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Sabour M, Agha-Rahimi A, Dehghani Ashkezari M, Seifati Sm, Kalantar Sm, Anbari F, Nabi A,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
Background: Polyvinylpyrrolidone (PVP) is a chemical used in intracytoplasmic sperm injection for sperm immobilization. In human sperm, PVP has been shown to damage sperm membranes, DNA integrity, mitochondrial membrane, and destroy axonal tubules and fibrous sheaths.
Objective: The aim of this study was to investigate the ideal time that sperm can be safely incubated in PVP with less possible damage.
Materials and Methods: Twenty-five normospermic samples were used. Sperm samples were prepared by swim-up method. Sperm samples incubated in 10% PVP at different time intervals (0, 15, 30, and 60 min). The effect of PVP was assessed on sperm structure, reactive oxyen species, acrosome reaction, Mitochondorial Membrane potential at different time intervals.
Results: Sperm parameters, DNA integrity and chromatin quality in 15, 30 and 60 min after incubation sperm with PVP were significantly changed compared to the 0 min. Moreover, in 30 and 60 min after incubation with PVP, above parameters were significantly changed compared to the 15 min. 60 min after incubation sperm with PVP, these parameters were significantly changed compared to the 30 min.
Conclusion: Sperm samples could be incubated with PVP for 15 min with less possible damage. While, prolonged incubation may damage the sperm parameters, DNA integrity and chromatin quality significantly.
Objective: The aim of this study was to investigate the ideal time that sperm can be safely incubated in PVP with less possible damage.
Materials and Methods: Twenty-five normospermic samples were used. Sperm samples were prepared by swim-up method. Sperm samples incubated in 10% PVP at different time intervals (0, 15, 30, and 60 min). The effect of PVP was assessed on sperm structure, reactive oxyen species, acrosome reaction, Mitochondorial Membrane potential at different time intervals.
Results: Sperm parameters, DNA integrity and chromatin quality in 15, 30 and 60 min after incubation sperm with PVP were significantly changed compared to the 0 min. Moreover, in 30 and 60 min after incubation with PVP, above parameters were significantly changed compared to the 15 min. 60 min after incubation sperm with PVP, these parameters were significantly changed compared to the 30 min.
Conclusion: Sperm samples could be incubated with PVP for 15 min with less possible damage. While, prolonged incubation may damage the sperm parameters, DNA integrity and chromatin quality significantly.
Nazari T, Ghasemi N, Ebrahimie E, Ebrahimi A, Talebi S, Tavakkoly Bazzaz J , Kalantar Sm,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
Background: Pregnancy loss is a significant health concern, especially in developing countries. Nearly 3-5% of couples trying to have children experience recurrent pregnancy loss (RPL). Unfortunately, advancing maternal age is highly associated with miscarriage while the available reproductive years are shortened, therefore determining why miscarriage occurs and how to prevent further miscarriages has become a major clinical and research focus. It has been estimated that the cause remains unexplained in more than 50% of cases, which strongly suggests that genetic factors may contribute towards the phenotype. This might be considered the fact that hundreds of genes and several critical pathways are involved in each physiological step necessary for guaranteeing reproductive success.
Objective: Mendelian forms of embryonic lethality offer a window into the essential genetic components of early embryonic development in humans. The study hypothesis was that exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL), which will further our understanding of human development at a molecular level.
Materials and Methods: This study involved 10 consanguineous couples having suffered at least 3 consecutive embryonic losses. Patients having risk factors such as abnormal karyotype, infectious disease during pregnancy, metabolic, autoimmune, endocrine disease, and uterine anomalies were excluded from the study. DNA was extracted from the tissue sample of ten aborted fetuses (probands) from ten different families with a history of idiopathic recurrent pregnancy loss. Parental peripheral blood samples were collected for confirmatory analysis and follow-up testing. Whole exome sequencing (WES) was performed using illumine HiSeq 2000 platform. Cytoscape 3.7.2 and BINGO were used for pathway and biological enrichment analysis of putatively pathogenic variants in miscarriages. All variants identified by exome sequencing were verified by Sanger sequencing in all parents.
