Hum. Reprod. Advance Access originally published online on September 14, 2006
Human Reproduction 2007 22(1):275-279; doi:10.1093/humrep/del367
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Factors determining early pregnancy loss in singleton and multiple implantations
Department of Obstetrics, Gynecology and Reproductive Medicine, Vrije University medical center (VUmc), Amsterdam, The Netherlands
1 To whom correspondence should be addressed at: Department of Obstetrics, Gynecology and Reproductive Medicine, Vrije University medical center (VUmc), PO Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail: mj.lambers{at}vumc.nl
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Abstract |
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BACKGROUND: The incidence of first trimester pregnancy loss is much lower in IVF twin pregnancies than in IVF singleton pregnancies. The objective of this study was to determine which embryonic and maternal factors contribute to this finding. METHODS: Retrospective data analysis of the outcome of 1593 pregnancies after day 3 double-embryo transfer (DET) after IVF or ICSI treatment. RESULTS: Of 1148 single implantations at 6 weeks, 936 (81.5%) were ongoing pregnancies. Of 445 multiple implantations at 6 weeks, 354 (79.6%) were ongoing multiple pregnancies, 80 (17.9%) were ongoing singleton pregnancies and 11 (2.5%) ended in a spontaneous abortion. Total pregnancy loss was 18.5 and 2.5% (P < 0.001) in singleton and twin gestations, respectively. Loss per gestational sac was 18.5 and 11.46% (P < 0.001), respectively. Determinants contributing to the continuation of gestation beyond 6 weeks were young maternal age, possibility to cryopreserve embryos and short GnRH agonist flare-up stimulation protocol. Whereas factors promoting multiple implantation at 6 weeks of gestation were young maternal age, high cumulative embryo score (CES), male infertility, long stimulation protocol and thick endometrium. CONCLUSIONS: Although multiple implantation at 6 weeks is predominantly determined by (morphological) embryo quality, the continuation of pregnancy beyond 6 weeks becomes more dependent on the combination of genetic and developmental potential of the embryo(s) and an optimal uterine milieu.
Key words: gestational sac/implantation/IVF/pregnancy loss/twin
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Introduction |
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The incidence of spontaneous abortion is the highest in the first trimester of pregnancy. Recent IVF studies showed a much lower incidence of pregnancy loss in pregnancies with twin nidation (Tummers et al., 2003
; La Sala et al., 2004; Zegers-Hochschild et al., 2004). Remarkably enough this finding does not only account for the incidence of total pregnancy loss; it was found that the incidence of loss per gestational sac in multiple pregnancies is also much lower compared with singleton pregnancies (Tummers et al., 2003).
It was suggested that embryos of twin pregnancies come from a better cohort and have better intrinsic potential (Tummers et al., 2003). Van Royen showed that top-quality embryos do have a better chance of implantation, but the question remains whether these embryos also have better chances of continuation of pregnancy (Van Royen et al., 1999). La Sala hypothesized that the embryonic potential for early development is not the same for twins and singletons (La Sala et al., 2005a). Finally, Zegers-Hochschild postulated that women with multiple gestation represent highly fertile individuals (Zegers-Hochschild et al., 2004).
Nevertheless, the studies published so far assumed rather than proved the dominant role of the embryo quality with regard to the observed better maintenance of pregnancy in case of multiple implantation with IVF. In the current study, we aimed to differentiate between embryonic quality and maternal factors contributing to the chance of multiple implantation and subsequent pregnancy loss. For this purpose, we performed logistic regression analyses of routinely registered variables with emphasis on the careful quality classification of the embryos.
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Materials and methods |
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We reviewed records of all IVF/ICSI patients treated in our centre between 1 January 2000 and 31 December 2004 and included all patients who met the following inclusion criteria: fresh IVF or ICSI treatment cycle, double-embryo transfer (DET) on day 3 after oocyte retrieval, positive serum pregnancy test on day 14–16 after oocyte retrieval and presence of one or more intrauterine gestational sac(s) on ultrasound at 6 weeks of gestation. Only one treatment cycle per patient was included. If patients had more cycles resulting in pregnancy within the study period, the first cycle was included. Pregnancies after cryopreservation, ectopic pregnancies and anembryonic pregnancies were excluded. Data regarding patient characteristics, treatment, embryo development and treatment outcome were collected.
