Educating and training a workforce for nutrition in a post-2015 world.

Nearly all countries in the world today are burdened with malnutrition, manifesting as undernutrition, micronutrient deficiencies, and/or overweight and obesity. Despite some progress, efforts to alleviate malnutrition are hampered by a shortage in number, skills, and geographic coverage, of a workforce for nutrition. Here, we report the findings of the Castel Gandolfo workshop, a convening of experts from diverse fields in March 2014 to consider how to develop the capacity of a global cadre of nutrition professionals for the post-2015 development era. Workshop participants identified several requirements for developing a workforce for nutrition, including an ability to work as part of a multisectoral team; communication, advocacy, and leadership skills to engage decision makers; and a set of technical skills to address future challenges for nutrition. Other opportunities were highlighted that could immediately contribute to capacity development, including the creation of a consortium to link global North and South universities, online training modules for middle managers, and practical, hands-on experiences for frontline nutrition workers. Institutional and organizational support is needed to enable workshop recommendations on education and training to be effectively implemented and sustained. The findings from the Castel Gandolfo workshop can contribute to the delivery of successful nutrition-relevant actions in the face of mounting external pressures and informing and attaining the forthcoming Sustainable Development Goals.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

First trimester histiotrophe shows altered sialylation compared with secretory phase glycoconjugates in human endometrium.

OBJECTIVES: Histiotrophe is now recognized as being an important feature of early human pregnancy, providing nutrients and growth factors to the developing embryo. Our aim was to examine the glycan composition of histiotrophe from first trimester decidua and to compare it with secretions present in endometrial glands from the late secretory phase of the menstrual cycle. STUDY DESIGN: Twenty samples of decidua from pregnancies between 8 and 11 weeks were processed into epoxy resin and sections stained with a panel of 22 lectins, together with six late secretory phase endometrial biopsies. MAIN OUTCOME MEASURES: Specimens were analysed using a semi-quantitative ranking system and the density of lectin binding to the glandular secretions and the epithelium assessed. RESULTS: With the onset of pregnancy, beta-galactose, alpha-N-acetyl galactosamine and N-Acetyl lactosamine bound by Arachis hypogaea, Glycine max, Helix pomatia and Erythrina crystagalli agglutinins appeared in terminal positions on oligosaccharide chains, suggesting loss of the capping sialic acid residues present in the non-pregnant state. CONCLUSIONS: Suppression of terminal sialylation is evident during early pregnancy, suggesting that modifications to endometrial glandular activity occur in response to placental signals. The changes may facilitate absorption of histiotrophe by the trophoblast and enhance availability of substrates for degradation.

Characterisation of Hofbauer cells in first and second trimester placenta: incidence, phenotype, survival in vitro and motility.

Macrophages, known as Hofbauer cells, are most abundant in placental villous stroma in the first and second trimesters. Their functions are not well defined. We have used a combination of in situ and in vitro methods to characterise these cells. Lectin histochemistry and immunohistochemistry were used to identify macrophages in situ. The lectin from Maclura pomifera (MPA) was found to mark cells bearing the CD68 antigen with optimal specificity and selectivity. MPA staining was used to show that they increase in number from mid first to mid second trimester, becoming much less abundant at term. The cells are absent from mesenchymal villi, being associated primarily with villous stroma containing the prominent interstitial channels characteristic of immature intermediate villi. A mixed stromal cell isolate was studied in monolayer culture, including the use of time-lapse microscopy. Cells from first or second trimester tissue contained a subpopulation of about 14-17% of cells that exhibited a macrophage-like morphology and expressed CD68 as well as MPA-binding glycans. These cells were short-lived in monoculture, but could persist in vitro in association with a fibroblast layer for several weeks. They could switch rapidly from a macrophage-like to a fibroblastic morphology, were highly motile and associated in clusters that rapidly formed and dissipated over periods of a few hours. These data suggest that Hofbauer cells play a role in the maturation of mesenchymal into immature intermediate-type stroma. They may be important in the excavation of stromal channels. Their prolonged viability in mixed cultures suggests a paracrine relationship with resident fibroblasts. Their location and migratory behaviour predict an ability to move rapidly around the villous stroma, perhaps within the channel system, and to make transient contacts both with other macrophages and stromal cells.

Dexamethasone stimulates placental system A transport and trophoblast differentiation in term villous explants.

