Human Reproduction vol.7 no.l pp.128-130, 1992
Placental protein 14 in human in-vitro fertilization early pregnancies
L.Nylund1, O.Gustafson, B.Lindblom, A.Pousette2, M.Seppala3, L.Riittinen3 and E.Akerlof2 Departments of Obstetrics and Gynaecology and 2Clinical Chemistry, Karolinska Institute, Huddinge University Hospital, 141 86 Huddinge, Sweden and 'Department I of Obstetrics and Gynaecology, Helsinki University Central Hospital, Helsinki, Finland 'To whom correspondence should be addressed
Placental protein 14 (PP14) and human chorionic gonadotrophin (HCG) were analysed in patients participating in an in-vitro fertilization-embryo transfer programme which did not include any kind of luteal support. Women with normal pregnancies, spontaneous abortions, ectopic pregnancies, biochemical pregnancies and non-pregnant women were compared. A combination of HCG and PP14 analyses distinguished between normal and abnormal implantation as early as 15 days after oocyte retrieval. The product of HCG (IU/1) and PP14 (/ig/1) concentrations differed significantly between normal pregnancy, spontaneous abortion and ectopic pregnancy (P = 0.0248). It is concluded that both endometrial (PP14) and trophoblastic (HCG) markers, when used in combination, exhibit changes in abnormal implantation which may be clinically useful. Key words: IVF/implantation/HCG/PP14/ectopic pregnancy
Introduction Several methods have been applied for differential diagnosis of early pregnancy. A tool for accurate recognition of spontaneous abortion and ectopic pregnancies would be of particular clinical interest in assisted reproduction which has a high proportion of abnormal pregnancies. The value of early identification of ectopic pregnancies is obvious, but early information about pregnancy outcome might also have psychological advantages for patients. Decidual function has received little attention in early pregnancy monitoring. The decidualized human endometrium synthesizes various glycoproteins, of which placental protein 14 (PP14) is the major one in the first trimester (Joshi et al., 1980; Julkunen et al., 1988; Seppala et al., 1988a,b,c; Fay et al. 1990). The serum concentration of PP14 increases progressively in normal pregnancies, peaks between the 8th and 10th weeks and gradually declines thereafter (Julkunen et al., 1985). We have studied PP14 levels in the serum of women undergoing in-vitro fertilization (TVF) and embryo transfer, and related their human chorionic gonadotrophin (HCG) and PP14 profiles to pregnancy outcome. 128
Materials and methods Blood samples were obtained from 34 women undergoing IVF/ET at Huddinge University Hospital, Stockholm, Sweden during the period 1986—1989. The programme included controlled hyperstimulation with clomiphene citrate 100 mg daily for 5 days, followed by intramuscular human menopausal gonadotrophin (HMG) injections 75-225 IU daily. HCG 10 000 IU was given 34.5 h before oocyte retrieval, performed either transvesically or transvaginally under ultrasound guidance. A maximum of four embryos were transferred on day 2, the day of oocyte retrieval being referred to as day 0. No luteal support was given in any of the cycles. Venous blood samples were drawn on days - 2 , 0, 2, 5, 8 and 15. The sera were frozen at -20°C for subsequent analyses. Further details were given by Lunell et al. (1989). HCG was analysed using a commercial radioimmunoassay kit from Diagnostic Product Corporation (Los Angeles, USA). This assay had a sensitivity of 5 IU/1. PP14 was measured with a specific radioimmunoassay as described previously (Seppala et al., 1988a). Normal pregnancy (n = 10) after FVF was defined as a pregnancy resulting in delivery of a normal infant after the 25th week. Spontaneous abortion (n = 6) was defined as an ultrasonographically verified intrauterine gestational sac and elevated HCG levels beyond day 8, followed by pregnancy loss before the 25th gestational week. Five cases of ectopic pregnancy occurred during the study period. All underwent laparotomy with histological confirmation of the diagnosis. Biochemical pregnancy was defined as a temporary elevation of serum HCG level > 15 IU/1 beyond day 8 and absence of ultrasonographic evidence of pregnancy (n = 3). For technical reasons, PP14 could be measured on days 8 and 15 in only one case of biochemical pregnancy. Patients who did not have HCG levels > 15 IU/1 at any time after day 8 were designated as 'non-pregnant' (n = 10).
