pernicious anaemia.
3. Vitamin B12 is found exclusively in animal products including meat, eggs, fish and milk. Excessive
vitamin B consumption may result in weight gain, which can potentially complicate labour.
Vitamin C
1. Vitamin C stimulates better absorption of iron and therefore helps to reduce the risk of maternal
anaemia. As an oxidant, it also guards the body against injurious free radicals. Combined with
other factors, deficiency of vitamin C is thought to result in difficult labour, but this is yet to be
established.
2. The recommended daily intake of vitamin C in pregnancy is 60 mg/day.22
Vitamin D
1. Vitamin D is a group of fat soluble prohormones that help to absorb calcium and phosphorous
from dietary intakes, which are required for stimulating skeleton formation of the fetus.
 44
2. Deficiency of vitamin D during pregnancy may result in an infant with rickets or type 1 diabetes
mellitus. The recommended daily intake of vitamin D during pregnancy and lactation is 5 µg/day.
However, excessive supplementation may be detrimental as it is associated with maternal fatigue
and loss of appetite.
Vitamin E
1. Vitamin E acts as an antioxidant in the lipid phase of cell membrane by protecting
polyunsaturated fatty acid from free radical damage. Higher intake of vitamin E has been found to
be associated with a decrease in cardiovascular risk, diabetic complications, and certain cancers
and cataracts.
2. Vitamin E deficiency may lead to peripheral neuropathy, spinocerebellar ataxia, skeletal
myopathy and pigmented retinopathy; however this has only been reported in association with
other genetic or malabsorption syndormes, not simply with a diet low in vitamin E.
3. The recommended daily intake for pregnant women is equivalent to the normal requirement in
women (7 mg/day), but higher during lactation (11 mg/day).

PAIN PATHWAY AND LABOUR ANALGESIA:
Introduction
1. The experience of labour is complex and subjective. Several factors affect a woman’s perception
of labour making each experience unique. However as a consistent finding, labour pain is ranked
high on the pain rating scale when compared to other painful life experiences.
2. The memory of this pain however is short lived and of parturients who experienced severe pain in
labour, 90% found the experience satisfactory three months later. This short term memory may
be related to the positive outcome that often occurs at the end of labour.
3. The pattern of labour pain differs between nulliparous and multiparous women and it is well
documented that pain scores are higher in the nulliparous compared to the multiparous woman
especially if there has been no antenatal education.
Mechanisms of Labour Pain
1. Pain Labour is the active process of delivering a foetus and is characterised by regular, painful
uterine contractions, which increase in frequency and intensity.
2. The pain of labour has two components: visceral and somatic, and its anatomy is well
documented. The cervix has a central role in both the first and second stage of labour.
Visceral pain
1. 1st stage of labour
2. Begins from onset of regular uterine contractions and ends at complete cervical dilatation. Pain is
caused by stretching of the lower uterine segment (LUS) and cervix, which stimulates the
mechanoreceptors. Noxious impulses are carried by sensory nerve fibers (Aδ and C), which
accompany sympathetic nerve endings, travel through paracervical ganglion and hypogastric
plexus to the lumbar sympathetic chain which enter the spinal cord at T10, T11, T12 and L1 spinal
segments.
3. Pain is visceral in nature i.e. transmitted slowly, poorly localized, primarily in the lower abdomen,
also referred to lumbosacral area, gluteal region and thighs.
Somatic pain
1. Second stage of labor
2. Begins from complete cervical dilatation and terminates with the delivery of the baby. Pain is
caused by distension of pelvic structures and perineum due to descent of the presenting part,
ischemia and frank injury and is carried by somatic afferent nerve fibers that transmit impulses
through pudendal nerve to the spinal cord at S2, S3, and S4 levels.
 45
3. Typical of somatic pain, it is sharp and well-localized.
4. However, Pain of 1st stage does not end with the beginning of 2nd stage but is superseded by
pain of 2nd stage.

Psychology of labour pain
1. Labour pain is an emotional experience and presents a psychological challenge for many
parturients. Indeed a recent Cochrane review concluded that women who had continuous intra-
partum support were less likely to have intra-partum analgesia or to report dissatisfaction.
2. Other groups have suggested that an underlying anxiety trait can both result in a higher uptake of
epidural analgesia as well as influence the analgesic effect of the epidural block. There is current
interest in determining whether the Pain Catastrophizing Score has any influence on either labour
outcome or analgesic uptake.
Adverse effects of labour pain:
Painful labor produces several adverse changes in maternal physiology, which have important
implications for the fetus too.

Management of labour pain
 46
Complementary and alternative therapies for pain management in labour
Complementary and alternative Pharmacological analgesia for labour pain
therapies for labour pain
Psychological e.g. hypnosis Inhalational analgesia e.g. entonox, isoflurane,
desflurane, sevoflurane (reduce pain perception)
Physical e.g. transcutaneous Parenteral opioids e.g. pethidine, morphine,
electrical nerve stimulation (TENS), diamorphine, fentanyl, remifentanil (reduce pain
acupuncture perception)
Other complimentary e.g. Regional analgesia e.g. epidural, combined spinal
aromatherapy, water immersion epidural, continuous spinal analgesia (reduce pain
transmission)

Water Immersion:
1. A systematic review of randomised controlled trials comparing water immersion during labour
with no immersion concluded that labouring in water in the first stage reduces the use of
analgesia and reported maternal pain, without adverse outcomes on labour duration, operative
delivery or neonatal outcomes. These conclusions however can only be accepted with caution
because the study sample sizes were small and blinding to the intervention was impossible.
2. Water immersion may be associated with increased uterine perfusion, shorter labours and a
decreased need for augmentation; the associated feeling of weightlessness also adds to comfort
and allows for easy change of position during labour.
3. Warm water, as well as inducing muscle relaxation and reducing anxiety may also decrease the
release of catecholamines and stimulate the release of endorphins.
TENS
1. There is conflicting evidence to support the efficacy of TENS in labour. The majority of trials report
it as being ineffective but the evidence for this is not strong, and in practice many parturients still
utilise TENS in labour.
2. The Cochrane collaboration are currently reviewing all randomised controlled trials involving the
use of TENS in labour to determine its effectiveness and safety.
Pharmacological analgesia for labour pain
Inhalational
Entonox
1. A 50:50 mixture of oxygen and nitrous oxide is the most widely available inhalational analgesia in
the United Kingdom.
2. Despite its widespread use, studies have shown that when used in labour for short periods, it is
not a potent analgesic but does provide some analgesia and is safe for labouring women and their
babies.
Isoflurane, Desflurane, Sevoflurane
1. These inhalational gases can be used with or without nitrous oxide to improve analgesic efficacy
during labour at sub anaesthetic concentrations. Compared to entonox, most women prefer
sevoflurane, and at a concentration of 0.8%, sevoflurane gives significantly better pain relief
scores but is associated with more sedation.
2. In practice, inhalational gases are not commonly utilised in labour due to technical difficulties in
their safe administration, scavenging, requirement of specific vaporisers and concerns about
atmospheric pollution.
Parenteral opioids
 47
Pethidine
1. Like entonox, pethidine is readily available and easy to administer.
2. In a survey by the UK National Birthday Trust (NBT), 16% of women receiving pethidine rated it as
‘helpful’ whereas 25% rated it as ‘unhelpful’. Interestingly, midwives rated pethidine higher than
parturients, interpreting sedation as analgesia. The optimal opioid for labour analgesia remains
unknown and a systematic review of the available choices concluded that no other opioid
appeared to be better than pethidine in providing pain relief in labour.
3. In practice, pethidine is still the most commonly utilised opioid as it can be easily administered by
midwives. The effects of systemic opioids in labour are predominantly sedative rather than
analgesic.
Remifentanil
1. This is increasingly being used due to its fast onset and short duration of action. A typical bolus
dose of 0.25 – 0.5 μg/kg with a lockout period of 1 – 2 minutes is currently being used. A small
double blind crossover study has suggested than intravenous remifentanil when compared to
entonox provides better pain relief.
Regional analgesia
Epidural
1. Epidural analgesia is thought to be the most effective method of providing pain relief in labour
and involves injecting local anaesthetic close to the nerves that transmit pain. It also gives the
option of providing regional anaesthesia for obstetric interventions such as forceps delivery and
caesarean sections, and obese and other parturients who are at risk of obstetric interventions
particularly benefit from an early epidural.
2. Low –dose local anaesthetic and opioid mixtures; typically 10–15 mls 0.1% bupivacaine with
2mcg/ml fentanyl are currently in use in most delivery units, however some units still use higher
concentrations of bupivacaine for labour analgesia.
3. Low-dose mixtures are thought to provide excellent analgesia while preserving motor function
with the mother more likely to mobilise during labour or deliver without assistance.
4. Maintenance of analgesia in labour is provided by intermittent top-up by midwives, by continuous
infusion or by use of patient-controlled epidural analgesia (PCEA). Continuous epidural infusions
are associated with more motor block than either top-ups or PCEA. PCEA in labour is set up with a
small background infusion and tops up that are smaller in volume than those usually administered
by midwives or anaesthetists, but which can be administered more often. It results in satisfactory
analgesia with reduced overall dose of bupivacaine and fentanyl.
5. Labour epidurals are associated with an increase in the duration of the second stage and an
increased risk of instrumental vaginal delivery. They are also associated with an increased need
for stimulation of labour contractions and may cause reduction in maternal blood pressure, and
fever.
6. They are the commonest cause of intrapartum pyrexia which is strongly associated with poor
neonatal outcome in the form of neonatal encephalopathy and cerebral palsy. The exact
mechanism is unknown but it is thought that an increased temperature of tissues increases their
metabolic rate and therefore their oxygen requirements, resulting in cerebral palsy.
7. The sympathetic blockade accompanying low-dose epidural top-ups is slow in onset and seldom
extensive. Maternal hypotension in these circumstances is usually associated with aorto-caval
compression.
8. Immediate serious complications of epidural analgesia include:
a. Massive misplaced injection intravascularly, intrathecally, or subdurally, high or total
spinal block (rare),
 48
b. hypotension, and
c. local anaesthetic induced convulsions and
d. cardiac arrest (rare).
9. Delayed complications include post dural puncture headache, transient backache, urinary
retention, epidural haematoma, abscess or meningitis (rare) and permanent neurological deficit
(rare). The majority of neurological injuries in this setting are not as a result of neuraxial analgesia
but are intrinsic to labour and delivery.
10. The amide local anaesthetic, levobupivacaine, is less cardiotoxic than racemic bupivacaine when
given intravenously. However, cardiotoxic doses of bupivacaine are unlikely to be reached if low-
dose infusions or top-ups are used. 20% lipid emulsion is now widely available as treatment for
cardiac arrest secondary to overdose of these long acting local anaesthetics.
Combined Spinal Epidural (CSE)
1. This involves an initial injection of a low dose of local anaesthetic and opioid (e.g. bupivacaine
2.5mg + fentanyl 15 – 25mcg) into the subarachnoid space followed by an epidural top-up. This
method is thought to provide more rapid and complete analgesia with less motor block compared
with the conventional epidural method.
2. In practice, traditional epidurals are performed far more frequently for labour analgesia than
CSEs. There appears to be little basis for offering CSEs over epidurals in labour and it is debatable
whether breaching the dura can ever be justified.
3. Evidence shows no difference in retrospective maternal satisfaction, ability to mobilise and
obstetric and neonatal outcome. Many units reserve this technique for those in rapidly advancing
labour when analgesia, particularly sacral is needed in the second stage of labour and for the
multiparous parturient who had an ineffective epidural on a previous occasion. Perceived risks of
the CSE technique include increased incidence of post-dural puncture headache, meningitis and
epidural catheter migration through the dural hole.
Continuous spinal analgesia (CSA)
1. This is typically provided by either intermittent bolus or continuous infusion techniques and may
be preferred in cases of accidental dural puncture.
2. It is used when other methods of pain relief are not available or in a mother in the very advanced
first stage of labour or for high risk maternities. CSA with microcatheters offers some advantages
over the single shot spinal or the continuous epidural techniques but it is associated with
complications such as cauda equina syndrome and may be inherently more dangerous than the
other two techniques.

WOUND HEALING:
Healing Definition:
1. Healing is the body response to injury in an attempt to restore normal structure and function.
Healing involves 2 processes:
2. Regeneration when healing takes place by proliferation of parenchymal cells and usually results in
complete restoration of the original tissues.
3. Repair when healing takes place by proliferation of connective tissue elements resulting in
fibrosis and scarring.
Regeneration
Some parenchymal cells are short-lived while others have a longer lifespan. In order to maintain
proper structure of tissues, these cells are under the constant regulatory control of their cell
cycle. These include growth factors such as: epidermal growth factor, fibroblast growth factor,
platelet derived growth factor, endothelial growth factor, transforming growth factor-β.
 49
Repair
Repair is the replacement of injured tissue by fibrous tissue. Two processes are involved in repair:
a. Granulation tissue formation;
b. Contraction of wounds.
Wound healing
Healing of skin wounds provides a classical example of combination of regeneration and repair
described above. Wound healing can be accomplished in one of the following two ways:
1. Healing by first intention (primary union)
2. Healing by second intention (secondary union).
Healing by First Intention (Primary Union)
This is defined as healing of a wound, which has the following characteristics:
1. Clean and uninfected;
2. Surgically incised;
3. Without much loss of cells and tissue; and
4. Edges of wound are approximated by surgical sutures.
The sequence of events in primary union:
1. Initial haemorrhage. Immediately after injury, the space between the approximated surfaces of
incised wound is filled with blood which then clots and seals the wound against dehydration and
infection.
2. Acute inflammatory response. This occurs within 24 hours with appearance of polymorphs from
the margins of incision. By 3rd day, polymorphs are replaced by macrophages.
3. Epithelial changes. The basal cells of epidermis from both the cut margins start proliferating and
migrating towards incisional space in the form of epithelial spurs. A well approximated wound is
covered by a layer of epithelium in 48 hours. The migrated epidermal cells separate the
underlying viable dermis from the overlying necrotic material and clot, forming scab which is cast
off. The basal cells from the margins continue to divide. By 5th day, a multilayered new epidermis
is formed which is differentiated into superficial and deeper layers.
4. Organisation. By 3rd day, fibroblasts also invade the wound area. By 5th day, new collagen fibrils
start forming which dominate till healing is completed. In 4 weeks, the scar tissue with scanty
cellular and vascular elements, a few inflammatory cells and epithelialised surface is formed.
5. Suture tracks. Each suture track is a separate wound and incites the same phenomena as in
healing of the primary wound
Healing by Second Intention (Secondary Union)
This is defined as healing of a wound having the following characteristics:
1. Open with a large tissue defect, at times infected;
2. Having extensive loss of cells and tissues; and
3. The wound is not approximated by surgical sutures but is left open.
The basic events in secondary union are similar to primary union but differ in having a larger tissue
defect which has to be bridged. Hence healing takes place from the base upwards as well as from the
margins inwards. The healing by second intention is slow and results in a large, at times ugly, scar as
compared to rapid healing and neat scar of primary union. The sequence of events in secondary union
is:
1. Initial haemorrhage. As a result of injury, the wound space is filled with blood and fibrin clot
which dries.
2. Inflammatory phase. There is an initial acute inflammatory response followed by appearance
of macrophages which clear off the debris as in primary union.
 50
3. Epithelial changes. As in primary healing, the epidermal cells from both the margins of wound
proliferate and migrate into the wound in the form of epithelial spurs till they meet in the
middle and re-epithelialise the gap completely. However, the proliferating epithelial cells do
not cover the surface fully until granulation tissue from base has started filling the wound
space. In this way, pre-existing viable connective tissue is separated from necrotic material
and clot on the surface, forming scab which is cast off. In time, the regenerated epidermis
becomes stratified and keratinised.
4. Granulation tissue. Main bulk of secondary healing is by granulations. Granulation tissue is
formed by proliferation of fibroblasts and neovascularisation from the adjoining viable
elements. The newly-formed granulation tissue is deep red, granular and very fragile. With
time, the scar on maturation becomes pale and white due to increase in collagen and
decrease in vascularity. Specialised structures of the skin like hair follicles and sweat glands
are not replaced unless their viable residues remain which may regenerate.
5. Wound contraction. Contraction of wound is an important feature of secondary healing, not
seen in primary healing. Due to the action of myofibroblasts present in granulation tissue, the
wound contracts to one-third to one fourth of its original size. Wound contraction occurs at a
time when active granulation tissue is being formed.
6. Presence of infection. Bacterial contamination of an open wound delays the process of
healing due to release of bacterial toxins that provoke necrosis, suppuration and thrombosis.
Surgical removal of dead and necrosed tissue, debridement, helps in preventing the bacterial
infection of open wounds.
Differences between Primary and Secondary Union of Wounds.
Feature Primary Union Secondary Union
1. Cleanliness of wound Clean Unclean
2. Infection Generally uninfected May be infected
3. Margins Surgical clean Irregular
4. Sutures Used Not used
5. Healing Scanty granulation tissue Exuberant granulation tissue
6. Outcome Neat linear scar Contracted irregular wound
7. Complications Infrequent, Suppuration, may require debridement
Epidermal inclusion cyst
formation
Complications of wound healing:
1. Infection of wound due to entry of bacteria delays the healing.
2. Implantation (epidermal) cyst formation may occur due to persistence of epithelial cells in the
wound after healing.
3. Pigmentation. Healed wounds may at times have rust-like colour due to staining with
haemosiderin. Some coloured particulate material left in the wound may persist and impart
colour to the healed wound.
4. Deficient scar formation. This may occur due to inadequate formation of granulation tissue.
5. Incisional hernia. A weak scar, especially after a laparotomy, may be the site of bursting open of a
wound (wound dehiscence) or an incisional hernia.
6. Hypertrophied scars and keloid formation. At times the scar formed is excessive, ugly and painful.
Excessive formation of collagen in healing may result in keloid (claw-like) formation, seen more
commonly in Blacks. Hypertrophied scars differ from keloid in that they are confined to the
borders of the initial wound while keloids have tumour-like projection of connective tissue.
 51
7. Excessive contraction. An exaggeration of wound contraction may result in formation of
contractures or cicatrisation e.g. Dupuytren’s (palmar) contracture, plantar contracture and
Peyronie’s disease (contraction of the cavernous tissues of penis).
8. Neoplasia. Rarely, scar may be the site for development of carcinoma later e.g. squamous cell
carcinoma in Marjolin’s ulcer i.e. a scar following burns on the skin.
Factors influencing or delaying wound healing:
Two types of factors influence the wound healing: those acting locally, and those acting in
general.
Local factors:
1. Infection is the most important factor acting locally which delays the process of healing.
2. Poor blood supply to wound slows healing e.g. injuries to face heal quickly due to rich blood
supply while injury to leg with varicose ulcers having poor blood supply heals slowly.
3. Foreign bodies including sutures interfere with healing and cause intense inflammatory reaction
and infection.
4. Movement delays wound healing.
5. Exposure to ionising radiation delays granulation tissue formation.
6. Exposure to ultraviolet light facilitates healing.
7. Type, size and location of injury determine whether healing takes place by resolution or
organisation.
Systemic factors:
1. Age. Wound healing is rapid in young and somewhat slow in aged and debilitated people due to
poor blood supply to the injured area in the latter.
2. Nutrition. Deficiency of constituents like protein, vitamin C (scurvy) and zinc delays the wound
healing.
3. Systemic infection delays wound healing.
4. Administration of glucocorticoids has anti-inflammatory effect.
5. Uncontrolled diabetics are more prone to develop infections and hence delay in healing.
6. Haematologic abnormalities like defect of neutrophil functions (chemotaxis and phagocytosis),
and neutropenia and bleeding disorders slow the process of wound healing.
Granulation tissue: The following 3 phases are observed in the formation of granulation tissue:
1. Phase of inflammation. Following trauma, blood clots at the site of injury there is acute
inflammatory response with exudation of plasma, neutrophils and some monocytes within 24
hours.
2. Phase of clearance. Combination of proteolytic enzymes liberated from neutrophils, autolytic
enzymes from dead tissues cells, and phagocytic activity of macrophages clear off the necrotic
tissue, debris and red blood cells.
3. Phase of ingrowth of granulation: This phase consists of 2 main processes: angiogenesis or
neovascularisation, and fibrogenesis.
i) Angiogenesis (neovascularisation).
a. Formation of new blood vessels at the site of injury takes place by proliferation of
endothelial cells from the margins of severed blood vessels. Soon, these blood vessels
differentiate into muscular arterioles, thin-walled venules and true capillaries. The
process of angiogenesis is stimulated with proteolytic destruction of basement
membrane.
b. Angiogenesis takes place under the influence of following factors:
i) Vascular endothelial growth factor (VEGF)
 52
ii) Platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), basic
fibroblast growth factor (bFGF) and surface integrins
ii) Fibrogenesis.
a. The new fibroblasts originate from fibrocytes as well as by mitotic division of
fibroblasts. Some of these fibroblasts have combination of morphologic and
functional characteristics of smooth muscle cells (myofibroblasts).
b. Collagen fibrils begin to appear by about 6th day. As maturation proceeds, more and
more of collagen is formed while the number of active fibroblasts and new blood
vessels decreases. This results in formation of inactive looking scar known as
cicatrisation.
Contraction of wounds:
1. The wound starts contracting after 2-3 days and the process is completed by the 14th day. During
this period, the wound is reduced by approximately 80% of its original size.
2. Contracted wound results in rapid healing since lesser surface area of the injured tissue has to be
replaced.
3. Mechanism of wound contraction:
i) Dehydration as a result of removal of fluid by drying of wound was first suggested but
without being substantiated.
ii) Contraction of collagen was thought to be responsible for contraction but wound
contraction proceeds at a stage when the collagen content of granulation tissue is very
small.
iii) Discovery of myofibroblasts appearing in active granulation tissue has resolved the
controversy surrounding the mechanism of wound contraction. These cells have features
intermediate between those of fibroblasts and smooth muscle cells. Their migration into
the wound area and their active contraction decreases the size of the defect.
iv) The evidences in support of this concept are both morphological as well as functional
characteristics of modified fibroblasts or myofibroblasts as under:
1. Fibrils present in the cytoplasm of these cells resemble those seen in smooth
muscle cells.
2. These cells contain actin-myosin similar to that found in non-striated muscle
cells.
3. Cytoplasm of these modified cells demonstrates immunofluorescent labelling
with anti-smooth muscle antibodies.
4. Nuclei of these cells have infoldings of nuclear membrane like in smooth
muscle cells.
5. These cells have basement membrane and desmosomes, which are not seen
in ordinary fibroblasts.
6. Drug response of granulation tissue is similar to that of smooth muscle.

KARYOTYPING IN GYNECOLOGY:
Definition:
1. Karyotypes describe chromosome count of an organism and what these chromosomes look like
(length, position of centromeres, banding pattern, differences between sex chromosomes, and
other physical characteristics) under a light microscope.
2. Karyotyping - process of pairing and ordering all chromosomes of an organism, thus providing a
genome-wide snapshot of an individual's chromosomes.
 53
3. Chromosomes are depicted (by rearranging a photomicrograph) in a standard format (in pairs,
ordered by size and position of centromere for chromosomes of the same size) known as a
Karyogram or Idiogram.
4. Clinical Cytogeneticists analyze human karyotypes to detect gross genetic changes.
Procedure:

1. The chromosomes of a cell are present within the nucleus. In normal stage they are indistinct and
cannot be studied.
2. During metaphase of cell division, they are separated and can be studied by a staining and
identification of individual chromosome under the microscope.
3. They are photographed and individual chromosomes are cut out and arranged in homologous
pairs in descending order of height. This photographic representation of the entire chromosome
complement of a cell is called karyotype.
4. The karyotyping is indicated to detect chromosomal anomalies (both structural and numerical).
5. Common Techniques for Studying Chromosomes
a. Karyotyping using Giemsa banding.
b. FISH (Fluroscent in situ Hybridization) is a rapid method as no culture is necessary.
Denver classification of chromosomes:
1. After identifying the chromosomes from their banding patterns, it is possible to arrange them in
seven groups according to their morphological features.
2. Group A consists of pairs of following chromosome numbers 1, 2 and 3 ( total chromosome
number being 6). They are the longest and metacentric.
3. Group B consists of pairs of chromosome numbers 5 and 6(total chromosome number being 4).
They are long and submetacentric.
4. Group C consists of pairs of chromosome numbers from 6 to 12 and X chromosome. They are of
medium size and submetacentric. The total number in this group varies with sex. In female, the
total number of chromosomes in C-group is 16 while in male it is 15.
5. Group D consists of pairs of chromosome number 13, 14 and 15. They are medium-sized
acrocentric chromosomes. They are SAT- chromosomes. Their total number is 6.
6. Group E consists of pairs of chromosome number 16, 17 and 18. They are shorter than the
previous group and submetacentric.
7. Group F consists of pairs of chromosome number 19 and 20. They are short metacentric.
 54
8. Group G consists of pairs of chromosomes 21and 22 in addition to Y chromosome (total no.5).

