Pregnancy Test

Galli Mainini test Mainini, Carlos Galli (1914-1961) • Born in the city of Buenos Aires, Argentina in 1914

• Graduated from Medical Faculty of Medicine of the University of Buenos Aires , specialized in Endocrinology • Earned a worldwide recognition with his method of detecting early biological pregnancy known as Galli Manini Reaction Test • Died on October 16, 1961 Galli Mainini’s Pregnancy Test • Also known as the Male Toad Pregnancy Test • used the male toad, Bufo arenarum, in a test for the diagnosis of pregnancy, based upon the expulsion of spermatozoa by the animal, after an injection of pregnancy urine. • "Classic" test to determine if someone is pregnant or not • Done by injecting subcutaneously the woman's fresh urine to the male toads, and after few hours, checking the frog's own urine for possible presence of sperms, which is an indicator for affirmative pregnancy • Cheap, reliable, relatively slow and laborious to perform • Comparable with that of the tests used today, but it was considerably longer and more labor-intensive • The test had an accuracy of 94 to 95% and both false positive and negative reactions • The over-sensitivity of males during the breeding season results in spontaneous spermiation and false positive results • Decreased sensitivity to HCG causes many false negative results during summer. • The most sensitive species was the tree frog, Hyla arborea, which will shed spermatozoa when injected with 1 i.u. • One of the least sensitive species was the edible frog, Rana esculenta, in which 16 to 30 i.u. are needed to produce spermiation. • The speed of the reaction is related to the amount of HCG injected and can occur within 15 min in Bufo bufo. • Provided an efficient and economical solution for early diagnosis of pregnancy and was used heavily in Argentina and Latin America for decades Galli Mainini’s real achievement in this test was discovering that male frogs and toads are suitable for this procedure. Previously, female rats, mice, rabbits and frogs were used, and they reacted much more

slowly. Furthermore, it was necessary to kill and dissect them to see the reaction. Principle: • The similarity between the pregnancy hormone Human Chorionic Gonadotrophin (HCG) and Luteinizing Hormone (LH). The brain uses LH, which is produced by the pituitary gland, to trigger ovulation and sperm production. • In pregnant women, high levels of HCG, which are made in the placenta, are present in the urine. In animals, this has the same effect as over-stimulation with the sex hormone LH, and produces the same reactions. Procedure: • Prepare a male frog for the experiment. The test also works on a female South African clawed frog to lay eggs within eight to 12 hours. The toads to be used for estimation should weigh between 15 and 30 g. (Frazer & Wohlzogen, 1950). When selecting these, the males may be identified by the following points:  Nuptial pads are present on the first and second fingers (P1. 1). The color of the nuptial pad varies according to the interval since the last moult. Shortly after the skin has been shed the pads are pale pink, and later darken gradually to a chocolate color.  The first and second fingers are thicker than the third and fourth.  There is strong muscular development on the forearms.  Croaking may be elicited easily in almost every male by gentle stroking of the ventral skin.

During the experiment, the toads should be kept in pairs in 400 ml. beakers, the bottoms of which should be covered with moist filterpaper. No special care should be taken to keep the toads in light of particular intensity, but direct sunlight should be avoided. The temperature during the experiments should approximately be at 200 C. The technique used is as follows. • Immediately before injection, a sample of toad's cloacal urine should be examined to check if spermatozoa are absent.

Forefeet of male (left) and female (right) Bufo bufo to show sexual characteristics.

Inject 1mL. of urine into the dorsal lymph sac of each male toad, particular care should be taken to see that there is no reflux of the injected solution by using a very fine hypodermic needle (no. 17), puncturing the dorsal skin anterior to the pelvis and passing the point of the needle back towards the tip of the urostyle, withdrawing the needle very slowly after injection, and handling the toads gently to keep them quiet. Struggling can often lead to the expulsion of some of the injection mass through the skin puncture. If reflux occurred in spite of these precautions, the toad should be rejected, as a loss of one drop (approx. 0 05 ml.) represents a loss of as much as 5% of the urine injected. (The frog feels no pain during this test, and after two weeks the animal can be used for another test.)

