Microglia Activation in the Midbrain of the Human Neonate: The Effect of Perinatal Hypoxic-Ischemic Injury.

Perinatal hypoxia-ischemia (PHI) is a major risk factor for the development of neuropsychiatric deficits later in life. We previously reported that after prolonged PHI, the dopaminergic neurons of the human neonate showed a dramatic reduction of tyrosine hydroxylase (TH) in the substantia nigra, without important signs of neuronal degeneration despite the significant reduction in their cell size. Since microglia activation could precede neuronal death, we now investigated 2 microglia activation markers, ionized calcium-binding adapter molecule 1 (Iba1), and the phagocytosis marker Cd68. The highest Iba1 immunoreactivity was found in neonates with neuropathological lesions of severe/abrupt PHI, while the lowest in subjects with moderate/prolonged or older PHI. Subjects with very severe/prolonged or chronic PHI showed an increased Iba1 expression and very activated microglial morphology. Heavy attachment of microglia on TH neurons and remarkable expression of Cd68 were also observed indicating phagocytosis in this group. Females appear to express more Iba1 than males, suggesting a gender difference in microglia maturation and immune reactivity after PHI insult. PHI-induced microglial "priming" during the sensitive for brain development perinatal/neonatal period, in combination with genetic or other epigenetic factors, could predispose the survivors to neuropsychiatric disorders later in life, possibly through a sexually dimorphic way.

Clinical symptoms associated with laboratory findings and placental histopathology in full-term, non-infected neonates born to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive mothers.

This study comprehensively examines clinical symptoms, laboratory findings, and placental pathology in 40 cases of singleton full-term SARS-CoV-2 negative neonates. Their mothers, previously healthy, with uncomplicated pregnancies, were infected peripartum and presented COVID-19 symptoms of various severity. Neonates had predominately diarrhea, the yet unreported absent sucking reflex, elevated COVID-19 inflammatory and ischemia/asphyxia markers as serum ferritin, interleukin-6 and cardiac troponin-T, while placentas demonstrated mild vascular and/or inflammatory lesions. We hypothesize that the above placental lesions may be associated with transient perinatal hypoxia resulting in absent sucking reflex, as well as with inflammatory cytokines transfer causing diarrhea.

Pharyngeal sampling for PCR-testing in the investigation of SARS-COV-2 vertical transmission in pregnancy.

The novel COVID-19 global pandemic has raised, among many others, major concerns regarding the impact of infection during pregnancy. Current evidence suggests that vertical transmission from mother to baby, antenatally or intrapartum, does occur, but is uncommon. According to the published reports of infants born to COVID-19-affected mothers, as well as the anecdotal experience of current practices worldwide, it appears that investigations regarding the potential of SARS-COV-2 vertical transmission in pregnancy have so far been based, to a large extent, on PCR testing of neonatal pharyngeal swab samples. Given that the transplacental route of intrauterine transmission for SARS-COV-2 is less likely to immediately involve the upper respiratory tract of the newborn, contrary to what happens after birth, it would be advisable to include appropriate biological samples, such as cord blood, placenta, amniotic fluid and neonatal blood, along with the pharyngeal samples, in order to contribute significantly to such investigations. It is important to point out that negative PCR tests of neonatal pharyngeal samples do not exclude the possibility of intrauterine viral transmission, while positive pharyngeal swabs are more likely to reflect intrapartum or postpartum contaminants, rather than antenatal intrauterine transmission, in the absence of other criteria. Revision and enhancement of the so far prevailing practices appear important, in order to facilitate the development of good clinical practice for managing neonates and ensuring safety of families and healthcare providers.

Apoptotic Markers in the Midbrain of the Human Neonate After Perinatal Hypoxic/Ischemic Injury.

