8 Characterization of the Developmental Stages

8

Characterization of the Developmental Stages

Day 3 p.c. Carnegie stage 2b: from 4 to about 16 cells;

diameter 0.1 to 0.2 mm.

Day 4 p.c. Carnegie stage 3: free blastocyst, from 16 to about 64 cells; diameter about 0.2 mm.

Day 5 to early day 6 p.c. Carnegie stage 4: blastocyst attached to the endometrium, from about 128 to about 256 cells; diameter 0.2 to 0.3 mm.

Late day 6 to early day 8 p.c. Carnegie stage 5a: implan- tation, prelacunar stage of the trophoblast; the fl attened blastocyst measures about 0.3 ¥ 0.3 ¥ 0.15 mm. The blas- tocyst is partially implanted. The implanted part of the blastocyst wall is considerably thickened, largely consist- ing of solid syncytiotrophoblast. The still not implanted, thin part of the blastocyst wall consists of a single layer of cytotrophoblast. The embryonic disk measures about 0.1 mm in diameter.

Late day 8 to day 12 p.c. Lacunar or trabecular stage.

Late day 8 to day 9 p.c. Carnegie stage 5b: diameter of chorionic sac 0.5 ¥ 0.5 ¥ 0.3 mm; embryonic disk about 0.1 mm. The syncytiotrophoblast at the implantation pole exhibits vacuoles as forerunners of the lacunar system.

Day 10 to day 12 p.c. Carnegie stage 5c: diameter of chorionic sac 0.9 ¥ 0.9 ¥ 0.6 mm. The vacuoles in the syn- cytiotrophoblast fuse to form the lacunar system; fi rst lacunae at the antiimplantation pole. First contact of lacunar system with eroded endometrial capillaries. Some maternal erythrocytes may be observed in the lacunae.

Around day 11, implantation is complete; the defect in the endometrial epithelium is closed by a blood coagu- lum and is covered by epithelium on day 12. At the implantation site, the endometrium measures 5 mm in thickness; fi rst signs of decidualization.

Day 13 to day 14 p.c. Carnegie stage 6, villous stage (fi rst free primary villi).

174

This chapter is a synopsis and presents brief descriptions of the average data of placenta and membranes through- out the single stages of placental development. Embryo- logic data concerning embryo and fetus are given only insofar as they are of importance for the defi nition of the stage. It is not the intention of this chapter to compare data of various sources on a scientifi c level but rather to present data that are directly applicable to the pathologic and histologic examination of human material. For this purpose, all data have been extrapolated and were stan- dardized where necessary.

The data are based on the following publications:

embryonic staging according to O’Rahilly (1973) and Boyd and Hamilton (1970): crown–rump length (CRL), embryonic and fetal weight, mean diameter of the chori- onic sac, placental diameter and thickness, placental weight according to Boyd and Hamilton (1970), O’Rahilly (1973), and Kaufmann (1981); placental and uterine thickness in vivo following Johannigmann et al. (1972);

length of umbilical cord according to Winckel (1893);

villous surfaces, villous volumes, and villous diameters following Hörmann (1951), Knopp (1960), Clavero-Nuñez and Botella-Llusia (1961, 1963), Aherne and Dunnill (1966), Kaufmann (1981), Schiemer (1981), and Gloede (1984); and mean trophoblastic thickness, distribution of villous cytotrophoblast, mean maternofetal diffusion distance according to Kaufmann (1972), Kaufmann and Stegner (1972), Gloede (1984), and Kaufmann (1981). For further details see the summarizing tables in Chapter 28.

Stages of Development

Day 1 p.c. (p.c. = postcoitus) Carnegie stage 1: one fer- tilized cell; diameter 0.1 mm.

Day 2 p.c. Carnegie stage 2a: from 2 to 4 cells; diameter

0.1 to 0.2 mm.

Day 13 p.c. The nearly round chorionic sac has a diam- eter of 1.2 to 1.5 mm; length of embryonic disk is 0.2 mm.

Day 14 p.c. Diameter of chorionic sac 1.6 to 2.1 mm;

length of embryonic disk 0.2 to 0.4 mm. First appearance of primitive streak and of yolk sac.

With the expansion of the lacunar system, the syncy- tiotrophoblast becomes reduced to radially oriented tro- phoblastic trabeculae, the forerunners of the stem villi.

After invasion of cytotrophoblast into the trabeculae, free trophoblastic outgrowths into the lacunae, the “free primary villi”, are formed. The trabeculae are now called villous stems. By defi nition, from this date onward, the lacunae are transformed into the intervillous space. Cyto- trophoblast from the former trabeculae penetrates the trophoblastic shell and invades the endometrium.

Days 15 to 18 p.c. Villous stage (secondary villi).

Days 15 to 16 p.c. Carnegie stage 7: diameter of cho- rionic sac about 5 mm; length of embryonic disk less than 0.9 mm; appearance of notochordal process and primitive node (Hensen).

Day 17 to 18 p.c. Carnegie stage 8: diameter of embry- onic sac less than 8 mm; length of chorionic disk less than 1.3 mm. On the germinal disk, the notochordal and neur- enteric canals, and primitive pit can be discerned.

Starting at the implantation pole and continuing all around the circumference to the antiimplantation pole, mesenchyme (derived from the extraembryonic meso- derm in the chorionic cavity) invades the villi, transform- ing them into secondary villi. The basal feet of the villous stems, connecting the latter with the trophoblastic shell, as well as some villous tips remain free of mesenchyme and thus persist in the primary villous stage (forerunners of the cell columns and cell islands).

Day 19 to 23 p.c. This is the beginning of the 2nd month postmenstruation (p.m.), villous stage (early tertiary villi).

