After the discovery of hormones in the early 20th century, new methods of pregnancy testing were developed. Some of these involved animal use, but how did the rabbit test work, and when did it get replaced?
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Welcome to Stuff you missed in History Class from how Stuff Works dot com. Hello, and welcome to the podcast. I'm Holly Frying and I'm Tracy d Wilson. So Tracy. This weekend, I was hanging out in another city with some friends. UM, and I made a joke referencing the movie The Rabbit Test. Do you know that movie? I know of it, but I don't think I've ever seen it. I will tell you a funny story after I tell you what the movie is about, because it evidences the mind of a child and how they process information. So in that movie, which is a comedy directed by the late great Joan Rivers, Billy Crystal stars as a man who gets pregnant and the title is a joke based on the now outdated practice of using rabbits as part of a medical test to determine if a woman was pregnant. Now, this movie came out, like I said, in seventy eight. So I was seven when it huh out, And I remember, Um, I had said something like I don't know if I had seen a poster or an ad or something, and I was like rabbits, Like I was excited to see this movie and my siblings, who are all a good bit older than me. We're like, oh, no, that movie is not for you, because of course it was about sex and reproduction. But my perception as a seven year old was that it must be a terrifying horror movies. I didn't know until I was in my twenties and it was a comedy. I was like, no, it's like a scary sci fi thriller, is what I made up in my head. Well, and and based on everything I know of you, Holly, I was genuinely astonished when I got this email from you with this outline last night, because it contains a number of things that are just not your bag, right, like talking about you know, pregnancy and um um, and I'm going to get to the animal testing things and we have a we have our our our opt out discussion in a moment. But my point in all of this is that I had had mentioned this movie to friends, and those friends are all a bit younger than my husband and I are, and they had never heard of the movie, and they had never heard of the use of rabbits in pregnancy testing. And I was trying to explain this to them with my not scientific knowledge and their eyes got really big and they seemed completely incredulous and thought maybe I was pulling one over on them. So, um, I was like, well, I know what I want to work on next, and it's this Because I thought everybody knew, they don't. Uh. If you also listen to the podcast saw Bones and you should, they indicated an episode on the history of pregnancy testing, and it mentions the rabbit test very briefly, but it's really more of a broad overview of how pregnancy testing has evolved over the centuries, and we are instead going to look really closely at how this particular test and one very like it both were developed in the twentieth century. So you may have surmised already, but heads up, this episode is going to involve a significant amount of discussion of animals being used in medical testing, with an outcome of mortality for the animal in most cases. Um, So if that is something that you would rather not hear about or rather not perhaps share with younger history buffs, you can tap right out of this one. Yeah. Yeah, I still have a whole layer of surprise of like, well, I love science, I know you do. It just seems like you know you're talking about the movie coming across as a terrifying horror movie. I feel like the subject is like a holly terrifying horror movie. Yeah. No, I mean it's not me. It's not me having a child, and it doesn't really talk about like any of the actual pregnancy, just the pathology and medical testing. So I think that's why it's a little easier for me to dig in on. And somehow I don't know why I seem to be able to handle this particular discussion of animal testing. Yeah, we'll see. I could start crying halfway through. We'll see how it goes. It's always different typing it than it is saying it. But definitely one of those subjects that is always on, like the right of first refusal list what I'm thinking of things to talk about. So, uh today, Home pregnancy tests they're inexpensive, very common. Usually the first way people find out if they are pregnant just go to the store and pick one up. But there have been plenty of folk medicine tests that people could try on their own over the years before they were actual working pregnancy tests and for a scientific confirmation of that pregnancy. Up until the seventies, you pretty much had to go to the doctor. You couldn't just pick up a home test. The use of urine to test for pregnancy was nothing new at all. It had been part of how people tested for pregnancy for centuries. Yeah, people had intuited that there was something going on with urine where uh it was a way to determine if someone was in fact carrying a child, But they were not necessarily rooted in science. Always they involved things like, you know, peeing on grain and see if it turned color. But a really major