1 Department of Obstetrics and Gynecology and 2 Department of Medical Statistics, University of Bonn, Sigmund-Freud-Straße 25, 53105 Bonn, Germany
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Abstract |
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Key words: HLA class II/ICSI/male infertility/MHC/spermatogenesis
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Introduction |
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There are two major lines of conceptual thinking about the involvement of MHC genes in reproduction. The first employs inadequate immunological interactions between partners or between mother and fetus as a potential cause of pregnancy failure (Beer et al., 1985; Gill, 1992
). MHC antigens, which are major determinants of acceptance or rejection of mammalian allografts in transplantation medicine, have consequently been investigated thoroughly in the context of infertility or pregnancy failure (reviewed by Ober and van der Ven, 1997). However, no convincing evidence could be accumulated for inadequately strong or weak maternal immune responses against fetal antigens as a general cause of pregnancy failure or idiopathic infertility.
The second concept about the involvement of MHC genes in control of pregnancy development employs a genetic basis. The existence of lethal and semilethal genes located within or in close neighbourhood to the MHC complex has been documented in the mouse (Frischauf, 1985; Yeom et al., 1992
), the rat (Gill, 1992
) and has also been proposed for humans (Ho et al., 1990
, 1991
, 1994
; Gill, 1994
; Jin et al., 1995
).
In wild mouse strains, 3040% of chromosomes contain the so-called T/t alleles, which are located within the murine homologue of the MHC complex on chromosome 17. Presence of T/t-alleles in homo- or heterozygosity can lead to a range of symptoms from embryonic death to reduced fertility or malformations (Frischauf, 1985). In the mouse, several genes which are expressed during spermatogenesis have been cloned within the T/t complex (Lader et al., 1989
; Ha et al., 1991
; Mazarakis et al., 1991
), which provides support for the thesis of a genetic basis of MHC-associated effects on fertility. Similar MHC-linked effects on fertility as described in mice could be observed in other species, e.g. in the rat, swine, chicken or horses (reviewed by Gill, 1994) and have also been suggested for humans (Gill, 1994
).
Studies on MHC-associated effects on reproduction in humans have with few exceptions (Christianssen et al., 1994) focused on sharing of HLA antigens in couples with unexplained infertility or recurrent spontaneous abortions and are in the majority based on the hypothesis of impaired maternal immune reactions against the fetus as a result of increased HLA similarity between partners (reviewed by Ober and van der Ven, 1997). However, more recent studies in couples with unexplained infertility (Ho et al., 1994
; Jin et al., 1995
), but also prospective studies in fertile populations (Ober et al., 1992
; Jin et al., 1995
) raise the possibility that homozygosity for genes which are closely linked to but not identical with certain HLA loci might be responsible for the increased fetal loss rates or prolonged pregnancy intervals observed in couples who share HLA alleles or haplotypes (Ober et al., 1992
, 1998
; Ober, 1995
).
However, it has not yet been investigated in detail if an influence of MHC genes on male fertility can be observed in humans which is analogous to the T/t associated effects described in mice.
In the light of recent findings of diploid HLA class I and II antigen expression on spermatozoa and HLA gene transcription during spermatogenesis in the human (Martin-Villa et al., 1996), we performed a comparative analysis of couples who underwent assisted reproduction treatment because of severe male factor infertility or tubal occlusion in the presence of normal semen parameters. We found significant differences of HLA class II allele and haplotype frequencies between males with normal spermatogenesis and males with andrological infertility. Differences in allele frequencies between the study groups were more pronounced when only males who could achieve pregnancies after assisted reproduction treatment were compared. No differences were observed in the corresponding female study groups. Our data suggest an influence of MHC-linked genes on male gamete quality and function in humans. Further investigations are needed to delineate if the observed associations are caused by immunological or other functions of the HLA genes or by putative HLA-linked T/t analogous developmental loci.
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Materials and methods |
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In the present investigation, 41 couples with recurrent spontaneous abortions (RSA) were evaluated as a second control group and were recruited from the outpatient clinic of the Women's Hospital of the University of Bonn.
Recruitment criteria for the different study groups were as follows.
