Chorionic villi are the exchange structures of the placenta where human fetuses receive oxygen and nutrients from maternal blood, this article reports an improvement of a published method to recover them from the blood of pregnant, women, quotes their number and illustrates their size and trypsin impregnation. The reasons to explain the presence of chorionic structures and villi in the circulating maternal blood, the possible significance of their existence in maternal blood and the mechanisms to remove them are discussed. Their size and trypsin impregnation are illustrated. This paper discusses the significance of the presence of such allogeneic structures in the circulating blood of pregnant women and presents a brief discussion on the role of trypsin and its inhibitor in pregnancy homeostasis

Nilo Pereira Luz

English

Nature Precedings

1 
CHORIONIC STRUCTURES IN MATERNAL BLOOD
Author: Nilo Pereira Luz*
Summary 
Chorionic villi are the exchange structures of the placenta where human 
fetuses receive oxygen and nutrients from maternal blood, This article 
reports an improvement of a published method to recover them  from  the 
blood of pregnant, women, quotes their number and illustrates their size 
and trypsin impregnation. The reasons to explain the presence of chorionic 
structures and villi in the circulating maternal blood, the possible 
significance of their existence in maternal blood and the mechanisms to 
remove them are discussed. Their size and trypsin  impregnation  are 
illustrated. This paper  discusses the  significance of the presence of such 
allogeneic structures in the circulating blood of pregnant women and 
presents a brief discussion on the role of trypsin and its inhibitor in 
pregnancy homeostasis.
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2MD, Professor of Obstetrics, Fellow of the International Academy of 
Cytology, Department of Obstetrics and Gynecology School of Medicine, 
Pontifical Catholic University, Porto Alegre, RS, Brazil
Correspondence address: av. Padre Cacique 266/301 Porto Alegre 90810-
240, RS, Brazil
e-mail address: nilopereiraluz@terra.com.br
Introduction
The embolization of pulmonary vessels by  chorionic  cells in women 
dying with eclampsia, has been reported by Schmorl1 in 1904 and Veit2  has 
described the deportation of chorionic villi to maternal blood. Even when 
these reports received only scant confirmations,  the deportation of 
chorionic villi to maternal blood was accepted as an actual fact by obstetric 
knowledge and treatises3.   Most of the published evidences on the subject 
reports only the migration of fetal cells or free fetal DNA to maternal 
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3blood, a feature observed in all pregnancies4.  Chorionic villi have been 
observed at uterine or pelvic veins but  not at peripheral blood 
simultaneously sampled5   Luz 6 has published a technique for their 
retrieval from maternal blood. Few studies have quantified the number of 
chorionic  cells or structures migrated maternal blood7, 8, 9.  Fetal DNA 
presence and fate in maternal plasma has been properly studied10, 11. A 
phase contrast microscope was used by Luz12 to study the number of 
chorionic villi found in fresh aprotinine (trasylol®Bayer)  treated blood 
samples, obtained from a group of 30 normal pregnant women at the end 
of pregnancy. 
In a study of this subject, Luz has  counted and measured the   chorionic 
villi  recovered from peripheral maternal blood, in a group of 10 normal 
pregnant women13.
  .   A mean of 5.76 ± 0.88 villus  and 15.4 ± 3.07 chorionic sheets > 100 
µm per ml of maternal blood have been found. Villus mean longitudinal 
size was 289.3 ± 13.05 mµ and mean transverse diameter was 116.4 ± 5.75 
µm. ( Figure 1).  The presence of trypsin at the syncytial envelopment of 
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4villi and in a sheet of trophoblast being detached is illustrated. (Figure 2). 
This article is intended to report an improvement of the published method 
to recover chorionic structures from maternal blood, to present the large 
dimensions of some of them  and to display their impregnation with 
trypsin.
These observations present new insights on the characteristics and size of 
chorionic structures at the circulating maternal blood. The recovery 
technique described in this article may help the persons interested on the 
subject to confirm  our reported observations.  New facts here reported 
could enhance  the understanding of some  unclear aspects on the 
immunology and the physiology of a pregnant women
Method
3 ml of maternal blood are obtained at a cubital vein, with a syringe soaked 
in heparin, immediately verted to a centrifuge tube containing methylic 
alcohol, gently agitated and kept in vertical position for 30 to 60 minutes, 
then centrifuged at 2,000 rpm by 30 minutes. The plasma over the sediment 
is removed with a small pipette leaving intact a linear portion of 3 to 5 mm 
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5directly over the sediment. The removed plasma is substituted by an equal 
amount of Bouin’s fixative, centrifuged again for 15 minutes at 2,000 rpm 
and kept at a vertical position for 6 hours. The centrifuge tube is next 
sectioned just above the sediment and split into two halfs by a vertical 
incision. The 2 complete, hardened pieces of the sediment are embedded in 
paraffin, sectioned at 3 µm and stained with HE or Masson’s trichrome. 
Microscope glass slides are scrutinized at 250x magnification.
Results
Large  pieces  of  trophoblastic  villi  and  sheets  of  detached 
syncytiotrophoblast  (Figure  1)  have  been  recovered  from  each  blood 
sample studied13. They present several degrees of degenerative alterations.. 
Their nuclei  are pale or not stained by hematoxylin. Our recent studies 
have  detected  trypsin  at  the  syncytial  envelopment  of  villi  and  in  the 
detached  chorionic  sheets  (Figure  2).  Monoclonal  antigens  specific  to 
