Andreas Neuer

Immune Recognition of the 60kD Heat Shock Protein: Implications for Subsequent Fertility

The 60kD heat shock protein (hsp60) is a highly conserved protein and a dominant antigen of most pathogenic bacteria. In some women, chronic or repeated upper genital tract infections with Chlamydia trachomatis, and possibly with other microorganisms, induces immune sensitization to epitopes of hsp60 that are present in both the microbial and human hsp60. Once a woman becomes sensitized to these conserved epitpes, any subsequent induction of human or bacterial hsp60 expression will reactivate hsp60-sensitized lymphocytes and initiate a pro-inflammatory immune response. Hsp60 is expressed during the early stages of pregnancy, by both the embryo and the maternal decidua. We examined, therefore, whether women who were sensitized to hsp60 experienced less successful pregnancy outcomes compared to women who were not sensitized to this antigen. In women undergoing in vitro fertilization (IVF), the presence of cervical IgA antibodies reactive with the C. trachomatis hsp60 correlated with implantation failure after embryo transfer. Further analysis revealed that an immunodominant epitope for these IgA antibodies was an hsp60 epitope shared between C. trachomatis and man. In subsequent studies of women not undergoing IVF, cervical IgA antibodies to the human hsp60 were identified in 13 of 91 reproductive age women. This antibody was most prevalent in those women with a history of primary infertility (p = 0.003). In addition, cervical anti-hsp60 IgA correlated with the detection of the pro-inflammatory cytokines interferon-γ (p = 0.001) and tumor necrosis factor-α (p = 0.02) in the cervix. Conversely, women with proven fertility had the highest prevalence of the anti-inflammatory cytokine, interleukin 10, in their cervices (p = 0.001). In an analysis of serum samples in a third study, women with a history of two or more consecutive first trimester spontaneous abortions had a higher prevalence (p = 0.01) of IgG antibodies to the human hsp60 (36.8%) than did age matched fertile women (11.1%) or women with primary infertility (11.8%). Immune sensitization to epitopes expressed by the human hsp60 may reduce the probability of a successful pregnancy outcome due to reactivation of hsp60-reactive lymphocytes, induction of a pro-inflammatory cytokine response and interference with early embryo development and/or implantation.

Antibody to the Chlamydia trachomatis 60 kDa heat shock protein in follicular fluid and in vitro fertilization outcome.

The association between 60 kDa Chlamydia trachomatis heat shock protein (CHSP60) antibodies and the etiology and outcomes of in vitro fertilization (IVF) outcomes is not well known.

Circulating Heat Shock Proteins in Women With a History of Recurrent Vulvovaginitis

OBJECTIVE: Predisposing factors influencing recurrences of bacterial vaginosis (BV) or vaginitis from Candida remain unidentified for most women. As a component of studies to determine host susceptibility factors to genital tract infections in women, we measured expression of the 60-kDa and 70-kDa heat shock proteins (hsp60 and hsp70, respectively) in the circulation of women with or without a history of recurrent BV or candidal vaginitis and with or without a current lower genital tract infection. Heat shock protein expression is associated with a down-regulation of pro-inflammatory immune responses that would inhibit microbial infection. METHOD: The investigators measured hsp60 and hsp70, antibodies to these proteins, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), and the anti-inflammatory cytokine interleukin-10 (IL-10) in sera by ELISA. The study population consisted of 100 women who attended a gynecology clinic in Campinas, Brazil. Of those, 55 had a history of recurrent vulvovaginitis (RV), while 45 were controls with no such history. Only women who were asymptomatic for at least 1 month were studied. RESULTS: Although all were asymptomatic, clinical and microbiological examination revealed that five of the women with a history of RV and two controls had a current candidal vaginal infection; 16 RV patients and 12 controls had BV; and six RV patients had both BV and candidiasis. Twenty-eight RV patients and 31 controls had no clinical or microbiological detectable vaginal infection. Among the RV patients, hsp60 and hsp70 were more prevalent in those with current BV (40.9% and 50.0%, respectively) or a candidal infection (45.5% and 54.5%) than in women with no current infection (21.4% and 17.9%). In the women with no history of RV, BV was not associated with a high prevalence of hsp60 (8.3%) or hsp70 (8.3%). Interleukin-10 and TNF were not more prevalent in vaginitis patients or controls with a current candidal infection or BV than in uninfected subjects. CONCLUSION: The high prevalence of circulating hsp60 and hsp70 in women with a history of RV and current BV or vaginal candidiasis, but not in women with no history of RV, suggests that differences in heat shock protein induction may be related to susceptibility to recurrent vaginal infections.

