In accordance with the relevant regulations and ethical principles of the Specification for the Allocation of Human Assisted Reproductive Technology and the Relevant Technical Specifications and Basic Standards for Human Sperm Bank, this study was carried out, and also approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University and the Ethics Committee of Shaanxi Maternal and Child Healthcare Hospital. Volunteers and embryo donors have signed the informed consent.
1.2 Study subjects
From May 2013 to July 2015, D3 fresh surplus embryos were collected from 120 infertile couples receiving routine IVF-ET treatment in the Reproductive Center of Shaanxi Maternal and Child Healthcare Hospital. The age of both husband and wife is less than 35 years and were not infected with HAV, HBV, HCV, HDV and HIV. They voluntarily donated discarded embryos. The quality of D3 embryo was evaluated by PETER cleavage stage embryos scoring system, and 238 embryos of grade I-III were selected from the collected fresh discarded embryos .
In vitro infection source of HBV came from the two treatment-naïve chronic hepatitis B patients, who were selected as volunteers in the outpatient department of Infectious Department of the First Affiliated Hospital of Xi'an Jiaotong University. HBV DNA quantitative of these two patients was 3.21×105IU/mL, and 2.89×108IU/mL respectively. The results of routine biochemical tests showed that the liver function of the patients was normal; HBsAg, HBeAg and HBcAb were positive; HBV genotype was type C; and HIV, HAV, HCV, HDV and EBV infections were excluded.
1.3 Main reagents
Cleavage embryo culture solution G-1TM PLUS, blastocyst culture solution G-2 TM PLUS and mineral oil for culture were purchased from Vitrolife Company, Sweden. SuperScript® III First-Strand Synthesis System was purchased from Thermo Fisher Company, USA. HBV DNA quantitative test kit was purchased from Shenzhen PG Biotechnology Co., Ltd. HBsAg, HBeAg and anti-HBs quantitative test kit was purchased from American Abbott Corporation. RNase inhibitors were purchased from American Invitrogen Company. dNTP（10mmol/L）Platinum Taq DNA polymerase was purchased from American Invitrogen Company. PCR primers were synthesized by Shanghai Sangon Biotech Co.,Ltd.
2 Experimental method
2.1 In vitro culture of early embryos
30μL micro drops were added into a 35mm culture dish, covered with mineral oil, and equalized overnight in a 6% CO2 incubator at 37 ℃. Fresh embryos were obtained every day and transferred into mineral oil-covered micro drops of embryo culture solution after the equalization overnight according to the experimental groups. On average, one embryo was put into each micro drop to observe the development of embryos and was cultured continuously in the incubator.
2.2 HBV infection in vitro
2.2.1 Source of HBV
15ml venous blood was drawn from the fasting CHB volunteers, and serum was separated. Then the serum was put in the sterile centrifuge tube, and in 56 ℃ water bath case for 30 minutes. After inactivation, they were sub-packaged, and the samples were taken for sterility test and kept in the refrigerator at - 80℃ for standby. The serum of the patient with HBV DNA 3.21×105IU/m was used as the source of low viral load infection in vitro, and the serum of the patient with HBV DNA 2.89×108IU/mL was used as the source of high viral load infection in vitro.
2.2.2 HBV infection in vitro
The inactivated HBV serum was mixed with embryo culture solution in a ratio of 1:3. Well-equalized embryo was transferred into the culture micro drop containing HBV particles by Pasteur pipette. After infection in 6% CO2 incubator at 37℃ for 24 hours, the embryo was removed and carefully washed with blastocyst culture solution for 8 times, and HBsAg quantity was detected in the last cleaning solution. If HBV DNA was negative, the embryos were transferred into the micro drops of blastocyst culture solution without HBV serum. After 24 hours, the supernatant was taken and stored at - 80℃ for unified detection.
2.2.3 Experimental groups
According to the principle of randomized grouping, the embryos were divided into four groups: group A1 was negative control group, and the culture solution was embryo culture solution for a total of 38 pieces; group A2 was healthy human serum group, and the culture solution was the mixture of healthy human serum and embryo culture solution for a total of 32 pieces; group A3 was low viral load serum group, and the culture solution was the mixture of low viral load serum and embryo culture solution for a total of 34 pieces; group A4 was high viral load serum group, and the culture solution was the mixture of high viral load serum and embryo culture solution for a total of 32 pieces. The mixture ratio of inactivated serum and embryo culture solution was 1:3.
2.2.4 Detection of culture supernatant
The supernatant of embryo culture was collected and tested. The supernatant which was less than 20μL in volume or polluted by mineral oil would be discarded. HBsAg quantity was detected by electrochemiluminescence. HBsAg<0.05 IU/mL was negative; HBsAg≥0.05 IU/mL was positive.
2.2.5 Detection of HBV mRNA expression in embryos by single-cell RT-PCR
220.127.116.11 Embryo cleavage
The embryos infected in vitro were washed with PBS buffer for 8 times and then placed in the embryo cleavage solution for decomposition for 2 hours. The last washing solution was reserved for the detection of HBsAg quantity and HBV DNA. The samples with both HBsAg quantity and HBV DNA negative could be used for subsequent experiments.
18.104.22.168 cDNA synthesis
(1) The specifications of Invitrogen's Super-script III First- Strand Synthesis reverse transcription kit was followed to operate. (2) The total volume of the tube was 20μL and heated at 50 ℃ for 50 min. cDNA was synthesized, and then the reaction was stopped by heating at 85 ℃ for 5 min. (3) After centrifugation, RNase H 1μL was added and heating lasted for 20 minutes at 37 ℃ to remove the non-transcribed RNA. The obtained cDNA was stored in -20 ℃ refrigerator, and β- actin was used as housekeeping gene.
22.214.171.124 Nested PCR
(1) According to the results of our previous experiments, we continued to use the primers designed by the mRNA gene sequence of HBV S region . Outer primer：the amplification length was 417 bp, and the nucleotide sequence was P1:5'-CATCTTCTTGTTGGTTCTTCTG-3'; P2:5'-TTAGGGTTTAAATGTATACCC-3'. Inner primer: the amplification length was 206 bp, nucleotide sequence was P3: 5'-TCTATGTTTCCCTCTTGTTGC-3'; P4:5'-TACCACATCATCCATATAACTG- 3'. (2) The nested PCR reaction system and the amplification conditions were the same as those of our published article .
2.3 Effect of HBV infection on embryo development
On D4 to D6 in the process of embryo culture in vitro, embryo development status was observed, recorded, and photographed every day. The blastocyst quality based on morphology parameters including cell count, fragmentation, and embryo expansion, was classified according to Gardner’s criteria with the grades (A, B, or C) describes both the trophectoderm and innercell mass of the embryo [13,14].
2.4 Statistical analysis
SPSS 16.0 software was applied for statistical analysis. The measurement data were expressed as x̅±s. Nonparametric test, Chi-square test or Fisher exact test was used to compare rates. Spearman correlation test was used for the correlation. The difference was statistically significant (p< 0.05).