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Urinary estrone glucuronide, pregnanediol glucuronide and human chorionic gonadotrophin in threatened abortion
Int. J. Gynecol. Obstet., International Federation
107
1988,21: 107-l 11 of Gynecology &Obstetrics
Urinary estrone glucuronide, pregnanediol glucuronide and human chorionic gonadotrophin in threatened abortion P.C. Ishwad,
S.M. Chitlange,
U.M. Joshi, V. Chowdhury”
and A.C. Mehta”
Institute for Research in Reproduction (ICMR) and “Nowrosjee Wadia Maternity Hospital, Parel, Bombay 400 012 (India) (Received April 2Oth, 1987) (Revised and accepted August 13th, 1987)
Abstract
Estimations of urinary estrone glucuronide, pregnanediol glucuronide and human chorionic gonadotrophin were carried out by ELBA to see their potential in predicting an abnormal outcome in cases with vaginal bleeding in early pregnancy. Reference values were set up with samples from women without bleeding in present or past pregnancies and with normal ultrasonic findings. None of the parameters were found to be sensitive enough to predict an abnormal outcome. However, predictability of an abnormal value was found to be 95% for estrone-3-glucuronide (E,G), 93 % for pregnanediol glucuronide (PdG) and 87% for human chorionic gonadotrophin (hCG). Keywords:
Abortion;
ELISA of urinary hor-
mones. Introduction
Bleeding in the first 20 weeks of pregnancy with/without lower abdominal cramps/backache is termed as threatened abortion. Beyond 24 gestational weeks, fetal death is termed as miscarriage, for which the reasons may be more than just hormonal. However, 0020-7292/88/$03.50 0 1988 International Federation of Gynecology&Obstetrics
chorionic tissue remaining functional, the pregnancy test remains positive. Using present clinical and laboratory parameters it is often difficult, until a late stage of the course to assign patients with threatened abortion for immediate curettage or continued treatment. A simple and rapid test for fetal viability is much awaited today to avoid delay, anxiety and prolonged costly hospitalization. Though ultrasonography diagnoses fetal status it is not prognostic. Besides it is expensive and not universally available. RIA for serum hormones involves an invasive technique and needs sophisticated facilities. Many articles have been published evaluating the levels of a variety of hormones in both blood and urine in normal and abortive pregnancies . Unfortunately, these reports are often contradictory in their results. The methods involved are in many cases laborious and require specialized equipment not readily available in a clinical situation. The method of analysis should be simple, inexpensive, reliable and rapid with a low analytical error. The half-life of the estimated hormone should be short to get a quick reflection of deterioration in placental and/or feto-placental function. We have used the simple technique of ELISA for estimating urinary hormones or their metabolites in pregnant patients. Clinical and Clinical Research
108
Ishwad et al.
The parameters estimated were estrone-3glucuronide (E,G), pregnanediol glucuronide (PdG) and human chorionic gonadotrophin (hCG). These parameters have been used to analyze human reproductive cycles and to monitor early trophoblastic growth [8]. E,G is a urinary metabolite mainly of estrone and partly of estradiol. It reflects the function of the feto-placental unit. PdG is the urinary metabolite of progesterone which is produced initially by the corpus luteum of pregnancy and later by the placenta. hCG has a trophoblastic origin and is excreted as such in the urine. Produced by the feto-placental unit, the estrogen level drops quickly after death of the conceptus, while hCG and progesterone produced by the placenta take longer to drop if placenta is not detached and hence still functioning and their assay, especially that of random urinary hCG is less reliable [3]. Materials and methods Two hundred forty-five subjects in different stages of pregnancy, with no vaginal bleeding during the present or past pregnancies and with positive fetal heart beats/movements, and fetal measurements normal for that stage of pregnancy were enrolled as the control group. One hundred thirty-five pregnant subjects with vaginal bleeding at the time of examination and with/without positive fetal heart beats/movements were enrolled as the study group. Early morning urine samples were collected from these women dating from 5 to 20 gestational weeks. Each sample was preserved with sodium azide as preservative at - 20°C till assayed within 1 month of collection. Ultrasonography was performed on the day of urine collection to ascertain fetal status. The enzyme-linked immunosorbant assay (ELBA), using penicillinase enzyme as a marker, was used to estimate E,G according to Shah et al. [l 11, PdG according to Khatkhatay et al. [5] and hCG according to Joshi et al. [7]. All three parameters were estimated by competitive ELBA. In brief, the Int JGynecol Obstet 27
