CD34 enrichment of umbilical cord blood.

OBJECTIVE: We describe the relation between the clamping time and blood volume collected, plus two enrichment systems for CD34+ stem cells from umbilical cord blood, to achieve an excellent recovery of cells with a high proliferative ability and bone marrow reconstitution. METHOD: After cord blood collection by an obstetrician, stem cell purification has been performed either with a combination of monoclonal antibodies, using the negative selection Stem Sep method, or with a positive cell selection based on their surface CD34 antigens, using the Mini Macs system. RESULTS: An excellent recovery of hematopoietic progenitors: Burst Forming Unit Erythroid; Colony Forming Unit Granulocyte and Macrophage; and Colony Forming Unit Granulocyte, Erythroid, Monocyte and Macrophage, inversely related to the rising of clamping time, has been achieved with the Mini Macs system (54 % of colonies, with 90 % purity). With the Stem Sep method, recovery of hematopoietic progenitors was 35 % (with 80 % purity). CONCLUSIONS: By an early clamping of umbilical cord blood, we obtained a higher number of CD34+ cells whose clonogenic activity increased with enrichment. This is particularly useful considering that the number of CD34+ stem cells contained in a unit of placental blood is enough for transplantation to a child, but not for an adult engraftment. Thus, using these methods, we can obtain a larger number of CD34+ stem cells, which increases the possibility of reducing graft-versus-host disease in adult patients, producing survival rates similar to those of transplantation of bone marrow from unrelated donors (Ref. 5).

Beta-Thalassemia major and pregnancy.

OBJECTIVE: It was studied the clinical management and the medical outcomes of 6 pregnancies in 5 women affected by Beta Thalassemia major, based on last guidelines and pharmacological treatments. BACKGROUND: Paediatric Department and Department of Obstetrics and Gynaecology of the University of Catania. METHODS: These patients were taken among a group of 116 women affected by beta-thalassemia major divided into three subgroups, according to the characteristics of their menstrual cycle: 1) women with primitive amenorrhoea, 2) women with secondary amenorrhoea and 3) women with normal menstruation. Only one woman, affected by primitive amenorrhoea, needed the induction of ovulation. An accurate and detailed pre-pregnancy assessment was effected before each conception. This was constituted by a series of essays, including checks for diabetes and hypothyroidism, for B and C hepatitis and for blood group antibodies. Moreover were evaluated: cardiac function, rubella immunity and transaminases. Other pregnancy monitoring, and cares during labour and delivery were effected according to usual obstetrics practice. RESULTS: All the women were in labour when they were 38 week pregnant, and the outcome were six healthy babies born at term. There were no complications related to the pregnancy and to the immediate outcome after delivery. CONCLUSIONS: The improvements of current treatments, especially in the management of iron deposits, the prolongation of survival rate, will result in a continuous increase of pregnancies in thalassemic women. Pregnancy is now a real possibility for women affected by such disease. Although numerous complications can occur, vigilant monitoring by both experienced obstetricians and hematologists can lead to successful pregnancy outcomes (Tab. 1, Fig. 1, Ref. 16).

Laparoscopic removal of a giant paratubal cyst complicated by hydronephrosis.

Paratubal cysts represent approximately 10% of all adnexal masses. In most cases they are very small, but very few cases are reported in the literature where they exceed 15 cm of diameter. Furthermore, giant paratubal cysts complicated by bilateral hydronephrosis are unique. The Authors describe a case of a huge paratubal cyst (30 cm in diameter), in a 14 year old obese girl, treated by complete laparoscopic enucleation.

[Innovative genetic, clinical and endocrinologic aspects in young females affected by premature ovarian insufficiency]. / Innovativi aspetti genetici, clinici ed endocrini in una giovane popolazione femminile affetta da insufficienza ovarica prematura.

