Pharmacologic ovarian preservation in young women undergoing chemotherapy.

The prognosis of malignancies in young women undergoing chemotherapy has dramatically improved recently, and more attention is given to the long term quality of life, including fertility and reproductive function preservation. Some chemotherapeutic drugs are known to be associated with gonadal toxicity (cyclophosphamide, L-phenylanine mustard, busulfan and nitrogen mustard) and others have less or un-quantified effects (doxorubicin, bleomycin, vinca alkaloids, as vincristine and vinblastin, cisplatin, nitrosoureas, cytosine arabinoside). Women are in need to identify best options to minimize ovarian damage during chemotherapy through the administration of protective drugs, better choice of therapy and with advocating oncofertility preservation. We reviewed the possible options focusing on the most studied gonadotrophin-releasing hormone agonists (GnRH-a) and the psychologically promising oral contraceptives (OC). Controversy exist on the benefit of gonadotrophin releasing hormone agonist (GnRH-a) or combined oral contraceptive administered at time of cancer therapy in preventing premature ovarian failure in women and the available data from both human and animal studies have been mixed. The best way to preserve fertility and ovarian function in young women undergoing chemotherapy still remains to be determined. In the absence of a best approach, each case should be evaluated individually, considering patient's wishes and expectations, the type of chemotherapy, age, obstetric history, ovarian reserve (combining multiple indicators such as basal hormone profile, anti müllerian hormone -AMH- and antral follicle count), family history of premature ovarian failure. We present a review of the available evidence on the value of administering GnRH-a and OC use to minimize or prevent the effect of chemotherapy agents on reproductive function.

Contribution of adenosine-producing ectoenzymes to the mechanisms underlying the mitigation of maternal-fetal conflicts.

The interactions taking place between mother and embryo have been the focus of detailed studies in recent years, where pregnancy is considered as an in vivo transplant. The immune systems of the mother and the embryo together establish a condition of tolerance, which lasts throughout the pregnancy. Alongside immunogenetic components, a contribution is provided by the ectoenzyme network, a chain of surface molecules mainly operating in closed environments and potentially providing inhibitory or activator signals. One of the soluble products of the ectoenzyme network with immunosuppressory potential is adenosine, a purine nucleoside that plays multiple roles in almost all tissues and organs. The hypothesis behind the work was studied in patients with recurrent pregnancy loss (RPL), an event which remains unexplained in over 50 percent of cases. To this aim, we analyzed the expression of CD39 (ectonucleoside triphosphate diphosphohydrolase 1, ENTPD1) and CD73 (ecto-5’-nucleotidase, NT5E), the main pathway for adenosine generation, in samples obtained from women with RPL. The study included the evaluation of the expression of TNF-alpha (a pro-inflammatory cytokine) and of an alternative pathway of adenosine generation run by CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) and PC-1 (ectonucleotide pyrophosphatase/phosphodiesterase 1, ENPP1). The results of this study highlight the existence of a network of surface enzymes expressed at the maternal/fetal interface and addressed to the production of adenosine. Perturbation of this network may induce a rescue pathway driven by CD38 and ENPP1. Ectoenzyme and inflammation may be considered now key elements in orchestrating the events leading to the interruption of pregnancy in the RPL sample analyzed and at the same potentially becoming therapeutic targets.

Human AB serum for generation of mesenchymal stem cells from human chorionic villi: comparison with other source and other media including platelet lysate.

OBJECTIVES: We have investigated foetal mesenchymal stem cells (MSCs) obtained from first-trimester chorionic villi (CV) and second-trimester amniotic fluid (AF), comparing them to adult bone marrow-derived MSCs. MATERIALS AND METHODS: We report on cell population growth in human allogeneic serum (HS) and platelet lysate (PL), immunophenotype, cytokine expression profile and immunoregulatory activity, of these foetal MSCs on stimulated peripheral blood mononuclear and lymphocyte subpopulations. RESULTS: Chorionic villi cells grow rapidly in HS, with 20 populations doublings (PDs) after 59 days (six passages), and also in animal serum, with 27 PDs after 65 days (seven passages). PL allowed for expansion in 60% of the samples tested, although it was lower than in HS. HS supported an average of 40 PDs of expansion in 20% of AF cells after 90 days, whereas animal serum supported 28.5 PDs in 66 days. CV and AF cells inhibited proliferation of stimulated T lymphocytes, suppressing population growth of both CD4+ and CD8+ T subpopulations and sometimes also, CD19+ cells. CONCLUSIONS: Our results indicate that CV would be an optimal source of MSCs with high expansion potential in a HS propagation system and immunoregulatory capacity of T and B lymphocytes. More than 90% of CV samples achieved large-scale expansion in HS, which is encouraging for potential clinical applications of these cells.

