Leiguang Wang

Measurement of sperm DNA fragmentation using bright-field microscopy: comparison between sperm chromatin dispersion test and terminal uridine nick-end labeling assay

OBJECTIVE To compare sperm chromatin dispersion (SCD) test with terminal deoxynucleotidyle transferase-mediated terminal uridine nick-end labeling (TUNEL) assay in assessing DNA fragmentation in human sperm through bright-field microscopy. DESIGN Prospectively designed, side-by-side comparative study. SETTING Medical genetics laboratory in a provincial research institution. PATIENT(S) Sixty male patients presented for infertility evaluation and 30 fertile, volunteer sperm donors. INTERVENTION(S) Semen analysis, SCD test, and TUNEL assay on the same semen sample and on the same spermatozoa. MAIN OUTCOME MEASURE(S) Sperm DNA fragmentation, determined by SCD test score or TUNEL assay score. RESULT(S) Sperm chromatin dispersion test and TUNEL assay identified similar proportions of sperm cells with DNA fragmentation in the same semen samples. When the SCD test and TUNEL assay were performed simultaneously on the same spermatozoa, TUNEL-negative sperm showed a large halo, whereas TUNEL-positive sperm showed no halo after Diff-Quik staining. However, in some sperm cells, DNA damage was detected by the SCD test but not by the TUNEL assay. CONCLUSION(S) Sperm chromatin dispersion test and TUNEL assay are both effective in detecting sperm DNA damage. Using bright-field microscopy, the SCD test appears to be more sensitive than the TUNEL assay.

Sperm chromatin integrity may predict future fertility for unexplained recurrent spontaneous abortion patients.

The pathogenesis of recurrent spontaneous abortion (RSA) is multi-factorial, complex and poorly understood. In the present study, semen parameters, including sperm chromatin integrity, sperm concentration, sperm motility and sperm morphology, were compared between 111 men whose partners had a history of unexplained RSA (RSA group) and 30 healthy fertile men (control group). The RSA group was further separated into three subgroups, depending on their reproductive outcome during the 12 months after they were enrolled in the study: the pregnancy subgroup consisted of 43 men whose partners achieved a successful pregnancy up to at least the 24th week of gestation; the abortion subgroup included 31 men whose partners experienced further abortions; and the infertile subgroup had 37 men whose partners did not have any positive pregnancy test after regular, unprotected intercourse. Significantly lower proportion of sperm with normal morphology was found in the abortion subgroup (14.7 ± 4.3%) than in the control group (17.5 ± 5.0%). Sperm concentrations were significantly lower in the infertile subgroup (55.7 ± 24.1%) than in the controls (68.6 ± 27.8%). The rates of abnormal sperm chromatin integrity were significantly higher in the abortion (16.7 ± 7.7%) and infertile (16.3 ± 6.6%) subgroups, compared to the control group (13.0 ± 4.4%). Logistic regression analysis showed that the subsequent reproductive outcome of the 111 RSA patients was negatively correlated to the rates of abnormal sperm chromatin integrity. In conclusion, sperm chromatin integrity, sperm morphology, and sperm concentration were associated with future reproductive outcome of RSA patients. The sperm chromatin integrity was a significant predictor for future abortion and infertility.

Sperm chromosomal aneuploidy and DNA integrity of infertile men with anejaculation

PurposeTo explore sperm chromosomal aneuploidy, sperm membrane and DNA integrity in infertile patients with anejaculation.MethodsSemen samples were collected from 18 infertile men with spinal cord injury (SCI) by penile vibratory stimulation (PVS) and from 14 psychogenic anejaculation (PA) patients by percutaneous vasal sperm aspiration (PVSA). These semen samples as well as samples from 16 donors were analyzed using the hypo-osmotic swelling (HOS) test, the sperm chromatin dispersion (SCD) test and multi-color fluorescence in situ hybridization (FISH) with probes specific for chromosomes 13, 18, 21, X and Y.ResultsThere were significant differences in the percentages of motile sperm, normal morphologic sperm and sperm DNA fragmentation between the infertile men with SCI and the control group (P < 0.05 and P < 0.01). The sperm motility was significantly greater in the PA-PVSA group than in the SCI-PVS group (P < 0.01). The number of round cells per mL of semen obtained from the 18 SCI patients by PVS was between 1 and 8 million. The rate of sperm DNA fragmentation in the SCI-PVS group was higher than that of the PA-PVSA group (P < 0.05). The aneuploidy rates for the SCI patients were 2.4-fold higher for chromosomes 13, 18 and 21 and 2.2-fold higher for chromosomes X and Y than for patients in the control group (P < 0.0001).ConclusionsThe semen quality is poorer, sperm DNA fragmentation and sperm chromosomal aneuploidies are seen at a higher rate for SCI patients compared to healthy, fertile and normospermic men. Whether the difference in yield is due to increased scrotal temperature, genitourinary infection, or other reasons requires further study.

