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Amniotic fluid and semen

16 min

Skills you'll build4 skills
  • Name the interferences that make a rupture-of-membranes test misleading
  • Decide whether a semen collection is complete and timely enough to analyze
  • Calculate total sperm number from concentration and ejaculate volume
  • Distinguish sperm immotility from loss of vitality

At the bench: Check specimen identity and suitability before analysis · Coordinate cell examination and chemistry for a body fluid specimen

Try first

Try first

A semen specimen arrives 20 minutes after collection. The man writes on the requisition that the first part of the ejaculate missed the container. What do you do?

Keep going to see the reasoning.

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Rupture-of-membranes tests

Nitrazine paper turns color in an alkaline sample, and amniotic fluid is alkaline. Blood, semen, cervical mucus, urine, antiseptics and some lubricants can also turn it positive. A fern pattern on a dried slide can be mimicked by cervical mucus, semen or blood, or obscured by a poorly prepared slide.1 Commercial tests that detect amniotic proteins are also adjuncts. The FDA has warned that using any of them alone can give false-negative or false-positive results.2 Each result is interpreted with the others and the clinical assessment.

A complete, timely semen collection

  1. Abstinence of 2 to 7 days, with the actual interval recorded.
  2. The whole ejaculate, by masturbation, in a container verified as nontoxic to sperm.
  3. Any lost portion recorded, with whether it was the first part.
  4. Transport at 20 to 37 °C, arriving preferably within 30 minutes and no later than 50 minutes, so testing starts within 60 minutes.

The first portion of the ejaculate is the richest in sperm. Losing it lowers the concentration and the total number. After an incomplete collection, a repeat collection usually comes before a low result is interpreted.3

Total sperm number

Total sperm number (million per ejaculate) = sperm concentration (million/mL) × whole-ejaculate volume (mL). Record an incomplete collection because it can change the result. Concentration is the number per milliliter. The total is the number in the whole ejaculate. The WHO lower reference limits are 16 million/mL for concentration and 39 million per ejaculate for the total. A result below a limit does not by itself classify a man as infertile.3

Sperm vitality

A sperm that does not move can still be viable. Vitality testing assesses the cell membrane. In an eosin-nigrosin preparation, a live sperm excludes eosin and stays unstained, and a dead sperm takes up the dye. Vitality is most useful when total motility is below 40%. Count at least 200 sperm and report the percentage live.3

Follow the bench demonstration

Sources for this step
References
  1. American College of Obstetricians and Gynecologists. Prelabor rupture of membranes: ACOG Practice Bulletin No. 217. Obstet Gynecol. 2020;135(3):e80-e97. Accessed September 27, 2026. https://www.acog.org/clinical/clinical-guidance/practice-bulletin/articles/2020/03/prelabor-rupture-of-membranes
  2. US Food and Drug Administration. FDA alerts healthcare providers, women about risks associated with improper use of rupture of membranes tests. Published August 8, 2018. Accessed September 27, 2026. https://www.fda.gov/news-events/press-announcements/fda-alerts-healthcare-providers-women-about-risks-associated-improper-use-rupture-membranes-tests
  3. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 6th ed. World Health Organization; 2021. Accessed September 27, 2026. https://www.who.int/publications/i/item/9789240030787

Watch one

Read each decision and its explanation.

A semen specimen arrives 25 minutes after collection, kept at body temperature. The requisition records 3 days of abstinence and no lost portion. The empty container weighed 35.2 g before collection and weighs 38.4 g with the specimen. The chamber count gives a concentration of 14 million/mL. What is the total sperm number?

  1. Check the collection: complete, 3 days of abstinence, delivered in 25 minutes at body temperature.

    A lost portion or a late delivery would affect every result.

  2. Find the volume: 38.4 g − 35.2 g = 3.2 g, so 3.2 mL.

    Volume by weight uses the recorded empty weight, and semen is assumed to be 1 g/mL.

  3. Multiply: 14 million/mL × 3.2 mL = 44.8 million.

    Concentration is per milliliter, so it must be multiplied by the whole volume.

  4. Compare with the WHO lower limits: the concentration is below 16 million/mL, and the total is above 39 million.

    Concentration and total are compared with their own limits.

