IFPA meeting 2008 workshops report.

Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.

Reference weights for placentas delivered before the 28th week of gestation.

CONTEXT: Very few studies have measured the weight of large numbers of placentas delivered before the 28th post-menstrual week. METHODS: We measured the weight of 930 singleton placentas delivered before the 28th post-menstrual week, and examined the distributions of weights in selected groups (week of gestation, reason for preterm birth, birth weight Z-score categories, placenta histology). We excluded 90 singleton placentas based on growth restriction as indicated by birth weight Z-score, resulting in a normative sample of 840 placentas. Weights for unfused twin placentas are also presented. RESULTS: Standard weights derived from our data set differ from those previously published, partly due to a larger sample size. Placenta weight varied with birth weight. Placentas from pregnancies ending due to preeclampsia, fetal indications or those showing evidence of poor perfusion on histology were among the smallest and their weights correlated with the smallest birth weights for gestational age. CONCLUSIONS: Placenta weights appear to be influenced by multiple maternal and fetal processes. We present a standard weight table for singleton placentas among live infants born between 23 and 27 completed weeks.

Placental 11 ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) expression very early during human pregnancy.

Maternal physiologic stress during gestation has been reported to be associated with negative developmental outcomes, including intra-uterine growth restriction and reduced birth weight, which can impact postnatal development, behavior and health. The human fetus is partially protected from elevated cortisol exposure by placental 11 ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), which oxidizes bioactive cortisol into bio-inactive cortisone. Importantly, despite the critical protective role hypothesized for 11ß-HSD2, the onset of its placental expression has yet to be clearly established. To this aim, we present immunocytochemical analysis of placentas collected 3-6 weeks post-conception. 11ß-HSD2 was present as early as 3 weeks post-conception in syncytiotrophoblasts, where most maternal-fetal exchange occurs, and in columnar epithelial cells encircling uterine endometrial glands, which provide early histiopathic nutrition to the embryo. 11ß-HSD2 expression in these critical maternal-fetal exchange areas is consistent with its hypothesized protective role. Future studies should investigate the mechanisms that may modulate embryonic glucocorticoid exposure earlier, immediately post-conception.

Glyceraldehyde-3-phosphate dehydrogenase of rat erythrocytes has no membrane component.

In the course of studying mammalian erythrocytes we noted prominent differences in the red cells of the rat. Analysis of ghosts by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed that membranes of rat red cells were devoid of band 6 or the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). Direct measurements of this enzyme showed that glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was about 25% of that in human cells; all of the glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was within the cytoplasm and none was membrane bound; and in the human red cell, about 1/3 of the enzyme activity was within the cytoplasm and 2/3 membrane bound. The release of glyceraldehyde-3-phosphate dehydrogenase from fresh rat erythrocytes immediately following saponin lysis was also determined using the rapid filtration technique recently described. The extrapolated zero-time intercepts of these reactions confirmed that, in the rat erythrocyte, none of the cellular glyceraldehyde-3-phosphate dehydrogenase was membrane bound. Failure of rat glyceraldehyde-3-phosphate dehydrogenase to bind to the membranes of the intact rat erythrocyte seems to be due to cytoplasmic metabolites which interact with the enzyme and render it incapable of binding to the membrane.

Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.

Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.

8-Bromo-3',5'-adenosine monophosphate stimulates the endocrine activity of human cytotrophoblasts in culture.

Cytotrophoblasts, purified from human term placentae, were cultured in the absence or presence of 8-bromo-cAMP or 8-bromo-cGMP. 8-Bromo-cAMP provoked a dose-dependent increase in the secretion of hCG and progesterone within 24 h. After 48 h, hCG secretion increased by more than 200-fold, and progesterone secretion increased nearly 5-fold. 8-Bromo-cGMP had no effect on hCG secretion. In culture in serum-supplemented medium, the mononuclear cytotrophoblasts aggregated and fused to form syncytia. This morphological transformation was not affected by 8-bromo-cAMP. Immunocytochemical studies of the alpha- and beta-subunits of hCG in control and 8-bromo-cAMP-stimulated cultures demonstrated that the cyclic nucleotide analog promoted the synthesis of both subunits in all cellular forms, including single mononuclear cells, cell aggregates, and syncytia. In serum-free medium, the cytotrophoblasts did not aggregate or form syncytia, yet they responded to 8-bromo-cAMP with an increase in hCG secretion. We conclude that the endocrine function of cytotrophoblasts can be stimulated by a cAMP-dependent mechanism which can be initiated independently of the formation of a syncytium.

