Stage 3 begins when a cavity first appears in the morula and ends after the zona (capsula) pellucida is shed when the embryo makes contact with the endometrial lining of the uterus. Stage 3 embryos are called free blastocysts. They have an estimated postfertilization age in vivo of between four and five days and measure 0.1 to 0.2 mm in diameter.
As there are only two stage 3 embryos in the Carnegie collection not all features of the stage can be seen in the serial sections. Therefore, we have supplemented the serial section databases with figures taken from descriptions of other stage 3 embryos. Because very few in vivo specimens have been described in the literature, most of the figures were obtained from in vitro specimens.
IN VIVO SPECIMENS
The serial sections of two stage 3 embryos from the Carnegie collection are included on this disk. They are embryo # 8794, a 58-cell blastocyst (approx. 96 hrs. or 4 days old), and embryo # 8663, a 107-cell blastocyst (approx. 108 hrs. or 4.5 days old). These embryos were first described by A.T. Hertig et al. in 1954.
IN VITRO SPECIMENS
Light micrographs of whole mount in vitro specimens are mainly from the atlas assembled by Veeck and Zaninovic (2003). Only specimen images which the authors considered normal and best showed developmental progress have been reproduced. Transmission electron micrographs (TEMs) are from Lopata, Kohlman and Kellow (1982), Gualtieri, Santella and Dale (1992), and Sathananthan, Menezes and Gunasheela (2003). Scanning electron micrographs (SEMs) are from Ménézo et al. (1999) and Makabe et al. (2001). Grateful appreciation is expressed to all these authors and their publishers for their generosity in permitting the use of their micrographs in the database.
A time-line for in vitro development shows that stage 3 begins at about 90 hours and by 144 hours a hatched embryo has formed. During this stage the embryo becomes divided into outer trophoblast cells and inner embryoblasts, or inner cell mass, that will develop into the embryo proper. There are several hypotheses as to how the fates of the morula blastomeres are determined and some of these are illustrated here.
In vitro stage 3 embryos go through four characteristic processes: 1) cavitation, 2) collapse/ expansion 3) hatching and 4) discarded cells. Each of these features is described below together with representative illustrations. Most of the figures include a measuring bar the length of which was calculated based on a diameter of an unhatched blastocyst, including the zona pellucida, of 175 µm before fixation.
The initiation of cavitation in the morula heralds the beginning of stage 3. Cavitation occurs in vitro between post insemination days 5 and 7. Cavity formation leads to the differentiation of the blastomeres into outer trophoblast cells and a collection of cells on the inside (embryoblasts) called the inner cell mass. The blastomeres exhibit a polarity that is thought to be regulated by their location within the embryo and the extent of their contact with other cells.
Cavitation results from the accumulation of fluid between the blastomeres. Since the total volume of the subzonal (perivitelline) cavity does not change substantially during cavity formation, initial fluid accumulation appears to be a metabolic product of the blastomeres rather than a transport from the outside.
Cavitation is divided into four phases in the database; 1) beginning, 2) early, 3) advanced and 4) final. Soon after compaction is completed a narrow, linear separation of a few cells signifies the beginning of the blastocystic cavity (Fig. 3). As the space appears the blastomere cell boundaries begin to be distinct again indicating the beginning of decompaction. Fluid-filled areas increase between the blastomeres during the second, early phase of cavitation and decompaction is completed (Fig. 4). The blastomeres once again have distinct cell boundaries but they differ in size and shape but a distinct inner cell mass is not yet apparent. Advanced cavitation occurs when blastocystic fluid accumulates to the degree that early morphological differences are apparent between the rounded cells of the inner cell mass and the elliptical cells of the trophoblast (Fig. 5). In this phase the blastocystic cavity occupies less than 50% of the blastocyst surface area. Final cavitation is achieved when the cavity occupies approximately 50% or more of the surface area and the blastomeres are separated clearly into trophoblast and inner cell mass groups (Fig. 6). The overall size of the blastocyst has not yet increased. Trophoblast cells are linked by dense arrays of gap junctions (Figs. 17-22, 51, 52, and 56).
The site of the inner cell mass determines the embryonic pole of the blastocyst (Fig. 12). The opposite side of the blastocyst becomes the abembryonic pole. Two axes can be distinguished; the polar (anterior-posterior) axis through the polar body and a perpendicular (dorso-ventral) axis at a right angle to the polar axis.
