Semen Formation and Physiology

Semen

• Semen contains spermatozoa suspended in seminal plasma
• Semen consists of composite solutions from the testes and male reproductive organs

Formation and Physiology

• Semen production and hormone creation are key aspects
• Follicle-stimulating hormone (FSH) acts on Sertoli cells of the testes and stimulates spermatogenesis.
• Luteinizing hormone (LH) impacts Leydig cells of the testes which promotes testosterone production
• Testosterone is the most potent androgen in males which helps with development and maintains the male reproductive system

Seminal Fluid Composition

• Seminal fluid accounts for 60-70%
• Spermatozoa only make up 5%
• Prostatic fluid counts for 20-30%
• Bulbourethral gland makes up the rest

Male Reproductive System

• Seminiferous tubules in testes host spermatogenesis
• Epididymis matures sperm where it develops flagella and becomes motile
• Prostate gland secretes acidic, milky fluid for liquefaction and produces citric acid, zinc, enzymes, and proteins
• Seminal vesicles produce alkaline, viscous fluid with fructose for energy and flavin for color
• Vas deferens transports sperm and is cut during vasectomy to propel sperm to ejaculatory duct
• The bulbourethral/Cowper's gland, secretes alkaline mucus to counteract vaginal acidity
• Sertoli cells are nurse cells for sperm
• Interstitial cells of Leydig secrete testosterone

Semen Collection Guidelines

• Sample should be complete from the very first to the last drop
• First part has a high sperm concentration
• Abstain from sex for 2-7 days where less than 2 days will decrease sperm count and more than 7 will decrease sperm motility
• Empty bladder before collection
• Use a sterile glass or plastic container, body temperature maintained at 37°C, and given to the lab within an hour

Semen Collection for Fertility Test

• 2-3 samples with 2 week intervals are required
• 2 abnormal samples are notable

Semen Collection for Fructose Test

• Transported on ice and frozen within 2 hours

Semen Collection Methods

• Avoid commercial condoms with spermicide
• Masturbation is advised
• Use silastic or non-lubricant rubber or polyurethane condoms
• Vaginal aspiration is for post-coital tests
• Coitus interruptus is not suggested as first portion is normally lost which leads to false low counts

Semen Analysis Collection Notes

• First portion of sample is sperm cells and prostatic fluid, if part is lost this can decrease sperm count, increase pH and fail to liquefy
• Last portion of ejaculate is mostly from the seminal vesicle, if part is missing semen volume will decrease, sperm count can be falsely increased and cause pH to decrease and will not clot

Sperm Gross Examination (Normal Values)

• Appearance: Grayish white, translucent
• Volume: 2-5 mL,
• Liquefaction Time: 30 minutes - 1 hour
• pH: 7.2 - 8.0
• Viscosity: Pours in droplets

Sperm Abnormalities

• Yellow is pyospermia, urine contamination, or prolonged abstinence
• Red is bleeding
• Turbid is infection
• Clear means azoospermia
• Less than 2mL is improper function of organs
• More than 5mL is prolonged abstinence
• More than 2 hours of liquefaction is a deficiency in prostatic enzymes
• Less than 7.2 pH is increased prostatic fluid
• More than 8.0 pH is infection
• Clumped is also a deficiency in enzymes

Semen Considerations

• Sperm motility testing is performed after liquefaction, done immediately
• Perform on well mixed, undiluted semen
• Evaluate speed and direction using 20 HPFs, and normal motility is more than 50% with a grade of at least 2
• Abnormal motility can be caused by midpiece and tail abnormalities and male antisperm antibodies

Sperm Motility Grading

• (A) 4.0 = Rapid straight line motility
• (B) 3.0 = Slower speed and some lateral movement
• (B) 2.0 = Slow progression + lateral movement
• (C) 1.0 = No forward progression
• (D) 0= No movement

Semen pH

• Less than 7.2 is increased prostatic fluid
• More than 8.0 is reproductive infection

Liquefaction Concerns

• Takes over 2 hrs, and becomes stringy/gel is an indication of prostatic insufficiency

Additional Info

• Progressive motility of 2, 3, or 4 is considered good
• Greater motile sperm = better fertilization

