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.

Endosulfan is toxic to the reproductive health of male freshwater fish, Cyprinion watsoni.

Endosulfan is an organochlorine pesticide that is toxic to aquatic life. Endosulfan might hamper the reproductive health of indigenous fish in agricultural areas of Pakistan where this pesticide is sprayed widely. The aim of the current study is to investigate the toxic effects of endosulfan on selected reproductive parameters of male freshwater fish, Cyprinion watsoni. Two concentrations of endosulfan (0.5 and 1 ppb for 30 days exposure) were tested for their effects on body weight, body length, and testicular weight, length, and width. Testicular testosterone was assayed from tissue extracts using enzyme immunoassay (EIA). A significant increase in the mortality rate was observed in both treated groups during both spawning and quiescent seasons. The overall behavior of fish in the aquarium was normal in all control and treated groups. However, the treated fish exhibited anxiety after treatment with endosulfan. The body weight and length, and testicular weight, length and width were not significantly different to the control group. The testicular testosterone concentrations were significantly lower in both endosulfan-treated groups compared to the control. The decrease was dose-dependent, with a significant difference between the two treated groups. The histomorphological results demonstrated various testicular alterations in the treated groups. These alterations included an increase in interlobular areas and clumping patterns in spermatocytes/spermatids. Because spermatids eventually differentiate into sperms, their low count will directly result in lower sperm count. Taken together, these results suggest that endosulfan is a toxicant that at least disturbs testosterone levels (possibly others) and negatively impacts the reproductive health of male freshwater fish.

Effect of gonadotrophins, oestradiol and insulin on cumulus expansion of Nili Ravi buffalo oocytes.

The objective of the present study was to investigate the cumulus expansions of Nili Ravi buffalo oocytes during cultured in TCM-199 supplemented with 2 µg/ml oestradiol (E(2)), 0.05 IU/ml recombinant human follicle stimulating hormone (rhFSH), 2IU/ml human chorionic gonadotrophin (hCG), and 0.12 IU/ml insulin (I). The cumulus oocytes complexes (COCs) were collected from 2-8mm follicles from local abattoir ovaries. Supplementation of medium with single hormones showed significant (P<0.0001) increase in mean diameter of COCs with rhFSH except E(2), hCG and insulin after 24 hours compared to the increase in the mean diameter of COCs matured in TCM-199 without any hormonal supplementation. With rhFSH even at 8th hour, significant increase (P<0.001) in cumulus expansion was observed. In combination of hormones the significant (P<0.0001) cumulus expansion was achieved in E(2)+rhFSH treatment group. The non significant (P>0.05) cumulus expansion was observed in treatment groups viz. E(2)+hCG, E(2)+Insulin, rhFSH+hCG, rhFSH+Insulin, hCG+Insulin, E(2)+rhFSH+hCG and E(2)+rhFSH+hCG+Insulin after 24 hours. In conclusion, supplementation of rhFSH alone and in combination with E(2)in TCM-199 has highly significant effect on cumulus expansion.

Effect of metformin hydrochloride in correcting hyperinsulinemia and high leptin levels in treatment of infertile polycystic patients.

BACKGROUND: Research conducted over PCOS by various groups in the world indicated the effect of Metformin on PCOS. Previous studies suggest that Glucophage by reducing hyper-insulinemia is clinically useful in the treatment of polycystic ovary syndrome (PCOS). Our Objective was to observe the role of Glucophage in treating infertility of polycystic patients within three months in Group A and six months in Group B patients. Another aim was to assess the decrease in hyper-insulinemia and Leptin levels by Glucophage in our population. METHODS: This study was carried out at the Department of Obstetrics and Gynaecology, Pakistan Institute of Medical Sciences, and Noor Specialised Clinic Islamabad, from Oct 2004 to Apr 2008. One hundred and seventy women in Group A and 145 in Group B fulfilling the clinical and biochemical criteria for PCOS were enrolled. Glucophage was started at an oral dose of 500 mg/day and maintained at 1500 mg for 3 months or 6 months in both Groups. Besides ultrasonography, serum FSH, LH, fasting insulin, fasting blood sugar, glucose insulin ratio, Quantitative Insulin Sensitivity Check Index (QUICKI) and leptin levels were performed. After three months in Group A and six months in Group B these criteria were again assessed. RESULTS: After 3 and 6 months of Metformin therapy, significant reduction in biochemical parameters was observed such as fasting glucose, insulin and leptin. Data were analysed using SPSS by paired t-test and ANOVA. CONCLUSION: Six months treatment with Glucophage proved to be better option for infertile PCOS.

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.

Morphological studies on crushed sciatic nerve of rabbits with electroacupuncture or diclofenac sodium treatment.

Sciatic nerves of 15 rabbits were crushed by Halsted straight mosquito hemostat with 8-11 Newton force for 60 seconds on a point 5 mm above the knee joint, and then the rabbits were equally divided into three groups. The acupuncture group was treated by electroacupuncture on Huan Tiao and Wei Zhong points (25 minutes/day) for 7 days. The medicine group was treated with intramuscular administration of dicofenac sodium (15 mg) daily for 7 days. The control group was not treated. After treatment, the distal parts of crushed nerve were examined under light microscope, the densities of normal myelinated fibers in 0.126 mm2 were counted, and the diameters of 20 normal myelinated fibers were measured for each animal. The results showed that the mean densities were 176.2 +/- 5.953 in the acupuncture group, 118.2 +/- 10.878 in the medicine group and 101.4 +/- 8.548 in the control group. The mean values were significantly different between the acupuncture and medicine groups (p < 0.01) and highly significant difference between the acupuncture and control groups (p < 0.001); but there was no significant difference between the medicine and control groups (p > 0.05). There are more small myelinated fibers (0-9 microm) in the acupuncture group than in the medicine and control groups (p = 0.0028). The results revealed and confirmed that acupuncture promotes nerve regeneration; diclofenac sodium did not show such an effect. The present study demonstrates the positive effect of acupuncture on regeneration of a crushed sciatic nerve in the rabbits. And acupuncture is a better treatment for regeneration of crushed nerve than diclofenac sodium.

Environmental pollutant Aroclor 1242 (PCB) disrupts reproduction in adult male rhesus monkeys (Macaca mulatta).

Adult male rhesus monkeys (Macaca mulatta) were given oral treatment of either Aroclor 1242 or vehicle (corn oil and glycerol) at a dose of 200 microg/kg body wt/day for 6 months to investigate the effects of the pollutant on plasma testosterone and the morphology of testes and accessory glands. Aroclor 1242 treatment significantly decreased testicular size and testosterone levels in plasma and adversely affected spermatogenic activity by disrupting epithelial organization. All components of the germinal epithelium were greatly reduced. The spermatogonia were either hypertrophied or had shrunken vesiculated cytoplasm with distorted mitochondria and nuclear pyknosis. Changes were milder in the Sertoli cells, where nuclear infoldings were reduced. Characteristic features of treated Leydig cells were the presence of electron-dense and electron-opaque zones, appearing as plaques, cell membrane abnormalities, and high variability in nuclear shape and heterochromatin distribution. All the Aroclor 1242-treated accessory glands contained more connective tissue than their vehicle-treated counterparts. The epithelium contained many layers of irregularly shaped necrotic cells possessing stereocilia in the epididymides, either hypochromic and hypertrophied or hyperchromic and hypotrophied cells in the prostate and shrunken cuboidal cells with elongated nuclei in the seminal vesicles. In conclusion, Aroclor 1242 treatment causes severe structural alterations on gonads and accessory organs in adult male rhesus monkeys, and these effects could be mediated through both estrogen and Ah receptors.

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.