Low maternal folate concentrations and maternal MTHFR C677T polymorphism are associated with an increased risk for neural tube defects in offspring: a case-control study among Pakistani case and control mothers.

BACKGROUND AND OBJECTIVES: There is considerable evidence that periconceptional maternal folate deficiency and coding variants in maternal genes coding for critical enzymes in the folate pathway are associated with neural tube defects (NTDs) in offspring. In a case-control study we investigated C677T polymorphism in the 5,10- methylenetetrahydrofolate reductase (MTHFR) gene in case and control mothers of Pakistani origin, and compared these with the respective maternal folate concentrations measured at the time of delivery. METHODS AND STUDY DESIGN: A case-control study was conducted among 109 case and 100 control mothers identified through the Holy Family Hospital Rawalpindi, Quaid-i-Azam University, Islamabad, Pakistan. Red blood cell (RBC) and serum folate concentrations and MTHFRC677T polymorphism were compared between case and control mothers. RESULTS: Mean RBC folate and serum folate concentrations were significantly lower in cases compared with control mothers (p<0.0001). Maternal MTHFR 677CT and 677TT genotypes were more common among cases compared with control mothers (CC vs TT p<0.0393 and CC/CT vs TT p<0.021). T-allele frequency was higher in cases compared with control mothers (C vs T p<0.017). Case mothers with 677CT or 677TT genotypes had significantly lower serum (p<0.0001) and RBC folate concentrations (p<0.0001) compared with control mothers. CONCLUSIONS: The present study provides further evidence that maternal folate deficiency and MTHFRC677T polymorphism might be associated with an increased risk for NTDs in offspring. Our results are limited by the fact that maternal folate concentrations were not obtained during the periconceptional period, but at delivery. Further analyses, including maternal folate levels during the periconceptional period, are warranted.

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).

Comparative study of intracytoplasmic sperm injection outcome from sperms retrieved by different procedures.

BACKGROUND: Different sperm retrieval procedures can help retrieve sperms that can be used for Intracytoplasmic Sperm Injection (ICSI) in azoospermic men. The objective was to compare the Intracytoplasmic sperm injection outcome, through different sperm retrieval procedures in azoospermic men. METHODS: A Retrospective Study was carried out at Islamabad Clinic Serving Infertile Couples from September 1999 to March 2005. The study includes 105 female subjects and 105 male azoospermic subjects. In 105 male subjects 50 subjects had Non-Obstructive Azoospermia (NOA) and 55 subjects had Obstructive azoospermia (OA). These subjects underwent surgical sperm retrieval procedures like Percutaneous Epididymal Sperm Aspiration (PESA) or Fine Needle Aspiration Biopsy. Intracytoplasmic sperm injection (ICSI) was performed from both and outcome was compared. All the values were expressed as Mean +/- SE. Limit of significance was set at (p < 0.05). Mean values were compared using unpaired Students t-test. RESULTS: Subjects who underwent ICSI procedure with motile sperms retrieved through Biopsy had significantly (p < 0.05) raised cleavage rate and live birth rate. CONCLUSION: No matter what ever the procedure of sperm retrieval used, the sperm motility has great significance. Significantly raised live birth rate was detected in subjects where sperms retrieved through biopsy were used for ICSI procedure.

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.