A monocentric analysis of the efficacy of extracellular cryoprotectants in unfrozen solutions for cleavage stage embryos.

BACKGROUND: In the absence of international guidelines indicating the usage of vitrification rather than slow-freezing, the study aim was to analyze a large cohort of slow-frozen/thawed embryos to produce a rationale supporting the standardization of IVF cryopreservation policy. METHODS: This retrospective analysis included 4779 cleavage stage embryos cryopreserved by slow-freezing/thawing from September 2009 to April 2017 at a single Center. Biological and clinical outcomes of three different commercial kits adopted sequentially, i.e. Vitrolife Cleave Kit® from Vitrolife (kit 1) vs. K-SICS-5000 Kit® and K-SITS-5000 Kit® from Cook Medical (kit 2) and Freeze/Thaw 1™ Kit® from Vitrolife (kit 3) were collected and compared in the light of cryoprotectants composition. RESULTS: Kit 3 compared to kit 1 and kit 2 showed significantly (P < 0.001) higher embryo survival (79.9% vs. 75.6 and 68.1%, respectively) and frozen embryo replacement (91.5% vs. 86.5 and 83.3%, respectively) rates, and significantly (P < 0.001) lower blastomere degeneration rate (41.5% vs. 43.6 and 52.4%, respectively). No significant difference for clinical outcomes was observed among kits. Only a slight positive trend was observed for kit 3 vs. kit 1 and kit 2 on delivery rate per thawing cycle (7.12% vs. 4.19 and 4.51%, respectively; P < 0.058) and live birth rate (3.07% vs. 2.59 and 1.93%, respectively, P < 0.069). Thawing solutions of kit 3 were similar to those of any warming protocol. CONCLUSIONS: A defined concentration of extracellular cryoprotectants in thawing/warming solutions had a beneficial effect on the embryo cryosurvival rate. Results could provide the rationale for the adoption of a single standardized warming protocol.

CFTR and FOXO1 gene expression are reduced and high mobility group box 1 (HMGB1) is increased in the ovaries and serum of women with polycystic ovarian syndrome.

We previously described increased HMGB1 and reduced FOXO1 dependent on CFTR loss of function in cystic fibrosis (CF) and we showed in vitro that HMGB1 was lowered by insulin. Reduced CFTR gene expression has been described in granulosa cells (GC) from PCOS-induced rats. We aimed at studying CFTR and FOXO1 gene expression in GC, HMGB1 concentrations in serum and follicular fluids (FF), and insulin and IL-6 in FF in PCOS women. Thirty PCOS and 36 non-PCOS women (CTRL) undergoing in vitro fertilization were enrolled. CFTR and FOXO1 gene expression were downregulated in PCOS (p ≤ .05). HMGB1 was higher in PCOS both in FF (p ≤ .05) and in serum (p < .005) whereas insulin was lower, and IL-6 was unchanged with respect to controls. 17-ß estradiol was higher in PCOS than in CTRL (p ≤ .005). HMGB1 correlated negatively with insulin in FF (p ≤ .005). The increase in HMGB1 both in FF and in serum, likely reflects both low grade inflammation and insulin sensitivity. IL-6 was unchanged possibly reflecting functions other than inflammation.

Development of a novel method for amniotic fluid stem cell storage.

BACKGROUND AIMS: Current procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. METHODS: We compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. RESULTS: hAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. CONCLUSIONS: This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks.

Cancer and fertility preservation: international recommendations from an expert meeting.

