Ewka C.M. Nelissen

Effect of in vitro culture of human embryos on birthweight of newborns

BACKGROUND In animal models, in vitro culture of preimplantation embryos has been shown to be a risk factor for abnormal fetal outcome, including high and low birthweight. In the human, mean birthweight of singletons after in vitro fertilization (IVF) is considerably lower than after natural conception, but it is not known whether culture conditions play a role in this. METHODS We compared pregnancy rates and perinatal outcomes from singleton pregnancies resulting from a total of 826 first IVF treatment cycles in which oocytes and embryos were randomly allocated to culture in either of two commercially available sequential media systems. RESULTS When the 110 live born singletons in the Vitrolife group were compared with the 78 singletons in the Cook group, birthweight +/- SEM (3453 +/- 53 versus 3208 +/- 61 g, P = 0.003), and birthweight adjusted for gestational age and gender (mean z-score +/- SEM: 0.13 +/- 0.09 versus -0.31 +/- 0.10, P = 0.001) were both significantly higher in the Vitrolife group. When analyzed by multiple linear regression together with several other variables that could possibly affect birthweight as covariates, the type of culture medium was significantly (P = 0.01) associated with birthweight. CONCLUSIONS In vitro culture of human embryos can affect birthweight of live born singletons.

Epigenetics and the placenta

BACKGROUND The placenta is of utmost importance for intrauterine fetal development and growth. Deregulation of placentation can lead to adverse outcomes for both mother and fetus, e.g. gestational trophoblastic disease (GTD), pre-eclampsia and fetal growth retardation. A significant factor in placental development and function is epigenetic regulation. METHODS This review summarizes the current knowledge in the field of epigenetics in relation to placental development and function. Relevant studies were identified by searching PubMed, Medline and reference sections of all relevant studies and reviews. RESULTS Epigenetic regulation of the placenta evolves during preimplantation development and further gestation. Epigenetic marks, like DNA methylation, histone modifications and non-coding RNAs, affect gene expression patterns. These expression patterns, including the important parent-of-origin-dependent gene expression resulting from genomic imprinting, play a pivotal role in proper fetal and placental development. Disturbed placental epigenetics has been demonstrated in cases of intrauterine growth retardation and small for gestational age, and also appears to be involved in the pathogenesis of pre-eclampsia and GTD. Several environmental effects have been investigated so far, e.g. ethanol, oxygen tension as well as the effect of several aspects of assisted reproduction technologies on placental epigenetics. CONCLUSIONS Studies in both animals and humans have made it increasingly clear that proper epigenetic regulation of both imprinted and non-imprinted genes is important in placental development. Its disturbance, which can be caused by various environmental factors, can lead to abnormal placental development and function with possible consequences for maternal morbidity, fetal development and disease susceptibility in later life.

Further evidence that culture media affect perinatal outcome: findings after transfer of fresh and cryopreserved embryos

BACKGROUND We have previously shown that the medium used for culturing IVF embryos affects the birthweight of the resulting newborns. This observation with potentially far-reaching clinical consequences during later life, was made in singletons conceived during the first IVF treatment cycle after the transfer of fresh embryos. In the present study, we hypothesize that in vitro culture of embryos during the first few days of preimplantation development affects perinatal outcome, not only in singletons conceived in all rank order cycles but also in twins and in children born after transfer of frozen embryos. Furthermore, we investigated the effect of culture medium on gestational age (GA) at birth. METHODS Oocytes and embryos from consecutive treatment cycles were alternately assigned to culture in either medium from Vitrolife or from Cook. Data on a cohort of 294 live born singletons conceived after fresh transfer during any of a patients IVF treatment cycles, as well as data of 67 singletons conceived after frozen embryo transfer (FET) and of 88 children of 44 twin pregnancies after fresh transfer were analysed by means of multiple linear regression. RESULTS In vitro culture in medium from Cook resulted in singletons after fresh transfer with a lower mean birthweight (adjusted mean difference, 112 g, P= 0.03), and in more singletons with low birthweight (LBW) <2500 g (P= 0.006) and LBW for GA ≥ 37 weeks (P= 0.015), when compared with singletons born after culture in medium from Vitrolife AB. GA at birth was not related to the medium used (adjusted difference, 0.05 weeks, P = 0.83). Among twins in the Cook group, higher inter-twin mean birthweight disparity and birthweight discordance were found. Z-scores after FET were -0.04 (± 0.14) in the Cook group compared with 0.18 (± 0.21) in the Vitrolife group (P> 0.05). CONCLUSIONS Our findings support our hypothesis that culture medium influences perinatal outcome of IVF singletons and twins. A similar trend is seen in case of singletons born after FET. GA was not affected by culture medium. These results indicate that in vitro culture might be an important factor explaining the poorer perinatal outcome after assisted reproduction technology (ART). Further research is needed to confirm this culture medium-induced effect in humans and to provide more insight into whether it is caused by epigenetic disturbance of imprinted genes in fetal or placental tissues. Moreover, embryo culture media and their effects need to be investigated thoroughly to select the best embryo culture medium in order to minimize or prevent short-term risks and maybe even long-term disease susceptibility.

