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Human Reproduction Update 2000, Vol. 6 No. 2 pp. 149–159 © European Society of Human Reproduction and Embryology The role of heat shock proteins in reproduction A.Neuer1,2,*, S.D.Spandorfer1, P.Giraldo1,3, S.Dieterle2, Z.Rosenwaks1 and S.S.Witkin1 1 Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, New York, USA, 2Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Witten/Herdecke, Germany, and 3 Department of Gynecology and Obstetrics, University of Campinas, Sao Paulo, Brazil Received on June 9, 1999; accepted on October 22, 1999 Heat shock proteins (HSP) were first identified in cells after exposure to elevated temperature. Subsequently HSP have been identified as a critical component of a very complex and highly conserved cellular defence mechanism to preserve cell survival under adverse environmental conditions. HSP are preferentially expressed in response to an array of insults, including hyperthermia, free oxygen radicals, heavy metals, ethanol, amino acid analogues, inflammation and infection. HSP interact with intracellular polypeptides and prevent their denaturation or incorrect assembly. In addition HSP are also involved in several processes essential for cellular function under physiological conditions. HSP production is enhanced during in-vitro embryo culture and they are among the first proteins produced during mammalian embryo growth. The spontaneous expression of HSP as an essential part of embryo development is well documented and the presence or absence of HSP influences various aspects of reproduction in many species. Finally, HSP are immunodominant antigens of numerous microbial pathogens, e.g. Chlamydia trachomatis, which have been recognized as the main cause of tubal infertility. Many couples with fertility problems have had a previous genital tract infection, have become sensitized to microbial HSP, and a prolonged and asymptomatic infection may trigger immunity to microbial HSP epitopes that are also expressed in man. Antibodies to both bacterial and human HSP are present at high titres in sera and hydrosalpinx fluid of many patients undergoing in-vitro fertilization (IVF). In a mouse in-vitro embryo culture model, these antibodies impaired the mouse embryo development at unique developmental stages. Recent studies indicate an association between a previous infection, immunity to HSP and reproductive failure. Key words: Chlamydia trachomatis/embryo development/heat shock proteins/infection and immunity/IVF TABLE OF CONTENTS Introduction General properties of heat shock proteins Reproductive functions of heat shock proteins Pathology of heat shock proteins: implications for reproductive outcome HSP60 and IVF outcome Acknowledgements References 149 150 150 153 154 157 157 Introduction Heat shock proteins (HSP) are highly conserved cellular stress proteins present in every organism from bacteria to man. The first description of a cellular heat stress response was made >37 years ago. It was first observed in 1962 that the salivary gland chromosomes of the fruit fly, Drosophila melanogaster, exhibited a characteristic puffing pattern after exposure to heat (Ritossa, 1962). The first gene products of this chromosomal puffing were identified 12 years later and the term ‘heat shock proteins’ was created (Tissiere et al., 1974). Since this time many other stimuli which induce a heat shock response have been identified. The chromosomal location of the genes coding for these proteins have been identified, the genes have been sequenced, the conformation of the resulting proteins have been described, and the mechanism of gene activation by nuclear heat shock transcription factors characterized (Westwood et al., 1991). Since the heat shock response is a vital cellular survival mechanism, it is understandable that HSP have gained considerable interest in almost every medical field including reproductive medicine, immunology and infectious diseases (Mizzen, 1998). Drugs modulating HSP expression (thus protecting integrity and homeostasis of cells and tissues) are undergoing clinical trials (Biro et al., 1996; Vigh et al., 1997). In the following paragraph a number of crucial characteristics that define this family of proteins are summarized. * To whom correspondence should be addressed at: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Witten/Herdecke, Olpe 19, 44135 Dortmund, Germany. Tel: (49) 231/5575 450; Fax: (49) 231/ 5575 4599; [email protected] 150 A.Neuer et al. General properties of heat shock proteins All organisms studied ranging from prokaryotic bacteria to mammals, including man, respond to an increase in temperature by switching off the synthesis of most proteins and commencing large-scale synthesis of a few HSP. Even thermophilic organisms, whose optimal growth temperature lies between 50–90°C, respond to a sudden temperature rise with a rapidly increased expression of HSP. The amino acid composition of HSP has not changed very much during evolution. HSP of highly divergent organisms are very similar to one another (their structure has been conserved). Some members of the HSP families are strictly inducible by stress, whereas others are constitutively expressed at normal temperature and are only slightly induced by heat shock. The term HSP refers to inducible protein products while HSC describes constitutively expressed HSP. HSP serve two major functions: firstly, under physiological conditions, they act as molecular chaperones (intracellular housekeeping proteins) which are involved in mediating the folding and transport of other intracellular proteins and in some cases their assembly into oligomeric structures. HSP act as chaperones by participating in the assembly of proteins without being part of the final protein structure (Ellis, 1987). Moreover they