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False Negative Pregnancy Test: How to Identify the Hook Effect

False negative results can be stressful part of your initial steps in pregnancy. You’re sure that you’re pregnant because you recently had a positive pregnancy test, but now a few weeks later, you see a faint pregnancy test line or no line at all. Should you worry?

Not yet.

If you take a pregnancy test after about week five of pregnancy, you might experience a type of false negative called the hook effect. To explain why this happens, this post will cover how a pregnancy test works, what the hook effect is, and what you can do.

How does a home pregnancy test work?

A home pregnancy test measures amount of hCG in your urine. This type of a test is a sandwich enzyme immunoassay. It sounds complicated, and the chemistry of it is, but if you’ve made a peanut butter and jelly sandwich before, then you’ll understand how this test works.

HCG is a really big molecule that is made of two parts: the alpha part and the beta part. Let’s call the alpha unit of hCG “the peanut butter” and the beta unit “the grape jelly”.  To detect both parts of hCG, a pregnancy test loads the stick with two different antibodies—let’s call them “the bread slices”. The first antibody gloms on to the alpha unit (“peanut butter”) and the second antibody gloms on to the beta unit (“the grape jelly”), which forms a chemical sandwich.

Once hCG is successfully sandwiched, when you take a pregnancy test this triggers release of a dye—that’s the dark line on a pregnancy test that means you’re pregnant! The first line on a pregnancy test is a control test that simply makes sure the antibodies and dye are working.

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Why can I get a positive pregnancy test then negative?

There are two reasons why you would get a positive pregnancy test and then a negative:

  1. Miscarriage/chemical pregnancy: With a miscarriage, hCG levels rise and then drop back down. As hCG levels decrease, you will notice the second line on a pregnancy tests getting lighter or disappearing completely.
  2. The hook effect: As your pregnancy progresses, changes in the type of hCG can cause false negative pregnancy test results. Read on to understand how and why this occurs.

You might expect that because the further along in pregnancy, the darker the line on your pregnancy test. But this is not the case, and it’s because of the antibodies loaded in the pregnancy test.

Just as you can have other variations of peanut butter and jelly sandwiches besides the one with classic grape jelly—say, with strawberry jam—there are also variations of hCG. Why does this matter? The relative amounts of these different variations change over the course of pregnancy, and this messes with the antibodies in your pregnancy test.

Pregnancy tests are good at measuring intact hCG—or when there’s peanut butter AND grape jelly—which is great during the first few weeks of pregnancy. However, around week five, the dominant form of hCG in your urine is hCG-βCF, which is a version of hCG that is all beta unit and no alpha, or all strawberry jam and no peanut butter.

That’s a problem for a pregnancy test. The test gets overwhelmed with all the beta fragments (or strawberry jam), which interferes with test’s ability to find intact hCG (or peanut butter and grape jelly).

Instead of a linear relationship where the line gets darker the more pregnant you are, the pattern actually looks more like a hook or curve, with a darkening line from weeks three to five of pregnancy, then a fading line thereafter.

This means it’s possible to get a positive pregnancy test, and then a few weeks later get a negative one—even though you’re still pregnant! Scientists have even tested this by taking a positive urine sample, immediately adding pure hCG-βCF, and then observing a negative result.

How can you tell if you’re experiencing the hook effect?

The hook effect can cause someone who has a healthy, normal pregnancy to get a negative pregnancy test. If you get a negative pregnancy test after a positive, how can you tell if it’s the hook effect?

There’s a simple test: all you have to do is dilute your urine, which prevents hCG-βCF excess. Here’s how:

  1. Collect your urine in a cup
  2. Dilute your urine with an equal quantity of water
  3. Re-take a pregnancy test using the diluted urine
  4. If you are experiencing the hook effect, the pregnancy test using diluted urine will be darker


Read more about pregnancy tests:

Should you take a pregnancy test?

Period late, but negative pregnancy test?

What’s the best pregnancy test?

Aarthi Gobinath, PhD

Aarthi Gobinath earned her PhD in neuroscience from the University of British Columbia. Her research covers the ways that stress affects the male and female brain differently.

She tackled the issue of sex bias in research by looking at why standard treatments for depression don't always work in the case of postpartum depression. Her work has been covered by Vice and Massive Science.

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