Influence of GnRH antagonist in reproductive women on in vitro fertilization and embryo transfer in fresh cycles.

The aim of the present study was to evaluate the influence of a gonadotropin-releasing hormone (GnRH) antagonist compared with a GnRH agonist on in vitro fertilization (IVF) cycle outcome in reproductive women. The characteristics of treatment and outcomes of pregnancy were retrospectively compared between the antagonist (GnRH-A, antagonist group) and agonist (GnRH-a, agonist group) regimens. The area under the curve (AUC) of receiver operating characteristic (ROC) curves was also used to evaluate whether the endometrial thickness (cm), progesterone (P) level (ng/ml) and estradiol (E2) level (pg/ml) on the day of human chorionic gonadotropin (hCG) administration (hCG day) had the ideal sensitivity and specificity for predicting clinical pregnancy. There were no significant differences in the baseline profiles of luteinizing hormone, E2 and P between the GnRH-A and GnRH-a groups (P=0.646, 0.224 and 0.119, respectively). However age, body mass index and follicle stimulating hormone (FSH) level significantly differed between the two groups (P<0.001, =0.025 and <0.001, respectively). Regarding treatment, there were significant differences in the stimulation duration (recombinant FSH days of usage), dose of gonadotrophins, E2, and P levels on hCG day, endometrial thickness on hCG day, mean number of total oocytes retrieved, mean number of two pronuclei oocytes, mean number of embryos available and mean number of embryos transferred (all P<0.001). The rate of clinical pregnancy was lower with the GnRH antagonist than with the GnRH agonist (P<0.001). Additionally, the live birth rate in the GnRH-A group was significantly lower than that in the GnRH-a group (P<0.001). The rate of ectopic pregnancy did not differ significantly between the treatment groups (P=0.840). However, the rate of ovarian hyperstimulation syndrome (OHSS) in group GnRH-A was significantly lower than that in group GnRH-a (P=0.039). Therefore, in the present series of patients who underwent IVF embryo transfer cycles, a GnRH antagonist protocol was associated with significantly lower rates of clinical pregnancy and live birth compared with a GnRH agonist protocol; however, the rate of OHSS was significantly lower with GnRH antagonist compared with GnRH agonist. Furthermore, the results of the influence of endometrial thickness on clinical pregnancy, based on the ROC curve (AUC), demonstrated that the AUC was 0.553 [95% confidence interval (CI): 0.521-0.585], and with a cutoff of 9.25 cm, the Youden index [sensitivity-(1-specificity)] was 0.085. The results of the influence of E2 level on hCG day on the clinical pregnancy rate revealed an AUC of 0.613 (95% CI: 0.581-0.644), and with a cutoff of 1,520 pg/ml, the Youden index was 0.184. The results of the influence of P level on hCG day (ng/ml) on the clinical pregnancy rate revealed an AUC of 0.526 (95% CI: 0.494-0.558), and with a cutoff of 0.415 ng/ml, the Youden index was 0.061. These results of the ROC curve analyses demonstrated that neither the endometrial thickness nor the E2 and P levels on hCG day had the ideal sensitivity or specificity for predicting clinical pregnancy.

Mutation analysis of the TUBB8 gene in primary infertile women with arrest in oocyte maturation.

Tubulin beta eight class VIII (TUBB8) is a subtype of ß-tubulin that only exists in primates. Mutations in the TUBB8 gene have been proven to cause oocyte maturation arrest. The aim of this study was to identify the new types of mutations in TUBB8. Six women (families) with oocyte maturation arrest and 100 healthy controls were recruited. The sequence of the TUBB8 gene was amplified and analyzed by Sanger sequencing, which revealed a de novo heterozygous variant c.292G > A (p.G98R) of TUBB8 in one affected individual. This TUBB8 variant was absent in the 100 fertile females and was predicted to be highly damaging to the function of the TUBB8 protein by SIFT and PolyPhen-2. This novel variant extends the spectrum of TUBB8 mutations and the presence of a TUBB8 mutation is being considered to be indicative of a poor prognosis for the success of assisted reproductive treatment.

Assessing predictors for the success of GnRH antagonist protocol in reproductive women in IVF/ICSI - in fresh cycles.