chorionic tissues recognized them only 33 % of cases, in a study performed 
by Bill Kalionis. Morphology has offered, up to now,  the best criteria for 
their identification.
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6Discussion
The first point to be stressed from this article is the fact these chorionic 
structures have been found and recovered from maternal blood in all 
studied cases. This was obtained sampling maternal blood with a protease 
inhibitor, or by the use of an immediate fixation of the blood sample. 
Douglas5 has recovered chorionic cells from uterine or pelvic vessels but 
none at the peripheral blood, even when simultaneously  sampled,. He 
believed this was caused by the trapping of the chorionic structures by the 
capillary network of the lungs. The presence of such large structures in 
peripheral blood of pregnant women, as reported in this article can be 
explained by the existence of arterio-venous shunts that are enlarged during 
pregnancy14, 15. 16, 17, allowing the bypass of large structures from arteries 
directly to veins (figure 1). Their absence as observed in most previous 
studies can be ascribed to their removal by maternal mechanism and  not to 
their capture by lung’s capillaries. Their constant presence in blood 
samples obtained by us6, 12, 13 can be explained by the use of an inhibitor of 
proteases or by the use of an immediate fixation, any one procedure 
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7interfering with the maternal removal mechanisms.  Other points be 
considered from our reported observations  are the number and the size of 
these structures  found in maternal blood12, 13.  A total of 15.4 ± 3.1 
syncytial sheets larger than 100 µm (range 22-114) and 5.8 ± 0.9 villi per 
ml of maternal blood have been found in a group of 10 normal pregnant 
women at the end of pregnancy. As the total volume of blood in a normal 
human pregnancy can be estimated as of 5,500 ml, our findings suggest the 
actual presence of 116,600 allogeneic structures in the  maternal blood, 
84,700 of them being chorionic sheets and 31,900 being  chorionic villi. 
They are large, in182  measured villi their mean longitudinal diameter was 
289.3µm ± 68.6 (range 68.6 to 1,372µm) and their mean transverse 
diameter was 116.4 ± 5.23µm (range 9,2 to 372.4 µm)).
 These data could be important to evaluate the actual amount of chorionic 
structures present in maternal blood and help to the understanding its 
possible significance.
Other point to be considered from our data,  is the immunologic 
significance of the presence of so many structures  the in circulating blood 
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8of pregnant women. Jerzak18  has   considered this as a currently neglected 
factor with influence on the development of the maternal tolerance to her 
fetus  Some studies have already associated the number of chorionic 
structures in maternal blood  with pregnancy diseases as pre-eclampsia 19, 20, 
21. 22, 23. 24
. The use of the  blood sampling method here reported may provide 
additional informations concerning  the amount of chorionic tissues 
circulating in maternal  blood in normal and abnormal pregnancies.  
The maternal mechanisms to remove them from circulating blood are 
aspects not currently studied. Thomas7 has studied in vitro the effects of 
trypsin upon chorionic villi. He has verified this enzyme, in vitro, at a 
concentration of 100 micrograms per ml will destroy fresh human 
chorionic villi in  3 to 4 minutes and promotes the detachment of the 
syncytial envelopment from the core of the villus. Few evidences have 
been  reported on the existence of these removal mechanisms.  Lo11 has 
described the short mean half-life of free fetal DNA in maternal plasma. 
Dhallan et al25, 26 have significantly increased the percentage of free fetal 
DNA from 6.3 to 34.7 % of total maternal plasma DNA, by the use of 
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9formaline in the treatment of the blood samples. This effect could be 
caused by a direct action of formaline, decreasing or stopping the maternal 
mechanism for the elimination of these allogeneic fetal/placental tissues 
from her blood. 
Impregnation with trypsin has been found by us in the syncytiotrophoblast 
envelopment of the villi and in other chorionic structures recovered from 
maternal blood (Figure 2).  
An important  point, quoted in this article, is the  biological effect of an 
urinary protease inhibitor in an experimental model of premature labor in 
mice. This substance has  significantly increased the duration of pregnancy 
in  induced premature labors in mice and has decreased the intensity of 
their placental apoptosis27, 28, 29, 30, 31, 32, 33, suggesting both, trypsin and its 
inhibitor are significant parameters for the maintenance of mice 
pregnancies. They are possible participants on the homeostasis of human 
pregnancy, as it is suggested by the in vitro effect of trypsin7 , by the 
trypsin presence in the chorionic tissues recovered from maternal blood, 
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10
here reported, and the evidences of their rapid removal from maternal 
blood25, 26..
Conclusions
1) a great number of large chorionic structure is daily deported to 
maternal blood
2)  they seem to be participants in the development of pregnant women 
immune tolerance to her fetus 
3) the possible action of trypsin and its inhibitor in  trophoblast could 
be  relevant to the physiology of human pregnancy
LEGENDS
Figure 1: A chorionic villus recovered from peripheral maternal blood.with 
a thickened syncytiotrophoblast and two detaching syncytial knots. The 
63x inserted at the left upper part of the figure demonstrates this villus is 
part of a much larger structure. Actual size can be estimated by the 50µm 
scale
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11
Figure 2: Trypsin impregnates on the syncytiotrophoblast from a 
degenerated villus and on a detaching sheet of trophoblast 
 
