Melinda J. Jasper

Seminal Fluid Induces Leukocyte Recruitment and Cytokine and Chemokine mRNA Expression in the Human Cervix after Coitus

In mice, seminal fluid elicits an inflammation-like response in the female genital tract that activates immune adaptations to advance the likelihood of conception and pregnancy. In this study, we examined whether similar changes in leukocyte and cytokine parameters occur in the human cervix in response to the male partner’s seminal fluid. After a period of abstinence in proven-fertile women, duplicate sets of biopsies were taken from the ectocervix in the periovulatory period and again 48 h later, 12 h after unprotected vaginal coitus, vaginal coitus with use of a condom, or no coitus. A substantial influx of CD45+ cells mainly comprising CD14+ macrophages and CD1a+ dendritic cells expressing CD11a and MHC class II was evident in both the stratified epithelium and deeper stromal tissue after coitus. CD3+CD8+CD45RO+ T cells were also abundant and increased after coitus. Leukocyte recruitment did not occur without coitus or with condom-protected coitus. An accompanying increase in CSF2, IL6, IL8, and IL1A expression was detected by quantitative RT-PCR, and microarray analysis showed genes linked with inflammation, immune response, and related pathways are induced by seminal fluid in cervical tissues. We conclude that seminal fluid introduced at intercourse elicits expression of proinflammatory cytokines and chemokines, and a robust recruitment of macrophages, dendritic cells, and memory T cells. The leukocyte and cytokine environment induced in the cervix by seminal fluid appears competent to initiate adaptations in the female immune response that promote fertility. This response is also relevant to transmission of sexually transmitted pathogens and potentially, susceptibility to cervical metaplasia.

Maternal tract factors contribute to paternal seminal fluid impact on metabolic phenotype in offspring

Significance Events at conception shape the future growth and health of offspring, to impact life course potential and disease susceptibility. The environment and experiences of both parents contribute to programming offspring phenotype through epigenetic modifications imparted before embryo implantation. How the father transmits this information remains elusive. Possible pathways include the sperm genome and epigenome, postejaculatory effects of seminal fluid on sperm, and indirect actions of seminal fluid on various female factors regulating embryo development. In this study, we provide evidence that seminal fluid acts to influence both sperm integrity and the balance of embryotrophic and embryotoxic signals in the female reproductive tract, in turn affecting embryo development and programming of future adiposity and metabolic phenotype in male offspring. Paternal characteristics and exposures influence physiology and disease risks in progeny, but the mechanisms are mostly unknown. Seminal fluid, which affects female reproductive tract gene expression as well as sperm survival and integrity, provides one potential pathway. We evaluated in mice the consequences for offspring of ablating the plasma fraction of seminal fluid by surgical excision of the seminal vesicle gland. Conception was substantially impaired and, when pregnancy did occur, placental hypertrophy was evident in late gestation. After birth, the growth trajectory and metabolic parameters of progeny were altered, most profoundly in males, which exhibited obesity, distorted metabolic hormones, reduced glucose tolerance, and hypertension. Altered offspring phenotype was partly attributable to sperm damage and partly to an effect of seminal fluid deficiency on the female tract, because increased adiposity was also evident in adult male progeny when normal two-cell embryos were transferred to females mated with seminal vesicle-excised males. Moreover, embryos developed in female tracts not exposed to seminal plasma were abnormal from the early cleavage stages, but culture in vitro partly alleviated this. Absence of seminal plasma was accompanied by down-regulation of the embryotrophic factors Lif, Csf2, Il6, and Egf and up-regulation of the apoptosis-inducing factor Trail in the oviduct. These findings show that paternal seminal fluid composition affects the growth and health of male offspring, and reveal that its impact on the periconception environment involves not only sperm protection but also indirect effects on preimplantation embryos via oviduct expression of embryotrophic cytokines.

