Amniotic Fluid and Semen Analysis

Amniotic fluid cushions the fetus, permits movement, helps maintain temperature, and supports lung development. Early in pregnancy, maternal plasma contributes much of the fluid. Fetal urine and lung fluid become major sources later. Fetal swallowing and intramembranous absorption (fluid taken up across the amnion into fetal vessels) remove fluid.^{11 McPherson RA, et al. Henry's Clinical Diagnosis and Management by Laboratory Methods. 2022.}

Ultrasound estimates volume because a sample from amniocentesis cannot show the total amount around the fetus. Polyhydramnios is commonly defined as a deepest vertical pocket of at least 8 cm or an amniotic fluid index of at least 24 cm. Oligohydramnios is generally defined as a deepest vertical pocket below 2 cm or an amniotic fluid index of 5 cm or less.^{2,32 Dashe JS, et al. SMFM Consult Series #46. 2018.3 American College of Obstetricians and…, et al. Indications for outpatient antenatal fetal surveillance. 2021.}

Collection and routing

Amniocentesis is performed under ultrasound guidance. Divide the specimen according to the ordered tests. For prenatal cytogenetic or molecular testing, many reference laboratories ask that the first 1 to 2 mL be discarded because it has the greatest risk of maternal cell contamination. Exact volumes, containers, and transport conditions come from the receiving laboratory.^{44 Mayo Clinic Laboratories. Chromosomal microarray, prenatal, amniotic fluid.}

Centrifugation depends on the test. Cell-culture and microbiology aliquots must retain the target cells or organisms. A bilirubin scan requires a cell-free supernatant. Process each aliquot according to the test ordered on it.

Record the specimen's color and visible blood before separating aliquots. Blood may indicate a traumatic collection or fetal bleeding and can interfere with AFP, AChE, bilirubin, and classical fetal lung maturity methods. Green or brown material may represent meconium. Cloudiness can reflect cells, debris, or contamination.^{11 McPherson RA, et al. Henry's Clinical Diagnosis and Management by Laboratory Methods. 2022.}

Suspected rupture of membranes

Prelabor rupture of membranes (PROM) is rupture before labor begins. Bedside evaluation usually combines the clinical history, sterile speculum examination for fluid pooling, nitrazine pH testing, and microscopic examination for a fern pattern.^{55 American College of Obstetricians and… Prelabor rupture of membranes. 2020.}

Nitrazine paper detects an alkaline sample. Blood, semen, cervical mucus, urine, antiseptics, and some lubricants can produce misleading results. Ferning can also be confused by cervical mucus, semen, blood, or a poorly prepared slide. Do not interpret either test alone.^{55 American College of Obstetricians and… Prelabor rupture of membranes. 2020.}

Commercial biomarker tests detect proteins such as placental alpha microglobulin-1, insulin-like growth factor binding protein-1, or a combination of placental protein 12 and AFP. They are adjuncts to the clinical assessment. The US Food and Drug Administration (FDA) has warned that using these tests by themselves can lead to false-negative or false-positive diagnoses and serious harm.^{6,7,8,96 US Food and Drug Administration. FDA alerts healthcare providers, women about risks associated… 2018.7 QIAGEN. AmniSure ROM Test.8 Clinical Innovations. ROM Plus Rapid Rupture of Fetal Membranes Test.9 Hologic. Actim PROM: Instructions for Use. 2024.}

Historical bilirubin testing for fetal anemia

Bilirubin absorbance at 450 nm, reported as delta OD450, was once used to estimate hemolysis in pregnancies affected by red-cell alloimmunization. The laboratory protected the sample from light, removed cells, scanned from 365 to 550 nm, and measured the excess absorbance above the baseline at 450 nm. The result was plotted against gestational age on Liley or Queenan zones.^{10,1110 Liley AW. Liquor amnii analysis in the management of the pregnancy… 1961.11 Queenan JT, et al. Deviation in amniotic fluid optical density at a wavelength of… 1993.}

The Hemolytic Disease of the Fetus and Newborn lesson covers the laboratory investigation of maternal red-cell antibodies.

