BGDa scenario 1 Prac Notes

BGD Prac Notes
http://web.med.unsw.edu.au/vslide/phase1/indexA2.asp
Prac 1: Histology of the Female Reproductive Tract
Ovary and Corpus Luteum
M
C
S
M
Te
Ti
TA
D
Co
Mf
Pm
O
C
Cr
Zp
G
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(G) Germinal epithelium – layer that covers the ovary
(TA) Tunica albuginea – superficial cortex that is more
fibrous
(C) Cortex – peripheral zone of the stroma in which the
follicles develop
(M) Medulla – central zone of the stroma which is
highly vascular
(S) CT stroma – spindle shaped cells, collagen fibres
that provide support network for ovary
(Pm) Primordial follicles – first stage of follicle/oocyte
development
(D) Developing follicles – second stage of
follicle/oocyte development
(Mf) Mature follicle – fully developed follicle/oocyte
that is in preparation for ovulation
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(O) Oocyte – cell that develops inside the follicle into
the ovum
(Zp) Zona Pellucida – a layer that encloses the oocyte
inside the follicle - glycoprotein
(Cr) Corona radiata – layer of cells that surrounds the
oocyte and breaks off with it upon ovulation
(Co) Cumulus oophorus – thickened cell layer that
surrounds the oocyte
(Ti) Theca interna – layer of cells outside the follicle
(Te) Theca externa – layer of cells outside the theca
interna
(Af) Atretic follicles – broken down follicles
Gl
Cl
Tl
Gl
Ca
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(Cl) Corpus luteum – end result of follicle ovulation
(Ca) Corpus albicans – break down of corpus luteum results in corpus albicans
(Gl) Granulosa lutein cells – granulosa cells increase in size and start producing progesterone
(Tl) Theca lutein cells – theca cells increase in size and continue to secrete estrogen
Uterine Tube
C
M
E
Lp
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(E) Simple columnar epithelium – covering the cilia (C)
(Lp) Lamina Propria – loose CT (collagen and reticular fibres) and blood vessels
(M) Muscularis – has a inner circular and outer longitudinal smooth muscle layers
Oviduct function – to push the ovum down the uterine tube to the uterus
• Uterus: proliferative and secretory phases
Proliferative
Sm
E
M
En
S
Ug
Secretory:
E
En
S
Ug
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(E) simple columnar epithelium, lines the endometrium (En)
(Ug) Uterine/endometrial glands – secrete nutrients like
glycogen
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(S) spiral arteries – allows constriction and therefore
menstrual flow
(Sm) smooth muscle – exist in bundles in the myometrium (M)
Cervix
Cg
Lp
Cc
V
Cc
Cg
M
V
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(Cc) Cervical Canal – has simple columnar epithelium
(V) Vagina – stratified squamous non-keratinising epithelium
(Cg) Cervical Glands (Nabothian) secreting mucus – highly viscous during proliferative phase and secretory
phase, low viscosity during ovulation
(Lp) Lamina propria containing CT, blood vessels, nerves and lymphatic tissue
(M) Muscularis layer of smooth muscle
Vagina
E
Vt
Sm
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(E) Stratified squamous non-keratinising epithelium
Vascular tissue (Vt), smooth muscle (Sm) but no glands
Mammary Glands: inactive and active
Inactive:
Sa
Se
Bv
A
Se
Sa
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(Sa) Secretory alveoli – hollow cavities of the secretory
lobules
(Se) Interlobular septa – CT that divides the lobules
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(La) Lactiferous ducts – ducts that bring the milk to the
surface from the secretory lobes
(A) Adipose tissue – collections of fat tissue
(Bv) Blood vessels
Active:
A
Se
Sa
Sa
Se
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•
•
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(Sa) Secretory alveoli – hollow cavities of the secretory lobules
(Se) Interlobular septa – CT that divides the lobules
(La) Lactiferous ducts – ducts that bring the milk to the surface from the secretory lobes
(A) Adipose tissue – collections of fat tissue
(Bv) Blood vessels
Practical 2: Female Reproductive System Anatomy
1. The Uterus
• Normal position: bent over the bladder, the cervix almost 90 degrees to the axis of the vagina
o Pear shaped, anteflex and anteversion
• Angles of flexion and version diagrams
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Made up of the fundus, body and isthmus
o Lined by the endometrium, myometrium and perimetrium
o Connects to the cervical canal by the internal os
Cervical canal is lined by palmate folds and nabothian glands
o Leads into the vagina by the external os
Uterus with uterine tubes and ovaries is like a bald man holding two footballs behind his back
o Dress robes form broad ligament
Medial section diagram
2. Clinical problems
• Per rectum, only the cervix can be felt
o Per vagina
o Supra pubic symphysis – requires an empty bladder/ stomach and relaxed patient
• Normal supports of the uterus:
o Round ligament, broad ligament, suspensory ligament of the ovary
3. The Ovaries
• Ovary is covered by the mesovarium
o suspended by the suspensory ligament of the ovary
• through this is the venous, arterial and nervous supply
o ovary is attached to the uterus by the ovarian ligament
• ovary is suspended in the peritoneal cavity, ova are released into the cavity where they are taken up by the
fimbriae of the uterine tubes
4. The Uterine Tubes
• Made up of the infundibulum, ampulla, isthmus and intramural
• Diagram of the uterine tubes
5. The Broad Ligament
• Made up of the:
o Mesometrium – covers the uterus
o Mesosalpinx – covers the uterine tubes
o Mesovarium – covers the ovaries
Diagram of broad ligament
6. Arterial, venous and lymphatic drainage
• Arteries/veins and lymph diagram
7. Radiographs
• Radiographs, white parts are radio-opaque
o Dye is injected per vagina (sheathe) using a speculum to dilate
o Catheter is connected to a syringe to inject dye into the cervix
Flows through uterine tubes into the peritoneal cavity
o Test determines blockages and asymmetric tubes
o Also shows up whether the uterus is shifted away from the midline, which it can be
• During pregnancy, the isthmus of the uterus becomes jelly-like and soft
o Can be felt by palpating supra pubic symphysis and per vagina
Fundus and body feel disconnected from the cervix
8. Female External Genitalia
• Region between the thighs – perineum
o Perineal body – between anus and posterior vagina
o Feels denser
• Muscle sheets deep to skin
o Transverse perineal muscles
o Levator ani muscles
• Urogenital triangle and anal triangle diagram
• External genitalia made up of:
o Mos pubis, labia majora, labia minora, frenulum, glans, prepuce of the clitoris, urethral orifice,
vaginal vestibule
• See diagrams from lecture
Practical 3: Embryology (fertilisation)
Oogenesis
• Number of eggs:
o Early in fetal development: 7 million
o Decreases by atresia (apoptotic cell death)
o At puberty: 400 000
10% of these released throughout reproductive life
• Follicles take 65 days to fully develop
