Genomic imprinting (also, namely, gene imprinting) is an epigenetic phenomenon inconsistent with the traditional Mendelian inheritance. The definition of genomic imprinting is that alleles from father or mother are modified when they transmit genetic information to offspring, resulting in only one of alleles from father or mother expressed in offspring [
Insulin-like growth factors (IGFs) are polypeptides that play an important role in cellular proliferation and differentiation. Insulin-like growth factor II (IGF-II) is an important factor of human early embryo and placenta development, and its roles are closely related with the gene imprinting status [
Case group: 47 pregnant women who visited outpatient clinic of family planning and eugenic genetics in First Affiliated Hospital of Sun Yat-sen University because of single gestation sac, empty embryo pregnancy, and embryonic development arrest from April 2002 to January 2004 were enrolled in this study. The informed consent was obtained from all subjects before participating in this study. The inclusion criteria of this group included: B-type ultrasonography that revealed embryonic growth arrest or no embryo observed by repeated B-type ultrasonography; regular menstrual cycle 3 months before pregnancy; 6–10 gestational weeks; no case history of endocrine diseases and cancer; no administration with hormone drugs during pregnancy; serum Toxplasma (TOX), Nubbavirs (RV), Cytomegalo virus (CMV), Herpes simplex virus (HSV) infection detection was negative. In this group, the mean age was
Control group: at the same time, 38 women of normal early pregnancy with single gestation sac were randomly selected. B-type ultrasonography revealed that embryonic development was normal as well as embryo and fetal heart beat. In this group, the mean age was
Chorionic villus cell culture: During the termination by artificial abortion, chorionic villi were collected under sterile conditions then dissected under a dissecting microscope. The dissected chorionic villi were separated and washed repeatedly with ice-cold normal saline to remove attached blood and deciduas, then shredded, digested with 0.25% trypsin for 5 min, followed by centrifugation at 1000 r/min for 10 min, and discarding supernatant. Subsequently, 1 ml collagenase II (sigma company, USA) was added into the sediments. 5 min later, supernatant was removed after centrifugation at 1000 r/min, and the sediments were resuspended in 2 ml complete medium (Complete Amnio Max, Invitrigen company, USA), and cell suspension was transferred into two flasks (about 1 ml). Cell growth was daily observed under inverted microscope. When cell fusion and good vitality were found in 40%–50% of cells, the cells were harvested for chromosome analysis.
G-banding chromosome preparation: Colchicine (0.15
Chorionic villus genomic DNA was extracted with saturated phenol and chloroform, and then IGF-II gene polymorphism was detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. The total volume of PCR reaction system was 50
Chorionic villus IGF-II gene imprinting status was detected by reverse transcription-polymerase chain reaction (RT-PCR) and ApaI restriction enzyme digestion. Heterozygous genomic DNA specimens (type AB) were selected to extract RNA. QIAGEN Poly
IGF-II gene imprinting status detected by RT-PCR.
All statistical analyses were performed by SPSS version 13.0 statistical software. The data in each group is presented as the
In this study, a total of 47 cases of chorionic villus were cultured in the abnormal embryonic development group, including culture failure in 6 cases, primary culture failure in 1 case because of villus degeneration, with a failure rate of 15%, while 38 cases in the normal embryonic development group were successfully cultured. The mean culture time in abnormal and normal embryonic development groups was
G-banding chromosome karyotype analysis of chorionic villus was normal in the normal embryonic development group while abnormal karyotype was 17 cases (42.5%) in the abnormal embryonic development group, including male karyotype in 6 cases, female karyotype in 5 cases, 45, XO in one case, 69, XXX in three cases and 69, XXY in 2 cases. One case of cat cry syndrome suffered from dead fetus at one time and induced abortion was performed in second pregnancy because cord blood chromosome analysis revealed cat cry syndrome. This unwanted pregnancy expressed embryonic development arrest, and its chorionic villus chromosomes were 46, XX and del (5) (p15). The detailed karyotypes were as shown in Table
Villus karyotype analysis of embryonic maldevelopment.