Day 19 to 21 p.c. Carnegie stage 9: diameter of cho- rionic sac less than 12 mm; length of embryonic disk equals the crown–rump length of the embryo, 1.5 to 2.5 mm, 1 to 3 somites. Neural folds appear; fi rst cardiac contractions.

Day 22 to 23 p.c. Carnegie stage 10: diameter of cho- rionic sac less than 15 mm; crown–rump length 2.0 to 3.5 mm; 4 to 12 somites. Neural folds start to fuse; two visceral arches.

The villous mesenchyme is characterized by the appear- ance of the fi rst fetal capillaries (formation of fi rst tertiary villi). The villous diameters are largely homogeneous, presenting two different-sized groups of villi. The larger villous stems and their branches exhibit diameter of 120 to 250 mm. Histologically, the stroma of both is mesenchymal in nature. Along their surfaces, one fi nds numerous small (diameters 30–60 mm) trophoblastic and villous sprouts.

Days 23 to 29 p.c. Early tertiary villus stage.

Days 23 to 26 p.c. Carnegie stage 11: diameter of the chorionic sac less than 18 mm; crown–rump length 2.5 to 4.5 mm; 13 to 20 somites; closure of the rostral neuropore;

optic vesicles identifi able.

Days 26 to 29 p.c. Carnegie stage 12: diameter of chorionic sac less than 21 mm; crown–rump length 3 to 5 mm; 21 to 29 somites; closure of the caudal neuropore;

three visceral arches; upper limb buds appear.

The length of villous stems between chorionic plate and trophoblastic shell varies from 1 mm (antiimplanta- tion pole) to 2 mm (implantation pole). The central two thirds are supplied with mesenchyme and capillaries (Fig.

8.1); the peripheral one third remains in the primary villous stage (cell columns). The villous calibers are similar to those described for the previous stage. The amount of trophoblastic and villous sprouts is reduced.

Most villi contain loose mesenchyme together with cen- trally positioned fetal capillaries (mesenchymal villi).

Peripherally, they continue via villous sprouts (with unvascularized mesenchymal core) into massive tropho- blastic sprouts. In the villous stems, vessels of larger caliber acquire the fi rst signs of a surrounding adventitia (beginning of formation of typical stem villi characterized by fi brous stroma). The villous trophoblastic surface is composed of an outer syncytiotrophoblast and complete inner layer of cytotrophoblast. Together they measure 20 to 30 mm in thickness.

The chorionic plate, consisting of fetal mesenchyme, cytotrophoblast, and syncytiotrophoblast, still lacks fi bri- noid. The trophoblastic shell is transformed into the basal plate by intense mixing of decidual and trophoblastic cells. Secretory activities or tissue necrosis of both cell types causes the appearance of the fi rst foci of Nitabuch fi brinoid. The superfi cial syncytiotrophoblastic layer of the basal plate, bordering the intervillous space, becomes locally replaced by Rohr fi brinoid.

Days 29 to 42 p.c. Late 2nd month p.m.

Days 29 to 32 p.c. Carnegie stage 13: diameter of chorionic sac less than 25 mm; crown–rump length 4 to 6 mm; 30 + somites; four limb buds and optic vesicle.

Days 32 to 35 p.c. Carnegie stage 14: diameter of chorionic sac less than 28 mm; crown–rump length 5 to 8 mm; fi rst appearance of lens pit and optic cup.

Days 35 to 37 p.c. Carnegie stage 15: diameter of chorionic sac less than 31 mm; crown–rump length 7 to 10 mm; closure of lens vesicle; clear evidence of cerebral vesicles; hand plates.

Days 37 to 42 p.c. Carnegie stage 16: diameter of chorionic sac less than 34 mm; crown–rump length 8 to 12 mm; embryonic weight about 1.1 g; retinal pigment visible; foot plates.

The net weight of the chorionic sac in stage 16 is about

6 to 10 g; thickness of the chorion at the implantation pole

Figure 8.2. Placental villi of the 8th week p.m. All villi are vascularized. As can be seen from the diffuse stromal structure, the villi still belong to the mesenchymal type. Paraffi n section. ¥125. (From Kaufmann, 1981, with permission.)

(p.m.). Note the thick trophoblastic covering, consisting of com- plete layers of cytotrophoblast and syncytiotrophoblast. Fetal capillaries are poorly developed or, in some places, are still

of a cell island can be seen attached to the villous surface. Paraffi n

section. ¥125.

is about 6 mm and at the antiimplantation pole about 3 mm. The uterine lumen is still open, and parietal and capsular decidua are not yet in contact.

The range of villous calibers changes slightly from the previous stage (Fig. 8.2). The largest stems reach diameters of less than 400 mm. A variety of medium- sized mesenchymal villi is found between the stem villi that measure about 200 mm in diameter and the small sprouts. The mean villous caliber is about 200 mm. The total placental villous surface is about 0.08 m

. The con- nective tissue layer of the chorionic plate is completely fi brosed, the fi brous tissue partly extending in the initial parts of the villous stems. The overwhelming share of the villous stroma is still mesenchymal in nature. The villous cytotrophoblastic layer is incomplete; 85% of the villous surface is double layered (cytotrophoblast plus syncytium). The thickness of the villous trophoblast varies between 10 and 30 mm (mean, 15.4 mm). Near the end of this period, most of the mesenchymal villi show increased numbers of macrophages, as well as the fi rst signs of reticular transformation of their stroma toward immature intermediate villi. Only 2.7% of the villous volume is occupied by fetal vascular lumens.

The mean maternofetal diffusion distance is more than 50 mm.

The villous stems are nearly completely occupied by connective tissue; basal segments, persisting in the primary villous stage, are the exception. Those segments now show the typical appearance of cell columns.

