Safety of embryo cryopreservation: insights from mid-term placental transcriptional changes.

BACKGROUND: In recent years, with benefits from the continuous improvement of clinical technology and the advantage of fertility preservation, the application of embryo cryopreservation has been growing rapidly worldwide. However, amidst this growth, concerns about its safety persist. Numerous studies have highlighted the elevated risk of perinatal complications linked to frozen embryo transfer (FET), such as large for gestational age (LGA) and hypertensive disorders during pregnancy. Thus, it is imperative to explore the potential risk of embryo cryopreservation and its related mechanisms. METHODS: Given the strict ethical constraints on clinical samples, we employed mouse models in this study. Three experimental groups were established: the naturally conceived (NC) group, the fresh embryo transfer (Fresh-ET) group, and the FET group. Blastocyst formation rates and implantation rates were calculated post-embryo cryopreservation. The impact of FET on fetal growth was evaluated upon fetal and placental weight. Placental RNA-seq was conducted, encompassing comprehensive analyses of various comparisons (Fresh-ET vs. NC, FET vs. NC, and FET vs. Fresh-ET). RESULTS: Reduced rates of blastocyst formation and implantation were observed post-embryo cryopreservation. Fresh-ET resulted in a significant decrease in fetal weight compared to NC group, whereas FET reversed this decline. RNA-seq analysis indicated that the majority of the expression changes in FET were inherited from Fresh-ET, and alterations solely attributed to embryo cryopreservation were moderate. Unexpectedly, certain genes that showed alterations in Fresh-ET tended to be restored in FET. Further analysis suggested that this regression may underlie the improvement of fetal growth restriction in FET. The expression of imprinted genes was disrupted in both FET and Fresh-ET groups. CONCLUSION: Based on our experimental data on mouse models, the impact of embryo cryopreservation is less pronounced than other in vitro manipulations in Fresh-ET. However, the impairment of the embryonic developmental potential and the gene alterations in placenta still suggested it to be a risky operation.

Msuite: A High-Performance and Versatile DNA Methylation Data-Analysis Toolkit.

DNA methylation is a pervasive and important epigenetic regulator in mammalian genome. For DNA methylome profiling, emerging bisulfite-free methods have demonstrated desirable superiority over the conventional bisulfite-treatment-based approaches, although current analysis software could not make full use of their advantages. In this work, we present Msuite, an easy-to-use, all-in-one data-analysis toolkit. Msuite implements a unique 4-letter analysis mode specifically optimized for emerging protocols; it also integrates quality controls, methylation call, and data visualizations. Msuite demonstrates substantial performance improvements over current state-of-the-art tools as well as fruitful functionalities, thus holding the potential to serve as an optimal toolkit to facilitate DNA methylome studies. Source codes and testing datasets for Msuite are freely available at https://github.com/hellosunking/Msuite/.

[Clinical and genetic analysis of childhood-onset myoclonus dystonia syndrome caused by SGCE variants].

Objective: To explore the clinical characteristics and genotyping results of childhood-onset myoclonus dystonia syndrome caused by SGCE variants. Methods: The clinical data of 9 children with SGCE-related myoclonus dystonia syndrome admitted at either the Department of Neurology, Beijing Children's Hospital, Capital Medical University or the Department of Pediatrics, Peking University First Hospital from May 2018 to October 2019 were collected and the patients were followed up. The definite diagnosis was made on the basis of whole exome sequencing and multiple ligation-dependent probe amplification. The clinical features and gene test results were analyzed retrospectively. Results: Data of 9 patients (4 boys and 5 girls) diagnosed as myoclonus dystonia syndrome caused by SGCE variants were collected. The onset age ranged from 1 year to 3 years and 2 months. The first symptom was myoclonus in 4 cases, while dystonia in the remaining 5 cases. In the course of the disease, 9 cases had myoclonus and 8 had dystonia. Myoclonic jerks were characterized by involuntary jerks in both upper limbs in 8 patients. Six patients had involuntary jerks of lower limbs, resulting in gait instability or even falling. The myoclonus was exacerbated during the fine motor activities, emotional stress or fatigue. Dystonia was characterized by abnormal gait, including 5 cases with right leg dystonia, and 3 cases with the left leg dystonia. Three probands had a positive family history. Intellectual development was normal in all cases. There was no obvious abnormality in video-electroencephalogram (EEG) during both ictal and interictal periods. Electromyography (EMG) and brain magnetic resonance imaging (MRI) of 9 patients were normal. Nine patients carried SGCE gene variants, including 3 frame shift variants, 2 nonsense variants, 2 missense variants, 1 fragment deletion variant and 1 splice site variant. Seven variants were inherited paternally, and 2 variants were de novo. Madopar was used in 8 patients, and nitrazepam in 4 patients, leading to the decrease in the myoclonus jerks and improvement of gait in 6 and 2 patients, respectively. Conclusions: SGCE gene variants can cause myoclonus dystonia syndrome. The onset of the disease may occur at infancy or preschool age, with either myoclonic jerks or dystonia as the initial symptom. Non-epileptic myoclonus is the prominent symptom, with upper limb mainly involved. Most of the patients have the accompanying symptoms of dystonia, and some of them may have spontaneous symptom relief. SGCE gene is imprinted maternally, and the inherited variants of SGCE are paternal in origin.