Placentas from pregnancies conceived by IVF/ICSI have a reduced DNA methylation level at the H19 and MEST differentially methylated regions

STUDY QUESTION Does IVF/ICSI have an effect on the epigenetic regulation of the human placenta? SUMMARY ANSWER We found a reduced DNA methylation level at the H19 and MEST differentially methylated regions (DMRs), and an increased RNA expression of H19 in placentas from pregnancies conceived by IVF/ICSI when compared with placentas from spontaneous conception. WHAT IS KNOWN ALREADY Changes in fetal environment are associated with adverse health outcomes. The placenta is pivotal for intrauterine environment. Animal studies show that epigenetic regulation plays an important role in these environment-induced phenotypic effects. Also, the preimplantation embryo environment affects birthweight as well as the risk of chronic adult diseases. Epigenetic processes are sensitive to the environment, especially during the period around conception. STUDY DESIGN AND PARTICIPANTS Placental tissue was collected from 35 spontaneously conceived pregnancies and 35 IVF/ICSI (5 IVF, 30 ICSI) derived pregnancies. We quantitatively analysed the DNA methylation patterns of a number of consecutive CpGs in the core regions of DMRs and other regulatory regions of imprinted genes, since these are involved in placental and fetal growth and development. METHODS By using pyrosequencing, the DNA methylation at seven germline-derived primary DMRs was analysed quantitatively. Five of these are maternally methylated (MEST isoform α and β, PEG3, KCNQ1OT1 and SNRPN) and two are paternally methylated [H19 DMR and the intergenic region between DLK1 and MEG3 (IG-DMR)]. The post-fertilization-derived secondary DMRs, IGF2 (DMR0 and 2) and IG-DMR (CG7, also called MEG3 DMR), and the MEG3 promoter region were examined as well. In case of differential methylation between the two groups, the effect on gene expression was assessed by quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE Both the promoter region of MEST isoform α and β and the 6th CTCF binding site within the H19 DMR were significantly hypomethylated in the IVF/ICSI group. The phenomenon was consistently observed over all CpG sites analysed and not restricted to single CpG sites. The other primary and secondary DMRs were not affected. Expression of H19 was increased in the IVF/ICSI group, while that of IGF2 and MEST remained similar. LIMITATIONS, REASONS FOR CAUTION In the IVF/ICSI group, mostly ICSI pregnancies were investigated. The ICSI technique or male subfertility could be a confounding factor. Therefore, our results are less generalizable to IVF pregnancies. WIDER IMPLICATIONS OF THE FINDINGS The clinical effects of the observed placental hypomethylations on the developmental programming of the IVF/ICSI progeny, if any, are as yet unknown. Whether the hypomethylation is an adaptation of the placenta to maintain fetal supply and ameliorate the effects of environmental cues, or whether it is a deregulation leading to deranged developmental programming with or without increased vulnerability for disease, consistent with the developmental origins of health and disease hypothesis, needs further investigation. STUDY FUNDING/COMPETING INTEREST(S) Partly funded by an unrestricted research grant by Organon BV (now MSD BV) without any role in study design, data collection and analysis, or preparation of the manuscript. No conflict of interests to declare. TRIAL REGISTRATION NUMBER Dutch Trial Registry (NTR) number 1298.