Our previous postmortem studies on neonates with neuropathological injury of perinatal hypoxia/ischemia (PHI) showed a dramatic reduction of tyrosine hydroxylase expression (dopamine synthesis enzyme) in substantia nigra (SN) neurons, with reduction of their cellular size. In order to investigate if the above observations represent an early stage of SN degeneration, we immunohistochemically studied the expression of cleaved caspase-3 (CCP3), apoptosis inducing factor (AIF), and DNA fragmentation by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin 3'-end-labeling (TUNEL) technique in the SN of 22 autopsied neonates (corrected age ranging from 34 to 46.5 gestational weeks), in relation to the severity/duration of PHI injury, as estimated by neuropathological criteria. No CCP3-immunoreactive neurons and a limited number of apoptotic TUNEL-positive neurons with pyknotic characteristics were found in the SN. Nuclear AIF staining was revealed only in few SN neurons, indicating the presence of early signs of AIF-mediated degeneration. By contrast, motor neurons of the oculomotor nucleus showed higher cytoplasmic AIF expression and nuclear translocation, possibly attributed to the combined effect of developmental processes and increased oxidative stress induced by antemortem and postmortem factors. Our study indicates the activation of AIF, but not CCP3, in the SN and oculomotor nucleus of the human neonate in the developmentally critical perinatal period.

The Effect of Perinatal Hypoxic/Ischemic Injury on Tyrosine Hydroxylase Expression in the Locus Coeruleus of the Human Neonate.

We have previously shown that perinatal hypoxic/ischemic injury (HII) may cause selective vulnerability of the mesencephalic dopaminergic neurons of human neonate. In the present study, we investigated the effect of perinatal HII on the noradrenergic neurons of the locus coeruleus (LC) of the same sample. We studied immunohistochemically the expression of tyrosine hydroxylase (TH, first limiting enzyme for catecholamine synthesis) in LC neurons of 15 autopsied infants (brains collected from the Greek Brain Bank) in relation to the neuropathological changes of acute or chronic HII of the neonatal brain. Our results showed that perinatal HII appears to affect the expression of TH and the size of LC neurons of the human neonate. In subjects with neuropathological lesions consistent with abrupt/severe HII, intense TH immunoreactivity was found in almost all neurons of the LC. In most of the neonates with neuropathological changes of prolonged or older injury, however, reduction in cell size and a decrease or absence of TH staining were observed in the LC. Intense TH immunoreactivity was found in the LC of 3 infants of the latter group, who interestingly had a longer survival time and had been treated with anticonvulsant drugs. Based on our observations and in view of experimental evidence indicating that the reduction of TH-immunoreactive neurons occurring in the LC after perinatal hypoxic insults persists into adulthood, we suggest that a dysregulation of monoaminergic neurotransmission in critical periods of brain development in humans is likely to predispose the survivors of perinatal HII, in combination with genetic susceptibility, to psychiatric and/or neurological disorders later in life.

A prenatally ascertained de novo terminal deletion of chromosomal bands 1q43q44 associated with multiple congenital abnormalities in a female fetus.

Terminal deletions in the long arm of chromosome 1 result in a postnatally recognizable disorder described as 1q43q44 deletion syndrome. The size of the deletions and the resulting phenotype varies among patients. However, some features are common among patients as the chromosomal regions included in the deletions. In the present case, ultrasonography at 22 weeks of gestation revealed choroid plexus cysts (CPCs) and a single umbilical artery (SUA) and therefore amniocentesis was performed. Chromosomal analysis revealed a possible terminal deletion in 1q and high resolution array CGH confirmed the terminal 1q43q44 deletion and estimated the size to be approximately 8 Mb. Following termination of pregnancy, performance of fetopsy allowed further clinical characterization. We report here a prenatal case with the smallest pure terminal 1q43q44 deletion, that has been molecularly and phenotypically characterized. In addition, to our knowledge this is the first prenatal case reported with 1q13q44 terminal deletion and Pierre-Robin sequence (PRS). Our findings combined with review data from the literature show the complexity of the genetic basis of the associated syndrome.

Insulin-like growth factors in embryonic and fetal growth and skeletal development (Review).

