Maternal serum hCG and alpha-fetoprotein levels in pregnancies conceived after IVF or ICSI with fresh and frozen–thawed embryos

P.W. Hui1,5, M.H.Y. Tang2, Y.H. Lam3, E.H.Y. Ng4, W.S.B. Yeung4 and P.C. Ho4

1 Department of Obstetrics and Gynaecology, Queen Mary Hospital, Hong Kong, 2 Prenatal Diagnostic and Counselling Department, Tsan Yuk Hospital, Hong Kong, 3 Hong Kong Prenatal Diagnosis Center, Hong Kong and 4 Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, China

5 To whom correspondence should be addressed at: 6/F, Professorial Block, Department of Obstetrics and Gynaecology, Queen Mary Hospital, 102 Pokfulam Road, Pokfulam, Hong Kong, China. e-mail: pwhui{at}hkusua.hku.hk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Studies have shown that levels of serum markers of Down’s syndrome were altered in pregnancies conceived after IVF, though the reason for this remains unknown. METHODS: Second-trimester maternal serum levels of hCG and alpha-fetoprotein (AFP) in pregnancies conceived with fresh and frozen–thawed embryos after assisted reproduction were compared with those conceived spontaneously. RESULTS: There were 203 pregnancies with fresh embryo transfers (130 IVF cases, 73 ICSI cases) and 98 pregnancies with frozen–thawed embryo transfers (61 IVF cases, 37 ICSI cases). The controls consisted of 17 145 spontaneous pregnancies. The median hCG multiples of the median (MoM) was significantly increased to 1.24 in 98 pregnancies conceived after frozen embryo transfer. This elevation was observed only in the IVF-frozen embryo transfer subgroup (P < 0.001), but not in the ICSI-frozen embryo transfer subgroup. The median AFP MoM for 203 pregnancies after fresh embryo transfer was 0.90. Among the subgroups, the median AFP MoM was significantly reduced to 0.90 and 0.86 in IVF-embryo transfer (P = 0.04) and ICSI-embryo transfer (P = 0.001) pregnancies respectively, and significantly raised to 1.20 in the IVF-frozen embryo transfer subgroup. CONCLUSIONS: The degree of alterations in maternal serum hCG and AFP levels varied between fresh and frozen–thawed embryos, and also between the mode of fertilization. Pregnancies resulting from ICSI or frozen embryo transfer should be regarded as distinct entities from those of IVF-embryo transfer.

Key words: alpha-fetoprotein/frozen–thawed embryo transfer/hCG/ICSI/IVF


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Maternal serum screening for Down’s syndrome is a well-established practice in many countries. Serum screening is commonly performed by combining maternal age with measurements of maternal serum hCG, alpha-fetoprotein (AFP) and unconjugated estriol (uE3) in the second trimester of pregnancy (Cuckle, 1992Go; Palomaki et al., 1993Go; Hsu et al., 1996Go; Onda et al., 1996Go; Lam et al., 1998Go). Previous studies have suggested that maternal serum hCG levels were elevated in pregnancies conceived after IVF, while AFP and uE3 levels were reduced (Barkai et al., 1996Go; Heinonen et al., 1996Go; Ribbert et al., 1996Go; Frishman et al., 1997Go; Lam et al., 1999Go). These alterations in the marker concentrations in IVF pregnancies led to a higher than expected screen-positive rate (Heinonen et al., 1996Go; Ribbert et al., 1996Go; Frishman et al., 1997Go; Lam et al., 1999Go; Wald et al., 1999Go). Adjustment of the marker levels is necessary to reduce unnecessary anxiety and amniocentesis. The pathogenesis of altered hCG and AFP levels in IVF pregnancies is unknown, but a suggestion has been made that this might be the result of the ovarian drive following controlled ovarian stimulation (Wald et al., 1999Go). However, recent findings showed a similar rise in hCG level in pregnancies conceived after transfer of frozen–thawed embryos; that is, without prior ovarian stimulation (Perheentupa et al., 2002Go; Raty et al., 2002Go). Pregnancies conceived after transfer of embryos resulting from ICSI appear to behave differently from those conceived after conventional IVF. It has been reported previously that maternal serum hCG levels were reduced rather than increased in pregnancies conceived after the transfer of fresh embryos from ICSI (Lam et al., 1999Go). However no data have been reported regarding analyte levels in pregnancies conceived after transfer of frozen–thawed embryos resulting from ICSI. In the present study, analyte levels in pregnancies conceived after the transfer of frozen–thawed embryos resulting from both conventional IVF and ICSI were reported.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
Chinese women with singleton pregnancies conceived after IVF or ICSI with either fresh or frozen–thawed embryos were recruited. An informed consent was obtained. The procedures of IVF, ICSI and frozen–thawed embryo transfers have been described previously (Ng et al., 2000Go). Maternal blood samples were taken between 15 and 20 weeks to measure levels of hCG and AFP. Women aged >=35 years at the expected date of confinement, or who had other risk factors for aneuploidies were offered the option of amniocentesis or chorionic villus sampling. The controls were selected from women with spontaneous conception who underwent serum screening for Down’s syndrome over the same time period. Only pregnancies with a known normal outcome were included in the final analysis.