Tubal infertility (n = 84)
Primary or secondary infertility due to tubal occlusion or severe tubal damage as documented by hysterosalpingography or laparoscopy with tubal chromopertubation; absence of additional endocrinological infertility factors; normal semen parameters according to WHO guidelines (WHO, 1994) in the male partner.
Male infertility (n = 104)
Severe male factor infertility (less than 5x106 progressively motile spermatozoa after semen preparation) and/or male factor with previous failure of fertilization in at least one conventional IVF attempt, exclusion of known causes of male infertility (e.g. congenital uni- or bilateral absence of the vas deferens (CBAVD or CUAVD), maldescensus testis, genital tract infections, abnormal karyotype, microdeletions of Yq), absence of infertility factors in the female partner. Patients with cryptorchism were excluded from our study group, because an association of uni- and bilateral cryptorchism with certain HLA-A alleles has been reported in the literature (Martinetti et al., 1992).
During the course of the study, each group of infertility patients was further subdivided into couples who achieved an ongoing pregnancy after IVF or ICSI treatment and those who did not. Ongoing clinical pregnancies were defined as pregnancies with ultrasonographically detectable heartbeats and regular embryonic development proceeding beyond 12 weeks of gestation.
Recurrent miscarriage (n = 41)
Three or more previous consecutive miscarriages with present partner, exclusion of known causes of miscarriage (genital tract infections, genetic, anatomic, endocrinological or autoimmunological factors), absence of additional infertility factors in both partners.
In addition to the specific selection criteria for each study group, the probands had to fulfil the following general selection criteria: no HLA-associated diseases in either partner (e.g. insulin-dependent diabetes mellitus, systemic lupus erythematodes, multiple sclerosis, myasthenia gravis), no consanguinity, both partners of middle or northern European origin.
We further used data from an ethnically matched published control sample (Begovich et al., 1992) to test for the normal distribution of class II alleles and three locus haplotypes in our population.
The study protocol was approved by the Ethics committee of the University of Bonn and all patients gave informed consent before participation in the study.
Laboratory methods
DNA-based typing for HLA DQA1, DQB1 and DRB1
DNA was isolated from 1015 ml whole blood by a modified salting-out procedure (Miller et al., 1988) or with the QIAamp Blood Kit® (Quiagen Inc., Hilden, Germany) and resuspended in 0.1xTE buffer (1 mmol/l Tris/HCl, 0.1 mmol/l EDTA, pH 8.0).
Patients were DNA-typed for the HLA class II loci DQA1, DQB1 and DRB1 with a PCR-based method using sequence-specific primers (PCR-SSP) as published (Olerup et al., 1993; Olerup and Zetterquist, 1992
). Briefly, oligonucleotide primers are designed to obtain amplification of specific alleles or groups of alleles. The method is based on the principle that a completely matched primer will be used more efficiently in a PCR-reaction than a primer with one or several mismatches. Assignment of alleles was based on the presence or absence of the amplified product after agarose gel electrophoresis and ethidium bromide staining. Primers for the third intron of the DRB1 gene, which is nonpolymorphic, were co-amplified in every PCR-reaction and served as internal positive amplification control (Olerup and Zetterquist, 1992
).
SSP-typing for HLA-DQA1 allows distinction of all nine expressed DQA1 alleles. For HLA-DQB1, typing was limited to 14 published primer pairs which will distinguish all 13 DQB1 alleles that have been observed in Caucasian populations, a second set of primers was used to distinguish between alleles DQB1 0301 and DQB1 0304. Similarly, DRB1 typing was limited to a low resolution typing set which identifies the correlates of the serological DR specificities DR1-DRw18. Specificities DR3, DRw13 and 14 were subtyped with specific primer mixes, thus allowing assignment of 16DRB1 alleles on DNA basis.
HLA DQA1, DQB1 and DRB1 alleles were assigned according to the nomenclature provided by the 1995 report of the WHO Nomenclature Committee (Bodmer et al., 1995). HLA class II haplotypes were assigned according to published haplotypes in middle European populations (Begovich et al., 1992
) and our own previous family studies and was not based on a formal computer program.
