1 Department of Obstetrics and Gynecology and 2 Department of Clinical Chemistry, Rikshospitalet,0860 Oslo, Norway
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Abstract |
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Key words: : clinical pregnancy/human chorionic gonadotrophin/in-vitro fertilization
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Introduction |
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Ultrasound (US) examination is effective for evaluation of ongoing pregnancies, but a gestational sac is not reliably visible until 3337 days after the luteinizing hormone (LH) surge (Shapiro et al ., 1992 ). As a result of this inability of US to identify very early pregnancy abnormalities, there is an ongoing effort to institute a method that can forecast pregnancy outcome. Several studies have investigated hormones like progesterone, oestradiol, relaxin, pregnancy-specific ß1-glycoprotein, inhibin, and CA-125 in predicting prognosis (Witt et al ., 1990
; Hahlin et al ., 1991
). The exact role of these markers and their clinical utility, however, has not yet been established. Researchers have reported the usefulness of early serum human chorionic gonadotrophin (HCG) (Confino et al ., 1986
; Dor et al ., 1988
; Heiner et al ., 1992
; Fridstrøm et al ., 1995
; Glatstein et al ., 1995
). One of these studies has reported clinically discriminatory HCG values on different days post embryo transfer (Glatstein et al ., 1995
), but none of them as early as on day 12.
The aim of this study was to investigate whether a single HCG value on day 12 after embryo transfer could be used as a marker to predict the outcome of a pregnancy after IVF.
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Materials and methods |
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In-vitro fertilization protocol
The patients underwent IVF and embryo transfer according to described protocols (Åbyholm et al ., 1990 ; Tanbo et al ., 1995
). Pituitary down regulation with a gonadotrophin releasing hormone agonist (GnRHa) (Suprefact ® ; Hoechest, Frankfurt am Main, Germany ) was used in all patients. Ovarian hyperstimulation was performed using human menopausal gonadotrophin (HMG) (Pergonal ® ; Serono, Aubonne, Switzerland ) and follicle stimulating hormone (FSH) (Fertinorm ® and Fertinorm HP ® ; Serono ). Transvaginal ultrasound (US) and serum oestradiol concentrations were used to monitor the IVF cycle. When three or more of the follicles had a diameter exceeding 18 mm, 10 000 IU units of HCG (Profasi ® ; Serono ) were administered s.c. to induce ovulation. Oocyte aspiration was performed approximately 3436 h after HCG was given using a standard transvaginal US-guided approach. Embryo transfer was performed 2 days after oocyte aspiration and a maximum of two embryos was transferred. All patients included in this series received progesterone support for 4 weeks, either as vaginal suppositories (600 mg daily) (Progestan ® ; Organon, Oss, The Netherlands ) or i.m. injections (25 mg daily) starting on the day prior to oocyte aspiration.
Serum HCG assay
Samples of venous blood were collected routinely on the 12th day after embryo transfer. Serum HCG concentrations were measured by microparticle enzyme immunoassay for the intact HCG molecule (Imx ® ; Abbot Laboratories, Abbot Park, IL, USA ) at the Central Chemical Laboratory of our hospital. The assay was calibrated using the World Health Organization (WHO) Third International Standard (75/537) (formerly designated the WHO First International Reference Preparation). The intra-assay coefficient of variation (CV) was 4.1% for a mean HCG of 23 IU/l; 3.9% for a HCG mean of 143 IU/l, and 3.4% for a mean HCG of 707 IU/l. The inter-assay CV was 4.6%, 6.1% and 9.6% respectively, and the sensitivity was 2 IU/l. Serum samples that were not obtained by day 12 after embryo transfer were discarded. As the HCG analyses were only performed on Monday, Wednesday and Friday, and not at weekends, some of the samples were stored for 1 to 2 days before being tested. If stored for more than 24 h, the samples were frozen at 24°C. Otherwise they were maintained at 4°C.
Pregnancy outcome
Pregnancy outcomes were divided into two groups: early pregnancy losses and vital pregnancies. Early losses included pregnancies classified as chemical (subclinical), ectopic gestations and first trimester spontaneous abortions. Vital pregnancies were defined as delivered singletons, multiple pregnancies or second trimester abortions occurring with a documented fetal heart, before the abortion. Our series contained five patients who experienced ectopic pregnancy. No heterotopic pregnancies were detected among our patients during the study period. The same study was performed with ectopic pregnancies excluded from the failures, and only singleton pregnancies were included in the successful group.
