Amniotic Fluid and Semen Analysis

Amniotic fluid and semen each carry laboratory results that depend on events before the specimen reaches the bench: an amniotic fluid bilirubin result survives only with immediate light protection, and a semen motility result survives only if the specimen stays warm and is examined on time. Both fluids also carry physiology into their chemistry. Amniotic fluid composition shifts with gestational age, and semen composition reflects four glandular fractions that must reach the container completely.

Amniotic fluid physiology and volume

Amniotic fluid fills the amnion surrounding the fetus. It exchanges water and solutes among the fluid, fetus, and maternal circulation, cushions the fetus mechanically and thermally, permits musculoskeletal movement, and allows lung development.¹ Volume reflects inflow (fetal urine and lung liquid) against outflow (fetal swallowing and intramembranous flow directly into fetal vessels). Early volume, about 35 mL, derives mainly from the maternal circulation, climbs to about 60 mL by 12 weeks, peaks at 800 to 1200 mL in the third trimester, and declines near term. After the first trimester, fetal urine dominates production and fetal swallowing balances it. From mid-pregnancy onward, the fetus also secretes lung liquid, and each fetal breathing movement washes pulmonary surfactants (lecithin, sphingomyelin, phosphatidylglycerol) into the fluid. These surfactants made amniotic fluid assessment of fetal lung maturity possible.¹

Ultrasound diagnoses polyhydramnios and oligohydramnios with gestational-age-specific criteria. The Society for Maternal-Fetal Medicine defines polyhydramnios in a singleton pregnancy as a deepest vertical pocket (DVP) of 8 cm or more, or an amniotic-fluid index of 24 cm or more.² Oligohydramnios is a DVP below 2 cm.³ Causes include failed fetal swallowing, gastrointestinal or neural anomalies, urinary tract malformation, membrane leakage, and increased absorption. Volume in a single aspirated specimen diagnoses neither condition and can distort analyte concentrations.

Fetal urine production shifts fluid chemistry as gestation proceeds: creatinine, urea, and uric acid rise while glucose and protein fall. Amniotic fluid creatinine has been used as a crude gestational-age indicator (1.5 to 2.0 mg/dL before 36 weeks, above 2.0 mg/dL after). Failure of neural tube closure lets fetal cerebrospinal fluid enter the sac directly, raising alpha-fetoprotein and acetylcholinesterase, the two markers used for neural tube defect detection.¹

Collection and handling

Amniocentesis is needle aspiration of amniotic fluid. The first 2 to 3 mL withdrawn, contaminated with maternal tissue fluid, blood, and cells, is discarded before the diagnostic specimen is collected.¹

Handling requirements are test-specific, and the requested test drives the routing:

Distinguishing amniotic fluid from maternal urine

A fluid recovered from the vagina when membrane rupture is suspected, or a bladder puncture during amniocentesis, raises the question of which fluid is on the swab.¹ Amniotic fluid creatinine stays at or below 3.5 mg/dL and urea at or below 30 mg/dL, versus up to 10 mg/dL creatinine and 300 mg/dL urea in maternal urine. Glucose and protein are weak discriminators because normal pregnancy itself can cause mild glycosuria and proteinuria.

Two physical tests support the chemistry. In the fern test, vaginal fluid is air-dried on a slide; amniotic fluid protein and sodium chloride crystallize into fern-like arborization that urine cannot form. In the nitrazine test, amniotic fluid’s near-neutral pH of 7.1 to 7.3 turns nitrazine paper blue above pH 6.0, versus normal vaginal pH of 4.5 to 6.0. Urine, blood, and semen also turn the paper blue, so nitrazine is a screening test only. Immunologic biomarker assays now confirm membrane rupture in equivocal cases.

Hemolytic disease and the delta OD450 scan

Hemolytic disease of the fetus and newborn (HDFN) begins when fetal red cells entering the maternal circulation provoke maternal IgG antibody, which crosses the placenta and destroys fetal red cells, releasing unconjugated bilirubin into the amniotic fluid. The immunohematology of antibody detection, titration, and surveillance is covered by the Hemolytic Disease of the Fetus and Newborn page.

