The epigenetic modifier ubiquitin-specific protease 22 (USP22) regulates embryonic stem cell differentiation via transcriptional repression of sex-determining region Y-box 2 (SOX2).

Pluripotent embryonic stem cells (ESCs) undergo self-renewal until stimulated to differentiate along specific lineage pathways. Many of the transcriptional networks that drive reprogramming of a self-renewing ESC to a differentiating cell have been identified. However, fundamental questions remain unanswered about the epigenetic programs that control these changes in gene expression. Here we report that the histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is a critical epigenetic modifier that controls this transition from self-renewal to differentiation. USP22 is induced as ESCs differentiate and is necessary for differentiation into all three germ layers. We further report that USP22 is a transcriptional repressor of the locus encoding the core pluripotency factor sex-determining region Y-box 2 (SOX2) in ESCs, and this repression is required for efficient differentiation. USP22 occupies the Sox2 promoter and hydrolyzes monoubiquitin from ubiquitylated histone H2B and blocks transcription of the Sox2 locus. Our study reveals an epigenetic mechanism that represses the core pluripotency transcriptional network in ESCs, allowing ESCs to transition from a state of self-renewal into lineage-specific differentiation programs.

Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success.

Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persist for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods.

Quantitative temporal proteomic analysis of human embryonic stem cell differentiation into oligodendrocyte progenitor cells.

Oligodendrocytes (OLs) are glial cells of the central nervous system, which produce myelin. Cultured OLs provide immense therapeutic opportunities for treating a variety of neurological conditions. One of the most promising sources for such therapies is human embryonic stem cells (ESCs) as well as providing a model to study human OL development. For these purposes, an investigation of proteome level changes is critical for understanding the process of OL differentiation. In this report, an iTRAQ-based quantitative proteomic approach was used to study multiple steps during OL differentiation including neural progenitor cells, glial progenitor cells and oligodendrocyte progenitor cells (OPCs) compared to undifferentiated ESCs. Using a 1% false discovery rate cutoff, ∼3145 proteins were quantitated and several demonstrated progressive stage-specific expression. Proteins such as transferrin, neural cell adhesion molecule 1, apolipoprotein E and wingless-related MMTV integration site 5A showed increased expression from the neural progenitor cell to the OPC stage. Several proteins that have demonstrated evidence or been suspected in OL maturation were also found upregulated in OPCs including fatty acid-binding protein 4, THBS1, bone morphogenetic protein 1, CRYAB, transferrin, tenascin C, COL3A1, TGFBI and EPB41L3. Thus, by providing the first extensive proteomic profiling of human ESC differentiation into OPCs, this study provides many novel proteins that are potentially involved in OL development.

Identification of RNA binding motif proteins essential for cardiovascular development.

BACKGROUND: We recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis with additional roles in vasculogenesis and angiogenesis. RESULTS: To determine the role of this gene in cardiac development, we have identified its zebrafish orthologs (rbm24a and rbm24b), and functionally evaluated them during zebrafish embryogenesis. Consistent with our underlying hypothesis, reduction in expression of either ortholog through injection of morpholino antisense oligonucleotides results in cardiogenic defects including cardiac looping and reduced circulation, leading to increasing pericardial edema over time. Additionally, morphant embryos for either ortholog display incompletely overlapping defects in the forming vasculature of the dorsal aorta (DA), posterior caudal vein (PCV) and caudal vein (CV) which are the first blood vessels to form in the embryo. Vasculogenesis and early angiogenesis in the trunk were similarly compromised in rbm24 morphant embryos at 48 hours post fertilization (hpf). Subsequent vascular maintenance was impaired in both rbm24 morphants with substantial vessel degradation noted at 72 hpf. CONCLUSION: Taken collectively, our functional data support the hypothesis that rbm24a and rbm24b are key developmental cardiac genes with unequal roles in cardiovascular formation.

Mouse ES cell-derived cardiac precursor cells are multipotent and facilitate identification of novel cardiac genes.

