The earliest written example of a test for diagnosing pregnancy comes from ancient Egypt. The Berlin medical papyrus, written around 1350 bc, contains multiple clinical pregnancy tests. One suggested counting the number of times a woman vomited when placed upon a mash of beer and dates.
Clearly, the aversion to strong aromatic odors with concomitant nausea and vomiting has been long viewed as a presumptive evidence of pregnancy. Another suggested:
Barley [and] wheat, let the woman water [them] with her urine every day with dates [and] the sand, in two bags. If they [both] grow, she will bear. If both do not grow, she will not bear at all.1
Variations on the latter theme existed in subsequent ancient texts, from Hippocrates to Galen, and are thought to be underpinned by the concept of analogy. Since a pregnant woman gives forth new life, her urine will similarly contain a vitalistic matter that will innervate anything it touches. Some may dismiss the results of these crude bioassays as nothing more than chance events; however, at least one study demonstrated a sensitivity of about 70% following ancient techniques.2
Later authorities described changes in the urine. "If the iridescence be quite distinct, it is a sign that conception is beginning. When it gives place to redness, it shows that impregnation is completed."1
Indeed, the visual examination of urine to diagnose pregnancy and a multitude of other conditions became quite popular during the Middle Ages (Figure 1). Such concepts continued in Western medicine until physicians had a greater understanding of the basic physiologic and hormonal underpinnings of ovulation and pregnancy. Until such a time, physicians, and their patients, were left with the largely subjective symptoms and signs that provided presumptive evidence of pregnancy: nausea with or without vomiting; disturbances in urination; fatigue; the perception of fetal movement; cessation of menses; changes in the breasts; discoloration of the vaginal mucosa; increased skin pigmentation and the development of abdominal striae; and the belief by the patient that she was pregnant.
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As a result of both the Industrial and Scientifi c Revolutions occurring in the 18th and 19th centuries, medicine was transformed from a profession that relied upon anecdotes to one that was increasingly based on scientifi c evidence. At the start of the 20th century, neither the term hormone nor its physiologic underpinnings were commonly understood. Indeed, in the realm of gynecology, not even the basic, cyclic changes of the endometrium in relation to ovarian function were comprehended with any certainty.
In May 1900, German gynecologist Ludwig Fraenkel (1870-1951) embarked upon a series of experiments exploring the role of the corpus luteum in rabbits, and by extension, humans. Th rough his rabbit experiments, Fraenkel identifi ed some of the hormones involved in female reproduction and named the hormone that promoted gestation "progesterone." Moreover, in later observations on women, Fraenkel discussed the physiologic timing of menstruation and ovulation, noting "ovulation is preceding the menstruation belonging to it for about one or two weeks."3
While Fraenkel experimented upon the corpus luteum and noted its eff ect on menstruation, others pondered the question of what caused the corpus luteum to form. One was Bernhard Zondek (1891-1966) (Figure 2). Zondek was intrigued by the problem of uterine and vaginal maturation. Observing that the administration of an ovarian extract in immature mice caused uterine enlargement, he postulated that an additional glandular source must exist in the body.
In collaboration with Selmar Asch heim, Zondek discovered that the anterior lobe of the pituitary contained 2 hormones that were found responsible for follicular maturation of the ovary (Prolan A, follicle-stimulating hormone [FSH]) and formation of the corpus luteum (Prolan B, luteinizing hormone [LH]). Th is was a crucial intellectual development, namely the concept of trophic hormones secreted by the body.
Concurrently, scientists in several laboratories around the world described the presence of a separate substance that promoted ovarian development and growth in rabbits and mice. Knowing that the anterior pituitary hypertrophies in pregnancy and having Zondek just demonstrate that a diff erent ovarian substance, folliculin, occurs in high concentration in bodily fl uids, Aschheim wondered if this new mystery hormone was also abundant in pregnancy, and in particular the urine of pregnant women.
