by Kelsey Tyssowski
figures by Olivia Foster

Let me look at your pee, and I’ll tell you your future.

For over 3000 years, women wondering if they’ll be mothers have heard some variation of this phrase. In the first known pregnancy tests, ancient Egyptian women urinated on barley or wheat seeds: quickly sprouting seeds indicated pregnancy. While this may sound like pseudoscience, several modern studies have shown that it works pretty well, correctly identifying 70-85% of pregnancies.

The ancient Egyptians were certainly onto something, as modern pregnancy tests work in much the same way: they detect a hormone in urine. But it’s still taken thousands of years of scientific progress to get us from peeing on seeds—and waiting a week to see if they sprout—to the fast, convenient pregnancy tests found in all pharmacies today.

“Piss Prophets”: Early history of the pregnancy test

Although it’s not intuitive that analysis of urine would be the key to pregnancy detection, most historical pregnancy tests have, for unknown reasons, focused on just that (Figure 1). In Europe, starting in the Middle Ages, “Piss Prophets” claimed to be able to predict pregnancy with a variety of bizarre urine tests. They believed that pregnant women’s urine would rust a nail, change the color of a leaf, or be home to tiny, living creatures. Based on what we know today, it’s unlikely that any of these tests were actually able to correctly detect pregnancy.

However, the first of the modern tests that could accurately detect pregnancy was no less bizarre. This test was developed in 1927 by German scientists, Aschheim and Zondek. They found that injecting a pregnant woman’s urine into sexually immature female mice would cause the mouse’s ovaries to grow and produce eggs.

Figure 1: A timeline of pregnancy testing. From ancient Egypt to today, the urine-based pregnancy test has improved in speed, accuracy, and feasibility. The oldest known test involved urinating on grain seeds and seeing if they sprouted. The late 1920s marked the first modern pregnancy tests, in which urine was injected into animals: pregnant women’s urine made them ovulate. These tests required shipping urine to a lab and took at least a week to get results. Starting in 1960, antibodies allowed testing for pregnancy to be done in doctor’s offices, making pregnancy testing faster and more routine. By 1971, an at-home version of this antibody-based test was available in Canada, although at-home testing didn’t reach the USA until 1977. The first stick tests similar to at-home tests on the market today were developed in 1988.

What’s so Special About Pregnant Women’s Urine?

If you’re like me, you’re now left with a burning question that you probably weren’t expecting to ask today: why does a pregnant lady’s pee make a juvenile mouse ovulate?

The short answer is that the urine of pregnant women has a unique blend of hormones (Figure 2). A woman (actually, anyone with a uterus, though throughout the article I’ll refer to women) becomes pregnant when an egg released from her ovary is fertilized by sperm. This fertilized egg then starts growing into an embryo and releases a hormone called hCG (a.k.a. human chorionic gonadotropin), which builds up in a woman’s body during early pregnancy. Like all hormones, hCG sends a signal. hCG’s particular job is to tell the uterus to stop the woman’s regular menstrual cycle and prepare itself for pregnancy.

Since hormones are excreted into the urine after they’re done sending their signals, the urine of pregnant women has hCG in it, whereas the urine of anyone who is not pregnant does not. hCG, it turns out, makes mice ovulate, allowing Aschheim and Zondek’s test to detect pregnancy. Since the invention of this test, all modern pregnancy tests have focused on detecting hCG.

Figure 2: Hormone levels during the menstrual cycle and pregnancy. The hormones, or signal-sending molecules, in a woman’s body change during the menstrual cycle and pregnancy. All successful urine-based pregnancy tests have detected hormones, which are secreted into the urine. During the menstrual cycle (right), the reproductive system changes throughout the month. Hormones signal for each of these changes to happen (left-top). Most notably, high levels of lutenizing hormone stimulate ovulation. When a woman becomes pregnant, her reproductive system must change in a different way to support the growing fetus. Upon pregnancy, a pregnancy-specific hormone, hCG, accumulates in a woman’s body (left-bottom). hCG is very similar to lutenizing hormone. Because of this similarity, hCG can send an ovulation signal, causing animals to ovulate when injected with the urine of pregnant women.

But why does hCG make young mice ovulate? hCG is very similar to another hormone called luteinizing hormone. In humans and in mice, luteinizing hormone stimulates the ovaries to release an egg every month of the menstrual cycle, and hCG can mimic that signal. Aschheim and Zondek’s test worked because the level of hCG in the urine of pregnant women is much higher than that of luteinizing hormone in non-pregnant, ovulating women: there’s never enough luteinizing hormone in urine to make a mouse ovulate.

Mice, Rabbits, and Frogs: Animal-based pregnancy tests

Although Aschheim and Zondek had developed an accurate test, it wasn’t exactly simple, especially compared to the drug store tests available today. They had to inject five mice per woman and wait about a week before getting the result. And even then, they could only detect the high levels of hCG that women have starting around 2 weeks after a missed period. In addition, the use of so many animals made it so that the test was expensive and relegated it to a few labs that received shipments of urine through the mail.

Today, women can know that they are pregnant as soon as 10 days after conception—a few days before a missed period—using tests they can buy for less than $10 in any drug store. But up through the 1930s, women had to wait until at least a month after conception, visit a doctor, mail her urine to a lab, and then wait at least another week to get the result of the test. Unsurprisingly, pregnancy testing in this era was not routine, used only by those who were wealthy or needed to know if they were pregnant for medical reasons.

The juvenile mouse test was slightly improved in 1931 by an American doctor, Maurice Freidman, who swapped juvenile mice for adult rabbits, which were easier to inject.

