Measuring early pregnancy loss: laboratory and field methods*

FERTILITY AND STERILITY Copyright < 1985 The American Fertility Society

Vol. 44, No.3, September 1985 Printed in U.SA.

Measuring early pregnancy loss: laboratory and field methods*

Allen J. Wilcox, M.D., Ph.D.t:!: Clarice R. Weinberg, Ph.D.t Robert E. Wehmann, M.D., Ph.D.§ E. Glenn Armstrong, Ph.D·11 Robert E. Canfield, M.D. ~ Bruce C. Nisula, M.D.# National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; Louisiana State University Medical Center, New Orleans, Louisiana; College of Physicians and Surgeons, Columbia University, New York, New York; and National Institute of Child Health and Human Development, Bethesda, Maryland

We intensively studied 30 women attempting pregnancy in order to lay groundwork for larger studies of early pregnancy loss. These women collected first morning urine specimens for up to 6 months after discontinuing use of birth control. Urine specimens were successfully collected for 98% of the woman-days in the study. Three assays for human chorionic gonadotropin (heG) were performed on each urine specimen. An immunoradiometric assay (IRMA) specific to the carboxyterminal peptide of the heG (3-chain proved to be more sensitive and more specific than two radioimmunoassays (RIAs). Using the IRMA, we found four cases in which heG rose and fell over successive days, consistent with early pregnancy loss. For three of these four cases, the level of heG was too 'low to be detectable with the RIAs. Among the control group of five women with tubal ligations, there was no detectable heG above threshold with the IRMA. Thus, the enhanced sensitivity and specificity of the IRMA allows very early pregnancy losses to be identified that would otherwise be undetectable. Furthermore, its effectiveness with small quantities of first morning urine makes the IRMA a useful tool for epidemiologic studies. Fertil Steril 44:366, 1985

Received May 2, 1985; revised and accepted June 19, 1985. *Supported by National Institutes of Health grants HD15455 and RR-00645. tNational Institute of Environmental Health Sciences, Epidemiology and Statistics Branches. :j:Reprint requests: Allen J. Wilcox, M.D., Ph.D., Mail Drop A3-02, NIEHS, P.O. Box 12233, Research Triangle Park, North Carolina 27709. §Department of Medicine, Louisiana State University Medical Center. IIDepartment of Pathology, College of Physicians and Surgeons, Columbia University. ~Department of Medicine, College of Physicians and Surgeons, Columbia University. #National Institute of Child Health and Human Development. 366

Wilcox et al. Measuring early pregnancy loss

Fifteen percent to 20% of recognized pregnancies end in spontaneous abortion. 1-3 However, less is known about how many pregnancies are lost before pregnancy is recognized and the extent to which such loss might contribute to clinical infertility.4 For many years the occurrence of early pregnancy loss could only be inferred from indirect evidence. 5 ,6 The refinement of radioimmunoassays (RIAs) in the early 1970s allowed the hormonal events of early pregnancy to be observed more directly. With the use of these assays, instances of very early pregnancy and subsequent loss could be documented. 7 , 8 Three recent studies Fertility and Sterility

1

.1

have extended these observations using RIAs to estimate the risk of early loss in groups of women. 9- 11 These studies measured human chorionic gonadotropin (hCG) in serum or urine as an indicator of pregnancy. Preliminary estimates of early loss obtained by these studies range from 8% to 57% of all pregnancies. Measuring the risk of early pregnancy loss involves issues of both immunochemistry and epidemiology. Before we could undertake a largescale study of early pregnancy loss in normal volunteers, it was necessary to determine the laboratory methods that could best be applied in an epidemiologic study and the field methods that would provide the specimens and data required. Specifically, we set out to address the following three questions: (1) Can hCG be measured sensitivelyand specifically in relatively small quantities of urine specimens collected daily? (2) Is it feasible to collect daily urine specimens and other daily information from volunteer women who are planning a pregnancy? (3) Can patterns of hCG rise and fall be detected in daily urine specimens that provide plausible evidence of early pregnancy loss?

