1 Department of Obstetrics and Gynecology, Assaf Harofe Medical Center, Zerifin and 2 Sheba Medical Center, Tel Hashomer and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
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Abstract |
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Key words: assisted reproduction/oocyte donation/triple test
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Introduction |
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It is well known that these serum analytes may be affected by various conditions, such as insulin-dependent diabetes mellitus (IDDM), multiple gestation, ethnic subgroups and wrong dates (Wald et al., 1997; Cuckle, 1998
). Therefore, adjustment criteria were introduced to correct both the FPR and the detection rate, thus providing a more accurate estimate of patient-specific calculated risk (Wald et al., 1997
; Cuckle, 1998
).
Over the past decade, both maternal age and the wide use of various assisted reproduction medications and technologies have risen dramatically (Salat-Baroux et al., 1988; Maymon et al., 1999a
), affecting mid-gestation serum markers in singletons (Barkai et al., 1996
; Heinonen et al., 1996
; Ribbert et al., 1996
; Frishman et al., 1997
; Hsu et al., 1999
; Wald et al., 1999
) as well as in twins (Maymon et al., 1999b
; Raty et al., 2000
).
The aim of the current study was to compare, for the first time, second trimester triple test serum screening in cases of oocyte donation (OD) with self oocyte IVF conceived singleton pregnancies. Both groups were followed from the time of their embryo transfer until delivery. The OD patients were used as a model because they lacked a corpus luteum and no induction of ovulation was carried out. Therefore, the second trimester serum markers in this group could be attributed almost entirely to the conceptus.
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Materials and methods |
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IVF protocol for donors and control cases
The procedure for assisted conception in our programme has been described previously (Shulman et al., 1999). Briefly, oocytes were obtained from patients undergoing IVF who agreed anonymously to donate their excess oocytes. Following initial pituitary suppression with a gonadotrophin-releasing hormone agonist, ovarian stimulation was achieved by the administration of human menopausal gonadotrophin (HMG) (Pergonal®; Teva Pharmaceutical Industries Ltd., Kfar Sava, Israel). According to sonographic criteria (leading follicle size >18 mm) and oestradiol concentration oocyte maturation was induced by the administration of human chorionic gonadotrophin (HCG) (10 000 IU) (Chorigon®; Teva Pharmaceutical Industries Ltd.). Under vaginal ultrasound-guided puncture oocytes were retrieved 36 h later. Embryo transfer was performed 4872 h later. Intravaginal natural micronized progesterone (Endometrin Slowris Co. Ltd., Gush Shegev, Israel) (200 mg divided two times a day) for luteal phase support was administered for 2 weeks, after which HCG concentrations were measured sequentially. Our policy is to perform transvaginal scan 4 weeks following embryo transfer. The scans were repeated weekly until fetal heart movement was detected. Supplemental vaginal natural micronized progesterone (Endometrin Slowris Co. Ltd) was administered until approximately 8 weeks from the date of embryo transfer and then tapered and discontinued within 1 week, as permitted according to the maintenance of serum concentrations.
Recipient protocol
During the transfer cycle, the women first received oestradiol valerate (Progyluton®; Schering AG, Berlin, Germany) in a fixed dose of 46 mg over a period varying between 12 and 30 days, depending on the individual patient. The daily dose was dictated by the endometrial width as measured by vaginal ultrasound, with the optimum measurement ranging between 812 mm. Intravaginal natural micronized progesterone (300 mg, divided, three times a day) was started once oocytes were available. Both the oestradiol valerate and natural micronized progesterone were continued for the next 57 weeks following embryo transfer.
All these women had a blood test for ß-HCG assessment between 12 and 14 days after embryo transfer and then a first-trimester scan. They later underwent second-trimester triple serum screening. The gestational age in the second trimester was calculated from the date of oocyte retrieval minus 2 weeks, or embryo transfer minus 16 days, to convert the menstrual dating. The serum samples of the women in both groups were tested in a routine analytical run together with regular maternal serum samples at the antenatal DS screening programme in Zer Medical Laboratories (ISO 9002 UK, and under the supervision of the Ministry of Health, Israel). Testing was carried out in a fashion blind to group classification. The measured serum concentrations of AFP, uE3 and HCG were expressed as multiples of the (unaffected) median (MoM) for each gestational week as established in Zer Medical Laboratories during the same period of time.
