Epigenetics and the placenta Nelissen, Ewka C M; van Montfoort, Aafke P A; Dumoulin, John C M ...
Human reproduction update,
05/2011, Letnik:
17, Številka:
3
Journal Article
Recenzirano
Odprti dostop
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.
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.
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.
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.
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 children's 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.
Abstract
STUDY QUESTION
Does embryo culture medium influence pregnancy and perinatal outcome in IVF?
SUMMARY ANSWER
Embryo culture media used in IVF affect treatment efficacy and the birthweight of ...newborns.
WHAT IS KNOWN ALREADY
A wide variety of culture media for human preimplantation embryos in IVF/ICSI treatments currently exists. It is unknown which medium is best in terms of clinical outcomes. Furthermore, it has been suggested that the culture medium used for the in vitro culture of embryos affects birthweight, but this has never been demonstrated by large randomized trials.
STUDY DESIGN, SIZE, DURATION
We conducted a multicenter, double-blind RCT comparing the use of HTF and G5 embryo culture media in IVF. Between July 2010 and May 2012, 836 couples (419 in the HTF group and 417 in the G5 group) were included. The allocated medium (1:1 allocation) was used in all treatment cycles a couple received within 1 year after randomization, including possible transfers with frozen–thawed embryos. The primary outcome was live birth rate.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Couples that were scheduled for an IVF or an ICSI treatment at one of the six participating centers in the Netherlands or their affiliated clinics.
MAIN RESULTS AND THE ROLE OF CHANCE
The live birth rate was higher, albeit nonsignificantly, in couples assigned to G5 than in couples assigned to HTF (44.1% (184/417) versus 37.9% (159/419); RR: 1.2; 95% confidence interval (CI): 0.99–1.37; P = 0.08). Number of utilizable embryos per cycle (2.8 ± 2.3 versus 2.3 ± 1.8; P < 0.001), implantation rate after fresh embryo transfer (20.2 versus 15.3%; P < 0.001) and clinical pregnancy rate (47.7 versus 40.1%; RR: 1.2; 95% CI: 1.02–1.39; P = 0.03) were significantly higher for couples assigned to G5 compared with those assigned to HTF. Of the 383 live born children in this trial, birthweight data from 380 children (300 singletons (G5: 163, HTF: 137) and 80 twin children (G5: 38, HTF: 42)) were retrieved. Birthweight was significantly lower in the G5 group compared with the HTF group, with a mean difference of 158 g (95% CI: 42–275 g; P = 0.008). More singletons were born preterm in the G5 group (8.6% (14/163) versus 2.2% (3/137), but singleton birthweight adjusted for gestational age and gender (z-score) was also lower in the G5 than in the HTF group (−0.13 ± 0.08 versus 0.17 ± 0.08; P = 0.008).
LIMITATIONS, REASONS FOR CAUTION
This study was powered to detect a 10% difference in live births while a smaller difference could still be clinically relevant. The effect of other culture media on perinatal outcome remains to be determined.
WIDER IMPLICATIONS OF THE FINDINGS
Embryo culture media used in IVF affect not only treatment efficacy but also perinatal outcome. This suggests that the millions of human embryos that are cultured in vitro each year are sensitive to their environment. These findings should lead to increased awareness, mechanistic studies and legislative adaptations to protect IVF offspring during the first few days of their existence.
STUDY FUNDING/COMPETING INTEREST(S)
This project was partly funded by The NutsOhra foundation (Grant 1203-061) and March of Dimes (Grant 6-FY13-153). The authors declare no conflict of interest.
TRIAL REGISTRATION NUMBER
NTR1979 (Netherlands Trial Registry).
TRIAL REGISTRATION DATE
1 September 2009.
DATE OF FIRST PATIENT'S ENROLMENT
18 July 2010.
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.
STUDY QUESTION
Do culture media influence birthweight of children born after IVF?
SUMMARY ANSWER
Some studies have observed a significant effect of culture media on birthweight, while others have ...not, but since most studies compared different culture media, conventional meta-analysis was not possible.
WHAT IS KNOWN ALREADY
Animal studies suggest that in vitro culture of embryos can have a significant effect on the birthweight of offspring when compared with in vivo developed embryos. The type of culture medium (or certain components of the medium) used is one of the causal factors.
