PREGNANCY TEST PAST AND PRESENT

It was the first of August 1959 I had just qualified as a doctor. My life’s ambition was achieved, not only have I become a doctor, that I stood first in a class of hundred and fifty men and women. I was on cloud nine. My first day at work, as a Dr started at 7 AM, Dr E called me into her office and asked me to go to the pathology for a pregnancy test. The pregnancy test was to be done for the princess of one of the independent states. There were some states still scattered throughout India after independence. This princess had never had a baby, as it was known that the prince was azospermic.  Maybe the princess had an indiscretion, on this occasion my boss had to know what was going on.  It was a hot day, and it was raining as well, she asked me to take her car which was outside with a chauffeur. For the pregnancy test we had to go to the pathology, it was about one kilometre. The car was a green Chevrolet. I was so delighted with this permission. I forgot all about the test and became very excited about the thought of sitting in this beautiful American car.  I had never sat in a car before. My dad was a lecturer at a prestigious university and we all lived on the university campus.  There was a school for the university staff children.  We never required a car. Sitting in the car, I was not thinking about the pregnancy test which I was thinking of earlier, but now I was thinking when I will be able to have a car. After I finished my training in obstetrics and gynaecology I got married. We went to the UK. My husband was already working there. The very first day we were standing on a bus stop, my husband noticed that I was shivering , he said to me he is coming in few minutes, when he returned, he was in a beautiful  blue  Beatle(VW)  Car . He said to me “let us go”. You cannot imagine my joy.  In my excitement of the car let me not forget the pregnancy test.  My very important job on day one, as a doctor was to get this pregnancy test done. I did not even know at this stage that these tests could be done at our college.  My bosses said go and do an (A Z) test.  She called it  Aschiem-Zondek .This is described further down in the text. There was a similar test, called the toad test which was introduced by, Lancelot Hogban looking for the pregnancy hormone called human chorionic gonadotrophin (hCG ). This was discovered in 1930.  It is produced by the trophoblastic (very young placental cells) cells of the fertilized egg. The hormone is excreted in the urine of the pregnant mothers. The urine from a pregnant woman was important; some ancient Egyptians used to do a very different pregnancy test with the urine. This was in 1350.BC. A woman who was supposed to be pregnant was made to pass urine on wheat and barley seeds over several days. If the seeds grew the woman was supposed to be pregnant. Not only that they declared the sex of the baby as well., however this was disputed, if the barley grew the baby was supposed to be male, if the wheat grew the baby was thought to be female and vice versa. The urine of men, and non pregnant women did not germinate the seeds.  It was speculated that the increased levels of oestrogens in the pregnant woman’s urine may be the cause of this success. The human chorionic  gonadotrophin,  the  pregnancy  hormone  was not discovered as yet. There were many weird tests for diagnosis of pregnancy using urine.  There was the Ribbon test, the ribbon was to be dipped in the urine, if the woman gagged or vomited smelling this ribbon she was considered to be pregnant. Then in 1500AD there was an eye test described by a Dr Jacques Gulliemeau  in which a pregnant woman’s eyes get deeply set with small pupils, drooping eyelids, swollen little veins at the corner of the eyes.By 1900 the piss became an important commodity. At this time the pregnancy hormone chorionic gonadotropin(hCG) was being discovered in the blood and the urine of the mother to be. Two scientists, Selmar Aschheim and  Bernhardt Zondek  invented a test in which the urine of the woman to be tested was injected in an immature rabbit, rat ,or mouse, several doses over many days , after many days the rodent was killed to see if it had ovulated , or ovaries have grown the corpus luteum  has developed , i.e. it is gone on heat  in spite  of the rodent being immature. If this happened the woman was supposed to be pregnant; it became a cliché to say that the rabbit died, which meant that the woman was pregnant. This test was good but expensive and the animal had to be killed. A -Z test did not last for long .By 1930 hCG was studied in detail, it was certain that this hormone is produced by the placenta and can be detected after the implantation of the pregnancy.  The Americans consider the pregnancy to start with implantation.  The toad test was being performed with a vengeance, both on male toads (bufo and female frog (south African clawed toads – xenopus). The male toad laid Sunitpermatzoa and the female toads laid ova. When my boss sent for this pregnancy test it was meant to be the toad test. When I arrived at the lab there was a technician with a few toads in a tank of water, the technician injected one of female frog with some urine, just under the skin. He asked me to come the following day. When I arrived nearly 24 hours later the tanks had a lot of frog’s eggs floating in the tank. This confirmed that the woman was indeed pregnant. She had a hysterectomy performed in the name of fibroid uterus. She indeed had fibroids as well which were causing her trouble. This saved the day for everybody concerned, particularly the princess.These tests for diagnosis of pregnancy were called bioassays the immunoassay tests started instead of bioassay test. Bioassay tests were cumbersome, expensive and required special space, staff and laboratory animals. It was often a false positive because of another hormone, Luteinising hormone ,produced from the pituitary which is produced in the normal menstrual cycle. By now many Hormones were identified, it was also discovered that hCG has a beta subunit fraction which is specific to pregnancy. Special monoclonal anti bodies(proteins specific to particular proteins) were also  discovered for the  beta subunit fraction of  hCG  . Judith Vaitukailis devised the first home based pregnancy test at the national institute of health in 1970 however; she missed out on its patent. These tests are based on antigen (a protein) and antibody reaction. An identifying medium is also added. Initially it was a radioimmunoassay, then they did it with blood, called  haemagglution inhibition test and then later it was a called a latex agglutination inhibition test. A dye was also added for identification. By  1978 all these tests became commercial with an annual sale of 20 million US dollars. In 1968 Margaret Crane got the US patent for 3 million US dollars.