In the last years, thanks to the improvement in the prognosis of cancer patients, a growing attention has been given to the fertility issues. International guidelines on fertility preservation in cancer patients recommend that physicians discuss, as early as possible, with all patients of reproductive age their risk of infertility from the disease and/or treatment and their interest in having children after cancer, and help with informed fertility preservation decisions. As recommended by the American Society of Clinical Oncology and the European Society for Medical Oncology, sperm cryopreservation and embryo/oocyte cryopreservation are standard strategies for fertility preservations in male and female patients, respectively; other strategies (e.g. pharmacological protection of the gonads and gonadal tissue cryopreservation) are considered experimental techniques. However, since then, new data have become available, and several issues in this field are still controversial and should be addressed by both patients and their treating physicians.In April 2015, physicians with expertise in the field of fertility preservation in cancer patients from several European countries were invited in Genova (Italy) to participate in a workshop on the topic of "cancer and fertility preservation". A total of ten controversial issues were discussed at the conference. Experts were asked to present an up-to-date review of the literature published on these topics and the presentation of own unpublished data was encouraged. On the basis of the data presented, as well as the expertise of the invited speakers, a total of ten recommendations were discussed and prepared with the aim to help physicians in counseling their young patients interested in fertility preservation.Although there is a great interest in this field, due to the lack of large prospective cohort studies and randomized trials on these topics, the level of evidence is not higher than 3 for most of the recommendations highlighting the need of further research efforts in many areas of this field. The participation to the ongoing registries and prospective studies is crucial to acquire more robust information in order to provide evidence-based recommendations.

"The More the Better" Paradox of Antenatal Ultrasound Examinations in Low-Risk Pregnancy.

Objective To investigate whether different antenatal care models could account for differences in operative delivery rates and adverse neonatal outcomes among low-risk pregnant women, and to identify independent variables associated with delivery modes and adverse neonatal outcomes. Study design Retrospective cohort from a single center of singleton, term, live births between January 2012 and June 2014. Rates of cesarean deliveries, operative vaginal deliveries, and neonatal morbidities were analyzed among women followed by private obstetrician-gynecologists versus national health system providers (certified nurse midwifes supervised by obstetrician-gynecologists), and adjusted for potential confounders. Results Among the 2,831 women in our cohort, obstetric and neonatal outcomes were independent of obstetric providers. After we controlled for confounders, private patients having more than four antenatal ultrasound examinations were more likely to undergo cesarean delivery than public patients with four or fewer sonographic assessments (five to eight prenatal scans: relative risk ratio, 3.3; 95% confidence interval [CI] 1.4-8; nine or more prenatal scans: relative risk ratio, 4.1; 95% CI 1.2-14). Conclusions Multiple prenatal ultrasound examinations in low-risk obstetric populations appear to be an independent and potentially modifiable risk factor for cesarean deliveries.

Human amniotic fluid stem cells: neural differentiation in vitro and in vivo.

The successful integration of stem cells after their implantation into the brain has become a central issue in modern neuroscience. In this study, we test the neural differentiation potential of c-Kit(+)/Oct-4(+) human amniotic fluid stem cells (hAFSCs) in vitro and their survival and integration in vivo. hAFSCs were induced towards neural differentiation and specific markers (GFAP, ß-III tubulin, CNPase, MAP2, NeuN, synapsines, S100, PMP22) were detected by immunofluorescence and Western blot analysis. Glial proteins were expressed as early as 2 weeks after the initial differentiation stimulus, whereas neuronal markers started to appear from the third week of differentiation under culturing conditions of high cell density. This timeline suggested that glial cells possessed a promoting role in the differentiation of hAFSCs towards a neuronal fate. hAFSCs were then implanted into the lateral ventricle of the brain of 1-day-old rats, since neuronal development occurs up to 1 month after birth in this animal model. Our data showed that hAFSCs survived for up to 6 weeks post-implantation, were integrated into various areas of the central nervous system and migrated away from the graft giving rise to mature neurons and oligodendrocytes. We conclude that hAFSCs are able to differentiate and integrate into nervous tissue during development in vivo.

Metformin and gonadotropins for ovulation induction in patients with polycystic ovary syndrome: a systematic review with meta-analysis of randomized controlled trials.