Granulocyte-Macrophage Colony-Stimulating Factor Promotes Glucose Transport and Blastomere Viability in Murine Preimplantation Embryos

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion from epithelial cells lining the female reproductive tract is induced during early pregnancy by ovarian steroid hormones and constituents of seminal plasma. In this study we have investigated the influence of GM-CSF on development of preimplantation mouse embryos. Blastocyst-stage embryos were found to specifically bind 125I-GM-CSF and analysis of GM-CSF mRNA receptor expression by reverse transcriptase-polymerase chain reaction indicated expression of the low-affinity α subunit of the GM-CSF receptor, but not the affinity-converting β subunit (βc), or GM-CSF ligand. GM-CSF receptor mRNA was present in the fertilized oocyte and all subsequent stages of development, and in blastocysts it was expressed in both inner cell mass and trophectoderm cells. In vitro culture of eight-cell embryos in recombinant GM-CSF accelerated development of blastocysts to hatching and implantation stages, with a maximum response at a concentration of 2 ng/ml (77 pM). Blastocysts recovered from GM-CSF-null mutant (GM−/−) mice on Day 4 of natural pregnancy or after superovulation showed retarded development, with the total cell number reduced by 14% and 18%, respectively, compared with GM+/+ embryos. Blastocysts generated in vitro from two-cell GM−/− and GM+/+ embryos were larger when recombinant GM-CSF was added to the culture medium (20% and 24% increases in total cell numbers in GM+/+ and GM−/− blastocysts, respectively). Incubation of blastocysts with recombinant GM-CSF elicited a 50% increase in the uptake of the nonmetabolizable glucose analogue, 3-O-methyl glucose. In conclusion, these data indicate that GM-CSF signaling through the low-affinity GM-CSF receptor in blastocysts is associated with increased glucose uptake and enhanced proliferation and/or viability of blastomeres. Together, the findings implicate a physiological role for maternal tract-derived GM-CSF in targeting the preimplantation embryo, and suggest that defective blastocyst development contributes to compromised pregnancy outcome in GM-CSF-null mutant mice.

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 1ḃ7‐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+ B220lo, 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‐γ 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.

Variations in peripheral blood levels of immunoreactive tumor necrosis factor α (TNFα) throughout the menstrual cycle and secretion of TNFα from the human corpus luteum

Abstract Objective: Several cytokines have been implicated as important mediators in the cyclic processes occurring in the reproductive organs. In the present study the peripheral blood concentrations of the cytokines interleukin(IL)-2, IL-6, and tumor necrosis factor (TNF) α, as well as the secretion of TNFα from the human corpus luteum were investigated. Study design: The study was undertaken at infertility clinics at large teaching hospitals. Eight women with unexplained infertility undergoing investigations with measurements of endocrine profiles throughout a cycle prior to IVF treatment were included in the study of blood concentrations of cytokines. Blood plasma were taken daily or every second day from a time 3–4 days before expected LH peak until menstruation. The levels of immunoreactive IL-2, IL-6 and TNFα were measured by ELISA technique and evaluated (repeated measures ANOVA and Scheffes test) in relation to levels on the day of the LH surge. To investigate a possible ovarian source of TNFα, corpus luteum (CL) tissue and cells obtained during the luteal phase from another group of women during abdominal surgery for benign uterine diseases, were cultured for 24 h to assess (ANOVA and Bonferroni test) the release of TNFα. Results: There were no significant fluctuations in the levels of IL-2 and IL-6 throughout the menstrual cycle. The concentration of TNFα showed significant fluctuations over the menstrual cycle. Compared to the values on the day of the LH surge, the concentrations were significantly increased during the late follicular phase and during the mid luteal phase. In the early luteal phase the levels were significantly decreased. Measurable levels of TNFα were found in the conditioned media from one out of three CL obtained from the early luteal phase, and in all media from CL obtained from mid- and late-luteal phases. Luteal cells in culture secreted TNFα, and the levels in the media were not influenced by the presence of hCG (100 IU/L). The conditioned media of luteal cells from late luteal phase contained higher levels than media of cells from early luteal phase, with the levels being higher in media of a mixture of all luteal cells, and large luteal cells as compared to small luteal cells. Conclusion: This study demonstrates that there are marked fluctuations of blood levels of TNFα during the menstrual cycle and that the human CL secretes TNFα, with indications of higher secretion during late luteal phase as compared to early luteal phase.

