EMBRYOLOGY

LP1 – YEAR 1Asist.Univ. Dr. Tulin Raluca

Covered Topics L.p.II. Primordial germ cells, spermatogensis, spermiogenesis - LECTUREL.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 – LECTUREL.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:

• 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

• 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 capacityTESTOSTERONE - LH –

secondary sexual characteristics, sex drive

Aplicatii cliniceMeasuring FSH + INHIBINA- determines if spermatogenesis is occurring (SERTOLI) INHIBINA – barometer of spermatogenesis (inhibits FSH and hypothalamic GnRH)Measruing LH + Testosterone – for sexual function (LEYDIG)

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

• 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: mlColor: opalescent pH: Lichefiere: completeTimp de lichefiere: 15minViscosity: normalConcentration: mil/mlTotal nr. of spz in sample: milMotility a+b: %Motility a+b+c: %

≥ 1,5mlOpalescent7,2-8,0Complete: distinct drops≤20 minNormal≥ 15 x 106 /ml or≥ 39 x 106 in sample≥32%≥40%

Results of analysis Normal valuesNormal appearance: %Abnormal appearance: %

Leukocites: 1%

> 4% normal appearance (based on WHO 2010 criteria) 1% leukocytes

Morphology

Halosperm is a diagnostic test based on the sperm chromatin dispersion technique of spermatozoa nuclei

Spermatozoa DNA fragmentation (SDF) – 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.

• 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

• 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• 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 (24-36 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 1-2 days before and after ovulation

• Contraception (Calendar method)• ART techniques (intrauterine insemination IUI-

following 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

Methods of contraception1. 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

3. 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 0,75 mg (1 tablet) repeat dose after 12-16 hours

Estro-progestativ Microgynon 4 tablets, repeat dose after 12 hours

Tabelul de mai jos furnizează informaţii detaliate privind formularea şi dozele pilulelor utilizate in prezent în contracepţia de urgenţă : 

Contraceptive (hormone) patch/Vaginal ringOrtho-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