Neurogenic potential of mesenchymal-like stem cells from human amniotic fluid: the influence of extracellular growth factors.

Amniotic fluids contain human stem cells, among which mesenchymal stem cells could be isolated. These cells have multipotent differentiation ability and no tumorigenic potential after transplantation in mice. These features make them good candidates for in vitro studies and for therapeutic purposes. The aim of this study was to isolate mesenchymal stem cell-like cultures from different amniotic fluids in order to study in vitro their neurogenic potential and assess if this process could be reproducible and standardized. We focused attention on the possible differential effects of soluble growth factors. Immunophenotypical and molecular characterization showed that the 31 amniotic fluid-derived cultures expressed mesenchymal markers as well as some stemness properties. These cells also appeared to be responsive to purines or acetylcholine showing an intracellular calcium increase, also reported for mesenchymal stem cells derived from other sources. Interestingly, in the presence of retinoic acid, these cells assumed a neuronal-like morphology. In addition, functional and molecular analyses revealed that retinoic acid-treated cells showed immature electric functional properties, the expression of neuronal markers and stemness genes. In conclusion, even if further investigations are required, the results presented here contribute to support the finding that amniotic fluid contains cells able to differentiate in vitro towards neural-like lineage in the presence of retinoic acid. The ability of retinoic acid to induce a possible neuronal progenitor culture makes the model useful to study a possible in vivo transplantation of these cells and to contribute to define the protocols for cell therapy.

Expression patterns of two serine protease HtrA1 forms in human placentas complicated by preeclampsia with and without intrauterine growth restriction.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that have in common abnormal placental implantation, a marked proliferation of villous cytotrophoblastic cells and focal necrosis of the syncytiotrophoblast. Several studies show an ischemic placenta with a high-resistance vasculature, which cannot deliver an adequate blood supply to the feto-placental unit. The cause of PE is a matter of debate, but recently studies in mice suggest that the primary feto-placental lesions are sufficient to initiate the disease. HtrA1, a member of the family of HtrA proteins, is a secreted multidomain protein with serine protease activity. It is expressed in first and third trimester of gestation. In specimens from the first trimester of gestation, immunostaining for HtrA1 is generally found in both layers of villous trophoblast, syncytiotrophoblast and cytotrophoblast. Cytoplasm of extravillous trophoblast and extracellular matrix of cell islands and cell columns are labeled for HtrA1. Specimens from third trimester of gestation show a more intense positivity for HtrA1 in the syncytiotrophoblast than in cytotrophoblast. The extravillous trophoblast and the decidual cells, is positive for HtrA1. The purpose of this study is to investigate the expression pattern of HtrA1 in placentas from PE without IUGR (maternal PE) and with IUGR (fetal PE) by quantitative western blotting and immunohistochemistry. By quantitative western blotting analysis we observed a significant upregulation of approximately 30 kDa HtrA1 form in PE. Differently, we detected a significant total HtrA1 down-regulation in PE-IUGR. Moreover, immunostaining for HtrA1 was positive in the villous trophoblast, in the syncytial knots and irregularly in the fetal vessel walls in PE placentas while immunostaining for HtrA1was present particularly in the syncytial knots in PE-IUGR placentas. In conclusion, we suggest that the approximately 30 kDa HtrA1 form can be correlated to maternal PE while that the significant down-regulation of total HtrA1 can be correlated to placental PE. These HtrA1 alterations could be considered as possible markers to discriminate placental PE from maternal PE.

Potential role of culture mediums for successful isolation and neuronal differentiation of amniotic fluid stem cells.

