Semen and Vaginal Fluid

Contents of semen from testes

sperm (2-5% of V)

Contents of semen from epididymis

K, Na, glycerylphosphorylcholine ( < 5% of V)

Contents of semen from ampulla

ergothioneine fructose ( < 5% of V)

Contents of semen from seminal vesicles

amino acids, flavin, P, K, fructose, prostaglandins (60% of V)

Contents of semen from prostate gland

citric acid, acid phosphatase, Ca, Na, Zn, K, prostate specific antigen (30% of V)

Contents of semen from bulbourethral glands

mucus ( < 5% of V)

Purpose of K, Na, and glycerylphosphorylcholine

energy for sperm

Purpose of ergothioneine fructose

antioxidant nourishment

Purpose of amino acids, flavin, P, K, fructose, and prostaglandins

suitable environment for sperm

Purpose of citric acid, acid phosphatase, Ca, Na, Zn, K, and prostate specific antigen

sperm motility

Purpose of mucus

lubricating protein

How much semen is needed for DNA analysis

1 ng / 480,000 ng DNA per ejaculate

Sperm survival time inside a woman’s body (typically)

1 to 2 days

Sperm survival time inside a woman’s body (during ovulation)

3 to 5 days

Sperm survival time inside a fallopian tube

7 days

Sperm survival time outside the body

a few mins to 2 hours

Typical ejaculation

releases 2 to 5 mLs seminal fluid and contains 200 to 500 million spermatozoa

Oligospermia

abnormally low sperm count

Azoospermia

no spermatozoa in seminal fluid

Aspermia

lack of semen with ejaculation

3 regions of spermatozoa

head, middle piece, tail

Head

acrosomal cap and nucleus, contains genetic material and enzymes for fertilization

Middle piece

contains mitochondria to create energy for sperm motility

Tail

sperm motility

Vasectomy

affects epididymis, no sperm in ejaculate

What is mostly tested for in semen?

sperm, flavin, acid phosphatase, and prostate specific antigen

What can still be detected in vasectomized males?

acid phosphatase, prostate specific antigen, and seminal vesicle specific antigen

Acid phosphatase (AP)

group of enzymes that hydrolyze phosphate esters / designed for an acidic pH / prostate is the most abundant source in humans (400x) / present in animal and plant species

Stability of acid phosphatase (AP)

6 months at 37^oC / 1 year at -20^oC / wet conditions decrease stability

Prostate specific antigen (PSA or P30)

molecular weight of 30kDa / protein present in seminal fluid (around 0.5 to 2.0 mg/mL) / responsible for hydrolyzing semenogelin

Stability of prostate specific antigen (P30)

3 years when dried at room temp / stability decreases when stored in wet conditions

Seminal vesicle specific antigen

two major semenogelins (semenogelin I and semenogelin II) / upon ejaculation, form a gel like layer around sperm to prevent capacitation / liquified after a few minutes / present in other male reproductive tissues / [SgI] and [SgII] are higher than prostate specific antigen and not typically found in other fluids

Types of evidence in sexual assult/rape cases

condoms, clothes, towels, sheets, tampons, rape kits

Sexual assault/rape evidence kit for victim

reference examples: pubic hair, head hair, blood / buccal sample / physical evidence: pubic combings, vaginal swab and smear, fingernail scrapings, all clothing, debris collection, urine

Sexual assault/rape evidence kit for suspect

reference sample: pubic hair, head hair, blood / buccal swab / physical evidence: pubic combings, penile swab, all clothing

Presumptive tests for ID of semen

visual examinations and acid phosphatase tests

Confirmatory tests for ID of semen

microscopic examination of sperm, identification of prostate specific antigens, seminal vesicle specific antigens, RNA based assays

Visual examination tests

lighting techniques typically used to search for stains / dried stains fluoresce under UV light, argon lasers, or alternate light sources (ALS) / based on the presence of flavins in the seminal fluid / common false + with sweat, vaginal fluid, urine, saliva / using ALS: 445 - 495 nm wavelength with filter (530 nm) / not specific for semen

Acid phosphatase color test

based on the ability of acid phosphatase to hydrolyze phosphate esters / can be used to search large areas for possible stains / common false + with vegetables, fruits, serum of male with prostate cancer, vaginal fluid, saliva, bacterial (lactobacillus acidophilus) / substrate reagent is alpha-naphthyl phosphate / color reagent is fast blue b to swab