Although the differentiation of ES cells to cardiomyocytes has been firmly established, the extent to which corresponding cardiac precursor cells can contribute to other cardiac populations remains unclear. To determine the molecular and cellular characteristics of cardiac-fated populations derived from mouse ES (mES) cells, we isolated cardiac progenitor cells (CPCs) from differentiating mES cell cultures by using a reporter cell line that expresses GFP under the control of a cardiac-specific enhancer element of Nkx2-5, a transcription factor expressed early in cardiac development. This ES cell-derived CPC population initially expressed genetic markers of both stem cells and mesoderm, while differentiated CPCs displayed markers of 3 distinct cell lineages (cardiomyocytes, vascular smooth muscle cells, and endothelial cells)--Flk1 (also known as Kdr), c-Kit, and Nkx2-5, but not Brachyury--and subsequently expressed Isl1. Clonally derived CPCs also demonstrated this multipotent phenotype. By transcription profiling of CPCs, we found that mES cell-derived CPCs displayed a transcriptional signature that paralleled in vivo cardiac development. Additionally, these studies suggested the involvement of genes that we believe were previously unknown to play a role in cardiac development. Taken together, our data demonstrate that ES cell-derived CPCs comprise a multipotent precursor population capable of populating multiple cardiac lineages and suggest that ES cell differentiation is a valid model for studying development of multiple cardiac-fated tissues.

Comparative proteomics of human embryonic stem cells and embryonal carcinoma cells.

Pluripotent human embryonic stem cells (ESCs) can be differentiated in vitro into a variety of cells which hold promise for transplantation therapy. Human embryonal carcinoma cells (ECCs), stem cells of human teratocarcinomas, are considered a close but malignant counterpart to human ESCs. In this study, a comprehensive quantitative proteomic analysis of ESCs and ECCs was carried out using the iTRAQ method. Using two-dimensional LC and MS/MS analyses, we identified and quantitated approximately 1800 proteins. Among these are proteins associated with pluripotency and development as well as tight junction signaling and TGFbeta receptor pathway. Nearly approximately 200 proteins exhibit more than twofold difference in abundance between ESCs and ECCs. Examples of early developmental markers high in ESCs include beta-galactoside-binding lectin, undifferentiated embryonic cell transcription factor-1, DNA cytosine methyltransferase 3beta isoform-B, melanoma antigen family-A4, and interferon-induced transmembrane protein-1. In contrast, CD99-antigen (CD99), growth differentiation factor-3, cellular retinoic acid binding protein-2, and developmental pluripotency associated-4 were among the highly expressed proteins in ECCs. Several proteins that were highly expressed in ECCs such as heat shock 27 kDa protein-1, mitogen-activated protein kinase kinase-1, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor like-2, and S100 calcium-binding protein-A4 have also been attributed to malignancy in other systems. Importantly, immunocytochemistry was used to validate the proteomic analyses for a subset of the proteins. In summary, this is the first large-scale quantitative proteomic study of human ESCs and ECCs, which provides critical information about the regulators of these two closely related, but developmentally distinct, stem cells.

Efficient differentiation of human embryonic stem cells into oligodendrocyte progenitors for application in a rat contusion model of spinal cord injury.

This study utilized a contusion model of spinal cord injury (SCI) in rats using the standardized NYU-MASCIS impactor, after which oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem cell (ESC) were transplanted into the spinal cord to study their survival and migration route toward the areas of injury. One critical aspect of successful cell-based SCI therapy is the time of injection following injury. OPCs were injected at two clinically relevant times when most damage occurs to the surrounding tissue, 3 and 24 hours following injury. Migration and survivability after eight days was measured postmortem. In-vitro immunofluorescence revealed that most ESC-derived OPCs expressed oligodendrocyte markers, including CNPase, GalC, Olig1, O4, and O1. Results showed that OPCs survived when injected at the center of injury and migrated away from the injection sites after one week. Histological sections revealed integration of ESC-derived OPCs into the spinal cord with contusion injury without disruption to the parenchyma. Cells survived for a minimum of eight days after injury, without tumor or cyst formation. The extent of injury and effect of early cell transplant was measured using behavioral and electrophysiological assessments which demonstrated increased neurological responses in rats transplanted with OPCs compared to controls.