In recent years, the use of stem cells has generated increasing interest in regenerative medicine and cancer therapies. The most potent stem cells derive from the inner cell mass during embryonic development and their use yields serious ethical and methodological problems. Recently, a number of reports suggests that another suitable source of multipotent stem cells may be the amniotic fluid. Amniotic fluid mesenchymal stem cells (AFMSCs) are capable of extensive self-renewal, able to differentiate in specialized cells representative of all three germ layers, do not show ethical restriction, and display minimal risks of teratomas and a very low immunogenity. For all these reasons, amniotic fluid appears as a promising alternative source for stem cell therapy. Their recent discovery implies a lack of knowledge of their specific features as well as the existence of a protocol universally recognized as the most suitable for their isolation, growth and long-term conservation. In this study, we isolated stem cells from six amniotic fluids; these cells were cultured with three different culture mediums (Mesenchymal Stem Cell Medium (MSCGM), PC-1 and RPMI-1640), characterized by cytofluorimetric analysis, and then either frozen or induced to neuronal differentiation. Even if the immunophenotype seemed not to be influenced by culture medium (all six samples cultured in the above-mentioned mediums expressed surface antigens commonly found on stem cells), cells showed different abilities to differentiate into neuron-like cells and to re-start the culture after short/long-term storage. Cells isolated and cultured in MSCGM showed the highest proliferation rate, and formed neuron-like cells when sub-plated with neuronal differentiation medium. Cells from PC-1, on the contrary, displayed an increased ability to re-start culture after short/long term storage. Finally, cells from RPMI-1640, even if expressing stem cells markers, were not able to differentiate in neuron-like cells. Further studies are still needed in order to assess the effective role of culture medium for a successful isolation, growth, differentiation and storage of AFMSCs, but our data underline the importance of finding a universally accepted protocol for the use of these cells.

Laparoscopic cryomyolysis: an alternative to myomectomy in women with symptomatic fibroids.

BACKGROUND: The purpose of this study was to determine whether cryomyolysis may present an alternative valid surgical procedure to hysterectomy or myomectomy for selected women with symptomatic fibroids who wish to preserve their uterus but do not desire future pregnancies. METHODS: Sixty-three women with symptomatic fibroids who refused either myomectomy or hysterectomy, requesting a conservative surgery for myomata, underwent laparoscopic cryomyolysis using a 3- to 5-mm or 8-mm cryoprobe (CRYOcare system). RESULTS: Our study showed a mean (+/-standard deviation) decrease of myoma volume of 60.3% (+/-20.7) and complete symptom relief in 83.6% (p < 0.001) of patients after a 12-month follow-up from cryomyolysis. No significant intra- or postoperative complications were noted. CONCLUSION: Cryomyolysis is an effective laparoscopic procedure for obtaining myoma shrinkage and symptom relief in women with symptomatic fibroids who desire to preserve their uterus.

Does previous surgery influence the asymmetric distribution of endometriotic lesions?

Our objective was to investigate the role of previous abdominal-pelvic surgery in the asymmetric distribution of pelvic endometriosic lesions. This was a retrospective study carried out at the Clinic of Obstetrics and Gynecology, University of Ancona, Italy, and included 238 patients with histological confirmation of endometriosis. The interventions were surgical treatment, at laparoscopy or laparotomy, for pelvic pain and endometriosis. The main outcome measure(s) were endometriotic lesions and adhesions in the pelvis found during surgery and the dinical records of the patients. We found unilateral lesions in 149 patients (62.6%): the right side of the pelvis affected in 55 patients (36.9%) and the left side in 94 patients (63.1%) (p < 0.01). In the group of patients who had undergone previous abdominal surgery, we found lesions on the right side in 26 cases (32.5%), and on the left in 54 cases (67.5%) (p < 0.01). We found that the patients who had undergone previous abdominal surgery had significantly more adhesions than those with no previous surgery (80/116 vs. 73/122, p = 0. 002). As a new finding, we have demonstrated that the left side of asymmetric distribution of intrapelvic macroscopic lesions is preserved and more evident in patients with previous abdominal surgery, including previous appendectomy. These data seem to be in agreement with our previous supposition of a possible interaction between previous abdominal surgery and the mechanisms of endometriosis development.

Amniotic levels of vascular endothelial growth factor and nitric oxide at the second trimester in Down's syndrome.

