Does underlying infertility in natural conception modify the epigenetic control of imprinted genes and transposable elements in newborns?

RESEARCH QUESTION: Does the epigenetic control of imprinted genes and transposable elements at birth differ according to time to conception in natural conception and after intrauterine insemination (IUI)? DESIGN: A total of 144 singletons were included in four groups: 50 natural pregnancies obtained within 6 months after stopping contraception (group 1); 34 natural pregnancies with infertility period between 6 and 12 months (group 2); 36 pregnancies with an infertility period of more than 12 months (group 3) and 24 pregnancies obtained after IUI (group 4). RESULTS: The placental DNA methylation levels of H19/IGF2 and KCNQ1OT1 were lower in groups 2, 3 and 4 than in group 1 (P = 0.025 in the overall comparison). The DNA methylation rate for LINE-1 was higher in placentas from group 2 than in group 1 (P = 0.022). In cord blood, DNA methylation levels were not significantly different between groups except for H19/IGF2 for which the DNA methylation levels were higher in group 2 than in group 1 (H19/IGF2-seq1 and seq2: P = 0.023 and P = 0.002, respectively). In placenta tissue, compared with group 1, relative expression for SNRPN and for LINE-1 was significantly higher in group 2 (P = 0.002 and P < 0.001, respectively). The relative expression of KCNQ1 in placenta was lower in group 4 than in group 1 (P = 0.013). In cord blood, compared with group 1, the relative expression for H19 was significantly higher in group 3 (P = 0.026), and the relative expression of LINE-1 was higher in groups 2 and 3 and in group 4 (P < 0.001). CONCLUSIONS: Infertility itself, and not only ART techniques, could contribute to potential epigenetic risks for children.

Placental volume and other first-trimester outcomes: are there differences between fresh embryo transfer, frozen-thawed embryo transfer and natural conception?

RESEARCH QUESTION: Does mode of conception influence placental volume and other first-trimester outcomes? DESIGN: This retrospective single-centre case-control study led in Dijon University Hospital included 252 singleton pregnancies (84 IVF with either fresh embryo transfer or frozen-thawed embryo transfer [FET] and 168 natural conceptions). First-trimester placental volume, uterine artery pulsatility index and maternal serum PAPP-A and beta-HCG were measured. Statistical analyses were adjusted for gestational age, the newborn's gender, maternal age, parity, body mass index and smoking status. RESULTS: Placental volume was significantly greater in the FET group than in the control group (P = 0.043) and fresh embryo transfer (P = 0.023) groups. At birth, fresh embryo transfer newborns were significantly smaller than controls (P = 0.01) and FET newborns (P = 0.008). Postpartum haemorrhage was far more frequent in FET than in controls and fresh embryo transfer group (38.1%, 2.6% and 1.9%, respectively; P < 0.0001). Placental volume positively correlated with PAPP-A, beta-HCG and the newborn's birth weight, and negatively correlated with uterine artery pulsatility index. CONCLUSIONS: Placental volume and other first-trimester parameters are modified by IVF with fresh embryo transfer and FET compared with natural conception, but with opposite trends. Given the different protocols used for these techniques, hormonal treatment per se may have a major effect on pregnancy outcomes through the modification of placental invasiveness.

Germline correction of an epimutation related to Silver-Russell syndrome.

Like genetic mutations, DNA methylation anomalies or epimutations can disrupt gene expression and lead to human diseases. However, unlike genetic mutations, epimutations can in theory be reverted through developmental epigenetic reprograming, which should limit their transmission across generations. Following the request for a parental project of a patient diagnosed with Silver-Russell syndrome (SRS), and the availability of both somatic and spermatozoa DNA from the proband and his father, we had the exceptional opportunity to evaluate the question of inheritance of an epimutation. We provide here for the first time evidence for efficient reversion of a constitutive epimutation in the spermatozoa of an SRS patient, which has important implication for genetic counseling.

Do frozen embryo transfers modify the epigenetic control of imprinted genes and transposable elements in newborns compared with fresh embryo transfers and natural conceptions?