method involves the following steps: immobilization of antibody on a polyvinyl chloride microtitre plate, incubating the sample and analysing penicillinase in the antibody immobilized well, measuring the bound enzyme activity by starch iodine as an indicator using either a calorimeter or ELISA reader. All assays are completed in 3 h. The creatinine was estimated in each urine sample and the value of the parameter expressed as concentration per milligram creatinine. Samples having creatinine < 100 pg/ml were considered unsuitable for analysis. All hormones could not be analysed in all samples. Number of cases and samples analysed are given in Table I and Fig. 1. All study cases were followed up till delivery or abortion. Those cases delivering after 36th week with babies > 2.2 kg were considered to have normal outcome. Cases with missed abortions, or abortions within 1 month from collection of urine were considered to have abnormal out-
Evaluation of E,G, PdG and hCG in missed and Table 1. threatened abortions for prognosis. Parameter
w
PdG
hCG
113 62 51 142
130 15 55 156
110 62 48 135
Normal values Total With normal outcome g)) With abnormal outcome (c)
61 26 35
112 60 52
90 44 46
Abnormal values Total (d) With abnormal outcome (e) With normal outcome (f) Sensitivity(e/c + e) Specifkity@/b + f)
81 II 4 69 86
44 41 3 44 95
45 39 6 46 88
Predictability of Normal value (b/a) Abnormal value (e/d)
43 95
54 93
49 87
Cases studied Missed abortions Threatened abortions No. of samples
Urinary hormones in threatened abortion
109
come. Cases delivering at term but weight of baby less than 2.2 kg and cases aborting after more than 6 weeks from urine collections were excluded from analysis. Thus only 130 out of 135 cases were included for statistical analysis.
looo~
Statistical analysis
(7) (36) (32) (34) (36) (44) (33) (24) , I I . . 5-6 7-6 S-40 H-i2 43.,415~(6,7.1649-M
40-
41
Since all hormone assays were evaluated to predict an abnormal outcome in the bleeding cases within 6 weeks of urine collection, an abnormal value was considered as positive and normal value as negative. True positives were those abnormal values which corresponded to an abnormal outcome. True negatives were those normal values corresponding to a normal outcome. Sensitivity was defined as the capacity of the test to predict abnormal outcome (or to detect true positives) and hence was equal to (true positive/true positive + false negative) X 100. Specificity was defined as the capacity of the test to avoid false positive and hence was equal to (true negative/true negative + false positive) X 100.
I
.
5-6
.
7-6
5-6
I
.
Results
.
S-,0 ,I-,2 (3-M ,5-% V-16 is-20
7-6
WEEKS
9-K) U-i2 ,3-14,546 17-1849.20
OF
GESTATION
*C=CENTILE
Fig. 1. Establishment of reference values for excretion of E,G, PdG and hCG in urine during pregnancy. Continuous lines show 10th. 50th and 90th centiles. Broken lines show lowest value recorded for the period. Numbers in parentheses indicate number of samples analysed.
Figure 1 gives the lOth, 50th and 90th centile as well as the lowest value obtained for particular weeks of pregnancy for the respective parameter. The patterns for the three parameters are similar to those reported in the literature for corresponding circulating hormones. The lowest values recorded during the study were considered as cut-off limits to avoid even a single, normal case being labelled as abnormal. Table I gives the detailed analysis for all the three parameters, E,G, PdG and hCG. For E,G, 142 samples from 113 cases were analysed. Sixty-two of the 113 cases were missed abortions while 51 were threatened abortions. Sixty-one normal values were obtained out of which 26 were compatible with normal outcome while 35 corresponded to an abnormal outcome. Eighty-one out of Clinical and Clinical Research
110
Ish wad et al.