Synthetic glucocorticoids (GCs) are given to women with threatened preterm labour but their administration has been linked to reduced infant birthweight. The underlying mechanisms are unknown, but impaired placental development and/or function has been implicated. The activity of the system A amino acid transporter is decreased in placentas from pregnancies complicated by fetal growth restriction. Whether GCs adversely affect placental amino acid transport is unknown. The objective of this study was to determine the regulatory effects of GCs on system A transport using a human in vitro placental explant model. Term explants (n=7) were treated with dexamethasone (DEX 10(-8)M or 10(-6)M) or vehicle for 48 h. System A activity was measured by the uptake of (14)C-N-methylated aminoisobutyric acid by explants. Explants were also processed for electron microscopy (EM), immunohistochemistry, and qRT-PCR. Lactate dehydrogenase (LDH), human chorionic gonadotropin (hCG) and human placental lactogen (hPL) release into the culture medium was measured. DEX (10(-6)M) stimulated system A activity compared to vehicle (p<0.05). System A transporter proteins were localized to the newly regenerating syncytiotrophoblast layer, but mRNA levels were unchanged with DEX treatment. DEX did not adversely affect explant viability as determined by reduced LDH release (p<0.05). DEX treatment was associated with morphological (accelerated apical microvilli formation, nuclear maturation, and increased cell organelle number) and functional (elevated hCG secretion, increased 11beta-HSD2 mRNA expression and reduced cytotrophoblast proliferation (p<0.05 for all)) markers of syncytiotrophoblast differentiation. These findings suggest that DEX stimulates system A activity and promotes syncytiotrophoblast differentiation and maturation.

Altered glycosylation in peri-implantation phase endometrium in women with stages III and IV endometriosis.

BACKGROUND: Endometriosis is a common cause of infertility and pelvic pain. Lectin histochemistry has shown that glycan expression is a sensitive marker of differentiation in the normal endometrium. Endometrial biopsies were taken during the implantation window from women with subfertility and advanced (stage III and IV) endometriosis to evaluate specific glycans bound by lectins from Dolichos biflorus agglutinin (DBA) and Vicia villosa agglutinin (VVA), which detect related but distinct glycan sequences regulated by progesterone action. METHODS: Endometrial tissue from 12 women with subfertility and advanced endometriosis and 11 healthy controls were taken on days 19-24 of the menstrual cycle and processed into either epoxy resin or paraffin wax. Lectin histochemistry was analysed using light microscopy to quantify the amount of glandular reaction product. RESULTS: There was a significant (P = 0.011) reduction in DBA binding to endometrium from patients with endometriosis compared with controls, which was not seen with VVA (P = 0.135). Three stage IV biopsies and one stage III biopsy completely failed to bind DBA and, of these, three showed moderate glandular binding of VVA. DBA and VVA binding differed significantly (P= 0.0039) in the endometriosis specimens whereas in controls no significant difference was detected (P = 0.812). CONCLUSION: Secretory phase glycosylation in women with advanced endometriosis differs from that in healthy women with a reduction in fucosylated N-acetylgalactosamine sequences bound by DBA. Shorter VVA-binding glycans are not significantly affected. In addition to indicating abnormalities of epithelial differentiation, these findings may be directly relevant to implantation failure, as blastocyst attachment requires a critical interaction with the epithelial glycocalyx.

Connexin expression pattern in the endometrium of baboons is influenced by hormonal changes and the presence of endometriotic lesions.

Experimentally induced endometriosis in baboons serves as an elegant model to discriminate between endometrial genes which are primarily associated with normal endometrial function and those that are changed by the presence of endometriotic lesions. Since connexin genes are characteristic of the hormonally regulated differentiation of the endometrium, we have examined connexin expression in baboon endometrium to delineate if they are altered in response to the presence of endometriotic lesions. Connexin expression in the endometrium of cycling baboons is similar to that of the human endometrium with Connexin(Cx)43 being primarily seen in the stromal compartment and Cx26 and Cx32 being present predominantly in the epithelium. Although Cx32 is up-regulated during the secretory phase, Cx26 and Cx43 are down-regulated. In the baboon model of induced endometriosis a change in connexin pattern was evident in the presence of endometriotic lesions. In the secretory phase, Cx26 and Cx32 are no longer present in the epithelium but Cx26 is now observed primarily in the stromal cells. Infusion of chorionic gonadotrophin in a manner that mimics blastocyst transit in utero failed to rescue the aberrant stromal expression of Cx26 that is associated with the presence of endometriotic lesions suggesting an impairment of the implantation process. The altered connexin pattern coupled with a loss of the channel protein in the epithelium and a gain of Cx26 in the stromal compartment suggests that the presence of lesions changes the uterine environment and thereby the differentiation programme. This aberrant expression of connexins may be an additional factor that contributes to endometriosis-associated infertility.

