British Journal of’Obstetrics and Gynaecology October 1911. Vol 84. pp 140-742
CIRCULATING LEVELS OF PREGNANCY-SPECIFIC &-GLYCOPROTEIN IN EARLY PREGNANCY BY
J. G. GRUDZINSKAS, W H O Research Fellow E. A, LENTON,* Biochemist
Y. B. GORDON, Lecturer I. M.
Research Fellow
&%SO,?
D. JEFFREY, Technician 0. SOBOWALE,* Registrar AND
T. CHARD, Professor Department of Reproductive Physiology, St Bartholomew’s Hospital Medical College and the London Hospital Medical College, London Summary Circulating levels of pregnancy-specific P,-glycoprotein (SP, or PSPG), luteinizing hormone (LH) and human chorionic gonadotrophin (HCG) were measured serially in 9 subjects immediately after conception. Ovulation occurred spontaneously in 3 subjects, or followed administration of clomiphene citrate (2 subjects) or bromocriptine (4 subjects). The timing of ovulation was determined by the appearance of the LH surge. Levels of HCG were detected 10 to 16 days, and SP,, 18 to 23 days after ovulation. These findings suggest that the measurement of plasma levels of SP, may provide valuable additional biochemical evidence of pregnancy.
THE measurement of proteins specifically produced by the placental trophoblast and secreted into the plasma and urine of pregnant women is widely used to detect pregnancy and also as an index of fetal wellbeing in late pregnancy. The isolation of a new trophoblast specific protein was described independently by Tatarinov and Masyukevich (1970) who called it trophoblast specific P-globulin (TBG), Bohn (1971) who called it ‘schwangerschafts~
~~
spezifisches PI-Glykoprotein’ (or SP,), and Lin et a1 (1974) who called it pregnancy associated plasma protein-C (PAPP-C). SP, has also been named pregnancy-specific P,-glycoprotein (PSPG) by Towler et a1 (1976). Since the development of a specific and sensitive radioimmunoassay, this material has been investigated as a potential diagnostic marker in early pregnancy. The radioimmunoassay for SP, (Grudzinskas et al, 1977) permits detection of nanogram concentrations, and preliminary data suggested that plasma levels of SP, could be detected at or within 7 to 14 days of implantation in some but not all pregnant women.
~~
* Present
address: Department of Obstetrics and Gynaecology, Jessop Hospital for Women, Sheffield. tPresent address: Royal North Shore Hospital, Sydney, N.S.W., Australia. 740
spl IN EARLY PREGNANCY
In this study we have measured the appearance of human chorionic gonadotrophin (HCG) and SP, in plasma from women in whom ovulation was precisely timed by the occurrence of the mid-cycle luteinising hormone (LH) surge. METHODS Plasma levels of SP, were measured using a modification of the radioimmunoassay described previously (Grudzinskas et al, 1977) : antibody bound and free antigen fractions were separated utilizing the second antibody technique. A final dilution of 1 : 1000 normal rabbit serum and 1 : 80 donkey anti-rabbit serum (Wellcome Reagents Ltd) were added, followed by 18 hours incubation before centrifugation. Over a period of two months the intra-assay variation was 4 per cent and the interassay variation 9 per cent. Circulating levels of LH were measured by radioimmunoassay and values expressed with reference to the second International Reference Preparation for human menopausal gonadotrophin, Medical Research Council Division of Biological Standards, Mill Hill, London. The antiserum for the LH assay was supplied by the National Pituitary Agency. As HCG cross-reacts with this antiserum, the apparent increase in plasma LH levels which occurs soon after implantation is interpreted as evidence of HCG production. Volunteers were recruited from a family planning and infertility clinic. In three subjects who ovulated spontaneously, an intrauterine contraceptive device had been recently removed. Two subjects received clomiphene citrate (50 mg daily for five days) as treatment for anovulation. The remaining four subjects had been treated with bromocriptine (3.75 mg daily) because of anovulation due to hyperprolactinaemia. The age distribution was 24 to 34 years. Venous blood was collected in heparinized tubes, the plasma separated by centrifugation, aliquoted, and stored at -20 "C.