the total 142 samples showed an abnormal value. Seventy-seven of these 81 were compatible with an abnormal outcome while 4 corresponded to a normal outcome. Thus sensitivity of E,G was calculated as 77/(77 + 35) = 69%. Specificity of E,G was 26/(26 + 4) = 86%. Predictability of a normal E,G value was found to be 26161 = 43% while that of an abnormal E,G value was found to be 77181 = 95%. Similarly, the sensitivities and specificities were calculated for PdG and hCG and the predictabilities of their normal and abnormal values were also obtained. Discussion The history of hormonal inter-relationships in threatened abortion is long and complex. The perfect hormonal test which predicts abortion and successful outcome accurately in 100% of cases is still not available. Opinions have varied on the prognostic value of hormone assays in patients with threatened abortions. According to Brown et al. [l] determination of total estrogen and of urinary pregnanediol was of no value. Nygren et al. [lo] studying the serum levels of hCG, progesterone and estradiol (E,) in patients admitted with threatened abortion reported that hCG level had the most prognostic value. Duff [3] has reported the predictive values of estrogen excretion to be 87% while that of urinary PdG to be 75%. Kunz and Keller [6] have stated that estimation of serum E, or progesterone affords approximately the same information. Hertz et al. [4] found good correlations (90070)between the 24-h urinary PdG levels and the outcome of pregnancy. Mendizabad et al. [9] found low levels of urinary PdG in women with apparent normal fetal development ultrasonographically but who later aborted. Recently, Davidson [2] recommended estimation of PdG for evaluation of threatened abortion. In the present study, E,G was a better parameter with respect to sensitivity (69%) while PdG was a better parameter with respect to
Int J Gynecol Obstet 27
specificity (95%), whereas hCG was a poor parameter with respect to both sensitivity and specificity. As none of the parameters could attain 80% sensitivity and 100% specificity, none of them qualify for a diagnostic/prognostic parameter in threatened abortion. Predictability of a normal value was poor in the cases of all three parameters showing thereby that a normal value did not always signify a normal outcome. The predictability of an abnormal vlaue was quite .high for all three parameters giving sufficiently clear indications of a jeopardised pregnancy. Duff [3] has made a similar observation that the accuracy of all urine tests in predicting abortion was high but there was quite a high failure rate in predicting continuing pregnancy. Therefore, it is felt that estimating hormonal parameters in a single urine sample fails to predict a normal outcome in threatened abortion cases. The combination of ultrasound to detect fetal status together with serial urinary estimations might help to improve the situation. With the present state of knowledge, neither ultrasound nor hormonal studies have a predictive value in threatened abortion. However, the diagnostic value of ultrasound is far superior to that of hormonal analysis. Acknowledgment The authors are grateful to the World Health Organization, Geneva, Dr. P. Samarajeeva, Courtauld Institute of Biochemistry, London and Dr. S.B. Moodbidri, Institute for Research in Reproduction for providing antisera to PdG, E,G and hCG, respectively. We wish to thank Dr. G.M. Sankolli, Mr. M.I. Khatkhatay and Mr. D.K. Pardhe for their assistance during the laboratory investigations. The assistance given by Mrs. V.P. Shenoy, Mrs. K.S. Patni, Mrs. N.S. Gurjar and Mrs. S.R. Baji during the study is appreciated. Thanks are also due to Mrs. Annette Fonseca for meticulous typing of the manuscript.
