Name: fertility preservation in patients with severe ovarian dysfunction
Uploaded: 2021-03-25T13:00:00
Description: Watch this Scientific Journal Video about Fertility Preservation in Patients with Severe Ovarian Dysfunction at JoVE.com

我们感谢伊哈纳、桥本太郎、高桥正彦、吉泽裕久、麻生太郎、藤田健太郎、久藤美子、久本裕久和武松木美子支持无毒品IVA程序，以及Aaron J.W. Hsueh教授（斯坦福大学医学院，加州斯坦福）对手稿的批判性阅读和编辑。我们也感谢丽贝卡杜鲁门和格雷戈里杜鲁门插入英语旁白。这项研究得到了日本科学促进会（JSPS）、科学研究B（19H03801）和具有挑战性的探索研究（18K19624）的支持。

从每个参加无药物IVA治疗的卵巢储备减少的 POR 患者那里获得书面知情同意。这项研究得到了国际卫生和福利大学伦理委员会（第17-S-21号）的批准。临床试验登记在编号为UMIN000034464，并根据《世界医学协会道德守则》（赫尔辛基宣言）进行。
1. 卵巢皮层提取
<ol><li>通过腹腔镜手术从一个或两个卵巢中取出部分卵巢皮层（10 x10毫米，厚度2-3毫米），如前所?...

<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. 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在这份手稿中，我们展示了无药物IVA的详细实验室协议。无药物IVA是一种新的不孕症治疗方法，为POR患者与减少卵巢储备，以促进次要卵泡生长，导致产生更成熟的卵母细胞卵巢刺激后，并在成功怀孕20增加。在15名卵巢储备减少的POR患者中，这种方法实现了一次自发怀孕，体外受精-胚胎移植允许4例活产，以及1例持续妊娠19例。
为了准备?...

卵巢功能在衰老期间逐渐下降，一些病理生理疾病，包括卡约型异常、自身免疫性疾病、化疗和放射治疗以及卵巢手术。生育力保护是患有严重卵巢功能障碍的未婚妇女保持未来怀孕潜力的最佳选择之一。在生育保护方面，目前有两种方法主要在生育领域。卵母细胞冷冻保存是一个成熟的生育保护程序，许多成功的病例已经报告1，2。另一方面，卵巢组织冷冻保存也建立了为癌症患者的生育保存，但它仍然是一个实验策略3，4。在这两种方法中，怀孕需要多个数量的成熟卵母细胞。一般来说，早产卵巢不足（POI）的患者在40岁之前成为闭经，卵巢储备低的中年妇女对卵巢刺激产生成熟的卵母细胞5、6、7的反应很差。此外，蚂蚁卵泡数量少的年轻患者也向卵巢刺激7显示POR。这些患者的可检索卵母细胞数量非常有限，即使经过适当的卵巢过度刺激，因此需要多个昂贵的程序，以确保足够的卵母细胞怀孕。
卵母细胞捐赠，其次是体外受精（IVF）与丈夫的精子和胚胎移植（ET）是这些POI和POOR患者谁很难获得自己的卵母细胞8，9，10的唯一选择。然而，卵母细胞捐赠是复杂的道德问题，以及自身免疫和怀孕并发症11，12，13，14。为了解决这些问题，需要建立使用患者自身卵母细胞的不孕症治疗。对于POI患者，我们开发了体外活化（IVA）方法，使卵泡生长成功，并产生成熟的卵母细胞，导致一些怀孕和分娩15。在IVA中，我们在腹腔镜手术切除卵巢后将卵巢皮层分割，并培养卵巢两天，用Akt刺激药物激活卵泡，然后进行异位移植回到第二次腹腔镜手术15下法洛皮管下的人工袋中。这个过程促进卵巢皮层分裂后原始、初级和次要卵泡的生长，促进河马信号中断16，随后两天培养与Akt信号刺激器17。
与严重的 POI 病例相比，卵巢储备减少的 POR 患者具有多个继发性毛囊。由于河马信号中断本身是有效的促进继发性毛囊生长16，我们最近证明成功的怀孕和分娩的POOR患者使用无药物IVA程序涉及皮质碎裂和正畸移植没有治疗Akt刺激药物。无药物IVA刺激卵巢卵泡的早期阶段，在仅进行一次手术后发育到前额卵泡阶段，并增加了为IVF-胚胎移植15，18检索卵母细胞的数量。无药物IVA方法与我们原来的IVA相比有几个优点，1） 避免培养过程中潜在的毛囊损失， 2） 尽量减少第二次手术的侵入性和成本， 3） 只涉及短期手术后卧床休息和 4） 由于正交移植而自发怀孕的可能性。我们最近发表了一篇视频文章，展示了无药物IVA19的外科手术和手术后卵巢刺激的详细协议20。在这里，我们介绍无药物IVA所需的实验室方法的详细信息。