fulfil crucial roles in intracellular transport, the maintenance of proteins in an inactive form and the prevention of protein degradation. Secondly, they are induced in response to cellular stresses which include changes in temperature, the presence of free oxygen radicals, viral and bacterial infections, heavy metals, ethanol, and ischaemia (Lindquist, 1986; Welch, 1992). The stress-elicited activation of heat shock genes is called the heat shock response. This heat shock response is frequently found in clinical situations, e.g. ischaemia, infection and circulatory and haemorrhagic shock. Cellular stress disturbs the tertiary structure of proteins and has adverse effects on cellular metabolism. However, pretreatment of cells with a mild stress, just sufficient to induce the expression of HSP, results in protection to subsequent insults. This phenomenon has been called ‘stress tolerance’ and is probably caused by the resolubilization of proteins that were denatured during the initial stress. In addition, it has been suggested that cellular structures like microfilaments and centrosomes but also cellular functions like transcription and translation are more stabilized during a second stressful event in stress tolerant cells. Due to their ubiquitous and essential role in the production, quality control and disposal of other proteins it is not surprising that HSP are among the most highly conserved gene products in nature. HSP are classified into different families according to their molecular weight measured in kDa rather than by their function. Most scientific knowledge has been accumulated on four families of HSP. These are the ‘small’ 27, 60, 70 and 90 kDa HSP. Recently the expression of another high molecular weight HSP during embryo development has been investigated (Hatayama et al., 1997). In the context of reproduction, HSP60 and HSP70 are most important. The HSP60 family consists of proteins that are highly expressed in a constitutive manner and are moderately stress inducible. The main localization of HSP60 is in the Figure 1. Heat shock protein (HSP) expression in human first trimester decidua. The epithelium of endometrial glands (arrow) and some stromal cells show an intense immunoreactivity for HSP60. Fresh frozen decidual tissue sections were immunostained by the avidin–biotin–peroxidase complex. 3,3′ diaminobenzidene was used as a chromogen. Original magnification ×200. mitochondria (Jindal et al., 1989). However, other loci including cell surface exposition of HSP60 has been documented (Soltys and Gupta, 1996). The HSP70 family comprises several proteins that are localized in distinct cellular compartments. The constitutively synthesized HSC70 is found in the cytosol and nuclei of cells and is only moderately stress inducible. The HSP70 family represents the most conserved group of proteins within the HSP superfamily (Hunt and Morimoto, 1985). For several reasons, the above described characteristic features of HSP increase their potential to become a target antigen in the pathogenesis of autoimmune diseases (Kaufmann, 1990). Firstly, HSP are phylogenetically conserved. In practical terms, there is a >50% sequence homology between prokaryotic HSP and HSP of mammalian cells (Lamb et al., 1989; Jones et al., 1993). Secondly, HSP are immunodominant antigens for many common microbes, which means that these infectious agents are mainly recognized by the immune system through recognition of their HSP epitopes. This is important for reproduction and assisted reproductive medicine, because many infertile couples have been sensitized during the course of a previous microbial infection. Finally, HSP are overexpressed at sites of acute and chronic inflammation (Van Eden, 1999). Thus, in a susceptible individual, exposure to an infectious agent could result in an immune response to the infecting agent’s HSP and/or could also cross-react with self directed, organ-specific proteins, resulting in an autoimmune disease. Reproductive functions of heat shock proteins Heat shock protein expression in reproductive tissue The presence of HSP has been demonstrated in different tissues relevant to human reproduction. Tabibzadeh and colleagues described the full complement of human HSP in the endometrium of healthy women (Tabibzadeh et al., 1996). Similarly, HSP expression can be detected in the decidua during the first trimester of pregnancy (Neuer et al., 1996, Figures 1 and 2). In an ongoing The role of heat shock proteins in reproduction 151 Figure 4. Heat shock protein (HSP) 27 expression in Fallopian tubes. Same patient as Figure 3. Original magnification ×400. Figure 2. Heat shock protein (HSP) expression during first trimester decidua. Decidual stromal cells immunoreactive for HSP70. Original magnification ×400. Figure 3. Heat shock protein (HSP) 27 expression in Fallopian tubes. Strong immunoreactivity in tubal epithelial cells of a pregnant patient with ectopic pregnancy. Original magnification ×200. study we have been able to demonstrate the presence and differential expression of HSP in Fallopian tube tissue of women with and without ectopic pregnancy (Figures 3 and 4). Maximum values of HSP27, HSP60 and HSC70 in the endometrium are expressed after ovulation and in the early secretory phase, which is the critical period of ‘endometrial receptivity’ for an implanting embryo. However, since oestrogen and progesterone receptors are associated with HSP, these HSP are constantly involved in the modulation of steroid function in the endometrium (Renoir et al., 1990; Bagchi et al., 1991). Recently the prevention of cytotoxic damage by cytokines has been proposed as another function of HSP in the endometrium (Tabibzadeh and Broome, 1999). In the endometrium leukocytes can produce high levels of reactive oxygen species and cytokines. Both products can