Laser-Assisted In Vitro Fertilization Facilitates Fertilization of Vitrified-Warmed C57BL/6 Mouse Oocytes with Fresh and Frozen-Thawed Spermatozoa, Producing Live Pups

The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762) of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1) LAIVF using V-W oocytes, 2) LAIVF using fresh oocytes, 3) conventional IVF using V-W oocytes and 4) conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml). LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298) and fresh oocytes (69%, 135/197), compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively). Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml) were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343) using V-W oocytes (P<0.05, compared to fresh spermatozoa), and 73% (195/266) using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168) and fresh (5%, 15/323) oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784), advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908). Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen-thawed spermatozoa, producing live pups. The ability to cryopreserve mouse gametes for LAIVF may facilitate management of large-scale transgenic mouse production facilities.National Institutes of Health (U.S.) (NIH National Research Service Award (T32-RR070036))National Institutes of Health (U.S.) (NIH National Cancer Institute Program Project (P01CA10451)

A Logistic Regression Model Including Dna Status and Morphology of Spermatozoa for Prediction of Fertilization in Vitro

To determine predictive values of routine semen analysis, sperm morphology evaluation using strict criteria and DNA status for in-vitro fertilization (IVF), 66 consecutive couples undergoing IVF in a university hospital IVF programme were prospectively investigated. Semen samples from 66 men were evaluated by routine semen analysis, morphology evaluation using strict criteria and acridine orange staining for determination of DNA status. A new technique is described for acridine orange scoring which consisted of evaluation of two smears per case, with and without heat treatment. Resistance to heat-provoked denaturation was determined by the difference between two evaluations. A logistic regression model was built and receiver operating characteristic curves were constructed to determine the threshold values and to compare diagnostic properties. Morphology evaluation using strict criteria and concentration of progressively motile spermatozoa were found to be the principal parameters determining the sperm fertilizing capacity in vitro. The logistic regression model composed of morphology evaluation using strict criteria and acridine orange score had a powerful diagnostic capability for prediction of fertilization in vitro.WoSScopu