infertility and sperm analysis

INFERTILITY AND SEMEN ANALYSIS

Dr VikashJR(Pathology)

IMS,BHU

Infertility-Apparent faliure of a couple to conceive

Sterility-Absolute inability to conceive.

If a couple fails to achieve pregnancy after 1 year of unprotected and regular intercourse , it is an indication to investigate the couple

Type

Primary- If conception has never occurred. Secondary

It may be physiological

Before puberty and after menopause

Over age of 40 year fertility reduced and increase risk for chromosomally and other malformed foetus.

Symptoms

Woman: abnormal menstrual periods Man: hormonal problems (changes in hair

growth or sexual function)

Infertility is rarely absolute so the term sub-fertility may be more appropriate

Spermatogonia--Mitosis--spermatocytes—Meiosis I-Haploid Secondary Spermatocytes---Meiosis II-Spermatid ( 74 days) Spermiogenesis:

differentiation of the round spermatid into a spermatozoon

This is the process in which sperm morphology is largely determined

What the spermatozoon looks like

The human sperm cell is about 70 µm long

The nucleus is in the head – contains the 23 chromosomes

It is the head which binds to the egg at fertilization

Midpiece: the energy for motility is generated

Tail: motility – the beat is initiated just behind the midpiece, and then propagated along the tail

BACKGROUND INFORMATION

At puberty there are 300,000 primordial follicles

Dominant follicle produces oestradiol which leads to LH surge

Ovulation occurs 24-36 hours later The fertilization life span of the ovum is

24-36 hours The receptivity of the endometrium is

days 16-19 of a 28 day cycle

Hypothalmic-Pitutary-Gonadal Axis

Causes of male infertility

Normal Semniferous tubule

Tubular atrophy

Causes of female infertility

Causes of female infertility Dyspareunia and Vaginal Causes Congenital Defect in genital tract-Absent or septate

vagina,Hypoplasia Infection- Chlamydia Cervicitis Cervical factor-cervical mucous Uterine causes-Hypoplasia,Malformed uterus and

incomplete os Tubal factor-Salpingitis,Gonorrhoea ir chlamydial

infection Ovaries-PCOD,LFD Peritoneal causes-Pelvic endometriosis Chronically ill health Hormonal pitutary dysfuction-

Hyperprolactinoma,Hypothalmic disease

Risk factors

Age Tobacco smoking Alcohol use Being overweight Too much exercise Caffeine intake

Tests and diagnosisTests for men General physical examination Semen analysis Hormone testing Transrectal and scrotal ultrasoundTests for women Ovulation testing Hysterosalpingography Laparoscopy Hormone testing Ovarian reserve testing Genetic testing Pelvic ultrasound

Tests History Examination Specific test- Hysterosalpingography(HSG) Laproscopic chromotubationSonosalpingography(SSG)Hysteroscopy and falloscopyAmpullary and fimbrial salpingoscopyEndometrial BiopsyFern TestUltrasoundHormonal Test

Hysterosalpingography(HSG)

Hydrosalpinx

FERN Test

Semen

A mixture of seminal plasma and cells Seminal plasma contains:

Prostatic fluid (~30% of the volume) Epididymal plasma (~5% of the volume) Seminal vesicle fluid (the remainder of the ejaculate)

The cells are: Spermatozoa Germ line cells Leukocytes of various types Bacteria Epithelial cells Occasional red cells

Semen Analysis

• There are several macroscopic evaluations which give useful diagnostic information about the sample:

–Appearance

–Odour

–Liquefaction

–Volume

–Viscosity

–pH

SEMEN ANALYSIS• pH is important because sperm die at pH < 6.9

• The pH of liquefied semen is normally determined using test strips (we use EM Science ColorpHast type, pH 6.5–10.0)

• We usually measure pH after volume and viscosity – by touching the “emptied” volumetric pipette to the test strip

• The normal pH range is 7.2–8.4• Inflammatory disorders of the accessory glands can take the pH outside of this range

Semen Analysis-Assesement

• The characteristics assessed are:– Motility– Sperm aggregation (random clumping) – “some” is normal,

but large clumps (each with hundreds of sperm) is abnormal

– Spermagglutination (between specific sites) – could suggest the presence of antisperm antibodies.

