Reasearch On The Relationship Between Y Chromosome Polymorphism And AZF Microdeletion, The Effects Of Methylene Blue On Sperm Quality

According to the World Health Organization, infertility affects about 10%-15% of couples in the world, male infertility accounts for about 50% of infertility couples. Male infertility is caused by complex factors, such as genetic factors, immunological factors, reproductive tract infections, varicoceles, exogenous factors and so on. In order to explore the genetic causes and exogenous factors on male infertility, the correlation between AZF polymorphism and Y chromosome microdeletion, as well as the effects of methylene blue (MB) on sperm quality and early embryo development were investigated in this paper.Microdeletions of the Y chromosome long arm azoospermia factor(AZF) is one of the major causes of severe oligozoospermia or azoospermia. AZFa microdeletions were screened in a group of XX patients with oligozoospermia or azoospermia, using nine sequence tag sites (STS) in Y chromosome. Our result displayed that out of 141 patients with sY86-present,5 cases showed sY84-absent (3.54%,5/141). DNA sequence revealed that a T-to-G transversion is located at the fifth base of the reverse sY84 primer, a single nucleotide polymorphism (SNP) named as rs72609647 in SNP databank from the NCBI website (http://www.ncbi.nlm.nih.gov/snp). To further study the correlation between SNP and male infertility, DNA sample were divided into 3 groups based on sperm density:healthy semen donors (≥60×106/mL); infertile males with normal sperm count (≥15×106/mL) and severe oligozoospermia group (< 5×106/mL) including normal AZF group and AZF microdeltion group. Allele-specific PCR results showed that about 10% incidence of AZFa-C genotype were observed not only in infertile men but also in healthy donors. However, only 4% incidence was found in AZF microdeltion group. Therefore, our findings indicated that the incidence of AZF microdeletion in AZFa-C genotype male is less than that in AZFa-A genotype male.Giving that exogenous parameters play important role on male infertility, the effects of methylene blue (MB) on sperm quality and early embryo development were studied. MB is classified as phenothiazine dyes. Recenty MB has been widely used for human disease diagnosis and treatment due to its low toxicity. However, it has been reported that MB can inhibit sperm motility in vitro. In this study, different doses of MB were incubated with human normal sperm for various time ranges from 30 minutes to 2 hour in vitro. The results showed that the sperm motility was decreased significantly after incubation with 0.1-0.01% MB for 30min, sperm DNA fragment index highly increased to 90%. However, there is no significant difference in sperm plasma memberane integrity. Mature male Sprague Dawley rats were administered MB intragastrically for two weeks. The results showed that the effects of MB on rat sperm motility are consistent to that observed in vitro. No impact on spermatogenesis, plasma membrane and sperm morphology were observed. In order to observe the affects of MB on sperm fertilization capacity, mouse spermatozoa incubated with 0.05%MB for 30min were introduced to normal egg through intracytoplasmic sperm injection. The results displayed that fertilization and cleavage rate were significantly decreased. Our findings in this study suggested that MB might inhibit sperm motility via sperm DNA damage and resulting in decreased sperm fertilization capacity.Conventionally, sperm plasma membrane integrity was detected using eosin staining or hypotonic swelling test. But their clinical application is limited due to the poor reproducibility of above methods. Cyanine dyes emerge as important organic functional dyes and have been utilized in biological field widely as a living cell fluroscent dyes in recent years. With the collaboration of Northwest University, China. 20 synthetic cyanine dyes (A1-A20) were tested for the biological applications, particularly in the field of detecting sperm plasma membrane integrity. Four dyes(cy1,cy2,cy11andcy13) showed potential candidates with membrane permeate, good affinity with sperm DNA, little fluorescence background. In conclusion, synthetic cyanine dyes are cheap and suitable for clinical analysis.