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1 ~ Developing Assays of Biologic Markers for Epidemiologic Studies: Experience with a Marker of Pregnancy and Early Loss Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted by the syncytiotrophoblast; it appears to enter the maternal circulation at the time of endometrial implantation of a fertil- ized ovum (Jaffe, 1986~. Its principal known role is to act at the ovarian corpus luteum to stimulate further secretion of progestins to support endometrial growth. Blood and urinary hCG concentrations rise rapidly and peak approximately 12 weeks after onset of the last menstrual period; thereafter, they slowly decline until the fetus and placenta are delivered. An addi- tional function of hCG may involve stimulus of steroid hormone production by the fetus. The period of maximum testosterone produc- tion by the fetal testis corresponds to the peak period of placental hCG produc- tion, and gaffers group has demonstrated that hCG binds to receptors in the fetal testis and stimulates the production of testosterone (Huhtanemi et al., 1977~. (Physiologic changes associated with the initiation of pregnancy are discussed in Chapter 20.) This chapter describes the development and application of an assay to assess uri- nary hCG content as a biologic marker. The purpose of describing the problems encountered in laboratory development and early use of inappropriate assays is 187 to make clear the need for complete devel- opment in the laboratory and preliminary studies in the field before assays are used in large epidemiologic studies. It is demonstrated that even with this widely studied hormone, there remains a need to improve the assays for use in field studies and a need for more extensive studies of normal populations. ASSAYS OF hCG In contrast with many other plasma pro- teins, hCG is excreted in the urine at a concentration approximately equal to that in blood. Marshall and colleagues (1968) demonstrated that to be the case throughout the first trimester, and Armstrong et al. ( 1984) verified that, even when minute quantities of serum hCG are present, the urine specimen is a ready source for assay and purification of hCG. . . ~ . . . . ~ However, it is also Hey that urine specimens contain partially degraded hormone fragments, and measurement of a serum specimen might be preferred for diagnostic interpreta- tion. Bioassays for hCG were developed by As- cheim and Zondek (1928) (the A-Z test) as a method to detect pregnancy several weeks after the first missed menstrual period. In the 1960s, immunologic assays

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188 for hCG replaced the more cumbersome bio- assays (Wide, 1962; Wide and Gemzell, 1960~; these added increased sensitivity and reproducibility, which permitted earlier diagnoses of pregnancy (Bell, 1969~. However, polyclonal (usually rab- bit) antibodies raised against hCG always cross-reacted with human luteinizing hormone (hLH) in the maternal serum sample or with hormone components in urine, owing to the high degree of homology between hCG and hLH. The sensitivity of these assays was limited by the inevitable presence of hLH immunoreactivity in maternal serum and also by either intact hLH or its frag- ments in urine. The relationship of all the homologous glycoprotein hormones became apparent . with the discovery that each comprised a single ax subunit and a single ~ subunit (Pierce et al., 1971~. The amino acid se- quences of the ~ subunits of hLH and hCG were shown to be identical, but minor dif- ferences were found in the structures of the ,B subunits (Bellisario et al., 1973; Morgan et al., 1973, 1975; Birken and Can- field, 1977; Kessler et al., 1979; Fiddes and Goodman, 1981; Pierce and Parsons, 1981~. When immunized with the purified ,6 subunit of hCG, rabbits occasionally made antibodies that were directed predom- inantly against this hormone and had lim- ited cross-reactivity with human hLH (Vai- tukaitis et al., 1972~; radioimmunoassays were developed that routinely detected hCG at 1 ng/ml in serum in the presence of circulating hLH. Radioimmunoassays with urine specimens were more complex, because of interfering substances and the presence FIGURE 15-1 Antibodies with specificity to ~ and B subunits of hCG. Shaded areas indicate regions that give rise to antibodies. Source: Canf~eld et al., 1987. FEAL9LE REPRODUCTIVE TOXICOLOaY of degradation products. Nonetheless, this assay method was used to develop non- radioisotopic home-testing kits for hCG in urine specimens to detect pregnancy with moderate reliability (Doshi, 1986) during the first 9 days after the first missed menstrual period. Two developments led to new hog assays with improved sensitivity and specifici- ty. First, monoclonal antibodies against the hCG ~ subunit could be produced, so that urine could be extracted efficiently by immunoaffinity adsorption. Second, pre- parations of the unique ,B COOH-terminal peptide region of hCG could be used as im- munogens to make high-affinity polyclonal antibodies (Birken et al., l 982~. Several laboratories have made prepara- tions of antibodies that recognize hCG and hLH to varied degrees (c.f., Vaitukai- tis et al., 1972; Birken et al., 1982; Thau et al., 1983; Ehrlich et al., 1985~. Of particular interest are antibodies that exhibit a high degree of specificity for hCG, that is, have very low reactivity with hLH. Four separate regions in hCG give rise to antibodies that may be used in assays with selectivity for hCG over hLH (Fig. 15- 1). Each region presumably reflects a conformational change resulting from structural differences. Region IV represents an antigenic deter- minant (epitope) that is not readily reac- tive in native hCG, but is present on the free ,B subunit and on forms of the degraded hCG' subunit excreted in human urine. That region has been most recently identified and affords the opportunity to distinguish B20 1 B202 \ ,:5 J Bl03 / a A 109' /~ A102 H 1 B107 B I 09 ~[B2 06 ( \ P3W80 tB108