The aim of the present study was to evaluate the factors that affect the success rate of gonadotropin-releasing hormone antagonist on in vitro fertilization/intracytoplasmic sperm injection cycles. Multivariate analysis was performed to assess the factors that influence the outcomes, such as oocytes retrieved, and the success of pregnancy. The results showed that E2, P on human chorionic gonadotropin (HCG) day and body mass index (BMI) were positively correlated with the number of oocytes retrieved (P=0.001, P=0.024, P=0.017, respectively). The duration of infertility as well as the luteinizing hormone on HCG day were negatively correlated with the number of oocytes (P=0.048, P=0.002, respectively). The age of the women and P on HCG day were negatively correlated with successful pregnancy (P<0.001, P=0.022). In conclusion, some parameters, such as E2, P, and LH on the HCG day, as well as age and BMI, may affect treatment outcomes.

GnRH antagonist for patients with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation for in vitro fertilization and embryo transfer in fresh cycles.

The aim of the present study was to evaluate the influence of a gonadotropin-releasing hormone (GnRH) antagonist compared with a GnRH agonist on the in vitro fertilization cycle outcome in patients with polycystic ovary syndrome. The outcomes of pregnancy were evaluated. The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was also used to evaluate whether the endometrial thickness (cm) and estradiol (E2) level (pg/ml) on the day of human chorionic gonadotropin (hCG) administration (the hCG day) had the best sensitivity and specificity for predicting a clinical pregnancy. The results demonstrated that there were significant differences in the E2 and progesterone levels between the two treatment groups on the hCG day. Furthermore, the mean number of total oocytes retrieved, mean number of 2 pronuclei oocytes, mean number of oocytes cleaved (P<0.05), mean number of embryos available (P=0.022) and mean number of embryos transferred (P=0.014) were significantly different. Additionally, the rates of ectopic pregnancy (P=0.984) and ovarian hyperstimulation syndrome (P=0.976) did not differ significantly between the treatment groups. Although the biochemical pregnancy (P=0.592), clinical pregnancy (P=0.617) and live birth (P=0.365) rates were lower with the GnRH antagonist than with the GnRH agonist, there were no significant differences in the outcomes between the two groups. Analysis of the influence of endometrial thickness with respect to the clinical pregnancy using the ROC (AUC) method revealed that when the best cutoff of 9.75 cm was used, the sensitivity was 62.5%, the specificity was 43.1% and the AUC was 0.53. Additionally, the Youden index was 0.056. Analysis of the influence of the E2 level on the hCG day on clinical pregnancy, using the ROC (AUC) method showed that the best cutoff was 2,984.5 pg/ml, which had a sensitivity of 68.8% and specificity of 52.9%, while the AUC was 0.573 (with a Youden index of 0.217). Furthermore, the results demonstrated that neither the endometrial thickness nor the E2 level on the hCG day had the best sensitivity and specificity for predicting a clinical pregnancy.

Outcomes of vitrified-warmed cleavage-stage embryo hatching after in vitro laser-assisted zona pellucida thinning in patients.

The aim of the present study was to determine whether the size of the zona pellucida (ZP) thinning area by laser-assisted hatching affected the potential development of vitrified-warmed embryos. A total of 196 vitrified-warmed cleavage-stage embryos (from 49 patients, four sister embryos per patient) were used in the study, i.e., four sister embryos from each patient were randomly assigned to four groups: a control group of embryos that were not zona-manipulated (zona intact, group A); one experimental group of embryos in which a quarter of the zona pellucida was thinned using laser-assisted ZP thinning (group B); a second experimental group of embryos in which half of ZP was thinned (group C); and a third group in which two-thirds of the ZP was thinned (group D). Subsequent blastocyst development was assessed. Microscopy was performed to study the hatching process of the embryos after zona thinning. The blastocyst formation rates were 71.43% in group A, 67.35% in group B, 65.31% in group C, and 51.02% in group D (groups B-D vs. group A, P=0.661, P=0.515, P=0.038, respectively). The rates of complete hatching were 30.61% in group A, 38.78% in group B, 61.22% in group C, and 48.98% in group D (groups B-D vs. group A, P=0.396, P=0.002, P=0.063, respectively). For a subgroup of patients, there was a significant difference in the complete hatching in all the groups for women aged <35 years (P=0.011), and there was a significant difference in the complete hatching in all the groups for secondary infertility women (P=0.022). There was no significant difference in the blastocyst formation rates in the different groups of women aged ≥35 years (P=0.340). In addition, there was no significant difference in the complete hatching in the different groups among women aged ≥35 years (P=0.492). The results of the present study showed that in vitrified-warmed embryo transfers at the cleavage-stage, and the two-thirds zona pellucida thinning group demonstrated a significantly decreased blastocyst formation rate compared with the control group, while the half zona pellucida thinning group demonstrated a significantly increased complete hatching rate compared with the control group, which may have a high value in clinical application.

