Noninvasive characterization of oocyte deformability in microconstrictions

Abstract

Oocytes naturally present mechanical defects that hinder their development after fertilization. Thus, in the context of assisted reproduction, oocyte selection based on their mechanical properties has great potential to improve the quality of the resulting embryos and the success rate of these procedures. However, using mechanical properties as a quantifiable selective criterion requires robust and nondestructive measurement tools. This study developed a constriction-based microfluidic device that monitors the deformation of mouse oocytes under controlled pressure. The device can distinguish mechanically aberrant oocyte groups from healthy control ones. On the basis of a mathematical model, we propose that deformability measurements infer both oocyte tension and elasticity, elasticity being the most discriminating factor in our geometry. Despite force transmission during oocyte deformation, no long-term damage was observed. This noninvasive characterization of mouse oocyte deformability in microconstrictions allows for a substantial advance in assessing the mechanical properties of mammalian oocytes and has potential application as a quantifiable selective criterion in medically assisted reproduction.

Fig. 1.. Constriction-based deformability assay for mouse oocytes.

(A) 3D design of the microfluidic device. The zoom shows the 54-μm square constriction in the center of the channel. (B) Image sequence of a representative passage of an oocyte through the constriction. Upper panel: image and annotated diagram of the oocyte before entering the constriction. Scale bar, 10 μm. The bottom panel shows the three phases of deformation we have identified: approach (1), entry (2), and transit (3). The first image of the sequence corresponds to zero flow in the channel (see fig. S1D). The images for which the front of the oocyte enters the constriction (X_Front = 0), the front of the oocyte reaches half the smallest dimension of the constriction (X_Front = d/2), and the rear of the oocyte enters the constriction (X_Rear = 0) are annotated. Scale bar, 50 μm; time in seconds and constriction highlighted in red. (C) Front (dotted line) and rear (solid line) position of the oocyte shown in (B) relative to the start of the constriction. The drawing shows the constriction in dotted red and the initial oocyte position in gray. The constriction is highlighted in red throughout the graph. The first horizontal dotted line indicates the time of zero flow in the channel. The following lines correspond to the annotated images in (B) X_Front = 0, X_Front = d/2, and X_Rear = 0. (D) Aspect ratio of the oocyte shown in (B) as a function of time (in seconds) since the start of the assay and corresponding applied pressure (in mbar). The lines show the aspect ratio and pressure for the three annotated images in (B) X_Front = 0, X_Front = d/2, and X_Rear = 0.

Fig. 2.. Measurement of pressure and aspect ratio during transit through the constriction for mechanically distinct groups of oocytes.

(A) Diagram of methods to obtain the four oocyte groups from oocytes arrested in prophase I. The double red bar indicates the resumption of meiotic division identified by NEBD. Early meiosis I corresponds to oocytes 2 to 3 hours after NEBD; late meiosis I to oocytes 6 to 8 hours after NEBD. cVCA oocytes are obtained by microinjection of cVCA mRNA in prophase I, and measured 4 hours after NEBD. (B) Oocyte aspect ratio as a function of applied pressure for a representative early meiosis I oocyte (gray) and a representative late meiosis I oocyte (orange). The lines show the aspect ratio and pressure (in mbar) for the three critical points: X_Front = 0 (X_f = 0), X_Front = d/2 (X_f = d/2), and X_Rear = 0 (X_r = 0) as described in Fig. 1. The filled-in area highlights the pressure difference between X_f = d/2 and X_r = 0. (C) Normalized pressure measured at the three critical points for early and late meiosis I oocytes and (D) median of normalized oocyte aspect ratio as a function of the median of the applied pressure for X_f = 0, X_f = d/2, and X_r = 0. n = 34 for early meiosis I and n = 35 for late meiosis I from two independent experiments. For each experiment, values are normalized to the median and interquartile range obtained for early meiosis I at X_f = 0. (E to G) Same as (B) to (D), respectively, for control (gray) and cVCA oocytes (blue). n = 46 for control and n = 43 for cVCA oocytes from four independent experiments. For each experiment, values are normalized to median and interquartile range obtained for control oocytes at X_f = 0. Error bars show median and interquartile range; ns, P > 0.05; *P = 0.0244, ***P = 0.0001, and ****P < 0.0001 to Mann-Whitney statistical test.

Fig. 3.. Modeling the deformation of a tensile elastic shell through the constriction.