IVF culture medium affects post-natal weight in humans during the first 2 years of life

STUDY QUESTION Is post-natal growth during the first 2 years of life in IVF singletons affected by type of medium used for culturing human embryos during an IVF treatment? SUMMARY ANSWER The in vitro culture of human embryos in medium from Cook resulted in singletons with a lower weight during the first 2 years of life compared with singletons born after embryo culture in medium from Vitrolife. WHAT IS KNOWN ALREADY In a previous study, we reported that type of medium used for culturing human IVF embryos during the first few days after fertilization until fresh embryo transfer significantly affects fetal growth and consequently birthweight of the resulting singletons. STUDY DESIGN, SIZE, DURATION From July 2003 to December 2006, a total of 1432 IVF treatment cycles with fresh embryo transfer were randomly allocated to have all embryos cultured in medium from Vitrolife AB (n = 715) or from Cook (n = 717). Two years after delivery, questionnaires were sent to the parents of all children requesting data about weight, height and head circumference around 1, 2, 3, 4, 6, 7.5, 9, 11, 14, 18 and 24 months of age. These measurements were collected as part of the childrens health programme at municipal infant welfare centres in the Netherlands by health professionals unaware of this study. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients requiring donor oocytes or applying for PGD were excluded from the study. From the 294 live born singletons that fulfilled our inclusion criteria, 29 were lost to follow-up. The remaining 265 singletons (Cook group: 117, Vitrolife group: 148) were included in the analysis. Data analysis included linear regression, to compare cross-sectionally weight standard deviation score (SDS), height SDS and head circumference, and the first order Berkey-Reed model for a longitudinal analysis of the growth data. MAIN RESULTS AND THE ROLE OF CHANCE Singletons in the Vitrolife group were heavier during the first 2 years of life compared with singletons in the Cook group. Cross-sectional analyses showed that adjusted weight SDS differed between groups at 1 (0.35 ± 0.14, P = 0.010), 2 (0.39 ± 0.14, P = 0.006), 3 (0.35 ± 0.14, P = 0.011), 4 (0.30 ± 0.13, P = 0.020), 11 (0.28 ± 0.13, P = 0.036), 14 (0.32 ± 0.13, P = 0.014) and 24 (0.39 ± 0.15, P = 0.011) months of age, while adjusted height SDS was only significantly different at 1 (0.21 ± 0.11, P = 0.048) month of age. Head circumference was similar between the two groups at all ages. Longitudinal analyses showed that both post-natal weight (P = 0.005) and height (P = 0.031) differed between the groups throughout the first 2 years of life, while the growth velocity was not significantly different between the two groups. LIMITATIONS, REASONS FOR CAUTION Factors that might influence post-natal growth were included in the analysis; however, it was not possible to include all such factors, for example childhood diseases or nutrition, as this information was not available. WIDER IMPLICATIONS OF THE FINDINGS The effect of culture medium during the first few days after fertilization on prenatal growth and birthweight persists during the first 2 years of life. This suggests that the human embryo is sensitive to its very early environment, and that the culture medium used in IVF may have lasting consequences. Further monitoring of the long-term growth, development and health of IVF children is therefore warranted. STUDY FUNDING/COMPETING INTEREST(S) W.V. was funded with an unrestricted research grant from the Stichting Fertility Foundation. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER Not applicable.