MATERIALS AND METHODS

We recruited 30 women in the Research Triangle area of North Carolina through ~ocal physicians, brochures, posters, and newspaper advertisements. Women were enrolled at the time they stopped using birth control to become pregnant. If either the woman or her partner had a history of chronic illness or fertility problems, the woman was excluded. Women were interviewed regarding pregnancy history and previous methods of birth control, as well as current medications; smoking, and environmental exposures. The women were asked to collect daily first morning urine specimens (30 ml) and store them frozen for pick-up every 1 or 2 weeks. Women used a daily record card to record menses, intercourse, and symptoms of pregnancy. Collection continued for 8 weeks into a clinical pregnancy or for 6 months if no pregnancy occurred. Women were paid $10 a week for the collection and storage of these specimens. Urine specimens were stored at - 20°C at the National Institute of Environmental Health Sciences until collection was completed and then shipped on dry ice to collaborating laboratories for analysis. Vol. 44, No.3, September 1985

As a control group, five women with tuballigations also collected urine specimens under this protocol. These were women with proven fertility, currently 20 to 35 years of age, who had been sterilized because they had achieved their desired family size. Each woman collected urine specimens for three complete menstrual cycles. Daily urine specimens were analyzed for hCG with three assays. Most assays were done in duplicate or triplicate, with the final value for each specimen calculated as an arithmetic mean. One assay was the RIA with the use of the SB6 antibody,12 as modified by Edmonds et al. lO Normal male urine was added to the standards to compensate for the nonspecific effects of urine in the unknown samples. The tracer was 125I_hCG (CR 121), which was iodinated, as described by Fraker and Speck. 13 The reference preparation was hCG (CR 121). A second RIA for hCG used an antiserum (R529) specific for the f3-carboxyterminal peptide region of hCG. 14 This assay was carried out at Louisiana State University (LSU) with the use of Con A Sepharose (Pharmacia, Piscataway, NJ) to extract hCG. The extraction reduces nonspecific interference from other substances in the urine. Furthermore, the R529 antiserum is able to detect hCG in the presence of human luteinizing hormone (hLH). By reducing interference from hLH and nonspecific sources, this assay permits hCG in urine to be measured more reliably at lower concentrations than with the SB6 RIA. Shortly after this project was begun, a new immunoradiometric assay (IRMA) for hCG was developed at Columbia University. 15, 16 This assay uses immunoaffinity purified and radiolabeled R525 antiserum, which offers the same specificity to the carboxyterminal peptide region of the hCG molecule, as does the R529 antiserum. In addition, the IRMA uses a solid-phase immobilized monoclonal antibody (B101) to extract hCG. We employed this assay in the pilot study for assess- . ment of its performance as an assay potentially more sensitive for hCG than the R529 RIA. Specimens were also analyzed in the LSU laboratory for hLH by the employment of an RIA with Con A Sepharose extraction. 17 Reagents for the assay were provided by the National Hormone and Pituitary Program. Results are reported in terms of International Units of the 2nd IRP human menopausal gonadotropin, which was crossreferenced to the working laboratory standard, LER-907. Wilcox et aI. Measuring early pregnancy loss

367

Table 1. Characteristics of Women in the Pilot Study (Excluding Two Women who were Pregnant at the Time of Enrollment) No.

%

24--36 years (mean, 29) 17/28 61

Age Prior pregnancy Outcome of most recent pregnancy Live birth Spontaneous abortion Induced abortion Most recent contraceptive Diaphragm Condom Jelly Rhythm Pill Intrauterine device Other Education High school College (at least 1 year) Graduate school (at least 1 year) Race White Asian

12117 3117 2117

71 18 12

10/28 5/28 4/28 4/28 2/28 1128 2/28

36 18 14 14 7 4 7

1128 16/30 11128

4 57 39

27/28 1128

96 4

standard curve that yielded a value B/Bo of < 0.90.) Criteria for the R529 RIA varied from 0.5 to 0.7 ng/ml (6.7 to 9.4 mIU/mI), depending on the run. The criterion for elevated hCG with the IRMA was set at 0.035 ng/ml (0.47 mIUlml). The IRMA is sensitive below 0.035 ng/ml, but 0.035 ng/ml has previously been shown to be a reasonable upper limit of hCG immunoreactivity in the urine of nonpregnant premenopausal women. 15