Middle cerebral artery peak systolic velocity measured by Doppler ultrasound has replaced serial delta OD450 testing for routine fetal anemia assessment. The Society for Maternal-Fetal Medicine (SMFM) recommends against using amniotic fluid delta OD450 to diagnose fetal anemia. The older scan and zone charts remain useful for understanding historical records. An unavailable or inconclusive Doppler assessment calls for specialist evaluation. It does not justify resuming routine delta OD450 testing.^{1212 Society for Maternal-Fetal Medicine. Clinical Guideline #8. 2015.}

Open fetal defect testing

Amniotic fluid AFP is interpreted as a multiple of the median for gestational age. A result at or above the laboratory's cutoff is followed by AChE testing when an open neural tube or ventral wall defect is suspected. Report visible blood, because fetal blood contains AChE and can produce a false-positive result. Interpret AFP, AChE, gestational age, blood contamination, and ultrasound findings together.^{1313 Mayo Clinic Laboratories. Alpha-fetoprotein, amniotic fluid.}

Fetal lung maturity tests

Classical fetal lung maturity tests measured surfactant activity or composition. These methods help in reading older records. Current obstetric guidance no longer uses them to time delivery.^{14,1514 American College of Obstetricians and…, et al. Medically indicated late-preterm and early-term deliveries. 2021.15 Association for Diagnostics & Laboratory… Fetal lung maturity testing. 2023.}

A semen analysis describes the ejaculate at a specific collection. Results depend on collection completeness, abstinence interval, temperature, time to examination, and the laboratory method. A single value does not establish fertility or infertility.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Spermatids form a head, acrosome, midpiece, and flagellum during spermiogenesis in the seminiferous tubules. Spermatozoa then acquire functional maturity and progressive motility during epididymal transit. Illness, fever, medication, toxic exposure, or another disruption during the preceding 2 to 3 months can affect the result.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

The ejaculate combines spermatozoa with secretions from several sites:^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Collection and timing

When volume will be determined by weight, record the empty collection container's weight before collection. Use that recorded tare with the filled-container weight and the validated calculation.

1. Ask the patient to maintain 2 to 7 days of ejaculatory abstinence and record the actual interval.
2. Collect the entire ejaculate by masturbation into a clean, wide-mouth container that has been verified as nontoxic to sperm. Record any lost portion and whether it was the first or later part of the ejaculate.
3. Collection near the laboratory is preferred. If collection occurs elsewhere, keep the closed container between 20 and 37 °C and deliver it preferably within 30 minutes and no later than 50 minutes so the examination can begin within 60 minutes.
4. A condom made and tested for semen collection may be used only when masturbation is not possible. Contraceptive condoms, lubricants, and saliva can impair sperm motility or contaminate the sample.
5. Record the collection time, receipt time, and start of analysis. Handle the specimen with standard precautions.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

The first portion of the ejaculate is sperm rich, so losing it can lower the measured concentration and total sperm number. After an incomplete collection, usually request a repeat collection before you interpret an unexpected low result.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Macroscopic examination

Normal liquefied semen is usually homogeneous and cream-gray or opalescent. A red or brown specimen suggests red blood cells. Yellow coloration may occur after prolonged abstinence, with jaundice, or from some medications. Record a strong urine-like or putrefactive odor because it may identify contamination or poor specimen condition. Odor alone does not diagnose infection.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Low volume can result from incomplete collection, retrograde ejaculation, ejaculatory duct obstruction, congenital absence of the vas deferens, or androgen deficiency. Interpret volume with the collection history, pH, sperm concentration, and clinical findings.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

The timing below keeps the initial examination within the World Health Organization (WHO) window:^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

The figure below connects the collection details and timed checks to the macroscopic and microscopic examination. It also shows two preanalytic problems that can change the result: an incomplete collection and liquefaction that prevents testing.

Assess viscosity after liquefaction by letting semen fall from a wide-bore pipette. A continuous thread longer than 2 cm indicates abnormal viscosity. Prolonged liquefaction and increased viscosity are different findings, so record them separately.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Measure pH after liquefaction, preferably at about 30 minutes and within 60 minutes. A pH below the laboratory's lower reference limit can support a distal tract obstruction or reduced seminal-vesicle contribution when the specimen also has low volume and few or no sperm. High pH has poor specificity, so it cannot identify infection by itself.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Microscopic examination

Mix the liquefied specimen before removing an aliquot. Use replicate preparations and follow the laboratory's written criteria for agreement between counts.

Sperm concentration and total number

An improved Neubauer hemocytometer remains a reference counting method. Prepare an appropriate dilution, load both chambers, and count at least 200 sperm in each replicate when possible. Calculate concentration from the dilution and chamber volume. The total sperm number is concentration multiplied by the measured ejaculate volume.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.} See Manual Cell-Counting Calculations for the chamber calculation.