STUDY DESIGN, SIZE, DURATION
We reviewed all available literature reporting on a relation between culture medium and birthweight in human studies and a selection of animal studies.
PARTICIPANTS/MATERIALS, SETTING, METHODS
An extensive literature search on Pubmed and Medline was performed with relevant search criteria relating to IVF, birthweight and culture medium.
MAIN RESULTS AND THE ROLE OF CHANCE
Eleven studies reporting on a relationship between culture medium and birthweight in human were included in this review. Five of these found significant differences in birthweight when offspring born after culture in different culture media were compared. The remaining studies did not find differences in birthweight after changing culture medium.
LIMITATIONS, REASONS FOR CAUTION
The number of human studies is limited and different culture media with different compositions are compared which makes a comparison between the studies difficult, if not impossible. Furthermore, most study designs were retrospective with consecutive use of different culture media and limited sample sizes, which makes bias of the results likely.
WIDER IMPLICATIONS OF THE FINDINGS
If it could be confirmed that the type of culture medium used does indeed influence phenotypic characteristics (such as birthweight) of children born after IVF, it would underline the importance of monitoring the health of IVF children in relation to aspects of the laboratory techniques used during embryo culture.
STUDY FUNDING/COMPETING INTEREST(S)
No funding was applicable to this study. No conflict of interest is declared.
Besides the established selection criteria based on embryo morphology and blastomere number, new parameters for embryo viability are needed to improve the clinical outcome of IVF and more particular ...of elective single-embryo transfer. Genome-wide gene expression in cumulus cells was studied, since these cells surround the oocyte inside the follicle and therefore possibly reflect oocyte developmental potential. Early cleavage (EC) was chosen as a parameter for embryo viability. Gene expression in cumulus cells from eight oocytes resulting in an EC embryo (EC-CC; n = 8) and from eight oocytes resulting in a non-EC (NEC) embryo (NEC-CC; n = 8) was analysed using microarrays (n = 16). A total of 611 genes were differentially expressed (P < 0.01), mainly involved in cell cycle, angiogenesis, apoptosis, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor signalling, general vesicle transport and chemokine and cytokine signalling. Of the 25 selected differentially expressed genes analysed by quantitative real-time PCR 15 (60%) genes could be validated in the original samples. Of these 8 (53%) could also be validated in 24 (12-EC-CC and 12 NEC-CC) extra independent samples. The most differentially expressed genes among these were CCND2, CXCR4, GPX3, CTNND1 DHCR7, DVL3, HSPB1 and TRIM28, which probably point to hypoxic conditions or a delayed oocyte maturation in NEC-CC samples. This opens up perspectives for new molecular embryo or oocyte selection parameters which might also be useful in countries where the selection has to be made at the oocyte stage before fertilization instead of at the embryonic stage.
STUDY QUESTION
When does a difference in human intrauterine growth of singletons conceived after IVF and embryo culture in two different culture media appear?
SUMMARY ANSWER
Differences in fetal ...development after culture of embryos in one of two IVF media were apparent as early as the second trimester of pregnancy.
WHAT IS KNOWN ALREADY
Abnormal fetal growth patterns are a major risk factor for the development of chronic diseases in adult life. Previously, we have shown that the medium used for culturing embryos during the first few days after fertilization significantly affects the birthweight of the resulting human singletons. The exact onset of this growth difference was unknown.
STUDY DESIGN, SIZE AND DURATION
In this retrospective cohort study, all 294 singleton live births after fresh embryo transfer in the period July 2003 to December 2006 were included. These embryos originated from IVF treatments that were part of a previously described clinical trial. Embryos were allocated to culture in either Vitrolife or Cook commercially available sequential culture media.
PARTICIPANTS/MATERIALS, SETTING, METHODS
We analysed ultrasound examinations at 8 (n = 290), 12 (n = 83) and 20 weeks’ (n = 206) gestation and used first-trimester serum markers pregnancy-associated plasma protein-A (PAPP-A) and free β-hCG. Differences between study groups were tested by the Student's t-test, χ2 test or Fisher's exact test, and linear multivariable regression analysis to adjust for possible confounders (for example, parity, gestational age at the time of ultrasound and fetal gender).