– Round cells: should be <1 per 40× field (~ 1 million/ml). If more abundant, a leukocyte test should be run

– Epithelial cells: usually present in small numbers– Erythrocytes: should not be present– Debris: particles smaller than sperm head, may be plentiful– Bacteria and protozoa: presence indicates infection

Semen Analysis-Motility Assessment

• % motile = the proportion of sperm with tail movement

• Progression rating = the grade of progression shown by the majority of the sperm: this can be from 0 (all immotile) to 4 (all with rapid progression); or from a (rapid progression) to d (all immotile)

• Differential motility count = proportion of sperm in each of 4 motility classes (rapid progressive; slow progressive; non-progressive; immotile

Motility Asses. Contd.

• Differential motility classification is based on the distance swum over time:

– Rapid progressive: > 25 µm/s

– Slow progressive: 5 – 25 µm/s

– Non-progressive: < 5 µm/s

– Immotile: no flagellar movement

Sperm Morphology Morphology is even more important than

motility and concentration Because of the small size of the human sperm

head, must use an air-dried smear which has been stained

The Papanicolaou method is best Prepared samples are assessed using a 100×

oil-immersion objective under bright field optics The WHO recommends that 200 spermatozoa

are counted per sample (and says that 2 × 200 is better)

Fields for counting must be selected at random When counting, remember about the normal

distribution

Morph. Contd.

Variations of norm al head shape

Sm all / large head Tapering heads

Pyriform heads Vacuolatedhead

Asym m etricinsertion

D istendedm idpiece

Thinm idpiece

C ytoplasm icdroplet

C oiledta il

Shortta il

H airp inta il

Bentta il

Term inal droplet

D uplicateta il

C onjo inedform

N on-insertedta il

Am orphous form s

C onstricted R educedacrosom e

D ensesta in ing

Sperm morphology – TZI The Teratozoospermic Index is an expression of the

average number of abnormalities per abnormal sperm Each sperm cell is assessed for an abnormality in the

head, neck/midpiece, or tail, and for a cytoplasmic droplet

If it does not have any of these abnormalities, it is “normal”

If it does have an abnormality, it is “abnormal”, and we score each abnormality. So, if a cell has an abnormal head and tail, it is counted as 1 cell, and 2 abnormalities

Then, (total # abnormalities) / (total # sperm) = TZI A TZI > 1.80 has been associated with poor sperm

fertilizing ability in vivo and in vitro

Semen Biochemistry Acid phosphatase: marker for prostatic function

Citric acid: can indicate prostatic function – low levels may indicate dysfunction or a prostatic duct obstruction

Zinc: marker for prostatic function – colorimetric assay (WHO)

Fructose: marker for seminal vesicle function, and is a substrate for sperm metabolism – spectrophotometric assay (WHO)

-Glucosidase: secreted exclusively by the epididymis and so is a marker for epididymal function – spectrophotometric assay (WHO)

Terminology

Aspermia-No semen Azoospermia-No sperm in semen Oligospermia-Low sperm count Asthenospermia-Dimnished Motility Necrospermia-Dead sperm Teratospermia-Abnormal Morphology

FERTILITY TREATMENT OPTIONS

If a partner is sterile (i.e. no gametes), then the couple would need donor gametes to achieve a pregnancy

If one or both partners are sub-fertile, then the treatment options are:

no treatment, or ovulation induction

intra-uterine insemination (+ ovulation induction)

in vitro fertilization (includes ICSI)

INTRA-UTERINE INSEMINATION (IUI)

IUI is the least invasive of all t/t - involves the selective washing of semen to isolate the motile spermatozoa (can’t put whole semen into the uterus)

Up to 15 million motile spermatozoa are inseminated

Advantages: relatively inexpensive – simple procedures minimal use of FSH can be used in consecutive cycles can usually start treatment virtually immediately

Disadvantages: lower success rate per cycle than other treatment

IN VITRO FERTILIZATION (IVF)Overview

There are many types of IVF

For virtually all types, the woman is treated with “fertility drugs” to stimulate the development of a group of eggs (the average is around 10 – but the range can be enormous)

Just prior to ovulation, the oocytes are retrieved

That afternoon, they are inseminated with prepared sperm

Inseminated eggs checked the next day for fertilization

The fertilized eggs are kept in culture for up to 5-6 days

Embryo transfer / possibly cryopreservation

IVF-Embryo development

IVF Contd.

One sperm is injected directly into an egg

Only mature eggs injected

After the insemination, the rest of the lab procedures are the same as for “standard” IVF

Thank you