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).

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
<ol>
	<li>Remove part of ovarian cortex (10 x 10...

<ol>
	<li>Liang, T., Motan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+Oocyte+Cryopreservation+for+Fertility+Preservation.">Mature Oocyte Cryopreservation for Fertility Preservation.</a> Advances in Experimental Medicine and Biology. 951, 155-161 (2016).</li><li>Yoon, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+births+after+vitrification+of+oocytes+in+a+stimulated+in+vitro+fertilization-embryo+transfer+program.">Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program.</a> Fertility and Sterility. 79 (6), 1323-1326 (2003).</li><li>Donnez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Livebirth+after+orthotopic+transplantation+of+cryopreserved+ovarian+tissue.">Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.</a> Lancet. 364 (9443), 1405-1410 (2004).</li><li>Meirow, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnancy+after+transplantation+of+cryopreserved+ovarian+tissue+in+a+patient+with+ovarian+failure+after+chemotherapy.">Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy.</a> New England Journal of Medicine. 353 (3), 318-321 (2005).</li><li>De Vos, M., Devroey, P., Fauser, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ovarian+insufficiency.">Primary ovarian insufficiency.</a> Lancet. 376 (9744), 911-921 (2010).</li><li>Scott, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicle-stimulating+hormone+levels+on+cycle+day+3+are+predictive+of+in+vitro+fertilization+outcome.">Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome.</a> Fertility and Sterility. 51 (4), 651-654 (1989).</li><li>Ferraretti, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESHRE+consensus+on+the+definition+of+'poor+response'+to+ovarian+stimulation+for+in+vitro+fertilization:+the+Bologna+criteria.">ESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertilization: the Bologna criteria.</a> Human Reproduction. 26 (7), 1616-1624 (2011).</li><li>Huhtaniemi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+Molecular+Pathophysiology,+Genetics,+and+Treatment+of+Primary+Ovarian+Insufficiency.">Advances in the Molecular Pathophysiology, Genetics, and Treatment of Primary Ovarian Insufficiency.</a> Trends in Endocrinology and Metabolism. 29 (6), 400-419 (2018).</li><li>M&#281;czekalski, B., Maciejewska-Jeske, M., Podfigurna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproduction+in+premature+ovarian+insufficiency+patients+-+from+latest+studies+to+therapeutic+approach.">Reproduction in premature ovarian insufficiency patients - from latest studies to therapeutic approach.</a> Prz Menopauzalny. 17 (3), 117-119 (2018).</li><li>Baker, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+plans+and+family-building+options+for+women+with+primary+ovarian+insufficiency.">Life plans and family-building options for women with primary ovarian insufficiency.</a> Seminars in Reproductive Medicine. 29 (4), 362-372 (2011).</li><li>Englert, Y., Govaerts, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+donation:+particular+technical+and+ethical+aspects.">Oocyte donation: particular technical and ethical aspects.</a> Human Reproduction. 13, 90-97 (1998).</li><li>Englert, Y., Rodesch, C., Laruelle, C., Govoerts, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+donation:+ethical+aspects+related+to+the+donor.">Oocyte donation: ethical aspects related to the donor.</a> Contraception, Fertilite, Sexualite. 25 (3), 251-257 (1997).</li><li>Storgaard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obstetric+and+neonatal+complications+in+pregnancies+conceived+after+oocyte+donation:+a+systematic+review+and+meta-analysis.">Obstetric and neonatal complications in pregnancies conceived after oocyte donation: a systematic review and meta-analysis.</a> BJOG: An International Journal of Obstetrics and Gynaecology. 124 (4), 561-572 (2017).</li><li>Storgaard, M., Malchau, S., Loft, A., Larsen, E., Pinborg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+donation+is+associated+with+an+increased+risk+of+complications+in+the+pregnant+woman+and+the+fetus.">Oocyte donation is associated with an increased risk of complications in the pregnant woman and the fetus.</a> Ugeskrift for Laeger. 179 (11), (2017).</li><li>Kawamura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippo+signaling+disruption+and+Akt+stimulation+of+ovarian+follicles+for+infertility+treatment.">Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.</a> Proceedings of the National Academy of Sciences. 110 (43), 17474-17479 (2013).</li><li>Hsueh, A. J., Kawamura, K., Cheng, Y., Fauser, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraovarian+control+of+early+folliculogenesis.">Intraovarian control of early folliculogenesis.</a> Endocrine Reviews. 36 (1), 1-24 (2015).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+dormant+ovarian+follicles+to+generate+mature+eggs.">Activation of dormant ovarian follicles to generate mature eggs.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (22), 10280-10284 (2010).</li><li>Suzuki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+fertility+preservation+following+ovarian+tissue+vitrification+in+patients+with+primary+ovarian+insufficiency.">Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency.</a> Human Reproduction. 30 (3), 608-615 (2015).</li><li>Tanaka, Y., Hsueh, A. J., Kawamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+approaches+of+drug-free+in+activation+and+laparoscopic+ovarian+incision+to+treat+patients+with+ovarian+infertility.">Surgical approaches of drug-free in activation and laparoscopic ovarian incision to treat patients with ovarian infertility.</a> Fertility and Sterility. , (2020).</li><li>Kawamura, K., Ishizuka, B., Hsueh, A. J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-free+in-vitro+activation+of+follicles+for+infertility+treatment+in+poor+ovarian+response+patients+with+decreased+ovarian+reserve.">Drug-free in-vitro activation of follicles for infertility treatment in poor ovarian response patients with decreased ovarian reserve.</a> Reproductive Biomedicine Online. 40 (2), 245-253 (2020).</li><li>Haino, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Follicular+Localization+in+Human+Ovarian+Cortex+for+Vitrification.">Determination of Follicular Localization in Human Ovarian Cortex for Vitrification.</a> Journal of Adolescent and Young Adult Oncology. 7 (1), 46-53 (2018).</li><li>Baird, D. T., Webb, R., Campbell, B. K., Harkness, L. M., Gosden, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+ovarian+function+in+sheep+after+ovariectomy+and+transplantation+of+autografts+stored+at+-196+C.">Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at -196 C.</a> Endocrinology. 140 (1), 462-471 (1999).</li><li>Qin, Y., Jiao, X., Simpson, J. L., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+primary+ovarian+insufficiency:+new+developments+and+opportunities.">Genetics of primary ovarian insufficiency: new developments and opportunities.</a> Human Reproduction Update. 21 (6), 787-808 (2015).</li><li>Domniz, N., Meirow, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+ovarian+insufficiency+and+autoimmune+diseases.">Premature ovarian insufficiency and autoimmune diseases.</a> Best Practice &amp; Research: Clinical Obstetrics &amp; Gynaecology. 60, 42-55 (2019).</li><li>Laven, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Ovarian+Insufficiency.">Primary Ovarian Insufficiency.</a> Seminars in Reproductive Medicine. 34 (4), 230-234 (2016).</li><li>Grynberg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+preservation+in+Turner+syndrome.">Fertility preservation in Turner syndrome.</a> Fertility and Sterility. 105 (1), 13-19 (2016).</li><li>Tomao, F., Spinelli, G. P., Panici, P. B., Frati, L., Tomao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+function,+reproduction+and+strategies+for+fertility+preservation+after+breast+cancer.">Ovarian function, reproduction and strategies for fertility preservation after breast cancer.</a> Critical Reviews in Oncology/Hematology. 76 (1), 1-12 (2010).</li><li>Kim, S., Lee, Y., Lee, S., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+tissue+cryopreservation+and+transplantation+in+patients+with+cancer.">Ovarian tissue cryopreservation and transplantation in patients with cancer.</a> Obstetrics &amp; Gynecology Science. 61 (4), 431-442 (2018).</li><li>Donnez, J., Dolmans, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+tissue+freezing:+current+status.">Ovarian tissue freezing: current status.</a> Current Opinion in Obstetrics and Gynecology. 27 (3), 222-230 (2015).</li><li>Wu, R. C., Kuo, P. L., Lin, S. J., Liu, C. H., Tzeng, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+mosaicism+in+patients+with+recurrent+abortion+or+premature+ovarian+failure.">X chromosome mosaicism in patients with recurrent abortion or premature ovarian failure.</a> Journal of the Formosan Medical Association. 92 (11), 953-956 (1993).</li><li>De Munck, N., Vajta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficiency+of+oocyte+vitrification.">Safety and efficiency of oocyte vitrification.</a> Cryobiology. 78, 119-127 (2017).</li><li>Argyle, C. E., Harper, J. C., Davies, M. 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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...