Urinary hormones in threatened abortion
References Brown JB, Evans JH, Beischer NA, Campbell DG, Fortune DW: Hormone levels in threatened abortion. J Obstet Gynaecol Br Communw 77: 690, 1970. Davidson BJ: Urinary pregnanediol-3glucuronide and estrone conjugates to creatinine ratios in early pregnancies complicated by vaginal bleeding. Fertil Steril 46(4): 610,1986. Duff GB: Prognosis in threatened abortion: a comparison between predictions made by sonar, urinary hormone assays and clinical judgement. Br J Obstet Gynecol 82: 858,197s. Hertz JB, Larsen JF, Arends J and Nielsen J: Progesterone and human chorionic gonadotrophin in serum and pregnanediol in urine in threatened abortion. Acta Obstet Gynecol Stand 59: 23,198O. Joshi UM, Shah HP, Sankolli GM and Khatkhatay MI: Development and application of penicillinase linked EIA in reproductive medicine. In: Clinical Biochemistry Principles and Practise (ed A.S. Eng, R.G. Webb), p 257. 1983. Khatkhatay MI, Sankolli GM, Meherji PK, Gogate J, Chowdhury V and Joshi UM: Application of penicillinase linked ELISA pf pregnanediol glucuronide for detection of ovulation and assessment of corpus luteum function. Endocrinol Jpn, in press. Kunz I and Keller PJ: HCG, HPL, oestradiol, progester-
8
9
10
11
111
one and AFP in serum in patients with threatened abortion. Br J Obstet Gynecol83: 640, 1976. Lesley BL, Stabenfeldt, GH, Overstreet JW, Hanson FW, Czekala N and Munro C: Urinary hormone levels at the time of ovulation and implantation. Fertil Steril 43: 861, 1985. Mendizabad AF, Quiroga S, Farinati Z, Lahoz M and Nagle C: Hormonal monitoring of early pregnancy by a direct radioimmunoassay of steroid glucuronides in first morning urine. Fertil Steri142: 737, 1984. Nygren KG, Johansson ED and Wide L: Evaluation of the prognosis of threatened abortion from the peripheral plasma levels of progesterone, estradiol and human chorionic gonadotrophin. Am J Obstet Gynecol 116: 916, 1973. Shah HP and Joshi UM: A simple, rapid and reliable enzyme linked immunosorbant assay for measuring estrone-3-glucuronide in urine. .I Steroid Biochem 16: 283, 1982.
Address for reprints: U.M. Joshi, PhD Deputy Director, Institute for Research in Reproduction, (ICMR), J.M. Street, Pnrel, Bombay 400 012, India
Clinical and Clinical Research
107
1988,21: 107-l 11 of Gynecology &Obstetrics
Urinary estrone glucuronide, pregnanediol glucuronide and human chorionic gonadotrophin in threatened abortion P.C. Ishwad,
S.M. Chitlange,
U.M. Joshi, V. Chowdhury”
and A.C. Mehta”
Institute for Research in Reproduction (ICMR) and “Nowrosjee Wadia Maternity Hospital, Parel, Bombay 400 012 (India) (Received April 2Oth, 1987) (Revised and accepted August 13th, 1987)
Abstract
Estimations of urinary estrone glucuronide, pregnanediol glucuronide and human chorionic gonadotrophin were carried out by ELBA to see their potential in predicting an abnormal outcome in cases with vaginal bleeding in early pregnancy. Reference values were set up with samples from women without bleeding in present or past pregnancies and with normal ultrasonic findings. None of the parameters were found to be sensitive enough to predict an abnormal outcome. However, predictability of an abnormal value was found to be 95% for estrone-3-glucuronide (E,G), 93 % for pregnanediol glucuronide (PdG) and 87% for human chorionic gonadotrophin (hCG). Keywords:
Abortion;
ELISA of urinary hor-
mones. Introduction
Bleeding in the first 20 weeks of pregnancy with/without lower abdominal cramps/backache is termed as threatened abortion. Beyond 24 gestational weeks, fetal death is termed as miscarriage, for which the reasons may be more than just hormonal. However, 0020-7292/88/$03.50 0 1988 International Federation of Gynecology&Obstetrics
chorionic tissue remaining functional, the pregnancy test remains positive. Using present clinical and laboratory parameters it is often difficult, until a late stage of the course to assign patients with threatened abortion for immediate curettage or continued treatment. A simple and rapid test for fetal viability is much awaited today to avoid delay, anxiety and prolonged costly hospitalization. Though ultrasonography diagnoses fetal status it is not prognostic. Besides it is expensive and not universally available. RIA for serum hormones involves an invasive technique and needs sophisticated facilities. Many articles have been published evaluating the levels of a variety of hormones in both blood and urine in normal and abortive pregnancies . Unfortunately, these reports are often contradictory in their results. The methods involved are in many cases laborious and require specialized equipment not readily available in a clinical situation. The method of analysis should be simple, inexpensive, reliable and rapid with a low analytical error. The half-life of the estimated hormone should be short to get a quick reflection of deterioration in placental and/or feto-placental function. We have used the simple technique of ELISA for estimating urinary hormones or their metabolites in pregnant patients. Clinical and Clinical Research
108
Ishwad et al.
