Genotyping: the HLA system and embryo development

Abstract

The human major histocompatibility complex (MHC), in addition to its role in the regulation of cell-cell interactions in the immune response, also influences reproductive success. Human leukocyte antigen-G (HLA-G) is an MHC class I gene of particular interest in reproductive biology because of its specific expression on fetal cytotrophoblast cells, and its reported involvement both in protection of the developing fetus from destruction by the maternal immune response and in the prevention of maternal pre-eclampsia. HLA-G has 15 known alleles at the DNA level, and allelic frequency varies among ethnic groups. This study describes the results of an inaugural attempt to correlate an HLA-G genetic polymorphism with pregnancy outcome in a patient population undergoing IVF. The study group was composed of 102 Caucasian women. A maternal HLA-G genetic polymorphism was investigated by polymerase chain reaction (PCR) analysis of DNA collected from granulosa cells surrounding oocytes harvested for the IVF procedure. While no statistically significant correlation was identified in this initial study, larger studies examining DNA from trios of mother, father and offspring are planned.

Figure 1

Structure of HLA-G at the DNA, RNA, and protein levels. At the DNA level, the exons are open boxes and the 3 ′UTR is a shaded, hatched box. The primary RNA transcript is larger than shown; the RNA transcript is shown only up to the stop codon in exon 6. Also shown is the location of the upper (U) and lower (L) primers used in this study.

Figure 2

Sequence alignment of the two most common Caucasian HLA-G alleles. The locations of the upper (U) and lower (L) primers are bold and underlined. (The actual primer sequences are shown in Table 2.) Similarities between the two alleles, G*01011 and G*01012, are shown by a dot. A two bp deletion and a 14 bp deletion in the G*01011 allele are denoted by asterisks (*). Ten by markers are denoted by a plus sign (+). The exon sequences are shown in upper case letters and the introns and 3′UT region sequences are shown in lower case letters. The termination codons in exons 6 and 8 are shown in bold letters. A single nucleotide polymorphism (SNP) is present at the location of the lower primer (position 3791) and is indicated by an arrow. The GenBank accession numbers for G*01011 and G*01012 are J03027 and S50740 respectively.

Figure 3

Sample gel showing the PCR products using primers for HLA-G. M = Molecular weight markers with an arrow pointing to the 700 bp marker; Lanes 11–18 represent patient samples that either had the presence or absence of a band for HLA-G.