Human Reproduction vol.13 no.1 pp.39–43, 1998

Parental human leukocyte antigens and implantation failure after in-vitro fertilization

Montserrat Creus1, Juan Balasch1,4, Francisco Fa´bregues1, Jaime Martorell2, Montserrat Boada3, Joana Pen˜arrubia1, Pedro N.Barri3 and Juan A.Vanrell1 1Department

of Obstetrics and Gynecology and 2Immunology Service, Faculty of Medicine, University of Barcelona, Hospital Clı´nic i Provincial, c/Casanova 143, 08036-Barcelona and 3Service of Reproductive Medicine, Institut Universitari Dexeus, Barcelona, Spain

4To

whom correspondence should be addressed

At present, it is well accepted that maternal recognition of paternally derived fetal antigens occurs during normal pregnancy and may be beneficial for implantation and maintenance of gestation. Thus, we have investigated the compatibility of human leukocyte antigens (HLA) in couples with successive failed in-vitro fertilization (IVF) cycles. Study group 1 included 50 couples with prior primary infertility who had not achieved a pregnancy after ù3 (range 3–7, mean 3.7) IVF cycles where at least two embryos (mean 3.3, range 2–4) were transferred in each attempt. An infertile control group (group 2) included 50 infertile couples undergoing IVF with the same indications as couples in group 1, who achieved a viable pregnancy with their first IVF attempt. The results were compared with those found in a population sample including 100 men and 100 women from the local population (group 3). We found a statistically significant (P < 0.05) excess of HLA sharing (ù2 antigens) between partners in group 1 as compared to groups 2. There was a trend toward increased HLA sharing in group 1 when groups 1 and 3 were compared. We conclude that some cases of implantation failure after IVF and embryo transfer might be caused by underlying close histocompatibility between partners. Key words: embryo transfer/histocompatibility/HLA antigens/ implantation failure/IVF

Introduction Early embryonic mortality in humans is very high and it has been postulated that the largest single cause of failed pregnancy is an error of implantation (Bulletti et al., 1996). The rate of spontaneous abortion may be as high as 60–78% if one takes into account those abortions occurring within the first month of conception which usually go undetected by patients (Bulletti et al., 1996). Similarly, implantation failure following embryo transfer is a major continuing problem in in-vitro fertilization (IVF). Thus, it has been disappointing that 85% of transferred © European Society for Human Reproduction and Embryology

human embryos resulting from IVF fail to implant in the uterus despite the selection of apparently normal embryos for transfer (Edwards, 1995a). Implantation is a brilliant example of successive interactions between two tissues, each genetically distinct from the other; thus, implantation involves a distinct immunological component (Edwards, 1995a, b). Antigenic differences between mother and fetus have traditionally been considered deleterious for viviparous reproduction, but it is now evident that maternal recognition of paternally derived fetal antigens not only occurs during normal pregnancy, but may be beneficial (Roberts et al., 1996). Clinical evidence pointing to the advantages of a wide antigenic difference between mother and fetus includes the high pregnancy rates reported after surrogate pregnancy involving embryo transfer or after embryo donation in IVF failure and repeated abortion patients, which involve the transfer of an embryo which is allogeneically distinct to both parents (Asch, 1992; Burton et al., 1992; Edwards, 1995a; Remohı´ et al., 1996, 1997). On the other hand, experimental studies in rodents have shown that genes in the region of the major histocompatibility complex (MHC) play an important role in reproduction, having effects on implantation, survival and growth of the embryos (Warner et al., 1988; Kurpisz and Fernandez, 1992; Jin et al., 1995). In the clinical setting, two previous reports have indicated that sharing of human leukocyte antigens (HLA) antigens may affect the success of IVF. However, the report by Ho et al. (1994) was of a Chinese population in Taiwan and included only patients with unexplained infertility undergoing their first IVF attempt, where women were treated by one of three different stimulation protocols, and underwent tubal embryo transfer by laparoscopy. Our previous report (Balasch et al., 1993) was a preliminary study including a small number of couples who failed three IVF cycles. In addition, in both previous HLA-sharing studies (Balasch et al., 1993; Ho et al., 1994) HLA typing was carried out only by conventional serological techniques which are now considered obsolete and up to 25% of serological HLA–DR typings may be incorrect when compared with DNA methods (Opelz et al., 1991; Christiansen, 1996). We therefore decided to investigate further the possible influence of the HLA system in Spanish couples failing repeated attempts of standard IVF.