MAIN RESULTS AND THE ROLE OF CHANCE
A total of 294 singleton pregnancies (Vitrolife group nVL = 168, Cook group: nC = 126) from 294 couples were included. At 8 weeks’ gestation, there was no difference between crown-rump length-based and ovum retrieval-based gestational age (ΔGA) (nVL = 163, nC = 122, adjusted mean difference, −0.04 days, P = 0.84). A total of 83 women underwent first-trimester screening at 12 weeks’ gestation (nVL = 45, nC = 38). ΔGA, nuchal translucency (multiples of median, MoM) and PAPP-A (MoM) did not differ between the study groups. Free β-hCG (MoM) ± SEM differed significantly (1.55 ± 0.19 in Vitrolife versus 1.06 ± 0.10 in Cook; P = 0.031, Student's t-test). At 20 weeks’ gestation, a more advanced GA, reflecting an increased fetal growth, was seen at ultrasound examination in the Vitrolife group (n = 115) when compared with the Cook group (n = 91). After adjustment for confounding factors, both the difference between GA based on three biparietal diameter dating formulas minus the actual (ovum retrieval based) GA (adjusted mean difference + 1.14 days (P = 0.04), +1.14 days (P = 0.04) and +1.36 days (P = 0.048)), as well as head circumference (HC) and trans-cerebellar diameter (TCD) were significantly higher in the Vitrolife group (HCvl 177.3 mm, HCc 175.9 mm, adjusted mean difference 1.8, P = 0.03; TCDvl 20.5 mm, TCDc 20.2 mm, adjusted mean difference 0.4, P = 0.008).
LIMITATIONS, REASONS FOR CAUTION
A first trimester (12 weeks) fetal screening was not yet offered routinely during the study period, therefore only 28% of women in our study participated in this elective screening programme. Although all sonographers were experienced and specially trained to perform these ultrasound examinations and were unaware of the randomization procedure, we cannot totally rule out possible intra- and inter-observer variability. Despite being indispensable in daily practice, sonographic weight formulas have a limited accuracy.
WIDER IMPLICATIONS OF THE FINDINGS
According to the fetal origins hypothesis, many adult diseases originate in utero owing to adaptations made by the fetus to the environment it encounters. This study indicates that the embryonic environment is already important for fetal development. Therefore, our study emphasizes the need to investigate fetal growth patterns after assisted reproduction technologies and long-term health outcomes of IVF children, especially in relation to the culture medium used during the first few days of preimplantation development.
TRIAL REGISTRATION NUMBER
Not applicable.
Abstract
Study question
Is there a difference in DNA methylation status of imprinted genes in placentas derived from IVF conceptions where embryo culture was performed in human tubal fluid (HTF) ...versus G5 culture medium?
Summary answer
We found no statistically significant differences in the mean DNA methylation status of differentially methylated regions (DMRs) associated with parentally imprinted genes in placentas derived from IVF conceptions cultured in HTF versus G5 culture medium.
What is known already
Animal studies indicate that the embryo culture environment affects the DNA methylation status of the embryo. In humans, birthweight is known to be affected by the type of embryo culture medium used. The effect of embryo culture media on pregnancy, birth and child development may thus be mediated by differential methylation of parentally imprinted genes in the placenta.
Study design, size, duration
To identify differential DNA methylation of imprinted genes in human placenta derived from IVF conceptions exposed to HTF or G5 embryo culture medium, placenta samples (n = 43 for HTF, n = 54 for G5) were collected between 2010 and 2012 s as part of a multi-center randomized controlled trial in the Netherlands comparing these embryo culture media. Placenta samples from 69 naturally conceived (NC) live births were collected during 2008–2013 in the Netherlands as reference material.
Participants/materials, setting, methods
To identify differential DNA methylation of imprinted genes, we opted for an amplicon-based sequencing strategy on an Illumina MiSeq sequencing platform. DNA was isolated and 34 DMRs associated with well-defined parentally imprinted genes were amplified in a two-step PCR before sequencing using MiSeq technology. Sequencing data were analyzed in a multivariate fashion to eliminate possible confounding effects.
