Contribution of chromosomal abnormalities and genes of the major histocompatibility complex to early pregnancy losses

Approximately 15 % of all clinically recognized pregnancies are spontaneously aborted. Miscarriage includes spontaneous abortions (SA) from the start to the 23-rd week of gestation [1, 2]. The most often cited preconditions for recurrent miscarriages (RM) are the antiphospholipid syndrome in mothers, genetic abnormalities in parents or in fetuses, anatomical and endocrine factors, inherited throm bophilias and various immunological causes [3, 4]. Among the most frequent risk factors for SA are the age of the mother and the pathology of the fetus. Pregnancy losses (PL) in 60 to 80 % of the cases are associated with chromosomal abnormalities (CA) UDC [576.316+575.113.2]:618.39-021.3


Introduction
Approximately 15 % of all clinically recognized pregnancies are spontaneously aborted.Miscarriage includes spontaneous abortions (SA) from the start to the 23-rd week of gestation [1,2].The most often cited preconditions for recurrent miscarriages (RM) are the antiphospholipid syndrome in mothers, genetic abnormalities in parents or in fetuses, anatomical and endocrine factors, inherited throm bophilias and various immunological causes [3,4].Among the most frequent risk factors for SA are the age of the mother and the pathology of the fetus.
In EPL occurring up to the 10th week of pregnancy, double and triple trisomies involving different chromosomes are often found.In most cases, autosomal double trisomies, regardless of the chromosomes involved, are lethal [17,19].Polyploidies are compatible with a fetal development in humans only as triploids 69, XXX and 69, XXY [12,20,21].
Information about the structure of CA in SA is gathered on the basis of numerous cytogenetic studies of various tissues using conventional cytogenetic analysis of metaphase chromosomes.However, as fetal tissue may be retained in utero for several days or weeks after the fetal demise, the tissue may be autolyzed and unresponsive to standard chromosome analysis.With the development of new methods like molecular cytogenetics, more opportunities ari se that enable the study of karyotypes in uncultured cells from SA [20].Interphase FISH allows identifying all types of numerical abnormalities (aneup loidy and polyploidy), including mosaic forms [18,[21][22][23].Consequently, studying the frequency and spectrum of CA in EPL is a key part of the investigations of reproductive failure in humans.
Immunologic factors contribute to PL especially at the early stages of gestation (up to 14 weeks).In particular, 45 % of cases of the EPL are accompanied by immunologic intolerance to the fetus.In such instances the immune system of the mother organism reacts to the fetus antigens as to foreign antigens and aims towards the destruction of the antigen source.The studies on misregulated interaction between the cells of the mother and the fetus are key issues for the identifi cation of factors involved in SA.
One of the main factors involved in immune responses is the major histocompatibility complex (MHC) encoded by human leukocyte antigen (HLA) gene locus.The HLA is an extended region of the genome that spans some 4 million base pairs (bp) on the short arm of human chromosome 6 between 6p21.31 and 6p21.32.The involvement of the HLA system in predisposition to RM has been analyzed through the identifi cation of particular HLA genes that are linked to reproductive losses [24][25][26], to similarities of HLA antigens between partners [27] and to genes involved in modulation of the HLA system in the gene expression network [28].
In this study we have investigated chromosome ab normalities in a series of SA material samples and have analyzed the prevalence of HLA-DRB1 and DQA1 alleles in the couples with RPL.

Materials and Methods.
Specimens from SA in the period from 4 to 14 weeks of gestation were obtained from 209 females aged 22 to 42 years.DNA for HLA analysis was extracted from peripheral blood leukocytes of 38 couples with recurrent miscarriage and 34 couples with the absence of miscarriages in anamnesis and having two or more children.All of the cases included in the present study were approved by the Ethics committee of the Institute of Hereditary Pathology NAMS of Ukraine.Informed consent for cytogenetic study and HLA-genotyping was obtained from all patients.

Banding cytogenetic processing
For SA material processing, the cells of the chorionic villi were separated from the decidual cells.We used the method of direct chromosome preparati on from chorion [29] and analyzed the samples cy togenetically using G-banded cytogenetic techni ques.Samples were visualized under a light microscope (Zeiss, Axioscope; Jena, Germany).A minimum of 10 metaphases were scored per sample.

