ART altered DNA methylation of the imprinted gene H19 in fetal tissue after multifetal pregnancy reduction.

PURPOSE: This study aimed to assess whether assisted reproductive technology alters DNA methylation levels at the H19 promoter and H19 imprinting control element (ICE) in fetal tissues obtained after multifetal pregnancy reduction. METHODS: Fetal tissues from multiple pregnancies were obtained, including fresh and frozen-thawed embryos: nine from conventional in vitro fertilization (c-IVF), four from intracytoplasmic sperm injection (ICSI), ten from cryopreserved IVF embryos (cryo-IVF), and six from cryopreserved ICSI (cryo-ICSI) embryos. Next-generation sequencing-based bisulfite PCR was used to determine the DNA methylation status of three CpG islands (H19-1, H19-2, and H19-3) in the H19 promoter and H19 ICE. The primary outcome was H19-1 DNA methylation status, whereas secondary outcomes assessed H19-2, H19-3, and ICE methylation. RESULTS: The ICSI (ß = -3.189, 95% CI = -5.034 to -1.345, p = 0.0026), cryo-IVF (ß = -2.150, 95% CI = -3.706 to -0.593, p = 0.0129), and cryo-ICSI (ß = -2.238, 95% CI = -3.817 to -0.659, p = 0.0110) groups exhibited significantly lower methylation levels in the primary outcome H19-1 region than the c-IVF group after adjustment. For the secondary outcome H19-2 region, significant decreases were observed in the cryo-IVF (ß = -2.132, 95% CI = -4.071 to -0.192, p = 0.0425) and cryo-ICSI groups (ß = -2.598, 95% CI = -4.566 to -0.630, p = 0.0168). CONCLUSIONS: These findings further indicate that embryo cryopreservation and potentially ICSI can lower the methylation level of the H19 promoter, advocating for careful use of these techniques when necessary.

Association between morphologic grading and implantation rate of Euploid blastocyst.

BACKGROUND: Standard morphologic evaluation has been the most widely adopted approach to embryo selection, and remains the most common strategy.The objective of the study to determine the association between the morphologic grading and implantation rate of euploid blastocysts in single frozen-thawed embryo transfer (SET) cycles. METHODS: A total of 271 patients aged 20-40 years undergoing euploid SET from January 2017 to December 2019 were included in retrospective cohort study.The cycles were divided into three groups based on their morphologic grading before cryopreservation: good-quality (n = 58), average-quality (n = 88) and poor-quality blastocysts (n = 125). The pregnancy outcome of the three morphologic groups were analyzed and a logistic regression of implantation rate was conducted. RESULTS: Good-quality blastocysts yielded statistically significantly higher implantation rates than poor-quality (79.31% vs. 48%; P<0.001). Planned subgroup analyses by age and the day of TE biopsy were conducted. Logistic regression analyses that adjusted for these variables identified higher implantation rates (adjusted odds ratio(aOR) = 4.083, 95% confidence interval (CI):1.836-9.082, P<0.001) for the good-quality blastocysts than for those that underwent poor-quality cycles in women aged < 35 years, but not in women aged ≥35 years (aOR = 6.074, 95% CI: 0.456-80.919, P = 0.172). The implantation rates were higher among women with good-quality blastocysts on both Day 5 and Day 6 of TE biopsy than among those with poor-quality blastocysts (Day 5, aOR = 3.294, 95% CI:1.260-8.616, P = 0.015; Day 6, aOR = 4.179, 95% CI:1.004 ~ 17.399, P = 0.049). Day 5 euploid blastocysts had no significant difference in implantation potential and early spontaneous abortion rate compared with similarly graded Day 6 euploid blastocysts. CONCLUSIONS: Blastocyst morphologic grading was associated with implantation rate for euploid embryo transfers after adjustment for potential confounders. These findings suggest that evaluating blastocyst morphology is critical when selecting the best euploid blastocyst.

Does the sex ratio of singleton births after frozen single blastocyst transfer differ in relation to blastocyst development?

