Expression of genes in the AKT signalling pathway in human oocytes from patients with polycystic ovaries.

Polycystic ovary syndrome is an endocrine disorder commonly found among females of reproductive age. Different factors have been correlated with this syndrome, although the aetiology of the disease is still unrecognized with both environmental and hereditary factors leading to the progression. Hormonal effects of the AKT pathway have made it an interesting study unit for PCOS cases. The aim of this study was to investigate the expression patterns of genes involved in the AKT pathway, including IRS1, IRS2, AKT1 and AKT2. In total, 13 human oocytes were collected for this study at the meiosis II stage, in which seven of them were collected from individuals with polycystic ovaries and the rest formed the control group of individuals with no signs of polycystic ovaries. RNA was extracted from oocytes and then the RNA was converted into cDNA for the real-time PCR process. Expression levels of four genes in the AKT pathway, in addition to housekeeping gene (ACTB), were evaluated. Expression levels of each gene were quantified using real-time PCR and statistical analysis was performed. The results of this study showed that there was no significant correlation between the expression of genes in oocyte samples obtained from patients with polycystic ovaries and the control group. This study is the first to evaluate the expression levels of genes involved in the AKT pathway in human oocyte samples. Therefore, it provides crucial information to form the basis of further studies.

Does Pten have an impact on oogenesis of PCOS mouse models?

Polycystic ovary syndrome (PCOS) is a complex disorder in which the aetiology is still not explained very well. The PI3K/PTEN (phosphatidylinositol 3-kinase/phosphatase and tensin homolog deleted on chromosome 10) pathway is an important pathway that is involved in many mechanisms, including proliferation, growth and motility. PTEN plays a role in granulosa cell proliferation and regulates the differentiation process. The aim of this study was to investigate the expression levels of Pten and Pik3ca in PCOS mouse models with and without any treatment procedures. Three groups of mouse models, PCOS, a PCOS group with clomiphene citrate treatment, and a PCOS group with the combination of clomiphene citrate, metformin and pioglitazone treatment, were established. Ovarian tissues, which were obtained from these groups and a control group with no PCOS, were embedded in paraffin and RNA was extracted. cDNA was synthesized and real-time PCR was conducted to evaluate the expression levels of Pten and Pik3ca. The results of this study showed that both Pten and Pik3ca genes were expressed in the ovarian tissues from the mouse models. Although one-way analysis of variance results showed that Pten was expressed significantly differently in the samples, individual Student's t-tests did not show any significantly different expression levels in each group. This study is important as it shows the expression patterns of two genes in PCOS mouse models with different treatment strategies, including clomiphene citrate, metformin and pioglitazone. The results of this study formed the basis of research studies and investigations into different genes within the PTEN pathway, as well as other pathways that are under investigation.

Investigation of BAK, BAX and MAD2L1 gene expression in human aneuploid blastocysts.

Maintaining genomic stability is crucial for normal development. At earlier stages of preimplantation development, as the embryonic genome activation is not fully completed, the embryos may be more prone to abnormalities. Aneuploidies are one of the most common genetic causes of implantation failure or first-trimester miscarriages. Apoptosis is a crucial mechanism to eliminate damaged or abnormal cells from the organism to enable healthy growth. Therefore, this study aimed to determine the relationship between the expression levels of genes involved in apoptosis in human aneuploid and euploid blastocysts. In total, 32 human embryos obtained from 21 patients were used for this study. Trophectoderm biopsies were performed and next-generation screening was carried out for aneuploidy screening. Total RNA was extracted from each blastocyst separately and cDNA was synthesized. Gene expression levels were evaluated using RT-PCR. The statistical analysis was performed to evaluate the gene expression level variations in the euploid and aneuploid embryos, respectively. The expression level of the BAX gene was significantly different between the aneuploid and euploid samples. BAX expression levels were found to be 1.5-fold lower in aneuploid cells. However, the expression levels of BAK and MAD2L1 genes were similar in each group. This study aimed to investigate the possible role of genes involved in apoptosis and aneuploidy mechanisms. The findings of this investigation revealed that the BAX gene was expressed significantly differently between aneuploid and euploid embryos. Therefore, it is possible that the genes involved in the apoptotic pathway have a role in the aneuploidy mechanism.

Molecular regulation of polycystic ovary syndrome: altered gene expression levels in mouse models pretreatment and post-treatment.