TaWe I. Serum placental protein 14 (PP14) concentrations Oig/1) relative to the day of ovum retrieval. Values are medians (range with no decimals) Day relative to oocyte retrieval
-2
0
Normal pregnancy (n = 10) Ectopic pregnancy (n = 5) Spontaneous abortion (n = 6) Biochemical (n = 3) Non-pregnant (n = 10)
14.7 (11-18) 14.5 (10-19) 13 (11-22) 8.6 (9-9) 16 8 (10-22)
12.5 (12-15) 15 (12-18) 12.4 (10-19) 17.2 (10-23) 15.1 (7-20)
15 12.9 (11-15) 17.8 (11-23) 11.5 (10-18) 16 1 (11-17) 13 7 (10-20)
15.1 (12-40) 15.5 (11-21) 17 4 (10-25) -
23.1 (17-107) 27.2 (21-34) 19 (18-25) 21.8
109 (46-147) 80 (32-93) 119 (49-211) 34
15 5 22.9 48.3 (10-23) (14-54) (40-77) © Oxford University Press
PP14 in human IVF early pregnancies Normal pregnancy
Spontaneous abortion
Ectoplc pregnancy 300
T
1-300
2 0 2 4 6 • 10 I2 14 1• Day rvlatlva t * aa«yta ratrhaval
- 2 0 Day
Biochemical pregnancy
2
4
ralallva
6 to
8
10121416
ao«y1a
ralrlvval
Non-pregnant
300
2 Day
0
2
4
relative
• to
8
101214
ata-ayi*
r*tri*val
Fig. 1. Median placental protein 14 (PP14) and human chononic gonadotrophin (HCG) levels in serum in different categories of women. Note the similarity of patterns prior to day 8, corresponding to the peri-implantation period. Thereafter, differentiation was detected between the various categories of women. Day 0 = day of ovum retrieval; day 2 = day of embryo transfer.
For statistical evaluation of the data median levels were calculated. The Kruskal—Wallis non-parametric test was used for comparisons between the groups. P-values of
Placental protein 14 in human in-vitro fertilization early pregnancies
L.Nylund1, O.Gustafson, B.Lindblom, A.Pousette2, M.Seppala3, L.Riittinen3 and E.Akerlof2 Departments of Obstetrics and Gynaecology and 2Clinical Chemistry, Karolinska Institute, Huddinge University Hospital, 141 86 Huddinge, Sweden and 'Department I of Obstetrics and Gynaecology, Helsinki University Central Hospital, Helsinki, Finland 'To whom correspondence should be addressed
Placental protein 14 (PP14) and human chorionic gonadotrophin (HCG) were analysed in patients participating in an in-vitro fertilization-embryo transfer programme which did not include any kind of luteal support. Women with normal pregnancies, spontaneous abortions, ectopic pregnancies, biochemical pregnancies and non-pregnant women were compared. A combination of HCG and PP14 analyses distinguished between normal and abnormal implantation as early as 15 days after oocyte retrieval. The product of HCG (IU/1) and PP14 (/ig/1) concentrations differed significantly between normal pregnancy, spontaneous abortion and ectopic pregnancy (P = 0.0248). It is concluded that both endometrial (PP14) and trophoblastic (HCG) markers, when used in combination, exhibit changes in abnormal implantation which may be clinically useful. Key words: IVF/implantation/HCG/PP14/ectopic pregnancy
Introduction Several methods have been applied for differential diagnosis of early pregnancy. A tool for accurate recognition of spontaneous abortion and ectopic pregnancies would be of particular clinical interest in assisted reproduction which has a high proportion of abnormal pregnancies. The value of early identification of ectopic pregnancies is obvious, but early information about pregnancy outcome might also have psychological advantages for patients. Decidual function has received little attention in early pregnancy monitoring. The decidualized human endometrium synthesizes various glycoproteins, of which placental protein 14 (PP14) is the major one in the first trimester (Joshi et al., 1980; Julkunen et al., 1988; Seppala et al., 1988a,b,c; Fay et al. 1990). The serum concentration of PP14 increases progressively in normal pregnancies, peaks between the 8th and 10th weeks and gradually declines thereafter (Julkunen et al., 1985). We have studied PP14 levels in the serum of women undergoing in-vitro fertilization (TVF) and embryo transfer, and related their human chorionic gonadotrophin (HCG) and PP14 profiles to pregnancy outcome. 128