Adhesion molecules in human trophoblast - a review. II. extravillous trophoblast.

At the tips of anchoring villi, cytotrophoblast (CTB) proliferation leads to a process of multilayering in which cells lose their attachment to the villous basement membrane and develop into columns, within which they adhere to one another using desmosomes, with associated intermediate filament bundles. Non-desmosomal cadherins, tight junction proteins and other adhesion molecules are also present, suggesting that actin-associated adhesions contribute to placental anchorage. In the distal columns, cell-cell interactions diminish, cells upregulate beta1 integrins and bind to a provisional fibrinoid extracellular matrix, eventually detaching to migrate into the decidual stroma and myometrium, where interstitial and endovascular extravillous trophoblast (EVT) populations show distinct repertoires of adhesion molecules.

Reproductive glycogenetics--a critical factor in pregnancy success and species hybridisation.

Hybridisation occurs rarely in nature and experiments using interspecific transfer of embryos generally result in implantation failure. Here we show that appropriate glycosylation of the apposing surfaces of endometrium and trophoblast probably is an important factor and may play a critical role in pregnancy success. Examination of closely related species shows that each has its own specific pattern of glycosylation, or glycotype, at the fetomaternal interface and that interacting surfaces appear to show complementarity, suggesting the existence of a glycocode. Studies on a camel/llama hybrid show that for successful implantation to occur, a hybrid must have a placental glycosylation pattern similar to that of the host species, suggesting that the glycocode and appropriate glycosylation may be critical factors in the establishment and maintenance of pregnancy. This new field of reproductive glycogenetics is not only relevant to the development of new species but may also have important implications in the area of human fertility.

Polymorphisms of the tumor suppressor gene LSAMP are associated with left main coronary artery disease.

Previous association mapping on chromosome 3q13-21 detected evidence for association at the limbic system-associated membrane protein (LSAMP) gene in individuals with late-onset coronary artery disease (CAD). LSAMP has never been implicated in the pathogenesis of CAD. We sought to thoroughly characterize the association and the gene. Non-redundant single nucleotide polymorphisms (SNPs) across the gene were examined in an initial dataset (168 cases with late-onset CAD, 149 controls). Stratification analysis on left main CAD (N = 102) revealed stronger association, which was further validated in a validation dataset (141 cases with left main CAD, 215 controls), a third control dataset (N = 255), and a family-based dataset (N = 2954). A haplotype residing in a novel alternative transcript of the LSAMP gene was significant in all independent case-control datasets (p = 0.0001 to 0.0205) and highly significant in the joint analysis (p = 0.00004). Lower expression of the novel alternative transcript was associated with the risk haplotype (p = 0.0002) and atherosclerosis burden in human aortas (p = 0.0001). Furthermore, silencing LSAMP expression in human aortic smooth muscle cells (SMCs) substantially augmented SMC proliferation (p<0.01). Therefore, the risk conferred by the LSAMP haplotype appears to be mediated by LSAMP down-regulation, which may promote SMC proliferation in the arterial wall and progression of atherosclerosis.

Placental expression of alpha2,6-linked sialic acid is upregulated in malaria.