<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

卵巢功能在衰老期间和某些病理生理条件下逐渐下降，包括卡约型异常、自身免疫性疾病、化疗和放射疗法以及卵巢手术。在患有严重卵巢功能障碍的未婚妇女中，生育能力的保存对于未来的怀孕非常重要。虽然卵母细胞冷冻保存是保存生育力的既定方法，但这些患者即使在卵巢过度刺激后也只能保留数量有限的卵母细胞，导致反复刺激，以确保卵母细胞有足够的保证未来怀孕。为了解决这个问题，我们最近开发了一种无药物体外活化（IVA）程序，使我们能够刺激卵巢卵泡的早期发育到前额卵泡阶段。这些前额叶囊可以响应独特的性腺激素刺激协议，导致每次卵巢刺激的检索卵母细胞数量增加，用于冷冻保存。无药物IVA包括手术方法和卵巢刺激。在腹腔镜手术中，我们从一个或两个卵巢中取出了部分皮层。卵巢皮质组织被切成小块，以破坏河马信号通路，刺激早期卵泡的发展。这些立方体被移植到剩余的卵巢以及两个输卵管的血清下。我们已经发布了无药物IVA的外科手术和随后的卵巢刺激协议，但在这里我们介绍了无药物IVA所需的实验室方法的细节。

在IVA方法15的第一版中，我们逐一移植卵巢皮质立方体到腹腔镜手术的移植部位（图1A）。由于使用了100-150个卵巢立方体，因此在腹腔镜手术下进行组织移植需要3-4个小时。此外，一些卵巢立方体在移植前丢失。由于IVA坎努拉可以一次将20-30个立方体转移到嫁接站点（图1B），因此我们可以将操作时间缩短到1-2小时。此外，IVA坎努拉可?...

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

我们为严重卵巢功能障碍患者提供卵巢卵泡无药物体外活化 （IVA） 的实验室程序详情。这种方法可以增加每次卵巢过度刺激的可检索卵母细胞的数量，并有利于这些患者的生育能力保护。

严重卵巢功能障碍患者的生育保护

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...

