Dean E. Morbeck

Fat tissue, aging, and cellular senescence.

Fat tissue, frequently the largest organ in humans, is at the nexus of mechanisms involved in longevity and age‐related metabolic dysfunction. Fat distribution and function change dramatically throughout life. Obesity is associated with accelerated onset of diseases common in old age, while fat ablation and certain mutations affecting fat increase life span. Fat cells turn over throughout the life span. Fat cell progenitors, preadipocytes, are abundant, closely related to macrophages, and dysdifferentiate in old age, switching into a pro‐inflammatory, tissue‐remodeling, senescent‐like state. Other mesenchymal progenitors also can acquire a pro‐inflammatory, adipocyte‐like phenotype with aging. We propose a hypothetical model in which cellular stress and preadipocyte overutilization with aging induce cellular senescence, leading to impaired adipogenesis, failure to sequester lipotoxic fatty acids, inflammatory cytokine and chemokine generation, and innate and adaptive immune response activation. These pro‐inflammatory processes may amplify each other and have systemic consequences. This model is consistent with recent concepts about cellular senescence as a stress‐responsive, adaptive phenotype that develops through multiple stages, including major metabolic and secretory readjustments, which can spread from cell to cell and can occur at any point during life. Senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells. Consistent with this, a senescent‐like state can develop in preadipocytes and fat cells from young obese individuals. Senescent, pro‐inflammatory cells in fat could have profound clinical consequences because of the large size of the fat organ and its central metabolic role.

Type of culture media does not affect embryo kinetics: a time-lapse analysis of sibling oocytes

STUDY QUESTION Are the morphokinetics of growing embryos affected by the type of culture media utilized? SUMMARY ANSWER Morphokinetic parameters used for embryo selection are not affected between the two different concept culture media analyzed. WHAT IS KNOWN ALREADY Studies on the effect of culture media on human embryos have focused on evaluating different in-house and commercially available media as well as comparing outcomes among different commercial media. Nonetheless, the evaluation of embryo development in these studies was based on static observations and very little is known from a dynamic point of view. STUDY DESIGN, SIZE, DURATION Prospective cohort study, October 2010 and April 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS University-affiliated infertility center. Patients undergoing egg donation (n = 75) in which embryos were cultured with two different types of media in a time-lapse system. Embryo development was analyzed with time-lapse imaging for single step media (Global®) and sequential media (Sage® Cleavage). Variables studied included the timing to two cells (t2), three cells (t3), four cells (t4) and five cells (t5) as well as the length of the second cell cycle (cc2 = t3 - t2) and the synchrony in the division from two to four cells (s2 = t4 - t3). Implantation and clinical pregnancy rates were also analyzed. MAIN RESULTS AND THE ROLE OF CHANCE No statistically significant differences were observed between the two media for all the variables analyzed. When analyzing the percentage of embryos falling within the optimal ranges proposed for s2, cc2 and t5, we did not find significant differences between the two media. Pregnancy and implantation rates were similar for the three types of transfers: 48.0% (CI 95% 28.4-67.6) and 42.0% (CI 95% 22.5-61.4) with Global media; 58.8% (CI 95% 35.4-82.2) and 38.2% (CI 95% 15.0-61.4) with Cleavage media; and 58.1% (CI 95% 40.7-75.4) and 37.1% (CI 95% 22.1-52.1) with mixed transferred, respectively. Multiple implantations (twins) were also similar among the three groups, with 24.0% (CI 95% 9.3-45.1) for transfers with embryos cultured in Global media, 17.6% (CI 95% 3.7-43.3) for transfers with embryos cultured in Cleavage media and 22.5% (CI 95% 9.5-41.0) with mixed transfers. LIMITATIONS, REASONS FOR CAUTION The study was not powered to test differences in pregnancy rates between the two culture media, as this was not the hypothesis tested. Results are based on observations with embryos from oocyte donors and need to be repeated with embryos from infertile patients of different ages. WIDER IMPLICATIONS OF THE FINDINGS The absence of differences in morphokinetics between two different media concepts validates the algorithm for embryo selection in diverse culture conditions. STUDY FUNDING/COMPETING INTEREST(S) No specific funding was obtained for this study; it was solely funded by IVI. None of the authors have any economic affiliation with Unisense Fertilitech A/S but IVI is a minor shareholder in Unisense Fertilitech A/S.

Cdc20 is critical for meiosis I and fertility of female mice.

Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.