OBJECTIVE: To determine whether the epigenetic control of imprinted genes (IGs) and transposable elements (TEs) differs at birth between fresh or frozen embryo transfers and natural conceptions. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): A total of 202 singleton births were divided into three groups: 84 natural pregnancies (controls), 66 in vitro fertilization/intracytoplasmic sperm injection with fresh embryo transfers, and 52 vitro fertilization/intracytoplasmic sperm injection with frozen embryo transfers. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pyrosequencing was used to assess the DNA methylation profiles of three IGs (H19/IGF2:IG-DMR [two sequences], KCNQ1OT1:TSS-DMR, and SNURF:TSS-DMR) and two TEs (LINE-1 and HERV-FRD) in cord blood and placenta. The quantitative reverse transcriptase polymerase chain reaction was used to study the transcription of three IGs (H19, KCNQ1, and SNRPN) and two TEs (LINE-1 and ORF2). RESULT(S): After adjustment, the placental DNA methylation levels of H19/IGF2 were lower in the fresh embryo transfer group than in the control (H19/IGF2-seq1) and frozen embryo transfer (H19/IGF2-seq2) groups. The DNA methylation rate for LINE-1 was lower in placentas from the fresh embryo transfer group than in placentas from the control and frozen embryo transfer groups and for HERV-FRD compared with controls. In cord blood, DNA methylation levels were not significantly associated with the mode of conception. The relative expression of LINE-1 and ORF2 was decreased in both cord blood and placental tissues from fresh embryo transfer conceptions compared with natural conceptions and frozen embryo transfer conceptions. CONCLUSION(S): Compared with natural conceptions and frozen embryo transfers, fresh embryo transfers were associated with methylation and/or transcription changes in some TEs and IGs, mostly in placental samples, which could indicate altered placental epigenetic regulation resulting from ovarian stimulation protocols.

What impact does oocyte vitrification have on epigenetics and gene expression?

Children conceived by assisted reproductive technologies (ART) have a moderate risk for a number of adverse events and conditions. The question whether this additional risk is associated with specific procedures used in ART or whether it is related to the intrinsic biological factors associated with infertility remains unresolved. One of the main hypotheses is that laboratory procedures could have an effect on the epigenome of gametes and embryos. This suspicion is linked to the fact that ART procedures occur precisely during the period when there are major changes in the organization of the epigenome. Oocyte freezing protocols are generally considered safe; however, some evidence suggests that vitrification may be associated with modifications of the epigenetic marks. In this manuscript, after describing the main changes that occur during epigenetic reprogramming, we will provide current information regarding the impact of oocyte vitrification on epigenetic regulation and the consequences on gene expression, both in animals and humans. Overall, the literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by oocyte vitrification, and it also underlines the need to improve our knowledge in this field.

The hypomethylation of imprinted genes in IVF/ICSI placenta samples is associated with concomitant changes in histone modifications.

Although more and more children are born by Assisted Reproductive Technologies (ART), ART safety has not fully been demonstrated. Notably, ART could disturb the delicate step of implantation, and trigger placenta-related adverse outcomes with potential long-term effects, through disrupted epigenetic regulation. We have previously demonstrated that placental DNA methylation was significantly lower after IVF/ICSI than following natural conception at two differentially methylated regions (DMRs) associated with imprinted genes (IGs): H19/IGF2 and KCNQ1OT1. As histone modifications are critical for placental physiology, the aim of this study was to profile permissive and repressive histone marks in placenta biopsies to reveal a better understanding of the epigenetic changes in the context of ART. Utilizing chromatin immunoprecipitation (ChIP) coupled with quantitative PCR, permissive (H3K4me3, H3K4me2, and H3K9ac) and repressive (H3K9me3 and H3K9me2) post-translational histone modifications were quantified. The analyses revealed a significantly higher quantity of H3K4me2 precipitation in the IVF/ICSI group than in the natural conception group for H19/IGF2 and KCNQ1OT1 DMRs (P = 0.016 and 0.003, respectively). Conversely, the quantity of both repressive marks at H19/IGF2 and SNURF DMRs was significantly lower in the IVF/ICSI group than in the natural conception group (P = 0.011 and 0.027 for H19/IGF2; and P = 0.010 and 0.035 for SNURF). These novel findings highlight that DNA hypomethylation at imprinted DMRs following ART is linked with increased permissive/decreased repressive histone marks, altogether promoting a more permissive chromatin conformation. This concomitant change in epigenetic state at IGs at birth might be an important developmental event because of ART manipulations.

Differences in expression rather than methylation at placenta-specific imprinted loci is associated with intrauterine growth restriction.