Macrophages regulate corpus luteum development during embryo implantation in mice

Macrophages are prominent in the uterus and ovary at conception. Here we utilize the Cd11b-Dtr mouse model of acute macrophage depletion to define the essential role of macrophages in early pregnancy. Macrophage depletion after conception caused embryo implantation arrest associated with diminished plasma progesterone and poor uterine receptivity. Implantation failure was alleviated by administration of bone marrow-derived CD11b+F4/80+ monocytes/macrophages. In the ovaries of macrophage-depleted mice, corpora lutea were profoundly abnormal, with elevated Ptgs2, Hif1a, and other inflammation and apoptosis genes and with diminished expression of steroidogenesis genes Star, Cyp11a1, and Hsd3b1. Infertility was rescued by exogenous progesterone, which confirmed that uterine refractoriness was fully attributable to the underlying luteal defect. In normally developing corpora lutea, macrophages were intimately juxtaposed with endothelial cells and expressed the proangiogenic marker TIE2. After macrophage depletion, substantial disruption of the luteal microvascular network occurred and was associated with altered ovarian expression of genes that encode vascular endothelial growth factors. These data indicate a critical role for macrophages in supporting the extensive vascular network required for corpus luteum integrity and production of progesterone essential for establishing pregnancy. Our findings raise the prospect that disruption of macrophage-endothelial cell interactions underpinning corpus luteum development contributes to infertility in women in whom luteal insufficiency is implicated.

Increased Adhesiveness in Cultured Endometrial-Derived Cells Is Related to the Absence of Moesin Expression

Abstract Human endometrial epithelial cells (EECs) are nonadhesive for embryos throughout most of the menstrual cycle. During the so-called implantation window, the apical plasma membrane of EECs acquire adhesive properties by undergoing a series of morphological and biochemical changes. The human endometrial-derived epithelial cell line, RL95-2, serves as an in vitro model for receptive uterine epithelium because of its high adhesiveness for trophoblast-derived cells. In contrast, the HEC-1-A cell line, which displays poor adhesive properties for trophoblast cells, is considered to be less receptive. The ezrin, radixin, and moesin protein family members, which are present underneath the apical plasma membrane, potentially act to link the cytoskeleton and membrane proteins. In the present study, we have further investigated the adhesive features in these two unrelated endometrial-derived cell lines using an established in vitro model for embryonic adhesion. We have also analyzed the protein pattern and mRNA expression of ezrin and moesin in RL95-2 cells versus HEC-1-A cells. The results demonstrate that RL95-2 cells were indeed more receptive (81% blastocyst adhesion) compared with HEC-1-A cells (46% blastocyst adhesion). An intermediate adhesion rate was found in primary EECs cultured on extracellular matrix gel, thus allowing a partial polarization of these cells (67% blastocyst adhesion). Furthermore, we found that moesin was absent from RL95-2 cells. In contrast, ezrin is expressed in both cell lines, yet it is reduced in adherent RL95-2 cells. Data are in agreement with the hypothesis that uterine receptivity requires down-regulation or absence of moesin, which is a less-polarized actin cytoskeleton.

Characterization of Ovarian Function in Granulocyte-Macrophage Colony-Stimulating Factor-Deficient Mice

Abstract During the estrous cycle and early pregnancy, lymphohe-mopoietic cytokines and chemokines contribute to the regulation of ovarian function by orchestrating the recruitment and activation of leukocytes associated with the ovulatory follicle and corpus luteum. The purpose of this study was to investigate the physiological role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the ovary, utilizing mice genetically deficient in GM-CSF. Our results show that the mean duration of the estrous cycle in GM-CSF-deficient (GM−/−) mice was extended by 1.5 days (mean ± SE, 4.9 ± 0.3 vs. 6.5 ± 0.5 days for GM+/+ and GM−/− mice, respectively). Similar ovulation rates were observed in immature superovulated mice (31.8 ± 7.7 vs. 28.9 ± 6.4 oocytes per mouse) and adult naturally cycling mice (10.4 ± 0.8 vs. 10.3 ± 0.8 oocytes per mouse). Furthermore, comparable numbers of oocytes were released from GM+/+ and GM−/− ovaries in an in vitro perfusion model. However, ovaries in pregnant GM−/− mice were found to comprise fewer cells and synthesize less progesterone (141.6 ± 10.3 vs. 116.5 ± 6 nM plasma), although the duration of pseudopregnancy was unaltered by GM-CSF deficiency (11.0 ± 0.2 vs. 11.0 ± 0.5 days). Immunohistochemical staining of leukocytes in the ovary during the periovulatory period indicated that the size and composition of ovarian leukocyte populations were unaltered in the absence of GM-CSF. However, an effect of GM-CSF deficiency on the activation phenotype of ovarian leukocytes was indicated by a 57% increase in mean secretion of nitric oxide in in vitro-perfused GM−/− ovaries, and diminished major histocompability complex (MHC) class II (Ia) expression in ovarian macrophages and/or dendritic cells (30.5 ± 7.2% vs. 9.1 ± 1.8% positive stain in GM+/+ and GM−/− ovaries, respectively). Furthermore, ovarian macrophages and neutrophils were diminished in number after parturition, with significantly decreased CD11b+ (Mac-1) staining in the stromal region of postpartum GM−/− ovaries (6.7 ± 0.6 vs. 3.6 ± 0.7% positive stain). In summary, GM-CSF does not appear to be essential for ovarian function but may play a role in fine-tuning the activation status and adhesive properties of ovarian myeloid leukocytes. Aberrant activation of these cells appears to compromise the luteinization process and the steroidogenic capacity of the corpus luteum during early pregnancy in GM-CSF-deficient mice.

