Implementation of an in-house flow cytometric analysis of DNA fragmentation in spermatozoa

An increased amount of DNA fragme ntation in the spermatozoa(SDF)is linked to male in fertility.The Sperm Chromati n Structure Assay(SCSA)is widely used for analysis of SDF.However,the current software(SCSASoftR)linked to this assay is licensed and often located within larger diagnostic centers.In this study,we present a protocol for using other types of software than SCSASoftR to determine the SDF index(DFI)with clinical relevance.This protocol is engineered after collect!ng and analyzing 254 samples from fertility patients and sperm donors over a 15-month period.DFI is analyzed using a strict protocol where the spermatozoa are treated with a strong acid(pH 1.2)followed by acridine orange.DFI is determined by a standard flow cytometric software,FACSDiva 6.1.3.Analysis of the outcome of the fertility treatment is included for 137 patients receiving either intrauterine inseminations(IUI)or timed coitus(TC).The results show that the chance of pregnancy decli nes as DFI in creases.We also found that the male DFI affects the chanee of pregnancy independent of the female age.We have shown that a standard flow cytometric software can be used when determi ning a clinical releva nt DFI.These findings are a sign ificant step toward impleme nting the an alysis as a part of the routi ne,in・house diag no sing of the male fertility patient and subseque ntly optimizing the treatme nt course of the couple with reduced human and financial costs.     

Association of XRCC1 and ERCC2 promoters’ methylation with chromatin condensation and sperm DNA fragmentation in idiopathic oligoasthenoteratozoospermic men

The aim of the study was to investigate whether the promoter methylation of XRCC1 and ERCC2 genes is associated with sperm DNA fragmentation and chromatin condensation in idiopathic oligoasthenoteratozoospermic men. This study involved 77 infertile men with idiopathic oligoasthenoteratozoospermia and 51 normozoospermic controls. The methylight method, TUNEL assay and aniline blue staining were used for the evaluation of XRCC1 and ERCC2 genes’ methylation, SDF and sperm chromatin condensation, respectively. SDF (p = .004) and XRCC1 methylation (p = .0056) were found to be significantly higher in men with idiopathic OAT than in the controls, while mature spermatozoa frequency was higher in controls as compared to infertile men (p < .0001). No significant association was found between SDF and methylation of XRCC1 and ERCC2 genes (p = .9277 and p = .8257, respectively). However, compared to the cut-off point obtained by receiver operating characteristic analysis, a significant association was found between SDF and XRCC1 methylation, positive and negative methylation groups, generated according to the cut-off value for XRCC1. XRCC1 methylation was found to have a significant effect on chromatin condensation (p = .0017). No significant difference was detected among ERCC2 methylation, male infertility and SDF. In conclusion, XRCC1 methylation may have a role in sperm chromatin condensation and idiopathic OAT.     

Can Melissa officinalis improve chromatin structure and sperm parameters in a rat model of varicocele?

Excessive production of reactive oxygen species is a central mechanism in the pathology of varicocele; Melissa officinalis (MO) is a medicinal plant from the Lamiaceae family that has antioxidant activity. In this study, we have investigated the potential therapeutic effect of MO on sperm parameters and chromatin structure in varicocelised rat. Male Wistar rats were randomly assigned to control, sham, varicocele, MO treatment and placebo groups. Rats in varicocele, MO treatment and placebo groups underwent left varicocele induction. Following the operation, rats in placebo and MO treatment groups received normal saline or MO extract, daily for 10 weeks respectively. At 10 weeks after varicocele induction, sperm chromatin and parameters were evaluated in all groups. Chromatin structure was evaluated by aniline blue, acridine orange and toluidine blue staining. All three staining outcomes were increased in varicocele and placebo compared control (and sham) groups (p < 0.05). Sperm count, motility, morphology and vitality were decreased between varicocele and control (and sham) group (p < 0.05). Sperm parameters and chromatin staining (else AB staining) outcomes improve in MO treatment compared to varicocele and placebo groups (p < 0.05). These findings suggest that MO ameliorate the harmful effect of varicocele on sperm parameters and chromatin structure.     

Are N‐glycolylneuraminic acid (Hanganutziu–Deicher) antigens important in pig‐to‐human xenotransplantation?

