Is there any correlation between sperm parameters and chromatin quality with embryo morphokinetics in patients with male infertility?

The main goal was to evaluate the correlation between sperm parameters and chromatin quality with embryo kinetics via time-lapse monitoring system (TLM). A total of 40 couples involved in the ICSI program as a result of male infertility. For assessment of sperm chromatin and DNA quality, we used aniline blue, toluidine blue, chromomycin A3, acridine orange and terminal transferase-mediated deoxyuridine triphosphate biotin end labelling assays. All mature oocytes were injected, and the generated zygotes (2PNs) were cultured in TLM. In day 3 after injection, single embryo transfer (SET) was carried out according to the morphology and morphokinetics. The patients were followed up until delivery. There were positive significant correlations between sperm count with CC2 (r = .330, p = .049), T4 (r = .329, p = .038), T6 (r = .342, p = .035) and T7 (r = .374, p = .025). Also, there were positive significant correlations between nonprogressive motility and T2 (r = 0.323, p = .042), T3 (r = .411, p = .013) and T4 (r = .418, p = .007). Regarding the sperm chromatin quality assays, there were negative significant correlations between CMA3 and CC2 (r = -.272, p = .049) and between acridine orange and T5 (r = -.221, p = .040). It seems that the abnormal sperm parameters and chromatin alteration affect the normal embryo kinetics in ICSI program.

Sperm chromatin quality and DNA integrity in partial versus total globozoospermia.

Globozoospermia is a severe form of teratozoospermia with low incidence in infertile patients, considered as one of the important causes of male infertility. The objective was to investigate the chromatin/DNA integrity as well as apoptosis in ejaculated spermatozoa of cases with partial or total globozoospermia. Fifty-seven semen samples were divided into three groups of partial globozoospermia (n = 17), total globozoospermia (n = 10) and normozoospermia (control; n = 30). Sperm chromatin condensation, DNA integrity and apoptosis were assessed using cytochemical assays. The results showed significant differences in sperm parameters of count and motility between two case groups versus controls. The percentages of spermatozoa with abnormal chromatin packaging and protamine deficiency were significantly higher in total and partial globozoospermic men compared to normozoospermic samples. Also, the rates of TUNEL-positive spermatozoa were significantly increased in both globozoospermic cases with respect to the control (18.3 ± 10.1 and 12.3 ± 9.2 versus 5.9 ± 3 respectively). However, no significant differences were noticed between two subgroups of patients with regard to sperm DNA denaturation, DNA fragmentation and apoptosis. Abnormal chromatin packaging, DNA damage and apoptosis were significantly higher in cases than controls. The sperm chromatin/DNA anomalies may be considered as one of the main aetiology of ART failure in globozoospermic patients.

Cryoprotectant-free vitrification of human spermatozoa in new artificial seminal fluid.

Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5 mg/mL human serum albumin and 0.25 mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09 ± 10.33 vs. 36.80 ± 13.75), grade A motility (36.59 ± 11.40 vs. 16.41 ± 11.24), and normal morphology (18.74 ± 8.35 vs. 11.85 ± 5.84) and viability (68.22 ± 10.83 vs. 60.86 ± 11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p = 0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p < 0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p < 0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF.

Are ART outcomes more successful in younger women? A retrospective study.

It is well known that with increasing age, fertility potential decreases in women. Since some of the women who undergo assisted reproductive technology (ART) treatments are very young patients or young donors, fecundity investigation seems necessary in this group. Data from patients who underwent in vitro fertilisation/intra-cytoplasmic sperm injection (IVF/ICSI) over 20 years were analysed retrospectively. The records of 407 infertile patients aged 17-25 years (study group) and 407 infertile patients aged 26-35 years (control group) were collected. The number of follicles > 14 mm, retrieved oocytes, MII oocytes, cleaved embryos in both IVF and ICSI cycles were significantly higher in the study group when compared with the control group (p˂0.001). However, the rates of chemical pregnancies were similar between the two groups. It therefore seems unlikely that younger age has a positive effect in predicting infertility treatment outcomes.

Prolonged incubation of processed human spermatozoa will increase DNA fragmentation.

