Avaliação da nested PCR em comparação aos testes sorológicos IDGA e ELISA para o diagnóstico da anemia infecciosa equina / Evaluation of nested PCR compared with AGID and ELISA serological tests for equine infectious anemia diagnosis

Comparou-se a técnica nested PCR (nPCR) com os testes sorológicos IDGA e ELISA para o diagnóstico da anemia infecciosa equina. Amostras do DNA provenientes das células mononucleares do sangue periférico foram submetidas à amplificação do gene gag pela nPCR, que apresentou valores de sensibilidade e especificidade relativas de 90 por cento e 52,9 por cento, respectivamente, em relação à IDGA, e valores de 85,7 por cento e 49 por cento, respectivamente, em relação ao ELISA. Considerando-se os fatores referentes às limitações de cada técnica, pode ser sugerido o uso da nPCR como teste de diagnóstico complementar para AIE em amostras brasileiras.

The nested polymerase chain reaction (nPCR) technique was compared to AGID and ELISA serological tests for the diagnosis of Equine Infectious Anemia. DNA samples from the peripheral blood mononuclear cells were subjected to the amplification of the gag gene by nPCR, which showed relative sensibility and specificity values of 90.0 percent and 52.9 percent respectively, compared to the AGID and values of 85.7 percent and 49.0 percent, respectively, as compared to ELISA. Considering the factors concerning the limitations of each technique, the use of nPCR can be suggested as a complementary diagnostic test for EIA in Brazilian samples.

Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.

We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from lung fragments, producing high DNA concentrations, with high sensitivity in PCR amplification (100%), followed by Protocols C and B. On the other hand, for PBMC samples, Protocol B gave the highest sensitivity in PCR amplification (100%), followed by Protocols C and A. We conclude that Protocol A should be used for PCR diagnosis from lung fragment samples, while Protocol B should be used for PBMC.

Avaliação do tratamento com isoniazida em bovinos infectados naturalmente pelo Mycobacterium bovis / Evaluation of the treatment with isoniazid carried out in bovines naturally infected with Mycobacterium bovis

Foi adotado o tratamento intermitente da tuberculose com isoniazida em 240 bovinos de um rebanho naturalmente infectado pelo M. bovis. No início do tratamento 36,6 por cento dos animais foram reagentes positivos e 2,9 por cento inconclusivos. Todos os animais do rebanho receberam isoniazida por via oral em doses de 25mg/kg de peso vivo, administrada três vezes por semana, às segundas, quartas e sextas-feiras, durante 10 meses, perfazendo 120 doses. A cura de 98,9 por cento dos animais tratados foi verificada por meio da dessensibilização alérgica realizada pela tuberculinização e controle bacteriológico de 39 animais abatidos. O tratamento neste rebanho não resultou em seleção de cepas resistentes a isoniazida, comprovada pelo teste de sensibilidade a essa droga, realizado em cultura de M. bovis isolada de um animal não curado.

A micro-anatomical model of the distribution of myocardial endomysial collagen.

Myocardial connective tissue probably provides passive support for regulating heart tensile strength and stiffness and ultimately for controlling heart mechanics through its endomysial part. However, endomysial collagen micro-arrangement is still a matter of debate. In order to define the fine distribution of left ventricle endomysial collagen, we applied the NaOH-scanning electron microscopy (SEM) maceration method (one of the techniques of choice for studying collagen micro-arrangement) to rabbit heart. Gomori-reticulum staining was used for correlated light microscopy (LM) observations. The SEM-NaOH method allowed isolation of collagen by removing other extracellular matrix components and cells and preserved collagen structure and position. Endomysial collagen appeared arranged in laminae that delimited the lacunae that were left empty by macerated myocytes and small vessels (mostly capillaries). These laminae were formed by reticular fibers, as confirmed by LM observations of Gomorireticulum-stained samples, and were organized in irregularly meshed networks made of thin (single) and thick (composed) filaments. In longitudinal views, collagen laminae extended the entire length of lacunae. In transversal views, the cut surface of the laminae appeared to be made of collagen bundles. These observations provide an updated microanatomical view of endomysial collagen distribution, which integrates previous studies. This model is based on the evidence that collagen laminae enveloped the surface of small vessels and myocytes. Thus, a type of myocyte-myocyte or capillary-myocyte "laminar connection" anchored to the entire cell length here is emphasized, rather than a type of "strut connection" anchored to defined loci, as usually described. This structure explains better how endomysium may provide the necessary support for heart compliance and protection against overstretch.

