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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 1  |  Page : 25-31

Effects of vitrification on the imprinted gene Snrpn in neonatal placental tissue


1 Center for Reproductive Medicine, Quanzhou Maternity and Child Healthcare Hospital, Quanzhou 362000, China
2 Health Screening Center, The First Affiliated Hospital, Fujian Medical University, Fuzhou 350004, China
3 Clinical Testing Center, The Second Affiliated Hospital, Fujian Medical University, Quanzhou 362000, China
4 Department of Obstetrics and Gynecology, The First Affiliated Hospital, Fujian Medical University, Fuzhou 350004, China
5 Center for Reproductive Medicine, The First Affiliated Hospital of Xiamen University, Xiamen 361003, China

Date of Submission06-May-2019
Date of Decision18-Jul-2019
Date of Acceptance15-Sep-2019
Date of Web Publication2-Apr-2020

Correspondence Address:
Ji-Feng Hu
The First Affiliated Hospital, Fujian Medical University, No. 20 Chazhong Road, Fuzhou 350004
China
You-Zhu Li
Center for Reproductive Medicine, The First Affiliated Hospital of Xiamen University, No. 55 Zhenhai Road, Xiamen 361003
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.281851

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  Abstract 


Objective: To investigate the effects of vitrification on the expression of the imprinted gene Snrpn in neonatal placental tissue.Methods: Neonatal placental tissue was collected from women with natural pregnancy (control group) and from women in assisted reproductive technology (ART) pregnancy group, following fresh and vitrified embryo transfer (fresh group and vitrified group, respectively). Snrpn mRNA expression and SNRPN protein levels in placental tissue from these three groups were assessed by real-time reverse transcription polymerase chain reaction and Western blot, respectively. DNA methylation in the Snrpn promoter region was analyzed by bisulfite-pyrosequencing.Results: The expression of Snrpn mRNA and SNRPN protein was found to be higher in placental tissue from the fresh and vitrified ART groups, compared to the control group. There was no significant difference in SNRPN gene or protein expression between the fresh and vitrified groups. DNA methylation at the Snrpn promoter region was not significantly different between these three groups.Conclusions: Human ART may alter the transcriptional expression and protein levels of the imprinted gene Snrpn. However, compared to other ART methods, vitrification may not aggravate or reduce this effect. Moreover, the altered expression of Snrpn is likely not directly related to DNA methylation of the Snrpn promoter region.

Keywords: Assisted Reproductive Technology, Polymerase Chain Reaction, Snrpn Vitrification, Western Blot


How to cite this article:
Yao JF, Huang YF, Huang RF, Lin SX, Guo CQ, Hua CZ, Wu PY, Hu JF, Li YZ. Effects of vitrification on the imprinted gene Snrpn in neonatal placental tissue. Reprod Dev Med 2020;4:25-31

How to cite this URL:
Yao JF, Huang YF, Huang RF, Lin SX, Guo CQ, Hua CZ, Wu PY, Hu JF, Li YZ. Effects of vitrification on the imprinted gene Snrpn in neonatal placental tissue. Reprod Dev Med [serial online] 2020 [cited 2020 May 26];4:25-31. Available from: http://www.repdevmed.org/text.asp?2020/4/1/25/281851




  Introduction Top


The first test-tube baby born in the UK represented a prelude to research into human reproductive technology. Assisted reproductive technologies (ARTs) have been used in clinical practice for more than 30 years. ART has not been found to directly cause significant defects in offspring. More than 5 million children have been born from ART-facilitated pregnancies and have become part of the world population. Nevertheless, the reproductive safety of ART has been widely discussed, due to its artificial nature and the lack of preclinical safety studies. Concern for the safety of children born from ART pregnancies has shifted from considerations of short-term security, such as birth defects, to rare diseases caused by defects in epigenetic and genetic imprinting.[1]

Cryopreservation of embryos is an important component of ARTs and contributes to the cumulative pregnancy rate of in vitro fertilization–embryo transfer (IVF-ET) treatment cycles. In 1985, vitrification was first reported for use in the preservation of mammalian embryos,[2] and in 1998, vitrification was successfully used for the cryopreservation of human cleavage embryos.[3] In the past 10 years, vitrification has become prominent in the ART field, due to its lower cost, rapid process, and high recovery rate, and has gradually replaced the traditional slow freezing technology.[4] Whether vitrification and freezing technology affect epigenetic modifications and embryonic development is of extreme interest to researchers, clinicians, and patients.