modulate the expression of HSP (Jaquire-Sarlin et al., 1994). Since leukocytes and cytokines, e.g. tumour necrosis factor-α (TNF-α), accumulate progressively during the secretory phase, it is possible that HSP protect endometrial cells from the adverse side-effects of this leukocyte accumulation and cytokine release (Tabibzadeh and Broome, 1999). Cells transfected with HSP70, for example, are protected from cytotoxic damage by TNF-α (Jaattela 1993). In addition it has been suggested that HSP70 can prevent DNA strand breaks, protect mitochondrial structure and function and thus inhibit apoptosis (Jaquire-Sarlin et al., 1994). Finally HSP are present in the human placenta (Divers et al., 1995; Ziegert et al., 1999). Immunohistochemical results revealed that HSP were more evident on the apical surface of the syncytiotrophoblasts than on stromal or muscle cells. Placental HSP expression did not differ between preterm and term pregnancies, indicating that their production was part of the physiological pregnancy process. However, immune complexes between immunoglobulin (Ig)G antibodies and HSP60 or HSP70 were detected only in the placentae of women who delivered preterm (Ziegert et al., 1999). This correlation suggests that autoimmunity to HSP might be involved in immune-mediated preterm labour. HSP expression and spermatogenesis During spermatogenesis, three distinct phases can be discerned: mitotic proliferation of spermatogonia; meiotic development of spermatocytes; and post-meiotic development of spermatids and maturation of the spermatozoon (Eddy et al., 1991). Since all these developmental stages represent situations where dramatic transformations and cellular differentiation take place, it is not surprising that spermatogenesis is accompanied by the expression of different HSP (Dix, 1997; Meinhardt et al., 1999). During mouse and rat spermatogenesis, the constitutive form of HSP70 (HSC70) accumulates (Allen et al., 1988a,b). Also mRNA coding for proteins related to HSP86 was found in rat and human testis (Lee, 1990). In infertile men it has been demonstrated that the number of HSP60-expressing spermatogonia paralleled the loss of spermatogenic function (Werner et al., 1997). These observations suggest that a low level of HSP60 expression in spermatogonia might lead to a decreased level of protection, which in turn could be involved in low spermatogenic efficiency. In a recent study, Dix et al. showed in a mouse model that the disruption of the HSP70-2 gene by gene targeting results in failed meiosis, germ cell apoptosis and male infertility (Dix et al., 1996). 152 A.Neuer et al. Spermatocytes of mice in which the HSP70-2 gene had been knocked out became arrested during meiosis. Morphological examination revealed that these animals had testes only one third the size of control mice. This failure of meiosis was associated with an increase in spermatocyte apoptosis (Mori et al., 1997). Thus, with minor exceptions (Mori et al., 1999), HSP70-2 participation during spermatogenesis is required for successful completion of meiosis in mouse spermatocytes. Induction of heat shock proteins by human semen The mRNA for one of the heat shock proteins, HSP70, has also been shown to be induced by cell-free seminal fluid as well as by isolated motile spermatozoa (Jeremias et al., 1997, 1998, 1999). In peripheral blood mononuclear cells and human cervical epithelial tumour cells (HeLa) in vitro, and in endocervical cells in vivo, exposure to semen resulted in transcription of the HSP70 gene. The mechanism of semen-induced HSP70 gene activation and the biological consequences of this activity remain a matter of speculation. Human semen is a rich source of prostaglandins, proteases, polyamines and other products which conceivably could induce a stress response in cells after physical contact. This response and HSP70 expression may activate lymphocytes that were previously sensitized to cross-reacting regions common to microbial HSP70s. By this mechanism, the immune system might initiate a rapid response to micro-organisms in semen, even to those organisms never previously encountered. HSP70 gene activation, by promoting suppression of pro-inflammatory immune responses (Cahill et al., 1996), may also inhibit an immune response to spermatozoa in the female reproductive tract. This, however, might contribute to the sexual transmission of disease pathogens, as suggested by Kelly et al. (1997) and Jeremias et al. (1997, 1998). HSP expression and oogenesis The female germ line like the mammalian male germ line is sensitive to hyperthermic as well as to other environmental stress factors. Similar to spermatogenesis, HSP expression is an integral process during oogenesis in a number of species. These include distant species like insects (Ambrosio and Schedl, 1984), fish and amphibians (Heikkila et al., 1985, 1997), but also mammals (Heikkila et al., 1986). The conservation of HSP expression in evolutionary diverse organisms supports the assumption of a fundamental role of HSP during germ cell development. HSP are found, for example, in ovarian nurse cells of Drosophila where they are subsequently transported to the oocyte (Zimmerman et al., 1983). In mammalian oocytes a ‘window’ for heat induction of HSP exists, which is regulated by the specific stage of oocyte development. In mouse oocytes, the heat shock response is maximized during the growth period of the oocyte and declines with acquisition of the full oocyte size. Finally it is shut off with the terminal oocyte and follicle differentiation (Curci et al., 1987, 1991). Thus, the ability of mouse oocytes to mount an inducible heat shock response is highest during early follicular growth and disappears prior to ovulation. Growing oocytes spontaneously express high levels of the constitutive 70 kDa HSP (HSC70). Thus HSC70 