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&#39;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&#39; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4.5 onz specimen container</td>
			<td>FALCON</td>
			<td>354013</td>
			<td>Other products may also be suitable</td>
		</tr>
		<tr>
			<td>60mm dish</td>
			<td>FALCON</td>
			<td>351007</td>
			<td>Other products may also be suitable</td>
		</tr>
		<tr>
			<td>50 x 50 cm sterile drape</td>
			<td>HOGY Medical</td>
			<td>SR-823</td>
			<td>Any type of sterile produsts may also be suitable</td>
		</tr>
		<tr>
			<td>Disposable pippete</td>
			<td>FALCON</td>
			<td>357575</td>
			<td>Other products may also be suitable</td>
		</tr>
		<tr>
			<td>Fine scissors, Curved</td>
			<td>WPI</td>
			<td>#14224-G</td>
			<td>Although other products may also be suitable, we strongly recommend use this products</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>TOKAI HIT</td>
			<td>TPiE-SP</td>
			<td>Use at operation room to maintain the temperature of dishes containing ovarian tissue before transplantation</td>
		</tr>
		<tr>
			<td>Human Serum Albumin Solution</td>
			<td>Irvine Scientific</td>
			<td>9988</td>
			<td>Medium for handling ovarian tissue</td>
		</tr>
		<tr>
			<td>IVA cannule</td>
			<td>KITAZATO</td>
			<td>446030 IVA-6030E</td>
			<td>Specific cannula for tissue autografting</td>
		</tr>
		<tr>
			<td>KAI medical Disposable scalpel</td>
			<td>WPI</td>
			<td>#5 10-A</td>
			<td>Although other products may also be suitable, we strongly recommend use this products</td>
		</tr>
		<tr>
			<td>Micro scissors, Curved</td>
			<td>WPI</td>
			<td>#503364</td>
			<td>Although other products may also be suitable, we strongly recommend use this products</td>
		</tr>
		<tr>
			<td>Modified HTF Medium-HEPES</td>
			<td>Irvine Scientific</td>
			<td>90126</td>
			<td>Medium for handling ovarian tissue</td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Osaki Medical</td>
			<td>15004</td>
			<td>Any type of sterile produsts may also be suitable</td>
		</tr>
		<tr>
			<td>Swiss Tweezers, Curved Tips</td>
			<td>KAI</td>
			<td>#504505</td>
			<td>Although other products may also be suitable, we strongly recommend use this products</td>
		</tr>
	</tbody>
</table>