Polycystic ovary syndrome (PCOS) is a complex disorder and genetic factors are believed to play a role. The main aim was to investigate expression levels of genes involved in PI3K/AKT signalling pathway pretreatment and post-treatment. Mouse models of PCOS were generated. Group one included control mice with no polycystic ovaries (n = 4), Group 2 included a PCOS mouse model (n = 8), Group 3 included PCOS mice treated with clomiphene citrate (n = 7) and Group 4 included PCOS mice treated with clomiphene citrate, metformin and pioglitazone (n = 8). Histochemical analyses were performed. Total RNA was extracted and cDNA was synthesized. Irs, Akt1 and Akt2, mTor and Pdpk1 gene expression levels were evaluated by RT-PCR amplification. In Group 1, cortex and medulla were evaluated as normal; in Group 2, ovarian cortex was composed of immature oocytes and cystic follicles with atretic follicles. In Groups 3 and 4, follicles were in the process of normal follicle differentiation. The expression levels of Akt1 and Pi3k were significantly different (P < 0.0001) between Groups 1 and 2. The significant differences in expression levels of Pi3k and Akt1 were also observed between the Group 1 and both Groups 3 and 4 (P < 0.0001). Furthermore, significant variations of the expression levels of mTor between Groups 1 and 4 were observed. The extrapolation of results of this study may imply that follicular development may be regulated by molecular pathways involving Pi3k, Akt1 and mTor expression. Therefore, genes in the PI3K/AKT pathway may have a direct regulatory role in the development of PCOS.

The effect of repeated controlled ovarian stimulation cycles on the gamete and embryo development.

The aim of this study was to investigate if there is an adverse effect of multiple controlled ovarian stimulation (COS) on the maturity of oocytes (MI and MII), fertilization rate, embryo developmental qualities and clinical pregnancy rates in donation cycles. In total, 65 patients undergoing oocyte donation cycles multiple times were included in this study. Patients were grouped as group A that consisted of donors with ≤2 stimulation cycles while B consisted of donors with ≥3 stimulation cycles; and group C included donors who had ≤15 oocytes, while group D had donors with ≥16 oocytes. Numbers of oocytes obtained, MI and MII oocytes, fertilization, embryo quality and clinical pregnancy outcomes were compared. Significant statistical differences were observed in total number of oocytes obtained, maturity of oocytes (MI and MII), fertilization rate, embryo qualities and clinical pregnancy outcomes of donors in groups A-D. Donors with ≤2 ovarian stimulation cycles had lower numbers of immature oocytes than donors with three or more stimulation cycles. However, donors with ≥3 stimulation cycles had higher numbers of mature oocytes, zygotes, with better day 3 embryo qualities and higher clinical pregnancy rates than donors with ≤2 stimulation cycles. Repeated COS does not seem to have any adverse effect on ovarian response to higher dose of artificial gonadotropin, as quality of oocytes collected and their embryological developmental potential were not affected by the number of successive stimulation cycles. The effect of multiple COS on the health of the oocyte donor needs to be assessed for future purpose.

Assessment of successful pregnancy using granular oocytes in ICSI treatments.

SummaryDifferent parameters affect the success of assisted reproduction technology (ART) treatments. One of the advantages of using intracytoplasmic sperm injection (ICSI) is that it also enables the assessment of the oocyte morphology. To date there has not been a clear conclusion on aberrant oocyte morphology and its consequences on the success of ART treatments. Therefore, in this study, we aimed to investigate the fertilization, embryo development and pregnancy rates in patients who have oocytes with granular cytoplasm. Additionally, we investigated if there were more aneuploid embryos obtained from abnormal cytoplasmic morphology. In total, 5704 oocytes were collected and, of these, 4036 were metaphase II (MII) oocytes. The morphology of these oocytes was assessed following denudation and 970 oocytes were observed to have granular cytoplasm. There was no difference in the fertilization rates between the oocytes with normal cytoplasm (89%) and oocytes with granular cytoplasm (72%). Cleavage of embryos and the number of embryos that reached the blastocyst stage were also similar in these two groups. The aneuploidy rates between the two groups were also similar. However, clinical pregnancies were significantly lower in embryos obtained from oocytes with granular cytoplasm (37.5% vs 70%, P<0.05). Therefore, the morphology of the oocyte is as important as morphology of the sperm. Even though normal fertilization and cleavage were achieved from oocytes with granular cytoplasm, their implantation potential was significantly compromised.

