First babies conceived with Automated Intracytoplasmic Sperm Injection.

RESEARCH QUESTION: Can an automated sperm injection robot perform Automated Intracytoplasmic Sperm Injection (ICSIA) for use in human IVF? DESIGN: The ICSIA robot automated the sperm injection procedure, including injection pipette advancement, zona pellucida and oolemma penetration with piezo pulses, and pipette removal after sperm release. The robot was first tested in mouse, hamster and rabbit oocytes, and subsequently using discarded human oocytes injected with microbeads. A small clinical pilot trial was conducted with donor oocytes to study the feasibility of the robot in a clinical setting. The ICSIA robot was controlled by engineers with no micromanipulation experience. Results were compared with those obtained with manual ICSI conducted by experienced embryologists. RESULTS: The ICSIA robot demonstrated similar results to the manual procedure in the different animal models tested as well as in the pre-clinical validations conducted in discarded human oocytes. In the clinical validation, 13 out of 14 oocytes injected with ICSIA fertilized correctly versus 16 out of 18 in the manual control; eight developed into good-quality blastocysts versus 12 in the manual control; and four were diagnosed as chromosomally normal versus 10 euploid in the manual control. Three euploid blastocysts from the ICSIA robot group have been transferred into two recipients, which resulted in two singleton pregnancies and two babies born. CONCLUSIONS: The ICSIA robot showed high proficiency in injecting animal and human oocytes when operated by inexperienced personnel. The preliminary results obtained in this first clinical pilot trial are within key performance indicators.

A single-cell assay for telomere DNA content shows increasing telomere length heterogeneity, as well as increasing mean telomere length in human spermatozoa with advancing age.

PURPOSE: The effect of age on telomere length heterogeneity in men has not been studied previously. Our aims were to determine the relationship between variation in sperm telomere length (STL), men's age, and semen parameters in spermatozoa from men undergoing in vitro fertilization (IVF) treatment. METHODS: To perform this prospective cross-sectional pilot study, telomere length was estimated in 200 individual spermatozoa from men undergoing IVF treatment at the NYU Fertility Center. A novel single-cell telomere content assay (SCT-pqPCR) measured telomere length in individual spermatozoa. RESULTS: Telomere length among individual spermatozoa within an ejaculate varies markedly and increases with age. Older men not only have longer STL but also have more variable STL compared to younger men. STL from samples with normal semen parameters was significantly longer than that from samples with abnormal parameters, but STL did not differ between spermatozoa with normal versus abnormal morphology. CONCLUSION: The marked increase in STL heterogeneity as men age is consistent with a role for ALT during spermatogenesis. No data have yet reported the effect of age on STL heterogeneity. Based on these results, future studies should expand this modest sample size to search for molecular evidence of ALT in human testes during spermatogenesis.

Robust measurement of telomere length in single cells.

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.

Telomeres and human reproduction.

Telomeres mediate biologic aging in organisms as diverse as plants, yeast, and mammals. We propose a telomere theory of reproductive aging that posits telomere shortening in the female germ line as the primary driver of reproductive aging in women. Experimental shortening of telomeres in mice, which normally do not exhibit appreciable oocyte aging, and which have exceptionally long telomeres, recapitulates the aging phenotype of human oocytes. Telomere shortening in mice reduces synapsis and chiasmata, increases embryo fragmentation, cell cycle arrest, apoptosis, spindle dysmorphologies, and chromosome abnormalities. Telomeres are shorter in the oocytes from women undergoing in vitro fertilization, who then produce fragmented, aneuploid embryos that fail to implant. In contrast, the testes are replete with spermatogonia that can rejuvenate telomere reserves throughout the life of the man by expressing telomerase. Differences in telomere dynamics across the life span of men and women may have evolved because of the difference in the inherent risks of aging on reproduction between men and women. Additionally, growing evidence links altered telomere biology to endometriosis and gynecologic cancers, thus future studies should examine the role of telomeres in pathologies of the reproductive tract.

Characterization of binding of folates and antifolates to brush-border membrane vesicles isolated from human kidney.

Antifolates such as methotrexate, raltitrexed, and pemetrexed are among the most effective and widely used anticancer drugs. The antifolates are also among the most unpredictable of anticancer drugs with respect to pharmacokinetics and toxicity. In this study, we assessed the binding of folates and antifolates to the folate receptors (FRs) of human proximal tubules and the effects of pH on binding. Binding of [(3)H]folic acid was pH-dependent, with maximal binding seen at pH 6. Equilibrium binding experiments with [(3)H]folic acid showed that K(d) values were unaffected, and B(max) values increased as the pH was decreased from 8.0 to 6.0. Increasing the osmolarity at pH 6.0 had no effect on intravesicular content, confirming that increased site-specific binding caused the observed changes in B(max) values. Enzymatic cleavage of glycosyl-phosphatidylinositol linkages abolished binding of [(3)H]folic acid to brush-border membrane vesicles, suggesting that [(3)H]folic acid was bound to FRs. In concentration-effect experiments conducted at different pH values, the antifolates raltitrexed and (2S)-2-[o-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl-methyl)-N-(prop-2-ynyl)amino]benzamido]-4-(tetrazol-5-yl) butyric acid (ZD9331) bound more tightly as pH increased from 6.0 to 8.0, whereas binding of 10-propargyl-5,8-dideazafolic acid (CB3717) was unchanged. The results obtained when K(i) values were converted to binding energies suggested that binding of some, but not all, antifolates and folates to FRs was pH-dependent, further indicating roles of luminal pH in renal reabsorption or secretion processes.