The parameters estimated were estrone-3glucuronide (E,G), pregnanediol glucuronide (PdG) and human chorionic gonadotrophin (hCG). These parameters have been used to analyze human reproductive cycles and to monitor early trophoblastic growth [8]. E,G is a urinary metabolite mainly of estrone and partly of estradiol. It reflects the function of the feto-placental unit. PdG is the urinary metabolite of progesterone which is produced initially by the corpus luteum of pregnancy and later by the placenta. hCG has a trophoblastic origin and is excreted as such in the urine. Produced by the feto-placental unit, the estrogen level drops quickly after death of the conceptus, while hCG and progesterone produced by the placenta take longer to drop if placenta is not detached and hence still functioning and their assay, especially that of random urinary hCG is less reliable [3]. Materials and methods Two hundred forty-five subjects in different stages of pregnancy, with no vaginal bleeding during the present or past pregnancies and with positive fetal heart beats/movements, and fetal measurements normal for that stage of pregnancy were enrolled as the control group. One hundred thirty-five pregnant subjects with vaginal bleeding at the time of examination and with/without positive fetal heart beats/movements were enrolled as the study group. Early morning urine samples were collected from these women dating from 5 to 20 gestational weeks. Each sample was preserved with sodium azide as preservative at - 20°C till assayed within 1 month of collection. Ultrasonography was performed on the day of urine collection to ascertain fetal status. The enzyme-linked immunosorbant assay (ELBA), using penicillinase enzyme as a marker, was used to estimate E,G according to Shah et al. [l 11, PdG according to Khatkhatay et al. [5] and hCG according to Joshi et al. [7]. All three parameters were estimated by competitive ELBA. In brief, the Int JGynecol Obstet 27
method involves the following steps: immobilization of antibody on a polyvinyl chloride microtitre plate, incubating the sample and analysing penicillinase in the antibody immobilized well, measuring the bound enzyme activity by starch iodine as an indicator using either a calorimeter or ELISA reader. All assays are completed in 3 h. The creatinine was estimated in each urine sample and the value of the parameter expressed as concentration per milligram creatinine. Samples having creatinine < 100 pg/ml were considered unsuitable for analysis. All hormones could not be analysed in all samples. Number of cases and samples analysed are given in Table I and Fig. 1. All study cases were followed up till delivery or abortion. Those cases delivering after 36th week with babies > 2.2 kg were considered to have normal outcome. Cases with missed abortions, or abortions within 1 month from collection of urine were considered to have abnormal out-
Evaluation of E,G, PdG and hCG in missed and Table 1. threatened abortions for prognosis. Parameter
w
PdG
hCG
113 62 51 142
130 15 55 156
110 62 48 135
Normal values Total With normal outcome g)) With abnormal outcome (c)
61 26 35
112 60 52
90 44 46
Abnormal values Total (d) With abnormal outcome (e) With normal outcome (f) Sensitivity(e/c + e) Specifkity@/b + f)
81 II 4 69 86
44 41 3 44 95
45 39 6 46 88
Predictability of Normal value (b/a) Abnormal value (e/d)
43 95
54 93
49 87
Cases studied Missed abortions Threatened abortions No. of samples
Urinary hormones in threatened abortion
109
come. Cases delivering at term but weight of baby less than 2.2 kg and cases aborting after more than 6 weeks from urine collections were excluded from analysis. Thus only 130 out of 135 cases were included for statistical analysis.
