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The Frog Test
Throughout history, there have been different frog tests with the aim of indicating the pregnancy status of women. The most well known frog test, is the Hogben test, which is a pregnancy testing method prevalent throughout the 1940s to 1960s , by using the underlying principle of hormones and its subsequent biological response in both genders of certain frog species. In South America, the Frog test was referred to as the Galli-Mainini test.

Introduction
Nowadays, the advancement in medical technology has enabled women to accurately check their pregnancy status by using ‘pee-on-a-stick’ pregnancy test kits at home. Before these accessible and convenient test kits were invented, scientists strived to discover a way in spotting pregnancy-related hormones by a natural, simple test, where animals were often included as clinical tools to facilitate the process. Frog test, or the frog pregnancy test, one of the past prevalent pregnancy scanning methods, will be introduced below, with an illustration of its history, development, related applications, and issues.

Definition and Background History
The species of frog Xenopus Laevis is also referred to as the African Clawed frog, which originated from sub-Saharan Africa. It was used as one of the most reliable and rapid pregnancy tests from the 1940s to the 1960s in a procedure called the ‘Hogben test’, named after the British zoologist Lancelot Hogben. Hogben researched endocrinology, Endocrinology, which is a study related to the endocrine system, and its secretion, known as hormones, which regulates humans’ growth, development, and metabolism. Using his knowledge of pituitary hormone, Hogben was dedicated to discovering an accurate method of testing for this pregnancy-revealing hormone.



Journey of the discovery
Hogben’s earlier works also revolved around pituitary hormones and frogs. Hogben validated his hypothesis that the removal of the pituitary gland would result in the white skin colour of frogs regardless of their growing environment. He observed that the skin colour of the adult frogs was dependent on their growing environment. The colour ranges from a dark environment which led to black skin colour, to a light environment where light-coloured frogs were found. This interesting phenomenon was the result of the pituitary gland’s presence. This discovery process was particularly significant as while he was studying the pituitary gland in frogs, an unexpected but game-changing side effect was the discovery of ovulation in female frogs after the injection of ox pituitary gland extracts into its dorsal lymph sac.

While he was carrying on with his work when he moved to South Africa in 1927, where his research revolved around injecting Xenopus Laevis with ox’ pituitary gland extracts, he accidentally discovered that Xenopus frogs would ovulate within a day if they were injected with pituitary extracts, as they were very sensitive to any hormonal changes. During this period, scientists knew that women contained pituitary hormones in their urine, and Hogben came to a realization that perhaps the presence of pituitary hormones in pregnant women’s urine could also be detected through an ovulating response in these frogs. Upon this unearthing, Hogben and another animal geneticist Francis Albert Eley Crew spent two years developing a way to raise and maintain these frogs in a lab setting, which led to a twenty-year boom in Hogben tests, which were claimed to be nearly 100% accurate.

Details of the test The Hogben test procedure consisted of injecting a sample of women’s urine into the skin on the back of the frog, specifically into the dorsal lymph sac. Around 12 hours later, results could be seen. If the woman was pregnant, then the frog would be ovulating, and a small cluster of eggs could be seen at the rear end of the frog. Interestingly, the same could be observed in the male species of Xenopus Laevis as well, and they were seen to produce sperm upon injection of a pregnant woman's urine. This mechanism is due to the pituitary hormone human chorionic gonadotropin (hCG), which is present only in a pregnant woman’s urine.

Advantageous Edge


Before this revolutionary discovery, pregnancy tests, called “A-Z tests” were troublesome and time-consuming. They consisted of injecting women’s urine twice a day, for three days, into five different mice. Then, the mice would have to be killed and scientists would examine if the mice had enlarged ovaries. This ovary growth is due to the hCG. Hogben tests were soon more popular and performed more widely than the A-Z tests because results could be seen in less than a day, and yielded highly accurate results. Moreover, Hogben tests did not involve killing the frogs, so each frog could be reused again, unlike in the A-Z tests where the rodents would have to be killed to examine ovary enlargement. Xenopus Laevis were also easier to maintain than rodents, and raising them came at a lower cost. This efficient and reliable Hogben test was used till the development of an even simpler and animal-free pregnancy test in the late 1960s.

Development of the application of the Frog Test
The frog test had been a highly dependent pregnancy testing method since the 1930s until the immunological test was presented in the 1960s. Pharmacists would inject the female’s early urine sample into the frogs and confirm their pregnancy with the spawning of eggs within 18 hours. However, there was a critical prerequisite for accurately performing the test. Women should wait for a few more days after the mark of their late menstruation. As the test was incredibly helpful and considerably time-effective, many countries started to import Xenopus laevis, which contributed to the spread of this species across Europe, Australia, Asia, North America, and almost everywhere around the globe. Despite its help in pregnancy scanning, an unexpected ecological catastrophe occurred after the introduction of this species across the globe.