Karyotyping in OG:
It is known that many reproductive system diseases are caused by chromosome abnormalities. There
is an association between chromosome karyotype abnormalities in peripheral blood lymphocytes and
azoospermia, oligospermia, amenorrhea, abnormal gonad development and adverse pregnancy
outcome.
Azoospermia or oligospermia and chromosome karyotype abnormalities
1. Sexual chromosome abnormalities are the most important contributor to azoospermia or
oligospermia. This phenotype is more common in patients with Klinefelter syndrome, which has
the chromosomal karyotype 47, XXY.
2. 10.55% of individuals with Klinefelter syndrome were azoospermic. Infertility in these patients is a
consequence of the direct harmful effect of an extra X chromosome, which causes a lethal gene
dosage effect in testicular cells that results in azoospermia .
3. Recent studies have shown that the absence of azoospermia factor (AZF) on the long arm of the Y
chromosome can also lead to azoospermia or severe oligozoospermia, which is closely related to
male infertility. AZF is a set of genes or gene clusters associated with spermatogenesis. Previous
studies revealed that Y chromosome microdeletions occurred in 1% to 55% of infertile men with
azoospermia or severe oligozoospermia.
Amenorrhea and chromosome karyotype abnormalities
1. Primary amenorrhea in women is caused by pituitary/ hypothalamic disorders (27.8%), gonadal
dysfunction (50.4%), or outflow tract abnormalities (21.8%). Thus, gonadal/ovarian disorders
make up half of the all PA cases. This category of etiology often stems from abnormal sex
chromosomes.
2. Turner syndrome (TS) is the most common chromosomal aneuploidy; it affects 1 in every 2,000
girls and is characterized by a short stature and gonadal dysgenesis in females who lack all or part
of one X chromosome.
Gonadal dysplasia and chromosome karyotype abnormalities
1. Disorders of sex development (DSD) are congenitally conditions with atypical chromosomal,
gonadal or anatomical sex development. Based on chromosomal classification, DSD is divided into
 55
sex chromosome abnormalities, 46,XY DSD and 46,XX DSD.
2. Sex chromosome abnormalities DSD include Klinefelter syndrome, Turner’s syndrome, super-male
syndrome, and ultra-female syndrome.
3. The XYY karyotype has an incidence of one in 1,000 male newborns and may result from a
nondisjunction in paternal meiosis II or postzygotic mitotic nondisjunction.
4. The 48,XXXY syndrome has an incidence of 1:50,000, and patients present as male with external
genital dysplasia.
5. 46,XY DSD and 46,XX DSD include additional marker chromosomes (mar), autosomal balanced
translocations, Robertsonian translocation, and sex reversal. Mar chromosomes are a structural
abnormal, and patients have the karyotype 48,XY,+mar1,+mar2. Although the source of the
chromosome cannot be determined, it is thought that it may carry decisive development-related
genes, thus affecting the development of the reproductive system.
6. Sex reversal includes 46,XY testicular feminization syndrome and 46,XX male syndrome. The
pathogenesis is associated with deletion, mutation, or translocation of the sex-determining region
Y gene (SRY) on the Y chromosome.
History of adverse pregnancy outcome and chromosome karyotype abnormalities
1. C h r o m o s o m a l a b n o r m a l i t i e s a r e m o r e c o m m o n in spontaneous abortion, patients with
congenital malformations or dysplasia, advanced maternal age pregnancy and infertile couples,
leading to spontaneous abortion, infertility, congenital malformations.
2. Balanced chromosomal translocation accounts for most cases; it has been observed in 0.6% of
infertile couples and in as many as 9.2% of couples with recurrent miscarriages.
3. Carriers of balanced chromosomal translocations are known to have high rates of unbalanced
gametes, exhibit impaired or reduced gametogenesis, produce large numbers of unbalanced
embryos and have a greater chance of being infertile and/or a high risk of conceiving
chromosomally abnormal pregnancies that lead to recurrent spontaneous abortions or children
with congenital anomalies. The most common disorders of Robertsonian translocation are
trisomy 13 and Down syndrome.
4. Chromosome inversion is also more common, and the main clinical manifestations are infertility,
abortion and stillbirth.
5. Chromosomal polymorphisms refer to the constant, small but non-pathological difference in the
structure and tinctorial strength of chromosomes between different individuals. It usually refers
to variation in the satellite zone of the D/G group chromosomes, insertion or deletion of the
secondary constriction of chromosome 1, 9, 16 and Y chromosome; and inversion of chromosome
9 and Y. When the homologous chromosomes with polymorphisms pair, the polymorphic section
cause difficulties in homologous chromosome pairing, which affects cell division, leading to
embryonic developmental disorders that result in abortion, embryonic death or chromosomal
abnormalities.
Conclusion:
1. The prognoses of different types of chromosome dysplasia are not the same, most are
undesirable. Therefore, prevention is particularly important. Preventive measures include the
implementation of chromosome counseling and chromosome detection.
2. The best preventative measure is third-generation of IVF (genetic screening) to prevent the birth
of fetuses with chromosomal abnormalities. Pregnant women should attend regular prenatal
checkups to identify fetal problems as soon as possible.

PULMMONARY SURFACTANAT:
Introduction:
 56
1. Surfactant is a surface acting material or agent that is responsible for lowering the surface tension
of a fluid.
2. Surfactant that lines the epithelium of the alveoli in lungs is known as pulmonary surfactant and it
decreases the surface tension on the alveolar membrane. It prevents the collapse of alveoli.
3. Source of secretion of pulmonary surfactant Pulmonary surfactant is secreted by two types of
cells:
a. Type II alveolar epithelial cells in the lungs, which are called surfactant secreting alveolar
cells or pneumocytes. Characteristic feature of these cells is the presence of microvilli on
their alveolar surface.
b. Clara cells, which are situated in the bronchioles. These cells are also called bronchiolar
exocrine cells
Composition of surfactant
1. Surfactant is a lipoprotein complex formed by lipids especially phospholipids, proteins and ions.
a. Phospholipids: Phospholipids form about 75% of the surfactant. Major phospholipid
present in the surfactant is dipalmitoylphosphatidylcholine (DPPC).
b. Other lipids: Other lipid substances of surfactant are triglycerides and
phosphatidylglycerol (PG)
c. Proteins: Proteins of the surfactant are called specific surfactant proteins. There are four
main surfactant proteins, called SPA, SPB, SPC and SPD. SP-A is a large glycoprotein and
has a collagen-like domain within its structure. It has multiple functions, including
regulation of the feedback uptake of surfactant by the type II alveolar epithelial cells that
secrete it. Surfactant becomes inactive in the absence of proteins.
d. Ions: Ions present in the surfactant are mostly calcium ions.
2. Formation of surfactant
a. Type II alveolar epithelial cells and Clara cells have a special type of membrane bound
organelles called lamellar bodies, which form the intracellular source of surfactant.
b. Laminar bodies contain surfactant phospholipids and surfactant proteins. These materials
are synthesized in endoplasmic reticulum and stored in laminar bodies.
c. By means of exocytosis, lipids and proteins of lamellar bodies are released into surface
fluid lining the alveoli. Here, in the presence of surfactant proteins and calcium, the
phospholipids are arranged into a lattice (meshwork) structure called tubular myelin.
d. Tubular myelin is in turn converted into surfactant in the form of a film that spreads over
the entire surface of alveoli. Most of the surfactant is absorbed into the type II alveolar
cells, catabolized and the products are loaded into lamellar bodies for recycling.
e. Formation of surfactant is facilitated by PG and calcium ions. Glucocorticoids play
important role in the formation of surfactant.

 57
3. Functions of surfactant
a. Surfactant reduces the surface tension in the alveoli of lungs and prevents collapsing
tendency of lungs.
b. Surfactant is responsible for stabilization of the alveoli, which is necessary to withstand
the collapsing tendency.
c. Another important function of surfactant is its role in defense within the lungs against
infection and inflammation. Hydrophilic proteins SPA and SPD destroy the bacteria and
viruses by means of opsonization.
4. Absence of surfactant in infants causes collapse of lungs and the condition is called respiratory
distress syndrome or hyaline membrane disease. Deficiency of surfactant occurs in adults also and
it is called Adult respiratory distress syndrome (ARDS)
Surfactant level in Newborn:
1. Surfactant synthesis begins at 24 weeks. It appears in amniotic fluid between 28 and 32 wk.
Mature levels of pulmonary surfactant are present in lung after 35 wk.
2. Surfctant level is increased by:
a. Antenatal corticosteroids
b. Stress induced by PIH, IUGR and twin gestation
3. Decrease in surfactant level:
a. Synthesis of surfactant depends in part on normal pH, temperature, and perfusion.
Asphyxia, hypoxemia, and pulmonary ischemia, particularly in association with
hypovolemia, hypotension, and cold stress, may suppress surfactant synthesis.
b. Fetal hyperinsulinemia ( maternal diabetes)
Hyaline membrane disease (Typpe II RDS):
1. In preterm, immature type II alveolar cells produce less surfactant, causing an increase in alveolar
surface tension and a decrease in compliance.
2. The resultant atelectasis causes pulmonary vascular constriction, hypoperfusion, and lung tissue
ischemia.
3. Pulmonary blood flow is reduced and ischemic injury both to the cells producing surfactant and to
the vascular bed results in an effusion of proteinaceous material into the alveolar spaces.
4. Hyaline membrane is formed through the combination of sloughed epithelium, protein, and
edema
5. This leads to hypoxia and respiratory failure leading to death.
Surfactant therapy:
1. Exosurf is a synthetic surfactant. Natural surfactants include Survanta (bovine), Infasurf (calf), and
Curosurf (porcine). Natural surfactants are superior because of their surfactant-associated protein
content, their more rapid onset, and their lower risk of pneumothorax and improved survival.
2. Types of surfactant therapy:
a. Prophylactic: surfactants within 15 mts of birth to all preterm < 30 weeks
b. Resque or relacement therapy: preferably within 2 hours after birth
c. Repeat: dosing is given via the endotracheal tube every 6-12 hr for a total of 2 to 4 doses
d. Initial and subsequent dose of Survanta is 100 mg/kg
e. Initial dose of Curosurf is 100-200 mg/kg and subsequent doses of 100 mg.
3. INSURE technique: Start resuscitation with CPAP of at least 5–6 cm water via mask or nasal prongs
to stabilize the airway and establish functional residual volume. Intubate àsurfactantà extubate
à CPAP (INSURE technique)
4. Side effects of surfactant therapy:
a. Small risk of pulmonary hemorrhage
 58
b. Secondary lung infection
c. Pneumothorax
d. Transient hypoxia,
e. Hypercapnia,
f. Bradycardia
g. Hypotension,
h. Blockage of the endotracheal tube
5. Contraindications:
a. Congenital anomalies incompatible with life
b. Respiratory distress in infants with laboratory evidence of lung maturity
6. Other uses of surfactant therapy:
a. Severe pneumonias
b. Meconium aspiration syndrome
c. Persistence of pulmonary hypertension
d. Pulmonary hemorrhage and adult RDS

PREVENTION OF PARENT TO CHILD TRANSMISSION (PPTCT)
Introduction:
1. Mother-to-child-transmission of HIV is a major route of HIV infection in children.
2. The National PPTCT programme recognises the four elements integral to preventing HIV
transmission among women and children. These are:
Prong 1: Primary prevention of HIV, especially among women of child bearing age.
Prong 2: Preventing unintended pregnancies among women living with HIV.
Prong 3: Prevent HIV transmission from pregnant women infected with HIV to their child.
Prong 4: Provide care, support and treatment to women living with HIV, her children and
women in child bearing age.
AN women attending AN clinic:
Four typical scenarios where pregnant women may attend the counselling and testing services
include:
1. Women attending ante natal clinics.
2. Pregnant spouse of HIV-positive men, or those with high risk behaviour.
3. Pregnant women screened at the Sub centre level by ANM/Nurse (whole blood finger
prick test) & Confirmation at ICTC.
4. Women presenting directly-in-labour (un-booked cases, require a HIV screening test
before delivery).
Process of Screening ANC Women
1. Women registered for ANC care would be screened for TB along with HIV and Syphilis by ANMs at
sub centre level.
2. ANM checks for TB symptoms (Refer Pregnant women to designated microscopic centre (DMC) at
PHC if there is a persistent cough of any duration. It may be accompanied by one or more of the
following symptoms such as weight loss, chest pain, tiredness, shortness of breath, fever,
particularly rise of temperature in the evening, in some cases there can be blood in the sputum,
loss of appetite and night sweats).
3. Refer all HIV positive pregnant women to RNTCP for TB diagnosis and treatment at the earliest
For HIV Negative Pregnant Women
1. Safe sex counselling
2. Couple counselling.
 59
3. Linkages to family planning services.
4. Free condoms.
5. Behaviour change communication (BCC) for high risk women and her partner.
6. Repeat HIV testing, considering window period if spouse is positive or s/he have high risk
behaviour.
7. Infant feeding and nutrition counselling
For Infected Pregnant Women
1. Ante-natal Care (ensure at least 4 visits)
2. Counselling on choices of continuation or medical termination of pregnancy (MTP)–to undertake
with in the first 3 months of pregnancy only.
3. Screening for TB (40 Gene-Xpert testing sites is being launched shortly) and other OIs.
4. Screening and syndromic treatment for STIs.
5. WHO clinical staging and CD4 testing.
6. Counselling on positive living, safe delivery, birth-planning and infant feeding options.
7. Couple and safe sex counselling and HIV testing of spouse and other living children.
8. Linkage to ART services.
9. Provide ART regardless of clinical stage and CD4 count
10. Nutrition counselling and linkages to Government/other nutrition programmes.
11. Family Planning Services.
12. EBF (expressed breast milk feeding) reinforcement/Infant feeding support through home visits.
13. Psycho-social support through follow-up counselling, home visits and support groups
For HIV Exposed Infant (HEI)
1. Exclusive breastfeeds up to 6 months (preferred Option-I WHO/NACO Guidelines 2010-11) and
continued breastfeeds in addition to complementary feeds after 6 months up to 1 year for EID
negative babies and up to 2 years for EID positive babies who receive Paediatric ART.
2. Postpartum ARV prophylaxis for infant for minimum 6 weeks.
3. Early infant diagnosis (EID) at 6 weeks of age; repeat testing at 6 months, 12 months & 6 weeks
after cessation of breastfeeds.
4. Co-trimoxazole prophylaxis from 6 weeks of age.
5. HIV care and Pediatric ART for infants and children diagnosed as HIV positive through EID.
6. Growth and nutrition monitoring.
7. Immunizations and routine infant care.
8. Gradual weaning after 6 months and introduction of complementary feeds from 6 months
onwards along with continuation of BF for at least 1 year for adequate growth & development of
the child..
9. Confirmation of HIV status of all babies at 18 months using all 3 Antibody (Rapid) Tests
Management of HIV positive AN/PN mother:

1. Initiate lifelong ART in all pregnant women with confirmed HIV infection regardless of WHO
clinical stage or CD4 cell count. ART shall be initiated only at ART centre
2. The recommended first-line regimen for HIV infected Pregnant Women is: Tenofovir (TDF) (300
mg) + Lamuvidine (3TC) (300 mg) + Efavirenz (EFV) (600 mg) (if there is no prior exposure to
NNRTIs (NVP/EFV) at any gestational age
3. Continue ART throughout pregnancy, delivery, breast feeding period and thereafter lifelong
4. Co-trimoxazole should be started if CD4 count is ≤ 250 cells/mm3 and continued through
pregnancy, delivery and breastfeeding as per national guidelines (Dose: Double strength tablet – 1
tab daily).
 60
5. Ensure that pregnant women take their folate supplements regularly
ARV Prophylaxis for Infants Born to Mothers Receiving Life-long ART
1. Birth to 6 weeks: (Infants with birth weight < 2000 gm) NVP 2 mg/kg once daily (0.2 ml/kg) for 6
weeeks; irrespective of whether exclusively breast fed or exclusively replacement fed. (may be
extended to 12 weeks, if mother has not received ART for adequate duration i.e atleast 24 weeks)
2. Birth to 6 weeks: (Infants with birth weight 2000-2500 gm) NVP 10 mg once daily (1 ml once
daily) for 6 weeeks; irrespective of whether exclusively breast fed or exclusively replacement fed.
(may be extended to 12 weeks, if mother has not received ART for adequate duration i.e atleast
24 weeks)
3. Birth to 6 weeks: (Infants with birth weight > 2500 gm) NVP 15 mg once daily (1.5ml/kg) for 6
weeeks; irrespective of whether exclusively breast fed or exclusively replacement fed. (may be
extended to 12 weeks, if mother has not received ART for adequate duration i.e atleast 24 weeks)
Interventions for Women Diagnosed with HIV Infection in Labour and Postpartum
1. There is a significant percentage of pregnant women with unknown HIV status presenting
directlyin- labour for delivery (un-booked cases). Any pregnant woman who presents in active
labour with unknown HIV status should be offered the routine screening of HIV, with opt-out
option as per National Guidelines. Screening using Whole Blood Finger Prick Test in the
delivery/labour ward should be undertaken.
2. Pregnant Women in Labour Who are Found Positive in HIV-screening Test should be Initiated on
ART (TDF+3TC+EFV) immediately.
3. The next day the Counsellor should visit the post-natal ward offer to ensure:
a. Pre-test Counselling,
b. Counsel and advise for exclusive breast feeding for first 6months, if she has already
started breast feeds; if not she must be counselled on option for breast vs replacement
feeding but must adhere to either exclusive breast feeding or exclusive replacement
feeding the first six months.
c. Confirm the HIV status by 3 rapid anti-body tests and CD4 testing of all HIV confirmed
cases by Lab tech
d. Post-partum mother and husband to reach the ART Centre within the next 2 days for Pre-
ART Registration and Adherence Counselling or atleast within 30 days.
e. No interruption in the continuation ART once the first dose was given to the HIV positive
pregnant women in the labour-room and the next day in the post-natal ward.
 61

Intra-partum Management:
1. Women on life-long ART should continue to receive ART as per the usual schedule including
during labour and delivery.
2. Caesarean section is not recommended for prevention of mother-to-child-transmission and only if
there is an Obstetric indication for the same.
3. For planned (elective) Caesarean sections, ART should be given prior to the operation.
4. In case of an emergency Caesarean section in pregnant women who are not on ART, ensure that
the women receive ART prior to the procedure and continues thereafter.
Safer Delivery Techniques:
1. Mother-to-child -transmission risk is increased by the prolonged rupture of membranes, repeated
P/V examinations, assisted instrumental delivery (vacuum or forceps), invasive foetal monitoring
procedures (scalp/foetal blood monitoring), episiotomy and prematurity.
2. Standard/Universal Work Precautions (UWP)
3. Do NOT rupture membranes artificially (keep membranes intact for as long as possible). The
membranes should be left intact as long as possible and artificial rupture of membrane reserved
for cases of foetal distress or delay in progress of labour.
4. Minimize vaginal examination and use aseptic techniques.
5. Avoid invasive procedures like foetal blood sampling, foetal scalp electrodes.
6. Avoid instrumental delivery as much as possible. Unless required in cases of foetal distress or
significant maternal fatigue to shorten labour or the duration of ruptured membranes. If
indicated, low-cavity outlet forceps is preferable to ventouse, as it is generally associated with
lower rates of foetal trauma than ventouse.
7. Avoid routine episiotomy
8. Avoid suctioning of NB unless meconium is present.
Safer surgical techniques are useful in conducting any operative procedures such as the Caesarean
section, repairing wounds/lacerations etc.
1. Use of ‘dry’ haemostatic techniques to minimize bleeding; i.e. good observation and following of
surgical fascial planes during dissection, judicious use of electro-cautery during Caesarean section
etc.
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2. During Caesarean section, wherever possible, the membranes are left intact until the head is
delivered through the surgical incision.
3. The cord should be clamped as early as possible after delivery;
4. Use of round-tip blunt needles for Caesarean section
5. Do not use fingers to hold the needle;
6. Use forceps to receive and hold the needle
7. Observe good practice when transferring sharps to surgical assistant eg. holding container for
sharps.
8. For disposal of tissues, placenta and other medical/infectious waste material from the delivery of
HIV-infected deliveries Standard waste disposal management guidelines should be followed.
The Post-partum Period
1. Infants born to HIV-infected mothers should receive NVP prophylaxis immediately after birth.
2. Skin contact with mother
3. Exclusive breast-feeding for 6 months and complementary feeding after that; no abrupt weaning.
4. Invove husband and family members
5. Assessment of maternal healing after delivery and evaluation for post partum infectious
complications.
6. Advise regular follow up and ART regularly
7. Family planning advice

LABORATORY DIAGNOSIS OF HIV INFECTION:
Diagnosis of HIV Infection
1. Like other infectious diseases, HIV diagnosis is made by either demonstrating the presence of
virus or viral products in the host, alternatively by detecting host response to the virus.
2. An HIV diagnosis is commonly made through serological assays to detect HIV specific antibodies
or by Nucleic Acid Amplification Test (NAAT) to detect HIV nucleic acids.
3. Serological Tests:
a. Enzyme linked immunosorbent assays (ELISAs),
b. Rapid tests and
c. Western blots (WBs) are the common tests for detecting HIV antibodies.
d. Additionally, Chemiluminescence Immunoassays (CIA), Immuno Floresent Assays and Line
Immunoassays are also available for specific HIV antibody detection.
e. Commercial assays are also available for P24 antigen detection.
4. NAAT:
a. These are sensitive tests for diagnosis of HIV infections. They use polymerase chain
reactions (PCRs) for the detecting various HIV structural genes (usually gag, pol and env).
PCRs are the test of choice in certain situations, such as early infant diagnosis and during
window period.
b. Branch DNA (bDNA) assays based on signal amplifications are also used.
5. Diagnosis in a child less than 18 months cannot be done using antibody based assays as maternal
antibodies may be present in the infant’s circulation. Therefore, up to the age of 18 months, the
diagnosis of HIV infection can only be reliably made by DNA PCR
Lab tests for Monitoring of Antiretroviral Therapy (ART)
1. CD4 T cell enumeration
2. HIV RNA load assays
3. Measurement of HIV p24,
4. Reverse Transcriptase (RT) activity assay.
 63
Prerequisites:
1. Pre-test Counselling
2. Confidentiality
SEROLOGICAL TESTS
Enzyme Linked Immunosorbent Assay (ELISA)
1. All ELISAs consist of either HIV antigens or antibodies (depending upon the principle), attached to
a solid phase (matrix or support), and incorporated with a conjugate and substrate detection
system.
2. Viral antigens may be whole viral lysates, recombinant, or synthetic peptides. The matrix can be
“wells” or “strips” of a microplate, plastic beads, or nitrocellulose paper.
3. Conjugates are most often antibodies (lgG, sometimes lgM and IgA) coupled to enzymes (alkaline
phosphatase or horseradish peroxidase), fluorochromes, or other reagents that will subsequently
bring about a reaction that can be detected. In case of enzyme conjugates, the signal generated is
a colour reaction and in case of fluorochrome, it is fluorescence.
4. The substrates used are 4-nitrophenylphosphate – for alkaline phosphatase and
ophenylenediamine dihydrochloride (OPD) and TMB – for horseradish peroxidase, which produce
colour on being acted upon by the respective enzymes. The colour can be measured on an ELISA
Reader as optical density (OD) values. ELISAs are suitable for use in laboratories where the
specimen load is high.
5. On the basis of the principle of the test, ELISA can be divided into:
a. Indirect
b. Competitive
c. Sandwich
d. Capture
6. Indirect is the common test; principle in the diagram

8. False Positive and False Negative ELISA Results
a. Autoimmune diseases
b. Alcoholic hepatitis
c. Primary biliary cirrhosis
d. Leprosy
e. Multiple pregnancies
9. Common causes of a false negative result:
a. Technical errors
b. During the window period and
c. During the end stage of the disease
Rapid Anti-HIV Tests
1. Several rapid tests have been developed using recombinant and/or synthetic antigens. The most
commonly employed rapid anti-HIV tests are based on the principle of Immunoconcentration/dot
blot immunoassay (vertical flow), Immunochromatographic (lateral flow), particle agglutination
(e.g., gelatine or latex), and Dipstick and Comb assay based on EIA.
Western Blot Test:
1. In the western blot, the various HIV specific recombinant or synthetic a n t i g e n s a r e a d s o r b e d o n t o
nitrocellulose paper. The antibody, when present, attaches to the antigen on the strip and the
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antigen antibody complex is then detected using enzyme conjugate and substrate. This is similar
to what is done in an ELISA test, except that the product is insoluble.
2. The test procedure should be carried out as per the kit insert. WB tests detect the presence of
antibodies against specific HIV proteins, which are seen as bands on the test strip. The test results
are interpreted as per kit instructions. WB tests are a highly specific conformational test.
Diagnosis of Paediatric HIV Infection (< 18 months)
1. Nucleic Acid Testing (NAT) can facilitate early infant diagnosis. NACO recommends the use of a
qualitative HIV-1 DNA PCR.
2. Steps for PCR:

NACO’s Strategy 2 B (used for diagnosis in symptomatic patients)
1. This strategy is used to determine the HIV status of a clinically symptomatic suspected AIDS cases
in which blood/serum/plasma is tested with a highly sensitive screening test.
2. The specimen is considered negative if the test gives a non-reactive result. In case the test result
is reactive the specimen is tested with another test kit (based on a different principle of test or
having a different antigen as compared to the first test).
3. If the result is also reactive with the second test kit, the specimen is considered to be positive for
HIV in a symptomatic AIDS case.