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MARKERS FOR EPIDEMIOLOGIC STUDIES between degraded hCG ~ subunit fragments and the intact hormone. The complete structure of the degraded ~ core fragment has been determined, and monoclonal an- tibodies that bind to Region IV have been developed (Birken et al., 1988; Krichevsky etal., 1988~. The importance of the four discrete re- gions of the hCG molecule is demonstrated by immunoradiometric assays (sandwich assays) that use two antibodies selected on the basis of binding to different hCG epitope regions. An early study found that one such assay exhibited an affinity far beyond that expected (Ehrlich et al., 1982~. A mathematical model was developed that accounted for that observation by predicting formation of a circular complex composed of one molecule of each antibody and two molecules of antigen (Moyle et al., 1983 a,b) (Fig. 15-2~. With those immunochemical reagents, a sensitive assay for hCG in urine was de- veloped that used an immunoradiometric assay (Wilcox et al., 1985~. The f-specif- ic monoclonal antibody B 101 is coupled to Sepharose beads, suspended in urine, spun down; the hCG binds to the antibody. To measure the amount of hCG captured, a Fc F(ab~/\\ F(ab) 189 i26I-labeled antibody from the rabbit (R525) is mixed with the resuspended Seph- arose-B 101 -hCG mixture. When this is spun down, amounts of radioactivity bound to the solid phase indicate quantities of hCG attached to the capture antibody. The assay permits hCG detection at concentra- tions approaching 0.01 ng/ml (highly puri- fied reference preparations of hCG have a bioassay value of approximately 13,000 IU/mg) and is 50-100 times more sensitive than any other existing method (Armstrong et al., 1984)(Fig. 15-3). When a conventional radioimmunoassay is performed on a urine specimen, contami- nating proteolytic enzymes in the urine can degrade the radiolabeled tracer during its incubation. This proteolytic artifact might lead to falsely increased measurements of hCG (Maruo et al., 1979~. In addition, other interfering urinary substances can add to the background noise and decrease the signal-to-noise ratio (Ayala et al., 1978~. In the immunoradio- metric assay, the radioactive tracer is introduced after proteases and interfer- ing substances have been washed away. SB- 6 radioimmunoassay in urine displays poor signal-to-noise ratio with wide variation FIGURE 15-2 High-af~mity circular complex that is formed in a A n t i b 0 d y "sandwich" assay. Source: Moyle et al., 1983b. B 1 02 h C G ~ ~) ()~ Im ~ ) 1 ~ ~ a F(ab) ~ 1 a ~ \\// ~// // F(ab) ~ , Fc Antibody _ B I O I \ hCG

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190 Figure 15-3 A. Schematic diagram of procedure for ~mmunoradiometr~c assay of hCG In unne; B. Sensitivity of hCG detection by ~mmunoradim metric assay. Source: Canfield et al., 1987. 10,000 - o at m 6000- :E between 1 and 10 ng/ml (Wilcox et al., 1985~. This contrasts with data from im- munoradiometric assay of same specimens, which displays greater sensitivity for hCG without widely fluctuating baseline. Those factors might have contributed to the differing results (8% to 57%) obtained in prior studies of fetal loss (Miller et al., 1980; Edmonds et al., 1982; Whittaker etal., 1983~. FIELD STUDIES OF EARLY FETAL LOSS: TESTING THE UTILITY OF THE hCG ASSAY A small trial was conducted to ascertain the feasibility of the study design and whether new assays with improved sensitiv- ity for hCG in urine would yield new data; specimens also were analyzed with earlier methods (Wehmann et al., 1981). FEMALE REPRODUCTIVE TOXICOLOGY A ~ , in, I m ' Lo CO -I< ~ + HOG I ) Wash to remove / 2) Incubate with rediolabel.d int~rtering substance: BCTP specific antibody ~. Washing and ~ centritugation ANTIGEN ANTIBODY "SANDWICH" ~ B 2000 Ouantitation of R525. a/ it HCG (ngJml)/ Urine ,6/ But ~ c, ' to HL~ (~g/ml ) Nonspeclf ic Blndlng 001 010 1 0 CONCENTRAT ION Paid volunteers in the field study col- lected approximately 1 oz of urine each morning and stored the containers frozen until the containers were picked up for assay (Wilcox et al., 1985). Three studies testing the utility of these assays have been completed. In the first study, 30 women collected daily urine specimens from the time they stopped contraception until they became pregnant or for 6 months if no pregnancy occurred (Wilcox et al., 1985~. The study showed the feasibility of the epidemiologic design and permitted investigators to detect several cycles with early fetal loss that would not have been detected with other available meth- ods. The second (control) group comprised women who had undergone tubal ligation; no patterns suggestive of pregnancy were seen. The study was performed blindly.

