Unraveling the dark matter, long non-coding RNAs, in male reproductive diseases: A narrative review

Recent advances in human transcriptome have revealed the fundamental and functional roles of long non-coding RNA in the susceptibility to diverse diseases and pathological conditions. They participate in wide range of biological processes such as the modulating of chromatin structure, transcription, translation, and posttranslation modification. In addition, based on their unique expression profiles and their association with clinical abnormalities such as those of related to male reproductive diseases, they can be used to develop therapeutic methods and biomarkers for screening of the diseases. In this study, we will review the identified lncRNAs and their molecular functions in the pathogenesis of male reproductive diseases such as prostate cancer, benign prostatic hyperplasia, prostatitis, testicular cancer, varicocele, and sperm abnormalities. Key words: Long noncoding RNA, Prostate cancer, Prostatic hyperplasia, Prostatitis, Varicocele, Sperm abnormalities

CAG repeat polymorphism in androgen receptor and infertility: A case-control study

Background: Androgens play a role in the development of male phenotype and spermatogenesis during puberty, the function of which is regulated by the androgen receptor (AR) gene. There is a polymorphism site in exon 1 of the gene encoding this receptor that can have different frequencies of CAG trinucleotide repeats and leads to the formation of polyglutamine chains of different lengths in the N-terminal domain of the AR protein and reduced sperm production by affecting spermatogenesis. Objective: To investigate whether the cause of a group of unexplained infertilities could be the increased frequency of CAG repeats in the AR gene of patients with oligozoospermia and azoospermia. Materials and Methods: In this case-control study, 84 men including 42 with unexplained infertility As a case group and 42 fertile men as a control group were selected. The frequency of CAG repeats was determined by the polymerase chain reaction method and then the difference in the frequency of these repeats was determined based on the difference in band size on the agarose gel. Results: The mean CAG repeat length in the azoospermia and oligozoospermia group was 17.5 ± 0.63 and in the fertile group it was 16.11 ± 0.75 (p = 0.46). In addition, most men (88.1% in the case group and 71.41% in the control group) had 13-23 repeats. Conclusion: No significant correlation was found between CAG repeat length and the risk of male factor infertility in an ethnically defined population of Iranian men. The role of regulatory factors and epigenetic changes should be taken into account too. Key words: Infertility, Azoospermia, Androgens, X chromosome, Spermatogenesis

Investigating the expressions of miRNA-125b and TP53 in endometriosis. Does it underlie cancer-like features of endometriosis? A case-control study

Background: Endometriosis is generally considered as a benign condition; however, there is a possibility for it to become cancerous. miR-125b is upregulated in both endometriotic tissues and serum samples of women with endometriosis but its potential targets in endometriosis are still not fully understood. Objective: The role of miR-125b in the regulation of TP53 expression in endometriosis was tested with a bioinformatics approach. In addition, the expression of miR-125b and TP53 in both eutopic (Eu-p) and ectopic endometrium (Ec-p) in the endometrium tissues of women with endometriosis was compared to those in the normal endometrium tissues of controls (Normal). Materials and Methods: In this case-control study, the Eu-p and Ec-p samples were collected from 20 women who underwent laparoscopic surgery, and the normal endometrium tissues were collected from 20 controls with no evidence of endometriosis. For bioinformatics approach, a protein-protein interaction network was constructed based on co-expressed potential targets of miR-125b. Quantitative polymerase chain reaction technique was used for the measurement of miR125b and TP53 expression. Results: Our results showed that miR-125b was significantly overexpressed in Ec-p (p-value: 0.021). In addition, there was a significant TP53 under expression in both the Ec-p and Eu-p samples compared with the Normal tissues (p-value: 0.003). Conclusion: The negative correlation between miR-125b and TP53 as well as a noticeable decreased expression of TP53 in both Ec-p and Eu-p samples may be interpreted as the roles of miR-125b/TP53 axis in the pathogenesis of endometriosis. In addition, these findings and bioinformatic analyses imply a possible role of miR-125b in cancer-like features of endometriosis. Key words: Endometriosis, TP53, miR-125b, Ectopic endometrium, Eutopic endometrium

Comparison of chromosomal instability of human amniocytes in primary and long-term cultures in AmnioMAX II and DMEM media: A cross-sectional study

Background: The genomic stability of stem cells to be used in cell therapy and other clinical applications is absolutely critical. In this regard, the relationship between in vitro expansion and the chromosomal instability (CIN), especially in human amniotic fluid cells (hAFCs) has not yet been completely elucidated. Objective: To investigate the CIN of hAFCs in primary and long-term cultures and two different culture mediums. Materials and Methods: After completing prenatal genetic diagnoses (PND) using karyotype technique and chromosomal analysis, a total of 15 samples of hAFCs from 650 samples were randomly selected and cultured in two different mediums as AmnioMAX II and DMEM. Then, proliferative cells were fixed on the slide to be used in standard chromosome G-banding analysis. Also, the senescent cells were screened for aneuploidy considering 8 chromosomes by FISH technique using two probe sets including PID I (X-13-18-21) & PID II (Y-15-16-22). Results: Karyotype and interphase fluorescence in situ hybridization (iFISH) results from 650 patients who were referred for prenatal genetic diagnosis showed that only 6 out of them had culture- derived CIN as polyploidy, including mosaic diploidtriploid and diploid-tetraploid. Moreover, the investigation of aneuploidies in senesced hAFCs demonstrated the rate of total chromosomal abnormalities as 4.3% and 9.9% in AmnioMAX- and DMEM-cultured hAFCs, respectively. Conclusion: hAFCs showed a low rate of CIN in two AmnioMAX II and DMEM mediums and also in the proliferative and senescent phases. Therefore, they could be considered as an attractive stem cell source with therapeutic potential in regenerative medicine. Key words: Human amniotic fluid cells, Chromosomal instability, Pseudomosaicism, Amniocentesis, Replicative senescence