A receptor binding site identified in the region 81–95 of the β-subunit of human luteinizing hormone (LH) and chorionic gonadotropin (hCG)

Two series of overlapping peptides comprising the entire sequences of the beta-subunits of human lutropin (LH) and choriogonadotropin (hCG) were prepared by a comprehensive synthetic strategy in order to identify all linear regions of the subunit that may participate in binding of the hormone to its receptor. Each series of peptides (15 residues in length) spanned the entire amino acid sequences of the two beta-subunits. The peptides were tested for their ability to inhibit the binding of 125I-labeled hCG or LH to rat ovarian membranes and for their ability to inhibit hCG-stimulated progesterone production in a Leydig cell bioassay. The most potent inhibitor of LH/hCG binding was a peptide containing the sequence beta 81-95, a receptor binding site of the LH/hCG beta subunit not previously described. The concentration at which LH/hCG binding was inhibited at 50% (IC50) was 20 microM and 30 microM for hCGbeta 81-95 and LH beta 81-95, respectively. These peptides also inhibited the stimulation of progesterone production by hCG in Leydig cell bioassays. In order to determine important residues that inhibit binding within this region, a third set of peptides was synthesized in which each residue of hCG beta 81-95 was sequentially replaced with the residue L-alanine. Five residues (Leu-86, Cys-88, Cys-90, Arg-94, and Arg-95) were critical for maximal inhibition of hCG binding by CG beta 81-95. In addition to site beta 81-95, other sites that inhibited hCG/LH binding but with significantly lower potencies included hCG beta 1-15, LH beta 41-55, and LH beta 91-105.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the microheterogeneity of the glycoprotein chorionic gonadotropin with high-performance capillary electrophoresis

Abstract Human chorionic gonadotropin (hCG) is a heteromeric glycoprotein hormone with a molecular mass of ca. 38 000. The carbohydrate side chains terminate with sialic acid and account for roughly 30% of the mass of the hormone. Glycoforms of hCG have been routinely identified with conventional methods of isoelectric focusing or chromatofocusing and exhibit varied bioactivity. In the present report, high-performance capillary electrophoresis (HPCE) was used to separate the glycoforms of hCG and its subunits. Optimal conditions for obtaining near-baseline resolution of the glycoforms were 25 mM borate, pH 8.8 containing 5 mM diaminopropane. The samples were separated in a 100 cm fused-silica capillary with an internal diameter of 50 μm at 25 kV and 28°C. In its native form, hCG migrated in less than 50 min as 8 distinct, highly resolved peaks. In the absence of diaminopropane, hCG migrated as a single, broad peak. When analyzed individually, the α subunit separated into four peaks and the β subunit resolved as seven peaks. The two subunits could also be separated when the heterodimer was incubated in 0.25% trifluoroacetic acid for 1 h prior to injection into the capillary. To illustrate the potential clinical application of this technique, four different sources of hCG were analyzed. The number of different isoforms was constant among the four samples; however, the relative concentration (amounts) of the isoforms varied. These results illustrate the potential utility of HPCE in the clinical diagnostic analysis of hCG microheterogeneity.

Composition of protein supplements used for human embryo culture

PurposeTo determine the composition of commercially available protein supplements for embryo culture media and test if differences in protein supplement composition are biologically relevant in a murine model.MethodsAmino acid, organic acid, ion and metal content were determined for 6 protein supplements: recombinant human albumin (AlbIX), human serum albumin (HSA and Buminate), and three complex protein supplements (SSS, SPS, LGPS). To determine if differences in the composition of these supplements are biologically relevant, mouse one-cell embryos were collected and cultured for 120 hours in each protein supplement in Global media at 5 and 20 % oxygen in an EmbryoScope time-lapse incubator. The compositions of six protein supplements were analyzed for concentrations of 39 individual amino acids, organic acids, ions and elements. Blastocyst development and cell cycle timings were calculated at 96-hours of culture and the experiments were repeated in triplicate. Blastocyst gene expression was analyzed.ResultsRecombinant albumin had the fewest undefined components , the lowest concentration of elements detected, and resulted in high blastocyst development in both 5 and 20 % oxygen. Buminate, LGPS and SPS had high levels of transition metals whereas SSS had high concentrations of amino acids. Pre-compaction mouse embryo development was delayed relative to embryos in AlbIX for all supplements and blastocyst formation was reduced in Buminate, SPS and SSS.ConclusionsThe composition of protein supplements are variable, consisting of previously undescribed components. High concentrations of pro-oxidant transition metals were most notable. Blastocyst development was protein dependent and showed an interaction with oxygen concentration and pro-oxidant supplements.

Washing mineral oil reduces contaminants and embryotoxicity

OBJECTIVE To determine if washing improves the quality of mineral oil used for embryo culture. DESIGN A 2 × 3 factorial experimental study. SETTING University hospital-based infertility center. ANIMAL(S) Mice. INTERVENTION(S) The chemical nature of contaminants present in two lots of mineral oil was determined. Effect of washing on toxicity and amount of toxin present in media was determined. MAIN OUTCOME MEASURE(S) The effect of washing was determined by a quality control bioassay or by directly determining the level of contaminant in oil-conditioned culture media. RESULT(S) Water, culture media, and media plus albumin were equally effective in reducing toxicity and concentration of toxin. Temperature did not affect washing results. Peroxide, aldehydes, and alkenals were present in one lot of oil, and Triton X-100 was identified in the other lot. Washed oil containing peroxide passed the one-cell mouse embryo bioassay, and washing reduced the amount of Triton X-100 by 25%. CONCLUSION(S) Mineral oil is the least defined component used for in vitro fertilization and embryo culture; therefore, it is important to determine if washing oil is beneficial. This study provides clear evidence that washing reduces toxicity of mineral oil.