is found at high levels in the pre-ovulatory oocyte (Curci et al., 1991). Later, its synthesis ceases shortly after germinal vesicle breakdown and it is undetectable in the ovulated oocyte at the time of fertilization. After meiosis, HSC70 synthesis has vanished completely. This is interesting to note, because it is known that mammalian oocytes are very heat sensitive. Since fully developed oocytes are unable to express the heat inducible HSP70 form, this could explain why mammalian oocytes exhibit an atypical and degenerate morphology after an exposure to hyperthermic stress (Baumgartner and Chrisman, 1981). The observed abnormalities included multinuclear eggs and an increase in size of the first polar body. In vitro, elevated temperature reduces the number of oocytes proceeding to metaphase II and decreases the rate of fertilization (Lenz et al., 1983). The above-described block of heat shock gene induction during oocyte differentiation seems to represent a general feature in oogenesis, even though it may follow different time schedules in different species. It is interesting to speculate about the role of HSP during the ovulation process. Since ovulation is characterized by the cardinal features of an inflammatory reaction (Espey, 1994), it is possible that HSP play a role in the ovulation process and the maintenance of the postovulatory metabolic activity and survival of the oocyte. The presence of HSP60 in human follicular fluid of patients undergoing in-vitro fertilization (IVF) has recently been demonstrated (Neuer et al., 1997). In the rat, HSP70 induction mediates luteal regression (Khanna et al., 1995). However at present no further detailed knowledge on the function of HSP during this time of the reproductive cycle exists. Heat shock proteins and embryo development The successful completion of the fertilization process and the initiation of the first cleavage steps mark the beginning of embryo development. Before questioning the role of HSP during embryo development it is important to realize that almost all of the present knowledge on the function and role of HSP in reproduction relies on information obtained from animal studies or from human somatic cell lines which can be induced to differentiate. Although data from animal models may suggest similar mechanisms in man, the exact role of HSP for human embryo development remains speculative. Due to technical difficulties and ethical restrictions most of the existing studies focus on HSP and early mammalian embryo development from a zygote up to the expanded trophoblast stage. Less experimental knowledge exists on the role of HSP for advanced embryo and organ development. Since the mouse is often used as a model in the study of mammalian development and since some of our own data are derived from a mouse embryo model, we will mainly focus on findings concerning the role of HSP in mouse embryo development. Although the HSP70 family has received special attention in this context, members of the 60 and 90 kDa HSP are also synthesized by the murine preimplantation embryos (Bensaude et al., 1983). Figure 5 shows an example of HSP60 expression in a murine 2-cell embryo. As for all mammals, the embryological development of the mouse can be subdivided in two main phases. The preimplantation period, which can be easily assessed in vitro and, secondly, the post-implantation period. On the role of HSP during early implantation and attachment to endometrial surfaces no information is presently available. The preimplantation period comprises the time span after ovulation and fertilization in the oviduct before complete implantation in the maternal uterus. In the mouse there is a 4–5 day preimplantation period. During this The role of heat shock proteins in reproduction Figure 5. Heat shock protein (HSP)60 expression in a murine 2-cell embryo. A scattered fine immunoreactivity is observed in the cytoplasm. The connective area between the two blastomeres is particularly positive. time embryos develop from the zygote to the blastocyst stage and migrate freely from the oviduct to the uterus. Distinctive features of HSP expression are directly linked to major events occurring during the preimplantation phase: (i) spontaneous, constitutive HSC70 expression begins with the onset of zygotic genome activity and at the early 2-cell stage. During the same period inducible HSP70 expression is still absent (Bensaude et al., 1983; Morange et al., 1984); (ii) the constitutive form, HSC70, is the predominant HSP expressed up to the blastocyst stage (Morange et al., 1984); (iii) in mouse embryos, the induction of HSP synthesis by heat shock begins at the blastocyst stage (Wittig et al., 1983); and (iv) since blastocyst formation marks the differentiation of two types of embryonic cells forming the inner cell mass and the outer cell mass, progressive acquisition of HSP inducibility is associated with continuing embryonic differentiation. Thus inducible HSP70 expression and the formation of heat shock protein expression appear to be developmentally regulated. It seems to be a common feature of mammalian embryos that very early stages of development are characterized by a lack of induced HSP synthesis. This inability generally reflects the absence of embryonic gene transcription. As soon as transcription resumes, most heat shock genes become stress inducible. Experiments using nuclei transfers have demonstrated that ageing of the egg cytoplasm directs the onset of heat shock gene transcription (Barnes et al., 1987; Howlett et al., 1987). At the 8-cell stage, the mouse embryo does not yet synthesize inducible HSP70 even after heat shock, but it does synthesize very high levels of the cognate HSC70. However, when an 8-cell stage nucleus is transferred into a 1-cell embryo devoid of its pronuclei the reconstructed 1-cell embryo does not synthesize any HSC70 in the first hours that follow the manipulation. However, after allowing time for cell division, the reconstructed embryos synthesize both the inducible HSP70 and the cognate HSC70 at the correct time relative to the development of the recipient cytoplast. Thus, distinct features of HSP expression are directly linked to major events occurring during the preimplantation phase (Dix et al., 1998). However, after implantation the expression of HSP is less co-ordinated and no uniformity exits in the HSP expression pattern of different tissues (Loones et al., 1997). 