Transforming growth factor-beta stimulates trophoblast oncofetal fibronectin synthesis in vitro: implications for trophoblast implantation in vivo.

In pregnancy tissues, oncofetal fibronectin (onfFN) has been localized specifically to the extracellular matrix (ECM) surrounding extravillous anchoring trophoblasts of the placental-uterine junction and chorion. When isolated from first or third trimester placentas, human cytotrophoblasts in culture secrete and deposit onfFN in the ECM. In addition, onfFN synthesis is significantly up-regulated in response to serum stimulatory factor(s). The goal of this study was to examine the role of transforming growth factor-beta (TGF beta), a cytokine present in uterine decidua, as a stimulator of trophoblast onfFN production. Our initial insight into the significance of TGF beta resulted from the serendipitous use of cord serum from a neonate with severe alloimmune thrombocytopenia. Trophoblasts cultured in medium containing this serum underwent normal morphological differentiation, but produced markedly less onfFN. In an analogous fashion, trophoblasts cultured in normal serum preincubated with anti-TGF beta neutralizing antibodies also produced significantly less onfFN. Exogenously added TGF beta 1 restored the ability of trophoblasts to produce onfFN by a factor of 4- to 5-fold in medium containing thrombocytopenic serum. In platelet-poor serum derived from human or bovine plasma, TGF beta 1 also induced onfFN synthesis, as assayed both in the conditioned medium and by immunocytochemical localization of onfFN in cell-associated ECM fibrils. Dose-response analysis demonstrated that the onfFN stimulatory response is sensitive to TGF beta, with an ED50 of 0.1-0.2 ng/ml. In a reciprocal fashion, TGF beta inhibited beta hCG secretion 3- to 4-fold. Our results demonstrate that TGF beta is a significant stimulator of trophoblast onfFN production. Furthermore, TGF beta appears to modulate trophoblast differentiation by up-regulating the expression of an anchoring trophoblast marker (onfFN) and down-regulating a phenotypic marker of villous syncytiotrophoblast (hCG beta). We speculate that trophoblast responsiveness to TGF beta in the implantation milieu contributes to trophoblast adhesion by stimulating the production of a trophoblast-derived implantation site fibronectin.

The effect of leukemia inhibitory factor (LIF) on trophoblast differentiation: a potential role in human implantation.

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein strongly associated with normal implantation in the mouse. We have recently determined that LIF is expressed in the human endometrium in a menstrual cycle dependent manner. Maximal expression is observed between days 19 and 25 of the menstrual cycle, coinciding with the time of human implantation. In this study we have utilized purified cultures of human cytotrophoblasts to examine the effects of LIF on several morphologic and biochemical markers of the trophoblastic differentiation. We purified human cytotrophoblasts from term placentae and cultured them with and without LIF (10 ng/mL). The secretion of human CG, oncofetal fibronectin, and progesterone were measured at 24, 48, 72, and 96 h. Northern blot analysis was used to assess messenger RNA (mRNA) expression of beta hCG and oncofetal fibronectin. We found that LIF markedly decreased trophoblast production of hCG protein at 72 and 96 h, as well as expression of beta hCG mRNA. LIF also significantly increased the expression of oncofetal fibronectin mRNA and secretion of the protein. LIF did not affect steroidogenic activity of cultured trophoblasts, as determined by progesterone production. These biochemical changes are characteristic of cytotrophoblast differentiation toward an anchoring extravillous phenotype. Thus, LIF appears to be an important regulator of human embryonic implantation by directly modulating trophoblast differentiation.

8-Bromo-adenosine 3',5'-monophosphate regulates expression of chorionic gonadotropin and fibronectin in human cytotrophoblasts.