The threshold value for recommending fetal karyotyping was 1:380. This is the equivalent risk of a term pregnancy 35 year old gravida, and the common practice in our national health care system.
The patient-specific risk estimation was derived from the combination of triple serum markers and maternal age calculated by a commercially available software program. For the OD the donors age was used to adjust for the DS risk.
Serum ß-HCG concentrations were assessed by enzyme immunoassay using Abbott IMX total ß-HCG assay distributed by Abbott GmbH Diagnostica, Wiesbaden Delkenheim, Germany. The IMX total ß-HCG assay is a microparticle enzyme immunoassay for the quantitative determination of mIU/l ß-HCG in human serum or plasma.
Assays for AFP, HCG and uE3 were performed as follows: intact HCG was measured using a commercial HCG immunoradiometric assay (IRMA) kit (ICN Pharmaceuticals, Costa Mesa, CA, USA). The kit is a solid phase component system using coated tubes. The standards were established according to the World Health Organization (WHO) third international standard 75/537. AFP was measured using the Byk Sangtec (Dietzenbach, Germany) coated tubes IRMA-mat assay, and uE3 was evaluated with the Diagnostic System Laboratories (Webster, TX, USA) ultra sensitive double antibody radioimmunoassay kit. The individual values were adjusted for maternal weight as well as for IDDM. The overall median MoM for the reference values was about 1 MoM for each marker (Table I).
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Statistical analysis
Descriptive parameters were expressed as mean ± SD. The mIU/l ß-HCG concentrations were transformed using natural logarithms, and their daily values for the IVF and OD groups were compared using Student's t-test. For the daily
ß-HCG in the first-trimester ln ß-HCG ratio of each pregnant woman within the two groups was calculated using the following equation:
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The marker levels are presented as median MoM. Means were calculated by taking log10 of the median MoM of each marker. One sample t-test was applied to compare AFP, HCG and uE3 in mean log10 values between the IVF, OD cases as well as the laboratory reference values. Pregnancy outcome results were compared using correlated Pearson's 2 statistics.
Statistical analysis was carried out in the statistical department of the Tel Aviv University using Statistics Package for Social Sciences software. P < 0.05 was considered significant.
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Results |
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The data on maternal age, number of embryos, daily ß-HCG concentrations, triple serum screening median MoM values and pregnancy outcome for both groups are presented in Table I.
The IVF women were slightly older than the donors, but this trend was not significant; however, the recipients were about 10 years older (P < 0.05).
A significantly higher number of embryos was transferred in the IVF cases (Table I). There was no significant difference between the two groups in
ß-HCG.
As seen in Table I, the median MoM HCG concentrations in both groups were higher than the reference values (though statistically insignificant). AFP was significantly higher in the OD group in comparison with the reference values and the self oocyte IVF cases (P < 0.002; t-test). Based on the triple serum screening results, a similar rate for the IVF group (11%) and for the OD group (13%) was defined as being screen positive (Table I
). Additionally, a similar rate of complicated obstetric outcome was found between the IVF and OD groups.
Six out of the seven cases (90%) and four out of seven (60%) from the IVF group and OD group respectively, which had adverse obstetric outcome, had HCG concentrations 1.2 MoM and five out of seven (70%) from the OD group had AFP > 1.2 MoM (Table II
).
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Discussion |
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Thus, the policy of providing women who conceived after assisted reproduction technology with the same antenatal screening approach as those who conceived spontaneously needs to be evaluated (Maymon et al., 1999a). Additionally the screening software is usually unable to allow the use of conception dates for estimating gestational age and calculation of the MoM values. This further limits the extent to which the laboratory can interpret the test results.
Another critical problem in the screening programme that needed to be addressed was the miscalculation of the duration of pregnancy. In previous reports of IVF singletons and twin pregnancies, the gestation was based on the oocyte retrieval date and further compared with either the first- and the second-trimester fetal biometrical measurement (Geirsson and Have, 1993; Wennerholm et al., 1998
; Tunon et al., 2000
). In those studies, ultrasound indicated that the pregnancy was about 0.92.1 days less advanced for singletons, and 1.6 days for twins. Thus, it may be postulated that this effect has only a minor contribution to the changes in the median MoM values reported in assisted reproduction technology pregnancies.
