Reanalysis of discarded blastocysts for autosomal aneuploidy after sex selection in cleavage-stage embryos / 대한생식의학회지

Objective@#The goal of the present study was to investigate the rate of chromosomal aneuploidies in surplus embryos after sex determination at the cleavage stage. Then, the same chromosomal aneuploidies were evaluated in blastocysts after extended culture. @*Methods@#Sixty-eight surplus embryos were biopsied at the cleavage stage and incubated for an additional 3 days to allow them to reach the blastocyst stage. The embryos were reanalyzed via fluorescence in situ hybridization (FISH) to examine eight chromosomes (13, 15, 16, 18, 21, 22, X, and Y) in both cleavage-stage embryos and blastocysts. @*Results@#Although the total abnormality rate was lower in blastocysts (32.35%) than in cleavage-stage embryos (45.58%), the difference was not significant (p=0.113). However, when we restricted the analysis to autosomal abnormalities, we observed a significant difference in the abnormality rate between the cleavage-stage embryos (44.11%) and the blastocysts (17.64%, p=0.008). A higher rate of sex chromosomal abnormalities was also observed in cleavage-stage embryos (29.4%) than in blastocysts (14.70%, p=0.038). @*Conclusion@#The data indicated that embryo biopsy should be conducted at the blastocyst stage rather than the cleavage stage. The results also emphasized that examination of common chromosomal aneuploidies apart from sex selection cycles can be conducted in the blastocyst stage with the FISH method.

Reanalysis of discarded blastocysts for autosomal aneuploidy after sex selection in cleavage-stage embryos / 대한생식의학회지

Objective@#The goal of the present study was to investigate the rate of chromosomal aneuploidies in surplus embryos after sex determination at the cleavage stage. Then, the same chromosomal aneuploidies were evaluated in blastocysts after extended culture. @*Methods@#Sixty-eight surplus embryos were biopsied at the cleavage stage and incubated for an additional 3 days to allow them to reach the blastocyst stage. The embryos were reanalyzed via fluorescence in situ hybridization (FISH) to examine eight chromosomes (13, 15, 16, 18, 21, 22, X, and Y) in both cleavage-stage embryos and blastocysts. @*Results@#Although the total abnormality rate was lower in blastocysts (32.35%) than in cleavage-stage embryos (45.58%), the difference was not significant (p=0.113). However, when we restricted the analysis to autosomal abnormalities, we observed a significant difference in the abnormality rate between the cleavage-stage embryos (44.11%) and the blastocysts (17.64%, p=0.008). A higher rate of sex chromosomal abnormalities was also observed in cleavage-stage embryos (29.4%) than in blastocysts (14.70%, p=0.038). @*Conclusion@#The data indicated that embryo biopsy should be conducted at the blastocyst stage rather than the cleavage stage. The results also emphasized that examination of common chromosomal aneuploidies apart from sex selection cycles can be conducted in the blastocyst stage with the FISH method.

Effects of vitamin A, C and E, or omega-3 fatty acid supplementation on the level of paraoxonase and arylesterase activity in streptozotocin-induced diabetic rats: an investigation of activities in plasma, and heart and liver homogenates

<p><b>INTRODUCTION</b>This study was designed and conducted to evaluate the effects of vitamin A, C and E supplementation, and omega-3 fatty acid supplementation on the activity of paraoxonase and arylesterase in an experimental model of diabetes mellitus.</p><p><b>METHODS</b>A total of 64 male Sprague Dawley® rats, each weighing 250 g, were randomly distributed into four groups: (a) normal control; (b) diabetic control; (c) diabetic with vitamin A, C and E supplementation; and (d) diabetic with omega-3 fatty acid supplementation. The animals were anaesthetised after four weeks of intervention, and paraoxonase and arylesterase activity in blood plasma, and liver and heart homogenates were measured.</p><p><b>RESULTS</b>Arylesterase activity in the heart and liver homogenates was significantly lower in the diabetic control group than in the normal control group (p < 0.01). Vitamin A, C and E supplementation, and omega-3 fatty acid supplementation significantly increased liver arylesterase activity (p < 0.05). No significant change was observed in paraoxonase activity and other investigated factors.</p><p><b>CONCLUSION</b>Vitamin A, C and E, or omega-3 fatty acid supplementation were found to increase liver arylesterase activity in streptozotocin-induced diabetic rats. These supplements may be potential agents for the treatment of diabetes mellitus complications.</p>