o Approximately 3 cycles, atresia can occur at any time
• Theca interna creates the hormones while theca externa processes the hormones
Ovulation
• Egg + zona pellucida + corona radiata are released
o Fimbriae take up egg
Ciliated epithelium move egg through the uterine tube
Possible ectopic pregnancy
Gametogenesis
• Gametes are formed in the gonads: testes/ovaries
• Females:
o Total number of eggs ever produced are those present in the newborn female
o Oocytes exist in an arrested state in the first meiotic division – prophase
After puberty, each cycle, pituitary gonadotrophin stimulates completion of meiosis I the day
before ovulation
• one daughter cell becomes the secondary oocyte, the other the first polar body
the secondary oocyte commences meiosis II and is arrested in metaphase which doesn’t
continue until fertilisation
• Male: occurs continuously in the seminiferous tubules
Fertilisation
• Sperm contributes nuclear material
o Doesn’t contribute mitochondria
• Sperm events:
o Capacitation – removal of glycoprotein coat and seminal proteins
o Acrosomal reaction – enzymes digest zona pellucida
o Membrane fusion – sperm and egg allowing sperm nuclei passage
• Egg events:
o Cortical reaction – prevents entry of sperm once fertilised
o 2nd meiotic division forming second polar body
• Male and female pronuclei form the first diploid cell
o Zygote
o Chromosomes pair off
Summary of Week 1
• Ovulation – the initial release of the oocyte
• Follicular fluid and fimbriae aid oocyte movement into the infundibulum, then the ampulla
• Sperm deposited in the vagina enter the uterus, mature (capacitation) and actively migrate along the uterine
tube
• Fertilisation normally occurs in the ampulla region
• The zygote undergoes mitosis to form a solid ball of cells (morula)
o A cavity forms (blastocyst)
• blastocyst is liberated from the zona pellucida allowing adplantation to the uterine wall
• follicle collapses and forms the corpus luteum
Early Cell division
• Blastocyst hatching:
o Blastocyst now consists of a ball of cells containing a large hollow fluid-filled (blastoceol) space
o Trophoblast layer – single layer of cells located at the periphery, pressed against the zona pellucida
wall – thus becoming squamous
o Inner cell mass – one wall of the blastoceol, there is a second layer of flattened cells
o Hatching:
Lysins from blastocyst/uterus and physical expansion cause a reduction in the thickness and
strength of the zona pellucida wall
Blastocyst typically hatches from a narrow opening (potentially the site of fertilisation)
o Assisted hatching – in vitro may need zona drilling, zona slitting or laser assisted hatching to
increase the probability of implantation
Implantation
• Uterine epithelium is invaded by syncitiotrophoblasts
o Bilaminar embryo forms – epiblast + hypoblast
o Blastoceol is covered in cytotrophoblast cells
o Amniotic cavity forms beneath the epiblast layer and above the cytotorophoblast layer
Spaces within the syncitiotrophoblast layer will with maternal blood and are called lacunae
o The extraembryonic mesoderm lines the cytotrophoblast cells on the blastoceol side
This forms the outer layer of the pre-yolk sac and later, the chorionic sac
Extra-embryonic spaces
• Inside the conceptus – amniotic sac, yolk sac, chorionic sac
Gastrulation
• Formation of the inner cell mass from the bilaminar embryo to the trilaminar embryo
o Endoderm and mesoderm migrate through the primitive streak from the epiblast layer
Once this is complete, the epiblast becomes the ectoderm
o These 3 germ layers will form the basis for all future tissue development
Notochord
• A midline column of cells running in a rostro-caudal direction within the mesoderm layer
• A transient developmental patterning structure
o Plays a role in :molecular signalling (patterning) and the direction of embryonic disk folding
(mechanical)
• Notochordal develops into a notochordal plate and then the notochord (axial mesoderm)
o Regulates differentiation of surrounding structures, including the ectoderm (neural plate) and
mesoderm (somites)
Folding
• At Carnegie stage 7, all edges of the embryonic disk will fold ventrally, forming a rostro-caudal C shaped
tube
o This encapsulates the endoderm allowing the tube-like gut to form
Prac 4: Female Reproductive Hormones and their Effects
Process of Vaginal Epithelial Cell Smears
• Cotton bud liberally soaked in saline is inserted into the vagina of the rat
• Cotton bud is rotated and removed
• Cotton bud is smeared across a glass slide
• Slide is fixed and soaked in various chemicals before examination under a microscope
Examination of vaginal epithelial cell smears
• Nucleated epithelial cells (blue-green with pink/mauve nuclei)
• Large squamous epithelial cells, ‘cornified’ (orange-red)
• Polymorphonuclear leucocytes (green, smaller than nucleated epithelial cells)
Analysis
• Oestrogens cause cornification of the vaginal epithelial cells – therefore presence of large, squamous orange
red cornified cells are present
• Progesterone inhibits this cornification – therefore nucleated blue green cells are visible
o Leukocytes may also be visible
Weight of the uterus
• The rat was dissected and the uterus extracted
o The uterus was weighed and expressed as a percentage of the rat weight
• Results:
o Oestrogen causes the uterus endometrium to proliferate and thus weight is greater
o Progesterone causes the endometrium to recede and thus the weight is less
o Untreated rat weight of uterus is dependant on their ovulatory stage
Pregnancy tests
• Two lines appear
o One line shows that the test has worked
o The other line shows if hCG is detected, often meaning the woman is pregnant
o Tests for presence, not level
• Uses monoclonal antibodies specific to the beta subunit of the hCG molecule
o Allows hCG to be detected independent of FSH, LH and TSH
o hCG is released by the blastocyst to keep the corpus luteum alive and producing progesterone so that
the endometrium is not lost and the pregnancy can continue
Expected results for Practical Class 4: Female Reproductive Hormones and their Effects.
Estrogen treatment encourages uterine growth and increases uterine fluid content and blood flow. The uterine weight
as a percentage of body weight should be the highest of the 3 groups of rats eg >0.5% of body weight. The uterine horns
look “water logged”. The vaginal smear contains mainly cornified epithelial cells (Fig 1).
Progesterone treatment ‐ inhibits uterine growth and slows down the effects of estrogen. The uterine weight as a
percentage of body weight is much lower than in the estrogen treated rats eg 0.1‐0.2%. The uterine horns look thin and
scrawny. The vaginal smear contains mainly nucleated epithelial cells (Fig 2).
Untreated rats
Results depend on the stage of the rat’s estrous cycle. See ntoes in prac book. Even if the rat is in Oestrus (as indicated by
mainly cornified epithelial cells) the uterine weight is usually not as high as that seen with exogenous estrogen treatment.
Fig 1 ‐ Vaginal smear from estrogen treated rat.