Chromosomal abnormality type | Caryotype | |
---|---|---|
Trisomy | 47, XX (XY), | 4 |
47, XX (XY), | 1 | |
47, XX (XY), | 2 | |
Triplont | 69, XXX | 3 |
69, XXY | 2 | |
Haplotype | 45, XO | 1 |
Other | 46, XY, t (9; 22) (p13; p12) | 1 |
46, XX/46, XY | 1 | |
46, XX, del (5) (p15) | 1 | |
46, XX, i(10)(qter→cen→qter) | 1 | |
Total | 17 |
In the abnormal embryonic development group, the imprinting loss rate of IGF-II gene was 44.7%, which was higher than 31.6% in the normal group, but without significant difference (
IGF-II gene imprinting status in the abnormal and normal embryonic development groups.
Group | Genotype AA/BB | AB | ||
% | % | |||
Normal group | 26 | 68.4 | 12 | 31.6 |
Abnormal group | 26 | 55.3 | 21 | 44.7 |
(chromosomal abnormality) | 6 | 35.3 | 11 | 64.7 |
A, B, and AB indicates three kinds of genetic imprinting status, AA/BB indicate normal imprinting, AB indicates gene imprinting loss.
The chromosomal abnormality rate was 42.5% in the abnormal embryonic development group. Of 17 specimens with abnormal karyotypes, 11 cases suffered from IGF-II gene imprinting loss, with the loss rate up to 64.7%. While, no abnormal villus chromosomal karyotype analysis was found in the normal embryonic development group. There was no significant difference between two groups (
The culture time of chorionic villus cells from abortion or dead fetus depended on cell survival rate. The culture time was up to 3 to 4 weeks if there were less active cells in tissues. Culture failure was defined as cell growth arrest for more than one month [
Villus culture in 40 cases of embryonic development arrest showed that chromosomal abnormality was 42.5%, indicating that nearly one half of fetus with chromosomal diseases (the majority was severe chromosomal abnormalities, resulting in severe imbalance of genetic materials) could not survive to birth, due to natural selection. Except chromosome 1, triplont was found in all human chromosomes. Of them, triplont in chromosome 1 accounted for one third of all triplonts and was one kind of highly lethal triplont. The other common triplonts included triplonts in chromosomes 21 and 22 [
The balance of cell apoptosis and proliferation was crucial to maintain pregnancy, and its functional regulation disorder might lead to early embryonic development arrest. Warner deemed that the dynamic balance of speed of development and apoptosis was an internal factor of embryo survival. In the process of embryonic development, development and apoptosis-related genes, cytokines and corresponding receptors played important roles in embryonic survival [
Imprinted genes of germ cells cycled with reproductive cycle through three phases of erase, formation, and maintenance. After fertilization, the majority of genes experienced re-demethylation, while imprinted genes maintained original methylation status before and after fertilization and nidation due to the protection of differentially methylated regions. Meanwhile, internal and external environment changes could lead to genetic alternations and gene imprinting loss. This study showed that IGF-II gene imprinting loss in the embryonic development arrest group was slightly higher than the normal group, without significant difference. However, IGF-II gene imprinting loss of 17 abnormal karyotypes in the abnormal embryonic development group was significantly higher than the normal embryonic development group. Thus, we speculated that chromosomal abnormalities of embryos could result in abnormal function expression of regulatory genes in early embryonic development, leading to increased IGF-II gene imprinting loss, which might destroy the balance between villus and deciduas, leading to shallow embryo implantation, spontaneous abortion, and embryonic development arrest. However, the collected data in this study was relatively less, and thus there should be a bigger sample size, to further discuss and explore whether IGF-2 imprinting loss was a new molecular genetic marker of spontaneous abortion or not.
Some scholars believe that IGF-II gene imprinting loss leads to overexpression [
There might be a lot of complicated middle links between IGF-II gene imprinting loss and IGF-II expression regulation.
This paper was supported by the National Natural Science Foundation of China (no. 30070788).