HLA nomenclature
As in other publications (Klitz et al., 1994), the HLA nomenclature given by the World Health Organization was simplified by giving only four digit allele names, whenever HLA class II haplotypes are indicated in tables or in the text. Alleles separated by hyphens indicate haplotypes. For example, the allele DRB1*1501 is written as 1501 and the haplotype DRB1*1501-DQA1*0102-DQB1*0602 is written as 1501102602 if not indicated otherwise.
Statistical methods
Because the evaluation of the data was mainly descriptive, corrections for multiple testing were only made to a certain degree. The heterogeneity of allelic distribution between different study groups was tested simultaneously (no separate testing of single alleles). The comparison of the allelic distributions between the study groups was performed using the 2 test. The same method was used for comparison of haplotype distributions in the different groups. All rows summing to less than three were combined in a separate allelic class to reduce the overall number of allelic comparisons.
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Results |
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However, significant differences of allele frequencies in all three class II loci (DQA1 P < 0.031; DQB1 P < 0.013; DRB1 P < 0.02) were found in males with andrological infertility when compared to the normozoospermic partners of couples with tubal infertility (Table I). Because it was unclear if the observed differences in allele frequencies were specific for andrological infertility, we chose husbands from couples with recurrent spontaneous abortions (RSA) with known normal semen parameters as a second fertile control group. Again, males with andrological infertility differed significantly from male partners of RSA couples in allele frequencies for all three HLA class II loci (HLA DQA1 P < 0.003, DQB1 P < 0.016; DRB1 P < 0.001). In contrast, allele frequencies in RSA males and normozoospermic males from couples with tubal infertility were highly congruent for HLA DQA1, DQB1 and DRB1 (Table I
). Differences between fertile and infertile males were most pronounced for the following HLA alleles: DRB1*0101, *01501 and *0701; DQA1*0101, *0102, *0201, DQB1*0201, *0501 and *0602 (Table I
). In conclusion, our data suggest that patients with male factor infertility and normozoospermia differ in their HLA class II allelic composition.
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Haplotype analysis
Differences in allele frequencies between fertile and infertile males were in the majority caused by relative decreases or increases of specific alleles in every HLA class II locus (Tables I and II), which raised the question of whether the observed changes occurred independently for every single allele or were caused by shifts in HLA class II haplotypes.
Because differences in allele frequencies had been strongest in patients with pregnancies after assisted reproduction treatment, we first compared HLA class II haplotype frequencies in those groups and in RSA males. Comparison of DRB1-DQA1-DQB1 three locus haplotype frequencies revealed a statistically significant difference between RSA males and males with andrological infertility (P < 0.037), which was stronger for the two locus DQA1-DQB1 (P < 0.009) and DRB1-DQA1 haplotypes (P < 0.009) (Table IV) than for the three locus haplotypes, whereas differences in DQB1-DRB1 haplotypes were smaller but nevertheless existent (P < 0.049). In contrast, comparison of three locus haplotype frequencies between males with andrological infertility and male partners of couples with tubal infertility did not reach significance (P < 0.087, Table IV
), although differences existed for DQA1-DQB1 (P < 0.019) and DQA1-DRB1 (P < 0.031) frequencies, but not for DQB1-DRB1 haplotypes (P < 0.112). In accordance with HLA class II allele distributions, haplotype frequencies in RSA males and males from couples with tubal infertility were almost equal.
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In summary, we could detect differences in HLA class II three locus haplotype frequencies in males with severe andrological infertility in comparison to normozoospermic males, which were caused by relative decreases or increases of four distinct HLA class II three locus haplotypes. Differences in haplotype frequencies only reached significance between the patient subgroups with pregnancies after assisted reproduction treatment and RSA males. However, more subtle shifts in haplotype frequencies which went in parallel with the changes observed in the pregnant subgroups could also be recognized in the total diagnostic groups (data not shown).
