Transferase-mediated dUDP Nick-end Labelling Research Articles

Effect of pesticide exposure on human sperm characteristics, genome integrity, and methylation profile analysis.

According to the United Nations' Food and Agriculture Organization, the quantities of pesticide used around the world have increased regularly since the 1990s. Given that pesticides may be classified as carcinogenic, mutagenic, neurotoxic, or toxic for reproduction, some have endocrine-disrupting properties that might be associated with a decline in sperm parameters in general and sperm DNA integrity in particular. These days, a sperm analysis is not enough to determine the etiology of male infertility. Genome integrity analysis is a key step in clarifying a large proportion of cases of male infertility. The objective of the present retrospective study was to assess the impact of self-reported pesticide exposure on sperm parameters and sperm DNA integrity in men consulting for infertility. In a retrospective study, a population of 671 men living in the Picardy region of France were assessed in a conventional sperm parameter analysis, Shorr staining, a DNA fragmentation assay (terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling), and chromatin decondensation with aniline blue staining. The exposed and the non-exposed groups did not differ significantly in some of the conventional sperm parameters (including volume, sperm count, and percent typical forms). However, vitality, progressive motility, and non-progressive motility were significantly lower in the exposed group. Levels of DNA fragmentation and chromatin decondensation were moderately higher in the exposed group.

Electroacupuncture of the Baihui and Shenting acupoints for vascular dementia in rats through the miR-81/IL-16/PSD-95 pathway

BackgroundThere is currently no effective treatment for vascular dementia (VaD). Scalp electroacupuncture (EA) has served clinically as an alternative treatment for VaD, but its mechanism is still unclear. In this study, we investigated the effect of EA at the Baihui (GV 20) and Shenting (GV 24) acupoints on spatial learning and memory ability, and the expression level of microRNA-81 (miR-81), interleukin-16 (IL-16), and postsynaptic density protein-95 (PSD-95) in the frontal cortex of VaD rats.MethodsMale Sprague-Dawley rats were randomly divided into four groups, sham, VaD, non-acupuncture (non-AP) and EA group. The VaD model was established by permanent bilateral occlusion of the common carotid arteries. Morris Water Maze was used to assess the rats’ spatial learning and memory. Immunochemistry (IHC), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blot analysis were performed to detect the expression level of miR-81, IL-16, and PSD-95. Finally, luciferase assay was used to determine the effect of miR-81 on IL-16 expression in PC12 cells.ResultsThe space exploration experiment of MWM showed the time and distance of the rat’s activities around the platform were decreased in the EA group. Compared to the VaD and non-AP group, the number of terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL)-positive frontal cortical neurons was significantly decreased in EA group. The number of the PSD-95-positive cells and the miR-81 expression level in the frontal cortical in the EA group was dramatically increased in comparison with the other groups. In the PC12 cell validation experiment, IL-16 expression level was reduced under the condition of the miR-81 mimic treatment, while increased in the miR-81 inhibitor group. The PSD-95 protein level was up-regulated in the small interfering (si)RNA-IL16 group compared to the NC-IL16 groups with or without oxygen/glucose deprivation/reperfusion (OGD/R) conditions (P<0.05). However, this was abolished by miR-81 mimic.ConclusionsIn VaD rats, EA may improve spatial learning and memory through miR-81/IL-16/PSD-95 pathway.

IVF/ICSI cumulative live birth rates per consumed oocyte remain comparable regardless of sperm DNA fragmentation by TUNEL

Research questionDoes sperm DNA fragmentation (SDF) affect reproductive success of IVF and intracytoplasmic sperm injection (ICSI) cycles measured as cumulative live birth rates (CLBR) in unselected couples? DesignClinical data from 1339 couples undergoing 2759 IVF/ICSI cycles using autologous oocytes with a SDF test by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay on their ejaculated spermatozoa were retrospectively evaluated. Main outcomes were calculated according to two different analyses: using 15% SDF as cut-off point (low ≤15% and high >15%); and categorizing participants based on four SDF ranges (<10%, 10– <20%, 20–30% and >30%). Live birth rate and CLBR per number of embryo transfers, per number of embryos replaced and consumed oocytes required to achieve the first live birth according to level of SDF were the main outcomes assessed. ResultsNo significant difference was found in clinical pregnancy rate and miscarriage rate between both groups. No differences in LBR per embryo transfer were found for the first or for all embryo transfers when comparing ≤15% and >15% sperm DNA fragmentation or by SDF ranges. The CLBR according to the number of embryo transfers and the number of embryos replaced showed no statistically significant differences between different SDF groups. When the same number of oocytes were inseminated, similar CLBR were obtained regardless of the degree of male sperm DNA fragmentation. ConclusionsHigh SDF did not impair live birth rates of unselected males undergoing IVF/ICSI cycles with autologous oocytes per transfer or the cumulative probability of a live birth.

