Role of epigenetic modifications in the aberrant CYP19A1 gene expression in polycystic ovary syndrome.

INTRODUCTION: In this study, the global DNA methylation, histone acetylation and methylation levels of cumulus cells (CCs) in infertile polycystic ovary syndrome (PCOS) patients and the correlation of these epigenetic modifications with the expression of the ovarian aromatase gene (as an important marker in the etiology of PCOS) were investigated. MATERIAL AND METHODS: A cross-sectional study was conducted on 24 patients (12 PCOS patients and 12 healthy women), who underwent ovarian stimulation. Nucleosome ELISA was performed, in order to identify the global occupancy level of Mecp2 (as a marker of DNA methylation) and H3K9me2/H3K9ac as histone modification markers in chromatin fractions obtained from CCs. The CYP19A1 gene expression was measured by qRT-PCR. The level of DNA incorporation of MeCP2, histone modification markers and binding of estrogen receptor ß (ERß) to CYP19A1 regulatory sequences were examined by ChIP-QPCR assay. RESULTS: The data demonstrate a significant increase in global occupancy levels of MeCP2 and H3K9ac markers and a decrease of H3K9me2 to chromatin in CCs of PCOS patients vs. control group. Furthermore, CYP19A1 gene expression, and the incorporation of H3K9ac in PII, PI.3, and PI.4 promoters of CYP19A1 in PCOS, were higher than those of controls. Also, significant hypomethylation of H3K9 at PII and DNA hypomethylated at PII and PI.3 promoters and differential binding of ERß to three promoters were observed in PCOS patients (p < 0.05). CONCLUSIONS: Aromatase expression can be affected by epigenetic modifications and differential ERß binding to the proximal CYP19A1 promoters. These mechanisms may be involved in the enhanced aromatase transcription during ovarian stimulation in PCOS patients.

Total Antioxidant Capacity; A Potential Biomarker for Non-Invasive Sex Prediction in Culture Medium of Preimplantation Human Embryos.

OBJECTIVE: The presence of a sex related metabolic difference in glucose utilization and, on the other hand, different developmental kinetic rates in human preimplantation embryos, has been previously observed, however, the correlation between these two events is unknown. Oxidative stress (OS) induced by higher glucose consumption appears to be a possible cause for the delayed development rate in female embryos. We examined the correlation between glucose consumption and total antioxidant capacity (TAC) concentration in individual embryo culture media for both male and female embryos. MATERIALS AND METHODS: In this cross-sectional study, we evaluated high quality embryos from 51 patients that underwent intracytoplasmic sperm injection (ICSI) and preimplantation genetic diagnosis (PGD) at the Royan Institute between December 2014 and September 2017. The embryos were individually cultured in G-2TM medium droplets at days 3-5 or 48 hours post PGD. We analysed the spent culture media following embryo transfer for total antioxidant capacity (TAC) and any remaining glucose concentrations through fluorometric measurement by chemiluminecence system which indirectly was used for measurement of glucose consumed by embryos. RESULTS: The results showed that female embryos consumed more glucose which was associated with decreased TAC concentration in their culture medium compared to male embryos. The mean of glucose concentration consumed by the female embryos (30.7 ± 4.7 pmol/embryo/hour) was significantly higher than that of the male embryos (25.3 ± 3.3 pmol/embryo/hour) (P<0.001). There were significantly lower levels of TAC in the surrounding culture medium of female embryos (22.60 ± 0.19 nmol/µl) compared with male embryos (24.74 ± 0.27 nmol/µl, P<0.01). CONCLUSION: This finding highlighted the utilization of sex dependent metabolic diversity between preimplantation embryos for non-invasive sex diagnosis and suggests the TAC concentration as a potential noninvasive biomarker for prediction of sex.

Oxidative Stress Statues in Serum and Follicular Fluid of Women with Endometriosis.

OBJECTIVE: This study aimed to evaluate the levels of two oxidative stress (OS) markers including lipid peroxide (LPO) and total antioxidant capacity (TAC) in both serum and follicular fluid (FF) of women with endometriosis after puncture. MATERIALS AND METHODS: In this cross-sectional study, a total number of sixty-three women younger than 40 years old with laparoscopy (gold standard for endometriosis diagnosis) indication underwent in vitro fertilization (IVF) program in the Royan Institute, Tehran, Iran from September 2013 to October 2014. About forty-three patients were diagnosed with endometriosis after laparoscopy. Blood and FF from the leading follicle in each stimulated ovary were obtained at the time of egg retrieval; samples were centrifuged and frozen until assessment. At the time of sample assessment, serum and FF samples were evaluated for the levels of LPO and TAC on spectrophotometery. RESULTS: We observed that women with endometriosis had significantly higher LPO and lower TAC levels in the serum and FF as compared with the control group (P<0.05). CONCLUSION: It has observed that FF of women with endometriosis, regardless of disease stage, increases the proliferation power of endometrial cells in vitro, we presume that inflammatory reactions-induced OS in ovary may be responsible for proliferation induction ability in FF obtained from women with endometriosis.