One of the causes of failure in ART is sperm DNA fragmentation which may be associated with long period of spermatozoa incubation at 37 °C. The objective was to evaluate the rate of sperm DNA fragmentation using the sperm chromatin dispersion (SCD) test after swim-up at different time intervals prior to use. In this prospective study, 21 normozoospermic specimens were analysed. The samples were incubated at 37 °C after preparation by direct swim-up. DNA fragmentation was assessed at different time intervals (0, 1, 2 and 3 h) using SCD test. Spermatozoa with no DNA fragmentation showed large- or medium-sized halos, and sperm cells with DNA fragmentation showed either a small halo or no halo. The rates of normal morphology and progressive motility after sperm processing were 72.33 ± 2.53% and 90 ± 1.02%, respectively. The rate of sperm DNA fragmentation was significantly higher after 2 h (8.81 ± 0.93%, P = 0.004) and 3 h (10.76 ± 0.89%, P < 0.0001) of incubation compared to 0 h (4.38 ± 0.8%). A positive correlation was found between the incubation time and sperm DNA damage (P < 0.0001). Prolonged incubation of prepared normozoospermic samples at 37 °C is associated with higher rates of sperm DNA fragmentation. Therefore, sperm samples intended for ART procedures should be used within 2 h of incubation at 37 °C.

Ultrastructure of human mature oocytes after vitrification.

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morphofunctional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microscopy has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.

Expression of the vascular endothelial growth factor receptor neuropilin-1 in the human endometrium.

Angiogenesis is a key process in the endometrium which undergoes dramatic changes during the menstrual cycle. Molecules such as vascular endothelial growth factor (VEGF), acting via two tyrosine kinase family receptors (VEGFR1 [Flt-1] and VEGFR2 [KDR/Flk-1]), are potent modulators of angiogenesis and vascular remodelling in the endometrium. Recently, neuropilin-1 (NRP-1) was shown to be expressed in endothelial cells binding VEGF(165) and therewith enhancing the binding of VEGF(165) to VEGFR2. This suggests that NRP-1, in addition to the known VEGF receptors, may play an important role in VEGF-induced angiogenesis. In this study, the expression of NRP-1 in the cycling human endometrium has been investigated by reverse transcription (RT)-polymerase chain reaction (RT-PCR), semi-quantitative competitive RT-PCR (RT-cPCR) and immunohistochemical staining. NRP-1 was expressed in all 32 endometrium samples throughout the menstrual cycle. However, samples from the proliferative phase showed significantly higher expression levels of NRP-1 mRNA compared to samples from the secretory phase (t/c-ratio 2.13 vs. 0.84, p<0.05). Immunohistochemistry confirmed the results showing increased NRP-1 staining in vascular endothelium, glandular epithelium and stromal cells of the proliferative phase endometrium. This study demonstrates mRNA and protein expression of NRP-1 in human endometrium samples throughout the menstrual cycle. The enhanced expression of NRP-1 in the proliferative phase suggests that it may participate in hormonally regulated changes of endometrial angiogenesis, preparing the endometrium for the implantation of an embryo. NRP-1 expression might act as a co-factor for VEGF(165) enhancing the angiogenic stimulus.

Characterization of infectivity of knob-modified adenoviral vectors in glioma.

Malignant glioma continues to be a major target for gene therapy and virotherapy due to its aggressive growth and the current lack of effective treatment. However, these approaches have been hampered by inefficient infection of glioma cells by viral vectors,particularly vectors derived from serotype 5 adenoviruses (Ad5). This results from limited cell surface expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on tumor cells. To circumvent this problem, Ad fiber pseudotyping,the genetic replacement of either the entire fiber or fiber knob domain with its structural counterpart from another human Ad serotype that recognizes a cellular receptor other than CAR, has been shown to enhance Ad infectivity in a variety of tumor types,including human glioma. Here, we have extended the paradigm of genetic pseudotyping to include fiber domains from non-human or"xenotype" Ads for infectivity enhancement of human glioma cell populations. In this study, we evaluated the gene transfer efficiency of a panel of Ad vectors which express one of five different "xenotype"fiber knob domains, including those derived from murine,ovine, porcine and canine species, in both human glioma cell lines as well as primary glioma tumor cells from patients. Adenovirus vectors displaying either canine Ad or porcine Ad fiber elements had the highest gene transfer to both glioma cell lines and primary tumor cells. The correlation between the viral infectivity of modified adenovirus vectors and expression of human CAR and CD46(an adenovirus type B receptor) on the surfaces of tumor cells was also analyzed. Taken together, human adenovirus vectors modified with "xenotype" fiber elements could be excellent candidates to target human glioma.