Long-time low-temperature transportation of human ovarian tissue before cryopreservation

Research questionCan the low-temperature transport time of removed human ovarian tissue be prolonged until cryopreservation? DesignFresh ovarian cortex from nine premenopausal patients was either slow-frozen immediately or stored at 4°C for 24 or 48 h before slow-freezing. The fresh and frozen–thawed biopsies were evaluated by follicle counting via calcein staining, histologic analyses via haematoxylin and eosin staining, and apoptosis via terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL). The fresh cortex was assessed by reactive oxygen species (ROS) and total antioxidant capacity (TAC) assay to detect oxidative stress. The frozen–thawed cortex biopsies were also evaluated by quantitative PCR for messenger RNA (mRNA) expression of BCL-2, BAX, TNFa, HIF-1a, BMP15 and GDF9, and Western blot for detection of BCL-2, BMP15, GDF9 and CASPASE-3. The frozen–thawed cortex was cultured in vitro for 4 days, anti-Müllerian hormone and glucose were assessed in the supernatant, and ROS and TAC assay detected any oxidative stress in the cortex. ResultsIn the fresh cortex, there were no significant differences between the three groups. In the frozen–thawed cortex, there were no significant differences between the three groups regarding follicle viability, TUNEL, mRNA expression of TNFa, HIF-1a or BMP15. GDF9 mRNA and BAX/BCL-2 were lower and higher at 48 h than at 0 h, respectively. However, the protein expression of BCL-2, CASPASE-3, GDF9 and BMP15 were no different. In the cultured cortex, ROS, TAC and glucose uptake were no different across the three groups. ConclusionOvarian tissue transportation was validated for 24 h in the procedure used in clinical practice. This study showed that 4–8°C transportation for 24 or 48 h does not seem to damage the ovarian tissue. However, ovarian tissue transportation beyond 48 h needs to be further studied for conclusions to be made.

Advanced glycation end products present in the obese uterine environment compromise preimplantation embryo development

Research questionProinflammatory advanced glycation end products (AGE), highly elevated within the uterine cavity of obese women, compromise endometrial function. Do AGE also impact preimplantation embryo development and function? DesignMouse embryos were cultured in AGE equimolar to uterine fluid concentrations in lean (1–2 µmol/l) or obese (4–8 µmol/l) women. Differential nuclear staining identified cell allocation to inner cell mass (ICM) and trophectoderm (TE) (day 4 and 5 of culture). Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling assay (day 5). Day 4 embryos were placed on bovine serum albumin/fibronectin-coated plates and embryo outgrowth assessed 93 h later as a marker of implantation potential. AGE effects on cell lineage allocation were reassessed following pharmacological interventions: either 12.5 nmol/l AGE receptor (RAGE) antagonist; 0.1 nmol/l metformin; or combination of 10 µmol/l acetyl-l-carnitine, 10 µmol/l N-acetyl-l-cysteine, and 5 µmol/l alpha-lipoic acid. Results8 µmol/l AGE reduced: hatching rates (day 5, P < 0.01); total cell number (days 4, 5, P < 0.01); TE cell number (day 5, P < 0.01), and embryo outgrowth (P < 0.01). RAGE antagonism improved day 5 TE cell number. ConclusionsAGE equimolar with the obese uterine environment detrimentally impact preimplantation embryo development. In natural cycles, prolonged exposure to AGE may developmentally compromise embryos, whereas following assisted reproductive technology cycles, placement of a high-quality embryo into an adverse ‘high AGE’ environment may impede implantation success. The modest impact of short-term RAGE antagonism on improving embryo outcomes indicates preconception AGE reduction via pharmacological or dietary intervention may improve reproductive outcomes for overweight/obese women.

Interaction of M2 macrophages and endometrial cells induces downregulation of GRIM-19 in endometria of adenomyosis

