Suzuki Nao, Yoshioka Nobuhito, Takae Seido, Sugishita Yodo, Tamura Midori, Hashimoto Shu, Morimoto Yoshiharu, Kawamura Kazuhiro
Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamaeku, Kawasaki, Kanagawa 216-8511, Japan.
IVF Namba Clinic, Osaka, Osaka 550-0015, Japan.
Hum Reprod. 2015 Mar;30(3):608-15. doi: 10.1093/humrep/deu353. Epub 2015 Jan 6.
Is ovarian tissue cryopreservation using vitrification followed by in vitro activation (IVA) of dormant follicles a potential approach for infertility treatment of patients with primary ovarian insufficiency (POI)?
Our vitrification approach followed by IVA treatment is a potential infertility therapy for POI patients whose ovaries contain residual follicles.
Akt (protein kinase B) stimulators [PTEN (phosphatase with TENsin homology deleted in chromosome 10) inhibitor and phosphatidyinositol-3-kinase (PI3 kinase) stimulator] activate dormant primordial follicles in vitro and ovarian fragmentation disrupts the Hippo signaling pathway, leading to the promotion of follicle growth. We treated POI patients with a combination of ovarian vitrification, fragmentation and drug treatment, followed by auto-transplantation, and reported successful follicle growth and pregnancies.
STUDY DESIGN, SIZE, DURATION: Prospective clinical study of 37 infertile women with POI between 12 August 2011 and 1 November 2013. We enrolled 10 new patients since the previous publication.
PARTICIPANTS/MATERIALS, SETTING, METHODS: POI patients were originally selected based on a history of amenorrhea for more than 1 year and elevated serum FSH levels of >40 mIU/ml (n = 31) but this was later changed to >4 months, age <40 years and serum FSH levels of >35 mIU/ml (n = 6) (mean 71.8 ± 30.8, range 35.5-197.6) so as to include patients with a shorter duration of amenorrhea. Under laparoscopic surgery, ovariectomy was performed and ovarian cortices were dissected into strips for vitrification. Some pieces were examined histologically. After warming, two to three strips were fragmented into smaller cubes before culturing with Akt stimulators for 2 days. After washing, ovarian cubes were transplanted beneath the serosa of Fallopian tubes under laparoscopic surgery. Follicle growth was monitored by ultrasound and serum estrogen levels. After oocyte retrieval from mature follicles, IVF was performed.
Among 37 patients, 54% had residual follicles based on histology. Among patients with follicles, 9 out of 20 showed follicle growth in auto-grafts with 24 oocytes retrieved from six patients. Following IVF and embryo transfer into four patients, three pregnancies were detected based on serum hCG, followed by one miscarriage and two successful deliveries. For predicting IVA success, we found that routine histological analyses of ovarian cortices and shorter duration from initial POI diagnosis to ovariectomy are valid parameters.
LIMITATIONS, REASONS FOR CAUTION: Although our findings suggest that the present vitrification protocol is effective for ovarian tissue cryopreservation, we have not compared the potential of vitrification and slow freezing in follicle growth after grafting. We chose the serosa of Fallopian tubes as the auto-grating site due to its high vascularity and the ease to monitor follicle growth. Future studies are needed to evaluate the best auto-grafting sites for ovarian tissues. Also, future studies are needed to identify biological markers to indicate the presence of residual follicles in POI to predict IVA treatment outcome.
In POI patients, ovarian reserve, namely the pool of residual follicles, continues to diminish with age. If one ovary is cryopreserved at an earlier stage of POI, patients could undergo additional non-invasive infertility treatments before the final decision for the IVA treatment. Furthermore, in the cases of unmarried POI patients, cryopreservation of ovarian tissues allows their fertility preservation until they desire to bear children.
STUDY FUNDING/COMPETING INTERESTS: This work was supported by Grant-In-Aid for Scientific Research (Research B: 24390376, Challenging Exploratory Research: 24659722, and Innovative Areas, Mechanisms regulating gamete formation in animals: 26114510) and by research funds from the Smoking Research Foundation, and the Takeda Science Foundation. None of the authors has a conflict of interest.
UMIN000010828.
对于原发性卵巢功能不全（POI）患者，采用玻璃化冷冻卵巢组织并随后对休眠卵泡进行体外激活（IVA）是否是一种潜在的不孕治疗方法？
我们的玻璃化冷冻方法结合IVA治疗对于卵巢中含有残余卵泡的POI患者是一种潜在的不孕治疗方法。
Akt（蛋白激酶B）刺激剂[PTEN（第10号染色体缺失的张力蛋白同源磷酸酶）抑制剂和磷脂酰肌醇-3-激酶（PI3激酶）刺激剂]可在体外激活休眠的原始卵泡，卵巢分割可破坏Hippo信号通路，从而促进卵泡生长。我们对POI患者进行了卵巢玻璃化冷冻、分割和药物治疗相结合的治疗，随后进行自体移植，并报告了卵泡成功生长和妊娠情况。
研究设计、规模、持续时间：2011年8月12日至2013年11月1日对37例POI不孕女性进行的前瞻性临床研究。自上次发表以来，我们招募了10名新患者。
参与者/材料、设置、方法：最初根据闭经1年以上且血清FSH水平>40 mIU/ml（n = 31）的病史选择POI患者，但后来改为闭经>4个月、年龄<40岁且血清FSH水平>35 mIU/ml（n = 6）（平均71.8±30.8，范围35.5 - 197.6），以便纳入闭经时间较短的患者。在腹腔镜手术下，进行卵巢切除术，将卵巢皮质切成条带进行玻璃化冷冻。部分组织块进行组织学检查。解冻后，将两到三条切成较小的方块，然后与Akt刺激剂一起培养2天。洗涤后，在腹腔镜手术下将卵巢方块移植到输卵管浆膜下。通过超声和血清雌激素水平监测卵泡生长。从成熟卵泡中取出卵母细胞后，进行体外受精。
37例患者中基于组织学检查54%有残余卵泡。在有卵泡的患者中，20例中有9例自体移植卵泡生长，从6例患者中取出24个卵母细胞。在对4例患者进行体外受精和胚胎移植后，根据血清hCG检测到3例妊娠，随后1例流产，2例成功分娩。对于预测IVA成功，我们发现卵巢皮质的常规组织学分析以及从最初POI诊断到卵巢切除术的较短时间是有效的参数。
局限性、谨慎理由：尽管我们的研究结果表明目前的玻璃化冷冻方案对卵巢组织冷冻保存有效，但我们尚未比较玻璃化冷冻和慢速冷冻在移植后卵泡生长方面的潜力。由于输卵管浆膜血管丰富且易于监测卵泡生长，我们选择其作为自体移植部位。未来需要研究评估卵巢组织的最佳自体移植部位。此外，未来需要研究确定生物标志物以指示POI中残余卵泡的存在，从而预测IVA治疗结果。
在POI患者中，卵巢储备即残余卵泡池会随着年龄持续减少。如果在POI的早期阶段冷冻一个卵巢，患者在最终决定进行IVA治疗之前可以接受额外的非侵入性不孕治疗。此外，对于未婚POI患者，冷冻卵巢组织可使其保留生育能力直至她们希望生育。
研究资金/利益冲突：本研究得到科学研究资助（研究B：24390376、挑战性探索性研究：24659722以及创新领域，动物配子形成的调控机制：26114510）、吸烟研究基金会研究资金以及武田科学基金会的支持。作者均无利益冲突。
UMIN000010828