Employment of liver tissue slice analysis to assay hepatotoxicity linked to replicative and nonreplicative adenoviral agents.

Whereas virotherapy has emerged as a novel and promising approach for neoplastic diseases, appropriate model systems have hampered preclinical evaluation of candidate conditionally replicative adenovirus agents (CRAds) with respect to liver toxicity. This is due to the inability of human viral agents to cross species. We have recently shown the human liver tissue slice model to be a facile means to validate adenoviral replication. On this basis, we sought to determine whether our ex vivo liver tissue slice model could be used to assess CRAd-mediated liver toxicity. We analyzed and compared the toxicity of a conditionally replicative adenovirus (AdDelta24) to that of a replication incompetent adenovirus (Adnull [E1-]) in mouse and human liver tissue slices. To accomplish this, we examined the hepatic apoptosis expression profile by DNA microarray analyses, and compared these results to extracellular release of aminotransferase enzymes, along with direct evidence of apoptosis by caspase-3 immunhistochemical staining and TUNEL assays. Human and mouse liver tissue slices demonstrated a marked increase in extracellular release of aminotransferase enzymes on infection with AdDelta24 compared to Adnull. AdDelta24-mediated liver toxicity was further demonstrated by apoptosis induction, as detected by caspase-3 immunohistochemical staining, TUNEL assay and microarray analysis. In conclusion, concordance of CRAd-mediated apoptosis in both the human and the mouse liver tissue slice models was demonstrated, despite the limited replication ability of CRAds in mouse liver slices. The results of this study, defining the CRAd-mediated apoptosis gene expression profiles in human and mouse liver, may lay a foundation for preclinical liver toxicity analysis of CRAd agents.

Gene therapy for carcinoma of the breast.

In view of the limited success of available treatment modalities for breast cancer, alternative and complementary strategies need to be developed. The delineation of the molecular basis of breast cancer provides the possibility of specific intervention by gene therapy through the introduction of genetic material for therapeutic purposes. In this regard, several gene therapy approaches for carcinoma of the breast have been developed. These approaches can be divided into six broad categories: (1) mutation compensation, (2) molecular chemotherapy, (3) proapoptotic gene therapy, (4) antiangiogenic gene therapy, (5) genetic immunopotentiation, and (6) genetic modulation of resistance/sensitivity. Clinical trials for breast cancer have been initiated to evaluate safety, toxicity, and efficacy. Combined modality therapy with gene therapy and chemotherapy or radiation therapy has shown promising results. It is expected that as new therapeutic targets and approaches are identified and advances in vector design are realized, gene therapy will play an increasing role in clinical breast cancer treatment.

A human adenoviral vector with a chimeric fiber from canine adenovirus type 1 results in novel expanded tropism for cancer gene therapy.

The development of novel therapeutic strategies is imperative for the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on 'xenotype' knob switching with non-human adenovirus have been exploited. Herein, we present the characterization of a novel vector derived from a recombinant Ad5 vector containing the canine adenovirus serotype 1 (CAV-1) knob (Ad5Luc1-CK1), the tropism of which has not been previously described. We compared the function of this vector with our other chimeric viruses displaying the CAV-2 knob (Ad5Luc1-CK2) and Ad3 knob (Ad5/3Luc1). Our data demonstrate that the CAV-1 knob can alter Ad5 tropism through the use of a CAR-independent entry pathway distinct from that of both Ad5Luc1-CK2 and Ad5/3-Luc1. In fact, the gene transfer efficiency of this novel vector in ovarian cancer cell lines, and more importantly in patient ovarian cancer primary tissue slice samples, was superior relative to all other vectors applied in this study. Thus, CAV-1 knob xenotype gene transfer represents a viable means to achieve enhanced transduction of low-CAR tumors.

Quantitative sports and functional classification (QSFC) for disabled people with spasticity.

Although sports classification for cerebral palsy has been in use for several years, it is complicated both for training and for scoring. People with cerebral palsy are difficult to fit into classification systems that are appropriate for other disability groups. The aim of this report is to describe the development of a framework for a simple quantitative classification of cerebral palsy. It was designed to be easily understood by all who are investigating, treating, training, coaching, and working with spastic disabled people. The scoring system is accurate and quick, so long as the definitions of items listed are adhered to.

From gene therapy to virotherapy for ovarian cancer.