Research questionDoes the aggregation of M2 macrophages affect the expression of gene associated with retinoid-interferon-induced mortality 19 (GRIM-19) in adenomyosis? DesignEndometrial tissues were collected from patients with (n = 15) and without (n = 15) adenomyosis. Tissues were analysed for GRIM-19 and toll-like receptor 4 (TLR4) expression by immunohistochemistry and western blotting. Apoptosis was analysed by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. Human endometrial stromal cells (HESC) were transfected with GRIM-19 small interfering RNA (SiRNA) to knockdown GRIM-19 expression. The HESC were co-cultured with M2 macrophages to detect the influence of M2 macrophages in HESC cells. Analyses included GRIM-19, caspase-3 and TLR4 expression by western blotting, and GRIM-19 and TLR4 by quantitative real-time polymerase chain reaction. Apoptosis was measured by flow cytometry and TUNEL assay. Cell proliferation (Cell Counting Kit-8 assay) and migration assays were carried out. ResultsThe expression of GRIM-19 was significantly lower in adenomyosis lesions compared with controls (P < 0.001). Deficiency of GRIM-19 induced by siRNA decreased apoptosis and increased proliferation and migration in HESC. A significant decrease in GRIM-19 expression occurred in HESC after co-culture with M2 macrophages (P = 0.018). After co-culture with M2 macrophage, apoptosis decreased and proliferation and cell invasion in HESC increased. Protein (P = 0.006) and mRNA (P = 0.013) expression of TLR4 in HESC also reduced after this co-culture. Up-regulation of GRIM-19 occurred in HESC treated with the activator TLR4 (P = 0.016). Up-regulation of GRIM-19 was significantly reversed in cells treated with the TLR4 inhibitor (P = 0.011). ConclusionM2 macrophages may be involved in regulating the expression of GRIM-19 partly through the TLR4 signalling axis in adenomyosis.

Association between sperm DNA fragmentation and idiopathic recurrent pregnancy loss: a systematic review and meta-analysis.

Sperm DNA fragmentation (sDF) has emerged as a valuable tool for evaluating male fertility, yet the relationship between DNA fragmentation in the male gamete and idiopathic recurrent pregnancy loss (RPL) remains a topic of ongoing debate. Hence, a meta-analysis was conducted of 12 prospective and 2 retrospective studies involving 530 men with a history of RPL who underwent sDF testing compared with 639 fertile control participants. The main outcome measures were sDF measured by comet assay, TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL), sperm chromatin dispersion (SCD) or sperm chromatin structure assay. Overall, couples with a history of idiopathic RPL demonstrated higher levels of sDF than fertile couples (average mean difference 11.98, P<0.001). Subgroup analysis demonstrated a similar average mean difference between the RPL and control groups using SCD compared with TUNEL, while mean paternal age and mean sperm motility in the RPL groups tested by meta-regression demonstrated no significant effect on the mean differences in sDF (P>0.10). These results support the diagnostic value of sDF over standard semen analysis, as well as a possible paternally derived genetic origin of unexplained RPL. Further prospective studies are required to further assess the predictive utility of sDF for assessing couples with unexplained RPL.

Development and characterization of a novel long-acting recombinant follicle stimulating hormone agonist by fusing Fc to an FSH-β subunit

Does a novel long-acting recombinant human FSH, KN015, a heterodimer composed of FSHα and FSHβ-Fc/Fc, offer a potential FSH alternative? KN015 had in vitro activity and superior in vivo bioactivity than recombinant human FSH (rhFSH), suggesting KN015 could serve as a potential FSH agonist for clinical therapy. rhFSH has very short half-life so that repeat injections are needed, resulting in discomfort and inconvenience for patients. The longest-acting rhFSH available in clinics is corifollitropin alpha (FSH-CTP), but its half-life is not long enough to sustain the whole therapy period, and additional injections of rhFSH are needed. Plasmids containing FSHα, FSHβ-Fc and Fc cDNA were transfected into Chinese hamster ovary (CHO) cells for KN015 production. The pharmacokinetics of KN015 was investigated in 6-week-old SD rats (n = 6/group) and healthy Cynomolgus monkeys in two different dose groups (n = 2/group). A series of experiments were designed for in vitro and in vivo characterization of the bioactivity of KN015 relative to rhFSH. The purity and molecular weight of KN015 were determined by reducing and non-reducing SDS-PAGE. To measure KN015 half-life, sera were collected at increasing time points and the remaining FSH concentration was measured by enzyme-linked immunosorbent assay. To assess the bioactivity of KN015 versus rhFSH in vitro, firstly cAMP production was assessed in CHO cells expressing FSH receptor (FSHR) with the treatment of Fc/Fc, rhFSH or KN015 at eight different doses (0.03, 0.09, 0.28, 0.83, 2.5, 7.5, 22.5, 67.5 nM), and secondly cumulus oocyte complexes (COCs; n = 20/group) of ICR mice (primed-PMSG 44 h before sacrificed) were collected and cultured in medium containing 1.25 pM Fc/Fc, rhFSH or KN015 at 37°C and then germinal vesicle breakdown (GVBD) and COC expansion were observed at 4 and 16 h, respectively. The in vivo activity of KN015 was compared with rhFSH by ovary weight gain and ovulation assays. In the former, ovary weight gains in 21-day-old female SD rats, after a single subcutaneous injection of KN015, were compared with those after several injections of rhFSH over a range of doses (n = 8/group). Sera were harvested for estradiol (E2) analysis, and the ovaries were processed for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL), RT-PCR and western blot. In the latter, 26-day-old female SD rats (n = 8/group) were injected with different doses of KN015 or rhFSH, and were sacrificed at 24 h after an injection of hCG (20 IU/rat). Moreover, the molecular responses stimulated by KN015 or rhFSH in the ovary were also analyzed through detecting expression of the FSH target genes (Cyp19a1, Fshr and Lhcgr) and phosphatidylinositide 3-kinase (PI3K) pathway activation. KN015 has a molecular weight of 82 kD and its half-life is 84 h in SD rats (10-fold longer than that of rhFSH) and 215 h in Cynomolgus monkeys. The EC50 value of the cAMP induction in CHO cells (KN015 versus rhFSH, 1.84 versus 0.87 nM), COC expansion and oocyte maturation assays showed KN015 had approximately half of rhFSH's activity in vitro. A single dose of KN015 (1.5 pmol/rat, 166.1 ± 19.7 mg, P < 0.01) stimulated significantly larger ovary weight gain than several injections of rhFSH (1.5 pmol/rat, 59.3 ± 28.1 mg, P < 0.01). The serum E2 level in the KN015 group was significantly higher than that in rhFSH group. The number of oocytes obtained by ovulation induction was comparable with or higher in the KN015 group than in the rhFSH group. KN015 was more effective than rhFSH in inducing FSH target genes (Cyp19a1, Fshr, Lhcgr) or activating the PI3K pathway in vivo. Moreover, a single injection of KN015 promoted granulosa cell proliferation and prevented follicle atresia to the same extent as several injections of rhFSH. All assays in this study were operated only in animals and clinical trials are needed to confirm they can be extrapolated to humans. KN015 is a valuable alternative to FSH and may have great potential for therapeutic applications. This study was supported by National Basic Research Program of China (2011|CB944504, 2012CB944403) and National Natural Science Foundation of China (81172473, 31371449). The authors have no conflicts of interest to declare.

