Trophoblast function is altered by decidual factors in gestational-dependant manner.

Inadequate implantation and placentation is associated with miscarriage and placental insufficiency. The decidual environment is thought to regulate trophoblast invasion, however this is poorly defined in humans. We aimed to determine the effect of decidualization on trophoblast function. In vitro decidualized primary human endometrial stromal cells (HESC) significantly enhanced first-trimester extravillous trophoblast (EVT) (6-8-weeks gestation) adhesion, outgrowth/invasion. In EVTs from 10 to 12-weeks gestation this effect was absent (adhesion, invasion) or reversed (outgrowth). HESC conditioned media had no effect on trophoblast MMP9 production/activity. Decidualization regulated EVT function in a gestational-dependent manner. This study highlights the importance of trophoblast-decidual synchrony.

Invasive trophoblast promote stromal fibroblast decidualization via Profilin 1 and ALOX5.

During the establishment of pregnancy, extravillous trophoblast (EVT) must invade into the uterine decidua to facilitate decidual artery remodelling to create the placental blood supply. The local decidual environment is thought to regulate trophoblast invasion, however these interactions are poorly defined in humans. Recent evidence in women suggests impaired decidualization is associated with miscarriage and preeclampsia. Primary human endometrial stromal cells (HESC) and first trimester extravillous trophoblast (EVTs) were used to assess the effect of EVT-secreted factors on HESC decidualization, adhesion, proliferation and migration. We determined the role of profilin (PFN)1, an EVT-secreted factor, on HESC function and identified a downstream target of PFN1. EVT-secreted factors induced HESC decidualization and enhanced decidualized HESC adhesion, proliferation and migration. Recombinant PFN1 enhanced methoxyprogesterone acetate-induced HESC decidualization and proliferation. PFN1 down-regulated the expression of lipoxygenase arachidonate 5-lipoxygenase (ALOX5) in HESC and THP-1 macrophages. ALOX5 localised to decidual cells and CD68+macrophages in 1st trimester decidua. This study demonstrated that EVT secretions, including PFN1, enhanced HESC decidualization and motility. This study has identified a new pathway that facilitates appropriate decidualization during the establishment of pregnancy.

Galectin-7 acts as an adhesion molecule during implantation and increased expression is associated with miscarriage.

INTRODUCTION: Galectins are expressed at the fetal-maternal interface and have multiple roles including during blastocyst implantation. The expression of galectin-7 however has not been investigated in the uterus. We aimed to localise galectin-7 to the endometrium of women with normal fertility and with a history of miscarriage and prospectively determine whether serum levels are altered in women who subsequently miscarry. We also investigated the role of galectin-7 on trophoblast-endometrial epithelial cell adhesion. METHODS: Immunohistochemistry localised galectin-7 to endometrium throughout the menstrual cycle in women (normal fertility or with history of miscarriage) and in first trimester implantation sites. Galectin-7 serum levels were determined by ELISA. We used both endometrial epithelial-trophoblast cell lines and primary cells for cell-cell adhesion experiments. RESULTS: Galectin-7 immunolocalized to endometrial luminal and glandular epithelium in normally fertile women and was upregulated in epithelium and stroma of women with a history of miscarriage. Similarly, galectin-7 serum levels were elevated at 6 weeks gestation in women who subsequently miscarried compared to gestation matched controls. Exogenous galectin-7 reduced endometrial epithelial-trophoblast adhesion in cell-line and primary cell assays. However, when endometrial epithelial cells were isolated from women with endometrial disorders, galectin-7 increased epithelial-trophoblast adhesion. CONCLUSIONS: Galectin-7 is produced by endometrial epithelium and is abnormally elevated in the endometrium of women with a history of miscarriage. Serum levels may be useful as a predictive biomarker of miscarriage. Our data suggests that galectin-7 facilitates adhesion of the embryo to the endometrium and elevated galectin-7 may result in abnormal adhesion.

Preimplantation human blastocysts release factors that differentially alter human endometrial epithelial cell adhesion and gene expression relative to IVF success.

