Any woman undergoing IVF/ICSI treatment receives hormone injections during the first 12 days of her menses cycle to make her produce at least more than 5 follicles resulting in increased chance of conceiving in that cycle. To monitor the development of ovarian follicles, Ultrasound examination is done 3-4 times in that cycle along with checking of estradiol level in her blood.Well stimulated ovary will look like this under trans-vaginal ultrasound examination.

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    Ultrasound Picture of Ovary with multiple follicles

About approximately 12-14 days after such hormone injections, patient is ready for egg collection. On the day of egg collection, patient is given mild sedation and local anaesthesia, follicular fluid is collected with the help of an aspiration pump and a special egg collection needle under trans-vaginal ultrasound guidance, taking care to keep the temperature of follicular fluid at 37’C.

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    Egg Collection showing Aspiration pump
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    Egg Collection needle US machine

Eggs are handled in sterile environment under bacteria-filtered positive air pressured room under a laminar air flow module.

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    Egg Identification in Laminar Air Flow
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    Clean Air positive pressure Module

The culture media used for growth of embryos are mostly ready-to-use imported material. As a quality control measure, the are media are checked regularly for pH and osmolarity using pH meter and osmometer.

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    Temperature is monitored by micro
    thermometer
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    Osmometer
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    pH meter reading the pH of special the medium
    in the incubator

Baheti Hospital’s IVF Unit is equipped with two CO2 Incubators and one recently added triple gas benchtop incubator. This triple gas incubator is specifically designed to provide better culture conditions compared to conventional CO2incubators. The low oxygen environment in this incubator helps grow better quality embryos.

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    Planer triple Gas Incubator
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    Binder CO2 Incubator
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    Forma CO2 Incubator

All are running simultaneously as a backup support in case any one of them fails. All the incubators and other IVF equipment are getting clean and uninterrupted power supply through online UPS. In case of equipment failure or CO2 gas exhaustion, an external siren gets activated to alert not only the Embryologist but all the staff of the Hospital to enable them to attend to the faults and rescue the precious eggs/embryos instantly. Besides the UPS, we do have the support of a generator to give us power supply during occasional long power failure.

Once the eggs are in the incubator, husband’s semen is washed and motile sperms are collected for further processing for either IVF or ICSI.

For fertilization, washed sperms from the husband’s semen are added in required number to the eggs in case of IVF.

In case, the sperms are low in number or are of poor motility grade, ICSI is done on eggs with the help of a micromanipulator. Size of an egg is approximately 0.15 mm and the size of a sperm is approximately 0.005 mm.

Hence, an inverted microscope is needed to magnify them to 200 to 400 times so that they can be visualized during the procedure. To do the high-precision ICSI work, this microscope has been improvised with a micromanipulator to hold an egg on one side and pick up and inject a single sperm into a single egg through the opposite side as shown below.

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    Nikon Narishige Micromanipulators
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    ICSI

After IVF/ICSI, cultured embryos are examined the next day morning for any sign of fertilization which is 2 PN (one pronucleus each from sperm and egg) appearing inside the egg. On further culturing, 2PN embryos will divide to become 4-cell by next day morning and 8 celled by day 3.

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    2 PN : the sign of fertilization on Day 1
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    4 celled embryo on Day 2

On further culturing, by Day 5, embryos will develop to a stage called Blastocyst. It is the stage when the the embryo enters and implants in mother's womb. Therefore, we prefer to transfer embryos at this stage to the patients' uterus

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    Blastocyst on Day 5
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    Hatching Blastocyst before implantation

Remaining surplus embryos are cryopreserved for future use for the same patient in case of failure of this fresh IVF/ICSI cycle. Embryos are frozen on small carriers which are labeled with patients name, date etc. and are stored under liquid nitrogen (-196’C). We use vitrification technique which is highly efficient technique to preserve cells.

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    Embryos stored in Liquid N2
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    Slow Freezing equipment