development took place in nineteen o two when E. H. Starling, who was a physician, and W. M. Bayliss, who was a physiologist, discovered secreton. And secreton is a polypeptide that is made up of more than two dozen amino acids. But what is really important is that it was the first hormone ever discovered. Secreton is a digestive hormone. It has nothing to do specifically with reproduction and Starling didn't start using the word hormone for several years. He first introduced it into medical nomenclature and a lecture that he gave at London's Royal College of Physicians in nineteen o five titled on the Chemical Correlation of the Functions of the Body, and in that lecture, Starling defined hormones as quote the chemical messengers which, speeding from cell to sell along the bloodstream, may coordinate the activities and growth of different parts of the body. And from the point where he established the term, all manner of discovery and research in the field of hormones began. Yeah, and that's still the pretty basic definition that you learn of what a hormone is in school. Yeah, at a very basic level. So from the nineteen teens, a number of researchers were examining the link between hormones and pregnancy. Through the introduction of extracts of human placenta into other animals in a variety of laboratory experience, it became clear that there were hormones in the mix that were related to reproduction. All of this sounds so obvious now, but at the time, I know, I think about like the shorthand of how people just attribute like people's urge to uh, you know, have romantic involvement with one another, they'll just go hormones. But like at the time, this was like mind blowing concept that that was what was driving your romantic interest. Yeah, And the hormone that was eventually identified in all of this was human choreonic gonadotropin, which is more commonly known as hCG, and that was the hormone that unlocked the door to pregnancy testing. So just for a little bit of clarity, but not too much, because I'm not going to give you the hard science because I will miss something up. But hCG is actually four independent molecules which each have their own functions in which are produced by separate cells. hCG is not only used as an indicator of pregnancy. Certain cancers can also be detected based on tests which analyze hc G content in a person's bloodstream. And hCG is present in women even when they are not pregnant, although it's a pretty small amount. They don't produce significant amounts unless they are crying a child. But pregnancy tests are designed to measure the levels of hCG and once that passes a certain threshold, it is a reliable indicator of pregnancy. In six two German doctors Selma Asheim and Bernhard Zondeck developed a pregnancy test that used mice. The preparation was described in a nine team thirty article in the journal California and Western Medicine by authors Herbert M. Evans, m d. And Miriam E. Simpson, m D. As follows the morning urine is sent into the laboratory and clean bottles. They meaning Ashaim and Zondeck recommend the addition of one drop of tri cresal per twenty five cubic centimeters of urine if it is necessary for the sample to be sent by mail. A group of five mice each wing six to eight grahams is used to test each urine specimen. So the mortality rate for mice just to test a single person sample was quite high at that point, but per Evans and Simpson, this test was far more reliable than any other options, and it produced results in as few as four days. It worked because the urine sample, which was injected into very young mice in a specified regiment. It was multiple injections over the course of several days would catalyze sexual maturation in the mice if the urine was from a pregnant woman. On the fifth day, between ninety six hundred hours after the start of the injection process, the mice were killed and then the crop seed based on an examination of the mice's ovaries, doctor could determine if the woman who had given the urine sample was pregnant. If the ovaries remained mostly small and smooth, the test was negative, but if there was enlargement and maturation beyond the normal development, it was positive. Ash I'man's on Deck also developed a variation on this test for cases where someone might more urgently need to know if they were pregnant. Although it came at a cost of more mice, They used more mice in the experiment and then that they dissected them earlier in the range past when they had begun the testing. That larger sample set made up for the shorter testing period. We are about to talk about a little variation on the method that they developed, but first we'll pause and have a quick word from one of the sponsors that keeps the show going. Other doctors took the work of the German team and the Asheim Zondeck test, which was often abbreviated to just called be called the AZ test, and they made slight tweaks to the method for what worked best for their laboratory situation and their staff at the University of California at Berkeley in the late nineteen twenties, researchers opted to use six rats for the procedure rather than mice, and they