PURPOSE: To investigate the associations between blastocyst development and the sex ratio (male:female) among singleton live births resulting from single-blastocyst frozen embryo transfer (FET) cycles. METHODS: Patients with singleton live births following the first autologous single FET of non- preimplantation genetic testing (PGT) blastocysts in a single reproductive medicine department between January 2015 and February 2019 were included in this retrospective study. The primary outcome measure was the singleton sex ratio. Multivariable logistic regression models were used to estimate the associations between blastocyst quality and singleton sex ratio after adjustment for some potential confounders. RESULTS: There were 638 high-quality and 572 poor-quality single blastocyst FETs, and the blastocysts were conceived via 855 IVF and 355 ICSI treatments. A total of 1210 singleton live births were assessed. High-quality single blastocyst FET resulted in a significantly higher sex ratio than did poor-quality single blastocyst FET (60% vs. 49.7%, P < 0.001). The infertility cause was not associated with sex ratio among singleton live births (P = 0.537). The results of a multivariate analysis revealed that a high-quality blastocyst has a 150% higher probability of being male than a poor-quality blastocyst (adjusted odds ratio (aOR) 1.57; 95% CI 1.24-2, P < 0.001). Among the three blastocyst morphological parameters, Grade B trophectoderm was significantly associated with a higher sex ratio than Grade C (aOR 1.71; 95% CI 1.33-2.21. P < 0.001). Neither expansion degree nor inner cell mass degree were significantly associated with the singleton sex ratio. CONCLUSIONS: A single high-quality blastocyst FET has a higher chance of resulting in a male infant than a female infant. The results demonstrate that grade B trophectoderm confers benefits in improving the implantation potential of male blastocysts.

Long-time vs. short-time insemination of sibling eggs.

This study aimed to evaluate the impact of a short insemination procedure on embryo development, assess the reliability of a fertilization check prior to the appearance of pronuclei and elucidate the role of rescue intracytoplasmic sperm injection (R-ICSI). Patients who had ≥9 eggs, were aged <38 years and had normal semen samples were included. The sibling eggs of each patient were divided into two groups; one half for conventional in vitro fertilization (IVF; insemination time, 20 h) and the other half for short insemination (insemination time, 5 h). R-ICSI was performed where total fertilization failure (TFF) was deemed to have occurred in the short insemination group. In total, 2,465 eggs were included. No significant differences were found in fertilization, abnormal fertilization, embryo quality, clinical pregnancy and implantation rates between the short insemination and conventional insemination groups. In the short insemination group, R-ICSI was performed in 11 cycles (6.7%); in 6 of these cycles, fertilization occurred in the patient's eggs in the 20 h insemination group, while in the other 5 cycles, the patient's eggs were not fertilized after 20 h insemination either. Following R-ICSI, 19 of 36 eggs were normally fertilized. Subsequently, 7 embryos were transferred in 4 fresh cycles resulting in one ectopic pregnancy. In conclusion, the short insemination procedure does not improve embryo development and pregnancy rates. Short insemination combined with early R-ICSI may be a method of preventing total fertilization failure, albeit with poor accuracy.

Outcomes of day 3 embryo transfer with vitrification using Cryoleaf: a 3-year follow-up study.

OBJECTIVE: To compare success rates of vitrified-warmed with fresh and frozen-thawed ETs DESIGN: Retrospective. SETTING: Public fertility center. PATIENT(S): Cryopreserved- thawed/warmed ETs were included in this study. Fresh cycles, in which supernumerary embryos were cryopreserved, were set as the fresh control group. INTERVENTION(S): Supernumerary day 3 embryos were cryopreserved by slow-freezing or vitrification and transferred after thawing or warming. MAIN OUTCOME MEASURE(S): Comparison of two cryopreservation techniques with respect to post-thaw survival of embryos, implantation and pregnancy rates, neonatal outcome, and congenital birth defects. RESULTS: A total of 962 fresh, 151 freezing-thawed and 300 vitrified-warmed cycles were included in this study. The survival and intact cell rates in the vitrification group were significantly higher compared with those in the slow freezing group (88.5 % vs 74.5 % and 86.6 % vs 64.0 %). The implantation, clinical pregnancy and live birth rates of the vitrification group were similar to the fresh and significant higher than slow freezing group. There were no significant differences in mean gestational age, birth weight, stillbirth, birth defects and the prevalence of neonatal diseases among three groups. CONCLUSION: Vitrified-warmed ETs yield comparable outcomes with fresh ETs and is superior to frozen-thawed ETs regarding the survival rate and clinical outcomes.