The current systematic review with meta-analysis of randomized controlled trials (RCTs) was aimed to evaluate the effects of metformin on reproductive outcomes in patients with polycystic ovary syndrome (PCOS) who receive gonadotropins for ovulation induction. After systematic review of electronic databases and websites for registration of RCTs, a total of 7 RCTs reporting data on 1023 cycles were included in the final analysis. Descriptive data showed an overall low studies' quality due to unclear sequence generation and allocation concealment, lack of blinding procedure, incomplete outcome data and several biases and/or confounders. Data synthesis showed that metformin improved live-birth (odds ratio [OR] = 1.94, 95% confidence interval [CI] 1.10 to 3.44; P = 0.020) and pregnancy (OR = 2.25, 95% CI 1.50 to 3.38; P < 0.0001) rates, without significant heterogeneity across the studies (P = 0.230, estimation of inconsistency = 30%; and P = 0.710, estimation of inconsistency = 0%, respectively, for live-birth and pregnancy rates). A significant reduction of cancellation rate was observed after metformin administration (OR = 0.41, 95% CI 0.24 to 0.72, P = 0.002) without significant heterogeneity across the studies (P = 0.500, estimation of inconsistency = 0%). Metformin administration influenced or did not influence other secondary endpoints assessed with a significant heterogeneity. In conclusion, metformin administration increases the live-birth and pregnancy rate in PCOS patients who receive gonadotropins for ovulation induction. Further well designed, blinded, placebo-controlled, and adequately powered RCTs are need to confirm that metanalytic results.

Single-incision mini-slings versus retropubic tension-free vaginal tapes: a multicenter clinical trial.

STUDY OBJECTIVE: To compare single-incision mini-slings (SIMSs) and retropubic tension-free vaginal tape (r-TVT) in terms of the long-term efficacy and safety for the treatment of female stress (SUI) or mixed urinary incontinence (MUI). DESIGN: Prospective multicenter cohort trial (registration number NCT00751088) (Canadian Task Force II). SETTINGS: Department of Obstetrics and Gynecology, Italy. PATIENTS: Two hundred-forty women with SUI/MUI. INTERVENTIONS: SIMS or r-TVT. MEASUREMENTS AND MAIN RESULTS: The operative time and the use of analgesic tablets were significantly (p < .001) higher and lower, respectively, in the r-TVT group versus the SIMS group. After 24 months of follow-up, no difference between the study arms was observed in terms of the complication rate (30/120 [25%] vs 19/120 [15.8%] for the r-TVT and SIMS arms, respectively; relative risk = 1.58; 95% confidence interval, 0.94-2.65; p = .083), whereas the subjective cure rate was significantly lower in the SIMS arm than in the r-TVT arm (57/103 [55.3%] vs 89/106 [84.0%] for the r-TVT and SIMS arms, respectively; relative risk = 0.66; 95% confidence interval, 0.54-0.80]; p < .001). The proportion of retreated patients for SUI/MUI was significantly higher in the SIMS arm than in the r-TVT arm (37/103 [34.9%] vs 12/106 [11.3%] for SIMS and r-TVT arm, respectively; p < .001). CONCLUSION: SIMS has no advantage in terms of safety over r-TVT and was found to be less effective than r-TVT. Thus, its use in the clinical practice should be questioned.

Analysis of pronuclear zygote configurations in 459 clinical pregnancies obtained with assisted reproductive technique procedures.