Macrophage-Derived LIF and IL1B Regulate Alpha(1,2)Fucosyltransferase 2 (Fut2) Expression in Mouse Uterine Epithelial Cells During Early Pregnancy

Macrophages accumulate within stromal tissue subjacent to the luminal epithelium in the mouse uterus during early pregnancy after seminal fluid exposure at coitus. To investigate their role in regulating epithelial cell expression of fucosylated structures required for embryo attachment and implantation, fucosyltransferase enzymes Fut1, Fut2 (Enzyme Commission number [EC] 2.4.1.69), and Fut4 (EC 2.4.1.214) and Muc1 and Muc4 mRNAs were quantified by quantitative real-time PCR in uterine epithelial cells after laser capture microdissection in situ or after epithelial cell coculture with macrophages or macrophage-secreted factors. When uterine macrophage recruitment was impaired by mating with seminal plasma-deficient males, epithelial cell Fut2 expression on Day 3.5 postcoitus (pc) was reduced compared to intact-mated controls. Epithelial cell Fut2 was upregulated in vitro by coculture with macrophages or macrophage-conditioned medium (MCM). Macrophage-derived cytokines LIF, IL1B, and IL12 replicated the effect of MCM on Fut2 mRNA expression, and MCM-stimulated expression was inhibited by anti-LIF and anti-IL1B neutralizing antibodies. The effects of acute macrophage depletion on fucosylated structures detected with lectins Ulex europaeus 1 (UEA-1) and Lotus tetragonolobus purpureas (LTP), or LewisX immunoreactivity, were quantified in vivo in Cd11b-dtr transgenic mice. Depletion of macrophages caused a 30% reduction in luminal epithelial UEA-1 staining and a 67% reduction in LewisX staining in uterine tissues of mice hormonally treated to mimic early pregnancy. Together, these data demonstrate that uterine epithelial Fut2 mRNA expression and terminal fucosylation of embryo attachment ligands is regulated in preparation for implantation by factors including LIF and IL1B secreted from macrophages recruited during the inflammatory response to insemination.

Endocrinology: Immunoactive interleukin-1β and tumour necrosis factor-α in thecal, stromal and granulosa cell cultures from normal and polycystic ovaries

Previous studies of follicular fluid from stimulated human ovaries have shown detectable amounts of immunoactive interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF). Other reports have shown the presence of IL-1 receptors, mRNA and antagonists for IL-1 in the human ovary. The aim of this study was to measure IL-1 and TNF concentrations in follicular fluid from unstimulated normal or polycystic ovaries (PCO) as well as concentrations in media conditioned by granulosa cells or thecal or stromal tissue. TNF concentrations were easily detected in follicular fluid (77 fmol/ml, range 20-95) and concentrations in PCO were similar to those in normal ovaries (70 versus 82 fmol/ml). TNF was virtually undetectable in all tissue culture media. IL-1 concentrations in all culture media were readily detected but showed no differences between different tissues or between PCO and normal ovaries. It was concluded that human ovaries readily produce immunoactive IL-1 in culture but produce less TNF despite detectable amounts of TNF in follicular fluid. There appears to be no difference between PCO and normal ovaries with respect to IL-1 and TNF.