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11) Lo, YM et al. Rapid clearance of fetal DNA from maternal plasma.

A method for the identification of chorionic villi in peripheral blood of pregnant women.

A noninvasive test for prenatal diagnosis based on fetal DNA present in maternal blood: a preliminary study. Lancet.369:474-81

Apoptosis in the first trimester human placenta: the role in maintaining immune privilege at the maternal/foetal interface and in trophoblast remodeling.

Elementos trofoblásticos no sangue periférico de gestantes.

Lipoteichoic acid induces preterm delivery in mice. J Pharmacol Toxicol Methods.

Methods to increase the percentage of free fetal DNA recovered from the maternal circulation.

N a t u r e P r e c e d i n g s : h d l :

Occurrence of trophoblasts in the blood of toxaemic patients.

Peripheral skin temperature and microcirculatory reactivity during pregnancy: a study with termography.

Pre-eclampsia: an excessive maternal inflammatory response to pregnancy.

Preventive effect of ritodrine hydrochloride and/or urinary trypsin inhibitor against lipopolysaccharide-induced preterm delivery in mice.

Repeated administration of low-dose lipopolysaccharide induces preterm delivery in mice: a model for human preterm parturition and for assesment of the therapeutic ability of drugs against preterm delivery.

Role of urinary trypsin inhibitor in the maintenance of pregnancy in mice. Obstet Gynecol.

Shedding of syncytiotrophoblast microvilli into the maternal circulation in preeclamptic pregnancies.

Shunting the intervillous space: new concepts in human uteroplacental vascularization.

Signes cutanées physiologiques au cours de la grossesse normale: étude de 60 femmes enceintes. Ann Dermatol Vénéréol.

The continuous migration of syncytial trophoblast from the fetal placenta into the maternal circulation. Trans Assoc Am Physicians.

Tocolytic effect of magnesium sulfate and/or urinary trypsin inhibitor against lipopolysaccharide-induced preterm delivery in mice. Acta Obstet Gynecol Scand.

Trophoblast cells in the blood stream in normal and abnormal pregnancy.

Trophoblast deportation in human pregnancy: its relevance to pre-eclampsia.

Trophoblast deportation in pre-eclamptic pregnancy.

Trophoblast turnover in health and disease.

Trophoblastic apoptosis in mice with preterm delivery and its suppression by urinary trypsin inhibitor. Obstet Gynecol.

Trophoblastic cells in the circulating blood during pregnancy. Am J Obstet Gynecol.

Über das Schiksal embolisch verschleppter Placentarzellen. Verh Deutsch Ges Pathol.

Ueber Deportation von Chorionzotten. Zentralbl Gynäkol.

Vascular changes of the skin in pregnancy: vascular spiders and palmar erythema. Surg Gynecol Obstet.

Villous cytotrophoblast regulation of syncytial apoptotic cascade in the human placenta. Histochem Cell

Vilosidades e estruturas recuperadas do sangue periférico de gestantes. Rev Bras Ginecol Obstet.

Chorionic Structures in Maternal Blood

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Chorionic Structures in Maternal Blood

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