BACKGROUND: Embryos selection is crucial to maintain high performance in terms of pregnancy rate, reducing the risk of multiple pregnancy during IVF. Pronuclear and nucleolar characteristics have been proposed as an indicator of embryo development and chromosomal complement in humans, providing information about embryo viability. METHODS: To correlate the zygote-score with the maternal age and the outcome of pregnancy, we analyzed the pronuclear and nucleolar morphology, the polar body alignment and the zygote configuration in 459 clinical pregnancies obtained by IVF and ICSI in our public clinic in Reggio Emilia, Italy. We derived odds ratios (OR) and Corenfield's 95% confidence intervals (CI). Continuous variables were compared with Student's t-test; P lower than .05 was considered statistically significant. RESULTS: We observed a significant increase of "A" pronuclear morphology configuration in 38-41 years old patients in comparison to that lower than or equal to 32 years old and a significant decrease of "B" configuration in 38-41 years old patients in comparison to that lower than or equal to 32 and in comparison to that of 33-37 years old. Related to maternal age we found no significant differences in P1 and in P2 configuration. We found no correlation between zygote-score, embryo cleavage and embryo quality. CONCLUSIONS: Our results confirm the limited clinical significance of zygote-score suggesting that it can not be associated with maternal age, embryo cleavage and embryo quality. The evaluation of embryo quality based on morphological parameters is probably more predictive than zygote-score.

Impact of insemination technique, semen quality and oocyte cryopreservation on pronuclear morphology of zygotes derived from sibling oocytes.

Pronuclear morphology seems to be an important predictive value of zygote development and integrity. In this study we want to evaluate the effect of insemination technique, male factor and oocyte cryopreservation on pronuclear morphology of zygotes derived from sibling oocytes in our Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova, Reggio Emilia, Italy. Subjects (n = 190) were submitted to IVF cycles with non-frozen and frozen sibling oocytes. Morphological evaluations were assessed using zygote pronuclear morphology (pronuclei, nucleoli and axis) in four groups: Group 1: 144 zygotes from 85 conventional IVF cycles with non-frozen oocytes; Group 2: 164 zygotes from 85 intracytoplasmic sperm injection (ICSI) cycles with Group 1 patients' sibling frozen oocytes; Group 3: 221 zygotes from 123 ICSI cycles with non-frozen oocytes; Group 4: 197 zygotes from 123 ICSI cycles with Group 3 patients' sibling frozen oocytes. No differences between Group 1 and Group 2 were seen. Group 3 was statistically different from Group 4 in relation to the nucleolar morphology. Oocyte cryopreservation procedure modified the nucleolar morphology of zygotes only in the presence of poor semen quality.

Human MATER localization in specific cell domains of oocytes and follicular cells.

MATER (Maternal Antigen That Embryos Require) is an oocyte-specific protein dependent on the maternal genome and required for early embryonic development. The gene products expressed in oocytes play important roles in folliculogenesis, fertilization and pre-implantation development. The aim of this study was to characterize the localization and distribution pattern of the human MATER protein during follicular development and after ovulation, to determine its functional role. Immunocytochemistry experiments coupled with confocal and electron microscopy analysis were carried out to determine the ultrastructural localization of MATER in human ovarian tissue and in isolated oocytes, obtained during IVF procedures. Human cumulus cells were cultured, with or without cycloheximide, to confirm endogenous biosynthesis of the protein. Human MATER is detectable at the onset of the follicular maturation process, suggesting this protein has a role at earlier stages in the human compared with other mammalian species. The presence of MATER is specific to the oocyte and follicular cells that, during maturation, are spatially and functionally associated with the oocyte. The nuclear, nucleolar and mitochondrial localization hints at a possible role in RNA processing and the metabolic activity of the cell.

Reference intervals for hemoglobin and hematocrit in a low-risk pregnancy cohort: implications of racial differences.