ART outcomes in GnRH antagonist protocol [flexible] and long GnRH agonist protocol during early follicular phase in patients with polycystic ovary syndrome: a randomized clinical trial

Since increased LH in the early follicular phase in PCOS patients especially in GnRH antagonist protocol could be associated with reduced oocyte quality and pregnancy and impared implantation. The current study was conducted to determine ART outcomes in GnRH antagonist protocol [flexible] and long GnRH agonist protocol and compare them with adding GnRH antagonist in GnRH antagonist [flexible] protocol during early follicular phase in patients with polycystic ovary syndrome undergoing ICSI. In this randomized clinical trial, 150 patients with polycystic ovary syndrome undergoing ICSI were enrolled from 2012 to 2014 and randomly assigned to receive either GnRH antagonist protocol during early and late follicular phase or GnRH antagonist protocol [flexible] or long GnRH agonist protocol. The clinical and laboratory pregnancy in three groups was determined and compared. In this context, the chi-square and Fisher's exact test and ANOVA were used for data analysis. Statistical significance was defined as p<0.05. There was no statistically significant difference with respect to chemical pregnancy and clinical pregnancy between the three groups. Also, other indices such as number and quality of oocytes and embryos were alike. Totally, according to our results, GnRH antagonist protocol during early and late follicular phase and GnRH antagonist protocol [flexible] and long GnRH agonist protocol in patients with polycystic ovary syndrome undergoing ICSI are similarly effective and use of each one based on patients' condition and physicians' opinion could be considered

Effect of administration of single dose GnRH agonist in luteal phase on outcome of ICSI-ET cycles in women with previous history of IVF/ICSI failure: a randomized controlled trial

GnRH agonist administration in the luteal phase has been suggested to beneficially affect the outcome of intracytoplasmic sperm injection [ICSI] and embryo transfer [ET] cycles. This blind randomized controlled study evaluates the effect of GnRH [Gonadotropine Releasing Hormone] agonist administration on ICSI outcome in GnRH antagonist ovarian stimulation protocol in women with 2 or more previous IVF/ICSI-ET failures. One hundred IVF failure women who underwent ICSI cycles and stimulated with GnRH antagonist ovarian stimulation protocol, were included in the study. Women were randomly assigned to intervention [received a single dose injection of GnRH agonist [0.1 mg of Decapeptil] subcutaneously 6 days after oocyte retrieval] and control [did not receive GnRH agonist] groups. Implantation and clinical pregnancy rates were the primary outcome measures. Although the age of women, the number of embryos transferred in the current cycle and the quality of the transferred embryos were similar in the two groups, there was a significantly higher rate of implantation [Mann Whitney test, p=0.041] and pregnancy [32.6% vs. 12.5%, p=0.030, OR=3.3, 95%CI, 1.08 to 10.4] in the in-tervention group. Our results suggested that, in addition to routine luteal phase support using progesterone, administration of 0.1 mg of Decapeptil 6 days after oocyte re-trieval in women with previous history of 2 or more IVF/ICSI failures led to a signif-icant improvement in implantation and pregnancy rates after ICSI following ovarian stimulation with GnRH antagonist protocol

Fourier transform infrared spectroscopy: a potential technique for noninvasive detection of spermatogenesis