Number of embryos biopsied as a predictive indicator for the outcome of preimplantation genetic diagnosis by fluorescence in situ hybridisation in translocation cases.

This study aimed to investigate the optimum number of embryos to be biopsied in order to increase the likelihood of obtaining a balanced/normal embryo following preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridisation (FISH) for translocation carriers. Patients with low number of fertilised oocytes (≤5) or low number of embryos available for PGD (<7) underwent multiple hormonal stimulation cycles and their embryos from each cycle were vitrified and accumulated to obtain at least three embryos for PGD. Fifty-seven PGD cycles were performed for translocation carriers by FISH on day 3 of embryo development. PGD and pregnancy outcomes were examined according to the number of embryos biopsied. The cancellation rates of embryo transfer for the reciprocal translocation carriers were 40% when more than eight embryos were biopsied and it was as high as 78% when low number of embryos (less than nine) were biopsied. For Robertsonian translocation carriers, when more than eight embryos were biopsied, there were no embryo transfer cancellations. This study showed that when there are more than nine embryos biopsied for PGD, the likelihood of obtaining a balanced embryo and positive pregnancy outcome is significantly higher (P < 0.05) in such the overall pregnancy rate was 63% for reciprocal and 86% for Robertsonian carriers. This was reduced to only 7% for reciprocal and 14% for Robertsonian translocation carriers when less than nine embryos were biopsied. One of the limitations of this study was that the analysis was performed by FISH and more studies should investigate the outcomes of embryo accumulation following comprehensive chromosome analysis.

Poor embryo development and preimplantation genetic diagnosis outcomes of translocations involving chromosome 10: Do we blame genetics?

Balanced reciprocal translocation carriers are usually phenotypically normal. Although the reproductive risk of these carriers varies, they generally have a lower chance to produce normal or balanced gametes. Preimplantation genetic diagnosis (PGD) is offered to these patients to increase their chances of becoming pregnant by selecting a balanced embryo for transfer. This study aimed to analyse the development and the PGD outcome of the embryos obtained from reciprocal translocation carriers focusing on ones with chromosome 10 rearrangements. In total, 27 reciprocal translocation carriers underwent 31 cycles of PGD. PGD was performed using multicolour fluorescence in situ hybridisation for 298 embryos and of these 136 were obtained from couples carrying translocations involving chromosome 10 rearrangements. Carriers of translocations involving chromosome 10 rearrangements have a lower chance of producing normal or balanced embryos compared with the carriers with other rearrangements. The development of embryos obtained from the patients with chromosome 10 rearrangements was impaired and only a limited number of embryos developed to the blastocyst stage.

Investigation of microRNA expression and DNA repair gene transcripts in human oocytes and blastocysts.

PURPOSE: The aim of the study is to investigate the regulation of DNA repair genes by microRNAs (miRNAs). miRNAs are short non-coding RNAs that regulate transcriptional and post-transcriptional gene silencing. Several miRNAs that are expressed during preimplantation embryo development have been shown or are predicted to target genes that regulate cell cycle checkpoints and DNA repair in response to DNA damage. METHODS: This study compares the expression level of 20 miRNAs and 9 target transcripts involved in DNA repair. The statistical significance of differential miRNA expression between oocytes and blastocysts was determined by t test analysis using the GraphPad Prism v6 software. The possible regulatory roles of miRNAs on their target messenger RNAs (mRNAs) were analysed using a Pearson correlation test. RESULTS: This study shows for the first time that several miRNAs are expressed in human oocytes and blastocysts that target key genes involved in DNA repair and cell cycle checkpoints. Blastocysts exhibited statistically significant lower expression levels for the majority of miRNAs compared to oocytes (p < 0.05). Correlation analyses showed that there was both inverse and direct association between miRNAs and their target mRNAs. CONCLUSIONS: miRNAs target many mRNAs including ones involved in DNA repair mechanisms. This study suggests that miRNAs and their target mRNAs involved in DNA repair are expressed in preimplantation embryos. Similar to the miRNAs expressed in adult tissues, these miRNAs seem to have regulatory roles on their target DNA repair mRNAs during preimplantation embryo development.

The potential for carbon dioxide removal by enhanced rock weathering in the tropics: An evaluation of Costa Rica.