However, a test using frogs, developed the British scientist Lancelot Hogben, marked the pinnacle of these animal tests. Frogs lay eggs, so they don’t need to be killed or dissected to assess ovulation and could therefore be reused, lowering test costs. This test also gave results faster: within twelve hours. The frog test increased the availability of pregnancy testing, but it still required shipping urine to a select number of frog labs.

Tens of thousands of frogs were injected with urine throughout the 1940s-1960s, but pregnancy testing in this era was still not the norm. Most labs would only test urine sent by a doctor, meaning that women had to rely on their doctors to get tested. And many doctors—and health insurance companies—would only grant a woman a test if she had some urgent medical reason that she needed to know she was pregnant. Most women instead relied on morning sickness and sore breasts as early clues to their pregnancy, not visiting a doctor to confirm until months after conception.

Blood and Test Tubes: The pee-on-a-stick precursors

This all changed in the 1960s when scientists began to develop pregnancy tests that didn’t require the use of live animals and could therefore be done in doctor’s offices. At first, these tests had similar sensitivity for hCG, but by the early 1970s, Drs. Vaitukaitis, Braunstein, and Ross had developed a test that could detect pregnancy just 2-3 weeks after conception—on the first day of a missed period. These new tests used antibodies.

Antibodies are molecules that recognize and stick to other molecules. In their usual job, they’re sticking to the molecules of bacteria or viruses to alert the immune system to these invaders. In pregnancy tests, however, scientists engineered antibodies that recognized and stuck to hCG. Armed with these hCG antibodies, the urine of pregnant women, and, strangely enough, sheep’s blood, we had a new pregnancy test.

To perform the test, scientists attached hCG to the outside of the sheep’s blood cells, decorating the blood cells with hCG. They then mixed these blood cells with the other test components: hCG antibodies and urine.

Based on the way that antibodies stick to hCG and to each other, the hCG-decorated blood cells would clump if they were mixed with the urine of a woman who was not pregnant. On the other hand, if the blood cells and hCG antibody were mixed with the urine of a woman who was pregnant, the blood cells would not clump. So by looking at the clumpiness of the blood cells, doctors could tell if a woman was pregnant (Figure 3).

Figure 3: Early antibody-based pregnancy tests. Early antibody-based pregnancy tests, including the first at-home tests, used hCG antibodies and hCG-decorated blood cells. The person performing the test would mix these two components with a woman’s urine. If she was not pregnant, the hCG antibodies would stick to the hCG-decorated blood cells and to each other, causing the blood cells to clump together (left). On the other hand, if the woman was pregnant, the high levels of hCG in her urine would stick to the hCG antibodies, preventing them from sticking to the blood cells (right). Therefore, if the woman was pregnant, her urine would prevent the blood cells from clumping. When reading the results of the test, the operator would look at whether the blood cells clumped to determine pregnancy.

These and other, similar antibody-based tests could give doctors results in a few minutes to hours and finally made pregnancy testing mainstream. At first, women still had to visit their doctor to get a pregnancy test.

But that, too, changed in the 1970s (1971 in Canada, 1977 in the US) when the first at-home pregnancy test hit the market, using this same blood-and-antibody technology. This test looked like a kid’s chemistry kit, including test tubes, droppers, dried capsules of sheep’s blood cells, and hCG antibody serum. Success in completing the test required carefully following a 10-step process all while keeping the test tubes in a place free from vibrations for two hours. But if used correctly, it was 97% accurate for a positive result and 80% accurate for a negative one. And for the first time, a woman could confirm that she was pregnant without ever contacting a doctor.

Pee on a Stick: The birth of the modern pregnancy test

Since the 1970s, at-home pregnancy tests have become the norm, with about 20 million sold per year in the US alone. Current stick tests, which were developed in 1988, are much easier to use, don’t involve sheep’s blood or test tubes, and are still extremely accurate: about 99% for a positive result. But they are doing the same thing we’ve been doing for thousands of years: detecting hormones in women’s urine. Modern pregnancy tests still use hCG antibodies, but instead of interacting with hCG-decorated blood cells, the antibodies interact with dye-activating molecules. These dye-activating molecules are what causes the blue line, or other symbol, to appear on positive pregnancy tests.

Women today usually find out that they’re pregnant just after (and sometimes before) missing a period in the comfort of their own bathroom without the involvement of a doctor. But as recently as the 1950s, women could not definitively confirm pregnancy until they started to noticeably gain weight or feel the fetus move—several months into the pregnancy. And until the 1970s, women relied on doctors to tell them that they were pregnant.

The development of the pregnancy test was not just a feat of scientific advance, but also allowed for a cultural revolution. The at-home pregnancy test became available a time when women were fighting back against the male-dominated medical establishment, trying to learn more about their health and their bodies. And it granted women more information about and control over their bodies. Women could use this information as they liked, without pressure from a doctor: they could decide for themselves to get a safe, early abortion or start prenatal care.

As a woman who is grateful for this cultural advance, I want to say thanks to the first person who thought, “I bet I can tell if she’s pregnant by looking at her pee.”

Kelsey Tyssowski is a graduate student in the Department of Genetics at Harvard Medical School. She is also a former editor-in-chief of the SITN Blog. You can find her on Twitter: @kelseytyss.

For more information:

  • A Thin Blue Line: a pregnancy test history put together by the NIH
  • An academic article about the science and culture of animal-based pregnancy tests
  • An excellent, but hard-to-access, academic review article about the history of the pregnancy test, and a less-descriptive, but similar, open-access article
  • An article in The Atlantic that describes the history of the pregnancy test from a more historical perspective
  • A video explaining the science behind modern pregnancy tests

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