Results: We were able to identify 32 variants (7 pathogenic, 9 likely pathogenic, and 16 VOUS) of which three genes are already known to be involved in lethal recessive disorders. These genes were compatible with the clinical phenotypes. In cases 1, 2,4 pathogenic variants were identified in know genes (CHRNG, RYR1) responsible for disorders with a clinical description that correlated with the phenotypic description. Of fetuses, in case 3,8,6 novel variants were identified in MYH3, ERBB3, FRAS1 responsible for muscular disorders and Fraser syndrome. Bioinformatics analysis of genes with mutation showed enrichment in biological processes of importance for embryonic development e.g. Fibrin clot formation complement, coagulation cascades, and Striated muscle contraction/muscle contraction, actin-myosin filament sliding. Variants with potential diagnostic value were reported to the patients referring physicians for genetic counseling and further diagnostic and reproductive action.
Conclusion: Next-generation sequencing (NGS) has been reported as being a useful tool for identifying variants in genes related to rare disorders leading to RPL. It has also helped to identify variants related to fetal molecular pathways in pregnancies having unexplained embryonic lethality or unexplained fetal malformations. This approach thus helps, genetic counseling regarding lethal fetal disorders of high-risk families and preimplantation genetic diagnosis. Future efforts should be directed towards increasing the number of sequencing families with RPL.
Objective: Mendelian forms of embryonic lethality offer a window into the essential genetic components of early embryonic development in humans. The study hypothesis was that exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL), which will further our understanding of human development at a molecular level.
Materials and Methods: This study involved 10 consanguineous couples having suffered at least 3 consecutive embryonic losses. Patients having risk factors such as abnormal karyotype, infectious disease during pregnancy, metabolic, autoimmune, endocrine disease, and uterine anomalies were excluded from the study. DNA was extracted from the tissue sample of ten aborted fetuses (probands) from ten different families with a history of idiopathic recurrent pregnancy loss. Parental peripheral blood samples were collected for confirmatory analysis and follow-up testing. Whole exome sequencing (WES) was performed using illumine HiSeq 2000 platform. Cytoscape 3.7.2 and BINGO were used for pathway and biological enrichment analysis of putatively pathogenic variants in miscarriages. All variants identified by exome sequencing were verified by Sanger sequencing in all parents.
Results: We were able to identify 32 variants (7 pathogenic, 9 likely pathogenic, and 16 VOUS) of which three genes are already known to be involved in lethal recessive disorders. These genes were compatible with the clinical phenotypes. In cases 1, 2,4 pathogenic variants were identified in know genes (CHRNG, RYR1) responsible for disorders with a clinical description that correlated with the phenotypic description. Of fetuses, in case 3,8,6 novel variants were identified in MYH3, ERBB3, FRAS1 responsible for muscular disorders and Fraser syndrome. Bioinformatics analysis of genes with mutation showed enrichment in biological processes of importance for embryonic development e.g. Fibrin clot formation complement, coagulation cascades, and Striated muscle contraction/muscle contraction, actin-myosin filament sliding. Variants with potential diagnostic value were reported to the patients referring physicians for genetic counseling and further diagnostic and reproductive action.
Conclusion: Next-generation sequencing (NGS) has been reported as being a useful tool for identifying variants in genes related to rare disorders leading to RPL. It has also helped to identify variants related to fetal molecular pathways in pregnancies having unexplained embryonic lethality or unexplained fetal malformations. This approach thus helps, genetic counseling regarding lethal fetal disorders of high-risk families and preimplantation genetic diagnosis. Future efforts should be directed towards increasing the number of sequencing families with RPL.