stored the urine samples at freezing temperature before injection. They of course heated it back up so that the animals were not going to shock after the injection. Using rats was actually more cost intensive than mice, but you see, Berkeley had already had an established rat colony for use in their laps. The primary benefit and using rats instead of mice was the fact that all of the rats usually survived the test at least the injection and sort of incubation portion of the test, but in mouse based testing, fift of the rodents died before they reached that day five exam a nation stage, and both tests the error rate was quite small, between one and two percent, and according to data collected in Ashiman Zondex lab, this test was also quite good at early detection. There were recorded instances of positive results before any other clinical signs of pregnancy were present, for example, just three to four days after the patient had missed a menstrual period. Results were most reliable though in cases of normal pregnancy, so in instances of more problematic situations like a tubal pregnancy. The results were, according to the German doctor's data, slightly less accurate. This was at a time in medical science when diagnostic tests weren't always common practice in Western medicine, and they certainly weren't common specifically for women. Additionally, a lot of doctors felt as though it was extraneous. They trusted their experience and diagnostic abilities to determine if someone was pregnant or not. There was concerned that testing labs removed for the relationship and the trust between the doctor and the patient. Yeah, this was particularly a problem UM in Great Britain. I was reading one article that UM mentioned that this one particular lab in Edinburgh had started taking mail in samples and people were like, but they're bypassing their doctors just to do a direct thing. Um. There were a lot of a lot of concerns. And this is at a point where there was insurance in Great Britain, but it didn't always cover women, and it certainly didn't cover them just wanting to find out if they were pregnant. That was usually only for like an emergency, which we'll talk about. Additionally, most of the time most women suspected they were pregnant already and they went to their doctor just to confirm what they already thought. And family practitioners used this as evidence that there was not a real need for such tests to become mainstream. In most cases, unless there was an urgent medical dean to know if a woman was pregnant. Health organizations and insurance companies where they existed, because insurance didn't really start until the late nineteen twenties, were unlikely to approve coverage for the cost of the test. Testing samples sent through the mail was particularly looked down upon us we just sort of alluded to. It was referred to as postal pathology. That's a a criticism that still exists today with all the various things that you can test at home and kind of mail away. But this test was recognized for its efficacy and it became more and more commonly administered. Both Selmer Asheim and Bernard Zondeck were Jewish, and both of them chose to leave Germany. I say chose, but they really had to for their lives in nineteen thirty three as the Nazi Party came into power, and they left their lab behind in the process. In thirty one, the landscape for pregnancy testing shifted when a similar test to the A Z test was introduced at the University of Pennsylvania by Maurice Harold Friedman and Maxwell Edward Lapham. Freedman, who was born on October three and Gary, Indiana, had been with the University of Pennsylvania as a faculty member since he had earned his bachelor's degree PhD and m d at the University of Chicago starting in nineteen nine, so he went into his undergrad at just sixteen, so he was still quite young when he developed the rabbit test, and Maxwell E. Lapham, who was born in newfe New York, was just three years older than his collaborator, and he was working at the university hospital at the time. And an article titled a Simple Rapid Procedure for the Laboratory Diagnosis of Early Pregnancies in the March issue of the American Journal of Obstetrics and Gynecology, Friedman and Lapham wrote about their choice to move away from mice for their test, starting with a reference to the as test, It said quote Yet this test admirable, though it is has some distinct disadvantages, one of which may make it impractical in a laboratory or hospital not closely affiliated with some university or institute. To perform the zondek Asheim test, it's essential to be in in a position to command a ready supply of amateur mice weighing from sixt grahams. If a large breeding colony of mice is not easily available, some difficulty might be encountered in procuring suitable animals at a time as test as desired. Moreover, even if one had at hand enough of the immature mice to answer the calculated requirements for a given week, and for some reason or other the number of samples submitted fell below expectations, the unused animals would soon mature beyond their usefulness, so that another group of the immature