Materials and methods Study group 1 included 50 couples with prior primary infertility who had not achieved a pregnancy after three or more (range 3–7, mean 3.7) IVF cycles. Patients in this experimental group had at least two embryos (mean 3.3, range 2–4) transferred in each attempt and none

39

M.Creus et al.

of them had a biochemical pregnancy or clinical spontaneous abortion after embryo transfer. As infertile control group (group 2) we included 50 primary infertility couples from the same geographical area as the couples in the experimental group to avoid any difference in ethnic composition between groups. Couples in group 2 were selected from those who had achieved a live birth with their first IVF attempt matching by indication for IVF and age (62 years). In order to compare the frequency of antigen compatibility within the HLA system in both infertility groups with expected figures in the general population assuming random mating, a second control group (group 3) included 100 men and 100 women from the local population who were healthy volunteers from the same local tissue-typing laboratory and who were randomly matched after tissue typing. A significant deviation at a given locus, from the frequency of sharing expected under the assumption of random mating, indicates a significant association between sharing of alleles at that locus and the criterion used to classify the couples into study groups (Jin et al., 1995). As in the two previous reports on the subject (Balasch et al., 1993; Ho et al., 1994), no increased HLA sharing was considered when a couple shared ø1 antigen, which is observed in three-quarters of control couples from the general population. All the IVFs were performed by the same previously reported protocol (Balasch et al., 1996) in which up to four embryos were replaced after gonadotrophin-releasing hormone agonist plus gonadotrophin ovarian stimulation followed by transvaginal oocyte retrieval. All women in groups 1 and 2 had both ovaries and no previous ovarian surgery. All individuals were studied during the same 18month period and all of them were tissue typed with the microlymphocytotoxicity test (class I HLA antigens) standardized by the National Institutes of Health (Bethesda, MD, USA; Darke and Dyer, 1993) and DNA-based techniques (PCR-SSCP®; Dynal, Oslo, Norway) (class II HLA antigens) as previously reported by us and others (Phelan et al., 1993; Vilardell et al., 1995). HLA-DR typing by polymerase chain reaction using sequence-specific primer mixes is a more reliable method than serology for the accurate identification of DR alleles and significantly reduces the incidence of phenotypic homozygosity (blanks) (Bryan et al., 1996). The genotyping technique used by us (Vilardell et al., 1995) identifies polymorphisms corresponding to serologically defined specificities DR1 to DR18, except for serological specificity DR3 whose splits DR17 and DR18 were combined with the main antigenic group for analysis (Bodmer et al., 1997). Clinical pregnancy was determined by ultrasonographic visualization of the gestational sac. The χ2 test and Student’s t-test were used for statistical analysis, as appropriate. Bonferroni’s correction was applied when multiple comparisons between groups were performed. Significance was defined as P , 0.05.

Results Patient characteristics were identical for both infertility groups studied. Mean ages in groups 1 and 2 were 33 6 3.9 and 32.5 6 2.4 years, and mean infertility duration 5.3 6 3.1 and 5.5 6 2.3 years, respectively. The indications for IVF in both groups 1 and 2 were tubal factor in 29 (58%) patients, unexplained infertility in 10 (20%), mild endometriosis in nine (18%), and male factor in two (4%). The number of embryos per replacement was 3.3 6 0.5 and 3.2 6 0.6 in groups 1 and 2, respectively. The frequency distribution of different HLA alleles found in the three groups studied (Table I) is similar to the allelic distribution reported in the Spanish general population (Imani40