Changes in Levels of Seminal Nitric Oxide Synthase, Macrophage Migration Inhibitory Factor, Sperm DNA Integrity and Caspase-3 in Fertile Men after Scrotal Heat Stress.

BackgroundThis study observes changes in levels of seminal nitric oxide (NO), nitric oxide synthase (NOS), macrophage migration inhibitory factor (MIF), sperm DNA integrity, chromatin condensation and Caspase-3in adult healthy men after scrotal heat stress (SHS).MethodsExposure of the scrotum of 25 healthy male volunteers locally at 40–43°C SHS belt warming 40 min each day for successive 2 d per week. The course of SHS was continuously 3 months. Routine semen analysis, hypo-osmotic swelling (HOS) test, Aniline blue (AB) staining, HOS/AB and terminal deoxynucleotidyl transferase-mediated d UDP nick-end labeling (TUNEL) were carried out before, during and after SHS. Seminal NO and NOS contents were determined by nitrate reduction method. The activated Caspase-3 levels of spermatozoa and MIF in seminal plasma were measured by the enzyme-linked immunosorbent assay (ELISA) method. Statistical significance between mean values was determined using statistical ANOVA tests.ResultsThe mean parameters of sperm concentration, motile and progressive motile sperm and normal morphological sperm were significantly decreased in groups during SHS 1, 2 and 3 months compared with those in groups of pre-SHS (P<0.001). Statistically significant differences of sperm DNA fragmentation, normal sperm membrane, and Caspase-3 activity as well as the level of NO, NOS and MIF in semen were observed between the groups before SHS and after SHS 3 months and the groups during SHS 1, 2 and 3 months (P<0.001). After three months of the SHS, various parameters recovered to the level before SHS. WBC in semen showed a positively significant correlation with the levels of NO, NOS, MIF and Caspase-3 activity. The percentage of abnormal sperm by using the test of HOS showed a positively significant correlation with that of HOS/AB.ConclusionsThe continuously constant SHS can impact the semen quality and sperm DNA and chromatin, which may be contributed to the high level of NO, NOS, MIF and Caspase-3 by SHS.

