Investigation of subfertility in the female Nsmf knockout mouse.

OBJECTIVE: To study if a pituitary or ovarian defect contributes to subfertility of the female Nsmf knockout (KO) mouse, an animal model of the hypogonadotropic hypogonadism gene NSMF. DESIGN: Analysis of hypothalamic, pituitary and ovarian gene expression at baseline, serum gonadotropin levels before and after gonadotropin-releasing hormone (GnRH) stimulation, ovarian response and implantation after superovulation, gonadotropin effects after ovariectomy, and ovarian NSMF protein expression. SETTING: University research laboratory. PATIENTS: None; mice were used. INTERVENTIONS: Gonadotropin-releasing hormone stimulation, superovulation, and ovariectomy in separate experiments. MAIN OUTCOME MEASURES: Gene expression in the hypothalamus, pituitary, and ovary; ovarian response and implantation after superovulation; serum gonadotropins after GnRH stimulation and ovariectomy; Western blot to measure ovarian NSMF expression. RESULTS: We found increased hypothalamic Kiss1, Gnrh1, and Jak2 mRNA expression in female Nsmf KO vs. wild type (WT) mice. However, pituitary gonadotropin, and GnRH receptor gene expression was not affected, and serum gonadotropin levels were normal. Gonadotropins increased after ovariectomy for both groups. Baseline Kiss1, Fshr, Prkaca, Prkar1a, and Gdf9 ovarian mRNA expression was increased and Cyp19a1 expression was decreased in Nsmf KO mice, while superovulated Nsmf KO mice had reduced ovarian Kiss1r, Prkar1a, and Fshr mRNA expression, 50% less oocytes, and normal implantation. Western blot demonstrated NSMF protein expression in the ovary of WT mice. CONCLUSIONS: Altered hypothalamic and ovarian gene expression was demonstrated in female Nsmf KO mice. It is possible that increased hypothalamic Gnrh1 and Kiss1 mRNA expression could compensate for reduced NSMF enabling a normal pituitary gonadotropin response. Impaired superovulation response, altered ovarian gene expression, and decreased number of oocytes indicate ovarian dysfunction, but a uterine factor cannot be excluded. These findings provide an anatomic basis for future mechanistic studies of subfertility in female Nsmf KO mice.

Photobiomodulation has rejuvenating effects on aged bone marrow mesenchymal stem cells.

The plasticity and proliferative capacity of stem cells decrease with aging, compromising their tissue regenerative potential and therapeutic applications. This decline is directly linked to mitochondrial dysfunction. Here, we present an effective strategy to reverse aging of mouse bone marrow mesenchymal stem cells (BM-MSCs) by restoring their mitochondrial functionality using photobiomodulation (PBM) therapy. Following the characterization of young and aged MSCs, our results show that a near-infrared PBM treatment delivering 3 J/cm2 is the most effective modality for improving mitochondrial functionality and aging markers. Furthermore, our results unveil that young and aged MSCs respond differently to the same modality of PBM: whereas the beneficial effect of a single PBM treatment dissipates within 7 h in aged stem cells, it is lasting in young ones. Nevertheless, by applying three consecutive treatments at 24-h intervals, we were able to obtain a lasting rejuvenating effect on aged MSCs. Our findings are of particular significance for improving autologous stem cell transplantation in older individuals who need such therapies most.

Chemically Defined and Xeno-Free Cryopreservation of Human-Induced Pluripotent Stem Cells.

Human-induced pluripotent stem cells (hiPSCs) can be derived from a variety of biopsy samples and have an unlimited capacity for self-renewal and differentiation into almost any cell type in the body. Therefore, hiPSCs offer unprecedented opportunities for patient-specific cell therapies, modeling of human diseases, biomarker discovery, and drug testing. However, clinical applications of hiPSCs require xeno-free and, ideally, chemically defined methods for their generation, expansion, and cryopreservation. In this chapter, we present a chemically defined and xeno-free slow freezing method for hiPSCs along with a chemically undefined protocol. Both approaches yield reasonable post-thaw viability and cell growth.

Can serum procalcitonin levels be useful in predicting spontaneous ureteral stone passage?