Three hours after injection, collect the second sample of urine from the male toad, examine microscopically and note the result as either + or - according to whether spermatozoa are present or absent. If it is positive, the injection of the female urine with HCG causes the maturation and expulsion of sperm by their Sertoli cells. In positive cases, the actual number of sperms present is


OTHER PREGNANCY TESTS Ascheim-Zondek test Procedure:

The Ascheim-Zondek test is based on the ‘prolan’ reaction in mice: a sample of the woman’s urine is injected into five immature mice. Each mouse is given an injection of urine twice a day for three consecutive days in different parts of its body; the fourth day is a ‘rest’ day. Exactly 100 hours after the first injection was administered, the animals are painlessly put to death before being fixed to a cork board. Their abdomens are cut open and the ovaries removed.

Results: • The changes observed confirm whether the woman is or is not pregnant: mature egg cells, corpora lutea, so called blood spots, an enlarged uterus and cell changes in the vagina (‘Schollenstadium’) are clear signs that the woman’s urine contains pregnancy hormones, indicating that she is pregnant.

Friedman’s Test Procedure: • The materials and equipment necessary for the performance of the prposed test are: (a) ordinary bed-pan specimen of urine, (b) a five cubic centimeter syringe, and (c) an unmated mature female rabbit. The urine is injected intravenously thrice daily for two days in four cubic centimeter doses. Forty eight hours after the first injection the rabbit is killed.

• •

Results: • • (+) - If the ovaries contain either corpora lutea or large bulging corpora lutea or large bulging corpora hemorrhagica (-) - If the ovaries contain neither corpora lutea nor corpora hemorrhagica but only clear unruptured follicles, regardless of their size

FALSE POSITIVES • A hydatid mole or chorio-epithelioma will give a positive reaction which quantitatively may be ten to fifty times as strong as that given by pregnancy. The reaction persists as long as any of the live tissue of either growth remains in contact with the maternal circulation. False positives from other sources are rare, and when carefully studied are usually accounted for by breaks in the technique of rabbit isolation or by mixed urine specimens. What may be called a false positive occurs in the case of incomplete abortion with retention of live placental tissue in contact with the maternal circulation. Bland, First, and Roeder 21 in their series reported 6.8 per cent false positives. "Especially," they say, "is this error likely to be encountered in women who are functionally sterile due to endocrine disturbances, or in women approaching the menopause. In these women a compensatory hypertrophy of the anterior hypophysis may produce an excessive quantity of hormone, which, finding its way into the urine, will render an incorrect positive." Pituitary hypertrophy following castration in the human being was noted by Tandler and Grosz, evidently an attempt to stimulate a poorly functioning ovary. Experimentally, Evans and Engle have shown that the hypophysis of gonadectomized animals possesses an activity five times greater than in the normal animal.

FALSE NEGATIVES • False negatives are more frequent, and may be accounted for by the use of rabbits less than twelve weeks old or rabbits of uncertain or poor stock, insufficient dosage of urine, insufficient time between the first injection and final reading of the reaction. Missed abortions and ectopics may give negative reactions if the fetus is dead, and no live placental cells are in contact with the maternal circulation.

No report on a urine specimen should state that a certain patient is pregnant or not pregnant unless a responsible person has secured the specimen by catheterization.

Hogben’s Test Xenopuslaevis • A female African clawed toad (Xenopuslaevis) is injected with urine (or an extract) into the dorsal lymph sac. Hogben showed that the injection of extracts of the anterior lobe of the ox pituitary (alp) stimulated ovulation andoviposition in Xenopuslaevis. It is confirmed that Xenopusinjected with alp will lay eggs even outside the normal breeding season.

Result • (+) - presence of five, six, or more eggs within four to twelve hours indicates pregnancy.

Male Frogs • A woman's urine or serum is injected into the dorsal lymph sac of two male frogs (Ranapipiens) or male toads (Bufomarinus).

Result • The presence of spermatozoa within four to twelve hours in the cloacal fluid of both animals is positive; in one animal, inconclusive; in neither animal, negative.