Table I. Human leukocyte antigens (HLA) sharing in the three groups studied Common HLA antigens

Locus Aa 1 2 3 11 24 29 30 33 Locus Ba 7 8 14 18 35 38 44 51 52 57 62 Locus DRa 1 3 4 7 11 12 13 15 16 ABDR ù 2b

Sharing of HLA Group 1 (n 5 50)

Group 2 (n 5 50)

Group 3 (n 5 100)

25 1 12 4 2 2 2 1 1 19 3 1 1 2 3 1 4 2 0 1 1 24 2 5 5 4 5 0 3 0 0 22

15 (30%) 1 7 2 2 1 2 0 0 11 (22%) 1 0 0 1 1 0 7 0 1 0 0 19 (38%) 1 2 0 4 7 1 2 2 0 11 (22%)

18 2 10 0 1 3 2 0 0 9 1 0 0 2 0 0 5 0 0 0 1 45 7 3 8 7 9 0 7 3 1 28

(50%)

(38%)

(48%)

(44%)

(36%)

(18%)

(45%)

(28%)

Group 1 5 failure with IVF; group 2 5 success with IVF; group 3 5 control group. aNo significant differences between groups 1, 2 and 3. bGroup 1 was significantly different from group 2 (P , 0.05).

shi et al., 1991; Vilardell et al., 1995). There was a statistically significant excess of HLA sharing in the couples who failed ù3 IVF treatment cycles (group 1) compared with couples in whom the first IVF attempt was successful (group 2) (P , 0.05). There was a trend (P 5 0.1) toward increased HLA sharing among couples in group 1 when compared with the population sample (group 3), differences not reaching statistical significance because of the relatively low number of cases in group 1. Twelve of 50 couples (24%) in group 1 shared no HLAs, whereas 16 of 50 (32%) shared one antigen, 15 of 50 (30%) shared two antigens, six of 50 (12%) shared three antigens, and one of 50 (2%) shared four antigens. In group 2, 19 of 50 couples (38%) shared no HLAs, 20 of 50 (40%) shared two antigens, eight of 50 (16%) shared two antigens, and three of 50 (6%) shared three antigens. Corresponding figures in group 3 were as follows: 32 of 100 couples (32%) shared no HLAs, 40 of 100 (40%) shared one antigen, and 26 of 100 (26%) shared two antigens. There was no significantly increased sharing of any specific HLA allele at a given locus but rather an increased sharing of ù2 of the HLA-A, B, DR antigens. There were no differences between groups 2 and 3

