High magnitude of light retardation by the zona pellucida is associated with conception cycles

Shen Y, Stalf T, Mehnert C, Eichenlaub-Ritter U, Tinneberg HR. High magnitude of light retardation by the zona pellucida is associated with conception cycles. HUMAN REPRODUCTION. 2005;20(6):1596-1606.BACKGROUND: Failures in expression of zona proteins correlate to subfertility in animals. Low expression of the zona proteins by the growing human oocyte may indicate reduced developmental potential. Therefore, we non-invasively analysed the thickness and the structure of the zona pellucida (ZP) of human oocytes with respect to embryo fate after ICSI. METHODS: Retardance magnitude and thickness of the inner, middle and outer layers of the ZP were quantitatively analysed by a Polscope in 166 oocytes selected for transfer after ICSI (63 patients; 32.8 +/- 4.4 years) on the basis of pronuclear score at day 1. Blastomere number was determined at day 2. Data were compared between conception cycles (CC; 65 oocytes/23 patients) and non-conception cycles (NCC; 101 oocytes/40 patients) and with respect to maternal age. RESULTS: The thickness was slightly elevated (P < 0.001), and the mean magnitude of light retardance was nearly 30% higher (P < 0.001) in the inner layer of the zona pellucida of oocytes contributing to CC compared to NCC. Embryos in the CC group tended to develop faster. CONCLUSIONS: The magnitude of light retardance by the zona pellucida inner layer appears to present a unique non-invasive marker for oocyte developmental potential

Use of failed-fertilized oocytes for diagnostic zona binding purposes after sperm binding improvement with a modified medium

Purpose: Because the availability of prophase oocytes for zona binding testing is limited, we compared sperm binding to the zona of failed- fertilized intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) oocytes after incubation in a standard IVF medium and a specially composed binding improvement medium. Methods: Semen samples from nine patients and nine fertile donors were separated in parallel by the standard swim-up method in both media. Subsequently, hemizona assays were performed with prophase, failed-fertilized ICSI and IVF oocytes. Results: Sperm separation resulted in a significantly higher sperm count (P < 0.01) and progressive motility (P = 0.018) in binding improvement medium. Moreover, spermatozoa coincubated with hemizonae (prophase, failed-fertilized ICSI and IVF oocytes) in binding improvement medium bound significantly more to hemizonae than in the controls (P < 0.01). However, the hemizona index did not differ. Conclusions: Thus, the limited number of human zonae can be increased by the use of oocytes that failed to fertilize during ICSI or IVF. This will lead to a qualitative improvement of the diagnostic spectrum in male-factor infertility.Articl

High magnitude of light retardation by the zona pellucida is associated with conception cycles

BACKGROUND: Failures in expression of zona proteins correlate to subfertility in animals. Low expression of the zona proteins by the growing human oocyte may indicate reduced developmental potential. Therefore, we noninvasively analysed the thickness and the structure of the zona pellucida (ZP) of human oocytes with respect to embryo fate after ICSI. METHODS: Retardance magnitude and thickness of the inner, middle and outer layers of the ZP were quantitatively analysed by a Polscope in 166 oocytes selected for transfer after ICSI (63 patients; 32.8 6 4.4 years) on the basis of pronuclear score at day 1. Blastomere number was determined at day 2. Data were compared between conception cycles (CC; 65 oocytes/23 patients) and non-conception cycles (NCC; 101 oocytes/40 patients) and with respect to maternal age. RESULTS: The thickness was slightly elevated (P &lt; 0.001), and the mean magnitude of light retardance was nearly 30% higher (P &lt; 0.001) in the inner layer of the zona pellucida of oocytes contributing to CC compared to NCC. Embryos in the CC group tended to develop faster. CONCLUSIONS: The magnitude of light retardance by the zona pellucida inner layer appears to present a unique non-invasive marker for oocyte developmental potential

Behavioral audiogram of two Arctic foxes (Vulpes lagopus)

With increased polar anthropogenic activity, such as from the oil and gas industry, there are growing concerns about how Arctic species will be affected. Knowledge of species’ sensory abilities, such as auditory sensitivities, can be used to mitigate the effects of such activities. Herein, behavioral audiograms of two captive adult Arctic foxes (Vulpes lagopus) were measured using a yes/no paradigm and descending staircase method of signal presentation. Both foxes displayed a typical mammalian U-shaped audiometric curve, with a functional hearing range of 125 Hz-16 kHz (sensitivity B 60 dB re: 20 μPa) and average peak sensitivity of 24 dB re: 20 μPa at 4 kHz. The foxes had a lower frequency range and sensitivity than would be expected when compared to previous audiograms of domestic dogs (Canis familiaris) and other carnivores. These differences indicate Arctic foxes (V. lagopus) may have a lower frequency range than previously expected, which was similar to the only other fox species tested to date, kit foxes (Vulpes macrotis). Alternatively, differences may be due to testing constraints, such as masking of test signals by ambient noise and/or an unintentionally trained conservative response bias, which most likely resulted in underestimated hearing curves. While results of this study should be interpreted with caution due to its limitations, findings indicate that foxes have a narrower frequency range than formerly presumed. Anthropogenic activities near fox habitats can mitigate their impacts by reducing noise at frequencies within the functional hearing range and peak sensitivities of this species

Behavioral audiogram of two Arctic foxes (Vulpes lagopus)

