Clinical outcomes of transfer of frozen and thawed single blastocysts derived from nonpronuclear and monopronuclear zygotes.

PURPOSE: In assisted reproductive technology, normal zygotes are bipronuclear (2PN) during fertilization confirmation; however, sometimes, nonpronuclear zygotes (0PN) and monopronuclear zygotes (1PN) are found during routine observations. METHODS: To elucidate the clinical usefulness of in vitro-fertilized embryos, we investigated the rates of clinical pregnancy, live birth, miscarriage, and congenital abnormality after transfer of frozen-thawed 1PN- and 0PN-derived single blastocysts at Denentoshi Ladies Clinic, Kanagawa, Japan. RESULTS: The rates of pregnancy and live birth for 1PN-derived blastocysts obtained by conventional in vitro fertilization were 37.5% and 27.1%, respectively, which was not significantly different from those for 2PN-derived blastocysts; however, the rates for 0PN-derived blastocysts were significantly lower. The pregnancy and live birth rates for 0PN-derived embryos obtained by intracytoplasmic sperm injection (ICSI) were 45.7% and 34.8%, respectively, which was not significantly different from those for 2PN-derived blastocysts; however, the rates for 1PN-derived blastocysts were significantly lower (4.0% for both) than those for 2PN- and 0PN-derived blastocysts. No congenital abnormalities were found in infants resulting from transfer of 0PN- or 1PN-derived blastocysts. CONCLUSIONS: Both 1PN- and 0PN-derived blastocysts can be used for embryo transfer; however, care should be taken in making decisions about 1PN-derived blastocysts, especially if they are obtained by ICSI.

Transcytosis of Human immunodeficiency virus 1 across the placenta is enhanced by treatment with tumour necrosis factor alpha.

The human placenta is relatively resistant to Human immunodeficiency virus 1 (HIV-1), but obstetric complications associated with inflammatory processes, including chorioamnionitis and spontaneous preterm delivery, are associated with increased rates of vertical transmission. It was hypothesized that the pro-inflammatory mediator tumour necrosis factor alpha (TNF-alpha), which promotes HIV-1 transmission across endothelial membranes, increases HIV-1 transmission across the placenta. Flow cytometry and immunostaining studies were performed, which demonstrated that the HIV-1 receptors CD4, CCR5 and CXCR4 were not expressed by villous trophoblast cells. Consequently, primary villous trophoblast cells were not infected with cell-free HIV-1 isolates, as measured by in situ PCR and quantitative PCR, but villous trophoblast cells were infected by HIV-1-infected peripheral blood mononuclear cells (PBMC). HIV-1 from infected PBMC was rapidly transported across confluent transformed trophoblast cell monolayers by transcytosis, and TNF-alpha significantly upregulated transcytosis of HIV-1 across the trophoblast layer without disrupting cell viability or confluency. Inhibitors of TNF-alpha (antibodies against TNF-alpha and TNF-alpha receptors) and an anti-inflammatory drug (tenidap) significantly reduced transcytosis rates. It was concluded that the villous trophoblast is resistant to infection by cell-free HIV-1 but susceptible to transcytosis of HIV-1 from infected PBMC, and inflammatory mediators such as TNF-alpha may play a critical role in promoting maternal-fetal transmission of HIV-1.

Haem oxygenase augments porcine granulosa cell apoptosis in vitro.

Haem oxygenases produce carbon monoxide, which, like nitric oxide, is a gaseous messenger molecule that is one of several important survival factors in ovarian follicles. However, little is known about the expression and possible functions of these enzymes in granulosa cells. The purpose of this study was to investigate the expression and possible role of haem oxygenases in porcine granulosa cells (PGCs). We obtained frozen sections of porcine ovaries and PGCs from ovarian follicles of various sizes by needle aspiration, and examined the expression of haem oxygenase-1 (HO-1; inducible type) and HO-2 (constitutive type) in PGCs by immunohistochemistry, RT-PCR, western blotting and flow cytometry. Both types of haem oxygenase were identified in PGCs throughout follicular development, but HO-1 was expressed primarily in granulosa cells in atretic follicles. We also investigated the effect of haem oxygenases on apoptosis of granulosa cells (flow cytometry to detect subdiploid DNA fluorescence) and on expression of Fas ligand (quantitative analysis of western blotting and flow cytometry). In tightly bound PGCs, the mean proportion of apoptotic cells treated with 1 microM haemin (a haem oxygenase substrate) was approximately 1.7-fold greater than that in untreated controls, and zinc protoporphyrin IX (ZnPP IX; a haem oxygenase inhibitor) completely inhibited the increase in apoptosis induced by haemin in 24-h culture. Conversely, in weakly associated PGCs, the proportion of apoptotic cells was not altered by haemin. The quantity of Fas ligand protein was increased in a dose-dependent manner in tightly bound PGCs treated with haemin compared with controls, and the haemin-induced increase in Fas ligand protein was inhibited by ZnPP IX. Thus we identified inducible HO-1 and constitutive HO-2 in PGCs throughout follicular development, and we conclude that products of reactions catalysed by haem oxygenases are likely to be important autocrine/paracrine factors that regulate apoptosis in PGCs.

Thrombin receptors and protease-activated receptor-2 in human placentation: receptor activation mediates extravillous trophoblast invasion in vitro.