RESULTS

We chose a criterion for elevated hCG for each assay. The criterion for the SB6 RIA was set at 3.5 ng/ml (47 mIU/mI), which is similar to the criterion for pregnancy chosen by Edmonds et al. lO for the same assay. This criterion does not exclude all instances of SB6 cross-reactivity with hLH in our samples. However, our separate measure ofhLH permits us to distinguish SB6 immunoreactivity due to hLH cross-reaction from immunoreactivity presumably due to hCG. The criterion for the R529 RIA was set at > 0.1 ng/ml above the least detectable concentration for the individual assay runs. ("Least detectable concentration" was the concentration of hCG in the

Characteristics of the pilot study women are shown in Table 1. Two women were pregnant at the time of enrollment and are excluded. Four women changed their mind about pregnancy before completion of the study and dropped out. Their cycles while they were in the study are included here. A total of 89 cycles were observed. Seventeen women had clinically confirmed pregnancies and 7 others went 6 months without clinical pregnancy (Table 2). Four of the 17 had a clinically apparent spontaneous abortion. (We define a clinically apparent abortion as one diagnosed by the woman's physician after a positive pregnancy test.) Urine specimens were successfully collected for 98% of the woman-days in the study. Specimens were frozen for up to 18 months before being analyzed. As a preliminary test of stability, we reanalyzed 88 urine specimens by the SB6 RIA 7 to 9 months after their first assay and 80 specimens by the IRMA 14 to 16 months after their first assay. The Pearson correlation was 0.93 for the SB6 RIA and 0.97 for the IRMA. In both, the hCG concentration was increased in the later assay, presumably because of water subli-

Table 2. Pregnancies and Drop-Outs During Each Cycle Observed (Excluding Two Women who were Pregnant at Enrollment) Cycle No. of women starting cycle No. of women becoming clinically pregnant No. of women with apparent early pregnancy loss No. of women dropping out after cycle No. of women finishing 6 months without clinical pregnancy Clinically pregnant (%)

Total

2

3

4

5

6

7

28

20

13

9

8

7

4

89

7

5

3

1

0

1

0

17

1

0

2

1

0

0

0

4

1

2

1

0

0

0

0

4

0

0

0

0

1

2

4

7

25

25

23

11

5a

aCumulative percent for cycles 5, 6, and 7. 368

Wilcox et aI. Measuring early pregnancy loss

Fertility and Sterility

Table 3. Number ofDays from First Day ofPrevious Menses to First Day of hCG Elevation for 15 Women with Clinically Recognized Pregnancies with Three Types of hCG Assays Women A

D

F

H

29 33 25

30 30 27

27 23 23

30 29 26

J

K

L

M

N

0

p

Q

s

T

32 29 27

33 32 28

24 22 19

32 29 28

21 22 19

29 25 23

29 27 26

29 28 26

37 33 32

27 24 23

Mean days

SB6 RIA R529 RIA IRMA

33 34 29

mation. This tendency was more marked for the longer time interval and was statistically significant for both. To test whether the two carboxyterminal peptide assays would be able to detect pregnancy earlier than the SB6 RIA, we compared the three assays in their detection of the hCG rise at the beginning of the 17 clinical pregnancies. Two women were missing at least one urine specimen at crucial days and are excluded. Data for the remaining 15 are shown in Table 3. The IRMA detected clinical pregnancy an average of2.6 days before the R529 RIA and 4.1 days before the SB6 RIA. We excluded the R529 RIA from further data analysis but retained the SB6 RIA in order to compare our findings with those of Edmonds et al.lO General observations about the pilot study data can be made with woman A as an example (Fig. 1). The lower panel of the figure shows data from the daily record card. Woman A entered the study during a menstrual period and participated for two complete cycles. Toward the end of her second cycle, she reported having symptoms of pregnancy; the length of the second cycle appears prolonged. She suspected she was pregnant and went to her physician for a pregnancy test, which was positive. When she subsequently miscarried, she was advised to return to birth control for several cycles, which ended her participation in our study. The second panel from the bottom shows her hLH data. A possible spike of hLH can be seen in the first cycle but not in the second. As illustrated here, the urinary assay for hLH with a single first morning sample was not completely dependable in showing clear midcycle spikes that would correspond with ovulation. The third panel from the bottom shows the results of daily hCG assays with the use of the IRMA. hCG data are plotted here on a logarithmic scale in order to accommodate the exponential rise in hCG that accompanies pregnancy. Although 0.035 ng/ml is the IRMA criterion for an Vol. 44, No.3, September 1985