Second-trimester serum assays
The total hCG and AFP serum levels were measured using a micro-particle enzyme immunoassay with the Abbott AXSYM autoanalyser (Abbott Laboratories Ltd, North Point, Hong Kong). Concentrations of the analytes were converted to multiples of the median (MoMs) of the gestational week and adjusted for maternal weight as described previously (Lam et al., 1999Go). Comparisons were made among pregnancies conceived after fresh embryo transfers, frozen embryo transfer and those conceived spontaneously. A further analysis was performed on the subgroups of pregnancies with conventional insemination (IVF) and ICSI. Not all of the serum marker MoMs fitted a parametric distribution; hence, a Wilcoxon rank-sum test was used to compare values between groups. A P-value < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS version 10.0 computer software.


    Results
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 Materials and methods
 Results
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Between May 1996 and May 2002, a total of 301 pregnancies after conventional IVF or ICSI was recruited into the study (Table I). These included 203 pregnancies with fresh embryo transfer (130 IVF cases, 73 ICSI cases) and 98 pregnancies with frozen embryo transfer (61 IVF cases, 37 ICSI cases). Spontaneous pregnancies (n = 17 145) with a known normal outcome were selected as controls. The mean maternal age of the controls was 30.5 years. Women in the assisted reproduction groups were significantly older than the controls (t-test, P < 0.05). The majority of the conventional IVF was performed for tuboperitoneal factor, followed by male factor. ICSI was mainly indicated in subfertility related to severe male problem. In the IVF-embryo transfer subgroup, there was no statistically significant difference in the marker levels among different causes of subfertility.


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Table I. Maternal age on the expected date of confinement, gestational age on the date of blood sampling and cause of subfertility of the studied population
 
The maternal serum hCG and AFP MoMs of controls (spontaneous pregnancies) and pregnancies following fresh embryo transfer and frozen embryo transfer are listed in Table II. For the controls, the median maternal serum hCG and AFP MoM were each 1.00. In pregnancies conceived after frozen embryo transfer, the median hCG MoM was significantly increased to 1.24 (P < 0.001) compared with 1.01 in those women conceived after fresh embryo transfer and 1.00 in those conceived spontaneously. The median AFP MoM for pregnancies conceived after transfer of fresh embryos was 0.90 and this was significantly lower (P < 0.001) than those conceived spontaneously and after transfer of a frozen–thawed embryo. The median AFP MoM for frozen embryo transfer (1.10) was not different from that of the controls.


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Table II. Maternal serum hCG and AFP MoMs in women with different subgroups of assisted reproduction
 
A further analysis of the maternal serum hCG and AFP MoMs for the different subgroups of pregnancies was also carried out. The median maternal serum hCG MoMs were 1.38 and 1.08 for pregnancies with IVF-frozen embryo transfer and ICSI-frozen embryo transfer respectively. A statistically significant increase was observed only in the IVF-frozen embryo transfer subgroup (P < 0.001), but not in ICSI-frozen embryo transfer subgroup. This marked increase in median hCG MoM in IVF-frozen embryo transfer pregnancies was also significant when compared with IVF-embryo transfer and ICSI-frozen embryo transfer pregnancies.

For median maternal serum AFP MoM, the results were consistently low among the subgroups of fresh embryo transfers. Values were significantly reduced to 0.90 MoM and 0.86 MoM in IVF-embryo transfer (P = 0.04) and ICSI-embryo transfer (P = 0.001) pregnancies respectively. However, the AFP MoM for IVF-frozen embryo transfer pregnancies was 1.20 and was significantly higher (P = 0.01) when compared with that in the controls and with the IVF-embryo transfer and ICSI-frozen embryo transfer subgroups.

The median hCG and AFP MoMs of the ICSI pregnancies were reduced when compared with the conventional IVF pregnancies in both fresh and frozen–thawed embryo transfer cycles.