153 Recently several studies focused on the molecular mechanisms regulating the expression of HSP, in particular on the properties of the two best studied heat shock transcription factors HSF1 and HSF2 (Christians et al., 1997a,b). Heat shock transcription factors (HSF) bind to the promoters of heat shock genes on conserved heat shock sequence elements (HSE). HSF1 is unable to bind to HSE in the absence of stress, while HSF2 is active under normal temperatures. HSF2 is believed to be the major factor for constitutive HSE binding activity. Studies in the mouse suggested that HSF2 might be involved in the control of heat shock gene expression during embryogenesis (Mezger et al., 1994a,b). Even earlier HSF1 is already present at the 1-cell stage. The relative abundance of HSF1 is correlated with the high amount of HSP70 gene expression at the 2-cell stage described previously (Christians et al., 1997b). Recently, additional HSFs, including a new human HSF have been discovered (for review, Morange et al., 1998). This multiplicity of HSF reflects the involvement of heat shock genes and their gene products in various essential cellular processes. Pathology of heat shock proteins: implications for reproductive outcome In addition to the above-described physiological properties of HSP in reproductive events, several pathological conditions have also been associated with HSP. The origin of the pathogenicity of HSP for reproduction is based on several mechanisms. Firstly, HSP can induce a persistent inflammatory response. Secondly, HSP molecules serve as antigenic targets for the immune system. Finally, the extensive amino acid sequence homology between human and microbial HSP could result in autoimmune mediated reproductive failure. Properties of bacterial HSP Members of the HSP60 and HSP70 families have been recognized as immunodominant antigens of many microbial pathogens. These include bacteria, e.g. Chlamydia trachomatis, one of the most frequently found sexually transmitted microbial pathogens in patients of reproductive age (Cerrone et al., 1991; Zhong and Brunham, 1992). Like eukaryotic cells, microbial pathogens express a constitutive level of HSP required for the maintenance of essential house keeping functions. During an infection the microbial stress protein synthesis is up-regulated and the increased amount of microbial HSP at sites of an infection can contribute to immunodominance of the microbial HSP. In recent years it has become clear that HSP play a major role in the acute inflammatory response and in the persistence of inflammatory reactions (Moseley, 1998). HSP released from infectious organisms or infected host cells can induce cytokine release and provoke an immune response (Tabona et al., 1998). After invasion into a host tissue, the pathogen is subjected to environmental conditions, such as elevated temperature, nutrient deprivation changes in pH and exposure to oxygen radicals, which induce a stress response. Thus, during an infection enhanced microbial HSP synthesis may be part of the protective response of the pathogen to host defences and can contribute to microbial virulence (Mizzen, 1998). This is important for reproduction since many couples seeking infertility treatment have had a previous exposure to microbial pathogens. C.trachomatis infections of the 154 A.Neuer et al. Table I. Properties of the 60 kDa family of heat shock proteins Detectable in all eukaryotic and prokaryotic organisms Essential chaperone proteins involved in transport, folding and assembly of protein subunits Production is elevated in response to environmental stress factors in order to minimize protein denaturation Amino acid sequence is highly conserved throughout evolution. The human and bacterial proteins share a sequence homology of ∼50% Immune responses to conserved regions of heat shock proteins have been implicated in autoimmune phenomena. HSP60 and IVF outcome IVF bypasses the requirement for patent Fallopian tubes and thus has become the treatment of choice for women with occluded or damaged Fallopian tubes. However it is only recently beginning to be appreciated that the cause of tubal blockage and sensitization to chlamydial and human HSP may also negatively influence postfertilization events (Spandorfer et al., 1999a; Moomjy et al., 1999). Based on the above-described model (Table II) we tried to elucidate the role of HSP in early embryo development and the consequences of immune sensitization to HSP60 for reproductive outcome of IVF patients. Immunity to chlamydial HSP60 and pregnancy outcome female genital tract for example are the major cause of infertility due to occluded Fallopian tubes. Several studies have revealed that sensitization to HSP60 of C.trachomatis and subsequent expression of the highly homologous human HSP60 can lead to unsuspected infertility problems. 