Kalantar Sm,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
About 15% of couples do not achieve pregnancy within one year and seek medical treatment for infertility. One in eight couples encounters problems when attempting to conceive a first child and one in six when attempting to conceive a subsequent child. Three percent of women remain involuntarily childless, while 6% of parous women are not able to have as many children as they would wish. Infertility affects both men and women. In 50% of involuntarily childless couples, a male-infertility-associated factor is found together with abnormal semen parameters. Male infertility is a common and severe health problem. Infertility not only affects one’s ability to have children, but also has emotional, psychological, family, and societal effects. The incidence may be increasing during the time of those affected, roughly 40% have idiopathic infertility. It is likely that the majority of those patients have genetic abnormalities that are the cause of their infertility. The understanding of the genes involved in spermatogenesis, sperm maturation, and normal sperm function is key, but we must also focus on better methods of accelerating advances into meaningful clinical diagnostic tests and therapies. During the past 30 years, significant improvements in technology have advanced the treatment of male infertility and Genetic evaluation as well. The primary advance has been intracytoplasmic sperm injection (ICSI) in conjunction with in vitro fertilization through ART cycles. Although this technological leap has allowed thousands of men to father a child who otherwise would have been unable to do so, the scientific study of the causes of male infertility has not kept pace. All urologists working in the field of Andrology must have an understanding of genetic abnormalities associated with infertility so that they can provide correct advice to couples seeking fertility treatment. Men with very low sperm counts can be offered a reasonable chance of paternity, using IVF, ICSI, and sperm harvesting from the testes in case of azoospermia
In fact, the clinical application of ICSI proceeded without sufficient scientific study of its safety to the offspring, or the future genetic ramifications several researchers and clinicians, and an international audience of experts in the field, reviewed the study of the genetics of male infertility, the tools available in the laboratory and clinic, the current state of knowledge, and the future of research and translation into clinical diagnostics and in this webinar the colleagues discussing the following aspects as:
The genetics of male infertility in the era of genomics
Important environmental factors on fertility potential
DNA damage how can affect the fertility potential of male
Methods and tools for the study of the genetics of male infertility
Clinical approach for evaluation of azoospermia men
Management of azoospermia due to microdeletions in y chromosome
Regeneration approach as new tools for now and near future to generate spermatogenesis.
In fact, the clinical application of ICSI proceeded without sufficient scientific study of its safety to the offspring, or the future genetic ramifications several researchers and clinicians, and an international audience of experts in the field, reviewed the study of the genetics of male infertility, the tools available in the laboratory and clinic, the current state of knowledge, and the future of research and translation into clinical diagnostics and in this webinar the colleagues discussing the following aspects as:
The genetics of male infertility in the era of genomics
Important environmental factors on fertility potential
DNA damage how can affect the fertility potential of male
Methods and tools for the study of the genetics of male infertility
Clinical approach for evaluation of azoospermia men
Management of azoospermia due to microdeletions in y chromosome
Regeneration approach as new tools for now and near future to generate spermatogenesis.
Hajimaqsoudi E, Darbeheshti F, Kalantar Sm, Javaheri A, Mirabutalebi Shr , Sheikhha Mh,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
Background: Endometriosis is generally considered as a benign condition, but there is a possibility for it to become cancerous. miR-125b was 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 endometrium (Eu-p) and ectopic endometrium (Ec-p) in endometrium tissues of patients with endometriosis was compared to these in the normal endometrium tissues of controls (Normal).
Materials and Methods: In this case-control study, the eutopic and ectopic samples were collected from 20 patients 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 PCR technique was used for measurement of miR125b and TP53 expression.
Results: Our results showed that miR-125b was significantly overexpressed in Ec-p. In addition, there was a significant TP53 underexpression in both Ec-p and Eu-p samples compared with normal tissues.
Conclusion: There was a negative correlation between miR-125b and TP53. In addition we observed a noticeable decreased expression of TP53 in both Ec-p and Eu-p samples. These findings may be interpreted as the roles of miR-125b/TP53 axis in the pathogenesis of endometriosis. With the help of bioinformatics analyses we conclude that there is a possible role of miR-125b in cancer-like features of endometriosis.
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 endometrium (Eu-p) and ectopic endometrium (Ec-p) in endometrium tissues of patients with endometriosis was compared to these in the normal endometrium tissues of controls (Normal).
Materials and Methods: In this case-control study, the eutopic and ectopic samples were collected from 20 patients 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 PCR technique was used for measurement of miR125b and TP53 expression.
Results: Our results showed that miR-125b was significantly overexpressed in Ec-p. In addition, there was a significant TP53 underexpression in both Ec-p and Eu-p samples compared with normal tissues.
Conclusion: There was a negative correlation between miR-125b and TP53. In addition we observed a noticeable decreased expression of TP53 in both Ec-p and Eu-p samples. These findings may be interpreted as the roles of miR-125b/TP53 axis in the pathogenesis of endometriosis. With the help of bioinformatics analyses we conclude that there is a possible role of miR-125b in cancer-like features of endometriosis.