HAART THERAPY:
1. The principle of combining several anti retroviral drugs is known as highly active antiretroviral therapy
(HAART)
2. Indications to start HAART
CD4 count (cells/mm3) Decision
Seroconversion Consider treatment
≥ 350 Monitor 3–6-monthly
201–350 Treat as soon as patient ready
 65
< 200 Treat as soon as possible
AIDS-defining diagnosis Treat as soon as possible
3. Types of antiretroviral drugs: There are four main antiretroviral drug classes currently used to
construct first-line treatment regimens;
i. Nucleoside and nucleotide reverse transcriptase inhibitors (NRTIS/NTRTIS), also called nucleoside
or nucleotide analogues:
1. 3TC (lamivudine, Epivir)
2. Abacavir
3. AZT (zidovudine, Retrovir)
ii. Non-nucleoside reverse transcriptase inhibitors (NNRTIS):
1. Efavirenz
2. Nevirapine
iii. Protease inhibitors (PIS):
1. Atazanavir
2. Darunavir
3. Fosamprenavir
iv. Integrase inhibitors:
1. Raltegravir
4. General Principles for the use of HAART
i. Combination of at least 3 drugs, usually: 2 NRTIS (the “NRTI backbone”), plus: NNRTI or 1-2 PIS
ii. Antiretroviral drugs and recommended combinations for therapy in treatment-naïve and
experienced patients:
Naïve patient:
1. ‘Combination antiretroviral therapy (ART) containing either efavirenz or boosted lopinavir
together with two NRTIs
2. Equivalent results are demonstrated with several first-line PIs; all protect against the
development of resistance.
Drug Experienced:
1. Regimen should include two fully active new agents from following different classes
2. PIs and NNRTIs
a. Darunavir
b. Tipranavir
c. Etravirine
3. Entry inhibitors
a. Maraviroc
b. Enfuvirtide (T-20)
4. Integrase inhibitors
a. Raltegravir

PCR AND ITS CLINICAL IMPLICATIONS:
INTRODUCTION:
1. PCR is an in vitro DNA amplification procedure in which millions of copies of a particular sequence
of DNA can be produced within a few hours. Starting material could be a single molecule of rRNA,
mRNA of DNA.
2. PCR uses DNA polymerase to repetitively amplify targeted portions of DNA. Each cycle of
amplification doubles the amount of DNA in the sample, leading to an exponential increase in
DNA with repeated cycles of amplification.
 66
3. The amplified DNA sequence can then be analyzed by gel electrophoresis, Southern blotting, or
direct sequence determination.
4. It can amplify the sequence, even when the targeted sequence makes up less than one part in a
million of the total initial sample.
5. The method can be used to amplify DNA sequences from any source—bacterial, viral, plant, or
animal.
The reaction cycle has the following steps:

1. For PCR, nucleic acid is first extracted (released) from the organism or a clinical sample potentially
containing the target organism by heat, chemical, or enzymatic methods.
2. Once extracted, this target nucleic acid is added to the reaction mix containing all the necessary
components for PCR (primers, nucleotides, covalent ions, buffer, and enzyme) and placed into a
thermal cycler to undergo amplification.
Steps in Amplification
Conventional PCR involves 25 to 50 repetitive cycles, with each cycle comprising three sequential
reactions:
a. Denaturation of target nucleic acid
b. Primer annealing to single-strand target nucleic acid extension of primer target duplex.
c. Extension of the primer-target duplex.
Denaturation
The reaction mixture is heated to 95°C for a short time period to denature the target DNA into single
strands that can act as templates for DNA synthesis.
Primer:
 67
1. It is necessary to know the nucleotide sequence of short segments on each side of the target
DNA. They are called flanking sequences and these sequences are used to prepare two, single-
stranded oligonucleotides which are called primers.
2. These two synthetic oligo nucleotides function as templates for DNA polymerase to selectively
amplify the target DNA.
3. Through complementary base pairing, one primer attaches to the top strand at one end of the
DNA segment of interest, and the other primer attaches to the bottom strand at the other end.
4. This would explain that we need two primers for PCR

5. Annealing (heating and then cooling) of primers to DNA: The primers are annealed by cooling to
50°C for 0.5 to 2 minutes. Then the primers pair with their complementary single stranded DNA
produced in the first step.
6. Extension:
a. DNA polymerase and deoxyribonucleoside triphosphates (in excess) are added to the
mixture to initiate the synthesis of two new strands complementary to the original DNA
strands.
b. DNA polymerase adds nucleotides to the 3'-hydroxyl end of the primer, and strand
growth extends across the target DNA, making complementary copies of the target.
c. At the completion of one cycle of replication, the reaction mixture is heated again to
separate the strands. Each strand binds a complementary primer, and the cycle of chain
extension is repeated.
d. DNA polymerase used for this reaction is derived from bacteria Thermus acquaticus. This
is a heat-stable DNA polymerase (Thermus aquaticus that normally lives at high
temperatures), the polymerase is not denatured and, therefore, does not have to be
added at each successive cycle.
7. Each newly synthesized strand can act as a template for the successive cycles. This leads to an
exponential increase in the amount of target DNA with each cycle hence the name “polymerase
chain reaction.”
8. After the amplification procedure, DNA hybridization technique or Southern blot analysis with a
suitable probe, shows the presence of the DNA in the sample tissue.
PCR types:
1. Reverse Transcriptase PCR (RTPCR):
a. m-RNA, r-RNA can be the starting material in such types of PCR. This is widely used in
expression mapping, determining when and where certain genes are expressed.
b. In ordinary PCR, DNA is detected; that DNA could be from a living or nonliving organism.
But in reverse PCR, mRNA is detected; that means, it is derived from a living organism.
Presence of HIV RNA in blood can be detected as early as 4 weeks after infection.
2. Nested PCR:
a. Nested PCR is intended to reduce the contamination in products due to the amplification
of unexpected primer binding sites.
b. Two sets of primers are used in two successive PCR runs, the second set intended to
 68
amplify a secondary target within the first run product. This is very successful, but
requires more detailed knowledge of the sequences involved.
3. Real Time PCR
a. By this method, quantitation of the number of virus present in a sample can be
calculated., e.g.,viral load in HIV or HBV.
b. So, the treatment modalities can be planned and the response to treatment could be
assessed.
4. Quantitative PCR
a. Q-PCR (Quantitative PCR) is used to rapidly measure the quantity of PCR product.
b. This is commonly used for the purpose of determining whether a sequence is present or
not, and if it is present the number of copies in the sample.
5. Quantitative Real time PCR (RQ-PCR)
This method is used to measure the amount of amplified product in real time.
6. RACE-PCR
a. RACE, or Rapid Amplification of cDNA Ends, is a technique used in molecular biology to
amplify the ends of messenger RNA (mRNA) transcripts.
7. Multiplex-PCR
By targeting multiple genes at once, additional information may be elicited from a single
test run that otherwise would require several times the reagents and technician time to
perform.
Clinical Applications of PCR
1. Diagnosis of bacterial and viral diseases:
a. PCR could detect even one TB bacillus present in the specimen. Any other bacterial
infection could also be detected similarly. The specific nucleotide sequences of the bacilli
are amplified by PCR and then detected by Southern blot analysis.
b. If reverse PCR is done, living organisms could be detected. This technique is widely used
in the diagnosis of viral infections like Hepatitis C, Cytomegalo virus and HIV.
c. PCR offers a rapid and sensitive method for detecting viral DNA sequences even when
only a small proportion of cells is harboring HIV virus.
2. Medicolegal cases:
a. PCR allows the DNA from a hair follicle or a blood cell to be analyzed. The restriction
analysis of DNA from the hair follicle from the crime scene is studied after PCR
amplification.
b. This pattern is then compared with that of various suspects; (DNA fingerprinting). This
helps to identify the criminal.
3. Diagnosis of genetic disorders:
a. The PCR technology has been widely used to amplify the gene segments that contain
known mutations for diagnosis of inherited diseases such as sickle cell anemia, beta
thalassemia, cystic fibrosis, etc.
b. PCR is especially useful for prenatal diagnosis of inherited diseases, where cells obtained
fromfetus by amniocentesis are very few.
4. Cancer detection:
a. PCR is widely used to monitor residual abnormal cells present in treated patients.
b. Similarly identification of mutations in oncosuppressor genes such as p53, retinoblastoma
gene, etc. can help to identify individuals at high risk of cancer.
5. Fossil studies: DNA can be isolated and PCR amplified from fossils and is used to study evolution
by comparing the sequences in the extinct and living organisms.
 69

ANTIMETABOLITES:
1. Folate antagonist Methotrexate (Mtx)
Pemetrexed
2. Purine antagonist 6-Mercaptopurine (6-MP),
6-Thioguanine (6-TG),
Azathioprine,
Fludarabine
3. Pyrimidine antagonist 5-Fluorouracil (5-FU),
Capecitabine,
Cytarabine (cytosine arabinoside)
ANTIMETABOLITES
1. These are analogues related to the normal components of DNA or of coenzymes involved in
nucleic acid synthesis. They competitively inhibit utilization of the normal substrate or get
themselves incorporated forming dysfunctional macromolecules.
1. Folate antagonist
Methotrexate (Mtx)
1. This folic acid analogue is one of the oldest and highly efficacious antineoplastic drugs which acts
by inhibiting dihydrofolate reductase (DHFRase)—blocking the conversion of dihydrofolic acid
(DHFA) to tetrahydrofolic acid (THFA).
2. Utilizing the folate carrier it enters into cells and is transformed to more active polyglutamate
form by the enzyme folypolyglutamate synthase (FPGS). Tetrahydrofolic acid is an essential
coenzyme required for one carbon transfer reactions in de novo purine synthesis and amino acid
interconversions. The inhibition is pseudoirreversible because Mtx has 50,000 times higher
affinity for the enzyme than the normal substrate.
3. Methotrexate has cell cycle specific action— kills cells in S phase; In addition to DHFRase it
inhibits thymidylate synthase as well so that DNA synthesis is primarily affected. However,
synthesis of RNA and protein also suffers.
4. It exerts major toxicity on bone marrow—low doses given repeatedly cause megaloblastic
anaemia, but high doses produce pancytopenia. Mucositis and diarrhoea are common side
effects. Desquamation and bleeding may occur in g.i.t.
5. Methotrexate is absorbed orally, 50% plasma protein bound, little metabolized and largely
excreted unchanged in urine. Salicylates, sulfonamides, dicumerol displace it from protein binding
sites. Aspirin and sulfonamides enhance toxicity of Mtx by decreasing its renal tubular secretion.
6. The toxicity of Mtx cannot be overcome by folic acid, because it will not be converted to the
active coenzyme form. However, Folinic acid (N5 formyl THFA, cirtrovorum factor) rapidly
reverses the effects. Thymidine also counteracts Mtx toxicity.
7. Methotrexate is apparently curative in choriocarcinoma: 15–30 mg/day for 5 days orally or 20–40
mg/m2 BSA i.m. or i.v. twice weekly. In a dose of 2.5–15 mg/day it is highly effective in
maintaining remission in children with acute leukaemias, but it is not good for inducing remission:
Mtx is widely used in non-Hodgkin lymphoma, breast, bladder, head and neck cancers, osteogenic
sarcoma, etc. It has prominent immunosuppressantproperty useful in rheumatoid arthritis,
psoriasis and many other antoimmune disorders
8. NEOTREXATE 2.5 mg tab, 50 mg/2 ml inj; BIOTREXATE 2.5 mg tab, 5, 15, 50 mg/vial inj.
9. The use of folinic acid rescue has permitted much higher doses of Mtx and has enlarged its scope
to many difficult-to-treat neoplasms. A nearly 100 times higher dose (250–1000 mg/m2 BSA) of
 70
Mtx is infused i.v. over 6 hours, followed by 3–15 mg i.v. calcium leucovorin within 3 hours,
repeated as required. This procedure can be repeated weekly.
Pemetrexed
1. This newer congener of Mtx primarily targets the enzyme thymidylate synthase. Though, it is also
a DHFRase inhibitor, the pool of THFA is not markedly reduced. Like Mtx it utilizes the folate
carrier to enter cells and requires transformation into polyglutamate form by FPGS for activity
enhancement.
2. Adverse effects (mucositis, diarrhoea, myelosuppression) are similar to Mtx, but a painful, itching
erythematous rash, mostly involving the hands and feet, ‘handfoot syndrome’ is quite common.
Dexamethasone can relieve it, and pretreatment can reduce its incidence. Low dose folic acid and
vit B12 pretreatment is recommended to limit pemetrexed induced myelosuppression. NSAIDs
should be avoided as they decrease pemetrexed clearance and may increase toxicity.
3. In combination with cisplatin, pemetrexed is approved for treatment of mesoepithelioma and
non-small cell lung carcinoma.
4. Dose: 500 mg/m2 i.v. every 3 weeks.
5. PEMEX 500 mg vial for i.v. inj.
2. Purine antagonists
Mercaptopurine (6-MP) and thioguanine (6-TG)
1. These are highly effective antineoplastic drugs. After synthesis in the body to the corresponding
monoribonucleotides, they inhibit the conversion of inosine monophosphate to adenine and
guanine nucleotides that are the building blocks for RNA and DNA. There is also feedback
inhibition of de novo purine synthesis. They also get incorporated into RNA and DNA which are
dysfunctional.
2. 6-MP and 6-TG are especially useful in childhood acute leukaemia, choriocarcinoma and have
been employed in some solid tumours as well. In acute leukaemia, both have been used in
combination regimens to induce remission and 6-MP for maintaining remission as well.
3. All antipurines are absorbed orally (though incompletely). Azathioprine and 6-MP are oxidised by
xanthine oxidase; their metabolism is inhibited by allopurinol; dose has to be reduced to ¼ - ½ if
allopurinol is given concurrently.
4. Thioguanine is not a substrate for xanthine oxidase; follows a different (S-methylation) metabolic
path and its dose need not be reduced if allopurinol is given. Methylation by thiopurine methyl
transferase (TPMT) is an additional pathway of 6-MP metabolism. Genetic deficiency of TPMT
makes the individual more susceptible to 6-MP induced myelosuppression, mucositis and gut
damage, while over expression of TPMT is an important mechanism of 6-MP resistance in acute
leukaemia cells. Toxicity of azathioprine is also enhanced in TPMT deficiency.
5. The main toxic effect of antipurines is bone marrow depression, which develops slowly.
Mercaptopurine causes more nausea and vomiting than 6-TG. It also produces a high incidence of
reversible jaundice. Hyperuricaemia occurs with both, and can be reduced by allopurinol.
6. Dose: 6-Mercaptopurine: 2.5 mg/kg/day, half dose for maintenance; PURINETHOL, EMPURINE, 6-
MP, 50 mg tab. 6-Thioguanine: 100–200 mg/m2/day for 5–20 days; 6-TG 40 mg tab.
Azathioprine
1. This antipurine acts by getting converted to 6-MP, but has more prominent immunosuppressant
action, probably because of selective uptake into immune cells and intracellular conversion to 6-
MP. This is further synthesized into the nucleic acid inhibitory neucleotides.
2. Azathioprine primarily suppresses cell mediated immunity (CMI) and is used mainly in
autoimmune diseases (rheumatoid arthritis, ulcerative colitis) as well as in organ transplantation.
3. Dose: Azathioprine 3–5 mg/kg/day, maintenance 1–2 mg/kg/ day; IMURAN, TRANSIMUNE,
 71
AZOPRINE 50 mg tab.
Fludarabine
1. This newer purine antimetabolite is phosphorylated intracellularly to the active triphosphate form
which then inhibits DNA polymerase and ribonucleotide reductase, interferes with DNA repair as
well as gets incorporated to form dysfunctional DNA.
2. Tumour cell apoptosis is promoted by multiple mechanisms confering activity even in slow
growing neoplasms. It is indicated in chronic lymphatic leukaemia and non-Hodgkin’s lymphoma
that have recurred after treatment.
3. Prominent adverse effects are chills, fever, myalgia, arthralgia and vomiting after injection,
myelosuppression and opportunistic infections (it is a potent suppressant of CMI).
4. Dose: 25 mg/m2 BSA daily for 5 days every 28 days by i.v. infusion FLUDARA 50 mg/vial inj.
3. Pyrimidine antagonists
Pyrimidine analogues have varied applications as antineoplastic, antifungal and antipsoriatic agents.
Fluorouracil (5-FU)
1. It is converted in the body to the corresponding nucleotide 5-fluoro- 2-deoxyuridine
monophosphate, which forms a covalent ternary complex with methyl-THFA and tymidylate
synthase (TS) resulting in irreversible inhibition of TS. Consequently conversion of deoxyuridilic
acid to deoxythymidylic acid is blocked. Selective failure of DNA synthesis occurs due to non-
availability of thymidylate. Accordingly, thymidine can partially reverse 5-FU toxicity.
2. 5-FU itself gets incorporated into RNA, interferes with RNA synthesis and function contributing to
its cytotoxicity. Even resting cells are affected, though rapidly multiplying ones are more
susceptible. Since inhibition of TS by 5-FU is dependednt on the prsence of THFA, concurrent
infusion of leucovorin enhances the efficacy of 5-FU. Cisplatin and oxaliplatin also synergise with
5-FU.
3. Most protocols now employ 5-FU along with leucovorin and cisplatin/ oxaliplatin. Currently, 5-FU
is a commonly used anticancer drug for many solid malignancies, especially of colon, rectum,
stomach, pancreas, liver, urinary bladder, head and neck. Oral absorption of 5-FU is unreliable. It
is primarily used by i.v. infusion. 5-FU is rapidly metabolized by dihydropyrimidine dehydrogenase
(DPD) resulting in a plasma tó of 15–20 min after i.v. infusion.
4. Genetic deficiency of DPD predisposes to severe 5-FU toxicity. Major toxicity of 5-FU is exerted on
the bone marrow and g.i.t. causing myelosuppression, mucositis, diarrhoea, nausea and vomiting.
Peripheral neuropathy (hand-foot syndrome) also occurs.
5. Dose: 500 mg/m2 i.v. infusion over 1–3 hours weekly for 6–8 weeks, or 12 mg/kg/day i.v. for 4
days followed by 6 mg/kg i.v. on alternate days, 3–4 doses. FLURACIL, FIVE FLURO, FIVOCIL 250
mg/5 ml and 500 mg/10 ml vial.
6. A 1% topical solution applied twice daily for 3–6 weeks has yielded gratifying results in superficial
basal cell carcinoma, and in actinic keratosis.
Capecitabine
1. It is an orally active prodrug of 5-FU. After absorption it is converted to deoxy-5-fluorouridine in
the liver and released in blood. Taken up by cells, it is hydrolysed to 5-FU by thymidine
phosphorylase. Because many breast and colorectal cancer cells express large quantity of this
enzyme, they generate more 5-FU and suffer higher toxicity than normal cells.
2. A combined regimen of capecitabine + oxaliplatin is frequently used in metastatic colorectal
cancer. It has also been utilized in 2nd line treatment of metastatic breast cancer along with
taxanes. Hand-foot syndrome and diarrhoea are prominent adverse effects, but bone marrow
depression and mucositis are less marked.
Cytarabine (Cytosine arabinoside)
 72
1. This cytidine analogue is phosphorylated in the body to the corresponding nucleotide which
inhibits DNA synthesis. The triphosphate of cytarabine is an inhibitor of DNA polymerase, as well
as blocks production of cytidilic acid. However, its incorporation into DNA is now considered to be
more important for the expression of its cytotoxicity. DNA repair is also affected. Cytarabine is cell
cycle specific and acts primarily during ‘S’ phase.
2. Cytarabine is useful only in leukaemias and lymphomas, and is not effective in solid tumours.
Primary use is induction of remission in acute myelogenous as well as lymphoblastic leukaemia in
children and in adults. It is also used for blast crisis in chronic myelogenous leukaemia and non-
Hodgkin’s lymphoma. Because cytarabine is rapidly deaminated and cleared from plasma, it is
administered either by rapid i.v. Injection (100 mg/m2) once or twice daily for 5–10 days, or by
continuous i.v. Infusion over 5–7 days. A high dose regimen of 1–3 g/day has also been used.
3. CYTABIN, CYTOSAR, BIOBIN, REMCYTA 100, 500, 1000 mg inj.
4. Major toxic effects are due to bone marrow suppression—leukopenia, thrombocytopenia,
anaemia and mucositis, diarrhoea.

SCREENING METHODS FOR OSTEOPOROSIS
Definition:
1. Osteoporosis is defined by the World Health Organization (WHO) as “a skeletal disease,
characterized by low bone mass and microarchitectural deterioration of bone tissue, with a
consequent increase in bone fragility and susceptibility to fracture”.
2. Histologically, the disorder is characterized by a decrease in cortical thickness and in the number
and size of the trabeculae of cancellous bone. Individual trabecular plates may be fractured or
trabecular connectivity may be reduced. There is no abnormality in the structure of organic
matrix (osteoid), nor is there any defect in mineralization.
Riggs and Melton classification
1. Type-I: Osteoclast-mediated: seen in recently postmenopausal women
2. Type-II: Osteoblast-mediated: osteoporosis affects women twice as frequently as men and is
related to:
a. Aging,
b. Chronic calcium deficiency,
c. Increased parathyroid hormone activity, and
d. Decreased bone formation.
3. An individual can display traits of both types of osteoporosis over time.
Screenig methods:
1. Standard radiography
2. Radiogrammetry
3. Photodensitometry
4. Compton scattering
5. Neutron activation analysis
6. Single and dual photon absorptiometry
7. Dual energy X-ray absorptiometry
8. Quantitative computed tomography
9. Quantitative ultrasound
10. Magnetic resonance imaging
11. Multidetector computed tomography.
Standard Radiography
1. Qualitative assessment of bone mineral content from standard radiographs of skeleton is usually
 73
based on subjective criteria. The assessment is commonly done on metacarpal, calcaneum, talus,
femoral neck, vertebral bodies, etc.
2. The findings are:
a. Generalized osteopenia, especially the vertebral column.
b. Thinning and accentuation of cortices.
c. Accentuation of primary trabeculae with thinning of secondary trabeculae.
d. Changes in vertebral body shape—wedge shape, biconcave and compression.
e. Insufficiency fractures due to normal stress on weakened bone. Sacrum, supra-acetabular
regions, os pubis, etc. are common sites. The fracture may be occult radiographically and
detected on CT or scintigraphy.
f. Fractures at other sites (especially wrist and hip).
3. The major disadvantage is that it is subjective and gets affected by body habitus, radiographic
exposure factors, the presence of overlying soft tissue and patient positioning.
4. Magnification radiography by either optical or directgeometric means has a small but significant
role in the assessment of osteoporosis. Subperiosteal and intracortical bone absorption, even
subtle, may be appreciated on magnification radiography.
Radiogrammetry
1. Radiogrammetry is one of the simplest methods of quantitative bone mineral assessment.
Conventional AP radiograph of a tubular bone (like metacarpal) is taken. The cortical thickness on
either side of medullary space is measured with a measuring device, and their sum is expressed as
the combined cortical thickness (CCT).
2. This technique is most commonly used at midshaft of second metacarpal. Cortical bone volume is
calculated from the CCT.
3. However, it does not reliably reflect absolute bone mineral content and does not measure
intracortical bone porosis.
4. This technique too, is imprecise and insensitive.
Photodensitometry
1. In this technique, images of bone of interest and of reference aluminum wedge of known density
are exposed on the same film. The optical density of the bone is then compared with that of the
wedge with photodensitometer.
2. This provides information comparable to that of single photon absorptiometry but with slightly
lower precision, sensitivity and accuracy.
Neutron Activation Analysis
1. In this method, neutrons from an accelerator or reactor bombard a small fraction of the total Ca
in the body, changing it to 49Ca, which is a radioactive isotope. By counting its activity, total
calcium content of the body is estimated.
2. Disadvantage is relatively large radiation dose (200 to 300 mrem).
Compton Scattering
1. This technique measures the absolute density of a volume of tissue based on its electron density
and is the only method other than computed tomography that can measure purely trabecular
bone.
2. It is based on measurement of scattered radiation from a source of 100 to 700 keV gamma rays
and is used for calcaneum, spine and radius.
3. The precision of this method is about 3 to 5 percent and radiation dose varies from 200 to 2000
mrem. At present, it is largely a research tool.
Single Energy Photon Absorptiometry
1. Single energy photon absorptiomerty (SPA) technique requires a gamma ray source (125I), a
 74
detector and intervening electronics that measure the beam attenuation through bone and
express the result in bone mineral per sq cm scanned.
2. A major clinical limitation of this technique, is that it reflects the status of peripheral long bone
and measures primarily the cortex.
Dual Energy Photon Absorptiometry
1. DPA technique utilizes radioisotope that emits photons at two different energy levels. The chief
advantage is that the gamma ray energy of the source is higher (153Gd with photons of 42 and
100 keV) and it has a uniform path length.
2. The lumbar spine from L1 to L4 is usually scanned. The major disadvantage is that vertebral
compression fractures with callus formation, kyphoscoliosis, articular facet hypertrophy,
diskogenic sclerosis, marginal osteophytosis and extraosseous calcification (aorta) are also
included in the integral measurement and may result in inaccurate and poorly reproducible
vertebral measurements.
Dual Energy X-ray Absorptiometry
1. DXA measurements are currently the standard of reference for the clinical diagnosis of
osteoporosis with bone densitometry. DXA makes use of an X-ray beam composed of two
different photon energies. The intensities of high-energy and low-energy photons are analyzed
separately after the protons have passed through bones and soft tissue. With a computing
algorithm, the attenuation values of bone are calculated.
2. Recent developments include fanbeam X-ray sources and a bank of detectors, that permit more
rapid scanning.
3. Dividing bone mineral content by the area yields bone mineral density (BMD) (in grams per
square centimeter). BMD is expressed in terms of standard deviation (SD) as a T-score and a Z-
score.
4. The T-score describes the difference between the BMD of the patient being examined and the
mean BMD of a standard young adult population (20–30 years of age), and refers to the peak of
bone mass.
5. The Z score shows the difference between the patient’s BMD and the mean BMD of age- and
gender-matched controls.
6. The WHO classifies BMD on the basis of the T- score:

7. Bone mineral density examinations have three principal roles, namely:
a. the diagnosis of osteoporosis,
b. the assessment of patients’ risk of fracture, and
c. monitoring response to treatment.
8. BMD is a strong predictor of fracture risk, accounting for 75 to 85 percent of bone strength. The
risk of fracture increases approximately 1.5-fold for each SD decrease from age-adjusted BMD.
9. Limitations:
a. It cannot help distinguish between cortical and trabecular bone and
b. cannot help diskriminate between changes due to bone geometry
Quantitative Computed Tomography
 75
1. Quantitative CT is different from DXA in that it provides separate estimates of trabecular and
cortical bone BMD as a true volumetric mineral density in milligrams per cubic centimeter.
2. A lateral computed radiograph (scout view) is first obtained for localization and then single axial
scans are obtained through the midplane of two to four lumbar vertebral bodies.
3. Quantitative readings are obtained from a region of interest over trabecular bone encompassing
3 to 4 cm3 of each vertebral body and from four different reference solutions in the phantom.
4. These readings are averaged and used to calculate the mineral density of trabecular bone in
mineral equivalents of K2HPO4 (mg/cm3)
5. Quantitative CT is excellent for predicting vertebral fractures in postmenopausal women and
serially measuring bone loss, generally with better sensitivity than projectional methods (such as
DXA)
6. The disadvantage of QCT is that accuracy of BMD decreases to 20 to 25 percent in elderly
osteoporosis populations.
Quantitative Ultrasound
1. QUS of bone is performed at substantially lower frequencies, i.e. between 200 kHz and 1.5 MHz
and signals are acquired in transmission mode. In addition to assessing bone density these
variables reflect parameters such as elasticity and microarchitecture which are important in
assessing fracture risk in osteoporotic subjects.
2. US is currently being performed at peripheral sites like patella, fingers, tibia and calcaneum. The
calcaneum is the most favored site as it is a weight bearing bone and composed almost entirely of
trabecular bone.
3. Quantitative US results can be expressed as absolute values or as T-scores and Z-scores.
4. Currently, US assessment is used as a screening tool, with confirmation of diagnosis by means of
DXA evaluation