(A) Diagram of the parameters used in the mathematical model. Shell parameters are shown in red: surface tension ${\mathrm{\tau }}_0$, elastic modulus E, mean shell thickness $t_{\mathrm{h}}$, volume conservation V = V₀. The geometric parameters of the constriction are in blue: minimal diameter d and inclination of the ramped segment α. Oocyte front (X_f) and rear (X_r) positions relative to the constriction entry (X₀) are in green. In black: R_f and R_r and P_f and P_r are the radii and pressures of the front and rear edges used to apply the Laplace law. In gray: $h_{\mathrm{f}}$ and $h_{\mathrm{r}}$ and $D_{\mathrm{f}} \text{and} D_{\mathrm{r}}$ are the heights of the front and rear caps and the channel diameters at the base of the front and rear caps used to calculate the equilibrium pressure difference associated with a given cell configuration (Materials and Methods). (B) Evolution of shell aspect ratio as a function of pressure gradient $P_{\mathrm{f}}-P_{\mathrm{r}}$ calculated with D₀ = 74.4 μm, E = 1.5 kPa, ${\mathrm{\tau }}_0$=1 nN·μm⁻¹, α = 9°, d = 50 μm, and t_h = 24.4 μm. The lines indicate the aspect ratio and pressure for the three critical points: X_Front = 0, X_Front = d/2, and the maximum pressure P_max reached with the modeling. The gray area indicates the pressure difference between P_Xf=d/2 and P_max. X_Rear = 0 corresponds to a hypothetical line and the striped area indicates the deformations observed in our experimental setup but not reached by the model. (C) Critical pressure at X_f = d/2 and (E) pressure difference between P_Xf=d/2 and P_max as a function of shell tension calculated for different elasticity values. m${\mathrm{\tau }}_0$ indicate the slope of the linear regression for the four elasticity conditions in (C). (D) Critical pressure at X_f = d/2 and (F) pressure difference between P_Xf=d/2 and P_max as a function of shell elasticity calculated for different tension values. m_E indicates the slope of the linear regression calculated for the four tension conditions in (D).

Fig. 4.. Meiotic spindle deformation and recovery after passage through constrictions.

(A) Control and cVCA oocytes approaching the constriction with insets encompassing their spindle; scale bars, 10 μm. The diagram illustrates measurements in (B) of spindle major axis and angle to channel axis. The sequences of images cropped from the left oocytes show an image every 15 (control) and 10 s (cVCA). Binarized images with the spindle contour in red are shown. Scale bars, 15 μm. Left: Percentage of spindle elongation relative to its length before oocyte deformation in the constriction. Right: Absolute value of the spindle long axis angle relative to the direction of the channel measured once the oocyte is fully deformed in the constriction (i.e., X_Rear = 0). n = 38 control and n = 33 cVCA from four independent experiments. **P = 0.0038 by t test; *P = 0.0388 by Kolmogorov-Smirnov test. (C) Spindles of control and cVCA oocytes 2 hours after passage through the device (right) or unmanipulated (left). Microtubules are in magenta, DNA in purple, cVCA in cyan, and bright-field images in gray. Scale bar, 10 μm. (D) Spindle length and metaphase plate width for control and cVCA oocytes measured from (C). No microfluidic: n = 12 control and n = 10 cVCA; microfluidic: n = 12 control and n = 20 cVCA from three independent experiments. ns, P > 0.05 for no-microfluidic versus microfluidic by two-way analysis of variance (ANOVA) test applied to spindle length and plate width. For all graphs, red bars represent the median. (E) Percentage of control and cVCA oocytes extruding a polar body (PBE) after recovery from the device or unmanipulated. No microfluidic: n = 22 control and n = 14 cVCA; microfluidic: n = 35 control and n = 25 cVCA from two independent experiments (represented by a dot). The bars represent the median.

Fig. 5.. Recovery of oocytes in meiosis II and zygotes after passage through constrictions.

(A) Left: Percentage of spindle elongation relative to its length before oocyte deformation in the constriction. Right: Absolute value of the spindle long axis angle relative to the direction of the channel measured once the oocyte is fully deformed in the constriction (i.e., X_Rear = 0). n = 23 oocytes in early meiosis I and n = 23 oocytes in meiosis II from three independent experiments. ****P < 0.0001 by t test; ns, P = 0.0591 by Kolmogorov-Smirnov test. (B) Spindles of oocytes in meiosis II 2 hours after passage through the device (right) or unmanipulated (left). Microtubules are in magenta, DNA in purple, cVCA in cyan, and bright-field images in gray. Scale bar, 10 μm. The diagram shows an oocyte in meiosis II with an extruded polar body and a spindle located beneath the cell cortex. (C) Spindle length and metaphase plate width for oocytes in meiosis II measured from (B). n = 7 for no microfluidic and n = 10 for microfluidic from one experiment. ns, P > 0.05 by Mann-Whitney test applied to spindle length and plate width. For all graphs, red bars represent the median. (D) Percentage of zygotes developing up to the blastocyst stage after recovery from the device, or unmanipulated. No microfluidic: n = 83, microfluidic: n = 87 from three independent experiments (two female mice per experiment) represented by a dot. The bars represent the median. (E) Embryos at different stages of development coming from zygotes recovered from the device (bottom) or unmanipulated (top). Scale bars, 30 μm.