Hojati S, Miresmaeili Sm, Montazeri F, Amiri S, Hoseini Sm, Kalantar Sm, Fesahat F,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
Background: Thin long tail deletion of Y chromosome is the most common molecular genetic cause of infertility. It is considered to be severe in men which occurs in the three region of the azoospermic factor; AZFa, AZFb and AZFc. These region contain multiple genes involved in spermatogenesis.
Objective: The aim of this study was to investigate the Y chromosome deletion pattern among infertile men with globozoospermic referring to Yazd Infertility Treatment Center.
Materials and Methods: 19 infertile men referred to Yazd Reproductive Science Institute with globozoospermia (from 2014 to 2016) were studied considering microdeletions in Y chromosome. Using multiplex PCR and six different STS (Sequence-Tagged Site) markers microdeletions of Y chromosome in AZFa, AZFb and AZFc regions was analysed.
Results: In our samples, the deletion of AZF regions of the Y chromosomes was not abserved in any blood sample of globozoospermic man.
Conclusion: In 19 samples, no defect was observed in the AZF regions of the Y chromosomes was not the cause of globozoospermia.
Objective: The aim of this study was to investigate the Y chromosome deletion pattern among infertile men with globozoospermic referring to Yazd Infertility Treatment Center.
Materials and Methods: 19 infertile men referred to Yazd Reproductive Science Institute with globozoospermia (from 2014 to 2016) were studied considering microdeletions in Y chromosome. Using multiplex PCR and six different STS (Sequence-Tagged Site) markers microdeletions of Y chromosome in AZFa, AZFb and AZFc regions was analysed.
Results: In our samples, the deletion of AZF regions of the Y chromosomes was not abserved in any blood sample of globozoospermic man.
Conclusion: In 19 samples, no defect was observed in the AZF regions of the Y chromosomes was not the cause of globozoospermia.
Yarahmadi G, Vahidi Mehrjardi My, Kalantar Sm,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
Background: Endometriosis, a relatively prevalent gynecologic disorder, affecting 6 to 10 percent of women in reproductive ages around the globe. Primary recognition can help to decrease its progression and morbidity. Many studies demonstrated that microRNA has a vital role in the pathogenesis of endometriosis. miR-1271 and its direct target gene, GRB2, expression have been studied in gynecologic cancers and found to be involved in cell proliferation, migration, and metastasis, while their role in endometriosis has not been studied.
Objective: In this study, we measured miR-1271 and GRB2 genes expression in the endometrial tissues of patients (eutopic and ectopic tissues) compared to the control samples.
Materials and Methods: In our study, the endometriosis tissue samples of 15 patients with endometriosis and 15 women without endometriosis were collected. We used quantitative polymerase chain reaction to check the level of miR-1271 and GRB2 genes expression in these samples.
Results: We observed a significant decrease in miR-1271 expression level in both ectopic and eutopic samples of patients with endometriosis compared with control samples, while there was a noticeable increase in the expression level of its target gene, GRB2, in tissues of endometriosis patients compared with normal control samples.
Conclusion: We discovered an inverse relationship between the reduction of miR-1271 expression level and increase in the expression level of GRB2. Therefore, increased GRB2 expression in endometriosis tissues can be due to decreased expression of this microRNA. Our findings suggested that miR-1271 maybe play the role as a biomarker in the diagnosis of patients with endometriosis.
Objective: In this study, we measured miR-1271 and GRB2 genes expression in the endometrial tissues of patients (eutopic and ectopic tissues) compared to the control samples.
Materials and Methods: In our study, the endometriosis tissue samples of 15 patients with endometriosis and 15 women without endometriosis were collected. We used quantitative polymerase chain reaction to check the level of miR-1271 and GRB2 genes expression in these samples.
Results: We observed a significant decrease in miR-1271 expression level in both ectopic and eutopic samples of patients with endometriosis compared with control samples, while there was a noticeable increase in the expression level of its target gene, GRB2, in tissues of endometriosis patients compared with normal control samples.
Conclusion: We discovered an inverse relationship between the reduction of miR-1271 expression level and increase in the expression level of GRB2. Therefore, increased GRB2 expression in endometriosis tissues can be due to decreased expression of this microRNA. Our findings suggested that miR-1271 maybe play the role as a biomarker in the diagnosis of patients with endometriosis.