animals would have to be gotten. Freedman and Lapham's test used rabbits instead of mice, and just as rats were more of a burden to support than mice, rabbits took a lot more effort and resources to keep in a lab setting. But there were some very significant benefits to rabbits over mice. For one, it required only one to two specimens rather than six. Aside from just the number of creatures harmed. This also just meant one or two necropsies rather than half a dozen, and unlike mice, rabbits only ovulate after mating. That meant they didn't need to use only very young animals. With mice, the very young specimens were used to avoid any confusion that might arise should Amounse just be developing a natural estrous cycle, which would look pretty similar to a positive result in the testing. As long as they knew the rabbit's history and it had been isolated because exposure to other female rabbits could cause estrus, it could stay viable as a test subject until it was needed, instead of aging out of usefulness for testing purposes. But even if a rabbit had not been fully isolated, it could still potentially be used per Friedman and Lapham quote. In case one has not had opportunity to isolate the rabbits for the desired period, and it is found necessary to perform a test, it is safe to use a rabbit that has been isolated in the laboratory for only eight or ten days, even if the rabbit in question had had coitus just before it was obtained. The corporal lutilla of pregnancy or pseudo pregnancy would then be least eight or ten days old and could not be confused with the fresh corporate lutia or corporal hemorrhagica produced by the injections of an active urine. Briefly, then one may safely use all rabbits that are not demonstrably pregnant at the end of three weeks of isolation. Pathologists also found the rabbits easier to handle, and it was easier to administer injections into the veins of their ears. They could perform the necropsy without the need for a microscope or a magnifying glass because the results were observable to the naked eye rather than needing to be magnified. Yeah, that was not the case with mice or you had to look very closely at their tiny, tiny organs. You could literally just do the necropcy and go yep, this one is positive. But the big, big difference was that a rabbit test could be turned around in forty eight hours rather than five days. While Lapham and Friedman tried a similar method of earlier necropsy that had been used in mice, as you remember, they had just up the number of mice used and then did a test doing sort of pattern recognition at a shorter interval. They actually found the results a lot less reliable when they try a similar method in rabbits. We're about to get into the numbers involved in the research that Freedman and Lapham used to test their rabbit method, but first we will take a quick sponsor break. To test their approach, the University of Pennsylvania team used a hundred and eleven samples. Three samples were deemed too toxic to use in the test, and the specific nature of the toxicity is not described in the Freedman and lap of paper, but a lab in Edinburgh, Scotland, which did quite a lot of pregnancy testing in the nineteen thirties, described sometimes getting samples of urine which were green because the women who had given them had used some sort of chemical means to try to avoid pregnancy. Those samples were lethal to mice, which was the testing animal that the Edinburgh lab used, so the test would not work. Freedman and Lapa mentioned as a desire to find a way to handle so called toxic samples. Writing quote zon Deck reports that about six percent of the samples submitted were too toxic. To be handled, and to obviate this difficulty, he has devised a method by which these toxic urines may be made innocuous. Since the appearance of this paper, we have not encountered a sample with which to test this procedure, so that left a hundred and eight samples for the study, and twenty five of those samples were from women who were in their last months of gestation, and all of their samples, as expected, yielded positive test results. Thirty two of the samples were from women who had gone to the clinic at the university hospital to determine if they were pregnant. Twenty five of them tested positive for pregnancy, and the research team was able to track twenty two of those women to further verify that they had been pregnant, although not all of them carried those pregnancies to full term. The remaining three did not remain in contact with the study. There were fifty one negative test results from this viable group of a hundred eight samples. Two of those samples were from men and were from women in the hospital who were known to have been not pregnant at the time of testing, including several with conditions that might clinically present as looking pregnant without a test, like they had nausea or their abdomens were swollen, something like that. Yes, so they wanted to include people that they absolutely knew were not pregnant to prove that the tests was accurate in both positive and negative and also people who might be told by a doctor who was just