AIM: Heterogeneity premature ovarian insufficiency (POI) is one of the reasons why there are different causes that contribute in determining this type of hormonal disorder. Although the causes have already been established for many types of premature ovarian failure, are still uncertain causes in most cases of idiopathic forms, despite the description of several candidate genes, including BMP-15 gene. The gene under study is precisely the BMP-15, which is part of the superfamily of Transforming Growth Factors-beta or the TGF-ß, which also belong to the growth differentiation factors (GDFs). METHODS: This study examined a sample of Sicilian women suffering from POI, carefully selected according to their age, since in these cases, the genetic factor probably has a greater impact. RESULTS AND CONCLUSION: Identify a mutant gene that causes ovarian failure may be important to make a diagnosis that can predict the possible future development of the disease. The outcome of the studies, however, has not found the gene in question, but it is hypothesized that this may be a direct consequence of the limited amount of women that was done the study, a case which may be rebutted by increasing the number of patients.

Protection of ovarian tissue from radiotherapy.

Advances researches in the diagnosis and treatment of childhood, adolescent and adult cancer have greatly increased the life expectancy of premenopausal women with cancer. However, one of the serious side effects of these treatments is the risk of damage to fertility. The ovaries are very sensitive to cytotoxic and radiotherapeutic treatment. The only established method of fertility preservation is embryo cryopreservation according to the Ethics Committee of the American Society for Reproductive Medicine (2005), but this option requires the patient to be of pubertal age, have a partner or use donor sperm, and be able to undergo a cycle of ovarian stimulation, which is not possible when the radiotherapy has to be initiated immediately or when stimulation is contraindicated according to the type of cancer. For patients who need immediate radiotherapy, cryopreservation of ovarian tissue is the only possible alternative. This manuscript reports the different techniques of cryopreservation and the results of transplantation of cryopreserved ovarian tissue. The current techniques allow cryopreservation of human ovarian fragments for a long time with good follicular survival rate after thawing. Numerous studies ultimately in this field have demonstrated to improve the survival rate of the oocytes and cryopreserved follicles. Moreover this manuscript includes a case of a 17-year-old girl who had to undergo pelvic irradiation for non-Hodgkin's lymphoma and the laparoscopic treatment to preserve the fertility (Fig. 2, Ref. 47).

Divergent effects of corticotropin releasing hormone on endothelial cell nitric oxide synthase are associated with different expression of CRH type 1 and 2 receptors.

1. Endothelium is a target for an array of factors involved in inflammation. Endothelial cells express receptors for CRH, a neuropeptide produced during inflammation. We report both the concentration-dependent inhibitory effect of CRH upon cytokine-stimulated nitrite release by H5V murine endothelioma cells, and its stimulatory one in HUVEC cells. 2. Western blot analysis showed that CRH inhibits cytokine-stimulated iNOS protein in H5V cells, and, instead, potentiated it in HUVEC cells. 3. H5V cells expressed both CRH receptors (CRH-R1 and R2) mRNAs, whereas HUVEC cells expressed the CRH-R2 mRNA solely. 4. CRH increased medium nitrites and iNOS protein expression in H5V cells pretreated with the selective CRH-R1 antagonist CP 154,526. However, the selective CRH-R2 antagonist anti-Svg-30 failed to produce similar effects. In fact, anti-Svg-30 inhibited CRH-induced increase of nitrite release and iNOS expression in HUVEC cells. 5. Our results confirm the activating role of CRH on endothelial cells, although it suggests its possible inhibitory role in the late phase of the inflammatory response. NO-mediated effects of CRH on endothelial cells could be exploited in therapeutic strategies related to inflammatory and/or degenerative diseases.

Induction of ovulation in thalassemic patients.

The authors studied 19 thalassemic patients: 14 patients with secondary amenorrhea (group A), 5 patients with primary amenorrhea (group B) and 1 patient with anovulation (group C). All the patients underwent three cycles of induction of ovulation with FSH. The level of 17 beta-estradiol and the diameter of follicles were evaluated every three days. Normal gonadal function was maintained in three patients with positive response at Step 1 of the treatment.

Osteopenia in female beta-thalassemic patients.

Sixty-four women with thalassemia major aged 13-28 years were studied. 49 patients had primary amenorrhea and 15 had secondary amenorrhea. Bone density of the spine was performed using lunar DPX. The Z score was used to evaluate the degree of osteopenia. In 82% of the patients a Z score less than -2 was found. BMD correlated negatively with the duration of amenorrhea and age of thalassemic patients.

Evaluation of bone mass in young amenorrheic women with anorexia nervosa.