OBJECTIVE: Since placentae in trisomy 21 show trophoblastic hypoplasia and hypovascularity, we investigated amniotic fluid vascular endothelial growth factor (VEGF) and nitric oxide (NO) in normal pregnancy and pregnancy complicated by trisomy 21. Furthermore, we investigated a possible role of NO in neurodegeneration of the brain in Down's syndrome. METHODS: We retrospectively assessed NO and VEGF on mid-trimester amniotic fluid from 15 women who had fetal Down's syndrome, and compared the results with those of 15 controls matched for age and gestation. RESULTS: In pregnancies complicated by trisomy 21, NO levels were significantly higher than in healthy controls (p < 0.001), whereas VEGF levels were significantly lower than in healthy controls (p < 0.05). CONCLUSIONS: Our results suggest that the high levels of NO and the low levels of VEGF observed in the amniotic fluid of fetuses with Down's syndrome may be a sign of an imbalance of placental vascularization and altered endothelial function. Overproduction of NO could contribute to pathological cell death in the central nervous system, a process that has been demonstrated in many neurodegenerative diseases.

Gestational hypertension plasma and human umbilical vein endothelial cells: an in vitro study.

BACKGROUND: The action of plasma from women affected by gestational hypertension (GH) on nitric oxide synthase (NOS) activity in cultured human umbilical vein endothelial cells (HUVECs) was evaluated in the present study, together with the effect on cytosolic calcium, on Na+, K+-ATPase activity and on membrane fluidity. METHODS: At 80% confluence, cultured HUVECs were incubated for 3 h at 37 degreesC with fresh culture medium (control samples) or with 20% (v/v) plasma (from five healthy non-pregnant women, five healthy pregnant women and five pregnant women affected by GH). RESULTS: After incubation with GH plasma, we observed a significant reduction in NOS activity, intracellular calcium concentrations and Na+, K+-ATPase activity. CONCLUSIONS: The present work gives further support to the hypothesis that a circulating factor in gestational hypertension, possibly produced by the fetoplacental unit, causes dysfunction of the vascular endothelial cells and NO reduction, resulting in a loss of vascular refractoriness to vasoactive agents.

Modifications induced by plasma of gestational hypertensive women on the Na+/K+-ATPase obtained from human placenta.

In order to investigate the molecular mechanisms of the inhibition of Na+/K+-ATPase in Gestational Hypertension (GH), we incubated Na+/K+-ATPase purified from human placenta of 6 healthy normotensive women with plasma from 6 GH women and 6 healthy controls. We determined the enzyme activity by the method of Esman, and the anthroyl-ouabain-binding capacity, dissociation constant (Kd) and average lifetime values (tau) by the static and dynamic fluorescence of anthroyl-ouabain. The lipid annulus of the enzyme was studied by static and dynamic fluorescence of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5- hexatriene (TMA-DPH). The addition of total and protein-free GH plasma to normal Na+/K+-ATPase significantly inhibited the enzymatic activity even at the lowest concentration studied (1:100), as well as the ouabain-binding capacity, Kd and tau. GH plasma significantly decreased the fluorescence polarization and lifetime values of TMA-DPH. These observations indicate that the inhibition caused by GH plasma on Na+/K+-ATPase might be due to a reduction of the number of active molecules or a modification of the ouabain-binding site suggesting the existence of digitalis-like factor. A link between the modification of the lipid moiety of the enzyme and the Na+/K+-ATPase inhibition might be hypothesized.

Transdermal estradiol and medroxyprogesterone acetate in hormone replacement therapy are both antioxidants.

We have evaluated the effects of the different components of hormone replacement therapy (HRT) on the production of free radicals in platelet membranes from menopausal women. The study included 12 women in menopause for at least 6 months to a maximum of 4 years. First, the effect was determined of progestin only during the administration of 20 mg/day medroxyprogesterone acetate for 5 days. The peroxide production level was measured on day 0 and day 5. The second set of experiments was carried out in the first month of cyclic HRT with transdermal estradiol 50 micrograms/day from day 1 to day 25 and medroxy-progesterone acetate from day 13 to day 25. In this experiment, the peroxide level was evaluated on days 0, 12 and 25. A significant reduction of peroxide level was observed after oral medroxyprogesterone acetate administration. During HRT, we observed a similar reduction in lipid oxidation at the peak of the estrogen effect, and a further decrease with the administration of medroxyprogesterone acetate. It is concluded that reduction of lipid peroxidation during HRT is not only due to estrogens, but also depends upon the combined action of sex steroids. This observation justifies not only the combined regimen (estrogens plus progestin) in HRT, but also the positive effects of progestins alone on patients who cannot use estrogens.