Ultrastructural morphodynamics of human Sertoli cells during testicular differentiation.

Our study reviews and ultrastructurally characterises human pre-Sertoli cells between the 6th and the 20th week of gestation by means of integrated light microscopy, transmission electron microscopy and high resolution scanning electron microscopy (standard or following ODO maceration). The morphofunctional differentiation of Sertoli cells defines testicular differentiation. These somatic cells are mostly of mesonephric origin and can be first morphologically recognised in 7 week-old embryos altogether with the formation of testicular cords. The latter organise as primordial germ cells surrounded by pre-Sertoli cells. Due to the great synthetic activity of pre-Sertoli cells the rough endoplasmic reticulum develops. The basal lamina of the cords becomes distinguishable at 7 to 8 weeks of gestation. Both prespermatogonia and pre-Sertoli cells actively proliferate but the latter greatly outnumber prespermatogonia. Many interdigitations and cytoplasmic processes are observed between neighbouring pre-Sertoli cells. Due to cell proliferation a sort of compartmentalisation is established inside the cords in which pre-Sertoli cells tend to localise closer to the basal membrane embracing prespermatogonia with long and thin cytoplasmic processes. One of the main typical features of differentiating pre-Sertoli cells is the irregular nucleus and the prominent nucleolus. When the embryo is 14 to 20 weeks-old pre-Sertoli cells maintain their general morphology whereas the most significant change is the maximum development of Leydig cells. Testicular cords do not show any lumen at all so they cannot be termed "tubules".

Extracellular fibrillar matrix architecture of human placental villi at term.

The spatial organisation of the extracellular fibrillar matrix of normal human placental villi at term can be directly visualised by scanning electron microscopy after 2N-NaOH maceration technique. By these methods, the extracellular fibrillar matrix of placental villi appears as a continuous network of isolated collagen fibrils and/or small fibrillar bundles interwoven each other. This sort of "collagenous fibrillar skeleton" forms the axis of chorionic villi and connects them with the basal plates running through the whole villous system of the placenta. Significant variations in the spatial arrangement as well as in the quantity of the extracellular matrix is observed at different levels of the villous ramification. Within the stem villi, the fibrillar extracellular matrix are abundant and, whereas the fibrils near the villous surface run parallel to the longitudinal axis of the villous (outer fibrils), those located in the inner core of the villous are arranged circularly around the wall of the fetal vessels (inner fibrils). In mature intermediate and terminal villi, viceversa, the extracellular fibrillar matrix is significandy reduced and the fibrils are mainly organised in a thin circular layer around the capillaries and sinusoids. The present study demonstrated the existence of a diverse spatial architecture of the extracellular matrix that results to be peculiar to the various levels of the ramification of the villous tree. Therefore, these morphological data strongly suggest a "compartmentalisation" of the villous tree as suggested by previous immunohistochemical study. Such a highly organised "collagenous fibrillar skeleton" stresses the important mechanical role of the extracellular matrix in sustaining the chorionic fetal vessels and the trophoblastic layer. Furthermore, the fine reticular-meshed network observed within the terminal villi suggests that at this level an additional role ensuring a favourable milieu for active feto-maternal exchanges may exist.

The human zona pellucida and scanning electron microscopy. Reality or artifacts?

The surface micro-morphology of the zona pellucida (ZP) was investigated in 158 inseminated but unfertilized mature human oocytes derived from assisted reproduction trials (ART) by means of traditional scanning electron microscopy (SEM) techniques (gold coating and conductive staining methods) and saponin-ruthenium red-osmium tetroxide-thiocarbohydrazide method (Sap-RR-Os-TC). The main aspect of the ZP by traditional SEM (122 oocytes) consisted in a porous, net-like structure (97 oocytes), whereas a nearly smooth or compact structure of ZP was detected in 25 oocytes (79.5% vs 20.5%). Using Sap RR-Os-TC method on 36 oocytes, 31 oocytes showed ZP with alternating tight and large meshed networks, whereas 5 oocytes displayed only tight meshed network (86.1% vs 13.9%). Due to our well standardized procedures, to the stabilizing action of the conductive staining on the zona material and similar results obtained with the use of Sap RR-Os-TC method, we confidentially regard the ZP changes, occurring in oocytes of various groups, as genuine features, likely related to their actual maturation status, rather than as artifacts. In addition, we emphasize the concept that a modern view of the ZP surface implies the best evidence of crossing filaments' network. We think that the ZP "spongy" or "compact" appearance is only the result of microfilaments network collapse, not the true three-dimensional (3-D) representation of ZP structure.