BACKGROUND: Medical expulsive therapy (MET) is recommended for ureteral stones when there is no indication for interventional treatment. Spontaneous passage (SP) may not always be perceived in patients undergoing MET. We aimed to demonstrate the effects of inflammatory factors on spontaneous ureteral stone passage in patients undergoing MET. METHODS: Our study was conducted between August and November, 2016, in healthy volunteers and patients with a single distal ureteral stone between 5 and 10 mm in diameter and no indications for interventional therapy. Blood and urine samples from all patients and healthy volunteers were tested. The patients were followed up every 2 weeks for 1 month unless emergency situations appeared. Patients with stone-free status at follow-up were concluded to have achieved complete stone passage [SP(+)], and failure [SP(-)] was concluded if the patient had not passed the stone by the end of the study. Blood samples of the patients and the control group were analyzed, recording WBC (white blood cell), CRP (c-reactive protein), SED (sedimentation), MPV (mean platelet volume), NLR (neutrophil-to-lymphocyte ratio), and serum procalcitonin levels. Abnormalities in urine samples were recorded. All patients received diclofenac sodium 75 mg/day, tamsulosin 0.4 mg/day, and at least 3 l/day fluid intake. Patients were followed for a month with kidney, ureter, bladder (KUB) plain films, ultrasonography (USG), and unenhanced abdominal CT scans while undergoing MET. Comparative statistical analyses were performed between the SP(+) and SP(-) groups. RESULTS: The procalcitonin levels of the SP(-) group were significantly higher (207 ± 145.1 pg/ml) than in the SP(+) group (132.7 ± 28.1 pg/ml) (p = 0.000). The leucocyturia rate of the SP(-) group was significantly higher than in the SP(+) group (p = 0.004). Based on the ROC curve analysis, 160 pg/ml (86.7% sensitivity, 70.8% specificity, p < 0.001; AUC: 0.788 95% CI (0.658-0.917) was identified as the optimal cut-off value for procalcitonin. In logistic regression analysis, a significant efficacy of procalcitonin and leucocyturia was observed in the univariate analysis on spontaneous passage. In the multivariate analysis, significant independent activity was observed with procalcitonin. (p < 0.05). CONCLUSION: Our findings suggest that high procalcitonin levels and the presence of leucocyturia have a strong negative effect on SP of ureteral stones between 5 and 10 mm in diameter. This relationship can be explained by stone impaction, possibly caused by increased mucosal inflammation.

Probing lasting cryoinjuries to oocyte-embryo transcriptome.

Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated legal and ethical issues, and delaying childbearing years. While the outcome of oocyte cryopreservation has recently been improved, currently used vitrification method still suffer from increased biosafety risk and handling issues while slow freezing techniques yield overall low success. Understanding better the mechanism of cryopreservation-induced injuries may lead to development of more reliable and safe methods for oocyte cryopreservation. Using the mouse model, a microarray study was conducted on oocyte cryopreservation to identify cryoinjuries to transcriptionally active genome. To this end, metaphase II (MII) oocytes were subjected to standard slow freezing, and then analyzed at the four-cell stage after embryonic genome activation. Non-frozen four-cell embryos served as controls. Differentially expressed genes were identified and validated using RT-PCR. Embryos produced from the cryopreserved oocytes displayed 200 upregulated and 105 downregulated genes, associated with the regulation of mitochondrial function, protein ubiquitination and maintenance, cellular response to stress and oxidative states, fatty acid and lipid regulation/metabolism, and cell cycle maintenance. These findings reveal previously unrecognized effects of standard slow oocyte freezing on embryonic gene expression, which can be used to guide improvement of oocyte cryopreservation methods.

Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer.

Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants.

A toxicity cost function approach to optimal CPA equilibration in tissues.

There is growing need for cryopreserved tissue samples that can be used in transplantation and regenerative medicine. While a number of specific tissue types have been successfully cryopreserved, this success is not general, and there is not a uniform approach to cryopreservation of arbitrary tissues. Additionally, while there are a number of long-established approaches towards optimizing cryoprotocols in single cell suspensions, and even plated cell monolayers, computational approaches in tissue cryopreservation have classically been limited to explanatory models. Here we develop a numerical approach to adapt cell-based CPA equilibration damage models for use in a classical tissue mass transport model. To implement this with real-world parameters, we measured CPA diffusivity in three human-sourced tissue types, skin, fibroid and myometrium, yielding propylene glycol diffusivities of 0.6 × 10-6 cm2/s, 1.2 × 10-6 cm2/s and 1.3 × 10-6 cm2/s, respectively. Based on these results, we numerically predict and compare optimal multistep equilibration protocols that minimize the cell-based cumulative toxicity cost function and the damage due to excessive osmotic gradients at the tissue boundary. Our numerical results show that there are fundamental differences between protocols designed to minimize total CPA exposure time in tissues and protocols designed to minimize accumulated CPA toxicity, and that "one size fits all" stepwise approaches are predicted to be more toxic and take considerably longer than needed.

