Endometrium immunomodulation by intrauterine insemination administration of treated peripheral blood mononuclear cell prior frozen/thawed embryos in patients with repeated implantation failure.

In assisted reproductive technology (ART) programmes, approximately 10% of infertile patients have at least two or three repeated implantation failures (RIFs) after an in vitro fertilization (IVF) protocol. Successful implantation mainly depends on local immune tolerance mechanisms involving a spectrum of cytokines, interleukins and growth factors. The latter have played pivotal roles in the recruitment of immune cells (and notably T-lymphocyte cells). In total, 250 couples participating in frozen-thawed embryo transfer programme were incorporated in a randomized clinical trial (peripheral blood mononuclear cells (PBMC) subgroup: n=122; control subgroup: n=128). In the PBMC group, a blood sample was collected 5 days before the scheduled frozen-thawed embryo transfer; PBMCs were isolated using Ficoll separation and then cultured for 72 h. Two days prior to embryo transfer, 0.4 ml of cultured PBMCs were transferred into the patient's uterus. Although the clinical pregnancy rate was higher in the PBMC group (34.4%) than in the control group (23.4%), this difference was not statistically significant (P=0.05 in a chi-squared test). Nevertheless, when we limited the analysis to patients with ≥3 RIFs (n=138), there was a significant difference in the clinical pregnancy rate between the PBMC group (38.6%) and the control group (19.7%; P=0.01). Our results imply that PBMC transfer can be part of effective fertility treatment for patients with RIF.

Female bed bugs (Cimex lectularius L) anticipate the immunological consequences of traumatic insemination via feeding cues.

Not all encounters with pathogens are stochastic and insects can adjust their immune management in relation to cues associated with the likelihood of infection within a life cycle as well as across generations. In this study we show that female insects (bed bugs) up-regulate immune function in their copulatory organ in anticipation of mating by using feeding cues. Male bed bugs only mate with recently fed females and do so by traumatic insemination (TI). Consequently, there is a tight temporal correlation between female feeding and the likelihood of her being infected via TI. Females that received predictable access to food (and therefore predictable insemination and infection cycles) up-regulated induced immunity (generic antibacterial activity) in anticipation of feeding and mating. Females that received unpredictable (but the same mean periodicity) access to food did not. Females that anticipated mating-associated immune insult received measurable fitness benefits (survival and lifetime reproductive success) despite laying eggs at the same rate as females that were not able to predict these cycles. Given that mating is a time of increased likelihood of infection in many organisms, and is often associated with temporal cues such as courtship and/or feeding, we propose that anticipation of mating-associated infection in females may be more widespread than is currently evidenced.

Estradiol impairs epithelial CXCL1 gradient in the cervix to delay neutrophil transepithelial migration during insemination.

Female reproductive mucosa must allow allogenic sperm survival whereas at the same time, avoid pathogen infection. To preserve sperm from neutrophil attack, neutrophils disappear from the vagina during the ovulatory phase (high estradiol); although the mechanisms that regulate neutrophil influx to the vagina during insemination remain controversial. We investigated the sex hormone regulation of the neutrophil migration through the cervix during insemination and revealed that ovulatory estradiol dose fades the CXCL1 epithelial expression in the ectocervix and fornix; hence, retarding neutrophil migration and retaining them in the epithelium. These mechanisms spare sperm from neutrophil attack to preserve reproduction, but might compromise immunity. However, luteal progesterone dose promotes the CXCL1 gradient expression to restore neutrophil migration, to eliminate sperm and prevent sperm associated pathogen dissemination. Surprisingly, these mechanisms are hormone dependent and independent of the insemination. Thus, sex hormones orchestrate tolerance and immunity in the vaginal lumen by regulating neutrophil transepithelial migration in the fornix and ectocervix.

Post-mating inflammatory responses of the uterus.

This review attempts to summarize the current knowledge on uterine inflammatory response after mating in horses, pigs and cattle. Post-mating endometritis has been extensively studied in horses as it has been considered to cause infertility. The inflammation is known to occur also in cattle, but it has not been investigated to a similar extent. There are a number of publications about mechanisms of post-mating uterine inflammation in pigs, which seem to resemble those in horses. The major focus of this review is the horse, but relevant literature is presented also on swine and cattle. Spermatozoa, seminal plasma and semen extenders play roles in the induction of inflammation. In addition, sperm numbers, concentration and viability, as well as the site of semen deposition may modulate the inflammatory response. Cytokines, polymorphonuclear leucocytes (PMN) and mononuclear cells represent the uterine inflammatory response to mating. Inflammation is the first line of defence against invasion and eliminates excess spermatozoa and bacteria. Semen deposition elicits a massive PMN invasion, followed by phagocytosis of sperm aided by the formation of neutrophil extracellular traps. Exposure of the female genital tract to semen is important also for endometrial receptivity and pre-implantation embryo development. Seminal plasma (SP) and inflammation elicit transient immune tolerance to antigens present in semen. SP contains immune-regulatory molecules that activate and control immune responses to antigens by stimulating expression of cytokines and growth factors and by initiating tissue remodelling. SP also regulates ovarian function. Effective elimination of excess sperm and inflammatory by-products and subsequent rapid return of the endometrium to the normal state is a prerequisite for pregnancy. Uterine backflow, driven by myometrial contractions and requiring a patent cervix, is an important physical tool in uterine drainage.

Utilising T cell receptor transgenic mice to define mechanisms of maternal T cell tolerance in pregnancy.

