DNA Methylation Status Of Imprinted H19,PEG1,KvDMR1 Genes In Human Sperm, IVM Oocytes And Preimplantaion Embryos

Assisted reproductive technology (ART) has been performed for over 20 years in China, and some derivative techniques, such as in vitro maturation (IVM), embryos freezing are also becoming popular in the clinical application. The offspring born after ART is becoming an important part of population, thus concerns about the safety of ART have been increasing. Many related studies have been performed but remain inconclusive. Recent studies suggest that in vitro fertilization (IVF)/ intracytoplasmic sperm injection may be associated with increased incidence of Angelman syndrome (AS) and Beckwith-Wiedemann syndrome (BWS). The majority of ART-conceived children with BWS and AS demonstrated imprinting defects, so Concerns about imprinting disorders linked to ART are therefore increasing. Many animal studies revealed that some steps of manipulation in the ART procedure, such as in vitro maturation (IVM), superovulation with high-dose hormone and in vitro embryo culture etc. may cause abnormal methylation profiles in oocytes or embryos. However, these conclusions cannot substitute for human studies, which are limited owing to ethical and legal restrictions. In this study, we collected human sperm, in vitro matured oocytes and fresh/frozen Day3 embryos produced during ART procedures and determined their methylation status of imprinted H19, PEGU1,KvDMR1 genes. We hope that our results could help to evaluate epigenetic risks linked to ART.PartⅠMethylation status of imprinted H19.PEG1,KvDMR1 genes in human sperm and embryos of ART patients[Objective] Previous studies revealed that men with oligospermia or azoospermia exhibited abnormal methylation in sperm cells, and the methylation errors could be transmitted to villus. In this study we collected abandoned embryos and sperm samples during IVF/ICSI procedures. We determined their H19, PEG1, and KvDMRl methylation status to investigate whether the imprinting errors can be transmitted to embryos, primarily evaluating epigenetic risks linked to sperm.[Methods] Patients undergoing ART procedures at the NanFang Hospital and Guiyang Maternal & Children Hospital from 2008-2011 were involved. The study protocol was approved by the Institutional Ethical Committee and Institutional Review Board. Informed consent was obtained from all patients. The sperm samples were collected after insemination, and Day3 embryos were collected after embryo transfer.(1) A total of 130 sperm samples were collected, and divided in four groups:A. Normospermia (sperm count> 20×106/mL):50; B. Moderate oligospermia (5×106/mL< sperm count< 20×106/mL):40 samples; C. Severe oligospermia (sperm count< 5×106/mL):25 samples; D. Azoospermioa:15 samples. The Combined Bisulfite Restriction Analysis, (COBRA) and Bisulfite Sequencing PCR (BSP) were used to determine H19, PEG1, and KvDMR1 methylation status in sperm cells.(2) According to the results of sperm methylation analysis, the Day3 embryos belonging to cases with abnormal methylation were examined for methylation status. Embryos belonging to cases with normospermia were used as control.60 embryos were used for H19 analysis,83 embryos for PEG1 and 58 embryos for KvDMRl.[Results](1) 18 of 130 sperm samples were found with abnormal errors, the total rate of aberration was 13.8%. The rates of abnormal methylation were 20.0% in Moderate oligospermia group (8/40),32.0% in severe oligospermia group (8/25), and 12.3% in TESE/PESE sperm group (1/15). The normospermia group also showed 2.0% abnormal methylation (1/50). Comparing the normospermia to aberrant-parameters sperm group (including moderate oligospermia, severe oligospermia and TESE/PESE sperm group), the rate of abnormal methylation was higher in aberrant-parameters sperm group (21.3%) (x2= 9.56, P= 0.002).(2) Compare the rate of aberrant methylation of different genes:aberrant methylation patterns of PEG1 were found in 12 sperm samples (9.2%, 12/130). The rate was significantly higher than H19 (2.3%) and KvDMR1 (2.3%) (χ2= 5.73, P= 0.017).(3) PCR amplification at single-embryo level:H19 gene was successfully amplified in 45 embryos (success rate:75.0%); PEG1 gene was successfully amplified in 56 embryos (success rate:67.5%); KvDMRl gene was successfully amplified in 40 embryos (success rate:69.0%).