In Africa, approximately 25 million pregnant women are at risk of Plasmodium falciparum infection each year, one in four has evidence of placental involvement and up to half of these may be associated with low birth weight outcomes. In infected pregnant women, the placenta is an ideal site for the accumulation of the parasites, and this reduces in extent in subsequent pregnancies. Recent data indicate that terminal alpha2,3 sialic acid-dependent routes are central to the efficient invasion of erythrocytes with P. falciparum, however, the role in placental malaria of sialylated, or other glycoconjugates, on syncytiotrophoblast has not previously been assessed. Placental biopsies from Zambian women showed the Neu5Ac(alpha2,6)Gal/GalNAc sequences bound by the lectin from Sambucus nigra (SNA-1) to have greatly increased expression on microvillous membranes in samples with chronic P. falciparum infection showing, by electronic image analysis, a significant trend (p=0.002) compared to samples with past or no infection. This suggests a specific placental membrane response to falciparum malaria. Expression of alpha2,6-linked sialic acid, demonstrated by the binding of SNA-1, has been associated with intercellular repulsion in tissues from patients with cancer, and such repulsion resulting from increased alpha2,6 sialylation of chorionic villi could influence intervillous placental parasite density. Sialic acid expression should be examined in placental malaria to identify if this is a malaria-specific phenomenon, and to determine its relation to placental inflammation and pregnancy outcomes.

Effects of oxygen on cell turnover and expression of regulators of apoptosis in human placental trophoblast.

Pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are associated with aberrant cell turnover, including increased apoptosis, in placental villous trophoblast. The increased apoptosis is associated with exaggerated expression of p53, which promotes cell cycle arrest or apoptosis via downstream proteins such as p21 or Bax. These changes in apoptosis and p53 expression are purported to result from exposure to altered oxygen tension. Using a model of villous trophoblast turnover, we examined the effect of 20%, 6% and 1% ambient oxygen (O(2)) on apoptosis, necrosis, proliferation and expression of p53 and related regulators of cell turnover, compared to both fresh tissue. Altered O(2) tension exerted an effect on cell turnover in cultured term villous tissue: cytotrophoblast proliferation was increased by culture in 20% O(2) and reduced in 1% O(2) (median proliferative index: fresh tissue=0.32%, 20% O(2)=0.9%, 6% O(2)=0.28%, 1% O(2)=0.07%). Apoptosis was increased in all culture environments, but was significantly enhanced by culture in 1% O(2) (median apoptotic index: fresh tissue=0.64%, 20% O(2)=2.96%, 6% O(2)=3.81%, 1% O(2)=9.2%). Necrotic cell death was also increased by culture in 1% O(2) compared to 6% and 20% O(2). The expression of p53, p21 and Mdm2 in both cytotrophoblast and stromal cells was increased following culture in 1% O(2). There was no alteration in the expression of Bax or Bcl-2. This study provides evidence that p53 is elevated in trophoblast following exposure to hypoxia. The potential role of the p53-pathway in the control of cell turnover in villous trophoblast and the regulation of p53 by altered O(2) tension merits further investigation.

Acute effects of caffeine and tobacco on arterial function and wave travel.

BACKGROUND: Caffeine and tobacco consumption are risk factors for heart failure, but their effects remain controversial. It has been hypothesized that they cause alterations in arterial stiffness and arterial wave travel which may increase ventricular loading. In this study the authors examined the influence of these widely used stimulants on wave intensity and arterial stiffness parameters using carotid wave intensity analysis. MATERIALS AND METHODS: A new Doppler-based ultrasound method was used to measure the acute effects of caffeine and tobacco on wave intensity in the right common carotid artery. The measurements enabled changes in arterial stiffness parameters to be recorded. RESULTS: In 17 subjects compared with 10 controls, caffeine increased blood pressure, early systolic wave intensity and wave speed, but late-systolic wave intensity and mid-systolic reflections were unchanged. In 11 smokers studied before and after smoking one cigarette, blood pressure and arterial stiffness increased but wave intensity was unchanged. No changes were observed in the controls. CONCLUSIONS: Increased wave intensity during ejection after caffeine suggested sympathomimetic effects on the left ventricular function. Increased wave speed in the common carotid artery implied augmented central loading after caffeine, but the absence of measurable changes in local arterial stiffness in the carotid artery suggested more complex and regional effects. Cigarette smoking acutely increased local arterial stiffness in the common carotid artery. These changes can be detected using wave intensity analysis.

Morphological and glycosylation changes associated with the endometrium and ectopic lesions in a baboon model of endometriosis.