Of all patients, the following information was collected for analysis: maternal age, duration of child wish, duration of infertility, previous pregnancy (yes/no), indication for treatment, type of treatment (IVF/ICSI), treatment protocol (short/long), duration of stimulation, endometrial thickness, level of estradiol (E2), number of follicles, number of oocytes, number of fertilized oocytes, number of embryos available for transfer, possibility of cryopreservation, number of embryos available for cryopreservation, morphological embryo score and pregnancy monitoring data.
Embryo development was evaluated shortly before embryo transfer, and the best two embryos were selected for transfer. Each embryo was scored for morphology (grade 1–4) according to its symmetry and the extent of fragmentation of the blastomeres (Rijnders and Lens, 1993; van Weering et al., 2002), an optimal quality embryo received score 1.
The cumulative embryo score (CES) was calculated according to the example of Steer et al. (1992), adjusted for day 3 embryos. For calculation of the CES, an embryo with grade 1 morphology received 4 points, grade 2 received 3 points, etc. Basically, the CES for day 2 embryos is obtained by multiplication of the morphology score with the number of blastomeres and summarizing the scores of all embryos transferred per patient. Seven or more blastomeres on day 3 is considered one of the criteria for a top-quality embryo (Van Royen et al., 1999). For day 3 embryos, the CES was adjusted as follows: embryos with 
7 blastomeres received 3 points, embryos with 5 or 6 blastomeres received 2 points and embryos with 
4 blastomeres received 1 point. In this study, the CES was obtained by multiplication of the score for morphology and number of blastomeres per embryo and summarizing the scores of both embryos transferred per patient.
A pregnancy test was performed 14–16 days after oocyte retrieval. Positive tests were followed by transvaginal ultrasonographic monitoring at 6, 9 and 12 weeks of gestation. If cardiac activity could not be diagnosed at 6 weeks of gestation, ultrasonographic monitoring of the pregnancy was repeated after a week. A clinical pregnancy was defined as a positive pregnancy test followed by intrauterine embryonic sac/parts at 6 weeks of gestation. An ongoing pregnancy was defined as an intrauterine pregnancy with one or two fetuses showing cardiac activity at 12 weeks of gestation. Spontaneous abortion was defined as intrauterine pregnancy with fetal cardiac activity at 6 weeks of gestation followed by fetal demise.
Statistical analysis
Statistical analyses were done by t-tests, 
2-tests and binary logistic regression analysis with backward likelihood ratio. In the regression analysis, the dependent variables were number of implantations at 6 weeks (single/multiple) and loss of gestational sac before 12 weeks (yes/no). Two-sided P < 0.05 was considered statistically significant.
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Results |
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Between 1 January 2000 and 31 December 2004, we performed 8552 ‘fresh’ IVF/ICSI treatment cycles in our centre. A total of 6959 cycles were excluded for the following reasons: embryo transfer was not performed on day 3 after oocyte retrieval (2546 cycles), treatment did not result in pregnancy (3924 cycles), patients had more than one treatment cycle resulting in pregnancy during the study period (245 cycles), there was single- or triple-embryo transfer (115 cycles), there was no gestational sac on ultrasound at 6 weeks of gestation (106 cycles), pregnancy was ectopic (17 cycles), no embryo transfer had taken place (4 cycles) or data were incomplete (2 cycles). A total of 1593 treatment cycles with clinical pregnancy remained for analysis.
The average age of the patients was 33.7 years. There were 864 pregnancies resulting from IVF treatment and 729 from ICSI treatment. At 6 weeks of gestation, there were 1148 pregnancies with single implantation and 445 pregnancies with multiple implantation. Of the pregnancies with single implantation, 936 (81.5%) were ongoing at 12 weeks. Of the pregnancies with multiple implantation, 354 (79.5%) were still multiple pregnancies at 12 weeks, 80 (18%) spontaneously reduced to a singleton pregnancy and 11 (2.5%) ended in a complete spontaneous abortion (Table I). The ongoing pregnancy rate for singleton and multiple pregnancies was 81.5 and 97.5% (P < 0.001), respectively, and the risk of loss per implanted gestational sac was 18.5 and 11.46% (P < 0.001), respectively (Table II).
