1 Department of Obstetrics and Gynecology, University of Innsbruck, Innsbruck, 2 Institute for Reproductive Medicine and Endocrinology, Bregenz and 3 Institute for Biostatistics and Documentation, University of Innsbruck, Innsbruck, Austria
4 To whom corresondence should be addressed at: Department of Obstetrics and Gynecology, University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria. Tel: +43 512 504 3051; Fax: +43 512 504 3055; Email: peter.schwarzler{at}uibk.ac.at
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Abstract |
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Key words: ART/birth defect/blastocyst transfer/early cleavage stage/pregnancy outcome
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Introduction |
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Although the human genome is activated between the 4 and 8 cell stages (Braude et al., 1988) there is only a minimal level of transcription during the initial stages of zygote formation and early cleavage divisions. Therefore, the mature oocyte must contain a storage pool of proteins and/or mRNA transcripts already at the time of ovulation. The cleavage cycle during which the zygote genome is activated (4 to 8 cells in humans) is the longest among pre-implantation development. Any developmental delay at this time will result in a decrease in the level of mRNA below a critical threshold. Correct timing of blastocyst formation is an initial indication of embryo quality as it shows that the embryos have passed the narrow gap of maternal to zygotic transition.
It was demonstrated that uterine contractility decreases at the time of blastocyst transfer when compared to early stage transfers on day 2 (Fanchin et al., 2001). The rhythm of contractions is reduced by half to 1.5 contractions per minute.
Furthermore, the endometrium on day 5 post-conception seems to be optimal for embryos to implant and mirrors the implantation time in natural conceptions (Gardner and Lane, 2003).
However, whilst many IVF (in vitro fertilization) clinics report their results in terms of pregnancy per oocyte pick-up or embryo transfer, patients frequently demand to know the take-home baby rate (THBR), which is defined as babies per implanted embryosand they want to evaluate the risks they and their children might face when they choose to use ART (Artificial Reproductive Techniques).
The purpose of this retrospective study was to evaluate differences in fertility and pregnancy rates and neonatal outcome when embryo transfer was performed either on day 23 (cleavage stage, CS-group) or on day 45 (blastocyst stage, BS-group).
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Materials and methods |
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Total pregnancy rate was recorded after serum pregnancy tests were performed 11 and 13 days after oocyte retrieval.
A total of 500 live born children resulted from these pregnancies, some of which were multiple birth deliveries. Forty-two women did not want to participate in the study and 19 women were lost for follow up.
Following written informed consent, patients' characteristics and medical history were evaluated by questionnaire after ART-treatment and delivery. Neonatal outcome of the two study populations was evaluated by collection and analysis of all birth records, in cases of referral to intensive care unit (ICU), neonatology discharge notes were reviewed. Endpoints for neonatal outcome were sex, birth weight in relation to term- and preterm delivery, Apgar-score, transfer rate to ICU and birth defects.
Variables of interest compared between the two study groups were pregnancy rate, number of vital pregnancies, rate of multiple pregnancies, sex, birth weight of the newborns and numbers of low-birth-weight infants, the latter were subdivided into term and preterm infants. Preterm delivery was defined as delivery at <36 completed weeks of gestation. Low birth weight was defined as 2500 g or less at term and preterm. Transfer rate to ICU and hospital stay of neonates and prevalence of major birth defects diagnosed in the first days after delivery was recorded. Birth defects were defined as abnormalities that are most likely of prenatal origin, including structural, chromosomal and genetic defects.
