Erik Høst

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).

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.

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.

Maternal serum supplementation in culture medium benefits maturation of immature human oocytes

This study compared the rates of maturation, fertilization, cleavage and pregnancy among oocytes matured in medium containing either human serum albumin (HSA) or maternal serum. Immature oocytes were obtained from 51 consecutive regularly cycling women <38 years of age. Immature oocytes were aspirated transvaginally on cycle day 8-9 after priming with FSH (Gonal-F 150 IU/day for 3 days, initiated on day 3). Oocytes were matured in Dyrkningsmedie til IVM supplemented with recombinant FSH (rFSH) 0.075 IU/ml and HCG 0.5 IU/ml for 28-30 h. In group I (n = 63 oocytes obtained from the first 23 cycles) the culture medium was supplemented with 2% (w/v) HSA. In group II (n = 74 oocytes obtained from the following 28 cycles) the medium was supplemented with 10% (v/v) heat-inactivated maternal serum. Intracytoplasmic sperm injection (ICSI) was performed on all methaphase II oocytes. Significantly increased rates of maturation 47/74 (63%) vs. 26/63 (41%) (P < 0.05), pregnancy 6/28 (21%) vs. 0/23 (0%) (P < 0.05) and implantation 6/20 (30%) vs. 0/15 (0%) (P < 0.05) were obtained from oocytes matured in culture medium with maternal serum supplementation compared with oocytes matured in medium supplemented with HSA. These results indicate that factors other than albumin in maternal serum play an important role in maturation and subsequent developmental capacity of human oocytes.

Time interval between FSH priming and aspiration of immature human oocytes for in-vitro maturation: a prospective randomized study

This prospective randomized controlled study was performed to examine the influence of coasting for 2 days versus 3 days following a fixed daily dose of FSH for 3 days. The outcome was 2-fold. In the first experiment (n = 50 cycles), the incidence of apoptosis in granulosa cells was compared. In the second experiment (n = 28 cycles), the rates of maturation, fertilization, cleavage, pregnancy and implantation were compared. In addition, clinical pregnancy rate per aspiration was registered. Granulosa cells were collected from follicular aspirates and pooled for each patient. The APOPTAG Detection Kit was used for staining of the granulosa cells and detection of apoptosis. Oocytes were matured in vitro for 28-30 h before intracytoplasmic sperm injection. The incidence of apoptosis in granulosa cells did not differ between granulosa cells obtained after 2 days coasting (n = 25 cycles) compared with granulosa cells obtained after 3 days coasting (n = 25 cycles) (26.2 versus 26.2%). When oocytes obtained after coasting for 2 days (n = 12 cycles) were compared with oocytes obtained after coasting for 3 days (n = 16 cycles), no significant difference was found between rates of maturation (63 versus 65%), fertilization (60 versus 68%), cleavage (86 versus 92%) or implantation [5/12; 42 versus 1/12 (8%)]. A higher clinical pregnancy rate per aspiration [5/16 (31%) versus 1/12 (8%)] was obtained after coasting for 3 days compared with coasting for 2 days. The difference was not significant. This randomized study showed no difference in apoptosis of granulosa cells and no difference in developmental competence of oocytes obtained after coasting for 3 days compared with 2 days coasting.

Morphology of spermatozoa used in IVF and ICSI from oligozoospermic men

The purpose of this study was to determine morphology in human spermatozoa from men with oligozoospermia and correlate with the fertilization rate, embryo score and pregnancy rate after IVF and intracytoplasmic sperm injection (ICSI) respectively. The study group consisted of 125 couples where the male partner suffered from oligozoospermia. Fifty of these had IVF (group A). Seventy-five couples in whom ICSI had been performed made up group B. Sperm samples were assessed according to the WHO manual. For each male, morphology of spermatozoa was judged according to Krügers strict criteria, WHO criteria and the teratozoospermia index (TZI). Oocyte monitoring was carried out according to a long down-regulation protocol using gonadotrophin-releasing hormone, recombinant FSH and human chorionic gonadotrophin. Embryos were transferred on day 2 after fertilization, with a maximum of three embryos. This study demonstrated no correlation between any of the morphological assessments of spermatozoa and the fertilization rate, embryo score and pregnancy rate, either after IVF or ICSI. Morphology in human spermatozoa according to Krügers strict criteria, WHO criteria and the TZI had no predictive value for the outcome after either IVF or ICSI.

Role of pituitary adenylate cyclase-activating peptide (PACAP) in the cyclic recruitment of immature follicles in the rat ovary

Following the midcyclic gonadotropin surge, PACAP is transiently expressed for approximately 12 h in the cyclic adult rat ovary. PACAP is observed in granulosa/lutein cells of the large mature follicles destined to ovulate and is believed to be a regulator of acute progesterone production and luteinization in these follicles. PACAP is also observed in solitary theca cells of immature follicles and in interstitial glandular cells intimately surrounding immature follicles. To examine if PACAP could be involved in the process of cyclic recruitment of such immature follicles, we primed immature granulosa cells from prepubertal ovaries with PACAP (1 nM and 100 nM) for 12 h. The treatment significantly stimulated the subsequent 24 h FSH-induced estradiol production (2.2 and 2.4 fold, respectively). The response seemed to be caused by a stimulation of aromatase activity. Estradiol production induced by testosterone was increased 2.4 and 2.6 fold, respectively, whereas functional FSH-receptors (cAMP production following FSH stimulation) or spontaneous apoptosis (immunohistochemical detection of DNA fragments) was unaffected. We conclude that PACAP priming of immature rat granulosa cells for 12 h increases subsequent FSH induced estradiol production and that PACAP could be involved in the cyclic recruitment of immature follicles in the adult rat ovary.