BACKGROUND: In the last 10 years an impressive increase in the frequency of food disorders (bulimia and anorexia) in teenagers has been recorded. Food disorders, present especially in girls, (part of more complex relational-familiar disorders) cause progressive repercussions on the general endocrine structure and in particular on the reproductive system of the subject. The most visible effect of this endocrine perturbation is the block of the ovarian function with secondary amenorrhoea and low levels of gonadotropins and estrogens due to the food disorder. In fact the qualitative and quantitative food deficit is responsible for the interruption of the normal hypothalamic and pituitary pulses regulating the correct monthly process of follicular growth. As in the physiological postmenopausal period, very low plasma levels of 17-b-estradiol (<30 pg/ml) can start the bone resorption with bone loss and/or osteoporosis related to the amount and the time of exposure to low levels of estrogens. OBJECTIVES: in our study we evaluate the influence of anorexia and amenorrhoea on bone mineral density. METHODS: We evaluate bone mineral density (BMD) with computerised bone mineralometry (MOC), a dual-energy-X-Ray (DEXA), in 18 women with anorexia nervosa and weight loss, 9-12 months amenorrhoea and with low plasma levels of 17-b-estradiol. The BMD was measured at the lumbar spine (L2-L4) and on total body in all subjects enrolled for this study. RESULTS: All the patients showed a decrease on the lumbar BMD with bone loss (49%) or osteoporosis (51%). On the other side the values of total body BMD were around the normal levels (86%) or showed bone loss (14%). CONCLUSIONS: Our data support the hypothesis that lower estrogenic levels associated with anorexia nervosa have an impact on BMD, especially at lumbar level, with early incidence for quantitative and qualitative lower food intake.

Uterine bleeding pattern during low dosage Noretisterone acetate and 17-b-Estradiol treatment in postmenopausal patients.

BACKGROUND: Recent years have been characterized by progressive optimization of postmenopausal hormonal replacement therapy. More physiological therapeutic protocols have been, in fact, proposed to control the possible symptomatology and to prevent the associated risks, with estro-progestinic compounds characterized by lower effective dosages and suitable for the single patient need. However this therapy is not widely accepted by the women from our country for the fears and the inconvenience raised around such side effects as abnormal uterine bleeding and spotting. AIM: to obtain a good compliance and clinical benefits a continuous administration protocol of the hormonal replacement therapy, alternatively to the sequential one has been proposed. METHODS: Our research group has been observing a sample of 42 patients for 12 months, taking oral 17-b-estradiol 1 mg/noretisterone 0.5 mg in continuous administration. All of them were aged from 42 to 63 years and had been in symptomatic menopause for at least 3 months. The characteristics, the onset and the trend of vaginal bleeding were registered in appropriate monthly diaries. Endometrial thickness was evaluated by transvaginal sonography before starting the administration, not exceeding 4 mm in all the women considered. RESULTS: The incidence of bleeding (calculated as a percent of women who experienced a vaginal bleeding for al least a day during a menstrual cycle) was from 26% to 32% in the 1st trimester, reducing during the following months. At 6 months of therapy only 5% of women reported evident vaginal bleeding; at 12 months 90% of women complained with absence of bleeding or spotting. At 12 months no women showed an endometrial thickness over 6 mm. CONCLUSIONS: This observational study suggests that the majority of treated patients proved to be positively responsive to the treatment and that the 17-b-estradiol 1 mg/noretisterone 0.5 mg association reduces the incidence of bleeding and spotting with a sufficient endometrial protection from hyperplasia.

Influence of sport and low animal saturated fats intake diet on lipid dismetabolisms in postmenopausal women.