Short portions of villous side branches, persisting in the primary villus stage and that are positioned some- where between chorionic and basal plate, may increase in size by continuous cell proliferation with subsequent fi brinoid degeneration; they thus establish the fi rst cell islands.

Third month p.m. 9th to 12th weeks p.m.; days 43 to 70 p.c.

Days 43 to 44 p.c. Carnegie stage 17: maximum diam- eter of chorionic sac 38 mm; crown–rump length 10 to 14 mm; fi nger rays.

Days 44 to 48 p.c. Carnegie stage 18: maximum diameter of chorionic sac 42 mm; crown–rump length 12 to 16 mm. Elbow region, toe rays, nipples, and eyelids appear.

Days 48 to 51 p.c. Carnegie stage 19: maximum diam- eter of chorionic sac 44 mm; crown–rump length 14 to 18 mm.

Days 51 to 53 p.c. Carnegie stage 20: maximum diam- eter of chorionic sac 47 mm; crown–rump length 17 to 22 mm. Upper limbs at the elbow region; fi rst signs of fi nger separation.

Days 53 to 54 p.c. Carnegie stage 21: maximum diam- eter of the oval chorionic sac 51 mm; crown–rump length 20 to 24 mm.

Day 54 to 56 p.c. Carnegie stage 22: maximum diam- eter of the oval chorionic sac 58 mm; crown–rump length 23 to 28 mm.

Days 56 to 60 p.c. Carnegie stage 23: maximum diam- eter of the oval chorionic sac 63 mm; crown–rump length 26 to 31 mm.

Days 61 to 70 p.c. Maximum diameter of the oval to irregular chorionic sac 68 mm; crown–rump length 26 to 31 mm.

Days 61 to 70 p.c. Maximum diameter of the oval to irregular chorionic sac 68 mm; crown–rump length 30 to 40 mm.

The embryonic weight increases throughout the 3rd month from 2 g to 17 g and the net weight of the chorionic sac from 10 g to 30 g. The chorionic sac is covered by villi over its surface; it is not yet subdivided into smooth chorion and placenta.

All villi are vascularized. Around the antiimplantation pole, the increased degenerative changes of villi and fi bri- noid deposition in the intervillous space indicate that the formation of the smooth chorion will commence soon.

Parietal and capsular decidua may come into contact locally, but they remain unfused.

The heterogeneity of villous diameters and villous structures increases. Fibrosis of the villous stems slowly extends into the more peripheral parts of the largest villi (diameters less than 500 mm). During the course of the 3rd month, most of the villi measuring between 100 and 400 mm establish the typical reticular appearance of immature intermediate villi (Fig. 8.3), characterized by numerous macrophages (Hofbauer cells). Small villi with diameters less than 100 mm show mesenchymal stroma.

Trophoblastic and villous sprouts are numerous. Total villous surface is about 0.3 m

. The trophoblastic thickness varies from 10 to 20 mm. Eighty percent of the villous surfaces are covered by cytotrophoblast. Fetal vessel lumens occupy about 4% of the villous volume. Some of the larger fetal vessels achieve a thick adventitia, consist- ing of fi brous stroma, which occupies larger parts of the villous stroma. The reticular stroma, as a sign of immatu- rity of the stem villi, is restricted to the superfi cial parts of the stroma positioned under the trophoblast.

In the previous stages fi brinoid was restricted to the cell islands and the basal plate, but now spot-like fi brinoid deposition at some of the villous surfaces can be observed.

Fibrinoid deposition at the intervillous surface of the chorionic plate is still an exception. The amnionic cavity has extended to such a degree that the amnionic meso- derm comes into contact with the connective tissue layer of the chorionic plate in many places.

Fourth month p.m. 13th to 16th week p.m.; 11th to 14th weeks p.c.

The shape of the chorionic sac becomes more and more

irregular because of compression between uterine wall

and fetus. Its maximum diameter increases throughout this period from 68 mm to 80 to 90 mm. The crown–rump length grows from 45 mm to 80 mm, and the fetal weight from 20 g to 70 g. The length of the umbilical cord is between 160 and 200 mm.

The continuous degeneration of placental villi at the antiimplantation pole, which is free of villi from the middle of the 4th month onward, as well as the villous proliferation at the implantation pole, initiates the differ- entiation of the chorionic sac into smooth chorion and placenta. The placental diameter increases from 50 mm to 75 mm at the end of this month and the placental weight from 30 g to 70 g. The maximal placental thickness in the delivered specimens is 10 to 12 mm. Numerous placental septa become visible. The cell columns become more deeply incorporated into the basal plate by fi brinoid deposition in their surrounding. The chorionic plate is in close contact with the amnion over its entire surface, giving it defi nite shape and layering. It consists of amnionic epithelium, amnionic mesoderm, chorionic mesoderm, a cytotrophoblast layer, and superfi cial syncytiotrophoblast.

The distribution of the villous calibers (Fig. 8.4) is similar to that described for the preceding month. The inhomogeneous mixture of villi is composed of stem villi

with diameters of 300 to 500 mm, the vascular adventitia of which occupies at least 75% of the villous stroma, and of immature intermediate villi with diameters of 100 to about 300 mm. Mesenchymal villi and sprouts, 40 to 80 mm in diameter, are numerous, but because of their size they occupy only a small proportion of the total villous volume.

Because the immature intermediate villi have the most characteristic reticular stroma at this stage and comprise the highest proportion of villi, one observes more villous macrophages (Hofbauer cells) than at all other stages of placental development. The total villous surface was mea- sured to be 0.5 to 0.6 m

. The share of fetal vessel lumens is increased to about 6%. Some of the capillaries establish contact with the villous trophoblast. In such places the syncytial nuclei are moved aside, resulting in the fi rst epithelial plates; therefore, the trophoblastic thickness varies between 2 and 12 mm (mean, 9.6 mm). As during the previous month, one observes cytotrophoblast on about 80% of the villous surface. Fibrinoid deposition becomes a usual fi nding on the villous surfaces.