Blastocysts in vitro typically undergo repeated collapse and expansion before they escape from the zona pellucida (Figs. 31, 32). Collapse is rapid, occurring in less than five minutes whereas complete re-expansion requires several hours. The phenomenon has been called blastocyst “breathing” and occurs just before hatching. It is not known whether or not it occurs in vivo .
Hatching is the process by which the expanded blastocyst breaks through and escapes from the zona pellucida. It must occur before implantation into the endometrium is possible. Hatching of healthy blastocysts in vitro usually occurs between post insemination days 7 and 8 and is divided into three phases in the database; 1) initiation (Fig. 38), 2) partial (Fig. 39) and 3) terminal (Fig. 40). The morphological changes occurring during hatching are shown schematically in Figure 36.
Hatching begins with the gradual accumulation of fluid in the blastocystic cavity and expansion of the blastocyst. The fluid accumulation causes increased pressure on both the trophoblast and the zona pellucida. Simultaneously, trophoblast cells proliferate to form a cohesive, single layer. The blastocyst contracts and expands intermittently. Proper expansion of the blastocyst by intake of fluid into the blastocystic cavity causes an increase in internal hydrostatic pressure that stretches the trophoblast epithelium. The blastocyst enlarges and its volume increases two- to three-fold causing the zona pellucida to thin. The blastocyst usually hatches out at the pole opposite the inner cell mass. The hatching locus is indicated by the appearance of a group of large, rounded, plump trophoblast cells called zona-breaker cells.
Usually the trophoblast cells begin to herniate through the zona pellucida first just prior to hatching (Fig. 38 ). Once an opening occurs, the blastocyst begins to protrude through. After about 50% of the blastocyst has hatched out, the remaining process is completed quickly. After the zona pellucida ruptures the eventual size of the opening produced is about one-quarter to one-third of the circumference of the capsule so that blastocyst escape is easy and rapid. During hatching and immediately afterwards, collapse and expansion of the blastocyst is a common phenomenon.
Hatching is complete when the blastocyst is totally outside the zona pellucida (Fig. 41). Once outside, the blastocyst enlarges substantially and the trophoblast cells thin and tight junctions form between them (Figs. 51- 52). After hatching, small fimbriated trophoblast projections appear that exhibit amoeboid movement (Fig. 53). The projections are not considered to be artifacts of in vitro culture since they have been observed also in hatched mammalian blastocysts in vivo. The length of the projections averages 27 µm. They may provide first contact between the blastocyst and the endometrial epithelium since they form in the precise region of the blastocyst where attachment usually occurs.
Examination of in vitro blastocysts with TEM reveals two types of cells that are apparently discarded by the developing embryo, 1) sequestered cells and 2) isolated cells. Sequestered cells (Figs. 54-57) are groups of cells located between the zona pellucida and the trophoblast. These cells are anuclear but contain in their cytoplasm extensive arrays of annulate lamellae, aggregates of dense granular material resembling chromatin, microtubules and free ribosomes. Isolated cells are found mainly in the blastocystic cavity and are rounded in shape and considerably more electron dense than cells of the trophoblast or inner cell mass. In some instances they show evidence of advanced degradation. They contain a nucleus and an extensive reticular nucleolus. Elements of rough endoplasmic reticulum, numerous ribosomes and small, well differentiated mitochondria are present in the cytoplasm.
Additional stage 3 embryos showing their morphological features in vitro are available at: www.sathembryoart.com
Buster, J.E., Bustillo, M., Redi. I.A., Cohen, S.W., Hamilton, M., Simon, J.A., Thorneycroft, I.H., and Marshall, J.R. (1985) Biologic and morphologic development of donated human ova recovered by nonsurgical uterine lavage. Am. J. Obstet. Gynecol. 153:211-217.
Hertig, A.T., Rock, J., Adams, E.C., and Mulligan, W.J. (1954) On the preimplantation stages of the human ovum: A description of four normal and four abnormal specimens ranging from the second to the fifth day of development. Carnegie Instn. Wash. Publ. 603, Contrib. Embryol. 35:199-220.
Lopata, A., Kohlman, D.J., and Kellow, G.N. (1982) The fine structure of human blastocysts developed in culture. Embryonic Development, Part B: Cellular Aspects, pp. 69-85. Alan R. Liss, Inc. New York .