Peroxides in mineral oil used for in vitro fertilization: defining limits of standard quality control assays

PurposeTo determine the relative sensitivities of the 1 and 2-cell mouse embryo assays (MEA) and the human sperm motility assay (HSMA) for peroxides in mineral oil. The effect of peroxide on blastocyst cell number and apoptosis was also studied.MethodsOne and two-cell MEA and HSMA were performed using mineral oil containing cumene hydroperoxide (CH).ResultsThe 1-cell MEA was twice as sensitive as the 2-cell MEA and 20-times more sensitive than the HSMA for CH in mineral oil. The sensitivity of the 1-cell MEA doubled when embryos were cultured individually versus group culture. CH decreased blastocyst cell number in a dose dependent manner.ConclusionsIndividually cultured 1-cell embryos had the highest sensitivity for peroxides in mineral oil. Current quality control assays, including group cultured murine embryos and human sperm motility, have limited sensitivity for peroxides in mineral oil and may not detect levels of peroxides that cause sub-lethal cellular damage.

Advances in quality control: mouse embryo morphokinetics are sensitive markers of in vitro stress

STUDY QUESTION Can time-lapse analysis of cell division timings [morphokinetics (MK)] in mouse embryos detect toxins at concentrations that do not affect blastocyst formation? SUMMARY ANSWER An MK algorithm enhances assay sensitivity while providing results 24–48 h sooner than the traditional mouse embryo assay (MEA). WHAT IS KNOWN ALREADY Current quality control testing methodology is sensitive but further improvements are needed to assure optimal culture conditions. MKs of embryo development may detect small variations in culture conditions. STUDY DESIGN Cross sectional—control versus treatment. Mouse embryo development kinetics of 466 embryos were analyzed according to exposure to various concentrations of toxins and toxic mineral oil. MATERIALS, SETTING, METHODS Cryopreserved 1-cell embryos from F1 hybrid mice were cultured with cumene hydroperoxide (CH) (0, 2, 4, 6 and 8 µM) and Triton X-100 (TX-100; 0, 0.0008, 0.0012, 0.0016 and 0.002%). Using the Embryoscope, time-lapse images were obtained every 20 min for 120 h in seven focal planes. End-points were timing and pattern of cell division and embryo development. The blastocyst rate (BR) was defined as the percentage of embryos that developed to the expanded blastocyst stage within 96 h. MAIN RESULTS AND THE ROLE OF CHANCE BR was not affected for embryos cultured in the three lowest concentrations of CH and the four lowest concentrations of TX-100. In contrast, a unique MK model detected all concentrations tested (P < 0.05). The MK model identified toxicity in two lots of toxic mineral oil that did not affect BR (P < 0.05). LIMITATIONS, REASONS FOR CAUTION A limited number of toxins were used so that the results may not apply to all potential embryo toxins. A larger sample size may also demonstrate other statistically significant developmental kinetic parameters. WIDER IMPLICATIONS OF THE FINDINGS MKs in mouse embryos are a sensitive and efficient method for quality control testing of in vitro culture conditions. BR, the end-point of traditional quality control assays, did not detect sublethal concentrations of toxins in the culture milieu in our study. This study demonstrates that temporal variation at key developmental stages reflects the quality of the culture environment. An MEA that incorporates MK will provide enhanced sensitivity and faster turn-around times. STUDY FUNDING/COMPETING INTEREST(S) The study was supported by Mayo Clinic Department of Obstetrics and Gynecology Small Grant Program. The authors have no competing interests to declare.

Variability in protein quality used for embryo culture: embryotoxicity of the stabilizer octanoic acid.

OBJECTIVE To screen human serum albumin (HSA) preparations for toxicity and investigate causes of variation. DESIGN Experimental laboratory study. SETTING University-based laboratory. ANIMAL(S) FVB and CF1 mice crossed to create embryos used in experiments. INTERVENTION(S) Mouse embryo assay performed with 5% or 15% HSA (100 mg/mL albumin) from three samples from three separate manufacturers (A, B, C). MAIN OUTCOME MEASURE(S) Blastocyst rates calculated at 96 hours of culture (experiments repeated in triplicate). RESULT(S) The HSA preparations were desalted to remove stabilizers added during HSA processing, then mass spectrometry was used to determine the relative variation in stabilizer concentrations; the effect of the stabilizer octanoic acid on embryo development was tested. At 5% HSA, all samples had blastocyst rates ≥ 70%; at 15% HSA, the blastocyst rates for samples B and C were <50%. Desalting did not affect sample B but did improve the blastocyst rates of sample C. Mass spectrometry revealed high levels of octanoic acid in sample C compared with sample A. The addition of octanoic acid to sample A produced toxicity similar to sample C. CONCLUSION(S) The stabilizer octanoic acid varies by lot and inhibits embryo development. Because octanoic acid is known to cause disruptions in mitochondrial bioenergetics, reduce intracellular pH, and induce oxidative damage in peripheral tissues, its use in embryo culture should be monitored and limited.