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Effect of cumulus cells and vitrification protocol on survival and subsequent development

Golestan jahromiPhD student

Introduction

Several lines of evidence indicate that

surrounding cumulus cells play a fundamental

role in the maturation process and full

development competence

Introduction

cumulus cells are beneficial to oocyte survival after cryopreservation

may minimize the release of cortical granules prevent premature zona hardening

maintaining fertilization capacity of cryopreserved oocytes

Introduction

Chian et al. reported that bovine oocytes matured without cumulus cells had a higher survival rate after vitrification.

Moreover, the rate of embryo development to the 8-cell stage in cumulus-cells free oocytes was significantly higher than that of cumulus cell-intact oocytes.

Introduction

Ice growth and recrystallization are considered to be important factors in determining vitrification outcomes. Synthetic ice blockers, which specifically inhibit the formation/emergence of ice nuclei and ice crystal growth, have recently been used to supplement vitrification solutions

Introduction

Unlike conventional cryoprotectants that inhibit freezing by interacting with water, ice blockers are believed to bind to the surface of growing ice crystals and inhibit the addition of any further water molecules in specific planes of growth This selective attraction to surfaces of ice growth permits ice blockers to exert significant effects even while present at very low concentrations.

Introduction

Small quantities of ice blocker can therefore modify the number and size of ice crystals and thereby change the vitrification tendency of a solution without adding additional toxicity

Introduction

The commercially available ice blockers are SuperCool X-1000 and SuperCool Z-1000.

To evaluate the effect of the presence of cumulus cells on the outcome of vitrification of GV or MII oocytes

The effect of adding ice blockers SuperCool X-1000 and SuperCool Z-1000 to vitrification media on oocyte survival and subsequent embryonic development

Materials & methods

Collection of oocytes

IVM

Vitrificationwarming

IVF

According to the manufacturer, the cooling and warming rates of the Cryotop are 23,000 and 42,000 °C/min, respectively

Motile spermatozoa were obtained by centrifugation of frozen–thawed semen

Day 2 after insemination

Day 8 after insemination

Cleavage rates proportion of oocytes developing to the blastocyst stage

Experiment 1COCs

GV

Cumulus- enclosed

partially-denuded oocytes

MII

Cumulus-enclosed

partially-denuded oocytes

Experiment 2: Effect of ice blocker X-1000 and Z-1000COCs

Control Basic media

Basic media + X-1000

Basic media + Z-1000

Basic media + X-1000 + Z-

1000

VS +1% (v/v) X-1000

VS +1% (v/v) Z-1000

VS +1% (v/v) Z-1000 and 1% (v/v) X-1000

Statistical analysis

The data for survival, cleavage and blastocyst rates were expressed as mean ± SD and analyzed using one-way ANOVA.

Differences were considered significant at a level of P < 0.05.

Results

development of bovine GV stageOocytes treated

N Survived, n (%)

Cleaved, n (%)

Blastocyst, n (%)

Blastocyst/cleavage (%)

Cumulus-enclosed control

141 141 (100 ± 0.0)

121 (86.3 ± 1.9)

47 (33.8 ± 1.8)

(39.5 ± 2.2)

Partially-denuded control

118 117 (99.3 ±0.8)

89 (75.8 ± 3.9)

14 (11.5 ± 4.2)

(14.8 ± 5.2)

Cumulus-enclosed vitrified

177 166 (93.8 ± 2.5)

108 (65.8 ± 5.6)

19 (11.3 ± 1.7)

(18.0 ± 3.5)b

Partially-denuded vitrified

143 117 (81.3 ± 3.6)c

56 (47.3 ± 4.0)c

4 (4.0 ± 2.3)c

(7.8 ± 4.5)b

embryo development of bovine MII stage

Oocytes treated

N Survived, n (%)

Cleaved, n (%)

Blastocyst, n (%) Blastocyst/cleavage (%)

Cumulus-enclosed control

130 126 (96.8 ± 1.5)a

115 (91.6 ± 2.5)a

45 (35.6 ± 2.8)a (39.0 ± 4.1)a

Partially-denuded control

122 119 (97.4 ± 1.1)a

92 (78.4 ± 4.7)b

32 (27.2 ± 2.3)b (34.6 ± 2.7) a,b

Cumulus-enclosed vitrified

158 147 (93.0 ± 2.3)a

51 (35.2 ± 4.6)c

7 (5.0 ± 4.3)c (12.6 ± 9.7) b,c

Partially-denuded vitrified

167 153 (91.8 ± 2.4)a

57 (36.8 ± 3.2)c

7 (4.4 ± 1.4)c (10.8 ± 3.5)c

development of bovine GV stage cumulus-enclosed oocytes vitrified with different ice blocker media