BACKGROUND: N-glycolylneuraminic acid (NeuGc) epitopes, so called Hanganutziu-Deicher (HD) antigens, which are widely expressed on endothelial cells of all mammals except humans, are considered to be potential targets for natural and elicited anti-nonGalalpha1-3 Gal (Gal) antibodies in humans. We previously reported that anti-NeuGc antibodies were not detected in healthy humans by enzyme-linked immunosorbent assay (ELISA) using NeuGc-GM3-coated plates, and no antibody production was observed in patients with a history of exposure to pig cells. However, a recent paper has revealed that (i) anti-NeuGc antibodies to porcine red blood cells (PRBC) are detectable in most healthy humans, and (ii) the majority of anti-nonGal antibodies are specific for NeuGc epitopes. The purpose of this study was to reassess whether NeuGc is important as an immunogenic nonGal epitope. METHODS: The binding of antibodies to PRBC and porcine aortic endothelial cells (PAEC) was compared. Cells were treated with (i) alpha-galactosidase, and then (ii) neuraminidase, which digests sialic acids, including NeuGc epitopes. Cells were incubated with human pooled sera, and applied to flow cytometric analysis. After enzyme digestion, almost complete reduction of Gal and NeuGc expression was confirmed by GS-IB4 and HU/Ch2-7 (a chicken monoclonal antibody against HD antigens), respectively. Trypsin, which removes membrane glycoproteins, and endoglycoceramidase which cleaves glycolipids, were used for differentiating between NeuGc-containing glycoproteins and glycolipids. RESULTS: Neuraminidase-treatment reduced the binding of immunoglobulin G (IgG) antibodies to PRBC; about half of the anti-nonGal IgG antibodies to PRBC were directed to NeuGc. In contrast, anti-nonGal antibodies to PAEC were not directed to NeuGc. Trypsin-treatment markedly reduced the expression of NeuGc only on PRBC. Endoglycoceramidase-treatment was followed by a greater reduction in NeuGc epitopes on PAEC than on PRBC. Most NeuGc on PRBC appeared to be linked to proteins, but most NeuGc on PAEC was expressed on glycolipids. CONCLUSIONS: Carbohydrate structures on PRBC are different from those on PAEC. Healthy human sera contain anti-nonGal IgG antibodies to NeuGc expressed on PRBC, but not on PAEC. We speculate that anti-nonGal IgG antibodies to PRBC can recognize both NeuGc and protein, and this may be the reason why such antibodies have not been detected by ELISA. A definite conclusion about the immunogenicity of NeuGc has not been obtained. More sera from patients (not from non-human primates) sensitized with porcine cells or organs need to be studied.     

Does sperm DNA fragmentation have negative impact on embryo morphology and morphokinetics in IVF programme?

Evaluation of sperm integrity may predict the in vitro fertilisation (IVF) outcomes. The aim was to evaluate the relationship between the sperm DNA fragmentation (sDNAf) with embryo morphology and morphokinetic using time-laps monitoring (TLM) and to select the best time points for normalisation in IVF setting. After evaluating the fertilisation and pronuclei (Z) scoring, 328 normally fertilised oocytes were assessed to time of pronuclei fading, time of 2 to 8 discrete cells (t2–t8) and abnormal cleavage patterns, such as multinucleation, direct cleavage, reverse cleavage and fragmentation. Sperm chromatin dispersion (SCD) assay was used for assessment of prepared sperm chromatin status. SCD was categorised into 4 groups of <6.5, 6.5–10.7, 10.7–20.1 and >20.1. The finding showed significant differences in t6 (p = .012), t7 (p = .045), t8 (p = .013) and s1 (p = .001) between 4 SCD groups. When morphokinetic variables were normalised to tPNf, this difference was observed in t2 (p = .003) and t6 (p = .017). Subsequently, the percentage of top quality embryos and Z₁ scoring were dependent to the sDNAf rate. In conclusion, tPNf was the best reference time point in IVF cycles. Also, we found high sDNAf rate had no negative impact on embryo morphology and morphokinetics in conventional IVF.     

DNA fragmentation in morphologically normal spermatozoa: how much should we be concerned in the ICSI era?

Intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility. However, there are still unanswered questions about the safety of this technique. During ICSI, only morphologically normal and motile spermatozoa are typically used to fertilize an oocyte. We recently reported that in infertile men, spermatozoa with apparently normal morphology may have DNA fragmentation. This finding consequently raised the possibility that spermatozoa with normal-shaped appearance but with DNA fragmentation could be mistakenly selected to fertilize oocytes during ICSI. This concern became more clinically significant following the subsequent finding that the presence of an increased proportion of normal spermatozoa with damaged DNA was negatively associated with embryo quality and pregnancy outcome after ICSI. Herein, we propose and discuss the hypothesis that the examination of DNA integrity in the subpopulation of highly motile (hence viable) and morphologically normal cells (and not in the total sperm population) may provide optimized information in prediction of ICSI success. More importantly, this new way of evaluation may provide reassurance about genomic normalcy and minimal risk of transmission of genetic disease and guide the development of improved methods of selection of spermatozoa with intact DNA to be used in assisted reproduction.     

Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection （ICSI） using ejaculated and testicular spermatozoa

AIM: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). METHODS: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. RESULTS: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. CONCLUSION: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.     

Presence of HHV‐6 genome in spermatozoa in a context of couples with low fertility: what type of infection?

Human herpesvirus‐6 (HHV‐6) is a betaherpesvirus whose genome may integrate into human chromosomes. Chromosomally integrated HHV‐6 (ciHHV‐6) may be transmitted vertically from parents to children. HHV‐6 DNA has been detected in semen, but its integrated or extrachromosomal status has not yet been characterised. The aim of this study was to determine the prevalence of HHV‐6 DNA and to search for ciHHV‐6 forms in spermatozoa purified from semen obtained from subjects explored for low fertility. A total of 184 sperm samples were purified using PureSperm^®. HHV‐6 viral load and species identification were performed by real‐time polymerase chain reaction. Of 179 sperm specimens analysed, three were positive for HHV‐6 (1.7%). Two samples (1.1%) had viral loads of 680 232 and 2 834 075 copies per million spermatozoa, compatible with loads expected for a ciHHV‐6 form. The viral load of the third positive sample (73 684 copies per million spermatozoa) was lower than would be expected for ciHHV‐6 infection, implying that the HHV‐6 DNA detected in spermatozoa corresponds mainly to ciHHV‐6. However, viral DNA may also be detected at a low level that is not in favour of the presence of ciHHV‐6. Further studies are necessary to determine the origin of detected viral genomes.     

Beyond Rabies: Are Free‐Ranging Skunks (Mephitis mephitis) in British Columbia Reservoirs of Emerging Infection?

Wild animal reservoirs are an important source of emerging and zoonotic infection. Skunks (Mephitis mephitis) are a reservoir of skunk strain rabies virus in Canada, with the exception of some areas including the province of British Columbia (BC). Beyond rabies, the reservoir status of skunks for emerging and zoonotic pathogens in BC is unknown. From March 2011 to February 2015, 50 free‐ranging skunks were necropsied and tested for 4 pathogens: influenza A, Aleutian disease virus (ADV), Leptospira spp. and Salmonella spp. Two skunks (4%) with respiratory disease caused by influenza A (H1N1) pdm09 were detected during the human flu season suggesting that skunks may represent a target population for reverse zoonosis of this strain of influenza A virus. High prevalence of ADV infection was detected (43/50, 86%). Two of the infected skunks exhibited Aleutian disease (AD) suggesting that skunks act as both a reservoir and a target population for the virus. Most studies of ADV have focused on the potential for infection of free‐ranging species living near mink farms. Our study suggests that urban skunks may be a primary host for the virus independent of domestic mink. Whether skunks act as a reservoir of ADV infection for other peridomestic species will depend on host specificity of the viral strains. Leptospira interrogans was detected in 18% (9/49) of the skunks. Identification of the serovar(s) detected is needed to determine any public health risk of leptospirosis following exposure to infected skunks. Salmonella spp. was isolated from three of 43 skunks (7%), specifically S. Typhimurium, S. Muenchen and S. Enteritidis. These serotypes cause disease in humans, but the low prevalence of infection suggests there is a low risk for zoonotic transmission.     

Oxidative stress‐induced alterations in seminal plasma antioxidants: Is there any association with keap1 gene methylation in human spermatozoa?

Kelch‐like ECH‐associated protein 1 (keap1)‐nuclear factor‐erythroid 2‐related factor 2 (Nrf2) pathway is one of the master regulators of cellular defence against oxidative stress. Epigenetic alterations like hypermethylation of keap1 gene impair keap1‐Nrf2 system in several oxidative stress–associated diseases. The objective of this study was to evaluate the epigenetic status of keap1 in sperm DNA of normozoospermic subjects, having different levels of reactive oxygen species (ROS) in seminal plasma. Semen samples were obtained from 151 apparently healthy male partners of couples who attended the Avicenna infertility clinic. Samples were categorised into four groups according to their ROS levels: group A (n = 39, ROS < 20 RLU/s per 10⁶ spermatozoa), group B (n = 38, 20 ≤ ROS < 40 RLU/s per 10⁶ spermatozoa), group C (n = 31, 40 ≤ ROS < 60 RLU/s per 10⁶ spermatozoa) and group D; (n = 43, ROS ≥ 60 RLU/s per 10⁶ spermatozoa). Keap1 methylation status was assessed using methylation‐specific PCR along with seminal total antioxidant capacity. The results showed no significant alterations in keap1 methylation in any groups, whereas the total antioxidant capacity enhanced with increasing levels of ROS exposure. These results indicate that keap1 was not methylated during ROS elevation and oxidative stress, suggesting that the cells have adopted other mechanisms to elevate antioxidant level.     