Snrpn is an imprinted gene that is maternally imprinted and paternally expressed, and is associated with cell differentiation, proliferation, and normal embryo development. Moreover, mutation in Snrpn and genomic imprinting disorders can lead to abnormal nervous system development.[5],[6],[7] In this study, we for the first time explore the effects of vitrification on the imprinted gene Snrpn in neonatal placental tissues from ART-facilitated pregnancy. We discuss the safety of vitrification on embryo epigenetics, which may improve birth quality in ART populations and fertility options for patients.


  Methods Top


Patients and ethical statement

A total of 95 women (average age: 25–38 years) with singleton pregnancies, resulting in live births without complications between March 2015 and October 2017, were included in this prospective cohort study. All cases represented full-term pregnancy and gave birth at the Quanzhou Maternity and Child Healthcare Hospital. Patients with hypertension, diabetes, heart disease, or other concurrent illness during pregnancy that could adversely affect the outcome of the pregnancy were excluded. All neonatal placental tissues were collected at the time of delivery and stored in liquid nitrogen until further use. Tissue specimens were divided into three groups: frozen group (29 cases), fresh group (31 cases), and control group (35 cases). All infertile patients received IVF-ET treatment at the Reproductive Center of Quanzhou Maternal and Child Healthcare Hospital. The most common causes of infertility in the study population were  Fallopian tube More Details problems in females or low sperm mobility in males. The frozen group represented ART pregnancy following vitrification and transfer of thawed embryos, and the fresh group represented ART pregnancy following fresh ET. The control group consisted of neonatal placental tissue acquired from the natural pregnancies delivered during the same period of time. All the basic information about maternity, including pregnancy outcomes, was recorded and compared among the three groups. This study was approved by the Ethics Committee of the Quanzhou Maternal and Child Health Hospital (Ethics of 2016: No. 1). All study participants provided written informed consent.

Instruments and reagents

All chemicals were purchased from Sigma-Aldrich, unless specified otherwise. The cDNA reverse transcription kit was purchased from TOYOBO Biotech (ReverTra Ace-α-TM, TOYOBO); the SYBR Green I fluorescence quantification kit was purchased from TAKARA (SYBR Premix Ex Taq™ TAKARA). Primers for quantitative polymerase chain reaction (PCR) analysis of Snrpn and β-actin mRNAs were synthesized by TAKARA. RNA quantitation was performed using an ultraviolet spectrophotometer (Thermo, ND-100), and the real-time quantitative PCR was performed using an ABI Step-one Plus real-time PCR instrument (Applied Biosystems).

Sample collection

Immediately after delivery, the placental leaflets located at the attachment of the umbilical cord were removed under sterile conditions, avoiding the calcified and necrotic areas. Then, the maternal aponeurosis layer was removed, and the placental villi were rinsed in ice-cold physiological saline solution. After washing, the tissue sample was stored in liquid nitrogen. After half an hour, the sample was transferred to a −80°C cryogenic refrigerator for further analysis.