Effects of the crude extract of Polygala tenuifolia Willd on human sperm in vitro

The aim of the present study is to analyze sperm membrane changes and the spermicidal effect in treatment with the crude extract from Polygala tenuifolia Willd (PTW) in vitro. The root of PTW was extracted in distilled water. Normal human spermatozoa were used to assess the spermicidal activity (Sander-Cramer assay) of the extract from the PTW root. The hypo-osmotic swelling (HOS) test and the eosin Y (EY) staining were used to detect the integrity of sperm membrane and vitality. The sperm chromatin dispersion (SCD) test was performed to determine sperm DNA integrity. N-9 was used as a reference standard and semen added to physiological saline was used as the control. Semen samples were donated by 42 healthy fertile men. The crude extract from the root of PTW could immobilize and kill 100% spermatozoa within 20 s in vitro at the concentrations of 20.0 and 10.0 mg/ml; at the concentration of 5.0 mg/ml, spermatozoa were immobilized in (39.5±3.2) s. In the groups of the crude extract from the root of PTW and N-9 solution, the rate of the normal HOS (tails swollen) and the white head (unstained) was 0%, and the rate of the abnormal HOS (tails unswollen) and red head (stained) was 100%. Sperm DNA fragmentation showed no change in exposure to the crude extract from the root of PTW and N-9 solution. The sperm revival test did not show any spermatozoa that recovered their motilities. The rapid spermicidal activity of the crude extract from the root of PTW in vitro may occur by the disruption of the sperm membrane integrity.

A new experimental three-dimensional, reticular intrauterine device (3-DRIUD) composed of nitinol and silicone rubber

BACKGROUND The aim of this study was to explore a new three-dimensional, reticular intrauterine device (3-DRIUD) composed of nitinol and silicone rubber and to observe the contraceptive effect of the device in rats. STUDY DESIGN Two contraceptive experiments were performed. In the first, female rats underwent bilateral placement of a 20.0-35.0-mm 3-DRUID (experimental group, n=30) via an abdominal incision or a sham operation with no IUD (control group, n=30). Two weeks after the operation was performed, the rats from either group were caged together with male rats. The contraceptive effects of the 3-DRIUD were observed at 1 to 3 months postoperation, after which the 3-DRIUDs were removed. One month after this second operation, the rats from the two groups were again coupled with fertile male rats. In a second experiment, female rats underwent bilateral placement of a 10.0-mm 3-DRUID (n=5) via an abdominal incision or a two-dimensional IUD (2-DIUD, n=20) and mated 1 month after surgery. The single-pipeline IUD was placed in 10 rats, while the enfolded-pipeline IUD was placed in 10 different rats. RESULTS In the first experiment, none of the females in the experimental 3-DRIUD group became pregnant (0/30, 0%) after 3 months, compared to 28/30 (93.3%, p<.0001) rats in the control group. After the 3-DRIUDs were removed from the experimental group after 3 months, 27/30 (90%) became pregnant, compared with 29/30 (97%, p>.05). The litter size (mean±SD) did not differ between groups (10.9±1.5 3-DRUID, 11.2±1.1 control, p>.05). In the second experiment, five rats had a 10.0-mm 3-DRUID (which was one third the length of one uterine horn) inserted into the bilateral uterine horns, and three of the five rats became pregnant. All 20 rats were pregnant 1 month after the insertion of the 2-DIUD. Thus, the contraceptive rate for the 2-DIUD group was 0. CONCLUSIONS The primary contraceptive mechanism effect of the new 3-DRIUD in rodents appears to be a result of occupying physical space in the uterus.