Expression of the chemokine eotaxin and its receptor, CCR3, in human endometrium

Eosinophils are present in human endometrium only immediately before and during menstruation, suggesting a role in that process. The expression of the eosinophil chemoattractant, eotaxin, and its receptor, CCR3, within the human endometrium were investigated by immunohistochemical analysis of tissue sections spanning the entire menstrual cycle. Eotaxin was localized to perivascular cells in the late secretory phase, and it was also identified in eosinophils. However, the highest levels of this chemokine were present in both luminal and glandular epithelial cells during the proliferative and secretory phases of the cycle. Treatment of endometrial tissue with monensin, which blocks protein secretion, increased epithelial immunoreactive eotaxin, substantiating synthesis in these cells. Although the CCR3 receptor was expressed by eosinophils, it was also strongly expressed by endometrial epithelial cells. The CCR3 receptor on purified, cultured endometrial epithelial cells was functional, as assessed by a transient Ca(2+) flux in response to eotaxin. These analyses demonstrate that eotaxin is expressed by endometrial cells and may therefore be involved in the recruitment of eosinophils into this tissue premenstrually. However, the observation that this chemokine and the CCR3 molecule are strongly expressed by epithelial cells throughout the cycle suggests that these proteins may have additional important functions within the endometrium.     

Patterns of chemokine receptor expression on peripheral blood gamma delta T lymphocytes: strong expression of CCR5 is a selective feature of V delta 2/V gamma 9 gamma delta T cells

Gammadelta T lymphocytes play an important role in the immune defense against infection, based on the unique reactivity of human Vdelta2Vgamma9 gammadelta T cells toward bacterial phosphoantigens. Chemokines and their corresponding receptors orchestrate numerous cellular reactions, including leukocyte migration, activation, and degranulation. In this study we investigated the expression of various receptors for inflammatory and homeostatic chemokines on peripheral blood gammadelta T cells and compared their expression patterns with those on alphabeta T cells. Although several of the analyzed receptors (including CCR6, CCR7, CXCR4, and CXCR5) were not differentially expressed on gammadelta vs alphabeta T cells, gammadelta T cells expressed strongly increased levels of the RANTES/macrophage inflammatory protein-1alpha/-1beta receptor CCR5 and also enhanced levels of CCR1-3 and CXCR1-3. CCR5 expression was restricted to Vdelta2 gammadelta T cells, while the minor subset of Vdelta1 gammadelta T cells preferentially expressed CXCR1. Stimulation with heat-killed extracts of Mycobacterium tuberculosis down-modulated cell surface expression of CCR5 on gammadelta T cells in a macrophage-dependent manner, while synthetic phosphoantigen isopentenyl pyrophosphate and CCR5 ligands directly triggered CCR5 down-modulation on gammadelta T cells. The functionality of chemokine receptors CCR5 and CXCR3 on gammadelta T cells was demonstrated by Ca(2+) mobilization and chemotactic response to the respective chemokines. Our results identify high level expression of CCR5 as a characteristic and selective feature of circulating Vdelta2 gammadelta T cells, which is in line with their suspected function as Th1 effector T cells.     

Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium

Matrix metalloproteinases (MMP) have specific spatial and temporal expression patterns in human endometrium and are critical for menstruation. Expression and activation mechanisms for proMMP-2 differ from other MMPs; in many cells proMMP-2 is specifically activated by membrane-type (MT)-MMPs. We examined the expression and localization of proMMP-2, MT1-MMP, and MT2-MMP in human endometrium across the menstrual cycle; and we examined the expression of MT1-MMP and activation of proMMP-2 in cultured endometrial stromal cells and their regulation by progesterone. MMP-2 was immunolocalized in 25 of 32 endometrial samples in all cellular compartments but with greatest intensity in degrading menstrual tissue. MT1-MMP mRNA was present throughout the cycle, and immunoreactive protein was detected in 24 of 32 samples, with the strongest staining in subsets of macrophages, neutrophils, and granular lymphocytes (but not mast cells or eosinophils) during the menstrual, mid-proliferative and mid-secretory phases. Patchy epithelial staining and staining of decidual cells, often periglandular in menstrual tissue, were also seen. MT2-MMP was more widespread than MT1-MMP without apparent cyclical variation and with maximal intensity in glandular epithelium. Cultured endometrial stromal cells released proMMP-2, and progesterone treatment significantly reduced the percentage level of its active (62 kDa) form (22.5 +/- 1.8% vs. 3.0 +/- 1.3%, without and with treatment, respectively, mean +/- SEM, P < 0.0001). This activation was blocked by a specific MMP inhibitor and restored following inhibitor removal. Progesterone also attenuated cell expression of MT1-MMP mRNA. We postulate that MT1-MMP activates proMMP-2 in endometrium, this activity being increased at the end of the cycle when progesterone levels fall, thus contributing to menstruation.     

