Hum. Reprod. Advance Access published online on January 24, 2008
Human Reproduction, doi:10.1093/humrep/dem409
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A family history of twinning in relation to multiple implantation
Department of Obstetrics, Gynecology and Reproductive Medicine, VU University Medical Center (VUmc), PO Box 7057, 1007 MB Amsterdam, The Netherlands
1 Correspondence address. Tel: +41 20 4440070; E-mail: mj.lambers{at}vumc.nl
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Abstract |
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BACKGROUND: A familial basis for dizygotic twinning is known for multiple ovulation. However, for multiple implantation this remains unclear. In IVF/intracytoplasmic sperm injection (ICSI) ‘multiple ovulation’ is artificially induced. If multiple implantation is not hereditary, the incidences of twins in families of patients with single and multiple implantation after IVF/ICSI with double embryo transfer (DET) should be comparable.
METHODS: A questionnaire study was conducted among patients with intra uterine pregnancy at 6 weeks of gestation, after IVF/ICSI treatment with DET 3 days after oocyte retrieval.
RESULTS: There were 940 patients who gave their informed consent. For women with single implantation (Group A), the incidence of one and of multiple twins among the family was 27.2 and 15.5%, respectively. For women with multiple implantation (Group B), this incidence was 29.5 and 17.8%, respectively, P = 0.424. The incidence of one and of multiple twins among first degree relatives was 10.6 and 1.1% in Group A; for Group B this was 8.7 and 1.9%, P = 0.469. Multivariate regression analysis also did not reveal ‘twins in family’ or ‘twins in first degree’ as an associated variable for multiple implantation at 6 weeks.
CONCLUSIONS: Incidences of twins in families of patients with single implantation and patients with multiple implantation after IVF/ICSI are comparable. Our data do not support the concept that multiple implantation is hereditary.
Key words: IVF/pregnancy/multiple implantation/twins
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is a familial basis for dizygotic twinning (Meulemans et al., 1996
; Lambalk, 2001). Mothers of dizygotic twins more often have twins among their siblings than mothers of singletons (Hemon et al., 1981; Hoekstra et al., 2007), and the risk of multiple pregnancy is increased in women reporting a history of multiple pregnancies in first degree relatives (Parazzini et al., 1996).The precise genetic basis of this hereditary twinning has not yet been elucidated, but it is known that in women from families with hereditary twinning multiple follicle growth and multiple ovulation occurs (Gilfillan et al., 1996; Lambalk and Schoemaker, 1997).
Multiple ovulation must be followed by multiple fertilization and multiple implantation to establish a multiple (dizygotic) pregnancy. It has been suggested that the endometrium of fertile women is very receptive and that this receptivity is a determinant for implantation and for multiple implantation (Matorras et al., 2005). This would imply that in women from families with twins the endometrium is also highly receptive for (multiple) embryo implantation. This would additionally contribute to a higher chance of multiple pregnancy. It is not known whether there is a familial basis for multiple implantation. In the case of hereditary multiple implantation, one should also expect some occurrence of familial monozygotic twinning for which there are no strong indications in the literature.
IVF/intracytoplasmic sperm injection (ICSI) can be regarded as a situation of artificially induced multiple ovulation and artificial multiple fertilization. With assisted reproduction treatments, there are often two embryos transferred [double embryo transfer (DET)] after in vitro fertilization or ICSI. In a previous study, we demonstrated that good embryo quality, presence of supernumerous embryos allowing cryo preservation, thick endometrium and young maternal age are positive determinants for the chance of multiple implantation (Lambers et al., 2007).
If multiple implantation is hereditary, we would expect a higher incidence of (any type of) twins in families of patients showing multiple implantation after IVF/ICSI with DET compared with families of patients showing single implantation after IVF/ICSI with DET.
In this study, we first investigated the incidences of twinning in families of women with multiple implantation after DET and in families of women with single implantation after DET. We then looked at whether a family history of twinning contributed to the chance of multiple implantation after DET in IVF/ICSI-treatment, which would indicate some hereditary basis. If having twins in the family is a reflection of a higher implantation potential, this may also lead to lower chances of early pregnancy loss. Therefore we also analysed whether spontaneous abortion rates between patients with and without a family history of twins were different.
