MEASURING THE REFRACTIVE INDEX OF OOCYTES BY

MEANS OF AN OPTICAL MICROSYSTEM

A Non Invasive Technique to Estimate the Evolution of Oocytes Maturity

Ioana R. Ivascu

Physics Department I, Applied Sciences Faculty, Bucarest “Politehnica” University

Splaiul Independentei, Bucuresti, 060042, Romania

Rabah Zeggari, Christian Pieralli, Bruno Wacogne

Institut FEMTO-ST, UMR CNRS 6174, Département d'Optique P. M. Duffieux, Faculté des Sciences et des Techniques

Route de Gray, 25030 Besançon Cedex, France

Christophe Roux

Service de Génétique Histologie Biologie du Développement et de la Reproduction

UMR645 – Interactions Hôte-Greffon et Ingénierie Cellulaire et Génique & IFR 133: IBCT, CHU St. Jacques

Université de Franche-Comté, 25030 Besançon, France

Keywords: In Vitro Fertilization, Maturation estimation, Optical microsystems, Refractive index measurement.

Abstract: In this paper, we present an optical microsystem used to measure the refractive index of oocytes. The

technique relies on oocytes refraction properties and on the optical coupling between optical fibers. A

theoretical model of the system is established by means of matrix optics. A numerical fitting allows

calculating the refractive index of the oocytes. In a first time we considered the oocytes like homogeneous

spheres. In a second time, we considered that the oocytes consist of a shell (the zona pellucida) and a core

(the cell). Experimental results showed that the refractive index of the oocytes increases during the

maturation process. In this position paper, we also discuss the temporal evolution of the oocytes maturity

and we point out that the maturation should not only be estimated visually but in a more continuous manner.

The device we propose could be a solution for such non invasive measurements.

1 INTRODUCTION

The ability of an oocyte to be fertilized and to

initiate an embryo development strongly depends on

its quality and maturity degree. Usually, the

maturation of an oocyte is estimated visually, by

direct observation with a microscope. The goal is to

observe the presence or the absence of particular

morphological features. The effectiveness of this

visual inspection is therefore highly biologist

dependent. Consequently, there is a crucial need for

more objective technique, and hopefully, more

effective methods.

Since the beginning of IVF (in vitro

fertilization), a few technological developments

have been investigated. They mainly concern the

preparation, the conditioning and the conservation of

gametes, as well as culture of the oocytes and

embryos (Suh, 2003). Some publications report

innovations in the technological domain. Some of

them concern the preparation, the conditioning and

the manipulation of the oocytes in micro-fluidic

systems (Beebe, 2002, Zeringue, 2001, Gauthier,

2002, Sadani, 2005, Zeggari, 2007, Kricka, 1997).

Other techniques are developed to estimate the

maturity of the semen (Danielli, 1952). The oocytes

maturity has also been investigated by means of

mechanical measurements. In this case they were

mainly conducted on animal oocytes (Ohtsubo,

1985, Sawai, 1974, Nakamura, 1978, Murayama,

2004). It must be noted that, to our knowledge, only

one conference (Inui, 2006) and one paper

(Wacogne, 2008) reports experiment on human

127

R. Ivascu I., Zeggari R., Pieralli C., Wacogne B. and Roux C. (2010).

MEASURING THE REFRACTIVE INDEX OF OOCYTES BY MEANS OF AN OPTICAL MICROSYSTEM - A Non Invasive Technique to Estimate the

Evolution of Oocytes Maturity.

In Proceedings of the Third International Conference on Biomedical Electronics and Devices, pages 127-132

DOI: 10.5220/0002756201270132

 SciTePress

oocytes. The invasive or non invasive aspect of the

techniques is probably an important issue.

Concerning non invasive techniques, optical

methods offer potential alternatives. They have been

tested on living cells (Shaked, 2009, Marquet, 2005)

or on oocytes (Zeggari, 2007, Pieralli, 2009). In this

paper, we present a simple micro-system used to

measure the refractive index of the oocytes. It is

based on work we have presented some time ago

(Zeggari, 2006). In this reference, we showed that

oocytes can optically act as microlens. Therefore, if

an oocyte is used as a coupling element between

optical fibers, it is possible to compute its refractive

index by analyzing the optical coupling. However, in

this feasibility study, refractive index measurement

was only demonstrated with one oocyte.