Dehghanian M, Vahidi Mehrjardi My, Kalantar Sm, Dehghani Mr,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
Background: Endometriosis, a common and multifactorial disease in women, has different symptoms such as pelvic pain and infertility. Recent studies demonstrated that genetic factors have an important role in its pathogenesis so that dysregulation of many genes and microRNAs have been reported in this disease. Based on previous studies, we know that decreased expression level of miR-337-3p in ovarian and cervical cancers can lead to increase its target genes like RAP1A, which plays role in the pathogenesis of these diseases. miR-337-3p expression also downregulated in serum samples of endometriosis patients. However, the role of miR-337-3p and its direct target gene RAP1A in endometriosis tissues have not been investigated.
Objective: The goal of this study was to compare the expression level of miR-337-3p and its direct target gene, RAP1A, in endometriosis tissues and control samples to find their relationship with pathogenesis of endometriosis.
Materials and Methods: We measured miR-337-3p and RAP1A expression levels by quantitative polymerase chain reaction (qRT-PCR) in 15 ectopic and eutopic tissue samples from patients with endometriosis and 15 normal endometrium tissue samples from women without endometriosis.
Results: The results showed a significant increase in the expression level of RAP1A gene in the endometriosis tissue samples (both of ectopic and eutopic tissues), while miR-337-3p expression level decreased significantly in these tissue samples compared with the normal endometrium samples.
Conclusion: In this study, we found an opposite relationship between miR-337-3p and RAP1A gene expression in endometriosis so that decrease in miR-337-3p expression can lead to increase in RAP1A gene expression in endometriosis tissues. Changes in the expression of these genes in our study can also interpret as the role of them in the pathogenesis and progression of endometriosis.
Objective: The goal of this study was to compare the expression level of miR-337-3p and its direct target gene, RAP1A, in endometriosis tissues and control samples to find their relationship with pathogenesis of endometriosis.
Materials and Methods: We measured miR-337-3p and RAP1A expression levels by quantitative polymerase chain reaction (qRT-PCR) in 15 ectopic and eutopic tissue samples from patients with endometriosis and 15 normal endometrium tissue samples from women without endometriosis.
Results: The results showed a significant increase in the expression level of RAP1A gene in the endometriosis tissue samples (both of ectopic and eutopic tissues), while miR-337-3p expression level decreased significantly in these tissue samples compared with the normal endometrium samples.
Conclusion: In this study, we found an opposite relationship between miR-337-3p and RAP1A gene expression in endometriosis so that decrease in miR-337-3p expression can lead to increase in RAP1A gene expression in endometriosis tissues. Changes in the expression of these genes in our study can also interpret as the role of them in the pathogenesis and progression of endometriosis.
Hossini Sm, Kalantar Sm, Aflatoonian B, Bahrami A, Montazeri F, Moghaddam Matin M,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
Background: The amniotic fluid contains a heterogeneous population of different cells that are produced prior to the gastrulation process. Therefore, it is expected that mesenchymal stem cells derived from the amniotic fluid will have high plasticity between mature and pluripotent stem cells. Due to unique features of these cells such as high cloning potential, high self-renewal capacity along with chromosomal stability and low immunogenicity as well as anti-inflammatory and immune-modulating properties, it has attracted more and more attention from researchers.
Objective: The aim of this study was to investigate the immunosuppressive genes in mesenchymal stem cells isolated from amniotic fluid of women with a history of recurrent pregnancy loss (RPL) and the effect of gamma interferon as an immunological stimulus on the expression of these genes.
Materials and Methods: The study group included pregnant women with a history of unexplained RPL. The control group consisted of pregnant women with at least one healthy child, no history of miscarriage, and normal hormonal and immunologic profiles. In this study, mesenchymal stem cells (MSCs) isolated from amniotic fluid from RPL and non-RPL women. On the other hand, each cell line was examined under 5 different treatment groups, control and 4 groups with 20 and 100 IU IFN-γ per ml of medium over two periods of 24 h and 72 h. Finally, the relative mRNA expression level of immune-suppressive/modulator gene including two indole amine-2 and 3-dioxygenase 1 and 2 in AF-MSCs in both groups were evaluated and compared using Q-PCR.
Results: The average expression of candidate gene IDO1 and IDO2 showed a significant increase in the RPL group rather than non-RPL, specially under treatment with 100 IU IFN-γ and after 24 h. Interestingly, expression of both genes IDO1 and IDO2 decrease after 72 h in RPL and non-RPL groups (p = 0.05).
