Maternal serum ß-HCG and {alpha}-fetoprotein concentrations in singleton pregnancies following assisted reproduction

A. Perheentupa1,4, A. Ruokonen2, L. Tuomivaara3, M. Ryynänen1 and H. Martikainen1

1 Department of Obstetrics and Gynecology, 2 Department of Clinical Chemistry, University of Oulu and 3 The Family Federation of Finland, Oulu, Finland


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The reason for the elevated levels of HCG in assisted reproduction pregnancies remains unknown. Our hypothesis was that this increase is caused by the ovarian superovulation therapy. METHODS: We compared the ß-HCG and {alpha}-fetoprotein (AFP) multiples of the median (MoM) in singleton pregnancies after IVF or ICSI with those achieved by frozen embryo transfer (FET) in spontaneous cycles. RESULTS: The HCG and AFP MoMs (± SEMs) of 59 FET pregnancies were compared with 144 IVF (including 48 ICSI) pregnancies. The maternal HCG of pregnancies following ovarian stimulation was 1.31 ± 0.08 MoM compared with 1.35 ± 0.12 MoM in the unstimulated ones. The values for AFP were 1.06 ± 0.05 versus 1.11 ± 0.05 respectively. No significant differences could be observed between pregnancies following stimulated IVF/ICSI and unstimulated FET cycles. CONCLUSIONS: Our results show that second trimester maternal serum HCG is also elevated in singleton pregnancies following spontaneous FET cycles. The increased maternal serum HCG in IVF pregnancies is thus not related to superovulation therapy. Because of the elevated maternal serum HCG levels, serum screening cannot be performed reliably in pregnancies following assisted reproduction technology. Ultrasonographic detection of the nuchal translucency is unaffected and should be used for this group of women undergoing assisted reproduction.

Key words: Down's syndrome/frozen embryo transfer/human chorionic gonadotrophin/ICSI/IVF


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of maternal serum HCG and {alpha}-fetoprotein (AFP) concentrations for screening fetal Down's syndrome has been soundly validated (Haddow et al., 1992Go; Phillips et al., 1992Go; Wald et al., 1992Go; Goodbum et al., 1994Go). Several conditions such as diabetes mellitus, multiple gestation and ethnic subgroups affect these markers, and adjustment criteria have therefore been introduced to improve the accuracy of the screening (Wald et al., 1997Go). Previous data suggest that maternal serum levels of HCG are elevated following IVF (Ribbert et al., 1996Go; Frishman et al., 1997Go). This leads to a higher than expected screen-positive rate for Down's syndrome. Although there are several reports on altered maternal serum HCG concentrations, no substantiated explanation has been given for this finding. IVF pregnancies are established shortly after ovarian hyperstimulation treatment. It would seem possible that the manipulation of the pituitary–ovarian axis by GnRH analogues and gonadotrophins could result in increased HCG concentrations in the pregnancy. Embryos fertilized identically are routinely frozen and transferred into the uterus during a natural cycle (frozen embryo transfer; FET). Comparing the maternal serum HCG concentrations in IVF/ICSI and FET pregnancies makes it possible to study whether ovarian stimulation results in elevated HCG levels.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
We collected all the available Down's syndrome screening data from women who achieved their pregnancies following IVF, ICSI or FET at a tertiary IVF referral centre (University Hospital of Oulu, Oulu, Finland) and a private institution (The Family Federation of Finland, Oulu Department, Oulu) during 1996–1999. Values for maternal serum HCG and AFP multiples of the median (MoM) were available from 218 women. These included 13 twin pregnancies, one spontaneous abortion and one induced abortion. Once these were excluded from the study, 59 FET, 48 ICSI and 96 IVF pregnancies were used for the statistical analysis.

Ovarian stimulation
Down-regulation with a GnRH agonist was started in the luteal phase of the preceding cycle. This was followed by gonadotrophin stimulation of the ovaries. Ovarian response was monitored with transvaginal ultrasound and the daily dose of gonadotrophin was adjusted accordingly. Ovulation was induced with 5000–10 000 IU HCG when at least two follicles measuring >18 mm in diameter were observed. Oocyte retrieval by transvaginal ultrasound-guided puncture was performed 36 h post-HCG administration.

