Cellular composition and viability of demi- and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro

The cellular composition and viability of intact, IVP embryos were compared with those of demi- and quarter-embryos produced by bisection of IVP morulae and blastocysts. Embryos were produced by established techniques from oocytes harvested from slaughterhouse ovaries. In Experiment 1, morulae at Day 6 or blastocysts at Day 7 were bisected on an inverted microscope using a microsurgical steel blade. Demi-embryos were then cultured without a zona pellucida until Day 8, when they were morphologically assessed for quality (viability). A higher proportion of demi-embryos made from blastocysts than from morulae were classified as viable (381/420, 91% vs 164/267, 61%; P < 0.001). In Experiment 2, only Day 7 blastocysts were bisected, and some of the resulting demi-embryos were bisected a second time 24 h later to produce quarter-embryos. The remaining demi-embryos, the quarter-embryos, and control intact embryos were cultured until Day 9, at which time they were assessed for quality and subjected to immunosurgery and differential staining to count inner cell mass (ICM) and trophectoderm cells. A higher proportion of demi-embryos than quarter-embryos was classified as viable (408/459, 89% vs 223/319, 70%, respectively; P < 0.001). Total cell numbers decreased with successive bisections, but the proportion of surviving cells found in the ICM was significantly (P < 0.05) higher in the best quality demi- and quarter-embryos (35 and 32%, respectively) than in the controls (22%). Transfer of all 12 quarter-embryos derived from 3 blastocysts, in pairs, into 6 recipient heifers resulted in 2 pregnancies, each with a single viable fetus at 90 d of gestation. The fetuses originated from 2 different blastocysts. The results suggest that bisection of intact IVP embryos into demi-embryos and bisection of those into quarter-embryos can increase the number of transferable embryos by as much as 178 and 235%, respectively.     

Production of monozygotic mouse twins from microsurgically bisected morulae

Mouse monozygotic twins were produced by bisection of the compacted morulae and transfer of the pairs of half-embryos after culture in vitro. The compacted morulae (about 16 cells) were microsurgically bisected, using a fine glass needle attached to a micromanipulator, without any supporting micro-instruments, after pretreatment for zona-softening and decompaction. About 80% of the morulae were bisected without visible cell damage. After 20 h in culture, the half-embryos were classified morphologically as eu-blastocysts, pseudo-blastocysts, or trophectodermal vesicles or non-integrated forms. After culture of 131 pairs of bisected morulae, 75 (57.3%) pairs of eu-blastocysts, 20 (15.3%) pairs comprising a eu-blastocyst and pseudo-blastocyst, and 9 (6.9%) pairs of pseudo-blastocysts, were obtained. The pseudo-blastocysts were considered to be derived from half-morulae in which some blastomeres were destroyed or dissociated as a result of micromanipulation. From 30 pairs of eu-blastocysts transferred to 21 recipients, 5 twin fetuses on Day 17 (18 pairs/9 recipients) and 3 twin male young (12 pairs/12 recipients) were obtained. Survival rate of the twin-embryo pairs was 27.8% at autopsy and 25.0% at term. None of the 20 pairs of pseudo-blastocysts transferred to 10 recipients gave rise to normal conceptuses.     

Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

Maintenance of the inner cell mass in human blastocysts from fragmented embryos

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.     

Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae

Pluripotency of isolated rabbit inner cell masses (ICMs) and cultured (3 days) inner cell mass (ICM) cells was tested by injecting these donor cells into day 3.5 blastocysts (experiment 1) or day 3 morulae (experiment 2) to produce chimeric embryos. Injected (n = 107) and noninjected (n = 103) embryos were transferred to the opposite uterine horns of the same recipient females. Chimerism was determined by adenosine deaminase (ADA) isozyme analysis on fetal tissue and by eye pigmentation at midgestation. In experiment 1, 53% and 64%, respectively, of blastocysts injected with ICMs or cultured ICM cells developed to midgestation, compared with 52% and 48% for controls. Of these fetuses, four (31%) and one (6%), respectively, had ADA chimerism. In experiment 2,38% and 62%, respectively, of the morulae injected with ICMs or cultured ICM cells developed to midgestation, compared with 46% and 56% for control morulae. Six (43%) chimeric fetuses from morulae injected with ICMs were detected by ADA analysis, but 12 (86%) chimeric fetuses were detected by eye pigmentation, indicating that eye pigmentation was a more sensitive marker for chimerism than our ADA assay. None of the 14 fetuses recovered after injecting morulae with cultured ICM cells were chimeric with either marker. No chimeras developed from control embryos. These studies demonstrate (1) that pregnancy rates are not compromised by injection of blastocysts or morulae with ICMs or cultured ICM cells, (2) that chimeric rabbit fetuses can be produced by injecting ICMs into either blastocysts or morulae, and (3) that cultured ICM cells can contribute to embryonic development when injected into blastocysts. © 1993 Wiley-Liss, Inc.     