Conclusion: Immunosuppression by MSCs, which is currently recognized as a powerful tool in preventing acute rejection, graft therapy, and regenerative medicine, is not an inherent potential but is induced by environmental factors. Various studies have identified that some potential causes of unexplained RPL are due to immunological factors. The results of this study, especially for indole amine-2 and 3-dioxygenase genes, do not rule out such a possibility. Despite the unknown role of AF-MSCs in the abortion mechanism, the results of this study suggest that there is a significant difference between the mRNA level of understudied genes between AF-MSCs in the RPL and non-RPL group. Due to the absence of such a similar study, it cannot be fully interpreted, however, these cells appear to represent genetic compartments of couples with a history of RPL that may defective in immunological factors. However, planning for further investigation of these uncertain immunological mechanisms appears to be valuable in the future.
Objective: The aim of this study was to investigate the immunosuppressive genes in mesenchymal stem cells isolated from amniotic fluid of women with a history of recurrent pregnancy loss (RPL) and the effect of gamma interferon as an immunological stimulus on the expression of these genes.
Materials and Methods: The study group included pregnant women with a history of unexplained RPL. The control group consisted of pregnant women with at least one healthy child, no history of miscarriage, and normal hormonal and immunologic profiles. In this study, mesenchymal stem cells (MSCs) isolated from amniotic fluid from RPL and non-RPL women. On the other hand, each cell line was examined under 5 different treatment groups, control and 4 groups with 20 and 100 IU IFN-γ per ml of medium over two periods of 24 h and 72 h. Finally, the relative mRNA expression level of immune-suppressive/modulator gene including two indole amine-2 and 3-dioxygenase 1 and 2 in AF-MSCs in both groups were evaluated and compared using Q-PCR.
Results: The average expression of candidate gene IDO1 and IDO2 showed a significant increase in the RPL group rather than non-RPL, specially under treatment with 100 IU IFN-γ and after 24 h. Interestingly, expression of both genes IDO1 and IDO2 decrease after 72 h in RPL and non-RPL groups (p = 0.05).
Conclusion: Immunosuppression by MSCs, which is currently recognized as a powerful tool in preventing acute rejection, graft therapy, and regenerative medicine, is not an inherent potential but is induced by environmental factors. Various studies have identified that some potential causes of unexplained RPL are due to immunological factors. The results of this study, especially for indole amine-2 and 3-dioxygenase genes, do not rule out such a possibility. Despite the unknown role of AF-MSCs in the abortion mechanism, the results of this study suggest that there is a significant difference between the mRNA level of understudied genes between AF-MSCs in the RPL and non-RPL group. Due to the absence of such a similar study, it cannot be fully interpreted, however, these cells appear to represent genetic compartments of couples with a history of RPL that may defective in immunological factors. However, planning for further investigation of these uncertain immunological mechanisms appears to be valuable in the future.
Jahanara M, Montazeri F, Sharifiyazdi H, Kalantar Sm, Hoseini Sm, Moshrefi M,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
Background: Chromosomal abnormalities are one of the most important causes of failure in in vitro fertilization. Preimplantation genetic testing can be a way to prevent the transfer of aneuploid embryos. It entails the use of invasive techniques to obtain embryonic DNA, with major technical limitations and ethical issues today. Therefore, the use of new non-invasive methods is a suitable solution to this problem. One of the non-invasive methods is to use the embryo spent culture medium. The origin of cell free DNA in embryo spent culture medium is trophectoderm cells and the internal cell mass.
Objective: Cell-free genomic DNA in the embryonic culture medium can be a non-invasive method for genetic assessment.
Materials and Methods: This study reviewed 25 spent embryo culture mediums. The spent culture medium used between day 3 and day 5 of embryonic development. Patients were undergoing intracytoplasmic sperm injection, and each embryo was in one drop of culture medium. We had two control samples: the culture medium contaminated with purified DNA from human blood and the culture medium without embryonic development. All samples were evaluated with nanodrop for dsDNA and ssDNA concentration. Among the collected medium, ten samples (group 1) concentrated by heating, then evaluating SRY and FMR1 genes with real-time polymerase chain reaction (RT-PCR) (group 1). Six samples were three days, and four samples were five days. The rest of the samples were classified into three groups. The cell-free DNA from the medium was purified with the blood DNA extraction kit. in group 2 with Genet bio kit, group 3 with YTzol pure DNA kit (yekta Tajhiz), and group 4 with High Pure Viral Nucleic Acid extraction kit (Roche). They evaluated by RT-PCR. Nine samples were three days, and six samples were five days.