Statistical analysis
The MannWhitney U test was used to compare the differences between the groups. A receiver operating characteristic (ROC) curve analysis (McNeil et al ., 1975 ; Hanley and McNeil, 1982
) was used to estimate the predictive power of the measured variables (Swets, 1979
), and to determine the HCG cut-off that optimally discriminated early pregnancy losses from vital pregnancy cycles. ROC curve was obtained by plotting the sensitivity of the HCG value versus 1-specificity for a series of multiple cut-off points on the 12th post embryo transfer day.
The cut-off point was chosen to maximize sensitivity as well as specificity. As a measure for the diagnostic ability of the cut-off to predict pregnancy outcome, the positive and negative predictive values were determined at the calculated cut-off value. As described (Glatstein et al ., 1995 ) the following definitions were used: sensitivity: the probability that a patient with an early pregnancy loss will have an HCG value less than the cut-off value, the true positive rate; specificity: the probability that a patient with a vital pregnancy will have HCG value greater than the cut-off value; positive predictive value: the probability that a patient with a HCG value less than the cut-off will have an early pregnancy loss; and negative predictive value: the probability that a patient with an HCG value greater than the cut-off will have a vital pregnancy. Then positive and negative post test probability were calculated and found to coincide with the positive and negative predictive value respectively. Unless otherwise stated, values are expressed as means ± SE of the mean with a probability value of P < 0.05.
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Results |
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Figure 3 illustrates the relative distribution of early pregnancy losses and vital pregnancies from the selected cut-off point of 55 IU/l to 100 IU/l. At the horizontal bar representing the chosen cut-off value at 55 IU/l 60% will have early pregnancy losses while 40% will have vital pregnancies. With increasing HCG cut-off values the figure shows that the relative distribution of vital pregnancies increases.
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Discussion |
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The present study shows that, by means of a single serum HCG determination on day 12 after embryo transfer, a group of patients can be identified that is at increased risk of carrying an abnormal pregnancy. At the cut-off value of 55 IU/l patients testing positively for HCG had a 61% risk of carrying an ectopic or non-viable intrauterine pregnancy. Therefore close monitoring of the course of the pregnancy is called for. Among those with a HCG value higher than 55 IU/l there was a 90% probability of a vital pregnancy. In total 26% of the patients in this study experienced early pregnancy loss while 74% had vital pregnancies. The latter group comprised one second trimester loss (16th week). Second trimester abortions are in this study regarded as successful implantation. As explained (Glatstein et al ., 1995 ) early HCG concentrations can predict implantation outcome, but it cannot invariably predict pregnancy outcome. Therefore, it would be unlikely that a single early HCG value could reliably predict the occurrence of a live birth, an event influenced by multiple factors other than implantation quality like a second or third trimester abortion due to an incompetent cervix.
The calculated cut-off reported in this study was chosen to best discriminate between early pregnancy losses and vital pregnancies. There is a limit to what can be achieved with biochemical diagnosis in early pregnancy, since pathological pregnancies often have a normal growth rate early in their course (e.g. blighted ovum, initially beating heart). The earlier the test is performed, the higher is the rate of false negative tests. By definition, there is a trade off between sensitivity and specificity. If one wishes to increase the HCG cut-off value on day 12 after embryo transfer from 55 IU/l to 100 IU/l there is a decline in positive predictive value (true spontaneous abortion) from 60% to 32% (Figure 3 ). At the same time the sensitivity increases from 73% to 90%, while the specificity falls from 83% to 35%. Among the 417 patients studied, there were only five (1.2%) who experienced ectopic pregnancy. Since there were very few ectopic pregnancies in the study, the main aim was to find a cut-off value that could discriminate between ordinary spontaneous and successful pregnancies. It is doubtful if any higher sensitivity (73%) than the one estimated at the cut-off value of 55 IU/l would be more suitable to detect ectopic pregnancies with enough certainty to be diagnostic. Mol et al . (1997) have reported on optimal HCG cut-off values for ectopic pregnancies in a group of 86 women where 24 (28%) experienced ectopic pregnancies (Mol et al ., 1997
). Their measurements were done on days 6, 9 and 15. The authors concluded that serum HCG measurement 9 days after embryo transfer could identify pregnancy failures with a 100% specificity at a cut-off value of 18 IU/l, but it could not identify patients with ectopic pregnancies with enough certainty to justify immediate treatment. Another recent study reported no difference in the measurable isoforms of HCG between normal intrauterine pregnancy and ectopic pregnancies (Mock et al ., 1998
). Abdominal pain or bleeding is usually an additional sign necessary to raise suspicion of ectopic pregnancy. Under these circumstances transvaginal sonography is of great diagnostic aid (Mol et al ., 1997
).
