investigation on mitochondrial DNA deletions and telomere shortening during multiple passages of adult stem cells

Limited resources for adult stem cells necessitate their in vitro culture prior to Clinical use. Investigating mitochondrial DNA [mtDNA] and telomere shortening has proved to be important indications of stem cell validity. This study was designed to investigate these indicators in multiple passages of three adult stem cell lines which were produced in our stem cell laboratory. In this study, Dental Pulp Stem Cells [DPSCs], Periapical Follicle Stem Cells [PAFSCs] and Human Foreskin Fibroblast [HFF] cell lines were expanded for 20 passages. After 1, 5, 10, 15 and 20 passages, expanded cells were harvested and DNA was extracted for further studies. Common mtDNA mutation was detected by multiplex PCR and telomere shortening was tested by Southern blot analysis. The common deletion was not detected in any of the stem cells or cell lines after several passages. In addition, Southern blot analysis indicated that the mean difference of telomere length between first and last passage was 0.25 kb in DPSC, 0.1 kb in PAFSC and 0.32 kb in HFF which indicates that the mean telomere length in various passages of the samples showed insignificant changes. Absence of mtDNA mutations in adult stem cell lines indicates good mitochondrial function even after 20 passages. In addition, absence of telomere shortening indicates stem cells validity after multiple passages. It is hoped this information could pave the way for using in vitro expansion of adult stem cells for future Clinical applications

Association between Y-chromosome AZFc region micro-deletions with recurrent miscarriage

In human, about 25% of implanted embryos are losing 1-2 week following attachment to the uterus. A subset of this population will have three or more consecutive miscarriages which define as repeated pregnancy loss [RPL]. Introducing the assisted reproductive technologies [ARTS] made a chance for infertile couples to solve their childless problem. This study was conducted to evaluate the incidence of Y-chromosome AZF region's micro-deletions in male partners of couples with recurrent miscarriage [RM]. Thirty male partner of couples with RM and thirty infertile males, who referred to the Yazd Research and Clinical Center for Infertility were recruited to this study. In addition, 30 healthy men were screened as a control group from the same center. After DNA extraction using salting out method, the multiplex-PCR was done for amplifying 8 known STSs proximal to the AZF region of the Y-chromosome. The results were compared between the groups using Fisher's exact t-test and p<0.05 was considered statistically significant. Of the 30 infertile males, 5 [16.6%] cases were associated with the AZF region micro-deletions of DYF87S, DYF84S1, DYF83S1 and DYF51S1, STSs. But in the fertile and RM male groups was found no deletions similar to those, of the infertile males [p=1.0]. Instead 4 [13.3%] cases of the RM group males had different micro-deletions included DYS220 [AZFb, sY129], DYS262, DYF8551, and DYF8651, STSs. The AZFc locus of Y-chromosome micro-deletions have a significant role in RM [p=0.045]. It seems that the Y-chromosome AZF region's micro-deletions are associated with RM, and we recommend adding this AZF region STSs into infertility analyzing panels.

Multiplex PCR screening of Y-chromosome microdeletions in azoospermic ICSI candidate men

It has been hypothesized that Y-q microdeletion can account for significant proportion of infertility in men. There are three nonoverlapping regions referred to as the "azoozpermia factors" AZFa, AZFb, and AZFc from proximal to distal part of Y-q. These have been defined as spermatogenesis loci, this region deletions have been shown to be involved in male azoospermic or severe oligoozospermic infertility. Evaluation the rate of Y-chromosome microdeletions in infertile men. In this case-control study, 25 azoospermic infertile men candidate for intracytoplasmic sperm injection [ICSI] were selected as case group. For control group, 25 normoozoospemric men were selected. All cases and controls had normal 46XY karyotype. DNA extraction and molecular analysis were done on blood samples. Multiplex-PCR method was done to identify the presence of microdeletion in AZFa, AZFb or AZFc loci. Eight STS primers that include two controls were selected to determine Y-chromosome microdeletions. 20% [5/25] of all patients have at least one microdeletion in more than one region of AZF loci. Totally 17 microdeletions was observed, one case had deletions in three AZF regions, and 4 cases had deletions in two AZF regions. The rate of deletions was 42% [7/17] for AZFc, 35% [6/17] for AZFa and 23% [4/17] for AZFb. The molecular DNA analysis could help us to know the real cause of infertility and can give good information for good decision for example in men whit microdeletions who want to undertake ICSI procedure the deletions will be passed to their son