After controlling for maternal age between the self oocyte IVF and the oocyte donors as well as correcting for gestational age by scanning, it is contended that the high FPR in the IVF and OD patients could be attributed to other factors directly associated with the pregnancy itself. Thus, there is a need to look carefully at the serum analyte profile which may be adjusted according to given conditions. Some authors (Muller et al., 1993; Lam et al., 1999
) did not find significantly increased HCG concentrations in IVF and IVFintracytoplasmic sperm injection (ICSI) singleton cases. However, Lam et al. (1999) indeed found a trend towards a 20% increase of the HCG in IVFICSI pregnancies. Six other groups (Barkai et al., 1996
; Heinonen et al., 1996
; Ribbert et al., 1996
; Frishman et al., 1997
; Hsu et al., 1999
; Wald et al., 1999
) have reported altered serum concentrations in assisted reproduction technology pregnant patients. All those authors have found that HCG concentrations were significantly higher than expected, while serum uE3 and AFP concentrations were found to be lower by some of them. It has recently been suggested (Wald et al., 1999
) that the high HCG concentration which is a placental marker may be explained by the progesterone concentrations remaining high in IVF pregnancies. The findings of the present study cannot support this hypothesis. Unlike what occurs in self oocyte IVF, where multiple corpora lutea may produce very high concentrations of progesterone (Shulman et al., 1994
), the progesterone concentrations in OD achieved pregnancies are exclusively from replacement treatment. This, according to our protocol, ends 57 weeks after embryo transfer. For the rest of the time, the progesterone originates exclusively from the placental steroidogenesis derived from the feto-placental compartment.
In the IVF group, the AFP serum concentrations were similar to the reference values, and this finding is in agreement with other reported IVF groups (Heinonen et al., 1996). The significantly increased AFP in the OD group found in this study is in agreement with a previous smaller report (Barkai et al., 1996
).
Although it has been reported that serum AFP concentration increases with maternal age by 4.4% per 10 years increment (Wald and Watt, 1996), other effects contributing to the 45% increase in AFP on average as currently measured in the OD group should be investigated. The uE3 serum concentrations which reflect the feto-placental compartment (Wald et al., 1997
) are similar both in the assisted reproduction groups as well as to our reference values, and are slightly higher than in other groups of IVF patients (Barkai et al., 1996
; Frishman et al., 1997
). The FPR in the study groups was similar to the complicated obstetric outcome rate and was probably indicative of those events rather than alerting for DS fetus. In this respect, IVF patients were reported to have delivered earlier and had smaller infants (Australian In Vitro Fertilization Collaborative Group, 1985
; Ribbert et al., 1996
) as well as having a high rate of pre-eclampsia (Heinonen et al., 1996
). In some cases, those obstetric complications are characterized by mid- gestation high serum HCG and AFP concentrations (Muller et al., 1993
; Ogle et al., 2000
). The rate of complicated obstetric outcome in our OD and IVF cases (19% and 15% respectively) is higher than the reported 10% FPR which finally ended with adverse perinatal outcome, in the general population (Ogle et al., 2000
). Various, subtle and often poorly defined mechanisms are involved in infertility, in which small and even intractable metabolic disturbances might be responsible. `Forcing' a pregnancy upon an infertile woman would not change those metabolic errors. Thus, the abnormal AFP and HCG serum concentrations might be characteristic of those pregnant women (Ribbert et al., 1996
). This theory can be supported by an observation that the false positive serum screening result tends to repeat itself in a subsequent pregnancy (Holding and Cuckle, 1994
). Therefore, it seems more reasonable to us that such assisted reproduction pregnancies would probably have another underlying pathology, affecting the serum screening results as well.
Women who conceive after assisted reproduction methods have on many occasions achieved their current pregnancy, which might even be their last one, after long-standing infertility. Until medicine discovers the exact reasons for the high FPR among infertile patients, the availability of a more precise algorithm is an important challenge in perinatology (Maymon et al., 1999a). Thus, it seems reasonable to justify correction of HCG MoM values in IVF cases and of AFP MoM values in OD patients. This approach may lower the FPR, thus offering them a more tailored screening programme, which takes into consideration their unique problems.
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Acknowledgements |
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Notes |
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References |
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Submitted on September 20, 2000; accepted on January 15, 2001.