Human chorionic gonadotropin increase in normal early pregnancy

Salim Daya, M.B., Ch.B. * Hamilton, Ontario, Canada

Serial determinations of human chorionic gonadotropin have been used in evaluating early pregnancies. This has been based on linear regression of logarithm-transformed hCG levels. However, these levels are changing in a curvilinear fashion as pregnancy advances. Consequently the simple linear model does not provide a good fit to the data. To define more precisely the normal values for hCG increase, serial determinations were performed on serum samples obtained from 29 patients who were carrying normally advancing pregnancies. Gestational age was established with basal body temperature records. Three gestational age periods were identified each with a linear increase in hCG. This was translated into hCG doubling times and percentage increase over time. These tables may be used to determine if an early pregnancy is advancing normally. (AM J OesTET GYNECOL 1987;156:286-90.)

Key terms: Pregnancy, normal, early chorionic gonadotropin levels

The development of laboratory assays for the detec­tion of pregnancy began almost 60 years ago with the report of a time-consuming bioassay performed in mice. 1 Since then it was discovered that human chori­onic gonadotropin (hCG) was the hormone produced in normal pregnancies and consequently a number of biologic, immunologic, and radioreceptor assays were developed to measure this hormone.2 One significant development was an antiserum specific for the 13-sub­unit of hCG, thereby allowing very low levels of the hormone to be detected without interference from lu­teinizing hormone.' This high level of sensitivity has enabled hCG to be detected by radioimmunoassay as early as 6 days after conception.• With increasing avail­ability of this assay the progress of early pregnancy can be monitored with serial determination of hCG.5

·8

The majority of the reports in the literature have been based on calculating predicted increases in hCG over time using linear regression of logarithmically transformed hCG levels in the first trimester. With use of this method it has been assumed that the rate of rise of hCG is constant in normal early intrauterine preg­nancies. However, hCG levels are constantly changing with the rate of rise decreasing as pregnancy advances until a plateau is reached at approximately 9 to I 0

From the Department of Obstetrics and Gynecology, McMaster Uni­versity.

Presented at the Forty-second Annual Meeting of The Society of Obstetricians and Gynaecologists of Canada, Charlottetown, Prince Edward Island, Canada, June 23-27, 1986.

Reprint requests: Dr. Salim Daya, Department of Obstetrics and Gy­necology, McMaster University, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5.

*Career Scientist of the Ontario Ministry of Health, Canada.

286

weeks' gestation. Simple linear regression of log hCG does not adequately represent this change. Conse­quently the predictive value for normal pregnancy with use of doubling times based on this method may lack sensitivity. An attempt to improve this has been made with use of smaller sampling intervals of hCG.8

Regression analysis is used to establish whether a re­lationship exists between two variables. For a particular value of the independent variable, the regression line helps to predict a corresponding best estimate of the dependent variable. By plotting a series of hCG levels for each gestational age, a simple linear regression model can provide a best estimate for hCG level at a particular gestational age. Since hCG levels are increas­ing in pregnancy in a nonlinear way, this model is not suitable in providing estimates of hCG increase over time without the knowledge of gestational age. However, this has been the main focus in the literature.

In this study, normal values for the rate of hCG in­crease in early pregnancy have been defined more pre­cisely and may improve the sensitivity and usefulness of serial determinations of hCG in the early detection of pathologic pregnancies.

Material and methods

Patients undergoing evaluation and treatment at the Infertility Clinic at Chedoke-McMaster Hospital, Ham­ilton, Canada, were selected for this study after giving informed consent. Serial hCG determinations were performed on serum samples obtained from 29 such patients who had conceived and were carrying normally advancing intrauterine pregnancies as confirmed by ul­trasonography. The mean sampling interval was 3.8 days. All these patients have since delivered healthy

Volume 156 Number 2

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Human chorionic gonadotropin increase in early pregnancy 287

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GESTATIONAL AGE (dlyt)

Fig. I. hCG increase in normal early pregnancy. Quadratic regression line equation is as follows: log hCG = -8.8695 + 0.6530 (gestational age) - 0.0052 (gestational age)2

• Symbols correspond to the indicated number of data points.

Table I. Polynomial regression analysis of early pregnancy hCG data*

Regression models

Straight line

Quadratic

Cubic

*Total plotted values = 165.