RESULTS The SP, radioimmunoassay had a minimum detection limit of 15 pg/l and a dynamic range of 15 to 500 pg/l. SP, was detected in the plasma
741
of all subjects studied within 23 days of the LH surge, the earliest being at 18 days (Fig. 1). The appearance of circulating levels of SP, was not related to the nature of ovulation, whether spontaneous or induced. In all subjects there was a sharp increase in SP, concentrations following the initial appearance of the protein. Plasma levels of HCG, implied by rising levels of LH, were detected within 10 to 16 days after the LH peak in all subjects. DISCUSS~ON The development of sensitive and specific assays for the detection of SP, has permitted the measurement of low concentrations of this protein in the circulation in early pregnancy (Grudzinskas et al, 1977), as well as the demonstration of SP, in the syncytiotrophoblast within 3 weeks of ovulation (Horne et al, 1977). Data from the former report describing the appearance of circulating levels of SP, at implantation, in some but not all women, have suggested that the measurement of plasma levels of SP, may provide additional biochemical evidence of very early pregnancy. However, the timing of ovulation was imprecisely defined, whereas in the present study the appearance of circulating levels of SP, has been accurately related to ovulation by measuring the LH surge in all subjects. Plasma levels of SP, were detected 18 to 23 days following ovulation. These findings are consistent with our earlier report and confirm the histochemical data of Horne et a1 (1977). Nevertheless, as controversy still exists regarding the biochemical characterisation of SP,, and the purity of reagents available, it is possible that the development of assays with increased sensitivity will permit the measurement of smaller quantities of this molecule, and therefore detection earlier in pregnancy. The interval between the appearance of HCG and SP, in plasma was not constant. This may be due to the normal variation in HCG production in early pregnancy, or to the non-specificity of the HCG-LH assay. The early appearance of SP, in the plasma of pregnant women establishes the measurement of SP, as an additional test of pregnancy with potential advantagessimilar to the HCG p-subunit
742
GRUDZINSKAS, LENTON, GORDON, KELSO, JEFFREY, SOBOWALE AND CHARD
500
50
20 14
21
28
Days after LH surge FIG.1 spontaneous ovulation (3 Plasma concentrations of SP1 in 9 subjects after conception. II-M subjects): 0-0 clomiphene citrate induced ovulation (2 subjects): A-A ovulation after bromocriptine administration (4 subjects).
assay. As the biochemical characteristics of SP, differ from LH and other pituitary hormones there would be no ambiguity with results at low concentrations because of cross-reaction with materials present in the non-pregnant state. Furthermore, in women treated with pituitary and chorionic gonadotrophins for ovulation induction and early pregnancy support, the detection of SP, may be the only unequivocal evidence of pregnancy available. Finally, the precise quantitation of SP, levels possible with radioimmunoassay may permit identification of abnormalities of early pregnancy such as anembryonic pregnancy and missed abortion when low levels are recorded (Grudzinskas and Gordon, 1977 unpublished observations).
ACKNOWLEDGEMENTS J.G.G. was supported by the World Health Organization. REFERENCES Bohn, H. (1971): Archiv fur Gynakologie, 210,440. Grudzinskas, J. G., Gordon, Y . B., Jeffrey, D., and Chard, T. (1977): Lancet, 1, 333. Horne, C. H. W., Towler, C. M., and Milne, G. D. (1977): Journal of Clinical Pathology, 30, 19. Lin, T. M., Halbert, S. P., Kiefer, D., Spellacy, W., and Gall, S. (1 974) : American Journal of Obstetrics and Gynecology, 118, 223. Tatarinov, Yu S., and Masyukcvich, V. N. (1970): Byulleten’ ‘tksperimental’ noi biologii i medicin;, 69, 66. Towler, C. M., Jandial, V., Horne, C . H. W., and Bohn, H. (1976): British Journal of Obstetrics and Gynaecology, 83, 368.