Functional platelet modifications induced by oral magnesium supplementation in normotensive and hypertensive pregnancy.

Platelet functionality alterations have been correlated to the onset of hypertension in pregnancy and oral Mg++ supplementation has been clinically postulated to counteract such alterations. We, therefore tested the effect of 4 weeks oral Mg++ pyrrolidone carboxylate supplementation on platelet function. Forty-eight pregnant women were enrolled in the study at the beginning of the third trimester (30-32 weeks). Twenty women were preeclamptic, while 28 remained normotensive and served as controls. All the women received 360 mg/day magnesium pyrrolidone carboxylate for 4 weeks. DPH fluorescence, Na+/K(+)-ATPase and Ca(++)-ATPase activity, intracellular free Ca++ concentrations were determined prior and after the 4-weeks supplementation. Oral Mg++ supplementation significantly increased platelet DPH fluorescence in both normotensive and preeclamptic women. In normotensive pregnant women, it also significantly increased the activity of Na+/K(+)-ATPase, the activity of Ca(++)-ATPase and reduced the concentration of intraplatelet free Ca++. In hypertensive pregnant women, Mg supplementation increases Na+/K(+)-ATPase activity and decreases intracellular free Ca++; this, in turn, contributes to reducing the activity of Ca(++)-ATPase. Magnesium supplementation to preventing hypertension in pregnancy seems to have a consistent biochemical and clinical background.

Natural killer cell activity in stage I endometrial carcinoma: correlation with nuclear grading, myometrial invasion, and immunoreactivity of proliferating cell nuclear antigen.

The purpose of this study was to examine the relationship between natural killer cell activity and biological behavior of tumor, expressed by architectural (FIGO) and nuclear grading, depth of myometrial invasion, and proliferating cell nuclear antigen (PCNA) index in patients with endometrial carcinoma. Forty patients with FIGO stage I endometrial carcinoma, treated with radical surgery, were included in this retrospective study. At the time of diagnosis, natural killer cell activity of peripheral blood was evaluated against K562 target tumor cells and correlated with architectural and nuclear grading, depth of myometrial invasion, and PCNA index of the tumor. Natural killer activity diminished with increasing nuclear grade of the tumor (P = 0.004); similarly, natural cytotoxicity decreased with myometrial invasion: for stages IC and IB endometrial carcinoma, the mean values of natural cytotoxicity were significantly lower than stage IA disease (P = 0.0001). Natural killer activity was significantly correlated with PCNA immunostaining of the tumor (r = -0.8). It is concluded that the natural immune reactivity seems to be related to the pathologic features of early stage endometrial carcinoma, showing a significant reduction in presence of nuclear pleomorphism and/or myometrial invasion, and also an inverse relationship with PCNA index.

Serum CA-125 concentration in endometriosis patients: role of pelvic and peritoneal irritation.

Serum CA-125 concentrations were investigated preoperatively in 91 consecutive women undergoing laparoscopy for infertility, pelvic pain and/or annexial cysts. The presence and extent of endometriosis were carefully assessed, including the American Fertility Society stage of disease, and implant and adhesion scores. Postoperative CA-125 measurements were obtained in 32 of 53 endometriosis patients and evaluated with respect to clinical evolution of the disease. Serum levels of CA-125 were significantly increased in patients with endometriosis (46.5 +/- 39.5 vs. 13.5 +/- 7.3 U/ml in controls, p < 0.001) and correlated with the severity of disease. A positive correlation (r = 0.7, p < 0.001) was observed between adhesion score and CA-125 levels, while the relationship with implant score was not significant (r = 0.3, p = 0.07). CA-125 level was also significantly increased in women with peritoneal endometriosis (70.7 +/- 47.3 vs. 33.5 +/- 25.6 U/ml for those with ovarian endometriosis), and in these patients the post-operative CA-125 level was significantly related to the clinical evolution of the disease, being higher in patients whose disease recurred compared to those with negative follow-up, irrespective of the adhesion score. We conclude that in endometriosis patients, serum CA-125 level is directly related to the adhesion score and peritoneal involvement, suggesting a central role of pelvic and peritoneal irritation in the increased level of this serum marker.