RNA extraction and cDNA synthesis

Total RNA was extracted from placental tissue using the RNAiso reagent (TAKARA) according to the manufacturer's instructions. Purity of the extracted RNA was measured at optical wavelengths of 230, 260, and 280 nm, using a NanoDrop Photometer (Thermo ND-100). RNA integrity was verified using ethidium bromide staining of the 28S and 18S ribosomal RNA bands on a 2% agarose gel. RNA was treated with PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time, TAKARA) to degrade any genomic DNA present in the sample. cDNA was generated from total RNA using RT Primer Mix (TOYOBO). The reaction conditions for cDNA synthesis were as follows: 2–4 μg total RNA, 2 μL 5 × DNA Eraser Buffer, 1 μL gDNA Eraser, 4 μL 5 × PrimeScript Buffer 2, 1 μL PrimeScript RT Enzyme Mix I, 1 μL RT Primer Mix, and RNase Free H2O added to a final volume of 20 μL. First-strand cDNA was synthesized at 37°C for 15 min. Next, mRNA-cDNA chains were denatured, and reverse transcriptase activity was arrested by heating samples to 85°C for 5 min, followed by cooling at 4°C for 5 min. Identical reactions were carried out without reverse transcriptase as a negative control. cDNA samples were stored at −20°C for further analysis.

Real-time reverse transcription polymerase chain reaction

Analysis of gene expression in each tissue sample was carried out using quantitative real-time PCR (qRT-PCR) of cDNA, the SYBR green method, and an ABI Prism 7500 instrument (Applied Biosystems). Two-step qRT-PCR using a double-curve standard was performed on the samples in triplicate, to control for PCR variations, according to the manufacturer's instructions. The quantity of each measured cDNA sample was normalized to the reference gene β-actin. Finally, the 2−ΔCT method was used to quantify relative mRNA levels.[8] Primers for amplification of Snrpn and β-actin are as follows: Snrpn F: 5'-CGAAGCAACCAGAGCGTGAA-3', R: 5'-GGTACCCGAGCAATGCCAGT-3', β-actin F: 5'-ACCATTGGCAATGAGCGGTT-3', R: 5'-GCGGATGTCCACGTCACACT-3'. Single product amplification was verified by solubility curve analysis and electrophoretic separation of qRT-PCR products on 1% agarose gels. The PCR efficiency for each single primer pair was determined using serial five-fold dilutions of cDNA transcripts. The linear correlation coefficient (R2), an indicator of best fit for the standard curve, was plotted to the standard data points of all genes and ranged from 0.998 to 0.999. Cycling conditions for qRT-PCR were 95°C for 10 s, followed by 40 cycles at 95°C for 5 s, then 60°C for 34 s. The dissociation cycle was 95°C for 15 s, followed by 60°C for 1 min, then 95°C for 15 s.

Western blot

Primary antibodies against human SNRPN (ab191439, 25 kDa), and secondary HRP-conjugated goat anti-rabbit IgG antibodies (PIERCE), were used. Briefly, 50 μL protein lysate containing 1% protease inhibitor to 10 mg placental tissue was fully homogenized in an ice bath. The protein content was determined using the BCA method. The quantified protein samples were denatured by boiling for 5 min and were then subjected to SDS/PAGE on a 12% gel. Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (MILLIPORE). The PVDF membranes were blocked with 5% nonfat dry milk at room temperature for 2 h and then incubated with primary anti-SNRPN antibody diluted 1:500 at 4°C overnight. After overnight incubation, membranes were washed three times with wash buffer (PBS containing 0.1% Tween 20). The dilution of the primary antibody against the internal reference protein β-ACTIN was 1:1,500. Membranes were then incubated with secondary antibody diluted 1:5,000 at room temperature for 2 h. After three washes with wash buffer, the PVDF membranes were treated with super ECL Plus (PIERCE). The chemiluminescent signals from the protein bands were visualized using radiography film. Signal intensities were quantified using ImageJ 1.37v software (National Institutes of Health, USA).

Analysis of CpG methylation of Snrpn promoter in placental tissues

The proportion of methylated CpG sites was determined using the bisulfite-pyrosequencing method.[9] Briefly, 500 ng DNA was treated with bisulfite using the EpiTect Bisulfite Kit (QIAGEN, NO.59104). PCR reactions amplifying the bisulfite-treated and purified DNA for subsequent pyrosequencing analysis were performed using PyroMark PCR Kit (QIAGEN, NO.978705) in a total volume of 20 μL. The pretreated PCR products were sequenced with PyroMark Q96 ID System (QIAGEN, Germany) using PyroMark Gold Q96 Reagent Kit (QIAGEN), in accordance with the manufacturer's instructions. The pyrosequencing primers were designed for 10 non-CpG loci using PyroMark Assay Design Software 2.0 (QIAGEN, Germany), and all the primers for Snrpn are listed in [Table 1]. The sequence used is from AF134832 (GenBank).
Table 1: Primer sequences for pyrosequencing

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Statistical analysis

All data were analyzed with SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Gender differences were compared using the Chi-squared test. Other data were analyzed using one-way analysis of variance and the S-N-K test. For all analyses, P = 0.05 was considered statistically significant.