BACKGROUND: Genome-wide studies have begun to link subtle variations in both allelic DNA methylation and parent-of-origin genetic effects with early development. Numerous reports have highlighted that the placenta plays a critical role in coordinating fetal growth, with many key functions regulated by genomic imprinting. With the recent description of wide-spread polymorphic placenta-specific imprinting, the molecular mechanisms leading to this curious polymorphic epigenetic phenomenon is unknown, as is their involvement in pregnancies complications. RESULTS: Profiling of 35 ubiquitous and 112 placenta-specific imprinted differentially methylated regions (DMRs) using high-density methylation arrays and pyrosequencing revealed isolated aberrant methylation at ubiquitous DMRs as well as abundant hypomethylation at placenta-specific DMRs. Analysis of the underlying chromatin state revealed that the polymorphic nature is not only evident at the level of allelic methylation, but DMRs can also adopt an unusual epigenetic signature where the underlying histones are biallelically enrichment of H3K4 methylation, a modification normally mutually exclusive with DNA methylation. Quantitative expression analysis in placenta identified two genes, GPR1-AS1 and ZDBF2, that were differentially expressed between IUGRs and control samples after adjusting for clinical factors, revealing coordinated deregulation at the chromosome 2q33 imprinted locus. CONCLUSIONS: DNA methylation is less stable at placenta-specific imprinted DMRs compared to ubiquitous DMRs and contributes to privileged state of the placenta epigenome. IUGR-associated expression differences were identified for several imprinted transcripts independent of allelic methylation. Further work is required to determine if these differences are the cause IUGR or reflect unique adaption by the placenta to developmental stresses.

Sperm imprinting integrity in seminoma patients?

BACKGROUND: Testicular germ cell tumor such as seminoma is strongly associated with male reproductive problems commonly associated with the alteration of sperm parameters as described in testicular dysgenesis syndrome. Interestingly, numerous studies have reported that the precursor of germ cell cancer, germ cell neoplasia in situ (GCNIS), present similarities to fetal gonocytes, specifically characterized by global DNA hypomethylation particularly on imprinting sequences. These disorders may have a common origin derived from perturbations of embryonal programming during fetal development. Presently, there is no available information concerning the sperm DNA methylation patterns of testicular cancer patients. For the first time, we evaluated the sperm imprinting of seminoma patients. A total of 92 cryopreserved sperm samples were included, 31 before seminoma treatment (S): 23 normozoospermic (SN) and 8 oligozoospermic (SO) and 61 sperm controls samples: 31 normozoospermic (N) and 30 oligozoospermic (O). DNA methylation levels of seven differentially methylated regions (DMRs) of imprinted genes [H19/IGF2: IG-DMR (CTCF3 and CTCF6 of H19 gene); IGF2-DMRs (DMR0 and DMR2); MEG3/DLK1:IG-DMR; SNURF:TSS-DMR; KCNQ1OT1:TSS-DMR] were assessed by pyrosequencing. All comparative analyses were adjusted for age. RESULTS: Comparisons of sperm DNA methylation levels between seminoma (S) and normozoospermic (N) samples showed a significant difference for the SNURF sequence (p = 0.017), but after taking into account the sperm parameters, no difference was observed. However, we confirmed a significant association between oligozoospermia (O) and imprinting defects for H19/IGF2-CTCF6 (p = 0.001), MEG3/DLK1 (p = 0.017), IGF2-DMR2 (p = 0.022), and SNURF (p = 0.032) in comparison with control groups (N). CONCLUSIONS: This study highlights the high risk of sperm imprinting defects in cases of oligozoospermia and shows for the first time that seminoma patients with normal spermatogenesis present sperm imprinting integrity. These data suggest a low probability of the involvement of a common imprinting defect in fetal cells leading to both TGCT and subfertility.

Singleton fetal growth kinetics depend on the mode of conception.