ENERGY SOURCES IN ENDOSCOPIC SURGERY:
1. There are a variety of electrosurgical instruments available to endoscopists to allow for a multitude of
procedures. As technology has improved, the increasing availability of endoscopic instruments has led
to the development of more complex endoscopic procedures.
2. Energy Sources
1. Monopolar cautery
2. Bipolar cautery
3. Ultrasonic Energy System
4. Harmonic scalpel
5. Laser
6. Argon Beam Coagulation
7. Radiofrequency electro-surgery
Monopolar electrosurgery
1. All electrosurgery is ‘bipolar’ in as much as the electrical current flows from one pole or electrode
to another. In monopolar electrosurgery, the active electrode is one pole and the patient return
electrode is the other. The main difference is that electrical current flows through the patient.
2. The tissue effects with monopolar electrosurgery include vaporisation (tissue destruction and
cutting), fulguration (tissue destruction and small vessel haemostasis), desiccation (cell wall
rupture and cytoplasm boiling) and coaptation (vessel sealing owing to denaturation and
renaturation of proteins)
3. These tissue effects are primarily achieved using the ‘cut’ or ‘coag’ electrosurgical unit (ESU)
settings while contacting the target tissue. Varying other parameters under the surgeon’s control,
 76
such as power setting, duration of activation and electrode configuration, can further modify the
desired tissue effect.
4. The scissors configuration in monopolar surgery allows the greatest tissue dissection capability,
adding tissue plane dissection and cold cutting to the repertoire.
5. Tissue temperatures with monopolar electrosurgery are ~100°C, 100–200°C and >200°C for
desiccation (and coaptation), vaporisation and fulguration, respectively.
Disadvantages:
1. The major disadvantage of monopolar electrosurgery is the rare but unavoidable risk of stray
current injury (SCI). These injuries are often not noticed during surgery as they usually occur
outside of the surgeon’s field of vision. They are not owing to surgeon error or lack of skill. It is
due to lateral thermal spread injury to adjacent structures with monopolar electrosurgery.
2. Smoke production during monopolar electrosurgery may be problematic, especially during
fulguration.
Advantages:
1. So, monopolar electrosurgery is a relatively inexpensive, readily available and versatile energy
source that yields the best range of tissue effects.
2. Despite the small risk of SCI, this modality remains in common use in conventional laparoscopy, as
well as in robot-assisted and single-port laparoscopy.
Conventional bipolar electrosurgery
1. In bipolar electrosurgery, the active and return electrodes are the two jaws of the energy
source. In bipolar electrosurgery, electrical current passes through the tissue held between the
jaws of the instrument, not through the patient, and results in tissue desiccation and vessel
coaptation in an analogous fashion to monopolar electrosurgery performed in contact mode.
2. Alternating current is standard output for ESUs and it is this physical property that results in
efficient sealing of vessels with bipolar electrosurgery.
Advantages:
3. A major advantage of conventional bipolar over monopolar electrosurgery is the ability to seal
(coapt) vessels up to ~5mm in diameter (monopolar contact mode and fulguration mode would
generally be reserved for vessels 1–2mm and <1mm in diameter, respectively).
4. The dissection capability of the bipolar forceps is good, especially in the Maryland configuration.
5. Bipolar electrosurgery is generally available and relatively inexpensive.
Disadvantages:
1. Lateral thermal spread that will continue until device activation is ceased; no audio signal from
the ESU to inform the surgeon when desiccation or coaptation is complete, which increases the
risk of injury from lateral thermal spread as well as tissue charring and tissue adherence to the
instrument jaws; and
2. The need for another instrument, such as a laparoscopic scissor, for tissue cutting.
Advanced bipolar electrosurgery
1. Advanced bipolar energy sources are revolutionary in several ways. Firstly, a ESU using a
computer-controlled tissue feedback system controls each device.
2. The tissue impedance is monitored with continuous adjustment of the generated voltage and
current to maintain the lowest possible power setting to achieve the desired tissue effect.
3. An audio signal alerts the surgeon that the endpoint has been reached. In this way, the risk of
lateral thermal spread as well as charring of the tissue and adherence of tissue to the device jaws
is minimised.
4. Secondly, these energy sources were the first to be approved to seal vessels up to 7mm in
diameter owing to technological advances such as:
 77
a. Tissue impedance monitoring up to 4000 times per second (LigaSure);
b. Temperature-sensitive material in the device jaws that optimises tissue temperatures at
~100 °C (EnSeal);
c. Delivery of pulsed energy with continuous feedback control to prevent tissue overheating
(PK System); and
d. Jaw design that optimises mechanical compression to the vascular pedicle.
5. Although the ability of these newer devices to seal vessels up to 7mm in diameter is
unquestioned, the expected minimisation of lateral thermal spread injuries owing to these
technologies has yet to be proven in clinical trials.
6. Some devices incorporate a cutting blade into the device jaws (LigaSure, EnSeal) that decreases
the need for a laparoscopic scissor.
Ultrasonic devices
1. The first ultrasonic energy source was described in 1993 by Amaral, called the ‘laparoscopic
scalpel’; it had the dual functionality of tissue cutting and vessel sealing.
2. Ultrasonic energy sources convert electrical energy into vibrations in the handpiece of the device
at frequencies more than 20 000 cycles per second, that is, above the audible range. These
vibrations oscillate the non-articulating jaw of the instrument.
3. Tissue is compressed between an articulating jaw and the nonarticulating jaw to impart the tissue
effects derived from combination of thermal and mechanical energy: desiccation and vessel
sealing is achieved at lower frequencies; and tissue cutting occurs at higher frequencies.
4. The tissue effects arise owing to the conversion of electrical energy into thermal energy for tissue
desiccation and vessel sealing, and also mechanical energy for the tissue cutting. As for the other
energy sources, the generated tissue temperature is ~100°C to achieve dessication and
coaptation; hence, lateral thermal spread injury is possible with ultrasonic energy sources.24
5. The tissue effects obtained with ultrasonic energy are essentially the same as those achieved with
contact monopolar electrosurgery or bipolar electrosurgery, with the added function of tissue
cutting. These tissue effects are achieved without the passage of electric current through the
patient or the tissue grasped by the device.
6. Advantages of ultrasonic devices include less instrument traffic, owing to the combination of
vessel-sealing and tissuecutting, and less smoke generation (a ‘mist’ of tissue debris and moisture
results rather than smoke per se). The dissection capability is good, but less than that of
monopolar scissors or Maryland bipolar forceps.
7. Disadvantages include the risk of lateral thermal spread injuries, as well as higher and more
prolonged instrument tip temperatures than other energy sources which could potentially result
in organ injury. Also, the cutting mode, in particular, requires training and experience. As for all
new-generation energy sources, ultrasonic devices are relatively expensive, but generally
accessible in most institutions.
Hybrid devices
1. Laparoscopic devices have recently been developed that combine several energy source
technologies. These include LigaSure Advance and Thunderbeat.
2. Incorporation of multiple functionalities into a single device may reduce instrument traffic and
the overall cost, although such benefits should be a secondary consideration if the individual
functionalities are compromised in the hybrid configuration.
3. Good-quality studies on the efficacy and safety of the hybrid devices are lacking.
The dangers of energy sources
1. Injuries owing to laparoscopic energy sources can be divided into two groups.
 78
2. Firstly, thereare injuries owing to surgical misadventure that occur within the surgeon’s field of
vision. These include iatrogenic injuries (that is, ‘I did it’ – a mistake by the surgeon) and lateral
thermal spread injuries (lateral thermal spread occurs with all energy sources).
3. Secondly, there are stray current injuries (SCIs) that may be owing to capacitive coupling,
insulation failure and direct coupling. These injuries occur outside the surgeon’s field of vision and
are not owing to surgeon error.
4. During the infancy of monopolar electrosurgery it was realised that the passage of alternating
current through the patient resulted in muscle contraction, cardiac arrhythmia and often death.
5. The French biophysicist D’Arsonoval subsequently discovered, in 1891, that it is possible to pass
high-frequency alternating current (<20 kHz) through the body without life-threatening
ramifications. In 1928, the American neurosurgeon Cushing and physicist Bovie introduced their
ESU that incorporated all the knowledge of the day about monopolar electrosurgery, namely,
electrical energy, high-frequency alternating current with continuous and interrupted waveforms,
time of application, power settings and electrode size. Their ESU remained essentially unchanged
until the addition of bipolar electrosurgery technology in the 1970s.
6. Although many energy sources have been introduced since the advent of laparoscopic surgery,
monopolar electrosurgery remains a commonly used and cost-effective modality. However, it is
unfortunately a not-infrequent cause of unexpected injury.
7. It is estimated that injuries owing to electrosurgery occur in 0.1– 0.5 per cent of laparoscopic
procedures.
8. Bowel injuries owing to laparoscopy are thought to have an incidence of 1.3 per 1000 cases and,
of these, half are thought to be owing to electrosurgery. Many, if not all, of these injuries could be
prevented if surgeons understood the physics of monopolar electrosurgery and used the available
technology.
Mechanisms of SCI
Capacitance coupling
1. The high-frequency alternating current used in monopolar electrosurgery and the relatively thin
instrument-insulation layer allows capacitance coupling to occur, where current from the active
electrode is transmitted across the intact insulation layer to another conductor nearby. Any
nearby tissues that contact the secondary conductor are at risk of SCI.
2. More concerning is with plastic laparoscopic ports: biological fluid coating the surface of the
insulation layer can act as the secondary conductor and the plastic port prevents the safe
dispersal of the capacitive current through the abdominal wall. In this situation, the risk of
capacitive coupling injury is theoretically higher.
Insulation failure
1. Only a thin insulation layer surrounding the active electrode of monopolar devices protects the
patient from SCI. Insulation failure occurs at sites of micro-fractures in the insulation layer, most
likely owing to capacitive coupling current and wear and tear, including the sterilisation process
for reusable devices. So, new instruments offer some, but not complete, protection against
insulation failure.
Direct coupling
1. Direct coupling injury occurs when stray current from capacitive coupling or insulation failure is
transmitted through another candidate to cause tissue damage.
2. In this scenario, the laparoscope is the most likely conductor to contact other instruments and
adjacent tissues.
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Robot-assisted and single-port laparoscopy
1. The popularity of monopolar electrosurgery has been maintained with the evolution of robot-
assisted and single-port laparoscopy.
2. The risk of SCI with single-port laparoscopy and robot-assisted (particularly single- versus multi-
site) laparoscopy is increased, primarily because of the surgeon’s decreased field of vision and
increased risk of instrument contact/clashing. The latter increases the risk of all of capacitive
coupling, insulation failure and, in particular, direct coupling SCIs.

ANTIMALARIALS IN PREGNANCY:
Introduction;
1. Although malaria is the most important parasitic disease of man there are very few studies of
antimalarials in human pregnancy. Almost nothing is known on the pharmacokinetics, safety and
efficacy of drugs used to treat pregnant women with malaria. This is because pregnant women,
even though a highly susceptible group, are systematically excluded from clinical trials. As a
result, there is very little evidence on which to base recommendations for the prevention and the
treatment of malaria during pregnancy.
2. There are data on antimalarials in animals but their relevance to human is questionable. The
problem is even more acute for the first trimester of pregnancy (when the risk of embryotoxcicity
is higher) and there are generally no agreed recommendations in the national policies for
antimalarial drug choice in asymptomatic parasitaemic women in the first trimester of pregnancy
Pharmacokinetics
1. The pharmacokinetics of antimalarials are altered in pregnancy. This is the consequence of
multiple factors: expansion of the volume of distribution, increase in clearance, changes in the
protein binding, lipid distribution and absorption of drugs, as well as influence of hormonal
changes on the drug metabolism.
2. These changes can result in lower plasma concentrations and AUCs and this can lead to reduced
efficacy.
Safety:
1. The antimalarials considered safe in the first trimester of pregnancy areQuinine, chloroquine,
proguanil, pyrimethamine and sulfadoxine–pyrimethamine. Of these, quinine remains the most
effective and can be used in all trimesters of pregnancy including the first trimester.
2. Amodiaquine, chlorproguanil- dapsone, halofantrine, lumefantrine and piperaquine have not
been evaluated sufficiently to permit positive recommendations.
3. Sulfadoxine–pyrimethamine is safe but may be ineffective in many areas because of increasing
resistance.
4. Primaquine and tetracyclines should not be used in pregnancy.
Recommended drugs:
Uncomplicated falciparum malaria
1. First trimester:
a. First episode: Quinine 10 mg/kg three times a day for 7 days with or without Clindamycin
5 mg/kg three times per day for 7 days.
b. Subsequent episodes: ACT (artemisinin based combination therapies) locally effective or
artesunate 2 mg/kg/d for 7 days with Clindamycin as above.
2. Second and third trimesters:
a. First episode: ACT locally effective or artesunate plus clindamycin as above.
b. Subsequent episodes: artesunate plus clindamycin as above. Or quinine plus clindamycin
as above.
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3. Prevention:
IPT(intermittent preventive therapy) with SP(Sulfadoxine and Pyrimethamine) where
efficacy remains.
Severe malaria
Artesunate 2.4 mg/kg IV at hour 0, 12 and 24 and continued every 24 hours until the patient can
tolerate oral artesunate 2 mg/kg/dose, for 7 days and clindamycin 5 mg/kg three times daily for 7
days.
OR
Quinine i.v: Loading dose (LD) 20 mg/kg given over four hours, then 10 mg given 8 hours after the
LD was started, followed by 10 mg/kg every 8 hours for 7d. Once the patient has recovered
sufficiently to tolerate oral medication both quinine 10 mg/kg and clindamycin 5 mg/kg, three
times daily should be continued daily for 7 days.
Non-falciparum malaria:
1. Chloroquine phosphate (1 tablet contains 250 mg salt, equivalent to 155.3 mg base). Dose is 10
mg/kg base once a day for 2 days followed by 5 mg/kg base on third day.
2. For chloroquine resistant P. vivax amodiaquine, quinine, mefloquine, artemisinin derivatives can
be used.
Prevention: chloroquine phosphate 300 mg on admission followed by 150 mg per week
Fron Dutta:
Management:
Prevention from mosquito bites is done using permethrin and pyrethroids-spray kill mosquitoes.
Electrically heated mats will kill mosquitoes in the room.
Chemoprophylaxis:
Chloroquine is given unless proved resistant. It is taken 300 mg base weekly, 2 weeks before
travel and covering the period of exposure and 4 weeks after leaving the endemic zone.
Mefloquine 5 mg/kg/week is the alternative drug in second and third trimesters when
chloroquine is found resistant.
Treatment:
1. Risks of malaria is life threatening in pregnancy. So benefits of treatment outweigh the potential
risk of antimalarial drugs.
2. Chloroquine—600 mg base P.O. followed by 300 mg 12 hours later. Then 300 mg daily for next 2
days. To prevent relapse during pregnancy, 300 mg is to be taken weekly until delivery. For radical
cure, primaquine should be postponed until pregnancy is over.
3. Parasites resistant to chloroquine should be given quinine (10 mg salt/kg P.O> every 8 hours for 7
days) under supervision. Patients with severe anemia may need blood transfusion. The
antimalarial drugs when given in therapeutic doses, have got no effect on uterine contraction
unless the uterus is irritable. Folic acid 10 mg should be given daily to prevent megaloblastic
anemia.
Complicated malaria:
1. Artesunate IV 2.4 mg/kg at 0, 12 and 24 hours, then daily thereafter. Oral therapy (2 mg/kg) is
started when the patient is stable. Alternatively Quinine IV followed by oral therapy is given.
Artesunate act very fast and resistance is rare. It is as effective as IV Quinine. Use is limited in the
second or third trimesters of pregnancy only when other drugs are found resistant.
2. Patient with severe malaria needs intensive care unit management. Supportive care is essential in
cases with hyperpyrexia, pulmonary edema, renal failure or DIC. Multidisciplinary team approach
is needed.

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KREB’S CITRIC ACID CYCLE:
1. The citric acid cycle is also known as the Tricarboxylic acid cycle (TCA cycle) and the Krebs cycle.
2. It is the final pathway where the oxidative metabolism of carbohydrates, amino acids, and fatty acids
converge, their carbon skeletons being converted to CO2 .
3. The TCA cycle is an aerobic pathway, because O2 is required as the final electron acceptor. This is in
contrast to glycolysis which operates in both aerobic and anaerobic conditions.
4. Location: It is located in the mitochondrial matrix in close proximity to electron transport chain.
5. Krebs cycle basically involves the combination of a two carbon acetyl CoA with a four carbon
oxaloacetate to produce a six carbon tricarboxylic acid, citrate. The two carbons are oxidized to CO2
and oxaloacetate is regenerated and recycled. Oxaloacetate is considered to play a catalytic role in
citric acid cycle.
6. The TCA cycle requires a large number of vitamins and minerals to function. These include niacin
(NAD+ ), riboflavin (flavin adenine dinucleotide [FAD] and flavin mononucleotide [FMN]), pantothenic
acid (coenzyme A), thiamine, Mg2+ , Ca2+ , Fe2+, and phosphate.
Preparatory steps:
1. Pyruvate is derived from Glycolysis and is transported by a carrier to mitochondria where it is
oxidatively decarboxylated to Acetyl CoA by pyruvate dehydrogenase complex.
2. Beta oxidation of fatty acids in mitochondria itself provide another source of Acetyl CoA.
3. All enzymes for TCA are present within mitochondria.
Pathway
Oxaloacetate
Citrate synthase
+ Acetyl CoA —> CoA-SH
Citric Acid
Aconitase
Cis-Aconitate + H2O
Aconitase
Isocitrate +NAD
Isocitrate dehydrogenase
Oxalosuccinate + NADH+H
Isocitarate dehydrogenase
Alpha keto glutarate + Co2
Alpha keto glutarate dehydrogenase
CoA-SH—> CO2
Succinyl CoA
succinyl thiokinase
CoA-SH
GTP+Pi —> GTP
Succinate
Succinate dehydrogenase
FAD —> FADH2

Fumarate
Fumarase
+H2O
Malate
Malate dehydrogenase
 82
NAD—> NADH + H
> Oxaloacetate
Steps:
1. 4 carbon oxaloacetate condense with 2 carbon Acetyl CoA, catalyzed by citrate synthase to form 6
carbon citrate.
2. Citrate is isomerized to isocitrate by aconitase ( Fe-S protein) in two step process of dehydration
followed by hydration through the formation of an intermediate cis-aconitate
3. In another two step process, isocitrate is dehydrogenated to form oxalosuccinate which
undergoes spontaneous decarboxylation to form alpha keto glutarate. The enzyme is isocitrate
dehydrogenase. NADPH is generated.
4. Alpha keto glutarate is oxidatively decarboxylated to form succinyl CoA by alpha keto glutarate
dehydrogenase, a multi enzyme complex.. NADH is generated. Co2 is removed.
5. Succinyl CoA is converted to succinate by succinate thiokinase. GTP is generated.
6. Succinate is dehydrogenated to Fumarate by succinate dehydrogenase. FADH2 is generated.
7. Fumarate is converted to L.malate by fumarase.
8. Malate is oxidized to oxaloacetate by malate dehydrogenase. NADH is generated.
9. Oxaloacetate again couples with acetyl CoA to restart the cycle
Summary:
Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2H2O ------>2C O2 + 3NADH + 3H+ + FADH2 + GTP + CoA
Significance of TCA cycle (Interpretation)
1. The TCA cycle is the final common pathway for the oxidation of carbohydrate, lipid, and protein
because glucose, fatty acids, and most amino acids are metabolized to acetyl-CoA. Final common
oxidative pathway for all major ingredients of food stuff.
2. It also has a central role in gluconeogenesis, lipogenesis, and interconversion of amino acids.
3. The citric acid cycle provides substrate for the respiratory chain
4. Many of these processes occur in most tissues, but the liver is the only tissue in which all occur to
a significant extent. Hence this cycle is impaired in liver diseases.
5. Complete oxidation of Acetyl CoA & ATP generation
6. It is an aerobic pathway:
a. There is no direct participation of oxygen in Krebs cycle. However, the cycle operates only
under aerobic conditions.
b. This is due to the fact that NAD+ and FAD (from NADH and FADH2, respectively) required
for the operation of the cycle can be regenerated in the respiratory chain only in the
presence of 02.
c. Therefore citric acid cycle is strictly aerobic in contrast to glycolysis which operates in
both aerobic and anaerobic conditions
Integration of major metabolic pathways:
1. The Citric Acid Cycle Takes Part in Gluconeogenesis, Transamination, & Deamination
2. Carbohydrates are metabolized through glycolytic pathway to pyruvate, and then converted to
acetyl CoA, which enters the citric acid cycle.
3. Fatty acids through beta oxidation, are broken down to acetyl CoA and then enter this cycle.
4. Glucogenic Amino acids after transamination enter at some points in this cycle. Ketogenic amino
acids are converted into acetyl CoA.
5. Excess Carbohydrates are converted to Neutral Fat. The pathway is glucose to pyruvate to acetyl
CoA to fatty acid.
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Energetics of TCA:
1. During the process of oxidation of acetyl CoA via citric acid cycle, 4 reducing equivalents (3 as
NADH and one as FADH2) are produced.
2. Oxidation of 3 NADH by electron transport chain coupled with oxidative phosphorylation results
in the synthesis of 3 x 2.5 = 7.5 ATP, whereas FADH2 leads to the formation of 1.5 ATP.
3. Besides there is one substrate level phosphorylation. Thus, a total of 10 ATP are produced from
one acetyl CoA.
a. 3 NADHx2.5 = 7.5 ATP
b. 1 FADH2x1.5 = 1.5
c. Substrate level = 1
Total = 10 ATP
Amphibolic pathway:
1. TCA cycle is both anabolic and catabolic and hence called Amphibolic. There is influx and efflux of
4 carbon units in the cycle.
2. TCA cycle is actively involved in gluconeogenesis, transamination and deamination
3. The anabolic reactions:
a. Oxaloacetate and α-ketoglutarate, respectively, serve as precursors for the synthesis of
aspartate and glutamate which, in turn, are required for the synthesis of other non-
essential amino acids, purines and pyrimidines.
b. Succinyl CoA —> Succinyl CoA is used for the synthesis of porphyins and heme.
c. Citrate —> acetyl CoA —> fatty acids and sterols.
4. Catabolic reaction: The two carbon compound acetyl-CoA produced from metabolism of
carbohydrates, Lipids and Proteins are oxidised in this cycle to produce CO2, H2O and energy as
ATP.
Anaplerotic role of TCA:
1. The citric acid cycle acts as a source of precursors of biosynthetic pathways, e.g. heme is
synthesized from succinyl CoA and aspartate from oxaloacetate.
2. To counterbalance such losses, and to keep the concentrations of the 4-carbon units in the cell,
anaplerotic reactions are essential. This is called anaplerotic role of TCA cycle (anaplerosis= fill
up)
3. Important Anaplerotic reactions:
a. Pyruvate is converted to oxaloacetate by pyruvate carboxylase.
b. Pyruvate is converted to malate by malate dehydrogenase.
c. Transamination reactions:
 84
i. Glutamate is converted to a-keto glutarate by glutamate dehydrogenase.
ii. Oxaloacetate is produced from aspartate by aspartate aminotransferase.
Inhibitors of TCA:
1. The important enzymes of TCA cycle inhibited by the respective inhibitors are:
i. Aconitase is inhibited by fluoro acetate.
ii. Alpha keto glutarate dehydrogenase is inhibited by arsenite.
iii. Succinate dehydrogenase is inhibited by malonate.
2. Fluoroacetate-a suicide substrate :
i. The inhibitor fluoroacetate is first activated to fluoroacetvl CoA which then condenses with
oxaloacetate to form fluorocitrate.
ii. TCA cycle (enzyme-aconitaseis) inhibited by fluorocitrate. The compound fluoroacetate, as
such, is a harmless substrate. But it is converted to a toxic compound (fluorocitrate) by
celIular metaboilsm. This is a suicide reaction committed by the cell, and thus fluoroacetate
is regarded as a suicide substrate.
Regulation of TCA:
1. Citrate synthase is inhibited by ATP, NADH, acyl CoA and succinyl CoA
2. Isocitarte dehydrogenase is activated by ADP; inhibited by ATP and NADH.
3. A-ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH
4. Availability of ADP is important for citric acid cycle to proceed.
Role of vitamins:
1. Four of the B vitamins are essential in the citric acid cycle and hence energy-yielding metabolism:
2. Riboflavin, in the form of flavin adenine dinucleotide (FAD), a cofactor for succinate
dehydrogenase;
3. Niacin, in the form of nicotinamide adenine dinucleotide (NAD), the electron acceptor for
isocitrate dehydrogenase, -ketoglutarate dehydrogenase, and malate dehydrogenase;
4. Thiamin(vitamin B1 ), as thiamin diphosphate, the coenzyme for decarboxylation in the -
ketoglutarate dehydrogenase reaction; and
5. Pantothenic acid, as part of coenzyme A, the cofactor attached to "active" carboxylic acid
residues such as acetyl-CoA and succinyl-CoA.
Metabolic defects of TCA:
1. Pyruvate dehydrogenase deficiency: leads to lactic acidosis and neurological disorders.
2. Acyl CA dehydrogenase deficiency: organic aciduria; glutaric aciduria; hypoglycemia and acidosis.
3. Pyruvate carboxylase deficiency: Lactic acidosis; hyperammonemia; hyperalaninemia.