Objective: As anemia in pregnancy is associated with adverse perinatal outcomes, we sought to define the mean and the fifth percentile of Hb and Ht using a contemporary multiethnic large cohort of low-risk pregnancies, and assess potential racial differences. Methods: We conducted a retrospective cohort study on women who delivered between 1 January 2008 and 31 December 2013 in Reggio Emilia County, Italy. Linear mixed effects models were used to describe changes in mean Hb and Ht, while quantile regression with matrix-design bootstrap defined changes in the fifth percentile of Hb and Ht, controlling for race, maternal age, smoking, and pregnancy number. Results: We analyzed 23,657 hemograms from 7318 pregnancies and 6870 women. Multivariate analysis showed that when compared to Caucasians', African women's mean Hb and Ht were respectively 0.24 (95%CI 0.3-0.17) g/dl and 0.7 (95%CI 0.8-0.5) % lower, while Asian mothers' were 0.11 (95%CI 0.19-0.03) g/dl and 0.3 (95%CI 0.5-0.1) % inferior. Similarly, both African and Asian women had lower fifth Ht percentiles (-1, 95%CI -1.3 to -0.6, and -0.4, 95%CI -0.7 to -0.04) than Caucasians, while African mothers also had lower fifth Hb percentile (0.3, 95%CI 0.5-0.1). The fifth percentile for Hb and Ht were, respectively, 11.3 (95%CI 11-11.5) g/dl and 32.8 (95%CI 32.3-33.4) % in the first trimester, 10.4 (95%CI 10.1-10.6) g/dl and 30.2 (95%CI 29.6-30.8) % in the second trimester, 10.1 (95%CI 9.8-10.3) g/dl and 30.6 (95%CI 30-31.1) % in the third trimester. Conclusions: We provided contemporary references to define anemia in pregnancy, and we confirmed that even in pregnancy, African and Asian women have lower Hb and Ht than Caucasian. Racial and population-specific references may have significant clinical and public health implication for more accurate disease diagnosis and appropriate treatment.

Uterine Papillary Serous Carcinoma Arising in a Polyp: A Multicenter Retrospective Analysis on 75 Patients.

OBJECTIVES: The objectives of this study were to evaluate whether the international recommendations on the management of uterine papillary serous carcinoma arising in a polyp are uniformly followed in Italian Oncologic Centers and whether the strategy adopted is effective. MATERIALS AND METHODS: Patients with uterine papillary serous carcinoma arising in a polyp and who had undergone a hysterectomy were identified in the 2003-2013 database of 7 Italian Gynecologic Oncology Centers. Clinical and pathologic characteristics and outcomes were compared between staging procedure types. Survival curves of the women were plotted using the Kaplan-Meier method and analyzed using Cox regression hazard model and the log-rank test. Associations between clinical parameters and the incidence of recurrence were assessed by generalized linear models and the Fisher test. RESULTS: A total of 75 patients met the inclusion criteria. Recurrence-free survival was affected positively by type of surgical staging and negatively by preoperative diagnosis of hypertension. The association between surgical staging and recurrence-free survival resulted significant at univariate survival analysis (P=0.048 and 0.045) and maintained a trend of significance (P=0.070) in multivariate analysis, whereas hypertension was demonstrated to be the principal influencing factor. CONCLUSIONS: The international recommendations on the management of uterine papillary serous carcinoma are not uniformly followed in daily practice, although the extension of the surgery seems to be associated with lower recurrence rates also when uterine papillary serous carcinoma is confined to a polyp or endometrial surface.

Psychological vulnerability of singleton children after the 'vanishing' of a co-twin following assisted reproduction.

We test the hypothesis that parents who conceived twins by assisted reproduction technology (ART), but experienced loss of one twin, have a different parental-child relationship compared with ART parents following a singleton pregnancy. We used the 1994-2005 ART database of the Centre for Infertility of the Arcispedale Santa Maria Nuova in Reggio Emilia, Italy to identify families of 53 singleton births after the 'vanishing' twin syndrome. The controls comprised 106 families who conceived and delivered singletons, matched for gestational age (>or= 28 or= 32 or= 37 weeks), maternal age (< 35, >or= 35 40 years), child's age (1-3, 4-6, and 7-11 years) and child's gender. We completed 3 tests: a sociodemographic questionnaire, the QUIT - Italian Questionnaires of Temperament - motor scale, and the Child Vulnerability Scale. We found that children in the study had significantly more difficulties at the beginning of nursery school (p = .002) and kindergarten (p = .0005), with more frequent anxiety of separation from the parents (nursery school, p = .009; kindergarten, p = .001). We found a lower mean QUIT motor score for the 7- to 11-year-old children when compared to the general Italian normative values, suggesting that parents perceived their children as having more motor difficulties. In contrast, analysis of the Child Vulnerability Scale showed that significantly more parents (15.1%) from the controls perceived their child as vulnerable compared to those from the study group (3.8%), p = .034. We conclude that despite the perceived motor difficulties and the difficulties in the process of individuation-separation that appear at the beginning of the different educational circumstances, parents of singletons following the 'vanishing' twin syndrome perceive their children as 'invincible', and thus less vulnerable compared to controls.