Female sexual function following a novel transobturator sling procedure without paraurethral dissection (modified-TOT)

ABSTRACT Purpose To determine whether there is a difference in sexual function after modified and classical TOT procedures. Materials and Methods Of the 80 sexually active women with SUI, 36 underwent an original outside-in TOT as described by Delorme, and 44 underwent modified TOT procedure, between 2011 and 2015. The severity of incontinence and sexual function were evaluated using International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) and Female Sexual Function Index (FSFI) questionnaires preoperatively and 3 months after surgery. Results The postoperative ICIQ-SF score was significantly lower than the preoperative ICIQ-SF score in both groups (p=0.004 for modified TOT and p=0.002 for classical TOT). There was no significant difference in the ICIQ-SF score reduction between the two groups (14.1±2.1 vs. 14.4±1.9; p=0.892). Complication rates according to the Clavien-Dindo classification were also similar in both groups. In both groups, difference between preoperative and postoperative FSFI scores revealed a statistically significant improvement in all domains. Comparison of postoperative 3-month FSFI scores of modified and classical TOT groups showed statistically significant differences in arousal, lubrication and orgasm domains. Desire, satisfaction, pain and total FSFI scores did not differ significantly between two groups. Conclusion The modified TOT technique is a simple, reliable and minimal invasive procedure. The cure rate of incontinence and complication rates are the same as those of the classical TOT technique. However, due to the positive effects of minimal tissue damage on sexual arousal and orgasmic function, modified TOT has an advantage over the classical TOT.

Female sexual function following a novel transobturator sling procedure without paraurethral dissection (modified-TOT).

PURPOSE: To determine whether there is a difference in sexual function after modified and classical TOT procedures. MATERIALS AND METHODS: Of the 80 sexually active women with SUI, 36 underwent na original outside-in TOT as described by Delorme, and 44 underwent modified TOT procedure, between 2011 and 2015. The severity of incontinence and sexual function were evaluated using International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) and Female Sexual Function Index (FSFI) questionnaires preoperatively and 3 months after surgery. RESULTS: The postoperative ICIQ-SF score was significantly lower than the preoperative ICIQ-SF score in both groups (p=0.004 for modified TOT and p=0.002 for classical TOT). There was no significant difference in the ICIQ-SF score reduction between the two groups (14.1±2.1 vs. 14.4±1.9; p=0.892). Complication rates according to the Clavien-Dindo classification were also similar in both groups. In both groups, difference between preoperative and postoperative FSFI scores revealed a statistically significant improvement in all domains. Comparison of postoperative 3-month FSFI scores of modified and classical TOT groups showed statistically significant differences in arousal, lubrication and orgasm domains. Desire, satisfaction, pain and total FSFI scores did not differ significantly between two groups. CONCLUSION: The modified TOT technique is a simple, reliable and minimal invasive procedure. The cure rate of incontinence and complication rates are the same as those of the classical TOT technique. However, due to the positive effects of minimal tissue damage on sexual arousal and orgasmic function, modified TOT has an advantage over the classical TOT.

Prognostic value of endocan in prostate cancer: clinicopathologic association between serum endocan levels and biochemical recurrence after radical prostatectomy.