Fig 2. Vaginal smear from progesterone treated rat.
Prac 5: Isozyme Electrophoresis
Introduction
• separation of a mixture of charged molecules can be achieved by electrophoresis
o electrophoresis is the migration caused by an electric field
this migration depends on:
• the strength of the field
• the size/shape and net charge of the molecules
• the composition of the conducting buffer
o in a polyacrylamide gel system, the pH determines the charge of the molecules and therefore the
electrophoretic mobility
proteins are amphoteric (act as an acid and a base) molecules with ionised side chains that
contribute to net charge depending on the pH
Electrophoresis of LDH Isoenzymes
• Here, we are using natural gel electrophoresis to identify the isoenzyme pattern of serum proteins
o Serum is often used for diagnostic purposes since its composition reflects the activity of the body
and is not distorted by its own metabolic activity
• LDH (lactate dehydrogenases) occur in several different tissues
o Thus, by identifying the isozymes that are found in various tissues, we can analyse the serum of a
patient to see which LDHs are present, and thus which organs may have been effected
o Gel can be stained specifically for LDH or for all proteins, depending on the stain
• LDH is tetrameric (4 monomers) and has 2 types of monomers – H (heart) and M (muscle)
o In the serum, different isozymes of LDH are present
o Thus, if there is a lesion on a particular organ, the LDH pattern of the serum reflects this affliction
o Extent of increase in particular subunits is proportional to the size of the damaged area
Duration of increase can also be useful
• Isozymes are first separated by native PAGE (polyacrylamide gel electrophoresis)
o LDH bands are visualised using tetrazolium salt
o Gel already contains lactate, NAD+ and bitro blue tetrazolium
LDH catalyses the oxidation of lactate and formation of NADH
• Phenazine methosulphate promotes transfer of reducing equivalents from NADH to
the tetrazolium compound, NAD+ regenerating and the formation of formazan dye (a
purple colour)
o Purple bands appear where this activity is present
• Coomassie Blue procedure stains all proteins present
Experiment
• 5µl Gel Loading Buffer (blue) was mixed with 5µ l sample on a parafilm using a pipettor
o Samples were loaded into onto the gel
o Samples: 1. Liver, 2. Heart, 3. Muscle, 4. LDH control, 5. Serum, 6. Serum from Patient A, 7.
Serum from Patient B, 8. Liver, 9. Heart, 10. Muscle
o Samples: 1. Liver protein, 2. Heart protein, 3. Muscle protein
• The electrophoresis apparatus was connected to the power rand the current adjusted to 10mA/gel (approx.
200V)
o Dye runs faster than proteins
o Proteins separate
Power turned off, gel removed
• The LDH gel was stained using the LDH staining solution and kept in the dark + incubated in a 37o water
bath
• The Protein gel was stained with Coomassie Blue R-250 dye
o Both gels were fixed and examined
See picture
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Prac 6: Embryology – implantation to 8 weeks
Stage 7
• Week 3, days 15-17, 0.4mm
• Primitive streak ending in the primitive node
o Gastrulation as cells migrate from the epiblast forming the mesoderm
• Axial process extends rostrally from the primitive node eventually forming the notocord
•
Connecting stalk becomes the placenta + umbilical cord
Stage 8
• Week 3, days 17-19, 1-1.5mm
• Folding of the flat disk
o Folds ventrally, away from the amniotic side
o Folds some of the yolk sac inwards, some stays outside
• Rapid growth of the brain plate causes ventral folding of the brain disk
• Rapid growth of the caudal end also causes curvature
• Neural plate folds up to form neural tube/groove
o Thus a valley in the dorsal side
• Notochord continues to form in the mesoderm, at either end the buccopharyngeal and cloacal membranes
will form
Buccopharyngeal
region
Neural groove
Amniotic sac
Folding
Notocord (beneath surface)
Primitive Streak
Stage 9
• Week 3, days 19-21, 1.5-2.5mm, 1-3 somites
• Ectoderm is very thin and thus we can see the white mesoderm
• Beginning of segmentation of the mesoderm
o Somitogenesis – increases to 40 somites over the whole development
Ectoderm (epidermis), cuboidal epithelium
Lateral plate mesoderm
Neural tube (columnar epithelium)
Endoderm (cuboidal epithelium)
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Endoderm forms the epithelium and glands of the gut
Intermediate mesoderm forms the kidneys etc
Splanchnic mesoderm forms the smooth muscle wall and CT of the gut
Coelom forms a horseshoe shape, forms the 3 cavities: pericardial, pleural and peritoneal
Somite (forms axial body plan):
o Somitoceol
o Becomes dermomytome by ectoderm signals and the sclerotome
Dermomytome becomes the dermatome and myotome
• Dermatome becomes the dermis
• Myotome becomes the muscles
Sclerotome becomes the vertebrae and vertebral bodies
Coelom diagrams
Stage 10:
• Week 4, days 22-23, 2-3.5mm, 4-12 somites
• Neural groove starts to fuse
• Somites increase in number
• Heart bulge becomes visible
• Brain folds
• Grows in size compared to the yolk sac
• Continues to fold
Stage 11:
• Week 4, days 23-26, 2.5-4.5mm, 13-20 somites
• Neural tube closes at multiple points zipping up to openings at either end
o Rostral neuropore closes
Failure of neural pores to close = anencephaly/spina bifida
• Heart tube bends – has the most birth defects
o Tubes extend cranially and caudally
• Fetal membranes: chorionic sac becomes grows chorionic villi
o Thickest part become the placenta
Heart diagram
Stage 12:
• Week 4, days 26-30, 3-5mm, 21-29 somites
• Brain develops into 3 vesicles: forebrain, midbrain and hindbrain
• Placodes develop – specialisations of tissue: eg: lens and otic (ear bud)
• Pharyngeal arches
o Form beneath brain
o Large to small, rostro-caudal
o Form facial and neck structures
o Contain all 3 embryonic layers
• Stomodeum – primitive mouth
• Caudal pore closes
• Liver visible
Stage 13:
• Week 5, days 28-32, 4-6mm, 30 somites
• Limb buds develop
o Forelimb bud forms first
• Lens placode, otic placode lost beneath the surface
• Pharyngeal arches develop
Stage 14:
• Week 5, days 31-35, 5-7mm
• Limb buds develop, start below the heart
• Cervical sinus, arches growing over smaller ones
• Lens placode
• Oral cavity
Stage 15:
• Week 5, days 35-38, 7-9mm
• Nasal placode
• 4th ventricle cavity
• somites hidden because dermis thickens and hides inside organs
*remember to label all diagrams
Prac 7: Polymerase Chain Reaction (PCR)
Week 1:
PCR Genetic Screening
• PCR is a common molecular technique that is used in medical diagnostics