Fifth month p.m. 17th to 20th week p.m.; 15 to 18th week p.c.

Because of the geometrically irregular outer shape of the fetus, the diameter of the chorionic sac cannot be Figure 8.3. Placental villi of the 12th week p.m. The larger villi

have achieved the reticular stroma of typical immature, inter- mediate villi. The smaller villi are mesenchymal in structure. The

fi rst small fetal arteries and veins can be seen. Paraffi n section.

¥125.

Figure 8.4. Placental villi of the 15th week p.m. The larger imma- ture intermediate villi exhibit the fi rst signs of central stromal fi brosis, originating from the larger fetal vessels, thus establishing the fi rst stem villi (SV). Several typical immature intermediate

villi (IV) and mesenchymal villi (MV) can be seen. As is typical for mesenchymal villi of the second and third trimester, they are associated with degenerating villi being more or less transformed into intravillous fi brinoid. Paraffi n section. ¥125.

estimated from this period onward. Over the course of the 5th month the crown–rump length increases from 80 mm to 130 mm and the fetal weight from 70 g to 290 g.

The placenta is clearly separated from the smooth chorion.

The placental diameter is between 75 mm and 100 mm, and the placental weight increases from 70 g to 120 g. The maximum placental thickness after delivery is about 12 to 15 mm; measured by ultrasonography and in situ, including the uterine wall, it is approximately 28 mm. The length of the umbilical cord varies between 200 and 315 mm.

The structure of the stem villi is nearly the same as in the previous month; however, their number is consider- ably increased throughout this month (Fig. 8.5). During the 20th week p.m., most of the large caliber villi (those exceeding 300 mm) have achieved the fi brous stroma of stem villi. The number of slender, long mesenchymal villi with diameters around 80 to 100 mm increases. The mean diameter of the remaining immature intermediate villi is slightly reduced to values around 150 mm. The mean villous diameter is 108 mm. The total villous surface is about 1.5 m

. Because the amount of villous cytotropho- blast is reduced to about 60% of the villous surface, the

extent of thin trophoblastic areas from 1 to 2 mm in thick- ness increases. Continuous development of fetal capillar- ies causes the reduction of mean maternofetal diffusion distance to about 22 mm.

Septa and cell islands that originally consisted mainly of accumulations of cells now grow considerably by apposition of fi brinoid. In their centers, cysts are often found.

Sixth month p.m. 21st to 24th week p.m.; 19th to 22nd week p.c.

The fetus grows from 130 mm to 180 mm in crown–

rump length. Its weight increases from 290 g to 600 g. The placental diameter is between 100 and 125 mm, and the placental weight increases form 120 g to 190 g. Placental thickness after delivery is 15 to 18 mm, and ultrasono- graphic measurements in situ, including the uterine wall, indicate a thickness of about 34 mm. The mean length of the cord is between 315 and 360 mm.

The histologic features change considerably. Most of

the immature intermediate villi become transformed into

stem villi of large caliber. More of the stem villi measure

about 200 mm in thickness, some achieving diameters of

more than 1000 mm. Their fi brous stroma still exhibits a small superfi cial rim of reticular connective tissue, indicating their immaturity (Fig. 8.6). Different from all earlier stages, increasing numbers of newly formed inter- mediate villi exhibit small calibers (only 100–150 mm).

Some of these villi are reticular in stromal structure, as are their parent villi, whereas others are slender, mature intermediate villi with poorly vascularized and poorly fi brosed, nonreticular stroma (80–120 mm) (Fig. 8.7). At their surfaces, the fi rst richly capillarized terminal villi are formed. They are diffi cult to identify in the large group of smallest villi, measuring 50 to 80 mm, because the other members of this group, the small mesenchymal villi and villous sprouts, exhibit structural features similar to those of the terminal villi in paraffi n sections. The total villous surface amounts to 2.8 m

. The mean trophoblastic thick- ness is reduced to 7.4 mm, and the mean maternofetal diffusion distance is about 22 mm.

Seventh month p.m. 25th to 28th week p.m.; 23rd to 26th week p.c.

When compared to the 6th month, there are only quan- titative changes. The crown–rump length increases from 180 mm to 230 mm and the fetal weight from 600 g to 1050 g.

The placental diameter is 125 to 150 mm, and the placental weight is increased to 190 to 260 g. The thickness of the

delivered placenta is 18 to 20 mm; by ultrasonography, including the uterine wall, it is 38 mm. The mean length of the umbilical cord increases from 360 to 410 mm.

The total villous surface of the placenta exceeds 4 m

. The distribution of structure and caliber of the villi are similar to what was seen during the 6th month. Only 45%

of the villous surface is covered by cytotrophoblast. The trophoblastic thickness varies between 0.5 and 8 mm (mean, 6.9 mm). The number of immature intermediate villi decreases in favor of stem villi and mature intermedi- ate and terminal villi. The lumens of fetal vessel amount to 9.1% of the villous volume.

Cell columns are surrounded by increasing amounts of fi brinoid and become deeply invaginated in the basal plate. The syncytiotrophoblastic covering of the chorionic plate begins to degenerate. It becomes replaced by an initially thin layer of fi brinoid that grows in thickness throughout the following weeks and forms the Langhans’

stria.

Eighth month p.m. 29th to 32nd week p.m.; 27th to 30th week p.c.

The crown–rump length is 230 to 280 mm, fetal weight

is 1050 to 1600 g. The placental diameter normally varies

between 150 and 170 mm; the placental weight increases

from 260 g to 320 g. The placental thickness after delivery

Figure 8.5. Placental villi of the 18th week p.m. This picture is comparable to that of the preceding stage. Formation of stem villi

with stromal fi brosis is somewhat more expressed. Paraffi n section. ¥125.