IVF, ICSI and FET
The semen and oocyte preparation, as well as ICSI and embryology techniques, have been described in detail previously (Tomas et al., 1998Go). In brief, for ICSI, the selected spermatozoon was immobilized and captured using a microinjection pipette (Swemed Lab International AB, Vastra Frolunda, Sweden). The spermatozoon was injected into an oocyte, which was held against a holding pipette (Swemed) by suction. Only mature oocytes which appeared to be morphologically normal and with a visible polar body were injected. After the injection procedure, the oocytes were rinsed and cultured overnight. The next morning, the medium was changed and the oocytes were checked for fertilization. Normally fertilized (two pronuclei) oocytes were cultured for a further 24 h and checked for cleavage. Embryo(s) were transferred into the uterine cavity 48 h after oocyte retrieval. Supernumerary embryos were frozen using a slow freezing protocol with 1,2-propanediol as the cryoprotectant (Testart et al., 1986Go).

Luteal phase support
Luteal support was given either orally or intravaginally using 600 (IVF/ICSI) or 400 mg/day (FET) natural micronized progesterone in three divided doses (Lugesteron; Leiras, Turku, Finland) for 2 weeks.

Screening for Down's syndrome
Blood for a serum sample was drawn from each pregnant woman at 14–17 weeks of pregnancy. Following assisted reproduction techniques (ART), the duration of the pregnancy was calculated from the fertilization of the ovum. No re-evaluation of the length of the pregnancy was carried out by ultrasonography. However, ultrasonography was used to screen for multiple gestations. Serum AFP and free ß-HCG were measured simultaneously by a dual-labelled time-resolved immunofluorometric assay (hAFP/free HCG ß Dual kit; Wallac, Turku, Finland). Median values for AFP and HCG have been calculated for each completed gestational week. In order to assure the comparability of the results, the values have been adjusted according to the weight of the mother. The reference values are expressed in multiples of medians (MoM), which were recalculated every 6 months.

Statistical analysis
Student's t-test was used to compare the HCG and AFP MoMs in stimulated and unstimulated cycles. Analysis of variance was used to compare levels between IVF, ICSI and FET groups. P < 0.05 was considered significant.


    Results
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 Materials and methods
 Results
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The mean (± SEM) age of the women was 31.4 ± 0.3 years. A mean of 2.0 ± 0.03 embryos were transferred. The length of pregnancy averaged 38.9 ± 0.2 weeks and weight of the baby at birth 3336 ± 46 g. All the groups studied were similar in these respects (Table IGo). However, the women in the stimulated group had a slightly higher body mass index than the ones in the unstimulated group (23.5 ± 0.3 versus 21.9 ± 0.5; P < 0.05).


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Table I. Maternal age, body mass index (BMI), number of embryos transferred, birthweight of the baby and gestational age in stimulated and unstimulated ART cycles. Values are means ± SEMs
 
Maternal serum HCG and AFP levels are given in Table IIGo. When all the ART serum screen levels were combined, HCG levels were elevated (1.32 ± 0.07 MoM) and AFP levels unaltered (1.07 ± 0.04). No significant difference was observed in the serum AFP or HCG levels between pregnancies following ovarian stimulation and FET. When IVF and ICSI groups were compared separately, AFP was significantly elevated following ICSI (1.22 ± 0.11 versus 0.98 ± 0.04; P < 0.05).


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Table II. Maternal serum HCG and {alpha}-fetoprotein (AFP) in stimulated and unstimulated ART cycles. Values are multiples of the medians (MoMs) ± SEMs
 

    Discussion
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 Materials and methods
 Results
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Our findings in the screening values of IVF pregnancies agree with the previous data from the Finnish population where maternal serum HCG concentrations were elevated significantly (1.52 MoM) while no change was observed in the AFP concentration (Heinonen et al., 1996Go). Several, but not all (Barkai et al., 1996Go), papers have described elevated maternal serum HCG levels during the second trimester in IVF pregnancies. The data on maternal serum AFP are less uniform and the possible alteration seems to be less in magnitude (Barkai et al., 1996Go; Heinonen et al., 1996Go; Ribbert et al., 1996Go; Frishman et al., 1997Go). However, the reasons for these findings have remained unresolved. These aberrant screening values may increase the number of invasive procedures performed to exclude Down's syndrome.

In this study, we showed for the first time that maternal serum HCG levels are increased also in pregnancies following FET in spontaneous cycles. Our data show that the maternal serum HCG levels are similar in pregnancies following stimulated (IVF/ICSI) and unstimulated (FET) cycles. Therefore, it can be concluded that the superovulation therapy is unlikely to be the cause of the elevated maternal serum HCG observed during the second trimester.