examining them that they were probably pregnant but were in fact not. Twenty four of the negative test results had been for women who had visited the clinic to determine if they were pregnant, and the study remained in contact with eighteen of those patients and they were verified as non pregnant. That left ninety two cases for which the doctors felt they had adequate data for the inclusion in the study statistical analysis, and that left them with an error rate of zero. But in their paper Forman and Lapham wrote, it is likely that if we had more material, we might have encountered an error or two. Yeah, they were not asserting that it was a percent effect of all the time, um, but of the ninety two that had been that it had been paired down to after they uh had to leave out the toxic urine samples and the ones that they lost track of it was accurate in that data. The Freedman Lapham test was adopted more readily than its predecessor, the mouse version, in part because the asy test had blazed a trail already, so people already had this idea that this test was worthwhile and was accurate, and also for all of the reasons that were laid out in the paper published by Freedman and Lapham. For example, hospitals found it much easier to set up a lab for a smaller number of rabbits than huge numbers of mice, which they would have to continually be breeding to get workable um animals that they could use in testing, even though it was still more expensive to house a rabbit, and the reliability and the speedier assessment during necropsy made this test much more appealing as well. And as a bit of myth busting, you may have heard the joke the rabbit died as a shorthand way to say that somebody is pregnant, and this has been a long a longstanding misconception connected to this test. I think both Holly and I at some point have probably said that I know I have repeated that myth, So apologies to anyone that heard that from my lips. You got misinformation. Yeah, the positive test result was not indicated by the rabbit dying. The rabbits had to be euthanized and necrop seed. The positive test result was definitely not the cause of the death. Correct. And while the rabbit test was more popular than the mouse test that preceded it, there was another way to test for pregnancy that followed in the nineteen thirties, so not very long after the rabbit test was introduced, called the frog test, and this one introduced into Western medicine by British biologist Lancelot hogban didn't kill the test animal, Xenopus levis. Frogs used in this test would begin ovulating and dropping eggs quite quickly after being injected with urine that contained hCG in the levels consistent with pregnancy. You will also sometimes hear this referred to as the Buffo test. That species was originally named Buffo levis before the name changed to Xenopus. So these frogs displayed their results in mere hours compared to the days that it took with previous methods of mice and rabbits, and the same frog could be used for repeated testing since there wasn't a necropsy needed to confirm these results. Yeah, this is also one of those things that has possibly led to some species integration in places it is not natural for it to exist, because it appears, at least based on what I read, that this really caused no harm to the frogs whatsoever. And so these labs would end up with lots and lots of frogs and eventually they would start letting some go. So they had originated in South Africa and then ended up up being let loose outside of labs throughout the Western world. We've done, uh like invasive species have come up on the show before, but I don't think I have ever heard of invasive pregnancy test frogs and the invasive frogs that have have gone on to retire from being pregnancy tests ers. They also eventually realized that they could use male frogs as well, and they would basically release sperm, and they actually had a much faster test result than the female frogs, So both both males and females of the species ended up being used. But hogg Ban did not get to retain a clear claim to this discovery. While he had apparently suggested that these frogs could be used to detect certain hormones and pregnant women. He didn't really land at the idea that the frogs could be used to design a pregnancy test, and he did not set up any of the testing around it. It was Hogben's student Hill L. Shapiro and Shapiro's research partner, Harry's Warrenstein who act took the idea from theoretical to practical by designing executing studies with the frogs. Of course, the frog test caught on because if its benefits over the other options, and it was used until the nineteen sixties, when pregnancy tests were developed that didn't involve the use of animals at all. After that, home kits eventually hit the market in the nineteen seventies, the first of which was called Predictor and which took two hours to offer a result. These were a lot more convoluted than today's where you just pe honest stick. If you have watched the Netflix series Glow, you've seen what one looks like. Yeah, yeah, And I should say that they there were other tests being developed that did not involve