o PCR facilitates the selective amplification of specific gene sequences in a DNA sample
o Can be used screen DNA samples for deletions in the Duchenne’s Muscular Dystrophy gene and
determine a family pedigree
• Process involves thermal cycling and DNA synthesis from oligonucleotide primers
o 3 different temperatures/cycle
1. denaturation step which separates DNA strands (92-95o)
2. oligonucleotide primers anneal to the DNA template (50-65o)
3. oligonucleotide primers are extended by Taq DNA polymerase (72o)
o usually 25-35 cycles used in each experiment
oligonucleotides provide specificity for experiment and allow specification to a certain part
of the DNA chain
• these are synthesised chemically in an automatic machine
• PCR exponentially amplifies a DNA sequence
o Each cycle doubles the number of DNA strands
o This allows enough DNA to be produced such that the DNA shows up on a gel
Experimental
• 20µ l of DNA was pipetted into a 0.2ml PCR tube
• 4 µl of PCR mix was added
o this contains: oligonucleotide primer (forward and backward), dATP, dCTP, dTTP, dGTP,
(NH4)2SO4, Tris-HCl, MgCl2
• 1 µl of Taq was added
• Contents of tube were mixed by flicking
• Tubes were labelled and placed in a PCR machine for thermal cycling
Preparation of genomic DNA
• 2ml of aqueous DNA solution was added to a glass test tube
o 4ml of cold isopropanol was added such that the isopropanol ran down the sides of the test tube
forming a layer about the aqueous DNA solution
• the pipette was used to stir the layer
• long fibrous DNA strands were observed
Week 2:
Background
• DNA molecules will migrate at different rates by electrophoresis due to their different sizes
o Performed in an agarose gel with an alkaline buffer
Agarose used because the pore size can be varied depending on the agarose percentage
• Ethidium bromide used to detect the DNA
o This is a planar molecule that intercalates between the stacked base pairs and
produces luminescence under UV light
o DNA fragments are negatively charged and migrate to the positive electrode
Process
• Gel was poured into the gel pouring apparatus and the comb added at the negative end
• The gel was left to set
• The apparatus was placed in an electrophoresis tank, the comb removed and this end placed near the –ve
terminal and buffer was added
• Samples were added using 20µl of sample and 5µl of gel loading buffer
• The gel was run at 100V
Observations/results
• DMD – Duchenne’s muscular dystrophy is an X-linked disease
Prac 8: Fetal Membranes and Placenta
Dr Murray Smith
Introduction
• There are 3 layers for nutrients such as O2 to pass through to get from the mother to the fetus
o Trophoblast layer, connective tissue and the endothelial cells of the fetal blood vessels
• Placenta – the fetal/maternal unit
o Greatest piece of intensive care equipment
o 9 month lifecycle, growth, development, maturation and death
• Birth
o Lungs need to mature, surfactant to free the alveoli
Museum and wet specimens
• Placenta at term is up to 20cm in diameter and 3cm thick (500-600grams)
o Chord is up to 60cm and has a twisted nature
o Fetal side of the placenta is smooth (amniochorionic membrane)
o Maternal side is rough and has 10-40 raised cobblestone areas called maternal lobes of the placenta
(cotyledons)
Umbilical Cord
• 60cm long, can be coiled and knotted
• outside bathed by amniotic fluid
• covered in amnion made up of amniotic ectoderm (amniotic epithelium) and its underlying CT: lamina
propria
o older fetuses: amniotic epithelium is stratified, basal cells distinct and cuboidal, more superficial
become like stratified squamous
• 2 arteries, and a vein – arteries coil like vines around the vein
o arteries take deoxygenated blood to the placenta
stop flow at birth so that the fetus doesn’t exsanguinate
• cord stretches and blood vessels are squeezed by lateral muscles
o small coiling muscles contract due to temperature decrease
o umbilical vein takes oxygenated blood back
o fetal Hb has a greater affinity for O2
o endothelium is the main part of the tunica intima (inside the vessel)
tunica media is made up of spiral smooth muscle bundles (fasicles)
• pale extracellular CT separates the muscle fibres
• Mucoid connective tissue or Wharton’s jelly (the tunica adventitia) – substance of the umbilical
o Like mesenchyme
o Includes the lamina propria and amniotic epithelium
o contains spindle and stellate-shaped fibroblasts, collagen, an amorphous ground substance and fluid
spaces
o doesn’t contain nerves or lymphatic vessels
• Allantois
o A diverticulum (blind ended duct), extends from the hindgut
o Lined by tall, epithelial cells derived from the endoderm
o Has a distinct lamina propria
o Acts as an excretory tube connected to the fetus of the bladder
• Outside layer – the amnion made up of extraembryonic mesoderm and ectoderm
Tm
Ti
Al
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M
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Mc
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Am
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(V) Vein
(A) Artery
(Al) Allantois
(Mc) Mucoid connective tissue
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(Am) Amnion
(Ti) Tunica intima
(Tm) Tunica media
(M) circular smooth muscle
Ti
M
A
Tm
Early Placenta
• There are two sides to the specimen – the thicker maternal side and the thinner fetal side
• Fetal side:
o Chorion (the chorionic plate) made up of two types of tissue:
Extra-embryonic mesoderm (mesoblast)
• Contains tributaries of umbilical vessels
Trophoblast (ectoblast)
o Lacunae (maternal blood spaces)
o Tertiary chorionic villi (floating and anchoring)
Have a core of mesoblast, veins, a layer of trophoblastic epithelium
• Outer, darker layer – syncytiotrophoblast
• Deeper, pale-staining layer – cytotrophoblast (Langhan’s cells)
• Fetal erythrocytes – some are nucleated
Anchoring villi are attached to the maternal tissue by cytotrophoblast cells known as
cytotrophoblastic columns
• Maternal side:
o Patches of endometrium containing endometrial glands lined by simple columnar epithelium
o Decidual cells (derived from connective tissue cells of the endometrial stroma
o Pink Extracellular protein (fibrinoid)
o Smooth muscle layer – myometrium – darker areas containing high levels of glucose
Mid-term Placenta
• Fetal side – smoother side faces the amniotic fluid
o Amnion has started to fuse to the chorion to form the amniochorionic membrane (chorionic plate)
o Blood vessels are present containing nucleated fetal blood
o Villi are bilaminar
Cytotrophoblast and Syncytiotrophoblast
o Lacunae
• Maternal side
o Large pale staining cells – decidual cells