Expression of pluripotent stem cell markers in the human fetal testis.

Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). However, the developmental potency of these cells in the fetal gonad still remains elusive. Thus, this study provides a comprehensive analysis of pluripotent and germ cell marker expression in human fetal testis 7-15 weeks postfertilization (pF) and compares this expression to their ability to derive EGCs. Although the majority of germ cells expressed stem cell markers stage-specific embryonic antigen (SSEA) 1, SSEA4, EMA-1, and alkaline phosphatase, only a small percentage of those (<1%) expressed OCT4, CKIT, and NANOG. Specifically, the number of OCT4(+)/CKIT(+)/NANOG(+) cells significantly increased in the developing cords during weeks 7-9, followed by a gradual decline into week 15 pF. By week 15 pF, the remaining OCT4(+)/CKIT(+)/NANOG(+) cells were found in the cords surrounding the periphery of the testis, and the predominant germ cells, CKIT(+) cells, no longer expressed OCT4 or NANOG. Based on morphology and early germ cell marker expression, including VASA, PUM2, and DAZL, we suggest these cells are mitotically active gonocytes or prespermatogonia. Importantly, the number of OCT4(+) cells correlated with an increase in the number of EGC colonies derived in culture. Interestingly, two pluripotent markers, Tra-1-60 and Tra-1-81, although highly expressed in EGCs, were not expressed by PGCs in the gonad. Together, these results suggest that PGCs maintain expression of pluripotent stem cell markers during and after sexual differentiation of the gonad, albeit in very low numbers.

SOX17 directly activates Zfp202 transcription during in vitro endoderm differentiation.

SOX17 is a SRY-related high-mobility group (HMG) box transcription factor that is necessary for endoderm formation in multiple species. Despite its essential function during endoderm formation and differentiation, few direct targets of SOX17 are known. To identify targets of SOX17, we isolated SOX17 binding sites with a chromatin immunoprecipitation (ChIP)-cloning screen. SOX17-ChIP identified zinc finger protein 202 (Zfp202) as a direct target of SOX17 during endoderm differentiation of F9 embryonal carcinoma cells. A sequence in the first intron of Zfp202 activated transcription in differentiated F9 cells, and overexpression of Sox17 increased the transcriptional activity of this sequence. SOX17 binds to a site within this sequence in electrophoretic mobility shift assays, and mutation of this site decreases the transcriptional activation. Zfp202 is induced concomitantly with Sox17 during endoderm differentiation of F9 cells. We also show that ZFP202 represses Hnf4a, which has been reported for the human ortholog ZNF202. Identifying targets of SOX17 will help to elucidate the molecular basis of endoderm differentiation and may provide a better understanding of the role of endoderm in patterning the other germ layers.

Expression of pluripotent stem cell markers in the human fetal ovary.

BACKGROUND: Human primordial germ cells (PGCs) can give rise to pluripotent stem cells such as embryonal carcinoma cells (ECCs) and embryonic germ cells (EGCs). METHODS: In order to determine whether PGCs express markers associated with pluripotency in EGCs and ECCs, the following study cross examines the expression patterns of multiple pluripotent markers in the human fetal ovary, 5.5-15 weeks post-fertilizaton (pF) and relates this expression with the ability to derive pluripotent EGCs in vitro. RESULTS: Specific subpopulations were identified which included OCT4(+)/Nanog(+)/cKIT(+)/VASA(+) PGCs and oogonia. Interestingly, these cells also expressed SSEA1 and alkaline phosphatase (AP) and SSEA4 expression occurred throughout the entire gonad. Isolation of SSEA1(+) cells from the gonad resulted in AP(+) EGC colony formation. The number of OCT4(+) or Nanog(+) expressing cells peaked by week 8 and then diminished after week 9 pF, as oogonia enter meiosis. In addition, the efficiency of EGC derivation was associated with the number of OCT4(+) cells. TRA-1-60 and TRA-1-81 were only detected in the lining of the mesonephric ducts and occasionally in the gonad. CONCLUSIONS: These results demonstrate that PGCs, a unipotent cell, express most, but not all, of the markers associated with pluripotent cells in the human fetal ovary.