Presentation and management of major complications of midurethral slings: Are complications under‐reported?

AIMS: Midurethral slings have become the mainstay of stress urinary incontinence (SUI) treatment due to their efficacy and low complication rates. The purpose of this study was to report the presentation and treatment of major complications from these minimally invasive treatments presented to a tertiary referral practice and to highlight a discrepancy in major complications between literature and the food and drug administration (FDA) device failure database. METHODS: From 2001 through 2005, we reviewed all cases of midurethral sling complications that presented to our institution. A literature review of all complications due to midurethral slings during the same time period was performed as was the FDA manufacturer and user facility device experience (MAUDE) database queried for self-reported complications. RESULTS: A total of 26 patients referred to UCLA with voiding dysfunction after sling placement was found to have mesh in the urethra or bladder. Treatments required a combination of urethrolysis with mesh removal, urethral reconstruction with graft, and bladder excision. These were compared to major complications reported in the world literature of <1%. The MAUDE database contained 161 major complications out of a total of 928 complications reported for suburethral slings. There was significantly more major complications reported in MAUDE than in published literature. CONCLUSIONS: Although rare, major complications of midurethral slings are more common than appear in literature. Devastating complications involving urethral and bladder perforations can present with mild urinary symptoms and thus are likely under-diagnosed and under-reported. Most of these cases need to be managed with additional reconstructive surgery.     

Protective effect of N-Acetylcysteine on exogenous hydrogen peroxide and endogenous superoxide anion induced DNA strand breakage in human spermatozoa

Aim: To study the protective effect of N-Acetylcysteine (NAC)on exogenous hydrogen peroxide- and endogenous superoxide an-ion-induced DNA strand breakage in human spermatozoa in vitro.     

What is behind phylogenetic analysis of hospital‐, community‐ and livestock‐associated methicillin‐resistant Staphylococcus aureus?

Methicillin‐resistant Staphylococcus aureus (MRSA) has been shown to be the predominant life‐threatening pathogen in Egypt. MRSA is a major cause of severe healthcare‐associated (HA) infections. During the last decades, the incidence of community‐associated (CA) MRSA infections has a complex epidemiology arising from the circulation of different strains in the general population. Moreover, livestock‐associated (LA) MRSA emerged recently becomes an emerging threat to public health. Therefore, it is important to illuminate the differences between CA‐, HA‐ and LA‐MRSA to shed light on their genetic diversity and evolution. This study presents the first data on analysing the correlation between CA‐, LA‐ and HA‐MRSA using antibiogram typing, molecular characteristics and antimicrobial resistance and virulence genes’ profiles. Overall, HA‐MRSA strains tended to be multidrug resistant and less virulent than both LA‐ and CA‐MRSA strains. Importantly, CA‐MRSA strains had a high homology with each of HA‐ and LA‐MRSA. However, no similarity was observed between HA‐ and LA‐MRSA. Our findings suggest that the epidemiological changes in genetic behaviour between HA‐ and LA‐MRSA are due to the presence of CA‐MRSA confirming that CA‐MRSA has created a public health crisis worldwide.     

Testicular sperm extraction after laparoscopic orchiectomy for bilateral postpubertal intra‐abdominal cryptorchidism: What chance of sperm retrieval?

Infertility occurs in up to 54% of men with bilateral undescended testes. Orchiectomy is considered the best therapeutic approach, especially when cryptorchidism is diagnosed in adulthood, due to a high risk of malignancy. A 33‐year‐old man was referred with a clinical presentation of empty scrotum and an ultrasonography and magnetic resonance imaging evaluation of intra‐abdominal bilateral cryptorchidism. Follicle‐stimulating hormone was 23.20 IU/L, luteinising hormone was 14.10 IU/L, total testosterone was 12.1 nmol/L, and 17‐beta‐oestradiol was 0.16 nmol/L. Semen analysis showed absolute azoospermia. Tumour marker levels were in the normal range. Testicular volume was 4.0 ml for right testis and 4.6 ml for left testis. The patient underwent a laparoscopy bilateral orchiectomy and subsequently a testicular sperm extraction (TESE), in the purpose to finding mature spermatozoa. The biological examination revealed the presence of immature sperm cells, not efficient for a cryopreservation. The histologic analyses show a pattern of Sertoli cell‐only syndrome and maturation arrest. TESE might be a good option for patients with absolute azoospermia and cryptorchidism, especially if bilateral. The procedure, performed after orchiectomy, is safe and does not have any impact on patient's health, although it is important to clarify the very low potential of sperm recovery.