CIRCULATING LEVELS OF PREGNANCY-SPECIFIC &-GLYCOPROTEIN IN EARLY PREGNANCY BY
J. G. GRUDZINSKAS, W H O Research Fellow E. A, LENTON,* Biochemist
Y. B. GORDON, Lecturer I. M.
Research Fellow
&%SO,?
D. JEFFREY, Technician 0. SOBOWALE,* Registrar AND
T. CHARD, Professor Department of Reproductive Physiology, St Bartholomew’s Hospital Medical College and the London Hospital Medical College, London Summary Circulating levels of pregnancy-specific P,-glycoprotein (SP, or PSPG), luteinizing hormone (LH) and human chorionic gonadotrophin (HCG) were measured serially in 9 subjects immediately after conception. Ovulation occurred spontaneously in 3 subjects, or followed administration of clomiphene citrate (2 subjects) or bromocriptine (4 subjects). The timing of ovulation was determined by the appearance of the LH surge. Levels of HCG were detected 10 to 16 days, and SP,, 18 to 23 days after ovulation. These findings suggest that the measurement of plasma levels of SP, may provide valuable additional biochemical evidence of pregnancy.
THE measurement of proteins specifically produced by the placental trophoblast and secreted into the plasma and urine of pregnant women is widely used to detect pregnancy and also as an index of fetal wellbeing in late pregnancy. The isolation of a new trophoblast specific protein was described independently by Tatarinov and Masyukevich (1970) who called it trophoblast specific P-globulin (TBG), Bohn (1971) who called it ‘schwangerschafts~
~~
spezifisches PI-Glykoprotein’ (or SP,), and Lin et a1 (1974) who called it pregnancy associated plasma protein-C (PAPP-C). SP, has also been named pregnancy-specific P,-glycoprotein (PSPG) by Towler et a1 (1976). Since the development of a specific and sensitive radioimmunoassay, this material has been investigated as a potential diagnostic marker in early pregnancy. The radioimmunoassay for SP, (Grudzinskas et al, 1977) permits detection of nanogram concentrations, and preliminary data suggested that plasma levels of SP, could be detected at or within 7 to 14 days of implantation in some but not all pregnant women.
~~
* Present
address: Department of Obstetrics and Gynaecology, Jessop Hospital for Women, Sheffield. tPresent address: Royal North Shore Hospital, Sydney, N.S.W., Australia. 740
spl IN EARLY PREGNANCY
In this study we have measured the appearance of human chorionic gonadotrophin (HCG) and SP, in plasma from women in whom ovulation was precisely timed by the occurrence of the mid-cycle luteinising hormone (LH) surge. METHODS Plasma levels of SP, were measured using a modification of the radioimmunoassay described previously (Grudzinskas et al, 1977) : antibody bound and free antigen fractions were separated utilizing the second antibody technique. A final dilution of 1 : 1000 normal rabbit serum and 1 : 80 donkey anti-rabbit serum (Wellcome Reagents Ltd) were added, followed by 18 hours incubation before centrifugation. Over a period of two months the intra-assay variation was 4 per cent and the interassay variation 9 per cent. Circulating levels of LH were measured by radioimmunoassay and values expressed with reference to the second International Reference Preparation for human menopausal gonadotrophin, Medical Research Council Division of Biological Standards, Mill Hill, London. The antiserum for the LH assay was supplied by the National Pituitary Agency. As HCG cross-reacts with this antiserum, the apparent increase in plasma LH levels which occurs soon after implantation is interpreted as evidence of HCG production. Volunteers were recruited from a family planning and infertility clinic. In three subjects who ovulated spontaneously, an intrauterine contraceptive device had been recently removed. Two subjects received clomiphene citrate (50 mg daily for five days) as treatment for anovulation. The remaining four subjects had been treated with bromocriptine (3.75 mg daily) because of anovulation due to hyperprolactinaemia. The age distribution was 24 to 34 years. Venous blood was collected in heparinized tubes, the plasma separated by centrifugation, aliquoted, and stored at -20 "C.