  Results Top


Clinical data analysis

There were no significant differences in maternal age, weight gain during pregnancy, fetal birth weight and length, umbilical cord length, or placental weight among the three groups [P > 0.05, [Table 2].
Table 2: Basic information of patients

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Comparison of Snrpn mRNA levels in placental tissues

To confirm whether the Snrpn transcript levels in neonatal placental tissue changed after vitrified frozen-thawed ET, placental tissues from the three different groups were collected at delivery. Real-time fluorescent quantitative PCR analysis showed that placental tissues from the fresh and vitrified group had higher Snrpn transcript levels than those from the control group (fresh: 0.0284 ± 0.0187, vitrified: 0.0325 ± 0.0108, and control: 0.0177 ± 0.0092, P < 0.001). Compared with the fresh group, Snrpn mRNA levels in the vitrified group showed a slight but not significant increase [P = 0.212; [Figure 1].
Figure 1: Relative expression of Snrpn mRNA in placental tissues. Real-time PCR using SYBR green was used on placental tissues from control, fresh, and vitrified groups. Standard errors of the means are indicated by bars. P < 0.05 was considered to indicate significance

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Effects of vitrification on SNRPN protein levels in placental tissues

To further confirm the effect of the vitrified frozen-thawed ET technique on SNRPN expression, we analyzed SNRPN protein levels in placental tissue by Western blot. The levels of SNRPN protein were significantly increased in the fresh and vitrified groups compared with the control group [fresh: 0.5513 ± 0.0168, vitrified: 0.5760 ± 0.0229, and control: 0.4450 ± 0.0249, P < 0.01; [Figure 2]. There was no significant difference in SNRPN protein expression in placental tissue between the fresh and vitrified groups [P = 0.286; [Figure 2].
Figure 2: The relative expression level of SNRPN protein in placental tissues analyzed by Western blotting. (a) Western blot analysis of SNRPN protein in placental tissues from the control, fresh, and vitrified groups. β-Actin served as a loading control. (b) Quantification of the intensities of Western blot signals. Intensity ratios represent the signal intensity of SNRPN relative to that of β-actin. The analysis was repeated three times and the quantitative value is expres sed as mean ± standard error of the mean. P < 0.05 was considered to indicate a significant difference

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Effects of vitrification on DNA methylation of the SNRPN promoter

DNA methylation is associated with the regulation of gene expression. We therefore analyzed the methylation status of CpG sites in the promoter CpG island region of the Snrpn gene in human placental tissue using the bisulfite-pyrosequencing method. Compared with the natural pregnancy control group, there was no statistically significant change in DNA methylation levels of the Snrpn promoter region in the fresh and vitrified groups [control: 29.97% ± 10.64%, fresh: 33.83% ± 16.63%, and vitrified: 33.86% ± 18.95%; P > 0.05; [Figure 3].
Figure 3: The methylation status of CpG sites in the promoter region of Snrpn in placental tissues using the bisulfite-pyrosequencing method. (a) Bisulfite-pyrosequencing profiles of Snrpn in placental tissues from the control, fresh, and vitrified groups. (b). Relative methylation level of Snrpn in placental tissues from the three groups. P < 0.05 was considered to indicate a significant difference

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  Discussion Top


Cryopreservation is an important process in ART.[10] With the help of IVF-ET technology, the first IVF baby was born after successful transfer of frozen-thawed embryos in 1983.[11] The first report of a successful pregnancy from a frozen egg was described in 1986.[12] In the past 30 years, cryopreservation technology has rapidly developed, and cryopreservation is widely used in ART laboratories as an integral part of ART. Cryopreservation of embryos can ensure the best utilization of the eggs and embryos obtained during the oocyte retrieval cycle, reducing the stress and burden of repeating ovarian stimulation on patients, and effectively reducing the risk of multiple pregnancies and ovarian hyperstimulation. However, the overall safety and risks of cryopreservation technology to improve fertility have not been determined.

