1 Department of Obstetrics and Gynecology, 2 Department of Clinical Chemistry, University of Oulu and 3 The Family Federation of Finland, Oulu, Finland
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Abstract |
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Key words: Down's syndrome/frozen embryo transfer/human chorionic gonadotrophin/ICSI/IVF
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Introduction |
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Materials and methods |
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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 500010 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., 1998). 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., 1986
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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 1417 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.
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Results |
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Discussion |
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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 frozenthawed 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., 1991), 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., 2000
). 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., 1999
). 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 1994).
Some evidence exists for late but successful implantation following IVF treatment (Ertzeid et al., 2000). 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 67. 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., 1993; Jauniaux et al., 1996
; van Rijn et al., 1999
). This may reflect suboptimal placentation (Wenstrom et al., 1994
). 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 1518 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., 1999
). Following intrauterine insemination, maternal serum midtrimester free ß-HCG levels were not elevated in a Taiwanese population (Hsu et al., 1999
). 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., 1999). 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.
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Notes |
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References |
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accepted on October 15, 2001.