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

Abstract

In the present report we studied the safety, efficacy and efficiency of using an infrared laser to facilitate IVF by assessing fertilization, development and birth rates after laser-zona drilling (LZD) in 30 subfertile genetically modified (GM) mouse lines. We determined that LZD increased the fertilization rate four to ten times that of regular IVF, thus facilitating the derivation of 26 of 30 (86.7%) GM mouse lines. Cryopreserved two-cell stage embryos derived by LZD-assisted IVF were recovered and developed to blastocysts in vitro at the same rate as frozen-thawed embryos derived by regular IVF. Surprisingly after surgical transfer to pseudopregnant recipients the birth rate of embryos derived by LZD-assisted IVF was significantly lower than that of embryos derived by regular IVF. However this result could be completely mitigated by the addition of 0.25 M sucrose to the culture medium during LZD which caused the oocyte to shrink in volume relative to the perivitelline space. By increasing the distance from the laser target site on the zona pellucida, we hypothesize that the hyperosmotic effect of sucrose reduced the potential for laser-induced cytotoxic thermal damage to the underlying oocytes. With appropriate preparation and cautious application, our results indicate that LZD-assisted IVF is a safe, efficacious and efficient assisted reproductive technology for deriving mutant mouse lines with male factor infertility and subfertility caused by sperm-zona penetration defects.

Figure 1

Laser beam targeted on the zona of a C57BL/6J oocyte showing the location of isotherm rings relative to the oolemma. (1) Zona thickness, (2) distance between the oolemma and the zona center (or laser drill center on the zona), (3) zona diameter, and (4) oocyte cell diameter. The laser isotherm rings appear as a series of six concentric rings of varying colors and indicate the maximum temperature reached at the ring diameter.

Figure 2

Diameters of holes after different durations of laser pulses at 300 mW in C57BL/6J mouse oocytes compared with the thickness of zona. Only at a pulse duration ≥230 µs was the drill hole size (diameter 10.2±0.14 µm) wider than the zona thickness (P<0.0001) and completely penetrated the zona.

Figure 3

Diameters of holes after different power levels (70–100%) of laser pulses at 230 µm duration compared with the thickness of the zona (ZP, zona pellucida). Only at 100% power (300 mW) and at least 230 µs duration was a laser pulse minimally effective to drill a hole significantly (P<0.01) large enough to completely penetrate the zona.

Figure 4

Comparisons of diameters of C57BL/6J oocytes in the absence (A) or presence of sucrose at 0.5 M (B) and 0.25 M (C). Bar=20 µm. Arrows, drilled holes.

Figure 5

Comparisons of oocyte and zona diameters at different concentrations of sucrose (0, 0.25 and 0.5 M). The oocyte cell diameters decreased significantly (P<0.05) when sucrose concentration was increased from 0 to 0.5 M, but the zona diameters remained unchanged (P>0.05).

Figure 6

Comparisons of the distance between the oolemma and laser drill center on the zona and between the oolemma and the 50 °C isotherm ring at different concentrations of sucrose (0, 0.25 and 0.5 M) with a laser pulse of 300 mW and duration 230 µs. In the absence of sucrose, the distance between the oolemma and the 50 °C ring was negative because the ring overlapped the oolemma at 1.7±0.6 µm. In the presence of 0.25 and 0.5 M sucrose, this distance was 7.9±0.9 and 11.2±0.6 respectively.

Figure 7

Comparisons of fertilization rates of LZD-assisted IVF vs regular IVF in four GM mouse lines using the same three males of each strain. Experiments were repeated three times, and over 150 oocytes were used for each IVF procedure. Regular vs laser-assisted, P<0.05 in each strain.

Figure 8

Comparisons of male-to-male fertilization rates of LZD-assisted IVF with unassisted cumulus-free IVF and regular IVF in nine GM mouse lines. (1) Regular IVF, (2) unassisted cumulus-free IVF, and (3) LZD-assisted IVF. Two replicates of experiments were performed for each of the three IVF types in each GM line, and over 100 oocytes were used for each IVF procedure. In all nine GM lines tested fertilization rates of LZD-assisted IVF vs regular IVF, P<0.05. Males from GM lines D, K, N and O showed no difference in fertilization rates between cumulus-free IVF and regular IVF, P>0.05. Males from GM lines R and S showed lower fertilization rate after cumulus-free IVF compared with that of regular IVF, P<0.05. Males from GM lines H and Q showed higher fertilization rates after cumulus-free IVF (P<0.05) compared with regular IVF but still lower than after LZD-assisted IVF (P<0.05). In only GM line P was the fertilization rate after cumulus-free IVF increased and equivalent to that of LZD-assisted IVF (P=0.63).