Effects of cryopreservation on sperm DNA integrity in normospermic and four categories of infertile males.

This study examined the effects of cryopreservation on DNA integrity of spermatozoa from 34 fertile subjects and 166 infertile subjects comprised of 80 teratospermic, 32 normospermic, 30 astheno-teratospermic, and 24 oligo-astheno-teratospermic individuals. Semen samples were prepared by swim-up and the Percoll density gradient centrifugation method (Pdgc) prior to freezing in liquid nitrogen. Neat and prepared samples were supplemented with cryoprotectant (SpermFreez) in cryoampoules and were frozen using the static phase vapor cooling procedure. Sperm DNA integrity of all thawed samples was determined using the alkaline comet assay. It was noticed that the sperm DNA integrity of frozen samples of fertile subjects was considerably higher than that of infertile subjects with greater catch-up integrity similar to the fresh samples. Freezing caused less chromatin damage to sperm of Pdgc samples from both fertile and infertile subjects as was compared to the neat and swim-up samples. It is concluded that the increase in comet frequency of frozen-thawed samples from infertile subjects was more prominent (8.25-22.78%; P<0.01) than in the fresh samples. Frozen-thawed samples from Ts (Teratospermic individuals) and ATs (Astheno-teratozoosspermic) showed higher level of OTM (Olive tail moment) indicating a higher level of chromatin fragmentation than fertile, Ns (Normospermics), and OATs (Oligo-astheno-teratozoospermics).

Adverse effects of arsenic exposure on uterine function and structure in female rat.

The present investigation examined the adverse effects of arsenic exposure on uterine function and structure of female rat at 56 days of age, exposed to different doses (50, 100, and 200ppm) of sodium arsenite in drinking water at immature age (28 days) for 28 days. Dose-dependent decrease (P<0.001) was observed in mean uterine weight and length in all treated groups compared to control. Higher arsenic deposition was found in uterine tissue against increased doses of arsenite. Arsenite treatment altered the histomormphology of the uterus. Uterine epithelium in 50ppm group was lined by cuboidal cells instead of columnar cells observed in control epithelium. In 100 and 200ppm groups, no demarcation was observed between epithelial cells and endometrial stroma. No basement membrane was seen in these groups; even in 50ppm, basement membrane was disturbed. The endometrial stroma in 100 and 200ppm groups was very dense in appearance and contained irregular-shaped cells. In myometrium, loosening of cells was observed in 100 and 200ppm groups. Dose-dependent decrease (P<0.001) was observed in mean uterine diameter, epithelial height, thickness of endometrium, myometrium, and in plasma levels of estradiol, progesterone, FSH and LH in all the treatment groups compared to control. In summary, arsenic is a major threat to female reproductive health acting as a reproductive toxicant and as an endocrine disruptor, restricted the function and structure of uterus, by altering the gonadotrophins and steroid levels, not only at high dose concentration but also at low (50ppm) levels, when they become mature.

Genotoxicity of sodium arsenite and DNA fragmentation in ovarian cells of rat.

Present study examined the genotoxic effects of arsenite in ovarian tissue of rat at 56 days of age. Immature (28 days old) female rats were exposed to different doses (50, 100, and 200 ppm) of sodium arsenite in drinking water for 28 days. DNA damage in ovarian tissue was measured by comet assay. All doses induced significant decrease in ovarian weight in a dose-dependent manner compared to control, more prominently at (P<0.001) 100 and 200 ppm. All the comet assay parameters showed significant difference with arsenite treatment compared to control group. In treatment groups, mean number of cells with intact DNA decreased while, mean comet number increased (P<0.001) in a dose-dependent manner compared to control. Significant decrease (P<0.05) was observed in mean comet length, height, comet head diameter and %DNA in comet head of high dose groups compared to control group. Dose dependent increase was found in mean comet tail length, %DNA in tail, tail moment and olive tail moment in high dose groups compared to control group. The study indicates that arsenic caused DNA damage to ovarian cells particularly at high doses and ensure comet assay as an effective method to detect DNA damage in tissue caused by metals.

Sperm preparation: DNA damage by comet assay in normo- and teratozoospermics.

The present study was carried out on semen samples of human fertile and infertile subjects, teratozoospermics (TZs) and idiopathics (IDs), with neat semen and sperm prepared by swim up or Percoll density gradient centrifugation procedures. Sperm morphology analysis revealed that only head and midpiece defects in TZs and IDs were significantly (P < 0.001) higher compared to fertile subjects. Infertile subjects indicated significantly higher (P < 0.001) sperm DNA damage compared to fertile subjects. Fertile subjects with sperm prepared from neat and Percoll density gradient centrifugation exhibited a comet tail DNA percentage of 20% and 15%, respectively. The TZs and IDs infertile subjects had higher levels of comet tail DNA of 33% and 25% and 25% and 19%, respectively. A significant (F = 24.01; P = 0.0059) decrease in mean comet head DNA percentage or sperm DNA integrity was observed in neat samples from fertile and infertile subjects by Repeated Measures ANOVA. In Percoll prepared samples from fertile, TZs, and IDs, there was a significant increase in sperm DNA integrity. Similarily, there was a decrease in abnormal sperm morphology in swim up and Percoll prepared sperm compared to neat samples. The Percoll density gradient centrifugation procedure yields sperm with an increase in sperm DNA integrity relative to swim up. Sperm DNA damage of TZs with both sperm preparation methods was significantly (P < 0.01) higher when compared to fertile and IDs. But the level of DNA damage was higher in IDs compared to fertile subjects. Compared to the other methods tested, the Percoll method yielded sperm with improved DNA integrity. In conjunction with semen analysis, the assessment of nuclear integrity improves the characterization of the semen sample and may be used as a tool for allocating the patients to specific assisted reproductive treatments.

Effect of hypothyroidism induced by propylthiouracil on ovarian function and structure in offspring from treated mothers (Rats).

The aim of the present study was to investigate the effects of hypothyroidism induced during the pre- and postnatal periods of life on ovarian function and structure in offspring (pups) 120 days of age. Three groups were used. In the prenatal group, treatment was given from conception to parturition. In the postnatal group, treatment was given from parturition to 25 days postpartum. Hypothyroidism was induced by administration of 0.1% 6-n-propyl-2-thiouracil (PTU) in the drinking water of mothers. Body weights of the offspring were measured weekly. In each group, ten offspring were sacrificed at 120 days of age. Postnatal PTU treated pups showed delay in eye opening, teething, fur development, and weaning (35-37 days) compared to control animals (28-30 days). Body weight of offspring in the postnatal PTU treatment group was significantly decreased (P < 0.001), while the prenatal PTU treatment group showed a significant increase (P < 0.0001) compared to control animals. There was a significant (P < 0.05) reduction in paired ovarian weight of offspring in the postnatal PTU treatment group compared to control animals. Diameter of the ovaries was not affected by any treatment. Regarding the morphometery, only offspring in the prenatal PTU treatment group showed a significant (P < 0.001) increase in the diameter of graafian follicles. No significant difference was observed in morphometery of the granulosa layer, primary, and developing follicles of control and all treated groups. Number of primary, developing, and graafian follicles of all the treated groups was similar to that of the control group. The corpora lutea of the postnatal PTU treated group contained a population of large numbers of luteal cells compared to the control group. The prenatal PTU treated group did not exhibit a profound effect on ovarian morphology, histology, and morphometery. No difference was found in the serum estradiol concentration of control and PTU treated groups. J. Exp. Zool. 293:407-413, 2002.