Christiaan Frederik Hoogendijk

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.

TUNEL assay and SCSA determine different aspects of sperm DNA damage.

For the determination of sperm DNA damage, different assays are used. However, no further distinction is made and the literature generally speaks about DNA damage. Thus, this study aimed at comparing the sperm chromatin structure assay (SCSA) and the TUNEL assay. In 79 patients, sperm DNA damage was determined flow cytometrically using the SCSA and the TUNEL assay. Moreover, normal sperm morphology was evaluated according to strict criteria. A statistical comparison of the two methods was performed using standard correlations, Bland and Altman plots, Passing–Bablok regressions and concordance correlation. Results show a significant difference between P‐ and G‐pattern morphology only for the mean channel fluorescence of the SCSA. Spearman’s rank correlations between the different parameters of both assays, SCSA and TUNEL, revealed significant associations between the parameters of the assays. However, when applying Bland and Altman plots, Passing–Bablok regression and concordance correlation results showed that these methods are not comparable. These different techniques determine different aspects of sperm DNA damage, i.e. ‘real’ DNA damage for the TUNEL assay and ‘potential’ DNA damage in terms of susceptibility to DNA denaturation for the SCSA. Thus, one should clearly distinguish between the different assays, not only practically and methodologically but also linguistically.

A novel approach for the selection of human sperm using annexin V-binding and flow cytometry

OBJECTIVE To develop a method whereby sperm with phosphatidylserine externalization can be separated from those without this feature. Because annexin V binds phosphatidylserine, this study is using this feature to select functional spermatozoa. In addition, the relationship between annexin V binding in human spermatozoa and normal sperm morphology according to strict criteria was to be assessed. DESIGN Prospective study. SETTING Department of Obstetrics and Gynaecology of Stellenbosch University at Tygerberg Academic Hospital, Tygerberg, South Africa. PATIENT(S) Semen from 14 healthy sperm donors. Exclusion criterion was the presence of less than 20 x 10(6)/mL total motile spermatozoa in the original sample. MAIN OUTCOME MEASURE(S) Annexin V-negative sperm, annexin V-positive sperm, normal sperm morphology. INTERVENTION(S) An aliquot of a semen sample after double density gradient centrifugation was incubated with annexin V fluorescein isothiocyanate conjugate (FITC). Cell fluorescence signals were determined using a FACScalibur flow cytometer equipped with a FACSSort fluidic sorting module. The sorting procedure delivered two sperm subpopulations: annexin V-negative and annexin V-positive. Morphology slides were made and the sperm morphology was assessed according to strict criteria. RESULT(S) There was a significant enrichment of annexin V-negative sperm as well as morphologically normal sperm in the annexin V-negative subgroup after separation with flow cytometry. The percentage of morphologically normal sperm increased from 8.3% in the control to 11.9% in the annexin V-negative fraction, whereas the percentage of annexin V-positive sperm decreased to 5.7%. CONCLUSION(S) The annexin V-negative sperm subpopulation had morphologically superior quality sperm compared to annexin V-positive sperm. It is important to select morphologically normal sperm during intracytoplasmic sperm injection (ICSI) as it may contribute to increased implantation and pregnancy rates (PR).

Allelic variation in the promoter region of the LDL receptor gene: analysis of an African-specific variant in the FP2 cis-acting regulatory element

DNA samples of 2303 individuals from nine different population groups were screened for variant -175g-->t in the promoter region of the low-density lipoprotein receptor (LDLR) gene. The -175g-->t variant detected at carrier frequencies of 3-10% in different African population groups was absent in the Caucasian and Asian (Chinese) individuals studied. In contrast to previous findings in Black South Africans where this polymorphism predominated in patients with familial hypercholesterolaemia (FH), it occurred at a significantly lower frequency in hypercholesterolaemics from the recently admixed Coloured population of South Africa compared with population-matched controls (P<0.0001). Haplotype and mutation analysis excluded the likelihood that this finding is due to association with a specific disease-related mutation in FH patients, although reversal of the positive association with FH observed in the Black population may, at least in part, be due to admixture linkage disequilibrium. Transient transfection studies in HepG2 cells demonstrated that the -175t allele is associated with a non-significant decrease ( approximately 7%) of LDLR transcription in the absence of sterols. The data presented in this study raise the possibility that the -175g-->t polymorphism may have subtle effects that become clinically important within certain genetic and/or environmental contexts.