Several other theories have been proposed to explain the increased serum HCG levels. Theoretically, embryos that initially implant into the endometrium but abort, could increase second trimester maternal HCG levels. However, several findings suggest the unlikelihood of this theory. The implantation potential of frozen–thawed embryos is lower than that of fresh ones, although there is no difference in the HCG levels of these groups. In our study, the number of embryos transferred did not vary between groups. In twin pregnancies, both HCG and AFP levels are twice those found in singleton pregnancies (Nebiolo et al., 1991Go), although in our study the AFP levels were unaltered (and lowered in some studied). In IVF twin pregnancies, second trimester maternal serum HCG levels were slightly higher than those in spontaneous pregnancies, and no significant elevation was observed in AFP levels (Räty et al., 2000Go). Furthermore, in multifetal pregnancies the reduction to twins during the first trimester resulted in clearly elevated AFP levels, while HCG levels were only modestly elevated in the second trimester (Rotmensch et al., 1999Go). As the transfer of a single embryo is gaining popularity, the relevance of early aborting embryos in elevated HCG levels can be studied in detail.

All groups in this study received luteal support. It is therefore impossible to evaluate the possible effect of progesterone on the second trimester levels of maternal serum HCG. This aspect remains unresolved and needs to be addressed in the future.

Untraceable metabolic disturbances responsible for the infertility could cause altered HCG (and AFP) levels during pregnancy. This theory is supported by Holding and Cuckle, who have shown that a screen-positive result is often repeated in the subsequent pregnancy (Holding and Cuckle 1994Go).

Some evidence exists for late but successful implantation following IVF treatment (Ertzeid et al., 2000Go). Since the serum HCG in our study was measured during the decreasing phase of the HCG curve, this possible difference in the length of the pregnancy could result in a difference in serum HCG similar to the one seen in our study. The length of the IVF/ICSI pregnancy is calculated from the fertilization date and confirmed in most cases by ultrasonography on week 6–7. However, the length of gestation in spontaneous pregnancies is based on the last menstrual period and the length of gestation may quite often be shorter than expected. This explanation for elevated HCG levels in ART pregnancies seems very unlikely.

Several authors have reported compromised pregnancy outcome associated with elevated maternal serum HCG levels in second trimester screening (Lieppman et al., 1993Go; Jauniaux et al., 1996Go; van Rijn et al., 1999Go). This may reflect suboptimal placentation (Wenstrom et al., 1994Go). Furthermore, increased incidence of vaginal bleeding in all stages of IVF/ICSI pregnancies has been recently observed (S.Koivurova, A.L.Hartikainen and L.Karinen, unpublished data). An association between a high level of maternal serum HCG at 15–18 weeks, the presence of an early diastolic notch in the uterine artery flow velocity waveform and adverse pregnancy outcome further support the explanation of poor uteroplacental circulation (Palacio et al., 1999Go). Following intrauterine insemination, maternal serum midtrimester free ß-HCG levels were not elevated in a Taiwanese population (Hsu et al., 1999Go). This may indicate that the elevated HCG levels in pregnancies following IVF/ICSI and FET may be related to artificial fertilization or the embryo culture.

In a small Asian patient population, reduced maternal serum AFP levels have been reported following ICSI (0.76 MoM), while HCG levels were slightly but not significantly reduced to 0.88 MoM (Lam et al., 1999Go). In contrast, in our Finnish population in the pregnancies following ICSI, both HCG and AFP were elevated (1.53 ± 0.21 and 1.22 ± 0.11 MoM respectively). The AFP levels in ICSI cycles were significantly higher compared with those measured in IVF pregnancies. The reason for this discrepancy remains unclear and studies on larger populations are needed.

In conclusion, maternal serum HCG during the second trimester is elevated in pregnancies following ART. Superovulation stimulation is unlikely to cause this alteration. Whether maternal characteristics and/or suboptimal placentation play a part in this phenomenon needs further elucidation. Investigation of this phenomenon is of importance in order to avoid unnecessary amniocentesis. The explanation for elevated HCG levels may shed light on the core problem of ART, i.e. poor implantation. For the time being, Down's syndrome screening of pregnancies following IVF/ICSI and FET is most efficiently carried out by nuchal translucency measurement.


    Notes
 
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, P.O.Box 5000, SF-90014, University of Oulu, Finland. E-mail: antper{at}utu.fi Back

Submitted on January 31, 2001


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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accepted on October 15, 2001.