Impact of XRCC1, GSTP1, and GSTM1 Polymorphisms on the Survival of Ovarian Carcinoma Patients Treated with Chemotherapy.

BACKGROUND: Single nucleotide polymorphic variants of DNA repair genes may improve drug efficacy through altering expression levels of the encoded proteins. This study evaluated the influence of genetic polymorphism GSTP1 Ile105Val, GSTM1 (null/non-null) and 2 XRCC1 polymorphisms (Arg194Trp and Arg399Gln) on the survival of ovarian carcinoma patients treated with chemotherapy. METHODS: 106 patients received treatment with a carboplatin-based or alternative chemotherapy. Polymorphisms were genotyped by pyrosequencing. RESULTS: The genotypes XRCC1 194Arg/Trp and XRCC1 194Trp/Trp conferred no significant risk of death when compared to 194Arg/Arg (hazard ratio (HR) 1.01, 95% confidence interval (CI) 0.33-3.09, and HR 0.89, 95% CI 0.31-2.57, respectively). Similarly, those carrying the XRCC1 399Arg/Gln genotype had no increased risk of death compared to the XRCC1 399Arg/Arg (HR 0.85, 95% CI 0.39-1.86); no homozygous carriers of the glutamine allele (XRCC1 399 Gln/Gln) were detected. The GSTP1 105Ile/Val had no increased risk of death compared to the GSTP1 105Ile/Ile (HR = 1.20, 95% CI = 0.55-2.63) and no homozygous carriers of the valine allele (GSTP1 105Val/Val) were detected in the study. Compared to the non-null genotype of GSTM1, the mortality rate was nonsignificantly reduced in patients with the null genotype (HR 1.07, 95% CI 0.48-2.42). However, overall survival of the patients treated with the carboplatin-based regimen was significantly longer than for those treated with alternative chemotherapy (plog-rank = 0.006). CONCLUSIONS: The present findings suggest that there are no correlations between genotypes and survival.

Single nucleotide polymorphisms in glutathione S-transferase P1 and M1 genes and overall survival of patients with ovarian serous cystadenocarcinoma treated with chemotherapy.

The effects of platinum-based drugs are controlled by genes that are involved in DNA detoxification, including glutathione S-transferase (GST)P1 and GSTM1, which have been associated with increased benefits in the chemotherapeutic treatment of patients with ovarian cancer. The present study assessed the effect of single nucleotide polymorphisms in GST genes on the overall survival (OS) of patients with ovarian serous cystadenocarcinoma that were treated with chemotherapy. A total of 95 patients received treatment with a carboplatin-based or alternative chemotherapy. Polymorphisms in the patients were genotyped using the following methods: Pyrosequencing, to identify GSTP1 Ile105Val; a relative quantification method, to identify the copy number variation in GSTM1; and polymerase chain reaction followed by gel electrophoresis, to identify the null vs. non-null genotypes of GSTM1. The association between genotypes and OS of patients was assessed using Kaplan-Meier survival curves and Cox proportional hazards regression analysis. The OS of patients treated with paclitaxel + carboplatin-based chemotherapy was significantly increased, compared with patients treated with alternative forms of chemotherapy (P=0.035). The OS of patients did not differ significantly between different GSTP1 genotypes (log-rank test, P=0.17). Cox proportional hazards regression analysis revealed that, since the start of the treatment, there was not a significant association between the GSTP1 isoleucine allele and the OS for heterozygous carriers of the isoleucine allele [hazards ratio (HR), 1.78; 95% confidence interval (CI), 0.77-4.12; P=0.18] and no homozygous carriers of the valine allele had been detected (HR, 0.00). There was no significant difference between GSTM1 genotypes, according to Kaplan-Meier survival analysis (log-rank test, P=0.83). Patients that possessed ≤1 copy of GSTM1 exhibited no decrease in OS (HR, 0.96; 95% CI, 0.37-2.51; P=0.94), compared with patients that possessed two copies of GSTM1 (HR, 0.71; 95% CI, 0.22-2.28; P=0.56). Overall, the present results suggest that there are no associations between polymorphisms in the GSTP1 and GSTM1 genes and the OS of patients with ovarian cancer following administration of adjuvant chemotherapy.

