Hypoxia enhances the expression of follistatin-like 3 in term human trophoblasts.

Hypoxic injury hinders placental differentiation and alters trophoblast gene expression. We tested the hypothesis that the expression of follistatin-like 3 (FSTL3), a member of the follistatin family of proteins, is modulated by hypoxia in primary human trophoblast (PHT). Using immunofluorescence of human term placental villi we detected the expression of FSTL3 protein in placental villi, primarily in trophoblasts. We verified this finding in cultured term PHT cells. Basal expression of FSTL3 transcript in cultured PHT cells, determined using quantitative PCR, was stable over the culture period. Importantly, when compared to culture in FiO(2)=20% or FiO(2)=8%, PHT cells cultured in FiO(2) <1% exhibited a 4-6 fold increase in FSTL3 mRNA expression as early as 4h in hypoxia. Whereas cellular FSTL3 protein was unchanged in hypoxia, we found that hypoxia increased the level of FSTL3 in the medium. Lastly, the exposure of PHT cells to either the hypoxia-mimetic cobalt chloride or the proline hydroxylase inhibitor dimethyloxaloylglycine upregulated the expression of FSTL3 transcript. Our data indicate that hypoxia enhances the expression of FSTL3 and its release from PHT cells. Our finding that hypoxia-mimetic agents enhance FSTL3 expression implicates HIF1alpha in this process.

Apoptosis in cryopreserved human ovarian tissue obtained from cancer patients: a tool for evaluating cryopreservation utility.

Fertility preservation is of major importance for women with cancer in whom ovarian function may be disturbed by the use of potentially sterilizing chemotherapeutic drugs and/or pelvic irradiation. Cryopreservation of ovarian cortical tissue is one of the potential options for preserving fertility among these women. Cryopreserved thawed human ovarian tissue can be autografted either orthotopically or heterotopically, but may also be transplanted first into an animal host with subsequent maturation and collection of oocytes. The objective of this study was to investigate the prevalence of ovarian follicular apoptosis in fresh and frozen/ thawed human ovarian tissue as a measure of follicular viability. The study group included 6 women with cancer who underwent ovarian tissue cryopreservation (OTCP). Ovarian tissue samples (n = 2) were obtained from each woman with one sample undergoing evaluation for apoptosis immediately following removal (control, group A) and the other evaluated for apoptosis following freezing/thawing (group B). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) and 4'6' diamido-2-phenylindole hydrochloride (DAPI) staining methods were used to investigate follicular apoptosis. Morphological changes in the same samples were evaluated in hematoxylin and eosin (H&E)-stained sections. In each slide, only primordial and primary follicles were evaluated for abnormal morphology and apoptosis. Abnormal morphology was demonstrated in 23.8+/-8.7% of group A follicles compared to 48.3+/-11.2% of group B follicles (p < 0.05). Apoptosis was demonstrated in 25.4+/-8.4% of group A follicles compared to 60.9+/-6.0% of group B follicles (p < 0.05). We have shown that the ovarian follicles in group B demonstrated a higher incidence of apoptosis compared to those of group A. Therefore, the data suggest that follicular apoptosis might be a consequence of the freezing and thawing procedure. This may be used as a method for evaluating and comparing the outcome of different freezing/thawing protocols.

Alternative pathways of ovarian apoptosis: death for life.

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrus/menstrual cycle by programmed cell death is essential for maintaining the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, play an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows for systematic studies of the mechanisms that control steroidogenesis and apoptosis of granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated for up to 24hr following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or tumor necrosis factor alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one that does not involve mitochondrial cytochrome C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and Affymetrix DNA microarray we discovered that Granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells, thereby allowing the apoptotic signals to bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures the cyclicity of estradiol and progesterone release in the estrus/menstrus cycle even during the initial stage of apoptosis.

Gonadotropin-induced gene regulation in human granulosa cells obtained from IVF patients: modulation of genes coding for growth factors and their receptors and genes involved in cancer and other diseases.