Oocytes treated N Survived, n (%)

Cleaved, n (%) Blastocyst, n (%)

Blastocyst/cleavage (%)

Control 110 104 (94.4 ± 1.9)a

77 (74.6 ± 3.2)

24(23.0 ± 10.4)

(31.2 ± 1.9)

Basic media 114 98 (86.0 ± 2.7)b

38 (38.0 ± 3.2)

2 (2.0 ± 1.3) (6.2 ± 4.1)

Basic media + X-1000

111 97 (88.4 ±2.9)

36 (37.2 ± 2.4)

3 (2.8 ± 1.2) (7.8 ± 3.2)

Basic media + Z-1000

110 93 (83.6 ± 4.8)

37 (40.0 ± 3.0)

2(2.2 ± 1.4) (4.8 ± 3.0)

Basic media + X-1000 + Z-1000

114 101 (88.0 ± 3.6)

43 (41.4 ± 6.7)

0 (0) (0)

Cumulus-enclosed oocytes vitrified at the GV

stage exhibited a significantly higher cleavage

rate and blastocyst rate than those vitrified at

MII stage (P < 0.05).

Why?

Discussi

on

Discussion

The role of the cumulus cells during vitrification of MII oocytes remains controversial. Some investigators reported that cumulus presence would protect MII oocytes against vitrification-induced damage.

Discussion

Zhang et al. found no difference in the development of vitrified ovine MII oocytes with or without cumulus cells. Gasparrini et al. reported that the presence of cumulus cells severely reduced the cleavage rate of MII buffalo oocytes following vitrification

Discussion

It is generally accepted that cumulus-oocyte

communication via an intact corona radiata is

necessary for oocytes to attain full

cytoplasmic maturation during IVM and

improve fertilization rates during IVF

Discussion

we established that the cleavage rate of

denuded (GV and MII) bovine oocytes was

significantly reduced compared to cumulus-

enclosed oocytes, and almost no denuded

bovine oocytes developed up to blastocyst

stage after in vitro fertilization.

In this study:

The survival, cleavage and blastocyst rate of cumulus-enclosed vitrified oocytes are significantly higher than that of partially-denuded vitrified oocytes.

Discussion

In the present study:No significant differences were detected between vitrified cumulus-enclosed and partially-denuded oocytes in the survival, cleavage and blastocyst rate.

Discussion

The possible explanation is the cumulus was detrimental to vitrification, which comprises the benefits of cumulus in IVF procedure. From another point of view, the intracytoplasmic sperm injection technique rather than conventional IVF has been used to achieve fertilization, which can circumvent the detrimental effects of removing the cumulus on subsequent zona penetrability.

Discussion

The cell cycle stage during meiosis appears to affect the results of bovine oocyte vitrification due to varying sensitivity to cooling procedures. Chilling injury is reported to be higher in vitrified immature oocytes, owing to low membrane stability and susceptibility of the cytoskeleton

Discussion

However, an increase in chromosomal abnormality has been observed in vitrified mature oocytes, owing to alterations in the meiotic spindle.

Discussion

The results indicate that cumulus-enclosed oocytes vitrified at the GV stage exhibited a significantly higher cleavage and blastocyst rate than those vitrified at MII.

Discussion This may be due to the increase in volume associated with cumulus expansion during maturation. It may also be due to the higher water permeability (Lp) and solute permeability (Ps) of MII than GV bovine oocytes. That means the changes of cell volume and intracellular CPA concentrations are more severe in MII than GV bovine oocytes during CPA addition and dilution process, which make it more sensitive.

Discussion

In the present study, we report for the first time the effect of ice blockers on the bovine oocytes, however, the results indicate that the survival rate and development competence of bovine oocytes vitrified in solutions supplemented with or without X-1000 and/or Z-1000 by Cryotop method are not significantly different.

Discussion

Ice blockers did not affect the survival rate and developmental competence of vitrified bovine oocytes.

Why?

DiscussionWhen the vitrification systems are large volume, such as organs, in which a large quantity of nucleators exit, ice blockers can suppress nucleation and recrystallization by binding to nucleators in solutions during vitrification and warming. Therefore, the ice growth was inhibited and damage to the systems was reduced.

Discussion

In this study, the combination of the two ice blocker agents inhibit blastocyst development maybe because 1% X-1000 and 1% Z-1000 is not the ideal balance, which shows less effective than either agent alone.

55

cumulus-enclosed GV bovine

oocytes survived vitrification and

subsequently developed at higher

rates than MII oocytes .

Ice blockers had no effect on

bovine oocyte vitrification.