Fertility Preservation in Patients with Severe Ovarian Dysfunction

Summary

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

Abstract

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

Introduction

Ovarian function declines progressively during aging and some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, and ovarian surgeries. Fertility preservation is one of the best options for unmarried women with severe ovarian dysfunction to preserve their potential for future pregnancy. For fertility preservation, currently two methods are available mostly in the onco-fertility field. Oocyte cryopreservation is a well-established procedure for fertility preservation and many successful cases have been reported^1,2. On the other hand, ovarian tissue cryopreservation was also established for fertility preservation in cancer patients but it is still an experimental strategy^3,4. In both methods, multiple numbers of mature oocytes are needed for pregnancy to occur. Generally, patients with premature ovarian insufficiency (POI), who become amenorrhea before 40 years of age, and middle-aged women with low ovarian reserve showed poor ovarian response (POR) to ovarian stimulation for yielding mature oocytes^5,6,7. Furthermore, young patients with a low number of antral follicles also showed POR to the ovarian stimulation⁷. These patients have very limited number of retrievable oocytes even after proper ovarian hyperstimulation, thus requiring multiple expensive procedures to ensure a sufficient number of oocytes for pregnancy.

Oocyte donation followed by in vitro fertilization (IVF) with husband's sperm and embryo transfer (ET) is the only option for these POI and POR patients who have difficulties obtaining their own oocytes^8,9,10. However, oocyte donation is complicated by ethical issues as well as autoimmune and pregnancy complications^11,12,13,14. To solve these issues, the establishment of infertility treatment using patients' own oocytes is desired. For POI patients, we have developed the in vitro activation (IVA) approach to allow successful follicle growth and the generation of mature oocytes, leading a number of pregnancies and deliveries¹⁵. In IVA, we fragmented ovarian cortexes after removal of ovaries under laparoscopic surgery and cultured them for two days to activate follicles by Akt-stimulating drugs and then perform heterotopic grafting back into artificial pouches made beneath the serosa of Fallopian tubes under second laparoscopic surgery¹⁵. This procedure promoted growth of primordial, primary and secondary follicles after ovarian cortex fragmentation to promote Hippo signaling disruption¹⁶, followed by two days culture with Akt signaling stimulators¹⁷.

In contrast to severe POI cases, POR patients with decreased ovarian reserve have multiple secondary follicles. Because Hippo signaling disruption alone is effective in promoting secondary follicle growth¹⁶, we recently demonstrated successful pregnancies and deliveries for POR patients using the drug-free IVA procedure involving cortical fragmentation and orthotopic grafting without treatment of the Akt stimulating drugs. Drug-free IVA stimulates early stage of ovarian follicles to develop to the preantral follicle stage after only one surgery and increased the number of retrieved oocytes for IVF-embryo transfer^15,18. The drug-free IVA approach has several advantages as compared with our original IVA by 1) avoiding potential follicle loss during culture, 2) minimizing invasiveness and costs of the second surgery, 3) involving only short-term post-surgery bed rest and 4) potential of spontaneous pregnancy due to orthotopic grafting. We recently published a video article showing the surgical procedures of drug-free IVA¹⁹ and detailed protocols of ovarian stimulation after the surgery²⁰. Here, we present details of laboratory methods required for drug-free IVA.

Protocol

Written informed consent was obtained from each POR patient with diminishing ovarian reserve who enrolled in the drug-free IVA treatment. This study was approved by ethical committee of International University of Health and Welfare (No. 17-S-21). Clinical trial was registered under number UMIN000034464 and carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).

1. Ovarian cortex extraction

1. Remove part of ovarian cortex (10 x 10 mm, 2-3 mm thickness) from one or both ovaries under general anesthesia through laparoscopic surgery as previous described^19,20.
2. Place the collected ovarian cortexes in a sterile container containing 10 mL of modified HTF (mHTF) at 37 °C.

2. Ovarian cortex fragmentation

NOTE: Prior to dissection of the ovarian cortex, warm up mHTF to 37 °C. Maintain sterile conditions throughout the procedure. All tools for this procedure are listed in Table of Materials.