Between the years of 1960 and 1980 ,Further improvement in Immunological techniques and our knowledge on  hormones  increased the bioassay done for pregnancy tests, were totally replaced by immunoassays  for pregnancy testing. Ultrasound also started  playing its role from 1960 onwards. Immunological tests are more sensitive and easier to do. These are a type of tests that measures a protein molecule with another substance for which we are looking for.This is a part of immunology it is called antigen antibody reaction. Proteins called Monoclonal antibodies specific for a particular protein (antigen) were also identified and manufactured.   There   are several thousand types of monoclonal antibodies which react with the special antigens you want them to react with .They are used in thousands of clinical situations in medicine. In pregnancy we want them to pick up  hCG.  Initially researchers identified it as a pregnancy hormone. However they kept getting false positive results. This was due to the presence of another hormone called luteinising hormone which is produced from the Pituitary during each menstrual cycle.  By 1972 it was identified that hCG has 2 fractions alfa and beta subunits. It is the beta sub-unit where the biological and immunological specificity resides as regards pregnancy tests. After all this, different types of pregnancy tests where manufactured, by 1978 the market was full of these tests when we look for the beta sub-unit of hCG the pregnancy tests are 99% accurate. After sexual intercourse the sperm can live in the fallopian tube up to 5 days waiting for the ovulation to occur when the egg   arrives is fertilized and it takes another 10 to12 days for implantation to take place. So the best time to test for pregnancy is about 17 days after sex. These pregnancy tests looking for beta subunit of chorionic gonadotropin, either in blood or urine are highly sensitive. They can be both qualitative  or quantitative. Quantitative  tests in blood can detect beta  hCG  as low as 1mIU/mL while urine test strips can  detect as low as 10mIU/mL  to 100mIU/mL ,depending on the brand of the strip .Most pregnancy  test have a threshold of 25 mIU/ mL  These pregnancy can be false positive. This is generally due to the drugs producing hCG, in accurate testing, some common medications EG; chlorpromazine, phenothiazine and methadone give false positive results Some cancers producing beta hCG, such as liver cancer, germ cell tumours of the ovary, trobhoblastic cancers( these are cancers from the placenta) again give false positive results Treatment of infertility when hCG injection is used for treatment also can give a false positive results . This can often be distressing, you are trying for a pregnancy and you get a false positive result.