fertility preservation in patients with severe ovarian dysfunction

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.

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...

Watch this Scientific Journal Video about Fertility Preservation in Patients with Severe Ovarian Dysfunction at JoVE.com

Fertility Preservation in Patients with Severe Ovarian Dysfunction

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, ...

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.

fertility-preservation-in-patients-with-severe-ovarian-dysfunction

Research

JoVE Journal

Medicine

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Introduction of Intracapsular Rotary-cut Procedures (IRCP): A Modified Hysteromyomectomy Procedures Facilitating Fertility Preservation

Uterine fibroids are common benign tumors in the female reproductive system. A hysterectomy is the most effective treatment for symptomatic fibroids. For patients desiring pregnancy, laparoscopic intracapsular myomectomy (LM) is an alternative surgery option. Although LM is widely accepted to treat fibroids, it is technically demanding with risk of excessive bleeding and difficult suturing, especially in cases with large fibroids or tumors in unusual locations. Therefore, we developed an intracapsular rotary-cut procedure (IRCP) as a modification of laparoscopic intracapsular myomectomy, with the intention to minimize risks of LM and help uterine healing. A summary of the improvements to the IRCP is described: 1) making an incision at the site of the fibroid with a length of one-third to one-half of the fibroid&#39;s diameter at a depth reaching the fibroid&#39;s surface; 2) holding the fibroid stably and making rotary cuts on the fibroid at a depth of 0.5&#8211;1 cm within its pseudo-capsule while pulling it outward slightly, making sure not to cut off any pieces of the fibroid; and 3) repeating the cutting-and-pulling until the longest dimension of the fibroid is outside the incision. The multiple cuts are to minimize the diameter and extend the length of the fibroid. When the multiple cuts cause half of the fibroid body to &#34;shrink&#34;, the fibroid is squeezed out by contraction of the surrounding myometrium. Evaluation of the outcomes of IRCP showed that the time of enucleation and suturing, intraoperative bleeding, and decline of hemoglobin were significantly lower in the IRCP group than the LM group. As for reproductive outcomes, the full-term live birth rate of the IRCP group was significantly higher than that of LM group. However, there was no difference in delivery modes between the two groups. In conclusion, IRCP significantly benefits fertility preservation by minimizing damage to the uterus, protecting myofibers of the pseudo-capsule, and resulting in a shallower residual cavity, which eases stitching and causes less bleeding. It is worthwhile to adopt IRCP in younger patients who desire preservation of their fertility.