looo~
Statistical analysis
(7) (36) (32) (34) (36) (44) (33) (24) , I I . . 5-6 7-6 S-40 H-i2 43.,415~(6,7.1649-M
40-
41
Since all hormone assays were evaluated to predict an abnormal outcome in the bleeding cases within 6 weeks of urine collection, an abnormal value was considered as positive and normal value as negative. True positives were those abnormal values which corresponded to an abnormal outcome. True negatives were those normal values corresponding to a normal outcome. Sensitivity was defined as the capacity of the test to predict abnormal outcome (or to detect true positives) and hence was equal to (true positive/true positive + false negative) X 100. Specificity was defined as the capacity of the test to avoid false positive and hence was equal to (true negative/true negative + false positive) X 100.
I
.
5-6
.
7-6
5-6
I
.
Results
.
S-,0 ,I-,2 (3-M ,5-% V-16 is-20
7-6
WEEKS
9-K) U-i2 ,3-14,546 17-1849.20
OF
GESTATION
*C=CENTILE
Fig. 1. Establishment of reference values for excretion of E,G, PdG and hCG in urine during pregnancy. Continuous lines show 10th. 50th and 90th centiles. Broken lines show lowest value recorded for the period. Numbers in parentheses indicate number of samples analysed.
Figure 1 gives the lOth, 50th and 90th centile as well as the lowest value obtained for particular weeks of pregnancy for the respective parameter. The patterns for the three parameters are similar to those reported in the literature for corresponding circulating hormones. The lowest values recorded during the study were considered as cut-off limits to avoid even a single, normal case being labelled as abnormal. Table I gives the detailed analysis for all the three parameters, E,G, PdG and hCG. For E,G, 142 samples from 113 cases were analysed. Sixty-two of the 113 cases were missed abortions while 51 were threatened abortions. Sixty-one normal values were obtained out of which 26 were compatible with normal outcome while 35 corresponded to an abnormal outcome. Eighty-one out of Clinical and Clinical Research
110
Ish wad et al.
the total 142 samples showed an abnormal value. Seventy-seven of these 81 were compatible with an abnormal outcome while 4 corresponded to a normal outcome. Thus sensitivity of E,G was calculated as 77/(77 + 35) = 69%. Specificity of E,G was 26/(26 + 4) = 86%. Predictability of a normal E,G value was found to be 26161 = 43% while that of an abnormal E,G value was found to be 77181 = 95%. Similarly, the sensitivities and specificities were calculated for PdG and hCG and the predictabilities of their normal and abnormal values were also obtained. Discussion The history of hormonal inter-relationships in threatened abortion is long and complex. The perfect hormonal test which predicts abortion and successful outcome accurately in 100% of cases is still not available. Opinions have varied on the prognostic value of hormone assays in patients with threatened abortions. According to Brown et al. [l] determination of total estrogen and of urinary pregnanediol was of no value. Nygren et al. [lo] studying the serum levels of hCG, progesterone and estradiol (E,) in patients admitted with threatened abortion reported that hCG level had the most prognostic value. Duff [3] has reported the predictive values of estrogen excretion to be 87% while that of urinary PdG to be 75%. Kunz and Keller [6] have stated that estimation of serum E, or progesterone affords approximately the same information. Hertz et al. [4] found good correlations (90070)between the 24-h urinary PdG levels and the outcome of pregnancy. Mendizabad et al. [9] found low levels of urinary PdG in women with apparent normal fetal development ultrasonographically but who later aborted. Recently, Davidson [2] recommended estimation of PdG for evaluation of threatened abortion. In the present study, E,G was a better parameter with respect to sensitivity (69%) while PdG was a better parameter with respect to
Int J Gynecol Obstet 27