The application of vitrification by ultra-fast cooling has promoted the development of refrigeration technology. Vitrification is the amorphous solidification of a supercooled liquid without ice crystal formation, resulting in better preservation of cell viability. During the process, the embryo is placed in a high concentration of vitrification cryoprotectant and undergoes super-fast cooling in the liquid. Vitrification converts the material into a glass or glass-like solid state free of any crystallized structure. Vitrification is simple, rapid, and economical and has a high embryo survival rate after resuscitation. Because of these advantages, vitrification has gradually replaced traditional slower freezing methods.[13],[14] As more and more children are born through ART method, increasing attention is being given to the effects of vitrification on adult health and disease that might be associated with the rapid cooling, rapid temperature rise and recovery, and cytotoxicity of cryoprotectants.

ART has become the standard treatment for infertile couples. Although ART technologies have solved many problems related to human infertility, deviations from the natural reproductive process and a lack of safety research before clinical application have raised questions about the safety of ART methods regarding genetic and epigenetic imprinting. The genome undergoes several phases of epigenetic programming during gametogenesis, egg fertilization, and early embryo development, which is when ART treatments are performed. During this period, major epigenetic reprogramming, which is crucial for normal embryo development, takes place. This epigenetic reprogramming is vulnerable to changes in environmental conditions encountered during the IVF process, which may possibly lead to disorders in epigenetic state. Imprinting disorders may be more common in children conceived by ART, and ART may cause epigenetic modifications in gametes or embryos, which could result in adverse health consequences in adulthood. Therefore, the long-term health of ART children are becoming of greater interest, causing a shift from perinatal birth defects to rare diseases in adulthood caused by epigenetic and genetic imprinting defects.[1],[15]

Imprinting refers to the differential expression of alleles depending on their parental origin, an important epigenetic phenomenon that strongly influences mammalian development.[16] Imprinted genes show monoallelic expression from either the paternal or maternal genome. Maternally imprinted, or silenced, genes are only transcribed from the paternal allele, whereas paternally imprinted genes are only transcribed from the maternal allele. Many imprinted genes play important roles in placental development and embryonic growth, as well as in a variety of processes after birth, and in nervous system health and behavioral ability in adulthood.[17],[18],[19]

The relationship between genetic imprinting and human diseases is attracting more and more attention. Imprinting function disorders can lead to embryonic tumors, stillbirth, and a variety of hereditary imprinting diseases. The embryonic stage at the time when ART methods are performed is a critical period of epigenetic state reconstruction. DNA has undergone a dramatic transformation of blocking and reestablishment. Many imprinted genes begin to express from either the paternal or the maternal allele and are sensitive to changes in environmental factors.[20],[21] In recent years, excluding parental infertility or multifetal factors, reports on the association of imprinted gene diseases and the use of ART technologies are increasing, and the potential adverse health risks in ART offspring are of increasing interest. ART could increase the risk of imprinting gene diseases.[22] Several studies have also confirmed that the ART procedure, such as superovulation and in vitro culture of oocytes, caused epigenetic changes in the embryos and offspring.[23],[24],[25] A recent study demonstrated that superovulation alters expression of genes critical to endometrial remodeling during early implantation.[26] Several case studies have suggested an association between ART and imprinting disorders.[15],[23],[27] Whether such early epigenetic alterations and changes can cause long-term diseases in children conceived by ART is still unknown. So far, there is no consensus as to whether ART increases the risk of imprinting diseases, especially as the freezing technology has not been well studied. Although vitrification currently provides good pregnancy outcomes, the possible chromosomal abnormalities and abnormal genetic imprinting that might be associated with vitrification need to be explored further.