OBJECTIVE: To study the impact of in vitro fertilization, with or without intracytoplasmic sperm injection (IVF/ICSI), frozen-embryo transfer (FET), and intrauterine insemination (IUI) on fetal growth kinetics throughout pregnancy and to compare the different modes of conception. DESIGN: Retrospective cohort study. SETTING: University. PATIENT(S): A total of 560 singleton pregnancies were included (96 IVF, 210 ICSI, 121 FET, and 133 IUI). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): We compared crown-rump length (CRL) at the first trimester (T1: 11-13 weeks of gestation [WG] + 6 days), estimated fetal weight (EFW) at the second (T2: 21-23 WG + 6 days) and third (T3: 31-33 WG + 6 days) trimesters, and birth weight (BW) z-scores with those in the reference curves (Papageorghiou for T1, and Ego M2 for T2, T3, and birth). Multivariate analyses were performed. RESULT(S): For T1, the CRL was longer than the reference curve whatever the assisted reproductive technique (ART). For T2, EFW was significantly greater for all groups compared with the reference curve, and for T3 only FET singletons had a greater EFW. ICSI, IVF, and IUI singletons had a significantly lower BW compared with reference curves. For all ART fetuses, growth kinetics differed from T2. Only FET fetuses maintained their significantly above-reference growth values. The proportion of fetuses for which at least one period of growth loss was observed from T2 to birth was higher after IVF, ICSI, and IUI than after FET. CONCLUSION(S): For the first time, we have highlighted that fetal growth kinetics differed from T2 depending on the ART protocols used. They could have an impact on trophoblastic invasiveness and might lead to long-term health effects.

Randomized controlled trial comparing embryo culture in two incubator systems: G185 K-System versus EmbryoScope.

OBJECTIVE: To study whether the closed culture system, as compared with a benchtop incubator with similar culture conditions, has a positive impact on intracytoplasmic sperm injection (ICSI) outcomes. DESIGN: Randomized controlled trial. SETTING: University hospital. PATIENT(S): A total of 386 patients undergoing ICSI cycles with at least six mature oocytes were randomized. INTERVENTION(S): Of these patients, 195 were assigned to the group with culture in a time-lapse imaging (TLI) system (EmbryoScope) and 191 to the group with culture in the G185 K-System (G185). MAIN OUTCOME MEASURE(S): Rate of implantation (primary endpoint) and embryo morphology grade. RESULT(S): No significant differences were found in the implantation rates. The proportion of high-grade embryos on day 2 was significantly higher in the TLI group compared with the G185 group (40.4% vs. 35.2%). The impact of the incubator on embryo morphology remained significant in multivariate analysis, which took into account the woman's age, the rank of attempt, and the smoking status (TLI vs. G185: odds ratio = 1.27; 95% confidence interval, [1.04-1.55]). No difference was found in the mean number of frozen embryos, even though the total proportion of frozen embryos was significantly higher in the TLI group than in the G185 group (29.5% vs. 24.8%). CONCLUSION(S): No difference in implantation rate was found between the two incubators for fresh cycles. It remains to be determined whether the observed differences in embryo morphology and the total number of embryos cryopreserved would translate into higher cumulative outcomes with subsequent frozen embryo transfers. CLINICAL TRIAL REGISTRATION NO: NCT02722252.

Severe ovarian hyperstimulation syndrome modifies early maternal serum beta-human chorionic gonadotropin kinetics, but obstetrical and neonatal outcomes are not impacted.

OBJECTIVE: To study the impact of severe ovarian hyperstimulation syndrome (OHSS) on beta-hCG kinetics and obstetrical and neonatal outcomes. DESIGN: Retrospective single-center case-control study. SETTING: University tertiary referral center. PATIENT(S): A total of 77 patients who presented a clinical pregnancy after IVF and had been hospitalized for severe OHSS were included in this study and compared with 231 controls presenting an IVF-induced clinical pregnancy without OHSS and matched for the year of pregnancy and the number of gestational sacs. INTERVENTION(S): None. MAIN OUTCOMES MEASURE(S): The outcome of pregnancy (miscarriage, medical abortion, or delivery), beta-hCG values, obstetrical, and neonatal outcomes. RESULT(S): After multivariate analysis adjusted for parity, tobacco smoking, presence of polycystic ovary syndrome, age, and body mass index, outcomes of pregnancies were not altered by OHSS. However, there was a trend toward a lower early miscarriage rate in the OHSS group (7.8%) than in the control group (16%). Maternal serum beta-hCG values at different time points of the pregnancy and fold changes of beta-hCG values were lower in OHSS than in controls (268 ± 160 vs. 389 ± 215 IU/L at day 16; and 4.8 ± 1.5 vs. 5.4 ± 1.4 fold change between day 16 and day 20). Beta-hCG also correlated negatively with the number of oocytes retrieved. Incidence of gestational diabetes, gestational hypertension, intrauterine growth restriction, premature birth, and low birth weight did not differ between groups. CONCLUSION(S): Although early maternal serum beta-hCG kinetics were modified in women after severe OHSS, the outcomes of these pregnancies remained comparable to those of IVF pregnancies without OHSS. According to these data, pregnancies after severe OHSS do not require particular care compared with IVF pregnancies, but differences in beta-hCG levels and kinetics should be taken into account when interpreting these results.