With increased polar anthropogenic activity, such as from the oil and gas industry, there are growing concerns about how Arctic species will be affected. Knowledge of species’ sensory abilities, such as auditory sensitivities, can be used to mitigate the effects of such activities. Herein, behavioral audiograms of two captive adult Arctic foxes (Vulpes lagopus) were measured using a yes/no paradigm and descending staircase method of signal presentation. Both foxes displayed a typical mammalian U-shaped audiometric curve, with a functional hearing range of 125 Hz-16 kHz (sensitivity B 60 dB re: 20 μPa) and average peak sensitivity of 24 dB re: 20 μPa at 4 kHz. The foxes had a lower frequency range and sensitivity than would be expected when compared to previous audiograms of domestic dogs (Canis familiaris) and other carnivores. These differences indicate Arctic foxes (V. lagopus) may have a lower frequency range than previously expected, which was similar to the only other fox species tested to date, kit foxes (Vulpes macrotis). Alternatively, differences may be due to testing constraints, such as masking of test signals by ambient noise and/or an unintentionally trained conservative response bias, which most likely resulted in underestimated hearing curves. While results of this study should be interpreted with caution due to its limitations, findings indicate that foxes have a narrower frequency range than formerly presumed. Anthropogenic activities near fox habitats can mitigate their impacts by reducing noise at frequencies within the functional hearing range and peak sensitivities of this species

Urogenital inflammation: Changes of leucocytes and ROS

The presence of excess leucocytes in the semen has been associated with male infertility. According to the WHO, concentrations of more than 10 leucocytes ml-1 are considered as leucocytospermia, indicating genital tract infections. Up to now, no consensus has been achieved on how leucocytes should be quantified in semen. Using the peroxidase staining and monoclonal antibodies to CD15, CD45 and CD68, we found significant differences between the detection methods. Only 47.4% of the semen samples that were assessed as leucocytospermic by CD45 were identified as such by peroxidase staining. The concentration of peroxidase-positive cells was significantly correlated with polymorphonuclear granulocyte (PMN) elastase (P &lt; 0.0001). However, a negative correlation of peroxidase-positive cells with the sperm concentration was only found in oligozoospermic patients (P &lt; 0.0001). Moreover, the slightly positive correlation with normal sperm morphology seems to be applicable only in cases of oligozoospermia. Significant negative correlation of the number of peroxidase-positive cells were found for both maximal inducible acrosome reaction (P = 0.0219) and the inducibility of acrosome reaction (P = 0.0370), indicating a rather deleterious effect of leucocytes on this important sperm function. Concerning the result in the in vitro fertilization programme, none of the examined parameters (PMN elastase, concentration of round cells and peroxidase-positive cells) showed a correlation with either fertilization or pregnancy. This result seems to be reasonable as severely damaged spermatozoa and leucocytes are eliminated from the ejaculate by different sperm separation methods. Interestingly, a significant negative correlation of the TUNEL assay as a measure of sperm DNA fragmentation was found only with pregnancy (P = 0.006) but not with fertilization. As DNA fragmentation can also be caused by ROS that are generated by leucocytes, this causality should not be neglected.Conference Pape

DNA fragmentation of spermatozoa and assisted reproduction technology

Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in the ejaculate and in the spermatozoa themselves. These data were correlated with fertilization and pregnancy data from IVF and intracytoplasmic sperm injection (ICSI) patients. Sperm DNA fragmentation did not correlate with fertilization rate, but there was a significantly reduced pregnancy rate in IVF patients inseminated with TUNEL-positive spermatozoa. ICSI patients exhibited the same tendency. This implies that spermatozoa with damaged DNA are able to fertilize an oocyte, but at the time the paternal genome is switched on, further development stops. The determination of ROS in the ejaculate and the percentage of ROS-producing spermatozoa revealed markedly stronger correlations between sperm functions (i.e. motility) and the percentage of ROS-producing spermatozoa. The influence of seminal leukocytes, known to produce large amounts of oxidants, on sperm DNA fragmentation should not be neglected.Articl

Influence of deoxyribonucleic acid damage on fertilization and pregnancy

Objective To investigate sperm DNA damage in relation to fertilization and pregnancy. Design Prospective study. Setting The Institute of Reproductive Medicine, Giessen, Germany. Patient(s) Semen collected from 249 patients attending the IVF program. Main outcome measure(s) The percentage of terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling- (TUNEL-), Fas-, and annexin-V-positive sperm and the proportion of green-fluorescing sperm in the acridine orange stain was determined and correlated with sperm concentration, motility, fertilization, and pregnancy. Result(s) Significant correlations with the concentration of motile sperm were only found for the acridine orange stain (before and after sperm separation) and for the TUNEL assay (after sperm separation). Moreover, patients whose sperm had a high percentage of DNA fragmentations showed significantly lower pregnancy rates (TUNEL assay: 19.05% vs. 34.65%; acridine orange stain: 24.58% vs. 37.93%). The apoptosis parameters (annexin V binding and Fas expression) showed no statistically significant differences. Conclusions Our data clearly demonstrate that DNA fragmentation, as determined by the TUNEL assay, is predictive for pregnancy in IVF. This implies that spermatozoa with DNA fragmentation can still fertilize an oocyte but that when paternal genes are "switched on," further embryonic development stops, resulting in failed pregnancy. It seems that, at least in the patients we analyzed, apoptosis in the sperm does not play a role for fertilization. This would imply that DNA fragmentation in human spermatozoa is caused by external factors, such as reactive oxygen species, rather than by apoptosis. © 2004 by American Society for Reproductive Medicine.Articl