29.5 28.0 25.4

elevation in hCG, we show data here down to a baseline of 0.010 ng/ml. Similarly, the baseline for the graphic display of the SB6 RIA data is down to 1.00 ng/ml (top panel). In order for us to keep track of the markedly different baselines of the two hCG assays, the baseline for the SB6 RIA is shown on the IRMA panel. The IRMA detects a rise ofhCG above the 0.035 ng/ml threshold in the luteal phase of the second cycle. The hCG fluctuates above threshold for almost 3 weeks and then falls. With the SB6 RIA (top panel), midcycle spikes occur in both cycles, consistent with the known cross-reactivity of the SB6 assay with hLH. During woman A's pregnancy, an elevation ofhCG above the SB6 criterion of 3.5 ng/ml occurred, although it lasted only for 5 of 8 consecutive days. Urinary SB6 reactivity during this pregnancy was scarcely higher than during ovulation. WOMAN A

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4

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Figure 1 Hormone profile for woman A. During the second cycle after stopping birth control, woman A experienced symptoms of pregnancy and a delayed onset of menses. She went to her physician for a pregnancy test, which was positive. Shortly after, she experienced bleeding that was diagnosed as a spontaneous abortion. She was advised by her physician to resume birth control for a few cycles, at which time she was no longer eligible for the study.

Wilcox et aI. Measuring early pregnancy loss

369

WOMAN B

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1Id ,I 1

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Figure 2 Hormone profile for woman B. Woman B suspected during her fourth cycle that she was pregnant. She went to her physician for pregnancy tests, which were negative. The IRMA shows a pattern of hCG rise and fall similar to that of woman A. Vaginal bleeding began shortly thereafter and was interpreted as menses. Woman B remained in the study until the end of her fifth cycle, having completed 6 months of urine collection without a clinical pregnancy. She subsequently conceived and delivered.

The SB6 RIA shows several brief midcycle eleva~ tions corresponding to hLH elevations, as well as a clear rise and fall with the apparent early loss. Several other aspects of woman B's record are worth noting. The hCG levels determined by the IRMA show an unusual pattern at the time she entered the study. She reported that she had had a spontaneous abortion and then had used a barrier method of birth control for one cycle just before entering the study. Whether this hCG pattern represents remnants of the previously recognized conception or a birth control failure with early loss cannot be determined with these data. The hLH assay shows midcycle spikes in every cycle except the one in which the woman apparently conceived. This is a further indication that our measurement of hLH in first morning urine specimens does not necessarily provide good information about midcycle hLH peaks. Finally, woman B reported symptoms of pregnancy in two cycles besides the one showing a probable early loss, demonstrating that symptoms are not entirely reliable as an indicator of early hCG production. A second early loss is apparent in data from woman C (Fig. 3). Like woman B, this volunteer

EARLY PREGNANCY LOSSES

We looked for evidence of early pregnancy loss among the 72 cycles that did not have clinically recognized conceptions. There were rare isolated spikes of hCG with the IRMA lasting only 1 day. Although these might reflect transient production of hCG, they are weak evidence for a pregnancy. Much stronger evidence is provided by a rise of hCG persisting over several days. There was a total of four cycles in which hCG was detected for more than a single day. These four cases are described in Figures 2 to 5. Woman B (Fig. 2) shows an apparent pregnancy loss in her fourth cycle, with a pattern of hCG very similar to that of woman A. Like woman A, woman B suspected she was pregnant and went to her physician for a pregnancy test. However, because her test was negative, her subsequent flow was regarded as a late period. The similarity of the hCG patterns of woman A and woman B demonstrates a grey area in the study of early pregnancy loss in which a transient pregnancy mayor may not be clinically recognized. Woman B continued in the study until she completed her 6 months of participation. After leaving the study, she became pregnant and has delivered. 370