DNA double strand breaks in human spermatozoa can be predictive for assisted reproductive outcome

Sperm DNA status has been reported to predict fertility outcomes in infertile men. The terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling test (TUNEL) is the most widely used method to evaluate this; however, its prognostic value is still debated. One hundred infertile men undergoing intracytoplasmic sperm injection (ICSI) and 61 fertile men were tested for sperm parameters, sex hormones and sperm DNA status by chromatin tests (acridine orange, aniline blue, decondensation) and by direct assays (TUNEL and phosphorylated histone H2AX-γH2AX). In both groups, the prognostic value of each parameter to predict assisted clinical pregnancy was compared. Sperm parameters (P < 0.05 or P < 0.01), FSH levels (P < 0.05) and DNA status (P < 0.05 to P < 0.001) were significantly different in participants compared with controls. Among infertile men, 47 had positive and 53 had ICSI outcome. Both chromatin analysis and TUNEL test were unable to distinguish individuals who had successful outcomes from those who failed ICSI treatments. γH2AX percentage and γH2AX fragmentation index were significantly higher in sperm from non-pregnant compared with pregnant couples (P < 0.05 and P < 0.01). γH2AX assay is more predictive of ICSI outcome than TUNEL in infertile couples with male factor infertility.

Gonadotrophin-releasing hormone agonists for fertility preservation: unraveling the enigma?

Can gonadotrophin-releasing hormone agonists (GnRH-a) preserve long-term fertility when administered prior to and concomitantly with chemotherapy? GnRH-a display a differential protective effect on fertility, depending upon the specific chemotherapy-induced mechanism of ovarian injury. The role of GnRH-a in fertility preservation has been constantly debated and their use is considered experimental due to conflicting clinical evidence and paucity of data regarding their mechanism for ovarian protection. In vivo model: 7-8 weeks old imprinting control region (ICR) mice were injected with GnRH-a (Leuprolide-acetate) or saline prior to and concomitantly with cyclophosphamide, doxorubicin or saline and sacrificed at various time-points on a longitudinal follow-up; 24 h (n = 36), 1 week (n = 40), 1 month (n = 36) and 9 months (n = 66) post chemotherapy treatment. Blood samples were drawn on Day 0 and on a monthly basis after chemotherapy treatment. On the day of sacrifice, blood samples were drawn and ovaries excised and processed for either immunohistochemistry (IHC), protein or RNA extraction. In vitro model: 21-23 days old Wistar-derived rats were sacrificed, their ovaries excised and primary granulosa cells (PGC) were either isolated for in vitro culture, or processed for immunofluorescence (IF) as well as for protein or RNA extraction. Ovarian reserve was estimated by serial measurements of serum anti-mullerian hormone (AMH), quantified by the AMH Gen II ELISA assay. Ovarian AMH and phosphorylated Akt (pAkt) were detected by immunoblotting. Vascular endothelial growth factor (VEGF) was measured by quantitative PCR. Ovarian GnRH receptor (GnRHR), AMH and CD34 were visualized by IHC, and apoptosis was evaluated using TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL). Cyclophosphamide-induced ovarian injury caused a prompt decrease in AMH level (P < 0.01) and a further long-term decline in serum AMH (P = 0.017), indicating damage to the ovarian reserve. Pretreatment with GnRH-a diminished AMH-decrease (P < 0.05) and maintained serum AMH level in the long run (P < 0.05). Doxorubicin-exerted ovarian-vascular-injury is also displayed by an acute increase in ovarian VEGF level (P < 0.05) and a sustained decrease in serum AMH level (P < 0.001). This was followed by ovarian recovery manifested by increased neovascularization. GnRH-a delayed the recovery in AMH level and decreased the level of VEGF (P < 0.001), thus interfering with the vascular recovery subsequent to doxorubicin-induced vascular damage. To portray the differential mechanism of each chemotherapy, cyclophosphamide and doxorubicin were given separately, whereas most of the clinical protocols include several types of chemotherapies. Thus, future study should explore a prospective evaluation of various chemotherapies, as well as combined chemotherapeutic protocols. Our study demonstrates that different chemotherapy agents affect the ovary via diverse mechanisms and thus the administration of GnRH-a concomitantly, could be beneficial to a subpopulation of patients treated with cyclophosphamide-based protocols. This work was partially supported by a grant from the Israel Science Foundation (ISF) to I.B.-A. The authors have no conflict of interest to disclose.

Mitogen-Activated Protein Kinase–Activated Protein Kinase 2 Mediates Apoptosis during Lung Vascular Permeability by Regulating Movement of Cleaved Caspase 3