STEM CELL BANKING AND STEMCELL THERAPY:
UMBILICAL CORD BLOOD BANKING
1. Cord blood banking of hemopoietic stem cells (HSC) from umbilical cord blood gives benefit to
store baby’s own HSC for a long time. This cord blood stem cell can be transplanted for the
treatment, in case that child or his/her siblings ever develop a metabolic, immunological,
hematological, neurological or cardiovascular disease.
2. Umbilical cord blood contains HSC that they have greater proliferative and colony forming
capacity and are more responsive to some growth factors. This cord blood is an useful source of
stem cells for mesenchymal (cartilage, fat, hepatic or cardiac) cells and neural precursor cells.
3. The major clinical use of umbilical cord blood is for hematological malignancy (leukemia) in
children. Compared to bone marrow transplant, the advantages of HSC transplant are:
a. Faster availability,
b. Better tolerance between donor and the recipient,
 85
c. Lower incidence and severity of graft versus host disease,
d. Low incidence of viral transmission (CMV, EBV),
e. Lack of donor attrition (bone marrow donor may not be available). Blood collection is
made from the ex-utero separated placenta.
STEM CELLS AND THERAPIES IN OBSTETRICS
1. Reproductive tissues are the important source of stem cells (progenitor cells). Stem cells have the
potential to be used in the field of regenerative medicine. A stem cell has the ability to renew
(reproduce) itself for long periods.
2. Potentials for the use of stem cells in Regenerative Medicine
a. Treatment of inherited genetic disorders
b. Treatment of hematological diseases.
Properties of Stem Cells
1. Ability to self-renew (undergoing numerous cell divisions) maintaining the undiff erentiated state.
2. Multipotency: Capacity to diff erentiate into a mature cell type.
Sources of Stem Cells
1. Embryonic tissues:
2. Fetal tissues:
3. Extrafetal tissues,
4. Adult gonads
A. Embryonic tissues: Inner cell mass (ICM) of the blastocyst, embryo and yolk sac.
B. Fetal Stem Cells: Human fetal hematopoietic stem cells (hf HSC) are primarily obtained from bone
marrow and liver. Virtually every part of the developing fetus has higher proliferative capacity. Th ese
cells have higher amount of telomerase activity and have longer telomeres compared to their adult
counterparts. Moreover, these tissues can diff erentiate effi ciently into neuronal, muscle and
osteogenic lineages. Primitive hf MSC are transduced by integrating vectors and they do not express
HLA-II. Th ey can be used for ex vivo gene therapy as well as postnatal bone tissue engineering.
C. Extrafetal tissues: Amniotic membranes, placenta, trophoblasts, amniotic fl uid cells, all contain
progenitor cells. These mesenchymal stem cells (MSC) can diff erentiate into most cell types of
mesodermal lineages.
Stem cell sample collection and banking
1. Currently the use of stem cells in regenerative medicine is regulated through institutional
regulatory boards.
2. Umbilical cord blood (UCB) collection and banking is an established source of HSC and MSC. Th is
is used fo treatment of hematological diseases like leukemia and bone marrow failure.
3. Fetal tissues can be obtained following medical termination of pregnancy. Stem cells from fetal
tissues can be harvested.
4. Intrauterine transplantation of Human fetal mesenchymal stem cells (hf MSC), collected from
liver, can be used for the treatment of hemoglobinopathies.
Clinical Applications:
1. Intrauterine stem cell transplantation (IUSCT) can be used to correct genetic disorders
(monogenic diseases).
2. Use of hf MSC has been explored for diseases having mesenchymal origin. hf MSC undergo site
specific differentiation and contribute to repair tissues in such diseases (muscular dystrophy,
osteogenesis imperfecta).
3. Hemoglobinopathies (α thalassemia, thalassemia, Sickle cell anemia).
4. Mucopolysaccharidoses (MPS)
5. Inherited immune deficiencies
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6. Allogeneic transplantation of HSC in the treatment of monogenic disorder has certain advantages.
It has high tolerance and less rejection rate as it is done before the onset of fetal immune
maturity (first trimester).
7. Autologous stem cells from fetal cord blood sampling or fetal liver biopsy in early pregnancy is
done and the cells are harvested. An ex-vivo gene transfer may be done thereafter. This also
reduces the risk of immune rejection.
8. However, fetal HSC in first trimester has favorable engraftment kinetics. In the first trimester fetal
hematopoietic stem cells are highly proliferative and they circulate in significant numbers.
Therefore these cells are the important source of autologous HSC.
9. Fetal mesenchymal stem cells can be bioengineered and used for the disease of bone, skin, liver
and heart.
10. The potential to use stem cells for the fabrication of tissues or organ implants may prove helpful
in the treatment of several diseases like genetic, immunodeficiency syndromes, urinary
incontinence, infertility and structural repair.
11. However, till date it is essential to understand its known limitations, putative benefits and the
unknown risks.
12. Until there is sufficient evidence on the efficacy of therapy, each case should be considered on an
individual basis.

OBSTETRIC OUTCOME IN MATERNAL PYELONEPHRITIS:
UTI in female: There is increased chance of urinary tract infection in females as compared to males due
to:
1. Short urethra (4 cm)
2. Close proximity of the external urethral meatus to the areas (vulva and lower third of vagina)
contaminated heavily with bacteria
3. Catheterization
4. Sexual intercourse.
Incidence: The overall incidence of pyelonephritis in pregnancy is between 1% and 3%.
Etiology:
1. It is more common in primigravidae than multiparae
2. Previous history of urinary tract infection increases the chance by 50%
3. Presence of asymptomatic bacteriuria increases the chance by 25% (4) Abnormality in the renal
tract is found in about 25%.
Physiologic changes responsible for acute pyelonephritis in pregnancy are:
1. Low ureteral peristalsis due to high progesterone levels.
2. Dextrorotation of gravid uterus causing compression of right ureter. Pyelonephritis occurs more
on the right side (70–80%) compared the left (10–15%).
Pathogenesis:
Predisposing factors:
1. Dilatation of the ureters and renal pelvis and stasis of the urine in the bladder and ureters are the
normal physiological changes during pregnancy.
2. The organisms responsible are E. coli (70%), Klebsiella pneumoniae (10%), Enterobacter, Proteus,
Pseudomonas and Staphylococcus aureus group.
3. About 10% of women develop bacteremia following acute pyelonephritis. 70–80% of
pyelonephritis occurs on the right side, 10–15% on the left side and only few are bilateral.
Clinical types:
Depending upon the mode of onset and the presenting features, the cases are grouped into :
 87
1. Acute or severe type
2. Chronic type
Clinical features:
1. The onset is acute and usually appears beyond the 16th week. The involvement is bilateral but if
unilateral, it is more frequent on the right side.
2. Clinical features are mainly due to endotoxemia. The chemical mediators (cytokines) released are:
IL-1, TNF and endogenous pyrogen. Important features are:
a. Acute aching pain over the loins, often radiating to the groin and costovertebral angle
tenderness, urgency, frequency, dysuria, hematuria.
b. Fever (spiky 40°C) with chills and rigor followed by hypothermia (34°C); anorexia, nausea,
vomiting and myalgias; respiratory distress and pulmonary edema (ARDS) due to
endotoxin induced alveolar injury.
Investigations:
Apart from the routine ones, serum level of creatinine, electrolytes and culture studies of urine
and blood should be done.
Differential diagnosis:
Includes (1) Acute appendicitis (2) Abruptio placentae (3) Red degeneration of fibroid (4) Acute
cholecystitis (5) Labor (6) Chorioamnionitis.
Complications:
1. Fetal: There may be increased fetal loss due to abortion, preterm labor, intrauterine fetal death
caused by hyperpyrexia and low birth weight babies (prematurity and dysmaturity).
2. Maternal: Anemia, Septicemia, septic shock, renal dysfunction and pulmonary insufficiency. ARDS
may develop due to endotoxin induced alveolar capillary membrane damage following
septicemia.
Management:
The outlines of management are:
1. Intravenous fluid (crystalloid) for adequate hydration.
2. Evaluate hemogram, serum electrolytes, creatinine.
3. Acetaminophen is given for the fever.
4. Monitor urine output (> 60 mL/hr), temperature and BP.
5. IV antibiotics—Cephalosporins, aminoglycosides (gentamicin), Cefazolin or Ceftriaxone, for 48
hours till culture report is available and then changed to oral therapy for another 10-14 days.
6. Repeat urine culture after 2 weeks of antimicrobial therapy and is repeated at each trimester of
pregnancy.
7. If the symptoms recur or the dip stick test for nitrate and leukocyte esterase is positive, urine
culture is repeated. The woman needs retreatment if the culture is positive.
8. Patient not responding with this therapy needs to be evaluated (sonography, CT scan,
radiography) for urinary tract obstruction.
9. Antimicrobial suppression therapy is continued till the end of pregnancy to prevent recurrence
30–40%). Nitrofurantoin 100 mg daily at bed time is effective.

GROUP B STREPTOCOCCI COLONIZATION IN PREGNANCY:
Introduction
1. Group B Streptococcus (GBS) or Streptococcus agalactiae is a gram-positive bacteria which
colonizes the gastrointestinal and genitourinary tract.
2. GBS is known to be the most common infectious cause of morbidity and mortality in neonates.
GBS is known to cause both early onset and late onset infections in neonates, but current
 88
interventions are only effective in the prevention of early-onset disease. Early onset GBS
infections occur within the first week of life, whereas late-onset disease occurs beyond the first
week of life.
Etiology
1. The main risk factor for early-onset GBS infection is colonization of the maternal genital tract with
Group B Streptococcus during labor. GBS is a normal flora of the gastrointestinal (GI) tract, which
is thought to be the main source for maternal colonization.
2. GBS cultures should be obtained with each pregnancy because colonization may be temporary.
Positive GBS urinary tract infection at any time during the pregnancy is a marker of
heavy colonization, and these patients should receive prophylaxis even if GBS culture is negative
between 35 to 37 weeks.
3. Additional risk factors for early onset GBS disease include young maternal age and black race.
4. Preterm labor (less than 37 weeks), maternal fever during labor (greater than 100.4 F or 36 C),
and prolonged rupture of membranes (greater than 18 hours) are also labor characteristics which
are risk factors for early-onset GBS disease. GBS colonization is has an incidence of 10-30% in
pregnancy. Without preventative measures, early onset GBS infection occurs in 1% to 2% of
neonates born to mothers with GBS colonization.
Epidemiology
1. GBS colonization has an incidence of 10-30% in pregnancy. Over the last 20 years, developments
in screening for GBS colonization, intrapartum prophylaxis, and secondary prevention of early-
onset GBS disease have resulted in a significant decrease in the incidence of early-onset GBS
infection.
2. In the early 1990s, there were approximately 1.7 cases of early-onset GBS infection per 1000 live
births. This has decreased to 0.34 to 0.37 per 1000 live births in recent years.
3. Seventy percent of cases of early-onset GBS infection are in term infants (greater than 37 weeks).
Interestingly, 60% of early-onset infections occur in patients with a negative rectovaginal GBS
culture between 35 to 37 weeks.
4. Group B streptococcus colonization in the rectovaginal area is discontinuous. Up to 33% of
patients whom have a positive GBS culture at 35-37 weeks, are not colonized at delivery. On the
contrary approximately 10% of women who are colonized at delivery will have a negative culture
at 35-37 weeks.
Pathophysiology
1. The principal route of neonatal early onset GBS infection is vertical transmission from colonized
mothers during passage through the vagina during labor and delivery. The majority of infants
exposed to GBS during delivery become colonized with GBS and do not develop signs or
symptoms of GBS infection.
2. The fetus is also susceptible to ascending infection into the amniotic fluid, with or without rupture
of membranes.
Evaluation
1. The principal defense against early-onset GBS infection is the administration of antibiotic
prophylaxis to mothers during labor and delivery. Identification of patients who will benefit from
intrapartum prophylaxis is an important aspect of routine prenatal care. The Center for Disease
Control and Prevention (CDC) recommends a universal culture-based screening.
2. Obstetrics providers should perform a rectovaginal culture for GBS in all patients between 35 and
37 weeks of gestation. Cultures are performed at this point in gestation because the negative
predictive value of the GBS culture is highest (95% to 99%) in the first 5 weeks after collection.
Patients who have an indication for preterm or early term induction of labor will benefit from GBS
 89
culture at or before 35 weeks, whereas nulliparous patients with unfavorable cervix may benefit
from GBS culture collection at 37 weeks.
3. Antibiotic susceptibility testing must be performed on all GBS cultures to guide antibiotic
prophylaxis in penicillin-allergic patients.
4. GBS bacteriuria is another marker of genital tract colonization. All pregnant patients should be
screened for asymptomatic bacteriuria during pregnancy, and all women with GBS bacteriuria at
any point during the pregnancy should receive intrapartum prophylaxis.
5. If GBS status is unknown, antibiotic prophylaxis should be initiated in patients with preterm labor
(less than 37 weeks gestation), maternal fever during labor (greater than 100.4 F or 38 C),
membranes ruptured greater than 18 hours, and/or in patients with a history of a previous child
with invasive early-onset GBS infection.
Management
1. Intravenous penicillin G is the treatment of choice for intrapartum antibiotic prophylaxis against
Group B Streptococcus. Penicillin G 5 million units intravenous is administered as a loading dose,
followed by 2.5 to 3 million units every 4 hours during labor until delivery.
2. Ampicillin is a reasonable alternative to penicillin G if penicillin G is unavailable. Ampicillin is
administered as a 2 gm intravenous loading dose followed by 1 gm intravenous every 4 hours
during labor until delivery.
3. Penicillin G and ampicillin should not be used in patients with penicillin allergy. Antibiotic
prophylaxis in patients with a history of anaphylaxis, angioedema, respiratory distress, or urticaria
following penicillin or cephalosporin is guided by antibiotic susceptibility testing.
4. If GBS is sensitive to both clindamycin and erythromycin, then clindamycin 900 mg intravenous
every 8 hours is recommended for GBS prophylaxis during labor until delivery[1]. Occasionally
GBS susceptibility testing will return susceptible to clindamycin but resistant to erythromycin.
Resistance to erythromycin can induce resistance to clindamycin even in the presence of a culture
that appears sensitive to clindamycin. For this reason, if the culture returns resistant to
erythromycin, vancomycin 1 gm intravenously every 12 hours is recommended for GBS
prophylaxis.
5. In patients without GBS susceptibility testing with penicillin allergy, vancomycin is recommended
for GBS prophylaxis with dosing as described above.
6. Cefazolin 2 gram intravenous loading dose followed by 1 gm every 8 hours may be used in
patients without a history of anaphylaxis, angioedema, respiratory distress, or urticaria following
penicillin or cephalosporin administration.
7. Initiating antibiotic prophylaxis greater than 4 hours before delivery is considered to be adequate
antibiotic prophylaxis and is effective in the prevention of transmission of GBS to the fetus.
However, antibiotic prophylaxis administered at a shorter interval will provide some protection. If
a patient presents in active labor and delivery is expected in less than 4 hours, antibiotic
prophylaxis should still be initiated.
8. GBS vaccines show promise to combat early-onset GBS infection, but there are currently no
approved GBS vaccines on the market.
Prognosis
1. Since the initiation of universal screening for GBS colonization and intrapartum antibiotic
prophylaxis, the incidence of early-onset GBS infection has decreased approximately 80%. Efficacy
of intrapartum antibiotic prophylaxis is estimated between 86% to 89%.
2. GBS culture screening during prenatal care will not identify all women with GBS colonization
during labor because genital tract colonization can be temporary. Approximately 60% of cases of
 90
early-onset GBS infection occur in neonates born to patients with negative GBS culture at 35 to 37
weeks.
3. Early-onset GBS infection typically presents in the first 24 to 48 hours of life. Symptoms include
respiratory distress, apnea, with signs of sepsis. Sepsis and pneumonia commonly result from
early-onset GBS infection, but rarely meningitis can occur. Mortality from early-onset GBS
infection is much higher in preterm infants than term infants. Preterm infants with early-onset
GBS infection have a case fatality rate between 20% to 30% compared to 2% to 3% in term
infants.

DISINFECTION OF OPERATION THEATER:

Introduction:
1. A clean operating environment is essential to prevent surgical site infection (SSI). The OT is
cleaned and disinfected to prevent microbial contamination.
2. Exogenous sources of infection in the OT are: people, anaesthesia equipment, surfaces such as
walls floors and furniture, air and dust, instruments supplies and medications.
3. There should be no dust in the OT; dust settling on the sterile field can carry microorganisms
particularly in operations of long duration.
4. Dust may be acquired from the outside environment due to defective filtration of air. Lint-
containing textiles can be a source of dust as also fl oor mops. Dust particles can be reduced by
good laundry practices to reduce the formation of lint and by the use of wet vacuum on the floor.
General principles
1. Surfaces must be routinely cleaned first with detergent to remove any foreign and organic matter.
Disinfection should follow cleaning. Do not apply disinfectant without cleaning as organic matter
such as pus, blood urine, amniotic fl uid, etc. inhibits the action of the disinfectant by protecting
microorganisms. A detergent disinfectant combination solution if available can be used for
convenience.
2. Spills must be cleaned immediately. Apply higher concentration of disinfectant to the spill, then
clean with detergent.
3. Disposable or freshly laundered washable cloths or mops should be used with freshly prepared
solution for each task.
4. OTs must be cleaned daily. This includes furniture, lights, equipment, windowsills, ledges, scrub
rooms and sinks. Thorough cleaning of the entire OT should be done once a week.
5. Wet vacuuming is the preferred method to clean the fl oors, wet mopping can be done if wet
vacuum is not available.
6. Collections of water should be dried immediately. Leaking faucets and sinks should be fixed as
wet areas encourage microbial growth and can be a source of infection.
Infrastructure of OTs
Location
To ensure a clean and uncontaminated environment, the OT should be located away from patient
care areas and patient traffic. For this reason, the OT is located at a higher level, preferably on the
top floor.
Ventilation and design requirements of the operation theatre are:
1. Airborne bacteria originate primarily from the skin of persons in the operation theatre (OT). The
bacteria carried on the skin reach the air through skin scales, which are constantly being shed by
the persons in the room. After remaining suspended in the air, the skin scales carrying bacteria
settle on various surfaces, equipment and on the fl oor of the OT. Appropriately circulated clean
filtered air will remove airborne organisms.
 91
2. Air should be circulated by positive pressure through high efficiency particulate air (HEPA) filters.
These are special fi lters, which can remove particles greater than 0.3 micron. Bacteria will be
removed, as the size of bacteria is 0.5–1 micron. HEPA filters should be monitored for effi ciency
on a regular basis and changed when required.
3. The ventilation rate is expressed as the number of air changes per hour. For an OT, the
recommended ventilation rate is 20 air changes per hour.
4. To prevent contamination of the clean zones, the direction of airfl ow should be from the ultra-
clean or aseptic zone to less clean zones in order of their cleanliness. This is achieved by an
appropriate pressure gradient. Highest positive pressure is maintained in the ultra-clean areas.
The higher pressure allows air to flow to less clean areas around the doors and openings and
prevents the entry of air from the less clean area.
5. Within the rooms air inlets should be at high level and outlets at low level so that the clean air
moves downwards through the room and towards the contaminated fl oor where it is exhausted
through the outlet.
6. The temperature in the OT should be between 18 and 24 °C. The room should have central air-
conditioning. Window air conditioners are a source of contamination and have no place in an OT.
7. The surfaces of floors and walls of the OT should be as hard, non-porous and smooth as possible.
Ceramic tiles are not ideal because the grouting between the tiles can harbour microorganisms.
There should be no cracks and crevices since cracks and crevices can harbour microorganisms and
cannot be cleaned properly. A special paint called epoxy paint is particularly suitable for the walls
of the OT. The joint between the walls and floor should be curved for easy cleaning.
8. The operation table should be placed away from the entrance.
9. The openings should be fitted with swing doors.
10. Since people are the main source of airborne contamination, the number of persons in the OT
should be kept to a minimum. Opening and closing of doors should be avoided as this interferes
with the direction of airflow.
Components of the OT
1. The OT is a multifunctional area. In this area patients are received and prepared for surgery, the
operation team prepares for surgery and the actual surgical procedures are carried out.
2. In many hospitals in the developing world, which do not have a CSSD, the OT may also include
equipment cleaning, processing and sterilization areas. In addition, there are areas for
administrative functions, sluicing and waste disposal. The OT areas are distributed into zones
depending upon the level of sterility and cleanliness required.
Concept of zoning
The features of the different zones in order of their cleanliness are:
1. Zone 3 is the cleanest or ultra-clean zone. It is also called the aseptic zone and includes the OT
and areas where the operation team and patient are prepared for surgery. The areas for
packaging and sterilizing surgical instruments are also included in this zone. The different areas in
this zone are physically separated from each other. Within this zone, the cleanest is the OT where
the patient’s tissues are exposed during surgery.
2. Zone 2 is the restricted zone where entry is restricted. It is the transitional area between the
outer zone and the aseptic zone. Persons entering this zone have to change to protective clothing
and footwear to prevent contamination of the surroundings.
3. Zone 1 is also called the outer zone and has similar level of cleanliness as other patient-care areas
in the hospital. It is the zone where patients are received and administrative functions are carried
out. Toilets are located in this zone.
 92
4. Zone 4 or disposal zone is a relatively dirty zone. Staff working in this area need to wear special
protective wear for their protection. There should be no movement of staff or equipment from
this zone to cleaner zones of the OT. This zone is connected by a separate corridor (also called
“dirty corridor”) leading out of the OT.
Daily cleaning procedure:
Before start of the first case, at least one hour before:
1. Damp dust with detergent–disinfectant all equipment, furniture and lights
2. Wipe surgical light refl ector again with 70% alcohol to remove the film left by the detergent
Between cases:
1. Place soiled towels, drapes and gowns in a clean laundry bag and send to laundry. Wet linen
should be placed in plastic container so that bacteria do not pass through the moist material.
2. Soiled instruments must be placed in disinfectant and then send to the cleaning area, this
prevents occupational hazard to the cleaner.
3. Wipe all used equipment, furniture and lights.
4. Move operating table to one side and wet vacuum or wet mop a 3–4 feet area around the
operating site.
5. Empty suction bottle and wash the suction bottle and tubing with detergent–disinfectant. Best is
disposable suction bottle.
Terminal daily cleaning after scheduled cases are over:
1. Remove all portable equipment from the room
2. Wipe windowsills, overhead lights, equipment, furniture and waste containers with a cloth soaked
in detergent disinfectant solution.
Wet vacuum or wet mop the entire fl oor area
1. Clean and disinfect the wheels/castors
2. Restock unsterile supplies
3. Check levels and dates of all sterile supplies and restock
4. Clean the air-conditioning grills
5. Clean scrub sinks with scouring powder
6. Empty all shelves, wipe with detergent–disinfectant and dry them before replacing the supplies.
Weekly general cleaning procedure
1. Remove all portable equipment. Clean lights and fi xtures with detergent disinfectant solution and
cloth.
2. Clean doors hinges and facings and rinse with solution.
3. Wipe down the walls with a mop soaked in detergent disinfectant solution.
4. Scrub the fl oor with fl oor cleaning machine and a phenol disinfectant detergent solution. Use a
wet vacuum to pick up the fl uid.
5. Replace clean portable equipment, clean wheels and castors by rolling them across a towel
saturated with detergent disinfectant.
6. Wash and dry all furniture and equipment including:
7. Operating room table
a. Suction holders
b. Foot and sitting stools
c. IV stands and all other stands
d. X-ray view boxes
e. All tables
f. Tubing to oxygen tanks
g. Waste containers and buckets
 93
Note: Thorough washing and cleaning is essential. Fumigation and fogging have no role in the
modern operation room. Fumigation with formalin is hazardous to persons and should not be
done. It can also harm sensitive equipment.