Interleukin-1 levels in the supernatant of conditioned media of embryos grown in autologous endometrial coculture: correlation with outcome after in vitro fertilization.

PROBLEM: To determine if interleukin (IL)‐1 produced by autologous endometrial coculture (AECC) was associated with outcome in patients with a history of multiple in vitro fertilization (IVF) failures. METHOD OF STUDY: The conditioned media (CM) from AECC cells exposed or non‐exposed to human embryos was analyzed for IL‐1. RESULTS: Embryos grown on AECC demonstrated a significant improvement in number of blastomeres and fragmentation (frag) when compared to embryos grown in conventional media without ECC (6.4±1.3 vs. 5.5±1.2 blastomeres and 14.6±9.3% vs. 18.4±9.8% frag; P<0.008 and 0.003, respectively). When IL‐1α and IL‐1β were undetectable in the CM, the embryos grown in ECC were of improved quality as compared to the embryos grown only in conventional media. Conversely, IL‐1ra levels in the CM were positively associated with embryo quality. Exposure or non‐exposure to an embryo did not result in differing levels of IL‐1α, IL‐1β, or IL‐1ra in the CM. IL‐1β levels were negatively associated with clinical pregnancy outcome (3.3 pg/mL (pregnant, n=12) vs. 27.1pg/mL (not pregnant, n=17); P=0.008, Mann–Whitney U‐test). IL‐1α and IL‐1ra levels were not associated with outcome. CONCLUSIONS: We have demonstrated a significant improvement in blastomere number and frag with ECC. The presence of IL‐1β in the CM was negatively associated with embryonic development and clinical pregnancy. The presence of IL‐1α in the CM was negatively associated with embryonic development and the presence of IL‐1ra in the CM was positively associated with embryonic development. Whether IL‐1β itself interferes with successful outcome after embryo transfer or if it is a marker for undetected endometritis in the biopsy specimens remains to be determined.

ORIGINAL ARTICLE: Antibody to the Chlamydia trachomatis 60 kDa Heat Shock Protein in Follicular Fluid and In Vitro Fertilization Outcome

The association between 60 kDa Chlamydia trachomatis heat shock protein (CHSP60) antibodies and the etiology and outcomes of in vitro fertilization (IVF) outcomes is not well known.

Chlamydia trachomatis Infection, Immunity, and Pregnancy Outcome

Chlamydia trachomatis can ascend from the cervix to the fallopian tubes and survive for long periods of time without causing symptoms. The immune response to infection clears the extracellular organisms but leads to development of a persistent intracellular infection. Repeated cycles of productive infection and persistence eventually induce tubal occlusion and infertility. Persistently infected cells continue to synthesize the chlamydial 60 kD heat shock protein (hsp60). Immunity to conserved regions of hsp60 may result in autoimmunity to human hsp60. Expression of hsp60 by the embryo and decidua during early pregnancy may reactivate hsp60-sensitized lymphocytes, disturb pregnancy-induced immune regulatory mechanisms, and lead to immune rejection of the embryo. Due to this mechanism women with tubal infertility who are sensitized to the human hsp60 may have a decreased probability of successful outcome after undergoing in vitro fertilization and embryo transfer.