Apoptosis is a key pathologic feature in acute lung injury. Animal studies have demonstrated that pathways regulating apoptosis are necessary in the development of acute lung injury, and that activation of p38 mitogen-activated protein kinase (MAPK) is linked to the initiation of the apoptotic cascade. In this study, we assessed the role of the MAPK-activated protein kinase (MK) 2, one of p38 MAPK's immediate downstream effectors, in the development of apoptosis in an animal model of LPS-induced pulmonary vascular permeability. Our results indicate that wild-type (WT) mice exposed to LPS demonstrate increased apoptosis, as evidenced by cleavage of caspase 3 and poly (ADP-ribose) polymerase 1 and increased deoxynucleotidyl transferase-mediated dUDP nick-end labeling staining, which is accompanied by increases in markers of vascular permeability. In contrast, MK2(-/-) mice are protected from pulmonary vascular permeability and apoptosis in response to LPS. Although there was no difference in activation of caspase 3 in MK2(-/-) compared with WT mice, interestingly, cleaved caspase 3 translocated to the nucleus in WT mice while it remained in the cytosol of MK2(-/-) mice in response to LPS. In separate experiments, LPS-induced apoptosis in human lung microvascular endothelial cells was also associated with nuclear translocation of cleaved caspase 3 and apoptosis, which were both prevented by MK2 silencing. In conclusion, our data suggest that MK2 plays a critical role in the development of apoptosis and pulmonary vascular permeability, and its effects on apoptosis are in part related to its ability to regulate nuclear translocation of cleaved caspase 3.

Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization

Sperm chromatin/DNA damage can be measured by a variety of assays. However, it has been reported that these tests may lose prognostic value in Assisted Reproductive Technology (ART) cycles when assessed in post-prepared samples, possibly due to the normalizing effect promoted by sperm preparation procedures. We have recently implemented a modified version of the Diff-Quik staining assay that allows for the evaluation of human sperm chromatin status in native samples, together with standard sperm morphology assessment. However, the value of this parameter in terms of predicting in vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) outcomes after sperm selection is unknown. In this study, data from 138 couples undergoing in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatments showed that sperm chromatin integrity was significantly improved after density gradient centrifugation and swim up (p<0.001), but no correlations were found with fertilization or embryo development rates (p>0.05). However, sperm samples presenting lower percentages of damaged chromatin were associated with better quality (Grade I) embryos in both ART procedures (p<0.05) and clinical pregnancy among IVF couples (p<0.05). Furthermore, regression analysis confirmed the clinical value of Diff-Quik staining in predicting IVF (but not ICSI) clinical pregnancy (OR: 0.927, 95% CI: 0.871-0.985, p=0.015), and a threshold value of 34.25% for this parameter was established. The proportion of IVF couples achieving a clinical pregnancy was reduced 1.9-fold when the percentage of abnormal dark staining was ≥34.25% (p=0.05). In conclusion, the Diff-Quik staining assay provides useful information regarding ART success, particularly in IVF cycles, where some degree of 'natural' sperm selection may occur; but not in ICSI, where sperm selection is operator dependent. This quick and low-cost assay is suggested as an alternative method to detect sperm chromatin status in minimal clinical settings, when no other well-established and robust assays (e.g. Sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling) are available.

Apoptosis of human ovarian tissue is not increased by either vitrification or rapid cooling

This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in vitrified and rapidly cooled human ovarian tissue. Apoptosis was assessed 30min and 24h after warming using transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation, as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryoprotectant solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis and either protocol could be an alternative to slow cooling of ovarian tissue.This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in human ovarian tissue cryopreserved using two different methods, i.e. vitrification and rapid cooling. Apoptosis was assessed in tissue 30min and 24h after warming using transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryopreservation solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis in human ovarian tissue and either protocol could be an alternative to slow cooling for the preservation of ovarian tissue.

Gonadotrophin-releasing hormone antagonist induces apoptosis in human decidual stromal cells: effect on GADD45  and MAPK signaling

The impact of gonadotrophin-releasing hormone (GnRH) antagonists used in IVF protocols on endometrial tissue remodeling, embryo implantation and the programming of early pregnancy is still unclear. Pregnancy and infant outcomes after treatment with GnRH antagonist for IVF are particular causes of concern. The purpose of this study was to investigate the mechanisms of GnRH antagonist-induced apoptosis of human decidual stromal cells and the effects of GnRH antagonist on the activation of ERK1/2, JNK and GADD45α signaling. Human decidual stromal cells were isolated from decidual tissue. The expression of GnRH-I receptor (GnRH-IR) was examined by immunoblot analysis and immunohistochemistry. The cells were treated with the GnRH antagonist, Cetrorelix. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay was used to examine cell viability. Cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling assay were used as indicators for cell apoptosis. Mitogen-activated protein kinase function was tested for the elucidation of intracellular signalings through the pre-treatment of stromal cells with ERK1/2 (U0126) and JNK (SP600125) inhibitors prior to the Cetrorelix treatment. To characterize the signaling pathway of GnRH antagonist, the endogenous GnRH-IR and GADD45α were knocked down by specific small-interfering RNA (siRNA). The GnRH-IR is expressed in human decidual stromal cells. Treatment with GnRH antagonist decreased cell viability, induced apoptosis and increased the phosphorylation of ERK1/2 and JNK. Cells pre-treated with U0126 and SP600125 were rescued from the GnRH antagonist-mediated inhibition of cell growth and did not exhibit GnRH antagonist-induced apoptosis and downstream GADD45α signaling. GnRH antagonist-mediated cell growth inhibition and apoptosis were also abolished by the knockdown of the endogenous GnRH-IR or GADD45α with siRNAs. The GnRH antagonist suppresses the growth of decidual stromal cells by inducing apoptosis through the GnRH-IR and through the ERK1/2 and JNK phosphorylation-dependent induction of GADD45α signaling. These results indicate that ERK1/2, JNK and GADD45α are coordinately regulated by the GnRH antagonist through the GnRH-IR to induce apoptosis in human decidual stromal cells, suggesting that the GnRH antagonist may play a role in decidual programming in human pregnancy.