Clinical and genetic analysis of childhood-onset myoclonus dystonia syndrome caused by SGCE variants / 中华儿科杂志

Objective@#To explore the clinical characteristics and genotyping results of childhood-onset myoclonus dystonia syndrome caused by SGCE variants.@*Methods@#The clinical data of 9 children with SGCE-related myoclonus dystonia syndrome admitted at either the Department of Neurology, Beijing Children′s Hospital, Capital Medical University or the Department of Pediatrics, Peking University First Hospital from May 2018 to October 2019 were collected and the patients were followed up. The definite diagnosis was made on the basis of whole exome sequencing and multiple ligation-dependent probe amplification. The clinical features and gene test results were analyzed retrospectively.@*Results@#Data of 9 patients (4 boys and 5 girls) diagnosed as myoclonus dystonia syndrome caused by SGCE variants were collected. The onset age ranged from 1 year to 3 years and 2 months. The first symptom was myoclonus in 4 cases, while dystonia in the remaining 5 cases. In the course of the disease, 9 cases had myoclonus and 8 had dystonia. Myoclonic jerks were characterized by involuntary jerks in both upper limbs in 8 patients. Six patients had involuntary jerks of lower limbs, resulting in gait instability or even falling. The myoclonus was exacerbated during the fine motor activities, emotional stress or fatigue. Dystonia was characterized by abnormal gait, including 5 cases with right leg dystonia, and 3 cases with the left leg dystonia. Three probands had a positive family history. Intellectual development was normal in all cases. There was no obvious abnormality in video-electroencephalogram (EEG) during both ictal and interictal periods. Electromyography (EMG) and brain magnetic resonance imaging (MRI) of 9 patients were normal. Nine patients carried SGCE gene variants, including 3 frame shift variants, 2 nonsense variants, 2 missense variants, 1 fragment deletion variant and 1 splice site variant. Seven variants were inherited paternally, and 2 variants were de novo. Madopar was used in 8 patients, and nitrazepam in 4 patients, leading to the decrease in the myoclonus jerks and improvement of gait in 6 and 2 patients, respectively.@*Conclusions@#SGCE gene variants can cause myoclonus dystonia syndrome. The onset of the disease may occur at infancy or preschool age, with either myoclonic jerks or dystonia as the initial symptom. Non-epileptic myoclonus is the prominent symptom, with upper limb mainly involved. Most of the patients have the accompanying symptoms of dystonia, and some of them may have spontaneous symptom relief. SGCE gene is imprinted maternally, and the inherited variants of SGCE are paternal in origin.

Detailed observation on expression dynamics of Polycomb group genes during rice early endosperm development in subspecies hybridization reveals their characteristics of parent-of-origin genes.

