Projects

REPRODUCTIVE IMMUNOLOGY Component

Introduction

The association of HLA with recurrent spontaneous abortions (RSA) is a long lasting story, which remains controversial. Since 1977, when Komlos et al reported increased HLA sharing between spouses in couples with RSA appeared, many studies have investigated this hypothesis as well as the possibility of specific HLA antigens or HLA haplotypes to be markers of RSAs, but their results are still conflicting. Discrepancies between studies are complicated by the inconsistent definitions of recurrent miscarriage and of control groups, the small numbers of populations sampled, the different tissue typing methodologies used and the exclusion of possibly relevant but still unknown loci.

To face all the above problems, a programmed multi-center study was included in the 11^th International Histocompatibility Workshop (Yokohama, Japan-1991), where 201 RSA couples (101 of them Japanese) were typed by either serological or molecular techniques. Although this study did not lead to a clear conclusion as to the HLA sharing of couples with primary or secondary abortions, several groups still include HLA typing in the investigation of aborting couples and use HLA sharing as a criterion for the selection of women for immunotherapy (lymphocyte immunization, intravenous immunoglobulin G administration). In addition, during the last years, there are studies suggesting that partners HLA homozygosity or sharing for specific HLA class II alleles (mainly DQA1) and haplotypes (DQA1/DQB1, DR/DQ) predispose to immune-mediated abortions and that maternal-fetal compatibility for these alleles is frequently observed in aborted fetuses. However, many other investigators doubt these suggestions.

Data from the 13^th IHW

Because of all the above data, a new study was proposed in the frame of the 13^th IHWG (Victoria, Canada-2002), where the possible role of HLA in the pathogenesis of RSAs would be revisited under the light of current knowledge and advanced technology:
a) Improved knowledge about the immunological mechanisms underlying abortions as well as their diagnosis and the discrimination of allo- and auto-immune-mediated miscarriages,
b) Availability of DNA-based typing techniques for detailed definitions (high resolution) of alleles for all HLA loci and
c) Indications about an involvement of HLA in fetus allorecognition through decidual natural killer (NK) cells, which recognize their HLA ligands on trophoblast (classical HLA-C and non classical HLA-G and HLAE molecules) by certain NK cell receptors (NKR).
An extended Workshop study was expected to give an answer to existing controversies, to possibly provide new evidence for the relationship of HLA to miscarriages and to help in the decision of considering or not HLA sharing as a marker of immunological abortions and as a criterion for the selection of women for immunotherapy. The study was planned to investigate whether:
a) A significant HLA sharing or an increased homozygosity exist in couples with abortions of immunological aetiology.
b) The aborter HLA phenotype is associated with repeated pregnancy losses.
c) Partners NKR repertoire (combined or not to their HLA phenotypes) is associated with abortions. In a second phase, the study was planned to be extended to couples following in vitro fertilization (IVF) programmes, where women younger than 35 years of age have failed to maintain a pregnancy after >5 embryo transfers (ET).

Unfortunately, because of various problems, this study was not completed. Although 23 laboratories have expressed relevant interest, finally we collected results from 3 groups only, so that the intended investigation and analysis were not possible and only preliminary observations could be made. However, in a 3-hour discussion meeting that took place in Victoria, 5 groups presented their own experience on the importance of HLA and HLA-related genes in the reproduction process and the diagnosis and treatment of RSA and other pregnancy problems (presentations to appear inHLA 2002-Joint Report -Proceedings of the IHWS). The presentations were followed by an interesting discussion, which led to a series of preliminary conclusions:
a) The "disease" definition (allo- or auto-immune- mediated abortions) is very important when studying HLA association with RSA as well as for the selection of women to be treated and for the choice of immunotherapy to be used.
b) Most individual studies have not revealed increased HLA sharing in couples with RSA. However, increased HLA class II allele sharing and/or homozygosity of specific HLA alleles (e.g. DQA*0505) may associate with some cases of autoimmune-mediated abortions.
c) HLA alleles/haplotypes appear to be associated with pregnancy-related diseases (e.g. preeclampsia).
d) HLA-linked genes (in particular olfactory receptor genes) are possibly involved in the reproductive process.
e) The KIR repertoire of women and mainly maternal inhibitory KIR / trophoblastic HLA-C epitope matching are possibly involved in the maintenance of pregnancy.
f) Treatment of RSA with paternal leukocyte immunization is effective in a subgroup of women with high number of miscarriages, where alloimmune phenomena are suspected (absence of anti-paternal antibodies included), other causes of miscarriages havebeen excluded and other pathologic factors have been eliminated.

The above conclusions/indications indicated the need for continuing the study. Future plans suggested by the participants of the meeting were the following:
a) To complete allele typing for all HLA loci in the cases already reported.
b) To motivate other groups to submit data and recruit additional participants in order to obtain a sufficient sample size and make possible a separate analysis for the different categories of abortions and for defined ethnic groups.
c) To continue the study within the framework of the next IHWC on well-defined allo/auto-immune-mediated abortions, on couples with repeated IVF failures and women with pregnancy-related disorders.
d) Try to establish a Reproduction and HLA data Registry.

Proposal for the 14th IHIWS

Goal:

The RSA project will investigate whether:
a) Increased HLA sharing or HLA allele homozygosity between partners predispose to immune-mediated abortions and repeated IVF/ET failures.
b) The aborters HLA phenotype is associated with repeated pregnancy losses/failures.
c) Maternal KIR / trophoblastic HLA-C epitope matching is involved in the maintenance of pregnancy.

Research plan:

Subjects:
a. Childless couples with 3 or more RSA
b. Couples with no more than one successful pregnancy and 2 or more consecutive RSAs
c. Women with <3 abortions, where histology / immunohistology of the products of the abortions has revealed immune abnormalities.
d. Couples with repeated IVF/ET failures (women younger than 35 years of age, >5 E/T failures.
e. Controls: Fertile couples with no history of abortions (ethnically matched).
Exclusions: In all cases, other than immune aetiology of pregnancys failures, ought to be excluded:

Stage I:

In the initial stage of the study the centers and laboratories interested in participation will be identified. As it is essential to have well defined groups of immunological abortions, participants have to ensure that they can follow the protocol that will be given to them for the identification and the investigation of the couples.

Stage II:

After the selection of couples, DNA samples will be collected from all partners for HLA-A, -B, -C, -G, -E, -DRB1, -DQA1, -DQB1, -DPA1, -DPB1 alleles typing by PCR-SSO. All individuals will be also typed for NKR polymorphisms following the protocol of the ³NK Receptors and HLA polymorphisms² component of the Workshop.
Typing results will be accepted by ASHI or EFI accredited laboratories. Laboratories with no accreditation will be accepted after successful typing of control DNA samples.
Laboratories can use their own reagents or those of the workshop (upon request and payment).

Stage III:

According to the results of the protocol immunological tests, couples will be divided to subgroups (cases with alloimmune, autoimmune, thrombophilic abnormalities) for final analysis (sharing, homozygosity, associations).

REPRODUCTIVE IMMUNOLOGY Component

If you are interested in the RSA component,

please complete the following form and send it to the following address:

Dr Marighoula Varla-Leftherioti

Department of Immunobiology, Helena Venizelou Maternity Hospital

2 Helens Venizelou sq., Athens 11521

Greece

Fax: 30-210-6402149, Tel: 30-210-6453759, e-mail: gr1bmdr@hol.gr

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if yes, please, mark in which of the following areas of the study you wish to participate:

HLA typing of RSA couples

NKR typing of RSA couples

HLA typing of IVF couples

NKR typing of IVF couples>

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