Proteolysis of the thrombin receptor, protease activated receptor-1 (PAR1), may enhance normal and pathological cellular invasion, and indirect evidence suggests that activation of PAR1 expressed by invasive extravillous trophoblasts (EVTs) influences human placentation. Here we describe PAR1, PAR2, and PAR3 protein distribution in the developing human placenta and implicate PAR1 and PAR2 activation in functions central to EVT invasion. PAR1, PAR2, and PAR3 are expressed in cultured 8- to 13-week-old EVTs, and in situ in 18- to 20-week-old placental syncytiotrophoblasts and invasive trophoblasts. Thrombin, but not the PAR2 agonist peptide SLIGKV, inhibited proliferation in cultured EVTs, although both agonists stimulated phosphoinositide hydrolysis and EVT invasion through Matrigel barriers. Thrombin-induced phosphoinositide hydrolysis was completely inhibited and the thrombin effect on proliferation was prevented when PAR1 cleavage was first blocked with specific monoclonal antibodies, indicating that PAR1 is the predominant thrombin receptor on EVTs. Together these results support a role for PAR1, and potentially PAR2 and PAR3 in the invasive phase of human placentation.

Placental extravillous cytotrophoblasts persistently express class I major histocompatibility complex molecules after human cytomegalovirus infection.

Human cytomegalovirus (HCMV) downregulates the class I major histocompatibility complexes (MHCs), HLA-A and -B, in infected fibroblasts to escape from antigen-specific cytotoxic T lymphocytes. The HCMV genes responsible for the downregulation of MHCs are US2, US3, US6, and US11, which encode type I membrane proteins working at the endoplasmic reticulum (ER). However, it is largely unknown whether HCMV downregulates the class I MHC molecules in placental extravillous cytotrophoblasts (EVT), which express HLA-C, -E, and -G to protect a semiallogenic fetus from maternal natural killer (NK) cells at the fetomaternal interface. Here, we report that differentiated EVT prepared from human first-trimester chorionic villi persistently express class I MHC molecules upon HCMV infection. When these US proteins were expressed in uninfected EVT, they were localized at the ER in the entire cytoplasm. However, subsequent HCMV infection resulted in dissociation of these US proteins from the ER, which relocated toward the cell membrane. In fibroblasts, these US proteins were localized at the ER before and after HCMV infection. These results suggest that the US gene products are not integrated into ER of HCMV-infected EVT and fail to downregulate class I MHC molecules.

Syncytiotrophoblast is a barrier to maternal-fetal transmission of herpes simplex virus.

Herpes simplex virus (HSV)-1 has been discovered in placental tissue from spontaneous miscarriages, but reports of transplacental transmission and fetal infection are extremely rare. Previously, we demonstrated that the villous syncytiotrophoblast, which forms a continuous layer between the maternal and fetal circulation, is resistant to HSV entry. Here, we tested our hypothesis that the villous syncytiotrophoblast prevents transplacental transmission of HSV secondary to decreased expression of HSV entry mediators (HveA, HveB, and HveC). In addition, we investigated the ability of HSV to infect extravillous trophoblast cells, which mediate placental attachment to the uterine wall, and the expression of HSV receptors in these cells. We performed fluorescence-activated cell sorting (FACS) analyses and immunostaining to demonstrate that HveA, HveB, and HveC were not expressed in third-trimester villous trophoblast cells. Consequently, villous explants obtained from third-trimester placentas were resistant to infection by a recombinant HSV-1 vector, HSV-1 KOS, but approximately 20% of mesenchymal cells within the villous core were infected when villous explants were pretreated with trypsin to disrupt the villous trophoblast layer. Conversely, FACS analysis and immunostaining demonstrated that extravillous trophoblast cells expressed HveA, HveB, and HveC, and these cells were efficiently infected by HSV vectors. Infection of extravillous trophoblast cells by HSV-1 was not reduced when the cells were pretreated with an antibody against HveA but was partially reduced when the cells were pretreated with antibodies directed against HveB and HveC. Thus, the decreased expression of herpesvirus entry mediators in villous syncytiotrophoblast prevents placental villous infection, thereby limiting maternal-fetal transmission of HSV.

Accumulation of CD16+ cells with secretion of Ksp37 in decidua at the end of pregnancy.

PROBLEM: Maternal cellular immunity is thought to be in a state of tolerance during pregnancy, but the precise mechanism of immunomodulation is not yet known. We investigated a novel serum protein, killer-specific secretory protein of 37 kDa (Ksp37), produced by cytotoxic lymphocytes, during pregnancy. METHOD OF STUDY: The level of Ksp37 was determined by enzyme linked immunosorbent assay (ELISA) in the sera of healthy pregnant women. Intracellular Ksp37 expression in mononuclear cells, isolated from peripheral blood and decidua at parturition, was examined with a flow cytometer. RESULTS: Serum Ksp37 levels significantly increased at late pregnancy, compared with non-pregnant controls and the first trimester of pregnancy. The flow cytometric analysis exhibited that Ksp37 was mainly expressed in CD16+ natural killer (NK) cells in decidua of term placenta. CONCLUSIONS: Serum Ksp37 level was elevated at late gestational period. CD16+ NK cells in decidua seem to be a main maternal source of Ksp37. Innate immunity, with CD16+ NK cells, may play important roles near parturition.

Viral infection of the trophoblast: time to take a serious look at its role in abnormal implantation and placentation?

Fragmentary evidence suggests that trophoblast viral infection may play a role in placental dysfunction, leading to complications including spontaneous miscarriage, preeclampsia, fetal growth restriction and preterm birth. Here, we review the mechanisms underlying differentiation and gestational age-dependent infection of trophoblast cells and the consequences of in vitro infection on trophoblast function. The relationship between trophoblast infection by common viruses and pregnancy outcomes is also analyzed. We conclude that there is sufficient evidence linking placental infection by common viruses, including viruses thought to be non-pathogenic or to have low pathogenicity, to indicate that this effect contributes to poor pregnancy outcome.