Interferon alpha 2b treatment of cervical intraepithelial neoplasia grade 2: modulation of natural killer cell.

The objective was to evaluate natural cytotoxicity of peripheral blood during interferon treatment in cervical intraepithelial neoplasia grade 2 (CIN2), as index of interferon activity. Twenty-one patients with CIN2, histologically proven, were treated with interferon alpha 2b (Intron A, Sheering-Plough Corp.), in a dose of 3,000,000 units three times per week for 8 weeks self-administered by intramuscular injection. The rate of remission to the interferon treatment was 38.1%. Eight healthy patients had a significant increase in natural killer activity during and after the treatment (p < 0.001), whereas the 13 nonresponder patients remained with low values of natural killer activity. By analyzing the basal natural killer activity before the treatment, the patients with clinicopathologic remission had a significantly higher mean value than nonresponder patients (p = 0.007). Notwithstanding the specimen exiguity, the individual basal natural killer activity seems to be a predictive parameter of interferon treatment response in patients with CIN2.

Endometrioid carcinoma of the ovary. Retrospective study.

From 1985 to 1991, 9 patients with endometrioid carcinoma of the ovary were treated and followed at the University of Ancona, Department of Gynecology and Obstetrics. Four patients (44.4%) had Stage I disease, 1 (11.1%) Stage II, 1 (11.1%) Stage III and 3 (33.3%) Stage IV. Six patients (66.6%) had grade 2 of the disease and 3 (33.3%) grade 3. Two of the patients (22.2%) had synchronous endometrial carcinoma while 3 had histologic evidence of endometriosis at the time of presentation. All the patients received treatment of combination of surgery, polychemotherapy, hormone and/or immunotherapy. The overall survival rate after a median follow up of 26.6 months was 66.6%. A high survival (100%) was observed for patients with associated endometriosis.

Cultured human trophoblast cells reproduce the initial events of placental biology.

OBJECTIVE: The objective of this study was to determine whether cultured trophoblast cells shared the same morphological and biological properties observed in trophoblast, in vivo. STUDY DESIGN: Trophoblast cells from human term placenta were cultured, morphologically, biochemically and immunochemically monitored for as long as 30 days. RESULTS: Single cells progressively aggregated and fused into a syncytio, the Ca2+ and the Ca(2+)-ATPase activity drooped, and the 72 kDa collagenase (MMP-2) was consistently expressed. CONCLUSIONS: Term placenta trophoblast cultures can be viewed and used as a model system mimicking morphological and biochemical events of placenta biology and differentiation.

Lymphocyte subset in HPV infection.

The outcome of HPV infection and its progression to neoplastic disease may be conditioned by the immune status of patients. In the present study we observed the systemic lymphocyte subset, with particular regard to NK cells and NK activity in 7 patients with HPV infection. The lymphocyte phenotypes in peripheral blood were studied using a panel of monoclonal antibodies, while NK activity was evaluated as percentage of lysis of K 562 target cells. We noticed a significant decrease of basal NK activity especially in patients with HPV 16-18 infection (p less than 0.001), without any difference in the absolute number of these cells.

Paget's disease of the vulva: advantages of demolitive surgery.

Four patients with vulvar Paget's disease were analysed retrospectively. In all cases the clinical manifestation was typical, with pruritus and visible lesion. None of the cases had a palpable cutaneous or subcutaneous mass but at the histologic exam a minimally invasive Paget's disease was detected. Treatment consisted of total vulvectomy and subsequent new reconstructive time; only one patient with clinically localized disease was treated with simple vulvectomy. Postoperative complications were minimal, thanks to the reconstructive time that assured a rapid recovery of the surgical injury and reconstruction of the urethral and vaginal meatuses. In all cases we obtained the complete removal of the lesion (negativity of resection margins), except in one case that was also the only one to have a recurrence after 40 months. The patients have been followed for an average of 58 months. Considering the results, we suggest demolitive surgery with subsequent reconstructive time in the treatment of vulvar Paget's disease.