Studies in mice demonstrate that the maternal T cell repertoire is aware of paternal antigens during pregnancy, but in healthy pregnancy reactive T cells do not mediate anti-fetal immunity. Mice expressing transgenic T cell receptors (TCRs) specific for paternal and conceptus antigens are powerful tools for elucidating the events surrounding paternal antigen presentation to the maternal T cell repertoire, the nature of the ensuing T cell response and the factors that skew the response towards immune tolerance to allow survival and development of the conceptus. While results from different transgenic TCR models are not always consistent, there is now sufficient data to allow a consensus interpretation that maternal antigen presenting cells present initially seminal fluid antigens and later placenta-derived antigens to both the CD4+ and CD8+ T cell repertoire. T cell proliferation is generally followed by entry into a state of anergy demonstrated by decreased cytokine production and hyporesponsiveness upon restimulation. Some models also demonstrate downregulation of the TCR and co-stimulatory molecules, clonal deletion of paternal antigen-reactive T cells, or alternatively T cell ignorance of paternal antigens. This review will summarise the range of transgenic TCR studies that have shed light on the events surrounding paternal antigen presentation and the various T cell responses to insemination and pregnancy. The benefits, limitations and caveats of these models, and their impact upon data interpretation, are discussed.

Mechanism of prolonged sperm storage and sperm survivability in hen oviduct: a review.

A unique property of the avian oviduct is to store sperm for a prolonged period. The sperm storage tubules (SST) are located in the utero-vaginal junction of the oviduct, where sperm can be stored and survived for a few weeks after insemination or natural mating. The immune system in the oviduct is essential to prevent tissue infection by various microorganisms, and it may also affect the fate and survivability of sperm in the oviduct. Anti-sperm immunoresponses including infiltration of leukocytes may be induced in the vagina of the oviduct. Sperm that will participate in fertilization may be selected by these immunoresponses. However, sperm stored in the SST may be protected from the immunoresponse by SST structures and transforming growth factor beta, whose expression is increased during sperm storage in the SST. In this review, the mechanism of sperm survivability with reference to the regulation of anti-sperm immunoresponses in hen oviduct is emphasized.

Semen activates the female immune response during early pregnancy in mice.

Insemination elicits inflammatory changes in female reproductive tissues, but whether this results in immunological priming to paternal antigens or influences pregnancy outcome is not clear. We have evaluated indices of lymphocyte activation in lymph nodes draining the uterus following allogeneic mating in mice and have investigated the significance of sperm and plasma constituents of semen in the response. At 4 days after mating, there was a 1b7-fold increase in the cellularity of the para-aortic lymph node (PALN) compared with virgin controls. PALN lymphocytes were principally T and B lymphocytes, with smaller populations of CD3(+) B220(lo), NK1.1(+) CD3(-) (NK) and NK1.1(+) CD3(+) (NKT) cells. CD69 expression indicative of activation was increased after mating and was most evident in CD3(+) and NK1.1(+) cells. Synthesis of cytokines including interleukin-2, interleukin-4 and interferon-gamma was elevated in CD3(+) PALN cells after exposure to semen, as assessed by intracellular cytokine fluorescence-activated cell sorting, immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction. Matings with vasectomized males indicated that the lymphocyte activation occurs independently of sperm. However, in contrast, males from which seminal vesicle glands were surgically removed failed to stimulate PALN cell proliferation or cytokine synthesis. Adoptive transfer experiments using radiolabelled lymphocytes from mated mice showed that lymphocytes activated at insemination home to embryo implantation sites in the uterus as well as other mucosal tissues and lymph nodes. These findings indicate that activation and expansion of female lymphocyte populations occurs after mating, and is triggered by constituents of seminal plasma derived from the seminal vesicle glands. Moreover, lymphocytes activated at insemination may help mediate maternal tolerance of the conceptus in the implantation site.

Role of uterine immune cells in early pregnancy in pigs.

The immune system discriminates 'self' from 'non-self', and eliminates that which it determines to be non-self. Mammalian pregnancy appears to represent a failure of self-non-self discrimination, yet it is a highly successful reproductive strategy. We present evidence that the immune system of the female pig responds to the challenges of both mating and the presence of conceptuses. Mating induces an influx of inflammatory leukocytes into the endometrial stroma and uterine lumen. This response, while partially under endocrine control, is amplified by as yet unidentified factors in seminal plasma. In addition to preventing microbial infection, this mating-induced immune response may enhance reproductive performance. During the first month of pregnancy when intimate contact between maternal and fetal tissues is being established, the number of uterine lymphocytes decrease in the luminal epithelium and increase in the endometrial stroma at sites of conceptus attachment. The majority of these lymphocytes express the CD2 and CD8 surface markers, consistent with either T or natural killer (NK) cell lineage. Dispersed endometrial cells obtained during early gestation exhibit a pregnancy-specific increase in NK-like lytic activity. Our ongoing efforts to determine whether these NK-like lymphocytes are the ones that localize to sites of conceptus attachment are discussed. We speculate on how the reactions of pig uterine leukocytes to seminal plasma and conceptuses might contribute to successful pregnancy.

Immunological role for seminal plasma in insemination and pregnancy.

Insemination confronts the female with paternally derived alloantigens and represents an immunological challenge preceding fertilization and implantation. Current evidence suggests a role for seminal plasma in regulating maternal immunity for insemination and pregnancy. In vitro seminal plasma has been shown to suppress T- and B-cell proliferation, neutrophil and macrophage phagocytic activity, as well as killer cell activity. Seminal plasma interacts with complement components C1 and C3 and contains factors that specifically bind the Fc region of IgG. These in vitro findings suggest possible seminal plasma-suppressive effects on female alloimmune responses after insemination. Seminal plasma also contains allotypic TLX antigens that could prime mothers prior to fertilization. Such priming effects for pregnancy acceptance are supported by improved implantation rates in controlled clinical trials using timed vaginal exposure to semen during in vitro fertilization or gamete intrafallopian transfer treatment cycles.