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MARKERS FOR EPIDEMIOLOGIC STUDIES In the third study, women using intra- uterine devices for contraception were evaluated to determine whether this con- traceptive technique prevented implanta- tion of the fertilized ovum. Urinary hCG was detected in only 1 of 107 menstrual cycles in this study. Wilcox et al. (1987a) concluded that implantation is infrequent among IUD users; the single pregnancy loss detected might have been a tubal implanta- tion. Figure 15-4 illustrates the types of findings that can be obtained in field studies. Three consecutive menstrual cycles are illustrated in which daily urine specimens were collected from one person during the second half of each cy- cle. During the first cycle, no conception occurred, and all hCG values were less than 0.01 ng/ml. During the second cycle, 3 weeks after the onset of the prior menstru- al period, urinary hCG rose for 6 days to 0.38 ng/ml. hCG steadily declined over the next 6 days and became undetectable. With the decline of hCG came the onset of menses-27 days after the onset of the pre- vious menses. The woman was unaware of her pregnancy or the episode of early fetal loss. However, the finding of 11 consecu- tive increases in urinary hCG above the normal background for that woman and the pattern of rise and fall leave little doubt 10 HUMAN 1 CHORIONIC GONADOTROPIN (ng/ml) 0 1 191 that a loss occurred. Three weeks after the apparent fetal loss, the woman again exhibited increasing urinary hCG. This time, she remained pregnant and delivered a normal, full-term infant (Wilcox et al., 1985~. FUTURE ASSAY DEVELOPMENTS Tests for urinary hCG have evolved from relatively insensitive bioassays to sensitive and specific immunoassays. The ectopic secretion of hCG by some tumors and its slight increase in postmenopausal urine specimens (Armstrong et al., 1984; Kuida et al., 1988) are unlikely to dimin- ish its epidemiologic value as the biologic marker of pregnancy in healthy women of reproductive age because the char- acter~st~c pregnancy-related changes in hCG concentration are not observed from tumor secretions. Other proteins that appear to be pregnancy-specific have shown less utility as markers in epidemio- logic studies; furthermore, they are usu- ally assayed in blood specimens, and their fate in urine is less well known. (An ex- tended discussion of markers of early preg- nancy is given in the Chapter 20.) Field trials involving regular hCG test- ing in women attempting to conceive have proved adaptable to large-scale epidemio- . . . . . ~ . . . . . . . . . . . . . . . ..... ~ . . ... .. ... . . - . . . . . ..... i. ..... _ ..... _ ..... r . . . . I . . . . . . . . . . . . . . I . . . .. I ..... 1 ..... ~ ..... ~ ~222"'"' ~ l 2 V"".2 I I ~ (~ by 1 :: \ ; 1 . . . I I / ~ . 1~ 1 ~ ~ ~ ~ I I 5 6 7 8 WEEKS (BARS INDICATE MENSES) 9 10 11 FIGURE 15~ hCG in urine during three consecutive menstrual cycles in human female unaware of pregnancy or earn fetal loss. Source: Wilcox et al., 1987b.

