Svend Lindenberg

The role of DNA strand breaks in human spermatozoa used for IVF and ICSI

Background. The objective of this study was to determine the incidence of spermatozoa with DNA strand breaks in four clinically different groups of infertile couples, and to correlate DNA damage with other semen analysis parameters, as well as fertilization rates and IVF outcome.

Apoptosis in human cumulus cells in relation to zona pellucida thickness variation, maturation stage, and cleavage of the corresponding oocyte after intracytoplasmic sperm injection

OBJECTIVE To assess the degree of apoptosis in the cumulus cells and the variation of the zona pellucida and the maturity and fertilization of the corresponding oocyte. DESIGN Retrospective study. SETTING Private fertility clinic. PATIENT(S) Fifty couples undergoing ICSI. INTERVENTION(S) ICSI. MAIN OUTCOME MEASURE(S) Correlation between apoptosis in the cumulus cells and the zona pellucida thickness variation, maturation stage, fertilization rate, and embryo score. RESULT(S) This study demonstrated no correlation between apoptosis in cumulus cells and the thickness and variation of the zona pellucida in oocytes and embryos. The incidence of apoptosis was significantly higher in cumulus cells from empty zona pellucidas and germinal vesicle stage and metaphase I oocytes compared with metaphase II oocytes. Non-fertilized metaphase II oocytes showed significantly higher incidence of apoptosis compared with fertilized metaphase II oocytes. There was a correlation between embryo score and the zona pellucida thickness variation. CONCLUSION(S) Apoptosis in cumulus cells had no impact on the zona pellucida thickness and variation in oocytes and embryos. The zona pellucida thickness variation was positively correlated to good embryo score. A higher degree of apoptosis was seen in cumulus cells from immature oocytes compared with mature oocytes. Furthermore, apoptosis in cumulus cells impaired the fertilization rate of metaphase II oocytes after ICSI.

DNA strand breaks in human spermatozoa: Correlation with fertilization in vitro in oligozoospermic men and in men with unexplained infertility

Background. The purpose of this study was to examine the correlation between DNA strand breaks in human spermatozoa and semen quality, fertilization rate and IVF outcome.

DNA strand breaks in human sperm cells, A comparison between men with normal and oligozoospermic sperm samples

BACKGROUND The aim of this report was to compare the degree of DNA strand breaks in known normal fertile men to those with oligozoospermia, and evaluate the presence of DNA strand breaks in normal raw sperm, after Percoll and swim-up and following conventional cryopreservation, as all these preparation methods might differ in selection of healthy sperm cells. METHODS Sperm samples from proven fertile sperm donors (n=20) and infertile men with oligozoospermia (n=33) were tested for the presence of DNA strand breaks in the spermatozoa, by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. A correlation to other sperm parameters, sperm counts, motility and Krügers strict criteria was performed. RESULTS DNA strand breaks were found significantly more often in sperm samples from men with oligozoospermia compared to sperm samples of normal fertile men. The degree of spermatozoa with DNA strand breaks was correlated proportional with the degree of morphological pathological sperm cells judged by Krügers strict criteria. The percentage of spermatozoa with DNA strand breaks in the samples was not influenced by procedures such as the swim-up technique, Percoll gradients or cryopreservation and thawing. CONCLUSION DNA strand breaks were found significantly more often in men with oligozoospermia compared to normospermic men. The DNA strand breaks might play an important role for the maturation process of the spermatozoa in the same way as apoptosis is controlling the number of early meiotic germ cells in the testis, and hereby play an important role in advanced fertility treatments (ICSI).

Embryo quality and developmental potential is compromised by age

Aims. The aim of this retrospective study was to assess whether and how the age of the woman affects the quality and developmental potential of the oocytes and embryos in an ART program.

Development of human oocytes matured in vitro for 28 or 36 hours

OBJECTIVE To compare the effects on human oocytes of in vitro maturation periods of 28 hours and 36 hours. DESIGN Retrospective analysis. SETTING University teaching hospital. PATIENT(S) A total of 48 infertile patients undergoing 55 cycles who volunteered for the experimental treatments. INTERVENTION(S) Immature oocytes were aspirated with use of transvaginal ultrasonography. Oocytes were matured, fertilized by intracytoplasmic sperm injection, and cultured for 2.5 or 3 days before being replaced into the uterus. MAIN OUTCOME MEASURE(S) Oocyte maturation, fertilization and cleavage rates, and clinical pregnancy rate. RESULT(S) Maturation of oocytes for either 28 hours or 36 hours before insemination resulted in similar rates of maturation, fertilization, and cleavage. The clinical pregnancy rate was similar in both groups (14%-15%). CONCLUSION(S) Shortening the in vitro maturation period does not compromise subsequent embryonic development.