Gonadotropins play a crucial role in ovarian homeostasis and fertilization. However, hypergonadotropin stimulation has been thought to increase the risk for ovarian cancer. Moreover, some correlation between high levels of gonadotropins in the circulation and Alzheimer's disease has been implicated, with no clear evidence on the molecular mechanism involved. Using DNA microarray technology and RNA from gonadotropin-stimulated human granulosa cells, which comprise the main bulk of the ovarian follicular somatic cells, we discovered that stimulation of cells with saturating doses of gonadotropins gives rise to the expression of genes coding for presenilin 1 and 2, along with the up-regulation of genes involved in steroidogenesis such as StAR, cytochrome P450scc enzyme system and aromatase. Moreover, gonadotropin stimulation in these cells dramatically elevates activity of genes coding for epiregulin and amphiregulin, which can bind and activate the EGF receptor and ERB4. These gene products may elevate the risk for ovarian, breast, endometrial and other non-gynecological cancers. Gene transcripts for oncogenes and tumor markers such as pleiomorphic adenoma gene-like 1 (Plagl1) tumor antigen (L6) and claudin 3 were markedly elevated following LH and FSH stimulation. In parallel, downregulation in ovarian cancer 1 (DOC1) and suppression of tumorigenicity (ST5) genes was observed, suggesting a potential increase for cancer development. In contrast, increase in tumor rejection antigen (gp96) 1 and decrease in connective tissue growth factor (CTGF), transforming growth factor-beta 1 induced transcript 1 (TGFB1Il), pim-1 oncogene (PIM1), v-maf musculoaponeurotic fibrosarcoma oncogene homologue (MAF) and CD24 antigen may be associated with a decreased risk for specific cancers. In conclusion, gonadotropin stimulation may modulate specific sets of gene transcripts that may either elevate or reduce the risk for specific diseases.

Cholesterol lowering in the older population: time for reassessment?

Hypercholesterolaemia is an established major risk factor for coronary heart disease (CHD) in the general population. In the vast majority of studies that focused on this particular age group and carefully eliminated other confounding factors such as co-morbid conditions, hypercholesterolaemia was a risk factor for CHD in the older population. Because the prevalence of CHD increases with advancing age, studies that consider not only the relative risk attributed to cholesterol but also the absolute numbers of people affected, show hypercholesterolaemia to be an even stronger risk factor in the elderly. Large primary and secondary prevention studies of HMG-CoA reductase inhibitors (statins) in the elderly have shown a reduction in major coronary events similar to that observed in the younger age group. The role of hypercholesterolaemia as a risk factor for stroke is less clear, and a major limitation is the heterogeneous nature of the disease. Nevertheless, most studies that evaluated non-haemorrhagic strokes separately showed a positive association with cholesterol levels, and statin therapy is effective in preventing stroke. These data provide a rationale for treating older hypercholesterolaemic people with statins, not only to prevent CHD, but also to prevent stroke.

Can creatinine clearance be accurately predicted by formulae in octogenarian in-patients?

BACKGROUND: As serum creatinine is relatively inaccurate for estimating renal function, prediction formulae are commonly used for more precise renal function estimation. However, these equations have not been studied in acutely hospitalized octogenarian patients. AIM: To compare three commonly used formulae for estimating GFR to measured creatinine clearance (CCR) in patients aged >/=80 years admitted to an acute geriatric department. DESIGN: Prospective, observational study. METHODS: Consecutive patients aged >/=80 years with urinary catheters, admitted over a 12-month period to the acute geriatric ward of a 600-bed university hospital, were enrolled in the study. All had an accurate 24-h urinary collection, as well as serum and urinary urea and creatinine determinations. CCR was calculated and compared with GFRs derived from the three formulae. RESULTS: Of the 154 patients enrolled in the study, 107 (69.5%) had normal serum creatinine (

Vertebral osteomyelitis: a rare complication of transvaginal ultrasound-guided oocyte retrieval.

OBJECTIVE: To present a case of vertebral osteomyelitis as a complication of transvaginal oocyte retrieval. DESIGN: Case report. SETTING: The IVF unit of a university-affiliated hospital. PATIENT(S): A 41-year-old woman who underwent IVF-ET treatment. INTERVENTION(S): Standard IVF-ET treatment cycles with the use of transvaginal ultrasound for oocyte retrieval and computed tomography-guided needle aspiration. MAIN OUTCOME MEASURE(S): Recovery of the patient, sequelae, and recurrence. RESULT(S): Vertebral osteomyelitis was diagnosed and treated with antibiotics. CONCLUSION(S): When severe low back pain occurs after ovum retrieval, vertebral osteomyelitis should be considered. Early diagnosis requires a high index of suspicion.

High incidence of preeclamptic toxemia in patients with polycystic ovarian disease.

The incidence of preeclamptic toxemia (PET) was investigated in 72 anovulatory, oligomenorrheic and previously nulliparous women who conceived after an induction of ovulation. One-thousand consecutive spontaneous pregnancies and 1,000 pregnancies of primiparae were used as control groups. The anovulatory group consisted of 33 consecutive well-documented cases of polycystic ovarian disease (PCO) and 39 anovulatory patients in whom PCO was excluded (A-NPCO). The results indicate that pregnancies after induction of ovulation are accompanied with a higher incidence of PET. The rate of this disorder was significantly higher in PCO groups than in A-NPCO women (28.5 vs. 4%), when calculated per number of all pregnancies. The difference between the incidence of PET in PCO, A-NPCO, control primiparae and normal control patients was even more pronounced when calculated on the basis of the number of patients (54.5, 12.5, 11 and 2.5%, respectively). Overproduction of steroid hormones, especially androgens, was suggested as the main factor for the appearance of PET in PCO patients.

