EMBRYOLOGY
LP1 – YEAR 1
Asist.Univ. Dr. Tulin Raluca
Covered Topics
L.p.I
I. Primordial germ cells, spermatogensis, spermiogenesis - LECTURE
L.P. 1. Regulating factors in spermatogenesis: FSH, LH
2. Leydig cells and Sertolig cells.
3. Blood-testis barrier.
4. Morphological characteristics of spermatozoa- abnormalities.
5. Clinical spermatogenetic tests: MAR test, halosperm, viability test,
spermiogram- definition, clinical use significance.
II. Ovogenesis, ovulation. Corpus luteum. Fertilization – LECTURE
L.P. 1. Follicular cycle
2. Uterine cycle (endometrial)
3. Methods for determining ovuliation- underlying principes behind
ovulation tests.
Microscope slides:
1. Ovary cross-section;
2. Testicle cross-section;
3. Morula. Blastula.
1. Celule germinale primordiale
• Endodermal cells
(originate from wall
of yolk sac)
• Formed during week
2, migrate (W4-W6)
to urogenital ridge
(W6)
• Essential for
gametogenesis- will
form ovocytes and
spermatozoa
Clinical implications
• Germinal cells may become stuck anywhere
along their migratory pathway where they
give rise to neoplasms down the median
sagittal line of the body:
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Pineal region 6%,
mediastinum 7%,
retroperitoneum 4%,
sacro-coccygeal region 42%,
ovaries 24%,
testicles 9%,
Other locations 8%
AFP and HCG are measured during clinical evaluation of the
disease
2. Spermatogenesis
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The formation process of spermatozoar
Begins at puberty
Continues throughout adult life
One spermatogenetic cycle takes 62 +/- 4 days
Takes pace in the seminiferous tubules in the
testicles
• Occurs through mitosis and meiosis
• Ends with spermiogenesis
Spermatogeneza
Testicular cells
• SERTOLI cells (sustentacular, supportive)
– Within seminiferous tubules
• ABP (androgen binding protein) indirectly stimulates
spermatogenesis by maintaining a high concentration of
testosterone around spermatogonia
• Inhibin – inhibits FSH and maintains the concentration of
spermatozoa
• LEYDIG cells (interstitial)
– Outside seminiferous tubules
• Secrete testosterone (levels are elevated during M3-M6 of fetal
life, during first year of infancy and throughout adult life post
puberty )
• Seminal cell line (spermatogonia---spermatozoa)
Hormonal regulation of
spermatogenesis
SPERMATOGENESIS – FSH –
reproductive capacity
TESTOSTERONE - LH –
secondary sexual
characteristics, sex drive
Aplicatii clinice
Measuring FSH + INHIBINA- determines if
spermatogenesis is occurring (SERTOLI)
INHIBINA – barometer of spermatogenesis (inhibits
FSH and hypothalamic GnRH)
Measruing LH + Testosterone – for sexual function
3. Blood-testes barrier
• Sertoli cells divide the seminiferous tubules into 2
compartments:
• BASAL – between basement membrane and Sertoli
intercellular junctions (encloses spermatogonia, primary
spermatocytes)
• LUMINAL – superior to Sertoli intercellular junctions
(blood-testes barrier)
• The main purpose of this barrier is to protect haploid cells
(n=23) against immunological recognition as non-self and
subsequent antibody-mediated attack.
Seminiferous tubules-adult testicle
Spermiogenesis
Spermiogenesis
• Last phase of spermatogenesis
• DOES NOT involve further cell division
• Spermatid (23 chromosomes) becomes
Spermatozoa (23 chromosomes)
• HEAD (GENETIC)- DNA + acrosome (hydrolytic enzymes)
• MIDDLE PIECE (METABOLIC) - mitochondria
• TAIL (ENGINE)
Spermiogram
• Is part of a male fertility analysis which checks the quantity and quality of
sperm
• Rezults
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NORMOSPERMIA – normal values
polyspermia = an above normal concentration of spermatozoa
oligospermia = a lower than normal concentration of spermatozoa
- hypospermia = semen volume < 1,5 mL
- hyperspermia = semen volume > 5 mL
- aspermia = an absence of semen
-azoospermia = an absence of spermatozoa
- piospermia = the presence of leukoctyes in semen
- hematospermia = the presence of RBCs in semen
- asthenozoospermia = motility < 40%
- teratozoospermia = > 96% of spermatozoa are morphologically abnormal
- necrozoospermia = spermatozoa are dead
- oligoasthenozoospermia = mobile spermatozoa < 15 mil./mL and motility< 40%
Spermiogram results
Results of analysis
Normal values
Volume: ml
Color: opalescent
pH:
Lichefiere: complete
Timp de lichefiere: 15min
Viscosity: normal
Concentration: mil/ml
Total nr. of spz in sample: mil
Motility a+b: %
Motility a+b+c: %
≥ 1,5ml
Opalescent
7,2-8,0
Complete: distinct drops
≤20 min
Normal
≥ 15 x 106 /ml or
≥ 39 x 106 in sample
≥32%
≥40%
Morphology
Results of analysis
Normal appearance: %
Abnormal appearance: %
Normal values
> 4% normal appearance
(based on WHO 2010 criteria)
Leukocites: 1%
1% leukocytes
Halosperm is a diagnostic test based on the sperm
chromatin dispersion technique of spermatozoa nuclei
Spermatozoa DNA fragmentation (SDF)
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Factors which may influence spermatozoa DNA fragmentaion: some medication with toxic effects,
fever, smoking, drugs, infectious diseases, age, varicocele, long-term abstinence.