Addition of 8-bromo-cAMP to primary cultures of human placental cytotrophoblasts results in significant alterations in the synthesis of secreted proteins, as detected by labeling with pulses of [35S]methionine. Using immunoprecipitation techniques, we demonstrated that exposure to 8-bromo-cAMP prevented the de novo synthesis and secretion of the extracellular matrix component fibronectin, but enhanced the production of hCG subunits. The effects of the cyclic nucleotide on synthesis and secretion of these proteins were evident within 24 h. 8-Bromo-cAMP increased the cellular content of mRNA encoding the hCG alpha- and beta-subunits and prevented the increase in fibronectin mRNA, as determined by blot hybridization analysis using specific cDNA probes. These findings demonstrate that cyclic nucleotides regulate the synthesis of several specific proteins in cultured human trophoblast by regulating levels of the mRNAs encoding the proteins. The actions of cyclic nucleotides in this regard may be essential for the normal expression of trophoblast endocrine function.

Fetal akinesia deformation sequence (Pena-Shokeir phenotype) associated with acquired intrauterine brain damage.

An infant with Pena-Shokeir phenotype was born to a cocaine-using mother. The pathologic findings included polyhydramnios, facial anomalies, arthrogryposis, camptodactyly, pulmonary hypoplasia, and tetralogy of Fallot. The neuropathologic findings were diffuse brainstem and spinal cord neuronal degeneration and focal cerebral infarction, consistent with acquired intrauterine ischemic damage.

Human trophoblast-endometrial interactions in an in vitro suspension culture system.

We developed an in vitro suspension co-culture system to examine the interaction of 1st, 2nd and 3rd trimester purified cytotrophoblasts with human endometrium. Endometrium explants were added to cytotrophoblast cell suspensions and placed on an angled gyrating platform in a 37 degrees C incubator. When endometrium was cultured alone it was able to remain viable for up to 3 days. When trophoblasts were cultured alone, they formed small and large aggregates, and occasionally spherical shells with hollow centers. When trophoblasts and endometrium were cultured together, the trophoblasts adhered to the exposed stromal surfaces of the tissue fragments. The surface epithelium was not receptive to trophoblast attachment except in one experiment when day 19 endometrium was used for the co-incubation, suggesting that surface attachment is usually restricted. A common finding was the presence of an acellular zone in the endometrium only adjacent to the attached trophoblasts. We speculate that this zone may be caused by proteolysis and resynthesis of ECM proteins by the trophoblasts. Based on our results, this in vitro suspension should prove useful for examining those factors which: (1) induce endometrial permissiveness, (2) promote paracrine effects on the endometrium, and (3) facilitate human trophoblast invasion.

Preeclampsia, trisomy 13, and the placental bed.

Genetic predisposition and abnormal trophoblastic function are thought to contribute to the development of preeclampsia. A multipara developed severe preeclampsia and subsequently delivered a live growth-retarded infant with trisomy 13. Biopsy of the placental bed taken immediately after delivery demonstrated inadequate trophoblastic remodeling of the maternal uterine vasculature, with an absence of normal physiologic changes in the spiral arteries. This case suggests that fetal trisomy 13 can be associated with preeclampsia in multiparous women and that abnormal trophoblastic invasion may contribute to the pathophysiology.

Endosalpingiosis found at laparoscopy for chronic pelvic pain.

OBJECTIVE: To assess a correlation between endosalpingiosis and pelvic pain. DESIGN: A retrospective analysis of every patient undergoing laparoscopy for chronic pelvic pain at Yale-New Haven Hospital by one surgeon from August 1992 through October 1993 was performed, focusing on those cases with endosalpingiosis. RESULTS: Of 51 laparoscopies performed for chronic pelvic pain, 37 demonstrated visual evidence of implants and pathology specimens were read as either endometriosis or endosalpingiosis in 23 cases. Of those 23 cases, 6 demonstrated endosalpingiosis, and 4 of those 6 demonstrated both endosalpingiosis and endometriosis. In all six cases endosalpingiosis was found in locations consistent with the patients' pelvic pain symptoms, and all six patients experienced relief from their pain symptoms after surgery. CONCLUSIONS: Endosalpingiosis may be found in association with chronic pelvic pain. The pelvic distribution of endosalpingiosis in patients with chronic pain is consistent with that generally found in endometriosis.