The chemokines, CX3CL1, CCL14, and CCL4, promote human trophoblast migration at the feto-maternal interface

Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. Chemokines, critical regulators of leukocyte migration, are abundant in endometrial epithelial and decidual cells at this time. We hypothesized that endometrial chemokines stimulate trophoblast invasion. Chemokine receptors CX3CR1 and CCR1 were immunolocalized in human first-trimester implantation sites, specifically to endovascular extravillous trophoblasts, but not to the invading interstitial EVTs (iEVTs), with weak staining also on syncytium. CCR3 was localized to invading iEVTs and to microvilli on the syncytial surface. Expression of CX3CL1 (fractalkine), CCL7 (MCP-3), and their receptors (CX3CR1, CCR1, CCR2, CCR3, and CCR5) mRNA was examined in cellular components of the maternal-embryonic interface by RT-PCR. Both chemokines were abundant in entire endometrium and placenta, endometrial cells (primary cultures and HES, a human endometrial epithelial cell line) and trophoblast cell lines (JEG-3, ACIM-88, and ACIM-32). Chemokine receptor mRNA was expressed by placenta and trophoblast cell lines: CCR1 by all trophoblast cell types, whereas CCR2, CCR3, and CX3CR1 were more variable. CX3CR1, CCR1, CCR2, and CCR5 were also expressed by endometrial cells. Migration assays used the trophoblast cell line most closely resembling extravillous cytotrophoblast (AC1M-88). Trophoblast migration occurred in response to CX3CL1, CCL14, and CCL4, but not CCL7. Endometrial cell-conditioned media also stimulated trophoblast migration; this was attenuated by neutralizing antibodies to CX3CL1 and CCL4. Thus, chemokines are expressed by maternal and embryonic cells during implantation, whereas corresponding receptors are on trophoblast cells. Promotion of trophoblast migration by chemokines and endometrial cell conditioned medium indicates an important involvement of chemokines in maternal-fetal communication.     

VEGF, bFGF, and their receptors in the endometrium of rhesus monkey during menstrual cycle and early pregnancy

A number of cytokines and growth factors are known to modulate proliferation and differentiation of human endometrium. In this study, the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and VEGF receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing region (KDR), and bFGF receptor 1 (Flg) were examined in the endometrium of rhesus monkey on Day 5, 10, 16, 20, 25 of menstrual cycle and on Day 19 of early pregnancy. Western blot analysis showed the specificity of the anti-human antibodies with the monkey tissue. The expression of mRNA and protein of VEGF was correlated with that of its receptor KDR, which was detected in epithelial, vascular, and myometrial cells. The localization of bFGF and its receptor Flg was similar to that of VEGF, except that the Flg was absent in the endothelial cells. Strong expression of VEGF and bFGF in the glandular epithelial cells was observed in the proliferative phase, declined in the secretory phase during the cycle. Stronger staining of these factors was also observed in the decidual cells of the pregnant uterus, as compared with the stromal cells of cycling uterus. No expression of Flt1 was detected in the tissue examined in this study. These data suggest that VEGF, bFGF, and their receptors play important roles in epithelial and stromal development, angiogenesis, and blood vessel function in the endometrium during the menstrual cycle and early pregnancy of the rhesus monkey.     

Expression of CXC and CC type of chemokines and its receptors in tuberculous and non-tuberculous effusions

Chemokines mediate their biological functions by transmigration of various immune cells to the site of infection. Tuberculous pleurisy provides an effective model to study the role of chemokines in the recruitment of immune cells to the pleura. Our aim was to understand the cumulative effect of chemokines (IP-10, MIG, IL-8, MCP-1, MIP-1α and RANTES) and its receptors (CXCR2, CXCR3, CCR1, CCR2, CCR5 and CCR7) in the recruitment of CD4⁺ T cells obtained from blood (BL) and pleural fluid (PF) of tuberculous (TB) and non-tuberculous (NTB) patients. We observed significant increase in CD4⁺ T cells in TB PF indicating lymphocytic rich effusion. All chemokines except RANTES were significantly high in PF compared to BL in TB group, whereas IL-8 and MCP-1 showed significant increase only in NTB PF. The significantly high levels of IFN-γ and ΤΝF-α in TB PF and their positive correlation with IP-10 and MIP-1α indicated their synergistic action to elicit a strong protective Th1 response. In spite of high levels of Th1 cytokines and chemokines in TB PF, significantly lower levels of RANTES indicated its limited role at the site. The CXC receptors in PF of both the groups and CC receptors except CCR5 in TB PF were significantly high compared to BL. Only CXCR2, CCR5 and CCR7 showed significant increase in TB compared to NTB. Thus a selective concentration of chemokines, cytokines and abundant expression of chemokine receptors confirm the accumulation of activated and memory T cells at the site of infection and help in polarizing Th1 immune response.     