Results: Although cell-free DNA was confirmed in the samples using nanodrop (with a range of 160 to 225 ng per microliter), the cycle of threshold did not observe in the RT-PCR product of group 1. The Purified samples were amplified in group 2,3 and 4 for SRY and FMR1 genes with RT-PCR and observed only acceptable cycle of threshold in the fourth group.
Conclusion: The high protein and solutes in the culture medium and the low amount and quality of DNA are restrictive. For better results, it is necessary to purify the genomic DNA and amplify it with precise kits. Our research is underway to improve DNA collection, amplification, and testing to isolate genomic DNA.
Objective: Cell-free genomic DNA in the embryonic culture medium can be a non-invasive method for genetic assessment.
Materials and Methods: This study reviewed 25 spent embryo culture mediums. The spent culture medium used between day 3 and day 5 of embryonic development. Patients were undergoing intracytoplasmic sperm injection, and each embryo was in one drop of culture medium. We had two control samples: the culture medium contaminated with purified DNA from human blood and the culture medium without embryonic development. All samples were evaluated with nanodrop for dsDNA and ssDNA concentration. Among the collected medium, ten samples (group 1) concentrated by heating, then evaluating SRY and FMR1 genes with real-time polymerase chain reaction (RT-PCR) (group 1). Six samples were three days, and four samples were five days. The rest of the samples were classified into three groups. The cell-free DNA from the medium was purified with the blood DNA extraction kit. in group 2 with Genet bio kit, group 3 with YTzol pure DNA kit (yekta Tajhiz), and group 4 with High Pure Viral Nucleic Acid extraction kit (Roche). They evaluated by RT-PCR. Nine samples were three days, and six samples were five days.
Results: Although cell-free DNA was confirmed in the samples using nanodrop (with a range of 160 to 225 ng per microliter), the cycle of threshold did not observe in the RT-PCR product of group 1. The Purified samples were amplified in group 2,3 and 4 for SRY and FMR1 genes with RT-PCR and observed only acceptable cycle of threshold in the fourth group.
Conclusion: The high protein and solutes in the culture medium and the low amount and quality of DNA are restrictive. For better results, it is necessary to purify the genomic DNA and amplify it with precise kits. Our research is underway to improve DNA collection, amplification, and testing to isolate genomic DNA.
Taheri F, Khalili Ma, Aflatoonian A, Kalantar Sm, Kariminejad R, Moshrefi M,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
Background: An association between morphology, and genetic integrity in human embryos is stablished, but this relationship is not absolute.
Objective: This study investigated the correlation between embryo morphological characteristics and chromosomal status in biopsied human embryos, using the array comparative genomic hybridization technique.
Materials and Methods: Preimplantation genetic testing for aneuploidy was performed on Day 3 embryos (n = 120) divided into two groups: 60 ‘deselected’ embryos (unsuitable for transfer or vitrification) versus 60 ‘selected’ embryos (suitable for transfer or vitrification). The morphological grading criteria, including blastomere number, symmetry, percentage of fragmentation rate, and zona pellucida appearance were correlated with array comparative genomic hybridization results.
Results: The incidence of chromosomal abnormalities was significantly higher in embryos with uneven blastomeres, fragmentations, and thick zona pellucida appearance.
Conclusion: In general, embryo selection based of morphological assessment cannot confirm the chromosomal integrity. However, some morphological parameters reflect the cytogenetic status of the deselected embryos.
Objective: This study investigated the correlation between embryo morphological characteristics and chromosomal status in biopsied human embryos, using the array comparative genomic hybridization technique.
Materials and Methods: Preimplantation genetic testing for aneuploidy was performed on Day 3 embryos (n = 120) divided into two groups: 60 ‘deselected’ embryos (unsuitable for transfer or vitrification) versus 60 ‘selected’ embryos (suitable for transfer or vitrification). The morphological grading criteria, including blastomere number, symmetry, percentage of fragmentation rate, and zona pellucida appearance were correlated with array comparative genomic hybridization results.
Results: The incidence of chromosomal abnormalities was significantly higher in embryos with uneven blastomeres, fragmentations, and thick zona pellucida appearance.