o Endometrial glands – the lumen is lined by simple columnar epithelium
Late Placenta
• Tertiary chorionic villi
o Syncytiotrophoblast layer, cytotrophoblast layer, core of blood vessels
Syncytiotrophoblasts may grow sprouts demonstrating active villous growth
• Decidua basalis – denser tissue on maternal side
o Composed of dark staining extracellular protein (fibrinoid)
o Decidual cells have a central pale nucleus and a basophilic cytoplasm
Placental barrier
• Made up of 5-6 layers depending on the presence of the cytotrophoblast layer
o Ie: between the lumen of the fetal capillary and the maternal blood, nutrients and gas pass through
Fetal capillary endothelium
Basement membrane
Extra-embryonic mesodermal tissue
Basement membrane of trophoblast
Cytotrophoblast layer
Syncytiotrophoblast layer
• Note: syncytiotrophoblasts secrete estrogen, progesterone and human chorionic gonadotrophin (maintains
the corpus luteum)
o Decidual cells develop from endometrial stroma cells upon implantation
Ventral to the embryo becomes the decidua basaslis
Around the embryo are the decidua capsularis
Others not in contact with the fetus are the decidua parietalis
Decidual cells produce hormones
E
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Sy
Cy
Cy
My
Sp
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(Sy) syncytiotrophoblasts
(Cy) cytotrophoblasts
(Ch) Chorionic plate
(V) Tertiary chorionic villi (anchoring)
(E) Nucleated erythrocytes
(Sb) Syncytiotrophoblast bleb
(Sp) Spiral artery
(My) Myometrium
(L) Lacunae
Sy
Em
Sy
G
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(Ch) Chorionic villi
(Sy) Syncytiotrophoblasts
(Cy) Cytotrophoblasts
(Em) Extraembryonic mesoderm
(D) decidua
(G) Gland
Ch
D
Cy
V
Cv
V
Em
L
•
•
•
•
•
(Cv) Chorionic villi
(V) blood vessel
(L) lacuna
(Sy) Syncytiotrophoblast
(Em) extraembryonic mesoderm
Sy
Prac 9: Concentration and response curves: actions of agonists and antagonists
Introduction
• Guinea-pig ileum is a smooth muscle preparation
o Muscle fibres contain many receptors that control muscle contraction
Eg: muscarinic receptors respond to acetylcholine
H2 receptors respond to histamine
• Magnitude of the response is thought to be proportional to the number of receptors occupied
• In the presence of a competitive antagonist the response of an agonist is decreased
o If the dose-response curve of the agonist and the antagonist present agonist were plotted
We have a parallel shift without a change in maximum response
Experimental
(Using a computer program to simulate the response of an isolated guinea pig ileum)
Preparation of guinea-pig ileum
• 2cm of guinea pig ileum is suspended in an organ bath
o one end is attached to a wire strut, the other to a cotton length
o cotton is attached to a transducer
• reservoir is filled with Krebs solution
o solution runs through to the organ bath using a clamp
• Carbogen is used for aeration of the tissue
o Temperature of the bath is kept at 32 degrees
• Drugs can be added to the bath by pipette and the response noted
o Bath can be washed out thoroughly to return the muscle to baseline activity
Dilution
• To work out the stock concentration required, c1v1 =c2v2 needs to be used
o Ie: stock concentration * volume to be added = final bath concentration * volume of bath
o = M * 0.1*10-3 = 10-7 * 10*10-3
o M = 10-5 M
Parts A and B
• Carbachol was added, and doses added in increasing concentrations with the response recorded (washing
between each)
• Histamine was added, and doses added in increasing concentrations with the response recorded (washing
between each)
o Results were recorded and %max response found for each
• Results were plotted on semi-log graph paper, %max response against concentration
• This EC50 was calculated
C and D
• Testing of atropine and mepyramine to see which inhibits carbachol and histamine
• It was found that atropine inhibits carbachol while mepyramine inhibits histamine
• What should happen:
o Carbachol in the presence of atropine, %max response vs concentration curve will have the same
max and undergo a parallel shift
o Same for histamine in the presence of mepyramine
Diagram of setup
Prac 10: Phenylketonuria (PKU)
Background
• Phenylketonuria is for the most part caused by a mutation in the gene for coding an enzyme: phenylalanine
hydroxylase
o This catalyses the conversion of phenylalanine to tyrosine
o Inactivation of this enzyme causes an accumulation of phenylalanine in body fluids
Blood levels are 20x higher than normal
Phenylpyruvate (a phenylketone) is excreted in the urine
• Initial tests involved filter paper that collected urine from nappies
o A reaction with FeCl3 turned the paper olive green in the presence of phenylpyruvate
Tests these days are based on a bacterial inhibition assay using blood from a prick test
• In NSW, PKU is detected by electrospray tandem mass spectrometry
o Accurate, rapid, requires little blood and can detect and quantify many metabolites simultatneously
Thus can include other diseases in the test
Worksheet
• PKU is an autosomal recessive disease
• It is caused by a deficiency in phenylalanine
o May also be caused by tetrahydrobioptein deficiency (an important co factor)
• Symptoms of the diseases are due to accumulation of the substrate, not a lack of the product
o Excess phenylalanine causes brain damage
• The disease is tested biochemically because there are many mutations of the genes
• Tandem mass spectrometry (MS/MS) separates molecules based on mass and structure (charge)
o Advantages:
Detects a large number of disorders with one test
Small amount of blood needed
Detects rare disorders
Fast, accurate and cheap
o Can detect 20 other disorders of amino acids and organic acid metabolism
• When a baby is found to have PKU, treatment involves elimination of phenylalanine from the diet (proteins)
until brain development has finished
o Some phenylalanine is required for function, so supplements are supplied as necessary
• Before a women with PKU gets pregnant, she must return to the lack of phenylalanine diet so that her baby
doesn’t contract the disease
• Diet coke contains phenylalanine as an artificial sugar and so people with PKU can’t have it
• In the nucleotide for the gene coding for phenylalanine hydroxylase, there are 13 exons and 79277
nucleotides
• Silent mutations are where the changing of an amino acid will not significantly change the function
o Eg: glycine to alanine is silent while cysteine to alanine isn’t
Experimental introduction
• Biological organisms depend on enzyme catalysed reactions for their existence