Acid phosphatase procedure

negative control: moisten a piece of filter paper, add 1 drop of AP spot test reagent (or 1 drop each A then B), observe color change / positive control: moisten a piece of filter paper and touch to reference sample, add 1 drop of AP spot test reagent (or 1 drop each A then B), observe color change / unknown sample: moisten a piece of filter paper and touch to unknown sample, add 1 drop of AP spot test reagent (or 1 drop each A then B), observe color change / read result within 1 min each time ( (-) = no color change or pink, and + = purple)

Microscopic examination of sperm

only need 1 intact sperm for identification / most common is christmas tree stain

Christmas tree stain

first, add nuclear fast red to sample (stains nucleus red) / second, add picroindigocarmine to sample (stains acrosomal cap, middle piece, and tail green)

Microscopic exam of sperm procedure (christmas tree stain)

negative control: apply nuclear fast red to clean microscope slide, allow to sit for 5 mins, rinse with DI water, apply picroindigocarmine stain, sit for 15 sec, rinse with ethanol, perform slide search / positive control: extract a small portion of reference stain with water, gently vortex, transfer suspension to a slide and evaporate at RT, apply nuclear fast red to slide, allow to sit for 5 mins, rinse with DI water, apply picroindigocarmine stain, sit for 15 sec, rinse with ethanol, perform slide search / unknown sample: extract a small portion of the unknown stain with water, gently vortex, transfer suspension to a slide and evaporate at RT, apply nuclear fast red to slide, allow to sit for 5 mins, rinse with DI water, apply picroindigocarmine stain, sit for 15 sec, rinse with ethanol, perform slide search / (-) = no sperm or no intact sperm, + = at least 1 intact sperm

ID of prostate specific antigen tests

ABAcard/immunochromatographic assay or ELISA

ABAcard/immunochromatographic assay

less sensitive / more specific / false (-) = high dose hook effect, false + = post ejaculatory urine

ELISA

more sensitive / less specific / false + = post ejaculatory urine, amniotic fluid, breast milk

ABAcard test for PSA/P30

immunochromatographic assay / add sample to test well / positive results may occur within 1 minute, read negative results after 10 minutes / false (-) = high dose hook effect

ELISA test procedure for PSA or P30

sample is added to well in which anti-PSA antibody is immobilized / add labeled monoclonal antihuman PSA antibody (labeled with horseradish peroxidase) / wash / add substrate (H₂O₂) and chromogen solution (TMB) / add stop solution (phosphoric acid) / positive result = color

ID of seminal vesicle specific antigen

options available include immunochromatographic assays and ELISA / procedures are similar to PSA test, but use anti-semenogelin antibody

Sample preparation procedure for ID of seminal vesicle specific antigen

cut a small portion of stain or swab / place sample in 200 to 300 microliters extraction buffer for 1 to 2 hours / place 20 to 30 microliters with running buffer and load into sample well

RNA based assays

based on mRNA expressed in spermatozoa or other cells specific to other male glands / use reverse transcriptase polymerase chain reaction / benefits: higher specificity, amendable to automation / drawbacks: RNA is unstable due to degredation by endogenous ribonucleases

Vaginal fluid

mixture of fluid secreted through vaginal walls, cervical mucus, and residual urine

Lugol’s iodone test (presumptive)

most common method based on detection of glycogenated epithelial cells / epithelial cells are large and irregular shape / glycogenated epithelieal cells are same but stain red or brown / varies with menstration, pregnancy, menopause, prepuberty / positive = > 50 epithelial cells and > 75% are glycogenated

Microflora DNA analysis (confirmatory)

characterization of microbial signature specific to female genital tract / use multiplex real time PCR for analysis

Lugol’s iodine test procedure

negative control: moisten a swab with DI water, rub on microscope slide, heat fix (1 min in oven), add 1 drop lugol’s iodine, perform slide search / positive control: moisten a swab with DI water, rub swab on reference stain, rub on microscope slide, heat fix (1 min in oven), add 1 drop lugol’s iodine, perform slide search / unknown sample: moisten a swab with DI water, run swab on unknown stain, rub on microscope slide, heat fix (1 min in oven), add 1 drop lugol’s iodine, perform slide search / + = > 50 epithelial cells and > 75% are glycogenated, (-) = < 50 epithelial cells and < 75% are glycogenated