Dr. Sai, Senior Embryologist of Malpani Infertility Clinic Pvt. Ltd explains how the magic of in-vitro maturation of oocytes works by giving some real-life experiences of patients.

The Magic of In-Vitro Maturation of oocytes

Dr. Sai, Senior Embryologist, Malpani Infertility Clinic Pvt. Ltd.

Mrs. Bhatt had very poor ovarian reserve. Her AMH level was 0.3 ng/ml and she had reached the oopause. We advised her to use donor eggs but she was quite certain she wanted to have a baby with her own eggs. We explained to her that her prognosis was bleak, but she was determined, and requested us to do our best to help her to have a baby with her own eggs.

We superovulated her aggressively using a letrozole - antagon protocol, with 750 IU of HMG daily. She had a very poor ovarian response as expected, and grew only one follicle. We advised her to cancel the cycle, but she was very keen on getting pregnant and requested us to proceed with the treatment.

Dr. Anjali did the egg collection and retrieved one oocycte cumulus complex from the follicle after flushing it multiple times.

When I stripped the oocyte, it unfortunately turned out to be immature - it was a germinal vesicle stage egg.

We decided to keep the egg for In vitro Maturation (IVM)

The egg matured exactly after 20 hours. I performed ICSI on that egg.

It fertilized and we transferred the embryo back into the uterus on day 2.

It was a gorgeous 4-Cell Embryo.

Even though we got only one embryo, the patient was very happy that at least we had helped her to reach this stage. She had been mentally prepared to get zero eggs and zero embryos, so this was quite a positive development from her point of view.

Thanks to the technique of In vitro Maturation, they got a beautiful embryo to transfer.

We kept our fingers crossed - and 14 days after the transfer, she was on top of the moon when the HCG result was positive, confirming that she was pregnant ! Her pregnancy is now progressing well !

So what is in vitro maturation ? and how do we do it ?

In vitro maturation, as the name suggests, refers to the process of maturing immature oocytes outside human ovaries, in the IVF lab.

Applications of In vitro maturationof oocytes :

• Oocyte donors, to preserve their eggs in egg bank.
• Fertility preservation for women with cancer who are undergoing gonadotoxic chemotherapy.
• Fertility preservation for young women without partners needing IVF treatment.
• Poor responders to ovarian stimulation.
• Patients with lots of immature eggs after egg collection.
• Patients with PCOS syndrome, leading to retrieval of lots of immature eggs, after being hyperstimulated. li>

Mature Oocyte Immature oocytes

In vitro Maturation medium is now commercially available.

At our centre we use "SAGE In vitro Maturation medium"

It is not a ready to use medium. One has to prepare it.

Maturation media is usually supplemented with recombinant FSH and hCG.

The protocol for preparation of In vitro maturation medium is as follows :

Solution A = 1 ml IVF culture medium

Solution B = We use Menogon (HMG). This powder contains a mixture of 75 IU
FSH and 75 IU LH. Dissolve this in 1 ml of IVF culture medium (A).

Solution C = 1 ml of Fresh Oocyte Maturation Medium in a test tube.

Solution D = Add 10 ul Solution B into Solution C

Solution D is now prepared Oocyte Maturation Medium.

In Vitro Maturation of Oocytes

In Vitro Maturation on cumulus-enclosed oocytes :

• Done on oocytes retrieved from small sized follicles.
• Done on oocytes with apparently compact cumulus complexes

Immature oocyte cumulus complex

• Immediately after retrieval, cumulus-enclosed immature oocytes are placed in a specialized IVM medium for 24-48 hours.
• Generally germinal vesicle-stage oocytes that matured within 30 hours of culture are developmentally more competent than are oocytes necessitating longer time to mature.
• After IVM, mature oocytes are transferred to traditional IVF media for insemination and embryo culture.
• Insemination of IVM-Mature oocytes can be done by either Conventional IVF technique or ICSI. ICSI has been our preferred method as oocytes are frequently denuded of granulosa cells for evaluation of maturational status. ICSI has been used to increase the chances of fertilization whether or not a male factor has been detected.

In Vitro Maturation on Stripped oocytes :

• Done on Germinal Vesicle stage oocytes (confirmed after denuding them of the surrounding cumulus cells)
• All Germinal Vesicle Stage oocytes are kept in Specialized IVM medium for 24-48 hours.
• After IVM, Mature (metaphase II) oocytes are transferred to traditional IVF Medium for ICSI.

Germinal Vesicle Stage oocyte. The germinal vesicle is the clear vacuole within the cytoplasm.

Photo of the egg after IVM. It has now become mature (metaphase II - MII) . You can see that the germinal vesicle has dissolved and the polar body can be seen at 12 o'clock.

IVM is not a panacea for all problems - and not all immature eggs will mature in vitro using this technique. However, it does allow us an additional option, and can be very helpful when treating poor ovarian responders !