HLA sharing and IVF

with respect to antigen distribution among the various HLA loci (Table I). Discussion Pregnancy presents an immunological challenge to a woman since half of the genes of the fetus derive from the father (Nelson, 1996). Human trophoblast cells may express a variety of non-MHC antigens such as CD46 [a complement-regulatory protein also called membrane co-factor protein (MCP)], heat shock protein and R80K, some of which are polymorphic (Clark, 1995; Vince and Johnson, 1995; Heyborne and Silver, 1996). There is currently no evidence to suggest that these proteins act as ligands for maternal immune recognition, although such recognition is theoretically possible (Clark, 1995; Vince and Johnson, 1995; Heyborne and Silver, 1996). Some of them (e.g. MCP) may protect trophoblasts against complement damage (Heyborne and Silver, 1996). HLA genes are of particular interest since they encode for molecules that are known to function as classical transplantation antigens and also govern immune responses. The classical HLA antigens are not expressed on trophoblast cells at the maternal–fetal interface, which instead express HLA-G, a class I molecule (Kovats et al., 1990). However, HLA-G has very limited polymorphism relative to classical HLA molecules (Nelson, 1996) and although both villous and extravillous cytotrophoblast cells transcribe HLA-G (Le Bouteiller, 1994; Vince and Johnson, 1995), no HLA-G was detected in the syncytiotrophoblast isolated from first trimester placenta (Chumbley et al., 1993) nor in the in-vitro differentiated syncytiotrophoblast from term placenta (Guillaudeux et al., 1995). According to recent studies it is possible that HLA-C is co-expressed with HLA-G in human extravillous trophoblast, but HLA-C is not present on villous cytotrophoblast and syncytiotrophoblast (King et al., 1996). In addition, low level of allogeneic polymorphism for HLA-C was found in comparison with HLA-A and -B (Zemmour and Parham, 1992). Therefore, these proteins are very unlikely to elicit ‘protection of damage’ maternal immune responses. Nevertheless, even in the absence of expression of classical HLA molecules at the maternal–fetal interface, maternal exposure clearly occurs either by fetal cells escaping into the maternal circulation or by other means. As previously reported by us, term pregnancy or abortion may sensitize the mother to fetal paternally derived HLA antigens, but the presence of HLA antibodies is not related to maternal complications in pregnancy and fetal wastage (Balasch et al., 1981; Gelabert et al., 1981). Rather, there is now little doubt that the expression of paternally inherited and fetal genes ensures that the conceptus is recognized as foreign by the mother and maternal recognition of pregnancy is considered beneficial for the survival of the embryo (Roberts et al., 1996). To explain the possible role of HLA in determining reproductive outcome, both immunological (Aksel, 1992) and genetic (Gill, 1994) hypotheses have been postulated. The first focuses on the nature and significance of the maternal immune response to the paternally derived antigens of the placenta and the fetus whereas the latter focuses on the influence of genes

that affect development, especially those that are linked to the MHC. Systems such as enhancement facilitation are believed to protect an allogeneically distinct fetus from its mother’s rejection responses, and a high degree of immunological compatibility between mother and fetus has been proposed as a cause of implantation failure or recurrent abortion (Edwards, 1995a). Conflicting results, however, have been obtained from different studies on the possible role of HLA in aetiopathogenesis of recurrent abortion and infertility. Increased compatibility for HLA-A, B, and DR locus between partners of recurrent spontaneous abortion compared to controls has been reported by several authors but also denied by others (for review, see Christiansen, 1996). Studies on HLA sharing in couples with recurrent spontaneous abortion suffer from certain limitations inherent in retrospective studies as well as from inadequate controls. In addition, none of them addresses the effects of parental HLA sharing in couples not selected on the basis of reproductive histories. To address these methodological limitations and to elucidate further the reproductive effects of maternal–fetal histocompatibility, Ober et al. (1985, 1988) performed prospective population-based studies of parental HLA sharing and reproductive outcome in the Hutterites, an isolated population that lives communally and proscribes contraception. The relationship between HLA sharing and reproductive outcome was examined in 111 Hutterite couples. Results obtained in these studies indicate that HLA sharing is associated with reduced fertility in Hutterites, as evidenced by more spontaneous abortions, longer intervals between births, and smaller completed family sizes among histocompatible couples. These data indicate a potentially important role for HLA genes in normal reproduction. Ober et al. (1988) suggest that longer intervals between births associated with HLA sharing may result from losses occurring early in gestation, before Hutterites would recognize pregnancy. If so, and considering the idea that unexplained infertility may be due to occult early abortions (Bloch, 1978; Edmonds et al., 1982) and that the condition includes a substantial proportion of couples with delayed conception (Collins et al., 1983; Wilcox et al., 1988), an increased frequency of HLA sharing should be found among those couples with normal infertility evaluation. Again results in the literature are contradictory. Thus, while some studies found no significant differences in the frequencies of HLA antigens between couples suffering from unexplained infertility and normal fertile couples (Nordlander et al., 1983; Persitz et al., 1985), others found a significantly higher frequency in HLA homozygosity among couples with unexplained infertility (Coulam et al., 1988). Similarly to unexplained infertility, some failures of embryo transfer after IVF, especially in cases with repeatedly unsuccessful embryo transfer, are thought to be because of occult abortion (Clark, 1989; Hasewaga et al., 1992). Curiously, however, HLA studies in IVF couples have been scanty (Balasch et al., 1993; Ho et al., 1994). In the study by Ho et al. (1994), 76 couples with unexplained infertility were typed for HLA and treated by IVF and tubal embryo transfer. There was a highly significant excess of HLA sharing (ù2 antigens) in the 36 couples who failed treatment. Similar 41