DNA strand breaks in human spermatozoa, a possible factor, to be considered in couples suffering from unexplained infertility

BACKGROUND The aim of this study was to assess the presence of DNA strand breaks and sperm cell morphology in men suffering from unexplained infertility, and to compare these results with normal fertile and oligozoospermic men. METHODS One fresh sperm sample from proven fertile sperm donors (n=20) and from infertile men with oligozoospermia, (<20 x 10(6)/ml sperm cells) (n=74), and men suffering from unexplained infertility (>20 x 10(6)/ml sperm cells) (n=39) who delivered two sperm samples with 24 hours interval, were tested for the presence of DNA strand breaks in the spermatozoa by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. Correlations to other sperm parameters, sperm cell counts, motility, activation and Krügers strict criteria were performed. RESULTS DNA strand breaks in sperm cell nuclei were found significantly more often in sperm samples from men suffering from unexplained infertility compared to those from normal fertile men, and significantly more rarely compared with sperm samples from men with oligozoospermia. The percentages of normal spermatozoa (Krügers strict criteria) were significantly lower in samples from men suffering from unexplained infertility compared to those of normal fertile men, but significantly higher compared to those of men with oligozoospermia. No difference was found between first and second day samples used for insemination, as regards DNA strand breaks, sperm cell morphology, total number of motile sperm cells, activation and motility degree. CONCLUSION The present data suggest that a subgroup of men suffering from unexplained infertility have DNA strand breaks in their sperm cell DNA. This group might suffer from the same malfunction as many men with oligozoospermia, however, their apoptotic activated sites in the testis are different. Delivery of sperm samples with 24 hours interval does not affect any sperm cell counts, CASA, DNA strand breaks or morphology findings in sperm samples from men suffering from unexplained infertility.

Monoclonal antibody 43-9F as a sensitive immunohistochemical marker of carcinoma in situ of human testis.

Invasive germ cell cancer can be prevented if the neoplasia is diagnosed at the stage of carcinoma in situ (CIS). In routine histologic examination CIS may be overlooked, particularly in prepubertal individuals. The detection of this early malignancy may be greatly facilitated by use of immunohistochemical staining. The authors investigated the sensitivity of an immunohistochemical staining procedure with monoclonal antibody 43‐9F in detection of CIS. Testicular specimens from 19 adult and two prepubertal individuals with CIS were tested. Positive staining reaction on the surface of malignant germ cells was encountered in all 21 specimens with CIS. The epithelial cells of the excretory ducts between testis and epididymis, including rete testis and canaliculi efferentes, reacted also positively with 43‐9F. No staining was observed in nonmalignant testicular cells including Sertoli cells or Leydig cells in any of these samples or in testicular biopsy specimens from 27 adult and 11 prepubertal subjects without evidence of testicular neoplasia. Monoclonal antibody 43‐9F was also found to react with cells of all four tested invasive testicular germ cell tumors. Thus, the monoclonal antibody 43‐9F is a sensitive immunohistochemical marker of CIS germ cells and may also be of potential value in detection of invasive testicular cancer.

Apoptosis in human cumulus cells in relation to maturation stage and cleavage of the corresponding oocyte

Background. The purpose of this study was to determine the incidence of apoptosis in cumulus cells, and correlate these findings with the maturation stage, fertilization rate and embryo score of the corresponding oocyte, in couples undergoing ICSI due to a male factor.

Differential expression of inflammation-related genes in the ovarian stroma and granulosa cells of PCOS women

Polycystic ovary syndrome (PCOS) is the most common female endocrine disorder. Ovarian changes in PCOS women are well characterized by ultrasound. However, the ovarian pathophysiology is not fully understood. The aim of this study was to characterize the expression, in both the central ovarian stroma and in granulosa cells (GCs), of a number of genes, including several inflammation-related genes, which have been hypothesized to be involved in the pathophysiology of PCOS. Biopsies of the central ovarian stroma were obtained from PCOS women (Rotterdam criteria) and from normally ovulating women in follicular phase. GCs were retrieved from PCOS-women and non-PCOS women, undergoing in vitro maturation. The expressions of 57 genes were analyzed by quantitative-PCR using a low-density-gene array. The main outcome measures were over-expression or under-expression of the specific genes. The results showed that in the central stroma of PCOS ovaries, five inflammation-related genes (CCL2, IL1R1, IL8, NOS2, TIMP1), the leukocyte marker CD45, the inflammation-related transcription factor RUNX2 and the growth factor AREG were under-expressed. The growth factor DUSP12 and the coagulation factor TFPI2 were over-expressed. In the GC of PCOS, all of the differentially expressed genes were over-expressed; the inflammation-related IL1B, IL8, LIF, NOS2 and PTGS2, the coagulation-related F3 and THBS1, the growth factors BMP6 and DUSP12, the permeability-related AQ3 and the growth-arrest-related GADD45A. In conclusion, the results indicate major alterations in the local ovarian immune system of PCOS ovaries. This may have implications for the PCOS-related defects in the inflammation-like ovulatory process and for the susceptibility to acquire the inflammatory state of ovarian hyperstimulation syndrome.