RESULTS The SP, radioimmunoassay had a minimum detection limit of 15 pg/l and a dynamic range of 15 to 500 pg/l. SP, was detected in the plasma
741
of all subjects studied within 23 days of the LH surge, the earliest being at 18 days (Fig. 1). The appearance of circulating levels of SP, was not related to the nature of ovulation, whether spontaneous or induced. In all subjects there was a sharp increase in SP, concentrations following the initial appearance of the protein. Plasma levels of HCG, implied by rising levels of LH, were detected within 10 to 16 days after the LH peak in all subjects. DISCUSS~ON The development of sensitive and specific assays for the detection of SP, has permitted the measurement of low concentrations of this protein in the circulation in early pregnancy (Grudzinskas et al, 1977), as well as the demonstration of SP, in the syncytiotrophoblast within 3 weeks of ovulation (Horne et al, 1977). Data from the former report describing the appearance of circulating levels of SP, at implantation, in some but not all women, have suggested that the measurement of plasma levels of SP, may provide additional biochemical evidence of very early pregnancy. However, the timing of ovulation was imprecisely defined, whereas in the present study the appearance of circulating levels of SP, has been accurately related to ovulation by measuring the LH surge in all subjects. Plasma levels of SP, were detected 18 to 23 days following ovulation. These findings are consistent with our earlier report and confirm the histochemical data of Horne et a1 (1977). Nevertheless, as controversy still exists regarding the biochemical characterisation of SP,, and the purity of reagents available, it is possible that the development of assays with increased sensitivity will permit the measurement of smaller quantities of this molecule, and therefore detection earlier in pregnancy. The interval between the appearance of HCG and SP, in plasma was not constant. This may be due to the normal variation in HCG production in early pregnancy, or to the non-specificity of the HCG-LH assay. The early appearance of SP, in the plasma of pregnant women establishes the measurement of SP, as an additional test of pregnancy with potential advantagessimilar to the HCG p-subunit
742
GRUDZINSKAS, LENTON, GORDON, KELSO, JEFFREY, SOBOWALE AND CHARD
500
50
20 14
21
28
Days after LH surge FIG.1 spontaneous ovulation (3 Plasma concentrations of SP1 in 9 subjects after conception. II-M subjects): 0-0 clomiphene citrate induced ovulation (2 subjects): A-A ovulation after bromocriptine administration (4 subjects).
assay. As the biochemical characteristics of SP, differ from LH and other pituitary hormones there would be no ambiguity with results at low concentrations because of cross-reaction with materials present in the non-pregnant state. Furthermore, in women treated with pituitary and chorionic gonadotrophins for ovulation induction and early pregnancy support, the detection of SP, may be the only unequivocal evidence of pregnancy available. Finally, the precise quantitation of SP, levels possible with radioimmunoassay may permit identification of abnormalities of early pregnancy such as anembryonic pregnancy and missed abortion when low levels are recorded (Grudzinskas and Gordon, 1977 unpublished observations).
ACKNOWLEDGEMENTS J.G.G. was supported by the World Health Organization. REFERENCES Bohn, H. (1971): Archiv fur Gynakologie, 210,440. Grudzinskas, J. G., Gordon, Y . B., Jeffrey, D., and Chard, T. (1977): Lancet, 1, 333. Horne, C. H. W., Towler, C. M., and Milne, G. D. (1977): Journal of Clinical Pathology, 30, 19. Lin, T. M., Halbert, S. P., Kiefer, D., Spellacy, W., and Gall, S. (1 974) : American Journal of Obstetrics and Gynecology, 118, 223. Tatarinov, Yu S., and Masyukcvich, V. N. (1970): Byulleten’ ‘tksperimental’ noi biologii i medicin;, 69, 66. Towler, C. M., Jandial, V., Horne, C . H. W., and Bohn, H. (1976): British Journal of Obstetrics and Gynaecology, 83, 368.