BACKGROUND: Endometriosis is one of the most common causes of infertility and pelvic pain. A baboon model has recently been developed whereby the intrapelvic injection of menstrual endometrium results in the induction of endometriotic lesions. We have used this model to investigate changes in ultrastructure and glycosylation of endometria from normal and diseased baboons. METHODS: Endometriosis was induced in eight female baboons; endometrial tissue and endometriotic lesions were removed on days 9-11 post ovulation between 3 and 16 months of disease and compared with endometrium from 17 control animals, using electron microscopy and lectin histochemistry. RESULTS: Ultrastructurally, diseased endometrial glands showed abnormalities in secretory vacuoles and an intracellular accumulation of glycogen; in later stages of the disease, glands resembled those of the late secretory phase endometrium. The abnormalities were mirrored by changes in glycan expression. In early disease, there was an increased binding of lectin from Dolichos biflorus agglutinin (DBA) to fucosylated N-acetylglucosamine residues, whereas in later stages, this binding generally decreased in association with the appearance of a late secretory phenotype. CONCLUSIONS: Endometriosis is accompanied by progressive changes in the gland architecture and biochemistry resulting in dyssynchrony within the window of uterine receptivity, which may result in the reduced fertility associated with this disease.

Ultrastructural aspects of long-term cat placental explant culture.

Changes in tissue architecture and ultrastructure in the cat placenta during long-term explant culture have been examined over an 11-day period. Pieces of cat placenta, dissected from the lamellar region, were cultured in CMRL-1066 medium and tissue was fixed for electron microscopy at 2, 5, 7, and 11 days' culture, as well as before culture was initiated (day 0). Four samples were examined at each time point. After two days, the trophoblast basal lamina and attached cytotrophoblast cells had begun to separate from the syncytium and the cytotrophoblasts were spreading over the surface of the exposed fetal stromal compartment, and by five days were showing signs of growth. At seven days' culture, cytotrophoblast multilayering was common, and vascular and stromal components were also well preserved with collagen biosynthesis evident. By 11 days, the centre of the culture was compacted and degenerate with loss of tissue architecture, but on the outside polyp-like growths could be seen, with a well-developed covering of trophoblast containing fat and secretory droplets, lining a connective tissue matrix and stromal components. The cat placenta, therefore, like the human, has the capacity for regrowth in explant culture, including both trophoblast and stromal components.

Characterization of the uterine phenotype during the peri-implantation period for LIF-null, MF1 strain mice.

Leukemia inhibitory factor plays a major role in the uterus and in its absence embryos fail to implant. Our knowledge of the targets for LIF and the consequences of its absence is still very incomplete. In this study, we have examined the ultrastructure of the potential implantation site in LIF-null MF1 female mice compared to that of wild type animals. We also compared expression of proteins associated with implantation in luminal epithelium and stroma. Luminal epithelial cells (LE) of null animals failed to develop apical pinopods, had increased glycocalyx, and retained a columnar shape during the peri-implantation period. Stromal cells of LIF-null animals showed no evidence of decidual giant cell formation even by day 6 of pregnancy. A number of proteins normally expressed in decidualizing stroma did not increase in abundance in the LIF-null animals including desmin, tenascin, Cox-2, bone morphogenetic protein (BMP)-2 and -7, and Hoxa-10. In wild type animals, the IL-6 family member Oncostatin M (OSM) was found to be transiently expressed in the luminal epithelium on late day 4 and then in the stroma at the attachment site on days 5-6 of pregnancy, with a similar but not identical pattern to that of Cox-2. In the LIF-null animals, no OSM protein was detected in either LE or stroma adjacent to the embryo, indicating that expression requires uterine LIF in addition to a blastocyst signal. Fucosylated epitopes: the H-type-1 antigen and those recognized by lectins from Ulex europaeus-1 and Tetragonolobus purpureus were enhanced on apical LE on day 4 of pregnancy. H-type-1 antigen remained higher on day 5, and was not reduced even by day 6 in contrast to wild type uterus. These data point to a profound disturbance of normal luminal epithelial and stromal differentiation during early pregnancy in LIF-nulls. On this background, we also obtained less than a Mendelian ratio of null offspring suggesting developmental failure.

Fetomaternal glycosylation of early placentation events in the African elephant Loxodonta africana.