In vitro fertilized human eggs. An electron microscopic study.

This paper describes by scanning and transmission electron microscopy the ultrastructure of the human fertilized egg and its vestments (cumulus oophorus and zona pellucida). Data are reported on the ultrastructure of a. conventional in vitro fertilized eggs (pronuclear eggs and cleaving eggs at two-to-four cell stage); b. eggs at the same developmental stage deriving fro intracytoplasmic sperm injection. The present results showed that: 1. The cumulus-enclosed fertilized egg is a highly dynamic structure in which egg vestments play a crucial role, positively affecting fertilization and healthy embryo development; 2. Intracytoplasmic sperm injection technique does not seem to significantly alter fertilized egg morphology.

Ultrastructural dynamic features of in vitro fertilization in humans.

The present study on in vitro fertilization in humans demonstrates that three-dimensional (3-D) fine morphology by high resolution scanning electron microscopy (SEM) combined with parallel light and transmission electron microscopy (TEM) can reveal a number of new cellular detailed findings which cannot be detected with other methods. In this study the following aspects have been investigated in early human embryos. 1. Micro-topographical features of the zona pellucida (ZP), surface blastomeres and polar body. 2. Intracytoplasmic features of mature and healthy oocyte, in vitro fertilized (IVF) oocyte and early embryo development. 3. Comparison of general views of intracytoplasmic sperm injection (ICSI) and conventional IVF (C-IVF) of early embryos. 4. Presence of unusual large tubular smooth endoplasmic reticulum (SER) aggregates in 4, 5, and 6-cell embryos after ICSI. 5. Inside views of 3-D blastocysts such as inner cell mass and trophoblast. To our knowledge, this is the first time that such images are reported by using these techniques.

Is there a mesonephric cell contribution to the gonadal primordium before sexual differentiation in humans?: An ultrastructural study.

Differentiation of the intermediate mesoderm involves a complex series of events that result in the formation of the rudiment of the entire adult renal system, gonads and gonoducts. This work, using light, transmission, and scanning electron microscopy describes in human embryos of different ages, the development and spatiotemporal organization of the mesonephric nephron, and the development of the gonadal primordium, with the purpose of knowing if and how these two blastemas contribute to the origin of the non germinal cell content of the gonad primordium. Our results show that between Carnegie stage 13 and 20, the mesonephric nephrons facing the gonadal area, are separated by a band of mesenchymal tissue from the gonad primordium and they retain their structural integrities at the level of epithelial wall and their basement membrane. The morphological stability of basement membranes of different nephric structures, as well as the mesonephric duct during the period studied, confirm the previous opinion that the mesonephros is functional. During the same period of time, the structural events underlying gonad development show that primordial germ cells (PGCs) first invade the gonadal area, and thereafter interact both with epithelial coelomic cells and mesenchymal cells. Both types of cells surround PGCs, initiating the growth and differentiation of the gonadal primordium. Therefore, a mesonephric cell contribution to the genesis of the somatic cell components of the gonadal primordium should be discarded in humans. The present work emphasizes the need for more research in this field.

A new morpho-functional classification of the Fallopian tube based on its three-dimensional myoarchitecture.

The recent direct observations, under scanning electron microscopy (SEM), of the three-dimensional architecture of myosalpinx in different mammals allows us classify salpinxes according to the myoarchitecture of their tubo-uterine junction (TUJ) and isthmus segments. Based upon the myoarchitecture of the outer wall of the TUJ we could find barrier-like species (rat and sow), sphincter-like species type a (rabbit and ewe) and sphincter-like species type b (cow and woman). The different architecture of TUJ can be explained by the different nature of the mating process. Based upon the myoarchitecture of the isthmus we could distinguish type 1 (rat) and type 2 (rabbit, ewe, sow, cow and woman) salpinxes. In the latter the close fusion of musculature deriving from the meso (extrinsic musculature) with the musculature of salpinx (intrinsic musculature) suggests the existence of a unique mesosalpinx contractile system. The myosalpinx is mostly made up of a single network of muscular fibers. Such a plexiform structure, owing to the uneven distribution of fibers, rather than producing a series of regular contraction waves, is more likely to generate random contraction waves. The random propagation of muscular network contraction may deform the plexiform wall of the myosalpinx causing the stirring of tubal contents. By such a stirring movement the contact between hormones and nutrients and the eggs or embryos is intensified, thus favoring a correct fertilization and early embryo development. Taken all together, these systematic results probably suggest an additional and rather new function for the musculature of the tube, namely to increase fertility in a large number of species.