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Materials and Methods |
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Until the end of 2004, DET was most common in our centre. All patients, who had an intact intrauterine pregnancy confirmed by ultrasonography at 6 weeks of gestation after DET on the third day after oocyte retrieval in a ‘fresh’ IVF/ICSI treatment at our centre between 1 January 2000 and 31 December 2004, were eligible for this questionnaire study. Pregnancies after cryo-preservation, ectopic pregnancies and anembryonic pregnancies were excluded. Data regarding patient characteristics, treatment, embryo development and treatment outcome had already been collected for previous analysis (Lambers et al., 2007
). Patients who had a spontaneous abortion and patients with an unknown pregnancy outcome after 12 weeks of gestation, first received a letter announcing this study with the request to reply regarding whether we could send them further information about the study along with the informed consent form, the questionnaire and a return envelope. Patients who were known to have an ongoing pregnancy directly received the information about the study along with the informed consent form, the questionnaire and a return envelope. Patients were requested to reply in all cases, whether they participated or refused to participate in the study. If we had not received a reply a month after the questionnaire had been sent, we contacted the patients to ask if they wanted to participate and if so, to ask them to return the informed consent form along with the answered questionnaire.
The questionnaire contained questions regarding family history of twinning (Hoekstra et al., 2004) and pregnancy outcome, smoking habits, and use of alcohol and medication, all before and during treatment and pregnancy.
All data collected by means of the questionnaire were combined with routinely registered data as described previously (Lambers et al., 2007): maternal age, duration of infertility, previous pregnancy (yes/no), indication for treatment, type of treatment (IVF/ICSI), treatment protocol (short/long), data on treatment monitoring, number of embryos available for transfer, possibility of cryo-preservation, number of embryos available for cryo-preservation, morphological embryo score and pregnancy monitoring data. Data were analysed using t-test, 
2-test and multivariate regression analysis where appropriate.
The study was approved by the Institutional Review Board of the VU University medical centre.
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Results |
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A total of 1593 questionnaires was sent to 1148 patients with single implantation (Group A) and 445 patients with multiple implantation (Group B). The response rate was 61.9%: 940 patients (59%) gave their informed consent (Group A: 660 patients, Group B: 280 patients), and 48 patients (3%) refused participation. Baseline characteristics were not different between the groups (Table I).
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There were 392 women (44.2%) who had one or more sets of twins in her family: 266 (42.8%) in Group A and 126 (47.5%) in Group B (P = 0.189). The incidences of one set of twins and of more than one set of twins in the family were 27.2 and 15.5%, respectively, in Group A and 29.5 and 17.8%, respectively, in Group B (P = 0.424). (Table II).
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There were 101 women (10.7%) who had one or more twins among first degree relatives: 73 (11.1%) in Group A and 28 (10%) in Group B. Incidences of one twin and multiple twins among first degree relatives were 10.6 and 1.1%, respectively, in Group A and 8.7 and 1.9%, respectively, in Group B (P = 0.469). (Table II).
There were 22 women who were part of a multiple pregnancy themselves: 6 of a monozygotic twin, 12 of a dizygotic twin and 4 of triplets. In Group A, the frequencies were 6 (0.9%), 6 (0.9%) and 4 (0.6%), respectively, and in Group B, the frequencies were 0 (0%), 6 (2.2%) and 0 (0%), respectively (P = 0.086). There were 38 women who reported that they had had another pregnancy, other than the pregnancy selected in our study period, that was a multiple pregnancy: 33 (5%) in Group A and 5 (1.8%) in Group B, P = 0.214.
There were 433 women (46.5%) who were smokers: 304 (46.6%) in Group A and 129 (46.2%) in Group B, P = 0.913. Of these smokers, 59.8% reported to have quit smoking entirely, 31.4% had quit for a certain period (usually during treatment and/or during pregnancy) and 7.8% had never quit smoking. Again there were no statistically significant differences between the groups.