The seminal plasma is an excellent source for noninvasive detection of spermatogenesis. The seminal plasma of normospermic and azoospermic men has been analyzed for detection of spermatogenesis. Optical spectroscopy [Attenuated Total Reflectance-Infrared spectroscopy [ATR-IR] and Fourier Transform infrared spectroscopy [FT-IR] has been used to analyze the seminal plasma and the metabolome of seminal plasma for detection of spermatogenesis. The seminal plasma of normospermic and azoospermic men has been analyzed by ATR-IR. The results show that there is a pattern variation in the azoospermic men compared to normospermic men. However, the seminal plasma is too complex to show significant pattern variation. Therefore, the metabolome which is a subcomponent of the seminal plasma was analyzed. The seminal plasma metabolome of normospermic and azoospermic men has been analyzed by FT-IR. A significant pattern change was observed. The data combined with chemometrics analysis showed that significant changes are observed at metabolome level. We suggest that FT-IR has the potential as a diagnostic tool instead of testicular biopsy

High omega-3: omega-6 fatty acids ratio increases fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in human ectopic endometrial cells

Endometriosis, a common chronic inflammatory disorder, is defined by the atypical growth of endometrium- like tissue outside of the uterus. Secretory phospholipase A2 group Ha [sPLA2-IIa] and fatty acid binding protein4 [FABP4] play several important roles in the inflammatory diseases, Due to reported potential anti-inflammatory effects of omega-3 and omega-6 fatty acids, the purpose of the present study was to investigate the effects of omega-3 and omega-6 polyunsaturated fatty acids [PUFAs] on fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in cultured endometrial cells. Ectopic and eutopic endometrial tissues obtained from 15 women were snap frozen. After thawing and tissue digestion, primary mixed stromal f and endometrial epithelial cell culture was performed for 8 days in culture mediums supplemented with normal and high ratios of omega-3 and omega-6 PUFA. sPLA2-IIa in the J culture medium and FABP4 level was determined using enzyme immuno assay [EIA] technique. Within ectopic endometrial cells group, the level of cellular FABP4 and extracellular sPLA2-IIa were remarkably increased under high omega-3 PUFA exposure compared with control condition [p=0.014 and p=0.04 respectively]. Omega-3 PUFAs may increase the level of cellular FABP4 and extracellular sPLA2-IIa in ectopic endometrial cells, since sPLAIIa and FABP4 may affect endometriosis via several mechanisms, more relevant studies are encouraged to know the potential effect of increased cellular FABP4 and extracellular sPLA2-IIa on endometriosis

FABP9 mutations are not detected in cases of infertility due to sperm morphological defects in Iranian men

Fatty acid binding proteins [FABPs] are members of the intracellular lipid binding protein [iLBPs] family and most of them show tissue specific expression. FABP9/PERF15 [Perforatorial15] is the male germ cell-specific fatty acid-binding protein. It was first identified as the major constituent of the murine sperm perforatorium and perinuclear theca. To date, investigations in mice have demonstrated that this protein has a role in the male reproductive system, especially in spermatogenesis. Also, it has been reported that FABP9 can protect sperm fatty acids from oxidative damage. Recently it was shown that it can affect sperm morphology in mice. Based on these findings, we designed a study to evaluate if mutations of this gene can affect sperm morphology in humans. In this case-control study, DNA was extracted from peripheral blood of 100 infertile males with normal sperm count but with a number of morphologically abnormal sperms in their semen that was above normal. Four exons and one intron of the FABP9 gene were amplified by polymerase chain reaction [PCR], re-sequenced and then analyzed for mutation detection. We did not detect any mutation in any area of the four exons, intron 3 and splice sites of FABP9 gene in any of the studied 100 samples. There was no mutation in the exonic regions and the poor sperm morphology. However, we didn't analyze the promoter, intron 1 and 2 to establish conclusions regarding the association of these genic regions and sperm dysmorphology