The insulin-like growth factors (IGF)-I and -II have a predominant role in fetal growth and development. IGFs are involved in the proliferation, differentiation and apoptosis of fetal cells in vitro and the IGF serum concentration has been shown to be closely correlated with fetal growth and length. IGF transcripts and peptides have been detected in almost every fetal tissue from as early in development as pre­implantation to the final maturation stage. Furthermore, IGFs have been demonstrated to be involved in limb morphogenesis. However, although ablation of Igf genes in mice resulted in growth retardation and delay in skeletal maturation, no impact on outgrowth and patterning of embryonic limbs was observed. Additionally, various molecular defects in the Igf1 and Igf1r genes in humans have been associated with severe intrauterine growth retardation and impaired skeletal maturation, but not with truncated limbs or severe skeletal dysplasia. The conflicting data between in vitro and in vivo observations with regard to bone morphogenesis suggests that IGFs may not be the sole trophic factors involved in fetal skeletal growth and that redundant mechanisms may exist in chondro- and osteogenesis. Further investigation is required in order to elucidate the functions of IGFs in skeletal development.

Mandibulofacial dysostosis (Treacher-Collins syndrome) in the fetus: novel association with Pectus carinatum in a molecularly confirmed case and review of the fetal phenotype.

BACKGROUND: Treacher Collins syndrome is the most common mandibulofacial dysostosis of autosomal dominant or, rarely, recessive inheritance. Affected fetuses may be identified by prenatal ultrasound or diagnosed at autopsy in case of perinatal death or pregnancy termination. METHODS: We describe the ultrasonographic, autopsy, and molecular findings in a 25-week-gestation affected fetus, and review the clinical, prenatal, and postmortem findings in 15 previously reported fetal and perinatal cases. RESULTS: A nearly complete spectrum of the typical facial characteristics can be present by the early second trimester of gestation, including subtle defects such as lower eyelid colobomas. Mandibular hypoplasia and bilateral auricle defects were constant findings in the affected fetal population. Downslanting palpebral fissures were the second more common feature, followed by midface hypoplasia, polyhydramnios, and ocular defects. Association with Pierre Robin sequence was common (38%) in the reviewed series. Previously unreported pectus carinatum was noted in our case bearing a heterozygous TCOF1 mutation. Other unique reported findings include salivary gland hyperplasia, single umbilical artery, and tracheo-esophageal fistula, all in molecularly unconfirmed cases. CONCLUSION: Treacher Collins syndrome can be prenatally detected by ultrasound and should be included in the wide range of genetic syndromes that can be diagnosed at perinatal autopsy. Affected fetuses tend to have a more severe phenotype than living patients. The reported association of Treacher Collins syndrome type 1 with pectus carinatum expands the phenotype, provides information on genotype-phenotype correlation, and suggests possible pathogenetic interactions between neural crest cell disorders and the formation of the sternum that merit investigation.

Immunohistochemical demonstration of urocortin 1 in Edinger-Westphal nucleus of the human neonate: colocalization with tyrosine hydroxylase under acute perinatal hypoxia.

Perinatal hypoxia could cause long-term disturbances of the dopaminergic (DA) systems, leading to behavioral and/or neurological deficits later in life. Increased expression of tyrosine hydroxylase (TH) was shown in the substantia nigra (SN) and ventral tegmental area (VTA) of human neonates that suffered severe/acute perinatal hypoxic insults, but also in all neurons of the Edinger-Westphal nucleus (EW). Since EW, in humans, contains urocortin 1 (UCN1)/centrally projecting neurons (EWcp), we investigated: (a) the development of UCN1-positive neurons and the possible effect of neonatal hypoxic/ischemic encephalopathy on UCN1 expression and (b) the possible colocalization of UCN1 with TH in neonates with histological signs of acute hypoxic injury. Our results showed that in EWcp of the human neonate, UCN1-immunoreactivity was already evident from 34 weeks of gestation onwards at very low levels. No UCN1-immunoreactivity was found in neurons of SN or VTA. In EWcp, a positive correlation was found between UCN1 expression and the age of the neonates, but not with hypoxia neuropathological grade. UCN1 was colocalized with TH in most EWcp neurons. Since UCN1 in EWcp may play a significant role in stress adaptation and consequently in stress-related disorders, the role of catecholamine synthesis in this nucleus under acute hypoxic conditions must be further investigated.