Alcohol use before treatment was also not different between the groups: 70.5% in Group A and 67.9% in Group B, P = 0.462. Almost all patients used folic acid: 96.4% in Group A and 99.3% in Group B, P = 0.083. The majority of the women (87.6%) started the use of folic acid from the start of the IVF/ICSI-treatment; this was not different between the groups: 86.3% in Group A and 87.5% in Group B, P = 0.632.
Multivariate regression analysis for multiple implantation revealed that the presence of a thick endometrium, application of a long GnRH-agonist stimulation protocol and high cumulative embryo score were variables associated with multiple implantation at 6 weeks (Table III). All other variables including ‘twins in the family’, ‘smoking’, ‘preconceptional use of folic acid’ and ‘alcohol use’ were not significantly associated with multiple implantation.
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When the multivariate regression analysis was repeated with ‘twins in first degree pedigrees’ instead of ‘twins in family’, the analysis revealed male factor infertility, thick endometrium, long GnRH-agonist stimulation protocol and high cumulative embryo score as variables associated with multiple implantation at 6 weeks (Table IV). Otherwise, all other variables remained not significantly associated with multiple implantation.
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The spontaneous abortion rate for patients with a family history of twins and for patients without a family history of twins was 10.2 and 12.1%, respectively. The percentage loss per gestational sac was 11.2 and 16.1%, respectively. These differences were not statistically different. Multivariate analysis also revealed that ‘twins in family’ was not associated with pregnancy loss between 6 and 12 weeks of gestation. The only variable associated with pregnancy loss was advanced maternal age (Table V). When the multivariate regression analysis was repeated with ‘twins in first degree pedigrees’ instead of ‘twins in family’, the analysis revealed that ‘twins in first degree’ was also not associated with pregnancy loss, but that possibility of cryopreservation was a variable positively associated with continuation of pregnancy (Table VI).
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Discussion |
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Dizygotic twinning is a biological process involving multiple ovulation, multiple fertilization and subsequent multiple implantation. Previous studies have demonstrated that multiple ovulation, among other causes, has a familial basis (Meulemans et al., 1996
; Lambalk, 2001), but it remains unknown whether the familial basis of dizygotic twinning additionally accounts for multiple implantation. Therefore, we investigated the incidence of twins in the families of women who became pregnant after IVF/ICSI-treatment with DET, which can be considered as an artificial situation of multiple ovulation and multiple fertilization.
Our study shows that there is no statistically significant difference in the incidence of twins in the family between patients who had multiple implantation after IVF/ICSI and those who showed single implantation after IVF/ICSI. Although in the general population the risk of multiple pregnancy is increased in women reporting a history of multiple pregnancies in first degree relatives (Parazzini et al., 1996), our findings make it unlikely that in IVF/ICSI-patients the chance of multiple implantation is influenced by their family history of twinning. The multivariate regression analysis demonstrated once more that a high cumulative embryo score, the application of a long stimulation protocol and thick endometrium are positively associated with multiple implantation. When the analysis was repeated with ‘twins with first degree pedigrees’ instead of ‘twins in family’, it revealed that male factor infertility is also positively associated with the chance of multiple implantation.
It was not surprising to find a high cumulative embryo score as the strongest contributing factor. Studies on the characterization of top-quality embryos (Van Royen et al., 1999, 2001) and studies comparing single embryo transfer and DET (Thurin et al., 2005) have already demonstrated the important role of good embryo quality in chances of multiple pregnancy. These results indicate that morphologically optimal embryos have high potential for further development and implantation. Our findings underline the fact that the embryo quality plays an essential part in the chances of pregnancy and of multiple implantation.
The finding that application of a long stimulation protocol was associated with multiple implantation can be explained both as being an embryonic and a maternal characteristic. In our clinic, the long GnRH-agonist stimulation protocol is, in general, applied in younger patients and patients with previous good ovarian response (Lambers et al., 2006). It has been demonstrated that younger patients have higher chances of pregnancy (Heffner, 2004) and higher chance of multiple implantation in case of DET (Roseboom et al., 1995; Stolwijk et al., 2000; Terriou et al., 2001). As such it can be regarded as a reflection of maternal characteristics. On the other hand, it can be a reflection of embryonic characteristics, since patients with good ovarian response to controlled ovarian hyperstimulation usually have around ten oocytes available for IVF/ICSI, increasing the chances of multiple fertilization and thereby creating the possibility of more optimal embryo selection. The possibility to select embryos and to cryo-preserve supernumerary embryos can be regarded as an indicator for good embryo quality, since the transferred embryos are selected from a group of embryos with more or less equal morphology scores.