Overall regression

985 (p <.0.0001)

1109 (p < 0.0001)

738 (p < 0.0001)

infants at term. Although some patients conceived spontaneously, most required therapeutic intervention in the form of homologous or heterologous insemi­nation, clomiphine citrate, human menopausal gonad­otropin, and/or human chorionic gonadotropin. Ges­tational age was established in all cases by the nadir on basal body temperature recordings.

Radioimmunoassay. Patient serum was separated from clotted blood samples and stored at - 20° C. Quantitative measurements of hCG were performed with use of a GammaDab M [iodine 125] ~-subunit of hCG radioimmunoassay kit (Travenol-Genentech Di­agnostics, Cambridge, Massachusetts). The procedure is a competitive binding assay that uses a precipitating antiserum reagent generated against the ~-subunit of the hCG hormone, 1251-labeled hCG, and a highly pu­rified preparation of hCG reagents as control (CA 2426), standard (CA 22002, 22003, 22004, 22006, and 22007) and calibrator/standard (CA 22005). The con-

F values

Lack of fit

2.22 (p < 0.005)

0.96 (p > 0.25)

0.96 (p > 0.25)

Improved predictive power

With use of With use quadratic vs. of cubic vs. straight line quadratic

176 (p < 0.001)

0.74 (p > 0.25)

trol reagent is calibrated at 90 mlU/ml, the standards at 5, 10, 25, 100, and 200 mlU/ml, and the calibrator/ standard at 50 mlU/ml. The standards and control reagents are calibrated to the World Health Organi­zation's immunoassay standard, the first international reference preparation 75/537 established in 1975, and labeled in international milliunits per milliliter. The values of the controls, standards, and patient serum can be converted directly from international milliunits per milliliter to international units per liter of standard international units. The calculated sensitivity of the as­say is 1 to 5 mlU/ml with <5 mlU/ml being taken as evidence for a nonpregnant state. The intrarun vari­ability determined from the mean of 20 simultaneous assays for three samples was 3.2% (range, 2.9% to 3.6%), and the interrun variability determined from the mean of the average of duplicates for 20 separate runs was 7.4% (range, 6.5% to 9.0%).

Statistics. Simple linear regression was performed

288 Daya

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on the hCG levels for each patient. Three gestational age ranges were specified (given in the Results section) and within each one the mean and lower two standard deviations (SD) were calculated for the slope parame­ters.

Results Since some of the patients had received hCG injec­

tions to induce ovulation, clearance studies were per­formed to determine how rapidly 10,000 IU of this hormone could be eliminated from the body. Daily blood samples were taken for hCG determinations from three such patients. It was observed that hCG was no longer detectable 12 days after the initial adminis­tration of this hormone. Subsequently in such patients, blood samples for hCG were not drawn earlier than 12 days after the last hCG dose.

Fig. I represents a scattergram of log hCG plotted against gestational age. It can be seen that as gestational age advances, the hCG level increases until a plateau is reached at approximately 9 weeks' gestation. The re­lationship appears to be a curvilinear one despite the logarithmic transformation. To determine which regression model would best fit the data, the following were tested: straight line, quadratic, and cubic. From Table I it is apparent that all models demonstrate a significant overall regression. However, when lack of

February 1987 Am J Obstet Gynecol

fit for each model is tested, the simple linear model is not appropriate (F.2.121 = 2.22, p < 0.005) whereas both the quadratic and cubic models are appropriate (F.1.121 = 0.956, p > 0.25, and F.0.121 = 0.961, p > 0.25, respectively). The quadratic model provides signifi­cantly more predictive power than that provided by the straight line model (partial F1.162 = 176, p < 0.001). Also the higher order cubic model does not improve this predictive power of the quadratic model (partial F1.161 = 0.738, p > 0.25) and is therefore not a suitable model to be used, since it is computationally more te­dious. The quadratic regression line is shown in Fig. I. Fig. 2 shows the residuals plotted against gestational age for the straight line and quadratic models and clearly confirms the superiority of the latter model. These statistical analyses confirm that the rate of rise of hCG is not linear but follows a quadratic curve, that is, it decreases as gestational age increases until a pla­teau is reached .