Mouse ascites golgi (MAG) mucin expression and regulation by progesterone in the rat uterus.

OBJECTIVE: To study the regulation of the blood group A-related high-molecular weight mucin glycoprotein epitope (mouse ascites golgi, MAG)-a menstrual cycle-dependent marker of endometrial receptivity-in a non-human endometrium model. METHODS: Immature Sprague-Dawley rats were injected with 1 microg of estradiol, 100 microg of testosterone, 100 microg of dexamethasone, 2.5 mg of progesterone (P), 0.325 mg of RU486, P and RU486, 100 microg of tamoxifen, or vehicle for 3 days, sacrificed, and the uteri were stained for MAG. Immunohistochemistry and blood analysis were the measurements used to compare the specimens from the exogenous hormonal and endogenous hormonal groups. Electron microscopy was used to locate the MAG epitope in one pseudopregnant adult Sprague-Dawley rat. RESULTS: The MAG epitope was present in endometrial glands of Sprague-Dawley rats, with maximal expression during proestrus and diestrus. Electron microscopy confirmed the Golgi location of this MAG epitope. In the untreated group, less than 0.5% of endometrial glands stained for MAG. The MAG was seen only in the glands of the P-treated rats and RU486 blunted this stimulatory effect by more than 95%. As little as 0.1 mg of P promoted MAG expression, with maximal response at 2.5 mg. Staining was seen 24 hours after P treatment, peaked at 72 hours, then declined. Induction of endogenous P by superovulation with pregnant mare serum gonadotropin (PMSG) and hCG (pseudopregnancy) also resulted in strong MAG glandular staining. CONCLUSION: Our results suggest that the MAG epitope is cyclically expressed and induced by P in rat endometrial glands.

Optimization of endometrial preparation results in a normal endometrial function test (EFT) and good reproductive outcome in donor ovum recipients.

PURPOSE: Numerous studies have investigated potential markers of endometrial receptivity as predictors of successful implantation. Cyclin E and p27 have recently been studied using the endometrial function test (EFT). Our objective is to determine the correlation between the expression of cyclin E and p27 and the adequacy of uterine preparation of recipients using donor oocytes. METHODS: Twenty recipients undergoing preparatory cycles with leuprolide acetate, estrogen, and progesterone. Endometrial biopsies were obtained 10-12 days after progesterone supplementation following the course of estrogen. The tissue was prepared for histological analysis and immunohistochemical staining for cyclin E assessment. The outcome of their subsequent ovum donation cycle was blinded to the reviewer of the EFT. RESULTS: All recipients showed normal luteal transformation. Nineteen (95%) of the recipients had a normal EFT. This is significantly higher than what we demonstrated, previously, in unexplained infertility patients, where only 40% of such patients had a normal EFT. Thirteen recipients with a normal EFT had a clinical pregnancy, while 6 did not become pregnant in their subsequent transfer cycles. The sole patient with an abnormal EFT did not conceive on 2 subsequent cycles. CONCLUSIONS: While a normal EFT does not guarantee a successful pregnancy, an abnormal EFT appears to be associated with pregnancy failure. This may be useful in identifying women who need adjustments to their stimulation protocols prior to progressing to a physically, emotionally, and financially costly cycle.

Association of glyceraldehyde-3-phosphate dehydrogenase with the human red cell membrane. A kinetic analysis.

A rapid filtration technique was used to analyze the kinetics of the binding reaction between glyceraldehyde-3-P dehydrogenase and the band 3 protein of the human erythrocyte membrane. Saponin was used to eliminate the membrane as a rate-limiting barrier. The re-equilibration of the enzyme following dilution of membranes in buffer took only a few seconds. Dissociation rates were greatly stimulated and association rates were reduced by increasing ionic strength. A Brönsted-Bjerrum analysis suggested that two or three charges of opposite sign on each protein were involved in binding. NADH appeared to elute the enzyme by interaction with its catalytic site, but the band 3 binding site extended beyond the NADH site, permitting the formation of a ternary NADH . enzyme . membrane complex. The release of the enzyme from fresh erythrocytes immediately following saponin lysis showed kinetics similar to the release of enzyme from ghosts. The extrapolated zero time intercepts of these reactions suggested that two-thirds of cellular glyceraldehyde-3-P dehydrogenase was membrane bound prior to hemolysis. This value is similar to that calculated for the association of the enzyme with band 3 in the intact red cell when the high ionic strength and enzyme-eluting compounds in the cytoplasm are taken into account.