(4) Methylation status in embryos:a. H19:5 embryos were found with hypomethylation (12.5%,5/40),4 of them belonged to patients with hypomethylation in sperm cells (30.8%,4/13), and 1 of them was from control cases (3.1%,1/32), and the difference was noted (χ2= 4.62, P= 0.031); b. PEG1:8 embryos were found with hypermethylation, and belonged to patients with hypermethylation in sperm cells (25.8%,8/31).2 embryos from control cases showed hypomethylation patterns. The rates of hypermethylation between two groups were significantly different (χ2 5.57, P= 0.018); c. KvDMR1:no methylation errors were found in embryos from patients with abnormal methylation, but 3 embryos from control cases showed hypomethylation patterns.[Conclusions](1) For men with oligospermia or azoospermia, the rate of abnormal methylation in sperm cells was higher than that of patients with normospermia; the abnormal methylation patterns may caused by abnormal alteration of DNA methyltransferases (Dnmts) during spermatogenesis, but further studies are needed to confirm this hypothesis. Sperm cells from normospermia patients could also exhibit aberrant methylation status, but the rate was much lower.(2) Methylation errors of sperm cells could be transmitted to embryos through ICSI technique. However, embryos produced by IVF showed no imprinting errors, and it is likely due to the fact that the sperm with methylation errors have lower ability of fertilization. So IVF application may have lower risks.(3) The absolute incidence of imprinting defects in babies conceived by ICSI is low, but it is still necessary to limit the indications of ICSI application for reduce the epigenetic risks.(4) The further work is to enlarge the sample size and gene quantity, confirming the hypothesis above.PartⅡThe effect of in vitro maturation and freezing on DNA methylation status of imprinted genes in human early embryosSectionⅠThe DNA methylation status of H19 and PEGl gene in human embryos after oocyte in vitro maturation[Objective] In vitro maturation (IVM) is a new ART technique. It is cost-effective and can reduce the incidence of OHSS. It is suitable for PCOS/PCO patients. Many babies have been born after IVM. Some studies suggested that IVM could cause abnormal imprinting in oocytes. In this study, we collected in vitro matured oocytes and corresponding embryos, and determine their methylation status of H19 and PEG1 genes, primarily evaluating epigenetic risks linked to IVM.[Methods](1) After getting the approval of Institutional Ethical Committee and informed consent of patients, PCOS/PCO patients undergoing IVM at the NanFang Hospital and Guiyang Maternal & Children Hospital from 2009-2011 were involved in the study. We collected the following samples:GV-arrested (Germinal vesicle) and MⅠ-arrested (MetaphaseⅠ) oocytes after 48h in vitro culture; abandoned Day3 embryos (2～9 cells,Ⅰ～Ⅳgrade); sperm samples (after insemination); embryos of routine IVF/ICSI patients was used as control (2～12 cells,Ⅰ-Ⅳgrade)(2) Methylation analysis:the Combined Bisulfite Restriction Analysis, (COBRA), pyrosequencing and Bisulfite Sequencing PCR (BSP) were used to determine H19 and PEG1 methylation status.[Results](1) PCR amplification at single-embryo level:a. H19 gene:H19 gene was successfully amplified in 52 IVM-embryos (success rate:75.0%,52/65) and 58 routine IVF/ICSI-embryos (success rate:82.9%,58/70); b. PEG1 gene: PEG1 gene was successfully amplified in 37 IVM- embryos (success rate: 74.0%,37/50) and 40 routine IVF/ICSI-embryos (success rate:69.0%, 40/58);(2) Methylation status in embryos:a. H19:the mean average methylation was 48.1%±5.8% for IVM group and 46.7%±6.0% for routine IVF/ICSI group and no difference were noted (t=1.98, P=0.258).5 embryos were found with abnormal methylation (9.6%,5/52) in IVM group. One embryo was with hypomethylation, and 4 embryos were with hypermethylation; 2 embryos were found with hypomethylation in routine IVF/ICSI group (3.4%, 2/58). The rates of abnormal methylation between two groups were not different (χ2= 0.87, P= 0.351); a. PEG1:the mean average methylation was 51.9%±4.3% for IVM group and 50.8%±5.7% for routine IVF/ICSI group and no difference were noted (t=1.99, P=0.411).5 embryos were found with hypomethylation (13.6%,5/37) in IVM group, and 3 embryos were found with hypomethylation (7.5%,3/40) in routine IVF/ICSI group. The rates of abnormal methylation between two groups were not different (χ2= 0.24, P= 0.623).