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Univariate analysis between single and multiple implantations at 6 weeks of gestation showed lower maternal age, more follicles at the time of oocyte retrieval, more fertilized oocytes, more embryos available for transfer, more embryos available for cryopreservation, thicker endometrium and higher CES for pregnancies with multiple implantation. These differences were statistically significant (Table III). All other variables were not significantly different.
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Multivariate regression analysis (Table IV) for multiple implantation revealed that the variables young maternal age, presence of male infertility, long stimulation protocol, a thick endometrium and high CES were independently positively associated with multiple implantation at 6 weeks. All other variables were not significantly associated to multiple implantation.
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Univariate analysis between pregnancies with and without loss of gestational sac(s) showed higher maternal age and lower percentage of cycles with cryopreservation for pregnancies with loss of gestational sac(s). These differences were statistically significant (Table V). All other variables were not significantly different.
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The multivariate regression analysis (Table VI) for pregnancy loss for all pregnancies revealed that the variables short stimulation protocol, possibility to cryopreserve embryos and young maternal age were independently positively associated with the continuation of pregnancy. All other variables, including CES, were not significantly associated to multiple implantation.
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Regression analysis for pregnancy loss for singletons showed exactly the same associated variables as regression analysis for pregnancy loss for all pregnancies, whereas regression analysis for pregnancy loss for multiples revealed only maternal age as an associated variable (Table VI).
Univariate analysis between patients treated with short- and long-stimulation protocol showed that patients treated with the short protocol were older, had less follicles, less oocytes, less embryos available for transfer and less embryos available for cryopreservation, but they had higher total levels of E2 and higher levels of E2 per oocyte (Table VII). All other variables were not significantly different.
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Discussion |
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Successful implantation and maintenance of implanted embryo(s) depend on the quality of the embryo, the quality of the implantation site and their interaction. Our work shows that the vanishing of one twin is a common feature of double nidation (Landy and Keith, 1998
; Pinborg, 2005), but on the other hand, it confirms the earlier observations by Tummers that embryos survive better after double implantation (Tummers et al., 2003). This remarkably better survival poses some urgent questions: Which of the aforementioned features of early pregnancy development predominate in determining the continuation of a pregnancy? Are these features the same as those that determine multiple implantation?
It has been suggested that in case of double implantation, continuation of pregnancy is more likely, because embryos were selected from a better cohort of embryos (Tummers et al., 2003). Our multiple logistic regression analysis indeed indicates good morphological embryo quality as the strongest factor determining the chance of double implantation. This is in agreement with studies characterizing top-quality embryos and studies comparing SET with DET (Van Royen et al., 1999; Van Royen et al., 2001; Thurin et al., 2005). These studies show that the morphological embryo score plays a pivotal role in the chance of pregnancy and multiple implantation rates. These studies do not show whether this morphological quality is also essential for the continuation of pregnancy.
From our analysis for chance of pregnancy loss, we found younger maternal age to be the strongest contributing factor. Furthermore, the possibility of cryopreservation and the application of the short GnRH-agonist protocol contributed positively to the continuation of pregnancy. Although embryo quality itself did not contribute significantly to the chance of pregnancy loss, we could ask ourselves to what extent the factors we found can be interpreted as parameters for embryo quality. For maternal age and the possibility to cryopreserve embryos, this could be the case.
There is strong correlation between increasing maternal age and the chance of pregnancy loss (Heffner, 2004), which is commonly attributed to the increasing rate of aneuploidy (Spandorfer et al., 2004). On the other hand, it was found for both older and younger groups of patients that only 36% of the morphologically good embryos were chromosomally normal after fluorescence in-situ hybridization (FISH) analysis of blastomeres (Staessen et al., 2004; Baart et al., 2006; Munne, 2006). An embryo that is morphologically optimal may have high potential of further development and subsequent implantation. But there still is a good possibility of chromosomal abnormality and therefore of spontaneous abortion, because approximately 50% of all first trimester abortions are associated with cytogenic abnormalities (Hassold et al., 1980).
Embryos for cryopreservation are supernumerary and must fulfil very strict morphological characteristics. In our clinic, there must remain at least 3 embryos after the selection of the embryos for transfer, each of them consisting of at least 6 blastomeres with morphology score 1 or 2 (not >10% fragmentation). These embryos are re-examined 1 day later and, in case of appropriate further development, selected for cryopreservation. Given these strict criteria, the possibility to cryopreserve can be regarded as an indicator for good embryo quality. However, it also depends on the quantitative ovarian response during hormonal stimulation, and as such, it represents maternal characteristics.