Tropical environments show great potential to sequester CO2 by enhanced rock weathering (ERW) of powdered mafic rocks applied to agricultural fields. This study seeks to assess carbon dioxide reduction (CDR) potential in the humid tropics (1) by experimental weathering of mafic rock powders in conditions simulating humid tropical soils, and (2) from weathering rates determined from a Holocene tropical soil chronosequence where parent material is andesitic sediments. Experimentally determined weathering rates by leaching of basaltic andesites from Costa Rica (Arenal and Barva) for 50 t ha-1 applications indicate potential sequestration of 2.4 to 4.5 t CO2 ha-1 yr-1, whereas the USGS basalt standard BHVO-1 yields a rate of 11.9 t ha-1 yr-1 (influenced by more mafic composition and finer particle size). The chronosequence indicates a rate of 1.7 t CO2 ha-1 yr-1. The weathering experiment consisted of 0.6 mm of powdered rock applied atop 12 mm of Ultisol at 35 °C. To simulate a tropical soil solution, 100-mL aliquots of a dilute solution of oxalic acid in carbonated DI water were rained onto soils over a 14-day period to simulate soil moisture in the humid tropics. Solutions were collected and analyzed by ICPMS for concentrations of leached cations. A potential ERW scenario for Costa Rica was assessed assuming that one-half of lowland agricultural kaolinitic soils (mainly Ultisols, common crop and pasture soils, excluding protected areas) were to receive 50 t ha-1 of annual or biennial applications of powdered mafic rock. With an experimentally determined humid tropical CDR rate for basaltic andesite (3.5 t ha-1 yr-1) and allowances for carbon costs (e.g. emissions from processing and delivery) that reduce CDR to a net 3.2 t ha-1 yr-1, potential annual CDR of this tropical nation is ∼2-4 million tons, amounting to ∼25-50 % of annual CO2 emissions (mainly from transportation in Costa Rica).

Differential expression of genes involved in steroidogenesis pathway in human oocytes obtained from patients with polycystic ovaries.

OBJECTIVE: Follicular development can be disturbed due to many factors, including having polycystic ovaries. Aberrant expression of genes involved in steroidogenesis pathway could lead to aberrant oocyte development. In this study, the gene expression levels of a number of genes that is functioning in steroidogenesis pathway were investigated. MATERIALS AND METHODS: The spare oocytes were collected from NEU Hospital IVF Center following controlled ovarian stimulation cycle. RNA was extracted using RNA/DNA Purification Kit (Norgen, Canada) and reverse transcription was performed using TruScript First Strand cDNA Synthesis Kit (Norgen, Canada). Real time PCR was conducted using LightCycler® 480 SYBR Green I Master (Roche, UK). RESULTS AND CONCLUSION: The expression levels of CYP11, CYP17, CYP19, HSD17B1, HSD3B2 and ACTB were detected in human MII stage oocytes obtained from oocyte donors aged between 18-30 years. The number of follicles and oocytes collected from the patients with polycystic ovaries were slightly higher compared to the control group. The expression level of CYP11A1 was shown to be statistically different in the oocytes obtained from the patients who do not have polycystic ovaries (p < 0.05), whereas statistically significant expression levels were observed for CYP17 in the oocytes obtained from patients with polycystic ovaries (p < 0.05). The expression level of HSD17B1 was also shown to be statistically different in the oocytes (p < 0.05). The extrapolation of the results indicates that the genes involved in steroidogenesis pathway are altered in cases of polycystic ovaries. Thus, it may have a role in the development of polycystic ovaries.

Thrombophilia associated gene polymorphisms: Does use of medication, including anti-coagulants, minerals or folic acid, prevent the miscarriages?

OBJECTIVE: Recurrent pregnancy loss (RPL) has been associated with thrombophilia. The use of prophylactic treatments against thrombophilia becomes necessary in order to increase the live birth rates in women with RPL. The aim of this study was to genotype thrombophilia associated polymorphisms and investigates the benefit of prophylactic treatment on the clinical pregnancy outcomes of women with specific genotypes of these polymorphisms. MATERIALS AND METHODS: A total of 62 women were included in this study. The polymorphisms associated with thrombophilia, including methyltetrahydrofolate reductase (MTHFR) 1298 and 677, Factor V Leiden (FVL) 1691, plasminogen activator inhibitor-1 (PA1-1) G/G and Factor II prothrombin 20,210, were genotyped using the real time PCR. The effect of prophylactic treatment using anti-coagulants of 0.4 mL dose of enoxaparin (3000-6000IU) and 75 mg dose of aspirin, 81 mg dose of aspirin, mineral of 15 mg dose of zinco c or10 mg dose of folic acid, was correlated with the genotypes of polymorphisms. RESULTS AND CONCLUSION: The clinical pregnancy outcomes were significantly improved in patients with MTHFR 677CC genotype when treated with zinco c. Furthermore, treatment with 75 mg of aspirin resulted in higher negative pregnancy rates in patients with MTHFR A1298C genotypes. Therefore, the results of this study should be used to re-evaluate the clinical applications in women with miscarriages.