BACKGROUND: Parent-of-origin gene expression and its role in seed development have drown a great attention in recent years. Genome-wide analysis has identified hundreds of candidate imprinted genes, a major type of parent-of-origin genes, in rice hybrid endosperms at the stage of 5 days after pollination (dap). However, the expression of these genes in early endosperm have been never confirmed due to technique limitations and the behavior of the imprinted genes in different rice hybridizations are still largely unknown. RESULTS: Here, based on our elaborate technique established previously, the expression patterns of PcG genes in the early stages of endosperm development (within 3 dap), were comprehensively analyzed. We revealed that the free nucleus stage of endosperm development is critical for parent-of-origin gene analysis. The expression of the imprinted genes are highly dynamic, likely corresponding to the critical developmental events during this period. Hybridizations between Oryza sativa japonica and indica showed that the expression patterns of the same imprinted gene could be varied by crossing with different parental cultivars, indicative of their parent-dependent character. There are strong alleles that often showed predominant expression over other alleles regardless of the parental origin, which provides a possible explanation for the cultivar-dependent predominant phenotype in crop hybridizations. In addition, we found that the transcripts of the same gene behave differently, with imprinting or non-imprinting patterns, suggesting the existence of not only imprinted and non-imprinted genes but also imprinted or non-imprinted transcripts, which reveals new aspects of the genomic imprinting. CONCLUSIONS: These findings on the characters of parent-of-origin genes shed light on the understanding the real role of gene imprinting in endosperm development.

Paternal benzo[a]pyrene exposure alters the sperm DNA methylation levels of imprinting genes in F0 generation mice and their unexposed F1-2 male offspring.

BACKGROUND: Benzo[a]pyrene (BaP) is an environmental pollutant known to cause teratogenesis. However, the mechanism underlying this teratogenic effect is not fully understood. Recently, the alteration of DNA methylation of imprinting genes has emerged as a specific epigenetic mechanism linking the impact of environmental pollutants on embryonic development to paternal exposures. The aim of this study was to investigate the transgenerational effects of paternal BaP exposure on the imprinting genes in mouse sperm DNA. METHODS: Male C57BL/6J mice received BaP (1.0 or 2.5 mg/kg) or olive oil twice a week for 12 weeks. The methylation status of 6 imprinting genes (H19, Meg3, Peg1, Peg3, Igf2 and Snrpn) was examined by bisulfite pyrosequencing of the sperm DNA of BaP-exposed F0 generation and their offspring. RESULTS: BaP exposure reduced the methylation levels in the imprinting genes H19 and Meg3 and increased the methylation levels of Peg1 and Peg3; however, no significant differences was observed for the methylation levels of Igf2 or Snrpn in the sperm DNA. Furthermore, BaP-exposed male mice were mated with unexposed female mice to generate F1-2 generations. The methylation levels of the 6 genes in the sperm DNA from F1-2 offspring showed a similar pattern as that of the F0 male. The effects were attenuated in F1-2 generations. CONCLUSIONS: Paternal BaP exposure altered the methylation levels of imprinting genes, implicating that imprinting genes are susceptible to environmental toxicants. Furthermore, a similar alteration was observed in the F1-2 generations although the attenuated in methylation in F2 generation, revealing a potential transgenerational effect.

Changes in DNA methylation and imprinting disorders in E9.5 mouse fetuses and placentas derived from vitrified eight-cell embryos.

Vitrification is increasingly used in assisted reproductive technology (ART) laboratories worldwide, and potential vitrification-induced risks require further exploration. The effect of vitrification on changes in DNA methylation and imprinting disorders was investigated in E9.5 mouse fetuses and placentas. Fetus and placental tissues were collected from the natural mating (nautural conception [NC]) group, in vitro culture (IVC) group and vitrified embryo transfer (VET) group. The fetal crown-rump length at E9.5 in both the IVC (0.210 ± 0.059 mm) and VET (0.205 ± 0.048 mm) groups was significantly reduced compared with the NC group (0.288 ± 0.083 mm). The global methylation levels of fetuses were decreased in the IVC group compared with the NC group and it was increased after vitrification compared with IVC (p < 0.05), similar to what was observed in the NC group (p > 0.05). The changes could be attributed to the disorders of DNA methyltransferases and ten-eleven translocations. In the IVC and VET fetuses, a majority of maternally expressed genes were upregulated, which repressed fetal growth. Furthermore, vitrification led to a change in the methylation level of KvDMR1, which resulted in the disturbance of gene imprinting. According to our results, vitrification could contribute to increased methylation compared with IVC and contributes to a gene imprinting disorder rather than recovery. Despite the routine use of embryo vitrification in clinical settings, the effect that this procedure may have on genomic imprinting deserves much greater attention.

Allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci using Staphylococcus aureus Cas9 (SaCas9).

Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions (DMRs), and aberrant genomic imprinted DNA methylation is associated with some human diseases, including Prader-Willi syndrome and cancer. Thus, the development of an effective strategy for the precise editing of allele-specific methylated genes is essential for the functional clarification of imprinting elements and the correction of imprinting disorders in human diseases. To discover a feasible allele-specific genome editing tool based on the CRISPR/Cas system, which is an efficient gene-targeting technique in various organisms, we examined the targeting efficiency of Staphylococcus aureus Cas9 (SaCas9) and Streptococcus pyogenes Cas9 (SpCas9) in response to DNA methylation interference. We found that the targeting efficiency of SaCas9, but not SpCas9, was enhanced by targeted DNA demethylation using the dCas9-Tet1 catalytic domain (CD) but suppressed by targeted DNA methylation using Dnmt3l-Dnmt3a-dCas9. An in vitro cleavage assay further demonstrated that SaCas9 nuclease activity was inhibited by 5-methylcytosine (5mC) in a synthesized CpG-containing context. Further analysis with ChIP-Q-PCR demonstrated that the non-methylated sequence targeting of SaCas9 depends on the binding preference of SaCas9 to non-methylated sequences. Taking advantage of this feature of SaCas9, we have successfully obtained non-methylated allele-biased targeted embryos/mice for two imprinting genes, H19 and Snrpn, with relatively high efficiencies of 28.6% and 47.4%, respectively. These results indicate that the targeting efficiency of SaCas9 was strongly reduced by DNA methylation. By using SaCas9, we successfully achieved allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci.

Effects of the HDAC inhibitor scriptaid on the in vitro development of bovine embryos and on imprinting gene expression levels.

This study examines the effects of the histone deacetylation inhibitor scriptaid (SCR) on preimplantation embryo development in vitro and on imprinting gene expression. We hypothesized that SCR would increase histone acetylation levels, enhance embryonic genome activation, and regulate imprinting and X-chromosome inactivation (XCI) in in vitro produced bovine embryos. Zygotes were cultured in vitro in presence or absence of SCR added at different time points. We assessed cleavage and blastocyst rates as well as the quality of blastocysts through: (i) differential cell counts; (ii) survival after vitrification/thawing and (iii) gene expression analysis -including imprinted genes. Blastocyst yields were not different in the control and experimental groups. While no significant differences were observed between groups in total cell or trophectoderm cell numbers, SCR treatment reduced the number of inner cell mass cells and improved the survival of vitrified embryos. Further, genes involved in the mechanism of paternal imprinting (GRB10, GNAS, XIST) were downregulated in presence of SCR compared with controls. These observations suggest SCR prevents deacetylation of paternally imprinting control regions and/or their up-regulation, as these events took place in controls. Whether or not such reductions in XIST and imprinting gene expression are beneficial for post implantation development remains to be clarified.

Effect of Intrauterine Malnutrition on Growth Characters and the DNA Methylation Level of H19 of Their Offspring / 医学研究杂志

