The goal of this movie is to demonstrate the procedure of our new approach to patients with ovarian dysfunction. Ovarian function declines progressively during aging and some pathophysiological conditions including karyotype abnormality, auto immune diseases, chemo and radiation therapies, and ovarian surgeries. Most of middle-aged women with a low ovarian reserve showed poor ovarian response to ovarian stimulation for yielding mature oocytes.
Furthermore, young patients with a low number of antral follicles also showed poor ovarian response to the ovarian stimulation. Fertility preservation is important to save the potential OCS for future pregnancy. For fertility preservation, oocyte cryopreservation was established.
However, these poor ovarian response patients could retrieve a very limited number of oocytes even after proper ovarian stimulation, leading to the requirement of repeated expensive procedures to ensure a sufficient number of oocytes to guarantee one baby. To solve these issues, we have recently developed the in vitro activation, IVA procedure, which enables us to stimulate the early stage of ovarian follicles to develop to pre-antral follicle stage. The original IVA procedure can promote primordial, primary, and secondary follicle growth by ovarian cortex fragmentation, leading to hippo signaling disruption.
This is then followed by two days of culturing with Akt signaling stimulator. This procedure is suitable for POI patients with few residual follicles. However, this procedure is an excessive treatment for the patients who have multiple secondary follicles.
For the patients who have some residual secondary follicles, we established the method called drug-free IVA and have already tested the possibility of promoting secondary follicle growth by only disrupting the hippo signaling pathway. We've also had some successful cases of delivery by drug-free IVA. We have already published the surgical procedure of the drug free IVA and the protocol of following ovarian stimulation.
In this video, we present the details of laboratory methods required for drug-free IVA. We recruited the patients for drug-free IVA based on the Bologna criteria. The patients could have multiple secondary follicles.
At first, part of the cortex was removed from one or both ovaries by laparoscopy. POR patients are those who had middle-size ovaries. In most of the patients, we extracted only part of the cortical tissues from the ovaries on one or both sides.
Extracted critical tissues were immediately transferred to a culture dish containing culture medium with HEPES buffer, and dissected this small tissue into small cubes for hippo signaling disruption. Details of the procedure will be shown in this video. Hippo signaling pathway is essential to maintain optimal organ size.
This pathway consists of several negative growth regulators. Once the hippo signaling is disrupted, phosphorylation levels of YAP, one of the negative growth regulators acting in a kinase cascade, is decreased and nuclear levels of YAP is increased. YAP acts in concert with TEAD transcriptional factors to increase downstream CCN growth factor.
On the other hand, actin polymerization of globular acting, G-actin, to the filamentous form, F-actin, is important for cell shape maintenance and locomotion. Previously reported induction of F-actin formation disrupts hippo signaling and induces overgrowth in human HeLa cells. We previously reported ovarian fragmentation increase F-actin formation, and it leads to the decrease of the level of phosphorylation of YAP.
It leads to increased expression of CCN growth factor. After cutting, these cubes are grafted into the remaining ovary. This procedure can promote the residual secondary follicle growth and lead to an increased number of matured oocyte.
Drug-free IVA consists of four steps. We have already published the surgical procedure for drug-free IVA. Please refer to that literature for ovarian cortex extraction and auto-grafting.
Here we show the detail procedures of laboratory methods required for drug-free IVA. In step one, we show how ovarian cortex fragmentation is performed. In step two, we show how to load the ovarian cortical cubes into the device for transplantation, Here, I will show ovarian cortex fragmentation.
This study was approved by ethical committee of our institute, and all procedures were carried out in accordance with the Code of Ethics of World Medical Association. Ovarian cortex fragmentation. The following tools required for this step:a dish, sterile gauze, fine scissors, fine forceps, and scalpel.
We usually use this type of dish and sterile gauze but other products like plastic Petri dish, glass dish, et cetera, may also be suitable for this procedure, and any type of sterile gauze is also suitable. We do however strongly recommend the use of this shape of forceps and scalpel. In addition, use the hot plate for maintaining the temperature of the dishes containing tissues Place under the sterile drape.
Cortical tissues are immediately transferred into the dish containing mHTF. Take a picture with patient's name for correlating with patient's data. Record the size, thickness, and condition of the tissue.
Remove residual medulla tissue. Before tissue fragmentation, remove the medulla tissue. Put the cortex on the sterile gauze.
As shown in the video, the gauze is moistened with medium. During this procedure, apply medium with a disposable pipette before the surface of cortex is dried out. Remove residual tissues with micro scissors until the thickness reaches one to two millimeters.
In POR patients, secondary follicles are located deeper than one millimeter from cortex surface, so do not make the tissues less than one millimeter in thickness. Cut one side of the cortex of each tissue into a strip measuring one by one by five millimeter for histological analysis to determine the presence of residual follicles. Put this strip into 1.5 milliliter tube with culture medium and keep at four degrees Celsius until fixing.
Preparation of ovarian cortical cubes. After removal of medulla tissue, ovarian cortical is fragmented for hippo signaling disruption. At first, cut the cortex into one by one by 10 millimeter strips using scalpel.
Then cut these strips into one millimeter cubed small cubes. In the medium, the tissue is slippery and difficult to cut. For easy cutting, cut the strips by pressing down on the scalpel rather than by pulling on it.
After cutting, count the number of cubes and prepare the auto-grafting. To avoid the changes of osmotic pressure in the medium, add the medium in moderation before tissue auto-grafting. For ovarian auto-grafting, use of a cannula-shaped device allows for easier loading.
This step contains two steps. Previously, ovarian fragments were transplanted one by one using forceps under laparoscopy. However, it took about three to four hours for surgery to be completed.
In this procedure, we use a cannular-shaped device made of an inner syringe and an outer cylinder. This is a simple shape and lightweight. In addition, the tip of this device is of a round shape.
There's no sharp points which avoids damage to the transplant site. Using this type of device allows bringing up to 20 to 30 cubes to implantation site at one time. This leads to efficient transplantation and reduction in the surgery time.
Loading ovarian cortical fragments. Preparation of IVA cannula. Prepare the IVA cannula and wash this device in mHTF.
Repeat aspirate and discharge medium several times. Fill the tip of a cannular by aspirating about 100 microliters of the medium. This is to avoid the cubes from drawing out during tissue fragment loading.
Loading cortical cubes. Pick a cube by fine forceps and put it into the tip of the cannular. The number of auto-grafted cubes depends on the size of the transplant site.
Repeat loading the cubes one by one until you reach the target number. After loading the cubes, pass the cannula to the surgeon carefully. If the grafting site is not ready, the part of the cannular that contains ovarian cubes is to be held by fingers to avoid a decrease in the temperature of cubes.
We have already reported the clinical results of drug-free IVA. Please refer to the published paper about clinical results. This laboratory protocol required 30 to 50 minutes for one patient.
The average time is for 10 to 20 minutes for ovarian cortex fragmentation, and 20 to 30 minutes for loading these fragments to IVA cannula. This depends on the tissue size, but almost all patients can be finished in this average time.Conclusions. Drug-free IVA is a new approach of infertility treatment for POR patients with diminishing ovarian reserve.
These laboratory protocol leads to reduction of both time required for surgery and the loss of fragments during transplantation. The laboratory technique of drug-free IVA could become the basis of future application of ovarian tissue culture to obtain mature oocytes without grafting.