Pregnancy complications in infertile patients with polycystic ovary syndrome: updated evidence.

Polycystic ovary syndrome (PCOS) is a common female disorder with a pathogenesis still today not completely known. To the present, PCOS is considered more than just a reproductive disorder since several metabolic consequences that could affect women's health during different stages of reproductive and post-reproductive life were reported. The aim of the current review was to evaluate present evidence-based data regarding the pregnancy complications in infertile patients with PCOS. An extensive literature search until February 2018 was performed in PubMed, Medline, the Cochrane Library and Web of Science. Outcomes were classified in: early pregnancy complications, late pregnancy complications, perinatal complications, offspring health and long-term offspring and maternal health. Even if the exact mechanisms involved are still unclear, women with PCOS have an increased risk of pregnancy-related complications, such as gestational diabetes mellitus (GDM), pregnancy-induced hypertension (PIH), preeclampsia (PE), premature delivery and caesarean section. Moreover, the offspring of women with PCOS are also at increased risk of congenital anomalies and hospitalization in childhood. Further studies are needed to study the mechanism underlying pregnancy complications in PCOS and to identify any interventions to reduce the risk of obstetric and neonatal risks in women affected by PCOS and in their offspring.

Amniotic fluid stem cell exosomes: Therapeutic perspective.

It is widely accepted that the therapeutic potential of stem cells can be largely mediated by paracrine factors, also included into exosomes. Thus, stem cell-derived exosomes represent a major therapeutic option in regenerative medicine avoiding, if compared to stem cells graft, abnormal differentiation and tumor formation. Exosomes derived from mesenchymal stem cells (MSC) induce damaged tissue repair, and can also exert immunomodulatory effects on the differentiation, activation and function of different lymphocytes. Therefore, MSC exosomes can be considered as a potential treatment for inflammatory diseases and also an ideal candidate for allogeneic therapy due to their low immunogenicity. Amniotic fluid stem cells (AFSCs) are broadly multipotent, can be expanded in culture, and can be easily cryopreserved in cellular banks. In this study, morphology, phenotype, and protein content of exosomes released into amniotic fluid in vivo and from AFSC during in vitro culture (conditioned medium) were examined. We found that AFSC-derived exosomes present different molecules than amniotic fluid ones, some of them involved in immunomodulation, such transforming growth factor beta and hepatic growth factors. The immunomodulatory effect of AFSC's exosomes on peripheral blood mononuclear cells stimulated with phytohemagglutinin was compared to that of the supernatant produced by such conditioned media deprived of exosomes. We present evidence that the principal effect of AFSC conditioned media (without exosomes) is the induction of apoptosis in lymphocytes, whereas exposure to AFSC-derived exosomes decreases the lymphocyte's proliferation, supporting the hypothesis that the entire secretome of stem cells differently affects immune-response. © 2017 BioFactors, 44(2):158-167, 2018.

Testicular sperm aspiration (TESA) in 327 ICSI cycles.