Objective To establish a mouse model of intrauterine malnutrition to investigate the growth characters and the DNA methylation level of H19 of the offspring. Methods The pregnancy ICR mice were randomly divided into the food restricted group and the control group. The food eaten by each pregnant mouse every day was monitored from d0. 5 to d12. 5. The food restricted pregnancy mice were given half amount food of the daily consumption during d0. 5-d12. 5. The mice in control group were given the normal food. The pregnant mice of both groups of were given normal food after delivery. The number, birth weight and sex ratio of the offspring were calculated. Furthermore, the body weight of the offspring was monitored at different growth stages. The oocytes of female offspring mice were obtained to detect the DNA methylation level of H19. Results (1) Compared to normal feeding pregnant mice, mice with food restricted were small and less active, and even some of them were died before delivery. The incidence of premature birth was significantly increased (P ＜0. 05). The number of abortion, stillbirth pregnant mice in the food restriction group were more than that in the control group, but the difference was not statistically significant (P ＞ 0. 05). (2) There was no significant difference in the number of offspring between pregnancy mice with food restricted or normal food (P ＞ 0. 05). As well as the number of male offspring between the two groups (P ＞ 0. 05). However, the offspring of the food restricted pregnant mice showed significant low birth weight (P ＜ 0. 05). (3) At the age of 3 weeks, there was still a low body weight in the offspring of the food restricted pregnant mice, but the difference had no significant difference between groups (P ＞ 0. 05). After weaning at 3 weeks, the offspring of food restricted pregnant mice began the catch-up growth. At the age of 6 weeks, the body weight of offspring of food restricted pregnant mice was higher than that of the normal offspring, but the difference was not statistically significant (P ＞ 0. 05). During the period of 6-8 weeks, the weight of offspring of normal feed pregnancy mice was increased slowly. But the body weight of food restriction offspring mice was increased rapidly. At the age of 8 weeks, the body weight of offspring of food restricted group was significantly higher than that in control group (P ＜ 0. 05). (4) The methylation level of H19 in oocyte from offspring of food restricted pregnancy mice was normal. Conclusion Food restriction at pregnancy period not only affect the outcome of pregnancy, but also increased the rate of premature birth. The offspring of food restriction pregnancy mice showed low birth weight. Until the age of 6 weeks, the offspring of food restriction pregnancy mice had lower body weight compared with the offspring with normal feeding pregnancy mice. The offspring of food restriction pregnancy mice demonstrated obesity at 8 weeks. But the H19 methylation levels of oocytes from offspring of pregnancy mice with food restriction was normal.

A Study on Methylation Status of Imprinted Gene H19 and PEG1 in Different Quality of Sperms and Embryos / 昆明医科大学学报

Objective By comparing the methylation status of imprinted gene H19 in three different quality spermatozoa and the methylation status of the embryos after the combination of spermatozoa, the sperm methylation status in the ART technique was preliminarily explored with the postnatal embryo methylation.Methods In this study, a total of 91 spermatozoa and 91 low－quality embryos were collected from IVF－ET in the First People's Hospital of Yunnan Province. Among them, sperm samples were divided into three groups according to the sperm concentration parameter in the WHO fourth edition,then the relationship between different quality spermatozoa and postnatal embryo methylation were analyzed.Results The abnormality rate of H19 methylation status in group B was significantly higher than that of the sperm group and the embryo group.Conclusions The abnormalities of H19 methylation mainly concentrated in the abnormal parameters of spermatozoa,suggesting that the abnormal state was related to the decrease of sperm quality;Through IVF, ICSI fertilized embryos have the same proportion of the corresponding abnormal state with no significant difference; Abnormal sperm embryos did not show abnormality, which may be related to the self－repair function of the embryo itself.

The emerging landscape of in vitro and in vivo epigenetic allelic effects.

Epigenetic mechanisms that cause maternally and paternally inherited alleles to be expressed differently in offspring have the potential to radically change our understanding of the mechanisms that shape disease susceptibility, phenotypic variation, cell fate, and gene expression. However, the nature and prevalence of these effects in vivo have been unclear and are debated. Here, I consider major new studies of epigenetic allelic effects in cell lines and primary cells and in vivo. The emerging picture is that these effects take on diverse forms, and this review attempts to clarify the nature of the different forms that have been uncovered for genomic imprinting and random monoallelic expression (RME). I also discuss apparent discrepancies between in vitro and in vivo studies. Importantly, multiple studies suggest that allelic effects are prevalent and can be developmental stage- and cell type-specific. I propose some possible functions and consider roles for allelic effects within the broader context of gene regulatory networks, cellular diversity, and plasticity. Overall, the field is ripe for discovery and is in need of mechanistic and functional studies.

Altered GNAS imprinting due to folic acid deficiency contributes to poor embryo development and may lead to neural tube defects.

