Initial Study On The Phenomenon And Mechanism Of Sex-specific Embryo Elimination In Spontaneous Abortion

Background and Objective:Spontaneous abortion (SA) is a common disease in gynaecology and obstetrics.The incidence rate is about 10-20% in all clinic abortion, and is increasing gradually in the recent years. Most of SA occurred in early pregnancy (pregnancy of 12 weeks or less). There are a number of possible causes of SA, which include genetic, anatomic, immune, endocrine, infectious, thrombophilia, as well as environmental and other unknown factors. To determine the cause of SA is very difficult, because the available clinical testing techniques are limited. Thus, the unexplained SA is up to 37-79% of the total SA cases.So far, genetic factors, particularly abnormal chromosom, are recognized as the most common predisposing factor in SA.), SA happened in less than 15 weeks of pregnancy, the incidence of chromosomal abnormalities is more than 50%(50-70%), tested by karyotype analysis. In recent years, studies have shown that, in addition to the high incidence of chromosomal abnormalities in SA (especially early SA), sex-specific embryo elimination is another common cause of SA. AS reported in other literature, in early SA, the ratio of female embryos to male embryos was about 3-4:1, which can fully ruled out mother-derived pollution. Our lab also found the gender-specific embryo elimination in early spontaneous abortion.Why are female embryos more prone to spontaneous abortion? As we known, the composition of sex chromosomes decides the gender of fetus. In the early embryo development, female embryos go through an event, XCI (X-chromosome inactivation), which does not take place in male embryos,. XCI occurs in the early embryo (started from the third day of the morula period after fertilixation). Female XCI is random, which means that, inactivated X-chromosome may be either prenatal or maternal for any cell. Theoretically, the ratio of the prenatal to maternal chromosome is close to 1:1. If the ratio of XCI deviated from 1:1, we will name it shewed X-chromosome inactivation. In sexual reproduction, in order to avoid being attacked from the mother's natural immunity, ectoblastic tissue only expresses maternal X chromosome from the beginning of implantation. In other words, in the villi cells, maternal X-chromosome will be remained active, because too much active paternal X chromosome may result in SA. This consumption has been confirmed by experiments carried out in mice from different labs, but no such findings was reported in villious of embryonic X chromosome inactivation of human spontaneous abortion.Based on these considerations, we will set up a diagnostic system, which include STR (short tandem repeat) loci for DHPLC (denature high performance liquid chromatography) technology to investigate the villi of XCI directly to test its X chromosome inactivation. X chromosome inactivation situation of mother somatic cells(peripheral blood), a comprehensive analysis of the mechanism of high-fat of female embryos in spontaneous abortion, will also be concerned in the diagnose and analysis of mechanism of sex-specific embryo elimination. We assume that, XCI can cause SA by three mechanisms:(1) mother's X chromosome was inactivated normally, but villi X chromosome was inactived abnormally. The inactivted maternal X chromosome or activated partenal X chromosome leads to SA; (2) villi X chromosome was inactivated normally, but mother's X chromosome was inactivated abnormally, thus lots of inactived X chromosome was passed to villus, which led to abortion.(3)both villi's and mother's X chromosome were inactivated abnormally, which caused abortion. Normal X chromosome inactivation has two stitutions:(1) villi X chromosome was inactivated normally, but mother's X chromosome was inactivted abnormally, which passed the high activation X chromosome to villus..(2) Both villus's and mother's X chromosome inactivated normally.Materials and methods1.The standards of sample choice:This study selected 167 early spontaneous abortion families(including villi tissue and peripheral blood of both parents),8～11 gestation,the average age of mothers was 30.84±5.28(21～40years)and 116 early induced abortion families(including villi tissue and peripheral blood of mother).8～11 gestation,the average age of mother was 29.60±4.47(23～48),excluding immunity,infection,trauma and other factors, collected during sterile curettage.2.Karyotype analysis of spontaneous abortion and induced abortion villi:the villi cell culture method or villus direct preparation method were used to obtain villi chromosome,the aim of G-banding karyotype analysis was to find the most common triggers of spontaneous abortion—chromosome number and structural abnormalities,and to determine the sex of embryos for the next study group basis.3. Establishment of DHPLC diagnosis of X chromosome inactivation:(1) Selected specific STR loci and design PCR primer. The first exon of the androgen receptor (human androgen receptor, AR) gene in X chromosome q11-12 had a three-nucleotide (CAG) STR loci, repeated 8 to 31 times, the heterozygosity was 88.8%. Designed primer, amplified PCR, fragment length was about 200～300bp.