Underlying basis involves differential DNA decondensation which results in a
characteristic halo around spermatozoal head with intact DNA and no halo around
spermatozoa with fragmented
Target population
• Infertile males with varicocele have a high percentage of spermatozoa with
fragmented DNA. The Halosperm test measures the degree of severity of this
condition
• In infections (especially with Chlamydia trachomatis and Mycoplasma), Halosperm
test allows doctors to monitor the effectiveness of antibiotic treatment and helps
them pick healthiest sample for assisted reproduction procedures;
Interpretation– reference values.
• SDF<30%  within normal range;
• SDF>30%  high rate of occurrence of fragmented DNA
MAR test
(Mixed Antiglobulin Reaction)
The direct MAR test detects the presence of antibodies bound to spermatozoa. A solution of
spermatozoa is mixed with a solution of latex beads bound to monoclonal anti- human IgA or
to monoclonal anti- human IgG.
Antisperm antibodies The presence of antibodies bound to membrane antigens on spermatozoa
is characteristic of immunologically derived infertility. This occurs in approximately 8% of
infertile males.
The limitations of the procedure. The direct MAR test can only be carried out on mobile
spermatozoa. Samples containing a low concentration of or weakly motile spermatozoa may
result in false negatives.
Interpretation- normal values.
When anti-sperm antibodies are present, mobile spermatozoa will bind to the latex beads in an
agglutination reaction. The resulting complexes will be as large as the magnitude of the
immunological reaction.
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Reference values:
• < 10% - negative;
• 10 – 39% - borderline (suspicion of immunologically derived infertility);
• > 40% - pozitive (high probability of immunologically derived infertility).
Clinical use
• Helps in choosing the most appropriate
method of assisted reproduction
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Azoospermia --- donor
OligoAsthenoTeratozoospermia --- classic IVF or ICSI
Normospermia ---Insemination (IUI)
Positive MAR (above 40%)---ICSI
Positive Halosperm (SDF above 30%)---ICSI or IVF
Ovogenesis
• Begins during intrauterine stage of life
• Is temporarily paused before birth
• Resumes at puberty
• BABY IS BORN WITH ALL THE OVOCYTES SHE WILL
EVER HAVE (1.000.000). Of these, 400.000 persist
until puberty and only 400 will make it to the
stage of secondary ovocyte (released during
ovulation). Ova are the result of fertilization.
The number of ova is equal to the number of
pregnancies.
Ovarian cycle
• Includes
– Follicular cycle (follicular phase)
– Ovulation
– Formation and development of corpus luteum si evolutia
corpului galben (luteal phase)
• On average, is 28 days long
• Begins at puberty
• Ends at menopause (50-52 years) = depletion of
ovarian store
• Remember! ----- The OVARIAN cycle is not the same
thing as the MENSTRUAL cycle.
Ovarian cycle vs. Uterine cycle
Clinical implications- follicular cycle
• Detecting ovarian stores (important in fertility practice)
– AMH (anti-Mullerian hormone)
• At the onset of puberty, AMH is produced by stratum granulosa cells of the growing
follicle. It is not produced by primordial follicles or mature Graffian follicles, which are
under the direct regulatory influence of FSH.
• AMH is expressed exclusively by stratum granulosua cells in unselected primary and
secondary follicles, this compound is the perfect marker for estimating ovarian reserves.
