Temperature and Environmental

Impact on Oocyte after OPU

Mulyoto Pangestu
Dept. Obstetrics and Gynaecology, Monash University
Fak. Peternakan Universitas Jenderal Soedirman
The challenge and changes

Apakah anda berani keluar

dari kandang?

In Vitro:
Temperature; Osmolarity; pH;
atmosphere (gas composition);
mechanics (suction pressure and
needle size)
 What is affected?
• Spindle:
• The human oocyte spindle is highly dynamic and susceptible to
irreversible damage during maturation, retrieval and handling.
• Meiotic spindles are composed of microtubules and are important
for chromosome alignment and separation of maternal
chromosomes during fertilization. The meiotic spindles of most
mammals are very sensitive to fluctuations in temperature.
• Temperature fluctuations during in-vitro manipulation may disrupt
the spindles and contribute to abnormal chromosome distribution
• Disrupted spindle may cause failed fertilization or abnormal
fertilization, such as aneuploidy
• Spindle imaging: a marker for embryo development and
implantation
– Cause: Temperature and Osmotic Shock
 Medium temperature during cooling and
rewarming of human oocytes.

Spindle dynamics after

cooling and rewarming in a
human oocyte.
Wei-Hua Wang et al. Hum. Reprod. 2001;16:2374-2378

© European Society of Human Reproduction and Embryology

 Spindle images in human oocytes after cooling and rewarming.

Wei-Hua Wang et al. Hum. Reprod. 2001;16:2374-2378

© European Society of Human Reproduction and Embryology

 Effect temperature on Spindle
• Spindles had completely disassembled by 5 min after
cooling and recovered by 20 min after rewarming to 37°C if
rewarming started soon after the oocyte's temperature
dropped to room temperature. However, when oocytes
were cooled and kept at 33, 28 or 25°C for 10 min and then
warmed, it was found that warming allowed 5/5, 2/5 and
0/5 oocytes of the spindles to recover respectively
• These results indicate that human meiotic spindles are
exquisitely sensitive to alterations in temperature.
• The maintenance of temperature at 37°C during in-vitro
manipulation is important for spindle integrity and,
therefore, is likely to be important for normal fertilization
and subsequent embryo development.

Wei-Hua Wang et al. Hum. Reprod. 2001;16:2374-2378

© European Society of Human Reproduction and Embryology
 Effect Osmotic Shock on Spindle
• Mature human oocytes were exposed to anisosmotic
sucrose solutions at concentrations of 35, 75, 150, 600,
1200, or 2400 (±5) milliosmolal (mOsm) at 37°C.
Control at 290 mOsm.
• Hyposmotic effects at 35, 75, and 150 mOsm resulted
in 100, 67, and 56% of oocytes having abnormal
spindles, respectively.
• Hyperosmotic effects at 600, 1200, and 2400 mOsm
resulted in 44, 44, and 100% of the spindles with
abnormal structure, respectively
• Anisosmotic conditions lead to disruption of the MII
spindle in human oocytes

Hum. Reprod. (2004) 19 (5): 1148-1154.

 pH and Oxygen
• Exposing oocyte to non physiological pH and
high oxygen may reduce the quality.
• pH may affect metabolic activity (?)
• Oxidative damage and change pH
 How to avoid it?
• Keep everything warm since oocyte come up
from the follicle until it reach the incubator.
• If oocyte have been exposed to lower
temperature (ie. Room Temperature), should be
returned to 37C immediately. These effects were
reversed when oocytes were returned to 37
degrees C after exposure to room temperature
for 2 min, but not for 10 and 30 minutes
• Uses the right media. Do not aspirate oocyte into
an empty tube.
 Warm
• Aspiration needle
• Buffer media for collection or flushing
• Test tube
• Plate
• Stage
• Work in a warm air and right gas mixture.
 OPU equipment

Ultrasound with
vaginal probe

Test tube warmers Vacuum pump

Humidicribs
 Mechanics

• Needle size 18-19 G

• Single lumen or double lumen
• Tubing length
• Aspiration pressure
 Ucapan terima kasih
• Panitia Workshop PIT POGI 22 tahun 2016
• Prof. Peter Temple-Smith; Direktur Education
Program in Reproduction and Development;
Dept. Obstetrics and Gynaecology, Monash
University
• Prof. Dr. M. Sodik; Dekan Fakultas Peternakan
Universitas Jenderal Soedirman Purwokerto.

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