Effect of oocyte dysmorphisms on intracytoplasmic sperm injection cycle outcomes in normal ovarian responders.

AIM: The aim of this study was to identify the influence of oocyte dysmorphisms on clinical outcomes after intracytoplasmic sperm injection cycle in normal responders. MATERIAL AND METHODS: In the prospective study, morphology of 1999 metaphase II oocytes retrieved from 316 intracytoplasmic sperm injection cycles was evaluated from March 2011 to March 2013 at Royan Institute. Controlled ovarian stimulation was performed by long standard agonist protocol. Oocyte morphology was assessed before sperm injection by one embryologist. The associations between fertilization rate, embryo quality and the independent variables were analyzed using odds ratio (OR) calculated with unconditional logistic regression test. RESULTS: From all retrieved oocytes, 1543 (77.1%) showed at least one morphologic aberration. Presence of cytoplasmic vacuoles and high cytoplasmic viscosity were associated with a significant decrease in the fertilization rate (OR: 0.5; P = 0.004 and OR: 0.6; P = 0.03, respectively). The results showed that oocyte morphology did not affect embryo quality. The number of gonadotrophin injections used showed a direct relation with presence of large perivitelline space. No significant difference was observed among four groups (women with total normal morphologic oocytes [group 1], women with total extracytoplasmic dysmorphic oocytes [group 2], women with total cytoplasmic dysmorphic oocytes [group 3] and women with total oocytes containing multiple dysmorphic features [group 4]) in terms of implantation and clinical pregnancy rates. CONCLUSIONS: Metaphase II oocyte morphology had minor impacts on fertilization rate, pronuclear morphology and embryo quality in women with normal ovarian response.

Zygote intrafallopian tube transfer versus intrauterine cleavage or blastocyst stage transfer after intracytoplasmic sperm injection cycles in patients with repeated implantation failure: A prospective follow-up study.

AIM: This study aimed to compare the outcomes between zygote intrafallopian transfer (ZIFT) with intrauterine day-3 (cleavage stage) embryo transfer and intrauterine day-5 (blastocyst stage) embryo transfer in patients undergoing intracytoplasmic sperm injection. MATERIAL AND METHODS: This prospective study was performed at Royan Institute, Tehran, Iran, between January 2012 and January 2014. Two hundred fifty women with more than three unexplained implantation failures were divided non-randomly into three groups according to embryonic age and methods used as follows: (i) intrauterine cleavage-stage embryo transfer (n = 100); (ii) intrauterine blastocyst-stage embryo transfer (n = 50); and (iii) ZIFT (n = 100). Implantation, clinical pregnancy, miscarriage and live birth rates were our main outcomes. RESULTS: Patients' characteristics and ovarian response were comparable among the three groups. Implantation rate (56.1% vs 27.9%) was significantly higher in the blastocyst group as compared to the ZIFT group; however, clinical pregnancy rate (38% vs 23%) was not statistically significantly different between the two groups, but due to the significantly higher miscarriage rate (34.7% vs 5.3%) in the ZIFT group, the live birth rate was significantly higher in the blastocyst group (P = 0.04). No significant differences were found between the cleavage-stage and blastocyst-stage groups in terms of implantation, clinical pregnancy, miscarriage and live birth rates. CONCLUSION: We do not recommend the use of the ZIFT procedure for patients with repeated implantation failures. It seems that replication of cleavage- or blastocyst-stage embryo transfer is more efficient and affordable.

Abdominal obesity can induce both systemic and follicular fluid oxidative stress independent from polycystic ovary syndrome.