Pluripotent stem cells from germ cells.

To date, stem cells have been derived from three sources of germ cells. These include embryonic germ cells (EGCs), embryonal carcinoma cells (ECCs), and multipotent germ line stem cells (GSCs). EGCs are derived from primordial germ cells that arise in the late embryonic and early fetal period of development. ECCs are derived from adult testicular tumors whereas GSCs have been derived by culturing spermatogonial stem cells from mouse neonates and adults. For each of these lines, their pluripotency has been demonstrated by their ability to differentiate into cell types derived from the three germ layers in vitro and in vivo and in chimeric animals, including germ line transmission. These germ line-derived stem cells have been generated from many species including human, mice, porcine, and chicken albeit with only slight modifications. This chapter describes general considerations regarding critical aspects of their derivation compared with their counterpart, embryonic stem cells (ESCs). Detailed protocols for EGC derivation and maintenance from human and mouse primordial germ cells (PGCs) will be presented.

Embryonic germ cells: when germ cells become stem cells.

Embryonic germ cells (EGCs) are pluripotent stem cells derived from primordial germ cells (PGCs). PGCs are progenitors of adult gametes, which diverge from the somatic lineage between late embryonic to early fetal development. First derived in the mouse, EGCs have also been derived from human, chicken, and pig. As pluripotent stem cells, EGCs demonstrate long-term self-renewal via clonal expansion in an undifferentiated state, and differentiate in vitro to form embryoid bodies containing cells that represent all three germ layers as well as mixed cell populations of less differentiated progenitors and precursors. This is also demonstrated in vivo by their formation into experimentally induced teratocarcinomas following transplantation. Furthermore, mice, pig, and chicken EGCs have also been shown to contribute to experimentally produced chimeric animals, including germline transmission. Importantly, EGCs demonstrate normal and stable karyotypes as well as normal patterns of genomic imprinting, including X-inactivation. Transplantation studies have begun in a variety of models in hopes of defining their potential use to treat a wide variety of human conditions, including diabetes and urological and neurological disorders.

Honoring the work and life of Leroy C. Stevens. A symposium as part of the International Stem Cell Initiative Workshop.

In 2016, a symposium was convened in Leroy C. Stevens' honor, in association with a meeting of the International Stem Cell Initiative (ISCI). ISCI, funded internationally, is composed of a group of ~100 scientists from many countries, under the leadership of Peter Andrews, who have worked together to characterize a significant number of human pluripotent stem cell lines, to monitor their genetic stability and their differentiation into mature cell types and tissues in vitro and in vivo. Those at the ISCI meeting puzzled through one of the thorniest problems in the therapeutic use of the differentiated derivatives of pluripotent stem cells for human therapy; namely, pluripotent stem cells can differentiate into any cell type in the adult organism, but they also have the capacity for unlimited self-renewal, hence if mutated they may have tumorigenic potential. The meeting considered how these cells might become genetically or epigenetically abnormal and how the safety of these cells for human therapeutic uses could be assessed and assured. The symposium was an opportunity to pay tribute to Leroy Stevens and to the basic science origins of this newest aspect of regenerative medicine. It was a time to reflect on the past and on how it can influence the future of our field.

Human embryonic germ cell derivatives facilitate motor recovery of rats with diffuse motor neuron injury.