technique for facile and precise transfer of mouse embryos

Successful Embryo Transfer [ET] technique is a fateful step of all efforts to achieve live births from in vitro produced embryos in assisted reproductive techniques or in knockout, transgenic or cloned animal projects. Small reproductive tract of mice and limitation of current techniques may not well satisfy the requirements for mass production of genetically modified mice. Genetic abnormalities of embryos, receptivity and uterine contractions, expulsion of embryos, blood, mucus or bacterial contamination on the transfer pipette tip, technical problems and even animal strain may affect embryo transfer outcome. In this study, two techniques of embryo transfer in mice were compared. In conventional technique the oviduct wall was punctured with a 30-gauge needle and the loaded Pasteur pipette with embryos and medium was inserted into the hole. In new technique, embryos that were loaded in modified micropipette with minimal medium were transferred directly to the oviduct by manual piston micro-pump easily. Embryo viability was evaluated considering the percentage of live healthy newborns. Results of the two techniques were compared by t-test within the NPAR1WAY procedure of SAS software [ver. 9.2]. The average live birth rates in the novel methods was significantly higher [42.4%] than the conventional method [21.7%, p<0.05]. In conclusion, using new embryo transfer technique improved birth rate by preventing embryos expulsion from the oviduct, saving time and easy transfer of embryos with minimum volume of medium

Propagation of human germ stem cells in long-term culture

Spermatogonial stem cells [SSCs], a subset of undifferentiated type A spermatogonia, are the foundation of complex process of spermatogenesis and could be propagated in vitro culture conditions for long time for germ cell transplantation and fertility preservation. The aim of this study was in vitro propagation of human spermatogonial stem cells [SSCs] and improvement of presence of human Germ Stem Cells [hGSCs] were assessed by specific markers POU domain, class 5, transcription factor 1 [POU5F1], also known as Octamer-binding transcription factor 4 [Oct-4] and PLZF [Promyelocytic leukaemia zinc finger protein]. Human testicular cells were isolated by enzymatic digestion [Collagenase IV and Trypsin]. Germ cells were cultured in Stem-Pro 34 media supplemented by growth factors such as glial cell line-derived neurotrophic factor, basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor to support self-renewal divisions. Germline stem cell clusters were passaged and expanded every week. Immunofluorecent study was accomplished by Anti-Oct4 antibody through the culture. The spermatogonial stem cells genes expression, PLZF, was studied in testis tissue and germ stem cells entire the culture. hGSCs clusters from a brain dead patient developed in testicular cell culture and then cultured and propagated up to 6 weeks. During the culture Oct4 were a specific marker for identification of hGSCs in testis tissue. Expression of PLZF was applied on RNA level in germ stem cells. hGSCs indicated by SSCs specific marker can be cultured and propagated for long-term in vitro conditions

Early fetal gender determination using real-time PCR analysis of cell-free fetal DNA during 6[th]-10[th] weeks of gestation

Nowadays, new advances in the use of cell free fetal DNA [cffDNA] in maternal plasma of pregnant women has provided the possibility of applying cffDNA in prenatal diagnosis as a non-invasive method. In contrary to the risks of invasive methods that affect both mother and fetus, applying cffDNA is proven to be highly effective with lower risk. One of the applications of prenatal diagnosis is fetal gender determination, which is important in fetuses at risk of sex-linked genetic diseases. In such cases by obtaining the basic information of the gender, necessary time management can be taken in therapeutic to significantly reduce the necessity of applying the invasive methods. Therefore in this study, the probability of detecting sequences on the human Y-chromosome in pregnant women has been evaluated to identify the gender of fetuses. Peripheral blood samples were obtained from 80 pregnant women with gestational age between 6[th] to 10[th] weeks and the fetal DNA was extracted from the plasma. Identification of SRY, DYS14 and DAZ sequences, which are not present in the maternal genome, was performed using Real-Time PCR. All the obtained results were compared with the actual gender of the newborns to calculate the test accuracy. Considerable 97.3% sensitivity and 97.3% specificity were obtained in fetal gender determination which is significant in the first trimester of pregnancy. Only in one case, false positive result was obtained. Using non-invasive method of cffDNAs in the shortest time possible, as well as avoiding invasive tests for early determination of fetal gender, provides the opportunity of deciding and employing early treatment for fetuses at risk of genetic diseases