Wilcox et al. Measuring early pregnancy loss

WOMAN C hCG: SB6 RIA

hCG: R525 IRMA

Figure 3 Hormone profile for woman C. Woman C experienced symptoms and appears to have had an early loss during her third cycle. She reported no symptoms of pregnancy and shows no signs of pregnancy during her other six cycles of data collection. Nonspecific immunoreactivity ofthe SB6 assay is apparent for much of that time. This reactivity cannot be attributed either to intact hCG (measured by the IRMA) or to hLH. This woman had been pregnant previously, and after the study she experienced a miscarriage and then a normal pregnancy and delivery. Fertility and Sterility

WOMAN D

~ ~'1JJ I I I I I 10

heG: R525 IRMA

I

~ b;.~~1ii.- ---t--1

= 0.1 0.01

Ji

-1 1..1-_

LH

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~ [-·:-f·· -j o

4

8

I

I

I

I

[

[

[

[

12

16

20

24

I [

28

WEEKS

Figure 4 Hormone profile for woman D. Woman D experienced no symptoms in association with an early pregnancy loss in her first cycle. She had a clinical pregnancy in the second cycle. That pregilancy proceeded normally to term. As in Figures 1 and 2, the cross-reactivity of the SB6 RIA with hLH is apparent in midcycle at the expected time of the hLH surge triggering ovulation.

was in the study for 6 months without a clinIcally diagnosed pregnancy. In the third cycle, hCG levels measured by the IRMA rose and fell over a 2-week period. The volunteer reported symptoms of pregnancy during this interval. Whereas the hCG level during this apparent pregnancy never reached a concentration that would allow it to be detected by the SB6 RIA, hCG levels measured by the SB6 assay were above our threshold for much of the 6 months that this woman was in the study. This is the only woman in the pilot study who showed a sustained elevation of SB6 immunoreactivity in the absence of either hLH or IRMA hCG immunoreactivity. This woman subsequently became pregnant and delivered. Figure 4 shows a third case of apparent pregnancy loss. Woman D became clinically pregnant in her second cycle after entering the study. Distinct midcycle hLH spikes are apparent in her two cycles with both the hLH assay and the SB6 assay. After the first hLH spike, there is a pattern of hCG rise and fall detected by the IRMA consistent with early pregnancy loss. No such pattern is apparent with the SB6 assay. This episode of hCG elevation occurs entirely during menses. The fourth instance of a possible early pregnancy loss is also the shortest duration of elevated hCG, not counting the I-day elevations. Woman E (Fig. 5) was in the study for 6 months Vol. 44, No.3, September 1985

without clinical pregnancy. There was an irregular pattern of hLH immunoreactivity with the hLH assay. hCG as measured by the IRMA was undetectable, except for an episode in which hCG rose above 0.035 ng/ml for 4 of 5 days. This episode occurred during menses. Among the remaining women, there was no instance ofIRMA reactivity above 0.035 ng/ml for longer than a single day. There were eight singleday spikes of hCG observed. These occurred in 6 of the remaining 68 cycles in the pilot study. These single values ranged from 0.035 to 0.173 ng/ml and were distributed sporadically throughout the menstrual cycle. There was no instance of an elevated hCG value in the 15 cycles contributed by the tubal ligation control subjects. In addition to the two apparent early losses that occurred during menses (Figs. 4 and 5), there was one clinical pregnancy (not shown) that was undetectable until the day before a 5-day menses. hCG rose steadily through the menses, and the pregnancy proceeded normally to term. DISCUSSION

We collected and analyzed daily urine specimens from a small group of women to provide the basis for designing larger studies of early pregnancy loss. The first question this study ad-

WOMAN E

Figure 5 Hormone profile for woman E. Woman E collected urine specimens for six cycles without symptoms of pregnancy. At the end of the third cycle there was a brief rise and fall of hCG consistent with a transient pregnancy and early loss. One year after she stopped birth control (6 months after leaving the study), she still had not become clinically pregnant. Wilcox et al. Measuring early pregnancy loss

371

Table 4. Summary of Previous Studies Estimating Occurrence of Early Pregnancy Loss Miller et al."