Vulnerability of the mesencephalic dopaminergic neurons of the human neonate to prolonged perinatal hypoxia: an immunohistochemical study of tyrosine hydroxylase expression in autopsy material.

Experimental studies indicate that hypoxia to the fetus, a common occurrence in many birth complications in humans, results in long-term disturbances of the central dopaminergic (DA) systems that persist in adulthood. Because dysregulation of DA systems is involved in the pathophysiology of many neurological and psychiatric disorders, we investigated the effects of perinatal hypoxia on the mesencephalic DA neurons of the human neonate using immunohistochemistry. We studied the expression of tyrosine hydroxylase (TH), the first and rate-limiting enzyme in catecholamine synthesis, in substantia nigra, and ventral tegmental area of 18 neonates in relation to the age and severity/duration of hypoxic injury estimated by neuropathological criteria. In severe/abrupt perinatal hypoxia, intense TH staining was observed in substantia nigra, ventral tegmental area, and, surprisingly, in the nonpreganglionic Edinger-Westphal nucleus. In severe/prolonged hypoxia, there was a striking reduction or even absence of TH immunoreactivity in all the mesencephalic nuclei. These observations suggest that at early states of perinatal hypoxia, there is a massive increase in dopamine synthesis and release that is followed by feedback blockage of dopamine synthesis through inhibition of TH by the end product dopamine. Early dysregulation of DA neurotransmission could predispose infant survivors of severe perinatal hypoxia to dopamine-related neurological and/or cognitive deficits later in life.

Expression of caspase-14 and keratin-19 in the human epidermis and appendages during fetal skin development.

Caspase-14 is a seemingly non-apoptotic caspase involved in keratinocyte differentiation and cornification of the skin. Keratin-19 is an epithelial marker and a potential marker of epidermal stem cells that is expressed during human fetal skin development. We examined the immunohistochemical expression of caspase-14 in relation to CK-19 in the human fetal skin during development and perinatally, to assess their role in human skin maturation. Skin samples were received at autopsy. In the fetal epidermis, caspase-14 was predominantly expressed in the more differentiated layers, gradually disappearing from the basal layer toward term. By contrast, keratin-19 expression gradually decreased with epidermal maturation through gestation (rho = -0.949; p = 0.0001) and was a marker of the germinative layers. Keratin-19 was preserved in scarce basal cell nests at term and postnatally. Caspase-14 and keratin-19 were inversely expressed in the differentiating epidermal layers through gestation (p < 0.0001). Concerning the appendages, in hair follicles and sebaceous glands, caspase-14 located preferentially in the more differentiated layers of the inner root sheath, whereas keratin-19 was expressed in the outer sheath. Eccrine sweat glands showed a variable pattern of caspase-14 and keratin-19 expression. In conclusion, caspase-14 emerged as a marker of human skin differentiation during development, while keratin-19 marked the germinative epithelial layers in the fetal epidermis and appendages and possibly the nests of epidermal stem cells.

Increased expression of tyrosine hydroxylase in the supraoptic nucleus of the human neonate under hypoxic conditions: a potential neuropathological marker for prolonged perinatal hypoxia.