M.Creus et al.

conclusions were found in our preliminary report where 15 couples treated with IVF because of tubal factor, endometriosis or unexplained infertility and who had three successive failed IVF cycles were compared with 15 IVF couples achieving a viable pregnancy with their first IVF attempt (Balasch et al., 1993). This is confirmed and expanded in the present study where the new HLA-DR typing by PCR amplification techniques and standard conditions in an IVF programme (common indications for IVF, gonadotrophin ovarian stimulation under pituitary suppression with a long protocol of GnRH-agonist administration, transvaginal oocyte recovery, and cervical embryo transfer of 2–4 embryos) were used. Moreover, in our study and in contrast with the report by Ho et al. (1994), couples in group 1 failed successive attempts (3–7) of embryo transfer. It is of note that none of the HLA studies on IVF couples, including the present report, identified any specific HLA antigen associated with failure of implantation after embryo transfer. Therefore, the HLA-sharing antigen itself probably does not influence the reproductive outcome; rather it may be a marker for the sharing of closely linked susceptibility genes or genetic defects. In fact, there is a substantial amount of evidence that the advantage of the hybrid fetus is not due to immune but genetic factors (Gill, 1994; Jin et al., 1995). Support for the genetic hypothesis for the cause of reproductive defects comes from the comparative genetics of the MHC. Genes influencing growth and reproduction in the MHC-linked region of the rat that are not class I or class II genes have been identified, and the presence between the HLA-B and DR chromosomal region of genes homologous to the grc region in the rat, which affects growth and development, and to the Ped locus in the mouse, which affects preimplantation embryonic development, has also been suggested in the human (Gill, 1994; Jin et al., 1995). In conclusion, our study shows that ù3 implantation failures after IVF are associated with ù2 shared HLAs between partners. Further studies on HLA sharing, including the HLAC recently discovered in trophoblast, are desirable in order to confirm our results. If so, it would be worthwhile to screen couples for HLA compatibility who fail multiple IVF attempts. Gamete or embryo donation would be a therapeutic alternative in these cases in order to provide the wide antigenic difference between mother and fetus necessary for successful implantation.

Acknowledgement We thank Mrs Paquita Antonell for her technical assistance.

References Aksel, S. (1992) Immunologic aspects of reproductive diseases. J. Am. Med. Assoc., 268, 2930–2934. Asch, R.H. (1992) High pregnancy rates after oocyte and embryo donation. Hum. Reprod., 7, 734. Balasch, J., Ercilla, G., Vanrell, J.A. et al. (1981) Effects of HLA antibodies on pregnancy. Obstet. Gynecol., 57, 444–446. Balasch, J., Jove´, I., Martorell, J. et al. (1993) Histocompatibility in in vitro fertilization couples. Fertil. Steril., 59, 456–458. Balasch, J., Fa´bregues, F., Creus, M. et al. (1996) Pure and highly purified follicle-stimulating hormone alone or in combination with human