To our surprise, we found that the application of a short GnRH agonist protocol was also positively associated with continuation of pregnancy. In our clinic, we apply this as treatment of first choice, when patients are older than 38 years or when there has been a previous poor response (Goverde et al., 2000). Intuitively, we rather expected an adverse implication of this characteristic on the likelihood of ongoing pregnancy. Comparing patients treated with a short protocol with patients treated with a long protocol, we found that, at the time of hCG injection, the total E2 levels and the E2 levels per retrieved oocyte were significantly higher (Table VII). E2 is the dominant hormone during the follicular phase and an important link in endometrial proliferation by up-regulation of progesterone receptors, vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I), heparin binding epidermal growth factor (HB-EGF) and L-selectin (Hoozemans et al., 2004). Therefore, we speculate, but can not prove, that because of the ‘flare up’ of endogenous gonadotrophin secretion in the short protocol, a more optimal endometrium develops, which may influence the quality of the implantation site.
From this, we hypothesize that the maternal age and the possibility of cryopreservation reflect embryo quality in genetic and in developmental potential respectively, whereas the type of stimulation protocol used reflects the potential of the implantation site to support a pregnancy.
With regression analysis for pregnancy loss for only singleton implantation, we found exactly the same associated factors as regression analysis for pregnancy loss for all pregnancies in more or less the same order of significance. The regression analysis for pregnancy loss for only multiple implantations showed maternal age as the sole associated factor. The stimulation protocol and the possibility of cryopreservation remained next in line but were no longer significantly associated. This loss of significance may be caused by the smaller group of multiples in combination with the much lower prevalence of pregnancy loss in multiple pregnancies.
There are other hypotheses that may explain why only maternal age remains significant for pregnancy loss in multiple implantations. It can be speculated that, in case of double implantation, the embryos collaborate. Matorras found the best mathematical model for the prediction of pregnancy and multiple pregnancy to be a ‘collaborative model’ based on the hypothesis that the implantation of one embryo facilitates the implantation of (an)other embryo(s) (Matorras et al., 2005). Through the embryonic–endometrial dialogue, specific endometrial molecules are activated (Hoozemans et al., 2004), optimizing the uterine environment for implantation. On the basis of this collaborative model, one could hypothesize that, in double implantation, the environment is optimized not only for the implantation of (an)other embryo(s) but also for survival of additional embryo(s). A second explanation may come from the increased placental mass in early multiple implantations (La Sala et al., 2005a,b). Twin placentas produce more hCG and progesterone than singleton placentas. Important factors in implantation, such as glycodeline A and MUC-1, are under progesterone control (Hoozemans et al., 2004). Through higher progesterone production in twin pregnancies, a superior hormonal support of the uterine milieu is created (La Sala et al., 2005b). Both the hypotheses suggest that, in multiple pregnancies, the uterine milieu is more optimal for the continuation of pregnancy, leaving (maternal age dependent) genetic potential as the sole determinant for pregnancy loss.
In contrast to its important role in determining chance of multiple implantation, the CES did not further contribute to the chance of continuation of pregnancy up to 12 weeks of gestation. However, one should bear in mind that the CES contributes to the chance of multiple implantation and that multiple implantations have a better chance of survival through the first trimester. Therefore, when two good quality embryos are transferred, patients have a higher chance of multiple implantation and, once there is multiple implantation, have lower chance of pregnancy loss. Other factors associated with multiple implantation were consistent with previous publications (Ziebe et al., 1997; Kovacs et al., 2003).
The likelihood of multiple implantation and the subsequent continuation of pregnancy in case of DET is dependent on a combined action of the embryo and the implantation site. Our analysis of data from women who became pregnant after DET on day 3 after oocyte retrieval indicates that occurrence of multiple implantation is predominantly dependent on (morphological) embryo quality, but the continuation of pregnancy is more dependent on the combination of genetic and developmental potential of the embryo(s) and an optimal uterine milieu.
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Submitted on June 7, 2006; resubmitted on August 11, 2006; accepted on August 16, 2006.
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