Main results and the role of chance
We found no statistically significant differences in the mean DNA methylation status of any of the imprinted DMRs in placentas derived from IVF conceptions cultured in HTF or G5 culture medium. We also did not observe any differences in the mean methylation status per amplicon nor in the variance in methylation per amplicon between the two culture medium
groups. A separate surrogate variable analysis also demonstrated that the IVF culture medium was not associated with the DNA methylation status of these DMRs. The mean methylation level and variance per CpG was equal between HTF and G5 placenta. Additional comparison of DNA methylation status of NC placenta samples revealed no statistically significant differences in mean amplicon and CpG methylation between G5, HTF and NC placenta; however, the number of placenta samples exhibiting outlier methylation levels was higher in IVF placenta compared to NC (P < 0.00001). Also, we were able to identify 37 CpG sites that uniquely displayed outlier methylation in G5 placentas and 32 CpG sites that uniquely displayed outlier methylation in HTF. In 8/37 (G5) and 4/32 (HTF) unique outliers CpGs, a medium-specific unique outlier could be directly correlated to outlier methylation of the entire amplicon.
Limitations, reasons for caution
Due to practical reasons, not all placentas were collected during the trial, and we collected the placentas from natural conceptions from a different cohort, potentially creating bias. We limited ourselves to the DNA methylation status of 34 imprinted DMRs, and we studied only the placenta and no other embryo-derived tissues.
Wider implications of the findings
It has often been postulated, but has yet to be rigorously tested, that imprinting mediates the effects of embryo culture conditions on pregnancy, birth and child development in humans. Since we did not detect any statistically significant effects of embryo culture conditions on methylation status of imprinted genes in the placenta, this suggests that other unexplored mechanisms may underlie these effects. The biological and clinical relevance of detected outliers with respect to methylation levels of CpGs and DMR require additional analysis in a larger sample size as well. Given the importance and the growing number of children born through IVF, research into these molecular mechanisms is urgently needed.
Study funding/competing interest(s)
This study was funded by the March of Dimes grant number #6-FY13-153. The authors have no conflicts of interest.
Trial registration number
Placental biopsies were obtained under Netherlands Trial Registry number 1979 and 1298.
STUDY QUESTION
Is gene expression in human preimplantation embryos affected by the medium used for embryo culture in vitro during an IVF treatment?
SUMMARY ANSWER
Six days of in vitro culture of ...human preimplantation embryos resulted in medium-dependent differences in expression level of genes involved in apoptosis, protein degradation, metabolism and cell-cycle regulation.
WHAT IS KNOWN ALREADY
Several human studies have shown an effect of culture medium on embryo development, pregnancy outcome and birthweight. However, the underlying mechanisms in human embryos are still unknown. In animal models of human development, it has been demonstrated that culture of preimplantation embryos in vitro affects gene expression. In humans, it has been found that culture medium affects gene expression of cryopreserved embryos that, after thawing, were cultured in two different media for 2 more days.
STUDY DESIGN, SIZE, DURATION
In a multicenter trial, women were randomly assigned to two culture medium groups G5 and human tubal fluid (HTF). Data on embryonic development were collected for all embryos. In one center, embryos originating from two pronuclei (2PN) zygotes that were not selected for transfer or cryopreservation on Day 2 or 3 because of lower morphological quality, were cultured until Day 6 and used in this study, if couples consented.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Ten blastocysts each from the G5 and HTF study groups, matched for fertilization method, maternal age and blastocyst quality, were selected and their mRNA was isolated and amplified. Embryos were examined individually for genome-wide gene expression using Agilent microarrays and PathVisio was used to identify the pathways that showed a culture medium-dependent activity.
MAIN RESULTS AND THE ROLE OF CHANCE
Expression of 951 genes differed significantly (P < 0.01) between the G5 and HTF groups. Eighteen pathways, involved in apoptosis, metabolism, protein processing and cell-cycle regulation, showed a significant overrepresentation of differentially expressed genes. The DNA replication, G1 to S cell-cycle control and oxidative phosphorylation pathways were up-regulated in the G5 group compared with the HTF group. This is in agreement with the morphological assessment of the 1527 embryos (originating from 2PN zygotes), which showed that embryos consisted of more cells on Day 2 (3.73 ± 1.30 versus 3.40 ± 1.35, P < 0.001) and Day 3 (7.00 ± 2.41 versus 5.84 ± 2.36, P < 0.001) in the G5 group when compared with the HTF group. Furthermore, the implantation rate was significantly higher in the G5 group compared with the HTF group (26.7% versus 14.7%, P = 0.002) after transfer on the second or the third day after fertilization.