42

menopausal gonadotrophin for ovarian stimulation after pituitary suppression in in-vitro fertilization. Hum. Reprod., 11, 2400–2404. Bloch, S.K. (1978) Occult pregnancy as a factor in unexplained infertility. J. Reprod. Med., 21, 251–253. Bodmer, J.G., Marsh, S.G.E., Albert, E.D. et al. (1997) Nomenclature for factors of the HLA system, 1996. Tissue Antigens, 49, 297–321. Bryan, C.F., Harrell, K.M., Nelson, P.W. et al. (1996) HLA-DR and DQ typing by polymerase chain reaction using sequence-primer mixes reduces the incidence of phenotypic homozygosity (blanks) over serology. Transplantation, 62, 1819–1824. Bulletti, C., Flamigni, C. and Giacomucci, E. (1996) Reproductive failure due to spontaneous abortion and recurrent miscarriage. Hum. Reprod. Update, 2, 118–136. Burton, G., Abdalla, H.I., Kirkland, A. and Studd, J.W.W. (1992) The role of oocyte donation in women who are unsuccessful with in-vitro fertilization treatment. Hum. Reprod., 7, 1103–1105. Christiansen, O.B. (1996) A fresh look at the causes and treatments of recurrent miscarriage, especially its immunological aspects. Hum. Reprod. Update, 2, 271–293. Chumbley, G., King, A., Holmes, N. and Loke, Y.W. (1993) In situ hybridization and Northern blot demonstration of HLA-G mRNA in human trophoblast populations by locus-specific oligonucleotide. Hum. Immunol., 37, 17–22. Clark, D.A. (1989) Current concepts of immunoregulation of implantation. In Chapman, M., Grudzinskas, G. and Chard, T. (eds), Implantation: Biological and Clinical Aspects. Springer Verlag, London, pp. 163–175. Clark, D.A. (1995) Immunobiological characterization of the trophoblastdecidual interface in human pregnancy. In Kurpisz, M. and Fernandez, N. (eds), Immunology of Human Reproduction. Bios Scientific, Oxford, pp. 301–311. Collins, J.A., Wrixon, W., Janes, L.B. and Wilson, E.H. (1983) Treatmentindependent pregnancy among infertile couples. N. Engl. J. Med., 309, 1201–1206. Coulam, C.B., Moore, B. and O’Fallon, W. (1988) Investigating unexplained infertility. Am. J. Obstet. Gynecol., 158, 1374–1381. Darke, C. and Dyer, P. (1993) Clinical HLA typing by cytotoxicity. In Dyer, P. and Middleton, D. (eds), Histocompatibility Testing. A Practical Approach. IRL Press, Oxford, pp. 51–80. Edmonds, D.K., Lindsay, K.S., Miller, J.F. et al. (1982) Early embryonic mortality in women. Fertil. Steril., 38, 447–453. Edwards, R.G. (1995a) Clinical approaches to increasing uterine receptivity during human implantation. Hum. Reprod., 10 (Suppl. 2), 60–66. Edwards, R.G. (1995b) Physiological and molecular aspects of human implantation. Hum. Reprod., 10 (Suppl. 2), 1–13. Gelabert, A., Balasch, J., Ercilla, G. et al. (1981) Abortion may sensitize the mother to HLA antigens. Tissue Antigens, 17, 353–356. Gill, T.J. III (1994) Reproductive immunology and immunogenetics. In Knobil, E. and Nell, J.D. (eds), The Physiology of Reproduction, 2nd edn. Raven, New York, vol. 2, pp. 783–812. Guillaudeux, T., Rodriguez, A.M., Girr, M. et al. (1995) Methylation status and transcriptional expression of the MHC class I loci in human trophoblast cells from term placenta. J. Immunol., 154, 3283–3299. Hasewaga, I., Tani, H., Takakuwa, K. et al. (1992) Immunotherapy with paternal lymphocytes preceding in vitro fertilization–embryo transfer for patients with repeated failure of embryo transfer. Fertil. Steril., 57, 445–447. Heyborne, K. and Silver, R.M. (1996) Immunology of postimplantation pregnancy. In Bronson, R.A., Alexander, N.J., Anderson, D. et al. (eds), Reproductive Immunology. Blackwell Science, Oxford, pp. 383–417. Ho, H.N., Yang, Y.S., Hsieh, R.P. et al. (1994) Sharing of HLA antigens in couples with unexplained infertility affects the success of in vitro fertilization and tubal embryo transfer. Am. J. Obstet. Gynecol., 170, 63–71. Imanishi, T., Akaza, T., Kimura, A. et al. (1992) Allele and haplotype frequencies for HLA and complement loci in various ethnic groups. In Tsuji, K., Aizawa, M. and Sasazuki, T. (eds), HLA 1991: Proceedings of the Eleventh International Histocompatibility Workshop and Conference. Oxford Science, Oxford, vol. 1, pp. 1065–1220. Jin, K., Ho, H.N., Speed, T.P. and Gill, T.J. III (1995) Reproductive failure and the major histocompatibility complex. Am. J. Hum. Genet., 56, 1456–1467. King, A., Boocock, C., Sharkey, A.M. et al. (1996) Evidence for the expression of HLA-C class I mRNA and protein by human first trimester trophoblast. J. Immunol., 156, 2068–2076. Kovats, S., Main, E.K., Librach, C. et al. (1990) A class I antigen, HLA-G, expressed in human trophoblast. Science, 248, 220–223.