NONIMMUNE HYDROPS FETALIS:
Introduction:
1. Nonimmune hydrops fetalis (NIHF) refers specifi cally to cases not caused by red cell
alloimmunization. With the development and widespread use of Rh(D) immune globulin, the
prevalence of Rh(D) alloimmunization and associated hydrops has dramatically decreased. As a
result, NIHF now accounts for almost 90% of cases of hydrops, with the prevalence in published
series reported as 1 in 1700-3000 pregnancies.
2. Hydrops fetalis is a Greek term that describes pathological fluid accumulation in fetal soft tissues
and serous cavities.
3. The features are detected by ultrasound, and are defined as the presence of > 2 abnormal fluid
collections in the fetus. These include ascites, pleural effusions, pericardial effusion, and
generalized skin edema (defined as skin thickness > 5 mm). Other frequent sonographic findings
include placental thickening (typically defined as a placental thickness > 4 cm in the second
trimester or > 6 cm in the third trimester) and polyhydramnios

Etiologies of nonimmune hydrops fetalis

Cause Cases Mechanism
1. Cardiovascular 17-35% Increased central venous pressure
2. Chromosomal 7-16% Cardiac anomalies, lymphatic dysplasia, abnormal myelopoiesis
3. Hematologic 4-12% Anemia, high output cardiac failure; hypoxia (alpha thalassemia)
4. Infectious 5-7% Anemia, anoxia, endothelial cell damage, and increased capillar
permeability
5. Thoracic 6% Vena caval obstruction or increased intrathoracic pressure with
impaired venous return

6. Twin-twin transfusion 3-10% Hypervolemia and increased central venous pressure

7. Urinary tract abnormalities 2-3% Urinary ascites; nephrotic syndrome with hypoproteinemia
8. Gastrointestinal 0.5-4% Obstruction of venous return; gastrointestinal obstruction and
infarction with protein loss and decreased colloid osmotic
pressure
9. Lymphatic dysplasia 5-6% Impaired venous return

10. Tumors; chorioangiomas 2-3% Anemia, high output cardiac failure, hypoproteinemia
11. Skeletal dysplasias 3-4% Hepatomegaly, hypoproteinemia, impaired venous return
12. Syndromic 3-4% Various

13. Inborn errors of metabolism 1-2% Visceromegaly and obstruction of venous return, decreased
erythropoiesis and anemia, and/or hypoproteinemia
14. Miscellaneous 3-15%

15. Unknown 15-25%

 94
Pathophysiology:
1. Overall, cardiovascular abnormalities are the most common cause of NIHF in most series,
accounting for about 20% of cases. NIHF can result from cardiac structural abnormalities,
arrhythmias, cardiomyopathy, cardiac tumors, or vascular abnormalities. In most cardiac cases,
hydrops is likely caused by increased central venous pressure due to a structural malformation or
from inadequate diastolic ventricular filling. The most common congenital heart defects reported
in association with NIHF are right heart defects. The prognosis of NIHF due to cardiac structural
abnormalities is poor, with combined fetal and infant mortality reported as 92%, largely due to
the severity of the heart defects that cause in utero congestive heart failure.
2. Chromosomal abnormalities, particularly Turner syndrome (45,X) and Down syndrome (trisomy
21) are also common causes of NIHF, accounting for 13% in a large systematic review. In prenatal
series, aneuploidy is the most common cause of NIHF, particularly when identified early in
gestation. Turner syndrome is associated with 50-80% of cases of cystic hygromas, which result
from a lack of communication between the lymphatic system and venous drainage in the neck.
Lymphatic dysplasia likely leads to the development of NIHF in these cases. NIHF has been
described in association with other aneuploidies, including trisomies 13 and 18, and triploidy.
3. Fetal anemia, which can result in immune hydrops if caused by blood group alloimmunization, can
also lead to NIHF. Etiologies include inherited conditions such as hemoglobinopathies, as well as
acquired conditions, such as hemolysis, fetomaternal hemorrhage, parvovirus infection, or red
cell aplasia. Among the hemoglobinopathies, the most common cause of NIHF is alpha
thalassemia. This autosomal recessive disorder is common in Southeast Asian populations, where
it accounts for 28- 55% of NIHF. The incidence in most other series of NIHF is about 10%..
Fetomaternal hemorrhage leading to hydrops may occur as either an isolated acute event, or as a
chronic, ongoing hemorrhage.
4. NIHF has been reported in association with a number of viral, bacterial, and parasitic infectious
diseases, including parvovirus, cytomegalovirus, syphilis, and toxoplasmosis. Parvovirus is the
most commonly reported infectious cause of NIHF.
5. Fetal thoracic abnormalities, including masses as well as congenital hydrothorax, can also be
associated with NIHF. The most frequent pulmonary lesion associated with NIHF is a congenital
pulmonary airway malformation (CPAM). The most common etiology of an isolated effusion
leading to NIHF is chylothorax, caused by lymphatic obstruction.
6. Twin-twin transfusion syndrome results from an imbalance in blood flow caused by anastomoses
in the placentas of monochorionic twin pregnancies. In severe cases, one or both twins may
develop NIHF, although more commonly the recipient twin is affected, likely due to hypervolemia
and increased central venous pressure.
7. Few primary abnormalities of the gastrointestinal tract have been associated with NIHF. Those
that have been reported include diaphragmatic hernia, midgut volvulus, gastrointestinal
obstruction, jejunal atresia, malrotation of the intestines, and meconium peritonitis. Hepatic
disorders such as cirrhosis, hepatic necrosis, cholestasis, polycystic disease of the liver, and biliary
atresia have been reported in association with NIHF, most likely due to hypoproteinemia.
8. Neoplastic diseases or fetal tumors can occur in utero and have been associated with NIHF.
Relatively common in this category are lymphangiomas, hemangiomas, sacrococcygeal,
mediastinal, and pharyngeal teratomas, and neuroblastomas. Many of these are very vascular and
lead to NIHF due to high output cardiac failure.
9. A large number of skeletal dysplasias have been associatedwith NIHF, including achondroplasia,
achondrogenesis, osteogenesis imperfecta, osteopetrosis, thanatophoric dysplasia, short-rib
polydactyly syndrome, and asphyxiating thoracic dysplasia. In all of these, the mechanism is
 95
unclear, although it has been proposed that hepatic enlargement occurs secondary to
intrahepatic proliferation of blood cell precursors to compensate for a small bone-marrow
volume. This may cause large vessel compression and lead to anasarca in these fetuses.
10. Inborn errors of metabolism and other genetic conditions are historically associated with 1-2% of
cases of NIHF, which may be transient or manifest as isolated ascites. Inherited metabolic
disorders that have been implicated as a cause of NIHF are most typically lysosomal storage
diseases such as various mucopolysaccharidoses, Gaucher disease, and Niemann-Pick disease.
11. Disorders associated with lymphatic dysfunction, such as Noonan and multiple pterygium
syndrome, present with cystic hygroma; idiopathic chylothorax, in which a local pleuromediastinal
lymph vessel disturbance occurs as the possible pathogenic mechanism for hydrops fetalis.
Evaluation:

1. Sonographic identifi cation of the hydropic fetus is not diffi cult. The diagnostic challenge is to
establish the etiology and determine the appropriate therapy (if available) and timing of delivery.
2. In a structurally normal fetus, the first step is to rule out alloimmunization as a cause. The
maternal blood type and Rh (D) antigen status are assessed as part of routine prenatal care, along
with an indirect Coombs test (an antibody screen) to evaluate for circulating red blood cell
antibodies.
3. Prenatal diagnosis with karyotype, fluorescence in situ hybridization, and/or chromosomal
microarray analysis.
4. Evaluation of the mean cell volume, which will be < 80 fL in thalassemia carriers. Fetal blood
sample can be evaluated for the presence of the abnormal Bart’ s hemoglobin.
5. Kleihauer-Betke smear will show the presence of fetal cells in the maternal peripheral blood in
fetomaternal hemorrhage.
6. If NIHF is chylothorax, caused by lymphatic obstruction. The fluid may be sampled at the time of
needle drainage or shunt placement, and the diagnosis is confi rmed by the finding of a fetal
pleural cell count with > 80% lymphocytes in the absence of infection.
 96
7. Careful histology of the placenta, liver, spleen, and bone marrow will often provide a clue that a
metabolic storage disorder was present. For many such disorders, testing is available to
determine a diagnosis and for prenatal diagnosis in a subsequent pregnancy.
8. Middle cerebral artery Doppler studies to assess for the presence of fetal anemia, which may be
treatable with intravascular transfusion.
9. A complete family history is thus imperative to rule out a known inherited disorder in the family
and to assess for consanguinity, which will increase the likelihood of a recessive disorder.
10. A fetal karyotype, fluorescence in situ hybridization studies, and/or chromosomal microarray
analysis should be offered in chromosomal defects.
11. Invasive testing also allows testing for lysosomal storage disorders, and polymerase chain
reaction studies for parvovirus, toxoplasmosis, and cytomegalovirus infection.
Maternal risks are associated with NIHF:
1. Women with NIHF may develop mirror syndrome, an uncommon complication in which the
mother develops edema that “ mirrors” that of her hydropic fetus.Mirror syndrome may
represent a form of preeclampsia, and is characterized by edema in approximately 90%,
hypertension in 60%, and proteinuria in 40% of cases.
2. Additional associated fi ndings with the syndrome include headache, visual disturbances, oliguria,
elevated uric acid, liver function tests, or creatinine levels, low platelets, anemia, and
hemodilution. In a study of 56 cases of mirror syndrome, the major maternal morbidity was
pulmonary edema, which occurred in 21%.
Obstetric complications:
1. Polyhydramnios and preterm birth occur frequently with NIHF.
2. Polyhydramnios is associated with maternal respiratory symptoms.
Prognosis of NIHF:
1. The prognosis of NIHF depends on the underlying etiology, gestational age at detection and
delivery, Apgar scores, extent of resuscitation in the delivery room, and whether the newborn
requires transport.
2. In one prenatal series, nearly half of those diagnosed< 24weeks had aneuploidy, with extremely
poor survival. However, even in the absence of a chromosomal abnormality, survival was < 50%.
3. Among liveborn infants, neonatal mortality with NIHF is reported to be as high as 60%.
4. Treatable causes of hydrops, such as fetal arrhythmia or infection with parvovirus B19, have a
better prognosis.
Management of NIHF
1. Cases generally fall into 1 of 3 categories: those amenable to fetal therapye which often require
urgent treatment or referral to a specialized center; those with a lethal prognosis, for whom
pregnancy termination or comfort care are the only options realistic to offer.
2. There is no evidence that elective preterm delivery will improve the outcome. In one
retrospective series, preterm birth < 34 weeks was a poor prognostic factor. Based on expert
opinion, development or worsening of NIHF in a pregnancy that has reached about 34 weeks
would seem a reasonable indication for delivery,
3. Fetal therapy: Therapy options may include intrauterine transfusion(s) for fetal anemia,
medications such as antiarrhythmic agents, drainage of large pleural effusions, corticosteroids for
CPAMs, or specialized procedures such as laser coagulation of placental anastomoses for twin-
twin transfusion syndrome.


 97
LYMPHATIC DRAINAGE OF VULVA:
Structures of the Vulva
The vulva is a collective term for several anatomical structures:
1. Mons pubis – a subcutaneous fat pad located anterior to the pubic symphysis. It formed by the
fusion of the labia majora.
2. Labia majora – two hair-bearing external skin folds.
a. They extend from the mons pubis posteriorly to the posterior commissure (a depression
overlying the perineal body).
b. Embryologically derived from labioscrotal swellings
3. Labia minora – two hairless folds of skin, which lie within the labia majora.
a. They fuse anteriorly to form the hood of the clitoris and extend posteriorly either side of
the vaginal opening.
b. They merge posteriorly, creating a fold of skin known as the fourchette.
c. Embryologically derived from urethral folds
4. Vestibule – the area enclosed by the labia minora. It contains the openings of the vagina (external
vaginal orifice, vaginal introitus) and urethra.
5. Bartholin’s glands – secrete lubricating mucus from small ducts during sexual arousal. They are
located either side of the vaginal orifice.
6. Clitoris – located under the clitoral hood. It is formed of erectile corpora cavernosa tissue, which
becomes engorged with blood during sexual stimulation. Embryologically derived from the genital
tubercle.
Lymphatic drainage of vulva:
1. There are dense lymphatic plexuses in the dermis of the vulva, which intercommunicate with
those of subcutaneous tissue.
2. The lymphatics of each side freely communicate with each of them
3. The lymphatics hardly cross beyond the labiocrural fold
4. The vulval lymphatics also anastomose with the lymphatics of the lower-third of the vagina and
drain into external iliac nodes
5. Lymphatics from the deep tissues of the vulva accompany the internal pudendal vessels to the
internal iliac nodes
6. Superficial inguinal lymph nodes are the primary lymph nodes that act as the sentinel nodes of
the vulva. Deep inguinal lymph nodes are secondarily involved. It is unusual to find positive pelvic
glands without metastatic disease in the inguinal nodes
7. Gland of Cloquet or Rosenmüller, which is the upper most deep femoral nodes is absent in about
50 percent of cases.
8. Labia majora (anterior half)
Lymphatics intercommunicate with the opposite side in the region of mons veneris →
Superficial inguinal nodes. Thus, there is bilateral and contralateral spread of metastasis in
malignancy affecting the areas.
9. Labia majora (posterior half)
Drains into → Superficial inguinal → Deep inguinal→ External iliac.
10. Labia minora and prepuce of clitoris:
Intercommunicating with the lymphatics of the opposite side in the vestibule and drains
into superficial inguinal nodes.
11. Glans of clitoris:
Drains directly into the deep inguinal and external iliac glands.
12. Bartholin’s glands:
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The lymphatics drain into superficial inguinal and anorectal nodes.
13. Node of Cloquet:
It was previously thought to be the main relay node through which the efferents from the
superficial inguinal nodes pass to the external iliac nodes. Recent study shows its
insignificant involvement in vulval malignancy, and thus, it is not considered to be the
relay node. The efferents from the superficial inguinal may reach the external iliac group
bypassing the node of Cloquet.
14. Applied anatomy (vulval malignancy)
a. Lymphatics: It is the commonest method of spread of lesion. It is estimated that in about
50 percent, the lymph glands are involved by the time the patient consults the physician.
The following facts are to be borne in mind:
b. The lymphatic spread is primarily by embolization and only at a late stage, the spread is
by permeation to fill the lymphatic channels.
c. Contralateral metastases are not infrequent (25%) as the lymphatics of the vulva cross
the midline.
d. When the ipsilateral nodes are not involved from a lesion located on one side, spread to
the contralateral groin node is very unlikely.
e. The lymph node involvement follows a sequential pattern. The lymphatics of labia →
superficial inguinal lymph nodes → deep inguinal lymph nodes → pelvic nodes.
f. Pelvic nodes are secondarily involved in about 20 percent with affected inguinal nodes.
The nodes involved are obturator, external iliac, hypogastric and common iliac.
g. Lymphatics of the clitoris, anus and rectovaginal septum may drain directly into the pelvic
lymph nodes.
h. Involvement of pelvic nodes, bypassing the inguinal lymph nodes, is less than 3 percent.
i. Incidence of lymph gland involvement is directly related to the site, size of the lesion and
the depth of stromal invasion. Chance of bilateral lymph node involvement also increases
when the midline structures (clitoris, perineum) are involved.

a. Histologically proved groin node involvement is present in 25 percent when missed on

clinical assessment. In almost 25 percent, the nodes are histologically negative when
clinically thought to be involved. Approximate incidence of lymph node involvement is
 99
given in Table 23.4.
b. Regional lymph nodes are assessed clinically and also by using MRI, sentinel node
lymphoscintigraphy, ultrasound and PET.

PHYSIOLOGY OF MICTURITION:
Introduction:
1. Micturition is the process by which the urinary bladder empties when it becomes filled.This
involves two main steps:
a. First, the bladder fills progressively until the tension in its walls rises above a threshold
level; this elicits the second step, which is:
b. a nervous reflex called the micturition reflex that empties the bladder or, if this fails, at
least causes a conscious desire to urinate.
2. Although the micturition reflex is an autonomic spinal cord reflex, it can also be inhibited or
facilitated by centers in the cerebral cortex or brain stem.
Physiologic Anatomy and Nervous Connections of the Bladder
1. The urinary bladder is a smooth muscle chamber composed of two main parts:
a. the body, which is the major part of the bladder in which urine collects, and
b. the neck, which is a funnel-shaped extension of the body, passing inferiorly and anteriorly
into the urogenital triangle and connecting with the urethra.The lower part of the bladder
neck is also called the posterior urethra because of its relation to the urethra.

2. The smooth muscle of the bladder is called the detrusor muscle. Contraction of the detrusor
muscle is a major step in emptying the bladder. Smooth muscle cells of the detrusor muscle fuse
with one another so that an action potential can spread throughout the detrusor muscle, from
one muscle cell to the next, to cause contraction of the entire bladder at once.
3. On the posterior wall of the bladder, lying immediately above the bladder neck, is a small
triangular area called the trigone. Each ureter, as it enters the bladder, courses obliquely through
the detrusor muscle and then passes another 1 to 2 centimeters beneath the bladder mucosa
before emptying into the bladder.
4. The bladder neck (posterior urethra) is 2 to 3 centimeters long, and its wall is composed of
detrusor muscle interlaced with a large amount of elastic tissue. The muscle in this area is called
the internal sphincter.
5. Beyond the posterior urethra, the urethra passes through the urogenital diaphragm, which
contains a layer of muscle called the external sphincter of the bladder. This muscle is a voluntary
skeletal muscle, in contrast to the muscle of the bladder body and bladder neck, which is entirely
 100
smooth muscle.
Innervation of the Bladder
1. The pelvic nerves, S-2 and S-3: Coursing through the pelvic nerves are both sensory nerve fibers
and motor nerve fibers. The motor nerves transmitted in the pelvic nerves are parasympathetic
fibers. These terminate on ganglion cells located in the wall of the bladder. Short postganglionic
nerves then innervate the detrusor muscle.
2. The skeletal motor fibers transmitted through the pudendal nerve to the external bladder
sphincter. These are somatic nerve fibers that innervate and control the voluntary skeletal muscle
of the sphincter.
3. Also, the bladder receives sympathetic innervation from the sympathetic chain through the
hypogastric nerves, connecting mainly with the L-2 segment of the spinal cord. These sympathetic
fibers stimulate mainly the blood vessels and have little to do with bladder contraction.
4. Some sensory nerve fibers also pass by way of the sympathetic nerves and may be important in
the sensation of fullness and, in some instances, pain.
Transport of Urine from the Kidney Through the Ureters and into the Bladder
1. The walls of the ureters contain smooth muscle and are innervated by both sympathetic and
parasympathetic nerves as well as by an intramural plexus of neurons and nerve fibers that
extends along the entire length of the ureters.
2. The ureters enter the bladder through the detrusor muscle in the trigone region of the bladder,
obliquely for several centimeters through the bladder wall. The normal tone of the detrusor
muscle in the bladder wall tends to compress the ureter, thereby preventing backflow of urine
from the bladder when pressure builds up in the bladder during micturition or bladder
compression. Each peristaltic wave along the ureter increases the pressure within the ureter so
that the region passing through the bladder wall opens and allows urine to flow into the bladder.
Filure of this mechanism can lead to vesicoureteral reflux.
Pain Sensation in the Ureters, and the Ureterorenal Reflex .
1. The ureters are well supplied with pain nerve fibers. When a ureter becomes blocked (e.g., by a
ureteral stone), intense reflex constriction occurs, associated with severe pain. Also, the pain
impulses cause a sympathetic reflex back to the kidney to constrict the renal arterioles, thereby
decreasing urine output from the kidney. This effect is called the ureterorenal reflex and is
important for preventing excessive flow of fluid into the pelvis of a kidney with a blocked ureter.

Filling of the Bladder and Bladder Wall Tone; the Cystometrogram
1. When there is no urine in the bladder, the intravesicular pressure is about 0, but by the time 30 to
50 milliliters of urine has collected, the pressure rises to 5 to 10 centimeters of water. Beyond
300 to 400 milliliters, collection of more urine in the bladder causes the pressure to rise rapidly.
 101
These pressure peaks are called micturition waves in the cystometrogram and are caused by the
micturition reflex.
Micturition Reflex
1. This is the result of a stretch reflex initiated by sensory stretch receptors in the bladder wall,
especially by the receptors in the posterior urethra when this area begins to fill with urine at the
higher bladder pressures.
2. Sensory signals from the bladder stretch receptors are conducted to the sacral segments of the
cord through the pelvic nerves and then reflexively back again to the bladder through the
parasympathetic nerve fibers by way of these same nerves.
3. As the bladder continues to fill, the micturition reflexes become more frequent and cause greater
contractions of the detrusor muscle. Once a micturition reflex begins, it is “self-regenerative.”
That is, initial contraction of the bladder activates the stretch receptors to cause a greater
increase in sensory impulses to the bladder and posterior urethra, which causes a further increase
in reflex contraction of the bladder; thus, the cycle is repeated again and again until the bladder
has reached a strong degree of contraction.
Facilitation or Inhibition of Micturition by the Brain
1. The micturition reflex is a completely autonomic spinal cord reflex, but it can be inhibited or
facilitated by centers in the brain. These centers include (1) strong facilitative and inhibitory
centers in the brain stem, located mainly in the pons, and (2) several centers located in the
cerebral cortex that are mainly inhibitory but can become excitatory.
2. The higher centers can prevent micturition, even if the micturition reflex occurs, by continual
tonic contraction of the external bladder sphincter until a convenient time presents itself.
Abnormalities of Micturition
Atonic Bladder Caused by Destruction of Sensory Nerve Fibers.
1. Micturition reflex contraction cannot occur if the sensory nerve fibers from the bladder to the
spinal cord are destroyed, thereby preventing transmission of stretch signals from the bladder.
When this happens, a person loses bladder control, despite intact efferent fibers from the cord to
the bladder and despite intact neurogenic connections within the brain.
2. Instead of emptying periodically, the bladder fills to capacity and overflows a few drops at a time
through the urethra. This is called overflow incontinence.
3. A common cause of atonic bladder is crush injury to the sacral region of the spinal cord.
Automatic Bladder Caused by Spinal Cord Damage Above the Sacral Region.
1. If the spinal cord is damaged above the sacral region but the sacral cord segments are still intact,
typical micturition reflexes can still occur. However, they are no longer controlled by the brain.
2. During the first few days to several weeks after the damage to the cord has occurred, the
micturition reflexes are suppressed because of the state of “spinal shock” caused by the sudden
loss of facilitative impulses from the brain stem and cerebrum.
3. However, if the bladder is emptied periodically by catheterization to prevent bladder injury
caused by overstretching of the bladder, the excitability of the micturition reflex gradually
increases until typical micturition reflexes return; then, periodic (but unannounced) bladder
emptying occurs.
Uninhibited Neurogenic Bladder Caused by Lack of Inhibitory Signals from the Brain.
1. Another abnormality of micturition is the so-called uninhibited neurogenic bladder, which results
in frequent and relatively uncontrolled micturition.
2. This condition derives from partial damage in the spinal cord or the brain stem that interrupts
most of the inhibitory signals.

 102
ROLE OF CYTOKINES IN PRETERM LABOUR:
Introduction:
1. Preterm Labour can occur spontaneously due to multiple aetiologies such as uterine
overdistension, as in multiple gestation, infection or inflammation.
2. Increased levels of inflammatory cytokines, have been reported in serum and/or amniotic fluid of
women with spontaneous preterm labour.
Organisms in genital tract
1. The vagina also harbors numerous microorganisms that exist in a regulated mutualistic
relationship with the host (the “microbiome”). Some of these microorganisms such
as Lactobacillus species reinforce the defense against invasion and colonization by opportunistic
pathogens. The composition of the vaginal microbiome is dynamic and undergoes changes
corresponding with hormonal fluctuations throughout the woman's reproductive life, i.e., from
puberty to menopause, and during pregnancy.
2. There is ample evidence that abnormal vaginal flora (albeit broadly and heterogeneously defined)
is associated with PTB
3. The most common microorganisms associated with PTB are genital Mycoplasma species, in
particular Ureaplasma urealyticum and Ureaplasma parvum. Others include
Leptotrichia/Sneathia, Atopobium vaginae, and bacterial vaginosis (BV)–associated bacteria types
1, 2, and 3, which are bacteria associated with BV, a condition in which the normal vaginal flora of
lactobacilli are replaced by other low- and high-grade pathogens. One could speculate that
immune system and hormonal changes that occur in pregnancy might alter vaginal microbiome
communities compared with the nonpregnant state.
4. Microorganisms are believed to ascend from the lower genital tract, cross the cervical barrier and
invade the decidua, chorioamniotic membranes, and amniotic fluid.
5. In some cases the umbilical cord and the fetus are infected. Infection is accompanied by a host-
inflammatory response, which involves accumulation of inflammatory cells in the chorioamniotic
membranes and expression of cytokines in the fetoplacental tissues.
6. The conventional paradigm is that the placenta is a sterile organ and any intrauterine infection
would be caused by ascending infection from the lower genital tract.
7. Indeed, there is abundant evidence that links infection and inflammation with preterm birth
(PTB), including associations with subclinical intrauterine infections, intra-amniotic infection, and
extrauterine maternal infections, such as pyelonephritis and periodontal disease.
8. Bacterial colonization of the human placenta has been found not only in patients with clinical
infections or in preterm births but also in normal pregnancy and term placentas. Both gram-
positive and gram-negative bacteria of diverse morphologic origins were present in a third of all
placentas from preterm and term pregnancies.
Inflammation in genital tract:
1. Considerable evidence now suggests that the proinflammatory cytokineprostaglandin cascade
plays a central role in the pathogenesis of infectionassociated preterm birth (Romero et al., 2005).
These inflammatory mediators are produced by macrophages, decidual cells, and fetal
membranes in response to bacteria or bacterial products.
2. Inflammation could trigger myometrial contractions, PPROM and cervical maturation leading to
preterm birth.
Inflammatory mediators and preterm birth (PTB)
3. A role for selected cytokines in preterm labor is based on the following observations:
a. Elevated concentrations of cytokines and prostaglandins in amniotic fluid are found in
patients with intra-amniotic infection and preterm labor;
 103
b. In vitro, bacterial products stimulate the production of proinflammatory cytokines by
human decidua; these cytokines, in turn, stimulate the production of prostaglandins by
the amnion and the decidua;
c. The administration of IL-1 to pregnant mice or nonhuman primates induces preterm
labor, which can be prevented by the administration of IL-1 receptor antagonist protein.
4. Gravett and colleagues (1994) have demonstrated in nonhuman primates that after experimental
intra-amniotic infection with group B streptococci there are sequential increases in the levels of
proinflammatory cytokines (IL-1β, TNF-α, IL-6, and IL-8), prostaglandins, and MMPs in amniotic
fluid that precede increases in uterine contractility by 24 to 48 hours and that result in preterm
delivery.
5. This model provides a characterization of the temporal relationships among infection,
inflammation, and labor. The observations presented above suggest that infection-associated
preterm birth is an acute event that occurs proximal to delivery.
6. Recent evidence, however, suggests that midtrimester amniotic fluid infection with Ureaplasma
urealyticum may result in preterm birth many weeks later (Greber et al., 2003; Gray et al., 1992).
Furthermore, elevated midtrimester concentrations of IL-6, a proinflammatory cytokine, in
amniotic fluid have been associated with preterm birth at 32 to 34 weeks of gestation (Wenstrom
et al., 1996).
7. Although the strongest evidence associating preterm birth with infection is derived from
intrauterine infections, considerable evidence also suggests that lower genital tract infections,
especially bacterial vaginosis, contribute to prematurity.
8. Thus, bacterial vaginosis represents an important and potentially preventable cause of
prematurity.
Periodental disease and PTB
1. Preterm birth has also been associated with maternal systemic infection (and is largely
attributable to the severity of maternal illness) and, more recently, with maternal periodontal
disease. Periodontal disease is an anaerobic bacterial infection of the mouth that affects up to 50
percent of the population, including pregnant women. Maternal periodontal disease has been
associated with several adverse pregnancy outcomes, including preterm birth, preeclampsia, and
fetal loss (Boggess et al., 2003; Jeffcoat et al., 2001a,b; Offenbacher et al., 1996). Three clinical
trials of periodontal treatment suggested a 50 percent reduction in the risk of preterm birth.
2. The gram-negative anaerobes associated with periodontitis may serve as a source of the
lipopolysaccharide endotoxin that increases the levels of proinflammatory mediators, including
cytokines and prostaglandins.
3. Hence, the host inflammatory response to a potential pathogen must play a critical role in
preterm birth. Cytokines (and some Toll-like receptors) are genetically very pleomorphic. It is
likely that genetic differences in inflammatory responsiveness play a major role in determining
whether or not a preterm birth occurs.
Cytokines:
1. Cytokines are small molecules produced by almost all nucleated cells. They play a key role in the
inflammatory process
2. Role of cytokines in normal labour:
a. Levels of cytokines in the IL-1 family rise during parturition. IL-1 increases leukocyte
trafficking in the endometrial tissue by enhancing vasodilatation, adding to the number of
cells producing labor-initiating cytokines and hormones.
b. IL-1β especially influences cervical remodeling and rhythmic contractions. IL-1 stimulates
endothelial cells to produce prostaglandin E2, a potent cervical dilator.
 104
c. IL-1 cytokines cause a marked, dose-dependent increase in plasma calcium, which may
stimulate contractions. IL-1β-induced changes in calcium homeostasis augment
myometrial contractility
d. IL-6 also acts as a growth factor and may stimulate the expression of oxytocin receptors
on myometrial cells to increase responsiveness to oxytocin
3. Role of cytokines in preterm labour:
a. One intriguing line of research suggests that the source of cytokines in normal full-term
labor differs from the source in PTL.
b. Placental cell cultures from laboring women without infections delivering preterm
produced significantly larger amounts of IL-1β, IL-6, and TNF-α than cells from
nonlaboring women at term.
Management of PTL due to inflamation:
1. In general, tocolytic therapies are largely ineffective at substan-tially delaying delivery and
reducing neonatal mortality.
2. As intrauterine infection is a common cause of PTL, antibioticshave previously been considered a
logical method of treatment. However, the use of antibiotics to prevent PTB has been associ-ated
with neonatal enterocolitis, as well as an increased risk of cerebral palsy.
3. As par-turition is associated with leukocyte influx into the intrauterinetissues, immunomodulation
has been investigated in mouse mod-els of PTB, using leukocyte depletion. Rinaldi et al. found
thatneutrophil depletion did not delay delivery.
4. Animalmodels are currently being utilised to investigate potential thera-peutic agents which may
target the intrauterine inflammation withthe intention of protecting against fetal injury.
5. Shynlova et al. (2014) targeted the activiationof murine peripheral maternal immune cells with a
broad-spectrum chemokine inhibitor. A reduced incidence of LPS-induced PTB was reported.
6. Folic acid, melatonin and statins, have recently been investigatedfor the treatment of PTB and
fetal loss in mouse models.
7. Using new ultrasound techniques, such as Tissue Doppler Imaging (TDI), to identify signs of fetal
inflammation is one strategy that shows promise.