60 kDa chlamydial and human HSP HSP60 is one of the best-characterized molecular chaperones of both eukaryotic and prokaryotic organisms. The major properties of HSP60 are delineated in Table I. Typically, during the course of an acute infection, immunity is restricted to HSP60 epitopes that are specific to the invading micro-organism, e.g. C.trachomatis. Most patients with infertility problems due to tubal occlusion have experienced a chronic persistent chlamydial infection (Witkin et al., 1997). In contrast to an acute infection, cells chronically infected with C.trachomatis synthesize only low levels of structural components but continue to produce chlamydial HSP60 at high levels (Beatty et al., 1993). Thus, some women with asymptomatic and untreated C.trachomatis infections and tubal infertility have experienced a long-term exposure to chlamydial HSP60. Since bacterial and human HSP share ∼50% amino acid sequence homology (Shinnik 1991), it has been proposed that a prolonged exposure of the immune system to chlamydial HSP60 and a concomitant exposure to both the chlamydial and human HSP60 may lead to autoantibody formation (Witkin et al., 1997). A possible model for impairment of early-stage pregnancy after immune sensitization to conserved regions of the C.trachomatis HSP60 has been outlined previously (Witkin et al., 1996) and is summarized in Table II. In one of our initial studies, the prevalence of IgA antibodies to chlamydial HSP60 in the cervix of 216 women undergoing IVF treatment was determined (Witkin et al., 1994). None of the investigated women ever had a recognized chlamydial infection. However, antichlamydial HSP60 IgA was identified in 41 (20.7%) patients. This is remarkable, because the presence of this antibody is considered to reflect an acute immune response to chlamydial HSP60 and was, in addition, associated with unsuccessful IVF outcome. Anti-HSP60 IgA was present in 26.3% women who did not become pregnant after transfer (P = 0.0007), 33.3% of women with only transient biochemical pregnancies, 30% of women with spontaneous abortions and only 7.3% of women with live births (Witkin et al., 1994). No relationship existed between HSP60 antibody status and the number of oocytes retrieved or fertilized. This suggested that some women undergoing IVF treatment were previously sensitized to chlamydial HSP and/or a previously undetected genital tract infection was still present in these women. The presence of the detected HSP antibodies was correlated with an adverse outcome after IVF treatment. The latter relationship becomes especially obvious, if one takes a closer look at couples where clinical sequelae of a previous infection like a hydrosalpinx are present. In a recent study, IVF patients with tubal occlusion, with or without hydrosalpinges, were tested for circulating antibodies to the chlamydial HSP10 which is known to be co-expressed with chlamydial HSP60 (Spandorfer et al., 1999a). Sera obtained from women whose male partners were infertile served as control. In this study clinical pregnancies were documented in 68% of the women with male factor infertility. This was significantly higher than the 43.1% rate in women with tubal occlusions (P = 0.04) and Table II. Suggested mechanism of 60 kDa heat shock protein (HSP60) immune mediated pregnancy failure A persistent infection (e.g. Chlamydia trachomatis) sensitizes a woman to HSP60 regions present in both microbes and man. Human (host) HSP60 is physiologically expressed during the pre- and peri-implantation stages of pregnancy by the embryo and the maternal decidua. Host HSP60 expression in early pregnancy reactivates lymphocytes previously sensitized to microbial (e.g. chlamydial) HSP60. The activated lymphocytes release pro-inflammatory cytokines, which induce also other lymphoid cells to release inflammatory and cytotoxic mediators. Cellular and humoral immune system activation disturbs immune regulatory mechanisms necessary to implantation and maintenance of the embryo. Antibodies to heat shock proteins impair embryo development. Embryos are less protected from adverse environmental conditions and are more likely to degenerate or undergo apoptosis. The role of heat shock proteins in reproduction the 41% rate in women with hydrosalpinx (P = 0.02). Simultaneously, antibodies to chlamydial HSP10 were more prevalent in women with hydrosalpinx (46.8%) than in women with tubal occlusion (15.5%; P = 0.0009) alone or male factor infertility (6%; P = 0.0001). Interestingly, antibodies to the human HSP60 were also more prevalent in women with tubal occlusion plus or minus hydrosalpinx than in women with male factor infertility. This again provided further proof for an autoimmune linkage between chlamydial and human HSP60. In another study 122 IVF subjects were screened for cervical IgA antibodies to synthetic peptides, which corresponded to the conserved epitopes of the chlamydial HSP60 (Witkin et al., 1996). Antibodies to a single epitope, corresponding to the amino acids 260–271 in the chlamydial HSP60 amino acid sequence, were found to be immunodominant in these patients. More importantly this epitope was present in both the chlamydial and human HSP60 (Yi et al., 1993). Once again women with this antibody had a significantly increased prevalence of only transient biochemical pregnancy (22.2%; P = 0.03) after embryo transfer than did antibody negative women (7.4%; Witkin et al., 1996). This again implied that cervical IgA antibody to conserved HSP60 epitopes expressed in both the human and chlamydial heat shock proteins and the failure of successful implantation after embryo transfer are interrelated. Finally, in a fourth study the detection of chlamydial IgA antibodies in follicular fluid of IVF patients are correlated with the presence of human HSP60 antigen in these secretions (Neuer et al., 1997). The analysis of the clinical diagnosis of these patients revealed that all women expressing human HSP60 had a tubal occlusion and failed to become pregnant in their IVF cycle. In conclusion, the summarized results of these studies revealed that a previous infection with C.trachomatis and a resulting immune sensitization to chlamydial heat shock protein