Probiotics improve bowel movements in hospitalized elderly patients--the PROAGE study.

OBJECTIVE: To determine the impact of probiotics on the prevention of problems with bowel movements malnutrition and infection. DESIGN: A randomized, double-blind, placebo-controlled trial. SETTING: Peripheral Geriatric Hospital. PARTICIPANTS: 243 elderly patients age ≥ 65 y who were hospitalized in a Geriatric Orthopedic Rehabilitation Department. INTERVENTION: Participants were randomized into treatment or control groups (daily probiotics or placebo for 45 consecutive days, respectively). MEASUREMENTS: The main outcomes were: number of days of constipation or diarrhea and the number of days of laxative use. Secondary measures were nutritional status and blood measurements. RESULTS: Of 599 patients admitted to the Geriatric Rehabilitation ward, 345 were eligible and agreed to participate. During a 7-day pre-trial period, 102 patients dropped out (45 and 57 in the probiotic and placebo groups respectively). Out of the 243 patients who entered the study, 28 dropped out during the study (11.5%), leaving 215 patients. Throughout the 45 days of follow-up, the incidence of diarrhea was significantly lower among the study group (HR=0.42, p=0.04) with a more pronounced difference among participants aged ≥ 80 y (HR=0.32, p=0.026). Laxative use (as an indicator of constipation severity) was significantly lower in the study group compared with the control group (HR=0.74, p=0.032). Serum albumin, prealbumin and protein increased significantly more in the treatment group compared with the control group among participants age ≥ 80 y (P=0.047, p=0.07, p=0.03 respectively) but not in the younger age group. CONCLUSION: We showed that probiotic supplements may have a positive effect on bowel movements among orthopedic rehabilitation elderly patients.

Management of Kell isoimmunization--evaluation of a Doppler-guided approach.

OBJECTIVE: To assess the role of peak systolic velocity in the middle cerebral artery (MCA-PSV) in the management of pregnancies complicated by Kell isoimmunization. METHODS: Sixteen fetuses were monitored by conventional protocol (Group 1) and eight fetuses by an MCA-PSV-guided protocol (Group 2). The conventional protocol included a weekly ultrasound evaluation and measurement of maternal anti-Kell titers every 4-6 weeks. In Group 2 Doppler assessment of the MCA-PSV was performed at intervals of 4 to 7 days and MCA-PSV>1.5 multiples of the median (MoM) was considered as an indication for fetal blood sampling (FBS). RESULTS: No parameter emerged as a reliable predictor of isoimmunization severity in Group 1. In Group 2, no FBS was necessary in one case since the MCA-PSV values obtained during the follow-up were <1.29 MoM. In two cases the first FBS was already indicated after 1 week of follow-up, but five other fetuses were followed for 3-9 weeks before FBS was indicated. All fetuses with MCA-PSV>1.5 MoM prior to intrauterine transfusion (IUT) had severe fetal anemia on FBS. In fetuses with severe anemia on the first FBS, the MCA-PSV values 7 days before the first FBS were <1.29 MoM (four cases), between 1.29 and 1.5 MoM (two cases) and >1.55 MoM (one case). CONCLUSIONS: In the management of Kell isoimmunization invasive procedures may be avoided by implementing MCA-PSV measurements. Delineation of appropriate intervals between reassessments, the reliability of MCA-PSV following repeated IUTs, and cut-off values for FBS await further study.

The safety and feasibility of percutaneous endoscopic gastrostomy placement by a single physician.

BACKGROUND AND STUDY AIMS: Percutaneous endoscopic gastrostomy (PEG) has become the procedure of choice for enteral feeding. However, the procedure usually requires two physicians, which makes it more difficult to schedule than procedures performed by a single physician. We investigated whether PEG insertion by a single physician could be done with the same safety and feasibility as by two physicians. PATIENTS AND METHODS: This study involved 339 consecutive patients who were referred for PEG. The same single physician, together with a nurse, performed the procedure in all patients, instead of the usual procedure performed by two physicians. Followed up of the patients for 1 month after the procedure was done. RESULTS: Minor complications occurred in 35 patients (10.3%), most frequently self-extubation and skin irritation. Eight patients (2.4%) had severe complications, including apnea in two, and wound infection that needed systemic antibiotics in another three patients. Three patients needed surgery because of peritonitis as a consequence of the procedure. There was no mortality in the first 48 hours after the procedure and only one patient death could be attributed to the procedure. CONCLUSIONS: The insertion of PEG by a single experienced physician is as safe as that described in the literature with two physicians. This should not replace the traditional approach with two physicians, but should be reserved for special situations when only one physician is available.