Interleukin-8-mediated heterologous receptor internalization provides resistance to HIV-1 infectivity. Role of signal strength and receptor desensitization

Human immunodeficiency virus type 1 (HIV-1) entry into CD4(+) cells requires the chemokine receptors CCR5 or CXCR4 as co-fusion receptors. We have previously demonstrated that chemokine receptors are capable of cross-regulating the functions of each other and, thus, affecting cellular responsiveness at the site of infection. To investigate the effects of chemokine receptor cross-regulation in HIV-1 infection, monocytes and MAGIC5 and rat basophilic leukemia (RBL-2H3) cell lines co-expressing the interleukin-8 (IL-8 or CXCL8) receptor CXCR1 and either CCR5 (ACCR5) or CXCR4 (ACXCR4) were generated. IL-8 activation of CXCR1, but not the IL-8 receptor CXCR2, cross-phosphorylated CCR5 and CXCR4 and cross-desensitized their responsiveness to RANTES (regulated on activation normal T cell expressed and secreted) (CCL5) and stromal derived factor (SDF-1 or CXCL12), respectively. CXCR1 activation internalized CCR5 but not CXCR4 despite cross-phosphorylation of both. IL-8 pretreatment also inhibited CCR5- but not CXCR4-mediated virus entry into MAGIC5 cells. A tail-deleted mutant of CXCR1, DeltaCXCR1, produced greater signals upon activation (Ca(2+) mobilization and phosphoinositide hydrolysis) and cross-internalized CXCR4, inhibiting HIV-1 entry. The protein kinase C inhibitor staurosporine prevented phosphorylation and internalization of the receptors by CXCR1 activation. Taken together, these results indicate that chemokine receptor-mediated HIV-1 cell infection is blocked by receptor internalization but not desensitization alone. Thus, activation of chemokine receptors unrelated to CCR5 and CXCR4 may play a cross-regulatory role in the infection and propagation of HIV-1. Since DeltaCXCR1, but not CXCR1, cross-internalized and cross-inhibited HIV-1 infection to CXCR4, the data indicate the importance of the signal strength of a receptor and, as a consequence, protein kinase C activation in the suppression of HIV-1 infection by cross-receptor-mediated internalization.     

Chemokine receptor expression and signaling in macaque and human fetal neurons and astrocytes: implications for the neuropathogenesis of AIDS.

Chemokines are believed to play a role in the neuropathogenesis of AIDS through their recruitment of neurotoxin-secreting, virally infected leukocytes into the CNS. Levels of chemokines are elevated in brains of patients and macaques with HIV/SIV-induced encephalitis. The chemokine receptors CCR3, CCR5, and CXCR4 are found on subpopulations of neurons in the cortex of human and macaque brain. We have developed an in vitro system using both macaque and human fetal neurons and astrocytes to further investigate the roles of these receptors in neuronal response to inflammation. Here we report the presence of functional HIV/SIV coreceptors CCR3, CCR5, and CXCR4 on fetal human and macaque neurons and CCR5 and CXCR4 on astrocytes immediately ex vivo and after several weeks in culture. Confocal imaging of immunostained neurons demonstrated different patterns of distribution for these receptors, which may have functional implications. Chemokine receptors were shown to respond to their appropriate chemokine ligands with increases in intracellular calcium that, in the case of neurons, required predepolarization with KCl. These responses were blocked by neutralizing chemokine receptor in mAbs. Pretreatment of neural cells with pertussis toxin abolished responses to stromal-derived factor-1alpha, macrophage inflammatory protein-1beta, and RANTES, indicating coupling of CCR5 and CXCR4 to a Gialpha protein, as in leukocytes. Cultured macaque neurons demonstrated calcium flux response to treatment with recombinant SIVmac239 envelope protein, suggesting a mechanism by which viral envelope could affect neuronal function in SIV infection. The presence of functional chemokine receptors on neurons and astrocytes suggests that chemokines could serve to link inflammatory and neuronal responses.     