Modern pregnancy test  are all immunoassay tests.  A scientist observed that people taking insulin for Diabetes developed antibodies. With this in mind ,researcher started to develop antibodies in animal models. After this immunology progressed; exponentially.  By 1972 the problem of luteinizing hormone was also sorted out as an alpha subfraction of hCG.  Improved prenatal care and legalization of abortion both made it urgent to make diagnosis of pregnancy as early as possible. Initially the immunoassay pregnancy tests were cumbersome needed, test tubes, chemical reagents, filters and so on. But as time moved on they became very simple.

By 1978 FDA approved these tests. Initially they required the help of a doctor or a lab but later 1979 home pregnancy tests were on the market. This gave woman a new opportunity to look after their health and keep the secrecy of their life style to themselves. By now the importance of folic acid in early pregnancy was also known. Further improvement in test techniques made the home pregnancy test very simple and fool proof.  As already mentioned earlier there accuracy was 99%.  They used special monoclonal antibody which reacted with any beat subfraction hCG present in the urine. This also had an agent to cause colour change if the level of hCG  was consistent with pregnancy levels . They are best done two weeks after the missed period. However the newer tests can give a positive reading even before you have missed a period. If you are fairly certain that you may be pregnant, and the test comes negative, you can repeat in few days. These tests come on a latex-coated test strip. This has been treated with different antibodies. The antibodies are placed in three distinct zones.  The first zone contains anti-a hCG which combines with any hCG in the urine and the other is immunoglobulin G (IgG). This a control to see that the test strip is working properly. Before the the urine flows to the second zone two things have happened 1) hCG in the urine had combined with anti-ahCG antibody forms a  complex  and 2)urine suspends the IgG. The urine then flows to the second zone carrying with it the IgG and the anti-a antibody complex. This second zone is the test zone contains anti-b hCG and a dye substrate, which reacts further to create a sandwich which turns bright blue in colour. This technique is referred as Elisa sandwich technique. The IgG from the first antibody zone goes into the third zone. This zone contains another antibody which reacts with IgG and forms another coloured line. This indicates that the test was properly done. The blue line in the test zone and another fainter line in the control zone indicate that the pregnancy test is positive, and the test was properly done. There are many different types of pregnancy tests with different designs and details. These tests have been passed by FDA. These tests have now become digital. On the test kits, it reads pregnant or not pregnant. It also indicates how many days of pregnancy. It is estimated that the home pregnancy market is over 200 million in sales per year. Why do we do these pregnancy tests?  These tests can be qualitative and quantitative. Quantitative test are always on the blood in complex situations of diagnosis and progress of malignant diseases. The urine pregnancy tests are done by almost everyone, women, students, teenagers, nurses and clinicians. These tests are very useful to assess the progress of pregnancy, is it alive and well ?failure of hCG levels to rise indicates that all is not well with the pregnancy. hCG levels double in 48 hours if pregnancy is progressing well . Quantitative measurements if small may indicate an ectopic pregnancy (pregnancy outside the uterus often in the Fallopian tubes). This diagnosis is often confirmed with the help of trans-vaginal  ultrasound. Ectopic pregnancy is a life threatening  condition .This requires urgent treatment The pregnancy test is often required after medical abortion to make sure that the abortion was complete especially if the woman continues to bleed or after removal of molar pregnancy (abnormal products of pregnancy these can sometimes turn into cancer.)Ultrasound also helps .Women with a positive pregnancy test, may also need medical check up in many areas, nutrition, sexually transmitted infections, antenatal care,  termination of pregnancy, contraception and psychosocial support.

It has been 56 years since; I first did the toad test for diagnosis of pregnancy. Today a wide range of tests are available for pregnancy and infertility, with the sale of these products exceeding 200 million American dollars. We have come a long way in 35 years or should I considerate 55years saving the martyrdom to poor rabbits.