In view of this curvilinear relationship, gestational age was divided into three phases (Fig. I) based on what appeared to be the most appropriate divisions when inspecting the scattergram. Polynomial regres­sion analysis for the best-fitting model was performed for each phase (Table II). In each of these phases, all three models demonstrated an overall regression that was significant and all models did not demonstrate a lack of fit indicating that all were appropriate. However, the higher order models did not improve the predictive power of the straight-line model. These results support the three divisions of gestational age and allow infer­ences to be made within each phase as hCG now in­creases in a linear fashion with gestational age. With this information, simple linear regression analysis was performed on hCG levels for each patient within each phase. The mean and lower 2 SD were calculated for the slope parameters in each phase (Table III). The mean and lower 2 SD of the doubling times were also calculated for each phase (Table IV) with use of the following formula:

Doubling time = log 2 slope

Doubling times can be translated into a percentage increase in hCG over time for each phase and thereby provide normal values that may be clinically useful (Table V).

Comment The application of radioimmunoassay to the detec­

tion of hCG has provided a very sensitive and specific test that allows the early detection of pregnancy. As this test is becoming more readily available, it is increasingly being used to determine whether an early pregnancy is advancing normally. This is particularly useful in

Volume 156 Number 2

Human chorionic gonadotropin increase in early pregnancy 289

Table II. Polynomial regression analysis of early pregnancy data per gestational phase

F vlaues

Improved predictive power with use of quadratic vs.

Linear regression models* Overall regression Lack of fit straight line

Phase It (n = 76) Straight line 384 1.53

(p < 0.0001) (p > 0.25) 0.19 Quadratic 190 0.97 (p > 0.25)

(p < 0.0001) (p > 0.25) Phase lit (n = 74)

Straight line 129 0.53 (p < 0.0001) (p > 0.25) 1.97

Quadratic 66 0.44 (p > 0.1) (p < 0.0001) (p > 0.25)

Phase Illt (n = 21) Straight line ·5.98 0.67

(p = 0.0244) (p >0.25) 0.17 Quadratic 2.95 0.77 (p > 0.25)

(p = 0.078) (p > 0.25)

*Cubic model was not included, since tolerance limits set at 0.0001 had been reached. tPhase I = Gestational age ""41 days. Phase II = 41-57 days. Phase III = 57-65 days.

Table III. Straight line slope parameters for each gestational phase

Mean slope Lower 2 SD

I

0.358 0.272

Phase

II

0.146 0.092

III

0.0263 0.0213

women who are at high risk for recurrent abortion and ectopic pregnancy and also in women who have symp­toms of uterine bleeding and/or lower abdominal pain. In these situations, serial hCG determinations are used as prognostic indicators of the status of the pregnancy. To distinguish between normal and ectopic pregnan­cies, it has been shown that measuring the rate of hCG increase in serum is a more effective method than use of hCG values alone.7 Several reports have indicated that the serum hCG levels in patients with ectopic preg­nancies are much lower than those seen in normal preg­nancies.6·9·11 However, this approach has significant lim­itations, since often the hCG levels may not be low but the rate of increase may be suboptimal.12 It would ap­pear that serial hCG determinations would provide more useful information in such cases.

The literature has focused on simple linear regres­sion of log-transformed hCG levels to evaluate early pregnancies. This has resulted in many normal preg­nancies being erroneously classified as advancing su­boptimally (Daya S, unpublished observations). For ex­ample, the straight-line model for the hCG levels in this paper (before subdivision into phases) had a slope of 0.199. The doubling time corresponding to this is 3.48

Table IV. Doubling times for each gestational phase

Phase

Doubling time Lower 2 SD

I

1.94 2.55

II

4.75 7.53

III

26.36 32.54

days. With use of this model, all of the patients who were sampled in phase 2 (gestational age, 41 to 57 days) would be considered as having abnormal hCG in­creases, since the mean doubling time in this phase was much longer (4.75 days). Similarly, Kadar et al. 12 pro­duced tables showing lower limits of the percentage increase in hCG between test samples drawn several days apart (for example, for a sampling interval of 2 days the percent increase in hCG equals 66%). With use of these tables, many patients sampled in phase 2 in this study again would be considered as having ab­normally advancing pregnancies. These interpretations are based on the assumption that hCG increases in early pregnancy at a constant rate. This study clearly shows that the rate of rise of hCG is not constant but is grad­ually decreasing with advancing pregnancy. The fact that the quadratic regression model provides a better fit for the data than the simple linear regression model supports this observation.