During implantation in the African elephant (Loxodonta africana), fetal trophoblast displaces the surface uterine epithelium and superficially penetrates the uterine glands. This limited invasion is followed by the upgrowth of blunt fingers of endometrial stroma, covered with trophoblast and containing capillaries that subsequently vascularize the growing placenta. We have used lectin histochemistry to compare the glycosylation of maternal endothelial cells in the endometrium with those growing within the trophoblastic processes of a 2 g embryo (approximately 125 days' gestation), and also examine changes in the endometrial glands associated with trophoblastic invasion. Maternal vessels at the apices of the trophoblast-covered stromal upgrowths showed increased expression of terminal N-acetyl galactosamine, N-acetyl glucosamine oligomers, some sialic acids, and tri/tetra-antennate non-bisected complex N-linked glycan, as indicated by increased lectin staining. The areas of increased staining were also more resistant to neuraminidase digestion. Invaded glands had distended walls composed of flattened epithelial cells, some of which showed heavy lectin staining suggestive of intracellular glycan accumulation. The vascular changes suggest that new maternal capillary growth is accompanied by alterations in surface glycosylation. This may be the result of increased glycosyl transferase activity associated with cell proliferation and may also indicate the expression of significantly increased anti-adhesive molecules preventing blood stasis and egress of maternal immunocompetent cells into the fetal compartment.

Apoptosis of terminal hypertrophic chondrocytes in an in vitro model of endochondral ossification.

It is widely accepted that growth plate chondrocytes undergo apoptosis when they reach the terminal hypertrophic stage of their differentiation during the process of endochondral ossification in vivo. In this report, an established chondrocyte cell culture model of mammalian endochondral ossification was utilized to investigate the fate of chondrocytes after they had entered hypertrophy in vitro. Fetal bovine epiphyseal chondrocytes were treated with the demethylating agent, 5-azacytidine, for 48 h and then cultured under azacytidine-depleted conditions. There was evidence for apoptosis in azacytidine-treated cells, as demonstrated by nuclear condensation and fragmentation (days 27 and 35) using transmission electron microscopy, and the detection of exposed phosphatidylserine on the plasma membrane surface of apoptotic chondrocytes (day 27) using fluorescence-labelled annexin V. Treated cultures on days 10 and 20 and untreated cultures at all corresponding time-points showed no morphological characteristics of apoptosis. In situ hybridization studies of treated cultures revealed that expression of the apoptotic suppressor, bcl-2, remained consistently high throughout the culture period, whilst the apoptotic inducer, bax, was not expressed until day 23. Quantification of these data showed a gradual shift in the ratio of the expression level of bcl-2 and bax in favour of bax with time in culture, particularly from day 23 onwards. Taken together, the results indicate that azacytidine-treated epiphyseal chondrocytes entered terminal hypertrophy from day 23 onwards in culture and died by apoptosis. This study confirms this culture system as a successful recapitulation of the entire mammalian chondrocyte differentiation pathway, including apoptosis. The culture model will prove valuable for studies of the apoptotic fate of terminally differentiated chondrocytes in the growth plate with a view to providing a better understanding of the underlying mechanisms of skeletal malformations and other pathological disorders such as osteoarthritis.

Developmental changes at the materno-embryonic interface in early pregnancy of the alpaca, Lamos pacos.

This study analyses the manner in which trophoblast cells adhere to uterine epithelium and the subsequent interactions that contribute to the establishment of epitheliochorial placentation in the alpaca Lama pacos. Specimens at the luteal and follicular phases and at 22, 26, 30 and 45 days of pregnancy (op) were processed for morphological studies. On day 15 op, the blastocysts are completely free within the uterine lumen, with implantation starting around day 20. On days 22 and 26 of gestation, the trophoblast is apposed to the epithelial surface of the uterus, with areas of contact and adhesion by means of complex interdigitation. Implantation sites occur prevalently in the left uterine horn, but an expanded trophoblast also occupies large extensions of the right horn, where the maternofetal interaction shows peculiar areas of apposition. As development continues, attachment areas become more extensive. On days 30 and 45, many secretory granules can be seen in the uterine epithelium, while giant multinucleate cells appear interposed between the remaining trophoblast cells, showing intense alkaline phosphatase activity, deposits containing iron and PAS-positive granules. Placental lactogen hormone is not present within the cytoplasm of the binucleate or multinucleate trophoblast cells. By day 30 of gestation, the trophoblast layer is lined by an extraembryonic connective tissue that by day 45 is well vascularized, thus indicating the starting point of placental formation. Fetal and maternal capillaries indent the epithelium and the trophoblast, narrowing the specialized areas of exchange, which occur along the entire maternofetal interface, characterizing the diffuse nature of this placenta.