The spectrophotometric bilirubin scan (delta OD450) is a historical amniotic-fluid bilirubin method. The specimen is protected from light, centrifuged clear, and scanned from 365 to 550 nm; the deviation of the absorbance curve at 450 nm above a straight line drawn between two reference points estimates bilirubin. When this method was in use, the deviation was plotted on the Liley curves (from 27 weeks) or the Queenan curves (from 14 weeks) to estimate the severity of hemolysis.⁴⁵ As little as 30 minutes of light exposure markedly lowers delta OD450, which required immediate light protection. Cells, hemoglobin, meconium, and other particulates distort the scan, so specimens were centrifuged immediately. Meconium-stained fluid was rejected because it falsely lowers the result. Bloody specimens were generally unacceptable because the 410-nm oxyhemoglobin peak overlaps the region of interest; chloroform extraction could remove this interference when unavoidable.¹

Current practice retired the method. The Society for Maternal-Fetal Medicine’s fetal-anemia guideline does not use delta OD450 to diagnose fetal anemia and names Doppler measurement of the middle cerebral artery peak systolic velocity (MCA-PSV) as the primary noninvasive test.⁶

Neural tube defect follow-up testing

Open neural tube disorders (anencephaly, open spina bifida) are screened with maternal serum alpha-fetoprotein (MSAFP), high-resolution ultrasound, and, when indicated, amniocentesis.⁷ Alpha-fetoprotein (AFP), the dominant early fetal liver protein, peaks at 12 to 15 weeks and declines thereafter. It reaches maternal serum through the coupled placental circulations and amniotic fluid mainly by diffusion and fetal urinary excretion; open neural tissue lets AFP leak abnormally into both compartments. Because normal levels vary weekly, results are expressed as multiples of the median (MoM) for that gestational week, and values above 2.0 MoM in either compartment are abnormal.¹ An elevated MSAFP prompts an immediate check for multiple pregnancy, the most common benign explanation, before ultrasound and, if indicated, amniotic fluid AFP measurement.

Every elevated amniotic fluid AFP is followed by amniotic fluid acetylcholinesterase (AChE), a neural-tissue enzyme more specific for neural tube defects because it has no other source in the fluid.⁷ The test is invalid on a bloody specimen, since blood itself carries AChE.

Fetal lung maturity methods

Fetal lung maturity (FLM) assays are no longer used to guide delivery timing. ACOG and SMFM guidance is explicit: when a maternal or fetal indication for delivery exists, delivery occurs regardless of FLM results, and a mature result is insufficient reason to deliver early.⁸ The Association for Diagnostics and Laboratory Medicine’s test-utilization guide states that FLM testing is not recommended in any clinical scenario and that no laboratory test is currently recommended to assess fetal maturity.⁹ Most commercial FLM assays have been withdrawn, although some reference laboratories still perform thin-layer chromatographic L/S ratios and hematology analyzers can still count lamellar bodies. The four classical methods retain teaching value because their interference profiles illustrate why surfactant specimens are handled the way they are.

• Lecithin:sphingomyelin (L/S) ratio compares lecithin, the dominant surfactant phospholipid that surges near 35 weeks, against sphingomyelin, produced at a steady rate from about 26 weeks. Blood or meconium contamination falsely raises the ratio because both fluids contain lecithin and sphingomyelin.
• Phosphatidylglycerol (PG) immunoassay detects a surfactant phospholipid that normally appears after 35 weeks. PG agglutination immunoassays tolerate blood and meconium contamination better than L/S chromatography.
• Foam (shake) test exploits surfactant’s detergent-like foam stability after ethanol addition and shaking. Blood and meconium can produce a falsely mature, stable-foam result.
• Lamellar body counts use the platelet channel of a hematology analyzer, because lamellar bodies overlap platelets in size. Blood, meconium, mucus, and clotting distort the count.