Ultrastructure and distribution of interstitial glandular cells and associated elements in human fetal ovaries.

In order to understand the fine structure and distribution of the interstitial glandular cells (IGCs) and associated elements in the human fetal ovary, we studied human fetal ovaries at 16 weeks post fertilization (p. f.) by transmission electron microscopy. Semithin sections revealed voluminous typical IGCs usually grouped in clusters, located in the interstitium among the ovigerous cords. Isolated primordial follicles were seen in the cords located close to the interstitium in which IGCs were present. Besides the main ultrastructural characteristics of steroid secreting cells, the IGCs showed lipofuscin granules and stacks of annulate lamellae in their cytoplasm. Fibrocytes, macrophages and mast cells were detected close to the IGCs. In particular, the fibrocytes were located around the IGCs, with which they occasionally formed focal cell contacts. Fibrocytes issued numerous long projections, which, together with collagen fibers, surrounded the clusters of IGCs and small vessels (mainly capillaries), often extending into the intercellular spaces among IGCs. These data indicated that, already at the initiation of folliculogenesis, the IGCs are present numerously in a close association with the ovigerous cords. The morphological aspects of IGCs were comparable to that of fetal testis interstitial (Leydig) cells and hilar cells in adult ovary, and suggest that fetal IGCs may be source of adult ovary hilar cells. In addition, we have here demonstrated for the first time that IGCs are associated with stromal cells whose distribution seems to support IGCs microtopography. Fetal ovarian fibrocytes revealed a structural arrangement similar to that of the "compartmentalizing cells" previously described in the adult testis. Macrophages and mast cells presumably have a role as local modulators of steroid synthesis. Mast cells may also affect fibrocyte organization and vascular permeability. We thus suggest that IGCs and associated cells may form a glandular unit in the human fetal ovary similar to that in the adult testis, and this structure is likely involved in early steroid secretion during gonadal differentiation.

Mitochondrial morphology in human fetal and adult female germ cells.

The aim of this study has been to observe, by electron microscopy, the morphological changes affecting mitochondria and associated organelles in the human female germ cell during oogenesis, maturation and fertilization. In the primordial germ cell (PGC), rounded mitochondria with a pale matrix and small vesicular cristae are disposed near the nucleus and significantly increase in number during PGC migration and settlement in the gonadal ridge, where they differentiate into oogonia. In these early stages of mammalian oogenesis, aggregates of mitochondria are typically clustered around or in close relationship with the nuage. In oocytes at early prophase stage, mitochondria proliferate while aligned along the outer surface of the nuclear membrane, contain a more dense matrix than before, and have lamellar cristae. Oocytes of primordial and primary follicles mostly contain round or irregular mitochondria whose matrix has become very light. These mitochondria show typical parallel, arched cristae, and are clustered near the nucleus with other organelles forming the Balbiani's vitelline body. When follicles grow, the mitochondria of the oocytes become even more numerous and are dispersed in the ooplasm. Both paranuclear accumulation and subsequent dispersion of mitochondria in the cytoplasm are likely to be regulated by microtubules. By ovulation, mitochondria are the most prominent organelles in the ooplasm. They form voluminous aggregates with smooth endoplasmic reticulum (SER) tubules and vesicles. These mitochondrial-SER aggregates (M-SER) and the mitochondrial-vesicle complexes (MV) could be involved in the production of a reservoir of substances or membranes anticipating subsequent fertilization and early embryogenesis. Just after fertilization, the mitochondria of the oocyte undergo a further substantial change in size, shape, and microtopography. In the pronuclear zygote, mitochondria concentrate around the pronuclei. During the first embryonic cleavage divisions, round or oval mitochondria with a dense matrix and few arched cristae are gradually replaced by elongated ones with a less dense matrix and numerous transverse cristae. A progressive reduction in size and number of M-SER aggregates and MV complexes also occurs. In summary, oocyte mitochondria show dynamic morphological changes as they increase in number and populate different cell domains within the oocyte. They form complex relationships with other cell organelles, according to the different energetic -metabolic needs of the cell during differentiation, maturation, and fertilization, and are ultimately inherited by the developing embryo, where they eventually assume a more typical somatic cell form.

