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Fertility Preservation in Patients with Severe Ovarian Dysfunction

Published: March 25th, 2021



1Advanced Reproduction Medicine Research Center, Department of Obstetrics and Gynecology, International University of Health and Welfare School of Medicine

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 tissu....

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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.......

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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.......

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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.......

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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).


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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

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