Genetic polymorphism in an inflammasome component, cervical mycoplasma detection and female infertility in women undergoing in vitro fertilization

The inflammasome is an inducible cytoplasmic structure that is responsible for production and release of biologically active interleukin-1 (IL-1). A polymorphism in the inflammasome component NALP3 has been associated with decreased IL-1 levels and increased occurrence of vaginal Candida infection. We hypothesized that this polymorphism-induced variation would influence susceptibility to infertility. DNA was obtained from 243 women who were undergoing in vitro fertilization (IVF) and tested for a length polymorphism in intron 2 of the gene coding for NALP3 (gene symbol CIAS1). At the conclusion of testing the findings were analyzed in relation to clinical parameters and IVF outcome. The frequency of the 12unit repeat allele, associated with maximal inflammasome activity, was 62.3% in cases of female infertility vs. 75.6% in cases where only the male partner had a detectable fertility problem (p=0.0095). Conversely, the frequency of the 7unit repeat allele was 28.9% in those with a female fertility problem, 17.0% in women with infertile males and 18.4% in idiopathic infertility (p=0.0124). Among the women who were cervical culture-positive for mycoplasma the frequency of the 7unit repeat was 53.7% as opposed to 19.5% in those negative for this infection (p<0.0001). We conclude that the CIAS1 7unit repeat polymorphism increases the likelihood of mycoplasma infection-associated female infertility.

Involvement of interleukin-1 and the interleukin-1 receptor antagonist in in vitro embryo development among women undergoing in vitro fertilization-embryo transfer.

AbstractPurpose: To examine the roles of Interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1ra), in in vitro embryo development and subsequent pregnancy outcome. Methods: Maternal serum utilized to supplement embryo growth in IVF cycles was analyzed for the presence of IL-1 cytokines. Results: The maternal serum that was utilized to supplement the embryo media was found to have measurable amounts of IL-1β and IL-1ra. Conclusions: Relative antagonism of the IL-1 system was positively associated with embryo development and pregnancy outcome.

Immune sensitization to the 60 kD heat shock protein and pregnancy outcome

Heat shock proteins are highly conserved proteins present in organisms ranging from bacteria to man. They are both dominant microbial immunogens and among the first proteins produced during mammalian embryo development. Since bacterial and human heat shock proteins share a high degree of amino acid sequence homology, it has been suggested that sensitization to bacterial heat shock proteins during an infection may result in autoimmunity to human heat shock proteins. Infertile couples seeking in vitro fertilization (IVF) may have been previously sensitized to bacterial heat shock proteins as a consequence of an asymptomatic upper genital tract infection. Due to daily clinical monitoring and precisely timed fertilization these patients are an ideal study group to investigate the effect of prior sensitization to heat shock proteins on preimplantation embryo development and implantation failure. Immune sensitization at the level of the cervix to the 60 kD heat shock protein (hsp60) has been associated with implantation failure in some IVF patients. Similarly, the highest prevalence of circulating hsp60 antibodies among IVF patients was found in the sera of women whose embryos failed to develop in vitro. To more directly assess whether humoral immunity to hsp60 influenced in vitro embryo development, a mouse embryo culture model was established. Monoclonal antibody to mammalian hsp60 markedly impaired mouse embryo development in vitro. These data suggest that immune sensitization to human hsp60, possibly developed as a consequence of infection, may adversely affect pregnancy outcome in some patients.

ORIGINAL ARTICLE: Antibody to the Chlamydia trachomatis 60 kDa Heat Shock Protein in Follicular Fluid and In Vitro Fertilization Outcome: CHLAMYDIAL HEAT SHOCK PROTEIN IN IVF PATIENTS

The association between 60 kDa Chlamydia trachomatis heat shock protein (CHSP60) antibodies and the etiology and outcomes of in vitro fertilization (IVF) outcomes is not well known.