Human trophoblast-extracellular matrix (ECM) interactions in vitro: ECM thickness modulates morphology and proteolytic activity.

Trophoblast invasion of the uterine extracellular matrix, a critical process for human implantation and uteroplacental vascular development, is a striking example of controlled invasiveness. To examine cellular behavior relevant to this process, human trophoblasts were cultured on (i) Millicell filters prelayered with Matrigel and (ii) coverslips precoated with a gentle slope of Matrigel (Matribeach). Histologic sections of the Millicell system demonstrated significant invasion. However, on Matribeach the cells exhibited markedly different characteristics depending on the thickness of the Matrigel. On zone 1 (1-4 microns thick), flat aggregates and syncytia were seen. In contrast, cells on zone 2 (4-14 microns) formed rounded aggregates with intercellular processes. In this zone, prominent degradation of pericellular Matrigel proteins was assessed by both light microscopy and scanning electron microscopy. Treatment with 8-bromo-cAMP inhibited this proteolytic process. On zone 3 (14-60 microns), unicellular trophoblasts or small aggregates caused minimal matrix degradation. JEG-3 human choriocarcinoma cells exhibited similar morphologic and degradative properties on Matribeach, but zone 2 proteolysis was not affected by 8-bromo-cAMP. Our results suggest that extracellular matrix thickness has profound effects on cellular morphology and proteolytic activity. Furthermore, while both normal and malignant human trophoblasts can degrade extracellular matrix proteins, only normal trophoblast extracellular matrix degradation is inhibited by 8-bromo-cAMP.

Is oncofetal fibronectin a trophoblast glue for human implantation?

Using an antibody probe specific for the class of fibronectins that contain the oncofetal domain, it was shown that oncofetal fibronectin (onfFN) is present wherever trophoblasts make contact with extraplacental extracellular matrix (ECM). In normal human implantation sites, onfFN was localized to a highly specific region-the ECM connecting extravilous trophoblasts and trophoblastic cell columns to the uterine decidua. This same zone of onfFN was present in an analogous location in extrauterine gestations. Like these in vivo extravillous trophoblasts, isolated cytotrophoblasts in primary culture synthesized and secreted onfFN as they underwent differentiation. Furthermore, when cocultured with an ECM gel, cytotrophoblast aggregates deposited onfFN at cell-ECM contact sites, resembling early implanting trophoblasts in vivo. In the presence of cyclic AMP agonists, onfFN synthesis was inhibited markedly. It is concluded from these results that onfFN is a trophoblast protein that, under cAMP regulation, could mediate implantation and placental-uterine attachment throughout gestation.

The efficacy of the placental biopsy.

OBJECTIVE: Our purpose was to determine the sensitivity and specificity of pathologic diagnoses made from a placental biopsy specimen compared with diagnoses made from a complete placental examination. STUDY DESIGN: Biopsy was performed on 200 singleton placentas with a 16-gauge Rutner biopsy needle shortly after delivery. The biopsy specimens and placentas were evaluated by standard placental pathologic criteria. RESULTS: The presence of villous edema on the biopsy specimen led to the diagnosis of placental villous edema with a sensitivity of 51% and specificity of 86%, yielding a positive predictive value of 0.97. The sensitivity of the biopsy diagnosis of "increased syncytial knots" was 86%, whereas the specificity was 82%, yielding a positive predictive value of 0.90. CONCLUSIONS: Because a placental biopsy specimen after delivery is reasonably sensitive for diagnosing villous abnormalities that reflect acute and chronic stresses to the placenta, it may be useful to develop a placental biopsy that can be performed safely during pregnancy. Such a biopsy could be the basis for the rational treatment of some diseases of pregnancy.