OBJECTIVE: The abdominal form of obesity is prevalent in women with polycystic ovary syndrome (PCOS). Visceral fat accumulation seems to play an important role in etiology of PCOS. In this cross-sectional study we evaluated the association of oxidative stress (OS) induced with PCOS and abdominal obesity in serum and follicular fluid (FF) of infertile women. STUDY DESIGN: A total of 80 women younger than 37 years old undergoing an IVF program were studied in the same period of time from September 2012 to October 2013. Blood serum and FF obtained from 40 women with PCOS (diagnosed by the Rotterdam 2004 criteria) and 40 women without PCOS undergoing IVF were evaluated for two OS markers: lipid peroxide (LPO) and total antioxidant capacity (TAC), after puncture. The patients were divided into 4 groups on the basis of presence of PCOS and waist-to-hip ratio (WHR) or abdominal obesity (OA). RESULTS: Healthy and PCOS women with abdominal obesity had significantly higher amounts of LPO in the serum and FF as compared with women without abdominal obesity. LPO concentration in FF was significantly lower than in serum and corroborates the hypothesis that the germinal cells have a potent antioxidant mechanism. We also found that LPO concentration in the PCOS group associated with AO had an increasing trend vs. those AO patients without PCOS but this difference was not significant, so the increase in LPO level was approximately independent of PCOS. Based on our results, the association and interaction between PCOS and AO can lead to TAC concentration reduction in patients. CONCLUSIONS: Abdominal obesity can induce local and systemic oxidative stress in PCOS and non-PCOS patients. We suggest that PCOS-induced disorders are likely to be exacerbated in the presence of abdominal obesity.

The Effects of ISM1 Medium on Embryo Quality and Outcomes of IVF/ICSI Cycles.

BACKGROUND: The aim of this study is to investigate the effect of ISM1 culture medium on embryo development, quality and outcomes of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles. This study compares culture medium commonly used in the laboratory setting for oocyte recovery and embryo development with a medium from MediCult. We have assessed the effects of these media on embryo development and newborn characteristics. MATERIALS AND METHODS: In this prospective randomized study, fertilized oocytes from patients were randomly assigned to culture in ISM1 (MediCult, cycles: n=293) or routine lab culture medium (G-1TM v5; Vitrolife, cycles: n=290) according to the daily media schedule for oocyte retrieval. IVF or ICSI and embryo transfer were performed with either MediCult media or routine lab media. Embryo quality on days 2/3, cleavage, pregnancy and implantation rates, baby take home rate (BTHR), in addition to the weight and length of newborns were compared between groups. RESULTS: There were similar cleavage rates for ISM1 (86%) vs. G-1TM v5 (88%). We observed a significantly higher percentage of excellent embryos in ISM1 (42.7%) compared to G-1TM v5 (39%, p<0.05). Babies born after culture in ISM1 had both higher birth weight (3.03 kg) and length (48.8 cm) compared to G-1TM v5 babies that had a birth weight of 2.66 kg and a length of 46.0 cm (p<0.001 for both). CONCLUSION: This study suggests that ISM1 is a more effective culture medium in generating higher quality embryos, which may be reflected in the characteristics of babies at birth.

Pyomyoma in a premenopausal woman with fever of unknown origin.

BACKGROUND: Pyomyoma (suppurative leiomyoma) is a rare disease that is a serious complication. Most cases have occurred in pregnant or postmenopausal women. CASE: A perimenopausal woman presented with fever and shoulder pain. She had no predisposing factors or history of leiomyoma. Ultrasonographic as well as abdominal and pelvic computed tomography scans showed an enlarged uterus with two large masses. Internal heterogeneous echogenicity was noted in the lower segment and body of the uterus. The elevated temperature continued despite a 3-day antibiotic course of clindamycin, ceftriaxone, and gentamicin. With a clinical impression of infected leiomyoma, she underwent a total abdominal hysterectomy and bilateral salpingo-oophorectomy. Pathological findings showed a leiomyoma with abscess formation. The patient responded well to surgery. CONCLUSION: Pyomyoma may be difficult to diagnose, especially in women with a nonspecific clinical presentation. Delayed diagnosis may result in serious complications, and surgery and broad spectrum antibiotics are indicated.

Effect of laser zona thinning on vitrified-warmed embryo transfer at the cleavage stage: a prospective, randomized study.

The aim of this study was to determine if laser zona thinning could improve the rates of pregnancy and implantation for vitrified-warmed embryo transfer at the cleavage stage. A total of 400 vitrified-warmed embryo transfer cycles were randomly assigned to either the test group or the control group. The zona pellucida of vitrified-warmed embryos in the patients of the control group was untreated, whereas in the test group it was partially thinned by laser just before embryo transfer. In the test group, the clinical pregnancy and implantation rates were significantly lower as compared with that of the control group (28.5 versus 43.0, P=0.002, and 11.2 versus 16.7, P=0.004, respectively). Therefore the results of this investigation show that laser zona thinning may have an unexpected adverse effect on the rates of clinical pregnancy and implantation of vitrified-warmed embryos at the cleavage stage.

Derivation of new human embryonic stem cell lines from preimplantation genetic screening and diagnosis-analyzed embryos.