We have investigated the potential of human pluripotent cells to restore function in rats paralyzed with a virus-induced motor neuronopathy. Cells derived from embryonic germ cells, termed embryoid body-derived (EBD) cells, introduced into the CSF were distributed extensively over the rostrocaudal length of the spinal cord and migrated into the spinal cord parenchyma in paralyzed, but not uninjured, animals. Some of the transplanted human cells expressed the neuroglial progenitor marker nestin, whereas others expressed immunohistochemical markers characteristic of astrocytes or mature neurons. Rare transplanted cells developed immunoreactivity to choline acetyltransferase (ChAT) and sent axons into the sciatic nerve as detected by retrograde labeling. Paralyzed animals transplanted with EBD cells partially recovered motor function 12 and 24 weeks after transplantation, whereas control animals remained paralyzed. Semi-quantitative analysis revealed that the efficiency of neuronal differentiation and extension of neurites could not account for the functional recovery. Rather, transplanted EBD cells protected host neurons from death and facilitated reafferentation of motor neuron cell bodies. In vitro, EBD cells secrete transforming growth factor-alpha (TGF-alpha) and brain-derived neurotrophic factor (BDNF). Neutralizing antibodies to TGF-alpha and to BDNF abrogated the ability of EBD-conditioned media to sustain motor neuron survival in culture, whereas neutralizing antibodies to BDNF eliminated the axonal outgrowth from spinal organotypics observed with direct coculture of EBD cells. We conclude that cells derived from human pluripotent stem cells have the capacity to restore neurologic function in animals with diffuse motor neuron disease via enhancement of host neuron survival and function.

Bone morphogenetic proteins produced by cells within embryoid bodies inhibit ventral directed differentiation by Sonic Hedgehog.

Mouse embryoid bodies (EBs) differentiate into dorsal spinal cord neural progenitors in response to retinoic acid (RA). Our data demonstrate that the addition of Sonic Hedgehog (Shh) directs towards a ventral spinal cord neural tube fate, but only at extremely high concentrations. One possible explanation is the presence of dorsal directing factors. Bone morphogenetic proteins (BMPs), known to direct dorsal spinal cord neural differentiation, were expressed in RA-treated EBs. Shh more potently directed ventral differentiation when combined with the BMP inhibitor Noggin. Further, when BMP7 was added, the ability of Shh to direct ventral differentiation was further mitigated.

Direct Reprogramming of Human Primordial Germ Cells into Induced Pluripotent Stem Cells: Efficient Generation of Genetically Engineered Germ Cells.

Primordial germ cells (PGCs) share many properties with embryonic stem cells (ESCs) and innately express several key pluripotency-controlling factors, including OCT4, NANOG, and LIN28. Therefore, PGCs may provide a simple and efficient model for studying somatic cell reprogramming to induced pluripotent stem cells (iPSCs), especially in determining the regulatory mechanisms that fundamentally define pluripotency. Here, we report a novel model of PGC reprogramming to generate iPSCs via transfection with SOX2 and OCT4 using integrative lentiviral. We also show the feasibility of using nonintegrative approaches for generating iPSC from PGCs using only these two factors. We show that human PGCs express endogenous levels of KLF4 and C-MYC protein at levels similar to embryonic germ cells (EGCs) but lower levels of SOX2 and OCT4. Transfection with both SOX2 and OCT4 together was required to induce PGCs to a pluripotent state at an efficiency of 1.71%, and the further addition of C-MYC increased the efficiency to 2.33%. Immunohistochemical analyses of the SO-derived PGC-iPSCs revealed that these cells were more similar to ESCs than EGCs regarding both colony morphology and molecular characterization. Although leukemia inhibitory factor (LIF) was not required for the generation of PGC-iPSCs like EGCs, the presence of LIF combined with ectopic exposure to C-MYC yielded higher efficiencies. Additionally, the SO-derived PGC-iPSCs exhibited differentiation into representative cell types from all three germ layers in vitro and successfully formed teratomas in vivo. Several lines were generated that were karyotypically stable for up to 24 subcultures. Their derivation efficiency and survival in culture significantly supersedes that of EGCs, demonstrating their utility as a powerful model for studying factors regulating pluripotency in future studies.

Early intervention for spinal cord injury with human induced pluripotent stem cells oligodendrocyte progenitors.