epitopes was associated with a poor prognosis for reproductive outcome and, in addition, impaired IVF results. Immunity to human HSP60 and pregnancy outcome To further elucidate the contribution of human anti-HSP60 antibodies to reproductive failure we determined the prevalence of IgG antibodies to the human HSP60 in maternal serum of patients undergoing infertility treatment. The results indicated that serum IgG antibodies to the human 60 kDa HSP were significantly more common in patients with arrested in-vitro embryo development than in IVF patients whose embryos continued to grow and were transferred to the uterus (Table III, Witkin et al., 1996). This finding seemed to be of major importance, since in IVF embryos are sometimes cultured in medium containing maternal serum. Thus, if a woman was already immunized to conserved HSP60 epitopes and would harbour HSP60 antibodies in her serum, this could interfere with the in-vitro development of the embryo. IVF culture and HSP expression Stressful manipulation of embryos in culture is a daily event in assisted animal reproductive technology. Embryos are transferred, cryopreserved, cloned, or microinjected with transgene constructs. As far as human IVF is concerned, one can also assume that stressful culture conditions in in-vitro culture enhance HSP expression in these embryos. Such embryos have to cope with 155 Table III. Relation between circulating immunoglobulin G antibodies to 60 kDa heat shock protein (HSP60) and the outcome of in-vitro fertilization (IVF). Sera from 155 women were tested IVF outcome No. subjects No. HSP60+ (%) No fertilization 14 1 (7.1) Arrested embryo development 13 6 (46.2)a Not pregnant 75 7(9.3) Pregnant 53 9 (17.0) Embryo transfer aP = 0.004 versus all others. handling, oxidative stress, variation of temperature and a completely altered cellular environment. Premature transfer to the uterus at the 4–8-cell stage may also lead to both nutritional and environmental stress. Consequently in the mouse HSP70 expression is found to be 5–15-fold higher in cultured embryos (Christians et al., 1995, 1997a). Induced expression of HSP due to environmental factors and constitutive HSP expression may both represent an essential requirement for successful embryo growth in an adverse environment. Overexpression of HSP in this situation is probably to the benefit of the developing embryo. However, on the contrary failed HSP induction and immunity to HSP could result in detrimental consequences for the growing embryo in vitro. In many IVF culture systems the in-vitro fertilized embryos are cultured in medium containing maternal serum. Pre-existing HSP antibodies in these sera at high titres could thus compromise the growth potential of developing embryos. In a recent study, antibodies to the most common mammalian HSP exerted a detrimental effect on mouse embryos at unique developmental stages (Neuer et al., 1998). In these experiments, 2-cell mouse embryos (B6D2F1) were cultured in the presence or absence of monoclonal antibodies specific for mammalian HSP60, HSP70 and HSP90. Embryo development was evaluated after 3, 5 and 7 days in culture by determining the number of blastocysts, hatched blastocysts and outgrown trophoblasts at the successive time points. Both anti-HSP60 and anti-HSP70 elicited a strong inhibitory effect on mouse embryo growth, but at unique development stages. At day 3, only 29% of the embryos cultured with HSP60 antibody developed to the blastocyst stage as compared with 67% of the embryos cultured with anti-HSP70, 72% cultured with anti-HSP90, and 79% in medium plus mouse monoclonal IgG1, which served as a control. By day 5, hatched embryos were present in 28% of the cultures containing antiHSP70, as opposed to 57% containing anti-HSP90 and 73% containing IgG1. At day 7, outgrown trophoblasts were observed in 9% of cultures containing anti-HSP70, 45% containing antiHSP90 and 66% cultured in medium plus IgG1. These results are shown in closer detail in Figure 6. In the presence of HSP antibodies these embryos became growth arrested and degenerated. Gross morphology of these embryos revealed irregular sized blastomeres and multiple fragments. An example of the gross morphology of these embryos is given in Figure 7. The observed embryos often contained 156 A.Neuer et al. Figure 7. Examples of degenerated murine embryos after culture in the presence of heat shock protein (HSP)60 immunoglobulin (Ig)G antibodies. Figure 6. Effect of antibodies to heat shock proteins (HSP) on developmental delay of mouse embryos after 3, 5 and 7 days in culture. Embryos were cultured in Roswell park Memorial Institiute (RPMI) 1640 medium/10% fetal calf serum (FCS) and monoclonal antibodies to human HSP90, HSP70 and HSP60 (100 µg/ml). Controls consisted of RPMI/10% FCS and mouse immunoglobulin (Ig)G1 (100 µg/ml). *P < 0.01 compared with controls; **P < 0.0001 compared with controls variable sized, degenerated blastomeres with multiple cellular fragments enclosed within the zonae pellucidae. Thus, these embryos resembled apoptotic cells. This is important to note, since both the production of HSP and programmed cell death are closely related to each other. Both systems are similarly considered as supporting systems responsible for the general well being of an organism and also as cellular responses to environmental insults. There is a surprising overlap between stressors inducing stress response and insults initiating apoptosis in different experimental systems (for review, see Punyiczki and Fesüs, 1998). Apoptosis is detrimental to blastocyst formation and leads to preimplantation embryo death (Jurisicova et al., 1996). The cellular morphology of apoptosis is characterized by cell shrinkage, chromatin condensation and membrane blebbing. In the final stages, the apoptotic cell becomes fragmented