Cytoplasmic projections of trophectoderm distinguish implanting from preimplanting and implantation-delayed mouse blastocytes

A scoring scheme was devised to characterize visually the morphological differentiation of whole-mount, unfixed mouse blastocysts. Embryos were recovered from groups of intact mice (implanting embryos) and mice ovariectomized on Day 3 of pregnancy (implantation-delayed embryos) every 3 h from 18:00 h on Day 4 until 12:00 h on Day 5. Blastocyst differentiation was assessed according to the presence of a zona pellucida, the appearance of the outer margin of trophectoderm cells, the visibility of the blastocoele and the relative size of the inner cell mass. The results obtained indicate that, during this period, implanting and implantation-delayed mouse blastocysts lose the zona as well as exhibit rounded trophectoderm cells, an enlarged inner cell mass and an increasing opacity of the blastocoele. In contrast, the trophectoderm cells of implanting blastocysts only exhibit extensive cytoplasmic projections, probably due to remodelling of the intracellular cytoskeleton. Growth of the inner cell mass appeared to precede the other morphological changes in the majority of blastocysts, and thus might be a prerequisite for further differentiation. The rate of blastocyst differentiation and the survival of embryos were adversely affected by the condition of delayed implantation, induced by ovariectomy. This study suggests that the appearance of cytoplasmic projections from trophectoderm cells is central to the control of blastocyst implantation.     

Integrity of the preimplantation pig blastocyst during expansion and loss of polar trophectoderm (Rauber cells) and the morphology of the embryoblast as an indicator for developmental stage

The embryonic ectoderm of the pig differentiated and became part of the outer barrier of the blastocyst (earlier formed by the trophectoderm alone) before shedding of the overlying polar trophectoderm around Day 10, thus securing the integrity of the rapidly expanding blastocyst. Ferritin, added to the medium of the blastocyst, was taken up rapidly by trophectoderm cells, but did not reach the blastocoele, and consequently no tracer was found within hypoblast cells. Embryonic ectoderm cells did not absorb the macromolecule, before or after loss of the polar trophectoderm. When ferritin was injected into the blastocoele, trophectoderm, hypoblast and embryoblast cells all absorbed the tracer. At Day 11, blastocyst diameter and embryoblast cell number varied widely and were hardly correlated. We suggest that embryoblast development may be a more reliable indicator for the developmental stage of a blastocyst than its diameter, which may merely be an indication of the viability of the trophoblast.     

A quantitative analysis of cell allocation to trophectoderm and inner cell mass in the mouse blastocyst

The allocation of cells to the trophectoderm and inner cell mass (ICM) in the mouse blastocyst has been examined by labelling early morulae (16-cell stage) with the short-term cell lineage marker yellow-green fluorescent latex (FL) microparticles. FL is endocytosed exclusively into the outside polar cell population and remains autonomous to the progeny of these blastomeres. Rhodamine-concanavalin A was used as a contemporary marker for outside cells in FL-labelled control (16-cell stage) and cultured (approximately 32- to 64-cell stage) embryos, immediately prior to the disaggregation and analysis of cell labelling patterns. By this technique, the ratio of outside to inside cell numbers in 16-cell embryos was shown to vary considerably between embryos (mean 10.8:5.2; range 9:7 to 14:2). In cultured embryos, the trophectoderm was derived almost exclusively (over 99% cells) from outside polar 16-cell blastomeres. The origin of the ICM varied between embryos; on average, most cells (75%) were descended from inside nonpolar blastomeres with the remainder derived from the outside polar lineage, presumably by differentiative cleavage. In blastocysts examined by serial sectioning, polar-derived ICM cells were localised mainly in association with trophectoderm and were absent from the ICM core. In nascent blastocysts with exactly 32 cells an inverse relationship was found between the proportion of the ICM descended from the polar lineage and the deduced size of the inside 16-cell population. From these results, it is concluded that interembryonic variation in the outside to inside cell number ratio in 16-cell morulae is compensated by the extent of polar 16-cell allocation to the ICM at the next division, thereby regulating the trophectoderm to ICM cell number ratio in early blastocysts.     