Materials and methods Blood samples were obtained from 34 women undergoing IVF/ET at Huddinge University Hospital, Stockholm, Sweden during the period 1986—1989. The programme included controlled hyperstimulation with clomiphene citrate 100 mg daily for 5 days, followed by intramuscular human menopausal gonadotrophin (HMG) injections 75-225 IU daily. HCG 10 000 IU was given 34.5 h before oocyte retrieval, performed either transvesically or transvaginally under ultrasound guidance. A maximum of four embryos were transferred on day 2, the day of oocyte retrieval being referred to as day 0. No luteal support was given in any of the cycles. Venous blood samples were drawn on days - 2 , 0, 2, 5, 8 and 15. The sera were frozen at -20°C for subsequent analyses. Further details were given by Lunell et al. (1989). HCG was analysed using a commercial radioimmunoassay kit from Diagnostic Product Corporation (Los Angeles, USA). This assay had a sensitivity of 5 IU/1. PP14 was measured with a specific radioimmunoassay as described previously (Seppala et al., 1988a). Normal pregnancy (n = 10) after FVF was defined as a pregnancy resulting in delivery of a normal infant after the 25th week. Spontaneous abortion (n = 6) was defined as an ultrasonographically verified intrauterine gestational sac and elevated HCG levels beyond day 8, followed by pregnancy loss before the 25th gestational week. Five cases of ectopic pregnancy occurred during the study period. All underwent laparotomy with histological confirmation of the diagnosis. Biochemical pregnancy was defined as a temporary elevation of serum HCG level > 15 IU/1 beyond day 8 and absence of ultrasonographic evidence of pregnancy (n = 3). For technical reasons, PP14 could be measured on days 8 and 15 in only one case of biochemical pregnancy. Patients who did not have HCG levels > 15 IU/1 at any time after day 8 were designated as 'non-pregnant' (n = 10).
TaWe I. Serum placental protein 14 (PP14) concentrations Oig/1) relative to the day of ovum retrieval. Values are medians (range with no decimals) Day relative to oocyte retrieval
-2
0
Normal pregnancy (n = 10) Ectopic pregnancy (n = 5) Spontaneous abortion (n = 6) Biochemical (n = 3) Non-pregnant (n = 10)
14.7 (11-18) 14.5 (10-19) 13 (11-22) 8.6 (9-9) 16 8 (10-22)
12.5 (12-15) 15 (12-18) 12.4 (10-19) 17.2 (10-23) 15.1 (7-20)
15 12.9 (11-15) 17.8 (11-23) 11.5 (10-18) 16 1 (11-17) 13 7 (10-20)
15.1 (12-40) 15.5 (11-21) 17 4 (10-25) -
23.1 (17-107) 27.2 (21-34) 19 (18-25) 21.8
109 (46-147) 80 (32-93) 119 (49-211) 34
15 5 22.9 48.3 (10-23) (14-54) (40-77) © Oxford University Press
PP14 in human IVF early pregnancies Normal pregnancy
Spontaneous abortion
Ectoplc pregnancy 300
T
1-300
2 0 2 4 6 • 10 I2 14 1• Day rvlatlva t * aa«yta ratrhaval
- 2 0 Day
Biochemical pregnancy
2
4
ralallva
6 to
8
10121416
ao«y1a
ralrlvval
Non-pregnant
300
2 Day
0
2
4
relative
• to
8
101214
ata-ayi*
r*tri*val
Fig. 1. Median placental protein 14 (PP14) and human chononic gonadotrophin (HCG) levels in serum in different categories of women. Note the similarity of patterns prior to day 8, corresponding to the peri-implantation period. Thereafter, differentiation was detected between the various categories of women. Day 0 = day of ovum retrieval; day 2 = day of embryo transfer.
For statistical evaluation of the data median levels were calculated. The Kruskal—Wallis non-parametric test was used for comparisons between the groups. P-values of