Introduction of Intracapsular Rotary-cut Procedures IRCP: A Modified Hysteromyomectomy Procedures Facilitating Fertility Preservation

Here, we present a protocol for performing an intracapsular rotary-cut procedure (IRCP), a modified laparoscopic intracapsular myomectomy that promotes fertility preservation.

Uterine fibroids are common benign tumors in the female reproductive system. A hysterectomy is the most effective treatment for symptomatic fibroids. For ...

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of disorders of consciousness, disrupted higher brain function, and verbal disturbances. As a consequence of an insufficient ability to communicate, objective evaluations are needed from family members, medical staff, and caregivers. One such evaluation is the assessment of functioning brain areas. Recently, multimodal brain imaging has been used to explore the function of damaged brain areas. [18F]-fluorodeoxyglucose positron emission tomography-computed tomography ([18F]FDG-PET/CT) is a successful tool for examining brain function. However, the assessment of brain glucose metabolism based on [18F]FDG-PET/CT is not standardized and depends on several varying parameters, as well as the patient&#39;s condition. Here, we describe a series of semiquantitative assessment protocols for a region-of-interest (ROI) image analysis using self-produced [18F]FDG tracers in patients with sTBI. The protocol focuses on screening the participants, preparing the [18F]FDG tracer in the hot lab, scheduling the acquisition of [18F]FDG-PET/CT brain images, and measuring glucose metabolism using the ROI analysis from a targeted brain area.

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

[18F]-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography is useful for studying glucose metabolism related to brain function. Here, we present a protocol for an [18F]FDG tracer set-up and semiquantitative assessment of the region-of-interest analysis for targeted brain areas associated with clinical manifestations in patients with severe traumatic brain injury.

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of ...

Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the human clinical condition and reproduction of inflammatory events corresponding to the onset of clinical biliary pancreatitis. The severity of pancreatic damage can be assessed by measuring the concentration, speed, and volume of the infused bile acid. This study provides an updated checklist of the materials and methods used in the protocol reproduction and shows the main results from this acute pancreatitis (AP) model. Most of the previous publications have limited themselves to reproducing this model in rats. We have applied this method in mice, which provides additional advantages (i.e., the availability of an arsenal of reagents and antibodies for these animals along with the possibility of working with genetically modified strains of mice) that may be relevant to the study. For acute pancreatitis induction in mice, we present a systematic protocol, with a defined dose of 2.5% sodium taurocholate at an infusion speed 10 &#181;L/min for 3 min in C57BL/6 mice that reaches its maximal level of severity within 12 h of induction and highlight results with outcomes that validate the method. With practice and technique, the total estimated time, from the induction of anesthesia to the completion of the infusion, is 25 min per animal.

Animal models for severe acute pancreatitis enable the study of pathophysiological changes at the initial stage, facilitating observation of the evolution of inflammatory events. Here we provide a protocol for the induction of severe acute biliary pancreatitis by retrograde infusion of sodium taurocholate into the pancreatic duct of anesthetized C57BL/6 mice.

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the ...

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety.

Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation treatments.

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation ...

Fertility Sparing Procedure using Carbon Dioxide Fiber Laser Vaporization of Ovarian Endometrioma

The surgical management of endometrioma is still a matter of debate. Cystectomy, which is recognized as the standard technique, seems to be associated with a potential reduction in the ovarian reserve due to the inadvertent removal and thermal damage of healthy ovarian tissue. New ablative techniques with reduced tissue penetration depth and less thermal spread to the surrounding parenchyma may represent a viable alternative to cystectomy. For these reasons, the aim of this manuscript is to demonstrate the ablation of the endometrioma capsule using a CO2 fiber laser technique and discuss the clinical outcomes. Once the cyst has been drained and washed, a biopsy is taken. After cyst eversion, vaporization of the inner surface of the cyst is performed using a CO2 fiber laser. The technique is simple and reproducible as even young surgeons without any surgical experience were more confident in performing laser CO2 vaporization instead of cystectomy. The positive effects of CO2 technology are reported in a randomized controlled trial, where the postoperative changes in the antral follicular count (AFC) and antimullerian hormone (AMH) levels were compared between patients who had their endometrioma excised (cystectomy) and those who had undergone endometrioma vaporization with CO2 laser. The patients treated with CO2 laser showed significantly increased AFC without a reduction in serum AMH levels as compared to the cystectomy group, in which both parameters were significantly reduced. The postoperative pregnancy rate was also assessed, and comparable pregnancy rates were found after both treatments. On the contrary, patients treated with the CO2 fiber laser technique had more favorable in-vitro fertilization (IVF) outcomes compared to cystectomy.
In conclusion, the CO2 fiber laser technique may represent a viable alternative to cystectomy in the surgical treatment of endometrioma in terms of ovarian preservation, pregnancy rates, and IVF outcomes. Moreover, it has the advantage of being independent of the surgeon's skills and personal experience.