Embryo multinucleation at the two-cell stage is an independent predictor of intracytoplasmic sperm injection outcomes.

OBJECTIVE: To determine the prognostic impact of the nuclear status at the two-cell stage on intracytoplasmic sperm injection (ICSI) outcomes. DESIGN: Retrospective study. SETTING: Hospital. PATIENT(S): Only ICSI cycles with time-lapse monitoring of transferred embryos with known implantation/delivery data from November 2012 to December 2014 were included. A total of 2,449 embryos were assessed for multinucleation rates at the two- and four-cell stage, and 608 transferred embryos were studied for ICSI outcomes. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation rate (IR) and live birth rate (LBR) according to the number of multinucleated blastomeres at the two-cell stage: none (Without-MNB2cell), one (MNB1/2cell), and two (MNB2/2cell); morphokinetics of MNB2cell embryos. RESULT(S): Embryos with MNB1/2cell led to lower IR (27.7%) and LBR (22.7%) than embryos Without-MNB2cell (33.4% and 29.8%, respectively). The MNB2/2cell embryos led to significantly lower IR (18.3%) and LBR (13.4%) than embryos Without-MNB2cell. This difference remained significant in multivariate analysis for implantation (odds ratio 0.57; 95% confidence interval 0.34-0.94) and birth (odds ratio 0.46; 95% confidence interval 0.26-0.80), independently of the other significant parameters (women's age, time of two-cell formation, and multinucleation at the four-cell stage). Among implanted MNB2cell, if cleavage into four cells occurred later than 37 hours after insemination, embryos were significantly more likely to lead to birth. CONCLUSION(S): The presence of multinucleation at the two-cell stage and more specifically in both blastomeres had a significant negative impact on birth potential. Thus, embryo multinucleation at the two-cell stage should be used as an additional noninvasive criterion for embryo selection.

The placenta: phenotypic and epigenetic modifications induced by Assisted Reproductive Technologies throughout pregnancy.

Today, there is growing interest in the potential epigenetic risk related to assisted reproductive technologies (ART). Much evidence in the literature supports the hypothesis that adverse pregnancy outcomes linked to ART are associated with abnormal trophoblastic invasion. The aim of this review is to investigate the relationship between epigenetic dysregulation caused by ART and subsequent placental response. The dialogue between the endometrium and the embryo is a crucial step to achieve successful trophoblastic invasion, thus ensuring a non-complicated pregnancy and healthy offspring. However, as described in this review, ART could impair both actors involved in this dialogue. First, ART may induce epigenetic defects in the conceptus by modifying the embryo environment. Second, as a result of hormone treatments, ART may impair endometrial receptivity. In some cases, it results in embryonic growth arrest but, when the development of the embryo continues, the placenta could bring adaptive responses throughout pregnancy. Amongst the different mechanisms, epigenetics, especially thanks to a finely tuned network of imprinted genes stimulated by foetal signals, may modify nutrient transfer, placental growth and vascularization. If these coping mechanisms are overwhelmed, improper maternal-foetal exchanges occur, potentially leading to adverse pregnancy outcomes such as abortion, preeclampsia or intra-uterine growth restriction. But in most cases, successful placental adaptation enables normal progress of the pregnancy. Nevertheless, the risks induced by these modifications during pregnancy are not fully understood. Metabolic diseases later in life could be exacerbated through the memory of epigenetic adaptation mechanisms established during pregnancy. Thus, more research is still needed to better understand abnormal interactions between the embryo and the milieu in artificial conditions. As trophectoderm cells are in direct contact with the environment, they deserve to be studied in more detail. The ultimate goal of these studies will be to render ART protocols safer. Optimization of the environment will be the key to improving the dialogue between the endometrium and embryo, so as to ensure that placentation after ART is similar to that following natural conception.