o Enzymes and isozymes are routinely used for diagnosis of diseases
the level of an enzyme’s activity can be measured
eg: after a heart attack, there is an increase in blood levels of enzymes important for repair
etc
• Certain temperatures and pH levels are vital for function
o This is because the enzymes function best in these ranges
• Here, acid phosphatase is used
o This catalyses p-nitrophenyl phosphate (p-NPP) into p-nitrophenol (p-NP)
Enzyme Assay
• The rate of an enzyme catalysed reaction is altered by pH, temperature, concentrations of substrate and
products etc
o Therefore, the initial rate of reaction is the most important measurement of activity
• Continuous assays require complex equipment but measure the entire product/substrate
formation/disappearance
o Stopped assay – reaction is allowed to run for 10 minutes and then the reaction stopped
Now, the substrates or products are measured and the data extrapolated
This assumes that the rate of reaction is linear
• Here p-NPP is catalysed into p-NP and stopped by NaOH
o Change can be measured by absorbance at 405nm since p-NP is a yellow coloured anion
Experiment
• Formula: A=ecl
o Absorbance = extinction coefficient (14000 l.Mol-1 cm-1) * concentration * path length (0.6cm)
• Part A: variation of reaction rate with time
o Results: the longer the time, the more substrate that is broken down and therefore the higher the
absorbance
• Part B: the effect of ph on the activity of acid phosphatase
o pH 5 – 5.5 is the optimum enzyme activity pH
• Part C: rate of reaction with increasing enzyme concentration
o Increased concentration of enzyme means a greater breakdown of substrate and therefore higher
absorbance
Prac 11: Embryology – stage 16 to fetus
Stage 16
• Week 6, 37-42 days, 8-11 mm
• Brain sections are visible (fore, mid, hind)
o 4th ventricle which develops into the cerebellum
visible
• Optic placode, nasal placode present
• Pharyngeal arches – cervical sinus site where the
pharyngeal arches are growing over inferior ones
• Rudimentary upper and lower limbs
o Paddle-like hand plate
o Move to the side of the body
Stage 17
• Week 6, days 42-44, 11-14mm
• Thickening of dermis
• 4th ventricle
• rays in upper limb buds (paddles)
• umbilical contains intestines
• head is large compared to the body
• nasal/optic placode present, otic lost to the surface
• auricular hillock – ridges on the 1st and 2nd pharyngeal
arches
• limb development over whole period:
o lateral to ventral movement + rotation
o upper rotates towards body, lower rotates away
Stage 18
• Week 7, days 44-48, 13-17mm
• Limb buds have grown and rotated
o Foot plate develops
• Solid ventral surface
• 4th ventricle is lost because of thickening of the dermis
• endochondral ossification (replacement of cartilage with
bone)
o afterwards, cartilage is left at the joints and at
epiphysial regions of long bones
Stage 19
• Week 7, days 48-51, 16-18mm
• Limbs rotate
• Apoptosis of webs on limbs
• Trunk elongates and straightens
• External ear develops (external acoustic meatus)
• Midgut still extends into the umbilicus
Stage 20
• Week 8, days 51-53, 18-22mm
• Vascular plexus develops
• Pigmentation of the eye
• Eyelid develops
Stage 21
• Week 8, days 53-54, 22-24mm
• External ear is placed at level of lower jaw
o Moves with facial/head development
o Abnormal in fetal alcohol syndrome
indicates neurological retardation
• limb/hand digits (webbed) development
Stage 22
• Vascular plexus
• upper/lower jaw (maxillary and mandibular)
• upper limb bud rotated
• toes
• ear more clear
Stage 23
• Week 8, days 56-60, 27-31mm
• External features developed
• Systems are underdeveloped:
o Brain/lungs/GIT/pulmonary system/cardiovascular
system
Early Fetus
• Week 10, 40mm
• Time of extensive growth in size and mass and differentiation of organ systems
• Brain continues to grow and develop, respiratory system differentiates, urogenital system differentiates into
male and female
o Endocrine and gastrointestinal tract begin to function
10 weeks
• Midgut still herniated
• Genital tubercle
12 weeks
• Basal bones of skull form by endochondral ossification
• Plate bones of skull form by intramembranous ossification
o Spaces in bones for teeth/gums
Fetal growth
• Huge growth in size
• 2 phases of growth
o 2nd trimester - length
o 3rd trimester – weight
• LBW = bad health outcomes
• Lungs – don’t mature until end of 3rd trimester
o Month 7, alveolar regions form
• Genital system
o Males – exterior gonads
• Gonadal descent 30 weeks
• Undescended testis, hyperspasia
• Nervous system
o Quite immature at birth
o Glial growth occurs post-natally
Defects
• Genetic and environmental
o Drugs, infections, temperature, maternal environment, radiation
Prac 12: Anatomical Framework of the Pelvis
Bones
Identify:
• Iliac crest
• tubercle of the iliac crest
• gluteal surface
• iliac surface
• auricular surface of the ilium
• anterior superior iliac spine
• anterior inferior iliac spine
• posterior superior iliac spine
• posterior inferior iliac spine
• acetabulum
• obturator foramen
• body of the pubis
• pubic tubercle
• pubic crest
• greater and lesser sciatic notches
Joints
Identify:
• lumbosacral joint
• sacroiliac joint
• pubic symphysis
• iliolumbar ligament
• sacroiliac ligament
• sacrotuberous ligament
• sacrospinous ligament
•
•
•
•
•
•
•
ischial spine
ischial
tuberosity
sacral
promontory
auricular surface
of the sacrum
pelvic sacral
foramina
ala of the
sacrum
ala of the
coccyx
Pelvis
• Diameters of the female pelvis at the inlet, medpelvis and outlet
• Sizes and shapes of female pelves
• Main features of the fetal head
• Differences between pelves of men and women
Muscles of the pelvic walls and floor
The perineal body, ischiorectal fossa
Pudendal nerve, internal pudendal artery, inferior rectal vessels, piriformis
Pelvic floor exercises
• Stop urination midstream
• Red light exercise
o Thus strengthening pelvic floor muscles so that tearing of muscles during parturition chance is lower
*************
Prac 13: Histology of the Male Reproductive Tract
Testis and rete testis
• Artery of the testes originates at the level of the kidney
o Spermatogenesis runs at 2-3 degrees lower than body
temperature
Pampiniform plexus is a plexus of veins that has a
heat exchange system
• Tunica albuginea (CT) runs into the core of the testis and
supports the rete testis
o All roads lead to Rome, septa lead to the mediastinum
• Spermatogenesis, sperm are produced in waves
o Mosaic of sperm development present in seminiferous
tubules
o Sertoli cells are stimulated by FSH and support the
spermatocytes
o Primary spermatocytes to secondary spermatocytes is
reduction of chromosomes