ANATOMICAL AND PHYSIOLOGICAL CHANGES OF RESPIRATORY SYSTEM IN PREGNANCY:

Introduction:
Major physiological and anatomical changes occur in the respiratory system during pregnancy due
to a combination of both hormonal and mechanical factors. Dyspnoea is a common complaint in
pregnancy affecting over half of women at some stage.
Anatomical chalenges:
1. Due to the effect of oestrogen, there is capillary engorgement of nasal, oropharyngeal and
laryngeal mucosa. Mucosal edema leads to greater resistance to flow
2. There is an increase in anteroposterior and transverse diameters of chest wall by 2 cm each and a
resultant increase in circumference by 5–7 cm.
3. Chest wall compliance decreases due to increased fat and abdominal content. Lung compliance
remains the same
4. The diaphragm is pushed up by 4cm
5. Diaphragmatic excursion increases by about 2cm
6. Rib cage expands: subcostal angle of the ribs at the xiphoidal level increases from 68.5° at the
beginning of pregnancy to 103.5° at term
7. Anatomical dead space increases by about 445% due to increased airway diameter late in
pregnancy
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8. Hydronephrosis during pregnancy occurs in 43% to 100% pregnant women, and it is more
prevalent with advancing trimester. The dilated collecting system can hold 200 to 300mL of urine,
leading to urinary stasis and a 40% increased risk for pyelonephritis in pregnant women with
asymptomatic bacteriuria versus nonpregnant women
Physiological Changes in lung mechanics:
1. Minute ventilation (MV) increases mainly due to increase in TV with minimal rise in respiratory
rate (1–2 breaths/min). There is a corresponding increase in alveolar ventilation.
2. Progesterone is a respiratory stimulant and sensitises chemoreceptors to carbon dioxide (CO2).
3. There is an increased production of CO2 (about 300 ml/min) and due to increased MV, PaCO2 falls
to 30–32 mmHg in first trimester and remains in this range throughout pregnancy. There is no
gradient between end-tidal CO2 and PaCO2 .
4. Respiratory alkalosis is incompletely compensated by reduction in serum bicarbonate levels to
about 20–21 mEq/L and resultant pH of 7.42–7.44. As a result of increased alveolar ventilation,
there is an increased PaO2 during pregnancy but after mid-gestation, PaO2 falls in supine position
as functional residual capacity (FRC) falls below closing capacity leading to closure of airways
during tidal volume breathing.
5. Oxygen delivery to foetus is increased by rightward shift in maternal oxygen dissociation curve
and an increase in P50 value is observed at term (30 vs. 26 mmHg). Foetal haemoglobin has a
higher affinity for oxygen and has a P50 of about 18 mmHg.
6. Despite the cephalad displacement of diaphragm, the excursion of diaphragm during breathing
increases by 2 cm while there is a reduction in chest wall excursion. No change is thus observed in
flow volume loops.
7. During labour, MV increases by 70–200%, PaCO2 decreases to 10–15 mmHg and oxygen
consumption increases by 40–75% due to increased metabolic demands. MV, TV and oxygen
consumption attain pre-pregnancy values by 6–8 weeks postpartum.
Other parameters:
1. Expiratory reserve volume ↓ 25%
2. Residual volume ↓ 15%
3. Functional residual capacity ↓ 20%
4. Tidal volume ↑ 45%
5. Inspiratory reserve volume ↑ 5%
6. Inspiratory capacity ↑ 15%
7. Vital capacity No change
8. Total lung capacity ↓ 5%
9. FEV1 No change
10. FEV1 /FVC No change
11. Closing capacity No change
Blood Gas analysis:

Gas exchange and gas transport

Blood gas tension PaCO2 decreases

PaO2 increases

Oxygen-carrying Maternal erythrocytes produce higher levels of 2,3-DPG

capacity p50 remains normal (by the combined effects of 2,3-DPG increase
and alkalosis)
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Control of ventilation

Central respiratory Progesterone-associated chronic respiratory alkalosis develops

control (due to increased sensitivity to CO2)

Respiratory workload and demand

Demands on the Increased body mass (increased weight plus one extra organism
respiratory system on board) = increased total body oxygen demand (by about
21%) and increased ventilatory requirements for the clearance of
the excess CO2
During labour, the oxygen consumption increases by about 60%.

Anaesthetic implications
1. Mallampati classification worsens during pregnancy and more so during labour. Upper airway
changes, enlarged breasts and obesity can make intubation difficult during pregnancy.
2. Laryngoscopes with short handles, smaller diameter endotracheal tubes and ramp position at the
head end might be needed in difficult scenarios.
3. Nasotracheal intubation should be avoided as there is an increased risk of nasal bleed during
pregnancy.
4. A decreased FRC and increased oxygen consumption can lead to rapid desaturation during
apnoea despite adequate pre-oxygenation.
5. An increased MV and low FRC result in faster de-nitrogenation (pre-oxygenation) and rapid
uptake of inhalational agents.
6. Hyperventilation should be avoided as it may cause respiratory alkalosis, leftward shift in oxygen
dissociation curve and decreased oxygen delivery to foetus.
7. Uncontrolled maternal pain during labour can further increase the metabolic demands with
resultant increase in maternal lactate levels indicating that oxygen requirements are increased
more than supply.
8. Despite enhanced response to hypoxic ventilatory drive, it is not possible to meet increased
oxygen demand without supplemental oxygen in susceptible parturient. Epidural analgesia is
beneficial by decreasing the metabolic demands during labour

FLUID AND ELECTROLYTE BALANCE IN LABOUR:
Introduction:
1. Normal pregnancy induces progressive adaptations in every system, and it is against this
background of altered maternal homeostasis that the giving of fluids during labour and delivery
must be considered. At the present time labour usually lasts for less than 24 hours in women
having their first baby and less than 12 hours in multigravid patients.
2. During such a short interval healthy mothers normally will not develop problems of fluid balance;
if problems do occur they are likely to follow the intravenous administration of excessive volumes
of sodium-free fluids. In addition to maternal complications, the biochemical consequences of
inappropriate fluid regimens will be reflected in the fetus although these may not become
obvious until some time after the infant is born.
Total body water:
1. Total body water increases progressively; Measurements using deuterium oxide suggest an
increase of 7-10 litres by term depending upon the degree of maternal oedema present
 107
2. Increase in extracellular water also occurs to about 6 litres but with a very wide range of
individual values.
3. This increase results from the amount of water present in the product of conception together
with that needed for the increase in maternal plasma volume, breasts and uterus. They usually
account for only 4 litres or so; thus about 2 litres remain to be accounted for. This 'excess' is
probably stored in the form of microdroplets in the mucopolysaccharides present in the ground
substance of connective tissue.
Total osmolality
1. During the first trimester of pregnancy total osmolality is reduced from about 290 to 280
mosmol/kg.
2. During the last trimester any increased fluid load is not excreted promptly, as might be expected,
but goes into the extravascular compartment.
Colloid osmotic pressure
1. There is an increase in total urinary protein and albumin excretion, especially notable after
20 weeks.
2. Plasma albumin concentration decreases by about 10 g/l during the first trimester, causing a fall
in colloid osmotic pressure of about 10 cmH20.
Plasma volume
There is a progressive increase in plasma volume between weeks 12 and 36 of pregnancy, the
peak values achieved being 35-45% above the non-pregnant average.
Renal function
1. The kidneys are the major controllers of fluid and electrolyte balance, and both renal plasma flow
(RPF) and glomerular filtration rate (GFR) are markedly increased during pregnancy. Yet urine
volume remains at about 1.5 litres. Hence, during pregnancy, water is absorbed with equal and
perhaps enhanced efficiency.
2. Glomerular filtration rate (GFR) increases 50% and renal plasma flow (RPF) increases up to 80% as
compared with nonpregnant levels.1 Tubular function and handling of water and electrolytes are
altered, leading to mild increases in proteinuria, glucosuria, lower serum osmolality, and
reductions in serum sodium levels. The kidneys are larger during pregnancy because of fluid
retention, and physiologic hydronephrosis is common.
Kidney Handling of Water and Electrolytes
1. The threshold for stimulating the osmoreceptors for antidiuretic hormone (ADH) and thirst are
lowered during pregnancy. Plasma osmolality approximates 270 mOsm/kg, and serum sodium
levels decrease an average of 4 to 5 mEq/L. This change may be mediated by increased β-human
chorionic gonadotropin, a pattern also seen to a lesser degree in menstruating women during the
luteal cycle.
2. It is hypothesized that the drop in serum sodium is related to the occurrence of vasodilation,
arterial underfilling, and subsequent ADH release.
3. Relaxin may play a role here because it has been shown to cause ADH secretion and water
drinking in animal studies, and levels are increased during human pregnancy.
4. Mild hyponatremia occurs at the same time there is a rise in aldosterone and its antinatriuretic
forces. Deoxycorticosterone also promotes sodium retention and upregulation of sodium pumps
across various membranes.
5. Total body potassium stores increase by approximately 320 mEq by the end of gestation. This
occurs despite the sodium retention from aldosterone because of the antikaliuretic effects of
progesterone. Potassium excretion is held constant throughout pregnancy, with changes in
tubular reabsorption adapting to alterations in filtered load.
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6. Typical laboratory values during pregnancy
Variable Average Values in Pregnancy

Plasma osmolality 270 mOsm/kg

Serum sodium 135 mEq/L

Serum potassium 3.8 mEq/L

Serum bicarbonate 18-20 mEq/L

Serum creatinine 0.5 mg/dL

Blood urea nitrogen 9.0 mg/dL

Uric acid 2-3 mg/dL

Fluid balance during labour
1. As has been discussed, a normal healthy woman at term has an abundance of body water and at
least 2 litres are 'stored' in her extravascular space; there is thus little reason to give large
volumes of intravenous fluids. Why then are intravenous fluids so often prescribed? There are
three major reasons: as vehicles for labour-inducing agents; to correct 'ketoacidosis'; and during
the maintenance of epidural analgesia.
2. Induction of labour: Labour can be induced or augmented by using 10 units of an oxytocic agent
in 500 ml of a 5 % dextrose solution, equivalent to 20 mu/ml. With modern, highly-accurate
pumps being capable of maintaining very slow infusion rates, it would seem more appropriate to
use smaller volumes of fluid with increased amounts of the oxytocic agent.

FLUID MANAGEMENT IN OBSTETRIC SHOCK:
Definition
Shock is a condition resulting from inability of the circulatory system to provide the tissues
requirements from oxygen and nutrients and to remove metabolites.
Types and Causes
1. Haemorrhagic shock excessive blood loss may be due to:
a. Causes of bleeding early in pregnancy.
b. Causes of antepartum haemorrhage.
c. Causes of postpartum haemorrhage.
2. Neurogenic shock painful conditions my be due to:
a. Disturbed ectopic pregnancy.
b. Concealed accidental haemorrhage.
c. Forceps or breech extraction before full cervical dilatation.
d. Rough internal version.
e. Crédé’s method.
f. Rupture uterus.
g. Acute inversion of the uterus.
h. Rapid evacuation of the uterine contents as in precipitate labour and rupture of
membranes in polyhydramnios. This is accompanied by rapid accumulation of blood in
the splanchnic area due to sudden relief of pressure (splanchnic shock).
3. Cardiogenic shock: ineffective contraction of the cardiac muscle due to
a. Myocardial infarction.
 109
b. Heart failure.
4. Endotoxic shock: generalised vascular disturbance due to release of toxins.
5. Anaphylactic shock: caused by sensitivity to drugs.
6. Other causes:
a. Embolism: amniotic fluid, air or thrombus.
b. Anaesthetic complications: as Mendelson's syndrome.
c. The shock may be caused by more than one factor as:
d. Incomplete abortion: leads to haemorrhagic and endotoxic shock.
e. Disturbed ectopic and rupture uterus: lead to haemorrhagic and neurogenic shock.
Classic Clinical Picture of Shock
1. Low blood pressure.
2. Rapid weak (thready) pulse.
3. Pallor.
4. Cold clammy sweat.
5. Cyanosis of the fingers.
6. Air hunger.
7. Dimness of vision.
8. Restlessness.
9. Oliguria or anuria.
HAEMORRHAGIC SHOCK
Classification of Haemorrhage
Class Blood Loss% Clinical Picture

I 15% Normal pulse & blood pressure.

Tilt test +ve .

II 20-25% Tachycardia.
Tachypnoea.
Pulse pressure (<30mmHg).
Low systolic pressure.
Delayed capillary filling.

III 30-35% Skin: cold, clammy and pale.

Severe drop in blood pressure.
Restlessness.
Oliguria (<30 ml/hour).
Metabolic acidosis (blood pH <7.5).

IV 40-45% Profound hypotension.

The carotid pulse is the only felt one.
Irreversible shock.
Tilt test
1. It is done in patient with considerable bleeding but the blood pressure and/ or pulse rate are
normal.
2. When this patient is in a sitting position, she develops hypotension and / or tachycardia.
Phases of Haemorrhagic Shock
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The normal pregnant woman can withstand blood loss of 500 ml and even up to 1000 ml during
delivery without obvious danger due to physiological cardiovascular and haematological
adaptations during pregnancy.
Phase of compensation
1. Sympathetic stimulation: It is the initial response to blood loss leading to peripheral
vasoconstriction to maintain blood supply to the vital organs.
2. Clinical picture: Pallor, tachycardia, tachypnoea.
Phase of decompensation
1. Blood loss exceeds 1000 ml in normal patient or less if other adverse factors are operating.
2. Clinical picture: is the classic clinical picture of shock (see before).
3. Adequate treatment at this phase improves the condition rapidly without residual adverse
effects.
Phase of cellular damage and danger of death
1. Inadequately treated haemorrhagic shock results in prolonged tissue hypoxia and damage with
the following effects:
2. Metabolic acidosis: due to anaerobic metabolism initiated after lack of oxygen.
3. Arteriolar dilatation: caused by accumulation of metabolites leading to pooling and stagnation of
blood in the capillaries and leakage of fluid into the tissues.
4. Disseminated intravascular coagulation: caused by release of thromboplastin from the damaged
tissues.
5. Cardiac failure: due to diminished coronary blood flow.
6. In this phase death is imminent, transfusion alone is inadequate and if recovery from acute phase
occurs residual tissue damage as renal and/ or pituitary necrosis will occur.
Management
Urgent interference is indicated as follow:
1. Detect the cause and arrest haemorrhage.
2. Establish an airway and give oxygen by mask or endotracheal tube.
3. Elevate the legs to encourage return of blood from the limbs to the central circulation.
4. Two or more intravenous ways are established for blood, fluids and drugs infusion which should
be given by IV route in shocked patient. If the veins are difficult to find a venous cut down or
intrafemoral canulation is done.
5. Restoration of blood volume by:
a. Whole blood: cross-matched from the same group if not available group O-ve may be
given as a life -saving.
b. Crystalloid solutions: as ringer lactate, normal saline or glucose 5%. They have a short half
life in the circulation and excess amount may cause pulmonary oedema.
c. Colloid solutions: as dextran 40 or 70, plasma protein fraction or fresh frozen plasma.
6. Drug therapy:
a. Analgesics: 10-15 mg morphine IV if there is pain, tissue damage or irritability.
b. Corticosteroids: Hydrocortisone 1gm or dexamethasone 20 mg slowly IV. Its mode of
action is controversial; it may decrease peripheral resistance and potentiate cardiac
response so it improves tissue perfusion.
c. Sodium bicarbonate: 100 mEq IV if metabolic acidosis is demonstrated.
d. Vasopressors: to increase the blood pressure so maintain renal perfusion.
i. Dopamine: 2.5m g/ kg/ minute IV is the drug of choice.
ii. ß -adrenergic stimulant: isoprenaline 1mg in 500 ml 5% glucose slowly IV infusion.
2. Monitoring:
 111
a. Central venous pressure (CVP): normal 10-12 cm water.
b. Pulse rate.
c. Blood pressure.
d. Urine output: normal 60 ml/hour.
e. Pulmonary capillary wedge pressure: Normal 6-18 Torr.
f. Clinical improvement in the: pallor, cyanosis, air hunger, sweating and consciousness.
Complications
1. Acute renal failure.
2. Pituitary necrosis (Sheehan’s syndrome).
3. Disseminated intravascular coagulation.
ENDOTOXIC (SEPTIC OR BACTERAEMIC) SHOCK
Obstetric Causes
1. Septic abortion.
2. Prolonged rupture of membranes.
3. Manipulations and instrumentations.
4. Trauma.
5. Retained placental tissues.
6. Puerperal sepsis.
7. Severe acute pyelonephritis.
Causative Organisms
1. Gram-negative bacilli: E.coli, proteus, pseudomonas and bacteroids. The endotoxin is a
phospholipopolysaccharide released by lysis of its cell envelope.
2. A similar picture is produced from exotoxin of ß-haemolytic streptococci, anaerobic streptococci
and clostridia.
Pathology
Release of endotoxin results in increased lysosomal permeability and cytotoxicity. The sequence
of events thereafter may occur in few minutes and include:
Stimulation of the adrenal medulla and sympathetic nervous system → constriction of arterioles
and venules → local acidosis → arteriolar dilatation but with continuing constriction of the
venules → capillary pooling of blood → haemorrhagic engorgement of bowel, liver, kidneys and
lungs.
There is associated extensive disseminated intravascular coagulation due to sudden massive
plasmin generation with which the antiplasmins cannot cope.
Clinical Features
Endotoxic shock passes with 2 main stages:
Reversible stage
It has 2 phases:
1. Early (warm) phase: there are;
a. hypotension,
b. tachycardia,
c. pyrexia,
d. rigors,
e. flushed skin,
f. patient is alert,
g. leucocytosis develops within hours.
2. Late (cold) phase: there are;
a. cold and clammy skin,
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b. mottled cyanosis,
c. purpura,
d. jaundice,
e. progressive mental confusion,
f. coma.
Irreversible stage
Prolonged cellular hypoxia leads to:
1. metabolic acidosis,
2. acute renal failure,
3. cardiac failure,
4. pulmonary oedema,
5. adrenal failure and ultimately death.
Differential Diagnosis
1. Amniotic fluid embolism.
2. Pulmonary embolism.
3. Pulmonary aspiration syndrome.
4. Myocardial infarction.
5. Incompatible blood transfusion.
Management
It includes 3 major lines of treatment:
Restoration of circulatory function and oxygenation
1. Replacement of blood loss: by whole blood, if not available start with colloids or crystalloids. The
CVP measurement is essential to guard against circulatory overload.
2. Corticosteroids: as;
a. Hydrocortisone 1gm IV / 6 hours or,
b. Dexamethasone 20 mg initially followed by 200 mg/day by IV infusion.
3. β-adrenergic stimulants: as isoprenaline cause arteriolar dilatation, increase heart rate and stroke
volume improving tissue perfusion. Blood volume must be normal prior to its administration.
4. Oxygen: if respiratory function is impaired.
5. Aminophylline: improves respiratory function by alleviating bronchospasm.
Eradication of infection
Antibiotic therapy:
1. Swabs for culture and sensitivity are taken first.
2. Antibiotic therapy is starting immediately till the result of culture and given by IV route. The
therapy should cover the wide range of organisms:
Antibiotic Acts upon Dose

Regimen 1 Ampicillin or Aerobic gram+ organisms and gram- 500-1000 mg/6 hours.
Cephalosporines cocci.

Gentamycin Aerobic gram- bacilli. 80 mg/ 8 hours.(not to be

given in the solutions).

Metronidazole Anaerobic. 500 mg/ 8 hours.

Regimen 2 Clindamycin Aerobic gram + organisms + gram- cocci + 600 mg/ 6 hours.
anaerobic organisms.
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Gentamycin Aerobic gram- bacilli. 80 mg/ 8 hours.

Surgical treatment:
is indicated when there is retained infected tissues as in septic abortion. It should be removed as soon as
antibiotic therapy and resuscitative measures have been started by:
1. Suction evacuation,
2. Digital evacuation, or
3. Hysterectomy in advanced infection with a gangrenous (clostridium welchii) or traumatised
uterus.
Correction of fluid and electrolyte deficits
Disseminated intravascular coagulation
Heparin therapy (see DIC) except if there is active bleeding where the condition is best treated by
fresh blood transfusion.
AMNIOTIC FLUID EMBOLISM
Definition
Passage of amniotic fluid into the maternal circulation leads to sudden collapse during labour but
can only be confirmed at necropsy.
Pathology
The condition is more common with strong uterine contraction, whether spontaneous or induced,
occurs after rupture of membranes particularly when there are open maternal blood vessels in
the placental site or in cervical lacerations.
The embolism passes to the pulmonary vessels leads to:
1. sudden death,
2. shock, or
3. Later death due to DIC and postpartum haemorrhage.
Clinical Picture
1. The onset is acute with sudden collapse, cyanosis and severe dyspnoea.
2. This is soon followed by twitching, convulsions and right side heart failure, with tachycardia,
pulmonary oedema and blood stained frothy sputum.
3. If death does not occur in this stage, DIC develops within 1 hour leading to generalised
bleeding.
Investigations
1. ECG: evidence of right side heart failure.
2. X-ray: non-specific mottled chest appearance.
3. Lung scan: with technetium-99m albumin shows perfusion defect.
4. Laboratory tests: evidence of DIC.
Differential Diagnosis
1. Acute pulmonary oedema.
2. Pulmonary aspiration (Mendelson’s) syndrome.
3. Other coagulation defects.
Treatment
Urgent treatment includes:
1. Oxygen: endotracheal intubation and positive pressure respiration is usually indicated as the
patient is often unconscious.
2. Aminophylline: 0.5 gm slowly IV to reduce bronchospasm.
3. Isoprenaline:0.1gm IV to improve pulmonary blood flow and cardiac activity.
 114
4. Digoxin and atropine: if central venous pressure is raised and pulmonary secretions are
excessive.
5. Hydrocortisone: 1 gm IV followed by slow IV infusion causes vasodilatation and improves
tissue perfusion.
6. Bicarbonate solution: if there is respiratory acidosis.
7. Low molecular weight dextran: reduces platelets aggregation in vital organs.
8. Heparin: for treatment of DIC if there is no active bleeding.
9. Vaginal delivery: is safer than C.S. if the baby is not yet delivered.
CARDIAC ARREST
Definition
Sudden circulatory collapse caused by sudden failure of the heart to pump the blood adequately.
Types
1. Complete cessation of mechanical and electrical activity: asystole.
2. Rapid ineffective activity: ventricular tachycardia and ventricular fibrillation.
3. Slow ineffective activity: sinus bradycardia and complete heart block.
In practice, asystole and ventricular fibrillation account for almost all cases of cardiac arrest.
Causes
Any cause of obstetric shock can end by cardiac arrest, the commonest of which are:
1. Severe haemorrhage.
2. Hypoxia due to eclampsia or anaesthesia.
3. Mendelson’s syndrome: gastric aspiration with pneumonitis.
4. Embolism of whatever the nature.
Diagnosis
1. Sudden collapse.
2. Loss of consciousness.
3. Absence of pulse including the carotid and femoral pulse.
4. Apnoea and cyanosis of variable degree.
5. Fixed dilatation of the pupils.
N.B. Attempts to auscultate the heart, to record blood pressure or ECG are only time wasting procedures
unless the patient is already being monitored during surgery.
Management
1. Urgent pairs of hands are needed to save the patient’s life.
2. Put the patient in the dorsal position onto a firm surface, even the floor.
3. A single firm thump with the closed fist over the lower sternum may be sufficient to correct
the condition otherwise,
4. The following ABC steps are carried out:
a. Airway:
i. Clear it: from vomitus, blood, teeth, foreign body ...etc.
ii. Maintain it: Pull mandible and tongue forward.
iii. Insert an airway.
iv. Endotracheal intubation as soon as possible.
b. Breathing:
i. One of the following is used:
ii. Mouth-to-mouth artificial respiration.
iii. Mask and ambubag with 100 % oxygen.
iv. Cuffed endotracheal tube with intermittent positive pressure of 100%
oxygen.
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c. Cardiac massage:
i. Using the heel of one hand, with the other on top, and with the arms
extended, apply pressure to the lower sternum using the full body weight.
ii. This should provide a palpable femoral or carotid pulse.
iii. The optimal compressions is 60 / minute in a ratio of 4:1 to ventillation.
d. Drip and Drugs:
i. Sodium bicarbonate 8.4% solution: to counteract metabolic acidosis. Give 100
ml initially and a further 10 ml for each subsequent minute of inadequate
circulation.
ii. Cardiac stimulants (inotropic drugs): can be given IV or intracardiac e.g.
1. Adrenaline 0.5-1.0 mg.
2. Atropine 0.6 mg.
3. Isoprenaline 4 mg in 500 ml solution.
4. Dopamine 500 mg in 500 ml solution (1-3 m g/ kg/ min).
5. Calcium chloride 10% solution.
e. Electrocardiogram:
i. to assess the condition and response to the therapy.
f. Fibrillation treatment
i. Direct current (DC) defibrillator is used.