The purpose of this study was to determine whether the increased expression of tyrosine hydroxylase (TH), the first and limiting enzyme in catecholamine synthesis in vasopressin (VP) neurons of the human neonate, represents a primary developmental phenomenon or reflects a secondary phenomenon related to the activation of VP systems due to perinatal hypoxia. Using immunohistochemistry, we investigated TH expression in the supraoptic nucleus (SON) of 15 human neonates at autopsy in relation to the age and severity/duration of hypoxic injury that was estimated on the basis of neuropathological criteria. Increased expression of TH was observed selectively in VP-synthesizing neurons of neonates who experienced prolonged perinatal hypoxia; was not related to the age, body weight/percentile, brain weight, or head perimeter of the subjects but depended on the neuropathological grade of the hypoxic injury (p < 0.01); and was found in VP-synthesizing neurons with increased cellular and nuclear size, that is, neurons with histological evidence of activation. Taken together, these observations indicate that increased expression of TH in VP neurons of SON is not developmentally determined but represents a response to hypoxic stress. We propose that increased TH expression in SON neurons of the human neonate may serve as a neuropathological marker of prolonged perinatal hypoxia in autopsy material.

Cranioectodermal dysplasia: a probable ciliopathy.

Cranioectodermal dysplasia (CED), also known as Sensenbrenner syndrome, is a rare autosomal recessive genetic disorder characterized by typical craniofacial, skeletal and ectodermal defects, and tubulointerstitial nephritis leading to early end-stage renal failure. We report on a new familial case of a 9-year-old patient and two fetuses of 23 and 19 weeks of gestation respectively. Hypohidrosis was an additional ectodermal finding is the patient with CED. Postmortem findings in the two fetuses included acromesomelic shortening, craniofacial characteristics with absence of craniosynostosis, small kidneys with tubular and glomerular microscopic cysts, persistent ductal plate with portal fibrosis in the liver, small adrenals and roughly unremarkable histopathology of the physeal growth plate. Posterior fossa anomalies were additional findings in this patient and included an enlarged cisterna magna and a posterior fossa cyst. The above findings, in association with renal cysts, persistent ductal plate and portal fibrosis, introduce CED, a nonlethal genetic skeletal disorder of yet unknown molecular origin, as a possible member of the expanding group of ciliopathies.

Genetic skeletal disorders of the fetus and infant: pathologic and molecular findings in a series of 41 cases.

BACKGROUND: Genetic skeletal disorders of the fetus and infant are a large group of genetic disorders, comprising the groups formerly assigned as skeletal dysplasias (osteochondrodysplasias), dysostoses, and malformation syndromes with a skeletal component. Genetic skeletal disorders may be prenatally detected by ultrasonography or result in intrauterine or early postnatal death, constituting one difficult diagnostic field met by the pathologist who performs the perinatal autopsy. METHODS: In this retrospective study, we have gathered radiologic, physical, histopathologic, and molecular data regarding 41 cases of genetic skeletal disorders diagnosed among 1980 fetal and perinatal autopsies over a 10-year period. RESULTS: Our series of cases were classified according to the 2006 Nosology and Classification of Genetic Skeletal Disorders. The overall frequency of genetic skeletal disorders was 1:48 autopsies. The FGFR3 group and osteogenesis imperfecta type 2 were the more frequently encountered disorders. The mean gestational age at autopsy was 21.9 weeks (range, 12-37 weeks). A final diagnosis was obtained in 95% of cases. Genetic skeletal disorders were detected by prenatal ultrasound in 90% of cases, with a correct typing of the disorder achieved in only 34%. Molecular analysis was confirmative in 5 cases. CONCLUSIONS: The central role of the perinatal pathologist in collaboration with specialized services is essential for the correct interpretation of the radiologic, physical, and histopathologic findings, to accurately classify specific types of genetic skeletal disorders and enable genetic counseling.

Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer.