Premature rupture of membranes

Premature rupture of membranes (PROM) is diagnosed from history, visible fluid pooling on speculum examination, and laboratory tests such as nitrazine pH and fern microscopy.¹⁰ Immunologic biomarker assays are adjuncts when the clinical examination is equivocal. Placental alpha-macroglobulin-1 (PAMG-1) and insulin-like growth factor binding protein-1 (IGFBP-1, also called placental protein 12) are the principal targets. Commercial devices include PAMG-1 swabs (AmniSure), IGFBP-1 swabs (Actim PROM), and a combined strip detecting AFP and IGFBP-1 (ROM Plus).¹¹¹²¹³

Contamination and false-positive rates vary by device and specimen conditions, including blood, urine, semen, antiseptics, and lubricants; each manufacturer validates its own tolerance limits, so collection and interpretation follow the validated product instructions. A combined-analyte device may detect AFP and IGFBP-1, but platforms differ in sensitivity, specificity, specimen stability, and contamination tolerance, so results require clinical correlation.

Semen physiology and specimen collection

Semen combines four fractions; complete mixing at ejaculation is required for a representative specimen.¹⁴

Spermatogenesis (mitosis and meiosis of germ cells, supported by Sertoli cells) in the seminiferous tubules produces immature, nonmotile spermatids, which then mature and grow flagella in the epididymis; the whole sequence spans roughly 90 days. A toxic exposure, febrile illness, or chemotherapy course during the preceding three months may therefore affect the current specimen and belongs in the interpretation of an abnormal analysis.

Because the four fractions differ in composition, losing part of the ejaculate distorts the analysis.¹⁴ Losing the first, sperm-rich portion lowers the sperm count, falsely raises pH, and impairs liquefaction. Losing the last portion lowers volume, falsely raises the sperm count, and falsely lowers pH. For fertility testing, the World Health Organization (WHO) recommends 2 to 7 days of sexual abstinence before collection. One examination may suffice to decide the next step; two or three ejaculates may be needed to define an individual baseline. Collect specimens by masturbation into a clean, sperm-compatible container, ideally on site. A home-collected specimen must stay at 20 to 37 °C, preferably close to the body, and reach the laboratory within 1 hour. Only special nonspermicidal, nonlubricant condoms may substitute for direct collection, because ordinary condoms contain spermicide. Coitus interruptus is unacceptable because the sperm-rich first fraction is easily lost and vaginal acidity damages sperm on contact. Treat all semen specimens as biohazardous because HIV, hepatitis, and herpes viruses are potential contaminants.

Macroscopic examination

Normal semen is gray-white and translucent, with a musty odor; very low sperm concentration can look nearly clear. Increased white turbidity suggests leukocytes and infection. If infection is suspected, culture precedes the rest of the analysis, and microbiology procedures cover the culture workup. Leukocytes must be distinguished microscopically from immature sperm. Red color indicates red cells and is always abnormal. Yellow color follows urine contamination (toxic to sperm and invalidating motility), prolonged abstinence, or certain medications. ASCP BOC examination ranges and WHO 6th-edition distribution centiles appear in the ASCP BOC Examination Reference Ranges page.

Volume is preferably measured by weighing the specimen in its collection container (WHO 6th edition).¹⁴ Decreased volume may accompany infertility, most commonly seminal vesicle dysfunction, or simply incomplete collection, which must always be ruled out first.

Liquefaction is normally complete within 15 to 30 minutes; the basic examination is performed between 30 and 60 minutes after collection, and liquefaction is recorded at 30 minutes and again at 60 minutes.¹⁴ A duration beyond 60 minutes suggests deficient prostatic proteolytic enzymes. A specimen still unliquefied at 2 hours is treated by 1:1 mixing with Dulbecco’s phosphate-buffered saline (DPBS) with repeated aspiration and dispensing, or by proteolytic digestion with bromelain.¹⁴ Both interventions can affect biochemical and motility or morphology results and must be documented. Any introduced dilution is carried into the sperm-concentration calculation.