Thick endometrium can be regarded as a reflection of hormonal levels and endogenous maternal environment. From IVF-studies, it is known that for a good chance of pregnancy the endometrium needs to reach a certain thickness (Kovacs et al., 2003). The positive association with multiple implantation that we found for male factor infertility can be explained by the fact that in these couples infertility is predominantly the result of low sperm quality. In most couples with severe male factor infertility, the female spouse has no factor of infertility at all (Bhattacharya et al., 2001). Therefore, these women have better chances of pregnancy and, in the case of DET, higher chances of multiple pregnancy (Lintsen et al., 2007). It remains unclear why this association was only found in the analysis with twins in first degree relatives.
Embryo implantation is an interplay between the embryo and endometrium and biologically it seems logical to assume both play essential parts in this process. Matorras et al. (2005) analysed several mathematical models for their predictive value for pregnancy and multiple implantation. The ‘collaborative model’, based on the hypothesis that implantation of one embryo facilitates implantation of (an)other embryo(s) was found to be the most accurate in prediction of pregnancy and multiple implantation. Prediction models principally based on maternal aspects were clearly less accurate. Their results indicate, in line with our findings regarding the determinants for implantation, that the leading part in (multiple) implantation is played by the embryos.
Since previous studies demonstrated that high folate status (Haggarty et al., 2006), and use of alcohol and smoking habits (Parazzini et al., 1996; Lintsen et al., 2005) may enhance chances of multiple pregnancies, our questionnaire also inquired after these compounds. Smoking and alcohol consumption have been shown to interfere with the estradiol feedback on FSH-production (Zenzes, 2000; Lintsen et al., 2005; Srivastava et al., 2005), resulting in an increased chance of multiple follicle growth and therefore multiple pregnancy. In our patient group, multiple follicle growth was induced by the IVF/ICSI treatment and we found no association with multiple implantation for smoking or alcohol consumption. One should keep in mind that behaviour like alcohol consumption is often underestimated (Feunekes et al., 1999) and that the majority of these patients quit smoking and alcohol consumption because of the treatment and their desire to become pregnant. But from these results, we hypothesize that smoking and alcohol consumption only contribute to an increased chance of multiple pregnancy because of an increased chance of multiple follicle growth.
A high folate status before pregnancy was recently found to increase the chance of twins (Haggarty et al., 2006). Although there is still discussion on this subject, it is known that the preimplantation embryo has an absolute need for folate for normal development (O'Neill, 1998). High levels of endogenous folate before conception may enhance normal development of the preimplantation embryo(s) and therefore increase the chance of twin pregnancies (Haggarty et al., 2006). In our study, almost all patients used supplementary folic acid, the majority already using it during treatment. This is a likely explanation why in our study the regression analysis did not reveal use of folic acid before conception as a variable associated with multiple implantation.
Spontaneous abortion rates were not different between patients with and without a family history of twinning and ‘twins in the family’ was also not revealed as an associated factor for pregnancy loss in the first trimester by multivariate regression analysis. The variables found from the regression analysis were the same as in our previous publication (Lambers et al., 2007). Since the group of patients in this study is a selection of the group we analysed for the previous publication, the current outcome underlines the association of these variables with pregnancy loss.
In summary, dizygotic twinning is primarily based on multiple ovulation and subsequent multiple implantation. From the artificial model of multiple ovulation, we learned that the incidence of twins in the family and twins among first degree relatives is equal for women who have multiple implantation after DET and women who have single implantation after DET. Multivariate regression analysis demonstrates that ‘twins in the family’ or ‘twins with first degree pedigrees’ are not associated with multiple implantation at 6 weeks gestation. Therefore it is unlikely that multiple implantation itself is hereditary and therefore it seems that the familial basis for twinning is restricted to multiple ovulation.
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Submitted on September 12, 2007; resubmitted on November 24, 2007; accepted on December 4, 2007.
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