这部电影的目标是展示我们治疗卵巢功能障碍患者的新方法的程序。卵巢功能在衰老期间逐渐下降，一些病理生理条件，包括卡约型异常、自身免疫性疾病、化疗和放射治疗以及卵巢手术。卵巢储备低的大多数中年妇女对卵巢刺激产生成熟卵母细胞的反应很差。此外，蚁泡数量少的年轻患者对卵巢刺激的卵巢反应也很差。生育力保护对于为未来怀孕保存潜在的 OCS 非常重要。为了保持生育能力，建立了卵母细胞冷冻保存。然而，这些卵巢反应不良的患者即使在适当的卵巢刺激后，也能检索到数量非常有限的卵母细胞，导致需要重复昂贵的手术，以确保有足够的卵母细胞来保证一个婴儿。为了解决这些问题，我们最近开发了体外活化，IVA程序，使我们能够刺激卵巢卵泡的早期阶段发展到前腹卵泡阶段。最初的IVA程序可以通过卵巢皮层分裂促进原始、原发和次生卵泡生长，导致河马信号中断。然后，用Akt信号刺激器进行两天的培养。此程序适用于残留卵泡较少的 POI 患者。然而，这个程序是一个过度治疗的患者谁有多个继发性毛囊。对于有一些残留的继发性卵泡的患者，我们建立了称为无药物IVA的方法，并且已经测试了仅仅通过破坏河马信号通路来促进二次卵泡生长的可能性。我们还成功通过无毒品 IVA 交付了一些案例。我们已经发布了无药物IVA的外科手术和遵循卵巢刺激的方案。在这段视频中，我们介绍了无药物IVA所需的实验室方法的细节。我们根据博洛尼亚标准招募了无药物IVA患者。患者可能有多个继发性毛囊。起初，部分皮层通过腹腔镜检查从一个或两个卵巢中去除。POR患者是那些有中等大小的卵巢。在大多数患者中，我们只从卵巢的一侧或两侧提取部分皮质组织。提取的关键组织立即被转移到含有 HEPES 缓冲器培养介质的培养皿中，并将这个小组织解剖成小立方体，以发出河马信号中断。此视频中将显示该过程的详细信息。河马信号通路对于保持最佳器官大小至关重要。这条途径由几个负增长的调节器组成。一旦河马信号中断，YAP的磷酸化水平就会降低，YAP的核水平就会增加， YAP 是基酶级联中的负增长调节器之一。YAP 与 TEAD 转录因子协同作用，以增加下游 CCN 增长因子。另一方面，球状作用物G-actin的电感聚合，以细丝形式F-actin，对细胞形状的维持和运动非常重要。先前报道的F-actin形成的诱导会破坏河马信号，并诱发人类 HeLa 细胞的过度生长。我们以前报告卵巢分裂增加F-actin的形成，它导致YYP磷酸化水平的降低。它导致CCN增长因子的表达增加。切割后，这些立方体被移植到剩余的卵巢中。这个过程可以促进残余的次生卵泡生长，并导致成熟卵母细胞的数量增加。无毒品 IVA 由四个步骤组成。我们已经发布了无药物IVA的外科手术。请参阅有关卵巢皮层提取和自动移植的文献。在这里，我们展示了无药物IVA所需的实验室方法的详细程序。在第一步，我们展示了卵巢皮层碎片是如何进行的。在第二步，我们展示如何将卵巢皮质立方体加载到设备中进行移植，在这里，我将显示卵巢皮层碎片。这项研究得到了我们研究所伦理委员会的批准，所有程序都是按照《世界医学会道德守则》进行的。卵巢皮层碎片。此步骤所需的以下工具：一盘、无菌纱布、细剪刀、细钳和手术刀。我们通常使用这种类型的菜和无菌纱布，但其他产品，如塑料培养皿，玻璃盘等，可能也适合这个程序，任何类型的无菌纱布也适合。然而，我们强烈建议使用这种形状的钳子和手术刀。此外，使用热板来维持无菌窗帘下含有组织位置的盘子的温度。皮质组织立即转移到含有 mHTF 的盘中。拍摄与患者姓名相关的照片，以与患者的数据相关联。记录组织的大小、厚度和状况。去除残留的美杜拉组织。在组织分裂之前，取出梅杜拉组织。将皮层放在无菌纱布上。如视频中所示，纱布用介质润湿。在此过程中，在皮层表面干燥之前，用一次性移液器涂抹介质。用微剪刀取出残留组织，直到厚度达到一到两毫米。在 POR 患者中，继发性毛囊位于离皮层表面一毫米以下的地方，因此不会使组织厚度小于一毫米。将每个组织皮层的一侧切成一个乘一个乘以五毫米的条状物，进行组织学分析，以确定残留卵泡的存在。将这条带子放入带培养介质的 1.5 毫升管中，保持在 4 摄氏度，直到固定。准备卵巢皮质立方体。去除梅杜拉组织后，卵巢皮质因河马信号中断而支离破碎。首先，用手术刀将皮层切成一个接一个的10毫米条。然后将这些条形切成一毫米立方体小立方体。在介质中，组织很滑，难以切割。为了便于切割，通过按下手术刀而不是拉扯来切割条带。切割后，数数立方体并准备自动移植。为了避免介质中渗透压力的变化，在组织自动移植之前适度添加介质。对于卵巢自动移植，使用管状设备可以更容易地加载。此步骤包含两个步骤。以前，卵巢碎片在腹腔镜下使用钳子逐一移植。然而，手术大约需要三到四个小时才能完成。在此过程中，我们使用由内注射器和外缸制成的罐状装置。这是一个简单的形状和轻量级。此外，此设备的尖端为圆形。没有可避免移植部位损坏的尖锐点。使用这种类型的设备允许一次将多达 20 到 30 个立方体带到植入站点。这导致有效的移植和减少手术时间。加载卵巢皮质碎片。IVA坎努拉的准备工作。准备IVA坎努拉，用mHTF清洗此设备。重复吸气和放电介质几次。通过吸气约100微升的介质来填充罐头。这是为了避免立方体在组织碎片加载过程中拔出。加载皮质立方体。用细钳挑选一个立方体，并将其放入罐头的尖端。自动移植立方体的数量取决于移植部位的大小。重复逐个加载立方体，直到您达到目标编号。加载立方体后，小心地将管子传递给外科医生。如果移植部位尚未准备好，则包含卵巢立方体的管状部分将由手指保持，以避免立方体温度降低。我们已经报告了无药物IVA的临床结果。请参阅已发表的关于临床结果的论文。此实验室协议要求一名患者需要 30 到 50 分钟。卵巢皮层碎片化的平均时间为10至20分钟，将这些碎片加载到IVA坎努拉的平均时间为20至30分钟。这取决于组织大小，但几乎所有的患者都可以在这个平均时间完成。结论。无药IVA是针对卵巢储备减少的 POR 患者的不孕症治疗新方法。这些实验室协议可减少手术所需的时间和移植过程中碎片的丢失。无毒IVA的实验室技术可以成为卵巢组织培养未来应用的基础，在不嫁接的情况下获得成熟的卵母细胞。

Research

JoVE Journal

Medicine

A Contusion Model of Severe Spinal Cord Injury in Rats

挫伤模型脊髓严重损伤的保护作用

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

科研

医学

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 ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

血管功能在慢性肾脏疾病的评估

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

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

功能人类肝脏保存和恢复由Subnormothermic机灌注手段

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

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

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

囊内旋切手术 (ircp) 的介绍: 一种改良子宫肌瘤切除术促进生育保护

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

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

[18f] fdg 示踪剂在重型颅脑损伤患者中的半定量评价

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

牛磺胆酸钠诱导C57BL / 6小鼠严重急性胰腺炎

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

Digital Handwriting Analysis of Characters in Chinese Patients with Mild Cognitive Impairment

中国轻度认知障碍患者人物数字笔迹分析

An increasing amount of evidence shows that cognitive deficits and movement dysfunctions are not separated. Patients with mild cognitive impairment (MCI) ...

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

通过卵母细胞维他化保存生育能力：临床和实验室视角

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

使用二氧化碳光纤激光汽化卵巢子宫内膜异位瘤的生育保留程序

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