Induced pluripotent stem (iPS) cells are at the forefront of research in regenerative medicine and are envisaged as a source for personalized tissue repair and cell replacement therapy. Here, we demonstrate for the first time that oligodendrocyte progenitors (OPs) can be derived from iPS cells generated using either an episomal, non-integrating plasmid approach or standard integrating retroviruses that survive and differentiate into mature oligodendrocytes after early transplantation into the injured spinal cord. The efficiency of OP differentiation in all 3 lines tested ranged from 40% to 60% of total cells, comparable to those derived from human embryonic stem cells. iPS cell lines derived using episomal vectors or retroviruses generated a similar number of early neural progenitors and glial progenitors while the episomal plasmid-derived iPS line generated more OPs expressing late markers O1 and RIP. Moreover, we discovered that iPS-derived OPs (iPS-OPs) engrafted 24 hours following a moderate contusive spinal cord injury (SCI) in rats survived for approximately two months and that more than 70% of the transplanted cells differentiated into mature oligodendrocytes that expressed myelin associated proteins. Transplanted OPs resulted in a significant increase in the number of myelinated axons in animals that received a transplantation 24 h after injury. In addition, nearly a 5-fold reduction in cavity size and reduced glial scarring was seen in iPS-treated groups compared to the control group, which was injected with heat-killed iPS-OPs. Although further investigation is needed to understand the mechanisms involved, these results provide evidence that patient-specific, iPS-derived OPs can survive for three months and improve behavioral assessment (BBB) after acute transplantation into SCI. This is significant as determining the time in which stem cells are injected after SCI may influence their survival and differentiation capacity.

The Role of E-cadherin in the Motility and Invasion of Implantation Site Intermediate Trophoblast.

During early pregnancy, intermediate (extravillous) trophoblast infiltrates the basal plate and invades the spiral arteries, a physiological process required to establish the maternal-fetal circulation. Immunostaining studies have shown that differentiation of trophoblast into this invasive subpopulation is associated with down-regulation of E-cadherin expression. To study the function of E-cadherin in trophoblast in vitro, we restored E-cadherin expression in an E-cadherin negative human implantation site intermediate trophoblastic cell line, IST-1, using a recombinant adenovirus, E-cad/Ad5 which constitutively expresses E-cadherin. In contrast to the control IST-1 cells which were individual and pleomorphic in shape, E-cad/Ad5 transduced cells were cohesive, uniform, and round. The motility and invasiveness of E-cad/Ad5 transduced IST-1 cells, as compared with the control cells, was significantly reduced. These effects were contact-dependent and were attenuated by a function-perturbing anti-E-cadherin antibody. In conclusion, our results indicate that expression of E-cadherin in IST-1 cells results in a contact-mediated inhibition of motility and invasion and suggest an important role for E-cadherin down-regulation in the intermediate trophoblast during implantation.

Decreased progesterone receptor expression in the intermediate trophoblastic cells of spontaneous abortions.

OBJECTIVE: To determine whether there are differences in the expression of progesterone receptor (PR) in intermediate trophoblastic cells of pregnancies ending in either spontaneous abortion (SAB) or elective abortion. DESIGN: Immunohistochemical identification of PR in intermediate trophoblastic cells. SETTING: Academic medical center. PATIENT(S): Subjects were 86 patients who either underwent first trimester SAB or elective abortion. INTERVENTION(S): All SAB and elective abortion specimens were serially sectioned and immunohistochemically stained for PR and for melanoma cell adhesion molecule. Melanoma cell adhesion molecule immunohistochemical staining was used as a sensitive and specific marker to identify intermediate trophoblastic cells on the adjacent tissue section. MAIN OUTCOME MEASURE(S): The PR staining of intermediate trophoblastic cells by semiquantitative immunostaining score. RESULT(S): The PR expression in intermediate trophoblastic cells was significantly greater in elective abortion specimens than in SAB specimens. When controlling for estimated gestational age, the difference in PR expression was even greater. CONCLUSION(S): The quantity of PR in intermediate trophoblastic cells is significantly less in SAB when compared to elective abortion pregnancies. Although it is unknown whether this is a primary or secondary event, this information may be an important finding in attempting to characterize both the molecular etiology of implantation and the molecular pathophysiology of SAB.