DOUBLE BHOR EFFECT:
Introduction:
1. Bohr effect enhances the exchange of oxygen and carbon dioxide across the placenta.
2. The Bohr effect describes the shift of the hemoglobin dissociation curve to the right by hydrogen
ions, which reduces the affinity of hemoglobin for oxygen.
Oxygen dissociation curve:
1. The binding ability of hemoglohin with 02 at different partial pressures of oxygen (pO2) can be
measured by a graphic representation known as 02 dissociation curve.
2. The oxygen dissociation curve for hemoglobin is sigmoidal in shape. A shift of the curve to right
means that Hb has reduced affinity and gives up O2 more to tissues.
3. The ability of hemoglobin to bind oxygen is affected by the following, which are collectively called
allosteric (“other site”) effectors.
a. The po2 (heme-heme interactions),
b. The ph of the environment,
c. The partial pressure of carbon dioxide
d. The availability of 2,3-bisphosphoglycerate.
Bhor effect:
1. The binding of oxygen to hemoglobin decreases with increasing H+ concentration (lower pH) or
when the hemoglobin is exposed to increased partial pressure of CO2 (pCO2). This phenomenon is
known as Bohr effect. lt is due to a change in the binding affinity of oxygen to hemoglobin. Bohr
effect causes a shift in the oxygen dissociation curve to the right.
2. Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion. Some
CO2 is carried as carbamate bound to the N-terminal aminogroups of hemoglobin (forming
carbaminohemoglobin)
3. When the pCO2 is high in tissues, CO2 diffuses into the red blood cells. The carbonic anhydrase in
the red cells favors the formation of carbonic acid (H2CO3). In RBC, the intracellular pH falls. This
reduces the affinity of Hb for O2 and O2 is released to the tissues.
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Carbonic anhydrase
CO2 + H2O ----------------- H2CO3 → H+ + HCO3 –
4. The binding of CO2 stabilizes the T (taut) or deoxy form of hemoglobin, resulting in a decrease in
its affinity for oxygen and a right shift in the oxygen dissociation.


















Double Bhor effect:
1. The 'double Bohr' effect helps to increase fetal oxygenation. The transfer of carbon dioxide from
fetal to maternal blood shifts the maternal oxyhaemoglobin curve to the right and the fetal curve
to the left, facilitating the transfer of oxygen across the placenta from mother to fetus
2. In the lungs, CO2 dissociates from the hemoglobin, and is released in the breath.
3. The carbon dioxide from the fetal side diffuses into the maternal blood, causing an increase in
maternal intervillous hydrogen ion, which reduces the affinity of maternal hemoglobin for
oxygen, increasing oxygen transfer to the fetus.
4. At the same time, the relative decrease in carbon dioxide on the fetal side causes the fetal blood
to become slightly more alkaline, increasing the fetal hemoglobin uptake of oxygen.
5. Since the Bohr effect occurs on both sides of oxygen delivery/uptake, it has been called the
double Bohr effect.
6. The double Bohr effect occurs functionally by the slight opening and closing of the hemoglobin
chain allowing or blocking entry of oxygen to the iron-heme–binding site. Carbon dioxide binding
to the sentinel histidine on the hemoglobin chain can block access of oxygen to the heme-binding
site.

SODIUM PUMP IN OBSTETRICS:
Introduction:
1. Multiple transport systems move sodium ions across the cell membrane. The primary sodium
transport system is termed the (Na,K) ATPase or sodium pump (SP).
2. It moves 3 sodium ions out of the cell for every 2 potassium ions moved in, maintaining the
appropriate ionic milieu in the cytosol and giving rise to the cell membrane potential.
Sodium Pump (SP) in Uncomplicated Pregnancy
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1. In normal pregnancy most studies report an increase in erythrocyte SP activity, lymphocyte Na
efflux, platelet Na efflux. One study reported an increased reticulocyte count, reticulocytes having
more SP units, but this appears to be variable. Moreover, the same changes are observed in blood
cells other than erythrocytes.
2. What purpose this apparent change in SP serves is unknown. One hypothetical benefit of
increased active sodium transport, with a consequent reduction in cell Na, would be a
hyperpolarization of mechanically active cells, as in the vascular smooth muscle (VSM), which
could reduce peripheral resistance and allow for greater blood flow to the fetal-placental unit. If
such changes were present in the uterus, it would tend to reduce the contractile activity of that
tissue and maintain it in a quiescent state. Indeed, the uterus is hyperpolarized during most of
pregnancy.
3. The increase in SP abundance in normal pregnancy may be mediated transcriptionally.
Aldosterone is known to upregulate SP mRNA levels in some cultured cells. Hence, increased
serum aldosterone in pregnancy might be predicted to increase expression of SP units in several
cell types, possibly peripheral blood cells. Progesterone may also increase SP mRNA, but this
hasn't been studied in pregnancy. There is an indication of altered myometrial SP isoform protein
abundance in human pregnancy.
CELL SODIUM TRANSPORT IN PREECLAMPSIA
1. The interest in sodium transport arises from the demonstration that increased vascular smooth
muscle (VSM) sodium, brought about by SP inhibition, increases the sensitivity of the vascular to
pressor substances. Moreover, if the cell sodium is sufficiently high, it leads to direct contraction
of VSM. Such a heightened contractile state of the vasculature in response to elevated cell Na is
mediated by a secondary increase in cytosolic, ionized calcium.
2. As mentioned, the hypertension manifest in PE (Pre eclampsia) is a result of increased peripheral
vascular resistance and consequently increased cell sodium represents one of several candidate
mechanisms to explain the increase in vessel tone and hypertension in PE.
Cell Sodium and Potassium in Preeclampsia
1. Most report significant increases in erythrocyte cell Na, lymphocyte cell Na, and leucocyte cell Na
in women having PE compared with normotensive pregnant women.
2. The studies do not allow for any firm conclusion regarding cell Na in PE, but more strongly
support an increase in cell Na. Hence, the hypothesis relating cell Na and hypertension remains
suggestive, but unconfirmed in PE. The study of cell K levels in PE has been less frequent. In
general those studies finding no change in cell Na also found no change in cell K and those studies
finding an increase in cell Na found a decrease in cell K.
Sodium Pump (SP) in Preeclampsia
3. SP status in women with PE is something of an open question. A major issue is the possible
presence of a circulating SP inhibitor in pregnant women that is significantly increased in PE.
4. Nevertheless, a majority of studies have found significant reductions in SP activity accompanying
PE as manifest in erythrocytes.
5. Study of the actual expression of the SP α-isoform (the functional unit of the SP) protein or mRNA
in PE has been very limited. But in one study there appeared to be decreases in both mRNA and
the α2 isoform of the SP in myometrium. There was also a reduction in mRNA of the a 1 and a 2
isoforms without reductions in the placental membrane protein expression of the two isoforms in
PE. This study also suggested that gestational age may be a very important confounding variable
for the evaluation of the SP.
6. Taken together, there is a suggestion that SP activity is reduced in PE. Some of this reduction may
be due to increased levels of a SP inhibitor, which may be elevated in the circulation of women
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with PE. Alternatively, the reductions may reflect decreased expression of one or more isoforms
of the SP.
7. Even small decreases in cell SP activity can have substantial physiologic effects. Hence, the actual
changes in SP activity may be small and yet pathologic. Patient to patient variability may be
substantial.
Sodium pump inhibitors in uncomplicated pregnancy
SP activity increases in uncomplicated pregnancy in comparison with the non-pregnant or post
partum state even though there may be modest increases in levels of an active SP inhibitor as
mentioned.
SODIUM PUMP INHIBITORS IN PREECLAMPSIA
1. There are several studies that report increased levels of a SP inhibitor as measured by digoxin
antibody, ouabain antibody, marinobufogenin antibody, by inhibition of SP in women with PE.
2. One of the most provocative set of results has been provided by the use of anti-digoxin antibodies
to treat PE. In these cases there was remarkable success in lowering blood pressure for several
hours. The Fab fragment has been used in hundreds of individuals without eliciting an immune
response and the antibody has almost no affinity for other steroidal compounds, consequently, in
those few cases, its ability to lower blood pressure suggests strongly that it binds a
hypertensinogenic, digitalis-like factor.
CONCLUSION AND PERSPECTIVE
Existing research points to sets of changes in Na handling during normal pregnancy with some confidence:
1. Serum Na concentration is significantly reduced during normal pregnancy and there is a profound,
progressive activation of the renin-angiotensin-aldosterone system during the course of
pregnancy.
2. Over this same interval there is an increase in SP number and activity and a reduction in cell Na
concentration manifest in peripheral blood cells with similar findings in a few studies of tissue or
tissue cells.
3. The strongest and most consistent findings indicate that there is an increase in cell Na, a decrease
in cell SP activity and higher levels of a digitalis-like SP inhibitor in PE.
4. Though limited, there are even studies that use an antibody fragment targeting such inhibitors to
treat effectively the hypertension of PE.

TORCH INFECTION:
1. “Vertical transmission” is a term that refers to the spread of infections from mother-to-baby. These
infections may occur while the foetus is still in the uterus (transplacenta), during labour and
delivery, or after delivery while breastfeeding. (Perinatal transmission)
2. Congenital infections (passed in utero): TORCH is an acronym for several of the more common
congenital infections. These are:
3. Across the placenta
a. Toxoplasmosis
b. Other infections (syphilis, hepatitis B, Coxsackie virus, Epstein-Barr virus,varicella-zoster
virus (chicken pox), and human parvovirus)
c. Rubella
d. Cytomegalovirus (CMV)
e. Herpes simplex virus
f. HIV
g. Listeria monocytogenes,
h. Plasmodium falciparum
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4. Perinatal infections (during labour and delivery)
a. Ascending maternal infection and chorioamnionitis causing fetal infection, usually
subsequent to prolonged rupture of membranes (PROM).
b. Perinatal infection acquired during birth via the haematogenous or genital route. These
include:
a. Gonorrhoea
b. Chlamydia
c. Herpes simplex virus
d. Human Papilloma Virus (genital warts)
e. Group B Streptococci (GBS)
f. Human immunodeficiency virus (HIV),
g. Herpes zoster virus (HZV),
h. Hepatitis B virus (HBV)
a. Postnatal infection transmitted via breastfeeding.
i. HIV

TOXOPLASMOSIS
Introduction:
1. Toxoplasmosis is a disease that results from infection with the Toxoplasma gondii parasite, one of
the world's most common parasites.
2. Toxoplasmosis may cause flu-like symptoms in some people, but most people affected never
develop signs and symptoms. For infants born to infected mothers and for people with weakened
immune systems, toxoplasmosis may cause serious complications.
3. Like CMV, toxoplasmosis is usually asymptomatic or has mild, non-specific symptoms and primary
infection during pregnancy can cause serious fetal effects. Unlike CMV, toxoplasmosis acquired
during pregnancy can be treated, reducing the fetal adverse effects.
Transmission:
1. Toxoplasmosis is acquired by eating raw or undercooked meat, contaminated salad or ingesting
soil contaminated with toxoplasma oocysts, which are excreted in the faeces of infected cats.
2. Pregnant women should be advised to avoid these exposures (wear gloves when gardening, avoid
handling cat litter). Direct contact with cats is rarely a source of infection - most acquire infection
as kittens and excrete oocysts for a short amount of time.
3. A third of infants become infected if their mother becomes infected during pregnancy, especially
in later pregnancy (but the severity of disease decreases).
4. About 75% pregnant women are susceptible but seroconversion during pregnancy is uncommon.
Clinical features:
1. There are many different presentations:
2. Systemic neonatal disease - rash, jaundice, thrombocytopenic purpura, hepatosplenomegaly,
pneumonia, progressive uveitis.
3. Neurological disease - hydrocephalus, microcephaly, microphthalmia, retinochoroiditis, cerebral
calcification.
4. Mild disease - isolated retinochoroiditis or mild cerebral calcification and no sign of cerebral
injury.
5. Sub-clinical - occurs in 70% of infected babies.
6. Relapsing - retinochoroiditis with flare-ups can occur at any age.
Diagnosis
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1. It is difficult: serological tests have poor sensitivity, false positive toxoplasma IgM is not
uncommon and low levels of IgM persist for many years following primary infection.
2. Confirmation of fetal infection is best done with amniotic fluid PCR.
Treatment:
Established congenital toxoplasmosis is treated with pyrimethamine, sulfadiazine and folic acid
from the second trimester until a year old.

CONGENITAL RUBELLA
I. Definition. Rubella is a viral infection capable of causing chronic intrauterine infection and damage to
the developing fetus.
II. Pathophysiology.
a. Rubella virus is an RNA virus with epidemic seasonal pattern
b. Fetal effects is greater the earlier in gestation that infection occurs, especially at 1-11 weeks,
when 90% of infected fetuses will be damaged; 24% at 15-16 weeks.
III. Clinical presentation.
a. Teratogenic effects. These include
a. Intrauterine growth retardation,
b. Congenital heart disease (patent ductus arteriosus or pulmonary artery stenosis),
c. Sensorineural hearing loss,
d. Cataracts or glaucoma,
e. Neonatal purpura, and
f. Dermatoglyphic abnormalities.
b. Systemic involvement can be manifested by:
1. Adenitis,
2. Hepatitis,
3. Hepatosplenomegaly,
4. Jaundice,
5. Anemia,
6. Decreased platelets with or without petechiae,
7. Myocarditis,
8. Eye lesions (iridocyclitis or retinopathy),
9. Pneumonia.
c. Late defects.
1. Including immunologic dyscrasias,
2. Hearing deficit,
3. Psychomotor retardation,
4. Autism,
5. Brain syndromes such as subacute sclerosing panencephalitis,
6. Diabetes mellitus, and
7. Thyroid disease.
Diagnosis: ELISA for IgM and IgG antibodies are the most commonly performed tests. Rubella is difficult
to differentiate clinically from other rash-causing infections such as measles and parvovirus BE19
Management.
1. There is no specific treatment for rubella.
2. Prevention consists of vaccination of the susceptible population (especially young
children).
3. Vaccine should not be given to pregnant women.
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Human immunodeficiency virus
Etiology
1. Family: Retroviridae
2. Genus: Lentivirus
3. HIV I: contains 2 copies of single stranded RNA; most common infection
4. HIV II: common among monkeys; rare in Ped.HIV difficult to identify with HIV I tests;
specific antibody test or III generation ELIZA should be used for testing
Mother To Child Transmission
1. Transmission rate is 12-30 %
2. Intra uterine: 30-40 %
3. Intrapartum: 60-70 %
4. Breastfeeding:
Transplacental
1. PCR is positive in fetal tissue by 10 weeks of gestation
2. In situ hybridization and immuno cytochemistry identify virus in fetal tissue in 1st
trimester
3. Viral detection soon after birth
Intrapartum
1. Infected blood, cervico vaginal secretions in birth canal à conjunctiva, skin abrasions,
gastric mucosa
2. Infected infant negative for viral detection in first week of life
3. First born twin 3 times more infected
Breast feeding
1. Both virus and viral laden cells are present breast milk
2. Mother infected before pregnancy: 14 %
3. Mother infected postnatally: 29 %
4. Benefit of breastfeeding outweighs the risk of HIV in developing countries
5. WHO: breastfeed first 6 months and rapid weaning thereafter
Risk factors for MTCT
1. Preterm < 34 weeks
2. Low CD4 count in mother
3. Birt weight < 2500 gms
4. Rupture of membranes for > 4 hours
5. Mother’s viral load > 1000 copies/mL
6. Vaginal delivery
7. Instrumental delivery
8. Caserian + ART to infant decrease transmission by 87 %
Diagnosis
1. Viral Culture: 100% specific; costly and needs sophisticated laboratory set up
2. Polymerase Chain Reaction (PCR) RNA/DNA:
1. Two PCRs have to be positive, done beyond one month and at least one of them
after age of 3 months.
2. Detect nearly 100% of infected newborns
3. Enzyme Linked Immunosorbent Assay (ELISA)
1. I Generation ELISA: antigen from crude viral lysate
2. II generation: part of virus
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3. III Generation ELISA: synthetic peptides as the antigen
4. Rapid tests
20. Management Care immediately after birth
1. Cord clamped soon; no milking;
2. Early baby bathing
3. Single dose NVP to mother during labour and to the baby within 72 hours after birth.
4. Exclusive replacement feeding is afass — acceptable, feasible, affordable, sustainable and
safe
5. Quick weaning at 6 mo
6. CTX prophylaxis from age of 4–6 weeks; 5 mg/kg/day
7. Growth monitoring
8. Screen for infections
21. When to start ART in children, guided by CD4
1. < 11 month infants : if CD4 < 1500 cells/mm3
2. 12–35 months : if CD4 < 750 cells/mm3
3. 36–59 months : if CD4 <350 cells/mm3
4. > 5 years old : < 350 cells/mm3 especially if symptomatic; or
5. Before CD4 drops below 200 cells/mm3.
ARV regimens
1. Zidovudine (AZT) + Lamivudine (3TC) + Nevirapine NVP
2. Stavudine (D4T) + Lamivudine (3TC) + Nevirapine (NVP)
3. Vaccine:
1. if the HIV-infected child is asymptomatic or mildly symptomatic – vaccinations should
be given.
2. Withold vaccine (live vaccines) for HIV-infected children who are symptomatic and
severely immuno-compromised.
Prevention
1. Transmission from the mother to child is likely to be about 15-45%.
2. There is 16.2% greater risk of mother-to-infant transmission of HIV when children are breast-
fed as opposed to formula-fed.
3. Single dose NVP 200mg given at the onset of labour and
4. single dose of syrup NVP 2mg/kg weight to the baby within 72 hours decreases risk of
transmission by 13.1% (breast feeding)..
5. Transmission of the HIV virus occurs most commonly during the first few months after birth
6. Avoid manipulations like amniocentesis and external cephalic version increase the risk of
transmission of HIV.
7. Long duration of rupture of membranes increase the transmission risk. It has been estimated
that with every hour, the risk of transmission increases by 2%.
8. Placental disruption and infections also adversely affect transmission.
9. Invasive fetal monitoring should be avoided, as should all invasive obstetric procedures.
10. Where facilities are available, elective LSCS should be offered.
11. If instrumental delivery is necessary, then forceps are a better option than vacuum suction
cup delivery.
12. Emergency LSCS is associated with high transmission
13. In India – normal delivery is recommended unless the woman has obstetric reasons
14. When replacement feeding (infant formula) is acceptable, feasible, affordable, sustainable
and clean water is available, HIV-infected mothers should avoid breastfeeding completely.
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15. Otherwise, exclusive breastfeeding is recommended during the first months of life, with early
abrupt weaning at 3-4 months or 6 months of age

Cytomegalovirus
Introduction:
1. Cytomegalovirus (CMV) is a DNA virus and a member of the herpesvirus group (human
herpesvirus 5). CMV is the most common cause of congenital infection.
2. More than 85% of infants born with CMV have a subclinical infection.
Transmission:
1. CMV is a ubiquitous virus that may be transmitted in secretions, including saliva, tears, semen,
urine, cervical secretions, blood (white blood cells), and breast milk.
2. CMV may also be transmitted to the infant intrapartum (through exposure to CMV in cervical
secretions), via breast milk, and via blood transfusion.
3. CMV is capable of penetrating the placental barrier as well as the blood–brain barrier. During
early pregnancy, CMV has a teratogenic potential in the fetus. In primary maternal infection the
virus is transmitted to the fetus in ~35% of cases.
4. Infection in early pregnancy causes more severe fetal infection with significant CNS sequelae.
Risk factors:
1. Risk factors for primary CMV infection during pregnancy include prolonged exposure to young
children (daycare workers, multiparous women) and
2. sexual contact (young maternal age, greater numbers of sexual partners, abnormal cervical
cytology, and having a sexually transmitted infection during pregnancy).
Prenatal presentation.
1. Pregnant women who acquire primary CMV may develop a mononucleosis-like illnesses.
2. Fetal anomalies consistent with congenital CMV infection that can be detected on prenatal
ultrasound examination include fetal growth restriction, cerebral periventricular echogenicity or
calcifications, cerebral ventriculomegaly, microcephaly, polymicrogyria, cerebellar hypoplasia,
hyperechogenic fetal bowel, hepatosplenomegaly, amniotic fluid abnormalities, ascites and/or
pleural effusion, and placental enlargement
3. Amniocentesis to perform polymerase chain reaction (PCR) for CMV DNA in amniotic fluid is the
preferred diagnostic approach for identifying an infected fetus.
Postnatal presentation
1. Subclinical infection. Occurs in 85–90% of cases. Despite being asymptomatic at birth, these
infants are at risk for sensorineural hearing loss (SNHL) during the first 6 years of life.
2. Low birthweight.
5. Classic CMV inclusion disease. Occurs in 10–15% of the cases and consists of intrauterine growth
restriction, hepatosplenomegaly with jaundice.
6. Neurologic complications include microcephaly, intracerebral calcifications (most
characteristically in the subependymal periventricular area), chorioretinitis,
Late sequelae.
1. SNHL occurs in 22–65% of symptomatic and 6–23% of asymptomatic infants
2. Visual impairment and strabismus are common in children with clinically symptomatic CMV
infection. Visual complications may occur, usually secondary to chorioretinitis, pigmentary
retinitis, macular scarring, optic atrophy, and central cortical defects.
Diagnosis
1. Culture for demonstration of the virus. The gold standard for the diagnosis of congenital CMV is
urine or saliva culture obtained before 3 weeks of age.
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2. Most urine specimens from infants with congenital CMV are positive within 48–72 hours,
especially if shell vial tissue culture techniques are used.
3. Polymerase chain reaction (PCR). PCR for CMV DNA is as sensitive as a urine culture for the
detection of CMV infection.
4. Serologic tests. Serologic tests based on the detection of immunoglobulin M (IgM) should not be
used to diagnose congenital CMV because they are less sensitive and more subject to false-
positive results than culture or PCR. Only 70% of neonates infected with congenital CMV have IgM
antibodies at birth.
5. Ultrasound or CT scans of the head may demonstrate characteristic periventricular calcifications.
Management
1. Prevention and treatment of maternal infection during pregnancy: frequent hand washing;
wearing gloves for specific child-care tasks; avoiding kissing children under age 6 on the mouth or
cheek; not sharing food, drinks, or oral utensils
2. Antiviral agents. No antiviral agent is yet approved for treatment of congenital CMV infection.
3. Valganciclovir is a prodrug of ganciclovir that is suitable for oral administration. Valganciclovir
administered orally to young infants at 16 mg/kg/dose, twice daily, provides the same systemic
ganciclovir exposure as does intravenous ganciclovir at 6 mg/kg/dose.
Prognosis.
Congenital CMV is the leading cause of SNHL regardless of the seroprevalence in the population.
For symptomatic infants at birth, mortality is up to 30%, and up to 90% will have late
complications (intellectual or developmental impairment, hearing loss, spasticity).

Congenital herpes simplex:
Neonatal HSV infection can be acquired in three distinct periods:
1. Rarely intrauterine, accounting for 1 in 250,000 deliveries
2. Most (85%) neonatal HSV infections are acquired perinatally, especially in maternal HSV infection,
longer membrane rupture duration, and the use of a fetal scalp monitor.
3. HSV can also be acquired postnatally in 10% of cases, especially when the the infant comes in
contact with a caretaker with herpes labialis infection.
4. Disseminated Neonatal HSV Infection
The disease involves the:
1. Central nervous system
2. Liver
3. Lungs
4. Adrenal glands
5. Skin
6. Eyes
7. Mucous membranes
Patients might present with:
1. Encephalitis
2. Hepatic failure
3. Respiratory failure
4. Skin rash
5. Disseminated intravascular coagulation
Central Nervous System (CNS) Neonatal HSV Infection
1. Seizures
2. Poor feeding
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3. Temperature instability
4. Lethargy
Diagnosis
1. The definitive method of diagnosing neonatal HSV infection is the isolation of HSV by culture.
HSV can be cultured from the conjunctivae, nasopharynx, mouth, or anus of the neonate. In
severe cases, a culture of the cerebrospinal fluid or blood might be prepared. However, the
diagnostic yield of skin and eye cultures is excellent and superior to any other site.
2. Neonates with central nervous system HSV infections should undergo a polymerase chain
reaction (PCR) test to confirm the diagnosis. PCR is superior to HSV cultures because it can
provide results much faster. The PCR’s sensitivity and specificity for CNS HSV infections is around
100%.
Management of Neonatal HSV Infections
Once the diagnosis of neonatal HSV infection is confirmed, antiviral therapy should be started.
Intravenous acyclovir at the dose of 20 mg/kg three times a day is the standard treatment for
neonatal HSV infections regardless of the form of the disease.
Screening
TORCH panel:
Tests Included (5 tests)
1. Cytomegalovirus IgG Antibody
2. Herpes Simplex Virus 1 IgG
3. Toxoplasma gondii - IgG
4. Herpes Simplex Virus 2 IgM
5. Rubella Virus – IgG
Interpretations
1. A normal result is negative antibody in the blood and means that it is unlikely that the person
tested has the infections
2. A positive result indicates high likelihood of infection with that microbe. IgM antibodies are
present when their is current or recent infection. However, further testing must be done to
confirm the results
3. The presence of IgG antibodies in pregnant woman usually indicate past infection
4. If both IgM and IgG antibodies are found in a newborn, it's probably because antibodies in the
mother have been transferred to the fetus through the placenta