DPBS preparation: to a 1-L volumetric flask add 750 mL purified water, 0.20 g KCl, 0.20 g KH₂PO₄, 0.10 g MgCl₂·6H₂O, 8.0 g NaCl, 2.16 g Na₂HPO₄·7H₂O, and 1.00 g D-glucose; separately dissolve 0.132 g CaCl₂·2H₂O in 10 mL purified water and add it slowly with continuous stirring to prevent precipitation; adjust to pH 7.4 with 1 mol/L NaOH; bring to 1000 mL with purified water. To liquefy a specimen, combine equal volumes of DPBS and semen and pipette repeatedly until liquefied.

Bromelain digestion: prepare a 10 IU/mL bromelain solution (1000 IU bromelain dissolved in about 60 mL DPBS over 15 to 20 minutes, then brought to 100 mL with DPBS); combine one part semen with one part of this solution for a total 1:2 dilution; stir; incubate at 37 °C for 10 minutes; mix well before analysis. Carry the 1:2 dilution into the sperm-concentration calculation.

Viscosity parallels liquefaction quality. Normal semen falls in small discrete droplets, and threads longer than 2 cm are abnormal (graded 0, watery, to 4, gel-like, or simply low, normal, or high). High viscosity interferes with motility estimation, concentration measurement, antisperm antibody testing, and biochemical assays.

pH must be read within 1 hour, since carbon dioxide escape drifts it upward over time. Clinical interest is in a low value: pH below 7.2 may indicate absent alkaline seminal-vesicle fluid or urine contamination (WHO 6th edition).¹⁴ Elevated pH suggests reproductive-tract infection.

Microscopic examination

Sperm concentration and total count are measured on an improved Neubauer hemacytometer with the method described for other body fluids, and the concentration calculation with its worked example is covered by the Manual Cell-Counting Calculations page. Dilution both fixes the counting arithmetic and immobilizes sperm on the grid. The 1:20 bicarbonate/formalin dilution counted in five small squares is a historical teaching and examination method. WHO 6th edition selects dilution and counted grids by concentration, aims for about 200 sperm per replicate, and judges replicate agreement with a count-dependent 95% confidence-limit table. The fixed 10% rule belongs to the historical method.¹⁴ Only fully developed sperm count toward concentration.

Immature spermatids and leukocytes, together called “round cells” because both lack flagella, are excluded from the sperm count but identified and quantified separately. A peroxidase-positive leukocyte concentration of 1.0 × 10⁶/mL or more meets the WHO consensus threshold for clinical significance and supports genital-tract inflammation. Infection is one possible cause, and targeted microbiology is performed when indicated. More than 1 million spermatids/mL signals disrupted spermatogenesis from viral infection, toxin exposure, or genetic disease.

Round-cell concentration can be back-calculated from a stained-smear differential. With N equal to round cells counted per 100 mature sperm and S equal to the already-determined sperm concentration in millions per mL:

round-cell concentration (million/mL) = N × S ÷ 100

A stained smear shows 3 round cells for every 100 sperm, and the measured sperm concentration is 48 million/mL. Round-cell concentration = 3 × 48 ÷ 100 = 1.44 million/mL. Because that exceeds the 1 million/mL threshold, a peroxidase stain is indicated to determine whether the round cells are predominantly leukocytes or spermatogenic cells.

Motility is assessed on a liquefied specimen within 1 hour, delivering a consistent volume (for example, 10 µL under a 22 × 22 mm coverslip) in duplicate, and either estimating the percentage moving forward across about 20 high-power fields or classifying 200 sperm per slide by category. WHO 6th edition uses four categories: rapidly progressive (RP), slowly progressive (SP), nonprogressive (NP), and immotile (IM).¹⁴ The older combined progressive-motility scheme and the a/b/c/d speed grades are historical. Computer-assisted semen analysis (CASA) systems provide the same information objectively and extend it to concentration and morphology, filtering out debris and detached flagella that would otherwise corrupt manual motion analysis.