Figure 8.6. Placental villi of the 21st week p.m. The stroma of the stem villi (SV) is largely fi brous. Only a discontinuous thin superfi cial rim of reticular connective tissue is reminiscent

of their derivation from immature intermediate villi. Paraffi n section. ¥125.

Figure 8.7. Placental villi of the 24th week p.m. Compared to the preceding stages, the variability in villous shapes and diam- eters is sharply increasing. The population of small slender villi

(X’s at lower left, lower right, and at center), originally referred

to as mesenchymal villi, has achieved structural characteristics

of mature intermediate villi. Paraffi n section. ¥125.

is about 20 to 22 mm; measured by ultrasonography in situ and including the uterine wall, it is 43 mm. The umbilical cord has a mean length between 410 and 455 mm.

Steeply increasing numbers of mature intermediate villi and terminal villi, both of which exhibit calibers of 40 to 100 mm, are the reason for considerably increased numbers of villous cross sections per square millimeter of histologic sections (Fig. 8.8). The total villous surface is increased to a mean of about 7 m

. In addition to the villi of small caliber that compose most of the villi, there are mainly stem villi of large caliber. Intermediate cali- bers of 100 to 200 mm are rare, causing a typical gap in the range of calibers. This gap may be evident as early as the second half of the 7th month. The few existing villi of this particular caliber, mostly immature intermediate villi, are grouped together in the centers of the villous trees.

Villous cytotrophoblast is reduced to about 35% of the villous surface. As a result of the beginning sinusoidal dilatation of the fetal capillaries in the newly formed terminal villi, the amount of vasculosyncytial membranes (epithelial plates) is increased, and the mean trophoblas- tic thickness reduced to about 6 mm.

Ninth month p.m. 33rd to 36th week p.m.; 31st to 34th week p.c.

The crown–rump length is 280 to 330 mm, fetal weight is 1600 to 2400 g, placental diameter is 170 to 200 mm, and the placental weight is 320 to 400 g. The placental thick- ness postpartum is 22 to 24 mm; by ultrasonography in situ, including the uterine wall, it is 45 mm. The mean cord length increases from 455 to 495 mm.

Histologically, the developmental processes described for the preceding month become even more prominent:

The total villous surface of the placenta is increased to about 10 m

. Capillary growth and continuous sinusoi- dal dilatation cause the mean maternofetal diffusion distance to decrease to less than 12 mm and the mean tro- phoblastic thickness to about 5 mm. Cytotrophoblast is found on only 25% of the villous surfaces. The largest stem villi reach 500 to 1500 mm in diameter. The small stromal rim, consisting of reticular connective tissue and indicating their immaturity, has normally disappeared to the credit of fi brous stroma that completely occupies the villous core (Fig. 8.9). The originally reticular superfi - cial zone of the stroma shows an increased number of connective tissue cells for a few weeks, compared to

Figure 8.8. Placental villi of the 29th week p.m. During this period, the mature intermediate villi and the stem villi (lower left) are the prevailing villous types. Immature intermediate villi

with typical reticular stroma (lower right) are less common.

Paraffi n section. ¥125.

Figure 8.9. Placental villi of the 33rd week p.m. The number of immature intermediate villi is further decreasing. Most villi are stem villi and mature intermediate villi; the latter are intermin- gled with the fi rst few terminal villi, which in paraffi n sections

(because of their similar diameters) are diffi cult to differentiate from mature intermediate villi. The stem villi are still not fully fi brosed; rather, they show a thin superfi cial layer that has few fi bers and is rich in connective tissue cells. Paraffi n section. ¥125.

the more central parts of the stem villi. This difference usually is no longer observed at term. Larger parts of the syncytiotrophoblast of the stem villi are replaced by fi bri- noid. Most villi are mature intermediate and terminal villi (Fig. 8.10). Small groups of immature intermediate villi, with calibers of 100 to 200 mm, can regularly be found in the centers of the villous trees, indicating placental growth is still active.

Tenth month p.m. 37th to 40th week p.m.; 35th to 38th week p.c.

The mean crown–rump length is increased from 330 mm to its fi nal value of 380 mm and the mean fetal weight from 2400 to 3400 g. The placental diameter during the last month of pregnancy varies between 200 and 220 mm, and the mean placental weight increases from 400 g to 470 g. There are considerable individual variations. Early clamping of the cord after delivery of the baby may even increase placental weight by as much as 100 g. The fi nal maximal placental thickness postpartum is about 25 mm;

measured by ultrasonography in situ, the uterine wall and placenta amount to 45 mm. The mean cord length at term is 495 to 520 mm.

The kind and amount of villous types differ from the foregoing stage in several aspects. There are considerably

increased numbers of terminal villi (about 40% of the total villous volume of the placenta) (Fig. 8.11) and a higher degree of capillarization of the latter, mainly because many of the capillary cross sections are dilated sinusoidally to maximally 40 mm. In well-preserved, early-fi xed placentas that are not suffering from fetal vessel collapse, the terminal fetal villous capillary lumens amount to 40% or more of the villous volume.