Treatment
Women undergoing assisted reproduction treatment received the combination of daily 0.1 mg triptorelin s.c. (Decapeptyl; IPSEN Biotech, Paris, France) starting in the midluteal phase of the preceding cycle. Three days after the onset of menstruation an ultrasound evaluation was performed. If downregulation (endometrium <3 mm, no ovarian cysts >10 mm) was demonstrated, 150 IU HP hMG (Menopur; Ferring Pharmaceuticals A/S, Ørestad City, Denmark) and 75 IU rFSH (Gonal-F; Serono Pharmaceuticals Ltd, Feltham, UK) were administered. When a cohort of >2 follicles reached a diameter of 19 mm, 10 000 IU hCG (Profasi; Serono Pharmaceuticals Ltd, Feltham, UK) were given. Luteal phase support consisted of administering 50 mg progesterone (Streuli, Richterswil, Switzerland) daily i.m. starting from the day of oocyte retrieval until 16 days thereafter. This was continued until pregnancy occurred, which was defined as the presence of gestational sac(s) with fetal heart activities at 30 days after transfer. Oocytes were collected 3435 h after the administration of 10 000 IU of hCG and were incubated in 0.5 ml IVF 20 medium (Scandinavian Science, AB products, Gothenborg, Sweden). In patients entering the IVF program for male factor or unexplained failure of fertilization, cumulus and corona cells were removed from the oocytes 23 h after oocyte retrieval by incubation in 25 IU/ml hyaluronidase (Type VIII; Sigma Chemical Co., St Louis, MO) and before performing ICSI. Further details are described elsewhere (Vanderzwalmen et al., 1996).
In vitro culture of embryos
Oocytes were incubated in IVF 20 medium (Scandinavian Science, Gothenborg, Sweden) and embryo culture was carried out in 4-well multi-dishes (Nunc, Roskilde, Denmark) containing each 500 µl of sequential media or in 40 µl microdrops under mineral oil (Cryo Bio System, Paris, France) at 37°C in a humidified atmosphere of 5% O2 in air. Sixteen to twenty hours after insemination or ICSI, the oocytes were assessed for the presence of two pronuclei and after rinsing they were transferred into G1-2 medium (Scandinavian Science, Gothenborg, Sweden) for an additional period of 48 h.
For blastocyst culture, embryos were placed in wells or micro-drops of CCM medium (Scandinavian Science) on day 3 and further cultured to the blastocyst stage.
Statistics
Associations between categorical variables were tested with the Pearson's chi square test and the Fisher's exact test. Differences between nonparametric distributed variables were examined with the MannWhitney U test. A P-value of <0.05 was considered statistically significant. Significance levels of multiple tests were adjusted using Bonferroni correction. All statistical calculations were performed using SPSS, version 11.0, for Windows.
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Results |
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Discussion |
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It could also be demonstrated that extending embryo culture to day 5 before transfer increased clinical and ongoing pregnancy rates for patients >35 years of age, whilst decreasing miscarriage rate (Levron et al., 2002; Wilson et al., 2002
). Our data showed a much higher pregnancy rate in the BS-group with 40% vs 25% in the CS-group (P<0.001). Accordingly, THBR were also significantly different with 37% and 22%, respectively (P<0.001). The group of patients for blastocyst culture in our study is younger as compared to the CS group, even though no significant difference is observed. However, with time we have enlarged the population of patients entering the blastocyst group. We observed that the numbers of oocytes and zygotes are factors that impaired the development of embryos to the blastocysts stage more than women's age. For example the pregnancy rate in a group of 40-year-old women producing an average of eight zygotes was 25%; in the same population but with a transfer on day 3, the pregnancy rate reached only 13% (data not shown).
Gardner (Gardner et al., 1998; Gardner, 2000
) reported significantly higher implantation rates of blastocysts compared to ET on day 23. Thus, acceptable pregnancy rates can be achieved by transferring fewer blastocysts. This was confirmed by a prospective randomized study by Van der Auwera (2002)
. However, other groups did not find a difference in implantation and pregnancy rates in patients receiving day 3 transfer and those receiving extended-culture embryo replacement (Coskun et al., 2000
; Levron et al., 2002
). Prolongation of embryo culture did not improve clinical outcome in terms of THBR (Lundqvist et al., 2002
). With regard to the implantation rate our results demonstrate the beneficial effect of blastocyst transfer, showing a higher percentage of embryos implanting in the BS-group. The higher pregnancy rate in this group might reflect the possibility of choosing intact embryos for transfer, since there was no difference in mean age of the parents, mean number of cycles, mean number of transferred embryos. However, this was a non-randomized retrospective cohort study and particularly at the beginning of the time line, patients with fewer embryos were certainly biased towards the CS-group.