Distribution of hyaluronan in the mouse endometrium during the periimplantation period of pregnancy

The tissue distribution of stromal hyaluronan (HA) in the periimplantation mouse uterus was studied histochemically using a biotin-labelled HA-binding complex from cartilage proteoglycan. HA is present around proliferating stromal cells in both the pregnant and pseudopregnant mouse uterus prior to their differentiation into the decidualized phenotype. Decidualization is accompanied by clearance of HA from the extracellular matrix (ECM). This clearing is part of an intrinsic developmental program of the differentiating deciduum. A specific embryonic signal from the implanting conceptus is not required for this phenomenon to occur, since a similar response could be induced in deciduoma produced by artificial stimulation of a receptive uterus. Clearing of HA from the antimesometrial stroma is consistent with the hypothesis that the HA-negative decidual cell may be involved in restricting the invasion of trophoblast cells during embryo implantation. Retention of HA within angiogenic regions of the decidua basalis implies a functional role for this molecule in placental vascularization.     

Decreased embryonic survival of in-vitro fertilized oocytes in rats is due to retardation of preimplantation development

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2 and allocated to 3 groups. On the evening of Day 0, rats in Groups I and II were allowed to mate. Embryos were collected on Day 4 (Group I, control morulae) or Day 5 (Group II, control blastocysts) and were transferred into the oviduct or uterine horn of Day-4 pregnant recipient rats. On the transfer side of the recipients, the bursa had been peeled from around the ovary to prevent endogenous oocytes from entering the oviduct. For Group III, unmated donors were killed 65-67 h after PMSG injection. Ovulated oocytes recovered from the oviducts were fertilized in vitro and transferred 16-18 h later. Embryos developing from in-vitro fertilized (IVF) oocytes were recovered on Day 5, separated into morulae (Group IIIm) and blastocysts (Group IIIb) and transferred into Day-4 pregnant recipients similar to control embryos. Some embryos from each group were used to determine the mean number of cells/embryo. Embryo recipients were killed on Day 20. After transfer, the development of IVF oocytes was retarded compared to control embryos. IVF morulae contained significantly fewer cells/embryo than did control morulae but were able to implant and grow to fetuses, in proportions similar to controls, if transferred into the oviduct of the recipients. These results suggest that the developmental potential of rat oocytes fertilized in vitro is limited due to asynchrony between the embryo and the uterine environment at the time of implantation, rather than possible defects incurred by the oocyte during the fertilization procedure.     

Reassortment of cells according to position in mouse morulae

Sixteen-cell mouse morulae were disaggregated and blastomeres originally occupying outer or inner positions were separated. Outer, inner, or unsorted populations of blastomeres were labeled with either trinitrobenzene sulphonic acid (TNP) or fluorescein isothiocyanate (FITC) and individual blastomeres aggregated to unlabelled partially decompact eight- to ten-cell morulae. After up to 6 h in culture, the positions of the labelled blastomeres within the aggregates were examined. The combined results demonstrated that between 86 and 92% of outer cells remained on the surface of the aggregate and flattened into extensive polygonal shapes, whereas 76-77% of the inner cells had become engulfed by the host morula cells and retained their initial spherical shape. Using unsorted cells, 33-37% were internalised, which is compatible with the most recent estimates of the presence of six to eight inner cells at the 16-cell stage. The possibility that differential adhesiveness of the outer and inner cells is involved in the allocation of cells to the trophectoderm and inner cell mass of the blastocyst is discussed.     