LIMITATIONS, REASONS FOR CAUTION
Despite careful matching of the embryos, it cannot be excluded that the differences observed between the study groups are caused by factors that we did not investigate. Extrapolation of these results to embryos used for transfer demands caution as in the present study embryos that were not selected for either embryo transfer or cryopreservation have been used for the culture experiment until Day 6.
WIDER IMPLICATIONS OF THE FINDINGS
This study shows that gene expression in human preimplantation embryos is altered by the culture medium used during IVF treatment and provides insight into the biological pathways that are affected. Whether these changes in gene expression have any long-term effects on children born after IVF remains unknown. However, it is possible that early adaptations of the preimplantation embryo to its environment persist during fetal and post-natal development.
STUDY FUNDING/COMPETING INTEREST(S)
No funding and no competing interests declared.
TRIAL REGISTRATION NUMBER
Not applicable.
Abstract
STUDY QUESTION
Can we detect DNA methylation differences between ART children that underwent embryo culture in different media?
SUMMARY ANSWER
We identified no significant differences in ...site-specific or regional DNA methylation between the different culture medium groups.
WHAT IS KNOWN ALREADY
Embryo culture in G3 or K-SICM medium leads to differences in embryonic, neonatal and childhood outcomes, including growth and weight. The methylome may mediate this association as the period of in vitro culture of ART treatments coincides with epigenetic reprogramming.
STUDY DESIGN, SIZE, DURATION
This study was conducted as a follow-up to a previous culture medium comparison study in which couples were pseudo-randomized to embryo culture in G3 or K-SICM medium. Of the resultant singletons, 120 (n = 65 G3, n = 55 K-SICM), were recruited at age 9.
PARTICIPANTS/MATERIALS, SETTING, METHODS
The ART children provided a saliva sample from which the methylome was analysed using the Infinium MethylationEPIC array. After quality and context filtering, 106 (n = 57 G3, n = 49 K-SICM) samples and 659 708 sites were retained for the analyses. Differential methylation analyses were conducted using mixed effects linear models corrected for age, sex, sample plate and cell composition. These were applied to all cytosine-guanine dinucleotide (CpG) sites, various genomic regions (genes, promoters, CpG Islands (CGIs)) and as a targeted analysis of imprinted genes and birth weight-associated CpG sites. Differential variance was assessed using the improved epigenetic variable outliers for risk prediction analysis (iEVORA) algorithm and methylation outliers were identified using a previously defined threshold (upper or lower quartile plus or minus three times the interquartile range, respectively).
MAIN RESULTS AND THE ROLE OF CHANCE
After correcting for multiple testing, we did not identify any significantly differentially methylated CpG sites, genes, promoters or CGIs between G3 and K-SICM children despite a lenient corrected P-value threshold of 0.1. Targeted analyses of (sites within) imprinted genes and birth weight-associated sites also did not identify any significant differences. The number of DNA methylation outliers per sample was comparable between the culture medium groups. iEVORA identified 101 differentially variable CpG sites of which 94 were more variable in the G3 group.
LARGE SCALE DATA
Gene Expression Omnibus (GEO) GSE196432
LIMITATIONS, REASONS FOR CAUTION
To detect significant methylation differences with a magnitude of <10% between the groups many more participants would be necessary; however, the clinical relevance of such small differences is unclear.
WIDER IMPLICATIONS OF THE FINDINGS
The results of this study are reassuring, suggesting that if there is an effect of the culture medium on DNA methylation (and methylation-mediated diseases risk), it does not differ between the two media investigated here. The findings concur with other methylome studies of ART neonates and children that underwent embryo culture in different media, which also found no significant methylome differences.
STUDY FUNDING/COMPETING INTEREST(S)
Study funded by March of Dimes (6-FY13-153), EVA (Erfelijkheid Voortplanting & Aanleg) specialty programme (grant no. KP111513) of Maastricht University Medical Centre (MUMC+) and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission. The authors do not report any conflicts of interest relevant to this study.
TRIAL REGISTRATION NUMBER
Dutch Trial register—NL4083
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