STUDY QUESTION: Do human blastocysts which subsequently implant release factors that regulate endometrial epithelial cell gene expression and adhesion to facilitate endometrial receptivity? SUMMARY ANSWER: Blastocysts which subsequently implanted released factors that altered endometrial epithelial gene expression and facilitated endometrial adhesion while blastocysts that failed to implant did not. WHAT IS KNOWN ALREADY: Human preimplantation blastocysts are thought to interact with the endometrium to facilitate implantation. Very little is known of the mechanisms by which this occurs and to our knowledge there is no information on whether human blastocysts facilitate blastocyst attachment to the endometrium. STUDY DESIGN, SIZE, DURATION: We used blastocyst-conditioned medium (BCM) from blastocysts that implanted (n = 28) and blastocysts that did not implant (n = 28) following IVF. Primary human endometrial epithelial cells (HEECs) (n = 3 experiments) were treated with BCM and the effect on gene expression and adhesion to trophoblast cells determined. We compared the protein production of selected genes in the endometrium of women with normal fertility (n = 40) and infertility (n = 6) during the receptive phase. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used real-time RT-PCR arrays containing 84 genes associated with the epithelial to mesenchymal transition. We validated selected genes by real-time RT-PCR (n = 3) and immunohistochemistry in the human endometrium (n = 46). Adhesion assays were performed using HEECs and a trophoblast cell line (n = 3). MAIN RESULTS AND THE ROLE OF CHANCE: Blastocysts that implanted released factors that differentially altered mRNA levels for six genes (>1.5 fold) compared with blastocysts that did not implant. A cohort of genes was validated at the protein level: SPARC and Jagged1 were down-regulated (P < 0.01), while SNAI2 and TGF-B1 were up-regulated (P < 0.05) by implanted compared with non-implanted BCM. Jagged-1 (P < 0.05) and Snai-2 protein (P < 0.01) showed cyclical changes in the endometrium across the cycle, and Jagged-1 staining differed in women with normal fertility versus infertility (only) (P < 0.01). HEEC adhesion to a trophoblast cell line was increased after treatment with implanted BCM compared with untreated control (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study and it would be beneficial to validate our findings using a physiological model, such as mouse. WIDER IMPLICATIONS OF THE FINDINGS: This new strategy has identified novel pathways that may be important for human preimplantation blastocyst-endometrial interactions and opens the possibility of examining and manipulating specific pathways to improve implantation and pregnancy success. STUDY FUNDING/COMPETING INTEREST: This study was supported by the National Health and Medical Research Council of Australia (Fellowship support #550905, #611827) and project grants by Monash IVF, Australia. There are no conflicts of interest to be declared.

Progesterone concentration in the marsupial Sminthopsis macroura: relationship with the conceptus, uterine glandular regeneration and body weight.

Close examination of hormonal profiles and uterine morphology in the marsupial reproductive cycle highlights significant differences between pregnant and non-pregnant cycles. In the polyovular dasyurid marsupial Sminthopsis macroura, we identified changes associated with gestation by comparing ovarian and plasma progesterone concentrations, uterine weights, uterine epithelial mitoses, body weights and gestation lengths between pregnant and non-pregnant luteal phases. The plasma progesterone profile of S. macroura was biphasic, peaking during unilaminar blastocyst expansion and on the day of implantation. Periods of rapid embryonic development were associated with increasing plasma progesterone concentrations and animal body weight. For the first time in a polyovular marsupial, we identified 1) a correlation between ovarian progesterone concentration and conceptus number during the luteal phase just prior to implantation (total ovarian progesterone), indicating a conceptus influence on progesterone concentration; 2) a pulse of uterine epithelial mitotic activity at the time of implantation and 3) increased mitotic activity in pregnant animals during unilaminar blastocyst formation compared with non-pregnant animals. Gestation length was reduced by up to 15%, due to the loss of, or reduction in, the four-cell arrest and more rapid definitive blastocyst expansion. This is the first time a conceptus influence on gestation length has been identified in a dasyurid. This study provides further evidence for the modification of the luteal phase by pregnancy in S. macroura.

Novel immunocontraceptive targets in mammals: uterine secretions and the conceptus; a marsupial approach.

We report the first immunocontraceptive trial in mammals using a uterine-secreted protein, the marsupial shell coat protein 4 (CP4). The marsupial shell coat, which surrounds the conceptus for 60-80% of gestation, is secreted by the uterine epithelium. Following immunization against glutathione S-transferase (GST)-CP4, the fertility of female common brushtail possums (n=6) was significantly reduced (P=0.000), and this reduction in fertility was positively correlated with the maximum GST-CP4 humoral immune response (P=0.025). Ultrastructural examination of the reproductive tract indicated that the cell-mediated immune response against GST-CP4 targeted the shell coat, the shell-free conceptus and the uterine glandular epithelium, thus preventing normal conceptus development and uterine secretion of shell coat proteins and nutrients. These results show that uterine-secreted proteins are promising immunocontraceptive targets, especially in pest mammal species, e.g. possum, rabbit and horse, that have uterine-secreted additions to embryonic coats, or that have late implantation requiring uterine nutrient provisioning from secretions.