Toll-like receptor 4 single-nucleotide polymorphisms Asp299Gly and Thr399Ile in ovarian cancers.

Toll-like receptor (TLR4) 4 is present in numerous cell types and serves as the first point of defense in the innate immune system. Single-nucleotide polymorphisms (SNPs) are present in a number TLR genes and have been associated with various infection and inflammation disorders. Asp299Gly and Thr399Ile, TLR4 SNPs, are associated with tumor progression. In the present study, cases of ovarian cancer were analyzed with regards to Asp299Gly and Thr399Ile of the TLR4 gene. Genotype analysis was performed using DNA from tissue samples from stage I-IV patients with ovarian cancer. DNA from tissue samples was extracted and analyzed by a pyrosequencing method following multiplex polymerase chain reaction. The genotypes of these SNPs were analyzed in the present study in a population of 105 patients, with different types of ovarian cancer, between 2004 and 2012. The allele frequencies for TLR4 Asp299Gly identified in this population were 1.00 (A) and 0.00 (G); for TLR4 Thr399Ile the allele frequencies were; 1.00 (C) and 0.00 (T). For TLR4 Asp299Gly the observed genotype frequency was 1.00 (AA), 0.00 (AG) and 0.0 (GG). In TLR4 Thr399Ile the observed genotype frequencies were 1.00 (CC), 0.00 (CT) and 0.00 (TT). TLR4 Asp299Gly and Thr399Ile alleles were not detected in the patients. These results indicated that the TLR4 299Gly and 399Ile alleles were exhibited at a lower frequency in the ovarian cancer patients that were examined.

TLR4 induces tumor growth and inhibits paclitaxel activity in MyD88-positive human ovarian carcinoma in vitro.

In ovarian cancer patients, chemotherapy resistance is the principal factor restricting long-term treatment. Paclitaxel (Pac) has been previously reported to be a ligand to Toll-like receptor 4 (TLR4). It was determined that TLR4 signaling is divided into the following two pathways: Myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent. The present study investigated the effect of TLR4 ligation by Pac in MyD88-positive (MyD88+) and MyD88-negative (MyD88-) human ovarian cancer cell lines. An RNA interference expression vector was specifically constructed to target TLR4 mRNA, which was stably transfected into the human ovarian cancer cell lines (SKOV3, OVCAR3, A2780 and 3AO). Cytokines, including interleukin (IL)-6 and IL-8, were detected. Cell proliferation and apoptosis were assessed in the cells transfected with scramble control and TLR4 shRNA to explore the possible functions of TLR4 in ovarian cancer cell growth. It was found that lipopolysaccharide and Pac significantly increase the secretion of IL-6 and IL-8 in the SKOV3 cell line. Similarly, Pac resulted in a significant upregulation of IL-6 and IL-8 in OVCAR3 cells, but not in A2780 and 3AO cells. These results suggested that in MyD88+ ovarian cancer cell lines, TLR4 depletion shows increased sensitivity to Pac treatment in inhibiting cell proliferation compared with in cells without TLR4 knockdown. On the contrary, such changes were not found in MyD88- cells (A2780 and 3AO). TLR4 negatively regulates Pac chemotherapy, particularly in terms of cell proliferation, and TLR4 may be a novel treatment target in Pac-resistant ovarian cancer.