Ovarian cancer has the highest mortality of all cancers of the female reproductive system. Although progress in conventional therapies (surgery, chemotherapy and irradiation) has been achieved, the 5-year survival rate for patients with advanced stage ovarian cancer is still low. On this basis it is clear that there is a need for novel therapeutic paradigms. Targeted approaches are based on the increasing knowledge of the molecular basics of ovarian cancer. In this regard, gene therapy is a novel targeted approach for the treatment of ovarian cancer. However, current gene therapy delivery systems (viral and non-viral vectors) have to address the issues of inefficient transduction of target ovarian cancer cells and/or ectopic non-target delivery with attendant toxicity. Of note, the limited tumor transduction associated with current gene therapy interventions is due, in large part, to the fact that the employed vectors have been replication-incompetent. In this regard, human clinical trials have shown that the approach of replication-incompetent vectors has yet to succeed in ovarian cancer patients. In contrast, replication-competent viruses offer a method to achieve efficient tumor cell oncolysis (virotherapy) in ovarian cancer. Thus, in this very promising approach of virotherapy the replicating virus itself is the anti-cancer agent. This review discusses the concepts of gene therapy and virotherapy as novel targeted therapeutic approaches for the treatment of ovarian cancer.

[Long-term follow-up of 700 trabeculectomies]. / Langzeituntersuchung von 700 Trabekulektomien.

BACKGROUND: A major focus of our study was to determine the value of postoperative intraocular pressure (IOP) in predicting the outcome of trabeculectomy (TE). PATIENTS AND METHODS: The medical charts of 547 patients undergoing glaucoma filtering surgery at the Department of Ophthalmology of the University of Cologne from 1987 to 1996 were reviewed. The eyes with congenital glaucoma and those treated with anti-metabolites were excluded. RESULTS: Defining the qualified criteria for success of trabeculectomy as an IOP

Trabeculectomy: a retrospective follow-up of 700 eyes.

A major focus of our study was to determine the value of postoperative intraocular pressure (IOP) in predicting the outcome of trabeculectomy (TE). The medical charts of 547 patients undergoing glaucoma filtering surgery at the Department of Ophthalmology of the University of Cologne from 1987 to 1996 were reviewed. The status of the visual field, level of visual acuity, appearance of the bleb, cup/disc ratio and IOP were studied. Pre- and post-operative glaucoma medication was recorded. The eyes with congenital glaucoma and those treated with antimetabolites were excluded. The results are presented with particular emphasis being placed not only on intraocular pressure (IOP) control but also on the progression of glaucomatous damage (deterioration of visual field or disc damage) and the decrease of visual acuity. The tonometric success rate of TE in controlling the IOP < 21 mmHg was 61%. Defining the rigid criteria for success of trabeculectomy as an IOP < 21 mmHg, no further visual field loss, no disc damage and no additionally required surgical intervention due to glaucoma, the success rate decreased to 44%. The results indicate that other factors than normalization of IOP determine the success rate of TE. Should trabeculectomy be the therapy of first choice in the early stage of glaucoma? Should trabeculectomy fail to control the IOP in the first eye, would this allow options, such as the use of antimetabolites in the second eye?

Failure of fertilization following intracytoplasmic injection of round-headed sperm.

BACKGROUND: Intracytoplasmic sperm injection (ICSI) is a very effective technique for the treatment of male factor infertility, even in severe cases. An exception to this rule is round-headed acrosomeless sperm, for which there is an extremely low success rate. The objective of this study was to report our first experience with ICSI using round-headed spermatozoa. MATERIALS AND METHODS: A total of four infertile males with globozoospermia underwent the conventional ICSI program during the period from 1995 to 1997. Semen parameters were evaluated according to the WHO criteria. Twenty-eight oocytes were collected following conventional ovarian superovulation. RESULTS: Volume, pH, and viscosity of the semen were within normal range. Sperm counts and progressive motility were from >1 to 90x10 6 mL, and from 0% to 60%, respectively. Distinct 100% acrosomeless morphology was observed with Giemsa staining. Twenty-two metaphase II oocytes (78.6% of the total) from four ICSI cases were injected with the husbandâs round-headed spermatozoa. Total fertilization failure was observed in all these cases. CONCLUSION: The results clearly demonstrate the important role of normal head morphology in activating the oocytes, as well as the need to develop a modified but safe ICSI technique, which will be effective in overcoming this severe type of male infertility.