About 20% of the villi are stem villi. In the fully matured placenta, the fi brous stroma reaches the trophoblastic or fi brinoid surface of the stem villi everywhere; a superfi cial reticular rim, or a superfi cial accumulation of fi broblasts, as during the 9th month, is usually absent at term. If not, it has to be interpreted as a sign of persist- ing immaturity. The syncytiotrophoblastic cover of the stem villi is degenerated in most places and often it is replaced by fi brinoid (Fig. 8.12). About 30% to 40% of the villous volume is made up of mature intermediate villi, which can be differentiated histologically from the terminal villi by their reduced degree of fetal capil- larization, and from the stem villi by the absence of large fetal vessels with media and adventitia identifi able by light microscopy. Maximally, only 10% of the total villous volume is of the immature, intermediate variety;

they normally appear as small, loosely arranged groups

Figure 8.11. Placental villi of the 38th week p.m. Dominating villous types are mature intermediate villi and terminal villi, both of small caliber. Several stem villi of varying caliber can be seen in between. As is typical for near-term placentas, the trophoblastic cover of the stem villi is partly replaced by

fi brinoid. The stromal core is completely fi brosed. Reticular stroma or cellular connective tissue (which as a typical sign of immaturity was visible below the trophoblast in earlier stages) is absent throughout the last few weeks. Paraffi n section.

¥125.

the preceding stage. Paraffi n section. ¥125.

Figure 8.12. Placental villi of the 40th week p.m. Caliber distri- bution is little different from that of the 38th week. Despite this fact, some remarkable changes exist: The fi brinoid deposits (homogeneously gray) around the larger stem villi and the number of terminal villi are considerably increased; also, because

of the irregular shapes of terminal villi at term, numerous fl at sections of villous surfaces can be seen. Here these structures appear as dark spots of seemingly accumulated nuclei (“tropho- blastic knotting”). Paraffi n section. ¥125.

in the centers of the villous trees, sometimes surrounding a central cavity.

The total villous volume of the placenta is about 12.5 m

. The mean trophoblastic thickness is reduced to about 4 mm and the mean maternofetal diffusion distance to less than 5 mm. About 20% of the villous surfaces are double layered, consisting of cytotrophoblast and syncy- tiotrophoblast; the remaining 80% are only covered by syncytium of highly varying thickness (0.5–10 mm). The cell bodies of the existing villous cytotrophoblast, however, are so thin at times that they may be diffi cult to identify; thus, many investigators tend to underesti- mate their quantity or even deny their existence. The amount of fi brinoid in and around the villi is variable.

We have never observed a complete absence of fi brinoid.

Amounts exceeding 10% of the total placental villous volume are an exception and likely to be a sign of disease processes. The amount of fi brinoid, inside and at the surfaces of the chorionic and the basal plate, is even more variable.

Table 8.1 summarizes the development of villous types throughout pregnancy.

NUCLEATED RED BLOOD CELLS

There is considerable confusion about the normal numbers of nucleated red blood cells (NRBCs) in neonatal blood and it is for this reason that this information is included here. In the truly normal term pregnancy very few, if any, red blood cells with nuclei are visible in the fetal blood during the microscopic placental examination. When NRBCs are seen in the fetal blood at term (and it is important that they be correctly diagnosed as such), this is a distinctly abnormal finding. The pathologist should endeavor to ascertain the reason for the presence of the NRBCs when they are seen in placental sections (Fig. 8.13). This is most con- veniently done by making a blood smear of neonatal blood.

An extensive literature exists on this topic and the findings in these contributions are not always in agreement. They have indeed led to considerable controversy as to how many NRBCs may be found in truly normal neonates. Most studies are based on neonatal blood smears, rather than placental sections. It is difficult to estimate the number accurately by studying placental slides. One reason for the results to be discrepant, especially in the older citations, is that the authors have not always stated the exact age of the neonates they studied. They have also not excluded infants who suffered from any of the many causes of hypoxia. Most reports made no reference to the possible existence of growth retardation and many other factors that are only now becoming known as causing fetal erythropoietin (EPO) release, presumably the main reason for secretion of NRBCs.

One of the earliest contributions to this topic is that by Geissler and

Japha (1901), who stated emphatically that “contrary to the dogma,”

T able 8.1. Structural characteristics of the fi ve villous types from the 4th to the 40th week p .m. W eek Stem villi Immature intermediate villi Mesenchymal villi Mature intermediate villi 4 Only mesenchymal villi (120– 250

m m) and trophoblastic sprouts 5 (30–60

m m) are present.

Large mesenchymal villi 6 (>200

m m) may show diffuse , moderate stromal fi brosis . 7 8 T he fi rst stromal channels appear within the mesenchymal villi. 9 Numerous immature Numerous short mesenchymal villi 10

intermediate villi (100–200

m m) (60–100

m m), partly continuous

11 with reticular stroma; caliber of with slim trophoblastic sprouts , the largest vessels is 20–30

m m. branch from the surfaces of immature intermediate villi. 12 Increasing amount and size of immature intermediate villi 13 (100–400

m m); caliber of stem vessels increased up to 100

m m; 14 vessel walls with two to three concentric layers of cells . 15 Light microscopically apparent bundles of collagen fi bers 16 arranged around vessel walls . 17 About 50% of all villi with calibers >150

m m show fusion of the T he numbers of mesenchymal villi fi brosed adventitial sheaths around the primitive arteries with those and trophoblastic sprouts are 18 of the veins . T his is the fi rst step toward formation of the fi brosed slowly decreasing . stromal core of stem villi. 19