FALSE POSITIVES • False positive results have been occasionally reported in the literature (Landgrebe, 1939). We have been unable to produce egg-laying in Xenopus with purified and biologically active extracts made from the urine of non-pregnant women or men (Hobson, 1958, 1965b). Shapiro (1936), Shapiro &Zwarenstein(1937) and Landgrebe (1939) found that certain steroids would induce ovulation and

oviposition in Xenopus. The doses required to effect this are larger than the amounts excreted by non-pregnant women. • Spontaneous oviposition may occur if the conditions under which the toads are kept in the laboratory are not standardized (Hobson, 1952a). The most likely reason for 'false positives' is that some toads will lay eggs up to 5 days after an injection, and the toads must be used systematically, and not haphazardly. Marks &Shackcloth (1963) have observed an occasional false positive when urines from non-pregnant women receiving chlorpromazine are tested on female Xenopus.

Hemagglutination inhibition Test • The initial success of the test developed by Wide and Gemzell has been substantiated by Wide (1962) and many other workers who prepared their own reagents for the haemagglutination inhibition reaction. Hamburger (1963) compared the immunological test with the male toad, Friedman and the AZ tests. In3000 cases the immunological test was correct in more than 99% of cases, and Hamburger concluded that it could replace both male toad and Friedman tests,but that it was not superior to the AZ test. Bertini(1965) examined 35,000 urines by the haemagglutination inhibition test, using reagents made in his laboratory.

FALSE POSITIVE • The sensitivity of his system was such that a minimum concentration of 600 i.u. hcg was detectable. The test was accurate in 97-7% of cases. There were 805 incorrect results and the majority of these were false negatives. Thirteen false positive results were obtained with urines from women with oligomenorrhoea, menopause praecox and post-partum amenorrhoea.

Modern Pregnancy Tests

Quickest result of pregnancy after fertilization can be detected by detecting EPF or Early Pregnancy Factor which remains in the blood within 48 hours of fertilization. Nevertheless, this procedure is comparatively expensive and time consuming. Most chemical tests for pregnancy look for the presence of the beta subunit of hCG or human chorionic gonadotropin in the blood or urine. hCG can be detected in urine or blood after implantation, which occurs six to twelve days after fertilization. Quantitative blood (serum beta) tests can detect hCG levels as low as 1 mIU/mL, while urine test strips have published detection thresholds of 20 mIU/mL to 100 mIU/mL, depending on the brand. Qualitative blood tests generally have a threshold of 25 mIU/mL, and so are less sensitive than some available home pregnancy tests. Most home pregnancy tests are based on lateral-flow technology. With obstetric ultrasonography the gestational sac sometimes can be visualized as early as four and a half weeks of gestation (approximately two and a half weeks after ovulation) and the yolk sac at about five weeks' gestation. The embryo can be observed and measured by about five and a half weeks. The heartbeat may be seen as early as six weeks, and is usually visible by seven weeks' gestation.

FALSE NEGATIVES • False negative readings can occur when testing is done too early. Quantitative blood tests and the most sensitive urine tests usually begin to detect hCG shortly after implantation, which can occur anywhere from 6 to 12 days after ovulation. hCG levels continue to rise through the first 20 weeks of pregnancy, so the chances of false test results diminish with time. Less sensitive urine tests and qualitative blood tests may not detect pregnancy until three or four days after implantation. Menstruation occurs on average 14 days after ovulation, so the likelihood of a false negative is low once a menstrual period is late.


False positive test results may occur for several reasons. These include: errors of test application, use of drugs containing the assay molecule, and non-pregnant production of the assay molecule. Spurious evaporation lines may appear on many home pregnancy tests if read after the suggested 3–5 minute window or reaction time, independent of an actual pregnancy. False positives may also appear on tests used past their expiration date. A woman who has been given an hCG injection as part of infertility treatment will test positive on pregnancy tests that assay hCG, regardless of her actual pregnancy status. However, some infertility drugs (e.g., clomid) do not contain the hCG hormone. 00431-0010.pdf

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