Development of bovine and porcine embryonic teratomas in athymic mice

Inner cell masses (ICM) and embryonic discs from bovine and porcine blastocysts of various ages were transplanted under the kidney capsule of athymic (nude) mice to evaluate growth of teratocarcinomas containing both differentiated tissues and undifferentiated stem cells. Inner cell masses were isolated immunosurgically from Day 8, Day 9 and Day 10 porcine blastocysts and from Day 8, Day 10 and Day 12 bovine blastocysts. Embryonic discs were mechanically dissected from Day 11 and Day 12 porcine embryos and from Day 14 bovine embryos. Day 6 egg cylinders were dissected from embryos and from hybrid embryos of a cross between BALB/C and an outbred strain of mouse. Two to four ICM, embryonic discs or egg cylinders were transplanted under the kidney capsule of each athymic host. After 8 weeks, graft hosts were killed and their tumors removed, fixed and prepared for histological and immunohistochemical examination. Embryonic teratomas developed at high frequency from murine egg cylinders and from Day 11 and Day 12 porcine and Day 14 bovine embryos. Tumors were observed only infrequently from younger bovine and porcine blastocysts. Murine embryonic tumors were composed of numerous differentiated cell types of ectodermal, mesodermal and endodermal origins, but representation of the three embryonic germ layers was somewhat more restricted in bovine and porcine embryonic tumors. No undifferentiated stem cells were detected in tumors of any of the three species. These results demonstrate that teratomas will develop from bovine and porcine embryos when grafted to an immunocompromised host, but the presence of undifferentiated teratocarcinoma stem cells from these species has yet to be achieved.     

Effects of human versus mouse leukemia inhibitory factor on the in vitro development of bovine embryos

Leukemia inhibitory factor (LIF) is a cytokine that shows conflicting effects on in vitro produced (IVP) bovine embryos. Bovine LIF (bLIF) has been cloned and used in culture, but there is no commercially available bLIF. Thus, researchers use human LIF (hLIF) to supplement the culture medium for bovine embryos because of its greater sequence homology compared to murine LIF (mLIF). We compared the effects of mLIF and hLIF on the development of bovine embryos in culture with the effects described for bLIF. Oocytes were matured and fertilized in vitro and cultured in modified synthetic oviduct fluid with BSA. On Day 6 post-insemination, morulae were cultured for 48h in the presence of: (1) mLIF, 100ngml(-1); (2) hLIF, 100ngml(-1); or (3) no LIF. Reduced blastocyst rates were observed on Day 8 for hLIF at the middle and expanded stages, while mLIF had no effect. In contrast, Day 8 blastocysts showed decreased cell counts both in terms of inner cell mass (ICM) and ICM/total cell proportions in the presence of mLIF, while hLIF had no effect. No changes were seen in trophectoderm (TE) and total cell counts. The increased hatching rates and TE cell counts previously described for bLIF, together with the disparate effects exhibited by hLIF and mLIF during blastocyst formation indicate these compounds are inappropriate to replace bLIF. We recommend that heterospecific LIF should not be used to supplement the culture medium for bovine embryo or embryonic stem cells.     

Effect of human leukemia inhibitory factor on in vitro development of IVF-derived bovine morulae and blastocysts

This study was conducted to investigate whether human leukemia inhibitory factor (hLIF) improves the subsequent development of IVF-derived bovine morulae and blastocysts. To obtain IVF-derived bovine morulae, ova were matured and fertilized in vitro and cultured in 0.5 ml of synthetic oviduct fluid (SOF) medium supplemented with 10% human serum (HS) for 5 d at 39 degrees C under a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2). Morulae and early blastocysts at Day 5 of culture were cultured in 0.5 ml of SOF medium with or without 5000 U/ml recombinant hLIF for 2 or 3 d (2 groups). To investigate the effect of addition of hLIF on the subsequent development of morulae, SOF medium was supplemented with 8 mg/ml BSA instead of HS. To test whether hLIF affects the subsequent development of IVF-derived bovine blastocysts, only good blastocysts that developed from SOF medium with or without hLIF at Days 7 and 8 of culture were frozen by a conventinal slow freezing method and again cultured in SOF medium with or without the addition of hLIF for 3 d after thawing (4 groups). Survival of frozen-thawed bovine embryos was evaluated for re-expansion and hatching of blastocysts during 3 d of culture. There was no significant difference in the developmental rate of Day 5 embryos to blastocysts between those cultured with (47.8%) and without (47.6%) addition of hLIF. However, the addition of hLIF before freezing significantly increased the hatching rate of IVF-derived bovine morulae (P < 0.05), whereas addition of hLIF after thawing did not increase the subsequent development of blastocysts. These results suggest that hLIF added at the Day 5 morula stage may contribute to bovine embryonic development through the hatching process.     

Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

Developmental potential of biopsied mouse blastocysts

The health of a preimplantation embryo can be diagnosed in one or more cells biopsied from the conceptus. Here, we tried to evaluate the impact of biopsy of some trophectoderm cells from hatching mouse blastocysts on their further in vitro implantation and early egg cylinder formation. Of 374 blastocysts evaluated 112 hours after hCG, 34% initiated hatching with a small number of mural, polar, or intermediate trophectoderm cells. Half of 59 embryos that underwent induction of hatching by zona puncturing herniated some cells through this opening. After removal of cells with a glass microneedle from spontaneously hatching blastocysts, viability assessed by vital FDA staining was impaired, as well as the in vitro zona pellucida shedding and implantation. When polar trophectoderm cells were biopsied, a significantly lower number of embryos reached the egg cylinder stage.     

Ability of outside cells from preimplantation mouse embryos to form inner cell mass derivatives

Previous studies have shown that inside cells in the preimplantation mouse embryo do not become committed to the formation of inner cell mass until after blastocyst formation. However, it is not yet clear whether outside cells are also labile late in preimplantation development or whether they become restricted to trophectoderm development at an earlier stage. The present study investigates the potency of outside cells isolated from late morulae just prior to blastocyst formation and shows that some, if not all, outside cells retain the potential to form inner cell mass derivatives in vitro and in vivo. This suggests that trophectoderm cells are not restricted in potential earlier than ICM cells and that all cells of the early embryo may be labile at least until blastulation.     

Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers

Horseradish peroxidase (HRP), together with Fast Green or rhodamine-conjugated dextran (RDX), was used as an intracellular lineage tracer to determine cell fate in the polar trophectoderm of 3.5-day-old mouse embryos. In HRP-injected midstage (approximately 39-cell) and expanded (approximately 65-cell) blastocysts incubated for 24 hr, the central polar trophectoderm cell was displaced from the embryonic pole an average of 20 micron (5% of blastocyst circumference) and 29 micron (6% of blastocyst circumference), respectively. Expanded blastocysts injected with HRP + Fast Green and incubated for 24 hr or with HRP + RDX and incubated for 48 hr showed a displacement of 24 micron (4% of blastocyst circumference) and 88 micron (14% of blastocyst circumference), respectively. Up to 10 HRP-positive trophectoderm cells were observed among embryos incubated for 48 hr, indicating that in those cases, the labeled progenitor cells had divided at least three times. Our observations show that the central polar trophectoderm cell divides in the plane of the trophectoderm in expanded blastocysts and, along with its descendants, is displaced toward the mural trophectoderm. The systematic tandem displacement of labeled cells and their descendants toward the abembryonic pole suggests the presence of a proliferative area at the embryonic pole of the blastocyst. Large shifts in inner cell mass (ICM) position in relation to the trophectoderm do not occur during blastocyst expansion. Furthermore, random movements within the polar trophectoderm population do not account for the replacement of labeled cells by unlabeled polar trophectoderm cells. Rather, we propose the hypothesis that the ICM contributes these replacement cells to the polar trophectoderm during blastocyst expansion.     

Localization of type I interferon in murine trophoblast and decidua during decidual formation.

Mouse trophoblast and decidua were examined by means of immunohistochemistry to define the localization of type I interferon. The decidua were stained for type I interferon at the time of implantation. The strong reaction was first observed in the primary decidual zone on day 5 and subsequently in the secondary decidual zone on day 6. After day 10, the decidua basalis and decidua capsularis showed a strong reaction. At the one-cell stage, embryos were weakly labelled, but a positive reaction was recognized in compacted morulae. Blastocysts on days 3 and 4 were positive in trophoblast and inner cell mass and a strong reaction was observed in the primitive endoderm on day 4. The visceral endoderm on day 5 and the trophoblast on day 6 were positive. After day 10, the trophoblast giant cells, labyrinth, visceral yolk sac and fetal blood cells gave a positive reaction. This study is the first demonstration of type I interferon localization in situ in mouse trophoblast and decidua during decidual formation.