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.
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.
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 reported1,2. On the other hand, ovarian tissue cryopreservation was also established for fertility preservation in cancer patients but it is still an experimental strategy3,4. 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 oocytes5,6,7. 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 oocytes8,9,10. However, oocyte donation is complicated by ethical issues as well as autoimmune and pregnancy complications11,12,13,14. 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 transfer15,18. 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.
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
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.
3. Auto-grafting of ovarian cortical fragments
4. Histological analysis of ovarian cortex
NOTE: To count the number of residual follicles, perform histological analysis as previous described15.
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 operation time to 1-2 hours. Furthermore, the IVA cannula could eliminate the loss of ovarian cortical cubes during surgery.
Figure 1: The effectiveness of the IVA cannula. (A) Previous method with transplanting ovarian cortical cubes one by one using a forceps. (B) The current method with transplanting 20-30 cubes at one time using the IVA cannula. Please click here to view a larger version of this figure.
Figure 2: Ovarian tissue dissection for drug-free IVA. (A) A piece of ovarian cortex is placed in a plastic dish containing mHTF at 37 °C. (B) To remove residual medulla tissues, the ovarian cortex is placed on a moistened gauze. (C) Medulla tissue is removed using a fine micro-scissor. (D) The thickness of cortex to be prepared is between 1 to 2 mm. (E) After removal of medulla tissue, a 1 mm x 1 mm x 5 mm of tissue piece from each ovarian cortical strip is dissected for histological analysis. (F) The dissected tissue is stored in 1.5 mL tube containing mHTF at 4 °C until fixing for histology. (G and H) The ovarian cortex is cut into 1 mm x 1 mm x 10 mm strips, and then further cut into 1 mm x 1 mm x 1 mm small cubes using a fine scalpel. Please click here to view a larger version of this figure.
Figure 3: Auto-grafting of ovarian cortical fragments. (A) Before loading cubes, 100-200 µL of mHTF is aspirated to fill the tip of the IVA cannula to avoid the cortical cubes from drying out during loading (B-E) Loading ovarian cortical cubes into the tip of IVA cannula. Please click here to view a larger version of this figure.
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 births, together with one ongoing pregnancy19.
For preparation of grafting ovarian cortex, medulla tissue was removed using a micro-scissors with 1-2 mm of thickness. Previously, we demonstrated that primordial, primary and majority of secondary follicles were located within 1 mm thickness from the surface of ovarian cortex in the patients with normal ovarian reserve21. In POI patients, we found that secondary follicles were located deeper than 1 mm but within 2 mm from the surface of ovarian cortex21. Antral follicles are known to locate in medulla tissues21, but no follicle was found in medulla tissues in POI patients21. Although POR patients with diminishing ovarian reserve could have antral follicles in medulla tissue, antral follicles are difficult to survive after grafting due to lack of blood vessel communication22. Therefore, we determined the thickness of ovarian cortex to be 1-2 mm and the critical step within the protocol is not making the cortical strips < 1-2 mm to avoid loss of precious residual follicles. During preparation of cortical strips and cubes, we used mHTF, but one can use similar medium which include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer.
In this procedure, we transplanted ovarian cortex after cutting it into 1 mm3 cubes15,20. Due to the difficulty in handling such small fragments for grafting, previous grafting ovarian cubes one by one under the laparoscopic surgery took 3-4 hours with some cubes occasionally got lost during surgery. After development of the IVA cannula, we could shorten the operation time to 1-2 hours, resulting in minimizing the invasiveness of the surgery to allow the drug-free IVA becoming a single day-surgery. Moreover, we could eliminate the loss of ovarian cortical cubes during surgery. Because of limited number of follicles in ovarian cortex in POR patients with diminishing ovarian reserve, it is important to avoid loss of ovarian cortical cubes. If the IVA cannula is not available, you may try to find a similar cannula to be able to use under laparoscopic surgery.
The decline of ovarian function is progressively caused by aging and some pathophysiological conditions 23,24,25. Thus, in unmarried women with severe ovarian dysfunction, fertility preservation is important to allow future pregnancy. For these patients, the methods of oocytes or ovarian tissue cryopreservation were reported26,27,28,29. Although oocyte cryopreservation is an established method for fertility preservation1,2, these patients could have very limited number of retrievable oocytes even after ovarian hyperstimulation due to low ovarian reserve. Furthermore, in aging patients with ovarian dysfunction, high incidence of chromosome abnormalities was reported in ovulated oocytes26,30. This leads to the requirement of multiple expensive procedures to ensure sufficient number of oocytes to guarantee pregnancy. To solve these problems, we have developed the drug-free IVA approach for increasing the number of mature oocytes for oocyte retrieval. Because oocyte cryopreservation is no longer an experimental method31,32, the drug-free IVA followed by oocyte cryopreservation is expected to be a promising method for fertility preservation in unmarried women with severe ovarian dysfunction. However, future clinical studies using the freeze-thawed oocytes obtained from the drug-free IVA will be required to determine the effectiveness of this approach for fertility preservation in unmarried women with severe ovarian dysfunction.
In conclusion, we developed the drug-free IVA as a new approach of infertility treatment for POR patient with diminishing ovarian reserve and showed the detail laboratory protocol to be able to repeat our procedures. The methodological limitations of drug-free IVA are difficulty in prediction of residual follicle number before the surgery and also inability to evaluate the graft survival. The laboratory technique of drug-free IVA could become the basis of future application of ovarian tissue culture to obtain mature oocytes without grafting.
The authors have nothing to disclose.
We thank Tatsuji Ihana, Sachiyo Kurimoto, Kazuko Takahasih, Yuki Yoshizawa, Maho Arashi, Kentaro Fujita, Erina Kudo, Yuka Kurimoto and Mayuko Wakatsuki for supporting the drug-free IVA procedure and Prof. Aaron J.W. Hsueh (Stanford University School of Medicine, Stanford, CA) for critical reading and editing of the manuscript. We also thank Rebecca Truman and Gregory Truman for inserting English narration. This study was supported by The Japan Society for the Promotion of Science (JSPS), Scientific Research B (19H03801), and Challenging Exploratory Research (18K19624).
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 |