BACKGROUND: An important aetiopathogenetic factor leading to menopausal age pathologies (such as hypertension, diabetes, cardiovascular diseases and so on) is certainly represented by the worsening of lipid dismetabolism. This condition is generally characterized by an increase in total mean cholesterol levels, LDL-cholesterol (low density lipoprotein-cholesterol), VLDL-cholesterol (very low density lipoprotein-cholesterol), triglycerides, with reduction of HDL-cholesterol (high density lipoprotein-cholesterol) mean levels. All these metabolic modifications are triggered or increased by the hypoestrogenemia typical of postmenopausal age. The aim of our study was to evaluate the influence on postmenopausal women's lipid metabolism of a low contribution of animal saturated fat diets, associated with physical training. METHODS: We enrolled in our study 36 mild hypercholesterolemic postmenopausal women (non smokers and not complaining of hypertension and diabetes) and we put them through aerobic training, consisting of 1 hour of a light continuous run, repeated 4 times per week. The final control of their metabolic status and of their lipid order was executed after 6 months, but during this period, all the women were observed bi-weekly or monthly, to assure a high uniform compliance in the group. None of the women enrolled took specific drugs for the lipid metabolism during the study. RESULTS: After 6 months from the beginning of the diet, with associated physical training, an important weight loss was observed. Increases of HDL-cholesterol mean levels (p<0.05) associated with a decrease of total cholesterol mean levels (p<0.05) were reported in all the subject examined. A statistically non-significant decrease in LDL and VLDL cholesterol mean levels was also noticed. CONCLUSIONS: A mild lipid dysmetabolism in postmenopause may be corrected advantageously by an opportune diet associated with moderate, but constant, physical training.

At term pregnancies in transfusion-dependent beta-thalassemic women.

The health background management and outcome of five pregnancies in women affected by Cooley Disease are described and the preconceptual guidance and care are considered. The patients were selected from a group of 103 thalassemic women divided into three subgroups according to their first and successive menstruation characteristics. Only one woman needed induction of ovulation. A precise and detailed pre-pregnancy assessment was done before each conception. All the women were in labour at 38 weeks' gestation, and five healthy babies were born at term, weighing between 2,600 and 3,200 g. The improvement in current treatments will result in a continuous increase in pregnancies in thalassemic women, making pregnancy a real eventuality for them. Furthermore, we are studying the possibility of collecting foetus umbilical cord blood after delivery, with the aim of obtaining complete marrow reconstitution in an attempt at heterologous transplantation to the mother.

Collection of fetal stem cells and newborn effects.

The aim of this work was to test fetal stem cells (FSC) number modification in relation to clamping time and newborn effect. The results show that a fast sample, between 20 and 40 seconds, from umbilical cord after fetus birth and before placental detachment assured a greater quantity of blood useful for the transplants; and that it was necessary to enrich the collected blood in CD34+ cells with specific clonogenic culture, as this is otherwise a small number for a donation to an adult. In the "new donors" the effects of the unconscious donation always depend on the clamping time, which should be the shortest possible to avoid blood overload, which is very dangerous in the presence of heart malformation.

Two enrichment methods to obtain CD34+ stem cells from umbilical cord blood.

We describe the relation between umbilical cord clamping time and two different enrichment system of CD34+ stem cells from umbilical cord blood with the proliferative ability and bone marrow reconstitution of the stem cells obtained. After an obstetrician performed the cord blood collection, the purification of stem cells was performed either with a combination of monoclonal antibodies (negative selections) using the Stem Sep method, or with a positive cells selection based on their surface CD34 antigens using the Mini Macs system. An excellent recovery of haematopoietic progenitors (Burst Forming Unit Erythroids (BFUE); Colony Forming Unit Granulocytes and Macrophages (CFU-GM); and Colony Forming Unit Granulocytes, Erythroids, Monocytes and Macrophages (CFU-GME)), inversely related to the increase in clamping time, was performed with the Mini Macs system (54% of colonies, with a 90% purity). With Stem Sep method, haematopoietic progenitors recovery was 35% (with a 80% purity). By applying early clamping of umbilical cord blood we obtained a greater number of CD34+ cells and their clonogenic activity was increased with enrichment. This is a useful technique considering that the number of CD34+ stem cells usually contained from a unit of placental blood is enough for the transplant to a child, but not for an adult. Thus, using these methods, we can get a larger number of CD34+ stem cells which reduces the risk of Graft versus Host Disease also in adult patients, producing survival rates similar to the those obtained with transplantation of bone marrow from unrelated donors.

Endocrine and metabolic effects of insulin sensitizers in the treatment of patients with polycystic ovary syndrome and hyperinsulinaemia.