1. Place the collected ovarian cortexes in a plastic dish containing 5-10 mL of mHTF at 37 °C and place them on the heat plate to maintain the tissue at 37 °C (Figure 2A).
2. Take a picture of each cortex using a digital camera with patient's name before starting next procedure to link patient background data.
3. Place an individual cortex on a sterile gauze moistened with mHTF (Figure 2B).
   NOTE: Apply additional mHTF with a disposable pipette on the gauze to prevent the surface of cortex being dried out during this procedure.
4. Remove residual medulla tissue where looks pink using a micro-scissors so that thickness of tissue reaches 1-2 mm (Figure 2C and D).
   NOTE: To avoid the loss of follicles at early stage, do not prepare the cortical strips less than 1 mm in thickness, because the early-stage follicles are located within 1-2 mm from the surface of ovarian cortex²¹.
5. Dissect a 1 mm x 1 mm x 5 mm of tissue piece from each ovarian cortical strip using a fine scalpel and subject it to histological analysis to determine the presence of residual follicles as previous described¹⁸ (Figure 2E).
   NOTE: The dissected tissues for histology are immersed in mHTF and keep at 4 °C until fixing (Figure 2F, see Step 4).
6. Cut the cortex into 1mm x 1 mm x 10 mm strips using a fine scalpel (Figure 2G).
   NOTE: It is easier to cut by pressing down on the scalpel than by pulling on it.
7. Cut these tissue strips into 1 mm x 1 mm x 1 mm small cubes (Figure 2H).
   NOTE: To avoid changes of osmotic pressure in the medium, add mHTF in moderation before tissue autografting.

3. Auto-grafting of ovarian cortical fragments

1. Open the package of the IVA cannula and put it on a sterile drape.
2. Rinse inside of the IVA cannula with mHTF by aspirating and discharging mHTF several times.
3. Aspirate 100-200 µL of mHTF to fill the tip of IVA cannula to avoid the cortical cubes from drying out during loading (Figure 3A).
4. Load the cortical cubes into the tip of IVA cannula using a fine tweezer (Figure 3B-E).
   NOTE: The number of cortical cubes for loading depends on the grafting site. Generally, 20-30 cubes can be transplanted into an orthotropic remaining ovary, whereas 10-15 cubes can be transplanted into a pouch beneath the serosa of the Fallopian tube.
5. After loading the cortical cubes, hold the tip of the cannula containing the cortical cubes by the fingers until transferring the IVA cannula to a surgeon. This will avoid the cube temperature to decreases.
   ​NOTE: The details of autografting procedure under laparoscopic surgery have been reported previously¹⁹. For grafting ovarian cortical cubes, a cannula-shaped device (IVA cannula) was developed by our collaborator, the Kitazato Corporation.

4. Histological analysis of ovarian cortex

NOTE: To count the number of residual follicles, perform histological analysis as previous described¹⁵.

1. Mark the cortical strip surface with a tissue marking dye before sample fixation. This will indicate the side of cortex in strip for histology.
2. Fix the tissue strips overnight at 4 °C using Bouin's solution.
   NOTE: To detect atretic follicles, we do not recommend using paraformaldehyde for fixing to avoid shrinkage of oocyte cytoplasm.
3. Dehydrate and embed the tissue strips in paraffin.
4. Serially section the paraffin block containing the tissue sample.
5. Stain each section using hematoxylin and eosin to visualize the follicles.
6. Count the follicles as previously described¹⁷.

Results

In the first publication of the IVA approach¹⁵, we transplanted the ovarian cortical cubes one by one into the grafting sites under laparoscopic surgery (Figure 1A). Because 100-150 ovarian cubes were used, it took 3-4 hours for tissue grafting under laparoscopic surgery. Also, some ovarian cubes were lost before grafting. Because the IVA cannula could transfer 20-30 cubes to a grafting site at one time (Figure 1B), we could shorten the o...

Discussion

In this manuscript, we showed a detailed laboratory protocol for drug-free IVA. The drug-free IVA is a new approach of infertility treatment for POR patient with diminishing ovarian reserve to promote secondary follicles growth, resulting in yielding more mature oocytes after ovarian stimulation and increasing in successful pregnancy²⁰. In 15 POR patients with diminishing ovarian reserve, this approach achieved one spontaneous pregnancy, in vitro fertilization-embryo transfer allowed four live bir...

Materials

Name	Company	Catalog Number	Comments
4.5 onz specimen container	FALCON	354013	Other products may also be suitable
60mm dish	FALCON	351007	Other products may also be suitable
50 x 50 cm sterile drape	HOGY Medical	SR-823	Any type of sterile produsts may also be suitable
Disposable pippete	FALCON	357575	Other products may also be suitable
Fine scissors, Curved	WPI	#14224-G	Although other products may also be suitable, we strongly recommend use this products
Hot plate	TOKAI HIT	TPiE-SP	Use at operation room to maintain the temperature of dishes containing ovarian tissue before transplantation
Human Serum Albumin Solution	Irvine Scientific	9988	Medium for handling ovarian tissue
IVA cannule	KITAZATO	446030 IVA-6030E	Specific cannula for tissue autografting
KAI medical Disposable scalpel	WPI	#5 10-A	Although other products may also be suitable, we strongly recommend use this products
Micro scissors, Curved	WPI	#503364	Although other products may also be suitable, we strongly recommend use this products
Modified HTF Medium-HEPES	Irvine Scientific	90126	Medium for handling ovarian tissue
Sterile gauze	Osaki Medical	15004	Any type of sterile produsts may also be suitable
Swiss Tweezers, Curved Tips	KAI	#504505	Although other products may also be suitable, we strongly recommend use this products