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= The Frog Test = The Frog test was a prevalent pregnancy testing method relying on frogs to show the pregnancy status of women. 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.

Throughout history, different frog tests were used to screen for pregnancy .They used the underlying principle of hormones and its subsequent biological response in both genders of certain frog species. The Hogben test was the most popular frog test. The Galli-Mainini test is another frog test developed based on similar principle of the Hogben test. The frog test was a highly dependent pregnancy testing method since the 1930s, until the immunological test was presented in the 1960s.

Underlying hormonal principle of the frog test: Human Chorionic Gonadotrophin
The Human Chorionic Gonadotrophin (hCG) is a hormone produced from the placenta in pregnant women, which is released into the bloodstream and can pass into the urine. It takes around 6 days after the implantation of the fertilized egg, to detect hCG levels in the blood. Normally, the hCG level increases and peaks at the first 14 weeks of pregnancy, and no hCG is detected after the delivery of the baby. The amount of hCG can also reflect the pregnancy. Less hCG is released in a single pregnancy than for twins or triplets. hCG blood tests can also screen for birth defects.

= The Hogben test =

Hogben Test as a frog test
The Hogben Test, named after the British zoologist Lancelot Hogben, was one of the most reliable and rapid pregnancy tests from the 1940s to the 1960s. The species of frog Xenopus laevis (African Clawed frog), originated from sub-Saharan Africa, was used. The Hogben test consists of using female frogs as the species in which urine samples are injected into. The Hogben test uses female frogs, unlike the Galli-Mainini test which uses male frogs.

Lancelot Hogben and his journey of discovery
Hogben researched endocrinology, and applied his knowledge to introduce an accurate method of testing for hCG, a pregnancy-revealing hormone. In his earlier works, his team observed that the skin colour of the adult frogs was dependent on their growing environment. A dark environment led to a black skin colour, a light environment led to light skin colour. Hogben hypothesized this as a result of the pituitary gland’s presence. Hogben validated his hypothesis by proving that the removal of the pituitary gland would result in the white skin colour of frogs regardless of their growing environment. During this study, he discovered that female frogs would ovulate after the injection of ox pituitary gland extracts into its dorsal lymph sac,

Hogben moved to South Africa in 1927. His research revolved around injecting Xenopus laevis with ox’ pituitary gland extracts, and he 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. This created a basis for the Hogben frog test as the presence of a pregnancy-dependent hormone in pregnant women’s urine could also be detected through an ovulating response in these frogs. Upon this unearthing, Hogben and 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 that were claimed to be nearly 100% accurate.

Testing Procedures for the Hogben 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. The urine should be collected a few days after the mark of a late menstruation.

= Advantageous Edge of Hogben Test = Before this revolutionary discovery, “A-Z tests” pregnancy 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 examined for enlarged ovaries. The enlargement is due to 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, each frog could be reused. Xenopus laevis were also easier to maintain than rodents, and raising them came at a lower cost.

= The Galli-Mainini Test as an alternative Frog Test to the Hogben Test = Carlos Galli Mainini (1914-1961), also specializing in endocrinology, strived to improve on the existing Hogben’s test, which took over 12 hours for early screening of pregnancy. He observed that male frogs or toads generate spermatozoa after long contact with female frogs, which was due to the secretion of hCG from the female's body. 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 hCG presence would lead to the release of sperms from the frogs.

This new testing method was 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.

Testing procedures for the Galli-Mainini Test
Similar to the Hogben test, a small amount of the women's urine would be injected into the dorsal lymph sac of the male frog. The presence of hCG in the injection of pregnant women's urine would cause the frog to release sperm within three hours. The sperm cells can be seen under a microscope. This procedure is painless for the frog, and the frog can be reused for another test after 2 weeks.

Ways to Improve Accuracy of the Male Frog Test (Galli-Mainini Test)
Researchers worked on validating the accuracy of the male frog 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 hCG.

= Advantages of Using Frogs As Test Animals = Many researchers could provide daily care by changing the water in the research tank. Frogs have been widely adopted as experimental animals because of their high ability to withstand prolonged hunger and their relative non-selectiveness towards food. In the frog tests, they could repeatedly used with at least one-week interval between each use, and checking that their urine does not contain any spermatozoa prior to the injection, which would invalidate the final result of testing.

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 biology, such as in frog-based diagnostic tools of polycystic kidney disease.

= An Unexpected Complication 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 deteriorated their skin function, killing the population within a week after infection. The 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 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, 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. 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. According to Professor Karen Lips from the University of Maryland, the best way to prevent further amphibian extinctions by the Batrachochytrium species of fungus is to limit international trade of amphibians, and instill 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 mentioned 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.

= References =