No. of women No. of cycles Source of hCG RIA Type of hCG RIA Collection schedule

Criterion of pregnancy

_Early-pregnancy losses (proportion of chemically detected pregnancies not becoming clinically apparent) Cycles.in which clinical pregnancies were conceived Clinically apparent spontaneous abortions, over all clini-cal pregnancies

Edm0'l~.

al.

et

82 207 Urine SB6 antibody Every other day from day 21 of the menstrual cycle until beginning of next menses 5 ng/ml on 1 56 mIU/ml day or 2 on 1 day ng/ml on 2 days 50/152 (33%) 67/l18 (57%) 197

91

623 Urine Ortho kit

226 Blood Amersham kit One sample at about the fourth week of each cycle 16 mIU/ml in one sample 7192 (8%)

102/623 (16%) 51/207 (25%)85/226 (38%)

15/102 (15%)

6/51 (12%)

11/85 (13%)

dressed was whether hCG could be measured sensitively and specifically in small quantities of urine collected as first morning voids. hCG is secreted by the trophoblast and has been detected in the medium of preimplantation embryos by the eighth day after fertilization. 18 One of the challenges of measuring hCG at low levels is to distinguish it from hLH, which is structurally similar tohCG and also present in every fertile cycle. Only recently have urinary assays been developed that are specific for hCG in the presence of-hLH. 14- 16 Comparisons of urine and plasma indicate that the concentration of hCG in urine is very similar to its concentration in plasma and that the clearance rate of hCG remains constant even at relatively low concentrations in the blood. 19 This is _important for epidemiologic studies, in that serial urine samples are far easier to collect than serial serum samples. Three studies have used RIAs of hCG to measure the risk of early pregnancy IOSS.9-11 Those studies are summarized in Table 4. Briefly, both Miller et al. 9 and Edmonds et al. 10 collected urine specimens every other day from the 21st day of the menstrual cycle until the onset of the next menses. Miller et a1. 9 used a commercial kit to assay hCG.Edmonds et a1. 10 assay-ed hCG with the use of the SB6 RIA and included a control group of 18 sterilized women for the determina372

Wilcox et aI. Measuring early pregnancy loss

tion of the assay criterion for pregnancy. Whittaker et al. 11 determined pregnancy on the basis of a single serum assay performed in the luteal phase of the cycle with a commercial kitY In our study, we compared three urinary hCG assays: the SB6 RIA used by Edmonds et al. 10 and two other assays with greater sensitivity and specificity. We found that the IRMA, which recognizes the carboxyterminal peptide region of the hCG j3-chain, could reliably measure hCG in daily urine specimens at very low levels without apparent hLH or nonspecific cross-reactivity. Among the control group of five women with tubal ligations, there was no hCG detected by the IRMA above the 0.035 ng/ml criterion. Thus, the IRMA can reliably detect hCG in urine at concentrations 100 times lower than the SB6 assay. With the IRMA we were able to detect clinical pregnancies on an average of 4 days sooner than with the SB6 RIA (Table 3). Our method for detection of early pregnancy loss depends on the measurement ofhCG in urine specimens stored frozen. hCG has been reported to be stable in frozen urine, even when repeatedly thawed and refrozen. 2o We tested the stability of hCG in frozen urine and found no evidence that hCG deteriorates over time. If anything, the concentration of hCG increased over time, presumably because of w~ter sublimation. The second question this study addressed was whether daily urine specimens and other daily information could feasibly be collected from normal women who are planning a pregnancy. On the average, women in the pilot study successfully collected urine specimens for all but 2% oftheir days in the study. We cannot be positive that every urine specimen was collected on the day specified; however, there was no day-to-day pattern of hLH or hCG that suggested otherwise. The high level of compliance suggested that payment might be superfluous for these volunteers. To determine whether this was the case, we sent anonymous questionnaires to volunteers asking how important payment was to their participation. Only 25% replied that they probably would have participated without payment. Collection and storage of daily urine specimens is manageable in field studies, but it is apparently inconvenient enough to discourage many volunteers if they are not paid. The third and final question this study addressed was whether there are patterns of hCG in daily urine specimens that can be plausibly inFertility and Sterility