OBJECTIVE: To evaluate the efficiency of testicular sperm recovery by testicular sperm aspiration (TESA) in an IVF program. DESIGN: Retrospective Data Analysis. SETTING: The Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova, Reggio Emilia, Italy. PATIENT(S): Couples undergoing TESA/ICSI for obstructive or nonobstructive azoospermia. INTERVENTION(S): ESA/ICSI. MAIN OUTCOME MEASURE(S): Efficiency of testicular sperm recovery, fertilization rate, implantation rate and clinical pregnancy rate. RESULT(S): Between March 1, 1997 and March 31, 2005, 327 cycles of TESA/ICSI were performed in couples in which the male had obstructive or nonobstructive azoospermia. The efficiency of testicular sperm recovery was 99.4% and 99.3%, fertilization rate 57.1% and 49.1%, implantation rate 5.7% and 6.2%, and the clinical pregnancy rate 12.9% and 15.4% in men with obstructive and nonobstructive infertility, respectively. CONCLUSION(S): The efficiency of TESA is very high in both obstructive and nonobstructive azoospermia. Because TESA is less invasive than TESE, it should be considered a valuable alternative to TESE in IVF programs, especially in setting where resources are limited.

NADPH oxidase-4 and MATER expressions in granulosa cells: Relationships with ovarian aging.

AIMS: Relevant roles in follicular development and ovulation are played by maternal antigen that embryos require (MATER), product of a maternal effect gene, and by reactive oxygen species (ROS), indispensable for the induction of ovulatory genes. At the moment, the relationship between these two biological systems and their involvement in the ovarian aging have not been still clarified. The aim of the current experimental study was to analyse the age-related changes of the MATER and NOX proteins. MATERIALS AND METHODS: MATER and ROS homeostasis was studied in granulosa cells (GCs) and cumulus cells (CCs) of infertile patients who undergone oocyte retrieval for in vitro fertilization cycles using Western blot and confocal immunofluorescence analysis. Samples were obtained from subjects with age≥40years (cases) and with age≤37years (controls). KEY FINDINGS: The expression pattern of MATER and NOX observed in GCs was not different from that observed in CCs. High levels of both proteins were detected in the control samples. A significant lower expression of both MATER and NOX4 was observed in the case versus control samples. SIGNIFICANCE: The expression of MATER and NOX4 proteins are closely related to the follicular development and ovulation with particular regard for ovarian aging.

Optimized Cryopreservation and Banking of Human Bone-Marrow Fragments and Stem Cells.

Adult mesenchymal stem cells are a promising source for cell therapies and tissue engineering applications. Current procedures for banking of human bone-marrow mesenchymal stem cells (hBM-MSCs) require cell isolation and expansion, and thus the use of large amounts of animal sera. However, animal-derived culture supplements have the potential to trigger infections and severe immune reactions. The aim of this study was to investigate an optimized method for cryopreservation of human bone-marrow fragments for application in cell banking procedures where stem-cell expansion and use are not immediately needed. Whole trabecular fragments enclosing the bone marrow were stored in liquid nitrogen for 1 year in a cryoprotective solution containing a low concentration of dimethyl sulfoxide and a high concentration of human serum (HuS). After thawing, the isolation, colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, cell senescence, apoptosis, and multi-lineage differentiation potential of hBM-MSCs were tested in media containing HuS compared with hBM-MSCs isolated from fresh fragments. Human BM-MSCs isolated from cryopreserved fragments expressed MSC markers until later passages, had a good proliferation rate, and exhibited the capacity to differentiate toward osteogenic, adipogenic, and myogenic lineages similar to hBM-MSCs isolated from fresh fragments. Moreover, the cryopreservation method did not induce cell senescence or cell death. These results imply that minimal processing may be adequate for the banking of tissue samples with no requirement for the immediate isolation and use of hBM-MSCs, thus limiting cost and the risk of contamination, and facilitating banking for clinical use. Furthermore, the use of HuS for cryopreservation and expansion/differentiation has the potential for clinical application in compliance with good manufacturing practice standards.