20 F irst true stem villi appear . Immature intermediate villi are Smaller ones (150–300

m m) show still the dominating villous type . 21 centrally fi brosed core with T hose with calibers of 100– arterial and venous adventitia 150

m m show no stromal fi brosis . 22 being fused. T hose >300

m m show T he larger ones show fi brosis a largely fi brosed core . of the walls of larger vessels . 23 T he number of mesenchymal villi with poorly fi brosed and poorly vascularized stroma, rich in cells , increases considerably . T hey grow in 24 Caliber >300

m m: completely Number and size of immature length and in width (calibers 80–150

m m) and show a continuous transition fi brosed stroma; caliber <300

m m: intermediate villi are decreasing . into mature intermediate villi. 25 superfi cial layer of reticular stroma below the trophoblast. 26 T ypical mesenchymal villi are rare Mature intermediate villi with dense Local spot-like groups of fi rst 27 and mostly located in the stroma, rich in stromal cells and poor typical terminal villi appear; surrounding of immature in fi bers , with calibers of 100– they show calibers of about 28 intermediate villi. 150

m m, comprise the dominating 60

m m, about half of their villous type . stromal volume being occupied 29 by capillary lumens . 30 Caliber >200

m m: completely 31 fi brosed stroma; Only a few evenly distributed Increasing amount of evenly caliber <200

m m: incomplete immature intermediate villi can distributed terminal villi with 32 superfi cial rim of reticular stroma. be found; the stroma is only sinusoidally dilated capillaries 33 partly reticular in nature . and few epithelial plates . 34 Mesenchymal villi are histologically 35 Usually , all stem villi are void of inconspicuous; the few identifi able T he relative number of mature T erminal villi are the dominating reticular stroma; however , below ones are usually located around the intermediate villi is decreasing to villous type; they amount to 36 the trophoblast still is a less T he few remaining immature central cavities . about 25% of total villous volume . about 40% of the total villous densely fi brosed rim, rich in intermediate villi are no longer T he caliber is reduced to 80–120

m m. volume . 37 fi broblasts . evenly dispersed but, rather , concentrated as small groups in 38 All stem villi (except a few around the centers of the villous trees the central cavity) are completely lining the central cavities . T h e 39 fi brosed. T he trophoblast of the extremely loose reticular stroma larger ones often is replaced by shows only a few typical stromal 40 fi brinoid. channels . Reproduced with permission of Kaufmann and Castellucci (Development and anatomy of the placenta. In: Haines’ s and T aylor’ s T ext book of Obstetrical and Gynaecological Pathology , 4th ed. H. F ox and M. W ells (eds .). Churchill Livingstone , Edinburgh, 1995).

NRBCs are not found in young children; it is possible, however, that they occur rarely in prematures. These authors were adamant in their opinion that the presence of NRBCs is to be viewed as “showing disease.” As many others, they did not specify the children’s ages or clinical conditions of their births. Lippman (1924) enumerated the NRBCs of neonatal blood, followed the children up to 5 days of life, and reviewed all prior literature. Lippman concluded that term neonates have an average of 3.2 NRBCs/100 white blood cells (WBCs). (To express the number of NRBCs per total WBC is a convenient and widely practiced way of enumerating these cells, but an alternate method is referred to below.) The number of NRBCs was found to fall rapidly after birth, and at the age of 5 days there were none left. The condition of the newborns is again not clearly stated in this paper, except that chil- dren with congenital syphilis were excluded; however, mothers with preeclampsia were not.

Ryerson and Sanes (1934) undertook one of the more incisive studies of placentally contained NRBCs. They were anxious to ascertain param- eters that allowed specifying the age of a gestation by histologic exami- nation of the placenta. They concluded that virtually all NRBCs had disappeared at the end of the third month of pregnancy. They suggested also that, if more than 1% NRBCs are found, this indicated prematurity (Fig. 8.13). Anderson (1941) made the next significant contribution and more or less confirmed the previous findings. He indicated that in approximately 16.5% of term placentas one or two NRBCs could be found among 1000 red blood cells (RBCs); in the remaining 83.5% there were none at term. Stillborns were observed to have greater numbers, and he stated that a “decided increase . . . points to pathologic states.”

Fox (1967) next published on this phenomenon; he related it to hypoxia or asphyxia. We concur with this interpretation of a relationship to fetal tissue hypoxia, and it is our practice to always take special note of the presence of NRBCs when examining placentas microscopically.

Green and Mimouni (1990) have addressed the question of NRBCs in newborns of diabetic mothers; their observation was that normative data are lacking in the literature. These authors stated the desirability to express NRBCs in absolute numbers, rather than as NRBCs/WBCs, as has been the common practice. One reason for so doing is that ele- vated WBC counts would lead to artificially low numbers of NRBCs when the usual method of enumeration (NRBC/100 WBCs) is employed.

Green and Mimouni also provided rigid criteria for the selection and suggested that “a value greater than 1 ¥ 10

/L should be considered as a potential index of intrauterine hypoxia.” Normally, there were no NRBCs in term neonates, and the 95th percentile was 1.7 in absolute counts. Diabetic mothers’ babies had increased numbers, as did infants with hypoxia and growth-retarded neonates. Shurin (1987) concluded

that there are about 200 to 600 NRBCs/mm

and 10,000 to 30,000 WBCs; she also stated that the normal infant has 4 to 5 NRBCs/100 WBCs in cord blood samples, which is higher than is our experience.

Shurin reflected that this number indicates the erythroid hyperplasia caused by high levels of EPO production.

Erythropoietin, initially made in liver and later in kidneys (Eckardt et al., 1992), is the principal agent in the secretion of NRBCs from the sites of hematopoiesis. It is now being measured with greater frequency in cord blood, and correlations are beginning to be made to perinatal circumstances. Maier et al. (1993) found that elevated levels of EPO in cord blood indicate prolonged fetal hypoxia and advocated that the determination of EPO levels might give the exact time course of events.

The EPO levels did not correlate with gestational age, meconium stain- ing, or Apgar scores, but they related to umbilical arterial pH level and were elevated in fetal growth retardation; they also quoted authors who found a relation to fetal death and cerebral palsy. These findings are of great significance to the perinatologist and pathologist with an inter- est in ascertaining more precisely than now possible the time of possible fetal hypoxia, especially its relation to possible problems during labor.