Replication protein A (RPA), a component of the origin recognition complex, is required for stabilization of single-stranded DNA at early and later stages of DNA replication being thus critical for eukaryotic DNA replication. Experimental studies in colon cancer cell lines have shown that RPA protein may be the target of cytotoxins designed to inhibit cellular proliferation. This is the first study to investigate the expression of RPA1 and RPA2 subunits of RPA protein and assess their prognostic value in colon cancer patients. We analyzed immunohistochemically the expression of RPA1 and RPA2 proteins in a series of 130 colon cancer resection specimens in relation to conventional clinicopathological parameters and patients' survival. Statistical significant positive associations emerged between: (a) RPA1 and RPA2 protein expressions (P=0.0001), (b) RPA1 and RPA2 labelling indices (LIs) and advanced stage of the disease (P=0.001 and 0.003, respectively), (c) RPA1 and RPA2 LIs and the presence of lymph node metastasis (P=0.002 and 0.004, respectively), (d) RPA1 LI and the number of infiltrated lymph nodes (P=0.021), (e) RPA2 LI and histological grade of carcinomas (P=0.05). Moreover, a statistical significant higher RPA1 LI was observed in the metastatic sites compared to the original ones (P=0.012). RPA1 and RPA2 protein expression associated with adverse patients' outcome in both univariate (log rank test: P<0.00001 and 0.00001, respectively) and multivariate (Cox model: P=0.092 and 0.0001, respectively) statistical analysis. Statistical significant differences according to the expression of RPA1 and RPA2 proteins were also noticed in the survival of stage II (P<0.00001 and 0.0016, respectively) and stage III (P=0.0029 and 0.0079, respectively) patients. In conclusion, RPA1 and RPA2 proteins appear to be useful prognostic indicators in colon cancer patients and attractive therapeutic targets for regulation by tumor suppressors or other proteins involved in the control of cell proliferation.

Frequent detection of cytomegalovirus in the intestine of patients with inflammatory bowel disease.

BACKGROUND: Although a growing number of reports have described inflammatory bowel disease (IBD) complicated with cytomegalovirus (CMV) infection, there are limited molecular studies that investigate CMV genome in intestinal sections of patients with IBD. METHODS: A cross-sectional prospective study was conducted between September 2000 and June 2003 in a cohort of 85 patients diagnosed with IBD (58 with ulcerative colitis and 27 with Crohn's disease) in two adult gastrointestinal referral centers in Athens, Greece. Prevalence of CMV infection was estimated by pathologic studies in intestinal sections and by molecular assays in blood and intestinal tissue samples and compared with a control group of 42 individuals with noninflammatory disease. RESULTS: Immunohistochemical staining showed CMV antigen in 10 IBD patients (7 with ulcerative colitis; 9 with severe disease), whereas CMV antigen was not detected in any of the controls. CMV genome in both the intestinal tissue and blood was found by polymerase chain reaction in 23 (27.1%) of the total IBD patients, in 18 (31.0%) of those with ulcerative colitis, and in 5 (18.5%) of those with Crohn's disease. In addition, five (5.9%) IBD patients (2 with ulcerative colitis and 3 with Crohn's disease) had detectable CMV genome in their intestinal samples but not in their blood. In the control group, five (11.9%) individuals had detectable CMV genome in their blood, but only one (2.2%) in his intestine. CONCLUSION: Patients with ulcerative colitis had more often detectable CMV genome in their blood as well as in their intestinal tissue samples as compared with controls (P = 0.022 and P < 0.0001, respectively). However, patients with Crohn's disease had more often detectable CMV genome only in their intestinal tissue samples as compared with controls (P = 0.001). Detection of CMV genome in blood or intestinal tissue was significantly associated with short duration of IBD (P = 0.0088 and 0.04, respectively) but not with age, sex, severity of the disease, activity at colonoscopy, pancolitis, administration of a specific treatment, and surgery. In this cross-sectional prospective study, detection of CMV genome or antigen in the intestine was commonly associated with IBD.

Myocardial ischemia in intestinal postischemic shock: the effect of hypoxemic reperfusion.