Disturbed epigenetic modifications have been linked to the pathogenesis of Neural Tube Defects (NTDs) in those with folate deficiency during pregnancy. However, evidence is lacking to delineate the critical region in epigenome regulated by parental folic acid and mechanisms by which folate deficiency affects normal embryogenesis. Our data from clinical samples revealed the presence of aberrant DNA methylation in GNAS imprinting cluster in NTD samples with low folate concentrations. Results from mouse models indicated that the establishment of GNAS imprinting was influenced by both maternal and paternal folate-deficient diets. Such aberrant GNAS imprinting was present prior to the gametogenesis period. Imprinting in Exon1A/GNAS gDMR was abolished in both spermatozoa and oocytes upon treating with a parental folate-deficient diet (3.6% in spermatozoa, 9.8% in oocytes). Interestingly, loss of imprinting in the GNAS gene cluster altered chromatin structure to an overwhelmingly open structure (58.48% in the folate-free medium group vs. 39.51% in the folate-normal medium group; P < 0.05), and led to a disturbed expression of genes in this region. Furthermore, an elevated cyclic AMP levels was observed in folate acid deficiency group. Our results imply that GNAS imprinting plays major roles in folic acid metabolism regulation during embryogenesis. Aberrant GNAS imprinting is an attribute to NTDs, providing a new perspective for explaining the molecular mechanisms by which folate supplementation in human pregnancy provides protection from NTDs.

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study.

Effect on the H19 gene methylation of sperm and organs of offspring after chlorpyrifos-methyl exposure during organogenesis period.

To elucidate the effect on the H19 gene methylation of sperm and organs in offspring by chlorpyrifos-methyl (CPM) exposure during organogenesis period, CPM was administered at doses of 4 (CPM4), 20 (CPM20), and 100 (CPM100) mg/kg bw/day from 7 days post coitum (d.p.c.) to 17 d.p.c. after mating CAST/Ei (♂) and B6 (♀). Anogenital distance (AGD) was measured at postnatal day (PND) 21. Clinical signs, body weights, feed and water consumption, organs weights, serum hormone values, and H19 methylation level of organ and sperm were measured at PND63. Body weights were significantly lower than control until PND6. AGD was significantly decreased in the CPM100 group in males and increased in the CPM20 group in females. The absolute weights of the thymus and epididymis were significantly increased for males in all of CPM treatment groups. In the CPM20 group, absolute weights of liver, kidney, heart, lung, spleen, prostate gland, and testes were significantly increased. Testosterone concentrations in serum were significantly increased by CPM treatment in males. H19 methylation level of liver and thymus showed decreased pattern in a dose-dependent manner in males. The levels of H19 methylation in sperm were 73.76 ± 7.16% (Control), 57.84 ± 12.94% (CPM4), 64.24 ± 3.79% (CPM20), and 64.24 ± 3.79% (CPM100). Conclusively, CPM exposure during organogenesis period can disrupt H19 methylation in sperm, liver, and thymus and disturb the early development of offspring.

Imprinting genes associated with endometriosis.

PURPOSE: Much work has been carried out to investigate the genetic and epigenetic basis of endometriosis and proposed that endometriosis has been described as an epigenetic disease. The purpose of this study was to extract the imprinting genes that are associated with endometriosis development. METHODS: The information on the imprinting genes can be accessed publicly from a web-based interface at http://www.geneimprint.com/site/genes-by-species. RESULTS: In the current version, the database contains 150 human imprinted genes derived from the literature. We searched gene functions and their roles in particular biological processes or events, such as development and pathogenesis of endometriosis. From the genomic imprinting database, we picked 10 genes that were highly associated with female reproduction; prominent among them were paternally expressed genes (DIRAS3, BMP8B, CYP1B1, ZFAT, IGF2, MIMT1, or MIR296) and maternally expressed genes (DVL1, FGFRL1, or CDKN1C). These imprinted genes may be associated with reproductive biology such as endometriosis, pregnancy loss, decidualization process and preeclampsia. DISCUSSION: This study supports the possibility that aberrant epigenetic dysregulation of specific imprinting genes may contribute to endometriosis predisposition.