CA 125 is an excretory product of human endometrial glands

The present investigation was undertaken to study the cellular localization and kinetics of synthesis of CA 125 in the endometrium. CA 125 was localized by immunohistochemistry to the infranuclear region of epithelial cells during the proliferative phase and to the apical luminal border during the secretory phase. In gestational endometrium, both the cytoplasm and the apical luminal border of epithelial cells were intensely positive. No staining was seen in endometrial stromal cells during the normal cycle or in decidualized endometria. Results obtained from in vitro cultures of separated glandular and stromal cells were similar to those obtained by immunohistochemistry. That is, epithelial cells released between 5 and 25 times more CA 125 into the culture medium than did stromal cells. The release of CA 125 was highest in epithelial and stromal cells obtained during the early secretory phase. CA 125 concentrations were markedly elevated in endometrial aspirations obtained during the secretory phase or in endometria with crumbling stroma compared to plasma levels. Plasma levels of CA 125 were slightly elevated during menses. These results suggest that CA 125 is an exocrine product of endometrial epithelial cells. Plasma levels of CA 125 may be of endometrial origin only when the membrane barriers, which normally prevent its entry into the circulation, are damaged.     

Localisation of the Notch family in the human endometrium of fertile and infertile women

To investigate the spatial and temporal immunolocalisation and staining intensity of the Notch signalling family in endometrium of fertile and infertile women, endometrial biopsies were collected by curettage from 25 fertile women across the menstrual cycle and 10 infertile women in the mid secretory phase of menstrual cycle. Immunohisotchemistry was completed for NOTCH1, -2, -3, -4, cleaved Notch, DLL1, -3, -4, JAGGED1, -2, HES and NUMB and immunostaining intensity measured in both the endometrial glandular and luminal epithelium. NOTCH1 and the ligands DLL1 and JAGGED1 were key proteins displaying increased staining intensity during the receptive phase of the menstrual cycle and dysregulated in infertile endometrium. Conversely, NUMB a negative regulator of Notch signalling was decreased in the mid secretory phase of the menstrual cycle in fertile women and increased with infertility.     

Macaque trophoblast migration is regulated by RANTES

In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade the endometrium, enter the uterine vasculature, and migrate within the arteries. The mechanisms that regulate this directional migration are unknown. We have used early gestation macaque trophoblasts to test the hypothesis that trophoblast migration is regulated by the chemokine, Regulated on Activation T-Cell Expressed and Secreted (RANTES). Immunohistochemical analysis of cryosections of endometrial tissue showed expression of RANTES by stromal cells and vascular cells. Isolated endothelial cells expressed RANTES as determined by immunocytochemistry and RT-PCR analyses. Immunohistochemical analysis of endometrial cryosections showed that the RANTES receptor, CCR5, was expressed by trophoblasts on anchoring villi and by cells within the trophoblastic shell. Cytokeratin-positive/CCR5-positive cells, consistent with trophoblasts, were also found scattered within the stroma and were often clustered around blood vessels. Isolated trophoblast cells expressed CCR5 as determined by immunocytochemistry and RT-PCR analyses. Isolated trophoblasts migrated towards RANTES when cultured in migration chambers and migration was reduced in the presence of anti-CCR5 antibody. When trophoblasts were cultured on dishes coated with recombinant RANTES, expression of beta1 integrin was increased. The RANTES-induced increase in beta1 integrin expression was inhibited by pertussis toxin. These data suggest a role for RANTES and CCR5 in the regulation of trophoblast migration within the endometrium and within the uterine vasculature.     

Paracrine regulation of endometrial function: interaction between progesterone and corticotropin-releasing factor (CRF) and activin A

Under the influence of ovarian steroid hormones, endometrial cells aer able to produce a wide variety of growth factors and peptide hormones that area believed to promote: (1) physiological growth and differentiation during the endometrial cycle; (2) decidualization, an essential preparative event for establishment of pregnancy; and (3) pathological growth and differentiation in endometriosis and cancer. Among the local factors produced by the human endometrium, corticotropin-releasing factor (CRF) and activin A have been evaluated in terms of localization and effects. CRF is a neuropeptide expressed by the epithelial and stromal cells of the human endometrium in increasing amounts from the endometrial proliferative to the secretory phase. CRF expression also increases in the pregnant endometrium, from early in the pregnancy until term. CRF-type 1 receptor mRNA is only expressed by stromal cells. Progesterone induces CRF gene expression and release from decidualized cells and CRF decidualizes cultured stromal endometrial cells. Urocortin, a CRF-related peptide, has been identified in endometrial epithelial and stromal cells, and its function is still under investigation. Activin A is a growth factor expressed in increasing amounts throughout endometrial phases by both epithelial and stromal cells. This growth factor is secreted into the uterine cavity with higher levels in the secretory phase. Maternal decidua expresses activin A mRNA in increasing amounts from early pregnancy until term. Human endometrium also expresses activin-A receptors and follistatin, its binding protein. Activin A decidualizes cultured human endometrial stromal cells (an effect reversed by follistatin) and modulates embryonic trophoblast differentiation and adhesion. Activin A is expressed in endometriosis and endometrial adenocarcinoma.     

Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.     

Aquaporin-2 expression in human endometrium correlates with serum ovarian steroid hormones

The aim of the present study was to examine the expression of aquaporin-2 (AQP2), a member of the water channel family aquaporins (AQPs), in human uterine endometrium and its modulation of ovarian steroid hormone at the proliferative and secretory phases. Western blot, immunohistochemistry, and RT-PCR were employed in the present study. Western blot revealed a 29-kDa band that represented AQP2 in human endometrium. The expression of AQP2 in endometrium was confirmed by RT-PCR and immunohistochemical results. The immunohistochemical analysis demonstrated that AQP2 was prominent in luminal and glandular epithelial cells of endometrium. The levels of endometrial AQP2 expression changed during the menstrual cycle and were higher in the secretory endometrium than in the proliferative endometrium. A significantly high level of AQP2 was detected at the mid-secretory phase. There was a positive correlation between the levels of the endometrial AQP2 expression and the concentrations of the serum 17beta-estradiol (E2) or/and progesterone (P4). These data for the first time corroborate that AQP2 is expressed in human endometrium and that the expression of AQP2 in human endometrium might be regulated by E2 or/and P4. The changed expression of AQP2 at different phases of the menstrual cycle may be essential to reproductive physiology in human. The high level of endometrial AQP2 expression was observed at the mid-secretory phase, the time of embryo implantation, suggesting that AQP2 might play physiological roles in the uterine receptivity.     

Progesterone-induced inhibition of chemokine receptor expression on peripheral blood mononuclear cells correlates with reduced HIV-1 infectability in vitro.

Recent studies have shown that progesterone, a sex steroid hormone, enhances the sexual transmission of various pathogens, including SIV. The goal of this study was to determine whether progesterone affects mechanisms underlying the sexual transmission of HIV-1. We first studied the effects of various physiologic concentrations of progesterone on the expression of chemokines and chemokine receptors by T cells and macrophages. Chemokines are involved in leukocyte recruitment to peripheral sites; in addition, the chemokine receptors CCR5 and CXCR4 are HIV-1 coreceptors, and their ligands can block HIV-1 infection. Progesterone treatment had no effect on constitutive expression of CCR5 and CXCR4 by nonactivated T cells and macrophages, but significantly inhibited IL-2-induced up-regulation of CCR5 and CXCR4 on activated T cells (p < 0.05). Progesterone also inhibited both mitogen-induced proliferation and chemokine secretion (macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, RANTES) by CD8+ T lymphocytes. Control and progesterone-treated PBMC cultures were also tested for susceptibility to infection by T cell-tropic (HIV-1MN) and macrophage-tropic (HIV-1JR-CSF) viral strains in vitro. Infection with low titers of HIV-1MN was consistently inhibited in progesterone-treated cultures; progesterone effects on infection with the HIV-1JR-CSF strain were more variable, but correlated with progesterone-induced reductions in CCR5 levels. These results indicate that progesterone treatment can inhibit mechanisms underlying HIV-1 transmission, including infection of CD4+ target cells via CXCR4/CCR5 coreceptors and effects on chemokine-mediated recruitment of lymphocytes and monocytes to mucosal epithelia.     

Distribution of hyaluronan in human endometrium across the menstrual cycle

Hyaluronan is a molecule with many known roles in cellular physiology and is often associated with tissue remodeling. The human endometrium undergoes dramatic remodeling during the course of the normal menstrual cycle but its regulation is not well understood. This study examined the levels of deposition of hyaluronan in human cycling endometrium by histochemical localization, using a highly specific hyaluronan-binding peptide. This was facilitated by avoiding conventional formalin fixation, which results in serious loss of the water-soluble hyaluronan. Peaks of hyaluronan deposition were observed in the stromal compartment during the mid-proliferative (days 5-10) and the mid-secretory phase (days 19-23) of the cycle. In physiological terms, the first phase corresponds to the time of rapid cellular proliferation of undifferentiated cells, whereas the second phase coincides with the time when the implantation of the conceptus would be initiated in a fertile cycle. By the time menstruation starts, very little hyaluronan remains in the stroma. In contrast with the stromal staining, hyaluronan deposition around blood vessels was constant throughout the cycle. The dramatic changes in hyaluronan deposition and their correlation with the cyclic growth and remodeling in the human endometrium suggest a major role for hyaluronan in the physiology of this tissue.