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 Materials and methods
 Results
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Several reports have described an elevated second-trimester maternal serum hCG level in pregnancies after assisted reproduction (Barkai et al., 1996Go; Ribbert et al., 1996Go; Wald et al., 1999Go), yet the exact mechanism involved remains controversial. It has been postulated to be due to the greater number of corpora lutea produced after controlled ovarian stimulation or to multiple early implantation sites not detected by ultrasound (Frishman et al., 1997Go; Wald et al., 1999Go). However, recent studies identified similar changes in second-trimester hCG and AFP levels in oocyte-donation pregnancies (Maymon and Shulman, 2001Go). The present study showed a significantly higher second-trimester maternal serum total hCG level in pregnancies conceived after frozen embryo transfer. A similar degree of elevation in free {beta}-hCG level in pregnancies conceived with frozen embryo transfer was also observed in two studies conducted in Finland (Perheentupa et al., 2002Go; Raty et al., 2002Go). In the frozen embryo transfer and oocyte-donation pregnancies there were no ovulation induction or controlled ovarian stimulation procedures to explain the elevation in hCG levels. In fact, this elevation in hCG might be related to an abnormal pathophysiology in the fetus or placenta after IVF. Since the serum hCG and AFP levels in IVF pregnancies were similar to those of natural pregnancies of a lesser maturity, the IVF process may have caused some form of delay in maturation of the fetus. Pregnancies from frozen embryo transfer differ from those achieved with fresh embryo transfer and oocyte donation with regard to the freezing and thawing procedures utilized. The marked increase in hCG level in pregnancies after frozen embryo transfer might result from an altered physiological process, and it is possible that this is related to the cryopreservation and thawing process.

Although the present study showed a significantly reduced level of maternal serum AFP in pregnancies after fresh embryo transfers, the data obtained on AFP level were more heterogeneous. The present findings were in agreement with observations made in a Caucasian population (Ribbert et al., 1996Go; Frishman et al., 1997Go), but differed from those found in other series (Barkai et al., 1996Go; Heinonen et al., 1996Go). To the present authors’ knowledge, there is no explanation to account for this alteration. The markedly reduced AFP level found in ICSI-embryo transfer pregnancy was also consistent with that of a previous report (Lam et al., 1999Go), but opposed the data obtained in the Finnish study (Perheentupa et al., 2002Go). The change in direction is towards an increased risk of Down’s syndrome, with a higher false-positive rate for second-trimester screening in IVF pregnancies being observed. In addition, a lower level of pregnancy-associated plasma protein-A was also found during the first trimester in these pregnancies (Maymon and Shulman, 2002Go). These findings suggested that there was an on-going yet persistent change in the physiology of the IVF pregnancies. Hence, an adjustment of the test results would be needed in order to reduce the unnecessary high false-positive rates for screening in both the first and second trimesters (Frishman et al., 1997Go; Lam et al., 1999Go; Maymon and Shulman, 2002Go).

In the present study, pregnancies conceived after frozen embryo transfer demonstrated elevated trends of hCG and AFP, and these results were similar to those reported in Finland (Perheentupa et al., 2002Go; Raty et al., 2002Go). However, when the maternal serum AFP of IVF-frozen embryo transfer and ICSI-frozen embryo transfer were analysed separately, it was found that the AFP MoM in the subgroup of IVF-frozen embryo transfer pregnancies was 20% higher than that of the controls, while the maternal serum AFP MoM of ICSI-frozen embryo transfer pregnancies was similar to that of the controls. The addition of freezing and thawing procedures in the embryos seems to result in elevation of both hCG and AFP levels as compared with fresh embryos. In contrast to the study in a Finnish population (Perheentupa et al., 2002Go), which reported higher hCG and AFP levels after ICSI, a trend towards a reduction in median hCG and AFP MoM was observed in pregnancies conceived after ICSI as compared with those after conventional IVF in the Chinese population. Maternal age is a potential confounding variable that may affect serum AFP concentration (Wald and Watt, 1996Go). In the present study population, a weak (but significant) negative correlation between maternal age and AFP MoM was found in the spontaneous pregnancies (Spearman’s {rho} = –0.025, P = 0.001), but not in assisted reproduction pregnancies. As the women undergoing frozen embryo transfer were older, maternal age could not account for the raised AFP levels in the frozen embryo transfer pregnancies.

To the best of the present authors’ knowledge, this is the first report on maternal serum hCG and AFP after ICSI-frozen embryo transfer, as in two previous investigations into frozen embryo transfer pregnancies (Perheentupa et al., 2002Go; Raty et al., 2002Go) the ICSI-frozen embryo transfer pregnancies were not analysed as a discrete subgroup. The median hCG and AFP MoM in ICSI-FET pregnancies were similar to those in controls; hence, the effects of freezing and thawing on hCG and AFP levels appear to be offset by the extra ICSI procedure. Although ICSI is mainly performed for subfertility related to severe male factor, the cause of infertility is unlikely to have a major impact on the serum marker levels. ICSI results in a tiny hole in the zona pellucida of the embryo, and this may have an adverse effect on the freezing and thawing process, as well as on any subsequent development after thawing.

Based on the results of the present study, it is possible that pregnancies conceived after assisted reproduction with a different mode of fertilization and embryo status behave differently from their spontaneous counterparts. Therefore, in future research and clinical management, pregnancies achieved after frozen embryo transfer and ICSI should be regarded as distinct entities from those achieved using fresh embryo transfer and IVF.


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 Materials and methods
 Results
 Discussion
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Submitted on September 27, 2002; accepted on December 13, 2002.