Interphase multicolor fl uorescence in situ hybridization (mFISH) analysis
Preliminary analysis was performed on interphase nuclei.The hybridization, post-hybridization washes and detection steps were done as described in [30].Image acquisition was performed using an Axioplan II microscope (Carl Zeiss Jena GmbH) equipped with fi lter sets for DAPI, FITC, TR, Cy3 and Cy5 fl uorescence channels.Image ana lysis was done with the help of Isis DGTSa, BGR-I and DGSoSa software (MetaSystems Hard & Soft ware GmbH, Altlussheim, Germany).Three ho me made probe sets were used as specifi ed below: All probe sets were fi rst tested separately on human metaphase spreads prior to use in probe mixes to evaluate quality and to exclude contamination.The region specifi c probes were mapped cytogenetically based on the inverted DAPI banding pattern of chromosomes.At least 100 interphase nuclei per sample were analyzed.

HLA analysis
DNA was extracted from peripheral blood leukocytes using the salting-out method.The genotyping of HLA-DRB and HLA-DQA1 was performed using polymerase chain reaction (PCR) in Terzik thermocycler (DNA-technology, Russian Federation (RF)) with automated regime according to the corresponding amplifi cation program.The ty ping of alleles of the above mentioned genes was conducted using the reagent kits designed for ge notyping via DNA amplifi cation by PCR with sequence-specifi c primers: «GenPak®HLA-DRB PCR test» and «GenPak®HLA-DQA1 PCR test» (Laboratory Isogen LLC, RF).PCR products of allelic variants were detected by 3 % agarose gel electrophoresis, stained with ethidium bromide in UV-light at 302 nm.
Statistical processing of results was conducted using Pearson criterion χ 2 .The critical level of significance for statistical criteria for all the types of analysis was defi ned as p < 0.05.The association of genotypes and alleles with the risk of reproductive failure was evaluated by calculating the odds ratio (OR) with 95 % confi dence interval.

Results and Discussion
We performed banding and molecular cytogenetic studies on 209 chorionic villus sampling (CVS) specimens from women with sonographically diagnosed MAs or blighted ovum (from 4 to 14 weeks of gestation).Banding cytogenetic results were obtainable in 120 cases.To test the products of conce ption where the banding analysis was impossible due to the absence of metaphases, the mFISH ana lysis was performed.mFISH has been described as the current gold standard to determine the numerical chromosome aneuploidies (NCA) and polyploidies.In contrast to karyotyping, it can be used on interphase nuclei, so it can be applied on specimens from CVS.The mFISH analysis added the va lue to the complex investigation performed in the cur rent study and contributed to the profi ling of NCA in tissues from EPL that is of great signifi cance for genetic counseling of the couples with RM as well as for human reproduction investigations.
Another factor of EPL according to a series of reports [27,28,30,31] is the homology of HLA-antigens between the partners or the presence of aggressor allele in the woman.To follow up on this information we have attempted to identify the immunological component of RM.We have selected 38 couples with RM that had a history of at least one miscarriage and karyotyped via cytogenetic and molecular-genetic methods.The control group consisted of 34 couples with normal obstetrics and genetic anamnesis having two or more healthy children.We have investigated 16 alleles of HLA-DRB1 and 10 alleles of the HLA-DQA1 genes, characterized the allelic polymorphisms and genotypes based on those genes and compared the data with the control group.
Compared to the control group the results showed different allele variant frequencies of the investigated genes in the couples with RPL (38 couples, 76 individuals, 152 alleles) and more specifi cally -in women with RPL (38 individuals, 76 alleles).We have observed an increased frequency of the DRB1*0301 allele in the couples with RPL (12.82 % versus 5.15 % in control group, Table 2) and in the women with RPL (14.1 % versus 4.41 %).Additiionally, in the women with RM the frequency of DRB1*1101-1104 and DQA1*0501 was higher than in the control group (20.51 % versus 8.82% and 38.46 % versus 22.06 % respectively, Table 3).
Odds ratio (OR) calculation shows that carrying DRB1*0301 allele increases the RPL risk in the couples by almost three times (OR = 2.79; CI -95 %: 1.14-  6.83), and carrying DQA1 *0501 allele increases the RPL risk in women by two times (OR =2.2; CI-95%: 1.06-4.59).The results of positive association of given alleles with RPL are concordant with other publications [33] and support our colleagues observations [33][34][35][36][37][38] that the presence of specifi c allelic variants of genes DR and DQ, that belong to HLA loci, in the mother can create the environment where the mother immune system does not recognize the fetus antigens and does not involve the mechanisms aimed at the protection of the fetus.The results were also analyzed from the perspective of similarity of allelic variants of genes HLA-DRB1 and HLA-DQA1 between the partners.Homology between the partners is considered to be critical if the allelic polymorphism homology is more than 50 % (when the partners have at least one similar allele of each locus).We have observed an increased frequency of homology of studied genes between partners in couples with RPL -42.10 % versus 17.65 % in control group (Table 4).
Odds ratio calculation showed a four-time increase of the RPL risk in the couples with the interpartner allelic variant homologies of genes HLA-DRB1 and HLA-DQA1 (OR = 3.60; CI -95 %: 1.22-10.68).
We have analyzed the results of the cytogenetic investigations of the RPL material in light of the inter-partner homology of the investigated HLA loci.In 36 couples we have cytogenetically investigated the samples from one SA, in two couples -from two SA.There were no signifi cant differences in frequency and spectrum of chromosomal anomalies dependent on the level of inter-partner HLA homology (Table 5).