Human embryo development and pregnancies in an homologous granulosa cell coculture system.

PURPOSE: Our purpose was to determine the effects of the coculture of embryos on human granulosa cells (GCs) in patients in the first cycle of IVF-ET treatment and in patients with repeated implantation failures and to investigate the presence of specific proteins in a 48-hr GC conditioned medium and the GC ultrastructural characteristics. METHODS: Eighteen patients with tubal or idiopathic infertility were enrolled in this study: 7 patients (Trial 1) were in the first cycle of IVF-ET treatment and 11 patients (Trial 2) had repeated implantation failures (one to five). Embryos from each patient were cocultured randomly either on homologous granulosa cells or on a conventional culture medium. RESULTS: At the end of the coculture period (day 5 or 6), 50% of the embryos (Trial 1) reached the blastocyst stage, with respect to 35% in Trial 2. The pregnancy rate per retrieval was 14.2 and 9%, respectively, in Trial 1 and in Trial 2. Many conditioned media showed proteins of 24-29 kDa. and some of them showed additional proteins of 90 kDa. The ultrastructural analysis of GCs showed healthy, metabolically active, protein-synthesizing, and mostly steroidogenic cells. CONCLUSIONS: GC cultures improve embryo development but not pregnancy rates both in Trial 1 and in Trial 2.

Bifurcation of the left superior pulmonary vein, associated with an anomalous connection with the left atrium.

An unusual case of bifurcation of the left superior pulmonary vein (LSPV) just before it enters the pericardium is described. The LSPV, which at the hilus of the lung originated from normal confluence of a superior and inferior root, bifurcated near the left atrium (LAt) of the heart into anterior (AB) and posterior (PB) branches that, separately invaginating the parietal pericardium, formed two individual serous sheaths. The PB coursed almost horizontally and opened, as usual, into the supero-dorsal wall of the LAt. The AB turned downward, reached the superior margin of the left auricle (LAu) and emptied into it. Thus, the AB was interposed between the pulmonary trunk and the LAt obstructing on the left side the communication between the transverse sinus of the pericardium and the pericardial cavity. The auricular opening of the AB was avalvular, but, unlike those of the normal pulmonary veins (PVs) which are surrounded by a large smooth inner surface, was, except for a narrow smooth-walled zone, close to the pectinate muscles. Moreover, an inferior muscular ridge at the inferior margin of its orifice of entrance into the LAu, separated it from the cavity of the LAt. It is well known that in development the PVs arise from convergence of capillaries belonging to the mediastinal part of the primitive splanchnic plexus and drain this into the systemic (cardinal and vitello-umbilical) veins of the embryo. As a consequence, it might be hypothesized that the AB of the LSPV probably represents a partial remnant either of a pulmonary-cardinal anastomotic mediastinal vein, or of a diverging vessel of the mediastinal plexus from which the PVs originate. In either case the AB became absorbed by the LAu, which, while it was developing on the left side of the primitive truncus arteriosus, drew the AB forward and downward, in the direction of its movement. The influence of such an anomaly of the PVs for altered intracardiac hemodynamics of the oxygenated blood flow has to be emphasized. Furthermore, the particular location of the AB, obstructing the communication between transverse sinus and pericardial cavity, can be a hindrance during cardio-pulmonary surgery.

The fetal development of the human uterine cervix from the 12th to the 31st postmenstrual week as revealed by scanning electron microscopy. Anatomical and clinical correlations.