In this study, we focused on the derivation of human embryonic stem cell (hESC) from preimplantation genetic screening (PGS)-analyzed and preimplantation genetic diagnosis (PGD)-analyzed embryos. Out of 62 fresh PGD/PGS-analyzed embryos, 22 embryos reached the blastocyst stage. From 12 outgrowth blastocysts, we derived four hESC lines onto a feeder layer. Surprisingly, karyotype analysis showed that hESC lines derived from aneuploid embryos had diploid female karyotype. One hESC line was found to carry a balanced Robertsonian translocation. All the cell lines showed hESC markers and had the pluripotent ability to differentiate into derivatives of the three embryonic germ layers. The established lines had clonal propagation with 22-31% efficiency in the presence of ROCK inhibitor. These results further indicate that hESC lines can be derived from PGD/PGS-analyzed embryos that are destined to be discarded and can serve as an alternative source for normal euploid lines.

Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos.

PURPOSE: The objective of this retrospective study was to evaluate the efficacy of vitrification and slow freezing for the cryopreservation of human cleavage stage embryos in terms of post-warming survival rate, post-warming embryo morphology and clinical outcomes. METHODS: The embryos of 305 patients at cleavage stages were cryopreserved either with vitrification (153 patients) or slow-freezing (152 patients) methods. After warming; the survival rate, post-warmed embryo morphology, clinical pregnancy and implantation rates were evaluated and compared between the two groups. RESULT(S): In the vitrification group versus slow freezing group, the survival rate (96.9% vs. 82.8%) and the post-warmed excellent morphology with all blastomeres intact (91.8% vs. 56.2%) were higher with an odds ratio of 6.607 (95% confidence interval; 4.184-10.434) and 8.769 (95% confidence interval; 6.460-11.904), respectively. In this group, the clinical pregnancy rate (40.5% vs. 21.4%) and the implantation rate (16.6% vs. 6.8%) were also higher with an odds ratio of 2.427 (95%confidence interval; 1.461-4.033) and 2.726 (95% confidence interval; 1.837-4.046), respectively. CONCLUSION(S): Vitrification in contrast to slow freezing is an efficient method for cryopreservation of human cleavage stage embryos. Vitrification provides a higher survival rate, minimal deleterious effects on post-warming embryo morphology and it can improve clinical outcomes.

Effect of laser zona pellucida opening on clinical outcome of assisted reproduction technology in patients with advanced female age, recurrent implantation failure, or frozen-thawed embryos.

OBJECTIVE: To determine whether laser-assisted hatching can improve clinical outcome of assisted reproductive techniques in patients with advanced female age, with recurrent implantation failure, or who are using frozen-thawed embryos. DESIGN: A prospective randomized study. SETTING: The infertility and IVF unit at a research facility in Iran. PATIENT(S): Four hundred ten patients with advanced female age (> or =37 y), 796 patients with recurrent implantation failure (for > or =2 cycles), and 180 patients with frozen-thawed embryos. INTERVENTION(S): Patients were divided equally into test and control groups. On the day of embryo transfer, the zona pellucida of the selected embryos in the test group were opened about 40 mum by using an infrared optical laser system, whereas in the control group they were all intact. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates and implantation rates. RESULT(S): In the patients with advanced female age or recurrent implantation failure, the clinical pregnancy and implantation rates were similar for the test and control groups. However, in the patients with frozen-thawed embryos, the rates were statistically significantly higher in the test group as compared with those of the control group (31.2% and 12.8% vs. 11.1% and 4.2%, respectively). CONCLUSION(S): The laser-assisted hatching improved the pregnancy and implantation rates in patients with frozen-thawed embryos but had no effect in patients with advanced female age or recurrent implantation failure.

Effect of fragment removal on blastocyst formation and quality of human embryos.

Blastomere fragmentation is one of the most significant defects in cleaving embryos. Scientists believed that removing the fragments was a possible way to reduce their unwanted effects. This hypothesis has been tested in some studies in which the development of human fragmented embryos was followed in vivo after all fragments were removed, but little is known about the potential for in-vitro development of such embryos, which is the subject of the present study. For this purpose, 4-6 cell surplus human embryos were scored according to the degree and pattern of fragmentation into four grades, allocated into two groups of control and fragmentation removal (experimental) and cultured sequentially. At the end of day 6 of culture, in the experimental group especially in grade IV blastocyst rate, size and number of blastomeres in each blastocyst were all improved compared with those of the control group (42.3 versus 20.0%; 19,205.7 +/- 1060.3 versus 15,825.9 +/- 448.7 microm(2) and 100.14 +/- 13.48 versus 63.75 +/- 19.79 respectively, P < 0.05). In the grade IV embryos, apoptotic index was also significantly reduced after embryo fragmentation removal (3.40 +/- 0.88 versus 22.99 +/- 4.45, P < 0.05). In conclusion, fragmentation removal had a positive effect on human fragmented embryos and produced the best quality blastocysts.