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terpreted as early pregnancy loss. Even when hCG can be sensitively and specifically assayed, there is a question of whether hCG is produced by transient pregnancy in a pattern that can be distinguished from other sources of hCG. Trophoblastic tissue is not the only producer of hCG; hCG has been detected in large quantities in the presence of tumors and certain diseases. 21 Menopausal women produce hCG, presumably from the pituitary.22 hCG can be detected at very low levels even among healthy nonpregnant women and healthy men. 23 We found four patterns ofhCG rise and fall in which hCG exceeded 0.035 ng/ml for at least 4 of 5 days. Excluding the initial samples from woman B, which represented events preceding entry to the study, all elevations besides those four patterns were either isolated I-day spikes or clinically recognized pregnancies. The four patterns ranged from a sustained elevation over several weeks similar to a clinical spontaneous abortion to a transient rise over only a few days. Three of these four patterns had peak hCG concentrations below the level detectable by the urinary SB6 RIA. Al,,::,mgh we cannot verify that pregnancy occurred in any of these four cases, neither can we explain the patterns in other ways. Secretion of hCG from non trophoblastic sources cannot be completely ruled out, but it seems unlikely that such secretion would account for the distinct episodes we observed. The negative results of the IRMA among the five women with tubal ligation further supports the interpretation that these hCG patterns represent early pregnancies. The eight I-day spikes do not have an obvious source. It is possible that very transient pregnancy could produce detectable hCG for only 1 day. It is also possible that these eight elevations are rare instances of contamination or laboratory error, occurring as they do against a background of more than 1500 hCG measurements below the threshold of the IRMA. The control group of five sterilized women is too small to rule out this possibility of a rare random artifact. The sporadic distribution of the I-day elevations in relation to th menstrual cycle makes us reluctant to interpret them as biologically meaningful. We collected and analyzed urine specimens for every day of each cycle because we could not be absolutely sure when during a cycle a pregnancy might first be detected. Even so, we did not expect to observe two apparent. early pregnancy losses Vol. 44, No.3, September 1985

within the time of menses. We also found one clinical pregnancy that was first detected at the time of menses. Evidently, a blastocyst can produce hCG during menses and even survive menses. We cannot look for confirmation in previous studies9 , 10 because those studies stopped specimen collection at the onset of menses. Although this pilot study was not designed to estimate the risk of early loss, the ratio of 4 early losses to 17 clinical pregnancies is considerably lower than two other estimates 9 , 10 based on hCG measurements in urine samples. (The data from Whittaker et al. 11 based on a single serum assay per cycle provide a lower estimate of early loss, but the infrequency of sampling makes this very likely an underestimate.) On the basis of four to seven urine samples per cycle, Edmonds et al. 1O found more early losses than clinical pregnancies. Our ratio is lower,even though we used daily specimens and employed a more sensitive and specific assay than has been previously available. This discrepancy may reflect differences in our assays or in our volunteer population. In our data, a criterion for the SB6 assay that is low enough to detect clinical spontaneous abortions also detects midcycle hLH. This suggests that false-positive values can bea problem when the urinary SB6 assay is used for detection of early pregnancy. Our volunteers tended to be well-educated women (Table 1); Edmonds et· al. 1O do not characterize their volunteers, but the tendency toward welleducated, middle-class volunteers is typical in this type of study. 9 Our data show distinct patterns ofhCG rise and fall among women attempting pregnancy. These patterns are not seen in a small group of sterilized women and are not explained by other known sources of hCG. We believe that these patterns represent early pregnancy loss, some of which . would be undetectable without the use of an assay having the properties of the IRMA. It appears feasible to conduct field studies to estimate the risk of early pregnancy loss by measuring hCG in first morning urine specimens with the IRMA.

Acknowledgments. We gratefully acknowledge the work of Ms. Joy Pierce, Ms. Ann Toledo, and Ms. Jeanne Warner of Survey Research Associates, who designed and supervised the field aspects of this study; Mr. John Schlatterer (Columbia) and Ms. Dawn Dobin· (LSU), who performed the laboratory analyses; Ms. Louise Horney and Mr. David Shore, who generated graphs and handled computer programming; the physicians in private practice who made our recruitment materials