Furthermore, no particular attention had been paid

on the coupling modeling. Recently, we set up a

more realistic model for the optical properties of the

oocytes and we computed refractive indices of a

larger number of oocytes. This is the subject of this

paper. Refractive indices are measured for 3 oocytes

maturation stages: VG (Germinal Vesicle), MI

(Metaphase I) and MII (Metaphase II). We show that

the refractive index evolves with the maturation of

the oocyte.

The micro-system as well as the experimental

protocol is presented in the next section of this

paper. Experimental results are the subject of section

3 while section 4 deals with a discussion we would

like to propose concerning the temporal aspect of the

oocyte maturation process. Then a conclusion will

be drawn in the last section of this article.

2 OPTICAL MICROSYSTEM AND

EXPERIMENTAL PROTOCOL

2.1 Experimental Set-up

A schematic diagram of the device is shown in

figure 1(a). It consists of a Deep Reactive Ion

Etched micro-machined piece of silicon anodic

bonded onto a glass substrate. The main features are

two U-grooves into which optical fibers are glued. In

this way, optical fibers are perfectly aligned. A

specific pigtailing arrangement was designed in

order to precisely adjust the distance between the

two fibers (500 µm). Fibers were conventional 125

µm diameter fibers. The core of one fiber is 50 µm

in diameter. It is the illumination fiber. The core of

the second fiber (the collection one) is 100 µm in

diameter.

The principle is to bring the oocyte between the

two optical fibers by means of the holding micro-

pipette commonly used in the conventional IVF

micro-manipulators. The idea is to analyse the

optical intensity collected for different positions of

the oocyte between the fibers. Figure 1(b) helps

understanding the principle of measurement. When

the cell is positioned close to the illumination fiber,

a large amount of light is collected due to the

refractive index of the cell that plays the role of a

microlens. Conversely, when the cell is positioned

close to the collection fiber, the amount of collected

light is much lower. The maximum of collected light

is obtained for a position between the two fibers.

Figure 1: Experimental set-up and principle of the

technique. (a) Schematic diagram of the device. (b)

Illustration of the coupling efficiency as a function of the

oocyte position. (c) Example of measurement.

2.2 Measurement Method

The method we used consists in measuring the

intensity for 3 positions of the cell: in contact with

the illumination fiber, right in the middle of the

fibers and in contact with the collection fiber. In this

way, 3 experimental points are obtained as it is

shown in figure 1(c). A computing model is then

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employed in order to fit theoretical data with

experimental points. We first consider that the

collected intensity strongly depends on the refractive

index of the oocyte and weakly on its absorption.

Indeed, absorption of the oocytes was estimated

by means of image processing performed on pictures

usually taken in the Besançon's IVF center

(comparison of the average grey level of the oocyte

and the grey level of the background). We found that

the absorption is about 6%.

From there, a Matlab programme is used to

compute the intensity collected as a function of the

refractive index and for the 3 above mentioned

positions of the oocyte. Iteration by iteration, a value

of the refractive index "n" is found when the average

distance between the experimental points and the

computed ones is minimum (least mean square

criterion).

2.3 Oocytes Preparation

Oocytes used in this study were excluded from

Assisted Reproductive Technology (ART) programs.

Their use has been approved by the clinical ethic

committee of the Besançon University Hospital the

of May 2004.

Oocytes are collected during attempts of ART

(by means of ICSI - Intracytoplasmic Sperm

Injection) or from follicular reduction. The complex

cumulus-oocyte are individualized under a binocular

microscope and transferred into a culture medium.

After 90 to 120 min of in vitro culture at 37 °C, the

cumulus is removed. This lead to a perfect

visualization of the oocytes under a microscope and

allows determining their maturity degree. Oocytes

are individually transferred into plastic boxes with

100 µl of culture medium. Their maturity degree is

defined by visual inspection with an inverted

microscope.

200 µl of IVF culture medium is deposited

between the two fibers of the device. One oocyte is

collected in the plastic box in 20 µl of IVF medium

and positioned against the emission fiber. The

holding micro-pipette is employed in order to

precisely position the oocyte. Collected intensity is

recorded and the oocyte is moved to the middle of

the two fibers where the second measurement is

made. Then, the oocyte is positioned against the

collection fiber for the last measurement.

Once the measurements are made, the oocytes

are replaced in the IVF medium in the plastic box.

