Safety, efficacy and efficiency of laser-assisted IVF in subfertile mutant mouse strains

Li, Ming Wen; Kinchen, Kristy L.; Vallelunga, Jadine M.; Young, Diana L.; Wright, Kaleb D K; Gorano, Lisa N.; Wasson, Katherine; Lloyd, Kevin C K

doi:10.1530/rep-12-0477

Cited by 10 publications

(12 citation statements)

References 35 publications

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“…This finding has also been demonstrated in other studies, where mouse embryos generated through laser-assisted zona drilling developed to the blastocyst stage in vitro at a rate similar to conventional IVF [13], [17], [18], but had significantly lower birth rates [11], [12], [17], [34], [35]. Although not ruled out here, neither parthenogenic activation, nor polyspermy appear to be problems of LAIVF with mouse gametes [11], [13], [16]–[18].…”

Section: Discussionsupporting

confidence: 88%

“…Whereas, LAIVF resulted in 42% two-cell embryos (n = 9,301/21,997), and was useful for obtaining live pups from 55 of 56 GM lines. Li et al found similarly low fertilization rates using fresh spermatozoa from subfertile GM C57BL/6 mice with conventional IVF (<15% two-cell embryos), while rates using LAIVF ranged from 25% to 85% two-cell embryos [12] .…”

Section: Discussionmentioning

confidence: 98%

“…Laser-assisted zona drilling offers promise for assisted reproduction in both humans and mice [11] – [17] . A recent study using laser-assisted IVF (LAIVF) to derive subfertile genetically-modified (GM) mouse lines achieved fertilization rates 4 to 10 times that of conventional IVF [12] . Further, fertilization rates have been shown to be higher with LAIVF in mice with poor quality sperm [11] , [13] , [16] – [19] .…”

Section: Introductionmentioning

confidence: 99%

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

Woods

Rosalia

et al. 2014

PLoS ONE

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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.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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

Woods

Rosalia

et al. 2014

PLoS ONE

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“…The thickness of the laser-perforated regions in the zona also impacts lentiviral gene delivery and embryo development (Li, Kinchen et al 2013). Larger laser-perforated regions result in higher number of blastocyst expressing the virally delivered gene.…”

Section: Resultsmentioning

confidence: 99%

En masse lentiviral gene delivery to mouse fertilized eggs via laser perforation of zona pellucida

et al. 2018

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Lentiviruses are highly efficient vehicles for delivering genes into cells. They readily transduce primary and immortalized cells in vivo and in vitro. Genes delivered by lentiviruses are incorporated and replicated as part of their host genome and therefore offer a powerful tool for creation of stable cell lines and transgenic animals. However, the zona pellucida surrounding the fertilized eggs acts as a barrier and hinders lentiviral transduction of embryos. Here, we utilize a laser, typically used to perforate the zona pellucida for in vitro fertilization, to permeabilize the zona for lentiviral gene delivery. A single hole in the zona is sufficient for the lentivirus to gain access to fertilized eggs without the need for microinjection for en masse gene delivery. Embryos generated by this method elicit no damage and can develop to term for creation of transgenic animals.

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“…Several approaches have been proposed to avoid the risk of laserinduced thermal damage to embryos. Among them are artificial shrinkage of oocytes (aimed at increasing the perivitelline space) during the IVF with laser-assisted zona drilling by adding sucrose to the culture medium [39][40][41]. Moreover, drilling location should be selected rather thoroughly at an area with no cells touching the zona pellucida.…”

Section: Discussionmentioning

confidence: 99%

Femtosecond laser is effective tool for zona pellucida engraving and tagging of preimplantation mammalian embryos

Ilina

Khramova

Filatov

et al. 2019

J Assist Reprod Genet

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Purpose Our purpose was to study whether application of femtosecond laser pulses for alphanumeric code marking in the volume of zona pellucida (ZP) could be effective and reliable approach for direct tagging of preimplantation embryos. Methods Femtosecond laser pulses (wavelength of 514 nm, pulse duration of 280 fs, repetition rate of 2.5 kHz, pulse energy of 20 nJ) were applied for precise alphanumeric code engraving on the ZP of mouse embryos at the zygote stage for individual embryo marking and their accurate identification. Embryo quality assessment every 24 h post laser-assisted marking as well as immunofluorescence staining (for ICM/TE cell number ratio calculation) were performed. Results Initial experiments have started with embryo marking in a single equatorial plane. The codes engraved could be clearly recognized until the thinning of the ZP prior to hatching. Since embryo may change its orientation during the ART cycle, multiplane code engraving seems to be more practical for simplifying the process of code searching and embryo identification. We have marked the ZP in three planes, and no decrease in developmental rates as well as no morphological changes of embryos post laser-assisted engraving have been observed as compared to control group embryos. Conclusions Our results demonstrate the suitability of femtosecond laser as a novel tool for noninvasive embryo tagging, enabling embryo identification from day 0.5 post coitum to at least early blastocyst stage. Thus, the versatility and the potential use of femtosecond lasers in the field of developmental biology and assisted reproduction have been shown.

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Safety, efficacy and efficiency of laser-assisted IVF in subfertile mutant mouse strains

Cited by 10 publications

References 35 publications

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

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

En masse lentiviral gene delivery to mouse fertilized eggs via laser perforation of zona pellucida

Femtosecond laser is effective tool for zona pellucida engraving and tagging of preimplantation mammalian embryos

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