o Secondary to spermatid is division
o Spermatid to spermatozoa is spermiogenesis
• Elongated myoid cells are contractile cells
1. Tunica albuginea, 2 Septula testis, 3 Lobulus testis, 4 Mediastinum testis, 5
Tubuli seminiferi contorti, 6 Tubuli seminiferi recti, 7 Rete testis, 8 Ductuli
efferentes testis, 9 Ductus epididymidis, 10 initial part of deferent duct
Tu
L
M
St
R
Se
(Tu) Tunica albuginea
(M) Mediastinum
(St) Seminiferous tubules
(Se) Interlobular septa
(L) Lobule
(R) Rete testis
R
S2
S3
S4
S5
St
S1
L
My
B
Se
(R) Rete testis
(Se) Seminiferous tubule
(St) Sertoli cell
(L) Leydig cells
(S1) Spermatogonia
(S2) Primary spermatocyte
(S3) Secondary spermatocyte
(S4) Spermatid
(S5) Spermatozoa
(B) Basement membrane
(My) Elongated myoid cells
St
S4
S5
S1
S2
Se
S1
S4
S3
Se
S2
(Se) Seminiferous tubule
(St) Sertoli cell
(L) Leydig cells
(S1) Spermatogonia
(S2) Primary spermatocyte
(S3) Secondary spermatocyte
(S4) Spermatid
(S5) Spermatozoa
(My) Elongated myoid cells
My
L
A
V
(A) Artery
(V) Veins: pampiniform plexus
Efferent Ductules and epididymis
• Efferent ductules have a cog wheel arrangement (columnar epithelium)
• Epididymis is formed by the mesonephric duct
o Develops under influence of testosterone
o Pseudo-stratified columnar epithelium with stereocilia
Giant microvilli, not motile, not cilia
E
D
(E) Epididymis
(D) Ductulus efferens
E
Ep
(E) Efferent ductules (ductulus efferens)
(Ep) Epididymis
Vas deferens
• Longitudinal circular and longitudinal band of muscle
Lo
C
V
L
M
E
L
(V) Vas deferens (ductus deferens)
(L) Lamina propria
(E) Pseudostratified columnar epithelium
(M) Muscle
(C) Circular muscle
(Lo) Longitudinal muscle
V
Seminal Vesicle
• Produces bulk of seminal fluid (fructose, prostaglandins, fibrinogen)
o Ejaculation is sympathetic but controlled by the parasympathetic
M
B
M
E
E
Sl
(Sl) Seminal vesicle lobules
(M) Muscle
(E) Columnar epithelium
(B) Blood vessels and nerves etc
Prostate
• Corpora amylacea may be present
o Concretions, concretionary bodies
o Condensations of salts, form a concentric ring pattern
o Characteristic of age
• Hypertrophy of mucosal and submucosal glands of prostate closes down prostatic urethra
o Prostate cancer cancer of the main glands
o Prostate is encapsulated, thus cancer doesn’t spread easily
• PSA – prostate specific antigen, raised levels may indicate cancer
Sm
Se
G
(Sm) Smooth muscle layers
(Se) Fibromuscular septa
(G) Gland
Se
Sm
U
M
Main
Sm
U
M
(U) Urethra
(M) Mucosa
(Sm) Submucosa
(Main) Main part
Penis
• Penis is the organ of urination and copulation
o Littre glands create a mucus lining to the penile urethra allowing safe sperm passage
• Corpus spongiosum has small venous sinuses because otherwise the urethra would close down
N
Cs
U
Ta
E1
Cc
Cc
E
B
T
Cc
Ta
N
Bv
B
Bv
(Cs) Corpus spongiosum
(N) Nerves
(Dv) Dorsal veins
(Cc) Corpus cavernosum
(E) Stratified squamous keratinising epithelium
(Ta) Tunica albuginea
(U) Urethra
(B) Blood sinuses
(L) Littre mucus gland
(T) Trabeculae
(Bv) Blood vessels
(E1) Stratified squamous epithelium
U
E1
Cs
L
B
E1
U
B
Ta
Dv
B
E
Prac 14: Organisms in Pelvic Inflammatory Disease
Background
• PID is a clinical syndrome associated with infection of pelvic organs
o Can be uni/poly-microbial
o Majority of infections are caused by Neisseria gonorrhoea and Chlamydia trachomatis or both
STIs, initially infect the vagina or urethra
• Multiply rapidly and spread to the cervix
• Cause local damage an an inflammatory response
Majority of cases are asymptomatic
• Unrecognised infection, therefore further transmission
• 30% of women, spreads further up genital tract causing PID
often symptoms are mild or non-specific (eg: abnormal bleeding, dysparenunia, vaginal
discharge)
• difficulty of diagnosis and potential for damage to reproductive health means often
treat when suspicious of the STIs
Diagnosis of Neisseria gonorrhoea and Chlamydia trachomatis
Gonorrhoea
• Gram negative diplococcus
o Gram staining will show pink cocci inside polymorphonuclear cells
• Requires an enriched media for growth
o Chocolate blood agar – heated red blood cells that become brown
Growth factors (X and V) are released
Useful for organisms such as Neisseria gonorrhoea and Haemophilus influenzae
o Gonococcal medium – nutrients like that of CBA but also antibiotics
Make the culture selective for Neisseria species
Used for isolation of N. gonorrhoeae from genital tract swabs
Contaminants like e. coli can often mask the presence of N gonorrhoea, and thus antibiotics
prevent these elements
• Oxidase test
o Detects oxidative enzymes involved in oxidative phosphorylation
o Growth is removed from medium with a platinum loop
Thus, placed on a filter paper streaked with oxidase reagent (tetramethyl—p-phenylene
diamine)
Purple colour development in 20 seconds indicates a positive test
• Carbohydrate fermentation test
o Bacteria can ferment a wide range of carbohydrates
o Fermentation pattern is characteristic of various bacteria
o Process:
A tube of carbohydrate broth is inoculated
• Contains a nutrient broth and 1% of the particular carbohydrate and an indicator
• Indicator reveals production of acid changing from purple to yellow
o Carbohydrate fermentation results in acid or acid and gas
A Durham tube is inverted in the tube to collect any gas produced
o Gonorrhoea – ferments glucose, but not maltose or sucrose
• Molecular tests
o PCR using urine
o Useful for mass screening programs in high-risk populations
o Information doesn’t provide information on antibiotic sensitivity of the organism
Often not a very sensitive test
Chlamydia
• Tissue culture
o Chlamydia is an obligate intracellular parasite
o Gold standard for diagnosis of C. trachomatis
o Slow
Requires specialised techniques since organism can only be grown in cell culture
Labour intensive
o Sensitivity may not be very high
Some swabs can be toxic
Transport medium may not be right
Freeze thaw cycles and other factors can influence the specimen
• ELISA, Direct fluorescent antibody staining and genetic probes
o ELISA – can’t detect whether infection is present or past
o Fluorescent antibody staining of the specimen using monoclonal antibodies
o Genetic probe that hybridises to the nucleic acid of Chlamydia trachomatis
Sensitivity is less than that of the tissue culture
• Molecular techniques
o PCR – sensitivity also higher than tissue culture
o Uses a urine sample
Can also use a cervical swab
o Fast, easy, can diagnose gonococcal infection at the same time
Smears and cultures from patients
• Microscopes with gram stains
o A: normal vaginal flora – pic
Gram positive rods (purple)
Yeast – candida?