(2) Detected and statisticsed the selected STR loci heterozygosity.422 female specimens, after chromosomal abnormalities were excepted, extract genomic DNA was,amplify PCR, then, the PCR products were detected on the non-denaturing DHPLC (50℃), statistics numbers of heterozygous peaks, calculate the actual heterozygosity. Heterozygosity= number of STR heterozygote/total number of samples.(3) Digested with methylation-sensitive restriction enzyme and set internal reference gene.With genomic DNA digested with HpaⅡ, and the MIC2 gene was used as an endogenous reference gene to determine the enzyme effect.(4) Established double PCR/DHPLC method. Established a double PCR system for STR and MIC2 gene locus; amplified the DNA samples before and after digested; optimized elution gradientaccording according to the fragment length of PCR produc and STR loci bases pairs under the DHPLC non-denaturing conditions.(5) Polyacrylamide gel electrophoresis and sequencing: 2 cases of selected samples for sequencing, select a family detected by non-denaturing polyacrylamide gel electrophoresis, in order to test the sensitivity of non-denaturing DHPLC.4. Study the relationship between spontaneous abortion women somatic X chromosome inactivation and spontaneous abortion: 165 cases of spontaneous abortion and 106 cases of induced abortion, extracted genomic DNA, digested with HpaⅡ, used the double PCR/DHPLC X-chromosome inactivation test.Inactivation ratio:(dl/ul)/(dl/ul+d2/u2) (d: digested the peak height, u: the peak height of undigested) were calculated, inactivation ratio of "proportion of more than 70% "and "proportion of more than 90%" as the strict standard to judge SXCI were used respectively.5. Study of the relationship between sex-specific embryo elimination in spontaneous abortion and X chromosome inactivation family: 105 female embryos of spontaneous abortion families (including villus and parents blood, of which 53 villi were normal karyotype,52 villi were abnormal karyotype).53 induced abortion female normal karyotype families (including villus and mother blood), extracted genomic DNA, digested with HpaⅡ, used double PCR/DHPLC detection of X-chromosome inactivation. Villi SXCI criterion: digested PCR amplification products still had the peak came from mother. Mother SXCI criterion: The inactivation ratio of "proportion of more than 70%" was used.Results and discussionIn the 167 cases of SA villus,165 (98.8%) cases were culture successfully.88 cases were tested to have abnormal chromosome, and the abnormal rate is 53.3%, which is consistent with abnormal rate reported in the early literature. This result indicated the importance of villi karyotype analysis in early spontaneous abortion. If villi chromosome could be tested, at least 50% of abortion could be diagnosed. With these specific diagnosesThese SA couples could be relieve from other unnecessary exams, and receive targeted specific fertility consultation. By karyotype analysis, we confirmed sex skewed XCI in SA. In the 165 successful villi cultures in our study, there werel05 female embryos and 60 male embryos (sex ratio is 1.8:1). In 106 induced abortion,53 cases were male and 53 were female embryos tested by karyotype analysis of villus, which include (male to female ratio was 1:1). The sex ratio of these two group has significantly different (P= 0.026).Using primers designed to detect the STR loci in AR gene showed that 346 cases were heterozygous loci in the 422 cases (82.0%) which is slightly lower than the 88.8% reported in the early literature. The MIC2 gene was used as an endogenous reference to determine whether enzyme effect was complete or not. We confirmed that the established double PCR/DHPLC detection system ris a relatively simple, sensitive and reliable system, which may have a prosperous future in research and clinical application.The results of the association between spontaneous abortion somatic SXCI and spontaneous abortion are inconsistent in the early literature. In the 165 cases of spontaneous abortion, the average age of mother was 30.84±5.28 (21～43 years). In the 106 cases of induced abortion, the average age of mother was 29.60±4.47 (23～48 years). No significant difference of age was found by statistic analysis (P= 0.077). and both groups were 8 to 11 weeks of gestation. The results showed that, using> 70% as the standard,21.9%(30/137) of spontaneous abortion women and 12.8 %(10/137) of induced abortion women were SXCI. But if increas the standard to a more stringent> 90%,8.0% (10/137) of spontaneous abortion women and2.3% (2/86) induced abortion women were SXCI. There were no difference of the SXCI incidence between the two groups (P=0.087, P=0.195) could be found by both of the two standards, Thus we cannot make the the conclusion that women SXCI is related with spontaneous abortion using women somatic SXCI as the only index, as. Meanwhile, in spontaneous abortion embryo from the SXCI women, female embryo accounted for 80.0%(24/30),while in embryo from the unshewed X inactivation SXCI women,female embryo accounted for 54.2%(58/107),which showed significant difference between the two groups(P= 0.011). This implied that women SXCI has relationship with specific gender embryo elimination, which means when mothers were SXCI, female embryos would be aborted more easily. The ratio of trisomy fetus in SXCI women (50%,15/30) was higher than the ratio of unshewed X inactivation women's (30%,32/107, P=0.040), suggesting that trisomy abortion was related wtih X chromosome inactivation.Study of X chromosome inactivation of spontaneous abortion from normal or abnormal female karyotype family, and induced abortion from normal female karyotype family showed that:in 53 spontaneous abortions from normal female karyotype family, the sources of X chromosome can be distinguished in 40 of them, and 85% (34/40) of these 40 cases had abnormal X chromosome inactivation (including 28 villus abnormalities only,4 abnormal villus and mother's X chromosome, two mother to villi X chromosome inactivation); in 52 spontaneous female abnormal karyotype families, the sources of X chromosomes can be distinguished in 36 of them, and 83.3% (30/36)of these 36 cases were abnormal X chromosome inactivation (including 19 villus abnormalities only,,6 abnormal villus and mother's X chromosome,5 mother to villi X chromosome inactivation); and in 53 induced abortion female normal karyotype families, the source of the abnormal X chromosome can be distinguished in 41 X chromosome, and the ratio of abnormal families was 39.0%(16/41), including 15 villous abnormality cases, and only one case of mother to villi X chromosome inactivation. Statistic analysis of these results showed that there were no significant difference (P> 0.0125) of the incidence of XCI between abnormal female karyotype group and normal female karyotype group in spontaneous abortion, but XCI in these two groups was significantly higher than that in the induced abortion normal female karyotype group (P=<0.00313), which indicated that female embryo abortion and abnormal X chromosome inactivation (especially abnormal villi X chromosome inactivation) are closely related, and that X chromosome inactivation may be an independent risk factor causing natural miscarriage, regardless of chromosomal condition.. In addition, in spontaneous abortion family,94.4%(17/18) SXCI women delivered high inactive X chromosome to embryos, only one passed her high active X-chromosome to embryo(1/18), while in the induced abortion group, only 3 SXCI women were SXCI(don't understand). Two of these X chromosome inactivation were caused by high active X chromosome being passed from mother., which indicated that high inactivation X chromosome being passed from SXCI women to embryo was one of the risk factor causing female embryo abortion. Moreover, we found that when analysingthe relationship between X chromosome inactivation and spontaneous abortion, it may be very important to determine if the SXCImother has passed the inactivation X chromosome to villi or not.ConclusionIn early spontaneous abortion, the phenomenon of specific gender embryo elimination was found, and X chromosome inactivation was a important step in embryo early develop. Through comprehensive comparison of villi from and X chromosome inactivation in peripheral blood of spontaneous abortion and induced abortion women, we found that female embryo spontaneous abortion are closely related to X chromosome inactivationespecially abnormal villi X chromosome inactivation., Abnormal X chromosome inactivation is likely to be a natural miscarriage risk factor independent of chromosomal abnormalities. To determine whether women SXCI is associated with female embryonic spontaneous abortion or not will depend on the delivery of the high inactivation X chromosome, to the embryo, which explains why there were more female embryos abortion in SXCI women. Meanwhile, the women SXCI may have relationship with trisomy incidence, cause the ratio of SXCI women abortion trisomy was significantly higher. In addition, the developed diagnosis system for X chromosome inactivation DHPLC method is highly sensitive, rapid, simple, convenient and economic. It is suitable for clinical application to find the cause of female embryos spontaneous abortion. With this technique, about 85% of women with female embryos spontaneous abortion were prospective to be found the root causes of the disease. Further exploration and study of the mechanisms of female embryo X chromosome inactivation abnormalities was needed to improve the applicability of this technique for research and clinical utility.