Conclusion: In general, embryo selection based of morphological assessment cannot confirm the chromosomal integrity. However, some morphological parameters reflect the cytogenetic status of the deselected embryos.
Jahanara M, Montazeri F, Moshrefi M, Sharifiyazdi H, Kalantar Sm,
Volume 19, Issue 5 (Suppl- 2021)
Volume 19, Issue 5 (Suppl- 2021)
Abstract
Background: It is essential to identify quality and healthy embryos for transfer in in vitro fertilisation successful. Research has shown that traditional morphological methods are not sufficient for diagnosis. The best embryo can select the best embryo with preimplantation genetic testing. With PNG be tested the severe inherited conditions or for chromosome abnormalities. It needs to biopsy from the embryo for genetic testing, includes separating one or more cells from the embryo, or collecting blastocoel fluid and embryo culture medium. Because of the risks of cell biopsy from embryos, it is necessary to find non-invasive methods for PNG. The research has shown, we can use genomic DNA in blastocoel fluid and embryo culture medium. It may be open a new way for selecting the best embryo.
Objective: This review tries to evaluate the results of research about non-invasive pre-implantation genetic testing. The most popular method is detecting and analyzing cell-free DNA in embryo culture medium for genetic testing. Of course, there is a long way to go to use this method for PNG. This article tries to examine its technical and biological problems.
Materials and Methods: Original research and review papers about non-invasive pre-implantation genetic testing were sourced by searching PubMed and Google Scholar databases until February 2021. The search included keywords: ‘spent culture media’; ‘cell-free DNA’; ‘non-invasive pre-implantation genetic testing’; ‘blastocentesis’; ‘blastocoel fluid’ and ‘pre-implantation genetic screening’.
Results: Available data suggested that blastocoel fluid and embryo spent culture medium, samples provide DNA suitable for genetic analysis and are a potential tool for preimplantation genetic testing. Embryonic DNA could be detected in the embryo spent culture media and blastocoel fluid. Primery studies have been successful in molecularly examining cell-free DNA, but the amount and quality of available DNA has varied. Reports of similarity in the results of free DNA genetic testing and embryo biopsy have been reported differently.
Conclusion: The reports have shown that results of trophectoderm biopsies may be different in cytogenetic data. It is likely for embryonic mosaicism or DNA contamination. It was said that aneuploid embryonic cells are removed from the embryo. Therefore, DNA distributes in the spent culture medium and blastocoel fluid. Of course, this point needs to check out more. The hard part is isolating and amplifying DNA to make an accurate clinical diagnosis. Some factors are distorting, and there are contaminants in this work, but generally, it is important and necessary that we have a non-invasive pre-implantation genetic testing.
Objective: This review tries to evaluate the results of research about non-invasive pre-implantation genetic testing. The most popular method is detecting and analyzing cell-free DNA in embryo culture medium for genetic testing. Of course, there is a long way to go to use this method for PNG. This article tries to examine its technical and biological problems.
Materials and Methods: Original research and review papers about non-invasive pre-implantation genetic testing were sourced by searching PubMed and Google Scholar databases until February 2021. The search included keywords: ‘spent culture media’; ‘cell-free DNA’; ‘non-invasive pre-implantation genetic testing’; ‘blastocentesis’; ‘blastocoel fluid’ and ‘pre-implantation genetic screening’.
Results: Available data suggested that blastocoel fluid and embryo spent culture medium, samples provide DNA suitable for genetic analysis and are a potential tool for preimplantation genetic testing. Embryonic DNA could be detected in the embryo spent culture media and blastocoel fluid. Primery studies have been successful in molecularly examining cell-free DNA, but the amount and quality of available DNA has varied. Reports of similarity in the results of free DNA genetic testing and embryo biopsy have been reported differently.
Conclusion: The reports have shown that results of trophectoderm biopsies may be different in cytogenetic data. It is likely for embryonic mosaicism or DNA contamination. It was said that aneuploid embryonic cells are removed from the embryo. Therefore, DNA distributes in the spent culture medium and blastocoel fluid. Of course, this point needs to check out more. The hard part is isolating and amplifying DNA to make an accurate clinical diagnosis. Some factors are distorting, and there are contaminants in this work, but generally, it is important and necessary that we have a non-invasive pre-implantation genetic testing.
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