Evidence that carbonyl stress by methylglyoxal exposure induces DNA damage and spindle aberrations, affects mitochondrial integrity in mammalian oocytes and contributes to oocyte ageing

Highly reactive carbonyl compounds formed during glycolysis, such as methylglyoxal (MG), can lead to the formation of 'advanced glycation end products' (AGE) and carbonyl stress. Toxic AGEs are suspected to accumulate and play a role in reducing quality and developmental potential of mammalian oocytes of aged females and in PCOS and diabetic patients. Whether and how MG and AGE affect young and aged oocytes at the cellular level is unknown. The study consists of three parts. In Part A expression of MG-detoxifying enzymes glyoxalases 1 and 2 was analysed by RT-PCR at different stages of maturation in denuded oocytes (DO), cumulus-enclosed oocytes (CEO) and metaphase (M)II oocytes of the CD-1 mouse to obtain information on stage-specific susceptibility to carbonyl stress. DO and CEO from young and aged females and from stimulated cycles were exposed to MG during maturation in vitro to assess also age-related changes in sensitivity to carbonyl stress induced by MG. Induction of apoptosis by MG on in vitro maturing DO was assessed by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling test. In Part B of the study, DO from large antral follicles of ovaries of adult, young MF-1 mice in late diestrous were exposed to MG to assess direct influences of MG and AGEs formed during continuous exposure to MG on rate and kinetics of maturation to MII, on DNA integrity (by γ-H2AX staining) in the germinal vesicle (GV) stage, and on spindle formation and chromosome alignment (by tubulin and pericentrin immunofluorescence and polarization microscopy), and chromosome segregation (by C-banding) during in vitro maturation. Since MG and AGEs can affect functionality of mitochondria in Part C, mitochondrial distribution and membrane potential was studied using JC-1 probe. Expression of a redox-sensitive mito-Grx1-roGFP2 protein in mitochondria of maturing oocytes by confocal laser scanning microscopy was employed to determine the inner mitochondrial glutathion (GSH)/glutathion disulfide (GSSG)-dependent redox potential. Part A revealed that mRNA for glyoxalases decreases during meiotic maturation. Importantly, cumulus from aged mice in CEO obtained from stimulated cycles does not protect oocytes efficiently from MG-induced meiotic arrest during in vitro maturation. Part B showed that the MG-induced meiotic delay or arrest is associated with significant rises in spindle aberrations, chromosome congression failure and aberrant telophase I in oocytes. MG exposure of meiotically arrested GV-stage oocytes significantly increases the numbers of γ-H2AX spots in the nucleus suggesting increased DNA damage, while MG exposure during maturation affects chromatin condensation and induces chromosome lagging at anaphase I. Moreover, Part C revealed that carbonyl stress by chronic exposure to MG is associated with delays in changes in mitochondrial distribution and altered inner-mitochondrial GSH/GSSG redox potential, which might be particularly relevant for cytoskeletal dynamics as well as processes after fertilization. Sensitivity to a meiotic block by MG appears dependent on the genetic background. The sensitivity to carbonyl stress by MG appears to increase with maternal age. Since MG-exposure induces DNA damage, meiotic delay, spindle aberrations, anaphase I lagging and epimutation, aged oocytes are particularly at risk for such disturbances in the absence of efficient protection by cumulus. Furthermore, disturbances in mitochondrial distribution and redox regulation may be especially critical for fertilization and developmental competence of oocytes exposed to MG and carbonyl stress before or during maturation, for instance, in aged females, or in PCOS or diabetic patients, in agreement with recent suggestions of correlations between poor follicular and embryonic development, lower pregnancy rate and presence of toxic AGEs in serum, irrespective of age.