Through a series of experiments, Aschheim and Zondek noted this substance specifi cally aff ected the formation of the corpus luteum. They initially thought that the gonadotropic substance was produced by the anterior pituitary. Through the research of Georgeanna Seegar Jones (1912- 2005), it was later found to be a product of the placenta, and subsequently named human chorionic gonadotropin (hCG).4 However, in 1928, Aschheim and Zondek published their work, explaining the principle of the pregnancy test.5 As originally described, the test is performed as outlined below:
Th e morning urine is sent into the laboratory in clean bottles. A group of fi ve mice, each weighing six to eight grams, is used to test each urine specimen. Th e total volume of urine injected into each mouse varies from 1.2 to 2.4 ccs. Th is total dosage is distributed in six doses during forty-eight hours. It is administered subcutaneously in increasing amounts of 0.2 to 0.4 ccs. On the fi fth day (ie. 96 to 100 hrs after injections are begun), the animals are autopsied and the genital system is examined. Th e ovaries of untreated animals of this age are always very small, smooth glandules, containing, at most, small follicles. In case the ovaries show further development, the type of reaction can be classifi ed as: Reaction I – Enlarged follicles Reaction II – Hemorrhagic follicles ("blood points") Reaction III – Corpora lutea Th e presence of Reaction II or III or both is an almost certain indication of the presence of the sex hormone in urine, characteristic of pregnancy in the case of humans. The reaction is considered positive if only one hemorrhagic follicle or one corpus luteum is observed in any one of the five animals.6
Within 2 years of their initial publication, published reports of more than 3,000 tests found an error rate of only 1% to 2%. It was hailed as a major breakthrough, not only because of its ability to diagnose pregnancy much earlier than ever before but also because of the ability to now diagnose molar pregnancies and choriocarcinoma. Additionally, the test was used to exclude pregnancy in cases of amenorrhea. Subsequently, numerous in vivo bioassays were developed for the qualitative detection of hCG through gonadotropin stimulation of the gonads with endpoints including ovulation in rabbits or frogs and spermiation in male toads.7-9
Unfortunately, numerous problems existed for these bioassays. First, they were costly to perform, requiring many animals for precision. Additionally, they took several hours to days to complete and were fraught with the imprecision associated with species diff erence and biologic variability. Additionally, they were relatively insensitive, needing anywhere from 100 mIU/mL to 3 IU/mL of hCG for a positive reaction. Th ese problems spurred research into more reliable and less costly alternatives. However, they fi rst needed to overcome the problem inherit in the hormone itself: the shared structure with the other gonadotropins.
Today we take for granted our understanding of hCG. We know that it is 1 of 4 glycoprotein hormones and is composed of an alpha and beta subunit. Th e hCG alpha subunit is identical to the alpha unit LH, FSH, and thyroid-stimulating hormone (TSH), whereas the beta subunit is unique for all 4 glycoprotein hormones. But in 1960 when the fi rst immunologic test by hemagglutination inhibition was developed by Wide and Gemzell, the tests could not accurately distinguish between hCG and LH. Consequently, while sensitivity was improved, specifi city was only marginally better than the original A-Z test.
It took the work of Judith Vaitukaitis and colleagues at the National Institutes of Health to create an antibody that would be specifi c to the beta-subunit of hCG, and therefore could be used in a radioimmunoassay. Th is was accomplished in 1972, when she was able to produce an antiserum from rabbits.10 Th is development improved the specifi city of the test, making possible accurate diagnosis as early as 8 days postovulation. Still, the tests were cumbersome and required trained offi ce or laboratory personnel to complete. Subsequently, all current pregnancy tests in hospitals or clinics or sold for home use rely on equivalent components, analytic methods, and performance, based upon the ability to detect hCG in a concentration of 25 mIU/ mL or greater in urine.
In March 1978, a "private little revolution" was announced to the readers of Vogue magazine. Th e e.p.t.® home pregnancy test was described in a full-page advertisement (Figure 3) and trumpeted a populist message that "at last early knowledge of pregnancy belongs easily and accurately to us all."11 Previously women were able to obtain reliable pregnancy testing at their physician's offi ce and hospitals. But such methods placed the patient in a dependent role of obtaining personal information about their well-being.
Th e e.p.t. advertisement appealed to the more modern sense of patient autonomy. However, there was an initial cultural backlash. In evaluating e.p.t. for its readers, Consumer Reports insinuated that the need for a home pregnancy test was evidence of promiscuity.11 Additionally, physicians expressed mixed opinions about the test, and most insisted on confi rming the diagnosis with an office pregnancy test. However, pharmaceutical companies believed the diagnosis of pregnancy was so personal and anxiety provoking that women would want to move this particular test from the public world of the doctor's office to their home. No longer would they be forced to deal with a middleman, but instead, they would be the first to know and determine with whom to share that knowledge.
Methods for diagnosing pregnancy have evolved over the past 4,000 years, from primitive urinary bioassays to mass-produced precision products. But at their core, these tests still answer a very personal and private question—Am I pregnant?—and ultimately will require an individual dedicated to women's health care to help deal with the answer.
The author reports no actual or potential conflict of interest in relation to this article.
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