According to the latest Society for Assisted Reproductive Technology (SART) report available, in 2001, multiple births accounted for 35.8% of all ART deliveries (Wright et al., 2004). Multiple pregnancies do not only mean an increased risk for mother and fetuses, they also are responsible for economic and social burden for the parents as well as for the health care system. Transferring embryos at the blastocyst stage has been hypothesized to increase pregnancy and implantation rates, while potentially increasing the multiple pregnancy rate. In a small randomized controlled trial in patients with good prognosis, Frattarelli could demonstrate superior implantation and pregnancy rates with blastocyst transfer compared to ET on day 3, even though fewer embryos were transferred (Frattarelli et al., 2003
). In our population, the BS-group yielded a significantly higher rate of twins (33% vs 16.5%), which reflects the higher implantation rate per embryo compared to the CS-group. The total higher number of multiple pregnancies in the BS-group (34% vs 17%) is mainly caused by the higher number of twins in this group. A possible explanation is that those embryos were chosen that have progressed past embryonic genome activation, thus believed to be more developmentally competent.
A main endpoint of this study was fetal outcome. Sex ratios, birth weight in relation to term- and preterm delivery, APGAR-scores, umbilical artery pH-values, transfer rate to intensive care unit, days of hospitalization and number of birth defects were compared in the two study populations: in some animal species (mice and calves) it has been observed that male embryos reach the blastocyst stage faster than female embryos, resulting in a higher proportion of male offspring (Tsunoda et al., 1985; Avery et al., 1991
). However, in humans, these findings could not be uniformly reproduced (Tarin et al., 1995
). Also, our data showed no statistically significant difference in male sex in the two study populations.
Previous data demonstrated a higher birth weight after blastocyst transfer in ruminants (Thompson et al., 1995); the authors speculate that the culture system possibly could have had an impact. Also, our data showed a trend towards higher birthweight in the BS-group, but without reaching significance.
Newborns with low birth weight (2500 g)either because of preterm delivery or because of fetal growth retardationare at increased risk for disabilities and death. The increased risk of low birth weight associated with the use of ART has been attributed largely to the higher rates of multiple pregnancies. Interestingly, singleton infants conceived with ART are also at increased risk for low birth weight at term, relative to singletons in the general population (Schieve et al., 2002
). The authors suggest that the 2.6-fold increased risk of low birth weight in those singletons may be directly related to treatments for infertility. Among twins conceived with ART, the risks of term and preterm low birth weight were similar to those in the general population of twins.
In general, studies have not shown an increased risk of major birth defects in children conceived after both IVF and ICSI (Van Steirteghem, 1998), but it seems that methodological problems may have led to an underestimation of the birth defect prevalence among children conceived by ART. Hansen et al. (2002)
reported, that by 1 year of age, a major birth defect had been identified in 9% of babies following ART, compared to only 4% for those conceived spontaneously. Differences were found in most categories, but were significant for musculoskeletal and cardiovascular defects assuming that infertility and not necessarily ART increases birth defects rates. A major flaw of the above cited study is the lack of an appropriate control group, however, a potential association of ART and a higher rate of birth defects cannot be ruled out.
Despite higher birth weight discordance and more ICU admissions among IVF/ICSI twins, neonatal outcome in IVF/ICSI twins seems to be comparable with that of non-IVF/ICSI twins, when only dizygotic twins were considered in the comparisons (Pinborg et al., 2004).
Our data, though small, do not give any evidence of a higher risk of congenital malformation by prolonging in vitro culture to day 5.
In conclusion, blastocyst transfer leads to increased implantation and pregnancy rates with an overall better THBR but with increased multiple pregnancies and preterm delivery rates compared to day 23 transfer. This might reflect increased competence of the blastocysts and better uterine synchronization. There were no differences observed in the two study groups in mean gestational age, in the rate of Caesarean section, Apgar and umbilical cord pH-values, total mean birth weight, percentage of low birth weight and admission to ICU. The incidence of minor and major birth defects in both study groups was equal but slightly higher to what is normally seen in the general population. However, final proof for the benefits of blastocyst transfer can only be obtained from prospective, randomized clinical trials.
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Submitted on December 22, 2003; resubmitted on March 17, 2004; accepted on June 9, 2004.