Investigation of potential biomarkers for thrombosis related diseases in Turkish Cypriot population.

Genetic and environmental factors are involved in development of many diseases. The allelic frequencies may differ in different populations and in different ethnic groups. The aim of this study was to investigate the genotypes of MTHFR and factor VII polymorphisms and to identify biomarkers for thrombosis related diseases in Turkish Cypriot population. The lipid profiles and genotypes of MTHFR polymorphisms (rs1801133, rs1801131) and factor VII (rs6046) genes were investigated for the first time in the Turkish Cypriot population. The heterozygosity for MTHFR (rs1801133, rs1801131) and FVII (rs6046) polymorphisms is high in Turkish Cypriot population. The heterozygosity for MTHFR C677T was 38%, MTHFR A1298C was 40% and factor VII G353A was 37%, respectively. Allelic frequencies between males and females were similar. There were no correlations between the genotypes of polymorphisms and the lipid profiles. This study is the first genetic epidemiology study that investigated the allelic frequencies of MTHFR and FVII polymorphisms associated with metabolic syndromes. This study proves to be a crucial analysis in order to use these polymorphisms as a predictor of disease development in the Turkish Cypriot community.

Investigation of KCNQ1 polymorphisms as biomarkers for cardiovascular diseases in the Turkish Cypriots for establishing preventative medical measures.

Potassium channels are important in transmitting electrical signals through potassium ions transport. These potassium channels are made from signals encoded by KCNQ1 gene. KCNQ1 polymorphisms were associated with many diseases, including many metabolic and cardiovascular diseases and therefore they can be employed as biomarkers. In this study we aimed to investigate KCNQ1 polymorphisms in the Turkish Cypriot population to reveal the allele frequencies specific for this population and use these polymorphisms as biomarkers to develop preventative medical measures. The genotypes of KCNQ1 polymorphisms (rs231361, rs231359, rs151290, rs2283228, rs2237895, rs2237896) were investigated for the first time in Turkish Cypriot population. The correlation between genotypes of these polymorphisms and plasma lipid levels in this population was also explored. The results of this study showed that there was significant differences of the allele frequencies of between rs2283228 allele of C and rs2237896 (P > 0.05) in Turkish Cypriot population. There was no association between the genotypes of the six polymorphisms and the lipid metabolism. This study is the first genetic epidemiology study that investigated the allelic frequencies of KCNQ1 polymorphisms associated with metabolic syndromes as well as cardiovascular diseases. This study proves to be crucial since the etiologic determinants and molecular pathology of cardiovascular diseases have not yet clearly understood. This study showed that genome wide association studies should be designed for preventative medicine in the Turkish Cypriot population.

Effect of a carbon fiber tabletop on the surface dose and attenuation for high-energy photon beams.

PURPOSE: The dose changes in the buildup region and beam attenuation by a carbon fiber tabletop were investigated for 6-and 18-MV photon beams. MATERIALS AND METHODS: Measurements were performed for 2 x 2 cm to 40 x 40 cm field sizes. The surface dose and percentage depth doses (PDD) were measured by a Markus parallel plate chamber. Attenuation measurements were made at the cylindrical phantom for 180 degrees rotation of the beam. RESULTS: A carbon fiber tabletop increases the surface dose from 7.5% to 63.0% and from 4% to 43% for small fields at 6 and 18 MV, respectively. The increase was nearly fivefold for the 10 x 10 cm field and nearly twofold for the 40 x 40 cm field. Beam attenuation of the tabletop varies from 3.0% to 5.6% for 180 degrees and 120 degrees gantry angles for 6 MV. CONCLUSION: The carbon fiber tabletop significantly decreases the skin-sparing effect. The dosimetric effect of the tabletop may be higher, especially for the intensity-modulated radiation therapy depending on the beam orientation. Attenuation should be considered and corrected such as any material under the patient at the treatment planning stage.