Effects of phosalone plant pesticide on sperm parameters and sexual hormone levels in Wistar rats: An experimental study

Background: Phosalone is an organophosphate insecticide, applied to control of plant pests. This compound has various side effects because it acts as an acetyl cholinesterase enzyme inhibitor. Objective: To investigate the effects of phosalone on the sperm parameters of and levels of sex hormones in adult male rats. Materials and Methods: In this experimental study, 16 adult (8-12 wk) male Wister rates (weighing 220-280 gr) were randomly assigned into 4 groups (n = 4/each). Group 1 (control) received only routine adequate water and food; Group 2, 3, and 4 received different low doses of phosalone (60, 90, and 120 mg/kg respectively). The rats were weighed and anesthetized after 48 days. Sperm parameters including number, motility, and viability as well as sex hormones (such as Luteinizing Hormone, Follicle Stimulating Hormone, and testosterone) were evaluated and compared after removing the epididymis tail. Results: Our results showed that phosalone decreased sperm motility, viability, and number in a dose-dependent manner. The level of FSH and LH was increased, and testosterone was decreased. Also, depending on the dose, phosalone decrease sperm motility and viability (p ≤ 0.001), while the level of FSH and LH was increased and testosterone was decreased (p = 0.861). Conclusion: Phosalone has negative effects on reproductive indices in male rats and can cause serious damage and decrease the number and sperms motility. It can also cause infertility due to changing the concentration of hormones. Key words: Organophosphate, Pesticides, Phosalone, Sperm, Sex hormones

Generation of viable blastocysts from discarded human cleavage embryos

AbstractBackgroundWhile a relationship between embryo morphology, developmental ability, and genetic integrity exists, the selection of embryos with higher implantation potential remains a major challenge in assisted reproductive technology (ART). This study investigated blastocyst developmental competence and euploidy status in human embryos that had been classed as too poor quality to transfer (ET) or cryopreserve at the cleavage stage.Embryos were divided into three groups. Group 1 (n= 41) included good quality embryos from candidates of preimplantation genetic testing for aneuploidy (PGT-A). Groups II and III were the "rejected" supernumerary embryos, defined as suboptimal for ET or vitrification after morphological examination, with embryos randomly divided between the groups. Group II embryos (n= 31) were cultured up to the day 3 cleavage stage, when they were biopsied and fixed. Group III embryos (n= 27) were cultured up to the day 5 blastocyst stage, when they were evaluated for morphology and chromosomal status. Chromosomal status in all groups was assessed by multi-color fluorescence in situ hybridization (FISH) for chromosomes 13, 18, 21, X, and Y.ResultsEuploidy rates in groups I, II, and III were 56.1%, 38.7%, and 55.5 %, respectively. Among the blastocysts that developed from "rejected" embryos, 59.3% were classed as good quality. The most frequent chromosomal aneuploidy was related to the sex chromosome (22.2%). The mosaicism rate was not significantly different between the group II and III embryos (25.8% vs. 37.0%,p= 0.28).ConclusionIn conclusion, surplus poor-quality embryos rejected from clinical utilization at the cleavage stage may develop into viable blastocysts with normal chromosomal status for at least 5 chromosomes. Recovery of euploidy during poor-quality embryo transition from cleavage stage to blastocyst could provide an alternative choice for ET

No mitochondrial DNA deletions but more D-loop point mutations in repeated pregnancy loss

Abstract Purpose Repeated pregnancy loss (RPL) occurs in 1 out of 300 couples, and the cause of about 50% of them remains idiopathic. Mitochondria have an important role in human development through ATP production and their involvement in apoptosis. Methods 96 RPL and 96 control females were used to investigate the frequency of deletions and point mutations in the displacement loop (D-loop) on mitochondria. Multiplex PCR and DNA sequencing methods were used to detect possible variations in the mitochondrial DNA (mtDNA). Results No deletions but a high frequency of point mutations were found in RPL females; among 129 variations observed in RPL, 22 mutations were significant (P<0.05) and the insertion of C in nucleotide 114 was novel. Conclusion High rate of mutations in D-loop of mtDNA was observed in maternal blood, a fact that may have a direct or indirect role in inducing RPL. The results can be used in the assessment of RPL and designing possible treatments for improving assisted reproduction

Evaluation of gametogenic potential of vitrified human umbilical cord Wharton’s jellyederived mesenchymal cells

Background aims. Vitrification as an advanced cryopreservation method is recommended for cell storage toward future applications. The purpose of this report was to appraise whether gametogenic potential of these cells is altered by vitrification. Methods. A two steps method was applied for hUCM cells vitrification. An n-hUCM group of hUCM cells served as control. In order to differentiation of hUCM cells into male germ cells, the cells were induced by retinoic acid, testosterone and testicular-cell-conditioned medium. To evaluate induced hUCM cells toward germ cells, we used immunocytochemistry and karyotyping methods. Results. v-hUCM cells similar to n-hUCM cells formed flat cells after gametogenic induction, and showed protein expression of germ-cell-specific markers DAZL, VASA (DDX4) and SCP3. Karyotyping pattern remained unchanged in the either groups. Conclusions. The analysis of these results demonstrates that vitrification does not alter differentiation potential of hUCMs to male germ like cells. These results may set an in vitro pattern to study germ-cell formation from hUCM cells and also as a potential source of sperms for male infertility