Elevated Adipsin and Reduced C5a Levels in the Maternal Serum and Follicular Fluid During Implantation Are Associated With Successful Pregnancy in Obese Women.

Introduction: Complement factors mediate the recruitment and activation of immune cells and are associated with metabolic changes during pregnancy. The aim of this study was to determine whether complement factors in the maternal serum and follicular fluid (FF) are associated with in vitro fertilization (IVF) outcomes in overweight/obese women. Methods: Forty overweight/obese (BMI = 30.8 ± 5.2 kg/m2) female patients, 33.6 ± 6.3 years old, undergoing IVF treatment for unexplained infertility were recruited. Baseline demographic information, including biochemical hormonal, metabolic, and inflammatory markers, and pregnancy outcome, was collected. Levels of 14 complement markers (C2, C4b, C5, C5a, C9, adipsin, mannose-binding lectin, C1q, C3, C3b/iC3b, C4, factor B, factor H, and properdin) were assessed in the serum and FF and compared to IVF outcome, inflammatory, and metabolic markers using multivariate and univariate models. Results: Out of 40 IVF cycles, 14 (35%) resulted in pregnancy. Compared to women with failed pregnancies, women with successful pregnancies had higher levels of adipsin in the serum and FF (p = 0.01) but lower C5a levels (p = 0.05). Serum adipsin levels were positively correlated with circulating levels of vitamin D (R = 0.5, p = 0.02), glucagon (R = 0.4, p = 0.03), leptin (R = 0.4, p = 0.01), resistin (R = 0.4, p = 0.02), and visfatin (R = 0.4, p = 0.02), but negatively correlated with total protein (R = -0.5, p = 0.03). Higher numbers of top-quality embryos were associated with increased levels of C3, properdin, C1q, factors H and B, C4, and adipsin, but with reduced C2 and C5a levels (p ≤ 0.01). Conclusions: Higher adipsin and lower C5a levels in the maternal serum during implantation are potential markers of successful outcome in obese women undergoing IVF-assisted pregnancies.

Early oocyte denudation does not compromise ICSI cycle outcome: a large retrospective cohort study.

This retrospective cohort study of 2051 consecutive fresh non-donor intracytoplasmic sperm injection (ICSI) cycles investigated whether time from oocyte retrieval to denudation, precisely measured and recorded by an operator-independent automated radiofrequency-based system, affected cycle outcome. ICSI cycles were divided into two groups: group I (denudation within <2 h of oocyte retrieval, n = 1118) and group II (denudation 2-5 h after oocyte retrieval, n = 933). Univariate analysis by two-sample t-test or Mann-Whitney test was used, as appropriate. Both groups were comparable with regards to mean number of oocytes retrieved and fertilized normally after ICSI. The mean number of embryos transferred and surplus embryos cryopreserved at the blastocyst stage were similar. There was no significant difference in fertilization, embryo implantation, pregnancy, clinical pregnancy or live birth rates between the groups. Analysis of group I ICSI outcome after subdivision into immediate (up to 30 min) and early (31-119 min) denudation showed no statistically significant differences between the two subgroups. In conclusion, early oocyte denudation within <2 h after retrieval does not appear to compromise ICSI cycle outcome, permitting more efficiency and flexibility in scheduling laboratory workload. As this was a retrospective observational study, further prospective studies are required to confirm the findings.

Human Embryos Created by Embryo Splitting Secrete Significantly Lower Levels of miRNA-30c.

Studies reporting term pregnancy and the production of genetically identical offspring from isolated blastomeres of early stage embryos have been carried out in small and large animals. However, very little is known about the effects of embryo splitting on the development and reproductive competency of human embryos. In this study, we investigated the effects of embryo splitting on profile of microRNAs (miRNAs) detected in their spent blastocyst medium (SBM) by comparative analysis of miRNA profiles in SBM of human twin embryos created by blastomere biopsy and SBM of blastocysts that resulted in a healthy pregnancy and live birth following embryo transfer. The profile of miRNA secretion in in vitro culture media consistently distinguishes twin from control embryos. We found that six miRNAs are significantly more abundant in SBM from twin embryos, while nine are significantly more abundant in SBM from euploid implanted blastocysts. These nine include miRNA-30c, a previously reported marker of blastocyst implantation potential. Furthermore, 22.9% of miRNAs secreted by twin embryos were never detected in SBM from normal reproductively competent blastocysts, or from trophectoderm (TE) samples from normal blastocysts donated for the research. The miRNA profile, unique to twin blastocysts, might be a result of differential lineage commitment in these embryos.

Developmental clock compromises human twin model created by embryo splitting.

STUDY QUESTION: Is the quality of the human embryos generated by twinning in vitro comparable to the quality of the embryos created by fertilization? SUMMARY ANSWER: Our data suggest that the human twin embryos created in vitro are unsuitable not only for clinical use but also for research purposes. WHAT IS KNOWN ALREADY: Pregnancy from in vitro generated monozygotic twins by embryo splitting or twinning leads to live birth of healthy animals. Similar strategies, however, have been less successful in primates. Recent reports suggest that the splitting of human embryos might result in viable, morphologically adequate blastocysts, although the qualitative analyses of the embryos created in such a way have been very limited. STUDY DESIGN, SIZE, DURATION: This study was a comparative analysis of embryos generated by twinning in vitro and the embryos created by in vitro fertilization. PARTICIPANTS/MATERIALS, SETTING, METHODS: We analysed morphokinetics and developmental competence of 176 twin embryos created by splitting of 88 human embryos from either early (2-5 blastomeres, n = 43) or late (6-10 blastomeres, n = 45) cleavage stages. We compared the data with morphometrics of embryos created by in vitro fertilization and resulting in pregnancy and live birth upon single blastocyst transfer (n = 42). MAIN RESULTS AND THE ROLE OF CHANCE: The morphokinetic data suggested that the human preimplantation development is subjected to a strict temporal control. Due to a 'developmental clock', the size of twin embryos was proportionate to the number of cells used for their creation. Furthermore, the first fate decision was somewhat delayed; the inner cell mass (ICM) became distinguishable later in the twin than in the normal blastocysts obtained through fertilization. If an ICM was present at all, it was small and of poor quality. The majority of the cells in the twin embryos expressed ICM and trophectoderm markers simultaneously. LIMITATIONS, REASONS FOR CAUTION: We created monozygotic twins by blastomere separation from cleavage stage embryos. Embryo twinning by blastocyst bisection may circumvent limitations set by the developmental clock. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, our data suggest that the human twin embryos created in vitro are unsuitable not only for clinical use but also for research purposes.

Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environment.

The protocols described here are comprehensive instructions for deriving human embryonic stem (hES) cell lines in xeno-free conditions from cryopreserved embryos. Details are included for propagation, cryopreservation and characterization. Initial derivation is on feeder cells and is followed by adaptation to a feeder-free environment; competent technicians can perform these simplified methods easily. From derivation to cryopreservation of fully characterized initial stocks takes 3-4 months. These protocols served as the basis for standard operating procedures (SOPs), with both operational and technical components, that we set to meet good manufacturing practice (GMP) and UK regulatory body requirements for derivation of clinical-grade cells. As such, these SOPs are currently used in our current GMP compliant facility to derive hES cell lines ab initio, in an animal product-free environment; these lines are suitable for research and potentially for clinical use in cell therapy. So far, we have derived eight clinical-grade lines, which will be freely available to the scientific community after submission/accession to the UK Stem Cell Bank.