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192 logic studies. Within a few days of the expected date of implantation of a fertil- ized ovum, hCG can be detected, as it can at least 2 days before the onset of the next expected menstrual period (Wilcox et al., 1985~. Early fetal loss has been detected by urinary hCG testing; this requires an hCG assay that is sensitive to a concentra- tion lower than 0.05 ng/ml. Much more needs to be done to advance methods in this field of reproductive biol- ogy, if large-scale epidemiologic studies are to be undertaken to search for environ- mental factors that alter the rate of early fetal loss. Any change in the rate of clini- cally apparent abortions potentially caused by environmental factors can be detected readily by routine hCG testing and by the clinical events that surround abortion. Improved Methods for hCG Detection To expand the present labor-intensive and time-consuming research methods for immunoradiometric measurement of hCG, several advances would be desirable. A simpler assay method is needed that preserves sensitivity and specificity for hCG while reducing the needs for labor, large urine specimens, and large quanti- ties of antibodies. These advances should be made in the direction of a non- radioactive-assay format. Ideally, many specimens could be processed accu- rately, rapidly, and inexpensively in the laboratories shortly after urine collec- tion. That would minimize the need for extensive deep-freeze storage space, because more than 90% of the specimens have negative results and would be discarded. Only urine specimens with positive screen- ing tests that suggest an episode of fetal loss would be shipped to a central assay laboratory. New Monoclonal Antibodies for hCG Detection In the quest for an improved assay for the urinary products of hCG, new antibod- ies with high affinities for the various forms of urinary hCG will be important. These should be monoclonal antibodies, FEMALE REPRODUCTIVE TOXICOLOGY to ensure a continuous source of complete- ly characterized immunochemical rea- gents. It is important to be able to char- acterize and detect not only the entire hormone, but also its degraded forms in urine; to maximize sensitivity; and to ensure that unusual biologic events, such as a selective decrease in the secretion of one of the subunits, do not lead to in- complete or inaccurate results. New and specific monoclonal antibody cell lines might be required to accomplish this task. Nonradioactive Methods for hCG Detection Many of the most sensitive research im- munoassays use radioactive iodine, but this approach has disadvantages, includ- ~ng: Radiolabeled reagents must be freshly prepared and characterized every few weeks and then shipped to the users. An ever-increasing problem with ra- dioactive waste disposal is of concern. Use of radioactive methods requires special training and surveillance in the laboratory. Epidemiologic research related to early fetal loss would be advanced substan- tially if a nonradioactive method to meas- ure hCG were devised in which the reagents had a shelf-life of 6 months to 1 year. The most widely used nonradioactive method is enzyme-linked immunosorbent assay (ELISA), which requires only basic instru- mentation (spectrophotometry) for quan- tification. The sensitivity of the assay depends on the enzyme amount and activity that can be specifically linked to the antigen or antibody measured. Several recent advances have been intro- duced to amplify the signal and increase sensitivity, including enzyme-antibody coupling through the avidin-biotin system (Fuccillo, 1985), lectin-carbohydrate coupling, and use of chimera antibodies of double specificity (Guesdon et al., 1983~. Those techniques involve noncova- lent linkage and therefore avoid the loss of enzyme catalytic activity due to chemi- cal modification. i

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MARKERS FOR EPIDEMIOLOGIC STUDIES Assays based on fluorescence or phos- phorescence depend on light absorption to provide the excitation energy to produce the emission. Assays based on chemilumine- scence depend on the energy from an oxida- tive chemical reaction to produce mole- cules in an electronically excited state; return to the ground state is accompanied by photon emission, which is detected pho- tometrically. Low quantum yields and high background interference limit the sen- sitivity of chemiluminescent, fluore- scent, and phosphorescent techniques. To avoid those problems, time-resolved fluorometric immunoassay has been devel- oped. Lanthanide chelates have a high quantum yield and a large Stokes shift (340-nm excitation wavelength and 614- nm emission wavelength). In addition to a ion' decay time for europium chelates ( 103- 10 ns), these properties permit an assay wherein a pulsed light of short dura- tion (compared with the decay time of the lanthanide chelates) is transmitted to the sample, and the interfering rapid decay fluorescence of other serum or uri- nary constituents is discriminated against by activating the detection system after a delay sufficient to complete the decay of naturally occurring fluoro- phores. Those techniques and others have the advantage of a nonradioactive detection system and increased reagent