PURPOSE: To assess the diagnostic capability of serum endocan level in association with clinicopathologic features and its impact on biochemical progression-free survival in patients with prostate cancer (PCa). METHODS: A total of 86 patients with localized prostate cancer were treated with open radical prostatectomy (RP). The control group included 80 patients who were referred to the urology outpatient clinic with normal rectal examination and prostate-specific antigen (PSA) levels. The patients' characteristics, baseline PSA value, and serum endocan levels were recorded. The patients were followed up with the measurement of PSA concentration every 3 months during the first year, thereafter every 6 months until 5 years, then yearly after surgery. The primary endpoint of follow-up was the time of biochemical recurrence. RESULTS: The median serum endocan levels were 3.14 ng/mL in the RP group and 2.98 ng/mL in the control group (p = 0.122). A total of 86 patients who underwent RP for PCa were divided into 2 groups based on a cutoff serum endocan level of 1.8 ng/mL. The distribution of Gleason score and biochemical failure rate were significantly higher in patients with serum endocan ≥1.8 ng/mL (p = 0.031 and p = 0.047). The biochemical recurrence-free time for endocan ≥1.8 ng/mL and <1.8 ng/mL were 38 and 56 months, respectively (p = 0.041). Spearman correlation analysis showed a linear relationship between endocan expression and Gleason score (p = 0.025, p = 0.511). Multivariate analysis revealed that elevated serum endocan level (≥1.8 ng/mL) was a significant predictor of biochemical progression-free survival (hazard ratio 2.44; 95% confidence interval 1.78-3.23; p = 0.001). CONCLUSIONS: The current study indicates that endocan has a close relationship with tumor recurrence in PCa.

Characterization of antioxidant system parameters in four freshwater fish species.

The potential use of antioxidant system parameters has gained considerable interest due to their pivotal role of detoxification mechanisms in environmental studies and culture fish point of view. Fish with different ecological needs may have different antioxidant capacity and response to environmental contaminants. Thus, the optimal working conditions and specific enzyme activities (Vmax and Km) of antioxidant system parameters (Superoxide dismutase, SOD; Catalase, CAT; Glutathione peroxidase, GPX; Glutathione reductase, GR and Glutathione S-transferase, GST) and glutathione (GSH) were determined in four commonly cultured freshwater fish species (tilapia; Oreochromis niloticus, carp; Cyprinus carpio, trout; Onchorhynchus mykiss and catfish; Clarias garipienus). Data showed that optimal concentrations of different buffers, pH and specific chemicals for each enzyme and GSH were similar in most cases for all fish species, except a few differences. The highest Vmax and Km values were found in carp for GPX and GST, though these values were the highest in tilapia, catfish and trout for CAT, SOD and GR, respectively. As a conclusion, optimization assays of these parameters in different bioindicator organisms based on their physiological and ecological differences may be useful for the aquatic ecosystem biomonitoring studies and also present fundamental data for utilization in aquaculture.

Response of antioxidant system of tilapia (Oreochromis niloticus) following exposure to chromium and copper in differing hardness.

Tilapias (Oreochromis niloticus) were exposed to copper or chromium in soft water (SW) (~80 mg CaCO3/L, conductivity 1.77 mS/cm) or hard water (HW) (~320 mg CaCO3/L, conductivity 5.80 mS/cm) using 2 exposure protocols (20 µM for 48 h and 10 µM for 144 h). Following the exposures, antioxidant enzyme activities [superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase; glutathione reductase; and glutathione S-transferase (GST)] and glutathione (GSH) levels were measured in the liver of fish. SOD and CAT activities of control fish kept in SW were significantly lower than control fish kept in HW. However, the other antioxidant indices (glutathione metabolism) of both control fish were unaffected from water hardness. Acute metal exposures did not alter the glutathione metabolism, whereas SOD activity in SW and CAT activity in both waters changed significantly. In subchronic duration, Cu exposure caused significant decreases in measured parameters, except for GST activity and GSH level. Similarly, GST activity and GSH level were unaffected from Cr exposure. This study showed that SOD and CAT were the most sensitive antioxidant indices, and that glutathione metabolism, in general, was not altered following metal exposures in different waters.

An essential role for heat shock transcription factor binding protein 1 (HSBP1) during early embryonic development.

Heat shock factor binding protein 1 (HSBP1) is a 76 amino acid polypeptide that contains two arrays of hydrophobic heptad repeats and was originally identified through its interaction with the oligomerization domain of heat shock factor 1 (Hsf1), suppressing Hsf1's transcriptional activity following stress. To examine the function of HSBP1 in vivo, we generated mice with targeted disruption of the hsbp1 gene and examined zebrafish embryos treated with HSBP1-specific morpholino oligonucleotides. Our results show that hsbp1 is critical for preimplantation embryonic development. Embryonic stem (ES) cells deficient in hsbp1 survive and proliferate normally into the neural lineage in vitro; however, lack of hsbp1 in embryoid bodies (EBs) leads to disorganization of the germ layers and a reduction in the endoderm-specific markers (such as α-fetoprotein). We further show that hsbp1-deficient mouse EBs and knockdown of HSBP1 in zebrafish leads to an increase in the expression of the neural crest inducers Snail2, Tfap2α and Foxd3, suggesting a potential role for HSBP1 in the Wnt pathway. The hsbp1-deficient ES cells, EBs and zebrafish embryos with reduced HSBP1 levels exhibit elevated levels of Hsf1 activity and expression of heat shock proteins (Hsps). We conclude that HSBP1 plays an essential role during early mouse and zebrafish embryonic development.