We evaluated 16 women with polycystic ovary syndrome (PCOS) and 67 controls, aged 18-34 years. All subjects had an oral glucose tolerance test (OGTT), showing a normal response. They were subdivided into 2 groups (normoinsulinaemic, hyperinsulinaemic) undergoing 7 days of octreotide treatment. This caused modifications in body mass index, waist-hip ratio and blood pressure. No modifications were observed in basal luteinizing hormone (LH), follicle-stimulating hormone, androgens, 17alpha-hydroxyprogesterone, oestradiol, sex-hormone-binding globulin (SHBG), prolactin and cortisol serum concentrations in normoinsulinaemic patients. A significant decrease in the concentration of LH, androgens, and a significant increase in SHBG were observed in the hyperinsulinaemic patients. In hyperinsulinaemic patients, the treatment restored a regular insulinaemic response to an OGTT. No modifications of glycaemic response were detected in normoinsulinaemic ones. In hyperinsulinaemic patients, a decompensation of the glycaemic response was observed. Several agents (insulin sensitizers) decrease insulin secretion and androgen concentration. They could be considered an approachable long-term therapy for hyperinsulinaemic hyperandrogenic patients. In the next few years the pharmacotherapy for PCOS could be greatly expanded, allowing some patients, such as hyperinsulinaemic ones, to be treated with insulin sensitizers.

Thalassaemic men affected by erectile dysfunction treated with transurethral alprostadil: case report.

The aim of this controlled clinical study, performed in a specialized institutional unit for thalassaemic men, was to consider the possibility of restoring erection in beta-thalassaemic patients with erectile dysfunction by administering E(1) prostaglandins (alprostadil) transurethrally. Four patients affected by beta-thalassaemia, aged between 32 and 52 years, and having an erectile dysfunction were included in the study. Each patient was given 500 microg alprostadil in the distal urethra. Response was evaluated by the erection assessment scale. The main outcome measures were: (i) the clinical study; (ii) FSH, LH, total and free testosterone plasma concentrations; and (iii) basal and dynamic Doppler sonography of cavernous arteries. The treatment produced a response of 3-4 on the erection assessment scale. Average minimum response time was 20 min, while average maximum response time was about 60 min. There was no evidence of significant side effects. Our hypothesis is that the delayed reaction was due to organ damage induced by iron load, causing a reduction or absence of elasticity in the interstitial tissue of the corpora cavernosa. Thus, we believe that treatment with alprostadil can be considered an effective, non-invasive therapy for thalassaemic patients with erectile dysfunction.

Adenomyosis and uterus rupture during labor.

A spontaneous uterus rupture occurred during the labour of a 37-week-pregnant woman showing a precocious rupture of membranes. It appeared enlarged and the cut surface of the myometrium showed coarse trabeculations. The histological examination showed a hypertrophic gravidic myometrium with heterogeneous areas of fibrosis and adenomyosis, necrotic decidual foci and hyperplastic cervical canal glands. Our experience suggests that a silent and spontaneous uterine rupture, is possible even in the absence of the principal risk factors.

Pregnancy outcome of a transfusion-dependent thalassemic woman.

A clinical case concerning a normal pregnancy outcome in a transfusion-dependent woman affected by homozygous beta thalassemia, whose partner was negative with regard to the "thalassemic trait", was reported. The patient showed no iron deposit problems, viral diseases that could have made the pregnancy management difficult or any complications during the gestation. Blood transfusion was not necessary during the following caesarean delivery. The outcome was a healthy female child, born at a gestational age of 38 weeks, showing neither malformations nor problems. This was possible due to a detailed preconceptual guidance and a pre-pregnancy assessment. The patient normally would have had a blood transfusion every 20 days and a strict desferrioxamine chelating therapy; however, this treatment was suspended during her pregnancy because of the well-recognised teratogenic effects of the drug. The average values of ferritin were just a little higher than before being pregnant. The foetus, due to her particular chelating activity, probably maintained these ferritin levels. A sample of 95 ml umbilical cord blood was taken during the delivery. It is well known that umbilical cord blood contains a good quantity of CD34+ stem cells, the haematopoietic progenitors. It was therefore collected for transplanting to the mother and for bone marrow reconstitution. Moreover, our experience suggests that desferrioxamine therapy during lactation does not alter iron excretion in breast milk. Therefore, women now affected by Cooley disease may possibly have a normal pregnancy without ovulation induction, intrauterine growth retardation, foetal loss and preterm labour.