Then, they are incubated in a Sanyo CO

incubator

(35 °C, 5% CO

). Their possible evolution (in terms

of maturity) is controlled 4 and 17 hours after the

first maturity evaluation with the microscope. If the

maturation stage of the oocytes has evolved, another

refractive index is measured.

2.4 Theoretical Models

As previously mentioned, a theoretical model of the

oocytes was defined in order to simulate their optical

properties. This model relies on the use of matrix

optics. Figure 2(a) shows a picture of an oocyte.

Two main features are visible: the cytoplasm and the

zona pellucida.

Figure 2: Description of the theoretical model used in this

study. (a) The two main features of the oocyte (zona

pellucida and cytoplasm). (b) Homogeneous model. (c)

Core-shell model.

In a first time, we simply considered the oocyte

as a homogeneous sphere whose refractive index is

(figure 2(b)). The center of the oocyte coincides

with the optical axis of the fibers. The refractive

index of the culture medium is n

. In a second time,

we constructed a more complex model where we

accounted for the "core-shell" structure of the oocyte

(figure 2(c)). Here, the refractive indices of the

cytoplasm and the zona pellucida are n

and n

respectively.

MEASURING THE REFRACTIVE INDEX OF OOCYTES BY MEANS OF AN OPTICAL MICROSYSTEM - A Non

Invasive Technique to Estimate the Evolution of Oocytes Maturity

129

3 EXPERIMENTAL RESULTS

Experiments were conducted with 30 oocytes. They

belong to 3 maturity classes: VG (10 oocytes), MI

(10 oocytes) and MII (10 oocytes). All of them were

collected at the VG maturation stage. After about 5

hours of incubation they evolved to the MI stage.

About 15 hours later, they became MII. Refractive

index measurements were made at each maturity

degree.

3.1 Preliminary Experiments

As previously stated, the absorption of the oocytes

was estimated by means of image processing. This is

described in figure 3. Figure 3(a) shows an oocyte.

The idea is to separate the part of the image

corresponding to the oocyte from the background.

To this end, a semi automatic thresholding is used.

The result is shown on figure 3(b). Once the oocyte

is isolated, the absorption is obtained by dividing the

average grey level of the oocyte by the average grey

level of the background. In our case, the average

absorption of the oocytes was 6%.

Figure 3: Image processing used to estimate the absorption

of the oocytes. (a) The initial image. (b) Result of the

image processing when the oocyte is isolated.

The second preliminary experiment concerned

the refractive index of the culture medium n

. To

measure it, we recorded the collected intensity

before and after the culture medium is deposited

between the fibers. Ray tracing is used to compute

the refractive index of the medium. In our case we

found n

=1.868.

3.2 Experimental Results

Two type of measurements were considered. In a

first time, we calculated the average refractive

indices in each maturity class. We did it with both

theoretical models. Results concerning the

homogeneous model are summarized in table 1

while those concerning the core-shell model are

presented in table 2.

Table 1: Average refractive index of the oocyte for the 3

maturation stages. Homogeneous model.

Stage Average n

VG 1.68

±0.03

M I 1.76

±0.04

M II 1.79

±0.03

Table 2: Average refractive index of the oocyte for the 3

maturation stages. Core-shell model.

Stage Average n

Avergae n

VG 1.79

±0.05

2.18

±0.02

M I 1.86

±0.06

2.10

±0.02

M II 1.88

±0.05

2.21

±0.02

In both cases, we observe that the refractive

index increases with the maturity degree of the

oocytes. In the case of the core-shell model, it seems

that the refractive index of the cytoplasm increases

while nothing can really be said about the refractive

index of the zona pellucida. Also, if we consider the

values of the standard deviations we can say that,

although the 3 maturation stages can be

individualized, it seems impossible to use this

optical technique to qualify the maturity of an

unknown oocyte.

However, is this result surprising? Indeed,

concerning the maturity degree of the oocytes, we

are analyzing cells that continuously evolve between

consecutive stages. Therefore, should we look for a

discrete separation between the optical properties of

the different stages? We would like to propose a

discussion on this aspect in part 4 of this paper.

We therefore considered the temporal evolution

of the refractive indices for each individual oocyte.

Results are presented in table 3 and 4. Table 3 shows

the results for the refractive index of the cytoplasm

while table 4 shows the result for the zona pellucida.