Effects of Cd, Zn and Cd + Zn combination on ATPase activitiy in the gill and muscle of tilapia (Oreochromis niloticus).

This study investigated the responses of Na(+)/K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase in the gill and muscle of a freshwater fish Oreochromis niloticus exposed to 1 µg/mL of Cd and Zn and their mixture for different periods (0, 7, 14, 21 and 28 days). At the end of experimental periods, the activities Na(+)/K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase in gill tissues and only Ca(2+)-ATPase activity in muscle tissues were measured. Gill Na(+)/K(+)-ATPase activity generally decreased following single metal exposures, whereas their combinations increased its activity. Gill Ca(2+)-ATPase activity decreased relative to the control at most exposure times for single exposures of Zn and Cd, as well as for the combined exposure. There was no gill Ca(2+)-ATPase activity after 28 days of exposure to Zn and Cd combined. Mg(2+)-ATPase activity was not affected significantly in gill tissue by exposure to Zn and Cd individually or in combination. Muscle Ca(2+)-ATPase activity also decreased significantly following metal exposure, but not as greatly as in the gill tissue. Tissue protein levels were mostly unaffected by metal exposures. This study showed that certain ATPases are highly sensitive to metal exposure whether the metals are essential or non essential, and suggests using gill tissue Na(+)/K(+)-ATPase and Ca(2+)-ATPase as sensitive biomarkers in metal contaminated waters.

Role of ART in imprinting disorders.

Assisted reproductive technologies (ART) offer revolutionary infertility treatments for millions of childless couples around the world. Currently, ART accounts for 1 to 3% of annual births in industrialized countries and continues to expand rapidly. Except for an increased incidence of premature births, these technologies are considered safe. However, new evidence published during the past decade has suggested an increased incidence of imprinting disorders in children conceived by ART. Specifically, an increased risk was reported for Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS), Silver-Russell syndrome, and retinoblastoma. In contrast, some studies have found no association between ART and BWS, AS, Prader-Willi syndrome, transient neonatal diabetes mellitus, and retinoblastoma. The variability in ART protocols and the rarity of imprinting disorders complicate determining the causative relationship between ART and an increased incidence of imprinting disorders. Nevertheless, compelling experimental data from animal studies also suggest a link between increased imprinting disorders and ART. Further comprehensive, appropriately powered studies are needed to better address the magnitude of the risk for ART-associated imprinting disorders. Large longitudinal studies are particularly critical to evaluate long-term effects of ART not only during the perinatal period but also into adulthood. An important consideration is to determine if the implicated association between ART and imprinting disorders is actually related to the procedures or to infertility itself.

Cryobanking of embryoid bodies to facilitate basic research and cell-based therapies.

Pluripotent stem cells offer unique opportunities for curing debilitating diseases. However, further comprehensive research is needed to better understand cell signaling during the differentiation of pluripotent cells into different cell lineages and accordingly to develop clinically applicable protocols. One of the limiting steps for differentiation studies is proper culture and expansion of pluripotent stem cells, which is labor intensive, expensive, and requires a great deal of expertise. This limiting step can be overcome by successful banking and distribution of embryoid bodies (EBs), which are aggregates of pluripotent stem cells and typically the starting point of differentiation protocols. The objective of this study was to investigate the feasibility of EB banking by studying survival and functionality of cryopreserved EBs. To this end, EBs were formed by culturing mouse 129 embryonic stem (ES) cells in the absence of leukemia inhibitory factor (LIF) in hanging drops and then subjected to different cryopreservation protocols. In a series of experiments, we first tested the postthaw survival of EBs as a function of dimethylsulfoxide (DMSO) and extracellular trehalose concentrations and cooling rates. Next, we studied the functionality of cryopreserved EBs by assessing their postthaw attachment, growth, and differentiation into various cell types. Higher (≥5%) DMSO concentrations alone or in combination with trehalose (0.1 M and 0.2 M) yielded good postthaw survival rates of >80%, whereas cooling of EBs at 1°C/min in the presence of 5% DMSO +0.1 M trehalose gave the best attachment and growth rates, with differentiation into cell lineages of three germ layers. Taken together, our results suggest that EBs are tolerant to cryopreservation-associated stresses and retain their differentiation potential after freezing and thawing. Furthermore, our experiments with dissociated EB cells and nondissociated EBs suggest that the extracellular matrix may play a beneficial role in the cryotolerance of EBs. Overall, our data support the feasibility of EB banking, which would facilitate advancement of cell-based therapies.