Prader–Willi syndrome (PWS) is the first congenital behavioral abnormality syndrome identified as a complex genetic imprinting dysfunction, characterized by childhood obesity, short stature, hypogonadism/hypogenitalism, hypotonia, cognitive impairment, and behavioral problems.[28] Usually, PWS occurs sporadically due to the loss of paternally expressed genes on chromosome 15 with the majority of individuals having the 15q11-q13 region deleted. Multiple imprinted genes have been identified in the 15q11-q13 region that are critical for the normal development of the nervous system. PWS appears to be a contiguous gene syndrome caused by the loss of at least two of a number of genes expressed exclusively from the paternal allele, including Snrpn, Mkrn3, Magel2,Ndn, and several snoRNAs, but it is not yet well known which specific genes in this region are associated with this syndrome.[29]Snrpn, located in 15q11-q13, is a polycistronic gene that encodes the SNRPN protein and is reported to be associated with PWS.[30]Snrpn is a maternally imprinted gene that is expressed from the paternal allele. It is the first imprinted gene identified to be associated with the pathogenesis of PWS and is closely related to embryonic development. Snrpn plays an essential role in gamete formation, oocyte maturation, sperm fertilization, and normal embryonic development. Mutation in Snrpn and its impaired imprinting can lead to abnormal nervous system development. The molecular mechanism of PWS is deficiency of paternally expressed gene or genes from the chromosome 15q11-q13, and reactivating expression of paternally expressed Snrpn and from the maternal chromosome might support a proof-of-principle for epigenetic-based therapy for the PWS in humans.[31] Compared to the tissue from the fetus, epigenetic modification abnormalities are more likely to occur in placental tissue due to interference from environmental factors in vitro. Therefore, in this study, we evaluated the expression of Snrpn gene and protein and DNA methylation levels at the Snrpn promoter in neonatal placental tissues from ART pregnancy following fresh and vitrified ET. Compared to the control group (neonatal placental tissues from natural pregnancy), we found that the expression levels of Snrpn mRNA and SNRPN protein are higher in placental tissue from the fresh and vitrified groups, but there was no significant difference between the fresh group and vitrified group. We speculate that IVF-ET technology including superovulation therapy may be associated to the altered expression of Snrpn. However, vitrification itself might not aggravate or reduce this effect compared to other ART methods. DNA methylation, specifically CpG methylation in promoter regions, plays a crucial role in regulating gene expression.[18],[21] We evaluated whether the expression of Snrpn could be directly affected by DNA methylation of the Snrpn promoter region. However, our results revealed no significant differences in CpG methylation at the Snrpn promoter among the three groups, and the differences in Snrpn expression levels could not be explained by promoter methylation. Therefore, we hypothesized that other epigenetic modifications, such as covalent histone modifications,[16] might be relevant in regulating Snrpn expression. Further research to investigate the effects of vitrification on imprinted genes, such as Snrpn, is warranted. Whether ART methodology, including vitrification, increases the risk of rare imprinting disorders in ART-conceived offspring is still unclear and merits further research.

Taken together, this study evaluated the safety of vitrification, focusing on changes in the expression of Snrpn within the PWS/AS imprinting region in neonatal placental tissue. The association between ART and congenital imprinting diseases and their pathogenesis are still understudied. This report was limited by a small size, and larger, more appropriately powered studies, including evaluating other imprinted genes, are required for a comprehensive analysis. Infertile couples always face the risk of failing to achieve a successful pregnancy or a dysfunctional childbirth. Larger longitudinal studies are required to determine the incidence of imprinting disorders in ART offspring and are critical for evaluating the long-term effects of ART during the perinatal period and into adulthood.

Financial support and sponsorship

This work was supported by the Quanzhou Science and Technology Plan Project in 2016 (No. 2016Z38) and the Natural Science Foundation Science and Technology Project Plan of Fujian in 2018 (No. 2018J01147).

Conflicts of interest

There are no conflicts of interest.



 
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