Complications of Xenopus importation


Starting from the 1970s, the global frog population began to vanish because of the spread of a pathogenic fungus, called chytrid, scientifically named Batrachochytrium dendrobatidis. This fungus was found to have originated from Korea during the mid 20th century. This fungus was a silent killer of thousands of frogs as it possessed the ability to suffocate the frogs by deteriorating their skin function, killing the population within a week after infection. The fungus mostly originated from the imported African Clawed Frogs, according to the researchers. International trade of the African Clawed Frogs for pregnancy tests was an indispensable cause for this, as it introduced invasive species to other ecological systems, harming the native species, which lack resistance to the fungus, in areas other than Africa. In 2004, a researcher from South Africa’s North-West university carried out an analysis on hundreds of Xenopus specimens to show that in their natural habitat, in Africa, they are occasionally infected by Batrachochytrium dendrobatidis. Whilst the Xenopus specimens can tolerate this infection, other frogs cannot. Researchers suggested that some frogs such as the Xenopus species may naturally have symbiotic microbes that live on their skin, and throughout evolution, have learnt to fight off the fungus. This invasive fungus has infected habitats everywhere, excluding Papua New Guinea and Antarctica. Jamie Voyles from the University of Nevada found that in Panama, there are certain areas where the fungus has lived for many years, and Atelopus varius which are also called Clown Frogs, were capable of fighting off this fungal infection. He comments that this indicates a shift from the epidemic stage of the fungus infection, to a more stabilized pandemic phase, as animals are seen to co-exist with the fungus and are more resistant to them. For now, the best way to prevent further amphibian extinctions by the Batrachochytrium species of fungus is to limit international trade of amphibians, which was the main cause of the Chytrid spread in the first place. For example, the state of California has been importing chytrid infected bullfrogs for the last decade, however, nothing has been done to stop the importations. Professor Karen Lips from the University of Maryland comments that the biggest precautionary steps are to limit this trade, as well as instil new laws to require testing and quarantine for imported animals. Dr Simon Clulow from the University of Newcastle has discovered in outdoor field experiments, that infection rates could be dramatically reduced if the salinity of the water was raised by 0.5 parts per trillion. He mentions that this slight increase in salinity may not work for all environments, but is a solution for ponds and artificial habitats. This method increases the survival rates of the frogs without having a significant effect on the rest of the ecosystem.

Today, Hogben’s test has become obsolete, however, it has made Xenopus frogs an important model and organism in the scientific community, and the Xenopus species are used to study a wide variety of diseases. Even the tadpoles of the Xenopus species have been incorporated as an organism used in the study of developmental biologies, such as in frog-based diagnostic tools of polycystic kidney disease.

An Alternative Frog Test to the Hogben Test
Provided that scientists often discover new things or make improvements by working further on previous scientists’ work, Carlos Galli Mainini (1914-1961), who also specializes in endocrinology, strived to improve on the existing Hogben’s test, which took over 12 hours for early screening of pregnancy. After making a careful observation that male frogs or toads generate spermatozoa after long contact with female frogs, the gonadotropic hormone would be secreted from the females’ bodies. He realized the female frogs used in Hogben’s test could be replaced by South American male frogs or toads. Women’s urine could be injected into the adult male frog’s dorsal lymph sac. The gonadotropic hormone in the urine of pregnant women, after injection, would lead to the release of sperms from the frogs. This new testing method was virally spread around the globe as it was even more efficient, with only 3 hours of waiting time for the results. He found different species suitable for this experiment, mostly indigenous frogs from Israel, like Batrachia, Bufo viridis, Rana ridibunda, and Hyla Arborea. Given that frogs are conveniently kept and raised by simple care, many researchers could provide daily care by changing the water in the research tank private practice. Frogs have been widely adopted as experimental animals because of their high ability to withstand prolonged hunger and their relative non-selectiveness towards edibles. In the frog tests, they could even be repeatedly used, with at least one-week interval and prior checking that their urine does not contain any spermatozoa before the test, which would invalidate the final result of testing.

Ways to Improve Accuracy of the Male Frog Test
After the suggestions of male frog species for pregnancy tests, researchers had been working on the validation of the accuracy of the test, by ruling out other possible sources that led to the presence of mature living spermatozoa in male frogs’ urine. Researchers successfully found, some of the dry frogs of the species Rana pipiens would be especially sensitive to distilled water and less sensitive to the gonadotropic hormone. They, moreover, suggested some feasible ways to increase the accuracy of the test, some are illustrated below. 1. 	Do not inject more than 5 cc. of fluid as a diluted solution or a large quantity of distilled water would trigger spermatozoa emission.

2. 	Always use Ringer’s solution, which is a salt solution having a similar salt concentration with human fluid, to avoid false positives of the test.

3. 	Avoid using dry frogs, which have lost 35% of weight because of drying, as they would be more sensitive to water, and release spermatozoa after having contact with water, regardless of the presence of the gonadotropic hormone.

4. 	Store the frogs in shallow water instead of room temperature to achieve maximum sensitiveness to the gonadotropic hormone in pregnant women’s urine.

Conclusion
The Frog test was a meaningful medical breakthrough that benefited women in the past and has led to further medical developments that are enhancing women’s sexual health today. From knowing the history of its development, we can appreciate the efforts made by many talented scientists, and their cross-decade collaboration to improve the accuracy and maximize its benefits to the public. Even though the frog test has eventually been replaced by faster pregnancy tests, it is unquestionably the root of today’s advanced tests.