stability. Further investigation is required to de- termine which approaches would provide the most satisfactory sensitivity and specificity, which until the present have been hallmarks of radioisotope - based technology. Requirement to Measure Other Biologic Markers Urinary hCG is a valuable biologic marker of implantation of a fertilized ovum, but it does not detect a fertilized ovum that does not implant. New methods to test for that should be developed. One approach would be a technique to collect uterine secretions and assay for the presence of hCG secreted by the nonimplanted ovum. Another approach involves developing adequate methods for the assay of other 193 pregnancy markers that do not depend on implantation. Although a wide variety of pregnancy-associated proteins have been described (Home and Nisbet, 1979), most appear to be associated with the later stages of pregnancy development. One ex- ception is the pregnancy-specific protein early pregnancy factor (EPF) (Morton et al., 1977~. EPE properties have been stud- ied in humans (Tinneberg et al., 1985), sheep (Morton et al., 1979) and mice (Morton et al., 1976), mainly by the ro- sette-inhibition assay. ~ ., Its appearance can be detected in maternal serum within 6-48 hours after fertilization and does not depend on implantation of the fertil- ized ovum for detection (Sinosich et al., 1985~. EPF persists in serum throughout the first two trimesters of pregnancy (Ca- vanagh et al., 1982) and rapidly disappears after embryo death or surgical removal (Nancarrow et al., 1979~. In a study of 13 multiparous women (Rolfe, 1982), EPF was detected within 48 hours of fertilization (ovulation dated by progesterone determination). Of cycles studied in which intercourse occurred at the time of ovulation, EPF was detected in 18 of 28 cycles, but con- tinued to be produced beyond 2 weeks in 4 instances; of these 4 subjects, only 2 proceeded to full-term pregnancy. In the remaining 14 of the 18 subjects, EPF became undetectable before the onset of the next menstrual cycle. The data suggest that many fertilized ova are lost before implantation. An assay less cumbersome and more sensitive than the rosette-in- hibition test should be investigated. EPF should be thoroughly elucidated, and the possible presence and assay of EPF or its metabolites in urine investigated. In epidemiologic studies of reproduc- tive function, improved markers of ovula- tion need to be developed in easily col- lected biologic specimenssaliva or urine. These improvements are needed to develop better methods to detect the vari- ous forms of the pituitary gonadotropins and also to detect various estrogen and progesterone derivatives throughout the menstrual cycle, to estimate the time of ovulation, and to document the formation of a corpus luteum.

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194 The requirement for new high-affinity antisera directed against hLH and FSH is based on the heterogeneity of the glycopro- tein hormones from the pituitary (Franchi- mont et al., 1972~. Isoelectric focusing data show that hLH from regularly cycling women is less acidic than that circulating in men and postmenopausal women, pre- sumably owing to a lower sialic acid con- tent (Wide, 1981~. Differences in sialic acid content are manifested not only in physicochemical properties, but also in immunoreactivity, receptor affinity, and biologic activity, even to the extent of having a variant of hLH that is immuno- logically reactive but devoid of biologic effect (Axelrod et al., 1979~. Multiple forms of FSH are also observed in other mammals, with as many as six immunoreactive forms present in the hamster (Ullsa- Aguirre and Chappel, 1982~. The different forms have the same molecular weight, but exhibit different affinities in lectin binding and have different bioactivity- to-immunoreactivity ratios when tested by radioreceptor assay. Those findings stress the importance of developing assays that measure the per- tinent forms of excreted pituitary gonado- tropins. Assays that- use antisera raised against antigens derived from postmeno- pausal or male sources might not be optimal for assays on normally cycling women and might have contributed to the primary lack of sensitivity in ovulation detection. Recent work documented that urinary FSH patterns closely resemble those found in serum FSH patterns when measured by granu- losa cell aromatase bioassay (Dahl et al., 1987~. Future work in gonadotropin-assay development should include assays for the intact hormone and its subunits, as well as determination of the correlation be- tween the immunoreactivity and bioactivi- ty of the substances being measured (e.g., gonadotropins isolated from normally cycling women), to monitor the validity of the assay. Two objectives are met by measuring cir- culating steroids or their metabolites. The first is to signal the onset of ovula- tion and thereby validate the accuracy of gonadotropin assays as an ovulation marker. The second is to assess the exis- FEA~ll;"E REPRODUCTIVE TOXICOLC)GY fence and adequacy of the corpus luteum. A variety of urinary estrogen metabolites have been assayed and their utility as markers of ovulation evaluated. Baker and colleagues ( 1979) assayed directly El-3-glucuronide, E2-3-glucuronide, E2- 