animals before the nineteen sixties, but they just you know, had not reached a level of reliability that they could supplant these animal based testing options. And as for the developers of the rabbit test, Maxwell E. Lapham wrote a book titled Maternity Care in Rural Communities in ninety eight, so later in that same decade after they introduced the rabbit test. The year before that, he had joined the faculty at Tulane University School of Medicine, and he actually stayed with Tulane until the end of his career. When he died at the age of eighty three, it was in Tulane University Hospital and per his New York Times obituary, during his time as dean of the medical school, he had significantly bolstered the financing of the school's research program, taking it from thirty thousand dollars to five point five million, and that was basically just through grant money. Maurice Freedman moved to the Washington, d c. Area and worked for the Beltsville Agricultural Research Center. He served in the Army Air Force in World War Two as a medical officer and opened a private practice after the war was over, specializing in internal medicine. He died of cancer in nine at the age of eighty seven, and one of Dr Friedman's more famous quotes regarding the rabbit test was quote, it's highly reliable. The only more reliable test is to wait nine months. That seems like a fun place to end it, uh, since it is a little bit to me, it's very fascinating, but you know, it does get into animal testing, which is not the most delightful topic, but it's an important part, I feel like of our scientific um history, particularly as it relates obviously to reproductive medicine, and it's something that people maybe don't realize that this was something that was that was not only commonplace, but it was also already being considered in terms of animal welfare. I know in Great Britain they already had laws in place by the time the mouse test was being used about how animals could be used for medical testing. That was one of the other things that led to some of the debates around whether people should adopt the test. It was it was like, yes, but we're harming animals and you would just realize you were pregnant in a couple of weeks anyway, right. Um, So there's a lot of a lot of interesting layers to it, and it kind of evidence is a discussion about animal rights that was happening I think earlier than we maybe think of it exists. Yeah. Well, and that conversation connects to something that listeners asked us to talk about before, which is the synthesis of insulince and how that was developed um, which is like, it's definitely on the very long topic idealist um and also connects to some of those questions about the ethics of of animals in medicine. Yeah. I have a fun postcard from Barcelona. Nice. It is from our listener Alex uh, and I believe the other name on here is Tari, although I'm not positive. Uh. It's written very very tiny and it has been through international mail. But Alex writes, Dear Holly and Tracy, Hello from Barcelona. My wife and I are traveling Europe to celebrate her finishing her master's degree in graphic design. Congratulations. I have been meaning to come here since first learning about Lisata familia in seventh grade, so this is something of a lifelong dream being fulfilled by the way. I know you two love to shout out educators, so I would like to thank my middle school Spanish teacher. I believe that's Senora Savera for inspiring this trip and for being an incredible teacher. So yes, of course, always always my it is off to educators. It was fortuitous timing for me to get to listen to your episode on Francisco Franco a few days before I left. I have always found the Spanish Civil War very interesting, but have only learned about it through fictional works such as Pans Labyrinth and for Whom the Bell Tolls. I actually got to discuss Franco a little bit with a tour guide at a winery, and as a Catalan he had an interesting perspective as well about how the Franco regime banned the Catalan language and suppressed the culture. Anyway, thank you for the great show. So it's a beautiful it's one of those um a sort of wide postcards, and it's a really beautiful perspective on the Sagarada Familia uh from inside and it's just absolutely lovely, So thank you, thank you again. I'm always wold that people want to stop while they're traveling beautiful places and write his postcard. It's quite flattering. Yeah. Uh, if you would like to direct to us. You can do so at History Podcast at how stuff works dot com. You can also find us pretty much everywhere on social media as missed in History, and you can go to missed in History dot com, which is our website where you will find every episode of the show that has ever existed, as well as show notes for the ones that Tracy and I have worked on. If you would like to subscribe to this podcast, I highly encourage it. That would be great. You could do that on the I Heart Radio app, at Apple Podcasts, or wherever you listen to podcasts. For more on this and thousands of other topics, visit how stuff Works dot com. 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