Gram negative rods
Gram positive cocci – strep?staph?
o B: Urethral discharge from male with gonococcal urethritis – pic Polymorphonuclear cells – multilobed nuclei
Extra and intra cellular diplococci
o C: cervical swab of female patient with gonococcal infection
PMNs
Diplococci + normal flora present
• Figures:
o 1: lifecycle of Chlamydia trachomatis
picture:
o 2: Picture of Chlamydia infecting epithelial cells
•
Culture plates
o Plate 1: gono. male, urethra, gonorrhoea
Pale yellow colonies of gonorrhoea
o Plate 2: CBA. Female, gonorrhoea
Pale yellow colonies of gonorrhoea and white normal flora
o Plate 3: gono. Female, Chlamydia
Nothing
Should see normal flora
o Plate 4: gono. Male, Chlamydia
Nothing
Case studies
Case 1: 23 woman, contacted by contact tracing, unprotected sex, no symptoms, white vaginal discharge
• Specimens
o Cervical swab
o Vaginal discharge sample
o Urine sample
• Tests
o PCR
o Gram stain
o Culture using gono-medium
• PCR results + culture results = Chlamydia infection
• This infection can lead to PID and thus infertility
o Also, can be asymptomatic
• Treatment:
o Antibiotics
o Education + barrier contraception
o Contact tracing of sexual partners + treat
o Notification of health department
Case 2: Peter 19 male, new girlfriend, unprotected sex, no symptoms
• Gram stain plates + other results:
o Gram negative stain + growth on plates
o PCR results
= gonorrhoea
• Management of case:
o Antibiotics
o Notification of health department
o Contact tracing
o Protection
Case 3: Mary 16 school girl, emergency room, 4 days of crampy abdominal pain + post-coital bleeding, no
nausea/vomiting, pain increased, sexually active, one male partner in 3 months, ‘uses condoms’
High temperature, tenderness of abdomen and cervical motion tenderness
• Differential diagnosis – typical presentation of PID
• Most likely causes – Chlamydia trachomatis and/or Neisseria gonorrhoea – unprotected sex
• Other organisms:
o Endogenous normal microflora
enteric organisms (of the intestines), anaerobic bacteria, clostridiums
• Tests:
o Gonococcus culture
o Oxidase test
o Tissue culture
o Carbohydrate fermentation test
o PCR
• Results:
o Gonorrhoea cultured
o Carbohydrate test indicates gonorrhoea (-ve is purple, +ve is yellow)
o Oxidase test is positive
o Gram negative stain present
o PCR indicates Chlamydia and gonorrhoea
• Consequences:
o Infertility
o Chronic pelvic pain
o Physical/psychosocial damage
o Ectopic pregnancies
Prac 15: Disorders associated with infertility in females
Endometriosis of the ovary
• Case 2:
o 32 year old, 4 year history of infertility
o 6 year history of lower abdominal pain pre-menstrually
o menstrual pain
o pain during intercourse
o tender, fixed adnexal mass was found
o laparoscopy revealed an enlarged right ovary and was adherent to the fallopian tube and broad
ligament
o dark brown cystic areas seen on the ovary, posterior uterus and the pouch of Douglas
• Clinical:
o Correct age group
o Infertility
o Lower abdominal pain
o Menstrual pain
o Coitus pain
o Palpation pain
Adnexal – appendages of the uterus
Inflammation of appendages
o Tissue of uterus lining grows somewhere else
o Torsion of loops of bowel volvulus
o Thus correlate with endometriosis
• Slide:
o Light pink stromal cells of the ovary
o dark purple cells of the endometrium present on the ovary
blood vessels and glands
o blood, necrotic tissue
o haemorrhage
o after menstruation
o prominent corpus albicans
Polycystic ovary (Stein-Leventhal syndrome)
• Case 4:
o 28 year old, infertility
o irregular periods
o hirsutism – hair growth on the chin, acne and oily skin, suggesting excess androgen
o serum testosterone and cortisol were assessed
testosterone was at the upper limit of normal
normal cortisol levels
o ultrasound described typical “pearl necklace” pattern
• Clinical:
o Hair growth, acne, oily skin, irregular menstrual flow
o Usually common in 10% of all reproductive women
• Diagnosis:
o History – general symptoms of polycystic ovary
o Ultrasound showed the “pearl necklace” pattern typical of polycystic ovary
o Ovary would also present enlarged
• Cortisol test
o Used to rule out adrenal hypoplasia: Cushing’s syndrome
o >5 cysts = abnormal
o androgens from in the theca cells of the follicles
• prognosis
o increased risk of type 2 diabetes
o obesity
o endometrial hyperplasia/carcinoma
o adverse pregnancy: hypertension, preterm/post-term birth, gestational diabetes
• treatment:
o treat symptoms
diet therapy to treat diabetes and weight, can be fertile
medication to regulate hormonal imbalance
• Slide:
o Chronic inflammation
o 2 large cysts
o follicles arrested in the antral stage
o thickening of the cortical region
o lack of appropriately developing follicles
o lots of blood vessels
Chronic Salpingitis
• Case 1:
o 23 year old, 3 years of infertility
o recurrent vaginal discharge
o occasional pelvic pain
o adnexal tenderness
o multiple sexual partners
o history of STDs + antibiotics
• Clinical:
o PID
Salpingitis
o Bacterial infection CT or GN invades upper genital tract
o Acute on chronic
o Abdominal pain, etc are all indicative of upper genital tract infection: salpingitis
• Symptoms:
o Fever, pain on urination, nausea, vomiting, dysmenuria, spotting
• Tests:
o Laparoscopy
o PCR
o Oxidase
o Carbohydrate
o Culture, gonomedium/CBA
o Gram stain
• Treatment:
o Antibiotics, contact tracing, notification, education
• Prognosis
o Infertility
o Ectopic pregnancy – rupture/haemorrhage
• Slide:
o Ulceration
o Neutrophils in clumps + pus
o Smaller lumen
o Dilated blood vessels
o Hypertrophy of muscles
Leiomyoma of the uterus
• Case 3:
o 31 yrs
o 10 year history of headaches
o bloating, crampy pain, irritable the week before her menstrual periods
o abdominal examination suggested uterine enlargement
• Clinical:
o Headaches due to increase in GnRH
o Uterine enlargement
o Leiomyoma can occur in 3 places: subserosal, intramural, submucosal
Often more than one
o Occurs in 30-50% of women in reproductive life
o Size compresses nerves and blood vessels + spze
• Diagnosed by transabdominal/transvaginal ultrasound
• Treatment
o Removal of fibroids
o Hormonal therapy, GnRH analogues to treat symptoms
o Severe lesions hysterectomy
• Slide:
o Subserosal, in capsulated, non-invasive
o Areas of fibrovascular tissue
o Neopoasm?
o Cytological features indicate a benign tumour
o Interlacing bundles of smooth muscle
Prac 16: Male Reproductive System
Testes
Diagrams
Prostate, seminal vesicle and ejaculatory ducts
Penis
Erection and Ejaculation
Blood/nerve/lymph supply
Cases