1 7,8-glucuronide, E5-3-glucuronide, and Es- 1 6a-glucuronide in urine and found that the E2-17,6-glucuronide assay was the most sensitive predictor of ovulation, fol- lowed closely by E,-3-glucuronide. The authors recommended the latter, because 5 times more of it is excreted than of the former and it can be detected in a 100-fold diluted urine specimen, in which poten- tially interfering substances would be diluted virtually to nonexistence. The Baker et al. study (1979) also indi- cated that random urine collections can be valid ovulation markers. Dividing the mass of steroid metabolite by the exact duration of collection gives the produc- tion rate in nanomoles per hour, and com- parison of this pattern for first-morning voids and 24-hour collections showed ex- cellent correlation. Results expressed as the steroid:creatinine ratio also cor- related highly with the 24-hour collection results. Thus, either method may be used in lieu of 24-hour collections. The E,- 3-glucuronide:pregnanediol 3a-glucuro- nide ratio is independent of urine volume and proved to be another valid ovulation marker. In a study of the urinary E~-3-glucuro- nide, hLH, and pregnanediol 3~-glucuro- nide as markers of ovarian function, Col- lins et al. (1979) found a good correlation between early-morning collection and 24- hour collections and found that the ratio of E~-3-glucuronide to pregnanediol 3a- glucuronide could be used to demarcate the duration of the fertile period. A multicenter study (WHO, 1 980b) evaluated the excretion pattern of E~- 3-glucuronide, E2- 1 7,8-glucuronide, E2- 3-glucuronide, E~- 1 6~-glucuronide, E~- 3-glucuronide, pregnanediol 3~-glucuron- ide, and pregnanetriol 3~-glucuronide throughout the menstrual cycle, to deter- mine which best indicated the fertile peri- od during the cycle; E~-3-glucuronide in the follicular phase provided a marker of ovulation 72 hours before ovulation,

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MARKERS FOR EPIDEMIOLOGIC STUDIES and assay of pregnanediol 3~-glucuronide was the best indicator of whether ovulation had occurred. The WHO study (1980b) also confirmed that first-morning voids yielded results as reliable as those obtained from 24- hour collections. OTHER CLINICAL OPPORTUNITIES If research is limited to young and otherwise normal women, unusual patterns might be missed. However, women with ab- normal physiology enrolled in an epidemio- logic study can lead to unexpected and inexplicable findings. Thus a wide spec- trum of clinical studies is important for epidemiologic research. Fecundity decreases with age in women. The rate of early fetal loss might differ between younger women and older women, and enrollment of older women in a study might affect the outcome; but no adequate data exist on the rate of early fetal loss in women older than 35 or 40 years. Effects of different types of prior con- traceptive use such as steroidal agents, IUDs, and spermicides should be studied. Furthermore, not all women have regular menstrual periods, and those with short and long luteal phases should be studied to determine, for example, how this affects the rate of fetal loss and timing of sneci- men collection to detect implantation and loss. The study of luteal phase charac- teristics has become feasible with the development of direct assays for urinary estrogen and progestin metabolites using nonradioisotopic assay systems. Postmenopausal women have a strong stim- ulus for pituitary gonadotropin secre- tion; to some extent, this appears to stim- ulate a minute degree of hCG synthesis and 195 secretion as well (Robertson et al., 1978; Armstrong et al., 1984~. Important ques- tions to study are whether this might be a problem when studying perimenopausal women who conceive, whether this is a pat- tern of hCG, and whether the degree of ovar- ian failure that leads to a slight amount of hCG secretion occurs only when a woman becomes infertile. The National Center for Health Statis- tics reports that 14% of couples in this country have a problem with infertility. Some men or women might have a genetic pre- disposition to a high rate of early fetal loss or failure of the fertilized ovum to implant. Artificial insemination pro- grams provide a fine opportunity to study this problem. It is also important to de- termine whether frozen and fresh semen specimens lead to different rates of con- ception and fetal loss. Although several studies have indicated that the use of frozen semen leads to diminished fecun- dability, at least one study reports that there is no difference in pregnancy rate whether fresh or frozen semen is employed (Trounson et al., 1980~. Epidemiologic research provides an opportunity to identify persons with un- usual reproductive patterns that might occur with low frequency in the population. Protocols should be designed that test the major epidemiologic hypotheses and also are sensitive to the occurrence of such unusual patterns. The research described above will in- crease the knowledge regarding the use of hCG as a marker of pregnancy and early fetal loss; that increased knowledge will benefit not only epidemiologic stud- ies, but also other research to detect abnormalities of human fertility.

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