Sperm Concentration and Count Dilution

• Use 1:20 with sodium bicarbonate and formalin or saline or cold water
• Charge both sides of hemocytometer
• Count cells in 2 WBC squares or 5 RBC squares
• Sperm concentration is per mL and sperm count is per ejaculate

Calculating Sperm

• Counted sperms * 10^5 or 10^6/ Sperm Concentration

Sperm Morphology

• Staining occurs on smear using Wright's, Giemsa, or Papanicolaou observing the head, neckpiece, midpiece, and tail
• Look under Oil Immersion Objective, evaluating 200 sperm

Sperm Morphology Considerations

• Head unites with ovum and tail is left out
• Mothers only pass on mitochondrial genes from the midpiece/tail
• Oval head is 3x5 um; tail is 4.5 um long, no big cytoplasmic droplets
• Acrosomes contain enzymes and encompass 50% of head or 2/3 of total nucleus
• Strict > 14%; Routine > 13%

Workup Tests

• Decreased Motility but Normal Count
  • Look for Dead Sperm: Viability tests with Eosin Nigrosin will stain dead sperm red
• Decreased Sperm Count
  • Check Support Medium: Resorcinol tests for Fructose Decreased Motility, Clumping - Look for Antibodies using serum incubation, mixed agglutination test and immunobead test
• Normal w/ continued infertility - Check female serum

Sterility Risk Factor

• Most common is Varicocele followed by:
  • Mumps and Klinefelter Syndrome (XXY) and Malignancy

Sperm Function Test Studies

• Hamster Egg Penetration to check fertilization
• In-Vitro Acrosome Reaction checks acrosomal enzymes
• Cervical Mucus Penetration test on vaginal aspirate
• Hypo-osmotic Swelling measures integrity and viability against Osmotic Challenge
• Round cell presence, can be spermatids (immature) or leukocytes which indicate peroxidase issues
• Microbial and Chemical Testing

Chemical Testing:

- Diagnose Epididymis and Prostatic Insufficiency Issues

Post Vasectomy Analysis:

- Check motility; Do at monthly intervals until two months with no sperm

Computer-assisted Semen Analysis (CASA)

- Provides data about velocity and direction of motion

Sugars

Fructose is high conc sugar in semen

Diluents

Formalin

Liquefaction

Should take place within 1-2 hours

Sperm Components

Mitochondrial sheath is midpiece

Additional Chemical Testing Markers

• Nuetral a-glucosidase at ≥ 20 mU
• Zinc at ≥ 2.4 umol
• Citric Acid at ≥ 52 umol
• Acid Phosphatase at > 200 units Are all used to detect issues.

Diarrhea

Increased H2O with stool where: - Osmotic can be malabsorption and maldigestion related - Secretory can be bacteria related.

Vaginal Samples and Concerns

Fern (protein/NaCl) indicate rupture Eval, Fibronectin = preterm, Creatinine = age

Amniotic Fluid

• Fluid found where fetus resides

Volumes by Trimester

• 1st Tri: 35mL
• 3rd Tri: 1L

Function

Cushion, Movement, Stabilize Temp

Contributor

Fluid from Fetus, Maternal contribution

Amniotic Fluid Concerns

Oligohydramnios vs. Polyhydramnios where: Less 800/1.2L fluid are urinary or fetal Swallowing defects

Samples

Fetal material collected using Amniocentesis, Transabdominal or Transvaginal method/routinely collects 30mL

Concerns based on sample.

Dark Green, Blood are Meconium and Trauma

Fetal Lung concerns are phospholipids, collected at 35th week

• Lecithin and Sphingomyelin
• Refrigerate at 72/1hr and transport in ice

HDN or lungs

HDN; Bilirubin Lungs; Phospholipids

Tests

Test with Lecithin is used to determine Fetal distress

Sterility risk factors

Klinefelter, Mumps.

Clinical Microscopy and Fetal Testing Additional Notes

Fetal Lung Maturity, or FLM

Phospholipids trapped air.

Lecithin/Sphingomyelin Ratio , or L/S Rate,

2: Mature, if not, use Antisera and use Amniostat-FLM

Bilirubin

Measured used light, in amber bottles with Liley Graph, and Acetylcholinesterase where: AChE: test if sample isn't RBC/blood in it.

Strained Feces

3+ Fetal Neutrophils for a positive result