OBJECTIVE: The circulatory shock following intestinal ischemia-reperfusion injury has been attributed to hypovolemia. The purpose of the current study is to clarify the pathophysiology of this type of shock and to test the hypothesis that hypoxemic compared with normoxemic reperfusion improves hemodynamics. DESIGN: Randomized animal study. SETTING: Medical school laboratory. SUBJECTS: Twenty-one pigs. INTERVENTIONS: Pigs were subjected to 120 mins of intestinal ischemia by clamping the superior mesenteric artery. Upon declamping, the animals were randomized into two groups: a group that received hypoxemic reperfusion (HR group, n = 8) with a PaO2 = 30-35 and a control group reperfused with PaO2 = 100 mm Hg (control group, n = 13). MEASUREMENTS AND MAIN RESULTS: Measurements included mean arterial pressure, cardiac index, pulmonary artery occlusion pressure, and requirements for fluids and epinephrine. Biopsies from the terminal ileal mucosa were taken for malondialdehyde measurements at baseline, at 120 mins of ischemia, and at 30 and 60 mins of reperfusion. A piece of left ventricle was obtained after 120 mins of reperfusion for histologic studies. Five of 13 animals of the control group died in intractable shock; no animal of the HR group died (p =.11). The decrease in the mean arterial pressure during reperfusion was more pronounced in the control group (p <.008) despite the larger doses of epinephrine administered, compared with the HR group (p <.02). During reperfusion, both groups exhibited a decrease in cardiac index; this was more pronounced in the control group (p =.0007). Pulmonary artery occlusion pressure increased during reperfusion in both groups and was more pronounced in the control group (p =.04 at 60 mins). Although mixed venous blood oxygen saturation of the control animals was higher at 30 mins of reperfusion (p =.005), it declined after 60 mins and became lower than that of HR animals at the end of reperfusion (p <.02). The myocardial histopathologic injury score was higher in the control group (2.0 +/- 0.69 and 3.4 +/- 0.89 for the HR and control groups, respectively; p <.03). The concentrations of intestinal mucosa malondialdehyde were significantly higher in the control group at 60 mins of reperfusion (p <.03). CONCLUSIONS: Acute myocardial ischemia and left heart failure significantly contribute to the circulatory shock that follows intestinal ischemia/reperfusion injury and are attenuated by hypoxemic reperfusion.

Reduced retinoblastoma gene protein to Ki-67 ratio is an adverse prognostic indicator for ovarian adenocarcinoma patients.

OBJECTIVE: Alterations in the retinoblastoma gene (RB-1) are common in human neoplasia. However, the clinical significance of the deregulated expression of RB-1 in ovarian cancer remains undefined. We therefore conducted a retrospective investigation to clarify the relationships of RB-1 gene protein (pRb) to the percentage of cycling cells, clinicopathologic variables, other G1 interacting proteins and prognosis of nonbenign epithelial ovarian tumors. METHODS: Paraffin-embedded tissue from 127 nonbenign epithelial ovarian tumors, including 44 of low malignant potential (LMP) and 83 primary ovarian adenocarcinomas, was stained immunohistochemically for pRb, p21(Cip1), p27(Kip1), p53, and Ki-67 antigen (a cell proliferation associated marker). Expression of these markers was correlated with clinicopathologic features and with overall survival of patients with adenocarcinomas. RESULTS: pRb levels were significantly lower in LMP tumors than in carcinomas (P = 0.027). In the latter group, pRb expression decreased with increasing grade (I-II vs III) (P = 0.010), advancing stage (I-II vs III) (P < 0.001), and bulk residual disease (P = 0.014). pRb was not related to Ki-67 expression (P > 0.10) or to overall survival (P > 0.10) but a low pRb to Ki-67 ratio emerged as an important indicator of poor survival in univariate analysis in the entire cohort (P = 0.0076) and in the platinum-treated patients (P = 0.0162) as well as in multivariate analysis, along with histologic type and FIGO stage. CONCLUSIONS: Diminished pRb levels are related to several clinicopathologic indicators of aggressiveness in ovarian adenocarcinomas. More importantly, pRb expression coupled with the percentage of Ki-67 positive cells is a better prognostic marker than pRb, Ki-67, or other G1 interacting proteins and supplements the information gained from traditional prognosticators.