Morphology is assessed on a thin, stained (Wright, Giemsa, Shorr, or Papanicolaou), oil-immersion smear of at least 200 sperm.¹⁴ Evaluate the ovoid head (about 5 µm × 3 µm), including its acrosomal cap, which covers roughly half the head surface and two-thirds of the nucleus and contains enzymes that permit ovum penetration. Also evaluate the neckpiece, midpiece (about 7 µm, the thickest region because its mitochondrial sheath powers tail movement), and tail. Head abnormalities compromise ovum penetration; neckpiece, midpiece, and tail abnormalities impair motility. Routine evaluation names double, giant, amorphous, pin, tapered, and constricted heads, and doubled, coiled, or bent tails. Kruger’s strict criteria add measured head, neck, and tail dimensions, acrosome size, and vacuole assessment with a stage micrometer or morphometry software. The WHO recommends these criteria, which are standard in assisted reproduction but not yet routine in general clinical laboratories. Normal-form thresholds depend on the criteria and named WHO edition applied, so both must appear on the morphology report.

Vitality distinguishes live but immotile sperm from dead sperm when concentration is normal but motility is markedly reduced. Perform an eosin-nigrosin stain within 1 hour of ejaculation and count at least 200 sperm. Living sperm exclude the dye and stay bluish-white; dead sperm take up the red eosin against the purple nigrosin background. Use the lower reference centile from the named current WHO manual edition.¹⁴ Vitality should track closely with motility. Many vital but immotile sperm point to a structural flagellar defect, and the vitality result separates that defect from simple cell death.

Seminal fructose and antisperm antibodies

Seminal fructose is evaluated when semen volume is low with azoospermia or acidic pH, especially when ejaculatory-duct obstruction, congenital bilateral absence of the vas deferens, or seminal-vesicle dysfunction is suspected. Low sperm concentration alone does not indicate fructose testing.¹⁴ For the resorcinol screen, dissolve 50 mg resorcinol in 33 mL concentrated hydrochloric acid, dilute to 100 mL with water, mix 1 mL semen with 9 mL reagent, and boil. Orange-red indicates fructose. Quantify fructose spectrophotometrically (normal 13 µmol/ejaculate or more). Because sperm metabolize fructose after collection, test specimens within 2 hours or freeze them.

Antisperm antibodies occur in either partner, most often the man. Disruption of the normally isolating blood-testes barrier by surgery, vasectomy reversal, trauma, or infection sensitizes the immune system to sperm antigens; damaged, antibody-coated sperm can also sensitize a female partner. Clumped sperm on a routine wet mount, graded few, moderate, or many, raise suspicion.¹⁴ The mixed agglutination reaction (MAR) screens chiefly for IgG by using antihuman globulin to bridge sperm-bound antibody to IgG-coated particles. The immunobead test uses polyacrylamide beads coated with anti-IgG, anti-IgM, or anti-IgA to localize binding to the head, neck, midpiece, or tail. Both tests detect and localize the antibody coating. WHO 6th edition gives no evidence-based reference values for either assay, so each laboratory validates its own range. A previous-edition threshold of 50% bound sperm has limited evidence, so results call for cautious interpretation; tail-tip-only binding is not pathologic. Head-bound antibodies impair mucus and ovum penetration, and tail-bound antibodies impair motility.

Post-vasectomy specimens

Post-vasectomy semen analysis follows the American Urological Association Vasectomy Guideline (2026).¹⁵ Submit the first post-vasectomy semen sample as early as 8 weeks, according to the surgeon’s protocol. Examine one fresh, well-mixed, uncentrifuged specimen within 2 hours of ejaculation. A single sample showing azoospermia, or 100,000 nonmotile sperm/mL or fewer, permits discontinuation of other contraception when the applicable AUA criteria are met. If examination occurs after 2 hours, require azoospermia before clearance. Continue contraception until clearance. Any motile sperm means clearance has not been achieved, and persistent motile sperm at 6 months warrants clinical review for repeat vasectomy.

Accessory gland markers and quality assurance

Biochemical markers tie each accessory gland’s contribution to a measured value.¹⁴

WHO 6th edition supplies lower reference limits for fructose, zinc, and neutral alpha-glucosidase; the citric acid and acid phosphatase thresholds are retained from earlier WHO editions and textbooks.