In this protocol, CO2 fiber laser technique is demonstrated for the surgical treatment of ovarian endometriosis, which represents a viable alternative in terms of fertility preservation with the major advantage of not being dependent on the surgeon's skills and personal experience.

The surgical management of endometrioma is still a matter of debate. Cystectomy, which is recognized as the standard technique, seems to be associated ...

Enema of Traditional Chinese Medicine for Patients with Severe Acute Pancreatitis

Severe acute pancreatitis (SAP) is characterized by high mortality rates, numerous complications, and extreme difficulties in treatment. A traditional Chinese medicine (TCM) enema has been widely utilized in clinical and experimental studies of SAP. It has been demonstrated to have beneficial effects in protecting pancreatic function and delaying disease progression. The procedure of TCM enema is to perfuse the Chinese medicinal liquid of raw rhubarb (Rheum officinale Baill. DaHuang) from the anus into the rectum and colon. Basic steps of the procedure include boiling the herbal solution in advance, using a disposable enema kit to assist patients in taking a left lying position, raising the patient's hip 10 cm, and carefully inserting the enema 30-35 cm into the anus, with a drip speed of 60-80 drops/min. The medicine liquid temperature should be equivalent to the patient's body temperature, typically between 37-39 &#176;C. The end of the enema operation should be slow, and Tui na (massage therapy) should be administered before extubation. The medication liquid should be kept in the rectum for more than 1 h. After issuing an enema, it is essential to observe the patient's feces and abdomen, and to evaluate the remission of abdominal signs and symptoms.

An enema is a type of traditional Chinese medicine (TCM) therapy used to treat severe acute pancreatitis (SAP). The therapy involves instilling a TCM liquid into the rectum and colon through the anus to keep the liquid in the intestine, absorbing through the intestinal mucosa. This study aims to standardize the enema procedure for SAP patients.

Severe acute pancreatitis (SAP) is characterized by high mortality rates, numerous complications, and extreme difficulties in treatment. A traditional ...

Tai Chi with Mindfulness Training to Manage Diabetic Peripheral Neuropathy

Effects of Mindfulness Training Combined with Tai Chi in Patients with Diabetic Peripheral Neuropathy

Diabetic peripheral neuropathy (DPN) is one of the most prevalent chronic complications of diabetes. One of its crucial therapy approaches is mind-body exercise. Recently, various exercise modalities, including stepping, resistance, aerobics, balance, and whole-body vibration, were investigated to construct the most suitable form of exercise for patients with DPN. The purpose of this study is to describe a standard protocol for mindfulness training combined with Tai Chi. The convenience sampling method was used to select 90 patients with DPN who met the inclusion and exclusion criteria from three communities. The three communities were randomly divided into the control group (CG), the Tai Chi group (TCG), and the mindfulness training combined with the Tai Chi group (MTCG). The CG was given routine health education guidance once a month, a total of three times. Based on the CG, the TCG practiced Tai Chi three times; the MTCG received mindfulness training combined with Tai Chi exercise a week for a total of 12 weeks. Before the intervention and 12 weeks after the intervention, the clinical symptoms, neurological function, attention awareness level, pain, and quality of life of the subjects were evaluated by Toronto Clinical Scoring System (TCSS), Mindful Attention Awareness Scale (MAAS), Visual analog scale (VAS), Diabetes Specificity Quality of life Scale (DSQL) and tumor necrosis factor-&#945;. Overall, the addition of mindfulness training to Tai Chi effectively enhances the exercise effects of Tai Chi. Therefore, mindfulness training combined with Tai Chi is worthy of promotion and application.

Author Spotlight: Integrating Tai Chi with Mindfulness Training to Achieve an Effective Mind-Body Exercise 

When studying exercise for treating diabetic peripheral neuropathy (DPN), achieving mind-body exercise is essential. This study proposes a protocol for mindfulness training combined with Tai Chi to achieve mind-body exercise.

Diabetic peripheral neuropathy (DPN) is one of the most prevalent chronic complications of diabetes. One of its crucial therapy approaches is mind-body ...