Conclusions
1. Cytogenetic and molecular-cytogenetic investigations of SA material identifi ed karyotype anomalies in 32.4 % of cases with prevalence of autosomal trisomy -42.65 %, triploidy -30.38 % and monosomy X -19.11 %.Among the miscarriages the sex ratio XY:XX was 1.2.
2. Complex analysis of frequency and distributi on of allelic variants of genes HLA-DRB1 and HLA-DQA1 permitted to establish the alleles DRB1*0301, DRB1*1101-1104 and DQA1*0501 to be aggressor ones in the women with RPL.The female carriers of the DQA1 *0501 allele have a twice higher RPL risk (OR = 2.2; CI -95 %: 1.06-4.59).
3. The cumulative homology of allelic polymorphism of more than 50 % of the HLA-DRB1 and HLA-DQA1 loci between the partners increases the RPL risk of by almost four times (OR = 3.60; CI -95 %: 1.22-10.68).
4. No differences in the frequency of chromosomal anomalies were found in the material of spontaneous abortions in couples with different level of homology of the HLA-DRB1 and HLA-DQA1 loci.

2 1
State Institution «Institute of Hereditary Pathology, NAMS of Ukraine» 31a, M. Lysenko Str., Lviv, Ukraine, 79008 2 Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics 10, Kollegiengasse, Jena, Germany, D-07743 huleyuk@yahoo.comAim.The determination of chromosomal abnormalities in samples from early pregnancy losses and allelic polymorphism of HLA-DRB1 and DQA1 genes in couples with recurrent miscarriage.Methods.Banding cytogenetic and interphase mFISH analysis, DNA extraction by salting method, PCR, agarose gel electrophoresis.Results.Cytogenetic and molecular-cytogenetic investigations of SA material identifi ed karyotype anomalies in 32.4 % of cases with prevalence of autosomal trisomy -42.65 %, triploidy -30.38 % and monosomy X -19.11 %.Complex analysis of frequency and distribution of allelic variants of genes HLA- DRB1 and HLA-DQA1 allowed establishing the alleles DRB1*0301, DRB1*1101-1104 and DQA1*0501 to be aggressor alleles in women with recurrent pregnancy loss (RPL).The cumulative homology of allelic polymorphism of more than 50 % of HLA-DRB1 and HLA-DQA1 loci between partners increases the risk of RPL by almost four times.Conclusion.The detected chromosome aneuploidies in the samples from products of conception and the changes in the major histocompatibility complex genes can cause the failure of a couples reproductive function and can lead to an early fetal loss.K e y w o r d s: pregnancy loss, chromosome abnormalities, cytogenetics, mFISH, chromosome, HLAgenotyping.

Table 3 . Identifi ed aggressor allele in women with RPL
N o t e: a quantity of alleles in group; b frequency of alleles in group.