It is hoped that the rapidity (or sluggishness) of the EPO response and the appearance of NRBCs will be further defined in the future so as to allow a better analysis of perinatal hypoxia. Because these phenomena require some significant metabolic steps in fetal performance, one would expect that very many hours must pass from an hypoxic event to significantly elevate the levels of EPO and NRBCs of the fetus; but we do not presently know the exact number of hours. We also do not know whether the fetal response with EPO and numbers of NRBCs secreted to different amounts of acute blood loss is the same or if it is a graded response. There are, however, occasionally specific cases that allow some insight into these questions. They need to be recorded for a better understanding of the response. It is then essential that the precise status of the gestation and the well-being of the fetus be known for accurate assessment. Thus, it would be impossible to compare accurately the levels of NRBCs found after a hemorrhage in a neonate with intrauterine growth restriction (IUGR) and those of a normal neonate; the former may have started at a higher baseline. Experimen- tal observations (presently known only from sheep) are insufficient for the clinical setting. Quantitative and temporal sequences by Shields et al. (1993) have reviewed what is known of this aspect of RBC restora- tion after experimental hemorrhage in sheep. Despite an initial rise in erythropoietin level, a significant hemorrhage (40%) is not followed by a significant increase in reticulocyte count, nor are the former blood volume and hematocrits restored before birth. Thus, the ovine model may not be adequate to settle these important questions.

Figure 8.13. Immature intermediate villus of the 6th week p.m.

The villus is covered by a uniform layer of syncytiotrophoblast lining the intervillous space (above), followed by a nearly com- plete layer of villous cytotrophoblast directly below. The villous

stroma is largely occupied by a fetal vessel containing numerous

nucleated red blood cells, as is typical for this stage of preg-

nancy. Hematoxylin and eosin (H&E) stain. ¥800.

Phelan et al. (1993) studied NRBCs in asphyxiated and normal neo- nates. They found that normal infants do not have NRBCs but that asphyxiated newborns have elevated counts. The most elevated NRBC counts were explicable only by assuming hypoxia to have occurred long before birth. Nicolini et al. (1990) made the observation that IUGR fetuses have elevated NRBC counts in cordocentesis samples.

The following observations may have relevance in this context.

1. A patient at term whom we have known had a major “gush of bright red blood” (later identified as fetal blood from disrupted vela- mentous vessels) upon insertion of an intrauterine pressure catheter. She was delivered by cesarean section 48 minutes later; the infant was pale but the placenta otherwise entirely normal. The cord arterial pH was 7.05, the fetal hemoglobin 14.6 g, hematocrit 44.8%, WBC 28,000, and platelets 120,000 and falling to 71,000. Three transfusions of packed RBCs were given to the newborn and the hemoglobin level was then only 10.3 g, hematocrit 29.9%. The first enumeration of NRBCs occurred at 1.5 hours as 19 NRBCs/100 WBCs; 6 hours later it was 32 NRBCs/100 WBCs. Thus, there was a continued hematologic response after the delivery of the anemic child, and the initial NRBC response was detectable already within 1 hour. This finding is contrary to the rapid decline of NRBCs postnatally when the hypoxic event has been more remote; under those circumstances most NRBCs are gone on the second day.

2. Another relevant case was a woman who had a car accident while wearing a lap belt and was at 28 weeks’ gestation. Approxi- mately 12 (± 1) hours later, the fetus was born with a hematocrit of 25% and an estimated 40 to 50 mL of fetal blood in the maternal cir- culation; 45 NRBCs/100 WBCs were then found. Moreover, villous edema indicated some degree of early fetal heart failure. This finding may be surprising with a hematocrit of 25% and suggests that final adjustment of blood volume had not yet been made.

3. A 24-year-old gravida 1 was involved in a car accident at 33 weeks. The baby was delivered 12 hours later by cesarean section.

There was a large retroplacental hematoma, consistent with age 12 hours. At 0.5 hour later, a complete blood count (CBC) showed 45 NRBCs/100 WBCs and a WBC count of 20,000; hematocrit of 39.7%.

Eleven hours later the number of NRBCs was 5/100 WBCs, but packed cells had been given; WBC was then 11,000. The child suffered cere- bral palsy.

4. A gravida 1 without toxemia, at 39 weeks and 4 days, with normal pregnancy awoke at 6:45 a.m. with a brief abdominal pain, finding many blood clots in the bed; she ruptured the membranes but stopped bleeding; there was no pain. At 8:12 a.m. she came to the hospital. Findings were fetal heart tone (FHT) 140 to 150; good fetal movements; speculum examination showed blood-tinged fluid in the vagina; no blood from the 1-cm cervix; nontender uterus. At 10:12 a.m. massive bradycardia occurred, followed by emergency cesarean section delivery at 10:37 a.m. Hematocrit was 19%, pH 6.77, PCO

125, PO

32, base deficit –20; NRBCs 18/100 WBCs; Apgar scores 0/0/0. Despite resuscitation and transfusion with packed cells, the child died the next day having a flat EEG. At autopsy, several myocar- dial infarcts, brain necroses, and infarcted adrenals, kidneys, spleen, and areas of liver were found. One velamentous vessel had ruptured, of a velamentous cord with bilobed placenta. This happened in bed, and bleeding was presumably stopped because the engaging head compressed the ruptured vessel. The brick-red myocardial necroses and the finding of 18 NRBCs at birth suggest that on admission the damage had already been done.

Through the courtesy of Dr. G. Altshuler, we provide here a more accurate means of assessing the precise number of NRBCs, as it is heavily infl uenced by WBC counts:

To calculate the absolute NRBC count from NRBCs/

WBCs (number/mm

), one fi rst needs to correct the machine-counted WBCs to a CWBC (corrected WBC) by the following formula:

CWBC= WBC

× 1000 100 + ( ^{NRBC /} 100 ^WBC )

Then: NRBC = WBC - CWBC, or the total NRBC count

= total CWBC ¥ NRBC/100 WBC.

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