Allelic effects
We next sought to determine if particular HLA alleles or rather the haplotypes which carry those specific alleles confer predisposition to male infertility. For this purpose, we compared the frequencies of the different allelic components of potentially protective and predisposing haplotypes in our study groups. As mentioned before, the DRB1-DQA1-DQB1 haplotypes 010101010501 and 070102010201/0303 were increased in patients with severe male factor infertility, suggesting that they confer susceptibility to male infertility. In contrast, the 150101020602 haplotype was decreased in male infertility patients. A comparison of the changes of HLA class II haplotypes and individual DRB1, DQA1 and DQB1 alleles showed that the majority of HLA class II alleles which were shifted in male infertility patients relative to controls were unique to a specific haplotype which was decreased or increased in the same direction (Table V). The results suggest that specific haplotypes rather than single alleles may be responsible for the observed association between HLA class II alleles and male infertility, although final statistical proof for this hypothesis still has to be provided.
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No significant differences in HLA class II antigen sharing could be observed between the total groups with tubal or male factor infertility for the three HLA class II loci that were examined.
Only when the subgroups of couples with male or tubal infertility and pregnancy success or RSA couples were compared were differences in allele sharing found for HLA-DRB1. A higher proportion of couples with male infertility shared two DRB1 alleles (18.6%) compared to couples with tubal infertility (2.33%) or recurrent spontaneous aborters (7.3%). Also, fewer couples with RSA shared one DRB1 allele (7.3%) compared to couples with male (25.6%) or tubal infertility (34.9%). No difference in allele sharing was found for the remaining HLA-loci DQA1 and DQB1. The results are summarized in Table VI, the observed differences were significant for comparison of male and tubal infertility (P < 0.044) and tubal infertility versus RSA (P < 0.038). Whereas there were no DRB1 alleles that were preferentially shared between partners with tubal infertility and RSA couples, sharing of DRB1*0701 was frequent in couples with male infertility (eight out of 20 DRB1 alleles that were shared, 40%).
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Discussion |
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In the present study, we observed significant differences in HLA class II allele frequencies between males with impaired spermatogenesis and males with normal spermatozoa. Our data suggest that genes located within the HLA class II region may be involved in the control of spermatogenesis in the human, a function distinct from regulation of gamete interactions via cell adhesion mechanisms. Expression of HLA antigens during spermatogenesis has recently been documented by Martin-Villa et al. (1996); their data indicate diploid expression of HLA class I and II antigens in purified spermatozoa and active ongoing translation of HLA proteins in germinal cells and also in spermatozoa. Other authors reported low levels of transcription of HLA class Ib genes in early spermatogenic stages (Guillaudeux et al., 1996; Fiszer et al., 1997
), e.g. spermatocytes and spermatids. Although those data do not allow conclusions with regard to potential functions of HLA molecules in spermatogenesis, they provide proof for active transcription and translation of HLA genes during this period. The correlation of increased frequencies of some HLA class II alleles with severe male factor infertility further suggests that different HLA alleles or haplotypes may have different effects at the cellular level. However, because the alleles and haplotypes which are elevated in male infertility are also present in patients with normozoospermia, albeit at a lower frequency, additional factors seem to play a role in impaired spermatogenesis in males carrying the `risk' alleles.
Because the couples suffering from severe male factor infertility in this study were without exception treated with ICSI, a method that circumvents the processes of spermatozoa/egg adhesion and penetration of the zona pellucida, we cannot determine whether impairment of gamete interactions or other substantial defects in sperm physiology are the basis of MHC-mediated effects on fertility.
The existence of spermatogenic genes within or close to the human HLA class II region in analogy to the murine T/t complex could explain the observed associations between HLA and male fertility. Indeed, human homologues of murine T/t complex genes which are expressed in testicular tissues have now been identified, some of which are close to the human MHC complex (Ragoussis et al., 1992; Tirosvoutis et al., 1995
). More detailed data on the organization of the class II region of the human MHC, especially a comparative analysis of sequencing data of single haplotypes, will probably reveal specific differences within this genetic region which may explain the role of certain HLA haplotypes in spermatogenesis (Beck and Trowsdale, 1999
).
Further studies, e.g. the analysis of extended haplotypes and their association with human sperm function as well as expression studies of potential human homologues of murine T/t loci, will clarify the role of the MHC complex and its components in the pathophysiology of male infertility.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 13, 1999; accepted on October 15, 1999.