To clarify the differentiation of the human uterine cervix, fetuses of the 12th, 15th, 18th, 20th, 21st, 22nd, and 31st postmenstrual week were studied by Scanning Electron Microscopy. At the 12th week the endocervical epithelium consisted of microvillous cells, often showing single cilia and anlages of tubular glands. At the 15th week the cervical canal was entirely formed and its surface cells appeared columnar. At the 18th week these cells were replaced by flat or slightly raised cells, provided with thin microplicae. At the 20th week the endocervical epithelium appeared pseudostratified with higher, apically-convex and shorter basal cells; glands developed in form of tubular invaginations of the luminal epithelium. At the 21st week in the lower part of the endocervix polymorphic, globose cells with short and stubby microvilli and others elongated, having short microplicae, were observed. These latter likely corresponded to the so-called columnar cells undergoing squamous metaplasia. Among microvillous and/or metaplastic cells, a number of apoptotic cells, as globose elements with a ruffled and invaginated surface, were also noted. At the 22nd week evident plicae palmatae were found, covered by microvillous secreting cells. These showed smooth bulged apices releasing droplets by a "micro-apocrine" mechanism. These features increased at the 31st week, when many droplets were noted also around the mouth of the cervical glands. Only at this phase of development fully ciliated cells were found often contacting secretory material. Mature squamous exfoliating cells with complex microplicae covered an hypertrophied portio vaginalis. The squamous cells extended toward a squamo-columnar junction in form of flat, tongue-like projections. Their tips consisted of immature squamous metaplastic cells, which were endocervical columnar progressively becoming elongated elements, exhibiting short microvilli. The above features are rather similar to those occurring during the adult reproductive age. Therefore, it might be hypothesized that, during pregnancy, a common gestational hormonal background may induce somewhat similar morpho-dynamic processes in the cells and tissues of the fetal reproductive tract mimicking what occurs in the adult female.

A three-dimensional study of human fetal endocervix with special reference to its epithelium.

The development of human fetal cervix has been systematically studied by SEM, obtaining a detailed map of its fine structure, particularly concerning the differentiation and maturation of the endocervical epithelium, including its "eversion" and "squamous metaplasia", normally occurring in postnatal life, but not yet observed in detail by electron microscopy in the fetus. Cervices from spontaneous abortion at 12, 15, 18, 20, 21 and 22 weeks and from intrauterine fetal death (hydrocephalus) at 31 weeks of development have been examined. At 12-15 weeks, as the canalization of the cervix proceeded, the endocervical epithelium consisted of high polyhedral cells, with regularly flattened or concave apices exhibiting scarce microvilli and often single primary cilia. Some narrow intercellular infoldings probably corresponded to primordial tubular glands. At the 18th week the epithelium was made up of a mosaic of flat or slightly raised polygonal cells, whose apical surface showed thin microplicae. At the 20th week a pseudostratified epithelium with many apically convex cells lined the cervical canal and the tubular glands. At 21 and 22 weeks "plicae palmatae" developed, covered by cells, often showing a smooth central area surrounded by microvilli, provided with a primary cilium and swollen by secretory material. This also formed rounded masses on the epithelium. In the lower part of the endocervix some very elongated cells showed short microplicae resulting from fusion of microvilli. At the 31st week secretion increased and its products spreading from the bottom of the glands contacted isolated ciliated cells at their openings and diffusely covered the surface epithelium. Most of the ectocervix exhibited squamous elements, with well-developed labyrinthine microplicae. These cells could overlap each other and also desquamate. The zone of the portio vaginalis around the os of the cervical canal appeared infolded and hypertrophic. Here, an indented squamo-columnar junction between the ectocervical and endocervical epithelium, caused by tongue-like prolongations of squamous epithelium directed toward the endocervix, was found. Their tips consisted of elongated cells, rich only in short microvilli. Our data indicate that the features of the microvillous cells are an expression of a hormone-dependent differentiative process. Thus, their secretion might be stimulated by progesterone. Similarly microplicae on the ectocervical epithelium (a sign of squamous maturation) might be promoted by estrogens. Furthermore, two aspects were significative: 1) the finding--in an early phase only (18th week)--of endocervically-located squamous cells, although devoid of microplicae; and 2) the occurrence--in the latest phase (31st week)--of an indented squamo-columnar junction on the surface of the portio. These features are in agreement with the caudal shift of the squamo-columnar junction near the uterine cavity to the ectocervix after cervico-vaginal demarcation; the squamous metaplasia of this everted endocervical epithelium has been reported by some authors. It is likely that these processes, occurring in fetal life as well as in pregnant women, are related to a common hormonal background, arising from the mother to her fetus.