If one was to select one group for closer follow up on basis of the HCG measured on day 12 after embryo transfer it ought to be the patients with values equal to or below 55 IU/l. The lower the HCG value (lower sensitivity) the higher is the likelihood of non-viable pregnancy (specificity).
To set a higher cut-off point in order to increase the sensitivity does not seem reasonable in as much as this would decrease the specificity considerably and not contribute to a better discrimination between vital and the potentially life-threatening ectopic pregnancies.
The HCG concentrations were significantly higher on day 12 after embryo transfer for vital pregnancy to early pregnancy losses. This confirms previous studies (Heiner et al ., 1992 ; Keith et al ., 1993
; Fridstrøm et al ., 1995
; Glatstein et al ., 1995
). By choosing day 12 as the test day, unnecessary delay of pregnancy diagnosis could be avoided, which is crucial both from a psychological and a medical point of view. Relief of tension might not only be important for the patient's well-being (Friedman, 1989
), but also for the outcome of pregnancy (Stray-Pedersen and Stray-Pedersen, 1984
). For patients using progesterone as luteal support, an early and reliable test for pregnancy is desirable in order to terminate luteal support in patients not achieving pregnancy.
It was shown in this series that multiple pregnancies had significantly higher HCG values compared with singletons on day 12 after embryo transfer. This finding is in agreement with previous reports (Heiner et al ., 1992 ; Fridstrøm et al ., 1995
; Glatstein et al ., 1995
) but ultrasound confirmation of the number of fetuses and their viability still should be used to make clinical recommendations to patients in any case of multiple pregnancy.
Further it was calculated that this series of singletons had significantly higher HCG on day 12 after embryo transfer than spontaneous abortions. Only very small changes in predictive values were observed after excluding multiple pregnancies from the group of vital pregnancies.
As explained (Fridstrøm et al ., 1995 ), several studies have shown that serial HCG determinations for differential diagnosis of early pregnancy are superior to single measurements with regard to diagnostic power (Marshall et al .,1968; Braunstein et al ., 1978
; Kadar et al , 1981
; Ankum et al ., 1993
). However, these studies concern patients presenting with actual suspicion of pathological pregnancy, in which active diagnostic measures are warranted. In women who experience recurrent abortions the most useful measurements in predicting the outcome in early pregnancy was recently reported to be ßHCG and gestation-sac diameter (Li et al ., 1998
). In contrast, more than 75% of the pregnant cycles in the present study led to successful implantation. It therefore seems reasonable to spare these patients the inconvenience of repeated blood sampling at short intervals.
As pointed out by Glatstein et al. (1995), ideally, each institution should analyse their own data to determine HCG cut-offs based on their own experience. Although quality control methods such as HCG reference preparations are standardized between clinical laboratories, minor differences in protocol may influence values. Glatstein et al . suggest a prospective study in multiple centres to confirm the validity of the cut-off values.
In summary, it can be concluded that HCG cut-off values determined by a ROC curve analysis is useful for discriminating between implantation successes and early pregnancy losses on day 12 after embryo transfer. The cut-off value might aid in the prognosis, clinical management and counselling of the patients. The lower the HCG values found, the greater is the need for repeated measurement and a closer follow up of the patients. Pregnant patients with HCG values greater than the cut-off value are followed with transvaginal US at 45 weeks post embryo transfer. Improved techniques for identification of pathological pregnancies after IVF will be of benefit for the patients and might also have cost saving consequences.
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Notes |
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References |
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Submitted on November 27, 1998; accepted on March 10, 1999.