• As age progresses, AMH levels gradually decrease
• Useful in monitoring patient’s response in assisted reproductive technology (ART)
• In males, levels of AMH are useful in distinguishing between
cryptoorchidism (Sertoli cells) and anorchia. In infants that present a genital
ambiguity, this marker serves in determination of the presence and proper
functioning of the testicles.
• Levels are normal throughout pregnancy
– INHIBIN B
• (produced by granulosa cells)- is measured during the follicular phase
and indicates the number of ovocytes that could be stimulated following
ART therapy
• Levels vary throughout ovarian cycle
• Ovarian cycle
• Follicular phase
• Ovulation
• Luteal phase
• Body temperature
• Gonadotropins (LH,
FSH)
• Sexual hormones
(E2, Progesterone)
• Uterine cycle
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Menstrual phase
Proliferative phase
Secretory phase
+/-Premenstrual
phase
Determination of ovulation
• Considering the fact that the length of an ovarian cycle is
28 days, ovulation should theoretically occur on day 14 (2436 hours after the LH peak, which triggers ovulation)
• Clinical signs
– Rise in body temperature
– Changes in cervical mucus (becomes more elastic and slipper to
ensure safe transportation of the sperm) that make it resemble
egg whites
• If an ovarian cycle is longer/shorter, ovulation is calculated
based on the luteal phase (14 days). This is known as the
“calendar method”
• Ex- ovarian cycle of 35 days – ovulation takes place on 35-14 = day 21
Clinical implications- ovulation
• Planning a pregnancy- most fertile period is 12 days before and after ovulation
• Contraception (Calendar method)
• ART techniques (intrauterine insemination IUIfollowing a natural cycle, without preceding
ovarian stimulation)
Ovulation tests
• Principle = measuring LH levels in urine
– LH reaches a peak
• Positive test
– If the test line (LH) is equal to or greater than the control line,
this indicate the LH peak and signals that ovulation will occur in
the next 24-28 hours
– MediTest, Oview, Minut, Ferlidona
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Methods of contraception
1.
HORMONAL
1.
Birth control pill (E2+P or only P) –INHIBIT OVULATION, cervical mucous becomes
impermeable, inhibits proliferation of uterine lining and thus prevents ovum implantation
2.
Hormone patch (5/5cm) –applied 3weeks/month with a 1 week break (E2+P)
3.
Vaginal ring – inserted 3weeks/month (by user) (E2+P)
4.
Injection every 3 months (high concentration of P) –parenteral administration, acts on
cervical mucous and ovulation
5.
Implant (high concentration of P) – inserted by doctor, in the arm- kept for 3 years
6.
Intrauterine device- inserted by doctor, kept for 5 years
2.
NONHORMONAL
1.
Intrauterine device
2.
Spermicides
3.
Condoms
4.
Diafragms
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NATURAL
1.
Body temperature- rises by 0.3-0.5 degrees after ovulation and remains elevated until the
menstrual phase
2.
Monitor change in cervical mucosa
4.
PERMANENT
1.
Tubal ligation/vasectomy
5.
EMERGENCY
1.
Pill with high concentration of P (Postinor)
2.
Pill with high concentration of E+P
3.
IUD inserted after 5 days max.
Emergency contraception
Nume
Commercial name
Recommended dose
Levonorgestrel
Postinor 2
Microgynon
0,75 mg (1 tablet) repeat dose after 12-16 hours
Estro-progestativ
4 tablets, repeat dose after 12 hours
Contraceptive (hormone)
patch/Vaginal ring
Ortho-Evra
Contraceptive (hormone) implant- 3
months
Intrauterine device
Assisted reproduction technology
(ART)
• Artificial insemination (introduction of semen directly into
uterine cavity) simple, inexpensive procedure that can be
done monthly
– May follow natural ovarian and uterine cycle
– May require ovarian stimulation
• In vitro fertilization (IVF) –ffertilization takes place outside
the woman’s body
– With ovarian stimulation + piercing of ovaries
– Incubated with prepared semen
– Resulting embryo )morula or blastocyst is implanted into uterine
cavity
• ICSI (intracytoplasmic sperm injection)
– Uses micromanipulation devices
– In cases where spermatozoa count is low or they are extracted
through biopsy
History of ART
• 1790 – birth of first baby after artificial
insemination using husband’s sperm in U.K.
• 1890 – birth of first baby after artificial
insemination with sperm from donor
• 1978 – Louise Brown – first baby conceived in a
test tube p
• 1984- first birth of frozen embryos
• 1992 – first birth after ICSI
• 1996 – first IVF baby in Romania