Pyrrolidine dithiocarbamate inhibits nuclear factor- B pathway activation, and regulates adhesion, migration, invasion and apoptosis of endometriotic stromal cells

The activation of nuclear factor-κB (NF-κB) has been implicated in the development and progression of endometriosis. The aim of this study is to investigate the potential application of pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, in the treatment of endometriosis. NF-κB-DNA-binding activity, IκB phosphorylation and expression of nuclear p65 protein in endometriotic ectopic stromal cells (EcSCs), endometriotic eutopic stromal cells (EuSCs) and normal endometrial stromal cells (NESCs) were detected by electrophoretic mobility shift assay and western blot analysis. Adhesion, migration, invasion and apoptosis of EcSCs were observed by means of adhesion, migration, invasion and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling assay, respectively. Gene and protein expressions of CD44s, matrix metalloproteinase (MMP)-2, MMP-9 and survivin in EcSCs were measured by RT-PCR and western blot analysis. The results showed that PDTC in the absence or presence of interleukin (IL)-1β showed stronger inhibitory effects on NF-κB-DNA-binding activity, IκB phosphorylation and expression of nuclear p65 protein in EcSCs than those in EuSCs or NESCs. PDTC enhanced apoptosis, and suppressed IL-1β-induced cellular adhesion, migration and invasion of EcSCs. Pretreatment of EcSCs with PDTC attenuated IL-1β-induced expressions of CD44s, MMP-2, MMP-9 and survivin at gene and protein levels. All these findings suggest that PDTC induces apoptosis and down-regulates adhesion, migration and invasion of EcSCs through the suppression of various molecules. Therefore, PDTC could be used as a therapeutic agent for the treatment of endometriosis.

Paternal age and sperm DNA decay: discrepancy between chromomycin and aniline blue staining

The effect of paternal age on sperm DNA fragmentation and decondensation was determined in a retrospective study involving 1769 patients. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay was used to assess fragmentation, and DNA decondensation was measured with either chromomycin or aniline blue staining. The impact of atypical forms was also analysed. DNA fragmentation increases with age, but is independent of the percentage of atypical forms. Both staining techniques revealed a negative correlation between the quality of sperm packaging and the percentage of atypical forms. Decondensation increases with increasing age and fragmentation when measured with chromomycin; however, an inverse relationship is observed when testing is performed using aniline blue. These observations are discussed in relation to the specificity of the dyes, the deposition of protamines and the impact of age and reactive oxygen species on protamine cross-linking.

Improvement in sperm DNA quality using an oral antioxidant therapy

Oxidative stress is now recognized as a common pathology that affects up to half of all infertile men. One of the principal mechanisms by which oxidative stress produces infertility is by damage to sperm DNA, either through direct oxidation of the DNA by reactive oxygen species (ROS) or by the initiation of apoptosis. The objective of this study was to determine if an oral antioxidant/mineral supplement could improve sperm DNA integrity in men with known oxidative stress. A total of 50 infertile men identified as exhibiting oxidative stress were administered oral antioxidant therapy for a period of 3 months. All participants were assessed at entry and exit for sperm DNA integrity with terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling, apoptosis with annexin V, protamination with chromomycin A(3) and ROS production with nitro blue tetrazolium assay. Sperm concentration, motility and morphology, together with assessment of serum male reproductive hormones (LH, FSH, testosterone, anti-Mullerian hormone), were also monitored. The principal finding that emerged from this study was that antioxidant therapy resulted in significant improvements in sperm DNA integrity (P = 0.002) and protamine packaging (P < 0.001), accompanied by a reduction in seminal ROS production (P = 0.027) and apoptosis (P = 0.004). No significant changes in routine sperm parameters (concentration, motility, morphology) or male reproductive hormones were observed.

Evaluation of human sperm function after being cryopreserved within the zona pellucida

To investigate the fertilization ability, chromatin structure, and DNA integrity of spermatozoa after being cryopreserved within an empty zona pellucida (ZP). Prospective study. Reproductive research center in a university-affiliated hospital. Normozoospermic patients. Spermatozoa were cryopreserved within the ZP or with traditional methods. The sperm recovery rate, sperm motility, fertilization ability, chromatin structure, and DNA integrity were assessed before and after cryopreservation. Significantly higher sperm recovery rate was identified for the spermatozoa cryopreserved within the ZP than those cryopreserved with traditional methods, but the motility recovery was similar. Frozen-thawed samples showed increased damage to the sperm chromatin and DNA, which were assessed by acridine orange test (AO) and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling assay; however, no difference of chromatin and DNA integrity was observed for spermatozoa cryopreserved within the ZP or with traditional methods. In addition, the fertilization ability, as assessed by injecting spermatozoa into hamster oocytes, was similar for spermatozoa cryopreserved with different cryopreservation methods. Cryopreservation of spermatozoa within an empty ZP results in higher sperm recovery rate, and the post-thaw sperm functions of spermatozoa cryopreserved within the ZP are comparable with spermatozoa cryopreserved with traditional methods.