Motility

Examine fresh wet preparations at a controlled temperature. Classify at least 200 sperm in each replicate as rapid progressive, slow progressive, non-progressive, or immotile. Report the categories used by the laboratory and confirm that the replicate counts agree within the validated limit.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Computer-assisted semen analysis can measure sperm kinematics when the system is carefully standardized and quality controlled. Debris can be mistaken for immotile sperm, so automated percentage motility is not automatically more reliable than a trained manual assessment.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Vitality

Vitality distinguishes live immotile sperm from dead sperm. It is especially useful when total motility is below 40%. Eosin-nigrosin staining is a common method: live sperm exclude eosin, and dead sperm take up the dye. Count at least 200 sperm and report the percentage viable.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Morphology

Prepare and stain a smear, preferably with a validated Papanicolaou method, then assess about 200 sperm under oil immersion. Apply strict criteria to the entire sperm: an oval head with a well-defined acrosomal region, an aligned slender midpiece without excessive residual cytoplasm, and a uniform uncoiled principal piece. Classify a sperm as normal only when all regions meet the laboratory's criteria. Other stains require laboratory-specific validation because fixation and staining change measured dimensions.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Round cells and other findings

Round cells include leukocytes and immature germ cells. They cannot be reliably separated by appearance alone in an unstained wet preparation. A peroxidase test identifies granulocytes. It does not detect every leukocyte type. A stained smear can support cell classification, and immunocytochemistry may be needed when the distinction matters. The WHO retains 1 million peroxidase-positive leukocytes per mL as a consensus decision limit. A count above that limit does not prove infection, and the limit is not a firm evidence-based cutoff for immature germ cells.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Record red blood cells, epithelial cells, sperm agglutination, and debris when present. Report them for interpretation with the clinical findings. Microscopy does not establish a diagnosis from them.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

WHO lower reference limits

The WHO sixth edition reports lower fifth-percentile values from men whose partners conceived within 12 months. These limits describe a reference population. A result below a limit does not classify a patient as infertile.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

These WHO clinical reference-population values differ from the ASCP BOC composite examination values. Use the named source for the question being answered.

Total motility is the sum of progressive and non-progressive sperm. Interpret the complete pattern, collection conditions, clinical question, and repeat results together. A single result cannot establish fertility or infertility.

Additional semen tests

Direct mixed antiglobulin reaction and immunobead tests detect antibodies attached to motile sperm, usually IgG or IgA. Report the immunoglobulin class, percentage of motile sperm with adherent particles, and binding site. Older manuals treated 50% antibody-coated motile sperm as clinically significant. The evidence for that threshold is limited, so use the laboratory's validated interpretive limit.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Accessory-gland marker reports give the total content per ejaculate. A concentration does not provide that total. The WHO sixth edition lists these lower reference limits:^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Separate sperm-free seminal plasma using the validated centrifugation method and store it according to the assay instructions. Citrate and acid phosphatase can also reflect prostatic secretion. No universal cutoff exists, so the laboratory must validate its own method and reference interval.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Post-vasectomy semen analysis

The 2026 American Urological Association guideline allows the first post-vasectomy semen analysis as early as 8 weeks. A fresh, well-mixed, uncentrifuged specimen examined within 2 hours may meet the laboratory criterion when it shows azoospermia or no more than 100,000 rare nonmotile sperm per mL. A specimen examined more than 2 hours after collection must show azoospermia because motility can no longer be interpreted reliably. The laboratory reports the finding and method. The urology service decides whether the patient may stop other contraception or needs another sample or procedure.^{1717 American Urological Association. Vasectomy: AUA Guideline (2026) Part I. 2026.}

Quality and regulatory control

Semen analysis needs documented training, competency assessment, calibrated pipettes and chambers, replicate-count rules, internal quality control, and external quality assessment where available. Control slides, video files, or other stable materials can be used for morphology and motility exercises. Biochemical assays require the same calibration and control practices as other quantitative laboratory tests.^{1616 World Health Organization. WHO Laboratory Manual for the Examination and Processing of… 2021.}

Under US Clinical Laboratory Improvement Amendments (CLIA) rules, a limited examination for sperm presence or absence and motility may qualify as provider-performed microscopy when an eligible provider performs it during the patient visit. Other semen test systems follow their assigned FDA complexity. A modified method or laboratory-developed test defaults to high complexity. The laboratory must verify the classification of each method it performs.^{18,1918 Centers for Disease Control and Prevention. Test complexities. 2024.19 Centers for Disease Control and Prevention. Provider-Performed Microscopy Procedures.}