Retrieval of rhesus monkey (Macaca mulatta) oocytes by ultrasound-guided needle aspiration: problems and solutions.

Oocytes of nonhuman primates such as rhesus monkeys are excellent models for diverse studies on developmental biology, epigenetics, human reproduction, and assisted reproductive technologies, as well as on transgenics. Such studies require numerous oocytes that can be retrieved after controlled ovarian stimulation. Currently, most primate centers use laparoscopic aspiration or laparotomy followed by aspiration to collect rhesus oocytes, although the ultrasound-guided needle aspiration is more advantageous due to reduced infection risk, less injury, and a shorter recovery period. Yet, some initial difficulties associated with the ultrasound-guided needle aspiration limit its broader application. The objective of the present study was to address these obstacles. By presenting practical solutions to the initial difficulties, results from our study show that it is possible to collect a mean number of 38 +/- 10 rhesus oocytes per hormonally stimulated female. These results compare favorably to the average number of rhesus oocytes collected using the laparoscopic approach and suggest that when initial obstacles are overcome, the ultrasound-guided oocyte retrieval represents a good alternative to more invasive approaches.

Application of intra- and extracellular sugars and dimethylsulfoxide to human oocyte cryopreservation.

PURPOSE: Oocyte cryopreservation may avoid many complications of human embryo freezing and provide future fertility for women undergoing cancer therapy. The objective of this study was to explore the application of intra- and extracellular sugars in combination with small amounts of dimethylsulfoxide (DMSO) to human oocyte cryopreservation as an alternative approach. METHODS: Discarded human oocytes that were obtained from IVF patients under informed consent and IRB approval, were cryopreserved by slow cooling to -196 degrees C after being randomly distributed into three groups: (i) DMSO control without intra- and extracellular sugar; (ii) extracellular sugar (raffinose) + DMSO; and (iii) intra- and extracellular sugar (trehalose and raffinose, respectively) + DMSO. Subsequently, all cryopreserved oocytes were thawed rapidly, and their survival was assessed by morphological criteria after 24 h of culture. RESULTS: A total of 71 oocytes were evaluated in three groups with survival rates of 88.5% (23/26), 68.2% (15/22), and 52.2% (12/23) for intra- and extracellular sugar+DMSO, extracellular sugar+DMSO, and DMSO control groups, respectively. CONCLUSION: These results support the use of intra- and extracellular sugars as an alternative approach for cryopreservation of human oocytes.

Laser-assisted intracytoplasmic sperm injection in human oocytes.

OBJECTIVE: To explore potential applications of a non-contact, 1,480-nm diode laser to intracytoplasmic sperm injection (ICSI) of human oocytes. STUDY DESIGN: Human oocytes obtained from in vitro fertilization (IVF) patients and failed to fertilize 24-48 hours after conventional IVF were used for ICSI along with discarded sperm. A noncontact, 1,480-nm diode laser was employed to immobilize sperm, to open a hole in the zona pellucida (ZP) and to perform ICSI through the hole. After ICSI and its simulation, oocytes were examined for formation of pronuclei, cleavage and normality of the cytoskeleton. RESULTS: The 1,480-nm diode laser permitted fast and easy sperm immobilization and microdrilling of ZP to facilitate microinjection. Of the 78 injected oocytes, 53 (68%) survived the procedure, and 13 (25%) of them formed two pronuclei by 18 hours. Further culture of two fertilized eggs resulted in cleavage up to the eight-cell stage before cease of culture. Four oocytes were fixed after simulation of the procedure without injecting sperm. None of them showed gross abnormalities in cytoskeletal organization. CONCLUSION: A noncontact, 1,480-nm diode laser can be used for the immobilization of sperm and for opening a hole in the ZP to facilitate ICSI, biopsy manipulation toward preimplantation genetic diagnosis and assisted hatching.