Here, we note that the general tendency is an

increase of the cytoplasm refractive index (except

for oocytes #6 and #9). Again, the case of the zona

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pellucida is no very clear. It should also be noted

that in some cases, the theoretical model did not

produce coherent values. This aspect is not yet

understood.

Table 3: Temporal evolution of the refractive index of the

cytoplasm.

at VG

at MI

at MII

oocyte #1 1.78 1.79 1.91

oocyte #2 1.83 1.93 -

oocyte #3 1.74 1.84 1.89

oocyte #4 1.745 1.85 1.89

oocyte #5 1.70 1.84 1.86

oocyte #6 1.84 1.79 1.79

oocyte #7 1.78 1.85 1.88

oocyte #8 1.83 1.84 1.87

oocyte #9 1.86 1.85 -

oocyte #10 1.88 1.915 1.924

Table 4: Temporal evolution of the refractive index of the

zona pellucida.

at VG n

at MI n

at MII

oocyte #1

1.95 1.81 2.26

oocyte #2

2.36 2.47 -

oocyte #3

1.99 2.03 2.4

oocyte #4

1.93 2.02 2.29

oocyte #5

1.82 2.20 2.13

oocyte #6

2.45 2.14 1.89

oocyte #7

1.94 2.07 2.24

oocyte #8

2.28 2.24 2.21

oocyte #9

2.42 2.22 -

oocyte #10

2.47 2.24 2.32

4 DISCUSSION

In this section, we would like to put to profit the fact

that this is a position paper. As it is mentioned

above, the 3 maturity classes are easily identified.

But it seems difficult to use the refractive index to

precisely define the maturation stage of an oocyte.

We think that this result is not very surprising

because the oocytes are subject to a continuous

maturation process from VG to MII. Therefore, it is

not likely that the refractive indices discretely differ

from one class to another one. In order illustrate this

let us remember that usually we see seven colors in

the rainbow. Apparently, the white light spectrum,

when visually observed, exhibits seven different

color classes. However, when a spectrogram is

recorded, the seven color classes cannot be

distinguished anymore. Probably, the qualification

of oocytes in 3 classes, visually identified by various

morphological characteristics, could be improved by

analyzing some of the oocytes optical properties on

a more continuous manner. The measurement of the

refractive indices may offer an answer to this

question.

The above mentioned remarks are obviously

opened to further discussions. But we think that the

oocytes should be chosen when they are not only

MII but also at the right moment when they are MII.

We believe that the use of new qualifying techniques

that allow taking into account the temporal evolution

of the oocytes would be profitable to IVF

techniques. At present, the device we present in this

paper should still be improved before it can be used

on a routine basis. However, an alternative technique

we presented recently (Pieralli, 2009) and based on

image processing could be envisaged.

5 CONCLUSIONS

In this paper we have presented a simple optical

micro-system used for measuring the refractive

index of oocytes. The device consists of a Deep

Reactive Ion Etched piece of silicon anodic bonded

onto a glass substrate. Two perfectly aligned

multimode optical fibers are attached to the device.

The oocyte is positioned between the two fibers by

means of the holding micro-pipette usually

employed in IVF centers. White light is launched

into one optical fiber and illuminates the oocyte

under test. Once the light has propagated through the

oocyte, it is collected by the second optical fiber and

the optical intensity is recorded. The collected light

is measured for 3 positions of the oocyte between

the fibers. In this way, and using a mathematical

fitting, refractive indices of the oocytes can be

measured.

Two theoretical models were developed. In one

case, the oocyte was considered as a homogeneous

sphere. In a second case, we took into account the

core-shell structure of the oocytes. In both cases, the

MEASURING THE REFRACTIVE INDEX OF OOCYTES BY MEANS OF AN OPTICAL MICROSYSTEM - A Non

Invasive Technique to Estimate the Evolution of Oocytes Maturity

131

refractive index of the cytoplasm tends to increase

with the degree of maturity. Although the maturity

classes can be visually distinguished, it seems

difficult to use the refractive index value to estimate

the maturity of a completely unknown oocyte. We

proposed a discussion on this issue. We think that,

and this is perhaps an interesting conclusion of this

work, oocytes should not only be qualified in terms

VG, MI and MII but also regarding their temporal

evolution over these maturation stages.

Studies on a much larger number of oocytes are

still required. Also, the investigation of other optical

characteristics should be envisaged. At the end, we

hope that these studies will help biologists in

choosing not only the right oocyte but also the right

oocyte at the right moment.

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