Total sperm number = 14 million/mL × 3.2 mL = 44.8 million per ejaculate.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A vaginal-fluid specimen tested for suspected rupture of membranes contains visible blood, and the nitrazine result is positive. Which limitation applies?

Incorrect. Blood is alkaline, so it pushes nitrazine toward a positive reaction and makes the result less reliable.

Correct. Nitrazine detects an alkaline sample, and blood, semen, cervical mucus, urine, antiseptics, and some lubricants can give a false-positive result, so interpret it with the fern test and the clinical assessment.

Incorrect. Nitrazine responds to pH. The commercial biomarker tests are the ones that detect amniotic-fluid proteins such as placental alpha microglobulin-1.

Hint
  1. Nitrazine responds to one property of the specimen.
  2. Consider whether blood shares that property with amniotic fluid.

Review Suspected rupture of membranes

Problem 2 of 3 · Hints available

An immotile sperm excludes eosin in a valid eosin-nigrosin vitality preparation. What does that finding support?

Incorrect. Immotility and loss of vitality are separate findings, so vitality testing is used to tell live immotile sperm from dead ones.

Incorrect. An intact membrane keeps eosin out, so an unstained sperm is live.

Correct. Live sperm exclude eosin and dead sperm take it up, so this sperm is live but immotile. Count at least 200 sperm and report the percentage viable.

Hint
  1. Eosin enters a sperm only through a damaged membrane.
  2. Consider what an unstained sperm shows about its membrane.

Review Vitality

Problem 3 of 3 · On your own

A complete ejaculate has a volume of 1.8 mL and a sperm concentration of 35 million/mL. What is the total sperm number per ejaculate, in millions?

Show the answer

63 million

Total sperm number = 35 million/mL × 1.8 mL = 63 million per ejaculate. The 35 million/mL is the concentration in 1 mL.

Review Sperm concentration and total number

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Tomasz Pelletier, 34, MRN 7731058, collects a semen specimen at home at 08:05 after 4 days of abstinence.
  • He carries it inside his jacket, and it arrives at 08:40.
  • He reports no lost portion.
TestResultReference intervalFlag
Volume2.0 mL≥1.4 mL≥1.4
Sperm concentration20 million/mL≥16 million/mL≥16
Total motility25 % Low≥42 %≥42Low
Vitality70 % live≥54 % live≥54

Specimen: Not measured on semen. Semen, complete collection, received 35 minutes after collection

Decision 1 of 3

Is the specimen suitable to analyze?

Arrival within 30 minutes is preferred. The limit is 50 minutes, and 35 minutes is within it.

The collection is complete, the jacket kept it between 20 and 37 °C, and testing can start within 60 minutes.

Home collection is allowed when the temperature and delivery time are met. Both are met here.

Review Collection and timing

Decision 2 of 3

What is the total sperm number?

That is the concentration in 1 mL. The ejaculate contains 2.0 mL.

Reported concentration as the total number

Concentration is per milliliter, and total sperm number multiplies it by the whole ejaculate volume. Reporting 24 million/mL as the total for a 2.5-mL ejaculate gives 24 million. The total is 24 million/mL × 2.5 mL = 60 million.

This divides the concentration by the volume. The total is the concentration multiplied by the volume.

20 million/mL × 2.0 mL = 40 million per ejaculate, just above the WHO lower limit of 39 million.

Review Sperm concentration and total number

Decision 3 of 3

Total motility is 25% and vitality is 70% live. How are the two results interpreted together?

Every motile sperm is alive, so 70% live minus 25% motile leaves about 45% live and immotile. Most of the immotile sperm are alive.

Immotile sperm can be alive. The eosin-nigrosin result measured 70% live, so only 30% are dead.

Assumed every immotile sperm is dead

Immotile sperm can be alive. Vitality testing checks membrane integrity, and live sperm exclude eosin. Reporting all immotile sperm as dead gives a vitality result that was never measured.

Vitality is most useful when total motility is below 40%, as it is here. It distinguishes live immotile sperm from dead sperm.

Review Vitality

The interpretation depends on the vitality result together with the motility. A complete, warm, timely specimen gave a total of 40 million, and 70% live with 25% motile shows that most of the immotile sperm are viable.

For reference

Sources checked