comparison of two enzyme treatments for the isolation of human follicles renee hodgson 1, kelly...

Comparison of two enzyme treatments for the isolation of

human folliclesRenee HODGSON1, Kelly LEWIS1, Debra GOOK 1,2, David

EDGAR1,2 1Reproductive Services and Melbourne IVF, Royal Women’s

Hospital, Parkville, Victoria, Australia.2 Department of Obstetrics & Gynaecology, University of

Melbourne, Parkville, Victoria, Australia.

1. Is it possible to isolate human follicles successfully using enzymatic digestion?

2. How does enzymatic digestion affect subsequent follicle development?

3. What are the challenges faced in isolating human follicles?

1. Is it possible to isolate human follicles successfully using enzymatic digestion?

Figure 1: Partially isolated human primordial follicle, stained with Neutral Red, surrounded by stromal cells and encapsulated in alginate.

• Enzymatic digestion appears to be an option to aid in isolation of human follicles.

• Both treatments in this experiment were successful in initial partial isolation of follicles.

1. Liberase DH,

2. Digestion cocktail:- Collagenase IV-S + DNase 1 + Protease Inhibitor.

2. How does digestion affect subsequent follicle development?

• Follicle survival following 15 days of culture was poor in both groups:

1. Liberase DH - 22% (11/50), and

2. Digestion cocktail - 19% (9/47).

• No significant difference between oxygen concentrations:

5% O2 = 22% live (n=49), and

20% O2 = 19% live (n=48).

• Selecting enzymatic digestion conditions that enable easy, further mechanical isolation, but do not damage individual follicles, appears to be the greatest challenge.

Since this experiment we have made modifications to the protocol such as:• Differing combinations and duration of enzyme exposure;• Increasing the number of washes to effectively arrest digestion; • Altering culture conditions to include 10% FCS;• Removing neutral red from digestion and culture.

• However, with all further modifications, no real success was achieved, apart from one primary follicle.

3. What are the challenges faced in isolating human follicles?

• A reliable method for the isolation of human preantral follicles that does not also negatively impact their subsequent development still remains to be established.

Figure 2: A day 7 human isolated primary follicle stained with Neutral Red.