Altered gene expression in human placentas after IVF/ICSI

STUDY QUESTION Is gene expression in placental tissue of IVF/ICSI patients altered when compared with a spontaneously conceived group, and are these alterations due to loss of imprinting (LOI) in the case of imprinted genes? SUMMARY ANSWER An altered imprinted gene expression of H19 and Pleckstrin homology-like domain family A member 2 (PHLDA2), which was not due to LOI, was observed in human placentas after IVF/ICSI and several biological pathways were significantly overrepresented and mostly up-regulated. WHAT IS KNOWN ALREADY Genomic imprinting plays an important role in placental biology and in placental adaptive responses triggered by external stimuli. Changes in placental development and function can have dramatic effects on the fetus and its ability to cope with the intrauterine environment. An increased frequency of placenta-related problems as well as an adverse perinatal outcome is seen in IVF/ICSI derived pregnancies, but the role of placental epigenetic deregulation is not clear yet. STUDY DESIGN AND PARTICIPANTS In this prospective cohort study, a total of 115 IVF/ICSI and 138 control couples were included during pregnancy. After applying several exclusion criteria (i.e. preterm birth or stillbirth, no placental samples, pregnancy complications or birth defects), respectively, 81 and 105 placentas from IVF/ICSI and control pregnancies remained for analysis. Saliva samples were collected from both parents. METHODS We quantitatively analysed the mRNA expression of several growth-related imprinted genes [H19, insulin-like growth factor 2 (IGF2), PHLDA2, cyclin-dependent kinase inhibitor 1C (CDKN1C), mesoderm-specific transcript homolog (MEST) isoform α and β by quantitative PCR] after standardization against three housekeeping genes [Succinate dehydrogenase A (SDHA), YWHAZ and TATA-binding protein (TBP)]. A quantitative allele-specific expression analysis of the differentially expressed imprinted genes was performed to investigate LOI, independent of the mechanism of imprinting. Furthermore, a microarray analysis was carried out (n = 10 in each group) to investigate the expression of non-imprinted genes as well. MAIN RESULTS AND THE ROLE OF CHANCE Both H19 and PHLDA2 showed a significant change, respectively, a 1.3-fold (P = 0.033) and 1.5-fold (P = 0.002) increase in mRNA expression in the IVF/ICSI versus control group. However, we found no indication that there is an increased frequency of LOI in IVF/ICSI placental samples. Genome-wide mRNA expression revealed 13 significantly overrepresented biological pathways involved in metabolism, immune response, transmembrane signalling and cell cycle control, which were mostly up-regulated in the IVF/ICSI placental samples. LIMITATIONS, REASONS FOR CAUTION Only a subset of samples was found to be fully informative, which unavoidably led to lower sample numbers for our LOI analysis. Our study cannot distinguish whether the reported differences in the IVF/ICSI group are exclusively attributable to the IVF/ICSI technique itself or to the underlying subfertility of the patients. WIDER IMPLICATIONS OF THE FINDINGS Whether these placental adaptations observed in pregnancies conceived by IVF/ICSI might be connected to an adverse perinatal outcome after IVF remains unknown. However, it is possible that these differences affect fetal development and long-term patterns of gene expression, as well as maternal gestational physiology. STUDY FUNDING/COMPETING INTERESTS Partly funded by an unrestricted research grant by Organon BV (now MSD BV) and GROW School for Oncology and Developmental Biology without any role in study design, data collection and analysis or preparation of the manuscript. No conflict of interests to declare. TRIAL REGISTRATION NUMBER Dutch Trial Registry (NTR) number 1298.

To do or not to do: IVF and ICSI in chronic hepatitis B virus carriers

Several assisted reproduction procedures, such as IVF and ICSI, are available for a variety of infertility problems. In fertility clinics, patients are screened for blood-borne viral infections, including hepatitis B virus (HBV). Reasons for screening are prevention of vertical transmission and laboratory safety. We present the case of a 26-year-old female patient with a chronic HBV infection, whose husband tested negative for hepatitis B. She and her husband were referred to our fertility clinic because of subfertility. Analysis of the husbands semen indicated the necessity of an ICSI procedure. The current Dutch guidelines advise against ICSI in chronic HBV carriers, since the risks and effects of chromosomal integration of HBV DNA in the fetus are not well-known. In this article, we review the scientific evidence for the risk of introducing HBV virus into the oocyte and subsequent integration of HBV DNA into the human genome, and debate the question of whether to do, or not to do, IVF and ICSI.