(3) Methylation status in IVM oocytes:considering that hypermethylation of H19 and hypomethylation of PEG1 in embryos may result from the methylation errors in oocytes, so we next examine the methylation status of IVM oocytes. a. H19 gene:H19 gene was successfully amplified in oocytes of 13 IVM paient (success rate:65.0%,13/20).5 cases (38.5%,5/13) showed hypermethylation patterns, and 2 of them had hypermethylation patterns in their corresponding embryos; b. PEG1 gene:PEG1 gene was successfully amplified in oocytes of 16 IVM paient (success rate:65.0%, 13/20). One case exhibited hypomethylation pattern, but the corresponding embryo showed normal methylation profile.(4) H19 methylation status in sperm cells:considering that the hypomethylation patterns in embryos of one IVM case and 2 routine IVF/ICSI cases may caused by abnormal methylation in sperm cells, we performed the methylation analysis for the3 sperm samples. The COBRA result showed the normal H19 methylation status in all 3 sperm samples.[Conclusions](1) There were no differences between IVM-embryos and routine IVF/ICSI embryos regarding the percent methylation and abnormal methylation rates of H19/PEG1 genes. The abnormal H19 hypermethylation patterns in embryos may be transmitted from imprinting errors in IVM oocytes; The abnormal PEG1 hypomethylation patterns in embryos may be the result of in vitro embryo culture.(2) M I-arrested could exhibit aberrant methylation profiles of H19/PEG1, and IVM process may be a possible cause.(3) IVF/ICSI application using rescue MⅡoocytes or immature MⅠoocytes may increase the imprinting defects risks for embryos or offspring, and should be carefully used. SectionⅡMethylation status of H19 gene in cryopreserved embryos[Objective] Embryos cryopreservation is very important in ART procedures. In China, the clinical pregnancy rate of Embryos cryopreservation is 40%-60%, and gave rise to the birth of many babies. The studies results about safety of embryo freezing are inconclusive. Freezing may theoretically affect the imprinting establishment/maintenance, but related studies are much fewer. In this study, we collected slow-freezing and vitrified Day3 embryos to determine their DNA methylation and evaluate the epigenetic risks link to embryo cryopreservation.[Methods](1) After getting the approval of Institutional Ethical Committee and informed consent of patients, patients undergoing IVF/ICSI at the NanFang Hospital and Guiyang Maternal & Children Hospital from 2009-2011 were involved in the study. After embryo transfer, Day3 embryos (2PN, bad quality) were collected and then cryo- preserved by slow-freezing protocol or vitrification. Methylation analyses were performed after embryos thawing.70 fresh embryos were used as controls;(2) Methylation analysis:the Combined Bisulfite Restriction Analysis, (COBRA) and Bisulfite Sequencing PCR (BSP) were used to determine H19 methylation status.[Results](1) PCR amplification at single-embryo level:a. slow-freezing:40 embryos were successfully amplified (success rate:51.3%,40/78); b. vitrification: 53 embryos were successfully amplified (success rate:71.6%,53/74); c. fresh embryos:58 embryos were successfully amplified (success rate: 82.9%,58/70). The success rate of PCR amplification for 3-type embryos was significant different (χ2= 17.58, P= 0.000), than vitrification embryos had a lower success rate (51.3%).(2) Methylation status in embryos:a. slow-freezing:the mean average methylation was 44.8%±6.4%, and 4 embryos showed hypomethylation (10.0%,4/40); b. vitrification:the mean average methylation was 47.5%±5.6%, and 7 embryos showed hypomethylation (13.2%,7/53); c. fresh embryos:the mean average methylation was 46.7%±6.0%, and 2 embryos showed hypomethylation (3.4%,2/58).(3) Compare the percent methylation:no difference in H19 methylation% was noted among slow-freezing, vitrification and fresh embryos (F= 2.09, P= 0.127)(4) The rate of abnormal methylation:no difference was noted among slow-freezing, vitrification and fresh embryos (χ2= 3.48, P= 0.175)[Conclusions](1) The success rate of PCR amplification for slow-freezing was lower, which may be due to the mechanical damage of cells;(2) The freezing may not increase the incidence of abnormal methylation in embryos;(3) The further work is to enlarge the sample size and gene quantity, confirming the hypothesis above.