specificity (95%), whereas hCG was a poor parameter with respect to both sensitivity and specificity. As none of the parameters could attain 80% sensitivity and 100% specificity, none of them qualify for a diagnostic/prognostic parameter in threatened abortion. Predictability of a normal value was poor in the cases of all three parameters showing thereby that a normal value did not always signify a normal outcome. The predictability of an abnormal vlaue was quite .high for all three parameters giving sufficiently clear indications of a jeopardised pregnancy. Duff [3] has made a similar observation that the accuracy of all urine tests in predicting abortion was high but there was quite a high failure rate in predicting continuing pregnancy. Therefore, it is felt that estimating hormonal parameters in a single urine sample fails to predict a normal outcome in threatened abortion cases. The combination of ultrasound to detect fetal status together with serial urinary estimations might help to improve the situation. With the present state of knowledge, neither ultrasound nor hormonal studies have a predictive value in threatened abortion. However, the diagnostic value of ultrasound is far superior to that of hormonal analysis. Acknowledgment The authors are grateful to the World Health Organization, Geneva, Dr. P. Samarajeeva, Courtauld Institute of Biochemistry, London and Dr. S.B. Moodbidri, Institute for Research in Reproduction for providing antisera to PdG, E,G and hCG, respectively. We wish to thank Dr. G.M. Sankolli, Mr. M.I. Khatkhatay and Mr. D.K. Pardhe for their assistance during the laboratory investigations. The assistance given by Mrs. V.P. Shenoy, Mrs. K.S. Patni, Mrs. N.S. Gurjar and Mrs. S.R. Baji during the study is appreciated. Thanks are also due to Mrs. Annette Fonseca for meticulous typing of the manuscript.
Urinary hormones in threatened abortion
References Brown JB, Evans JH, Beischer NA, Campbell DG, Fortune DW: Hormone levels in threatened abortion. J Obstet Gynaecol Br Communw 77: 690, 1970. Davidson BJ: Urinary pregnanediol-3glucuronide and estrone conjugates to creatinine ratios in early pregnancies complicated by vaginal bleeding. Fertil Steril 46(4): 610,1986. Duff GB: Prognosis in threatened abortion: a comparison between predictions made by sonar, urinary hormone assays and clinical judgement. Br J Obstet Gynecol 82: 858,197s. Hertz JB, Larsen JF, Arends J and Nielsen J: Progesterone and human chorionic gonadotrophin in serum and pregnanediol in urine in threatened abortion. Acta Obstet Gynecol Stand 59: 23,198O. Joshi UM, Shah HP, Sankolli GM and Khatkhatay MI: Development and application of penicillinase linked EIA in reproductive medicine. In: Clinical Biochemistry Principles and Practise (ed A.S. Eng, R.G. Webb), p 257. 1983. Khatkhatay MI, Sankolli GM, Meherji PK, Gogate J, Chowdhury V and Joshi UM: Application of penicillinase linked ELISA pf pregnanediol glucuronide for detection of ovulation and assessment of corpus luteum function. Endocrinol Jpn, in press. Kunz I and Keller PJ: HCG, HPL, oestradiol, progester-
8
9
10
11
111
one and AFP in serum in patients with threatened abortion. Br J Obstet Gynecol83: 640, 1976. Lesley BL, Stabenfeldt, GH, Overstreet JW, Hanson FW, Czekala N and Munro C: Urinary hormone levels at the time of ovulation and implantation. Fertil Steril 43: 861, 1985. Mendizabad AF, Quiroga S, Farinati Z, Lahoz M and Nagle C: Hormonal monitoring of early pregnancy by a direct radioimmunoassay of steroid glucuronides in first morning urine. Fertil Steri142: 737, 1984. Nygren KG, Johansson ED and Wide L: Evaluation of the prognosis of threatened abortion from the peripheral plasma levels of progesterone, estradiol and human chorionic gonadotrophin. Am J Obstet Gynecol 116: 916, 1973. Shah HP and Joshi UM: A simple, rapid and reliable enzyme linked immunosorbant assay for measuring estrone-3-glucuronide in urine. .I Steroid Biochem 16: 283, 1982.
Address for reprints: U.M. Joshi, PhD Deputy Director, Institute for Research in Reproduction, (ICMR), J.M. Street, Pnrel, Bombay 400 012, India
Clinical and Clinical Research