Neonatal lupus erythematosus in offspring of mothers with experimental systemic lupus erythematosus.

Neonatal lupus erythematosus (NLE) syndrome is a result of the transfer of autoantibodies produced by the mother, across the placenta, to the fetus. NLE is characterized by a transient dermatitis, a variety of systemic and hematological abnormalities, and isolated cases of congenital heart block. The latter has been reported to be due to the presence of autoantibodies specific to La (SS-B) and/or Ro (SS-A). As female mice with experimental SLE, induced by immunization with the monoclonal anti-DNA 16/6 Id, produce a variety of autoantibodies including anti-Ro and anti-La antibodies, we examined the relevance of NLE in the murine system. Offspring of SLE-afflicted BALB/c mothers possessed antibody titers to the 16/6 Id, ssDNA, and nuclear extract, which gradually declined until reduced to normal levels by day 60 after delivery. Antibody titers in the sera of the mothers remained elevated throughout this period. Electrocardiograms were recorded from groups of neonates from mothers with experimental SLE. The results indicated that a high percentage of the offspring had defects in their conduction system including first, second, and third degree heart block; significant bradycardia; and wide QRS complex. Normal patterns were observed in offspring of healthy mothers. Experiments done with mice that were exposed to SLE-related autoantibodies early in their development indicated that offspring to mothers with experimental SLE were neither protected nor more susceptible to disease induction by the 16/6 Id.

Acute hepatitis C during pregnancy. Case report and review of the literature.

Treatment of chronic hepatitis C with interferon is well established (Fried 1995, Zein 1995) though not very successful. We report a case of a pregnant doctor who developed acute hepatitis C six weeks after accidental needle-stick injury from a hepatitis C virus positive patient. We review the literature that deals with such cases.

Neonatal lupus erythematosus with cardiac involvement in offspring of mothers with experimental systemic lupus erythematosus.

Neonatal lupus erythematosus (NLE) syndrome is characterized by a transient dermatitis, a variety of systemic and hematological abnormalities, and isolated cases of congenital complete heart block. The latter has been reported to be due to the presence of autoantibodies specific to La (SS-B) and/or Ro (SS-A). As female mice with experimental systemic lupus erythematosus (SLE) induced by immunization with the human monoclonal anti-DNA antibody bearing the 16/6 Id produce variety of autoantibodies including anti-Ro and anti-La antibodies, we looked for NLE related symptoms in the murine model. Offspring of BALB/c mice with SLE possessed high levels of autoantibodies that declined gradually till reduced to normal levels at day 60 after delivery. Electrocardiograms recorded in groups of offspring from mothers with experimental SLE indicated that a high percentage of the offspring had defects in their conductive system including first-, second-, and third-degree heart block, significant bradycardia, and a wide QRS complex. In contrast, a normal pattern was observed in offspring of healthy mothers.

Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases.

Gonadotrophins exert a major effect on ovarian development and on the control of fertilization. By stimulating cells with forskolin (FK), it is possible to study which genes are activated by gonadotrophins via the cAMP cascade, and which by alternative pathways. Using RNA isolated from stimulated cells, we found that 59% of the total genes modulated by LH were also modulated by FK, while 69% of the genes modulated exclusively by FSH were also modulated by FK. Gene transcripts involved in steroidogenesis/progesterone production were highly elevated, while 17beta-hydroxysteroid dehydrogenase was down-regulated. This suggests that a decrease in the conversion of androstenedione to testosterone and estrone to estradiol occurs during luteinization. Down-regulation of genes coding for actin cytoskeleton proteins and cytokeratin 18 was observed in response to gonadotrophin and cAMP stimulation. Several of the genes coding for the microtubule network were also modulated, implying that rearrangement of the cytoskeletal proteins permits better coupling between organelles involved in steroidogenesis. A dramatic change in gene transcripts coding for signalling enzymes was observed following LH stimulation. This includes the down-regulation of adenylyl cyclase 7 and 9, elevation of cAMP-dependent phosphodiesterase, and the up-regulation of a negative regulator of G-protein signalling (RGS16) that may negate gonadotrophin signalling via guanine nucleotide binding proteins. Thus luteinized cells, despite increased gene transcripts to LH/chorionic gonadotrophin (CG) receptors, respond inefficiently to gonadotrophin stimulation, due to attenuation of signal transduction in the cAMP cascade at multiple steps. Novel genes involved in the regulation of apoptosis were found for the first time to be up-regulated by gonadotrophin stimulation, including: BAX inhibitor-1, granulysin and apoptosis repressor with caspase recruitment domain (ARC). These proteins may be involved in a unique alternative pathway of ovarian cell death. Such a pathway could temporarily preserve the mitochondria and progesterone production during the initial stages of granulosa cell apoptosis.