Wilcox et aI. Measuring early pregnancy loss

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available to their patients; Donna Baird, Ph.D., Curtis Eshelman, M.D., Dale Sandler, Ph.D., and Walter Rogan, M.D., who commented on prior drafts of this article; and most importantly, the 30 anonymous women who conscientiously provided daily records and urine specimens for our analysis. REFERENCES 1. French FE, Bierman JM: Probabilities of fetal mortality. Public Health Rep 77:835, 1962 2. Leridon H: Intrauterine mortality. In Human Fertility: The Basic Components~ Chicago, University of Chicago Press, 19.77, p 48 3. Wilcox AJ, Treloar AE, Sandler DP: Spontaneous abortion over time: comparing occurrence in two cohorts of women a generation apart. Am J Epidemiol114:548, 1981 4. Bloch SK: Occult pregnancy as a factor in unexplained infertility. J ReprodMed 21:251, 1978 5. Hertig AT, Rock J, Adams EC, Menkin MC: Thirty-four fertilized human ova, good, bad and indifferent, recovered from 210 women of known fertility. Pediatrics 23:202, 1959 6. Roberts CJ, Lowe CR: Where have all the conceptions gone? Lancet 1:498, 1975 7. Braunstein GD, Karow WG, Gentry WD, Wade ME: Subclinical spontaneous abortion. Obstet Gynecol (SupplJ 50:41s, 1977 8. Chartier M, Roger M, Barrat J, Michelon B: Measurement of plasma human chorionic gonadotropin (hCG) and (3-hCG activities in the late luteal phase: evidence of the occurrence of spontaneous menstrual abortions in infertile women. Fertil Steril31:134, 1979 9. Miller JF, Williamson E, Glue J, Gordon YB, Grudzinskas JG, Sykes A: Fetal loss after implantation: a prospective study. Lancet 2:554, 1980 10. Edmonds DK, Lindsay KS, Miller JF, Williamson E, Wood PJ: Early embryonic mortality in women. Fertil Steril 38:447, 1982 11. Whittaker PG, Taylor A, Lind T: Unsuspected pregnancy loss in healthy women. Lancet 1:1126, 1983 12. Vaitukaitis JL, Braunstein GD, Ross GT: A radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone. Am J Obstet Gynecol 113:751, 1972

374

Wilcox et al. Measuring early pregnancy loss

13. Fraker PJ, Speck JC Jr: Protein and cell membrane iodinations with a sparingly soluble chloromide, 1,3,4,6-tetrachloro-3a,6a-diphenyl-glycouril. Biochem Biophys Res Commun 80:849, 1972 14. Wehmann RE, Harman SM, Birken S, Canfield RE, Nisula BC: Convenient radioimmunoassay that measures urinary human choriogonadotropin in the presence of urinary human lutropin. Clin Chern 27:1997, 1981 15. Armstrong EG, Ehrlich PH, Birken S, Schlatterer JP, Siris E, Hembree W, Canfield RE: Use of a highly sensitive and specific immunoradiometric assay for detection of human chorionic gonadotropin in urine of normal, nonpregnant and pregnant individuals. J Clin Endocrinol Metab 59:867, 1984 16. Ehrlich PH, Moustafa ZA, Krichevsky A, Birken S, Armstrong EG, Canfield RE: Characterization and relative orientation of epitopes for monoclonal antibodies and antisera to human chorionic gonadotropin. Am J Reprod Immunol Microbiol. In press 17. Wehmann RE, Blackman MR, Harman SM: Metabolic clearance rates of luteinizing hormone in women during different phases of the menstrual cycle and while taking an oral contraceptive. J Clin Endocrinol Metab 55:654, 1982 18. Fishel SB, Edwards RG, Evans CJ: Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro. Science 223:816, 1984 19. Wehmann RE, Nisula BC: Metabolic and renal clearance rates of purified human chorionic gonadotropin. J Clin Invest 68:184, 1981 20. McCready J, Braunstein GD, Helm D, Wade ME: Modification of the choriogonadotropin beta-subunit radioimmunoassay for determination of urinary choriogonadotropin. Clin Chern 24:1958, 1978 21. Vaitukaitis JL: Human chorionic gonadotropin-a hormone secreted for many reasons. N Engl J Med 301:324, 1979 22. Suginami H, Kawaoi A: Immunohistochemical location of a human chorionic gonadotropin-like substance in the human pituitary gland. J Clin Endocrinol Metab 55:1161, 1982 23. Borkowski A, Muquardt C: Human chorionic gonadotropin in the plasma of normal, nonpregnant subjects. N Engl J Med 301:298, 1979

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