Complexity categorization follows the CLIA framework: qualitative semen analysis limited to the presence or absence of sperm is a moderate-complexity test, detection of motility qualifies as a provider-performed microscopy procedure, and comprehensive manual semen analyses are typically laboratory-developed or modified procedures that default to high complexity.¹⁶ Because control materials are limited and motility and morphology assessment are inherently subjective, standardized WHO procedures, commercial controls and training aids, and CAP and American Association of Bioanalysts proficiency testing support quality assurance. CASA reduces inter-operator variability.

References

1. McPherson RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory Methods. 24th ed. Elsevier; 2022.
2. Dashe JS, Pressman EK, Hibbard JU; Society for Maternal-Fetal Medicine. SMFM Consult Series #46: Evaluation and management of polyhydramnios. Am J Obstet Gynecol. 2018;219(4):B2-B8. doi:10.1016/j.ajog.2018.07.016.
3. American College of Obstetricians and Gynecologists, Society for Maternal-Fetal Medicine. Indications for outpatient antenatal fetal surveillance: ACOG Committee Opinion No. 828. Obstet Gynecol. 2021;137(6):e177-e197. doi:10.1097/AOG.0000000000004407.
4. Liley AW. Liquor amnii analysis in the management of the pregnancy complicated by rhesus sensitization. Am J Obstet Gynecol. 1961;82:1359-1370.
5. Queenan JT, Tomai TP, Ural SH, King JC. Deviation in amniotic fluid optical density at a wavelength of 450 nm in Rh-immunized pregnancies from 14 to 40 weeks' gestation: a proposal to standardize the use of the delta OD 450. Am J Obstet Gynecol. 1993;168(5):1370-1376. doi:10.1016/S0002-9378(11)90771-0.
6. Society for Maternal-Fetal Medicine. Clinical Guideline #8: the fetus at risk for anemia: diagnosis and management. Am J Obstet Gynecol. 2015;213(5):697-709. Reaffirmed 2025. SMFM Clinical Guideline #8.
7. American College of Obstetricians and Gynecologists. Neural tube defects: ACOG Practice Bulletin No. 187. Obstet Gynecol. 2017;130:e279-e290. ACOG Practice Bulletin No. 187.
8. American College of Obstetricians and Gynecologists, Society for Maternal-Fetal Medicine. Medically indicated late-preterm and early-term deliveries: ACOG Committee Opinion No. 831. Obstet Gynecol. 2021;138(1):e35-e39. Interim update February 2024. doi:10.1097/AOG.0000000000004447.
9. Association for Diagnostics and Laboratory Medicine. Fetal lung maturity testing. In: Optimal Testing: ADLM's Guide to Lab Test Utilization. December 12, 2023. Accessed August 31, 2026. ADLM test utilization guide.
10. American College of Obstetricians and Gynecologists. Prelabor rupture of membranes: ACOG Practice Bulletin No. 217. Obstet Gynecol. 2020;135:e80-e97. Reaffirmed 2026. ACOG Practice Bulletin No. 217.
11. QIAGEN. AmniSure ROM Test: Instructions for Use. Accessed August 31, 2026. AmniSure instructions for use.
12. Clinical Innovations. ROM Plus Rapid Rupture of Fetal Membranes Test: Instructions for Use. ART-0087, Rev 02. Accessed August 31, 2026. ROM Plus instructions for use.
13. Hologic. Actim PROM: Instructions for Use. 2024. Accessed August 31, 2026. Actim PROM instructions for use.
14. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 6th ed. World Health Organization; 2021. Accessed August 31, 2026. WHO laboratory manual.
15. American Urological Association. Vasectomy: AUA Guideline (2026) Part I. J Urol. 2026. doi:10.1097/JU.0000000000004861.
16. Centers for Disease Control and Prevention. Test complexities. Clinical Laboratory Improvement Amendments. Updated 2024. Accessed August 31, 2026. CDC CLIA test complexities.