HLA sharing and IVF Kurpisz, M. and Fernandez, N. (1992) Main histocompatibility complex and reproductive system. Am. J. Reprod. Immunol., 28, 19–30. Le Bouteiller, P. (1994) HLA class I chromosomal region, genes and products: facts and questions. CRC Crit. Rev. Immunol., 14, 89–129. Nelson, J.L. (1996) Maternal-fetal immunology and autoimmune disease. Arthritis Rheum., 39, 191–194. Nordlander, C., Fuchs, T., Hammarstro¨m, L. and Smith, C.I.E. (1983) Human leukocyte antigens group A in couples with unexplained infertility. Fertil. Steril., 40, 60–65. Ober, C.L., Hauck, W.W., Kostyu, D.D. et al. (1985) Adverse effects of human leukocyte antigen-DR sharing on fertility: a cohort study in a human isolate. Fertil. Steril., 44, 227–232. Ober, C., Elias, S., O’Brien, E. et al. (1988) HLA sharing and fertility in Hutterite couples: evidence for prenantal selection against compatible fetuses. Am. J. Reprod. Immunol. Microbiol., 18, 111–115. Opelz, G., Mytilineos, J., Scherer, S. et al. (1991) Survival of DNA HLA-DR typed and matched cadaver kidney transplants. Lancet, 338, 461–463. Persitz, E., Oksenberg, J.R., Margoliath, E.J. et al. (1985) Histoincompatibility in couples with unexplained infertility. Fertil. Steril., 43, 733–738. Phelan, D.L., Mickelson, E.M., Noreen, H.S. et al. (1993) Laboratory Manual, 3rd edn. American Society for Histocompatibility and Immunogenetics, Lenexa. Remohı´, J., Gallardo, E., Levy, M. et al. (1996) Oocyte donation in women with recurrent pregnancy loss. Hum. Reprod., 11, 2048–2051. Remohı´, J., Gartner, B., Gallardo, E. et al. (1997) Pregnancy and birth rates after oocyte donation. Fertil. Steril., 67, 717–723. Roberts, R.M., Xie, S. and Mathialagan, N. (1996) Maternal recognition of pregnancy. Biol. Reprod., 54, 294–302. Vilardell, C., Isart, F., Maso´, M. et al. (1995) DRβ1 allelic distribution in the Spanish population. Transplant Proc., 27, 2413–2414. Vince, G.S. and Johnson, P.M. (1995), Materno-fetal immunobiology in normal pregnancy and its possible failure in recurrent spontaneous abortion? Hum. Reprod., 10 (Suppl. 2), 107–113. Warner C.M., Brownell, M.S. and Ewoldsen, M.A. (1988) Why aren’t embryos immunologically rejected by their mothers? Biol. Reprod., 38, 17–29. Wilcox, A.J., Weinberg, C.R., O’Connor, J.F. et al. (1988) Incidence of early loss of pregnancy. N. Engl. J. Med., 319, 189–194. Zemmour, J. and Parham, P. (1992) Distinctive polymorphism at the HLA-C locus: implications for the expression of HLA-C. J. Exp. Med., 176, 937–950. Received on July 21, 1997; accepted on September 30, 1997

43