Many of the reports in the literature have used a cohort of patients to provide several samples in order to determine the rate of rise of hCG. This introduces both intrapatient and interpatient variability. The method used in this paper has attempted to reduce

290 Daya

Table V. Percentage increase in hCG over time*

I (""Al days)

Time interoal

I betwee tests Increase Lower

(days) (%) 2 SD

1 43 30 2 103 73 3 193 127 4 320 197 5 500 290 6 757 410 7 1127 570

intrapatient variability by use of the slope of hCG in­

crease in each patient to determine the normal values.

This allows a more sensitive method of interpreting

hCG increases in the pregnant population without hav­

ing the bias of intrapatient variability.

Division of the gestational age (from day 20 to 66)

into three phases has provided a method of evaluating

the progress in early pregnancy without violating the

principles of regression analysis. The rate of rise is

relatively constant in each of these phases. It is impor­

tant to note that with use of regression analysis, one

cannot ignore the gestational age in predicting the rate

of rise of hCG. This is especially important in following

the cases of women at high risk for spontaneous abor­

tion and ectopic pregnancy so that important prog­

nostic information can be provided. Similarly it may be

especially relevant in infertile women who are known

to be at high risk of having pathologic pregnancies and

in whom gestational age is often known from basal body

temperature graphs. In such patients the normal values

presented in this paper would be very useful. To de­

termine the minimum acceptable change in hCG re­

quired to classify the pregnancy as one that is advancing

normally, receiver operating characteristics curve anal­

ysis will have to be performed. With use of this method

the rate of change in pathologic pregnancies can be

compared with normal pregnancies to determine the

best "cut-off' that will be clinically useful as a prognostic

indicator.

REFERENCES

l. Ascheim S, Zondek B. Hypophysenvorderlappenhormon und Ovarialhormon. Klin Wochenschr 1927;6:1322.

Phase

II

February 198 7 Am J Obstet Gynecol

lII (41-57 days) (57-65 days)

I ) Increase Lower Increase Lower

(%) 2 SD (%) 2 SD

17 10 2.7 2 33 20 5.3 4.3 53 33 8.3 6.7 80 43 l l 9

107 60 14 l 1.3 140 73 17 13.7 177 90 20.3 16

2. Sheehan C. Current status of pregnancy testing. Am J Med Tech l 983;49:485.

3. Vaitukaitis JL, Braunstein GD, Ross GT. A radioimmu­noassay which specifically measures human chorionic go­nadotropin in the presence of human luteinizing hor­mone. AM J 0BSTET GYNECOL 1972;113:751.

4. Braunstein GD, Rasor J, Adler D, Danzer H, Wade ME. Serum and human chorionic gonadotropin levels throughout normal pregnancy. AM J 0BSTET GYNECOL 1976;126:678.

5. Batzer FR, Schlaff S, Goldfarb AF, Corson SL. Serial J3-subunit human chorionic gonadotropin doubling time as a prognosticator of pregnancy outcome in an infertile population. Fertil Steril 1981;35:307.

6. Braunstein GD, Karow WG, Gentry WC, Rasor J, Wade ME. First-trimester chorionic gonadotropin measure­ments as an aid in the diagnosis of early pregnancy dis­orders. AM j 0BSTET GYNECOL 1978; 13 l :25.

7. Kadar N, DeCherney AH, Romero R. Receiver operating characteristic (ROC) curve analysis of the relative efficacy of single and serial chorionic gonadotropin determina­tions in the early diagnosis of ectopic pregnancy. Fertil Steril 1982;37:542.

8. Pittaway DE, Reish RL, Wentz AC. Doubling times of hu­man chorionic gonadotropin increase in early viable in­trauterine pregnancies. AM j 0BSTET GYNECOL 1985; 152:299.

9. Kosasa TS, Taymor ML, Goldstein DP, Levesque LA. Use of a radioimmunoassay specific for human chorionic go­nadotropin in the diagnosis of early ectopic pregnancy. Obstet Gynecol 1973;42:868.

10. Rasor RL, Braunstein GD. A rapid modification of the beta-hCG radioimmunoassay: use as an aid in the diag­nosis of ectopic pregnancy. Obstet Gynecol 1977;50:553.

11. Saxena BB, Landesman R. The use of a radioreceptor assay of human chorionic gonadotropin for the diagnosis and management of ectopic pregnancy. Fertil Steril l 975;26:397.

12. Kadar N, Caldwell BV, Romero R. A method of screening for ectopic pregnancy and its indications. Obstet Gynecol 1981;58:162.