into apoptotic bodies, which are rapidly eliminated by phagocytes. However in the early stages of apoptosis extensive DNA degradation occurs. Cleavage of the DNA may yield doublestranded, low molecular weight fragments (mono-and oligonucleosomes) as well as single strand breaks (‘nicks’) in the high molecular weight DNA. Those DNA strand breaks can be detected by enzymatic labelling with modified nucleotides (dUTP). Terminal deoxynuleotidyl transferase (TdT) labels blunt ends of double-stranded DNA breaks. The end-labelling method has also been termed TUNEL (TdT-mediated X-dUTP nick-end labelling). The use of fluorescein-dUTP to label the DNA strand breaks allows the detection of the incorporated nucleotides directly with a fluorescence microscope. Mouse in-vitro co-culture studies To further assess the impact of HSP antibodies on embryo development we TUNEL stained murine embryos that had been grown in an endometrial co-culture system in the presence of varying concentrations (10, 50 and 100 µg/ml) of monoclonal antibodies to HSP60. In this culture model, a total of 160 2-cell murine embryos from B6D2F1 mice were grown under two sets of conditions. Half of the embryos were grown using 10% fetal calf serum (FCS) in Roswell Park Memorial Institute (RPMI) 1640 culture medium in varying concentrations of antibodies to mammalian HSP60. The rest were grown in an endometrial coculture (ECC) system in addition to the same medium and the same antibody concentrations. Endometrial co-culture tissue was obtained from a fertile patient and consisted of an equal mixture of stromal and glandular cells. Embryonic development to the blastocyst stage (B), hatching stage (H) and outgrowth stage (O) was analysed. The control embryos (not grown in antibodies to HSP60) progressed to the B, H and O stages in 95, 70 and 70% of cases respectively. In the study group without ECC, embryo growth was inhibited at a concentration of 100 µg/ml anti-HSP60 antibodies at each stage of development (B 25%, H 15% and O 15%, P < 0.001). Utilizing the ECC system, control embryos (not grown with antibodies to HSP60) progressed to the B, H and O stages in 100% of cases for each stage, respectively. In this ECC model, toxicity was only evident at a concentration of 100 µg/ml of HSP60 antibody (B 80%, not significant; H 70%, P = 0.02; and O 60%, P = 0.003). At the highest concentration of antibodies used, the growth inhibition exhibited was always significantly less in the ECC model. In addition TUNEL positivity was more frequent in embryos exposed to antibodies to HSP60 than in unexposed embryos (30/43 versus 6/17, P = 0.03). In Figure 8, an example of TUNEL positivity in a murine blastocyst after exposure to HSP60 antibodies is shown. Recently in another set of experiments the above-described invitro model was applied to further assess the role of hydrosalpinx fluid on embryo growth (Spandorfer et al., 1999b). This experimental design was chosen because hydrosalpinx formation is very prevalent in women with previous infections and tubal occlusion. In addition it has been suggested that hydrosalpinx formation influences pregnancy rates after IVF. Thus we speculated that hydrosalpinx fluid might contain antibodies to HSP60. Table IV displays the HSP antibody, cytokine and chlamydial antibody content of hydrosalpinx fluid. Interestingly The role of heat shock proteins in reproduction 157 Figure 8. Example of TdT-mediated X-dUTP nick-end labelling (TUNEL)-positive fluorescence. This murine blastocyst was cultured in the presence of 100 µg/ ml heat shock protein (HSP)60 antibodies. Distinct foci of TUNEL-positive fluorescence with varying intensity are visible. Figure on left shows TUNEL-stained blastocyst; figure on right shows light microscopy. Original magnification ×200 Table IV. Detection of antibodies and cytokines in hydrosalpinx fluids from 16 women. Values in parentheses are percentages Compound No. positive (pg/ml) Human HSP60 IgG 6 (37.5) Chlamydia HSP10 IgG 9 (56.3)a Chlamydia IgG 2 (12.5) Human HSP60 IgA 2 (12.5) Chlamydia HSP10 IgA 1 (6.3) Chlamydia IgA 5 (31.3) Mean 11 (68.8)b 79 c 10 (62.5) 533 Interleukin-1β 9 (56.3) 24 Interleukin-6 3 (18.8) 42 Interleukin-10 3 (18.8) 10 Interferon-γ Interleukin-1 receptor antagonist Ig = immunoglobulin. = 0.02 versus Chlamydia immunoglobulin (Ig)A; bP = 0.01 versus interleukin (IL)-6, IL-10; c P = 0.02 versus IL-6, IL-10. aP both human HSP60 and chlamydial HSP are present in hydrosalpinx fluid. In a mouse model we have previously shown that anti-HSP60 and anti-HSP70 antibodies exhibited a growth inhibiting effect at unique developmental stages of murine embryos (Neuer et al., 1998). In this model, we used an endometrium co-culture model that mimics the in-utero conditions of IVF patients with hydrosalpinx. Since hydrosalpinx formation is most often the sequela of a previous or repeated infection, antibodies to HSP60 are a possible factor in the toxicity exhibited by hydrosalpinx fluid. Women with tubal occlusion undergoing IVF treatment with or without hydrosalpinx harbour bacterial and human HSP antibodies in their sera at a high rate (Spandorfer et al., 1999a). The endometrium appears to reduce the toxicity of these antibodies. As demonstrated by TUNEL staining, a possible mechanism of this toxicity may involve the induction of apoptosis. HSP are, as outlined above, essential for successful completion of the single developmental stages of an embryo. The specific expression pattern of HSP may thus play both an essential role in differentiation and a protective role against apoptosis (Mailhos et al., 1993). Since embryos exposed to anti-HSP60 stained TUNEL-positive more often than unexposed embryos it is possible that HSP antibodies may render an embryo more susceptible to apoptosis. 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